Thesis (Ph. D.)--University of Washington, 2004. / Vita. Includes bibliographical references (leaves 138-149).

Src Homology 3 (SH3) domains are small protein- protein interaction domains that bind to proline-rich peptides, mediating a range of important biological processes. Because the deregulation of events involving SH3 domains forms the basis of many human diseases, the SH3 domains are appealing targets for the development of potential therapeutics. Previously in the field, no examples of entirely small-molecule ligands for the SH3 domains have been identified. However, in our research group, we have discovered a class of heterocyclic compounds that bind to the Tec SH3 domain at conserved residues in the proline-rich peptide binding site, with weak to moderate affinity. The highest affinity of these was 2- aminoquinoline (Kd = 125 mM). In this thesis, a range of approaches are described, that were intended to contribute towards development of higher affinity small-molecule ligands for the Tec SH3 domain. Preliminary experiments, involving testing a variety of compounds structurally related to 2- aminoquinoline, provided new structure activity information, and led to a better understanding of the 2-aminoquinoline/SH3 domain binding event. The major component of this thesis is a thorough investigation into the synthesis of a range of 2- aminoquinoline derivatives. N-Substituted- 2-aminoquinolines were synthesised, however these compounds bound the SH3 domain with slightly lower affinity than 2-aminoquinoline. 6- Substituted-2-aminoquinolines were subsequently prepared, and ligands were identified with up to six-fold improved affinity relative to 2-aminoquinoline, and enhanced selectivity for the Tec SH3 domain. The techniques used for the ligand binding studies were Nuclear Magnetic Resonance (NMR) chemical shift perturbation and Fluorescence Polarisation (FP) peptide displacement assays. As part of the ligand binding studies, it was intended that the 3D tructure of a 2- aminoquinoline ligand/SH3 complex would be obtained using NMR methods, provided that a ligand was identified that bound the SH3 domain in slow exchange on the NMR timescale. However, this goal was not fulfilled. Despite this, the work presented in this thesis provides a solid foundation for the development of potent 2-aminoquinoline ligands for SH3 domains, with engineered specificity. / Thesis (Ph.D.)--School of Molecular and Biomedical Science, 2005.

Thesis (M.Sc.) - Carleton University, 2005. / Includes bibliographical references (p. 134-145). Also available in electronic format on the Internet.

Thesis (M.Sc.) - Carleton University, 2005. / Includes bibliographical references (p. 98-108). Also available in electronic format on the Internet.

Thesis (Ph.D.)--University of Alberta, 2010. / A thesis submitted to the Faculty of Graduate Studies and Research in partial fulfillment of the requirements for the degree of Doctor of Philosophy, Medical Sciences, Biomedical Engineering, Department of Electrical and Computer Engineering. Title from pdf file main screen (viewed on April 11, 2010). Includes bibliographical references.

Thesis (Ph. D.)--University of Washington, 1999. / Vita. Includes bibliographical references (leaves 110-113).

Thesis (Ph. D.)--Carleton University, 2001. / Also available in electronic format on the Internet.

Thesis (Ph.D.)--Hong Kong University of Science and Technology, 2005. / Includes bibliographical references (leaves 217-253). Also available in electronic version.

Amino acid deprivation of Escherichia coli results in the accumulation of guanosine 5'-triphosphate 3'-diphosphate and guanosine 3', 5'-bispyrophosphate, collectively designated (p)ppGpp. These nucleotides are synthesized by a ribosome-associated enzyme encoded by the relA gene and are thought to represent starvation stress signal molecules. They may mediate the global reorganization of cellular metabolism, known as the stringent response, that is characteristic of starving bacteria and which apparently represents a survival strategy. In this dissertation, the following aspects of the stringent response are characterized: (i) the temperature phenotypes of relA mutants; (ii) the C-terminal domain of RelA; and (iii) the role of RelC (ribosomal protein L11) in the regulation of RelA. All three of the commonly used relA mutant alleles of E. coli, relA1, relA2, and ∆relA251::kan, conferred temperature-sensitive (ts) phenotypes. The temperature sensitivity was associated with decreased thermotolerance, and relA mutants were killed at temperatures as low as 42°C. The ts phenotypes were suppressed by increasing the osmolarity of growth media and by certain mutant alleles of rpoB, the gene encoding the β-subunit of RNA polymerase, suggesting a defect in transcription. DNA in heat-shocked wild type bacteria was initially relaxed but the normal level of negative supercoiling was restored within 10 min after heat shock. In contrast, DNA in heat-shocked relA mutants remained relaxed. This relA-associated defect in DNA negative supercoiling was suppressed by increased medium osmolarity. Furthermore, the re/A-mediated ts phenotype was suppressed by low concentrations of novobiocin, a specific inhibitor of the B subunit of DNA gyrase. Moreover, low concentrations of novobiocin restored DNA negative supercoiling in the relA mutant at high temperature. Based on previous reports, it is proposed that low concentrations of novobiocin induce the synthesis of the DNA gyrase A and B subunits, and the resulting increase in DNA gyrase activity restores normal supercoiling at high temperature. Collectively, the data suggest that relA mutants are unable to efficiently transcribe key genes required for thermotolerance, and this defect is related to their inability to restore negative supercoiling of DNA at higher temperatures. In addition, the proposed defect in transcription may be related to the observation that ppGpp binds to the p-subunit of RNA polymerase. The portion of relA encoding the C-terminal half of RelA (starting at amino acid 455), designated 'RelA, was subcloned. Overexpression of 'RelA relaxed the stringent response by inhibiting (p)ppGpp synthesis during amino acid deprivation. 'RelA represented the ribosome-binding domain, and when overexpressed, 'RelA somehow replaced RelA on ribosomes. The 'RelA ribosome-binding domain was further localized to a region between amino acids 455 to 682 with the main binding activity in a fragment extending from amino acids 560 to 682. Several criteria were used to establish the fact that 'RelA also mediated the formation of homodimers. These included co-purification of RelA and 'RelA, glutaraldehyde protein crosslinking, and analysis by nondenaturing polyacrylamide gel electrophoresis. The dimerization domain overlapped with the ribosome-binding domain. Affinity blotting assays using 'RelA as a probe revealed RelA and 'RelA as the only proteins in crude cell extracts that bound 'RelA. Therefore, these studies failed to identify the ribosomal components that interact with RelA. Amino add-deprived rplK (previously known as relC) mutants of E. coli cannot activate ribosome-bound RelA and consequently exhibit relaxed phenotypes. The rplK gene encodes ribosomal protein L11, suggesting that L11 is involved in regulating the activity of RelA. The overexpression of derivatives of rplK that contained short N-terminal deletions that eliminated the proline-rich helix resulted in relaxed phenotypes. In contrast, bacteria overexpressing normal L11 exhibited a typical stringent response. The L11 mutant proteins were incorporated into ribosomes. A derivative in which Pro22 was changed to Leu22 was constructed by site-directed mutagenesis. This amino add substitution was sufficient to confer a relaxed phenotype when it was overexpressed. A variety of methods were used in attempts to demonstrate a direct interaction between L11 and RelA, but all yielded negative results. These results indicate that the N-terminal proline-rich helix, and Pro22 in particular, is directly involved in activating RelA activity during amino acid deprivation. The mechanism apparently does not involve a direct interaction between RelA and L11 and is presumably mediated by another ribosomal component. / Graduate

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