Amy K. Sheaffer

Preclinical Profile and Characterization of the Hepatitis C Virus NS3 Protease Inhibitor Asunaprevir (BMS-650032)

ABSTRACT Asunaprevir (ASV; BMS-650032) is a hepatitis C virus (HCV) NS3 protease inhibitor that has demonstrated efficacy in patients chronically infected with HCV genotype 1 when combined with alfa interferon and/or the NS5A replication complex inhibitor daclatasvir. ASV competitively binds to the NS3/4A protease complex, with Ki values of 0.4 and 0.24 nM against recombinant enzymes representing genotypes 1a (H77) and 1b (J4L6S), respectively. Selectivity was demonstrated by the absence of any significant activity against the closely related GB virus-B NS3 protease and a panel of human serine or cysteine proteases. In cell culture, ASV inhibited replication of HCV replicons representing genotypes 1 and 4, with 50% effective concentrations (EC50s) ranging from 1 to 4 nM, and had weaker activity against genotypes 2 and 3 (EC50, 67 to 1,162 nM). Selectivity was again demonstrated by the absence of activity (EC50, >12 μM) against a panel of other RNA viruses. ASV exhibited additive or synergistic activity in combination studies with alfa interferon, ribavirin, and/or inhibitors specifically targeting NS5A or NS5B. Plasma and tissue exposures in vivo in several animal species indicated that ASV displayed a hepatotropic disposition (liver-to-plasma ratios ranging from 40- to 359-fold across species). Twenty-four hours postdose, liver exposures across all species tested were ≥110-fold above the inhibitor EC50s observed with HCV genotype-1 replicons. Based on these virologic and exposure properties, ASV holds promise for future utility in a combination with other anti-HCV agents in the treatment of HCV-infected patients.

Herpes Simplex Virus DNA Cleavage and Packaging Proteins Associate with the Procapsid prior to Its Maturation

ABSTRACT Packaging of DNA into preformed capsids is a fundamental early event in the assembly of herpes simplex virus type 1 (HSV-1) virions. Replicated viral DNA genomes, in the form of complex branched concatemers, and unstable spherical precursor capsids termed procapsids are thought to be the substrates for the DNA-packaging reaction. In addition, seven viral proteins are required for packaging, although their individual functions are undefined. By analogy to well-characterized bacteriophage systems, the association of these proteins with various forms of capsids, including procapsids, might be expected to clarify their roles in the packaging process. While the HSV-1 UL6, UL15, UL25, and UL28 packaging proteins are known to associate with different forms of stable capsids, their association with procapsids has not been tested. Therefore, we isolated HSV-1 procapsids from infected cells and used Western blotting to identify the packaging proteins present. Procapsids contained UL15 and UL28 proteins; the levels of both proteins are diminished in more mature DNA-containing C-capsids. In contrast, UL6 protein levels were approximately the same in procapsids, B-capsids, and C-capsids. The amount of UL25 protein was reduced in procapsids relative to that in more mature B-capsids. Moreover, C-capsids contained the highest level of UL25 protein, 15-fold higher than that in procapsids. Our results support current hypotheses on HSV DNA packaging: (i) transient association of UL15 and UL28 proteins with maturing capsids is consistent with their proposed involvement in site-specific cleavage of the viral DNA (terminase activity); (ii) the UL6 protein may be an integral component of the capsid shell; and (iii) the UL25 protein may associate with capsids after scaffold loss and DNA packaging, sealing the DNA within capsids.

Isolation of Herpes Simplex Virus Procapsids from Cells Infected with a Protease-Deficient Mutant Virus

ABSTRACT Herpes simplex virus type 1 (HSV-1) capsid proteins assemble in vitro into spherical procapsids that differ markedly in structure and stability from mature polyhedral capsids but can be converted to the mature form. Circumstantial evidence suggests that assembly in vivo follows a similar pathway of procapsid assembly and maturation, a pathway that resembles those of double-stranded DNA bacteriophages. We have confirmed the above pathway by isolating procapsids from HSV-1-infected cells and characterizing their morphology, thermal sensitivity, and protein composition. Experiments were carried out with an HSV-1 mutant (m100) deficient in the maturational protease for which it was expected that procapsids—normally, short-lived intermediates—would accumulate in infected cells. Particles isolated from m100-infected cells were found to share the defining properties of procapsids assembled in vitro. For example, by electron microscopy, they were found to be spherical rather than polyhedral in shape, and they disassembled at 0°C, unlike mature capsids, which are stable at this temperature. A three-dimensional reconstruction computed at 18-Å resolution from cryoelectron micrographs showed m100 procapsids to be structurally indistinguishable from procapsids assembled in vitro. In both cases, their predominant components are the four essential capsid proteins: the major capsid protein (VP5), the scaffolding protein (pre-VP22a), and the triplex proteins (VP19C and VP23). VP26, a small, abundant but dispensable capsid protein, was not found associated withm100 procapsids, suggesting that it binds to capsids only after they have matured into the polyhedral form. Procapsids were also isolated from cells infected at the nonpermissive temperature with the HSV-1 mutant tsProt.A (a mutant with a thermoreversible lesion in the protease), and their identity as procapsids was confirmed by cryoelectron microscopy. This analysis revealed density on the inner surface of the procapsid scaffolding core that may correspond to the location of the maturational protease. Upon incubation at the permissive temperature, tsProt.A procapsids transformed into polyhedral, mature capsids, providing further confirmation of their status as precursors.

Resistance Analysis of the Hepatitis C Virus NS3 Protease Inhibitor Asunaprevir

ABSTRACT Asunaprevir (BMS-650032) is a potent hepatitis C virus (HCV) NS3 protease inhibitor demonstrating efficacy in alfa interferon-sparing, direct-acting antiviral dual-combination regimens (together with the NS5A replication complex inhibitor daclatasvir) in patients chronically infected with HCV genotype 1b. Here, we describe a comprehensive in vitro genotypic and phenotypic analysis of asunaprevir-associated resistance against genotypes 1a and 1b using HCV replicons and patient samples obtained from clinical studies of short-term asunaprevir monotherapy. During genotype 1a resistance selection using HCV replicons, the primary NS3 protease substitutions identified were R155K, D168G, and I170T, which conferred low- to moderate-level asunaprevir resistance (5- to 21-fold) in transient-transfection susceptibility assays. For genotype 1b, a higher level of asunaprevir-associated resistance was observed at the same selection pressures, ranging from 170- to 400-fold relative to the wild-type control. The primary NS3 protease substitutions identified occurred predominantly at amino acid residue D168 (D168A/G/H/V/Y) and were associated with high-level asunaprevir resistance (16- to 280-fold) and impaired replication capacity. In asunaprevir single-ascending-dose and 3-day multiple-ascending-dose studies in HCV genotype 1a- or 1b-infected patients, the predominant pre-existing NS3 baseline polymorphism was NS3-Q80K. This substitution impacted initial virologic response rates in a single-ascending-dose study, but its effects after multiple doses were more ambiguous. Interestingly, for patient NS3 protease sequences containing Q80 and those containing K80, susceptibilities to asunaprevir were comparable when tested in an enzyme assay. No resistance-associated variants emerged in these clinical studies that significantly impacted susceptibility to asunaprevir. Importantly, asunaprevir-resistant replicons remained susceptible to an NS5A replication complex inhibitor, consistent with a role for asunaprevir in combination therapies.

The Discovery of Asunaprevir (BMS-650032), An Orally Efficacious NS3 Protease Inhibitor for the Treatment of Hepatitis C Virus Infection

The discovery of asunaprevir (BMS-650032, 24) is described. This tripeptidic acylsulfonamide inhibitor of the NS3/4A enzyme is currently in phase III clinical trials for the treatment of hepatitis C virus infection. The discovery of 24 was enabled by employing an isolated rabbit heart model to screen for the cardiovascular (CV) liabilities (changes to HR and SNRT) that were responsible for the discontinuation of an earlier lead from this chemical series, BMS-605339 (1), from clinical trials. The structure-activity relationships (SARs) developed with respect to CV effects established that small structural changes to the P2* subsite of the molecule had a significant impact on the CV profile of a given compound. The antiviral activity, preclincial PK profile, and toxicology studies in rat and dog supported clinical development of BMS-650032 (24).

Evidence for Controlled Incorporation of Herpes Simplex Virus Type 1 UL26 Protease into Capsids

ABSTRACT Herpes simplex virus type 1 (HSV-1) capsids are initially assembled with an internal protein scaffold. The scaffold proteins, encoded by overlapping in-frame UL26 and UL26.5 transcripts, are essential for formation and efficient maturation of capsids. UL26 encodes an N-terminal protease domain, and its C-terminal oligomerization and capsid protein-binding domains are identical to those of UL26.5. The UL26 protease cleaves itself, releasing minor scaffold proteins VP24 and VP21, and the more abundant UL26.5 protein, releasing the major scaffold protein VP22a. Unlike VP21 and VP22a, which are removed from capsids upon DNA packaging, we demonstrate that VP24 (containing the protease domain) is quantitatively retained. To investigate factors controlling UL26 capsid incorporation and retention, we used a mutant virus that fails to express UL26.5 (ΔICP35 virus). Purified ΔICP35 B capsids showed altered sucrose gradient sedimentation and lacked the dense scaffold core seen in micrographs of wild-type B capsids but contained capsid shell proteins in wild-type amounts. Despite C-terminal sequence identity between UL26 and UL26.5, ΔICP35 capsids lacking UL26.5 products did not contain compensatory high levels of UL26 proteins. Therefore, HSV capsids can be maintained and/or assembled on a minimal scaffold containing only wild-type levels of UL26 proteins. In contrast to UL26.5, increased expression of UL26 did not compensate for the ΔICP35growth defect. While indirect, these findings are consistent with the view that UL26 products are restricted from occupying abundant UL26.5 binding sites within the capsid and that this restriction is not controlled by the level of UL26 protein expression. Additionally, ΔICP35 capsids contained an altered complement of DNA cleavage and packaging proteins, suggesting a previously unrecognized role for the scaffold in this process.

Combinations of Lambda Interferon with Direct-Acting Antiviral Agents Are Highly Efficient in Suppressing Hepatitis C Virus Replication

ABSTRACT The clinical efficacy of a pegylated form of human lambda 1 interferon (IFN-λ1; also referred to herein as lambda) has been demonstrated in patients chronically infected with hepatitis C virus (HCV) representing genotypes 1 through 4. In these proof-of-concept studies, lambda showed an improved safety profile compared to the pegylated form of alfa interferon (referred to herein as alfa). In the study described in this report, an assessment of the in vitro antiviral activity of type III IFNs toward different HCV replicons revealed that the unpegylated recombinant form of IFN-λ1 (rIFN-λ1) exerted the most robust effect, while rIFN-λ3 exhibited greater activity than rIFN-λ2. More importantly, cross-resistance to rIFN-λ1 was not observed in replicon cell lines known to have reduced susceptibility to investigational direct-acting antiviral (DAA) agents targeting the essential HCV nonstructural protein NS3, NS5A, or NS5B. When combined with either rIFN-α, the NS3 protease inhibitor (NS3 PI) asunaprevir (ASV), the NS5A replication complex inhibitor (NS5A RCI) daclatasvir (DCV), or the NS5B polymerase site I inhibitor (NS5B I) BMS-791325, rIFN-λ1 displayed a mixture of additive and synergistic effects. In three-drug combination studies, inclusion of lambda with ASV and DCV also yielded additive to synergistic effects. In line with these observations, it was demonstrated that a regimen that used a combination of rIFN-λ1 with one or two DAAs was superior to an IFN-free regimen in clearing HCV RNA in genotype 1a cell lines representing wild-type and NS3 protease inhibitor-resistant sequences. Overall, these data support further clinical development of lambda as part of alternative combination treatments with DAAs for patients chronically infected with HCV.

Single- and Multiple-Ascending-Dose Studies of the NS3 Protease Inhibitor Asunaprevir in Subjects with or without Chronic Hepatitis C

ABSTRACT Hepatitis C virus (HCV) protease inhibitors combined with pegylated alfa interferon-ribavirin have demonstrated improved efficacy compared with pegylated alfa interferon-ribavirin alone for the treatment of chronic hepatitis C. Asunaprevir (BMS-650032), a novel HCV NS3 protease inhibitor in clinical development, was evaluated for safety, antiviral activity, and resistance in four double-blind, placebo-controlled, sequential-panel, single- and multiple-ascending-dose (SAD and MAD) studies in healthy subjects or subjects with chronic HCV genotype 1 infection. In SAD studies, subjects (healthy or with chronic HCV infection) were randomized to receive asunaprevir in dose groups of 10 to 1,200 mg or a placebo. In MAD studies, healthy subjects were randomized to receive asunaprevir in dose groups of 10 to 600 mg twice daily or a placebo for 14 days; subjects with HCV infection received asunaprevir in dose groups of 200 to 600 mg twice daily, or a placebo, for 3 days. Across all four studies, headache and diarrhea were the most frequent adverse events in asunaprevir recipients. Asunaprevir at doses of 200 to 600 mg resulted in rapid HCV RNA decreases from the baseline; maximal mean changes in HCV RNA over time were 2.7 and 3.5 log10 IU/ml in the SAD and MAD studies, respectively. No enrichment of signature asunaprevir-resistant viral variants was detected. In conclusion, the novel NS3 protease inhibitor asunaprevir, when administered at single or multiple doses of 200 to 600 mg twice daily, is generally well tolerated, achieving rapid and substantial decreases in HCV RNA levels in subjects chronically infected with genotype 1 HCV.

Development of a chimeric replicon system for phenotypic analysis of NS3 protease sequences from HCV clinical isolates.

BACKGROUND To support clinical development of HCV non-structural protein (NS) 3 protease inhibitors (PIs), phenotypic monitoring of patient isolates is a prerequisite for understanding the emergence of resistance. HCV isolates typically fail to replicate in cell culture, necessitating the use of alternative phenotyping methods. METHODS An NS3 protease chimeric replicon system was developed to monitor the phenotype of clinical isolates. The transfer of NS3 protease domain sequences from HCV-infected patients to the background of genotype (Gt) 1a-H77c, 1b-Con1 and 2a-JFH-1 lab strain replicons adapted to high-level cell culture replication was investigated. RESULTS NS3 protease sequences derived from HCV Gt 1a or Gt 1b infected patients were transferred into Gt 1a and 1b replicons, respectively. Replication was detected for 20% of Gt 1a and 75% of Gt 1b sequences. Incorporation of known cell culture adaptive change NS3-E176G improved replication of Gt 1b but not of Gt 1a sequences. Transfer of Gt 1a clinical sequences into the Gt 1b background enhanced replication and allowed phenotypic analysis of all sequences. A correlation was observed between clinical isolate sequence polymorphisms and reduced susceptibility to NS3 PI. In mixed populations containing known NS3 PI resistance changes NS3-R155K or D168E/V, sensitivity of resistance detection was ≥ 10%. CONCLUSIONS An HCV replicon capable of supporting phenotypic characterization of patient-derived HCV NS3 protease sequences was developed. Pre-existence of amino acid changes associated with NS3 PI resistance highlights the need for combination therapies in the treatment of HCV.

Characterization of monoclonal antibodies recognizing amino- and carboxy-terminal epitopes of the herpes simplex virus UL42 protein

A panel of monoclonal antibodies (MAbs) directed against the herpes simplex virus type 1 (HSV-1) DNA polymerase (Pol) accessory protein, UL42, was developed and characterized. Thirteen different MAbs were isolated which exhibited varied affinities for the protein. All MAbs reacted with UL42 in ELISA, Western blot and immunoprecipitation analyses. Competitive ELISA was used to show that 6 different epitopes within UL42 were recognized by the MAbs. Immunoprecipitation of amino- and carboxy-terminal truncations of UL42 mapped the epitopes to regions containing amino acids 1-10, 10-108, 338-402, 402-460, and 460-477. All but one of these epitopes were outside the minimal active portion of the protein previously mapped to amino acids 20-315. None of these MAbs, alone or in combination, specifically neutralized the ability of UL42 to stimulate Pol activity in vitro. These results are consistent with structure-function studies that showed that N- and C-terminal regions of the UL42 protein, those recognized by the MAbs, are not involved in UL42 function in vitro.