Application Notes

Fast molecular interaction screening of epigenetic gene regulator G9a with fragments from a large chemical space

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APPLICATION NOTE 9 ©2019 NanoTemper Technologies, Inc. South San Francisco, CA, USA. All Rights Reserved. Discussion Dianthus NT.23PicoDuo was used to test the binding of a 2,490-fragment library to the cancer target G9a. By using TRIC as the biophysical technology to first detect and then quantify interactions, novel candidates were identified with high micromolar affinities for G9a from a library of fragments with a broad range of molecular mass and complexity. The entire fragment library was screened in duplicates (5,784 data points including controls) in less than 8 hours of instrument time, using less than 20 µg of target protein. Not only were novel G9a ligands identified in the single-dose experiments, but straightforward additional testing in the affinity screen step was used to further expand the hit list and investigate interesting compound-induced effects, such as aggregation. However, further testing and especially optimization of the identified interactors would be necessary in order to increase compound affinity, which can be performed with the same system and the same advantages shown for lead ID. Using Dianthus NT.23PicoDuo saved many days of screening time compared to other high throughput screening systems by at least a factor of 6 in some cases. Combining its speed, flexible throughput and ability to screen fragments from a large chemical space, Dianthus is the perfect fit and primary choice for fragment-based lead discovery. Materials and Methods G9a was labeled following the labeling protocol as specified in NanoTemper Technologies RED-NHS 2nd Generation labeling kit. The assay buffer for all experiments was 50 mM HEPES, pH 8.0, 150 mM NaCl, 2 mM GSH, 0.005% TWEEN® 20 and a final volume of 20 µL per datapoint was used for both the single-dose and the affinity screen in Dianthus 384-well microwell plates. The fragment library was dissolved in 100% DMSO at a concentration of 100 mM and SAM was dissolved in water to a concentration of 10 mM. In all experiments with SAM as a ligand, a final amount of 1% DMSO was added to mimic the conditions for fragment dilutions. We used a HAMILTON STARlet system for liquid handling. First, we diluted the fragments from the 100 mM stock solution into assay buffer to 4 mM and 4% DMSO. In a second step, the fragments were further diluted into a G9a- containing solution to a final concentration of 1 mM and 1%DMSO. Labeled G9a was used at a final concentration of 5 nM. The first dilution was into a conventional microwell plate, while the second dilution step occurred in the Dianthus 384-well microplate, where the measurements took place. For affinity measurements, fragments were pre- diluted to 2 mM stocks in assay buffer, and 0.5-fold dilution were prepared in assay buffer containing

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