Elevated plasma levels of triglyceride-rich ApoBCLs (ApoB-Containing Lipoproteins) constitute a major component of the residual risk for coronary heart disease (CHD) in patients who have been treated with statins and/or PCSK9 inhibitors. To mitigate the ApoBCL risk component, we need a deeper understanding of the machinery that controls the production of ApoBCLs. This machinery is comprised of two membrane proteins, Scap and Sterol Regulatory Element-Binding Proteins (SREBPs). Scap is a protein embedded in the endoplasmic reticulum (ER) membrane through eight transmembrane helices. Scap forms complexes with SREBPs, which are also bound to the ER membrane through two transmembrane helices. SREBPs contain transcription factor domains that control the synthesis of fatty acids, triglycerides, and cholesterol, which form the lipid component of ApoBCLs. Activation of SREBPs requires their transport by Scap to the Golgi where two proteases release their transcription factor domain that can now enter the nucleus for target gene activation. When cholesterol in the ER rises and binds to Scap, it traps SREBPs in the ER, thus preventing proteolytic cleavage and nuclear entry. A major hurdle in understanding Scap’s switch-like molecular mechanism and how it controls SREBPs and ApoBCL production is the lack of a soluble cholesterol-mimetic compound that specifically binds and inhibits Scap. This proposal is based on a recent breakthrough in our laboratory involving the development of a novel high- throughput and rapid screening protocol, which has identified the first small molecule that binds specifically to Scap’s cholesterol-binding site and blocks activation of SREBPs. Scap contains two large loops (Loop1 and Loop7) that extend into the lumen of the ER bind each other when cholesterol in the ER is low. When ER cholesterol rises, it binds to Loop1, causing Loop1 to dissociate from Loop7, trapping the Scap/SREBP complex in the ER and blocking the transcriptional activation of all SREBP target genes. In Aim 1, we outline studies to improve the potency of our recently discovered cholesterol-mimetic Scap inhibitor and will use these inhibitors to understand how Loop1 dissociates from Loop7. The inhibitors will also be used as stabilizing agents to enable structural determination of Scap, which will elucidate the cholesterol binding mechanism at an atomic level. In Aim 2, we outline our approach to optimizing the in vivo pharmacokinetic properties of our various Scap inhibitors, after which we will explore their effects in inhibiting Scap and SREBP target genes in livers of mice under different metabolic conditions. Our previous studies involving genetically altered mice revealed that inhibition of Scap in the liver blocks SREBPs and markedly reduces synthesis of fatty acids, triglycerides, and cholesterol, dramatically lowering ApoBCL production in these models and also in wild-type mice fed a high fat diet and in hamsters fed a high carbohydrate diet. If thes...