Antituberculosis Screening Compound Libraries

Tuberculosis (TB) is an infectious disease that has persistently accompanied humanity since its inception and throughout society's evolution [1-2]. Tuberculosis remains in the top 10 causes of death worldwide, while its treatment is a long process that has many side effects. Therefore, the discovery and development of new effective drugs against tuberculosis that target novel biochemical pathways and treat drug-resistant forms of the disease present an urgent need worldwide.

From the molecular-biological point of view, the most appropriate strategy for structure-based drug design (SBDD) is the identification and development of new medicines that target unique proteins of Mycobacterium tuberculosis, which participate in the most fundamental processes within mycobacteria, retaining maximum conservatism while having no direct homologs in humans and animals.³ This approach improves the chances of overcoming side effects related to the inhibition of similar protein targets of the host.

Life Chemicals has designed two Antituberculosis Screening Libraries of over 9,200 drug-like screening compounds aiming at key protein targets to facilitate high-throughput screening (HTS) programs focused on anti-TB drug discovery research:

• Antituberculosis Focused Library by 2D Similarity (4,200 compounds)
• Antituberculosis Docking Library by Structure-based Approaches (5,000 compounds)

The compound selection can be customized based on your requirements, cherry picking is available.

Please, contact us at orders@lifechemicals.com for any additional information and price quotations.

Antituberculosis Focused Library by 2D Similarity

This Screening Library was developed by a 2D fingerprint similarity search against the reference set of 23,734 biologically active compounds (IC₅₀, K_i, etc., less than 10 μM, Inhibition > 25 %), extracted from the Binding and ChEMBL databases. Therapeutically relevant viral assays data both for the Mycobacterium tuberculosis bacterium and the relevant protein targets, protein families were employed:

Over 4,200 unique structurally diverse small-molecule compounds were selected from the Company’s proprietary HTS Compound Collection by filtering and merging their activity type data (Fig. 1).

Figure 1. Compound distribution targeting organism and single protein targets within the Antituberculosis Library.

Antituberculosis Docking Library by Structure-based Approaches

The Screening Set contains 5,000 structurally-diverse screening molecules picked out by virtual molecular screening against the following tuberculosis-focused drug targets:

• 2-trans-enoyl-acyl carrier protein reductase (InhA)
• dTDP-6-deoxy-d-xylo-4-hexulose 3,5-epimerase (RmlC)
• Decaprenylphosphoribose-2-epimerase (DprE1 and DprE2)

2-trans-enoyl-acyl carrier protein reductase (InhA)

The 2-trans-enoyl-acyl carrier protein reductase (InhA) catalyzes the biosynthesis of one of the main components of the Mycobacterium tuberculosis cell wall – mycolic fatty acid, which is essential to the survival of microorganisms by providing inherent resistance [4]. This NADH-dependent enzyme is the key target for antitubercular agents, such as isoniazid (INH), 4-hydroxy-2-pyridones, gallic acid formazans, which inhibit InhA by forming a covalent adduct with the NAD cofactor [5, 6]. INH, the most prescribed antituberculosis drug, requires to be activated by catalase-peroxidase enzyme KatG, whose point mutations lead to the creation of new multidrug-resistant TB (MDR-TB) strains. Therefore, the drug development has focused on finding more advanced compounds, such as triclosan derivatives [7, 8], that possess significant Mtb InhA inhibitory activity and do not require prior activation by KatG, demonstrating their efficacy both in vitro and in vivo.

Designed via a receptor-based approach, this Library comprises potential inhibitors of InhA enzyme, M. tuberculosis-specificprotein responsible for bacteria cell wall synthesis that is not present in mammals.

The structure of InhA protein and the binding mode of its known inhibitors were studied based on the analysis of crystal structure records in PDB. This information has provided a detailed understanding of the protein-ligand interaction mechanism.

The Life Chemicals HTS Compound Collection was processed according to ADME requirements, and all undesirable chemical groups were filtered out. The resulting drug-like set of compounds was screened by molecular docking using the Glide program (Schrödinger software). 3FNH and 2H7I PDB entries were selected for the docking studies for the most favorable ligand binding and high resolution of the crystal structures. The reference set of active ligands was used for the evaluation of the docking procedure [4, 5]. The presence of the NAD⁺ coenzyme was taken into account in virtual screening as it is involved in ligand binding.

A set of around 4,200 potential antituberculosis agents capable of binding with InhA protein has been obtained based on the docking results (Fig.2). Compounds have been selected by ligand efficacy and predicted binding mode.

Key features:

• Method: high-throughput virtual screening (docking), molecular fitting
• X-Ray data used: 3FNH, 2H7I
• Constraints: no
• Filters used: PAINS, toxic, reactive
• Number of compounds selected: 4160

Figure 2. Spatial structure binding site of the complex of InhA with lead docking molecule F2269-0228

dTDP-6-deoxy-d-xylo-4-hexulose 3,5-epimerase (RmlC)

dTDP-6-deoxy-d-xylo-4-hexulose 3,5-epimerase (RmlC), a saccharide component essential for the virulence of pathogenic bacteria, represents one of the 4 isomerase enzymes (RmlA, RmlB, RmlC, and RmlD) in the deoxythymidine triphosphate (dTDP)-L-rhamnose pathway of Mycobacterium tuberculosis.

RmlC takes part in the intermediate stage by inverting the hydroxyls at the 3’’ and 5’’ positions, and thus creating an unstable flipping ring structure [9]. The dimeric structure, in which each monomer is a beta-sandwich of 2 beta-sheets, distinguishes RmlC from other enzymes. This enzyme was shown to be a promising tuberculosis-related drug target due to its uniqueness, specificity, and lack of need to bind to a cofactor [10]. In addition to M. tuberculosis, this enzyme is commonly found in pathogens such as Salmonella enterica Serovar Typhimurium, Coxiella burnetii, Streptococcus suis, and Prymnesium parvum, some of which may infect both people and animals [11, 12].

A set of around 200 potential antituberculosis agents capable of binding with RmlC protein was obtained based on the docking results (Fig.2). Compounds have been selected by ligand efficacy and predicted binding mode.

Key features:

• Method: high-throughput virtual screening (docking), molecular fitting
• X-Ray data used: 2IXC
• Constraints: no
• Filters used: PAINS, toxic, reactive
• Number of compounds selected: 194

Figure 3. Spatial structure binding site of the complex of RmlC with lead docking molecule F2880-1322

Decaprenylphosphoribose-2-epimerase (DprE1 and DprE2)

Decaprenylphosphoryl-β-D-ribose-2-epimerase (DprE) is a heterodimeric enzyme comprising DprE1 and DprE2 proteins. DprE1 catalyzes the conversion of decaprenyl-phospho-ribose (DPR) into decaprenyl-phospho-arabinose (DPA). DPA is the precursor for the synthesis of arabinogalactan and lipoarabinomannan, structural components of the mycobacterial cell wall [13, 14]. The main benefits of this enzyme, which make it an excellent tuberculosis drug target, are its prominent metabolic turnover and its location in the periplasm, which limits the influence of such factors as the action of efflux pumps or cytoplasmic inactivation mechanisms [15]. However, the most important characteristic of DprE1 is the presence of nucleophiles at its active sites and binding with inhibitors both covalently and non-covalently. So far, four new drugs, namely, BTZ-043, PBTZ-169, OPC-167832, and TBA-7371 (non-covalent) have been formally tested in the clinical trials [16].

A set of about 650 potential antituberculosis agents capable of binding with DprE protein was obtained based on the docking results (Fig. 2). Compounds have been selected by ligand efficacy and predicted binding mode.

Key features:

• Method: high-throughput virtual screening (docking), molecular fitting
• X-Ray data used: 4P8C, 4NCR, 4P8N
• Constraints: no
• Filters used: PAINS, toxic, reactive
• Number of compounds selected: 649

Figure 4. Spatial structure binding site of the complex of DprE1 with lead docking molecule F0913-3597

References

1. https://www.who.int/tb/publications/2019/consolidated-guidelines-drug-resistant-TB-treatment/en/
2. Stephani Joy Y. Macalino, Junie B. Billones, Voltaire G. Organo, Maria Constancia O. Carrillo. In Silico Strategies in Tuberculosis Drug Discovery., Molecules. 2020 Feb; 25(3): 665. Published online.
3. Xiaocui Wu, Jinghui Yang, Guangkun Tan, Haican Liu, Yin Liu, Yinjuan Guo, Rongliang Gao, Baoshan Wan, Fangyou Yu. Drug Resistance Characteristics of Mycobacterium tuberculosis Isolates From Patients With Tuberculosis to 12 Antituberculous Drugs in China., Front Cell Infect Microbiol. 2019; 9: 345. Published online
4. Prasad, M. S., Bhole, R. P., Khedekar, P. B., Chikhale, R. V. Mycobacterium enoyl acyl carrier protein reductase (InhA): A key target for antitubercular drug discovery. Bioorganic Chemistry. 2021;115,105242. doi:10.1016/j.bioorg.2021.105242.
5. de Ávila M. B., Bitencourt-Ferreira G., de Azevedo W. F. Structural Basis for Inhibition of Enoyl-[Acyl Carrier Protein] Reductase (InhA) from Mycobacterium tuberculosis. Curr Med Chem. 2020;27(5):745-759. doi: 10.2174/0929867326666181203125229.
6. Kamsri, P., Hanwarinroj, Ch., Phusi, N. Punkvang Discovery of New and Potent InhA Inhibitors as Anti-tuberculosis Agents: Structure Based Virtual Screening Validated by Biological Assays and X-ray Crystallography. Journal of Chemical Information and Modeling. 2019; doi:10.1021/acs.jcim.9b00918
7. Chetty, S., Armstrong, T., Sharma Kharkwal, S. New InhA Inhibitors Based on Expanded Triclosan and Di-Triclosan Analogues to Develop a New Treatment for Tuberculosis. Pharmaceuticals (Basel). 2021;14;14(4):361. doi: 10.3390/ph14040361.
8. Campaniço, A., Moreira, R., Lopes, F. Drug discovery in tuberculosis. New drug targets and antimycobacterial agents. Eur J Med Chem. 2018;150:525-545. doi: 10.1016/j.ejmech.2018.03.020.
9. Belete TM. Recent Progress in the Development of Novel Mycobacterium Cell Wall Inhibitor to Combat Drug-Resistant Tuberculosis. Microbiology Insights. 2022. doi:10.1177/11786361221099878
10. Sunilkumar, B., Basheera, Sh. Virtual screening for identifying a putative inhibitor of rmlc, a major target protein in tuberculosis disease. International Journal of Pharma and Bio Sciences. 2015. 6. 616-628.
11. Cross, A., Roy S. Spinning sugars in antigen biosynthesis: characterization of the Coxiella burnetii and Streptomyces griseus TDP-sugar epimerases, Journal of Biological Chemistry. 2022. 298. 5. 101903. ISSN 0021-9258. https://doi.org/10.1016/j.jbc.2022.101903.
12. Dong, C., Major L. L., Allen A., Blankenfeldt W. High-resolution structures of RmlC from Streptococcus suis in complex with substrate analogs locate the active site of this class of enzyme. Structure. 2003. 11(6):715-23. doi: 10.1016/s0969-2126(03)00098-4. PMID: 12791259.
13. 1. Imran, M., A S, A., Thabet, H. K., Abida, & Afroz Bakht, M. Synthetic molecules as DprE1 inhibitors: A patent review. Expert opinion on therapeutic patents. 2021;31(8), 759–772. https://doi.org/10.1080/13543776.2021.1902990.
14. 2. Cole Stewart T. 2016 Inhibiting Mycobacterium tuberculosis within and without Phil. Trans. R. Soc. B3712015050620150506. https://doi.org/10.1098/rstb.2015.0506.
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