Mazzei Luca, Ranieri Sofia, Silvestri Davide, Greene-Cramer Rebecca, Cioffi Christopher, Montelione Gaetano T, Ciurli Stefano
Laboratory of Bioinorganic Chemistry, Department of Pharmacy and Biotechnology, University of Bologna, 40127, Bologna, Italy.
Center for Biotechnology and Interdisciplinary Sciences, Rensselaer Polytechnic Institute, Troy, NY, 12180, USA.
Sci Rep. 2024 Dec 31;14(1):32175. doi: 10.1038/s41598-024-81990-y.
This manuscript details the application of Isothermal Titration Calorimetry (ITC) to characterize the kinetics of 3CL, the main protease from the Severe Acute Respiratory Syndrome CoronaVirus-2 (SARS-CoV-2), and its inhibition by Ensitrelvir, a known non-covalent inhibitor. 3CL is essential for producing the proteins necessary for viral infection, which led to the COVID-19 pandemic. The ITC-based assay provided rapid and reliable measurements of 3CL activity, allowing for the direct derivation of the kinetic enzymatic constants K and k by monitoring the thermal power required to maintain a constant temperature as the substrate is consumed. The manuscript highlights several advantages of the proposed ITC-based assay over traditional methods used to study 3CL, such as Förster Resonance Energy Transfer (FRET) and Liquid Chromatography-Mass Spectrometry (LC-MS) and overcomes the need for non-biological substrates or discontinuous post-reaction steps. The ease of application of the ITC method allowed for the determination of the temperature dependence of the catalytic constants, enabling the estimation of the reaction activation energy. Additionally, the assay was used to determine the inhibition mode and kinetic parameters for 3CL inhibition by Ensitrelvir. This molecule was revealed to act as a slow- and tight-binding inhibitor that forms an initial E•I complex (K = 9.9 ± 0.7 nM) quickly transitioning to a tighter E•I* assembly (K* = 1.1 ± 0.2 nM). This versatile calorimetric method is proposed for general use in the discovery and development of drugs targeting 3CL.
本手稿详细介绍了等温滴定量热法（ITC）在表征严重急性呼吸综合征冠状病毒2（SARS-CoV-2）的主要蛋白酶3CL的动力学及其被已知非共价抑制剂恩昔瑞韦抑制方面的应用。3CL对于产生病毒感染所需的蛋白质至关重要，正是这种蛋白质导致了COVID-19大流行。基于ITC的检测方法能够快速、可靠地测量3CL的活性，通过监测底物消耗过程中维持恒定温度所需的热功率，可以直接推导动力学酶常数K和k。该手稿强调了所提出的基于ITC的检测方法相对于用于研究3CL的传统方法（如荧光共振能量转移（FRET）和液相色谱-质谱联用（LC-MS））的几个优点，并克服了对非生物底物或不连续反应后步骤的需求。ITC方法的易于应用使得能够确定催化常数的温度依赖性，从而能够估算反应活化能。此外，该检测方法还用于确定恩昔瑞韦对3CL抑制的抑制模式和动力学参数。结果表明，该分子作为一种慢结合和紧密结合抑制剂，首先快速形成初始的E•I复合物（K = 9.9 ± 0.7 nM），然后迅速转变为更紧密的E•I组装体（K = 1.1 ± 0.2 nM）。本文提出这种通用的量热方法可普遍用于靶向3CL的药物发现和开发。
