Novel Small-Molecule Inhibitors of Hepatitis C Virus Entry Block Viral Spread and Promote Viral Clearance in Cell Culture

Combinations of direct-acting anti-virals offer the potential to improve the efficacy, tolerability and duration of the current treatment regimen for hepatitis C virus (HCV) infection. Viral entry represents a distinct therapeutic target that has been validated clinically for a number of pathogenic viruses. To discover novel inhibitors of HCV entry, we conducted a high throughput screen of a proprietary small-molecule compound library using HCV pseudoviral particle (HCVpp) technology. We independently discovered and optimized a series of 1,3,5-triazine compounds that are potent, selective and non-cytotoxic inhibitors of HCV entry. Representative compounds fully suppress both cell-free virus and cell-to-cell spread of HCV in vitro. We demonstrate, for the first time, that long term treatment of an HCV cell culture with a potent entry inhibitor promotes sustained viral clearance in vitro. We have confirmed that a single amino acid variant, V719G, in the transmembrane domain of E2 is sufficient to confer resistance to multiple compounds from the triazine series. Resistance studies were extended by evaluating both the fusogenic properties and growth kinetics of drug-induced and natural amino acid variants in the HCVpp and HCV cell culture assays. Our results indicate that amino acid variations at position 719 incur a significant fitness penalty. Introduction of I719 into a genotype 1b envelope sequence did not affect HCV entry; however, the overall level of HCV replication was reduced compared to the parental genotype 1b/2a HCV strain. Consistent with these findings, I719 represents a significant fraction of the naturally occurring genotype 1b sequences. Importantly, I719, the most relevant natural polymorphism, did not significantly alter the susceptibility of HCV to the triazine compounds. The preclinical properties of these triazine compounds support further investigation of entry inhibitors as a potential novel therapy for HCV infection

Potent Antiviral Synergy between Monoclonal Antibody and Small-Molecule CCR5 Inhibitors of Human Immunodeficiency Virus Type 1

The chemokine receptor CCR5 provides a portal of entry for human immunodeficiency virus type 1 (HIV-1) into susceptible CD4(+) cells. Both monoclonal antibody (MAb) and small-molecule CCR5 inhibitors have entered human clinical testing, but little is known regarding their potential interactions. We evaluated the interactions between CCR5 MAbs, small-molecule CCR5 antagonists, and inhibitors of HIV-1 gp120, gp41, and reverse transcriptase in vitro. Inhibition data were analyzed for cooperative effects using the combination index (CI) method and stringent statistical criteria. Potent, statistically significant antiviral synergy was observed between the CCR5 MAb PRO 140 and the small-molecule CCR5 antagonists maraviroc (UK-427,857), vicriviroc (SCH-D), and TAK-779. High-level synergy was observed consistently across various assay systems, HIV-1 envelopes, CCR5 target cells, and inhibition levels. CI values ranged from 0.18 to 0.64 and translated into in vitro dose reductions of up to 14-fold. Competition binding studies revealed nonreciprocal patterns of CCR5 binding by MAb and small-molecule CCR5 inhibitors, suggesting that synergy occurs at the level of receptor binding. In addition, both PRO 140 and maraviroc synergized with the chemokine RANTES, a natural ligand for CCR5; however, additive effects were observed for both small-molecule CCR5 antagonists and PRO 140 in combination with other classes of HIV-1 inhibitors. The findings provide a rationale for clinical exploration of MAb and small-molecule CCR5 inhibitors in novel dual-CCR5 regimens for HIV-1 therapy

Feasibility of Conducting a Trial Assessing Benefits and Risks of Planned Caesarean Section Versus Planned Vaginal Birth: A Cross-Sectional Study

Introduction: Though interest is growing for trials comparing planned delivery mode (vaginal delivery [VD]; cesarean section [CS]) in low-risk nulliparous women, appropriate study design is unclear. Our objective was to assess feasibility of three designs (preference trial [PCT], randomized controlled trial [RCT], partially randomized patient preference trial [PRPPT]) for a trial comparing planned delivery mode in low-risk women. Methods: A cross-sectional survey of low-risk, nulliparous pregnant women (N = 416) and healthcare providers (N = 168) providing prenatal care and/or labor/delivery services was conducted in Argentina (2 public, 2 private hospitals). Proportion of pregnant women and providers willing to participate in each design and reasons for not participating were determined. Results: Few women ( 85%). Believing randomization unacceptable (RCT, PRPPT) and desiring choice of delivery mode (RCT) were women’s reasons for not participating. For providers, commonly cited reasons for not participating included unacceptability of performing CS without medical indication, difficulty obtaining informed consent, discomfort enrolling patients (all designs), and violating women’s right to choose (RCT). Conclusions for Practice: Important limitations were found for each trial design evaluated. The necessity of stronger evidence regarding delivery mode in low-risk women suggests consideration of additional designs, such as a rigorously designed cohort study or an RCT within an obstetric population with equivocal CS indications.Fil: Amyx, Melissa Michele. Instituto de Efectividad Clínica y Sanitaria; Argentina. University of Tulane; Estados UnidosFil: Althabe, Fernando. University of Tulane; Estados Unidos. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Centro de Investigaciones en Epidemiología y Salud Pública. Instituto de Efectividad Clínica y Sanitaria. Centro de Investigaciones en Epidemiología y Salud Pública; ArgentinaFil: Rivo, Julie. Instituto de Efectividad Clínica y Sanitaria; ArgentinaFil: Pingray, María Verónica. University of Duke; Estados UnidosFil: Minckas, Nicole. Instituto de Efectividad Clínica y Sanitaria; ArgentinaFil: Belizán, María. Instituto de Efectividad Clínica y Sanitaria; ArgentinaFil: Gibbons, Luz. Instituto de Efectividad Clínica y Sanitaria; ArgentinaFil: Murga, Gerardo T.. Instituto de Maternidad Y Ginecología Nuestra Señora de Las Mercedes; ArgentinaFil: Fiorillo, Angel Eduardo. Centro de Educación Médica e Investigaciones Clínicas "Norberto Quirno"; ArgentinaFil: Malamud, Julio D.. Centro de Educación Medica E Invest.clinicas; ArgentinaFil: Casale, Roberto A.. Sanatorio de la Mujer.; ArgentinaFil: Cormick, Gabriela. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Centro de Investigaciones en Epidemiología y Salud Pública. Instituto de Efectividad Clínica y Sanitaria. Centro de Investigaciones en Epidemiología y Salud Pública; Argentina. Hospital Nacional Profesor Alejandro Posadas; ArgentinaFil: Belizan, Jose. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Centro de Investigaciones en Epidemiología y Salud Pública. Instituto de Efectividad Clínica y Sanitaria. Centro de Investigaciones en Epidemiología y Salud Pública; Argentin

Selection of viral variants with reduced susceptibility to triazines.

<p>Cell cultures that were ∼30% infected with cell culture-derived GT 1a/2a HCV were established and treated with 1 µM PRO0371155 or DMSO in the passage control. Cultures were split twice weekly to maintain sub-confluent levels. The percentage of HCV+ cells as a function of time was determined at 3–4 day intervals. Cells were subjected to staining with anti-NS3 antibodies and analysis by flow cytometry as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035351#s2" target="_blank">Materials and Methods</a>.</p

Effect of drug-induced and natural amino acid variants at position 719 on the growth kinetics of HCV genotype 1/2a in cell culture.

<p>The drug resistant variant, V719G and three natural amino acid polymorphisms, I719, L719, A719, were introduced into genotype 1a and 1b genetic backgrounds using the QuikChange II XL site-directed mutagenesis kit (Stratagene). Full-length HCV RNAs bearing various polymorphisms at position 719 were prepared and electroporated into naïve target cells. Infected cell cultures were maintained for up to 2.5 months. Every 3–4 days, viral supernatants were harvested and subsequently used to infect naïve target cells. After 72 hrs, <i>Renilla</i> luciferase activity was measured and viral titer/infectivity was expressed in R.L.U. (relative light units). Changes in viral titers were plotted on a logarithmic scale as a function of time. (A) A comparison of the growth kinetics of GT 1a/2a HCV (diamonds) with the drug-induced variant, GT 1a/2a-V719G (squares) (B) A comparison of the growth kinetics of GT 1a/2a HCV (diamonds) with a potential polymorphism, GT 1a/2a-V719A (triangles). (C) A comparison of the growth kinetics of GT 1b/2a HCV (diamonds) with the drug-induced polymorphism, GT 1b/2a-V719G (squares). (D) A comparison of the growth kinetics of GT 1b/2a HCV (diamonds) with a natural polymorphism, GT 1b/2a-V719I (circles) and two potential polymorphisms GT 1b/2a-V719L (squares) and GT 1b/2a-V719A (triangles).</p

Spectrum of activity of representative triazines against envelopes isolated from HCV⁺ patient sera.

<p>HCV RNA was isolated and purified from sera obtained from individuals infected with different strains of HCV, representing genotypes 1a, 1b, 2a, 2b. Envelopes representing genotypes 3, 4, and 6 were described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035351#s2" target="_blank">Materials and Methods</a> and published elsewhere <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035351#pone.0035351-Lavillette1" target="_blank">[33]</a>. Genotype specific primers were used to amplify sequences encoding the E1/E2 glycoprotein from each of the strains by RT-PCR. Envelope sequences were ligated into expression constructs as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035351#s2" target="_blank">Materials and Methods</a>. Unique HCVpp, representing different genotypes, were produced in 293T cells and validated with the anti-CD81 mAb, JS-81. Individual HCVpp were normalized based on total infectivity and added to Hep3B cells in the presence of various concentrations of PRO0371155 (Panel A) or PRO0502797 (Panel B) in 384-well microplates. Luciferase activity was measured 72 hrs post-infection using Bright Glo reagent (Promega). The potency of PRO0371155 and PRO0502797 against each fusogenic envelope was expressed as a mean EC₅₀. Each data point represents the average of multiple dose response experiments (n>3). The genotype specific panel consisted of the following strains: genotype 1a (n = 22), genotype 1b (n = 19), genotype 2a (n = 2), genotype 2b (n = 1), genotype 3, (n = 1), genotype 4, n = 1), genotype 6 (n = 1). Actual number of strains tested for each compound is summarized in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035351#pone-0035351-t001" target="_blank">Table 1</a>. The box extends from the 25^th to the 75^th percentile, with a line at the median EC₅₀. The whiskers mark the full range from the lowest to the highest EC₅₀.</p

PRO0371155 inhibits HCV infection by both cell-free and cell-cell modes of transmission.

<p>The anti-viral activity of PRO0371155 was evaluated in an assay that measures both cell-free and cell-cell transmission of virus <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035351#pone.0035351-Brimacombe1" target="_blank">[37]</a>. To evaluate transmission of GT 1a/2a HCV in culture, infected cells (90% HCV+) were stained with CMFDA green, according to the manufacturer's instructions (Invitrogen), and mixed at a ratio of 5∶1 with non-stained naïve cells. Mixed infected and naïve cells (7.5×10⁵ total) were seeded in T-75 flasks and subjected to treatment with 1 µM of PRO0371155 or DMSO vehicle for 72 h at 37°C. As a positive control for cell-cell transmission, PA-25, a mouse monoclonal antibody raised against sE2 was also evaluated in the viral spread assay at a single concentration of 10 µg/mL. This concentration is approximately 20-fold above its EC₅₀ concentration and is sufficient to completely inhibit HCV entry in a single-cycle infection assay. Cells were detached, fixed and permeabilized with BD Cytofix/cytoperm (BD Biosciences). Infected cells were stained with anti-NS3 antibody 1847 (Virostat) or mIgG1 (BD Biosciences) and counter-stained with 1 µg/mL AlexFluor 647 (Invitrogen). Single- and dual-labeled cells were quantified by flow cytometry.</p

Long term treatment of HCV infected cell cultures with a triazine compound promotes viral clearance.

<p>PRO0371155 was evaluated for its inhibitory activity against GT 1a/2a HCV over multiple weeks in culture. (A) Cell cultures were ∼90% infected with cell culture-derived GT 1a/2a HCV. Cultures were treated with 1 µM PRO0371155 or DMSO in the passage control. At 3–4 day intervals, cells were subjected to staining with anti-NS3 antibodies and analysis by flow cytometry as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035351#s2" target="_blank">Materials and Methods</a>. The percentage of HCV+ cells as a function of time was plotted for each experiment. (B) Results obtained in the protein expression analysis were confirmed and extended by qRT-PCR analysis of HCV RNA isolated from HCV infected cultures treated with PRO0371155 or DMSO control at various time points. HCV RNA was normalized to GAPDH mRNA and expressed in copies/cell. The limit of quantification of the qRT-PCR assay was found to be ∼0.5 copies/cell. (C) Cell cultures were ∼20% infected with cell culture-derived GT 1a/2a HCV. Cultures were treated with 1 µM PRO0371155, 10 IU/mL IFN-alpha (Sigma) or DMSO in the passage control. At 3–4 day intervals, cells were subjected to staining with anti-NS3 antibodies and analysis by flow cytometry as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035351#s2" target="_blank">Materials and Methods</a>. The percentage of HCV+ cells as a function of time was plotted for each experiment. (D) Data shown in panel C were re-plotted on a different scale to accentuate the differences between IFN-alpha and PRO0371155 treated HCV+ cultures.</p

Various triazine compounds exhibit broad activity against HCV genotype 1a and 1b strains.

<p>Various triazine compounds exhibit broad activity against HCV genotype 1a and 1b strains.</p