title: Distant homology modeling of LCAT and its validation through in silico targeting and in vitro and in vivo assays.
abstract: LCAT (lecithin:cholesterol acyltransferase) catalyzes the transacylation of a fatty acid of lecithin to cholesterol, generating a cholesteryl ester and lysolecithin. The knowledge of LCAT atomic structure and the identification of the amino acids relevant in controlling its structure and function are expected to be very helpful to understand the enzyme catalytic mechanism, as involved in HDL cholesterol metabolism. However - after an early report in the late '90 s - no recent advance has been made about LCAT three-dimensional structure. In this paper, we propose an LCAT atomistic model, built following the most up-to-date molecular modeling approaches, and exploiting newly solved crystallographic structures. LCAT shows the typical folding of the α/β hydrolase superfamily, and its topology is characterized by a combination of α-helices covering a central 7-strand β-sheet. LCAT presents a Ser/Asp/His catalytic triad with a peculiar geometry, which is shared with such other enzyme classes as lipases, proteases and esterases. Our proposed model was validated through different approaches. We evaluated the impact on LCAT structure of some point mutations close to the enzyme active site (Lys218Asn, Thr274Ala, Thr274Ile) and explained, at a molecular level, their phenotypic effects. Furthermore, we devised some LCAT modulators either designed through a de novo strategy or identified through a virtual high-throughput screening pipeline. The tested compounds were proven to be potent inhibitors of the enzyme activity.
authors.affiliation: Laboratorio di Biochimica e Biofisica Computazionale, Università degli Studi di Milano, Milano, Italia.
authors.forename: Cristina
authors.initials: C
authors.lastname: Sensi
authors.affiliation: Centro Enrica Grossi Paoletti, Dipartimento di Scienze Farmacologiche e Biomolecolari, Università degli Studi di Milano, Milano, Italia.
authors.forename: Sara
authors.initials: S
authors.lastname: Simonelli
authors.affiliation: Dipartimento di Farmacia, Università Degli Studi di Parma, Parma, Italia.
authors.forename: Ilaria
authors.initials: I
authors.lastname: Zanotti
authors.affiliation: Dipartimento di Scienze Veterinarie e Sanità Pubblica, Università degli Studi di Milano, Milano, Italia.
authors.forename: Gabriella
authors.initials: G
authors.lastname: Tedeschi
authors.affiliation: Dipartimento di Farmacia, Università Degli Studi di Parma, Parma, Italia.
authors.forename: Giulia
authors.initials: G
authors.lastname: Lusardi
authors.affiliation: Centro Enrica Grossi Paoletti, Dipartimento di Scienze Farmacologiche e Biomolecolari, Università degli Studi di Milano, Milano, Italia.
authors.forename: Guido
authors.initials: G
authors.lastname: Franceschini
authors.affiliation: Centro Enrica Grossi Paoletti, Dipartimento di Scienze Farmacologiche e Biomolecolari, Università degli Studi di Milano, Milano, Italia.
authors.forename: Laura
authors.initials: L
authors.lastname: Calabresi
authors.affiliation: Laboratorio di Biochimica e Biofisica Computazionale, Università degli Studi di Milano, Milano, Italia.
authors.forename: Ivano
authors.initials: I
authors.lastname: Eberini
mesh_terms: D000818:Animals; D001665:Binding Sites; D002384:Catalysis; D004789:Enzyme Activation; D006801:Humans; D008297:Male; D051379:Mice; D008958:Models, Molecular; D062105:Molecular Docking Simulation; D009154:Mutation; D007862:Phosphatidylcholine-Sterol O-Acyltransferase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011487:Protein Conformation; D013379:Substrate Specificity
chemical_list: D007862:Phosphatidylcholine-Sterol O-Acyltransferase
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