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• W2085191002 abstract "Binary complex formation between the immunosuppressive drug cyclosporin A (CsA) and cyclophilin 18 is the prerequisite for the ability of CsA to inhibit the protein phosphatase activity of calcineurin, a central mediator of antigen-receptor signaling. We show here that several CsA derivatives substituted in position 3 can inhibit calcineurin without prior formation of a complex with cyclophilin 18. [Methylsarcosine3]CsA was shown to inhibit calcineurin, either in its free form with an IC50 value of 10 μm, or in its complex form with cyclophilin 18 with an IC50 of 500 nm. [Dimethylaminoethylthiosarcosine3]CsA ([Dat-Sar3]CsA) was found to inhibit calcineurin on its own, with an IC50 value of 1.0 μm, but was not able to inhibit calcineurin after forming the [Dat-Sar3]CsA-cyclophilin 18 binary complex. Despite their different inhibitory properties, both CsA and [Dat-Sar3]CsA suppressed T cell proliferation and cytokine production mainly through blocking NFAT activation and interleukin-2 gene expression. Furthermore, to demonstrate that [Dat-Sar3]CsA can inhibit calcineurin in a cyclophilin-independent manner in vivo, we tested its effect in a Saccharomyces cerevisiae strain (Δ12), in which all the 12 cyclophilins and FKBPs were deleted. [Dat-Sar3]CsA, but not CsA, bypassed the requirement for cellular cyclophilins and caused growth inhibition in the salt-stressed Δ12 strain. Binary complex formation between the immunosuppressive drug cyclosporin A (CsA) and cyclophilin 18 is the prerequisite for the ability of CsA to inhibit the protein phosphatase activity of calcineurin, a central mediator of antigen-receptor signaling. We show here that several CsA derivatives substituted in position 3 can inhibit calcineurin without prior formation of a complex with cyclophilin 18. [Methylsarcosine3]CsA was shown to inhibit calcineurin, either in its free form with an IC50 value of 10 μm, or in its complex form with cyclophilin 18 with an IC50 of 500 nm. [Dimethylaminoethylthiosarcosine3]CsA ([Dat-Sar3]CsA) was found to inhibit calcineurin on its own, with an IC50 value of 1.0 μm, but was not able to inhibit calcineurin after forming the [Dat-Sar3]CsA-cyclophilin 18 binary complex. Despite their different inhibitory properties, both CsA and [Dat-Sar3]CsA suppressed T cell proliferation and cytokine production mainly through blocking NFAT activation and interleukin-2 gene expression. Furthermore, to demonstrate that [Dat-Sar3]CsA can inhibit calcineurin in a cyclophilin-independent manner in vivo, we tested its effect in a Saccharomyces cerevisiae strain (Δ12), in which all the 12 cyclophilins and FKBPs were deleted. [Dat-Sar3]CsA, but not CsA, bypassed the requirement for cellular cyclophilins and caused growth inhibition in the salt-stressed Δ12 strain. Both immunosuppressive drugs cyclosporin A (CsA) 1The abbreviations used are: CsA, cyclosporin A; FK506, tacrolimus; PPIases, peptidyl-prolyl cis/trans isomerases; Cyp18, cyclophilin 18; FKBP12, FK506-binding protein 12; PP1, Ser/Thr protein phosphatase type 1; PP2A, protein phosphatase type 2A; PP2B, protein phosphatase type 2B or calcineurin; PP2C, protein phosphatase 2C; pNPP, p-nitrophenyl phosphate; RII phosphopeptide, 19-residue phosphopeptide of the regulatory subunit of type II cAMP-dependent protein kinase; PBMCs, peripheral blood mononuclear cells; NFAT, nuclear factor of activated T cell; OVA, ovalbumin; [Dat-Sar3]CsA, [(R)-α-N,N-dimethylaminoethylthiosarcosine3]cyclosporin A methanesulfonate; [Me-Sar3]CsA, [(R)-α-methyl sarcosine3]cyclosporin A; IL, interleukin; PMA, phorbol 12-myristate 13-acetate; GST, glutathione S-transferase; TCR, T cell receptor; Me, methyl; wt, wild-type; I, inhibitor. and tacrolimus (FK506) are believed to inhibit antigen-specific activation of lymphocytes by blocking T cell receptor (TCR) signaling cascades through targeting the Ca2+/calmodulin-dependent Ser/Thr phosphatase calcineurin (1.Liu 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 (3634) Google Scholar, 2.Schreiber S.L. Crabtree G.R. Immunol. Today. 1992; 13: 136-142Abstract Full Text PDF PubMed Scopus (1971) Google Scholar), which represents a specific bottleneck of antigen-receptor signaling. The drugs alone do not inhibit the enzymatic activity of calcineurin. CsA and FK506 bind to and inhibit calcineurin only after interaction with their respective peptidyl-prolyl cis/trans isomerases (PPIases) (EC 5.2.1.8), cyclophilins (Cyps), and FK506-binding proteins (FKBPs) through a gain-of-function mechanism. Therefore, these particular PPIases were also termed immunophilins. Among the many known human PPIases, the most abundant cytosolic members cyclophilin 18 (Cyp18) and FKBP12 were found as major receptor proteins for CsA and FK506, respectively (2.Schreiber S.L. Crabtree G.R. Immunol. Today. 1992; 13: 136-142Abstract Full Text PDF PubMed Scopus (1971) Google Scholar, 3.Fischer G. Angew. Chem. Int. Ed. Engl. 1994; 33: 1415-1436Crossref Scopus (329) Google Scholar). Based on the structures of FKBP12-FK506-calcineurin (4.Griffith J.P. Kim J.L. Kim E.E. Sintchak M.D. Thomson J.A. Fitzgibbon M.J. Fleming M.A. Caron P.R. Hsiao K. Navia M.A. Cell. 1995; 82: 507-522Abstract Full Text PDF PubMed Scopus (774) Google Scholar, 5.Kissinger C.R. Parge H.E. Knighton D.R. Lewis C.T. Pelletier L.A. Tempczyk A. Kalish V.J. Tucker K.D. Showalter R.E. Moomaw E.W. Gastinel L.N. Habuka N. Chen X. Maldonado F. Barker J.E. Bacquet R. Villafranca J.E. Nature. 1995; 378: 641-644Crossref PubMed Scopus (699) Google Scholar) and Cyp18-CsA-calcineurin complexes (6.Huai Q. Kim H.Y. Liu Y. Zhao Y. Mondragon A. Liu J.O. Ke H. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 12037-12042Crossref PubMed Scopus (232) Google Scholar, 7.Jin L. Harrison S.C. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 13522-13526Crossref PubMed Scopus (199) Google Scholar), it is proposed that the inhibition of calcineurin results from blockage of access for protein substrates to the active site by the drug-immunophilin complexes, but does not involve a direct contact to the catalytic center of the protein phosphatase. The inertness of the inhibitor to the catalytic subunit provides the basis for the high specificity of the drug-immunophilin complexes for calcineurin inhibition, as compared with the inhibition of other Ser/Thr protein phosphatases by their corresponding inhibitors. CsA derivatives could be grouped according to their inhibitory properties toward Cyp18 and calcineurin (8.Sigal 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 (229) Google Scholar, 9.Nelson P.A. Akselband Y. Kawamura A. Su M. Tung R.D. Rich D.H. Kishore V. Rosborough S.L. DeCenzo M.T. Livingston D.J. Harding M.W. J. Immunol. 1993; 150: 2139-2147PubMed Google Scholar). Whereas CsA inhibits Cyp18 and the CsA-Cyp18 complex inhibits calcineurin, some CsA derivatives, e.g. [N-methyl-Ala6]CsA, neither in their uncomplexed forms, nor in the complex with Cyp18, are able to inhibit calcineurin (8.Sigal 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 (229) Google Scholar). The studies with these compounds helped to reveal the immunosuppressive mechanism of CsA, as well as the signal transduction pathways in activated T cells, in which the calcineurin phosphatase activity is involved (8.Sigal 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 (229) Google Scholar, 10.Liu 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). For instance, the activation of one of the major transcription factors of antigen-receptor signaling in lymphocytes, NFAT (nuclear factor of activated T cells), is among the known activities of calcineurin. NFAT is a substrate of calcineurin and directly dephosphorylated at multiple sites (11.Okamura H. Aramburu J. Garcia-Rodriguez C. Viola J.P. Raghavan A. Tahiliani M. Zhang X. Qin J. Hogan P.G. Rao A. Mol. Cell. 2000; 6: 539-550Abstract Full Text Full Text PDF PubMed Scopus (387) Google Scholar). Recent genetic studies have suggested the different importance of two major isoforms of calcineurin in T cell development and function, because of their different expression levels in T cells (12.Bueno 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, 13.Zhang B.W. Zimmer G. Chen J. Ladd D. Li E. Alt F.W. Wiederrecht G. Cryan J. O'Neill E.A. Seidman C.E. Abbas A.K. Seidman J.G. J. Exp. Med. 1996; 183: 413-420Crossref PubMed Scopus (140) Google Scholar). The phenotypic analyses of calcineurin Aβ-deficient mice demonstrated a defective immune response that was similar to CsA treatment, whereas only a modest immunologic phenotype was observed in calcineurin Aα-deficient mice. Whereas targeted disruption of the gene of single calcineurin isoform greatly improved our knowledge concerning calcineurin participation in immunosuppression, the indirect mode of action of CsA via a presenter protein renders the drug effect less clear in vivo. For instance, both calcineurin Aα–/– and calcineurin Aβ–/– T cells remained sensitive to CsA inhibition (12.Bueno 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, 13.Zhang B.W. Zimmer G. Chen J. Ladd D. Li E. Alt F.W. Wiederrecht G. Cryan J. O'Neill E.A. Seidman C.E. Abbas A.K. Seidman J.G. J. Exp. Med. 1996; 183: 413-420Crossref PubMed Scopus (140) Google Scholar). Fortunately, chemical genetic studies would provide an alternative approach to answer these questions (14.Schreiber S.L. Bioorg. Med. Chem. 1998; 6: 1127-1152Crossref PubMed Scopus (365) Google Scholar). Small molecule compounds with inhibitory potency and specificity toward Cyp18 and calcineurin different from that of CsA could help us to distinguish between the Cyp18 and calcineurin inhibition involved in cellular effects of CsA. As a first step, the design of CsA derivatives that could inhibit calcineurin in a Cyp18-independent manner, and thus render calcineurin inhibition independent of the intrinsic cyclophilins of the targeted cells, would seem to be ideal to dissect the numerous biological effects of CsA. Here we describe CsA derivatives, in which a single hydrogen atom of sarcosine at position 3 (Sar3) has been replaced by either alkyl or alkylthio groups (15.Seebach D. Beck A.K. Bossler H.G. Gerber C. Ko S.Y. Murtiashaw C.W. Naef R. Shoda S. Thaler A. Krieger M. Wenger R. Helv. Chim. Acta. 1993; 76: 1564-1590Crossref Scopus (84) Google Scholar), bypassing the requirement for Cyp18 binding in calcineurin inhibition. Sar3 had been chosen for modification because of its importance in the control of the CsA conformation (16.Wenger R.M. France J. Bovermann G. Walliser L. Widmer A. Widmer H. FEBS Lett. 1994; 340: 255-259Crossref PubMed Scopus (78) Google Scholar). [Dimethylaminoethylthiosarcosine3]CsA ([Dat-Sar3]CsA) was found to inhibit calcineurin on its own in the low micromolar range, but was unable to inhibit calcineurin after forming the [Dat-Sar3]CsA-Cyp18 binary complex. [Methylsarcosine3]CsA ([Me-Sar3]CsA) was shown to inhibit calcineurin, either in its complex form with Cyp18, or in its free form. To the known CsA derivatives, we added two new types of compounds with different inhibitory properties. Like CsA, [Dat-Sar3]CsA suppressed T cell proliferation and cytokine production, and blocked the activation of NFAT. Calcineurin is conserved in eukaryotes, from yeast to humans. Calcineurin also functions in fungi to control a myriad of physiological processes including cell cycle progression, cation homeostasis, and morphogenesis (17.Fox D.S. Heitman J. Bioessays. 2002; 24: 894-903Crossref PubMed Scopus (115) Google Scholar). CsA and FK506 exert potent antifungal effects against a variety of pathogenic fungi. In Saccharomyces cerevisiae, calcineurin null mutants fail to recover from α-factor pheromone-induced G1 cell cycle arrest and are sensitive to high concentrations of Na+, Li+, or Mn2+ ions (18.Hemenway C.S. Heitman J. Cell Biochem. Biophys. 1999; 30: 115-151Crossref PubMed Scopus (195) Google Scholar). To demonstrate that [Dat-Sar3]CsA can inhibit calcineurin in a Cyp-independent manner in vivo, we took advantage of yeast genetics (19.Cardenas M.E. Lorenz M. Hemenway C. Heitman J. Perspect. Drug Discov. Des. 1994; 2: 103-126Crossref Scopus (31) Google Scholar) and tested its effect in a S. cerevisiae strain (Δ12) (20.Dolinski K. Muir S. Cardenas M. Heitman J. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 13093-13098Crossref PubMed Scopus (251) Google Scholar), which lacks all cyclophilins and FKBPs. We could demonstrate that [Dat-Sar3]CsA, but not CsA, bypassed the requirement for cellular Cyps and caused growth inhibition in salt-stressed Δ12 strain. Expression and purification of recombinant human calcineurin was performed as published (21.Mondragon A. Griffith E.C. Sun L. Xiong F. Armstrong C. Liu J.O. Biochemistry. 1997; 36: 4934-4942Crossref PubMed Scopus (87) Google Scholar). Protein phosphatase 1 (recombinant rabbit muscle α-isoform) was purchased from Calbiochem (Bad Soden, Germany) and protein phosphatase 2C (recombinant human α-isoform) from Upstate Biotechnology (Biomol, Hamburg, Germany). Protein phosphatase 2A was isolated from porcine brain and purified to homogeneity as described (22.Tung H.Y. Alemany S. Cohen P. Eur. J. Biochem. 1985; 148: 253-263Crossref PubMed Scopus (129) Google Scholar). The catalytic subunit of bovine heart cAMP-dependent protein kinase and IL-7 were obtained from Roche Diagnostics. Calmodulin, buffers, and salts were purchased from Sigma. Recombinant human Cyp18 and human FKBP12 have been prepared as described elsewhere (23.Tradler T. Stoller G. Rucknagel K.P. Schierhorn A. Rahfeld J.U. Fischer G. FEBS Lett. 1997; 407: 184-190Crossref PubMed Scopus (60) Google Scholar). The biotinylated and nonbiotinylated 19-residue peptides of a partial sequence of the RII subunit of the bovine protein kinase A (DLDVPIPGRFDRRVSVAAE-OH) were synthesized by solid phase peptide synthesis. The cytokines IL-15 and IL-7 were from R&D Systems (Wiesbaden, Germany). Cell media and all supplements were purchased from Biochrom (Berlin, Germany). The wild-type strain JK93da (wt) and isogenic strain KDY98.4a (Δ12) of S. cerevisiae were kindly provided by J. Heitman (20.Dolinski K. Muir S. Cardenas M. Heitman J. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 13093-13098Crossref PubMed Scopus (251) Google Scholar). The synthesis was performed by a method previously described (15.Seebach D. Beck A.K. Bossler H.G. Gerber C. Ko S.Y. Murtiashaw C.W. Naef R. Shoda S. Thaler A. Krieger M. Wenger R. Helv. Chim. Acta. 1993; 76: 1564-1590Crossref Scopus (84) Google Scholar). In brief, a series of CsA position 3 side chain derivatives were synthesized by generation of an enolate at the sarcosine residue and reaction with various electrophiles. The reactions occurred in the presence or absence of LiCl for obtaining S or R diastereoisomers selectively. [N-Methyl-Asp(O-tert-But)3]CsA (Table I, entry 6) is a mixture of S and R diastereoisomers. The identities of products were confirmed with ESI-MS. We analyzed the samples with reverse phase analytic high performance liquid chromatography and the purities of all CsA derivatives are higher than 95%.Table IInhibition constants of CsA derivatives for inhibition of peptidyl-prolyl cis/trans isomerase activity of Cyp18 and of CsA derivatives or CsA derivative-Cyp18 complexes for inhibition of phosphatase activity of calcineurinInhibition of enzyme activities by CsA derivativesEntryAbbreviationsCompoundCyp18aS.D. ≤ 20%.KiCalcineurin (in the presence of Cyp18)bS.D. ≤ 10%.,cNI, ≤ 10% inhibition of calcineurin by a preformed complex of CsA derivative-Cyp18 (10 μm/10 μm). IC50Calcineurin (without Cyp182)bS.D. ≤ 10%.,dNI, ≤ 10% inhibition of calcineurin by a CsA derivative (10 μm). IC50nmnmμmCsACyclosporin A0.5100NI1[Dat-Sar3]CsA[(R)-α-N,N-Dimethylaminoethylthiosarcosine3])CsA methanesulfonate0.1NI1.02[(3R, 4R)-3-Hydroxy-N,4-dimethyl-L-2-aminooctanoic acid1] [(R) α-N, N-dimethylaminoethylthiosarcosine3]CsA methanesulfonate4.3NI1.53[Me-Sar3]CsA[(R)-α-Methylsarcosine3]CsA≤0.5500104[(R)-α-Methylthiosarcosine3]CsA0.51000105[(S)-α-Methylsarcosine3]CsA≤0.5(∼45% inhibition at 5 μm Cyp)NI6[N-Methyl-Asp(O-tert-But)3]CsA1180 nmNIa S.D. ≤ 20%.b S.D. ≤ 10%.c NI, ≤ 10% inhibition of calcineurin by a preformed complex of CsA derivative-Cyp18 (10 μm/10 μm).d NI, ≤ 10% inhibition of calcineurin by a CsA derivative (10 μm). Open table in a new tab RII Phosphopeptide-based Calcineurin Activity Assay—The biotinylated and nonbiotinylated 19-residue peptides of a partial sequence of the RII subunit of the bovine protein kinase A were phosphorylated at residue 15 as previously described (24.Baumgrass R. Weiwad M. Erdmann F. Liu J.O. Wunderlich D. Grabley S. Fischer G. J. Biol. Chem. 2001; 276: 47914-47921Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar). For all calcineurin activity assays, the 33P-labeled RII phosphopeptide was used. Calcineurin activity was measured using a scintillation proximity assay (24.Baumgrass R. Weiwad M. Erdmann F. Liu J.O. Wunderlich D. Grabley S. Fischer G. J. Biol. Chem. 2001; 276: 47914-47921Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar). In brief, preincubation of calmodulin (50 nm), calcineurin (1.32 nm), and inhibitor at the required concentrations in the phosphatase assay buffer (40 mm Tris/HCl, pH 7.5, 100 mm NaCl, 6 mm MgCl2, 0.5 mm dithiothreitol, 1 mm CaCl2, 0.1 mg/ml bovine serum albumin) was carried out at 22 °C for 30 min in a 96-well microtiter plate (Costar, Bodenheim, Germany). 10 pmol of biotinylated [33P]RII phosphopeptide was added to each well in a total assay volume of 100 μl. After dephosphorylation of RII phosphopeptide by calcineurin at 30 °C for 20 min, 90 μl of the reaction mixture were transferred to a scintillation well coated with streptavidin (PerkinElmer Life Sciences). Biotinylated RII phosphopeptide was allowed to bind to streptavidin for 20 min at 22 °C. The well was washed once with water, and the RII phosphopeptide associated 33P was measured in a MicroBeta top counter (Wallac, Turku, Finland). The obtained data were fitted and computed with the SigmaPlot program (SPSS Inc., San Rafael, CA). The calcineurin inhibition was measured at a concentration range of 0.5 to 30 μm CsA (AWD, Dresden, Germany) or CsA derivatives. For determination of the calcineurin inhibition by drug-Cyp18 complexes, the assay was also carried out in the presence of varying concentrations of Cyp18 and 10 μm CsA or CsA derivatives. For competition experiments, the mixture of calcineurin and [Dat-Sar3]CsA was pre-equilibrated in the assay buffer at 22 °C for 30 min. Subsequent use of the incubation mixture in the RII phosphopeptide calcineurin activity assay yielded a final concentration of 1.32 nm calcineurin, 10 μm [Dat-Sar3]CsA. Increasing concentrations of Cyp18 from 0.01 to 10 μm were used to compete with calcineurin for [Dat-Sar3]CsA. Simultaneous inhibition of calcineurin by FK506 (1 μm) and [Me-Sar3]CsA (5 μm) or [Dat-Sar3]CsA (0.75 μm) was examined dependent on the FKBP12 concentration (0.5–250 nm). For kinetic studies, biotinylated RII phosphopeptide and nonbiotinylated RII phosphopeptide were mixed at a molar ratio of 1:200 in assay buffer. A total RII phosphopeptide concentration of 5 to 15 μm was used for each concentration of [Dat-Sar3]CsA (0.25–1.5 μm) with a calcineurin concentration of 0.66 nm. p-Nitrophenyl Phosphate (pNPP)-based Calcineurin Activity Assay— Phosphatase activity was measured at room temperature using pNPP as substrate in phosphatase assay buffer (see above). After preincubation of calcineurin with the inhibitor in phosphatase assay buffer at room temperature for 20 min, the reaction was initiated by the addition of pNPP to a final concentration of 10 mm. The release of p-nitrophenol was continuously measured using a Dynatec MR7000 microtiter plate reader at 410 nm for 45 min. Inhibition of Other Protein Ser/Thr Phosphatases—The activities of the protein phosphatases PP2A and PP2C were measured with RII phosphopeptide, as described for calcineurin. For assaying PP2C a final concentration of 30 mm MgCl2 was additionally included. The protein phosphatase concentrations were adjusted to an activity level of ∼80% dephosphorylation of 10 pmol of RII phosphopeptide within 20 min. Protein phosphatase 1 and PP2A were assayed by using 32P-labeled phosphorylase a as substrate (Sigma) (25.Pelech S. Cohen P. Eur. J. Biochem. 1985; 148: 245-251Crossref PubMed Scopus (72) Google Scholar) and 32P-labeled casein was used for calcineurin and PP2C as previously described (26.Waelkens E. Goris J. Di Salvo J. Merlevede W. Biochem. Biophys. Res. Commun. 1984; 120: 397-404Crossref PubMed Scopus (14) Google Scholar). The protein phosphatase inhibition was evaluated for [Me-Sar3]CsA and [Dat-Sar3]CsA in a concentration range of 1 to 100 μm. Peptidyl-prolyl cis/trans-Isomerase Assay—PPIase activity was determined with oligopeptide substrates using protease-coupled assays as described elsewhere (27.Fischer G. Wittmann-Liebold B. Lang K. Kiefhaber T. Schmid F.X. Nature. 1989; 337: 476-478Crossref PubMed Scopus (1212) Google Scholar). Typically, experiments were performed with PPIase concentrations in the low nanomolar range and CsA or CsA derivatives within the assay. The effect of the inhibitors on the PPIase activity was calculated from the remaining activity after preincubation of the enzyme and the inhibitor at 22 °C for 20 min. The pre-equilibrated mixture of [Dat-Sar3]CsA and calcineurin (50 μm/13.2 nm stock solution) in assay buffer containing calmodulin (50 nm) was used to examine the reversibility of inhibition by dialysis experiments. The mixture was placed on a Pierce system microdialyzer equipped with a Mr 3000 cut-off dialysis membrane and dialyzed against the assay buffer at 4 °C. Reference activity was determined with a calcineurin sample treated similarly but without addition of [Dat-Sar3]CsA. Aliquots of 10 μl of dialyzed enzyme or [Dat-Sar3]CsA-enzyme complex were assayed using RII phosphopeptide substrate. Cyp18 was fused to the C-terminal of GST. The fusion protein (25 μm) was incubated with calcineurin (50 μm) in the presence of Me2SO, CsA, or [Dat-Sar3]CsA (25 μm) in 200 μl at 22 °C for 30 min with gentle agitation. The GST-Cyp18 and all bound proteins were precipitated with 50 μl of glutathione-Sepharose. The proteins were eluted from the Sepharose beads with 100 μl of SDS-PAGE sample buffer by heating at 90 °C for 10 min after the beads were washed three times with RIPA buffer (150 mm NaCl, 1% Nonidet P-40, 0.5% deoxycholate, 0.1% SDS, 50 mm Tris/HCl, pH 7.5). The supernatants after centrifugation were subjected to SDS-PAGE. Proteins were transferred from gel onto nitrocellulose membrane by the tank blotting procedure and visualized by the enhanced chemiluminescent detection system (Amersham Biosciences) using polyclonal antibodies specific for calcineurin or Cyp18. Human peripheral blood mononuclear cells (PBMCs) were obtained from healthy volunteers using Ficoll-Hypaque gradient centrifugation. The cells from one buffy coat (∼2 × 108 cells) were cultured overnight in RPMI 1640 supplemented with 2 mm l-glutamine, heat-inactivated 10% fetal calf serum, and 1% penicillin/streptomycin mixture. The washed cells were homogenized in 7 ml of buffer (25 mm Tris/HCl, pH 7.4, 3 mm CaCl2, 0.5 mm dithiothreitol, 0.1 mm EDTA, 500 nm okadaic acid, and a protease inhibitor mixture) on ice, with a Teflon homogenizer. The homogenate was incubated with 200 μl of calmodulin-Sepharose 4B beads (Amersham Biosciences) with constant agitation for 60 min at 4 °C. The beads were pelleted by gravitation and washed twice with the homogenizing buffer. The beads were resuspended in 0.5 ml of assay buffer (see above) and immediately used to assay calcineurin. The final amount of enzyme isolated from 106 cells gave an activity of 0.087 pmol min–1 using the RII phosphopeptide substrate. T Cell Separation and Culture—CD4+ and CD8+ T cells were isolated by positive selection from human PBMCs using magnetic Multisort MicroBeads (Miltenyi Biotech, Bergisch Gladbach, Germany) (28.Miltenyi S. Muller W. Weichel W. Radbruch A. Cytometry. 1990; 11: 231-238Crossref PubMed Scopus (1464) Google Scholar) with a purity of >97%. The cells were cultured at 3 × 107/ml in RPMI 1640 supplemented with 2 mm l-glutamine, heat-inactivated 10% fetal calf serum, and 1% penicillin-streptomycin mixture overnight. Splenic cells were prepared from mice homozygously transgenic (tg) 1 mice, homozygous for the DO11.10 a/b-TCR (OVA-TCRtg/tg) (29.Murphy K.M. Heimberger A.B. Loh D.Y. Science. 1990; 250: 1720-1723Crossref PubMed Scopus (1653) Google Scholar) on BALB/c background (gift from Dennis Y. Loh, Washington University School of Medicine, St. Louis, MO). The mice were bred and maintained under specific pathogen-free conditions. Proliferation—PBMCs were labeled with 5-(and 6-)carboxyfluorescein-diacetate-succinimidylester before stimulation with plate-bound anti-CD3 plus anti-CD28 ABs (2 × 106 cells/ml) (30.Richter A. Lohning M. Radbruch A. J. Exp. Med. 1999; 190: 1439-1450Crossref PubMed Scopus (173) Google Scholar) in the presence of inhibitors, or Me2SO as control in 24-well plates. Stock solutions of all inhibitors were in Me2SO (Sigma, Deisenhofen, Germany) and added to a final concentration of 0.5% Me2SO in each cell sample. The cells were cultured for 5 days and directly analyzed on viable cells by flow cytometry. OVA-specific splenic cells were stimulated with the antigenic peptide OVA323–339 in the presence of inhibitors or Me2SO as control in 96-well plates and cultured for 4 days. For experiments with additional cytokines, sorted T cells were used and IL-2 (10 IU/ml), IL-7 (10 ng/ml), and IL-15 (10 ng/ml) were added just before stimulation. Intracellular Cytokine Production—PBMCs (2 × 106/ml) were preincubated with one of the following compounds at various concentrations as indicated: CsA (AWD, Dresden, Germany), [Dat-Sar3]CsA, [Me-Sar3]CsA, or Me2SO at 37 °C for 10 min in 24-well plates. Stock solutions of the inhibitors in Me2SO (Sigma) were added to a final concentration of 0.5% Me2SO in each cell sample. PBMCs were stimulated with 10 ng/ml PMA and 1 μg/ml ionomycin (both from Sigma) for 5 h with the addition of brefeldin A (5 μg/ml) for the last 3 h of stimulation. Cells were then fixed in 2% paraformaldehyde for 20 min, permeabilized by washing in phosphate-buffered saline supplemented with 0.5% saponin and 1% fetal calf serum, incubated with anti-IL-2 phycoerythrin-conjugated antibody, and measured by flow cytometry. Sorted T cells were used in experiments with exogenous cytokines, and IL-2 (10 IU/ml), IL-7 (10 ng/ml), and IL-15 (10 ng/ml) were added just before stimulation. Sorted T cells transfected with the NFAT-reporter gene plasmid (Stratagene, Netherlands) by electroporation (Amaxa, Cologne, Germany) were cultured in RPMI 1640 with 10% fetal calf serum and 2 mm l-glutamine for 16 h at 37 °C in 5% CO2. The cells were preincubated with inhibitors for 30 min and then stimulated with 10 ng/ml PMA and 1 μg/ml ionomycin for 5 h. After cell lysis the level of the extracted luciferase from these cells was determined by bioluminescence measurement using the luciferase assay system (Promega, Mannheim, Germany). In addition, a β-galactosidase plasmid was cotransfected as internal standard. To investigate cyclophilin-independent relative sensitivity of cells to the growth inhibition by [Dat-Sar3]CsA, the wild-type strain JK93da (wt) and the isogenic strain KDY98.4a (Δ12) lacking the 12 authentic immunophilins were used (20.Dolinski K. Muir S. Cardenas M. Heitman J. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 13093-13098Crossref PubMed Scopus (251) Google Scholar, 31.Breuder T. Hemenway C.S. Movva N.R. Cardenas M.E. Heitman J. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 5372-5376Crossref PubMed Scopus (125) Google Scholar). Samples of cell suspensions with similar A600 of the strains to be tested were used in the presence of CsA or [Dat-Sar3]CsA under lithium cation stress. The yeast strains were grown to early log phase at 30 °C. Serial 100-fold dilutions were spotted onto YPD medium with or without 100 mm LiCl and with or without 10 μm inhibitor (32.Gautschi M. Mun A. Ross S. Rospert S. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 4209-4214Crossref PubMed Scopus (129) Google Scholar). Stock solutions of the inhibitors were prepared in ethanol. All media contained 1% ethanol (31.Breuder T. Hemenway C.S. Movva N.R. Cardenas M.E. Heitman J. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 5372-5376Crossref PubMed Scopus (125) Google Scholar). Incubation was performed at 22 °C for 5 days. Biochemical Characterization of Sar3-modified CsA Derivatives—CsA derivatives with a modified Sar3 residue were prepared by established chemical procedures (Fig. 1) (15.Seebach D. Beck A.K. Bossler H.G. Gerber C. Ko S.Y. Murtiashaw C.W. Naef R. Shoda S. Thaler A. Krieger M. Wenger R. Helv. Chim. Acta. 1993; 76: 1564-1590Crossref Scopus (84) Google Scholar). All compounds showed low nanomolar affinity for Cyp18 (Table I), the prerequisite for calcine" @default.
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