Expanding the results of a high throughput screen against an isochorismate-pyruvate lyase to enzymes of a similar scaffold or mechanism

Abstract

Antibiotic resistance is a growing health concern, and new avenues of antimicrobial drug design are being actively sought. One suggested pathway to be targeted for inhibitor design is that of iron scavenging through siderophores. Here we present a high throughput screen to the isochorismatepyruvate lyase of Pseudomonas aeruginosa, an enzyme required for the production of the siderophore pyochelin. Compounds identified in the screen are high nanomolar to low micromolar inhibitors of the enzyme and produce growth inhibition in PAO1 P. aeruginosa in the millimolar range under iron-limiting conditions. The identified compounds were also tested for enzymatic inhibition of E. coli chorismate mutase, a protein of similar fold and similar chemistry, and of Y. enterocolitica salicylate synthase, a protein of differing fold but catalyzing the same lyase reaction. In both cases, subsets of the inhibitors from the screen were found to be inhibitory to enzymatic activity (mutase or synthase) in the micromolar range and capable of growth inhibition in their respective organisms (E. coli or Y. enterocolitica).

An Open and Shut Case: The Interaction of Magnesium with MST Enzymes

“…The MST enzymes have been investigated previously, with considerable interest as to their promise in the development of new antimicrobials. There is a wealth of literature describing the development of inhibitors, both designed rationally (as substrate and potential transition state analogues) ,,,− and by high-throughput screening methodologies. , While some inhibitors have been developed with micro- to nanomolar affinities, the failures to some extent highlight our lack of understanding of the kinetic and chemical mechanisms of these enzymes. Mechanistic studies of these enzymes have been limited primarily to mutational analysis and structural biology approaches.…”

“…Large diamond crystals (500 μm × 300 μm × 300 μm) formed after 3 days. The low-magnesium crystals were initially intended to provide an inhibitor-bound structure and as such were soaked with 16 mM 4,6-dinitro-2-oxo-1,3-benzoxathiol-5-yl methyl carbonate (inhibitor 10 in ref ( 36 )) for 45 min before flash cooling at −160 °C; however, none of the compound was evident in the electron density maps. High-magnesium EntC crystals were grown similarly to the low-magnesium crystals in a solution containing 0.1 M MES (pH 6.2), 50% PEG 200, and 0.05 M ammonium chloride.…”

Unraveling the Structure and Mechanism of the MST(ery) Enzymes

“…A subsequent study produced similar findings. A high-throughput screen against the isochorismate-pyruvate lyase PchB from P. aeruginosa was conducted in 2014[30]. PchB is not an MST enzyme, but is a lyase only enzyme that removes the pyruvylenol tail from chorismate, like the salicylate synthases do.…”

Breaking a pathogen's iron will: Inhibiting siderophore production as an antimicrobial strategy

“…A second enzyme in the pathway, PchB, is an isochorismate-pyruvate lyase performing the pericyclic elimination reaction (Figure 9A, green ) [85]. A high throughput screen was designed that exploits salicylate fluorescence, and the hits were tested for inhibition in PchB, the Y. enterocolitica salicylate synthase Irp9 ( Section 7.1 ) and also E. coli chorismate mutase (EcCM) [86]. The rationale was that Irp9 performs the same reaction, and EcCM is a structural homologue performing a similar reaction with a similar transition state.…”

“…[82] D. Isochorismate-pyruvate lyase inhibitor from HTS that also is effective against salicylate synthase and chorismate mutase. [86]. E. The structure of the salicylate synthase from Y. enterocolitica (Irp9, purple cartoon, PDB ID: 2FN1) with the catalytic Mg ion (green sphere) and products salicylate and pyruvate (purples sticks) was used to align two inhibitor-bound structures of the salicylate synthase from M. tuberculosis, MbtI.…”