FLIM-FRET Protein-Protein Interaction Assay.

Fluorescence lifetime imaging performed under FRET conditions between two interacting molecules is a sensitive and robust way to quantify intermolecular interactions in cells. The fluorescence lifetime, an inherent property of the fluorophore, remains unaffected by factors such as concentration, laser intensity, and other photophysical artifacts. In the context of FLIM-FRET, the focus lies on measuring the fluorescence lifetime of the donor molecule, which diminishes upon interaction with a neighboring acceptor molecule. In this study, we present a step-by-step experimental protocol for applying FLIM-FRET to investigate protein-protein interactions involving various RAS isoforms and RAS effectors at the live cell's plasma membrane. By utilizing the FRET pair comprising enhanced green fluorescent protein (eGFP) and fluorescent mCherry, we demonstrate that the proximity and possible nanoclustering of eGFP-tagged KRAS4b G12D and mCherry-tagged KRAS4b WT led to a reduction in the donor eGFP's fluorescence lifetime. The donor lifetime of eGFP-tagged KRAS decreases even further when treated with a dimer-inducing small molecule, or in the presence of RAF proteins, suggesting a greater FRET efficiency, and thus less distance, between donor and acceptor.

Virtual screening of ultra-large chemical libraries identifies cell-permeable small-molecule inhibitors of a "non-druggable" target, STAT3 N-terminal domain.

STAT3 N-terminal domain is a promising molecular target for cancer treatment and modulation of immune responses. However, STAT3 is localized in the cytoplasm, mitochondria, and nuclei, and thus, is inaccessible to therapeutic antibodies. Its N-terminal domain lacks deep pockets on the surface and represents a typical "non-druggable" protein. In order to successfully identify potent and selective inhibitors of the domain, we have used virtual screening of billion structure-sized virtual libraries of make-on-demand screening samples. The results suggest that the expansion of accessible chemical space by cutting-edge ultra-large virtual compound databases can lead to successful development of small molecule drugs for hard-to-target intracellular proteins.