title: High throughput screen identifies small molecule inhibitors specific for Mycobacterium tuberculosis phosphoserine phosphatase.
abstract: The emergence of drug-resistant strains of Mycobacterium tuberculosis makes identification and validation of newer drug targets a global priority. Phosphoserine phosphatase (PSP), a key essential metabolic enzyme involved in conversion of O-phospho-l-serine to l-serine, was characterized in this study. The M. tuberculosis genome harbors all enzymes involved in l-serine biosynthesis including two PSP homologs: Rv0505c (SerB1) and Rv3042c (SerB2). In the present study, we have biochemically characterized SerB2 enzyme and developed malachite green-based high throughput assay system to identify SerB2 inhibitors. We have identified 10 compounds that were structurally different from known PSP inhibitors, and few of these scaffolds were highly specific in their ability to inhibit SerB2 enzyme, were noncytotoxic against mammalian cell lines, and inhibited M. tuberculosis growth in vitro. Surface plasmon resonance experiments demonstrated the relative binding for these inhibitors. The two best hits identified in our screen, clorobiocin and rosaniline, were bactericidal in activity and killed intracellular bacteria in a dose-dependent manner. We have also identified amino acid residues critical for these SerB2-small molecule interactions. This is the first study where we validate that M. tuberculosis SerB2 is a druggable and suitable target to pursue for further high throughput assay system screening.
authors.affiliation: From the Vaccine and Infectious Disease Research Centre, Translational Health Science and Technology Institute, Gurgaon 122016, Haryana, India, the Symbiosis School of Biomedical Sciences, Symbiosis International University, Lavale, Maharashtra 412115, India.
authors.forename: Garima
authors.initials: G
authors.lastname: Arora
authors.affiliation: From the Vaccine and Infectious Disease Research Centre, Translational Health Science and Technology Institute, Gurgaon 122016, Haryana, India.
authors.forename: Prabhakar
authors.initials: P
authors.lastname: Tiwari
authors.affiliation: the Biomedical Informatics Center, National Institute of Cholera and Enteric Diseases, Kolkata, West Bengal 700010, India.
authors.forename: Rahul Shubhra
authors.initials: RS
authors.lastname: Mandal
authors.affiliation: the CSIR-Institute of Microbial Technology, Chandigarh, 160036, India, and.
authors.forename: Arpit
authors.initials: A
authors.lastname: Gupta
authors.affiliation: the CSIR-Institute of Microbial Technology, Chandigarh, 160036, India, and.
authors.forename: Deepak
authors.initials: D
authors.lastname: Sharma
authors.affiliation: the Bioinformatics Centre, Bose Institute, Kolkata, West Bengal 700054, India.
authors.forename: Sudipto
authors.initials: S
authors.lastname: Saha
authors.affiliation: From the Vaccine and Infectious Disease Research Centre, Translational Health Science and Technology Institute, Gurgaon 122016, Haryana, India, ramandeep@thsti.res.in.
authors.forename: Ramandeep
authors.initials: R
authors.lastname: Singh
mesh_terms: D000595:Amino Acid Sequence; D000995:Antitubercular Agents / Q000737:chemistry / Q000494:pharmacology; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D045744:Cell Line, Tumor; D004791:Enzyme Inhibitors / Q000737:chemistry / Q000494:pharmacology*; D057166:High-Throughput Screening Assays / Q000379:methods*; D006801:Humans; D007700:Kinetics; D008826:Microbial Sensitivity Tests; D008958:Models, Molecular; D008969:Molecular Sequence Data; D015394:Molecular Structure; D009169:Mycobacterium tuberculosis / Q000187:drug effects / Q000201:enzymology* / Q000235:genetics; D009675:Novobiocin / Q000031:analogs & derivatives / Q000737:chemistry / Q000494:pharmacology; D010744:Phosphoric Monoester Hydrolases / Q000037:antagonists & inhibitors* / Q000145:classification / Q000235:genetics; D010802:Phylogeny; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D012394:Rosaniline Dyes / Q000737:chemistry / Q000494:pharmacology; D017386:Sequence Homology, Amino Acid; D054852:Small Molecule Libraries / Q000737:chemistry / Q000494:pharmacology
chemical_list: D000995:Antitubercular Agents; D001426:Bacterial Proteins; D004791:Enzyme Inhibitors; D012394:Rosaniline Dyes; D054852:Small Molecule Libraries; D009675:Novobiocin; C006260:clorobiocin; C025485:basic fuchsin; D010744:Phosphoric Monoester Hydrolases; C022394:phosphoserine phosphatase
keywords: Bacterial Protein Phosphatase; Enzyme Kinetics; High Throughput Screening (HTS); Molecular Docking; Mycobacterium tuberculosis