Medical Research Council Mitochondrial Biology Unit, University of Cambridge, Keith Peters Building, Cambridge Biomedical Campus, Hills Road, Cambridge, CB2 0XY, United Kingdom.
Medical Research Council Mitochondrial Biology Unit, University of Cambridge, Keith Peters Building, Cambridge Biomedical Campus, Hills Road, Cambridge, CB2 0XY, United Kingdom.
Mol Metab. 2022 Jun;60:101469. doi: 10.1016/j.molmet.2022.101469. Epub 2022 Mar 10.
The mitochondrial pyruvate carrier (MPC) has emerged as a promising drug target for metabolic disorders, including non-alcoholic steatohepatitis and diabetes, metabolically dependent cancers and neurodegenerative diseases. A range of structurally diverse small molecule inhibitors have been proposed, but the nature of their interaction with MPC is not understood, and the composition of the functional human MPC is still debated. The goal of this study was to characterise the human MPC protein in vitro, to understand the chemical features that determine binding of structurally diverse inhibitors and to develop novel higher affinity ones.
We recombinantly expressed and purified human MPC hetero-complexes and studied their composition, transport and inhibitor binding properties by establishing in vitro transport assays, high throughput thermostability shift assays and pharmacophore modeling.
We determined that the functional unit of human MPC is a hetero-dimer. We compared all different classes of MPC inhibitors to find that three closely arranged hydrogen bond acceptors followed by an aromatic ring are shared characteristics of all inhibitors and represent the minimal requirement for high potency. We also demonstrated that high affinity binding is not attributed to covalent bond formation with MPC cysteines, as previously proposed. Following the basic pharmacophore properties, we identified 14 new inhibitors of MPC, one outperforming compound UK5099 by tenfold. Two are the commonly prescribed drugs entacapone and nitrofurantoin, suggesting an off-target mechanism associated with their adverse effects.
This work defines the composition of human MPC and the essential MPC inhibitor characteristics. In combination with the functional assays we describe, this new understanding will accelerate the development of clinically relevant MPC modulators.
线粒体丙酮酸载体 (MPC) 已成为代谢紊乱（包括非酒精性脂肪性肝炎和糖尿病、代谢依赖性癌症和神经退行性疾病）的有前途的药物靶点。已经提出了一系列结构多样的小分子抑制剂，但它们与 MPC 的相互作用方式尚不清楚，功能性人 MPC 的组成仍存在争议。本研究的目的是在体外表征人 MPC 蛋白，了解决定结构多样抑制剂结合的化学特征，并开发新型高亲和力抑制剂。
我们通过建立体外转运测定、高通量热稳定性偏移测定和药效团建模，重组表达和纯化了人 MPC 异源复合物，并研究了它们的组成、转运和抑制剂结合特性。
我们确定人 MPC 的功能单位是异源二聚体。我们比较了所有不同类别的 MPC 抑制剂，发现三个紧密排列的氢键受体后面是一个芳香环，这是所有抑制剂的共同特征，也是高活性的最低要求。我们还证明了高亲和力结合不是归因于先前提出的与 MPC 半胱氨酸形成共价键。根据基本药效团特性，我们鉴定出 14 种新型 MPC 抑制剂，其中一种比 UK5099 强十倍。两种是常用的药物恩他卡朋和呋喃妥因，这表明它们的不良反应与非靶向机制有关。
这项工作定义了人 MPC 的组成和必需的 MPC 抑制剂特征。结合我们描述的功能测定，这一新认识将加速具有临床相关性的 MPC 调节剂的开发。
