Preclinical Characterization of BI 201335, a C-Terminal Carboxylic Acid Inhibitor of the Hepatitis C Virus NS3-NS4A Protease

Abstract

BI 201335 is a hepatitis C virus (HCV) NS3-NS4A (NS3 coexpressed with NS4A) protease inhibitor that has been shown to have potent clinical antiviral activity. It is a highly optimized noncovalent competitive inhibitor of full-length NS3-NS4A proteases of HCV genotypes 1a and 1b with K i values of 2.6 and 2.0 nM, respectively. K i values of 2 to 230 nM were measured against the NS3-NS4A proteases of HCV genotypes 2 to 6, whereas it was a very weak inhibitor of cathepsin B and showed no measurable inhibition of human leukocyte elastase. BI 201335 was also shown to be a potent inhibitor of HCV RNA replication in vitro with 50% effective concentrations (EC 50 s) of 6.5 and 3.1 nM obtained in genotype 1a and 1b replicon assays. Combinations of BI 201335 with either interferon or ribavirin had additive effects in replicon assays. BI 201335 had good permeability in Caco-2 cell assays and high metabolic stability after incubation with human, rat, monkey, and dog liver microsomes. Its good absorption, distribution, metabolism, and excretion (ADME) profile in vitro, as well as in rat, monkey, and dog, predicted good pharmacokinetics (PK) in humans. Furthermore, drug levels were significantly higher in rat liver than in plasma, suggesting that distribution to the target organ may be especially favorable. BI 201335 is a highly potent and selective NS3-NS4A protease inhibitor with good in vitro and animal ADME properties, consistent with its good human PK profile, and shows great promise as a treatment for HCV infection. Chronic hepatitis C virus (HCV) infection affects 130 to 170 million individuals worldwide (14). The etiologic agent is a small enveloped single-stranded RNA virus belonging to the Flaviviridae family, Hepacivirus genus (32). Although present in human populations for thousands of years, it was discovered only 20 years ago as the causative agent of non-A, non-B hepatitis (6). The HCV genome consists of approximately 9,600 bases, encoding a single polyprotein of approximately 3,000 amino acids, flanked by conserved 5Ј and 3Ј untranslated regions (UTRs). The viral polyprotein comprises four structural proteins followed by six nonstructural (NS) proteins that play essential roles in viral replication (25).One of the best-studied nonstructural proteins is NS3, a bifunctional protein that consists of an N-terminal protease domain and a C-terminal helicase domain (9). The protease domain has a trypsin-like fold with a flat and solvent-exposed substrate binding site (11,21). The central portion of the NS4A protein is integrated into the protein fold of the NS3 protease domain and is required for full activity (3). The NS3-NS4A (NS3 coexpressed with NS4A) protease plays a critical role in the maturation of the viral polyprotein precursor and was recognized early on as potential target for antiviral drugs (2). Indeed, the first direct acting antiviral agent to be studied in humans was the protease inhibitor BILN 2061, and two other protease inhibitors, telaprevir and boceprevir, are currently in phase III trials (10,1...

STARTVerso1: A randomized trial of faldaprevir plus pegylated interferon/ribavirin for chronic HCV genotype-1 infection

“…Faldaprevir (BI 201335) is a potent HCV NS3/4A PI administered once daily [10][11][12]. Four phase 2 studies evaluated the efficacy and safety of faldaprevir with PegIFN alfa-2a plus RBV [13][14][15][16].…”

Combined X-ray, NMR, and Kinetic Analyses Reveal Uncommon Binding Characteristics of the Hepatitis C Virus NS3-NS4A Protease Inhibitor BI 201335

“…In our case, the bromine appears to impart a more modest affinity improvement, possibly because of the shifts in both the ligand and receptor that are required to adopt the correct geometry. Nonetheless, the overall benefit of the bromine to BI 201335 as a drug is even greater because it also imparts desirable antiviral potency and pharmacokinetic properties (12,15).…”

“…We also present NMR evidence that shielding of the catalytic triad by the multi aromatic ring P2 substituent of BI 201335 perturbs the protonation states of its Asp-His pair, which may in turn favor the binding of the carboxylate to the active site. Finally, we elaborate on previously reported steady-state kinetic analyses that indicated BI 201335 is a competitive inhibitor of NS3-NS4A protease activity (12) by reporting the individual association and dissociation rate constants determined by stopped flow methods.…”

“…However, distinct from other compounds presently in the clinic, BI 201335 possesses a C-terminal carboxylic acid that contributes significantly to the specificity of the compound for the viral protease (13). Furthermore, the amino acid side chains and N-terminal capping group of BI 201335 have been optimized for potency in both cellular and biochemical assays and to provide good absorption, distribution, metabolism, elimination, and pharmacokinetics properties allowing efficient uptake and distribution of the drug to the target organ, the liver (12). During this optimization, multi-ring aromatic systems extending from the P2 proline were found to be particularly beneficial to C-terminal carboxylate-containing molecules (standardized terminology for protease substrate amino acids (P1-P4) and substrate side chain binding sites (S1-S4) is used throughout (14)).…”

“…1a) is a promising drug candidate that potently and selectively targets the viral NS3-NS4A protease (12). Similar to other protease inhibitors, BI 201335 features a peptidic backbone such that it can form extended, substrate-like interactions with an exposed ␤-strand of the enzyme substrate-binding site.…”