Rational Design of Hit Compounds Targeting Staphylococcus aureus Threonyl-tRNA Synthetase
- Mariia Yu. RybakMariia Yu. RybakDepartment of Protein Synthesis Enzymology, Institute of Molecular Biology and Genetics National Academy of Sciences of Ukraine, 150 Zabolotnogo Street, Kyiv 03143, UkraineMore by Mariia Yu. Rybak,
- Olga I. GudzeraOlga I. GudzeraDepartment of Protein Synthesis Enzymology, Institute of Molecular Biology and Genetics National Academy of Sciences of Ukraine, 150 Zabolotnogo Street, Kyiv 03143, UkraineMore by Olga I. Gudzera,
- Oksana B. GorbatiukOksana B. GorbatiukDepartment of Cell Regulatory Mechanisms, Institute of Molecular Biology and Genetics National Academy of Sciences of Ukraine, 150 Zabolotnogo Street, Kyiv 03143, UkraineMore by Oksana B. Gorbatiuk,
- Mariia O. UsenkoMariia O. UsenkoDepartment of Cell Regulatory Mechanisms, Institute of Molecular Biology and Genetics National Academy of Sciences of Ukraine, 150 Zabolotnogo Street, Kyiv 03143, UkraineMore by Mariia O. Usenko,
- Sergiy M. YarmolukSergiy M. YarmolukDepartment of Medicinal Chemistry, Institute of Molecular Biology and Genetics National Academy of Sciences of Ukraine, 150 Zabolotnogo Street, Kyiv 03143, UkraineMore by Sergiy M. Yarmoluk,
- Michael A. TukaloMichael A. TukaloDepartment of Protein Synthesis Enzymology, Institute of Molecular Biology and Genetics National Academy of Sciences of Ukraine, 150 Zabolotnogo Street, Kyiv 03143, UkraineMore by Michael A. Tukalo, and
- Galyna P. Volynets*Galyna P. Volynets*Email: [email protected]Department of Medicinal Chemistry, Institute of Molecular Biology and Genetics National Academy of Sciences of Ukraine, 150 Zabolotnogo Street, Kyiv 03143, UkraineThe Scientific-Services Company “OTAVA”, 150 Zabolotnogo Street, Kyiv 03143, UkraineMore by Galyna P. Volynets
Copyright © 2021 The Authors. Published by American Chemical Society
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Abstract
Staphylococcus aureus is one of the most dangerous nosocomial pathogens which cause a wide variety of hospital-acquired infectious diseases. S. aureus is considered as a superbug due to the development of multidrug resistance to all current therapeutic regimens. Therefore, the discovery of antibiotics with novel mechanisms of action to combat staphylococcal infections is of high priority for modern medicinal chemistry. Nowadays, aminoacyl-tRNA synthetases are considered as promising molecular targets for antibiotic development. In the present study, we used for the first time S. aureus threonyl-tRNA synthetase (ThrRS) as a molecular target. Recombinant S. aureus ThrRS was obtained in the soluble form in a sufficient amount for inhibitor screening assay. Using the molecular docking approach, we selected 180 compounds for investigation of inhibitory activity toward ThrRS. Among the tested compounds, we identified five inhibitors from different chemical classes decreasing the activity of ThrRS by more than 70% at a concentration of 100 μM. The most active compound 2,4-dibromo-6-{[4-(4-nitro-phenyl)-thiazol-2-yl]-hydrazonomethyl}-phenol has an IC50 value of 56.5 ± 3.5 μM. These compounds are not cytotoxic toward eukaryotic cells HEK293 (EC50 > 100 μM) and can be useful for further optimization and biological research.
Introduction
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Staphylococcus aureus is an opportunistic Gram-positive pathogen that causes a wide range of hospital-acquired human diseases ranging from skin infections and abscesses to much more severe endocarditis, osteomyelitis, pneumonia, meningitis, sepsis, and so forth. (1−8) The major problem of staphylococcal infection treatment is the multidrug resistance to all antibiotics currently used in clinic, including methicillin, vancomycin, daptomycin, and linezolid. (9−15) Therefore, the development of antistaphylococcal agents with novel mechanisms of action is of urgent need.
Nowadays, aminoacyl-tRNA synthetases are recognized as promising molecular targets for antibiotic development. (16−18) Aminoacyl-tRNA synthetases are key enzymes in protein synthesis which ligate amino acids to cognate transfer RNAs being involved in the early stages of translation of the genetic code. Most living cells possess 20 aminoacyl-tRNA synthetases for each of the standard amino acids. These enzymes are divided into two classes, class I and class II, which have different folds of catalytic domain and different preferences for the hydroxyl group of the tRNA. (19,20) Aminoacyl-tRNA synthetases possess some structural divergence in prokaryotic and eukaryotic organisms which increases the possibility for development of selective inhibitors toward enzymes from pathogenic microorganisms in comparison with human homologues. Aminoacyl-tRNA synthetases are conservative among bacteria, suggesting that compounds targeting these enzymes may demonstrate a wide range of antibacterial activity. A number of available crystal structures of aminoacyl-tRNA synthetases provide a basis for receptor-based drug design and virtual screening. Aminoacyl-tRNA synthetases are well expressed in the soluble form and can be used for high-throughput screening. Threonyl-tRNA synthetase (ThrRS) is a promising molecular target for malaria treatment. (21,22) For example, borrelidin, polyketide macrolide, isolated from Streptomyces species, is a potent inhibitor of ThrRS from Plasmodium falciparum. (23,24) Due to high toxicity of this compound, a series of borrelidin derivatives with lower toxicity has been synthesized. (22,25,26) The inhibitors of ThrRS from Haemophilus influenza, Escherichia coli, and Burkholderia thailandensis possessing antibacterial activity have been reported among sulfonamide derivatives. (27) Recently, the inhibitors of Salmonella enterica ThrRS among quinazolinone derivatives have been identified using fragment-based target hopping assay by Guo et al. (28) The most active compound demonstrates an IC50 value of 1.4 μM toward Salmonella enterica ThrRS. It should be noted that this compound revealed antibacterial activity toward Escherichia coli ATCC25922 and S. enterica with an MIC value of 16 μg/mL and toward S. aureus ATCC29213, S. aureus R3708, and Enterococcus faecalis ATCC29212 with an MIC value of 32 μg/mL. (28) Furthermore, these authors performed structure-based optimization of quinazolinone-threonine hybrids and discovered the inhibitor of S. enterica ThrRS with an IC50 value of 0.5 μM and MIC values of 16–32 μg/mL toward the tested bacterial strains. (29)
For the best of our knowledge, none of the small-molecular inhibitors of ThrRS from S. aureus have been reported in scientific literature so far. Therefore, the aim of this study is to obtain recombinant S. aureus ThrRS and identify inhibitors of this enzyme with molecular docking into the available crystal structure of ThrRS. (30)
Results and Discussion
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Cloning and Purification of S. aureus Threonyl-tRNA Synthetase
The gene encoding S. aureus ThrRS (1938 bp) with restriction sites for NcoI and HindIII was synthesized by ATG/biosynthetics GmbH (Merzhausen, Germany). To obtain the required amount of the S. aureus ThrRS gene for ligation, we performed preparative restriction of 5 μg of plasmid DNA pGE-ThrRS(NcoI-HindIII) from the clones of E. coli Top10 cells with NcoI and HindIII. The linear DNA fragment was purified from gel using the NucleoSpin gel extraction kit (Macherey-Nagel) protocol.
The ThrRS gene fragment was ligated with a dephosphorylated vector pET28b linearized with NcoI and HindIII. E. coli cells TOP10 were electroporated with the plasmid pET28b-ThrRS. Plasmid DNA of clones with the correct fragments was prepared with a GeneJET Plasmid Miniprep Kit (Thermo Scientific). Transformation efficiency was analyzed with NcoI and HindIII. All tested plasmids contained a fragment of the expected size.
The most optimal conditions for expression of recombinant S. aureus ThrRS in the soluble fraction are the following: E. coli BL21(DE3)pLysS cells in LB medium, with induction by 0.25 mM isopropyl β-d-1-thiogalactopyranoside (IPTG) for 3 h at 37 °C or E. coli Lemo21(DE3) cells in 2YT medium supplemented with 250 μM l-rhamnose at 30 °C during 16–18 h. The procedure for selection of optimal expression conditions for S. aureus ThrRS is available in Supporting Information (Figures S1–S10).
The protein was purified by DEAE Sepharose anion chromatography using a salt gradient from 25 to 500 mM NaCl (Figures S11, S12). The resulting eluate was concentrated and loaded onto a pre-equilibrated Heparin Sepharose 6 Fast Flow column and then eluted using the KCl gradient (from 0 to 300 mM) (Figure S13). The protein was concentrated to 3.5 mg/mL and stored with 50% glycerol at −80 °C. The total yield of purified ThrRS (>95% purity) is 10 mg from 1 L starting bacterial culture. This protocol of purification was used for obtaining the required amount of recombinant S. aureus ThrRS for compound screening.
Rational Design of S. aureus ThrRS Inhibitors
In order to identify small-molecular inhibitors of S. aureus ThrRS, we have performed molecular docking of OTAVA compound library containing 124,831 compounds. (31) According to molecular docking results, we have selected 180 compounds for investigation of their inhibitory activity toward S. aureus ThrRS in an aminoacylation assay. The inhibitory activity for all tested compounds (%) (measured at least in duplicates) is available in Supporting Information (Table S1). Among the tested compounds, we identified active compounds belonging to five chemical classes—the derivatives of 2-(benzothiazol-2-ylsulfanyl)-1-phenyl-ethanone, 2-phenoxy-N-phenyl-acetamide, 4-phenyl-2-propionylamino-thiophene-3-carboxylic acid ethyl ester, 1-phenyl-pyrrolidine-2,5-dione, and N-benzylidene-N′-(4-phenyl-thiazol-2-yl)-hydrazine.
The compound 1-(4-methoxy-phenyl)-2-(6-nitro-benzothiazol-2-ylsulfanyl)-ethanone (compound 1) inhibits S. aureus ThrRS by 88.47% at a concentration of 100 μM. The IC50 value for this compound is 158 ± 72 μM (measured in duplicates). According to molecular docking results, this compound interacts simultaneously with adenine-binding and amino acid-binding regions of ThrRS (Figure 1). The superposition of compound 1 and the threonyl adenylate analogue, extracted from the crystal structure of ThrRS (PDB ID: 1NYQ), in the active site is presented in Figure 2. As it can be seen in Figure 1, 4-methoxy-phenyl interacts with amino acid residues in the adenine-binding site and forms a hydrogen bond with Arg377, the benzothiazol ring interacts with the amino acid residues in the threonyl-binding region, and the nitro group at the C6 position of this heterocycle builds hydrogen bonds with Cys336 and Tyr468.
Figure 1
Figure 1. Chemical structure of 1-(4-methoxy-phenyl)-2-(6-nitro-benzothiazol-2-ylsulfanyl)-ethanone (compound 1) (a). The complex of compound 1 with amino acid residues in the active site of S. aureus ThrRS. The hydrogen bonds are shown by green dashed lines with the distances indicated in Å, the hydrophobic interactions are presented by magenta dashed lines, π-sulfur interactions are shown by yellow dashed lines, and π-cation interactions are indicated with orange dashed lines (b).
Figure 2
Figure 2. Superposition of the threonyl adenylate analogue, extracted from the crystal structure with the PDB accession code: 1NYQ (carbon atoms are labeled with magenta color) and compound 1 (carbon atoms are labeled with blue color) in the active site of S. aureus threonyl—tRNA synthetase.
In the present study, we tested six derivatives of 2-phenoxy-N-phenyl-acetamide for inhibitory activity toward S. aureus ThrRS. As it can be noticed from Table 1, the most active compound 2-(2-tert-butyl-phenoxy)-N-(2-methyl-5-nitro-phenyl)-acetamide (compound 2) inhibits S. aureus ThrRS by 85.59% at a concentration of 100 μM. According to molecular docking results, tert-butyl of this compound interacts with the amino acid residues in the adenine-binding region of ThrRS, the nitro-phenyl ring interacts with the amino acid residues in the threonyl-binding region, and the nitro group forms the metal-acceptor bond with Zn (Figure 3).
Figure 3
Figure 3. Complex of 2-(2-tert-butyl-phenoxy)-N-(2-methyl-5-nitro-phenyl)-acetamide (compound 2) with amino acid residues in the active site of S. aureus ThrRS. The hydrophobic interactions are indicated with magenta dashed lines, π-cation interactions are shown by orange dashed lines, and the metal-acceptor bond is presented by the gray dashed line.
Table 1. Structures and In Vitro Inhibitory Activity of 2-Phenoxy-N-phenyl-acetamide Derivatives toward S. aureus ThrRS
We have made superposition of the most active compounds 1 and 2 in the aminoacyl-adenylate binding site of ThrRS (Figure S14). It was found that these ligands have a similar binding mode. Both compounds 1 and 2 interact simultaneously with adenine- and threonyl-binding regions and form π-cation interactions with the same residues Arg365 and Arg526. Possibly, the formation of hydrogen bonds of the nitro group in compound 1 is more profitable for inhibitory activity than the formation of the metal-acceptor bond of the nitro group in compound 2 with Zn.
In this research, three derivatives of 4-phenyl-2-propionylamino-thiophene-3-carboxylic acid ethyl ester were tested for inhibitory activity toward S. aureus ThrRS. As it can be seen from Table 2, the most active compound—4-(4-chloro-phenyl)-2-[2-(2,6-dimethyl-morpholin-4-yl)-acetylamino]-thiophene-3-carboxylic acid ethyl ester (compound 8), inhibits S. aureus ThrRS by 78.33%. According to molecular docking results, 2,6-dimethyl-morpholine heterocycle of this compound interacts with the amino acid residues in the adenine-binding region of ThrRS and forms a hydrogen bond with Arg377 and 4-chloro-phenyl interacts with the threonyl-binding region (Figure 4).
Figure 4
Figure 4. Complex of 4-(4-chloro-phenyl)-2-[2-(2,6-dimethyl-morpholin-4-yl)-acetylamino]-thiophene-3-carboxylic acid ethyl ester (compound 8) with amino acid residues in the active site of S. aureus ThrRS. The hydrogen bonds are shown by green dashed lines with the distances indicated in Å, the hydrophobic interactions are indicated with magenta dashed lines, π-sulfur interaction is presented by yellow dashed lines, and the halogen bond is shown by blue dashed lines.
Table 2. Structures and In Vitro Inhibitory Activity of 4-Phenyl-2-propionylamino-thiophene-3-carboxylic Acid Ethyl Ester Derivatives toward S. aureus ThrRS
Among the investigated compounds, we have found two inhibitors of S. aureus ThrRS belonging to 1-phenyl-pyrrolidine-2,5-dione derivatives (Table 3). The compound 2-[1-(4-iodo-2-methyl-phenyl)-2,5-dioxo-pyrrolidin-3-ylsulfanyl]-nicotinic acid (compound 11) inhibits ThrRS by 78.62% at a concentration of 100 μM. According to molecular docking results, the nicotinic acid moiety of this compound interacts with the adenine-binding region of ThrRS and forms a hydrogen bond with Arg377 and 1-iodo-3-methyl-phenyl is located in the threonyl-binding region (Figure 5).
Figure 5
Figure 5. Complex of 2-[1-(4-iodo-2-methyl-phenyl)-2,5-dioxo-pyrrolidin-3-ylsulfanyl]-nicotinic acid (compound 11) with amino acid residues in the active site of S. aureus ThrRS. The hydrogen bonds are shown by green dashed lines with the distances indicated in Å, the hydrophobic interactions are presented by magenta dashed lines, π-cation interactions are indicated with orange dashed lines, and the metal-acceptor bond is shown by gray dashed lines.
Table 3. Structures and In Vitro Inhibitory Activity of 1-Phenyl-pyrrolidine-2,5-dione Derivatives toward S. aureus ThrRS
According to biochemical testing, N-benzylidene-N′-(4-phenyl-thiazol-2-yl)-hydrazine derivatives possess inhibitory activity toward S. aureus ThrRS. It should be noted that earlier, the compounds from this chemical class were reported by us as the inhibitors of M. tuberculosis leucyl-tRNA synthetase and methionyl-tRNA synthetase. (32,33) Therefore, the derivatives of N-benzylidene-N′-(4-phenyl-thiazol-2-yl)-hydrazine inhibit both classes of aminoacyl-tRNA synthetases.
As it can be seen from Table 4, among the 13 tested N-benzylidene-N′-(4-phenyl-thiazol-2-yl)-hydrazine derivatives, the most active compound −2,4-dibromo-6-{[4-(4-nitro-phenyl)-thiazol-2-yl]-hydrazonomethyl}-phenol (compound 13) inhibits ThrRS by 72.65%. The IC50 value for this compound is 56.5 ± 3.5 μM (measured in duplicates). Molecular docking results demonstrate that nitro-phenyl interacts with the amino acid residues in the threonyl-binding region of ThrRS and 2,4-dibromophenyl is located in the adenine-binding region of S. aureus ThrRS (Figure 6).
Figure 6
Figure 6. Complex of 2,4-dibromo-6-{[4-(4-nitro-phenyl)-thiazol-2-yl]-hydrazonomethyl}-phenol (compound 13) with amino acid residues in the active site of S. aureus ThrRS. The hydrogen bonds are shown by green dashed lines with the distances indicated in Å, the hydrophobic interactions are presented by magenta dashed lines, π-cation interaction is indicated with orange dashed lines, π-sulfur interaction is presented by yellow dashed lines, and the metal-acceptor bond is shown by gray dashed lines.
Table 4. Structures and In Vitro Inhibitory Activity of N-Benzylidene-N′-(4-phenyl-thiazol-2-yl)-hydrazine Derivatives toward S. aureus ThrRS
We have tested five hit compounds for cytotoxicity toward the human cell line HEK293 using standard MTT assay. (34) It was found that compounds 1, 2, 8, and 13 are not cytotoxic at a concentration of 100 μM and only compound 11 reveals slight toxicity, decreasing the growth of HEK293 cells by 30% at a concentration of 100 μM (EC50 > 100 μM).
Conclusions
ARTICLE SECTIONS
Using the rational design approach, we identified five hit compounds from different chemical classes, inhibiting S. aureus threonyl-tRNA synthetase (ThrRS) by more than 70% at a concentration of 100 μM. The most active compound 2,4-dibromo-6-{[4-(4-nitro-phenyl)-thiazol-2-yl]-hydrazonomethyl}-phenol has an IC50 value of 56.5 ± 3.5 μM. According to the results of molecular docking, the inhibitors interact simultaneously with adenine- and threonyl-binding regions of S. aureus ThrRS. These compounds are not cytotoxic toward the human cell line HEK293 and can be used for further chemical optimization and biological research. It should be noted that the derivatives of N-benzylidene-N′-(4-phenyl-thiazol-2-yl)-hydrazine inhibit both classes of aminoacyl-tRNA synthetases from different microorganisms and can be considered for the development of broad spectrum antibiotics.
Methods
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Molecular Docking
Semiflexible molecular docking of the small-molecular compounds into the active site of the S. aureus ThrRS crystal structure (PDB ID: 1NYQ) was performed with the program DOCK 4.0. (35−38) The molecules of 5′-O-(N-(l-threonyl)-sulfamoyl)adenosine and water were removed from the crystal structure, but the Zn ion was kept in the active site.
The ligand geometry was calculated using the YFF force field. (39) The partial atomic charges for the ligands were assigned with the Kirchhoff method. (40) The partial atomic charges for the receptor were set with the Amber force field. The spheres of the active site were predicted using sphgen software from the DOCK package. The spheres which were outside of the aminoacyl-adenylate binding site of ThrRS were deleted manually. The energy grids of the receptor were generated using the grid program from the DOCK package.
The docking of the compound library into the aminoacyl-adenylate binding site of ThrRS was performed using the parameters described earlier. (32)
After the docking procedure, we have performed complex analysis to select compounds for in vitro screening. At first, compounds were ranged based on score values (less than −40 kcal/mol). Then, the compounds were evaluated for the ability to form hydrogen bonds with amino acid residues in the aminoacyl adenylate binding site of S. aureus ThrRS, such as Tyr468, Met334, Gln490, Asp385, Arg365, Ser522, Thr523, and Val378 using our in-house program y_hbonds, which is based on the analysis of distances and angles between the respective donor and acceptor atoms. Considering these criteria, we have chosen about 12,000 compounds for further visual inspection of receptor–ligand complexes. During visual analysis, we evaluated correctness of torsion angles, stacking interactions, complementarity of ligand and receptor surfaces, and so forth. Taking into account all these parameters, we have selected 180 compounds for investigation of their inhibitory activity toward recombinant S. aureus ThrRS.
Visual analysis of the receptor–ligand complexes was performed using Discovery Studio Visualizer 4.0. (41)
Cloning of ThrRS
The gene encoding ThrRS with restriction sites for NcoI and HindIII was synthesized by ATG/biosynthetics GmbH (Merzhausen, Germany) based on sequence information of the S. aureus ThrRS gene (GenBank ID: ABD30857.1). The linearized DNA fragment pGE(NcoI-HindIII)-ThrRS-S was ligated to the plasmid vector pET28b (Novagen) which was previously dephosphorylated with phosphatase in 1× reaction buffer (Roche). Ligation was performed with T4 DNA ligase in 1× ligase buffer (Promega) for 2 h at 23 °C. E. coli TOP10 cells (Invitrogen) were transformed by electroporation using a Bio-Rad Gene Pulsar. The screening of positive clones was performed using the GeneJET Plasmid Miniprep Kit (Thermo Scientific). Positive clones were identified using restrictases NcoI and HindIII FastDigest (Thermo Scientific).
ThrRS Expression
The plasmid pET28b-ThrRS was transformed into E. coli BL21(DE3)pLysS and Lemo21(DE3) competent cells. The level of expression was analyzed in LB (Lauria-Broth), TB (Terrific-Broth), phosphate, and 2×YT media with 50 μg/mL kanamycin. The bacterial growth was continued until the OD600 reached around 0.6. The protein synthesis was induced by IPTG in the range of concentration from 0.025 mM to 1 mM at 18, 25, and 37 °C.
Aliquots after 3–4 h and overnight induction were analyzed for solubility according to the protocol of Zerbs et al. (42)
For ThrRS expression, a modified (43) autoinduction protocol (44) was also used. E. coli BL21(DE3)pLysS and Lemo21(DE3) cells harboring the plasmid pET28b-ThrRS were incubated at 37 °C overnight in 2 mL of 2×YT medium containing 50 μg/mL kanamycin and 1% glucose. 1:1000 dilutions of the overnight culture were taken for inoculation of 2×YT medium supplemented with 40 μg/mL kanamycin, 25 mM (NH4)2SO4, 50 mM KH2PO4, 50 mM Na2HPO4, 1 mM MgSO4, 0.05% glucose, 0.2% α-lactose, and 0.5% glycerol. The cultures were incubated at 18/30/37 °C for 16–18 h under conditions of intensive aeration.
Purification of S. aureus ThrRS
E. coli cells BL21(DE3)pLysS were grown in the medium LB until the OD600 reached 0.6, and the expression of ThrRS was induced by 0.25 mM IPTG for 3 h at 37 °C. The cells were pelleted by centrifugation (15 min at 6000 g at 4 °C), and the pellets were stored at −80 °C. The bacterial pellet was dissolved in buffer A containing 100 mM Tris-HCl (pH 8.0), 300 mM NaCl, 10 mM DTT, 5 mM PMSF, 4% glycerol, 1 mM MgCl2, 0.01 mg/mL lysozyme, and protease inhibitor cocktail tablet, EDTA-free. The cells were incubated on ice for 30 min and sonicated for 5 min with four sonicated bursts of 25 s followed by interval of 60 s for cooling. The cell pellet was precipitated by centrifugation at 20000g for 25 min at 4 °C. The supernatant was dialyzed in buffer B containing 20 mM Tris-HCl (pH 8.0), 2 mM DTT, 0.1 mM PMSF, and 5 mM MgCl2 for 3 h at 4 °C. Then, the buffer was changed and dialyzed overnight. After dialysis, the lysate was loaded onto a DEAE Sepharose column (Amersham Biosciences) (V = 6.5 mL), washed at a flow rate of 1 mL/min, and eluted with a gradient of NaCl from 25 to 500 mM (2 × 50 mL). Fractions were analyzed by the Bradford method (45) and SDS-PAGE. Fractions containing ThrRS were joined and concentrated on an Amicon Ultra-4 at 10 K MWCO spin column (Millipore, Billerica, MA) at 4 °C to remove salt impurities and loaded onto the Heparin Sepharose 6 Fast Flow column (GE Healthcare) (0.5 × 10 cm), washed with buffer B and eluted with a gradient of KCl from 0 to 300 mM (2 × 25 mL). The peak fractions, containing ThrRS, were joined and concentrated on Amicon Ultra-4 (7000 g, 4 °C).
Protein concentrations were determined by the Bradford method. (45) The absorption coefficient at λ = 280 nm (ε280 = 73355 M–1 cm–1) and the absorption of 0.1% solution (A280 (1 mg/mL) = 0.985 mg–1 mL) were calculated from the S. aureus ThrRS amino acid sequence using the ExPASy ProtParam tool (46) and taken for determination of enzyme concentration.
In Vitro Aminoacylation Assay
The standard aminoacylation assay was performed in the reaction mixture (20 μM) containing 50 mM HEPES-NaOH (pH 7.5), 20 mM MgCl2, 5 mM β-mercaptoethanol, 30 mM KCl, 100 μg/mL BSA, 4 mg/mL of the total E. coli MRE600 tRNA, 27 μM [14C]-L-Thr, and 500 nM recombinant S. aureus ThrRS with appropriate concentrations of the compound (dissolved in DMSO). The reactions were initiated by the addition of 10 mM ATP and incubated for 10 min at 37 °C. The reaction was stopped by the addition of 10% trichloroacetic acid and was loaded on the GF/C filter, washed with 5% trichloroacetic acid, dried, and counted using a scintillation counter. Aliquots were quenched with 10% trichloracetic acid, and the level of tRNA aminoacylation was measured using a scintillation counter [Hidex 600 SL liquid scintillation analyzer (Finland)].
MTT Assay
The toxicity of compounds toward the HEK293 cell was examined using a standard MTT assay. (35) The cells were cultivated in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum, 100 mg/mL streptomycin, and 100 mg/mL penicillin in humidified air with 5% CO2 at 37 °C. The cells were seeded into 96-well plates at a concentration of 2 × 105 cells/mL and grown for 24 h. Then, the cells were supplemented with compounds in DMSO solution (final DMSO concentration was less than 0.5%) at different concentrations. After 72 h of incubation, the cells were treated with 15 μL of MTT solution (5 mg/mL) for 4 h at 37 °C, 5% CO2. The formazan precipitates were dissolved in 200 μL of DMSO, and the absorbance at λ = 540 nm was measured with spectrofluorometer MR 700 (Dynatech). The cell viability was calculated as a percentage relative to intact control cells.
Supporting Information
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The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsomega.1c03789.
Analysis of ThrRS expression in E. coli BL21(DE3)pLysS cells; analysis of expression of the ThrRS soluble fraction with induction by 0.5 or 1 mM IPTG concentrations at 37 °C; analysis of expression of the ThrRS soluble fraction with induction by 0.25, 0.5, or 1 mM IPTG concentrations at 25 °C; analysis of ThrRS expression at 0.1 and 0.25 mM IPTG concentration and in the medium with 3% ethanol after induction for 3 h at 37 °C; expression of the ThrRS soluble fraction with induction by 0.1, 0.25, and 1 mM IPTG concentration and in the medium with 3% ethanol for 3 h at 18 and 25 °C; expression of the ThrRS soluble fraction after overnight induction at 18 and 25 °C; expression of the ThrRS soluble fraction in Lemo21(DE3) E. coli cells in LB medium after 4 h of induction at 30 °C with 0.4 mM IPTG and various concentrations of l-rhamnose; expression of the ThrRS soluble fraction in Lemo21(DE3) E. coli cells in LB medium after overnight induction at 30 °C; expression of S. aureus ThrRS using the autoinduction protocol; SDS-PAGE analysis of soluble and insoluble fractions of E. coli proteins after induction expression of S. aureus ThrRS by autoinduction conditions (10 μL of culture per lane was loaded); elution profile (absorbance at 280 nm versus elution volume) of the anion exchanger DEAE-Sepharose; purification of S. aureus ThrRS using the DEAE-Sepharose column; purification of S. aureus ThrRS using the heparin-Sepharose column; superposition of compound 1 (carbon atoms are labeled with blue color) and compound 2 (carbon atoms are labeled with magenta color) in the active site of S. aureus threonyl-tRNA synthetase; and structures and in vitro inhibitory activity toward S. aureus ThrRS for compounds, selected according to molecular docking results (PDF)
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Author Information
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- Galyna P. Volynets - Department of Medicinal Chemistry, Institute of Molecular Biology and Genetics National Academy of Sciences of Ukraine, 150 Zabolotnogo Street, Kyiv 03143, Ukraine; The Scientific-Services Company “OTAVA”, 150 Zabolotnogo Street, Kyiv 03143, Ukraine;
https://orcid.org/0000-0002-0166-2642;
- Mariia Yu. Rybak - Department of Protein Synthesis Enzymology, Institute of Molecular Biology and Genetics National Academy of Sciences of Ukraine, 150 Zabolotnogo Street, Kyiv 03143, Ukraine;https://orcid.org/0000-0002-0478-0222
Acknowledgments
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This work was supported by the NAS of the Ukraine grant for young scientists (project no 0120U000079) for 2020–2021.
References
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This article references 46 other publications.
- 1Krishna, S.; Miller, L. S. Host-pathogen interactions between the skin and Staphylococcus aureus. Curr. Opin. Microbiol. 2012, 15, 28– 35, DOI: 10.1016/j.mib.2011.11.003[Crossref], [PubMed], [CAS], Google Scholar1https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BC387mvFGhsA%253D%253D&md5=478c2056528833971c2fc0ac792684ceHost-pathogen interactions between the skin and Staphylococcus aureusKrishna Sheila; Miller Lloyd SCurrent opinion in microbiology (2012), 15 (1), 28-35 ISSN:.Staphylococcus aureus is responsible for the vast majority of bacterial skin infections in humans. The propensity for S. aureus to infect skin involves a balance between cutaneous immune defense mechanisms and virulence factors of the pathogen. The tissue architecture of the skin is different from other epithelia especially since it possesses a corneal layer, which is an important barrier that protects against the pathogenic microorganisms in the environment. The skin surface, epidermis, and dermis all contribute to host defense against S. aureus. Conversely, S. aureus utilizes various mechanisms to evade these host defenses to promote colonization and infection of the skin. This review will focus on host-pathogen interactions at the skin interface during the pathogenesis of S. aureus colonization and infection.
- 2Welte, T.; Kantecki, M.; Stone, G. G.; Hammond, J. Ceftaroline fosamil as a potential treatment option for Staphylococcus aureus community-acquired pneumonia in adults. Int. J. Antimicrob. Agents 2019, 54, 410– 422, DOI: 10.1016/j.ijantimicag.2019.08.012[Crossref], [PubMed], [CAS], Google Scholar2https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1MXhslens7jN&md5=7658ca7aa0d179ba432198480eafe160Ceftaroline fosamil as a potential treatment option for Staphylococcus aureus community-acquired pneumonia in adultsWelte, Tobias; Kantecki, Michal; Stone, Gregory G.; Hammond, JenniferInternational Journal of Antimicrobial Agents (2019), 54 (4), 410-422CODEN: IAAGEA; ISSN:0924-8579. (Elsevier B.V.)A review. Staphylococcus aureus (S. aureus), including methicillin-resistant S. aureus (MRSA), is an important etiol. cause of community-acquired pneumonia (CAP) and assocd. with significant morbidity and mortality. Empiric therapy for CAP frequently consists of β-lactam monotherapy or β-lactam/macrolide combination therapy. However, such agents are often ineffective against S. aureus and do not reflect the emergence and increasing prevalence of MRSA in the community setting. Ceftaroline fosamil is a fifth-generation parenteral cephalosporin with broad-spectrum activity against Gram-pos. pathogens - such as S. aureus (including MRSA), Streptococcus pneumoniae and Streptococcus pyogenes - and typical Gram-neg. pathogens, including Haemophilus influenzae and Moraxella catarrhalis. The approval of ceftaroline fosamil in the United States and Europe for the treatment of adults with moderate-to-severe CAP was based on two phase 3 trials (FOCUS 1 and 2), which demonstrated that ceftaroline fosamil was non-inferior to ceftriaxone, a std. empiric treatment for CAP, while exhibiting a comparable safety profile. Although head-to-head trials of ceftaroline fosamil vs. comparators against MRSA CAP are lacking, the effectiveness of ceftaroline fosamil in subpopulations of patients not covered by phase 3 trials (e.g. those with MRSA CAP or severe renal impairment) has been demonstrated in the Clin. Assessment Program and Teflaro Utilization Registry (CAPTURE) study. As ineffective empiric therapy is assocd. with adverse outcomes, including mortality and increased costs, ceftaroline fosamil, with its extended spectrum of activity, is an attractive alternative to std. antibiotic CAP regimens.
- 3Jang, Y.-R.; Kim, T.; Kim, M.-C.; Sup Sung, H.; Kim, M.-N.; Kim, M. J.; Kim, S. H.; Lee, S.-O.; Choi, S.-H.; Woo, J. H.; Kim, Y. S.; Chong, Y. P. Sternoclavicular septic arthritis caused by Staphylococcus aureus: excellent results from medical treatment and limited surgery. Infect. Dis. 2019, 51, 694– 700, DOI: 10.1080/23744235.2019.1639810[Crossref], [CAS], Google Scholar3https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1MXhtlKjsL%252FN&md5=394959812dc09f323db66d2d3865751aSternoclavicular septic arthritis caused by Staphylococcus aureus: excellent results from medical treatment and limited surgeryJang, Young-Rock; Kim, Taeeun; Kim, Min-Chul; Sung, Heung Sup; Kim, Mi-Na; Kim, Min Jae; Kim, Sung Han; Lee, Sang-Oh; Choi, Sang-Ho; Woo, Jun Hee; Kim, Yang Soo; Chong, Yong PilInfectious Diseases (2019), 51 (9), 694-700CODEN: IDNIAU; ISSN:2374-4243. (Taylor & Francis Ltd.)Aggressive surgery such as en bloc joint resection is favored for treating uncommon sternoclavicular (SC) septic arthritis, based on expert opinion and small case series. It analyzed the clin. characteristics and treatment outcomes of patients with Staphylococcus aureus SC septic arthritis treated medically or with limited surgery. All adult patients with this septic arthritis at the Asan Medical Center between Sept. 2009 and Dec. 2016 were reviewed. Limited surgery was defined as simple incision, drainage, and debridement of the infected joint. Of 22 patients enrolled, 11 received medical treatment only, and 11 underwent limited surgery, and none underwent aggressive surgery. Most patients (73%) had underlying predisposing conditions such as infection at a distant site, diabetes and liver cirrhosis, and none had i.v. drug abuse or HIV infection. Complications such as chest wall and/or neck abscess, clavicular and/or sternal osteomyelitis were identified in 18 patients (82%). Patients with chest wall and/or neck abscesses tended more often to undergo limited surgery than patients without such abscesses (73% vs. 27%, p = .09). The median duration of i.v. antibiotics was 35 days (IQR, 25-46 days). Treatment was successful in all cases. In a median 53-wk follow-up (IQR, 8-171 wk), there was no relapse of arthritis or joint deterioration. Medical treatment alone or with limited surgery could be successful therapeutic strategies for complicated S. aureus SC septic arthritis in selected patients.
- 4Tascini, C.; Attanasio, V.; Ripa, M.; Carozza, A.; Pallotto, C.; Bernardo, M.; Francisci, D.; Oltolini, C.; Palmiero, G.; Scarpellini, P. Ceftobiprole for the treatment of infective endocarditis: a case series. J. Glob. Antimicrob. Resist. 2020, 20, 56– 59, DOI: 10.1016/j.jgar.2019.07.020[Crossref], [PubMed], [CAS], Google Scholar4https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BB3MvhvVKqtw%253D%253D&md5=1ca81aee6c5fc65f045b9aad96e35208Ceftobiprole for the treatment of infective endocarditis: A case seriesTascini Carlo; Attanasio Vittorio; Palmiero Giulia; Ripa Marco; Oltolini Chiara; Scarpellini Paolo; Carozza Antonio; Pallotto Carlo; Bernardo Mariano; Francisci DanielaJournal of global antimicrobial resistance (2020), 20 (), 56-59 ISSN:.OBJECTIVES: Ceftobiprole is a relatively new cephalosporin with broad-spectrum activity and good tolerability. Despite its promising characteristics, to our knowledge, only two case reports, previously published also by some of us, is available concerning its administration for the treatment of infective endocarditis. Hereby we report our experience in this field. METHODS: All the patients with infective endocarditis treated with ceftobiprole were enrolled. RESULTS: 12 cases of endocarditis were treated with ceftobiprole, 11/12 in combination with daptomycin and 1/12 as monotherapy. Gram-positive bacteria were isolated in 12/12 patients; 3 cases were polymicrobial. Cure rate was 83% (10/12 patients). In 9/12 (75%) cases, patients were switched to ceftobiprole following failure of previous antimicrobial regimen. In 3/3 patients in which ceftobiprole was administered because of persistently positive blood culture, bacteraemia clearance was rapidly achieved. CONCLUSIONS: Ceftobiprole, especially in combination, could be a promising alternative treatment for infective endocarditis.
- 5Dugourd, P.-M.; Dupont, A.; Hubiche, T.; Chiaverini, C.; Alkhalifa, A.; Roudiere, L.; Tristan, A.; Gustave, C.-A.; Del Giudice, P. Érythème généralisé fébrile et choc : choc toxinique staphylococcique. Ann. Dermatol. Venereol. 2019, 146, 287– 291, DOI: 10.1016/j.annder.2018.12.002[Crossref], [PubMed], [CAS], Google Scholar5https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BB3cjmt1Omtw%253D%253D&md5=861a8d08009f3c3b3d64d79cc95614baStaphylococcal toxic shock syndrome should be considered in the event of diffuse erythema with fever and shockDugourd P-M; Dupont A; Hubiche T; Del Giudice P; Chiaverini C; Alkhalifa A; Roudiere L; Tristan A; Gustave C-AAnnales de dermatologie et de venereologie (2019), 146 (4), 287-291 ISSN:0151-9638.BACKGROUND: Toxic shock syndrome (TSS) was first described by Todd in 1978. The relevant Lancet publication reported 7 cases of children with fever, exanthema, hypotension and diarrhoea associated with multiple organ failure. An association between TSS and use of hyper-absorbent tampons in menstruating women was discovered in the 1980s. Following the market withdrawal of such tampons, TSS virtually disappeared. Herein we report a new case of TSS in a 15-year-old girl. PATIENTS AND METHODS: A 15-year-old patient was admitted to intensive care for severe sepsis and impaired consciousness associated with diffuse abdominal pain. Dermatological examination revealed diffuse macular exanthema. Laboratory tests showed hepatic cytolysis (ASAT 101 U/L, ALAT 167 U/L, total bilirubin 68μmol/L) and an inflammatory syndrome. Lumbar puncture and blood cultures were sterile while thoraco-abdomino-pelvic and brain scans were normal. The patient was menstruating and had been using a tampon over the previous 24hours. Vaginal sampling and tampon culture revealed TSST-1 toxin-producing S. aureus. Management consisted of intensive care measures and treatment with amoxicillin-clavulanic acid and clindamycin for 10 days. CONCLUSION: In case of septic shock associated with diffuse macular exanthema a diagnosis of TSS must be envisaged, particularly in menstruating women.
- 6Toledo, A. G.; Golden, G.; Campos, A. R.; Cuello, H.; Sorrentino, J.; Lewis, N.; Varki, N.; Nizet, V.; Smith, J. W.; Esko, J. D. Proteomic atlas of organ vasculopathies triggered by Staphylococcus aureus sepsis. Nat. Commun. 2019, 10, 4656, DOI: 10.1038/s41467-019-12672-x[Crossref], [PubMed], [CAS], Google Scholar6https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BB3MnntFWkug%253D%253D&md5=01131ef2560d996b312ea20f6c540b4aProteomic atlas of organ vasculopathies triggered by Staphylococcus aureus sepsisToledo Alejandro Gomez; Golden Gregory; Esko Jeffrey D; Toledo Alejandro Gomez; Golden Gregory; Varki Nissi; Esko Jeffrey D; Campos Alexandre Rosa; Cuello Hector; Sorrentino James; Lewis Nathan; Lewis Nathan; Lewis Nathan; Varki Nissi; Nizet Victor; Nizet Victor; Smith Jeffrey WNature communications (2019), 10 (1), 4656 ISSN:.Sepsis is a life-threatening condition triggered by a dysregulated host response to microbial infection resulting in vascular dysfunction, organ failure and death. Here we provide a semi-quantitative atlas of the murine vascular cell-surface proteome at the organ level, and how it changes during sepsis. Using in vivo chemical labeling and high-resolution mass spectrometry, we demonstrate the presence of a vascular proteome that is perfusable and shared across multiple organs. This proteome is enriched in membrane-anchored proteins, including multiple regulators of endothelial barrier functions and innate immunity. Further, we automated our workflows and applied them to a murine model of methicillin-resistant Staphylococcus aureus (MRSA) sepsis to unravel changes during systemic inflammatory responses. We provide an organ-specific atlas of both systemic and local changes of the vascular proteome triggered by sepsis. Collectively, the data indicates that MRSA-sepsis triggers extensive proteome remodeling of the vascular cell surfaces, in a tissue-specific manner.
- 7Bergin, S. P.; Holland, T. L.; Fowler, V. G., Jr.; Tong, S. Y. C. Bacteremia, sepsis, and infective endocarditis associated with Staphylococcus aureus. Curr. Top. Microbiol. Immunol. 2015, 409, 263– 296, DOI: 10.1007/82_2015_5001
- 8Zhang, Y.; Zhang, J.; Chen, W.; Angsantikul, P.; Spiekermann, K. A.; Fang, R. H.; Gao, W.; Zhang, L. Erythrocyte membrane-coated nanogel for combinatorial antivirulence and responsive antimicrobial delivery against Staphylococcus aureus infection. J. Controlled Release 2017, 263, 185– 191, DOI: 10.1016/j.jconrel.2017.01.016[Crossref], [PubMed], [CAS], Google Scholar8https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXhtFehs78%253D&md5=9fee13f24eabeaf6a9b91f5d0cefe98dErythrocyte membrane-coated nanogel for combinatorial antivirulence and responsive antimicrobial delivery against Staphylococcus aureus infectionZhang, Yue; Zhang, Jianhua; Chen, Wansong; Angsantikul, Pavimol; Spiekermann, Kevin A.; Fang, Ronnie H.; Gao, Weiwei; Zhang, LiangfangJournal of Controlled Release (2017), 263 (), 185-191CODEN: JCREEC; ISSN:0168-3659. (Elsevier B.V.)We reported an erythrocyte membrane-coated nanogel (RBC-nanogel) system with combinatorial antivirulence and responsive antibiotic delivery for the treatment of methicillin-resistant Staphylococcus aureus (MRSA) infection. RBC membrane was coated onto the nanogel via a membrane vesicle templated in situ gelation process, whereas the redox-responsiveness was achieved by using a disulfide bond-based crosslinker. We demonstrated that the RBC-nanogels effectively neutralized MRSA-assocd. toxins in extracellular environment and the toxin neutralization in turn promoted bacterial uptake by macrophages. In intracellular reducing environment, the RBC-nanogels showed an accelerated drug release profile, which resulted in more effective bacterial inhibition. When added to the macrophages infected with intracellular MRSA bacteria, the RBC-nanogels significantly inhibited bacterial growth compared to free antibiotics and non-responsive nanogel counterparts. These results indicate the great potential of the RBC-nanogel system as a new and effective antimicrobial agent against MRSA infection.
- 9Gardete, S.; Tomasz, A. Mechanisms of vancomycin resistance in Staphylococcus aureus. J. Clin. Invest. 2014, 124, 2836– 2840, DOI: 10.1172/jci68834[Crossref], [PubMed], [CAS], Google Scholar9https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BC2cfosFektw%253D%253D&md5=65c16134b7595389112856744ca0a9e4Mechanisms of vancomycin resistance in Staphylococcus aureusGardete Susana; Tomasz AlexanderThe Journal of clinical investigation (2014), 124 (7), 2836-40 ISSN:.Vancomycin is a glycopeptide antibiotic used for the treatment of Gram-positive bacterial infections. Traditionally, it has been used as a drug of last resort; however, clinical isolates of methicillin-resistant Staphylococcus aureus (MRSA) strains with decreased susceptibility to vancomycin (vancomycin intermediate-resistant S. aureus [VISA]) and more recently with high-level vancomycin resistance (vancomycin-resistant S. aureus [VRSA]) have been described in the clinical literature. The rare VRSA strains carry transposon Tn1546, acquired from vancomycin-resistant Enterococcus faecalis, which is known to alter cell wall structure and metabolism, but the resistance mechanisms in VISA isolates are less well defined. Herein, we review selected mechanistic aspects of resistance in VISA and summarize biochemical studies on cell wall synthesis in a VRSA strain. Finally, we recapitulate a model that integrates common mechanistic features of VRSA and VISA strains and is consistent with the mode of action of vancomycin.
- 10Kali, A. Antibiotics and bioactive natural products in treatment of methicillin resistant Staphylococcus aureus: A brief review. Pharmacogn. Rev. 2015, 9, 29– 34, DOI: 10.4103/0973-7847.156329[Crossref], [PubMed], [CAS], Google Scholar10https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXoslSls70%253D&md5=bcb5673a9b6e44a95abe2a8fb10f18d7Antibiotics and bioactive natural products in treatment of methicillin-resistant Staphylococcus aureus: a brief reviewKali, ArunavaPharmacognosy Reviews (2015), 9 (17), 29-34CODEN: PRHEEV; ISSN:0973-7847. (Medknow Publications and Media Pvt. Ltd.)A review. Infections caused by Staphylococcus aureus strains with Methicillin resistance are assocd. with increased mortality and morbidity, aggressive course, multiple drug resistance and hospital outbreaks. Several first and second line antibiotics are rapidly becoming ineffective for treatment due to emergence of resistance. Exts. of medicinal plants are rich source of unique phytochems. Plants used in traditional medicine have been reported to have significant anti-MRSA activity. The objective of this review is to provide a brief overview of antibiotics as well as anti-MRSA natural products and their future prospect.
- 11Kaur, D.; Chate, S. Study of antibiotic resistance pattern in methicillin resistant staphylococcus aureus with special reference to newer antibiotic. J. Global Infect. Dis. 2015, 7, 78– 84, DOI: 10.4103/0974-777x.157245[Crossref], [PubMed], [CAS], Google Scholar11https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXitVGjtrrJ&md5=ff4ea27e6f3506b9c7d9cd3af6e425d1Study of antibiotic resistance pattern in methicillin resistant staphylococcus aureus with special reference to newer antibioticKaur, Dardi Charan; Chate, Sadhana SanjayJournal of Global Infectious Diseases (2015), 7 (2), 78-84CODEN: JGIDAM; ISSN:0974-8245. (Medknow Publications and Media Pvt. Ltd.)The worldwide epidemic of antibiotic resistance is in danger of ending the golden age of antibiotic therapy. Resistance impacts on all areas of medicine, making successful empirical therapy much more difficult to achieve. Staphylococcus aureus demonstrates a unique ability to quickly respond to each antibiotic with the development of a resistance mechanism, starting with penicillin,until the most recent, linezolid and daptomycin. Methicillin resistant aureus (MRSA) has become endemic today in hospitals worldwide. Resistance to the newer antimicrobial-agents - linezolid, vancomycin, teicoplanin, daptomycin are been reported and also the fear of pandrug-resistance. This study was carried out to know the antimicrobial resistant pattern of MRSA to newer antibiotic, to know any isolates are extensively-drug resistant (XDR)/pandrug resistant (PDR), inducible macrolide-lincosamide streptogramin B (iMLSB), mupirocin resistance. Thirty-six MRSA isolates resistant to the routinely tested antibiotic were further tested for list of antibiotic by a group of international experts. Isolates were tested for iMLSB and mupirocin resistance by the disk diffusion method. Of 385 MRSA, 36 (9.35%) isolates of MRSA were resistant to routinely tested antibiotic. Among these 36 MRSA isolates, none of our isolates were XDR/PDR or showed resistant to anti-MRSA cephalosporins (ceftaroline), phosphonic acids, glycopeptides, glycylcyclines, and fucidanes. Lower resistance was seen in oxazolidinones (2.78%), streptogramins (5.56%), lipopeptide (5.56%). Thirty-four (94.44%) isolates showed constitutive MLSB (cMLSB) resistance and two (5.56%) iMLSB phenotypes. High- and low-level mupirocin resistance were in 13 (36.11%) and six (16.67%), resp. In our study, none of our isolates were XDR or PDR. No resistance was obsd. to ceftaroline, telavancin, teicoplanin, and vancomycin; but the presence of linezolid resistance (1, 2.28%) and daptomycin resistance (2, 5.56%) in our rural set-up is a cause of concern.
- 12Arunkumar, V.; Prabagaravarthanan, R.; Bhaskar, M. Prevalence of methicillin-resistant Staphylococcus aureus (MRSA) infections among patients admitted in critical care units in a tertiary care hospital. Int. J. Res. Med. Sci. 2017, 5, 2362– 2366
- 13McGuinness, W. A.; Malachowa, N.; DeLeo, F. R. Vancomycin Resistance in Staphylococcus aureus. Yale J. Biol. Med. 2017, 90, 269– 281[PubMed], [CAS], Google Scholar13https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1MXhslOktr0%253D&md5=56ebbfb1e8a82bb29e528b54ea328cc5Vancomycin resistance in Staphylococcus aureusMcGuinness, Will A.; Malachowa, Natalia; DeLeo, Frank R.Yale Journal of Biology and Medicine (2017), 90 (2), 269-281CODEN: YJBMAU; ISSN:0044-0086. (Yale Journal of Biology and Medicine)The evolution of Staphylococcus aureus during the modern antibiotic era has been delineated by distinct strain emergence events, many of which include acquisition of antibiotic resistance. The relative high burden of methicillin-resistant S. aureus (MRSA) in healthcare and community settings is a major concern worldwide. Vancomycin, a glycopeptide antibiotic that inhibits cell wall biosynthesis, remains a drug of choice for treatment of severe MRSA infections. S. aureus strains exhibiting increased resistance to vancomycin, known as vancomycin intermediate-resistant S. aureus (VISA) (MIC = 4-8 μg/mL), were discovered in the 1990s. The mol. basis of resistance in VISA is polygenic and involves stepwise mutations in genes encoding mols. predominantly involved in cell envelope biosynthesis. S. aureus isolates with complete resistance to vancomycin (MIC ≥ 16 μg/mL) are termed vancomycin-resistant S. aureus (VRSA)-they were first reported in the U.S. in 2002. Resistance in VRSA is conferred by the vanA gene and operon, which is present on a plasmid. Although treatment of VRSA infections is challenging, the total no. of human VRSA infections to date is limited (14 in the U.S.). By comparison, the burden of VISA is relatively high and the mol. mechanisms of resistance are less well-defined. VISA are assocd. with persistent infections, vancomycin treatment failure, and poor clin. outcomes. Here, we review in brief progress made toward understanding the acquisition of antibiotic resistance in S. aureus, with an emphasis on the mol. mechanisms underlying vancomycin resistance.
- 14Marty, F. M.; Yeh, W. W.; Wennersten, C. B.; Venkataraman, L.; Albano, E.; Alyea, E. P.; Gold, H. S.; Baden, L. R.; Pillai, S. K. Emergence of a clinical daptomycin-resistant Staphylococcus aureus isolate during treatment of methicillin-resistant Staphylococcus aureus bacteremia and osteomyelitis. J. Clin. Microbiol. 2006, 44, 595– 597, DOI: 10.1128/jcm.44.2.595-597.2006[Crossref], [PubMed], [CAS], Google Scholar14https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD28XitFSgsro%253D&md5=a7cd9c5b21a552e05b5a92d203336163Emergence of a clinical daptomycin-resistant Staphylococcus aureus isolate during treatment of methicillin-resistant Staphylococcus aureus bacteremia and osteomyelitisMarty, Francisco M.; Yeh, Wendy W.; Wennersten, Christine B.; Venkataraman, Lata; Albano, Esperanza; Alyea, Edwin P.; Gold, Howard S.; Baden, Lindsey R.; Pillai, Satish K.Journal of Clinical Microbiology (2006), 44 (2), 595-597CODEN: JCMIDW; ISSN:0095-1137. (American Society for Microbiology)The emergence of a clin. daptomycin-resistant Staphylococcus aureus isolate occurred during treatment of methicillin-resistant S. aureus bacteremia and probable vertebral osteomyelitis. The breakthrough isolate was indistinguishable from pretreatment daptomycin-susceptible isolates by pulsed-field gel electrophoresis. Daptomycin nonsusceptibility was confirmed by MIC and time-kill curve analyses.
- 15Ikeda-Dantsuji, Y.; Hanaki, H.; Sakai, F.; Yanagisawa, C.; Nakae, T.; Sakai, F.; Tomono, K.; Takesue, Y.; Honda, J.; Nonomiya, Y.; Suwabe, A.; Nagura, O.; Yanagihara, K.; Mikamo, H.; Fukuchi, K.; Kaku, M.; Kohno, S.; Yoshida, K.; Niki, Y. Linezolid-resistant Staphylococcus aureus isolated from 2006 through 2008 at six hospitals in Japan. J. Infect. Chemother. 2011, 17, 45– 51, DOI: 10.1007/s10156-010-0085-1[Crossref], [PubMed], [CAS], Google Scholar15https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3MXhvV2iu74%253D&md5=7265ce121b0824d409caa05821b971caLinezolid-resistant Staphylococcus aureus isolated from 2006 through 2008 at six hospitals in JapanIkeda-Dantsuji, Yurika; Hanaki, Hideaki; Sakai, Fuminori; Tomono, Kazunori; Takesue, Yoshio; Honda, Junichi; Nonomiya, Yuriko; Suwabe, Akira; Nagura, Osanori; Yanagihara, Katsunori; Mikamo, Hiroshige; Fukuchi, Kunihiko; Kaku, Mitsuo; Kohno, Shigeru; Yanagisawa, Chie; Nakae, Taiji; Yoshida, Koichiro; Niki, YoshihitoJournal of Infection and Chemotherapy (2011), 17 (1), 45-51CODEN: JICHFN; ISSN:1341-321X. (Springer Japan)Limited use of linezolid for treating methicillin-resistant Staphylococcus aureus (MRSA) infection was approved in Japan in 2006. We report here the status of linezolid-resistant MRSAs in Japan. Eleven linezolid-resistant clin. isolates from 11 patients at six hospitals were collected from 2006 through 2008. The minimal inhibitory concn. (MIC) of linezolid in these strains varied from 8 to 64 μg/mL. All strains had at least one G2576T mutation in the chromosomal gene(s) encoding domain V of the 23S rRNA (rRNA). Chromosomal DNA encoding five copies of the domain V region was analyzed by polymerase chain reaction (PCR). Strains with the linezolid MICs of 64, 32, 16, and 8 μg/mL had the G2576T mutation(s) in four, three (or four), two, and one copy of the 23S rRNA genes, resp. These results suggest that the level of linezolid resistance seems to be roughly correlated with the no. of mutations in the genes encoding 23S rRNA. DNA samples from all 11 strains were subjected to pulsed-field gel electrophoresis and were classified into seven independent clones having >92% identity. Among the 11 patients, five had been treated with linezolid and the remainder, in two hospitals, had no history of prior linezolid use. The results suggested possible nosocomial infections by linezolid-resistant MRSA.
- 16Pang, L.; Weeks, S. D.; Van Aerschot, A. Aminoacyl-tRNA synthetases as valuable targets for antimicrobial drug discovery. Int. J. Mol. Sci. 2021, 22, 1750, DOI: 10.3390/ijms22041750[Crossref], [PubMed], [CAS], Google Scholar16https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3MXotVKqt70%253D&md5=b732971a63f06fe876036b21b8e3f546Aminoacyl-tRNA synthetases as valuable targets for antimicrobial drug discoveryPang, Luping; Weeks, Stephen d.; Van aerschot, ArthurInternational Journal of Molecular Sciences (2021), 22 (4), 1750CODEN: IJMCFK; ISSN:1422-0067. (MDPI AG)A review. Aminoacyl-tRNA synthetases (aaRSs) catalyze the esterification of tRNA with a cognate amino acid and are essential enzymes in all three kingdoms of life. Due to their important role in the translation of the genetic code, aaRSs have been recognized as suitable targets for the development of small mol. anti-infectives. In this review, following a concise discussion of aaRS catalytic and proof-reading activities, the various inhibitory mechanisms of reported natural and synthetic aaRS inhibitors are discussed. Using the expanding repository of ligand-bound X-ray crystal structures, we classified these compds. based on their binding sites, focusing on their ability to compete with the assocn. of one, or more of the canonical aaRS substrates. In parallel, we examd. the determinants of species-selectivity and discuss potential resistance mechanisms of some of the inhibitor classes. Combined, this structural perspective highlights the opportunities for further exploration of the aaRS enzyme family as antimicrobial targets.
- 17Kwon, N. H.; Fox, P. L.; Kim, S. Aminoacyl-tRNA synthetases as therapeutic targets. Nat. Rev. Drug Discovery 2019, 18, 629– 650, DOI: 10.1038/s41573-019-0026-3[Crossref], [PubMed], [CAS], Google Scholar17https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1MXptlajtrY%253D&md5=dd9c680cd873d35c8ab10350cd7fb044Aminoacyl-tRNA synthetases as therapeutic targetsKwon, Nam Hoon; Fox, Paul L.; Kim, SunghoonNature Reviews Drug Discovery (2019), 18 (8), 629-650CODEN: NRDDAG; ISSN:1474-1776. (Nature Research)A review. Aminoacyl-tRNA synthetases (ARSs) are essential enzymes for protein synthesis with evolutionarily conserved enzymic mechanisms. Despite their similarity across organisms, scientists have been able to generate effective anti-infective agents based on the structural differences in the catalytic clefts of ARSs from pathogens and humans. However, recent genomic, proteomic and functionomic advances have unveiled unexpected disease-assocd. mutations and altered expression, secretion and interactions in human ARSs, revealing hidden biol. functions beyond their catalytic roles in protein synthesis. These studies have also brought to light their potential as a rich and unexplored source for new therapeutic targets and agents through multiple avenues, including direct targeting of the catalytic sites, controlling disease-assocd. protein-protein interactions and developing novel biologics from the secreted ARS proteins or their parts. This Review addresses the emerging biol. and therapeutic applications of human ARSs in diseases including autoimmune and rare diseases, and cancer.
- 18Ho, J. M.; Bakkalbasi, E.; Söll, D.; Miller, C. A. Drugging tRNA aminoacylation. RNA Biol. 2018, 15, 667– 677, DOI: 10.1080/15476286.2018.1429879[Crossref], [PubMed], [CAS], Google Scholar18https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BC1MvhvVCgug%253D%253D&md5=129b1a19b33997a85f6af42a4f575341Drugging tRNA aminoacylationHo Joanne M; Bakkalbasi Erol; Miller Corwin A; Soll Dieter; Soll DieterRNA biology (2018), 15 (4-5), 667-677 ISSN:.Inhibition of tRNA aminoacylation has proven to be an effective antimicrobial strategy, impeding an essential step of protein synthesis. Mupirocin, the well-known selective inhibitor of bacterial isoleucyl-tRNA synthetase, is one of three aminoacylation inhibitors now approved for human or animal use. However, design of novel aminoacylation inhibitors is complicated by the steadfast requirement to avoid off-target inhibition of protein synthesis in human cells. Here we review available data regarding known aminoacylation inhibitors as well as key amino-acid residues in aminoacyl-tRNA synthetases (aaRSs) and nucleotides in tRNA that determine the specificity and strength of the aaRS-tRNA interaction. Unlike most ligand-protein interactions, the aaRS-tRNA recognition interaction represents coevolution of both the tRNA and aaRS structures to conserve the specificity of aminoacylation. This property means that many determinants of tRNA recognition in pathogens have diverged from those of humans-a phenomenon that provides a valuable source of data for antimicrobial drug development.
- 19Ribas de Pouplana, L.; Schimmel, P. Two classes of tRNA synthetases suggested by sterically compatible dockings on tRNA acceptor stem. Cell 2001, 104, 191– 193, DOI: 10.1016/s0092-8674(01)00204-5[Crossref], [PubMed], [CAS], Google Scholar19https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BD3M7mslajtw%253D%253D&md5=f91390856e9ce2ee119d926cea3ea8a7Two classes of tRNA synthetases suggested by sterically compatible dockings on tRNA acceptor stemRibas de Pouplana L; Schimmel PCell (2001), 104 (2), 191-3 ISSN:0092-8674.There is no expanded citation for this reference.
- 20Eriani, G.; Delarue, M.; Poch, O.; Gangloff, J.; Moras, D. Partition of tRNA synthetases into two classes based on mutually exclusive sets of sequence motifs. Nature 1990, 347, 203– 206, DOI: 10.1038/347203a0[Crossref], [PubMed], [CAS], Google Scholar20https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK3MXpslensg%253D%253D&md5=3b8c5ed8a05996c6d9e97ebcb3b1b7ecPartition of tRNA synthetases into two classes based on mutually exclusive sets of sequence motifsEriani, Gilbert; Delarue, Marc; Poch, Olivier; Gangloff, Jean; Moras, DinoNature (London, United Kingdom) (1990), 347 (6289), 203-6CODEN: NATUAS; ISSN:0028-0836.Out of 18 known aminoacyl-tRNA synthetases (aaRS), only 9 referred to as class I synthetases (GlnRS, TyrRS, MetRS, GluRS, ArgRS, ValRS, IleRS, LeuRS, TrpRS), display 2 short common consensus sequences. The sequence of Escherichia coli ProRS, a dimer of relative mol. mass 127.402, which is homologous to both ThrRS and SerRS, is reported. These 3 latter aaRS share 3 new sequence motifs with AspRS, AsnRS, LysRS, HisRS, and the B subunit of PheRS. These 3 motifs (motifs 1, 2, and 3), in a search through the entire data bank, proved to be specific for this set of aaRS (referred to as class II). Class II may also contain AlaRS and GlyRS, because these sequences have a typical motif 3. Surprisingly, this partition of aaRS in 2 classes is strongly correlated on the functional level with the acylation occurring either on the 2' OH (class I) or 3' OH (class II) of the ribose of the last nucleotide of tRNA.
- 21Novoa, E. M.; Camacho, N.; Tor, A.; Wilkinson, B.; Moss, S.; Marín-García, P.; Azcárate, I. G.; Bautista, J. M.; Mirando, A. C.; Francklyn, C. S.; Varon, S.; Royo, M.; Cortés, A.; Ribas de Pouplana, L. Analogs of natural aminoacyl-tRNA synthetase inhibitors clear malaria in vivo. Proc. Natl. Acad. Sci. U.S.A. 2014, 111, E5508– E5517, DOI: 10.1073/pnas.1405994111[Crossref], [PubMed], [CAS], Google Scholar21https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2cXitVClu7nL&md5=9878f848d956e30171b3356867dd33f6Analogs of natural aminoacyl-tRNA synthetase inhibitors clear malaria in vivoNovoa, Eva Maria; Camacho, Noelia; Tor, Anna; Wilkinson, Barrie; Moss, Steven; Marin-Garcia, Patricia; Azcarate, Isabel G.; Bautista, Jose M.; Mirando, Adam C.; Francklyn, Christopher S.; Varon, Sonia; Royo, Miriam; Cortes, Alfred; Ribas de Pouplana, LluisProceedings of the National Academy of Sciences of the United States of America (2014), 111 (51), E5508-E5517CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)Malaria remains a major global health problem. Emerging resistance to existing antimalarial drugs drives the search for new antimalarials, and protein translation is a promising pathway to target. Here we explore the potential of the aminoacyl-tRNA synthetase (ARS) family as a source of antimalarial drug targets. First, a battery of known and novel ARS inhibitors was tested against Plasmodium falciparum cultures, and their activities were compared. Borrelidin, a natural inhibitor of threonyl-tRNA synthetase (ThrRS), stands out for its potent antimalarial effect. However, it also inhibits human ThrRS and is highly toxic to human cells. To circumvent this problem, we tested a library of bioengineered and semisynthetic borrelidin analogs for their antimalarial activity and toxicity. We found that some analogs effectively lose their toxicity against human cells while retaining a potent antiparasitic activity both in vitro and in vivo and cleared malaria from Plasmodium yoelii-infected mice, resulting in 100% mice survival rates. Our work identifies borrelidin analogs as potent, selective, and unexplored scaffolds that efficiently clear malaria both in vitro and in vivo.
- 22Saint-Léger, A.; Sinadinos, C.; Ribas de Pouplana, L. The growing pipeline of natural aminoacyl-tRNA synthetase inhibitors for malaria treatment. Bioengineered 2016, 7, 60– 64, DOI: 10.1080/21655979.2016.1149270[Crossref], [PubMed], [CAS], Google Scholar22https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28XlslWgsLo%253D&md5=c1495beaba0de9abc1150af425db0350The growing pipeline of natural aminoacyl-tRNA synthetase inhibitors for malaria treatmentSaint-Leger, Adelaide; Sinadinos, Christopher; Ribas de Pouplana, LluisBioengineered (2016), 7 (2), 60-64CODEN: BIOEGL; ISSN:2165-5987. (Taylor & Francis, Inc.)Malaria remains a major global health problem. Parasite resistance to existing drugs makes development of new antimalarials an urgency. The protein synthesis machinery is an excellent target for the development of new anti-infectives, and aminoacyl-tRNA synthetases (aaRS) have been validated as antimalarial drug targets. However, avoiding the emergence of drug resistance and improving selectivity to target aaRS in apicomplexan parasites, such as Plasmodium falciparum, remain crucial challenges. Here we discuss such issues using examples of known inhibitors of P. falciparum aaRS, namely halofuginone, cladosporin and borrelidin (inhibitors of ProRS, LysRS and ThrRS, resp.). Encouraging recent results provide useful guidelines to facilitate the development of novel drug candidates which are more potent and selective against these essential enzymes.
- 23Hütter, R.; Poralla, K.; Zachau, H. G.; Zähner, H. Metabolic products of microorganisms. 5l. On the mechanism of action of borrelidin-inhibition of the threonine incorporation in sRNA. Biochem. Z. 1966, 344, 190– 196[PubMed], [CAS], Google Scholar23https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaF28XpsFKgsA%253D%253D&md5=e090e8ea25a54b9c860c7f64e1b79b22Metabolic products of microorganisms. LI. Mechanism of action of borrelidin. Inhibition of the incorporation of threonine into sRNA (soluble RNA)Huetter, R.; Poralla, K.; Zachau, H. G.; Zehner, H.Biochemische Zeitschrift (1966), 344 (2), 190-6CODEN: BIZEA2; ISSN:0366-0753.cf. CA 64, 15963e. The mode of action of the antibiotic borrelidin has been studied. The expts. indicate that borrelidin inhibits esp. the incorporation of threonine into sRNA.
- 24Paetz, W.; Nass, G. Biochemical and Immunological Characterization of Threonyl-tRNA Synthetase of Two Borrelidin-Resistant Mutants of Escherichia coli K12. Eur. J. Med. Chem. 1973, 35, 331– 337, DOI: 10.1111/j.1432-1033.1973.tb02843.x
- 25Wilkinson, B.; Gregory, M. A.; Moss, S. J.; Carletti, I.; Sheridan, R. M.; Kaja, A.; Ward, M.; Olano, C.; Mendez, C.; Salas, J. A.; Leadlay, P. F.; van Ginckel, R.; Zhang, M.-Q. Separation of anti-angiogenic and cytotoxic activities of borrelidin by modification at the C17 side chain. Bioorg. Med. Chem. Lett. 2006, 16, 5814– 5817, DOI: 10.1016/j.bmcl.2006.08.073[Crossref], [PubMed], [CAS], Google Scholar25https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD28XhtVCrt7zI&md5=9a7c1cc1b881e7c6e395d8e527d48031Separation of anti-angiogenic and cytotoxic activities of borrelidin by modification at the C17 side chainWilkinson, Barrie; Gregory, Matthew A.; Moss, Steven J.; Carletti, Isabelle; Sheridan, Rose M.; Kaja, Andrew; Ward, Michael; Olano, Carlos; Mendez, Carmen; Salas, Jose A.; Leadlay, Peter F.; van Ginckel, Rob; Zhang, Ming-QiangBioorganic & Medicinal Chemistry Letters (2006), 16 (22), 5814-5817CODEN: BMCLE8; ISSN:0960-894X. (Elsevier Ltd.)A set of novel borrelidin analogs have been prepd. by precursor-directed biosynthesis. Structure-activity relationship anal. suggests that steric structural arrangement within the C17 side chain is important for differentiating cytotoxic and antiangiogenic activities. A C17-cyclobutyl analog 3 was found to have markedly increased selectivity for in vitro angiogenesis inhibition over cytotoxicity and is therefore potentially useful as an anticancer agent.
- 26Sugawara, A.; Tanaka, T.; Hirose, T.; Ishiyama, A.; Iwatsuki, M.; Takahashi, Y.; Otoguro, K.; O̅mura, S.; Sunazuka, T. Borrelidin analogues with antimalarial activity: design, synthesis and biological evaluation against Plasmodium falciparum parasites. Bioorg. Med. Chem. Lett. 2013, 23, 2302– 2305, DOI: 10.1016/j.bmcl.2013.02.075[Crossref], [PubMed], [CAS], Google Scholar26https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3sXktFWqur8%253D&md5=afa2cf528c533554131c7cf81095c906Borrelidin analogues with antimalarial activity: Design, synthesis and biological evaluation against Plasmodium falciparum parasitesSugawara, Akihiro; Tanaka, Toshiaki; Hirose, Tomoyasu; Ishiyama, Aki; Iwatsuki, Masato; Takahashi, Yoko; Otoguro, Kazuhiko; Omura, Satoshi; Sunazuka, ToshiakiBioorganic & Medicinal Chemistry Letters (2013), 23 (8), 2302-2305CODEN: BMCLE8; ISSN:0960-894X. (Elsevier B.V.)Borrelidin, a structurally unique 18-membered macrolide, was found to express antimalarial activity against drug-resistant Plasmodium falciparum malaria parasites, with IC50 value of 0.93 ng/mL. However, it also displays strong cytotoxicity against human diploid embryonic MRC-5 cells. To investigate the issue of the cytotoxicity of borrelidin, borrelidin-based analogs were synthesized and their anti-Plasmodium properties were evaluated. In this communication, we report that novel borrelidin analog I, bearing the CH2SPh moiety via a triazole linkage, was found to retain a potent antimalarial activity, against drug-sensitive and drug-resistant parasite strains, but possess only weak cytotoxicity against human cells.
- 27Teng, M.; Hilgers, M. T.; Cunningham, M. L.; Borchardt, A.; Locke, J. B.; Abraham, S.; Haley, G.; Kwan, B. P.; Hall, C.; Hough, G. W.; Shaw, K. J.; Finn, J. Identification of bacteria-selective threonyl-tRNA synthetase substrate inhibitors by structure-based design. J. Med. Chem. 2013, 56, 1748– 1760, DOI: 10.1021/jm301756m[ACS Full Text
], [CAS], Google Scholar27https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3sXhvVCjt7w%253D&md5=6619e410c2680cd7670f3652593ce58cIdentification of Bacteria-Selective Threonyl-tRNA Synthetase Substrate Inhibitors by Structure-Based DesignTeng, Min; Hilgers, Mark T.; Cunningham, Mark L.; Borchardt, Allen; Locke, Jeffrey B.; Abraham, Sunny; Haley, Gregory; Kwan, Bryan P.; Hall, Courtney; Hough, Grayson W.; Shaw, Karen J.; Finn, JohnJournal of Medicinal Chemistry (2013), 56 (4), 1748-1760CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)A series of potent and bacteria-selective threonyl-tRNA synthetase (ThrRS) inhibitors have been identified using structure-based drug design. These compds. occupied the substrate binding site of ThrRS and showed excellent binding affinities for all of the bacterial orthologs tested. Some of the compds. displayed greatly improved bacterial selectivity. Key residues responsible for potency and bacteria/human ThrRS selectivity have been identified. Antimicrobial activity has been achieved against wild-type Haemophilus influenzae and efflux-deficient mutants of Escherichia coli and Burkholderia thailandensis. - 28Guo, J.; Chen, B.; Yu, Y.; Cheng, B.; Cheng, Y.; Ju, Y.; Gu, Q.; Xu, J.; Zhou, H. Discovery of novel tRNA-amino acid dual-site inhibitors against threonyl-tRNA synthetase by fragment-based target hopping. Eur. J. Med. Chem. 2020, 187, 111941, DOI: 10.1016/j.ejmech.2019.111941[Crossref], [PubMed], [CAS], Google Scholar28https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1MXitlGhtLbM&md5=5ebcdf329ae1afa5cee3cf5799b3ec6bDiscovery of novel tRNA-amino acid dual-site inhibitors against threonyl-tRNA synthetase by fragment-based target hoppingGuo, Junsong; Chen, Bingyi; Yu, Ying; Cheng, Bao; Cheng, Yanfang; Ju, Yingchen; Gu, Qiong; Xu, Jun; Zhou, HuihaoEuropean Journal of Medicinal Chemistry (2020), 187 (), 111941CODEN: EJMCA5; ISSN:0223-5234. (Elsevier Masson SAS)Threonyl-tRNA synthetase (ThrRS) is a key member of the aminoacyl-tRNA synthetase (aaRS) family that plays essential roles in protein biosynthesis, and ThrRS inhibitors have potential in the therapy of multiple diseases, such as microbial infections and cancers. Based on a unique tRNA-amino acid dual-site inhibitory mechanism identified recently with the herb-derived prolyl-tRNA synthetase (ProRS) inhibitor halofuginone (HF), a series of compds. have been designed and synthesized by employing a fragment-based target hopping approach to simultaneously target the tRNAThr and L-threonine binding pockets of ThrRS. Among them, compd. 30d showed an IC50 value of 1.4μM against Salmonella enterica ThrRS (SeThrRS) and MIC values of 16-32μg/mL against the tested bacterial strains. The cocrystal structure of SeThrRS in complex with 30d was detd. at high resoln., revealing that 30d simultaneously occupies both binding pockets for the nucleotide A76 of tRNAThr and L-threonine in an ATP-independent manner, a novel mechanism compared to all other reported ThrRS inhibitors. Our study provides a new class of ThrRS inhibitors, and more importantly, it presents the first exptl. evidence that the tRNA-amino acid dual-site inhibitory mechanism could apply to other aaRSs beyond ProRS, thus providing great opportunities for designing new mechanistic inhibitors for aaRS-based therapeutics. oxoquinazolin-.
- 29Guo, J.; Chen, B.; Yu, Y.; Cheng, B.; Ju, Y.; Tang, J.; Cai, Z.; Gu, Q.; Xu, J.; Zhou, H. Structure-guided optimization and mechanistic study of a class of quinazolinone-threonine hybrids as antibacterial ThrRS inhibitors. Eur. J. Med. Chem. 2020, 207, 112848, DOI: 10.1016/j.ejmech.2020.112848[Crossref], [PubMed], [CAS], Google Scholar29https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXitVOqsLzI&md5=3127bd6273e7959271edbf6a789fbefcStructure-guided optimization and mechanistic study of a class of quinazolinone-threonine hybrids as antibacterial ThrRS inhibitorsGuo, Junsong; Chen, Bingyi; Yu, Ying; Cheng, Bao; Ju, Yingchen; Tang, Jieyu; Cai, Zhengjun; Gu, Qiong; Xu, Jun; Zhou, HuihaoEuropean Journal of Medicinal Chemistry (2020), 207 (), 112848CODEN: EJMCA5; ISSN:0223-5234. (Elsevier Masson SAS)Aminoacyl-tRNA synthetases (aaRSs) are an attractive class of antibacterial drug targets due to their essential roles in protein translation. While most traditional aaRS inhibitors target the binding pockets of substrate amino acids and/or ATP, we recently developed a class of novel tRNA-amino acid dual-site inhibitors including inhibitor 3 ((2S,3R)-2-amino-N-((E)-4-(6,7-dichloro-4-oxoquinazolin-3(4H)-yl)but-2-en-1-yl)-3-hydroxybutanamide) against threonyl-tRNA synthetase (ThrRS). Here, the binding modes and structure-activity relationships (SARs) of these inhibitors were analyzed by the crystal structures of Salmonella enterica ThrRS (SeThrRS) in complex with three of them. Based on the cocrystal structures, twelve quinazolinone-threonine hybrids were designed and synthesized, and their affinities, enzymic inhibitory activities, and cellular potencies were evaluated. The best deriv. 8g achieved a Kd value of 0.40 μM, an IC50 value of 0.50 μM against SeThrRS and MIC values of 16-32 μg/mL against the tested bacterial strains. The cocrystal structure of the SeThrRS-8g complex revealed that 8g induced a bended conformation for Met332 by forming hydrophobic interactions, which better mimicked the binding of tRNAThr to ThrRS. Moreover, the inhibitory potency of 8g was less impaired than a reported ATP competitive inhibitor at high concns. of ATP, supporting our hypothesis that tRNA site inhibitors are likely superior to ATP site inhibitors in vivo, where ATP typically reaches millimolar concns.
- 30Torres-Larios, A.; Sankaranarayanan, R.; Rees, B.; Dock-Bregeon, A.-C.; Moras, D. Conformational movements and cooperativity upon amino acid, ATP and tRNA binding in threonyl-tRNA synthetase. J. Mol. Biol. 2003, 331, 201– 211, DOI: 10.1016/s0022-2836(03)00719-8[Crossref], [PubMed], [CAS], Google Scholar30https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD3sXls1eru7Y%253D&md5=1b9322b3cafb3a1137175e3c0ab86112Conformational movements and cooperativity upon amino acid, ATP and tRNA binding in threonyl-tRNA synthetaseTorres-Larios, Alfredo; Sankaranarayanan, Rajan; Rees, Bernard; Dock-Bregeon, Anne-Catherine; Moras, DinoJournal of Molecular Biology (2003), 331 (1), 201-211CODEN: JMOBAK; ISSN:0022-2836. (Elsevier Science Ltd.)The crystal structures of threonyl-tRNA synthetase (ThrRS) from Staphylococcus aureus, with ATP and an analog of threonyl adenylate, are described. Together with the previously detd. structures of Escherichia coli ThrRS with different substrates, they allow a comprehensive anal. of the effect of binding of all the substrates: threonine, ATP and tRNA. The tRNA, by inserting its acceptor arm between the N-terminal domain and the catalytic domain, causes a large rotation of the former. Within the catalytic domain, four regions surrounding the active site display significant conformational changes upon binding of the different substrates. The binding of threonine induces the movement of as much as 50 consecutive amino acid residues. The binding of ATP triggers a displacement, as large as 8 A at some Cα positions, of a strand-loop-strand region of the core β-sheet. Two other regions move in a cooperative way upon binding of threonine or ATP: the motif 2 loop, which plays an essential role in the first step of the aminoacylation reaction, and the ordering loop, which closes on the active site cavity when the substrates are in place. The tRNA interacts with all four mobile regions, several residues initially bound to threonine or ATP switching to a position in which they can contact the tRNA. Three such conformational switches could be identified, each of them in a different mobile region. The structural anal. suggests that, while the small substrates can bind in any order, they must be in place before productive tRNA binding can occur.
- 31We used a database of commercially available compounds; Otava Ltd., http://www.otavachemicals.com, (accessed April, 2020).
- 32Kovalenko, O. P.; Volynets, G. P.; Rybak, M. Y.; Starosyla, S. A.; Gudzera, O. I.; Lukashov, S. S.; Bdzhola, V. G.; Yarmoluk, S. M.; Boshoff, H. I.; Tukalo, M. A. Dual-target inhibitors of mycobacterial aminoacyl-tRNA synthetases among N-benzylidene-N′-thiazol-2-yl-hydrazines. Medchemcomm 2019, 10, 2161– 2169, DOI: 10.1039/c9md00347a[Crossref], [PubMed], [CAS], Google Scholar32https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1MXit1GlsbrN&md5=524015cced076511f6bb4aa7424766d8Dual-target inhibitors of mycobacterial aminoacyl-tRNA synthetases among N-benzylidene-N'-thiazol-2-yl-hydrazinesKovalenko, Oksana P.; Volynets, Galyna P.; Rybak, Mariia Yu.; Starosyla, Sergiy A.; Gudzera, Olga I.; Lukashov, Sergiy S.; Bdzhola, Volodymyr G.; Yarmoluk, Sergiy M.; Boshoff, Helena I.; Tukalo, Michael A.MedChemComm (2019), 10 (12), 2161-2169CODEN: MCCEAY; ISSN:2040-2503. (Royal Society of Chemistry)Effective treatment of tuberculosis is challenged by the rapid development of Mycobacterium tuberculosis (Mtb) multidrug resistance that presumably could be overcome with novel multi-target drugs. Aminoacyl-tRNA synthetases (AARSs) are an essential part of protein biosynthesis machinery and attractive targets for drug discovery. Here, we exptl. verify a hypothesis of simultaneous targeting of structurally related AARSs by a single inhibitor. We previously identified a new class of mycobacterial leucyl-tRNA synthetase inhibitors, N-benzylidene-N'-thiazol-2-yl-hydrazines. Mol. docking of a library of novel N-benzylidene-N'-thiazol-2-yl-hydrazine derivs. into active sites of M. tuberculosis LeuRS (MtbLeuRS) and MetRS (MtbMetRS) resulted in a panel of the best ranking compds., which were then evaluated for enzymic potency. Screening data revealed 11 compds. active against MtbLeuRS and 28 compds. active against MtbMetRS. The hit compds. display dual inhibitory potency as demonstrated by IC50 values for both enzymes. Compd. I is active against Mtb H37Rv cells in in vitro bioassays.
- 33Gudzera, O. I.; Golub, A. G.; Bdzhola, V. G.; Volynets, G. P.; Lukashov, S. S.; Kovalenko, O. P.; Kriklivyi, I. A.; Yaremchuk, A. D.; Starosyla, S. A.; Yarmoluk, S. M.; Tukalo, M. A. Discovery of potent anti-tuberculosis agents targeting leucyl-tRNA synthetase. Bioorg. Med. Chem. 2016, 24, 1023– 1031, DOI: 10.1016/j.bmc.2016.01.028[Crossref], [PubMed], [CAS], Google Scholar33https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28XhsVKksro%253D&md5=5bce1d6fbf6dda8394233e4b0dbafb83Discovery of potent anti-tuberculosis agents targeting leucyl-tRNA synthetaseGudzera, Olga I.; Golub, Andriy G.; Bdzhola, Volodymyr G.; Volynets, Galyna P.; Lukashov, Sergiy S.; Kovalenko, Oksana P.; Kriklivyi, Ivan A.; Yaremchuk, Anna D.; Starosyla, Sergiy A.; Yarmoluk, Sergiy M.; Tukalo, Michail A.Bioorganic & Medicinal Chemistry (2016), 24 (5), 1023-1031CODEN: BMECEP; ISSN:0968-0896. (Elsevier B.V.)Tuberculosis is a serious infectious disease caused by human pathogen bacteria Mycobacterium tuberculosis. Bacterial drug resistance is a very significant medical problem nowadays and development of novel antibiotics with different mechanisms of action is an important goal of modern medical science. Leucyl-tRNA synthetase (LeuRS) has been recently clin. validated as antimicrobial target. Here the authors report the discovery of small-mol. inhibitors of M. tuberculosis LeuRS. Using receptor-based virtual screening the authors have identified six inhibitors of M. tuberculosis LeuRS from two different chem. classes. The most active compd. I inhibits LeuRS with IC50 of 6 μM. A series of derivs. has been synthesized and evaluated in vitro toward M. tuberculosis LeuRS. It was revealed that the most active compd. II inhibits LeuRS with IC50 of 2.27 μM. All active compds. were tested for antimicrobial effect against M. tuberculosis H37Rv. The compd. I seems to have the best cell permeability and inhibits growth of pathogenic bacteria with IC50 = 10.01 μM and IC90 = 13.53 μM.
- 34Mosmann, T. Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J. Immunol. Methods 1983, 65, 55– 63, DOI: 10.1016/0022-1759(83)90303-4[Crossref], [PubMed], [CAS], Google Scholar34https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADyaL2c%252FovFSmtw%253D%253D&md5=c1116f4ca7a7d2b79fab2a0bd2c2d439Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assaysMosmann TJournal of immunological methods (1983), 65 (1-2), 55-63 ISSN:0022-1759.A tetrazolium salt has been used to develop a quantitative colorimetric assay for mammalian cell survival and proliferation. The assay detects living, but not dead cells and the signal generated is dependent on the degree of activation of the cells. This method can therefore be used to measure cytotoxicity, proliferation or activation. The results can be read on a multiwell scanning spectrophotometer (ELISA reader) and show a high degree of precision. No washing steps are used in the assay. The main advantages of the colorimetric assay are its rapidity and precision, and the lack of any radioisotope. We have used the assay to measure proliferative lymphokines, mitogen stimulations and complement-mediated lysis.
- 35Bodian, D. L.; Yamasaki, R. B.; Buswell, R. L.; Stearns, J. F.; White, J. M.; Kuntz, I. D. Inhibition of the fusion-inducing conformational change of influenza hemagglutinin by benzoquinones and hydroquinones. Biochemistry 1993, 32, 2967– 2978, DOI: 10.1021/bi00063a007[ACS Full Text], [CAS], Google Scholar35https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK3sXksVSgsrk%253D&md5=a44f15d36e94e05892e5231512efd553Inhibition of the fusion-inducing conformational change of influenza hemagglutinin by benzoquinones and hydroquinonesBodian, Dale L.; Yamasaki, R. Bryan; Buswell, Richard L.; Stearns, Jay F.; White, Judith M.; Kuntz, I. D.Biochemistry (1993), 32 (12), 2967-78CODEN: BICHAW; ISSN:0006-2960.Influenza hemagglutinin (HA) undergoes a conformational change that is required for viral entry. The rearrangement includes exposure of the fusion peptide, a hydrophobic segment buried in the trimer interface of the native protein. Since fusion peptide release triggers the membrane fusion event crucial for viral replication, inhibition of fusion peptide exposure should prevent infection. The authors reasoned that small mols. that bind to HA and stabilize its nonfusogenic conformation would block viral activity. A computer-assisted method was used to select putative HA ligands. One of the selected compds., 4A,5,8,8A-tetrhydro-5,8-methano-1,4-naphthoquinone, prevented the conversion of X31 HA to a conformation recognized by α-fusion peptide antisera. Several derivs. of this compd., including both benzoquinones and hydroquinones, also showed inhibition. The most effective compds. tested have IC50s between 1 and 20 μM. Representation compds. also inhibited virus-induced syncytia formation, HA-mediated hemolysis, and viral infectivity in vitro. The inhibitors are attractive leads for the development of antiviral drugs and can serve as probes of the mechanism of the conformational change of HA.
- 36Ewing, T. J. A.; Makino, S.; Skillman, A. G.; Kuntz, I. D. DOCK 4.0: search strategies for automated molecular docking of flexible molecule databases. J. Comput.-Aided Mol. Des. 2001, 15, 411– 428, DOI: 10.1023/a:1011115820450[Crossref], [PubMed], [CAS], Google Scholar36https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD3MXktVSlsbc%253D&md5=a76436739db67e4908845f123710179eDOCK 4.0: search strategies for automated molecular docking of flexible molecule databasesEwing, Todd J. A.; Makino, Shingo; Skillman, A. Geoffrey; Kuntz, Irwin D.Journal of Computer-Aided Molecular Design (2001), 15 (5), 411-428CODEN: JCADEQ; ISSN:0920-654X. (Kluwer Academic Publishers)The search strategies developed for docking flexible mols. to macromol. sites that are incorporated into the widely distributed DOCK software version 4.0, are described. The search strategies include incremental construction and random conformation search and utilize the existing Coulombic and Lennard-Jones grid-based scoring function. The incremental construction strategy is tested with a panel of 15 crystallog. test cases, created from 12 unique complexes whose ligands vary in size and flexibility. For all test cases, at least one docked position is generated within 2 Å of the crystallog. position. For 7 of 15 test cases, the top scoring position is also within 2 Å of the crystallog. position. The algorithm is fast enough to successfully dock a few test cases within seconds and most within 100 s. The incremental construction and the random search strategy are evaluated as database docking techniques with a database of 51 mols. docked to two of the crystallog. test cases. Incremental construction outperforms random search and is fast enough to reliably rank the database of compds. within 15 s per mol. on an SGI R10000 cpu.
- 37Ring, C. S.; Sun, E.; McKerrow, J. H.; Lee, G. K.; Rosenthal, P. J.; Kuntz, I. D.; Cohen, F. E. Structure-based inhibitor design by using protein models for the development of antiparasitic agents. Proc. Natl. Acad. Sci. U.S.A. 1993, 90, 3583– 3587, DOI: 10.1073/pnas.90.8.3583[Crossref], [PubMed], [CAS], Google Scholar37https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK3sXktVShu7o%253D&md5=a52b220512a0f2e2325383dd317b1503Structure-based inhibitor design by using protein models for the development of antiparasitic agentsRing, Christine S.; Sun, Eugene; McKerrow, James H.; Lee, Garson K.; Rosenthal, Philip J.; Kuntz, Irwin D.; Cohen, Fred E.Proceedings of the National Academy of Sciences of the United States of America (1993), 90 (8), 3583-7CODEN: PNASA6; ISSN:0027-8424.The lack of an exptl. detd. structure of a target protein frequently limits the application of structure-based drug design methods. In an effort to overcome this limitation, we have investigated the use of computer model-built structures for the identification of previously unknown inhibitors of enzymes from two major protease families, serine and cysteine proteases. We have successfully used our model-built structures to identify computationally and to confirm exptl. the activity of nonpeptidic inhibitors directed against important enzymes in the schistosome [2-(4-methoxybenzoyl)-1-naphthoic acid, Ki = 3 μM] and malaria {oxalic bis[(2-hydroxy-1-naphthylmethylene)hydrazide], IC50 = 6 μM} parasite life cycles.
- 38Shoichet, B. K.; Stroud, R. M.; Santi, D. V.; Kuntz, I. D.; Perry, K. M. Structure-based discovery of inhibitors of thymidylate synthase. Science 1993, 259, 1445– 1450, DOI: 10.1126/science.8451640[Crossref], [PubMed], [CAS], Google Scholar38https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK3sXktF2qsLc%253D&md5=a2e9380f78c3dd0f213443df91cefb75Structure-based discovery of inhibitors of thymidylate synthaseShoichet, Brian K.; Stroud, Robert M.; Santi, Daniel V.; Kuntz, Irwin D.; Perry, Kathy M.Science (Washington, DC, United States) (1993), 259 (5100), 1445-50CODEN: SCIEAS; ISSN:0036-8075.A mol. docking computer program (DOCK) was used to screen the Fine Chem. Directory, a database of com. available compds., for mols. that are complementary to thymidylate synthase (TS), a chemotherapeutic target. Besides retrieving the substrate and several known inhibitors, DOCK proposed putative inhibitors previously unknown to bind to the enzyme. Three of these compds. inhibited Lactobacillus casei TS at submillimolar concns. One of these inhibitors, sulisobenzone, crystd. with TS in two configurations that differed from the DOCK-favored geometry: a counterion was bound in the substrate site, which resulted in a 6 to 9 angstrom displacement of the inhibitor. The structure of the complexes suggested another binding region in the active site that could be exploited. This region was probed with mols. sterically similar to sulisobenzone, which led to the identification of a family of phenolphthalein analogs that inhibit TS in the 1 to 30 micromolar range. These inhibitors do not resemble the substrates of the enzyme. A crystal structure of phenolphthalein with TS shows that it binds in the target site in a configuration that resembles the one suggested by DOCK.
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- 40Yakovenko, O.; Oliferenko, A. A.; Bdzhola, V. G.; Palyulin, V. A.; Zefirov, N. S. Kirchhoff atomic charges fitted to multipole moments: Implementation for a virtual screening system. J. Comput. Chem. 2008, 29, 1332– 1343, DOI: 10.1002/jcc.20892[Crossref], [PubMed], [CAS], Google Scholar40https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1cXlvFWksbo%253D&md5=a05b4cdbc8814396086c1ac996ceeb4bKirchhoff atomic charges fitted to multipole moments: implementation for a virtual screening systemYakovenko, Olexander; Oliferenko, Alexander A.; Bdzhola, Volodymyr G.; Palyulin, Vladimir A.; Zefirov, Nikolai S.Journal of Computational Chemistry (2008), 29 (8), 1332-1343CODEN: JCCHDD; ISSN:0192-8651. (John Wiley & Sons, Inc.)The Kirchhoff charge model is a viable method of generating inexpensive and electrostatically reasonable at. charges for mol. mech. force fields. The charging method uses a computationally fast algorithm based on the principle of electronegativity relaxation. Parameters of the method, orbital electronegativities and hardnesses, are fitted to reproduce ref., ab initio calcd. dipole and quadrupole moments of a representative training set of neutral and charged org. mols. covering most medicinal chem. relevant bonding situations. Transferability and accuracy of the derived parameters are confirmed on an external test set. Comparisons to other charge models are made. Implementation of the new Kirchhoff charges into a virtual screening engine is elucidated.
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- 43Gorbatiuk, O. B.; Tsapenko, M. V.; Pavlova, M. V.; Okunev, O. V.; Kordium, V. A. Bioaffinity sorbent based on immobilized protein A Staphylococcus aureus: development and application. Biopolym. Cell 2012, 28, 141– 148, DOI: 10.7124/bc.000041[Crossref], [CAS], Google Scholar43https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC38XosFSktb0%253D&md5=804a529193ac7689fe83d702612d0f1aBioaffinity sorbent based on immobilized protein A Staphylococcus aureus: development and applicationGorbatiuk, O. B.; Tsapenko, M. V.; Pavlova, M. V.; Okunev, O. V.; Kordium, V. A.Biopolymers and Cell (2012), 28 (2), 141-147CODEN: BCIEBC ISSN:. (Institut Molekulyarnoi Biologii i Genetiki NAN Ukraini)Aim. The obtaining of bioaffinity sorbent based on the immobilized protein A of S. aureus (SPA) using two cellulose-binding domains (CBD), and its application for purifn. of antibodies. Methods. The DNA sequences encoding SPA and two CBD were genetically fused, expressed in the high-productive Escherichia coli system and the protein SPA-CBD2 was obtained in a sol. form. The SPA-CBD2 fusion protein was affinity immobilized on the microcryst. cellulose. Results. Capacity of bioaffinity sorbent (1 mg SPA-CBD2/1 mL CC31-cellulose), dynamic capacity (3 mg mouse IgG/1 mL bioaffinity sorbent), efficiency and stability during prolonged storage were detd. The bio-affinity sorbent was used for purifn. of antibodies. The purity of antibodies in eluted fractions was more than 95%. The purified antibodies detected target antigens with a high sensitivity. Conclusions. The designed bioaffinity sorbent provides obtaining pure poly- and monoclonal antibodies in functionally active form and can be useful for the fractionation of mouse IgG.
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- 45Bradford, M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 1976, 72, 248– 254, DOI: 10.1016/0003-2697(76)90527-3[Crossref], [PubMed], [CAS], Google Scholar45https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaE28XksVehtrY%253D&md5=43f388a493becc4193c7a49d9de0e1dbA rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye bindingBradford, Marion M.Analytical Biochemistry (1976), 72 (1-2), 248-54CODEN: ANBCA2; ISSN:0003-2697.A protein detn. method that involves the binding of coomassie Brilliant Blue G 250 to protein is described. The binding of the dye to protein causes a shift in the absorption max. of the dye from 465 to 595 nm, and it is the increase in absorption at 595 nm that is monitored. This assay is very reproducible and rapid with the dye binding process virtually complete in ∼ 2 min with good color stability for 1 hr. There is little or no interference from cations such as Na+ or K+ nor from carbohydrates such as sucrose. A small amt. of color is developed in the presence of strongly alk. buffering agents, but the assay may be run accurately by the use of proper buffer controls. The only components found to give excessive interfering color in the assay are relatively large amts. of detergents such as Na dodecyl sulfate, Triton X 100, and commercial glassware detergents. Interference by small amts. of detergent may be eliminated by the use of proper control.
- 46Gasteiger, E.; Gattiker, A.; Hoogland, C.; Ivanyi, I.; Appel, R. D.; Bairoch, A. ExPASy: The proteomics server for in-depth protein knowledge and analysis. Nucleic Acids Res. 2003, 31, 3784– 3788, DOI: 10.1093/nar/gkg563[Crossref], [PubMed], [CAS], Google Scholar46https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD3sXltVWjtLc%253D&md5=adf2bae943800f2b00d9e475e5968f36ExPASy: the proteomics server for in-depth protein knowledge and analysisGasteiger, Elisabeth; Gattiker, Alexandre; Hoogland, Christine; Ivanyi, Ivan; Appel, Ron D.; Bairoch, AmosNucleic Acids Research (2003), 31 (13), 3784-3788CODEN: NARHAD; ISSN:0305-1048. (Oxford University Press)The ExPASy (the Expert Protein Anal. System) World Wide Web server, is provided as a service to the life science community by a multidisciplinary team at the Swiss Institute of Bioinformatics (SIB). It provides access to a variety of databases and anal. tools dedicated to proteins and proteomics. ExPASy databases include SWISS-PROT and TrEMBL, SWISS-2DPAGE, PROSITE, ENZYME and the SWISS-MODEL repository. Anal. tools are available for specific tasks relevant to proteomics, similarity searches, pattern and profile searches, post-translational modification prediction, topol. prediction, primary, secondary and tertiary structure anal. and sequence alignment. These databases and tools are tightly interlinked: a special emphasis is placed on integration of database entries with related resources developed at the SIB and elsewhere, and the proteomics tools have been designed to read the annotations in SWISS-PROT in order to enhance their predictions. ExPASy started to operate in 1993, as the first WWW server in the field of life sciences. In addn. to the main site in Switzerland, seven mirror sites in different continents currently serve the user community.
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Abstract
Figure 1
Figure 1. Chemical structure of 1-(4-methoxy-phenyl)-2-(6-nitro-benzothiazol-2-ylsulfanyl)-ethanone (compound 1) (a). The complex of compound 1 with amino acid residues in the active site of S. aureus ThrRS. The hydrogen bonds are shown by green dashed lines with the distances indicated in Å, the hydrophobic interactions are presented by magenta dashed lines, π-sulfur interactions are shown by yellow dashed lines, and π-cation interactions are indicated with orange dashed lines (b).
Figure 2
Figure 2. Superposition of the threonyl adenylate analogue, extracted from the crystal structure with the PDB accession code: 1NYQ (carbon atoms are labeled with magenta color) and compound 1 (carbon atoms are labeled with blue color) in the active site of S. aureus threonyl—tRNA synthetase.
Figure 3
Figure 3. Complex of 2-(2-tert-butyl-phenoxy)-N-(2-methyl-5-nitro-phenyl)-acetamide (compound 2) with amino acid residues in the active site of S. aureus ThrRS. The hydrophobic interactions are indicated with magenta dashed lines, π-cation interactions are shown by orange dashed lines, and the metal-acceptor bond is presented by the gray dashed line.
Figure 4
Figure 4. Complex of 4-(4-chloro-phenyl)-2-[2-(2,6-dimethyl-morpholin-4-yl)-acetylamino]-thiophene-3-carboxylic acid ethyl ester (compound 8) with amino acid residues in the active site of S. aureus ThrRS. The hydrogen bonds are shown by green dashed lines with the distances indicated in Å, the hydrophobic interactions are indicated with magenta dashed lines, π-sulfur interaction is presented by yellow dashed lines, and the halogen bond is shown by blue dashed lines.
Figure 5
Figure 5. Complex of 2-[1-(4-iodo-2-methyl-phenyl)-2,5-dioxo-pyrrolidin-3-ylsulfanyl]-nicotinic acid (compound 11) with amino acid residues in the active site of S. aureus ThrRS. The hydrogen bonds are shown by green dashed lines with the distances indicated in Å, the hydrophobic interactions are presented by magenta dashed lines, π-cation interactions are indicated with orange dashed lines, and the metal-acceptor bond is shown by gray dashed lines.
Figure 6
Figure 6. Complex of 2,4-dibromo-6-{[4-(4-nitro-phenyl)-thiazol-2-yl]-hydrazonomethyl}-phenol (compound 13) with amino acid residues in the active site of S. aureus ThrRS. The hydrogen bonds are shown by green dashed lines with the distances indicated in Å, the hydrophobic interactions are presented by magenta dashed lines, π-cation interaction is indicated with orange dashed lines, π-sulfur interaction is presented by yellow dashed lines, and the metal-acceptor bond is shown by gray dashed lines.
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ARTICLE SECTIONSThis article references 46 other publications.
- 1Krishna, S.; Miller, L. S. Host-pathogen interactions between the skin and Staphylococcus aureus. Curr. Opin. Microbiol. 2012, 15, 28– 35, DOI: 10.1016/j.mib.2011.11.003[Crossref], [PubMed], [CAS], Google Scholar1https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BC387mvFGhsA%253D%253D&md5=478c2056528833971c2fc0ac792684ceHost-pathogen interactions between the skin and Staphylococcus aureusKrishna Sheila; Miller Lloyd SCurrent opinion in microbiology (2012), 15 (1), 28-35 ISSN:.Staphylococcus aureus is responsible for the vast majority of bacterial skin infections in humans. The propensity for S. aureus to infect skin involves a balance between cutaneous immune defense mechanisms and virulence factors of the pathogen. The tissue architecture of the skin is different from other epithelia especially since it possesses a corneal layer, which is an important barrier that protects against the pathogenic microorganisms in the environment. The skin surface, epidermis, and dermis all contribute to host defense against S. aureus. Conversely, S. aureus utilizes various mechanisms to evade these host defenses to promote colonization and infection of the skin. This review will focus on host-pathogen interactions at the skin interface during the pathogenesis of S. aureus colonization and infection.
- 2Welte, T.; Kantecki, M.; Stone, G. G.; Hammond, J. Ceftaroline fosamil as a potential treatment option for Staphylococcus aureus community-acquired pneumonia in adults. Int. J. Antimicrob. Agents 2019, 54, 410– 422, DOI: 10.1016/j.ijantimicag.2019.08.012[Crossref], [PubMed], [CAS], Google Scholar2https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1MXhslens7jN&md5=7658ca7aa0d179ba432198480eafe160Ceftaroline fosamil as a potential treatment option for Staphylococcus aureus community-acquired pneumonia in adultsWelte, Tobias; Kantecki, Michal; Stone, Gregory G.; Hammond, JenniferInternational Journal of Antimicrobial Agents (2019), 54 (4), 410-422CODEN: IAAGEA; ISSN:0924-8579. (Elsevier B.V.)A review. Staphylococcus aureus (S. aureus), including methicillin-resistant S. aureus (MRSA), is an important etiol. cause of community-acquired pneumonia (CAP) and assocd. with significant morbidity and mortality. Empiric therapy for CAP frequently consists of β-lactam monotherapy or β-lactam/macrolide combination therapy. However, such agents are often ineffective against S. aureus and do not reflect the emergence and increasing prevalence of MRSA in the community setting. Ceftaroline fosamil is a fifth-generation parenteral cephalosporin with broad-spectrum activity against Gram-pos. pathogens - such as S. aureus (including MRSA), Streptococcus pneumoniae and Streptococcus pyogenes - and typical Gram-neg. pathogens, including Haemophilus influenzae and Moraxella catarrhalis. The approval of ceftaroline fosamil in the United States and Europe for the treatment of adults with moderate-to-severe CAP was based on two phase 3 trials (FOCUS 1 and 2), which demonstrated that ceftaroline fosamil was non-inferior to ceftriaxone, a std. empiric treatment for CAP, while exhibiting a comparable safety profile. Although head-to-head trials of ceftaroline fosamil vs. comparators against MRSA CAP are lacking, the effectiveness of ceftaroline fosamil in subpopulations of patients not covered by phase 3 trials (e.g. those with MRSA CAP or severe renal impairment) has been demonstrated in the Clin. Assessment Program and Teflaro Utilization Registry (CAPTURE) study. As ineffective empiric therapy is assocd. with adverse outcomes, including mortality and increased costs, ceftaroline fosamil, with its extended spectrum of activity, is an attractive alternative to std. antibiotic CAP regimens.
- 3Jang, Y.-R.; Kim, T.; Kim, M.-C.; Sup Sung, H.; Kim, M.-N.; Kim, M. J.; Kim, S. H.; Lee, S.-O.; Choi, S.-H.; Woo, J. H.; Kim, Y. S.; Chong, Y. P. Sternoclavicular septic arthritis caused by Staphylococcus aureus: excellent results from medical treatment and limited surgery. Infect. Dis. 2019, 51, 694– 700, DOI: 10.1080/23744235.2019.1639810[Crossref], [CAS], Google Scholar3https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1MXhtlKjsL%252FN&md5=394959812dc09f323db66d2d3865751aSternoclavicular septic arthritis caused by Staphylococcus aureus: excellent results from medical treatment and limited surgeryJang, Young-Rock; Kim, Taeeun; Kim, Min-Chul; Sung, Heung Sup; Kim, Mi-Na; Kim, Min Jae; Kim, Sung Han; Lee, Sang-Oh; Choi, Sang-Ho; Woo, Jun Hee; Kim, Yang Soo; Chong, Yong PilInfectious Diseases (2019), 51 (9), 694-700CODEN: IDNIAU; ISSN:2374-4243. (Taylor & Francis Ltd.)Aggressive surgery such as en bloc joint resection is favored for treating uncommon sternoclavicular (SC) septic arthritis, based on expert opinion and small case series. It analyzed the clin. characteristics and treatment outcomes of patients with Staphylococcus aureus SC septic arthritis treated medically or with limited surgery. All adult patients with this septic arthritis at the Asan Medical Center between Sept. 2009 and Dec. 2016 were reviewed. Limited surgery was defined as simple incision, drainage, and debridement of the infected joint. Of 22 patients enrolled, 11 received medical treatment only, and 11 underwent limited surgery, and none underwent aggressive surgery. Most patients (73%) had underlying predisposing conditions such as infection at a distant site, diabetes and liver cirrhosis, and none had i.v. drug abuse or HIV infection. Complications such as chest wall and/or neck abscess, clavicular and/or sternal osteomyelitis were identified in 18 patients (82%). Patients with chest wall and/or neck abscesses tended more often to undergo limited surgery than patients without such abscesses (73% vs. 27%, p = .09). The median duration of i.v. antibiotics was 35 days (IQR, 25-46 days). Treatment was successful in all cases. In a median 53-wk follow-up (IQR, 8-171 wk), there was no relapse of arthritis or joint deterioration. Medical treatment alone or with limited surgery could be successful therapeutic strategies for complicated S. aureus SC septic arthritis in selected patients.
- 4Tascini, C.; Attanasio, V.; Ripa, M.; Carozza, A.; Pallotto, C.; Bernardo, M.; Francisci, D.; Oltolini, C.; Palmiero, G.; Scarpellini, P. Ceftobiprole for the treatment of infective endocarditis: a case series. J. Glob. Antimicrob. Resist. 2020, 20, 56– 59, DOI: 10.1016/j.jgar.2019.07.020[Crossref], [PubMed], [CAS], Google Scholar4https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BB3MvhvVKqtw%253D%253D&md5=1ca81aee6c5fc65f045b9aad96e35208Ceftobiprole for the treatment of infective endocarditis: A case seriesTascini Carlo; Attanasio Vittorio; Palmiero Giulia; Ripa Marco; Oltolini Chiara; Scarpellini Paolo; Carozza Antonio; Pallotto Carlo; Bernardo Mariano; Francisci DanielaJournal of global antimicrobial resistance (2020), 20 (), 56-59 ISSN:.OBJECTIVES: Ceftobiprole is a relatively new cephalosporin with broad-spectrum activity and good tolerability. Despite its promising characteristics, to our knowledge, only two case reports, previously published also by some of us, is available concerning its administration for the treatment of infective endocarditis. Hereby we report our experience in this field. METHODS: All the patients with infective endocarditis treated with ceftobiprole were enrolled. RESULTS: 12 cases of endocarditis were treated with ceftobiprole, 11/12 in combination with daptomycin and 1/12 as monotherapy. Gram-positive bacteria were isolated in 12/12 patients; 3 cases were polymicrobial. Cure rate was 83% (10/12 patients). In 9/12 (75%) cases, patients were switched to ceftobiprole following failure of previous antimicrobial regimen. In 3/3 patients in which ceftobiprole was administered because of persistently positive blood culture, bacteraemia clearance was rapidly achieved. CONCLUSIONS: Ceftobiprole, especially in combination, could be a promising alternative treatment for infective endocarditis.
- 5Dugourd, P.-M.; Dupont, A.; Hubiche, T.; Chiaverini, C.; Alkhalifa, A.; Roudiere, L.; Tristan, A.; Gustave, C.-A.; Del Giudice, P. Érythème généralisé fébrile et choc : choc toxinique staphylococcique. Ann. Dermatol. Venereol. 2019, 146, 287– 291, DOI: 10.1016/j.annder.2018.12.002[Crossref], [PubMed], [CAS], Google Scholar5https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BB3cjmt1Omtw%253D%253D&md5=861a8d08009f3c3b3d64d79cc95614baStaphylococcal toxic shock syndrome should be considered in the event of diffuse erythema with fever and shockDugourd P-M; Dupont A; Hubiche T; Del Giudice P; Chiaverini C; Alkhalifa A; Roudiere L; Tristan A; Gustave C-AAnnales de dermatologie et de venereologie (2019), 146 (4), 287-291 ISSN:0151-9638.BACKGROUND: Toxic shock syndrome (TSS) was first described by Todd in 1978. The relevant Lancet publication reported 7 cases of children with fever, exanthema, hypotension and diarrhoea associated with multiple organ failure. An association between TSS and use of hyper-absorbent tampons in menstruating women was discovered in the 1980s. Following the market withdrawal of such tampons, TSS virtually disappeared. Herein we report a new case of TSS in a 15-year-old girl. PATIENTS AND METHODS: A 15-year-old patient was admitted to intensive care for severe sepsis and impaired consciousness associated with diffuse abdominal pain. Dermatological examination revealed diffuse macular exanthema. Laboratory tests showed hepatic cytolysis (ASAT 101 U/L, ALAT 167 U/L, total bilirubin 68μmol/L) and an inflammatory syndrome. Lumbar puncture and blood cultures were sterile while thoraco-abdomino-pelvic and brain scans were normal. The patient was menstruating and had been using a tampon over the previous 24hours. Vaginal sampling and tampon culture revealed TSST-1 toxin-producing S. aureus. Management consisted of intensive care measures and treatment with amoxicillin-clavulanic acid and clindamycin for 10 days. CONCLUSION: In case of septic shock associated with diffuse macular exanthema a diagnosis of TSS must be envisaged, particularly in menstruating women.
- 6Toledo, A. G.; Golden, G.; Campos, A. R.; Cuello, H.; Sorrentino, J.; Lewis, N.; Varki, N.; Nizet, V.; Smith, J. W.; Esko, J. D. Proteomic atlas of organ vasculopathies triggered by Staphylococcus aureus sepsis. Nat. Commun. 2019, 10, 4656, DOI: 10.1038/s41467-019-12672-x[Crossref], [PubMed], [CAS], Google Scholar6https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BB3MnntFWkug%253D%253D&md5=01131ef2560d996b312ea20f6c540b4aProteomic atlas of organ vasculopathies triggered by Staphylococcus aureus sepsisToledo Alejandro Gomez; Golden Gregory; Esko Jeffrey D; Toledo Alejandro Gomez; Golden Gregory; Varki Nissi; Esko Jeffrey D; Campos Alexandre Rosa; Cuello Hector; Sorrentino James; Lewis Nathan; Lewis Nathan; Lewis Nathan; Varki Nissi; Nizet Victor; Nizet Victor; Smith Jeffrey WNature communications (2019), 10 (1), 4656 ISSN:.Sepsis is a life-threatening condition triggered by a dysregulated host response to microbial infection resulting in vascular dysfunction, organ failure and death. Here we provide a semi-quantitative atlas of the murine vascular cell-surface proteome at the organ level, and how it changes during sepsis. Using in vivo chemical labeling and high-resolution mass spectrometry, we demonstrate the presence of a vascular proteome that is perfusable and shared across multiple organs. This proteome is enriched in membrane-anchored proteins, including multiple regulators of endothelial barrier functions and innate immunity. Further, we automated our workflows and applied them to a murine model of methicillin-resistant Staphylococcus aureus (MRSA) sepsis to unravel changes during systemic inflammatory responses. We provide an organ-specific atlas of both systemic and local changes of the vascular proteome triggered by sepsis. Collectively, the data indicates that MRSA-sepsis triggers extensive proteome remodeling of the vascular cell surfaces, in a tissue-specific manner.
- 7Bergin, S. P.; Holland, T. L.; Fowler, V. G., Jr.; Tong, S. Y. C. Bacteremia, sepsis, and infective endocarditis associated with Staphylococcus aureus. Curr. Top. Microbiol. Immunol. 2015, 409, 263– 296, DOI: 10.1007/82_2015_5001
- 8Zhang, Y.; Zhang, J.; Chen, W.; Angsantikul, P.; Spiekermann, K. A.; Fang, R. H.; Gao, W.; Zhang, L. Erythrocyte membrane-coated nanogel for combinatorial antivirulence and responsive antimicrobial delivery against Staphylococcus aureus infection. J. Controlled Release 2017, 263, 185– 191, DOI: 10.1016/j.jconrel.2017.01.016[Crossref], [PubMed], [CAS], Google Scholar8https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXhtFehs78%253D&md5=9fee13f24eabeaf6a9b91f5d0cefe98dErythrocyte membrane-coated nanogel for combinatorial antivirulence and responsive antimicrobial delivery against Staphylococcus aureus infectionZhang, Yue; Zhang, Jianhua; Chen, Wansong; Angsantikul, Pavimol; Spiekermann, Kevin A.; Fang, Ronnie H.; Gao, Weiwei; Zhang, LiangfangJournal of Controlled Release (2017), 263 (), 185-191CODEN: JCREEC; ISSN:0168-3659. (Elsevier B.V.)We reported an erythrocyte membrane-coated nanogel (RBC-nanogel) system with combinatorial antivirulence and responsive antibiotic delivery for the treatment of methicillin-resistant Staphylococcus aureus (MRSA) infection. RBC membrane was coated onto the nanogel via a membrane vesicle templated in situ gelation process, whereas the redox-responsiveness was achieved by using a disulfide bond-based crosslinker. We demonstrated that the RBC-nanogels effectively neutralized MRSA-assocd. toxins in extracellular environment and the toxin neutralization in turn promoted bacterial uptake by macrophages. In intracellular reducing environment, the RBC-nanogels showed an accelerated drug release profile, which resulted in more effective bacterial inhibition. When added to the macrophages infected with intracellular MRSA bacteria, the RBC-nanogels significantly inhibited bacterial growth compared to free antibiotics and non-responsive nanogel counterparts. These results indicate the great potential of the RBC-nanogel system as a new and effective antimicrobial agent against MRSA infection.
- 9Gardete, S.; Tomasz, A. Mechanisms of vancomycin resistance in Staphylococcus aureus. J. Clin. Invest. 2014, 124, 2836– 2840, DOI: 10.1172/jci68834[Crossref], [PubMed], [CAS], Google Scholar9https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BC2cfosFektw%253D%253D&md5=65c16134b7595389112856744ca0a9e4Mechanisms of vancomycin resistance in Staphylococcus aureusGardete Susana; Tomasz AlexanderThe Journal of clinical investigation (2014), 124 (7), 2836-40 ISSN:.Vancomycin is a glycopeptide antibiotic used for the treatment of Gram-positive bacterial infections. Traditionally, it has been used as a drug of last resort; however, clinical isolates of methicillin-resistant Staphylococcus aureus (MRSA) strains with decreased susceptibility to vancomycin (vancomycin intermediate-resistant S. aureus [VISA]) and more recently with high-level vancomycin resistance (vancomycin-resistant S. aureus [VRSA]) have been described in the clinical literature. The rare VRSA strains carry transposon Tn1546, acquired from vancomycin-resistant Enterococcus faecalis, which is known to alter cell wall structure and metabolism, but the resistance mechanisms in VISA isolates are less well defined. Herein, we review selected mechanistic aspects of resistance in VISA and summarize biochemical studies on cell wall synthesis in a VRSA strain. Finally, we recapitulate a model that integrates common mechanistic features of VRSA and VISA strains and is consistent with the mode of action of vancomycin.
- 10Kali, A. Antibiotics and bioactive natural products in treatment of methicillin resistant Staphylococcus aureus: A brief review. Pharmacogn. Rev. 2015, 9, 29– 34, DOI: 10.4103/0973-7847.156329[Crossref], [PubMed], [CAS], Google Scholar10https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXoslSls70%253D&md5=bcb5673a9b6e44a95abe2a8fb10f18d7Antibiotics and bioactive natural products in treatment of methicillin-resistant Staphylococcus aureus: a brief reviewKali, ArunavaPharmacognosy Reviews (2015), 9 (17), 29-34CODEN: PRHEEV; ISSN:0973-7847. (Medknow Publications and Media Pvt. Ltd.)A review. Infections caused by Staphylococcus aureus strains with Methicillin resistance are assocd. with increased mortality and morbidity, aggressive course, multiple drug resistance and hospital outbreaks. Several first and second line antibiotics are rapidly becoming ineffective for treatment due to emergence of resistance. Exts. of medicinal plants are rich source of unique phytochems. Plants used in traditional medicine have been reported to have significant anti-MRSA activity. The objective of this review is to provide a brief overview of antibiotics as well as anti-MRSA natural products and their future prospect.
- 11Kaur, D.; Chate, S. Study of antibiotic resistance pattern in methicillin resistant staphylococcus aureus with special reference to newer antibiotic. J. Global Infect. Dis. 2015, 7, 78– 84, DOI: 10.4103/0974-777x.157245[Crossref], [PubMed], [CAS], Google Scholar11https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXitVGjtrrJ&md5=ff4ea27e6f3506b9c7d9cd3af6e425d1Study of antibiotic resistance pattern in methicillin resistant staphylococcus aureus with special reference to newer antibioticKaur, Dardi Charan; Chate, Sadhana SanjayJournal of Global Infectious Diseases (2015), 7 (2), 78-84CODEN: JGIDAM; ISSN:0974-8245. (Medknow Publications and Media Pvt. Ltd.)The worldwide epidemic of antibiotic resistance is in danger of ending the golden age of antibiotic therapy. Resistance impacts on all areas of medicine, making successful empirical therapy much more difficult to achieve. Staphylococcus aureus demonstrates a unique ability to quickly respond to each antibiotic with the development of a resistance mechanism, starting with penicillin,until the most recent, linezolid and daptomycin. Methicillin resistant aureus (MRSA) has become endemic today in hospitals worldwide. Resistance to the newer antimicrobial-agents - linezolid, vancomycin, teicoplanin, daptomycin are been reported and also the fear of pandrug-resistance. This study was carried out to know the antimicrobial resistant pattern of MRSA to newer antibiotic, to know any isolates are extensively-drug resistant (XDR)/pandrug resistant (PDR), inducible macrolide-lincosamide streptogramin B (iMLSB), mupirocin resistance. Thirty-six MRSA isolates resistant to the routinely tested antibiotic were further tested for list of antibiotic by a group of international experts. Isolates were tested for iMLSB and mupirocin resistance by the disk diffusion method. Of 385 MRSA, 36 (9.35%) isolates of MRSA were resistant to routinely tested antibiotic. Among these 36 MRSA isolates, none of our isolates were XDR/PDR or showed resistant to anti-MRSA cephalosporins (ceftaroline), phosphonic acids, glycopeptides, glycylcyclines, and fucidanes. Lower resistance was seen in oxazolidinones (2.78%), streptogramins (5.56%), lipopeptide (5.56%). Thirty-four (94.44%) isolates showed constitutive MLSB (cMLSB) resistance and two (5.56%) iMLSB phenotypes. High- and low-level mupirocin resistance were in 13 (36.11%) and six (16.67%), resp. In our study, none of our isolates were XDR or PDR. No resistance was obsd. to ceftaroline, telavancin, teicoplanin, and vancomycin; but the presence of linezolid resistance (1, 2.28%) and daptomycin resistance (2, 5.56%) in our rural set-up is a cause of concern.
- 12Arunkumar, V.; Prabagaravarthanan, R.; Bhaskar, M. Prevalence of methicillin-resistant Staphylococcus aureus (MRSA) infections among patients admitted in critical care units in a tertiary care hospital. Int. J. Res. Med. Sci. 2017, 5, 2362– 2366
- 13McGuinness, W. A.; Malachowa, N.; DeLeo, F. R. Vancomycin Resistance in Staphylococcus aureus. Yale J. Biol. Med. 2017, 90, 269– 281[PubMed], [CAS], Google Scholar13https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1MXhslOktr0%253D&md5=56ebbfb1e8a82bb29e528b54ea328cc5Vancomycin resistance in Staphylococcus aureusMcGuinness, Will A.; Malachowa, Natalia; DeLeo, Frank R.Yale Journal of Biology and Medicine (2017), 90 (2), 269-281CODEN: YJBMAU; ISSN:0044-0086. (Yale Journal of Biology and Medicine)The evolution of Staphylococcus aureus during the modern antibiotic era has been delineated by distinct strain emergence events, many of which include acquisition of antibiotic resistance. The relative high burden of methicillin-resistant S. aureus (MRSA) in healthcare and community settings is a major concern worldwide. Vancomycin, a glycopeptide antibiotic that inhibits cell wall biosynthesis, remains a drug of choice for treatment of severe MRSA infections. S. aureus strains exhibiting increased resistance to vancomycin, known as vancomycin intermediate-resistant S. aureus (VISA) (MIC = 4-8 μg/mL), were discovered in the 1990s. The mol. basis of resistance in VISA is polygenic and involves stepwise mutations in genes encoding mols. predominantly involved in cell envelope biosynthesis. S. aureus isolates with complete resistance to vancomycin (MIC ≥ 16 μg/mL) are termed vancomycin-resistant S. aureus (VRSA)-they were first reported in the U.S. in 2002. Resistance in VRSA is conferred by the vanA gene and operon, which is present on a plasmid. Although treatment of VRSA infections is challenging, the total no. of human VRSA infections to date is limited (14 in the U.S.). By comparison, the burden of VISA is relatively high and the mol. mechanisms of resistance are less well-defined. VISA are assocd. with persistent infections, vancomycin treatment failure, and poor clin. outcomes. Here, we review in brief progress made toward understanding the acquisition of antibiotic resistance in S. aureus, with an emphasis on the mol. mechanisms underlying vancomycin resistance.
- 14Marty, F. M.; Yeh, W. W.; Wennersten, C. B.; Venkataraman, L.; Albano, E.; Alyea, E. P.; Gold, H. S.; Baden, L. R.; Pillai, S. K. Emergence of a clinical daptomycin-resistant Staphylococcus aureus isolate during treatment of methicillin-resistant Staphylococcus aureus bacteremia and osteomyelitis. J. Clin. Microbiol. 2006, 44, 595– 597, DOI: 10.1128/jcm.44.2.595-597.2006[Crossref], [PubMed], [CAS], Google Scholar14https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD28XitFSgsro%253D&md5=a7cd9c5b21a552e05b5a92d203336163Emergence of a clinical daptomycin-resistant Staphylococcus aureus isolate during treatment of methicillin-resistant Staphylococcus aureus bacteremia and osteomyelitisMarty, Francisco M.; Yeh, Wendy W.; Wennersten, Christine B.; Venkataraman, Lata; Albano, Esperanza; Alyea, Edwin P.; Gold, Howard S.; Baden, Lindsey R.; Pillai, Satish K.Journal of Clinical Microbiology (2006), 44 (2), 595-597CODEN: JCMIDW; ISSN:0095-1137. (American Society for Microbiology)The emergence of a clin. daptomycin-resistant Staphylococcus aureus isolate occurred during treatment of methicillin-resistant S. aureus bacteremia and probable vertebral osteomyelitis. The breakthrough isolate was indistinguishable from pretreatment daptomycin-susceptible isolates by pulsed-field gel electrophoresis. Daptomycin nonsusceptibility was confirmed by MIC and time-kill curve analyses.
- 15Ikeda-Dantsuji, Y.; Hanaki, H.; Sakai, F.; Yanagisawa, C.; Nakae, T.; Sakai, F.; Tomono, K.; Takesue, Y.; Honda, J.; Nonomiya, Y.; Suwabe, A.; Nagura, O.; Yanagihara, K.; Mikamo, H.; Fukuchi, K.; Kaku, M.; Kohno, S.; Yoshida, K.; Niki, Y. Linezolid-resistant Staphylococcus aureus isolated from 2006 through 2008 at six hospitals in Japan. J. Infect. Chemother. 2011, 17, 45– 51, DOI: 10.1007/s10156-010-0085-1[Crossref], [PubMed], [CAS], Google Scholar15https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3MXhvV2iu74%253D&md5=7265ce121b0824d409caa05821b971caLinezolid-resistant Staphylococcus aureus isolated from 2006 through 2008 at six hospitals in JapanIkeda-Dantsuji, Yurika; Hanaki, Hideaki; Sakai, Fuminori; Tomono, Kazunori; Takesue, Yoshio; Honda, Junichi; Nonomiya, Yuriko; Suwabe, Akira; Nagura, Osanori; Yanagihara, Katsunori; Mikamo, Hiroshige; Fukuchi, Kunihiko; Kaku, Mitsuo; Kohno, Shigeru; Yanagisawa, Chie; Nakae, Taiji; Yoshida, Koichiro; Niki, YoshihitoJournal of Infection and Chemotherapy (2011), 17 (1), 45-51CODEN: JICHFN; ISSN:1341-321X. (Springer Japan)Limited use of linezolid for treating methicillin-resistant Staphylococcus aureus (MRSA) infection was approved in Japan in 2006. We report here the status of linezolid-resistant MRSAs in Japan. Eleven linezolid-resistant clin. isolates from 11 patients at six hospitals were collected from 2006 through 2008. The minimal inhibitory concn. (MIC) of linezolid in these strains varied from 8 to 64 μg/mL. All strains had at least one G2576T mutation in the chromosomal gene(s) encoding domain V of the 23S rRNA (rRNA). Chromosomal DNA encoding five copies of the domain V region was analyzed by polymerase chain reaction (PCR). Strains with the linezolid MICs of 64, 32, 16, and 8 μg/mL had the G2576T mutation(s) in four, three (or four), two, and one copy of the 23S rRNA genes, resp. These results suggest that the level of linezolid resistance seems to be roughly correlated with the no. of mutations in the genes encoding 23S rRNA. DNA samples from all 11 strains were subjected to pulsed-field gel electrophoresis and were classified into seven independent clones having >92% identity. Among the 11 patients, five had been treated with linezolid and the remainder, in two hospitals, had no history of prior linezolid use. The results suggested possible nosocomial infections by linezolid-resistant MRSA.
- 16Pang, L.; Weeks, S. D.; Van Aerschot, A. Aminoacyl-tRNA synthetases as valuable targets for antimicrobial drug discovery. Int. J. Mol. Sci. 2021, 22, 1750, DOI: 10.3390/ijms22041750[Crossref], [PubMed], [CAS], Google Scholar16https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3MXotVKqt70%253D&md5=b732971a63f06fe876036b21b8e3f546Aminoacyl-tRNA synthetases as valuable targets for antimicrobial drug discoveryPang, Luping; Weeks, Stephen d.; Van aerschot, ArthurInternational Journal of Molecular Sciences (2021), 22 (4), 1750CODEN: IJMCFK; ISSN:1422-0067. (MDPI AG)A review. Aminoacyl-tRNA synthetases (aaRSs) catalyze the esterification of tRNA with a cognate amino acid and are essential enzymes in all three kingdoms of life. Due to their important role in the translation of the genetic code, aaRSs have been recognized as suitable targets for the development of small mol. anti-infectives. In this review, following a concise discussion of aaRS catalytic and proof-reading activities, the various inhibitory mechanisms of reported natural and synthetic aaRS inhibitors are discussed. Using the expanding repository of ligand-bound X-ray crystal structures, we classified these compds. based on their binding sites, focusing on their ability to compete with the assocn. of one, or more of the canonical aaRS substrates. In parallel, we examd. the determinants of species-selectivity and discuss potential resistance mechanisms of some of the inhibitor classes. Combined, this structural perspective highlights the opportunities for further exploration of the aaRS enzyme family as antimicrobial targets.
- 17Kwon, N. H.; Fox, P. L.; Kim, S. Aminoacyl-tRNA synthetases as therapeutic targets. Nat. Rev. Drug Discovery 2019, 18, 629– 650, DOI: 10.1038/s41573-019-0026-3[Crossref], [PubMed], [CAS], Google Scholar17https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1MXptlajtrY%253D&md5=dd9c680cd873d35c8ab10350cd7fb044Aminoacyl-tRNA synthetases as therapeutic targetsKwon, Nam Hoon; Fox, Paul L.; Kim, SunghoonNature Reviews Drug Discovery (2019), 18 (8), 629-650CODEN: NRDDAG; ISSN:1474-1776. (Nature Research)A review. Aminoacyl-tRNA synthetases (ARSs) are essential enzymes for protein synthesis with evolutionarily conserved enzymic mechanisms. Despite their similarity across organisms, scientists have been able to generate effective anti-infective agents based on the structural differences in the catalytic clefts of ARSs from pathogens and humans. However, recent genomic, proteomic and functionomic advances have unveiled unexpected disease-assocd. mutations and altered expression, secretion and interactions in human ARSs, revealing hidden biol. functions beyond their catalytic roles in protein synthesis. These studies have also brought to light their potential as a rich and unexplored source for new therapeutic targets and agents through multiple avenues, including direct targeting of the catalytic sites, controlling disease-assocd. protein-protein interactions and developing novel biologics from the secreted ARS proteins or their parts. This Review addresses the emerging biol. and therapeutic applications of human ARSs in diseases including autoimmune and rare diseases, and cancer.
- 18Ho, J. M.; Bakkalbasi, E.; Söll, D.; Miller, C. A. Drugging tRNA aminoacylation. RNA Biol. 2018, 15, 667– 677, DOI: 10.1080/15476286.2018.1429879[Crossref], [PubMed], [CAS], Google Scholar18https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BC1MvhvVCgug%253D%253D&md5=129b1a19b33997a85f6af42a4f575341Drugging tRNA aminoacylationHo Joanne M; Bakkalbasi Erol; Miller Corwin A; Soll Dieter; Soll DieterRNA biology (2018), 15 (4-5), 667-677 ISSN:.Inhibition of tRNA aminoacylation has proven to be an effective antimicrobial strategy, impeding an essential step of protein synthesis. Mupirocin, the well-known selective inhibitor of bacterial isoleucyl-tRNA synthetase, is one of three aminoacylation inhibitors now approved for human or animal use. However, design of novel aminoacylation inhibitors is complicated by the steadfast requirement to avoid off-target inhibition of protein synthesis in human cells. Here we review available data regarding known aminoacylation inhibitors as well as key amino-acid residues in aminoacyl-tRNA synthetases (aaRSs) and nucleotides in tRNA that determine the specificity and strength of the aaRS-tRNA interaction. Unlike most ligand-protein interactions, the aaRS-tRNA recognition interaction represents coevolution of both the tRNA and aaRS structures to conserve the specificity of aminoacylation. This property means that many determinants of tRNA recognition in pathogens have diverged from those of humans-a phenomenon that provides a valuable source of data for antimicrobial drug development.
- 19Ribas de Pouplana, L.; Schimmel, P. Two classes of tRNA synthetases suggested by sterically compatible dockings on tRNA acceptor stem. Cell 2001, 104, 191– 193, DOI: 10.1016/s0092-8674(01)00204-5[Crossref], [PubMed], [CAS], Google Scholar19https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BD3M7mslajtw%253D%253D&md5=f91390856e9ce2ee119d926cea3ea8a7Two classes of tRNA synthetases suggested by sterically compatible dockings on tRNA acceptor stemRibas de Pouplana L; Schimmel PCell (2001), 104 (2), 191-3 ISSN:0092-8674.There is no expanded citation for this reference.
- 20Eriani, G.; Delarue, M.; Poch, O.; Gangloff, J.; Moras, D. Partition of tRNA synthetases into two classes based on mutually exclusive sets of sequence motifs. Nature 1990, 347, 203– 206, DOI: 10.1038/347203a0[Crossref], [PubMed], [CAS], Google Scholar20https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK3MXpslensg%253D%253D&md5=3b8c5ed8a05996c6d9e97ebcb3b1b7ecPartition of tRNA synthetases into two classes based on mutually exclusive sets of sequence motifsEriani, Gilbert; Delarue, Marc; Poch, Olivier; Gangloff, Jean; Moras, DinoNature (London, United Kingdom) (1990), 347 (6289), 203-6CODEN: NATUAS; ISSN:0028-0836.Out of 18 known aminoacyl-tRNA synthetases (aaRS), only 9 referred to as class I synthetases (GlnRS, TyrRS, MetRS, GluRS, ArgRS, ValRS, IleRS, LeuRS, TrpRS), display 2 short common consensus sequences. The sequence of Escherichia coli ProRS, a dimer of relative mol. mass 127.402, which is homologous to both ThrRS and SerRS, is reported. These 3 latter aaRS share 3 new sequence motifs with AspRS, AsnRS, LysRS, HisRS, and the B subunit of PheRS. These 3 motifs (motifs 1, 2, and 3), in a search through the entire data bank, proved to be specific for this set of aaRS (referred to as class II). Class II may also contain AlaRS and GlyRS, because these sequences have a typical motif 3. Surprisingly, this partition of aaRS in 2 classes is strongly correlated on the functional level with the acylation occurring either on the 2' OH (class I) or 3' OH (class II) of the ribose of the last nucleotide of tRNA.
- 21Novoa, E. M.; Camacho, N.; Tor, A.; Wilkinson, B.; Moss, S.; Marín-García, P.; Azcárate, I. G.; Bautista, J. M.; Mirando, A. C.; Francklyn, C. S.; Varon, S.; Royo, M.; Cortés, A.; Ribas de Pouplana, L. Analogs of natural aminoacyl-tRNA synthetase inhibitors clear malaria in vivo. Proc. Natl. Acad. Sci. U.S.A. 2014, 111, E5508– E5517, DOI: 10.1073/pnas.1405994111[Crossref], [PubMed], [CAS], Google Scholar21https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2cXitVClu7nL&md5=9878f848d956e30171b3356867dd33f6Analogs of natural aminoacyl-tRNA synthetase inhibitors clear malaria in vivoNovoa, Eva Maria; Camacho, Noelia; Tor, Anna; Wilkinson, Barrie; Moss, Steven; Marin-Garcia, Patricia; Azcarate, Isabel G.; Bautista, Jose M.; Mirando, Adam C.; Francklyn, Christopher S.; Varon, Sonia; Royo, Miriam; Cortes, Alfred; Ribas de Pouplana, LluisProceedings of the National Academy of Sciences of the United States of America (2014), 111 (51), E5508-E5517CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)Malaria remains a major global health problem. Emerging resistance to existing antimalarial drugs drives the search for new antimalarials, and protein translation is a promising pathway to target. Here we explore the potential of the aminoacyl-tRNA synthetase (ARS) family as a source of antimalarial drug targets. First, a battery of known and novel ARS inhibitors was tested against Plasmodium falciparum cultures, and their activities were compared. Borrelidin, a natural inhibitor of threonyl-tRNA synthetase (ThrRS), stands out for its potent antimalarial effect. However, it also inhibits human ThrRS and is highly toxic to human cells. To circumvent this problem, we tested a library of bioengineered and semisynthetic borrelidin analogs for their antimalarial activity and toxicity. We found that some analogs effectively lose their toxicity against human cells while retaining a potent antiparasitic activity both in vitro and in vivo and cleared malaria from Plasmodium yoelii-infected mice, resulting in 100% mice survival rates. Our work identifies borrelidin analogs as potent, selective, and unexplored scaffolds that efficiently clear malaria both in vitro and in vivo.
- 22Saint-Léger, A.; Sinadinos, C.; Ribas de Pouplana, L. The growing pipeline of natural aminoacyl-tRNA synthetase inhibitors for malaria treatment. Bioengineered 2016, 7, 60– 64, DOI: 10.1080/21655979.2016.1149270[Crossref], [PubMed], [CAS], Google Scholar22https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28XlslWgsLo%253D&md5=c1495beaba0de9abc1150af425db0350The growing pipeline of natural aminoacyl-tRNA synthetase inhibitors for malaria treatmentSaint-Leger, Adelaide; Sinadinos, Christopher; Ribas de Pouplana, LluisBioengineered (2016), 7 (2), 60-64CODEN: BIOEGL; ISSN:2165-5987. (Taylor & Francis, Inc.)Malaria remains a major global health problem. Parasite resistance to existing drugs makes development of new antimalarials an urgency. The protein synthesis machinery is an excellent target for the development of new anti-infectives, and aminoacyl-tRNA synthetases (aaRS) have been validated as antimalarial drug targets. However, avoiding the emergence of drug resistance and improving selectivity to target aaRS in apicomplexan parasites, such as Plasmodium falciparum, remain crucial challenges. Here we discuss such issues using examples of known inhibitors of P. falciparum aaRS, namely halofuginone, cladosporin and borrelidin (inhibitors of ProRS, LysRS and ThrRS, resp.). Encouraging recent results provide useful guidelines to facilitate the development of novel drug candidates which are more potent and selective against these essential enzymes.
- 23Hütter, R.; Poralla, K.; Zachau, H. G.; Zähner, H. Metabolic products of microorganisms. 5l. On the mechanism of action of borrelidin-inhibition of the threonine incorporation in sRNA. Biochem. Z. 1966, 344, 190– 196[PubMed], [CAS], Google Scholar23https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaF28XpsFKgsA%253D%253D&md5=e090e8ea25a54b9c860c7f64e1b79b22Metabolic products of microorganisms. LI. Mechanism of action of borrelidin. Inhibition of the incorporation of threonine into sRNA (soluble RNA)Huetter, R.; Poralla, K.; Zachau, H. G.; Zehner, H.Biochemische Zeitschrift (1966), 344 (2), 190-6CODEN: BIZEA2; ISSN:0366-0753.cf. CA 64, 15963e. The mode of action of the antibiotic borrelidin has been studied. The expts. indicate that borrelidin inhibits esp. the incorporation of threonine into sRNA.
- 24Paetz, W.; Nass, G. Biochemical and Immunological Characterization of Threonyl-tRNA Synthetase of Two Borrelidin-Resistant Mutants of Escherichia coli K12. Eur. J. Med. Chem. 1973, 35, 331– 337, DOI: 10.1111/j.1432-1033.1973.tb02843.x
- 25Wilkinson, B.; Gregory, M. A.; Moss, S. J.; Carletti, I.; Sheridan, R. M.; Kaja, A.; Ward, M.; Olano, C.; Mendez, C.; Salas, J. A.; Leadlay, P. F.; van Ginckel, R.; Zhang, M.-Q. Separation of anti-angiogenic and cytotoxic activities of borrelidin by modification at the C17 side chain. Bioorg. Med. Chem. Lett. 2006, 16, 5814– 5817, DOI: 10.1016/j.bmcl.2006.08.073[Crossref], [PubMed], [CAS], Google Scholar25https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD28XhtVCrt7zI&md5=9a7c1cc1b881e7c6e395d8e527d48031Separation of anti-angiogenic and cytotoxic activities of borrelidin by modification at the C17 side chainWilkinson, Barrie; Gregory, Matthew A.; Moss, Steven J.; Carletti, Isabelle; Sheridan, Rose M.; Kaja, Andrew; Ward, Michael; Olano, Carlos; Mendez, Carmen; Salas, Jose A.; Leadlay, Peter F.; van Ginckel, Rob; Zhang, Ming-QiangBioorganic & Medicinal Chemistry Letters (2006), 16 (22), 5814-5817CODEN: BMCLE8; ISSN:0960-894X. (Elsevier Ltd.)A set of novel borrelidin analogs have been prepd. by precursor-directed biosynthesis. Structure-activity relationship anal. suggests that steric structural arrangement within the C17 side chain is important for differentiating cytotoxic and antiangiogenic activities. A C17-cyclobutyl analog 3 was found to have markedly increased selectivity for in vitro angiogenesis inhibition over cytotoxicity and is therefore potentially useful as an anticancer agent.
- 26Sugawara, A.; Tanaka, T.; Hirose, T.; Ishiyama, A.; Iwatsuki, M.; Takahashi, Y.; Otoguro, K.; O̅mura, S.; Sunazuka, T. Borrelidin analogues with antimalarial activity: design, synthesis and biological evaluation against Plasmodium falciparum parasites. Bioorg. Med. Chem. Lett. 2013, 23, 2302– 2305, DOI: 10.1016/j.bmcl.2013.02.075[Crossref], [PubMed], [CAS], Google Scholar26https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3sXktFWqur8%253D&md5=afa2cf528c533554131c7cf81095c906Borrelidin analogues with antimalarial activity: Design, synthesis and biological evaluation against Plasmodium falciparum parasitesSugawara, Akihiro; Tanaka, Toshiaki; Hirose, Tomoyasu; Ishiyama, Aki; Iwatsuki, Masato; Takahashi, Yoko; Otoguro, Kazuhiko; Omura, Satoshi; Sunazuka, ToshiakiBioorganic & Medicinal Chemistry Letters (2013), 23 (8), 2302-2305CODEN: BMCLE8; ISSN:0960-894X. (Elsevier B.V.)Borrelidin, a structurally unique 18-membered macrolide, was found to express antimalarial activity against drug-resistant Plasmodium falciparum malaria parasites, with IC50 value of 0.93 ng/mL. However, it also displays strong cytotoxicity against human diploid embryonic MRC-5 cells. To investigate the issue of the cytotoxicity of borrelidin, borrelidin-based analogs were synthesized and their anti-Plasmodium properties were evaluated. In this communication, we report that novel borrelidin analog I, bearing the CH2SPh moiety via a triazole linkage, was found to retain a potent antimalarial activity, against drug-sensitive and drug-resistant parasite strains, but possess only weak cytotoxicity against human cells.
- 27Teng, M.; Hilgers, M. T.; Cunningham, M. L.; Borchardt, A.; Locke, J. B.; Abraham, S.; Haley, G.; Kwan, B. P.; Hall, C.; Hough, G. W.; Shaw, K. J.; Finn, J. Identification of bacteria-selective threonyl-tRNA synthetase substrate inhibitors by structure-based design. J. Med. Chem. 2013, 56, 1748– 1760, DOI: 10.1021/jm301756m[ACS Full Text], [CAS], Google Scholar27https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3sXhvVCjt7w%253D&md5=6619e410c2680cd7670f3652593ce58cIdentification of Bacteria-Selective Threonyl-tRNA Synthetase Substrate Inhibitors by Structure-Based DesignTeng, Min; Hilgers, Mark T.; Cunningham, Mark L.; Borchardt, Allen; Locke, Jeffrey B.; Abraham, Sunny; Haley, Gregory; Kwan, Bryan P.; Hall, Courtney; Hough, Grayson W.; Shaw, Karen J.; Finn, JohnJournal of Medicinal Chemistry (2013), 56 (4), 1748-1760CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)A series of potent and bacteria-selective threonyl-tRNA synthetase (ThrRS) inhibitors have been identified using structure-based drug design. These compds. occupied the substrate binding site of ThrRS and showed excellent binding affinities for all of the bacterial orthologs tested. Some of the compds. displayed greatly improved bacterial selectivity. Key residues responsible for potency and bacteria/human ThrRS selectivity have been identified. Antimicrobial activity has been achieved against wild-type Haemophilus influenzae and efflux-deficient mutants of Escherichia coli and Burkholderia thailandensis.
- 28Guo, J.; Chen, B.; Yu, Y.; Cheng, B.; Cheng, Y.; Ju, Y.; Gu, Q.; Xu, J.; Zhou, H. Discovery of novel tRNA-amino acid dual-site inhibitors against threonyl-tRNA synthetase by fragment-based target hopping. Eur. J. Med. Chem. 2020, 187, 111941, DOI: 10.1016/j.ejmech.2019.111941[Crossref], [PubMed], [CAS], Google Scholar28https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1MXitlGhtLbM&md5=5ebcdf329ae1afa5cee3cf5799b3ec6bDiscovery of novel tRNA-amino acid dual-site inhibitors against threonyl-tRNA synthetase by fragment-based target hoppingGuo, Junsong; Chen, Bingyi; Yu, Ying; Cheng, Bao; Cheng, Yanfang; Ju, Yingchen; Gu, Qiong; Xu, Jun; Zhou, HuihaoEuropean Journal of Medicinal Chemistry (2020), 187 (), 111941CODEN: EJMCA5; ISSN:0223-5234. (Elsevier Masson SAS)Threonyl-tRNA synthetase (ThrRS) is a key member of the aminoacyl-tRNA synthetase (aaRS) family that plays essential roles in protein biosynthesis, and ThrRS inhibitors have potential in the therapy of multiple diseases, such as microbial infections and cancers. Based on a unique tRNA-amino acid dual-site inhibitory mechanism identified recently with the herb-derived prolyl-tRNA synthetase (ProRS) inhibitor halofuginone (HF), a series of compds. have been designed and synthesized by employing a fragment-based target hopping approach to simultaneously target the tRNAThr and L-threonine binding pockets of ThrRS. Among them, compd. 30d showed an IC50 value of 1.4μM against Salmonella enterica ThrRS (SeThrRS) and MIC values of 16-32μg/mL against the tested bacterial strains. The cocrystal structure of SeThrRS in complex with 30d was detd. at high resoln., revealing that 30d simultaneously occupies both binding pockets for the nucleotide A76 of tRNAThr and L-threonine in an ATP-independent manner, a novel mechanism compared to all other reported ThrRS inhibitors. Our study provides a new class of ThrRS inhibitors, and more importantly, it presents the first exptl. evidence that the tRNA-amino acid dual-site inhibitory mechanism could apply to other aaRSs beyond ProRS, thus providing great opportunities for designing new mechanistic inhibitors for aaRS-based therapeutics. oxoquinazolin-.
- 29Guo, J.; Chen, B.; Yu, Y.; Cheng, B.; Ju, Y.; Tang, J.; Cai, Z.; Gu, Q.; Xu, J.; Zhou, H. Structure-guided optimization and mechanistic study of a class of quinazolinone-threonine hybrids as antibacterial ThrRS inhibitors. Eur. J. Med. Chem. 2020, 207, 112848, DOI: 10.1016/j.ejmech.2020.112848[Crossref], [PubMed], [CAS], Google Scholar29https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXitVOqsLzI&md5=3127bd6273e7959271edbf6a789fbefcStructure-guided optimization and mechanistic study of a class of quinazolinone-threonine hybrids as antibacterial ThrRS inhibitorsGuo, Junsong; Chen, Bingyi; Yu, Ying; Cheng, Bao; Ju, Yingchen; Tang, Jieyu; Cai, Zhengjun; Gu, Qiong; Xu, Jun; Zhou, HuihaoEuropean Journal of Medicinal Chemistry (2020), 207 (), 112848CODEN: EJMCA5; ISSN:0223-5234. (Elsevier Masson SAS)Aminoacyl-tRNA synthetases (aaRSs) are an attractive class of antibacterial drug targets due to their essential roles in protein translation. While most traditional aaRS inhibitors target the binding pockets of substrate amino acids and/or ATP, we recently developed a class of novel tRNA-amino acid dual-site inhibitors including inhibitor 3 ((2S,3R)-2-amino-N-((E)-4-(6,7-dichloro-4-oxoquinazolin-3(4H)-yl)but-2-en-1-yl)-3-hydroxybutanamide) against threonyl-tRNA synthetase (ThrRS). Here, the binding modes and structure-activity relationships (SARs) of these inhibitors were analyzed by the crystal structures of Salmonella enterica ThrRS (SeThrRS) in complex with three of them. Based on the cocrystal structures, twelve quinazolinone-threonine hybrids were designed and synthesized, and their affinities, enzymic inhibitory activities, and cellular potencies were evaluated. The best deriv. 8g achieved a Kd value of 0.40 μM, an IC50 value of 0.50 μM against SeThrRS and MIC values of 16-32 μg/mL against the tested bacterial strains. The cocrystal structure of the SeThrRS-8g complex revealed that 8g induced a bended conformation for Met332 by forming hydrophobic interactions, which better mimicked the binding of tRNAThr to ThrRS. Moreover, the inhibitory potency of 8g was less impaired than a reported ATP competitive inhibitor at high concns. of ATP, supporting our hypothesis that tRNA site inhibitors are likely superior to ATP site inhibitors in vivo, where ATP typically reaches millimolar concns.
- 30Torres-Larios, A.; Sankaranarayanan, R.; Rees, B.; Dock-Bregeon, A.-C.; Moras, D. Conformational movements and cooperativity upon amino acid, ATP and tRNA binding in threonyl-tRNA synthetase. J. Mol. Biol. 2003, 331, 201– 211, DOI: 10.1016/s0022-2836(03)00719-8[Crossref], [PubMed], [CAS], Google Scholar30https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD3sXls1eru7Y%253D&md5=1b9322b3cafb3a1137175e3c0ab86112Conformational movements and cooperativity upon amino acid, ATP and tRNA binding in threonyl-tRNA synthetaseTorres-Larios, Alfredo; Sankaranarayanan, Rajan; Rees, Bernard; Dock-Bregeon, Anne-Catherine; Moras, DinoJournal of Molecular Biology (2003), 331 (1), 201-211CODEN: JMOBAK; ISSN:0022-2836. (Elsevier Science Ltd.)The crystal structures of threonyl-tRNA synthetase (ThrRS) from Staphylococcus aureus, with ATP and an analog of threonyl adenylate, are described. Together with the previously detd. structures of Escherichia coli ThrRS with different substrates, they allow a comprehensive anal. of the effect of binding of all the substrates: threonine, ATP and tRNA. The tRNA, by inserting its acceptor arm between the N-terminal domain and the catalytic domain, causes a large rotation of the former. Within the catalytic domain, four regions surrounding the active site display significant conformational changes upon binding of the different substrates. The binding of threonine induces the movement of as much as 50 consecutive amino acid residues. The binding of ATP triggers a displacement, as large as 8 A at some Cα positions, of a strand-loop-strand region of the core β-sheet. Two other regions move in a cooperative way upon binding of threonine or ATP: the motif 2 loop, which plays an essential role in the first step of the aminoacylation reaction, and the ordering loop, which closes on the active site cavity when the substrates are in place. The tRNA interacts with all four mobile regions, several residues initially bound to threonine or ATP switching to a position in which they can contact the tRNA. Three such conformational switches could be identified, each of them in a different mobile region. The structural anal. suggests that, while the small substrates can bind in any order, they must be in place before productive tRNA binding can occur.
- 31We used a database of commercially available compounds; Otava Ltd., http://www.otavachemicals.com, (accessed April, 2020).
- 32Kovalenko, O. P.; Volynets, G. P.; Rybak, M. Y.; Starosyla, S. A.; Gudzera, O. I.; Lukashov, S. S.; Bdzhola, V. G.; Yarmoluk, S. M.; Boshoff, H. I.; Tukalo, M. A. Dual-target inhibitors of mycobacterial aminoacyl-tRNA synthetases among N-benzylidene-N′-thiazol-2-yl-hydrazines. Medchemcomm 2019, 10, 2161– 2169, DOI: 10.1039/c9md00347a[Crossref], [PubMed], [CAS], Google Scholar32https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1MXit1GlsbrN&md5=524015cced076511f6bb4aa7424766d8Dual-target inhibitors of mycobacterial aminoacyl-tRNA synthetases among N-benzylidene-N'-thiazol-2-yl-hydrazinesKovalenko, Oksana P.; Volynets, Galyna P.; Rybak, Mariia Yu.; Starosyla, Sergiy A.; Gudzera, Olga I.; Lukashov, Sergiy S.; Bdzhola, Volodymyr G.; Yarmoluk, Sergiy M.; Boshoff, Helena I.; Tukalo, Michael A.MedChemComm (2019), 10 (12), 2161-2169CODEN: MCCEAY; ISSN:2040-2503. (Royal Society of Chemistry)Effective treatment of tuberculosis is challenged by the rapid development of Mycobacterium tuberculosis (Mtb) multidrug resistance that presumably could be overcome with novel multi-target drugs. Aminoacyl-tRNA synthetases (AARSs) are an essential part of protein biosynthesis machinery and attractive targets for drug discovery. Here, we exptl. verify a hypothesis of simultaneous targeting of structurally related AARSs by a single inhibitor. We previously identified a new class of mycobacterial leucyl-tRNA synthetase inhibitors, N-benzylidene-N'-thiazol-2-yl-hydrazines. Mol. docking of a library of novel N-benzylidene-N'-thiazol-2-yl-hydrazine derivs. into active sites of M. tuberculosis LeuRS (MtbLeuRS) and MetRS (MtbMetRS) resulted in a panel of the best ranking compds., which were then evaluated for enzymic potency. Screening data revealed 11 compds. active against MtbLeuRS and 28 compds. active against MtbMetRS. The hit compds. display dual inhibitory potency as demonstrated by IC50 values for both enzymes. Compd. I is active against Mtb H37Rv cells in in vitro bioassays.
- 33Gudzera, O. I.; Golub, A. G.; Bdzhola, V. G.; Volynets, G. P.; Lukashov, S. S.; Kovalenko, O. P.; Kriklivyi, I. A.; Yaremchuk, A. D.; Starosyla, S. A.; Yarmoluk, S. M.; Tukalo, M. A. Discovery of potent anti-tuberculosis agents targeting leucyl-tRNA synthetase. Bioorg. Med. Chem. 2016, 24, 1023– 1031, DOI: 10.1016/j.bmc.2016.01.028[Crossref], [PubMed], [CAS], Google Scholar33https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28XhsVKksro%253D&md5=5bce1d6fbf6dda8394233e4b0dbafb83Discovery of potent anti-tuberculosis agents targeting leucyl-tRNA synthetaseGudzera, Olga I.; Golub, Andriy G.; Bdzhola, Volodymyr G.; Volynets, Galyna P.; Lukashov, Sergiy S.; Kovalenko, Oksana P.; Kriklivyi, Ivan A.; Yaremchuk, Anna D.; Starosyla, Sergiy A.; Yarmoluk, Sergiy M.; Tukalo, Michail A.Bioorganic & Medicinal Chemistry (2016), 24 (5), 1023-1031CODEN: BMECEP; ISSN:0968-0896. (Elsevier B.V.)Tuberculosis is a serious infectious disease caused by human pathogen bacteria Mycobacterium tuberculosis. Bacterial drug resistance is a very significant medical problem nowadays and development of novel antibiotics with different mechanisms of action is an important goal of modern medical science. Leucyl-tRNA synthetase (LeuRS) has been recently clin. validated as antimicrobial target. Here the authors report the discovery of small-mol. inhibitors of M. tuberculosis LeuRS. Using receptor-based virtual screening the authors have identified six inhibitors of M. tuberculosis LeuRS from two different chem. classes. The most active compd. I inhibits LeuRS with IC50 of 6 μM. A series of derivs. has been synthesized and evaluated in vitro toward M. tuberculosis LeuRS. It was revealed that the most active compd. II inhibits LeuRS with IC50 of 2.27 μM. All active compds. were tested for antimicrobial effect against M. tuberculosis H37Rv. The compd. I seems to have the best cell permeability and inhibits growth of pathogenic bacteria with IC50 = 10.01 μM and IC90 = 13.53 μM.
- 34Mosmann, T. Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J. Immunol. Methods 1983, 65, 55– 63, DOI: 10.1016/0022-1759(83)90303-4[Crossref], [PubMed], [CAS], Google Scholar34https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADyaL2c%252FovFSmtw%253D%253D&md5=c1116f4ca7a7d2b79fab2a0bd2c2d439Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assaysMosmann TJournal of immunological methods (1983), 65 (1-2), 55-63 ISSN:0022-1759.A tetrazolium salt has been used to develop a quantitative colorimetric assay for mammalian cell survival and proliferation. The assay detects living, but not dead cells and the signal generated is dependent on the degree of activation of the cells. This method can therefore be used to measure cytotoxicity, proliferation or activation. The results can be read on a multiwell scanning spectrophotometer (ELISA reader) and show a high degree of precision. No washing steps are used in the assay. The main advantages of the colorimetric assay are its rapidity and precision, and the lack of any radioisotope. We have used the assay to measure proliferative lymphokines, mitogen stimulations and complement-mediated lysis.
- 35Bodian, D. L.; Yamasaki, R. B.; Buswell, R. L.; Stearns, J. F.; White, J. M.; Kuntz, I. D. Inhibition of the fusion-inducing conformational change of influenza hemagglutinin by benzoquinones and hydroquinones. Biochemistry 1993, 32, 2967– 2978, DOI: 10.1021/bi00063a007[ACS Full Text], [CAS], Google Scholar35https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK3sXksVSgsrk%253D&md5=a44f15d36e94e05892e5231512efd553Inhibition of the fusion-inducing conformational change of influenza hemagglutinin by benzoquinones and hydroquinonesBodian, Dale L.; Yamasaki, R. Bryan; Buswell, Richard L.; Stearns, Jay F.; White, Judith M.; Kuntz, I. D.Biochemistry (1993), 32 (12), 2967-78CODEN: BICHAW; ISSN:0006-2960.Influenza hemagglutinin (HA) undergoes a conformational change that is required for viral entry. The rearrangement includes exposure of the fusion peptide, a hydrophobic segment buried in the trimer interface of the native protein. Since fusion peptide release triggers the membrane fusion event crucial for viral replication, inhibition of fusion peptide exposure should prevent infection. The authors reasoned that small mols. that bind to HA and stabilize its nonfusogenic conformation would block viral activity. A computer-assisted method was used to select putative HA ligands. One of the selected compds., 4A,5,8,8A-tetrhydro-5,8-methano-1,4-naphthoquinone, prevented the conversion of X31 HA to a conformation recognized by α-fusion peptide antisera. Several derivs. of this compd., including both benzoquinones and hydroquinones, also showed inhibition. The most effective compds. tested have IC50s between 1 and 20 μM. Representation compds. also inhibited virus-induced syncytia formation, HA-mediated hemolysis, and viral infectivity in vitro. The inhibitors are attractive leads for the development of antiviral drugs and can serve as probes of the mechanism of the conformational change of HA.
- 36Ewing, T. J. A.; Makino, S.; Skillman, A. G.; Kuntz, I. D. DOCK 4.0: search strategies for automated molecular docking of flexible molecule databases. J. Comput.-Aided Mol. Des. 2001, 15, 411– 428, DOI: 10.1023/a:1011115820450[Crossref], [PubMed], [CAS], Google Scholar36https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD3MXktVSlsbc%253D&md5=a76436739db67e4908845f123710179eDOCK 4.0: search strategies for automated molecular docking of flexible molecule databasesEwing, Todd J. A.; Makino, Shingo; Skillman, A. Geoffrey; Kuntz, Irwin D.Journal of Computer-Aided Molecular Design (2001), 15 (5), 411-428CODEN: JCADEQ; ISSN:0920-654X. (Kluwer Academic Publishers)The search strategies developed for docking flexible mols. to macromol. sites that are incorporated into the widely distributed DOCK software version 4.0, are described. The search strategies include incremental construction and random conformation search and utilize the existing Coulombic and Lennard-Jones grid-based scoring function. The incremental construction strategy is tested with a panel of 15 crystallog. test cases, created from 12 unique complexes whose ligands vary in size and flexibility. For all test cases, at least one docked position is generated within 2 Å of the crystallog. position. For 7 of 15 test cases, the top scoring position is also within 2 Å of the crystallog. position. The algorithm is fast enough to successfully dock a few test cases within seconds and most within 100 s. The incremental construction and the random search strategy are evaluated as database docking techniques with a database of 51 mols. docked to two of the crystallog. test cases. Incremental construction outperforms random search and is fast enough to reliably rank the database of compds. within 15 s per mol. on an SGI R10000 cpu.
- 37Ring, C. S.; Sun, E.; McKerrow, J. H.; Lee, G. K.; Rosenthal, P. J.; Kuntz, I. D.; Cohen, F. E. Structure-based inhibitor design by using protein models for the development of antiparasitic agents. Proc. Natl. Acad. Sci. U.S.A. 1993, 90, 3583– 3587, DOI: 10.1073/pnas.90.8.3583[Crossref], [PubMed], [CAS], Google Scholar37https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK3sXktVShu7o%253D&md5=a52b220512a0f2e2325383dd317b1503Structure-based inhibitor design by using protein models for the development of antiparasitic agentsRing, Christine S.; Sun, Eugene; McKerrow, James H.; Lee, Garson K.; Rosenthal, Philip J.; Kuntz, Irwin D.; Cohen, Fred E.Proceedings of the National Academy of Sciences of the United States of America (1993), 90 (8), 3583-7CODEN: PNASA6; ISSN:0027-8424.The lack of an exptl. detd. structure of a target protein frequently limits the application of structure-based drug design methods. In an effort to overcome this limitation, we have investigated the use of computer model-built structures for the identification of previously unknown inhibitors of enzymes from two major protease families, serine and cysteine proteases. We have successfully used our model-built structures to identify computationally and to confirm exptl. the activity of nonpeptidic inhibitors directed against important enzymes in the schistosome [2-(4-methoxybenzoyl)-1-naphthoic acid, Ki = 3 μM] and malaria {oxalic bis[(2-hydroxy-1-naphthylmethylene)hydrazide], IC50 = 6 μM} parasite life cycles.
- 38Shoichet, B. K.; Stroud, R. M.; Santi, D. V.; Kuntz, I. D.; Perry, K. M. Structure-based discovery of inhibitors of thymidylate synthase. Science 1993, 259, 1445– 1450, DOI: 10.1126/science.8451640[Crossref], [PubMed], [CAS], Google Scholar38https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK3sXktF2qsLc%253D&md5=a2e9380f78c3dd0f213443df91cefb75Structure-based discovery of inhibitors of thymidylate synthaseShoichet, Brian K.; Stroud, Robert M.; Santi, Daniel V.; Kuntz, Irwin D.; Perry, Kathy M.Science (Washington, DC, United States) (1993), 259 (5100), 1445-50CODEN: SCIEAS; ISSN:0036-8075.A mol. docking computer program (DOCK) was used to screen the Fine Chem. Directory, a database of com. available compds., for mols. that are complementary to thymidylate synthase (TS), a chemotherapeutic target. Besides retrieving the substrate and several known inhibitors, DOCK proposed putative inhibitors previously unknown to bind to the enzyme. Three of these compds. inhibited Lactobacillus casei TS at submillimolar concns. One of these inhibitors, sulisobenzone, crystd. with TS in two configurations that differed from the DOCK-favored geometry: a counterion was bound in the substrate site, which resulted in a 6 to 9 angstrom displacement of the inhibitor. The structure of the complexes suggested another binding region in the active site that could be exploited. This region was probed with mols. sterically similar to sulisobenzone, which led to the identification of a family of phenolphthalein analogs that inhibit TS in the 1 to 30 micromolar range. These inhibitors do not resemble the substrates of the enzyme. A crystal structure of phenolphthalein with TS shows that it binds in the target site in a configuration that resembles the one suggested by DOCK.
- 39Yakovenko, O. Y.; Oliferenko, A.; Golub, A.; Bdzhola, V.; Yarmoluk, S. The new method of distribution integrals evaluations for high throughput virtual screening. Ukr. Bioorg. Acta 2007, 1, 52– 62
- 40Yakovenko, O.; Oliferenko, A. A.; Bdzhola, V. G.; Palyulin, V. A.; Zefirov, N. S. Kirchhoff atomic charges fitted to multipole moments: Implementation for a virtual screening system. J. Comput. Chem. 2008, 29, 1332– 1343, DOI: 10.1002/jcc.20892[Crossref], [PubMed], [CAS], Google Scholar40https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1cXlvFWksbo%253D&md5=a05b4cdbc8814396086c1ac996ceeb4bKirchhoff atomic charges fitted to multipole moments: implementation for a virtual screening systemYakovenko, Olexander; Oliferenko, Alexander A.; Bdzhola, Volodymyr G.; Palyulin, Vladimir A.; Zefirov, Nikolai S.Journal of Computational Chemistry (2008), 29 (8), 1332-1343CODEN: JCCHDD; ISSN:0192-8651. (John Wiley & Sons, Inc.)The Kirchhoff charge model is a viable method of generating inexpensive and electrostatically reasonable at. charges for mol. mech. force fields. The charging method uses a computationally fast algorithm based on the principle of electronegativity relaxation. Parameters of the method, orbital electronegativities and hardnesses, are fitted to reproduce ref., ab initio calcd. dipole and quadrupole moments of a representative training set of neutral and charged org. mols. covering most medicinal chem. relevant bonding situations. Transferability and accuracy of the derived parameters are confirmed on an external test set. Comparisons to other charge models are made. Implementation of the new Kirchhoff charges into a virtual screening engine is elucidated.
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- 42Zerbs, S.; Frank, A. M.; Collart, F. R. Chapter 12 Bacterial Systems for Production of Heterologous Proteins. Methods Enzymol. 2009, 463, 149– 168, DOI: 10.1016/s0076-6879(09)63012-3[Crossref], [PubMed], [CAS], Google Scholar42https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3cXmt12jtg%253D%253D&md5=7d56562cdee6f23602aa9f55845c6b88Bacterial systems for production of heterologous proteinsZerbs, Sarah; Frank, Ashley M.; Collart, Frank R.Methods in Enzymology (2009), 463 (Guide to Protein Purification), 149-168CODEN: MENZAU; ISSN:0076-6879. (Elsevier Inc.)Proteins are the working mols. of all biol. systems and participate in a majority of cellular chem. reactions and biol. processes. Knowledge of the properties and function of these mols. is central to an understanding of chem. and biol. processes. In this context, purified proteins are a starting point for biophys. and biochem. characterization methods that can assist in the elucidation of function. The challenge for the prodn. of proteins at the scale and quality required for exptl., therapeutic, and com. applications has led to the development of a diverse set of methods for heterologous protein prodn. Bacterial expression systems are commonly used for protein prodn. as these systems provide an economical route for protein prodn. and require minimal tech. expertise to establish a lab. protein prodn. system.
- 43Gorbatiuk, O. B.; Tsapenko, M. V.; Pavlova, M. V.; Okunev, O. V.; Kordium, V. A. Bioaffinity sorbent based on immobilized protein A Staphylococcus aureus: development and application. Biopolym. Cell 2012, 28, 141– 148, DOI: 10.7124/bc.000041[Crossref], [CAS], Google Scholar43https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC38XosFSktb0%253D&md5=804a529193ac7689fe83d702612d0f1aBioaffinity sorbent based on immobilized protein A Staphylococcus aureus: development and applicationGorbatiuk, O. B.; Tsapenko, M. V.; Pavlova, M. V.; Okunev, O. V.; Kordium, V. A.Biopolymers and Cell (2012), 28 (2), 141-147CODEN: BCIEBC ISSN:. (Institut Molekulyarnoi Biologii i Genetiki NAN Ukraini)Aim. The obtaining of bioaffinity sorbent based on the immobilized protein A of S. aureus (SPA) using two cellulose-binding domains (CBD), and its application for purifn. of antibodies. Methods. The DNA sequences encoding SPA and two CBD were genetically fused, expressed in the high-productive Escherichia coli system and the protein SPA-CBD2 was obtained in a sol. form. The SPA-CBD2 fusion protein was affinity immobilized on the microcryst. cellulose. Results. Capacity of bioaffinity sorbent (1 mg SPA-CBD2/1 mL CC31-cellulose), dynamic capacity (3 mg mouse IgG/1 mL bioaffinity sorbent), efficiency and stability during prolonged storage were detd. The bio-affinity sorbent was used for purifn. of antibodies. The purity of antibodies in eluted fractions was more than 95%. The purified antibodies detected target antigens with a high sensitivity. Conclusions. The designed bioaffinity sorbent provides obtaining pure poly- and monoclonal antibodies in functionally active form and can be useful for the fractionation of mouse IgG.
- 44Studier, F. W. Protein production by auto-induction in high-density shaking cultures. Protein Expression Purif. 2005, 41, 207– 234, DOI: 10.1016/j.pep.2005.01.016[Crossref], [PubMed], [CAS], Google Scholar44https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2MXjsFCktLw%253D&md5=d31b76747c808ce6fbb27c7a1192b6b8Protein production by auto-induction in high-density shaking culturesStudier, F. WilliamProtein Expression and Purification (2005), 41 (1), 207-234CODEN: PEXPEJ; ISSN:1046-5928. (Elsevier)Inducible expression systems in which T7 RNA polymerase transcribes coding sequences cloned under control of a T7lac promoter efficiently produce a wide variety of proteins in Escherichia coli. Investigation of factors that affect stability, growth, and induction of T7 expression strains in shaking vessels led to the recognition that sporadic, unintended induction of expression in complex media, previously reported by others, is almost certainly caused by small amts. of lactose. Glucose prevents induction by lactose by well-studied mechanisms. Amino acids also inhibit induction by lactose during log-phase growth, and high rates of aeration inhibit induction at low lactose concns. These observations, and metabolic balancing of pH, allowed development of reliable non-inducing and auto-inducing media in which batch cultures grow to high densities. Expression strains grown to satn. in non-inducing media retain plasmid and remain fully viable for weeks in the refrigerator, making it easy to prep. many freezer stocks in parallel and use working stocks for an extended period. Auto-induction allows efficient screening of many clones in parallel for expression and soly., as cultures have only to be inoculated and grown to satn., and yields of target protein are typically several-fold higher than obtained by conventional IPTG induction. Auto-inducing media have been developed for labeling proteins with selenomethionine, 15N or 13C, and for prodn. of target proteins by arabinose induction of T7 RNA polymerase from the pBAD promoter in BL21-AI. Selenomethionine labeling was equally efficient in the commonly used methionine auxotroph B834(DE3)(found to be metE) or the prototroph BL21(DE3).
- 45Bradford, M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 1976, 72, 248– 254, DOI: 10.1016/0003-2697(76)90527-3[Crossref], [PubMed], [CAS], Google Scholar45https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaE28XksVehtrY%253D&md5=43f388a493becc4193c7a49d9de0e1dbA rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye bindingBradford, Marion M.Analytical Biochemistry (1976), 72 (1-2), 248-54CODEN: ANBCA2; ISSN:0003-2697.A protein detn. method that involves the binding of coomassie Brilliant Blue G 250 to protein is described. The binding of the dye to protein causes a shift in the absorption max. of the dye from 465 to 595 nm, and it is the increase in absorption at 595 nm that is monitored. This assay is very reproducible and rapid with the dye binding process virtually complete in ∼ 2 min with good color stability for 1 hr. There is little or no interference from cations such as Na+ or K+ nor from carbohydrates such as sucrose. A small amt. of color is developed in the presence of strongly alk. buffering agents, but the assay may be run accurately by the use of proper buffer controls. The only components found to give excessive interfering color in the assay are relatively large amts. of detergents such as Na dodecyl sulfate, Triton X 100, and commercial glassware detergents. Interference by small amts. of detergent may be eliminated by the use of proper control.
- 46Gasteiger, E.; Gattiker, A.; Hoogland, C.; Ivanyi, I.; Appel, R. D.; Bairoch, A. ExPASy: The proteomics server for in-depth protein knowledge and analysis. Nucleic Acids Res. 2003, 31, 3784– 3788, DOI: 10.1093/nar/gkg563[Crossref], [PubMed], [CAS], Google Scholar46https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD3sXltVWjtLc%253D&md5=adf2bae943800f2b00d9e475e5968f36ExPASy: the proteomics server for in-depth protein knowledge and analysisGasteiger, Elisabeth; Gattiker, Alexandre; Hoogland, Christine; Ivanyi, Ivan; Appel, Ron D.; Bairoch, AmosNucleic Acids Research (2003), 31 (13), 3784-3788CODEN: NARHAD; ISSN:0305-1048. (Oxford University Press)The ExPASy (the Expert Protein Anal. System) World Wide Web server, is provided as a service to the life science community by a multidisciplinary team at the Swiss Institute of Bioinformatics (SIB). It provides access to a variety of databases and anal. tools dedicated to proteins and proteomics. ExPASy databases include SWISS-PROT and TrEMBL, SWISS-2DPAGE, PROSITE, ENZYME and the SWISS-MODEL repository. Anal. tools are available for specific tasks relevant to proteomics, similarity searches, pattern and profile searches, post-translational modification prediction, topol. prediction, primary, secondary and tertiary structure anal. and sequence alignment. These databases and tools are tightly interlinked: a special emphasis is placed on integration of database entries with related resources developed at the SIB and elsewhere, and the proteomics tools have been designed to read the annotations in SWISS-PROT in order to enhance their predictions. ExPASy started to operate in 1993, as the first WWW server in the field of life sciences. In addn. to the main site in Switzerland, seven mirror sites in different continents currently serve the user community.
Supporting Information
Supporting Information
ARTICLE SECTIONSThe Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsomega.1c03789.
Analysis of ThrRS expression in E. coli BL21(DE3)pLysS cells; analysis of expression of the ThrRS soluble fraction with induction by 0.5 or 1 mM IPTG concentrations at 37 °C; analysis of expression of the ThrRS soluble fraction with induction by 0.25, 0.5, or 1 mM IPTG concentrations at 25 °C; analysis of ThrRS expression at 0.1 and 0.25 mM IPTG concentration and in the medium with 3% ethanol after induction for 3 h at 37 °C; expression of the ThrRS soluble fraction with induction by 0.1, 0.25, and 1 mM IPTG concentration and in the medium with 3% ethanol for 3 h at 18 and 25 °C; expression of the ThrRS soluble fraction after overnight induction at 18 and 25 °C; expression of the ThrRS soluble fraction in Lemo21(DE3) E. coli cells in LB medium after 4 h of induction at 30 °C with 0.4 mM IPTG and various concentrations of l-rhamnose; expression of the ThrRS soluble fraction in Lemo21(DE3) E. coli cells in LB medium after overnight induction at 30 °C; expression of S. aureus ThrRS using the autoinduction protocol; SDS-PAGE analysis of soluble and insoluble fractions of E. coli proteins after induction expression of S. aureus ThrRS by autoinduction conditions (10 μL of culture per lane was loaded); elution profile (absorbance at 280 nm versus elution volume) of the anion exchanger DEAE-Sepharose; purification of S. aureus ThrRS using the DEAE-Sepharose column; purification of S. aureus ThrRS using the heparin-Sepharose column; superposition of compound 1 (carbon atoms are labeled with blue color) and compound 2 (carbon atoms are labeled with magenta color) in the active site of S. aureus threonyl-tRNA synthetase; and structures and in vitro inhibitory activity toward S. aureus ThrRS for compounds, selected according to molecular docking results (PDF)
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