Discovery of Potent, Orally Bioavailable Inhibitors of Human Cytomegalovirus.

A high-throughput screen based on a viral replication assay was used to identify inhibitors of the human cytomegalovirus. Using this approach, hit compound 1 was identified as a 4 µM inhibitor of HCMV that was specific and selective over other herpes viruses. Time of addition studies indicated compound 1 exerted its antiviral effect early in the viral life cycle. Mechanism of action studies also revealed that this series inhibited infection of MRC-5 and ARPE19 cells by free virus and via direct cell-to-cell spread from infected to uninfected cells. Preliminary structure-activity relationships demonstrated that the potency of compound 1 could be improved to a low nanomolar level, but metabolic stability was a key optimization parameter for this series. A strategy focused on minimizing metabolic hydrolysis of the N1-amide led to an alternative scaffold in this series with improved metabolic stability and good pharmacokinetic parameters in rat.

Hepatitis C virus serine protease: synthesis of radioactive and stable isotope-labeled potent inhibitors.

Drug candidates labeled with radioactive and stable isotopes are required for absorption, distribution, metabolism, and excretion (ADME) studies, pharmacokinetics, autoradiography, bioanalytical, and other research activities. The findings from these studies are crucial in the development of a drug candidate and its approval for human use. Herein, we report the synthesis of potent and selective hepatitis C virus serine protease inhibitors related to BILN 2061 and BI 201335 labeled with radioactive and stable isotopes. Synthetic efforts were focused on the common substituted thiazole moiety, which is easily accessible via a Hantzsch's reaction of α-bromoketones and mono-substituted thioureas. In the radioactive synthesis, commercially available carbon-14 thiourea was utilized to prepare mono-substituted thioureas, which upon condensation with α-bromoketones in isopropanol followed by ester hydrolysis gave the desired carbon-14-labeled protease inhibitors. The same strategy was used to prepare these inhibitors labeled with stable isotopes. Mono-substituted thioureas were obtained from commercially available deuterium-labeled intermediates and then condensed with α-bromoketones followed by ester hydrolysis to give the deuterium-labeled inhibitors.

Discovery of hepatitis C virus NS3-4A protease inhibitors with improved barrier to resistance and favorable liver distribution.

Given the emergence of resistance observed for the current clinical-stage hepatitis C virus (HCV) NS3 protease inhibitors, there is a need for new inhibitors with a higher barrier to resistance. We recently reported our rational approach to the discovery of macrocyclic acylsulfonamides as HCV protease inhibitors addressing potency against clinically relevant resistant variants. Using X-ray crystallography of HCV protease variant/inhibitor complexes, we shed light on the complex structural mechanisms by which the D168V and R155K residue mutations confer resistance to NS3 protease inhibitors. Here, we disclose SAR investigation and ADME/PK optimization leading to the identification of inhibitors with significantly improved potency against the key resistant variants and with increased liver partitioning.

Molecular dynamics simulations and structure-based rational design lead to allosteric HCV NS5B polymerase thumb pocket 2 inhibitor with picomolar cellular replicon potency.

The design and preliminary SAR of a new series of 1H-quinazolin-4-one (QAZ) allosteric HCV NS5B thumb pocket 2 (TP-2) inhibitors was recently reported. To support optimization efforts, a molecular dynamics (MD) based modeling workflow was implemented, providing information on QAZ binding interactions with NS5B. This approach predicted a small but critical ligand-binding induced movement of a protein backbone region which increases the pocket size and improves access to the backbone carbonyl groups of Val 494 and Pro 495. This localized backbone shift was consistent with key SAR results and was subsequently confirmed by X-ray crystallography. The MD protocol guided the design of inhibitors, exploiting novel H-bond interactions with the two backbone carbonyl groups, leading to the first thumb pocket 2 NS5B inhibitor with picomolar antiviral potency in genotype (gt) 1a and 1b replicons (EC50 = 120 and 110 pM, respectively) and with EC50 ≤ 80 nM against gt 2-6.

Peptide backbone replacement of hepatitis C virus NS3 serine protease C-terminal cleavage product analogs: discovery of potent succinamide inhibitors.

A number of potent peptidic inhibitors of the NS3 protease have been described in the literature based on a substrate-based approach. In an on-going effort to reduce the peptidic character of this class of inhibitors, two novel series of analogs have been prepared in which the usual P3 amino acid residue is replaced by a succinamide fragment. This new backbone modification not only reduces the peptidic nature of traditional inhibitors but also provides new SAR opportunities for the capping group. Optimization of each of these two series resulted in inhibitors with sub-nanomolar potencies.

Synthesis and optimization of a novel series of HCV NS3 protease inhibitors: 4-arylproline analogs.

In this report we describe the synthesis and evaluation of diverse 4-arylproline analogs as HCV NS3 protease inhibitors. Introduction of this novel P2 moiety opened up new SAR and, in combination with a synthetic approach providing a versatile handle, allowed for efficient exploitation of this novel series of NS3 protease inhibitors. Multiple structural modifications of the aryl group at the 4-proline, guided by structural analysis, led to the identification of analogs which were very potent in both enzymatic and cell based assays. The impact of this systematic SAR on different drug properties is reported.

Structure-based design of novel HCV NS5B thumb pocket 2 allosteric inhibitors with submicromolar gt1 replicon potency: discovery of a quinazolinone chemotype.

We describe the structure-based design of a novel lead chemotype that binds to thumb pocket 2 of HCV NS5B polymerase and inhibits cell-based gt1 subgenomic reporter replicons at sub-micromolar concentrations (EC50<200nM). This new class of potent thumb pocket 2 inhibitors features a 1H-quinazolin-4-one scaffold derived from hybridization of a previously reported, low affinity thiazolone chemotype with our recently described anthranilic acid series. Guided by X-ray structural information, a key NS5B-ligand interaction involving the carboxylate group of anthranilic acid based inhibitors was replaced by a neutral two-point hydrogen bonding interaction between the quinazolinone scaffold and the protein backbone. The in vitro ADME and in vivo rat PK profile of representative analogs are also presented and provide areas for future optimization of this new class of HCV polymerase inhibitors.

Molecular mechanism by which a potent hepatitis C virus NS3-NS4A protease inhibitor overcomes emergence of resistance.

Although optimizing the resistance profile of an inhibitor can be challenging, it is potentially important for improving the long term effectiveness of antiviral therapy. This work describes our rational approach toward the identification of a macrocyclic acylsulfonamide that is a potent inhibitor of the NS3-NS4A proteases of all hepatitis C virus genotypes and of a panel of genotype 1-resistant variants. The enhanced potency of this compound versus variants D168V and R155K facilitated x-ray determination of the inhibitor-variant complexes. In turn, these structural studies revealed a complex molecular basis of resistance and rationalized how such compounds are able to circumvent these mechanisms.

Cross-species absorption, metabolism, distribution and pharmacokinetics of BI 201335, a potent HCV genotype 1 NS3/4A protease inhibitor.

The present study describes the cross-species absorption, metabolism, distribution and pharmacokinetics of BI 201335, a potent HCV protease inhibitor currently in phase III clinical trials. BI 201335 showed a good Caco-II permeability (8.7 × 10(-6) cm/sec) and in vitro metabolic stability (predicted hepatic clearence (CL(hep)) <19% Q(h) in all species tested). Single dose PK revealed a clearance of 17, 3.0 and 2.6 mL/min/kg in rat, monkey and dog respectively, with a corresponding oral bioavailability of 29.1, 25.5 and 35.6%. Comparative plasma and liver PK profile in rodents showed a high liver Kp in the rat (42-fold), suggesting high target tissue distribution. Simple allometry based on animal PK predicted a human oral CL/F of 168 mL/min, within two-fold of the observed value (118 mL/min) at 240 mg in healthy volunteers. Allometry of volume of distribution generated a low exponent of 0.59, and a much lower predicted Vss/F (5-fold less than observed). Several different approaches of Vss/F prediction were evaluated and compared with the value observed in human. The averaged Vss/F from preclinical animals provides the best estimation of the observed human value (169 L vs. 175 L). Corresponding human "effective" t(1/2) values were also compared. The predicted human t(1/2) based on the CL from allometry with metabolic corrections and the averaged animal Vss represented the best estimation of the clinical data (12.1 vs. 17.2 hr). The present study demonstrated that the good preclinical ADMEPK profile of BI 201335 is consistent with that observed in the clinic. While preclinical data accurately predicted the human CL, the prediction of human Vss seems to be more challenging. The averaged Vss/F from all tested preclinical animals provided the best prediction of human Vss and the resulting "effective" t(1/2).

In vitro resistance profile of the hepatitis C virus NS3 protease inhibitor BI 201335.

The in vitro resistance profile of BI 201335 was evaluated through selection and characterization of variants in genotype 1a (GT 1a) and genotype 1b (GT 1b) replicons. NS3 R155K and D168V were the most frequently observed resistant variants. Phenotypic characterization of the mutants revealed shifts in sensitivity specific to BI 201335 that did not alter susceptibility to alpha interferon. In contrast to macrocyclic and covalent protease inhibitors, changes at V36, T54, F43, and Q80 did not confer resistance to BI 201335.

Combined X-ray, NMR, and kinetic analyses reveal uncommon binding characteristics of the hepatitis C virus NS3-NS4A protease inhibitor BI 201335.

Hepatitis C virus infection, a major cause of liver disease worldwide, is curable, but currently approved therapies have suboptimal efficacy. Supplementing these therapies with direct-acting antiviral agents has the potential to considerably improve treatment prospects for hepatitis C virus-infected patients. The critical role played by the viral NS3 protease makes it an attractive target, and despite its shallow, solvent-exposed active site, several potent NS3 protease inhibitors are currently in the clinic. BI 201335, which is progressing through Phase IIb trials, contains a unique C-terminal carboxylic acid that binds noncovalently to the active site and a bromo-quinoline substitution on its proline residue that provides significant potency. In this work we have used stopped flow kinetics, x-ray crystallography, and NMR to characterize these distinctive features. Key findings include: slow association and dissociation rates within a single-step binding mechanism; the critical involvement of water molecules in acid binding; and protein side chain rearrangements, a bromine-oxygen halogen bond, and profound pK(a) changes within the catalytic triad associated with binding of the bromo-quinoline moiety.

Preclinical characterization of BI 201335, a C-terminal carboxylic acid inhibitor of the hepatitis C virus NS3-NS4A protease.

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.

Discovery of a potent and selective noncovalent linear inhibitor of the hepatitis C virus NS3 protease (BI 201335).

C-Terminal carboxylic acid containing inhibitors of the NS3 protease are reported. A novel series of linear tripeptide inhibitors that are very potent and selective against the NS3 protease are described. A substantial contribution to the potency of these linear inhibitors arises from the introduction of a C8 substituent on the B-ring of the quinoline moiety found on the P2 of these inhibitors. The introduction of a C8 methyl group results not only in a modest increase in the cell-based potency of these inhibitors but more importantly in a much better pharmacokinetic profile in rats as well. Exploration of C8-substitutions led to the identification of the bromo derivative as the best group at this position, resulting in a significant increase in the cell-based potency of this class of inhibitors. Structure-activity studies on the C8-bromo derivatives ultimately led to the discovery of clinical candidate 29 (BI 201335), a very potent and selective inhibitor of genotype1 NS3 protease with a promising PK profile in rats.

The effect of the P1 side chain on the binding of optimized carboxylate and activated carbonyl inhibitors of the hepatitis C virus NS3 protease.

Peptidyl inhibitors of the hepatitis C virus NS3 protease hold much promise as direct-acting antiviral agents against hepatitis C infection. The optimization of N-terminal cleavage products, found to exhibit activity (product inhibition) against the enzyme, has led to potent tripeptide inhibitors that bear free C-terminal carboxylate groups. An analogous activated carbonyl compound (pentafluoroethyl ketone) bearing a P1 norvaline (Nva) was found to possess comparable activity against hepatitis C virus protease. However, an analogue bearing an aminocyclopropylcarboxylic acid (Acca) P1 residue exhibited very poor activity. (19)F-NMR studies indicate that the propensity of the Acca-derived activated carbonyl to form hemiketals is only slightly reduced compared with that of a P1 Nva equivalent. These results, as well as molecular modeling studies, argue against steric hindrance of the nucleophilic attack of Ser-139 accounting for the poor mechanism-based inhibition by the former. We hypothesize that the conformational properties of the respective C-termini in the context of an adaptable active site account for the divergent P1 structure-activity relationships.

Dimethylthiazolidine carboxylic acid as a rigid p3 unit in inhibitors of serine proteases: application to two targets.

Serine proteases are a very large class of enzymes, many of which represent important targets for therapeutic agents against a wide variety of disease states. The similarity in active site architecture for these proteases has often allowed inhibitor design strategies for a particular target to be successfully applied to other enzymes in the class. In many cases, the presence of a bulky P3 amino acid residue in peptide-based inhibitors is central to conferring an extended peptide conformation, critical to binding of the ligands to serine protease active sites. The dimethylthiazolidine carboxylic acid 'residue' was found to be effective as a novel P3 replacement in peptidomimetic inhibitors of two distinct serine proteases, the hepatitis C NS3 protease and the human cytomegalovirus maturational protease. An array of NMR methods was used to confirm that the dimethylthiazolidine carboxylic acid unit indeed confers conformational and dynamic properties very similar to that of the rigidified parent structures.

Use of the fused NS4A peptide-NS3 protease domain to study the importance of the helicase domain for protease inhibitor binding to hepatitis C virus NS3-NS4A.

The NS3 protein of hepatitis C virus is unusual because it encodes two unrelated enzymatic activities in linked protease and helicase domains. It has also been intensively studied because inhibitors targeting its protease domain have potential to significantly improve treatment options for those infected with this virus. Many enzymological studies and inhibitor discovery programs have been carried out using the isolated protease domain in complex with a peptide derived from NS4A which stimulates activity. However, some recent publications have suggested that the NS3 helicase domain may influence inhibitor binding and thus suggest work should focus on the full-length NS3-NS4A protein. Here we present the characterization of a single-chain protease in which the NS4A peptide activator is linked to the N-terminus of the NS3 protease domain. This protein behaves well in solution, and its protease activity is very similar to that of full-length NS3-NS4A. We find that this fusion protein, as well as the noncovalent complex of the NS4A peptide with NS3, gives similar Ki values, spanning 3 orders of magnitude, for a set of 25 structurally diverse inhibitors. We also show that simultaneous mutation of three residues on the surface of the helicase domain which has been hypothesized to interact with the protease does not significantly affect enzymatic activity or inhibitor binding. Thus, the protease domain with the NS4A peptide, in a covalent or noncovalent complex, is a good model for the protease activity of native NS3-NS4A.

Potent triazolyl-proline-based inhibitors of HCV NS3 protease.

The design and synthesis of tripeptide-based inhibitors of the HCV NS3 protease containing a novel P2-triazole is described. Replacement of the P2 quinoline with a triazole moiety provided a versatile handle which could be expediently modified to generate a diverse series of inhibitors. Further refinement by the incorporation of an aryl-substituted triazole and replacement of the P1 acid with an acyl sulfonamide ultimately provided inhibitors with interesting cellular activity.

The therapeutic potential of NS3 protease inhibitors in HCV infection.

Hepatitis C virus (HCV) infection is a serious cause of chronic liver disease worldwide and afflicts > 170 million people. The HCV-encoded NS3 protease is essential for viral replication and has long been recognised as a prime target for antiviral drugs. However, the peculiar active site structure of this enzyme, a shallow dent on the surface of the protein, has rendered the development of small-molecule inhibitors a highly challenging task. Nevertheless, perseverance and creativity has led to significant progress in this field over the last few years resulting in three compounds that are reported to enter the clinic. The impressive reduction of HCV RNA plasma levels observed with two of these inhibitors (ciluprevir and VX-950) in clinical trials has undoubtedly illustrated the potential of this viral enzyme-targeted drug discovery approach.