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Group Meeting Literature Report Brief Introduction on Mechanism of Cyclosporin Speaker :Geng Hao Supervisor:Prof. Yun-Dong Wu Nov.22th 2013 1 Outline • • • • The Discovery of Cyclosporin A and Cyclophilin Calcineurin and CsA-CyP complex The mechanism of immunosuppressant-CsA Cyclosporin analogs 2 The Discovery of Cyclosporin A Cyclosporin A (often shortened to CsA) was initially isolated from the fungus Tolypocladium inflatum (多孔木霉), found in a soil sample obtained in 1969 from Mountain Hardangervidda, Norway by Dr. Hans Peter Frey, a Sandoz biologist. Cyclosporin A Mountain Hardangervidda(哈当厄高原) No antibacterial activity. The immunosuppressive effect of cyclosporin was discovered on 31 January 1972 by employees of Sandoz (now Novartis) in Basel, Switzerland, in a screening test on immune suppression designed and implemented by Hartmann F. Stähelin, M.D. Tolypocladium inflatum (多孔木霉) Svarstad, 2000,From Norway to Novartis: Cyclosporin from Tolypocladium inflatum in an open access bioprospecting regime. Biodiversity and Conservation 9 (11): 1521–1541. 3 The Discovery of Cyclosporin A Most peptides are synthesized by ribosomes, but cyclosporin is a cyclic nonribosomal peptide of 11 amino acids. 7 N-Methylations 3 uncommon amino acid (contains a single D-amino acid) at 1, 2, 8 position respectively Very famous immunosuppresant! The first patient, on 9 March 1980, was a 28-year-old woman. Cyclosporin was subsequently approved for use in 1983. Since then, it has been used to prevent and treat graft-versus-host reactions in bone-marrow transplantation and to prevent rejection of kidney, heart, and liver transplants. And it raised the survival rate from 50% to 70% after internal organ transplants. Borel JF, 2002. History of the discovery of cyclosporin and of its early pharmacological development. Wien. Klin. Wochenschr. 114 (12): 433–7 4 The Discovery of Cyclophilin What’s the target of CsA ? 1984 1991 1993 5 The Discovery of Cyclophilin Cyclophilins(Cyp) are a family of proteins that bind to cyclosporine. Originally discovered as a specific ligand of Cyclosporin and named based on this. CypA is the most famous one which has a beta barrel structure with two alpha helices and a betasheet. Cyclophilin is not born to bind to CsA Cyclophilin A is a cytosolic and abundant protein (0.1% of total cellular protein). Possesses a peptidyl prolyl isomerase(PPIase) activity, which catalyzes the isomerization of peptide bonds from trans form to cis form at proline residues and facilitates protein folding. Has been proposed to act as a chaperone(分 子伴侣) in protein trafficking. Play important role for some virus replication.(HIV,HCV) Watashi K, et a1. , 2003, 38:1282-1288 Fischer G, Aumuller T; Rev Physiol Biochem Pharmaeol, 2003, 148: 105—150 6 Calcineurin and CsA-CyP complex What happened after CsA bind to Cyclophilin A ? 1984 2002 7 The mechanism of immunosuppressant-CsA Calcineurin (CN) is a enzyme involved in the T-cell activation. Known as calcium-dependent serine-threonine phosphatase, Calcineurin activates nuclear factor of activated T cell(NFATc), a transcription factor, by dephosphorylating it. The activated NFATc is then translocated into the nucleus, where it upregulates the expression of interleukin 2 (IL2), which, in turn, stimulates the growth and differentiation of T cell response. interleukin 2 Majed M. Hamawy, et al .Transplantation Review, 2003, 17, 165-171 8 The mechanism of immunosuppressant-CsA Cyclosporine (CsA) binds to cyclophylin (CpN), forming a complex. The CsA–CpN complex binds and blocks the function of the enzyme calcineurin (CaN), As a result, CaN fails to dephosphorylate the nuclear factor of activated T cells (NFATc), and thereby blocks the transport of NF-ATc to the nucleus and the binding of NF-ATc to the its partner(NF-ATn). No complex bind to the promoter of the interleukin 2 (IL-2) gene and initiate IL-2 production. Consequently, T cells do not produce IL-2, which is necessary for full T-cell activation. Therefore the CsA inhibits T cell activation. T Cell activation Expression of IL-2 et al cell factor Proliferation and differentiat ion of T cell CsA-CypA complex Steffan Ho, et al. Clinical Immunology and immunopathogy, 1996, 80, 40-45 9 The mechanism of immunosuppressant-CsA Cyclophilin A(CypA) binding domain Calcineurin binding domain Qing Huai, Hengming Ke;PNAS, 2002, 99, 12037-12042 10 The mechanism of immunosuppressant-CsA The CyPA-CsA-CN structure revealed many hydrogen bond and hydrophobic interactions between CypA-CsA complex and CN. CyPA-CsA interacts with Arg122 at the active site of CN, implying direct involvement of CyPA-CsA in the regulation of CN catalysis. Interfacial interactions between CN and CsA-CyP. A total of 25 residues of CN are involved in interaction with CyPA-CsA. Red balls represent residues interacting with CyPA and green balls represent residues interacting with CsA (green sticks). Qing Huai, Hengming Ke;PNAS, 2002, 99, 12037-12042 11 Crystal structure of Cyclosporin A 1984 HR Loosli, H Kessler, H Oschkina,; HELV CHIM ACTA, 1985, 68, 682-704 12 Crystal structure of CsA and stucture when binding with Cyclophilin Cyclosporin A- Cyclophilin C Complex Cyclosporin A -Cyclophilin D Complex 2-Val Cyclosporin A -Cyclophilin D Complex Crystal Structure of human calcineurin complexed with cyclosporin A and human cyclophilin (U-13C)Cyclosporin A bound to Cyclophilin [d-(cholinylester)ser8]-cyclosporin Cyclophilin B Complex Cyclosporin A -Cyclophilin G Complex Cyclosporin A- Cyclophilin J Complex Cyclosporin A - murine cyclophilin C complexed Overlay of Crystal structure of CsA from different CsA-CyP complexes 2ESL 2Z6W 1CWF 1MF8 1CYA 1CYN 2WFJ 2OJU 2RMC Amide nitrogen toward the inside direction N-methylation toward the outside Beneficial to pass through the membrane Conformation of CsA in 1MF8 No intermolecular H-bond 13 Calcineurin and CsA-CyP complex x 14 Non-Immunosuppressive Cyclosporin Analogs Cyclosporin A NIM-811 DEBIO-025 SCY-635 15 Non-Immunosuppressive Cyclosporin Analogs NIM 811 Inhibition of Human Immunodeficiency VirusType1 Replication by SDZ NIM 811 , a Nonimmunosuppressive Cyclosporine Analog. ANTIMICROBLAL AGENTS AND CHEMOTHERAPY,Aug. 1994,p. 1763-1772 Debio-025 In vitro blood distribution and protein binding of a new anti-HIV drug, DEBIO-025. DRUG METABOLISM REVIEWS,2005, 37, p.29-29 SCY-635 Preclinical evaluation of SCY-635, a cyclophilin inhibitor with potent anti-HCV activity. HEPATOLOGY, 2006,44,p.534-535 Gallay PA, Clin. Liver Dis., 2009 16 What are we interested in • The effect of different substituents on the conformation of Cyclosporin • How the conformation affect the binding of Cyclosporin and Cyclophilin /CsA-CyP-Calcinurin • Design new modification for better different kinds of activities. First step : A good computational method to study it. 17 Preview Cyclophilin What are the different roles they play. How CypA bound to HIV-1 Capsid Anti-HCV Mechanisms of CypA Welcome to the next literature report! 18 19