Inhibition and Binding Kinetics of the Hepatitis C Virus NS3 Protease Inhibitor ITMN-191 Reveals Tight Binding and Slow Dissociative Behavior

Small-molecule hepatitis C virus (HCV) NS3 protease inhibitors such as boceprevir (SCH 503034) have been shown to have antiviral activity when they are used as monotherapy and in combination with pegylated alpha interferon and ribavirin in clinical trials. Improvements in inhibitor potency and pharmacokinetic properties offer opportunities to increase drug exposure and to further increase the sustained virological response. Exploration of the structure-activity relationships of ketoamide inhibitors related to boceprevir has led to the discovery of SCH 900518, a novel ketoamide protease inhibitor which forms a reversible covalent bond with the active-site serine. It has an overall inhibition constant (K i * ) of 7 nM and a dissociation half-life of 1 to 2 h. SCH 900518 inhibited replicon RNA at a 90% effective concentration (EC 90 ) of 40 nM. In biochemical assays, SCH 900518 was active against proteases of genotypes 1 to 3. A 2-week treatment with 5؋ EC 90 of the inhibitor reduced the replicon RNA level by 3 log units. Selection of replicon cells with SCH 900518 resulted in the outgrowth of several resistant mutants (with the T54A/S and A156S/T/V mutations). Cross-resistance studies demonstrated that the majority of mutations for resistance to boceprevir and telaprevir caused similar fold losses of activity against all three inhibitors; however, SCH 900518 retained more activity against these mutants due to its higher intrinsic potency. Combination treatment with alpha interferon enhanced the inhibition of replicon RNA and suppressed the emergence of resistant replicon colonies, supporting the use of SCH 900518-pegylated alpha interferon combination therapy in the clinic. In summary, the results of the preclinical characterization of the antiviral activity of SCH 900518 support its evaluation in clinical studies.Hepatitis C virus (HCV) is a major cause of chronic hepatitis worldwide. Despite recent advances, the eradication of chronic HCV infection remains challenging. Approximately 50% of patients infected with the most prevalent form of the virus (genotype 1) fail to respond to treatment with the current standard of care of pegylated alpha interferon in combination with ribavirin (4, 13). Thus, there is a great unmet medical need for the development of new agents with activity against HCV to improve the sustained virological response (SVR). The HCV genome is translated as a single polyprotein precursor which is processed by cellular and viral proteases into structural proteins (the C, E1, and E2 proteins), followed by nonstructural (NS) proteins (the p7, NS2, NS3, NS4A, NS4B, NS5A, and NS5B proteins). The NS3 protein is multifunctional, with the N-terminal region (amino acids 1 to 181) encoding a serine protease and the remaining polypeptide encoding a helicase. The NS3 protease noncovalently binds to the cofactor NS4A, which contributes to the formation and stability of the active site and helps to anchor the NS3/NS4A complex to the membrane. The NS3 protease is responsible for the processing of the HCV polyp...

Section: Discussionmentioning

confidence: 90%

Preclinical Characterization of the Antiviral Activity of SCH 900518 (Narlaprevir), a Novel Mechanism-Based Inhibitor of Hepatitis C Virus NS3 Protease

Tong¹,

Arasappan

Bennett

et al. 2010

“…In addition, the single mutant A156T or A156V or the double mutant at positions 36/155 or 36/156, all conferring high-level resistance, were observed in some VX-950-treated patients. Similarly, samples from SCH-503034-treated patients revealed the emergence of the low-to moderate-level resistance mutations T54A, V170A, and A156S and, less commonly, the high-level resistance mutant A156T (27, 28).More recently, other HCV PIs, including , have progressed to the early stages of clinical evaluation (12,15,19,20,23,29). These compounds are structurally related macrocyclic inhibitors that are chemically distinct from VX-950 and SCH-503034.…”

mentioning

confidence: 99%

“…More recently, other HCV PIs, including , have progressed to the early stages of clinical evaluation (12,15,19,20,23,29). These compounds are structurally related macrocyclic inhibitors that are chemically distinct from VX-950 and SCH-503034.…”

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confidence: 99%

Susceptibility of Treatment-Naive Hepatitis C Virus (HCV) Clinical Isolates to HCV Protease Inhibitors

Bae

Sun

et al. 2010

In order to assess the natural variation in susceptibility to hepatitis C virus (HCV) NS3 protease inhibitors (PIs) among untreated HCV patient samples, the susceptibilities of 39 baseline clinical isolates were determined using a transient-replication assay on a panel of HCV PIs, including two ␣-ketoamides (VX-950 and SCH-503034) and three macrocyclic inhibitors (MK-7009, ITMN-191, and TMC-435350). Some natural variation in susceptibility to all HCV PIs tested was observed among the baseline clinical isolates. The susceptibility to VX-950 correlated strongly with the susceptibility to SCH-503034. A moderate correlation was observed between the susceptibilities to ITMN-191 and MK-7009. In contrast, the phenotypic correlations between the ␣-ketoamides and macrocyclic inhibitors were significantly lower. This difference is partly attributable to reduced susceptibility of the HCV variants containing the NS3 polymorphism Q80K (existing in 47% of genotype 1a isolates) to the macrocyclic compounds but no change in the sensitivity of the same variants to the ␣-ketoamides tested. Our results suggest that the natural variation in baseline susceptibility may contribute to different degrees of antiviral response among patients in vivo, particularly at lower doses.Hepatitis C virus (HCV) is characterized by a high degree of genetic diversity because of its rapid replication rate and turnover, combined with the poor fidelity of the HCV RNA-dependent RNA polymerase (RdRp) (3, 5, 32). The nucleotide sequences among the six different genotypes (GTs) differ at 30 to 35% of nucleotide sites (25,26). Each of the six major GTs of HCV contains a series of closely related subtypes whose nucleotide sequences typically differ from each other by 20 to 25%. Furthermore, 5 to 8% sequence divergence is present between individual strains (variants) of HCV within a given subtype. A comprehensive analysis of HCV NS3 sequences from a larger number of GT-1 isolates found that amino acid polymorphisms were detected all along the protease sequence, including residues associated either with resistance to HCV protease inhibitors (PIs) (V36, I170, and D168) or with compensatory mutations (I72, T72, Q86, and I153) (1, 2, 30). However, many questions remain, including whether natural variation in the NS3 protease sequence impacts the susceptibility of HCV to PIs currently in development and whether there are any relationships among the chemotypes of the PIs and their baseline susceptibilities.Hepatitis C virus NS3/4A serine PIs have demonstrated potent antiviral activity in subjects infected with HCV GT-1 by specifically blocking NS3/4A protease-dependent HCV polyproprotein processing. Among these PIs, VX-950 (telaprevir) and SCH-503034 (boceprevir), the two most clinically advanced NS3/4A serine PIs, are both ␣-ketoamide compounds that covalently bind to the active-site serine of the protease (6,9,10,14,16,22). These drugs also have similar resistance profiles. Mutations V36A/M, T54A, R155K, and A156S in the NS3 protease gene, conferring a low level ...

“…Collectively, our data suggest that ITMN-8187 could be developed for use in once-daily treatment regimens in combination with other directly acting antiviral agents. Biochemical inhibition studies (data not shown) indicate that ITMN-8187 associates with genotype 1a and 1b NS3/4A proteases through a slow/tight binding mechanism with slow dissociation (off-rate) similar to that noted previously for danoprevir (26). While the importance of increased drug target residence times to efficacy has been demonstrated in other systems (32), its contribution here is uncertain given the absence of reliable measures of HCV protein turnover and synthesis rates (26).…”

Section: Discussionmentioning

confidence: 67%

Preclinical Characterization and Human Microdose Pharmacokinetics of ITMN-8187, a Nonmacrocyclic Inhibitor of the Hepatitis C Virus NS3 Protease

Rajagopalan¹,

Pan²,

Schaefer³

et al. 2017

Self Cite

The current paradigm for the treatment of chronic hepatitis C virus (HCV) infection involves combinations of agents that act directly on steps of the HCV life cycle. Here we report the preclinical characteristics of ITMN-8187, a nonmacrocyclic inhibitor of the NS3/4A HCV protease. X-ray crystallographic studies of ITMN-8187 and simeprevir binding to NS3/4A protease demonstrated good agreement between structures. Low nanomolar biochemical potency was maintained against NS3/4A derived from HCV genotypes 1, 2b, 4, 5, and 6. In cell-based potency assays, halfmaximal reduction of genotype 1a and 1b HCV replicon RNA was afforded by 11 and 4 nM doses of ITMN-8187, respectively. Combinations of ITMN-8187 with other directly acting antiviral agents in vitro displayed additive antiviral efficacy. A 30-mg/kg of body weight dose of ITMN-8187 administered for 4 days yielded significant viral load reductions through day 5 in a chimeric mouse model of HCV. A 3-mg/kg oral dose administered to rats, dogs, or monkeys yielded concentrations in plasma 16 h after dosing that exceeded the half-maximal effective concentration of ITMN-8187. Human microdose pharmacokinetics showed low intersubject variability and prolonged oral absorption with first-order elimination kinetics compatible with once-daily dosing. These preclinical characteristics compare favorably with those of other NS3/4A inhibitors approved for the treatment of chronic HCV infection.