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dc.contributor.advisorKrause, Kurt
dc.contributor.authorScaletti, Emma Rose
dc.date.available2014-07-01T20:59:31Z
dc.date.copyright2014
dc.identifier.citationScaletti, E. R. (2014). S. aureus alanine racemase: ‘A target for structure-based drug design’ (Thesis, Doctor of Philosophy). University of Otago. Retrieved from http://hdl.handle.net/10523/4879en
dc.identifier.urihttp://hdl.handle.net/10523/4879
dc.description.abstractStaphylococcus 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 over-expressed in E. coli, following which it was purified to homogeneity by IMAC and size-exclusion chromatography. DadXSas was crystallized, however these crystals were severely twinned and diffracted poorly, and thus a structure did not result from this research. Kinetic characterization of DadXSas was performed and compared to other alanine racemases in the literature. DadXSas was one of the slowest enzymes, and had the weakest affinity for both enantiomers of alanine out of all the published alanine racemases. Interestingly, the enzyme differed markedly to AlrSas, having a reaction rate two orders of magnitude lower. Analysis of the nine HTS molecules against AlrSas indicated that only six compounds inhibited the enzyme to an acceptable level (BAS, JFD, S14, TCRS-151, 1944, and TO5-13). These compounds inhibited the enzyme in the low micromolar range. Further IC50 determinations to test for the possibility of non-specific inhibition indicated that of the six compounds JFD and TO5-13 were the most likely to be inhibiting AlrSas by promiscuous mechanisms. X-ray crystallography studies did not yield the structure of AlrSas in complex with any of the six HTS compounds. Preliminary screening of AlrSas via DSF against the MayBridge fragment library identified 28 fragments which either significantly increased or decreased the Tm of the enzyme. Subsequent IC50 analysis against AlrSas indicated that three of these compounds inhibited the enzyme in the low millimolar range (CC42223, GK00199, and CC42201) and thus were significantly weaker inhibitors than the HTS compounds analyzed. Promiscuous inhibition tests indicated that the fragments were not eliciting their inhibitory effects by general protein aggregation. X-ray crystallography studies identified promising electron density consistent with the presence of these fragments, which were subsequently modeled into the structures. CC42201 and CC42223 were both located in the active site, where they made similar interactions. In contrast, the fragment GK00199 did not bind in the active site of AlrSas, but was instead located in a small cavity on the surface of the protein. The location of this fragment could represent a possible allosteric site on the enzyme. Overall, this study demonstrated fragment-based drug discovery to be a viable strategy for identifying non substrate-like inhibitors of AlrSas.
dc.language.isoen
dc.publisherUniversity of Otago
dc.rightsAll items in OUR Archive are provided for private study and research purposes and are protected by copyright with all rights reserved unless otherwise indicated.
dc.subjectalanine
dc.subjectracemase
dc.subjectcrystallography
dc.subjectfragment-screening
dc.titleS. aureus alanine racemase: "A target for structure-based drug design"
dc.typeThesis
dc.date.updated2014-07-01T05:01:58Z
dc.language.rfc3066en
thesis.degree.disciplineBiochemistry
thesis.degree.nameDoctor of Philosophy
thesis.degree.grantorUniversity of Otago
thesis.degree.levelDoctoral
otago.interloanyes
otago.openaccessAbstract Only
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