The CoviDrop™ SARS-CoV-2 Targeted Proprotein Convertase Activity/Inhibition Assay Kit is a complete set of optimized buffers and reagents designed for measuring SARS-CoV-2-targeted proprotein convertase (PC) cleavage activity and inhibition using various human samples so that the functional PC activity targeting SARS-CoV-2 S1/S2 site cleavage can be identified. The kit has the following advantages:
Background
COVID-19 is an infectious disease caused by SARS-CoV-2, a new member of the same coronavirus family that caused SARS and MERS. It was found that the SARS-CoV-2 spike glycoprotein harbors a furin/PC cleavage site at the boundary between the S1/S2 subunits, which could be cleaved by furin and/or furin-like PCs secreted from host cells and bacteria in the airway epithelium [1, 2]. Unlike SARS-CoV, cell entry of SARS-CoV-2 is pre-activated by furin and/or furin-like PCs, reducing its dependence on target cell proteases for entry [3]. The cleavage activation of S-protein is well demonstrated to be essential for SARS-CoV-2 spike-mediated viral binding to ACE2, cell-cell fusion, and viral entry into human lung cells [4, 5]. It was also observed that other viruses containing a furin/PC cleavage site, such as H5N1, increased replicates and developed higher pathogenicity [6]. The SARS-CoV-2 furin/PC cleavage site has been with one core region SPRRAR│SV (eight amino acids, P6–P2′). The core region is very unique, as its P2 or P3 position is a positively charged residue (Arg), and another residue is hydrophobic (Ala). These factors allow this site to be cleaved by furin or furin-like PC and the cleavage efficiency to be facilitated by other serine proteases targeting dibasic amino acid sites such as matriptase, plasmin, human airway trypsin (HAT), and TMPRSS2. Furthermore, a serine at P6 could also highly increase the cleavage efficiency, causing increased viral replication, unrestricted organ tropism, virulence, and mortality rate as proven in H5N1 infection in mice [7]. The importance of measuring SARS-CoV-2S1/S2 site targeted cleavage PC or facilitating protease activity is emphasized by that the PC-based SARS-CoV-2 S1/S2 cleavage increases SARS-Cov-2 entry into cells and replication and eventually develops higher pathogenicity of COVID-19.
Fig 1. SARS-CoV-2 spike protein cleavage by proprotein convertases and other proteases during entry into cells.
Principles and ProcedureThis kit contains all reagents necessary for screening various inhibitors against SARS-CoV-2-targeted cleavage PCs and facilitating proteases. In this assay, a SARS-CoV-2 specific substrate is tagged with polyhistidine at the N-terminal and biotin at the C-terminal and bound onto microplate wells through histidine/Ni-NTA at its N-terminal. The cleavage of the substrate at S1/S2 site will remove the S2 part (C-terminal) of the substrate after washing, which causes the decrease in signal generated by avidin/biotin binding after adding Cleavage Detection Solution (CDS). The inhibition of the cleavage by inhibitors will block the reduction of the signals. The signal intensity is measured through an ELISA-like reaction by reading the absorbance in a microplate spectrophotometer at a wavelength of 450 nm. The more the substrate is cleaved, the lower the signal that will be generated. Thus, the PC cleavage activity is inversely proportional to the signal intensity.
Starting MaterialsInput materials can be cell/tissue extracts, serum, plasma, swab samples, and various body fluids.
References:1. Andersen KG et al: Nat Med. 26: 450-452, 2020. 2. Coutard B. et al: Antiviral Research, 176, 2020.3. Shang J et al: Proc Natl Acad Sci. 117: 11727-11734, 2020.4. Hoffmann M et al: Mol Cell, 78:779-784, 2020.5. Hoffmann M et al: Cell. 181:271-280, 2020.6. Decha P. et al: Biophys J. 95: 128–134, 2008.7. Zhang Y et al: J Virol. 86: 6924–6931, 2012.
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