Covidyte™ ED450
Coronaviruses (CoVs) can infect humans and multiple species of animals, causing a wide spectrum of diseases. In late 2019, a novel coronavirus, termed severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was determined as a cause for several cases of respiratory disease (Covid-19). Even though most infected patients only suffer from mild symptoms such as fever and cough associated with a good prognosis, the disease can progress into fatal cases of pneumonia and acute respiratory failure, especially in older males with comorbidities. The virus rapidly spread worldwide. It has infected more than a million people, and Covid-19 has claimed more than seventy thousand fatalities (as of April 6, 2020). Currently, there are not any specific and effective options available for treating Covid-19. At present the clinical treatment of Covid-19 is mainly symptomatic combined with repurposing of already marketed antiviral drugs such as Remdesivir and antibiotics to treat secondary infections. There is an extremely urgent need for the development of specific antiviral therapeutics and vaccines against SARS-CoV-2. The coronavirus main protease, which plays a pivotal role in viral gene expression and replication through the proteolytic processing of replicase polyproteins, is an attractive target for anti-CoV drug design. The inhibition of viral proteases necessary for proteolytic processing of polyproteins has been a successful strategy in the treatment of human immunodeficiency virus (HIV) and hepatitis C respectively, proving the potential of protease inhibitors for the treatment of viral infections. Similarly, the main protease of SARS-CoV-2 is thought to be essential for viral replication and, therefore, is regarded as promising target for antiviral therapy of Covid-19. Covidyte™ ED450 is a peptide substrate containing 12 amino acid sequence (VNSTLQSGLRKM) that can be cleaved by coronavirus proteases. The dark-FRET peptide contains Dabcyl (quencher) and Edans (donor) on the C-and N-terminals respectively where the fluorescence of Edans is effectively quenched by Dabcyl when the peptide is intact. When the peptide is hydrolyzed by coronavirus proteases, the Edans fragment generates significantly enhanced fluorescence since its fluorescence is no longer quenched by Dabcyl. The activity of coronavirus proteases can be effectively monitored by the fluorescence intensity of Edans. Covidyte™ ED450 is a convenient tool for screening inhibitors of coronavirus proteases.
Example protocol
PREPARATION OF STOCK SOLUTIONS
Unless otherwise noted, all unused stock solutions should be divided into single-use aliquots and stored at -20 °C after preparation. Avoid repeated freeze-thaw cycles.
Note Make single use aliquots and store at -20 °C.
Covidyte™ ED450 stock solution (200X)
Add 25 µL (For cat# 13537) or 250 µL (For cat# 13538) DMSO to Covidyte™ ED450 vial.Note Make single use aliquots and store at -20 °C.
PREPARATION OF WORKING SOLUTION
1. Covidyte™ ED450 working solution
Dilute substrate stock solution at 1:200 in 20 mM Tris buffer (pH 7.5) or buffer of your choice. Use 50 μL of substrate solution per assay in a 96-well plate.2. Coronavirus proteases dilution
Dilute the coronavirus proteases as desired.SAMPLE EXPERIMENTAL PROTOCOL
Sample Protocol for One 96-well plate
- Add 50 μL of EACH protease dilution to respective wells of the assay plate.
- Add 50 μL of Covidyte™ ED450 working solution to each protease dilution.
- Monitor the fluorescence increase with a fluorescence plate reader at Ex/Em = 350/460 nm (cutoff 420nm).
For end-point reading: Incubate the reaction at a desired temperature for 30 to 120 minutes, protected from light. Then measure the fluorescence intensity.
Spectrum
Open in Advanced Spectrum Viewer
Product family
Name | Excitation (nm) | Emission (nm) | Extinction coefficient (cm -1 M -1) | Quantum yield | Correction Factor (280 nm) |
Covidyte™ EN450 | 336 | 455 | 5900 | - | 0.107 |
Covidyte™ TF670 | 649 | 663 | 250000 | 0.271 | 0.027 |
Covidyte™ IF670 | 656 | 670 | 2500001 | 0.251 | 0.03 |
References
View all 3 references: Citation Explorer
Synthesis, modification and docking studies of 5-sulfonyl isatin derivatives as SARS-CoV 3C-like protease inhibitors.
Authors: Liu, Wei and Zhu, He-Min and Niu, Guo-Jun and Shi, En-Zhi and Chen, Jie and Sun, Bo and Chen, Wei-Qiang and Zhou, Hong-Gang and Yang, Cheng
Journal: Bioorganic & medicinal chemistry (2014): 292-302
Authors: Liu, Wei and Zhu, He-Min and Niu, Guo-Jun and Shi, En-Zhi and Chen, Jie and Sun, Bo and Chen, Wei-Qiang and Zhou, Hong-Gang and Yang, Cheng
Journal: Bioorganic & medicinal chemistry (2014): 292-302
Synthesis and evaluation of pyrazolone compounds as SARS-coronavirus 3C-like protease inhibitors.
Authors: Ramajayam, R and Tan, Kian-Pin and Liu, Hun-Ge and Liang, Po-Huang
Journal: Bioorganic & medicinal chemistry (2010): 7849-54
Authors: Ramajayam, R and Tan, Kian-Pin and Liu, Hun-Ge and Liang, Po-Huang
Journal: Bioorganic & medicinal chemistry (2010): 7849-54
Evaluation of metal-conjugated compounds as inhibitors of 3CL protease of SARS-CoV.
Authors: Hsu, John T-A and Kuo, Chih-Jung and Hsieh, Hsing-Pang and Wang, Yeau-Ching and Huang, Kuo-Kuei and Lin, Coney P-C and Huang, Ping-Fang and Chen, Xin and Liang, Po-Huang
Journal: FEBS letters (2004): 116-20
Authors: Hsu, John T-A and Kuo, Chih-Jung and Hsieh, Hsing-Pang and Wang, Yeau-Ching and Huang, Kuo-Kuei and Lin, Coney P-C and Huang, Ping-Fang and Chen, Xin and Liang, Po-Huang
Journal: FEBS letters (2004): 116-20
Page updated on October 30, 2024