Structure-Guided In Vitro to In Vivo Pharmacokinetic Optimization of Propargyl-Linked Antifolates

Abstract

Pharmacokinetic/pharmacodynamic properties are strongly correlated with the in vivo efficacy of antibiotics. Propargyl-linked antifolates, a novel class of antibiotics, demonstrate potent antibacterial activity against both Gram-positive and Gram-negative pathogenic bacteria, including multidrug-resistant Staphylococcus aureus. Here, we report our efforts to optimize the pharmacokinetic profile of this class to best match the established pharmacodynamic properties. High-resolution crystal structures were used in combination with in vitro pharmacokinetic models to design compounds that not only are metabolically stable in vivo but also retain potent antibacterial activity. The initial lead compound was prone to both N-oxidation and demethylation, which resulted in an abbreviated in vivo half-life (∼20 minutes) in mice. Stability of leads toward mouse liver microsomes was primarily used to guide medicinal chemistry efforts so robust efficacy could be demonstrated in a mouse disease model. Structure-based drug design guided mitigation of N-oxide formation through substitutions of sterically demanding groups adjacent to the pyridyl nitrogen. Additionally, deuterium and fluorine substitutions were evaluated for their effect on the rate of oxidative demethylation. The resulting compound was characterized and demonstrated to have a low projected clearance in humans with limited potential for drug-drug interactions as predicted by cytochrome P450 inhibition as well as an in vivo exposure profile that optimizes the potential for bactericidal activity, highlighting how structural data, merged with substitutions to introduce metabolic stability, are a powerful approach to drug design.

Folic Acid Antagonists: Antimicrobial and Immunomodulating Mechanisms and Applications

“…The other approach which has also gained popularity in the recent years focuses on targeting DHFRs, being useful for both Gram-positive and Gram-negative bacteria, including TMP-resistant species too. Propargyl-linked antifolates consists of a highly conserved diaminopyrimidine ring linked through an acetylenic group to a biaryl system which allows the tunability of the compounds as shown in Figure 10 [119,120]. The success of these drugs on the resistant DHFR enzymes is based on their shared action mechanism, where the diaminopyrimidine ring binds a conserved acidic residue of the enzyme allowing the rest of the molecule to penetrate into the active site [119].…”

“…Propargyl-linked antifolates consists of a highly conserved diaminopyrimidine ring linked through an acetylenic group to a biaryl system which allows the tunability of the compounds as shown in Figure 10 [119,120]. The success of these drugs on the resistant DHFR enzymes is based on their shared action mechanism, where the diaminopyrimidine ring binds a conserved acidic residue of the enzyme allowing the rest of the molecule to penetrate into the active site [119]. These novel drugs have achieved inhibitory potencies in the nanomolar scale and therefore are promising candidates for antibiotic purposes [8,115].…”

Synthesis and Characterization of Functionalized Amino Dihydropyrimidines Toward the Analysis of their Antibacterial Structure–Activity Relationships and Mechanism of Action

“…Attempts to cocrystallize or soak 4 also failed to return any structures containing density indicative of 4 binding. The Sa DHFR structure was previously determined with NADPH, TMP, and other folate-mimicking derivatives. − The inclusion of 4 at saturating concentrations with Sa DHFR did not yield crystals under previously determined conditions, and broad screens for new conditions did not successfully produce visible protein crystals. Using the previous conditions (10 mg/mL SaDHFR), we grew crystals with NADPH (1.1 mM) and TMP (saturating) in addition to 4 at saturating conditions.…”

Design, Synthesis, Biological Evaluation, and Molecular Docking Studies of Pleuromutilin Derivatives Containing Thiazole

“…The antibacterial effect of time-dependent drugs could not be enhanced by increasing concentrations, compared to concentrationdependent drugs. 31 For time-dependent antibacterial agents, treatment outcomes could be improved by extending the treatment course. These results showed that compounds 14 and 15 had a bacteriostatic effect at high concentrations and displayed very promising antimicrobial activity against MRSA.…”