| 1) Exposure at the site of action |
• On- and off-target affinity and selectivity |
| 2) Target engagement |
• Solubility and toxicity profile |
| 3) Functional pharmacological activity |
• Chemical and/or metabolic stability |
| Ensure requirements are fulfilled in all assays: biochemical, in-cell assays (in vitro) and animal models (in vivo). Confirm that the phenotype is relevant to human disease. |
• Cell permeability/in vivo activity (including active efflux) |
| • Lipophilicity (calculated or measured via clog P) |
| • Assay interference and/or promiscuous binding |
| • Structural integrity and purity (particularly important for purchased compounds from commercial vendors) |
