An isothermal calorimetry assay for determining steady state kinetic and Ensitrelvir inhibition parameters for...

This manuscript details the application of Isothermal Titration Calorimetry (ITC) to characterize the kinetics of 3CL^pro, 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^pro 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^pro activity, allowing for the direct derivation of the kinetic enzymatic constants K_M and k_cat 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^pro, 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^pro inhibition by Ensitrelvir. This molecule was revealed to act as a slow- and tight-binding inhibitor that forms an initial E•I complex (K_I = 9.9 ± 0.7 nM) quickly transitioning to a tighter E•I* assembly (K_I* = 1.1 ± 0.2 nM). This versatile calorimetric method is proposed for general use in the discovery and development of drugs targeting 3CL^pro.