Computational Design and Experimental Validation of an Orthogonal X-bond/H-bond System to Improve the Binding Affinity and Recognition Specificity between Anesthetic Protein Kinase Cι and its Pseudosubstrate Peptide

Abstract

Protein kinase C isoform [Formula: see text] (PKC[Formula: see text]) is an essential regulator of diverse neurobiological signaling pathways, which has been implicated in general anesthetic action and a variety of nervous neoplasms. Traditional efforts have been primarily addressed on the development of small-molecule inhibitors to target the catalytic or regulatory domain of this kinase. Instead, we herein reported a rational chemical modification of Par3 pseudosubstrate peptide to target the kinase domain with improved affinity and specificity. Structural and energetic analysis suggested that the Par3 peptide can be divided into two binding-independent sections; they are separately anchored to the substrate-binding pocket of PKC[Formula: see text] with two aromatic anchor residues Phe818 and Phe823. The side-chain phenyl group of these two aromatic residues was systematically substituted by different halogen moieties at [Formula: see text]-, [Formula: see text]- and [Formula: see text]-positions of the phenyl group. It is revealed that the [Formula: see text]-bromination of Par3 Phe818 and the [Formula: see text]-iodinization of Par3 Phe823 residue can separately form two geometrically and energetically satisfactory orthogonal halogen (X)-bond/hydrogen (H)-bond [ortX/H] motifs across Par3 complex interface with PKC[Formula: see text]; they co-define a so-called ortX/H system and were demonstrated to improve both the binding affinity and recognition specificity of the kinase–peptide interaction by integrating in silico analysis and in vitro assay. The peptide affinity promotes 23.5-fold and 6.6-fold upon the formation of ortX/H motif-I and motif-II, respectively, which further increases to 43.5-fold by forming the complete ortX/H system, indicating cooperativity between the two motifs in the system. In addition, we also demonstrated that the ortX/H system can considerably enhance the peptide selectivity for its cognate PKC[Formula: see text] and noncogante isoforms PKC[Formula: see text] and PKC[Formula: see text].