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 isochorismate-pyruvate 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 Escherichia coli chorismate mutase, a protein of similar fold and similar chemistry, and of Yersinia 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).

Keywords: Chorismate mutase; Isochorismate-pyruvate lyase; Salicylate synthase; Siderophore.

Figure 1. Salicylate-capped Siderophores and the Enzymes Targeted

a. The salicylate-capped siderophores generated by P. aeruginosa (pyochelin), Yersinia spp. (yersiniabactin) and M. tuberculosis (mycobactin). b. The isochorismate-pyruvate lyase from Pseudomonas aeruginosa (PchB) is the specific enzyme of screen, and performs the pericyclic cleavage of isochorismate 1 to form salicylate 2 and pyruvate 3. Inhibitors were also tested against the salicylate synthase from Yersinia enterocolitica (Irp9), which is a chorismate-utilizing enzyme and a structural homologue of the isochorismate synthase PchA from P. aeruginosa. Irp9 and PchA catalyze the conversion of chorismate 4 to isochorismate 1. Irp9 subsequently catalyzes the same lyase reaction as PchB from an isochorismate intermediate, whereas PchA does not. The compounds were also tested for inhibition of E. coli chorismate mutase (EcCM), also a chorismate-utilizing enzyme, which performs a pericyclic reaction similar to that catalyzed by PchB, generating prephenate 5.

Figure 2. Enzyme scaffolds and transition states

The X-ray crystallographic structures for PchB (PBD code: 3REM) with salicylate and pyruvate bound, EcCM (1ECM) with Bartlett’s TSA inhibitor bound, and Irp9 (2FN1) with Mg²⁺, salicylate and pyruvate are depicted as cartoons. Each of these enzymes is a homodimer, with one monomer shaded darker than the other, and the active sites identified by the ligands shown as sticks. PchB and EcCM share the same fold – they are AroQ enzymes, whereas Irp9 is in the MST family of enzymes. The transition states for the reactions catalyzed, isochorismate-pyruvate lyase (left) and chorismate mutase (right), are shown below. The transition states are similar, differing only in the alignment of the pyruvylenol tail over the ring to make a cycle at the 1 (mutase) or 2 (lyase) carbon. PchB and Irp9 perform the same chemistry using different scaffolds, whereas PchB and EcCM perform related reactions in the same scaffold. It should be noted that PchB has adventitious mutase activity, albeit very low.

Figure 3. Compounds selected from screen for further evaluation

Figure 4. Growth inhibition of Pseudomonas aeruginosa, Yersinia enterocolitica and E. coli growth

a. P. aeurginosa growth inhibition by compound 6. b. P. aeurginosa growth inhibition by compound 10. For both A and B, left: WT PAO1 strain; middle: ΔpvdA PAO1, a pyoverdin minus strain; right: ΔpchE PAO1, a pyochelin minus strain. c. Y. enterocolitica growth inhibition by compound 6 (left) and compound 10 (right). For A–C: symbols: ∘ iron poor, ∙ iron rich. d. E. coli growth inhibition by compound 6 (∘) and compound 10 (∙) under iron rich conditions.