An Endoperoxide Reactivity-Based FRET Probe for Ratiometric Fluorescence Imaging of Labile Iron Pools in Living Cells

Abstract

Iron is essential for sustaining life, as its ability to cycle between multiple oxidation states is critical for catalyzing chemical transformations in biological systems. However, without proper regulation, this same redox capacity can trigger oxidative stress events that contribute to aging along with diseases ranging from cancer to cardiovascular and neurodegenerative disorders. Despite its importance, methods for monitoring biological iron bound weakly to cellular ligands−the labile iron pool−to generate a response that preserves spatial and temporal information remain limited, owing to the potent fluorescence quenching ability of iron. We report the design, synthesis, and biological evaluation of FRET Iron Probe 1 (FIP-1), a reactivity-based probe that enables ratiometric fluorescence imaging of labile iron pools in living systems. Inspired by antimalarial natural products and related therapeutics, FIP-1 links two fluorophores (fluorescein and Cy3) through an Fe(II)-cleavable endoperoxide bridge, where Fe(II)-triggered peroxide cleavage leads to a decrease in fluorescence resonance energy transfer (FRET) from the fluorescein donor to Cy3 acceptor by splitting these two dyes into separate fragments. FIP-1 responds to Fe(II) in aqueous buffer with selectivity over competing metal ions and is capable of detecting changes in labile iron pools within living cells with iron supplementation and/or depletion. Moreover, application of FIP-1 to a model of ferroptosis reveals a change in labile iron pools during this form of cell death, providing a starting point to study iron signaling in living systems.

In vivo bioluminescence imaging of labile iron accumulation in a murine model of Acinetobacter baumannii infection

“…1B). A modest response is observed with free copper salts, as is similarly observed for the related fluorescence probe FIP-1 (15). However, as a typical eukaryotic cell exhibits a ∼10-fold higher level of iron over copper coupled with the high buffering capacity of copper with glutathione and metallochaperones (picomolar to femtomolar K d values) (55)(56)(57)(58)(59)(60), the modest response to free copper salts suggests that ICL-1 should have sufficient selectivity to detect alterations in biological ferrous iron levels.…”

“…In ICL-1, we caged D-aminoluciferin with an Fe 2+ -reactive endoperoxide trigger (15,17,51) inspired by antimalarial agents that exhibit Fe 2+ -dependent pharmacology (52,53). ICL-1 was designed to undergo metal-and redox-specific Fe 2+ -dependent cleavage to generate D-aminoluciferin, which can interact with the firefly luciferase enzyme to produce red light output through a catalytic bioluminescent reaction.…”

“…In this regard, detection of iron with both metal and oxidation state specificity is of central importance, because while iron is stored primarily in the ferric oxidation state, a ferrous iron pool loosely bound to cellular ligands, defined as the labile iron pool (LIP), exists at the center of highly regulated networks that control iron acquisition, trafficking, and excretion. Indeed, as a weak binder on the Irving-Williams stability series (13), Fe 2+ provides a challenge for detection by traditional recognition-based approaches (14), and as such we (15)(16)(17) and others (18)(19)(20) have pursued activity-based sensing approaches to detect labile Fe 2+ stores in cells (21)(22)(23)(24)(25). These tools have already provided insights into iron biology, as illustrated by the direct identification of elevations in LIPs during ferroptosis (26,27), an emerging form of cell death, using the ratiometric iron indicator FIP-1 (15).…”

Recent Progress in Ferroptosis Inducers for Cancer Therapy

“…F) Fluorescence images of live HEK 293T cells stained with 10 × 10 −6 m FIP‐1 after treatment with bathophenanthroline disulfonate (BPS, ferrous iron chelator, 1 × 10 −3 m ); deferoxamine (DFO, ferric iron chelator, 0.25 × 10 −3 m ); vehicle (DMSO, control) and Fe(NH 4 ) 2 (SO 4 ) 2 (H 2 O) 6 (ferroptosis inducer, 0.1 × 10 −3 m ), respectively. (Scale bar: 25 µm) Reproduced with permission . Copyright 2016, American Chemical Society.…”

Analytical Methods for Imaging Metals in Biology: From Transition Metal Metabolism to Transition Metal Signaling

“…FIP-1 is a FRET-based probe that uses an endoperoxide trigger 135 to cleave a linker between 5-aminomethyl coumarin and fluorescein in the presence of Fe 2+ 136 . This direct and oxygen-independent 2-component reaction proceeds rapidly in the presence of Fe 2+ and results in a 2-fold FRET change.…”