Fluorescent PyrAte-(S)-citalopram conjugates enable imaging of the serotonin transporter in living tissue

Fluorescent labeling techniques have enabled the visualization of various biomolecules, cellular structures, and their associated physiological processes. At the same time, there remains a demand for developing novel fluorescent compounds possessing unique chemical properties for biological imaging. A recently developed class of fluorophores, termed PyrAtes, displays optimal brightness and large Stokes shifts that are ideal for fluorescence microscopy. Herein, we report the development of PyrAte-based fluorescently labeled ligands that bind to the serotonin transporter (SERT), a membrane transport protein important for neurotransmitter homeostasis, which hitherto has not been visualized in its native environment using fluorescent small molecules. The design of a PyrAte fluorophore attached to (S)-citalopram, a selective serotonin reuptake inhibitor, resulted in the synthesis of two fluorescent drug conjugates varying in linker length: PYR-C6-CIT and PYR-C3-CIT. Docking and molecular dynamics experiments are performed to estimate their binding affinities to SERT. Our in vitro experiments confirm both compounds are effectively binding to SERT overexpressed in human embryonic kidney 293 cells, with the shorter conjugate displaying improved SERT affinity and membrane staining properties. Furthermore, ex vivo imaging of endogenous SERT was demonstrated in acute mouse brain slices using two-photon microscopy. The large Stokes shift of the PyrAte fluorophore enables simultaneous detection of its own fluorescence signal at 500 nm along with that of a yellow fluorescent protein-based serotonergic marker. Our findings provide novel tools for unprecedented SERT visualization and establish the utility of PyrAtes for the selective staining of membrane proteins in live cells and tissue.