Cyclophilins inhibition

In cyclophilin inhibition assays (Table 1), a comparison of the fluoroolefin and olefin isosteres illustrated the subtle differences between the two closely related isosteres and thereby presumably the importance of the added electrostatic and electronic interactions derived from fluorination. Note in particular that the c/s 1 (H) disastereomer was a more potent inhibitor than the cis 1 (F) and that similarly trans 2 (F) was more effective than trans 2 (H). 

Halestrap AP, et al. Cyclosporin A binding to mitochondrial cyclophilin inhibits the permeability transition pore and protects hearts from ischaemia/reperfusion injury. Mol. Cell Biochem. 1997 174 167-172. 

Ciclosporin binds to the cytosolic protein cyclophilin (immunophilin) of immunocompetent lymphocytes, especially T lymphocytes. This complex of ciclosporin and cyclophilin inhibits calcineurin, which activates transcription of interleukin-2 and inhibits lymphokine production and interleukin release, leading to reduced function of effector T cells [1 ]. 

Cyclosporine A (CsA) is a water-insoluble cyclic peptide from a fungus composed of 11 amino acids. CsA binds to its cytosolic receptor cyclophilin. The CsA/cyclophilin complex reduces the activity of the protein phosphatase calcineurin. Inhibition of this enzyme activity interrupts antigen receptor-induced activation and translocation of the transcription factor NEAT to the nucleus which is essential for the induction of cytokine synthesis in T-lymphocytes. 

Liu S, Asparuhova M, Brondani V, Ziekau I, Klimkait T, Schumperh D (2004) Inhibition of HlV-1 multiplication by antisense U7 snRNAs and siRNAs targeting cyclophilin A, Nucleic Acids Res 32 3752-3759... 

A cytosolic binding protein for CsA was first isolated in 1984 and named cyclophilin, later cyclophilin A (CyPA), in reference to its high affinity for CsA (18). CyPA is a basic, abundant protein with a mass of 18 kDa, and it is found in a variety of tissues. The first clue to its function came in 1989 when two independent groups isolated the enzyme that catalyzes pepti-dyl proline isomerization/peptidylprolyl cis-trans isomerase (EC 5.2.1.8 PPIase), in protein chains and (re)discovered CyPA (19, 20). CyPA is a potent PPIase, and its enzymatic activity is strongly inhibited by CsA. 

Immunophilin is the generic term for a binding protein for an immunosuppressive agent, and all currently known immunophilins belong to the cyclophilin or FKBP families. The two families of immunophilins— like the small molecules they bind—don t have any readily apparent relationship. FKBP12 and CyPA have no sequence similarity, CsA does not inhibit FKBP12, and FK506 does not inhibit CyPA. 

Kiessig et al. studied the interaction of the immunosuppressant drug cyclosporin A and some derivatives to its cellular receptor protein cyclo-philin (26). The cyclophilin-cyclosporin A complex mediates the immunosuppressive action of cyclosporin A by inhibition of the phosphatase calci-neurin. Using ACE in the equilirium-mixture mode, a separation of the cyclophilin-cyclosporin A complex and the unbound cyclophilin was achieved. The approach allowed a qualitative estimation of the binding affinity of cyclosporin A derivatives compared to cyclosporin A. For the calculation of binding constants, electrophoretically mediated microanalysis was applied. 

Another drug whose immunosuppressive action is mediated by the complexation with a cellular protein of the immunophilins is FK506. FK506 binds to proteins of the FKBP family. The complex formed is involved in the inhibition of the phosphatase calcineurin, similar to the mode of action of CsA after binding to cyclophilin. The ACE method was used to detect... 

The preparation of the fluoroolefin amide isosteres is reviewed. The incorporation of the amide isosteres in peptidomimetics and the influence of that isosteric substitution on biological activity on inhibition of peptidyl prolyl isomerases cyclophilin (CyP) and Pini, dipeptidyl peptidase IV/CD26 (DPP IV) and thermolysin is described. In addition, select fiuoroolefination procedures which may have utility in the construction of fluoroolefin amide isosteres are illustrated. 

F.A. Etzkorn, L.A. Stolz, Z.Y. Chang, C.T. Walsh, Role of the cyclosporin-A-cyclophilin complex, FK506-FK506-binding protein complex and calcineurin in the inhibition of T-cell signal transduction, Curr. Opin. Struct. Biol. 3 (1993) 929-933. 

The immimosuppressive effect of cyclosporin and FK506 could not initially be explained by these observations. Only with the discovery that cyclosporin and FK506 achieve their immunosuppressive effect via inhibition of calcineurin did it become clear that the immimosuppression is mediated by a complex reaction chain involving calcineurin. It was shown that the complexes of cyclosporin/cyclophilin and FK506/ FK506 binding protein bind to calcineurin and inhibit the phosphatase activity of the latter. 

Tacrolimus (FK 506) is an immunosuppressant macrolide antibiotic produced by Streptomyces tsukubaensis. It is not chemically related to cyclosporine, but their mechanisms of action are similar. Both drugs bind to cytoplasmic peptidyl-prolyl isomerases that are abundant in all tissues. While cyclosporine binds to cyclophilin, tacrolimus binds to the immunophilin FK-binding protein (FKBP). Both complexes inhibit calcineurin, which is necessary for the activation of the T-cell-specific transcription factor NF-AT. 

Fig. 4.1 Mechanism of action of cyclosporine. Cyclosporine readily diffuses into the cytoplasm of the target cells where it binds to cyclophilins. The cyclosporine-cyclophilin complex stably associates with calcineurin and inhibits calcineurin activity. Calcineurin is a Ca2+-dependent enzyme— serine/threonine phosphatase— which after activation by Ca2+, dephosphorylates a cytosolic component of NFAT (NFATc, cytosolic factor of activated T cells). After dephosphorylation, NFATc migrates from the cytoplasm to the nucleus where it associates with NFATn and induces transcription of several cytokine genes including IL-2. Cyclosporine inhibits calcineurin activity after associating with cyclophilins, resulting in the inhibition of IL-2 production and other cytokines (see Color Insert)...

Sirolimus binds to the cytosolic protein FK-binding protein R (FKBP-12) but does not block calcineurin activity. It does not bind to cyclophilins, which are cytosolic receptors for cyclosporine. Unlike cyclosporine and tacrolimus, sirolimus does not inhibit the activation of NFAT responsive genes. After binding to its cytosolic receptors, sirolimus inhibits a protein kinase, the mammalian target of rapamycin (mTOR) pathway, via suppression of PP2-A. When mTOR is inhibited, the cells will not proceed to the S phase, and the cell cycle will be blocked (Fig. 4.3). As a result, sirolimus blocks T-cell proliferation but its effects are downstream of the IL-2 receptors. IL-2 binding to its receptors activates intracellular protein kinases that in turn activate gene transcription and T-cell proliferation. 

The molecular basis of CsA s biological activity is the formation of a complex with the immunophilin cyclophilin A (CyPA) in such a way that the CsA-CyPA complex can associate with calcineurin (CN), which is a Ca2+/calmodulin-dependent Ser/Thr phosphatase. As a result, dephosphorylation of a nuclear factor of activated T-cells (NFAT) is inhibited. 

Ciclosporin is of fungal origin it is a cyclic peptide composed of 11, in part atypical, amino acids. Therefore, orally administered ciclosporin is not degraded by gastrointestinal proteases. InT-helper cells, it inhibits the production of interleukin-2 by interfering at the level of transcriptional regulation. Normally, nuclear factor of activated T cells, (NFAT) promotes the expression of interleu-kin-2. This requires dephosphorylation of the precursor, phosphorylated NFAT, by the phosphatase calcineurin, enabling NFAT to enter the cell nucleus from the cytosol. Ciclosporin binds to the protein cyclophilin in the cell interior the complex inhibits calcineurin, hence the production of interleukin-2. 

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