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Bioluminescence-Based Determination of Cytosolic Accumulation of Antibiotics in Escherichia coli
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Bioluminescence-Based Determination of Cytosolic Accumulation of Antibiotics in Escherichia coli
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ACS Infectious Diseases

Cite this: ACS Infect. Dis. 2024, 10, 5, 1602–1611
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https://doi.org/10.1021/acsinfecdis.3c00684
Published April 9, 2024

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Abstract

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Antibiotic resistance is an alarming public health concern that affects millions of individuals across the globe each year. A major challenge in the development of effective antibiotics lies in their limited ability to permeate cells, noting that numerous susceptible antibiotic targets reside within the bacterial cytosol. Consequently, improving the cellular permeability is often a key consideration during antibiotic development, underscoring the need for reliable methods to assess the permeability of molecules across cellular membranes. Currently, methods used to measure permeability often fail to discriminate between the arrival within the cytoplasm and the overall association of molecules with the cell. Additionally, these techniques typically possess throughput limitations. In this work, we describe a luciferase-based assay designed for assessing the permeability of molecules in the cytosolic compartment of Gram-negative bacteria. Our findings demonstrate a robust system that can elucidate the kinetics of intracellular antibiotic accumulation in live bacterial cells in real time.

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Antibiotic resistance is a growing global problem that directly leads to increased risks associated with bacterial infections. Recent data reveal that antibiotic resistance was responsible for nearly 5 million fatalities in 2019. (1) A primary driver of the resistance phenotype is the overuse and misuse of antibiotics in human medicine and agriculture, as well as the lack of development of new antibiotics. (2) Infections that are highly resistant can lead to prolonged illness, increased healthcare costs, and higher mortality rates. Urgent measures, including responsible antibiotic stewardship, innovative drug development, and public awareness, are essential to combating this pressing threat to modern medicine. Consequently, it is important to prioritize strategies aimed at circumvention of antibiotic resistance.
Among the primary hurdles in the development of effective antibiotics is their general lack of cellular permeability. (3) This challenge is particularly pronounced when targeting Gram-negative bacteria and mycobacteria due to their additional membranes that pose barriers to molecular entry. Compounding this issue is the fact that some of the most crucial drug targets are situated within the bacterial cytosol, emphasizing the need for permeable antibiotics in combatting bacterial infections. (4) In the pursuit of novel antibiotics, it becomes critically important to develop methodologies that can reliably report on molecule accumulation in bacteria with high efficiency. (5−12) Currently, several methods are available to evaluate the permeability of molecules in Gram-negative bacteria. The most widely used method of LC–MS/MS does not require a chemical tag to be added to the test molecule. (13,14) However, its widespread adoption has been significantly hampered by inherent throughput limitations, limiting its broad application in the field. Another widely used approach involves optical analysis of cells that are treated with compounds chemically modified with a fluorophore to track their entry into the cell. (15,16) Critically, these methods often fail to report on whether the molecules arrive within the cytoplasmic space and, instead, provide information on the total association of the molecules with the target cells. (17)
Our group has recently described methods to interrogate the accumulation of molecules onto the surface of Gram-positive bacteria and past the outer membrane of diderm bacteria using a combination of click chemistry (18,19) and HaloTag. (20) Herein, we sought to establish a luciferase-based assay (21) to determine the accumulation of molecules to the cytosol of Gram-negative bacteria in real time. In this assay, luciferase-expressing bacteria in the presence of 6-hydroxy-2-cyanobenzothiazole (CBT–OH) are incubated with test molecules tagged via a disulfide bond to d-cysteine (d-cys) (22) (Figure 1a). Upon the arrival of the conjugate in the reducing environment of the cytosol, (23) reduction of the disulfide bond to generate d-cys in the cytosol enables a fast and biorthogonal (24) recombination of CBT and d-cys to generate intracellular d-luciferin that is rapidly processed by luciferase to generate light (Figures 1a; S1). (25) We showed that the system was robust, displayed a high signal-to-noise ratio, and revealed the kinetics of the intracellular accumulation of antibiotics.

Figure 1

Figure 1. (a) Schematic showing the workflow of DCCAA. (b) SDS-PAGE analysis of luciferase protein expression in E. coli at 2 or 26 h post IPTG induction. A molecular weight ladder with sizes in kilodaltons (kDa) is shown. The expected molecular weight of luciferase is 62 kDa.

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Results and Discussion

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The reaction between aminothiols (including cysteines) and CBT exhibits numerous favorable characteristics, including reaction speed, ease of use, and exceptional specificity. This reaction has been leveraged in a variety of applications including protein labeling, (26−28) molecular imaging, (29) and to investigate the permeability of peptides into mammalian cells. (21) Alternatively, mice expressing luciferase have been used to detect endogenous d-cys in the brain of animals. (30,31) We identified many advantages of the d-cys cytosolic accumulation assay (DCCAA). DCCAA generates real time measurement upon singular cellular treatment (no washing steps or chase treatment required), thereby reducing the number of assay manipulation steps. Additionally, the assay workflow is compatible with multiwell plates thus enabling high-throughput analysis. Finally, bioluminescence signals are more compatible with bacterial species that have intrinsic fluorescence, which can introduce a high background noise in fluorescence-based assays.
The first step was to evaluate the expression of luciferase in Escherichia coli. Bacterial cells transformed with the luciferase-expressing plasmid were grown to mid log phase and induced with isopropyl-β-D-1-thiogalactopyranoside (IPTG). Two conditions were tested: a 2 h short induction and a 26 h long induction. (32,33) Both induced and uninduced samples were collected, and protein expression was evaluated via sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The presence of a band consistent with the molecular weight of luciferase (62 kDa) was identified (Figure 1b). Our results showed similar expression levels for both time points; therefore, the shorter incubation time (2 h) was selected for subsequent assays.
An assessment of the difference in luminescence signal between induced and uninduced samples in DCCAA was then performed. Bioluminescence imaging of a multiwell plate containing luciferase-expressing E. coli cells treated with d-luciferin also revealed a notable increase in luminescence upon induction (Figure 2a). E. coli cells harboring the luciferase plasmid were coincubated with CBT–OH and d-cystine for one h, during which luminescence was recorded. Here, d-cystine served as a surrogate for a test molecule (Figure 2b). Kinetic analysis of the cellular treatment revealed a pronounced increase in the luminescence signal over time. The signal response was IPTG-dependent, which is consistent with the proposed luciferase mediated processing of d-luciferin upon generation of d-cys in the cytosol.

Figure 2

Figure 2. (a) Bioluminescence image of a 96-well plate containing E. coli treated with100 μM d-luciferin in the presence and absence of IPTG induction. (b) Chemical structures of the building blocks used for the assay. (c) Bioluminescence analysis of luciferase-expressing E. coli cells incubated with 100 μM CBT–OH and 100 μM d-cystine for 30 min, followed by centrifugation and separation of supernatant and pellet at which point luminescence measurements were initiated and continued for 60 min. Noncentrifuged cells (CBT–OH + d-cystine) were used as a control. Inset at the top displays a magnified portion of the graph, highlighting the PBS and supernatant traces. (d) Luciferase-expressing E. coli cells treated with 100 μM CBT–OH and 100 μM d-cystine in the presence and absence of IPTG induction, over 60 min. (e) Luciferase-expressing E. coli cells treated with 100 μM CBT–OH or 100 μM CBT–NH2 and 100 μM d-cystine over 60 min. Cells treated with PBS and CBT only (CBT–OH or CBT–NH2) were used as controls wherever appropriate. Data are represented as mean ± SD (n = 3 independent samples in a single experiment).

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Next, we set out to establish whether the luminescence signal generation was confined to within the cellular structure instead of the extracellular recombination of CBT/d-Cys. Luciferase-expressingE. coli cells were first coincubated with CBT–OH and d-cystine for 30 min, after which, the cells were subjected to centrifugation, leading to the separation of the supernatant from the cellular pellet. The supernatant was then resuspended in phosphate-buffered saline (PBS). Luminescence measurements were then performed for an additional 60 min (Figure 2c). Consistent with the expected cytosolic localization of the luciferase, the luminescence signal detected in the supernatant was minimal, whereas the signal originating from the cellular pellet was significantly more pronounced. These observations are consistent with the intracellular nature of the signal and provide evidence that the luciferase enzyme remains localized within the cytoplasm for the entire duration of the assay.
We appreciated that the physicochemical properties of CBT could potentially be subjected to its own cellular accumulation barriers. We therefore sought to test two versions of CBT that have been described as compatible with recombination with d-cys to form luciferin. We evaluated CBT–OH and 6-amino-2-cyanobenzothiazole (CBT–NH2) (34) as potential substrates for the click reaction (Figure 2b,e). Notably, the in vitro rate constants for the reactions of the hydroxy- and amino-cyanobenzothiazole with l-cys have previously been reported to be 3.2 and 2.6 M–1 s–1, respectively. (35) Briefly, luciferase-expressing E. coli cells were coincubated with the CBT variants and d-cystine as described before. Our results showed that cells treated with CBT–NH2 produced a higher luminescence signal than those treated with CBT–OH (Figure 2d). Interestingly, CBT–OH outperformed CBT–NH2 in an in vitro cell-free setup consistent with prior reports (Figure S2). We pose that CBT–NH2 may have higher levels of accumulation relative to its hydroxy counterpart, and it was therefore selected as the preferred CBT variant for subsequent experiments.
We then set out to empirically determine the optimum concentration of CBT–NH2 for DCCAA. Briefly, following IPTG induction, bacterial cells were washed and either coincubated with varying concentrations of CBT–NH2 and 50 μM d-cystine or incubated with CBT–NH2 alone. A concentration of 100 μM was considered to have a sufficient signal-to-noise ratio necessary for the assay and was selected for subsequent experiments (Figure 3a). This concentration of CBT–NH2 was also found to not alter the cellular viability as determined by colony forming units (CFUs) analysis (Figure S3). Next, a similar titration experiment was performed with d-cystine by using a constant level of CBT–NH2. Our results showed that both 50 and 100 μM d-cystine exhibited a favorable signal-to-noise ratio (Figure 3b).

Figure 3

Figure 3. (a) Bioluminescence analysis of luciferase-expressing E. coli cells treated with 50 μM d-cystine and varying concentrations of CBT–NH2 over 60 min at the 60 min time point. (b) Luciferase-expressing E. coli cells treated with 100 μM NH2–CBT and varying concentrations of d-cystine at the 60 min time point. (c) Luciferase-expressing E. coli cells (left) or a cell-free assay with luciferase enzyme (right) treated with 10 μM l-luciferin, 10 μM d-luciferin or 10 μM d-cystine with 100 μM CBT–NH2 (cell assay), over 60 min. (d) Luciferase-expressing E. coli cells treated with 100 μM d-cystine and 100 μM CBT–NH2 or 100 μM CBT–NH2 only, in the presence and absence of a 30 min pretreatment with 100 μM NEM, over 60 min. (e) Luciferase-expressing E. coli cells treated with 100 μM d-cystine or 100 μM d-cystine-ME and 100 μM CBT–NH2 at the 60 min time point. (f) Luciferase-expressing E. coli cells treated with 100 μM d-cystine and 100 μM CBT–NH2 in the absence and presence of a 30 min pretreatment with PMBN at different concentrations at the 60 min time point. Cells treated with only PBS and CBT–NH2 were used as controls, wherever appropriate. Data are represented as mean ± SD (n = 3 independent samples in a single experiment). Statistical analysis performed by two-tailed t-test with Welch’s correction, *p ≤ 0.01, **p ≤ 0.01, ***p ≤ 0.001, ns = not significant.

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Through our initial assay development efforts, the background signal was higher than we had anticipated; therefore, we sought to investigate its potential source. We considered that it could be from intracellular pools of l-cys combining with CBT–NH2 to form l-luciferin (Figure S4). (21,29,36−38) Previous reports have shown that l-luciferin can undergo epimerization in the presence of firefly luciferase and then act as a substrate for the enzyme. (39) Indeed, upon coincubation of luciferase-expressing E. coli with L-luciferin, a signal lower than that of d-luciferin, yet significantly higher than the background, was observed (Figure 3c). This observation suggests that l-luciferin undergoes epimerization within the cytoplasm of E. coli. In a cell free experiment, l-luciferin exhibited a baseline luminescence signal in the presence of purified luciferase, whereas d-luciferin exhibited a considerably elevated signal intensity (Figure 3c). Notably, this observation is in agreement with the essential role of Coenzyme A (CoA) in the epimerization process of l-luciferin to d-luciferin catalyzed by luciferase (Figure S5). (37,40)
We next sought to evaluate the necessity for d-cys to be uncoupled prior to signal generation. At first, a cell free set up was evaluated by incubating d-cystine with luciferase in the presence or absence of the reducing agent tris(2-carboxyethyl) phosphine (TCEP) (Figure S6). The inclusion of TCEP was found to be essential for signal generation, providing evidence for the requirement of a reducing environment to promote signal production. A similar requirement for a reducing environment was next evaluated in the cell. For these experiments, E. coli was treated with N-ethylmaleimide (NEM), which covalently modifies cellular thiols and is expected to reduce the cellular pool of reducing agents including glutathione and l-cys. (23,41) Luciferase-expressing cells were pretreated with NEM or PBS, followed by incubation with d-cystine, as described previously. Pretreatment with NEM resulted in a complete shutdown of the signal (Figure 3d). These results demonstrate that the reducing environment of the cell is required for signal generation in a manner that is consistent with the levels of cellular thiols. Moreover, we tested the effect of NEM on the background signal that emerged solely from the addition of CBT–NH2. Our findings demonstrate that NEM also effectively abolished the signal originating from CBT–NH2 (Figure 3d), indicating that the presence of l-cys most likely contributes to the observed background signal.
Considering the inherent characteristics of our initial test compound, d-cystine, containing two carboxylic acid moieties, we explored the possibility of masking these groups to enhance the accumulation. Masking negatively charged carboxylic acids through esterification is widely used as a permeability strategy to enhance the lipophilicity and passive membrane permeability of molecules, particularly, therapeutic agents with intracellular targets. (42,43) Once inside the cell, the ester may be enzymatically hydrolyzed to the acid, resulting in conversion to the parent compound. It is noteworthy that the introduction of a methyl group at the carboxylic position in d-luciferin, as seen in d-luciferin-methyl-ester, results in its failure to be recognized by the firefly luciferase enzyme. (44) While the findings of Antonczak and colleagues (45) suggest an absence of methyl esterases in E. coli, subsequent research by the Grimes laboratory (46) utilizing methyl ester NAM derivatives in their peptidoglycan labeling approach, lends support to the presence of methyl esterases in E. coli.
To test the masking ability of methyl ester, we used DCCAA to compare d-cystine and d-cystine-methyl ester (d-cystine-ME) (Figure S7). Our results showed that cellular treatment with d-cystine-ME led to signals well above the background, suggestive of esterase unmasking of the methyl ester (Figure 3e). Interestingly, the cellular signals were ca. 30% lower than that of unmasked d-cystine. This could indicate that d-cystine may be actively transported by an importer (47,48) or the esterase processing is inherently slow. When the experiment was conducted in a cell-free system, the signal from d-cystine-ME remained at the background level (Figure S8). These findings provide support for the processing of ester groups in E. coli cells. The presence of esterases in E. coli bears relevance in the realm of drug development, especially in the potential utilization of a prodrug approach for antibiotics. Nevertheless, we believe that DCCAA can be generally leveraged to gain further insight into the substrate specificity and enzymatic activity of E. coli esterases.
The diderm cell envelope structure in Gram-negative bacteria, particularly the outer membrane, serves as a substantial barrier to the permeation of antibiotics with intracellular targets. Therefore, there is a critical need to identify and develop molecules that can disrupt this accumulation barrier. Such molecules can potentially be used as antibiotic adjuvants that can broadly improve activity. One example of a molecule capable of perturbing the outer membrane is polymyxin B nonapeptide (PMBN), which is a modified form of Polymyxin B lacking the fatty acid tail. PMBN can permeabilize the outer membrane of E. coli at low, nontoxic concentrations. (49) Moreover, it has also demonstrated the ability to enhance the efficacy of erythromycin and provide protection against Gram-negative bacteria in mice. (50) We posed the question of whether the inclusion of PMBN in our assay would increase the permeability of bacterial cells to d-cystine, leading to an amplified signal response.
To test the potential impact of PMBN, luciferase expressing E. coli cells were pretreated with PMBN at increasing concentrations and the assay was carried out as described previously (Figure 3f). Unexpectedly, a dose-dependent decrease in the luminescence signal with increasing concentrations of PMBN was observed. This trend was also observed when cells were, instead, treated with d-luciferin (Figure S9). Crucially, we noted no loss of cellular viability with the same concentrations of PMBN (Figure S10). We wondered whether PMBN could be leading to the release of components critical to DCCAA. We observed a dose-dependent increase in outer membrane permeation as measured by nitrocefin (Figure S11). A similar pattern was observed in the case of a SYTOX Green assay, which is a high-affinity nucleic acid stain typically used to assess membrane integrity (Figure S12). Notably, treatment of E. coli cells with PMBN has been previously reported to cause the release of intracellular, low-molecular weight substances such as free amino acids and uracil. (51) Hence, we hypothesize that the observed reduction in signal may be due to the leakage of substrates, specifically the release of ATP from the cells or the in cyto produced d-luciferin which is integral for the oxidation of d-luciferin by luciferase. (52,53)
Having optimized the assay parameters of DCCAA and shown its ability to respond to the model molecule d-cystine, we next leveraged this assay to evaluate the accumulation of structural motifs that are relevant for clinical applications. For this, a small panel of antibiotics (including ciprofloxacin, puromycin, linezolid, and rifamycin B) were synthesized and tagged with a disulfide-linked d-cys (Figure 4a). In all, this panel included a range of molecules that varied in physicochemical properties. Before performing the cellular assay, we aimed to test the release of d-cys in a cell-free experiment. It is noteworthy that in the absence of a permeability barrier, when exposed to a reducing agent, all four compounds were expected to generate an identical luminescence signal at the same concentration. This should be because of the release of an equal number of d-cys molecules from each compound.

Figure 4

Figure 4. (a) Structures of the antibiotic conjugates tagged with a d-cys moiety via a disulfide linkage. (b) Bioluminescence analysis of a cell-free assay with luciferase enzyme treated with 25 μM d-cystine or 25 μM antibiotic conjugates and 25 μM CBT–NH2 over 60 min and (c) at the 60 min time point upon treatment with 1 mM TCEP. (d) Bioluminescence analysis of luciferase-expressing E. coli cells treated with 50 μM d-cystine or 50 μM antibiotic conjugates with 100 μM CBT–NH2 over 60 min and (e) at the 60 min time point. The inset at the top of (d) provides a zoomed-out view of the graph, highlighting the d-cystine signal. Cells treated with PBS and CBT only (CBT–NH2) were used as controls wherever appropriate. Data are represented as mean ± SD (n = 3 independent samples in a single experiment). Statistical analysis performed by two-tailed t-test with Welch’s correction, **p ≤ 0.01, ***p ≤ 0.001, ns = not significant.

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Interestingly, our cell-free results revealed an unexpected result (Figure 4b). While there was no significant difference in the luminescence signal for puromycin, rifamycin B, and linezolid conjugates, the signal for ciprofloxacin was much lower (Figure 4c). We hypothesized that the residual covalently tagged d-cys in the ciprofloxacin conjugate after the breakage of the disulfide bond, could potentially undergo a nonproductive click reaction with CBT–NH2 (Figure S13) thereby reducing the effective concentration of CBT. Nonetheless, all four antibiotics were evaluated in the cellular assay. As before, the evaluation of these molecules was conducted by coincubating luciferase-expressing bacterial cells with CBT–NH2 and the antibiotic conjugates, and subsequent luminescence measurements were made. It is noteworthy that all four conjugates demonstrated noticeable permeation exceeding the baseline signal, as seen in Figure 4d, and as indicated by the luminescence signals observed at the 60 min time point (Figure 4e). A similar pattern was also observed when the observation window was extended to 120 min (Figure S14) and we demonstrate that the assay is concentration dependent (Figure S15). Notably, addition of the antibiotic conjugates to the luciferase expressing E. coli cells was found to be minimally disruptive to their cell envelope structure, as evidenced by a SYTOX Green assay (Figure S16).
We then proceeded to develop a kinetic model for the molecular uptake of the conjugated antibiotics. The model serves as the foundation for a nonlinear least-squares analysis of our data. Specifically, the model accounts for the accumulation of each molecule in the cytoplasm of E. coli and the subsequent click and luciferase-based bioluminescence enzymatic reactions. Of significance, the time-dependent luminescence response (ILum.) can be characterized by eq 1, where ε0 and I0 respectively represent the luminescence coefficient factor and baseline signal in our experiments. d-Luciferin (*) denotes the oxidized d-luciferin molecule that is associated with the luminescence signal.
ILum.=ε0×[D−luciferin(*)]+I0
(1)
Time-dependent luminescence signals are then utilized to derive a solution with physical significance for the specified differential equations, as detailed in Supporting Information. Special attention is given to estimating the accumulation rate constant, denoted as kacc (min–1), and the reduction rate constant, denoted as kred (min–1). Values for the click reaction rate constant, kclick (156 M–1min–1) (35) and the enzymatic reaction rate constant, kenz (96 min–1) (54) were adopted from the literature.
To illustrate how kacc and kred affect time-dependent luminescence signals, we selectively adjusted the associated transport rates in a set of simulated kinetic responses. In Figure 5a, a sequence of simulated responses is presented, incorporating various kacc values ranging from 0.01 to 1.00 min–1, paired with each kred value set at 0.01, 0.10, and 0.50 min–1. Importantly, an offset of 3 min was introduced in the experimental data to account for the time delay between the beginning of the assay and the first luminescence reading, owing to logistics of pipetting, mixing, etc. Additionally, all signals, experimental and simulated, were normalized with respect to their values at 63 min for the purpose of this model. As depicted, irrespective of the kred values, the curves exhibit exponential behavior at lower kacc values, transitioning gradually into a semilinear shape at higher kacc values.

Figure 5

Figure 5. (a) Simulated luminescence traces, based upon the model for molecular accumulation in bacteria, with gradually increasing rate constants kacc and kred. (b) Experimental time-resolved luminescence traces illustrating the accumulation of test molecules in E. coli. The best-fit lines from simulations are overlaid in black. The corresponding values for kacc and kred for each, are depicted in (c).

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Next, we sought optimal fits for each time-resolved signal, representing the averages from four independent trials. In Figure 5b, the most accurate fitting curves are presented, superimposed on each signal. Notably, for the ciprofloxacin-methyl-ester, linezolid, and puromycin conjugates, the best-estimated kacc values were respectively determined to be 0.05, 0.05, and 0.25 min–1, each accompanied by a kred of 0.10 min–1 (Figure 5c). These findings imply that the membrane transport rates for conjugated ciprofloxacin methyl ester and linezolid are five times lower than that of conjugated puromycin. Significantly, despite linezolid being known for its vulnerability to efflux pump activity, we observed its accumulation behavior in our assay. This may be attributed to the possibility that our luminescence readout process operates on a faster time scale than its efflux out of the cell or that efflux pumps do not effectively recognize linezolid when tagged with a d-cys moiety. As far as d-cys is concerned, as expected, it displayed a higher kacc value of 1.00 min–1 and kred value of 0.50 min–1, suggesting faster accumulation and reduction processes, compared to the conjugated antibiotics (Figure S17). Unexpectedly, rifamycin B, when compared to the other three components, displayed slightly distinct kinetics. The best-fitted curve revealed a rate constant similar to that of d-cystine (i.e., kacc = 1.00 min–1; kred = 0.50 min–1). This may be due to a difference in its membrane transport mechanism or as a result of the property of the molecule itself given its larger size. Significantly, molecules with comparable molecular weights to our antibiotic conjugates, such as malachite green (329.46 Da), have been documented to exhibit a transport rate of approximately 4.2 min–1 and 0.013 min–1 through the outer- and inner-membranes of E. coli, respectively. (55) In contrast, a similar molecule without a permanent dipole moment, namely crystal violet (372.54 Da; net charge of 1+), displays inner-membrane transport rates that can be orders of magnitude smaller. (56)

Discussion and Conclusion

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Quantifying intracellular compound accumulation within the bacterial cytosol, especially during the development phase of antibiotics with cytosolic targets, is crucial for the development of efficacious drugs with useable potency against their targets. Here, we present DCCAA, a luminescence-based method enabling the real-time monitoring of antibiotic intracellular accumulation in live Gram-negative bacterial cells. In this technique, the molecule of interest is conjugated with a d-cys moiety through a disulfide bond, which is susceptible to reduction within the cytosolic milieu. The liberated d-cys then engages in a click reaction with CBT, coadministered with the tagged molecule, resulting in the generation of d-luciferin. This substrate undergoes oxidation by luciferase, emitting light in the process, as a measurable indicator of intracellular antibiotic accumulation dynamics (Figure 1a). Additionally, our results showed compelling evidence supporting the presence of methyl esterases in E. coli. This investigation highlights the potential use of DCCAA to decipher the presence and substrate-specificity of esterases in different bacterial species. We were also able to elucidate the membrane disruption activity of an antimicrobial compound. Specifically, we investigated the activity of PMBN, a known Gram-negative outer membrane disrupting agent, via DCCAA. Lastly, through DCCAA we were able to monitor the intracellular accumulation of the d-cys conjugates of ciprofloxacin methyl ester, linezolid, puromycin, and rifamycin B.
We found all four antibiotic conjugates tested to display a signal above the background, indicating accumulation in the cytosol of bacteria. Through kinetic analysis, we found that the puromycin conjugate displayed a higher transport rate than the linezolid and ciprofloxacin-methyl-ester conjugates, while the rifamycin B conjugate displayed a distinct kinetic profile, unlike the other compounds, likely owing to a distinct membrane transport process or its larger size. We note that DCCAA does require the conjugation of a d-cysteine to the test molecule to make it compatible with luciferin generation. There must be some consideration of the location of this tag within the molecule (antibiotic) of interest. One option is to modify an existing amino group to then have the terminal amine of d-cysteine effectively replace that property within the molecules. Similar to the use of the fluorophore tag, one also has to consider the site within the molecule in consideration of the biological activity of the agent. Naturally, the physiochemical properties of the molecule could change with the addition of the tag, and these considerations are made in light of the ability to determine cytosolic arrival.
In conclusion, we propose that DCCAA can serve not only to elucidate the real-time intracellular accumulation dynamics of compounds tagged with a d-cys molecule in live bacterial cells but also to potentially unveil the membrane disruption abilities of untagged compounds. Furthermore, DCCAA can also be employed to reveal the presence and substrate specificity of esterase activity in live bacterial cells.

Methods

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Transformation of FLUC2 pET28a into E. coli

For expression of the luciferase protein in E. coli, the FLUC2 pET28a plasmid was first transformed into DH5α E. coli cells for amplification. In short, 50 μL of competent DH5α E. coli cells and 2–5 μL of plasmid were added to an Eppendorf tube and kept on ice. After 30 min, a water bath was heated to 42 °C and the tube containing the cells and plasmid was placed in the bath for 30 s followed by another 2 min on ice. One mL of sterile LB medium was added to the tube, mixed, and transferred to a culture tube which was incubated at 37 °C for 1 h. Subsequently, 25–200 μL of cells from this tube were grown on LB/agar plates with kanamycin (50 μg/mL) at 37 °C overnight. Individual colonies were then picked from these plates and grown overnight in LB broth with 50 μg/mL kanamycin at 37 °C. The following day, a ZymoPURE Plasmid Miniprep Kit was used to extract the plasmid from the DH5α E. coli cells. A transformation was then performed as described above with competent BL21(DE3) E. coli cells for optimal expression. Glycerol stocks of both BL21 and DH5α E. coli cells were prepared by mixing 1 mL of overnight growth with 1 mL of 60% glycerol in water.

Luciferase Protein Expression in E. coli

Sterile culture tubes each containing 3 mL of LB medium and 50 μg/mL kanamycin were inoculated with a stab of the transformed BL21 luciferase-expressing E. coli glycerol stock and incubated at 37 °C overnight. The next morning, the cells were diluted at a 1:10 ratio into fresh LB broth containing 50 μg/mL kanamycin and were grown at 37 °C for 3 h, or until the optical density at 600 nm reached a value between 0.6 and 0.8. Cultures were then induced with 1 mM isopropyl β-d-1-thiogalactopyranoside (IPTG) at 37 °C for 2 h in a shaker incubator to induce protein expression.

Evaluation of Luciferase Protein Expression via SDS-PAGE

Overnight cultures of luciferase-expressing E. coli were grown and diluted the next morning as described above. When the diluted cells reached an OD600 between 0.6 and 0.8, IPTG was added to one culture tube to induce protein expression. Both plus and minus IPTG cultures were then incubated at 37 °C for 2 or 26 h. On the same day that these cells were induced, another two overnight cultures were grown. The next day, these cells were diluted, and IPTG was used to induce protein expression of one culture for 2 h at 37 °C. One mL of media was collected from the plus and minus IPTG samples from both time points. These were centrifuged at 3300g for 3 min in a HERAEUS multicentrifuge ×1 centrifuge (Thermo Fisher Scientific). The supernatant was discarded, and the cells were resuspended in 400 μL 1× phosphate buffered saline (PBS) at pH 7.2.40 μL of the cell resuspension was mixed with 10 μL of a 5X SDS-PAGE sample loading buffer. This mixture was then boiled for 5 min to denature the proteins. To run the gel, 8 μL of Thermo Scientific PageRuler Plus Prestained Protein Ladder, 10 to 250 kDa, was added to the first well. The samples were loaded at 20 μL, and then gel was run at 240 V for 30 min.

Bioluminescence-Based Permeability Assays in Luciferase Expressing E. coli

Overnight cultures of luciferase-expressing E. coli were grown, and luciferase protein expression was induced as above. Subsequently, washing was performed by first pelleting the cells in the HERAEUS multicentrifuge ×1 centrifuge (Thermo Fisher Scientific) at 3300g for 3 min. The supernatant was then discarded, and the cells were resuspended in 1× PBS (volume same as original culture). This was repeated 2 times. The cells were resuspended after the last wash in 1× PBS. In NEM/PMBN pretreatment experiments, a 30 min incubation with the appropriate compound was performed at this point at the desired concentrations (100 μM for NEM; 0.5, 1, and 3 μM for PMBN). Cells were then washed and resuspended in 1× PBS in the same manner as described above. In a 96-well, black, flat-bottomed plate, the molecules of interest were then added to obtain the final desired concentration in a total volume of 100 μL. Note that stock solutions of molecules of interest were prepared in 1× PBS. 1× PBS was also added to any blank and negative control wells, to ensure the same total volume and number of cells in all the wells. A stock solution of CBT (6-Amino-2-cyanobenzothiazole) was prepared in N,N-Dimethylformamide (DMF), and appropriate volumes were added to the culture tubes containing cells to obtain the desired concentrations. Then, the same volume of cells was pipetted into each well, mixed, and placed in the BioTek Synergy H1Microplate Reader. The instrument was set to run on the end point/kinetic luminescence setting. Luminescence was set to read every 5 min for the run, usually for 60 min (or 120 min, for the extended time-course analysis). For the assay to test the luminescence of the pellet and supernatant of centrifuged E. coli cells, the plate was read for 30 min following which a set of wells treated with CBT and d-cystine were moved to another plate and pelleted as described above. The supernatant was then carefully separated and readded to the plate. The pellet was resuspended and also readded to the plate. Nonpelleted cells treated with CBT and d-cystine served as one of the controls. Luminescence in this case was then read for another 60 min. For all luminescence readings via the BioTek Synergy H1Microplate Reader, luminescence fiber was the optics type. The gain was set to 240, and the integration time was set to 0:01:00. The temperature was set to 37 °C.

Cell-free Luciferase Assays

The 14.9 mg/mL luciferase enzyme stock received from Promega was diluted in 25 mM Tris buffer (pH 8) to prepare 0.4 mg/mL aliquots (40 μL each), which were then stored in −80 °C. All assays were performed in 25 mM Tris buffer (pH 8) in 96-well, black, flat-bottomed plates. Molecules of interest were added to obtain the final desired concentration in a total volume of 100 μL. Additionally, appropriate amounts of MgSO4 (100 mM), freshly made ATP (20 mM) and Luciferase stocks (0.4 mg/mL) prepared in 25 mM Tris buffer (pH 8) were added to the wells to yield final concentrations of 5 mM, 1 mM and 20 μg/mL, respectively. Wherever appropriate, a TCEP (20 mM) stock prepared in 25 mM Tris buffer (pH 8) was added to a final concentration of 1 mM. Similarly, wherever appropriate, a Coenzyme A (10 mM) stock prepared in 25 mM Tris buffer (pH 8) was added to a final concentration of 0.5 mM. In the experiments testing the effect of Coenzyme A, a porcine liver esterase stock [10 mg/mL in 25 mM Tris buffer (pH 8)] was added to yield a final concentration of 0.5 mg/mL. The plates were read using the BioTek Synergy H1Microplate Reader. The instrument was set to run on the end point/kinetic luminescence setting. Luminescence was set to read every 5 min for the run, usually between 30 min to 2 h. Luminescence fiber was the optics type. The gain was set to 240, and the integration time was set to 0:01:00. The temperature was set to 37 °C.

CFU Analysis

Overnight cultures of luciferase-expressing E. coli were grown, and luciferase protein expression was induced as described above. Subsequently, washing was performed by first pelleting the cells in the HERAEUS Multicentrifuge ×1 centrifuge (Thermo Fisher Scientific) at 3300g for 2–3 min. The supernatant was then discarded, and the cells were resuspended in 1× PBS (volume same as original culture). This was repeated 2 times. The cells were resuspended after the last wash in 1× PBS. Resuspended cells were then incubated with the test compounds (CBT or PMBN) at the indicated concentrations for 30 min (PMBN) or 1 h (CBT). Cells were then washed and resuspended as described above. Serial dilutions of the cells were then carried out in PBS up to a dilution factor of 10–8. 80 μL of chosen dilutions was then plated on LB agar plates with 50 μg/mL kanamycin and incubated at 37 °C for 16 h. Subsequently, the number of colonies was manually counted.

Nitrocefin Assay

Overnight cultures of luciferase-expressing E. coli were grown, and luciferase protein expression was induced as above to mimic conditions of the cells used in the bioluminescence assay. Cells were then washed twice and resuspended in 1× PBS and then incubated with the different concentrations of PMBN (same as those concentrations used in the bioluminescence assays) with 50 μg/mL nitrocefin for 30 min. As a negative control, cells were treated with PBS, and as a positive control, cells were treated with 10 mM EDTA for 30 min. At the end of the incubation period, the absorbance of the cells was read at 486 nm using the BioTek Synergy H1Microplate Reader. Untreated cells with no nitrocefin were treated as the blank. Wherever presented, the absorbances were blank-subtracted.

SYTOX Green Assay

Overnight cultures of luciferase-expressing E. coli were grown, and luciferase protein expression was induced as above to mimic conditions of the cells used in the bioluminescence assay. Cells were then washed twice and resuspended in 1× PBS and then incubated with the test compounds (same concentrations as those used in the bioluminescence assays) for the appropriate time durations (30 min for PMBN and 1 h for antibiotic conjugates). Cells were then washed twice again and resuspended in 1× PBS. The experiment was then conducted as per the manufacturer’s protocol. As a negative control, cells were treated with PBS, and as a positive control, cells were treated with 10 mM EDTA. Cells were analyzed using an Attune NxT flow cytometer equipped with a 488 nm laser and 525/40 nm bandpass filter. The data were analyzed using the Attune NxT Software, where populations were gated and no less than 10,000 events per sample were recorded. Wherever presented, the mean fluorescence intensity (MFI) is the ratio of fluorescence levels above the negative control treatment.

Supporting Information

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The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsinfecdis.3c00684.

  • Additional figures, tables, and materials/methods are included in the Supporting Information file (PDF)

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Author Information

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  • Corresponding Author
  • Authors
    • Rachita Dash - Department of Chemistry, University of Virginia, Charlottesville, Virginia 22904, United States;  Orcidhttps://orcid.org/0009-0008-1830-1318
    • Kadie A. Holsinger - Department of Chemistry, University of Virginia, Charlottesville, Virginia 22904, United States
    • Mahendra D. Chordia - Department of Chemistry, University of Virginia, Charlottesville, Virginia 22904, United States
    • Mohammad Sharifian Gh. - Department of Chemistry, University of Virginia, Charlottesville, Virginia 22904, United States
  • Notes
    The authors declare no competing financial interest.

Acknowledgments

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This study was supported by the NIH grant GM124893-01 (M.M.P.).

References

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This article references 56 other publications.

  1. 1
    Murray, C. J. L.; Ikuta, K. S.; Sharara, F.; Swetschinski, L.; Robles Aguilar, G.; Gray, A.; Han, C.; Bisignano, C.; Rao, P.; Wool, E.; Johnson, S. C.; Browne, A. J.; Chipeta, M. G.; Fell, F.; Hackett, S.; Haines-Woodhouse, G.; Kashef Hamadani, B. H.; Kumaran, E. A. P.; McManigal, B.; Achalapong, S.; Agarwal, R.; Akech, S.; Albertson, S.; Amuasi, J.; Andrews, J.; Aravkin, A.; Ashley, E.; Babin, F.-X.; Bailey, F.; Baker, S.; Basnyat, B.; Bekker, A.; Bender, R.; Berkley, J. A.; Bethou, A.; Bielicki, J.; Boonkasidecha, S.; Bukosia, J.; Carvalheiro, C.; Castañeda-Orjuela, C.; Chansamouth, V.; Chaurasia, S.; Chiurchiù, S.; Chowdhury, F.; Clotaire Donatien, R.; Cook, A. J.; Cooper, B.; Cressey, T. R.; Criollo-Mora, E.; Cunningham, M.; Darboe, S.; Day, N. P. J.; De Luca, M.; Dokova, K.; Dramowski, A.; Dunachie, S. J.; Duong Bich, T.; Eckmanns, T.; Eibach, D.; Emami, A.; Feasey, N.; Fisher-Pearson, N.; Forrest, K.; Garcia, C.; Garrett, D.; Gastmeier, P.; Giref, A. Z.; Greer, R. C.; Gupta, V.; Haller, S.; Haselbeck, A.; Hay, S. I.; Holm, M.; Hopkins, S.; Hsia, Y.; Iregbu, K. C.; Jacobs, J.; Jarovsky, D.; Javanmardi, F.; Jenney, A. W. J.; Khorana, M.; Khusuwan, S.; Kissoon, N.; Kobeissi, E.; Kostyanev, T.; Krapp, F.; Krumkamp, R.; Kumar, A.; Kyu, H. H.; Lim, C.; Lim, K.; Limmathurotsakul, D.; Loftus, M. J.; Lunn, M.; Ma, J.; Manoharan, A.; Marks, F.; May, J.; Mayxay, M.; Mturi, N.; Munera-Huertas, T.; Musicha, P.; Musila, L. A.; Mussi-Pinhata, M. M.; Naidu, R. N.; Nakamura, T.; Nanavati, R.; Nangia, S.; Newton, P.; Ngoun, C.; Novotney, A.; Nwakanma, D.; Obiero, C. W.; Ochoa, T. J.; Olivas-Martinez, A.; Olliaro, P.; Ooko, E.; Ortiz-Brizuela, E.; Ounchanum, P.; Pak, G. D.; Paredes, J. L.; Peleg, A. Y.; Perrone, C.; Phe, T.; Phommasone, K.; Plakkal, N.; Ponce-de-Leon, A.; Raad, M.; Ramdin, T.; Rattanavong, S.; Riddell, A.; Roberts, T.; Robotham, J. V.; Roca, A.; Rosenthal, V. D.; Rudd, K. E.; Russell, N.; Sader, H. S.; Saengchan, W.; Schnall, J.; Scott, J. A. G.; Seekaew, S.; Sharland, M.; Shivamallappa, M.; Sifuentes-Osornio, J.; Simpson, A. J.; Steenkeste, N.; Stewardson, A. J.; Stoeva, T.; Tasak, N.; Thaiprakong, A.; Thwaites, G.; Tigoi, C.; Turner, C.; Turner, P.; van Doorn, H. R.; Velaphi, S.; Vongpradith, A.; Vongsouvath, M.; Vu, H.; Walsh, T.; Walson, J. L.; Waner, S.; Wangrangsimakul, T.; Wannapinij, P.; Wozniak, T.; Young Sharma, T. E. M. W.; Yu, K. C.; Zheng, P.; Sartorius, B.; Lopez, A. D.; Stergachis, A.; Moore, C.; Dolecek, C.; Naghavi, M. Global Burden of Bacterial Antimicrobial Resistance in 2019: A Systematic Analysis. Lancet 2022, 399 (10325), 629– 655,  DOI: 10.1016/S0140-6736(21)02724-0
    Google Scholar
    1
    Global burden of bacterial antimicrobial resistance in 2019: a systematic analysis
    Murray, Christopher J. L.; Ikuta, Kevin Shunji; Sharara, Fablina; Swetschinski, Lucien; Aguilar, Gisela Robles; Gray, Authia; Han, Chieh; Bisignano, Catherine; Rao, Puja; Wool, Eve; et al.
    Lancet (2022), 399 (10325), 629-655CODEN: LANCAO; ISSN:0140-6736. (Elsevier Ltd.)
    Antimicrobial resistance (AMR) poses a major threat to human health around the world. Previous publications have estd. the effect of AMR on incidence, deaths, hospital length of stay, and health-care costs for specific pathogen-drug combinations in select locations. To our knowledge, this study presents the most comprehensive ests. of AMR burden to date. We estd. deaths and disability-adjusted life-years (DALYs) attributable to and assocd. with bacterial AMR for 23 pathogens and 88 pathogen-drug combinations in 204 countries and territories in 2019. We obtained data from systematic literature reviews, hospital systems, surveillance systems, and other sources, covering 471 million individual records or isolates and 7585 study-location-years. We used predictive statistical modeling to produce ests. of AMR burden for all locations, including for locations with no data. Our approach can be divided into five broad components: no. of deaths where infection played a role, proportion of infectious deaths attributable to a given infectious syndrome, proportion of infectious syndrome deaths attributable to a given pathogen, the percentage of a given pathogen resistant to an antibiotic of interest, and the excess risk of death or duration of an infection assocd. with this resistance. Using these components, we estd. disease burden based on two counterfactuals: deaths attributable to AMR (based on an alternative scenario in which all drug-resistant infections were replaced by drug-susceptible infections), and deaths assocd. with AMR (based on an alternative scenario in which all drug-resistant infections were replaced by no infection). We generated 95% uncertainty intervals (UIs) for final ests. as the 25th and 975th ordered values across 1000 posterior draws, and models were cross-validated for out-of-sample predictive validity. We present final ests. aggregated to the global and regional level. On the basis of our predictive statistical models, there were an estd. 4.95 million (3.62-6.57) deaths assocd. with bacterial AMR in 2019, including 1.27 million (95% UI 0.911-1.71) deaths attributable to bacterial AMR. At the regional level, we estd. the all-age death rate attributable to resistance to be highest in western sub-Saharan Africa, at 27.3 deaths per 100 000 (20.9-35.3), and lowest in Australasia, at 6.5 deaths (4.3-9.4) per 100 000. Lower respiratory infections accounted for more than 1.5 million deaths assocd. with resistance in 2019, making it the most burdensome infectious syndrome. The six leading pathogens for deaths assocd. with resistance (Escherichia coli, followed by Staphylococcus aureus, Klebsiella pneumoniae, Streptococcus pneumoniae, Acinetobacter baumannii, and Pseudomonas aeruginosa) were responsible for 929 000 (660 000-1 270 000) deaths attributable to AMR and 3.57 million (2.62-4.78) deaths assocd. with AMR in 2019. One pathogen-drug combination, meticillin-resistant S aureus, caused more than 100 000 deaths attributable to AMR in 2019, while six more each caused 50 000-100 000 deaths: multidrug-resistant excluding extensively drug-resistant tuberculosis, third-generation cephalosporin-resistant E coli, carbapenem-resistant A baumannii, fluoroquinolone-resistantE coli, carbapenem-resistant K pneumoniae, and third-generation cephalosporin-resistant K pneumoniae. To our knowledge, this study provides the first comprehensive assessment of the global burden of AMR, as well as an evaluation of the availability of data. AMR is a leading cause of death around the world, with the highest burdens in low-resource settings. Understanding the burden of AMR and the leading pathogen-drug combinations contributing to it is crucial to making informed and location-specific policy decisions, particularly about infection prevention and control programs, access to essential antibiotics, and research and development of new vaccines and antibiotics. There are serious data gaps in many low-income settings, emphasizing the need to expand microbiol. lab. capacity and data collection systems to improve our understanding of this important human health threat.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB38XpvFGrtb0%253D&md5=b99b0063434bbae5c4d799ab977e973c
  2. 2
    Hutchings, M. I.; Truman, A. W.; Wilkinson, B. Antibiotics: Past, Present and Future. Curr. Opin. Microbiol. 2019, 51, 72– 80,  DOI: 10.1016/j.mib.2019.10.008
    Google Scholar
    2
    Antibiotics: past, present and future
    Hutchings, Matt; Truman, Andrew; Wilkinson, Barrie
    Current Opinion in Microbiology (2019), 51 (), 72-80CODEN: COMIF7; ISSN:1369-5274. (Elsevier Ltd.)
    A review. The first antibiotic, salvarsan, was deployed in 1910. In just over 100 years antibiotics have drastically changed modern medicine and extended the av. human lifespan by 23 years. The discovery of penicillin in 1928 started the golden age of natural product antibiotic discovery that peaked in the mid-1950s. Since then, a gradual decline in antibiotic discovery and development and the evolution of drug resistance in many human pathogens has led to the current antimicrobial resistance crisis. Here we give an overview of the history of antibiotic discovery, the major classes of antibiotics and where they come from. We argue that the future of antibiotic discovery looks bright as new technologies such as genome mining and editing are deployed to discover new natural products with diverse bioactivities. We also report on the current state of antibiotic development, with 45 drugs currently going through the clin. trials pipeline, including several new classes with novel modes of action that are in phase 3 clin. trials. Overall, there are promising signs for antibiotic discovery, but changes in financial models are required to translate scientific advances into clin. approved antibiotics.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1MXitV2msbjP&md5=cdb3445303ef36cbe05f5ec1d2a0e37a
  3. 3
    Zgurskaya, H. I.; Rybenkov, V. V. Permeability Barriers of Gram-Negative Pathogens. Ann. N.Y. Acad. Sci. 2020, 1459 (1), 5– 18,  DOI: 10.1111/nyas.14134
    Google Scholar
    3
    Permeability barriers of Gram-negative pathogens
    Zgurskaya Helen I; Rybenkov Valentin V
    Annals of the New York Academy of Sciences (2020), 1459 (1), 5-18 ISSN:.
    Most clinical antibiotics do not have efficacy against Gram-negative pathogens, mainly because these cells are protected by the permeability barrier comprising the two membranes with active efflux. The emergence of multidrug-resistant Gram-negative strains threatens the utility even of last resort therapeutic treatments. Significant efforts at different levels of resolution are currently focused on finding a solution to this nonpermeation problem and developing new approaches to the optimization of drug activities against multidrug-resistant pathogens. The exceptional efficiency of the Gram-negative permeability barrier is the result of a complex interplay between the two opposing fluxes of drugs across the two membranes. In this review, we describe the current state of understanding of the problem and the recent advances in theoretical and empirical approaches to characterization of drug permeation and active efflux in Gram-negative bacteria.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BB3M3jsVWgsg%253D%253D&md5=fd30e9ef5963606be429019846a1b1fa
  4. 4
    Kohanski, M. A.; Dwyer, D. J.; Collins, J. J. How Antibiotics Kill Bacteria: From Targets to Networks. Nat. Rev. Microbiol. 2010, 8 (6), 423– 435,  DOI: 10.1038/nrmicro2333
    Google Scholar
    4
    How antibiotics kill bacteria: from targets to networks
    Kohanski, Michael A.; Dwyer, Daniel J.; Collins, James J.
    Nature Reviews Microbiology (2010), 8 (6), 423-435CODEN: NRMACK; ISSN:1740-1526. (Nature Publishing Group)
    A review. Antibiotic drug-target interactions, and their resp. direct effects, are generally well characterized. By contrast, the bacterial responses to antibiotic drug treatments that contribute to cell death are not as well understood and have proven to be complex as they involve many genetic and biochem. pathways. In this review, we discuss the multilayered effects of drug-target interactions, including the essential cellular processes that are inhibited by bactericidal antibiotics and the assocd. cellular response mechanisms that contribute to killing. We also discuss new insights into these mechanisms that have been revealed through the study of biol. networks, and describe how these insights, together with related developments in synthetic biol., could be exploited to create new antibacterial therapies.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3cXlsFWit74%253D&md5=716c7004637b4255e529f90263b2483f
  5. 5
    Kojima, S.; Nikaido, H. Permeation Rates of Penicillins Indicate That Escherichia Coli Porins Function Principally as Nonspecific Channels. Proc. Natl. Acad. Sci. U.S.A. 2013, 110 (28), E2629– E2634,  DOI: 10.1073/pnas.1310333110
    Google Scholar
    5
    Permeation rates of penicillins indicate that Escherichia coli porins function principally as nonspecific channels
    Kojima, Seiji; Nikaido, Hiroshi
    Proceedings of the National Academy of Sciences of the United States of America (2013), 110 (28), E2629-E2634, SE2629/1-SE2629/3CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)
    Small, hydrophilic compds. such as β-lactams diffuse across the outer membrane of Gram-neg. bacteria through porin channels, which were originally thought to be nonspecific channels devoid of any specificity. However, since the discovery of an ampicillin-binding site within the OmpF channel in 2002, much attention has been focused on the potential specificity of the channel, where the binding site was assumed either to facilitate or to retard the penetration of β-lactams. Since the earlier studies on porin permeability were done without the knowledge of the contribution of multidrug efflux pumps in the overall flux process across the cell envelope, in this study we have carefully studied both the porin permeability and active efflux of ampicillin and benzylpenicillin. It was found that the influx occurs apparently by a spontaneous passive diffusion without any indication of specific binding within the concn. range relevant to the antibiotic action of these drugs, and that the higher permeability for ampicillin is totally as expected from the gross property of this drug as a zwitterionic compd. The active efflux by AcrAB was more effective for benzylpenicillin due to the stronger affinity and high degree of pos. cooperativity. The data now give a complete quant. picture of the influx, efflux, and periplasmic degrdn. (catalyzed by AmpC β-lactamase) of these two compds., and correlate closely with the susceptibility of Escherichia coli strains used here.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3sXht1Gls7vK&md5=aadfe456c8ba34fd3b8522143313d41d
  6. 6
    June, C. M.; Vaughan, R. M.; Ulberg, L. S.; Bonomo, R. A.; Witucki, L. A.; Leonard, D. A. A fluorescent carbapenem for structure function studies of penicillin-binding proteins, β-lactamases, and β-lactam sensors. Anal. Biochem. 2014, 463, 70– 74,  DOI: 10.1016/j.ab.2014.07.012
    Google Scholar
    6
    A fluorescent carbapenem for structure function studies of penicillin-binding proteins, β-lactamases, and β-lactam sensors
    June, Cynthia M.; Vaughan, Robert M.; Ulberg, Lucas S.; Bonomo, Robert A.; Witucki, Laurie A.; Leonard, David A.
    Analytical Biochemistry (2014), 463 (), 70-74CODEN: ANBCA2; ISSN:0003-2697. (Elsevier B.V.)
    By reacting fluorescein isothiocyanate with meropenem, the authors prepd. a carbapenem-based fluorescent β-lactam. Fluorescein-meropenem binds both penicillin-binding proteins and β-lactam sensors and undergoes a typical acylation reaction in the active site of these proteins. The probe binds the class D carbapenemase OXA-24/40 with close to the same affinity as meropenem and undergoes a complete catalytic hydrolysis reaction. The visible light excitation and strong emission of fluorescein render this mol. a useful structure-function probe through its application in sodium dodecyl sulfate-PAGE assays as well as soln.-based kinetic anisotropy assays. Its classification as a carbapenem β-lactam and the position of its fluorescent modification render it a useful complement to other fluorescent β-lactams, most notably Bocillin FL. The authors show the utility of fluorescein-meropenem by using it to detect mutants of OXA-24/40 that arrest at the acyl-intermediate state with carbapenem substrates but maintain catalytic competency with penicillin substrates.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2cXht1yntrrK&md5=4e334a7676582ff6b55ca99adfdda986
  7. 7
    Davis, T. D.; Gerry, C. J.; Tan, D. S. General Platform for Systematic Quantitative Evaluation of Small-Molecule Permeability in Bacteria. ACS Chem. Biol. 2014, 9 (11), 2535– 2544,  DOI: 10.1021/cb5003015
    Google Scholar
    7
    General Platform for Systematic Quantitative Evaluation of Small-Molecule Permeability in Bacteria
    Davis, Tony D.; Gerry, Christopher J.; Tan, Derek S.
    ACS Chemical Biology (2014), 9 (11), 2535-2544CODEN: ACBCCT; ISSN:1554-8929. (American Chemical Society)
    The chem. features that impact small-mol. permeability across bacterial membranes are poorly understood, and the resulting lack of tools to predict permeability presents a major obstacle to the discovery and development of novel antibiotics. Antibacterials are known to have vastly different structural and physicochem. properties compared to nonantiinfective drugs, as illustrated herein by principal component anal. (PCA). To understand how these properties influence bacterial permeability, the authors have developed a systematic approach to evaluate the penetration of diverse compds. into bacteria with distinct cellular envelopes. Intracellular compd. accumulation is quantitated using LC-MS/MS, then PCA and Pearson pairwise correlations were used to identify structural and physicochem. parameters that correlate with accumulation. An initial study using 10 sulfonyladenosines in Escherichia coli, Bacillus subtilis, and Mycobacterium smegmatis has identified nonobvious correlations between chem. structure and permeability that differ among the various bacteria. Effects of cotreatment with efflux pump inhibitors were also studied. This sets the stage for use of this platform in larger prospective analyses of diverse chemotypes to identify global relations between chem. structure and bacterial permeability that would enable the development of predictive tools to accelerate antibiotic drug discovery.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2cXhsV2hurzP&md5=25a114aafaf5ded965923e963010afaa
  8. 8
    Ghai, I.; Winterhalter, M.; Wagner, R. Probing transport of charged β-lactamase inhibitors through OmpC, a membrane channel from E. coli. Biochem. Biophys. Res. Commun. 2017, 484 (1), 51– 55,  DOI: 10.1016/j.bbrc.2017.01.076
    Google Scholar
    There is no corresponding record for this reference.
  9. 9
    Kaščáková, S.; Maigre, L.; Chevalier, J.; Réfrégiers, M.; Pagès, J. M. Antibiotic Transport in Resistant Bacteria: Synchrotron UV Fluorescence Microscopy to Determine Antibiotic Accumulation with Single Cell Resolution. PLoS One 2012, 7 (6), e38624  DOI: 10.1371/journal.pone.0038624
    Google Scholar
    9
    Antibiotic transport in resistant bacteria: synchrotron UV fluorescence microscopy to determine antibiotic accumulation with single cell resolution
    Kascakova, Slavka; Maigre, Laure; Chevalier, Jacqueline; Refregiers, Matthieu; Pages, Jean-Marie
    PLoS One (2012), 7 (6), e38624CODEN: POLNCL; ISSN:1932-6203. (Public Library of Science)
    A mol. definition of the mechanism conferring bacterial multidrug resistance is clin. crucial and today methods for quant. detn. of the uptake of antimicrobial agents with single cell resoln. are missing. Using the naturally occurring fluorescence of antibacterial agents after deep UV (DUV) excitation, we developed a method to non-invasively monitor the quinolones uptake in single bacteria. Our approach is based on a DUV fluorescence microscope coupled to a synchrotron beamline providing tuneable excitation from 200 to 600 nm. A full spectrum was acquired at each pixel of the image, to study the DUV excited fluorescence emitted from quinolones within single bacteria. Measuring spectra allowed us to sep. the antibiotic fluorescence from the autofluorescence contribution. By performing spectroscopic anal., the quantification of the antibiotic signal was possible. To our knowledge, this is the first time that the intracellular accumulation of a clin. antibiotic could be detd. and discussed in relation with the level of drug susceptibility for a multiresistant strain. This method is esp. important to follow the behavior of quinolone mols. at individual cell level, to quantify the intracellular concn. of the antibiotic and develop new strategies to combat the dissemination of MDR-bacteria. In addn., this original approach also indicates the heterogeneity of bacterial population when the same strain is under environmental stress like antibiotic attack.
    >> More from SciFinder ®
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  10. 10
    Zhou, Y.; Joubran, C.; Miller-Vedam, L.; Isabella, V.; Nayar, A.; Tentarelli, S.; Miller, A. Thinking Outside the “Bug”: A Unique Assay To Measure Intracellular Drug Penetration in Gram-Negative Bacteria. Anal. Chem. 2015, 87 (7), 3579– 3584,  DOI: 10.1021/ac504880r
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    10
    Thinking Outside the "Bug": A Unique Assay To Measure Intracellular Drug Penetration in Gram-Negative Bacteria
    Zhou, Ying; Joubran, Camil; Miller-Vedam, Lakshmi; Isabella, Vincent; Nayar, Asha; Tentarelli, Sharon; Miller, Alita
    Analytical Chemistry (Washington, DC, United States) (2015), 87 (7), 3579-3584CODEN: ANCHAM; ISSN:0003-2700. (American Chemical Society)
    Significant challenges are present in antibiotic drug discovery and development. One of these is the no. of efficient approaches Gram-neg. bacteria have developed to avoid intracellular accumulation of drugs and other cell-toxic species. In order to better understand these processes and correlate in vitro enzyme inhibition to whole cell activity, a better assay to evaluate a key factor, intracellular accumulation of the drug, is urgently needed. Here, we describe a unique liq. chromatog. (LC)-mass spectrometry (MS) approach to measure the amt. of cellular uptake of antibiotics by Gram-neg. bacteria. This method, which measures the change of extracellular drug concn., was evaluated by comparing the relative uptake of linezolid by Escherichia coli wild-type vs. an efflux pump deficient strain. A higher dosage of the drug showed a higher accumulation in these bacteria in a dosing range of 5-50 ng/mL. The Escherichia coli efflux pump deficient strain had a higher accumulation of the drug than the wild-type strain as predicted. The approach was further validated by detg. the relative meropenem uptake by Pseudomonas aeruginosa wild-type vs. a mutant strain lacking multiple porins. These studies show great promise of being applied within antibiotic drug discovery, as a universal tool to aid in the search for compds. that can easily penetrate bacterial cells.
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  11. 11
    Cama, J.; Bajaj, H.; Pagliara, S.; Maier, T.; Braun, Y.; Winterhalter, M.; Keyser, U. F. Quantification of Fluoroquinolone Uptake through the Outer Membrane Channel OmpF of Escherichia Coli. J. Am. Chem. Soc. 2015, 137 (43), 13836– 13843,  DOI: 10.1021/jacs.5b08960
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    11
    Quantification of fluoroquinolone uptake through the outer membrane channel OmpF of Escherichia coli
    Cama, Jehangir; Bajaj, Harsha; Pagliara, Stefano; Maier, Theresa; Braun, Yvonne; Winterhalter, Mathias; Keyser, Ulrich F.
    Journal of the American Chemical Society (2015), 137 (43), 13836-13843CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
    Decreased drug accumulation is a common cause of antibiotic resistance in microorganisms. However, there are few reliable general techniques capable of quantifying drug uptake through bacterial membranes. The authors present a semiquant. optofluidic assay for studying the uptake of autofluorescent drug mols. in single liposomes. They studied the effect of the Escherichia coli outer membrane channel OmpF on the accumulation of the fluoroquinolone antibiotic, norfloxacin, in proteoliposomes. Measurements were performed at pH 5 and pH 7, corresponding to two different charge states of norfloxacin that bacteria are likely to encounter in the human gastrointestinal tract. At both pH values, the porins significantly enhance drug permeation across the proteoliposome membranes. At pH 5, where norfloxacin permeability across pure phospholipid membranes is low, the porins increase drug permeability by 50-fold on av. The authors est. a flux of about 10 norfloxacin mols. per s per OmpF trimer in the presence of a 1 mM concn. gradient of norfloxacin. They also performed single channel electrophysiol. measurements and found that the application of transmembrane voltages causes an elec. field driven uptake in addn. to concn. driven diffusion. The authors propose a phys. mechanism for the pH mediated change in bacterial susceptibility to fluoroquinolone antibiotics.
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  12. 12
    Cinquin, B.; Maigre, L.; Pinet, E.; Chevalier, J.; Stavenger, R. A.; Mills, S.; Réfrégiers, M.; Pagès, J. M. Microspectrometric Insights on the Uptake of Antibiotics at the Single Bacterial Cell Level. Sci. Rep. 2015, 5 (1), 17968,  DOI: 10.1038/srep17968
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    There is no corresponding record for this reference.
  13. 13
    Geddes, E. J.; Li, Z.; Hergenrother, P. J. An LC-MS/MS Assay and Complementary Web-Based Tool to Quantify and Predict Compound Accumulation in E. Coli. Nat. Protoc. 2021, 16 (10), 4833– 4854,  DOI: 10.1038/s41596-021-00598-y
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    13
    An LC-MS/MS assay and complementary web-based tool to quantify and predict compound accumulation in E. coli
    Geddes, Emily J.; Li, Zhong; Hergenrother, Paul J.
    Nature Protocols (2021), 16 (10), 4833-4854CODEN: NPARDW; ISSN:1750-2799. (Nature Portfolio)
    A review. Novel classes of broad-spectrum antibiotics have been extremely difficult to discover, largely due to the impermeability of the Gram-neg. membranes coupled with a poor understanding of the physicochem. properties a compd. should possess to promote its accumulation inside the cell. To address this challenge, numerous methodologies for assessing intracellular compd. accumulation in Gram-neg. bacteria have been established, including classic radiometric and fluorescence-based methods. The recent development of accumulation assays that utilize liq. chromatog.-tandem mass spectrometry (LC-MS/MS) have circumvented the requirement for labeled compds., enabling assessment of a substantially broader range of small mols. Our unbiased study of accumulation trends in Escherichia coli using an LC-MS/MS-based assay led to the development of the eNTRy rules, which stipulate that a compd. is most likely to accumulate in E. coli if it has an ionizable Nitrogen, has low Three-dimensionality and is relatively Rigid. To aid in the implementation of the eNTRy rules, we developed a complementary web tool, eNTRyway, which calcs. relevant properties and predicts compd. accumulation. Here we provide a comprehensive protocol for anal. and prediction of intracellular accumulation of small mols. in E. coli using an LC-MS/MS-based assay (which takes ∼2 d) and eNTRyway, a workflow that is readily adoptable by any microbiol., biochem. or chem. biol. lab.
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  14. 14
    Iyer, R.; Ye, Z.; Ferrari, A.; Duncan, L.; Tanudra, M. A.; Tsao, H.; Wang, T.; Gao, H.; Brummel, C. L.; Erwin, A. L. Evaluating LC-MS/MS To Measure Accumulation of Compounds within Bacteria. ACS Infect. Dis. 2018, 4 (9), 1336– 1345,  DOI: 10.1021/acsinfecdis.8b00083
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    14
    Evaluating LC-MS/MS To Measure Accumulation of Compounds within Bacteria
    Iyer, Ramkumar; Ye, Zhengqi; Ferrari, Annette; Duncan, Leonard; Tanudra, M. Angela; Tsao, Hong; Wang, Tiansheng; Gao, Hong; Brummel, Christopher L.; Erwin, Alice L.
    ACS Infectious Diseases (2018), 4 (9), 1336-1345CODEN: AIDCBC; ISSN:2373-8227. (American Chemical Society)
    A general method for detg. bacterial uptake of compds. independent of antibacterial activity would be a valuable tool in antibacterial drug discovery. LC-MS/MS assays have been described, but it has not been shown whether the data can be used directly to inform medicinal chem. We describe the evaluation of an LC-MS/MS assay measuring assocn. of compds. with bacteria, using a set of over a hundred compds. (inhibitors of NAD-dependent DNA ligase, LigA) for which in vitro potency and antibacterial activity had been detd. All compds. were active against an efflux-deficient strain of Escherichia coli with reduced LigA activity (E. coli ligA251 ΔtolC). Testing a single compd. concn. and incubation time, we found that, for equipotent compds., LC-MS/MS values were not predictive of antibacterial activity. This indicates that measured bacteria-assocd. compd. was not necessarily exposed to the target enzyme. Our data suggest that, while exclusion from bacteria is a major reason for poor antibacterial activity of potent compds., the distribution of compd. within the bacterial cell may also be a problem. The relative importance of these factors is likely to vary from one chem. series to another. Our observations provide directions for further study of this difficult issue.
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  15. 15
    Jones, S. W.; Christison, R.; Bundell, K.; Voyce, C. J.; Brockbank, S. M. V.; Newham, P.; Lindsay, M. A. Characterisation of Cell-Penetrating Peptide-Mediated Peptide Delivery. Br. J. Pharmacol. 2005, 145 (8), 1093– 1102,  DOI: 10.1038/sj.bjp.0706279
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    15
    Characterization of cell-penetrating peptide-mediated peptide delivery
    Jones, Simon W.; Christison, Richard; Bundell, Ken; Voyce, Catherine J.; Brockbank, Sarah M. V.; Newham, Peter; Lindsay, Mark A.
    British Journal of Pharmacology (2005), 145 (8), 1093-1102CODEN: BJPCBM; ISSN:0007-1188. (Nature Publishing Group)
    Cell-penetrating peptides such as antennapedia, TAT, transportan and polyarginine have been extensively employed for in vitro and in vivo delivery of biol. active peptides. However, little is known of the relative efficacy, toxicity and uptake mechanism of individual protein transduction domain-peptide conjugates, factors that will be crit. in detg. the most effective sequence. In the present study, we show by FACS anal. that unconjugated antennapedia, TAT, transportan and polyarginine demonstrate similar kinetic uptake profiles, being maximal at 1-3 h and independent of cell type (HeLa, A549 and CHO cell lines). A comparison of the magnitude of uptake of cell-penetrating peptide conjugates demonstrated that polyarginine = transportan>antennapedia > TAT. However, examn. of cellular toxicity showed that antennapedia < TAT < transportan < ∩ polyarginine, with antennapedia-peptide conjugates having no significant toxicity even at 100 μM. Confocal studies of the mechanism of antennapedia- and TAT-peptide uptake showed that the time course of uptake and their cellular distribution did not correlate with transferrin, a marker of clathrin-mediated endocytosis. In contrast, the peptides co-localized with a marker of lipid rafts domains, cholera toxin, which was attenuated following the disruption of these domains using methyl-β-cyclodextrin. Overall, comparison of the uptake and toxicity suggests that antennapedia provides the optimal cell-penetrating peptide for peptide delivery in vitro and that both antennapedia- and TAT-mediated peptide delivery occurs predominantly via lipid raft-dependent but clathrin-independent endocytosis.
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  16. 16
    Fischer, R.; Waizenegger, T.; Köhler, K.; Brock, R. A Quantitative Validation of Fluorophore-Labelled Cell-Permeable Peptide Conjugates: Fluorophore and Cargo Dependence of Import. Biochim. Biophys. Acta, Biomembr. 2002, 1564 (2), 365– 374,  DOI: 10.1016/S0005-2736(02)00471-6
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    16
    A quantitative validation of fluorophore-labelled cell-permeable peptide conjugates: fluorophore and cargo dependence of import
    Fischer, Rainer; Waizenegger, Thomas; Kohler, Karsten; Brock, Roland
    Biochimica et Biophysica Acta, Biomembranes (2002), 1564 (2), 365-374CODEN: BBBMBS; ISSN:0005-2736. (Elsevier B.V.)
    Cell-permeable peptides were evaluated for a quant. controlled import of small mols. The dependence of the import efficiency on the fluorophore, on the position of the fluorophore as well as on the nature of the cargo were addressed. Cellular uptake was quantitated by flow cytometry and fluorescence correlation microscopy (FCM). Fluorophores with different spectral characteristics, covering the whole visible spectral range, were selected in order to enable the simultaneous detection of several cell-permeable peptide constructs. The transcytosis sequences were based either on the sequence of the Antennapedia homeodomain protein (AntpHD)-derived penetratin peptide or the Kaposi fibroblast growth factor (FGF)-derived membrane translocating sequence (MTS)-peptide. In general, the AntpHD-derived peptides had a three- to fourfold higher import efficiency than the MTS-derived peptides. In spite of the very different physicochem. characteristics of the fluorophores, the import efficiencies for analogs labeled at different positions within the sequence of the import peptides showed a strong pos. correlation. However, even for peptide cargos of very similar size, pronounced differences in import efficiency were obsd. The use of cell-permeable peptide/cargo constructs for intracellular analyses of structure-function relationships therefore requires the detn. of the intracellular concns. for each construct individually.
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  17. 17
    Illien, F.; Rodriguez, N.; Amoura, M.; Joliot, A.; Pallerla, M.; Cribier, S.; Burlina, F.; Sagan, S. Quantitative Fluorescence Spectroscopy and Flow Cytometry Analyses of Cell-Penetrating Peptides Internalization Pathways: Optimization, Pitfalls, Comparison with Mass Spectrometry Quantification. Sci. Rep. 2016, 6 (1), 36938,  DOI: 10.1038/srep36938
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    17
    Quantitative fluorescence spectroscopy and flow cytometry analyses of cell-penetrating peptides internalization pathways: optimization, pitfalls, comparison with mass spectrometry quantification
    Illien, Francoise; Rodriguez, Nicolas; Amoura, Mehdi; Joliot, Alain; Pallerla, Manjula; Cribier, Sophie; Burlina, Fabienne; Sagan, Sandrine
    Scientific Reports (2016), 6 (), 36938CODEN: SRCEC3; ISSN:2045-2322. (Nature Publishing Group)
    The mechanism of cell-penetrating peptides entry into cells is unclear, preventing the development of more efficient vectors for biotechnol. or therapeutic purposes. Here, we developed a protocol relying on fluorometry to distinguish endocytosis from direct membrane translocation, using Penetratin, TAT and R9. The quantities of internalized CPPs measured by fluorometry in cell lysates converge with those obtained by our previously reported mass spectrometry quantification method. By contrast, flow cytometry quantification faces several limitations due to fluorescence quenching processes that depend on the cell line and occur at peptide/cell ratio >6.108 for CF-Penetratin. The anal. of cellular internalization of a doubly labeled fluorescent and biotinylated Penetratin analog by the two independent techniques, fluorometry and mass spectrometry, gave consistent results at the quant. and qual. levels. Both techniques revealed the use of two alternative translocation and endocytosis pathways, whose relative efficacy depends on cell-surface sugars and peptide concn. We confirmed that Penetratin translocates at low concn. and uses endocytosis at high μM concns. We further demonstrate that the hydrophobic/hydrophilic nature of the N-terminal extremity impacts on the internalization efficiency of CPPs. We expect these results and the assocd. protocols to help unraveling the translocation pathway to the cytosol of cells.
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  18. 18
    Liu, Z.; Lepori, I.; Chordia, M. D.; Dalesandro, B. E.; Guo, T.; Dong, J.; Siegrist, M. S.; Pires, M. M. A Metabolic-Tag-Based Method for Assessing the Permeation of Small Molecules Across the Mycomembrane in Live Mycobacteria**. Angew. Chem., Int. Ed. 2023, 62 (20), e202217777  DOI: 10.1002/anie.202217777
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    There is no corresponding record for this reference.
  19. 19
    Kelly, J. J.; Dalesandro, B. E.; Liu, Z.; Chordia, M. D.; Ongwae, G. M.; Pires, M. M. Measurement of Accumulation of Antibiotics to Staphylococcus Aureus in Phagosomes of Live Macrophages. Angew. Chem. 2024, 136 (3), e202313870  DOI: 10.1002/ange.202313870
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    There is no corresponding record for this reference.
  20. 20
    Ongwae, G. M.; Lepori, I.; Chordia, M. D.; Dalesandro, B. E.; Apostolos, A. J.; Siegrist, M. S.; Pires, M. M. Measurement of Small Molecule Accumulation into Diderm Bacteria. ACS Infect. Dis. 2023, 9 (1), 97– 110,  DOI: 10.1021/acsinfecdis.2c00435
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    20
    Measurement of Small Molecule Accumulation into Diderm Bacteria
    Ongwae, George M.; Lepori, Irene; Chordia, Mahendra D.; Dalesandro, Brianna E.; Apostolos, Alexis J.; Siegrist, M. Sloan; Pires, Marcos M.
    ACS Infectious Diseases (2023), 9 (1), 97-110CODEN: AIDCBC; ISSN:2373-8227. (American Chemical Society)
    Some of the most dangerous bacterial pathogens (Gram-neg. and mycobacterial) deploy a formidable secondary membrane barrier to reduce the influx of exogenous mols. For Gram-neg. bacteria, this second exterior membrane is known as the outer membrane (OM), while for the Gram-indeterminate Mycobacteria, it is known as the "myco" membrane. Although different in compn., both the OM and mycomembrane are key structures that restrict the passive permeation of small mols. into bacterial cells. Although it is well-appreciated that such structures are principal determinants of small mol. permeation, it has proven to be challenging to assess this feature in a robust and quant. way or in complex, infection-relevant settings. Herein, we describe the development of the bacterial chloro-alkane penetration assay (BaCAPA), which employs the use of a genetically encoded protein called HaloTag, to measure the uptake and accumulation of mols. into model Gram-neg. and mycobacterial species, Escherichia coli and Mycobacterium smegmatis, resp., and into the human pathogen Mycobacterium tuberculosis. The HaloTag protein can be directed to either the cytoplasm or the periplasm of bacteria. This offers the possibility of compartmental anal. of permeation across individual cell membranes. Significantly, we also showed that BaCAPA can be used to analyze the permeation of mols. into host cell-internalized E. coli and M. tuberculosis, a crit. capability for analyzing intracellular pathogens. Together, our results show that BaCAPA affords facile measurement of permeability across four barriers: the host plasma and phagosomal membranes and the diderm bacterial cell envelope.
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  21. 21
    Karatas, H.; Maric, T.; D’Alessandro, P. L.; Yevtodiyenko, A.; Vorherr, T.; Hollingworth, G. J.; Goun, E. A. Real-Time Imaging and Quantification of Peptide Uptake in Vitro and in Vivo. ACS Chem. Biol. 2019, 14 (10), 2197– 2205,  DOI: 10.1021/acschembio.9b00439
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    There is no corresponding record for this reference.
  22. 22
    Godinat, A.; Bazhin, A. A.; Goun, E. A. Bioorthogonal Chemistry in Bioluminescence Imaging. Drug Discovery Today 2018, 23 (9), 1584– 1590,  DOI: 10.1016/j.drudis.2018.05.022
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    There is no corresponding record for this reference.
  23. 23
    Carmel-Harel, O.; Storz, G. Roles of the Glutathione- and Thioredoxin-Dependent Reduction Systems in the Escherichia Coli and Saccharomyces Cerevisiae Responses to Oxidative Stress. Annu. Rev. Microbiol. 2000, 54 (1), 439– 461,  DOI: 10.1146/annurev.micro.54.1.439
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    There is no corresponding record for this reference.
  24. 24
    Patterson, D. M.; Nazarova, L. A.; Prescher, J. A. Finding the Right (Bioorthogonal) Chemistry. ACS Chem. Biol. 2014, 9 (3), 592– 605,  DOI: 10.1021/cb400828a
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    24
    Finding the Right (Bioorthogonal) Chemistry
    Patterson, David M.; Nazarova, Lidia A.; Prescher, Jennifer A.
    ACS Chemical Biology (2014), 9 (3), 592-605CODEN: ACBCCT; ISSN:1554-8929. (American Chemical Society)
    A review. Bioorthogonal chemistries can be used to tag diverse classes of biomols. in cells and other complex environments. With over 20 unique transformations now available, though, selecting an appropriate reaction for a given expt. is challenging. The authors compare and contrast the most common classes of bioorthogonal chemistries and provide a framework for matching the reactions with downstream applications. The authors also discuss ongoing efforts to identify novel biocompatible reactions and methods to control their reactivity. The continued expansion of the bioorthogonal toolkit will provide new insights into biomol. networks and functions and thus refine the authors' understanding of living systems.
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  25. 25
    Liang, G.; Ren, H.; Rao, J. A Biocompatible Condensation Reaction for Controlled Assembly of Nanostructures in Living Cells. Nat. Chem. 2010, 2 (1), 54– 60,  DOI: 10.1038/nchem.480
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    25
    A biocompatible condensation reaction for controlled assembly of nanostructures in living cells
    Liang, Gaolin; Ren, Hongjun; Rao, Jianghong
    Nature Chemistry (2010), 2 (1), 54-60CODEN: NCAHBB; ISSN:1755-4330. (Nature Publishing Group)
    Through controlled synthesis and mol. assembly, biol. systems are able to organize mols. into supramol. structures that carry out sophisticated processes. Although chemists have reported a few examples of supramol. assembly in, the controlled covalent synthesis of large mols. and structures in vivo has remained challenging. Here the authors report a condensation reaction between 1,2-aminothiol and 2-cyanobenzothiazole that occurs in vitro and in living cells under the control of either pH, disulfide redn. or enzymic cleavage. In vitro, the size and shape of the condensation products, and the nanostructures subsequently assembled, were different in each case and could thus be controlled by tuning the structure of the monomers. Direct imaging of the products obtained in the cells revealed their locations-near the Golgi bodies under enzymic cleavage control-demonstrating the feasibility of a controlled and localized reaction in living cells. This intracellular condensation process enabled the imaging of the proteolytic activity of furin.
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  26. 26
    Nguyen, D. P.; Elliott, T.; Holt, M.; Muir, T. W.; Chin, J. W. Genetically Encoded 1,2-Aminothiols Facilitate Rapid and Site-Specific Protein Labeling via a Bio-Orthogonal Cyanobenzothiazole Condensation. J. Am. Chem. Soc. 2011, 133 (30), 11418– 11421,  DOI: 10.1021/ja203111c
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    26
    Genetically Encoded 1,2-Aminothiols Facilitate Rapid and Site-Specific Protein Labeling via a Bio-orthogonal Cyanobenzothiazole Condensation
    Nguyen, Duy P.; Elliott, Thomas; Holt, Matthew; Muir, Tom W.; Chin, Jason W.
    Journal of the American Chemical Society (2011), 133 (30), 11418-11421CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
    The authors report evolved orthogonal pyrrolysyl-tRNA synthetase/tRNACUA pairs that direct the efficient, site-specific incorporation of Nε-L-thiaprolyl-L-lysine, Nε-D-cysteinyl-L-lysine, and Nε-L-cysteinyl-L-lysine into recombinant proteins in Escherichia coli. The authors demonstrate that the unique 1,2-aminothiol introduced by the authors' approach can be efficiently, rapidly, and specifically labeled via a cyanobenzothiazole condensation to quant. introduce biophys. probes into proteins. Moreover, the authors show that, in combination with cysteine labeling, this approach allows the dual labeling of proteins with distinct probes at two distinct, genetically defined sites.
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  27. 27
    Ramil, C. P.; An, P.; Yu, Z.; Lin, Q. Sequence-Specific 2-Cyanobenzothiazole Ligation. J. Am. Chem. Soc. 2016, 138 (17), 5499– 5502,  DOI: 10.1021/jacs.6b00982
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    27
    Sequence-Specific 2-Cyanobenzothiazole Ligation
    Ramil, Carlo P.; An, Peng; Yu, Zhipeng; Lin, Qing
    Journal of the American Chemical Society (2016), 138 (17), 5499-5502CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
    The use of small, natural chem. reporters in conjunction with catalyst-free bioorthogonal reactions will greatly streamline protein labeling in a cellular environment with min. perturbation to their function. Here we report the discovery of a 2-cyanobenzothiazole (CBT)-reactive peptide tag, CX10R7, from a cysteine-encoded peptide phage library using the phage-assisted interrogation of reactivity method. Fusion of CX10R7 with a protein of interest allows site-specific labeling of the protein with CBT both in vitro and on the surface of E. coli cells. Mutagenesis studies indicated that the reactivity and specificity of CX10R7 are attributed to the sequence environment, in which the residues surrounding cysteine help to stabilize the ligation product.
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  28. 28
    Ren, H.; Xiao, F.; Zhan, K.; Kim, Y.-P.; Xie, H.; Xia, Z.; Rao, J. A Biocompatible Condensation Reaction for the Labeling of Terminal Cysteine Residues on Proteins. Angew. Chem., Int. Ed. 2009, 48 (51), 9658– 9662,  DOI: 10.1002/anie.200903627
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    28
    A Biocompatible Condensation Reaction for the Labeling of Terminal Cysteine Residues on Proteins
    Ren, Hongjun; Xiao, Fei; Zhan, Ke; Kim, Young-Pil; Xie, Hexin; Xia, Zuyong; Rao, Jianghong
    Angewandte Chemie, International Edition (2009), 48 (51), 9658-9662, S9658/1-S9658/16CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)
    A protein-labeling method based on the use of a single amino acid tag - an N-terminal cysteine residue - and small-mol. probes contg. a cyanobenzothiazole (CBT) unit has been used for the specific fluorescence labeling of proteins in vitro and at the surface of live cells. This simple ligation reaction proceeds with a high degree of specificity under physiol. conditions.
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  29. 29
    Van de Bittner, G. C.; Bertozzi, C. R.; Chang, C. J. Strategy for Dual-Analyte Luciferin Imaging: In Vivo Bioluminescence Detection of Hydrogen Peroxide and Caspase Activity in a Murine Model of Acute Inflammation. J. Am. Chem. Soc. 2013, 135 (5), 1783– 1795,  DOI: 10.1021/ja309078t
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    29
    Strategy for Dual-Analyte Luciferin Imaging: In Vivo Bioluminescence Detection of Hydrogen Peroxide and Caspase Activity in a Murine Model of Acute Inflammation
    Van de Bittner, Genevieve C.; Bertozzi, Carolyn R.; Chang, Christopher J.
    Journal of the American Chemical Society (2013), 135 (5), 1783-1795CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
    In vivo mol. imaging holds promise for understanding the underlying mechanisms of health, injury, aging, and disease, as it can detect distinct biochem. processes such as enzymic activity, reactive small-mol. fluxes, or post-translational modifications. Current imaging techniques often detect only a single biochem. process, but, within whole organisms, multiple types of biochem. events contribute to physiol. and pathol. phenotypes. The authors present a general strategy for dual-analyte detection in living animals that employs in situ formation of firefly luciferin from two complementary caged precursors that can be unmasked by different biochem. processes. To establish this approach, the authors have developed Peroxy Caged Luciferin-2 (PCL-2, I), a H2O2-responsive boronic acid probe that releases 6-hydroxy-2-cyanobenzothiazole (HCBT) upon reacting with this reactive oxygen species, as well as a peptide-based probe, z-Ile-Glu-ThrAsp-D-Cys (IETDC), which releases D-cysteine in the presence of active caspase 8. Once released, HCBT and D-cysteine form firefly luciferin in situ, giving rise to a bioluminescent signal if and only if both chem. triggers proceed. This system thus constitutes an AND-type mol. logic gate that reports on the simultaneous presence of H2O2 and caspase 8 activity. Using these probes, chemoselective imaging of either H2O2 or caspase 8 activity was performed in vitro and in vivo. Moreover, concomitant use of PCL-2 and IETDC in vivo establishes a concurrent increase in both H2O2 and caspase 8 activity during acute inflammation in living mice. Taken together, this method offers a potentially powerful new chem. tool for studying simultaneous oxidative stress and inflammation processes in living animals during injury, aging, and disease, as well as a versatile approach for concurrent monitoring of multiple analytes using luciferin-based bioluminescence imaging technologies.
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  30. 30
    Roychaudhuri, R.; Gadalla, M. M.; West, T.; Snyder, S. H. A Novel Stereospecific Bioluminescent Assay for Detection of Endogenous D-Cysteine. ACS Chem. Neurosci. 2022, 13 (23), 3257– 3262,  DOI: 10.1021/acschemneuro.2c00528
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    30
    A Novel Stereospecific Bioluminescent Assay for Detection of Endogenous D-Cysteine
    Roychaudhuri, Robin; Gadalla, Moataz M.; West, Timothy; Snyder, Solomon H.
    ACS Chemical Neuroscience (2022), 13 (23), 3257-3262CODEN: ACNCDM; ISSN:1948-7193. (American Chemical Society)
    The presence of endogenous D-stereoisomers of amino acids in mammals dispels a long-standing dogma about their existence. D-Serine and D-aspartate function as novel neurotransmitters in mammals. However, the stereoisomer with the fastest, spontaneous in vitro racemization rate, D-cysteine, has not been reported. We utilized a novel, stereospecific, bioluminescent assay to identify endogenous D-cysteine in substantial amts. in the eye, brain, and pancreas of mice. D-Cysteine is enriched in mice embryonic brains at day E9.5 (4.5 mM) and decreases progressively with development (μM levels). D-Cysteine is also present in significantly higher amts. in the human brain white matter compared with gray matter. In the luciferase assay, D-cysteine conjugates with cyano hydroxy benzothiazole in the presence of a base and reducing agent to form D-luciferin. D-Luciferin, subsequently, in the presence of firefly luciferase and ATP, emits bioluminescence proportional to the concn. of D-cysteine. The assay is stereospecific and allows the quant. estn. of endogenous D-cysteine in tissues in addn. to its specificity for D-cysteine. Future efforts aimed at bioluminescent in vivo imaging of D-cysteine may allow a more noninvasive means of its detection, thereby elucidating its function.
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  31. 31
    Semenza, E. R.; Harraz, M. M.; Abramson, E.; Malla, A. P.; Vasavda, C.; Gadalla, M. M.; Kornberg, M. D.; Snyder, S. H.; Roychaudhuri, R. D-Cysteine Is an Endogenous Regulator of Neural Progenitor Cell Dynamics in the Mammalian Brain. Proc. Natl. Acad. Sci. U.S.A. 2021, 118 (39), e2110610118  DOI: 10.1073/pnas.2110610118
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    31
    D-cysteine is an endogenous regulator of neural progenitor cell dynamics in the mammalian brain
    Semenza, Evan R.; Harraz, Maged M.; Abramson, Efrat; Malla, Adarsha P.; Vasavda, Chirag; Gadalla, Moataz M.; Kornberg, Michael D.; Snyder, Solomon H.; Roychaudhuri, Robin
    Proceedings of the National Academy of Sciences of the United States of America (2021), 118 (39), e2110610118CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)
    D-amino acids are increasingly recognized as important signaling mols. in the mammalian central nervous system. However, the d-stereoisomer of the amino acid with the fastest spontaneous racemization ratein vitro in vitro, cysteine, has not been examd. in mammals. Using chiral high-performance liq. chromatog. and a stereospecific luciferase assay, we identify endogenous d-cysteine in the mammalian brain. We identify serine racemase (SR), which generates the N-methyl-d-aspartate (NMDA) glutamate receptor coagonist d-serine, as a candidate biosynthetic enzyme for d-cysteine. d-cysteine is enriched more than 20-fold in the embryonic mouse brain compared with the adult brain. d-cysteine reduces the proliferation of cultured mouse embryonic neural progenitor cells (NPCs) by ∼50%, effects not shared with d-serine or l-cysteine. The antiproliferative effect of d-cysteine is mediated by the transcription factors FoxO1 and FoxO3a. The selective influence of d-cysteine on NPC proliferation is reflected in overgrowth and aberrant lamination of the cerebral cortex in neonatal SR knockout mice. Finally, we perform an unbiased screen for d-cysteine-binding proteins in NPCs by immunopptn. with a d-cysteine-specific antibody followed by mass spectrometry. This approach identifies myristoylated alanine-rich C-kinase substrate (MARCKS) as a putative d-cysteine-binding protein. Together, these results establish endogenous mammalian d-cysteine and implicate it as a physiol. regulator of NPC homeostasis in the developing brain.
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  32. 32
    Zhang, B. S.; Jones, K. A.; McCutcheon, D. C.; Prescher, J. A. Pyridone Luciferins and Mutant Luciferases for Bioluminescence Imaging. ChemBioChem 2018, 19 (5), 470– 477,  DOI: 10.1002/cbic.201700542
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    There is no corresponding record for this reference.
  33. 33
    Xiong, Y.; Zhang, Y.; Li, Z.; Reza, M. S.; Li, X.; Tian, X.; Ai, H. Engineered Amber-Emitting Nano Luciferase and Its Use for Immunobioluminescence Imaging In Vivo. J. Am. Chem. Soc. 2022, 144 (31), 14101– 14111,  DOI: 10.1021/jacs.2c02320
    Google Scholar
    There is no corresponding record for this reference.
  34. 34
    White, E. H.; Wörther, H.; Seliger, H. H.; McElroy, W. D. Amino Analogs of Firefly Luciferin and Biological Activity Thereof1. J. Am. Chem. Soc. 1966, 88 (9), 2015– 2019,  DOI: 10.1021/ja00961a030
    Google Scholar
    There is no corresponding record for this reference.
  35. 35
    Godinat, A.; Budin, G.; Morales, A. R.; Park, H. M.; Sanman, L. E.; Bogyo, M.; Yu, A.; Stahl, A.; Dubikovskaya, E. A. A Biocompatible “Split Luciferin” Reaction and Its Application for Non-Invasive Bioluminescent Imaging of Protease Activity in Living Animals. Curr. Protoc. Chem. Biol. 2014, 6 (3), 169– 189,  DOI: 10.1002/9780470559277.ch140047
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    There is no corresponding record for this reference.
  36. 36
    Nakamura, M.; Niwa, K.; Maki, S.; Hirano, T.; Ohmiya, Y.; Niwa, H. Construction of a New Firefly Bioluminescence System Using L-Luciferin as Substrate. Tetrahedron Lett. 2006, 47 (7), 1197– 1200,  DOI: 10.1016/j.tetlet.2005.12.033
    Google Scholar
    There is no corresponding record for this reference.
  37. 37
    Niwa, K.; Nakamura, M.; Ohmiya, Y. Stereoisomeric Bio-Inversion Key to Biosynthesis of Firefly d-Luciferin. FEBS Lett. 2006, 580 (22), 5283– 5287,  DOI: 10.1016/j.febslet.2006.08.073
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    37
    Stereoisomeric bio-inversion key to biosynthesis of firefly D-luciferin
    Niwa, Kazuki; Nakamura, Mitsuhiro; Ohmiya, Yoshihiro
    FEBS Letters (2006), 580 (22), 5283-5287CODEN: FEBLAL; ISSN:0014-5793. (Elsevier B.V.)
    The chirality of the luciferin substrate is crit. to the luciferin-luciferase reaction producing bioluminescence. In firefly, the biosynthetic pathway of D-luciferin is still unclear, although it can be synthesized in vitro from D-cysteine. Here, we show that the firefly produces both D- and L-luciferin, and that the amt. of active D-luciferin increases gradually with maturation stage. Studies of firefly body exts. indicate the possible conversion of L-cysteine via L-luciferin into D-luciferin, suggesting that the biosynthesis is enzymically regulated by stereoisomeric bio-inversion of L-luciferin. We conclude that the selection of chirality in living organisms is not as rigid as previously thought.
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  38. 38
    Ren, Y.; Qiang, Y.; Zhu, B.; Tang, W.; Duan, X.; Li, Z. General Strategy for Bioluminescence Sensing of Peptidase Activity In Vivo Based on Tumor-Targeting Probiotic. Anal. Chem. 2021, 93 (9), 4334– 4341,  DOI: 10.1021/acs.analchem.1c00093
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    38
    General strategy for bioluminescence sensing of peptidase activity in vivo based on tumor-targeting probiotic
    Ren, Yiqian; Qiang, Yao; Zhu, Beibei; Tang, Wei; Duan, Xinrui; Li, Zhengping
    Analytical Chemistry (Washington, DC, United States) (2021), 93 (9), 4334-4341CODEN: ANCHAM; ISSN:0003-2700. (American Chemical Society)
    The abnormally expressed peptidases in human tissues are assocd. with many kinds of cancers. Monitoring of endogenous peptidase activity could allow us for pathophysiol. elucidation and early clin. diagnosis. Herein, we developed a general strategy for bioluminescence (BL) sensing of peptidase activity in vivo based on tumor-targeting probiotics. The probiotic that harbored a luciferase-encoding plasmid was used to target and colonize tumor and provide luciferase for BL imaging. The peptide-based probes Lc and GPc were applied to track leucine aminopeptidase (LAP) and dipeptidyl peptidase IV (DPPIV) activity, resp., by simply adding L-leucine and Gly-Pro dipeptides at the N-terminus of D-cysteine, which were specifically controlled by peptidase cleavage and released free D-cysteine to conduct a subsequent click condensation reaction with 2-cyano-6-hydroxybenzothiazole (HCBT) to produce firefly luciferin in situ, giving rise to a strong BL signal. Neither gene modification of cells of interest nor complicated synthesis was required in this BL system. Encouraged by these advantages, we successfully used our probes to monitor LAP and DPPIV activities in vitro and in vivo, resp. A good linearity between BL and peptidase was obtained in the concn. range of 2.5-40.0 mU/mL with a limit of detection (LOD) of 1.1 mU/mL (55 ng/mL) for LAP and 2.0-40.0 mU/mL with a LOD of 0.78 mU/mL (1.15 ng/mL) for DPPIV, resp. Addnl., approx. 5-fold (LAP) and 10-fold (DPPIV) differences in the BL signal before and after treatment with a specific inhibitor were also obtained for in vivo BL imaging. All these results reflected the potential application value of our probes in BL sensing of peptidase activity. We envision that our strategy may be a useful approach for monitoring a wide range of peptidases in tumors, esp. in primary tumors.
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  39. 39
    Niwa, K.; Nakajima, Y.; Ohmiya, Y. Applications of Luciferin Biosynthesis: Bioluminescence Assays for l-Cysteine and Luciferase. Anal. Biochem. 2010, 396 (2), 316– 318,  DOI: 10.1016/j.ab.2009.09.014
    Google Scholar
    There is no corresponding record for this reference.
  40. 40
    Nakamura, M.; Maki, S.; Amano, Y.; Ohkita, Y.; Niwa, K.; Hirano, T.; Ohmiya, Y.; Niwa, H. Firefly Luciferase Exhibits Bimodal Action Depending on the Luciferin Chirality. Biochem. Biophys. Res. Commun. 2005, 331 (2), 471– 475,  DOI: 10.1016/j.bbrc.2005.03.202
    Google Scholar
    40
    Firefly luciferase exhibits bimodal action depending on the luciferin chirality
    Nakamura, Mitsuhiro; Maki, Shojiro; Amano, Yoshiharu; Ohkita, Yutaka; Niwa, Kazuki; Hirano, Takashi; Ohmiya, Yoshihiro; Niwa, Haruki
    Biochemical and Biophysical Research Communications (2005), 331 (2), 471-475CODEN: BBRCA9; ISSN:0006-291X. (Elsevier)
    Firefly luciferase is able to convert L-luciferin into luciferyl-CoA even under ordinary aerobic luciferin-luciferase reaction conditions. The luciferase is able to recognize strictly the chirality of the luciferin structure, serving as the acyl-CoA synthetase for L-luciferin, whereas D-luciferin is used for the bioluminescence reaction. D-Luciferin inhibits the luciferyl-CoA synthetase activity of L-luciferin, whereas L-luciferin retards the bioluminescence reaction of D-luciferin, meaning that both enzyme activities are prevented by the enantiomer of its own substrate.
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  41. 41
    Fahey, R. C.; Brown, W. C.; Adams, W. B.; Worsham, M. B. Occurrence of Glutathione in Bacteria. J. Bacteriol. 1978, 133 (3), 1126– 1129,  DOI: 10.1128/jb.133.3.1126-1129.1978
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    41
    Occurrence of glutathione in bacteria
    Fahey, Robert C.; Brown, Willie C.; Adams, William B.; Worsham, Michael B.
    Journal of Bacteriology (1978), 133 (3), 1126-9CODEN: JOBAAY; ISSN:0021-9193.
    Glutathione and sol. thiol content were examd. in a broad spectrum of bacteria. Significant sol. thiol was present in all cases. The thiol compd. was glutathione in most of the gram-neg. bacteria but not in most of the gram-pos. bacteria studied. Glutathione was absent in 4 anaerobes and 1 microaerophile but was present in a blue-green bacterium. The glutathione content of Escherichia coli increased significantly during transition from exponential to stationary phase.
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  42. 42
    Taylor, M. D. Improved Passive Oral Drug Delivery via Prodrugs. Adv. Drug Delivery Rev. 1996, 19 (2), 131– 148,  DOI: 10.1016/0169-409X(95)00104-F
    Google Scholar
    There is no corresponding record for this reference.
  43. 43
    Beaumont, K.; Webster, R.; Gardner, I.; Dack, K. Design of Ester Prodrugs to Enhance Oral Absorption of Poorly Permeable Compounds: Challenges to the Discovery Scientist. Curr. Drug Metab. 2003, 4 (6), 461– 485,  DOI: 10.2174/1389200033489253
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    43
    Design of ester prodrugs to enhance oral absorption of poorly permeable compounds: Challenges to the discovery scientist
    Beaumont, Kevin; Webster, Robert; Gardner, Iain; Dack, Kevin
    Current Drug Metabolism (2003), 4 (6), 461-485CODEN: CDMUBU; ISSN:1389-2002. (Bentham Science Publishers Ltd.)
    A review and discussion. Many drugs are administered at sites that are remote from their site of action. The most common route of drug delivery is the oral route. The optimal physicochem. properties to allow high transcellular absorption following oral administration are well established and include a limit on mol. size, hydrogen bonding potential and adequate lipophilicity. For many drug targets, synthetic strategies can be devised to balance the physicochem. properties required for high transcellular absorption and the SAR for the drug target. However, there are drug targets where the SAR requires properties at odds with good membrane permeability. These include a requirement for significant polarity and groups that exhibit high hydrogen bonding potential such as carboxylic acids and alcs. In such cases, prodrug strategies have been employed. The rationale behind the prodrug strategy is to introduce lipophilicity and mask hydrogen bonding groups of an active compd. by the addn. of another moiety, most commonly an ester. An ideal ester prodrug should exhibit the following properties:. (1) Weak (or no) activity against any pharmacol. target,. (2) Chem. stability across a pH range,. (3) High aq. soly.,. (4) Good transcellular absorption,. (5) Resistance to hydrolysis during the absorption phase,. (6) Rapid and quant. breakdown to yield high circulating concns. of the active component post absorption. This paper will review the literature around marketed prodrugs and det. the most appropriate prodrug characteristics. In addn., it will examine potential discovery approaches to optimizing prodrug delivery and recommend a strategy for prosecuting an oral prodrug approach.
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  44. 44
    Mohammad, I.; Liebmann, K. L.; Miller, S. C. Firefly Luciferin Methyl Ester Illuminates the Activity of Multiple Serine Hydrolases. Chem. Commun. 2023, 59 (55), 8552– 8555,  DOI: 10.1039/D3CC02540C
    Google Scholar
    There is no corresponding record for this reference.
  45. 45
    Antonczak, A. K.; Simova, Z.; Tippmann, E. M. A Critical Examination of Escherichia Coli Esterase Activity *. J. Biol. Chem. 2009, 284 (42), 28795– 28800,  DOI: 10.1074/jbc.M109.027409
    Google Scholar
    There is no corresponding record for this reference.
  46. 46
    Brown, A. R.; Wodzanowski, K. A.; Santiago, C. C.; Hyland, S. N.; Follmar, J. L.; Asare-Okai, P.; Grimes, C. L. Protected N-Acetyl Muramic Acid Probes Improve Bacterial Peptidoglycan Incorporation via Metabolic Labeling. ACS Chem. Biol. 2021, 16 (10), 1908– 1916,  DOI: 10.1021/acschembio.1c00268
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    46
    Protected N-Acetyl Muramic Acid Probes Improve Bacterial Peptidoglycan Incorporation via Metabolic Labeling
    Brown, Ashley R.; Wodzanowski, Kimberly A.; Santiago, Cintia C.; Hyland, Stephen N.; Follmar, Julianna L.; Asare-Okai, PapaNii; Grimes, Catherine Leimkuhler
    ACS Chemical Biology (2021), 16 (10), 1908-1916CODEN: ACBCCT; ISSN:1554-8929. (American Chemical Society)
    Metabolic glycan probes have emerged as an excellent tool to investigate vital questions in biol. Recently, methodol. to incorporate metabolic bacterial glycan probes into the cell wall of a variety of bacterial species has been developed. In order to improve this method, a scalable synthesis of the peptidoglycan precursors is developed here, allowing for access to essential peptidoglycan immunol. fragments and cell wall building blocks. The question was asked if masking polar groups of the glycan probe would increase overall incorporation, a common strategy exploited in mammalian glycobiol. Here, we show, through cellular assays, that E. coli do not utilize peracetylated peptidoglycan substrates but do employ Me esters. The 10-fold improvement of probe utilization indicates that (i) masking the carboxylic acid is favorable for transport and (ii) bacterial esterases are capable of removing the Me ester for use in peptidoglycan biosynthesis. This investigation advances bacterial cell wall biol., offering a prescription on how to best deliver and utilize bacterial metabolic glycan probes.
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  47. 47
    Korshunov, S.; Imlay, K. R. C.; Imlay, J. A. Cystine Import Is a Valuable but Risky Process Whose Hazards Escherichia Coli Minimizes by Inducing a Cysteine Exporter. Mol. Microbiol. 2020, 113 (1), 22– 39,  DOI: 10.1111/mmi.14403
    Google Scholar
    There is no corresponding record for this reference.
  48. 48
    Chonoles Imlay, K. R.; Korshunov, S.; Imlay, J. A. Physiological Roles and Adverse Effects of the Two Cystine Importers of Escherichia Coli. J. Bacteriol. 2015, 197 (23), 3629– 3644,  DOI: 10.1128/JB.00277-15
    Google Scholar
    There is no corresponding record for this reference.
  49. 49
    French, S.; Farha, M.; Ellis, M. J.; Sameer, Z.; Côté, J. P.; Cotroneo, N.; Lister, T.; Rubio, A.; Brown, E. D. Potentiation of Antibiotics against Gram-Negative Bacteria by Polymyxin B Analogue SPR741 from Unique Perturbation of the Outer Membrane. ACS Infect. Dis. 2020, 6 (6), 1405– 1412,  DOI: 10.1021/acsinfecdis.9b00159
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    49
    Potentiation of Antibiotics against Gram-Negative Bacteria by Polymyxin B Analogue SPR741 from Unique Perturbation of the Outer Membrane
    French, Shawn; Farha, Maya; Ellis, Michael J.; Sameer, Zaid; Cote, Jean-Philippe; Cotroneo, Nicole; Lister, Troy; Rubio, Aileen; Brown, Eric D.
    ACS Infectious Diseases (2020), 6 (6), 1405-1412CODEN: AIDCBC; ISSN:2373-8227. (American Chemical Society)
    Therapeutics targeting Gram-neg. bacteria have the challenge of overcoming a formidable outer membrane (OM) barrier. We characterize the action of SPR741, a novel polymyxin B (PMB) analog shown to potentiate several large-scaffold antibiotics in Gram-neg. pathogens. Probing the surface topol. of Escherichia coli using at. force microscopy revealed substantial OM disorder at concns. of SPR741 that lead to antibiotic potentiation. Conversely, very little cytoplasmic membrane depolarization was obsd. at these same concns., indicating that SPR741 acts predominately on the OM. Truncating the LPS core with genetic perturbations uniquely sensitized E. coli to SPR741, suggesting that LPS core residues keep SPR741 at the OM where it can potentiate a co-drug, rather than permit its entry to the cytoplasmic membrane. Further, a promoter activity assay revealed that SPR741 challenge induced the expression of RcsAB, a stress sensor for OM perturbation. Together these results indicate that SPR741 interacts predominately with the OM, in contrast to the dual action of PMB and colistin at both the outer and cytoplasmic membranes.
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  50. 50
    Ofek, I.; Cohen, S.; Rahmani, R.; Kabha, K.; Tamarkin, D.; Herzig, Y.; Rubinstein, E. Antibacterial Synergism of Polymyxin B Nonapeptide and Hydrophobic Antibiotics in Experimental Gram-Negative Infections in Mice. Antimicrob. Agents Chemother. 1994, 38 (2), 374– 377,  DOI: 10.1128/AAC.38.2.374
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    50
    Antibacterial synergism of polymyxin B nonapeptide and hydrophobic antibiotics in experimental gram-negative infections in mice
    Ofek, Izhak; Cohen, Sofie; Rahmani, Rita; Kabha, Kisra; Tamarkin, Dove; Herzig, Yaacov; Rubinstein, Ethan
    Antimicrobial Agents and Chemotherapy (1994), 38 (2), 374-7CODEN: AMACCQ; ISSN:0066-4804.
    Polymyxin B nonapeptide, derived by cleavage of the fatty acyl diaminobutryic acid from polymyxin B, is considerably less toxic, less bactericidal activity, and retains its ability to render gram-neg. bacteria susceptible to several antibiotics by permeabilizing their outer membranes. The peptide rendered all 53 polymyxin-susceptible strains tested more susceptible to novobiocin, lowering the MIC of novobiocin 8-fold or more. The combination of polymyxin B nonapeptide with novobiocin or with erythromycin administered i.p. in multiple doses synergistically protected mice infected with gram-neg. bacteria. This combination may be clin. useful because of the apparent rarity of the acquisition of resistance.
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  51. 51
    Dixon, R. A.; Chopra, I. Polymyxin B and Polymyxin B Nonapeptide Alter Cytoplasmic Membrane Permeability in Escherichia Coli. J. Antimicrob. Chemother. 1986, 18 (5), 557– 563,  DOI: 10.1093/jac/18.5.557
    Google Scholar
    There is no corresponding record for this reference.
  52. 52
    Lomakina, G. Yu.; Ugarova, N. N. Kinetics of the Interaction of Colistin with Live Escherichia Coli Cells by the Bioluminescence Method. Mosc. Univ. Chem. Bull. 2022, 77 (1), 42– 47,  DOI: 10.3103/S0027131422010059
    Google Scholar
    There is no corresponding record for this reference.
  53. 53
    Ihssen, J.; Jovanovic, N.; Sirec, T.; Spitz, U. Real-Time Monitoring of Extracellular ATP in Bacterial Cultures Using Thermostable Luciferase. PLoS One 2021, 16 (1), e0244200  DOI: 10.1371/journal.pone.0244200
    Google Scholar
    There is no corresponding record for this reference.
  54. 54
    Branchini, B. R.; Magyar, R. A.; Murtiashaw, M. H.; Anderson, S. M.; Zimmer, M. Site-Directed Mutagenesis of Histidine 245 in Firefly Luciferase: A Proposed Model of the Active Site. Biochemistry 1998, 37 (44), 15311– 15319,  DOI: 10.1021/bi981150d
    Google Scholar
    54
    Site-directed mutagenesis of histidine 245 in firefly luciferase: A proposed model of the active site
    Branchini, Bruce R.; Magyar, Rachelle A.; Murtiashaw, Martha H.; Anderson, Shannon M.; Zimmer, Marc
    Biochemistry (1998), 37 (44), 15311-15319CODEN: BICHAW; ISSN:0006-2960. (American Chemical Society)
    Firefly luciferase (I) catalyzes the highly efficient emission of yellow-green light from the substrate, luciferin, by a sequence of reactions that require Mg-ATP and O2. The authors previously reported that 2-(4-benzoylphenyl)thiazole-4-carboxylic acid (BPTC), a firefly luciferin analog, was a potent photoinactivation reagent for I. A I tetrapeptide [244HHGF247] was identified, the degrdn. of which was directly correlated to the photooxidn. process. Here, the authors report the construction and purifn. of wild-type (WT) I and mutants H244F, H245F, H245A, and H245D. The results of photoinactivation and kinetic and bioluminescence studies with these proteins were consistent with His-245 being the primary functional target of BPTC-catalyzed enzyme inactivation. The possibility that His-245 is oxidized to Asp during the photooxidn. reaction was supported by the extremely low specific activity (∼300-fold lower than WT I) of the H245D mutant. Using the previously reported crystal structures of I without substrates and the functionally related phenylalanine-activating subunit of gramicidin synthetase 1 as a starting point, the authors performed mol. modeling studies and propose here a model for the I active site with substrates, luciferin and Mg-ATP, bound. This model was used to provide a structure-based interpretation of the role of peptide 244HHGF247 in firefly bioluminescence.
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  55. 55
    Wilhelm, M. J.; Sharifian Gh, M.; Dai, H.-L. Chemically Induced Changes to Membrane Permeability in Living Cells Probed with Nonlinear Light Scattering. Biochemistry 2015, 54 (29), 4427– 4430,  DOI: 10.1021/acs.biochem.5b00600
    Google Scholar
    There is no corresponding record for this reference.
  56. 56
    Wilhelm, M. J.; Sharifian Gh, M.; Dai, H.-L. Influence of Molecular Structure on Passive Membrane Transport: A Case Study by Second Harmonic Light Scattering. J. Chem. Phys. 2019, 150 (10), 104705,  DOI: 10.1063/1.5081720
    Google Scholar
    56
    Influence of molecular structure on passive membrane transport: A case study by second harmonic light scattering
    Wilhelm, Michael J.; Sharifian Gh., Mohammad; Dai, Hai-Lung
    Journal of Chemical Physics (2019), 150 (10), 104705/1-104705/8CODEN: JCPSA6; ISSN:0021-9606. (American Institute of Physics)
    We present an exptl. study, using the surface sensitive technique, second harmonic light scattering (SHS), to examine the influence of structure on the propensity of a mol. to passively diffuse across a phospholipid membrane. Specifically, we monitor the relative tendency of the structurally similar amphiphilic cationic dyes, malachite green (MG) and crystal violet (CV), to transport across membranes in living cells (E. coli) and biomimetic liposomes. Despite having nearly identical mol. structures, mol. wts., cationic charges, and functional groups, MG is of lower overall symmetry and consequently has a symmetry allowed permanent dipole moment, which CV does not. The two mols. showed drastically different interactions with phospholipid membranes. MG is obsd. to readily cross the hydrophobic interior of the bacterial cytoplasmic membrane. Conversely, CV does not. Furthermore, expts. conducted with biomimetic liposomes, constructed from the total lipid ext. of E. coli and contg. no proteins, show that while MG is able to diffuse across the liposome membrane, CV does not. These observations indicate that the SHS results measured with bacteria do not result from the functions of efflux pumps, but suggests that MG possesses an innate mol. property (which is absent in CV) that allows it to passively diffuse across the hydrophobic interior of a phospholipid membrane. (c) 2019 American Institute of Physics.
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Cite this: ACS Infect. Dis. 2024, 10, 5, 1602–1611
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  • Abstract

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    Figure 1

    Figure 1. (a) Schematic showing the workflow of DCCAA. (b) SDS-PAGE analysis of luciferase protein expression in E. coli at 2 or 26 h post IPTG induction. A molecular weight ladder with sizes in kilodaltons (kDa) is shown. The expected molecular weight of luciferase is 62 kDa.

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    Figure 2

    Figure 2. (a) Bioluminescence image of a 96-well plate containing E. coli treated with100 μM d-luciferin in the presence and absence of IPTG induction. (b) Chemical structures of the building blocks used for the assay. (c) Bioluminescence analysis of luciferase-expressing E. coli cells incubated with 100 μM CBT–OH and 100 μM d-cystine for 30 min, followed by centrifugation and separation of supernatant and pellet at which point luminescence measurements were initiated and continued for 60 min. Noncentrifuged cells (CBT–OH + d-cystine) were used as a control. Inset at the top displays a magnified portion of the graph, highlighting the PBS and supernatant traces. (d) Luciferase-expressing E. coli cells treated with 100 μM CBT–OH and 100 μM d-cystine in the presence and absence of IPTG induction, over 60 min. (e) Luciferase-expressing E. coli cells treated with 100 μM CBT–OH or 100 μM CBT–NH2 and 100 μM d-cystine over 60 min. Cells treated with PBS and CBT only (CBT–OH or CBT–NH2) were used as controls wherever appropriate. Data are represented as mean ± SD (n = 3 independent samples in a single experiment).

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    Figure 3

    Figure 3. (a) Bioluminescence analysis of luciferase-expressing E. coli cells treated with 50 μM d-cystine and varying concentrations of CBT–NH2 over 60 min at the 60 min time point. (b) Luciferase-expressing E. coli cells treated with 100 μM NH2–CBT and varying concentrations of d-cystine at the 60 min time point. (c) Luciferase-expressing E. coli cells (left) or a cell-free assay with luciferase enzyme (right) treated with 10 μM l-luciferin, 10 μM d-luciferin or 10 μM d-cystine with 100 μM CBT–NH2 (cell assay), over 60 min. (d) Luciferase-expressing E. coli cells treated with 100 μM d-cystine and 100 μM CBT–NH2 or 100 μM CBT–NH2 only, in the presence and absence of a 30 min pretreatment with 100 μM NEM, over 60 min. (e) Luciferase-expressing E. coli cells treated with 100 μM d-cystine or 100 μM d-cystine-ME and 100 μM CBT–NH2 at the 60 min time point. (f) Luciferase-expressing E. coli cells treated with 100 μM d-cystine and 100 μM CBT–NH2 in the absence and presence of a 30 min pretreatment with PMBN at different concentrations at the 60 min time point. Cells treated with only PBS and CBT–NH2 were used as controls, wherever appropriate. Data are represented as mean ± SD (n = 3 independent samples in a single experiment). Statistical analysis performed by two-tailed t-test with Welch’s correction, *p ≤ 0.01, **p ≤ 0.01, ***p ≤ 0.001, ns = not significant.

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    Figure 4

    Figure 4. (a) Structures of the antibiotic conjugates tagged with a d-cys moiety via a disulfide linkage. (b) Bioluminescence analysis of a cell-free assay with luciferase enzyme treated with 25 μM d-cystine or 25 μM antibiotic conjugates and 25 μM CBT–NH2 over 60 min and (c) at the 60 min time point upon treatment with 1 mM TCEP. (d) Bioluminescence analysis of luciferase-expressing E. coli cells treated with 50 μM d-cystine or 50 μM antibiotic conjugates with 100 μM CBT–NH2 over 60 min and (e) at the 60 min time point. The inset at the top of (d) provides a zoomed-out view of the graph, highlighting the d-cystine signal. Cells treated with PBS and CBT only (CBT–NH2) were used as controls wherever appropriate. Data are represented as mean ± SD (n = 3 independent samples in a single experiment). Statistical analysis performed by two-tailed t-test with Welch’s correction, **p ≤ 0.01, ***p ≤ 0.001, ns = not significant.

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    Figure 5

    Figure 5. (a) Simulated luminescence traces, based upon the model for molecular accumulation in bacteria, with gradually increasing rate constants kacc and kred. (b) Experimental time-resolved luminescence traces illustrating the accumulation of test molecules in E. coli. The best-fit lines from simulations are overlaid in black. The corresponding values for kacc and kred for each, are depicted in (c).

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  • References

    This article references 56 other publications.

    1. 1
      Murray, C. J. L.; Ikuta, K. S.; Sharara, F.; Swetschinski, L.; Robles Aguilar, G.; Gray, A.; Han, C.; Bisignano, C.; Rao, P.; Wool, E.; Johnson, S. C.; Browne, A. J.; Chipeta, M. G.; Fell, F.; Hackett, S.; Haines-Woodhouse, G.; Kashef Hamadani, B. H.; Kumaran, E. A. P.; McManigal, B.; Achalapong, S.; Agarwal, R.; Akech, S.; Albertson, S.; Amuasi, J.; Andrews, J.; Aravkin, A.; Ashley, E.; Babin, F.-X.; Bailey, F.; Baker, S.; Basnyat, B.; Bekker, A.; Bender, R.; Berkley, J. A.; Bethou, A.; Bielicki, J.; Boonkasidecha, S.; Bukosia, J.; Carvalheiro, C.; Castañeda-Orjuela, C.; Chansamouth, V.; Chaurasia, S.; Chiurchiù, S.; Chowdhury, F.; Clotaire Donatien, R.; Cook, A. J.; Cooper, B.; Cressey, T. R.; Criollo-Mora, E.; Cunningham, M.; Darboe, S.; Day, N. P. J.; De Luca, M.; Dokova, K.; Dramowski, A.; Dunachie, S. J.; Duong Bich, T.; Eckmanns, T.; Eibach, D.; Emami, A.; Feasey, N.; Fisher-Pearson, N.; Forrest, K.; Garcia, C.; Garrett, D.; Gastmeier, P.; Giref, A. Z.; Greer, R. C.; Gupta, V.; Haller, S.; Haselbeck, A.; Hay, S. I.; Holm, M.; Hopkins, S.; Hsia, Y.; Iregbu, K. C.; Jacobs, J.; Jarovsky, D.; Javanmardi, F.; Jenney, A. W. J.; Khorana, M.; Khusuwan, S.; Kissoon, N.; Kobeissi, E.; Kostyanev, T.; Krapp, F.; Krumkamp, R.; Kumar, A.; Kyu, H. H.; Lim, C.; Lim, K.; Limmathurotsakul, D.; Loftus, M. J.; Lunn, M.; Ma, J.; Manoharan, A.; Marks, F.; May, J.; Mayxay, M.; Mturi, N.; Munera-Huertas, T.; Musicha, P.; Musila, L. A.; Mussi-Pinhata, M. M.; Naidu, R. N.; Nakamura, T.; Nanavati, R.; Nangia, S.; Newton, P.; Ngoun, C.; Novotney, A.; Nwakanma, D.; Obiero, C. W.; Ochoa, T. J.; Olivas-Martinez, A.; Olliaro, P.; Ooko, E.; Ortiz-Brizuela, E.; Ounchanum, P.; Pak, G. D.; Paredes, J. L.; Peleg, A. Y.; Perrone, C.; Phe, T.; Phommasone, K.; Plakkal, N.; Ponce-de-Leon, A.; Raad, M.; Ramdin, T.; Rattanavong, S.; Riddell, A.; Roberts, T.; Robotham, J. V.; Roca, A.; Rosenthal, V. D.; Rudd, K. E.; Russell, N.; Sader, H. S.; Saengchan, W.; Schnall, J.; Scott, J. A. G.; Seekaew, S.; Sharland, M.; Shivamallappa, M.; Sifuentes-Osornio, J.; Simpson, A. J.; Steenkeste, N.; Stewardson, A. J.; Stoeva, T.; Tasak, N.; Thaiprakong, A.; Thwaites, G.; Tigoi, C.; Turner, C.; Turner, P.; van Doorn, H. R.; Velaphi, S.; Vongpradith, A.; Vongsouvath, M.; Vu, H.; Walsh, T.; Walson, J. L.; Waner, S.; Wangrangsimakul, T.; Wannapinij, P.; Wozniak, T.; Young Sharma, T. E. M. W.; Yu, K. C.; Zheng, P.; Sartorius, B.; Lopez, A. D.; Stergachis, A.; Moore, C.; Dolecek, C.; Naghavi, M. Global Burden of Bacterial Antimicrobial Resistance in 2019: A Systematic Analysis. Lancet 2022, 399 (10325), 629– 655,  DOI: 10.1016/S0140-6736(21)02724-0
      1
      Global burden of bacterial antimicrobial resistance in 2019: a systematic analysis
      Murray, Christopher J. L.; Ikuta, Kevin Shunji; Sharara, Fablina; Swetschinski, Lucien; Aguilar, Gisela Robles; Gray, Authia; Han, Chieh; Bisignano, Catherine; Rao, Puja; Wool, Eve; et al.
      Lancet (2022), 399 (10325), 629-655CODEN: LANCAO; ISSN:0140-6736. (Elsevier Ltd.)
      Antimicrobial resistance (AMR) poses a major threat to human health around the world. Previous publications have estd. the effect of AMR on incidence, deaths, hospital length of stay, and health-care costs for specific pathogen-drug combinations in select locations. To our knowledge, this study presents the most comprehensive ests. of AMR burden to date. We estd. deaths and disability-adjusted life-years (DALYs) attributable to and assocd. with bacterial AMR for 23 pathogens and 88 pathogen-drug combinations in 204 countries and territories in 2019. We obtained data from systematic literature reviews, hospital systems, surveillance systems, and other sources, covering 471 million individual records or isolates and 7585 study-location-years. We used predictive statistical modeling to produce ests. of AMR burden for all locations, including for locations with no data. Our approach can be divided into five broad components: no. of deaths where infection played a role, proportion of infectious deaths attributable to a given infectious syndrome, proportion of infectious syndrome deaths attributable to a given pathogen, the percentage of a given pathogen resistant to an antibiotic of interest, and the excess risk of death or duration of an infection assocd. with this resistance. Using these components, we estd. disease burden based on two counterfactuals: deaths attributable to AMR (based on an alternative scenario in which all drug-resistant infections were replaced by drug-susceptible infections), and deaths assocd. with AMR (based on an alternative scenario in which all drug-resistant infections were replaced by no infection). We generated 95% uncertainty intervals (UIs) for final ests. as the 25th and 975th ordered values across 1000 posterior draws, and models were cross-validated for out-of-sample predictive validity. We present final ests. aggregated to the global and regional level. On the basis of our predictive statistical models, there were an estd. 4.95 million (3.62-6.57) deaths assocd. with bacterial AMR in 2019, including 1.27 million (95% UI 0.911-1.71) deaths attributable to bacterial AMR. At the regional level, we estd. the all-age death rate attributable to resistance to be highest in western sub-Saharan Africa, at 27.3 deaths per 100 000 (20.9-35.3), and lowest in Australasia, at 6.5 deaths (4.3-9.4) per 100 000. Lower respiratory infections accounted for more than 1.5 million deaths assocd. with resistance in 2019, making it the most burdensome infectious syndrome. The six leading pathogens for deaths assocd. with resistance (Escherichia coli, followed by Staphylococcus aureus, Klebsiella pneumoniae, Streptococcus pneumoniae, Acinetobacter baumannii, and Pseudomonas aeruginosa) were responsible for 929 000 (660 000-1 270 000) deaths attributable to AMR and 3.57 million (2.62-4.78) deaths assocd. with AMR in 2019. One pathogen-drug combination, meticillin-resistant S aureus, caused more than 100 000 deaths attributable to AMR in 2019, while six more each caused 50 000-100 000 deaths: multidrug-resistant excluding extensively drug-resistant tuberculosis, third-generation cephalosporin-resistant E coli, carbapenem-resistant A baumannii, fluoroquinolone-resistantE coli, carbapenem-resistant K pneumoniae, and third-generation cephalosporin-resistant K pneumoniae. To our knowledge, this study provides the first comprehensive assessment of the global burden of AMR, as well as an evaluation of the availability of data. AMR is a leading cause of death around the world, with the highest burdens in low-resource settings. Understanding the burden of AMR and the leading pathogen-drug combinations contributing to it is crucial to making informed and location-specific policy decisions, particularly about infection prevention and control programs, access to essential antibiotics, and research and development of new vaccines and antibiotics. There are serious data gaps in many low-income settings, emphasizing the need to expand microbiol. lab. capacity and data collection systems to improve our understanding of this important human health threat.
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    2. 2
      Hutchings, M. I.; Truman, A. W.; Wilkinson, B. Antibiotics: Past, Present and Future. Curr. Opin. Microbiol. 2019, 51, 72– 80,  DOI: 10.1016/j.mib.2019.10.008
      2
      Antibiotics: past, present and future
      Hutchings, Matt; Truman, Andrew; Wilkinson, Barrie
      Current Opinion in Microbiology (2019), 51 (), 72-80CODEN: COMIF7; ISSN:1369-5274. (Elsevier Ltd.)
      A review. The first antibiotic, salvarsan, was deployed in 1910. In just over 100 years antibiotics have drastically changed modern medicine and extended the av. human lifespan by 23 years. The discovery of penicillin in 1928 started the golden age of natural product antibiotic discovery that peaked in the mid-1950s. Since then, a gradual decline in antibiotic discovery and development and the evolution of drug resistance in many human pathogens has led to the current antimicrobial resistance crisis. Here we give an overview of the history of antibiotic discovery, the major classes of antibiotics and where they come from. We argue that the future of antibiotic discovery looks bright as new technologies such as genome mining and editing are deployed to discover new natural products with diverse bioactivities. We also report on the current state of antibiotic development, with 45 drugs currently going through the clin. trials pipeline, including several new classes with novel modes of action that are in phase 3 clin. trials. Overall, there are promising signs for antibiotic discovery, but changes in financial models are required to translate scientific advances into clin. approved antibiotics.
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    3. 3
      Zgurskaya, H. I.; Rybenkov, V. V. Permeability Barriers of Gram-Negative Pathogens. Ann. N.Y. Acad. Sci. 2020, 1459 (1), 5– 18,  DOI: 10.1111/nyas.14134
      3
      Permeability barriers of Gram-negative pathogens
      Zgurskaya Helen I; Rybenkov Valentin V
      Annals of the New York Academy of Sciences (2020), 1459 (1), 5-18 ISSN:.
      Most clinical antibiotics do not have efficacy against Gram-negative pathogens, mainly because these cells are protected by the permeability barrier comprising the two membranes with active efflux. The emergence of multidrug-resistant Gram-negative strains threatens the utility even of last resort therapeutic treatments. Significant efforts at different levels of resolution are currently focused on finding a solution to this nonpermeation problem and developing new approaches to the optimization of drug activities against multidrug-resistant pathogens. The exceptional efficiency of the Gram-negative permeability barrier is the result of a complex interplay between the two opposing fluxes of drugs across the two membranes. In this review, we describe the current state of understanding of the problem and the recent advances in theoretical and empirical approaches to characterization of drug permeation and active efflux in Gram-negative bacteria.
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    4. 4
      Kohanski, M. A.; Dwyer, D. J.; Collins, J. J. How Antibiotics Kill Bacteria: From Targets to Networks. Nat. Rev. Microbiol. 2010, 8 (6), 423– 435,  DOI: 10.1038/nrmicro2333
      4
      How antibiotics kill bacteria: from targets to networks
      Kohanski, Michael A.; Dwyer, Daniel J.; Collins, James J.
      Nature Reviews Microbiology (2010), 8 (6), 423-435CODEN: NRMACK; ISSN:1740-1526. (Nature Publishing Group)
      A review. Antibiotic drug-target interactions, and their resp. direct effects, are generally well characterized. By contrast, the bacterial responses to antibiotic drug treatments that contribute to cell death are not as well understood and have proven to be complex as they involve many genetic and biochem. pathways. In this review, we discuss the multilayered effects of drug-target interactions, including the essential cellular processes that are inhibited by bactericidal antibiotics and the assocd. cellular response mechanisms that contribute to killing. We also discuss new insights into these mechanisms that have been revealed through the study of biol. networks, and describe how these insights, together with related developments in synthetic biol., could be exploited to create new antibacterial therapies.
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    5. 5
      Kojima, S.; Nikaido, H. Permeation Rates of Penicillins Indicate That Escherichia Coli Porins Function Principally as Nonspecific Channels. Proc. Natl. Acad. Sci. U.S.A. 2013, 110 (28), E2629– E2634,  DOI: 10.1073/pnas.1310333110
      5
      Permeation rates of penicillins indicate that Escherichia coli porins function principally as nonspecific channels
      Kojima, Seiji; Nikaido, Hiroshi
      Proceedings of the National Academy of Sciences of the United States of America (2013), 110 (28), E2629-E2634, SE2629/1-SE2629/3CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)
      Small, hydrophilic compds. such as β-lactams diffuse across the outer membrane of Gram-neg. bacteria through porin channels, which were originally thought to be nonspecific channels devoid of any specificity. However, since the discovery of an ampicillin-binding site within the OmpF channel in 2002, much attention has been focused on the potential specificity of the channel, where the binding site was assumed either to facilitate or to retard the penetration of β-lactams. Since the earlier studies on porin permeability were done without the knowledge of the contribution of multidrug efflux pumps in the overall flux process across the cell envelope, in this study we have carefully studied both the porin permeability and active efflux of ampicillin and benzylpenicillin. It was found that the influx occurs apparently by a spontaneous passive diffusion without any indication of specific binding within the concn. range relevant to the antibiotic action of these drugs, and that the higher permeability for ampicillin is totally as expected from the gross property of this drug as a zwitterionic compd. The active efflux by AcrAB was more effective for benzylpenicillin due to the stronger affinity and high degree of pos. cooperativity. The data now give a complete quant. picture of the influx, efflux, and periplasmic degrdn. (catalyzed by AmpC β-lactamase) of these two compds., and correlate closely with the susceptibility of Escherichia coli strains used here.
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    6. 6
      June, C. M.; Vaughan, R. M.; Ulberg, L. S.; Bonomo, R. A.; Witucki, L. A.; Leonard, D. A. A fluorescent carbapenem for structure function studies of penicillin-binding proteins, β-lactamases, and β-lactam sensors. Anal. Biochem. 2014, 463, 70– 74,  DOI: 10.1016/j.ab.2014.07.012
      6
      A fluorescent carbapenem for structure function studies of penicillin-binding proteins, β-lactamases, and β-lactam sensors
      June, Cynthia M.; Vaughan, Robert M.; Ulberg, Lucas S.; Bonomo, Robert A.; Witucki, Laurie A.; Leonard, David A.
      Analytical Biochemistry (2014), 463 (), 70-74CODEN: ANBCA2; ISSN:0003-2697. (Elsevier B.V.)
      By reacting fluorescein isothiocyanate with meropenem, the authors prepd. a carbapenem-based fluorescent β-lactam. Fluorescein-meropenem binds both penicillin-binding proteins and β-lactam sensors and undergoes a typical acylation reaction in the active site of these proteins. The probe binds the class D carbapenemase OXA-24/40 with close to the same affinity as meropenem and undergoes a complete catalytic hydrolysis reaction. The visible light excitation and strong emission of fluorescein render this mol. a useful structure-function probe through its application in sodium dodecyl sulfate-PAGE assays as well as soln.-based kinetic anisotropy assays. Its classification as a carbapenem β-lactam and the position of its fluorescent modification render it a useful complement to other fluorescent β-lactams, most notably Bocillin FL. The authors show the utility of fluorescein-meropenem by using it to detect mutants of OXA-24/40 that arrest at the acyl-intermediate state with carbapenem substrates but maintain catalytic competency with penicillin substrates.
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    7. 7
      Davis, T. D.; Gerry, C. J.; Tan, D. S. General Platform for Systematic Quantitative Evaluation of Small-Molecule Permeability in Bacteria. ACS Chem. Biol. 2014, 9 (11), 2535– 2544,  DOI: 10.1021/cb5003015
      7
      General Platform for Systematic Quantitative Evaluation of Small-Molecule Permeability in Bacteria
      Davis, Tony D.; Gerry, Christopher J.; Tan, Derek S.
      ACS Chemical Biology (2014), 9 (11), 2535-2544CODEN: ACBCCT; ISSN:1554-8929. (American Chemical Society)
      The chem. features that impact small-mol. permeability across bacterial membranes are poorly understood, and the resulting lack of tools to predict permeability presents a major obstacle to the discovery and development of novel antibiotics. Antibacterials are known to have vastly different structural and physicochem. properties compared to nonantiinfective drugs, as illustrated herein by principal component anal. (PCA). To understand how these properties influence bacterial permeability, the authors have developed a systematic approach to evaluate the penetration of diverse compds. into bacteria with distinct cellular envelopes. Intracellular compd. accumulation is quantitated using LC-MS/MS, then PCA and Pearson pairwise correlations were used to identify structural and physicochem. parameters that correlate with accumulation. An initial study using 10 sulfonyladenosines in Escherichia coli, Bacillus subtilis, and Mycobacterium smegmatis has identified nonobvious correlations between chem. structure and permeability that differ among the various bacteria. Effects of cotreatment with efflux pump inhibitors were also studied. This sets the stage for use of this platform in larger prospective analyses of diverse chemotypes to identify global relations between chem. structure and bacterial permeability that would enable the development of predictive tools to accelerate antibiotic drug discovery.
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    8. 8
      Ghai, I.; Winterhalter, M.; Wagner, R. Probing transport of charged β-lactamase inhibitors through OmpC, a membrane channel from E. coli. Biochem. Biophys. Res. Commun. 2017, 484 (1), 51– 55,  DOI: 10.1016/j.bbrc.2017.01.076
      There is no corresponding record for this reference.
    9. 9
      Kaščáková, S.; Maigre, L.; Chevalier, J.; Réfrégiers, M.; Pagès, J. M. Antibiotic Transport in Resistant Bacteria: Synchrotron UV Fluorescence Microscopy to Determine Antibiotic Accumulation with Single Cell Resolution. PLoS One 2012, 7 (6), e38624  DOI: 10.1371/journal.pone.0038624
      9
      Antibiotic transport in resistant bacteria: synchrotron UV fluorescence microscopy to determine antibiotic accumulation with single cell resolution
      Kascakova, Slavka; Maigre, Laure; Chevalier, Jacqueline; Refregiers, Matthieu; Pages, Jean-Marie
      PLoS One (2012), 7 (6), e38624CODEN: POLNCL; ISSN:1932-6203. (Public Library of Science)
      A mol. definition of the mechanism conferring bacterial multidrug resistance is clin. crucial and today methods for quant. detn. of the uptake of antimicrobial agents with single cell resoln. are missing. Using the naturally occurring fluorescence of antibacterial agents after deep UV (DUV) excitation, we developed a method to non-invasively monitor the quinolones uptake in single bacteria. Our approach is based on a DUV fluorescence microscope coupled to a synchrotron beamline providing tuneable excitation from 200 to 600 nm. A full spectrum was acquired at each pixel of the image, to study the DUV excited fluorescence emitted from quinolones within single bacteria. Measuring spectra allowed us to sep. the antibiotic fluorescence from the autofluorescence contribution. By performing spectroscopic anal., the quantification of the antibiotic signal was possible. To our knowledge, this is the first time that the intracellular accumulation of a clin. antibiotic could be detd. and discussed in relation with the level of drug susceptibility for a multiresistant strain. This method is esp. important to follow the behavior of quinolone mols. at individual cell level, to quantify the intracellular concn. of the antibiotic and develop new strategies to combat the dissemination of MDR-bacteria. In addn., this original approach also indicates the heterogeneity of bacterial population when the same strain is under environmental stress like antibiotic attack.
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    10. 10
      Zhou, Y.; Joubran, C.; Miller-Vedam, L.; Isabella, V.; Nayar, A.; Tentarelli, S.; Miller, A. Thinking Outside the “Bug”: A Unique Assay To Measure Intracellular Drug Penetration in Gram-Negative Bacteria. Anal. Chem. 2015, 87 (7), 3579– 3584,  DOI: 10.1021/ac504880r
      10
      Thinking Outside the "Bug": A Unique Assay To Measure Intracellular Drug Penetration in Gram-Negative Bacteria
      Zhou, Ying; Joubran, Camil; Miller-Vedam, Lakshmi; Isabella, Vincent; Nayar, Asha; Tentarelli, Sharon; Miller, Alita
      Analytical Chemistry (Washington, DC, United States) (2015), 87 (7), 3579-3584CODEN: ANCHAM; ISSN:0003-2700. (American Chemical Society)
      Significant challenges are present in antibiotic drug discovery and development. One of these is the no. of efficient approaches Gram-neg. bacteria have developed to avoid intracellular accumulation of drugs and other cell-toxic species. In order to better understand these processes and correlate in vitro enzyme inhibition to whole cell activity, a better assay to evaluate a key factor, intracellular accumulation of the drug, is urgently needed. Here, we describe a unique liq. chromatog. (LC)-mass spectrometry (MS) approach to measure the amt. of cellular uptake of antibiotics by Gram-neg. bacteria. This method, which measures the change of extracellular drug concn., was evaluated by comparing the relative uptake of linezolid by Escherichia coli wild-type vs. an efflux pump deficient strain. A higher dosage of the drug showed a higher accumulation in these bacteria in a dosing range of 5-50 ng/mL. The Escherichia coli efflux pump deficient strain had a higher accumulation of the drug than the wild-type strain as predicted. The approach was further validated by detg. the relative meropenem uptake by Pseudomonas aeruginosa wild-type vs. a mutant strain lacking multiple porins. These studies show great promise of being applied within antibiotic drug discovery, as a universal tool to aid in the search for compds. that can easily penetrate bacterial cells.
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    11. 11
      Cama, J.; Bajaj, H.; Pagliara, S.; Maier, T.; Braun, Y.; Winterhalter, M.; Keyser, U. F. Quantification of Fluoroquinolone Uptake through the Outer Membrane Channel OmpF of Escherichia Coli. J. Am. Chem. Soc. 2015, 137 (43), 13836– 13843,  DOI: 10.1021/jacs.5b08960
      11
      Quantification of fluoroquinolone uptake through the outer membrane channel OmpF of Escherichia coli
      Cama, Jehangir; Bajaj, Harsha; Pagliara, Stefano; Maier, Theresa; Braun, Yvonne; Winterhalter, Mathias; Keyser, Ulrich F.
      Journal of the American Chemical Society (2015), 137 (43), 13836-13843CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
      Decreased drug accumulation is a common cause of antibiotic resistance in microorganisms. However, there are few reliable general techniques capable of quantifying drug uptake through bacterial membranes. The authors present a semiquant. optofluidic assay for studying the uptake of autofluorescent drug mols. in single liposomes. They studied the effect of the Escherichia coli outer membrane channel OmpF on the accumulation of the fluoroquinolone antibiotic, norfloxacin, in proteoliposomes. Measurements were performed at pH 5 and pH 7, corresponding to two different charge states of norfloxacin that bacteria are likely to encounter in the human gastrointestinal tract. At both pH values, the porins significantly enhance drug permeation across the proteoliposome membranes. At pH 5, where norfloxacin permeability across pure phospholipid membranes is low, the porins increase drug permeability by 50-fold on av. The authors est. a flux of about 10 norfloxacin mols. per s per OmpF trimer in the presence of a 1 mM concn. gradient of norfloxacin. They also performed single channel electrophysiol. measurements and found that the application of transmembrane voltages causes an elec. field driven uptake in addn. to concn. driven diffusion. The authors propose a phys. mechanism for the pH mediated change in bacterial susceptibility to fluoroquinolone antibiotics.
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    12. 12
      Cinquin, B.; Maigre, L.; Pinet, E.; Chevalier, J.; Stavenger, R. A.; Mills, S.; Réfrégiers, M.; Pagès, J. M. Microspectrometric Insights on the Uptake of Antibiotics at the Single Bacterial Cell Level. Sci. Rep. 2015, 5 (1), 17968,  DOI: 10.1038/srep17968
      There is no corresponding record for this reference.
    13. 13
      Geddes, E. J.; Li, Z.; Hergenrother, P. J. An LC-MS/MS Assay and Complementary Web-Based Tool to Quantify and Predict Compound Accumulation in E. Coli. Nat. Protoc. 2021, 16 (10), 4833– 4854,  DOI: 10.1038/s41596-021-00598-y
      13
      An LC-MS/MS assay and complementary web-based tool to quantify and predict compound accumulation in E. coli
      Geddes, Emily J.; Li, Zhong; Hergenrother, Paul J.
      Nature Protocols (2021), 16 (10), 4833-4854CODEN: NPARDW; ISSN:1750-2799. (Nature Portfolio)
      A review. Novel classes of broad-spectrum antibiotics have been extremely difficult to discover, largely due to the impermeability of the Gram-neg. membranes coupled with a poor understanding of the physicochem. properties a compd. should possess to promote its accumulation inside the cell. To address this challenge, numerous methodologies for assessing intracellular compd. accumulation in Gram-neg. bacteria have been established, including classic radiometric and fluorescence-based methods. The recent development of accumulation assays that utilize liq. chromatog.-tandem mass spectrometry (LC-MS/MS) have circumvented the requirement for labeled compds., enabling assessment of a substantially broader range of small mols. Our unbiased study of accumulation trends in Escherichia coli using an LC-MS/MS-based assay led to the development of the eNTRy rules, which stipulate that a compd. is most likely to accumulate in E. coli if it has an ionizable Nitrogen, has low Three-dimensionality and is relatively Rigid. To aid in the implementation of the eNTRy rules, we developed a complementary web tool, eNTRyway, which calcs. relevant properties and predicts compd. accumulation. Here we provide a comprehensive protocol for anal. and prediction of intracellular accumulation of small mols. in E. coli using an LC-MS/MS-based assay (which takes ∼2 d) and eNTRyway, a workflow that is readily adoptable by any microbiol., biochem. or chem. biol. lab.
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    14. 14
      Iyer, R.; Ye, Z.; Ferrari, A.; Duncan, L.; Tanudra, M. A.; Tsao, H.; Wang, T.; Gao, H.; Brummel, C. L.; Erwin, A. L. Evaluating LC-MS/MS To Measure Accumulation of Compounds within Bacteria. ACS Infect. Dis. 2018, 4 (9), 1336– 1345,  DOI: 10.1021/acsinfecdis.8b00083
      14
      Evaluating LC-MS/MS To Measure Accumulation of Compounds within Bacteria
      Iyer, Ramkumar; Ye, Zhengqi; Ferrari, Annette; Duncan, Leonard; Tanudra, M. Angela; Tsao, Hong; Wang, Tiansheng; Gao, Hong; Brummel, Christopher L.; Erwin, Alice L.
      ACS Infectious Diseases (2018), 4 (9), 1336-1345CODEN: AIDCBC; ISSN:2373-8227. (American Chemical Society)
      A general method for detg. bacterial uptake of compds. independent of antibacterial activity would be a valuable tool in antibacterial drug discovery. LC-MS/MS assays have been described, but it has not been shown whether the data can be used directly to inform medicinal chem. We describe the evaluation of an LC-MS/MS assay measuring assocn. of compds. with bacteria, using a set of over a hundred compds. (inhibitors of NAD-dependent DNA ligase, LigA) for which in vitro potency and antibacterial activity had been detd. All compds. were active against an efflux-deficient strain of Escherichia coli with reduced LigA activity (E. coli ligA251 ΔtolC). Testing a single compd. concn. and incubation time, we found that, for equipotent compds., LC-MS/MS values were not predictive of antibacterial activity. This indicates that measured bacteria-assocd. compd. was not necessarily exposed to the target enzyme. Our data suggest that, while exclusion from bacteria is a major reason for poor antibacterial activity of potent compds., the distribution of compd. within the bacterial cell may also be a problem. The relative importance of these factors is likely to vary from one chem. series to another. Our observations provide directions for further study of this difficult issue.
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    15. 15
      Jones, S. W.; Christison, R.; Bundell, K.; Voyce, C. J.; Brockbank, S. M. V.; Newham, P.; Lindsay, M. A. Characterisation of Cell-Penetrating Peptide-Mediated Peptide Delivery. Br. J. Pharmacol. 2005, 145 (8), 1093– 1102,  DOI: 10.1038/sj.bjp.0706279
      15
      Characterization of cell-penetrating peptide-mediated peptide delivery
      Jones, Simon W.; Christison, Richard; Bundell, Ken; Voyce, Catherine J.; Brockbank, Sarah M. V.; Newham, Peter; Lindsay, Mark A.
      British Journal of Pharmacology (2005), 145 (8), 1093-1102CODEN: BJPCBM; ISSN:0007-1188. (Nature Publishing Group)
      Cell-penetrating peptides such as antennapedia, TAT, transportan and polyarginine have been extensively employed for in vitro and in vivo delivery of biol. active peptides. However, little is known of the relative efficacy, toxicity and uptake mechanism of individual protein transduction domain-peptide conjugates, factors that will be crit. in detg. the most effective sequence. In the present study, we show by FACS anal. that unconjugated antennapedia, TAT, transportan and polyarginine demonstrate similar kinetic uptake profiles, being maximal at 1-3 h and independent of cell type (HeLa, A549 and CHO cell lines). A comparison of the magnitude of uptake of cell-penetrating peptide conjugates demonstrated that polyarginine = transportan>antennapedia > TAT. However, examn. of cellular toxicity showed that antennapedia < TAT < transportan < ∩ polyarginine, with antennapedia-peptide conjugates having no significant toxicity even at 100 μM. Confocal studies of the mechanism of antennapedia- and TAT-peptide uptake showed that the time course of uptake and their cellular distribution did not correlate with transferrin, a marker of clathrin-mediated endocytosis. In contrast, the peptides co-localized with a marker of lipid rafts domains, cholera toxin, which was attenuated following the disruption of these domains using methyl-β-cyclodextrin. Overall, comparison of the uptake and toxicity suggests that antennapedia provides the optimal cell-penetrating peptide for peptide delivery in vitro and that both antennapedia- and TAT-mediated peptide delivery occurs predominantly via lipid raft-dependent but clathrin-independent endocytosis.
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    16. 16
      Fischer, R.; Waizenegger, T.; Köhler, K.; Brock, R. A Quantitative Validation of Fluorophore-Labelled Cell-Permeable Peptide Conjugates: Fluorophore and Cargo Dependence of Import. Biochim. Biophys. Acta, Biomembr. 2002, 1564 (2), 365– 374,  DOI: 10.1016/S0005-2736(02)00471-6
      16
      A quantitative validation of fluorophore-labelled cell-permeable peptide conjugates: fluorophore and cargo dependence of import
      Fischer, Rainer; Waizenegger, Thomas; Kohler, Karsten; Brock, Roland
      Biochimica et Biophysica Acta, Biomembranes (2002), 1564 (2), 365-374CODEN: BBBMBS; ISSN:0005-2736. (Elsevier B.V.)
      Cell-permeable peptides were evaluated for a quant. controlled import of small mols. The dependence of the import efficiency on the fluorophore, on the position of the fluorophore as well as on the nature of the cargo were addressed. Cellular uptake was quantitated by flow cytometry and fluorescence correlation microscopy (FCM). Fluorophores with different spectral characteristics, covering the whole visible spectral range, were selected in order to enable the simultaneous detection of several cell-permeable peptide constructs. The transcytosis sequences were based either on the sequence of the Antennapedia homeodomain protein (AntpHD)-derived penetratin peptide or the Kaposi fibroblast growth factor (FGF)-derived membrane translocating sequence (MTS)-peptide. In general, the AntpHD-derived peptides had a three- to fourfold higher import efficiency than the MTS-derived peptides. In spite of the very different physicochem. characteristics of the fluorophores, the import efficiencies for analogs labeled at different positions within the sequence of the import peptides showed a strong pos. correlation. However, even for peptide cargos of very similar size, pronounced differences in import efficiency were obsd. The use of cell-permeable peptide/cargo constructs for intracellular analyses of structure-function relationships therefore requires the detn. of the intracellular concns. for each construct individually.
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    17. 17
      Illien, F.; Rodriguez, N.; Amoura, M.; Joliot, A.; Pallerla, M.; Cribier, S.; Burlina, F.; Sagan, S. Quantitative Fluorescence Spectroscopy and Flow Cytometry Analyses of Cell-Penetrating Peptides Internalization Pathways: Optimization, Pitfalls, Comparison with Mass Spectrometry Quantification. Sci. Rep. 2016, 6 (1), 36938,  DOI: 10.1038/srep36938
      17
      Quantitative fluorescence spectroscopy and flow cytometry analyses of cell-penetrating peptides internalization pathways: optimization, pitfalls, comparison with mass spectrometry quantification
      Illien, Francoise; Rodriguez, Nicolas; Amoura, Mehdi; Joliot, Alain; Pallerla, Manjula; Cribier, Sophie; Burlina, Fabienne; Sagan, Sandrine
      Scientific Reports (2016), 6 (), 36938CODEN: SRCEC3; ISSN:2045-2322. (Nature Publishing Group)
      The mechanism of cell-penetrating peptides entry into cells is unclear, preventing the development of more efficient vectors for biotechnol. or therapeutic purposes. Here, we developed a protocol relying on fluorometry to distinguish endocytosis from direct membrane translocation, using Penetratin, TAT and R9. The quantities of internalized CPPs measured by fluorometry in cell lysates converge with those obtained by our previously reported mass spectrometry quantification method. By contrast, flow cytometry quantification faces several limitations due to fluorescence quenching processes that depend on the cell line and occur at peptide/cell ratio >6.108 for CF-Penetratin. The anal. of cellular internalization of a doubly labeled fluorescent and biotinylated Penetratin analog by the two independent techniques, fluorometry and mass spectrometry, gave consistent results at the quant. and qual. levels. Both techniques revealed the use of two alternative translocation and endocytosis pathways, whose relative efficacy depends on cell-surface sugars and peptide concn. We confirmed that Penetratin translocates at low concn. and uses endocytosis at high μM concns. We further demonstrate that the hydrophobic/hydrophilic nature of the N-terminal extremity impacts on the internalization efficiency of CPPs. We expect these results and the assocd. protocols to help unraveling the translocation pathway to the cytosol of cells.
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    18. 18
      Liu, Z.; Lepori, I.; Chordia, M. D.; Dalesandro, B. E.; Guo, T.; Dong, J.; Siegrist, M. S.; Pires, M. M. A Metabolic-Tag-Based Method for Assessing the Permeation of Small Molecules Across the Mycomembrane in Live Mycobacteria**. Angew. Chem., Int. Ed. 2023, 62 (20), e202217777  DOI: 10.1002/anie.202217777
      There is no corresponding record for this reference.
    19. 19
      Kelly, J. J.; Dalesandro, B. E.; Liu, Z.; Chordia, M. D.; Ongwae, G. M.; Pires, M. M. Measurement of Accumulation of Antibiotics to Staphylococcus Aureus in Phagosomes of Live Macrophages. Angew. Chem. 2024, 136 (3), e202313870  DOI: 10.1002/ange.202313870
      There is no corresponding record for this reference.
    20. 20
      Ongwae, G. M.; Lepori, I.; Chordia, M. D.; Dalesandro, B. E.; Apostolos, A. J.; Siegrist, M. S.; Pires, M. M. Measurement of Small Molecule Accumulation into Diderm Bacteria. ACS Infect. Dis. 2023, 9 (1), 97– 110,  DOI: 10.1021/acsinfecdis.2c00435
      20
      Measurement of Small Molecule Accumulation into Diderm Bacteria
      Ongwae, George M.; Lepori, Irene; Chordia, Mahendra D.; Dalesandro, Brianna E.; Apostolos, Alexis J.; Siegrist, M. Sloan; Pires, Marcos M.
      ACS Infectious Diseases (2023), 9 (1), 97-110CODEN: AIDCBC; ISSN:2373-8227. (American Chemical Society)
      Some of the most dangerous bacterial pathogens (Gram-neg. and mycobacterial) deploy a formidable secondary membrane barrier to reduce the influx of exogenous mols. For Gram-neg. bacteria, this second exterior membrane is known as the outer membrane (OM), while for the Gram-indeterminate Mycobacteria, it is known as the "myco" membrane. Although different in compn., both the OM and mycomembrane are key structures that restrict the passive permeation of small mols. into bacterial cells. Although it is well-appreciated that such structures are principal determinants of small mol. permeation, it has proven to be challenging to assess this feature in a robust and quant. way or in complex, infection-relevant settings. Herein, we describe the development of the bacterial chloro-alkane penetration assay (BaCAPA), which employs the use of a genetically encoded protein called HaloTag, to measure the uptake and accumulation of mols. into model Gram-neg. and mycobacterial species, Escherichia coli and Mycobacterium smegmatis, resp., and into the human pathogen Mycobacterium tuberculosis. The HaloTag protein can be directed to either the cytoplasm or the periplasm of bacteria. This offers the possibility of compartmental anal. of permeation across individual cell membranes. Significantly, we also showed that BaCAPA can be used to analyze the permeation of mols. into host cell-internalized E. coli and M. tuberculosis, a crit. capability for analyzing intracellular pathogens. Together, our results show that BaCAPA affords facile measurement of permeability across four barriers: the host plasma and phagosomal membranes and the diderm bacterial cell envelope.
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    21. 21
      Karatas, H.; Maric, T.; D’Alessandro, P. L.; Yevtodiyenko, A.; Vorherr, T.; Hollingworth, G. J.; Goun, E. A. Real-Time Imaging and Quantification of Peptide Uptake in Vitro and in Vivo. ACS Chem. Biol. 2019, 14 (10), 2197– 2205,  DOI: 10.1021/acschembio.9b00439
      There is no corresponding record for this reference.
    22. 22
      Godinat, A.; Bazhin, A. A.; Goun, E. A. Bioorthogonal Chemistry in Bioluminescence Imaging. Drug Discovery Today 2018, 23 (9), 1584– 1590,  DOI: 10.1016/j.drudis.2018.05.022
      There is no corresponding record for this reference.
    23. 23
      Carmel-Harel, O.; Storz, G. Roles of the Glutathione- and Thioredoxin-Dependent Reduction Systems in the Escherichia Coli and Saccharomyces Cerevisiae Responses to Oxidative Stress. Annu. Rev. Microbiol. 2000, 54 (1), 439– 461,  DOI: 10.1146/annurev.micro.54.1.439
      There is no corresponding record for this reference.
    24. 24
      Patterson, D. M.; Nazarova, L. A.; Prescher, J. A. Finding the Right (Bioorthogonal) Chemistry. ACS Chem. Biol. 2014, 9 (3), 592– 605,  DOI: 10.1021/cb400828a
      24
      Finding the Right (Bioorthogonal) Chemistry
      Patterson, David M.; Nazarova, Lidia A.; Prescher, Jennifer A.
      ACS Chemical Biology (2014), 9 (3), 592-605CODEN: ACBCCT; ISSN:1554-8929. (American Chemical Society)
      A review. Bioorthogonal chemistries can be used to tag diverse classes of biomols. in cells and other complex environments. With over 20 unique transformations now available, though, selecting an appropriate reaction for a given expt. is challenging. The authors compare and contrast the most common classes of bioorthogonal chemistries and provide a framework for matching the reactions with downstream applications. The authors also discuss ongoing efforts to identify novel biocompatible reactions and methods to control their reactivity. The continued expansion of the bioorthogonal toolkit will provide new insights into biomol. networks and functions and thus refine the authors' understanding of living systems.
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    25. 25
      Liang, G.; Ren, H.; Rao, J. A Biocompatible Condensation Reaction for Controlled Assembly of Nanostructures in Living Cells. Nat. Chem. 2010, 2 (1), 54– 60,  DOI: 10.1038/nchem.480
      25
      A biocompatible condensation reaction for controlled assembly of nanostructures in living cells
      Liang, Gaolin; Ren, Hongjun; Rao, Jianghong
      Nature Chemistry (2010), 2 (1), 54-60CODEN: NCAHBB; ISSN:1755-4330. (Nature Publishing Group)
      Through controlled synthesis and mol. assembly, biol. systems are able to organize mols. into supramol. structures that carry out sophisticated processes. Although chemists have reported a few examples of supramol. assembly in, the controlled covalent synthesis of large mols. and structures in vivo has remained challenging. Here the authors report a condensation reaction between 1,2-aminothiol and 2-cyanobenzothiazole that occurs in vitro and in living cells under the control of either pH, disulfide redn. or enzymic cleavage. In vitro, the size and shape of the condensation products, and the nanostructures subsequently assembled, were different in each case and could thus be controlled by tuning the structure of the monomers. Direct imaging of the products obtained in the cells revealed their locations-near the Golgi bodies under enzymic cleavage control-demonstrating the feasibility of a controlled and localized reaction in living cells. This intracellular condensation process enabled the imaging of the proteolytic activity of furin.
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    26. 26
      Nguyen, D. P.; Elliott, T.; Holt, M.; Muir, T. W.; Chin, J. W. Genetically Encoded 1,2-Aminothiols Facilitate Rapid and Site-Specific Protein Labeling via a Bio-Orthogonal Cyanobenzothiazole Condensation. J. Am. Chem. Soc. 2011, 133 (30), 11418– 11421,  DOI: 10.1021/ja203111c
      26
      Genetically Encoded 1,2-Aminothiols Facilitate Rapid and Site-Specific Protein Labeling via a Bio-orthogonal Cyanobenzothiazole Condensation
      Nguyen, Duy P.; Elliott, Thomas; Holt, Matthew; Muir, Tom W.; Chin, Jason W.
      Journal of the American Chemical Society (2011), 133 (30), 11418-11421CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
      The authors report evolved orthogonal pyrrolysyl-tRNA synthetase/tRNACUA pairs that direct the efficient, site-specific incorporation of Nε-L-thiaprolyl-L-lysine, Nε-D-cysteinyl-L-lysine, and Nε-L-cysteinyl-L-lysine into recombinant proteins in Escherichia coli. The authors demonstrate that the unique 1,2-aminothiol introduced by the authors' approach can be efficiently, rapidly, and specifically labeled via a cyanobenzothiazole condensation to quant. introduce biophys. probes into proteins. Moreover, the authors show that, in combination with cysteine labeling, this approach allows the dual labeling of proteins with distinct probes at two distinct, genetically defined sites.
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    27. 27
      Ramil, C. P.; An, P.; Yu, Z.; Lin, Q. Sequence-Specific 2-Cyanobenzothiazole Ligation. J. Am. Chem. Soc. 2016, 138 (17), 5499– 5502,  DOI: 10.1021/jacs.6b00982
      27
      Sequence-Specific 2-Cyanobenzothiazole Ligation
      Ramil, Carlo P.; An, Peng; Yu, Zhipeng; Lin, Qing
      Journal of the American Chemical Society (2016), 138 (17), 5499-5502CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
      The use of small, natural chem. reporters in conjunction with catalyst-free bioorthogonal reactions will greatly streamline protein labeling in a cellular environment with min. perturbation to their function. Here we report the discovery of a 2-cyanobenzothiazole (CBT)-reactive peptide tag, CX10R7, from a cysteine-encoded peptide phage library using the phage-assisted interrogation of reactivity method. Fusion of CX10R7 with a protein of interest allows site-specific labeling of the protein with CBT both in vitro and on the surface of E. coli cells. Mutagenesis studies indicated that the reactivity and specificity of CX10R7 are attributed to the sequence environment, in which the residues surrounding cysteine help to stabilize the ligation product.
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    28. 28
      Ren, H.; Xiao, F.; Zhan, K.; Kim, Y.-P.; Xie, H.; Xia, Z.; Rao, J. A Biocompatible Condensation Reaction for the Labeling of Terminal Cysteine Residues on Proteins. Angew. Chem., Int. Ed. 2009, 48 (51), 9658– 9662,  DOI: 10.1002/anie.200903627
      28
      A Biocompatible Condensation Reaction for the Labeling of Terminal Cysteine Residues on Proteins
      Ren, Hongjun; Xiao, Fei; Zhan, Ke; Kim, Young-Pil; Xie, Hexin; Xia, Zuyong; Rao, Jianghong
      Angewandte Chemie, International Edition (2009), 48 (51), 9658-9662, S9658/1-S9658/16CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)
      A protein-labeling method based on the use of a single amino acid tag - an N-terminal cysteine residue - and small-mol. probes contg. a cyanobenzothiazole (CBT) unit has been used for the specific fluorescence labeling of proteins in vitro and at the surface of live cells. This simple ligation reaction proceeds with a high degree of specificity under physiol. conditions.
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      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1MXhsFGru7zP&md5=d63d17244172c6ae5bdfb9ed9b4fc6aa
    29. 29
      Van de Bittner, G. C.; Bertozzi, C. R.; Chang, C. J. Strategy for Dual-Analyte Luciferin Imaging: In Vivo Bioluminescence Detection of Hydrogen Peroxide and Caspase Activity in a Murine Model of Acute Inflammation. J. Am. Chem. Soc. 2013, 135 (5), 1783– 1795,  DOI: 10.1021/ja309078t
      29
      Strategy for Dual-Analyte Luciferin Imaging: In Vivo Bioluminescence Detection of Hydrogen Peroxide and Caspase Activity in a Murine Model of Acute Inflammation
      Van de Bittner, Genevieve C.; Bertozzi, Carolyn R.; Chang, Christopher J.
      Journal of the American Chemical Society (2013), 135 (5), 1783-1795CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
      In vivo mol. imaging holds promise for understanding the underlying mechanisms of health, injury, aging, and disease, as it can detect distinct biochem. processes such as enzymic activity, reactive small-mol. fluxes, or post-translational modifications. Current imaging techniques often detect only a single biochem. process, but, within whole organisms, multiple types of biochem. events contribute to physiol. and pathol. phenotypes. The authors present a general strategy for dual-analyte detection in living animals that employs in situ formation of firefly luciferin from two complementary caged precursors that can be unmasked by different biochem. processes. To establish this approach, the authors have developed Peroxy Caged Luciferin-2 (PCL-2, I), a H2O2-responsive boronic acid probe that releases 6-hydroxy-2-cyanobenzothiazole (HCBT) upon reacting with this reactive oxygen species, as well as a peptide-based probe, z-Ile-Glu-ThrAsp-D-Cys (IETDC), which releases D-cysteine in the presence of active caspase 8. Once released, HCBT and D-cysteine form firefly luciferin in situ, giving rise to a bioluminescent signal if and only if both chem. triggers proceed. This system thus constitutes an AND-type mol. logic gate that reports on the simultaneous presence of H2O2 and caspase 8 activity. Using these probes, chemoselective imaging of either H2O2 or caspase 8 activity was performed in vitro and in vivo. Moreover, concomitant use of PCL-2 and IETDC in vivo establishes a concurrent increase in both H2O2 and caspase 8 activity during acute inflammation in living mice. Taken together, this method offers a potentially powerful new chem. tool for studying simultaneous oxidative stress and inflammation processes in living animals during injury, aging, and disease, as well as a versatile approach for concurrent monitoring of multiple analytes using luciferin-based bioluminescence imaging technologies.
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    30. 30
      Roychaudhuri, R.; Gadalla, M. M.; West, T.; Snyder, S. H. A Novel Stereospecific Bioluminescent Assay for Detection of Endogenous D-Cysteine. ACS Chem. Neurosci. 2022, 13 (23), 3257– 3262,  DOI: 10.1021/acschemneuro.2c00528
      30
      A Novel Stereospecific Bioluminescent Assay for Detection of Endogenous D-Cysteine
      Roychaudhuri, Robin; Gadalla, Moataz M.; West, Timothy; Snyder, Solomon H.
      ACS Chemical Neuroscience (2022), 13 (23), 3257-3262CODEN: ACNCDM; ISSN:1948-7193. (American Chemical Society)
      The presence of endogenous D-stereoisomers of amino acids in mammals dispels a long-standing dogma about their existence. D-Serine and D-aspartate function as novel neurotransmitters in mammals. However, the stereoisomer with the fastest, spontaneous in vitro racemization rate, D-cysteine, has not been reported. We utilized a novel, stereospecific, bioluminescent assay to identify endogenous D-cysteine in substantial amts. in the eye, brain, and pancreas of mice. D-Cysteine is enriched in mice embryonic brains at day E9.5 (4.5 mM) and decreases progressively with development (μM levels). D-Cysteine is also present in significantly higher amts. in the human brain white matter compared with gray matter. In the luciferase assay, D-cysteine conjugates with cyano hydroxy benzothiazole in the presence of a base and reducing agent to form D-luciferin. D-Luciferin, subsequently, in the presence of firefly luciferase and ATP, emits bioluminescence proportional to the concn. of D-cysteine. The assay is stereospecific and allows the quant. estn. of endogenous D-cysteine in tissues in addn. to its specificity for D-cysteine. Future efforts aimed at bioluminescent in vivo imaging of D-cysteine may allow a more noninvasive means of its detection, thereby elucidating its function.
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    31. 31
      Semenza, E. R.; Harraz, M. M.; Abramson, E.; Malla, A. P.; Vasavda, C.; Gadalla, M. M.; Kornberg, M. D.; Snyder, S. H.; Roychaudhuri, R. D-Cysteine Is an Endogenous Regulator of Neural Progenitor Cell Dynamics in the Mammalian Brain. Proc. Natl. Acad. Sci. U.S.A. 2021, 118 (39), e2110610118  DOI: 10.1073/pnas.2110610118
      31
      D-cysteine is an endogenous regulator of neural progenitor cell dynamics in the mammalian brain
      Semenza, Evan R.; Harraz, Maged M.; Abramson, Efrat; Malla, Adarsha P.; Vasavda, Chirag; Gadalla, Moataz M.; Kornberg, Michael D.; Snyder, Solomon H.; Roychaudhuri, Robin
      Proceedings of the National Academy of Sciences of the United States of America (2021), 118 (39), e2110610118CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)
      D-amino acids are increasingly recognized as important signaling mols. in the mammalian central nervous system. However, the d-stereoisomer of the amino acid with the fastest spontaneous racemization ratein vitro in vitro, cysteine, has not been examd. in mammals. Using chiral high-performance liq. chromatog. and a stereospecific luciferase assay, we identify endogenous d-cysteine in the mammalian brain. We identify serine racemase (SR), which generates the N-methyl-d-aspartate (NMDA) glutamate receptor coagonist d-serine, as a candidate biosynthetic enzyme for d-cysteine. d-cysteine is enriched more than 20-fold in the embryonic mouse brain compared with the adult brain. d-cysteine reduces the proliferation of cultured mouse embryonic neural progenitor cells (NPCs) by ∼50%, effects not shared with d-serine or l-cysteine. The antiproliferative effect of d-cysteine is mediated by the transcription factors FoxO1 and FoxO3a. The selective influence of d-cysteine on NPC proliferation is reflected in overgrowth and aberrant lamination of the cerebral cortex in neonatal SR knockout mice. Finally, we perform an unbiased screen for d-cysteine-binding proteins in NPCs by immunopptn. with a d-cysteine-specific antibody followed by mass spectrometry. This approach identifies myristoylated alanine-rich C-kinase substrate (MARCKS) as a putative d-cysteine-binding protein. Together, these results establish endogenous mammalian d-cysteine and implicate it as a physiol. regulator of NPC homeostasis in the developing brain.
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    32. 32
      Zhang, B. S.; Jones, K. A.; McCutcheon, D. C.; Prescher, J. A. Pyridone Luciferins and Mutant Luciferases for Bioluminescence Imaging. ChemBioChem 2018, 19 (5), 470– 477,  DOI: 10.1002/cbic.201700542
      There is no corresponding record for this reference.
    33. 33
      Xiong, Y.; Zhang, Y.; Li, Z.; Reza, M. S.; Li, X.; Tian, X.; Ai, H. Engineered Amber-Emitting Nano Luciferase and Its Use for Immunobioluminescence Imaging In Vivo. J. Am. Chem. Soc. 2022, 144 (31), 14101– 14111,  DOI: 10.1021/jacs.2c02320
      There is no corresponding record for this reference.
    34. 34
      White, E. H.; Wörther, H.; Seliger, H. H.; McElroy, W. D. Amino Analogs of Firefly Luciferin and Biological Activity Thereof1. J. Am. Chem. Soc. 1966, 88 (9), 2015– 2019,  DOI: 10.1021/ja00961a030
      There is no corresponding record for this reference.
    35. 35
      Godinat, A.; Budin, G.; Morales, A. R.; Park, H. M.; Sanman, L. E.; Bogyo, M.; Yu, A.; Stahl, A.; Dubikovskaya, E. A. A Biocompatible “Split Luciferin” Reaction and Its Application for Non-Invasive Bioluminescent Imaging of Protease Activity in Living Animals. Curr. Protoc. Chem. Biol. 2014, 6 (3), 169– 189,  DOI: 10.1002/9780470559277.ch140047
      There is no corresponding record for this reference.
    36. 36
      Nakamura, M.; Niwa, K.; Maki, S.; Hirano, T.; Ohmiya, Y.; Niwa, H. Construction of a New Firefly Bioluminescence System Using L-Luciferin as Substrate. Tetrahedron Lett. 2006, 47 (7), 1197– 1200,  DOI: 10.1016/j.tetlet.2005.12.033
      There is no corresponding record for this reference.
    37. 37
      Niwa, K.; Nakamura, M.; Ohmiya, Y. Stereoisomeric Bio-Inversion Key to Biosynthesis of Firefly d-Luciferin. FEBS Lett. 2006, 580 (22), 5283– 5287,  DOI: 10.1016/j.febslet.2006.08.073
      37
      Stereoisomeric bio-inversion key to biosynthesis of firefly D-luciferin
      Niwa, Kazuki; Nakamura, Mitsuhiro; Ohmiya, Yoshihiro
      FEBS Letters (2006), 580 (22), 5283-5287CODEN: FEBLAL; ISSN:0014-5793. (Elsevier B.V.)
      The chirality of the luciferin substrate is crit. to the luciferin-luciferase reaction producing bioluminescence. In firefly, the biosynthetic pathway of D-luciferin is still unclear, although it can be synthesized in vitro from D-cysteine. Here, we show that the firefly produces both D- and L-luciferin, and that the amt. of active D-luciferin increases gradually with maturation stage. Studies of firefly body exts. indicate the possible conversion of L-cysteine via L-luciferin into D-luciferin, suggesting that the biosynthesis is enzymically regulated by stereoisomeric bio-inversion of L-luciferin. We conclude that the selection of chirality in living organisms is not as rigid as previously thought.
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    38. 38
      Ren, Y.; Qiang, Y.; Zhu, B.; Tang, W.; Duan, X.; Li, Z. General Strategy for Bioluminescence Sensing of Peptidase Activity In Vivo Based on Tumor-Targeting Probiotic. Anal. Chem. 2021, 93 (9), 4334– 4341,  DOI: 10.1021/acs.analchem.1c00093
      38
      General strategy for bioluminescence sensing of peptidase activity in vivo based on tumor-targeting probiotic
      Ren, Yiqian; Qiang, Yao; Zhu, Beibei; Tang, Wei; Duan, Xinrui; Li, Zhengping
      Analytical Chemistry (Washington, DC, United States) (2021), 93 (9), 4334-4341CODEN: ANCHAM; ISSN:0003-2700. (American Chemical Society)
      The abnormally expressed peptidases in human tissues are assocd. with many kinds of cancers. Monitoring of endogenous peptidase activity could allow us for pathophysiol. elucidation and early clin. diagnosis. Herein, we developed a general strategy for bioluminescence (BL) sensing of peptidase activity in vivo based on tumor-targeting probiotics. The probiotic that harbored a luciferase-encoding plasmid was used to target and colonize tumor and provide luciferase for BL imaging. The peptide-based probes Lc and GPc were applied to track leucine aminopeptidase (LAP) and dipeptidyl peptidase IV (DPPIV) activity, resp., by simply adding L-leucine and Gly-Pro dipeptides at the N-terminus of D-cysteine, which were specifically controlled by peptidase cleavage and released free D-cysteine to conduct a subsequent click condensation reaction with 2-cyano-6-hydroxybenzothiazole (HCBT) to produce firefly luciferin in situ, giving rise to a strong BL signal. Neither gene modification of cells of interest nor complicated synthesis was required in this BL system. Encouraged by these advantages, we successfully used our probes to monitor LAP and DPPIV activities in vitro and in vivo, resp. A good linearity between BL and peptidase was obtained in the concn. range of 2.5-40.0 mU/mL with a limit of detection (LOD) of 1.1 mU/mL (55 ng/mL) for LAP and 2.0-40.0 mU/mL with a LOD of 0.78 mU/mL (1.15 ng/mL) for DPPIV, resp. Addnl., approx. 5-fold (LAP) and 10-fold (DPPIV) differences in the BL signal before and after treatment with a specific inhibitor were also obtained for in vivo BL imaging. All these results reflected the potential application value of our probes in BL sensing of peptidase activity. We envision that our strategy may be a useful approach for monitoring a wide range of peptidases in tumors, esp. in primary tumors.
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    39. 39
      Niwa, K.; Nakajima, Y.; Ohmiya, Y. Applications of Luciferin Biosynthesis: Bioluminescence Assays for l-Cysteine and Luciferase. Anal. Biochem. 2010, 396 (2), 316– 318,  DOI: 10.1016/j.ab.2009.09.014
      There is no corresponding record for this reference.
    40. 40
      Nakamura, M.; Maki, S.; Amano, Y.; Ohkita, Y.; Niwa, K.; Hirano, T.; Ohmiya, Y.; Niwa, H. Firefly Luciferase Exhibits Bimodal Action Depending on the Luciferin Chirality. Biochem. Biophys. Res. Commun. 2005, 331 (2), 471– 475,  DOI: 10.1016/j.bbrc.2005.03.202
      40
      Firefly luciferase exhibits bimodal action depending on the luciferin chirality
      Nakamura, Mitsuhiro; Maki, Shojiro; Amano, Yoshiharu; Ohkita, Yutaka; Niwa, Kazuki; Hirano, Takashi; Ohmiya, Yoshihiro; Niwa, Haruki
      Biochemical and Biophysical Research Communications (2005), 331 (2), 471-475CODEN: BBRCA9; ISSN:0006-291X. (Elsevier)
      Firefly luciferase is able to convert L-luciferin into luciferyl-CoA even under ordinary aerobic luciferin-luciferase reaction conditions. The luciferase is able to recognize strictly the chirality of the luciferin structure, serving as the acyl-CoA synthetase for L-luciferin, whereas D-luciferin is used for the bioluminescence reaction. D-Luciferin inhibits the luciferyl-CoA synthetase activity of L-luciferin, whereas L-luciferin retards the bioluminescence reaction of D-luciferin, meaning that both enzyme activities are prevented by the enantiomer of its own substrate.
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    41. 41
      Fahey, R. C.; Brown, W. C.; Adams, W. B.; Worsham, M. B. Occurrence of Glutathione in Bacteria. J. Bacteriol. 1978, 133 (3), 1126– 1129,  DOI: 10.1128/jb.133.3.1126-1129.1978
      41
      Occurrence of glutathione in bacteria
      Fahey, Robert C.; Brown, Willie C.; Adams, William B.; Worsham, Michael B.
      Journal of Bacteriology (1978), 133 (3), 1126-9CODEN: JOBAAY; ISSN:0021-9193.
      Glutathione and sol. thiol content were examd. in a broad spectrum of bacteria. Significant sol. thiol was present in all cases. The thiol compd. was glutathione in most of the gram-neg. bacteria but not in most of the gram-pos. bacteria studied. Glutathione was absent in 4 anaerobes and 1 microaerophile but was present in a blue-green bacterium. The glutathione content of Escherichia coli increased significantly during transition from exponential to stationary phase.
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    42. 42
      Taylor, M. D. Improved Passive Oral Drug Delivery via Prodrugs. Adv. Drug Delivery Rev. 1996, 19 (2), 131– 148,  DOI: 10.1016/0169-409X(95)00104-F
      There is no corresponding record for this reference.
    43. 43
      Beaumont, K.; Webster, R.; Gardner, I.; Dack, K. Design of Ester Prodrugs to Enhance Oral Absorption of Poorly Permeable Compounds: Challenges to the Discovery Scientist. Curr. Drug Metab. 2003, 4 (6), 461– 485,  DOI: 10.2174/1389200033489253
      43
      Design of ester prodrugs to enhance oral absorption of poorly permeable compounds: Challenges to the discovery scientist
      Beaumont, Kevin; Webster, Robert; Gardner, Iain; Dack, Kevin
      Current Drug Metabolism (2003), 4 (6), 461-485CODEN: CDMUBU; ISSN:1389-2002. (Bentham Science Publishers Ltd.)
      A review and discussion. Many drugs are administered at sites that are remote from their site of action. The most common route of drug delivery is the oral route. The optimal physicochem. properties to allow high transcellular absorption following oral administration are well established and include a limit on mol. size, hydrogen bonding potential and adequate lipophilicity. For many drug targets, synthetic strategies can be devised to balance the physicochem. properties required for high transcellular absorption and the SAR for the drug target. However, there are drug targets where the SAR requires properties at odds with good membrane permeability. These include a requirement for significant polarity and groups that exhibit high hydrogen bonding potential such as carboxylic acids and alcs. In such cases, prodrug strategies have been employed. The rationale behind the prodrug strategy is to introduce lipophilicity and mask hydrogen bonding groups of an active compd. by the addn. of another moiety, most commonly an ester. An ideal ester prodrug should exhibit the following properties:. (1) Weak (or no) activity against any pharmacol. target,. (2) Chem. stability across a pH range,. (3) High aq. soly.,. (4) Good transcellular absorption,. (5) Resistance to hydrolysis during the absorption phase,. (6) Rapid and quant. breakdown to yield high circulating concns. of the active component post absorption. This paper will review the literature around marketed prodrugs and det. the most appropriate prodrug characteristics. In addn., it will examine potential discovery approaches to optimizing prodrug delivery and recommend a strategy for prosecuting an oral prodrug approach.
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    44. 44
      Mohammad, I.; Liebmann, K. L.; Miller, S. C. Firefly Luciferin Methyl Ester Illuminates the Activity of Multiple Serine Hydrolases. Chem. Commun. 2023, 59 (55), 8552– 8555,  DOI: 10.1039/D3CC02540C
      There is no corresponding record for this reference.
    45. 45
      Antonczak, A. K.; Simova, Z.; Tippmann, E. M. A Critical Examination of Escherichia Coli Esterase Activity *. J. Biol. Chem. 2009, 284 (42), 28795– 28800,  DOI: 10.1074/jbc.M109.027409
      There is no corresponding record for this reference.
    46. 46
      Brown, A. R.; Wodzanowski, K. A.; Santiago, C. C.; Hyland, S. N.; Follmar, J. L.; Asare-Okai, P.; Grimes, C. L. Protected N-Acetyl Muramic Acid Probes Improve Bacterial Peptidoglycan Incorporation via Metabolic Labeling. ACS Chem. Biol. 2021, 16 (10), 1908– 1916,  DOI: 10.1021/acschembio.1c00268
      46
      Protected N-Acetyl Muramic Acid Probes Improve Bacterial Peptidoglycan Incorporation via Metabolic Labeling
      Brown, Ashley R.; Wodzanowski, Kimberly A.; Santiago, Cintia C.; Hyland, Stephen N.; Follmar, Julianna L.; Asare-Okai, PapaNii; Grimes, Catherine Leimkuhler
      ACS Chemical Biology (2021), 16 (10), 1908-1916CODEN: ACBCCT; ISSN:1554-8929. (American Chemical Society)
      Metabolic glycan probes have emerged as an excellent tool to investigate vital questions in biol. Recently, methodol. to incorporate metabolic bacterial glycan probes into the cell wall of a variety of bacterial species has been developed. In order to improve this method, a scalable synthesis of the peptidoglycan precursors is developed here, allowing for access to essential peptidoglycan immunol. fragments and cell wall building blocks. The question was asked if masking polar groups of the glycan probe would increase overall incorporation, a common strategy exploited in mammalian glycobiol. Here, we show, through cellular assays, that E. coli do not utilize peracetylated peptidoglycan substrates but do employ Me esters. The 10-fold improvement of probe utilization indicates that (i) masking the carboxylic acid is favorable for transport and (ii) bacterial esterases are capable of removing the Me ester for use in peptidoglycan biosynthesis. This investigation advances bacterial cell wall biol., offering a prescription on how to best deliver and utilize bacterial metabolic glycan probes.
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    47. 47
      Korshunov, S.; Imlay, K. R. C.; Imlay, J. A. Cystine Import Is a Valuable but Risky Process Whose Hazards Escherichia Coli Minimizes by Inducing a Cysteine Exporter. Mol. Microbiol. 2020, 113 (1), 22– 39,  DOI: 10.1111/mmi.14403
      There is no corresponding record for this reference.
    48. 48
      Chonoles Imlay, K. R.; Korshunov, S.; Imlay, J. A. Physiological Roles and Adverse Effects of the Two Cystine Importers of Escherichia Coli. J. Bacteriol. 2015, 197 (23), 3629– 3644,  DOI: 10.1128/JB.00277-15
      There is no corresponding record for this reference.
    49. 49
      French, S.; Farha, M.; Ellis, M. J.; Sameer, Z.; Côté, J. P.; Cotroneo, N.; Lister, T.; Rubio, A.; Brown, E. D. Potentiation of Antibiotics against Gram-Negative Bacteria by Polymyxin B Analogue SPR741 from Unique Perturbation of the Outer Membrane. ACS Infect. Dis. 2020, 6 (6), 1405– 1412,  DOI: 10.1021/acsinfecdis.9b00159
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      Potentiation of Antibiotics against Gram-Negative Bacteria by Polymyxin B Analogue SPR741 from Unique Perturbation of the Outer Membrane
      French, Shawn; Farha, Maya; Ellis, Michael J.; Sameer, Zaid; Cote, Jean-Philippe; Cotroneo, Nicole; Lister, Troy; Rubio, Aileen; Brown, Eric D.
      ACS Infectious Diseases (2020), 6 (6), 1405-1412CODEN: AIDCBC; ISSN:2373-8227. (American Chemical Society)
      Therapeutics targeting Gram-neg. bacteria have the challenge of overcoming a formidable outer membrane (OM) barrier. We characterize the action of SPR741, a novel polymyxin B (PMB) analog shown to potentiate several large-scaffold antibiotics in Gram-neg. pathogens. Probing the surface topol. of Escherichia coli using at. force microscopy revealed substantial OM disorder at concns. of SPR741 that lead to antibiotic potentiation. Conversely, very little cytoplasmic membrane depolarization was obsd. at these same concns., indicating that SPR741 acts predominately on the OM. Truncating the LPS core with genetic perturbations uniquely sensitized E. coli to SPR741, suggesting that LPS core residues keep SPR741 at the OM where it can potentiate a co-drug, rather than permit its entry to the cytoplasmic membrane. Further, a promoter activity assay revealed that SPR741 challenge induced the expression of RcsAB, a stress sensor for OM perturbation. Together these results indicate that SPR741 interacts predominately with the OM, in contrast to the dual action of PMB and colistin at both the outer and cytoplasmic membranes.
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    50. 50
      Ofek, I.; Cohen, S.; Rahmani, R.; Kabha, K.; Tamarkin, D.; Herzig, Y.; Rubinstein, E. Antibacterial Synergism of Polymyxin B Nonapeptide and Hydrophobic Antibiotics in Experimental Gram-Negative Infections in Mice. Antimicrob. Agents Chemother. 1994, 38 (2), 374– 377,  DOI: 10.1128/AAC.38.2.374
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      Antibacterial synergism of polymyxin B nonapeptide and hydrophobic antibiotics in experimental gram-negative infections in mice
      Ofek, Izhak; Cohen, Sofie; Rahmani, Rita; Kabha, Kisra; Tamarkin, Dove; Herzig, Yaacov; Rubinstein, Ethan
      Antimicrobial Agents and Chemotherapy (1994), 38 (2), 374-7CODEN: AMACCQ; ISSN:0066-4804.
      Polymyxin B nonapeptide, derived by cleavage of the fatty acyl diaminobutryic acid from polymyxin B, is considerably less toxic, less bactericidal activity, and retains its ability to render gram-neg. bacteria susceptible to several antibiotics by permeabilizing their outer membranes. The peptide rendered all 53 polymyxin-susceptible strains tested more susceptible to novobiocin, lowering the MIC of novobiocin 8-fold or more. The combination of polymyxin B nonapeptide with novobiocin or with erythromycin administered i.p. in multiple doses synergistically protected mice infected with gram-neg. bacteria. This combination may be clin. useful because of the apparent rarity of the acquisition of resistance.
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    51. 51
      Dixon, R. A.; Chopra, I. Polymyxin B and Polymyxin B Nonapeptide Alter Cytoplasmic Membrane Permeability in Escherichia Coli. J. Antimicrob. Chemother. 1986, 18 (5), 557– 563,  DOI: 10.1093/jac/18.5.557
      There is no corresponding record for this reference.
    52. 52
      Lomakina, G. Yu.; Ugarova, N. N. Kinetics of the Interaction of Colistin with Live Escherichia Coli Cells by the Bioluminescence Method. Mosc. Univ. Chem. Bull. 2022, 77 (1), 42– 47,  DOI: 10.3103/S0027131422010059
      There is no corresponding record for this reference.
    53. 53
      Ihssen, J.; Jovanovic, N.; Sirec, T.; Spitz, U. Real-Time Monitoring of Extracellular ATP in Bacterial Cultures Using Thermostable Luciferase. PLoS One 2021, 16 (1), e0244200  DOI: 10.1371/journal.pone.0244200
      There is no corresponding record for this reference.
    54. 54
      Branchini, B. R.; Magyar, R. A.; Murtiashaw, M. H.; Anderson, S. M.; Zimmer, M. Site-Directed Mutagenesis of Histidine 245 in Firefly Luciferase: A Proposed Model of the Active Site. Biochemistry 1998, 37 (44), 15311– 15319,  DOI: 10.1021/bi981150d
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      Site-directed mutagenesis of histidine 245 in firefly luciferase: A proposed model of the active site
      Branchini, Bruce R.; Magyar, Rachelle A.; Murtiashaw, Martha H.; Anderson, Shannon M.; Zimmer, Marc
      Biochemistry (1998), 37 (44), 15311-15319CODEN: BICHAW; ISSN:0006-2960. (American Chemical Society)
      Firefly luciferase (I) catalyzes the highly efficient emission of yellow-green light from the substrate, luciferin, by a sequence of reactions that require Mg-ATP and O2. The authors previously reported that 2-(4-benzoylphenyl)thiazole-4-carboxylic acid (BPTC), a firefly luciferin analog, was a potent photoinactivation reagent for I. A I tetrapeptide [244HHGF247] was identified, the degrdn. of which was directly correlated to the photooxidn. process. Here, the authors report the construction and purifn. of wild-type (WT) I and mutants H244F, H245F, H245A, and H245D. The results of photoinactivation and kinetic and bioluminescence studies with these proteins were consistent with His-245 being the primary functional target of BPTC-catalyzed enzyme inactivation. The possibility that His-245 is oxidized to Asp during the photooxidn. reaction was supported by the extremely low specific activity (∼300-fold lower than WT I) of the H245D mutant. Using the previously reported crystal structures of I without substrates and the functionally related phenylalanine-activating subunit of gramicidin synthetase 1 as a starting point, the authors performed mol. modeling studies and propose here a model for the I active site with substrates, luciferin and Mg-ATP, bound. This model was used to provide a structure-based interpretation of the role of peptide 244HHGF247 in firefly bioluminescence.
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    55. 55
      Wilhelm, M. J.; Sharifian Gh, M.; Dai, H.-L. Chemically Induced Changes to Membrane Permeability in Living Cells Probed with Nonlinear Light Scattering. Biochemistry 2015, 54 (29), 4427– 4430,  DOI: 10.1021/acs.biochem.5b00600
      There is no corresponding record for this reference.
    56. 56
      Wilhelm, M. J.; Sharifian Gh, M.; Dai, H.-L. Influence of Molecular Structure on Passive Membrane Transport: A Case Study by Second Harmonic Light Scattering. J. Chem. Phys. 2019, 150 (10), 104705,  DOI: 10.1063/1.5081720
      56
      Influence of molecular structure on passive membrane transport: A case study by second harmonic light scattering
      Wilhelm, Michael J.; Sharifian Gh., Mohammad; Dai, Hai-Lung
      Journal of Chemical Physics (2019), 150 (10), 104705/1-104705/8CODEN: JCPSA6; ISSN:0021-9606. (American Institute of Physics)
      We present an exptl. study, using the surface sensitive technique, second harmonic light scattering (SHS), to examine the influence of structure on the propensity of a mol. to passively diffuse across a phospholipid membrane. Specifically, we monitor the relative tendency of the structurally similar amphiphilic cationic dyes, malachite green (MG) and crystal violet (CV), to transport across membranes in living cells (E. coli) and biomimetic liposomes. Despite having nearly identical mol. structures, mol. wts., cationic charges, and functional groups, MG is of lower overall symmetry and consequently has a symmetry allowed permanent dipole moment, which CV does not. The two mols. showed drastically different interactions with phospholipid membranes. MG is obsd. to readily cross the hydrophobic interior of the bacterial cytoplasmic membrane. Conversely, CV does not. Furthermore, expts. conducted with biomimetic liposomes, constructed from the total lipid ext. of E. coli and contg. no proteins, show that while MG is able to diffuse across the liposome membrane, CV does not. These observations indicate that the SHS results measured with bacteria do not result from the functions of efflux pumps, but suggests that MG possesses an innate mol. property (which is absent in CV) that allows it to passively diffuse across the hydrophobic interior of a phospholipid membrane. (c) 2019 American Institute of Physics.
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