Tesis: "Serine protease inhibitor"

1

Gariani, Talal. "Design of serine protease inhibitor peptides". Thesis, Imperial College London, 1997. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.267244.

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2

Combe, Caroline Jane. "Hepatic receptor(s) for serine protease-inhibitor complexes". Thesis, University of Aberdeen, 1995. http://digitool.abdn.ac.uk/R?func=search-advanced-go&find_code1=WSN&request1=AAIU549619.

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A number of questions about the hepatic mechanisms of tissue-type plasminogen activator (t-PA) clearance still remain unanswered. Although certain liver endothelial cell receptors have been implicated, the parenchymal cell system, which is responsible for most clearance, still remains a mystery. The aim of this project, in the most simple terms, was to solve this mystery. The foundation upon which this project was built was that t-PA is cleared, by a hepatic receptor, in complex with its primary inhibitor, plasminogen activator inhibitor type 1 (PAI-1). The affinity of binding was estimated to be 0.8-1.0 nM and the number of binding sites per cell, 35 000-70 000. Affinity chromatography and chemical cross-linking resulted in a band of A?70 kDa which was presumed to be the receptor. This project was designed to characterize this hepatic receptor for t-PA-PAI-1 and determine whether plasmin-2-antiplasmin (PAP) is recognised by the same receptor. Characterizing the receptor was attempted initially by employing cell binding assays using the human hepatoma cell line, Hep G2. This methodology required the formation and characterization of pure pre-formed ligands which was achieved by overcoming preliminary problems. The binding assays showed that competition between t-PA-PAI-1 and PAP was occurring but that high non-specific binding and error between duplicate samples suggested that this system was not suitable for characterization of the receptor. The data accumulated in this study suggested that LRP was primarily responsible for hepatic uptake of t-PA and that proteases were recognised preferentially in complex with their inhibitors.

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3

Swedberg, Joakim Erik. "Rational design of serine protease inhibitors". Thesis, Queensland University of Technology, 2011. https://eprints.qut.edu.au/48131/1/Joakim_Swedberg_Thesis.pdf.

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Proteases regulate a spectrum of diverse physiological processes, and dysregulation of proteolytic activity drives a plethora of pathological conditions. Understanding protease function is essential to appreciating many aspects of normal physiology and progression of disease. Consequently, development of potent and specific inhibitors of proteolytic enzymes is vital to provide tools for the dissection of protease function in biological systems and for the treatment of diseases linked to aberrant proteolytic activity. The studies in this thesis describe the rational design of potent inhibitors of three proteases that are implicated in disease development. Additionally, key features of the interaction of proteases and their cognate inhibitors or substrates are analysed and a series of rational inhibitor design principles are expounded and tested. Rational design of protease inhibitors relies on a comprehensive understanding of protease structure and biochemistry. Analysis of known protease cleavage sites in proteins and peptides is a commonly used source of such information. However, model peptide substrate and protein sequences have widely differing levels of backbone constraint and hence can adopt highly divergent structures when binding to a protease’s active site. This may result in identical sequences in peptides and proteins having different conformations and diverse spatial distribution of amino acid functionalities. Regardless of this, protein and peptide cleavage sites are often regarded as being equivalent. One of the key findings in the following studies is a definitive demonstration of the lack of equivalence between these two classes of substrate and invalidation of the common practice of using the sequences of model peptide substrates to predict cleavage of proteins in vivo. Another important feature for protease substrate recognition is subsite cooperativity. This type of cooperativity is commonly referred to as protease or substrate binding subsite cooperativity and is distinct from allosteric cooperativity, where binding of a molecule distant from the protease active site affects the binding affinity of a substrate. Subsite cooperativity may be intramolecular where neighbouring residues in substrates are interacting, affecting the scissile bond’s susceptibility to protease cleavage. Subsite cooperativity can also be intermolecular where a particular residue’s contribution to binding affinity changes depending on the identity of neighbouring amino acids. Although numerous studies have identified subsite cooperativity effects, these findings are frequently ignored in investigations probing subsite selectivity by screening against diverse combinatorial libraries of peptides (positional scanning synthetic combinatorial library; PS-SCL). This strategy for determining cleavage specificity relies on the averaged rates of hydrolysis for an uncharacterised ensemble of peptide sequences, as opposed to the defined rate of hydrolysis of a known specific substrate. Further, since PS-SCL screens probe the preference of the various protease subsites independently, this method is inherently unable to detect subsite cooperativity. However, mean hydrolysis rates from PS-SCL screens are often interpreted as being comparable to those produced by single peptide cleavages. Before this study no large systematic evaluation had been made to determine the level of correlation between protease selectivity as predicted by screening against a library of combinatorial peptides and cleavage of individual peptides. This subject is specifically explored in the studies described here. In order to establish whether PS-SCL screens could accurately determine the substrate preferences of proteases, a systematic comparison of data from PS-SCLs with libraries containing individually synthesised peptides (sparse matrix library; SML) was carried out. These SML libraries were designed to include all possible sequence combinations of the residues that were suggested to be preferred by a protease using the PS-SCL method. SML screening against the three serine proteases kallikrein 4 (KLK4), kallikrein 14 (KLK14) and plasmin revealed highly preferred peptide substrates that could not have been deduced by PS-SCL screening alone. Comparing protease subsite preference profiles from screens of the two types of peptide libraries showed that the most preferred substrates were not detected by PS SCL screening as a consequence of intermolecular cooperativity being negated by the very nature of PS SCL screening. Sequences that are highly favoured as result of intermolecular cooperativity achieve optimal protease subsite occupancy, and thereby interact with very specific determinants of the protease. Identifying these substrate sequences is important since they may be used to produce potent and selective inhibitors of protolytic enzymes. This study found that highly favoured substrate sequences that relied on intermolecular cooperativity allowed for the production of potent inhibitors of KLK4, KLK14 and plasmin. Peptide aldehydes based on preferred plasmin sequences produced high affinity transition state analogue inhibitors for this protease. The most potent of these maintained specificity over plasma kallikrein (known to have a very similar substrate preference to plasmin). Furthermore, the efficiency of this inhibitor in blocking fibrinolysis in vitro was comparable to aprotinin, which previously saw clinical use to reduce perioperative bleeding. One substrate sequence particularly favoured by KLK4 was substituted into the 14 amino acid, circular sunflower trypsin inhibitor (SFTI). This resulted in a highly potent and selective inhibitor (SFTI-FCQR) which attenuated protease activated receptor signalling by KLK4 in vitro. Moreover, SFTI-FCQR and paclitaxel synergistically reduced growth of ovarian cancer cells in vitro, making this inhibitor a lead compound for further therapeutic development. Similar incorporation of a preferred KLK14 amino acid sequence into the SFTI scaffold produced a potent inhibitor for this protease. However, the conformationally constrained SFTI backbone enforced a different intramolecular cooperativity, which masked a KLK14 specific determinant. As a consequence, the level of selectivity achievable was lower than that found for the KLK4 inhibitor. Standard mechanism inhibitors such as SFTI rely on a stable acyl-enzyme intermediate for high affinity binding. This is achieved by a conformationally constrained canonical binding loop that allows for reformation of the scissile peptide bond after cleavage. Amino acid substitutions within the inhibitor to target a particular protease may compromise structural determinants that support the rigidity of the binding loop and thereby prevent the engineered inhibitor reaching its full potential. An in silico analysis was carried out to examine the potential for further improvements to the potency and selectivity of the SFTI-based KLK4 and KLK14 inhibitors. Molecular dynamics simulations suggested that the substitutions within SFTI required to target KLK4 and KLK14 had compromised the intramolecular hydrogen bond network of the inhibitor and caused a concomitant loss of binding loop stability. Furthermore in silico amino acid substitution revealed a consistent correlation between a higher frequency of formation and the number of internal hydrogen bonds of SFTI-variants and lower inhibition constants. These predictions allowed for the production of second generation inhibitors with enhanced binding affinity toward both targets and highlight the importance of considering intramolecular cooperativity effects when engineering proteins or circular peptides to target proteases. The findings from this study show that although PS-SCLs are a useful tool for high throughput screening of approximate protease preference, later refinement by SML screening is needed to reveal optimal subsite occupancy due to cooperativity in substrate recognition. This investigation has also demonstrated the importance of maintaining structural determinants of backbone constraint and conformation when engineering standard mechanism inhibitors for new targets. Combined these results show that backbone conformation and amino acid cooperativity have more prominent roles than previously appreciated in determining substrate/inhibitor specificity and binding affinity. The three key inhibitors designed during this investigation are now being developed as lead compounds for cancer chemotherapy, control of fibrinolysis and cosmeceutical applications. These compounds form the basis of a portfolio of intellectual property which will be further developed in the coming years.

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4

Wångsell, Fredrik. "Design and Synthesis of Serine and Aspartic Protease Inhibitors". Licentiate thesis, Linköping University, Linköping University, Organic Chemistry, 2006. http://urn.kb.se/resolve?urn=urn:nbn:se:liu:diva-7372.

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This thesis describes the design and synthesis of compounds that are

intended to inhibit serine and aspartic proteases. The first part of the text deals with preparation of inhibitors of the hepatitis C virus (HCV) NS3 serine protease. Hepatitis C is predominantly a chronic disease that afflicts about 170 million people worldwide. The NS3 protease, encoded by HCV, is essential for replication of the virus and has become one of the main targets when developing drugs to fight HCV. The inhibitors discussed here constitute surrogates for the widely used N-acyl-hydroxyproline isostere designated 4-hydroxy-cyclopentene. The stereochemistry of the 4-hydroxy-cyclopentene scaffold was determined by nuclear overhauser effect spectroscopy (NOESY) and the regiochemistry by heteronuclear multiple bond correlation (HMBC). The scaffold was decorated with different substituents to obtain both linear and macrocyclic HCV NS3 protease inhibitors that display low nanomolar activity. The second part of the thesis describes the design and synthesis of potential aspartic protease inhibitors. The hydroxyethylene motif was used as a noncleavable transition state isostere. The synthetic route yielded a pivotal intermediate with excellent stereochemical control, which was corroborated by NOESY experiments. This intermediate can be diversified with different substituents to furnish novel aspartic protease inhibitors.

Report code: LIU-TEK-LIC-2006:45

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5

Poliakov, Anton. "Peptide-Based Inhibitors of Hepatitis C Virus NS3 Serine Protease: Kinetic Aspects and Inhibitor Design". Doctoral thesis, Uppsala universitet, Institutionen för naturvetenskaplig biokemi, 2004. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-4127.

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Hepatitis C is a serious disease that affects about 200 million people worldwide. No anti-HCV vaccine or specific anti-viral drugs are available today. Non-structural protein 3 (NS3) of HCV is a bifunctional serine protease/helicase, and the protease has become a prime target in the search for anti-HCV drugs. In this work, the complete HCV NS3 gene has been cloned and expressed, and the protein has been purified using affinity chromatography. An assay for measuring the protease activity of full-length NS3 protease has been developed and used for inhibition studies. A series of peptide-based inhibitors of NS3 protease varying in length, the composition of the side-chain and the N- and C-terminal groups have been studied. Potent tetra-, penta- and hexapeptide inhibitors of the NS3 protease were discovered. Hexapeptides with an acyl sulfonamide C-terminal residue were the most potent inhibitors of the NS3 protease, having nanomolar Ki-values. The selectivity of the inhibitors was assessed using other serine and cysteine proteases. NS3 protease inhibitors with electrophilic C-terminal groups were non-selective while those comprising a C-terminal carboxylate or acyl sulfonamide group were selective. All inhibitors with a small hydrophobic P1 side-chain residue were non-selective for the NS3 protease, being good inhibitors of human leukocyte elastase. This result highlights the importance of the P1 residue for inhibitor selectivity, which stems from the major role of this residue in determining substrate specificity of serine proteases. Electrophilic inhibitors often cause slow-binding inhibition of serine and cysteine proteases. This was observed with other proteases used in our work but not with NS3 protease, which indicates that mechanism of inhibition of NS3 protease by electrophilic inhibitors may not involve formation of a covalent bond. The structure-activity relationships obtained in this work can be used for improvement of peptide-based inhibitors of HCV NS3 protease towards higher inhibitory potency and selectivity.

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6

Reinhard, Eva. "Cell and tissue localization of glia derived nexin, a serine protease inhibitor /". [S.l.] : [s.n.], 1989. http://e-collection.ethbib.ethz.ch/show?type=diss&nr=9040.

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7

Eriksson, Röhnisch Kajsa. "Purification and Technical Application of a Serine Protease Inhibitor from Solanum tuberosum". Thesis, Uppsala universitet, Institutionen för biologisk grundutbildning, 2016. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-275284.

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8

elMasry, Nadia Farida. "Folding studies on mutants of Chymotrypsin Inhibitor 2". Thesis, University of Cambridge, 1993. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.309338.

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9

Forney, John Russell. "Interaction of the Human Serine Protease Inhibitor Alpha-1-antitrypsin with Cryptosporidium parvum". DigitalCommons@USU, 1997. https://digitalcommons.usu.edu/etd/3961.

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The human serine protease inhibitor (serpin) alpha-1-antitrypsin (AAT) was studied for potential interaction with components of the protozoan parasite Cryptosporidium parvum. A homogenate prepared from C. parvum oocysts was incubated with purified human AAT, and complexes formed between the serpin and components of the homogenate were detected using an enzyme-linked immunosorbent assay (ELISA). Serpin:parasite infections were effectively blocked by preincubating AAT with a cognate target enzyme, porcine pancreatic elastase, prior to performing the ELISA on the homogenate. Incubation of a mixture of C. parvum oocysts and sporozoites with AAT demonstrated preferential fluorescence labeling of the sporozoite surface membrane by indirect immunofluorescence assay. Localization of serpin complexes on sporozoites was confirmed by immunogold electron microscopy. AAT was evaluated for in vitro anticryptosporidial activity in a bovine fallopian tube epithelial (BFTE) cell culture system using both oocysts and filter purified sporozoites as inocula. Serial dilutions of AAT were mixed with oocysts (or sporozites) and used to inoculate BFTE cell monolayers. Inoculted cells were maintained at 37ºC/5% CO2 and collected at 24-,48-,72-, and 96-hr post-inoculation intervals. The addition of AAT and other select protease inhibitors (i.e.,antipain, aprotinin, leupeptin, soybean trypsin inhibitor, and phenylmethylsulfonyl floride) significantly inhibited parasite infection (P<0.01) in a concentration- and time-dependent manner when bleach-decontaminated oocysts were used in the inoculum. The anticryptosporidial activity of AAT is postulated to be linked to an antagonistic effect on oocyst excystation and, putatively, the forced expenditure of bioenergetic reserves prior to host cell invasion. This postulate was supported by the observations that serpin activity had no statistically significant effect on reducing established in vitro infections (i.e., 24 hr post-inoculation prior to addition of AAT) and did not inhibit infection of BFTE cells when inoculted with sporozoite preparations. The combined application of AAT and the aminoglycoside paromomycin demonstrated a synergistic anticryptosporidial effect on in vitro infection and suggested the basis for a multi-agent therapeutic protocol in preventing cryptosporidosis. These studies collectively demonstrated an inticryptosporidial potential for serine protease inhibitors, in particular for AAT, and suggest an alternative approach to conventional therapeutic strategies.

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10

Ferreira, Graziele Cristina. "Purificação e caracterização de um inibidor de elastase de neutrófilos do feijão-caupi (Vigna unguiculata L Walp)". reponame:Repositório Institucional da UFABC, 2017.

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Orientador: Prof. Dr. Sergio Daishi Sasaki
Dissertação (mestrado) - Universidade Federal do ABC, Programa de Pós-Graduação em Biossistemas, 2017.
O Feijão Caupi (Vigna unguiculata (L.) Walp) é uma leguminosa com importante representatividade econômica e nutricional, especialmente no Brasil. Inibidores de serino proteases, como a tripsina, já foram descritos na espécie, assim como em outras plantas. No entanto, nesta espécie, ainda não foram identificados inibidores que apresentem atividade sobre a elastase de neutrófilos humana (HNE), protease envolvida em muitos processos patológicos, como na instalação e progressão da doença pulmonar obstrutiva crônica (DPOC). Nesse estudo, purificamos um inibidor a partir do extrato protéico de Vigna unguiculata que apresenta atividade sobre HNE. Inicialmente, foi realizado o processo de extração alcalina de proteínas, seguido de três passos cromatográficos distintos, utilizando as colunas Hitrap-Q (Troca-iônica), Source15RPC (Fase-Reversa) e ACE18 (Fase-Reversa). Essas etapas foram acompanhadas por testes de atividade inibitória, utilizando os substratos fluorogênicos Meo-Suc-Ala-Ala-Pro-Val-MCA (Elastase) e Z-Phe-Arg-MCA (Tripsina), além de ensaios da quantificação de concentração total de proteínas. Para determinar a massa do inibidor, foram utilizadas as técnicas de espectrometria de massa por MALDI-TOF e SDS-PAGE, o inibidor apresenta massa molecular de 10,99 KDa. O Ki para HNE foi determinado no valor de 9 pM. O inibidor não apresentou atividade inibitória sobre tripsina e trombina, porém foi observada atividade sobre subtilisina e quimotripsina. Estes dados indicam que o inibidor purificado trata-se de uma molécula ainda não caracterizada, devido às suas atividades inibitórias o nomeamos de Vigna unguiculata Elastase Inhibitor (VuEI).
The cowpea (Vigna unguiculata (L.) Walp) is a legume of important economic and nutritional representativeness, especially in Brazil. Serine protease inhibitors, such as trypsin, have been described in many species, as well as in other plants. In this specie an inhibitor with activity on human neutrophil elastase (HNE) has not yet been identified. This protease is involved in many pathological processes, such as the onset and progression of chronic obstructive pulmonary disease (COPD). We purified and characterized an inhibitor from the protein extract of Vigna unguiculata presenting activity towards HNE. Firstly, we performed the alkaline extraction procedure for proteins followed by three different chromatographic steps using Hitrap Q (ion exchange), Source15RPC (Reversed-Phase) and ACE18 (Reversed Phase) columns. These steps were followed by the inhibitory activity tests using fluorogenic substrates, MeO-Suc-Ala-Ala-Pro-Val-MCA (elastase) and Z-Phe-Arg-MCA (trypsin), and quantitation assays of protein concentration. To determinate the size of the molecule, we used MALDI-TOF mass spectrometry and SDS-PAGE. The molecular mass of the inhibitor was 10,99 kDa. The dissociation constant (Ki) toward HNE was 9 pM. HNE inhibitor showed no inhibitory activities toward trypsin and thrombin. However, the inhibitor presented activity toward subtilisin and chymotrypsin. These datas indicate that this molecule is a novel inhibitor to HNE and we named it Vigna unguiculata Elastase Inhibitor (VuEI).

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11

Pearson, David Scott. "Reversible Photoregulation of Binding of the Serine Protease α-Chymotrypsin to a Functional Surface". Thesis, University of Canterbury. Chemistry, 2007. http://hdl.handle.net/10092/2508.

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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.

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12

Posarac, Vesna. "Characterization and anti-HIV activity of the proprotein convertase-directed serine protease inhibitor, Spn4A". Thesis, University of British Columbia, 2008. http://hdl.handle.net/2429/3434.

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HIV/AIDS is a global health problem of immense magnitude, with 33 million people living with HIV and 2 million AIDS-related deaths per year. As the development of drug resistance undermines treatment efficacy, the long-term success of anti-retroviral therapy depends upon the introduction of novel drugs aimed at additional targets essential for the viral life cycle. With a critical role in many viral diseases including the proteolytic maturation of the HIV-1 envelope glycoprotein gp160, the secretory pathway proprotein convertases (PCs) represent a potential anti-viral target. Our laboratory has reported the identification of Spn4A, a potent naturally occurring secretory pathway serine protease inhibitor directed at the prototype PC member, furin. Because of the requirement for the PCs in the production of infectious HIV-1, we hypothesized that strategic manipulation of PC activity by Spn4A and Spn4A-engineered variants would provide a means of effectively limiting HIV-1 infection. This thesis details the investigation of the anti-proteolytic activities and anti-HIV-1 properties of recombinant adenoviruses expressing Spn4A and Spn4A bio-engineered variants, including a secreted recombinant Spn4A (Spn4A S). Our data shows that the expression of Spn4A S in MAGI-CCR5 cells and furin-deficient LoVo cells inhibited the PC-dependent processing of the HIV-1 envelope precursor gp160. Furthermore, inhibition of processing resulted in a nearly complete reduction of productive HIV-1 infection as determined by HIV-1 Tat-driven β-galactosidase activity and multinuclear activation of a galactosidase indicator (MAGI) assays. Complementing the previously described anti-furin activity of Spn4A, our studies indicate that Spn4A S inhibits additional PCs involved in gp160 maturation, and that PC inhibition can serve as an effective means of limiting HIV-1 infection. With the central role of the PCs in the replication and pathogenesis of numerous infectious agents, the identification of Spn4A S as an efficacious HIV inhibitor establishes Spn4A as a prospective broad-based agent for the inhibition of PC-related diseases.

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13

Kruger, Sarah Jane y n/a. "Characterisation of Proteins from Grevillea robusta and NMR Studies of the Serine Protease Inhibitor". Griffith University. School of Science, 2004. http://www4.gu.edu.au:8080/adt-root/public/adt-QGU20040618.150708.

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Proteins that recognise the sugar surface structures on cells have an enormous potential to be used as tools in the characterisation of these structures. A group of proteins, called lectins, have been identified that can bind to carbohydrate complexes on the receptors of cells. The crude extract from Grevillea robusta seeds was found to contain lectin-like proteins that were different from most other lectins, as they would specifically target the receptors of white blood cells and not those found on red blood cells. Therefore, the lectin isolated from G.robusta could be used as a tool to identify the specific surface structures on white blood cells. The lectin was isolated using affinity chromatography where a complex (oligosaccharide) matrix was used. Agglutination, binding and sugar inhibition assays confirmed the isolated protein was a lectin. The lectin was found in low amounts (up to 5% of the total protein content) within the seeds of G.robusta. As a result of this low yield, the identification of the lectin by PAGE was difficult because the levels of protein were beyond the detection limit of the commercial staining reagents. The lectin was called the GR2 protein and was characterised as a monocot mannose binding lectin based on its sugar specificity for only mannose. A serine protease inhibitor was isolated from the seeds of G.robusta using two different chromatography methods, reverse phase HPLC (GR1.HPLC) and gel filtration chromatography (GR1.GF). Ion exchange chromatography was used to initially separate the proteins in the crude extract and the fraction containing the GR1 protein was further purified using reverse phase HPLC (GR1.HPLC). N-terminal sequencing results of the GR1.HPLC protein, showed evidence of proteolytic cleavage during the extraction process, which lead to the second purification method being established. Protease inhibitors were added to the buffers prior to being purified by gel filtration chromatography, which resulted in the GR1 protein being isolated from the crude extract without the presence of the contaminating protein. Mass spectroscopy identified the molecular weight of the GR1 protein to be 6669Da and the full amino acid sequence was derived by cDNA techniques. Sequence alignment studies of the GR1 protein showed significant similarities with the Bowman-Birk inhibitor. The positioning of the cysteine residues were conserved throughout the Bowman-Birk superfamily, however these residues were not conserved within the GR1 protein. Competitive inhibition assays on the GR1 protein revealed the protein could inhibit both trypsin and chymotrypsin at similar levels to that seen for the Bowman-Birk inhibitor. Therefore, the GR1 protein was characterised as a member of the Bowman-Birk superfamily of serine protease inhibitors. The three-dimensional structure of the GR1 protein was determined using two-dimensional NMR spectroscopy. Computer programs such as XEASY, DYANA and SYBYL® were used to tabulate the information taken from the 2D experiments, generate structures and minimise these structures respectively. The solution structure of the GR1 protein was found to contain a region of antiparallel β-sheet structure that corresponded to the trypsin binding site and the remainder of the protein consisted of loops and turns that were held together by disulfide bridges (the chymotrypsin-binding region). Structural similarities between the GR1 protein and the Bowman-Birk inhibitor existed only in the trypsin-binding site of the Bowman-Birk inhibitor. The GR1 protein is the first member of the Proteaceae family to be characterised as a Bowman-Birk inhibitor. This thesis outlines the isolation and biochemical characterisation of the two proteins found within Grevillea robusta and also describes the steps involved and results obtained in determining the three-dimensional structure of the GR1 protein.

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14

Kruger, Sarah Jane. "Characterisation of Proteins from Grevillea robusta and NMR Studies of the Serine Protease Inhibitor". Thesis, Griffith University, 2004. http://hdl.handle.net/10072/366534.

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Proteins that recognise the sugar surface structures on cells have an enormous potential to be used as tools in the characterisation of these structures. A group of proteins, called lectins, have been identified that can bind to carbohydrate complexes on the receptors of cells. The crude extract from Grevillea robusta seeds was found to contain lectin-like proteins that were different from most other lectins, as they would specifically target the receptors of white blood cells and not those found on red blood cells. Therefore, the lectin isolated from G.robusta could be used as a tool to identify the specific surface structures on white blood cells. The lectin was isolated using affinity chromatography where a complex (oligosaccharide) matrix was used. Agglutination, binding and sugar inhibition assays confirmed the isolated protein was a lectin. The lectin was found in low amounts (up to 5% of the total protein content) within the seeds of G.robusta. As a result of this low yield, the identification of the lectin by PAGE was difficult because the levels of protein were beyond the detection limit of the commercial staining reagents. The lectin was called the GR2 protein and was characterised as a monocot mannose binding lectin based on its sugar specificity for only mannose. A serine protease inhibitor was isolated from the seeds of G.robusta using two different chromatography methods, reverse phase HPLC (GR1.HPLC) and gel filtration chromatography (GR1.GF). Ion exchange chromatography was used to initially separate the proteins in the crude extract and the fraction containing the GR1 protein was further purified using reverse phase HPLC (GR1.HPLC). N-terminal sequencing results of the GR1.HPLC protein, showed evidence of proteolytic cleavage during the extraction process, which lead to the second purification method being established. Protease inhibitors were added to the buffers prior to being purified by gel filtration chromatography, which resulted in the GR1 protein being isolated from the crude extract without the presence of the contaminating protein. Mass spectroscopy identified the molecular weight of the GR1 protein to be 6669Da and the full amino acid sequence was derived by cDNA techniques. Sequence alignment studies of the GR1 protein showed significant similarities with the Bowman-Birk inhibitor. The positioning of the cysteine residues were conserved throughout the Bowman-Birk superfamily, however these residues were not conserved within the GR1 protein. Competitive inhibition assays on the GR1 protein revealed the protein could inhibit both trypsin and chymotrypsin at similar levels to that seen for the Bowman-Birk inhibitor. Therefore, the GR1 protein was characterised as a member of the Bowman-Birk superfamily of serine protease inhibitors. The three-dimensional structure of the GR1 protein was determined using two-dimensional NMR spectroscopy. Computer programs such as XEASY, DYANA and SYBYL® were used to tabulate the information taken from the 2D experiments, generate structures and minimise these structures respectively. The solution structure of the GR1 protein was found to contain a region of antiparallel β-sheet structure that corresponded to the trypsin binding site and the remainder of the protein consisted of loops and turns that were held together by disulfide bridges (the chymotrypsin-binding region). Structural similarities between the GR1 protein and the Bowman-Birk inhibitor existed only in the trypsin-binding site of the Bowman-Birk inhibitor. The GR1 protein is the first member of the Proteaceae family to be characterised as a Bowman-Birk inhibitor. This thesis outlines the isolation and biochemical characterisation of the two proteins found within Grevillea robusta and also describes the steps involved and results obtained in determining the three-dimensional structure of the GR1 protein.
Thesis (PhD Doctorate)
Doctor of Philosophy (PhD)
School of Science
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15

De, Veer Simon J. "Development of novel protease inhibitors for epidermal kallikrein proteases". Thesis, Queensland University of Technology, 2014. https://eprints.qut.edu.au/114508/1/Simon_de%20Veer_Thesis.pdf.

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Preserving the integrity of the skin's outermost layer (the epidermis) is vital for humans to thrive in hostile surroundings. Covering the entire body, the epidermis forms a thin but impenetrable cellular cordon that repels external assaults and blocks the escape of water and electrolytes from within. This structure exists in a perpetual state of repair and regeneration where the production of new cellular subunits (keratinocytes) at the base of the epidermis is offset by the gradual release of terminally differentiated corneocytes from the surface. It is increasingly clear that proteinases (hereafter termed proteases) are essential for assembling and maintaining the epidermal barrier. More than thirty proteases are expressed by keratinocytes or immune cells that infiltrate the skin, and the activity of each must be maintained within narrow limits and confined to the correct time and place. Accordingly, dysregulated proteolytic activity is a common factor in a multitude of skin disorders that range in severity from relatively mild to life-threatening. Serine proteases from the kallikrein-related peptidase (KLK) family are widely recognised as key modulators of epidermal barrier function. For several decades, KLK proteases have been regarded as major contributors to the natural shedding of corneocytes from the skin's surface by degrading (corneo)desmosomes in the stratum corneum. Additionally, controlled KLK proteolytic activity influences the step-wise processing of key molecules involved in hydration and acidification (pro-filaggrin), and anti-microbial defence (cathelicidins). Further, KLK proteases have prominent roles in the response to barrier disruption that involve stimulating inflammation and alerting the immune system. Thus, failure to properly control KLK proteolytic activity represents a significant threat to epidermal barrier function, as seen in a spectrum of skin disorders, including Netherton syndrome and atopic dermatitis. The focus of this study was to develop novel inhibitors for three KLK proteases with the strongest links to epidermal (patho)physiology (KLK5, KLK7 and KLK14) by engineering the naturally occurring cyclic peptide, sunflower trypsin inhibitor-1 (SFTI-1). Initially, each target protease was screened against a dedicated library of individually synthesised peptide substrates (sparse matrix library) to identify sequence combinations that bound to the active site with high affinity. This approach yielded a series of efficient tetrapeptide substrates for each protease that outperformed existing substrates from conventional screening methods, such as positional scanning and phage display. Strikingly, the optimal substrates for KLK5, KLK7 and KLK14 each displayed a unique physicochemical signature, indicating that the active site of each protease was configured to recognise distinct cleavage motifs. This phenomenon was explored in more detail by analysing structures of diverse serine proteases from the S1A (chymo)trypsin fold, which identified several points of high sequence variation in the active site cleft that likely contribute to divergent cleavage site specificities. Subsequently, favoured cleavage sequences for KLK5, KLK7 and KLK14 were substituted into the contact β-strand of SFTI-1 to generate inhibitor variants with improved affinity and selectivity. Whereas most inhibitor design strategies based on Laskowski inhibitors (including SFTI-1) focus mainly on optimising the interaction between protease and inhibitor, this study paired binding loop modifications with a second step that aimed to refine the intramolecular hydrogen bond network, as shown recently for engineered SFTI variants targeting KLK4. Like the previous study, improving the tendency for an inhibitor to form intramolecular hydrogen bonds generally led to improvements in inhibitory activity. However, it was also evident that certain hydrogen bonds were detrimental, revealing that the quantity of hydrogen bonds should not be the only criteria during this screening process. Additionally, the iterative optimisation of inhibitors for KLK5 highlighted the need to consider both sides of the reactive site when engineering Laskowski inhibitors as generating a selective KLK5 variant was dependent on substituting the P2′ residue. Using this design process, it was possible to successfully re-direct the inhibitory activity of SFTI-1 to three separate protease targets that showed varying affinity for the wild-type inhibitor. To delve further into the Laskowski mechanism, SFTI-1 was used as a model system to explore the molecular basis for Laskowski inhibitor potency and specificity. Here, inhibitor association and dissociation kinetics were characterised for a series of variants with different binding sequences and hydrogen bond tendencies. These analyses revealed that the primary determinant for rapid association was the pre-organised conformation of the inhibitor binding loop rather than its sequence, whereas coordinated inter- and intramolecular interactions promoted efficient religation and slow dissociation. As the conformation of the binding loop is conserved, inhibitor selectivity dictated by the binding sequence was found to arise from modulating the off-rate. Performing additional analyses on eight fold-divergent inhibitor families revealed that these concepts were generally applicable to Laskowski inhibitors, providing broad new insights on protease inhibitor function and design. These findings were subsequently applied to engineer an additional series of potent inhibitors with broad-range activity. Finally, the substrates and inhibitors developed for KLK5, KLK7 and KLK14 were used in biological assays to investigate the activity and significance of each target protease in healthy and diseased skin. KLK peptide substrates were applied to profile KLK hyperactivity in skin extracts from transgenic KLK5 mice and Spink5-/- mice. Additionally, KLK inhibitors were used to sequentially block different KLKs in gel-based and in situ zymography experiments. Selective and broad-range inhibitors were also evaluated in ex vivo desquamation assays to examine the relative importance of KLK5, KLK7 and KLK14 during corneocyte shedding, which revealed a major role for KLK7. Collectively, these findings shed light on the individual contributions of KLK proteases to maintaining the epidermal barrier and identify a series of therapeutic leads for further development as novel treatments for skin disorders associated with dysregulated KLK proteolytic activity.

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16

Wångsell, Fredrik. "Design and Synthesis of Aspartic and Serine Protease Inhibitors : Targeting the BACE-1 and the HCV NS3 Protease". Doctoral thesis, Uppsala universitet, Institutionen för läkemedelskemi, 2009. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-108985.

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This thesis describes work done to design and synthesize protease inhibitors, with the intention of developing therapeutic agents for Alzheimer’s disease (AD) and the chronic liver condition caused by infection of the hepatitis C virus (HCV). AD is the most common form of dementia, and HCV infection is the primary reason for liver transplantation in industrialized countries. Today, these two illnesses affect 24 and 170 million people, respectively. It has been shown that the human aspartic protease BACE-1 plays an important role in the development of AD, and thus inhibition of BACE-1 may offer a way to improve the quality of life of individuals afflicted with the disease. Furthermore, it is known that the serine protease NS3 is a vital component in the replication of HCV. Several novel potent BACE-1 inhibitors encompassing different transition state mimics were prepared. First, a hydroxyethylene moiety encompassing a secondary hydroxyl group was evaluated as a transition state analogue, producing inhibitors in the low nanomolar range. Various tertiary hydroxyl isosteres were also investigated as the central core, with the aim of shielding the pivotal hydroxyl group. These transition state isosteres consisted of tertiary hydroxyl analogues of previously used secondary hydroxyl containing norstatine, statine, and hydroxyethylamine isosteres. Several tertiary hydroxyl-containing inhibitors were found to be active in the low micromolar range. In addition, two inhibitors were co-crystallized with the BACE-1 enzyme to provide X-ray crystal structures, which furnished valuable binding information for further design of improved BACE-1 inhibitors. The goal in the HCV NS3 protease inhibitor project was to design, synthesize and evaluate a novel hydroxycyclopentene bioisostere to the previously used acyl-hydroxyproline moiety. The investigation revealed that it was possible to synthesize inhibitors containing this new bioisostere that were potent in the low nanomolar range. Further optimization by rigidification of the most active inhibitor resulted in equipotent macrocyclic compounds.

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Kent, Patricia. "Hepatic iron metabolism: studies on the regulation and function of Serine Protease Inhibitor clade B3 and Hemojuvelin". Thesis, McGill University, 2014. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=123313.

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In the human body as in all mammals, iron is an essential trace element. Despite its required presence for many biological functions, it is also toxic because of its labile oxidation states and ability to catalyze reactions. For this reason, tight control must be exerted to maintain iron in a bioavailable yet redox inert state. Also since mammals possess no regulated iron excretion mechanism, the dietary uptake of the metal must be tightly controlled as well. This ensures that there is enough iron for the body's needs yet not too much as to result in iron overload and the complications which arise from it.On a systemic level, mechanisms have evolved to safely move iron throughout the body, between cells that utilize, recycle, and store it. At the forefront of this process is hepcidin, a peptide hormone orchestrating the body's systemic iron homeostasis. Hepcidin is in turn controlled via iron, such that a balance is achieved between utilization and storage of the metal. Despite the number of safeguards that have evolved to maintain homeostasis, complications can arise, which allow researchers to ask questions and find answers.It was investigated in Chapter 2 how a serine protease inhibitor, SERPIN B3, is affected by iron in vitro. A causative link was determined in vivo in a mouse model between iron overload and hepatic SERPIN B3 expression. However, in vitro, the results could not be recapitulated at the level of transcription, despite looking at both primary and secondary effects of iron. Furthermore, it was not possible to prove or disprove that SERPIN B3 plays a role in hepcidin expression.In Chapter 3, the investigation of the role of two proteins which are well established in the hepcidin regulatory pathways was conducted. A novel knockout mouse was generated combining deletions in both the hereditary hemochromatosis (HFE) and hemojuvelin (HJV) proteins and iron accumulation and hepcidin expression in this double knockout (DKO) were investigated. It was shown that the DKO mice are phenotypically like HJV single knockout mice and that there is crosstalk between the two iron sensing pathways.
Comme chez tous les mammifères, le fer est un élément essentiel au corps humain. Malgré sa présence nécessaire à de nombreuses fonctions biologiques, il est également potentiellement toxique à cause de ses états d'oxydation labiles et de sa capacité à catalyser des réactions. Pour cette raison un contrôle sévère doit être exercé pour maintenir le fer dans un état redox inerte tout en étant disponible biologiquement. Les mammifères ne possédant pas de mécanisme régulé d'excrétion du fer, l'apport alimentaire de ce métal doit également être étroitement contrôlé. Cela permet de combler adéquatement les besoins du corps en fer tout en évitant la surcharge en fer et les complications qui en résultent.Au niveau systémique, des mécanismes ont évolué pour déplacer le fer correctement partout dans le corps entre des cellules qui utilisent, recyclent ou stockent cet élément. En première ligne de ce processus se trouve l'hepcidine, un peptide hormone, orchestrant l'homéostasie du fer au niveau systémique. L'hepcidine est à son tour régulée par l'intermédiaire du fer, de manière telle que la balance est atteinte entre l'utilisation et le stockage de ce métal. Malgré la présence de nombreux systèmes de contrôle responsables de la maintenance de l'homéostasie, des complications peuvent apparaître qui permettent aux chercheurs de poser des questions et de trouver des réponses.Dans le chapitre 2, il a été étudié comment la SERPINE B3, un inhibiteur de protéase à serine, est affectée in vitro par le fer. Un lien de cause à effet avait été déterminé entre la surcharge en fer et l'expression hépatique de la SERPINE B3 in vivo dans un modèle murin. Cependant, les résultats au niveau de la transcription n'ont pas pu être récapitulés in vitro, malgré la recherche d'effets primaires et secondaires du fer. De plus, il n'a pas été possible de prouver ou d'invalider que la SERPINE B3 joue un rôle dans l'expression de l'hepcidine.Dans le chapitre 3, l'investigation du rôle de deux protéines qui sont bien établies dans les voies de régulation de l'hepcidine a été conduite. Un nouveau modèle de souris knock-out a été généré combinant les délétions de HFE (héréditaire hemochromatose protein) et de l'hémojuveline (HJV). L'accumulation du fer et l'expression de l'hepcidine ont été étudiées dans ce double knock-out (DKO). Il a été montré que les souris DKO sont phénotypiquement similaires aux souris knockout HJV et qu'il existe un lien entre les deux systèmes de détection du fer.

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Gulley, Melissa M. "Biochemical characterization of serpins in the malaria vector, Anopheles gambiae". Thesis, Kansas State University, 2013. http://hdl.handle.net/2097/15870.

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Master of Science
Division of Biology
Kristin Michel
To date malaria is the most important tropical disease, which is caused by Plasmodium sp. and vectored by anopheline mosquitoes. The mosquito’s immune system is one of the limiting factors of malaria transmission. Immune reactions, such as the prophenoloxidase (PPO) pathway result in the melanization of pathogens, and are effective at limiting parasite numbers. Novel strategies for malaria control aim to exploit the immune system to interrupt parasite transmission by boosting the immune responses in the mosquito vector. Serpins play a crucial role in regulating protease cascades involved in immunity of arthropods. In Anopheles gambiae, the major malaria vector in Sub-Saharan Africa, 18 SRPN genes encoding 23 distinct proteins have been identified. So far, two are identified as active inhibitors, and both affect parasite survival. This research aims to identify additional inhibitory serpins in An. gambiae and elucidate their potential function. Identification of such serpins will enhance our understanding of the immune system of this important vector species and may identify immunoregulators to be used in malaria control. SRPN7, 9, and 18 were tested for their ability to inhibit commercial proteases in vitro. Recombinant SRPN18 had no inhibitory activity, while SRPN7 and 9 inhibited several serine proteases. SRPN7, 9 and 18 were tested against two recombinant An. gambiae clip serine proteases (CLIPBs) that are required for activation of phenoloxidase and thus regulate melanization. Only SRPN9 strongly inhibited CLIPB9 in vitro, suggesting that this serpin is a potential negative regulator of melanization. This hypothesis is further supported by the finding that SRPN9 can inhibit PO activity in insect hemolymph, ex vivo. Taken together, this research identifies SRPN18 as the first non-inhibitory serpin described in mosquitoes. Additionally, this study describes the larval-specific SRPN7 as a functional inhibitor. Future studies on these proteins will elucidate their precise physiological functions. Finally, this thesis provides strong evidence that SRPN9 is a negative regulator of melanization in An. gambiae and may therefore affect pathogen survival within this important vector species.

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19

Lundmark, Kristoffer. "Characterisation of free and conjugated protease inhibitors from Solanum tuberosum". Thesis, Uppsala universitet, Institutionen för kemi - BMC, 2017. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-313835.

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The main purpose of the master thesis project is to investigate the influence of selected serine protease inhibitors (SPI) on the catalytic action of the serine proteases chymotrypsin and trypsin, in a conjugated and non-conjugated state. The inhibitors included for this study were extracted from Solanum tuberosum, i.e.common potato. The purification method included in this study consist of crude extraction by mixer, followed by a salt-out procedure with ammonium sulphate. Further purification steps were cation exchange chromatography and, finally, gel filtration to obtain SPI of high purity. The purified sample was then characterized by SDS-page and kinetic activity measurement of trypsin and chymotrypsin action on synthetic substrate derivate, N-Benzoyl-DL-arginine-4-nitroanilide hydrochloride (BAPA) and N-Succinyl-L-phenylalanine-p-nitroaniline (SFpNA) respectively. The characterization showed inhibitory inactivation of both pancreatic proteases. This would indicate successful extraction of SPI. To investigate inhibitory action in a conjugated state, either enzyme or inhibitor was immobilized onto aluminium oxide membranes. Then two different experimental setups were tested, called experiment 1 and 2. In experiment 1, the inhibitor was immobilized and the interaction was monitored from a retention shift of enzyme flow-through compared to a blank column, using detection at 280 nm of the enzyme. In experiment 2 the enzyme was instead immobilized and a mixture of inhibitor and substrate was circulated with monitoring of the catalytic activity. The main goal was thus to measure the effects on the kinetics in the conjugated state compared to enzyme and inhibitor in the free state. The result from both experiment 1 and 2 did not yield consistent and reliable result so the discussed method should be regarded as preliminary results. The study also includes investigation of inhibitor-enzyme interaction as revealed by molecular mass data to determine complex formation. This part was conducted with static light scattering analysis to determine the stoichiometry for the interaction between pancreas proteases and the inhibitor. Results from light scattering showed promising indication of many-to-one interaction between enzyme and inhibitor, which have been seen by previous studies. It should be considered a preliminary result as complex formation does not exclude aggregation of enzymes or inhibitor in the solution.

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20

Tshidino, Shonisani Cathphonia. "Purification and partial characterization of a Myofibril-Bound Serine Protease and its endogenous inhibitor from skeletal muscle of the ostrich". Thesis, Nelson Mandela Metropolitan University, 2008. http://hdl.handle.net/10948/703.

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The ostrich is becoming an important source of meat for humans in developed and developing countries. This study was conducted to purify and characterize myofibrilbound serine protease (MBSP) and its endogenous inhibitor (MBSPI) from skeletal muscle of the ostrich. It is well documented that MBSP is tightly bound to myofibrils and its endogenous inhibitor has been purified from the same tissue of other studied mammalian species. Literature supports an association of MBSP and its endogenous inhibitor with the degradation of myofribrillar proteins, resulting in the softening of muscle that lead to the conversion of muscle into meat with the control of the inhibitor. MBSP was successfully dissociated from washed myofibrils by 40 percent ethylene glycol at pH 8.5. Following centrifugation, MBSP was partially purified in two chromatographic steps, namely Toyopearl Super Q 650S and p-aminobenzamidine-Agarose. On the other hand, MBSPI was fractionated from the sarcoplasmic fraction using 75 percent ammonium sulfate saturation, followed by centrifugation and partially purified by three chromatographic steps, namely Toyopearl Super Q 650S, Superdex 200 and HiTrap SP HR. Ostrich MBSP was physicochemically and kinetically characterized, while MBSPI was only physicochemically characterized. Ostrich MBSP revealed an Mr of 21 kDa, cleaving synthetic fluorogenic substrates specifically at the carboxyl side of arginine residues. Optimum pH and temperature of ostrich MBSP were 8.0 and 40˚C, respectively. Kinetic parameters (Km and Vmax values) were calculated from Lineweaver-Burk plots. The characteristics of ostrich MBSP were compared to the values obtained for commercial bovine trypsin in this study, as well as that obtained for MBSP from various fish species and mouse. The results suggest that ostrich MBSP is a trypsin-like serine protease, thereby confirming the existence of MBSP in ostrich skeletal muscle. Partially purified ostrich MBSPI (Mr 17 kDa) (one form) shares 100 percent identity to myoglobin from the same species, while 2 other forms of MBSPI (Mr values of 35 and 36 kDa) exhibited high sequence identity to glyceraldehyde 3- phosphate dehydrogenase (GAPDH) (76 percent) from human and rat.

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21

Florencio, Ariana Corrêa. "Efeitos do inibidor específico para serinoprotease rBmTI-A em modelo experimental de inflamação pulmonar alérgica crônica em camundongos Balb/c". Universidade de São Paulo, 2018. http://www.teses.usp.br/teses/disponiveis/5/5165/tde-13062018-094113/.

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INTRODUÇÃO: A asma ainda acomete um número crescente de indivíduos, podendo ser muito grave e, algumas vezes, fatal. A despeito da melhor eficiência diagnóstica e eficácia terapêutica, a maioria dos asmáticos graves não obtém controle total dos sintomas com as terapias disponíveis. Alguns estudos sugerem a atuação de inibidores de serinoproteases em diversos processos inflamatórios, entre estes inibidores encontra-se o Boophilus microplus trypsin Inhibitor (BmTI-A). OBJETIVO: Avaliar se o recombinante do inibidor de serinoproteases rBmTI-A modula a hiperresponsividade brônquica à metacolina, inflamação e remodelamento das vias aéreas em um modelo experimental de inflamação pulmonar alérgica crônica. MÉTODOS: Camundongos Balb-c foram divididos em 4 grupos: SAL (salina), OVA (sensibilizados com ovoalbumina), SAL+rBmTI-A (controle tratados com rBmTI-A ) e OVA+rBmTI-A (sensibilizados com ovoalbumina e tratados com rBmTI-A). Nos dias 0 e 14 do protocolo, os animais receberam injeção intraperitoneal (i.p) de salina (0,9% NaCl) (SAL e SAL+rBmTI-A) e ovoalbumina (50 ug/mL) (OVA e OVA+rBmTI-A). Nos dias 22, 24, 26 e 28 foram submetidos à inalação com salina (0,9% NaCl) ou ovoalbumina (10 mg/ml) e foram tratados com rBmTI-A (35,54 pmol em 50 uL de NaCl) ou apenas salina, via instilação nasal, nos dias 22 e 28. No dia 29, foram realizadas as seguintes análises: hiper-responsividade à metacolina e respostas máximas de resistência e elastância do sistema respiratório; quantificação do número total de células, macrófagos, linfócitos e polimorfonucleares no fluido do lavado broncoalveolar (FLBA); determinação da concentração das citocinas IL-4, IL-5, IL-10, IL-13, IL-17A e IFN-y no FLBA por citometria de fluxo (Cytometric Bead Array - CBA); avaliação da expressão de IL-4, IL-5, IL-10, IL-13, IL-17, MMP-9 e TIMP-1 nas vias aéreas; análise histopatológica do pulmão para quantificação de eosinófilos, fibras colágenas e elásticas e avaliação da atividade proteolítica de tripsina-like, MMP-1 e MMP9. A significância foi considerada p < 0,05. RESULTADOS: O tratamento com rBmTI-A nos animais sensibilizados reduziu a atividade proteolítica de tripsina no tecido pulmonar; a resposta máxima de Rrs e Ers; o número de polimorfonucleares e a concentração de IL-5, IL-10, IL-13 e IL-17A no FLBA; a expressão de IL-5, IL-13, IL-17, MMP-9 e TIMP-1 nas vias aéreas; o número de eosinófilos e a fração de fibras colágenas e elásticas nas vias aéreas do grupo OVA+rBmTI-A comparado ao grupo OVA (p < 0,05). CONCLUSÃO: O rBmTI-A atenuou a hiper-responsividade brônquica, a inflamação e o remodelamento nesse modelo experimental de inflamação pulmonar alérgica crônica. Embora mais estudos precisem ser realizados, este inibidor pode contribuir como potencial ferramenta terapêutica para o tratamento de asma
INTRODUCTION: Asthma still affects an increasing number of individuals and can be very serious and sometimes fatal. Despite the improved diagnostic efficiency and therapeutic efficacy, most severe asthmatics do not have complete symptom control with available therapies. Some studies suggest the role of serine protease inhibitors in various inflammatory processes, such as Boophilus microplus trypsin inhibitor (BmTIA). AIMS: To evaluate whether rBmTI-A serine protease inhibitor recombinant modulates bronchial hyperresponsiveness to methacholine, airway inflammation and remodeling in an experimental model of chronic allergic lung inflammation. METHODS: Balb/c mice were divided in four groups: SAL (saline), OVA (sensitized with ovalbumin), SAL+rBmTI-A (control treated with rBmTI-A) and OVA+rBmTI-A (sensitized with ovalbumin and treated with rBmTI-A). On days 0 and 14 of the protocol the animals received intraperitoneal injection (i.p) of saline (0.9% NaCl) (SAL and SAL+rBmTI-A) and ovalbumin (50 ug/mL) (OVA and OVA+rBmTI-A). On days 22, 24, 26 and 28 the groups were submitted to inhalations with saline (0.9% NaCl) or ovalbumin (10 mg/ml) and were treated with a rBmTI-A (35.54 pmol in 50 uL of saline or just saline) by nasal instillation, on the days 22 and 28. On day 29, the following analysis were performed: hyperresponsiveness to methacholine and the maximal resistance and elastance responses of the respiratory system were obtained; quantification of the total number of cells, macrophages, lymphocytes and polymorphonuclear in bronchoalveolar lavage fluid (FLBA); determination of the cytokines IL-4, IL-5, IL-10, IL-13, IL-17A and IFN-y concentration in FLBA by Cytometric Bead Array (CBA); IL4, IL-5, IL-10, IL-13, IL-17, MMP-9 and TIMP-1 expression in the airways; histopathological analysis of the lung for quantification of eosinophils, collagen and elastic fibers and evaluation of trypsin-like, MMP-1 and MMP9 proteolytic activity. Significance was considered when p < 0.05. RESULTS: The treatment with rBmTI-A in sensitized animals reduced: the proteolytic activity of trypsinlike in lung tissue, the maximum response of Rrs and Ers, the number of polymorphonuclear cells and the concentration of IL-5, IL-10, IL-13 and IL-17A in FLBA, the expression of IL-5, IL-13, IL-17, MMP-9 and TIMP-1 in the airways, the number of eosinophils and the fraction of collagen and elastic fibers in the airways of the OVA + rBmTI-A group compared to the OVA group (p < 0.05). CONCLUSION: rBmTI-A attenuated bronchial hyperresponsiveness, inflammation and remodeling in this experimental model of chronic allergic pulmonary inflammation. Although more studies need to be performed, this inhibitor may contribute as a potential therapeutic tool for the asthma treatment

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22

Mkaouar, Héla. "Rôle des serpines, inhibiteurs de protéases à serine, du microbiote digestif humain dans les maladies inflammatoires de l'intestin". Thesis, Université Paris-Saclay (ComUE), 2019. http://www.theses.fr/2019SACLS108.

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Les inhibiteurs des protéases à sérine (Serpins) constituent une classe d'enzymes très peu étudiée chez les bactéries. Dans ce travail de thèse nous nous sommes intéressés à l'étude des serpins provenant du microbiote intestinal et l'investigation de leur potentiel anti-inflammatoire pour le traitement des maladies inflammatoires chroniques de l'intestin (MICI) chez l'homme. Pour cela nous avons identifié les serpins provenant du microbiote intestinal humain et analysé leur diversité ainsi que leur distribution entre les individus malades et sains. Ces données nous ont permis d'isoler les serpins significativement associées aux MICI. La purification de quarte d'entre elles nous a amené à démontrer qu'elles inhibent les protéases humaines impliquées dans les MICI. L'analyse biochimique et cinétique approfondie de ces protéines a montré qu'elles possèdent des propriétés originales notamment leur efficacité d'inhibition élevée. L'étude de l'effet protecteur de trois serpins chez un modèle animal de colite a démontré pour la première fois l'efficacité des serpins in vivo démontrant ainsi leur potentiel thérapeutique
Serine protease inhibitors (Serpins) are a class of proteins that reamin poorly studied in bacteria. In this thesis we are interested in the study of serpins originating from the intestinal microbiota and the investigation of their anti-inflammatory potential for the treatment of inflammatory bowel diseases (IBD) in humans. For this we have identified serpins from the human gut microbiota and analyzed their diversity as well as their distribution between healthy and IBD patients. These data allowed isolating serpins significantly associated with IBD. The purification of four of them led us to demonstrate that they inhibit human proteases involved in IBD. Biochemical and kinetic analysis of these proteins showed that they exhibit original properties, in particular their high inhibition efficiency. The study of the protective effect of three serpins in an animal model of colitis demonstrated for the first time the efficacy of serpins in vivo demonstrating thus their therapeutic potential

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GRAVELEAU-BILLEMAZ, LAURE. "Topologie de la forme fonctionnelle des serpines (serine protease inhibitor) : etude par l'analyse des processus de repliement in vitro des formes intacte et proteolyse de l'alpha#1 antiprotease humaine". Paris 11, 1994. http://www.theses.fr/1994PA112082.

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L'alpha-1 antiprotease humaine, inhibiteur de la famille des serpines a ete l'objet de nos etudes. Ce substrat suicide des proteases a serine subit un rearrangement structural important apres proteolyse de la liaison peptidique reactive met 358-ser 359, le rendant inactif. En utilisant les signaux intrinseques de la proteine (principalement l'emission de fluorescence des residus aromatiques et l'ellipticite) nous avons cherche a preciser l'ampleur des rearrangements structuraux induits par la proteolyse de la boucle reactive et l'insertion de sa partie n-terminale dans le feuillet beta a. Les resultats obtenus indiquent que, dans la conformation de l'inhibiteur proteolyse, l'etendue des surfaces hydrophobes accessibles a l'acide 8-anilino-naphtalene sulfonique est plus faible que celle de l'inhibiteur natif. L'environnement des residus aromatiques est aussi modifie: ce rearrangement se reflete non seulement par la modification conformationnelle des segments proches du feuillet beta a mais aussi de la partie proche du nouveau c-terminal, de l'helice f et du feuillet beta b. Ces modifications structurales ont ete mises en evidence par les modifications des pk#a#p#p d'ionisation des tyrosines ainsi que par la modification des transitions de denaturation-renaturation caracterisant chacune des deux formes de l'inhibiteur. L'etude du repliement de l'inhibiteur a ph 8,5 et a ph 10,6 nous a permis de discuter le role de la tyr 244 dans les modifications de l'environnement du trp 194, ainsi que la participation possible de la tyr 187 dans la restructuration du feuillet beta a. La conformation metastable de la structure native de l'inhibiteur maintenue par la boucle reactive specifique ainsi que le processus de reorganisation du feuillet beta a contribuant a la stabilisation de la proteine apres proteolyse, constituent un modele d'etude particulierement interessant pour la comprehension du role de cette etape ultime du repliement de l'alpha-1 antiprotease et plus generalement des mecanismes de regulation caracteristiques des inhibiteurs plasmatiques des proteases

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24

Leahy, Darren. "Probing the role of methionine oxidation in substrate and inhibitor interactions with native and recombinant Human Neutrophil Elastase". Thesis, Queensland University of Technology, 2020. https://eprints.qut.edu.au/204141/1/Darren_Leahy_Thesis.pdf.

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This thesis was an exploration of how Human Neutrophil Elastase (HNE) activity can be modulated by oxidation of methionine residues located on substrates and inhibitors. Research focused on producing a molecular toolbox of innovative HNE substrates and inhibitors specifically engineered to include methionine, then assessing the mechanism by which oxidation leads to targeted interaction with HNE. This may be an important biochemical process in chronic obstructive pulmonary disease, which is linked to HNE destruction of elastic lung tissue together with oxidative damage by cigarette smoke and neutrophil-mediated inflammation.

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25

Reid, Janet C. "Identification and characterization of novel proteolytic interactions of prostate cancer-expressed kallikrein-related peptidases, type II transmembrane serine proteases and matrix metalloproteinases". Thesis, Queensland University of Technology, 2015. https://eprints.qut.edu.au/81594/1/Janet_Reid_Thesis.pdf.

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This study investigated interactions of protein-cleaving enzymes (or proteases) that promote prostate cancer progression. It provides the first evidence of a novel regulatory network of protease activity at the surface of cells. The proteases kallikrein-related peptidases 4 and 14, and matrix metalloproteinases 3 and 9 are cleaved at the cell surface by the cell surface proteases hepsin and TMPRSS2. These cleavage events potentially regulate activation of downstream targets of kallikrein 4 and 14 such as cell surface signalling via the protease-activated receptors (PARs) and cell growth-promoting factors such as hepatocyte-growth factor.

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26

Kretzschmar, Tim [Verfasser]. "Effects of a synthetic serine protease inhibitor, camostat mesilate (FOY-305), on markers of pancreatic acinar cell damage, inflammation, and fibrosis in dogs with suspected naturally occuring chronic pancreatitis / Tim Kretzschmar". Berlin : Freie Universität Berlin, 2015. http://d-nb.info/1072410664/34.

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27

Bhatia, Harminder Singh. "Bacterial expression, purification and characterization of human alpha 2 antiplasmin". VCU Scholars Compass, 2006. http://scholarscompass.vcu.edu/etd_retro/170.

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The serpin antiplasmin (APL) is the primary inhibitor of plasmin, a proteinase that digests fibrin, the main component of blood clots. Most serpins are serine protease inhibitors, which undergo dramatic conformational change in forming a tight covalent complex with the target protease. Plasmin has been shown to be angiogenic through its protease activity, but it is also angiostatic, being the source of angiostatin, which inhibits angiogenesis. The main objective of our study is to obtain antiplasmin in large amounts, for crystallization and structure determination of APL and of its complex with plasmin, and for solution studies of the complex. Bacterially expressed APL will not be glycosylated, an advantage in crystallization trials.Bacterial expression of rAPL has been problematic. We have found that it can be greatly enhanced through the use of host E.coli cells that carry extra copies of genes for tRNAs coding for rarely used codons in E.coli that occur in high frequency in eukaryotic genes. Several vectors were screened for rAPL expression (pET19b, pET20b and pET28b). rAPL is expressed in high yield from a pET28b construct in host BL-21 RIPL codon plus cells. rAPL thus expressed accumulates as inclusion bodies, but can be solubilized using N-lauroyl sarcosine at pH11. Refolding and purification of rAPL is achieved by using a sizing column followed by a Nickel His-tag affinity column with an imidazole gradient. rAPL fractions thus obtained are stable at 4Â°C in the presence of EDTA. However, no inhibitor activity of this rAPL towards trypsin was observed, nor did it form inhibition complex with trypsin. The presence of trace protease and/or failure to fold correctly may be preventing recovery of inhibitory activity. A screen of various refolding buffers failed to yield soluble, stable APL.

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28

Costa, Marianges Zadrozny Gouvêa da. "Frequência de tabagismo e das mutações N34S e P55S do gene Serine Protease Inhibitor Kazal-Type 1 (SPINK1) e da mutação R254W do gene Quimotripsina C (CTRC) em pacientes portadores de pancreatite crônica e em controle". Universidade de São Paulo, 2015. http://www.teses.usp.br/teses/disponiveis/5/5168/tde-06112015-160722/.

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A pancreatite crônica é uma desordem complexa, na qual a interação entre fatores ambientais e genéticos resulta na enfermidade. O presente estudo incluiu 148 pacientes com diagnóstico de pancreatite crônica, 110 etilistas crônicos e 297 controles sadios com o objetivo de investigar a frequência de tabagismo e das mutações N34S e P55S do gene SPINK1 e R254W do gene CTRC nesta população. Foi aplicado questionário presencial e realizada reação de sequenciamento para a pesquisa das mutações genéticas, após assinatura do Termo de Consentimento Livre e Esclarecido. Os portadores de pancreatite crônica possuíam etiologia alcoólica em 74% das vezes e idiopática em 26%. A pancreatite alcoólica apresentou-se de maneira distinta da pancreatite crônica idiopática, sendo que o primeiro grupo é composto por maior prevalência do gênero masculino (88,18% versus 34,21%), por maior média de idade (55,64 anos versus 45,20 anos), menor frequência de caucasianos (63,89% versus 84,21%), menor escolaridade (23,30% concluíram ensino médio ou superior versus 57,89%) e maior frequência de repercussões da doença, como diarréia (54,21% versus 24,24%), emagrecimento (56,07% versus 24,24%), diabete melito (57,94% versus 36,36%) e ocorrência de pseudocistos pancreáticos (31,78% versus 12,12%), repercussões estas que não foram acompanhadas de maior frequência de alterações morfológicas, como calcificações pancreáticas ou dilatação do ducto pancreático principal. A frequência de tabagismo foi significativamente maior em pacientes com pancreatite crônica alcoólica do que em etilistas sem pancreatite crônica, podendo ser considerado cofator de risco para o desenvolvimento da pancreatite crônica entre alcoolistas (p = 0,002); a frequência da mutação N34S do gene SPINK1 em pacientes com pancreatite crônica foi de 3,38%, maior do que a frequência de 0,49% encontrada nos grupos controle (p = 0,016); a frequência de 2,03% da mutação P55S do gene SPINK1 e a frequência de 0,67% da mutação R254W do gene CTRC, encontradas nos pacientes com pancreatite crônica, não diferiram estatisticamente quando comparadas às frequências, de 0,49% de ambas mutações, encontradas nos grupos controle. (p = 0,120 e 0,751). Pela investigação da associação de tabagismo e da mutação N34S do gene SPINK1 com as características clínicas e morfológicas da pancreatite crônica, verificou-se que a mutação N34S não se associou a maior gravidade da apresentação clínica ou morfológica da pancreatite crônica; no entanto o tabagismo associou-se a maior frequência de diabete melito entre os portadores de pancreatite crônica. Concluiuse que o tabagismo e a mutação N34S do gene SPINK1 podem ser considerados cofatores de risco para o desenvolvimento da pancreatite crônica
Chronic pancreatitis is a complex disorder in which the interaction between environmental and genetic factors results in the disease. This study included 148 patients with chronic pancreatitis, 110 chronic alcoholics and 297 healthy controls in order to investigate the frequency of smoking and N34S and P55S mutation of SPINK1 gene and R254W of CTRC gene in this population. A questionnaire was applied and gene sequencing was done, after having the Informed Consent Statement. Those with chronic pancreatitis had alcoholic etiology in 74% of cases and idiopathic in 26%. Alcoholic pancreatitis presented in a distinct way of idiopathic chronic pancreatitis. The first group is composed of a higher prevalence of males (88.18% versus 34.21%), by higher mean age (55.64 years versus 45.20 years), lower frequency of Caucasians (63.89% versus 84.21%), lower education (23.30% completed secondary or higher education versus 57.89%) and worst impact from the disease such as diarrhea (54.21% versus 24.24%), weight loss (56.07% versus 24.24%), diabetes mellitus (57.94% versus 36.36%) and occurrence of pancreatic pseudocysts (31.78% versus 12 , 12%). These effects were not accompanied by increased frequency of morphological changes, such as pancreatic calcifications or dilation of the main pancreatic duct. The frequency of smoking was significantly higher in patients with alcoholic pancreatitis than in alcoholics without chronic pancreatitis, therefore tabagism may be considered as a cofactor for the development of chronic pancreatitis among alcoholics (p = 0.002); the frequency of N34S mutation of SPINK1 gene in patients with chronic pancreatitis was 3.38%, higher than the rate of 0.49% found in the control groups (p = 0.016); the frequency of 2.03% of the P55S mutation of SPINK1 gene and the frequency of 0.67% of the CTRC gene R254W mutation found in patients with chronic pancreatitis were not statistically different when compared to the frequencies of 0.49% of both mutations, found in the control groups. (p = 0.120 and 0.751) For the investigation of the association of smoking and N34S mutation of SPINK1 gene with the clinical and morphological features of chronic pancreatitis, it was noticed that the N34S mutation did not determine a greatest severity in the presentation of chronic pancreatitis, however smoking was associated with a higher frequency of diabetes mellitus in patients with chronic pancreatitis. It was concluded that smoking and the N34S mutation of SPINK1 gene are positively correlated with chronic pancreatitis

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29

Marr, Sharon Ann. "The synthesis of potential serine protease inhibitors". Thesis, University of Hull, 1998. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.310261.

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Leung, Donmienne Doen Mun. "Studies of serine and cysteine protease inhibitors /". St. Lucia, Qld, 2001. http://www.library.uq.edu.au/pdfserve.php?image=thesisabs/absthe16491.pdf.

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31

Farady, Christopher. "The mechanism of inhibition of antibody-based inhibitors of the serine protease MT-SP1". Diss., Search in ProQuest Dissertations & Theses. UC Only, 2008. http://gateway.proquest.com/openurl?url_ver=Z39.88-2004&rft_val_fmt=info:ofi/fmt:kev:mtx:dissertation&res_dat=xri:pqdiss&rft_dat=xri:pqdiss:3311347.

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32

Gan, Xiangdong Groutas William C. "Novel mechanism-based inhibitors of serine proteases". Diss., Access through your commercial service, 2005. http://il.proquest.com/products_umi/dissertations/.

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Thesis (M.S.)--Wichita State University, College of Liberal Arts and Sciences, Dept. of Chemistry.
"December 2005." Title from PDF title page (viewed on February 6, 2007). UMI Number: 1436556 Thesis adviser: William C. Groutas. Includes bibliographic references (leaves 54-59).

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33

Johansson, Per-Ola. "Design and synthesis of inhibitors that target the serine protease thrombin, the malarial aspartyl proteases plasmepsin I and II, and the hepatitis C virus NS3 serine protease /". Linköping : Linköpings universitet, 2005. http://www.bibl.liu.se/liupubl/disp/disp2005/tek981s.pdf.

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34

Wångsell, Fredrik. "Design and synthesis of serine and aspartic protease inhibitors /". Linköping : Linköpings universitet, 2006. http://urn.kb.se/resolve?urn=urn:nbn:se:liu:diva-7372.

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35

Rukamp, Karrie Eileen Adlington. "Design and synthesis of inhibitors for serine and cysteine proteases". Diss., Available online, Georgia Institute of Technology, 2004:, 2003. http://etd.gatech.edu/theses/available/etd-04082004-180343/unrestricted/rukamp%5Fkarrie%5Fe%5Fa%5F200312%5Fphd.pdf.

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36

Tarling, Chris Andrew. "Studies towards the synthesis of potential serine protease inhibitors". Thesis, University of Cambridge, 2000. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.621643.

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37

Villegas, Gonzalo Jose Domingo. "Cyclic peptides as inhibitors or substrates of serine proteases". Thesis, Imperial College London, 1995. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.281780.

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38

Ålander, Cecilia. "Kinetic studies of serine protease inhibitors in 'active barrier' model systems". Thesis, Uppsala universitet, Institutionen för kemi - BMC, 2015. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-256910.

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39

Koot, Gretchen E. "Serine and cysteine protease inhibitors for blockade of cell mediated cytotoxicity /". abstract and full text PDF (UNR users only), 2002. http://0-gateway.proquest.com.innopac.library.unr.edu/openurl?url_ver=Z39.88-2004&rft_val_fmt=info:ofi/fmt:kev:mtx:dissertation&res_dat=xri:pqdiss&rft_dat=xri:pqdiss:3121138.

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40

Rukamp, Brian John. "Design, synthesis, and evaluation of novel thiobenzyl ester substrates and aza-peptide inhibitors for serine and cysteine proteases". Diss., Available online, Georgia Institute of Technology, 2004:, 2003. http://etd.gatech.edu/theses/available/etd-04072004-180202/unrestricted/rukamp%5Fbrian%5Fj%5F200312%5Fphd.pdf.

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41

Lauro, Andrea Marie. "The design and synthesis of novel serine proteinase inhibitors". Diss., Georgia Institute of Technology, 1989. http://hdl.handle.net/1853/30032.

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42

Maharjan, Ashok. "Characterization and Gene Expression Analysis of Kazal-Type Serine Protease Inhibitors of Globisporangium ultimum". Bowling Green State University / OhioLINK, 2021. http://rave.ohiolink.edu/etdc/view?acc_num=bgsu1626616718512491.

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43

Wångsell, Fredrik. "Design and Synthesis of Aspartic and Serine Protease Inhibitors targeting the BACE-1 and the HCV NS3 Protease /". Uppsala : Acta Universitatis Upsaliensis, 2009. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-108985.

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44

Mauricio, Anna Theresa. "Heterocyclic alpha-aminoalkylphosphonate diphenyl esters as inhibitors of serine proteases : Part II: Basic alpha-aminoalkylphosphonate diphenyl esters as inhibitors of cathepsin G". Diss., Georgia Institute of Technology, 1996. http://hdl.handle.net/1853/27157.

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45

ZAHEDI, RANA. "Analyse de la relation structure-fonction de cl inhibiteur (inhibiteur de protease a serine)". Paris 7, 1996. http://www.theses.fr/1996PA077335.

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Le cl inhibiteur (clinh) est le seul inhibiteur des serines proteases de la voie classique due complement et l'inhibiteur majeur des facteurs de la phase contracte, le facteur ixx et la kallikreine. La deficience ou la dysfonction de cl inhibiteur est associee a des symptomes d'angio-oedeme hereditaire. Le deficit en cl inhibiteur, resultat de l'activation incontrolee du cl, est accompagne par un niveau serique reduit de c4 et de c2. Ce deficit predispose au devloppement des maladies auto-immunes : glomerulonephrite ou lupus erythemateux. Des travaux recent ont suggere un role therapeutique pour le clinh dans le traitement des etats septiques graves. Ce travail analyse la relation structure-fonction de clinh dans le but de mieux comprendre le mecanisme d'action de cet inhibiteur et d'entrevoire de meilleurs therapies pour les conditions cliniques qui lui sont associee.

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46

Crowther, Damian C. "The bioengineering of targeted serpins". Thesis, University of Cambridge, 1992. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.260598.

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47

Crawley, James Thomas Blick. "Tissue factor pathway inhibitors in atherosclerosis and vascular bleeding". Thesis, Imperial College London, 2001. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.250653.

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48

West, Andrew. "Investigations by mass spectrometry of the interactions of novel serine protease inhibitors with herpes simplex virus type 2 and human cytomegalovirus proteases". Thesis, University of Warwick, 1999. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.343830.

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49

Moore, Michael John Brian. "The design and synthesis of mechanism-based inhibitors of serine proteases". Thesis, University of Canterbury. Chemistry, 1998. http://hdl.handle.net/10092/7301.

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Serine proteases are involved in a number of physiological processes and have proved to be a valuable therapeutic target in the treatment of disease states resulting when the above processes move beyond homeostasis. The investigation of low molecular weight compounds as irreversible and reversible inhibitors of serine proteases has been fuelled by the possibility of rational drug design and their use as mechanistic probes of enzyme action. As introduced in Chapter one particular attention has been focused on mechanism-based inactivators as these elicit clinically desirable specific, efficient and irreversible inhibition. The synthesis and assay of funtionalised imide mechanism-based inhibitors of the serine protease α-chymotrypsin is the subject of this thesis. N-[(Sulfonyl)oxy]succinimides of type 1.41 (L=SO₂R') are known mechanism-based inhibitors of α-chymotrypsin operating via a Lossen rearrangement that unmasks an inactivating isocyanate species. Inhibitory activity has been found to be mediated by the nature of the R substituent and the R' substituent of the L group. Structure activity relationships were investigated by preparing a number of derivatives of type 1.41. The design of the derivatives prepared focused on modulating the R' substituent to interact with extended binding sites of α-chymotrypsin, a strategy that would enhance inhibitory activity. Chapter 2 describes the synthesis of 1.41 and 2.1. Retrosynthetic analysis identified a route involving N-hydroxyimides to be favoured. A synthesis of 1.41 and 2.1 via this key intermediate required reaction between hydroxylamine and succinic and glutaric acid derivatives respectively. These derivatives were prepared using literature methods employing Guareschi, Michael and malonate ester reactions. A systematic study of the synthesis of succinic acids found the optimum route to involve the Stobbe condensation however a short synthesis employing amide base alkylation of succinimide was undertaken and this methodology may prove to be the ideal general route. An aromatic series of derivatives, 1.41f-h was therefore synthesised using the methodology above as were "dimeric" inhibitors 1.41m and n capable of releasing two equivalents of inactivating species during inhibition. Succinimides with 3-C phenyl substituent rather than the benzyl substituent of the derivatives above, were prepared and a series of N-[(sulfonyl)oxy]glutarimides 2.1a, c-e where the extent and type of substitution were varied were prepared. N-[(Acyl)oxy]imides 1.41r-o and 2.1b (L=C(O)R') may also inhibit α-chymotrypsin and these too were investigated. Chapter 3 discusses the assay of inhibitory activity of compounds of type 1.41 and 2.1 against α-chymotrypsin. All the synthesised derivatives, excepting a series of N[(acyl)oxy]succinimides 1.41o-r, were found to be active to such a degree that all but one of the active compounds could not be assayed using sampling techniques. These potent inhibitors were then assayed using the progress curve method. Three compounds 1.41g and hand 2.1c were of such potency that the rate at which they inhibited achymotrypsin could not be measured even with the progress curve method. All three of these compounds possessed a benzyl substituent which was found to be a requiremnent for the exhibition of mechanism-based inhibition in the succinimide series. Compounds 1.41g and 1.41h owed their potency to being able to interact favourably with the Sn' subsites of α-chymotrypsin by containing aromatic substitution. Chapter 4 discusses the use of Evan's oxazolidinone chemistry in the preparation of chiral succinates from which an enantiopure inhibitor of type 1.41 was prepared. Preliminary inhibition studies showed that (R)-1.41f was less active than its racemate indicating more activity resided in the (S)-enantiomer. Chapter 5 discusses the design and synthesis of a potential novel imide mechanism-based inhibitor thought to act by unmasking a quinone imine methide in the active site of α-chymotrypsin. Although the compound released reactivity on hydrolysis it was not found to inhibit α-chymotrypsin significantly.

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50

Bastianelli, Giacomo. "Computational design of protein-based serine proteases inhibitors : tools and applications". Paris 7, 2009. http://www.theses.fr/2009PA077175.

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PfSUBl et PfSUB2 sont deux régulateurs de l'étape érythrocytaires du parasite et représentent de nouvelles cibles thérapeutiques intéressantes pour le développement de nouvelles familles de composés contre le paludisme. La limite majeure pour un tel développement rationnel de molécule sur les PFSUBs reste l'absence de structures expérimentales et des difficultés à exprimer l'enzyme recombinante active en grande quantité. L'utilisation d'un criblage à haut débit n'est donc pas envisageable à ce jour. Afin de contourner ces problèmes, nous avons mis en place une stratégie de recherche rationnelle d'inhibiteurs protéiques à l'aide d'outils in silico. Cette thèse met l'accent sur la validation et l'application d'un ensemble de d'outils bioinformatiques pour effectuer du « protein design ». Nous avons utilisé ces outils afin de modifier la spécificité d'une structure existante contre une enzyme de malaria en identifiant un mutant de EETI-II qui inhibe PvSUBl avec un Ki de 86 μM. Notre approche a aussi été appliquée au « reverse-engineer » de PcFKl, une petite protéine de venin d'araignée qui inhibe le cycle érythrocytaire de P. Falciparum. Cette hypothèse basée sur nos prédictions a été confirmée par des tests in vitro sur PfSUBl
PfSUBl and PfSUB2 are two key regulators of the erythrocytic stage of the parasite and are interesting drug targets for developing new leading compounds against malaria. The major limitations to the drug discovery on PfSUBs are the absence of an experimental structure and the difficulties of expressing large quantities of the active enzymes, restricting the use of high-throughput screening of compounds. To overcome these obstacles, we set up a discovery process based on the computational design of protein-based inhibitors. The thesis focused on developing, validating and applying a series of bioinformatics tools to use in computational protein design. We used these tools to change the specificity of an existing scaffold towards a malaria enzyme, identifying a EETI-II mutant that inhibits PvSUBl with a Ki of 86 μM. Our computational protein design approach was also applied to reverse-engineer PcFKl, a spider-venom derived small protein that inhibits the erythrocytic stage of P. Falciparum. The hypothesis we made using these tools was experimentally confirmed by the in-vitro enzymatic testing on PfSUBl. Despite the challenges we faced, mostly due to the lack of a expérimental structure of PvSUBl, we successfully designed the first protein-based inhibitor of SUBI. The reverse-engineering we performed on PcFKl further confirms the reliability of thèse structural bioinformatics methods

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