意大利巴里大学Ciro Leonardo Pierri等研究人员合作发现，计算机模拟与体外实验的结合方法识别与早发型Leigh综合征相关的肉碱O-乙酰转移酶（CRAT）p.Tyr110Cys变异活性的调节因子。2024年12月16日，《中国药理学报》在线发表了这项成果。

研究人员采用了结合计算机模拟与体外实验的综合方法，通过计算机对化学文库进行虚拟筛选，并进行后续的体外验证。该研究旨在识别小分子调节剂，其能够调控野生型（WT）肉碱O-乙酰转移酶（CRAT）和与早发型Leigh综合征相关的p.Tyr110Cys（Y110C）变异的活性。

通过3D分子建模、对接模拟和化学文库的虚拟筛选，研究人员根据化合物与WT-CRAT和p.Tyr110Cys-CRAT突变体3D模型的预测结合亲和力及相互作用来优先筛选潜在的CRAT调节剂。所进行的计算机分析通过对纯化的重组CRAT蛋白和来自对照成纤维细胞及CRAT缺乏症患者（该患者CRAT基因存在复合杂合错义突变p.Tyr110Cys和p.Val569Met）的细胞裂解液进行体外检测进行了验证。

通过应用筛选策略及上述标准，研究人员测试了三种市售批准药物（这些药物也已知可能与线粒体相互作用），即格列美脲、青蒿素和多佐胺，以及已知能与线粒体蛋白相互作用的苏拉明。结果表明，苏拉明（1−1000 μM）剂量依赖性地抑制了WT-CRAT和p.Tyr110Cys_CRAT变异体的活性。

青蒿素（0.1–200 μM）剂量依赖性地刺激了重组p.Tyr110Cys CRAT突变体的活性，而格列美脲和多佐胺未改变这些蛋白对乙酰辅酶A的活性。该研究证明了结合计算机模拟与体外实验的方法在识别调节CRAT酶活性的潜在化合物方面的有效性，为针对CRAT相关疾病的潜在治疗干预提供了宝贵的见解。

研究人员表示，CRAT是参与线粒体能量代谢的关键酶。CRAT活性的改变已成为Leigh综合征及相关线粒体疾病发病机制的重要因素。

附：英文原文

Title: 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

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

Issue&Volume: 2024-12-16

Abstract: 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 (11000μM) dose-dependently inhibited the activity of both WT-CRAT and p.Tyr110Cys_CRAT variant. Artemisinin (0.1–200 μM) 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 therapeutic interventions targeting CRAT-related disorders.

DOI: 10.1038/s41401-024-01435-0

Source: https://www.nature.com/articles/s41401-024-01435-0