Rac1 (IC50 = 1.1 μM)

In this study, researchers 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 IC50 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.[1]

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EHop-016's inhibition of Rac causes a compensatory mechanism that increases the activity of Rho GTPase RhoA, which is closely related to Rac. EHop-016 (2–5) μM decreases Rac-regulated cell functions, such as lamellipodia formation and cell migration, and inhibits the association of active Vav2 with Rac1(G15A) mutant fusion protein in MDA-MB-435 cells. EHop-016 also has an IC50 of 10 μM, which inhibits the viability of MDA-MB-435 cells.[1]In SM and AML patient-derived cells, EHop-016 also prevents KITD814V-induced growth.[2]

EHop-016 treatment of KITD814V-bearing cells dramatically increases leukemic mice's survival rate. Consistent with the in vitro findings described above, the treatment of KITD814V-bearing cells with EHop-016 versus NSC23766 significantly enhanced the survival of leukemic mice, as demonstrated by reduced spleen size and PB counts [2]

Rac Activity Assays [1]
Rac activity was determined from lysates of the MDA-MB-435 and MDA-MB-231 human metastatic cancer cell lines (from ATCC). Cancer cells in culture medium (DMEM, 10% FBS, pH 7.5) were treated with vehicle (0.1% DMSO) or varying concentrations of EHop-016 (0–10 μm) for 24 h. Rac1 activity was determined as described previously, using the G-LISA Rac1 activation assay kit. For generation of IC50 curves for each inhibitor (EHop-016 or NSC23766), data from three independent duplicate experiments were pooled, and four-parameter dose-response curves were fitted using the non-linear regression function of GraphPad Prism®.

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Rho GTPase Activity Assays[1]
Rho, Rac, and Cdc42 activities were analyzed from MDA-MB-435 and MDA-MB-231 cell lysates by pull-down assays following treatment with EHop-016 for 24 h. The GST-Rho binding domain from rhotekin was used to isolate active GTP-bound Rho, and a GST-Cdc42 and Rac interactive binding (CRIB) domain of PAK1 was used to isolate active Rac-GTP or Cdc42-GTP, as described previously. Active and total Rho GTPases were identified by Western blotting with specific antibodies.

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Interaction of Tiam-1 DH/PH Domain with Rac1(G15A) [1]
His-tagged Tiam-1 DH-PH pET construct was transformed into Rosetta DE3 Escherichia coli cells, and clarified lysates were purified by batch affinity chromatography using His-Select nickel affinity gel. Tiam-1 was eluted with 300 mm imidazole and separated by an FPLC size exclusion Superdex 200 column. Purity of the Tiam-1 fraction at 1.7 mg/ml was observed to be >95% by SDS-PAGE. GST-Rac1(G15A) glutathione-agarose or glutathione-agarose beads alone were preincubated with varying concentrations of EHop-016 or NSC23766 for 1 h in lysis buffer (1% Igepal, 20 mm HEPES, 150 mm NaCl, 5 mm MgCl2, pH 7.5). Purified His-Tiam-1 DH/PH domain was added at a concentration of 2:1 Rac1(G15A)/Tiam-1 and incubated for another 1 h at 4 °C. Pull-downs were washed three times in 1% Igepal buffer and 1 time in HEPES buffer and Western blotted with an anti-His antibody to visualize His-Tiam-1 DH/PH domain protein.

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The MDA-MB-435 and MDA-MB-231 human metastatic cancer cell lines' lysates are used to measure Rac activity. For a full day, cancer cells grown in culture medium (DMEM, 10% FBS, pH 7.5) are exposed to either a vehicle (0.1% DMSO) or different concentrations of EHop-016 (0–10 μM). With the G-LISA Rac1 activation assay kit, Rac1 activity is measured.

Fluorescence Microscopy [1]
As described previously, MDA-MB-435 or MDA-MB-231 cells in culture medium were treated with vehicle (0.1% DMSO) or EHop-016 at 2 and 4 μm for 24 h. Cells were fixed, permeabilized, and stained with rhodamine phalloidin to visualize F-actin. Fluorescence micrographs were acquired at ×600 magnification in an Olympus BX40 fluorescence microscope using a Spot digital camera.

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Cell Migration Assays [1]
As described previously, quiescent MDA-MB-435 cells were treated with vehicle or varying concentrations of EHop-016 (0–5 μm) for 24 h. Exactly 2 × 105 cells were placed on the top well of Transwell chambers with culture medium containing 10% FBS in the bottom well. The number of cells that migrated to the underside of the membrane following a 4-h incubation was quantified for each treatment. Fixed cells were stained with propidium iodide to visualize nuclei. For each treatment (three biological experiments with two technical replicates each), cells in 20 microscopic fields were quantified at ×200 magnification in a Olympus CKX41 inverted fluorescence microscope.

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Cell Viability Assays [1]
As described previously, MDA-MB-231, MDA-MB-435, or MCF-10A mammary epithelial cells (from ATCC) were incubated in vehicle (0.1% DMSO) or varying concentrations of EHop-016 (0–10 μm) for 24 h. Cell viability was measured using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide cell survival and proliferation kit according to the manufacturer's instructions.

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For 24 hours, mammary epithelial cells (MDA-MB-231, MDA-MB-435, or MCF-10A) are cultured in 0.1% DMSO vehicle or different concentrations of EHop-016 (0–10 μM). Following the manufacturer's instructions, the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide cell survival and proliferation kit is used to measure cell viability.

KITD814V-bearing mice.
2.5 μM
KITD814V-bearing 32D cells with EHop-016 are administered by i.v. injection.

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Transplantation into C3H/HeJ mice was carried out by administering a single i.v. injection of 2 × 106 32D cells bearing WT KIT or KITD814V with or without RacN17 or PakK299R, or 1 × 106 KITD814V-bearing 32D cells cultured overnight with DMSO (vehicle), 25 μM NSC23766, or 2.5 μM EHop-016. Mice were harvested at the time of moribundity, and PB, femurs, spleen, lungs, and liver were collected for histopathological and flow cytometric analysis.[2]

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

[2]. J Clin Invest . 2013 Oct;123(10):4449-63.

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.[1]

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An acquired somatic mutation at codon 816 in the KIT receptor tyrosine kinase is associated with poor prognosis in patients with systemic mastocytosis and acute myeloid leukemia (AML). Treatment of leukemic cells bearing this mutation with an allosteric inhibitor of p21-activated kinase (Pak) or its genetic inactivation results in growth repression due to enhanced apoptosis. Inhibition of the upstream effector Rac abrogates the oncogene-induced growth and activity of Pak. Although both Rac1 and Rac2 are constitutively activated via the guanine nucleotide exchange factor (GEF) Vav1, loss of Rac1 or Rac2 alone moderately corrected the growth of KIT-bearing leukemic cells, whereas the combined loss resulted in 75% growth repression. In vivo, the inhibition of Vav or Rac or Pak delayed the onset of myeloproliferative neoplasms (MPNs) and corrected the associated pathology in mice. To assess the role of Rac GEFs in oncogene-induced transformation, we used an inhibitor of Rac, EHop-016, which specifically targets Vav1 and found that EHop-016 was a potent inhibitor of human and murine leukemic cell growth. These studies identify Pak and Rac GTPases, including Vav1, as potential therapeutic targets in MPN and AML involving an oncogenic form of KIT.[2]

C25H30N6O

430.55

430.248

C, 69.74; H, 7.02; N, 19.52; O, 3.72

1380432-32-5

51031035

Light yellow to yellow solid powder

1.27

3.263

2

6

8

32

573

0

O1C([H])([H])C([H])([H])N(C([H])([H])C1([H])[H])C([H])([H])C([H])([H])C([H])([H])N([H])C1=NC([H])=C([H])C(=N1)N([H])C1C([H])=C([H])C2=C(C=1[H])C1=C([H])C([H])=C([H])C([H])=C1N2C([H])([H])C([H])([H])[H]

AFTZZRFCMOAFCR-UHFFFAOYSA-N

InChI=1S/C25H30N6O/c1-2-31-22-7-4-3-6-20(22)21-18-19(8-9-23(21)31)28-24-10-12-27-25(29-24)26-11-5-13-30-14-16-32-17-15-30/h3-4,6-10,12,18H,2,5,11,13-17H2,1H3,(H2,26,27,28,29)

4-N-(9-ethylcarbazol-3-yl)-2-N-(3-morpholin-4-ylpropyl)pyrimidine-2,4-diamine

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

Solubility in Formulation 1: ≥ 2.5 mg/mL (5.81 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.5 mg/mL (5.81 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 3: ≥ 2.5 mg/mL (5.81 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 4: 2% DMSO +30% PEG 300 +5% Tween 80 +ddH2O: 5mg/mL

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Solubility in Formulation 5: 10 mg/mL (23.23 mM) in 50% PEG300 50% Saline (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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 (Please use freshly prepared in vivo formulations for optimal results.)