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In vitro kinetic profiling by enzymatic approaches to select FAPIs with a long residence time
Emile Verhulst1, Pawel Brzeminski2, Anke de Groot1
1Laboratory of Medical Biochemistry, Faculty of Pharmaceutical, Biomedical and Veterinary Sciences, University of Antwerp, Wilrijk, 2610, Belgium.
European Journal of Medicinal Chemistry
|December 27, 2025
Summary
Developing new methods to measure how long cancer drug candidates bind to their targets is crucial. This study introduces a kinetic assay to better rank fibroblast activation protein alpha inhibitors for improved theranostic radioligand therapy.
Area of Science:
- Biochemistry and Molecular Biology
- Pharmacology and Pharmaceutical Sciences
- Oncology
Background:
- Fibroblast activation protein alpha (FAP) is a key target in cancer-associated fibroblasts, crucial for tumor progression and fibrosis.
- Current FAP inhibitors (FAPIs) for imaging have rapid dissociation, limiting tumor retention and theranostic applications.
- A validated in vitro method to assess FAPI-target residence time is needed for effective preclinical evaluation.
Purpose of the Study:
- To establish a medium-throughput in vitro assay for determining FAPI dissociation rate constants (koff) and drug-target residence time.
- To enable kinetic structure-activity relationship (SAR) analysis for rational FAPI design.
- To improve the selection and prioritization of FAPIs for theranostic radioligand therapy.
Main Methods:
- Utilized a jump-dilution assay to measure the dissociation rate constant (koff) of FAPIs under tight-binding conditions.
- Integrated progress-curve assays to determine association rate constants (kon) and inhibition constants (Ki).
- Performed kinetic SAR analysis and molecular docking to correlate binding kinetics with FAP structure.
Main Results:
- Successfully determined koff, kon, and Ki for various FAPIs, enabling direct calculation of drug-target residence time (τ = 1/koff).
- Demonstrated that kinetic profiling provides finer discrimination among FAPIs compared to conventional IC50 screening.
- Identified key structural features and warhead chemistries influencing FAPI binding kinetics and residence time.
Conclusions:
- The developed kinetic assay provides a robust method for estimating FAPI-target residence time, crucial for theranostic development.
- Kinetic profiling facilitates data-driven ranking of FAPIs, optimizing lead selection for preclinical studies.
- This approach supports the rational design of FAPIs with enhanced tumor retention for improved theranostic radioligand therapy.
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