Research Article

Realtime, continuous assessment of complex-mixture protease and protease inhibitor activity

¹ Department of Chemical & Biomolecular Engineering, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA
² New Liberty Proteomics Corporation, New Liberty, KY 40355, USA

^* Corresponding author: rcperkins.nlp@gmail.com

Received: 6 May 2022
Accepted: 18 May 2022

Abstract

Recently the treatment PAXLOVID™ (nirmatrelvir co-packaged with ritonavir) was authorized for use as a treatment for COVID-19. The presumed mechanism of action of the treatment, an inhibitor of a Sars-Cov-2 “3CL” protease, continues decades-long interest in viral protease inhibition in the fight against pathogenic viruses (e.g., HIV protease inhibitors). Proteolysis assay methods vary widely, roughly bounded by interrogation of basic biochemistry and high-throughput, early-stage drug screening. Reported here are methods that provide unique and biologically relevant characterization of proteolysis and protease inhibition. A companion report provides evidence that these methods show promise for drug and basic biological discovery, especially for early detection of potential side effects. Electron spin resonance spectroscopy and spin labeling (ESR_SL) of whole proteins are leveraged to monitor reactants and products of whole-protein digestion through differentiation of angular mobility of those products and reactants. These proof-of-concept data demonstrate consistency with prior art for all possible combinations of four proteases, two whole-protein substrates and three inhibitors. Thus, ESR_SL is shown to uniquely and widely interrogate proteolysis of natural, whole-protein, substrates insuring the biological relevance of results.