Identifying Ortholog Selective Fragment Molecules for Bacterial Glutaredoxins by NMR and Affinity Enhancement by Modification with an Acrylamide Warhead

Abstract

Illustrated here is the development of a new class of antibiotic lead molecules targeted at Pseudomonas aeruginosa glutaredoxin (PaGRX). This lead was produced to (a) circumvent efflux-mediated resistance mechanisms via covalent inhibition while (b) taking advantage of species selectivity to target a fundamental metabolic pathway. This work involved four components: a novel workflow for generating protein specific fragment hits via independent nuclear magnetic resonance (NMR) measurements, NMR-based modeling of the target protein structure, NMR guided docking of hits, and synthetic modification of the fragment hit with a vinyl cysteine trap moiety, i.e., acrylamide warhead, to generate the chimeric lead. Reactivity of the top warhead-fragment lead suggests that the ortholog selectivity observed for a fragment hit can translate into a substantial kinetic advantage in the mature warhead lead, which bodes well for future work to identify potent, species specific drug molecules targeted against proteins heretofore deemed undruggable.

Using Structure-guided Fragment-Based Drug Discovery to Target Pseudomonas aeruginosa Infections in Cystic Fibrosis

“…Hence glutaredoxins are considered as potential drug targets. An NMR-led FBDD campain targeted the P. aeruginosa glutaredoxin ( Pa GRX) ( Khattri et al, 2020 ). This work involved: 1) generating Pa GRX-specific fragment hits by screening 463 fragment molecules using independent STD NMR measurements, 2) NMR-based modeling of Pa GRX, 3) NMR-guided docking of hits and 4) derivatising the fragment hit with a vinyl cysteine trap moiety (acrylamide warhead with strong tendency to form alkylated cysteine adducts) to generate the chimeric lead ( Figure 6 ).…”

“…A vinyl cysteine trap moiety resulted in the development of chimeric lead molecule with enhanced reactivity and selectivity to Pa GRX over human GRX1 (hGRX1). Putative model Pa GRX and FTMap identified epitope maps of Pa GRX in this figure are adopted from ( Khattri et al, 2020 ) with permission from the authors.…”