Roland M. Wenger

Immunosuppressive and Nonimmunosuppressive Cyclosporine Analogs Are Toxic to the Opportunistic Fungal Pathogen Cryptococcus neoformans via Cyclophilin-Dependent Inhibition of Calcineurin

ABSTRACT Cyclosporine (CsA) is an immunosuppressive and antimicrobial drug which, in complex with cyclophilin A, inhibits the protein phosphatase calcineurin. We recently found that Cryptococcus neoformansgrowth is resistant to CsA at 24°C but sensitive at 37°C and that calcineurin is required for growth at 37°C and pathogenicity. Here CsA analogs were screened for toxicity against C. neoformans in vitro. In most cases, antifungal activity was correlated with cyclophilin A binding in vitro and inhibition of the mixed-lymphocyte reaction and interleukin 2 production in cell culture. Two unusual nonimmunosuppressive CsA derivatives, (γ-OH) MeLeu4-Cs (211-810) and D-Sar (α-SMe)3Val2-DH-Cs (209-825), which are also toxic to C. neoformans were identified. These CsA analogs inhibit C. neoformans via fungal cyclophilin A and calcineurin homologs. Our findings identify calcineurin as a novel antifungal drug target and suggest nonimmunosuppressive CsA analogs warrant investigation as antifungal agents.

Inhibition of Human Immunodeficiency Virus Type 1 Replication in Human Cells by Debio-025, a Novel Cyclophilin Binding Agent

ABSTRACT Debio-025 is a synthetic cyclosporine with no immunosuppressive capacity but a high inhibitory potency against cyclophilin A (CypA)-associated cis-trans prolyl isomerase (PPIase) activity. A lack of immunosuppressive effects compared to that of cyclosporine was demonstrated both in vitro and in vivo. For three cyclosporines, the inhibitory potential against PPIase activity was quantitatively correlated with that against human immunodeficiency virus type 1 (HIV-1) replication. Debio-025 selectively inhibited the replication of HIV-1 in a CD4+ cell line and in peripheral blood mononuclear cells: potent activity was demonstrated against clinical isolates of various HIV-1 subtypes, including isolates with multidrug resistance to reverse transcriptase and protease inhibitors. Simian immunodeficiency virus and HIV-2 strains were generally resistant to inhibition by Debio-025; however, some notable exceptions of sensitive HIV-2 clinical isolates were detected. In two-drug combination studies, additive inhibitory effects were found between Debio-025 and 19 clinically used drugs of different classes. Clinical HIV-1 isolates that are naturally resistant to Debio-025 and that do not depend on CypA for infection were identified. Comparison of the amino acid sequences of the CypA binding domain of the capsid (CA) protein from Debio-025-sensitive and -resistant HIV-1 isolates indicated that resistance was mostly associated with an H87Q/P exchange. Mechanistically, cyclosporines competitively inhibit the binding of CypA to the HIV-1 CA protein, which is an essential interaction required for early steps in HIV-1 replication. By real-time PCR we demonstrated that early reverse transcription is reduced in the presence of Debio-025 and that late reverse transcription is almost completely blocked. Thus, Debio-025 seems to interfere with the function of CypA during the progression/completion of HIV-1 reverse transcription.

The 3D structure of a cyclosporin analogue in water is nearly identical to the cyclophilin-bound cyclosporin conformation

The conformation of [d‐MeSer3‐d‐Ser‐(O‐Gly8]CS, a water soluble cyclosporin derivative, has been determined in (D6)DMSO and in water using NMR. In these polar solvents the conformation is identical and very similar to the structure found in the cyclophilin‐cyclosporin complex. However, it differs significantly from its conformation in deuterated chloroform. This demonstrates unambiguously that the large structure change is induced primarily by the polar solvent rather than by complex formation with cyclophilin.

Cyclosporine: Chemistry, Structure-Activity Relationships and Mode of Action

Cyclosporine is a cyclic peptide with a selective immunosuppressive action. One of its 11 amino acids is a novel amino acid, (4R)-4-((E)-2-butenyl-4,N-dimethyl-L-threonine, the preparation of which allowed the synthesis of cyclosporine and specifically modified analogues. Structure-activity relationships emerging from the study of modified cyclosporins suggest that a large portion of the cyclosporine molecule is involved in interactions with its lymphocyte receptor and that this includes amino acids 1, 2, 3 and 11.

Synthetic routes to NEtXaa4-cyclosporin A derivatives as potential anti-HIV I drugs

An efficient synthesis in 10 steps and overall yields up to 27% of NEtXaa(4)-cyclosporin A derivatives (Xaa = Leu, Val, Ile, Thr) starting from cyclosporin A is described. Biological activities of the new analogues show promising results for the design of cyclosporin derivatives exhibiting non-immunosuppressive and anti-HIV activity

Identification of several cyclosporine binding proteins in lymphoid and non-lymphoid cells in vivo

The immunosuppressant cyclosporine A (CSA) has been shown to bind to the ubiquitous cellular protein, cyclophilin, and to inhibit its rotamase activity. In the present study, 3H-cyclosporine diazirine analogue was used to photolabel viable human cells of lymphoid and fibroblast origin in order to identify the intracellular targets for the drug. While cyclophilin was strongly labeled in situ, additional minor cyclosporine-protein complexes of 25, 40, 46 and 60 kDa were identified in the T cell leukemia cell line Jurkat. These proteins bound specifically, since only active CSA but not inactive CSH or FK506 competed for binding. Photolabeling of MRC5 cells, a CSA resistant human fibroblast cell line, revealed a 25 kDa complex as the major product, while the 46 and 60 kDa bands were not detectable and cyclophilin labeling was only faint, even though both MRC5 and Jurkat cells contain similar cyclophilin concentrations. Thus, our data suggest that the intracellular targets of CSA and/or the accessibility to cyclophilin varies considerably in drug sensitive and resistant cell types, which may contribute to explaining the lymphocyte selectivity of the drug.

Interaction of cyclosporin A and two cyclosporin analogs with cyclophilin: relationship between structure and binding

The immunosuppressant drug cyclosporin A exists as various conformers in water. Up to 1 h is needed to reach maximum complex formation after mixing the drug with its receptor, cyclophilin, or an antibody, indicating that only a fraction of the conformers in aqueous solution adopts a conformation suitable for binding. In the present study we compare the binding behavior of cyclosporin to that of two analogs, using a biosensor instrument (BIAcore, Pharmacia). The amount of complex formation was measured as a function of time after adding the peptides to cyclophilin. The equilibrium affinity constants of cyclophilin for these analogs have been measured. The slow binding of cyclosporin to cyclophilin compared to the instant binding of the cyclosporin analogs supports the hypothesis that cyclophilin recognizes a well defined conformation of cyclosporin that exists in water prior to binding.

A comparison of cyclosporine binding by cyclophilin and calmodulin and the identification of a novel 45 KD cyclosporine-binding phosphoprotein in Jurkat cells.

Cyclosporine mediates its immunosuppressive effect by preventing the synthesis of lymphokine mRNA during the process of T lymphocyte activation. Although the detailed molecular mechanism by which CsA achieves this effect is unknown, two proteins have been identified as putative intracellular CsA‐receptor proteins. One of these, calmodulin, is an important Ca++‐binding protein and enzyme cofactor and the other, cyclophilin, is a novel protein that is reported to have protein kinase activity. In this study the CsA‐binding capacity of both these proteins has been assessed using CsA‐coated ELISA plates and CsA‐affinity gel matrices. CsA binding was shown by cyclophilin whereas no CsA‐calmodulin binding could be detected under identical conditions. However, it was not possible to demonstrate any cyclophilin‐associated protein kinase activity. Jurkat cells were probed for the presence of CsA‐binding proteins using the CsA‐affinity gel matrix; a 17 KD protein, most probably cyclophilin, was identified as the major CsA‐binding protein. In addition, a previously unidentified CsA‐binding 45 KD phosphoprotein was precipitated from 32P‐labeled Jurkat cells. These results would support cyclophilin as the major, if not only, intracellular receptor protein for CsA. However, the relationship between binding of CsA to cyclophilin and/or the 45 KD phosphoprotein and the immunosuppressive effects of CsA is still unknown.

IMMUNOCHEMICAL STUDY OF CYCLOSPORINE CONFORMATION IN AQUEOUS MEDIUM

ABSTRACT The fine specificity of monoclonal antibodies for different residues of cyclosporine (Cs) has been studied. The results indicate that the conformation of Cs in aqueous medium corresponds to that observed in crystals rather than that found in aprotic solvents.

New Synthetic Routes to NEtXaa4-Cyclosporin Derivatives as Potential Anti-HIV Drugs

Since the discovery of the immunosuppresive activity of Cyclosporin A (CsA), considerable work has been devoted to the chemical synthesis of analogues. More recently, the finding of potential anti-HIV I activity of CsA evoked interest for the design of more selective cyclosporins active against HIV I but devoid of immunosuppresive activity. Based on previous observations that a N-methyl group at residue 4 is involved in one of the main metabolic degradation pathways [1], the synthesis of CsA analogues disposing various N-ethyl substituted residues at position 4 appeared particularly appealing for developing potential anti-HIV drugs [2]. Here we present the synthesis of new NEtXaa4CsA derivatives and their biological activities.