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Introduction <p>Human protein kinase C (PKC) is a family of signal transducing isozymes involved in various cellular physiological and pathological processes. The atypical PKC isozyme ζ (PKCζ) is expressed abundantly in the central nervous system and has been implicated in anesthetic preconditioning cascades. Previous efforts have primarily focused on the discovery and development of small-molecule chemical agents to target PKCζ active site.</p> Methods <p>We attempted to perform rational molecular design of potent peptidic inhibitors to competitively disrupt the native interaction between the PKCζ kinase domain and its natural substrates. A peptide segment covering the phosphorylated Thr595 residue of substrate protein PAR1b was derived, which, however, can only bind to PKCζ in a weak potency due to lacking of protein context support. Nevertheless, the peptide affinity can be significantly improved by rationally introducing a so-called “designer halogen(X)/hydrogen(H)-bonding (<i>dgr</i>[XHB]) system” across its complex interface with PKCζ. Structural analysis revealed that the binding mode of PAR1b peptide is arranged into a twisted conformation that packs tightly against the kinase protein surface separately at its two anchor residues Phe585 and Phe591.</p> Results <p>The <i>ortho</i>-, <i>meta</i>- and <i>para</i>-positions of the side-chain phenyl group of the two phenylalanine residues were systematically substituted by four halogens (X = F, Cl, Br and I), unraveling that <i>para</i>-substitution of Phe585 and <i>meta</i>-substitution of Phe591 with bromine (–Br) can form two geometrically and energetically satisfactory halogen bonds (XBs) with the backbone carbonyl oxygen (=O) of PKCζ Met344 residue and the side-chain amide oxygen (=O) of PKCζ Asn461 residue, respectively; they are also shared by two hydrogen bonds (HBs) separately with PKCζ Arg348 and Tyr465 residues to create two “orthogonal X/H-bonding (<i>otg</i>[XHB]) motifs”. The <i>dgr</i>[XHB] system was defined to consist of the two <i>otg</i>[XHB]-1 and <i>otg</i>[XHB]-2 motifs, which can considerably improve the binding affinity of PAR1b peptide to PKCζ kinase domain and potently compete with natural substrate for the domain.</p> Conclusion <p>A synergetic effect was observed between the <i>otg</i>[XHB]-1 and <i>otg</i>[XHB]-2, indicating that the two motifs can co-work together in <i>dgr</i>[XHB] to promote the kinase–peptide interaction in a cooperative manner. The substrate-derived peptides can be regarded as potential leading entities to develop competitive peptidic inhibitors with the cognate substrates of PKCζ isozyme.</p>

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Competitive Disruption of the Intermolecular Interaction Between Anesthetic PKC Isozyme ζ and its Cognate Substrates using Halogenated Peptidic Inhibitors with Affinity Improved by a Designer Halogen(X)/Hydrogen(H)-Bonding (dgr[XHB]) System

  • 期刊论文

  • 发表日期：

    2025年2月22日

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  • Fang Lian,
  • Zhong Wang,
  • Zhidong Zhou,
  • Xiaohong Du

摘要

Introduction

Human protein kinase C (PKC) is a family of signal transducing isozymes involved in various cellular physiological and pathological processes. The atypical PKC isozyme ζ (PKCζ) is expressed abundantly in the central nervous system and has been implicated in anesthetic preconditioning cascades. Previous efforts have primarily focused on the discovery and development of small-molecule chemical agents to target PKCζ active site.

Methods

We attempted to perform rational molecular design of potent peptidic inhibitors to competitively disrupt the native interaction between the PKCζ kinase domain and its natural substrates. A peptide segment covering the phosphorylated Thr595 residue of substrate protein PAR1b was derived, which, however, can only bind to PKCζ in a weak potency due to lacking of protein context support. Nevertheless, the peptide affinity can be significantly improved by rationally introducing a so-called “designer halogen(X)/hydrogen(H)-bonding (dgr[XHB]) system” across its complex interface with PKCζ. Structural analysis revealed that the binding mode of PAR1b peptide is arranged into a twisted conformation that packs tightly against the kinase protein surface separately at its two anchor residues Phe585 and Phe591.

Results

The ortho-, meta- and para-positions of the side-chain phenyl group of the two phenylalanine residues were systematically substituted by four halogens (X = F, Cl, Br and I), unraveling that para-substitution of Phe585 and meta-substitution of Phe591 with bromine (–Br) can form two geometrically and energetically satisfactory halogen bonds (XBs) with the backbone carbonyl oxygen (=O) of PKCζ Met344 residue and the side-chain amide oxygen (=O) of PKCζ Asn461 residue, respectively; they are also shared by two hydrogen bonds (HBs) separately with PKCζ Arg348 and Tyr465 residues to create two “orthogonal X/H-bonding (otg[XHB]) motifs”. The dgr[XHB] system was defined to consist of the two otg[XHB]-1 and otg[XHB]-2 motifs, which can considerably improve the binding affinity of PAR1b peptide to PKCζ kinase domain and potently compete with natural substrate for the domain.

Conclusion

A synergetic effect was observed between the otg[XHB]-1 and otg[XHB]-2, indicating that the two motifs can co-work together in dgr[XHB] to promote the kinase–peptide interaction in a cooperative manner. The substrate-derived peptides can be regarded as potential leading entities to develop competitive peptidic inhibitors with the cognate substrates of PKCζ isozyme.