Hepatit C är en leversjukdom som orsakas av hepatit C viruset (HCV). Sjuk- domen kan ha akuta och kroniska former och sjukdomsförloppet kan variera från några veckor till att vara livslångt. Ofta är infektionen asymtomatisk, vilket gör det svårt att upptäcka sjukdomen tidigt. Detta leder till en allvarlig sjukdomsprogression med cirros och levercancer som resultat. HCV- infekt- ioner är idag den huvudsakliga orsaken till levertransplantationer. Viruset överförs via blod, främst via osteriliserad medicinsk utrustning. Det beräknas att 130 till 150 miljoner människor i världen lider av en kronisk HCV infekt- ion. Idag finns det inget vaccin mot viruset, men forskning på detta område pågår. HCV finns över hela världen. Det finns sex genotyper och olika sub- typer av viruset. De vanligaste genotyperna i världen är genotyp 1 och 3.
Tills nyligen var standardbehandlingen av en HCV infektion interferon och ribavirin. Tyvärr ger behandlingen önskad effekt hos endast 50% av patienterna och ger ofta svåra bieffekter. Med hjälp av nya läkemedel har andelen lyckade behandlingar stigit till 90%. Trots detta behövs nya läkeme- del då viruset muterar snabbt, vilket orsakar uppkomsten av läkemedelsresi- stenta virusformer. Dessutom har de flesta nya läkemedlen utvecklats för att behandla genotyp 1 infektioner. Behovet av en fortsatt läkemedelsutveckling mot HCV är alltså stort.
Vid infektion av leverceller bildar viruset ett effektivt replikationskom- plex som består av flera virala proteiner. Proteinerna kodas av virusets en- kelsträngade RNA-genom. Vissa HCV proteiner är enzymer som är vitala för virusets förökning och överlevnad. Våra studier fokuserades på NS3-4A proteaset och NS5B polymeraset som båda är viktiga läkemedelsmål vid behandling av HCV infektioner.
I denna avhandling beskrivs hur vi utvärderade substanser som skiljer sig strukturellt från redan godkända och utvecklande läkemedel mot NS3 pro- teaset. Vi identifierade nya pyrazinonbaserade föreningar som uppvisar en inhiberande effekt mot genotyp 1 (både ursprungliga och läkemedelsresi- stenta varianter av viruset) och genotyp 3. Dessa föreningar har potential att utvecklas till läkemedelskandidater med aktivitet mot ovan nämnda enzym- varianter.
Eliminering av HCV hos en patient kan lättast uppnås genom att använda en kombination av olika antivirala läkemedel med skilda läkemedelsmål. Därför har vi även studerat ett annat av HCVs essentiella enzymer: NS5B – ett RNA-beroende RNA-polymeras.
Två typer av NS5B hämmare (inhibitorer) förekommer: allosteriska inhi- bitorer och de som verkar direkt via det aktiva sätet. Verkningsmekanismen hos de direkt verkande polymerashämmarna är ganska okomplicerad. De förhindrar att nya RNA-strängar kan bildas genom att blockera påbyggnaden av strängen (så kallad kedjeterminering). Mekanismerna bakom allosteriska inhibitorer är däremot inte lika väl förstådd. Med ett instrument baserast på ytplasmonresonansteknik (eng. surface plasmon resonanse, SPR) studerade vi interaktionsmekanismer och egenskaper hos allosteriska inhibitorer till NS5B-polymeras av genotyp 1 och 3. Detta resulterade i några viktiga insik- ter. Vi förväntar oss att våra studier kommer att få konsekvenser för selekt- ionen och optimeringen av nya allosteriska NS5B-hämmare.
För att mäta aktiviteten hos NS5B-polymeras och studera dess hämmare etablerade vi en SPR-baserad assay som ger möjligheten att övervaka poly- meriseringsreaktionen och dess hämning i realtid. Denna assay kan imple- menteras i läkemedelsutvecklingen av nya preparat mot HCV.
Slutligen genomförde vi en screening av en samling (ett så kallat biblio- tek) av små kemiska föreningarna, som kallas fragment, för att identifiera de små molekyler som kan användas som utgångspunkt för utveckling av nya allosteriska inhibitorer mot NS5B-polymeras. Vi identifierade flera fragment som efter ytterligare utveckling kan bli nya potenta allosteriskt verkande läkemedelskandidater.
Vi hoppas att våra studier kommer att bidra till upptäckten av nya läke- medel mot HCVs olika genotyper och deras läkemedelsresistenta varianter.
Acknowledgments
First of all, I would like to express my sincere gratitude to my supervisor
Helena Danielson for accepting me as a PhD student, for being inspiring and
supportive throughout my study. You have been a great mentor and teacher for me!
I thank Mikael Widersten for being my co-supervisor and for sharing your knowledge in biochemistry.
I would like to thank Gunnar Johansson for giving advises and answering my endless questions, Doreen Dobritzsch for helping with crystallography work, valuable discussions, and also for very tasty cakes, Ylva Ivarsson for your organizational skills and a lot of fun in the lab, Francoise Raffalli-
Mathieu for your teaching advises and encouraging me to speak Swedish. Gun Stenberg, your molecular biology knowledge and skills are admirable! I
have very much appreciated that you shared them with me. Thank you for all your help and assistance!
Gunnar Svensson, Bo Fredriksson and Inger Hermanson for being always
helpful and friendly.
I also want to thank my collaborators and co-authors from the Department of Medicinal Chemistry: Anna-Karin Belfrage, Eva Åkerblom and Anja Sand-
ström, former Medivir co-workers: Vera Baraznenok, Elizabeth Hamelink
and Lotta Vrang, from GE Healthcare: Veronica Fridh and Olof Karlsson. Thank you all for fruitful collaboration and interesting project discussions. During my PhD I had outstanding project students that I would like to thank:
Arne, Laura, Andrea, Caroline, Darius and Lisa, thank you very much for
your great work, valuable help and your contribution to the projects!
Further, I would like to express my gratitude to present and former co- workers of HD group. Sara, thank you for joining HCV project, it was a great pleasure to work with you in the lab, thank you for your continuous help and support as well as being a wonderful friend. Vladimir, I am im- pressed how quickly you grasp new knowledge, you are genius! Thanks for a lot of fun in and outside the lab! Alice, you brought your great spirit to our
lab and to me. Thank you for encouragement and support during my thesis writing. Helena N, thank you for sharing your Biacore expertise and lending T200 machine. Christian, thank you for introducing me to LaTeX and Inkscape. Although, this thesis is written in Word, all the figures were made in Inkscape, which was extremely useful. I am also thankful for the discus- sion about Biacore experiments, for skiing lesson and for keeping my Ger- man in shape. Angelica, as someone has already mentioned that lab without you became too calm. It’s true! Thanks for introducing me into the NS3 pro- ject, you are also a brilliant teacher! Tony, you are a great SPR expert. Sofia, you possess great organizational skills and you are a talented movie-maker.
Johan, you have been my best lab supervisor ever since I started my research
training in this lab several years ago. I am very grateful for your introduction to the SPR technique, to the NS5B project and for continuous help. And thank you for correcting my Swedish summary! Malin, your endless source of energy is encouraging. Thank you for taking me into salsa classes, it was a great fun! Ikram, your kindness and calmness is remarkable, I am very thankful for your great collaboration and for all your help, especially in dif- ficult times!
Dirk, my office mate, thank you for your help and efforts for trying to crys-
tallize our proteins, for our chit-chat about football and Bundesliga. I also very much appreciated that you never spoke English to me! Erika, always a smiley girl, thanks for inquiring about my writings, I hope one day I can join your fitness class. Emil, it was great to have lab teaching with you and thanks for organizing our biochemistry meetings. Huan, it was always nice to meet you in the lab and have funny talks during the lunch. Åsa, tjenamors din gamla galosch! I thought we would finish this journey together, but I can’t wait. Thilak, for all funny conversations and tasty Indian food.
Cissi, Vikash, Ali, Gustav and Ilaria, for a pleasant working environment and
social activities outside the lab.
Maxim and Aleksandra thank you for being wonderful friends and for enjoy-
able times together. Aleksandra B, for our pleasant lunch discussions and for providing solutions to improve my experiments.
I want to thank my friend Nikolaus, or Nikolaus von Myra – a patron saint of students, for all your help and support, without you I would never achieve this. Thank you!
Finally, in life, nobody except your parents will care to overlook who you have become and continue believing in what you can still be. I thank my
Mother for inestimable support, encouragement and endless love! My Fa- ther, for continuous support and inspiration.
References
1. World Health Organization (2015) Hepatitis C, Fact sheet N°164.
2. Choo Q-L, et al. (1989) Isolation of a cDNA clone derived from a blood-borne non-A, non-B viral hepatitis genome. Science 244(4902):359-362.
3. Shors T (2011) Understanding viruses (Jones & Bartlett Publishers). 4. Inokuchi M, et al. (2009) Infection of B cells with hepatitis C virus for the
development of lymphoproliferative disorders in patients with chronic hepatitis C. Journal of Medical Virology 81(4):619-627.
5. Lindenbach BD & Rice C (2001) Flaviviridae: the viruses and their replication.
Fields virology 1:991-1041.
6. Moradpour D, Penin F, & Rice CM (2007) Replication of hepatitis C virus. Nat
Rev Micro 5(6):453-463.
7. Davis GL (1999) Hepatitis C virus genotypes and quasispecies. The American
journal of medicine 107(6):21-26.
8. Butkiewicz NJ, et al. (1996) Enhancement of Hepatitis C Virus NS3 Proteinase Activity by Association with NS4A-Specific Synthetic Peptides: Identification of Sequence and Critical Residues of NS4A for the Cofactor Activity. Virology 225(2):328-338.
9. Koch JO, Lohmann V, Herian U, & Bartenschlager R (1996) In VitroStudies on the Activation of the Hepatitis C Virus NS3 Proteinase by the NS4A Cofactor.
Virology 221(1):54-66.
10. Grakoui A, McCourt DW, Wychowski C, Feinstone SM, & Rice CM (1993) Characterization of the hepatitis C virus-encoded serine proteinase:
determination of proteinase-dependent polyprotein cleavage sites. Journal of
Virology 67(5):2832-2843.
11. Kim JL, et al. (1996) Crystal Structure of the Hepatitis C Virus NS3 Protease Domain Complexed with a Synthetic NS4A Cofactor Peptide. Cell 87(2):343- 355.
12. Brass V, et al. (2008) Structural determinants for membrane association and dynamic organization of the hepatitis C virus NS3-4A complex. Proceedings of
the National Academy of Sciences of the United States of America
105(38):14545-14550.
13. Schechter I & Berger A (1967) On the size of the active site in proteases. I. Papain. Biochemical and Biophysical Research Communications 27(2):157-162. 14. Hesson T, Mannarino A, & Cable M (2000) Probing the relationship between
RNA-stimulated ATPase and helicase activities of HCV NS3 using 2'-O-methyl RNA substrates. Biochemistry 39(10):2619-2625.
15. Beran RKF, Serebrov V, & Pyle AM (2007) The Serine Protease Domain of Hepatitis C Viral NS3 Activates RNA Helicase Activity by Promoting the Binding of RNA Substrate. Journal of Biological Chemistry 282(48):34913- 34920.
16. Beran RKF & Pyle AM (2008) Hepatitis C Viral NS3-4A Protease Activity Is Enhanced by the NS3 Helicase. Journal of Biological Chemistry
283(44):29929-29937.
17. Llinàs-Brunet M, et al. (2000) Highly potent and selective peptide-based inhibitors of the hepatitis C virus serine protease: towards smaller inhibitors.
Bioorganic & Medicinal Chemistry Letters 10(20):2267-2270.
18. Lin C, Kwong A, & Perni R (2006) Discovery and development of VX-950, a novel, covalent, and reversible inhibitor of hepatitis C virus NS3. 4A serine protease. Infectious Disorders-Drug Targets (Formerly Current Drug Targets-
Infectious Disorders) 6(1):3-16.
19. Colarusso S, et al. (2003) Phenethyl Amides as Novel Noncovalent Inhibitors of Hepatitis C Virus NS3/4A Protease: Discovery, Initial SAR, and Molecular Modeling. Journal of Medicinal Chemistry 46(3):345-348.
20. Hagel M, et al. (2011) Selective irreversible inhibition of a protease by targeting a noncatalytic cysteine. Nat Chem Biol 7(1):22-24.
21. Saalau-Bethell SM, et al. (2012) Discovery of an allosteric mechanism for the regulation of HCV NS3 protein function. Nat Chem Biol 8(11):920-925. 22. Tsantrizos YS (2004) The design of a potent inhibitor of the hepatitis C virus
NS3 protease: BILN 2061—From the NMR tube to the clinic. Peptide Science 76(4):309-323.
23. Reiser M, et al. (2005) Antiviral efficacy of NS3-serine protease inhibitor BILN-2061 in patients with chronic genotype 2 and 3 hepatitis C. Hepatology 41(4):832-835.
24. Vertex Pharmaceuticals Incorporated (2014) Discontinuation of INCIVEK® (telaprevir) tablets in the United States.
25. Rosenquist Å, et al. (2014) Discovery and Development of Simeprevir
(TMC435), a HCV NS3/4A Protease Inhibitor. Journal of Medicinal Chemistry 57(5):1673-1693.
26. Romano KP, Ali A, Royer WE, & Schiffer CA (2010) Drug resistance against HCV NS3/4A inhibitors is defined by the balance of substrate recognition versus inhibitor binding. Proceedings of the National Academy of Sciences of
the United States of America 107(49):20986-20991.
27. Pawlotsky J-M (2011) Treatment failure and resistance with direct-acting antiviral drugs against hepatitis C virus. Hepatology 53(5):1742-1751.
28. Lenz O, et al. (2013) Virologic response and characterisation of HCV genotype 2–6 in patients receiving TMC435 monotherapy (study TMC435-C202).
Journal of Hepatology 58(3):445-451.
29. Wang QM, et al. (2002) Oligomerization and Cooperative RNA Synthesis Activity of Hepatitis C Virus RNA-Dependent RNA Polymerase. Journal of
Virology 76(8):3865-3872.
30. Behrens SE, Tomei L, & De Francesco R (1996) Identification and properties of the RNA-dependent RNA polymerase of hepatitis C virus. The EMBO journal 15(1):12.
31. Kao CC, et al. (2000) Template Requirements for RNA Synthesis by a Recombinant Hepatitis C Virus RNA-Dependent RNA Polymerase. Journal of
Virology 74(23):11121-11128.
32. Appleby TC, et al. (2015) Structural basis for RNA replication by the hepatitis C virus polymerase. Science 347(6223):771-775.
33. Le Pogam S, et al. (2008) Existence of hepatitis C virus NS5B variants naturally resistant to non-nucleoside, but not to nucleoside, polymerase inhibitors among untreated patients. Journal of Antimicrobial Chemotherapy 61(6):1205-1216.
34. Yi G, et al. (2012) Biochemical Study of the Comparative Inhibition of Hepatitis C Virus RNA Polymerase by VX-222 and Filibuvir. Antimicrobial
Agents and Chemotherapy 56(2):830-837.
35. Gentile I, Buonomo AR, Zappulo E, & Borgia G (2014) Discontinued drugs in 2012 – 2013: hepatitis C virus infection. Expert Opinion on Investigational
Drugs 24(2):239-251.
36. Kati W, et al. (2015) In Vitro Activity and Resistance Profile of Dasabuvir, a Nonnucleoside Hepatitis C Virus Polymerase Inhibitor. Antimicrobial Agents
and Chemotherapy 59(3):1505-1511.
37. Lam AM, et al. (2012) Genotype and Subtype Profiling of PSI-7977 as a Nucleotide Inhibitor of Hepatitis C Virus. Antimicrobial Agents and
Chemotherapy 56(6):3359-3368.
38. Dutartre H, Bussetta C, Boretto J, & Canard B (2006) General Catalytic Deficiency of Hepatitis C Virus RNA Polymerase with an S282T Mutation and Mutually Exclusive Resistance towards 2′-Modified Nucleotide Analogues.
Antimicrobial Agents and Chemotherapy 50(12):4161-4169.
39. Gane EJ, et al. (2013) Nucleotide Polymerase Inhibitor Sofosbuvir plus Ribavirin for Hepatitis C. New England Journal of Medicine 368(1):34-44. 40. Lawitz EJ, et al. (2013) All-oral therapy with nucleotide inhibitors sofosbuvir
and GS-0938 for 14 days in treatment-naive genotype 1 hepatitis C (NUCLEAR). Journal of Viral Hepatitis 20(10):699-707.
41. Anderson AC (2003) The Process of Structure-Based Drug Design. Chemistry
& Biology 10(9):787-797.
42. Lipinski CA (2004) Lead- and drug-like compounds: the rule-of-five revolution.
Drug Discovery Today: Technologies 1(4):337-341.
43. Lin K, Perni RB, Kwong AD, & Lin C (2006) VX-950, a Novel Hepatitis C Virus (HCV) NS3-4A Protease Inhibitor, Exhibits Potent Antiviral Activities in HCV Replicon Cells. Antimicrobial Agents and Chemotherapy 50(5):1813- 1822.
44. Venkatraman S, et al. (2006) Discovery of (1R,5S)-N-[3-Amino-1- (cyclobutylmethyl)-2,3-dioxopropyl]- 3-[2(S)-[[[(1,1-
dimethylethyl)amino]carbonyl]amino]-3,3-dimethyl-1-oxobutyl]- 6,6-dimethyl- 3-azabicyclo[3.1.0]hexan-2(S)-carboxamide (SCH 503034), a Selective, Potent, Orally Bioavailable Hepatitis C Virus NS3 Protease Inhibitor: A Potential Therapeutic Agent for the Treatment of Hepatitis C Infection. Journal of
Medicinal Chemistry 49(20):6074-6086.
45. Kempf DJ, et al. (1995) ABT-538 is a potent inhibitor of human
immunodeficiency virus protease and has high oral bioavailability in humans.
Proceedings of the National Academy of Sciences of the United States of America 92(7):2484-2488.
46. Vacca JP, et al. (1994) L-735,524: an orally bioavailable human
immunodeficiency virus type 1 protease inhibitor. Proceedings of the National
Academy of Sciences of the United States of America 91(9):4096-4100.
47. Varghese JN (1999) Development of neuraminidase inhibitors as anti-influenza virus drugs. Drug Development Research 46(3-4):176-196.
48. Congreve M, Carr R, Murray C, & Jhoti H (2003) A ‘Rule of Three’ for fragment-based lead discovery? Drug Discovery Today 8(19):876-877. 49. Hopkins AL, Groom CR, & Alex A (2004) Ligand efficiency: a useful metric
for lead selection. Drug Discovery Today 9(10):430-431.
50. Hopkins AL, Keseru GM, Leeson PD, Rees DC, & Reynolds CH (2014) The role of ligand efficiency metrics in drug discovery. Nat Rev Drug Discov 13(2):105-121.
51. Scott DE, Coyne AG, Hudson SA, & Abell C (2012) Fragment-Based Approaches in Drug Discovery and Chemical Biology. Biochemistry 51(25):4990-5003.
52. Tsai J, et al. (2008) Discovery of a selective inhibitor of oncogenic B-Raf kinase with potent antimelanoma activity. Proceedings of the National Academy of
Sciences 105(8):3041-3046.
53. Otto A (1968) Excitation of nonradiative surface plasma waves in silver by the method of frustrated total reflection. Z. Physik 216(4):398-410.
54. Liedberg B, Nylander C, & Lunström I (1983) Surface plasmon resonance for gas detection and biosensing. Sensors and Actuators 4:299-304.
55. Jönsson U, et al. (1991) Real-time biospecific interaction analysis using surface plasmon resonance and a sensor chip technology. Biotechniques 11(5):620-627. 56. Langmuir I (1918) THE ADSORPTION OF GASES ON PLANE SURFACES
OF GLASS, MICA AND PLATINUM. Journal of the American Chemical
Society 40(9):1361-1403.
57. Bochud P-Y, et al. (2009) Genotype 3 is associated with accelerated fibrosis progression in chronic hepatitis C. Journal of Hepatology 51(4):655-666. 58. Nkontchou G, et al. (2011) HCV genotype 3 is associated with a higher
hepatocellular carcinoma incidence in patients with ongoing viral C cirrhosis.
Journal of Viral Hepatitis 18(10):e516-e522.
59. Kanwal F, Kramer JR, Ilyas J, Duan Z, & El-Serag HB (2014) HCV genotype 3 is associated with an increased risk of cirrhosis and hepatocellular cancer in a national sample of U.S. Veterans with HCV. Hepatology 60(1):98-105. 60. De Clercq E (2014) Current race in the development of DAAs (direct-acting
antivirals) against HCV. Biochemical Pharmacology 89(4):441-452. 61. Hopkins C, et al. (2004) Novel pyrazinone inhibitors of mast cell tryptase:
synthesis and SAR evaluation. Bioorganic & Medicinal Chemistry Letters 14(19):4819-4823.
62. Jones DE & South MS (2010) Synthesis of a versatile 2 (1H)-pyrazinone core for the preparation of Tissue Factor-Factor VIIa inhibitors. Tetrahedron 66(14):2570-2581.
63. Zhang X, Schmitt AC, Jiang W, Wasserman Z, & Decicco CP (2003) Design and Synthesis of Potent, Non-peptide Inhibitors of HCV NS3 Protease.
Bioorganic & Medicinal Chemistry Letters 13(6):1157-1160.
64. Tyndall JDA & Fairlie DP (1999) Conformational homogeneity in molecular recognition by proteolytic enzymes. Journal of Molecular Recognition 12(6):363-370.
65. Pawar VG & De Borggraeve WM (2006) 3,5-Dihalo-2(1H)-pyrazinones: Versatile Scaffolds in Organic Synthesis. Synthesis 2006(17):2799-2814. 66. Gising J, et al. (2014) Achiral Pyrazinone-Based Inhibitors of the Hepatitis C
Virus NS3 Protease and Drug-Resistant Variants with Elongated Substituents Directed Toward the S2 Pocket. Journal of Medicinal Chemistry 57(5):1790- 1801.
67. Rönn R, et al. (2008) Hepatitis C virus NS3 protease inhibitors comprising a novel aromatic P 1 moiety. Bioorganic & medicinal chemistry 16(6):2955-2967. 68. Trozzi C, et al. (2003) In Vitro Selection and Characterization of Hepatitis C
Virus Serine Protease Variants Resistant to an Active-Site Peptide Inhibitor.
Journal of Virology 77(6):3669-3679.
69. Dahl G, Sandström A, Åkerblom E, & Danielson UH (2007) Resistance profiling of hepatitis C virus protease inhibitors using full-length NS3. Antiviral
70. Ludmerer SW, et al. (2005) Replication Fitness and NS5B Drug Sensitivity of Diverse Hepatitis C Virus Isolates Characterized by Using a Transient
Replication Assay. Antimicrobial Agents and Chemotherapy 49(5):2059-2069. 71. Källberg M, et al. (2012) Template-based protein structure modeling using the