Cytosolic glutathione transferases (GSTs) are a large family of enzymes that play an important role in detoxification of xenobiotics. They catalyse the conjugation of the glutathione tripeptide (GSH) to a wide range of toxic electrophilic acceptors. The overall 3D folds and architectures of the catalytic sites of many GSTs are conserved. They are composed of a well conserved glutathione binding site (G-site) and a promiscuous hydrophobic binding site (H-site). The 3D structure and ligand specificity has allowed the sub-classification of the multiple isoforms within the soluble GST superfamily. GSTs are involved in the drug detoxification and so are the target of medicinal chemistry programmes but it has proven difficult to generate isoform-specific inhibitors due to their inherent promiscuity. In this project, Venughopal Bhat (University of Edinburgh, laboratory of Dr. Mike Greaney) and I have explored a new platform to probe enzyme specificity. Protein-directed dynamic combinatorial chemistry (DCC) allows the assembly and amplification of a ligand within the confines of a binding site. DCC was used as a tool to explore the promiscuous H-site of four eukaryotic GSTs. I purified recombinant forms of SjGST, hGST P1-1, mGST M1-1 and mGST A4-4 from E. coli and assayed them with the universal, synthetic GST substrate 1-chloro-2,4-dinitrobenzene (CDNB). Venughopal Bhat prepared a ten-member, thermodynamically-controlled, dynamic combinatorial library (DCL) of acyl hydrazones from a 1-chloro-2-nitrobenzene aldehyde and ten acylhydrazides. This DCL was incubated with each of the four GST isozymes (spanning diverse classes) and distinct amplification effects were observed for SjGST and hGST P1-1. I subsequently carried out several biophysical experiments in an attempt to rank each of the ligands. These experiements, coupled with molecular modelling, provided insight into the basis of the observed selectivity. Bacterial GSTs are thought to play a role in primary metabolism and display a different GSH-conjugation mechanism compared to the eukaryotic GSTs. A recombinant form of the beta-class GST from the pathogenic bacterium Burkholderia cenocepacia was isolated, purified and biochemically characterised. The same ten-member acylhydrazone DCL was interfaced with the bacterial GST which was shown to amplify a hydrophobic library member that shared structural features with the known substrate 2-hydroxy-6-oxo-6-phenyl-2,4-dienoate (HOPDA). With the collaboration of Venughopal Bhat, I attempted to explore the putative active site of a GST-like protein with an unknown function using the same DCL. Although no amplification was observed, a new aldehyde template was suggested for future DCC experiments on this protein. GSTs are widely employed in biotechnology as protein fusion tags to enhance target protein solubility coupled with a facile enzyme assay. Manish Gupta and Juan Mareque-Rivas (University of Edinburgh) used the N-terminal, hexahistidine-tagged SjGST to demonstrate that quantum dots (QDs) coated with nitrilotriacetic acid (NTA) bound to Ni2+ ions can be used to reversibly and selectively bind, purify, and fluorescently label a His6-tagged GST in one step with retention of enzymatic activity. For this prupose, I purified and characterized both the untagged and hexahistidinetagged – SjGST prior to their experiments.

The first half of this dissertation describes the synthesis and biological activities of a series of amino aldehydes; which were derivatives of the basic amino acids, arginine, lysine and ornithine. The synthesis of the amino aldehydes was complicated by the difficulty of producing an intermediate oxidation state (the aldehyde) in the presence of two other functional groups (the α-amino, and the side chain functionality). The amino aldehydes were of biological interest due to the fact that they were inhibitors of the proteolytic enzymes called kallikreins. The kallikreins are known to be involved with the renin-angiotensin system, arginine vasopressin, and the prostaglandins, in the regulation of blood pressure. The aldehydes were assayed for their ability to inhibit the kallikrein-mediated production of kinins, and by the inhibition of the cleavage of Nᵅ-tosyl arginine methyl ester (TAME) to the carboxylic acid. Two of the amino aldehydes (Nᵅ-t-Boc-Nᴳ-nitro-L-argininal and Nᵅ-t-Boc-Nᴳ-tosyl-L-argininal) were effective inhibitors in both bioassays at micromolar concentrations. The second part of the dissertation details the development of two syntheses of β-substituted analogues of cysteine. The first method was based on sulfenylation of Nᵅ-formyl-α, β-dehydro amino acid esters, followed by protection of the sulfhydryl group as the benzyl or para-methylbenzyl thioether. The Nᵅ-formyl and ester groups were cleaved by acidic hydrolysis, and the amino group was then blocked as the t-butyloxycarbonyl derivative. This procedure gave cysteine analogues which were suitable for direct use in solid phase peptide synthesis. A second, more efficient preparation of the cysteine analogues was based on the conjugate addition of lithium benzylthiolate (or lithium para-methylbenzylthiolate) to the Nᵅ-formyl-α, β-dehydroamino acid esters. This synthesis was more efficient since the cysteine analogues were generated directly in S-protected form. The fully protected intermediates were deprotected at the amino and carboxyl groups, followed by treatment with di-tert-butyl dicarbonate. The Nᵅ- t-Boc-β-S-benzyl cysteine analogues (or Nᵅ -t-Boc-β-S-para-methylbenzyl) also were suitable for direct use in solid phase peptide synthesis.

This thesis describes the synthesis and preliminary enzymatic study of glutaryl-S-(8-aminooctyl)-L-cysteinylglycine and glutaryl-S-(10-aminodecyl)-L-cysteinylglycine as inhibitors of glyoxalase I. These analogs of glutathione were prepared as potential ligands for affinity chromatography purification of glyoxalase I. The compounds were synthesized by a seven-step procedure in overall yields of 24% for the octyl analog and 33% for the decyl analog. Both compounds exhibited mixed type inhibition of the enzyme, with the decyl derivative being more inhibitory than the octyl derivative. The inhibition was nonlinear (parabolic) for both compounds. Although less inhibitory than the corresponding S-substituted glutathione derivatives, these analogs are promising candidates for affinity chromatography ligands. Such compounds may also be useful in studying the mechanism of glyoxalase I.

Fung Pik Ha. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1994. / Includes bibliographical references (leaves 116-121). / Acknowledgments --- p.i / Table of Contents --- p.ii / List of Figures --- p.v / List of Tables --- p.x / Abstract --- p.xi / Chapter Chapter I --- Introduction --- p.1 / Chapter Chapter II --- Literature Reviews / Chapter II.l --- Glycosidase --- p.3 / Chapter II.2 --- Biosynthesis of N-linked Glycoprotein --- p.4 / Chapter II.3 --- Mechanism of Enzyme Catalysed Reaction --- p.8 / Chapter II.4 --- Types of Glycosidase Inhibitors --- p.12 / Chapter II.5 --- Cyclophellitol and Aminocyclitols / Chapter II.5.1 --- General background on cyclophellitol --- p.17 / Chapter II.5.2 --- Mode of inhibition of cyclophellitol --- p.20 / Chapter II.5.3 --- General background on aminocyclitols --- p.24 / Chapter Chapter III --- Characterization of Synthetic Glycosidase Inhibitors / Chapter III.1 --- Covalent-based Inactivator (Cyclophellitol and its Analogues) / Chapter III.1.1 --- Introduction --- p.28 / Chapter III.1.2 --- Materials --- p.32 / Chapter III.1.3 --- Methods / Chapter III.1.3.1 --- Inhibitory assay of commercially available glycosidases --- p.33 / Chapter III.1.3.2 --- Partial purification of β-D-mannosidase from A. oryzae --- p.34 / Chapter III.1.3.3 --- Protein assay in purification of β-D-mannosidase --- p.38 / Chapter III.1.3.4 --- Inhibitory assay for partially purified β-D- mannosidase (A . oryzae) --- p.38 / Chapter III.1.3.5 --- Influence of dialysis on glycosidase inhibition --- p.39 / Chapter III.1.3.6 --- Inactivation experiment on glycosidases --- p.39 / Chapter III.1.4 --- Results / Chapter III.1.4.1 --- Inhibitory activities of cyclophellitol and its analogues against glycosidases --- p.41 / Chapter III.1.4.2 --- Effect of dialysis on glycosidase inhibition --- p.44 / Chapter III.1.4.3 --- The kinetic studies of glycosidase inactivation --- p.47 / Chapter III. 1.5 --- Discussion --- p.50 / Chapter III.1.6 --- Further studies --- p.55 / Chapter III.2 --- Reversible Competitive Inhibitors (Aminocyclitols) / Chapter III.2.1 --- Introduction --- p.56 / Chapter III.2.2 --- Materials --- p.58 / Chapter III.2.3 --- Methods / Chapter III.2.3.1 --- Assay of glucoside hydrolase inhibition activity --- p.60 / Chapter III.2.3.2 --- Glucose oxidase method for determination of released D-glucose --- p.60 / Chapter III.2.3.3 --- Inhibitory assay of aminocyclitols on other glycosidases --- p.61 / Chapter III.2.3.4 --- Influence of dialysis on the glycosidase inhibition --- p.62 / Chapter III.2.3.5 --- Lineweaver-Burk plot --- p.63 / Chapter III.2.4 --- Results / Chapter III.2.4.1 --- Inhibitory activities of valiolamine and related aminocyclitols against six glycosidases --- p.64 / Chapter III.2.4.2 --- Characterization the aminocyclitols as reversible competitive inhibitors --- p.69 / Chapter III.2.5 --- Discussion --- p.80 / Chapter Chapter IV --- Isolation of the Naturally Occurring Glycosidase Inhibitor from Mushrooms / Chapter IV.1 --- Introduction --- p.83 / Chapter IV.2 --- Materials --- p.84 / Chapter IV.3 --- Methods / Chapter IV.3.1 --- Preparation of Ganoderma lucidum --- p.86 / Chapter IV.3.2 --- Preparation of V. volvacea --- p.86 / Chapter IV.3.3 --- Inhibitory assay of aqueous extract of mushrooms on glycosidases --- p.87 / Chapter IV.3.4 --- Anthrone method for determination of reducing sugars --- p.87 / Chapter IV.3.5 --- Flash liquid chromatography for purification of putative inhibitors in G. lucidum --- p.88 / Chapter IV.4 --- Results / Chapter IV.4.1 --- Prescreening of Inhibitory effects of Various Fungal Extracts --- p.90 / Chapter IV.4.2 --- Inhibitory Effects of Partially Purified G. lucidum Extract on Glycosidase --- p.92 / Chapter IV.4.3 --- Effect of Endogenous Substrates on Glycosidase Activities --- p.93 / Chapter IV.4.4 --- Results of Liquid Column Chromatography --- p.93 / Chapter IV.4.5 --- Structure Determination and Characterization of purified compounds --- p.95 / Chapter IV.4.6 --- Inhibitory Activities of Compounds A and B against Brewers yeast a- glucosidase --- p.96 / Chapter IV.5 --- Discussion --- p.98 / Chapter Chapter V --- Conclusions --- p.113 / References --- p.116

Thesis (Ph.D.)--Massachusetts Institute of Technology, Dept. of Biology, 1981. / MICROFICHE COPY AVAILABLE IN ARCHIVES AND SCIENCE. / Vita. / Includes bibliographical references. / by Thomas Clifford Alber. / Ph.D.

Acquired Immunodeficiency Syndromes (AIDS), a disease caused by the infection of human immunodeficiency virus (HIV), is still incurable to date. Various types of anti-viral drugs have been developed and most of these drugs are targeted on HIV reverse transcriptase and protease. Highly active antiretroviral therapy (HAART) has been used on AIDS treatment recently. However, new drugs are required to delay the resistance onset and to maximize the effectiveness of combination therapy by inhibiting a variety of targets simultaneously. / In the second part, the possibility of using the vector-based approach of RNA interference (RNAi) to reduce the expression of HIV-1 integrase and HIV replication in mammalian cells was examined. RNAi suppressed protein synthesis through the induction of sequence-specific gene silencing of 21-25 nucleotides (nt) double stranded RNA fragments, termed small interfering RNA (siRNA). pSilencer series vectors with different promoters (p Silencer 1.0-U6, pSilencer 2.0-U6, pSilencer 3.0-H1) were used on shRNA expression inside HeLa cells. Four different hairpin constructs containing the 19-nt corresponding to the nucleotide sequence of HIV integrase at positions 19-27, 79-96, 158-176 and 495-513 were generated for RNAi study. (Abstract shortened by UMI.) / Integrase is one of the important enzymes on HIV infection. It acts by integrating viral RNA to host DNA and this is one of the ideal targets for therapeutic intervention. Previous results in our laboratory demonstrated that luffin, a type-I ribosome inactivating protein (RIP), had high potency on integrase inhibition. In the first part of this thesis, alpha-luffin cDNA was cloned from the seed of Luffa cylindrica. Three different sets of expression vectors were used to produce recombinant luffin. Different deletion mutants of luffin were also generated for structural analysis on integrase inhibition. Recombinant alpha-luffin and its various deletion mutants were expressed exclusively in the form of inclusion bodies despite different expression conditions had been attempted. Various refolding strategies and conditions were carried out but the problem of insolubility was consistently found after removal of the denaturing reagents. The problem of insolubility was improved by using the maltose binding protein (MBP) luffin fusion construct. However, there is evidence that this soluble MBP-luffin formed a multimeric fusion protein complex rather than monomer and removal of MBP tag resulted in the precipitation of luffin. / Lau Tat San. / "August 2005." / Adviser: C. C. Wan. / Source: Dissertation Abstracts International, Volume: 67-07, Section: B, page: 3594. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2005. / Includes bibliographical references (p. 202-225). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract in English and Chinese. / School code: 1307.

Orientador: Adelino Vieira de Godoy Netto / Co-orientador: Antonio Eduardo Mauro / Banca: Isabele Rodrigues Nascimento / Banca: Victor Marcelo Deflon / Banca: Ana Maria da Costa Ferreira / Banca: Adriano Bof de Oliveira / Resumo: Nos últimos anos o interesse na obtenção de novos fármacos a base de metais vem aumentando consideravelmente. Desde a descoberta da atividade antitumoral da cisplatina na década de 60 inúmeros complexos de Pt(II) foram sintetizados, mas poucos chegaram aos testes clínicos. Por este motivo, o íon Pd(II) vem sendo usado sistematicamente no planejamento de novos compostos biologicamente ativos, pois apresenta a configuração eletrônica d8 e a mesma geometria quadrado planar dos compostos de platina, além de exibir outros modos de ação frente aos seus alvos biológicos. Neste trabalho foram sintetizados e caracterizados novos complexos de paládio(II) do tipo [PdX2(TAA)(PPh3)] e [PdX(L)(PPh3)]X [X = Cl-, Br-, I-, SCN-; TAA = tioacetamida; L = 4-metil-3-tiossemicarbazida (4-MeT) ou 4-fenil-3-tiossemicarbazida (4-PhT)]. Os complexos foram caracterizados pelas técnicas de IV, RMN, análise elementar e análise termogravimétrica. As estruturas moleculares dos complexos [PdI(4-MeT)(PPh3)]I (3), [Pd(SCN)(4-MeT)(PPh3)](SCN) (4) e [PdI(4-PhT)(PPh3)]I (7) foram determinadas via difração de raios X de monocristal, indicando um ambiente quadrático plano ao redor do metal, com seus sítios de coordenação ocupados pela trifenilfosfina, pela tiossemicarbazida coordenada de maneira bidentada e o I ou SCN. A citotoxicidade in vitro de todos os complexos foi avaliada pelo método do MTT, frente as culturas celulares de tumores murinos LM3 (adenocarcinoma mamário) e LP07 (adenocarcinoma pulmonar). Os compostos mais promissores tiveram sua capacidade de interação com o DNA investigada. Os resultados demostraram que o DNA não é o alvo principal, uma vez que os complexos só interagiram com a biomolécula em altas concentrações. Diante disto, outros possíveis alvos foram investigados. A capacidade dos complexos em inibir as enzimas topoisomerases foi avaliada pela técnica de eletroforese em gel de agarose e os dados mostraram que quase todos... / Abstract: In the last years the interest in obtaining new metal drugs has increased considerably. Since the discovery of the antitumor activity of cisplatin in the 60's, many Pt(II) complexes were synthesized, but only few reached the clinical trials. For this reason, the Pd(II) ion has been used systematically in the design of new biologically active compounds. Palladium complexes display the same electron configuration and square planar geometry of platinum compounds, furthermore these complexes can interact through different way towards pharmacological targets. In this work new palladium(II) complexes of the type [PdX2(TA)(PPh3)] and [PdX(L)(PPh3)]X [X = Cl-, Br-, I-, SCN-; TAA = thioacetamide , L = 4-methyl-3-thiosemicarbazide (4-MeT) or 4-phenyl-3-thiosemicarbazide (4-PhT)], were synthesized and characterized. All the compounds were characterized by infrared, nuclear magnetic resonance spectroscopy, elemental analysis and thermogravimetric analysis. The structures of three complexes [(PdI(4-MeT)(PPh3)]I, [Pd(SCN)(4-MeT)(PPh3)](SCN) and [(PdI(4-PhT)(PPh3)]I were determined by single crystal X-ray diffraction and was observerd a square planar environment around the metal center, with the coordination sites occupied by triphenylphosphine, the N,S-donors ligand and iodine atom or thiocyanate group. The in vitro cytotoxicity of the complexes were evaluated against the murine tumor cells LM3 (breast adenocarcinoma) and LP07 (lung adenocarcinoma). The most promising compounds were further evaluated by their ability to interact with a purine base and the DNA. The results showed that DNA is not the primary target of these compounds because they only interacted with this biomolecule at high concentrations. Thus, others potential targets were investigated . The capacity of the complexes to inhibit topoisomerase enzymes was evaluated by electrophoresis, and the data showed that almost all the compounds inhibit this enzyme at a concentration rage... / Doutor

Orientador: Reinaldo Marchetto / Banca: Saulo Santesso Garrido / Banca: Flávio Henrique da Silva / Banca: Clovis Ryuichi Nakaie / Banca: Hérida Regina Nunes Salgado / Resumo: YacG é uma pequena proteína membro da família dos zinc-fingers, descoberta em E. coli. Sua função biológica está ligada a inibição da atividade de DNA girase e ao metabolismo do stress em procariotos. YacG atua na inibição da atividade de DNA girase por um mecanismo bipartido, a região do domínio zinc-finger atua impedindo a ligação do DNA à subunidade B e a região c-terminal liga-se a subunidade A. Embora a literatura cite YacG como sendo um inibidor de DNA girase, nada foi observado a respeito da atividade de inibição de YacG de E.coli e de outras linhagens de micro-organismos frente a outras DNA topoisomerases e não foi realizado nenhum trabalho envolvendo fragmentos peptídicos desta proteína, e tampouco ensaios de inibição do crescimento celular por estes. Sendo assim YacG de Klebsiella pneumoniae foi obtida por expressão heteróloga e uma série de peptídeos derivados desta foram sintetizados pelo método de fase sólida. Os peptídeo sintéticos foram projetados de forma a manter a região zinc-finger nativa (YacGAG1), substituídos os resíduos de cisteína por serina (YacGAG4), alanina (YacGAG5), histidina (YacGAG6 e YacGAG8) e também sintetizada a sua região c-terminal (YacGAG7). Os ensaios de inibição da atividade de DNA girase pelos peptídeos sintéticos e proteína, mostraram que YacG, YacGAG4, YacGAG5 e YacGAG7 conseguem inibir a atividade desta enzima, ao contrario do fragmento nativo YacGAG1. Por outro lado YacGAG1 conseguiu inibir a atividade de Topoisomerase IIα humana juntamente com os demais peptídeos YacGAG4, YacGAG5 e YacGAG7 e proteína YacG. Os ensaios aqui apresentados corroboram com os dados apresentados na literatura onde a região c-terminal por si só é capaz de inibir a atividade de DNA girase. Os ensaios de anisotropia de fluorescência demonstraram que a interação dos peptídicos sintéticos com DNA girase se dá pela interação destes com... / Abstract: YacG belongs to zinc-finger's protein family discovered in E. coli. Its biological function is connected to the catalytic inhibition of DNA gyrase and stress metabolism in prokaryotes. This protein inhibits DNA gyrase (gyrase) through a bipartite mechanism where the zinc-finger domain prevents the DNA ligation to the B subunit (GyrB) and the C-terminal bindings to the A subunit (GyrA). Although it is classified as a gyrase inhibitor neither the inhibition activity of YacG from E. coli and other microorganisms against other DNA topoisomerases nor the biological activity of fragments in cellular assays has been evaluated until present. In this study, YacG from Klebsiella pneumoniae was obtained by heterologous expression and derivative peptides from this protein were synthesized by the solid-phase method. The synthetic peptides were designed to keep the native region of zinc-finger (YacGAG1), to substitute cysteines to serines (YacGAG4), to alanine (YacGAG5), to histidine (YacGAG6 and YacGAG8) and also having only the C-terminal region (YacGAG7). The inhibition assays of gyrase by the peptides and the native protein revealed that YacG, YacGAG4, YacGAG4 e YacGAG7 inhibited this enzyme and the human topoisomerase IIα (htopoIIα). However, the same was not observed for the native fragment YacGAG1, which inhibited only the htopoIIα. The results are in agreement with literature showing that solely the C-terminal region is able to inhibit the gyrase. The assays of fluorescence anisotropy indicated that these synthetic peptides interact with GyrA. Besides inhibiting the gyrase and the htopoIIα, these peptides also revealed bacteriostatic activity against gram-negative and gram-positive bacteria. A computational study by applying a molecular dynamics protocol highlighted the importance of residues I47, R46 and W40 from YacG in the process of molecular recognition and interaction with GyrB. The results... / Doutor

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