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Discovery and Characterization of Synthesized and FDA-Approved Inhibitors of Clostridial and Bacillary Collagenases
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  • Alaa Alhayek
    Alaa Alhayek
    Helmholtz Institute for Pharmaceutical Research Saarland (HIPS), Helmholtz Center for Infection Research (HZI), Campus Building E8.1, 66123 Saarbrücken, Germany
    Department of Pharmacy, Saarland University, Campus Building C2. 3, 66123 Saarbrücken, Germany
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  • Ahmed S. Abdelsamie
    Ahmed S. Abdelsamie
    Helmholtz Institute for Pharmaceutical Research Saarland (HIPS), Helmholtz Center for Infection Research (HZI), Campus Building E8.1, 66123 Saarbrücken, Germany
    Department of Chemistry of Natural and Microbial Products, Institute of Pharmaceutical and Drug Industries Research, National Research Centre, El-Buhouth St., Dokki, 12622 Cairo, Egypt
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  • Esther Schönauer
    Esther Schönauer
    Department of Biosciences and Medical Biology, University of Salzburg, Hellbrunner Str. 34, 5020 Salzburg, Austria
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  • Virgyl Camberlein
    Virgyl Camberlein
    Helmholtz Institute for Pharmaceutical Research Saarland (HIPS), Helmholtz Center for Infection Research (HZI), Campus Building E8.1, 66123 Saarbrücken, Germany
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  • Evelyn Hutterer
    Evelyn Hutterer
    Department of Biosciences and Medical Biology, University of Salzburg, Hellbrunner Str. 34, 5020 Salzburg, Austria
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    Orcidhttps://orcid.org/0000-0001-8622-4758
  • Gernot Posselt
    Gernot Posselt
    Department of Biosciences and Medical Biology, University of Salzburg, Hellbrunner Str. 34, 5020 Salzburg, Austria
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  • Jamil Serwanja
    Jamil Serwanja
    Department of Biosciences and Medical Biology, University of Salzburg, Hellbrunner Str. 34, 5020 Salzburg, Austria
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  • Constantin Blöchl
    Constantin Blöchl
    Department of Biosciences and Medical Biology, University of Salzburg, Hellbrunner Str. 34, 5020 Salzburg, Austria
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  • Christian G. Huber
    Christian G. Huber
    Department of Biosciences and Medical Biology, University of Salzburg, Hellbrunner Str. 34, 5020 Salzburg, Austria
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  • Jörg Haupenthal
    Jörg Haupenthal
    Helmholtz Institute for Pharmaceutical Research Saarland (HIPS), Helmholtz Center for Infection Research (HZI), Campus Building E8.1, 66123 Saarbrücken, Germany
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  • Hans Brandstetter
    Hans Brandstetter
    Department of Biosciences and Medical Biology, University of Salzburg, Hellbrunner Str. 34, 5020 Salzburg, Austria
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  • Silja Wessler*
    Silja Wessler
    Department of Biosciences and Medical Biology, University of Salzburg, Hellbrunner Str. 34, 5020 Salzburg, Austria
    *Email: [email protected]
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  • Anna K. H. Hirsch*
    Anna K. H. Hirsch
    Helmholtz Institute for Pharmaceutical Research Saarland (HIPS), Helmholtz Center for Infection Research (HZI), Campus Building E8.1, 66123 Saarbrücken, Germany
    Department of Pharmacy, Saarland University, Campus Building C2. 3, 66123 Saarbrücken, Germany
    *Email: [email protected]
    More by Anna K. H. Hirsch
    Orcidhttps://orcid.org/0000-0001-8734-4663
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Journal of Medicinal Chemistry

Cite this: J. Med. Chem. 2022, 65, 19, 12933–12955
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https://doi.org/10.1021/acs.jmedchem.2c00785
Published September 26, 2022

Copyright © 2022 The Authors. Published by American Chemical Society. This publication is licensed under

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Abstract

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In view of the worldwide antimicrobial resistance (AMR) threat, new bacterial targets and anti-infective agents are needed. Since important roles in bacterial pathogenesis have been demonstrated for the collagenase H and G (ColH and ColG) from Clostridium histolyticum, collagenase Q1 and A (ColQ1 and ColA) from Bacillus cereus represent attractive antivirulence targets. Furthermore, repurposing FDA-approved drugs may assist to tackle the AMR crisis and was addressed in this work. Here, we report on the discovery of two potent and chemically stable bacterial collagenase inhibitors: synthesized and FDA-approved diphosphonates and hydroxamates. Both classes showed high in vitro activity against the clostridial and bacillary collagenases. The potent diphosphonates reduced B. cereus-mediated detachment and death of cells and Galleria mellonella larvae. The hydroxamates were also tested in a similar manner; they did not have an effect in infection models. This might be due to their fast binding kinetics to bacterial collagenases.

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Introduction

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Bacterial resistance is on the rise, and a global economic and public health crisis might be on the horizon due to the high incidence of deaths caused by multidrug-resistant bacteria. (1,2) This also comes together with the slow discovery of new antibiotics. If this trend continues, mild infections of today might become lethal in the future. (1,2) To combat the rise in antibiotic resistance, alternative, nonantibiotic treatment approaches are urgently needed. Antivirulence agents (also called “pathoblockers”), which selectively inhibit pathogenicity factors of bacteria, and hence prevent or delay infection, are one potential strategy. (3,4) This could─without exerting selective pressure─harness the host’s immune system to fight the infection. (3,4) Finding new applications for existing FDA-approved drugs is gaining popularity to tackle the current antimicrobial-resistance crisis as it reduces the cost, time, and effort required for the regular discovery of new antibiotics. (5) Pentamidine is an antiprotozoal medicine and an example of an FDA-approved drug that has been repurposed for antibacterial use. (6) This approach holds promise as it may overcome the slow development of new antibiotics.
Clostridium histolyticum (C. histolyticum) and Bacillus cereus (B. cereus) are Gram-positive bacteria and the epitome of many serious opportunistic infections, including gas gangrene as well as wound, corneal, and gastro-intestinal infections. (7,8) As these bacteria resist the activity of many antibiotics (such as penicillin), they are still susceptible to the activity of gentamycin, vancomycin, erythromycin, and other antibiotics. (9,10) The pathogenicity of these microorganisms is linked to their secreted toxins and proteases, which assist them to elude defensive mechanisms, reach deep locations for nourishment, and consequently replicate and persist at the infection site. (11) This might also enhance bacterial histotoxicity by promoting toxin diffusion. (11) Bacterial collagenases are calcium- and zinc-dependent metalloproteases and the etiologic feature of the aforementioned pathogens; they destroy tissues by demolishing extracellular matrix (ECM) collagen. (12,13) Fibrillar collagen is the most common protein in the ECM (up to 90%). It has a stable structure that resists proteolysis and can only be broken down by true bacterial and mammalian collagenases. (12,13) Bacterial collagenases process triple helical collagen under physiological conditions into small peptides and amino acids by targeting multiple sites. (12,13) Mammalian collagenases, on the other hand, such as the collagenolytic matrix metalloproteinases (MMPs), cleave collagen at a single site to generate the characteristic 3/4 and 1/4 fragments. After this initial cut, other enzymes assist in the further proteolysis of the collagen fragments. (13) Collagen is involved in many vital physiological processes such as tissue regeneration and wound healing, in addition to its tissue-supporting functions. (13) Therefore, any bacterial collagenase-induced imbalance in collagen structure or quantity will have detrimental effects on tissue regeneration and wound healing besides the formation of voids in the ECM, which allow the bacteria to invade and gain access to anaerobic areas. (14) Consequently, protecting collagen from bacterial collagenase represents a promising approach for developing antivirulence agents that can be used to treat collagenase-positive infections.
In contrast to the homologous B. cereus collagenases, C. histolyticum collagenases have been well studied. They are composed of two main structural modules: the collagenase unit and collagen recruitment domains. The collagenase unit is composed of an activator and peptidase domains. (15,16) In the latter, two histidine residues in a HEXXH motif and a downstream glutamate coordinate the catalytic zinc ion. The general-acid base glutamate and the zinc ion polarize the water molecule in the active site and activate it for the nucleophilic attack. (15−18) The collagen recruitment domains are suggested to be involved in collagen swelling and binding to fibrillar and insoluble collagen. (15−18) High-resolution crystal structures are available for C. histolyticum collagenases but not for B. cereus collagenases, which share a high sequence similarity (i.e., 70%). (17,19−21)
We focused in this study on C. histolyticum collagenases H and G (ColH and ColG), as well as B. cereus collagenases Q1 and A (ColQ1 and ColA). These bacterial collagenases are attractive candidates for the development of antivirulence drugs due to their role in bacterial pathogenicity as well as their extracellular localization. (14) The penetration of the bacterial cell wall is generally a key challenge for the development of antibacterial agents; however, in this instance, it can be avoided.
Distinct collagenase inhibitors have been identified to date, the majority of which include a zinc-binding group (ZBG) that binds to the catalytic zinc ion, displacing the catalytic water molecule from the coordination sphere and rendering the enzyme inactive. Figure 1a shows examples of known ColH inhibitors. (22−24) Their lack of selectivity over human MMPs is the primary drawback that prevents further development of these existing potent inhibitors.

Figure 1

Figure 1. Examples of known bacterial collagenase H and Q1 (ColH and ColQ1) inhibitors. (a) Structures of bacterial ColH inhibitors. (22−24) (b) Structures of the recently identified inhibitors of ColH and ColQ1. (25−28) Zinc-binding groups are highlighted by dashed rectangles. n.d.: not determined.

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Recently, we were able to develop selective inhibitors of bacterial collagenases. Compound 4a, a thiocarbamate, serves as a prodrug. By conversion into a free thiol 4b, it can bind to the zinc ion (Figure 1b). (25) The rest of the molecule binds to the conserved clostridial nonprimed edge strand, explaining the selectivity for the bacterial collagenases over the human off-target MMPs. (25) Succinimide 5 is a more rigid derivative of compound 4b (Figure 1b). (26) Similarly to thiocarbamates, this class has been validated for its high selectivity for several bacterial collagenases, including ColH and ColG from C. histolyticum, ColT from C. tetani, and ColQ1 from B. cereus, over the unwanted inhibition of human MMPs. The main drawback of these two compound classes is their chemical instability due to the oxidation of the thiol group to the corresponding disulfide, which leads to the loss of their activity.
Replacing the thiol group with another stable ZBG to maintain the chemical stability of the inhibitor is necessary, in addition to maintaining the high selectivity toward bacterial metalloproteases. The first stable and selective inhibitor (i.e., compound 6) (Figure 1b) of ColH was recently reported. (27,28) Among various ZBGs, a phosphonate group has high stability and selectivity but moderate activities on ColH and ColQ1. (27,28)
In this work, we aimed to find chemically stable, potent, and selective bacterial collagenase inhibitors. Furthermore, we set out to characterize a range of compounds bearing several different ZBGs. We identified two chemical classes, namely, diphosphonates (including FDA-approved drugs) and hydroxamates with excellent selectivity, low cytotoxicity, and remarkable micro- to submicromolar activity in vitro against B. cereus ColQ1, ColA, and C. histolyticum ColH and ColG. This finding demonstrates the potential of these compounds to exert a broad-spectrum activity toward a variety of disease-causing bacteria. In addition, we show their activity in increasingly complex whole-cell assays and efficacy in a simple Galleria mellonella infection model. Hydroxamates were tested analogously. Surprisingly, they did not demonstrate the same inhibitory impact in the infection models, despite similar potencies in enzyme-inhibition studies.

Results and Discussion

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Screening of Compounds with Various ZBGs on ColQ1 and ColH

To discover new small-molecule inhibitors with a stable ZBG, a total of 38 compounds with distinct ZBGs were tested at 100 μM in an in vitro peptidolytic assay using a custom-made collagenase-specific quenched fluorescent substrate (Table S1 and Figure S1). Two compounds (13 and 27) showed strong inhibition against the collagenase unit of ColQ1 (COlQ1-CU) (98 ± 1% inhibition and 97 ± 2% inhibition) and the peptidase unit of ColH (ColH-PD) (83 ± 9% inhibition and 84 ± 2% inhibition), respectively (Figure 2). Both compounds were selected for further studies, and a small library based on 13 and 27 structures was designed and synthesized (i.e., 16 diphosphonates and 6 hydroxamates).

Figure 2

Figure 2. Chemical structures of compounds 13 and 27 and their activities against the collagenase unit (CU) of ColQ1 and peptidase domain (PD) of ColH.

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Synthesis of New Anticollagenase Pathoblocker Agents

Diphosphonate Synthesis

To get access to the different diphosphonate compounds, a synthetic route composed of four steps starting from the corresponding diamine was implemented. As depicted in Scheme 1, the synthesis of the diphosphonate compounds 7–12 started by refluxing the corresponding diamine with oxalic acid in 4 N hydrochloric acid to achieve the 1,4-dihydroquinoxaline-2,3-diones 7a–12a. These quinoxaline-dione intermediates were then reacted with POCl3 in DMF to provide the 2,3-dichloroquinoxalines 7b–12b, which were used in the following step without additional purification. (29,30) The resulting dichloroquinoxalines 7b–12b were converted via an Arbuzov reaction to the corresponding diethyl phosphonate esters 7c–12 using triethyl phosphite and heated in a sealed tube at 150 °C. TMSBr was then utilized to cleave the diethyl phosphonate esters 7c–12c to their corresponding phosphonic acids 7–12 in good yields.

Scheme 1

Scheme 1. Synthesis of Compounds 7–12a
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aReagents and conditions: (a) oxalic acid, 4 N HCl, reflux, 6 h; (b) POCl3, DMF, 50 °C, 6 h; (c) triethyl phosphite, sealed tube, 150 °C, 18 h; and (d) bromotrimethylsilane, dry DCM, stirring, room temperature, 18 h.

Compound 13 was synthesized using the same general procedure for the synthesis of the diphosphonates (Scheme 2). To get further access to the monophosphonate compound 16, intermediate 13b was converted into 16a using LiOH in a dioxane/water mixture, as described by Yang et al. (Scheme 2). (30) The latter was then subjected to the Arbuzov reaction followed by ester hydrolysis using TMSBr to afford the corresponding phosphonic acid 16.

Scheme 2

Scheme 2. Synthesis of Compounds 13 and 16a
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aReagents and conditions: (a) oxalic acid, 4 N HCl, reflux, 6 h; (b) POCl3, DMF, 50 °C, 6 h; (c) triethyl phosphite, sealed tube, 150 °C, 18 h; (d) bromotrimethylsilane, dry DCM, stirring, room temperature, 18 h; (e) dioxane/H2O (1:1), LiOH, 55 °C, 24 h; and (f) POCl3, DMF, 0 °C to room temperature, 5 min.

Refluxing of 3,4-diaminobenzoic acid and oxalic acid in 4 N hydrochloric acid yielded 14a, which reacted with POCl3 in DMF to afford the corresponding dichloro derivative 14b. Compound 14c was synthesized by reacting 2,3-dichloroquinoxaline-6-carboxylic acid 14b with 3,4-dichloroaniline in DCM at room temperature for 18 h using EDC·HCl as a coupling reagent. This was followed by the Arbuzov reaction and ester hydrolysis to achieve the desired compound 14 (Scheme 3).

Scheme 3

Scheme 3. Synthesis of Compound 14a
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aReagents and conditions: (a) oxalic acid, 4 N HCl, reflux, 6 h; (b) POCl3, DMF, 50 °C, 6 h; (c) 3,4-dichloroaniline, EDC.HCl, DCM, 18 h; (d) triethyl phosphite, sealed tube, 150 °C, 18 h; and (e) bromotrimethylsilane, dry DCM, stirring, room temperature, 18 h.

Compound 15a was obtained via a Suzuki cross-coupling reaction by reacting 4-bromobenzene-1,2-diamine and (4-chlorophenyl)boronic acid in the presence of 2 M sodium carbonate and (1,1′-bis(diphenylphosphino)ferrocene)palladium(II) dichloride [Pd(dppf)Cl2] as a catalyst in a mixture of dioxane/H2O (4:1) under microwave irradiation (150 °C, 150 W) for 20 min. (31) This was followed by the general procedure for the diphosphonate synthesis to afford compound 15 (Scheme 4).

Scheme 4

Scheme 4. Synthesis of Compound 15a
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aReagents and conditions: (a) (4-chlorophenyl)boronic acid, dioxane/H2O (4:1), Na2CO3 (2 M), Pd(PPh3)4, microwave, 20 min; (b) oxalic acid, 4 N HCl, reflux, 6 h; (c) POCl3, DMF, 50 °C, 6 h; (d) triethyl phosphite, sealed tube, 150 °C, 18 h; and (e) bromotrimethylsilane, dry DCM, stirring, room temperature, 18 h.

Hydroxamate Synthesis

As described in Scheme 5, the hydroxamates 27–33 were synthesized in six steps. The monocarboxylic acids 22a and 22b were first obtained through a monosaponification using sodium hydroxide in a mixture of ethanol and water. These intermediates were then activated using EDC·HCl and HOBt, in dichloromethane with diisopropylethylamine, to form the desired amides 23a and 23b by reacting them with the free amine. Then, an addition of hydrochloric acid (4 N in dioxane) afforded the free amines 24a and 24b, and these were reacted with the diazo transfer reagent to form the azides 25a and 25b. The ethyl esters were engaged in a KCN-catalyzed aminolysis reaction, which led to the formation of the azido hydroxamic acids 26a and 26b. The final step was a copper-catalyzed Huisgen 1,3-dipolar cycloaddition to give the desired 1,4-disubstituted 1,2,3-triazoles 27–33, the needed alkynes 34a–34d being previously synthesized by the nucleophilic substitution of phenols or thiophenol on propargyl bromide.

Scheme 5

Scheme 5. Synthesis of Hydroxamic Acid Compoundsa
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aReagents and conditions: (a) NaOH, EtOH/H2O (4:1), rt., 18 h; (b) tert-butyl N-(2-aminoethyl)carbamate, EDC.HCl, HOBt, DIPEA, CH2Cl2, rt., 18 h; (c) 4 N HCl, EtOH, 0 °C to rt., 18 h; (d) azide-N-diazoimidazole-1-sulfonamide hydrogen sulfate, K2CO3, ZnCl2, DIPEA, MeOH, rt., 18 h; (e) aq. hydroxylamine (50% in water w/w), KCN (cat.), MeOH, rt., 18 h; and (f) alkyne 34a–34d, prop-2-ynoxybenzene or prop-2-ynylsulfanylbenzene, CuSO4 (5H2O), NaAsc, N,N-dimethylformamide/H2O (1.2:1), rt., 18 h.

Activity on B. cereus ColQ1

Structure–Activity Relationships of the Synthesized and FDA-Approved Diphosphonate Compounds on ColQ1

The initial testing of the diphosphonate 13 and its synthesized derivatives using ColQ1-CU showed that the presence of both phosphonic acid groups is indispensable for inhibition (Table 1), indicating that the phosphonate groups act as ZBG to the catalytic zinc ion. The unsubstituted diphosphonic acid quinoxaline 7 showed lower activity than the 3,4-dichloro analogue 13 (Table 1). Introducing an electron-withdrawing group such as fluorine at position 6 of the quinoxaline moiety of 8 led to no significant change in activity. Interestingly, increasing the lipophilicity by adding chlorine or bromine at position 6 of the quinoxaline of 9, 10, and 13 led to a significant boost in activity in comparison with the unsubstituted 7, indicating the importance of the lipophilic groups at this part of the inhibitor (Table 1). This finding prompted us to further explore variable lipophilic moieties at this part of the molecule in an attempt to enhance the activity. Compounds 14 and 15 were synthesized and tested yielding less potent inhibitors, which could be an indication of inappropriate bulky groups at that part of the molecule. Adding an electron-donating group at position 6 of the quinoxaline moiety showed a slight improvement in the activity, as shown by the methyl derivative 11, while the methoxy derivative 12 resulted in a decrease in activity (Table 1).
Table 1. Percent Inhibition (at a Concentration of 100 μM) and IC50 Values of Diphosphonate Derivatives against ColQ1-CUa
a

Means and SD of three independent experiments, n.i.: no inhibition (percent inhibition < 5%).

In the case of ColG-PD (a close homologue of ColQ1), the SAR could be rationalized by a crystal structure in complex with compound 13 determined at 1.95 Å resolution (Figure 3 and Table S2). In addition to a nonfunctional binding site at the rear of the peptidase domain, we could detect 13 in the primed binding pocket, albeit at relatively low occupancy/high mobility. The inhibitor could be modeled into the active site with the help of a polder map. One of the phosphonate groups acted as ZBG and simultaneously interacted with Glu555 and Tyr607, while the aromatic scaffold of 13 and the chlorides established hydrophobic interactions in the primed binding pockets with Phe515, His523, and Ile576.

Figure 3

Figure 3. Crystal structure of ColG-PD in complex with 13 solved at a resolution of 1.95 Å. Close-up view of the active site in ball-and-stick representation. The inhibitor (cyan) is shown in sticks with a polder map contoured at 2.5 σ above the background. The catalytic zinc ion (dark gray) and the calcium ion (green) are shown as spheres (PDB code: 7ZBV).

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As drug repurposing is gaining attraction these days, especially for finding new antimicrobial agents, (5) we tested a number of FDA-approved diphosphonates regarding their effect on ColQ1-CU (Table 2). Tiludronate disodium and alendronate sodium resulted in a moderate inhibition of ColQ1-CU (63 ± 3 and 76 ± 1%, respectively) at a concentration of 100 μM. Diphosphonate FDA-approved drugs are routinely used in the treatment of bone diseases and were never reported to have antibacterial activity. (32) This calls for testing more diphosphonate FDA-approved drugs for their activity on bacterial collagenases to find potent inhibitors with known data regarding their safety, efficacy, and pharmacokinetics.
Table 2. Percent Inhibition of ColQ1-CU by Selected FDA-Approved Diphosphonates at a Concentration of 100 μMa
a

Means and SD of three independent experiments, n.i.: no inhibition (percent inhibition < 5%).

Structure–Activity Relationships of Hydroxamate Compounds on ColQ1

An initial screening led to the identification of hydroxamate 27; the SAR study was performed using COLQ1-CU, which is articulated around a 1,4-disubstituted 1,2,3-triazole ring (Figure S2 and Table 3). To explore the ortho-acetamide phenoxy group, four derivatives were synthesized (28–31). The move of the ortho-acetamide to meta- and para-positions as in 28 and 29, respectively, led to a slight decrease in activity, while its removal (compound 31) or its replacement by a 2-fluorine motif (compound 30) produced similar activities. The phenol replacement of 31 by thiophenol (compound 32) led to a lower ColQ1-CU inhibition. Interestingly, compound 33 demonstrated that bulky substituents in the α-position of the hydroxamate could be tolerated.
Table 3. Percent Inhibition (at a Concentration of 100 μM) or IC50 Values of Hydroxamates against ColQ1-CUa
a

Means and SD of three independent experiments.

Next, we evaluated the potential of the most effective compounds 27 and 33 as broad-spectrum inhibitors of bacterial collagenases and determined the inhibition constants using ColG-CU, ColH-PD, ColA-CU (from B. cereus strain ATCC14579), and ColQ1-CU. The results revealed that both compounds inhibited clostridial and bacillary collagenases in the low micro- to submicromolar range (Table 4).
Table 4. Inhibition Constant (Ki) of 27 and 33 against Bacterial Collagenasesa
  2733
bacteriaproteinKi (μM)Ki (μM)
C. histolyticumColH-PD11.6 ± 0.41.7 ± 0.2
ColG-CU31 ± 118.4 ± 0.6
B. cereusColQ1-CU0.10 ± 0.020.82 ± 0.07
ColA-CU3.4 ± 44.7 ± 0.3
a

Means and SD of three independent experiments.

We rationalized the screening results based on the crystal structure of 27 in complex with the ColG-PD, solved at a 1.80 Å resolution (Figure 4 and Table S2). The bound inhibitor occupied the active site from the S3 pocket to the S2′ binding site. In the S3 binding pocket, the ortho-acetamide group directly interacted with the edge strand via a hydrogen bond to the backbone amide of Glu498, while the aromatic phenoxy ring established π–π stacking interactions with the side chain of Trp539. This explains the observed preference for the ortho-configuration of the acetamide group.

Figure 4

Figure 4. Crystal structure of ColG-PD in complex with 27 solved at 1.80 Å resolution. Close-up view of the active site in ball-and-stick representation. The inhibitor (cyan) is shown in sticks with the maximum likelihood weighted 2Fo–Fc electron density map contoured at 1σ. The catalytic zinc ion (dark gray) and the calcium ion (green) and water molecules (red) are shown as spheres (PDB code: 7Z5U).

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The central triazole ring hydrogen bonded with Glu555 and formed a π–hydrogen interaction with Tyr599 in the S2 binding pocket. The hydroxamate group coordinated, as expected, the catalytic zinc ion. The isobutyl group in the α-position formed hydrophobic interactions with Tyr607 and His523 in the S2′ pocket. The hydrophobic benzyl group of 33 can be expected to fit similarly into the S2′ pocket.

Selectivity over Human MMPs and Activity against Other Bacterial Collagenases

To assess the selectivity of the compounds over human MMPs and on other bacterial collagenases, selected compounds were tested against ColG-CU and ColH-PD from C. histolyticum and ColA-CU from B. cereus strain ATCC14579. In addition, the compounds were tested on catalytic domains of three human MMPs, which are characterized by different depths of the S1′ binding pocket (MMP-1 (shallow), −2 (intermediate), and −3 (deep)). They were also tested against other important human off-targets, which are involved in gene expression and the processing of TNF-α; these include HDAC-3, -8, TACE (ADAM-17), (33,34) and COX-1.
Our data showed that the compounds have high activity against most tested bacterial collagenases, which confirms their broad-spectrum inhibitory potency against bacterial targets (Table 5). This broad activity is comparable to that previously observed for compounds carrying thiol or phosphonate ZBGs. (25−27)
Table 5. Percent Inhibition of ColA-CU, ColH-PD, and ColG-CU in the Presence of 100 μM of Compounds 13 14, 15, 27, Tiludronate Disodium, and Alendronate Sodiuma
classcpd.ColA-CUColH-PDColG-CU
synthesized diphosphonates1371 ± 483 ± 968 ± 4
1482 ± 296 ± 272 ± 3
1586 ± 897 ± 570 ± 8
FDA-approved diphosphonatestiludronate disodium72 ± 291 ± 228 ± 7
alendronate sodium18 ± 425 ± 528 ± 7
hydroxamate2793 ± 284 ± 271 ± 7
a

Means and SD of at least two independent experiments.

On the other hand, the compounds possess a high selectivity over most of the tested human off-targets (except for 13 against the tested MMPs and tiludronate disodium against MMP-1) (Tables 6 and S3).
Table 6. Inhibition of Compounds 13 14, 15, 27, Tiludronate Disodium, and Alendronate Sodium against Three MMPsa
IC50 (μM)
classcpd.MMP-1MMP-2MMP-3
synthesized diphosphonates1353 ± 379 ± 233 ± 11
14>100>100>100
15>100>100>100
FDA-approved diphosphonatestiludronate disodium50 ± 10>100>100
alendronate sodium>100>100>100
hydroxamate27>100>100>100
a

Means and SD of two independent experiments, >100: IC50 is higher than 100 μM.

Cytotoxicity against Mammalian Cell Lines

Besides the selectivity, cytotoxicity is also an important criterion, especially when it comes to a potential therapeutic application in humans. In this context, we evaluated the cytotoxicity of 13, 14, 15, and 27 against four mammalian cell lines, comprising HepG2 (hepatocellular carcinoma), HEK293 (embryonal kidney), NHDF (normal human dermal fibroblasts), and MDCK II (Madin–Darby canine kidney II) cells. Interestingly, the compounds did not show cytotoxic effects (IC50 values > 100 μM or 200 μM) against these cell lines (Table S4). This makes them suitable for further investigation to determine their ADMET profile.

Small-Molecule Collagenase Inhibitors Prevent Collagen I Cleavage

We examined collagen I (Col I) cleavage induced by the full-length ColQ1-FL with and without inhibitors to investigate whether the compounds have an anticollagenolytic effect on the collagenase’s natural substrate. After 4 h of co-incubation of ColQ1-FL with Col I, in the absence and presence of inhibitor, Col I breakdown was investigated. On reducing polyacrylamide gels, the hallmark bands of Col I (i.e., α1, α2, and β chains) were clearly visible. Compared to the negative control (no inhibitor), 13 and 27 displayed considerable anti-ColQ1 activity and maintained the chains of Col I, as shown at concentrations above 3 and 0.8 μM, respectively. Below these concentrations, substantial chain disintegration was detected (Figure 5). The diphosphonate derivatives 11, 10, 14, and 15 inhibited the collagenase activity at all concentrations tested (12.5–1.5 μM) (except for 10, which showed inhibition only at 12.5 μM) and protected Col I chains from cleavage (Figure 4). Similar findings were observed for other diphosphonate derivatives 7, 9, and 11 and the hydroxamate derivative 33 (Figure S3). In contrast, severe degradation was visible at all tested concentrations (12.5–1.5 μM) of diphosphonate16 and the hydroxamate derivatives 28, 29, 31, and 32 (Figure S3), indicating their low activity on ColQ1.

Figure 5

Figure 5. Activity of ColQ1 inhibitors against the collagenolytic activity of the full-length (FL) ColQ1. Inhibitors prevented the cleavage of 1 mg/mL of Col I chains (i.e., β, α-1, and α-2). The Bacillus cereus ColQ1-FL (50 ng) was incubated with 1 mg/mL Col I for 3 h, and the degradation was then visualized by 12% SDS-PAGE. Col I: 1 mg/mL Col I without any protease. M (kDa): molecular weight standards, Col I: type I collagen, ColQ1: collagenase Q1.

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These findings are corroborated by the previously determined inhibition data obtained with the peptidolytic assay. This highlights that several of our hydroxamate and diphosphonate compounds are capable in vitro of preventing cleavage of the large, structurally more complex physiological substrate of collagenases, i.e., Col I, which accounts for 80–90% of the collagen in the body. (35) Based on these findings, we next investigated whether this protective effect would also prevail in a more complex cellular setting.

Small-Molecule Inhibitors Reduce Collagenase Activity and Preserve Fibroblast Cell Integrity

Collagenase Release during B. cereus Infection of NHDF Cells

To investigate the potential antivirulence activity of ColQ1 inhibitors, we developed an in vitro infection model using the common connective tissue cell line NHDF in the ECM. The fibroblasts have crucial functions in the ECM, as they synthesize their main components (such as collagen) and maintain their homeostasis. (36) Previous reports revealed that bacillary ColQ1 and ColA have a prominent collagenolytic activity that is greater than or similar to the well-studied clostridial ColG and ColH. (20) Therefore, B. cereus collagenases were used as model proteases to further evaluate the inhibitors’ effects in infection settings.
To establish the model, the release of B. cereus collagenases was investigated to determine the duration needed for the AH187 strain (expresses two collagenases: B7HV61 (sequence identical to ColQ1) and B7HZW5 (ColA)) to secrete considerable amounts of collagenase into the surrounding DMEM medium of NHDF cells. To inspect the release of collagenase, the DMEM medium was collected at various time points and investigated by gelatin zymography. The zymography analysis revealed that the bacteria required at least 4 h to release substantial amounts of collagenases, which increased over time, as seen in Figure S4b. In the zymograms, besides the bands of the full-length ColQ1 and ColA (109 kDa), also truncated isoforms (100–40 kDa) were detected (Figure S4b). This phenomenon has been previously reported for other bacterial collagenases. (19) After 4 h of infection, we could also observe a massive reduction (>50%) in fibrillar collagen in the ECM quantified with the picrosirius red assay (Figure S4d). (37,38) Concomitantly, NHDF cells started to detach and their morphology changed from spindle-shaped to round, as evidenced by light microscopy and SDS-PAGE analysis of the cell lysate (Figure S4a,c). In addition, we monitored the release of lactate dehydrogenase (LDH) (39) into the DMEM to detect cells undergoing cell death. Significant amounts of LDH were excreted after 4 h, and the excretion increased over time (Figure S4e). Studies showed that B. cereus protein complexes hemolysin BL and nonhemolytic enterotoxin induced cytotoxicity and cell detachment, which means not only collagenases may induce cytotoxicity but other toxins also contribute. (40,41) These results suggest that bacterial collagenases may play a role in inducing cellular necrosis. Based on these findings, we chose a time window of 4–6 h for testing the efficacy of collagenase inhibitors in the NHDF infection model.

Collagenase Inhibitors Suppress the Gelatinolytic Activity of B. cereus Collagenases Released in the NHDF Infection Model

We investigated the most potent compounds from our in vitro cleavage assays in the NHDF infection model at concentrations ranging from 0, 25, 50, 100–200 μM. We observed a dose-dependent inhibitory effect on the activity of the secreted collagenases into the supernatant of infected cells detected by gelatin zymography. At 200 μM, 13 completely suppressed the gelatinolytic activity, while for 14 a complete inhibition was already observed at a concentration of 100 μM (Figures 6 and S5). The other diphosphonate derivatives (10, 11, and 15) were also evaluated. Compound 15 also demonstrated a clear dose-dependent inhibitory effect on gelatin turnover (Figure S6), while this was less evident for 10 and 11, which inhibited the collagenase activity toward gelatin only marginally at 100 μM (Figure S6).

Figure 6

Figure 6. Activities of FDA-approved tiludronate disodium and compound 14 on the fibroblast (NHDF) cells infected with Bacillus cereus. (a) The antigelatinolytic activities of compounds tiludronate disodium and 14 against B. cereus collagenases. The DMEM medium of the infected NHDF cells was applied to the zymograms. Clear regions against blue background indicate that gelatin in the gel has been cleaved. (b) The amount of fibrillar collagens maintained by tiludronate disodium and 14 in the infected NHDF cells (highlighted in the yellow background). (c) The cytotoxicity of B. cereus infection (highlighted in the yellow background) in NHDF cells treated with and without tiludronate disodium and 14. Ctrl represents the noninfected cells (gray column) and the infected cells and nontreated with inhibitors (red column). Statistical analysis was performed with one-way ANOVA, and statistical significance was analyzed by the Tukey test. Significance was calculated by comparing nontreated vs treated cells with compounds (mean ± SD, ****p < 0.0001, **p < 0.01, *p ≤ 0.05, ns: nonsignificant). Ctrl: control. M (kDa): molecular weight marker.

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The most promising FDA-approved diphosphonate drugs from the in vitro enzyme assays were also tested. Tiludronate disodium and alendronate sodium could completely inhibit gelatin turnover in the zymography at a concentration of 100 μM and 200 μM, respectively (Figures 6 and S9). Interestingly, the hydroxamate compounds (27, 28, 29, 30, 31, 32, and 33) showed no inhibitory effect on the collagenase in the zymography at all tested concentrations (Figures S11 and S12).

Collagenase Inhibitors Prevent NHDF Cell Detachment and Cleavage of Fibrillar Collagens of the ECM

We further evaluated the effect of the diphosphonate and hydroxamate compounds on cell morphology, the fibrillar collagen content of the ECM, and cell viability in the infection model. As shown in Figures 6 and S5, the diphosphate compounds 13 and 14 showed a dose-dependent effect on the infected NHDF cells. Above a concentration of 25 μM, they were both able to sustain the cells during infection. The cells stayed attached and maintained their spindle shape (Figures S7 and S8), and above 50 μM, we observed a significant reduction in LDH release, indicating higher cell viability. The diphosphonate derivative 15 and the FDA-approved diphosphonate drugs tiludronate disodium and alendronate sodium also showed dose-dependent effects on NHDF morphology, fibrillar ECM collagen content, and LDH release, however, at a concentration of ≥100 μM (Figures 6 and S8–S10). In contrast, diphosphonates 10 and 11 showed a smaller effect at all concentrations tested (except for 10, which resulted in higher cell viability and attachment at 100 μM) (Figures S6 and S8).
We also examined the hydroxamate compounds 27, 28, 29, 31, 32, and 33. Interestingly, none of them showed the expected effects. They were neither able to rescue cell morphology nor able to maintain the collagen content of the ECM. Also, no inhibition of the collagenase in the gelatin zymography was detected at all concentrations tested (Figures S11 and S12). Intrigued by the results of the hydroxamate compounds, we investigated their stability with LC-MS in the conditions of the NHDF infection model to obtain a potential explanation for the observed findings. We used 27 as a representative example for this compound class. The LC-MS spectra (Figure S13) confirmed, however, the stability of 27 under the assay conditions.
To sum up, in the NHDF infection model, the potent diphosphonate compounds from the in vitro enzyme assays were shown to have anticollagenolytic activity by inactivating the bacterial collagenases. Furthermore, they reduced the cytotoxicity induced directly or indirectly by the released collagenases and maintained the spindle-shaped morphology of the cells. In contrast, the hydroxamate-based compounds displayed almost no anticollagenolytic activity in the infection model, despite their inhibitory activity in the in vitro enzyme assays.

Rapid, Slow, or Very Slow Reversibility of Diphosphonate Inhibitors Depends on Target Collagenase

Next, we compared the mechanism inhibition of the diphosphonate and the hydroxamate compounds to the bacterial collagenases. For this purpose, we performed rapid dilution assays to test the reversibility of compound inhibition with ColA-CU and ColQ1-CU from B. cereus and ColH-PD and ColG-CU from C. histolyticum. (36) Upon rapid dilution, rapidly reversible inhibitors quickly dissociate from the enzyme and progress curves similar to the uninhibited control are observed, while irreversible or very slowly dissociating inhibitors remain bound to the enzyme, which only very gradually recovers activity.
Intriguingly, as demonstrated in Figure S14, we observed clear differences between the compound classes. While hydroxamates 27 and 33 behaved toward both bacillary and clostridial collagenases like rapidly reversible inhibitors─as expected from active-site directed competitive inhibitors─the diphosphonate compounds showed a more varied response. In the case of ColQ1-CU, both 13 and 15 displayed progress curves with approximately less than 9% residual activity, which is typical for irreversible or very slowly dissociating inhibitors. (36) Therefore, we examined ColQ1-CU treated overnight with 13 by mass spectrometry of the intact protein and could confirm that the compound did not result in a covalent modification (Figure S15). This leads to the conclusion that 13 must be a very slowly dissociating reversible binder of ColQ1. In the case of ColA-CU, the rapid dilution assay revealed only a minimal inhibition in the presence of 13, but 15 displayed a progress curve typical for slowly dissociating inhibitors. In the case of ColH, the effect of 13 and 15 was reversed compared to ColA, and in the case of ColG, both diphosphonates behaved like rapidly reversible inhibitors (Figure S14).
Slowly and very slowly dissociating inhibition are interesting mechanisms for reducing enzyme activity, as they increase the lifetime of the enzyme–inhibitor complex. Its advantages appear in the latter phases of drug development when pharmacological properties (i.e., dosing interval and patient safety) need to be optimized. (36,37) We speculate that the differential behavior observed in the infection models between the diphosphonate and hydroxamate compounds is caused by their different dissociation properties, which results in different enzyme–inhibitor complex lifetimes. The exact mechanism underlying the slow dissociation behavior, however, remains currently elusive, as we could not get a crystal structure of a diphosphonate inhibitor complex with ColA or ColQ1. Other reasons could also be related to the limited activity of the hydroxamate in the infection experiments. For instance, the accumulation of the hydroxamates inside the infected cells or binding to the large substrate of collagenase (i.e., collagen), which subsequently results in a reduced concentration available to bind with the extracellular bacterial collagenases.

Small-Molecule Inhibitors Reduce Collagenase Activity and Maintain Epithelial Cell Integrity

Collagenase Inhibitors Maintain the TEER of MDCK II Cells

Epithelial cells form intercellular tight junctions establishing a sealed epithelium to control the diffusion of membrane proteins, the uptake of small molecules, and protect the body from hazardous substances. (42,43)B. cereus-mediated cell detachment severely destroys the epithelial barrier function, allowing bacterial access to deeper areas of the tissue. To quantify the effect on the epithelial barrier function, we investigated selected compounds regarding their effect on the transepithelial electrical resistance (TEER) of polarized MDCK II cells. We treated MDCK II cells with our target compounds followed by the infection with the B. cereus AH187 or coincubation with the supernatant from AH187 strain culture, which contains ColQ1 and ColA. In comparison to the control without inhibitor, the TEER of the infected MDCK cells (Figures 7 and S16) was maintained with compounds 14, 15, tiludronate disodium, and alendronate sodium at 100 μM. On the other hand, compounds 13 and 10 failed to sustain the TEER of the infected cells at 200 μM (Figure S16) but they retain the TEER of the challenged cells with 50% (v/v) supernatant (Figure 7b).

Figure 7

Figure 7. Change in the transepithelial electrical resistance (TEER) of the Madin–Darby canine kidney II (MDCK II) cells challenged with Bacillus cereus bacteria or 50% (v/v) culture supernatant and treated with or without collagenase inhibitors. (a) 14 and 15 preserve the TEER value of the MDCK II infected with B. cereus compared with the nontreated conditions with inhibitor. (b) Compounds 11, 13, 15, and tiludronate disodium maintained the TEER of MDCK II cells challenged with B. cereus supernatant. Each curve represents the average ± standard deviation of at least three independent experiments.

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This discrepancy might be due to the lower amounts of collagenases secreted into the supernatant compared to the very high bacterial densities during infection that lead to a high collagenase secretion. This might have disrupted the optimum inhibitor/collagenase ratio and resulted in lower efficacy of some inhibitors. Thus, the effect of some inhibitors might be less in this case. Despite these variations in effect, our findings support the notion that collagenases are involved in attacking epithelial barriers and that our inhibitors can help to preserve the cellular junctions, thereby reducing bacterial invasion. These findings corroborate the theory that collagenases are one of the factors that might be involved in disturbing the TEER of epithelial cells. Our collagenase inhibitors proved their potential to preserve the cell attachment and the junction between them, subsequently reducing bacterial invasion.

Compounds Do Not Interfere with B. cereus Growth

The aim of this work was to develop an antivirulence agent that prevents bacteria from causing damage rather than killing them. (3) Therefore, we tested the compounds on B. cereus growth to rule out antibacterial activity and ensure that the effects shown in the in vitro infection models were not caused by influencing bacterial viability. For this purpose, we selected the most potent compounds and tested them against the AH187 strain. As shown in Table S5, the compounds had no effect on AH187 growth and their minimum inhibitory concentration (MIC50) was >200 or >100 μM. These data support that the antivirulence activity, and not an antibacterial activity, was responsible for the observed effect in the infection experiments.

ColQ1 Inhibitors Maintain the Survival of G. mellonella Larvae

Finally, we evaluated selected compounds in an in vivo infection model. G. mellonella larvae are one of the most frequently used models to evaluate the effectiveness of newly discovered inhibitors and are well established for assessing B. cereus cytotoxicity. (44) We reported previously that treating the G. mellonella larvae with ColQ1 caused death of the larvae. (28) We established B. cereus infection of G. mellonella by injecting AH187 strain into the larvae in the presence or absence of our compounds. The larvae were incubated at 37 °C throughout the experiment, and their survival was monitored twice a day. Three synthesized inhibitors and two FDA-approved drugs were tested. Compounds 13, 14, 15, tiludronate disodium, and alendronate sodium ameliorated the survival of the larvae in a dose-dependent manner when compared to a control where no inhibitor was administered. At 100 μM, compounds 14 and 15 boosted the survival by around 50% and 35%, respectively. At lower concentration (i.e., 25 μM), both had a reduced effect (Figures 8 and S17). The FDA-approved tiludronate disodium and alendronate sodium increased the survival by 40% at 100 and 200 μM, respectively (Figures 8 and S17). A concentration of 200 μM of 13 showed the highest effect, increasing the survival by about 35%, while 50 μM showed the smallest effect and only improved survival by 5% (Figure S17). Overall, the data revealed the protective effect of the collagenase inhibition during B. cereus infection. As a result, these antivirulence compounds may be evaluated as a promising therapeutic agent in the future.

Figure 8

Figure 8. Survival analysis of Galleria mellonella larvae treated with Bacillus cereus AH187 with and without 14 and tiludronate disodium. Each curve represents results of three independent experiments; the statistical difference between groups treated with 100, 50, and 25 μM of compound 14 and B. cereus AH187 and with the group treated only with B. cereus AH187 is p < 0.0001, p = 0.0039, and p = 0.0173, respectively. The statistical difference between groups treated with 100 μM tiludronate disodium and with B. cereus AH187 is p = 0.0032 (log-rank test). The survival rate for the larvae treated with compound 14 and tiludronate disodium in PBS was 100%.

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Conclusions

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To tackle the AMR crisis and the slow discovery of new anti-infectives, non-traditional therapies and FDA-approved drugs repurposing are promising strategies. One potential non-traditional approach is the use of antivirulence agents, which inhibit the pathogenicity factors of bacteria and thus prevent or delay infection without exerting selective pressure. Bacterial collagenases are one of the antivirulence targets that are currently gaining wide attention. In this study, we identified two classes of inhibitors (synthesized and FDA-approved diphosphonates and hydroxamates) that target clostridial and bacillary collagenases. Among these, compounds, 13, 14, 15, tiludronate disodium, and alendronate sodium of the diphosphonate class and 27 and 33 of the hydroxamate class displayed high and broad-spectrum in vitro inhibition of clostridial collagenases ColH and ColG and on bacillary collagenases ColA and ColQ1. Furthermore, the majority of them demonstrated adequate selectivity over human MMPs and other off-targets. These compounds showed no cytotoxicity in four mammalian cell lines. We also studied the biological effects of these compounds in infection models using B. cereus as a representative bacterium-producing collagenase. In this context, we developed fibroblast- and epithelial cell infection models to characterize the effect of B. cereus collagenases and their inhibition. Our findings suggest that the inhibition of B. cereus collagenases by the most potent diphosphonate compounds maintained the fibroblast cell attachment, cell morphology, and cell viability, preserved the fibrillar collagen content, and sustained the TEER of epithelial cells. We also tested the compounds in vivo in the G. mellonella larvae infection model, where they enhanced the survival rate. The hydroxamates did not display any inhibition in the infection models; however, they showed similar potency as diphosphonates in the enzyme cleavage assays. This could be explained by their quick dissociation from the clostridial and bacillary collagenases. In contrast, the most active diphosphonate demonstrated high potency in both enzyme assays and infection models, which might be due to their slow to very slow dissociation from the bacillary collagenases. These findings offer insight into the role of bacterial collagenases in infections and the significance of their inhibition with small-molecule inhibitors and FDA-approved drugs, which might represent a potential treatment strategy in the future.

Materials and Methods

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Chemistry

Chemical names follow the IUPAC nomenclature. Starting materials were purchased from Chempur, Sigma-Aldrich, Acros, Combi-Blocks, or Fluorochem and were used without purification. Column chromatography was performed using the automated flash chromatography system Combiflash Rf+ (Teledyne Isco) equipped with RediSepRf silica columns. The final products were dried in high vacuum. 1H NMR and 13C NMR spectra were measured on a Bruker AM500 spectrometer (at 500 and 125 MHz, respectively) at 300 K and on a Bruker Fourier 300 (at 300 and 75 MHz, respectively) at 300 K. Chemical shifts are reported in δ (parts per million: ppm) by reference to the hydrogenated residues of deuterated solvent as the internal standard: 2.05 ppm (1H NMR), 29.8, and 206.3 ppm (13C NMR) for acetone-d6, 2.50 ppm (1H NMR) and 39.52 ppm (13C NMR) for DMSO-d6. Signals are described as br (broad), s (singlet), d (doublet), t (triplet), dd (doublet of doublets), ddd (doublet of doublet of doublets), dt (doublet of triplets), and m (multiplet). All coupling constants (J) are given in Hertz (Hz). Mass spectrometry was performed on a TSQ Quantum (Thermo Fisher, Dreieich, Germany). The triple quadrupole mass spectrometer was equipped with an electrospray interface (ESI). Purity of compounds was determined by LC-MS using the area percentage method on the UV trace recorded at a wavelength of 254 nm and found to be >95%. The Surveyor-LC-system consisted of a pump, an auto sampler, and a PDA detector. The system was operated by the standard software Xcalibur. An RP C18 NUCLEODUR 100-5 (3 mm) column (Macherey-Nagel GmbH) was used as the stationary phase. All solvents were HPLC grade. In a gradient run, using acetonitrile and water, the percentage of acetonitrile (containing 0.1% trifluoroacetic acid) was increased from an initial concentration of 0% at 0 min to 100% at 13 min and kept at 100% for 2 min. The injection volume was 15 μL, and the flow rate was set to 800 μL/min. MS analysis was carried out at a needle voltage of 3000 V and a capillary temperature of 350 °C. Mass spectra were acquired in positive mode, using the electron spray ionization method, from 100 to 1000 m/z, and UV spectra were recorded at a wavelength of 254 nm and in some cases at 360 nm. High-resolution mass spectrometry (HRMS) measurements were recorded on a SpectraSystems-MSQ LC-MS system (Thermo Fisher).

Experimental Procedures of Diphosphonates

The following compounds were prepared according to previously described procedures: 7a–16a and 7b–14b. (29−31)

General Procedure A: Preparation of 1,4-Dihydro-2,3-quinoxaline-dione Derivatives 7a–14a and 15b

A mixture of 1,2-phenylenediamine derivative (1 equiv) and oxalic acid (1.2 equiv) was refluxed in 4 N HCl (20 mL) for 6 h, cooled to RT, poured over ice, and filtered. The product was washed with water and dried to give the title compound. The product was used in the next step without further purification.

General Procedure B: Preparation of 2,3-Dichloroquinoxaline Derivatives 7b–14b and 15c

A mixture of 1,4-dihydro-2,3-quinoxalinediones 7a–14a and 15b (2 mmol) and POCl3 (10 mL) was stirred at 50 °C in DMF (30 mL) for 2 h, cooled to RT, poured over ice, and filtered. The product was washed with water and dried to give the title compound. The product was used in the next step without further purification.

General Procedure C: Preparation of Diethyl Phosphonate Derivatives 7c–13c, 14d, 15d, and 16b

2,3-Dichloroquinoxaline derivatives 7b–14b, 15c, and 16a (1 equiv) were suspended in triethyl phosphite (10 equiv) and heated to 150 °C in a sealed tube for 18 h. Most of the unreacted triethyl phosphite was evaporated in vacuo, and the resultant oil was purified by column chromatography.

General Procedure D: Preparation of Phosphonic Acid Derivatives 7–16

To a solution of diethyl phosphonate (1 equiv) in dry DCM, bromotrimethylsilane (10 equiv) was added dropwise over a period of 15 min. The reaction mixture was stirred at RT overnight. Then, MeOH was added and stirred at RT for 30 min. Solvents were concentrated in vacuo, and the resultant oil was purified by preparative HPLC.
6-(4-Chlorophenyl)-1,4-dihydroquinoxaline-2,3-dione (15b)
Compound 15b was synthesized according to general procedure A, using 4′-chloro-[1,1′-biphenyl]-3,4-diamine 15a (110 mg, 0.5 mmol) and oxalic acid (54 mg, 0.6 mmol). The product was obtained as a yellow solid (90 mg, 66%). 1H NMR (500 MHz, DMSO-d6) δ: 11.96 (d, J = 10.7 Hz, 2H), 7.57 (d, J = 8.7 Hz, 2H), 7.53 (d, J = 8.7 Hz, 2H), 7.41 (dd, J = 8.5, 1.9 Hz, 1H), 7.37 (d, J = 1.9 Hz, 1H), 7.22 (d, J = 8.5 Hz, 1H). 13C NMR (126 MHz, DMSO-d6) δ: 155.1, 155.5, 138.4, 133.9, 132.1, 129.0, 127.8, 126.6, 126.2, 122.0, 116.4, 113.1. MS (ESI+) m/z 273 [M + H]+.
Tetraethyl Quinoxaline-2,3-diylbis(phosphonate) (7c)
Compound 7c was synthesized according to general procedure C, using 2,3-dichloroquinoxaline 7b (90 mg, 0.45 mmol) and triethyl phosphite (819 μL, 4.5 mmol). The residue was purified by automated column chromatography (Hex/EtOAc = 1/1) to give the desired product (white solid, 129 mg, 71%). 1H NMR (500 MHz, CDCl3) δ 8.12 (dd, J = 6.4, 3.5 Hz, 2H), 7.83 (dd, J = 6.4, 3.4 Hz, 2H), 4.56–3.98 (m, 8H), 1.37 (t, J = 7.1 Hz, 12H). 13C NMR (126 MHz, CDCl3) δ 149.9 (dd, JC-P = 226.4, 30.0 Hz), 140.9–140.1 (m), 132.4, 129.8, 63.9 (t, JC-P = 3.2 Hz), 19.3–9.9 (m). 31P NMR (202 MHz, CDCl3) δ 6.83. MS (ESI+) m/z 403.05 [M + H]+.
2,3-Dichloro-N-(3,4-dichlorophenyl)quinoxaline-6-carboxamide (14c)
2,3-Dichloroquinoxaline-6-carboxylic acid (122 mg, 0.50 mmol) was dissolved in DCM (20 mL). EDC·HCl (191 mg, 1 mmol) was added, followed by 3,4-dichloroaniline (162 mg, 1 mmol). The reaction was stirred at room temperature for 24 h. The crude product was purified using column chromatography DCM to DCM/MeOH 1%. The product was obtained as a white solid (107 mg, 55%). 1H NMR (500 MHz, DMSO) δ 10.87 (s, 1H), 8.71 (s, 1H), 8.39 (d, J = 6.7 Hz, 1H), 8.29–8.13 (m, 2H), 7.74 (dd, J = 70.6, 7.2 Hz, 2H). 13C NMR (126 MHz, DMSO) δ 164.7, 147.0, 146.4, 141.8, 139.8, 139.4, 136.9, 131.4, 131.1, 130.6, 128.8, 127.9, 126.0, 122.0, 120.8. MS (ESI+) m/z 387.96 [M + H]+.
2,3-Dichloro-6-(4-chlorophenyl)quinoxaline (15c)
Compound 15c was synthesized according to general procedure B, using 6-(4-chlorophenyl)-1,4-dihydroquinoxaline-2,3-dione 15b (85 mg, 0.31 mmol) and POCl3 (5 mL). The product was obtained as a white solid (69 mg, 72%). 1H NMR (500 MHz, DMSO) δ 8.38 (d, J = 1.9 Hz, 1H), 8.28 (dd, J = 8.8, 2.0 Hz, 1H), 8.16 (d, J = 8.8 Hz, 1H), 7.94 (d, J = 8.5 Hz, 2H), 7.61 (d, J = 8.5 Hz, 2H). 13C NMR (126 MHz, DMSO) δ 145.6, 145.0, 142.1, 140.8, 139.9, 137.2, 134.2, 131.0, 129.7, 129.6, 128.9, 125.3.
Quinoxaline-2,3-diylbis(phosphonic acid) (7)
Compound 7 was synthesized according to general procedure D, using tetraethyl quinoxaline-2,3-diylbis(phosphonate) 7c (120 mg, 0.29 mmol), bromotrimethylsilane (444 μL, 2.9 mmol), and DCM (15 mL). The reaction was stirred at RT overnight. The crude product was purified using preparative HPLC (CH3CN (HCOOH 0.05%)/H2O (HCOOH 0.05%) = 0.2:9.8 to 10:0). The product was obtained as a white solid (53 mg, 61%). 1H NMR (500 MHz, DMSO) δ 8.23 (dd, J = 6.4, 3.4 Hz, 2H), 8.04 (dd, J = 6.4, 3.4 Hz, 2H). 13C NMR (126 MHz, DMSO) δ 153.1 (dd, JC-P = 203.1, 30.4 Hz), 140.6 (dd, JC-P = 20.1, 4.5 Hz), 133.0, 129.7. 31P NMR (202 MHz, DMSO) δ 5.24. Purity: 100%. HRMS (ESI–) calculated for C8H7N2O6P2 [M – H]− 288.9784, found 288.9785.
(6-Fluoroquinoxaline-2,3-diyl)bis(phosphonic acid) (8)
Compound 8 was synthesized according to general procedure D, using tetraethyl (6-fluoroquinoxaline-2,3-diyl)bis(phosphonate) 8c (120 mg, 0.29 mmol), bromotrimethylsilane (444 μL, 2.9 mmol), and DCM (15 mL). The reaction was stirred at RT overnight. The crude product was purified using preparative HPLC (CH3CN (HCOOH 0.05%)/H2O (HCOOH 0.05%) = 0.2:9.8 to 10:0). The product was obtained as a white solid (63 mg, 72%). 1H NMR (500 MHz, MeOD) δ 8.31 (dd, J = 9.3, 5.7 Hz, 1H), 7.91 (dd, J = 8.9, 2.7 Hz, 1H), 7.85 (td, J = 8.8, 2.8 Hz, 1H). 13C NMR (126 MHz, MeOD) δ 165.6 (d, JC-F = 255.4 Hz), 153.5 (dd, JC-P,C-P = 208.9, 30.7 Hz), 151.9 (ddd, JC-P,C-P = 210.6, 30.4, 3.3 Hz), 143.2 (ddd, JC-P,C-F = 19.1, 13.8, 2.5 Hz), 139.7 (dd, JC-P,C-F = 18.8, 2.2 Hz), 133.4 (d, JC-F = 10.3 Hz), 124.3 (d, JC-F = 26.9 Hz), 113.8 (d, JC-F = 22.1 Hz). 31P NMR (202 MHz, MeOD) δ 6.89 (d, J = 7.1 Hz), 6.66 (d, J = 6.9 Hz). Purity: 96%. HRMS (ESI–) calculated for C8H6FN2O6P2 [M– H]− 306.9690, found 306.9690.
(6-Chloroquinoxaline-2,3-diyl)bis(phosphonic acid) (9)
Compound 9 was synthesized according to general procedure D, using tetraethyl (6-chloroquinoxaline-2,3-diyl)bis(phosphonate) 9c (110 mg, 0.25 mmol), bromotrimethylsilane (326 μL, 2.5 mmol), and DCM (15 mL). The reaction was stirred at RT overnight. The crude product was purified using preparative HPLC (CH3CN (HCOOH 0.05%)/H2O (HCOOH 0.05%) = 0.2:9.8 to 10:0). The product was obtained as a white solid (48 mg, 59%). 1H NMR (500 MHz, DMSO) δ 8.28 (d, J = 2.3 Hz, 1H), 8.24 (d, J = 9.0 Hz, 1H), 8.04 (dd, J = 9.0, 2.3 Hz, 1H). 13C NMR (126 MHz, DMSO) δ 154.3 (dd, JC-P = 201.5, 30.2 Hz), 153.6 (dd, JC-P = 202.3, 30.3 Hz), 140.9 (dd, JC-P = 18.4, 2.6 Hz), 139.3 (dd, JC-P = 18.3, 2.7 Hz), 137.3, 133.5, 131.6, 128.3. 31P NMR (202 MHz, DMSO) δ 4.87 (d, J = 5.3 Hz), 4.72 (d, J = 6.1 Hz). Purity: 95%. HRMS (ESI–) calculated for C8H6ClN2O6P2 [M – H]− 322.9395, found 322.9395.
(6-Bromoquinoxaline-2,3-diyl)bis(phosphonic acid) (10)
Compound 10 was synthesized according to general procedure D, using tetraethyl (6-bromoquinoxaline-2,3-diyl)bis(phosphonate) 10c (80 mg, 0.16 mmol), bromotrimethylsilane (209 μL, 1.6 mmol), and DCM (15 mL). The reaction was stirred at RT overnight. The crude product was purified using preparative HPLC (CH3CN (HCOOH 0.05%)/H2O (HCOOH 0.05%) = 0.2:9.8 to 10:0). The product was obtained as a white solid (46 mg, 75%). 1H NMR (500 MHz, DMSO) δ 8.42 (s, 1H), 8.14 (d, J = 0.8 Hz, 2H). 13C NMR (126 MHz, DMSO) δ 154.4 (dd, JC-P = 200.9, 30.1 Hz), 153.8 (dd, JC-P = 201.7, 30.0 Hz), 141.1 (dd, JC-P = 18.3, 2.4 Hz), 139.5 (dd, JC-P = 18.1, 2.2 Hz), 136.0, 131.6, 131.5, 126.1. 31P NMR (202 MHz, DMSO) δ 4.90 (d, J = 7.1 Hz), 4.71 (d, J = 7.1 Hz). Purity: 100%. HRMS (ESI–) calculated for C8H6BrN2O6P2 [M – H]− 366.8890, found 366.8890.
(6-Methylquinoxaline-2,3-diyl)bis(phosphonic acid) (11)
Compound 11 was synthesized according to general procedure D, using tetraethyl (6-methylquinoxaline-2,3-diyl)bis(phosphonate) 11c (110 mg, 0.26 mmol), bromotrimethylsilane (340 μL, 2.6 mmol), and DCM (15 mL). The reaction was stirred at RT overnight. The crude product was purified using preparative HPLC (CH3CN (HCOOH 0.05%)/H2O (HCOOH 0.05%) = 0.2:9.8 to 10:0). The product was obtained as a white solid (64 mg, 80%). 1H NMR (500 MHz, DMSO) δ 8.10 (d, J = 8.6 Hz, 1H), 7.98 (s, 1H), 7.87 (dd, J = 8.6, 1.7 Hz, 1H), 2.63 (s, 3H). 13C NMR (126 MHz, DMSO) δ 152.9 (dd, JC-P = 203.4, 30.5 Hz), 151.9 (dd, JC-P = 204.2, 30.5 Hz), 143.6, 140.7 (dd, JC-P = 17.4, 1.4 Hz), 139.2 (dd, JC-P = 17.5, 1.4 Hz), 135.2, 129.2, 128.2, 21.9. 31P NMR (202 MHz, DMSO) δ 5.60 (d, J = 5.2 Hz), 5.48 (d, J = 7.0 Hz). Purity: 95%. HRMS (ESI–) calculated for C9H9N2O6P2 [M – H]− 302.9941, found 302.9941.
(6-Methoxyquinoxaline-2,3-diyl)bis(phosphonic acid) (12)
Compound 12 was synthesized according to general procedure D, using tetraethyl (6-methoxyquinoxaline-2,3-diyl)bis(phosphonate) 12c (90 mg, 0.20 mmol), bromotrimethylsilane (260 μL, 2.0 mmol), and DCM (15 mL). The reaction was stirred at RT overnight. The crude product was purified using preparative HPLC (CH3CN (HCOOH 0.05%)/H2O (HCOOH 0.05%) = 0.2:9.8 to 10:0). The product was obtained as a white solid (46 mg, 70%). 1H NMR (500 MHz, DMSO) δ 8.11 (d, J = 9.2 Hz, 1H), 7.66 (dd, J = 9.2, 2.6 Hz, 1H), 7.52 (d, J = 2.4 Hz, 1H), 4.02 (s, 3H). 13C NMR (126 MHz, MeOD) δ 163.1, 152.9 (dd, JC-P = 203.0, 30.3 Hz), 151.9 (dd, JC-P = 204.1, 30.3 Hz), 142.6 (dd, JC-P = 17.1, 1.4 Hz), 139.2 (dd, JC-P = 17.4, 1.4 Hz), 130.3, 126.1, 106.0, 55.3. 31P NMR (202 MHz, DMSO) δ 5.99 (d, J = 4.6 Hz), 5.68 (d, J = 6.0 Hz). Purity: 100%. HRMS (ESI–) calculated for C9H9N2O7P2 [M – H]− 318.9890, found 318.9890.
(6,7-Dichloroquinoxaline-2,3-diyl)bis(phosphonic acid) (13)
Compound 13 was synthesized according to general procedure D, using tetraethyl (6,7-dichloroquinoxaline-2,3-diyl)bis(phosphonate) 13c (80 mg, 0.17 mmol), bromotrimethylsilane (222 μL, 1.7 mmol), and DCM (15 mL). The reaction was stirred at RT overnight. The crude product was purified using preparative HPLC (CH3CN (HCOOH 0.05%)/H2O (HCOOH 0.05%) = 0.2:9.8 to 10:0). The product was obtained as a white solid (39 mg, 65%). 1H NMR (500 MHz, DMSO) δ 8.51 (s, 2H). 13C NMR (126 MHz, DMSO) δ 155.1 (dd, JC-P = 199.4, 29.9 Hz), 139.6 (dd, JC-P = 19.9, 3.9 Hz), 135.58, 130.57. 31P NMR (202 MHz, DMSO) δ 4.49. Purity: 98%. HRMS (ESI–) calculated for C8H5Cl2N2O6P2 [M – H]− 356.9005, found 356.9005.
(6-((3,4-Dichlorophenyl)carbamoyl)quinoxaline-2,3-diyl)bis(phosphonic acid) (14)
Compound 14 was synthesized according to general procedure D, using tetraethyl (6-((3,4-dichlorophenyl)carbamoyl)quinoxaline-2,3-diyl)bis(phosphonate) 14d (100 mg, 0.17 mmol), bromotrimethylsilane (222 μL, 1.7 mmol), and DCM (15 mL). The reaction was stirred at RT overnight. The crude product was purified using preparative HPLC (CH3CN (HCOOH 0.05%)/H2O (HCOOH 0.05%) = 0.2:9.8 to 10:0). The product was obtained as a white solid (43 mg, 53%). 1H NMR (500 MHz, DMSO) δ 10.99 (s, 1H), 8.87 (d, J = 1.8 Hz, 1H), 8.45 (dd, J = 8.8, 1.8 Hz, 1H), 8.33 (d, J = 8.8 Hz, 1H), 8.25 (d, J = 2.4 Hz, 1H), 7.84 (dd, J = 8.8, 2.4 Hz, 1H), 7.68 (d, J = 8.8 Hz, 1H). 13C NMR (126 MHz, DMSO) δ 164.9, 155.6 (dd, JC-P = 70.9, 29.7 Hz), 154.0 (dd, JC-P = 71.9, 30.0 Hz), 141.8 (dd, JC-P = 18.3, 2.7 Hz), 139.9 (dd, JC-P = 18.4, 2.6 Hz), 139.5, 137.3, 131.4, 131.2, 131.1, 130.1, 129.3, 126.0, 122.0, 120.8. 31P NMR (202 MHz, DMSO) δ 4.95 (d, J = 4.8 Hz), 4.71 (d, J = 6.0 Hz). Purity: 98%. HRMS (ESI–) calculated for C15H10Cl2N3O7P2 [M – H]− 475.9376, found 475.9376.
(6-(4-Chlorophenyl)quinoxaline-2,3-diyl)bis(phosphonic acid) (15)
Compound 15 was synthesized according to general procedure D, using tetraethyl (6-(4-chlorophenyl)quinoxaline-2,3-diyl)bis(phosphonate) 15d (70 mg, 0.13 mmol), bromotrimethylsilane (170 μL, 1.3 mmol), and DCM (15 mL). The reaction was stirred at RT overnight. The crude product was purified using preparative HPLC (CH3CN (HCOOH 0.05%)/H2O (HCOOH 0.05%) = 0.2:9.8 to 10:0). The product was obtained as a white solid (24 mg, 44%). 1H NMR (500 MHz, DMSO) δ 8.45 (d, J = 1.9 Hz, 1H), 8.37 (dd, J = 8.8, 2.0 Hz, 1H), 8.29 (d, J = 8.8 Hz, 1H), 8.04–7.96 (m, 2H), 7.67–7.59 (m, 2H). 13C NMR (126 MHz, DMSO) δ 154.4 (dd, JC-P = 88.9, 30.6 Hz), 152.8 (dd, JC-P = 89.0, 29.9 Hz), 142.7, 140.9 (dd, JC-P = 20.3, 5.2 Hz), 140.0 (dd, JC-P = 20.4, 5.1 Hz), 137.4, 134.3, 131.8, 130.3, 129.8, 129.7, 126.5. 31P NMR (202 MHz, DMSO) δ 5.31, 5.27. Purity: 98%. HRMS (ESI–) calculated for C14H10ClN2O6P2 [M – H]− 398.9708, found 398.9707.
(6,7-Dichloro-3-oxo-3,4-dihydroquinoxalin-2-yl)phosphonic Acid (16)
Compound 16 was synthesized according to general procedure D, using diethyl (6,7-dichloro-3-oxo-3,4-dihydroquinoxalin-2-yl)phosphonate 16b (90 mg, 0.25 mmol), bromotrimethylsilane (326 μL, 2.5 mmol), and DCM (15 mL). The reaction was stirred at RT overnight. The crude product was purified using preparative HPLC (CH3CN (HCOOH 0.05%)/H2O (HCOOH 0.05%) = 0.2:9.8 to 10:0). The product was obtained as a white solid (32 mg, 43%). 1H NMR (500 MHz, DMSO) δ 8.10 (s, 1H), 7.48 (s, 1H). 13C NMR (126 MHz, DMSO) δ 159.9 (d, JC-P = 213.5 Hz), 154.0 (d, JC-P = 28.6 Hz), 134.4, 132.9 (d, JC-P = 2.3 Hz), 131.2 (d, JC-P = 24.4 Hz), 130.7, 125.7, 117.2. 31P NMR (202 MHz, DMSO) δ 1.54. Purity: 95%. HRMS (ESI–) calculated for C8H4Cl2N2O4P [M – H]− 292.9291, found 292.9290.

Experimental Procedures of Hydroxamates

General Procedure A2: Monosaponification

Malonate diester (1 equiv) was dissolved in a mixture of ethanol/water (4:1, 0.43–0.47 M), and sodium hydroxide (1.2 equiv) was added. The reaction mixture was stirred at RT overnight. Then, solvents were evaporated under reduced pressure, and the aqueous mixture remaining was diluted with sat. aq. NaHCO3 and washed with CH2Cl2. The aqueous layer was then carefully acidified (pH ∼ 1) with aq. HCl and extracted with CH2Cl2. The combined organic layers were dried over MgSO4, filtered, and concentrated under reduced pressure affording the desired product.

General Procedure B2: Amide Formation

Carboxylic acid (1 equiv) and amine (1.1 equiv) were dissolved in CH2Cl2 (0.21 M). HOBt (0.1 equiv), EDC.HCl (1.5 equiv), and diisopropylethylamine (3 equiv) were then added, and the mixture was stirred at RT overnight. The mixture was then washed with diluted aq. HCl (1 M), sat. aq. NaHCO3, and sat. aq. NaCl. The combined organic layers were dried over MgSO4, filtered, and concentrated under reduced pressure or the residue was finally purified by flash chromatography to give the desired amide.

General Procedure C2: Boc Deprotection

The Boc-protected intermediate was dissolved in a mixture of ethanol and dichloromethane (1:1, 0.08–0.14 M), and the reaction was cooled down to 0 °C before the addition of 4 N HCl in dioxane (0.17–0.28 M). The mixture was stirred at RT overnight. Then, solvents were evaporated to give the desired compound.

General Procedure D2: Diazo Transfer

A suspension of amine (1 equiv), ZnCl2 (0.06 equiv), and K2CO3 (1 equiv) in ethanol (0.8–0.93 M) under an inert atmosphere was cooled down to 0 °C with an ice bath. Separately, diisopropylethylamine (1.1–3.5 equiv) was slowly added to a solution of 1H-imidazole-1-sulfonyl azide; hydrogen chloride (1.2 equiv) was dissolved in ethanol (0.33–0.74 M) under an inert atmosphere (solution A). The azide-containing solution A was immediately added dropwise to the first mixture at 0 °C. Then, the cooling bath was removed, and the white mixture was stirred at RT overnight. The mixture was then cooled down to 0 °C, diluted with water, and carefully acidified to pH = 2 with 1 N aq. HCl. It was finally extracted with ethyl acetate. The combined organic layers were dried over MgSO4, filtered, and concentrated under reduced pressure or the residue was finally purified by flash chromatography to give the desired azide.

General Procedure E2: Aminolysis

To an ester solution (1 equiv) in methanol (0.15–0.19 M) were added aq. hydroxylamine (50% w/w in water, 0.15–0.19 M) and KCN (0.1 equiv). The mixture was stirred overnight. Then, the solvents were removed at RT under reduced pressure and the residue was purified by flash chromatography to afford the desired hydroxamate.

General Procedure F2: Copper-Catalyzed Click Reaction

Azide (1 equiv) and alkyne (1.0 equiv) were dissolved in N,N-dimethylformamide or dioxane (0.05–0.08 M) before the addition of copper sulfate pentahydrate (0.2 equiv) in water (0.1–0.15 M) and sodium ascorbate (0.5 equiv). The resulting mixture was stirred at room temperature overnight. The mixture was then diluted in water and extracted with ethyl acetate. The combined organic layers were dried over MgSO4, filtered, and concentrated under reduced pressure. The residue was finally purified by flash chromatography affording the desired 1,4-triazole.

General Procedure G2: Alkyne Formation

To a phenol solution (1 equiv) in N,N-dimethylformamide (0.2–0.5 M) were added K2CO3 (2 equiv) and propargyl bromide (1.1 equiv). The resulting solution was heated to 60 °C and stirred overnight. The mixture was then diluted in water and extracted three times with ethyl acetate. The organic layers were combined, and solvents were evaporated under reduced pressure affording the desired alkyne.
2-Ethoxycarbonyl-4-methyl-pentanoic Acid (22a)
Compound 22a was synthesized according to the general procedure A2, using diethyl isopropylmalonate (4000 mg, 18.5 mmol) and sodium hydroxide (888 mg, 22.2 mmol) in EtOH/H2O (40 mL, 32:8 v/v) overnight. Compound 22a was obtained as a colorless oil (2400 mg, 68%) and was used in the next step without further purification. 1H NMR (500 MHz, DMSO-d6) δ: 12.80 (br s, 1H), 4.10 (q, J = 7.1 Hz, 2H), 3.36–3.33 (m, 1H), 1.65–1.59 (m, 2H), 1.49 (sep, J = 6.7 Hz, 1H), 1.17 (t, J = 7.1 Hz, 3H), 0.86 (d, J = 6.6 Hz, 3H), 0.86 (d, J = 6.6 Hz, 3H). 13C NMR (126 MHz, DMSO-d6) δ: 170.6, 169.6, 60.7, 49.8, 37.2, 25.7, 22.2, 22.1, 14.0. MS (ESI+): m/z = 189 [M + H]+.
2-Benzyl-3-ethoxy-3-oxo-propanoic Acid (22b)
Compound 22b was synthesized according to the general procedure A2, using diethyl benzylmalonate (1070 mg, 4.3 mmol) and sodium hydroxide (205 mg, 5.1 mmol) in EtOH/H2O (10 mL, 8:2 v/v) overnight. Compound 22b was obtained as a colorless oil (694 mg, 72%) and was used in the next step without further purification. 1H NMR (500 MHz, CDCl3) δ: 10.23 (br s, 1H), 7.36–7.31 (m, 2H), 7.30–7.26 (m, 3H), 4.22 (q, J = 7.1 Hz, 2H), 3.76 (t, J = 7.7 Hz, 1H), 3.30 (dd, J = 7.7 and 2.8 Hz, 2H), 1.26 (t, J = 7.1 Hz, 3H). 13C NMR (126 MHz, CDCl3) δ: 174.3, 168.9, 137.4, 128.9 (2C), 128.7 (2C), 127.1, 62.0, 53.6, 34.9, 14.1. MS (ESI+): m/z = no ionization [M + H]+.
Ethyl 2-[2-(tert-Butoxycarbonylamino)ethylcarbamoyl]-4-methyl-pentanoate (23a)
Compound 23a was synthesized according to the general procedure B2, using carboxylic acid 22a (2000 mg, 10.6 mmol), tert-butyl N-(2-aminoethyl)carbamate (1876 mg, 11.6 mmol), HOBt (162.8 mg, 1.0 mmol), EDC.HCl (3060 mg, 16.0 mmol), and diisopropylethylamine (5560 μL, 31.8 mmol) in CH2Cl2 (50 mL) overnight. The crude product was purified by flash chromatography on silica gel (cHex to cHex/EtOAc 6:4) affording compound 23a as a yellowish solid (1868 mg, 53%). 1H NMR (DMSO-d6) δ: 8.13 (t, J = 5.3 Hz, 1H), 6.71 (t, J = 5.4 Hz, 1H), 4.10–4.01 (m, 2H), 3.29–3.26 (m, 1H), 3.15–3.01 (m, 2H), 2.98–2.95 (m, 2H), 1.64–1.53 (m, 2H), 1.45–1.40 (m, 1H), 1.37 (s, 9H), 1.15 (t, J = 7.1 Hz, 3H), 0.85 (d, J = 6.6 Hz, 3H), 0.84 (d, J = 6.6 Hz, 3H). 13C NMR (DMSO-d6) δ: 170.0, 168.4, 155.6, 77.7, 60.4, 50.2, 39.52, 38.8, 37.4, 28.2 (3C), 25.5, 22.6, 22.0, 14.0. MS (ESI+): m/z = 331 [M + H]+, 275 [M–tBu+H]+, 231 [M–Boc+H]+.
2-[(2-Ethoxycarbonyl-4-methyl-pentanoyl)amino]ethylammonium Chloride (24a)
Compound 24a was synthesized according to the general procedure C2, using the Boc-protected intermediate 23a (1855 mg, 5.6 mmol) and 4 N HCl in dioxane (20 mL) in a mixture of CH2Cl2/EtOH (40 mL, 20:20 v/v) overnight. Compound 24a was obtained as a colorless oil (1500 mg, quant. yield) and was used in the next step without further purification. 1H NMR (300 MHz, DMSO-d6) δ: 8.53 (t, J = 5.5 Hz, 1H), 8.13 (br s, 3H), 4.09 (dd, J = 7.2 and 1.8 Hz, 1H), 4.04 (dd, J = 7.2 and 1.8 Hz, 1H), 3.35–3.28 (m, 3H), 2.89–2.77 (m, 2H), 1.65–1.59 (m, 2H), 1.44 (sep, J = 6.7 Hz, 1H), 1.16 (t, J = 7.1 Hz, 3H), 0.86 (d, J = 6.5 Hz, 3H), 0.85 (d, J = 6.5 Hz, 3H). 13C NMR (75 MHz, DMSO-d6) δ: 169.9, 168.8, 60.5, 50.2, 38.2, 37.3, 36.6, 25.6, 22.4, 22.2, 14.0. MS (ESI+): m/z = 231 [M + H]+.
Ethyl 2-(2-Azidoethylcarbamoyl)-4-methyl-pentanoate (25a)
Compound 25a was synthesized according to the general procedure D2, using amine 24a (1500 mg, 5.6 mmol), ZnCl2 (46 mg, 0.34 mmol), K2CO3 (778 mg, 5.6 mmol), diisopropylethylamine (3420 μL, 19.6 mmol), and diazo transfer reagent (1415 mg, 6.7 mmol) in EtOH (15 mL). The mixture was stirred at room temperature overnight. The crude product was purified by flash chromatography on silica gel (cHex/EtOAc 9:1 to 6:4) affording compound 25a as a colorless oil (1430 mg, quant. yield). 1H NMR (500 MHz, CDCl3) δ: 6.85–6.81 (m, 1H), 4.23–4.16 (m, 2H), 3.44–3.42 (m, 4H), 3.33–3.30 (m, 1H), 1.83–1.71 (m, 2H), 1.57 (sep, J = 6.7 Hz, 1H), 1.28 (t, J = 7.1 Hz, 3H), 0.93 (d, J = 6.6 Hz, 3H), 0.92 (d, J = 6.6 Hz, 3H). 13C NMR (126 MHz, CDCl3) δ: 172.6, 169.2, 61.7, 51.7, 50.9, 40.4, 39.3, 26.5, 22.6, 22.1, 14.2. MS (ESI+): m/z = 257 [M + H]+.
N-(2-Azidoethyl)-2-(hydroxycarbamoyl)-4-methyl-pentanamide (26a)
Compound 26a was synthesized according to the general procedure E2, using ester 25a (600 mg, 2.3 mmol), KCN (30.5 mg, 0.47 mmol), and aq. NH2OH (12 mL, 50% w/w in water) in MeOH (12 mL) overnight. The crude product was purified by flash chromatography on silica gel (CH2Cl2 to CH2Cl2/MeOH 9:1) affording compound 26a as a white solid (366 mg, 65%). 1H NMR (500 MHz, DMSO-d6) δ: 10.50 (s, 1H), 8.93 (s, 1H), 7.85 (t, J = 5.1 Hz, 1H), 3.36–3.34 (m, 2H), 3.25–3.22 (m, 2H), 3.00–2.97 (m, 1H), 1.64–1.58 (m, 1H), 1.56–1.51 (m, 1H), 1.43 (sep, J = 6.6 Hz, 1H), 0.84 (d, J = 6.4 Hz, 6H). 13C NMR (126 MHz, DMSO-d6) δ: 169.4, 166.6, 49.9, 49.0, 38.6, 38.4, 25.6, 22.6, 22.1. MS (ESI+): m/z = 243 [M + H]+.
N-(2-Prop-2-ynoxyphenyl)acetamide (34a)
Compound 34a was synthesized according to the general procedure G2, using N-(2-hydroxyphenyl)acetamide (150 mg, 0.99 mmol), K2CO3 (274 mg, 1.98 mmol), and propargyl bromide (103 μL, 1.09 mmol) in DMF (5 mL). The resulting solution was heated to 60 °C and stirred overnight. The mixture was then diluted in water and extracted three times with ethyl acetate. Organic layers were combined, and solvents were evaporated under reduced pressure affording compound 34a as a brown solid (182 mg, 96%). 1H NMR (500 MHz, DMSO-d6) δ: 9.13 (s, 1H), 7.91 (d, J = 7.5 Hz, 1H), 7.12–7.11 (m, 1H), 7.06 (t, J = 7.5 Hz, 1H), 6.93 (t, J = 7.5 Hz, 1H), 4.86 (d, J = 2.0 Hz, 2H), 3.60 (t, J = 2.2 Hz, 1H), 2.08 (s, 3H). 13C NMR (126 MHz, DMSO-d6) δ: 168.5, 147.6, 127.9, 124.1, 122.6, 121.0, 112.9, 79.2, 78.6, 56.0, 23.8. MS (ESI+): m/z = 190 [M + H]+.
N-[2-[4-[(2-Acetamidophenoxy)methyl]triazol-1-yl]ethyl]-2-(hydroxycarbamoyl)-4-methyl-pentanamide (27)
Compound 27 was synthesized according to the general procedure F2, using azide 26a (88 mg, 0.36 mmol), alkyne 34a (69 mg, 0.36 mmol), copper (II) sulfate pentahydrate (18.1 mg, 0.07 mmol), and sodium ascorbate (35.8 mg, 0.18 mmol) in DMF (5.5 mL) and H2O (4.5 mL). The mixture was stirred at room temperature overnight. The crude product was purified by preparative HPLC (H2O + 0.05% FA/CAN + 0.05% FA 95:5 to 5:95) affording 27 as a white solid after lyophilization (82 mg, 51%). 1H NMR (500 MHz, DMSO-d6) δ: 10.52 (s, 1H), 9.01 (s, 1H), 8.95 (s, 1H), 8.15 (s, 1H), 7.91–7.90 (m, 1H), 7.84 (t, J = 5.7 Hz, 1H), 7.24–7.23 (m, 1H), 7.07–7.04 (m, 1H), 6.92–6.89 (m, 1H), 5.20 (s, 2H), 4.44 (t, J = 6.1 Hz, 2H), 3.52–3.50 (m, 2H), 2.94 (t, J = 7.7 Hz, 1H), 2.07 (s, 3H), 1.57–1.46 (m, 2H), 1.35 (sep, J = 6.6 Hz, 1H), 0.80 (d, J = 6.6 Hz, 3H), 0.79 (d, J = 6.6 Hz, 3H). 13C NMR (126 MHz, DMSO-d6) δ: 169.4, 168.4, 166.6, 148.5, 142.7, 127.9, 124.8, 124.2, 122.3, 120.8, 113.2, 62.3, 49.0, 48.6, 39.0, 38.4, 25.5, 23.9, 22.4, 22.1. Purity: 100%. HRMS–ESI+ (m/z): calculated for C20H29N6O5 [M + H]+ 433.2199, found 433.2190.
N-[2-[4-[(3-Acetamidophenoxy)methyl]triazol-1-yl]ethyl]-2-(hydroxycarbamoyl)-4-methyl-pentanamide (28)
Compound 28 was synthesized according to the general procedure F2, using azide 26a (55 mg, 0.23 mmol), alkyne 34b (43 mg, 0.23 mmol), copper (II) sulfate pentahydrate (11.3 mg, 0.05 mmol), and sodium ascorbate (22.4 mg, 0.11 mmol) in DMF (3 mL) and H2O (2 mL). The mixture was stirred at RT overnight. The crude product was purified by flash chromatography on silica gel (CH2Cl2 to CH2Cl2/MeOH 9:1) affording 28 as a white solid after lyophilization (22 mg, 22%). 1H NMR (500 MHz, DMSO-d6) δ: 10.50 (s, 1H), 9.91 (s, 1H), 8.94 (s, 1H), 8.13 (s, 1H), 7.85 (t, J = 5.3 Hz, 1H), 7.34 (s, 1H), 7.21–7.18 (m, 1H), 7.12–7.10 (m, 1H), 6.73–6.72 (m, 1H), 5.07 (s, 2H), 4.43 (t, J = 5.2 Hz, 2H), 3.53–3.49 (m, 2H), 2.94 (t, J = 7.6 Hz, 1H), 2.03 (s, 3H), 1.57–1.47 (m, 2H), 1.38–1.30 (m, 1H), 0.81 (d, J = 6.2 Hz, 3H), 0.80 (d, J = 6.2 Hz, 3H). 13C NMR (126 MHz, DMSO-d6) δ: 169.4, 168.3, 166.6, 158.3, 142.5, 140.5, 129.5, 124.7, 111.7, 108.9, 105.8, 61.0, 48.9, 48.6, 39.0, 38.3, 25.5, 24.1, 22.4, 22.2. Purity: 97%. HRMS–ESI+ (m/z): calculated for C20H29N6O5 [M + H]+ 433.2199, found 433.2190.
N-[2-[4-[(4-Acetamidophenoxy)methyl]triazol-1-yl]ethyl]-2-(hydroxycarbamoyl)-4-methyl-pentanamide (29)
Compound 29 was synthesized according to the general procedure F2, using azide 26a (55 mg, 0.23 mmol), alkyne 34c (43 mg, 0.23 mmol), copper (II) sulfate pentahydrate (11.3 mg, 0.05 mmol), and sodium ascorbate (22.4 mg, 0.11 mmol) in DMF (3 mL) and H2O (2 mL). The mixture was stirred at RT overnight. The crude product was purified by flash chromatography on silica gel (CH2Cl2 to CH2Cl2/MeOH 9:1) affording 29 as a white solid after lyophilization (43 mg, 43%). 1H NMR (500 MHz, DMSO-d6) δ: 10.52 (s, 1H), 9.81 (s, 1H), 8.97 (s, 1H), 8.13 (s, 1H), 7.87 (t, J = 5.5 Hz, 1H), 7.48 (d, J = 8.9 Hz, 2H), 6.96 (d, J = 8.9 Hz, 2H), 5.06 (s, 2H), 4.43 (t, J = 5.7 Hz, 2H), 3.52–3.48 (m, 2H), 2.94 (t, J = 7.6 Hz, 1H), 2.00 (s, 3H), 1.56–1.46 (m, 2H), 1.38–1.31 (m, 1H), 0.81 (d, J = 6.3 Hz, 3H), 0.80 (d, J = 6.3 Hz, 3H). 13C NMR (126 MHz, DMSO-d6) δ: 169.5, 167.8, 166.7, 153.9, 142.7, 132.9, 124.8, 120.5 (2C), 114.7 (2C), 61.3, 48.9, 48.7, 39.0, 38.4, 25.5, 23.9, 22.4, 22.2. Purity: 98%. HRMS–ESI+ (m/z): calculated for C20H29N6O5 [M + H]+ 433.2199, found 433.2166.
N-[2-[4-[(2-Fluorophenoxy)methyl]triazol-1-yl]ethyl]-2-(hydroxycarbamoyl)-4-methyl-pentanamide (30)
Compound 30 was synthesized according to the general procedure F2, using azide 26a (70 mg, 0.29 mmol), alkyne 34d (37 μL, 0.29 mmol), copper (II) sulfate pentahydrate (14.4 mg, 0.06 mmol), and sodium ascorbate (28.5 mg, 0.14 mmol) in dioxane (4 mL) and H2O (3 mL). The mixture was stirred at RT overnight. The crude product was purified by flash chromatography on silica gel (CH2Cl2 to CH2Cl2/MeOH 9:1) affording 30 as a white solid after lyophilization (74 mg, 65%). 1H NMR (500 MHz, DMSO-d6) δ: 10.52 (s, 1H), 8.97 (s, 1H), 8.17 (s, 1H), 7.86 (t, J = 5.5 Hz, 1H), 7.38–7.35 (m, 1H), 7.23–7.19 (m, 1H), 7.16–7.13 (m, 1H), 6.97–6.93 (m, 1H), 5.19 (s, 2H), 4.44 (t, J = 5.2 Hz, 2H), 3.53–3.49 (m, 2H), 2.93 (t, J = 7.6 Hz, 1H), 1.56–1.45 (m, 2H), 1.37–1.29 (m, 1H), 0.80 (d, J = 6.4 Hz, 3H), 0.79 (d, J = 6.4 Hz, 3H). 13C NMR (126 MHz, DMSO-d6) δ: 169.5, 166.6, 151.8 (d, J = 243.5 Hz), 146.0 (d, J = 10.6 Hz), 142.1, 125.2, 124.9 (d, J = 3.6 Hz), 121.4 (d, J = 6.0 Hz), 116.1 (d, J = 17.5 Hz), 115.4, 61.9, 48.9, 48.7, 39.0, 38.4, 25.5, 22.4, 22.2. 19F NMR (471 MHz, DMSO-d6) δ: −134.7. Purity: 100%. HRMS–ESI+ (m/z): calculated for C18H25FN5O4 [M + H]+ 394.1890, found 394.1862.
2-(Hydroxycarbamoyl)-4-methyl-N-[2-[4-(phenoxymethyl)triazol-1-yl]ethyl]pentanamide (31)
Compound 31 was synthesized according to the general procedure F2, using azide 26a (70 mg, 0.29 mmol), prop-2-ynoxybenzene (37 μL, 0.29 mmol), copper (II) sulfate pentahydrate (14.4 mg, 0.06 mmol), and sodium ascorbate (28.5 mg, 0.14 mmol) in dioxane (4 mL) and H2O (3 mL). The mixture was stirred at RT overnight. The crude product was purified by flash chromatography on silica gel (CH2Cl2 to CH2Cl2/MeOH 9:1) affording 31 as a white solid after lyophilization (56 mg, 51%). 1H NMR (500 MHz, DMSO-d6) δ: 10.51 (br s, 1H), 8.97 (s, 1H), 8.14 (s, 1H), 7.87 (t, J = 5.5 Hz, 1H), 7.32–7.28 (m, 2H), 7.04–7.02 (m, 2H), 6.96–6.93 (m, 2H), 5.10 (s, 2H), 4.43 (t, J = 5.1 Hz, 2H), 3.52–3.49 (m, 2H), 2.94 (t, J = 7.6 Hz, 1H), 1.57–1.46 (m, 2H), 1.38–1.30 (m, 1H), 0.81 (d, J = 6.4 Hz, 3H), 0.80 (d, J = 6.4 Hz, 3H). 13C NMR (126 MHz, DMSO-d6) δ: 169.5, 166.6, 158.1, 142.6, 129.6 (2C), 124.9, 120.9, 114.6 (2C), 61.0, 48.9, 48.7, 39.0, 38.4, 25.5, 22.4, 22.2. Purity: 100%. HRMS–ESI+ (m/z): calculated for C18H26N5O4 [M + H]+ 376.1985, found 376.1956.
2-(Hydroxycarbamoyl)-4-methyl-N-[2-[4-(phenylsulfanylmethyl)triazol-1 yl]ethyl]pentanamide (32)
Compound 32 was synthesized according to the general procedure F2, using azide 26a (70 mg, 0.29 mmol), prop-2-ynylsulfanylbenzene (43 mg, 0.29 mmol), copper (II) sulfate pentahydrate (14.4 mg, 0.06 mmol), and sodium ascorbate (28.5 mg, 0.14 mmol) in dioxane (4 mL) and H2O (3 mL). The mixture was stirred at RT overnight. The crude product was purified by flash chromatography on silica gel (CH2Cl2 to CH2Cl2/MeOH 95:5) affording 27 as a light-green solid after lyophilization (64 mg, 56%). 1H NMR (500 MHz, DMSO-d6) δ: 10.49 (s, 1H), 8.94 (s, 1H), 7.89 (s, 1H), 7.80 (t, J = 5.9 Hz, 1H), 7.38–7.36 (m, 2H), 7.33–7.30 (m, 2H), 7.20–7.17 (m, 1H), 4.38–4.36 (m, 2H), 4.26 (s, 2H), 3.48–3.44 (m, 2H), 2.93 (t, J = 7.6 Hz, 1H), 1.57–1.45 (m, 2H), 1.40–1.32 (m, 1H), 0.82–0.81 (m, 6H). 13C NMR (126 MHz, DMSO-d6) δ: 169.4, 166.6, 143.2, 136.1, 129.0 (2C), 128.0 (2C), 125.8, 123.5, 48.9, 48.5, 38.9, 38.4, 27.2, 25.5, 22.4, 22.1. Purity: 100%. HRMS–ESI+ (m/z): calculated for C18H26N5O3S [M + H]+ 392.1756, found 392.1727.
N-[2-[4-[(2-Acetamidophenoxy)methyl]triazol-1-yl]ethyl]-2-benzyl-3-(hydroxyamino)-3-oxo-propanamide (33)
Compound 33 was synthesized according to the general procedure F2, using azide 21b (45 mg, 0.1 mmol), alkyne 29a (43 mg, 0.1 mmol), copper (II) sulfate pentahydrate (5 mg, 0.02 mmol), and sodium ascorbate (9.9 mg, 0.05 mmol) in dioxane (2 mL) and H2O (1 mL). The mixture was stirred at RT overnight. The crude product was purified by flash chromatography on silica gel (CH2Cl2 to CH2Cl2/MeOH: 9/1) affording 28 as a white solid after lyophilization (7 mg, 15%). 1H NMR (500 MHz, DMSO-d6) δ: 10.45 (s, 1H), 9.00–8.93 (m, 2H), 8.04–7.91 (m, 3H), 7.24–6.91 (m, 8H), 5.19 (s, 2H), 4.39 (br s, 2H), 3.49 (br s, 2H), 3.21–3.17 (m, 1H), 2.99–2.96 (br s, 2H), 2.06 (s, 3H). 13C NMR (126 MHz, DMSO-d6) δ: 168.7, 168.4, 165.6, 148.5, 142.7, 138.8, 128.7 (2C), 128.2 (2C), 127.9, 126.2, 124.9, 124.2, 122.3, 120.8, 113.2, 62.2, 52.2, 48.6, 39.0, 34.6. Purity: 96%. HRMS–ESI+ (m/z): calculated for C23H27N6O5 [M + H]+ 467.2047, found 467.2013.

Expression and Purification of ColQ1

ColQ1 was produced and purified in its complete length and collagenase unit from B. cereus strain Q1 (Uniprot: B9J3S4; Tyr94-Gly765), as previously described. (20)

In Vitro FRET-Based Proteolytic Assay (ColQ1, ColA, ColG, and ColH)

For all targets, the percent of inhibition and IC50 measurements were carried out as previously described. (20,25,26) Experiments were performed in triplicate, and the results are provided as means ± standard deviation. For the determination of the inhibition constant (Ki), similar assay conditions were chosen. However, nominal final enzyme concentrations of 1 nM ColQ1-CU, 10 nM ColH-PD, 35 nM ColA, and 60 nM ColG were used and the reactions were monitored for 2 min 24 s. Regression analysis was performed using GraphPad Prism v 9.0.0 (GraphPad Software, San Diego, CA). The experiments were performed under first-order conditions ([S0] ≪ KM), which resulted in an approximation of the Kiapp to the true inhibition constant (Ki); therefore, the results are reported as Ki values.

Reversibility Assays by Rapid Dilution

The recovery of enzymatic activity after a rapid large dilution was performed following Copeland, 2013. (32) In short, ColA, ColG, ColH, and ColQ1 were incubated for 30 min at 100-fold the concentration required for the activity assay (i.e., 4.375, 7.500, 1.250, and 0.125 μM, respectively) with a concentration of inhibitor equivalent to 10-fold the IC50. The mixture was then diluted 100-fold into the reaction buffer. The reaction was immediately initiated by the addition of the quenched-fluorescent substrate FS1-1 at a final concentration of 2 μM. The reaction was monitored for 2 min (excitation: 328 nm, emission: 392 nm) at 25 °C in an Infinite M200 plate reader (Tecan, Grödig, Austria).

Mass Spectrometric Analysis of Collagenase–Ligand Interactions

The collagenase subunit of ColQ1 incubated with either Ilomastat, 13, or no inhibitor was investigated by high-performance liquid chromatography coupled to mass spectrometry (HPLC-MS). Prior to HPLC-MS measurements, ColQ1-CU samples were buffer-exchanged to 150 mmol·L–1 ammonium acetate on 3 kDa molecular weight cutoff centrifugal filters (Amicon Ultra 0.5 mL, Merck, Darmstadt, Germany) and reconstituted to a concentration of 0.5 mg·mL–1. ColQ1-CU samples were separated on a capillary HPLC instrument (UltiMate U3000 RSLC, Thermo Fisher Scientific, Germering, Germany) equipped with a Supelco Discovery C18 column (150 × 2.1 mm2 i.d., 3 μm particle size, 300 Å pore size; Sigma-Aldrich, Vienna, Austria). The column was operated at a flow rate of 150 μL·min–1 and a column oven temperature of 70 °C. Using in-line split-loop mode, 7 microliters of sample [0.5 mg mL–1] was injected in triplicate. The separation was carried out employing a gradient of mobile phase A (H2O + 0.10% formic acid (98–100%; Sigma-Aldrich)) and B (acetonitrile (HiPerSolv; VWR chemicals, Radnor, PA) + 0.10% formic acid) as follows: 25.0% B for 5.0 min, 25.0–90.0% B in 20 min, 95.0% B for 5.0 min, and 25.0% B for 10 min. Water (H2O) was purified by a MilliQ Integral 3 system (Merck Millipore, Burlington, MA). Mass spectrometric data was acquired on a Thermo Scientific QExactive benchtop quadrupole-Orbitrap mass spectrometer employing an Ion Max source with a heated electrospray ionization probe (both from Thermo Fisher Scientific, Bremen, Germany) and an MXT715-000─MX Series II Switching Valve (IDEX Health & Science, LLC, Oak Harbor, WA), as described earlier. (45) MS settings were optimized for the ColQ1-CU protein: the resolution was set to 17,500 at m/z 200 in a mass range of m/z 500–2700, the source heater temperature was lowered to 200 °C, and the in-source collision-induced dissociation was increased to 30.0 eV. Data was acquired between minutes 5 and 35. Mass spectra were deconvoluted employing the ReSpect algorithm implemented in the Chromeleon Chromatography Data System, version 7.2.10 (Thermo Fisher Scientific, Waltham, MA).

Selectivity toward Human MMPs

The MMP inhibition assay (Sigma-Aldrich, Saint Louise, MO) was performed as previously reported and in accordance with the manufacturer’s instructions. The fluorescence signals were measured in a CLARIOstar plate reader (BMG LABTECH, Ortenberg, Germany) at a concentration of 100 μM.

Compound Toxicity

Cytotoxicity assays on HepG2, HEK293, NHDF, and MDCK II cells were carried out as described previously. (25)

In Vitro Collagen Cleavage Assay

The experiments were done as described before. (20,28) Briefly, in a buffer containing 250 mM HEPES, 150 mM NaCl, 5 mM CaCl2, 5 μM ZnCl2, pH 7.5, 1 mg/mL acid-soluble type I collagen from bovine tail (Thermo Fischer Scientific) was incubated with 50 ng full-length ColQ1. Compounds were evaluated at various concentrations and incubated together with collagen and ColQ1 at 25 °C for 3 h. After stopping the reaction with 50 mM EDTA, the mixture was loaded on 12% SDS-PAGE gel and stained with colloidal coomassie G-250 dye. (46) Two separate experiments were carried out for each compound.

In Vitro NHDF Infection Model

NHDF cells (1 × 105, Promo Cell C-12302) per well were seeded in 24-well plates (Greiner) with DMEM medium (Gibco) containing 10% (v/v) fetal calf serum (FCS, Gibco) and 1% (v/v) glutamine (Gibco). Prior to the treatment, the cells were cultured at 37 °C for 24 h with 5% CO2. In brain heart infusion (BHI) medium, B. cereus AH187 bacteria were cultivated to a mid-exponential phase. The culture was centrifuged at 4000g for 7 min at RT before being rinsed and diluted in PBS. Then, B. cereus suspension was added to the cells to give an MOI (multiplicity of infection) of 0.03. Before the infection, cells were starved for 1 h with DMEM containing only 1% (v/v) glutamine and no FCS. The cells were infected with B. cereus for 1, 2, 4, and 6 h to examine the kinetics of collagenase release. The DMEM supernatant was collected and harvested cleared by centrifugation at 4000g at 4 °C for 10 min. After the cells were washed, they were lysed using a lysis buffer (20 mM tris pH 7.5, 1 mM EDTA, 100 mM NaCl, 1% Triton 100, 0.5% Na-deoxycholate, 0.1% SDS, 1 × PIT, 1 mM Na3VO4, 1 mM Na-molybdate, 20 mM NaF, 20 mM β-glycerophosphate). Cell debris was removed by centrifugation with 12,000g at 4 °C for 10 min. The DMEM supernatants and cell lysates were stored at −80 °C for further investigation. The cell morphology was monitored with a light microscope (Olympus) using a 20X objective. The kinetic study was performed in three independent experiments. To study the behavior of ColQ1 inhibitors in this model, the experiment was done as stated above with a few changes; the compounds were added to the NHDF cells along with the bacterial suspension, and all were incubated at 37 °C for 5 h and 5% CO2. Two controls were considered in each experiment, uninfected cells, and infected cells without inhibitor. Each experiment was repeated three times in total.

Nonreducing Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE) and Zymography

To perform the zymography, we collected supernatants from the NHDF infection experiments and mixed them with 1% nonreducing loading buffer. They were then electrophoretically separated after loading onto 10% SDS-PAGE gels containing 0.1% gelatin (Roth, Karlsruhe, Germany). Following separation, the gel was incubated at 4 °C for 2 × 30 min with gentle agitation in a renaturation buffer (50 mM HEPES pH 7.5, 200 mM NaCl, 10 mM CaCl2, 10 μM ZnCl2, 2.5% Triton X-100). The gel was then treated in a developing buffer (50 mM HEPES pH 7.5, 200 mM NaCl, 10 mM CaCl2, 10 μM ZnCl2, 0.02% Brij-35) at 37 °C overnight. By staining the gel with 0.1% Coomassie brilliant blue R-250 dye overnight, transparent bands of gelatinolytic activity could be seen. The ChemiDoc XRS+ imaging system (Biorad) was used to scan the gels, and image analysis was performed with Image lab software (Li-Cor Biosciences).

Reducing Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis

The cell lysate was placed onto a 12% SDS-PAGE gel with similar total protein content and stained overnight with Coomassie brilliant blue G-250. The gel was then visualized with ChemiDoc XRS+ imaging system (Biorad), and the signal analysis was exerted with Image lab software.

Picrosirius Red Assay

After infection, the NHDF cells were washed 3 × with PBS and then incubated with Bouin solution (Sigma-Aldrich) at RT for 20 min. The cells were incubated with 0.1% Picrosirius red dye (ab150681) at RT for 2 h. Then, they were washed 1 × with 0.01 N HCl and the matrix was dissolved in 0.01 N NaOH. The absorption was measured at 570 nm using a Tecan Infinite M200 plate reader (Tecan, Grödig, Austria). By dividing the absorbance of each sample by the absorbance of the healthy sample, the relative collagen quantity was determined. For each condition, the experiment was performed three times.

Lactate Dehydrogenase (LDH) Release Assay

The manufacturer’s procedure was followed to measure the released LDH amount in the supernatant of NHDF cells. Briefly, in a 96-well plate (Grenier), 50 μL of the supernatant was combined with 50 μL of substrate. The plate was incubated at RT for 30 min in the dark, and then the reaction was stopped with 50 μL of the stop solution. The absorbance was measured at 490 nm with a Tecan Infinite M200 plate reader (Tecan, Grödig, Austria). The cytotoxicity was calculated relative to the control (no inhibitor).

Stability of 27 with LC-MS

A concentration of 200 μM of compound 27 was incubated with DMEM medium at 37 °C for 5 h and 5% CO2. After the incubation, 2 μL of the compound was transferred to LC-MS vials containing 200 μL of acetonitrile and LC-MS spectra were measured. Three controls were included: (i) 27 in DMEO, (ii) 27 in DMEM without incubation, and (iii) DMEM medium.

Transepithelial Electric Resistance (TEER) Experiment

MDCK II cells were seeded at a density of 3 × 104 cells/mL onto a Millipore hanging cell culture insert at 37 °C for 12 days with 5% CO2. On day 5, the medium was changed. Prior to treatment, the cells were starved for 16 h in FCS-free RPMI medium (Gibco). The bacteria or bacterial-free supernatant was prepared; the bacteria was used at an MOI of 0.03, while 50% (v/v) of supernatant was added. The ColQ1 inhibitors were added into the inner compartment. The TEER of the cells was measured with Millicell ERS-2 (Electrical Resistance System) over time. Three readings were recorded for each well, and the unit area resistance (UAR) was calculated using the mean values of the TEER following the equation
Changes in TEER were normalized to the initial UAR (t = 0), which was set to 100%.

B. cereus Supernatant Production

B. cereus AH187 strain was cultured in FCS-free DMEM medium at 37 °C. The supernatant was harvested by centrifugation and kept at −80 °C until needed. The supernatant was sterile-filtered with a 0.22 μm filter (Greiner).

B. cereus Growth Inhibition Assay

The effect of the compounds on B. cereus growth was carried out by growing the bacteria in BHI medium until the mid-growth phase. Next, the bacterial suspension was diluted until OD600 nm was 0.2 and combined with compounds in a range of 200–3 μM in a 96-well plate. The plates were subsequently incubated at 37 °C for 48 h in a Tecan Infinite M200 plate reader (Tecan, Grödig, Austria). The MIC values presented are the average of at least two independent determinations.

In vivo Galleria Mellonella Infection Model

G. mellonella larvae were purchased from a fishing store. Injections were carried out using an LA120 syringe pump (Landgraf Laborsysteme, Langenhagen, Germany) equipped with 1 mL Injekt-F tuberculin syringes (B. Braun, Melsungen, Germany) and Sterican 0.30 × 12 mm2, 30 G × 1.5 needles (B. Braun). The larvae were divided into five groups depending on their treatment: (i) sterile PBS, (ii) no injection, (iii) only compound, (iv) BC AH187 bacterial suspension, and (v) BC AH187 bacterial suspension with compound. Larvae were incubated at 37 °C for 3 days and inspected twice daily. The total larvae used in all three experiments were 40 larvae per group. When the larvae became black and did not move when simulated with a tweezer, they were deemed dead.

Crystallization, X-ray Data Collection, and Analysis

Crystals of ColG-PD were grown in 0.1 M tris-Bicine pH 8.5, 0.04 M pentaethylene glycol, 0.04 M diethylene glycol, 0.04 M triethylene glycol, 0.04 M tetraethylene glycol, 10% (w/v) poly(ethylene glycol) 20,000, and 20% (v/v) poly(ethylene glycol) 550 monomethyl ether in sitting-vapor diffusion plates. Crystals were soaked with 10 mM 27 and 13 for 2 weeks. The crystals were cryoprotected with MiTeGen LV Cryo-oil (MiTeGen, Ithaca, NY) and immediately flash-frozen in liquid nitrogen. X-ray diffraction data was collected on beamline ID30 at the European Synchrotron Radiation Facility (ESRF) in Grenoble, France. The data sets were indexed, integrated, and scaled using XDS (47) and AIMLESS. (48) Molecular replacement was performed with PHASER (49) using as search model PDB entry 2y6i (ligand and activator domain deleted). Ligand coordinates and restraints were generated using the Grade Web Server. (50) Final structures were obtained using PHENIX (51) together with model building in WinCoot. (52) PyMOL v 4.0.0 was used for figure generation (The PyMOL Molecular Graphics System, Version 4.0.0 Schrödinger, LLC). The final refined structures were deposited in the Protein Data Bank (PDB) as entries 7Z5U and 7ZBV. Data collection and refinement statistics are listed in Table S2.

Statistical Analysis

Graphical data in the manuscript is presented as the means ± SDs. Statistical comparisons are performed by Tukey one-way ANOVA test, which shows significant differences between conditions. Parametric/nonparametric statistical analysis used in the study was based on normality and homogeneity of variance. A value of p ≤ 0.05 was considered statistically significant, while p > 0.05 was considered nonsignificant. The normalized measurements were statistically compared between treated and nontreated groups using the generalized estimating equation model to account for correlated data arising from repeated measures. The survival of G. mellonella was computed using the Kaplan–Meier method, and the log-rank test was applied to calculate the significance of differences between conditions.

Supporting Information

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The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acs.jmedchem.2c00785.

  • Molecular Formula Strings (CSV)

  • Screening results for ColH and ColQ1, inhibition of MMPs and other off-targets by selected compounds, and cytotoxicity and antibacterial activity of selected compounds (Tables S1–S5); inhibition of the screened compounds on ColQ1 and ColH, activity of the compounds on Col I cleavage, on NHDF infected cells, on TEER of MDCK II cells, and on G. mellonella infection model; 1H NMR and 13C NMR and LC-MS spectra for final compounds and data collection and refinement statistics (Figures S4–S17) (PDF)

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Author Information

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  • Corresponding Authors
    • Silja Wessler - Department of Biosciences and Medical Biology, University of Salzburg, Hellbrunner Str. 34, 5020 Salzburg, Austria;  Email: [email protected]
    • Anna K. H. Hirsch - Helmholtz Institute for Pharmaceutical Research Saarland (HIPS), Helmholtz Center for Infection Research (HZI), Campus Building E8.1, 66123 Saarbrücken, Germany;  Department of Pharmacy, Saarland University, Campus Building C2. 3, 66123 Saarbrücken, Germany;  Orcidhttps://orcid.org/0000-0001-8734-4663;  Email: [email protected]
  • Authors
    • Alaa Alhayek - Helmholtz Institute for Pharmaceutical Research Saarland (HIPS), Helmholtz Center for Infection Research (HZI), Campus Building E8.1, 66123 Saarbrücken, Germany;  Department of Pharmacy, Saarland University, Campus Building C2. 3, 66123 Saarbrücken, Germany
    • Ahmed S. Abdelsamie - Helmholtz Institute for Pharmaceutical Research Saarland (HIPS), Helmholtz Center for Infection Research (HZI), Campus Building E8.1, 66123 Saarbrücken, Germany;  Department of Chemistry of Natural and Microbial Products, Institute of Pharmaceutical and Drug Industries Research, National Research Centre, El-Buhouth St., Dokki, 12622 Cairo, Egypt
    • Esther Schönauer - Department of Biosciences and Medical Biology, University of Salzburg, Hellbrunner Str. 34, 5020 Salzburg, Austria
    • Virgyl Camberlein - Helmholtz Institute for Pharmaceutical Research Saarland (HIPS), Helmholtz Center for Infection Research (HZI), Campus Building E8.1, 66123 Saarbrücken, Germany
    • Evelyn Hutterer - Department of Biosciences and Medical Biology, University of Salzburg, Hellbrunner Str. 34, 5020 Salzburg, Austria;  Orcidhttps://orcid.org/0000-0001-8622-4758
    • Gernot Posselt - Department of Biosciences and Medical Biology, University of Salzburg, Hellbrunner Str. 34, 5020 Salzburg, Austria
    • Jamil Serwanja - Department of Biosciences and Medical Biology, University of Salzburg, Hellbrunner Str. 34, 5020 Salzburg, Austria
    • Constantin Blöchl - Department of Biosciences and Medical Biology, University of Salzburg, Hellbrunner Str. 34, 5020 Salzburg, Austria
    • Christian G. Huber - Department of Biosciences and Medical Biology, University of Salzburg, Hellbrunner Str. 34, 5020 Salzburg, Austria
    • Jörg Haupenthal - Helmholtz Institute for Pharmaceutical Research Saarland (HIPS), Helmholtz Center for Infection Research (HZI), Campus Building E8.1, 66123 Saarbrücken, Germany
    • Hans Brandstetter - Department of Biosciences and Medical Biology, University of Salzburg, Hellbrunner Str. 34, 5020 Salzburg, Austria
  • Author Contributions

    A.A. and A.S.A. contributed equally to this work.

  • Funding

    Helmholtz Association’s Initiative and Networking Fund European Research Council (ERC): ERC starting grant 757913. Austrian Science Fund (FWF): P 31843, W 1213, and I 4360. Open Access is funded by the Austrian Science Fund (FWF).

  • Notes
    The authors declare the following competing financial interest(s): The authors declare the following financial interest(s): A.S.A, V.C., J.H., and A.K.H.H are co-inventors on an international patent application WO2022/043322 that incorporates compounds outlined in the library screening in the supplementary information.

    PDB codes: compound 13 (7ZBV) and compound 27 (7Z5U). The authors will release the atomic coordinates upon article publication.

Acknowledgments

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The authors are grateful to Jeannine Jung, Selina Wolter, Isabella Widlroither, Martina Wiesbauer, and Melanie Anja Schwarz for their technical support. The authors acknowledge financial support from the Austrian Science Fund (FWF) grant P 31843 (E.S.), grant W 1213 (C.B. and C.G.H.), and grant I 4360 (S.W.), the European Research Council (ERC starting grant 757913, A.K.H.H.), and by the Helmholtz Association’s Initiative and Networking Fund (A.K.H.H.).

Abbreviations Used

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BHI

brain heart infusion medium

Col I

collagen I

ColQ1 and ColA

collagenases from Bacillus cereus AH187 and Q1 strains

ColH

ColG Clostridium histolyticum collagenases

CU

collagenase unit

DMEM

Dulbecco’s modified Eagle’s medium

ECM

extracellular matrix

FCS

fetal calf serum

FRET

fluorescence resonance energy transfer

HEK293

embryonal kidney cell

HepG2

hepatocellular carcinoma cell

IC50

the half-maximal inhibitory concentration

LDH

lactate dehydrogenase

M (kDa)

molecular weight standards in kilo Dalton

MDCK II

Madin–Darby canine kidney II

MIC

minimum inhibitory concentration

MMPs

human matrix metalloproteases

NHDF

normal human dermal fibroblast cell

PD

peptidase unit

RPMI

Roswell Park Memorial Institute Medium

SDS-PAGE

sodium dodecyl sulfate polyacrylamide

TEER

transepithelial electrical resistance

ZBG

zinc-binding group

References

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    N-Aryl-3-mercaptosuccinimides as Antivirulence Agents Targeting Pseudomonas aeruginosa Elastase and Clostridium Collagenases
    Konstantinovic, Jelena; Yahiaoui, Samir; Alhayek, Alaa; Haupenthal, Joerg; Schoenauer, Esther; Andreas, Anastasia; Kany, Andreas M.; Mueller, Rolf; Koehnke, Jesko; Berger, Fabian K.; Bischoff, Markus; Hartmann, Rolf W.; Brandstetter, Hans; Hirsch, Anna K. H.
    Journal of Medicinal Chemistry (2020), 63 (15), 8359-8368CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)
    In light of the global antimicrobial-resistance crisis, there is an urgent need for novel bacterial targets and antibiotics with novel modes of action. It has been shown that Pseudomonas aeruginosa elastase (LasB) and Clostridium histolyticum (Hathewaya histolytica) collagenase (ColH) play a significant role in the infection process and thereby represent promising antivirulence targets. Here, we report novel N-aryl-3-mercaptosuccinimide inhibitors that target both LasB and ColH, displaying potent activities in vitro and high selectivity for the bacterial over human metalloproteases. Addnl., the inhibitors demonstrate no signs of cytotoxicity against selected human cell lines and in a zebrafish embryo toxicity model. Furthermore, the most active ColH inhibitor shows a significant redn. of collagen degrdn. in an ex vivo pig-skin model.
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    Voos, K.; Schönauer, E.; Alhayek, A.; Haupenthal, J.; Andreas, A.; Müller, R.; Hartmann, R. W.; Brandstetter, H.; Hirsch, A. K. H.; Ducho, C. Phosphonate as a Stable Zinc-Binding Group for “Pathoblocker” Inhibitors of Clostridial Collagenase H (ColH). ChemMedChem 2021, 1– 12,  DOI: 10.1002/cmdc.202000994
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    Alhayek, A.; Khan, E. S.; Schönauer, E.; Däinghaus, T.; Shafiei, R.; Voos, K.; Han, M. K. L.; Ducho, C.; Posselt, G.; Wessler, S.; Brandstetter, H.; Haupenthal, J.; del Campo, A.; Hirsch, A. K. H. Inhibition of Collagenase Q1 of Bacillus Cereus as a Novel Antivirulence Strategy for the Treatment of Skin-Wound Infections. Adv. Ther. 2022, 2100222  DOI: 10.1002/adtp.202100222
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    Inhibition of Collagenase Q1 of Bacillus cereus as a Novel Antivirulence Strategy for the Treatment of Skin-Wound Infections
    Alhayek, Alaa; Khan, Essak S.; Schonauer, Esther; Dainghaus, Tobias; Shafiei, Roya; Voos, Katrin; Han, Mitchell K. L.; Ducho, Christian; Posselt, Gernot; Wessler, Silja; Brandstetter, Hans; Haupenthal, Jorg; del Campo, Aranzazu; Hirsch, Anna K. H.
    Advanced Therapeutics (Weinheim, Germany) (2022), 5 (3), 2100222CODEN: ATWGAP; ISSN:2366-3987. (Wiley-VCH Verlag GmbH & Co. KGaA)
    Despite the progress in surgical techniques and antibiotic prophylaxis, opportunistic wound infections with Bacillus cereus remain a public health problem. Secreted toxins are one of the main factors contributing to B. cereus pathogenicity. A promising strategy to treat such infections is to target these toxins and not the bacteria. Although the exoenzymes produced by B. cereus are thoroughly investigated, little is known about the role of B. cereus collagenases in wound infections. In this report, the collagenolytic activity of secreted collagenases (Col) is characterized in the B. cereus culture supernatant (csn) and its isolated recombinantly produced ColQ1 is characterized. The data reveals that ColQ1 causes damage on dermal collagen (COL). This results in gaps in the tissue, which might facilitate the spread of bacteria. The importance of B. cereus collagenases is also demonstrated in disease promotion using two inhibitors. Compd. 2 shows high efficacy in peptidolytic, gelatinolytic, and COL degrdn. assays. It also preserves the fibrillar COLs in skin tissue challenged with ColQ1, as well as the viability of skin cells treated with B. cereus csn. A Galleria mellonella model highlights the significance of collagenase inhibition in vivo.
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    Zhang, P.-M.; Li, Y.-W.; Zhou, J.; Gan, L.-L.; Chen, Y.-J.; Gan, Z.-J.; Yu, Y. A One-Pot Facile Synthesis of 2,3-Dihydroxyquinoxaline and 2,3-Dichloroquinoxaline Derivatives Using Silica Gel as an Efficient Catalyst. J. Heterocycl. Chem. 2018, 55, 1809– 1814,  DOI: 10.1002/jhet.3224
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    A One-pot Facile Synthesis of 2,3-Dihydroxyquinoxaline and 2,3-Dichloroquinoxaline Derivatives Using Silica Gel as an Efficient Catalyst
    Zhang, Pei-Ming; Li, Yao-Wei; Zhou, Jing; Gan, Lin-Ling; Chen, Yong-Jie; Gan, Zong-Jie; Yu, Yu
    Journal of Heterocyclic Chemistry (2018), 55 (7), 1809-1814CODEN: JHTCAD; ISSN:1943-5193. (Wiley-Blackwell)
    An efficient one-pot reaction has been developed for the synthesis of 2,3-dichloroquinoxaline derivs. I (R1 = H, H3CO, Cl, etc.; R2 = H, F, CH3, etc.; R3 = H, CH3; R1R2 = CH=CH-CH=CH). The reaction was performed in two steps via a silica gel catalyzed tandem process from o-phenylenediamines 2-H2N-3-R3-4-R1-5-R2C6HNH2 and oxalic acid, followed by addn. of phosphorus oxychloride (POCl3). A variety of 2,3-dichloroquinoxalines I have been obtained in good to excellent overall yields.
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    Yang, Y.; Zhang, S.; Wu, B.; Ma, M.; Chen, X.; Qin, X.; He, M.; Hussain, S.; Jing, C.; Ma, B.; Zhu, C. An Efficient Synthesis of Quinoxalinone Derivatives as Potent Inhibitors of Aldose Reductase. ChemMedChem 2012, 7, 823– 835,  DOI: 10.1002/cmdc.201200054
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    An Efficient Synthesis of Quinoxalinone Derivatives as Potent Inhibitors of Aldose Reductase
    Yang, Yanchun; Zhang, Shuzhen; Wu, Bobin; Ma, Mingming; Chen, Xin; Qin, Xiangyu; He, Minlan; Hussain, Saghir; Jing, Chaojun; Ma, Bing; Zhu, Changjin
    ChemMedChem (2012), 7 (5), 823-835CODEN: CHEMGX; ISSN:1860-7179. (Wiley-VCH Verlag GmbH & Co. KGaA)
    A novel and facile synthesis of quinoxalinone derivs. was developed in which a wide range of 3-chloroquinoxalin-2(1H)-ones as key intermediates can be generated chemo- and regioselectively in good yields from corresponding quinoxaline-2,3(1H,4H)-diones. This new protocol is arguably superior, as it allows the design and prepn. of a variety of bioactive quinoxaline-based compds., which are particularly effective in the treatment of diabetes and its complications. Through this procedure, a new class of quinoxalinone-based aldose reductase inhibitors were synthesized successfully. Most of the inhibitors, with an N1-acetic acid head group and a substituted C3-phenoxy side chain, proved to be potent and selective. Their IC50 values ranged from 11.4 to 74.8 nM. Among them, 2-(3-(4-bromophenoxy)-7-fluoro-2-oxoquinoxalin-1(2H)-yl)acetic acid and 2-(6-bromo-3-(4-bromophenoxy)-2-oxoquinoxalin-1(2H)-yl)acetic acid were the most active. Structure-activity relationship and mol. docking studies highlighted the importance of the ether spacer in the C3-phenoxy side chains, and provided clear guidance on the contribution of substitutions both at the core structure and the side chain to activity.
  31. 31
    Rangarajan, M.; Kim, J. S.; Sim, S.-P.; Liu, A.; Liu, L. F.; LaVoie, E. J. Topoisomerase I Inhibition and Cytotoxicity of 5-Bromo- and 5-Phenylterbenzimidazoles. Bioorg. Med. Chem. 2000, 8, 2591– 2600,  DOI: 10.1016/S0968-0896(00)00188-7
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    Topoisomerase I inhibition and cytotoxicity of 5-bromo- and 5-phenylterbenzimidazoles
    Rangarajan, Meera; Kim, Jung Sun; Sim, Sai-Peng; Liu, Angela; Liu, Leroy F.; LaVoie, Edmond J.
    Bioorganic & Medicinal Chemistry (2000), 8 (11), 2591-2600CODEN: BMECEP; ISSN:0968-0896. (Elsevier Science Ltd.)
    Topoisomerase I is an enzyme that is essential for maintaining the three-dimensional structure of DNA during the processes of transcription, translation and mitosis. With the introduction of new clin. agents that are effective in poisoning topoisomerase I, this enzyme has proved to be an attractive mol. target in the development of anticancer drugs. Several terbenzimidazoles have been identified as potent topoisomerase I poisons. Structure-activity data on various terbenzimidazoles have revealed that the presence of lipophilic substituents at the 5-position of various terbenzimidazoles correlates with enhanced cytotoxicity. While the effect of having substituents at both the 5- and 6-positions had not been evaluated, previous studies did indicate that the presence of a fused benzo-ring at the 5,6-position results in a significant decrease in topoisomerase I poisoning activity and cytotoxicity. In the present study we investigated whether substituents at both the 5- and 6-positions of varied terbenzimidazoles would allow for retention of topo I poisoning activity. The 6-bromo, 6-methoxy, or 6-Ph derivs. of both 5-bromo- and 5-phenylterbenzimidazole were synthesized and evaluated for topo I poisoning activity, as well as their cytotoxicity toward human lymphoblastoma cells. The data indicate that such derivs. do retain similar topo I poisoning activity and possess cytotoxicity equiv. to either 5-bromo- or 5-phenylterbenzimidazole. Significant enhancement in the topoisomerase I poisoning activity and cytotoxicity of 5-phenylterbenzimidazole is obsd. when the 2"-position is substituted with either a chloro or trifluoromethyl substituent. The influence of such substituents on the biol. activity of 5,6-dibromoterbenzimidazole (I) was also explored. In the case of either 2"-chloro-5,6-dibromoterbenzimidazole or 2"-trifluoromethyl-5,6-dibromoterbenzimidazole (II), topoisomerase I poisoning was not enhanced relative to I. While cytotoxicity toward RPMI 8402 was also not significantly affected comparative studies performed against several solid human tumor cell lines did reveal a significant increase in cytotoxicity obsd. for II as compared to I.
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    Lozano-Calderon, S. A.; Colman, M. W.; Raskin, K. A.; Hornicek, F. J.; Gebhardt, M. Use of Bisphosphonates in Orthopedic Surgery. Orthop. Clin. North Am. 2014, 45, 403– 416,  DOI: 10.1016/j.ocl.2014.03.006
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    Use of bisphosphonates in orthopedic surgery: pearls and pitfalls
    Lozano-Calderon Santiago A; Colman Matthew W; Raskin Kevin A; Hornicek Francis J; Gebhardt Mark
    The Orthopedic clinics of North America (2014), 45 (3), 403-16 ISSN:.
    Bisphosphonates are medications known to decrease bone resorption by inhibiting osteoclastic activity. They are the first-line therapy for the treatment of osteoporosis because a significant body of literature has proved their efficacy in reducing the risk of fracture in the hip, spine and other nonvertebral osseous sites. In addition, the use of bisphosphonates has significantly decreased morbidity and increased survival, and they have also proved to be cost-effective. Unexpected adverse effects have been reported recently, but the benefit of bisphosphonates use outweighs the risks. This article reviews the current use of bisphosphonates in orthopedic surgery.
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    Gooz, M. ADAM-17: The Enzyme That Does It All. Crit. Rev. Biochem. Mol. Biol. 2010, 45, 146– 169,  DOI: 10.3109/10409231003628015
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    ADAM-17: the enzyme that does it all
    Gooz, Monika
    Critical Reviews in Biochemistry and Molecular Biology (2010), 45 (2), 146-169CODEN: CRBBEJ; ISSN:1040-9238. (Informa Healthcare)
    A review. This review focuses on the role of ADAM-17 in disease. Since its debut as the tumor necrosis factor converting enzyme (TACE), ADAM-17 has been reported to be an indispensable regulator of almost every cellular event from proliferation to migration. The central role of ADAM-17 in cell regulation is rooted in its diverse array of substrates: cytokines, growth factors, and their receptors as well as adhesion mols. are activated or inactivated by their cleavage with ADAM-17. It is therefore not surprising that ADAM-17 is implicated in numerous human diseases including cancer, heart disease, diabetes, rheumatoid arthritis, kidney fibrosis, Alzheimer's disease, and is a promising target for future treatments. The specific role of ADAM-17 in the pathophysiol. of these diseases is very complex and depends on the cellular context. To exploit the therapeutic potential of ADAM-17, it is important to understand how its activity is regulated and how specific organs and cells can be targeted to inactivate or activate the enzyme.
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    Ropero, S.; Esteller, M. The Role of Histone Deacetylases (HDACs) in Human Cancer. Mol. Oncol. 2007, 1, 19– 25,  DOI: 10.1016/j.molonc.2007.01.001
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    The role of histone deacetylases (HDACs) in human cancer
    Ropero, Santiago; Esteller, Manel
    Molecular Oncology (2007), 1 (1), 19-25CODEN: MOONC3; ISSN:1574-7891. (Elsevier B.V.)
    A review. The balance of histone acetylation and deacetylation is an epigenetic layer with a crit. role in the regulation of gene expression. Histone acetylation induced by histone acetyl transferases (HATs) is assocd. with gene transcription, while histone hypoacetylation induced by histone deacetylase (HDAC) activity is assocd. with gene silencing. Altered expression and mutations of genes that encode HDACs have been linked to tumor development since they both induce the aberrant transcription of key genes regulating important cellular functions such as cell proliferation, cell-cycle regulation and apoptosis. Thus, HDACs are among the most promising therapeutic targets for cancer treatment, and they have inspired researchers to study and develop HDAC inhibitors.
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    Gelse, K.; Pöschl, E.; Aigner, T. Collagens - Structure, Function, and Biosynthesis. Adv. Drug Deliv. Rev. 2003, 55, 1531– 1546,  DOI: 10.1016/j.addr.2003.08.002
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    Collagens - structure, function, and biosynthesis
    Gelse, K.; Poschl, E.; Aigner, T.
    Advanced Drug Delivery Reviews (2003), 55 (12), 1531-1546CODEN: ADDREP; ISSN:0169-409X. (Elsevier Science B.V.)
    A review. The extracellular matrix represents a complex alloy of variable members of diverse protein families defining structural integrity and various physiol. functions. The most abundant family is the collagens with >20 different collagen types identified so far. Collagens are centrally involved in the formation of fibrillar and microfibrillar networks of the extracellular matrix, basement membranes as well as other structures of the extracellular matrix. This review focuses on the distribution and function of various collagen types in different tissues. It introduces their basic structural subunits and points out major steps in the biosynthesis and supramol. processing of fibrillar collagens as prototypical members of this protein family. A final outlook indicates the importance of different collagen types not only for the understanding of collagen-related diseases, but also as a basis for the therapeutic use of members of this protein family.
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    Healing, W. The Fibroblast. Proc. R. Soc. Med. 1967, 60, 778– 782,  DOI: 10.1177/003591576706000829
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    Lattouf, R.; Younes, R.; Lutomski, D.; Naaman, N.; Godeau, G.; Senni, K.; Changotade, S. Picrosirius Red Staining: A Useful Tool to Appraise Collagen Networks in Normal and Pathological Tissues. J. Histochem. Cytochem. 2014, 62, 751– 758,  DOI: 10.1369/0022155414545787
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    Picrosirius red staining: a useful tool to appraise collagen networks in normal and pathological tissues
    Lattouf, Raed; Younes, Ronald; Lutomski, Didier; Naaman, Nada; Godeau, Gaston; Senni, Karim; Changotade, Sylvie
    Journal of Histochemistry and Cytochemistry (2014), 62 (10), 751-758, 8 pp.CODEN: JHCYAS; ISSN:0022-1554. (Sage Publications)
    Specific staining of the extracellular matrix components is esp. helpful in studying tissue remodeling, particularly in the case of connective tissue pathologies. As developed by Junqueira and colleagues in 1979, specific staining by Picrosirius red is one of the most important stains to study collagen networks in different tissues. Under polarized light, collagen bundles appear green, red or yellow, and are easily differentiated from the black background, thus allowing for quant. morphometric anal. As Junqueira and colleagues point out, many studies use color staining to differentiate collagen bundles and to specify collagen types, yet other studies report that polarized colors only reflect fiber thickness and packing. Using a simple histol. example, our study illustrates the inability of Picrosirius red staining to differentiate collagen types, since the absorbed amt. of polarized light by this dye strictly depends on the orientation of the collagen bundles.
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    Junqueira, L. C. U.; Cossermelli, W.; Brentani, R. Differential Staining of Collagens Type I, II and III by Sirius Red and Polarization Microscopy. Arch. Histol. Jpn. 1978, 41, 267– 274,  DOI: 10.1679/aohc1950.41.267
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    Differential staining of collagens type I, II and III by Sirius Red and polarization microscopy
    Junqueira L C; Cossermelli W; Brentani R
    Archivum histologicum Japonicum = Nihon soshikigaku kiroku (1978), 41 (3), 267-74 ISSN:0004-0681.
    Organs of fish, amphibian, reptile, bird and mammals when stained by Sirius Red and studied with polarization microscopy present different colors in regions where collagens I, II and III have been described. Collagen type I presented a yellow, orange or red color while collagen type III appeared green. Collagen type II, present in cartilage and chondrosarcoma showed a variable color according to the tissue and the species. Its color and morphology however always permitted its clear distinction from collagens type I and type III.
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    Das, C.; Lucia MS, H. K.; T, J. Quantification of Lactate Dehydrogenase for Cell Viability Testing Using Cell Lines and Primary Cultured Astrocytes. Physiol. Behav. 2017, 176, 139– 148,  DOI: 10.1002/cptx.21.Quantification
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    Lindbäck, T.; Hardy, S. P.; Dietrich, R.; Sødring, M.; Didier, A.; Moravek, M.; Fagerlund, A.; Bock, S.; Nielsen, C.; Casteel, M.; Granum, P. E.; Märtlbauer, E. Cytotoxicity of the Bacillus Cereus Nhe Enterotoxin Requires Specific Binding Order of Its Three Exoprotein Components. Infect. Immun. 2010, 78, 3813– 3821,  DOI: 10.1128/IAI.00247-10
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    Cytotoxicity of the Bacillus cereus Nhe enterotoxin requires specific binding order of its three exoprotein components
    Lindback, Toril; Hardy, Simon P.; Dietrich, Richard; Soedring, Marianne; Didier, Andrea; Moravek, Maximilian; Fagerlund, Annette; Bock, Stefanie; Nielsen, Carina; Casteel, Maximilian; Granum, Per Einar; Martlbauer, Erwin
    Infection and Immunity (2010), 78 (9), 3813-3821CODEN: INFIBR; ISSN:0019-9567. (American Society for Microbiology)
    This study focuses on the interaction of the three components of the Bacillus cereus Nhe enterotoxin with particular emphasis on the functional roles of NheB and NheC. The results demonstrated that both NheB and NheC were able to bind to Vero cells directly while NheA lacked this ability. It was also shown that Nhe-induced cytotoxicity required a specific binding order of the individual components whereby the presence of NheC in the priming step as well as the presence of NheA in the final incubation step was mandatory. Priming of cells with NheB alone and addn. of NheA plus NheC in the second step failed to induce toxic effects. Furthermore, in soln., excess NheC inhibited binding of NheB to Vero cells, whereas priming of cells with excess NheC resulted in full toxicity if unbound NheC was removed before addn. of NheB. By using mutated NheC proteins where the two cysteine residues in the predicted β-tongue were replaced with glycine (NheCcys-) or where the entire hydrophobic stretch was deleted (NheChr-), the predicted hydrophobic β-tongue of NheC was found essential for binding to cell membranes but not for interaction with NheB in soln. All data presented here are compatible with the following model. The first step in the mode of action of Nhe is assocd. with binding of NheC and NheB to the cell surface and probably accompanied by conformational changes. These events allow subsequent binding of NheA, leading to cell lysis.
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    Tausch, F.; Dietrich, R.; Schauer, K.; Janowski, R.; Niessing, D.; Märtlbauer, E.; Jessberger, N. Evidence for Complex Formation of the Bacillus Cereus Haemolysin BL Components in Solution. Toxins 2017, 9, 1– 18,  DOI: 10.3390/toxins9090288
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    Chen, S.; Einspanier, R.; Schoen, J. Transepithelial Electrical Resistance (TEER): A Functional Parameter to Monitor the Quality of Oviduct Epithelial Cells Cultured on Filter Supports. Histochem. Cell Biol. 2015, 144, 509– 515,  DOI: 10.1007/s00418-015-1351-1
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    Transepithelial electrical resistance (TEER): a functional parameter to monitor the quality of oviduct epithelial cells cultured on filter supports
    Chen, Shuai; Einspanier, Ralf; Schoen, Jennifer
    Histochemistry and Cell Biology (2015), 144 (5), 509-515CODEN: HCBIFP; ISSN:0948-6143. (Springer)
    Cultivation of oviduct epithelial cells on porous filters fosters in vivo-like morphol. and functionality. However, due to the optical properties of the filter materials and the cells' columnar shape, cell quality is hard to assess via light microscopy. The authors aim to evaluate transepithelial elec. resistance (TEER) measurement as a prognostic quality indicator for the cultivation of porcine oviduct epithelial cells (POEC). POEC were maintained in four different types of media for 3 and 6 wk to achieve diverse culture qualities, and TEER was measured before processing samples for histol. Culture quality was scored using morphol. criteria (presence of cilia, confluence and cell polarity). The authors furthermore analyzed the correlation between cellular height (as a measure of apical-basal polarization) and TEER in fully differentiated routine cultures (biol. variation) and in cultures with altered cellular height due to hormonal stimulation. Fully differentiated cultures possessed a moderate TEER between 500 and 1100 Ω*cm2. Only 5% of cultures which exhibited TEER values in this defined range had poor quality. Sub-differentiated cultures showed either very low or excessively high TEER. The authors unveiled a highly significant (P < 0.0001) neg. linear correlation between TEER and epithelial height in well-differentiated cultures (both routine and hormone stimulated group). This may point toward the interaction between tight junction assembly and epithelial apical-basal polarization. In conclusion, TEER is a straightforward quality indicator which could be routinely used to monitor the differentiation status of oviduct epithelial cells in vitro.
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    Srinivasan, B.; Shuler, L.; Hickman, J. J. TEER Measurement Techniques for in Vitro Barrier Model Systems. SLAS Technol. 2015, 20, 107– 126,  DOI: 10.1177/2211068214561025.TEER
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    Ramarao, N.; Nielsen-Leroux, C.; Lereclus, D. The Insect Galleria Mellonella as a Powerful Infection Model to Investigate Bacterial Pathogenesis. J. Vis. Exp. 2012, 70, e4392,  DOI: 10.3791/4392
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    Blöchl, C.; Regl, C.; Huber, C. G.; Winter, P.; Weiss, R.; Wohlschlager, T. Towards Middle-up Analysis of Polyclonal Antibodies: Subclass-Specific N-Glycosylation Profiling of Murine Immunoglobulin G (IgG) by Means of HPLC-MS. Sci. Rep. 2020, 10, 18080  DOI: 10.1038/s41598-020-75045-1
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    Towards middle-up analysis of polyclonal antibodies: subclass-specific N-glycosylation profiling of murine immunoglobulin G (IgG) by means of HPLC-MS
    Blochl Constantin; Regl Christof; Huber Christian G; Wohlschlager Therese; Regl Christof; Huber Christian G; Wohlschlager Therese; Winter Petra; Weiss Richard
    Scientific reports (2020), 10 (1), 18080 ISSN:.
    In recent years, advanced HPLC-MS strategies based on intact protein ("top-down") or protein subunit ("middle-up/middle-down") analysis have been implemented for the characterization of therapeutic monoclonal antibodies. Here, we assess feasibility of middle-up/middle-down analysis for polyclonal IgGs exhibiting extensive sequence variability. Specifically, we addressed IgGs from mouse, representing an important model system in immunological investigations. To obtain Fc/2 portions as conserved subunits of IgGs, we made use of the bacterial protease SpeB. For this purpose, we initially determined SpeB cleavage sites in murine IgGs. The resulting Fc/2 portions characteristic of different subclasses were subsequently analysed by ion-pair reversed-phase HPLC hyphenated to high-resolution mass spectrometry. This enabled simultaneous relative quantification of IgG subclasses and their N-glycosylation variants, both of which influence IgG effector functions. To assess method capabilities in an immunological context, we applied the analytical workflow to polyclonal antibodies obtained from BALB/c mice immunized with the grass pollen allergen Phl p 6. The study revealed a shift in IgG subclasses and Fc-glycosylation patterns in total and antigen-specific IgGs from different mouse cohorts, respectively. Eventually, Fc/2 characterization may reveal other protein modifications including oxidation, amino acid exchanges, and C-terminal lysine, and may thus be implemented for quality control of functional antibodies.
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    Dyballa, N.; Metzger, S. Fast and Sensitive Colloidal Coomassie G-250 Staining for Proteins in Polyacrylamide Gels. J. Vis. Exp. 2009, 30, 2– 5,  DOI: 10.3791/1431
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    Kabsch, W. XDS. Acta Crystallogr., Sect. D: Biol. Crystallogr. 2010, 66, 125– 132,  DOI: 10.1107/S0907444909047337
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    Software XDS for image rotation, recognition and crystal symmetry assignment
    Kabsch, Wolfgang
    Acta Crystallographica, Section D: Biological Crystallography (2010), 66 (2), 125-132CODEN: ABCRE6; ISSN:0907-4449. (International Union of Crystallography)
    The usage and control of recent modifications of the program package XDS for the processing of rotation images are described in the context of previous versions. New features include automatic detn. of spot size and reflecting range and recognition and assignment of crystal symmetry. Moreover, the limitations of earlier package versions on the no. of correction/scaling factors and the representation of pixel contents have been removed. Large program parts have been restructured for parallel processing so that the quality and completeness of collected data can be assessed soon after measurement.
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    Evans, P. R.; Murshudov, G. N. How Good Are My Data and What Is the Resolution?. Acta Crystallogr., Sect. D: Biol. Crystallogr. 2013, 69, 1204– 1214,  DOI: 10.1107/S0907444913000061
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    How good are my data and what is the resolution?
    Evans, Philip R.; Murshudov, Garib N.
    Acta Crystallographica, Section D: Biological Crystallography (2013), 69 (7), 1204-1214CODEN: ABCRE6; ISSN:0907-4449. (International Union of Crystallography)
    Following integration of the obsd. diffraction spots, the process of data redn.' initially aims to det. the point-group symmetry of the data and the likely space group. This can be performed with the program POINTLESS. The scaling program then puts all the measurements on a common scale, avs. measurements of symmetry-related reflections (using the symmetry detd. previously) and produces many statistics that provide the first important measures of data quality. A new scaling program, AIMLESS, implements scaling models similar to those in SCALA but adds some addnl. analyses. From the analyses, a no. of decisions can be made about the quality of the data and whether some measurements should be discarded. The effective resoln.' of a data set is a difficult and possibly contentious question (particularly with referees of papers) and this is discussed in the light of tests comparing the data-processing statistics with trials of refinement against obsd. and simulated data, and automated model-building and comparison of maps calcd. with different resoln. limits. These trials show that adding weak high-resoln. data beyond the commonly used limits may make some improvement and does no harm.
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    McCoy, A. J. Solving Structures of Protein Complexes by Molecular Replacement with Phaser. Acta Crystallogr., Sect. D: Biol. Crystallogr. 2007, 63, 32– 41,  DOI: 10.1107/S0907444906045975
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    Solving structures of protein complexes by molecular replacement with Phaser
    McCoy, Airlie J.
    Acta Crystallographica, Section D: Biological Crystallography (2007), 63 (1), 32-41CODEN: ABCRE6; ISSN:0907-4449. (International Union of Crystallography)
    Mol. replacement (MR) generally becomes more difficult as the no. of components in the asym. unit requiring sep. MR models (i.e. the dimensionality of the search) increases. When the proportion of the total scattering contributed by each search component is small, the signal in the search for each component in isolation is weak or non-existent. Maximum-likelihood MR functions enable complex asym. units to be built up from individual components with a 'tree search with pruning' approach. This method, as implemented in the automated search procedure of the program Phaser, has been very successful in solving many previously intractable MR problems. However, there are a no. of cases in which the automated search procedure of Phaser is suboptimal or encounters difficulties. These include cases where there are a large no. of copies of the same component in the asym. unit or where the components of the asym. unit have greatly varying B factors. Two case studies are presented to illustrate how Phaser can be used to best advantage in the std. 'automated MR' mode and two case studies are used to show how to modify the automated search strategy for problematic cases.
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    Maximum-likelihood X-ray macromol. structure refinement in BUSTER has been extended with restraints facilitating the exploitation of structural similarity. The similarity can be between two or more chains within the structure being refined, thus favoring NCS, or to a distinct 'target' structure that remains fixed during refinement. The local structural similarity restraints (LSSR) approach considers all distances less than 5.5 Å between pairs of atoms in the chain to be restrained. For each, the difference from the distance between the corresponding atoms in the related chain is found. LSSR applies a restraint penalty on each difference. A functional form that reaches a plateau for large differences is used to avoid the restraints distorting parts of the structure that are not similar. Because LSSR are local, there is no need to sep. out domains. Some restraint pruning is still necessary, but this has been automated. LSSR have been available to academic users of BUSTER since 2009 with the easy-to-use and options. The use of LSSR is illustrated in the re-refinement of PDB entries , where enables the correct ligand-binding structure to be found, and , where contributes to the location of an addnl. copy of the cyclic peptide ligand.
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  • Abstract

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    Figure 1

    Figure 1. Examples of known bacterial collagenase H and Q1 (ColH and ColQ1) inhibitors. (a) Structures of bacterial ColH inhibitors. (22−24) (b) Structures of the recently identified inhibitors of ColH and ColQ1. (25−28) Zinc-binding groups are highlighted by dashed rectangles. n.d.: not determined.

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    Figure 2

    Figure 2. Chemical structures of compounds 13 and 27 and their activities against the collagenase unit (CU) of ColQ1 and peptidase domain (PD) of ColH.

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    Scheme 1

    Scheme 1. Synthesis of Compounds 7–12a
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    aReagents and conditions: (a) oxalic acid, 4 N HCl, reflux, 6 h; (b) POCl3, DMF, 50 °C, 6 h; (c) triethyl phosphite, sealed tube, 150 °C, 18 h; and (d) bromotrimethylsilane, dry DCM, stirring, room temperature, 18 h.

    Scheme 2

    Scheme 2. Synthesis of Compounds 13 and 16a
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    aReagents and conditions: (a) oxalic acid, 4 N HCl, reflux, 6 h; (b) POCl3, DMF, 50 °C, 6 h; (c) triethyl phosphite, sealed tube, 150 °C, 18 h; (d) bromotrimethylsilane, dry DCM, stirring, room temperature, 18 h; (e) dioxane/H2O (1:1), LiOH, 55 °C, 24 h; and (f) POCl3, DMF, 0 °C to room temperature, 5 min.

    Scheme 3

    Scheme 3. Synthesis of Compound 14a
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    aReagents and conditions: (a) oxalic acid, 4 N HCl, reflux, 6 h; (b) POCl3, DMF, 50 °C, 6 h; (c) 3,4-dichloroaniline, EDC.HCl, DCM, 18 h; (d) triethyl phosphite, sealed tube, 150 °C, 18 h; and (e) bromotrimethylsilane, dry DCM, stirring, room temperature, 18 h.

    Scheme 4

    Scheme 4. Synthesis of Compound 15a
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    aReagents and conditions: (a) (4-chlorophenyl)boronic acid, dioxane/H2O (4:1), Na2CO3 (2 M), Pd(PPh3)4, microwave, 20 min; (b) oxalic acid, 4 N HCl, reflux, 6 h; (c) POCl3, DMF, 50 °C, 6 h; (d) triethyl phosphite, sealed tube, 150 °C, 18 h; and (e) bromotrimethylsilane, dry DCM, stirring, room temperature, 18 h.

    Scheme 5

    Scheme 5. Synthesis of Hydroxamic Acid Compoundsa
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    aReagents and conditions: (a) NaOH, EtOH/H2O (4:1), rt., 18 h; (b) tert-butyl N-(2-aminoethyl)carbamate, EDC.HCl, HOBt, DIPEA, CH2Cl2, rt., 18 h; (c) 4 N HCl, EtOH, 0 °C to rt., 18 h; (d) azide-N-diazoimidazole-1-sulfonamide hydrogen sulfate, K2CO3, ZnCl2, DIPEA, MeOH, rt., 18 h; (e) aq. hydroxylamine (50% in water w/w), KCN (cat.), MeOH, rt., 18 h; and (f) alkyne 34a–34d, prop-2-ynoxybenzene or prop-2-ynylsulfanylbenzene, CuSO4 (5H2O), NaAsc, N,N-dimethylformamide/H2O (1.2:1), rt., 18 h.

    Figure 3

    Figure 3. Crystal structure of ColG-PD in complex with 13 solved at a resolution of 1.95 Å. Close-up view of the active site in ball-and-stick representation. The inhibitor (cyan) is shown in sticks with a polder map contoured at 2.5 σ above the background. The catalytic zinc ion (dark gray) and the calcium ion (green) are shown as spheres (PDB code: 7ZBV).

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    Figure 4

    Figure 4. Crystal structure of ColG-PD in complex with 27 solved at 1.80 Å resolution. Close-up view of the active site in ball-and-stick representation. The inhibitor (cyan) is shown in sticks with the maximum likelihood weighted 2Fo–Fc electron density map contoured at 1σ. The catalytic zinc ion (dark gray) and the calcium ion (green) and water molecules (red) are shown as spheres (PDB code: 7Z5U).

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    Figure 5

    Figure 5. Activity of ColQ1 inhibitors against the collagenolytic activity of the full-length (FL) ColQ1. Inhibitors prevented the cleavage of 1 mg/mL of Col I chains (i.e., β, α-1, and α-2). The Bacillus cereus ColQ1-FL (50 ng) was incubated with 1 mg/mL Col I for 3 h, and the degradation was then visualized by 12% SDS-PAGE. Col I: 1 mg/mL Col I without any protease. M (kDa): molecular weight standards, Col I: type I collagen, ColQ1: collagenase Q1.

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    Figure 6

    Figure 6. Activities of FDA-approved tiludronate disodium and compound 14 on the fibroblast (NHDF) cells infected with Bacillus cereus. (a) The antigelatinolytic activities of compounds tiludronate disodium and 14 against B. cereus collagenases. The DMEM medium of the infected NHDF cells was applied to the zymograms. Clear regions against blue background indicate that gelatin in the gel has been cleaved. (b) The amount of fibrillar collagens maintained by tiludronate disodium and 14 in the infected NHDF cells (highlighted in the yellow background). (c) The cytotoxicity of B. cereus infection (highlighted in the yellow background) in NHDF cells treated with and without tiludronate disodium and 14. Ctrl represents the noninfected cells (gray column) and the infected cells and nontreated with inhibitors (red column). Statistical analysis was performed with one-way ANOVA, and statistical significance was analyzed by the Tukey test. Significance was calculated by comparing nontreated vs treated cells with compounds (mean ± SD, ****p < 0.0001, **p < 0.01, *p ≤ 0.05, ns: nonsignificant). Ctrl: control. M (kDa): molecular weight marker.

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    Figure 7

    Figure 7. Change in the transepithelial electrical resistance (TEER) of the Madin–Darby canine kidney II (MDCK II) cells challenged with Bacillus cereus bacteria or 50% (v/v) culture supernatant and treated with or without collagenase inhibitors. (a) 14 and 15 preserve the TEER value of the MDCK II infected with B. cereus compared with the nontreated conditions with inhibitor. (b) Compounds 11, 13, 15, and tiludronate disodium maintained the TEER of MDCK II cells challenged with B. cereus supernatant. Each curve represents the average ± standard deviation of at least three independent experiments.

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    Figure 8

    Figure 8. Survival analysis of Galleria mellonella larvae treated with Bacillus cereus AH187 with and without 14 and tiludronate disodium. Each curve represents results of three independent experiments; the statistical difference between groups treated with 100, 50, and 25 μM of compound 14 and B. cereus AH187 and with the group treated only with B. cereus AH187 is p < 0.0001, p = 0.0039, and p = 0.0173, respectively. The statistical difference between groups treated with 100 μM tiludronate disodium and with B. cereus AH187 is p = 0.0032 (log-rank test). The survival rate for the larvae treated with compound 14 and tiludronate disodium in PBS was 100%.

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      Bacterial collagenases - A review
      Duarte Ana Sofia; Correia Antonio; Esteves Ana Cristina
      Critical reviews in microbiology (2016), 42 (1), 106-26 ISSN:.
      Bacterial collagenases are metalloproteinases involved in the degradation of the extracellular matrices of animal cells, due to their ability to digest native collagen. These enzymes are important virulence factors in a variety of pathogenic bacteria. Nonetheless, there is a lack of scientific consensus for a proper and well-defined classification of these enzymes and a vast controversy regarding the correct identification of collagenases. Clostridial collagenases were the first ones to be identified and characterized and are the reference enzymes for comparison of newly discovered collagenolytic enzymes. In this review we present the most recent data regarding bacterial collagenases and overview the functional and structural diversity of bacterial collagenases. An overall picture of the molecular diversity and distribution of these proteins in nature will also be given. Particular aspects of the different proteolytic activities will be contextualized within relevant areas of application, mainly biotechnological processes and therapeutic uses. At last, we will present a new classification guide for bacterial collagenases that will allow the correct and straightforward classification of these enzymes.
    14. 14
      Harrington, D. J. Bacterial Collagenases and Collagen-Degrading Enzymes and Their Potential Role in Human Disease. Infect. Immun. 1996, 64, 1885– 1891
      14
      Bacterial collagenases and collagen-degrading enzymes and their potential role in human disease
      Harrington, Dean J.
      Infection and Immunity (1996), 64 (6), 1885-1891CODEN: INFIBR; ISSN:0019-9567. (American Society for Microbiology)
      A review with 82 refs. The aim of this review is to provide an overview of the bacterial pathogens that have been shown to elaborate enzymes capable of degrading collagen and to discuss the potential roles of these proteinases in the pathogenesis of the human diseases with which these organisms are assocd.
    15. 15
      Bauer, R.; Wilson, J. J.; Philominathan, S. T. L.; Davis, D.; Matsushita, O.; Sakona, J. Structural Comparison of ColH and ColG Collagen-Binding Domains from Clostridium Histolyticum. J. Bacteriol. 2013, 195, 318– 327,  DOI: 10.1128/JB.00010-12
      15
      Structural comparison of ColH and ColG collagen-binding domains from Clostridium histolyticum
      Bauer, Ryan; Wilson, Jeffrey J.; Philominathan, Sagaya Theresa Leena; Davis, Dan; Matsushita, Osamu; Sakon, Joshua
      Journal of Bacteriology (2013), 195 (2), 318-327CODEN: JOBAAY; ISSN:0021-9193. (American Society for Microbiology)
      Clostridium histolyticum secretes collagenases, ColG and ColH, that cause extensive tissue destruction in myonecrosis. The C-terminal collagen-binding domain (CBD) of collagenase is required for insol. collagen fibril binding and subsequent collagenolysis. The high-resoln. crystal structures of ColG-CBD (s3b) and ColH-CBD (s3) are reported. The new x-ray structure of s3 was solved at 2.0-Å resoln. (R = 17.4%; Rfree = 23.3%), while the resoln. of the previously detd. s3b was extended to 1.4 Å (R = 17.9%; Rfree = 21.0%). Despite sharing only 30% sequence identity, the mols. resemble one another closely (root mean square deviation [RMSD] Cα = 1.5 A). All but one residue, whose side chain chelates with Ca2+, are conserved. The dual Ca2+ binding site in s3 is completed by an unconserved aspartate. Differential scanning calorimetric measurements showed that s3 gains thermal stability, comparable to s3b, by binding to Ca2+ (holo Tm = 94.1°; apo Tm = 70.2°C). Holo s3 is also stabilized against chem. denaturants urea and guanidine HCl. The three most crit. residues for collagen interaction in s3b are conserved in s3. The general shape of the binding pocket is retained by altered loop structures and side chain positions. Small-angle x-ray scattering data revealed that s3 also binds asym. to minicollagen. Besides the calcium-binding sites and the collagen-binding pocket, architecturally important hydrophobic residues and the hydrogen-bonding network around the cis-peptide bond are well conserved within the metallopeptidase subfamily M9B. CBDs were previously shown to bind to the extracellular matrix of various tissues. Compactness and extreme stability in physiol. Ca2+ concn. possibly make both CBDs suitable for targeted growth factor delivery.
    16. 16
      Eckhard, U.; Schönauer, E.; Ducka, P.; Briza, P.; Nüss, D.; Brandstetter, H. Biochemical Characterization of the Catalytic Domains of Three Different Clostridial Collagenases. Biol. Chem. 2009, 390, 11– 18,  DOI: 10.1515/BC.2009.004
      16
      Biochemical characterization of the catalytic domains of three different clostridial collagenases
      Eckhard, Ulrich; Schoenauer, Esther; Ducka, Paulina; Briza, Peter; Nuess, Dorota; Brandstetter, Hans
      Biological Chemistry (2009), 390 (1), 11-18CODEN: BICHF3; ISSN:1431-6730. (Walter de Gruyter GmbH & Co. KG)
      Clostridial collagenases are used for a broad spectrum of biotechnol. applications and represent prime target candidates for both therapy and diagnosis of clostridial infections. In this study, we biochem. characterized the catalytic domains of three clostridial collagenases, collagenase G (ColG) and H (ColH) from Clostridium histolyticum, and collagenase T (ColT) from C. tetani. All protein samples showed activity against a synthetic peptidic substrate (furylacryloyl-Leu-Gly-Pro-Ala, FALGPA) with ColH showing the highest overall activity and highest substrate affinity. Whereas the Km values of all three enzymes were within the same order of magnitude, the turnover rate kcat of ColG decreased 50- to 150-fold when compared to ColT and ColH. It is noteworthy that the protein N-terminus significantly impacts their substrate affinity and substrate turnover as well as their inhibition profile with 1,10-phenanthroline. These findings were complemented with the discovery of a strictly conserved double-glycine motif, positioned 28 amino acids upstream of the HEXXH zinc binding site, which is crit. for enzymic activity. These observations have consequences with respect to the topol. of the N-terminus relative to the active site as well as possible activation mechanisms.
    17. 17
      Eckhard, U.; Schönauer, E.; Nüss, D.; Brandstetter, H. Structure of Collagenase G Reveals a Chew-and-Digest Mechanism of Bacterial Collagenolysis. Nat. Struct. Mol. Biol. 2010, 18, 1109– 1114,  DOI: 10.1038/nsmb.2127
      There is no corresponding record for this reference.
    18. 18
      Matsushita, O.; Koide, T.; Kobayashi, R.; Nagata, K.; Okabe, A. Substrate Recognition by the Collagen-Binding Domain of Clostridium Histolyticum Class I Collagenase. J. Biol. Chem. 2001, 276, 8761– 8770,  DOI: 10.1074/jbc.M003450200
      18
      Substrate recognition by the collagen-binding domain of Clostridium histolyticum class I collagenase
      Matsushita, Osamu; Koide, Takaki; Kobayashi, Ryoji; Nagata, Kazuhiro; Okabe, Akinobu
      Journal of Biological Chemistry (2001), 276 (12), 8761-8770CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)
      Clostridium histolyticum type I collagenase (ColG) has a segmental structure, S1+S2+S3a+S3b. S3a and S3b bound to insol. collagen, but S2 did not, thus indicating that S3 forms a collagen-binding domain (CBD). Because S3a+S3b showed the most efficient binding to substrate, cooperative binding by both domains was suggested for the enzyme. Monomeric (S3b) and tandem (S3a+S3b) CBDs bound to atelocollagen, which contains only the collagenous region. However, they did not bind to telopeptides immobilized on Sepharose beads. These results suggested that the binding site(s) for the CBD is(are) present in the collagenous region. The CBD bound to immobilized collagenous peptides, (Pro-Hyp-Gly)n and (Pro-Pro-Gly)n, only when n is large enough to allow the peptides to have a triple-helical conformation. They did not bind to various peptides with similar amino acid sequences or to gelatin, which lacks a triple-helical conformation. The CBD did not bind to immobilized Glc-Gal disaccharide, which is attached to the side chains of hydroxylysine residues in the collagenous region. These observations suggested that the CBD specifically recognizes the triple-helical conformation made by three polypeptide chains in the collagenous region.
    19. 19
      Abfalter, C. M.; Schönauer, E.; Ponnuraj, K.; Huemer, M.; Gadermaier, G.; Regl, C.; Briza, P.; Ferreira, F.; Huber, C. G.; Brandstetter, H.; Posselt, G.; Wessler, S. Cloning, Purification and Characterization of the Collagenase ColA Expressed by Bacillus Cereus ATCC 14579. PLoS One 2016, 11, 1– 19,  DOI: 10.1371/journal.pone.0162433
      There is no corresponding record for this reference.
    20. 20
      Hoppe, I. J.; Brandstetter, H.; Schönauer, E. Biochemical Characterisation of a Collagenase from Bacillus Cereus Strain Q1. Sci. Rep. 2021, 11, 4187  DOI: 10.1038/s41598-021-83744-6
      20
      Biochemical characterisation of a collagenase from Bacillus cereus strain Q1
      Hoppe, Isabel J.; Brandstetter, Hans; Schoenauer, Esther
      Scientific Reports (2021), 11 (1), 4187CODEN: SRCEC3; ISSN:2045-2322. (Nature Research)
      Abstr.: Collagen is the most abundant protein in higher animals and as such it is a valuable source of amino acids and carbon for saprophytic bacteria. Due to its unique amino acid compn. and triple-helical tertiary structure it can however only be cleaved by specialized proteases like the collagenases secreted by some bacteria. Among the best described bacterial collagenases are ColG and ColH from Clostridium histolyticum. Many Bacillus species contain homologues of clostridial collagenases, which play a role in some infections caused by B. cereus. Detailed biochem. and enzymic characterizations of bacillial collagenases are however lacking at this time. In an effort to close this gap in knowledge we expressed ColQ1 from B. cereus strain Q1 recombinantly, investigated its metal dependency and performed peptide, gelatin and collagen degrdn. assays. Our results show that ColQ1 is a true collagenase, cleaving natively folded collagen six times more efficiently than ColG while at the same time being a similarly effective peptidase as ColH. In both ColQ1 and ColG the rate-limiting step in collagenolysis is the unwinding of the triple-helix. The data suggest an orchestrated multi-domain mechanism for efficient helicase activity.
    21. 21
      Eckhard, U.; Schönauer, E.; Brandstetter, H. Structural Basis for Activity Regulation and Substrate Preference of Clostridial Collagenases G, H, and T. J. Biol. Chem. 2013, 288, 20184– 20194,  DOI: 10.1074/jbc.M112.448548
      21
      Structural basis for activity regulation and substrate preference of clostridial collagenases G, H, and T
      Eckhard, Ulrich; Schoenauer, Esther; Brandstetter, Hans
      Journal of Biological Chemistry (2013), 288 (28), 20184-20194CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)
      Clostridial collagenases are among the most efficient enzymes to degrade by far the most predominant protein, collagen, in the biosphere. Here, the authors present crystal structures of 3 clostridial collagenase isoforms (ColG, ColH, and ColT). The comparison of unliganded and liganded structures revealed quaternary subdomain dynamics. In the unliganded ColH structure, this globular dynamics was modulated by an Asp switch motion that bound to the catalytic Zn2+. The authors further identified a Ca2+-binding site in proximity to the catalytic Zn2+. Both ions were required for full activity, explaining why Ca2+ critically affected the enzymic activity of clostridial collagenases. These studies further revealed that loops close to the active site thus serve as a characteristic substrate selectivity filter. These elements explained the distinct peptidolytic and collagenolytic activities of these enzymes and provided a rational framework to engineer collagenases with customized substrate specificity as well as for inhibitor design.
    22. 22
      Oshima, N.; Narukawa, Y.; Takeda, T.; Kiuchi, F. Collagenase Inhibitors from Viola Yedoensis. J. Nat. Med. 2013, 67, 240– 245,  DOI: 10.1007/s11418-012-0665-8
      22
      Collagenase inhibitors from Viola yedoensis
      Oshima, Naohiro; Narukawa, Yuji; Takeda, Tadahiro; Kiuchi, Fumiyuki
      Journal of Natural Medicines (2013), 67 (1), 240-245CODEN: JNMOBN; ISSN:1861-0293. (Springer Japan)
      Fractionation of acetone and methanol exts. of Viola yedoensis, under the guidance of inhibition against Clostridium histolyticum collagenase (ChC), resulted in the isolation of esculetin (1) (IC50 12 μM) and scopoletin (2) (IC50 1.8 μM) as the active constituents, together with trans-p-coumaric acid (3), cis-p-coumaric acid (4), 3-O-β-d-glucosyl-7-O-α-L-rhamnosylkaempferol (5), rutin (6), isovitexin (7), isoorientin (8), vicenin-2 (9), isoscoparin (10), vanillic acid (11) and adenosine (12). Modification of phenolic hydroxy groups of 1 showed that small O-alkyl groups largely increased the activity, whereas larger O-alkyl groups decreased the activity, and 6,7-dimethoxycoumarin (scoparone 13) potently inhibited ChC (IC50 24 nM).
    23. 23
      Scozzafava, A.; Supuran, C. T. Protease Inhibitors: Synthesis of Matrix Metalloproteinase and Bacterial Collagenase Inhibitors Incorporating 5-Amino-2-Mercapto-1,3,4-Thiadiazole Zinc Binding Functions. Bioorganic Med. Chem. Lett. 2002, 12, 2667– 2672,  DOI: 10.1016/S0960-894X(02)00564-4
      23
      Protease inhibitors: Synthesis of matrix metalloproteinase and bacterial collagenase inhibitors incorporating 5-amino-2-mercapto-1,3,4-thiadiazole zinc binding functions
      Scozzafava, Andrea; Supuran, Claudiu T.
      Bioorganic & Medicinal Chemistry Letters (2002), 12 (19), 2667-2672CODEN: BMCLE8; ISSN:0960-894X. (Elsevier Science Ltd.)
      Matrix metalloproteinase (MMP)/bacterial collagenase inhibitors incorporating 5-amino-2-mercapto-1,3,4-thiadiazole zinc binding functions are reported. A series of compds. was prepd. by reaction of arylsulfonyl isocyanates or arylsulfonyl halides with phenylalanyl-alanine, followed by coupling with 5-amino-2-mercapto-1,3,4-thiadiazole in the presence of carbodiimides. These new compds. were assayed as inhibitors of human MMP-1, MMP-2, MMP-8 and MMP-9, and of the collagenase isolated from the anaerobe Clostridium histolyticum (ChC). The new derivs. proved to be powerful inhibitors of these metalloproteases, with activities in the low micromolar range for some of the target enzymes, depending on the substitution pattern at the arylsulfonyl(ureido) moieties.
    24. 24
      Scozzafava, A.; Supuran, C. T. Protease Inhibitors: Synthesis of Matrix Metalloproteinase and Bacterial Collagenase Inhibitors Incorporating 5-Amino-2-Mercapto-1,3,4-Thiadiazole Zinc Binding Functions. Bioorganic Med. Chem. Lett. 2000, 12, 2667– 2672,  DOI: 10.1016/S0960-894X(02)00564-4
      There is no corresponding record for this reference.
    25. 25
      Schönauer, E.; Kany, A. M.; Haupenthal, J.; Hüsecken, K.; Hoppe, I. J.; Voos, K.; Yahiaoui, S.; Elsässer, B.; Ducho, C.; Brandstetter, H.; Hartmann, R. W. Discovery of a Potent Inhibitor Class with High Selectivity toward Clostridial Collagenases. J. Am. Chem. Soc. 2017, 139, 12696– 12703,  DOI: 10.1021/jacs.7b06935
      25
      Discovery of a Potent Inhibitor Class with High Selectivity toward Clostridial Collagenases
      Schonauer Esther; Hoppe Isabel J; Elsasser Brigitta; Brandstetter Hans; Kany Andreas M; Haupenthal Jorg; Husecken Kristina; Yahiaoui Samir; Hartmann Rolf W; Voos Katrin; Ducho Christian; Hartmann Rolf W
      Journal of the American Chemical Society (2017), 139 (36), 12696-12703 ISSN:.
      Secreted virulence factors like bacterial collagenases are conceptually attractive targets for fighting microbial infections. However, previous attempts to develop potent compounds against these metalloproteases failed to achieve selectivity against human matrix metalloproteinases (MMPs). Using a surface plasmon resonance-based screening complemented with enzyme inhibition assays, we discovered an N-aryl mercaptoacetamide-based inhibitor scaffold that showed sub-micromolar affinities toward collagenase H (ColH) from the human pathogen Clostridium histolyticum. Moreover, these inhibitors also efficiently blocked the homologous bacterial collagenases, ColG from C. histolyticum, ColT from C. tetani, and ColQ1 from the Bacillus cereus strain Q1, while showing negligible activity toward human MMPs-1, -2, -3, -7, -8, and -14. The most active compound displayed a more than 1000-fold selectivity over human MMPs. This selectivity can be rationalized by the crystal structure of ColH with this compound, revealing a distinct non-primed binding mode to the active site. The non-primed binding mode presented here paves the way for the development of selective broad-spectrum bacterial collagenase inhibitors with potential therapeutic application in humans.
    26. 26
      Konstantinović, J.; Yahiaoui, S.; Alhayek, A.; Haupenthal, J.; Schönauer, E.; Andreas, A.; Kany, A. M.; Müller, R.; Koehnke, J.; Berger, F. K.; Bischoff, M.; Hartmann, R. W.; Brandstetter, H.; Hirsch, A. K. H. N-Aryl-3-Mercaptosuccinimides as Antivirulence Agents Targeting Pseudomonas Aeruginosa Elastase and Clostridium Collagenases. J. Med. Chem. 2020, 63, 8359– 8368,  DOI: 10.1021/acs.jmedchem.0c00584
      26
      N-Aryl-3-mercaptosuccinimides as Antivirulence Agents Targeting Pseudomonas aeruginosa Elastase and Clostridium Collagenases
      Konstantinovic, Jelena; Yahiaoui, Samir; Alhayek, Alaa; Haupenthal, Joerg; Schoenauer, Esther; Andreas, Anastasia; Kany, Andreas M.; Mueller, Rolf; Koehnke, Jesko; Berger, Fabian K.; Bischoff, Markus; Hartmann, Rolf W.; Brandstetter, Hans; Hirsch, Anna K. H.
      Journal of Medicinal Chemistry (2020), 63 (15), 8359-8368CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)
      In light of the global antimicrobial-resistance crisis, there is an urgent need for novel bacterial targets and antibiotics with novel modes of action. It has been shown that Pseudomonas aeruginosa elastase (LasB) and Clostridium histolyticum (Hathewaya histolytica) collagenase (ColH) play a significant role in the infection process and thereby represent promising antivirulence targets. Here, we report novel N-aryl-3-mercaptosuccinimide inhibitors that target both LasB and ColH, displaying potent activities in vitro and high selectivity for the bacterial over human metalloproteases. Addnl., the inhibitors demonstrate no signs of cytotoxicity against selected human cell lines and in a zebrafish embryo toxicity model. Furthermore, the most active ColH inhibitor shows a significant redn. of collagen degrdn. in an ex vivo pig-skin model.
    27. 27
      Voos, K.; Schönauer, E.; Alhayek, A.; Haupenthal, J.; Andreas, A.; Müller, R.; Hartmann, R. W.; Brandstetter, H.; Hirsch, A. K. H.; Ducho, C. Phosphonate as a Stable Zinc-Binding Group for “Pathoblocker” Inhibitors of Clostridial Collagenase H (ColH). ChemMedChem 2021, 1– 12,  DOI: 10.1002/cmdc.202000994
      There is no corresponding record for this reference.
    28. 28
      Alhayek, A.; Khan, E. S.; Schönauer, E.; Däinghaus, T.; Shafiei, R.; Voos, K.; Han, M. K. L.; Ducho, C.; Posselt, G.; Wessler, S.; Brandstetter, H.; Haupenthal, J.; del Campo, A.; Hirsch, A. K. H. Inhibition of Collagenase Q1 of Bacillus Cereus as a Novel Antivirulence Strategy for the Treatment of Skin-Wound Infections. Adv. Ther. 2022, 2100222  DOI: 10.1002/adtp.202100222
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      Inhibition of Collagenase Q1 of Bacillus cereus as a Novel Antivirulence Strategy for the Treatment of Skin-Wound Infections
      Alhayek, Alaa; Khan, Essak S.; Schonauer, Esther; Dainghaus, Tobias; Shafiei, Roya; Voos, Katrin; Han, Mitchell K. L.; Ducho, Christian; Posselt, Gernot; Wessler, Silja; Brandstetter, Hans; Haupenthal, Jorg; del Campo, Aranzazu; Hirsch, Anna K. H.
      Advanced Therapeutics (Weinheim, Germany) (2022), 5 (3), 2100222CODEN: ATWGAP; ISSN:2366-3987. (Wiley-VCH Verlag GmbH & Co. KGaA)
      Despite the progress in surgical techniques and antibiotic prophylaxis, opportunistic wound infections with Bacillus cereus remain a public health problem. Secreted toxins are one of the main factors contributing to B. cereus pathogenicity. A promising strategy to treat such infections is to target these toxins and not the bacteria. Although the exoenzymes produced by B. cereus are thoroughly investigated, little is known about the role of B. cereus collagenases in wound infections. In this report, the collagenolytic activity of secreted collagenases (Col) is characterized in the B. cereus culture supernatant (csn) and its isolated recombinantly produced ColQ1 is characterized. The data reveals that ColQ1 causes damage on dermal collagen (COL). This results in gaps in the tissue, which might facilitate the spread of bacteria. The importance of B. cereus collagenases is also demonstrated in disease promotion using two inhibitors. Compd. 2 shows high efficacy in peptidolytic, gelatinolytic, and COL degrdn. assays. It also preserves the fibrillar COLs in skin tissue challenged with ColQ1, as well as the viability of skin cells treated with B. cereus csn. A Galleria mellonella model highlights the significance of collagenase inhibition in vivo.
    29. 29
      Zhang, P.-M.; Li, Y.-W.; Zhou, J.; Gan, L.-L.; Chen, Y.-J.; Gan, Z.-J.; Yu, Y. A One-Pot Facile Synthesis of 2,3-Dihydroxyquinoxaline and 2,3-Dichloroquinoxaline Derivatives Using Silica Gel as an Efficient Catalyst. J. Heterocycl. Chem. 2018, 55, 1809– 1814,  DOI: 10.1002/jhet.3224
      29
      A One-pot Facile Synthesis of 2,3-Dihydroxyquinoxaline and 2,3-Dichloroquinoxaline Derivatives Using Silica Gel as an Efficient Catalyst
      Zhang, Pei-Ming; Li, Yao-Wei; Zhou, Jing; Gan, Lin-Ling; Chen, Yong-Jie; Gan, Zong-Jie; Yu, Yu
      Journal of Heterocyclic Chemistry (2018), 55 (7), 1809-1814CODEN: JHTCAD; ISSN:1943-5193. (Wiley-Blackwell)
      An efficient one-pot reaction has been developed for the synthesis of 2,3-dichloroquinoxaline derivs. I (R1 = H, H3CO, Cl, etc.; R2 = H, F, CH3, etc.; R3 = H, CH3; R1R2 = CH=CH-CH=CH). The reaction was performed in two steps via a silica gel catalyzed tandem process from o-phenylenediamines 2-H2N-3-R3-4-R1-5-R2C6HNH2 and oxalic acid, followed by addn. of phosphorus oxychloride (POCl3). A variety of 2,3-dichloroquinoxalines I have been obtained in good to excellent overall yields.
    30. 30
      Yang, Y.; Zhang, S.; Wu, B.; Ma, M.; Chen, X.; Qin, X.; He, M.; Hussain, S.; Jing, C.; Ma, B.; Zhu, C. An Efficient Synthesis of Quinoxalinone Derivatives as Potent Inhibitors of Aldose Reductase. ChemMedChem 2012, 7, 823– 835,  DOI: 10.1002/cmdc.201200054
      30
      An Efficient Synthesis of Quinoxalinone Derivatives as Potent Inhibitors of Aldose Reductase
      Yang, Yanchun; Zhang, Shuzhen; Wu, Bobin; Ma, Mingming; Chen, Xin; Qin, Xiangyu; He, Minlan; Hussain, Saghir; Jing, Chaojun; Ma, Bing; Zhu, Changjin
      ChemMedChem (2012), 7 (5), 823-835CODEN: CHEMGX; ISSN:1860-7179. (Wiley-VCH Verlag GmbH & Co. KGaA)
      A novel and facile synthesis of quinoxalinone derivs. was developed in which a wide range of 3-chloroquinoxalin-2(1H)-ones as key intermediates can be generated chemo- and regioselectively in good yields from corresponding quinoxaline-2,3(1H,4H)-diones. This new protocol is arguably superior, as it allows the design and prepn. of a variety of bioactive quinoxaline-based compds., which are particularly effective in the treatment of diabetes and its complications. Through this procedure, a new class of quinoxalinone-based aldose reductase inhibitors were synthesized successfully. Most of the inhibitors, with an N1-acetic acid head group and a substituted C3-phenoxy side chain, proved to be potent and selective. Their IC50 values ranged from 11.4 to 74.8 nM. Among them, 2-(3-(4-bromophenoxy)-7-fluoro-2-oxoquinoxalin-1(2H)-yl)acetic acid and 2-(6-bromo-3-(4-bromophenoxy)-2-oxoquinoxalin-1(2H)-yl)acetic acid were the most active. Structure-activity relationship and mol. docking studies highlighted the importance of the ether spacer in the C3-phenoxy side chains, and provided clear guidance on the contribution of substitutions both at the core structure and the side chain to activity.
    31. 31
      Rangarajan, M.; Kim, J. S.; Sim, S.-P.; Liu, A.; Liu, L. F.; LaVoie, E. J. Topoisomerase I Inhibition and Cytotoxicity of 5-Bromo- and 5-Phenylterbenzimidazoles. Bioorg. Med. Chem. 2000, 8, 2591– 2600,  DOI: 10.1016/S0968-0896(00)00188-7
      31
      Topoisomerase I inhibition and cytotoxicity of 5-bromo- and 5-phenylterbenzimidazoles
      Rangarajan, Meera; Kim, Jung Sun; Sim, Sai-Peng; Liu, Angela; Liu, Leroy F.; LaVoie, Edmond J.
      Bioorganic & Medicinal Chemistry (2000), 8 (11), 2591-2600CODEN: BMECEP; ISSN:0968-0896. (Elsevier Science Ltd.)
      Topoisomerase I is an enzyme that is essential for maintaining the three-dimensional structure of DNA during the processes of transcription, translation and mitosis. With the introduction of new clin. agents that are effective in poisoning topoisomerase I, this enzyme has proved to be an attractive mol. target in the development of anticancer drugs. Several terbenzimidazoles have been identified as potent topoisomerase I poisons. Structure-activity data on various terbenzimidazoles have revealed that the presence of lipophilic substituents at the 5-position of various terbenzimidazoles correlates with enhanced cytotoxicity. While the effect of having substituents at both the 5- and 6-positions had not been evaluated, previous studies did indicate that the presence of a fused benzo-ring at the 5,6-position results in a significant decrease in topoisomerase I poisoning activity and cytotoxicity. In the present study we investigated whether substituents at both the 5- and 6-positions of varied terbenzimidazoles would allow for retention of topo I poisoning activity. The 6-bromo, 6-methoxy, or 6-Ph derivs. of both 5-bromo- and 5-phenylterbenzimidazole were synthesized and evaluated for topo I poisoning activity, as well as their cytotoxicity toward human lymphoblastoma cells. The data indicate that such derivs. do retain similar topo I poisoning activity and possess cytotoxicity equiv. to either 5-bromo- or 5-phenylterbenzimidazole. Significant enhancement in the topoisomerase I poisoning activity and cytotoxicity of 5-phenylterbenzimidazole is obsd. when the 2"-position is substituted with either a chloro or trifluoromethyl substituent. The influence of such substituents on the biol. activity of 5,6-dibromoterbenzimidazole (I) was also explored. In the case of either 2"-chloro-5,6-dibromoterbenzimidazole or 2"-trifluoromethyl-5,6-dibromoterbenzimidazole (II), topoisomerase I poisoning was not enhanced relative to I. While cytotoxicity toward RPMI 8402 was also not significantly affected comparative studies performed against several solid human tumor cell lines did reveal a significant increase in cytotoxicity obsd. for II as compared to I.
    32. 32
      Lozano-Calderon, S. A.; Colman, M. W.; Raskin, K. A.; Hornicek, F. J.; Gebhardt, M. Use of Bisphosphonates in Orthopedic Surgery. Orthop. Clin. North Am. 2014, 45, 403– 416,  DOI: 10.1016/j.ocl.2014.03.006
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      Use of bisphosphonates in orthopedic surgery: pearls and pitfalls
      Lozano-Calderon Santiago A; Colman Matthew W; Raskin Kevin A; Hornicek Francis J; Gebhardt Mark
      The Orthopedic clinics of North America (2014), 45 (3), 403-16 ISSN:.
      Bisphosphonates are medications known to decrease bone resorption by inhibiting osteoclastic activity. They are the first-line therapy for the treatment of osteoporosis because a significant body of literature has proved their efficacy in reducing the risk of fracture in the hip, spine and other nonvertebral osseous sites. In addition, the use of bisphosphonates has significantly decreased morbidity and increased survival, and they have also proved to be cost-effective. Unexpected adverse effects have been reported recently, but the benefit of bisphosphonates use outweighs the risks. This article reviews the current use of bisphosphonates in orthopedic surgery.
    33. 33
      Gooz, M. ADAM-17: The Enzyme That Does It All. Crit. Rev. Biochem. Mol. Biol. 2010, 45, 146– 169,  DOI: 10.3109/10409231003628015
      33
      ADAM-17: the enzyme that does it all
      Gooz, Monika
      Critical Reviews in Biochemistry and Molecular Biology (2010), 45 (2), 146-169CODEN: CRBBEJ; ISSN:1040-9238. (Informa Healthcare)
      A review. This review focuses on the role of ADAM-17 in disease. Since its debut as the tumor necrosis factor converting enzyme (TACE), ADAM-17 has been reported to be an indispensable regulator of almost every cellular event from proliferation to migration. The central role of ADAM-17 in cell regulation is rooted in its diverse array of substrates: cytokines, growth factors, and their receptors as well as adhesion mols. are activated or inactivated by their cleavage with ADAM-17. It is therefore not surprising that ADAM-17 is implicated in numerous human diseases including cancer, heart disease, diabetes, rheumatoid arthritis, kidney fibrosis, Alzheimer's disease, and is a promising target for future treatments. The specific role of ADAM-17 in the pathophysiol. of these diseases is very complex and depends on the cellular context. To exploit the therapeutic potential of ADAM-17, it is important to understand how its activity is regulated and how specific organs and cells can be targeted to inactivate or activate the enzyme.
    34. 34
      Ropero, S.; Esteller, M. The Role of Histone Deacetylases (HDACs) in Human Cancer. Mol. Oncol. 2007, 1, 19– 25,  DOI: 10.1016/j.molonc.2007.01.001
      34
      The role of histone deacetylases (HDACs) in human cancer
      Ropero, Santiago; Esteller, Manel
      Molecular Oncology (2007), 1 (1), 19-25CODEN: MOONC3; ISSN:1574-7891. (Elsevier B.V.)
      A review. The balance of histone acetylation and deacetylation is an epigenetic layer with a crit. role in the regulation of gene expression. Histone acetylation induced by histone acetyl transferases (HATs) is assocd. with gene transcription, while histone hypoacetylation induced by histone deacetylase (HDAC) activity is assocd. with gene silencing. Altered expression and mutations of genes that encode HDACs have been linked to tumor development since they both induce the aberrant transcription of key genes regulating important cellular functions such as cell proliferation, cell-cycle regulation and apoptosis. Thus, HDACs are among the most promising therapeutic targets for cancer treatment, and they have inspired researchers to study and develop HDAC inhibitors.
    35. 35
      Gelse, K.; Pöschl, E.; Aigner, T. Collagens - Structure, Function, and Biosynthesis. Adv. Drug Deliv. Rev. 2003, 55, 1531– 1546,  DOI: 10.1016/j.addr.2003.08.002
      35
      Collagens - structure, function, and biosynthesis
      Gelse, K.; Poschl, E.; Aigner, T.
      Advanced Drug Delivery Reviews (2003), 55 (12), 1531-1546CODEN: ADDREP; ISSN:0169-409X. (Elsevier Science B.V.)
      A review. The extracellular matrix represents a complex alloy of variable members of diverse protein families defining structural integrity and various physiol. functions. The most abundant family is the collagens with >20 different collagen types identified so far. Collagens are centrally involved in the formation of fibrillar and microfibrillar networks of the extracellular matrix, basement membranes as well as other structures of the extracellular matrix. This review focuses on the distribution and function of various collagen types in different tissues. It introduces their basic structural subunits and points out major steps in the biosynthesis and supramol. processing of fibrillar collagens as prototypical members of this protein family. A final outlook indicates the importance of different collagen types not only for the understanding of collagen-related diseases, but also as a basis for the therapeutic use of members of this protein family.
    36. 36
      Healing, W. The Fibroblast. Proc. R. Soc. Med. 1967, 60, 778– 782,  DOI: 10.1177/003591576706000829
      There is no corresponding record for this reference.
    37. 37
      Lattouf, R.; Younes, R.; Lutomski, D.; Naaman, N.; Godeau, G.; Senni, K.; Changotade, S. Picrosirius Red Staining: A Useful Tool to Appraise Collagen Networks in Normal and Pathological Tissues. J. Histochem. Cytochem. 2014, 62, 751– 758,  DOI: 10.1369/0022155414545787
      37
      Picrosirius red staining: a useful tool to appraise collagen networks in normal and pathological tissues
      Lattouf, Raed; Younes, Ronald; Lutomski, Didier; Naaman, Nada; Godeau, Gaston; Senni, Karim; Changotade, Sylvie
      Journal of Histochemistry and Cytochemistry (2014), 62 (10), 751-758, 8 pp.CODEN: JHCYAS; ISSN:0022-1554. (Sage Publications)
      Specific staining of the extracellular matrix components is esp. helpful in studying tissue remodeling, particularly in the case of connective tissue pathologies. As developed by Junqueira and colleagues in 1979, specific staining by Picrosirius red is one of the most important stains to study collagen networks in different tissues. Under polarized light, collagen bundles appear green, red or yellow, and are easily differentiated from the black background, thus allowing for quant. morphometric anal. As Junqueira and colleagues point out, many studies use color staining to differentiate collagen bundles and to specify collagen types, yet other studies report that polarized colors only reflect fiber thickness and packing. Using a simple histol. example, our study illustrates the inability of Picrosirius red staining to differentiate collagen types, since the absorbed amt. of polarized light by this dye strictly depends on the orientation of the collagen bundles.
    38. 38
      Junqueira, L. C. U.; Cossermelli, W.; Brentani, R. Differential Staining of Collagens Type I, II and III by Sirius Red and Polarization Microscopy. Arch. Histol. Jpn. 1978, 41, 267– 274,  DOI: 10.1679/aohc1950.41.267
      38
      Differential staining of collagens type I, II and III by Sirius Red and polarization microscopy
      Junqueira L C; Cossermelli W; Brentani R
      Archivum histologicum Japonicum = Nihon soshikigaku kiroku (1978), 41 (3), 267-74 ISSN:0004-0681.
      Organs of fish, amphibian, reptile, bird and mammals when stained by Sirius Red and studied with polarization microscopy present different colors in regions where collagens I, II and III have been described. Collagen type I presented a yellow, orange or red color while collagen type III appeared green. Collagen type II, present in cartilage and chondrosarcoma showed a variable color according to the tissue and the species. Its color and morphology however always permitted its clear distinction from collagens type I and type III.
    39. 39
      Das, C.; Lucia MS, H. K.; T, J. Quantification of Lactate Dehydrogenase for Cell Viability Testing Using Cell Lines and Primary Cultured Astrocytes. Physiol. Behav. 2017, 176, 139– 148,  DOI: 10.1002/cptx.21.Quantification
      There is no corresponding record for this reference.
    40. 40
      Lindbäck, T.; Hardy, S. P.; Dietrich, R.; Sødring, M.; Didier, A.; Moravek, M.; Fagerlund, A.; Bock, S.; Nielsen, C.; Casteel, M.; Granum, P. E.; Märtlbauer, E. Cytotoxicity of the Bacillus Cereus Nhe Enterotoxin Requires Specific Binding Order of Its Three Exoprotein Components. Infect. Immun. 2010, 78, 3813– 3821,  DOI: 10.1128/IAI.00247-10
      40
      Cytotoxicity of the Bacillus cereus Nhe enterotoxin requires specific binding order of its three exoprotein components
      Lindback, Toril; Hardy, Simon P.; Dietrich, Richard; Soedring, Marianne; Didier, Andrea; Moravek, Maximilian; Fagerlund, Annette; Bock, Stefanie; Nielsen, Carina; Casteel, Maximilian; Granum, Per Einar; Martlbauer, Erwin
      Infection and Immunity (2010), 78 (9), 3813-3821CODEN: INFIBR; ISSN:0019-9567. (American Society for Microbiology)
      This study focuses on the interaction of the three components of the Bacillus cereus Nhe enterotoxin with particular emphasis on the functional roles of NheB and NheC. The results demonstrated that both NheB and NheC were able to bind to Vero cells directly while NheA lacked this ability. It was also shown that Nhe-induced cytotoxicity required a specific binding order of the individual components whereby the presence of NheC in the priming step as well as the presence of NheA in the final incubation step was mandatory. Priming of cells with NheB alone and addn. of NheA plus NheC in the second step failed to induce toxic effects. Furthermore, in soln., excess NheC inhibited binding of NheB to Vero cells, whereas priming of cells with excess NheC resulted in full toxicity if unbound NheC was removed before addn. of NheB. By using mutated NheC proteins where the two cysteine residues in the predicted β-tongue were replaced with glycine (NheCcys-) or where the entire hydrophobic stretch was deleted (NheChr-), the predicted hydrophobic β-tongue of NheC was found essential for binding to cell membranes but not for interaction with NheB in soln. All data presented here are compatible with the following model. The first step in the mode of action of Nhe is assocd. with binding of NheC and NheB to the cell surface and probably accompanied by conformational changes. These events allow subsequent binding of NheA, leading to cell lysis.
    41. 41
      Tausch, F.; Dietrich, R.; Schauer, K.; Janowski, R.; Niessing, D.; Märtlbauer, E.; Jessberger, N. Evidence for Complex Formation of the Bacillus Cereus Haemolysin BL Components in Solution. Toxins 2017, 9, 1– 18,  DOI: 10.3390/toxins9090288
      There is no corresponding record for this reference.
    42. 42
      Chen, S.; Einspanier, R.; Schoen, J. Transepithelial Electrical Resistance (TEER): A Functional Parameter to Monitor the Quality of Oviduct Epithelial Cells Cultured on Filter Supports. Histochem. Cell Biol. 2015, 144, 509– 515,  DOI: 10.1007/s00418-015-1351-1
      42
      Transepithelial electrical resistance (TEER): a functional parameter to monitor the quality of oviduct epithelial cells cultured on filter supports
      Chen, Shuai; Einspanier, Ralf; Schoen, Jennifer
      Histochemistry and Cell Biology (2015), 144 (5), 509-515CODEN: HCBIFP; ISSN:0948-6143. (Springer)
      Cultivation of oviduct epithelial cells on porous filters fosters in vivo-like morphol. and functionality. However, due to the optical properties of the filter materials and the cells' columnar shape, cell quality is hard to assess via light microscopy. The authors aim to evaluate transepithelial elec. resistance (TEER) measurement as a prognostic quality indicator for the cultivation of porcine oviduct epithelial cells (POEC). POEC were maintained in four different types of media for 3 and 6 wk to achieve diverse culture qualities, and TEER was measured before processing samples for histol. Culture quality was scored using morphol. criteria (presence of cilia, confluence and cell polarity). The authors furthermore analyzed the correlation between cellular height (as a measure of apical-basal polarization) and TEER in fully differentiated routine cultures (biol. variation) and in cultures with altered cellular height due to hormonal stimulation. Fully differentiated cultures possessed a moderate TEER between 500 and 1100 Ω*cm2. Only 5% of cultures which exhibited TEER values in this defined range had poor quality. Sub-differentiated cultures showed either very low or excessively high TEER. The authors unveiled a highly significant (P < 0.0001) neg. linear correlation between TEER and epithelial height in well-differentiated cultures (both routine and hormone stimulated group). This may point toward the interaction between tight junction assembly and epithelial apical-basal polarization. In conclusion, TEER is a straightforward quality indicator which could be routinely used to monitor the differentiation status of oviduct epithelial cells in vitro.
    43. 43
      Srinivasan, B.; Shuler, L.; Hickman, J. J. TEER Measurement Techniques for in Vitro Barrier Model Systems. SLAS Technol. 2015, 20, 107– 126,  DOI: 10.1177/2211068214561025.TEER
      There is no corresponding record for this reference.
    44. 44
      Ramarao, N.; Nielsen-Leroux, C.; Lereclus, D. The Insect Galleria Mellonella as a Powerful Infection Model to Investigate Bacterial Pathogenesis. J. Vis. Exp. 2012, 70, e4392,  DOI: 10.3791/4392
      There is no corresponding record for this reference.
    45. 45
      Blöchl, C.; Regl, C.; Huber, C. G.; Winter, P.; Weiss, R.; Wohlschlager, T. Towards Middle-up Analysis of Polyclonal Antibodies: Subclass-Specific N-Glycosylation Profiling of Murine Immunoglobulin G (IgG) by Means of HPLC-MS. Sci. Rep. 2020, 10, 18080  DOI: 10.1038/s41598-020-75045-1
      45
      Towards middle-up analysis of polyclonal antibodies: subclass-specific N-glycosylation profiling of murine immunoglobulin G (IgG) by means of HPLC-MS
      Blochl Constantin; Regl Christof; Huber Christian G; Wohlschlager Therese; Regl Christof; Huber Christian G; Wohlschlager Therese; Winter Petra; Weiss Richard
      Scientific reports (2020), 10 (1), 18080 ISSN:.
      In recent years, advanced HPLC-MS strategies based on intact protein ("top-down") or protein subunit ("middle-up/middle-down") analysis have been implemented for the characterization of therapeutic monoclonal antibodies. Here, we assess feasibility of middle-up/middle-down analysis for polyclonal IgGs exhibiting extensive sequence variability. Specifically, we addressed IgGs from mouse, representing an important model system in immunological investigations. To obtain Fc/2 portions as conserved subunits of IgGs, we made use of the bacterial protease SpeB. For this purpose, we initially determined SpeB cleavage sites in murine IgGs. The resulting Fc/2 portions characteristic of different subclasses were subsequently analysed by ion-pair reversed-phase HPLC hyphenated to high-resolution mass spectrometry. This enabled simultaneous relative quantification of IgG subclasses and their N-glycosylation variants, both of which influence IgG effector functions. To assess method capabilities in an immunological context, we applied the analytical workflow to polyclonal antibodies obtained from BALB/c mice immunized with the grass pollen allergen Phl p 6. The study revealed a shift in IgG subclasses and Fc-glycosylation patterns in total and antigen-specific IgGs from different mouse cohorts, respectively. Eventually, Fc/2 characterization may reveal other protein modifications including oxidation, amino acid exchanges, and C-terminal lysine, and may thus be implemented for quality control of functional antibodies.
    46. 46
      Dyballa, N.; Metzger, S. Fast and Sensitive Colloidal Coomassie G-250 Staining for Proteins in Polyacrylamide Gels. J. Vis. Exp. 2009, 30, 2– 5,  DOI: 10.3791/1431
      There is no corresponding record for this reference.
    47. 47
      Kabsch, W. XDS. Acta Crystallogr., Sect. D: Biol. Crystallogr. 2010, 66, 125– 132,  DOI: 10.1107/S0907444909047337
      47
      Software XDS for image rotation, recognition and crystal symmetry assignment
      Kabsch, Wolfgang
      Acta Crystallographica, Section D: Biological Crystallography (2010), 66 (2), 125-132CODEN: ABCRE6; ISSN:0907-4449. (International Union of Crystallography)
      The usage and control of recent modifications of the program package XDS for the processing of rotation images are described in the context of previous versions. New features include automatic detn. of spot size and reflecting range and recognition and assignment of crystal symmetry. Moreover, the limitations of earlier package versions on the no. of correction/scaling factors and the representation of pixel contents have been removed. Large program parts have been restructured for parallel processing so that the quality and completeness of collected data can be assessed soon after measurement.
    48. 48
      Evans, P. R.; Murshudov, G. N. How Good Are My Data and What Is the Resolution?. Acta Crystallogr., Sect. D: Biol. Crystallogr. 2013, 69, 1204– 1214,  DOI: 10.1107/S0907444913000061
      48
      How good are my data and what is the resolution?
      Evans, Philip R.; Murshudov, Garib N.
      Acta Crystallographica, Section D: Biological Crystallography (2013), 69 (7), 1204-1214CODEN: ABCRE6; ISSN:0907-4449. (International Union of Crystallography)
      Following integration of the obsd. diffraction spots, the process of data redn.' initially aims to det. the point-group symmetry of the data and the likely space group. This can be performed with the program POINTLESS. The scaling program then puts all the measurements on a common scale, avs. measurements of symmetry-related reflections (using the symmetry detd. previously) and produces many statistics that provide the first important measures of data quality. A new scaling program, AIMLESS, implements scaling models similar to those in SCALA but adds some addnl. analyses. From the analyses, a no. of decisions can be made about the quality of the data and whether some measurements should be discarded. The effective resoln.' of a data set is a difficult and possibly contentious question (particularly with referees of papers) and this is discussed in the light of tests comparing the data-processing statistics with trials of refinement against obsd. and simulated data, and automated model-building and comparison of maps calcd. with different resoln. limits. These trials show that adding weak high-resoln. data beyond the commonly used limits may make some improvement and does no harm.
    49. 49
      McCoy, A. J. Solving Structures of Protein Complexes by Molecular Replacement with Phaser. Acta Crystallogr., Sect. D: Biol. Crystallogr. 2007, 63, 32– 41,  DOI: 10.1107/S0907444906045975
      49
      Solving structures of protein complexes by molecular replacement with Phaser
      McCoy, Airlie J.
      Acta Crystallographica, Section D: Biological Crystallography (2007), 63 (1), 32-41CODEN: ABCRE6; ISSN:0907-4449. (International Union of Crystallography)
      Mol. replacement (MR) generally becomes more difficult as the no. of components in the asym. unit requiring sep. MR models (i.e. the dimensionality of the search) increases. When the proportion of the total scattering contributed by each search component is small, the signal in the search for each component in isolation is weak or non-existent. Maximum-likelihood MR functions enable complex asym. units to be built up from individual components with a 'tree search with pruning' approach. This method, as implemented in the automated search procedure of the program Phaser, has been very successful in solving many previously intractable MR problems. However, there are a no. of cases in which the automated search procedure of Phaser is suboptimal or encounters difficulties. These include cases where there are a large no. of copies of the same component in the asym. unit or where the components of the asym. unit have greatly varying B factors. Two case studies are presented to illustrate how Phaser can be used to best advantage in the std. 'automated MR' mode and two case studies are used to show how to modify the automated search strategy for problematic cases.
    50. 50
      Smart, O. S.; Womack, T. O.; Flensburg, C.; Keller, P.; Paciorek, W.; Sharff, A.; Vonrhein, C.; Bricogne, G. Exploiting Structure Similarity in Refinement: Automated NCS and Target-Structure Restraints in BUSTER. Acta Crystallogr., Sect. D: Biol. Crystallogr. 2012, 68, 368– 380,  DOI: 10.1107/S0907444911056058
      50
      Exploiting structure similarity in refinement: automated NCS and target-structure restraints in BUSTER
      Smart, Oliver S.; Womack, Thomas O.; Flensburg, Claus; Keller, Peter; Paciorek, Wlodek; Sharff, Andrew; Vonrhein, Clemens; Bricogne, Gerard
      Acta Crystallographica, Section D: Biological Crystallography (2012), 68 (4), 368-380CODEN: ABCRE6; ISSN:0907-4449. (International Union of Crystallography)
      Maximum-likelihood X-ray macromol. structure refinement in BUSTER has been extended with restraints facilitating the exploitation of structural similarity. The similarity can be between two or more chains within the structure being refined, thus favoring NCS, or to a distinct 'target' structure that remains fixed during refinement. The local structural similarity restraints (LSSR) approach considers all distances less than 5.5 Å between pairs of atoms in the chain to be restrained. For each, the difference from the distance between the corresponding atoms in the related chain is found. LSSR applies a restraint penalty on each difference. A functional form that reaches a plateau for large differences is used to avoid the restraints distorting parts of the structure that are not similar. Because LSSR are local, there is no need to sep. out domains. Some restraint pruning is still necessary, but this has been automated. LSSR have been available to academic users of BUSTER since 2009 with the easy-to-use and options. The use of LSSR is illustrated in the re-refinement of PDB entries , where enables the correct ligand-binding structure to be found, and , where contributes to the location of an addnl. copy of the cyclic peptide ligand.
    51. 51
      Adams, P. D.; Afonine, P. V.; Bunkóczi, G.; Chen, V. B.; Davis, I. W.; Echols, N.; Headd, J. J.; Hung, L.-W.; Kapral, G. J.; Grosse-Kunstleve, R. W.; McCoy, A. J.; Moriarty, N. W.; Oeffner, R.; Read, R. J.; Richardson, D. C.; Richardson, J. S.; Terwilliger, T. C.; Zwart, P. H. PHENIX: A Comprehensive Python-Based System for Macromolecular Structure Solution. Acta Crystallogr., Sect. D: Biol. Crystallogr. 2010, 66, 213– 221,  DOI: 10.1107/S0907444909052925
      51
      PHENIX: a comprehensive Python-based system for macromolecular structure solution
      Adams, Paul D.; Afonine, Pavel V.; Bunkoczi, Gabor; Chen, Vincent B.; Davis, Ian W.; Echols, Nathaniel; Headd, Jeffrey J.; Hung, Li Wei; Kapral, Gary J.; Grosse-Kunstleve, Ralf W.; McCoy, Airlie J.; Moriarty, Nigel W.; Oeffner, Robert; Read, Randy J.; Richardson, David C.; Richardson, Jane S.; Terwilliger, Thomas C.; Zwart, Peter H.
      Acta Crystallographica, Section D: Biological Crystallography (2010), 66 (2), 213-221CODEN: ABCRE6; ISSN:0907-4449. (International Union of Crystallography)
      A review. Macromol. X-ray crystallog. is routinely applied to understand biol. processes at a mol. level. However, significant time and effort are still required to solve and complete many of these structures because of the need for manual interpretation of complex numerical data using many software packages and the repeated use of interactive three-dimensional graphics. PHENIX has been developed to provide a comprehensive system for macromol. crystallog. structure soln. with an emphasis on the automation of all procedures. This has relied on the development of algorithms that minimize or eliminate subjective input, the development of algorithms that automate procedures that are traditionally performed by hand and, finally, the development of a framework that allows a tight integration between the algorithms.
    52. 52
      Emsley, P.; Lohkamp, B.; Scott, W. G.; Cowtan, K. Features and Development of Coot. Acta Crystallogr., Sect. D: Biol. Crystallogr. 2010, 66, 486– 501,  DOI: 10.1107/S0907444910007493
      52
      Features and development of Coot
      Emsley, P.; Lohkamp, B.; Scott, W. G.; Cowtan, K.
      Acta Crystallographica, Section D: Biological Crystallography (2010), 66 (4), 486-501CODEN: ABCRE6; ISSN:0907-4449. (International Union of Crystallography)
      Coot is a mol.-graphics application for model building and validation of biol. macromols. The program displays electron-d. maps and at. models and allows model manipulations such as idealization, real-space refinement, manual rotation/translation, rigid-body fitting, ligand search, solvation, mutations, rotamers and Ramachandran idealization. Furthermore, tools are provided for model validation as well as interfaces to external programs for refinement, validation and graphics. The software is designed to be easy to learn for novice users, which is achieved by ensuring that tools for common tasks are 'discoverable' through familiar user-interface elements (menus and toolbars) or by intuitive behavior (mouse controls). Recent developments have focused on providing tools for expert users, with customisable key bindings, extensions and an extensive scripting interface. The software is under rapid development, but has already achieved very widespread use within the crystallog. community. The current state of the software is presented, with a description of the facilities available and of some of the underlying methods employed.

  • Supporting Information

    Supporting Information

    The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acs.jmedchem.2c00785.

    • Molecular Formula Strings (CSV)

    • Screening results for ColH and ColQ1, inhibition of MMPs and other off-targets by selected compounds, and cytotoxicity and antibacterial activity of selected compounds (Tables S1–S5); inhibition of the screened compounds on ColQ1 and ColH, activity of the compounds on Col I cleavage, on NHDF infected cells, on TEER of MDCK II cells, and on G. mellonella infection model; 1H NMR and 13C NMR and LC-MS spectra for final compounds and data collection and refinement statistics (Figures S4–S17) (PDF)


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