Cat. No.: N3CLP-100 (for 100T)

Cat. No.: N3CLP-500 (for 500T)

Description

2019-nCoV Mpro/3CLpro Inhibitor Screening Kit is a reagent kit used for high-throughput screening of inhibitors targeting the main protease (Mpro) or 3C-like protease (3CLpro) of the novel coronavirus (2019-nCoV). The kit utilizes fluorescence detection and offers a simple, rapid, and sensitive method for inhibitor screening. The protein sequence of the novel coronavirus Mpro/3CLpro protease used in this kit is identical to the natural sequence of the main protease (Mpro) found in the novel coronavirus, without any additional labels or amino acids, ensuring more authentic and reliable screening results.

At the end of 2019, a pneumonia outbreak caused by the novel coronavirus emerged and rapidly escalated into a global pandemic from early 2020 onwards, attracting worldwide attention. This virus was named 2019-nCoV by the World Health Organization (WHO) and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) by the International Committee on Taxonomy of Viruses. The pneumonia resulting from novel coronavirus infection was named Coronavirus Disease 2019 (COVID-19) by WHO, commonly referred to as "COVID-19" or "novel coronavirus pneumonia."

The novel coronavirus (2019-nCoV) belongs to the single-stranded RNA virus group and shares a high degree of homology with SARS-CoV and MERS-CoV. After infecting host cells, the viral RNA's ORF1a/b is translated to produce two polyproteins (pp1a and pp1ab) with the help of host cell machinery. These polyproteins undergo intramolecular cleavage by the main protease (Mpro) and papain-like proteases to generate multiple non-structural proteins. Mpro is also known as 3C-like protease (3CLpro) due to its similarity to the 3C protease of picornaviruses in terms of cleavage site specificity. The non-structural proteins derived from polyprotein processing are involved in the production of viral subgenomic RNAs and four structural proteins (envelope/E protein, membrane/M protein, spike/S protein, and nucleocapsid/N protein), facilitating the replication and release of progeny viruses. Because Mpro plays a crucial role in the virus lifecycle and has no homologous protein in the human body, it serves as an ideal target for antiviral drug development. Since the SARS outbreak in 2003, there has been extensive research on SARS drugs based on the Mpro structure. HIV drugs like Lopinavir and Ritonavir also target similar protease sites and have been clinically validated. Moreover, Mpro is highly conserved among β-coronaviruses, suggesting that Mpro inhibitors could exhibit broad-spectrum antiviral activity against coronaviruses, potentially extending to related animal diseases like swine coronaviruses.

The novel coronavirus (2019-nCoV) Mpro/3CLpro inhibitor screening kit employs the fluorescence resonance energy transfer (FRET) technique. Edans serves as the fluorescence donor (Donor), and Dabcyl acts as the fluorescence acceptor (Acceptor) or quencher. The absorption spectra of these two fluorescence groups partially overlap, and when they are at an appropriate distance (generally 7-10 nm), fluorescence energy transfers from the donor to the acceptor, leading to a decrease in donor fluorescence intensity. Edans and Dabcyl are attached to both ends of the natural substrate of the 2019-nCoV Mpro/3CLpro protease, namely Dabcyl-KTSAVLQSGFRKME-Edans. When the substrate is uncut by the protease, the two groups are close enough for fluorescence resonance energy transfer to occur, resulting in the quenching of Edans fluorescence by Dabcyl, leading to no detectable fluorescence. After the substrate is cleaved by the 2019-nCoV Mpro/3CLpro protease, the peptide's two ends separate, and the two groups move apart. Edans fluorescence is no longer quenched by Dabcyl, allowing Edans fluorescence to be detected. This fluorescence-based detection method enables the sensitive measurement of the enzymatic activity of the 2019-nCoV Mpro/3CLpro protease. In the presence of an inhibitor targeting the 2019-nCoV Mpro/3CLpro protease in the reaction system, fluorescence generation is suppressed, and the fluorescence intensity is inversely proportional to the inhibitory effect of the inhibitor, allowing the detection of the inhibitor's inhibitory effect. Edans has a maximum excitation wavelength of 340 nm and a maximum emission wavelength of 490 nm.

This kit includes 2019-nCoV Mpro/3CLpro, substrate, and a positive control 2019-nCoV Mpro/3CLpro inhibitor (Ebselen). The quantities of 2019-nCoV Mpro/3CLpro and substrate are optimized in the kit. This ensures detection not only of inhibitors with very low IC50 values but also of inhibitors with higher IC50 values. The kit provides the positive control Ebselen, with publicly available data indicating an in vitro IC50 of around 0.67 μM; however, actual measured data might show slight variation.

When used for testing in a 96-well plate format, the small package P0312S of this kit allows for 100 tests, and the medium package P0312M allows for 500 tests.

Features

• The results of inhibitor screening from this kit are more reliable and trustworthy. There is a difference of only 12 amino acids between 2019-nCoV Mpro/3CLpro and SARS-CoV's Mpro/3CLpro, with a homology of over 96%. Their structures are nearly identical. Therefore, for the screening of 2019-nCoV Mpro/3CLpro inhibitors, it is crucial to use a recombinant protein with a sequence that is identical to the natural 2019-nCoV Mpro/3CLpro protein. We have developed the novel coronavirus (2019-nCoV) Mpro/3CLpro Inhibitor Screening Kit utilizing 2019-nCoV Mpro/3CLpro and its specific substrate. The 2019-nCoV Mpro/3CLpro used in this kit is purified using our proprietary protein expression technology platform. This ensures that the expressed recombinant 2019-nCoV Mpro/3CLpro protein has no additional labels or extra amino acids, maintaining complete sequence identity with the Mpro/3CLpro amino acid sequence of the 2019-nCoV virus. Using this kit for screening coronavirus inhibitors ensures the obtained inhibitors are more authentic and trustworthy, avoiding false positives due to the presence of additional amino acids.
• The compatibility of this kit is strong. Common solvents like DMSO, anhydrous ethanol, and glycerol have minimal impact on the test results of this kit. Tested reaction systems with DMSO or anhydrous ethanol, glycerol content reaching 10% had minimal impact on the test results, and when reaching 20%, the signal decrease after 10 minutes of incubation was no more than 10%. Deionizing agents like Triton X-100 at 0.4% had virtually no effect after 10 minutes of incubation, and at 1%, the signal decrease after 10 minutes of incubation was no more than 25%.
• This kit demonstrates good signal stability and strength. After 5 minutes of incubation, the signal becomes stable and remains relatively unchanged within 20 minutes. Moreover, the signal for 100% enzyme activity is about 20 times that of the background signal, ensuring a sufficient signal range for inhibitor screening.

Storage

Store at -20°C, valid for one year. The substrate should be stored away from light.

Precaution

• Assay Buffer, Substrate, and Ebselen (10mM) must be completely thawed and equilibrated to room temperature before use, as using them otherwise can impact the detection results. When working with 2019-nCoV Mpro/3CLpro, it should be kept on ice. After use, each reagent should be immediately stored under the conditions specified in the kit instructions.
• Ensure that the sample pH is between 7 and 8, or ensure that the pH of the reaction system is within this range after adding the sample. Deviations from this pH range may affect the signal values and stability of the detection results.
• The solvent of the test inhibitor samples may interfere with the detection. It is recommended to use the provided Assay Buffer as the solvent for diluting samples. If other solvents must be used, perform some tests and add an equal volume of solvent as 100% enzyme activity control. It has been tested that concentrations of up to 10% DMSO or anhydrous ethanol in the reaction system do not affect the detection results. However, it's still recommended to conduct preliminary experiments and minimize the concentrations of DMSO, anhydrous ethanol, and other solvents in the reaction system.
• Due to the limited water solubility of the positive control inhibitor Ebselen (10mM), it should be diluted using DMSO.
• For reagents with small volumes, it's advised to centrifuge briefly to ensure that the liquid settles at the bottom of the tube before use. Frozen reagents must be completely thawed and mixed before use.
• For detection, it's recommended to use a 96-well black plate. 96-Well Black Opaque Plates are recommended.
• This product is intended for scientific research by professionals only. It must not be used for clinical diagnosis or treatment, food, or drugs, and should not be stored in regular households.
• For your safety and health, wear appropriate lab attire and disposable gloves while handling the reagents.