Ensuring the availability of new antibiotics to eradicate resistant pathogens is a critical issue, but very few new antibacterials have been recently commercialized. In an effort to rationalize their discovery process, the industry has utilized chemical library and high-throughput approaches already applied in other therapeutical areas to generate new antibiotics. This strategy has turned out to be poorly adapted to the reality of antibacterial discovery. Commercial chemical libraries contain molecules with specific molecular properties, and unfortunately systemic antibacterials are more hydrophilic and have more complex structures. These factors are critical, since hydrophobic antibiotics are generally inactive in the presence of serum. Here, we review how the skewed distribution of systemic antibiotics in chemical space influences the discovery process.
The pharmacologic effect of an antibiotic is directly related to its unbound concentration at the site of infection. Most commercial antibiotics have been selected in part for their low propensity to interact with serum proteins. These nonspecific interactions are classically evaluated by measuring the MIC in the presence of serum. As higher-throughput technologies tend to lose information, surface plasmon resonance (SPR) is emerging as an informative medium-throughput technology for hit validation. Here we show that SPR is a useful automatic tool for quantification of the interaction of model antibiotics with serum proteins and that it delivers precise real-time kinetic data on this critical parameter.Serum proteins play an important role in binding to many drugs, including antibiotics. In general, serum proteins decrease the free fraction of antibiotic available for the elimination of bacteria, since only the non-protein-bound molecules are pharmacologically active. The proteins involved in this sequestration are mainly human serum albumin (HSA), the most abundant serum protein (4% wt/vol); ␣-1-acid glycoprotein (AGP); and gamma globulin (5, 7, 11).Currently, most of the reports of the inhibitory effects of serum proteins on antibiotics are derived from in vitro studies that have employed the MIC method (3, 9) or time-killing curves (16). The findings described in those reports correlate well with in vivo data (13) and are useful for evaluation of the potential of a new drug candidate. However, it is also necessary to rapidly and precisely characterize how a molecule binds to serum proteins in terms of affinity constants to drive the synthesis of new and more efficient analogs. A variety of physical techniques for measurement of the levels of protein binding have been proposed. The most classical are ultracentrifugation (3) and dialysis (3,8); but other alternative techniques have been used, like circular dichroism analysis (1) and extrinsic fluorescence analysis (15). More recently, surface plasmon resonance (SPR) was proposed as a medium-to high-throughput alternative for evaluation of the kinetics of relatively lipophilic drugs that bind to human serum proteins in real time (14).Antibiotics are characterized as having a relatively high hydrophilicity compared to the hydrophilicities of other drug classes. Consequently, antibiotics have lower affinities for serum proteins. Because the development of fast analytical methods that allow the measurement of antibiotic-serum protein interaction kinetics with a small amount of sample is desirable, we have evaluated if SPR can measure low affinities and how SPR can be used to prescreen rapidly libraries of antibiotic candidates for their propensity to bind to serum proteins. MATERIALS AND METHODSBacterial strains, antimicrobial agents, and media. Staphylococcus aureus reference strain CIP 76.25 (ATCC 25923) was used. S. aureus was grown, subcultured, and quantified in Mueller-Hinton broth and on Mueller-Hinton agar (Difco Laboratories, Detroit, MI). Antimicrobial ag...
The ketoacyl-acyl carrier protein (ACP) reductase FabG catalyzes the NADPH/NADH dependent reduction of β-ketoacyl-ACP substrates to β-hydroxyacyl-ACP products, the first reductive step in the fatty acid biosynthesis elongation cycle. FabG proteins are ubiquitous in bacteria and are part of the type II fatty acid synthase system. Mining the genome uncovered several putative FabG-like proteins. Among them, we identified MSMEG_6753 whose gene was found adjacent to , encoding a recently characterized enoyl-CoA dehydratase, and to, encoding another potential reductase. Recombinantly expressed and purified MSMEG_6753 exhibits ketoacyl reductase activity in the presence of acetoacetyl-CoA and NADPH. This activity was subsequently confirmed by functional complementation studies in a thermosensitive mutant. Furthermore, comparison of the and the NADP-bound MSMEG_6753 crystal structures showed that cofactor binding induces a closed conformation of the protein. A Δ deletion mutant could be generated in , indicating that this gene is dispensable for mycobacterial growth. Overall, these results showcase the diversity of FabG-like proteins in mycobacteria and new structural features regarding the catalytic mechanism of this important family of enzymes that may be of importance for the rational design of specific FabG inhibitors.
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