Cyclophilin Inhibitors Research Articles

Influences of cyclosporin A and non-immunosuppressive derivatives on cellular cyclophilins and viral nucleocapsid protein during human coronavirus 229E replication

The well-known immunosuppressive drug cyclosporin A inhibits replication of various viruses including coronaviruses by binding to cellular cyclophilins thus inactivating their cis-trans peptidyl-prolyl isomerase function. Viral nucleocapsid proteins are inevitable for genome encapsidation and replication. Here we demonstrate the interaction between the N protein of HCoV-229E and cyclophilin A, not cyclophilin B. Cyclophilin inhibitors abolish this interaction. Upon infection, cyclophilin A stays evenly distributed throughout the cell, whereas cyclophilin B concentrates at ER-bleb-like structures. We further show the inhibitory potential of non-immunosuppressive CsA derivatives Alisporivir, NIM811, compound 3 on HCoV-229E-GFP and -Luciferase replication in human Huh-7.5 hepatoma cells at 18 and 48 h time points post infection with EC50 s at low micromolar ranges. Thus, non-immunosuppressive CsA derivatives effectively inhibit HCoV-229E replication suggesting them as possible candidates for the treatment of HCoV infection. The interruption of interaction between CypA and N protein by CsA and its derivatives suggest a mechanism how CypA inhibitors suppress viral replication.

Cyclophilin Inhibitor NV556 Reduces Fibrosis and Hepatocellular Carcinoma Development in Mice With Non-Alcoholic Steatohepatitis.

Hepatocellular carcinoma (HCC), the third major cause of cancer mortality, can result from non-alcoholic steatohepatitis (NASH). Due to limited efficacy of drugs approved for HCC and no drug available yet for NASH, identification of new effective treatments is crucial. Here, we investigated whether NV556, a cyclophilin inhibitor derived from sanglifehrins, would decrease the development of NASH and HCC in a preclinical mouse model. For our experiment, male mice were administered streptozotocin to disrupt pancreatic cells and nourished with high-fat diet since 3 weeks of age. Afterward, NV556 or vehicle was orally administered daily for 6 weeks before the 14-week-old time point for the development of NASH, or 10 weeks before the 30-week-old time point for the establishment of HCC. Body weight, blood glucose level, and liver weight were recorded. Moreover, for NASH, livers were histologically examined for inflammation and steatosis. Collagen was measured by Sirius Red staining of hepatic tissues. Systemic cytokine levels in serum were detected by multiplex assays. For HCC, nodules of livers were measured and scored according to a developed system with number and size of nodules as criteria. NV556 significantly decreased collagen deposition (p = 0.0281), but did not alter inflammation, steatosis, body and liver weight, and systemic cytokine production compared to control mice with NASH symptoms. For HCC, NV556 statistically reduced the number (p = 0.0091) and diameter of tumorous nodules (p = 0.0264), along with liver weight (p = 0.0026) of mice.Our study suggests NV556 as a promising candidate for treatment of NASH-derived fibrosis and HCC.

Adaptive Mutations in Replicase Transmembrane Subunits Can Counteract Inhibition of Equine Arteritis Virus RNA Synthesis by Cyclophilin Inhibitors.

Previously, the cyclophilin inhibitors cyclosporine (CsA) and alisporivir (ALV) were shown to inhibit the replication of diverse RNA viruses, including arteriviruses and coronaviruses, which both belong to the order Nidovirales In this study, we aimed to identify arterivirus proteins involved in the mode of action of cyclophilin inhibitors and to investigate how these compounds inhibit arterivirus RNA synthesis in the infected cell. Repeated passaging of the arterivirus prototype equine arteritis virus (EAV) in the presence of CsA revealed that reduced drug sensitivity is associated with the emergence of adaptive mutations in nonstructural protein 5 (nsp5), one of the transmembrane subunits of the arterivirus replicase polyprotein. Introduction of singular nsp5 mutations (nsp5 Q21R, Y113H, or A134V) led to an ∼2-fold decrease in sensitivity to CsA treatment, whereas combinations of mutations further increased EAV's CsA resistance. The detailed experimental characterization of engineered EAV mutants harboring CsA resistance mutations implicated nsp5 in arterivirus RNA synthesis. Particularly, in an in vitro assay, EAV RNA synthesis was far less sensitive to CsA treatment when nsp5 contained the adaptive mutations mentioned above. Interestingly, for increased sensitivity to the closely related drug ALV, CsA-resistant nsp5 mutants required the incorporation of an additional adaptive mutation, which resided in nsp2 (H114R), another transmembrane subunit of the arterivirus replicase. Our study provides the first evidence for the involvement of nsp2 and nsp5 in the mechanism underlying the inhibition of arterivirus replication by cyclophilin inhibitors.IMPORTANCE Currently, no approved treatments are available to combat infections with nidoviruses, a group of positive-stranded RNA viruses, including important zoonotic and veterinary pathogens. Previously, the cyclophilin inhibitors cyclosporine (CsA) and alisporivir (ALV) were shown to inhibit the replication of diverse nidoviruses (both arteriviruses and coronaviruses), and they may thus represent a class of pan-nidovirus inhibitors. In this study, using the arterivirus prototype equine arteritis virus, we have established that resistance to CsA and ALV treatment is associated with adaptive mutations in two transmembrane subunits of the viral replication machinery, nonstructural proteins 2 and 5. This is the first evidence for the involvement of specific replicase subunits of arteriviruses in the mechanism underlying the inhibition of their replication by cyclophilin inhibitors. Understanding this mechanism of action is of major importance to guide future drug design, both for nidoviruses and for other RNA viruses inhibited by these compounds.

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 STATEMENTCyclophilin 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.

Cyclophilin A allows the allosteric regulation of a structural motif in the disordered domain 2 of NS5A and thereby fine-tunes HCV RNA replication

Implicated in numerous human diseases, intrinsically disordered proteins (IDPs) are dynamic ensembles of interconverting conformers that often contain many proline residues. Whether and how proline conformation regulates the functional aspects of IDPs remains an open question, however. Here, we studied the disordered domain 2 of nonstructural protein 5A (NS5A-D2) of hepatitis C virus (HCV). NS5A-D2 comprises a short structural motif (PW-turn) embedded in a proline-rich sequence, whose interaction with the human prolyl isomerase cyclophilin A (CypA) is essential for viral RNA replication. Using NMR, we show here that the PW-turn motif exists in a conformational equilibrium between folded and disordered states. We found that the fraction of conformers in the NS5A-D2 ensemble that adopt the structured motif is allosterically modulated both by the cis/trans isomerization of the surrounding prolines that are CypA substrates and by substitutions conferring resistance to cyclophilin inhibitor. Moreover, we noted that this fraction is directly correlated with HCV RNA replication efficiency. We conclude that CypA can fine-tune the dynamic ensemble of the disordered NS5A-D2, thereby regulating viral RNA replication efficiency.

The cyclophilin inhibitor CRV431 inhibits liver HBV DNA and HBsAg in transgenic mice.

Hepatitis B virus (HBV) infection is a major health burden worldwide with 240 million chronically infected individuals. Nucleos(t)ide analogs and interferons are the current standards of care due to their suppression of HBV replication, but the treatments rarely eradicate HBV from individuals. Similar to current treatments for human immunodeficiency virus type-1 (HIV-1) and hepatitis C virus (HCV) patients, improved HBV therapies will require the combination of multiple drugs which target distinct steps of the HBV life cycle. In this study, we tested the potential of a cyclophilin inhibitor, CRV431, to affect HBV replication in transgenic mice. We found that oral treatment with CRV431 (50 mg/kg/day) for a period of 16 days significantly reduced liver HBV DNA levels and moderately decreased serum HBsAg levels. We observed an additive inhibitory effect on liver HBV DNA levels in mice treated with a combination of low doses of CRV431 (10 mg/kg/day) and the nucleotide prodrug, tenofovir exalidex (TXL), (5 mg/kg/day). No toxicity was observed in CRV431-treated mice. Although it is well known that CRV431 neutralizes the peptidyl-prolyl isomerase activity of cyclophilins, its anti-HBV mechanism(s) of action remains unknown. Nevertheless, this study provides the first demonstration of a beneficial effect of a cyclophilin inhibitor in vivo in an HBV transgenic mouse model. Altogether our data reveal the potential of CRV431 to be part of improved new therapies for HBV patients.

A computationally designed binding mode flip leads to a novel class of potent tri-vector cyclophilin inhibitors.

Cyclophilins (Cyps) are a major family of drug targets that are challenging to prosecute with small molecules because the shallow nature and high degree of conservation of the active site across human isoforms offers limited opportunities for potent and selective inhibition. Herein a computational approach based on molecular dynamics simulations and free energy calculations was combined with biophysical assays and X-ray crystallography to explore a flip in the binding mode of a reported urea-based Cyp inhibitor. This approach enabled access to a distal pocket that is poorly conserved among key Cyp isoforms, and led to the discovery of a new family of sub-micromolar cell-active inhibitors that offer unprecedented opportunities for the development of next-generation drug therapies based on Cyp inhibition. The computational approach is applicable to a broad range of organic functional groups and could prove widely enabling in molecular design.

Delayed Wound Healing in Mice with Impaired Procoagulant Platelet Formation

Platelets are well recognized as a key player in primary hemostasis and thrombosis, but their role in the subsequent initiation of tissue repair is less well characterized. Within the hemostatic thrombus, ballooned platelets reminiscent of the procoagulant or necrotic platelet are first observed in the periphery (extravascular and in contact with collagen) and subsequently throughout the platelet plug. The key role of cyclophilin D (CypD) in the regulation of platelet mitochondrial permeability transition pore (mPTP) and procoagulant platelet formation is well established. Furthermore, the cyclophilin inhibitor cyclosporine is known to both block procoagulant platelet formation in vitroand to result in delayed wound healing in patients. While the consequences of CypD's absence have been studied in vivo in models of venous and arterial thrombosis, the effects on wound healing and tissue repair have not been investigated.Here we investigated the role of CypD-dependent events in the setting of cutaneous wound healing. Punch biopsies (6 mm) were placed and wound diameter (days 0, 1,2, 5,7,10,14) was examined. In mice with a global absence of CypD and in mice with platelet/megakaryocyte-specific CypD deficiency, wound healing was substantially and similarly delayed (n=9 -11 for each group). This demonstrates that platelet CypD-mediated processes play a key role in the initiation of hemostatic thrombus resolution and subsequent wound healing. The initial resolution of wound healing was delayed by ~ 2-3 days. This resulted in a significant difference at days 4 and 7 post-injury in the size of the wounds of these mice (p<0.05). To elaborate possible mechanisms by which platelet CypD-deficiency resulted in delayed wound healing, skin from the area of the wound was harvested at key early time points (day 0,1,2,5) and histological examination was performed. Early in the initiation of wound resolution, polymorphonuclear cells (PMNs) successively infiltrate the platelet plug. In mice lacking CypD, either globally or platelet-specific, polymorphonuclear cell (PMN) infiltration of the resolving thrombus on days 1 and 2 was markedly impaired. However, in all mice, PMNs amassed at the edges of the wound at 2 days post-injury. Furthermore, subsequent wound resolution was dysregulated in the absence of platelet CypD. Wounds from these mice (day 5) demonstrated marked blebbing and structural abnormalities. Remarkably, these delays in wound healing and PMN succession occurred in the absence of either gross or microscopic bleeding consistent with the absence of a defect in initial hemostasis previously reported in platelet-CypD deficient mice.These results indicate that platelets, independent of their hemostatic role, play a key role in the initiation of wound resolution. And that, subsequent to hemostasis, a platelet CypD-mediated process, presumably the procoagulant platelet transition, is required for the timely and orchestrated initiation of wound resolution and successive wound PMN infiltration. These results are consistent with the established in vitro role of CypD in mediating the procoagulant platelet morphologic transition and the procoagulant platelet's known role in mediating neutrophil activation. Finally, our results identify a key platelet-dependent mechanism by which therapy with the immunosuppressant cyclosporine can result in delayed wound healing. DisclosuresJobe:CSL: Consultancy; Shire: Consultancy; Octapharma: Consultancy.

Transmembrane member 16A participates in hydrogen peroxide-induced apoptosis by facilitating mitochondria-dependent pathway in vascular smooth muscle cells.

Transmembrane member 16A (TMEM16A), an intrinsic constituent of the Ca2+ -activated Cl- channel, is involved in vascular smooth muscle cell (VSMC) proliferation and hypertension-induced cerebrovascular remodelling. However, the functional significance of TMEM16A for apoptosis in basilar artery smooth muscle cells (BASMCs) remains elusive. Here, we investigated whether and how TMEM16A contributes to apoptosis in BASMCs. Cell viability assay, flow cytometry, Western blot, mitochondrial membrane potential assay, immunogold labelling and co-immunoprecipitation (co-IP) were performed. Hydrogen peroxide (H2 O2 ) induced BASMC apoptosis through a mitochondria-dependent pathway, including by increasing the apoptosis rate, down-regulating the ratio of Bcl-2/Bax and potentiating the loss of the mitochondrial membrane potential and release of cytochrome c from the mitochondria to the cytoplasm. These effects were all reversed by the silencing of TMEM16A and were further potentiated by the overexpression of TMEM16A. Endogenous TMEM16A was detected in the mitochondrial fraction. Co-IP revealed an interaction between TMEM16A and cyclophilin D, a component of the mitochondrial permeability transition pore (mPTP). This interaction was up-regulated by H2 O2 but restricted by cyclosporin A, an inhibitor of cyclophilin D. TMEM16A increased mPTP opening, resulting in the activation of caspase-9 and caspase-3. The results obtained with cultured BASMCs from TMEM16A smooth muscle-specific knock-in mice were consistent with those from rat BASMCs. These results suggest that TMEM16A participates in H2 O2 -induced apoptosis via modulation of mitochondrial membrane permeability in VSMCs. This study establishes TMEM16A as a target for therapy of several remodelling-related diseases.

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.

Characterization of the Anti-Hepatitis C Virus Activity of New Nonpeptidic Small-Molecule Cyclophilin Inhibitors with the Potential for Broad Anti-Flaviviridae Activity.

ABSTRACTAlthough members of the Flaviviridae display high incidence, morbidity, and mortality rates, the development of specific antiviral drugs for each virus is unlikely. Cyclophilins, a family of host peptidyl-prolyl cis-trans isomerases (PPIases), play a pivotal role in the life cycles of many viruses and therefore represent an attractive target for broad-spectrum antiviral development. We report here the pangenotypic anti-hepatitis C virus (HCV) activity of a small-molecule cyclophilin inhibitor (SMCypI). Mechanistic and modeling studies revealed that the SMCypI bound to cyclophilin A in competition with cyclosporine (CsA), inhibited its PPIase activity, and disrupted the CypA-nonstructural protein 5A (NS5A) interaction. Resistance selection showed that the lead SMCypI hardly selected amino acid substitutions conferring low-level or no resistance in vitro. Interestingly, the SMCypI selected D320E and Y321H substitutions, located in domain II of the NS5A protein. These substitutions were previously associated with low-level resistance to cyclophilin inhibitors such as alisporivir. Finally, the SMCypI inhibited the replication of other members of the Flaviviridae family with higher 50% effective concentrations (EC50s) than for HCV. Thus, because of its chemical plasticity and simplicity of synthesis, our new family of SMCypIs represents a promising new class of drugs with the potential for broad-spectrum anti-Flaviviridae activity as well as an invaluable tool to explore the role of cyclophilins in viral life cycles.

Modulation of the Circadian Period: Searching for Isoform-Selective Cyclophilin Inhibitors

A dynamic, transcription-based oscillator coordinates physiological and behavioral processes with specific times in the 24-hour day, ultimately acting as a cellular clock. The transcription factor complex known as CLOCK:BMAL1 is at the core of this oscillator that generates circadian (about a day) rhythms. BMAL1 possesses a transactivation domain (TAD) that acts as a molecular switch to control the activation or repression state of CLOCK:BMAL1 and modulate circadian period. Recent findings from our lab have demonstrated that cis/trans isomerization about a Trp-Pro imide bond in the TAD is necessary for proper 24-hour circadian timekeeping. Members of the cyclophilin family, which possess peptidyl-prolyl isomerase (PPIase) activity, typically catalyze this intrinsically slow process in vivo. Use of the broad specificity cyclophilin inhibitor Cyclosporin A lengthens circadian rhythms and its persistent use as an immunosuppressant in transplant recipients is associated with deregulation of circadian rhythms. We found that a number of nuclear-localized cyclophilins exhibit robust catalytic activity on the BMAL1 TAD in vitro, yet the absence of isoform-selective inhibitors has made it difficult to tease out their respective contributions to circadian timekeeping. Here we present our initial studies on three nuclear-localized PPIases: PPIA, PPIE, and PPIH. Fluorescence polarization binding assays have allowed us to explore substrate binding in conjunction with 15N/1H ZZ NMR exchange assays that probe catalytic activity. Utilizing in silico ligand docking against high-resolution x-ray structures and NMR-based fragment-based drug discovery (FBDD), we aim to find isoform-selective inhibitors to better understand their role in circadian rhythms.

Discovery of non-peptidic small molecule inhibitors of cyclophilin D as neuroprotective agents in Aβ-induced mitochondrial dysfunction.

Cyclophilin D (CypD) is a mitochondria-specific cyclophilin that is known to play a pivotal role in the formation of the mitochondrial permeability transition pore (mPTP).The formation and opening of the mPTP disrupt mitochondrial homeostasis, cause mitochondrial dysfunction and eventually lead to cell death. Several recent studies have found that CypD promotes the formation of the mPTP upon binding to β amyloid (Aβ) peptides inside brain mitochondria, suggesting that neuronal CypD has a potential to be a promising therapeutic target for Alzheimer's disease (AD). In this study, we generated an energy-based pharmacophore model by using the crystal structure of CypD-cyclosporine A (CsA) complex and performed virtual screening of ChemDiv database, which yielded forty-five potential hit compounds with novel scaffolds. We further tested those compounds using mitochondrial functional assays in neuronal cells and identified fifteen compounds with excellent protective effects against Aβ-induced mitochondrial dysfunction. To validate whether these effects derived from binding to CypD, we performed surface plasmon resonance (SPR)-based direct binding assays with selected compounds and discovered compound 29 was found to have the equilibrium dissociation constants (KD) value of 88.2nM. This binding affinity value and biological activity correspond well with our predicted binding mode. We believe that this study offers new insights into the rational design of small molecule CypD inhibitors, and provides a promising lead for future therapeutic development.

Alisporivir rescues defective mitochondrial respiration in Duchenne muscular dystrophy

Duchenne muscular dystrophy (DMD) is a severe muscle disease of known etiology without effective, or generally applicable therapy. Mitochondria are affected by the disease in animal models but whether mitochondrial dysfunction is part of the pathogenesis in patients remains unclear. We show that primary cultures obtained from muscle biopsies of DMD patients display a decrease of the respiratory reserve, a consequence of inappropriate opening of the permeability transition pore (PTP). Treatment with the cyclophilin inhibitor alisporivir − a cyclosporin A derivative that desensitizes the PTP but does not inhibit calcineurin − largely restored the maximal respiratory capacity without affecting basal oxygen consumption in cells from patients, thus reinstating a normal respiratory reserve. Treatment with alisporivir, but not with cyclosporin A, led to a substantial recovery of respiratory function matching improved muscle ultrastructure and survival of sapje zebrafish, a severe model of DMD where muscle defects are close to those of DMD patients. Alisporivir was generally well tolerated in HCV patients and could be used for the treatment of DMD.

Development of a cyclosporin A derivative with excellent anti-hepatitis C virus potency

Synthetic modification of cyclosporin A at P3-P4 positions led to the discovery of NIM258, a next generation cyclophilin inhibitor with excellent anti-hepatitis C virus potency, with decreased transporter inhibition, and pharmacokinetics suitable for coadministration with other drugs. Herein is disclosed the evolution of the synthetic strategy to from the original medicinal chemistry route, designed for late diversification, to a convergent and robust development synthesis. The chiral centers in the P4 fragment were constructed by an asymmetric chelated Claisen rearrangement in the presence of quinidine as the chiral ligand. Identification of advanced crystalline intermediates enabled practical supply of key intermediates. Finally, macrocyclization was carried out at 10% weight concentration by a general and unconventional “slow release” concept.

Abstract 4313: Protein synthesis modulates paraptotic death induced by inhibition of cyclophilins in glioblastoma (GBM) cells

Abstract In this study, we investigated the mechanisms underlying cell death induced by the small molecule cyclophilin inhibitor NIM811. NIM811 effectively killed GBM cells through a non-apoptotic pathway, as there was no nuclear shrinkage or caspase activation. Instead, death was preceded by dramatic cytosolic vacuolization, stemming from enlargement of the endoplasmic reticulum (ER). RNA-seq revealed upregulation of genes pertinent to the unfolded protein response (UPR) and autophagy. Western blotting for UPR and autophagy mediators also supported a transient UPR signaling and compromised autophagy during prolonged NIM811 treatment. By utilizing a bicistronic reporter plasmid, we independently quantitated Cap-dependent and Cap-independent translation. We further assessed the long-term effects mediated by different short-term paraptosis inhibitors via colony formation assay. We find that NIM811 initiates paraptosis in GBM cells, due to an abnormal upregulation of protein translation that caused accumulation of misfolded or unfolded proteins in ER. Transient activation of pro-survival autophagy and UPR signals were shutdown during prolonged treatment with NIM811, thus allowing cell death to occur. Cycloheximide was previously reported by others to suppress paraptosis. In our studies, we find instead that it temporarily delayed vacuole formation, but actually enhanced paraptotic cell death in the long term. The mTOR inhibitors rapamycin or torin-2 rescued cells from NIM811 induced paraptosis by sustaining autophagy and the UPR, and specifically restrained cap-dependent translation. We propose that ER vacuolization could be a protective response generated by cells to isolate defective proteins within the ER in order to prevent misfolded aggregates to interfere with cellular homeostasis. For that reason, simply inhibiting vacuole formation without clearing protein accumulation in the ER was insufficient to prevent cell death. These findings aid our understanding of cellular mechanisms that contribute to non-apopotic death. Moreover, this work underlines the importance of evaluating cellular responses in cell death experiments over multiple time frames to judge the true impact. Citation Format: Lin Wang, Justin Gundelach, Richard Bram. Protein synthesis modulates paraptotic death induced by inhibition of cyclophilins in glioblastoma (GBM) cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4313. doi:10.1158/1538-7445.AM2017-4313

Drugs in Development for Hepatitis B.

With high morbidity and mortality worldwide, there is great interest in effective therapies for chronic hepatitis B (CHB) virus. There are currently several dozen investigational agents being developed for treatment of CHB. They can be broadly divided into two categories: (1) direct-acting antivirals (DAAs) that interfere with a specific step in viral replication; and (2) host-targeting agents that inhibit viral replication by modifying host cell function, with the latter group further divided into the subcategories of immune modulators and agents that target other host functions. Included among the DAAs being developed are RNA interference therapies, covalently closed circular DNA (cccDNA) formation and transcription inhibitors, core/capsid inhibitors, reverse transcriptase inhibitors, hepatitis B surface antigen (HBsAg) release inhibitors, antisense oligonucleotides, and helioxanthin analogues. Included among the host-targeting agents are entry inhibitors, cyclophilin inhibitors, and multiple immunomodulatory agents, including Toll-like receptor agonists, immune checkpoint inhibitors, therapeutic vaccines, engineered T cells, and several cytokine agents, including recombinant human interleukin-7 (CYT107) and SB 9200, a novel therapy that is believed to both have direct antiviral properties and to induce endogenous interferon. In this review we discuss agents that are currently in the clinical stage of development for CHB treatment as well as strategies and agents currently at the evaluation and discovery phase and potential future targets. Effective approaches to CHB may require suppression of viral replication combined with one or more host-targeting agents. Some of the recent research advances have led to the hope that with such a combined approach we may have a functional cure for CHB in the not distant future.

A homolog of cyclophilin D is expressed in Trypanosoma cruzi and is involved in the oxidative stress\u2013damage response

Mitochondria have an important role in energy production, homeostasis and cell death. The opening of the mitochondrial permeability transition pore (mPTP) is considered one of the key events in apoptosis and necrosis, modulated by cyclophilin D (CyPD), a crucial component of this protein complex. In Trypanosoma cruzi, the protozoan parasite that causes Chagas disease, we have previously described that mitochondrial permeability transition occurs after oxidative stress induction in a cyclosporin A-dependent manner, a well-known cyclophilin inhibitor. In the present work, a mitochondrial parasite cyclophilin, named TcCyP22, which is homolog to the mammalian CyPD was identified. TcCyP22-overexpressing parasites showed an enhanced loss of mitochondrial membrane potential and loss of cell viability when exposed to a hydrogen peroxide stimulus compared with control parasites. Our results describe for the first time in a protozoan parasite that a mitochondrial cyclophilin is a component of the permeability transition pore and is involved in regulated cell death induced by oxidative stress.

Feline coronavirus replication is affected by both cyclophilin A and cyclophilin B.

Feline coronavirus (FCoV) causes the fatal disease feline infectious peritonitis, which is currently incurable by drug treatment, and no effective vaccines are available. Cyclosporin A (CsA), a cyclophilin (Cyp) inhibitor, inhibits the replication of FCoV in vitro and in vivo as well as the replication of human and animal coronaviruses. However, the mechanism underlying the regulation of coronavirus replication by CsA is unknown. In this study, we analysed the role of Cyps in FCoV replication using knockdown and knockout cells specific to Cyps. Inhibition of CypA and CypB reduced FCoV replication, with replication in knockout cells being much less than that in knockdown cells. Furthermore, the proteins expressed by CypA and CypB harbouring mutations in their respective predicted peptidyl-prolyl cis–transisomerase active sites, which also alter the affinities between Cyps and CsA, inhibited FCoV replication. These findings indicate that the peptidyl-prolyl cis–transisomerase active sites of Cyps might be required for FCoV replication.

Discovery of Potent Cyclophilin Inhibitors Based on the Structural Simplification of Sanglifehrin A.

Cyclophilin inhibition has been a target for the treatment of hepatitis C and other diseases, but the generation of potent, drug-like molecules through chemical synthesis has been challenging. In this study, a set of macrocyclic cyclophilin inhibitors was synthesized based on the core structure of the natural product sanglifehrin A. Initial compound optimization identified the valine-m-tyrosine-piperazic acid tripeptide (Val-m-Tyr-Pip) in the sanglifehrin core, stereocenters at C14 and C15, and the hydroxyl group of the m-tyrosine (m-Tyr) residue as key contributors to compound potency. Replacing the C18-C21 diene unit of sanglifehrin with a styryl group led to potent compounds that displayed a novel binding mode in which the styrene moiety engaged in a π-stacking interaction with Arg55 of cyclophilin A (Cyp A), and the m-Tyr residue was displaced into solvent. This observation allowed further simplifications of the scaffold to generate new lead compounds in the search for orally bioavailable cyclophilin inhibitors.