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A specific small-molecule inhibitor of protein kinase C?I activity improves metabolic dysfunction in human adipocytes from obese individuals.

ABSTRACT: The metabolic consequences and sequelae of obesity promote life-threatening morbidities. PKC?I is an important elicitor of inflammation and apoptosis in adipocytes. Here we report increased PKC?I activation via release of its catalytic domain concurrent with increased expression of proinflammatory cytokines in adipocytes from obese individuals. Using a screening strategy of dual recognition of PKC?I isozymes and a caspase-3 binding site on the PKC?I hinge domain with Schrödinger software and molecular dynamics simulations, we identified NP627, an organic small-molecule inhibitor of PKC?I. Characterization of NP627 by surface plasmon resonance (SPR) revealed that PKC?I and NP627 interact with each other with high affinity and specificity, SPR kinetics revealed that NP627 disrupts caspase-3 binding to PKC?I, and in vitro kinase assays demonstrated that NP627 specifically inhibits PKC?I activity. The SPR results also indicated that NP627 affects macromolecular interactions between protein surfaces. Of note, release of the PKC?I catalytic fragment was sufficient to induce apoptosis and inflammation in adipocytes. NP627 treatment of adipocytes from obese individuals significantly inhibited PKC?I catalytic fragment release, decreased inflammation and apoptosis, and significantly improved mitochondrial metabolism. These results indicate that PKC?I is a robust candidate for targeted interventions to manage obesity-associated chronic inflammatory diseases. We propose that NP627 may also be used in other biological systems to better understand the impact of caspase-3-mediated activation of kinase activity.

SUBMITTER: Sparks R

PROVIDER: S-EPMC6791326 | biostudies-literature | 2019 Oct

REPOSITORIES: biostudies-literature

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A specific small-molecule inhibitor of protein kinase CδI activity improves metabolic dysfunction in human adipocytes from obese individuals.

Sparks Robert R Lui Ashley A Bader Deena D Patel Rekha R Murr Michel M Guida Wayne W Fratti Rutilio R Patel Niketa A NA

The Journal of biological chemistry 20190814 41

The metabolic consequences and sequelae of obesity promote life-threatening morbidities. PKCδI is an important elicitor of inflammation and apoptosis in adipocytes. Here we report increased PKCδI activation via release of its catalytic domain concurrent with increased expression of proinflammatory cytokines in adipocytes from obese individuals. Using a screening strategy of dual recognition of PKCδI isozymes and a caspase-3 binding site on the PKCδI hinge domain with Schrödinger software and mol ...[more]

PMID: 31413114

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Project description:One of the most clinically relevant molecular aberrations in breast cancer is overexpression of human epidermal growth factor receptor type 2 (HER2). We aimed to develop a radiolabeled tyrosine kinase inhibitor for HER2-targeted breast cancer imaging. In this study, a radioiodinated analog (125/131I-IBA-CP) of the HER2-selective inhibitor CP724,714 was prepared and evaluated in HER2-positive or -negative subcutaneous human breast cancer xenografts. Methods: The CP724,714 analog IBA-CP was synthesized and assayed for its inhibitory activities against HER2 and 6 other tyrosine kinases. 125/131I-IBA-CP was prepared using a copper-mediated radioiodination method with enhanced labeling yield and molar activity. In vitro biologic activity, including specific and nonspecific binding of 131I-IBA-CP to its HER2 kinase target, was assessed in different cell lines. In vivo small-animal 125I-IBA-CP SPECT imaging and biodistribution studies were conducted on mice bearing HER2-positive, HER2-negative, or epidermal growth factor receptor (EGFR)-positive tumors. Nonradioactive IBA-CP and the EGFR inhibitor erlotinib were used as blocking agents to investigate the binding specificity and selectivity of 125/131I-IBA-CP toward HER2 in vitro and in vivo. Additionally, 125/131I-ICP was prepared by direct radioiodination of CP724,714 for comparison with 125/131I-IBA-CP. Results: IBA-CP displayed superior in vitro inhibitory activity (half-maximal inhibitory concentration, 16 nM) and selectivity for HER2 over 6 other cancer-related tyrosine kinases. 125/131I-IBA-CP was prepared in a typical radiochemical yield of about 65% (decay-corrected), radiochemical purity of more than 98%, and molar activity of 42 GBq/?mol at the end of synthesis. SPECT imaging revealed significantly higher uptake of 125I-IBA-CP than of 125I-ICP in the HER2-positive MDA-MB-453 tumors. Uptake in the HER2-negative MCF-7 tumors was much lower. Binding of 125I-IBA-CP in the MDA-MB-453 tumors was blocked by coinjection with an excess amount of IBA-CP, but not by erlotinib. Conclusion: The radiolabeled HER2-selective inhibitor 125/131I-IBA-CP is a promising probe for in vivo detection of HER2-positive tumors.

Dataset Information

A specific small-molecule inhibitor of protein kinase C?I activity improves metabolic dysfunction in human adipocytes from obese individuals.

Publications

A specific small-molecule inhibitor of protein kinase CδI activity improves metabolic dysfunction in human adipocytes from obese individuals.

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