Cyclophilin Inhibitors Research Articles

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.

Use of a Real-Time Fluorescence Monitoring System for High-Throughput Screening for Prolyl Isomerase Inhibitors

Cyclophilin is a ubiquitous peptidyl prolyl cis/trans isomerase that plays critical roles in many biological processes. A number of cyclophilin inhibitors have been designed based on the structure of the immunosuppressant cyclosporin A. To discover inhibitors that have other structures, the authors established the high-throughput screening (HTS) method using FDSS6000 real-time fluorescence detector. The inhibitors identified with this HTS showed significant correlation with direct interaction as measured by surface plasmon resonance. This high-throughput assay system is a powerful tool for the discovery of peptidylprolyl isomerase inhibitors.

The cyclophilin inhibitor Debio 025 combined with PEG IFNα2a significantly reduces viral load in treatment-naïve hepatitis C patients #

The anti-hepatitis C virus (HCV) effect and safety of three different oral doses of the cyclophilin inhibitor Debio 025 in combination with pegylated interferon-alpha2a (PEG IFN-alpha2a) were investigated in a multicenter, randomized, double-blind, placebo-controlled escalating dose-ranging phase II study in treatment-naïve patients with chronic hepatitis C. Doses of 200, 600, and 1,000 mg/day Debio 025 in combination with PEG IFN-alpha2a 180 microg/week for 4 weeks were compared with monotherapy with either 1,000 mg/day Debio 025 or 180 microg/week PEG IFN-alpha2a. In patients with genotypes 1 and 4, the 600- and 1,000-mg combination treatments induced a continuous decay in viral load that reached -4.61 +/- 1.88 and -4.75 +/- 2.19 log(10) IU/mL at week 4, respectively. In patients with genotypes 2 and 3, HCV RNA levels at week 4 were reduced by -5.91 +/- 1.11 and -5.89 +/- 0.43 log(10) IU/mL, respectively, with the same treatment regimens. Adverse events were comparable between treatment groups apart from a higher incidence of neutropenia associated with PEG IFN-alpha2a and an increased incidence of isolated hyperbilirubinemia at the highest dose of Debio 025 (1,000 mg/day). These results confirm that Debio 025 has a potent activity and an additive effect on HCV RNA reduction in genotype 1 and 4 patients at 600 and 1,000 mg/day when combined with PEG IFN-alpha2a.

Refinement and 3D‐QSAR Studies of Inhibitors of Cyclophilin A Containing Amide Linker

Cyclophilin A (CypA) is a ubiquitous cellular enzyme playing essential role in many biological processes, and the discovery of CypA inhibitor is now of special interest in the treatment of immunological disorders. In this work, molecular modeling studies were performed to develop a predictive Common Pharmacophore Hypothesis (CPH) and use it for alignment in 3D‐QSAR studies using CoMFA and CoMSIA. A total of 30 compounds containing an amide fragment as the key linker, consisting of 17 of our previously discovered CypA inhibitors and 13 other inhibitors reported in the literature, were selected for pharmacophore refinement and 3D‐QSAR studies. The best pharmacophore hypothesis AADR, which had two hydrogen bond acceptors, a hydrogen bond donor, and an aromatic ring, was obtained and used for the alignment of molecules in CoMFA and CoMSIA model development. The models showed a good r 2 value of 0.992 and 0.949 for CoMFA and CoMSIA, respectively. The contour maps of the models were analyzed to give structural insight for activity improvement of future novel CypA inhibitors. The CPH can also provide a powerful template for virtual screening and design of new CypA inhibitors.

Gene-mediated Restoration of Normal Myofiber Elasticity in Dystrophic Muscles

Dystrophin mediates a physical link between the cytoskeleton of muscle fibers and the extracellular matrix, and its absence leads to muscle degeneration and dystrophy. In this article, we show that the lack of dystrophin affects the elasticity of individual fibers within muscle tissue explants, as probed using atomic force microscopy (AFM), providing a sensitive and quantitative description of the properties of normal and dystrophic myofibers. The rescue of dystrophin expression by exon skipping or by the ectopic expression of the utrophin analogue normalized the elasticity of dystrophic muscles, and these effects were commensurate to the functional recovery of whole muscle strength. However, a more homogeneous and widespread restoration of normal elasticity was obtained by the exon-skipping approach when comparing individual myofibers. AFM may thus provide a quantification of the functional benefit of gene therapies from live tissues coupled to single-cell resolution.

An evaluation of the cyclophilin inhibitor Debio 025 and its potential as a treatment for chronic hepatitis C

Debio 025 is a cyclophilin (Cyp) inhibitor without calcineurin-binding properties. The drug inhibits viral replication of genotype 1b and 2a replicons in nanomolar concentrations and shows an additive to synergistic antiviral effect with interferon, ribavirin, and specifically targeted antiviral therapy for hepatitis C (STAT-C) drugs. There is no cross-resistance with protease and polymerase inhibitors. In humans, Debio 025 has shown activity against genotypes 1, 2, 3, and 4, and displays an additive antiviral effect with pegylated interferon (peg-IFN)α2a in genotype 1 and 4 patients. The most prominent side effect is reversible hyperbilirubinaemia caused by inhibition of biliary transporters. Debio 025 is a potent anti-HCV drug, with a novel mechanism of action and an efficacy profile that makes it an attractive candidate for combination with current and future HCV treatments.

Perspectives in therapy for hepatitis C

Background: Chronic hepatitis C virus (HCV) infection is a worldwide health problem. Response rates to current standard of treatment (pegylated IFNs and ribavirin) are low in patients with the prevailing genotype 1. Objective: To review papers published or presented at recent international meetings showing the results of trials of new anti-HCV drugs. Methods: Literature search using the terms ‘antivirals’, ‘interferon’, ‘pegylated interferon’, ‘ribavirin’, ‘polymerase inhibitors’, ‘protease inhibitors’, ‘cyclophilin inhibitors’ and ‘hepatitis C virus’. Search of abstracts containing the same terms in the title and presented at the American Association for the Study of Liver Diseases 2007 and at the European Association for the Study of the Liver 2008 meetings. Results/conclusion: Preliminary results of Phase II studies of new compounds look promising, although side effects are higher than with current standard of treatment.

Interferon-based therapy for chronic hepatitis C: current and future perspectives

Pegylated interferon alpha (peginterferon alpha) plus ribavirin is the current mainstay of treatment for patients with chronic HCV infection. When peginterferon alpha plus ribavirin is administered for the standard duration, a sustained virological response is achieved in around 50% of patients infected with HCV genotype 1 and around 80% of patients infected with HCV genotype 2 or 3. Data now suggest that treatment duration can be shortened or lengthened depending on baseline viral load and/or early on-treatment viral kinetics, offering the prospect of individualizing therapy further to improve response or to prevent treatment from being unnecessarily extended. Further efforts to optimize therapy are likely to involve the use of new anti-HCV agents, several of which are currently in the early stages of development. These agents include HCV protease inhibitors (particularly those against NS3-4A protease), HCV polymerase inhibitors (including both nucleoside and non-nucleoside analogs) and cyclophilin inhibitors. These compounds will be used, at least initially, in combination with peginterferon alpha plus ribavirin, extending the pivotal role of interferon-based therapy in the management of chronic hepatitis C.

Investigation of Debio 025, a cyclophilin inhibitor, in the dystrophic mdx mouse, a model for Duchenne muscular dystrophy

Duchenne muscular dystrophy (DMD) is a severe muscle wasting disorder caused by the absence of the cytoskeletal protein dystrophin. This leads to muscle cell death accompanied by chronic inflammation. Cyclosporin A (CsA) is a powerful immunosuppressive drug, which has been proposed for DMD treatment. CsA also directly regulates the mitochondrial permeability transition pore (mPTP), which participates in cell death pathways through the inhibition of cyclophilin D. Here, we evaluated whether Debio 025, a cyclophilin inhibitor with no immunosuppressive activity, improves the dystrophic condition in a mouse model of DMD, through regulation of mPTP. The potency of Debio 025 to protect mouse dystrophic cells against mitochondria-mediated death was assessed by caspase-3 activity and calcium retention capacity assays. Mdx(5Cv) mice (3-week-old) were treated daily by gavage for 2 weeks with Debio 025 (10, 30 or 100 mg kg(-1)), CsA (10 mg kg(-1)) or placebo. The effects on muscle necrosis and function were measured. In vitro investigations showed protective effect of low concentrations of Debio 025 against cell death. Histology demonstrated that Debio 025 partially protected the diaphragm and soleus muscles against necrosis (10 and 100 mg kg(-1), respectively). Hindlimb muscles from mice receiving Debio 025 at 10 mg kg(-1) relaxed faster, showed alteration in the stimulation frequency-dependent recruitment of muscle fibres and displayed a higher resistance to mechanical stress. Debio 025 partially improved the structure and the function of the dystrophic mouse muscle, suggesting that therapies targeting the mPTP may be helpful to DMD patients.

Mitochondrial Defects and Dysfunction in Calcium Regulation in Glaucomatous Trabecular Meshwork Cells

Disruption in intracellular calcium ion (Ca(2+)) homeostasis has major effects on health. Persistent Ca(2+) overload induces mitochondrial permeability transition pore (MPTP) opening, which prompts mitochondrial release of calcium (mCICR) and reactive oxygen species (ROS) into the cytosol which, in turn, compromises mitochondrial function. This study was conducted to examine intracellular Ca(2+) levels and mitochondrial vulnerability to Ca(2+) stress in trabecular meshwork (TM) of individuals with primary open-angle glaucoma (POAG). Primary cultures of TM cells from POAG (GTM) and age-matched, nondiseased (NTM) eyes, obtained from postmortem donors eyes by standard surgical trabeculectomy, were treated with the following calcium regulators: the mitochondrial respiratory chain I inhibitor rotenone (ROT); the mitochondrial permeability transition pore (MPTP) inhibitors cyclosporine (Cys) and aristolochic acid (ArA); the Ca(2+) chelators BAPTA/AM or EDTA; the mitochondrial Ca(2+) uniporter inhibitor ruthenium red (RR); the Ca(2+)/Na(+) exchanger inhibitor trifluoperazine; and the inositol 1,4,5-triphosphate receptor type 3 (IP3R) inhibitors 2-aminoethoxydiphenyl borane (2-APB) and xestospongin C (Xe-C). Ca(2+) concentrations in the cytoplasm ([Ca(2+)](c)) and mitochondria ([Ca(2+)](m)) were determined by confocal microscopy and flow cytometry with the fluorescent Ca(2+) indicators fluo-3/AM and rhod-2/AM, respectively. Mitochondrial membrane potential (DeltaPsim) was examined with the fluorescent probe tetramethylrhodamine ethyl ester (TMRE). The expression of cyclophilin D, a protein that induces MPTP opening was also measured. There was increased [Ca(2+)](c), [Ca(2+)](m), mCICR, MPTP opening, and expression of cyclophilin D and decreased DeltaPsim in POAG TM cells compared with control cells. ROT artificially exacerbated these conditions in GTM cells. Chelation of [Ca(2+)](c) and inhibition of IP3R and MPTP opening suppressed mitochondrial dysfunction and reduced the additional effects of ROT in GTM cells. POAG TM cells have defective mitochondrial function, which causes them to be abnormally vulnerable to Ca(2+) stress. The dysfunction in calcium regulation by these cells may contribute to the failure of this tissue to control IOP. Pharmacologic inhibitors of IP3R, MPTP opening, and cyclophilin D could have clinical implications for primary open-angle glaucoma.

Combinations of Cyclophilin Inhibitor NIM811 with Hepatitis C Virus NS3-4A Protease or NS5B Polymerase Inhibitors Enhance Antiviral Activity and Suppress the Emergence of Resistance

Chronic hepatitis C virus (HCV) infection remains a major global health burden while current interferon-based therapy is suboptimal. Efforts to develop more effective antiviral agents mainly focus on two viral targets: NS3-4A protease and NS5B polymerase. However, resistant mutants against these viral specific inhibitors emerge quickly both in vitro and in patients, particularly in the case of monotherapy. An alternative and complementary strategy is to target host factors such as cyclophilins that are also essential for viral replication. Future HCV therapies will most likely be combinations of multiple drugs of different mechanisms to maximize antiviral activity and to suppress the emergence of resistance. Here, the effects of combining a host cyclophilin inhibitor NIM811 with other viral specific inhibitors were investigated in vitro using HCV replicon. All of the combinations led to more pronounced antiviral effects than any single agent, with no significant increase of cytotoxicity. Moreover, the combination of NIM811 with a nucleoside (NM107) or a non-nucleoside (thiophene-2-carboxylic acid) polymerase inhibitor was synergistic, while the combination with a protease inhibitor (BILN2061) was additive. Resistant clones were selected in vitro with these inhibitors. Interestingly, it was much more difficult to develop resistance against NIM811 than viral specific inhibitors. No cross-resistance was observed among these inhibitors. Most notably, NIM811 was highly effective in blocking the emergence of resistance when used in combination with viral protease or polymerase inhibitors. Taken together, these results illustrate the significant advantages of combining inhibitors targeting both viral and host factors as key components of future HCV therapies.

The oral cyclophilin inhibitor DEBIO 025* has a "significant antiviral effect" against different genotypes of the hepatitis C virus

The oral cyclophilin inhibitor DEBIO 025* has a "significant antiviral effect" against different genotypes of the hepatitis C virus

Genetic and pharmacologic inhibition of mitochondrial-dependent necrosis attenuates muscular dystrophy.

Muscular dystrophies comprise a diverse group of genetic disorders that lead to muscle wasting and, in many instances, premature death. Many mutations that cause muscular dystrophy compromise the support network that connects myofilament proteins within the cell to the basal lamina outside the cell, rendering the sarcolemma more permeable or leaky. Here we show that deletion of the gene encoding cyclophilin D (Ppif) rendered mitochondria largely insensitive to the calcium overload-induced swelling associated with a defective sarcolemma, thus reducing myofiber necrosis in two distinct models of muscular dystrophy. Mice lacking delta-sarcoglycan (Scgd(-/-) mice) showed markedly less dystrophic disease in both skeletal muscle and heart in the absence of Ppif. Moreover, the premature lethality associated with deletion of Lama2, encoding the alpha-2 chain of laminin-2, was rescued, as were other indices of dystrophic disease. Treatment with the cyclophilin inhibitor Debio-025 similarly reduced mitochondrial swelling and necrotic disease manifestations in mdx mice, a model of Duchenne muscular dystrophy, and in Scgd(-/-) mice. Thus, mitochondrial-dependent necrosis represents a prominent disease mechanism in muscular dystrophy, suggesting that inhibition of cyclophilin D could provide a new pharmacologic treatment strategy for these diseases.

The cyclophilin inhibitor Debio-025 shows potent anti–hepatitis C effect in patients coinfected with hepatitis C and human immunodeficiency virus

Debio-025 is an oral cyclophilin (Cyp) inhibitor with potent anti-hepatitis C virus activity in vitro. Its effect on viral load as well as its influence on intracellular Cyp levels was investigated in a randomized, double-blind, placebo-controlled study. Mean hepatitis C viral load decreased significantly by 3.6 log(10) after a 14-day oral treatment with 1200 mg twice daily (P < 0.0001) with an effect against the 3 genotypes (1, 3, and 4) represented in the study. In addition, the absence of viral rebound during treatment indicates that Debio-025 has a high barrier for the selection of resistance. In Debio-025-treated patients, cyclophilin B (CypB) levels in peripheral blood mononuclear cells decreased from 67 +/- 6 (standard error) ng/mg protein (baseline) to 5 +/- 1 ng/mg protein at day 15 (P < 0.01). Debio-025 induced a strong drop in CypB levels, coinciding with the decrease in hepatitis C viral load. These are the first preliminary human data supporting the hypothesis that CypB may play an important role in hepatitis C virus replication and that Cyp inhibition is a valid target for the development of anti-hepatitis C drugs.

Chemical biology for revealing a life cycle of hepatitis C virus

It is one of the major subjects in the virology field to develop novel anti-viral strategies as well as to reveal the mechanism of viral replication. We have developed a method of chemical compound-directed analysis on viral replication. Until now, we rediscovered cyclosporin A and tamoxifen as anti-hepatitis C virus (HCV) compounds. Through analysis using these compounds, it was revealed that host cell factors cyclophilin (CyP) and estrogen receptor were important for HCV replication. CyP was demonstrated to serve as a new target of the development for anti-HCV agents and CyP inhibitors are now under clinical trials. Thus, an application of chemical biology to virology would provide not only mechanistic aspects of viral life cycles but also new anti-viral strategies.

Structure-based discovery of a family of synthetic cyclophilin inhibitors showing a cyclosporin-A phenotype in Caenorhabditis elegans

Cyclophilins, which are found in all cellular compartments and with diverse biological roles, are now drug targets for a number of diseases including HIV infection, malaria and ischaemia. We used the database-mining program LIDAEUS and in silico screening to discover the dimedone family of inhibitors which show a conserved ‘ball and socket’ binding mode with a dimethyl group in the hydrophobic binding pocket of human cyclophilin A (CypA) mimicking a key interaction of the natural inhibitor cyclosporin A (CsA). The most potent derivative binds CypA with a K d of 11.2 ± 9.2 μM and an IC 50 for activity against Caenorhabditis elegans (C. elegans) of 190 μM compared to 28 μM for CsA. These dimedone analogues provide a new scaffold for the synthesis of families of peptidomimetic molecules with potential activity against HIV, malaria, and helminth parasite infections.

Cyclophilin inhibitors in hepatitis C viral infection

Cyclophilins (Cyps) are proteins that are ubiquitously present with peptidyl-prolyl cis-trans isomerase activity and play an important role in de novo protein folding and in isomerization of native proteins in several cellular systems. There is growing evidence that indicates CypB is a positive modulator of the HCV RNA-dependent RNA polymerase in the replication complex. Early in vitro and animal data with selective Cyp inhibitors show a potent anti-HCV effect. This anti-HCV effect was confirmed in the first patient study with the selective Cyp inhibitor Debio-025. Preclinical data suggest that Cyp inhibitors may present a higher barrier to the selection of resistance than protease and polymerase inhibitors and that a combination of Cyp inhibitors with either of these drugs or interferon results in additive or synergistic anti-HCV activity. By interfering at the level of host–viral interaction, Cyp inhibition may open the way for a novel approach to anti-HCV treatment that could be complementary, not only to interferon-based treatment, but also to future treatments that directly target HCV replication enzymes such as protease and polymerase inhibitors.

Methamphetamine and 3,4-methylenedioxymethamphetamine interact with central nicotinic receptors and induce their up-regulation

Previous work from our group indicated that α7 nicotinic acetylcholine receptors (α7 nAChR) potentially play a role in methamphetamine (METH) and 3,4-methylenedioxymethamphetamine (MDMA) neurotoxicity. The aims of the present study were two-fold: (1) to demonstrate the interaction of METH and MDMA with homomeric α7 nAChR ([ 3H]methyllycaconitine binding) and other heteromeric subtypes ([ 3H]epibatidine binding); and (2) to show the effects of amphetamine derivative pretreatment on the density of binding sites. METH and MDMA displaced [ 3H]methyllycaconitine and [ 3H]epibatidine binding in membranes from NGF-differentiated PC 12 cells and mouse brain, with K i values in the micromolar range, MDMA revealing a greater affinity than METH. In addition, METH and MDMA induced a time- and concentration-dependent increase in [ 3H]methyllycaconitine and [ 3H]epibatidine binding; which had already been apparent after 6 h of pretreatment, and which peaked in differentiated PC 12 cells after 48 h. The highest increases were found in [ 3H]epibatidine binding, with MDMA inducing higher increases than METH. Treatment with METH and MDMA increased B max of high-affinity sites for both radioligands without affecting K d. The heightened binding was inhibited by pretreatment with cycloheximide, suggesting the participation of newly synthesised proteins while inhibition of protein trafficking to plasma membrane did not block up-regulation. The effects of protein kinase and cyclophilin inhibitors on such up-regulation were explored, revealing a rapid, differential and complex regulation, similar to that described for nicotinic ligands. All of these results demonstrate that METH and MDMA have affinity for, and can interact with, nAChR, inducing their up-regulation, specially when higher doses are used. Such effects may have a role in METH- and MDMA-induced neurotoxicity, cholinergic neurotransmission, and in processes related to addiction and dependence.

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.

NIM811, A Cyclophilin Inhibitor, and NM107, An HCV Polymerase Inhibitor, Synergistically Inhibits HCV Replication and Suppresses the Emergence of Resistance In Vitro

NIM811, A Cyclophilin Inhibitor, and NM107, An HCV Polymerase Inhibitor, Synergistically Inhibits HCV Replication and Suppresses the Emergence of Resistance In Vitro