In the assay set up phase, Exquiron first provided uniformity plates and serial dilutions of a reference inhibitor for potency determination on both targets. Next, 10,000 compounds were provided at two different concentrations for a pilot screen to determine compound concentration for HTS.
After determining an appropriate screening concentration of 10 µM, the remaining 240,000 compounds from the Exquiron collection were plated and shipped to SAMDI Tech in less than three weeks. The HTS was completed in four weeks, including data analysis and hit calling. The assays were very robust with average Z’ of 0.82, and only one plate (from 740 plates) failed the Z’ quality criterion of >0.6 (Figure B). Given the low plate failure rate, compounds from this plate were repeated in the hit confirmation phase.
For activity confirmation of putative hits, 6,600 compounds were cherry-picked in duplicates and were provided to SAMDI Tech within one week of hit calling decision. The same compound set was picked and tested in parallel in a counter assay performed at Exquiron.
Finally, 692 confirmed active compounds were picked and formatted as serial dilutions in quadruplicates, and provided to SAMDI Tech for potency determination. In parallel, the same set of compounds was tested in the counter assay at Exquiron, and purity was determined by LCMS analysis. The entire data analysis and curve fitting was carried out at Exquiron, with IC50 values, graphical overlay plots and results of purity determination provided 4 weeks after initiation of this phase.
A summary of the project with phases and timelines is shown in the table.
|Project Phase||No. Compound Plates||No. Compounds||No. Data Points||Average Z'||Turnaround (Weeks)*|
|Pilot Screen||60 (2x 30)||10,000||40,000||0.80||3|
|Hit Confirmation||76 (2x 38)||6,600||26,400||0.84 (enzymes)
|IC50 Determination||132 (3x 34)||692||41,520||0.79 (enzymes)
*All timelines include preparation and shipment of assay-ready compound plates to SAMDI Tech in the United States.