A Fundamental Source of Misclassification in Single-Concentration Binding Screens

Area of Science:

• Biochemistry
• Chemical Biology
• Drug Discovery

Background:

• High-throughput screening (HTS) is crucial for identifying drug candidates and chemical probes.
• Primary HTS assays typically use a single compound concentration to identify potential hits.
• Hits advance to secondary assays for detailed characterization, including equilibrium dissociation constant (Kd) determination.

Purpose of the Study:

• To identify a fundamental vulnerability in single-concentration binding screens.
• To analyze how signal nonadditivity amplifies errors in hit calling.
• To develop strategies to mitigate misclassification risks in HTS.

Main Methods:

• First-principles analysis of compound-target binding signals.
• End-to-end simulations with known Kd values and signal transformations.
• Mapping of operating regimes where signal amplification is most severe.

Main Results:

• Signal nonadditivity, arising from nonlinear signal generation or processing, can significantly amplify small errors in compound and target concentrations.
• This amplification leads to inaccurate single-point readouts, causing false negatives (missed binders) and potentially penalizing true high-affinity binders.
• Simulations identified specific regimes where this distortion is most pronounced.

Conclusions:

• Single-concentration screens possess a fundamental vulnerability related to signal additivity.
• A practical workflow is proposed to address this: define Kd range, use ligand-excess design, control concentrations, test readout additivity/monotonicity, and assess misclassification risk.
• Implementing these strategies can improve the reliability of hit identification in drug discovery screening.