However, the high effective concentrations (IC50 > 75 μM) of NSC23766 limit its use as a therapeutic agent [36]. Other known Rac inhibitors also have IC50s of 10 to 50 μM [44] and [45]; including the recently published Rac inhibitors

AZA1, ZINC69391, and IA-116 [46] and [47]. At concentrations ranging from 5 to 20 μM, AZA1 acted as a dual inhibitor for Rac and Cdc42, and blocked prostate cancer cell proliferation, cell migration, and reduced Cyclin D1, and PAK and Akt activities [46]. Another compound ZINC69361, which inhibited Rac activity with an IC50 of 61 μM and reduced lung metastasis, was used as a lead to derive IA-116, which was selective for Rac and inhibited the FRAX597 ic50 interaction between Rac and the Rac GEF p-Rex1; albeit, at μM effective concentrations [47]. Recent studies have also shown the utility of the NSC23766 derivative

AZA197, which was identified as a selective inhibitor for the closely related Rac homolog Cdc42. AZA197, at 1 to 10 μM, inhibited the Cdc42 GEF Dbs activity, PAK and ERK activities, and reduced Cyclin D levels, colon cancer cell proliferation, AC220 and cancer progression in a mouse model [48]. The potency of this inhibitor is similar to that of ML141 (CID2950007), another Cdc42 selective inhibitor with an IC50 ~ 3 to 5 μM [49], that was shown to inhibit melanoma cell migration [50]. These data demonstrate the utility of developing chemical probes to target both Rac and Cdc42 in malignant cancer. To improve the efficacy of NSC23766 and its derivatives, we developed a panel of related compounds [51], and identified EHop-016 as a Rac inhibitor that is 100 times more potent than NSC23766, and binds to the effector domain of Rac1 with a tighter interaction [52]. To our knowledge, EHop-016 is one of the most potent Rac inhibitors that has been published, and is an effective

tool for probing Rac function in cell and mouse models; as has been shown by us and others, in studies using breast cancer cell lines, leukemia, melanoma, and T lymphocytes [50], [52], [53] and [54]. We reported that EHop-016 inhibits the Rac activity of metastatic cancer cells with an IC50 of 1 μM by blocking the specific interaction of Rac with the Rac GEF and oncogene Vav. EHop-016 Adenosine also inhibits the activity of the Rac downstream effector PAK, lamellipodia extension, and cell migration in metastatic cancer cells at concentrations less than 10 μM, while concentrations ≥ 10 μM inhibits the activity of the close Rac homolog Cdc42, and cell viability [52] and [53]. The aim of this study was to test the In Vivo effects of EHop-016 in cancer progression. We used metastatic cancer cell lines and a mouse model of experimental metastasis to demonstrate the efficacy of EHop-016 at reducing mammary fat pad tumor growth, metastasis, and angiogenesis.