Towards the understanding of resistance mechanisms in clinically isolated trimethoprim-resistant, methicillin-resistant Staphylococcus aureus dihydrofolate reductase

Abstract

Resistance to therapeutics such as trimethoprim-sulfamethoxazole has become an increasing problem in strains of methicillin-resistant Staphylococcus aureus (MRSA). Clinically isolated trimethoprim-resistant strains reveal a double mutation, H30N/F98Y, in dihydrofolate reductase (DHFR). In order to develop novel and effective therapeutics against these resistant strains, we evaluated a series of propargyl-linked antifolate lead compounds for inhibition of the mutant enzyme. For the propargyl-linked antifolates, the F98Y mutation generates minimal (between 1.2- and 6-fold) losses of affinity and the H30N mutation generates greater losses (between 2.4- and 48-fold). Conversely, trimethoprim affinity is largely diminished by the F98Y mutation (36-fold) and is not affected by the H30N mutation. In order to elucidate a mechanism of resistance, we determined a crystal structure of a complex of this double mutant with a lead propargyl-linked antifolate. This structure suggests a resistance mechanism consistent both for the propargyl-linked class of antifolates and for trimethoprim that is based on the loss of a conserved water-mediated hydrogen bond.

Keywords: antifolates; dihydrofolate reductase; methicillin-resistant Staphylococcus aureus; trimethoprim.

Figure 1

a) 2 Fo–Fc density (1.6 σ, gray) for compound 2 and residues Leu 5, Asp 27, Asn 30, Thr 111, and water molecule (red sphere), b) Omit 2 Fo–Fc density (1.3 σ, blue) for compound 2 and residues Leu 5, Asp 27, Asn 30, Phe 92, Thr 111 and a water molecule (red sphere),c) compound 2 and the residues in the active site.

Figure 2

Residues in the active site that form hydrogen bonds to the diaminopyrimidine ring of compound 2 (lavender) for: (a) Sa (wt) DHFR (green) (PDB ID: 3FQ0); (b) Sa (F98Y) DHFR (cyan) (PDB ID: 3FQO); and (c) Sa (H30N, F98Y) DHFR (pink) (PDB ID: 3I8A). Water molecules are shown as colored spheres to match the enzyme. Interactions that are within hydrogen bonding distance are represented by dashed lines in black. Weakened hydrogen bonds are represented by dashed lined in red.