PROJECT SUMMARY/ABSTRACT Serum low-density lipoprotein (LDL) causes atherosclerotic heart disease. As the LDL receptor (LDLR) on the liver clears LDL from the blood, upregulating hepatic LDLR reduces both LDL and cardiovascular events. The self-cleaving protease PCSK9 (proprotein convertase subtilisin/kexin type 9) is a validated therapeutic target; it chaperones the LDLR for lysosomal degradation, downregulating its function. Antibodies against PCSK9 lower LDL and improve clinical outcomes, but cost and administration requirements illustrate a need for alternatives. Liver-targeted siRNA also robustly lowers LDL, but unlike the well-tolerated genetic variants, it removes all PCSK9 from the cell, raising safety concerns. Though the mechanistic basis for PCSK9's effect on the hepatic LDLR is well understand, its functions outside this canonical pathway are less clear. Therefore, the overall goal of our proposal is to develop biochemical tools to mechanistically dissect the biology of PCSK9, so as to 1) anticipate the long-term effects of current anti-PCSK9 therapies and 2) develop the proofs-of-principle for novel PCSK9-targeting strategies. To this end, we have identified three promising lead compounds that specifically target intracellular PCSK9. These compounds both inhibit PCSK9 self-proteolysis and also upregulate LDLR expression. In Aim 1, we will chemically optimize these compounds, confirm the mechanistic basis of their effects, and validate their function on PCSK9 and the LDLR in an in vivo model. This will establish novel chemical probes which can be used to study the poorly known function of intracellular PCSK9, as well as serve as the starting point for an alternative small molecule therapeutic to upregulate the LDLR. We have also found that additional factors, including heparan sulfate proteoglycans (HSPGs), interact with PCSK9 to modulate its trafficking to the lysosome, suggesting that there are additional drug targets in this pathway. In Aim 2, we will evaluate the contribution of HSPGs as mediators of the PCSK9 endocytic trafficking required for LDLR downregulation, both in vitro and in the human. These results will elucidate whether heparins, which are already approved therapies, could be repurposed to inhibit PCSK9 function. In Aim 3, we will identify and validate as yet unknown regulators of PCSK9 endocytic trafficking via an unbiased, genome-wide CRISPR interference screen. These results will help mechanistically define how PCSK9 is trafficked to the lysosome, provide answers to why PCSK9 only affects certain tissues, and offer potential novel therapeutic targets which would be anticipated to have similar therapeutic windows to PCSK9 itself. Overall, we expect our study to provide key information on the safety of long-term PCSK9 inhibition, lay the groundwork for novel treatments, and identify new therapeutic targets against atherosclerosis.