Selective Modulation of Nuclear Factor of Activated T-Cell Function in Restenosis by a Potent Bipartite Peptide InhibitorNovelty and Significance
Rationale: Nuclear factor of activated T-cells (NFAT) is importantly implicated in pathological cardiac remodeling and vascular lesion formation. NFAT functionality is mainly regulated by calcineurin, a Ca2+-dependent multi-effector phosphatase. Calcineurin inhibitors such as cyclosporine A (CsA) were shown to be effective in the treatment of restenosis and vascular inflammation but with adverse side effects.
Objective: This prompted the design of more selective inhibitors such as VIVIT and inhibitors of NFAT-calcineurin association, which unfortunately have a poor potency precluding clinical use.
Methods and Results: Here, we describe the rational design of a potent bipartite inhibitor of NFAT–calcineurin interaction, MCV1, which targets two separate calcineurin docking motifs. Modeling, site-directed mutagenesis, and functional studies demonstrated that MCV1 acts by allosteric modulation of calcineurin. Comparable to CsA, MCV1 prevents NFAT activation at nanomolar potency without impairing calcineurin phosphatase activity, nuclear factor-κB nuclear import, and general cell signaling. In contrast, CsA but not MCV1-activated basal level extracellular signal-regulated kinases activity and prevented nuclear import of calcineurin, independent of NFAT activation. In vivo MCV1 abrogated NFAT-mediated T-cell activation in a model of PMA-elicited peritonitis, whereas topical application of MCV1 markedly reduced neointima formation in a mouse model of restenosis.
Conclusions: We designed a bipartite NFAT inhibitor that is more potent than VIVIT and more selective than CsA. MCV1 constitutes not only a powerful tool to unravel NFAT function but also a potential drug candidate for the treatment of diseases implicating NFAT activation.
Transcription factors of the nuclear factor of activated T-cells (NFAT; C1–C5) family are expressed in T-cells, B-cells, NK-cells, and mast cells, as well as in all major nonimmune cell types relevant to cardiovascular diseases, including cardiomyocytes, vascular smooth muscle cells (VSMCs), endothelial cells, and macrophages.1 NFAT functionality is tightly regulated by calcineurin, a calmodulin-dependent calcium-activated phosphatase. On activation, calcineurin binds and dephosphorylates cytoplasmic NFAT, which then translocates to the nucleus of activated cells to induce cytokine expression. Except for its key role in immunity, NFAT has been importantly implicated in osteoclast differentiation, muscle fiber-type specialization, cardiac valve development, myocardial hypertrophy, heart failure, and restenosis.1–9
Given the key role of calcineurin–NFAT signaling in various physiological and immunologic processes, its inhibition has long been considered a powerful therapeutic modality in the treatment of graft transplant rejection, autoimmune diseases, and cardiovascular disorders. The traditional immunosuppressants cyclosporine A (CsA) and FK506 disrupt calcineurin phosphatase activity to inhibit all of its downstream effectors, including NFAT. However, their application was associated with adverse side effects such as nephrotoxicity, hypertension, and malignancy.10–12 In search of more selective and less toxic NFAT inhibitors, recent attention has been given to calcineurin–NFAT docking inhibitors that block calcineurin–NFAT interactions rather than phosphatase activity of calcineurin. These efforts were given further impetus to the identification of VIVIT peptide (MAGPHPVIVITGPHEE),13 LxVP peptide,14,15 and a series of synthetic leads, termed inhibitors of NFAT–calcineurin association (INCA).16 However, the low potency of VIVIT (≈30–100 μmol/L in vascular cells17), the cytotoxicity of INCA compounds, or the broad inhibition of calcineurin phosphatase activity by LxVP peptide13,16,18 disqualifies these compounds for direct clinical use. Selective and potent inhibitors of the calcineurin–NFAT cascade are still eagerly awaited.17
In this study, we pursued a two-step strategy to optimize VIVIT in search of a selective NFAT inhibitor with superior potency. First, the minimal essential motif of VIVIT was defined. Second, this motif was conjugated to an INCA analog creating a potent bipartite compound, which simultaneously targets two calcineurin docking sites and selectively inhibits NFAT at low nanomolar potency without affecting calcineurin phosphatase activity. Given its favorable features, we propose that this conjugate has great potential in the treatment of immune-related and cardiovascular disorders such as restenosis, cardiac hypertrophy, and transplant rejection.
Detailed Methods are provided in the Online Supplement at http://circres.ahajournals.org.
Transient Transfection and Dual Luciferase Assay
Cells were seeded in 24-well plates. After 24 hours, cells were cotransfected with pNFAT-Luc reporter and pRL-CMV plasmid with FuGene 6 transfection reagent according to the manufacturer's instructions.
Custom-designed murine cytokine antibody arrays were performed according to the manufacturer's instructions (RayBiotech, UK).
C57BL/6 mice were obtained from Charles River Laboratories, Maastricht, the Netherlands. To measure T-cell activation in vivo, mice were first injected intraperitoneally with either phosphate-buffered saline control, MCV1, or CsA (both 50 μmol, n=5). After 30 minutes, the mice were injected intraperitoneal with PMA (100 ng) or phosphate-buffered saline and, after 2 hours, blood and peritoneal leukocytes were collected.
To induce neointimal lesions, 9-week-old apolipoprotein E−/− mice were fed a western-type diet ad libitum for 1 week before injury and throughout the experiment. Transluminal wire injury of the left common carotid artery was performed as described previously.32 Immediately after denudation, a 25% F-127 pluronic gel (Sigma-Aldrich, Zwijndrecht, the Netherlands) containing phosphate-buffered saline, MCV1 (100 μmol/L), or CsA (100 μmol/L) was applied to the adventitia of the left common carotid artery (10 μL/mouse). After 1 or 4 weeks, the mice were anesthetized, in situ fixed through the left cardiac ventricle, and the carotid arteries were isolated.
Identify the Essential Motif of VIVIT for NFAT Inhibition
Because both SPRIEIT and truncated VIVIT peptides were able to inhibit NFAT activation,13,16,19 it is conceivable that not all of the amino acids of VIVIT are essential for the blockade of calcineurin–NFAT docking. We first sought to define the minimal essential motif of VIVIT via alanine scanning and stepwise truncation. A NFAT1 (also known as NFATc2 or NFATp) reporter gene assay in murine macrophage RAW cells or proliferation assay in murine VSMCs was used to assess NFAT inhibition by the synthetic peptides.20 Stepwise truncation and alanine scan studies confirmed the critical importance of the GxHPVIVIx core motif, where “x” is any amino acid (Figure 1A, B). Second, our lead peptides HPVIVI, HPVIVIT, GPHPVIVI, and GPHPVIVIT were synthesized, which showed similar inhibition of NFAT transcriptional activation in RAW cells at 100 μmol/L (data not shown). We therefore examined their effect on the inhibition of platelet-derived growth factor-BB–mediated VSMC proliferation with titrated concentration. HPVIVIT and GPHPVIVIT were much more potent than HPVIVI or GPHPVIVI, suggesting that threonine in the PxIxIT motif is strictly required. HPVIVIT was 10-fold more potent than GPHPVIVIT and was thus chosen as the minimal essential motif of VIVIT (Figure 1C).
Bipartite Conjugates of INCA and HPVIVIT Are More Potent Than VIVIT in NFAT Inhibition
We next took advantage of recent findings that INCA compounds inhibit NFAT–calcineurin interactions by reaction to sulfhydryl groups near the putative VIVIT binding cleft.18 We conjugated several INCA mimetics to the N-terminal amino group of HPVIVIT (Figure 2A), arguing that bipartite antagonists of NFAT should bind more avidly to calcineurin by simultaneously interacting with the VIVIT and the INCA binding clefts. Flexible 8- to 27.6-Å linker arms were introduced to stretch the estimated 15-Å gap between the two binding sites. Two of these maleimido-conjugated VIVIT motifs, MCV1 and MCV2, were able to inhibit NFAT activation to basal levels at 100 nmol/L, whereas HPVIVIT or INCA12 were completely ineffective at this concentration. Although MCV3 and MCV4 appeared to be more potent than HPVIVIT, they were unable to totally prevent ionomycin/PMA-stimulated NFAT activation (Online Figure IA). The IC50 values of MCV1 and MCV2 were 61.7 nmol/L and 127.7 nmol/L, respectively (Figure 2B), which is a 1000-fold more potent than the parent peptide VIVIT (IC50=30–100 μmol/L).13,20 MCV1 is at least equally potent as CsA (IC50=29.3 nmol/L) and was used as the lead peptide.
We next tested the ability of MCV1 to compete with purified GST-NFATc2 for binding to calcineurin in vitro in a pull-down assay (Figure 2C). MCV1 was partly effective at 100 nmol/L and completely disrupted calcineurin–NFAT interactions at 10 μmol/L, showing similar inhibitory effect to VIVIT in calcineurin binding to GST-NFAT. In keeping, pull-down assay with full-length calcineurin showed that MCV1 partially inhibited calcineurin–NFAT interaction at 1 μmol/L and completely disrupted the binding at 10 μmol/L (Online Figure II). Moreover, MCV1 completely inhibited NFAT dephosphorylation at 1 μmol/L, whereas INCA12, the VIVIT parent peptide, and HPVIVIT were completely ineffective at this concentration (Figure 2D). Quantitative analyses of Western blots are given inFigures 2E and 2F, respectively (n=3). We next addressed NFAT inhibition by MCV1 in different cells relevant to vasculopathies such as restenosis. MCV1 but not VIVIT at 1 μmol/L could significantly inhibit NFAT transcriptional activation not only in macrophages as shown in Figure 2B but also in endothelial cells, VSMCs, and cardiomyocytes (Online Figure III). Furthermore, MCV1 at 100 nmol/L was seen to abrogate nuclear translocation of NFAT1-GFP after ionomycin stimulation, whereas HPVIVIT showed only weak inhibition even at a 100-fold higher concentration (Figure 2G, 2H). Nuclear distribution of GFP protein was confirmed with DAPI counterstain (Online Figure IV). Remarkably, MCV2 showed potent inhibition of NFAT nuclear import at 1 μmol/L but lost its inhibitory capacity at 10 μmol/L. The mechanism underlying this biphasic activity is unclear.
The inhibitory effect of MCV1 was persistent (Online Figure VA), with an estimated half-life of approximately 36 hours. In addition, MCV1 did not show overt signs of cytotoxicity in murine VSMCs cells as assessed by Trypan blue exclusion assay at concentrations of up to 10 μmol/L (Online Figure VB). Flow cytometry analysis of VSMC apoptosis by Annexin V–FITC/propedium iodine showed that both MCV1 and CsA did not induce VSMC apoptosis or necrosis. (Online Figure VI).
MCV1 Is More Selective Than CsA in NFAT Inhibition
The currently available inhibitors of calcineurin–NFAT signaling such as CsA generally cause quite severe side effects, such as renal dysfunction and hypertension. Of note, the toxicity of CsA not only is attributable to disruption of calcineurin–NFAT signaling but also may do so by perturbating other signaling pathways, such as MAP kinase or nuclear factor κB signaling, or by activating endothelial functionality through activation of endothelin-1 (ET-1) or nitric oxide signaling. We first addressed the selectivity of MCV1 over CsA in calcineurin–NFAT signaling. MCV1 at concentrations of up to 10 μmol/L did not inhibit calcineurin phosphatase activity, indicating that MCV1 does not target the calcineurin catalytic site, despite effectively inhibiting calcineurin–NFAT interaction and NFAT dephosphorylation. In contrast, 1 μmol/L CsA inhibited calcineurin phosphatase by approximately 90% (Figure 3A). Similar results were observed after PMA/ionomycin stimulation in the presence of MCV1 or CsA (Online Figure VII). Because CsA-induced hypertension and kidney dysfunction were seen to proceed through ET-1–dependent vasorestriction,33–34 we next examined the release of ET-1 in murine endothelial EOMA cells treated with CsA or MCV1 by enzyme-linked immunosorbent assay, in which CsA but not MCV1 at 10 μmol/L showed a significant increase in ET-1 secretion (Figure 3B).
In addition to NFAT family members, calcineurin also binds endogenous regulators such as A-kinase anchoring protein 79 (AKAP 79) and calcineurin-binding protein-1/calcineurin inhibitor (Cabin-1/Cain), which carry motifs with similarity to the PxIxIT consensus motif of NFAT21,22 (Online Figure IB). As shown by Figure 3C, binding of calcineurin to either AKAP 79 or Cabin-1 was not affected by MCV1 at concentrations that suffice for effective disruption of calcineurin–NFAT interaction. We also examined YFP-p65 protein nuclear translocation because nuclear factor κB is another downstream effector of calcineurin (Figure 3D). CsA completely disrupted p65 activation and prevented its nuclear import to basal levels, whereas MCV1 showed no significant effect (Figure 3F).
Calcineurin was seen to translocate to the nucleus together with NFAT as a complex.23–26 We observed a partial nuclear co-import of endogenous calcineurin with NFAT1-GFP on ionomycin stimulation (Figure 3E). CsA at 1 μmol/L completely blocked the nuclear co-import of calcineurin and NFAT. Surprisingly, MCV1 only blunted nuclear import of NFAT (Figure 3G) but not of calcineurin. Therefore, Ca2+-mediated nuclear import of calcineurin is involved in but not directly linked to NFAT activation. Furthermore, we tested if MCV1 impaired general cellular signaling over extracellular signal-regulated kinases (ERK) activation (Figure 3H). Pretreatment with either MCV1 or CsA did not alter PMA-induced ERK activation, and pretreatment with MCV1 alone had no effect on endogenous ERK activity. Interestingly, although CsA did not affect PMA-stimulated ERK activity, it markedly increased phospho-ERK levels in nonstimulated cells at concentrations as low as 0.1 μmol/L. Thus, CsA not only prevented dephosphorylation of NFAT but also facilitated its phosphorylation via activation of the ERK pathway in an unknown mechanism. This dual mode of action of CsA may be instrumental in the high potency of CsA and its off-target immunotoxicity. CsA was shown to be able to dose-dependently activate ERK, reaching a maximal plateau of ERK phosphorylation under 1 to 10 μmol/L of CsA stimulation (Online Figure VIII).
Molecular modeling studies of MCV1 interaction with calcineurin were performed to provide a mechanistic basis for the profound gain in affinity of MCV1 versus the separate building blocks HPVIVIT and MPB. The most favorable configuration with the lowest energy (Figure 4A) shows that MCV1 has a distinct binding site from the catalytic site of calcineurin and should not impair the calcineurin phosphatase activity. The hydrophobic side chains of MCV1 are in close contact with the β11 loop, whereas the maleimido group is accommodated in the groove formed by the β11-β12 and β13-β14 turns. No solutions in the 20 runs with low energies were found to support a direct contact between the maleimido unit and Cys266, which actually was partially protected by the β11-β12 loop. The minimal binding energy of MCV1 was −12.59 kcal/mol, which is comparable to that reported for PVIVIT (−13 kcal/mol).27 To assess the hypothesis that there is a covalent reaction between maleimido group of MCV1 and sulfhydryl group, NFAT translocation inhibitory capacity of MCV1 was compared to that of MCV1 that had been preincubated with DTT. Pretreatment with DTT totally blunted the inhibitory effect of MCV1 (Figure 4B). Likewise, preincubation cells with sulfhydryl-modifying reagents IAM or INCA12 dose-dependently prevented the MCV1-induced inhibition of NFAT translocation (Figure 4C). To firmly establish that MCV1 covalently binds to calcineurin, purified calcineurin protein was incubated in vitro with MCV1 in the presence or absence of DTT. After native polyacrylamide gel electrophoresis (10%; none denaturing), binding characteristics were analyzed by Western blotting against calcineurin. A progressive portion of full-length calcineurin, size 81 kDa, appeared to migrate faster after incubation with increasing concentration of MCV1 than untreated protein, possibly because of MCV-induced conformational changes in calcineurin or changes in surface-exposed charge. Importantly, preincubation with equimolar of DTT negated the quicker migration pattern of calcineurin caused by MCV1 at 10 μmol/L, pointing to covalent binding between calcineurin and MCV1 (Online Figure IV). Furthermore, site-directed mutation of Cys266 of calcineurin catalytic unit led to impaired displacement of NFAT by MCV1 but not VIVIT (Figure 4D), supporting a covalent interaction between MCV1 and calcineurin. On the basis of these studies, we propose the model for MCV1-mediated NFAT inhibition depicted in Figure 4E.
MCV1 Abrogates NFAT-Mediated T-Cell Activation, VSMC Proliferation, and Reduces Neointima Formation in a Mouse Model of Restenosis
To define the therapeutic potential of MCV1 in a PMA-elicited peritonitis model in vivo, we inoculated mice with MCV1 or CsA. Intraperitoneal pretreatment with both MCV1 and CsA inhibited PMA-elicited CD4+ T-cell activation as demonstrated by expression of CD69, CD40, and CD86. No effect was observed on the CD8+ T-cell activation for both compounds (Figure 5A). Total blood cell numbers and pattern remained unaffected (data not shown). ANOVA analysis of creatinin/aspartate amino transferase/alanine amino transferase/lactate dehydrogenase concentrations in PMA stimulated plasma under CsA/MCV1 treatment did not reveal any statistically significant differences between groups (Online Figure X). All other liver toxicity data between groups were negative, which suggests that MCV1 should not have an unexpected side effect. However, unlike MCV1, CsA was seen to stimulate IL-6 secretion, pointing to increased NFκB activation, in accordance with previous findings in Figure 3 (Figure 5B).
NFAT has been shown to regulate myocardial hypertrophy and neointimal formation after vascular injury.17 Previously, we showed that FK506 blocked VSMC hyperplasia and plaque development in atherosclerosis.28 We therefore examined whether selective NFAT inhibition was equally potent as CsA in preventing VSMC proliferation in vitro and neointimal formation in vivo. MCV1 at 10 μmol/L showed similar potency to CsA (Online Figure XI). We have examined the antihyperplastic effect of MCV1 on neointimal formation in a mouse model of carotid denudation at 4 weeks after surgery. Interestingly, after 4 weeks of lesion development, MCV1 and CsA treatment resulted in a significant 50% reduction in neointima formation in denuded carotid arteries of apolipoprotein E−/− mice compared to a mock-treated control (P=0.02; Figure 6A). Whereas no significant changes in macrophage and collagen content were noted, α-smooth muscle actin-positive VSMCs were significantly increased both in the MCV1 and the CsA-treated groups, indicating a distinct regulation of VSMC proliferation and differentiation by calcineurin–NFAT signaling (Figure 6B). To determine whether either MCV1 or CsA affected the initial process after vascular injury, we also analyzed lesion formation at 1 week after surgery. Topical MCV1 and CsA treatment did not affect reendothelialization, as judged by CD31 staining (Online Figure XII) and by quantitative analysis of Evans Blue-stained vessel area (Online Figure XIV). Adequate carotid endothelial denudation in our mouse model was demonstrated by performing Evans Blue staining at day 1 after injury, in which more than 80% of injured left common carotid artery but not the right common carotid artery showed positive staining (Online Figure XV). Macrophage content also did not differ between the groups (Online Figure XIII). The vessel areas of media plus intima and lesion cellularity were unaltered at that time point as well as examined by hematoxyline and eosin staining. Similarly, as in the 4-week study, smooth muscle α-actin-positive VSMC content was increased, particularly in the MCV1-treated mice. Thus, MCV1 seems to preferentially inhibit injury-induced smooth muscle cell differentiation from a contractile to a proliferative phenotype. No significant differences were observed in the morphology of the contralateral right common carotid artery either 1 or 4 weeks after surgery (Online Figure XVI).
NFAT is deemed to play a pivotal role in the treatment of transplant rejection, autoimmune diseases, and cardiovascular disorders such as restenosis and cardiac hypertrophy. As primary and the most thoroughly characterized substrate of calcineurin, NFAT is regarded as the best candidate target for therapy. Previously, we and others have shown that selective NFAT inhibition by the peptide antagonist VIVIT is an effective strategy to coordinately target inflammatory and VSMC hyperplastic responses that underlie restenosis.17 However, the suboptimal pharmacological features of VIVIT and particularly its low micromolar potency will hamper its direct clinical application. Recently identified small organic molecule inhibitors, INCAs, could selectively inhibit calcineurin–NFAT interaction at micromolar potency16 in vitro. These quinine-based or quinoneimine-based compounds were, however, found to be rather cytotoxic, disqualifying them for therapeutic use.18 Therefore, small molecule inhibitors of NFAT with improved potency are eagerly awaited.
Previous attempts to improve cellular entry and thus potency of VIVIT by conjugating a cell-permeable peptide tag to the N-terminal end of VIVIT did display an increased potency, but the gain in affinity was only limited.29 We therefore sought to optimize the peptide by modifying the terminal ends of the central motif with a maleimido entity that was previously shown to allosterically interfere with VIVIT binding to calcineurin, aiming at the generation of a bipartite inhibitor. By stepwise stripping, we identified the shortest motif of VIVIT, HPVIVIT, as an effective inhibitor of NFAT-dependent transcriptional activation and VSMC proliferation. Subsequent conjugation of HPVIVIT at its N-terminal end to a maleimide unit led to the successful design of a maleimido-conjugated peptide with low nanomolar potency for NFAT inhibition. In fact, the potency of the resultant conjugate, MCV1, was almost 1000-fold higher than that of the parental peptide VIVIT. In comparison with INCAs, MCV1 showed no overt cytotoxicity both in vivo and in vitro. We also show that MCV1 does not display the NFAT-independent effects associated with CsA and establishes its efficacy in vivo in a model of peritonitis as well as of restenosis.
Conceivably, the remarkably high affinity of MCV1 results from the simultaneous targeting of two separate NFAT docking sites of calcineurin.30 However, the binding energy for MCV1 and for PVIVIT are rather similar and combined with the observed DTT-induced loss in affinity, this led us to hypothesize that the high affinity of MCV1 is likely attributable to covalent interaction with calcineurin. The binding model presented in this study may thus reflect the initial step in MCV1 binding to calcineurin, inducing a reconfiguration of the β11-β12 loop and rendering the Cys266 more accessible to the C-terminal maleimide. A second beneficial factor may be that in contrast to the highly hydrophilic characteristic of VIVIT, the MCV analogues are rather lipophilic, favoring cellular uptake. Furthermore, MCV1 contains the central HPVIVIT motif of VIVIT, which was shown capable of inhibiting all four members of the cytoplasmic NFAT family (NFATc1-c4). Combined with the fact that NFATc1, NFATc2, and NFATc3 are coexpressed both in immune cells and in VSMCs,17 the observed potent NFAT inhibition by MCV1 in various cell types suggests that the conjugate targets all cytosolic isoforms of NFAT.
Nuclear co-import of calcineurin and NFAT may potentially help to maintain a certain level of NFAT activation. Nuclear calcineurin could be detected in more than 90% of cardiomyocytes after chronic stimulation by angiotensin II for 6 hours or longer. In contrast, fewer cells with calcineurin nuclear import were seen within 2 hours of treatment.23 Interestingly, in the same study inhibition of nuclear import of calcineurin was seen to blunt NFAT activation rather than to prevent NFAT dephosphorylation, indicating that the former is regulated by calcineurin nuclear co-import. In agreement, we observed partial (15%–20%) nuclear colocalization of calcineurin and NFAT after acute stimulation by ionomycin for 30 minutes. Here, we extend these findings by demonstrating that calcineurin nuclear co-import is probably independent of NFAT activation because MCV1 specifically interferes with calcineurin docking to NFAT, leaving calcineurin phosphates activity unchanged.
Calcineurin was shown to be a multifunctional regulator of various downstream signaling pathways. Except for its major substrate NFAT, calcineurin is able to activate nuclear factor κB31 and dephosphorylate BAD.32 Thus, CsA or FK506 treatment will simultaneously inhibit NFAT activity and that of other downstream substrates of calcineurin. The clinical application of CsA and FK506 is accompanied by undesirable side effects such as nephrotoxicity, hypertension, cancer, and renal dysfunction.10–12 So far, it is still unclear to what extent their toxicity is associated with disruption of calcineurin targets other than NFAT, with calcineurin-independent activation of transforming growth factor-β or synergistic activation of multiple signaling pathways. A selective NFAT inhibitor MCV1 may shed light on this issue and constitute a novel drug candidate and may be a possibly less toxic alternative to CsA in the treatment of NFAT-mediated diseases. With the help of MCV1, we were able to more selectively examine the downstream pathway of calcineurin and to compare its inhibitory pattern with CsA. As summarized in Figure 4E, CsA is able not only to quench calcineurin phosphatase activity but also to facilitate MAP kinase-mediated NFAT rephosphorylation in VSMCs. The observed dual mode of action of CsA could at least partly explain the off-target effects of this immunosuppressant. Further study will be needed to elucidate the location and underlying pathways of CsA-induced NFAT hyperphosphorylation.
In conclusion, we identify MCV1 as a synthetic peptide inhibitor of NFAT with nanomolar potency that is not only more potent than VIVIT but also more selective than CsA in inhibiting NFAT signaling. MCV1 represents a novel tool for probing NFAT function in vitro and in vivo in various cell types, and it may assist in clarifying the role of NFAT in these diseases. Its high and selective immunosuppressant capacity in vivo indicates that MCV1 holds promise for clinical use in the treatment of transplant rejection, autoimmune disorders, and cardiovascular diseases implicating NFAT activation, such as restenosis and potentially cardiac hypertrophy.
Sources of Funding
This study was financially supported by grants LFA5952 from Dutch Technology Foundation (S.T.W.). This study was also supported by grants 2003T.201 from the Netherlands Heart Foundation. E.B., T.v.B., and M.R.B. belong to the European Vascular Genomics Network (http://www.evgn.org), a Network of Excellence supported by the European Community's Sixth Framework Program for Research Priority 1 (Life Sciences, Genomics, and Biotechnology for Health; contract LSHM-CT-2003-503254). Dr I. Bot is funded by grant 916.86.046 from the Netherlands Organization for Scientific Research.
The authors are grateful to Dr A. Rao (Harvard Medical School, Boston, MA) for providing the NFAT1-GFP plasmid, to Dr de Windt ( Utrecht University Medical Center, the Netherlands) for the NFAT-luc reporter plasmid, and to Dr M. D. Lopez and Dr J. M. Redondo (Madrid Heart Center, Spain) and Dr. Yanchao Huang (Leiden University Medical center, the Netherlands) for their kind help with pull-down assays. Dr Chi-wing Chow (Albert Einstein College of Medicine, New York, NY) is acknowledged for the stimulating discussion and helpful comments.
In October 2011, the average time from submission to first decision for all original research papers submitted to Circulation Research was 15 days.
Non-standard Abbreviations and Acronyms
- cytoplasmic nuclear factor of activated T-cells
- casein kinase 1
- cyclosporin A
- Down syndrome critical region gene 1
- extracellular signal regulated kinase
- FK506 binding protein 12
- glycogen synthase kinase-3
- left common carotid artery
- nuclear factor of activated T-cells
- cytoplasmic nuclear factor of activated T-cells
- nuclear nuclear factor of activated T-cells
- nuclear factor kappa B
- protein kinase C
- phospholipase C-γ
- phorbol 12-myristate 13-acetate
- vascular smooth muscle cell
- Received January 16, 2011.
- Revision received November 9, 2011.
- Accepted November 16, 2011.
- © 2012 American Heart Association, Inc.
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Novelty and Significance
What Is Known?
Nuclear factor of activated T-cells (NFAT) is known to play a pivotal role in autoimmune diseases and cardiovascular disorders.
Calcineurin phosphatase inhibitor cyclosporin A (CsA) is a rather nonspecific inhibitor of NFAT activation and its use is associated with adverse side effects.
Recently designed NFAT inhibitory peptides (VIVIT) or imidazole-based small molecules are more selective but are toxic or of low potency.
What New Information Does This Article Contribute?
We have developed a potent VIVIT-based bipartite inhibitor of NFAT–calcineurin interaction, MCV1, which targets two separate calcineurin docking motifs.
MCV1 is shown to be more potent than VIVIT peptide and more selective than CsA in NFAT inhibition.
MCV1 inhibits vascular smooth muscle cell proliferation and neointima formation in a carotid injury mouse model.
NFAT is importantly implicated in autoimmune diseases, transplant rejection, and cardiovascular disorders. Calcineurin inhibitors such as CsA generally lack NFAT specificity and cause adverse side effects, whereas selective NFAT inhibitors such as VIVIT and inhibitors of NFAT–calcineurin association (INCA) have poor potency, precluding clinical use. We describe the rational design of a potent bipartite inhibitor of NFAT–calcineurin interaction, MCV1, which targets two separate calcineurin docking motifs. Functional and modeling studies demonstrated that MCV1 acts by allosteric modulation of calcineurin. MCV1 prevents NFAT activation at nanomolar potency, without impairing calcineurin phosphatase activity, nuclear factor-κB nuclear impor,t and general cell signaling. In contrast, CsA, but not MCV1, activated basal level extracellular signal-regulated kinase activity and prevented nuclear import of calcineurin, independent of NFAT activation. In vivo, MCV1 abrogated NFAT-mediated T-cell activation in a model of PMA-elicited peritonitis, whereas topical application of MCV1 markedly reduced neointima formation in a mouse model of restenosis. Thus, the designed bipartite compound is more potent than VIVIT and more selective than CsA in NFAT inhibition. We envisage that MCV1 constitutes not only a powerful tool to unravel NFAT function but also a potential drug candidate for the treatment of diseases implicating NFAT activation.