Chitosan- and chitosan-polaxamer 407 based nanoparticles: green nanotechnology tools for biomedical applications

Abstract

The chymotrypsin-like main protease (3CLpro) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is responsible for cleavage of two viral polyproteins into 16 nonstructural pro- teins. Its catalytic activity is critical for the life cycle of the SARS-CoV-2, since the inhibition of the 3CLpro can effectively block the whole replication of the virus. The 3CLpro has a bell- shaped activity-pH profile with a peak performance set at a value of approximately 7.4. Molecular dynamics and X-ray crystallog- raphy suggested that the enzyme undergoes some conformational changes due to the pH change of the environment. Our goal was to characterize the effects of pH on structure and dynamics of the 3CLpro in solution using nuclear magnetic resonance (NMR) spectroscopy. We obtained high quality NMR spectra recorded at pH between 5.2 and 8.5 for perdeuterated protein samples, selectively 1H, 13C-labelled on the methyl groups of Ile, Leu, Val and Met (ILVM) residues. Moreover, selective isotopic labelling of the histidine residues provided us with the residue specific pKa values for the individual imidazole moieties within this 66 kDa protein molecule. Since four out of seven histidines are located within the enzyme active site, these data allowed us to correlate the protonation states of the His side-chains with the proteolytic activity of the 3CLpro. Interestingly, we found that the proton- ation state of the histidines distant from the catalytic center also affects the 3CLpro proteolytic activity, leading to the discovery of the new allosteric pathway. Taken together, our results improve our understanding of the 3CLpro regulation and may facilitate the design of new potent inhibitors.