High-Throughput Screening for Small-Molecule Inhibitors of the Ga12/13/RhoA Signaling Pathway.

Abstract

Cancer metastasis is a major health problem that involves movement of tumor cells from their primary tissue site to a secondary tissue site. Cellular functions involved in metastasis include cell proliferation, adhesion, migration,invasion, survival, and angiogenesis. The Galpha12/13–RhoA-Serum Response Factor (SRF) transcriptional signaling pathway is involved in metastasis for a variety of malignancies. Serum and lysophosphatidic acid receptor activation can stimulate activation of this pathway leading to gene expression. Gene expression is required for various steps of metastasis. Guanosine nucleotide exchange factor (GEF) activation of RhoA is also important for metastasis. RhoA activation is known for its cytosekeletal effects, which are required for cell motility. Within this pathway, Rho is activated by the regulators of G-protein signaling homology domain (RH) family of GEFs. I have focused on leukemia-associated RhoGEF (LARG) due to its identification in leukemia and link to cancer. As a result, inhibitors of this pathway should serve as useful pharmacological tools and potential therapeutics. Therefore, I undertook a cell-based high-throughput screen (HTS) using a modified Rho-specific serum response element (SRE)-luciferase reporter to identify novel inhibitors of this pathway. In a separate project, I undertook a molecular HTS using a fluorescence polarization guanosine nucleotide RhoA binding assay to identify inhibitors of the interaction between LARG and RhoA. In the cell-based screen, I identified two inhibitors (IC50s - ~1-2 uM), CCG-977 and CCG-1423, whereas in the molecular screen I identified five inhibitors (IC50s - ~ 4-40 uM). In follow-up studies, I determined CCG-1423 to modulate some aspect of megakaryoblastic leukemia 1 (MKL1)/SRF-mediated transcription. Also, CCG-1423 inhibited LPA-mediated DNA synthesis, cell growth, survival, and invasion. Structure-activity relationship studies for CCG-1423 yielded promising analogs that were less potent, but more selective and less toxic. CCG-1423 regulation of global gene expression identified 4 metastasis-related genes (RGS4, RGS7, CTGF, and SOX9) that may be involved in prostate cancer metastasis. Future mechanistic studies will be pursued for compounds identified here. Also, the functional role of the CCG-1423-regulated genes in cancer biology will be investigated. Overall, the compounds identified here should serve as useful starting points for the development of novel Rho pathway inhibitors.