title: 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.
authors.affiliation: Department of Pharmaceutical Sciences, University of Connecticut, 69 N. Eagleville Rd., Storrs, CT 06269, USA.
authors.forename: Kathleen M
authors.initials: KM
authors.lastname: Frey
authors.forename: Michael N
authors.initials: MN
authors.lastname: Lombardo
authors.forename: Dennis L
authors.initials: DL
authors.lastname: Wright
authors.forename: Amy C
authors.initials: AC
authors.lastname: Anderson
mesh_terms: D003001:Cloning, Molecular; D003460:Crystallization; D005493:Folic Acid Antagonists / Q000494:pharmacology; D016106:Methicillin Resistance / Q000235:genetics* / Q000502:physiology; D008958:Models, Molecular; D015394:Molecular Structure; D020125:Mutation, Missense / Q000235:genetics; D013211:Staphylococcus aureus / Q000201:enzymology* / Q000502:physiology; D013762:Tetrahydrofolate Dehydrogenase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D014295:Trimethoprim / Q000494:pharmacology; D014296:Trimethoprim Resistance / Q000235:genetics* / Q000502:physiology
chemical_list: D005493:Folic Acid Antagonists; D014295:Trimethoprim; D013762:Tetrahydrofolate Dehydrogenase