METTL3 cellular target engagement

Cellular target engagement of STM2457 was measured by thermal shift using the InCell Pulse Assay (DiscoverX). HeLa cells were transfected with pICP-hMETTL3-eLP (human METTL3 Met1-Leu580) or pICP-mMETTL3-eLP (mouse METTL3 Met1-Leu580), using Fugene HD (Promega, #E2311) following the supplier’s protocol. 24 hours post transfection, cells were frozen and stored in liquid nitrogen until the day of the assay. On the day of the assay 100 nL compound dilutions (11 point 3-fold dilutions, top concentration in assay: 25 μM) were spotted into the assay plate (Greiner Dilution, 384-well, PP, transparent, #784201). The transfected cells were thawed in a water bath at 37°C and cryoprotectant was exchanged with Opti-MEM lacking phenol red (Gibco, Cat #11058-021). 20 μL of the cell suspension was added to each well of the assay plate to give a final cell number of 1360 cells/well. Plates were sealed with aluminium foil and incubated for 1 hour at 37°C. Afterwards, plates were incubated for 15 minutes upside down in a water bath at 45°C, further incubated for 10 minutes at room temperature, and finally centrifuged briefly to gather the liquid in the bottom of each well. Subsequently, 25 μL of detection solution (InCELL Hunter Detection Kit, DiscoverX, #96-0079L; working solution: 16.7 v/v EA reagent, 16.7 v/v lysis buffer and 66.7% substrate reagent) was added to each well and the solution was transferred to the measurement plate (Corning, 384-well, PS, black, Flat Bottom, #3575). The plate was sealed with aluminium foil and incubated at room temperature and slow shaking for 30 minutes. Finally, the assay plate was centrifuged for 1 minute at 100 x g and room temperature and luminescence was measured using an EnVision multimode plate reader (Perkin Elmer). Dose response curves were obtained from three biological replicates. All data in this section were plotted using GraphPad Prism (Version 9).