Activatable Fluorescence Probe via Self-Immolative Intramolecular Cyclization for Histone Deacetylase Imaging in Live Cells and Tissues

Abstract

Histone deacetylases (HDACs) play essential roles in transcription regulation and are valuable theranostic targets. However, there are no activatable fluorescent probes for imaging of HDAC activity in live cells. Here, we develop for the first time a novel activatable two-photon fluorescence probe that enables in situ imaging of HDAC activity in living cells and tissues. The probe is designed by conjugating an acetyl-lysine mimic substrate to a masked aldehyde-containing fluorophore via a cyanoester linker. Upon deacetylation by HDAC, the probe undergoes a rapid self-immolative intramolecular cyclization reaction, producing a cyanohydrin intermediate that is spontaneously rapidly decomposed into the highly fluorescent aldehyde-containing two-photon fluorophore. The probe is shown to exhibit high sensitivity, high specificity, and fast response for HDAC detection in vitro. Imaging studies reveal that the probe is able to directly visualize and monitor HDAC activity in living cells. Moreover, the probe is demonstrated to have the capability of two-photon imaging of HDAC activity in deep tissue slices up to 130 μm. This activatable fluorescent probe affords a useful tool for evaluating HDAC activity and screening HDAC-targeting drugs in both live cell and tissue assays.

One-Atom Substitution Enables Direct and Continuous Monitoring of Histone Deacylase Activity

“…The release of radioisotopically labeled acetate was used to analyze enzymatic reactions. − More recently, acetate could be captured by coupling to an enzymatic reaction and a chemical reaction was used to trap the HDAC8-mediated release of thioacetate yielding a chromophore . Reagents for the detection of the generated primary amine in the peptide product could either be added chemicals, like biotin-containing compounds or fluorescent dyes, reacting with the lysine side chain , or intramolecular reactions, like transesterification with a coumarine dye, which is possible only if the lysine side chain is released by HDAC activity. − Additionally, aggregation-induced emission , and modulation of binding to DNA , were used to probe HDAC activity.…”

Optical Imaging of Epigenetic Modifications in Cancer: A Systematic Review

“…Using stochastic optical reconstruction microscopy, Xu et al were able to characterize the three high-order chromatin structures in different epigenomic states in MCF10A human breast epithelial cells, which may affect gene transcription in space (Xu and Liu 2019;Xu et al 2018). Liu et al developed a novel activatable two-photon fluorescence probe to image HDAC activity (Liu et al 2018) (Fig. 3a, b).…”

Fluorogenic probes for detecting deacylase and demethylase activity towards post-translationally-modified lysine residues

“…Other groups subsequently reported different probes for the uorescent detection of lysine deacetylase activity using reactions that are based on imine formation, 17,18 nucleophilic aromatic substitution reactions to generate changes in a probe uorescence response, 19 or self-immolative intramolecular cyclization reactions. 20 Förster resonance energy transfer (FRET) based probes that contain quencher and uorophore fragments attached to the 3-lysine side chain of non-natural substrates have also been described. These probes rely on enzymatic cleavage of either their quencher or uorophore fragments to produce a uorescence reponse.…”