Metal impurities cause false positives in high-throughput screening campaigns

Area of Science:

• Drug discovery
• Chemical biology
• Assay development

Background:

• Organic impurities are known causes of false positives in screening campaigns.
• However, not all false positives can be attributed to organic contaminants.
• Inorganic impurities represent an underappreciated source of assay interference.

Purpose of the Study:

• To identify inorganic impurities in screening libraries that cause false-positive results.
• To investigate the mechanism of interference by specific inorganic impurities.
• To develop a method for detecting and mitigating inorganic impurity interference.

Main Methods:

• Screening of compound libraries for unexpected assay signals.
• Targeted investigation of inorganic compounds, specifically zinc.
• Development and application of a counter screen using the chelator TPEN.
• Retrospective analysis of high-throughput screening (HTS) data.

Main Results:

• Discovery of inorganic impurities, including zinc, causing false positives across various assay types (biochemical, biosensor).
• Demonstration that zinc can interfere with multiple targets and readout systems.
• Validation of TPEN as an effective chelator to identify zinc-induced false positives.
• Identification of zinc as a common interferent in historical HTS screens.

Conclusions:

• Inorganic impurities are a significant, often overlooked, source of false positives in drug discovery screening.
• Zinc is a prevalent inorganic impurity that can confound assay results.
• The proposed TPEN counter screen provides a robust method to eliminate zinc-related false positives, improving screening reliability.