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 β-lactamases， the antifolate trimethoprim， fluoroquinolones， and aminoglycosides. Specifically of interest to the study here， a prevalent gene (dfrA1) 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 DfrA1 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.