Resistance Mechanism to an Uncompetitive Inhibitor of a Single-Substrate, Single-Product Enzyme: A Study of Helicobacter Pylori Glutamate Racemase

Abstract

Two independent series of inhibitors of Helicobacter pylori glutamate racemase (MurI) were characterized for their kinetic mechanism, and one was used to generate resistant mutants in vitro. Mutant MurI enzymes from these strains were characterized by structural, genetic, kinetic and biophysical methods. Both inhibitor series, pyrazolopyrimidinediones and benzodiazepines, are uncompetitive with respect to the glutamate substrate, and the resistance mutations were found to act by reducing the affinity of MurI for substrate, thereby reducing the pool of enzyme-substrate complex available for binding inhibitor, while still allowing sufficient glutamate racemase activity for peptidoglycan construction. Uncompetitive inhibitors of a single-substrate, single-product enzyme are rare, and this work gives insight into an remarkable resistance mechanism. This article will discuss the projected clinical impact of H. pylori MurI resistance on these types of inhibitors.

Pyridodiazepine Amines Are Selective Therapeutic Agents for Helicobacter pylori by Suppressing Growth through Inhibition of Glutamate Racemase but Are Predicted To Require Continuous Elevated Levels in Plasma To Achieve Clinical Efficacy

“…Similar to the pyrazolopyrimidinediones (12), resistant mutants were easily generated, and mutations were found across MurI (A35T, A75T, and C162Y) outside residues that interact with the compound (9,14). Structural biology and biochemical studies have provided a rationale as to why these mutations confer resistance against both series of MurI inhibitors (9,24), and it is likely that future inhibitors of H. pylori MurI will suffer from the same issue. While the frequencies of spontaneous resistance were improved for compound A compared to those of the HTS hit, possibly as a result of the potency improvement, they remained relatively high enough to warrant clinical development.…”

“…Although these types of experiments might point to a resistance mechanism rather than to the target of a compound, the fact that the compound inhibits MurI and that the biochemical potency of the inhibitor was decreased against the purified mutant proteins (24) shows that MurI must be the target. This was backed up by peptidoglycan precursor analyses of H. pylori treated with the pyridodiazepine amine that showed a profile expected for the inhibition of MurI.…”

Flavodoxins as Novel Therapeutic Targets against Helicobacter pylori and Other Gastric Pathogens

“…Oxidative decarboxylation of pyruvate [26,33,[44][45][46] Flavodoxin:quinone reductase (FqrB) Oxidative decarboxylation of pyruvate [33,42,46] 2-oxoglutarate:acceptor oxidoreductase, subunits A (OorA), B (OorB), C (OorC) and D (OorD) Succinyl-CoA production [26,33,45] Cell Wall Structure N-succinyl-L,L-diaminopimelic acid desuccinylase, SDAP-deacylase (DapE) Succinylase pathway (lysine biosynthesis) [17,47,48] Glutamate racemase MurI Peptidoglycan biosynthesis [49,50] cag pathogenicity island (translocation of bacterial factors (e.g., Cag A and peptidoglycan)…”

“…Peptidoglycan, synthesized by Mur enzymes in a multistep pathway, is an essential component of the bacterial cell wall. MurA, acting on the first step, is targeted by Fosfomycin [50]. For glutamate racemase MurI, transforming L-Glu into D-Glu, several inhibitors have been described [49,50].…”

“…MurA, acting on the first step, is targeted by Fosfomycin [50]. For glutamate racemase MurI, transforming L-Glu into D-Glu, several inhibitors have been described [49,50]. The widely used β-lactam antibiotics inhibit peptidoglycan cross-linking which is carried out in the last steps of the pathway [17,49,50].…”