Tissue transglutaminase (TG2) is a calcium-dependent enzyme that natively catalyses the formation of isopeptidic bonds between protein- or peptide-bound glutamine and lysine residues. Physiologically, it is ubiquitously expressed in tissues, with roles in cellular differentiation, extracellular matrix stabilisation, and apoptosis, among others. However, its unregulated activity has been associated with various pathologies including fibrosis, cancer and celiac disease. Since most pathologies are associated with an increased transamidation activity, efforts have been directed towards the development of TG2 inhibitors. In this context, the work described in this thesis is centred on reversible inhibitors, building on recent work done within the Keillor group in two directions, namely localisation and potency. In a localisation-driven approach, we developed a photolabile derivative of a known reversible inhibitor, in order to form a covalent bond with the enzyme and determine the inhibitor’s binding site. In tandem, we optimised a protocol for the expression of TG2 incorporating ArgΔ10 and LysΔ8, amino acids that are 13C- and 15N-labelled to provide a mass shift of 10 and 8 Da, respectively, compared to the corresponding unlabelled amino acids. This “heavy” TG2 was developed as a tool for reference in the analysis of the tryptic digest of labelled protein. In a potency-driven approach, based on the observation that previous trans cinnamoyl inhibitor scaffolds were susceptible to nucleophilic attack by glutathione, we developed a bis(triazole) scaffold with reduced electrophilicity. The preparation of a small library of compounds showed that this scaffold demonstrates a preference for electron-withdrawing substituents, such as nitro groups. Continuing in a potency-driven approach, and inspired by work done in the identification of glutathione-resistant scaffolds, we studied a new alkynyl scaffold. While still susceptible to glutathione addition, these compounds showed a marked improvement in potency, with the lead compound having an IC50 of 930 nM and being established as a competitive inhibitor with a Ki of 420 nM, our most potent reversible inhibitor to date. Furthermore, this scaffold also produced an inhibitor lacking nitro groups (to limit eventual cellular toxicity), but maintaining good potency, with an IC50 value of 3.03 μM.

京都大学 / 0048 / 新制・課程博士 / 博士(理学) / 甲第19883号 / 理博第4210号 / 新制||理||1605(附属図書館) / 32960 / 京都大学大学院理学研究科化学専攻 / (主査)教授 三木 邦夫, 教授 杉山 弘, 教授 藤井 紀子 / 学位規則第4条第1項該当 / Doctor of Science / Kyoto University / DGAM

The biodesulfurization 4S-pathway is a promising complementary enzymatic approach to remove sulfur from recalcitrant thiophenic derivatives in petroleum products that remain from conventional hydrodesulfurization method without diminishing the calorific value of oil. The final step of this pathway involves the carbon-sulfur bond cleavage from HBPS, and the production of the final products 2-hydroxybiphenyl (HBP) and sulfite, has been recognized as the rate-limiting step, partially as a result of product inhibition. However, the mechanisms and factors responsible for product inhibition in the last step have not been fully understood. In this work, we proposed a computational investigation using molecular dynamic simulations and free energy calculations on 2’-hydroxybiphenyl-2-sulfinate (HBPS) desulfinase (DszB) with different bound ligands as well as different solvent conditions to develop a fundamental understanding of the molecular-level mechanism responsible for product inhibition. Based on available crystal structures of DszB and biochemical characterization, we proposed a “gate” area close to substrate binding site of DszB is responsible for ligand egress and plays a role in product inhibition. We have conducted biphasic molecular dynamic simulations to evaluate the proposed gate area functionality. Non-bonded interaction energy analysis shows that hydrophobic residues around the gate area produce van der Waals interactions inhibiting translocation through the gate channel, and therefore, the molecules are easily trapped inside the binding site. Umbrella sampling molecular dynamics was performed to obtain the energy penalty associated with gate conformational change from open to close, which was 2.4 kcal/mol independent of solvent conditions as well as bound ligands. Free energy perturbation calculations were conducted for a group of six selected molecules bound to DszB. The selections were based on functional group representation and to calculate binding free energies that were directly comparable to experimental inhibition constants, KI. Our work provides a fundamental molecular-level analysis on product inhibition for the biodesulfurization 4S-pathway.

How the linear protein chains fold into their three-dimensional active conformation is one of the remaining puzzles of modern science. Other than molecular chaperones, ribosome - the cellular protein synthesis machinery, has also been implicated in protein folding. The active site of protein folding activity of the ribosome (PFAR) is in the domain V of the main RNA component of the large ribosomal subunit, which also constitutes the peptidyl transferase center. We have characterized the mechanism of PFAR using ribosomes or ribosome-borne folding modulators (RFMs) and human carbonic anhydrase I (HCA) as a model system. RFMs from all three kingdoms of life showed PFAR.  By multiple addition of the denatured protein in the refolding assay we demonstrate that the RFMs can recycle efficiently to assist refolding of a new batch of denatured protein. The turnover of the RFMs, which includes release of the protein substrate, takes milliseconds. Furthermore, fast kinetics of HCA refolding suggests that an early folding intermediate is the substrate for PFAR. Our results demonstrate for the first time that PFAR is catalytic. It was shown that two anti-prion drugs 6AP and GA specifically inhibit PFAR by binding to the domain V of the 23S / 25S rRNA. Using UV-crosslinking followed by primer extension we have identified the interaction sites of 6AP on domain V of 23S rRNA, which overlap with the protein binding sites, and are sensitive to mutagenesis. We find that 6AP and GA inhibit PFAR by direct competition with the substrate protein for the binding sites. Also, 6AP derivatives inhibit PFAR in the same order as their antiprion activity, 6AP8CF3 > 6AP8Cl > 6AP > 6APi. These results suggest involvement of PFAR in prion processes. To clarify the role of PFAR in prion processes, we studied HET-s prion aggregation in the presence of domain V/ IV/II of rRNA. The rRNAs, especially domain V rRNA not only reduced HET-s aggregation, but also changed the morphology of the HET-s fibrils, which became shorter and less compact. These results show that PFAR actively prevents large amyloid aggregation and thus, possibly influence prion propagation.

The prokaryotic Erwinia chrysanthemi ligand-gated ion channel (ELIC) is competitively inhibited by acetylcholine (Pan et al., 2012). Acetylcholine is the native agonist of the structurally related family of eukaryotic acetylcholine receptors, which like ELIC are pentameric ligand-gated ion channels. To understand the opposite effect upon acetylcholine binding between ELIC and acetylcholine receptors, we used statistical coupling analysis to predict mutations necessary for installing acetylcholine agonism into ELIC. Statistical coupling analysis was performed on the acetylcholine binding protein from Lymnaea stagnalis. This protein is a structural surrogate for the agonist binding domain of acetylcholine receptors, for which a high-resolution structure in complex with acetylcholine is available. Our analysis identified a group of statistically coupled residues that comprises several amino acids previously implicated in acetylcholine agonism of acetylcholine receptors. Mapping these residues onto ELIC revealed 15 residue discrepancies, 4 of which were chosen for initial mutagenesis based upon their proximity to the known agonist binding site. Electrophysiological characterization of ELIC mutants indicates that the potency of the native agonist, cysteamine, is decreased, highlighting the optimized role wild-type residues serve in native agonism. None of the mutants were activated by acetylcholine, however the double mutant A75D/F133W abolished competitive antagonism by acetylcholine, and instead led to acetylcholine dependent potentiation of cysteamine-induced currents. This work demonstrates the ability of statistical coupling analysis to identify functionally important residues in pentameric ligand-gated ion channels and reveals that acetylcholine can be converted from a competitive antagonist into a potentiator, by installing two residues present in acetylcholine receptors.

No / The protein O6-alkylguanine-DNA alkyltransferase (Atase) is responsible for the repair of DNA lesions generated by several clinically important anti-cancer drugs; this is manifest as active resistance in those cancer cell lines proficient in Atase expression. Novel O6-substituted guanine analogues have been synthesized, bearing acidic, basic and hydrogen bonding functional groups. In contrast to existing O6-modified purine analogues, such as methyl or benzyl, the new compounds were found to resist repair by Atase even when tested at concentrations much higher than O6-benzylguanine, a well-established Atase substrate active both in vitro and in vivo. The inactivity of the new purines as covalent substrates for Atase indicates that agents to deliver these groups to DNA would represent a new class of DNA-modifying drug that circumvents Atase-mediated resistance.

Very little is known about why and how the Parkinson's disease (PD) neurodegenerative process begins and progresses. In the course of developments for treatment of PD, the discovery of the inhibition of monoamine oxidase (MAO B) was a conceptual breakthrough, and has now been firmly established. MAO B has also been implicated in the neurodegenerative processes resulting from exposure to xenobiotic amines. For example, MAO B catalyzes the first step of the bioactivation of the parkinsonian inducing pro-neurotoxin, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). Additional insight into the mechanism of catalysis of MAO B and the mechanism of neurotoxicity by MPTP is therefore very valuable in the pursuit of the treatment of PD. / Thesis (Ph.D. (Pharmaceutical Chemistry))--North-West University, Potchefstroom Campus, 2008.

Very little is known about why and how the Parkinson's disease (PD) neurodegenerative process begins and progresses. In the course of developments for treatment of PD, the discovery of the inhibition of monoamine oxidase (MAO B) was a conceptual breakthrough, and has now been firmly established. MAO B has also been implicated in the neurodegenerative processes resulting from exposure to xenobiotic amines. For example, MAO B catalyzes the first step of the bioactivation of the parkinsonian inducing pro-neurotoxin, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). Additional insight into the mechanism of catalysis of MAO B and the mechanism of neurotoxicity by MPTP is therefore very valuable in the pursuit of the treatment of PD. / Thesis (Ph.D. (Pharmaceutical Chemistry))--North-West University, Potchefstroom Campus, 2008.

Very little is known about why and how the Parkinson's disease (PD) neurodegenerative process begins and progresses. In the course of developments for treatment of PD, the discovery of the inhibition of monoamine oxidase (MAO B) was a conceptual breakthrough, and has now been firmly established. MAO B has also been implicated in the neurodegenerative processes resulting from exposure to xenobiotic amines. For example, MAO B catalyzes the first step of the bioactivation of the parkinsonian inducing pro-neurotoxin, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). Additional insight into the mechanism of catalysis of MAO B and the mechanism of neurotoxicity by MPTP is therefore very valuable in the pursuit of the treatment of PD. / Thesis (Ph.D. (Pharmaceutical Chemistry))--North-West University, Potchefstroom Campus, 2008.

The glucose transport protein, GLUT1, is highly expressed in rapidly proliferating cells, including cancer cells, while decreased GLUT1 levels are found in diseases such as GLUT1 deficiency syndrome and Alzheimer’s. There is increased interest in developing GLUT1 inhibitors as novel anticancer therapeutics, and the discovery of compounds that directly stimulate GLUT1 function. This work investigates how small molecules stimulate and/or inhibit GLUT1-mediated glucose transport, either directly or through the AMPK pathway. Using sugar transport assays and docking analyses to explore Ligand–GLUT1 interactions and specificity of binding, we show that: 1) Ligands inhibit GLUT1 by competing with glucose for binding to the exofacial or endofacial sugar binding sites; 2) Subsaturating inhibitor concentrations stimulate sugar uptake; 3) Ligands inhibit GLUT1–, GLUT3– and GLUT4–mediated sugar uptake in HEK293 cells; and 4) Inclusion of a benzonitrile head group on endofacial GLUT1 inhibitors confers greater inhibitory potency. Furthermore, we investigated AMPK-regulated GLUT1 trafficking in cultured blood-brain barrier endothelial cells, and show that inhibition of GLUT1 internalization is not responsible for increased cell surface levels of GLUT1 observed with AMPK activation in these cells. This study provides a framework for screening candidate GLUT1 inhibitors for specificity, and for optimizing drug design and delivery. Our data on transport stimulation at low inhibitor concentrations support the idea that GLUT1 functions as a cooperative oligomer of allosteric alternating access subunits.