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An isoform-selective, small-molecule inhibitor targets the autoregulatory mechanism of p21-activated kinase.

ABSTRACT: Autoregulatory domains found within kinases may provide more unique targets for chemical inhibitors than the conserved ATP-binding pocket targeted by most inhibitors. The kinase Pak1 contains an autoinhibitory domain that suppresses the catalytic activity of its kinase domain. Pak1 activators relieve this autoinhibition and initiate conformational rearrangements and autophosphorylation events leading to kinase activation. We developed a screen for allosteric inhibitors targeting Pak1 activation and identified the inhibitor IPA-3. Remarkably, preactivated Pak1 is resistant to IPA-3. IPA-3 also inhibits activation of related Pak isoforms regulated by autoinhibition, but not more distantly related Paks, nor >200 other kinases tested. Pak1 inhibition by IPA-3 in live cells supports a critical role for Pak in PDGF-stimulated Erk activation. These studies illustrate an alternative strategy for kinase inhibition and introduce a highly selective, cell-permeable chemical inhibitor of Pak.

SUBMITTER: Deacon SW

PROVIDER: S-EPMC4353635 | biostudies-literature | 2008 Apr

REPOSITORIES: biostudies-literature

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An isoform-selective, small-molecule inhibitor targets the autoregulatory mechanism of p21-activated kinase.

Deacon Sean W SW Beeser Alexander A Fukui Jami A JA Rennefahrt Ulrike E E UE Myers Cynthia C Chernoff Jonathan J Peterson Jeffrey R JR

Chemistry & biology 20080401 4

Autoregulatory domains found within kinases may provide more unique targets for chemical inhibitors than the conserved ATP-binding pocket targeted by most inhibitors. The kinase Pak1 contains an autoinhibitory domain that suppresses the catalytic activity of its kinase domain. Pak1 activators relieve this autoinhibition and initiate conformational rearrangements and autophosphorylation events leading to kinase activation. We developed a screen for allosteric inhibitors targeting Pak1 activation ...[more]

PMID: 18420139

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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/μmol 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.

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An isoform-selective, small-molecule inhibitor targets the autoregulatory mechanism of p21-activated kinase.

Publications

An isoform-selective, small-molecule inhibitor targets the autoregulatory mechanism of p21-activated kinase.

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