Displacement Induced Off–On Fluorescent Biosensor Targeting IDO1 Activity in Live Cells

Abstract

We show how the macrocyclic host cucurbit[8]­uril (CB[8]) and a fluorescent dye form a biosensing ensemble while its cavity simultaneously traps tryptophan, the upstream substrate of IDO1 enzymes, therefore providing a label-free method to monitor the activity of IDO1 in real time. Incubation of malignant HeLa and HepG2 cells overexpressing IDO1 with the associative biosensor resulted in its spontaneous uptake and a fluorescence switch-on response in situ, which can be traced to the displacement of tryptophan from CB[8] upon IDO1-catalyzed oxidation. The results, for the first time, establish a supramolecular sensing concept for the detection of intracellular enzymatic activity in live cells, thus allowing direct cell-based analysis and inhibitor screening compatible with commercial instruments including microplate reader, fluorescent microscopy, and flow cytometry.

Molecular Probes, Chemosensors, and Nanosensors for Optical Detection of Biorelevant Molecules and Ions in Aqueous Media and Biofluids

“…49,222 Di, Xu, and Hu utilized the CB8•MDPP chemosensor (C2.13) to visualize the enzymatic activity of cytosolic enzyme indoleamine 2,3-dioxygenase 1 (IDO1) in living cells. 269 This enzyme catalyzes the oxidation of tryptophan into N′-formylkynurenine, which is a much weaker binder and poorer fluorescence quencher for the chemosensor. Thus, the enzymatic activity of IDO1 in malignant HeLa and HepG2 cells overexpressing IDO1 was readily monitored by following the emission turn-on response of the chemosensor upon consumption of Trp.…”

“…In contrast, the CB8•PMI complex ( C2.12 ) dissociates upon addition of micromolar quantities of Phe-containing peptides, providing an indicator displacement assay with emission quenching in aqueous media . High binding affinities for Trp- and Phe-type compounds and useful spectroscopic responses (emission, absorbance, CD) can be achieved with CB8-based chemosensors that utilize the methylated diazaperoperylenium (MDPP) as dye. , Di, Xu, and Hu utilized the CB8•MDPP chemosensor ( C2.13 ) to visualize the enzymatic activity of cytosolic enzyme indoleamine 2,3-dioxygenase 1 (IDO1) in living cells . This enzyme catalyzes the oxidation of tryptophan into N ′-formylkynurenine, which is a much weaker binder and poorer fluorescence quencher for the chemosensor.…”

Cucurbit [8] uril-based supramolecular fluorescent biomaterials for cytotoxicity and imaging studies of kidney cells

“…From the structural point of view, the rigid cavity of Q [ n ]s derives the classical Q [ n ]-based host-guest chemistry, the rich carbonyl oxygen of its portal yields the Q [ n ]-based coordination chemistry, and positively density outer surface of the Q [ n ] rich in methane breeds the novel outer surface interaction of Q [ n ] ( Ni et al, 2014 ). Therefore, compared with other macrocycles ( Liu Z. et al, 2017 ; Murray et al, 2017 ), Q [ n ]s distinguish themselves via the excellent ability to form inclusion complexes with various guest molecules through host-guest, coordination, and out surface interactions ( Assaf and Nau, 2015 ; Barrow et al, 2015 ; Liu J. et al, 2017 ; Jia et al, 2019 ; Lin et al, 2020 ; Yang et al, 2021 ; Zhang et al, 2021 ; Wang et al, 2022 ).…”

Cell‐Based Identification of New IDO1 Modulator Chemotypes

“…Currently employed cell-based assays monitoring IDO1 activity utilize p-dimethylamino benzaldehyde (p-DMAB), [11] high-performance liquid chromatography (HPLC), [12] NFK Green [13] or cucurbit [8]uril-dimethyldiazaper-opyrenium dication complex (MP&CB [8]). [14] HPLCbased techniques or p-DMAB require a transfer of cell supernatant [11] which is technologically challenging in screening assays. NFK Green displays a low dynamic detection range in cells [13] and MP&CB [8]-mediated detection of Trp requires homogenous diffusion across cell membranes.…”

“…NFK Green displays a low dynamic detection range in cells [13] and MP&CB [8]-mediated detection of Trp requires homogenous diffusion across cell membranes. [14] Thus, there is a high demand for new cell-based screening assays for identification of IDO1 inhibitor classes.…”