AAT BIOQUEST COVID-19

Research Solutions for Coronavirus – AAT Bioquest

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Covidyte™ Peptide Substrate Series Developed For Researchers In The Fight Against COVID-19

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.

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.

Multiple peptide substrates have been developed for monitoring the intensity of coronavirus proteases via FRET.

The Covidyte™ ED450, Covidyte™ EN450, Covidyte™ TF670, and Covidyte™ IF670 peptide substrates provide strong, extended fluorescent signal for searching inhibitors of coronavirus proteases with high sensitivity.

The papain-like proteases (PLpro) of coronaviruses carry out proteolytic maturation of non-structural proteins that play a role in replication of the virus and performs deubiquitination of host cell factors to scuttle antiviral responses. The newly-developed Covipyte™ EN450 is a peptide substrate containing the 9 amino acid sequence (RELNGGAPI) that can be cleaved by coronavirus PLpro, used for screening and studying the kinetics of PLpro inhibitors.

Additionally, Z-KAGG-AMC, Z-LRGG-AMC, and Z-KKAG-AMC are cleaved by papain-like proteases to give the highly fluorescent AMC product, to measure protease activity, and might be used for screening and studying kinetics of PLpro inhibitors.

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.

Tide Quencher™ dyes have been proven to be the extremely effective quenchers for developing FRET protease substrates for high throughput screening applications together with our bright Tide Fluor™ and iFluor™ dyes.

Covidicyte™ fluorescent donors Tide Fluor™ 5WS and Edans spectrum curves, visualized using AAT Bioquest Advanced Spectrum Viewer

Covipyte™ EN450
Peptide substrate containing a 9 amino acid sequence (RELNGGAPI) that can be cleaved by coronavirus PLpro
The dark-FRET peptide contains Edans (donor) and Dabcyl (quencher) on the N-and C-terminals respectively where the fluorescence of Edans is effectively quenched by Dabcyl when the peptide is intact. 

Z-KAGG-AMC
Protease substrate that is cleaved by papain-like proteases to give the highly fluorescent AMC product.
The fluorescence intensity of released AMC is proportional to the protease activity.

Z-KKAG-AMC
Protease substrate that is cleaved by papain-like proteases to give the highly fluorescent AMC product.
The fluorescence intensity of released AMC is proportional to the protease activity.

Z-LRGG-AMC
Protease substrate that is cleaved by papain-like proteases to give the highly fluorescent AMC product.
The fluorescence intensity of released AMC is proportional to the protease activity.

Covidyte™ IF670

Peptide substrate containing a 14 amino acid sequence that can be cleaved by coronavirus proteases

The dark-FRET peptide contains Tide Quencher™ 5 (TQ5) as a quencher and iFluor™ 670 as a fluorescent donor on the N-and C-terminals
respectively

When the peptide is hydrolyzed by coronavirus proteases, the iFluor™ 670 fragment generates significantly enhanced fluorescence since its fluorescence is no longer quenched by TQ5

Covidyte™ TF670

Peptide substrate containing a 14 amino acid sequence that can be cleaved by coronavirus proteases

The dark-FRET peptide contains Tide Quencher™ 5 (TQ5) as a quencher and Tide Fluor™ 5 (TF5) as a fluorescent donor on the N-and C-terminals respectively

When the peptide is hydrolyzed by coronavirus proteases, the TF5 fragment generates significantly enhanced fluorescence since its fluorescence is no longer quenched by TQ5

Covidyte™ ED450

Peptide substrate containing an 11 amino acid sequence that can be cleaved by coronavirus proteases

Covidyte™ EN450

Peptide substrate containing a 14 amino acid sequence that can be cleaved by coronavirus proteases
  • For both Covidyte™ ED450 and Covidyte™ EN450, the dark-FRET peptide contains Dabcyl (quencher) and Edans (donor) on the N-and C-terminals respectively.
  • When the peptide is hydrolyzed by coronavirus proteases, the Edans fragment generates significantly enhanced fluorescence since its fluorescence is no longer quenched by Dabcyl.

Coronavirus (SARS-CoV-2)

As the coronavirus pandemic (COVID-19) becomes more widespread, the urgency to develop diagnostic tools for viral screening, and the advancement of viral therapeutics and vaccine candidates becomes increasingly apparent. Here at AAT Bioquest we are committed to providing reliable and sensitive solutions for the advancement of COVID-19 research. Browse our comprehensive portfolio of research solutions from virus discovery, pathogen detection and research surveillance to vaccine development.

1) Intro to COVID-19

The coronavirus disease 2019 (COVID-19) is an infectious disease caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). It was initially discovery in December 2019, in Wuhan, China, and since then COVID-19 has infected more than 2.6 million people in 185 countries, resulting in over 182,000 fatalities (as of April 22, 2020). While most infected patients suffer from mild symptoms characterized by fever, cough and shortness of breath, the disease can progress into fatal cases of pneumonia and acute respiratory failure, notably in older males with comorbidities.

1.1 SARS-CoV-2 Structure: RNA & Protein Components

Identification and sequencing of the SARS-CoV-2 full-length genome demonstrated that this novel CoV shares a 79.5% sequence identity with the SARS-CoV that occurred in 2002. Similar to SARS-CoV, the SARS-CoV-2 genome encodes four key structural proteins: spike proteins (S), membrane glycoproteins (M), envelope proteins (E) and nucleocaspid proteins (N). The spike protein, which is responsible for facilitating the entry of SARS-CoV-2 into the host cell, utilizes the same host receptor Angiotensin-Converting Enzyme 2(ACE2) as SARS-CoV for cell entry. Such similarities allude to the possibility that therapies developed during the SARS-CoV outbreak could help prevent or serve as a viable treatment for COVID-19.

For more information regarding the functionality of SARS-CoV-2 proteins and its RNA components, please refer to the table below.

Figure 1. Illustration of the SARS-CoV-2 structure (figure made in BioRender).

Table 1. Overview of SARS-CoV-2 protein and RNA components.

ComponentFunction
Spike ProteinsSpike proteins (S-proteins) are large, heavily-glycosylated transmembrane proteins that form the distinctive “corona” or crown-like appearance on the surface of the virus. The ectodomain of S-proteins are comprised of an N-terminal and a C-terminal domain known as the S1 and S2 subunits, respectively. The S1 subunit, which contains the receptor binding domain, is responsible for recognizing and binding to the host cell receptor. The S2 subunit, which contains a fusion peptide and two heptad repeats (HR1 and HR2), are responsible for virus-cell membrane fusion. From a therapeutic stand point, S-proteins are promising targets for SARS-CoV-2 vaccine and anti-viral development of therapies (e.g. antibodies) capable of blocking virus binding and fusion.
Membrane ProteinsMembrane proteins (M-proteins), which are the most abundant of all the structural proteins, define the shape of the viral envelope. M-proteins are considered to be the principal coordinator of SARS-CoV-2 assembly, as it interacts with all of the major coronavirus structural proteins. Interaction of M-proteins with S-proteins promotes retention of S-proteins in the ER-Golgi compartment and its integration into new virions. M-protein and N-protein interactions stabilize the nucleocapsid and facilitate completion of viral assembly. M-protein and E-protein interactions form the viral envelop which assist in the production and release of virus-like particles.
Envelope ProteinsEnvelope proteins (E-proteins) are the smallest of the structural proteins. They are vital for virus production and maturation and facilitate in the release of the virus. A small portion of E-proteins are integrated into the virion envelope, while the majority localizes at the site of intracellular trafficking (e.g. ER-Golgi).
Nucleocapsid ProteinsNucleocapsid proteins (N-proteins), which are heavily phosphorylated, function primarily to bind the SARS-CoV-2 RNA genome and encapsulate it within the viral envelope forming a ribonucleocapsid complex. This capsid is essential for viral self-assembly and replication. N-proteins also affect host cell responses to viral infection.
RNA GenomeRNA is the genome of the virus. Once SARS-CoV-2 enters host cells, it replicates a genomic RNA to produce smaller subgenomic RNA fragments. These fragments are used to synthesize essential proteins (e.g. S-, M-, N- and E-proteins) for viral infection.

2) Research solutions for virus discovery

Having the right tools is essential for accelerating scientific discovery for COVID-19 and SARS-CoV-2. From genomic tools for viral characterization and vaccine development to immunoassay solutions for the development of rapid diagnostic kits, AAT Bioquest offers comprehensive solutions to advance your research.

2.1 Discovering viral genome by Sanger Sequencing

Sanger sequencing by capillary electrophoresis is a chain termination method used to determine the nucleotide order of DNA. Sanger sequencing selectively incorporates chain-terminating dideoxynucleotides (ddNTPs) by DNA polymerase during in vitro DNA replication. The extension products are then separated by capillary electrophoresis and the molecules are injected by an electrical current into a long glass capillary filled with a gel polymer. Although newer NGS technologies are becoming increasingly common in clinical research labs for their higher throughput capabilities and lower costs per sample, Sanger sequencing is still the “gold standard” for clinical research sequencing with 99.99% efficacy rate. In addition to its role in identifying SARS-CoV-2, Sanger sequencing by capillary electrophoresis can be used to verify reverse transcriptase-polymerase chain reaction results and provide accurate discernment of SARS-CoV-2 from other pathogens.

Table 2. Available MagaDye™ fluorescent ddNTPs for Sanger sequencing.

Product Name Nucleotide Ex (nm) Em (nm) Abs(nm) Unit Size Cat No.
MagaDye™ 535-ddGTP Guanine 503 nm 536 nm 503 nm 5 nmoles 17063
MagaDye™ 535-ddGTP Guanine 503 nm 536 nm 503 nm 50 nmoles 17067
MagaDye™ 561-ddATP Adenine 498 nm 561 nm 498 nm 5 nmoles 17062
MagaDye™ 561-ddATP Adenine 498 nm 561 nm 498 nm 50 nmoles 17066
MagaDye™ 588-ddTTP Thymine 498 nm 588 nm 498 nm 5 nmoles 17061
MagaDye™ 588-ddTTP Thymine 498 nm 588 nm 498 nm 50 nmoles 17065
MagaDye™ 613-ddCTP Cytosine 498 nm 614 nm 498 nm 5 nmoles 17060
MagaDye™ 613-ddCTP Cytosine 498 nm 614 nm 498 nm 50 nmoles 17064

Table 3. Specifications for MagaDye™ 4 Color Sanger Sequencing Terminator Kit For Research solutions for virus discovery

Product Name ddNTPs Ex (nm) Em (nm) Abs (nm) Unit Size Cat No.
MagaDye™ 4 Color Sanger Sequencing Terminator Kit ddGTP ddATP ddTTP ddCTP 488 nm 536 nm 561 nm 588 nm 614 nm 498 nm 5 nmoles 17068

3) Research solutions for COVID-19 pathogen detection

Nucleic acid analysis by RT-PCR and serologic assays are two techniques extensively used for the detection of SARS-CoV-2. RT-PCR is the most widely used platform and is recommended for screening or diagnosis of early infection by SARS-CoV-2. RT-PCR works to detect the presence of viral RNA prior to seroconversion, the time period during which a specific antibody develops against the pathogen and becomes detectable in the blood. Serological assays such as the enzyme-linked immunosorbent assay (ELISA) and the lateral flow immunoassay (LFIA) detect for the presence of antibodies in serum that are produced in response to viral infection.

Figure 1. Fluorescence images of live and fixed HeLa cells stained with StrandBrite RNA Green (Green) and counter-stained with Hoechst 33342. Fluorescence signal were measured using a fluorescence microscope with FITC filter.

Table 4. RNA quantification and PCR reagents

Product Name Ex (nm) Em (nm) Unit Size Cat No.
StrandBrite™ Green Fluorimetric RNA Quantitation Kit *High Selectivity* 490 nm 540 nm 100 Tests 17657
StrandBrite™ Green RNA Quantifying Reagent 490 nm 525 nm 1 mL 17610
StrandBrite™ Green RNA Quantifying Reagent 490 nm 525 nm 10 mL 17611
Portelite™ Fluorimetric RNA Quantitation Kit 490 nm 525 nm 100 Tests 17658
Portelite™ Fluorimetric RNA Quantitation Kit 490 nm 525 nm 500 Tests 17659
Cyber Green™ [Equivalent to SYBR® Green] *20X Aqueous PCR Solution* 498 nm 522 nm 5 x 1 mL Tests 17591
Cyber Green™ [Equivalent to SYBR® Green] *20X Aqueous PCR Solution* 498 nm 522 nm 1 mL 17592
Cyber Green™ Nucleic Acid Gel Stain [Equivalent to SYBR® Green] 498 nm 522 nm 100 µL 17604

4) Research solutions for COVID-19 vaccine development

Proteases play essential roles in protein activation, cell regulation and cell signaling, as well as, in the generation of amino acids for protein synthesis or utilization in other metabolic pathways. 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 (Mpro or 3CLpro) is thought to be essential for viral gene expression and replication and, therefore, is regarded as a major target for anti-CoV drug design. AAT Bioquest offers a variety of dark-FRET peptide substrates and fluorogenic substrates which can be effectively used to screen for inhibitors of coronavirus proteases, papin-like proteases and more.

Assay Principle for Dark-FRET Protease Substrates

Figure 3. FRET mechanisms of Covidyte™ and Covipyte™ COVID-19 peptide substrates (figure made in BioRender).. 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 (figure drawn in BioRender).

4.1 Covidyte™ SARS-CoV-2 substrates

Covidyte™ dark-FRET peptide substrates are robust high throughput screening tools for determining inhibitors of coronavirus proteases. Covidyte™ substrates contain either an 11 or 14 amino acid sequence that can be cleaved by coronavirus proteases. Modified to the terminal ends of each amino acid sequence is a fluorescent donor and non-fluorescent quencher dye. In its intact conformation, substrate fluorescence is quenched by the two dyes being in close proximity of one another. When the internally quenched Covidyte™ substrate is hydrolyzed by coronavirus proteases, a highly fluorescent peptide fragment is produced, and the fluorescence increase is proportional to the coronavirus proteases activity.

Figure 4. Chemical structure for Covidyte™ EN450.

Table 5. Covidyte™ substrates for screening coronavirus protease inhibitors.

Product Name Sequence Length Amino Acid Sequence Target Ex (nm) Em (nm) ε1 Unit Size Cat No.
Covidyte™ EN450 14 amino acids KTSAVLQSGFRKME Coronavirus proteases 336 445 5900 100 Tests 13535
Covidyte™ EN450 14 amino acids KTSAVLQSGFRKME Coronavirus proteases 336 445 5900 1000 Tests 13536
Covidyte™ ED450 12 amino acids VNSTLQSGLRKM Coronavirus proteases 336 445 5900 100 Tests 13537
Covidyte™ ED450 12 amino acids VNSTLQSGLRKM Coronavirus proteases 336 445 5900 1000 Tests 13538
Covidyte™ TF670 14 amino acids KTSAVLQSGFRKME Coronavirus proteases 649 664 250,000 100 Tests 13540
Covidyte™ TF670 14 amino acids KTSAVLQSGFRKME Coronavirus proteases 649 664 250,000 1000 Tests 13541
Covidyte™ IF670 12 amino acids VNSTLQSGLRKM Coronavirus proteases 656 670 250,000 100 Tests 13542
Covidyte™ IF670 12 amino acids VNSTLQSGLRKM Coronavirus proteases 656 670 250,000 1000 Tests 13543
Covipyte™ EN450 9 amino acids RELNGGAPI Papin-like proteases 336 445 5900 100 Tests 13545
  1. ε = Extinction coefficient at their maximum absorption wavelength. The units of extinction coefficient are cm-1M-1.

4.2 Covipyte™ SARS-CoV-2 substrates

The Covipyte™ EN450 dark-FRET peptide substrate contains a 9 amino acid sequence (RELNGGAPI) specific for coronavirus papin-like proteases (PLpro). Conjugated to the N- and C-terminals, respectively, are an Edans donor molecule and a Dabcyl quencher molecule. In its intact conformation, substrate fluorescence is quenched by the donor and quencher molecule being in close proximity of one another. When hydrolyzed by coronavirus PLpro, a highly fluorescent Edans fragment is produced and the fluorescence increase is proportional to the coronavirus proteases activity making it a convenient tool for screening and studying the kinetics of PLpro inhibitors. PLpro of coronaviruses carries out proteolytic maturation of non-structural proteins that play a role in replication of the virus and performs deubiquitination of host cell factors to scuttle antiviral responses.

Additionally, AAT Bioquest offers 3 tetra-peptide substrates, Z-KAGG-AMC, Z-KKAG-AMC, and Z-LRGG-AMC, for screening and evaluating coronavirus PLpro inhibitors. Upon hyrolysis by PLpro, these substrates generate highly fluorescent AMC products (Ex/Em = 341/441 nm).

 

Table 6. Covipyte™ substrates for screening coronavirus protease inhibitors.

Product Target Ex (nm) Em (nm) Unit Size Cat No.
Z-LRGG-AMC Papin-like proteases 341 445 1 mg 13550
Z-KAGG-AMC Papin-like proteases 341 445 1 mg 13552
Z-KKAG-AMC Papin-like proteases 341 445 1 mg 13552

Note

  1. Ext. Coeff. = Extinction coefficient at their maximum absorption wavelength. The units of extinction coefficient are cm-1M-1.

4.3 ACE2 Receptor Recognition by SARS-CoV-2

The binding of viruses to receptors on the surface of target cells is a critical first step in the infectious viral life cycle as it facilitates the entry of the virus into the host cells. Recently, it has been discovered that the spike protein of SARS-CoV-2 utilizes the ACE2 surface receptor on host cells to promote cellular entry. The significance of this discovery recognizes ACE2 as a promising therapeutic target for SARS-CoV-2. To screen and evaluate ACE2 inhibitors AAT Bioquest offers Mca-APK(Dnp), a fluorogenic ACE2 substrate. Upon enzymatic cleavage at the proline-lysine residue, the Dnp quencher is removed and fluorescence of Mca is restored. Mca-APK(Dnp) ACE2 substrates have an excitation and emission maxima of 322 nm and 381 nm, respectively, and are easily adaptable for high through-put screening.

Figure 5. ACE2 dose response was measure with Mca-APK(Dnp) substrate.

5) ACE2 substrates for screening ACE2 inhibitors.

Name Ex (nm) Em (nm) Unit Size Cat No.
Mca-APK(Dnp) ACE2 substrate 322 381 1 mg 13555
Mca-APK(Dnp) ACE2 substrate 322 381 10 mg 13556

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