Development of antibacterial compounds that block evolutionary pathways to resistance

Abstract

Antibiotic resistance is a worldwide challenge. A potential approach to block resistance is to simultaneously inhibit WT and known escape variants of the target bacterial protein. Here we applied an integrated computational and experimental approach to discover compounds that inhibit both WT and trimethoprim (TMP) resistant mutants of E. coli dihydrofolate reductase (DHFR). We identified a novel compound (CD15-3) that inhibits WT DHFR and its TMP resistant variants L28R, P21L and A26T with IC50 50-75 micromole against WT and TMP-resistant strains. Resistance to CD15-3 was dramatically delayed compared to TMP in in vitro evolution. Whole genome sequencing of CD15-3 resistant strains showed no mutations in the target folA locus. Rather, gene duplication of several efflux pumps gave rise to weak (about twofold increase in IC50) resistance against CD15-3. Altogether, our results demonstrate the promise of strategy to develop evolution drugs - compounds which block evolutionary escape routes in pathogens.

Empowering systems-guided drug target discovery with metabolic and structural analysis

“…Of the assessed proteins besides folA (shown here for consistency on Fig. 6A and (Zhang et al, 2020)) only overexpression of folK showed clear sign of rescue from growth inhibition. folK encodes for 6-Hydroxymethyl-7,8-dihydropterin pyrophosphokinase (HPPK).…”

“…In the present study we deployed global metabolomics, gene over-expression, and growth recovery studies analysed in the context of the metabolic network to search for the cellular target of a novel "anti-evolution" lead CD15-3. We observed that CD15-3 markedly delays the emergence of antibiotic resistant bacterial phenotypes (Zhang et al, 2020). This could be attributed to factors like; 1) CD15-3 was designed to interact with WT as well as TMP resistant mutants which arise under conditions of TMP selection.…”

“…Regulated overexpression of candidate genes viz., folK, folP, folC, folD, metF and pabC using the pBAD promoter with 0.1% arabinose induction was carried out to determine whether any of the overexpressed genes show recovery from CD15-3 induced growth inhibition and thus may be the non-DHFR target of CD15-3. In our previous study (Zhang et al, 2020) we showed that overexpression of folA (encoding DHFR) partially rescued CD15.3 induced toxicity at lower concentrations of CD15.3. However folA overexpression from pBAD was restricted to 0.005% arabinose induction to avoid overexpression induced toxicity (Bhattacharyya et al, 2016) which would complicate interpretation of the rescue experiments.…”

“…In a previous study, we showed that cellular overexpression of DHFR helped cells to partially recover from CD15-3-induced growth inhibition (Zhang et al, 2020). As full rescue of growth at all concentrations of CD15-3 was not observed with overexpression of DHFR, we went on to explore possible non-DHFR intracellular targets of CD15-3.…”

“…In our previous study we presented a new compound, CD15-3, as an in-house developed “evolution-drug” lead that significantly delays the emergence of resistant phenotypes. CD15-3 was designed to interact with wild type DHFR (dihydrofolate reductase) and its trimethoprim (TMP) resistant mutants (Zhang et al, 2020). TMP is a well-known antifolate antibiotic that inhibits bacterial growth by selectively blocking bacterial DHFR - a critical enzyme in bacterial one-carbon metabolism.…”