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Crystal Structures of Trimethoprim-Resistant DfrA1 Rationalize Potent Inhibition by Propargyl-Linked Antifolates
Michael N Lombardo1, Narendran G-Dayanandan1, Dennis L Wright1
1Department of Pharmaceutical Sciences, University of Connecticut , 69 North Eagleville Road, Storrs, Connecticut 06269, United States.
Abstract:
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.
Insights
New antifolates, propargyl-linked antifolates (PLAs), effectively inhibit trimethoprim-resistant dihydrofolate reductase (DHFR) enzymes in key bacteria like E. coli and K. pneumoniae. Crystal structures guide the design of broadly active inhibitors against both wild-type and resistant DHFR forms.
Area of Science:
- Microbiology and Molecular Biology
- Drug Discovery and Development
- Structural Biology
Background:
- Multidrug-resistant Enterobacteriaceae, including E. coli and K. pneumoniae, pose significant global health threats.
- Class I and II integrons contribute to resistance against critical antibiotics like carbapenems, fluoroquinolones, and trimethoprim.
- The dfrA1 gene confers high-level trimethoprim resistance by encoding an altered dihydrofolate reductase (DHFR) enzyme.
Purpose of the Study:
- To develop novel antifolates targeting both wild-type and trimethoprim-resistant DHFR enzymes.
- To evaluate the efficacy of propargyl-linked antifolates (PLAs) against resistant DHFR.
- To elucidate the structural basis for PLA inhibition of DfrA1 DHFR.
Main Methods:
- Biochemical assays to assess enzyme inhibition.
- X-ray crystallography to determine high-resolution protein-ligand complex structures.
- Structure-activity relationship (SAR) analysis.
Main Results:
- Propargyl-linked antifolates (PLAs) demonstrated activity against both wild-type and DfrA1 DHFR.
- Two high-resolution crystal structures of DfrA1 complexed with potent PLAs were determined.
- These structures provide insights into the binding interactions and mechanisms of inhibition.
Conclusions:
- PLAs are promising candidates for combating trimethoprim-resistant Enterobacteriaceae infections.
- Structural data will facilitate the rational design of next-generation antifolates with broad activity.
- Targeting DHFR remains a critical strategy for overcoming antimicrobial resistance.
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