Antiviral Approaches Against HCV • PEG-INF + Ribavirin Therapy. • Host / Viral Factors Affecting Treatment Response. Adverse Effect of Therapy. • Sofosbuvir + Ledipasvir Therapy.
Targets for Antiviral Therapy • Polymerase • Protease
HCV Polymerase & Drug Screening YasirWaheed
Structure of HCV genome: HCV encodes a long open reading frame of about 3011 amino acids which is divided into structural and non-structural proteins flanked by 5’ and 3’ untranslated regions. HCV structural proteins are core E1, E2, p7 while the non-structural proteins are NS2, NS3, NS4A, NS4B, NS5A and NS5B. Waheed et al., 2013
HCV Polymerase • HCV polymerase (NS5B) possess the structure of a typical polymerase with fingers, palm and thumb subdomains. • The fingers subdomain interacts with the incoming nucleoside triphosphate. • The palm subdomain is the catalytic center for the nucleotidyl transfer reaction and the thumb subdomain plays a role in positioning the RNA for initiation and elongation. • NS5B is a potent target for the design of antiviral strategies (Waheed et al., 2012).
Structure of HCV Polymerase: HCV polymerase is divided in to fingers, palm and thumb domains. The palm region contains five highly conserved motifs named from A to E (Waheed et al., 2013). Motif A: Binding with Mg++ ions Motif B: Sugar selection Motif C: GDD Motif D: Forms the palm core region Motif E: Interaction of palm with thumb Motif F: Forms Interconnecting loop Waheed et al., 2012
Drug Screening • Biochemical Assay • Cell Based Assay • Animal Trials • Human Trials
Cell Based Assay (5BR Assay) Transfection of HCV Polymerase HCV Polymerase uses cellular RNAs as template and forms double stranded RNAs Double Stranded RNAs will activate RIG-I signaling RIG-I will activate luciferase linked with INF-beta Ratio of luciferase will reflect the activity of HCV Polymerase Ranjith-Kumar et al., 2011: Plos ONE
Figure 1: Effect of different compounds on HCV RNA polymerase in 5BR assay: Eight different compounds were used in 5BR assay to evaluate their effect on the activity of HCV RNA polymerase. All the compounds were dissolved in 100% DMSO and 10 µM of each compound was used in the assay. X-axis shows the name of different inhibitors used along with DMSO (control) while Y-axis shows the percentage activity of the compounds in 5BR assay. Number above the graph shows the percentage activity of compounds with means and standard deviations of three assays.
Figure: Effect of HCV 796 in 5BR assay: Different concentrations of HCV 796 were used in 5BR assay and their effect on the activity of HCV RNA polymerase was evaluated. X-axis shows the different concentrations of HCV 796 used along with DMSO (control) while Y-axis shows the percentage activity at different concentrations in 5BR assay. Number above the graph shows the percentage activity of HCV RNA polymerase in the presence of different concentrations of HCV 796 with means and standard deviations of three assays. The EC50 calculated is shown above the graph.
Biochemical Replication Assay NS 5B-21 Cloning Expression in bacteria PET - 22 Expression HCV Polymerase Synthetic template of Poly A Replication Assay Mutations in Active site Site Directed Mutagenesis Screening of Replication inhibitors
Biochemical Assay • HCV Polymerase protein expressed in bacteria with His tag. • Synthetic RNA template: LE19 & PE46 • ATP, [α-32P]CTP, GTP & UTP • Buffer Incubate at 30oC for 1h
Dmso 40nM 200nM 500nM 1uM 2uM 10uM 20uM 50uM -PE -DN Figure: In vitro RdRp assay by using wild type HCV polymerase with increased concentration of HCV-796 drug. The RNA product was separated on 20% polyacrylamide 7.5 M urea gel. The radioactive signal was detected and quantified by using PhosphorImager. The de novo initiation (DN) and primer extension (PE) RNA products, by using different HCV-796 drug concentrations are shown in gel.
Site Directed Mutagenesis When HCV-796 was added to cell lines harboring HCV replicon, the major resistant mutant arises was C316Y. We did site directed mutagenesis and generate C316Y mutant.
Effect of HCV 796 on C316Y mutant in 5BR assay: Different concentrations of HCV 796 were used in 5BR assay and their effect on the activity of C316Y mutant HCV RNA polymerase was evaluated. x-axis shows the different concentrations of HCV 796 used along with DMSO (control) while y-axis shows the percentage activity at different concentrations in 5BR assay. Number above the graph shows the percentage activity of C316Y mutant HCV RNA polymerase in the presence of different concentrations of HCV 796 with means and standard deviations of three assays. The EC50 calculated is shown above the graph.
D 0.002 0.01 0.05 0.1 0.5 1 2.5 5 10 25 50 100 500uM Conc in uM PE DN Figure: In vitro RdRp assay by using C316Y mutant HCV polymerase with increased concentration of HCV-796 drug. The RNA product was separated on 20% polyacrylamide 7.5 M urea gel. The radioactive signal was detected and quantified by using PhosphorImager. The de novo initiation (DN) and primer extension (PE) RNA products, by using different HCV-796 drug concentrations are shown in gel.
Figure: DSF of wild type and C316Y mutant polymerase with HCV 796 drug. Considerable shift in protein denaturation profile was observed when wild type polymerase protein was used with HCV 796 drug. C316Y mutant polymerase did not showed any difference in protein denaturation profile in the presence and absence of HCV 796 drug.
Effect of HCV-796 compound on HCV RNA polymerases from GN 1-6 in 5BR assay: HCV polymerases from genotype 1-6 were used in 5BR assay and effect of HCV-796 compound was evaluated. The compound was dissolved in 100% DMSO and 10 µM of compound was used in the assay. X-axis shows the name of different HCV genotype polymerase used along with DMSO (control) while Y-axis shows the percentage activity of the polymerases in 5BR assay. Number above the graph shows the percentage activity of compounds with means and standard deviations of three assays.
Interaction of HCV NS5B with HCV-796: HCV NS5B protein is shown in blue, HCV-796 compound is shown in red, Interacting amino acids are shown green.
Amino acid comparison of interacting residues of HCV NS5B of genotypes 1-6 with HCV-796 compound.
Site directed mutagenesis of genotype 4a polymerase: HCV genotype 4a polymerase is mutated V414M and effect of HCV-796 compound is analyzed. Effect of HCV-796 is also analyzed on genotype 1b and wild type polymerase as control.
HCV Polymerase Inhibitors • Nucleoside Inhibitors NIs bind at the enzyme active site and compete with their natural NTP counterparts for incorporation. • Non-Nucleoside Inhibitors Non-nucleoside inhibitors target the HCV polymerase at four different sites. The NNI I and NNI II target the HCV polymerase at thumb site while the NNI III and NNI IV target the palm domain.
Nucleoside Inhibitors MK0608 Chimeric mice were treated with MK0608 along with telaprevir and Interferon. The mice treated with triple therapy of MK0608, telaprevir and interferon became HCV negative soon after the commencement of therapy and remained HCV RNA negative after 12 weeks of cessation of therapy. Mice treated with high dose of telaprevir and MK0608 became HCV RNA negative after one week and remained negative after 18 weeks [Ohra et al., 2011]. RG7128 RG7128 was administered to HCV genotype 1 treatment naïve patients at the dose of 1500 mg b.i.d. in combination with Peg-INF and ribavirin, the reduction in viral RNA was 5 log10 at 4 week with 85% of patients achieving RVR [Lalezari et al., 2008].
Non-Nucleoside Inhibitors Class I • Benzimidazole and Indole derivatives. • They mostly inhibit the initiation of HCV RNA synthesis by NS5B. MK-3281 • MK-3281 produces promising results in chimeric mouse model. MK-3281 was administered as a dose of 10 mg/kg and 50 mg/kg b.i.d., the reduction in viral titer was 1.4 log10 and 3.1 log10respectively [Narjes et al., 2010].
Non-Nucleoside Inhibitors Class II • Phenlyalanines, Thiophenes, Pyranoindoles and Hydroxypyranones derivatives. • Binds in a shallow hydrophobic pocket situated at the base of thumb domain. Filibuvir • Filibuvirshows the EC50 of 70 nM by using HCV replicon and IC50 of 73 nMin RdRp assay [Yi et al., 2012]. • Filibuvirinduce mutations at the HCV NS5B residue M423. This mutation is observed in 76% of patients receiving ≥ 300 mg bid dose of Filiburvir [Troke et al., 2012].
Non-Nucleoside Inhibitors Class III • Benzothiadiazine and Acylpyrrolidinesderivatives. • Binds near the active site at inner thumb/palm domain. ANA-598 • In phase II trials, the ANA598 was administered with Peg-INF and ribavirin. 56% of HCV patients achieved RVR at 4rth week as compared to 20% RVR in patients receive only Peg-INF and ribavirin therapy. When the drug was administered 200 mg twice a day with Peg-INF and ribavirin, 73% of patients achieved undetectable HCV RNA at 12th week [Abravanel et al., 2010].
Non-Nucleoside Inhibitors Class IV • Benzofuran derivatives. • Binds in a large hydrophobic pocket present in the palm domain. HCV 796 • HCV 796 is a benzofuran derivative which binds in the primer grip site and showed EC50 of 9 nMin cell lines by using HCV replicon [Keneteman et al., 2009]. • Resistant mutations arises were C316Y and S365T[Hang et al., 2009]. The C316Y mutant has EC50 of more than 100 times higher than the wild type NS5B in cell based assay [Waheed et al unpublished data].
Viral Resistance • The major problem for the development of drug resistance in HCV patients is the presence of high genetic diversity in viral genome. The diversity is linked with two parameters: (a) The estimated half-life of HCV is 2 – 5 hours, with the production and clearance of 1010 – 1012 /day in infected patients [Hermann et al., 2000; Neumann et al., 1998] (b) HCV polymerase lacks the proof reading ability with 10-3 to 10-5mutations per nucleotide per genomic replication. These features support the production of large viral variants during infection and selection of resistant variants during treatment [Bartenschlager et al., 2000; Legrand-Abravanel et al., 2010]. • The generation of resistant mutant is dependent upon the drug target site. The non-nucleoside inhibitor HCV 796 targets the Palm II site and generates resistant mutants C316Y. The C316Y mutant is 30% less active then the wild type. The nucleoside inhibitors target the active site and they generate the resistant mutants which are replication defective e.g. the NIs R1479 and PSI-6130 generates the S96T and S282T resistant mutants respectively. The mutants S282T and S96T are 85% and 96% respectively less active then the wild type replicon [McCown et al., 2008]. • The mutation rate of HCV polymerase is very high; everyday large numbers of mutants with single or double nucleotide substitution are generated. Sequence comparison of 507 HCV treatment naïve patients shows that 8.6% of HCV genotype 1a and 1.4% of HCV genotype 1b patients possess at least one drug resistant mutation and only 2 cases possess multiple drug resistant mutations. Viral titer in majority of patients was very high suggesting that drug resistant strains might have attained the replication levels comparable to nonresistant viruses [Kuntzen et al., 2008].
Combination Therapy • In a study by Le-Pogam et al., two non-nucleoside inhibitors; one targeting the thumb domain and second targeting the palm domain were employed in a combination therapy study by using HCV replicon. The double mutant M414L and M423T were predominantly selected, although the frequency of occurrence of double mutant was only 0.006% as compared with 1.3% for single mutant. The double mutant showed 81% reduced replication capacity compared to wild type replicon [Le-Pogam et al., 2006].
Conclusion • Different NIs and NNIs are being used to target the HCV RNA dependent RNA polymerase. The major advantage of the NIs is their pan-genotypic effect and they shows sound barrier to the emergence of resistant mutant. They target the active site of HCV polymerase and resistant mutants generated by them are replication defective. The major disadvantage of the NIs inhibitors is that they are normally delivered in prodrug form and they are dependent upon cellular kinase for their conversion into active form. The NIs also faces the problem of competition for incorporation with cellular NTPs, which are normally present in higher concentrations in the cell. • The non-nucleoside inhibitors target different sites on HCV polymerase. The effect of most of NNIs is different on different genotypes; they do not face the problem of competition as the NIs. The major disadvantage of these inhibitors is their week barrier for the emergence of resistant variants. • The current Interferon plus ribavirin therapy is for longer duration, has number of side effects and large number of patients are non-responder to the therapy. The aim of the future therapy is to develop drugs which are more potent, less toxic, allow a shorter duration for therapy and produces pan-genotypic response.