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Kinetic Studies of NS3 and NS5B from Hepatitis C Virus

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Effects of protease inhibition by modification of helicase residues in hepatitis C virus nonstructural protein 3. FEBS J. 2007; 274: 5979-5986. III Nurbo, J., Peterson, S.D., Dahl, G., Danielson U.H., Karlén, A. and Sandström, A. Beta-amino acid substitutions and structure-based CoMFA modeling of hepatitis C virus NS3 protease inhibitors. Bioorg. Med. Chem. 2008; 16: 5590-5605. IV Dahl, G., Gutiérrez-Arenas, O., and Danielson U.H. Hepatitis C virus NS3 protease is activated by low concentrations of pro- tease inhibitors. Submitted. V Geitmann, M., Dahl, G., Lohmann, V., Friebe, P., Paeshuyse, J., Herdewjin, P., Puerstinger, G., Bartenschlager, R., Neyts, J. and Danielson U.H. Kinetic, mechanistic and chemodynamic characterisation of non-nucleoside hepatitis C virus NS5B polymerase inhibitors using SPR biosensor technology. Submitted. VI Geitmann, M., Dahl, G. and Danielson U.H. Kinetic characteriz- ation of HCV NS3 interactions with NS4A and inhibitors. Submitted. Contents Introduction .................................................................................................. 11 Proteins, enzymes, proteases and polymerases ....................................... 11 Hepatitis C .............................................................................................. 12 The disease ......................................................................................... 12 HCV Genotypes ................................................................................. 12 HCV life cycle .................................................................................... 13 The viral proteins ............................................................................... 14 HCV Model systems .......................................................................... 16 Combating HCV ..................................................................................... 17 Current treatment ................................................................................ 17 HCV drug discovery ........................................................................... 18 Drug resistance ................................................................................... 20 Immune escape ................................................................................... 20 HCV NS3 ................................................................................................ 22 Structure and function ........................................................................ 22 NS3 as a drug target ........................................................................... 23 Model systems of NS3 ....................................................................... 25 HCV NS5B ............................................................................................. 26 Structure and function ........................................................................ 26 NS5B as a drug target ......................................................................... 27 Aims ............................................................................................................. 30 Experimental procedures ............................................................................. 31 Cloning, expression and purification of NS3 .......................................... 31 Activity and inhibition measurements of NS3 ........................................ 31 Biosensor assays for NS5B and NS3 ...................................................... 32 Results ......................................................................................................... 33 Improving expression and purification of NS3 ....................................... 33 NS3 protease inhibitor resistance (Paper I) ............................................ 34 Helicase can influence protease inhibition (Paper II) ............................. 37 Novel NS3 inhibitor designs (Paper III) ................................................. 38 Inhibitor activation of NS3 (Paper IV) ................................................... 38 Studies of interaction between NS5B and inhibitors (Paper V) .............. 40 Characterization of NS3 inhibitors using a biosensor (Paper VI) ........... 41 Perspectives ................................................................................................. 44 Summary ................................................................................................. 44 The future ................................................................................................ 44 Sammanfattning ........................................................................................... 46 Hepatit C ................................................................................................. 46 Proteiner, enzymer och läkemedelsutveckling ........................................ 46 Att slå ut hepatit C viruset ...................................................................... 47 Modellsystem .......................................................................................... 47 Min forskning .......................................................................................... 48 Acknowledgements ..................................................................................... 50 References ................................................................................................... 52 Abbreviations ADME Adsorption distribution metabolism excretion ATP Adenosine tri-phosphate Cardif CARD-containing adaptor protein CHAPS 3-[(3-Cholamidopropyl)dimethylammonio]-1-propane- sulfonate CoMFA Comparative molecular field analysis DABCYL 4-((4-(dimethylamino)phenyl)azo)benzoic acid DMSO Dimethyl sulfoxide DTT Dithiothreitol E1 and E2 Envelope protein 1 and Envelope protein 2 EDC 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydro chloride EDANS 5-((2-Aminoethyl)amino)naphthalene-1-sulfonic acid EMCV Encephalomyocarditis virus ER Endoplasmatic reticulum FRET Förster resonance energy transfer F-protein Frameshift protein HCV Hepatitis C virus HEPES 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid HIV Human immunodeficiency virus Huh-7 Human hepatoma cell line 7 IMAC Immobilized metal ion affinity chromatography IPS-1 IFN- promoter stimulator 1 IRES Internal ribosome entry site JFH-1 Japanese fulminant hepatitis 1 MAVS Mitochondrial antiviral signaling protein MuLV Murine leukemia virus NA Nucleoside analogue Neo Neomycin NHS N-hydroxysuccinimide NNA Non-nucleoside analogue NS Non-structural protein NTP Nucleotide triphosphate PCR Polymerase chain reaction PEG Polyethylene glycol PKR Protein kinase R RNA/DNA Ribonucleic acid/Dehydroxy ribonucleic acid SPR Surface plasmon resonance TLR3 Toll-like receptor 3 TRIF Toll/Interleukin-1-receptor-domain-containing adaptor-inducing interFeron-! VISA Virus-induced signaling adaptor Introduction Biochemistry teaches us about the chemistry of life; How does life work? The more you dig into the mountain of knowledge amassed through the his- tory of natural sciences and learn how things were, are and relate to one an- other, one cannot avoid being amazed of the complex chemical processes that happen all around us at all times. However, the more you learn, the more you realize how little is actually known. The task for a scientist is to contin- ue the search for knowledge, giving his or her contribution, and by doing so make the world a better place. Even though the practical use for scientific work is sometimes hard to see, not even the wisest can predict the usage of the knowledge obtained. When my grandfather Olle Dahl defended his thesis in 1958 (Dahl 1958), the applications and benefits that mankind have today, just 50 years later, thanks to biochemistry and biotechnology, was unimaginable. Proteins, enzymes, proteases and polymerases Proteins are composed of long chains of amino acids. Shorter chains of amino acids are called peptides. There are 20 natural amino acids with different physico-chemical properties and since proteins can contain from a few to sev- eral thousand amino acids, the variation of functionality in proteins is tre- mendous. Proteins exist in all forms of life and are responsible for essentially all processes taking place; be it the collagen holding your body together or the enzymes responsible for digesting that cinnamon roll you ate yesterday.
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