Effect of calmodulin on CN585-mediated calcineurin inhibition. Calcineurin inhibition by various CN585 concentrations was measured in the presence of 50 nm (square), 100 nm (diamond), and 200 nm (triangle) calmodulin using the RII phosphopeptide substrate. Initial rates of calcineurin activity were plotted against the inhibitor concentration. The data presented are the means ± S.D. of three independent experiments.

Analysis of the inhibition type and putative calcineurin binding site. Inhibition type of CN585 was evaluated using substrate concentration from 2.1 to 20.1 μm RII phosphopeptide mixture containing 100 nm biotinylated and 0.5–20 μm nonbiotinylated RII phosphopeptide. The inhibition mode and the K_i value of CN585 were determined by plotting 1/v against CN585 (Dixon plot) (A) and by plotting [S]/v against CN585 (B). C, to obtain indications about the CN585 binding site on calcineurin, the phosphatase was simultaneously inhibited with CN585 and increasing concentrations of the Cyp18·CsA complex. Measurements were performed using ³³P-labeled RII phosphopeptide, 3 μm CN585, 10 μm CsA, and various Cyp18 concentrations. Because of the high affinity between Cyp18 and CsA, the concentration of the Cyp18·CsA complex is identical to the applied Cyp18 concentration. Moreover, by using DynaFit software, two theoretical binding curves were calculated according to a model of identical binding sites (long dash) and a model of different binding sites (dash-dot-dot) of CN585 and Cyp18·CsA. The shape of the measured curves (solid line) clearly indicates a binding on identical or overlapping binding sites of both inhibitors. Moreover, the concentration-dependent inhibition of calcineurin by Cyp18·CsA and CN585 alone is also displayed in the figure. All experimental data presented are the means ± S.D. of three independent experiments.

Influence of CN585 on NFAT translocation in HeLa cells. HeLa cells were transfected with a green fluorescent protein-NFAT^2–417 expression plasmid by electroporation. Ten hours after transfection cells were incubated for 45 min with CN585, CN675, or CsA as positive and [MeVal]⁴CsA as negative controls. Then cells were stimulated with PMA/ionomycin for 1 h, paraformaldehyde-fixed, and 4′,6-diamidino-2-phenylindole-stained. The fluorescence images were recorded with a CCD camera upon Nikon TE200 epifluorescence microscope. The experiment was repeated two times with similar results.

Influence of CN585 on proliferation of stimulated PBMC. Human PBMC were prepared using Ficoll density gradient, labeled with 5,6-carboxyfluorescein diacetate succinimidyl ester, and incubated with 1 μm CsA, various CN585 concentrations, 7 μm CN675, or DMSO as control. Afterward, the PMBC (2 × 10⁶ cells/ml) were stimulated with plate-bound anti-CD3/anti-CD28 antibodies and cultured for 5 days. All samples were directly analyzed on viable cells by flow cytometry, and individual generations were calculated using ModFitLT software. The experiment was repeated three times with similar results.

Influence of CN585 and CN675 on calcineurin phosphatase activity. Calcineurin activity was measured in phosphatase assays using ³³P-labeled RII phosphopeptide (A) or pNPP (B) as substrates in the presence of increasing concentrations of CN585 (closed circle) or CN675 (open circle). The data presented are the percent activity of a DMSO-containing control as means ± S.D. of three independent measurements.

Calcineurin inhibition by CN585 is reversible. A, a preincubated mixture of 15 nm calcineurin and 40 μm CN585 in assay buffer containing 100 nm calmodulin was used to examine the reversibility of inhibition in a dialysis experiments. The mixture and a non-inhibited calcineurin control were placed in a 3-kDa MicroDialyzer and dialyzed. Initial rates of calcineurin activity of the dialyzed enzyme or CN585-enzyme complex were determined using RII phosphopeptide substrate. B, additionally, initial rates of 4–28 nm calcineurin preincubated with calmodulin, 1–10 μm CN585, and ³³P-labeled RII phosphopeptide were measured at 30 °C. V_max was plotted versus the concentration of calcineurin. In the case of a reversible inhibitor, all straight lines have a common point of intersection. The data shown are the means ± S.D. of three independent experiments.

Coprecipitation of recombinant and endogenous calcineurin using CN778 affinity matrix. A, biotinylated CN585 derivative, CN778, was immobilized onto streptavidin-Sepharose. Beads were incubated with recombinant calcineurin or Jurkat cell lysate in the presence and absence of CN585 or CN675 for 3 h at 4 °C. Recombinant calcineurin (CaN) and endogenous calcineurin of Jurkat cells interacts with the affinity matrix. CN585 interferes with calcineurin binding, whereas the non-inhibitory compound CN675 has no effect on calcineurin interaction. B, binding of calcineurin subunits onto the CN778 matrix is shown. The catalytic subunit of calcineurin A (CaN A) and the regulatory subunit B (CaN B) were incubated separately or together with the immobilized CN778. The regulatory subunit shows no interaction with the inhibitor, whereas the catalytic subunit interacts with the inhibitor matrix. C, shown is competition of calcineurin interaction to CN585 by CsA·Cyp18 and FK506·FKBP12 complexes. CN778 affinity matrix was incubated with Jurkat cell lysate, and after several washing steps, CsA·Cyp18 and FK506·FKBP12 complexes were added. In the case of CsA·Cyp18, calcineurin binding to the beads was strongly diminished, whereas the FK506·FKBP12 had no effect on calcineurin interaction. Biotin-saturated streptavidin-Sepharose was used as control. Subsequently, the matrix was washed with PBS and boiled in SDS sample buffer, and bound proteins were separated by SDS-PAGE. The proteins were blotted onto nitrocellulose membrane, probed with anti-calcineurin antibody, and analyzed by ECL reaction.

CN585 decreases NFAT reporter gene activity and reduces intracellular IL-2 production of stimulated PBMC. A, Jurkat cells were transfected with a NFAT-luciferase reporter plasmid by electroporation. The cells were preincubated with the compounds for 30 min and then stimulated with PMA/ionomycin for 5 h. After cell lysis the level of extracted luciferase was determined by using luciferase assay system. Additionally, pcDNA-lacZ was cotransfected, and β-galactosidase activity was measured as internal standard. B, Ficoll-isolated human PBMC (5 × 10⁶ cells/ml) were preincubated with various concentrations of CN585, 15 μm CN675, or 1 μm CsA at 37 °C for 30 min in a 24-well plate. Subsequently, cells were stimulated with PMA/ionomycin (iono) for 5 h and for the last 3 h in the presence brefeldin A. PBMC were fixed in paraformaldehyde, permeabilized with 0.5% saponin in PBS/fetal calf serum, incubated with anti-IL-2 fluorescein isothiocyanate-conjugated antibody, and analyzed by flow cytometry. The data presented are the means ± S.D. of three independent experiments.