Analogues of Nitrofuran Antibiotics are Potent GroEL/ES Pro-drug Inhibitors with Efficacy against Enterococcus Faecium, Staphylococcus Aureus, and Escherichia Coli

Abstract

In 2019, the ESKAPE pathogens were highlighted by the World Health Organization as some of the most prominent threats to human health, capable of developing significant antibiotic resistance. These pathogens contribute to over 2 million annual infections and ~23,000 annual deaths in the U.S. In addition, various strains of E. coli have also shown a proclivity to develop resistance against common drug classes. The prevalence of these infections highlights an urgency to discover new antibiotics that target previously unexploited pathways essential to bacterial survival. The bacterial GroEL/ES chaperonin system is viewed as a viable antibiotic target. It has been proven essential to bacterial survival and homeostasis under all conditions. Previous studies identified hit GroEL/ES inhibitors with potential antibiotic activity. One promising hit (1) was shown to be a moderate but selective GroEL/ES inhibitor with antibacterial effects against Gram-negative pathogens (K. pneumoniae and A. baumannii). The structural similarity of 1 to known antibiotics – nitroxoline, nifuroxazide, and nitrofurantoin – prompted me to develop two series of hydroxyquinoline and nitrofuran-based analogs. I then assessed these compounds’ abilities to inhibit in vitro GroEL/ES activity, as well as to selectively target ESKAPE/E. coli bacteria over human cells. Initially, I found the nitrofuran analogs were stronger inhibitors of bacterial growth than the hydroxyquinolines, but were weaker at blocking GroEL/ES functions. However, considering nitrofuran-based antibiotics behave as pro-drugs, it was found that they became much more effective GroEL/ES inhibitors when E. coli NfsB nitroreductase was introduced into the GroEL/ES-dMDH refolding assay, metabolizing the nitro groups to their active species. Importantly, lead analogs that potently inhibited bacterial growth exhibited low cytotoxicity to human colon and intestine cells. Although I found E. coli were able to generate varying degrees of irreversible resistance to nifuroxazide, nitrofurantoin, and lead inhibitor 17, perhaps through mutations known to effect NfsA and NfsB nitroreductases, the resulting strains were not necessarily cross-resistant to the other inhibitors. Thus, combination therapy may help bypass these resistance mechanisms. In summary, this study identified key structure-activity relationships to selectively inhibit GroEL/ES and the growth of several bacterial species. Results from this study will aid future efforts to improve inhibitor potency.