S. aureus alanine racemase: "A target for structure-based drug design"
|Institution:||University of Otago|
|Keywords:||alanine; racemase; crystallography; fragment-screening|
|Full text PDF:||http://hdl.handle.net/10523/4879|
Staphylococcus aureus is an opportunistic Gram-positive bacterium which causes a wide variety of diseases ranging from minor skin infections to potentially fatal conditions such as pneumonia, meningitis and septicemia. The pathogen is a leading cause of nosocomial acquired infection, a problem exacerbated by the existence of methicillin and glycopeptide antibiotic resistant strains which are becoming exceedingly difficult to treat. Alanine racemase (Alr) is a pyridoxal-5’-phosphate dependent enzyme which catalyzes reversible racemization between enantiomers of alanine. As D-alanine is an essential component of the bacterial cell wall peptidoglycan, inhibition of Alr is lethal to prokaryotes. Additionally, while ubiquitous amongst bacteria, this enzyme is absent in humans and most eukaryotes, making it an excellent antibiotic drug target. There are two alanine racemase isozymes in S. aureus; Alr, which is constitutively expressed, and a catabolic alanine racemase (DadX), which is inducible by L-alanine. Current Alr inhibitors such as D-cycloserine are reminiscent of the alanine substrate and inhibit the enzyme by binding covalently to the enzymes’ PLP cofactor. They therefore non-specifically target other enzymes that utilize this cofactor and are associated with severe toxicity during treatment. This emphasizes the need for the identification of new non substrate-like Alr inhibitors. This research focused on the use of S. aureus Alr (AlrSas) as a template for structure-based drug design studies. This first involved solving the native structure, and kinetically characterizing the enzyme. Following structure solution, enzyme inhibition and X-ray crystallographic studies of AlrSas with nine compounds identified through high-throughput screening (HTS) (Anthony et al., 2011; Ciustea et al., 2012; Lee et al., 2013) were performed. In addition, fragment-based screening studies using differential scanning fluorimetry (DSF) were performed to identify new non substrate-like Alr inhibitors. To our knowledge this is the first reported use of this drug discovery method being applied to an alanine racemase. AlrSas from the highly antibiotic resistant Mu50 strain was over-expressed in E. coli, following which it was purified using anion-exchange, hydrophobic interaction and size-exclusion chromatography. Following crystallization, the native structure was solved to 2.15 Å resolution. Comparison of AlrSas with various alanine racemases demonstrated a conserved overall fold with the enzyme sharing most similarity with those from other Gram-positive bacteria. Structural examination indicated that the active site binding pocket, dimer interface and active site entryway of the enzyme were potential targets for structure-aided inhibitor design. AlrSas was kinetically characterized, and a comparison with selected alanine racemases indicated three orders of magnitude difference in their kinetic constants. In addition, the second alanine racemase isozyme from S. aureus (DadXSas) was studied. DadXSas from the MRSA252 strain was…