Discovery of novel antituberculosis agents among 3-phenyl-5-(1-phenyl-1H-[1,2,3]triazol-4-yl)-[1,2,4]oxadiazole derivatives targeting aminoacyl-tRNA synthetases

Antibiotic resistance is a major problem of tuberculosis treatment. This provides the stimulus for the search of novel molecular targets and approaches to reduce or forestall resistance emergence in Mycobacterium tuberculosis. Earlier, we discovered a novel small-molecular inhibitor among 3-phenyl-5-(1-phenyl-1H-[1,2,3]triazol-4-yl)-[1,2,4]oxadiazoles targeting simultaneously two enzymes—mycobacterial leucyl-tRNA synthetase (LeuRS) and methionyl-tRNA synthetase (MetRS), which are promising molecular targets for antibiotic development. Unfortunately, the identified inhibitor does not reveal antibacterial activity toward M. tuberculosis. This study aims to develop novel aminoacyl-tRNA synthetase inhibitors among this chemical class with antibacterial activity toward resistant strains of M. tuberculosis. We performed molecular docking of the library of 3-phenyl-5-(1-phenyl-1H-[1,2,3]triazol-4-yl)-[1,2,4]oxadiazole derivatives and selected 41 compounds for investigation of their inhibitory activity toward MetRS and LeuRS in aminoacylation assay and antibacterial activity toward M. tuberculosis strains using microdilution assay. In vitro screening resulted in 10 compounds active against MetRS and 3 compounds active against LeuRS. Structure-related relationships (SAR) were established. The antibacterial screening revealed 4 compounds active toward M. tuberculosis mono-resistant strains in the range of concentrations 2–20 mg/L. Among these compounds, only one compound 27 has significant enzyme inhibitory activity toward mycobacterial MetRS (IC50 = 148.5 µM). The MIC for this compound toward M. tuberculosis H37Rv strain is 12.5 µM. This compound is not cytotoxic to human HEK293 and HepG2 cell lines. Therefore, 3-phenyl-5-(1-phenyl-1H-[1,2,3]triazol-4-yl)-[1,2,4]oxadiazole derivatives can be used for further chemical optimization and biological research to find non-toxic antituberculosis agents with a novel mechanism of action.


Results
To find novel MetRS and LeuRS inhibitors with the antituberculosis activity we performed molecular docking of the pre-selected compound library of 3-phenyl-5-(1-phenyl-1H- [1,2,3]triazol-4-yl)- [1,2,4]oxadiazole derivatives into active sites of investigated enzymes. According to molecular docking results and visual analysis of the bestscored complexes, we selected 41 compounds for investigation of their inhibitory activity toward recombinant mycobacterial LeuRS and MetRS. The results of testing are presented in Table 1.
According to the data from Tables 3, 4 compounds have activity between 2 and 20 mg/L toward several monoresistant strains. Among these compounds, only one compound 27 (with growth inhibition cutoff 5.17-51.7 µM) has significant enzyme inhibitory activity toward mycobacterial MetRS. Compounds 40 (  www.nature.com/scientificreports/ determined after incubation for 7 and 14 days, correspondingly. As it can be seen from the Table 4, compound 27 inhibits the growth of M. tuberculosis H37Rv in the medium containing glucose as a carbon source with 1-week MIC value of 12.5 µM and 2-week MIC value of 25 µM and in the medium containing DPPC as a carbon source with 1-week and 2-week MIC values of 25 µM. In the media containing BSA, the compounds did not inhibit the growth of M. tuberculosis H37Rv. It may be explained by the potency them to bind with hydrophobic pockets of free BSA which leads to the decrease of the efficient concentrations of compounds capable to bind with aminoacyl-tRNA synthetases. Similar insights were shown with molecular simulations of human serum albumin (HSA) and three clinically promising squalenoylated drugs (gemcitabine-squalene, adenine-squalene, and doxorubicin-squalene). Data suggest that these drugs may accumulate by HSA and inside low-density lipoproteins 23 . DPPC forms spontaneously vesicle-like structures 24 , thus it may act as the scavenger for different hydrophobic and amphiphilic molecules, similar to lipoproteins 23 .
We have tested compound 27 for cytotoxicity toward human cell lines HEK293 (human embryonic kidney 293) and HepG2 (human hepatocellular carcinoma cell line) using standard MTT assay. According to the results of in cellulo testing, this compound did not affect cell viability in the range of concentrations from 3.125 µM to 50 µM, suggesting that CC 50 , concentration to cause 50% cytotoxicity, for HEK293 and HepG2 > 50 µM.

Discussion
In this study, we have investigated 41 derivatives of 3-Phenyl-5-(1-phenyl-1H-[1,2,3]triazol-4-yl)-[1,2,4]oxadiazole for inhibitory activity toward M. tuberculosis MetRS and LeuRS and established several structure-activity relationships (SAR). It has been observed that the nature of R 1 substituent significantly influences the compound's inhibitory activity toward M. tuberculosis LeuRS and MetRS. We found that compounds with R 1 = bromine atom are more active than those with an ethoxy group, fluorine, or hydrogen in this position. It can be seen from the comparison of activity for compounds 34, 39, 23, and 1 and compound pairs such as 36, 30, and 35, 26. From the obtained results, the order of potency for the substituent R 1 could be proposed as following: Br ≥ CH 3 CH 2 COOH > F > H. Fluorine and bromine atoms have almost equal activity toward mycobacterial LeuRS and MetRS for compound pair 12 and 32. In the case of compound pair 15 and 33, the bromine atom is more favored than the fluorine atom for inhibitory activity toward M. tuberculosis MetRS but in a case of mycobacterial LeuRS, a fluorine atom is more profitable.
It was revealed that the R 3 significantly affects the compound's inhibitory activity toward mycobacterial MetRS and LeuRS. The introduction of fluorine atom instead of Hydrogen in this position leads to a significant decrease of inhibitory activity toward LeuRS and MetRS. To see this effect one can refer to compound pair 2 and 3.
We have investigated that the substituent R 4 has also influenced the inhibitory activity toward mycobacterial LeuRS and MetRS. For a pair of compounds with R 1 = F and R 7 = Cl, R 4 = methoxy group (27) is more favored than the methyl group (25). For a pair of compounds with R 1 = F and R 7 = H, R 4 = ethoxy group (30) is more profitable than the methoxy group (26) for both enzymes. In a series of compounds with R 1 = Br (34)(35)(36), the order of inhibitory activity for both enzymes is the following: F < OCH 3 < OCH 2 CH 3 . For a pair of compounds with R 1 = OCH 2 CH 3 (39)(40), the Cl atom at the position R 4 plays an important role for dual-targeted inhibition activity toward LeuRS and MetRS and consequently for antibacterial activity toward M. tuberculosis resistant strains.
It was found that R 5 also impacts the compound's inhibitory activity toward mycobacterial MetRS and LeuRS. The presence of a methoxy group (compound 20) is more favored than the methyl group (compound 18) in this position for inhibitory activity toward both enzymes. The introduction of substituents CF 3 (19) or Cl (16) in this position leads to an increase of inhibitory activity toward MetRS and almost complete loss of activity or even activation of LeuRS and vice versa, the fluorine atom (15) in this position causes increase of inhibitory activity toward LeuRS and activation of MetRS. Therefore, the order of potency of substituent R 5 for MetRS could be proposed as following: CF 3 < Cl < OCH 3 < CH 3 < F and for LeuRS as following: F < OCH 3 < CH 3 < CF 3 < Cl. It should be noted, that the R 5 = methoxy group (compound 20) is important for membrane permeability since the substitution of OCH 3 with any other group such as CH 3 , CF 3 , F or Cl leads to complete loss of antibacterial activity toward M. tuberculosis resistant strains.
According to structure-activity relationship (SAR) studies of compounds 9-14, the order of R 6 substituent inhibitory efficiency for MetRS is following: F < CH 3 CH 2 < OCH 3 < H < Cl < Br, and for LeuRS: OCH 3 < CH 3 CH 2 < F < H < Cl < Br. For a pair of compounds with R 5 = OCH 3 (20,21), the introduction of R 6 = methoxy group instead of Hydrogen leads to complete loss, even activation of both LeuRS and MetRS. Moreover, this substitution causes a complete loss of antibacterial activity toward M. tuberculosis resistant strain. The nature of the R 7 substituent for compounds 26-29 has a similar effect for both enzymes: Cl < CH 3 < OCH 3 Fig. 1a and Fig. 1b respectively. Despite compound 27 has better inhibitory activity toward MetRS, than toward LeuRS, binding modes for both enzymes are similar: 2-methoxy-5-chloro-phenyl interacts with the adenine-binding region and 4-fluoro phenyl ring binds to the amino acid binding pocket (Fig. 1).
We have calculated free energy binding (ΔG) for this compound with aminoacyl adenylate binding pocket of mycobacterial MetRS and LeuRS using umbrella sampling algorithm and Weighted Histogram Analysis Method (WHAM). ΔGb has been determined from the PMF curve as the difference between the PMF with the ligandbound minus the ligand when it is unbound (Fig. 2).
According to umbrella sampling calculations, the free energy binding of compound 27 with MetRS is-2 kcal/ mol, while for LeuRS it was impossible to build the PMF curve. The profiles of PMF curves for MetRS and LeuRS are available as Supplementary Notes. It seems that the umbrella sampling algorithm is more sensitive in silico  www.nature.com/scientificreports/ method to predict the binding affinity of ligands with receptors than docking and can be a useful approach for structure-based optimization of compounds within one chemical class.

Materials and methods
Molecular docking. Molecular docking of the ligands into the aminoacyl-adenylate binding sites of MetRS crystal structure (PDB ID: 6AX8) and LeuRS homology model, which was obtained by us earlier 5 , was performed with DOCK 4.0 program [25][26][27][28] . The water and ligand molecules were deleted from MetRS PDB-file. Ligand geometry was evaluated in the YFF force field 29 . Partial atomic charges for compounds were set using Kirchhoff method 30 . Docking of the ligands into MetRS and LeuRS active sites was carried out using previously described parameters 21 . The complexes of ligands with MetRS and LeuRS active sites were visually analyzed by Discovery Studio Visualizer 31 .
Free energy calculation by umbrella sampling. Molecular dynamics simulations were carried out with GROMACS v.4.5 [32][33][34] . The umbrella sampling algorithm and Weighted Histogram Analysis Method (WHAM) 35 were used to calculate the free energy profile for the separation of MetRS-inhibitor and LeuRS-inhibitor complexes. The starting coordinates used for simulations were from docking complexes. The topology file for ligand was generated using the web-site Automated Topology Builder (ATB) 36 . Topology files for aminoacyl-tRNA synthetases have been generated from PDB-files using pdb2gmx command. The system was set up using the Gromos96 53a6 force field and solvated with the SPC water model.
The center of mass of the receptor-ligand complex has been placed at (4.0, 4.0, 4.0) in a box of dimensions 12 × 12 × 12 using editconf command. Then, the system was solvated with genbox command and neutralized using Na + or Clions according to the charge of the system using genion command. The periodic boundary conditions and the particle mesh Ewald method were used with a nonbonded cutoff of 9 Å. Each system was first energy minimized using 5000 steps of steepest descent method followed by NPT equilibration for 100 ps. Using the make_ndx command we have defined a custom index group for pulling simulation. The pulling of ligands for each system has been performed in Y-dimension using a force constant of 1000 kJ/(mol nm 2 ). A series of configurations along the Y-axis has been generated corresponding to each of the frames saved in the continuous pulling simulation. To measure the distance between protein and ligand on all of these frames, we have used Perl script to iteratively call the g_dist command. The total path with a length of 4.5 nm was divided into 0.1 nm wide equidistant windows. Each coordinate file that is required to obtain 0.1-nm spacing has been prepared for umbrella sampling simulations. At first, NPT equilibration in each window was performed. Then, each input file was passed to the umbrella sampling simulation. Then, using WHAM we have extracted the potential of mean force (PMF), which yields the ΔG for the binding/unbinding process.
The Growth inhibition determination against M. tuberculosis H37Rv and clinically relevant resistant strains. Percent growth inhibition of compounds was determined against M. tuberculosis strains using detection of reduction in resazurin (calorimetric growth indicator) compared to untreated growth controls. MIC was determined using optical density (OD) and a calorimetric growth indicator. Compound concentration cutoff values for all test compounds that showed > 80% growth reduction when compared to untreated growth control.
Antibacterial activity of compounds was determined in Middlebrook 7H9 complete medium which was prepared by the following procedure: 4.7 g of 7H9 powder (Difco; Becton Dickinson) was supplemented with 2 mL glycerol (Fisher) and with Milli-Q-water to the final volume 900 mL and mixed until dissolved. To this volume 100 ml of ADC (5 g bovine serum albumin (BSA) (Sigma), 2 g dextrose (Fisher), and 3 mg catalase (Sigma) dissolved in Milli-Q-water to final volume 100 mL) was added. Then, 10  Compounds were dissolved in 100% DMSO. Three drug concentrations were prepared -20 mg/L, 2 mg/L, and 0.2 mg/L. Assay plates (96-well clear round-bottom plates) were prepared by adding 2 µL of the compound solution to each well at a specific concentration leaving two empty wells untreated growth controls. 100 µL of inoculum was added to all wells. Sealed assay plates were incubated at 37 ºC. On day 7, 10 µL Alamar Blue dye was added to each analytical well. Plates were incubated for 3 days and OD readings were taken at 570 nm and 600 nm. The following formula was used to determine % growth inhibition: Cytotoxicity assay. Compound cytotoxicity was determined by standard MTT assay 37 using human embryonic kidney 293 (HEK293) and human hepatocellular carcinoma (HepG2) cell lines according to the method described previously 38 . HEK 293 cell line was obtained from the Russian Cell Culture Collection (Institute of Cytology of the Russian Academy of Science, St.Petersburg, Russia) and HepG2 -from the Bank of Cell Lines from human and animal tissue (R. E. Kavetsky Institute of Experimental Pathology, Oncology and Radiobiology, NAS of Ukraine, Kyiv, Ukraine).