Post-infection treatment with the E protein inhibitor BIT225 reduces disease severity and increases survival of K18-hACE2 transgenic mice infected with a lethal dose of SARS-CoV-2.

The Coronavirus envelope (E) protein is a small structural protein with ion channel activity that plays an important role in virus assembly, budding, immunopathogenesis and disease severity. The viroporin E is also located in Golgi and ER membranes of infected cells and is associated with inflammasome activation and immune dysregulation. Here we evaluated in vitro antiviral activity, mechanism of action and in vivo efficacy of BIT225 for the treatment of SARS-CoV-2 infection. BIT225 showed broad-spectrum direct-acting antiviral activity against SARS-CoV-2 in Calu3 and Vero cells with similar potency across 6 different virus strains. BIT225 inhibited ion channel activity of E protein but did not inhibit endogenous currents or calcium-induced ion channel activity of TMEM16A in Xenopus oocytes. BIT225 administered by oral gavage for 12 days starting 12 hours before infection completely prevented body weight loss and mortality in SARS-CoV-2 infected K18 mice (100% survival, n = 12), while all vehicle-dosed animals reached a mortality endpoint by Day 9 across two studies (n = 12). When treatment started at 24 hours after infection, body weight loss, and mortality were also prevented (100% survival, n = 5), while 4 of 5 mice maintained and increased body weight and survived when treatment started 48 hours after infection. Treatment efficacy was dependent on BIT225 dose and was associated with significant reductions in lung viral load (3.5 log10), virus titer (4000 pfu/ml) and lung and serum cytokine levels. These results validate viroporin E as a viable antiviral target and support the clinical study of BIT225 for treatment and prophylaxis of SARS-CoV-2 infection.

Preclinical Considerations for Long-acting Delivery of Tenofovir Alafenamide from Subdermal Implants for HIV Pre-exposure Prophylaxis.

PURPOSE: Long-acting formulations of the potent antiretroviral prodrug tenofovir alafenamide (TAF) hold potential as biomedical HIV prevention modalities. Here, we present a rigorous comparison of three animal models, C57BL/6 J mice, beagle dogs, and merino sheep for evaluating TAF implant pharmacokinetics (PKs). METHODS: Implants delivering TAF over a wide range of controlled release rates were tested in vitro and in mice and dogs. Our existing PK model, supported by an intravenous (IV) dosing dog study, was adapted to analyze mechanistic aspects underlying implant TAF delivery. RESULTS: TAF in vitro release in the 0.13 to 9.8 mg d-1 range with zero order kinetics were attained. Implants with equivalent fabrication parameters released TAF in mice and sheep at rates that were not statistically different, but were 3 times higher in dogs. When two implants were placed in the same subcutaneous pocket, a two-week creep to Cmax was observed in dogs for systemic drug and metabolite concentrations, but not in mice. Co-modeling IV and TAF implant PK data in dogs led to an apparent TAF bioavailability of 9.6 in the single implant groups (compared to the IV group), but only 1.5 when two implants were placed in the same subcutaneous pocket. CONCLUSIONS: Based on the current results, we recommend using mice and sheep, with macaques as a complementary species, for preclinical TAF implant evaluation with the caveat that our observations may be specific to the implant technology used here. Our report provides fundamental, translatable insights into multispecies TAF delivery via long-acting implants.

A Pan-Cyclophilin Inhibitor, CRV431, Decreases Fibrosis and Tumor Development in Chronic Liver Disease Models.

Previous studies show that cyclophilins contribute to many pathologic processes, and cyclophilin inhibitors demonstrate therapeutic activities in many experimental models. However, no drug with cyclophilin inhibition as the primary mode of action has advanced completely through clinical development to market. In this study, we present findings on the cyclophilin inhibitor, CRV431, that highlight its potential as a drug candidate for chronic liver diseases. CRV431 was found to potently inhibit all cyclophilin isoforms tested-A, B, D, and G. Inhibitory constant or IC50 values ranged from 1 to 7 nM, which was up to 13 times more potent than the parent compound, cyclosporine A (CsA), from which CRV431 was derived. Other CRV431 advantages over CsA as a nontransplant drug candidate were significantly diminished immunosuppressive activity, less drug transporter inhibition, and reduced cytotoxicity potential. Oral dosing to mice and rats led to good blood exposures and a 5- to 15-fold accumulation of CRV431 in liver compared with blood concentrations across a wide range of CRV431 dosing levels. Most importantly, CRV431 decreased liver fibrosis in a 6-week carbon tetrachloride model and in a mouse model of nonalcoholic steatohepatitis (NASH). Additionally, CRV431 administration during a late, oncogenic stage of the NASH disease model resulted in a 50% reduction in the number and size of liver tumors. These findings are consistent with CRV431 targeting fibrosis and cancer through multiple, cyclophilin-mediated mechanisms and support the development of CRV431 as a safe and effective drug candidate for liver diseases. SIGNIFICANCE STATEMENT: Cyclophilin inhibitors have demonstrated therapeutic activities in many disease models, but no drug candidates have yet advanced completely through development to market. In this study, CRV431 is shown to potently inhibit multiple cyclophilin isoforms, possess several optimized pharmacological properties, and decrease liver fibrosis and tumors in mouse models of chronic liver disease, which highlights its potential to be the first approved drug primarily targeting cyclophilin isomerases.

Systems Biology Reveals NS4B-Cyclophilin A Interaction: A New Target to Inhibit YFV Replication.

Yellow fever virus (YFV) replication is highly dependent on host cell factors. YFV NS4B is reported to be involved in viral replication and immune evasion. Here interactions between NS4B and human proteins were determined using a GST pull-down assay and analyzed using 1-DE and LC-MS/MS. We present a total of 207 proteins confirmed using Scaffold 3 Software. Cyclophilin A (CypA), a protein that has been shown to be necessary for the positive regulation of flavivirus replication, was identified as a possible NS4B partner. 59 proteins were found to be significantly increased when compared with a negative control, and CypA exhibited the greatest difference, with a 22-fold change. Fisher's exact test was significant for 58 proteins, and the p value of CypA was the most significant (0.000000019). The Ingenuity Systems software identified 16 pathways, and this analysis indicated sirolimus, an mTOR pathway inhibitor, as a potential inhibitor of CypA. Immunofluorescence and viral plaque assays showed a significant reduction in YFV replication using sirolimus and cyclosporine A (CsA) as inhibitors. Furthermore, YFV replication was strongly inhibited in cells treated with both inhibitors using reporter BHK-21-rep-YFV17D-LucNeoIres cells. Taken together, these data suggest that CypA-NS4B interaction regulates YFV replication. Finally, we present the first evidence that YFV inhibition may depend on NS4B-CypA interaction.

The role of immunophilins in viral infection.

BACKGROUND: Tremendous progress has been made in the past 20 years in understanding the roles played by immunophilins, and in particular the cyclophilins, in supporting the replication cycles of human viruses. A growing body of genetic and biochemical evidence and data from clinical trials confirm that cyclophilins are essential cofactors that contribute to establishing a permissive environment within the host cell that supports the replication of HIV-1 and HCV. Cyclophilin A regulates HIV-1 replication kinetics and infectivity, modulates sensitivity to host restriction factors, and cooperates in the transit of the pre-integration complex into the nucleus of infected cells. Cyclophilin A is an essential cofactor whose expression supports HCV-specific RNA replication in human hepatocytes. GENERAL SIGNIFICANCE: Peptidyl-prolyl isomerase inhibitors have been used in clinical trials to validate cyclophilins as antiviral targets for the treatment of HIV-1 and Chronic Hepatitis C virus infection and as molecular probes to identify the roles played by immunophilins in supporting the replication cycles of human viruses. SCOPE OF REVIEW: This review summarizes emerging research that defines the functions of immunophilins in supporting the replication cycles of HIV-1, HCV, HBV, coronaviruses, and other viral pathogens and describes new information that suggests a role for immunophilins in regulating innate immune responses against chronic viral infection. MAJOR CONCLUSIONS: The dependence on cyclophilins by evolutionarily distinct viruses for accomplishing various steps in replication such as viral entry, initiation of genomic nucleic acid replication, viral genome uncoating, nuclear import and nuclear entry, emphasizes the potential of cyclophilin inhibitors as therapeutic agents. This article is part of a Special Issue entitled Proline-directed Foldases: Cell Signaling Catalysts and Drug Targets.

C5A Protects Macaques from Vaginal Simian-Human Immunodeficiency Virus Challenge.

A safe and effective vaginal microbicide could decrease human immunodeficiency virus (HIV) transmission in women. Here, we evaluated the safety and microbicidal efficacy of a short amphipathic peptide, C5A, in a rhesus macaque model. We found that a vaginal application of C5A protects 89% of the macaques from a simian-human immunodeficiency virus (SHIV-162P3) challenge. We observed no signs of lesions or inflammation in animals vaginally treated with repeated C5A applications. With its noncellular cytotoxic activity and rare mechanism of action, C5A represents an attractive microbicidal candidate.

Alisporivir inhibition of hepatocyte cyclophilins reduces HBV replication and hepatitis B surface antigen production.

BACKGROUND & AIMS: Cyclophilins are host factors required for hepatitis C virus replication. Cyclophilin inhibitors such as alisporivir have shown strong anti-hepatitis C virus activity in vitro and in clinical studies. However, little is known about whether hepatocyte cyclophilins are involved in the hepatitis B virus (HBV) life cycle. We investigated the effects of 2 cyclophilin inhibitors (alisporivir and NIM811) on HBV replication and hepatitis B surface antigen (HBsAg) production in cell lines. METHODS: Liver-derived cell lines producing full-length HBV and HBsAg particles, owing to stable (HepG2215) or transient (HuH-7) transfection, or infected with HBV (HepaRG cells; Invitrogen [Carlsbad, CA]), were incubated with alisporivir or NIM811 alone, or alisporivir in combination with a direct antiviral (telbivudine). The roles of individual cyclophilins in drug response was evaluated by small interfering RNA knockdown of cyclophilin (CYP)A, CYPC, or CYPD in HepG2215 cells, or CYPA knockdown in HuH-7 cells. The kinetics of antiviral activity were assessed based on levels of HBV DNA and HBsAg and Southern blot analysis. RESULTS: In HepG2215, HuH-7, and HepaRG cells, alisporivir reduced intracellular and secreted HBV DNA, in a dose-dependent manner. Knockdown of CYPA, CYPC, or CYPD (reduced by 80%) significantly reduced levels of HBV DNA and secreted HBsAg. Knockdown of CYPA significantly reduced secretion of HBsAg, leading to accumulation of intracellular HBsAg; the addition of alisporivir greatly reduced levels of HBsAg in these cells. The combination of alisporivir and telbivudine had greater antiviral effects than those of telbivudine or alisporivir alone. CONCLUSIONS: Alisporivir inhibition of cyclophilins in hepatocyte cell lines reduces replication of HBV DNA and HBsAg production and secretion. These effects are potentiated in combination with direct antiviral agents that target HBV-DNA polymerase.

Cyclophilin and NS5A inhibitors, but not other anti-hepatitis C virus (HCV) agents, preclude HCV-mediated formation of double-membrane-vesicle viral factories.

Although the mechanisms of action (MoA) of nonstructural protein 3 inhibitors (NS3i) and NS5B inhibitors (NS5Bi) are well understood, the MoA of cyclophilin inhibitors (CypI) and NS5A inhibitors (NS5Ai) are not fully defined. In this study, we examined whether CypI and NS5Ai interfere with hepatitis C virus (HCV) RNA synthesis of replication complexes (RCs) or with an earlier step of HCV RNA replication, the creation of double-membrane vesicles (DMVs) essential for HCV RNA replication. In contrast to NS5Bi, both CypI and NS5Ai do not block HCV RNA synthesis by way of RCs, suggesting that they exert their antiviral activity prior to the establishment of enzymatically active RCs. We found that viral replication is not a precondition for DMV formation, since the NS3-NS5B polyprotein or NS5A suffices to create DMVs. Importantly, only CypI and NS5Ai, but not NS5Bi, mir-122, or phosphatidylinositol-4 kinase IIIα (PI4KIIIα) inhibitors, prevent NS3-NS5B-mediated DMV formation. NS3-NS5B was unable to create DMVs in cyclophilin A (CypA) knockdown (KD) cells. We also found that the isomerase activity of CypA is absolutely required for DMV formation. This not only suggests that NS5A and CypA act in concert to build membranous viral factories but that CypI and NS5Ai mediate their early anti-HCV effects by preventing the formation of organelles, where HCV replication is normally initiated. This is the first investigation to examine the effect of a large panel of anti-HCV agents on DMV formation, and the results reveal that CypI and NS5Ai act at the same membranous web biogenesis step of HCV RNA replication, thus indicating a new therapeutic target of chronic hepatitis C.

Evaluation of PD 404,182 as an anti-HIV and anti-herpes simplex virus microbicide.

PD 404,182 (PD) is a synthetic compound that was found to compromise HIV integrity via interaction with a nonenvelope protein viral structural component (A. M. Chamoun et al., Antimicrob. Agents Chemother. 56:672-681, 2012). The present study evaluates the potential of PD as an anti-HIV microbicide and establishes PD's virucidal activity toward another pathogen, herpes simplex virus (HSV). We show that the anti-HIV-1 50% inhibitory concentration (IC50) of PD, when diluted in seminal plasma, is ∼1 µM, similar to the IC50 determined in cell culture growth medium, and that PD retains full anti-HIV-1 activity after incubation in cervical fluid at 37°C for at least 24 h. In addition, PD is nontoxic toward vaginal commensal Lactobacillus species (50% cytotoxic concentration [CC50], >300 µM), freshly activated human peripheral blood mononuclear cells (CC50, ∼200 µM), and primary CD4(+) T cells, macrophages, and dendritic cells (CC50, >300 µM). PD also exhibited high stability in pH-adjusted Dulbecco's phosphate-buffered saline with little to no activity loss after 8 weeks at pH 4 and 42°C, indicating suitability for formulation for transportation and storage in developing countries. Finally, for the first time, we show that PD inactivates herpes simplex virus 1 (HSV-1) and HSV-2 at submicromolar concentrations. Due to the prevalence of HSV infection, the ability of PD to inactivate HSV may provide an additional incentive for use as a microbicide. The ability of PD to inactivate both HIV-1 and HSV, combined with its low toxicity and high stability, warrants additional studies for the evaluation of PD's microbicidal candidacy in animals and humans.

The combination of alisporivir plus an NS5A inhibitor provides additive to synergistic anti-hepatitis C virus activity without detectable cross-resistance.

Alisporivir (ALV), a cyclophilin inhibitor, is a host-targeting antiviral (HTA) with multigenotypic anti-hepatitis C virus (HCV) activity and a high barrier to resistance. Recent advances have supported the concept of interferon (IFN)-free regimens to treat chronic hepatitis C. As the most advanced oral HTA, ALV with direct-acting antivirals (DAAs) represents an attractive drug combination for IFN-free therapy. In this study, we investigated whether particular DAAs exhibit additive, synergistic, or antagonistic effects when combined with ALV. Drug combinations of ALV with NS3 protease, NS5B polymerase, and NS5A inhibitors were investigated in HCV replicons from genotypes 1a, 1b, 2a, 3, and 4a (GT1a to -4a). Combinations of ALV with DAAs exerted an additive effect on GT1 and -4. A significant and specific synergistic effect was observed with ALV-NS5A inhibitor combination on GT2 and -3. Furthermore, ALV was fully active against DAA-resistant variants, and ALV-resistant variants were fully susceptible to DAAs. ALV blocks the contact between cyclophilin A and domain II of NS5A, and NS5A inhibitors target domain I of NS5A; our data suggest a molecular basis for the use of these two classes of inhibitors acting on two distinct domains of NS5A. These results provide in vitro evidence that ALV with NS5A inhibitor combination represents an attractive strategy and a potentially effective IFN-free regimen for treatment of patients with chronic hepatitis C. Due to its high barrier and lack of cross-resistance, ALV could be a cornerstone drug partner for DAAs.

Mice lacking cyclophilin B, but not cyclophilin A, are protected from the development of NASH in a diet and chemical-induced model.

Cyclophilins are a diverse family of peptidyl-prolyl isomerases (PPIases) of importance in a variety of essential cellular functions. We previously reported that the pan-cyclophilin inhibitor drug reconfilstat (CRV431) decreased disease in mice under the western-diet and carbon tetrachloride (CCl4) non-alcoholic steatohepatitis (NASH) model. CRV431 inhibits several cyclophilin isoforms, among which cyclophilin A (CypA) and B (CypB) are the most abundant. It is not known whether simultaneous inhibition of multiple cyclophilin family members is necessary for the observed therapeutic effects or if loss-of-function of one is sufficient. Identifying the responsible isoform(s) would enable future fine-tuning of drug treatments. Features of human liver fibrosis and complete NASH can be reliably replicated in mice by administration of intraperitoneal CCl4 alone or CCl4 in conjunction with high sugar, high cholesterol western diet, respectively. Here we show that while wild-type (WT) and Ppia-/- CypA KO mice develop severe NASH disease features under these models, Ppib-/- CypB KO mice do not, as measured by analysis of picrosirius red and hematoxylin & eosin-stained liver sections and TNFα immuno-stained liver sections. Cyclophilin inhibition is a promising and novel avenue of treatment for diet-induced NASH. In this study, mice without CypB, but not mice without CypA, were significantly protected from the development of the characteristic features of NASH. These data suggest that CypB is necessary for NASH disease progression. Further investigation is necessary to determine whether the specific role of CypB in the endoplasmic reticulum secretory pathway is of significance to its effect on NASH development.

Cyclophilin D knockout significantly prevents HCC development in a streptozotocin-induced mouse model of diabetes-linked NASH.

A family of Peptidyl-prolyl isomerases (PPIases), called Cyclophilins, localize to numerous intracellular and extracellular locations where they contribute to a variety of essential functions. We previously reported that non-immunosuppressive pan-cyclophilin inhibitor drugs like reconfilstat (CRV431) or NV556 decreased multiple aspects of non-alcoholic fatty liver disease (NAFLD) in mice under two different non-alcoholic steatohepatitis (NASH) mouse models. Both CRV431 and NV556 inhibit several cyclophilin isoforms, among which cyclophilin D (CypD) has not been previously investigated in this context. It is unknown whether it is necessary to simultaneously inhibit multiple cyclophilin family members to achieve therapeutic benefits or if loss-of-function of one is sufficient. Furthermore, narrowing down the isoform most responsible for a particular aspect of NAFLD/NASH, such as hepatocellular carcinoma (HCC), would allow for more precise future therapies. Features of human diabetes-linked NAFLD/NASH can be reliably replicated in mice by administering a single high dose of streptozotocin to disrupt pancreatic beta cells, in conjunction with a high sugar, high fat, high cholesterol western diet over the course of 30 weeks. Here we show that while both wild-type (WT) and Ppif-/- CypD KO mice develop multipe severe NASH disease features under this model, the formation of HCC nodules was significantly blunted only in the CypD KO mice. Furthermore, of differentially expressed transcripts in a qPCR panel of select HCC-related genes, nearly all were downregulated in the CypD KO background. Cyclophilin inhibition is a promising and novel avenue of treatment for diet-induced NAFLD/NASH. This study highlights the impact of CypD loss-of-function on the development of HCC, one of the most severe disease outcomes.

Correlation between NS5A dimerization and hepatitis C virus replication.

Hepatitis C virus (HCV) is the main agent of acute and chronic liver diseases leading to cirrhosis and hepatocellular carcinoma. The current standard therapy has limited efficacy and serious side effects. Thus, the development of alternate therapies is of tremendous importance. HCV NS5A (nonstructural 5A protein) is a pleiotropic protein with key roles in HCV replication and cellular signaling pathways. Here we demonstrate that NS5A dimerization occurs through Domain I (amino acids 1-240). This interaction is not mediated by nucleic acids because benzonase, RNase, and DNase treatments do not prevent NS5A-NS5A interactions. Importantly, DTT abrogates NS5A-NS5A interactions but does not affect NS5A-cyclophilin A interactions. Other reducing agents such as tris(2-carboxyethyl)phosphine and 2-mercaptoethanol also abrogate NS5A-NS5A interactions, implying that disulfide bridges may play a role in this interaction. Cyclophilin inhibitors, cyclosporine A, and alisporivir and NS5A inhibitor BMS-790052 do not block NS5A dimerization, suggesting that their antiviral effects do not involve the disruption of NS5A-NS5A interactions. Four cysteines, Cys-39, Cys-57, Cys-59, and Cys-80, are critical for dimerization. Interestingly, the four cysteines have been proposed to form a zinc-binding motif. Supporting this notion, NS5A dimerization is greatly facilitated by Zn(2+) but not by Mg(2+) or Mn(2+). Importantly, the four cysteines are vital not only for viral replication but also critical for NS5A binding to RNA, revealing a correlation between NS5A dimerization, RNA binding, and HCV replication. Altogether our data suggest that NS5A-NS5A dimerization and/or multimerization could represent a novel target for the development of HCV therapies.

HCV NS5A and IRF9 compete for CypA binding.

BACKGROUND & AIMS: Cyclophilin A (CypA) is vital for HCV replication. Cyp inhibitors successfully decrease viral loads in HCV-infected patients. However, their mechanisms of action remain unknown. Since interferon (IFN) can also suppress HCV replication, we asked whether a link between CypA and the IFN response exists. METHODS: We used cellular and recombinant pulldown approaches to investigate the possibility of a specific association of CypA with host ligands. RESULTS: We found for the first time that CypA binds to a major component of the IFN response - the IFN regulatory factor 9 (IRF9). IRF9 is the DNA-binding component of the transcriptional IFN-stimulated gene factor 3 (ISGF3). CypA binds directly to IRF9 via its peptidyl-prolyl isomerase (PPIase) pocket. Cyp inhibitors such as cyclosporine A (CsA) or non-immunosuppressive derivates such as alisporivir and SCY-635, prevent IRF9-CypA complex formation. CypA binds to the C-terminal IRF-association-domain (IAD), but not to the DNA-binding or linker domains of IRF9. Remarkably, CypA associates with the multimeric ISGF3 complex. We also obtained evidence that CypA neutralization enhances IFN-induced transcription. Interestingly, the hepatitis C virus (HCV) non-structural 5A (NS5A) protein, which is known to modulate the IFN response, competes with IRF9 for CypA binding and can prevent the formation of IRF9-CypA complexes. CONCLUSIONS: This study demonstrates for the first time that CypA binds specifically to a component of the Janus kinase/signal transducer and activator of transcription (JAK/STAT) pathway, IRF9. This study also reveals a novel opportunity of HCV to modulate the IFN response via NS5A.

Acute antagonism in three-drug combinations for vaginal HIV prevention in humanized mice.

Adolescent girls and young women in low- to middle-income countries are disproportionately at risk of becoming HIV-1 infected. New non-vaccine biomedical products aimed at overcoming this global health challenge need to provide a range of safe, effective, and discreet dosage forms based on the delivery of one or more antiviral compounds. An overarching strategy involves vaginal drug administration through inserts/tablets, gels, films, and intravaginal rings. The approach derives its appeal from being women-controlled and topical, there-by potentially minimizing systemic exposure to the agents and their metabolites. Oral regimens based on tenofovir disoproxil fumarate (TDF) and emtricitabine (FTC) are established and effective in HIV-1 pre-exposure prophylaxis (PrEP), and form a promising basis for vaginal PrEP. Here, we used bone marrow/liver/thymus humanized mice to measure the in vivo efficacy against HIV-1 of single and combination antiviral compounds applied vaginally, coupled with data analysis using the Chou-Talalay mathematical model to study the dose-effect characteristics. Unexpectedly, strong antagonism was observed in drug combinations composed of TDF-FTC coupled with a third agent using a different mode of action against HIV-1. The antagonistic effect was remedied when TDF was omitted from the regimen. Our approach provides a translational template for the preclinical, rational, and systematic evaluation of drug combinations for the prevention of HIV-1, and other viral diseases.

The Cyclophilin Inhibitor Rencofilstat Decreases HCV-induced Hepatocellular Carcinoma Independently of Its Antiviral Activity.

There is an urgent need for the identification of new drugs that inhibit HCV-induced hepatocellular carcinoma (HCC). Our work demonstrates that cyclophilin inhibitors (CypI) represent such new drugs. We demonstrated that the non-immunosuppressive cyclosporine A (CsA) analog (CsAa) rencofilstat possesses dual therapeutic activities for the treatment of HCV infection and HCV-induced HCC. Specifically, we showed that HCV infection of humanized mice results in the progressive development of HCC. This was true for four genotypes tested (1 to 4). Remarkably, we demonstrated that rencofilstat inhibits the development of HCV-induced HCC in mice even when added 16 weeks post-infection when HCC is well established. Importantly, we showed that rencofilstat drastically reduces HCC progression independently of its anti-HCV activity. Indeed, the CypI rencofilstat inhibits HCC while other anti-HCV agents such as NS5A (NS5Ai) and NS5B (NS5Bi) fail to reduce HCC. In conclusion, this study shows for the first time that the CypI rencofilstat represents a potent therapeutic agent for the treatment of HCV-induced HCC.

The Cyclophilin Inhibitor Rencofilstat Decreases HCV-Induced Hepatocellular Carcinoma Independently of Its Antiviral Activity.

There is an urgent need for the identification of new drugs that inhibit HCV-induced hepatocellular carcinoma (HCC). Our work demonstrates that cyclophilin inhibitors (CypIs) represent such new drugs. We demonstrate that the nonimmunosuppressive cyclosporine A (CsA) analog (CsAa) rencofilstat possesses dual therapeutic activities for the treatment of HCV infection and HCV-induced HCC. Specifically, we show that the HCV infection of humanized mice results in the progressive development of HCC. This is true for the four genotypes tested (1 to 4). Remarkably, we demonstrate that rencofilstat inhibits the development of HCV-induced HCC in mice even when added 16 weeks after infection when HCC is well established. Importantly, we show that rencofilstat drastically reduces HCC progression independently of its anti-HCV activity. Indeed, the CypI rencofilstat inhibits HCC, while other anti-HCV agents such as NS5A (NS5Ai) and NS5B (NS5Bi) fail to reduce HCC. In conclusion, this study shows for the first time that the CypI rencofilstat represents a potent therapeutic agent for the treatment of HCV-induced HCC.

The cyclophilin inhibitor SCY-635 suppresses viral replication and induces endogenous interferons in patients with chronic HCV genotype 1 infection.

BACKGROUND & AIMS: SCY-635 is a non-immunosuppressive analog of cyclosporin A that inhibits cyclophilins A and B and hepatitis C virus (HCV) replication in vitro. In a phase 1b multi-dose escalation study, we evaluated the safety, plasma pharmacokinetics, and antiviral activity of 15 days of monotherapy with SCY-635 in adults with chronic genotype 1 HCV infection. METHODS: Twenty adults with chronic HCV genotype 1 were randomized to SCY-635 oral doses of 100, 200, or 300 mg three times daily for 15 days. RESULTS: No dose-limiting clinical or laboratory toxicities were identified. On day 15, the mean decline in plasma viremia was 2.24±1.74 log(10) IU/ml with SCY-635 900 mg/d. Individual antiviral responses correlated with host IL28B genotype. Post hoc analyses indicated treatment with SCY-635 increased plasma protein concentrations of interferon α (IFNα), IFNs λ(1) and λ(3), and 2'5' oligoadenylate synthetase 1 (2'5'OAS-1), with the greatest increases in IL28B CC and CT subjects. Changes in plasma concentrations for all markers were coincident with changes in the plasma concentration of SCY-635. Peaks of IFNs α, λ(1), and λ(3) and 2'5'OAS-1 were observed within 2 h after drug administration. In replicon cells, SCY-635 enhanced secretion of type I and type III IFNs and increased the expression of IFN-stimulated genes (ISG). CONCLUSIONS: These studies establish clinical proof of concept for SCY-635 as a novel antiviral agent and suggest that restoration of the host innate immune response to chronic hepatitis C infection may represent a major mechanism through which cyclophilin inhibitors exert clinical antiviral activity.

Sublimable C5A delivery provides sustained and prolonged anti-HIV microbicidal activities.

We have identified a short amphipathic helical peptide, called C5A, which exhibits potent microbicidal activities in vitro and which offers protection from vaginal HIV transmission in vivo in a humanized mouse model. However, there are many obstacles to overcome before C5A can be considered a true microbicidal candidate. First, it must be stabilized against enzymatic degradation in a continuously warm and moist environment. Second, it must be delivered in a controlled manner to achieve long-term and coitally independent efficacy. We demonstrate in this in vitro study that the combination of two matrices with different subliming properties ((hexamethylcyclotrisiloxane [HMCS] and cyclododecane [CDD]) containing 10% labile C5A yielded the best results in terms of controlled release and preserved anti-HIV activity of the peptide when pre-exposed to cell-free medium or cell culture at body temperature for up to 2 months.

PD 404,182 is a virocidal small molecule that disrupts hepatitis C virus and human immunodeficiency virus.

We describe a virucidal small molecule, PD 404,182, that is effective against hepatitis C virus (HCV) and human immunodeficiency virus (HIV). The median 50% inhibitory concentrations (IC(50)s) for the antiviral effect of PD 404,182 against HCV and HIV in cell culture are 11 and 1 µM, respectively. The antiviral activity of PD 404,182 is due to the physical disruption of virions that is accompanied to various degrees (depending on the virus and exposure temperature/time) by the release of viral nucleic acids into the surrounding medium. PD 404,182 does not directly lyse liposomal membranes even after extended exposure, and it shows no attenuation in antiviral activity when preincubated with liposomes of various lipid compositions, suggesting that the compound inactivates viruses through interaction with a nonlipid structural component of the virus. The virucidal activity of PD 404,182 appears to be virus specific, as little to no viral inactivation was detected with the enveloped Dengue and Sindbis viruses. PD 404,182 effectively inactivates a broad range of primary isolates of HIV-1 as well as HIV-2 and simian immunodeficiency virus (SIV), and it does not exhibit significant cytotoxicity with multiple human cell lines in vitro (50% cytotoxic concentration, >300 µM). The compound is fully active in cervical fluids, although it exhibits decreased potency in the presence of human serum, retains its full antiviral potency for 8 h when in contact with cells, and is effective against both cell-free and cell-associated HIV. These qualities make PD 404,182 an attractive candidate anti-HIV microbicide for the prevention of HIV transmission through sexual intercourse.