Collaborative hit discovery project with SAMDI Tech



Exquiron and SAMDI Tech worked jointly on a Client’s hit finding campaign in which two related enzymes (Enz1 and Enz2) were targeted. In a duplexed assay format, the two distinct peptide substrates (S1 and S2) and products (P1 and P2) were detected and quantified by the SAMDI label-free assay technology (Figure A, courtesy of SAMDI Tech, Inc.). Being a mass spectrometry-based platform, SAMDI allows multi-analyte analysis without the need for antibodies, fluorophores, or radioactivity. Exquiron provided assay-ready compound plates, SAMDI Tech developed and optimized the assays, performed HTS using its platform, and provided data files to Exquiron for data evaluation. Exquiron compiled data and determined hits using specific criteria, and provided characterization of selected compounds in a counter-screening assay with fluorescence readout.

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)*
Assay Development 18 - - N/A 3
Pilot Screen 60 (2x 30) 10,000 40,000 0.80 3
HTS 740 240,000 480,000 0.82 7
Hit Confirmation 76 (2x 38) 6,600 26,400 0.84 (enzymes)
0.90 (counter)
IC50 Determination 132 (3x 34) 692 41,520 0.79 (enzymes)
0.89 (counter)

*All timelines include preparation and shipment of assay-ready compound plates to SAMDI Tech in the United States.