Crystal Structures of Trimethoprim-Resistant DfrA1 Rationalize Potent Inhibition by Propargyl-Linked Antifolates

Multidrug-resistant Enterobacteriaceae, notably Escherichia coli and Klebsiella pneumoniae, have become major health concerns worldwide. Resistance to effective therapeutics is often carried by class I and II integrons that can confer insensitivity to carbapenems, extended spectrum beta-lactamases, the antifolate trimethoprim, fluoroquinolones, and aminoglycosides. Specifically of interest to the study here, a prevalent gene (dfrAl) coding for an insensitive dihydrofolate reductase (DHFR) confers 190- or 1000 fold resistance to trimethoprim for K. pneumoniae and E. coli, respectively. Attaining inhibition of both the wild -type and resistant forms of the enzyme is critical for new antifolates. For several years, we have been developing the propargyl-linked antifolates (PLAs) as effective inhibitors against trimethoprim-resistant DHFR enzymes. Here, we show that the PLAs are active against both the wild -type and DfrA1 DHFR proteins. We report two high -resolution crystal structures of DfrAl bound to potent PLAs. The structure activity relationships and crystal structures will be critical in driving the design of broadly active inhibitors against wild -type and resistant DHFR.