Structure-Based Drug Design for Carbonic Anhydrases & Membrane Interactions of Human Visinin-Like Protein-1 (VILIP-1)
MetadataShow full item record
Part A: Structure-Based Drug Design for Carbonic Anhydrases In humans there are twelve carbonic anhydrase (CA) isozymes that possess catalytic activity for the reversible hydration of carbon dioxide (Supuran, & Scozzafava, 2007). Carbonic anhydrases (CAs) underpin vital physiological and pathological processes and are so pharmaceutical targets for a variety of diseases. The recent findings in CA research were the validation of transmembrane human CA IX and human XII proteins as targets for cancer chemotherapy. Studies have shown that the specific targeting of CA IX (or XII) can lead to an effective anti-cancer therapy, especially for hypoxic tumours. Chapter one of this thesis is the introduction to carbonic anhydrases and the catalytic and inhibition mechanisms of different CA families. It has appeared within the literature, highlighting the involvement of CAs in a wide range of physiological functions, which makes them notable as targets for clinical inhibitors (Supuran & Scozzafava, 2007; Liljas, Hakansson, Jonson & Xue, 1994; Hilvo et al, 2008). This chapter introduces the classical recognition moiety needed for the inhibitor compounds in order to recognise and bind to the active site of CA, which is the aromatic or heteroaromatic sulfonamide moiety (R-SO2NH2). They operate through coordination of the sulfonamide anion to the active site zinc cation, replacing thez inc-bound hydroxyl anion, thereby impeding the endogenous reaction. Typically, 'tail' groups are appended to the aromatic sulfonamide anchor with the aim of improving potency and optimise d pharmacokinetic properties of CA inhibitors. Varying tail moieties can be appended to th e aromatic sulfonamide anchor via a triazole linker. Chapter two is a brief description of the objectives of this thesis. This chapter tries to convey an idea about the proteins involved in the thesis and reasons as to why these enzymes were investigated, with detailed descriptions presented in the relevant chapters. Chapter three presents a thorough study of the X-ray crystallographic structures of different CA II- inhibitor complexes. The three-dimensional structures of these CA inhibitor compounds with human CA II enabled us to understand their interactions with the active site residues of CA II, and thus to determine the structural features of th e ligands responsible for their weak or strong CA inhibition.T he interactions of all of these compounds with CA II were compared to the interactions and properties of topirama te with CA II active site residues. Topiramate is a low nanomolar inhibitor of human CA I I, and a well-established anti-epileptic drug. This is to compare the active site binding properties of the new CA ligands with those of topiramate when inside the human CA II active site. To obtain a preliminary idea on the stability of different CA II: ligand complexes, thermal denaturation studies of these complexes were performed using circular dichroism (CD) spectroscopy. Since the recent findings in CA research has shown the specific targeting o f transmembrane CA IX (or XII) leading to an effective anti-cancer therapy, ou r collaborators started working on the aspect to differentiate the inhibition of cancer-related , transmembrane CAs such as CA IX, CA XII from cytosolic CAs like CA I, CA II. Th e strategy was to attach a carbohydrate moiety to the high affinity zinc-binding aromat ic sulfonamide CA moiety leading to the formation of sulfonamide glycoconjugates with a sugar-aromatic-SO2NH2 motif. The -SO2NH2 zinc binding moiety of the glycoconjugates plays a key role in CA enzyme recognition and is essential for efficacy. The sugar tail is responsible for the high-water solubility of the compound and this hydrophilic group impairs the ability of the sulfonamide glycoconjugates to passively diffuse through lipid membranes, and this facilitates a selective or preferential inhibition of transmembran e CAs over cytosolic CAs, which would help in treating cancer. The stereochemical and structural variations of the carbohydrate moiety provide the opportunity for exploring th e differences among different CA active site architecture, thus yielding a neutral and wate r soluble CA inhibitor that has excellent potential as an isozyme selective inhibitor. Three different aromatic sulfonamides carrying a thio, sulfinyl or sulfonyl glucoside triazole tail moiety on the benzenesulfonamide CA pharmacophore have been cocrystallised with human CA II. Additional sugar-containing derivatives of similar topology have been co-crystallised with CA II to assess the effects of length variation and acetylation. Compounds different from the typical aromatic sulfonamide CA inhibitors were also co-crystallised with CA II, where the classic aromatic moiety of th e zinc-binding sulfonamide CA inhibitors is absent from these compounds, and instead a hydrophilic monosaccharide or disaccharide moiety has been introduced directly to th e primary sulfonamide group to get sugar-SO2NH2 motif. The fourth set of CA inhibitors used for co-crystallisation was sulfamate compounds (R-OSO2NH2). Sulfamates, like sulfonamides, are a group of strong CA inhibitors. While direct interactions between th e compounds and the protein were identified only in few cases, the current structure s provide clues as to where and how to extend the compounds in order to increase direc t interactions, and thus obtain an isozyme-specific inhibitor with improved pharmacologic al properties. Chapter four explores two other CA isozymes; human CA IX and carbonic anhydras e from Plasmodium falciparum. This chapter also present the attempts to successfully express and purify these proteins. Part B: Membrane Interactions of Human Visinin-like Protein-1 (VILIP-1) VILIPs are part of the subfamily of neuronal calcium sensor (NCS) protein. All members of NCS protein family are EF-hand proteins, and they share the characteristic feature of N-terminal myristoylation as well as the calcium-myristoyl switch. When calcium levels elevate, NCS proteins undergo the calcium-myristoyl switch which is the central mechanism of their involvement in cellular calcium signalling. Previous studies have shown that the membrane association of proteins by a myristoyl group alone is weak and requires other interactions between the protein and phospholipid on the membrane for stability. In addition to increasing the strength of the protein-membrane association, protein-phospholipid interactions also help to target proteins to different subcellullar domains (Braunewell et al, 2010). Chapter one provides a background to NCS proteins, particularly VILIP-1. The chapter highlights the functional and structural aspects of the proteins in this family. Chapter two presents the objectives of this part of the thesis. The aims include expression and purification of VILIP-1 proteins (unmyristoylated, myristoylated and VILIP-1 mutant S6A/K7A), protein folding experiments of these proteins in the absence and presence of calcium ions, and the monolayer adsorption experiments using Langmuir surface film balance. Chapter three explains the expression and purification details of the recombinant VILIP-1 proteins; unmyristoylated VILIP-1, myristoylated VILIP-1 and VILIP-1 mutant S6A/K7A. Ser6 and Lys7 are two of the main N-terminal residues found to be involved in forming interactions with membrane phospholipids, which in turn, help in protein-membrane association. These two residues have been replaced using the neutralcharged alanine to investigate the importance and the effects on the protein-membrane association in their absence. Chapter four of this thesis explores the thermal stability of VILIP-1 proteins. Four separate experiments were performed; unmyristoylated VILIP-1 in the absence of calcium, unmyristoylated VILIP-1 in the presence of calcium, myristoylated VILIP-1 in the absence of calcium, and myristoylated VILIP-1 in the presence of calcium. Quaternary structure of VILIP-1 mutant S6A/K7A protein in solution was also determined using SEC-MALLS. Chapter five experimentally determines the postulated interaction of VILIP-1 with different PIP derivatives. Studies have shown VILI...
Master of Philosophy (MPhil)
School of Biomolecular and Physical Sciences
Item Access Status