title: Characterization of EHop-016, novel small molecule inhibitor of Rac GTPase.
abstract: The Rho GTPase Rac regulates actin cytoskeleton reorganization to form cell surface extensions (lamellipodia) required for cell migration/invasion during cancer metastasis. Rac hyperactivation and overexpression are associated with aggressive cancers; thus, interference of the interaction of Rac with its direct upstream activators, guanine nucleotide exchange factors (GEFs), is a viable strategy for inhibiting Rac activity. We synthesized EHop-016, a novel inhibitor of Rac activity, based on the structure of the established Rac/Rac GEF inhibitor NSC23766. Herein, we demonstrate that EHop-016 inhibits Rac activity in the MDA-MB-435 metastatic cancer cells that overexpress Rac and exhibits high endogenous Rac activity. The IC(50) of 1.1 μM for Rac inhibition by EHop-016 is ∼100-fold lower than for NSC23766. EHop-016 is specific for Rac1 and Rac3 at concentrations of ≤5 μM. At higher concentrations, EHop-016 inhibits the close homolog Cdc42. In MDA-MB-435 cells that demonstrate high active levels of the Rac GEF Vav2, EHop-016 inhibits the association of Vav2 with a nucleotide-free Rac1(G15A), which has a high affinity for activated GEFs. EHop-016 also inhibits the Rac activity of MDA-MB-231 metastatic breast cancer cells and reduces Rac-directed lamellipodia formation in both cell lines. EHop-016 decreases Rac downstream effects of PAK1 (p21-activated kinase 1) activity and directed migration of metastatic cancer cells. Moreover, at effective concentrations (<5 μM), EHop-016 does not affect the viability of transformed mammary epithelial cells (MCF-10A) and reduces viability of MDA-MB-435 cells by only 20%. Therefore, EHop-016 holds promise as a targeted therapeutic agent for the treatment of metastatic cancers with high Rac activity.
authors.affiliation: Department of Biochemistry, School of Medicine, University of Puerto Rico, Medical Sciences Campus, San Juan, Puerto Rico.
authors.forename: Brenda L
authors.initials: BL
authors.lastname: Montalvo-Ortiz
authors.forename: Linette
authors.initials: L
authors.lastname: Castillo-Pichardo
authors.forename: Eliud
authors.initials: E
authors.lastname: Hernández
authors.forename: Tessa
authors.initials: T
authors.lastname: Humphries-Bickley
authors.forename: Alina
authors.initials: A
authors.lastname: De la Mota-Peynado
authors.forename: Luis A
authors.initials: LA
authors.lastname: Cubano
authors.forename: Cornelis P
authors.initials: CP
authors.lastname: Vlaar
authors.forename: Suranganie
authors.initials: S
authors.lastname: Dharmawardhane
mesh_terms: D000634:Aminoquinolines / Q000494:pharmacology; D001665:Binding Sites / Q000187:drug effects; D001943:Breast Neoplasms / Q000188:drug therapy* / Q000201:enzymology / Q000473:pathology; D002227:Carbazoles / Q000138:chemical synthesis / Q000494:pharmacology*; D045744:Cell Line, Tumor; D002470:Cell Survival / Q000187:drug effects; D015195:Drug Design; D004791:Enzyme Inhibitors / Q000138:chemical synthesis* / Q000494:pharmacology*; D005260:Female; D006801:Humans; D011743:Pyrimidines / Q000138:chemical synthesis / Q000494:pharmacology*; D011993:Recombinant Fusion Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics; D020830:rac1 GTP-Binding Protein / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000235:genetics; D020741:rho GTP-Binding Proteins / Q000037:antagonists & inhibitors
chemical_list: D000634:Aminoquinolines; D002227:Carbazoles; D004791:Enzyme Inhibitors; C572775:N4-(9-ethyl-9H-carbazol-3-yl)-N2-(3-morpholin-4-ylpropyl)-pyrimidine-2,4-diamine; C487513:NSC 23766; D011743:Pyrimidines; D011993:Recombinant Fusion Proteins; D020830:rac1 GTP-Binding Protein; D020741:rho GTP-Binding Proteins