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Covidyte™ ED450

Proteases play essential roles in protein activation, cell regulation and signaling, as well as in the generation of amino acids for protein synthesis or utilization in other metabolic pathways. FRET protease substrates are widely used for detecting protease activities, in particular, for virus protease that often require a long peptide sequence for optimal binding such as coronavirus, HIV and HCV proteases. The internally quenched FRET peptide substrate is digested by a protease to generate the highly fluorescent peptide fragment. The fluorescence increase is proportional to the protease activity. EDANS and DABCYL are a common pair for developing FRET protease substrates.
Proteases play essential roles in protein activation, cell regulation and signaling, as well as in the generation of amino acids for protein synthesis or utilization in other metabolic pathways. FRET protease substrates are widely used for detecting protease activities, in particular, for virus protease that often require a long peptide sequence for optimal binding such as coronavirus, HIV and HCV proteases. The internally quenched FRET peptide substrate is digested by a protease to generate the highly fluorescent peptide fragment. The fluorescence increase is proportional to the protease activity. EDANS and DABCYL are a common pair for developing FRET protease substrates.
Proteases play essential roles in protein activation, cell regulation and signaling, as well as in the generation of amino acids for protein synthesis or utilization in other metabolic pathways. FRET protease substrates are widely used for detecting protease activities, in particular, for virus protease that often require a long peptide sequence for optimal binding such as coronavirus, HIV and HCV proteases. The internally quenched FRET peptide substrate is digested by a protease to generate the highly fluorescent peptide fragment. The fluorescence increase is proportional to the protease activity. EDANS and DABCYL are a common pair for developing FRET protease substrates.
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Physical properties
Molecular weight2034.36
SolventDMSO
Spectral properties
Correction Factor (280 nm)0.107
Extinction coefficient (cm -1 M -1)5900
Excitation (nm)336
Emission (nm)455
Storage, safety and handling
H-phraseH303, H313, H333
Hazard symbolXN
Intended useResearch Use Only (RUO)
R-phraseR20, R21, R22
StorageFreeze (< -15 °C); Minimize light exposure
UNSPSC12171501

OverviewpdfSDSpdfProtocol


Molecular weight
2034.36
Correction Factor (280 nm)
0.107
Extinction coefficient (cm -1 M -1)
5900
Excitation (nm)
336
Emission (nm)
455
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.

Platform


Fluorescence microplate reader

Excitation350 nm
Emission460 nm
Cutoff420 nm
Recommended plateSolid black

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.

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
  1. Add 50 μL of EACH protease dilution to respective wells of the assay plate.
  2. Add 50 μL of Covidyte™ ED450 working solution to each protease dilution.
  3. Monitor the fluorescence increase with a fluorescence plate reader at Ex/Em = 350/460 nm (cutoff 420nm). 
For kinetic reading: Immediately start measuring fluorescence intensity continuously and record data every 5 minutes for 30-120 minutes.
For end-point reading: Incubate the reaction at a desired temperature for 30 to 120 minutes, protected from light. Then measure the fluorescence intensity.

Calculators


Common stock solution preparation

Table 1. Volume of DMSO needed to reconstitute specific mass of Covidyte™ ED450 to given concentration. Note that volume is only for preparing stock solution. Refer to sample experimental protocol for appropriate experimental/physiological buffers.

0.1 mg0.5 mg1 mg5 mg10 mg
1 mM49.156 µL245.778 µL491.555 µL2.458 mL4.916 mL
5 mM9.831 µL49.156 µL98.311 µL491.555 µL983.11 µL
10 mM4.916 µL24.578 µL49.156 µL245.778 µL491.555 µL

Molarity calculator

Enter any two values (mass, volume, concentration) to calculate the third.

Mass (Calculate)Molecular weightVolume (Calculate)Concentration (Calculate)Moles
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Spectrum


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spectrum

Spectral properties

Correction Factor (280 nm)0.107
Extinction coefficient (cm -1 M -1)5900
Excitation (nm)336
Emission (nm)455

Product Family


NameExcitation (nm)Emission (nm)Extinction coefficient (cm -1 M -1)Correction Factor (280 nm)
Covidyte™ EN45033645559000.107
Covidyte™ TF6706496642500000.027
Covidyte™ IF67065667025000010.03

Images


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
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
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