The screening effort progressed through stages including:
- Structure-based virtual screening. The purpose of which is to employ structure-based virtual screening methods to select approximately 500 compounds capable of inhibiting Fyn-kinase from a broad diversity set.
- Purity analysis and IC50 determination of Fyn-kinase hits. To perform verification and validation of active compounds (IC50 determination) as well as to determine purity of selected active compounds by LC-MS analysis.
- Hit expansion. To select 2,000 structurally related compounds, which will be screened in the primary assay, and perform validation of actives (IC50 determination, up to 200 compounds), plus determine the purity of all compounds progressed to potency determination by LC-MS analysis.
- Active-to-hit studies. The goal is to deliver at least two distinct chemical series suitable for progression into a hit-to-lead phase.
- Hit identification phase. Resulted in 116 compounds spread over five SFK (sarc family kinase) chemical series libraries that are effective at inhibiting Fyn kinase and are thus effective at activating the target LKB-1 AMPK pathway.