Abstract 1214: Rational Design of a Potent Bipartite Inhibitor of Nuclear Factor of Activated T Cells (NFAT), a Key Regulator of Cardiovascular Hyperplasia

Abstract

Purpose: Targeted intervention in calcineurin/NFAT signaling is an effective strategy in the treatment of vascular smooth muscle cell (vSMC) hyperplasia and restenosis. We have recently shown that VIVIT, a novel peptide inhibitor of NFAT, is more selective and less toxic than cyclosporine A (CsA) and holds promise in antirestenotic therapy. However, its suboptimal pharmacological features preclude direct clinical use. In this study, we sought to overcome these setbacks by stepwise optimization of VIVIT. Methods and Results: In the first stage stepwise truncation and alanine scan studies were performed to establish the critical importance of the HPVIVIT motif for NFAT inhibition. In order to create a non-toxic bipartite compound that simultaneously targets two recently identified NFAT docking sites on calcineurin, the HPVIVIT motif was conjugated to a maleimide derivative via a series of flexible spacers of 8–28 Å. One of the conjugates, maleimide conjugated HPVIVIT or MCV, was 10,000 more potent in inhibiting NFAT (IC50=1.9 nM) than the parent peptide. MCV was able to abrogate ionomycin induced nuclear translocation of NFAT1-GFP at 100 nM and this effect persisted for >36h. Moreover, it profoundly inhibited platelet-derived growth factor-BB stimulated vSMC proliferation at concentrations as low as 10 nM (P=0.04; ~80%). MCV (100 nM) selectively disrupted calcineurin NFAT interaction in a GST pull-down assay but did not affect that of other binding partners (AKAP-79/Cain). It was at least equally potent as CsA in inhibiting ionomycin induced NFAT dephosphorylation in COS-1 cells. Unlike CsA however, MCV appeared to be non-toxic and did not impair the activity of calcineurin phosphatase. Moreover, in contrast to CsA MCV did not affect mitogen-activated protein kinase activity after PMA stimulation in COS-1 cells at concentrations up to 10 μM nor did it influence NFκB activity. Finally, CsA prevented nuclear accumulation of calcineurin, which remained unaffected by MCV1 (1 μM). Conclusion : The potency, chemical features and superior selectivity of the bipartite NFAT inhibitor MCV render this compound an excellent lead in the treatment of inflammatory disorders implicating NFAT activation, including myocardiac hypertrophy and restenosis.