Recent progress in the application of fluorescence polarization assays to small molecule screening

Fluorescence polarization (FP) is a homogeneous, separation-free technique for characterizing intermolecular interactions. By virtue of its low cost, simplicity, and high reproducibility, FP is inherently amenable to high-throughput screening (HTS) of small molecules, rendering it an indispensable platform in drug discovery.

This review collects literature from original publications, standard sources, and PubMed, with relevant compound information verified in PubChem. The authors summarize the small molecules identified by FP assay from 2023 to the present according to a target classification scheme, while briefly analyzing application trends. The authors also introduce several newly constructed FP assay platforms that have not yet been deployed for large-scale screening.

To enable larger-scale, higher-precision screening and expand FP into emerging target classes, ongoing efforts have focused on probe design, signal-to-noise ratio enhancement, and automated instrumentation, and have successfully achieved several technological innovations. Nevertheless, given the intrinsic limitations of FP--insufficient sensitivity for detecting weak interactions, difficulty in obtaining high-affinity probes and stable buffer conditions, pervasive false positives--it is necessary to consider complementary approaches and employ multilevel orthogonal validation strategies. Future development is shifting toward the integration of FP with diverse technologies: the introduction of data-driven algorithms and deep learning is expected to more effectively mitigate false positives, and the convergence of time-resolved fluorescence anisotropy imaging (TR-FAIM) strategy holds promise for establishing an integrated universal platform that couples small molecule screening and validation, fully grounded in the FP principle.