Selective Phosphorylation Inhibitor of Delta Protein Kinase C–Pyruvate Dehydrogenase Kinase Protein–Protein Interactions: Application for Myocardial Injury in Vivo

Abstract

Protein kinases regulate numerous cellular processes, including cell growth, metabolism and cell death. Because the primary sequence and the three-dimensional structure of many kinases are highly similar, the development of selective inhibitors for only one kinase is challenging. Furthermore, many protein kinases are pleiotropic, mediating diverse and sometimes even opposing functions by phosphorylating multiple protein substrates. Here, we set up to develop an inhibitor of a selective protein kinase phosphorylation of only one of its substrates. Focusing on the pleiotropic delta protein kinase C (δPKC), we used a rational approach to identify a distal docking site on δPKC for its substrate, pyruvate dehydrogenase kinase (PDK). We reasoned that an inhibitor of PDK's docking should selectively inhibit the phosphorylation of only PDK without affecting phosphorylation of the other δPKC substrates. Our approach identified a selective inhibitor of PDK docking to δPKC with an in vitro Kd of ~50 nM and reducing cardiac injury IC 50 of ~5 nM. This inhibitor, which did not affect the phosphorylation of other δPKC substrates even at 1 µM, demonstrated that PDK phosphorylation alone is critical for δPKC-mediated injury by heart attack. The approach we describe is likely applicable for the identification of other substrate-specific kinase inhibitors. Graphical Abstract *Corresponding Author. mochly@stanford.edu. ASSOCIATED CONTENT Supporting Information. Experimental details, additional figures and data are available free of charge via the Internet at http:// pubs.acs.org. Author ContributionsAll authors have given approval to the final version of the manuscript.The authors declare that no competing interests exist. HHS Public AccessAuthor manuscript J Am Chem Soc. Author manuscript; available in PMC 2016 October 14. Author Manuscript Author ManuscriptAuthor ManuscriptAuthor Manuscript INTORDUCTIONThe protein kinases super family accounts for approximately 2% of the eukaryotic genes and about 518 protein kinases are predicted in the human kinome. 1 Protein kinases catalyzed phosphorylation, the transfer of the γ-phosphoryl group from adenosine triphosphate (ATP) to the hydroxyl group of defined amino acid, which regulated many biological processes, including metabolism, transcription, cell cycle progression, and differentiation. Phosphorylation is the most widespread type of post-translational modification in signal transduction with over 500,000 potential phosphorylation sites for any given kinase in the human proteome and 25,000 phosphorylation events described for 7,000 human proteins. 2,3 Phosphorylation is mediated by the catalytic domain that consists of a small N-terminal lobe of β-sheets, a larger C-terminal lobe of α-helices, and the ATP binding site in a cleft between the two lobes. 4 Many kinase inhibitors target the highly conserved ATP-binding pocket. 5 However, since the catalytic domain of most eukaryotic kinases is structurally similar, developing specific protein kinase inhibitors that target the c...

Switching off IMMP2L signaling drives senescence via simultaneous metabolic alteration and blockage of cell death

“…PKC-δ has previously been shown to directly phosphorylate pyruvate dehydrogenase kinase (PDK), leading to phosphorylation-dependent inhibition of mitochondrial pyruvate dehydrogenase (PDH), a key regulator mediating pyruvate's entrance into the tricarboxylic acid (TCA) cycle. 53,54 Notably, enhanced PDH activity due to its dephosphorylation is recognized as the causative event critically implicated in the execution of oncogene-induced senescence to suppress tumor initiation as well as cancer progression. 55 Accordingly, we explored the possibility that IMMP2L signaling-mediated recruitment of PKC-δ to the mitochondria may serve as the upstream event responsible for modulating PDH phosphorylation in response to oncogenic insults and tumorigenesis.…”

Engineered Substrate‐Specific Delta PKC Antagonists to Enhance Cardiac Therapeutics

“…[1] This isozyme is involved in various signal transduction pathways,r egulating both physiological and pathological conditions including cancer, [2] neurodegenerative diseases, [3] and ischemic heart disease [4] by phosphorylating multiple protein substrates.H ere we describe ar ational approach to developing inhibitors of dPKC that selectively abrogate its interaction with and phosphorylation of one protein substrate at atime. [5] We designed three peptides to inhibit the docking and phosphorylation of three dPKC substrates:myristoylated alanine-rich C-kinase substrate (MARCKS), dynaminrelated protein 1( Drp1) and insulin receptor substrate 1( IRS1). These peptide inhibitors,w hich bind to dPKC with high affinity in vitro,s electively inhibit the phosphorylation of only the corresponding substrate and differentially affect dPKC function in both in culture and ex vivo models for myocardial infarction (MI).…”

Correcting mitochondrial fusion by manipulating mitofusin conformations

“…Mfn2 was bound to carboxylic acid sites of AGILE (Nanomedical Diagnostics, San Digeo CA) graphene chips 23 as previously described 24 . Minipeptides were applied at varying concentrations (500 nM – 1 mM) and the change in sensor chip charge recorded for 15 min.…”