Background: Rac is a central regulator of cancer cell migration/invasion and metastasis. Results: EHop-016 inhibits Rac activity with an IC 50 of 1 M. EHop-016 blocks Rac interaction with the Rac exchange factor Vav2, lamellipodia extension, and cell migration. Conclusion: EHop-016 is an effective Rac inhibitor. Significance: EHop-016 has potential as a metastasis therapeutic and for investigations of Rac-regulated cellular responses.

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 nM) 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 IC50s of 103 nM and 78 nM 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 signal transducer and activator of transcription (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 h) in MBQ-167 decreases metastatic cancer cell viability with a GI50 of ~130 nM, without affecting non-cancer 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 ~90%. In conclusion, MBQ-167 is 10X more potent than other currently available Rac/Cdc42 inhibitors, and has potential to be developed as an anticancer drug, as well as a dual inhibitory probe for the study of Rac and Cdc42.

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.

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 × 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 (R2> 0.995) over the range of 5 – 1000 ng/mL (1/x2 weighting). Pharmacokinetic parameters were obtained by non-compartmental analysis using WinNonlin®. The area under the curve (AUC0-∞) ranged from 328 – 1869 ng·hr/mL and 133 – 487 ng·hr/mL for IP and PO dosing respectively. The elimination half-life (t1/2) ranged from 3.8 – 5.7 hours and 3.4 – 26.8 hours 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% - 40%. These results support further preclinical evaluation of EHop-016 as a new anti-cancer therapy.

Most people develop acute hepatitis B virus (HBV)-related hepatitis that is controlled by both humoral and cellular immune responses following acute infection. However, a number of individuals in HBV-endemic areas fail to resolve the infection and consequently become chronic carriers. While a vaccine is available and new antiviral drugs are being developed, elimination of persistently infected cells is still a major issue. Standard treatment in HBV infection includes IFN-α, nucleoside, or nucleotide analogs, which has direct antiviral activity and immune modulatory capacities. However, immunological control of the virus is often not durable. A robust T-cell response is associated with control of HBV infection and liver damage; however, HBV-specific T cells are deleted, dysfunctional, or become exhausted in chronic hepatitis patients. As a result, efforts to restore virus-specific T-cell immunity in chronic HBV patients using antiviral therapy, immunomodulatory cytokines, or therapeutic vaccination have had little success. Adoptive cell transfer of T cells with specificity for HBV antigen+ cells represents an approach aiming to ultimately eliminate residual hepatocytes carrying HBV covalently closed circular DNA (cccDNA). Here, we discuss recent findings describing HBV immunopathology, model systems, and current therapies.