title: A high-throughput screening assay for simultaneous selection of inhibitors of Mycobacterium tuberculosis 1-deoxy-D-xylulose-5-phosphate synthase (Dxs) or 1-deoxy-D-xylulose 5-phosphate reductoisomerase (Dxr).
abstract: 1-deoxy-D-xylulose 5-phosphate reductoisomerase (Dxr) is involved in the synthesis of isoprenoids by the methylerythritol phosphate pathway. Dxr is essential in Mycobacterium tuberculosis (Mtu), absent in humans and amenable to structure-aided design. To further assess the druggability of the enzyme, the energetics of binding of fosmidomycin to Mtu Dxr was studied by isothermal calorimetry. Binding was enhanced by nicotinamide adenine dinucleotide phosphate hydrogen (NADPH) and driven by enthalpy (ΔH -10.2 kcal/mol, ΔS 1.1 cal mol(-1)K(-1)). This suggests the possibility of finding novel inhibitors that bind enthalpically, making Dxr an attractive target. The cost of the Dxr substrate, 1-deoxy-D-xylulose-5-phosphate, for high-throughput screening (HTS) is prohibitive. Hence, an HTS assay that couples Dxr to the upstream enzyme 1-deoxy-D-xylulose-5-phosphate synthase (Dxs), also a valid target, was developed. A high concentration of NADPH was used to bias it to detect Dxr inhibitors that bind like fosmidomycin. The assay Z' was 0.75. It was equally sensitive to inhibitors of Dxs and Dxr, that is, fosmidomycin and fluropyruvate inhibited it with IC(50)s similar to that in the individual enzyme assays (79 vs 54 nM for fosmidomycin). To distinguish inhibitors of Dxs from Dxr, individual enzyme assays and a microplate thermofluor binding assay were developed. The assay simultaneously screens two targets and is cost-effective.
authors.affiliation: AstraZeneca India Pvt. Ltd., Hebbal, Bangalore, India.
authors.forename: Vaishali
authors.initials: V
authors.lastname: Humnabadkar
authors.forename: Ramesh K
authors.initials: RK
authors.lastname: Jha
authors.forename: Nuzhat
authors.initials: N
authors.lastname: Ghatnekar
authors.forename: Sunita M
authors.initials: SM
authors.lastname: De Sousa
mesh_terms: D019747:Aldose-Ketose Isomerases / Q000037:antagonists & inhibitors*; D000900:Anti-Bacterial Agents / Q000378:metabolism / Q000494:pharmacology; D004789:Enzyme Activation / Q000187:drug effects*; D004791:Enzyme Inhibitors / Q000302:isolation & purification / Q000494:pharmacology*; D005578:Fosfomycin / Q000031:analogs & derivatives / Q000378:metabolism / Q000494:pharmacology; D057166:High-Throughput Screening Assays; D009097:Multienzyme Complexes / Q000037:antagonists & inhibitors*; D009169:Mycobacterium tuberculosis / Q000187:drug effects / Q000201:enzymology; D010088:Oxidoreductases / Q000037:antagonists & inhibitors*; D011485:Protein Binding; D015203:Reproducibility of Results; D014166:Transferases / Q000037:antagonists & inhibitors*
chemical_list: D000900:Anti-Bacterial Agents; D004791:Enzyme Inhibitors; D009097:Multienzyme Complexes; D005578:Fosfomycin; C024640:fosmidomycin; D010088:Oxidoreductases; C114231:1-deoxy-D-xylulose 5-phosphate reductoisomerase; D014166:Transferases; C109461:deoxyxylulose-5-phosphate synthase; D019747:Aldose-Ketose Isomerases