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Nonstructural protein 5A (NS5A) represents a novel target for the treatment of hepatitis C virus (HCV). Daclatasvir, recently reported by Bristol-Myers-Squibb, is a potent NS5A inhibitor currently under investigation in phase 3 clinical trials. While the performance of daclatasvir has been impressive, the emergence of resistance could prove problematic and as such, improved analogues are being sought. By varying the biphenyl-imidazole unit of daclatasvir, novel inhibitors of HCV NS5A were identified with an improved resistance profile against mutant strains of the virus while retaining the picomolar potency of daclatasvir. One compound in particular, methyl ((S)-1-((S)-2-(4-(4-(6-(2-((S)-1-((methoxycarbonyl)-L-valyl)pyrrolidin-2-yl)-1H-imidazol-5-yl)quinoxalin-2-yl)phenyl)-1H-imidazol-2-yl)pyrrolidin-1-yl)-3-methyl-1-oxobutan-2-yl)carbamate (17), exhibited very promising activity and showed good absorption and a long predicted human pharmacokinetic half-life. This compound represents a promising lead that warrants further evaluation.

A high throughput screen of the Pfizer compound collection was carried out using a hepatitis C virus (HCV) genotype 1b subgenomic replicon cell line. Those confirmed hits that demonstrated broad spectrum activity without overt cytotoxicity were further evaluated, leading to the identification of a series of pyrrolopyridines with excellent antiviral activity in a fully infectious HCV cell-based assay and pharmacokinetic properties.

The nonnucleoside reverse transcriptase inhibitors (NNRTIs) are key components of highly active antiretroviral therapy (HAART) for the treatment of human immunodeficiency virus type 1 (HIV-1). A major problem with the first approved NNRTIs was the emergence of mutations in the HIV-1 reverse transcriptase (RT), in particular K103N and Y181C, which led to resistance to the entire class. We adopted an iterative strategy to synthesize and test small molecule inhibitors from a chemical series of pyrazoles against wild-type (wt) RT and the most prevalent NNRTI-resistant mutants. The emerging candidate, lersivirine (UK-453,061), binds the RT enzyme in a novel way (resulting in a unique resistance profile), inhibits over 60% of viruses bearing key RT mutations, with 50% effective concentrations (EC50s) within 10-fold of those for wt viruses, and has excellent selectivity against a range of human targets. Altogether lersivirine is a highly potent and selective NNRTI, with excellent efficacy against NNRTI-resistant viruses.

Maraviroc is a CCR5 antagonist in clinical development as one of a new class of antiretrovirals targeting human immunodeficiency virus type 1 (HIV-1) coreceptor binding. We investigated the mechanism of HIV resistance to maraviroc by using in vitro sequential passage and site-directed mutagenesis. Serial passage through increasing maraviroc concentrations failed to select maraviroc-resistant variants from some laboratory-adapted and clinical isolates of HIV-1. However, high-level resistance to maraviroc was selected from three of six primary isolates passaged in peripheral blood lymphocytes (PBL). The SF162 strain acquired resistance to maraviroc in both treated and control cultures; all resistant variants were able to use CXCR4 as a coreceptor. In contrast, maraviroc-resistant virus derived from isolates CC1/85 and RU570 remained CCR5 tropic, as evidenced by susceptibility to the CCR5 antagonist SCH-C, resistance to the CXCR4 antagonist AMD3100, and an inability to replicate in CCR5 Δ32/Δ32 PBL. Strain-specific mutations were identified in the V3 loop of maraviroc-resistant CC1/85 and RU570. The envelope-encoding region of maraviroc-resistant CC1/85 was inserted into an NL4-3 background. This recombinant virus was completely resistant to maraviroc but retained susceptibility to aplaviroc. Reverse mutation of gp120 residues 316 and 323 in the V3 loop (numbering from HXB2) to their original sequence restored wild-type susceptibility to maraviroc, while reversion of either mutation resulted in a partially sensitive virus with reduced maximal inhibition (plateau). The plateaus are consistent with the virus having acquired the ability to utilize maraviroc-bound receptor for entry. This hypothesis was further corroborated by the observation that a high concentration of maraviroc blocks the activity of aplaviroc against maraviroc-resistant virus.

In ongoing studies towards novel hepatitis C virus (HCV) therapeutics, inhibitors of nonstructural protein 5A (NS5A) were evaluated. Specifically, starting from previously reported lead compounds, peripheral substitution patterns of a series of biaryl-linked pyrrolidine NS5A replication complex inhibitors were probed and structure-activity relationships were elucidated. Using molecular modelling and a supercritical fluid chromatographic (SFC) technique, intramolecular H-bonding and peripheral functional group topology were evaluated as key determinants of activity and membrane permeability. The novel compounds exhibited retained potency as compared with the lead compounds, and also showed promising results against a panel of resistance viruses. Together, the results of the study take us a step closer towards understanding the potency of daclatasvir, a clinical candidate upon which the compounds were based, and to designing improved analogues as second-generation antiviral agents targeting NS5A.

The current standard of care for hepatitis C virus (HCV) patients is cotreatment with human alpha interferon (IFN-α) and ribavirin. The host factor USP18 functions to regulate the interferon signaling pathway by acting as an off-switch. In order to understand whether the inhibition of USP18 represents a valid target for the enhancement of interferon treatment for chronic viral diseases, we have used a wide range of RNA interference (RNAi) reagents to suppress USP18 gene expression in Huh7 cell lines. We demonstrate that a USP18 knockdown results in IFN-α2a signaling (measured by increased IFN-stimulated response element [ISRE] reporter gene activity, 2′,5′-oligoadenylate synthetase [2-5 OAS] expression, and ISG15 induction) that is increased by ∼100-fold, whereas the antiviral (AV) potency in both the Huh7 HCV subgenomic replicon assay and the Huh7.5 HCV infectious virus assay increased by ∼3-fold. While the degree of the USP18 knockdown of USP18 elicited by the different RNAi reagents correlated with the enhancement of IFN-α2a signaling, it did not correlate with the enhancement of AV activity. The failure of increased IFN-α2a signaling to fully translate into increased AV potency was also observed for encephalomyocarditis virus (EMCV) assays using Huh7.5 cells. These data suggest that the IFN-mediated AV response in Huh7.5 cells has only a limited dependence on USP18 activity.

Maraviroc (UK-427,857) is a selective CCR5 antagonist with potent anti-human immunodeficiency virus type 1 (HIV-1) activity and favorable pharmacological properties. Maraviroc is the product of a medicinal chemistry effort initiated following identification of an imidazopyridine CCR5 ligand from a high-throughput screen of the Pfizer compound file. Maraviroc demonstrated potent antiviral activity against all CCR5-tropic HIV-1 viruses tested, including 43 primary isolates from various clades and diverse geographic origin (geometric mean 90% inhibitory concentration of 2.0 nM). Maraviroc was active against 200 clinically derived HIV-1 envelope-recombinant pseudoviruses, 100 of which were derived from viruses resistant to existing drug classes. There was little difference in the sensitivity of the 200 viruses to maraviroc, as illustrated by the biological cutoff in this assay (= geometric mean plus two standard deviations [SD] of 1.7-fold). The mechanism of action of maraviroc was established using cell-based assays, where it blocked binding of viral envelope, gp120, to CCR5 to prevent the membrane fusion events necessary for viral entry. Maraviroc did not affect CCR5 cell surface levels or associated intracellular signaling, confirming it as a functional antagonist of CCR5. Maraviroc has no detectable in vitro cytotoxicity and is highly selective for CCR5, as confirmed against a wide range of receptors and enzymes, including the hERG ion channel (50% inhibitory concentration, >10 μM), indicating potential for an excellent clinical safety profile. Studies in preclinical in vitro and in vivo models predicted maraviroc to have human pharmacokinetics consistent with once- or twice-daily dosing following oral administration. Clinical trials are ongoing to further investigate the potential of using maraviroc for the treatment of HIV-1 infection and AIDS.

HIV-1 proteinase activity is thought to occur primarily post-integration by cleaving the viral Gag and Gag-Pol polyproteins. Its role in the pre-integration stages of viral replication, however, has not been studied in detail. Here we report that a synthetic peptide analogue, UK-88,947, which is a specific inhibitor of purified HIV-1 proteinase, inhibits the processing of the viral polyproteins in cultures of HIV-1 infected cells and prevents the formation of mature, infectious virions. Analysis of DNA from HIV-1 infected cells treated with UK-88,947 showed that viral DNA synthesis was inhibited when the compound was added to cultures one hour before infection. Similar results were obtained when AZT was used. Neither HIV-1 reverse transcriptase or the replication of FIV are inhibited by UK-88,947.