Emerging role of cyclophilin A in HIV-1 infection: from producer cell to the target cell nucleus.

The HIV-1 genome encodes a small number of proteins with structural, enzymatic, regulatory, and accessory functions. These viral proteins interact with a number of host factors to promote the early and late stages of HIV-1 infection. During the early stages of infection, interactions between the viral proteins and host factors enable HIV-1 to enter the target cell, traverse the cytosol, dock at the nuclear pore, gain access to the nucleus, and integrate into the host genome. Similarly, the viral proteins recruit another set of host factors during the late stages of infection to orchestrate HIV-1 transcription, translation, assembly, and release of progeny virions. Among the host factors implicated in HIV-1 infection, Cyclophilin A (CypA) was identified as the first host factor to be packaged within HIV-1 particles. It is now well established that CypA promotes HIV-1 infection by directly binding to the viral capsid. Mechanistic models to pinpoint CypA's role have spanned from an effect in the producer cell to the early steps of infection in the target cell. In this review, we will describe our understanding of the role(s) of CypA in HIV-1 infection, highlight the current knowledge gaps, and discuss the potential role of this host factor in the post-nuclear entry steps of HIV-1 infection.

Cyclosporin A-Based PROTACs Can Deplete Abundant Cellular Cyclophilin A without Suppressing T Cell Activation.

Cyclophilin A (CypA), the cellular receptor of the immunosuppressant cyclosporin A (CsA), is an abundant cytosolic protein and is involved in a variety of diseases. For example, CypA supports cancer proliferation and mediates viral infections, such as the human immunodeficiency virus 1 (HIV-1). Here, we present the design of PROTAC (proteolysis targeting chimera) compounds against CypA to induce its intracellular proteolysis and to investigate their effect on immune cells. Interestingly, upon connecting to E3 ligase ligands, both peptide-based low-affinity binders and CsA-based high-affinity binders can degrade CypA at nM concentration in HeLa cells and fibroblast cells. As the immunosuppressive effect of CsA is not directly associated with the binding of CsA to CypA but the inhibition of phosphatase calcineurin by the CypA:CsA complex, we investigated whether a CsA-based PROTAC compound could induce CypA degradation without affecting the activation of immune cells. P3, the most efficient PROTAC compound discovered from this study, could deplete CypA in lymphocytes without affecting cell proliferation and cytokine production. This work demonstrates the feasibility of the PROTAC approach in depleting the abundant cellular protein CypA at low drug dosage without affecting immune cells, allowing us to investigate the potential therapeutic effects associated with the endogenous protein in the future.

Cell-free DNA Release in the Plasma of Patients with Cardiac Disease is Associated with Cell Death Processes.

Cell-free DNA (cfDNA) is released into the plasma of patients with cardiac disease. Here, the source and mechanism of plasma cfDNA release in patients with myocardial infarction (MI) and other cardiac diseases (n = 59) were investigated. Plasma levels of various markers including M30 (apoptosis), M65 (apoptosis and necrosis), cyclophilin A (CyPA) (necrosis), and myeloperoxidase (MPO) (neutrophil activation) were assayed. The plasma cfDNA concentrations in MI and other cardiac diseases were significantly higher than that in the healthy control subjects. Significant differences were not observed among the cardiac disease patients (MI and other cardiac diseases) and healthy control subjects in M30, M65, and CyPA levels. In contrast,the MPO levels were significantly elevated in cardiac disease patients when compared to control groups, and MPO levels in MI patients were significantly higher than other cardiac diseases patients. These results suggest that cfDNA is mainly released by neutrophils via NETosis in addition to apoptosis except for epithelial apoptosis in patients with cardiac disease and the degree is greater in MI patients. The results from this study provide basic information for diagnosis marker of MI.

Cyclophilin A Inhibits Human Respiratory Syncytial Virus (RSV) Replication by Binding to RSV-N through Its PPIase Activity.

Human respiratory syncytial virus (hRSV) is the most common pathogen which causes acute lower respiratory infection (ALRI) in infants. Recently, virus-host interaction has become a hot spot of virus-related research, and it needs to be further elaborated for RSV infection. In this study, we found that RSV infection significantly increased the expression of cyclophilin A (cypA) in clinical patients, mice, and epithelial cells. Therefore, we evaluated the function of cypA in RSV replication and demonstrated that virus proliferation was accelerated in cypA knockdown host cells but restrained in cypA-overexpressing host cells. Furthermore, we proved that cypA limited RSV replication depending on its PPIase activity. Moreover, we performed liquid chromatography-mass spectrometry, and the results showed that cypA could interact with several viral proteins, such as RSV-N, RSV-P, and RSV-M2-1. Finally, the interaction between cypA and RSV-N was certified by coimmunoprecipitation and immunofluorescence. Those results provided strong evidence that cypA may play an inhibitory role in RSV replication through interaction with RSV-N via its PPIase activity. IMPORTANCE RSV-N, packed in the viral genome to form the ribonucleoprotein (RNP) complex, which is recognized by the RSV RNA-dependent RNA polymerase (RdRp) complex to initiate viral replication and transcription, plays an indispensable role in the viral biosynthesis process. cypA, binding to RSV-N, may impair this function by weakening the interaction between RSV-N and RSV-P, thus leading to decreased viral production. Our research provides novel insight into cypA antiviral function, including binding to viral capsid protein to inhibit viral replication, which may be helpful for new antiviral drug exploration.

Microplitis bicoloratus bracovirus regulates cyclophilin A-apoptosis-inducing factor interaction to induce cell apoptosis in the insect immunosuppressive process.

Microplitis bicoloratus bracovirus (MbBV) induces apoptosis in hemocytes of the host (Spodoptera litura) via the cyclophilin A (CypA)-mediated signaling pathway. However, the mechanisms underlying CypA-mediated signaling during apoptosis remain largely unknown. Therefore, in this study, we investigated how CypA and apoptosis-inducing factor (AIF) interact during MbBV-mediated apoptosis. Our findings showed that MbBV induces apoptosis through the CypA-AIF axis of insect immune suppression. In MbBV-infected Spli221 cells, both the expression of the cypa gene and the release of AIF from the mitochondria increased the number of apoptotic cells. CypA and AIF underwent concurrent cytoplasm-nuclear translocation. Conversely, blocking of AIF release from mitochondria not only inhibited the CypA-AIF interaction but also inhibited the cytoplasmic-nuclear translocation of AIF and CypA. Importantly, the survival of the apoptotic phenotype was significantly rescued in MbBV-infected Spli221 cells. In addition, we found that the cyclosporine A-mediated inhibition of CypA did not prevent the formation of the CypA and AIF complex; rather, this only suppressed genomic DNA fragmentation. In vitro experiments revealed direct molecular interactions between recombinant CypA and AIF. Taken together, our results demonstrate that the CypA-AIF interaction plays an important role in MbBV-induced innate immune suppression. This study will help to clarify aspects of insect immunological mechanisms and will be relevant to biological pest control.

The adhesion protein of Mycoplasma genitalium inhibits urethral epithelial cell apoptosis through CypA-CD147 activating PI3K/ Akt/NF-κB pathway.

By interacting with the receptor on the host cells membrane, Mycoplasma genitalium, a prokaryotic bacterium primarily transmitted through sexual contact, can adhere to and even enter cells. The adhesion protein of M. genitalium (MgPa) plays a critical function in the adhering and subsequent invasion into host cells. Our prior studies verified that cyclophilin A (CypA) was the receptor of MgPa on human urethral epithelial cells (SV-HUC-1) membrane and could induce pro-inflammatory cytokines production through the CypA-CD147-ERK-NF-κB pathway. This research aims to understand how MgPa interacts with its membrane receptor CypA to cause apoptosis in host cells. We employed flow cytometry to see if MgPa prevents or enhances apoptosis of SV-HUC-1 cells. The apoptosis-related proteins such as Bax, caspase-3, and cleaved caspase-3 were assayed using Western blot. Results suggested that MgPa could inhibit the apoptosis of SV-HUC-1 cells. And we demonstrated that interference with the expression of CypA or CD147 significantly reversed the inhibitory effect of MgPa on SV-HUC-1 cells apoptosis, indicating that MgPa inhibited urothelial cells apoptosis through CypA/CD147. Furthermore, we discovered that MgPa regulates the PI3K/Akt/NF-κB pathway through CypA/CD147 to inhibit SV-HUC-1 cells apoptosis. Ultimately, the inhibitory effect of MgPa on the apoptosis of the urothelial epithelial cells extracted from CypA-knockout mice was validated by Annexin V/PI assay. The results corroborated that MgPa could also inhibit mouse urothelial epithelial cells apoptosis. In summary, we demonstrated that MgPa could inhibit SV-HUC-1 cells apoptosis via regulating the PI3K/Akt/NF-κB pathway through CypA/CD147, providing experimental evidence for elucidating the survival strategies of M. genitalium in host cells. KEY POINTS: • M. genitalium protein of adhesion inhibited human urethral epithelial cells apoptosis through CypA-CD147 activating the signal pathway of PI3K/Akt/NF-κB • The knockdown of CypA and CD147 could downregulate the M. genitalium -activated PI3K/Akt/NF-κB pathway in SV-HUC-1 cells • MgPa could inhibit the apoptosis of normal C57BL mouse primary urethral epithelial cells, but not for CypA-knockout C57BL mouse primary urethral epithelial cells.

Purification and characterization of Cyclophilin: a protein associated with protein folding in Salmonella Typhimurium.

Salmonella Typhimurium (ST) is a Gram-negative zoonotic pathogenic bacterium that causes infectious disease in humans as well as in animals. It causes foodborne diarrheal or gastrointestinal illness and fever called salmonellosis, which is a leading cause of millions of deaths worldwide. Salmonellaenterica serovar Typhimurium (S. Typhimurium) during its pathogenesis take away the actin cytoskeleton of their host cells and this is the crucial step of its infection cycle. Cyclophilin A, a type of peptidyl-prolyl isomerase that's encoded by the ppiA gene in ST, plays pleiotropic roles in maintaining bacterial physiology. In this investigation, the proteomic characterization of the peptidyl-prolyl cis-trans isomerase- A (Cyclophilin A) from Salmonella Typhimurium is reported. Cyclophilin A (CypA) protein from Salmonella Typhimurium proved to be highly conserved and homologous protein sequence compared to other organisms. This protein was expressed in Escherichia coli followed by its purification in a recombinant form protein exhibited a characteristic PPIases activity (Vmax = 0.8752 ± 0.13892 µmoles/min, Km = 0.9315 ± 0.5670 µM) in comparison to control. The mass spectrometry analysis of Cyp A protein-peptide showed a highest sequence similarity with the cyclophilin protein of Salmonella. PPIases proteins (enzyme) data suggest that Ppi-A has roles in the protein folding that may be contributing to the virulence of Salmonella by isomerization of protein outline. These results suggest an active and vital role of this protein in protein folding along with regulation in Salmonella Typhimurium.

Cyclophilin A: a key player for etiological agent infection.

Cyclophilin A (CypA), a key member of the immunophilin family, is the most abundantly expressed isozyme of the 18 known human cyclophilins. Besides acting as an intracellular receptor for cyclosporine A, CypA plays a vital role in microorganismal infections, cardiovascular diseases, liver diseases, kidney diseases, neurodegeneration, cancer, rheumatoid arthritis, periodontitis, sepsis, asthma, and aging. This review focuses on the pivotal roles of CypA in the infection of etiological agents, which manifests mainly in promoting or inhibiting viral replication based on the host cell type and viral species. CypA can interact with viral proteins and thus regulate the replication cycle of the virus. CypA is involved in pathogenic bacterial infections by regulating the formation of host actin skeleton or membrane translocation of bacterial toxins, or mediated the adhesion of Mycoplasma genitalium during the infection processes by acting as a cellular receptor of M. genitalium. CypA also plays a critical role in infection or the life cycle of certain parasites or host immune regulation. Moreover, we summarized the current understanding of CypA inhibitors acting as host-targeting antiviral agents, thus opening an avenue for the treatment of multiple viral infections due to their broad antiviral effects and ability to effectively prevent drug resistance. Therefore, the antiviral effect of CypA has the potential to promote CypA inhibitors as host-targeting drugs to CypA-involved etiological agent infections and human diseases. KEY POINTS: • CypA is involved in the replication and infection of several viruses, pathogenic bacteria, mycoplasma, and parasites. • CypA inhibitors are in a strong position to inhibit the infection of viruses, bacterial, and mycoplasma.

Relevance of AIF/CypA Lethal Pathway in SH-SY5Y Cells Treated with Staurosporine.

The AIF/CypA complex exerts a lethal activity in several rodent models of acute brain injury. Upon formation, it translocates into the nucleus of cells receiving apoptotic stimuli, inducing chromatin condensation, DNA fragmentation, and cell death by a caspase-independent mechanism. Inhibition of this complex in a model of glutamate-induced cell death in HT-22 neuronal cells by an AIF peptide (AIF(370-394)) mimicking the binding site on CypA, restores cell survival and prevents brain injury in neonatal mice undergoing hypoxia-ischemia without apparent toxicity. Here, we explore the effects of the peptide on SH-SY5Y neuroblastoma cells stimulated with staurosporine (STS), a cellular model widely used to study Parkinson's disease (PD). This will pave the way to understanding the role of the complex and the potential therapeutic efficacy of inhibitors in PD. We find that AIF(370-394) confers resistance to STS-induced apoptosis in SH-SY5Y cells similar to that observed with CypA silencing and that the peptide works on the AIF/CypA translocation pathway and not on caspases activation. These findings suggest that the AIF/CypA complex is a promising target for developing novel therapeutic strategies against PD.

Human MxB Inhibits the Replication of Hepatitis C Virus.

Type I interferon (IFN) inhibits viruses by inducing the expression of antiviral proteins. The IFN-induced myxovirus resistance B (MxB) protein has been reported to inhibit a limited number of viruses, including HIV-1 and herpesviruses, but its antiviral coverage remains to be explored further. Here we show that MxB interferes with RNA replication of hepatitis C virus (HCV) and significantly inhibits viral replication in a cyclophilin A (CypA)-dependent manner. Our data further show that MxB interacts with the HCV protein NS5A, thereby impairing NS5A interaction with CypA and NS5A localization to the endoplasmic reticulum, two events essential for HCV RNA replication. Interestingly, we found that MxB significantly inhibits two additional CypA-dependent viruses of the Flaviviridae family, namely, Japanese encephalitis virus and dengue virus, suggesting a potential link between virus dependence on CypA and virus susceptibility to MxB inhibition. Collectively, these data have identified MxB as a key factor behind IFN-mediated suppression of HCV infection, and they suggest that other CypA-dependent viruses may also be subjected to MxB restriction.IMPORTANCE Viruses of the Flaviviridae family cause major illness and death around the world and thus pose a great threat to human health. Here we show that IFN-inducible MxB restricts several members of the Flaviviridae, including HCV, Japanese encephalitis virus, and dengue virus. This finding not only suggests an active role of MxB in combating these major pathogenic human viruses but also significantly expands the antiviral spectrum of MxB. Our study further strengthens the link between virus dependence on CypA and susceptibility to MxB restriction and also suggests that MxB may employ a common mechanism to inhibit different viruses. Elucidating the antiviral functions of MxB advances our understanding of IFN-mediated host antiviral defense and may open new avenues to the development of novel antiviral therapeutics.

The Three-Fold Axis of the HIV-1 Capsid Lattice Is the Species-Specific Binding Interface for TRIM5α.

Rhesus TRIM5α (rhTRIM5α) potently restricts replication of human immunodeficiency virus type 1 (HIV-1). Restriction is mediated through direct binding of the C-terminal B30.2 domain of TRIM5α to the assembled HIV-1 capsid core. This host-pathogen interaction involves multiple capsid molecules within the hexagonal HIV-1 capsid lattice. However, the molecular details of this interaction and the precise site at which the B30.2 domain binds remain largely unknown. The human orthologue of TRIM5α (hsTRIM5α) fails to block infection by HIV-1 both in vivo and in vitro This is thought to be due to differences in binding to the capsid lattice. To map the species-specific binding surface on the HIV-1 capsid lattice, we used microscale thermophoresis and dual-focus fluorescence correlation spectroscopy to measure binding affinity of rhesus and human TRIM5α B30.2 domains to a series of HIV-1 capsid variants that mimic distinct capsid arrangements at each of the symmetry axes of the HIV-1 capsid lattice. These surrogates include previously characterized capsid oligomers, as well as a novel chemically cross-linked capsid trimer that contains cysteine substitutions near the 3-fold axis of symmetry. The results demonstrate that TRIM5α binding involves multiple capsid molecules along the 2-fold and 3-fold interfaces between hexamers and indicate that the binding interface at the 3-fold axis contributes to the well-established differences in restriction potency between TRIM5α orthologues.IMPORTANCE TRIM5α is a cellular protein that fends off infection by retroviruses through binding to the viruses' protein shell surrounding its genetic material. This shell is composed of several hundred capsid proteins arranged in a honeycomb-like hexagonal pattern that is conserved across retroviruses. By binding to the complex lattice formed by multiple capsid proteins, rather than to a single capsid monomer, TRIM5α restriction activity persists despite the high mutation rate in retroviruses such as HIV-1. In rhesus monkeys, but not in humans, TRIM5α confers resistance to HIV-1. By measuring the binding of human and rhesus TRIM5α to a series of engineered HIV-1 capsid mimics of distinct capsid lattice interfaces, we reveal the HIV-1 capsid surface critical for species-specific binding by TRIM5α.

Spodoptera litura cyclophilin A is required for Microplitis bicoloratus bracovirus-induced apoptosis during insect cellular immune response.

Microplitis bicoloratus bracovirus (MbBV) is a polydnavirus found in the parasitic wasp M. bicoloratus. Although MbBV is a known inducer of apoptosis in host hemocytes, the mechanism by which this occurs remains elusive. In this study, we found that expression of cyclophilin A (CypA) was significantly upregulated in Spodoptera litura hemocytes at 6-day post-parasitization. Similar results were reported in High Five cells (Hi5 cells) infected by MbBV, suggesting that the upregulation of CypA is linked to MbBV infection in insect cells. cDNA encoding CypA was cloned from parasitized hemocytes of S. litura, and bioinformatic analyses showed that S. litura CypA belongs to the cyclophilin family of proteins. Overexpression of S. litura CypA in Hi5 cells revealed that the protein promotes MbBV-induced apoptosis in vitro. Conversely, suppression of the expression and activity of CypA protein significantly rescued the apoptotic phenotype observed in MbBV-infected Hi5 cells, suggesting that it plays a key role in this process. MbBV infection also promoted the cytoplasmic-nuclear translocation of CypA in Hi5 cells. Taken together, these results suggest that MbBV infection upregulates the expression of CypA, which is required for MbBV-mediated apoptosis. Our findings provide insight into the role that CypA plays in insect cellular immune response.

Binding mode of AIF(370-394) peptide to CypA: insights from NMR, label-free and molecular docking studies.

The complex formation between the proteins apoptosis-inducing factor (AIF) and cyclophilin A (CypA) following oxidative stress in neuronal cells has been suggested as a main target for reverting ischemia-stroke damage. Recently, a peptide encompassing AIF residues 370-394 has been developed to target the AIF-binding site on CypA, to prevent the association between the two proteins and suppress glutamate-induced cell death in neuronal cells. Using a combined approach based on NMR spectroscopy, synthesis and in vitro testing of all Ala-scan mutants of the peptide and molecular docking/molecular dynamics, we have generated a detailed model of the AIF (370-394)/CypA complex. The model suggests us that the central region of the peptide spanning residues V374-K384 mostly interacts with the protein and that for efficient complex inhibition and preservation of CypA activity, it is bent around amino acids F46-G75 of the protein. The model is consistent with experimental data also from previous works and supports the concept that the peptide does not interfere with other CypA activities unrelated to AIF activation; therefore, it may serve as an ideal template for generating future non-peptidic antagonists.

Global Profiling of PknG Interactions Using a Human Proteome Microarray Reveals Novel Connections with CypA.

Mycobacterium tuberculosis (Mtb) serine/threonine kinase PknG plays an important role in the Mtb-host interaction by facilitating the survival of Mtb in macrophages. However, the human proteins with which the PknG interacts, and the underlying molecular mechanisms are still largely unknown. In this study, a HuProt array is been applied to globally identify the host proteins to which PknG binds. In this way, 125 interactors are discovered, including a cyclophilin protein, CypA. This interaction between PknG and CypA is validated both in vitro and in vivo, and functional studies show that PknG significantly reduces the protein levels of CypA through phosphorylation, which consequently inhibit the inflammatory response through downregulation of NF-κB and ERK1/2 pathways. Phenotypically, overexpression of PknG reduces cytokine levels and promotes the survival of Mycobacterium smegmatis (Msm) in macrophages. Overall, it is expected that the PknG interactors identified in this study will serve as a useful resource for further systematic studies of the roles that PknG plays in the Mtb-host interactions.

The roles of CyPA and CD147 in cardiac remodelling.

Cyclophilin A (CyPA), an abundantly expressed protein belonging to the immunophilin family, is involved in a variety of physiological and pathological activities together with its extracellular receptor, CD147. Many studies have provided compelling evidence supporting critical roles of CyPA in immune and infectious diseases and malignant tumours. Recent studies have revealed that both CyPA and CD147 strongly promote cardiovascular inflammation, myocardial ischaemia-reperfusion injury, and myocardial remodelling processes. Here, we review the potential roles of CyPA and CD147 in cardiac remodelling and their implications for the development of novel pharmacological therapies for heart failure.

Active Site Crowding of Cytochrome P450 3A4 as a Strategy To Alter Its Selectivity.

Substrate-promiscuous enzymes are a promising starting point for the development of versatile biocatalysts. In this study, human cytochrome P450 3A4, known for its ability to metabolise hundreds of drugs, was engineered to alter its regio- and stereoselectivity. Rational mutagenesis was used to introduce steric hindrance in a specific manner in the large active site of P450 3A4 and to favour oxidation at a more sterically accessible position on the substrate. Hydroxylation of a synthetic precursor of (R)-lisofylline, a compound under investigation for its anti-inflammatory properties, was chosen as a first proof-of-principle application of our protein engineering strategy. In a second example, increasing active site crowding led to an incremental shift in the selectivity of oxidation from an internal double bond to a terminal phenyl group in a derivative of theobromine. The same correlation between crowding and selectivity was found in a final case focused on the hydroxylation of the steroid sex hormone progesterone.

Melatonin attenuates angiotensin II-induced cardiomyocyte hypertrophy through the CyPA/CD147 signaling pathway.

Melatonin is well known for its cardioprotective effects; however, whether melatonin exerts therapeutic effects on cardiomyocyte hypertrophy remains to be investigated, as do the mechanisms underlying these effects, if they exist. Cyclophilin A (CyPA) and its corresponding receptor, CD147, which exists in a variety of cells, play crucial roles in modulating reactive oxygen species (ROS) production. In this study, we explored the role of the CyPA/CD147 signaling pathway in angiotensin II (Ang II)-induced cardiomyocyte hypertrophy and the protective effects exerted by melatonin against Ang II-induced injury in cultured H9C2 cells. Cyclosporine A, a specific CyPA/CD147 signaling pathway inhibitor, was used to manipulate CyPA/CD147 activity. H9C2 cells were then subjected to Ang II or CyPA treatment in either the absence or presence of melatonin. Our results indicate that Ang II induces cardiomyocyte hypertrophy through the CyPA/CD147 signaling pathway and promotes ROS production, which can be blocked by melatonin pretreatment in a concentration-dependent manner, in cultured H9C2 cells and that CyPA/CD147 signaling pathway inhibition protects against Ang II-induced cardiomyocyte hypertrophy. The protective effects of melatonin against Ang II-induced cardiomyocyte hypertrophy depend at least partially on CyPA/CD147 inhibition.

Keeping your armour intact: how HIV-1 evades detection by the innate immune system: HIV-1 capsid controls detection of reverse transcription products by the cytosolic DNA sensor cGAS.

HIV-1 infects dendritic cells (DCs) without triggering an effective innate antiviral immune response. As a consequence, the induction of adaptive immune responses controlling virus spread is limited. In a recent issue of Immunity, Lahaye and colleagues show that intricate interactions of HIV capsid with the cellular cofactor cyclophilin A (CypA) control infection and innate immune activation in DCs. Manipulation of HIV-1 capsid to increase its affinity for CypA results in reduced virus infectivity and facilitates access of the cytosolic DNA sensor cGAS to reverse transcribed DNA. This in turn induces a strong host response. Here, we discuss these findings in the context of recent developments in innate immunity and consider the implications for disease control and vaccine design.

Cyclophilin A regulates JNK/p38-MAPK signaling through its physical interaction with ASK1.

Cyclophilin A (CypA), a member of the immunophilin family, is predominantly localized in the cytoplasm. The peptidylprolyl isomerase (PPIase) activity of CypA has been demonstrated to be involved in diverse cellular processes, including intracellular protein trafficking, mitochondrial function, pre-mRNA processing, and maintenance of multiprotein complex stability. In this study, we have demonstrated that CypA regulates apoptosis signaling-regulating kinase 1 (ASK1) through its direct binding. ASK1 is a member of MAPK kinase kinase (MAP3K) family, and selectively activates both JNK and p38 MAPK pathways. Here, we also report that CypA negatively regulates phosphorylation of ASK1 at Ser966, and that CypA reduces ASK1 and its downstream kinases of the JNK and p38 signaling. ASK1 is known to induce caspase-3 activation and apoptosis, and CypA inhibited ASK1-mediated apoptosis by decrease in caspase-3 activity under cellular stress conditions. Overall, we conclude that CypA negatively regulates ASK1 functions by its physical interaction with ASK1.

Discovery of Novel Small Molecule Anti-HCV Agents via the CypA Inhibitory Mechanism Using O-Acylation-Directed Lead Optimization.

In this work, the relationship between cyclophilin A (CypA) and HCV prompted us to screen a series of small molecule CypA inhibitors which were previously reported by our group. Among them, compound 1, discovered as a non-immunosuppressive anti-HCV agent with an EC50 value of 0.67 µM in a virus assay, was selected for further study. Subsequent chemical modification by O-acylation led to a novel class of molecules, among which compound 25 demonstrated the most potent anti-HCV activity in the virus assay (EC50 = 0.19 µM), but low cytotoxicity and hERG cardiac toxicity. The following studies (a solution stability assay and a simple pharmacokinetic test together with a CypA enzyme inhibition assay) preliminarily indicated that 25 was a prodrug of 1. To the best of our knowledge, 25 is probably the most potent currently reported small molecule anti-HCV agent acting via the CypA inhibitory mechanism. Consequently, our study has provided a new potential small molecule for curing HCV infection.