Abstract 226: Mechanism of LCAT Activation by Compound A

Background: Lecithin:cholesterol acyltransferase (LCAT) catalyzes cholesteryl ester (CE) production from free cholesterol (FC) and phosphatidylcholine (lecithin), promoting HDL formation. Objective: To investigate activation of LCAT by Compound A (Amgen), a previously described small-molecule activator of LCAT, with the ultimate goal of developing novel LCAT activators for therapeutic use. Methods: LCAT activity in plasma from Familial LCAT Deficiency (FLD) patients with different mutations was quantitated by TLC before and after addition of Compound A. HEK293 cells were transiently transfected with plasmids containing wild-type (WT) or mutant LCAT cDNA. Media was then incubated with either vehicle or Compound A and LCAT activity was quantitated using a novel plate assay utilizing Methylumbelliferyl Palmitate as a substrate. Results: Compound A increased LCAT activity for a subset of FLD mutations to a level above which renal disease may occur. Mutations of Cys31 in vitro strongly affected basal LCAT activity as well as activation by Compound A. Charged residues at position 31 profoundly decreased activity whereas bulky hydrophobic groups increased LCAT activity up to 3-fold (p < 0.005, all). Mass spectrometry of WT LCAT incubated with Compound A revealed a +103.017 m/z adduct to the tryptic peptide containing Cys31, indicative of a cyanopyrazine adduct to LCAT Cys31. Molecular modeling identified potential binding sites of Compound A to LCAT. Conclusions: Our findings yield important mechanistic insight into LCAT activation that can be used to design novel LCAT activators for therapeutic use.