AN IN SILICO STUDY OF NOVEL FLUOROQUINOLONES AS INHIBITORS OF DNA GYRASE OF STAPHYLOCOCCUS AUREUS

Objective: This study is an attempt to identifying an effective fluoroquinolones (FQ) s against STAPHYLOCOCCUS AUREUS (S. aureus) by in silico analysis of 150 (FQ) compounds using iGemDock v2.1 tool.Methods: Structure of DNA gyrase (2XCT) was retrieved from the Protein Data Bank (PDB) and the structures of (FQ) compounds were selected from literature survey of 400 novel compounds and the physical, chemical and molecular characteristics of each compound were obeyed for drug-relevant properties based on Lipinski's rule of five, then a total of 150 (FQ)s were docked against the protein of the 2XCT enzyme.Results: From this study, it was found that the compound (1) [(3R,7E)-9-fluoro-7-(isonicotinoylhydrazono)-3-methyl-10-(4-methylpiperazin-1-yl)-2,3-dihydro-7H-[1,4]oxazino[2,3,4-ij]quinoline-6-carboxylic acid] and the compound (2) [1-cyclopropyl-6-fluoro-7-{4-[(8-hydroxyquinolin-2-yl)methyl]piperazin-1-yl}-4-oxo-1,4-dihydroquinoline-3-carboxylic acid] showed the best interaction value against 2XCT enzyme, the binding energy was (-104. 58 kcal/mol), (-26. 5kcal/mol) respectively whereas the reference ciprofloxacin (CIP) was (-74. 33 kcal/mol).Conclusion: Further in vitro studies of these compounds against the enzyme will lead a new pathway to drug discovery.Â

Antituberculosis Drug Repurposing: A New Hope for Tackling Multi-Challenging TB in Timely Manner

Tuberculosis still stands as the world’s leading infectious disease as 1/4th of the world’s population harbors Latent TB infection (LTBI) > 10 million develops active TB and ~ 1.5 million people die per year. Approximately 4,65,000 people fell ill with multidrug or rifampicin-resistant tuberculosis (MDR/RR-TB)/year. This deadly TB scenario demands new TB drug regimens to tackle global infection reservoir, and worldwide spread of drug resistance and DS TB. Successful entry of single new drug into market is much complicated mission owing to time, cost, efficacy, and safety issues. Therefore, drug repurposing seems one reliable hope to meet the challenges of modern TB drug discovery timely, as it starts with examining market acclaimed drugs against other diseases for their efficacies against tuberculosis avoiding several lengthy and costly steps required for new molecules. Several drugs have been identified, which show potential for TB treatment. There is need for careful consideration of various trial designs to ensure that TB phase III trials are initiated for fruitful development of new TB treatment regimens. TB drug repurposing will not only give fast track novel drugs but will also serve to identify new targets for future development in cost-effective manner

Discovery of OJT008 as a Novel Inhibitor of Mycobacterium tuberculosis

Despite recent progress in the diagnosis of Tuberculosis (TB), the chemotherapeutic management of TB is still challenging. Mycobacterium tuberculosis (Mtb) is the etiological agent of TB, and TB is classified as the 13th leading cause of death globally [WHO 2021]. 558,000 people were reported to develop multi-drug resistant TB globally [WHO 2020]. Our research focuses on targeting Methionine Aminopeptidase (MetAP), an essential protein for the viability of Mtb. MetAP is a metalloprotease that catalyzes the removal of N-terminal methionine (NME) during translation of protein [Giglione et al., 2003]. This essential role of MetAPs makes this enzyme an auspicious target for the development of novel therapeutic agents for the treatment of TB. Mtb possesses two MetAP1 isoforms: MtMetAP1a and MtMetAP1c, which are vital for Mtb viability, hence a promising chemotherapeutic target for Mtb infection [Zhang et al., 2009; Olaleye et al., 2010; Griffin et al., 2011; Vanunu et al., 2019]. In our study, we cloned, overexpressed recombinant MtMetAP1c, and investigated the in vitro inhibitory effect of OJT008 on cobalt and nickel ion activated MtMetAP1c. The compound’s potency against replicating and multidrug-resistant (MDR) Mtb strains was also investigated. The induction of the overexpressed recombinant MtMetAP1c was optimized at hours with a final concentration of 1mM Isopropyl β-D-1-thiogalactopyranoside. The average yield for MtMetAP1c was 4.65 mg/L of Escherichia coli culture. A preliminary MtMetAP1c metal dependency screen showed optimum activation with nickel and cobalt ions at 100µM. The half-maximal inhibitory concentration (IC50) values of OJT008 against MtMetAP1c activated with CoCl2 and NiCl2 were in the micromolar range. Our in silico study showed OJT008 strongly binds to both metal activated MtMetAP1c, as evidenced by strong molecular interactions and higher binding score thereby corroborating our result. Thus, validating the pharmacophore’s metal specificity. The potency of OJT008 against both active and multidrug-resistant (MDR) Mtb was in the low micromolar concentrations, correlating well with our biochemical data on MtMetAP1c inhibition. These results suggest that OJT008 is a potential lead compound for the pre-clinical development of novel small molecules for the therapeutic management of TB

Identification of selective novel hits against Mycobacterium tuberculosis KasA potential allosteric sites using bioinformatics approaches

Tuberculosis (TB) is a global health threat that has led to approximately 1.5 million deaths annually. According to the World Health Organization (WHO), TB is among the top ten deadly diseases and is the leading cause of death due to a single infectious agent. The main challenge in the effective treatment and control of TB is the ongoing emergence of resistant strains of Mycobacterium tuberculosis (Mtb) which lead to multi-drug resistant (MDR) and extensive-drug resistant (XDR) TB. Hence, the identification and characterization of novel drug targets and drugs that modulate the activity of the pathogen are an urgent priority. The current situation even necessitates the reengineering or repurposing of drugs in order to achieve effective control. The β-ketoacyl-acyl carrier protein synthase I (KasA) of Mycobacterium tuberculosis is an essential enzyme in the mycobacterial fatty acid synthesis (FAS-II) pathway and is believed to be a promising target for drug discovery in TB. It is one of the five main proteins of the FAS-II pathway and catalyzes a key condensation reaction in the synthesis of meromycolate chains, the precursors of mycolic acids involved in cell wall formation. Although this protein has been extensively studied, little research has been devoted to the allosteric inhibition of potential drug compounds. The main aim of this research was to identify the allosteric sites on the protein that could be involved in the inhibition of substrate binding activities and novel drug compounds that bind to these sites by use of in-silico approaches. The bioinformatics approaches used in this study were divided into four main objectives namely identification of KasA homolog sequences, sequence analysis and protein characterization, allosteric site search and lastly virtual screening of DrugBank compounds via molecular docking. Fifteen homolog sequences were identified from the BLASTP analysis and were derived from bacteria, fungi and mammals. In order to discover important residues and regions within the KasA proteins, sequence alignment, motif analysis and phylogenetic studies were performed using Mtb KasA as a reference. Sequence alignment revealed conserved residues in all KasA proteins that have functional importance such as the catalytic triad residues (Cys171, His311 and His345). Motif analysis identified 18 highly conserved motifs within the KasA proteins with structural and functional roles. In addition, motifs unique to the Mtb KasA protein were also identified and explored for inhibitor drug design purposes. Phylogenetic analysis of the homolog sequences showed a distinct clustering of prokaryotes and eukaryotes. A distinctive clustering was also observed for species belonging to the same genus. Since the mechanism of action of most drugs involves the active site, allosteric site search was conducted on Mtb KasA and the human homolog protein using a combination of pocket detection algorithms with the aim of identifying sites that could be utilized in allosteric modulator drug discovery. This was followed by the virtual screening of 2089 FDA approved DrugBank compounds against the entire protein surfaces of Mtb KasA and Hsmt KasA, performed via molecular docking using AutoDock Vina. Screening of the compounds was based on the binding energies, with more focus on identifying ligands that bound exclusively to the acyl-binding tunnel of Mtb KasA. This reduced the data set to 27 promising drug compounds with a relatively high binding affinity for Mtb KasA, however, further experiments need to be performed to validate this result. Among these compounds were DB08889, DB06755, DB09270, DB11226, DB00392, DB12278, DB08936, DB00781, DB13720 and DB00392, which displayed relatively low binding energies for Mtb KasA when compared to the human homolog protein.Thesis (MSc) -- Faculty of Science, Biochemistry and Microbiology, 202

In silico identification of selective novel hits against the active site of wild type mycobacterium tuberculosis pyrazinamidase and its mutants

The World Health Organization declared Tuberculosis a global health emergency and has set a goal to eradicate it by 2035. However, effective treatment and control of the disease is being hindered by the emerging Multi-Drug Resistant and Extensively Drug Resistant strains on the most effective first line prodrug, Pyrazinamide (PZA). Studies have shown that the main cause of PZA resistance is due to mutations in the pncA gene that codes for the target protein Pyrazinamidase (PZase). Therefore, this study aimed to identify novel drug compounds that bind to the active site of wild type PZase and study the dynamics of these potential anti-TB drugs in the mutant systems of PZase. This approach will aid in identifying drugs that may be repurposed for TB therapy and/or designed to counteract PZA resistance. This was achieved by screening 2089 DrugBank compounds against the whole wild type (WT) PZase protein in molecular docking using AutoDOCK4.2. Compound screening based on docking binding energy, hydrogen bonds, molecular weight and active site proximity identified 47 compounds meeting all the set selection criteria. The stability of these compounds were analysed in Molecular Dynamic (MD) simulations and were further studied in PZase mutant systems of A3P, A134V, A146V, D8G, D49A, D49G, D63G, H51P, H137R, L85R, L116R, Q10P, R140S, T61P, V139M and Y103S. Generally, mutant-ligand systems displayed little deviation from the WT systems. The compound systems remained compact, with less fluctuations and more hydrogen bond interactions throughout the simulation (DB00255, DB00655, DB00672, DB00782, DB00977, DB01196, DB04573, DB06414, DB08981, DB11181, DB11760, DB13867, DB13952). From this research study, potential drugs that may be repurposed for TB therapy were identified. Majority of these drugs are currently used in the treatment of hypertension, menopause disorders and inflammation. To further understand the mutant-ligand dynamic systems, calculations such as Dynamic Residue Network (DRN) may be done. Also, the bioactivity of these drugs on Mycobacterium tuberculosis may be studied in wet laboratory, to understand their clinical impart in vivo experiments.Thesis (MSc) -- Faculty of Science, Biochemistry and Microbiology, 202

The activity of nybomycin against mycobacterium tuberculosis.

Doctoral Degree. University of KwaZulu-Natal, Durban.Nybomycin was discovered in 1955, but was never developed for clinical use. The compound was noticed again in recent years when it displayed bactericidal activity against certain fluoroquinolone-resistant bacterial species. The work presented here aims chiefly at describing the effect of nybomycin on Mycobacterium tuberculosis. The study is made up of three parts. In the first part, in vitro nybomycin susceptibility testing was conducted with various fluoroquinolone-susceptible and fluoroquinolone-resistant bacterialspecies. All M. tuberculosis isolates displayed low nybomycin inhibitory concentrations regardless of fluoroquinolone resistance. Similar susceptibility results were obtained for N. gonorrhoeae isolates, but results obtained with other bacterial species were less promising. In the second part, in silico investigations were conducted to elucidate the mechanism of action of nybomycin in M. tuberculosis. Results show that nybomycin binds to M. tuberculosis gyrase enzyme with an affinity at least similar to that of fluoroquinolones. No clear differences in binding affinity were observed when gyrA mutations, commonly associated with fluoroquinolone resistance, were considered. The results suggest that the mechanism of action of nybomycin against M. tuberculosis involves inhibition of gyrase enzyme. In the third part, M. tuberculosis mutants with increased nybomycin minimum inhibitory concentrations were selected and compared with the wild type organism through whole genome sequencing. None of the isolates harbored any mutations commonly linked to known drug resistance mechanisms. This indicates that M. tuberculosis likely employs a novel mechanism of resistance against nybomycin. This may further signify that nybomycin has an additional mechanism of action against M. tuberculosis, besides the action on gyrase enzyme, as suggested by the in silico results from this study. Twenty-two genes were identified through whole genome sequencing that may potentially be linked to the mechanism of resistance and possibly an additional mechanism of action

Drug Repurposing

This book focuses on various aspects and applications of drug repurposing, the understanding of which is important for treating diseases. Due to the high costs and time associated with the new drug discovery process, the inclination toward drug repurposing is increasing for common as well as rare diseases. A major focus of this book is understanding the role of drug repurposing to develop drugs for infectious diseases, including antivirals, antibacterial and anticancer drugs, as well as immunotherapeutics