Project 3 - Ronen Marmorstein The RAS-RAF-MEK-ERK (MARK) and PI3K-AKT signaling pathways are constitutively activated through multiple mechanisms in melanoma implicating the kinases in these pathways as important therapeutic targets. In particular, the BRAF kinase, part of the MARK pathway, plays an important role in melanoma as BRAF somatic mutations have been found in about two-thirds of malignant melanomas with the majority of these mutations resulting in elevated kinase activity from a V600E substitution in the kinase domain (BRAFV600E). In addition, members of our program project have found that 57% of melanomas have highly amplified levels of wild-type BRAF. Within the PI3K/AKT signaling pathway, the AKT kinase, which is activated by PISKa, is constitutively activated in most metastatic melanomas. Taken together, BRAF, BRAFV600E and PISKa are important oncoproteins associated with melanoma and are therefore attractive targets for inhibition for melanoma treatment. Although, small molecule inhibitors for both BRAF and PISKa have been developed, they suffer from a lack of suitable potency and specificity. The overall goal of project 3 is to prepare improved BRAF-, BRAFV600E- and PISKa - specific inhibitors that can be used to specifically interrogate the RAS-RAF-MEK-ERK (MAPK) and PI3K-AKT signaling pathways in melanoma and that can be further developed into drugs for the treatment of melanoma. For our studies we will combine biochemical, chemical, structural and computational methodologies towards the structure-based design of kinase-type specific inhibitors with the following Specific Aims: (1) Develop potent and specific BRAF and BRAFV600E inhibitors, and (2) Develop potent and specific PISKag inhibitors. We have generated significant preliminary data supporting the feasibility of completing each of our aims and our studies will be highly synergistic with each of the other projects of the program to synthesize and screen for novel organometallic BRAF, BRAFV600E and PISKa inhibitors (Project 4), and to test these inhibitors using complex in vitro and orthotopic in vivo (Project 1) and immunological (Project 2) models. These studies will also employ Core C to test inhibitors on melanoma cells for quality control and for screening against a panel of cell lines. Together with the other projects of the program, our studies will contribute to the mechanistic understanding of the roles played by the RAS-RAF-MEK-ERK (MAPK) and PI3K-AKT signaling pathways in melanoma and to the development of novel strategies for the treatment of melanoma.