Viaud J, Peterson JR.. An allosteric kinase inhibitor binds the p21-activated kinase autoregulatory domain covalently. Mol Cancer Ther 8: 2559-2565

Fox Chase Cancer Center, 333 Cottman Avenue, Philadelphia, PA 19111, USA.
Molecular Cancer Therapeutics (Impact Factor: 5.68). 10/2009; 8(9):2559-65. DOI: 10.1158/1535-7163.MCT-09-0102
Source: PubMed


Kinases are important therapeutic targets in oncology due to their frequent deregulation in cancer. Typical ATP-competitive kinase inhibitors, however, also inhibit off-target kinases that could lead to drug toxicity. Allosteric inhibitors represent an alternative approach to achieve greater kinase selectivity, although examples of such compounds are few. Here, we elucidate the mechanism of action of IPA-3, an allosteric inhibitor of Pak kinase activation. We show that IPA-3 binds covalently to the Pak1 regulatory domain and prevents binding to the upstream activator Cdc42. Preactivated Pak1, however, is neither inhibited nor bound significantly by IPA-3, demonstrating exquisite conformational specificity of the interaction. Using radiolabeled IPA-3, we show that inhibitor binding is specific and reversible in reducing environments. Finally, cell experiments using IPA-3 implicate Pak1 in phorbol-ester-stimulated membrane ruffling. This study reveals a novel allosteric mechanism for kinase inhibition through covalent targeting of a regulatory domain.

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Available from: Julien Viaud, Jun 10, 2014
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    • "Pharmacological inhibition of Pak1 reverses EMT-associated morphological changes, cell scattering, and motility in prostate cancer cells Since Pak1-mediated cytoskeletal assembly is essential for prostate cancer cell motility, we assumed that Pak1 activation induced by TGFβ1 may be necessary for the promotion of EMT. To confirm this, we treated both PC3 and DU145 cells with a selective Pak1 inhibitor (IPA 3), which works through covalent binding to the Pak1 regulatory domain and preventing the binding of the upstream activators (Rac1/ cdc42) [49]. Cells treated with IPA 3 were associated with significant inhibition of TGFβ1-mediated cell scattering and morphological changes similar to mesenchymal cells (Fig. 5A and B). "
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    ABSTRACT: Transforming growth factor beta (TGFβ) is believed to play a dual role in prostate cancer. Molecular mechanism by which TGFβ1 suppresses early prostate tumor growth and induces epithelial-to-mesenchymal transition (EMT) in advanced stages is not known. We determined if P21-activated kinase1 (Pak1), which mediates cytoskeletal remodeling is necessary for the TGFβ1 induced prostate cancer EMT. Effects of TGFβ1 on control prostate cancer PC3 and DU145 cells and those with IPA 3 and siRNA mediated Pak1 inhibition were tested for prostate tumor xenograft in vivo and EMT in vitro. TGFβ1 inhibited PC3 tumor xenograft growth via activation of P38-MAPK and caspase-3, 9. Long-term stimulation with TGFβ1 induced PC3 and DU145 cell scattering and increased expression of EMT markers such as Snail and N-cadherin through tumor necrosis factor receptor-associated factor-6 (TRAF6)-mediated activation of Rac1/Pak1 pathway. Selective inhibition of Pak1 using IPA 3 or knockdown using siRNA both significantly inhibited TGFβ1-induced prostate cancer cell EMT and expression of mesenchymal markers. Our study demonstrated that TGFβ1 induces apoptosis and EMT in prostate cancer cells via activation of P38-MAPK and Rac1/Pak1 respectively. Our results reveal the potential therapeutic benefits of targeting TGFβ1-Pak1 pathway for advanced-stage prostate cancer. Copyright © 2015. Published by Elsevier B.V.
    Biochimica et Biophysica Acta (BBA) - Molecular Cell Research 03/2015; 1853(5). DOI:10.1016/j.bbamcr.2015.02.023 · 5.02 Impact Factor
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    • "PAK1 was also identified as a major mediator of resistance to phosphoinositide 3-kinase inhibitors in lymphoma cell lines [15]. Attempts to develop a specific small molecule PAK inhibitor resulted in the discovery of IPA-3, an allosteric inhibitor of group I PAK activation [16]–[18] which is suitable for studies of PAK functions although its properties preclude its use in the clinical practice. We have previously reported that IPA-3 treatment of human leukemic JURL-MK1 cells reduced their ability to bind to fibronectin, one of the major components of the bone marrow extracellular matrix [19] and we have also noted IPA-3 toxicity for hematopoietic cells. "
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    ABSTRACT: P21-activated kinases (PAKs) are involved in the regulation of multiple processes including cell proliferation, adhesion and migration. However, the current knowledge about their function is mainly based on results obtained in adherent cell types. We investigated the effect of group I PAK inhibition using the compound IPA-3 in a variety of human leukemic cell lines (JURL-MK1, MOLM-7, K562, CML-T1, HL-60, Karpas-299, Jurkat, HEL) as well as in primary blood cells. IPA-3 induced cell death with EC50 ranging from 5 to more than 20 μM. Similar range was found for IPA-3-mediated dephosphorylation of a known PAK downstream effector, cofilin. The cell death was associated with caspase-3 activation, PARP cleavage and apoptotic DNA fragmentation. In parallel, 20 μM IPA-3 treatment induced rapid and marked decrease of the cell adhesivity to fibronectin. Per contra, partial reduction of PAK activity using lower dose IPA-3 or siRNA resulted in a slight increase in the cell adhesivity. The changes in the cell adhesivity were also studied using real-time microimpedance measurement and by interference reflection microscopy. Significant differences in the intracellular IPA-3 level among various cell lines were observed indicating that an active mechanism is involved in IPA-3 transport.
    PLoS ONE 03/2014; 9(3):e92560. DOI:10.1371/journal.pone.0092560 · 3.23 Impact Factor
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    • "Previous studies demonstrated that IPA-3 prevented Cdc42-induced PAK1 autophosphorylation on T423 and significantly inhibited PAK1 catalytic activity [8], [9]. The inhibitory action of IPA-3 is achieved in part by binding covalently to the regulatory domain of PAK1 which in turn prevented the physical interaction with Cdc42 or other GTPase activators [9]. IPA-3-targeting regulatory domain is less conserved within kinases, thus confers a remarkably high selectivity to this inhibitor [8]. "
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    ABSTRACT: Hepatocellular carcinoma (HCC) is one of the major malignancies worldwide and is associated with poor prognosis due to the high incidences of metastasis and tumor recurrence. Our previous study showed that overexpression of p21-activated protein kinase 1 (PAK1) is frequently observed in HCC and is associated with a more aggressive tumor behavior, suggesting that PAK1 is a potential therapeutic target in HCC. In the current study, an allosteric small molecule PAK1 inhibitor, IPA-3, was evaluated for the potential in suppressing hepatocarcinogenesis. Consistent with other reports, inhibition of PAK1 activity was observed in several human HCC cell lines treated with various dosages of IPA-3. Using cell proliferation, colony formation and BrdU incorporation assays, we demonstrated that IPA-3 treatment significantly inhibited the growth of HCC cells. The mechanisms through which IPA-3 treatment suppresses HCC cell growth are enhancement of apoptosis and blockage of activation of NF-κB. Furthermore, our data suggested that IPA-3 not only inhibits the HCC cell growth, but also suppresses the metastatic potential of HCC cells. Nude mouse xenograft assay demonstrated that IPA-3 treatment significantly reduced the tumor growth rate and decreased tumor volume, indicating that IPA-3 can suppress the in vivo tumor growth of HCC cells. Taken together, our demonstration of the potential preclinical efficacy of IPA-3 in HCC provides the rationale for cancer therapy.
    PLoS ONE 07/2013; 8(7):e68843. DOI:10.1371/journal.pone.0068843 · 3.23 Impact Factor
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