Identification of Novel Antimicrobial Compounds Targeting Mycobacterium tuberculosis S-Adenosyl-L-Homocysteine Hydrolase Using Dual Hierarchical In Silico Structure-Based Drug Screening.

The emergence of multidrug-resistant and extensively drug-resistant Mycobacterium tuberculosis (M. tuberculosis) has become a major medical problem. S-adenosyl-L-homocysteine hydrolase (MtSAHH) was selected as the target protein for the identification of novel anti-TB drugs. Dual hierarchical in silico Structure-Based Drug Screening was performed using a 3D compound structure library (with over 150 thousand synthetic chemicals) to identify compounds that bind to MtSAHH's active site. In vitro experiments were conducted to verify whether the nine compounds selected as new drug candidates exhibited growth-inhibitory effects against mycobacteria. Eight of the nine compounds that were predicted by dual hierarchical screening showed growth-inhibitory effects against Mycobacterium smegmatis (M. smegmatis), a model organism for M. tuberculosis. Compound 7 showed the strongest antibacterial activity, with an IC50 value of 30.2 µM. Compound 7 did not inhibit the growth of Gram-negative bacteria or exert toxic effects on human cells. Molecular dynamics simulations of 40 ns using the MtSAHH-Compound 7 complex structure suggested that Compound 7 interacts stably with the MtSAHH active site. These in silico and in vitro results suggested that Compound 7 is a promising lead compound for the development of new anti-TB drugs.

Identification of novel antimicrobial compounds targeting Mycobacterium tuberculosis shikimate kinase using in silico hierarchical structure-based drug screening.

The development of new anti-TB drugs to prevent the spread of multidrug-resistant Mycobacterium tuberculosis (Mtb) strains is imperative. Mtb shikimate kinase (MtSK) was selected as the target protein to screen for new anti-TB drugs. We performed hierarchical in silico screening using a library of 154,118 compounds to search for novel compounds that could bind to the active site of MtSK. The growth-inhibitory effects of the candidate compounds on Mycobacterium smegmatis were evaluated in vitro. Nine of the 11 candidate compounds exhibited inhibitory effects against mycobacteria in vitro. The inhibitory activity of Compound 2 (IC50 = 1.39 µM) was higher than that of isoniazid, the first-line drug for TB treatment. Moreover, Compound 2 did not exhibit toxicity against mammalian cells and Escherichia coli. Molecular dynamics simulations using the MtSK-Compound 2 complex structure in a timeframe of 100 ns suggested that Compound 2 could stably bind to MtSK. The binding free energy of Compound 2 was estimated to be -37.96 kcal/mol using the MM/PBSA method, demonstrating that Compound 2 can stably bind to MtSK. These in silico and in vitro results indicated that Compound 2 is a promising hit compound for the development of novel anti-TB drugs.

Computational Screening and Experimental Validation of Inhibitor Targeting the Complex Formation of Grb14 and Insulin Receptor.

The development of drugs targeting gene products associated with insulin resistance holds the potential to enhance our understanding of type 2 diabetes mellitus (T2DM). The virtual screening, based on a three-dimensional (3D) protein structure, is a potential technique to accelerate the development of molecular target drugs. Among the targets implicated in insulin resistance, the genetic characterization and protein function of Grb14 have been clarified without contradiction. The Grb14 gene displays significant variations in T2DM, and its gene product is known to inhibit the function of the insulin receptor (IR) by directly binding to the tyrosine kinase domain. In the present study, a virtual screening, based on a 3D structure of the IR tyrosine kinase domain (IRß) in complex with part of Grb14, was conducted to find compounds that can disrupt the complex formation between Grb14 and IRß. First, ten compounds were selected from 154,118 compounds via hierarchical in silico structure-based drug screening, composed of grid docking-based and genetic algorithm-based programs. The experimental validations suggested that the one compound can affect the blood glucose level. The molecular dynamics simulations and co-immunoprecipitation analysis showed that the compound did not completely suppress the protein-protein interaction between Grb14 and IR, though competitively bound to IR with the tyrosine kinase pseudosubstrate region in Grb14.

Identification of novel inhibitors for mycobacterial polyketide synthase 13 via in silico drug screening assisted by the parallel compound screening with genetic algorithm-based programs.

Identifying small compounds capable of inhibiting Mycobacterium tuberculosis polyketide synthase 13 (Pks13), in charge of final step of mycolic acid biosynthesis, could lead to the development of a novel antituberculosis drug. This study screened for lead compounds capable of targeting M. tuberculosis Pks13 from a chemical library comprising 154,118 compounds through multiple in silico docking simulations. The parallel compound screening (PCS), conducted via two genetic algorithm-based programs was applied in the screening strategy. Out of seven experimentally validated compounds, four compounds showed inhibitory effects on the growth of the model mycobacteria (Mycobacterium smegmatis). Subsequent docking simulation of analogs of the promising leads with the assistance of PCS resulted in the identification of three additional compounds with potent antimycobacterial effects (compounds A1, A2, and A5). Further, molecular dynamics simulation predicted stable interaction between M. tuberculosis Pks13 active site and compound A2, which showed potent antimycobacterial activity comparable to that of isoniazid. The present study demonstrated the efficacy of in silico structure-based drug screening through PCS in antituberculosis drug discovery.

Computer-assisted screening of mycobacterial growth inhibitors: Exclusion of frequent hitters with the assistance of the multiple target screening method.

Background: The emergence of frequent hitters (FHs) remains a challenge in drug discovery. We have previously used in silico structure-based drug screening (SBDS) to identify antimycobacterial candidates. However, excluding FHs has not been integrated into the SBDS system. Methods: A dataset comprising 15,000 docking score (protein-compound affinity matrix) was constructed by multiple target screening (MTS): DOCK-GOLD two-step docking simulations with 154,118 compounds versus the 30 target proteins essential for mycobacterial survival. After extraction of 141 compounds from the protein-compound affinity matrix, compounds determined to be FHs or false positives were excluded. Antimycobacterial properties of the top nine compounds selected through SBDS were experimentally evaluated. Results: Nine compounds designated KS1-KS9 were selected for experimental evaluation. Among the selected compounds, KS3, identified as adenosylhomocysteinase inhibitor, showed a potent inhibitory effect on antimycobacterial growth (inhibitory concentration [IC]50 = 1.2 M). However, the compound also showed potent cytotoxicity. Conclusion: The MTS method is applicable in SBDS for the identification of enzyme-specific inhibitors.

Structural modification of a novel inhibitor for mycobacterium enoyl-acyl carrier protein reductase assisted by In silico structure-based drug screening.

Background: Mycobacterium tuberculosis enoyl-acyl carrier protein reductase (mtInhA) is involved in the biosynthesis of mycolic acids, a major component of mycobacterial cell walls, and has been targeted in the development of anti-tuberculosis (TB) drugs. In our previous in silico structure-based drug screening study, we identified KES4, a novel class of mtInhA inhibitor. KES4 is composed of four ring structures (A-D-rings) and molecular dynamic simulation predicted that the D-ring is essential for the interaction with mtInhA. Methods: The structure-activity relationship study of the D-ring was attempted and aided by in silico docking simulations to improve the mtInhA inhibitory activity of KES4. A virtual chemical library of the D-ring-modified KES4 was then constructed and subjected to in silico docking simulation against mtInhA using the GOLD program. The candidate compound showing the highest GOLD score, referred to as KEN1, was synthesized, and its biological properties were compared with those of the lead compound KES4. Results: We achieved the synthesis of KEN1 and evaluated its effects on InhA activity, mycobacterial growth, and cytotoxicity. The antimycobacterial activity of KEN1 was comparable to that of the lead compound (KES4), although it exhibited superior activity in mtInhA inhibition. \. Conclusions: We obtained a KES4 derivative with high mtInhA inhibitory activity by in silico docking simulation with a chemical library consisting of a series of D-ring-modified KES4.

In silico structure-based drug screening of novel antimycobacterial pharmacophores by DOCK-GOLD tandem screening.

BACKGROUND: Enzymes responsible for cell wall development in Mycobacterium tuberculosis are considered as potential targets of anti-tuberculosis (TB) agents. Mycobacterial cyclopropane mycolic acid synthase 1 (CmaA1) is essential for mycobacterial survival because of its critical role in synthesizing mycolic acids. MATERIALS AND METHODS: We screened compounds that were capable of interacting with the mycobacterial CmaA1 active site using a virtual compound library with an in silico structure-based drug screening (SBDS). Following the selection of such compounds, their antimycobacterial activity was examined. RESULTS: With the in silico SBDS, for which we also used DOCK-GOLD programs and screening methods that utilized the structural similarity between the selected active compounds, we identified two compounds with potent inhibitory effects on mycobacterial growth. The antimycobacterial effect of the compounds was comparable to that of isoniazid, which is used as a first-line anti-TB drug. CONCLUSION: The compounds identified through SBDS were expected to be a novel class of anti-TB pharmacophores.

Identification of compounds with potential antibacterial activity against Mycobacterium through structure-based drug screening.

To identify novel antibiotics against Mycobacterium tuberculosis, we performed a hierarchical structure-based drug screening (SBDS) targeting the enoyl-acyl carrier protein reductase (InhA) with a compound library of 154,118 chemicals. We then evaluated whether the candidate hit compounds exhibited inhibitory effects on the growth of two model mycobacterial strains: Mycobacterium smegmatis and Mycobacterium vanbaalenii. Two compounds (KE3 and KE4) showed potent inhibitory effects against both model mycobacterial strains. In addition, we rescreened KE4 analogs, which were identified from a compound library of 461,383 chemicals through fingerprint analysis and genetic algorithm-based docking simulations. All of the KE4 analogs (KES1-KES5) exhibited inhibitory effects on the growth of M. smegmatis and/or M. vanbaalenii. Based on the predicted binding modes, we probed the structure-activity relationships of KE4 and its analogs and found a correlative relationship between the IC50 values and the interaction residues/LogP values. The most potent inhibitor, compound KES4, strongly and stably inhibited the long-term growth of the model bacteria and showed higher inhibitory effects (IC50 = 4.8 µM) than isoniazid (IC50 = 5.4 µM), which is a first-line drug for tuberculosis therapy. Moreover, compound KES4 did not exhibit any toxic effects that impede cell growth in several mammalian cell lines and enterobacteria. The structural and experimental information of these novel chemical compounds will likely be useful for the development of new anti-TB drugs. Furthermore, the methodology that was used for the identification of the effective chemical compound is also likely to be effective in the SBDS of other candidate medicinal drugs.

Identification of novel antimycobacterial chemical agents through the in silico multi-conformational structure-based drug screening of a large-scale chemical library.

The increasing prevalence of drug-resistant tuberculosis, which is resistant to effective multiple antibiotic, presents a major global health threat. The thymidine monophosphate kinase (TMPK) of Mycobacterium tuberculosis (M. tuberculosis), which is an essential enzyme for the maintenance of the thymidine triphosphate pools, is considered an attractive target for the development of effective antibiotics against tuberculosis. In this study, we attempted to identify novel chemical compounds that specifically target the M. tuberculosis TMPK (mtTMPK). We performed in silico structure-based drug screening using the crystal structure data of mtTMPK and a large-scale virtual compound library, which is composed of 6,192,930 chemicals. Through a three-step screening method using the DOCK and GOLD, we identified ten chemical compounds that were predicted to have high binding affinity to the active site cleft of the mtTMPK. We then evaluated the antibiotic effects of these chemical compounds on model mycobacteria strains. As a result, we found that a chemical compound, K10, completely inhibited the growth of Mycobacterium vanbaalenii (M. vanbaalenii) and Mycobacterium smegmatis (M. smegmatis). Moreover, K10 does not exhibit any toxic effects on the growth of enterobacteria and mammalian cells. The structural and experimental information regarding this novel chemical compound, K10, is likely to be useful for the hit-to-lead optimization of new antibiotics for the treatment of tuberculosis.

Identification of novel potential antibiotics for tuberculosis by in silico structure-based drug screening.

The enoyl-acyl carrier protein reductase of Mycobacterium tuberculosis (MTB) is a key enzyme of the type II fatty acid synthesis system. It is involved in the production of mycolic acid and is a known target for isoniazid, an effective antibiotic for tuberculosis treatment. The increasing prevalence of tuberculosis in many areas of the world, which is associated with the rise of drug-resistant MTB strains, presents a major global health threat. In this study, we attempted to identify novel antibiotics specifically targeting the MTB enoyl-acyl carrier protein reductase. We performed in silico structure-based drug screening using the crystal structure data for the MTB enoyl-acyl carrier protein reductase (PDB code; 2H7I) and a virtual compound library, which includes 152,102 chemicals. By a two-step screening method using DOCK (first screening) and GOLD (second screening), we identified 5 chemical compounds expected to have high binding affinity to the active center of the MTB enoyl-acyl carrier protein reductase. Moreover, we examined the antibiotic effects of these chemical compounds on model bacterial strains by in vitro experiments. We found that a chemical compound, which has a basic skeleton comprised of dibenzofuran, acetoamide, trizol, furyl and methylphenyl groups, completely inhibited the growth of Mycobacterium vanbaalenii and had no toxic effects on enterobacteria and cultured mammalian cells. Therefore, the chemical compound is likely to be useful in the research and development of new antibiotics for tuberculosis.

Identification of a novel chemical potentiator and inhibitors of UCH-L1 by in silico drug screening.

Ubiquitin-C-terminal hydrolase L1 (UCH-L1) is a de-ubiquitinating enzyme expressed in the brain and reproductive tissues as well as certain cancers. The hydrolase activity of UCH-L1 has been implicated in Alzheimer's disease and cancer invasion; therefore, it may represent a therapeutic target for these diseases. The present study was undertaken to identify novel chemical modulators for the hydrolase activity of UCH-L1. To identify chemicals that bind to the active site of UCH-L1, we carried out in silico structure-based drug screening using human UCH-L1 crystal structure data (PDB ID: 2ETL) and virtual compound libraries containing 26,891 and 304,205 compounds. Among the compounds with the highest binding scores, we identified one that potentiates the hydrolase activity of UCH-L1, and six that inhibit the activity in enzymatic assays. These compounds may be useful for research on UCH-L1 function, and could lead to candidate therapeutics for UCH-L1-associated diseases.

Identification of novel chemical inhibitors for ubiquitin C-terminal hydrolase-L3 by virtual screening.

UCH-L3 (ubiquitin C-terminal hydrolase-L3) is a de-ubiquitinating enzyme that is a component of the ubiquitin-proteasome system and known to be involved in programmed cell death. A previous study of high-throughput drug screening identified an isatin derivative as a UCH-L3 inhibitor. In this study, we attempted to identify a novel inhibitor with a different structural basis. We performed in silico structure-based drug design (SBDD) using human UCH-L3 crystal structure data (PDB code; 1XD3) and the virtual compound library (ChemBridge CNS-Set), which includes 32,799 chemicals. By a two-step virtual screening method using DOCK software (first screening) and GOLD software (second screening), we identified 10 compounds with GOLD scores of over 60. To address whether these compounds exhibit an inhibitory effect on the de-ubiquitinating activity of UCH-L3, we performed an enzymatic assay using ubiquitin-7-amido-4-methylcoumarin (Ub-AMC) as the substrate. As a result, we identified three compounds with similar basic dihydro-pyrrole skeletons as UCH-L3 inhibitors. These novel compounds may be useful for the research of UCH-L3 function, and in drug development for UCH-L3-associated diseases.