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

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.

Molecular Pathways: Targeting the Kinase Effectors of RHO-Family GTPases

“…Further, this LIMK1/2 inhibitor was found to synergize with salirasib, a RAS inhibitor, to inhibit tumor cell growth and destabilization of actin cytoskeleton; together these findings suggest that this drug combination could be considered to treat neurofibromatosis-1 (64). …”

JG6, a novel marine-derived oligosaccharide, suppresses breast cancer metastasis via binding to cofilin

“…Molecular imaging of JG6 by conjugating to fluorescein could be an option. Our findings may highlight the strategy of targeting actin-binding proteins, such as cofilin or cofilin regulators such as LIMK1/2[33, 34], rather than actin itself for cancer therapy.…”

The NF1 gene revisited - from bench to bedside

“…Inhibition of LIM kinase in the Rho-ROCK-LIMK-cofilin pathway regulated by neurofibromin is another potential strategy. In Nf1 −/− MEFs, novel LIMK inhibitors blocked the phosphorylation of cofilin, resulting in actin severance and inhibition of cell migration and growth [185]. The utility of these drugs in NF1-deficient tumours may be worth investigating, especially in combination with Ras or AURKA inhibitors, which may have synergistic effects [185, 186].…”

“…In Nf1 −/− MEFs, novel LIMK inhibitors blocked the phosphorylation of cofilin, resulting in actin severance and inhibition of cell migration and growth [185]. The utility of these drugs in NF1-deficient tumours may be worth investigating, especially in combination with Ras or AURKA inhibitors, which may have synergistic effects [185, 186]. Improved understanding of the biology of NF1 and neurofibromin in normal cells and cancer is critical for the development of novel treatment strategies.…”