Nitrofurans: Revival of an “old” drug class in the fight against antibiotic resistance

Antibiotic resistance is one of the greatest contemporary threats to the human health, which has increasingly been undermining the effectiveness of existing antimicrobial therapies.Development of novel antibiotics is inarguably the key to combat this threat, yet this is a lengthy process (10 to 15 years) at a hefty price tag of approximately US$1.3 billion for development of an approved drug [1,2].A new drug, once introduced into the market, also faces the risk of drug resistance emergence.It is, therefore, essential to diversify therapeutic strategies, including revival and reintroduction of "old" antibacterials for treating multidrug-resistant pathogens [3].Nitrofuran class of synthetic molecules, introduced in the 1940s and 1950s, belongs to this category [4].Several nitrofurans are currently on the market: nitrofurazone for topical infections and urinary catheter coating, nitrofurantoin for urinary tract infections, and furazolidone for bacterial diarrhea and Helicobacter pylori infections.Here, we highlight aspects of this drug class that have recently been unraveled, laying foundation for future improvements and judicial uses of nitrofurans against ever-expanding antibiotic-resistant bacteria. Nitrofuran-activating enzymes in Escherichia coliNitrofuran compounds are prodrugs.In E. coli, they are activated via reduction by 2 type I oxygen-insensitive nitroreductases, NfsA and NfsB.These enzymes catalyze a stepwise 2-electron reduction of the nitro moiety into reactive nitroso and hydroxylamino derivatives, one of which is considered responsible for the antibacterial activity of nitrofurans (Fig 1A) [5][6][7].Peterson and colleagues reported the existence of type II nitroreductase activity in E. coli that reduces nitrofuran by 1-electron transfer mechanism and is sensitive to molecular oxygen (Fig 1A) [8].The identity of enzymes catalyzing the oxygen-sensitive nitrofuran reduction has not been revealed until 2019, when we identified a novel nitrofuran-activating enzyme by selecting for furazolidone-resistant mutants in an nfsA nfsB E. coli double knock-out strain.A total of 15 independently isolated mutants resistant to bactericidal concentration of furazolidone contained mutations in the ahpF gene, which encodes a component of the antioxidant alkyl hydroperoxide reductase [9].Subsequent enzymatic assays of purified AhpF protein determined that this enzyme is a type II oxygen-sensitive nitroreductase [9].The antibacterial mechanism of the nitrofuran derivatives, once activated by nitroreductases, is ill defined.Multiple effects have been observed, including DNA lesions and oxidative stress and inhibition of the RNA and protein biosynthesis [10][11][12].However, it remains to be clarified which of these affected targets (DNA, RNA, and protein) are directly attacked by the nitroreductase-activated nitrofuran derivatives and which are simply downstream effects of the interaction between these derivatives and bacterial essential targets.We hypothesize that the nitro moiety at the C 5 of the furan ring, once reduced by nitroreductases, acts as a warhead