Intergenotypic Comparison of Various Antivirals and Immunomodulators Against Hepatitis C Virus

Abstract

Worldwide, 170 million people are chronically infected with hepatitis C virus (HCV). HCV is a major cause of potentially life-threatening liver diseases such as fibrosis, cirrhosis, and hepatocellular carcinoma. The development of prophylactic vaccines for the prevention of HCV infections has been challenging. In recent years, a number of highly potent drugs have been developed to treat HCV infections. Direct acting antivirals (DAAs) used in combination with IFN and/or ribavirin are either protease inhibitors such as telaprevir, boceprevir or simeprevir (available as Incivek, Victrelis and Olysio respectively) or a nucleotide NS5B polymerase inhibitor sofosbuvir (available as Sovaldi®). Recently, the Food and Drug Administration (FDA) has approved the combination, sofosbuvir with the NS5A inhibitor ledipasvir (Harvoni®), to treat patients infected with HCV. The treatment response of various drugs, however, is found to closely correlate with different HCV genotypes. This variation in treatments responses prompted the current study. The current study was carried out in order to compare the anti-HCV activity of various classes of HCV inhibitors (both DAA and non DAA/immunomodulators) against HCV genotypes 1a, 1b, 2a and 4a (using the respective subgenomic replicons) in vitro cell culture system. Both FDA approved reference compounds along with those undergoing the clinical trials either for the treatment of HCV infections or host-targetting immunomodulators such as the cholesterol lowering statins) reported to have anti-HCV activity were included in the study. Besides reference compounds, six novel synthetic compounds were also evaluated for their anti-HCV activity against four different HCV subgenomic replicons 1a, 1b, 2a and 4a (using the respective subgenomic replicons). For most genotypes similar antiviral activities were obtained for the selected panel of antiviral agents. Among the reference pannel, boceprevir (NS3 inhibitor), clemizole (NS4B inhibitor), sofobuvir, JT16 (NS5B inhibitors), alisporivir and AL 9 (HTAs) were almost equipotent against all the HCV replicons used in the study. Drugs also known to have immunomodulatory effect and to treat other etiologies (such as fluvustatin and nitazoxanide) and food additives (eg. EGCG) proved much less active (sometimes inactive) compared to the DAA and HTA (host targeting antivirals). Among the novel HCV inhibitors, RSHCNS5B_0058 and RSHCNS5B_0066 exhibited panogenotypic anti-HCV activity against the Abstract xi genotypes used in the study. Furthermore, inhibitor RSHCNS5B_0003 was equipotent against 1a, 1b and 4a whearase antiviral activity could not be dectect against 2a. The NS4B of HCV is a multifunctional protein and is an iducer of membraneous web which serves as a platform for HCV replication. In the present study, two NS4B inhibitors clemizole and anguizole were assessed for their anti HCV activities. Clemizole was active at high concentration wherase anguizole proved to be inactive against HCV 2a replicon. In order to design the NS4B inhibitors in future with pan-genotypic potential, an in silico study was also planned to generate global consensus sequence of 701 HCV NS4B protein sequences reported from different regions of world. In future, this would be very helpful to design novel pangenotypic NS4B inhibitors using global consensus sequence as a representative of all HCV genotypes. During global consensus of NS4B, the C-terminal domain (CTD) of NS4B was found to be relatively more conserved. Aromatic residues of N-terminal domain F49, W50, W55, F57, and Y63, hydrophobic leucine residues L237, L240, L245 and positively charged R248 and H250 of CTD, which are involved in the membrane interaction of NS4B were highly conserved among all HCV genotypes. Dimerization motif of transmembrane domain 3 (TMD3) (G143YGAG147) was also highly conserved among all HCV genotypes. The surrounding phenylalanine residues F118 & F155 of dimerization motifs and residues T87, S88 and T95 of TMD1 Ser/Thr cluster which are involved in the hydrogen (H) bond interactions were well conserved as well. One of the two palmitoylation sites (C257 and C261), C257 was highly variable. Interestingly, the NS4B sequences of 3a Pakistani isolates did not cluster together in phylogenetic tree due to high degree of variability among them. From the highly conserved region of the NS4B, five peptides were developed that could serve as peptide vaccines in future. In conclusion, the drugs (cholesterol, nitazoxanide and tizoxanide) used for other indications and plant extracts (silymarin, silibinin and EGCG) proved to have very limited antiviral activity and for some even non-existing as compared to the DAAs and known HTAs (alisporivir and AL-9). Furthermore, global consensus sequence of HCV NS4B and five short peptides were developed that could be used in future for the development of NS4B inhbitors and peptides vaccines repectively.