In vitro kinetic profiling by enzymatic approaches to select FAPIs with a long residence time

Fibroblast activation protein α (FAP) is a transmembrane serine protease overexpressed in cancer-associated fibroblasts and implicated in tumor progression and fibrosis. Although several FAP inhibitors (FAPIs) show excellent tumor uptake for positron emission tomography (PET) imaging, rapid target dissociation often limits tumor retention and constrains their use in theranostic radioligand therapy. Considerable effort is directed toward FAPI radioligands with prolonged tumor residence time, yet a validated, medium-throughput in vitro method to estimate FAPI-target residence time remains lacking, hindering correct prioritization of lead radioligands for preclinical evaluation. In this study, we employ a jump-dilution assay to determine the dissociation rate constant (k_off) of FAPIs under tight-binding conditions, providing a direct handle on drug-target residence time (τ = 1/k_off). We combine this assay with progress-curve assays to determine the association rate constant (k_on) and inhibition constant (Kᵢ). By resolving binding kinetics, especially k_off, this approach addresses limitations of conventional IC₅₀-based screening, offering a finer discrimination among leads and enabling data-driven ranking based on target residence time. We demonstrate the utility of this workflow through a kinetic structure-activity relationship (SAR) analysis on FAPIs with varying structural features and warhead chemistries (carbonitrile, α-ketoamide). Molecular docking studies allowed us to correlate experimental kinetic parameters with predicted binding modes within the three-dimensional structure of FAP, focusing on critical interactions within the active site. These findings highlight the value of kinetic profiling in FAPI development and support the rational design of theranostic agents with prolonged target retention.