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Assays for COVID-19 Drug Discovery

Accelerate COVID-19 Drug Discovery and Development Programs Targeting Proinflammatory Cytokines & Others with Qualified Assays

The World Health Organization (WHO) declared COVID-19 as a pandemic in 2020. Although COVID-19 cases have significantly declined since then, medical science experts project the disease and its aftermath will continue as long as the virus continues to spread and mutate – and will continue to pose a danger to human health.

 

After years of intense study the disease is now better understood, as is its transmission when the SARS-CoV-2 virus infects the respiratory tract and, in severe cases, leads to acute respiratory distress syndrome (ARDS). The high mortality rate in COVID-19 patients has been linked to cytokine release syndrome (CRS), commonly referred to as cytokine storm, which is an excessive and dysregulated production of proinflammatory cytokines by the body’s immune system. Currently, active clinical programs are implemented that focus on developing and repurposing therapeutic drugs for treatment of COVID-19.

 

Implement Eurofins DiscoverX® cell-based assays to accelerate your COVID-19 drug discovery from basic research through QC lot release programs, particularly in managing proinflammatory cytokines associated with high mortality rate in COVID-19 patients.

 

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Advantages of Cell-Based Assays for COVID-19 Drug Discovery

  • Target Specific – Mechanism-of-action (MOA)-based, functional assays for drugs targeting proinflammatory cytokines such as IL-1, IL-6, IL-7, GM-CSF, and TNFα that are implicated in COVID-19 pathology
  • Qualified Bioassays – Accelerate your drug release program into QC lot release with bioassays qualified using approved therapeutics like Actemra® (tocilizumab), Kineret® (anakinra), Leukine® (sargramostim), and Humira® (adalimumab)
  • Complete Understanding – Evaluate both receptor-proximal and -distal responses for a comprehensive understanding of the biologic and small molecule therapeutics MOA
  • Complete Solutions – Utilize Eurofins DiscoverX’s Custom Capabilities for the creation of other COVID-19-relevant receptor assays such as ACE2, TMPRSS2, and more

Implications of Cytokine Storm in COVID-19 Patients

Implications of Cytokine Storm in COVID-19 Patients

CRS is a proinflammatory immune response that causes hyperinflammation and includes various cytokines such as interleukins (ILs), GM-CSF, chemokines, TNFα and more. This reaction remains one of the main reasons behind the severe implications of COVID-19. Even when the patients survive, there are long-lasting implications associated with tissue injury caused by the immune cells. Patients prone to getting secondary infections may suffer multiple organ failures leading to death.

Key Resources

 

Actemra is a registered trademark of Chugai Seiyaku Kabushiki Kaisha Corp., a member of the Roche Group; Kineret is a registered trademark, licensed by Swedish Orphan Biovitrum AB and marketed by Sobi, Inc.; Leukine is a registered trademark licensed to Genzyme Corporation; and Humira is a registered trademark AbbVie, Inc.

Eurofins DiscoverX offers a variety of cell-based assays for COVID-19 drug discovery and development programs. Contact custom assay development for additional assays or visit Eurofins Discovery to learn more about cell-based and phenotypic assay services.

Cell-Based Assays for COVID-19 Drug Discovery

Therapeutic Type or MOA Therapeutic Target Recommended Cell-Based Assay
Monoclonal antibodies; to be used in combination with protease inhibitors Adalimumab TNFα PathHunter® Adalimumab Bioassay Kit (Cat. No. 93-0538B15-00132)
Combination studies Adalimumab + Tocilizumab TNFα + IL-6
Varies Anakira IL-1β PathHunter Anakinra Bioassay Kit (Cat. No. 93-1032Y3-00106)
Varies Angiotensin Converting Enzyme (ACE) inhibitors ACE2 Contact Custom Assay Development (Request Early Access)
Varies Angiotensin receptor blockers AGTR1 PathHunter CHO-K1 AGTR1 Bioassay Kit (Cat. No. 93-0312Y2-00126)
Monoclonal antibodies; to be used in combination with protease inhibitors Camrelizumab PD-1
  • PathHunter Jurkat PD-1 (SHP1) Signaling Cell Line (Cat. No. 93-1104C19)
  • PathHunter PD-1 (SHP1) Signaling Bioassay Kit (Cat. No. 93-1104Y19-00118)
Monoclonal antibodies; to be used in combination with protease inhibitors Eculizumab C5AR1
  • PathHunter CHO-K1 C5AR1 β-Arrestin Cell Line (Cat. No. 93-0557C2)
  • PathHunter C5AR1 Activated GPCR Internalization U2OS Cell Line (Cat. No. 93-0777C3)
  • cAMP Hunter™ CHO-K1 C5R1 Gi Cell Line (Cat. No. 95-0079C2)
  • PathHunter CHO-K1 C5AR1 Bioassay Kit (Cat. No. 93-0557Y2-0006)
Nonspecific anti-inflammatory and immunisuppressive drugs Fingolimod S1P1; EDG1 Sphingosine 1-phosphate receptor 1 (S1P1; EDG1)
  • PathHunter CHO-K1 Eine (Cat. No. 93-0207C2)
  • PathHunter EDG1 Total GPCR Internalization HEK 293 Cell Line (Cat. No. 93-0784C1)
  • cAMP Hunter™ CHO-K1 EDG1 Gi Cell Line (Cat. No. 95-0142C2)DG1 β-Arrestin Cell L
Nonspecific anti-inflammatory and immunosuppressive drugs Fingolimod S1P2; EDG5 Sphingosine 1-phosphate receptor 2 (S1P2; EDG5)
  • PathHunter CHO-K1 EDG5 β-Arrestin Cell Line (Cat. No. 93-0256C2)
  • PathHunter EDG5 Activated GPCR Internalization U2OS Cell Line (Cat. No. 93-0735C3)
Nonspecifc anti-inflammatory and immunosuppressive drugs Fingolimod S1P3; EDG3 Sphingosine 1-phosphate receptor 3 (S1P3; EDG3)
  • PathHunter HEK 293 EDG3 β-Arrestin Cell Line (Cat. No. 93-0217C1)
  • PathHunter CHO-K1 EDG3 β-Arrestin Cell Line (Cat. No. 93-0217C2)
  • PathHunter EDG3 Activated GPCR Internalization U2OS Cell Line (Cat. No. 93-0663C3)
  • CHO-K1 EDG3 Gq Cell Line (Cat. No. 95-1007C2)
Nonspecific anti-inflammatory and immunosuppressive drugs Fingolimod S1P4; EDG6 Sphingosine 1-phosphate receptor 4 (S1P4; EDG6)
  • PathHunter CHO-K1 EDG6 β-Arrestin Cell Line (Cat. No. 93-0227C2)
Antiviral drugs, non-specific Interleukin-2 IL-2
  • PathHunter DLD1 IL7R/IL2RG Dimerization Cell Line (Cat. No. 93-0997C13)
  • PathHunter HEK 293 IL9R/IL2RG Dimerization Cell Line (Cat. No. 93-1036C1)
  • PathHunter U2OS IL21R/IL2RG Dimerization Cell Line (Cat. No. 93-1035C3)
  • PathHunter U2OS IL2RB/IL2RG Dimerization Cell Line (Cat. No. 93-0998C3)
  • PathHunter U2OS IL2RB/IL2RG/IL2RA Dimerization Cell Line (Cat. No. 93-1003C3)
  • PathHunter U2OS IL4R/IL2RG Dimerization Cell Line (Cat. No. 93-0988C3)
  • PathHunter U2OS IL2RB/IL2RG/IL2RA Dimerization Bioassay Kit (Cat. No. 93-1003Y3-00092)
Kinase inhibitors Jakotinib hydrochloride STAT3 PathHunter HepG2 STAT3 Pathway Reporter Cell Line (Cat. No. 93-1144C44)
Nonspecifc anti-inflammatory and immunosuppresive drugs Leflunomide   Contact Custom Assay Development
Monoclonal antibodies; to be used in combination with protease inhibitors Meplazumab   Contact Custom Assay Development
Monoclonal antibodies; to be used in combination with protease inhibitors PD-1 mAb PD-1
  • PathHunter Jurkat PD-1 (SHP1) Signaling Cell Line (Cat. No. 93-1104C19)
  • PathHunter PD-1 (SHP1) Signaling Bioassay Kit (Cat. No. 93-1104Y19-00118)
Miscellaneous others Pirfenidone TGFBR1
PDGFRa
PDGFRb
  • PathHunter U2OS TGFBR1/TGFBR2 Dimerization Cell Line (Cat. No. 93-0889C3)
  • PathHunter U2OS PDGFRa Functional Assay (Cat. No. 93-23C308)
  • PathHunter U2OS PDGFRb Functional Assay (Cat. No. 93-0493C3)
  • PathHunter U2OS PDGFRa Bioassay Kit (Cat. No. 93-0823Y3-00072)
  • PathHunter U2OS PDGFRb Bioassay Kit (Cat. No. 93-0493Y3-00074)
Miscellaneous others rhGM-CSF GM-CSF PathHunter Sargramostim Bioassay Kit (Cat. No. 93-1078Y3-00112)
Kinase inhibitors Ruxolitinib STAT3 PathHunter HepG2 STAT3 Pathway Reporter Cell Line (Cat. No. 93-1144C44)
Nonspecific anti-inflammatory and immunosuppressive drugs Thalidomide Varies SPRINTer™ Protein Turnover Biosensor Platform Contact Custom Assay Development
Monoclonal antibodies; to be used in combination with protease inhibitors Tocilizumab IL-6 PathHunter Tocilizumab Bioassay Kit (Cat. No. 93-1045B3-00110)

Additional cell lines and kits (and alternative sizes) are available for the therapeutics indicated above. Please contact technical support for more information

Cell-based assays to study therapeutic candidates associated with COVID-19 research are based on Eurofins DiscoverX's Enzyme Fragment Complementation (EFC) technology. Figures below highlight select assay designs for some widely investigated therapeutic targets implicated in cytokine storm in COVID-19 patients.

PathHunter® Tocilizumab Bioassay Assay Principle

PathHunter® Tocilizumab Bioassay Assay Principle

Measure IL-6 induced heterodimerization of IL-6 receptor (IL-6R) with the IL-6 Signal Transducer protein (IL-6ST; gp130) using the PathHunter Tocilizumab Bioassay (Cat. No. 93-1045B3-00110) qualified with the FDA approved therapeutic Actemra (tocilizumab, an anti-IL-6R antibody being evaluated as potential therapeutic for COVID-19). The assay principle involves the complementing of enzyme donor ProLink™ (PK) and enzyme acceptor (EA) EFC fragments tagged to IL-6ST and IL-6R, respectively. In presence of IL-6, the receptor units dimerize and there is an increase in signal detected upon substrate addition. However, in the presence of tocilizumab, the binding of IL-6 is inhibited and the signal decreases. Actemra® a registered trademark of Chugai Seiyaku Kabushiki Kaisha Corp., a member of the Roche Group.

PathHunter Anakinra Bioassay Assay Principle

PathHunter Anakinra Bioassay Assay Principle

Measure IL-1β induced heterodimerization of IL-1R1 and IL-1RAP receptors using the PathHunter Anakinra Bioassay (Cat. No. 93-1032Y3-00106) qualified with the FDA approved therapeutic Kineret (anakinra, an IL-1R antagonist being evaluated as potential therapeutic for COVID-19). The assay principle involves the complementing of PK and EA EFC fragments tagged to IL-1RAP and IL-R1, respectivitly. In presence of IL-1β, the receptors dimerize and there is an increase in signal detected upon substrate addition. However, in the presence of anakinra, the binding of IL-1β is inhibited and the signal decreases. Kineret® is a registered trademark, licensed by Swedish Orphan Biovitrum AB and marketed by Sobi, Inc.

PathHunter Sargramostim Bioassay Assay Principle

PathHunter Sargramostim Bioassay Assay Principle

Measure GM-CSF induced heterodimerization of CSF2RA with CSF2RB PathHunter Sargramostim Bioassay Kit (Cat. No. 93-1078Y3-00112) qualified with the FDA approved therapeutic Leukin (sargramostim), which is being evaluated as potential therapeutic for COVID-19. The assay principle involves the complementing of PK and EA EFC fragments tagged to CSF2RB and CSF2RA, respectivitly. In presence of GM-CSF or sargramostim, the receptors dimerize and there is an increase in signal detected upon substrate addition. Leukine® is a registered trademark licensed to Genzyme Corporation.

PathHunter Adalimumab Bioassay Assay Principle

PathHunter Adalimumab Bioassay Assay Principle

Detect IkB protein degradation as a result of TNFα-mediated activation of the NF-kB signaling pathway using PathHunter Adalimumab Bioassay Kit (Cat. No. 93-0538B15-00132) qualified with the FDA approved therapeutic Humira (adalimumab), which is being evaluated as potential therapeutic for COVID-19. The assay principle involves the enzyme donor ProLabel® (PL) tagged to phosphorylated IkB, and the IκB levels can then be measured by the addition of EA, which forces complementation of the two EFC fragments. The resulting active enzyme hydrolyzes the substrate to generate the signal that is proportional to the degree of IκB stabilization. In presence of TNFα, which activates the NF-kB signaling pathway, proteasome-mediated IκB degradation occurs and there is a loss of signal. In the presence of both TNFα and adalimumab, which blocks the binding of TNFα, degradation of IκB is avoided, and the EFC complementation occurs leading to an increase in signal upon substrate addition. Humira® is a registered trademark AbbVie, Inc.

Qualify COVID-19 Therapeutics with Receptor Dimerization Bioassays

Reproducibly measure ligand-mediated receptor dimerization for potency and stability testing of therapeutics such as Kineret® (anakinra) implemented in QC lot release programs.

Qualify COVID-19 Therapeutics with Receptor Dimerization Bioassays

 

The PathHunter Anakinra Bioassay (Cat. No. 93-1032Y3-00106) was used to quantify an inhibitory response from Kineret (anakinra, an IL-1R antagonist being evaluated as potential therapeutic for COVID-19). [A.] In the presence of anakinra, there is a dose dependent inhibition (signal-to-background (S/B) 4.2; IC50 302 ng/mL) of IL-R1 and IL-1RAP dimerization. In comparison with agonist IL-1β, the receptors dimerize and there is an increase in signal detected (S/B 5.3; EC50 4.2 ng/mL). [B.] Dilutional linearity derived from the relative potency experiments performed over a range of 50-150%. Overall, the bioassay measures a robust response to both anakinra and IL-1β with a high accurate potency of over 99.4%. Kineret is a registered trademark, licensed by Swedish Orphan Biovitrum AB and marketed by Sobi, Inc.

Measure TNFα-Mediated Activation of the NF-κB Signaling Pathway with MOA-Reflective, IkB Protein Degradation Bioassays

>Measure TNFα-Mediated Activation of the NF-κB Signaling Pathway with MOA-Reflective, IkB Protein Degradation Bioassays

 

The PathHunter Adalimumab Bioassay Kit (Cat. No. 93-0538B15-00132) was used to measure IκB protein degradation as a result of TNFα-mediated activation of the NF-kB signaling pathway. [A.] In the presence of adalimumab (Humira®), there is a dose dependent inhibition of TNFα-mediated activation IkB protein degradation and an increased signal (S/B 6.5; IC50 5.45 ng/mL). In comparison with TNFα alone, a decrease in signal detected (S/B 10.3; IC50 53.9 ng/mL). [B.] Dilutional linearity derived from the relative potency experiments performed over a range of 50-150%. Overall, the bioassay measures a robust response to both Humira and TNFα with an excellent accurate potency of over 99.99%. Humira is a registered trademark AbbVie, Inc.