Cyclosporin A (CsA, 1) is the drug of choice for preventing rejection of transplanted human organs. Mechanism of action studies have shown that CsA binds to cyclophilins A and B (CyP A; CyP B) and that the CsA-CyP complex binds to and inhibits calcineurin (CaN), a calmodulin-dependent serine/threonine protein phosphatase. Although much is known about this property of CsA, only a limited number of CsA analogs have been synthesized and evaluated for immunosuppression, due to the complexity of the total synthesis of derivatives. In addition to immunosuppression, CsA is implicated in several other biological activities including inhibition of HIV replication in acute and chronically infected T-lymphocytes, and binding to hsp7O with dissociation constants near 50 nM. Hsp7O appears to be important in progression of cancer, where it has been implicated in resistance to immunological defense mechanisms and to the onset of drug resistance to certain antitumor drugs. Hsp7O also has been shown to be in the rabies virion and to be a component of the hepatitis B virion. Nothing is known about the effect of CsA structure on these later activities. We have achieved the first synthesis of a CsA analog by solid phase peptide synthesis during the past grant period. We propose to develop the chemistry to carry out solid phase synthesis of CsA itself and its analogs, and to apply this technology to the synthesis of CsA libraries by combinatorial methods. Screening methods for detecting and quantitating binding to cyclophilin, calcineurin, hsp7O, HIV protease and other proteins will be developed. Solution conformations of certain interesting compounds will be determined by NMR. These studies will greatly increase the data-base for correlating the biological activities of CsA with structure, and may provide novel approaches and lead compounds for new therapeutic agents.

Agency
National Institute of Health (NIH)
Institute
National Institute of Arthritis and Musculoskeletal and Skin Diseases (NIAMS)
Type
Research Project (R01)
Project #
2R01AR032007-13
Application #
2078794
Study Section
Bio-Organic and Natural Products Chemistry Study Section (BNP)
Project Start
1983-03-01
Project End
1999-03-31
Budget Start
1995-06-05
Budget End
1996-03-31
Support Year
13
Fiscal Year
1995
Total Cost
Indirect Cost
Name
University of Wisconsin Madison
Department
Type
Schools of Pharmacy
DUNS #
161202122
City
Madison
State
WI
Country
United States
Zip Code
53715
Kuzmic, P (1996) Program DYNAFIT for the analysis of enzyme kinetic data: application to HIV proteinase. Anal Biochem 237:260-73
Kuzmic, P; Peranteau, A G; Garcia-Echeverria, G et al. (1996) Mechanical effects on the kinetics of the HIV proteinase deactivation. Biochem Biophys Res Commun 221:313-7
Hu, M K; Badger, A; Rich, D H (1995) Cyclosporin analogs modified in the 3,7,8-positions: substituent effects on peptidylprolyl isomerase inhibition and immunosuppressive activity are nonadditive. J Med Chem 38:4164-70
Bartz, S R; Hohenwalter, E; Hu, M K et al. (1995) Inhibition of human immunodeficiency virus replication by nonimmunosuppressive analogs of cyclosporin A. Proc Natl Acad Sci U S A 92:5381-5
Peranteau, A G; Kuzmic, P; Angell, Y et al. (1995) Increase in fluorescence upon the hydrolysis of tyrosine peptides: application to proteinase assays. Anal Biochem 227:242-5
Kuzmic, P; Garcia-Echeverria, C; Rich, D H (1993) Stabilization of HIV proteinase dimer by bound substrate. Biochem Biophys Res Commun 194:301-5
Garcia-Echeverria, C; Kofron, J L; Kuzmic, P et al. (1993) A continuous spectrophotometric direct assay for peptidyl prolyl cis-trans isomerases. Biochem Biophys Res Commun 191:70-5
Kuzmic, P (1993) Kinetic assay for HIV proteinase subunit dissociation. Biochem Biophys Res Commun 191:998-1003
Moss, M L; Palmer, R E; Kuzmic, P et al. (1992) Identification of actin and HSP 70 as cyclosporin A binding proteins by photoaffinity labeling and fluorescence displacement assays. J Biol Chem 267:22054-9
Kuzmic, P; Moss, M L; Kofron, J L et al. (1992) Fluorescence displacement method for the determination of receptor-ligand binding constants. Anal Biochem 205:65-9

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