Discovery of a latent calcineurin inhibitory peptide from its auto inhibitory domain by docking, dynamic simulation and <i>in vitro</i> methods

Auto Inhibitory Domain (AID) of calcineurin (CN) was discovered two decades ago. Fewer investigations are reported to find out shortest possible peptide from the AID for CN inhibition. Hence, the current study has focused on screening of nearly 150 peptide fragments derived from the AID using in silico method. Therefore, we have employed docking studies, aiming to analyze the best pose of AID derived peptides on CN active site. We also analyzed binding free energy (ΔG) of docked complex using Molecular Mechanics/Generalized Born Surface Area (MM/GBSA). MM/GBSA predicts two short peptides P1and P2 found to be lowest binding free energy. Two peptides exhibit better binding affinity with CN, suggests that the possible candidates for potential CN inhibition. Further, the stability of the docked complex was analyzed using Molecular Dynamic (MD) simulation. MD study shows that CNA:P2 is most stable complex than CN A:P1 and CN A:AID. Besides, we have synthesized and purified P1 and P2 peptides over High Performance Liquid Chromatography (HPLC) found to be 90.31% and 98.93% of purity respectively. In addition, AID peptides were characterized over mass spectral analysis. Peptides were subjected to CN inhibitory assay using malachite green method. Where, P1 and P2 exhibit CN inhibition better than AID. In particular, shortest peptide P2 shows highest inhibitory activity than AID. Enzyme assay reveals CN inhibitory activity of P2 peptide is consistent with in silico results. In silico and in vitro results corroborated each other to confirm short peptide P2 can be used as a potential CN inhibitor.