Potential conflict of interest: Nothing to report.
Cyclosporin A (CsA) inhibits the in vitro replication of HCV subgenomic replicons. We here report on the potent anti-HCV activity of the non-immunosuppressive cyclosporin DEBIO-025. The 50% effective concentration for inhibition of HCV subgenomic replicon replication in Huh 5-2 cells (luciferase assay) by DEBIO-025 was 0.27 ± 0.03 μg/mL and for CsA 2.8 ± 0.4 μg/mL. The concentration that reduced the growth of exponentially proliferating Huh 5-2 cells by 50% was greater than 27 μg/mL for DEBIO-025 and 12 ± 6 μg/mL for CsA, resulting in a selectivity index of approximately 900 for DEBIO-025 and 40 for CsA. The superior activity of DEBIO-025, as compared with CsA, was corroborated by monitoring HCV RNA levels in Huh 5-2, two other HCV subgenomic replicon-containing cell lines, and by monitoring the luciferase signal and viral antigen production in hepatoma cells that had been infected with an infectious full-length chimeric HCV construct. The combination of interferon alpha 2a with either CsA or DEBIO-025 resulted in an additive to slightly synergistic antiviral activity. DEBIO-025, at concentrations of 0.5 and 1 μg/mL, was able to clear cells from their HCV replicon within three to four passages, whereas treatment with CsA at the same concentrations for seven consecutive passages did not result in clearance of the HCV replicon. In conclusion, DEBIO-025, a compound that is also endowed with potent anti-HIV activity and is well tolerated in animals and humans, may form an attractive new option for the therapy of HCV infections, particularly in HCV/HIV co-infected patients. (HEPATOLOGY 2006;43:761–770.)
Hepatitis C virus (HCV) is an enveloped single-stranded (+) RNA virus that belongs to the separate genus Hepacivirus of the family Flaviviridae.1 HCV causes acute and chronic liver disease, including chronic hepatitis, cirrhosis, and hepatocellular carcinoma.2 Worldwide more than 170 million people are chronically infected with HCV and are thus at increased risk of developing serious life-threatening liver disease. Current standard therapy for chronic hepatitis C consists of the combination of pegylated interferon alpha (IFN-α) 2a in combination with ribavirin.3
Cyclosporin A (CsA) (Fig. 1) is an 11–amino acid (AA) cyclic peptide from which seven AA are N-methylated and three AA are uncommon, that is, (4R)-4-[(E)-2-butenyl-4-methyl-L-threonine] in position 1, (L)-alpha-amino-butyric acid in postion 2, and (D)-alanine in position 8. CsA, a secondary metabolite of Tolypocladium sp., exerts potent immunosuppressive activity and is used to prevent organ transplant rejection.4 In addition to its immunosuppressive effect, CsA has been reported to be a potent inhibitor of the replication of HIV-1.5, 6 The compound is also endowed with anti-schistosomal activity7–12 and is an inhibitor of Plasmodium falciparum13–15 and Plasmodium vivax.16
Recently CsA was shown to exhibit anti-HCV activity in vitro.17, 18 In 1988, Teraoka and colleagues19 reported that CsA had a beneficial effect on non-A, non-B hepatitis in two chimpanzees chronically infected with HCV. After treatment with CsA was terminated, the disease recurred in both chimpanzees.19 After these early reports, three clinical trials were initiated in which the effect of CsA (in combination with IFN) was studied in patients with chronic hepatitis C. The results of these trials showed some on-treatment virological response in a small pilot study and a higher sustained virological response rate for IFN–CsA combination therapy as compared with IFN monotherapy.20–22 Blood CsA concentrations were related to the virological response.20, 21 The immunosuppressive properties of CsA would be undesirable if the compound would be used as an antiviral for the treatment of a chronic viral infection.
We report on a novel synthetic non-immunosuppressive cyclosporin, DEBIO-025, that is a more potent inhibitor of HCV replication than CsA, both in subgenomic HCV replicon-containing hepatoma cells and in productively HCV-infected cells. Because DEBIO-025 is also a potent inhibitor of HIV-1 replication, and has an excellent safety profile, it may be an attractive drug candidate for the treatment of HCV infections, particularly in HIV-1/HCV–co-infected patients.
The chemical structure of DEBIO-025 is depicted in Fig. 1A. The preparation of DEBIO-025 was based on the synthetic strategy for D-MeAla3-EtXaa4-cyclosporin analogs described previously.23 CsA was purchased from Fluka Chemie GmbH (9471 Buchs, Switzerland), 2′-C-methyl-Cytidine,24 JT (2-[4-[[4- (acetylamino)-4′-chloro-[1,1′-biphenyl]-2-yl]methoxy]phenyl]-1-cyclohexyl-1H-benzimidazole-5-carboxylic acid),25 and VX-95026–28 were synthesized by standard methods.
Cells and Viruses.
Cell clones Huh 7.5 (kindly provided by Dr. Charles M. Rice, Rockefeller University New York) and the highly permissive cell clone Huh 7-Lunet (V. Lohmann and R. Bartenschlager, unpublished) as well as Huh 7 cells containing subgenomic HCV replicons I389luc-ubi-neo/NS3-3′/5.1 (Huh 5-2), I377NS3-3′/wt (Huh 9–13) or I389/hygro-ubi-NS3-3 ′/5.1 have been described recently.29–32 Cells were grown in Dulbecco's modified Eagle's Medium (DMEM; Gibco, Merelbeke, Belgium) supplemented with 10% heat-inactivated fetal bovine serum (Integro, Zaandam, The Netherlands), 1× non-essential amino acids (Gibco), 100 IU/mL penicillin (Gibco), 100 μg/mL streptomycin (Gibco), and 250 μg/mL Geneticin (G418, Gibco) for Huh 5-2 cells, 1,000 μg/mL G418 for Huh 9–13 cells or 25 μg/ml hygromycin (Gibco) for Huh mono cells.
Anti-HCV Assay in Huh 5-2 Cells.
Huh 5-2 cells were seeded at a density of 5 × 103 per well in a tissue culture–treated white 96-well view plate (Packard, Canberra, Canada) in complete DMEM supplemented with 250 μg/mL G418. After incubation for 24 hours at 37°C (5% CO2) medium was removed and 3-fold serial dilutions in complete DMEM (without G418) of the test compounds were added in a total volume of 100 μL. After 4 days of incubation at 37°C, cell culture medium was removed and luciferase activity was determined using the Steady-Glo luciferase assay system (Promega, Leiden, The Netherlands); the luciferase signal was measured using a Luminoskan Ascent (Thermo, Vantaa, Finland). The 50% effective concentration (EC50) was defined as the concentration of compound that reduced the luciferase signal by 50%.
Anti-HCV Assay in Huh 9–13 and Huh-Mono Cells.
Huh 9–13 or Huh mono cells were seeded at a density of 5 × 103 cells per well in 96-well cell culture plates in complete DMEM supplemented with 1,000 μg/mL G418 for Huh 9–13 cells or 25 μg/mL hygromycin for Huh mono. After 24 hours incubation at 37°C, cell culture medium was removed and 3-fold serial dilutions of the test compounds in complete DMEM without G418 or hygromycin were added in a total volume of 100 μL. After 4 days incubation at 37°C, cell culture fluid was removed and monolayers were washed once with phosphate-buffered saline. Cells were lysed in 350 μL RLT buffer (Qiagen, Venlo, The Netherlands) according to the manufacturer's instruction. Lysates were stored at −80°C until further use.
Production of Infectious HCV in Cell Culture.
A full-length chimeric genome between HCV strains J6 and JFH1 was generated. This construct was modified as described recently33 to obtain a bicistronic genome carrying a luciferase reporter gene (Jc1-Luc). Huh 7.5 cells were transfected by electroporation by using RNA transcripts of the genomes, and cell culture supernatant was collected between 24 and 96 hours after transfection. Supernatants were filtered (0.45 μmol/L), and limiting dilution assays were performed as described recently.34 The cell culture infectious dose 50 per milliliter were 1.3 × 105 and 4.2 × 103 for Jc1 and Jc1-Luc, respectively.
Antiviral Assay in Infected Target Cells.
Huh 7-Lunet and Huh 7.5 cells were seeded at a density of 2 × 104 or 4 × 104 cells per well of a 12-well plate or in case of immunofluorescence analysis, per 24-well plate containing a glass coverslip, respectively. Twenty-four hours later, the medium was replaced by 0.5 mL Jc1-Luc virus stock (12-well plates) or 0.25 mL Jc1 virus stock (12-well plates). Four hours later, the virus inoculum was replaced by medium containing different concentrations of CsA or DEBIO-025. Duplicate wells (or more) were incubated for an additional 72 hours, and cells were then harvested for the luciferase assay and immunofluorescence analysis, respectively.
Luciferase Assay on HCV-Infected Huh 7-Lunet or Huh 7.5 Cells.
Infected cells in cultures of 12-well plates were washed twice with phosphate-buffered saline and lysed in 350 μL/well luciferase lysis buffer (1% Triton X-100, 25 mmol/L glycylglycine, 15 mmol/L MgSO4, 4 mmol/L ethylene guanosine tetra-acetic acid, and 1 mmol/L dithiothreitol). Firefly luciferase activity was measured as described previously.35 Briefly, after one freeze/thaw cycle, cells were resuspended and 100 μL cell lysate was mixed with 360 μL assay buffer [25 mmol/L glycylglycine, 15 mmol/L MgSO4, 1 mmol/L dithiothreitol, 2 mmol/L adenosine triphosphate (ATP), 15 mmol/L potassium phosphate buffer, pH 7.8] and 200 μL substrate solution (200 mmol/L luciferin, 25 mmol/L glycylglycine). Finally, luminescence was measured by using a Lumat LB9507 luminometer (Berthold, Freiburg, Germany) for 20 samples. All luciferase assays were performed in duplicate.
Immunofluorescence Analysis of HCV-Infected Huh 7-Lunet or Huh 7.5 Cells.
Cells grown on glass coverslips were fixed with 3% paraformaldehyde and permeabilized with 0.5% Triton X-100. Immunostaining of NS3 was performed according to standard protocols by using rabbit anti-NS3 sera and goat antibodies conjugated to Alexa Fluor 488 (Molecular Probes, Eugene, OR). Nuclear DNA was stained with DAPI (Molecular Probes).
A 25 μL reverse transcription quantitative polymerase chain reaction (RT-qPCR) reaction contained 12.5 μL 2× reaction buffer (Eurogentec, Seraing, Belgium), 6.3 μL H2O, 5 μL total cellular RNA extract, and in the case of Huh 9–13 samples, 300 nmol/L neo-forward primer [5′-CCG GCT ACC TGC CCA TTC-3′], 300 nmol/L neo-reverse primer [5′-CCA GAT CAT CCT GAT CGA CAA G-3′], 300 nmol/L neo-probe [5′-FAM-ACA TCG CAT CGA GCG AGC ACG TAC-TAMRA-3′] or for Huh mono samples 300 nmol/L UTR-forward primer [5′-ACG CAG AAA GCG TCT AGC CAT GGC GTT AGT-3′], 300 nmol/L UTR-reverse primer [5′-TCC CGG GGC ACT CGC AAG CAC CCT ATC AGG-3′], 300 nmol/L UTR-probe [5′-FAM-TGG TCT GCG GAA CCG GTG AGT ACA CC-TAMRA-3′]. The RT step was performed at 48°C for 30 minutes, 15 minutes at 95°C, and subsequent PCR amplification of 40 cycles of denaturation at 94°C for 20 seconds and annealing and extension at 60°C for 1 minute in an ABI 7000 sequence detector. All samples were analyzed in three replicate reactions.
Huh 5-2, Huh 9-13, or Huh mono cells were seeded at a density of 5 × 103 cells per well of a 96-well plate in complete DMEM with the appropriate concentrations of G418 or hygromycin. Serial dilutions of the test compounds in complete DMEM without or G418 or hygromycin were added 24 hours after seeding. Cells were allowed to proliferate for 3 days at 37°C, after which the cell number was determined by means of the (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium)/phenazinemethosulfate method (Promega). The 50% cytostatus concentration (CC50) was defined as the concentration that inhibited the proliferation of exponentially growing cells by 50%.
The effects of drug–drug combinations were evaluated using the method of Prichard and Shipman.36 In brief, the theoretical additive effect is calculated from the dose–response curves of individual compounds by the equation Z = X + Y(1 − X), where X represent the inhibition produced by DEBIO-025 or CsA alone and Y represents IFN-α 2a alone. Z represents the effect produced by the combination of either DEBIO-025 or CsA with IFN-α 2a. The theoretical additive surface is subtracted from the actual experimental surface, resulting in a horizontal surface that equals the zero plane when the combination is additive, a surface that lies above the zero plane indicates a synergistic effect of the combination, and a surface below the zero plane indicates antagonism. The antiviral assay was carried out essentially as described for Huh 5-2 cells except that compounds were added in checkerboard format. For each compound, three replicate plates were used to measure the dose–response curve of each individual compound. The data obtained from all three plates were used to calculate the theoretical additive surface. Combination studies for each pair of compounds were also done in triplicate. Data were analyzed for variance by the ANOVA test.
HCV Replicon Clearance and Rebound.
Huh 9–13 cells were seeded in a 25-cm2 T-flask at a density of 3 × 105 cells per culture flask in complete DMEM (without G418) that contained either no antiviral compounds or contained CsA, or DEBIO-025 (both at 0.5 or 1 μg/mL). Cells were grown until 90% confluency was reached (on average about 4 days later). Subsequently, cells were trypsinized, their numbers counted with a Coulter counter, after which 3 × 105 cells were seeded in a new 25-cm2 T-flask containing the same concentration of compound; 1.5 × 105 cells from each flask were lysed in RLT buffer and stored at −80°C until further use. In total, cells were passaged 7 consecutive times in the presence or absence of compound; after which all cultures were further passaged two more times in complete DMEM (without CsA or DEBIO-025) that contained 1,000 μg/mL G418. Again 3 × 105 cells were seeded in a new T-flask and 1.5 × 105 cells were lysed in RLT buffer. After all samples had been collected, RNA was extracted and samples were analyzed by RT-qPCR for their replicon content.
Structure of DEBIO-025.
The chemical structure of DEBIO-025 is depicted in Fig. 1A. DEBIO-025 is a cyclosporin differing from the parent cyclic undecapeptide at position 3, with sarcosine having been replaced by Me-Alanine, at position 4 with leucine having been replaced by valine and with the nitrogen being N-ethylated instead of N-methylated.
Antiviral Activity of DEBIO-025 in HCV Subgenomic Replicon Containing Huh 5-2 Cells.
Both CsA (Fig. 2B), and DEBIO-025 (Fig. 2A) inhibit HCV replication (as monitored by measuring the luciferase activity) in a concentration-dependent manner, in Huh 5-2 cells. The 50% effective concentration (EC50) of DEBIO-025 was calculated at 0.27 ± 0.03 μg/mL, which is 10-fold lower than the EC50 (2.8 ± 0.4 μg/mL) obtained for CsA in the same cell line (Table 1). When the effect of both compounds was assessed on exponentially growing Huh 5-2 cells, DEBIO-025 proved to be less cytostatic (CC50 ≥ 27 μg/mL) than CsA (CC50 = 12 ± 6 μg/mL). A selectivity index (CC50/EC50) of approximately 900 was calculated for DEBIO-025, whereas this value was approximately 40 for CsA. For comparative reasons, the antiviral activity of both a nucleoside and a non-nucleoside HCV polymerase inhibitor, 2′-C-methyl-cytidine (24), 2-[4-[[-(acetylamino)-4′-chloro-[1,1′-biphenyl]-2-yl]methoxy]phenyl-1-cyclohexyl-1H-benzimidazole-5-carboxylic acid25 and an HCV protease inhibitor (VX-950)26–28 was determined in Huh 5-2 cells. The EC50 values for these three compounds were, respectively, 0.1 ± 0.06 μg/mL, 0.66 ± 0.09 μg/mL, and 0.4 ± 0.06 μg/mL. DEBIO-025 proved also more potent (4-fold) than CsA in Huh 5-2 cells, when the antiviral activity was assessed by means of RT-qPCR assay (Fig. 3). The EC50 values obtained using this latter assay were somewhat higher than when the antiviral activity was assessed by the luciferase assay (Table 1).
Table 1. Inhibitory Effect of DEBIO-025 and CsA on Various HCV Replicons
0.27 ± 0.03
2.8 ± 0.4
12 ± 6
0.27 ± 0.05
1.2 ± 0.2
12 ± 6
0.12 ± 0.01
26 ± 3
0.7 ± 0.3
13 ± 4
0.08 ± 0.02
27 ± 11
0.3 ± 0.1
5.5 ± 0.5
The potent activity of DEBIO-025 was also confirmed in Huh 9–13 cells. Huh 9–13 replicons have the same genetic makeup as Huh 5-2 replicons, but (1) carry different adaptive mutations and (2) a higher HCV replicon RNA content than Huh 5-2 cells. Also in Huh 9–13 cells, DEBIO-025 resulted in a concentration-dependent decrease in HCV RNA levels as measured by RT-qPCR (Fig. 3B) and proved about 6-fold more potent than CsA (Table 1).
To exclude that the antiviral activity of DEBIO-025 or CsA was caused by inhibition of the activity of the internal ribosomal entry site (IRES) of the encephalomyocarditis virus (ECMV), the effect of DEBIO-025 and CsA was also evaluated in Huh mono cells containing a monocistronic HCV replicon that lacks an ECMV IRES. In addition, this replicon carries a hygromycin phosphotransferase gene instead of the neo-gene of Huh 5-2 and Huh 9-13 replicons. Again DEBIO-025 resulted in a concentration-dependent reduction of viral RNA levels (Fig. 3C) with an EC50 of 0.08 ± 0.02 μg/mL (CC50: 27 ± 11 μg/mL) and proved, also in this cell line, more potent (4-fold) than CsA (Table 1).
Antiviral Activity of DEBIO-025 in Cultures Infected With HCV.
The anti-HCV activity of CsA and DEBIO-025 was next studied in either Huh 7-Lunet or Huh 7.5 cells that had been infected with cell culture–grown HCV designated Jc1, or the analogous virus that was modified to carry a genome with an integrated luciferase receptor gene (Jc1-Luc). After infection with HCV, cultures were incubated for 72 hours, in the presence of serial dilutions of CsA or DEBIO-025, after which time cells that had been infected with the Jc1-Luc virus were analyzed for luciferase activity (Fig. 4A–B), and cells that had been infected with the Jc1 virus were stained with an anti-NS3 antibody, and antigen expression was visualized by an immunofluorescence assay (Fig. 5). At the highest concentration tested (10 μg/mL), both compounds reduced somewhat the total cell number (Fig. 5A–B). Treatment of Jc1-Luc–infected Huh 7-Lunet or Huh 7.5 cells with either CsA or DEBIO-25 resulted in a dose-dependent inhibition of luciferase activity (Fig. 4A–B). The luciferase activity in Huh 7-Lunet cells was reduced to background levels at a concentration of 0.37 μg/mL; an approximately 10-fold higher concentration of CsA was needed to result in the same antiviral effect (Fig. 4A). In Huh 7.5 cells, DEBIO-025 proved more effective than CsA (Fig. 4B). Similarly, when either Huh 7-Lunet or Huh 7.5 cells were infected with the Jc1 virus, DEBIO-025 proved invariably more potent in inhibiting viral replication as assessed by monitoring viral antigen production in infected cells (Fig. 5A–B).
The combined antiviral effects of IFN-α 2a with either DEBIO-025 or CsA in Huh 5-2 cells were studied. A slight synergistic activity was noted at the highest concentrations of IFN-α 2a used, but overall the combined anti-HCV activity of either DEBIO-025 or CsA with IFN-α 2a can be considered as additive (Fig. 6).
HCV Replicon Clearance and Rebound.
To study whether the more potent anti-HCV activity of DEBIO-025 as compared with CsA is also reflected in the ability of the compound to more efficiently clear Huh 9–13 cells from their replicon, these latter cells were cultured (without G418 pressure) in the presence of either CsA or DEBIO-025 (both at 0.5 or 1 μg/mL) or were left untreated for seven consecutive passages (Fig. 7). One extra culture was grown in the absence of CsA or DEBIO-025 but under selective pressure of G418. After seven passages, no significant difference in HCV replicon content was noted between Huh 9–13 cells that were grown (without antiviral) under, or without selective pressure of, G418. After G418 selection was reinitiated, both cultures continued to grow normally, indicating that culturing Huh 9–13 for seven passages without G418 pressure does not result in a drop in the replicon content. In Huh 9–13 cells that had been treated with CsA, in the absence of G418, a rapid decrease in HCV replicon content was already noted after the first passage. Another six consecutive passages of these cells in the presence of CsA did not result in a further reduction of their replicon content. Treatment with DEBIO-025 resulted in a more pronounced drop in HCV replicon content during the first passage, and after three consecutive passages in the presence of DEBIO-025, HCV replicon levels dropped below the detection limit. To confirm that Huh 9–13 cells that had been treated for 7 days with DEBIO-025 were indeed cleared from their replicon, G418 selection was restarted for two more passages. Only those cells that still carry the HCV replicon will be able to proliferate under these conditions, and cells without replicon will die in the presence of G418. Indeed, when the DEBIO-025–treated Huh 9–13 cells were again cultured in the presence of G418, the cultures died (Fig. 7). DEBIO-025 was able to clear Huh 9–13 cells from their HCV replicon. Both cultures that had been treated with CsA for 7 consecutive passages were able to proliferate in the presence of G418.
CsA was recently shown to inhibit HCV replication in vitro.17, 18 Here we report on the potent anti-HCV activity of DEBIO-025, a non-immunosuppressive cyclosporin. Neither CsA nor DEBIO-025 proved active against other members of the family of the Flaviviridae, such as a pestvirus (the bovine viral diarrhea virus in MDBK) and a flavivirus [the yellow fever virus in Vero or HepG2 cells] (our unpublished data). We initially identified DEBIO-025 as a more potent inhibitor of HCV replication using (a luciferase containing) HCV subgenomic replicon. The more pronounced antiviral activity of DEBIO-025 as compared with CsA was confirmed using two other HCV replicon constructs. These included a construct that lacks the ECMV IRES and thus excludes the possibility of observed activity being caused by inhibition of this non-HCV IRES. Recently, robust HCV cell culture systems have been elaborated.33, 34, 37 The activity of DEBIO-025 and CsA was studied in hepatoma cells infected with a full-length infectious chimeric genome between HCV strains J6 and JFH1.33 In this assay, both DEBIO-025 and CsA resulted in a dose-dependent antiviral activity, whereby DEBIO-025 again proved superior over CsA, corroborating the data obtained with the subgenomic replicons.
We next studied whether DEBIO-025 and CsA are able to clear cells from their HCV replicon. A compound that is able to clear cells from their replicon in cell culture may have the potential to eradicate the virus from the hepatocytes of infected patients and thus result in a sustained virological response. Whereas DEBIO-025 rapidly and efficiently cleared cells from their replicon, CsA at equimolar concentrations was unable to do so. CsA also may have the capacity to clear cells from their replicon at higher drug concentrations. However, because CsA exhibits some cytostatic effect at a concentrations of 10 μg/mL and because replication of subgenomic HCV replicons is influenced by cytostatic activities, higher concentrations of CsA were not tested in this assay.
If DEBIO-025 were used in the clinical setting for the treatment of HCV infections, it may be in combination with interferon alpha 2a. We therefore studied the anti-HCV activity of this combination, which proved, as did the combination of IFN-α 2a and CsA, to be mostly additive. This is not unexpected for two drugs that have a different mode of action and for which it may be assumed that one does not interfere with the functioning of the other and vice versa.
DEBIO-025 is a chemically modified cyclosporin and contains, compared with CsA, three distinct modifications. Two modifications are located in the presumed calcineurin-binding domain of DEBIO-025 and result in a low affinity of DEBIO-025 for calcineurin. As a consequence, DEBIO-025 lacks an immunosuppressive effect, as evidenced from the following observations: (data not shown) (i) in vitro, DEBIO-025 is 7.000-fold less active than CsA in inhibiting interleukin-2 production by Jurkat cells; (ii) DEBIO-025 is inactive in an in vitro model of the proliferative response of CD4+ peripheral blood mononuclear cells, whereas CsA, tested in parallel at the same concentrations, results in a clear immunosuppressive activity; (iii) daily administration of DEBIO-025 to rats for 4 consecutive weeks has no effect on the IgM immune response to Keyhole Limpet hemocyanin, whereas treatment with CsA, at the same dosages, results in a marked decrease in IgM production; (iv) treatment of rats with CsA, but not DEBIO-025, results in a significant decrease in cytotoxic T cells. Whereas the calcineurin binding domain of DEBIO-025 contains two modifications as compared with CsA, only one modification is found in the cyclophilin binding domain; that is, the glycine residue at position 3 in CsA is modified to an alanine residue in DEBIO-025. This results in an approximately threefold increase in the affinity of DEBIO-025 for cyclophilin A (to be reported elsewhere).
Like CsA, DEBIO-025 is also endowed with anti-HIV-1 activity and is approximately 10-fold more potent than CsA against HIV-1.38 Indeed, during the HIV-1 replication cycle, cyclophilin A is recruited to the HIV-1 capsid and saturates the presumed binding sites for TRIM5α/Ref1 host cell restriction factor on the Gag protein. CsA, and also DEBIO-025, block the binding of cyclophilin A to the capsid of HIV-1 and hence are expected to enable the TRIM5α/Ref1 host cell restriction factor to exert its action.5 Also, the CsA analog NIM 811 ([4-methyl-Ile]-cyclosporin) exhibits anti–HIV-1 activity, without being immunosuppressive.39–43
Our data, as well as the findings of Watashi and colleagues,18 provide compelling evidence that the anti-HCV activity of CsA is indeed independent from its immunosuppressive activity. Because DEBIO-025 inhibits the replication of HCV subgenomic replicons, which do not encode structural proteins and thus do not form particles, the mode of action of DEBIO-025 against HCV cannot be linked to an effect on structural proteins and is thus different from the mechanism used by the compound to exhibit its anti–HIV-1 activity. The most likely mechanism for CsA and DEBIO-025 inhibition of HCV replication is via inhibition of the PPIase (peptidyl-propyl cis/trans isomerase) activity of cyclophilins. The anti-HCV activity of CsA was shown not to involve the calcineurin/NFAT-mediated pathway that the compound shares with FK506, an immunosuppressive drug lacking anti-HCV activity.44 Cyclosporin D (CsD), an analog of CsA with weak immunosuppressive activity but retaining the ability to bind cyclophilins, was also able to inhibit HCV replication,44 and NIM811 also retains activity against HCV in vitro.18
Watashi and colleagues reported recently that cyclophilin B (CypB) acts as a functional regulator of the HCV RNA-dependent RNA polymerase and that the anti-HCV activity of CsA is mediated by disturbing this interaction.45 This is in line with the observations of Nakagawa and colleagues,44 who observed a reduction in HCV replication after the knockdown of cyclophilin A, B, or C.44 The precise binding affinity of DEBIO-025 for Cyp B will be the subject of further study as well as the ability of the compound to disturb the interaction between Cyp B and the RNA-dependent RNA polymerase of HCV.
The fact that both CsA and DEBIO-025 are inactive against other members of the family of the Flaviridae, (i.e., pesti- and flaviviruses) indicates that cyclophilins are, in contrast to the situation for HCV, not essential for the replication of these viruses. Alternatively, cyclophilins may not be the target of CsA and DEBIO-025.
In conclusion, we report on the potent anti-HCV activity of the non-immunosuppressive cyclosporin DEBIO-025. In humans, DEBIO-025 has linear dose-dependent plasma pharmacokinetics.46 Plasma levels up to 10- to 50-fold the EC50 value for inhibition of HCV replicon replication in vitro can be achieved in humans.46 Tissue distribution, performed by whole body autoradiography in rats showed high tissue-to-blood radioactivity ratios, favoring liver penetration (data not shown). Sufficiently high, antivirally active, concentrations of DEBIO-025 likely can be achieved in the human liver. Moreover, DEBIO-025 is endowed with a strong anti-HIV activity. Combined with the favorable pharmacokinetic and toxicity profile in initial clinical trials, the compound may prove an interesting candidate drug for the treatment of HCV infections, particularly in HIV-1/HCV–co-infected patients.
The authors thank Katrien Geerts for excellent technical assistance and Dominique Brabants, Chantal Biernaux and Christiane Callebaut for dedicated editorial help.