Description:
<jats:title>Abstract</jats:title>
<jats:p>Metastasis continues to be the primary cause of cancer-related death. Therefore, we focus on designing small molecules inhibitors to block metastasis. The Rho GTPase family members Rac1, and Cdc42 are critical regulators of cancer cell migration and invasion, and thus, metastasis. Rac1 is activated by Guanine Nucleotide Exchange Factors (GEFs), which promote the exchange of GDP to GTP. In most cancers, the homologous Rac1 and Cdc42 are hyperactivated due to the overexpression of GEFs. Therefore, we designed inhibitors to block the activation and interaction of Rac1 and Cdc42 with their GEFs. Our lead compound MBQ-167, is a dual inhibitor of Rac1 and Cdc42 with IC50s of 103 nM and 78 nM, respectively. MBQ-167 inhibited the viability and migration of metastatic cancer cells without affecting non-metastatic or non-cancer cells. Moreover, MBQ-167 reduced mammary tumor growth and metastasis of HER2 positive and triple-negative breast cancer cells in immunocompromised mice by ~ 90%. To further elucidate the mechanism of action of MBQ-167, we tested the hypothesis that MBQ-167 blocks the interaction of specific Rac1/Cdc42 GEFs by interaction with particular amino acid residues in the switch I and II homologous regions of Rac1 and Cdc42 proteins. Therefore, to achieve the objective of elucidating the interaction site of MBQ-167 on Rac1 and Cdc42, we used in silico analysis and Nuclear Magnetic Resonance (NMR) of MBQ-167 with Rac1 in a GDP bound state. These results were biochemically validated by pulldown assays using a G15A nucleotide-free mutant predicted to interact tightly with Rac1/Cdc42 GEFs. In silico, using Autodock Tools, MBQ-167 was docked to the GEF binding domain of Rac1 and Cdc42 with low binding energy, indicating a tight interaction. For the NMR structural analyses, Rac1 was isotopically labeled with 15N and titrated with several concentrations of MBQ-167, at ratios of 1:1, 1:2, 1:3, and 1:4. We were able to detect chemical shift perturbations in key residues in the switch I and II regions that interact with Rac1/Cdc42 GEFs and regulate the activity. Moreover, these perturbations were more intense with increasing amounts of MBQ-167, confirming the specificity of the interaction. Therefore, our data signify the direct interaction of MBQ-167 with Rac1, thus validating the role of MBQ-167 as a specific Rac1/Cdc42 inhibitor.</jats:p>
<jats:p>Citation Format: Julia I. Medina, Marvin Bayro, Eliud Hernandez, Cornelis Vlaar, Ricardo Gonzalez, Suranganie Dharmawardhane. Interaction site elucidation of the Rac1/Cdc42 inhibitor, MBQ-167 [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4018.</jats:p>