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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    • "We used IPA-3, a chemical inhibitor that specifically targets inactive group 1 PAKs (Deacon et al., 2008;Viaud and Peterson, 2009) by preferentially binding to the inactive conformation of the PAKs' regulatory domain. Accordingly , it has been observed to inhibit Rac1-mediated PAK activation dose dependently, whereas IPA-3 has a substantially reduced effect on already-active PAK (Deacon et al., 2008;Viaud and Peterson, 2009). To determine the half-life of intercellular IPA-3, we treated the cells with IPA-3 and replaced the media after 20 min of incubation. "
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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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    • "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.
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