Journal of Antimicrobial Chemotherapy Advance Access published May 9, 2015
J Antimicrob Chemother doi:10.1093/jac/dkv110
Identification of an anti-TB compound targeting the tyrosyl-tRNA synthetase Ningyu Zhu1†, Yuan Lin2†, Dongsheng Li1, Nana Gao1, Chang Liu1, Xuefu You1, Jiandong Jiang1,2, Wei Jiang1‡ and Shuyi Si1*‡ 1 2
Institute of Medicinal Biotechnology, Peking Union Medical College and Chinese Academy of Medical Sciences, Beijing 100050, China; State Key Laboratory of Bioactive Substances and Function of Natural Medicine, Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100050, China Downloaded from http://jac.oxfordjournals.org/ at University of Connecticut on May 11, 2015
*Corresponding author. Tel: +86-10-63180604; Fax: +86-10-63180604; E-mail: [email protected] †N. Z. and Y. L. contributed equally to this work. ‡W. J. and S. S. contributed equally to this work.
Received 25 November 2014; returned 13 January 2015; revised 20 March 2015; accepted 1 April 2015 Objectives: Drug-resistant Mycobacterium tuberculosis poses a great threat to human health. Tyrosyl-tRNA synthetase (TyrRS) is one of the aminoacyl tRNA synthetases that catalyse the attachment of amino acids to their cognate tRNAs and are essential for protein synthesis. There are several distinctive differences between bacterial and human TyrRS and therefore it could be a potential target for developing antimicrobial agents. This study aimed to identify a new anti-TB agent targeting M. tuberculosis TyrRS (MtTyrRS). Methods: We first used Mycobacterium smegmatis for a phenotypic screening of 20 000 compounds. The hit compounds were then screened with MtTyrRS. The interaction between hit compound IMB-T130 and the target protein was analysed by surface plasmon resonance (SPR) assay and molecular docking experiments. The target of IMB-T130 was further confirmed by the overexpression of the target protein. The antibacterial activity of IMB-T130 against various standard and clinical drug-resistant M. tuberculosis strains was evaluated using the microplate Alamar blue assay. Results: Compound IMB-T130 was identified as a hit compound that inhibits the growth of M. smegmatis and the in vitro activity of MtTyrRS. The interaction between IMB-T130 and MtTyrRS was confirmed by SPR assay and molecular docking analysis. The higher MIC for a strain overexpressing the target protein also suggests that MtTyrRS is likely to be the target of IMB-T130. IMB-T130 shows excellent anti-TB activity and low cytotoxicity. Conclusions: IMB-T130 inhibits the growth of MDR-TB and XDR-TB by targeting MtTyrRS. Because of its low cytotoxicity against mammalian cells, IMB-T130 is a promising new agent against drug-resistant M. tuberculosis. Keywords: Mycobacterium tuberculosis, anti-tuberculosis drug screen, SPR assay, molecular docking
Introduction TB, which is caused by Mycobacterium tuberculosis, is a widespread infectious disease leading to 1.5 million deaths each year.1 In addition, antibiotic resistance is a growing problem for TB infections, with MDR- and XDR-TB infections being reported worldwide.2 Unfortunately, only two new clinically relevant drugs, bedaquiline and delamanid, have been introduced to the market in the past 50 years.3,4 Therefore, there is an urgent need to develop new anti-TB drugs for the treatment of infections caused by drug-resistant M. tuberculosis. Aminoacyl tRNA synthetases (AaRS) catalyse the attachment of amino acids to their cognate tRNAs and are essential for protein synthesis.5,6 The aminoacylation of tRNA is carried out in two
steps. First, an amino acid residue forms an aminoacyl-AMP complex with ATP, releasing pyrophosphate. The aminoacyl-tRNA complex is then assembled by esterification at the acceptor arm of the cognate tRNA.7 The inhibition of AaRS activity blocks protein synthesis, which consequently impairs many important physiological processes and stops bacterial growth.8 Therefore, AaRS could be a class of targets for the development of new antibacterial agents. Mupirocin, an inhibitor of isoleucyl-tRNA synthetase, is a successful example.9 Tyrosyl-tRNA synthetase (TyrRS) is one type of AaRS that was reported to be a target of a series of anti-Gram-positive bacterial compounds.10 – 12 However, there is no such report of anti-TB compounds targeting M. tuberculosis TyrRS (MtTyrRS). Comparison of amino acid sequences of TyrRS from M. tuberculosis and human cytoplasm indicates that the
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Materials and methods Materials ATP, IPTG, HEPES and trichloroacetic acid (TCA) were purchased from Sigma. L-[3,5-3H]tyrosine was purchased from PerkinElmer. Escherichia coli MRE 600 tRNA was purchased from Roche. Compound IMB-T130 was purchased from J&K Chemical (synthesized by Enamine, Kyiv, Ukraine). The bacterial strains used in the MIC test were clinical isolates or were purchased from ATCC. Acid– albumin – dextrose–citric acid (ADC) was purchased from BD Biosciences. All other chemicals used were of analytical grade.
Methods Expression and purification of TyrRS M. tuberculosis H37Rv genomic DNA was provided by the Beijing Research Institute for Tuberculosis Control. The TyrRS gene was amplified using primers designed by Primer 5.0 software: 5′ -TCTTGCCATATGTCTGGCATGATCCT CGATG-3′ (NdeI, sense) and 5′ -GGTGGCTCCTCGAGGCCAATCCGTTCC-3′ (XhoI, anti-sense).13 The PCR product was cloned into vector pET30a using the NdeI and XhoI restriction enzyme sites. The resulting pET30a-TyrRS plasmid contains the TyrRS gene fused with a 6×His tag at the C-terminal. The plasmid was transformed into an E. coli BL21 (DE3) strain. A single clone was picked and grown at 378C in LB medium supplemented with 100 mg/L kanamycin for cloning and maintenance. TyrRS was expressed by the addition of 1 mM IPTG and incubation at 208C for 10 h. TyrRS, present in the supernatant, was collected and loaded onto an Ni2+ His Trap chelating column (GE Healthcare). The loading buffer comprised 25 mM Tris, 500 mM NaCl and 30 mM imidazole (pH 7.8). The target protein was eluted using a stepwise gradient of imidazole in elution buffer (25 mM Tris, 500 mM NaCl and 50 – 500 mM imidazole, pH 7.8). Eluted
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fractions were analysed using 12% (w/v) SDS – PAGE followed by Coomassie blue staining and the purity was also determined. The TyrRS concentration was measured by the Bradford method using BSA as a standard. The purified MtTyrRS protein was stored at 2808C.
Phenotypic screening of compound library using M. smegmatis As M. tuberculosis grows slowly, we used M. smegmatis, a non-pathogenic bacterial species that is biologically close to M. tuberculosis, to screen a library of 20 000 compounds. These compounds included a combination of some natural products (plant, actinomycetes and fungi) from the Institute of Medicinal Biotechnology and synthetic products (synthesized by Enamine). The M. smegmatis strain was grown in Middlebrook 7H9 medium in 96-well plates and compounds from the library were added at 5 mg/L. To each well, except the blank control, 199 mL of 7H9 medium (containing 20% ADC and 1% inoculum from the seed culture) and 1 mL of the test compound (1000 mg/L in DMSO) was added. Each plate contained 88 wells as the sample group with 5 mg/L of the test compounds and the remaining 8 wells were the control group, to which 1 mL of DMSO was added instead of the test compound. The plates were incubated at 378C for 48 h and then the OD600 was measured.13,14
Enzyme assay and TyrRS inhibitor screening TyrRS activity was measured by aminoacylation. The reaction was performed in a 96-well plate at 378C. A 50 mL mixture containing 100 mM Tris, 10 mM MgCl2, 40 mM KCl, 1000 mg/L BSA, 1 mM dithiothreitol (DTT), 0.04 mM tRNA, 2 mM ATP, 0.3 mM L-[ring-3,5-3H]tyrosine, 10 mM L-tyrosine and 5 mg/L TyrRS was added to each well. TyrRS protein was pre-incubated with 1 mL of test compound (10 mg/L) at 48C for 30 min in 40 mL of buffer (125 mM Tris, 12.5 mM MgCl2, 50 mM KCl, 1250 mg/L BSA and 1.25 mM DTT); 1 mL of DMSO was added for the control group. Then, the reaction was initiated by the addition of 10 mL of substrate (0.2 mM tRNA, 10 mM ATP, 1.5 mM L-[ring-3,5-3H]tyrosine and 50 mM L-tyrosine). After incubation at 378C for 15 min, the reaction was stopped by the addition of 20 mL of pre-chilled 5% TCA solution (containing 0.02 M sodium pyrophosphate) at 08C. The reaction mixture was then transferred onto filter papers cut to 800×800 mm squares. The filter papers were washed twice with TCA solution, once with ethanol and then dried on a heated plate.15 Next, they were placed into 24-well plates and 1 mL of scintillation liquid was added to each well. The amount of radioactivity was determined using a scintillation counter.
Inhibitory effect of compound IMB-T130 on TyrRS TyrRS (5 mg/L) was pre-incubated with gradient concentrations (0.16–40 mg/L) of IMB-T130, or 1% DMSO as a control, in 40 mL of buffer (125 mM Tris, 12.5 mM MgCl2, 50 mM KCl, 1250 mg/L BSA and 1.25 mM DTT) at 48C for 30 min. The reaction conditions and detection method were the same as for the TyrRS inhibitor screening described above.
Confirmation of the interaction between IMB-T130 and TyrRS by SPR assay SPR is widely used for measuring the interaction between small molecules and proteins. To detect the interaction between IMB-T130 and TyrRS, Biacore sensor chips were coated with TyrRS and then exposed to IMB-T130 at a gradient of concentrations (3.12 – 25 mM). The measurements were performed using a Biacore T100 system (GE Healthcare) at 258C in a PBS-P+ running buffer (20 mm phosphate buffer, 2.7 mM KCl, 137 mM NaCl, 0.05% surfactant P20, pH 7.4 and 5% DMSO). Purified His-tagged TyrRS (20 mg/L) was immobilized onto a CM5 sensor chip using an N-hydroxysuccinimide/1-ethyl-3-(3-dimethylperpyl)-carboiimide
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identity is only 15.6%. Because of the apparent sequence difference as well as the essential role of TyrRS for the growth of M. tuberculosis, we consider TyrRS to be a promising new anti-TB target. Taking into account that the identified compounds based on the enzyme assay may not show antibacterial activity, we performed a two-step screening including a phenotype screening and a targeted screening. Mycobacterium smegmatis mc2155, a non-pathogenic strain, was chosen to substitute for M. tuberculosis to examine the antibacterial activity of compounds. The TyrRS amino acid sequences from M. tuberculosis and M. smegmatis exhibit 79% sequence identity and the important amino acids in the active centre are same. Therefore, we inferred that an anti-TB agent targeting TyrRS might also inhibit the growth of M. smegmatis in a similar way. In this study, we identified 78 hit compounds through a phenotype screening of 20000 compounds with M. smegmatis. Only compound IMB-T130 {5-chloro-N-[4-(pyridin-2-yl)-1,3-thiazol-2-yl]thiophene-2-carboxamide} showed potent inhibitory activity of TyrRS in vitro at 10 mg/L. IMB-T130 showed similar antibacterial potency as first-line anti-TB drugs and even better inhibitory effects against drug-resistant strains. Furthermore, we performed a surface plasmon resonance (SPR) assay between IMB-T130 and TyrRS, an overexpressing experiment of MtTyrRS in M. smegmatis and a molecular docking analysis in silico between IMB-T130 and the active centre of MtTyrRS to confirm if TyrRS is the target of IMB-T130. A series of tests suggested that TyrRS is likely to be the target of IMB-T130.
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amine coupling kit in 10 mM sodium acetate buffer at pH 4.5. The protein density is 5000 response units (RU). To determine the binding affinity of His-tagged TyrRS and IMB-T130, solutions of compound (5% DMSO) at different concentrations (3.12–25 mM) were injected into the TyrRS immobilized chambers. The surface was regenerated using Gly-HCl (pH 3.0). IMB-T130 was proven to bind to TyrRS by a measurable dose-dependent change in RU.16,17
Determination of the MIC for M. smegmatis overexpressing TyrRS
Molecular docking between IMB-T130 and TyrRS To determine how the structure of IMB-T130 contributes to its inhibitory activity against TyrRS, a molecular docking analysis between IMB-T130 and TyrRS was performed. The crystal structure of TyrRS solved at a 2.9 A˚ resolution was retrieved from the Protein Data Bank (ID code 2JAN). Because there are no cocrystallized molecules in this crystal, the docking pocket needs to be generated by key amino acids of the active sites. As the different bacterial TyrRS have homologous catalytic domains, their active sites are similar.19 There is a Rossmann fold in the catalytic domain of MtTyrRS and HIGH and KMSKS (KFGKS in MtTyrRS) motifs exist in the active centre.20 Besides MtTyrRS, the three-dimensional structures of TyrRS from E. coli,21 Staphylococcus aureus,22 Bacillus stearothermophilus 23 and Thermus thermophilus 24 were also solved by X-ray crystallography. According to a recent study, the most important amino acid residues in the MtTyrRS active centre are Tyr36, Gly38, Phe39, Asp40, His47, Gly49, His50, Tyr171, Gln175, Asp178, Gln191, Gly193, Gly194, Gln197, Leu223, Val224, Lys231, Phe232, Gly233 and Lys234.25,26 The Discovery Studio 4.1 program (State Key Laboratory of Bioactive Substances and Function of Natural Medicine, Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, China) was used to perform molecular docking.27 The binding pocket was generated based on 20 important amino acids. Three conformations of the IMB-T130 molecular structure were generated after optimization and then docking with the active pocket was carried out using the CDOCKER program. According to the scores of – CDOCKER energy and – CDOCKER interaction energy, the best 10 results were retained.
Anti-TB activity of IMB-T130 TyrRS is known to be an essential enzyme for ligating tyrosine to its cognate tRNA. It plays an important role in the production of proteins. Thus, functional inhibition of this enzyme is expected to inhibit the growth of M. tuberculosis. The anti-TB activity of IMB-T130 was tested using the microplate Alamar blue assay (MABA). The inhibitory activity was tested against standard strain H37Rv (ATCC 27294), susceptible clinical strains STB-FJ05060 and STB-FJ05349 and drug-resistant clinical isolates MDR-FJ05120, MDR-FJ05189 and XDR-FJ05195. The first-line anti-TB
Cell cytotoxicity evaluation To test the cytotoxic effect of IMB-T130, a range of different cell types were used. Cells were seeded in triplicate in 96-well culture plates at a density of 5×103 cells per well. Then, the cells were incubated with IMB-T130 at a gradient of concentrations from 1.56 to 200 mM. After incubation for 24 h, thiazolyl blue tetrazolium bromide reagent was added. After further incubation for 4 h, 50 mL of DMSO was added to each well. The absorbance was measured at 490 nm and the average values were calculated for triplicate wells. The IC50 values were calculated on a concentration–response curve.
Susceptibility of other bacterial strains to IMB-T130 The MICs of compound IMB-T130 for other representative bacteria from ATCC and clinical isolates except M. tuberculosis were determined using agar dilution method recommend by the CLSI. Inoculations were adjusted to yield 104 cfu/spot by a multipoint inoculator (Bolney, Sussex, UK). The bacteria were incubated at 358C for 18 h. The MICs were determined as the lowest concentration of the compound that inhibited the growth of bacteria on the plate. Levofloxacin was used as a reference drug.13
Results Expression and purification of TyrRS TyrRS can ligate tyrosine to its cognate tRNA. In this study, active TyrRS enzyme was required to screen the compound library. The recombinant protein was purified and the purification was confirmed by the appearance of a single band at 47.5 kDa after SDS – PAGE and Coomassie blue staining (Figure S1, available as Supplementary data at JAC Online).
Compounds that inhibit M. smegmatis and MtTyrRS The library containing 20 000 compounds was screened by growing M. smegmatis in 96-well plates, adding the compounds at 5 mg/L. Each plate contained 88 wells for the test compounds and the remaining 8 wells were the control group. For the control, 1 mL of DMSO was added into the wells instead of the test compounds. The plates were incubated at 378C for 48 h and then the OD600 was measured. After screening, 78 compounds were identified with an OD600 of ,50% of the control and were inferred to have possible anti-TB activity. These compounds were further tested for their ability to inhibit TyrRS at a concentration of 10 mg/L. We measured the amount of radioactivity after 15 min of incubation in the reaction mixture using the in vitro assay system described in the Materials and methods section and found that compound IMB-T130 could suppress .50% of TyrRS activity at a concentration of 10 mg/L. The molecular weight of IMB-T130 is 321.81 Da and its structure is shown in Figure 1(a).
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For the overexpression of the MtTyrRS protein in M. smegmatis, we constructed the plasmid pMV261-eGFP by inserting the eGFP gene into the pMV261 vector with ClaI and HpaI restriction enzyme sites. After that, the MtTyrRS gene was inserted between the promoter and the eGFP gene using the BamHI and HindIII sites in a way that TyrRS is in frame with eGFP. The resulting pMV261:TyrRS-eGFP plasmid was amplified by E. coli and then transformed into competent M. smegmatis. PCR was used to identify strains expressing eGFP, which indicated the expression of the MtTyrRS-eGFP fusion protein. Expression of the recombinant protein was further confirmed by fluorescence microscopy and western blotting with anti-eGFP antibody. The MICs of IMB-T130 for M. smegmatis with a control vector (pMV261-eGFP plasmid) and strains overexpressing a nonrelated gene (M. tuberculosis ribosomal protein L12) or TyrRS-eGFP were determined by the conventional plate dilution method.16,18
drugs rifampicin and isoniazid were also included in the analysis for comparison. The gradient concentrations of compounds varied from 0.01 to 128 mg/L. All M. tuberculosis strains were cultured at 378C in Middlebrook 7H9 broth supplemented with 0.2% glycerol and 10% ADC until the mid-log phase of growth. The cells were diluted in Middlebrook 7H9 broth to 106 cfu/mL with gradient concentrations of compounds. Then, the MIC was measured in sterile 96-well plates with a final volume of 100 mL per well. The MIC was defined as the lowest drug concentration that prevented the colour change of Alamar blue reagent from blue to pink.28
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Next, the inhibition ratios at different concentrations of IMB-T130 (40, 20, 10, 5, 2.5, 1.25, 0.625, 0.31 and 0.16 mg/L) were determined. The activity of MtTyrRS was inhibited by
Confirming the interaction between IMB-T130 and TyrRS
O
(a) S
NH
S Cl
N N IMB-T130
Inhibition ratio
Higher MIC for a strain overexpressing TyrRS
IC50 = 2.619 mg/L = 8.138 mM
0.6
0.4
0.2
0.0
–1
0 1 Log (IMB-T130, mg/L)
2
Figure 1. (a) Structure of IMB-T130 {5-chloro-N-[4-(pyridin-2-yl)1,3-thiazol-2-yl]thiophene-2-carboxamide}. (b) Inhibition of aminoacylation activity by IMB-T130. TyrRS (5 mg/L) was incubated with different concentrations (0.16 – 40 mg/L) of IMB-T130 at 48C for 30 min and the aminoacylation activity was then determined as detailed in the Materials and methods section. The IC50 value is plotted as the ratio of the radioactivity signal over the concentration of compound (log plot) that fits to a variable-slope dose – response equation. The experiment was repeated three times.
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To examine the interaction between compound IMB-T130 and the target protein, an SPR assay was performed. Biacore sensor chips were coated with purified TyrRS and then exposed to IMB-T130 at different concentrations. The RU were measured at four different concentrations (3.12 –25 mM) of IMB-T130 (concentrations .25 mM could not be used due to the poor water solubility of IMB-T130). The results showed a significant dose-dependent response at the concentrations tested (Figure 2).
If IMB-T130 inhibits the growth of mycobacteria by targeting TyrRS, the antibacterial activity of IMB-T130 should be lower for a strain overexpressing the TyrRS protein. Therefore, we constructed an expression plasmid containing TyrRS-eGFP. This plasmid was transformed into M. smegmatis and expression of the recombinant protein was confirmed by the appearance of a GFP signal (Figure 3) and western blotting with anti-eGFP antibody (Figure S2). The MIC of IMB-T130 for an M. smegmatis strain with a PMV261 control plasmid is 0.312 mg/L. The MIC of IMB-T130 for a strain overexpressing TyrRS is 1.25 mg/L, which is 4-fold higher than the MIC for the control strain. In contrast, expression of the MtL12 gene in M. smegmatis did not increase the MIC of IMB-T130 (Table 1). This result showed that the TyrRS protein is likely to be the target of IMB-T130 in vivo.
Analysis of the molecular docking results Ten results were reserved after the molecular docking experiments between the three conformations of IMB-T130 and the active pocket of TyrRS. The result giving the highest score for both – CDOCKER energy and – CDOCKER interaction energy indicates the most probable docking model of the complex. This
MtTyrRS immobilized IMB-T130
RU
20
25 mM
10
12.5 mM 6.25 mM
0
3.12 mM
–10 –20 –20
0
20
40
60 Time (s)
80
100
120
140
Figure 2. Demonstration of the interaction between TyrRS and compound IMB-T130 by SPR. A sensor chip coated with purified TyrRS was exposed to various concentrations (3.12– 25 mM) of IMB-T130. The change in RU is shown.
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(b)
IMB-T130 in a dose-dependent manner with an IC50 value of 8.138 mM (Figure 1b). The result showed that IMB-T130 is a potent competitive inhibitor of TyrRS.
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(a)
(a)
(b)
Leu223 Gly194
Vector control
Val224
TyrRS-eGFP
Figure 3. Fluorescence signal in M. smegmatis with a control vector or an overexpression plasmid for TyrRS-eGFP. (a) M. smegmatis cells carrying plasmid pMV261. (b) M. smegmatis cells carrying plasmid pMV261:TyrRS-eGFP. This figure appears in colour in the online version of JAC and in black and white in the print version of JAC.
Gly49
(b)
Leu223
Strain
MIC (mg/L) of IMB-T130
Control vector (PMV261 plasmid) Overexpressing MtTyrRS Overexpressing MtL12
Gly194 Val224
0.312 1.25 0.312
Gly49
model showed that IMB-T130 inserts into the pocket, interacting with four important amino acid residues of the MtTyrRS active centre (Figure 4). The chlorine atom formed a halogen bond with Val224, a type of non-covalent intermolecular interaction. There was a hydrophobic interaction between the thiophene of IMB-T130 and the alkyl group of Leu223. The thiophene also had intermolecular hyperconjugation with the carbonyl group of Gly49. The N atoms of thiazole formed a conventional hydrogen bond with Gly194. Based on this docking model, IMB-T130 occupied the centre position in the active pocket of MtTyrRS; therefore, it might be able to prevent the two substrates from binding to the enzyme simultaneously. Especially, the Gly194 and Val224 played important roles in the binding of the tyrosyl-adenylate intermediate.
Anti-TB activity of compound IMB-T130 The anti-TB activity of IMB-T130 was tested using a MABA against strain H37Rv and the MIC was found to be 0.08 mg/L. For WT clinical strains FJ05349 and FJ05060, the MIC was also 0.08 mg/L. For MDR clinical strains MDR-FJ05120 and MDR-FJ05189, the MICs of IMB-T130 were 0.25 and 4 mg/L, respectively. For the XDR clinical strain XDR-FJ05195, the MIC was 0.25 mg/L. These data indicate that IMB-T130 has comparable anti-TB activity of susceptible strains to the first-line drugs such as rifampicin (0.0625 –0.125 mg/L) and isoniazid (0.0625 –0.125 mg/L).29 For MDR strains, IMB-T130 performed better than rifampicin and similar to isoniazid. For XDR strains, it performed substantially better than rifampicin and isoniazid. These results indicate that IMB-T130 may inhibit the growth of M. tuberculosis by targeting TyrRS, which is a distinct mechanism from the first-line anti-TB antibiotics.
Susceptibility of other bacterial species to IMB-T130 MtTyrRS was the screening target in our experiment. Because of the conservative nature of this protein among various bacterial
Figure 4. Molecular docking of TyrRS and IMB-T130. (a) Overview of the active pocket of TyrRS bound to IMB-T130. IMB-T130 is represented by a stick model (grey, carbon atoms; blue, nitrogen atoms; deep red, oxygen atoms; yellow, sulphur atoms; green, chlorine atoms). The red sticks represent the amino acid residues interacting with IMB-T130. (b) The halogen bond formed between the chlorine atom and Val224 (red line), the hydrophobic interaction between the thiophene of IMB-T130 and the alkyl group of Leu223 (purple line), the intermolecular hyperconjugation between the carbonyl group of Gly49 and the thiophene (blue line) and the conventional hydrogen bond formed by the N atom of thiazole and the hydrogen atoms of Gly194 (green line).
species, this identified compound might also inhibit the activity of homologous proteins in other bacteria. We therefore examined the growth inhibition of eight representative bacterial strains (four Gram-positive and four Gram-negative) by IMB-T130. The results showed that IMB-T130 exhibited high MICs for these Gram-positive and Gram-negative bacteria (Table 2). Moreover, the IC50 of cytotoxicity for human embryonic kidney 293 cells was found to be 22 mM and for HeLa cancer cells was .100 mM. These results indicate that compound IMB-T130 has a selective and strong anti-TB effect with low cytotoxicity.
Discussion AaRS catalyse the attachment of amino acids to their cognate tRNAs and are essential for protein synthesis. AaRS represent a class of attractive targets for developing new antibiotics, because they are essential for microbial survival and share low homology with their eukaryotic counterparts. For example, mupirocin is a successful marketed antibiotic that targets AaRS. TyrRS is one type of AaRS. Compounds that inhibit the function of TyrRS would kill bacteria by disrupting protein synthesis and would have low toxicity against humans. In order to find a lead
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Table 1. Antibacterial activity of IMB-T130 against M. smegmatis with a control vector or an overexpression plasmid for MtTyrRS or MtL12
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Table 2. MICs of IMB-T130 for various bacterial strains MIC (mg/L) Strain
rifampicin
isoniazid
levofloxacin
0.08 0.08 0.08 0.25 4 0.25 32 32 64 128 256 128 128 128
0.125 ,0.5 ,0.5 .256 .256 4 — — — — — — — —
0.125 ,0.5 ,0.5 4 ,0.5 .256 — — — — — — — —
— — — — — — 0.25 0.25 0.25 0.5 ≤0.03 0.06 1 0.5
STB, clinically susceptible strain of M. tuberculosis; MDR, MDR M. tuberculosis; XDR, XDR M. tuberculosis. Strains FJ05060, FJ05120, FJ05189 and FJ05195 are clinical isolates of M. tuberculosis. a Methicillin-susceptible S. epidermidis. b MSSA. c MRSA. d Vancomycin-susceptible Enterococcus. e ESBL negative. f ESBL positive.
compound targeting TyrRS with antibacterial activity, we performed a two-step screening including a phenotype screening and a targeted screening. As an alternative to M. tuberculosis, we chose M. smegmatis to determine the antibacterial activity of compounds because it is non-pathogenic and fast growing. Comparison of the TyrRS amino acid sequences from M. tuberculosis and M. smegmatis revealed 79% sequence identity. The 20 important amino acids comprising the active centre of TyrRS from these two species are identical. Although the drug susceptibility of these two strains are not fully parallel, our experiments showed that it is feasible to screen an anti-TB agent using M. smegmatis as replacement at some level. It is more suitable for high-throughput screening. In this study, by screening a library of compounds, we identified only one compound (IMB-T130) that could inhibit the growth of M. smegmatis and the activity of MtTyrRS. Additionally, we confirmed the interaction between IMB-T130 and TyrRS by SPR assay and analysed this interaction using a molecular docking program. The higher MIC for a strain overexpressing TyrRS also suggested that TyrRS is likely to be the target of IMB-T130. Importantly, IMB-T130 showed anti-TB potency similar to firstline drugs and was even more effective against drug-resistant strains. IMB-T130 therefore represents a new anti-TB compound targeting MtTyrRS. The significant dose-dependent signal measured in our SPR experiments confirmed the direct interaction between TyrRS and compound IMB-T130. Based on this result, we analysed how the structure of IMB-T130 contributes to its inhibitory activity against TyrRS by molecular docking analysis. The active pocket comprises
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20 amino acid residues of MtTyrRS, as indicated by recent studies. 19,25 The highest scoring docking model showed that IMB-T130 forms interactions with four important amino acid residues of MtTyrRS. IMB-T130 occupied the centre position of the active pocket and inhibited the function of the protein. The increased MIC of IMB-T130 for a TyrRS-overexpressing strain verified that the protein might be the target of compound IMB-T130. TyrRS is expressed in almost all prokaryotic and eukaryotic cells. TyrRS proteins from prokaryotic bacteria exhibit similar structures. Previous studies have shown that TyrRS belongs to class I of the AaRS, with highly conserved active sites, especially among prokaryotes. But IMB-T130 was found to be a selective inhibitor of M. tuberculosis, only showing weak inhibitory activity against Staphylococcus epidermidis and S. aureus. Based on the molecular docking results, we thought that the large active pocket may be one of the reasons for this phenomenon. The active pocket of TyrRS was found to be much larger than the molecular size of IMB-T130. We believe that IMB-T130 binds to the active centre of TyrRS from various bacteria in different ways because of the non-conserved sequences. This might affect the inhibition efficiency towards TyrRS from different sources and explain the differences in MICs for different bacteria. Additionally, the cell wall differences between M. tuberculosis and the other bacteria might be another reason for the distinct MICs. Compound IMB-T130 showed strong selective anti-TB activity and there was no significant cross-resistance between IMB-T130 and the first-line anti-TB drugs. We speculated that compound IMB-T130 may inhibit the growth of M. tuberculosis via a
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H37Rv STB-FJ05349 STB-FJ05060 MDR-FJ05120 MDR-FJ05189 XDR-FJ05195 Staphylococcus epidermidis 12228a Staphylococcus aureus 29213b Staphylococcus aureus 33591c Enterococcus faecalis 29212d Escherichia coli 25922e Klebsiella pneumoniae 7e Klebsiella pneumoniae 700603f Pseudomonas aeruginosa 27853
IMB-T130
Anti-TB agent targeting tyrosyl-tRNA synthetase
Acknowledgements We thank Professor Kanglin Wan from the Chinese Center for Disease Control and Prevention (CCDC) for helping us test the anti-TB activity of compound. We thank Dr Kate Fox of Edanz Writing Company (Liwen Bianji, Beijing), Professor Yanchang Wang and Mr Michael Bokros from Florida State University, Medicine College, for helping us to check and polish the English expression and edit the style of the manuscript.
Funding This work was supported by the Foundation for Innovative Research Groups (grant number 81321004), the Funds for International Cooperation Exchange between China-Sweden (grant number 81361138020), the National Natural Science Foundation of China (grant numbers 81072672 and 81302816), grants from the State Mega Programs (2012ZX09301002-003/ 006; 2015ZX09102007-008/016) and a grant from the Beijing science and technology projects (Z141102004414065).
Transparency declarations None to declare.
Supplementary data Figures S1 and S2 are available as Supplementary data at JAC Online (http://jac.oxfordjournals.org/).
an individual patient data meta-analysis. Clin Infect Dis 2014; 59: 1364– 74. 3 Blair HA, Scott LJ. Delamanid: a review of its use in patients with multidrug-resistant tuberculosis. Drugs 2015; 75: 91– 100. 4 Nagabushan H, Roopadevi HS. Bedaquiline: a novel antitubercular drug for multidrug-resistant tuberculosis. J Postgrad Med 2014; 60: 300–2. 5 Gadakh B, Van Aerschot A. Aminoacyl-tRNA synthetase inhibitors as antimicrobial agents: a patent review from 2006 till present. Expert Opin Ther Pat 2012; 22: 1453 –65. 6 Yanagisawa T, Sumida T, Ishii R et al. A paralog of lysyl-tRNA synthetase aminoacylates a conserved lysine residue in translation elongation factor P. Nat Struct Mol Biol 2010; 17: 1136–43. 7 Delarue M. Aminoacyl-tRNA synthetases. Curr Opin Struct Biol 1995; 5: 48– 55. 8 Van de Vijver P, Vondenhoff GH, Kazakov TS et al. Synthetic microcin C analogs targeting different aminoacyl-tRNA synthetases. J Bacteriol 2009; 191: 6273– 80. 9 Yanagisawa T, Lee JT, Wu HC et al. Relationship of protein structure of isoleucyl-tRNA synthetase with pseudomonic acid resistance of Escherichia coli. A proposed mode of action of pseudomonic acid as an inhibitor of isoleucyl-tRNA synthetase. J Biol Chem 1994; 269: 24304 – 9. 10 Wang XD, Deng RC, Dong JJ et al. 3-Aryl-4-acyloxyethoxyfuran-2(5H)ones as inhibitors of tyrosyl-tRNA synthetase: synthesis, molecular docking and antibacterial evaluation. Bioorg Med Chem 2013; 21: 4914–22. 11 Xiao ZP, Ouyang H, Wang XD et al. 4-Alkoxy-3-arylfuran-2(5H)-ones as inhibitors of tyrosyl-tRNA synthetase: synthesis, molecular docking and antibacterial evaluation. Bioorg Med Chem 2011; 19: 3884 –91. 12 Xiao ZP, Ma TW, Liao ML et al. Tyrosyl-tRNA synthetase inhibitors as antibacterial agents: synthesis, molecular docking and structure–activity relationship analysis of 3-aryl-4-arylaminofuran-2(5H)-ones. Eur J Med Chem 2011; 46: 4904– 14. 13 Lin Y, Zhu N, Han Y et al. Identification of anti-tuberculosis agents that target the cell-division protein FtsZ. J Antibiot (Tokyo) 2014; 67: 671– 6. 14 Xu S, Pei L, Li D et al. Synthesis and antimycobacterial evaluation of natural oridonin and its enmein-type derivatives. Fitoterapia 2014; 99: 300–6. 15 Werner RG, Thorpe LF, Reuter W et al. Indolmycin inhibits prokaryotic tryptophanyl-tRNA ligase. Eur J Biochem 1976; 68: 1 –3. 16 Lin Y, Li Y, Zhu Y et al. Identification of antituberculosis agents that target ribosomal protein interactions using a yeast two-hybrid system. Proc Natl Acad Sci USA 2012; 109: 17412 –7. 17 Neumann T, Junker HD, Schmidt K et al. SPR-based fragment screening: advantages and applications. Curr Top Med Chem 2007; 7: 1630 – 42. 18 Lin Y, Li Y, Zhu N et al. The antituberculosis antibiotic capreomycin inhibits protein synthesis by disrupting interaction between ribosomal proteins L12 and L10. Antimicrob Agents Chemother 2014; 58: 2038–44. 19 Odynets KO, Korneliuk OI. A model of three-dimensional structure of Mycobacterium tuberculosis tyrosyl-tRNA synthetase. Ukr Biokhim Zh 2008; 80: 62– 75. 20 Bonnefond L, Giege R, Rudinger-Thirion J. Evolution of the tRNA(Tyr)/ TyrRS aminoacylation systems. Biochimie 2005; 87: 873– 83.
References 1 Zumla A, George A, Sharma V et al. The WHO 2014 global tuberculosis report: further to go. Lancet Glob Health 2015; 3: e10–2.
21 Kobayashi T, Takimura T, Sekine R et al. Structural snapshots of the KMSKS loop rearrangement for amino acid activation by bacterial tyrosyl-tRNA synthetase. J Mol Biol 2005; 346: 105–17.
2 Bastos ML, Hussain H, Weyer K et al. Treatment outcomes of patients with multidrug-resistant and extensively drug-resistant tuberculosis according to drug susceptibility testing to first- and second-line drugs:
22 Qiu X, Janson CA, Smith WW et al. Crystal structure of Staphylococcus aureus tyrosyl-tRNA synthetase in complex with a class of potent and specific inhibitors. Protein Sci 2001; 10: 2008 –16.
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mechanism distinct from that of existing anti-TB drugs. The result of the SPR assay confirmed the interaction between IMB-T130 and TyrRS. We believe that the inhibitory activity of IMB-T130 against MtTyrRS generated its anti-TB activity without cross-resistance of existing anti-TB drugs. The higher MIC for a strain overexpressing TyrRS also supports our opinion. However, it seemed that the concentration of IMB-T130 required to inhibit the growth of M. tuberculosis was lower than that required to inhibit the aminoacylation activity of MtTyrRS in vitro. One possible reason is the difference between the extracellular and intracellular environment and the IC50 value we detected in vitro might deviate from its actual value in vivo. Another possible reason is that IMB-T130 may be a multitarget compound.30 In this study, we report that compound IMB-T130 inhibits the aminoacylation activity of MtTyrRS. This compound showed selective inhibition against M. tuberculosis and displayed low toxicity against mammalian cells. Therefore, chemical modification of IMB-T130 to further optimize its bactericidal capacity, increase its water solubility (log P¼4.1) and reduce its toxicity may result in a new promising anti-TB drug highly effective against drug-resistant M. tuberculosis.
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23 Brick P, Bhat TN, Blow DM. Structure of tyrosyl-tRNA synthetase refined at 2.3 A˚ resolution. Interaction of the enzyme with the tyrosyl adenylate intermediate. J Mol Biol 1989; 208: 83 –98.
27 Al-Balas QA, Sowaileh MF, Hassan MA et al. Novel N-substituted aminobenzamide scaffold derivatives targeting the dipeptidyl peptidase-IV enzyme. Drug Des Devel Ther 2014; 8: 129– 63.
24 Yaremchuk A, Kriklivyi I, Tukalo M et al. Class I tyrosyl-tRNA synthetase has a class II mode of cognate tRNA recognition. EMBO J 2002; 21: 3829– 40.
28 Collins L, Franzblau SG. Microplate alamar blue assay versus BACTEC 460 system for high-throughput screening of compounds against Mycobacterium tuberculosis and Mycobacterium avium. Antimicrob Agents Chemother 1997; 41: 1004 –9.
25 Mykuliak VV, Kornelyuk AI. The mechanisms of substrates interaction with the active site of Mycobacterium tuberculosis tyrosyl-tRNA synthetase studied by molecular dynamics simulations. Biopolym Cell 2014; 30: 157–62. 26 Mykuliak VV, Dragan AI, Kornelyuk AI. Structural states of the flexible catalytic loop of M. tuberculosis tyrosyl-tRNA synthetase in different enzyme– substrate complexes. Eur Biophys J 2014; 43: 613– 22.
29 Franzblau SG, Witzig RS, McLaughlin JC et al. Rapid, low-technology MIC determination with clinical Mycobacterium tuberculosis isolates by using the microplate Alamar Blue assay. J Clin Microbiol 1998; 36: 362– 6. 30 Simithy J, Reeve N, Hobrath JV et al. Identification of shikimate kinase inhibitors among anti-Mycobacterium tuberculosis compounds by LC-MS. Tuberculosis (Edinb) 2014; 94: 152–8.
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J Antimicrob Chemother doi:10.1093/jac/dkv110
Identification of an anti-TB compound targeting the tyrosyl-tRNA synthetase Ningyu Zhu1†, Yuan Lin2†, Dongsheng Li1, Nana Gao1, Chang Liu1, Xuefu You1, Jiandong Jiang1,2, Wei Jiang1‡ and Shuyi Si1*‡ 1 2
Institute of Medicinal Biotechnology, Peking Union Medical College and Chinese Academy of Medical Sciences, Beijing 100050, China; State Key Laboratory of Bioactive Substances and Function of Natural Medicine, Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100050, China Downloaded from http://jac.oxfordjournals.org/ at University of Connecticut on May 11, 2015
*Corresponding author. Tel: +86-10-63180604; Fax: +86-10-63180604; E-mail: [email protected] †N. Z. and Y. L. contributed equally to this work. ‡W. J. and S. S. contributed equally to this work.
Received 25 November 2014; returned 13 January 2015; revised 20 March 2015; accepted 1 April 2015 Objectives: Drug-resistant Mycobacterium tuberculosis poses a great threat to human health. Tyrosyl-tRNA synthetase (TyrRS) is one of the aminoacyl tRNA synthetases that catalyse the attachment of amino acids to their cognate tRNAs and are essential for protein synthesis. There are several distinctive differences between bacterial and human TyrRS and therefore it could be a potential target for developing antimicrobial agents. This study aimed to identify a new anti-TB agent targeting M. tuberculosis TyrRS (MtTyrRS). Methods: We first used Mycobacterium smegmatis for a phenotypic screening of 20 000 compounds. The hit compounds were then screened with MtTyrRS. The interaction between hit compound IMB-T130 and the target protein was analysed by surface plasmon resonance (SPR) assay and molecular docking experiments. The target of IMB-T130 was further confirmed by the overexpression of the target protein. The antibacterial activity of IMB-T130 against various standard and clinical drug-resistant M. tuberculosis strains was evaluated using the microplate Alamar blue assay. Results: Compound IMB-T130 was identified as a hit compound that inhibits the growth of M. smegmatis and the in vitro activity of MtTyrRS. The interaction between IMB-T130 and MtTyrRS was confirmed by SPR assay and molecular docking analysis. The higher MIC for a strain overexpressing the target protein also suggests that MtTyrRS is likely to be the target of IMB-T130. IMB-T130 shows excellent anti-TB activity and low cytotoxicity. Conclusions: IMB-T130 inhibits the growth of MDR-TB and XDR-TB by targeting MtTyrRS. Because of its low cytotoxicity against mammalian cells, IMB-T130 is a promising new agent against drug-resistant M. tuberculosis. Keywords: Mycobacterium tuberculosis, anti-tuberculosis drug screen, SPR assay, molecular docking
Introduction TB, which is caused by Mycobacterium tuberculosis, is a widespread infectious disease leading to 1.5 million deaths each year.1 In addition, antibiotic resistance is a growing problem for TB infections, with MDR- and XDR-TB infections being reported worldwide.2 Unfortunately, only two new clinically relevant drugs, bedaquiline and delamanid, have been introduced to the market in the past 50 years.3,4 Therefore, there is an urgent need to develop new anti-TB drugs for the treatment of infections caused by drug-resistant M. tuberculosis. Aminoacyl tRNA synthetases (AaRS) catalyse the attachment of amino acids to their cognate tRNAs and are essential for protein synthesis.5,6 The aminoacylation of tRNA is carried out in two
steps. First, an amino acid residue forms an aminoacyl-AMP complex with ATP, releasing pyrophosphate. The aminoacyl-tRNA complex is then assembled by esterification at the acceptor arm of the cognate tRNA.7 The inhibition of AaRS activity blocks protein synthesis, which consequently impairs many important physiological processes and stops bacterial growth.8 Therefore, AaRS could be a class of targets for the development of new antibacterial agents. Mupirocin, an inhibitor of isoleucyl-tRNA synthetase, is a successful example.9 Tyrosyl-tRNA synthetase (TyrRS) is one type of AaRS that was reported to be a target of a series of anti-Gram-positive bacterial compounds.10 – 12 However, there is no such report of anti-TB compounds targeting M. tuberculosis TyrRS (MtTyrRS). Comparison of amino acid sequences of TyrRS from M. tuberculosis and human cytoplasm indicates that the
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Materials and methods Materials ATP, IPTG, HEPES and trichloroacetic acid (TCA) were purchased from Sigma. L-[3,5-3H]tyrosine was purchased from PerkinElmer. Escherichia coli MRE 600 tRNA was purchased from Roche. Compound IMB-T130 was purchased from J&K Chemical (synthesized by Enamine, Kyiv, Ukraine). The bacterial strains used in the MIC test were clinical isolates or were purchased from ATCC. Acid– albumin – dextrose–citric acid (ADC) was purchased from BD Biosciences. All other chemicals used were of analytical grade.
Methods Expression and purification of TyrRS M. tuberculosis H37Rv genomic DNA was provided by the Beijing Research Institute for Tuberculosis Control. The TyrRS gene was amplified using primers designed by Primer 5.0 software: 5′ -TCTTGCCATATGTCTGGCATGATCCT CGATG-3′ (NdeI, sense) and 5′ -GGTGGCTCCTCGAGGCCAATCCGTTCC-3′ (XhoI, anti-sense).13 The PCR product was cloned into vector pET30a using the NdeI and XhoI restriction enzyme sites. The resulting pET30a-TyrRS plasmid contains the TyrRS gene fused with a 6×His tag at the C-terminal. The plasmid was transformed into an E. coli BL21 (DE3) strain. A single clone was picked and grown at 378C in LB medium supplemented with 100 mg/L kanamycin for cloning and maintenance. TyrRS was expressed by the addition of 1 mM IPTG and incubation at 208C for 10 h. TyrRS, present in the supernatant, was collected and loaded onto an Ni2+ His Trap chelating column (GE Healthcare). The loading buffer comprised 25 mM Tris, 500 mM NaCl and 30 mM imidazole (pH 7.8). The target protein was eluted using a stepwise gradient of imidazole in elution buffer (25 mM Tris, 500 mM NaCl and 50 – 500 mM imidazole, pH 7.8). Eluted
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fractions were analysed using 12% (w/v) SDS – PAGE followed by Coomassie blue staining and the purity was also determined. The TyrRS concentration was measured by the Bradford method using BSA as a standard. The purified MtTyrRS protein was stored at 2808C.
Phenotypic screening of compound library using M. smegmatis As M. tuberculosis grows slowly, we used M. smegmatis, a non-pathogenic bacterial species that is biologically close to M. tuberculosis, to screen a library of 20 000 compounds. These compounds included a combination of some natural products (plant, actinomycetes and fungi) from the Institute of Medicinal Biotechnology and synthetic products (synthesized by Enamine). The M. smegmatis strain was grown in Middlebrook 7H9 medium in 96-well plates and compounds from the library were added at 5 mg/L. To each well, except the blank control, 199 mL of 7H9 medium (containing 20% ADC and 1% inoculum from the seed culture) and 1 mL of the test compound (1000 mg/L in DMSO) was added. Each plate contained 88 wells as the sample group with 5 mg/L of the test compounds and the remaining 8 wells were the control group, to which 1 mL of DMSO was added instead of the test compound. The plates were incubated at 378C for 48 h and then the OD600 was measured.13,14
Enzyme assay and TyrRS inhibitor screening TyrRS activity was measured by aminoacylation. The reaction was performed in a 96-well plate at 378C. A 50 mL mixture containing 100 mM Tris, 10 mM MgCl2, 40 mM KCl, 1000 mg/L BSA, 1 mM dithiothreitol (DTT), 0.04 mM tRNA, 2 mM ATP, 0.3 mM L-[ring-3,5-3H]tyrosine, 10 mM L-tyrosine and 5 mg/L TyrRS was added to each well. TyrRS protein was pre-incubated with 1 mL of test compound (10 mg/L) at 48C for 30 min in 40 mL of buffer (125 mM Tris, 12.5 mM MgCl2, 50 mM KCl, 1250 mg/L BSA and 1.25 mM DTT); 1 mL of DMSO was added for the control group. Then, the reaction was initiated by the addition of 10 mL of substrate (0.2 mM tRNA, 10 mM ATP, 1.5 mM L-[ring-3,5-3H]tyrosine and 50 mM L-tyrosine). After incubation at 378C for 15 min, the reaction was stopped by the addition of 20 mL of pre-chilled 5% TCA solution (containing 0.02 M sodium pyrophosphate) at 08C. The reaction mixture was then transferred onto filter papers cut to 800×800 mm squares. The filter papers were washed twice with TCA solution, once with ethanol and then dried on a heated plate.15 Next, they were placed into 24-well plates and 1 mL of scintillation liquid was added to each well. The amount of radioactivity was determined using a scintillation counter.
Inhibitory effect of compound IMB-T130 on TyrRS TyrRS (5 mg/L) was pre-incubated with gradient concentrations (0.16–40 mg/L) of IMB-T130, or 1% DMSO as a control, in 40 mL of buffer (125 mM Tris, 12.5 mM MgCl2, 50 mM KCl, 1250 mg/L BSA and 1.25 mM DTT) at 48C for 30 min. The reaction conditions and detection method were the same as for the TyrRS inhibitor screening described above.
Confirmation of the interaction between IMB-T130 and TyrRS by SPR assay SPR is widely used for measuring the interaction between small molecules and proteins. To detect the interaction between IMB-T130 and TyrRS, Biacore sensor chips were coated with TyrRS and then exposed to IMB-T130 at a gradient of concentrations (3.12 – 25 mM). The measurements were performed using a Biacore T100 system (GE Healthcare) at 258C in a PBS-P+ running buffer (20 mm phosphate buffer, 2.7 mM KCl, 137 mM NaCl, 0.05% surfactant P20, pH 7.4 and 5% DMSO). Purified His-tagged TyrRS (20 mg/L) was immobilized onto a CM5 sensor chip using an N-hydroxysuccinimide/1-ethyl-3-(3-dimethylperpyl)-carboiimide
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identity is only 15.6%. Because of the apparent sequence difference as well as the essential role of TyrRS for the growth of M. tuberculosis, we consider TyrRS to be a promising new anti-TB target. Taking into account that the identified compounds based on the enzyme assay may not show antibacterial activity, we performed a two-step screening including a phenotype screening and a targeted screening. Mycobacterium smegmatis mc2155, a non-pathogenic strain, was chosen to substitute for M. tuberculosis to examine the antibacterial activity of compounds. The TyrRS amino acid sequences from M. tuberculosis and M. smegmatis exhibit 79% sequence identity and the important amino acids in the active centre are same. Therefore, we inferred that an anti-TB agent targeting TyrRS might also inhibit the growth of M. smegmatis in a similar way. In this study, we identified 78 hit compounds through a phenotype screening of 20000 compounds with M. smegmatis. Only compound IMB-T130 {5-chloro-N-[4-(pyridin-2-yl)-1,3-thiazol-2-yl]thiophene-2-carboxamide} showed potent inhibitory activity of TyrRS in vitro at 10 mg/L. IMB-T130 showed similar antibacterial potency as first-line anti-TB drugs and even better inhibitory effects against drug-resistant strains. Furthermore, we performed a surface plasmon resonance (SPR) assay between IMB-T130 and TyrRS, an overexpressing experiment of MtTyrRS in M. smegmatis and a molecular docking analysis in silico between IMB-T130 and the active centre of MtTyrRS to confirm if TyrRS is the target of IMB-T130. A series of tests suggested that TyrRS is likely to be the target of IMB-T130.
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amine coupling kit in 10 mM sodium acetate buffer at pH 4.5. The protein density is 5000 response units (RU). To determine the binding affinity of His-tagged TyrRS and IMB-T130, solutions of compound (5% DMSO) at different concentrations (3.12–25 mM) were injected into the TyrRS immobilized chambers. The surface was regenerated using Gly-HCl (pH 3.0). IMB-T130 was proven to bind to TyrRS by a measurable dose-dependent change in RU.16,17
Determination of the MIC for M. smegmatis overexpressing TyrRS
Molecular docking between IMB-T130 and TyrRS To determine how the structure of IMB-T130 contributes to its inhibitory activity against TyrRS, a molecular docking analysis between IMB-T130 and TyrRS was performed. The crystal structure of TyrRS solved at a 2.9 A˚ resolution was retrieved from the Protein Data Bank (ID code 2JAN). Because there are no cocrystallized molecules in this crystal, the docking pocket needs to be generated by key amino acids of the active sites. As the different bacterial TyrRS have homologous catalytic domains, their active sites are similar.19 There is a Rossmann fold in the catalytic domain of MtTyrRS and HIGH and KMSKS (KFGKS in MtTyrRS) motifs exist in the active centre.20 Besides MtTyrRS, the three-dimensional structures of TyrRS from E. coli,21 Staphylococcus aureus,22 Bacillus stearothermophilus 23 and Thermus thermophilus 24 were also solved by X-ray crystallography. According to a recent study, the most important amino acid residues in the MtTyrRS active centre are Tyr36, Gly38, Phe39, Asp40, His47, Gly49, His50, Tyr171, Gln175, Asp178, Gln191, Gly193, Gly194, Gln197, Leu223, Val224, Lys231, Phe232, Gly233 and Lys234.25,26 The Discovery Studio 4.1 program (State Key Laboratory of Bioactive Substances and Function of Natural Medicine, Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, China) was used to perform molecular docking.27 The binding pocket was generated based on 20 important amino acids. Three conformations of the IMB-T130 molecular structure were generated after optimization and then docking with the active pocket was carried out using the CDOCKER program. According to the scores of – CDOCKER energy and – CDOCKER interaction energy, the best 10 results were retained.
Anti-TB activity of IMB-T130 TyrRS is known to be an essential enzyme for ligating tyrosine to its cognate tRNA. It plays an important role in the production of proteins. Thus, functional inhibition of this enzyme is expected to inhibit the growth of M. tuberculosis. The anti-TB activity of IMB-T130 was tested using the microplate Alamar blue assay (MABA). The inhibitory activity was tested against standard strain H37Rv (ATCC 27294), susceptible clinical strains STB-FJ05060 and STB-FJ05349 and drug-resistant clinical isolates MDR-FJ05120, MDR-FJ05189 and XDR-FJ05195. The first-line anti-TB
Cell cytotoxicity evaluation To test the cytotoxic effect of IMB-T130, a range of different cell types were used. Cells were seeded in triplicate in 96-well culture plates at a density of 5×103 cells per well. Then, the cells were incubated with IMB-T130 at a gradient of concentrations from 1.56 to 200 mM. After incubation for 24 h, thiazolyl blue tetrazolium bromide reagent was added. After further incubation for 4 h, 50 mL of DMSO was added to each well. The absorbance was measured at 490 nm and the average values were calculated for triplicate wells. The IC50 values were calculated on a concentration–response curve.
Susceptibility of other bacterial strains to IMB-T130 The MICs of compound IMB-T130 for other representative bacteria from ATCC and clinical isolates except M. tuberculosis were determined using agar dilution method recommend by the CLSI. Inoculations were adjusted to yield 104 cfu/spot by a multipoint inoculator (Bolney, Sussex, UK). The bacteria were incubated at 358C for 18 h. The MICs were determined as the lowest concentration of the compound that inhibited the growth of bacteria on the plate. Levofloxacin was used as a reference drug.13
Results Expression and purification of TyrRS TyrRS can ligate tyrosine to its cognate tRNA. In this study, active TyrRS enzyme was required to screen the compound library. The recombinant protein was purified and the purification was confirmed by the appearance of a single band at 47.5 kDa after SDS – PAGE and Coomassie blue staining (Figure S1, available as Supplementary data at JAC Online).
Compounds that inhibit M. smegmatis and MtTyrRS The library containing 20 000 compounds was screened by growing M. smegmatis in 96-well plates, adding the compounds at 5 mg/L. Each plate contained 88 wells for the test compounds and the remaining 8 wells were the control group. For the control, 1 mL of DMSO was added into the wells instead of the test compounds. The plates were incubated at 378C for 48 h and then the OD600 was measured. After screening, 78 compounds were identified with an OD600 of ,50% of the control and were inferred to have possible anti-TB activity. These compounds were further tested for their ability to inhibit TyrRS at a concentration of 10 mg/L. We measured the amount of radioactivity after 15 min of incubation in the reaction mixture using the in vitro assay system described in the Materials and methods section and found that compound IMB-T130 could suppress .50% of TyrRS activity at a concentration of 10 mg/L. The molecular weight of IMB-T130 is 321.81 Da and its structure is shown in Figure 1(a).
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For the overexpression of the MtTyrRS protein in M. smegmatis, we constructed the plasmid pMV261-eGFP by inserting the eGFP gene into the pMV261 vector with ClaI and HpaI restriction enzyme sites. After that, the MtTyrRS gene was inserted between the promoter and the eGFP gene using the BamHI and HindIII sites in a way that TyrRS is in frame with eGFP. The resulting pMV261:TyrRS-eGFP plasmid was amplified by E. coli and then transformed into competent M. smegmatis. PCR was used to identify strains expressing eGFP, which indicated the expression of the MtTyrRS-eGFP fusion protein. Expression of the recombinant protein was further confirmed by fluorescence microscopy and western blotting with anti-eGFP antibody. The MICs of IMB-T130 for M. smegmatis with a control vector (pMV261-eGFP plasmid) and strains overexpressing a nonrelated gene (M. tuberculosis ribosomal protein L12) or TyrRS-eGFP were determined by the conventional plate dilution method.16,18
drugs rifampicin and isoniazid were also included in the analysis for comparison. The gradient concentrations of compounds varied from 0.01 to 128 mg/L. All M. tuberculosis strains were cultured at 378C in Middlebrook 7H9 broth supplemented with 0.2% glycerol and 10% ADC until the mid-log phase of growth. The cells were diluted in Middlebrook 7H9 broth to 106 cfu/mL with gradient concentrations of compounds. Then, the MIC was measured in sterile 96-well plates with a final volume of 100 mL per well. The MIC was defined as the lowest drug concentration that prevented the colour change of Alamar blue reagent from blue to pink.28
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Next, the inhibition ratios at different concentrations of IMB-T130 (40, 20, 10, 5, 2.5, 1.25, 0.625, 0.31 and 0.16 mg/L) were determined. The activity of MtTyrRS was inhibited by
Confirming the interaction between IMB-T130 and TyrRS
O
(a) S
NH
S Cl
N N IMB-T130
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IC50 = 2.619 mg/L = 8.138 mM
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0 1 Log (IMB-T130, mg/L)
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Figure 1. (a) Structure of IMB-T130 {5-chloro-N-[4-(pyridin-2-yl)1,3-thiazol-2-yl]thiophene-2-carboxamide}. (b) Inhibition of aminoacylation activity by IMB-T130. TyrRS (5 mg/L) was incubated with different concentrations (0.16 – 40 mg/L) of IMB-T130 at 48C for 30 min and the aminoacylation activity was then determined as detailed in the Materials and methods section. The IC50 value is plotted as the ratio of the radioactivity signal over the concentration of compound (log plot) that fits to a variable-slope dose – response equation. The experiment was repeated three times.
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To examine the interaction between compound IMB-T130 and the target protein, an SPR assay was performed. Biacore sensor chips were coated with purified TyrRS and then exposed to IMB-T130 at different concentrations. The RU were measured at four different concentrations (3.12 –25 mM) of IMB-T130 (concentrations .25 mM could not be used due to the poor water solubility of IMB-T130). The results showed a significant dose-dependent response at the concentrations tested (Figure 2).
If IMB-T130 inhibits the growth of mycobacteria by targeting TyrRS, the antibacterial activity of IMB-T130 should be lower for a strain overexpressing the TyrRS protein. Therefore, we constructed an expression plasmid containing TyrRS-eGFP. This plasmid was transformed into M. smegmatis and expression of the recombinant protein was confirmed by the appearance of a GFP signal (Figure 3) and western blotting with anti-eGFP antibody (Figure S2). The MIC of IMB-T130 for an M. smegmatis strain with a PMV261 control plasmid is 0.312 mg/L. The MIC of IMB-T130 for a strain overexpressing TyrRS is 1.25 mg/L, which is 4-fold higher than the MIC for the control strain. In contrast, expression of the MtL12 gene in M. smegmatis did not increase the MIC of IMB-T130 (Table 1). This result showed that the TyrRS protein is likely to be the target of IMB-T130 in vivo.
Analysis of the molecular docking results Ten results were reserved after the molecular docking experiments between the three conformations of IMB-T130 and the active pocket of TyrRS. The result giving the highest score for both – CDOCKER energy and – CDOCKER interaction energy indicates the most probable docking model of the complex. This
MtTyrRS immobilized IMB-T130
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Figure 2. Demonstration of the interaction between TyrRS and compound IMB-T130 by SPR. A sensor chip coated with purified TyrRS was exposed to various concentrations (3.12– 25 mM) of IMB-T130. The change in RU is shown.
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(b)
IMB-T130 in a dose-dependent manner with an IC50 value of 8.138 mM (Figure 1b). The result showed that IMB-T130 is a potent competitive inhibitor of TyrRS.
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(a)
(b)
Leu223 Gly194
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Val224
TyrRS-eGFP
Figure 3. Fluorescence signal in M. smegmatis with a control vector or an overexpression plasmid for TyrRS-eGFP. (a) M. smegmatis cells carrying plasmid pMV261. (b) M. smegmatis cells carrying plasmid pMV261:TyrRS-eGFP. This figure appears in colour in the online version of JAC and in black and white in the print version of JAC.
Gly49
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Gly194 Val224
0.312 1.25 0.312
Gly49
model showed that IMB-T130 inserts into the pocket, interacting with four important amino acid residues of the MtTyrRS active centre (Figure 4). The chlorine atom formed a halogen bond with Val224, a type of non-covalent intermolecular interaction. There was a hydrophobic interaction between the thiophene of IMB-T130 and the alkyl group of Leu223. The thiophene also had intermolecular hyperconjugation with the carbonyl group of Gly49. The N atoms of thiazole formed a conventional hydrogen bond with Gly194. Based on this docking model, IMB-T130 occupied the centre position in the active pocket of MtTyrRS; therefore, it might be able to prevent the two substrates from binding to the enzyme simultaneously. Especially, the Gly194 and Val224 played important roles in the binding of the tyrosyl-adenylate intermediate.
Anti-TB activity of compound IMB-T130 The anti-TB activity of IMB-T130 was tested using a MABA against strain H37Rv and the MIC was found to be 0.08 mg/L. For WT clinical strains FJ05349 and FJ05060, the MIC was also 0.08 mg/L. For MDR clinical strains MDR-FJ05120 and MDR-FJ05189, the MICs of IMB-T130 were 0.25 and 4 mg/L, respectively. For the XDR clinical strain XDR-FJ05195, the MIC was 0.25 mg/L. These data indicate that IMB-T130 has comparable anti-TB activity of susceptible strains to the first-line drugs such as rifampicin (0.0625 –0.125 mg/L) and isoniazid (0.0625 –0.125 mg/L).29 For MDR strains, IMB-T130 performed better than rifampicin and similar to isoniazid. For XDR strains, it performed substantially better than rifampicin and isoniazid. These results indicate that IMB-T130 may inhibit the growth of M. tuberculosis by targeting TyrRS, which is a distinct mechanism from the first-line anti-TB antibiotics.
Susceptibility of other bacterial species to IMB-T130 MtTyrRS was the screening target in our experiment. Because of the conservative nature of this protein among various bacterial
Figure 4. Molecular docking of TyrRS and IMB-T130. (a) Overview of the active pocket of TyrRS bound to IMB-T130. IMB-T130 is represented by a stick model (grey, carbon atoms; blue, nitrogen atoms; deep red, oxygen atoms; yellow, sulphur atoms; green, chlorine atoms). The red sticks represent the amino acid residues interacting with IMB-T130. (b) The halogen bond formed between the chlorine atom and Val224 (red line), the hydrophobic interaction between the thiophene of IMB-T130 and the alkyl group of Leu223 (purple line), the intermolecular hyperconjugation between the carbonyl group of Gly49 and the thiophene (blue line) and the conventional hydrogen bond formed by the N atom of thiazole and the hydrogen atoms of Gly194 (green line).
species, this identified compound might also inhibit the activity of homologous proteins in other bacteria. We therefore examined the growth inhibition of eight representative bacterial strains (four Gram-positive and four Gram-negative) by IMB-T130. The results showed that IMB-T130 exhibited high MICs for these Gram-positive and Gram-negative bacteria (Table 2). Moreover, the IC50 of cytotoxicity for human embryonic kidney 293 cells was found to be 22 mM and for HeLa cancer cells was .100 mM. These results indicate that compound IMB-T130 has a selective and strong anti-TB effect with low cytotoxicity.
Discussion AaRS catalyse the attachment of amino acids to their cognate tRNAs and are essential for protein synthesis. AaRS represent a class of attractive targets for developing new antibiotics, because they are essential for microbial survival and share low homology with their eukaryotic counterparts. For example, mupirocin is a successful marketed antibiotic that targets AaRS. TyrRS is one type of AaRS. Compounds that inhibit the function of TyrRS would kill bacteria by disrupting protein synthesis and would have low toxicity against humans. In order to find a lead
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Table 1. Antibacterial activity of IMB-T130 against M. smegmatis with a control vector or an overexpression plasmid for MtTyrRS or MtL12
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Table 2. MICs of IMB-T130 for various bacterial strains MIC (mg/L) Strain
rifampicin
isoniazid
levofloxacin
0.08 0.08 0.08 0.25 4 0.25 32 32 64 128 256 128 128 128
0.125 ,0.5 ,0.5 .256 .256 4 — — — — — — — —
0.125 ,0.5 ,0.5 4 ,0.5 .256 — — — — — — — —
— — — — — — 0.25 0.25 0.25 0.5 ≤0.03 0.06 1 0.5
STB, clinically susceptible strain of M. tuberculosis; MDR, MDR M. tuberculosis; XDR, XDR M. tuberculosis. Strains FJ05060, FJ05120, FJ05189 and FJ05195 are clinical isolates of M. tuberculosis. a Methicillin-susceptible S. epidermidis. b MSSA. c MRSA. d Vancomycin-susceptible Enterococcus. e ESBL negative. f ESBL positive.
compound targeting TyrRS with antibacterial activity, we performed a two-step screening including a phenotype screening and a targeted screening. As an alternative to M. tuberculosis, we chose M. smegmatis to determine the antibacterial activity of compounds because it is non-pathogenic and fast growing. Comparison of the TyrRS amino acid sequences from M. tuberculosis and M. smegmatis revealed 79% sequence identity. The 20 important amino acids comprising the active centre of TyrRS from these two species are identical. Although the drug susceptibility of these two strains are not fully parallel, our experiments showed that it is feasible to screen an anti-TB agent using M. smegmatis as replacement at some level. It is more suitable for high-throughput screening. In this study, by screening a library of compounds, we identified only one compound (IMB-T130) that could inhibit the growth of M. smegmatis and the activity of MtTyrRS. Additionally, we confirmed the interaction between IMB-T130 and TyrRS by SPR assay and analysed this interaction using a molecular docking program. The higher MIC for a strain overexpressing TyrRS also suggested that TyrRS is likely to be the target of IMB-T130. Importantly, IMB-T130 showed anti-TB potency similar to firstline drugs and was even more effective against drug-resistant strains. IMB-T130 therefore represents a new anti-TB compound targeting MtTyrRS. The significant dose-dependent signal measured in our SPR experiments confirmed the direct interaction between TyrRS and compound IMB-T130. Based on this result, we analysed how the structure of IMB-T130 contributes to its inhibitory activity against TyrRS by molecular docking analysis. The active pocket comprises
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20 amino acid residues of MtTyrRS, as indicated by recent studies. 19,25 The highest scoring docking model showed that IMB-T130 forms interactions with four important amino acid residues of MtTyrRS. IMB-T130 occupied the centre position of the active pocket and inhibited the function of the protein. The increased MIC of IMB-T130 for a TyrRS-overexpressing strain verified that the protein might be the target of compound IMB-T130. TyrRS is expressed in almost all prokaryotic and eukaryotic cells. TyrRS proteins from prokaryotic bacteria exhibit similar structures. Previous studies have shown that TyrRS belongs to class I of the AaRS, with highly conserved active sites, especially among prokaryotes. But IMB-T130 was found to be a selective inhibitor of M. tuberculosis, only showing weak inhibitory activity against Staphylococcus epidermidis and S. aureus. Based on the molecular docking results, we thought that the large active pocket may be one of the reasons for this phenomenon. The active pocket of TyrRS was found to be much larger than the molecular size of IMB-T130. We believe that IMB-T130 binds to the active centre of TyrRS from various bacteria in different ways because of the non-conserved sequences. This might affect the inhibition efficiency towards TyrRS from different sources and explain the differences in MICs for different bacteria. Additionally, the cell wall differences between M. tuberculosis and the other bacteria might be another reason for the distinct MICs. Compound IMB-T130 showed strong selective anti-TB activity and there was no significant cross-resistance between IMB-T130 and the first-line anti-TB drugs. We speculated that compound IMB-T130 may inhibit the growth of M. tuberculosis via a
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H37Rv STB-FJ05349 STB-FJ05060 MDR-FJ05120 MDR-FJ05189 XDR-FJ05195 Staphylococcus epidermidis 12228a Staphylococcus aureus 29213b Staphylococcus aureus 33591c Enterococcus faecalis 29212d Escherichia coli 25922e Klebsiella pneumoniae 7e Klebsiella pneumoniae 700603f Pseudomonas aeruginosa 27853
IMB-T130
Anti-TB agent targeting tyrosyl-tRNA synthetase
Acknowledgements We thank Professor Kanglin Wan from the Chinese Center for Disease Control and Prevention (CCDC) for helping us test the anti-TB activity of compound. We thank Dr Kate Fox of Edanz Writing Company (Liwen Bianji, Beijing), Professor Yanchang Wang and Mr Michael Bokros from Florida State University, Medicine College, for helping us to check and polish the English expression and edit the style of the manuscript.
Funding This work was supported by the Foundation for Innovative Research Groups (grant number 81321004), the Funds for International Cooperation Exchange between China-Sweden (grant number 81361138020), the National Natural Science Foundation of China (grant numbers 81072672 and 81302816), grants from the State Mega Programs (2012ZX09301002-003/ 006; 2015ZX09102007-008/016) and a grant from the Beijing science and technology projects (Z141102004414065).
Transparency declarations None to declare.
Supplementary data Figures S1 and S2 are available as Supplementary data at JAC Online (http://jac.oxfordjournals.org/).
an individual patient data meta-analysis. Clin Infect Dis 2014; 59: 1364– 74. 3 Blair HA, Scott LJ. Delamanid: a review of its use in patients with multidrug-resistant tuberculosis. Drugs 2015; 75: 91– 100. 4 Nagabushan H, Roopadevi HS. Bedaquiline: a novel antitubercular drug for multidrug-resistant tuberculosis. J Postgrad Med 2014; 60: 300–2. 5 Gadakh B, Van Aerschot A. Aminoacyl-tRNA synthetase inhibitors as antimicrobial agents: a patent review from 2006 till present. Expert Opin Ther Pat 2012; 22: 1453 –65. 6 Yanagisawa T, Sumida T, Ishii R et al. A paralog of lysyl-tRNA synthetase aminoacylates a conserved lysine residue in translation elongation factor P. Nat Struct Mol Biol 2010; 17: 1136–43. 7 Delarue M. Aminoacyl-tRNA synthetases. Curr Opin Struct Biol 1995; 5: 48– 55. 8 Van de Vijver P, Vondenhoff GH, Kazakov TS et al. Synthetic microcin C analogs targeting different aminoacyl-tRNA synthetases. J Bacteriol 2009; 191: 6273– 80. 9 Yanagisawa T, Lee JT, Wu HC et al. Relationship of protein structure of isoleucyl-tRNA synthetase with pseudomonic acid resistance of Escherichia coli. A proposed mode of action of pseudomonic acid as an inhibitor of isoleucyl-tRNA synthetase. J Biol Chem 1994; 269: 24304 – 9. 10 Wang XD, Deng RC, Dong JJ et al. 3-Aryl-4-acyloxyethoxyfuran-2(5H)ones as inhibitors of tyrosyl-tRNA synthetase: synthesis, molecular docking and antibacterial evaluation. Bioorg Med Chem 2013; 21: 4914–22. 11 Xiao ZP, Ouyang H, Wang XD et al. 4-Alkoxy-3-arylfuran-2(5H)-ones as inhibitors of tyrosyl-tRNA synthetase: synthesis, molecular docking and antibacterial evaluation. Bioorg Med Chem 2011; 19: 3884 –91. 12 Xiao ZP, Ma TW, Liao ML et al. Tyrosyl-tRNA synthetase inhibitors as antibacterial agents: synthesis, molecular docking and structure–activity relationship analysis of 3-aryl-4-arylaminofuran-2(5H)-ones. Eur J Med Chem 2011; 46: 4904– 14. 13 Lin Y, Zhu N, Han Y et al. Identification of anti-tuberculosis agents that target the cell-division protein FtsZ. J Antibiot (Tokyo) 2014; 67: 671– 6. 14 Xu S, Pei L, Li D et al. Synthesis and antimycobacterial evaluation of natural oridonin and its enmein-type derivatives. Fitoterapia 2014; 99: 300–6. 15 Werner RG, Thorpe LF, Reuter W et al. Indolmycin inhibits prokaryotic tryptophanyl-tRNA ligase. Eur J Biochem 1976; 68: 1 –3. 16 Lin Y, Li Y, Zhu Y et al. Identification of antituberculosis agents that target ribosomal protein interactions using a yeast two-hybrid system. Proc Natl Acad Sci USA 2012; 109: 17412 –7. 17 Neumann T, Junker HD, Schmidt K et al. SPR-based fragment screening: advantages and applications. Curr Top Med Chem 2007; 7: 1630 – 42. 18 Lin Y, Li Y, Zhu N et al. The antituberculosis antibiotic capreomycin inhibits protein synthesis by disrupting interaction between ribosomal proteins L12 and L10. Antimicrob Agents Chemother 2014; 58: 2038–44. 19 Odynets KO, Korneliuk OI. A model of three-dimensional structure of Mycobacterium tuberculosis tyrosyl-tRNA synthetase. Ukr Biokhim Zh 2008; 80: 62– 75. 20 Bonnefond L, Giege R, Rudinger-Thirion J. Evolution of the tRNA(Tyr)/ TyrRS aminoacylation systems. Biochimie 2005; 87: 873– 83.
References 1 Zumla A, George A, Sharma V et al. The WHO 2014 global tuberculosis report: further to go. Lancet Glob Health 2015; 3: e10–2.
21 Kobayashi T, Takimura T, Sekine R et al. Structural snapshots of the KMSKS loop rearrangement for amino acid activation by bacterial tyrosyl-tRNA synthetase. J Mol Biol 2005; 346: 105–17.
2 Bastos ML, Hussain H, Weyer K et al. Treatment outcomes of patients with multidrug-resistant and extensively drug-resistant tuberculosis according to drug susceptibility testing to first- and second-line drugs:
22 Qiu X, Janson CA, Smith WW et al. Crystal structure of Staphylococcus aureus tyrosyl-tRNA synthetase in complex with a class of potent and specific inhibitors. Protein Sci 2001; 10: 2008 –16.
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mechanism distinct from that of existing anti-TB drugs. The result of the SPR assay confirmed the interaction between IMB-T130 and TyrRS. We believe that the inhibitory activity of IMB-T130 against MtTyrRS generated its anti-TB activity without cross-resistance of existing anti-TB drugs. The higher MIC for a strain overexpressing TyrRS also supports our opinion. However, it seemed that the concentration of IMB-T130 required to inhibit the growth of M. tuberculosis was lower than that required to inhibit the aminoacylation activity of MtTyrRS in vitro. One possible reason is the difference between the extracellular and intracellular environment and the IC50 value we detected in vitro might deviate from its actual value in vivo. Another possible reason is that IMB-T130 may be a multitarget compound.30 In this study, we report that compound IMB-T130 inhibits the aminoacylation activity of MtTyrRS. This compound showed selective inhibition against M. tuberculosis and displayed low toxicity against mammalian cells. Therefore, chemical modification of IMB-T130 to further optimize its bactericidal capacity, increase its water solubility (log P¼4.1) and reduce its toxicity may result in a new promising anti-TB drug highly effective against drug-resistant M. tuberculosis.
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23 Brick P, Bhat TN, Blow DM. Structure of tyrosyl-tRNA synthetase refined at 2.3 A˚ resolution. Interaction of the enzyme with the tyrosyl adenylate intermediate. J Mol Biol 1989; 208: 83 –98.
27 Al-Balas QA, Sowaileh MF, Hassan MA et al. Novel N-substituted aminobenzamide scaffold derivatives targeting the dipeptidyl peptidase-IV enzyme. Drug Des Devel Ther 2014; 8: 129– 63.
24 Yaremchuk A, Kriklivyi I, Tukalo M et al. Class I tyrosyl-tRNA synthetase has a class II mode of cognate tRNA recognition. EMBO J 2002; 21: 3829– 40.
28 Collins L, Franzblau SG. Microplate alamar blue assay versus BACTEC 460 system for high-throughput screening of compounds against Mycobacterium tuberculosis and Mycobacterium avium. Antimicrob Agents Chemother 1997; 41: 1004 –9.
25 Mykuliak VV, Kornelyuk AI. The mechanisms of substrates interaction with the active site of Mycobacterium tuberculosis tyrosyl-tRNA synthetase studied by molecular dynamics simulations. Biopolym Cell 2014; 30: 157–62. 26 Mykuliak VV, Dragan AI, Kornelyuk AI. Structural states of the flexible catalytic loop of M. tuberculosis tyrosyl-tRNA synthetase in different enzyme– substrate complexes. Eur Biophys J 2014; 43: 613– 22.
29 Franzblau SG, Witzig RS, McLaughlin JC et al. Rapid, low-technology MIC determination with clinical Mycobacterium tuberculosis isolates by using the microplate Alamar Blue assay. J Clin Microbiol 1998; 36: 362– 6. 30 Simithy J, Reeve N, Hobrath JV et al. Identification of shikimate kinase inhibitors among anti-Mycobacterium tuberculosis compounds by LC-MS. Tuberculosis (Edinb) 2014; 94: 152–8.
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