Particularly for hits from cell-based HTS, it is important to decipher the actual mode of action of your hits. Follow-up assays that reveal the nature of compound–target interactions, such as cellular binding or in-cell residence time assays, are important tools to help with making decisions. It is also important to know if an observed activity of a hit is driven by the desired mechanism or cellular pathway.
In these instances, there are necessary specialized assays that can be used to narrow an existing list of hits or to deeper characterize a selected number of hits that are already advanced in the hit-to-lead process.
Advanced Cell-Based Assays to Shape the Profile of Your Hits:
- Off-target and species selectivity assays.
- NanoBRET/NanoGlo target engagement for a wide variety of membrane or intracellular assays.
- Tag-Lite binding for GPCRs.
- Cellular thermal shift assays (CETSA®) to test cellular label-free target engagement.
- Kinetic probe competition assay (kPCA) for different target types, such as enzymes, protein–protein interactions, or GPCRs (Schiele, et al., Anal Biochem (2015)).
- Residence time assays using high-content analysis.
- Rescue experiments (different assay platforms); example for a protein degrader project: Is the observed protein level decrease related to proteasomal degradation? This question can be answered if the degradation assay is run in the presence of specific proteasome or E3-ligase inhibitors.
Specialized Phenotypic Cell-Based Assays:
- Cell viability
- Proliferation
- DNA damage
- Apoptosis
- RT-qPCR
- Sequencing
- Cytokine panel
