Lecithin:Cholesterol Acyltransferase Activation by Sulfhydryl-Reactive Small Molecules: Role of Cysteine-31

Abstract

Lecithin:cholesterol acyltransferase (LCAT) catalyzes plasma cholesteryl ester formation and is defective in familial lecithin:cholesterol acyltransferase deficiency (FLD), an autosomal recessive disorder characterized by low high-density lipoprotein, anemia, and renal disease. This study aimed to investigate the mechanism by which compound A [3-(5-(ethylthio)-1,3,4-thiadiazol-2-ylthio)pyrazine-2-carbonitrile], a small heterocyclic amine, activates LCAT. The effect of compound A on LCAT was tested in human plasma and with recombinant LCAT. Mass spectrometry and nuclear magnetic resonance were used to determine compound A adduct formation with LCAT. Molecular modeling was performed to gain insight into the effects of compound A on LCAT structure and activity. Compound A increased LCAT activity in a subset (three of nine) of LCAT mutations to levels comparable to FLD heterozygotes. The site-directed mutation LCAT-Cys31Gly prevented activation by compound A. Substitution of Cys31 with charged residues (Glu, Arg, and Lys) decreased LCAT activity, whereas bulky hydrophobic groups (Trp, Leu, Phe, and Met) increased activity up to 3-fold ( < 0.005). Mass spectrometry of a tryptic digestion of LCAT incubated with compound A revealed a +103.017 adduct on Cys31, consistent with the addition of a single hydrophobic cyanopyrazine ring. Molecular modeling identified potential interactions of compound A near Cys31 and structural changes correlating with enhanced activity. Functional groups important for LCAT activation by compound A were identified by testing compound A derivatives. Finally, sulfhydryl-reactive-lactams were developed as a new class of LCAT activators. In conclusion, compound A activates LCAT, including some FLD mutations, by forming a hydrophobic adduct with Cys31, thus providing a mechanistic rationale for the design of future LCAT activators.

Interaction of lecithin:cholesterol acyltransferase with lipid surfaces and apolipoprotein A-I-derived peptides

“…Thus, the atomistic free-energy calculations and CG simulations showed that the diffusion of lipids, especially in the case of UC molecules, to the active site of LCAT is not directly assisted by apoA-I. Interestingly, a recent research study revealed that molecular agents targeted for the lipid binding site of LCAT can increase the V max of LCAT ( 66 ). Surprisingly, the compound also activated LCAT deficiency causing mutants, which raises hopes for treating these disorders in the future.…”

Interaction of lecithin-cholesterol acyltransferase with lipid surfaces and apolipoprotein A-I derived peptides: implications for the cofactor mechanism of apolipoprotein A-I

“…Thus, the atomistic free-energy calculations and CG-simulations showed that the diffusion of lipids, especially in the case of CHOL molecules, to the active site of LCAT is not directly assisted by apoA-I. Interestingly, a recent research study revealed that molecular agents targeted for the lipid binding site of LCAT can increase the V max of LCAT (Freeman et al, 2017). Surprisingly, the compound also activated LCAT deficiency causing mutants which raises hopes for treating these disorders in the future.…”

Complete and Partial Lecithin:Cholesterol Acyltransferase Deficiency Is Differentially Associated With Atherosclerosis

“…These include both enzyme replacement therapy to treat FLD 10 but, more importantly, small molecules aimed at increasing LCAT activity to decrease risk of atherosclerosis. 11 In this light, we have tested the hypothesis that FLD and FED are two disease entities with different 10.1161/CIRCULATIONAHA.118.034706 associations with atherosclerosis. Our results highlight remarkable discrepancies between the two LCAT disease entities in a first head to head comparison.…”