Antituberculosis Screening Compound Libraries

Tuberculosis (TB) is an infectious disease that has persistently accompanied humanity since its inception and throughout humankind’s evolution [1-2]. Tuberculosis remains in the top 10 causes of death worldwide, and its treatment is a long process with many side effects. Therefore, the discovery and development of new effective drugs against tuberculosis that open up novel biochemical pathways and treat drug-resistant forms 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 advanced medicines that target unique proteins of Mycobacterium tuberculosis, which participate in the most fundamental processes within mycobacteria, retaining maximum conservatism due to the fact that they have 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 11,800 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 (7,600 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

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 upon targeting organism and single protein targets within the Antituberculosis Focused Library.

Representative compounds from the Antituberculosis-focused Screening Set

F6395-1845

F6660-0772

F3360-0001

F6559-1641

F5577-0010

F0612-0802

F0526-1426

F0612-0096

F6064-1766

F0161-0047

F6660-0774

F2701-0571

Antituberculosis Docking Library by Structure-based Approaches

The Screening Set contains 7,600 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)
• Phosphoserine phosphatase (SerB2)
• Phosphoserine aminotransferase

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, and gallic acid formazans, which inhibit InhA by forming a covalent adduct with the NAD cofactor [5, 6]. The INH, the most prescribed antituberculosis drug, requires it 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 by means of a receptor-based approach, this Library comprises potential inhibitors of the InhA enzyme, an M. tuberculosis-specificprotein responsible for bacteria cell wall synthesis that is not present in mammals.

The structure of the 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 set of drug-like compounds was screened by molecular docking using the Glide program (Schrödinger software). The 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 to evaluate the docking procedure [4, 5]. The presence of the NAD⁺ coenzyme as the factor involved in ligand binding was considered in virtual screening.

Based on the docking results, around 4,200 potential antituberculosis agents capable of binding with the InhA protein have been obtained (Fig.2). The 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: QikProp properties and descriptors
• 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)

The 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 no need to bind to a cofactor [10]. In addition to M. tuberculosis, it is commonly found in other 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. 3). 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: QikProp properties and descriptors
• 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)

The 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 attractive features of this enzyme, which make it an excellent tuberculosis drug target, are its prominent metabolic turnover and 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 formally been tested in clinical trials [16].

As a result of our research work, a set of about 650 potential antituberculosis agents capable of binding with DprE protein was obtained based on the docking data (Fig. 4). 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: QikProp properties and descriptors
• Number of compounds selected: 649

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

Phosphoserine aminotransferase (SerC)

The Phosphoserine aminotransferase (SerC) is an important enzyme in the biosynthesis of serine, an essential component of proteins and other biomolecules. In Mycobacterium tuberculosis (Mtb), SerC plays a crucial role in virulence and survival in host cells. Therefore, it is not surprising that this enzyme affects Mtb pathogenesis and overall cell survival [17]. Its inhibitors can lead to a decrease in serine biosynthesis or even the death of the bacterial cell [18]. Thus, the development of new inhibitors of this enzyme is a promising approach as a new therapeutic strategy against tuberculosis.

Proceeding from this, we designed our docking set, having analyzed the phosphoserine aminotransferase structures 3VOM and 2FYF, which correspond to the sequence of P9WQ73. The structures of 3VOM and 2FYF consist of 376 amino acid residues and the N-terminal end of 22 amino acids not included in the models of 3VOM and 2FYF. The Alpha Fold model has a very high quality (> 90 %), with only the first five amino acids demonstrating their quality as being less than 70 %. Since the binding site is located close to the N-terminal part of the protein, it was decided to use the Alpha Fold model, including the N-terminal fragment of the sequence, which is absent in the structures of PDB. In addition, the Alpha Fold model had better statistical data and sites on the sitemap (Fig. 5).

Key features:

• Method: glide ligand docking (standard precision)
• Docked in the site2 of AlphaFold model
• Constraints: no
• Filters used: QikProp properties and descriptors
• Number of compounds selected: 1,330

Figure 6. Spatial structure binding site of the complex of SerC with the lead docking molecule F0520-3446.

Phosphoserine phosphatase (SerB2)

SerB2 phosphoserine phosphatase is one of the key enzymes of the HAD family of Mycobacterium tuberculosis (Mtb). SerB2 contains two domains: the PSP domain, which is characteristic of many phosphoserine phosphatases, and the ACT domain, as well as cofactors in the form of divalent metals [19]. SerB2 catalyzes the dephosphorylation of O-phospho-L-serine to L-serine [20]. This process is critical for the growth and survival of the bacterial cell, as L-serine is an essential amino acid. Since this function is fundamental to cell life, it is obvious that the enzyme in question is involved in a number of cardinal processes, such as cell wall synthesis and modulation of the host immune response [21]. The Mtb cell wall plays an important role in the pathogenesis of the bacterium and its ability to evade the host immune response, making SerB2 an important therapeutic target, as changes in the composition of the bacterial cell wall directly affect virulence and drug resistance [22].

Since there are no experimentally confirmed crystal structures for SerB (UniProt ID: O53289), the spatial structure model of the protein was reconstructed de novo using the AlphaFold and I-Tasser. The binding sites on SerB surface were predicted (Fig. 7-8). The selected site includes the key amino acid residues for binding [23] and has the best score.

Figure 7. The AlphaFold model sitemap.

Key features:

• Method: glide ligand docking (standard precision)
• Docked in the site4 of AlphaFold model and I-Tasser model
• Constraints: no
• Filters used: QikProp properties and descriptors
• Number of compounds selected: 1,313

Figure 8. Spatial structure binding site of the complex of SerB with the lead docking molecule F3406-6059

References

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