Thesis (MScMed)--University of Stellenbosch, 2007. / Bibliography. Also available via the Internet.

Thesis (Ph. D.)--Columbia University, 1924. / Vita. eContent provider-neutral record in process. Description based on print version record. Bibliography: p. 23.

Thesis (M.SC.) - Carleton University, 2007. / Includes bibliographical references (p. 101-106). Also available in electronic format on the Internet.

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.

The blastoderm embryo of Drosophila melanogaster consists of a unicellular syncytium with a large number of peripheral nuclei. The cytoplasm surrounding each peripheral nucleus is compartmentalised into apical periplasm above each nucleus and basal periplasm below it. The expression of different genes in the syncytial blastoderm is crucial for the genetic control of development. The pair-rule genes are involved in controlling the pattern of metamerisation of the embryo. Pair-rule mRNAs are expressed in alternate metameres, in a pattern of stripes. Within each stripe, mRNA is found in the apical periplasm of the syncytial blastoderm. By analysing the distribution of mRNA of a number of hybrid constructs, I show that the 3' untranslated part of three pair-rule genes are required for the apical localisation of their transcripts. A 1.2kb region in the 3' end of fushi tarazu (ftz), a 700bp region in the 3' end of hairy (h) and a 160bp fragment of the 3' untranslated part of the even-skipped (eve) pair-rule gene are shown to contain apical localisation signals. I show that the mechanism of apical localisation is unlikely to involve a cytoplasmic process and that the 3' untranslated part of the bicoid (bed) gene contains sequences necessary for apical localisation. I propose that apical localisation involves a nuclear mechanism which exports mRNA from the apical side of the nuclear membrane. I demonstrate that apical localisation is achieved by an RNA-mediated process and not by a DNA-mediated mechanism. Finally, I demonstrate that the intracellular localisation of transcripts encoding cytoplasmic proteins influences the distribution of the protein in the periplasm. I propose that the function of apical localisation is to limit the diffusion of pair-rule proteins so that the pattern of protein expression resembles precisely the transcriptional domain.

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. 98-108). Also available in electronic format on the Internet.

Thesis (Ph.D. (Biochemistry and Microbiology)) - Rhodes University, 2008

Ligand-observed NMR spectroscopy is frequently employed in early-stage drug discovery, often as an initial screen to narrow the field of potential drug-like molecules. However, its use is limited to this early stage. More information regarding binding mode can be extracted from these experiments via quantification, and this should help extend the remit of these experiments beyond simple screening functions. Initially, it was shown that the amount of signal that could be produced from an STD NMR experiment could be dramatically increased by careful consideration of the selective saturation pulse. By systematically shortening the Gaussian pulse and positioning it at specific offset positions, it was shown that these dramatic increases in signal are genuine and need not result in false positives. Quantitative STD NMR spectroscopy as applied to Hsp90 and a series of small fragment ligands provided evidence to suggest that the precise inter-atomic distances between a protein and ligand within a crystal structure correlate with both initial rates of STD build up, and T1-adjusted STD values. This precise correlation has implications for chemotype clustering and initial binding mode selection, something which should be useful in the absence of a crystal structure. Taking the same quantitative principles and applying to LOGSY experiments elucidated another, discrete property of protein-ligand binding. Examining the ‘LOGSY difference’ signal for protons of a ligand allows us to see what protons are in close proximity to conserved, bound water at the protein-ligand binding interface. This is fundamentally different to the information gained from STD experiments. Applying the insights to a protein of a different nature, Ras, it was shown that quantitative STD can be applied to proteins of both different size and structure. Furthermore, more evidence was acquired to suggest that conserved, bound water in the binding site really is responsible for generating LOGSY signal. In the absence of these molecules, as in Ras, proximity of a proton to an exchangeable tends to dominate. In addition we were able to show that these quantitative methods can be used together to help eliminate incorrect computationally generated docking poses. The work presented in this thesis provides evidence for the advantages of STD and LOGSY NMR spectroscopy in fragment-based drug discovery. The information that can be extracted from relatively simple ligand-observed NMR experiments should be used to provide more evidence at an earlier stage of the drug discovery process, hopefully reducing late-stage attrition and helping us get to the therapeutic drug molecules we need a little more quickly.

<p>ZMPSTE24 is a seven transmembrane domain zinc metalloprotease that resides in the ER and inner nuclear membranes of mammalian cells. The crystal structures of both the mammalian and yeast homologs, ZMPSTE24 and Ste24, respectively, were solved recently and revealed a common novel structure. Both structures contain a large chamber of mixed hydrophobicity that is capped on both sides. The canonical catalytic HExxH zinc-binding motif lies inside the chamber. Defects in the enzymatic function of human ZMPSTE24 have been shown to cause premature aging disorders. In addition to the well-defined role ZMPSTE24 and Ste24 play in the maturation of prelamin A in mammals and <b>a</b>-factor in yeast, both proteins have been proposed to play protective roles in Type 2 diabetes and viral infections by interactions with the cellular translocon. ZMPSTE24 can also be inhibited by several common HIV aspartyl protease inhibitors, possibly causing the frequent and common side-effects of these prescribed drugs. As of now, no precise location for substrate binding has been identified in either ZMPSTE24 or Ste24. Thus, the goal of this project is to localize residues in the enzyme that are important for substrate binding. The yeast homolog Ste24 was used as a model system as it functionally complements the mammalian enzyme and can be reliably cloned, overexpressed, and purified in an active form. </p> <p>Three approaches were taken to directly determine the <i>K_D </i>values for substrates of Ste24. The ability to perform a direct analysis of <i>K_D</i> values of Ste24 mutations was successfully optimized using microscale thermophoresis. Through <i>K_D</i> analysis, the Ste24 mutation G255A, while completely inactive, does not prevent substrate binding. Alternatively, L441A and L410A mutations showed both an increase in thermal stability and a decrease in binding affinity, that could explain their lower activity levels. A photoaffinity labeling-based proteomics experiment was utilized to precisely locate the site of the prenyl group to a hydrophobic patch lying just under a side portal of Ste24, near K234, during the -aaXing cleavage of <b>a</b>-factor maturation. To assess the method of inhibition of HIV protease inhibitors on Ste24 the conserved aspartate mutants were explored. All mutations of these aspartate residues resulted in a severe loss of Ste24 function and instability of the protein.</p>