Abstract 10516: Identification of a PCSK9-LDLR Disruptor Macrocycle with in vivo Function

Elevated LDL-C is a major risk factor for atherosclerotic cardiovascular disease, the leading cause of death worldwide. Proprotein convertase subtilisin/kexin type 9 (PCSK9) has pronounced effects on LDL-C levels via its modulation of hepatic LDL receptors (LDLR), the main pathway for cholesterol removal from the circulation. The epidermal growth factor precursor homology domain A (EGF-A) of the LDLR serves as a primary contact with PCSK9 via a flat interface, presenting a challenge for identifying small molecule PCSK9-LDLR disruptors. We employed an affinity-based screen of 10 13 in vitro -translated macrocyclic peptides to identify high affinity PCSK9 ligands that utilize a novel, induced-fit pocket and partially disrupt the PCSK9-LDLR interaction (Hit 1 - PCSK9-LDLR FRET, IC 50 2 nM, A max 41%). Structure-based design led to 13 PCSK9i , a molecule with enhanced in vitro function (PCSK9-LDLR FRET, IC 50 2 nM, A max 78%) and pharmacokinetic properties suitable for in vivo evaluation. To determine if 13 PCSK9i ’s functional activity in vitro would translate in vivo , C57BL/6 mice were dosed with vehicle or 13 PCSK9i via subcutaneous injection twice-daily for 3 days, and blood and liver were collected for the measurement of plasma total cholesterol (TC) and hepatic LDLR density. A PCSK9 antibody was included as a positive control. As illustrated in the figure, 13 PCSK9i increased hepatic LDLR density in a dose-dependent manner. Despite being 1/100 th its size, 13 PCSK9i (MW = 1.65 kDa) increased hepatic LDLR density on par with a PCSK9 antibody (MW = 144 kDa). Plasma TC levels were significantly reduced versus vehicle in all 13 PCSK9i dose groups, with similar reductions noted in the 13 PCSK9i 30 mg/kg and PCSK9 antibody groups (44% and 48%, respectively). In summary, 13 PCSK9i breaks new ground with its previously undescribed allosteric mechanism and, in turn, as the smallest molecule identified to date with in vivo PCSK9-LDLR disruptor function.