Developing new inhibitors of site-one protease to prevent and treat cancers

About This Grant

Project Summary: Increased lipid production is a hallmark of cancer cells, which must support rapid cell growth, maintain membrane homeostasis, and induce lipid-dependent signaling pathways. Targeting lipid production is emerging as a vulnerability for cancers. For example, clear cell renal cell carcinoma (ccRCC) stimulates lipid overproduction downstream of oncogenic HIF-2a signaling; and hepatic cell carcinoma (HCC), is major risk factor for Metabolic Dysfunction-Associated Steatotic Liver Disease (MASLD). Lipogenesis is globally regulated by the sterol regulatory element binding proteins (SREBPs). SREBPs are produced as membrane anchored transcription factors that require proteolytic liberation to traffic to the nucleus and increase lipogenic transcription. Site-one protease (S1P) is the serine protease necessary for the activation of SREBPs and presents a target for anti-cancer therapies. While a small molecule inhibitor of S1P termed PF-429242 has been previously developed, it was not suitable for clinical application. Additionally, development of S1P inhibitors has been stymied by the low activity and poor behavior of this enzyme when it is purified. Recent work by our group and others revealed that S1P is activated by a co-factor, SREBP regulating gene (SPRING). Our group established that S1P could be purified in its active form through its interaction with SPRING, with which it forms a stable complex. We discovered that SPRING enables S1P activity by displacing the inhibitory pro-domain of S1P. Building on this, we determined the structural basis for the inhibition of S1P by PF-429242 and used this information to elucidate how S1P recognizes substrate peptides. However, critical questions about the scope of SPRING activities remain, and whether it is required for all S1P-related functions is controversial. Answering these questions requires the development of tool molecules specific for the S1P- SPRING complex. Here, we propose to leverage our biochemical, structural, and cellular expertise to identify small drug-like molecules that inhibit the active form of S1P when it is in complex with SPRING. We will use an unbiased screen of a large chemical library and counter-screen with orthogonal high-throughput biochemical and cell-based assays to select molecules that are highly selective for S1P/SPRING and efficacious at inhibiting S1P- dependent cancer cell growth, proliferation, and migration. By utilizing a mutant S1P we developed that is resistant to PF-429242, we will prioritize molecules with new mode-of-action. In Aim 1, we will implement a primary assay that measures S1P kinetics and test the 350,000 compounds in our chemical library. In Aim 2, we will efficiently and effectively prioritize compounds to identify hits with specific, on-target activity. In Aim 3, we will use cell-based activity assays and mode-of-action studies to prioritize top hits and develop lead compounds that are potent and specific inhibitors of S1P/SPRING activity in cancer cell lines.