Viral Proteases as Targets for Coronavirus Disease 2019 Drug Development

Abstract

Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), continues to be a global threat since its emergence. Although several COVID-19 vaccines have become available, the prospective timeframe for achieving effective levels of vaccination across global populations remains uncertain. Moreover, the emergence of SARS-CoV-2 variants presents continuing potential challenges for future vaccination planning. Therefore, development of effective antiviral therapies continues to be an urgent unmet need for COVID-19. Successful antiviral regimens for the treatment of human immunodeficiency virus and hepatitis C virus infections have established viral proteases as validated targets for antiviral drug development. In this context, we review protease targets in drug development, currently available antiviral protease inhibitors, and therapeutic development efforts on SARS-CoV-2 main protease and papain-like protease. SIGNIFICANCE STATEMENT: Coronavirus disease 2019 (COVID-19) continues to be a global threat since its emergence. The development of effective antiviral therapeutics for COVID-19 remains an urgent and long-term need. Because viral proteases are validated drug targets, specific severe acute respiratory syndrome coronavirus 2 protease inhibitors are critical therapeutics to be developed for treatment of COVID-19.

Fig. 1.

Scheme of SARS-CoV-2 genome. ORF1a and ORF1b occupying two-thirds of the 30-kb RNA genome directly translate to 2 polyproteins, pp1a and pp1ab. The polyproteins are processed to produce 16 nonstructure proteins by 3CLpro and PLpro. Other ORFs encode four structural proteins and nine accessory factors of SARS-CoV-2. E, envelope protein; M, membrane protein; N, nucleocapsid protein.

Fig. 2.

Biochemical assays for identification of protease inhibitors. (A) In fluorescence assay, donor fluorescence in the substrate is quenched by an acceptor in intact peptide. The presence of protease cleaves the peptide and releases a high-fluorescence fragment. (B) The substrate in a luminescence assay has an aLuc linked to a peptide, which is inactive in the substrate. The cleavage under protease releases aLuc, which can further elicit glow in the presence of luciferin detection reagent.

Fig. 3.

Design strategies of cell-based assay for SARS-CoV-2 protease. (A) FlipGFP reporter has parallel β10-11 strands, which are incompatible with the rest of the GFP fragments. 3CLpro cuts a linker between β10 and β11 to allow reorientation of β11, which results in the formation of fluorescent GFP. (B) NanoBit, a luciferase complementation reporter, has a peptide connecting Large BiT (LgBit) and Small BiT (SmBit) with high luciferase activity. The cleavage of connecting peptide by 3CLpro increases the proximity between LgBit and SmBit, resulting in low luciferase activity. (C) FLuc reporter is inactive as expressed. PLpro cleaves the target peptide to allow FLuc domain dimerization with catalytic activity.