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Engineered Substrate-Specific Delta PKC Antagonists to Enhance Cardiac Therapeutics.

ABSTRACT: Most protein kinases phosphorylate multiple substrates, each of which induces different and sometimes opposing functions. Determining the role of phosphorylation of each substrate following a specific stimulus is challenging but is essential to elucidate the role of that substrate in the signaling event. Here we describe a rational approach to identify inhibitors of delta protein kinase C (?PKC), each inhibiting the phosphorylation of only one of ?PKC's substrates. ?PKC regulates many signaling events and we hypothesized that a docking inhibitor of a given substrate to ?PKC should selectively abrogate the phosphorylation of only that substrate, without affecting the phosphorylation of the other ?PKC substrates. Here we report the development of selective inhibitors of three ?PKC substrates (in vitro Kd ?3?nm); two greatly reduced ischemia-induced cardiac injury with an IC50 of ?200?nm and the third had no effect, indicating that its respective substrate phosphorylation by ?PKC has no role in the response to cardiac ischemia and reperfusion. The three inhibitors are highly specific; even at 1??m, the phosphorylation of other ?PKC protein substrates was unaffected. The rationale we describe is likely applicable for the development of other substrate-specific inhibitors as well.

SUBMITTER: Qvit N

PROVIDER: S-EPMC5308906 | biostudies-literature | 2016 Dec

REPOSITORIES: biostudies-literature

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Engineered Substrate-Specific Delta PKC Antagonists to Enhance Cardiac Therapeutics.

Qvit Nir N Kornfeld Opher S OS Mochly-Rosen Daria D

Angewandte Chemie (International ed. in English) 20161117 50

Most protein kinases phosphorylate multiple substrates, each of which induces different and sometimes opposing functions. Determining the role of phosphorylation of each substrate following a specific stimulus is challenging but is essential to elucidate the role of that substrate in the signaling event. Here we describe a rational approach to identify inhibitors of delta protein kinase C (δPKC), each inhibiting the phosphorylation of only one of δPKC's substrates. δPKC regulates many signaling ...[more]

PMID: 27860071

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Project description:ObjectivesThe goal of this study was to demonstrate the enhancement of human cardiac progenitor cell (hCPC) reparative and regenerative potential by genetic modification for the treatment of myocardial infarction.BackgroundRegenerative potential of stem cells to repair acute infarction is limited. Improved hCPC survival, proliferation, and differentiation into functional myocardium will increase efficacy and advance translational implementation of cardiac regeneration.MethodshCPCs isolated from the myocardium of heart failure patients undergoing left ventricular assist device implantation were engineered to express green fluorescent protein (hCPCe) or Pim-1-GFP (hCPCeP). Functional tests of hCPC regenerative potential were performed with immunocompromised mice by using intramyocardial adoptive transfer injection after infarction. Myocardial structure and function were monitored by echocardiographic and hemodynamic assessment for 20 weeks after delivery. hCPCe and hCPCeP expressing luciferase were observed by using bioluminescence imaging to noninvasively track persistence.ResultshCPCeP exhibited augmentation of reparative potential relative to hCPCe control cells, as shown by significantly increased proliferation coupled with amelioration of infarction injury and increased hemodynamic performance at 20 weeks post-transplantation. Concurrent with enhanced cardiac structure and function, hCPCeP demonstrated increased cellular engraftment and differentiation with improved vasculature and reduced infarct size. Enhanced persistence of hCPCeP versus hCPCe was revealed by bioluminescence imaging at up to 8 weeks post-delivery.ConclusionsGenetic engineering of hCPCs with Pim-1 enhanced repair of damaged myocardium. Ex vivo gene delivery to modify stem cells has emerged as a viable option addressing current limitations in the field. This study demonstrates that efficacy of hCPCs from the failing myocardium can be safely and significantly enhanced through expression of Pim-1 kinase, setting the stage for use of engineered cells in pre-clinical settings.

Dataset Information

Engineered Substrate-Specific Delta PKC Antagonists to Enhance Cardiac Therapeutics.

Publications

Engineered Substrate-Specific Delta PKC Antagonists to Enhance Cardiac Therapeutics.

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