No description available.

Rapid, sensitive and quantitative assays for proteases are of great significance for drug development and in diagnosis of diseases. Herein, we describe work towards a novel assay for the multiplexed detection of proteases using ICP-MS. Protease substrates were synthesized containing a diethylenetriaminepentaaceticacid(DTPA) ligand to chelate lanthanide metal ions at the N-terminus, providing a distinct tag for each substrate. A biotin label was appended to the C-terminus allowing separation of uncleaved peptide from the digestion. The enzymatic activities can then be determined by detecting the lanthanide signal of the peptide cleavage products by ICP-MS. Substrates synthesized include DTPA-Gln-Val-Tyr-Gly-Nle-Nle-Lys(biotin)-amide, DTPA-Asp-Gln-Val-Asp-Gly-Lys(biotin)-amide and DTPA-Gly-Pro-Gln-Gly-Leu-Glu-Ala-Lys-Lys(biotin)-amide for calpain-1, caspase-3 and MMP-9 They were loaded with terbium, holmium and praseodymium respectively. As a proof-of-concept, α-chymotrypsin assays were carried out using DTPA-Asp-Leu-Leu-Val-Tyr-Asp-Lys(Biotin) loaded with lutetium, as a substrate. Calpain-1 assays were also performed. Parallel assays with commercially available fluorogenic substrates for both the enzymes were performed for comparison.

Rapid, sensitive and quantitative assays for proteases are of great significance for drug development and in diagnosis of diseases. Herein, we describe work towards a novel assay for the multiplexed detection of proteases using ICP-MS. Protease substrates were synthesized containing a diethylenetriaminepentaaceticacid(DTPA) ligand to chelate lanthanide metal ions at the N-terminus, providing a distinct tag for each substrate. A biotin label was appended to the C-terminus allowing separation of uncleaved peptide from the digestion. The enzymatic activities can then be determined by detecting the lanthanide signal of the peptide cleavage products by ICP-MS. Substrates synthesized include DTPA-Gln-Val-Tyr-Gly-Nle-Nle-Lys(biotin)-amide, DTPA-Asp-Gln-Val-Asp-Gly-Lys(biotin)-amide and DTPA-Gly-Pro-Gln-Gly-Leu-Glu-Ala-Lys-Lys(biotin)-amide for calpain-1, caspase-3 and MMP-9 They were loaded with terbium, holmium and praseodymium respectively. As a proof-of-concept, α-chymotrypsin assays were carried out using DTPA-Asp-Leu-Leu-Val-Tyr-Asp-Lys(Biotin) loaded with lutetium, as a substrate. Calpain-1 assays were also performed. Parallel assays with commercially available fluorogenic substrates for both the enzymes were performed for comparison.

G-NNACI, a Naja naja atra chymotrypsin inhibitor consists of 57 amino acid residues cross-linked by three disulfide bridges and belongs to the Kunitz/BPTI superfamily, has been successfully cloned and expressed in our laboratory. Since snake venom non-neurotoxic Kunitz/BPTI inhibitors are most conserved in the core and in the N-terminal surface area, Ala-screening mutagenesis, deletion and Domain swapping on the N-terminus were carried out in this study to assess the role of N-terminus in G-NNACI. G-NNACI mutants with single amino acid substitution and deleted mutants were prepared. The secondary structure of all mutated proteins did not significantly alter as evidenced by CD spectra. Although all mutants are found to be functionally active as an inhibitor, their inhibitory potency against chymotrypsin differed. In contrast to G-NNACI and other mutants, R1A¡BP2A and ¡µN3 mutants had a propensity to alter their disulfide linkages under basic conditions. The results of thermal and urea denaturation suggested that amino acid substitution and deletion at the N-terminus lead to a change in the structural stability of G-NNACI. Consequently, the inhibitory potency of G-NNACI mutants along with time was affected. B chain of

175 leaves ; 26 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Thesis (Ph.D.1973) from the Dept. of Organic Chemistry, University of Adelaide

This thesis examines the design, synthesis and biological applications of a series of inhibitors which incorporate an azobenzene photoswitch, a peptidyl backbone and a trifluoromethyl ketone warhead. The photoswitch can be isomerised by irradiation with UV or visible light and has been employed to modulate the reactivity of the enzyme. Chapter one gives a brief outline of some of the important areas related to this work. Examples of previously utilised photoswitches as well as some background on serine protease and the uses of fluorine in medicine has been covered. Chapter two outlines the synthesis of the key trifluoromethyl carbinol 2.6 by two different methods. The condensation of a fluorinated aldehyde with a nitroalkane affords an α-nitro trifluoromethyl carbinol which can be reduced to give the desired amine 2.6. Treatment of oxazolones with trifluoroacetic anhydride via a modified Dakin-West reaction gives trifluoromethyl ketones which can be reduced to give trifluoromethyl carbinols. Chapter three investigate the synthesis of substituted stilbenes and phenanthrenes as alternative molecular switches to azobenzenes. Molecular modelling of phenanthrenes suggests they may be suitable mimics of E-azobenzenes. Chapter four outlines the synthesis of a series of mono and disubstituted azobenzenes by direct sulfonation of azobenzene or by condensation of nitroso arenes with aryl amines. The switches incorporate one or two peptidyl residues designed to improve specificity towards the enzyme. Chapter five examines the photoisomerisation of eight potential inhibitors by irradiating with UV or visible light. Irradiation with UV light enriches the sample to give 78-93 % of the Z-isomer. Irradiation with visible light gave photostationary states with 14-21 % Z-isomer. Ambient photostationary states are ca. 22 % Z-isomer. Chapter six looks at the testing of five trifluoromethyl ketones as potential inhibitors ofα-chymotrypsin. The inhibitors vary in substituents, substitution patterns and chain length. The inhibitors were tested at both ambient and Z-enriched photostationary states and were found to exhibit slow binding kinetics. In all cases the Z-enriched inhibitor solution was at least 3-fold more potent than the ambient solution. Chapter seven is an experimental chapter and outlines the synthesis of the compounds prepared in this thesis.

This thesis presents the first example of reversible photoregulation of the binding of a protease, α-chymotrypsin, to a surface. A modular approach is used involving the azobenzene photoswitch group, a surface linker and an enzyme binding group. This approach is designed to be easily extended to the photoregulation of binding of other proteases to surfaces by use of enzyme binding groups selective to these proteases. Chapter one gives a brief outline of some of the important areas involved in to this work, including molecular switches, proteases and surface modification. Chapter two describes the synthesis of azobenzene-containing boronate esters designed as photoswitch inhibitors of α-chymotrypsin. Boronate esters were prepared containing the aminophenylboronate group or the peptidomimetic borophenylalanine group for enzyme binding and a range of substituents designed for enzyme affinity and/or surface attachment. Syntheses primarily involved peptide coupling reactions and azobenzene formation by condensation of nitrosobenzenes and anilines. Coupling reactions were successfully carried out using EDCI or isobutyl chlorofomate in several cases where other reagents gave unacceptable decomposition. Chapter three describes the syntheses and HPLC stability studies of derivatives of a noncovalent α-chymotrypsin inhibitor. Several dipeptide-based compounds containing either an amide group for surface attachment or an azobenzene group for photoswitching were prepared, primarily using peptide coupling reactions. Each compound was incubated with α-chymotrypsin to assess its stability, and all were found by HPLC monitoring to be stable to α-chymotrypsin catalysed hydrolysis. Chapter four describes syntheses of azobenzene-containing trifluoromethylketones and α-ketoesters designed as photoswitch inhibitors of α-chymotrypsin. Trifluoromethylketones/α-ketoesters containing amine groups for surface attachment were prepared, primarily using peptide coupling reactions, but could not be isolated due to the incompatibility of the electrophilic ketone and primary amine groups. Trifluoromethylketones/α-ketoesters containing terminal alkynes for surface attachment were prepared either by the attachment of an alkyne substituent group to a symmetrical azobenzene core or by Pd-catalysed reaction of a protected alkyne with an azobenzene having a halide substitutent. Chapter five describes syntheses of sulfur-containing surface linkers for use in surface attachment of the photoswitch inhibitors described in chapters 2-4. A range of compounds containing disulfide or protected thiol groups for surface attachment and azide or carboxylic acid groups for inhibitor attachment were prepared. Syntheses primarily involved coupling of functionalised alcohols/amides to carboxylic acid-containing disulfides/thioacetates. Selected linkers were attached to azobenzenes by amide coupling or azide-alkyne cycloaddition for surface attachment, photoswitching and/or enzyme assay. Azide-alkyne cycloaddition yields were initially poor, but were improved by use of stoichiometric amounts of copper catalyst. Chapter six describes UV/vis photoisomerisation studies and enzyme assays carried out to assess enzyme photoswitching of the compounds described in chapters 2-5. The trifluoromethylketones and α-ketoesters described in chapter 4 gave the best results, with moderate inhibition of α-chymotrypsin (µM affinity constants) and up to 5.3 fold changes in inhibition on UV/vis irradiation. Many of the boronate esters described in chapter 2 were found to inhibit α-chymotrypsin, but were somewhat unstable to irradiation. The dipeptide-based compounds described in chapter 3 were inactive against α-chymotrypsin. Good photoisomerisation was obtained for an azobenzene containing a symmetrical disulfide surface linker and poor photoisomerisation was obtained for an azobenzene containing a lipoic acid surface linker. Chapter seven describes surface attachment of selected photoswitch inhibitors and studies of photoregulated enzyme binding to the resultant functional surfaces. Self assembled monolayers (SAMs) of disulfides were formed on gold surfaces and characterised by electrochemistry and contact angle measurements. Binding of α-chymotrypsin to SAMs containing a photoswitch inhibitor was detected by quartz crystal microbalance (QCM), but was found to be largely irreversible. An alkyne-containing photoswitch inhibitor was attached to a surface plasmon resonance (SPR) chip in a two step procedure involving generation of an azide modified surface followed by azide-alkyne cycloaddition. Binding of α-chymotrypsin to the resultant modified surface was detected by SPR and successfully regulated by UV/vis irradiation. Chapter eight provides conclusions for the work described in this thesis and suggests future directions. Chapter nine gives experimental details for the work described in this thesis.

Thesis (Ph.D. 1973) from the Dept. of Organic Chemistry, University of Adelaide.

Universiẗat, Veterinärmedizinische Fakultät, Diss., 2005--Leipzig.

by Yuen-Kam Tsoi. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2003. / Includes bibliographical references (leaves 160-173). / Abstracts in English and Chinese. / Abstract --- p.i / 論文摘要 --- p.iv / List of figures --- p.vii / List of tables --- p.x / Abbreviations --- p.xi / Chapter Chapter 1 --- Purification and characterization of the chymotrypsin inhibitor (MCCI) from the seeds of Momordica cochinchinensis / Chapter 1.1 --- Introduction l / Chapter 1.1.1 --- Classification of protease inhibitor --- p.2 / Chapter 1.1.2 --- Therapeutic potential of protease inhibitors --- p.4 / Chapter 1.2 --- Rationale of the present study --- p.6 / Chapter 1.3 --- Materials and methods / Chapter 1.3.1 --- Materials --- p.9 / Chapter 1.3.2 --- Preparation of chymotrypsin-Sepharose 4B affinity column --- p.10 / Chapter 1.3.3 --- Protein extraction --- p.11 / Chapter 1.3.4 --- Chymotrypsin-Sepharose 4B affinity chromatography --- p.12 / Chapter 1.3.5 --- Reversed phase high pressure liquid chromatography --- p.12 / Chapter 1.3.6 --- Assays for protease inhibitory activities --- p.14 / Chapter 1.3.6.1 --- Assay for chymotrypsin activity --- p.15 / Chapter 1.3.6.2 --- Assay for trypsin activity --- p.15 / Chapter 1.3.6.3 --- Assay for elastase activity --- p.16 / Chapter 1.3.6.4 --- Assay for subtilisin activity --- p.16 / Chapter 1.3.7 --- Determination of protein concentration --- p.17 / Chapter 1.3.8 --- Titration of chymotrypsin --- p.17 / Chapter 1.3.9 --- Sodium dodecyl sulfate polyacrylamide gel electrophoresis --- p.18 / Chapter 1.3.10 --- Determination of molecular weight by mass spectrometry --- p.19 / Chapter 1.3.11 --- Partial amino acid sequencing --- p.20 / Chapter 1.3.12 --- Effects of chymotrypsin on MCCI --- p.20 / Chapter 1.3.13 --- Stability assay --- p.21 / Chapter 1.4 --- Results --- p.21 / Chapter 1.4.1 --- Isolation of MCCI from the seeds of Momordica cochinchinensis --- p.21 / Chapter 1.4.2 --- N-terminal amino acid sequencing --- p.27 / Chapter 1.4.3 --- Determination of molecular weight --- p.31 / Chapter 1.4.4 --- Inhibitory activity of MCCI towards different proteases --- p.33 / Chapter 1.4.5 --- Effects of chymotrypsin on MCCI --- p.37 / Chapter 1.4.6 --- Stability of MCCI on heating and at different pH --- p.37 / Chapter 1.5 --- Discussion --- p.42 / Chapter Chapter 2 --- Immunomodulatory effect of MCCI / Chapter 2.1 --- Introduction of the immune system and protease inhibitors --- p.51 / Chapter 2.2 --- Rationale of the present study --- p.55 / Chapter 2.3 --- Materials and methods --- p.56 / Chapter 2.3.1 --- Materials --- p.56 / Chapter 2.3.2 --- Isolation of different types of immune cells --- p.57 / Chapter 2.3.3 --- Determination of cell proliferation --- p.60 / Chapter 2.3.4 --- Determination of H2O2 formation --- p.61 / Chapter 2.3.5 --- Assay of interleukin-2 --- p.61 / Chapter 2.3.6 --- Determination of cell viability --- p.62 / Chapter 2.4 --- Results --- p.63 / Chapter 2.4.1 --- Murine splenocytes --- p.63 / Chapter 2.4.1.1 --- In vitro effect of MCCI on the proliferation of murine splenocytes --- p.63 / Chapter 2.4.1.2 --- Effect of MCCI on cytokine production --- p.63 / Chapter 2.4.2 --- Murine lymphocytes --- p.66 / Chapter 2.4.2.1 --- In vitro effect of MCCI on the proliferation of lymphocytes --- p.66 / Chapter 2.4.2.2 --- Effect of MCCI on cytokine production --- p.66 / Chapter 2.4.3 --- Murine bone marrow cells --- p.69 / Chapter 2.4.3.1 --- Effect of MCCI on the growth of murine bone marrow cells --- p.69 / Chapter 2.4.4 --- Murine neutrophills --- p.69 / Chapter 2.4.4.1 --- Effect of MCCI on H2O2 formation --- p.69 / Chapter 2.4.5 --- Murine macrophages --- p.71 / Chapter 2.4.5.1 --- Effect of MCCI on the growth of macrophages --- p.71 / Chapter 2.4.5.2 --- Effect of external ATP on the growth of macrophages --- p.71 / Chapter 2.4.5.3 --- Effect of ATP on the growth of macrophages pre-treated with MCCI --- p.76 / Chapter 2.4.5.4 --- Effect of MCCI on the growth of macrophages pre-treated with ATP --- p.76 / Chapter 2.4.5.5 --- Effect of MCCI on H201production --- p.79 / Chapter 2.5 --- Discussion --- p.82 / Chapter Chapter 3 --- Anti-oxidative effect of MCCI in primary rat hepatocytes culture / Chapter 3.1 --- Introduction --- p.91 / Chapter 3.1.1 --- Liver disease and protease inhibitors --- p.91 / Chapter 3.1.2 --- Primary rat hepatocyte as a pharmacological model --- p.93 / Chapter 3.1.3 --- tert-Butyl hydroperoxide as an oxidative stress inducer --- p.94 / Chapter 3.1.4 --- Endogenous antioxidant enzymes against ROS --- p.96 / Chapter 3.2 --- Rationale of the present study --- p.99 / Chapter 3.3 --- Materials and methods --- p.101 / Chapter 3.3.1 --- Materials --- p.101 / Chapter 3.3.2 --- Isolation of primary rat hepatocytes --- p.101 / Chapter 3.3.2.1 --- Liver perfusion --- p.101 / Chapter 3.3.2.2 --- Preparation of collagen pre-coated culture plates --- p.103 / Chapter 3.3.2.3 --- Hepatocytes culturing --- p.103 / Chapter 3.3.3 --- Drug treatment and oxidative stress induction --- p.104 / Chapter 3.3.4 --- Cytotoxicity assessment --- p.105 / Chapter 3.3.5 --- Cellular GSH content determination --- p.105 / Chapter 3.3.6 --- Protein determination by Lowry's method --- p.106 / Chapter 3.3.7 --- Medium MDA determination --- p.106 / Chapter 3.3.8 --- Medium GSSG determination --- p.107 / Chapter 3.3.9 --- Antioxidant enzymes measurement --- p.108 / Chapter 3.3.9.1 --- Catalase measurement --- p.108 / Chapter 3.3.9.2 --- SOD measurement --- p.109 / Chapter 3.3.9.3 --- GST measurement --- p.109 / Chapter 3.3.9.4 --- GR measurement --- p.110 / Chapter 3.3.10 --- Statistical analysis --- p.110 / Chapter 3.4 --- Results --- p.111 / Chapter 3.4.1 --- Cytotoxicity of MCCI on rat hepatocytes --- p.111 / Chapter 3.4.2 --- Effect of tBHP and MCCI on hepatocytes viability --- p.111 / Chapter 3.4.3 --- Effects of tBHP and MCCI on hepatocytes GSH and GSSG content --- p.117 / Chapter 3.4.4 --- Effect of MCCI on lipid peroxidation of hepatocytes --- p.121 / Chapter 3.4.5 --- Effect of MCCI on antioxidant enzymes activities --- p.121 / Chapter 3.4.6 --- Comparison with typical antioxidants --- p.125 / Chapter 3.5 --- Discussion --- p.127 / Chapter Chapter 4 --- Cytotoxicity of MCCI on tumor cell lines / Chapter 4.1 --- Introduction --- p.134 / Chapter 4.1.1 --- Relationship between protease inhibitors and cancer --- p.134 / Chapter 4.1.2 --- Cell cycle and apoptosis --- p.137 / Chapter 4.2 --- Rationale of the present study --- p.140 / Chapter 4.3 --- Materials and methods --- p.141 / Chapter 4.3.1 --- Materials --- p.141 / Chapter 4.3.2 --- Cell culture --- p.141 / Chapter 4.3.3 --- MTT assay --- p.142 / Chapter 4.3.4 --- Cell cycle analysis --- p.142 / Chapter 4.3.5 --- DNA fragmentation --- p.143 / Chapter 4.4 --- Results --- p.136 / Chapter 4.4.1 --- Cytotoxicity of MCCI --- p.144 / Chapter 4.4.2 --- Cell cycle and apoptosis analysis --- p.147 / Chapter 4.5 --- Discussion --- p.152 / Conclusion and future perspectives --- p.157 / References --- p.160