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 (P < 0.005). Mass spectrometry of a tryptic digestion of LCAT incubated with compound A revealed a +103.017 m/z 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 β-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.
|Number of pages||13|
|Journal||Journal of Pharmacology and Experimental Therapeutics|
|State||Published - Aug 1 2017|
Bibliographical noteFunding Information:
This research was supported [in part] by the Intramural Research Program of the National Institutes of Health [National Heart, Lung, and Blood Institute and National Center for Advancing Translational Sciences]. This research was also supported by the National Institutes of Health National Heart, Lung, and Blood Institute [Grants R01-HL071818 and R01-HL122416 (to J.J.G.T.) and Ruth L. Kirschstein National Research Service Award F32HL131288 (to K.A.M.)]. The authors thank Dr. Tim Hanusa for advice on the Hartree–Fock modeling studies and Dr. Alisa Glukhova for construction of the WT-LCAT-6-His pcDNA4 plasmid.
© 2017, American Society for Pharmacology and Experimental Therapy. All rights reserved.
ASJC Scopus subject areas
- Molecular Medicine