|Year : 2018 | Volume
| Issue : 1 | Page : 31-38
Identification and development of oxoquinazoline derivatives as novel mycobacterial inhibitors targeting cell wall synthesis enzyme
Prasanthi Malapati, Krishna Siva Vagolu, Dhramarajan Sriram
Department Pharmacy, Birla Institute of Technology and Science, Hyderabad, Telangana, India
|Date of Web Publication||5-Mar-2018|
Prof. Dhramarajan Sriram
Department of Pharmacy, Birla Institute of Technology and Science, Pilani - Hyderabad Campus, Shameerpet, Jawahar Nagar, Ranga Reddy, Hyderabad - 500 078, Telangana
Source of Support: None, Conflict of Interest: None
Background: Tuberculosis (TB) still remains the leading cause of death worldwide and was unanswered till date. Available treatment strategies have many drawbacks such as longer treatment period, side effects, and drug interactions, which result in patient noncompliance. In the present work, we thrived to develop inhibitors against unexplored key target glutamate racemase. Methods: Lead was identified from in-house database using differential scanning fluorimetry, inhibitors were developed by lead derivatization technique and evaluated them by various biological assays. Results: In oxoquinazoline series, compounds 18 (10.1 ± 0.62 μM) and 22 (5.23 ± 0.34 μM) were found to be the most promising potent inhibitors among all. These compounds also showed their inhibition on replicating and nonreplicating bacteria. Conclusion: Our attempt to develop the potent novel inhibitors against Mycobacterium tuberculosis resulted in developing few promising inhibitors, yet these compounds need further studies to answer all questions in drug discovery. Further optimization of compounds can result in still better compounds for treating TB.
Keywords: Biofilm, nutrient starvation model, oxoquinazoline, tuberculosis
|How to cite this article:|
Malapati P, Vagolu KS, Sriram D. Identification and development of oxoquinazoline derivatives as novel mycobacterial inhibitors targeting cell wall synthesis enzyme. Biomed Biotechnol Res J 2018;2:31-8
|How to cite this URL:|
Malapati P, Vagolu KS, Sriram D. Identification and development of oxoquinazoline derivatives as novel mycobacterial inhibitors targeting cell wall synthesis enzyme. Biomed Biotechnol Res J [serial online] 2018 [cited 2021 Oct 18];2:31-8. Available from: https://www.bmbtrj.org/text.asp?2018/2/1/31/226572
| Introduction|| |
Tuberculosis (TB) is a well-known air-borne deadly disease in the world. It is caused by Mycobacterium tuberculosis (Mtb). Each year, millions of new cases and death have been reporting all over the globe due to this disease. The potent drugs and available treatment regimen were not successful in eradicating the disease. Owing to the development of drug tolerance, there is need for exploring substitution targets against Mtb. The complex lipid cell wall of Mtb is responsible for its virulence nature. Majority drugs reported act on cell wall target formation of outer layers. Formation of monolayer of peptidoglycan (which occurs inside in cytosol) was least explored in the current research. Glutamate racemase (GR) is an important racemization enzyme for conversion of L-glutamate (L-glu) to D-glutamate (D-glu). D-glu is very important for the cell wall formation in most of the bacterial species; this shows the essentiality of the enzyme for bacterial survival. Inhibitors against GR in other bacteria such as 8-benzyl pteridine-6,7-diones and pyridodiazepine amines acting against Staphylococcus aureus and Helicobacter pylori, respectively, were reported.,
In this study, we have developed the novel inhibitors against GR using design and synthetic logics, followed by thorough biological validation. Our design part of inhibitors started with employing the popular thermal shift assay as an inhibitor screening tool. Although its major application is in characterizing stability of macromolecules, it became prominent as a screening technique owing to its advantages over others. Our in-house database containing diverse-structured compounds were screened using this technique.
| Methods|| |
Thermal shift assay
Stability studies were performed in duplicates through thermal shift assay/differential scanning fluorimetry (DSF) using real-time polymerase chain reaction (PCR) thermal cycler (Bio-Rad). The reaction mixture containing GR, D-glu, test compound, SYPRO orange dye, and buffer was set for gradient heating from 20°C to 80°C. Changes in dye fluorescence determine the unfolding/stability pattern of protein. Tm values were obtained from the minima of the first derivative (−dF/dt) plots of unfolding protein plots using an inbuilt function in Bio-Rad prime PCR software.
All the commercially obtained reagents were used directly. For all the compounds, H and 13 C NMR spectra were recorded on a Bruker AM-400 NMR spectrometer, Bruker BioSpin Corp., Germany. Tetramethylsilane is the internal standard. Temperatures are reported in degree Celsius and are uncorrected. Compounds were analyzed for C, H, and N, and analytical results obtained were within ±0.4% of the calculated values for the formula shown. Molecular weights of the synthesized compounds were checked by (Shimadzu, LCMS-2020) ESI-MS method [Supplementary].[Additional file 1]
In vitro glutamate racemase activity assay
The recombinant genes coding for GR in Mtb H37Rv and Bacillus subtilis subsp. subtilis 168 were PCR amplified with N-terminal histidine tags using expression plasmid and vector pET28a+ and E. coli BL21 (DE3) cells, respectively. Ni ± NTA metal-affinity column (His-bind resin, Novagen) was used for the purification of enzyme GR. Purity of protein was established using SDS-PAGE., The protein was stored in aliquots at −80°C, and single time thawed protein was used for activity studies. A reaction mixture of 100 μl consisting of 100 mM Tris–HCl pH 8.0, 5 mM NAD+, 1 mM D-glu, 10 U/mL L-glu dehydrogenase (chemicals were procured from Sigma), and enzyme concentration of 1 μM Bsb GR at different concentrations (0.5 μM to 50 μM) of compounds was monitored using a microplate spectrophotometer (Spectromax M4, Molecular Devices). Half maximal inhibitory concentration (IC50) was determined by measuring the formation of NADH at 340 nm.
Molecular docking simulations
Computer-aided studies on D-glu bound GR of Mtb (PDB ID: 5HJ7) and Bsb (PDB ID: 1ZUW) were performed using Schrodinger software. Sitemap module was utilized to generate possible drug-binding sites. The protein was prepared; optimization and grid generation were done using modules Protein Preparation Wizard, OPLS_2005 force field and Glide grid, respectively. The ligands were energy minimized using Ligprep module. Docking studies were performed using Glide XP module docking calculations. Molecular dynamics (MD) simulations for a timeframe of 10 ns were run in Desmond using OPLS_2005 force field to study the stability pattern of macromolecule in solvent system environment. Defining simulation parameters and calculation of RMSD and RMSF plots were carried as reported previously.,
Mycobacterium tuberculosis susceptibility assay
Synthesized molecules were screened against replicating Mtb H37Rv bacteria using microplate Alamar blue assay method (MABA).Mtb culture having A590 1.0 in Middlebrook 7H9 broth (MB) with 10% oleic acid-dextrose-catalase (OADC) (Himedia) was diluted in ratio of 1:20 from which 100 μl cultures were used as inoculums for assay. Each thawed test compound solution was diluted in 100 μl of MB serially in sterile 96-well microtiter plates. Positive and negative controls were plated along with standards rifampicin (RIF), ethambutol, and isoniazid (INH). The plates were incubated at 37°C for about 1 week followed by addition of 30 μl Alamar blue solution to each well and incubation for 12 h. Viability is indicated by a color change. The lowest concentration of each inhibitor that prevented the color change was noted as its minimum inhibitory concentration (MIC). Inhibitor susceptibility testing in the presence of pump inhibitors verapamil and piperine was done with concentrations 50 μg/mL and 8 μg/mL, respectively, using as above described.
Nutrient starvation assay
Mtb culture starved to attain dormancy in phosphate buffered saline (PBS) for 6 weeks. Later, bacteria were tested with test and standard compounds and incubated for 7 days at a concentration of 10 μg/mL. After incubation period, cell suspensions were diluted by 10-fold using MB with 10% OADC; further, 100 μl of each dilution was plated in sterile 48-well plate containing 450 μl of MB in triplicates. The plates were kept for incubation at 37°C for 4 weeks. Bacterial count by the most probable number (MPN) values was calculated using the standard statistical methods.
Kill kinetics assay
Mtb culture with A590 0.6–1.0 was centrifuged and obtained cell pellet was diluted with 10% PBS until the A590 reaches 0.1, followed by incubation for 2 weeks. Tubes were labeled as control (dimethyl sulfoxide [DMSO]) and variable concentrations of inhibitor (5, 10, and 20 μM). To each tube, 5 mL of PBS and 50 μl of culture were added. To test drug tubes, 100 μl of stock solution (50x) of compound was added to attain desired final concentration. To the control tube, 100 μl DMSO was added. The contents in tubes were mixed and incubated at 37°C. The treated cell suspensions were plated for testing activity at 0, 7, 14, and 21 day intervals for each test drug concentration. The bacterial count was calculated using standard statistical methods employing MPN assay.
Mtb culture with A590 between 0.7 and 1.0 was incubated in Sautons media (Himedia) for 5 weeks at 37°C to develop biofilm. To the matured biofilm, test compounds were added at desired concentration and swirled and sealed to incubate for a week. After incubation period, Tween-80 (0.1% v/v) was added and the contents of each well were centrifuged and pellet was washed with5 mL of wash buffer (PBS with 10% glycerol and 0.05% Tween-80). Finally, the cell pellet was suspended in 5 mL of wash buffer and kept on rocking at room temperature for 12 h. The perseverance of bacteria in the biofilm population was determined by comparing standard treated plates with positive control plates using MPN assay.
The cell toxicity testing was performed for all the synthesized compounds on mouse macrophage cell line (RAW 264.7). In a sterile 96-well microtiter plate, each well was added with 5 × 103 cells and incubated at different test drug concentrations for 48 h at 37°C. After the incubation period was completed, 10 μl of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT reagent) (10 mg/mL) was added and incubated for 3 h more. Later, media were removed and 200 μl of DMSO was added to each well. The reading at A560 was recorded using Perkin Elmer Victor X3 microplate reader against the blank. Whole assay was performed in triplicates. The cytotoxicity was represented as % inhibition at each particular concentration for each test drug.
| Results|| |
Identification of lead
Virtual screening flow of our in-house database with chemically diverse structures using DSF and enzyme assay to identification of lead 1 inhibiting Mtb GR is depicted in [Figure 1].
|Figure 1: (a) Differential scanning fluorimetry plots of a Mycobacterium tuberculosis glutamate racemase native, with D-glutamate and lead 1. (b) The plot of derivative fluorescent-based signal against temperature; Tm and ΔTm (shift) can be measured from the minimum of the plot. (c) Structure of identified lead 1 with Tm and IC50 value in Bsb|
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Chemical synthesis and characterization
The target molecules are synthesized using following procedure depicted in [Scheme 1],[Scheme 2],[Scheme 3].
Identification and characterization of inhibitors using enzyme inhibitory assay, molecular docking, and protein thermal stability assay
All the synthesized compounds were tested for their inhibition by enzyme assay and there after performed molecular docking and simulation studies followed by mode of inhibition characterization through DSF [Table 1] and [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6], [Figure 7].
|Figure 2: Log dose–response curve of compound 22 with different log concentrations (X-axis) versus percentage inhibition (Y-axis)|
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|Figure 3: Docking pose of active molecule 18 in site 1 of Mycobacterium tuberculosis (5HJ7) and Bsb (1ZUW) in superimposed view. Yellow color represents protein and ligand in 5HJ7 whereas blue represents 1ZUW|
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|Figure 4: Binding pose and interaction pattern of compound 18 in (a) Mycobacterium tuberculosis and (b) Bsb|
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|Figure 5: RMSD plots of glutamate racemase inbound state with compounds 18 as a function of time in Mycobacterium tuberculosis (a) and (b) Bsb|
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|Figure 6: RMSF plots of glutamate racemase inbound state with compounds 18 as a function of time in Mycobacterium tuberculosis (a) and (b) Bsb|
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|Figure 7: Thermal stability curves of Mycobacterium tuberculosis glutamate racemase depicting the noncompetitive inhibition by compound 22|
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Nutrient starved model
Considering the IC50 and MIC values, compounds 18 and 22 were selected to determine their inhibitory activity on nonreplicating Mtb [Figure 8].
|Figure 8: Activity profile of compounds in the nutrient starvation model. Bacterial count (mean ± standard error, n = 3) for control and treated groups was estimated through most probable number assay|
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Kill kinetics study
It is essential to analyze the kill of drug molecule at different concentrations against time. Compound 22 was selected for kill kinetic study [Figure 9].
|Figure 9: Kill kinetic curve of compound 22 depicting bacteriostatic inhibition|
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Compounds were tested for inhibition on persistent bacterial biofilms [Figure 10].
|Figure 10: Comparative biofilm inhibitory activity plots of compounds 18 and 22 against Mtb along with standards. Bacterial count estimation (Mean ± S.D., n = 4) for control and treated groups was conducted by using the MPN assay|
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| Discussion|| |
In this study, we did a medium throughput screening using DSF and enzyme assays. A comparative study of melt temperature (Tm) revealed that unbound protein showed Tm~43.1°C, whereas in-bound state with D-glu showed Tm~44.3°C approximately. Database compounds showed a broad range of Tm. Among all the library compounds, lead 1 showed a significant ΔTm. When assay performed in the presence of D-glu, lead 1 showed Tm~46.6°C inferring that protein is more stable in the presence of lead 1 than with D-glu and rest of the library as well. Recent reports have stated that it is not possible to obtain catalytically active form of protein in solution state. Therefore, we have considered performing activity assay on GR of Bsb having 40% and 56% of respective sequence identity and similarity with Mtb and carried out the enzyme inhibitory assay, following the recent reports. Lead 1 also showed good inhibitory activity on GR of Bsb with an IC50 of 21.45 ± 0.58 μM [Figure 1]. Hence, we have considered lead 1 for further optimization and characterization through synthetic strategies [Supplementary].
We have performed the inhibitory enzyme assay for all the synthesized compounds using GR of Bsb. The inhibitory activity results of compounds are listed in [Table 1]. Among all, four compounds have shown inhibitory activity around 10 μM which is better than lead 1 molecule (IC50 21.45 ± 0.58 μM). Compound 22 has shown an IC50 of 5.23 ± 0.76 μM which is five times more potent than lead 1 [Figure 2].
We performed docking and MD of molecules in the crystal structures of Mtb and Bsb GR in complex with D-glu (PDB ID: 5HJ7, 1ZUW, respectively). The inhibitors were not able to dock their active sites; hence, we have tried docking in allosteric sites generated using sitemap. Based on site scores, site 1 in both the proteins was selected based on site scores for further docking studies. Here, we discuss the binding and interaction pattern of active compound 18; the superimposition of both proteins and compound 18 in its binding mode are shown in [Figure 3]. The 2D-binding orientation of compound 18 within two proteins is represented in [Figure 4]. The bound conformation of the compound 18 showed interactions with the side chain of the Glu153 in both proteins. The compound 18 was fit into the allosteric sites of the proteins with docking scores of 2.598 kcal/mol and −4.245 kcal/mol in 5HJ7 and 1ZUW, respectively. This shows the confirmation of inhibitory activity in both the organisms. Compound 18 in protein complexes was subjected to a 10 ns simulation study. The RMSD plots for the protein–ligand complexes have been carried out to measure the distance between atoms 10 ns time frame [Figure 5]. RMSD for 1ZUW, both Cα̨ and ligand was within the average of ~1.5Š and ~0.9Š, and RMSD for 5HJ7 both Cα̨ and ligand was within the average of ~1.5Š and ~0.8Š, respectively. The RMSF analysis shows the fluctuation range undergone by every residue in the protein during simulation. RMSF plot for compound 18 in both protein complexes is shown in [Figure 6]. The complex shows the least fluctuations with an average RMSF value of ~1.3Š and 1.2Š, respectively, for 5HJ7 and 1ZUW.
Mode of inhibition was determined using DSF. The thermal shift analysis of active molecule 18 indicated a noncompetitive mode of inhibition with a shift in ΔTm by ~1.5°C with unbound protein, and when tested along with protein–substrate complex, there was a shift by ~5.6°C with unbound protein [Figure 7]. This indicates that the protein and protein–substrate complex were more stabilized by compound 17 compared to lead 1.
Synthesized and standard drugs were tested for determining MIC using MABA with drug concentrations from 50 μg/mL to 0.78 μg/mL [Table 1]. None of the compounds has shown inhibition against whole cell bacteria. Assuming efflux of the drug by bacteria as the major reason for their inactivity, compounds 18, 22, 29, and 32 active in enzyme assay were tested for susceptibility in the presence of efflux pump inhibitors such as verapamil and piperine. The compounds 18 and 22 have shown improvement in activity in the presence of verapamil and no change with piperine [Table 1]. In nutrient starvation model, starved culture was treated with the compounds 18 and 22 and with standards INH, RIF, and moxifloxacin (MOXI). INH, RIF, and MOXI have shown an inhibition of 1, 1.8, and 2.2 log reduction, respectively, compared with control [Figure 8]. Test compounds 18 and 22 have shown a log reduction of 1.7 and 1.6, respectively. This indicates that test compounds were showing better activities than INH on dormant culture.
We have evaluated compounds for determining their kill kinetics at different concentrations against bacteria at 0, 7, 14, and 21 days after drug treatment. Compounds 18 and 22 showing good inhibition in above study on nutrient starved culture have shown minimum bacterial concentration more than fourfold of their MIC values, indicating that they are bacteriostatic. The kinetic graph of compound 22 is shown in [Figure 9].
Compounds 18 and 22 were evaluated for their inhibitory potency on persistent biofilm-forming bacteria. Compounds 18 and 22 and MOXI have shown a log reduction of 1, 1.2, and 1.8, respectively, compared to control. Test compounds 18 and 22 have shown log reduction of 0.5 and 2, respectively, as shown in [Figure 10]. All the compounds were tested at a concentration of 25 μM, and the percentage inhibition was found to be in varied ranges. However, most of the compounds were not toxic at 25 μM [Table 1].
| Conclusion|| |
We were able to identify efficient compounds against both replicating and nonreplicating stages of Mtb. Compounds 18 and 22 results show that they are equipotent to standards against active bacteria; in addition, they are potent against persistent bacteria. As the urge for new antitubercular drugs is increasing day by day, the present class of drugs would be a suitable class for further drug development studies.
Prasanthi Malapati and Vagolu Siva Krishna are thankful to Department of Science and Technology, Government of India, for the Inspire fellowship. Dharmarajan Sriram is thankful to Department of Biotechnology, Government of India, for the Tata innovation fellowship (BT/HRD/35/01/04/2015).
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
| References|| |
Israyilova A, Buroni S, Forneris F, Scoffone VC, Shixaliyev NQ, Riccardi G, et al.
Biochemical characterization of glutamate racemase-A new candidate drug target against Burkholderia cenocepacia
infections. PLoS One 2016;11:e0167350.
Geng B, Basarab G, Comita-Prevoir J, Gowravaram M, Hill P, Kiely A, et al.
Potent and selective inhibitors of Helicobacter pylori
glutamate racemase (MurI): Pyridodiazepine amines. Bioorg Med Chem Lett 2009;19:930-6.
Ciulli A. Biophysical screening for the discovery of small-molecule ligands. Methods Mol Biol 2013;1008:357-88.
Rudolf AF, Skovgaard T, Knapp S, Jensen LJ, Berthelsen J. A comparison of protein kinases inhibitor screening methods using both enzymatic activity and binding affinity determination. PLoS One 2014;9:e98800.
Poen S, Nakatani Y, Opel-Reading HK, Lassé M, Dobson RC, Kraus KL. Glutamate racemase is the primary target of β-chloro-D-alanine in Mycobacterium tuberculosis.
Biochem J 2016;473:1267-80.
Prosser GA, Rodenburg A, Khoury H, de Chiara C, Howell S, Snijders AP, et al
. Exploring the structure of glutamate racemase from Mycobacterium tuberculosis
as a template for anti-mycobacterial drug discovery. Antimicrob Agents Chemother 2016;606:6091-9.
Schrödinger Suite 2012 Protein Preparation Wizard. Epik, v2.2, Impact, v5.7, Prime, v2.3. New York, NY: Schrödinger, LLC; 2012.
Kräutler V, van Gunsteren WF, Hünenberger PH. A fast SHAKE algorithm to solve distance constraint equations for small molecules in molecular dynamics simulations. J Comput Chem 2001;22:501-8.
Suryadevara P, Yogeeswari P, Soni V, Devi PB, Nandicoori VK, Sriram D, et al.
Computational sampling and simulation based assessment of novel Mycobacterium tuberculosis
glutamine synthetase inhibitors: Study involving structure based drug design and free energy perturbation. Curr Top Med Chem 2016;16:978-95.
Reck F, Alm R, Brassil P, Newman J, Dejonge B, Eyermann CJ, et al.
Novel N-linked aminopiperidine inhibitors of bacterial topoisomerase type II: Broad-spectrum antibacterial agents with reduced hERG activity. J Med Chem 2011;54:7834-47.
Alibert S, Pages JM. Efflux pump inhibitors in bacteria. Expert Opin TherPat2007;17:883-8.
Betts JC, Lukey PT, Robb LC, McAdam RA, Duncan K. Evaluation of a nutrient starvation model of Mycobacterium tuberculosis
persistence by gene and protein expression profiling. Mol Microbiol 2002;43:717-31.
Kulka K, Hatfull G, Ojha AK. Growth of Mycobacterium tuberculosis
biofilms. J Vis Exp 2012.pii: 3820.
Cronan MR, Tobin DM. Fit for consumption: Zebrafish as a model for tuberculosis. Dis Model Mech 2014;7:777-84.
Reck F, Alm RA, Brassil P, Newman JV, Ciaccio P, McNulty J, et al.
Novel N-linked aminopiperidine inhibitors of bacterial topoisomerase type II with reduced pK(a): Antibacterial agents with an improved safety profile. J Med Chem 2012;55:6916-33.
Charifson PS, Grillot AL, Grossman TH, Parsons JD, Badia M, Bellon S, et al.
Novel dual-targeting benzimidazole urea inhibitors of DNA gyrase and topoisomerase IV possessing potent antibacterial activity: Intelligent design and evolution through the judicious use of structure-guided design and structure-activity relationships. J Med Chem 2008;51:5243-63.
Venkatraman J, Bhat J, Solapure SM, Sandesh J, Sarkar D, Aishwarya S, et al.
Screening, identification, and characterization of mechanistically diverse inhibitors of the Mycobacterium tuberculosis
enzyme, pantothenate kinase (CoaA). J Biomol Screen 2012;17:293-302.
[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6], [Figure 7], [Figure 8], [Figure 9], [Figure 10]