Derivation of Self-inhibitory Helical Peptides to Target Rho-kinase Dimerization in Cerebrovascular Malformation: Structural Bioinformatics Analysis and Peptide Binding Assay

Rho‐kinase dimerization is essential for its kinase activity and biological function; disruption of the dimerization has recently been established as a new and promising therapeutics strategy for cerebrovascular malformation (CM). Based on Rho‐kinase dimer crystal structure we herein combined in silico analysis and in vitro assay to rationally derive self‐inhibitory peptides from the dimerization interface. Three peptides namely Hlp1, Hlp2 and Hlp3 were successfully designed that have potential capability to rebind at the dimerization domain of Rho‐kinase. Molecular dynamics (MD) simulations revealed that these peptides are helically structured when bound to Rho‐kinase, but exhibit partially intrinsic disorder in unbound state. Binding free energy (BFE) analysis suggested that the peptides have a satisfactory energetic profile to interact with Rho‐kinase. The computational findings were then substantiated by fluorescence anisotropy assays, conforming that the helical peptides can bind tightly to Rho‐kinase with affinity KD at micromolar level. These designed peptides are considered as lead molecular entities that can be further modified and optimized to obtain more potent peptidomimetics as self‐competitors to disrupt Rho‐kinase dimerization in CM.