Staphylococcus aureus resistance is a consistent problem with a large impact on the health care system. Infections with resistant S. aureus can cause serious adverse effects and can result in death.

Fatty acid biosynthetic enzymes exploit the reactivity of acyl- and malonyl-thioesters for catalysis. Here we synthesize acetyl/malonyl-CoA analogs with esters or amides in place of the thioester and characterize their behavior as substrates or inhibitors of the E. coli FabH ketosynthase. The acetyl- and malonyl-oxa(dethia)CoA analogs undergo extremely slow hydrolysis in the presence of FabH or C112Q mutant, which mimics the acyl-enzyme intermediate. Decarboxylation of malonyl-oxa(dethia)CoA by FabH or C112Q mutant was not detected. The amide analogs were completely stable to enzyme activity as expected. In enzyme assays, acetyl-oxa(dethia)CoA is surprisingly slightly activating, while acetyl-aza(dethia)CoA is a moderate inhibitor. The malonyl-oxa/aza(dethia)CoAs are inhibitors with Ki’s near the Km of malonyl-CoA. For comparison, we determine the FabH catalyzed decomposition rates for acetyl/malonyl-CoA, revealing some fundamental catalytic traits of FabH. The stable and inhibitory properties of the substrate analogs makes them promising for structure-function studies to undercover the basis of FabH cooperativity and enzyme:substrate interactions.

Tuberculosis (TB) is among the greatest public health and safety concerns in the 21st century Mycobacterium tuberculosis, which causes TB, infects alveolar macrophages and uses these cells as one of its primary sites of replication. The current TB treatment regimen, which consists of chemotherapy involving a combination of 3-4 antimicrobials for a duration of 6-12 months, is marked with significant side effects, toxicity, and poor compliance. Targeted drug delivery offers a strategy that can overcome many of the problems of current TB treatment by specifically targeting infected macrophages. Recent advances in nanotechnology and material science have opened an avenue to explore drug carriers that actively and passively target macrophages. This approach can increase the drug penetration into macrophages by using ligands on the nanocarrier that interact with specific receptors for macrophages. This review encompasses the recent development of drug carriers specifically targeting macrophages actively and passively. Future directions and challenges associated with development of effective TB treatment are also discussed.

Fatty acid and polyketide biosynthetic enzymes exploit the reactivity of acyl- and malonyl-thioesters for catalysis. A prime example is FabH, which initiates fatty acid biosynthesis in many bacteria and plants. FabH performs an acyltransferase reaction with acetyl-CoA to generate an acetyl-S-FabH acyl-enzyme intermediate and subsequent decarboxylative Claisen-condensation with a malonyl-thioester carried by an acyl carrier protein (ACP). We envision that crystal structures of FabH with substrate analogues can provide insight into the conformational changes and enzyme/substrate interactions underpinning the distinct reactions. Here, we synthesize acetyl/malonyl-CoA analogues with esters or amides in place of the thioester and characterize their stability and behavior as Escherichia coli FabH substrates or inhibitors to inform structural studies. We also characterize the analogues with mutant FabH C112Q that mimics the acyl-enzyme intermediate allowing dissection of the decarboxylation reaction. The acetyl- and malonyl-oxa(dethia)CoA analogues undergo extremely slow hydrolysis in the presence of FabH or the C112Q mutant. Decarboxylation of malonyl-oxa(dethia)CoA by FabH or C112Q mutant was not detected. The amide analogues were completely stable to enzyme activity. In enzyme assays with acetyl-CoA and malonyl-CoA (rather than malonyl-ACP) as substrates, acetyl-oxa(dethia)CoA is surprisingly slightly activating, while acetyl-aza(dethia)CoA is a moderate inhibitor. The malonyl-oxa/aza(dethia)CoAs are inhibitors with K i’s near the K m of malonyl-CoA. For comparison, we determine the FabH catalyzed decomposition rates for acetyl/malonyl-CoA, revealing some fundamental catalytic traits of FabH, including hysteresis for malonyl-CoA decarboxylation. The stability and inhibitory properties of the substrate analogues make them promising for structure–function studies to reveal fatty acid and polyketide enzyme/substrate interactions.