Discovery of novel brain permeable human ACSS2 inhibitors for blocking breast cancer brain metastatic growth

Abstract Breast-cancer brain metastasis (BCBM) poses a significant clinical challenge, resulting in an end-stage diagnosis and hindered by limited therapeutic options. The blood-brain barrier (BBB) acts as an anatomical and physiological hurdle for therapeutic compounds, restricting the effective delivery of therapies to the brain. In order to grow and survive in a nutrient-poor environment, tumors in the brain must adapt to their metabolic needs, becoming highly dependent on acetate. These tumors rely on the conversion of acetate to acetyl-CoA by the enzyme Acetyl-CoA synthetase 2 (ACSS2), a key metabolic enzyme involved in regulating fatty acid synthesis and protein acetylation in tumor cells. ACSS2 has emerged as a crucial enzyme required for the growth of tumors in the brain. Here, we utilized a computational pipeline, combining pharmacophore-based shape screen methodology with ADME property predictions to identify novel brain-permeable ACSS2 inhibitors. From a small molecule library, this approach identified 30 potential ACSS2 binders, from which two candidates, AD-5584 and AD-8007, were validated for their binding affinity, predicted metabolic stability, and, notably, their ability to traverse the BBB. We show that treatment of BCBM cells, MDA-MB-231BR, with AD-5584 and AD-8007 leads to a significant reduction in lipid storage, reduction in colony formation, and increase in cell death in vitro . Utilizing an ex vivo orthotopic brain-slice tumor model, we show that treatment with AD-8007 and AD-5584 significantly reduces tumor size and synergizes with radiation in blocking BCBM tumor growth ex vivo. Importantly, we show that following intraperitoneal injections with AD-5584 and AD-8007, we can detect these compounds in the brain, confirming their BBB permeability. Thus, we have identified and validated novel ACSS2 inhibitor candidates for further drug development and optimization as agents for treating patients with breast cancer brain metastasis.