ASSAYS FOR COVID-19 DRUG DISCOVERY

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.

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

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

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

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

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.

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

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.