DOCSLIB.ORG

Identification of Rabggtase Inhibitors and Mechanistic Studies of Rab Gtpase Prenylation

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Identification of Rabggtase Inhibitors and Mechanistic Studies of Rab Gtpase Prenylation Identification of RabGGTase inhibitors and mechanistic studies of Rab GTPase prenylation and recycling using fluorescent sensors Dissertation zur Erlangung des akademischen Grades eines Doktors der Naturwissenschaften (Dr. rer. nat.) des Fachbereichs Chemie der Technischen Universität Dortmund Angefertigt am Max-Planck-Institut für molekulare Physiologie in Dortmund vorgelegt von Yaowen Wu aus China Dortmund, Dezember 2007 1. Gutachter : Prof. Dr. R.S. Goody 2. Gutachter : Prof. Dr. H. Waldmann Erklärung/Declaration Die vorliegende Arbeit wurde in der Zeit von August 2004 bis December 2007 am Max-Planck-Institut für molekulare Physiologie in Dortmund unter der Anleitung von Prof. Dr. Roger S. Goody, Dr. Kirill Alexandrov und Prof. Dr. Herbert Waldmann durchgeführt. Hiermit versichere ich an Eides statt, dass ich die vorliegende Arbeit selbständig und nur mit den angegebenen Hilfsmitteln angefertigt habe The present work was accomplished from August 2004 to Dezember 2007 at Max- Planck-Institute for molecular physiology in Dortmund under the guidance of Prof. Dr. Roger S. Goody, Dr. Kirill Alexandrov and Prof. Dr. Herbert Waldmann. I hereby declare that I performed the work presented independently and did not use any other but the indicated aids. Dortmund, December 2007 Yaowen Wu Contents Abbreviations V Zusammenfassung VII 1. Abstract 1 2. Introduction 5 2.1 Protein prenylation and prenyltransferases .......................................................... 5 2.1.1 Discovery of protein prenylation............................................................ 5 2.1.2 Protein prenyltransferases and their substrates.................................... 7 2.1.3 Mechanism of protein prenylation ....................................................... 10 2.1.4 Post-prenylation modification .............................................................. 12 2.2 Function of small GTPases..................................................................................16 2.2.1 GTPase cycle...................................................................................... 16 2.2.2 The structural basis of Rab GTPases function.................................... 18 2.2.3 Rabs function in vesicular transport .................................................... 24 2.2.4 Regulation of Rab membrane localization and recycling..................... 33 2.3 Protein prenyltransferase inhibitors .....................................................................39 2.3.1 Farnesyl transferase inhibitors (FTIs).................................................. 39 2.3.2 Mechanism of FTIs action ................................................................... 40 2.3.3 GGTase-I and RabGGTase as therapeutic targets............................. 43 2.4 Chemical protein ligation .....................................................................................46 2.4.1 Native chemical ligation ...................................................................... 46 2.4.2 Expressed protein ligation................................................................... 47 2.4.3 Protein trans-splicing........................................................................... 49 3. Aims of this work 53 4. Results and Discussion 57 I 4.1 Development of fluorescent sensors for Rab prenylation ....................................57 4.1.1 Strategy for the construction of fluorescent sensors based on Rab proteins ........................................................................................................ 58 4.1.2 Semi-synthesis of Rab7 fluorescent conjugates.................................. 61 4.1.2.1 Expression and purification of Rab7 C-terminal α-thioesters..... 61 4.1.2.2 Characterization of Rab7 α-thioesters ....................................... 64 4.1.2.3 Fluorophore conjugation and protein ligation ............................. 69 4.1.2.4 Characterization of Rab7 fluorescent conjugates....................... 79 4.1.3 NBD-FPP as a sensitive fluorescent sensor for Rab prenylation ........ 88 4.1.3.1 A sensitive, real-time fluorometric assay for RabGGTase activity ............................................................................................................... 88 4.1.3.2 Characterization of NBD-farnesyltation...................................... 92 4.1.3.3 Steady-state kinetics of the NBD-farnesylation reaction .......... 101 4.2 Identification of RabGGTase inhibitors ..............................................................105 4.2.1 High-throughput screening for RabGGTase inhibitors ...................... 105 4.2.2 In silico screening of RabGGTase inhibitors ..................................... 107 4.2.3 Structure and activity relationship (SAR) analysis of peptide inhibitors for RabGGTase.......................................................................................... 110 4.2.4 Quantitative analysis of the interaction of identified inhibitors with prenyltransferases...................................................................................... 116 4.2.5 Selectivity of inhibitors towards RabGGTase, FTase and GGTase-I 121 4.2.6 Inhibition of RabGGTase in vivo by peptide inhibitors....................... 126 4.3 The mechanistic basis of Rab prenylation .........................................................129 4.3.1 Simulation of the Rab C-terminus in the ternary protein complex ..... 129 4.3.2 Rab C-terminus plays multiple roles in Rab prenylation.................... 131 4.3.2.1 Engineering of the Rab C-terminus based on simulation and binary complex structures.................................................................... 131 4.3.2.2 CBR interacting motif is essential for Rab prenylation in vitro.. 135 4.3.2.3 Effects of sequence downstream CIM on Rab prenylation ...... 138 4.3.2.4 Rab C-terminus modulates affinity of Rab:REP binary complex and Rab:REP:RabGGTase ternary complex ....................................... 140 4.4 The thermodynamic basis of Rab recycling.......................................................149 4.4.1 The functional segregation of REP and GDI ..................................... 149 II 4.4.2 Development of a fluorescent sensor for the interaction of prenylated Rab with REP and GDI .............................................................................. 151 4.4.3 Construction and solubilization of semi-synthetic geranylgeranylted Rab7 .......................................................................................................... 158 4.4.4 Quantitative analysis of the interaction of mono- and digeranylgeranylated Rab7 with REP and GDI .......................................... 165 4.4.5 A model for Rab recycling ................................................................. 170 5. Materials and Methods 175 5.1 Materials............................................................................................................175 5.1.1 Chemicals ......................................................................................... 175 5.1.2 Other chemicals from collaborators................................................... 177 5.1.3 Other materials.................................................................................. 178 5.1.4 Instruments ....................................................................................... 179 5.1.5 Buffers and growth media ................................................................. 180 5.2 Molecular cloning methods................................................................................181 5.2.1 Plasmids and bacterial strains........................................................... 181 5.2.2 Preparation of competent cells.......................................................... 182 5.2.3 Preparative PCR ............................................................................... 183 5.2.4 Purification of PCR products by agarose gel electrophoresis ........... 184 5.2.5 Subcloning ........................................................................................ 184 5.2.6 Chemical transformation ................................................................... 185 5.2.7 Colony PCR screen........................................................................... 186 5.2.8 Preparation of plasmid DNA.............................................................. 186 5.2.9 DNA sequencing ............................................................................... 187 5.2.10 Transformation by electroporation................................................... 188 5.3 Protein expression and purification methods.....................................................189 5.3.1. Expression and purification of RabGGTase, REP-1, RabGDI, Rab7wt and Rab7 mutants...................................................................................... 189 5.3.2 Expression and purification of Rab-thioester proteins ....................... 191 5.4 Analytical methods ............................................................................................193 5.4.1 Denaturing SDS-PAGE ..................................................................... 193 5.4.2 MALDI-TOF-mass spectrometry ....................................................... 194 III 5.4.3 LC-ESI-mass spectrometry ............................................................... 194 5.4.4 Analytical reversed-phase (RP)- and gel filtration (GF)-HPLC .......... 195 5.4.5 Ion exchange chromatography.......................................................... 196 5.5 Biochemical methods.........................................................................................197
Load more

Recommended publications
  • Understanding Drug-Drug Interactions Due to Mechanism-Based Inhibition in Clinical Practice
    pharmaceutics Review Mechanisms of CYP450 Inhibition: Understanding Drug-Drug Interactions Due to Mechanism-Based Inhibition in Clinical Practice Malavika Deodhar 1, Sweilem B Al Rihani 1 , Meghan J. Arwood 1, Lucy Darakjian 1, Pamela Dow 1 , Jacques Turgeon 1,2 and Veronique Michaud 1,2,* 1 Tabula Rasa HealthCare Precision Pharmacotherapy Research and Development Institute, Orlando, FL 32827, USA; [email protected] (M.D.); [email protected] (S.B.A.R.); [email protected] (M.J.A.); [email protected] (L.D.); [email protected] (P.D.); [email protected] (J.T.) 2 Faculty of Pharmacy, Université de Montréal, Montreal, QC H3C 3J7, Canada * Correspondence: [email protected]; Tel.: +1-856-938-8697 Received: 5 August 2020; Accepted: 31 August 2020; Published: 4 September 2020 Abstract: In an ageing society, polypharmacy has become a major public health and economic issue. Overuse of medications, especially in patients with chronic diseases, carries major health risks. One common consequence of polypharmacy is the increased emergence of adverse drug events, mainly from drug–drug interactions. The majority of currently available drugs are metabolized by CYP450 enzymes. Interactions due to shared CYP450-mediated metabolic pathways for two or more drugs are frequent, especially through reversible or irreversible CYP450 inhibition. The magnitude of these interactions depends on several factors, including varying affinity and concentration of substrates, time delay between the administration of the drugs, and mechanisms of CYP450 inhibition. Various types of CYP450 inhibition (competitive, non-competitive, mechanism-based) have been observed clinically, and interactions of these types require a distinct clinical management strategy. This review focuses on mechanism-based inhibition, which occurs when a substrate forms a reactive intermediate, creating a stable enzyme–intermediate complex that irreversibly reduces enzyme activity.
    [Show full text]
  • Pharmacological Targeting of the Mitochondrial Phosphatase PTPMT1 by Dahlia Doughty Shenton Department of Biochemistry Duke
    Pharmacological Targeting of the Mitochondrial Phosphatase PTPMT1 by Dahlia Doughty Shenton Department of Biochemistry Duke University Date: May 1 st 2009 Approved: ___________________________ Dr. Patrick J. Casey, Supervisor ___________________________ Dr. Perry J. Blackshear ___________________________ Dr. Anthony R. Means ___________________________ Dr. Christopher B. Newgard ___________________________ Dr. John D. York Dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the Department of Biochemistry in the Graduate School of Duke University 2009 ABSTRACT Pharmacological Targeting of the Mitochondrial Phosphatase PTPMT1 by Dahlia Doughty Shenton Department of Biochemistry Duke University Date: May 1 st 2009 Approved: ___________________________ Dr. Patrick J. Casey, Supervisor ___________________________ Dr. Perry J. Blackshear ___________________________ Dr. Anthony R. Means ___________________________ Dr. Christopher B. Newgard ___________________________ Dr. John D. York An abstract of a dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the Department of Biochemistry in the Graduate School of Duke University 2009 Copyright by Dahlia Doughty Shenton 2009 Abstract The dual specificity protein tyrosine phosphatases comprise the largest and most diverse group of protein tyrosine phosphatases and play integral roles in the regulation of cell signaling events. The dual specificity protein tyrosine phosphatases impact multiple
    [Show full text]
  • Content by Dr. Vishvanath Tiwari 1 E-PG Pathshala for Biophysics
    e-PG Pathshala for Biophysics, MHRD project, UGC PI: Prof M.R. Rajeswari, A.I.I.M.S., New Delhi Paper 05: Molecular Enzymology and Protein Engineering Module No. 13: Mechanism and Kinetics of Competitive inhibition Content writer: Dr. Vishvanath Tiwari Department of Biochemistry, Central University of Rajasthan, Ajmer-305817 Objective: Objective of the present module is to understand the mechanism and kinetics of the competitive inhibition as well as role of dissociation constant of inhibitor in drug designing. We will also discuss the different examples of the competitive inhibition. This module is divided into following sections- 1. Introduction 2. Competitive inhibition 2.1 Mechanism of competitive inhibitor 2.2 Kinetics of competitive inhibitor 2.3 Determination of dissociation constant for competitive inhibitor 2.4 Examples of competitive inhibitors 4. Significance of the competitive inhibition in drug designing 3. Summary 4. Question 5. Resources and suggested reading 1. Introduction: The negative regulator or enzyme inhibitor can reduce the rate of enzyme-catalyzed reaction. Inhibition of the enzyme could be significant in term of inhibiting the crucial Content by Dr. Vishvanath Tiwari 1 enzymatic pathways. Enzyme inhibitions are irreversible, suicide, feedback and reversible inhibition. Reversible inhibition involves weak non-covalent interactions between enzyme and inhibitors. The non-covalent interaction involves hydrogen bonding, hydrophobic interactions, van der Waal’s forces and salt bridges. The cumulative effects of these interactions result into strong interactions. Because of weak non-covalent interactions, reversible inhibitor can be separated from the enzymes therefore the name reversible inhibition is given. On the basis of effect of varying concentration of enzyme’s substrate on the inhibitor, Dr.
    [Show full text]
  • Inhibition of Monoamine Oxidase Activity by Antidepressants and Mood Stabilizers
    Biogenic Amines Vol. 25, issue 1 (2011), pp. 59–81 BIA250111A02 Inhibition of monoamine oxidase activity by antidepressants and mood stabilizers Reprinted from: Neuroendocrinology Letters 2010; 31(5): 645–656. Zdeněk Fišar, Jana Hroudová, Jiří Raboch Department of Psychiatry, First Faculty of Medicine, Charles University in Prague and General University Hospital in Prague, Prague, Czech Republic. Key words: antidepressive agents; monoamine oxidase inhibitors; mood stabilizers Abstract Monoamine oxidase (MAO), the enzyme responsible for metabolism of mono- amine neurotransmitters, has an important role in the brain development and function, and MAO inhibitors have a range of potential therapeutic uses. We investigated systematically in vitro effects of pharmacologically different antide- pressants and mood stabilizers on MAO activity. Effects of drugs on the activity of MAO were measured in crude mitochondrial fraction isolated from cortex of pig brain, when radiolabeled serotonin (for MAO-A) or phenylethylamine (for MAO-B) was used as substrate. The several antidepressants and mood stabilizers were compared with effects of well known MAO inhibitors such as moclobemide, iproniazid, pargyline, and clorgyline. In general, the effect of tested drugs was found to be inhibitory. The half maximal inhibitory concentration, parameters of enzyme kinetic, and mechanism of inhibi- tion were determined. MAO-A was inhibited by the following drugs: pargyline > clorgyline > iproniazid > fluoxetine > desipramine > amitriptyline > imipramine > citalopram > venlafaxine > reboxetine > olanzapine > mirtazapine > tianeptine > moclobemide, cocaine >> lithium, valproate. MAO-B was inhibited by the following drugs: pargyline > clorgyline > iproniazid > fluoxetine > venlafaxine > amitriptyline > olanzapine > citalopram > desipramine > reboxetine > imipramine > tianeptine > mirtazapine, cocaine >> moclobemide, lithium, valproate. The mechanism of inhibition of MAOs by several antidepressants was found various.
    [Show full text]
  • Chemical Evidence for Potent Xanthine Oxidase Inhibitory Activity of Ethyl Acetate Extract of Citrus Aurantium L
    molecules Article Chemical Evidence for Potent Xanthine Oxidase Inhibitory Activity of Ethyl Acetate Extract of Citrus aurantium L. Dried Immature Fruits Kun Liu, Wei Wang *, Bing-Hua Guo, Hua Gao, Yang Liu, Xiao-Hong Liu, Hui-Li Yao and Kun Cheng School of Pharmacy, Qingdao University, Qingdao 266021, Shandong, China; [email protected] (K.L.); [email protected] (B.-H.G.); [email protected] (H.G.); [email protected] (Y.L.); [email protected] (X.-H.L.); [email protected] (H.-L.Y.); [email protected] (K.C.) * Correspondence: [email protected]; Tel./Fax: +86-532-869-91172 Academic Editor: Isabel C. F. R. Ferreira Received: 24 January 2016 ; Accepted: 29 February 2016 ; Published: 2 March 2016 Abstract: Xanthine oxidase is a key enzyme which can catalyze hypoxanthine and xanthine to uric acid causing hyperuricemia in humans. Xanthine oxidase inhibitory activities of 24 organic extracts of four species belonging to Citrus genus of the family Rutaceae were assayed in vitro. Since the ethyl acetate extract of C. aurantium dried immature fruits showed the highest xanthine oxidase inhibitory activity, chemical evidence for the potent inhibitory activity was clarified on the basis of structure identification of the active constituents. Five flavanones and two polymethoxyflavones were isolated and evaluated for inhibitory activity against xanthine oxidase in vitro. Of the compounds, hesperetin showed more potent inhibitory activity with an IC50 value of 16.48 µM. For the first time, this study provides a rational basis for the use of C. aurantium dried immature fruits against hyperuricemia. Keywords: Citrus; xanthine oxidase; aurantii fructus immaturus; hesperetin 1.
    [Show full text]
  • Structural and Inhibition Studies on Udp-Galactopyranose Mutase
    STRUCTURAL AND INHIBITION STUDIES ON UDP-GALACTOPYRANOSE MUTASE A Thesis Submitted to the College of Graduate Studies and Research in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy In the Department of Chemistry University of Saskatchewan By Sarathy Karunan Partha © Copyright Sarathy Karunan Partha, October 2010. All rights reserved Permission to Use In presenting this thesis in partial fulfilment of the requirements for a Postgraduate degree from the University of Saskatchewan, I agree that the Libraries of this University may make it freely available for inspection. I further agree that permission for copying of this thesis in any manner, in whole or in part, for scholarly purposes may be granted by the professor or professors who supervised my thesis work or, in their absence, by the Head of the Department or the Dean of the College in which my thesis work was done. It is understood that any copying or publication or use of this thesis or parts thereof for financial gain shall not be allowed without my written permission. It is also understood that due recognition shall be given to me and to the University of Saskatchewan in any scholarly use which may be made of any material in my thesis. Requests for permission to copy or to make other use of material in this thesis in whole or part should be addressed to: Head of the Department of Chemistry University of Saskatchewan Saskatoon, Saskatchewan (S7N 5C9) i ABSTRACT UDP-galactopyranose mutase (UGM) is a flavoenzyme which catalyzes the interconversion of UDP-galactopyranose (UDP-Gal p) and UDP-galactofuranose (UDP- Gal f).
    [Show full text]
  • Ethanol, Catecholamines and Alkaloids: Interface of Neurochemistry and Alcoholism
    Loyola University Chicago Loyola eCommons Dissertations Theses and Dissertations 1974 Ethanol, Catecholamines and Alkaloids: Interface of Neurochemistry and Alcoholism Joel Allen Rubenstein Loyola University Chicago Follow this and additional works at: https://ecommons.luc.edu/luc_diss Part of the Pharmacology, Toxicology and Environmental Health Commons Recommended Citation Rubenstein, Joel Allen, "Ethanol, Catecholamines and Alkaloids: Interface of Neurochemistry and Alcoholism" (1974). Dissertations. 1428. https://ecommons.luc.edu/luc_diss/1428 This Dissertation is brought to you for free and open access by the Theses and Dissertations at Loyola eCommons. It has been accepted for inclusion in Dissertations by an authorized administrator of Loyola eCommons. For more information, please contact [email protected]. This work is licensed under a Creative Commons Attribution-Noncommercial-No Derivative Works 3.0 License. Copyright © 1974 Joel Allen Rubenstein ETHANOL, CATECHOLAMINES AND ALKALOIDS : INTERFACE OF NEUROCHEMISTRY AND ALCOHOLISM by Joel Allen Rubenstein A Dissertation Submitted to the Faculty of the Graduate School of Loyola University of Chicago in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy June 1974 Joel Allen Rubenstein Loyola University of Chicago ETHANOL, CATECHOI.AMINES AND ALKALOIDS: INTERFACE Of NEUROCHEMISTRY AND ALCOHOLISM The theory has been advanced that tetrahydroisoquinoline (TIQ) alkaloids may form in neuronal and chrornaffin cells during alcohol metab­ olism via condensation of catecholamines (CAs) and alcohol-derived aldehydes. The cyclic CA-type alkaloids could assume a physiological role in the development of alcohol dependence (G.Cohen and M. Collins, Science 167: 1749-1751). As groundwork for in vivo studies on TIQs during alcoholism, the following questions required study.
    [Show full text]
  • Development of a Novel, High-Affinity Ssdna Trypsin Inhibitor
    Journal of Enzyme Inhibition and Medicinal Chemistry ISSN: 1475-6366 (Print) 1475-6374 (Online) Journal homepage: https://www.tandfonline.com/loi/ienz20 Development of a novel, high-affinity ssDNA trypsin inhibitor Stanislaw Malicki, Miroslaw Ksiazek, Pawel Majewski, Aleksandra Pecak, Piotr Mydel, Przemyslaw Grudnik & Grzegorz Dubin To cite this article: Stanislaw Malicki, Miroslaw Ksiazek, Pawel Majewski, Aleksandra Pecak, Piotr Mydel, Przemyslaw Grudnik & Grzegorz Dubin (2019) Development of a novel, high-affinity ssDNA trypsin inhibitor, Journal of Enzyme Inhibition and Medicinal Chemistry, 34:1, 638-643, DOI: 10.1080/14756366.2019.1569648 To link to this article: https://doi.org/10.1080/14756366.2019.1569648 © 2019 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group. View supplementary material Published online: 06 Feb 2019. Submit your article to this journal Article views: 514 View related articles View Crossmark data Full Terms & Conditions of access and use can be found at https://www.tandfonline.com/action/journalInformation?journalCode=ienz20 JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY 2019, VOL. 34, NO. 1, 638–643 https://doi.org/10.1080/14756366.2019.1569648 SHORT COMMUNICATION Development of a novel, high-affinity ssDNA trypsin inhibitor Stanislaw Malickia,b, Miroslaw Ksiazeka,b,c, Pawel Majewskib, Aleksandra Pecaka,b, Piotr Mydelb,d, Przemyslaw Grudnika and Grzegorz Dubina,b aMalopolska Centre of Biotechnology, Jagiellonian University, Krakow, Poland; bDepartment of Microbiology, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Krakow, Poland; cDepartment of Oral Immunology and Infectious Diseases, University of Louisville School of Dentistry, Kentucky, USA; dDepartment of Clinical Science, Broegelmann Research Laboratory, University of Bergen, Bergen, Norway ABSTRACT ARTICLE HISTORY Inhibitors of serine proteases are not only extremely useful in the basic research but are also Received 3 October 2018 applied extensively in clinical settings.
    [Show full text]
  • Spring 2013 Lecture 16 & 17
    CHM333 LECTURES 16 & 17: 2/22 – 25/13 SPRING 2013 Professor Christine Hrycyna ENZYME INHIBITION - INHIBITORS: • Interfere with the action of an enzyme • Decrease the rates of their catalysis • Inhibitors are a great focus of many drug companies – want to develop compounds to prevent/control certain diseases due to an enzymatic activity 1. e.g. AIDS and HIV protease inhibitors • HIV protease essential for processing of proteins in virus • Without these proteins, viable viruses cannot be released to cause further infection - Inhibitors can be REVERSIBLE or IRREVERSIBLE • Irreversible Inhibitors o Enzyme is COVALENTLY modified after interaction with inhibitor o Derivatized enzyme is NO longer a catalyst – loses enzymatic activity o Original activity cannot be regenerated o Also called SUICIDE INHIBITORS § e.g. nerve gas, VX gas § Aspirin! Acetylates Ser in active site of cyclooxygenase (COX) enzyme • Reversible Inhibitors o Bind to enzyme and are subsequently released o Leave enzyme in original condition o Three subclasses: § Competitive Inhibitors § Non-competitive Inhibitors § Uncompetitive Inhibitors o Can be distinguished by their kinetics of inhibition How are inhibitors characterized experimentally? • First, perform experiment without inhibitor o Measure velocity at different substrate concentrations, keeping [E] constant. Choose values of [S] o Get [S] and V values from experiment o Take reciprocal of [S] and V values (“1 over”) o Plot on graph 1/[S] (x) vs. 1/V (y) – don’t use Michaelis-Menten – not reliable • Second, do the SAME experiment in parallel using a fixed amount of inhibitor and same values for E and S • Get V values and plot together with uninhibited reaction • Depending on how the graph looks and using the subsequent equation of the line we can: o Determine type of inhibition (competitive vs.
    [Show full text]
  • Inhibition of Trypsin by Heparin and Dalteparin, a Low Molecular Weight Heparin OLIVERA M
    J. Serb. Chem. Soc. 74 (4) 379–388 (2009) UDC 577.112.383+66.097.8+547.995 JSCS–3839 Original scientific paper Inhibition of trypsin by heparin and dalteparin, a low molecular weight heparin OLIVERA M. BOSNIĆ1, KRISTINA R. GOPČEVIĆ2*#, MIROSLAV M. VRVIĆ3# and IVANKA M. KARADŽIĆ2# 1School of Medicine, Department of Physiology, University of Belgrade, Višegradska 26, 11000 Belgrade, 2School of Medicine, Department of Chemistry, University of Belgrade, Višegradska 26, 11000 Belgrade, and 3Faculty of Chemistry, University of Belgrade, Studentski trg 12–16, 11000 Belgrade, Serbia (Received 28 August, revised 19 September 2008) Abstract: The interaction between trypsin, a prototype S1 serine protease, with heparin and its low molecular weight derivative dalteparin were investigated. Direct inhibition of the proteolytic activity of trypsin by heparin and dalteparin, used in concentrations typical for their clinical application, was detected. The half-maximum inhibition of the trypsin activity was achieved at 15.25±1.22 μg/mL for heparin and was estimated to be at 58.47±15.20 μg/mL for dalte- parin. Kinetic analyses showed that heparin and its low molecular weight deri- vative dalteparin inhibited trypsin by occupation of an exosite, producing non- competitive and mixed inhibition, respectively. Heparin as a noncompetitive inhibitor with constant of inhibition Ki1,2 = 0.151±0.019 μM and dalteparin with Ki1 = 0.202±0.030 μM and Ki2 = 0.463±0.069 μM in mixed inhibition both represent moderate inhibitors of serine protease trypsin. The obtained con- stants of inhibition indicate that under the clinically applied concentrations of heparin and dalteparin, trypsins and their homolog S1 serine proteases could be directly inhibited, influencing the delicate control of proteolytic reactions in homeostasis.
    [Show full text]
  • Screening of Prenyldiphosphate Mimetics For
    SCREENING OF PRENYLDIPHOSPHATE MIMETICS FOR THEIR POTENTIAL TO INHIBIT 4-HYDROXYBENZOATE OLIGOPRENYL TRANSFERASE Dissertation To obtain the Doctoral Degree in Natural Sciences (Dr. rer. nat.) Submitted to The Faculty I of Natural Sciences (Biological Sciences) Martin Luther University Halle-Wittenberg (MLU), Germany Institute of Pharmacy by Amina Msonga Born on 16.01.1980 in Nzega, Tanzania Halle (Saale), Germany SCREENING OF PRENYLDIPHOSPHATE MIMETICS FOR THEIR POTENTIAL TO INHIBIT 4-HYDROXYBENZOATE OLIGOPRENYL TRANSFERASE Dissertation zur Erlangung des Doktorgrades der Naturwissenschaften (Dr. rer. nat.) der Naturwissenschaftlichen Fakultät I – Biowissenschaften der Martin-Luther-Universität Halle-Wittenberg Institut für Pharmazie vorgelegt von Frau Amina Msonga geboren am 16.01.1980 in Nzega, Tanzania Gutachter: Prof. Dr. L. A. Wessjohann (Martin-Luther-University, Halle-Wittenberg (& Leibniz-IPB)) Prof. Dr. M. Pietzsch (Martin-Luther-University, Halle-Wittenberg) Prof. Dr. O. Kayser (Technical University, Dortmund) Verteidigt am: 25.01.2017 ii ACKNOWLEDGEMENT Accomplishment of this work has been contributed by direct or indirect support of some people who helped me in one-way or another. Firstly, I would like to express my heartfelt appreciations to my supervisor; Prof. Dr. L. A. Wessjohann for welcoming me at IPB and accepting to supervise me. His kind guidance, continuous support, patience, availability for consultation and for giving me opportunities to attend workshops and a conference are higly appreciated. Furthermore, his valuable advice, suggestions, comments, constructive criticism and encouragement led to successful completion of this study and made it meaningful to the society. Secondly, I would like to thank Prof. Dr. M. Pietzsch for accepting to be my mentor. My sincere acknowledgement goes to the DAAD/MoEVT and IPB for financial support of the study.
    [Show full text]
  • Inhibition of Trypsin by Heparin and Dalteparin, a Low Molecular Weight Heparin OLIVERA M
    J. Serb. Chem. Soc. 74 (4) 379–388 (2009) UDC 577.112.383+66.097.8+547.995 JSCS–3839 Original scientific paper Inhibition of trypsin by heparin and dalteparin, a low molecular weight heparin OLIVERA M. BOSNIĆ1, KRISTINA R. GOPČEVIĆ2*#, MIROSLAV M. VRVIĆ3# and IVANKA M. KARADŽIĆ2# 1School of Medicine, Department of Physiology, University of Belgrade, Višegradska 26, 11000 Belgrade, 2School of Medicine, Department of Chemistry, University of Belgrade, Višegradska 26, 11000 Belgrade, and 3Faculty of Chemistry, University of Belgrade, Studentski trg 12–16, 11000 Belgrade, Serbia (Received 28 August, revised 19 September 2008) Abstract: The interaction between trypsin, a prototype S1 serine protease, with heparin and its low molecular weight derivative dalteparin were investigated. Direct inhibition of the proteolytic activity of trypsin by heparin and dalteparin, used in concentrations typical for their clinical application, was detected. The half-maximum inhibition of the trypsin activity was achieved at 15.25±1.22 μg/mL for heparin and was estimated to be at 58.47±15.20 μg/mL for dalte- parin. Kinetic analyses showed that heparin and its low molecular weight deri- vative dalteparin inhibited trypsin by occupation of an exosite, producing non- competitive and mixed inhibition, respectively. Heparin as a noncompetitive inhibitor with constant of inhibition Ki1,2 = 0.151±0.019 μM and dalteparin with Ki1 = 0.202±0.030 μM and Ki2 = 0.463±0.069 μM in mixed inhibition both represent moderate inhibitors of serine protease trypsin. The obtained con- stants of inhibition indicate that under the clinically applied concentrations of heparin and dalteparin, trypsins and their homolog S1 serine proteases could be directly inhibited, influencing the delicate control of proteolytic reactions in homeostasis.
    [Show full text]