Binding affinity of penicillin-binding proteins (PBP) of Staphylococcus aureus for several beta-lactam antibiotics was examined in a methicillin-susceptible strain and in two methicillin-resistant strains, one heterogeneous and one homogeneous, to determine relationships between PBP binding and expression of resistance. PBPs 1-4 had similar affinities in all three strains. PBP 2a affinities were similar in both resistant strains. For the susceptible strain and for the susceptible subpopulation of cells in the heterogeneous strain, growth inhibition correlated with binding to PBPs 1-3, suggesting that these PBPs mediate susceptibility in both. For resistant cells, growth inhibition correlated only with binding to PBP 2a, suggesting that this is the target in resistant cells. Determination of binding affinity for PBP 2a or the concentration that inhibits growth of the most resistant subpopulation of cells should be included in evaluation of beta-lactam antibiotics for potential activity against methicillin-resistant strains of S. aureus.

A higher rate of clinical failures in patients treated with daptomycin (2 mg/kg of body weight, given once daily) compared with rates in patients treated with conventional regimens caused early termination of this comparative clinical trial. One explanation for these failures could be that daptomycin is highly protein bound and that the concentration of the unbound active drug is too low for antibacterial activity. To assess this explanation, we studied the binding of daptomycin to proteins by using an ultrafiltration method. pH (7.0 to 7.4), temperature (25 or 37°C), or daily freezing and thawing over 2 months had no effect on binding of daptomycin to proteins. We found that daptomycin was bound to albumin (90%) at 4 g/100 ml. Binding of daptomycin was not concentration dependent (2.5 to 80 ,ug/ml). In human serum samples spiked with daptomycin, average binding was 94% 2.4%. In 6 subjects given an intravenous infusion of daptomycin (3 mg/kg), average binding was 90% + 2.1%. Susceptibility studies showed that a concentration in serum 20 times the unbound concentration was needed to equal the MIC of the total drug. These results indicate that daptomycin is highly bound (90 to 94%) to albumin and that clinical failure to daptomycin can in part be explained by the low concentration of the unbound drug.

Clostridium difficile (C. difficile) is a Gram positive, anaerobic bacterium that infects the lumen of the large intestine and produces toxins. This results in a range of syndromes from mild diarrhea to severe toxic megacolon and death. Alarmingly, the prevalence and severity of C. difficile infection are increasing; thus, associated morbidity and mortality rates are rising. 4-Aminothiazolyl analogues of the antibiotic natural product GE2270 A (1) were designed, synthesized, and optimized for the treatment of C. difficile infection. The medicinal chemistry effort focused on enhancing aqueous solubility relative to that of the natural product and previous development candidates (2, 3) and improving antibacterial activity. Structure-activity relationships, cocrystallographic interactions, pharmacokinetics, and efficacy in animal models of infection were characterized. These studies identified a series of dicarboxylic acid derivatives, which enhanced solubility/efficacy profile by several orders of magnitude compared to previously studied compounds and led to the selection of LFF571 (4) as an investigational new drug for treating C. difficile infection.