Charged Propargyl-Linked Antifolates Reveal Mechanisms of Antifolate Resistance and Inhibit Trimethoprim-Resistant MRSA Strains Possessing Clinically Relevant Mutations

Drug-resistant
enzymes must balance catalytic function with inhibitor
destabilization to provide a fitness advantage. This sensitive balance,
often involving very subtle structural changes, must be achieved through
a selection process involving a minimal number of eligible point mutations.
As part of a program to design propargyl-linked antifolates (PLAs)
against trimethoprim-resistant dihydrofolate reductase (DHFR) from Staphylococcus aureus, we have conducted a thorough study
of several clinically observed chromosomal mutations in the enzyme
at the cellular, biochemical, and structural levels. Through this
work, we have identified a promising lead series that displays significantly
greater activity against these mutant enzymes and strains than TMP.
The best inhibitors have enzyme inhibition and MIC values near or
below that of trimethoprim against wild-type S. aureus. Moreover, these studies employ a series of crystal structures of
several mutant enzymes bound to the same inhibitor; analysis of the
structures reveals a more detailed molecular understanding of drug
resistance in this important enzyme.