A rebinding-assay for measuring extreme kinetics using label-free biosensors.

Area of Science:

• Biochemistry
• Pharmacology
• Chemical Biology

Background:

• In vitro kinetic measurements are crucial for drug discovery, aiding in target binding confirmation and property optimization.
• Early-stage drug candidates often display transient kinetics, posing measurement challenges in standard drug discovery workflows.
• Traditional methods for irreversible inhibitors and rapid association processes have limitations in resolving fundamental kinetic parameters.

Purpose of the Study:

• To introduce a practical kinetic rebinding assay for the screening and characterization of drug compounds.
• To address limitations in measuring transient kinetics and rapid association rates in drug discovery.
• To enable the estimation of kinetic interaction constants, including the association rate constant, decoupled from dissociation.

Main Methods:

• Development of a probe-based assay utilizing mixed-phase partitioning in a flow-injection configuration.
• Implementation of label-free biosensing technology for kinetic measurements.
• Application of a finite element analysis-based biosensor model to simulate rebinding inhibition and generate surrogate data.
• Validation of an algebraic model for estimating kinetic interaction constants using surrogate and experimental data.

Main Results:

• The developed assay successfully measures challenging kinetic interactions, including transient kinetics and rapid association rates.
• A validated algebraic model allows for the estimation of kinetic interaction constants.
• Experimental proof-of-principle demonstrated the ability to estimate the association rate constant independently of the dissociation process.
• The assay provides enhanced capabilities for kinetic screening and characterization of compounds in drug discovery.

Conclusions:

• The introduced kinetic rebinding assay offers a practical and effective tool for characterizing drug-binding kinetics, particularly for challenging interactions.
• This method advances drug discovery by providing accurate kinetic data crucial for lead optimization and pre-clinical development.
• The assay's ability to decouple association and dissociation rates offers deeper mechanistic insights into drug-target interactions.