Development of small molecule selective inhibitors of neuronal nitric oxide synthase (nNOS) for melanoma treatment - SUMMARY Cutaneous melanoma is the most aggressive form of skin malignancy. Despite the revolutionary developments of treatment using immunotherapy and targeted therapy, the 5-year survival rate of metastatic disease remains low (~32%). A significant proportion of melanoma patients do not respond to the existing therapies, and the treatment options become even more limited when patients develop resistance over time. Our interdisciplinary team has demonstrated that neuronal nitric oxide synthase (nNOS)-mediated nitric oxide (NO) signaling plays an essential role in melanoma progression and immune checkpoint PD-L1-mediated immunosuppression. Inhibition of nNOS showed great promise in inhibiting melanoma progression and tumor growth. Despite its clear potential as a therapeutic target, few successes have been achieved in developing selective nNOS inhibitors, given the high structural similarity among the three isoforms of the nitric oxide synthase enzymes. With two decades of continuous effort, we successfully elucidated the structural basis for a group of highly selective nNOS inhibitors; Using our approach, two compounds, HH044 and MAC-3-190, demonstrated potent anti-melanoma activity in vivo, both when used alone and in combination with immune checkpoint inhibitors. Our central hypothesis is that orally bioavailable small molecule inhibitors targeting nNOS represent a novel strategy for melanoma treatment, particularly in combination with immune checkpoint blockade (ICB). The proposed study aims to design and synthesize novel nNOS inhibitors with improved inhibition potency, nNOS selectivity, and pharmacological properties, and evaluate their therapeutic potential and underlying anti- melanoma mechanisms. In Aim 1, we will use a structure-based approach to develop more potent and selective nNOS inhibitors with improved oral bioavailability, followed by screening for anti-melanoma activities. In Aim 2, we will conduct mechanistic studies to determine the on-target effects of nNOS inhibitors and to understand the molecular mechanisms of nNOS inhibition in melanoma cells with distinct genomic mutation backgrounds. Aim 3 will explore the mechanisms and therapeutic utility of nNOS inhibitors as a sensitizing therapy to improve checkpoint blockade immunotherapy using a syngeneic, immunologically responsive mouse melanoma model and patient- derived melanoma organoids. Successful completion of the proposed study will develop structure-based, mechanism-driven, first-in-class orally bioavailable small molecules targeting nNOS for melanoma therapy alone or in combination with ICB.
