An isothermal calorimetry assay for determining steady state kinetic and enzyme inhibition parameters for SARS-CoV-2 3CL-protease.

This manuscript describes the application of Isothermal Titration Calorimetry (ITC) to characterize the kinetics of 3CL pro from the Severe Acute Respiratory Syndrome CoronaVirus-2 (SARS-CoV-2) and its inhibition by Ensitrelvir, a known non-covalent inhibitor. 3CL pro is the main protease that plays a crucial role of producing the whole array of proteins necessary for the viral infection that caused the spread of COVID-19, responsible for millions of deaths worldwide as well as global economic and healthcare crises in recent years. The proposed calorimetric method proved to have several advantages over the two types of enzymatic assays so far applied to this system, namely Förster Resonance Energy Transfer (FRET) and Liquid Chromatography-Mass Spectrometry (LC-MS). The developed ITC-based assay provided a rapid response to 3CL pro activity, which was used to directly derive the kinetic enzymatic constants K M and k cat reliably and reproducibly, as well as their temperature dependence, from which the activation energy of the reaction was obtained for the first time. The assay further revealed the existence of two modes of inhibition of 3CL pro by Ensitrelvir, namely a competitive mode as previously inferred by crystallography as well as an unprecedented uncompetitive mode, further yielding the respective inhibition constants with high precision. The calorimetric method described in this paper is thus proposed to be generally and widely used in the discovery and development of drugs targeting 3CL pro .