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Title: Gossypol inhibits calcineurin phosphatase activity at multiple sites

item Dowd, Michael

Submitted to: European Journal of Pharmacology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 10/19/2006
Publication Date: 11/27/2006
Citation: Carruthers, N.J., Dowd, M.K., Stemmer, P.M. 2006. Gossypol inhibits calcineurin phosphatase activity at multiple sites. European Journal of Pharmacology. 555(2-3):106-114.

Interpretive Summary: Gossypol inhibits the activity of the calcineurin, a phosphatase enzyme that is a target of immunosuppressant drugs. This work explores the mechanism by which this inhibition occurs. The results indicate that the interactions between gossypol and the enzyme are complicated, but suggest that gossypol is not acting in any specific manner. The lack of specificity indicates that gossypol may not be useful as an immune system suppressant. The work will be of interest to researchers working to understand gossypol’s biological activity.

Technical Abstract: Calcineurin, the calcium/calmodulin dependant serine/threonine phosphatase is the target for the immunosuppressant drugs FK506 and cyclosporine A. These calcineurin inhibitors each require an immunophilin protein cofactor. Gossypol, a polyphenol produced by the cotton plant, inhibits calcineurin, in a noncompetitive, reversible manner, and is independent of any cofactor. We found that gossypol acts by at least two mechanisms to inhibit calcineurin phosphatase activity. A calmodulin-independent form of calcineurin was less sensitive to inhibition by gossypol than native calcineurin, indicating that gossypol may interfere with calmodulin binding. A fluorescence polarization based assay demonstrated that 100 µM gossypol reduced the affinity of calmodulin for calcineurin. Inhibition of calcineurin phosphatase activity by gossypol could not be overcome by adding excess calmodulin or by testing the inhibition toward a calmodulin-independent calcineurin, indicating that gossypol acts at a site different from the calmodulin-binding site. Gossypol decreased the affinity of calcineurin for immunosuppressant/immunophilin complexes only in the presence of calmodulin, indicating that gossypol blocks effects of calmodulin binding to calcineurin. In addition, gossypol had a stimulatory effect on native calcineurin in the absence of calmodulin, possibly indicating a calmodulin mimetic effect. Gossypol exists in two enantiomeric forms which are reported to have different potency for cell toxicity. (+)- and (-)-gossypol had equivalent potency for inhibition of native and calmodulin-independent calcineurin phosphatase activity, and for inhibition of calmodulin binding. The inhibition of calcineurin by gossypol via multiple binding sites without stereo-specificity indicates that gossypol is not a specific calcineurin inhibitor.