ORCID Profile
0000-0001-6015-3904
Current Organisation
Prince of Songkla University
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Publisher: Wiley
Date: 22-05-2023
DOI: 10.1002/PRO.4654
Abstract: Methylenetetrahydrofolate reductase (MTHFR) is a key metabolic enzyme in colonization and virulence of Neisseria meningitidis , a causative agent of meningococcal diseases. Here, the biochemical and structural properties of MTHFR from a virulent strain of N. meningitidis serogroup B ( Nm MTHFR) were characterized. Unlike other orthologs, Nm MTHFR functions as a unique homohexamer, composed of three homo‐dimerization partners, as shown in our 2.7 Å resolution crystal structure. Six active sites were formed solely within monomers and located away from the oligomerization interfaces. Flavin adenine dinucleotide cofactor formed hydrogen bonds with conserved sidechains, positioning its isoalloxazine ring adjacent to the overlapping binding sites of nicotinamide adenine dinucleotide (NADH) coenzyme and CH 2 ‐H 4 folate substrate. Nm MTHFR utilized NADH ( K m = 44 μM) as an electron donor in the NAD(P)H‐CH 2 ‐H 4 folate oxidoreductase assay, but not nicotinamide adenine dinucleotide phosphate (NADPH) which is the donor required in human MTHFR. In silico analysis and mutagenesis studies highlighted the significant difference in orientation of helix α7A (Phe215–Thr225) with that in the human enzyme. The extended sidechain of Met221 on helix α7A plays a role in stabilizing the folded structure of NADH in the hydrophobic box. This supports the NADH specificity by restricting the phosphate group of NADPH that causes steric clashes with Glu26. The movement of Met221 sidechain allows the CH 2 ‐H 4 folate substrate to bind. The unique topology of its NADH and CH 2 ‐H 4 folate binding pockets makes Nm MTHFR a promising drug target for the development of new antimicrobial agents that may possess reduced off‐target side effects.
Publisher: Cold Spring Harbor Laboratory
Date: 27-03-2023
DOI: 10.1101/2023.03.26.531482
Abstract: We previously reported potent ligands and inhibitors of Mycobacterium tuberculosis dethiobiotin synthetase ( Mt DTBS), a promising target for antituberculosis drug development (Schumann et al., ACS Chem Biol. 2021, 16, 2339-2347) here the unconventional origin of the fragment compound they were derived from is described for the first time. Compound 1 (9b-hydroxy-6b,7,8,9,9a,9b-hexahydrocyclopenta[3,4]cyclobuta[1,2-c]chromen-6(6a H )-one), identified by in silico fragment screen, was subsequently shown by surface plasmon resonance to have dose-responsive binding ( K D 0.6 mM). Clear electron density was revealed in the DAPA substrate binding pocket, when 1 was soaked into Mt DTBS crystals, but the density was inconsistent with the structure of 1 . Here we show the lactone of 1 hydrolyses to carboxylic acid 2 under basic conditions, including those of the crystallography soak, with subsequent ring-opening of the component cyclobutane ring to form cyclopentylacetic acid 3 . Crystals soaked directly with authentic 3 produced electron density that matched that of crystals soaked with presumed 1 , confirming the identity of the bound ligand. The synthetic utility of fortuitously formed 3 enabled subsequent compound development into nanomolar inhibitors. Our findings represent an ex le of chemical modification within drug discovery assays and demonstrate the value of high-resolution structural data in the fragment hit validation process. A molecule flagged in an in silico docking screen against Mt DTBS, was inadvertently hydrolysed in the crystal conditions used for hit validation. The resulting fragment-sized molecule bound to the DAPA substrate binding pocket of the target enzyme ( Mt DTBS) with millimolar affinity, as measured by surface plasmon resonance, but was later modified to a highly potent (nanomolar) ligand and promising lead for the development of novel tuberculosis treatments.
Location: Thailand
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