Target and Pathway Engagement Assays

While biophysical and biochemical assays are best suited to drive the earlier stages of the drug development cascade, as confidence in a drug candidate grows it is important to measure its activity within a more complex cellular environment, providing a more accurate assessment of drug behavior.  

We have extensive experience in identifying and executing the correct cell-based target engagement assays to support drug discovery projects. Examples of our target and pathway engagement assays include, but are not limited to: 

• Cyclic AMP (cAMP) and Cyclic GMP (cGMP) assays (e.g., Glo^TM assays, NanoBret, AlphaScreen) 
• Calcium assays (e.g., Calcium imaging, FLIPR) 
• Inositol triphosphate (IP3) assays (e.g., IP-one assay) 
• Quantification of total and phosphorylated proteins (e.g., HTRF, AlphaLISA, Jess, western blotting, flow cytometry) 
• Caspase-1 activation assays (e.g., Caspase-GLOTM assay) 
• β-arrestin activation assays (e.g., HTRF) 

Why are target and pathway engagement assays important? 

The drug discovery pipeline faces a well-known problem with the very high attrition rate of potential drug candidates. A contributing factor to this is the lack of on-target engagement and target specificity, leading to potential off-target toxicity.  

Thus, proving the target engagement of a lead compound early in a drug discovery project is imperative. This approach ensures that project resources are best utilized on the correct compounds, maximizing the chances of successful clinical progression. 

Example applications of target and pathway engagement assays 

The NLRP3 inflammasome is a vital component of the innate immune system, responsible for Caspase-1 activation and the secretion of proinflammatory cytokines. We have developed a robust assay system designed for screening NLRP3 inhibitors. Using biologically relevant THP-1 cells and a highly sensitive Caspase-1 readout, this system enables efficient high-throughput compound screening.