MOLECULAR GENETICS OF FLUOROQUINOLONE RESISTANCE AND THE STRUCTURES OF KEY PYRIMIDINE BIOSYNTHESIS PATHWAY ENZYMES FROM MYCOBACTERIUM TUBERCULOSIS

Ph.DDOCTOR OF PHILOSOPH

Enhanced penetration of pro-apoptotic and anti-angiogenic micellar nanoprobe in 3D multicellular spheroids for chemophototherapy

Chi-Hwa Wang is supported by National Additive Manufacturing Innovation Cluster @ National University of Singapore. Nitish V. Thakor is supported by Singapore National Research Foundation, Award No.: NRF-CRP15- 201 5-04. Vishnu Sunil greatly appreciates the National University of Singapore Research Scholarship for the funding of his Ph.D. studies in the National University of Singapore.Peer reviewedPostprin

Redox regulation of cell state and fate

10.1016/j.redox.2018.11.014Redox Biology25101056

Crystallization of uracil phosphoribosyltransferase (MtUPRT) from Mycobacterium tuberculosis

Exploring new drug targets in parallel to designing strategies for rational use of existing drugs would greatly aid the Tuberculosis (TB) drug development program. The key enzymes involved in the essential metabolic and regulatory pathways are usually sought for in the pursuit of potential drug targets. Likewise, uracil phosphoribosyltransferase (UPRT) is a key enzyme in the synthesis uridine 5’-monophosphate (UMP), the precursor of the pyrimidine nucleotides. It has been recently shown to be the probable target of 5-fluorouracil in Mycobacterium tuberculosis (Mtb). Here we report the purification, characterization and crystallization of the full length UPRT from Mtb (MtUPRT) encoded by the gene upp (Rv3309c). The MtUPRT was overexpressed in BL21 (DE3) E.coli expression system followed by three step chromatographic purification procedures. The purified MtUPRT was concentrated to 8mg/ml; single crystals were obtained using the sitting drop vapour diffusion method. The crystals were diffracted to 3.0 Å resolution and belonged to the space group P32 with unit cell parameters a = b = 118.09, c = 77.88 Å and four monomers in the asymmetric unit. Understanding the three dimensional structure of this essential enzyme will greatly help in screening of appropriate inhibitors of MtUPRT and thus assist in TB drug development

Structure and mapping of spontaneous mutational sites of PyrR from Mycobacterium tuberculosis

The emergence of resistant Mycobacterium tuberculosis (Mtb) infection and the dearth of drugs against tuberculosis have made it imperative to identify and validate novel targets and classes of drugs for treatment. The pyrimidine operon regulatory protein (PyrR), a regulator of de novo pyrimidine synthesis, is an essential enzyme and a probable 5-fluorouracil (5-FU) target in Mtb, with mutations in PyrR attributable to 5-FU resistance. Here we report, for the first time, the co-crystal structure of the PyrR-5-FU complex along with mapping of spontaneous mutational sites of PyrR. A cluster of mutations in the presence of the drug usually indicates a plausible region of drug-target interaction. Notably, we observed that three of the mutated PyrR residues lie in close proximity to the 5-FU binding site, including the amino acid Val178, which is involved in water mediated hydrogen bonding contact with 5-FU. Computational modeling of the PyrR-5'-phosphoribosyl-α-1'-pyrophosphate (PRPP) complex revealed the location of several other mutations at the PRPP binding site of PyrR, indicating their probable role in resistance. Indeed, 5-FU-resistant strains harboring these mutations exhibited decreased susceptibility to 5-FU. Considering that pyrimidine analogs are predominantly regarded to inhibit PyrR, the present studies will be beneficial for the screening of appropriate inhibitors of PyrR and help provide insight into future TB drug design and development