The Structure and Competitive Substrate Inhibition of Dihydrofolate Reductase from Enterococcus faecalis Reveal Restrictions to Cofactor Docking

Abstract

We are addressing bacterial resistance to antibiotics by repurposing a well-established classic antimicrobial target, the dihydrofolate reductase (DHFR) enzyme. In this work, we have focused on Enterococcus faecalis, a nosocomial pathogen that frequently harbors antibiotic resistance determinants leading to complicated and difficult-to-treat infections. An inhibitor series with a hydrophobic dihydrophthalazine heterocycle was designed from the anti-folate trimethoprim. We have examined the potency of this inhibitor series based on inhibition of DHFR enzyme activity and bacterial growth, including in the presence of the exogenous product analogue folinic acid. The resulting preferences were rationalized using a cocrystal structure of the DHFR from this organism with a propyl-bearing series member (RAB-propyl). In a companion apo structure, we identify four buried waters that act as placeholders for a conserved hydrogen-bonding network to the substrate and indicate an important role in protein stability during catalytic cycling. In these structures, the nicotinamide of the nicotinamide adenine dinucleotide phosphate cofactor is visualized outside of its binding pocket, which is exacerbated by RAB-propyl binding. Finally, homology models of the TMPR sequences dfrK and dfrF were constructed. While the dfrK-encoded protein shows clear sequence changes that would be detrimental to inhibitor binding, the dfrF-encoded protein model suggests the protein would be relatively unstable. These data suggest a utility for anti-DHFR compounds for treating infections arising from E. faecalis. They also highlight a role for water in stabilizing the DHFR substrate pocket and for competitive substrate inhibitors that may gain advantages in potency by the perturbation of cofactor dynamics.

Synthesis and Characterization of Novel N‐(Phenyl, Benzyl, Hetaryl)‐2‐([1,2,4]Triazolo[1,5‐c]Quinazolin‐2‐ylthio)Acetamides by Spectral Data, Antimicrobial Activity, Molecular Docking and QSAR Studies

“…Dihydrofolate reductase (DHFR) is a classic drug target, because it promotes the NADPH‐dependent reduction of 7,8‐dihydrofolate to yield 5,6,7,8‐tetrahydrofolate, which is involved in the biosynthesis of purines, thymidylate, and several amino acids in microbial cells . Moreover, investigation of hydrophobic heterocyclic dihydrophthalazines series, which were designed from antifolate drug trimethoprim, the propyl and trifluoropropyl substituents had an important role in protein stability during catalytic cycling, due to flexibility – an important determinant to fit into the inhibitor, thereby allowing it to take advantage of any available subpockets of the binding site .…”

“…The crystal structure of the enzyme E . faecalis DHFR (4M7U.pdb) was obtained from the protein data bank, and Trimetoprim was used as reference .…”

Evaluation of New Dihydrophthalazine-Appended 2,4-Diaminopyrimidines against Bacillus anthracis: Improved Syntheses Using a New Pincer Complex

“…Measurements of the MIC and the K i utilized a racemic mixture, unless otherwise noted, of each compound, as described earlier [ 8 , 13 , 27 , 36 ]. In brief, MIC values were based on standardized cultures of B. anthracis Sterne strain as prescribed by the CLSI [ 29 ].…”

Hemisyntheses and In-silico Study of New Analogues of Carlina Oxide from Carthamus Caeruleus Roots

“…As a result, the complex formed between DHFR and the IM9 analogue has the lowest energy (- [33,34]. Inhibition of this enzyme blocks DNA synthesis and cell division, leading to cell death.…”