Study of calcineurin interaction with inhibitor-1 and natural products. Towards novel calcineurin inhibitors

Calcineurin (CaN) is an eukaryotic Ser/Thr protein phosphatase that plays an important role in lymphocyte activation. Inhibition of CaN leads to immune system suppression, a necessary procedure in organ transplantation. However, use of CaN inhibitors results in toxic side effects. The search for more specific immunosuppressants is therefore an important and ongoing endeavor. The focus of this thesis was to investigate methods that could lead to the discovery of novel CaN inhibitors.;CaN is closely related to protein phosphatase-1 (PP-1) but both phosphatases retain distinct substrates, regulatory proteins, and inhibitors. Two such inhibitors, okadaic acid (OA) and microcystin-LR (MCLR), potently inhibit PP-1 but are markedly less effective against CaN. Mutagenesis of CaN was undertaken to generate a form of CaN more sensitive to OA and MCLR. The optimal construct was a Y159I:F160Y:L312C:Y315L quadruple point mutant that showed 600-fold and 37-fold increased sensitivity to MCLR and OA, respectively. These studies provide the basis for chemical engineering to generate analogs of OA and MCLR that are CaN specific.;Multiple proteins use the PXIXIT amino acid sequence to interact with CaN, most notably the nuclear factor of activated T-cells (NFAT). NFAT dephosphorylation by CaN is necessary for immune system activation. The second chapter of results demonstrates that inhibitor-1 (I-1), a CaN substrate, contains the PXIXIT motif used for CaN binding. Disruption of the PXIXIT motif of I-1 reduced its dephosphorylation by CaN. I-1 therefore is a suitable substrate for identification of novel immunosuppressants that could block the interaction between CaN and the PXIXIT motif of NFAT proteins.;A new method of bioassay-guided isolation of novel CaN inhibitors from extracts of marine organisms was established in the final chapter of results. Monitoring the inhibition of CaN activity using the I-1 substrate during extract purification led to identification of three compounds: halisulfate-7, hipposulfate C, and a novel one, irregularsulfate. These compounds were identified as equipotent microM inhibitors of CaN and PP-1. The method of bioassay-guided isolation of CaN inhibitors is applicable to searching for potential drugs that could block access to the active site of CaN, or to the PXIXIT motif binding groove.