Trihydroxybenzaldoximes are redox cycling inhibitors of ThDP-dependent DXP synthase.

Pathogenic bacteria must swiftly adapt to dynamic infection environments in order to survive and colonize in the host. 1-Deoxy-d-xylulose-5-phosphate synthase (DXPS) is thought to play a critical role in bacterial adaptation during infection and is a promising drug target. DXPS utilizes a thiamine diphosphate (ThDP) cofactor to catalyze the decarboxylative condensation of pyruvate and D-glyceraldehyde-3-phosphate (d-GAP) to form DXP, a precursor to isoprenoids and B vitamins. DXPS follows a ligand-gated mechanism in which pyruvate reacts with ThDP to form a long-lived lactyl-ThDP (LThDP) adduct which is coordinated by an active-site network of residues. d-GAP binding ostensibly disrupts this network to activate LThDP for decarboxylation. Our lab previously reported trihydroxybenzaldoximes inhibitors which are competitive with respect to D-GAP, and uncompetitive with respect to pyruvate, suggesting they bind after E-LThDP complex formation. Here, we conducted mechanistic studies to determine if these compounds inhibit DXPS by preventing LThDP activation or if they act as inducers of LThDP activation. We discovered that the catechol moiety of the trihydroxybenzaldoxime scaffold undergoes oxidation under alkaline aerobic conditions, and inhibitory potency is reduced under oxygen restriction. Leveraging long range (1)H-(15)N HSQC NMR and electrochemical measurements, we demonstrated that the oxidized form of the trihydroxybenzaldoxime induces LThDP decarboxylation. The oxime moiety accepts electrons from the resulting carbanion, resulting in formation of acetyl-ThDP which hydrolyzes to form acetate. SAR studies revealed that the catechol attenuates the redox activity of the oxime moiety, and under aerobic conditions these compounds are oxidized and thus act as redox cycling inhibitors of DXPS. Further exploration of redox active DXPS probes may provide new insights for inhibition strategies and selective probe development.