Antiviral Activity and Mode of Action of TMC647078, a Novel Nucleoside Inhibitor of the Hepatitis C Virus NS5B Polymerase

Abstract

Chronic infection with hepatitis C virus (HCV) is a major global health burden and is associated with an increased risk of liver cirrhosis and hepatocellular carcinoma. Current therapy for HCV infection has limited efficacy, particularly against genotype 1 virus, and is hampered by a range of adverse effects. Therefore, there is a clear unmet medical need for efficacious and safe direct antiviral drugs for use in combination with current treatments to increase cure rates and shorten treatment times. The broad genotypic coverage achievable with nucleosides or nucleotides and the high genetic barrier to resistance of these compounds observed in vitro and in vivo suggest that this class of inhibitors could be a valuable component of future therapeutic regimens. Here, we report the in vitro inhibitory activity and mode of action of 2-deoxy-2-spirocyclopropylcytidine (TMC647078), a novel and potent nucleoside inhibitor of the HCV NS5B RNA-dependent RNA polymerase that causes chain termination of the nascent HCV RNA chain. In vitro combination studies with a protease inhibitor resulted in additive efficacy in the suppression of HCV RNA replication, highlighting the potential for the combination of these two classes in the treatment of chronic HCV infection. No cytotoxic effects were observed in various cell lines. Biochemical studies indicated that TMC647078 is phosphorylated mainly by deoxycytidine kinase (dCK) without inhibiting the phosphorylation of the natural substrate, and high levels of triphosphate were observed in Huh7 cells and in primary hepatocytes in vitro. TMC647078 is a potent novel nucleoside inhibitor of HCV replication with a promising in vitro virology and biology profile.Infection with hepatitis C virus (HCV), the causative agent of hepatitis C, is an important global health burden, with an estimated 120 to 170 million persons chronically infected (7,11). Chronic HCV infection can lead to liver cirrhosis and hepatocellular carcinoma and is the leading cause of liver transplantation (10). The virus is transmitted mainly via bloodblood contact, and spontaneous virus clearance has been estimated to be achieved for 26% of infected subjects (29).HCV is a member of the Flaviviridae family of viruses in the genus Hepacivirus, comprising at least six major genotypes and multiple subtypes. The 9.6-kb positive-sense, single-stranded RNA genome of HCV encodes four structural proteins (the core protein, the envelope glycoproteins E1 and E2, and p7) and six nonstructural proteins responsible for viral replication (NS2, NS3, NS4A, NS4B, NS5A, and NS5B). The nonstructural protein NS5B is an RNA-dependent RNA polymerase (RdRp) responsible for the amplification of the HCV RNA and the assembly of the replicase complex at the endoplasmic reticulum membrane (3, 25).Currently, HCV patients are treated with a combination of weekly pegylated alpha interferon (IFN-␣) injections and twice-daily ribavirin, which has considerable tolerability issues (28) and results in a sustained viral response in only 40 to 50% of patient...

Broad-spectrum agents for flaviviral infections: dengue, Zika and beyond

“…Furthermore, the phosphorylation efficiency of kinases is influenced by several factors, resulting in variations of the EC 50 (effector concentration for half-maximum response) value of a nucleoside inhibitor in different cell types used for the assay. For example, EC 50 values were reportedly higher in immortalized cell lines, such as the frequently used Huh-7 cells, compared with primary hepatic cells 107 . Based on these considerations, the tropism exerted by different flaviviruses may also have an important role in the potency of nucleoside inhibitors.…”

The search for nucleoside/nucleotide analog inhibitors of dengue virus

“…For instance, where is a significant difference between the efficiency of triphosphate conversion when comparing Huh7 (human liver cell line) and human primary hepatocytes (Berke et al, 2011).…”

Nucleosides and Nucleotides for the Treatment of Viral Diseases

“…63 A unique series of 1 0 -substituted C-nucleosides possessing a 4-aza-7,9dideaza-adenosine base 56 were shown to have modest activity against HCV with improved potency upon preparation of their S-acyl-2-thioethyl prodrugs 57. [67][68][69][70] In the 2 0 -cyclopropyl series, TMC647078 (59) was active in the replicon assay (EC 50 ¼ 7.3 μM) across all GTs, but exhibited reduced activity against NS5B S282T. Preparation of the 2 0 -C-methyl-1 0 -cyano derivatives eliminated the cytotoxicity and subsequent development of the 5 0 -phosphoramidate prodrug of the 1 0 -cyano analog resulted in the clinical candidate GS-6620 58.…”

“…64 However, they also demonstrated substantial cytotoxicity. 67,68 Low plasma exposure in rats led to the preparation of the ester prodrugs 60 and 61, which resulted in reduced replicon activity (EC 50 ¼ 42.8 and 20.3 μM, respectively) but improved in vivo exposure relative to the parent nucleoside. 65 Like many other HCV nucleos(t)ide inhibitors, GS-6620 contained a 2 0 -C-methylribosyl core and maintained a pan-genotypic profile.…”

“…7-Heterocyclic substituted 7-deaza-adenine derivatives containing either the 2 0 -F-2 0 -C-methyl or 2 0 -OH-2 0 -C-methyl substitution (65,66) demonstrated modest HCV replicon potency and modest liver triphosphate levels when administered in vivo to rats. 71 In a similar fashion, an extensive series of 6-substituted-7-heteroaryl-7-deazapurine ribonucleosides (68)(69)(70) were studied, but replicon activity was generally correlated with cytotoxicity. 71 In a similar fashion, an extensive series of 6-substituted-7-heteroaryl-7-deazapurine ribonucleosides (68)(69)(70) were studied, but replicon activity was generally correlated with cytotoxicity.…”