Prospective Screening of Novel Antibacterial Inhibitors of Dihydrofolate Reductase for Mutational Resistance

Abstract

Resistance to trimethoprim (TMP) resulting from point mutations in the enzyme drug target dihydrofolate reductase (DHFR) drives the development of new antifolate inhibitors effective against methicillin-resistant Staphlococcus aureus (MRSA). For the past several years we have used structure-based design to create propargyl-linked antifolates that are highly potent antibacterial agents. In order to focus priority on the development of lead compounds with a low propensity to induce resistance, we prospectively evaluated resistance profiles for two of these inhibitors in an MRSA strain. By selection with the lead inhibitors, we generated resistant strains that contain single point mutations F98Y and H30N associated with TMP resistance and one novel mutation, F98I, in DHFR. Encouragingly, the pyridyl propargyl-linked inhibitor selects mutants at low frequency (6.85 ؋ 10 ؊10 to 1.65 ؋ 10 ؊9 ) and maintains a low MIC (2.5 g/ml) and a low mutant prevention concentration (1.25 g/ml), strongly supporting its position as a lead compound. Results from this prospective screening method inform the continued design of antifolates effective against mutations at the Phe 98 position. Furthermore, the method can be used broadly to incorporate ideas for overcoming resistance early in the development process.

Protein design algorithms predict viable resistance to an experimental antifolate

“…The addition of the Val31Leu mutation confers an additional 8-fold loss for compound 1 and a 4-fold loss for TMP, resulting in total 256-and 128-fold losses, respectively. As observed previously, the Sa(F98Y)DHFR mutation does not reduce the fitness of the strain (11,12). The presence of the V31L/F98Y mutation reported here results in minimal (14%) loss of fitness.…”

“…Because of observed conformations of the escape mutations to antifolate inhibitors described in ref. 11, we modeled ∼10,000 possible binding conformations of compound 1 to DHFR. These binding poses were first filtered by OSPREY's MinDEE/A* algorithms (18) that searched for the lowest energy conformation of any of the mutants to each of the binding conformations.…”

Nonracemic Antifolates Stereoselectively Recruit Alternate Cofactors and Overcome Resistance in S. aureus

“…Using a structure-based approach, we have developed an advanced lead series of inhibitors that displays low nanomolar inhibition of the wild-type DHFR and potent activity against a range of MRSA strains (MIC values 0.04–0.72 µg/mL) and other important Gram-positive pathogens. 8–11 This compound class is characterized by a unique propargylic linker between the polar diaminopyrimidine head group and a hydrophobic biaryl domain as exemplified in Figure 1. Alkyne functionality is unique and structurally distinct from other unsaturated units in that the linear, cylindrical nature of the group allows it to fit through very narrow passages in a binding site such as in the case of ponatinib binding the mutant form of Bcr-Abl.…”

Crystal Structures of Klebsiella pneumoniae Dihydrofolate Reductase Bound to Propargyl-Linked Antifolates Reveal Features for Potency and Selectivity

“…For several years, we have been optimizing the propargyl-linked antifolates to inhibit the growth of Gram-positive bacteria by inhibiting the DHFR enzymes from these bacteria (24,25,31,32). Relatively hydrophobic biphenyl PLAs (compounds 1 and 2) potently inhibit methicillin-resistant Staphylococcus aureus and Streptococcus pyogenes, but they do not inhibit the growth of Gram-negative species such as Klebsiella pneumoniae, despite apparent inhibition of the K. pneumoniae DHFR enzyme (Table 1).…”