Preclinical characterization of a non-peptidomimetic HIV protease inhibitor with improved metabolic stability.

Protease inhibitors (PIs) remain an important component of antiretroviral therapy for the treatment of HIV-1 infection due to their high genetic barrier to resistance development. Nevertheless, the two most commonly prescribed HIV PIs, atazanavir and darunavir, still require co-administration with a pharmacokinetic boosting agent to maintain sufficient drug plasma levels which can lead to undesirable drug-drug interactions. Herein, we describe GS-9770, a novel investigational non-peptidomimetic HIV PI with unboosted once-daily oral dosing potential due to improvements in its metabolic stability and its pharmacokinetic properties in preclinical animal species. This compound demonstrates potent inhibitory activity and high on-target selectivity for recombinant HIV-1 protease versus other aspartic proteases tested. In cell culture, GS-9770 inhibits Gag polyprotein cleavage and shows nanomolar anti-HIV-1 potency in primary human cells permissive to HIV-1 infection and against a broad range of HIV subtypes. GS-9770 demonstrates an improved resistance profile against a panel of patient-derived HIV-1 isolates with resistance to atazanavir and darunavir. In resistance selection experiments, GS-9770 prevented the emergence of breakthrough HIV-1 variants at all fixed drug concentrations tested and required multiple protease substitutions to enable outgrowth of virus exposed to escalating concentrations of GS-9770. This compound also remained fully active against viruses resistant to drugs from other antiviral classes and showed no in vitro antagonism when combined pairwise with drugs from other antiretroviral classes. Collectively, these preclinical data identify GS-9770 as a potent, non-peptidomimetic once-daily oral HIV PI with potential to overcome the persistent requirement for pharmacological boosting with this class of antiretroviral agents.

Expanded profiling of Remdesivir as a broad-spectrum antiviral and low potential for interaction with other medications in vitro.

Remdesivir (GS-5734; VEKLURY) is a single diastereomer monophosphoramidate prodrug of an adenosine analog (GS-441524). Remdesivir is taken up by target cells and metabolized in multiple steps to form the active nucleoside triphosphate (GS-443902), which acts as a potent inhibitor of viral RNA-dependent RNA polymerases. Remdesivir and GS-441524 have antiviral activity against multiple RNA viruses. Here, we expand the evaluation of remdesivir's antiviral activity to members of the families Flaviviridae, Picornaviridae, Filoviridae, Orthomyxoviridae, and Hepadnaviridae. Using cell-based assays, we show that remdesivir can inhibit infection of flaviviruses (such as dengue 1-4, West Nile, yellow fever, Zika viruses), picornaviruses (such as enterovirus and rhinovirus), and filoviruses (such as various Ebola, Marburg, and Sudan virus isolates, including novel geographic isolates), but is ineffective or is significantly less effective against orthomyxoviruses (influenza A and B viruses), or hepadnaviruses B, D, and E. In addition, remdesivir shows no antagonistic effect when combined with favipiravir, another broadly acting antiviral nucleoside analog, and has minimal interaction with a panel of concomitant medications. Our data further support remdesivir as a broad-spectrum antiviral agent that has the potential to address multiple unmet medical needs, including those related to antiviral pandemic preparedness.

Discovery of velpatasvir (GS-5816): A potent pan-genotypic HCV NS5A inhibitor in the single-tablet regimens Vosevi® and Epclusa®.

Direct-acting antiviral inhibitors have revolutionized the treatment of hepatitis C virus (HCV) infected patients. Herein is described the discovery of velpatasvir (VEL, GS-5816), a potent pan-genotypic HCV NS5A inhibitor that is a component of the only approved pan-genotypic single-tablet regimens (STRs) for the cure of HCV infection. VEL combined with sofosbuvir (SOF) is Epclusa®, an STR with 98% cure-rates for genotype 1-6 HCV infected patients. Addition of the pan-genotypic HCV NS3/4A protease inhibitor voxilaprevir to SOF/VEL is the STR Vosevi®, which affords 97% cure-rates for genotype 1-6 HCV patients who have previously failed another treatment regimen.

In Vitro Antiviral Activity and Resistance Profile Characterization of the Hepatitis C Virus NS5A Inhibitor Ledipasvir.

Ledipasvir (LDV; GS-5885), a component of Harvoni (a fixed-dose combination of LDV with sofosbuvir [SOF]), is approved to treat chronic hepatitis C virus (HCV) infection. Here, we report key preclinical antiviral properties of LDV, including in vitro potency, in vitro resistance profile, and activity in combination with other anti-HCV agents. LDV has picomolar antiviral activity against genotype 1a and genotype 1b replicons with 50% effective concentration (EC50) values of 0.031 nM and 0.004 nM, respectively. LDV is also active against HCV genotypes 4a, 4d, 5a, and 6a with EC50 values of 0.11 to 1.1 nM. LDV has relatively less in vitro antiviral activity against genotypes 2a, 2b, 3a, and 6e, with EC50 values of 16 to 530 nM. In vitro resistance selection with LDV identified the single Y93H and Q30E resistance-associated variants (RAVs) in the NS5A gene; these RAVs were also observed in patients after a 3-day monotherapy treatment. In vitro antiviral combination studies indicate that LDV has additive to moderately synergistic antiviral activity when combined with other classes of HCV direct-acting antiviral (DAA) agents, including NS3/4A protease inhibitors and the nucleotide NS5B polymerase inhibitor SOF. Furthermore, LDV is active against known NS3 protease and NS5B polymerase inhibitor RAVs with EC50 values equivalent to those for the wild type.

Robust and persistent replication of the genotype 6a hepatitis C virus replicon in cell culture.

Genotype 6 (GT6) hepatitis C virus (HCV) is prevalent in Southeast Asia and southern China, where it can constitute up to 50% of HCV infections. Despite this, no direct-acting antivirals are approved to treat GT6 HCV infection, and no cell culture systems have been described. In this study, we aimed to develop a GT6 HCV subgenomic replicon to facilitate the identification and development of new HCV therapies with pan-genotype activity. A subgenomic replicon cDNA encoding a GT6a consensus sequence plus an NS5A amino acid substitution (S232I) was synthesized. Electroporation of RNA encoding the GT6a replicon into Huh-7-derived cells consistently yielded 20 to 100 stable replicon colonies. Genotypic analyses of individual replicon colonies revealed new adaptive mutations across multiple viral nonstructural proteins. The E30V and K272R mutations in NS3 and the K34R mutation in NS4A were observed most frequently and were confirmed to enhance GT6a replicon replication in the presence of the NS5A amino acid substitution S232I. These new adaptive mutations allowed establishment of robust luciferase-encoding GT6a replicons for reproducible quantification of HCV replication, and the luciferase-encoding replicons enabled efficient determinations of antiviral activity for HCV inhibitors in a 384-well assay format. While nucleoside/nucleotide NS5B inhibitors and cyclophilin A inhibitors had similar antiviral activities against both GT6a and GT1b replicons, some nonnucleoside NS5B inhibitors, NS3 protease inhibitors, and NS5A inhibitors had less antiviral activity against GT6a replicons. In conjunction with other genotype replicons, this robust GT6a replicon system will aid in the development of pan-genotypic HCV regimens.

In vitro efficacy of approved and experimental antivirals against novel genotype 3 hepatitis C virus subgenomic replicons.

Infection with genotype 3 hepatitis C virus (HCV) is common throughout the world, however no direct-acting antiviral (DAA) has been approved to treat this genotype. We therefore attempted to develop novel genotype 3 replicons to facilitate the discovery and development of new HCV therapies. A novel Huh-7-derived cell line 1C but not Lunet cells enabled the selection of a few stable colonies of a genotype 3a subgenomic replicon (strain S52). Genotypic analysis revealed a mutation of P89L in the viral NS3 protease domain, which was confirmed to enhance genotype 3a RNA replication and enable the establishment of highly replicating luciferase-encoding replicons. Secondary adaptive mutations that further enhanced RNA replication were identified in the viral NS3 and NS4A proteins. In addition, cell lines that were cured of genotype 3a replicons demonstrated higher permissiveness specifically to genotype 3a HCV replication. These novel replicons and cell lines were then used to study the activity of approved and experimental HCV inhibitors. NS3 protease and non-nucleoside NS5B polymerase inhibitors often demonstrated substantially less antiviral activity against genotype 3a compared to genotype 1b. In contrast, nucleoside analog NS5B inhibitors and host-targeting HCV inhibitors showed comparable antiviral activity between genotypes 3a and 1b. Overall, the establishment of this novel genotype 3a replicon system, in conjunction with those derived from other genotypes, will aid the development of treatment regimens for all genotypes of HCV.

High-content, high-throughput analysis of cell cycle perturbations induced by the HSP90 inhibitor XL888.

BACKGROUND: Many proteins that are dysregulated or mutated in cancer cells rely on the molecular chaperone HSP90 for their proper folding and activity, which has led to considerable interest in HSP90 as a cancer drug target. The diverse array of HSP90 client proteins encompasses oncogenic drivers, cell cycle components, and a variety of regulatory factors, so inhibition of HSP90 perturbs multiple cellular processes, including mitogenic signaling and cell cycle control. Although many reports have investigated HSP90 inhibition in the context of the cell cycle, no large-scale studies have examined potential correlations between cell genotype and the cell cycle phenotypes of HSP90 inhibition. METHODOLOGY/PRINCIPAL FINDINGS: To address this question, we developed a novel high-content, high-throughput cell cycle assay and profiled the effects of two distinct small molecule HSP90 inhibitors (XL888 and 17-AAG [17-allylamino-17-demethoxygeldanamycin]) in a large, genetically diverse panel of cancer cell lines. The cell cycle phenotypes of both inhibitors were strikingly similar and fell into three classes: accumulation in M-phase, G2-phase, or G1-phase. Accumulation in M-phase was the most prominent phenotype and notably, was also correlated with TP53 mutant status. We additionally observed unexpected complexity in the response of the cell cycle-associated client PLK1 to HSP90 inhibition, and we suggest that inhibitor-induced PLK1 depletion may contribute to the striking metaphase arrest phenotype seen in many of the M-arrested cell lines. CONCLUSIONS/SIGNIFICANCE: Our analysis of the cell cycle phenotypes induced by HSP90 inhibition in 25 cancer cell lines revealed that the phenotypic response was highly dependent on cellular genotype as well as on the concentration of HSP90 inhibitor and the time of treatment. M-phase arrest correlated with the presence of TP53 mutations, while G2 or G1 arrest was more commonly seen in cells bearing wt TP53. We draw upon previous literature to suggest an integrated model that accounts for these varying observations.

Itraconazole, a commonly used antifungal that inhibits Hedgehog pathway activity and cancer growth.

In a screen of drugs previously tested in humans we identified itraconazole, a systemic antifungal, as a potent antagonist of the Hedgehog (Hh) signaling pathway that acts by a mechanism distinct from its inhibitory effect on fungal sterol biosynthesis. Systemically administered itraconazole, like other Hh pathway antagonists, can suppress Hh pathway activity and the growth of medulloblastoma in a mouse allograft model and does so at serum levels comparable to those in patients undergoing antifungal therapy. Mechanistically, itraconazole appears to act on the essential Hh pathway component Smoothened (SMO) by a mechanism distinct from that of cyclopamine and other known SMO antagonists, and prevents the ciliary accumulation of SMO normally caused by Hh stimulation.

Hedgehog signal transduction via Smoothened association with a cytoplasmic complex scaffolded by the atypical kinesin, Costal-2.

The seven-transmembrane protein Smoothened (Smo) transduces extracellular activation of the Hedgehog (Hh) pathway by an unknown mechanism to increase transcriptional activity of the latent cytoplasmic transcription factor Ci (Cubitus interruptus). Here, we present evidence that Smo associates directly with a Ci-containing complex that is scaffolded and stabilized by the atypical kinesin, Costal-2 (Cos2). This complex constitutively suppresses pathway activity, but Hh signaling reverses its regulatory effect to promote Ci-mediated transcription. In response to Hh activation of Smo, Cos2 mediates accumulation and phosphorylation of Smo at the membrane as well as phosphorylation of the cytoplasmic components Fu and Su(fu). Positive response of Cos2 to Hh stimulation requires a portion of the Smo cytoplasmic tail and the Cos2 cargo domain, which interacts directly with Smo.

A defective response to Hedgehog signaling in disorders of cholesterol biosynthesis.

Smith-Lemli-Opitz syndrome (SLOS), desmosterolosis and lathosterolosis are human syndromes caused by defects in the final stages of cholesterol biosynthesis. Many of the developmental malformations in these syndromes occur in tissues and structures whose embryonic patterning depends on signaling by the Hedgehog (Hh) family of secreted proteins. Here we report that response to the Hh signal is compromised in mutant cells from mouse models of SLOS and lathosterolosis and in normal cells pharmacologically depleted of sterols. We show that decreasing levels of cellular sterols correlate with diminishing responsiveness to the Hh signal. This diminished response occurs at sterol levels sufficient for normal autoprocessing of Hh protein, which requires cholesterol as cofactor and covalent adduct. We further find that sterol depletion affects the activity of Smoothened (Smo), an essential component of the Hh signal transduction apparatus.

Hedgehog-mediated patterning of the mammalian embryo requires transporter-like function of dispatched.

The dispatched (disp) gene is required for long-range Hedgehog (Hh) signaling in Drosophila. Here, we demonstrate that one of two murine homologs, mDispA, can rescue disp function in Drosophila and is essential for all Hh patterning activities examined in the early mouse embryo. Embryonic fibroblasts lacking mDispA respond normally to exogenously provided Sonic hedgehog (Shh) signal, but are impaired in stimulation of other responding cells when expressing Shh. We have developed a biochemical assay that directly measures the activity of Disp proteins in release of soluble Hh proteins. This activity is disrupted by alteration of residues functionally conserved in Patched and in a related family of bacterial transmembrane transporters, thus suggesting similar mechanisms of action for all of these proteins.