Non-immunosuppressive Cyclophilin Inhibitor Research Articles

Structurally distinct cyclosporin and sanglifehrin analogs CRV431 and NV556 suppress established HCV infection in humanized-liver mice.

We previously reported that the non-immunosuppressive cyclophilin inhibitors (CypIs)—cyclosporin A analog CRV431 and sanglifehrin analog NV556—efficiently inhibit HCV replication in vitro. In this study, we asked whether they can also reduce HCV replication in vivo. We found that a single oral administration of CRV431 and NV556 to HCV-infected humanized-liver mice drastically reduced HCV blood levels. The antiviral effect was observed when CRV431 or NV556 were each individually administered with HCV, 3, 6 weeks or even 3 months post-infection when viral replication is robust. These results were confirmed in chimeric mice implanted with human hepatocytes isolated from three distinct donors. Remarkably, no viral rebound was observed 5 months after a single dose administration of 50 mg/kg of CRV431 or NV556 four weeks post-HCV infection, indicating the possibility of suppression of an established viral infection. Since we recently demonstrated that both CRV431 and NV556 also inhibit the development of liver fibrosis and hepatocellular carcinoma in nonviral-induced non-alcoholic steatohepatitis mouse models, our present data suggest that the two entirely structurally different CypIs—CRV431 and NV556—derived from unrelated natural products, represent attractive partners to current direct-acting agent (DAA) regimens for the treatment of hepatitis C and liver diseases.

Discovery of a novel 9-position modified second-generation anti-HCV candidate via bioconversion and semi-synthesis of FR901459

Evidence that hepatitis C virus (HCV) utilizes cellular cyclophilin proteins in the virus replication cycle has increased attention on cyclophilin inhibitors as attractive therapeutic targets in the treatment of HCV. Previous reports have described a number of non-immunosuppressive cyclophilin inhibitors, most of which require many synthetic steps for their preparation. Sasamura et al. have previously reported the isolation of bioconversion derivative 4. This analog is a convenient starting point for optimization due to the presence of the readily modifiable primary hydroxyl group and because it shows moderate anti-HCV activity and decreased immunosuppressive activity. We have also established an efficient C-alkylation reaction at the 3-position. Through a detailed structure-activity relationship study, we discovered a new type of clinical candidate 14 which requires a short synthetic process and has potent anti-HCV activity and reduced immunosuppressive activity, as well as improved aqueous solubility and pharmacokinetics.

Discovery of ASP5286: A novel non-immunosuppressive cyclophilin inhibitor for the treatment of HCV

Evidence indicates that hepatitis C virus (HCV) utilizes cellular cyclophilin proteins in its replication, and cyclophilin inhibitors represent a new class of anti-HCV agents. We have established an efficient synthetic methodology to generate FR901459 derivatives via N, O-acyl migration reaction while avoiding total synthesis. Through a detailed structure–activity relationship study, we improved anti-HCV activity while decreasing immunosuppressive activity. Additionally, we discovered the importance of substitution at the 3 position for not only improving anti-HCV activity but also pharmacokinetic profile. Finally, by striking an appropriate balance between potency, solubility, and permeability, we discovered ASP5286 (13) as a potential clinical candidate for anti-HCV therapy.

Discovery of a Potent and Orally Bioavailable Cyclophilin Inhibitor Derived from the Sanglifehrin Macrocycle.

Cyclophilins are a family of peptidyl-prolyl isomerases that are implicated in a wide range of diseases including hepatitis C. Our aim was to discover through total synthesis an orally bioavailable, non-immunosuppressive cyclophilin (Cyp) inhibitor with potent anti-hepatitis C virus (HCV) activity that could serve as part of an all oral antiviral combination therapy. An initial lead 2 derived from the sanglifehrin A macrocycle was optimized using structure based design to produce a potent and orally bioavailable inhibitor 3. The macrocycle ring size was reduced by one atom, and an internal hydrogen bond drove improved permeability and drug-like properties. 3 demonstrates potent Cyp inhibition ( Kd = 5 nM), potent anti-HCV 2a activity (EC50 = 98 nM), and high oral bioavailability in rat (100%) and dog (55%). The synthetic accessibility and properties of 3 support its potential as an anti-HCV agent and for interrogating the role of Cyp inhibition in a variety of diseases.

The cyclophilin-inhibitor alisporivir stimulates antigen presentation thereby promoting antigen-specific CD8+ T cell activation

Cyclophilin-inhibitors have potent antiviral activity against Hepatitis C virus (HCV) and are promising candidates for broad-spectrum antiviral therapy. Cyclosporine A (CsA) acts immunosuppressive by blocking T cell activation and antigen presentation. Alisporivir, a non-immunosuppressive CsA analog in clinical development, does not inhibit T cell activation. In this study we explored the impact of alisporivir on antigen presentation. Hepatoma cells endogenously expressing the epitope-restricting major histocompatibility complex-class I (MHC-I) allele HLA-A2 and constitutively expressing a viral antigen were established to study the impact of cyclophilin-inhibitors on antigen presentation. Antigen-specific CD8(+) T cell activation and MHC-I surface expression were measured to quantify antigen presentation. Our work establishes a novel cell culture model to study antigen presentation in liver-derived cells. Authentic regulation of antigen presentation was ensured by the action of pro- and anti-inflammatory cytokines. Alisporivir pretreatment stimulated antigen presentation by hepatoma target cells, leading to enhancement of antigen-specific CD8(+) T cell activation by 40%. Alisporivir, as well as a panel of other cyclophilin-inhibitors, induced an increase of MHC-I and beta-2 microglobulin on the surface of several cell lines. The drug neither enhanced MHC-I transcript or protein levels nor affected surface expression of other proteins or protein trafficking in general. Proteasome-inhibitors completely blocked the alisporivir-directed enhancement of surface MHC-I, suggesting an influence of the drug on peptide-availability. Alisporivir stimulates antigen presentation by inducing enhanced MHC-I surface expression, thereby promoting antigen-specific CD8(+) T cell activation. This immunostimulatory function might further contribute to the antiviral activity of non-immunosuppressive cyclophilin-inhibitors.

Melanocytes from Patients Affected by Ullrich Congenital Muscular Dystrophy and Bethlem Myopathy have Dysfunctional Mitochondria That Can be Rescued with Cyclophilin Inhibitors.

Ullrich congenital muscular dystrophy and Bethlem myopathy are caused by mutations in collagen VI (ColVI) genes, which encode an extracellular matrix protein; yet, mitochondria play a major role in disease pathogenesis through a short circuit caused by inappropriate opening of the permeability transition pore, a high-conductance channel, which causes a shortage in ATP production. We find that melanocytes do not produce ColVI yet they bind it at the cell surface, suggesting that this protein may play a trophic role and that its absence may cause lesions similar to those seen in skeletal muscle. We show that mitochondria in melanocytes of Ullrich congenital muscular dystrophy and Bethlem myopathy patients display increased size, reduced matrix density, and disrupted cristae, findings that suggest a functional impairment. In keeping with this hypothesis, mitochondria (i) underwent anomalous depolarization after inhibition of the F-ATP synthase with oligomycin, and (ii) displayed decreased respiratory reserve capacity. The non-immunosuppressive cyclophilin inhibitor NIM811 prevented mitochondrial depolarization in response to oligomycin in melanocytes from both Ullrich congenital muscular dystrophy and Bethlem myopathy patients, and partially restored the respiratory reserve of melanocytes from one Bethlem myopathy patient. These results match our recent findings on melanocytes from patients affected by Duchenne muscular dystrophy (Pellegrini et al., 2013), and suggest that skin biopsies may represent a minimally invasive tool to investigate mitochondrial dysfunction and to evaluate drug efficacy in ColVI-related myopathies and possibly in other muscle wasting conditions like aging sarcopenia.

Potent nonimmunosuppressive cyclophilin inhibitors with improved pharmaceutical properties and decreased transporter inhibition.

Nonimmunosuppressive cyclophilin inhibitors have demonstrated efficacy for the treatment of hepatitis C infection (HCV). However, alisporivir, cyclosporin A, and most other cyclosporins are potent inhibitors of OATP1B1, MRP2, MDR1, and other important drug transporters. Reduction of the side chain hydrophobicity of the P4 residue preserves cyclophilin binding and antiviral potency while decreasing transporter inhibition. Representative inhibitor 33 (NIM258) is a less potent transporter inhibitor relative to previously described cyclosporins, retains anti-HCV activity in cell culture, and has an acceptable pharmacokinetic profile in rats and dogs. An X-ray structure of 33 bound to rat cyclophilin D is reported.

From Chemical Tools to Clinical Medicines: Nonimmunosuppressive Cyclophilin Inhibitors Derived from the Cyclosporin and Sanglifehrin Scaffolds

The cyclophilins are widely expressed enzymes that catalyze the interconversion of the cis and trans peptide bonds of prolines. The immunosuppressive natural products cyclosporine A and sanglifehrin A inhibit the enzymatic activity of the cyclophilins. Chemical modification of both the cyclosporine and sanglifehrin scaffolds has produced many analogues that inhibit cyclophilins in vitro but have reduced immunosuppressive properties. Three nonimmunosuppressive cyclophilin inhibitors (alisporivir, SCY-635, and NIM811) have demonstrated clinical efficacy for the treatment of hepatitis C infection. Additional candidates are in various stages of preclinical development for the treatment of hepatitis C or myocardial reperfusion injury. Recent publications suggest that cyclophilin inhibitors may have utility for the treatment of diverse viral infections, inflammatory indications, and cancer. In this review, we document the structure-activity relationships of the nonimmunosuppressive cyclosporins and sanglifehrins in clinical and preclinical development. Aspects of the pharmacokinetic behavior and chemical biology of these drug candidates are also described.

NIM811, a cyclophilin inhibitor without immunosuppressive activity, is beneficial in collagen VI congenital muscular dystrophy models

Ullrich congenital muscular dystrophy (UCMD) and Bethlem myopathy (BM) are inherited muscle diseases due to mutations in the genes encoding the extracellular matrix protein collagen (Col) VI. Opening of the cyclosporin A-sensitive mitochondrial permeability transition pore (PTP) is a causative event in disease pathogenesis, and a potential target for therapy. Here, we have tested the effect of N-methyl-4-isoleucine-cyclosporin (NIM811), a non-immunosuppressive cyclophilin inhibitor, in a zebrafish model of ColVI myopathy obtained by deletion of the N-terminal region of the ColVI α1 triple helical domain, a common mutation of UCMD. Treatment with antisense morpholino sequences targeting col6a1 exon 9 at the 1-4 cell stage (within 1 h post fertilization, hpf) caused severe ultrastructural and motor abnormalities as assessed by electron and fluorescence microscopy, birefringence, spontaneous coiling events and touch-evoked responses measured at 24-48 hpf. Structural and functional abnormalities were largely prevented when NIM811--which proved significantly more effective than cyclosporin A--was administered at 21 hpf, while FK506 was ineffective. Beneficial effects of NIM811 were also detected (i) in primary muscle-derived cell cultures from UCMD and BM patients, where the typical mitochondrial alterations and depolarizing response to rotenone and oligomycin were significantly reduced; and (ii) in the Col6a1(-/-) myopathic mouse model, where apoptosis was prevented and muscle strength was increased. Since the PTP of zebrafish shares its key regulatory features with the mammalian pore, our results suggest that early treatment with NIM811 should be tested as a potential therapy for UCMD and BM.

Cyclophilins as Modulators of Viral Replication

Cyclophilins are peptidyl‐prolyl cis/trans isomerases important in the proper folding of certain proteins. Mounting evidence supports varied roles of cyclophilins, either positive or negative, in the life cycles of diverse viruses, but the nature and mechanisms of these roles are yet to be defined. The potential for cyclophilins to serve as a drug target for antiviral therapy is evidenced by the success of non-immunosuppressive cyclophilin inhibitors (CPIs), including Alisporivir, in clinical trials targeting hepatitis C virus infection. In addition, as cyclophilins are implicated in the predisposition to, or severity of, various diseases, the ability to specifically and effectively modulate their function will prove increasingly useful for disease intervention. In this review, we will summarize the evidence of cyclophilins as key mediators of viral infection and prospective drug targets.

Cyclophilin Inhibitors: An Emerging Class of Therapeutics for the Treatment of Chronic Hepatitis C Infection

The advent of the replicon system together with advances in cell culture have contributed significantly to our understanding of the function of virally-encoded structural and nonstructural proteins in the replication cycle of the hepatitis C virus. In addition, in vitro systems have been used to identify several host proteins whose expression is critical for supporting such diverse activities as viral entry, RNA replication, particle assembly, and the release of infectious virions. Among all known host proteins that participate in the HCV replication cycle, cyclophilins are unique because they constitute the only host target that has formed the basis of pharmaceutical drug discovery and drug development programs. The introduction of the nonimmunosuppressive cyclophilin inhibitors into clinical testing has confirmed the clinical utility of CsA-based inhibitors for the treatment of individuals with chronic hepatitis C infection and has yielded new insights into their mechanism(s) of action. This review describes the biochemical evidence for the potential roles played by cyclophilins in supporting HCV RNA replication and summarizes clinical trial results obtained with the first generation of nonimmunosuppressive cyclophilin inhibitors.

The Cyclophilin Inhibitor SCY-635 Disrupts Hepatitis C Virus NS5A-Cyclophilin A Complexes

The nonimmunosuppressive cyclophilin (Cyp) inhibitor SCY-635 blocks hepatitis C virus (HCV) replication both in vitro and in vivo and represents a novel potent anti-HCV agent. However, its mechanism of action remains to be fully elucidated. A growing body of evidence suggests that cyclophilin A (CypA) is absolutely necessary for HCV replication and that the HCV nonstructural 5A (NS5A) protein serves as a main viral ligand for CypA. In this study, we examined the effect of SCY-635 on HCV replication. Specifically, we asked whether SCY-635 blocks HCV replication by targeting CypA-NS5A interactions. We also investigated the possibility that HCV can escape SCY-635 selection pressure and whether this resistance influences either CypA-NS5A interactions or the dependence of HCV on CypA. We found not only that SCY-635 efficiently inhibits HCV replication, but it is sufficient alone to clear HCV replicon-containing cells. We found that SCY-635 prevents CypA-NS5A interactions in a dose-dependent manner. SCY-635 prevents the contact between CypA and NS5A derived from genotypes 1 to 3. Together, these data suggest that NS5A-CypA interactions control HCV replication and that SCY-635 blocks viral replication by preventing the formation of these complexes. We also found that NS5A mutant proteins found in SCY-635-resistant HCV replicons behave similarly to wild-type NS5A in terms of both CypA binding and SCY-635-mediated dissociation and inhibition of CypA binding. However, the NS5A mutations found in SCY-635-resistant HCV replicons rescued viral replication in CypA-knockdown cells, suggesting that the NS5A mutations, which arose in vitro under SCY-635 selection, do not alter the binding affinity of CypA for NS5A. These specific mutations in NS5A eliminate the dependence of HCV RNA replication on the expression of host CypA.

Abstract 3836: Enhancement of doxorubicin cytotoxicity in breast cancer by NIM811, A non-immunosuppressive cyclosporine A analog

Abstract Cyclophilin A (CypA) is a member of the immunophilin family of peptidyl prolyl isomerases (PPI) that catalyze the cis-trans interconversion of proline imide bonds of peptides. In this role, cyclophilins have been found to function as signaling switches, regulating the activity of receptors, kinases and transcription factors. The PPI activity of CypA is inhibited by the immunosuppressive drug cyclosporine A (CsA), and in turn the CypA-CsA complex inhibits calcineurin-mediated NFAT activation. Our laboratory has demonstrated that CypA is necessary for the prolactin (PRL)-induced activation of Jak2/Stat5 signaling and gene expression (Cancer Res 68:7769, 2008). In addition, we have also shown that CsA inhibits the in vitro and in vivo growth and progression of both ER+ and ER- breast cancer cell lines. Here, we show that the non-immunosuppressive CsA analog NIM811 blocked PRL-stimulated activation of the Jak2/Stat5, PI3K/AKT and MAPK pathways in T47D cells. NIM811 also inhibited ER+, ER-, and Her2+ breast cancer cell proliferation, survival, motility and anchorage independent growth in a dose-dependent manner. NIM811 inhibited PRL induced gene expression, such as CISH and Cyclin D1. NIM811 also blocked the PRL-induced association between Stat5 and cMyb, and at higher doses, triggered PARP cleavage and activate Caspase 3. Furthermore, NIM811 increased the effects of doxorubicin in inhibiting monolayer cell growth and soft agar colony growth in vitro. In vivo, NIM811 alone significantly induced the primary tumor central necrosis and inhibited lymph node metastasis; combining NIM811 with a low dose of doxorubicin significantly inhibited MDA-MB-231 breast cancer xenograft tumor growth and distant organ metastasis. In summary, these results indicate that non-immunosuppressive cyclophilin inhibitor NIM811 has significant potential as both a single agent or in combination with cytotoxic agents for breast cancer therapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3836. doi:1538-7445.AM2012-3836

The cyclophilin inhibitor alisporivir prevents hepatitis C virus–mediated mitochondrial dysfunction

Alisporivir (Debio-025) is an analogue of cyclosporine A and represents the prototype of a new class of non-immunosuppressive cyclophilin inhibitors. In vitro and in vivo studies have shown that alisporivir inhibits hepatitis C virus (HCV) replication, and ongoing clinical trials are exploring its therapeutic potential in patients with chronic hepatitis C. Recent data suggest that the antiviral effect is mediated by inhibition of cyclophilin A, which is an essential host factor in the HCV life cycle. However, alisporivir also inhibits mitochondrial permeability transition by binding to cyclophilin D. Because HCV is known to affect mitochondrial function, we explored the effect of alisporivir on HCV protein-mediated mitochondrial dysfunction. Through the use of inducible cell lines, which allow to investigate the effects of HCV polyprotein expression independent from viral RNA replication and which recapitulate the major alterations of mitochondrial bioenergetics observed in infectious cell systems, we show that alisporivir prevents HCV protein-mediated decrease of cell respiration, collapse of mitochondrial membrane potential, overproduction of reactive oxygen species and mitochondrial calcium overload. Strikingly, some of the HCV-mediated mitochondrial dysfunctions could even be rescued by alisporivir. These observations provide new insights into the pathogenesis of HCV-related liver disease and reveal an additional mechanism of action of alisporivir that is likely beneficial in the treatment of chronic hepatitis C.

Safety, pharmacokinetics, and antiviral activity of the cyclophilin inhibitor NIM811 alone or in combination with pegylated interferon in HCV-infected patients receiving 14 days of therapy

Background Cyclophilin inhibitors have shown activity against a variety of viruses, including HCV. NIM811, a novel, non-immunosuppressive cyclophilin inhibitor was studied in ascending doses in a randomized, double-blind, placebo-controlled 14-day trial in genotype 1 HCV patients. Doses of 10 up to 600 mg were given orally once or twice daily as monotherapy (9:3 randomization of NIM811:placebo). 600 mg or placebo bid for 14 days was then co-administered with pegylated interferon alpha (PEG-IFN-α) administered on days 1 and 8 to genotype 1 relapsers. Results NIM811 was well tolerated at all doses. Although lack of antiviral effect was noted in the monotherapy arms, liver transaminase normalization occurred at doses over 75 mg. Mild, clinically non-significant elevations of bilirubin, and significant declines in platelet numbers were observed in the 400 and 600 mg bid groups. In the combination group, the mean HCV RNA decline was 2.85 log, compared to a 0.56 log in the PEG-IFN alone arm. The mean ALT (alanine transaminase) declined significantly by day 14 in the combination, but was unchanged in the PEG-IFN alone group. In the combination therapy group, the mean platelets were 203 × 10 9/L at baseline and fell to 105 × 10 9/L by day 14; for patients treated with PEG-IFN the values were 177 × 10 9/L and 139 × 10 9/L. There was a significant increase in bilirubin, although this did not reach clinically concerning levels. There were no severe or serious adverse events. The pharmacokinetics in both monotherapy and combination arms were dose linear and not affected by PEG-INF. Conclusion NIM811 monotherapy resulted in a normalization of liver transaminases in the absence of significant virological response. The combination of NIM811 and pegylated interferon alpha showed significant antiviral activity compared to interferon alone in genotype 1 HCV relapsers. The use of oral cyclophilin inhibitors as part of a combination regime for treatment of hepatitis C, especially to deter resistance, holds promise.

Abstract 3564: Anti-breast cancer activity of NIM811, a nonimmunosuppressive cyclosporine A analogue

Abstract Cyclophilin A (CypA) is a member of the immunophilin family of peptidyl prolyl isomerases (PPI) that catalyze the cis-trans interconversion of proline imide bonds of peptides. In this role, cyclophilins have been found to function as signaling switches, regulating the activity of receptors, kinases and transcription factors. The PPI activity of CypA is inhibited by the immunosuppressive drug cyclosporine A (CsA), and in turn the CypA-CsA complex inhibits calcineurin-mediated NFAT activation. Our laboratory has recently demonstrated that CypA is necessary for the prolactin (PRL)-induced activation of Jak2/Stat5 signaling and gene expression (Ca Res 68:7769, 2008). In addition, we have also shown that CsA inhibits the in vitro and in vivo growth and progression of both ER+ and ER- breast cancer cell lines. Here, we show that the non-immunosuppressive CsA analog NIM811 blocked PRL-stimulated activation of the Jak2/Stat5 pathway, and the kinases AKT and MAPK in T47D cells. NIM811 also inhibited ER+, ER-, and Her2+ breast cancer cell proliferation, survival and anchorage independent growth in a dose-dependent manner. NIM811 also blocked the PRL-induced association between Stat5 and cMyb, and at higher doses, triggered PARP cleavage. In summary, these results indicate that non-immunosuppressive cyclophilin inhibitor NIM811 has significant potential as a novel for breast cancer therapeutic. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3564.

666 THE EFFECTS OF SCY-635 A NON-IMMUNOSUPPRESSIVE CYCLOSPORIN ANALOG ON STELLATE CELL PROLIFERATION, COLLAGEN SYNTHESIS, TIMP-1 AND COLLAGENASE PRODUCTION

Aims: The non-immunosuppressive cyclophilin inhibitor SCY-635 is a potent inhibitor of HCV-specific RNA replication in genotype 1 derived HCV replicons. Clinical studies indicate that acute administration of SCY-635 significantly reduces plasma viremia in patients with chronic hepatitis C virus infection. Recent data have shown that cyclosporin and nonimmunosuppressive analogues such as NIM-811 can suppress the proliferation of, and collagen production in, hepatic stellate cells (HSC) (Kohjima et al. Liver Int., 2007). To examine the potential effects of SCY-635 on liver fibrosis and apoptosis, we have investigated the effects of SCY-635 on cell growth, collagen synthesis, TIMP-1 and collagenase levels and TGF-b signaling in HSCs. Methods: Human HSC-derived LI-90 cells either resting or stimulated with TGFb1 were used for these studies. The levels of collagen, matrix metalloproteinase 1 (MMP-1), tissue inhibitor of metalloproteinase-1 (TIMP-1) and levels of phosphorylation of p38 and Smad2 were evaluated by ELISA. Cell proliferation (BrdU incorporation) and apoptosis (ApoGlow) were measured. Apoptosis was evaluated using primary rat hepatocytes after treatment with Ad5IkB adenovirus followed by overnight incubation with TNF-α. Results: SCY-635 and cyclosporin (CsA) decreased the synthesis of collagen. At a clinically relevant concentration of 0.25 μM in non-stimulated or in TGFβ1 stimulated LI-90 cells, SCY-635 significantly reduced the collagen production by more than 70%. There was a parallel decrease in TIMP-1 secretion into the HSC medium and an increase in the production of MMP-1. SCY-635 inhibited proliferation without induction of apoptosis. Concentrations of SCY-635 ranging from 0.1 to 5 μM enhanced the phosphorylation of p38 and suppressed the phosphorylation of Smad2. SCY-635 inhibition of liver apoptosis was equipotent with CsA exhibiting IC50 values of 2.8 ± 0.3 and 3.6 ± 0.3 μM, respectively. Conclusions: These findings demonstrated that SCY-635 suppressed cell growth, reduced type I collagen synthesis and TIMP-1 secretion, and enhanced MMP-1 production suggesting that SCY-635 may exert an anti-fibrogenic effect. These effects may be mediated through the TGF-β signaling pathway. Taken together, these data suggest that SCY-635 may not only reduce the replication of HCV but also reduce the fibrosis associated with chronic hepatitis C virus infection.

The Isomerase Active Site of Cyclophilin A Is Critical for Hepatitis C Virus Replication

Cyclosporine A and nonimmunosuppressive cyclophilin (Cyp) inhibitors such as Debio 025, NIM811, and SCY-635 block hepatitis C virus (HCV) replication in vitro. This effect was recently confirmed in HCV-infected patients where Debio 025 treatment dramatically decreased HCV viral load, suggesting that Cyps inhibitors represent a novel class of anti-HCV agents. However, it remains unclear how these compounds control HCV replication. Recent studies suggest that Cyps are important for HCV replication. However, a profound disagreement currently exists as to the respective roles of Cyp members in HCV replication. In this study, we analyzed the respective contribution of Cyp members to HCV replication by specifically knocking down their expression by both transient and stable small RNA interference. Only the CypA knockdown drastically decreased HCV replication. The re-expression of an exogenous CypA escape protein, which contains escape mutations at the small RNA interference recognition site, restored HCV replication, demonstrating the specificity for the CypA requirement. We then mutated residues that reside in the hydrophobic pocket of CypA where proline-containing peptide substrates and cyclosporine A bind and that are vital for the enzymatic or the hydrophobic pocket binding activity of CypA. Remarkably, these CypA mutants fail to restore HCV replication, suggesting for the first time that HCV exploits either the isomerase or the chaperone activity of CypA to replicate in hepatocytes and that CypA is the principal mediator of the Cyp inhibitor anti-HCV activity. Moreover, we demonstrated that the HCV NS5B polymerase associates with CypA via its enzymatic pocket. The study of the roles of Cyps in HCV replication should lead to the identification of new targets for the development of alternate anti-HCV therapies.

Evaluation of putative inhibitors of mitochondrial permeability transition for brain disorders — Specificity vs. toxicity

Inhibition of mitochondrial permeability transition (mPT) has emerged as a promising approach for neuroprotection and development of well-tolerated mPT inhibitors with favorable blood-brain barrier penetration is highly warranted. In a recent study, 28 clinically available drugs with a common heterocyclic structure were identified as mPT inhibitors e.g. trifluoperazine, promethazine and nortriptyline. In addition, neuroprotection by structurally unrelated drugs e.g. neurosteroids, 4-hydroxy-tamoxifen and trimetazidine has been attributed to direct inhibition of mPT. The regulation of mPT is complex and highly dependent on the prevailing experimental conditions. Several features of mPT, such as swelling, depolarization or NADH oxidation, can also occur independently of the mPT phenomenon. Here, in isolated rodent brain-derived and human liver mitochondria, we re-evaluate drugs promoted as potent mPT inhibitors. We address the definition of an mPT inhibitor and present strategies to reliably detect mPT inhibition in vitro. Surprisingly, none of the 12 compounds tested displayed convincing mPT inhibition or effects comparable to cyclophilin D inhibition by the non-immunosuppressive cyclophilin inhibitor D-MeAla 3-EtVal 4-Cyclosporin (Debio 025). Propofol and 2-aminoethoxydiphenyl borate (2-APB) inhibited swelling in de-energized mitochondria but did not increase calcium retention capacity (CRC). Progesterone, trifluoperazine, allopregnanolone and 4-hydroxy-tamoxifen dose-dependently reduced CRC and respiratory control and were thus toxic rather than beneficial to mitochondrial function. Interestingly, topiramate increased CRC at high concentrations likely by a mechanism separate from direct mPT inhibition. We conclude that a clinically relevant mPT inhibitor should have a mitochondrial target and increase mitochondrial calcium retention at concentrations which can be translated to human use.

Evaluation of a cyclophilin inhibitor in hepatitis C virus–infected chimeric mice in vivo†

Cyclosporin A (CsA) inhibits replication of the HCV subgenomic replicon, and this effect is believed to not be mediated by its immunosuppressive action. We found that DEBIO-025, a novel non-immunosuppressive cyclophilin inhibitor derived from CsA, inhibited HCV replication in vitro more potently than CsA. We also examined the inhibitory effect of DEBIO-025 on naive HCV genotypes 1a or 1b in vivo using chimeric mice with human hepatocytes. These mice were treated for 14 days with DEBIO-025, pegylated-interferon alpha-2a (Peg-IFN), a combination of either drugs, or CsA in combination with Peg-IFN. In mice treated with Peg-IFN, serum HCV RNA levels decreased approximately 10-fold whereas DEBIO-025 treatment alone did not induce any significant change. In mice treated with both DEBIO-025 and Peg-IFN, HCV RNA levels decreased more than 100-fold. All mice treated with Peg-IFN combined with CsA died within 4 days. The combination treatment of DEBIO-025 and Peg-IFN reduced HCV RNA levels and core protein expression in liver, indicating that the HCV RNA levels reduction in serum was attributable to intrahepatic inhibition of HCV replication. We demonstrated that DEBIO-025 was better tolerated than CsA, and that its anti-HCV effect appeared to be synergistic in combination with Peg-IFN in vivo.