Protein kinases are important regulatory enzymes in signal transduction and in cell regulation. Understanding inhibition mechanisms of kinases is important for the further development of new therapies for cancer and inflammatory diseases. I have developed a statistical approach based on the Mantel test to find the relationship between the shapes of ATP binding sites and their affinities for inhibitors. My shape-based dendrogram shows clustering of the kinases based on similarity in shape. I investigate the pocket in terms of conservation of surrounding amino acids and atoms in order to identify the key determinants of ligand binding. I find that the most conserved regions are the main chain atoms in the hinge region and I show that the tetrahydropyran ring of staurosporine causes induced-fit of the glycine rich loop. I apply multiple linear regression to select distances measured between the distinctive parts of residues which correlate with the binding constants. This method allows me to understand the importance of the size of the gatekeeper residue and the closure between the first glycine of the GXGXXG motif and the aspartate of the DFG loop, which act together to promote tight binding to staurosporine. I also find that the greater the number of hydrogen bonds made by the kinase around the methylamine group of staurosporine, the tighter the binding to staurosporine. The website I have developed allows a better understanding of cross reactivity and may be useful for narrowing down the options for a synthetic strategy to design kinase inhibitors.

The chloride/proton antiporter ClC-5 is primarily expressed in the kidney where it aids in re-absorption of proteins from the glomerular filtrate. Functional disruption of ClC-5 causes Dent’s Disease – a renal condition characterized by proteinuria and kidney failure in a third of all cases. The majority of disease-causing mutations translate into premature truncations of the carboxy-terminal (Ct) region of ClC-5 and are predicted to disrupt the protein-protein interactions mediated by this domain. In this thesis, direct ATP binding to the two cystathionine β-synthase (CBS) domains of ClC-5 was demonstrated. ATP binding enhanced the global compactness of the ClC-5 Ct region likely through a clamping motion of the CBS domains around the nucleotide. Along with ATP, the sodium proton exchange regulatory factor 2 (NHERF2) also binds ClC-5; however, the molecular mechanism behind this interaction was unknown as ClC-5 lacked the PDZ binding motif traditionally localized at the Ct end of bait proteins. Here, we also identified a class I PDZ binding motif (657-660; TSII) within the internal sequence of ClC-5. Despite the buried position of this motif in the Ct peptide’s X-ray crystal structure (PDB: 2J9L), the high propensity of this region for dynamic flexibility prompted us to test whether it could mediate NHERF2 interactions. In support of this hypothesis, we demonstrated that the motif is transiently available to interact directly with NHERF2 in vivo and to enable an enhancement in receptor-mediated endocytosis in mammalian cells. Collectively, these results gave further evidence that the intracellular Ct region of ClC-5 serves as a hub to mediate interactions essential for its maturation, stability, and trafficking in renal epithelium, as well as providing further insights into the molecular basis of Dent’s Disease.

The chloride/proton antiporter ClC-5 is primarily expressed in the kidney where it aids in re-absorption of proteins from the glomerular filtrate. Functional disruption of ClC-5 causes Dent’s Disease – a renal condition characterized by proteinuria and kidney failure in a third of all cases. The majority of disease-causing mutations translate into premature truncations of the carboxy-terminal (Ct) region of ClC-5 and are predicted to disrupt the protein-protein interactions mediated by this domain. In this thesis, direct ATP binding to the two cystathionine β-synthase (CBS) domains of ClC-5 was demonstrated. ATP binding enhanced the global compactness of the ClC-5 Ct region likely through a clamping motion of the CBS domains around the nucleotide. Along with ATP, the sodium proton exchange regulatory factor 2 (NHERF2) also binds ClC-5; however, the molecular mechanism behind this interaction was unknown as ClC-5 lacked the PDZ binding motif traditionally localized at the Ct end of bait proteins. Here, we also identified a class I PDZ binding motif (657-660; TSII) within the internal sequence of ClC-5. Despite the buried position of this motif in the Ct peptide’s X-ray crystal structure (PDB: 2J9L), the high propensity of this region for dynamic flexibility prompted us to test whether it could mediate NHERF2 interactions. In support of this hypothesis, we demonstrated that the motif is transiently available to interact directly with NHERF2 in vivo and to enable an enhancement in receptor-mediated endocytosis in mammalian cells. Collectively, these results gave further evidence that the intracellular Ct region of ClC-5 serves as a hub to mediate interactions essential for its maturation, stability, and trafficking in renal epithelium, as well as providing further insights into the molecular basis of Dent’s Disease.

Malaria is a major public health problem that affects millions of lives globally. The increased burden of malaria requires new interventions that will address the eradication of the disease. Current interventions include vector control by using insecticide-treated bed nets and indoor residual spraying, and antimalarial drugs to control the parasite. Parasite resistance has been reported for the currently used effective antimalarial drugs. To pre-empt the impact of parasite resistance a continued development of new antimalarial drugs that have novel mechanisms of action should be pursued. Antimalarial drug discovery requires that potential antimalarial drugs should have different drug targets to those already targeted, to lower the chances of resistance. Potential antimalarial drugs should preferably provide a single radical cure to prevent reproduction at all life cycle stages. This study tested the effects of in silico designed compounds targeting plasmodial Ca2+- dependent protein kinases (CDPK) 1 & 4, FIKK kinases and bromodomain proteins on the Plasmodium parasite. These enzymes are involved in gene regulation and are important factors during gene transcription. In P. falciparum the gatekeeper kinases contain small hydrophobic pockets near the ATP-binding site. These hydrophobic pockets allow for selective inhibition of these proteins at the ATP-binding site. The compounds were tested in vitro to determine their antiplasmodial activity. These compounds are shown to be potential inhibitors of the intra-erythrocytic P. falciparum parasites as three of the compounds showed selective cytotoxic activity at less than 1 μM against the chloroquine sensitive laboratory strains (3D7 and NF54). Even though the proteins targeted by these compounds have been previously indicated to play a role at specific stages during the parasite’s life cycle, the compounds tested here were not able to target the sexual gametocyte stages of the Plasmodium parasite. Further optimisation of these compounds should be performed to improve activity against both the asexual and sexual stages of the parasites. / Dissertation (MSc)--University of Pretoria, 2017. / Pharmacology / MSc / Unrestricted

7 ABSTRACT Crystallization of the zebrafish P2X4 receptor in both open and closed states revealed conformational differences in the ectodomain structures, including the dorsal fin and left flipper domains. The role of these domains in forming of ATP-binding pocket and receptor function was investigated by using alanine scanning mutagenesis of the R203- L214 (dorsal fin) and the D280-N293 (left flipper) sequences of the rat P2X4 receptor and by examination of the responsiveness to ATP and orthosteric analog agonists 2- (methylthio)adenosine 5'-triphosphate, adenosine 5'-(γ-thio)triphosphate, 2'(3'-O-(4- benzoylbenzoyl)adenosine 5'-triphosphate, and α,β-methyleneadenosine 5'- triphosphate. ATP potency/efficacy was reduced in 15 out of 26 alanine mutants. The R203A, N204A, and N293A mutants were essentially non-functional, but receptor function was restored by ivermectin, an allosteric modulator. The I205A, T210A, L214A, P290A, G291A, and Y292A mutants exhibited significant changes in the responsiveness to orthosteric analog agonists. In contrast, the responsiveness of L206A, N208A, D280A, T281A, R282A, and H286A mutants to analog agonists was comparable to that of the wild type receptor. These experiments, together with homology modeling, indicate that residues of the first group located in the upper part of...