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PHM142 Fall 2016 Coordinator: Dr. Jeffrey Henderson Instructor: Dr. David Hampson Newest Drugs for Hepatitis C Matthew Luu, Yuan Fang (Pearl) Cai, Shiming Sun, Ziyi Cheng What is Hepatitis C? • Flaviviridae virus family • Liver cells are primary host cells • (+) single-stranded lipid-enveloped RNA virus • • Genotype variants and sub-variants (eg. genotype 1,2) 60-85% Chronicity • Liver cirrhosis → Hepatic Failure → Hepatocellular Carcinoma Hepatitis C Life Cycle • Flaviviridae binds hepatocytes to trigger receptor-mediated endocytosis and undergoes decapsidation in cytoplasm • Translation of viral genome in rough ER produces a polyprotein • Cleaved by cellular and viral proteases to structural and nonstructural proteins (NS) functioning in viral replication • NS5A forms membranous web • NS5B RNA-dependent RNA polymerase • NS3/4A serine protease • Core protein binds lipid droplet for assembly • Assembled virion bud out of cell via membrane Bartenschlager, R., Cosset, F., & Lohmann, V. (2010). Hepatitis C virus replication cycle. Journal of Hepatology, 53(3), 583-585. NS5A Protein: Overview • Interact with other nonstructural proteins • Consists of a lead sequence followed by three domains • The α-helix anchor NS5A into cell membrane • A pivotal component of membranous web replication complex • All three domains necessary for viral replication https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3961994/ NS5A protein: Structure • Domain I contains Zinc-binding and RNA-binding motif • Domain II and III are unfolded • Domain I is essential for HCV replication. • Domain II is associated with RNA replication and Domain III is important for virus assembly • Dimeric structure is consistent with surface interactions with other proteins, RNA molecules and cell membranes Crystal Structure of NS5A Dimer Domain I Genotype 1a (A), and 1B (B) https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4093949/ NS5A inhibitors • Exact inhibitory mechanisms of these molecules on HCV is unknown • Conserved symmetrical molecules • Suggest that they act on dimer of NS5A proteins • Examples include Daklinza and Ledipasvir https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4093949/ Daklinza • Highly specific and potent NS5A inhibitor • Directly binds to HCV NS5A at dimer interface • Induces an alteration in the subcellular localization of HCV NS5A proteins, which inhibits the formation and activation of HCV replication complexes • Blocks intracellular HCV RNA synthesis, virus assembly and secretion http://pillsgermany.com/tovar/daklinza/ Sofosbuvir: NS5B Inhibitors • In Canada, sofosbuvir is the only available nucleoside inhibitor of NS5B • There are 3 therapies: 1. Genotypes 1 and 4: Sofosbuvir + Peginterferon + Ribavirin 2. Genotypes 2 and 3: Sofosbuvir + Ribavirin 3. Most recently, genotype 1: Sofosbuvir + Ledipasvir Ledipasvir inhibits HCV RNA replication and virion production by targeting the HCV NS5A protein. Mechanism: Sofosbuvir • Sofosbuvir: prodrug active antiviral agent 2'-deoxy-2'-αfluoro-β-C-methyluridine-5'triphosphate. • The triphosphate acts as a defective substrate for the NS5B protein. • NS5B is the viral RNA polymerase, thus sofosbuvir acts as an inhibitor of viral RNA synthesis. Drug-drug Interactions: Sofosbuvir + Ledipasvir • Sofosbuvir and ledipasvir: substrates of drug transporter P-gp. • P-gp/MDR1: ATP-dependent efflux pump; expressed in the intestinal apical side; pumps drugs back into the intestinal lumen • P-gp inducers decrease sofosbuvir and ledipasvir plasma concentration; reduced therapeutic effect; loss of virologic response. • P-gp inhibitors may be coadministered with sofosbuvir or ledipasvir http://www.cyprotex.com/useruploads/Transporters_inte stines.jpg NS3/4A NS3 • Protein involved in viral replication with both a protease and helicase domain • Protease activity - important for generating viral non-structural proteins that are required for HCV replication • • Cleavage of four sites of the HCV polypeptides (post-translational processing) - essential of viral replication Helicase activity - important for RNA replication NS4A • Cofactor involved in enhancing the activity of NS3 NS3/4A Inhibitors • Serine Protease Inhibitors • Linear alpha-ketoamide derivatives that covalently bind to the active site (located between the His57 and Asp81 of the N terminal, and Ser139 of the C terminal). I.e. Boceprevir, Telaprevir • Prevents the cleavage of the HCV polypeptide at four sites: NS4A,NS4B, NS5A, NS5B • Used in combination with other HCV drugs http://www.nature.com/nbt/journal/v29/n1 1/full/nbt.2031.html Zepatier • Fixed-dose combination drug containing elbasvir (NS5A inhibitor) and grazoprevir (NS3/4A inhibitor) for treatment of HCV genotype 1 and 4. • Works by stopping the viral replication and assembly. Summary • Hepatitis C is a (+) single-stranded RNA virus from the Flaviviridae family • Translation in host cell (hepatocytes) RER creates polyprotein which is cleaved into structural and non-structural proteins by host and viral proteases • Treatments of HepC targets NS5A, NS5B and NS3/4A protease • Effect of compounds targeting NS5A on HCV replication is unknown • Daclinza is a potent NS5A inhibitor and is most effective and resistant to mutations when used with other HCV drugs like Harvon • NS5B Inhibitor Sofosbuvir acts as a defective substrate which binds with RNA polymerase. By blocking RNA polymerase, the RNA synthesis and life cycle of HCV is stopped. • Sofosbuvir and ledipasvir are substrates of efflux transporter P-gp. To maintain effective plasma concentration of these drugs, we may need to co-administer P-gp inhibitors. • It is highly anticipated that sofosbuvir may play a key role in future all-oral regimens for the treatment of genotype 1 HCV infections • N3/4A serine protease inhibitors works by inhibiting the formation of new replication sites • NS3/4A serine protease inhibitors works in combination of other HCV drugs • Zepatier is a combination of Elbasvir (NS5A inhibitor) and Grazoprevir (NS3/4A inhibitor) References Akiyama S (Dec 2001). "[Mechanisms of drug resistance and reversal of the resistance]". Human Cell. 14 (4): 257–60. PMID 11925925 Drugs, N. E. W. (2015). Daclatasvir. Australian Prescriber, 38(6), 217–219. Bartenschlager, R., Cosset, F., & Lohmann, V. (2010). Hepatitis C virus replication cycle. Journal of Hepatology, 53(3), 583-585. Carrion, A., Martin, P. (2016). Safety and efficacy of elbasvir and grazoprevir for treatment of hepatitis C. Expert Opinion on Drug Safety, 15(6): 883-890. Dugum M, O'Shea R. Hepatitis C virus: here comes all-oral treatment. Cleve Clin J Med 2014;81(3):159-72. Lambert, S. M., Langley, D. R., Garnett, J. A., Angell, R., Hedgethorne, K., Meanwell, N. A., & Matthews, S. J. (2014). The crystal structure of NS5A domain 1 from genotype 1a reveals new clues to the mechanism of action for dimeric HCV inhibitors. Protein Science : A Publication of the Protein Society, 23(6), 723–734. Liang, J., Ghany, MG. (2013). Current and Future Therapies for Hepatitis C Virus Infection. The New England Journal of Medicine, 368:1907-1917 McGivern DR, Masaki T, Lovell W, Hamlett C, Saalau-Bethell S, Graham B. (2015). Protease Inhibitors Block Multiple Functions of the NS3/4A Protease-Helicase during the Hepatitis C Virus Life Cycle. Nakamoto, S., Kanda, T., Wu, S., Shirasawa, H., & Yokosuka, O. (2014). Hepatitis C virus NS5A inhibitors and drug resistance mutations. World Journal of Gastroenterology : WJG, 20(11), 2902–2912. Salam, KA., Akimitsu, N. (2013). Hepatitis C Virus NS3 Inhibitors: Current and Future Perspectives. BioMed Research International. 467869, 9 pages. Nakamoto, S., Kanda, T., Wu, S., Shirasawa, H., & Yokosuka, O. (2014). Hepatitis C virus NS5A inhibitors and drug resistance mutations. World Journal of Gastroenterology, 20(11), 2902–12. https://doi.org/10.3748/wjg.v20.i11.2902 Obrien, C., & Agresti, N. (2012). NS5A inhibitors. Current Hepatitis Reports. https://doi.org/10.1007/s11901-012-0138-2