Computer-Based Identification of a Novel LIMK1/2 Inhibitor that Synergizes with Salirasib to Destabilize the Actin Cytoskeleton

Efrat Mashiach-Farkash *,1 , Roni Rak *,3 , Galit Elad-Sfadia 3 , Roni Haklai 3 , Shmuel Carmeli 2 , Yoel Kloog 3 and Haim J. Wolfson 1 1 The Blavatnik School of Computer Science, Raymond and Beverly Sackler Faculty of Exact Sciences, Tel-Aviv University, Tel Aviv, Israel 2 School of Chemistry, Raymond and Beverly Sackler Faculty of Exact Sciences, Tel-Aviv University, Tel Aviv, Israel 3 Department of Neurobiology, The George S. Wise Faculty of Life Sciences, Tel-Aviv University, Tel Aviv, Israel * denotes equal contributors Received: June 21, 2012; Accepted: July 06, 2012; Published: July 07, 2012; Keywords: Cofilin, Ras, Rac, Rho, LIMK. Abbreviations: ADF, actin-depolymerizing factor; CSRD, cysteine/serine-rich domain; CTD, C-terminal domain; FCS, fetal calf serum; FTS, S-trans, trans-farnesyl thiosalicyclic acid; GAP, GTPase activating protein; GRD, GAP-related domain; LIMK, LIM kinase; MEF, Mouse embryonic fibroblast; NF1, neurofibromin; NF1-/-, neurofibromin deficient ;Pak1, p21-activated kinase 1; PI3K, phospatidylinositol 3-kinase. Correspondence: Yoel Kloog, email: // // Abstract Neurofibromin regulates cell motility via three distinct GTPase pathways acting through two different domains, the Ras GTPase-activating protein-related domain (GRD) and the pre-GRD domain. First, the GRD domain inhibits Ras-dependent changes in cell motility through the mitogen activated protein cascade. Second, it also regulates Rho-dependent (Ras-independent) changes by activating LIM kinase 2 (LIMK2), an enzyme that phosphorylates and inactivates cofilin (an actin-depolymerizing factor). Third, the pre-GRD domain acts through the Rac1 GTPase, that activate the P21 activated kinase 1 (PAK1)-LIMK1-cofilin pathway. We employed molecular modeling to identify a novel inhibitor of LIMK1/2. The active sites of an ephrin-A receptor (EphA3) and LIMK2 showed marked similarity (60%). On testing a known inhibitor of EphA3, we found that it fits to the LIMK1/2-ATP binding site and to the latter’s substrate-binding pockets. We identified a similar compound, T56-LIMKi, and found that it inhibits LIMK1/2 kinase activities. It blocked the phosphorylation of cofilin which led to actin severance and inhibition of tumor cell migration, tumor cell growth, and anchorage-independent colony formation in soft agar. Because modulation of LIMK by neurofibromin is not affected by the Ras inhibitor Salirasib, we examined the combined effect of Salirasib and T56-LIMKi each of which can affect cell motility by a distinct pathway. We found that their combined action on cell proliferation and stress-fiber formation in neurofibromin-deficient cells was synergistic. We suggest that this drug combination may be developed for treatment of neurofibromatosis and cancer.