Purification and inhibitor screening of the full-length SARS-CoV-2 nucleocapsid protein

Abstract Since the end of 2020, the SARS-CoV-2 strain has undergone several mutations, and new variants continue to emerge. The immune escape ability of the mutants has enhanced, showing robust transmissibility. The neutralizing ability of the neutralizing antibodies produced during previous infections decreased in some mutants. This poses a severe challenge to the preventive and therapeutic effectiveness of vaccines and antibody drugs. Nucleocapsid protein, one of the main structural proteins of the coronavirus, plays an important role in the life cycle of the novel coronavirus and has proven to be one of the key targets for drug development. The first key step in drug development is to obtain pure nucleocapsid proteins. However, as nucleocapsid proteins have a nucleic acid binding function and can automatically undergo liquid–liquid phase separation and agglomerate, the purification of full-length nucleocapsids is extremely challenging. In this study, a set of easy-to-operate processes for the purification of nucleocapsid protein was developed. Finally, a pure full-length nucleocapsid protein without nucleic acid contamination was obtained. Subsequently, we also targeted the nucleic acid binding domain of the nucleocapsid protein and screened for potential SARS-CoV-2 inhibitors using virtual screening and biolayer interferometry (BLI) technology. Finally, a small molecule inhibitor, Light Green SF ( K D = 19.9 µM), which can bind to nucleocapsid protein, was identified. In the future, we will continue to conduct more in-depth research and attempt to develop drugs that possess a good inhibitory effect on the current novel coronavirus mutants.