Project Summary – Project 3 HSP70 is a key cancer-critical survival protein that is necessary to maintain proper protein folding in a stressed cell. The HSP70 family contains more than eight family members. The one under study here is the major stress- induced family member HSPA1A, hereafter HSP70. Unlike other family members, this protein is overexpressed in over 75% of metastatic melanoma, and is associated with drug resistance and poor survival. In the past ten years we have developed a series of inhibitors that target the stress-induced form of HSP70 but not other family members like Hsc70 and GRP75. Additionally, we recently noted a significant fraction of HSP70 located at the mitochondria of tumor but not normal cells, and we coupled our inhibitors to a triphenylphosphonium moiety that helps direct these compounds to mitochondria. In the past funding cycle we tested this compound against melanoma, and solved the crystal structure and mechanism of action. In the current funding cycle, we take newer and more potent derivatives of our novel mitochondria-directed HSP70 inhibitors to the toughest-to-treat subtypes of melanoma: those that are resistant to BRAF/MEK inhibitors, those that have wild type BRAF and NRAS (WT/WT), and those that have metastasized to the brain (melanoma brain metastases, or MBMs). Targeting these three categories has the potential for significant clinical impact, and our preliminary data support the use of HSP70i for these tumor sub-types. In the proposed work we collaborate extensively with our P01 colleagues to explore for the first time the impact of HSP70i on the stressed tumor microenvironment, including cancer associated fibroblasts and the aged micro-environment. We also focus on several new HSP70 clients, including ErbB3, ID3 and GPX4. To succeed in these goals, we have accrued a team that is expert in HSP70 and proteostasis (Murphy and George), medicinal chemistry (Salvino) and the brain metastatic micro- environment (Chen). We expect the proposed research to yield candidate HSP70i for clinical development, and to provide an important therapeutic option for the toughest-to-treat melanoma sub-types.