EHop-016

EHop-016 is a novel and specific inhibitor of Rac1 and Rac3 activity (IC50= 1.1 μm).

Rac is a member of the RAS superfamily of small GTPase. It functions in various cellular processes including regulation of secretory processes, membrane ruffles, cell polarization and phagocytosis of apoptotic cells etc. It is contributed to malignant progression.

Ehop-016 inhibited Rac activity in metastatic cancer cell MDA-MB-435 which overexpressed Rac and inhibited high endogenous Rac activity as well. In metastatic breast cancer cell MDA-MB-231, Ehop-016 blocked the Rac activity and reduced Rac-mediated lamelipodia formation. It also attenuated the effects of Rac downstream protein p21-activated kinase 1 and directed migration of metastatic cancer cells. [1]

In C3H/HeJ mice carrying KITD814V following overnight treatment of 2.5 μM EHop-016 or 25 μM NSC23766, EHop-016 reduced spleen size and PB counts thus significantly promoted the survival of leukemic mice compared with NSC23766. [2]

References:
1.  Montalvo-Ortiz BL, Castillo-Pichardo L, Hernández E et al. Characterization of EHop-016, novel small molecule inhibitor of Rac GTPase. J Biol Chem. 2012 Apr 13;287(16):13228-38.
2.  Martin H, Mali RS, Ma P et al. Pak and Rac GTPases promote oncogenic KIT-induced neoplasms. J Clin Invest. 2013 Oct;123(10):4449-63.

Pharmacokinetics of Rac inhibitor EHop-016 in mice by ultra-performance liquid chromatography tandem mass spectrometry.[Pubmed:25594952]

J Chromatogr B Analyt Technol Biomed Life Sci. 2015 Feb 15;981-982:19-26.

The Rho GTPase Rac is an important regulator of cancer cell migration and invasion; processes required for metastatic progression. We previously characterized the small molecule EHop-016 as a novel Rac inhibitor in metastatic breast cancer cells and recently found that EHop-016 was effective at reducing tumor growth in nude mice at 25 mg/kg bodyweight (BW). The purpose of this study was to compare the pharmacokinetics and bioavailability of EHop-016 at different dosages in a single dose input scheme (10, 20 and 40 mg/kg BW) following intraperitoneal (IP) and oral gavage (PO) administration to nude mice. We developed and validated a rapid and sensitive method for the quantitation of EHop-016 in mouse plasma by ultra high performance liquid chromatography coupled with electrospray ionization tandem mass spectrometry (UPLC/MS/MS). Separation was carried out on an Agilent Poroshell 120 EC-C18 column (3.0 mm x 50 mm) using organic and aqueous mobile phases. EHop-016 was identified from its accurate mass and retention time from the acquired full-scan chromatogram and quantified by its peak area. The validated method was linear (R(2)>0.995) over the range of 5-1000 ng/mL (1/x(2) weighting). Pharmacokinetic parameters were obtained by non-compartmental analysis using WinNonlin. The area under the curve (AUC(0)-infinity) ranged from 328 to 1869 ng h/mL and 133-487 ng h/mL for IP and PO dosing, respectively. The elimination half-life (t(1)/(2)) ranged from 3.8-5.7 h to 3.4-26.8 h for IP and PO dosing, respectively. For both IP and PO administration, the AUC(0)-infinityvalues were proportional to the tested doses demonstrating linear PK profiles. The relative bioavailability of EHop-016 after oral gavage administration ranged from 26% to 40%. These results support further preclinical evaluation of EHop-016 as a new anti-cancer therapy.

Characterization of EHop-016, novel small molecule inhibitor of Rac GTPase.[Pubmed:22383527]

J Biol Chem. 2012 Apr 13;287(16):13228-38.

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 muM for Rac inhibition by EHop-016 is approximately 100-fold lower than for NSC23766. EHop-016 is specific for Rac1 and Rac3 at concentrations of 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 muM), 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.

The Rac Inhibitor EHop-016 Inhibits Mammary Tumor Growth and Metastasis in a Nude Mouse Model.[Pubmed:25389450]

Transl Oncol. 2014 Oct 24;7(5):546-55.

Metastatic disease still lacks effective treatments, and remains the primary cause of cancer mortality. Therefore, there is a critical need to develop better strategies to inhibit metastatic cancer. The Rho family GTPase Rac is an ideal target for anti-metastatic cancer therapy, because Rac is a key molecular switch that is activated by a myriad of cell surface receptors to promote cancer cell migration/invasion and survival. Previously, we reported the design and development of EHop-016, a small molecule compound, which inhibits Rac activity of metastatic cancer cells with an IC50 of 1 muM. EHop-016 also inhibits the activity of the Rac downstream effector p21-activated kinase (PAK), lamellipodia extension, and cell migration in metastatic cancer cells. Herein, we tested the efficacy of EHop-016 in a nude mouse model of experimental metastasis, where EHop-016 administration at 25 mg/kg body weight (BW) significantly reduced mammary fat pad tumor growth, metastasis, and angiogenesis. As quantified by UPLC MS/MS, EHop-016 was detectable in the plasma of nude mice at 17 to 23 ng/ml levels at 12 h following intraperitoneal (i.p.) administration of 10 to 25 mg/kg BW EHop-016. The EHop-016 mediated inhibition of angiogenesis In Vivo was confirmed by immunohistochemistry of excised tumors and by In Vitro tube formation assays of endothelial cells. Moreover, EHop-016 affected cell viability by down-regulating Akt and Jun kinase activities and c-Myc and Cyclin D expression, as well as increasing caspase 3/7 activities in metastatic cancer cells. In conclusion, EHop-016 has potential as an anticancer compound to block cancer progression via multiple Rac-directed mechanisms.

Development of EHop-016: a small molecule inhibitor of Rac.[Pubmed:25033803]

Enzymes. 2013;33 Pt A:117-46.

The Rac inhibitor EHop-016 was developed as a compound with the potential to inhibit cancer metastasis. Inhibition of the first step of metastasis, migration, is an important strategy for metastasis prevention. The small GTPase Rac acts as a pivotal binary switch that is turned "on" by guanine nucleotide exchange factors (GEFs) via a myriad of cell surface receptors, to regulate cancer cell migration, survival, and proliferation. Unlike the related GTPase Ras, Racs are not usually mutated, but overexpressed or overactivated in cancer. Therefore, a rational Rac inhibitor should block the activation of Rac by its upstream effectors, GEFs, and the Rac inhibitor NSC23766 was developed using this rationale. However, this compound is ineffective at inhibiting the elevated Rac activity of metastatic breast cancer cells. Therefore, a panel of small molecule compounds were derived from NSC23766 and screened for Rac activity inhibition in metastatic cancer cells. EHop-016 was identified as a compound that blocks the interaction of Rac with the GEF Vav in metastatic human breast cancer cells with an IC50 of ~1muM. At higher concentrations (10muM), EHop-016 inhibits the related Rho GTPase Cdc42, but not Rho, and also reduces cell viability. Moreover, EHop-016 inhibits the activation of the Rac downstream effector p21-activated kinase, extension of motile actin-based structures, and cell migration. Future goals are to develop EHop-016 as a therapeutic to inhibit cancer metastasis, either individually or in combination with current anticancer compounds. The next generation of EHop-016-based Rac inhibitors is also being developed.

EHop-016 is a novel potent and selective inhibitor of Rac GTPase; inhibits Rac1 activity in MDA-MB-435 cells with an IC50 of 1.1 uM.

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