Inserting “OFF-to-ON” BODIPY Tags into Cytokines: A Fluorogenic Interleukin IL-33 for Real-Time Imaging of Immune Cells

Abstract

The essential functions that cytokine/immune cell interactions play in tissue homeostasis and during disease have prompted the molecular design of targeted fluorophores to monitor their activity in real time. Whereas activatable probes for imaging immune-related enzymes are common, many immunological functions are mediated by binding events between cytokines and their cognate receptors that are hard to monitor by live-cell imaging. A prime example is interleukin-33 (IL-33), a key cytokine in innate and adaptive immunity, whose interaction with the ST2 cell-surface receptor results in downstream signaling and activation of NF-κB and AP-1 pathways. In the present work, we have designed a chemical platform to site-specifically introduce OFF-to-ON BODIPY fluorophores into full cytokine proteins and generate the first nativelike fluorescent analogues of IL-33. Among different incorporation strategies, chemical aminoacylation followed by bioorthogonal derivatization led to the best labeling results. Importantly, the BODIPY-labeled IL-33 derivatives�unlike IL-33-GFP constructs�exhibited ST2-specific binding and downstream bioactivity profiles comparable to those of the wild-type interleukin. Real-time fluorescence microscopy assays under no wash conditions confirmed the internalization of IL-33 through ST2 receptors and its intracellular trafficking through the endosomal pathway. We envision that the modularity and versatility of our BODIPY labeling platform will facilitate the synthesis of minimally tagged fluorogenic cytokines as the next generation of imaging reagents for real-time visualization of signaling events in live immune cells.

Rational Design of Pyrido[3,2-b]indolizine as a Tunable Fluorescent Scaffold for Fluorogenic Bioimaging

“…Beyond fluorescence labeling, fUAAs with environment-sensing fluorogenicity, namely fluorogenic unnatural amino acids (FUAAs), further enable background-free visualization of biomolecular interactions, especially for protein–ligand binding or PPIs. Despite several applications of fluorogenic cores (e.g., BODIPY and NBD) and their analogs in FUAAs, , the development of small and biocompatible fluorogenic unnatural amino acids remains in high demand but still presents significant challenges.…”

Brought to Light: A Fluorogenic Probe to Monitor Immunosuppressants

“…Vendrell et al already have experience in developing fluorogenic peptide probes for in vivo labeling. For instance, they showed efficient linker-free coupling of BODIPY to tryptophane. − This time they envisaged a fluorogenic protein probe for immunosuppressive drug monitoring using the immunophilins peptidylprolyl isomerase A (PPIA) and FK506-binding protein (FKBP12) as protein receptors that tightly bind immunosuppressive drugs such as tacrolimus. A key aspect of the design was a short linker between the fluorophore and the protein so that the binding of a relatively small ligand, such as the cyclic peptide tacrolimus, would result in the desired turn-on fluorescence.…”