(343) Biological evaluation of a library of thiamine-like 1,2,3-triazoles towards the inhibition of DXP synthase

Objective : The non-mevalonate pathway (MEP pathway) in bacteria is responsible for synthesizing DMAPP and IPP, critical precursors in the biosynthesis of isoprenoids. DXP synthase is a key enzyme in this pathway, and its absence in humans makes it a promising target in the development of potentially newer antibiotics. Thiamine diphosphate (ThDP; Vitamin B1) is a cofactor responsible for the C-C bond formation between pyruvate and glyceraldehyde-3-phosphate to yield 1-Deoxy-D-xylulose 5-phosphate (DXP). ThDP is the template for inhibitor design.

Methods: A library of modified 1,2,3-triazole molecules were tested using kinetic enzyme assays and DXS-DXR (DXP synthase-DXP reductase; Echelon BioSciences)-coupled assays to evaluate their inhibitory potential. Compounds were tested at 1 mM to 10 nM concentration range. The compounds were dissolved in DMSO, and then added to a 96-well plate. Subsequently, pyruvate (3 mM) and glyceraldehyde-3-phosphate (600 µM) were added, followed by NADPH (225 µM). Incubation at 25°C for 5 minutes with reaction buffer was done, followed by initiating the enzyme reaction by adding DXS (67 nM) and DXR (7 nM) enzymes. The kinetic spectrophotometric readings were then captured at 340 nm over a 15-minute interval. Ketoclomazone (KCZ) was used as a control. KCZ is an herbicide, and inhibitor of DXP synthase via a yet-unclear mechanism.

Results: SRM-4, SRM-5, SRM-7, SRM-13 and KCZ had docking (binding) scores of -9.5, -9.7, -9.1, -9.2, and -7.8 kcal/mol, respectively. SRM-4, SRM-5, SRM-7, SRM-13 and KCZ exhibited IC50 values of 0.128, 0.314, 27.82, 9.824, and 1.62 µM respectively.

Conclusions: We have obtained two inhibitors in the nanomolar range i.e., SRM-4 and SRM-5. This is supported by their binding energy scores. As such, SRM-4 is the most potent inhibitor of DXP synthase.