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Chasing Red Herrings: Palladium Metal Salt Impurities Feigning KRAS Activity in Biochemical Assays
Thomas Gerstberger1, Helmut Berger1, Frank H Büttner2
1Boehringer Ingelheim RCV GmbH & Co. KG, Dr. Boehringer Gasse 5-11, A-1121 Vienna, Austria.
Journal of Medicinal Chemistry
|July 15, 2024
Summary
Assay artifacts from metal salts, specifically palladium impurities, falsely indicated KRAS inhibitors in a high-throughput screen. A Metal Ion Interference Set (MIIS) was developed to identify and mitigate such interferences in biochemical assays.
Area of Science:
- Medicinal Chemistry
- Biochemistry
- Oncology
Background:
- Developing small molecule inhibitors for the active, GTP-loaded KRAS on-state is crucial for clinical oncology.
- Identifying reliable chemical starting points for KRAS inhibitors presents a significant medicinal chemistry challenge.
- Initial high-throughput screening efforts for KRAS inhibitors yielded promising hits.
Purpose of the Study:
- To report learnings from a KRAS hit-finding campaign.
- To address the issue of assay artifacts in biochemical screening.
- To develop and validate a method for assessing metal ion interference in assays.
Main Methods:
- A KRAS hit-finding campaign utilizing biochemical high-throughput screening.
- Investigation of compound potencies and identification of assay artifacts.
- Development and application of a Metal Ion Interference Set (MIIS) for assay validation.
- Profiling the MIIS across 74 diverse assays, including label-free biophysical assays.
Main Results:
- Initial KRAS inhibitor hits were found to be artifacts caused by metal salts interfering with the AlphaScreen assay technology.
- Palladium impurities from chemical synthesis were identified as the source of apparent KRAS inhibition.
- Label-free biophysical assays demonstrated reduced susceptibility to metal ion interference compared to luminescence- and fluorescence-based assays.
- General estimates for metal salt interference in luminescence- and fluorescence-based assays were established.
Conclusions:
- Careful assay development and validation are essential to avoid false positives in drug discovery screening.
- The Metal Ion Interference Set (MIIS) is a valuable tool for up-front assay characterization and mitigation of metal ion interference.
- Understanding assay technology limitations, such as metal salt interference, is critical for reliable identification of potential drug candidates.
