Combined in silico/in vitro approaches for identifying modulators of the activity of the p.Tyr110Cys Carnitine O-Acetyltransferase(CRAT)variant associated to an early onset case of Leigh syndrome  

作　　者：Lucas Cafferati Beltrame Maria Noemi Sgobba Luna Laera Valeria Scaglione Sabino Todisco Serena Barile Anna Lucia Francavilla Danila Imperia De Luca Michele Montaruli Vito Porcelli Lorenzo Guerra Anna De Grassi Mariateresa Volpicella Ciro Leonardo Pierri 

机构地区：[1]Department of Biosciences,Biotechnologies and Environment,University of Bari“Aldo Moro”,70125,Bari,Italy [2]Laboratory of Biochemistry,Structural and Molecular Biology,Department of Pharmacy–Pharmaceutical Sciences,University of Bari“Aldo Moro”,70125,Bari,Italy

出　　处：《Acta Pharmacologica Sinica》2025年第4期1123-1136,共14页中国药理学报(英文版)

基　　金：supported by EU funding within the MUR PNRR Extended Partnership initiative on Emerging Infectious Diseases(Project No.PE00000007,INF‐ACT);MUR PNRR National Center for Gene Therapy and Drugs based on RNA Technology(Project No.CN_00000041);The authors would also like to thank for the IT resources made available by ReCaS(https://www.recas-bari.it/index.php/en/),a project funded by the MIUR(Italian Ministry for Education,University and Research)in the“PON Ricerca e Competitivita 2007-2013‐Azione I-Interventi di rafforzamento strutturale”PONa3_00052,Avviso 254/Ric,University of Bari.

摘　　要：Carnitine O-acetyltransferase(CRAT)is a crucial enzyme involved in mitochondrial energy metabolism.Alterations in CRAT activity have emerged as significant contributors to the pathogenesis of Leigh syndrome and related mitochondrial disorders.In this study we employed an integrated approach combining in silico docking analysis and virtual screening of chemical libraries with subsequent in vitro validation to identify small molecule modulators of the activity of the wild type(WT)CRAT and the p.Tyr110Cys(Y110C)variant associated to an early onset case of Leigh syndrome.Through 3D molecular modeling,docking simulations,and virtual screening of chemical libraries,potential CRAT modulators were prioritized based on their predicted binding affinities and interactions with the 3D models of the WT-CRAT and of the p.Tyr110Cys-CRAT mutant.The performed in silico analyses were validated through in vitro assays on the purified recombinant CRAT proteins and cell-lysates from control fibroblasts and the fibroblasts of a patient with genetic diagnosis of CRAT-deficiency,carrying the compound heterozygous missense variants in the CRAT gene,namely p.Tyr110Cys and p.Val569Met.Based on the above screening by applying the indicated filtering strategy and mentioned criteria,3 commercially available approved drugs(also known for their possible interactions with mitochondria)namely glimepiride,artemisinin and dorzolamide,as well as suramin(already known for its ability to interact with mitochondrial proteins)were tested in in vitro assays.We found that suramin(1−1000μM)dose-dependently inhibited the activity of both WT-CRAT and p.Tyr110Cys_CRAT variant.Artemisinin(0.1–200μM)dose-dependently stimulated the activity of the recombinant p.Tyr110Cys CRAT mutant,whereas glimepiride and dorzolamide did not change the activity of these proteins towards acetyl-CoA.This study demonstrates the effectiveness of this combined approach in identifying novel compounds for modulating CRAT enzyme activity,providing valuable insights for potential

关 键 词：carnitine acetyltransferase Leigh-like syndrome mitochondrial encephalomyopathy 3D modeling and virtual screening of chemical libraries SURAMIN ARTEMISININ 

分 类 号：R73[医药卫生—肿瘤]