Inhibition of the Calcineurin-NFAT Interaction by Small Organic Molecules Reflects Binding at an Allosteric Site (2005)

Transcriptional signaling from the Ca2+-calmodulin-activated phosphatase calcineurin to its substrate NFAT (nuclear factor of activated T cells, also termed NFATc) is critically dependent on a protein-protein docking interaction between calcineurin and the PXIXIT motif in NFAT. Several inhibitors of NFAT-calcineurin association (INCA compounds) prevent binding of NFAT or the peptide ligand PVIVIT to calcineurin. Here we show that the binding site on calcineurin for INCA1, INCA2, and INCA6 is centered on cysteine 266 of calcineurin Aα and does not coincide with the core PXIXIT-binding site. Although ample evidence indicates that INCA1 and INCA2 react covalently with cysteine 266, covalent derivatization alone is not sufficient for maximal inhibition of the calcineurin-PVIVIT interaction, because the maleimide INCA12 reacts with the same site and produces only very modest inhibition. Thus, inhibition arises through an allosteric change affecting the PXIXIT docking site, which may be assisted by covalent binding but depends on other specific features of the ligand. The spatial arrangement of the binding sites for PVIVIT and INCA makes it probable that the change in conformation involves the β11-β12 loop of calcineurin. The finding that an allosteric site controls NFAT binding opens new alternatives for inhibition of calcineurin-NFAT signaling. Transcriptional signaling from the Ca2+-calmodulin-activated phosphatase calcineurin to its substrate NFAT (nuclear factor of activated T cells, also termed NFATc) is critically dependent on a protein-protein docking interaction between calcineurin and the PXIXIT motif in NFAT. Several inhibitors of NFAT-calcineurin association (INCA compounds) prevent binding of NFAT or the peptide ligand PVIVIT to calcineurin. Here we show that the binding site on calcineurin for INCA1, INCA2, and INCA6 is centered on cysteine 266 of calcineurin Aα and does not coincide with the core PXIXIT-binding site. Although ample evidence indicates that INCA1 and INCA2 react covalently with cysteine 266, covalent derivatization alone is not sufficient for maximal inhibition of the calcineurin-PVIVIT interaction, because the maleimide INCA12 reacts with the same site and produces only very modest inhibition. Thus, inhibition arises through an allosteric change affecting the PXIXIT docking site, which may be assisted by covalent binding but depends on other specific features of the ligand. The spatial arrangement of the binding sites for PVIVIT and INCA makes it probable that the change in conformation involves the β11-β12 loop of calcineurin. The finding that an allosteric site controls NFAT binding opens new alternatives for inhibition of calcineurin-NFAT signaling. Ca2+-calcineurin signaling serves vital purposes in mammalian cells, among them the provision of a direct link between the mobilization of cytoplasmic Ca2+ and gene expression (1Aramburu J. Rao A. Klee C.B. Curr. Top. Cell. Regul. 2000; 36: 237-295Crossref PubMed Scopus (276) Google Scholar, 2Rusnak F. Mertz P. Physiol. Rev. 2000; 80: 1483-1521Crossref PubMed Scopus (1109) Google Scholar). In the physiological activation of T cells, calcineurin acts through NFAT 4The abbreviations used are: NFATnuclear factor of activated T cellsCsAcyclosporin ADTTdithiothreitolIAMiodoacetamideINCAinhibitor of NFAT-calcineurin associationMALDI-TOFmatrix-assisted laser desorption ionization time-of-flightNEMN-ethylmaleimidePVIVIT14-mer peptide MAGPHPVIVITGPHEE-amide family transcription factors and other transcriptional effectors (3Rao A. Luo C. Hogan P.G. Annu. Rev. Immunol. 1997; 15: 707-747Crossref PubMed Scopus (2223) Google Scholar, 4Serfling E. Berberich-Siebelt F. Chuvpilo S. Jankevics E. Klein-Hessling S. Twardzik T. Avots A. Biochim. Biophys. Acta. 2000; 1498: 1-18Crossref PubMed Scopus (170) Google Scholar, 5Crabtree G.R. Olson E.N. Cell. 2002; 109: S67-S79Abstract Full Text Full Text PDF PubMed Scopus (1097) Google Scholar, 6Hogan P.G. Chen L. Nardone J. Rao A. Genes Dev. 2003; 17: 2205-2232Crossref PubMed Scopus (1557) Google Scholar). Levels of calcineurin in T cells are limiting, and upward or downward modulation of calcineurin enzymatic activity is directly reflected in increased or decreased transcription from cytokine gene promoters (7Fruman D.A. Klee C.B. Bierer B.E. Burakoff S.J. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 3686-3690Crossref PubMed Scopus (755) Google Scholar, 8O'Keefe S.J. Tamura J. Kincaid R.L. Tocci M.J. O'Neill E.A. Nature. 1992; 357: 692-694Crossref PubMed Scopus (788) Google Scholar, 9Clipstone N.A. Crabtree G.R. Nature. 1992; 357: 695-697Crossref PubMed Scopus (1476) Google Scholar, 10Tsuboi A. Masuda E.S. Naito Y. Tokumitsu H. Arai K. Arai N. Mol. Biol. Cell. 1994; 5: 119-128Crossref PubMed Scopus (34) Google Scholar, 11Fruman D.A. Pai S.Y. Burakoff S.J. Bierer B.E. Mol. Cell. Biol. 1995; 15: 3857-3863Crossref PubMed Google Scholar, 12Batiuk T.D. Pazderka F. Enns J. DeCastro L. Halloran P.F. J. Clin. Investig. 1995; 96: 1254-1260Crossref PubMed Scopus (88) Google Scholar, 13Batiuk T.D. Kung L. Halloran P.F. J. Clin. Investig. 1997; 100: 1894-1901Crossref PubMed Scopus (77) Google Scholar, 14Bueno O.F. Brandt E.B. Rothenberg M.E. Molkentin J.D. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 9398-9403Crossref PubMed Scopus (158) Google Scholar). The immunosuppressive drugs cyclosporin A (CsA) and FK506 owe their clinical effectiveness to the ability of CsA-cyclophilin or FK506-FKBP12 complexes to inhibit calcineurin in cells of the immune system (15Liu J. Farmer Jr., J.D. Lane W.S. Friedman J. Weissman I. Schreiber S.L. Cell. 1991; 66: 807-815Abstract Full Text PDF PubMed Scopus (3620) Google Scholar, 16Liu J. Albers M.W. Wandless T.J. Luan S. Alberg D.G. Belshaw P.J. Cohen P. MacKintosh C. Klee C.B. Schreiber S.L. Biochemistry. 1992; 31: 3896-3901Crossref PubMed Scopus (502) Google Scholar). However, the beneficial actions of CsA and FK506 are counterbalanced by serious toxicities attributed at least in part to their interference with calcineurin signaling in other cells and tissues (17Sigal N.H. Dumont F. Durette P. Siekierka J.J. Peterson L. Rich D.H. Dunlap B.E. Staruch M.J. Melino M.R. Koprak S.L. Williams D. Witzel B. Pisano J.M. J. Exp. Med. 1991; 173: 619-628Crossref PubMed Scopus (230) Google Scholar, 18Dumont F.J. Staruch M.J. Koprak S.L. Siekierka J.J. Lin C.S. Harrison R. Sewell T. Kindt V.M. Beattie T.R. Wyvratt M. Sigal N.H. J. Exp. Med. 1992; 176: 751-760Crossref PubMed Scopus (232) Google Scholar). nuclear factor of activated T cells cyclosporin A dithiothreitol iodoacetamide inhibitor of NFAT-calcineurin association matrix-assisted laser desorption ionization time-of-flight N-ethylmaleimide 14-mer peptide MAGPHPVIVITGPHEE-amide The centrality of calcineurin and the calcineurin-NFAT pathway to immune responses has suggested that interrupting signaling at any of several points could provide a therapeutic alternative to current immunosuppressive drugs. Promising target points are the Ca2+ signal that activates calcineurin (19Serafini A.T. Lewis R.S. Clipstone N.A. Bram R.J. Fanger C. Fiering S. Herzenberg L.A. Crabtree G.R. Immunity. 1995; 3: 239-250Abstract Full Text PDF PubMed Scopus (58) Google Scholar, 20Venkatesh N. Feng Y. DeDecker B. Yacono P. Golan D. Mitchison T. McKeon F. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 8969-8974Crossref PubMed Scopus (45) Google Scholar), the protein-protein interaction of calcineurin and NFAT (21Loh C. Shaw K.T.-Y. Carew J. Viola J.P.B. Luo C. Perrino B.A. Rao A. J. Biol. Chem. 1996; 271: 10884-10891Abstract Full Text Full Text PDF PubMed Scopus (266) Google Scholar, 22Wesselborg S. Fruman D.A. Sagoo J.K. Bierer B.E. Burakoff S.J. J. Biol. Chem. 1996; 271: 1274-1277Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar, 23Aramburu J. García-Cozár F. Raghavan A. Okamura H. Rao A. Hogan P.G. Mol. Cell. 1998; 1: 627-637Abstract Full Text Full Text PDF PubMed Scopus (258) Google Scholar, 24Aramburu J. Yaffe M.B. López-Rodríguez C. Cantley L.C. Hogan P.G. Rao A. Science. 1999; 285: 2129-2133Crossref PubMed Scopus (521) Google Scholar, 25Roehrl M.H.A. Kang S. Aramburu J. Wagner G. Rao A. Hogan P.G. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 7554-7559Crossref PubMed Scopus (139) Google Scholar), and the cooperative binding of NFAT and AP1 on DNA (26Jain J. McCaffrey P.G. Valge-Archer V.E. Rao A. Nature. 1992; 356: 801-804Crossref PubMed Scopus (428) Google Scholar, 27Northrop J.P. Ullman K.S. Crabtree G.R. J. Biol. Chem. 1993; 268: 2917-2923Abstract Full Text PDF PubMed Google Scholar, 28Chen L. Glover J.N. Hogan P.G. Rao A. Harrison S.C. Nature. 1998; 392: 42-48Crossref PubMed Scopus (413) Google Scholar, 29Peterson B.R. Sun L.J. Verdine G.L. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 13671-13676Crossref PubMed Scopus (51) Google Scholar, 30Macián F. García-Rodríguez C. Rao A. EMBO J. 2000; 19: 4783-4795Crossref PubMed Scopus (263) Google Scholar). Each strategy has the potential to be more selective and, hence, less toxic than treatment with CsA or FK506, but none has yet advanced to the stage of small nonpeptide inhibitors that can be tested in vivo. There is considerable evidence that targeting the calcineurin-NFAT protein-protein interaction will produce a selective inhibition. We have demonstrated that the calcineurin-NFAT interaction is based on recognition of a PXIXIT motif in NFAT and that this recognition is essential for efficient signaling (23Aramburu J. García-Cozár F. Raghavan A. Okamura H. Rao A. Hogan P.G. Mol. Cell. 1998; 1: 627-637Abstract Full Text Full Text PDF PubMed Scopus (258) Google Scholar). Peptides that compete for binding at the PXIXIT recognition site both inhibit the calcineurin-NFAT interaction in vitro and selectively inhibit calcineurin-NFAT signaling in cells (23Aramburu J. García-Cozár F. Raghavan A. Okamura H. Rao A. Hogan P.G. Mol. Cell. 1998; 1: 627-637Abstract Full Text Full Text PDF PubMed Scopus (258) Google Scholar, 24Aramburu J. Yaffe M.B. López-Rodríguez C. Cantley L.C. Hogan P.G. Rao A. Science. 1999; 285: 2129-2133Crossref PubMed Scopus (521) Google Scholar, 31Liu J. Arai K. Arai N. J. Immunol. 2001; 167: 2677-2687Crossref PubMed Scopus (35) Google Scholar). A recently published study (32Noguchi H. Matsushita M. Okitsu T. Moriwaki A. Tomizawa K. Kang S. Li S.-T. Kobayashi N. Matsumoto S. Tanaka K. Tanaka N. Matsui H. Nat. Med. 2004; 10: 305-309Crossref PubMed Scopus (234) Google Scholar) shows that a competitor peptide modified to promote its uptake into cells can prevent heterologous graft rejection in mice. Further, high throughput screening of a library of organic compounds has led to identification of nonpeptide inhibitors of NFAT-calcineurin association (INCA compounds) (25Roehrl M.H.A. Kang S. Aramburu J. Wagner G. Rao A. Hogan P.G. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 7554-7559Crossref PubMed Scopus (139) Google Scholar). These compounds interfere with calcineurin-NFAT signaling in cells, motivating a continued search for inhibitors that have higher affinity and reduced nonspecific toxicity and that are suitable for in vivo administration to animals. The further development of inhibitors can be guided by structural information about the sites of protein-protein interaction and inhibitor binding. To this end, we have determined the structure of the NFAT docking site on in which the PXIXIT recognition peptide of NFAT in an and of the peptide directly calcineurin H. Rao A. Hogan P.G. J. Mol. Biol. 2004; PubMed Scopus Google Scholar). Here we the structural by a binding site for INCA compounds at a cysteine to the PXIXIT peptide docking site. is covalent binding or binding of the INCA compounds at this site that recognition of PXIXIT peptide and NFAT. to the that are to be in In the that calcineurin activity Klee C.B. Nature. 1996; PubMed Scopus Google Scholar, F. T. Sci. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar, D. J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar, D. S. Biophys. 2002; PubMed Scopus Google Scholar), may be by and expression for and the of calcineurin Aα with the and has J. Yaffe M.B. López-Rodríguez C. Cantley L.C. Hogan P.G. Rao A. Science. 1999; 285: 2129-2133Crossref PubMed Scopus (521) Google Scholar). with or or with the to for and to at 266, is to by with and In the of the in by at the of from the by affinity on and in and with by and at In the of in the reduced to of the and the further into any interference of with the INCA In in of The PVIVIT 14-mer peptide J. Yaffe M.B. López-Rodríguez C. Cantley L.C. Hogan P.G. Rao A. Science. 1999; 285: 2129-2133Crossref PubMed Scopus (521) Google Scholar) at in a of of and of in of The peptide by high The and at and at INCA2, and INCA12 from at in and at of at for to at to on the on in a the at at in an Each and other for of the by In binding calcineurin at or in direct binding calcineurin used at in for the signal to a by of the same with iodoacetamide N-ethylmaleimide or INCA12 for to from direct with calcineurin have to a for binding to a of sites J. Hogan P.G. Proc. Natl. Acad. Sci. U. S. A. 1994; PubMed Scopus Google Scholar). and for INCA compounds have to a because the are not sufficient to for covalent and of the INCA The of association of and INCA2 to produce an of PVIVIT binding. To of the by with of for and to the of only calcineurin and PVIVIT on peptide binding and not prevent of peptide by of only the INCA to with calcineurin and PVIVIT demonstrated that of INCA2 from the To of the PVIVIT and calcineurin with or and PVIVIT binding the the calcineurin in is than the of the calcineurin-PVIVIT interaction, of is a for the of in and in for at with INCA1, INCA2, or the of this into and or further for the of to into and with for and with INCA for and by binding the to in a and with and with in and and the target an in with of at at and of the A in the peptide a of is the peptide in that with INCA1, INCA2, or or in to with INCA6 to the to by the same the in A of the of the both in peptide and in that with or INCA The or of with peptide the that of the compounds with the cysteine of nuclear of and of cytokine in T cells in (23Aramburu J. García-Cozár F. Raghavan A. Okamura H. Rao A. Hogan P.G. Mol. Cell. 1998; 1: 627-637Abstract Full Text Full Text PDF PubMed Scopus (258) Google Scholar, 24Aramburu J. Yaffe M.B. López-Rodríguez C. Cantley L.C. Hogan P.G. Rao A. Science. 1999; 285: 2129-2133Crossref PubMed Scopus (521) Google Scholar, 25Roehrl M.H.A. Kang S. Aramburu J. Wagner G. Rao A. Hogan P.G. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 7554-7559Crossref PubMed Scopus (139) Google Scholar). activity in a D.A. Pai S.Y. Klee C.B. Burakoff S.J. Bierer B.E. 1996; PubMed Scopus Google Scholar) with peptide T cells on by at and in and, on and to an of and with in of The by the of for at and the by the of Each to a and the and its of determined by based on the of calcineurin Aα from and J.P. M.J. K. Cell. 1995; Full Text PDF PubMed Scopus Google Scholar, Lewis L.A. A. N. Chen F. R. Nature. 1995; PubMed Scopus Google Scholar). The of PVIVIT peptide from Li H. Rao A. Hogan P.G. J. Mol. Biol. 2004; PubMed Scopus Google Scholar). The compounds INCA1, INCA2, and INCA12 have to inhibit recognition of NFAT by calcineurin by the binding of the PVIVIT peptide to calcineurin. Several have the that a covalent is in the inhibition of PVIVIT peptide binding. the inhibitors or compounds that are and that are to the of binding at of an INCA for INCA1 with a of inhibition only of at is of the of a covalent INCA1, INCA2, and INCA6 inhibitors in the binding by with and not could of the compounds by to a less or covalent with of are sites of covalent by with this of calcineurin with the INCA2 of calcineurin by In more of calcineurin with the maleimide INCA12 by INCA1, INCA2, and INCA6 and not of calcineurin with the and INCA12 reduced the calcineurin-PVIVIT signal in the of an INCA competitor and not of INCA12 sufficient to the calcineurin-PVIVIT but high not the signal from PVIVIT for high of and the calcineurin-PVIVIT signal to a that the signal of peptide The for this is that calcineurin the ability to PVIVIT with reduced In the of the nonspecific and could be to of binding or to of the the PVIVIT peptide is The be in a in the maximal signal by PVIVIT with or calcineurin and a in the of the of the binding In the for calcineurin change in but to an with a in affinity of the interaction and are inhibitors of the calcineurin-PVIVIT interaction, and that at the same site the INCA of a activity to and the that compounds a cysteine of calcineurin. the of several in calcineurin on the activity of INCA2 to the and a more study that the to the of INCA2 demonstrated that this also the effectiveness of INCA1, and the other cysteine not for the of INCA The structure of calcineurin shows cysteine in the and We calcineurin in which cysteine to to calcineurin with the same a of PVIVIT to the However, of the other than not the effectiveness of INCA1, INCA2, or the in the of other the of the inhibitors and not The in this also the of These that the of is and sufficient for a of PVIVIT binding by INCA of a tested the ability of the INCA compounds to react with the calcineurin peptide The peptide is with a to the of a covalent at that demonstrated that INCA1 and INCA2, at react covalently with the calcineurin peptide in by with that the and not to react with the a with INCA1 and INCA2 and that the INCA compounds the the peptide is to have a conformation in the show that INCA1 and INCA2 are to cysteine an INCA6 more with of the peptide several by with The that covalent with in calcineurin higher of a that the of the or a interaction that in targeting INCA6 to the site. of of calcineurin not provide evidence of an the same peptide by with of in the of and calcineurin. However, it has to in of other that are to be covalently modified by because the is M. S.Y. Biochemistry. 2001; PubMed Scopus Google Scholar, J.J. S. M. J. P.J. Chem. Biol. 2003; PubMed Scopus Google Scholar). of the not prevent its in the with the because in the INCA in and the directly by of by association of INCA compounds with calcineurin has demonstrated by and by (25Roehrl M.H.A. Kang S. Aramburu J. Wagner G. Rao A. Hogan P.G. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 7554-7559Crossref PubMed Scopus (139) Google Scholar). further that of INCA2, it is with for not PVIVIT binding with the INCA the INCA2, not to into of calcineurin with at an that the site on not to any of PVIVIT binding the The by INCA2 is because treatment with the of INCA2 in of INCA1 and INCA2 has the of their for In INCA6 the of NFAT and calcineurin-NFAT signaling calcineurin enzymatic activity (25Roehrl M.H.A. Kang S. Aramburu J. Wagner G. Rao A. Hogan P.G. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 7554-7559Crossref PubMed Scopus (139) Google Scholar). INCA1 and INCA2 not at that because of their and the less INCA6 toxicity in of to the of INCA2 in T cells, for which INCA2 is not used at INCA2 to of the of the nuclear of and the of and and and not However, that the physiological with a inhibition of calcineurin activity A is of calcineurin through INCA2 or from INCA2 that are by INCA2 into to produce of the in These new the (25Roehrl M.H.A. Kang S. Aramburu J. Wagner G. Rao A. Hogan P.G. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 7554-7559Crossref PubMed Scopus (139) Google Scholar) that current INCA compounds are to the calcineurin-NFAT interaction in vitro and that the of compounds to inhibit the calcineurin-NFAT pathway in cells The of this study has to the binding site on calcineurin for the calcineurin-NFAT signaling inhibitors INCA1, INCA2, and The of the site in the of of calcineurin and its spatial to the NFAT docking site on calcineurin are in A and B. The from of to the of the PVIVIT peptide is Thus, to the INCA inhibitors not PVIVIT peptide and NFAT by binding in the core PXIXIT recognition site but by an allosteric change in the site. The probable of the allosteric change is the β11-β12 loop of which and to the and of the site H. Rao A. Hogan P.G. J. Mol. Biol. 2004; PubMed Scopus Google Scholar). and other in or are in with the that both of the β11-β12 loop makes with the of and and a for the peptide from to and are in with and is in a on the of and in with the of and of a ligand in direct with the binding is covalent or is to a structural and could the conformation of the β11-β12 In to any in the of of could also in the and its and its to of the A of the of INCA compounds is that of at a structural that the PVIVIT docking site and that of a covalent part of the for the In of the INCA compounds have the potential for covalent binding. INCA1 and INCA2, in are with the cysteine specific on PVIVIT derivatization of could be a of their or could be a interaction that the compounds to the of to covalent binding. INCA6 is less of a covalent through targeting of INCA6 to the site or through of the The also that INCA6 is not a covalent A more of the with calcineurin will of the that physiological and other in the that from of the with peptide in the to the INCA2 to could a in of S. Y. K. K. Chen T. S. T. K. K. Proc. Natl. Acad. Sci. U. S. A. 2001; PubMed Scopus Google Scholar, A. J.K. I. B. I. A. T. S. J. K. K. J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar) and for other M. S.Y. Biochemistry. 2001; PubMed Scopus Google Scholar, J.J. S. M. J. P.J. Chem. Biol. 2003; PubMed Scopus Google Scholar, Mol. Google Scholar, J. S. 1993; PubMed Scopus Google Scholar, J. M. Chem. 1997; 10: PubMed Scopus Google Scholar, R.J. L.A. Chem. 2000; PubMed Scopus Google Scholar), or an at the in the of with J.P. R. A. C. Biochemistry. 1999; PubMed Scopus (77) Google Scholar). are in the Chem. 1994; PubMed Scopus Google Scholar) and could further alternative of of is the of D. L. J. R. C. M.J. Biochemistry. 2004; PubMed Scopus Google Scholar), which in their react with cysteine to of or D. L. J. R. C. M.J. Biochemistry. 2004; PubMed Scopus Google Scholar, A. J.N. D. Nature. 2003; PubMed Scopus Google Scholar). However, a interaction between calcineurin and INCA compounds (25Roehrl M.H.A. Kang S. Aramburu J. Wagner G. Rao A. Hogan P.G. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 7554-7559Crossref PubMed Scopus (139) Google Scholar), and the with peptide evidence that the INCA compounds cysteine A to is that the covalent inhibitors and INCA12 react with and the affinity of calcineurin for PVIVIT but less than INCA1, INCA2, and finding the of an change than covalent derivatization of in inhibition. further serves an that other inhibitors of calcineurin-PVIVIT binding by high throughput screening (25Roehrl M.H.A. Kang S. Aramburu J. Wagner G. Rao A. Hogan P.G. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 7554-7559Crossref PubMed Scopus (139) Google Scholar) may covalently or at this allosteric site. The current a the identification of inhibitors of calcineurin-NFAT signaling. of the PXIXIT-binding site H. Rao A. Hogan P.G. J. Mol. Biol. 2004; PubMed Scopus Google Scholar) has a structural for the of inhibitors that compete directly with the NFAT docking binding at the site will a less for the structure of the complexes has suggested by are further high throughput screening with of the screening of calcineurin or of library compounds with or that will the in the of inhibitors and of D.A. Annu. Rev. Biophys. 2004; PubMed Scopus Google Scholar) on the site we have it may to covalent that are to the site by a interaction and are with nonspecific sites Curr. Chem. Biol. 2000; PubMed Scopus Google Scholar, G. A. D. D. Y. D. J. Chem. 2003; PubMed Scopus Google Scholar). The of covalent inhibitors is an in because of the of between a in the of the interaction and an in the of a site of interaction, of cysteine and may provide an this The of affinity at the PXIXIT-binding site to at and the an for of calcineurin signaling in has on the that calcineurin activity is by the physiological of and of at the site Klee C.B. Nature. 1996; PubMed Scopus Google Scholar, F. T. Sci. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar, D. J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar, D. S. Biophys. 2002; PubMed Scopus Google Scholar, K. M. J. Immunol. 1999; Google Scholar, F. J. Biol. Chem. 2002; PubMed Scopus Google Scholar, H. T. J. 2002; PubMed Scopus Google Scholar). or in cells also at the site, could modulation of calcineurin activity with a of selective on We the of and for to the with