Efficacy and delivery strategies of the dual Rac/Cdc42 inhibitor MBQ-167 in HER2 overexpressing breast cancer.

Trastuzumab and trastuzumab-based treatments are the standard of care for breast cancer patients who overexpress the human epidermal growth factor receptor 2 (HER2). However, patients often develop resistance to trastuzumab via signaling from alternative growth factor receptors that converge to activate guanine nucleotide exchange factors (GEFs) that in turn activate the Rho GTPases Rac and Cdc42. Since Rac and Cdc42 have been implicated in high tumor grade and therapy resistance, inhibiting the activity of Rac and Cdc42 is a rational strategy to overcome HER2-targeted therapy resistance. Therefore, our group developed MBQ-167, a dual Rac/Cdc42 inhibitor with IC50s of 103 nM and 78 nM for Rac and Cdc42, respectively, which is highly effective in reducing cell and tumor growth and metastasis in breast cancer cell and mouse models. Herein, we created a trastuzumab resistant variant of the SKBR3 HER2 positive breast cancer cell line and show that Rac activation is a central mechanism in trastuzumab resistance. Next, we tested the potential of targeting MBQ-167 to HER2 overexpressing trastuzumab-resistant cell lines in vitro, and show that MBQ-167, but not trastuzumab, reduces cell viability and induces apoptosis. When MBQ-167 was targeted to mammary fatpad tumors established from HER2 overexpressing cells via immunoliposomes functionalized with trastuzumab, MBQ-167 and MBQ-167-loaded liposomes show equal efficacy in reducing the viability of trastuzumab-resistant cells, inhibiting tumor growth in mouse xenografts, and reducing metastasis to lungs and liver. This study demonstrates the efficacy of MBQ-167 as an alternative therapeutic in HER2 overexpressing cancers, delivered either in free form or in liposomes.

New insights into the role of VKORC1 polymorphisms for optimal warfarin dose selection in Caribbean Hispanic patients through an external validation of a population PK/PD model.

Warfarin, an oral anticoagulant, has been used for decades to prevent thromboembolic events. The complex interplay between CYP2C9 and VKORC1 genotypes on warfarin PK and PD properties is not fully understood in special sub-groups of patients. This study aimed to externally validate a population pharmacokinetic/pharmacodynamic (PK/PD) model for the effect of warfarin on international normalized ratio (INR) and to evaluate optimal dosing strategies based on the selected covariates in Caribbean Hispanic patients. INR, and CYP2C9 and VKORC1 genotypes from 138 patients were used to develop a population PK/PD model in NONMEM. The structural definition of a previously published PD model for INR was implemented. A numerical evaluation of the parameter-covariate relationship was performed. Simulations were conducted to determine optimal dosing strategies for each genotype combinations, focusing on achieving therapeutic INR levels. Findings revealed elevated IC50 for G/G, G/A, and A/A VKORC1 haplotypes (11.76, 10.49, and 9.22 mg/L, respectively), in this population compared to previous reports. The model-guided dosing analysis recommended daily warfarin doses of 3-5 mg for most genotypes to maintain desired INR levels, although subjects with combination of CYP2C9 and VKORC1 genotypes * 2/* 2-, * 2/* 3- and * 2/* 5-A/A would require only 1 mg daily. This research underscores the potential of population PK/PD modeling to inform personalized warfarin dosing in populations typically underrepresented in clinical studies, potentially leading to improved treatment outcomes and patient safety. By integrating genetic factors and clinical data, this approach could pave the way for more effective and tailored anticoagulation therapy in diverse patient groups.

The Rac inhibitor HV-107 as a potential therapeutic for metastatic breast cancer.

BACKGROUND: The significant challenge in treating triple-negative breast cancer (TNBC) lies in its high rate of distant metastasis. To address this, inhibiting metastasis formation in TNBC is vital. Rac is a key player in cancer metastasis. Previously, we developed Ehop-016, a Rac inhibitor that successfully reduced tumor growth and metastasis in mice. In this study, we assessed the effectiveness of HV-107, a derivative of Ehop-016, in inhibiting TNBC metastasis at lower doses. METHODS: Rho GTPases activity assays were performed with the use of GST-PAK beads and Rac, Rho, and Cdc42 GLISA. Cell viability was assessed through trypan blue exclusion and MTT assays. Cell cycle analysis was conducted using flow cytometry. To evaluate invading capabilities, transwell assays and invadopodia formation assays were performed. Metastasis formation studies were conducted using a breast cancer xenograft mouse model. RESULTS: HV-107 inhibited Rac activity by 50% in MDA-MB-231 and MDA-MB-468 cells at concentrations of 250-2000 nM, leading to a 90% decrease in invasion and invadopodia activity. Concentrations of 500 nM and above caused dose-dependent reductions in cell viability, resulting in up to 20% cell death after 72 h. Concentrations exceeding 1000 nM upregulated PAK1, PAK2, FAK, Pyk2, Cdc42, and Rho signallings, while Pyk2 was downregulated at 100-500 nM. Through in vitro experiments, optimal concentrations of HV-107 ranging from 250 to 500 nM were identified, effectively inhibiting Rac activity and invasion while minimizing off-target effects. In a breast cancer xenograft model, administration of 5 mg/kg HV-107 (administered intraperitoneally, 5 days a week) reduced Rac activity by 20% in tumors and decreased metastasis by 50% in the lungs and liver. No observed toxicity was noted at the tested doses. CONCLUSION: The findings indicate that HV-107 exhibits promising potential as a therapeutic medication utilizing Rac inhibition mechanisms to address metastasis formation in TNBC.

Characterization of Novel Derivatives of MBQ-167, an inhibitor of the GTP-binding proteins Rac/Cdc42.

Rac and Cdc42, are homologous GTPases that regulate cell migration, invasion, and cell cycle progression; thus, representing key targets for metastasis therapy. We previously reported on the efficacy of MBQ-167, which blocks both Rac1 and Cdc42 in breast cancer cells and mouse models of metastasis. To identify compounds with increased activity, a panel of MBQ-167 derivatives was synthesized, maintaining its 9-ethyl-3-(1H-1,2,3-triazol-1-yl)-9H-carbazole core. Similar to MBQ-167, MBQ-168 and EHop-097, inhibit activation of Rac and Rac1B splice variant and breast cancer cell viability, and induce apoptosis. MBQ-167 and MBQ-168 inhibit Rac and Cdc42 by interfering with guanine nucleotide binding, and MBQ-168 is a more effective inhibitor of PAK (1,2,3) activation. EHop-097 acts via a different mechanism by inhibiting the interaction of the guanine nucleotide exchange factor (GEF) Vav with Rac. MBQ-168 and EHop-097 inhibit metastatic breast cancer cell migration, and MBQ-168 promotes loss of cancer cell polarity to result in disorganization of the actin cytoskeleton and detachment from the substratum. In lung cancer cells, MBQ-168 is more effective than MBQ-167 or EHop-097 at reducing ruffle formation in response to EGF. Comparable to MBQ-167, MBQ-168 significantly inhibits HER2+ tumor growth and metastasis to lung, liver, and spleen. Both MBQ-167 and MBQ-168 inhibit the cytochrome P450 (CYP) enzymes 3A4, 2C9, and 2C19. However, MBQ-168 is ~10X less potent than MBQ-167 at inhibiting CYP3A4, thus demonstrating its utility in relevant combination therapies. In conclusion, the MBQ-167 derivatives MBQ-168 and EHop-097 are additional promising anti metastatic cancer compounds with similar and distinct mechanisms.

Efficacy of Rac and Cdc42 Inhibitor MBQ-167 in Triple-negative Breast Cancer.

Triple-negative breast cancer (TNBC) is an aggressive form of breast cancer, with a high predisposition for locally invasive and metastatic cancer. With the objective to reduce cancer metastasis, we developed small molecule inhibitors to target the drivers of metastasis, the Rho GTPases Rac and Cdc42. Of these, MBQ-167 inhibits both Rac and Cdc42 with IC50s of 103 and 78 nmol/L, respectively; and consequently, inhibits p21-activated kinase (PAK) signaling, metastatic cancer cell proliferation, migration, and mammosphere growth; induces cell-cycle arrest and apoptosis; and decreases HER2-type mammary fatpad tumor growth and metastasis (Humphries-Bickley and colleagues, 2017). Herein, we used nuclear magnetic resonance to show that MBQ-167 directly interacts with Rac1 to displace specific amino acids, and consequently inhibits Rac.GTP loading and viability in TNBC cell lines. Phosphokinome arrays in the MDA-MB-231 human TNBC cells show that phosphorylation status of kinases independent of the Rac/Cdc42/PAK pathway are not significantly changed following 200 nmol/L MBQ-167 treatment. Western blotting shows that initial increases in phospho-c-Jun and phospho-CREB in response to MBQ-167 are not sustained with prolonged exposure, as also confirmed by a decrease in their transcriptional targets. MBQ-167 inhibits tumor growth, and spontaneous and experimental metastasis in immunocompromised (human TNBC) and immunocompetent (mouse TNBC) models. Moreover, per oral administration of MBQ-167 at 100 mg/kg body weight is not toxic to immunocompetent BALB/c mice and has a half-life of 4.6 hours in plasma. These results highlight the specificity, potency, and bioavailability of MBQ-167, and support its clinical potential as a TNBC therapeutic.

Polymorphism in early development: The account of MBQ-167.

With McCrone's famous statement in mind, we set out to investigate the polymorphic behavior of a small-molecule dual inhibitor of Rac and Cdc42, currently undergoing preclinical trials. Herein, we report the existence of two polymorphs for 9-ethyl-3-(5-phenyl-1H-1,2,3-triazol-3-yl)-9H-carbazole (MBQ-167). These were characterized by differential scanning calorimetry, thermogravimetric analysis, Raman and Infrared spectroscopy, as well as powder and single crystal X-ray diffraction. The results obtained from the thermal analysis revealed that MBQ-167 form II undergoes an exothermic phase transition to form I, making this the thermodynamically stable form. An examination of the Burger-Ramberger rules for assigning thermodynamic relationships in polymorphic pairs indicate that this system is monotropic. The structure elucidation reveals that these forms crystallize in the orthorhombic (Pbca) and monoclinic (P21/n) space groups. A conformational analysis shows that the metastable form (form II) presents the most planar conformation along the significant torsion angles identified. Hirshfeld surface analysis confirms that van der Waals contacts are the primary interactions and only subtle differences in short contacts help differentiate each form. These findings support the notion that polymorphism is prevalent in organic molecules and that one should invest time and money probing possible polymorphs, particularly in early development as in the case of MBQ-167.

Solubility Measurements and Correlation of MBQ-167 in Neat and Binary Solvent Mixtures.

MBQ-167 is a novel, small-molecule dual inhibitor of Rac and Cdc42, small GTPases that are involved in cytoskeletal organization, cell cycle progression, and cell migration. In an in vivo mouse model, MBQ-167 has been shown to significantly reduce mammary tumor growth and metastasis and is currently undergoing preclinical studies for the treatment of metastatic cancer. To date, no solubility data have been reported for this compound. For this reason, the present study aims to determine the solubility of this compound in eight neat solvents (acetonitrile, 1-butanol, 2-butanol, ethanol, ethyl acetate, methanol, 1-propanol, and 2-propanol) and two binary solvent mixtures [ethyl acetate (2) + heptane (3) and ethanol (2) + water (3)] between the temperatures of 278.15 and 333.15 K. The results obtained employing the polythermal method show that the solubility of MBQ-167 increases with an increase in temperature in all neat solvents used within this study. Moreover, in the two binary solvent mixtures, the solubility of this compound increases with increasing temperature and decreases with an increasing mass fraction of the antisolvent (heptane or water). The experimental solubility data were correlated using the modified Apelblat and λh model equations. The predicted solubility data acquired from the Apelblat and λh model equations correlate well with the experimental solubility data as indicated by the low ARD % (≤1.8304 and ≤6.5366, respectively). No solvent-mediated polymorphic phase transitions were observed while performing the solubility studies, and no other solid forms were detected after the recrystallization in the solvents and solvent mixtures. The solubility data determined here can offer pathways to develop pharmaceutical crystallization processes that can further the translation of MBQ-167 into a clinical setting.

Genotype-driven pharmacokinetic simulations of warfarin levels in Puerto Ricans.

Objectives The inter-individual variability of warfarin dosing has been linked to genetic polymorphisms. This study was aimed at performing genotype-driven pharmacokinetic (PK) simulations to predict warfarin levels in Puerto Ricans. Methods Analysis of each individual dataset was performed by one-compartmental modeling using WinNonlin®v6.4. The k e of warfarin given a cytochrome P450 2C9 (CYP2C9) genotype ranged from 0.0189 to 0.0075 h-1. K a and V d parameters were taken from literature. Data from 128 subjects were divided into two groups (i.e., wild-types and carriers) and statistical analyses of PK parameters were performed by unpaired t-tests. Results In the carrier group (n=64), 53 subjects were single-carriers and 11 double-carriers (i.e., *2/*2, *2/*3, *2/*5, *3/*5, and *3/*8). The mean peak concentration (Cmax) was higher for wild-type (0.36±0.12 vs. 0.32±0.14 mg/L). Likewise, the average clearance (CL) parameter was faster among non-carriers (0.22±0.03 vs. 0.17±0.05 L/h; p=0.0001), with also lower area under the curve (AUC) when compared to carriers (20.43±6.97 vs. 24.78±11.26 h mg/L; p=0.025). Statistical analysis revealed a significant difference between groups with regard to AUC and CL, but not for Cmax. This can be explained by the variation of k e across different genotypes. Conclusions The results provided useful information for warfarin dosing predictions that take into consideration important individual PK and genotyping data.

Genotype-driven pharmacokinetic simulations of warfarin levels in Puerto Ricans.

OBJECTIVES: The inter-individual variability of warfarin dosing has been linked to genetic polymorphisms. This study was aimed at performing genotype-driven pharmacokinetic (PK) simulations to predict warfarin levels in Puerto Ricans. METHODS: Analysis of each individual dataset was performed by one-compartmental modeling using WinNonlin®v6.4. The ke of warfarin given a cytochrome P450 2C9 (CYP2C9) genotype ranged from 0.0189 to 0.0075 h-1. Ka and Vd parameters were taken from literature. Data from 128 subjects were divided into two groups (i.e., wild-types and carriers) and statistical analyses of PK parameters were performed by unpaired t-tests. RESULTS: In the carrier group (n=64), 53 subjects were single-carriers and 11 double-carriers (i.e., *2/*2, *2/*3, *2/*5, *3/*5, and *3/*8). The mean peak concentration (Cmax) was higher for wild-type (0.36±0.12 vs. 0.32±0.14 mg/L). Likewise, the average clearance (CL) parameter was faster among non-carriers (0.22±0.03 vs. 0.17±0.05 L/h; p=0.0001), with also lower area under the curve (AUC) when compared to carriers (20.43±6.97 vs. 24.78±11.26 h mg/L; p=0.025). Statistical analysis revealed a significant difference between groups with regard to AUC and CL, but not for Cmax. This can be explained by the variation of ke across different genotypes. CONCLUSIONS: The results provided useful information for warfarin dosing predictions that take into consideration important individual PK and genotyping data.

Pharmacokinetics of the Rac/Cdc42 Inhibitor MBQ-167 in Mice by Supercritical Fluid Chromatography-Tandem Mass Spectrometry.

The Rho GTPases Rac and Cdc42 are potential targets against metastatic diseases. We characterized the small molecule MBQ-167 as an effective dual Rac/Cdc42 inhibitor that reduces HER2-type tumor growth and metastasis in mice by ∼90%. This study reports the pharmacokinetics and tissue distribution of MBQ-167 following intraperitoneal and oral single-dose administrations. We first developed and validated a bioanalytical method for the quantitation of MBQ-167 in mouse plasma and tissues by supercritical fluid chromatography coupled with electrospray ionization tandem mass spectrometry. MBQ-167 was rapidly distributed into the kidneys after intraperitoneal dosing, whereas oral administration resulted in higher distribution to lungs. The elimination half-lives were 2.17 and 2.6 h for the intraperitoneal and oral dosing, respectively. The relative bioavailability of MBQ-167 after oral administration was 35%. This investigation presents the first analysis of the pharmacokinetics of MBQ-167 and supports further preclinical evaluation of this drug as a potential anticancer therapeutic.

Design, synthesis and biological evaluation of new carbazole derivatives as anti-cancer and anti-migratory agents.

Based on the efficacy of EHop-016 as an inhibitor of migration and Rac1 activation, a new series of carbazole derivatives has been synthesized. Cytotoxic and anti-migratory effects of these compounds were evaluated in MCF-7 and MDA-MB-231 breast cancer cell lines. Preliminary investigations of their anticancer activity demonstrated that several compounds have moderate antiproliferative effects on cancer cell lines with GI50 values in the range of 13-50 µM. Furthermore, compounds 3b and 11b inhibit migration activity of metastatic cell line MDA-MB-231 by 32% and 34%, respectively. Compound 11b was shown to inhibit activation of the Rho GTPase Rac1 by 55% at 250 nM in both MDA-MB-231 and MDA-MB-435 cell lines. Compared with the IC50 of Rac1 inhibition by lead compound EHop-016 of 1.1 µM, compound 11b demonstrates 4X improved in vitro efficacy.

Characterization of a Dual Rac/Cdc42 Inhibitor MBQ-167 in Metastatic Cancer.

The Rho GTPases Rac (Ras-related C3 botulinum toxin substrate) and Cdc42 (cell division control protein 42 homolog) regulate cell functions governing cancer malignancy, including cell polarity, migration, and cell-cycle progression. Accordingly, our recently developed Rac inhibitor EHop-016 (IC50, 1,100 nmol/L) inhibits cancer cell migration and viability and reduces tumor growth, metastasis, and angiogenesis in vivo Herein, we describe MBQ-167, which inhibits Rac and Cdc42 with IC50 values of 103 and 78 nmol/L, respectively, in metastatic breast cancer cells. Consequently, MBQ-167 significantly decreases Rac and Cdc42 downstream effector p21-activated kinase (PAK) signaling and the activity of STAT3, without affecting Rho, MAPK, or Akt activities. MBQ-167 also inhibits breast cancer cell migration, viability, and mammosphere formation. Moreover, MBQ-167 affects cancer cells that have undergone epithelial-to-mesenchymal transition by a loss of cell polarity and inhibition of cell surface actin-based extensions to ultimately result in detachment from the substratum. Prolonged incubation (120 hours) in MBQ-167 decreases metastatic cancer cell viability with a GI50 of approximately 130 nmol/L, without affecting noncancer mammary epithelial cells. The loss in cancer cell viability is due to MBQ-167-mediated G2-M cell-cycle arrest and subsequent apoptosis, especially of the detached cells. In vivo, MBQ-167 inhibits mammary tumor growth and metastasis in immunocompromised mice by approximately 90%. In conclusion, MBQ-167 is 10× more potent than other currently available Rac/Cdc42 inhibitors and has the potential to be developed as an anticancer drug, as well as a dual inhibitory probe for the study of Rac and Cdc42. Mol Cancer Ther; 16(5); 805-18. ©2017 AACR.

VAV2, a guanine nucleotide exchange factor for Rac1, regulates glucose-stimulated insulin secretion in pancreatic beta cells.

AIMS/HYPOTHESIS: Rho GTPases (Ras-related C3 botulinum toxin substrate 1 [Rac1] and cell division cycle 42 [Cdc42]) have been shown to regulate glucose-stimulated insulin secretion (GSIS) via cytoskeletal remodelling, trafficking and fusion of insulin-secretory granules with the plasma membrane. GTP loading of these G proteins, which is facilitated by GDP/GTP exchange factors, is a requisite step in the regulation of downstream effector proteins. Guanine nucleotide exchange factor VAV2 (VAV2), a member of the Dbl family of proteins, has been identified as one of the GDP/GTP exchange factors for Rac1. Despite recent evidence on the regulatory roles of VAV2 in different cell types, roles of this guanine nucleotide exchange factor in the signalling events leading to GSIS remain undefined. Using immunological, short interfering RNA (siRNA), pharmacological and microscopic approaches we investigated the role of VAV2 in GSIS from islet beta cells. METHODS: Co-localisation of Rac1 and VAV2 was determined by Triton X-114 phase partition and confocal microscopy. Glucose-induced actin remodelling was quantified by live cell imaging using the LifeAct-GFP fluorescent biosensor. Rac1 activation was determined by G protein linked immunosorbent assay (G-LISA). RESULTS: Western blotting indicated that VAV2 is expressed in INS-1 832/13 beta cells, normal rat islets and human islets. Vav2 siRNA markedly attenuated GSIS in INS-1 832/13 cells. Ehop-016, a newly discovered small molecule inhibitor of the VAV2-Rac1 interaction, or siRNA-mediated knockdown of VAV2 markedly attenuated glucose-induced Rac1 activation and GSIS in INS-1 832/13 cells. Pharmacological findings were recapitulated in primary rat islets. A high glucose concentration promoted co-localisation of Rac1 and VAV2. Real-time imaging in live cells indicated a significant inhibition of glucose-induced cortical actin remodelling by Ehop-016. CONCLUSIONS/INTERPRETATION: Our data provide the first evidence to implicate VAV2 in glucose-induced Rac1 activation, actin remodelling and GSIS in pancreatic beta cells.

Phagocyte-like NADPH oxidase (Nox2) promotes activation of p38MAPK in pancreatic ß-cells under glucotoxic conditions: Evidence for a requisite role of Ras-related C3 botulinum toxin substrate 1 (Rac1).

It is well established that glucotoxicity (caused by high glucose concentrations; HG) underlies pathogenesis of islet dysfunction in diabetes. We have recently demonstrated that Nox2 plays a requisite role in the generation of reactive oxygen species (ROS) under HG conditions, resulting in mitochondrial dysregulation and loss of islet ß-cell function. Herein, we investigated roles of Nox2 in the regulation of downstream stress kinase (p38MAPK) activation under HG conditions (20mM; 24h) in normal rodent islets and INS-1 832/13 cells. We observed that gp91-ds-tat, a specific inhibitor of Nox2, but not its inactive analog, significantly attenuated HG-induced Nox2 activation, ROS generation and p38MAPK activation, thus suggesting that Nox2 activation couples with p38MAPK activation. Since Rac1, is an integral member of the Nox2 holoenzyme, we also assessed the effects of Rac1 inhibitors (EHT 1864, NSC23766 and Ehop-016) on HG-induced p38MAPK activation in isolated ß-cells. We report a significant inhibition of p38MAPK phosphorylation by Rac1 inhibitors, implying a regulatory role for Rac1 in promoting the Nox2-p38MAPK signaling axis in the ß-cell under the duress of HG. 2-Bromopalmitate, a known inhibitor of protein (Rac1) palmitoylation, significantly reduced HG-induced p38MAPK phosphorylation. However, GGTI-2147, a specific inhibitor of geranylgeranylation of Rac1, failed to exert any significant effects on HG-induced p38MAPK activation. In conclusion, we present the first evidence that the Rac1-Nox2 signaling module plays novel regulatory roles in HG-induced p38MAPK activation and loss in glucose-stimulated insulin secretion (GSIS) culminating in metabolic dysfunction and the onset of diabetes.

Pharmacokinetics of Rac inhibitor EHop-016 in mice by ultra-performance liquid chromatography tandem mass spectrometry.

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 × 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 (AUC0-∞) ranged from 328 to 1869 ng h/mL and 133-487 ng h/mL for IP and PO dosing, respectively. The elimination half-life (t1/2) 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 AUC0-∞values 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.

The Rac Inhibitor EHop-016 Inhibits Mammary Tumor Growth and Metastasis in a Nude Mouse Model.

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 µM. 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.

Pak and Rac GTPases promote oncogenic KIT-induced neoplasms.

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.

Development of EHop-016: a small molecule inhibitor of Rac.

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 ~1µM. At higher concentrations (10µM), 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.

Characterization of EHop-016, novel small molecule inhibitor of Rac GTPase.

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.

Novel inhibitors of Rac1 in metastatic breast cancer.

OBJECTIVE: Rho family GTPases are molecular switches that control signaling pathways regulating a myriad of cellular functions. Rac1, a Rho family member, plays a critical role in several aspects of tumorigenesis, cancer progression, invasion, and metastasis. Rac proteins are not mutated in most invasive human cancers but are found to be overactive or over-expressed. Since Rho GTPases are activated by guanine nucleotide exchange factors (GEFs), inhibition of the interaction of Rac with its GEFs is a targeted strategy for blocking Rac activation. METHODS: The IC50 of NSC23766, an inhibitor of the interaction of Rac1 with a subset of GEFs, is too high for therapeutic use and more efficacious inhibitors are desired. Therefore, we initiated the synthesis of new derivatives of NSC23766 with modifications of the substituents connected to the central pyrimidine ring, and tested their Rac1 inhibitory activity. RESULTS: Several of the NSC23766 derivatives were shown to inhibit Rac1 activity of cancer cells with higher efficiency (20-50% more) than NSC23766. The new compounds are not toxic to normal mammary epithelial cells and are more efficient (60-70%) than NSC23766 in inhibiting cell migration and reducing cell spreading and extension of lamellipodia, cell functions regulated by Rac that contribute to cancer invasion. CONCLUSION: Based on the results, we conclude that the novel compounds show promise of further development as small molecule inhibitors of invasive breast cancer progression.