Biological and Medicinal Chemistry

N-Acryloylindole-alkyne (NAIA) enables profiling new ligandable hotspots in chemoproteomics experiments and imaging thiol oxidation

Authors

Abstract

We report a new class of compounds, N-acryloylindole-alkynes (NAIAs), as promising cysteine-reactive probes for proteome-wide cysteine profiling and imaging of thiol oxidative modifications. NAIAs showed superior cysteine reactivity owing to delocalization of π electrons of the acrylamide warhead over the whole indole scaffold, resulting in its activation for faster reaction with cysteines. This allows NAIAs to ligand functional cysteines more effectively than IAA, as well as to image oxidized thiols in cells facing oxidative stress by confocal fluorescence microscopy. In MS-based ABPP experiments, NAIAs successfully captured a new pool of ligandable cysteines and proteins even compared to the current state-of-the-art cysteine profiling data. Competitive ABPP experiments further demonstrate the ability of NAIA to discover hit compounds targeting these new cysteines and proteins. This work should initiate development of new cysteine-reactive probes, particularly those with activated acrylamide, for advancing cysteine imaging and profiling, and covalent ligand screening for drug research.

Content

Thumbnail image of NAIA_MS_20221001.pdf

Supplementary material

Thumbnail image of NAIA_SI_20221001.pdf
Supporting Information
Experimental details about chemical synthesis and characterization, supporting figures
Thumbnail image of NAIA_Supplementary Data 1_HepG2 cell lysates.xlsx
Supplementary Data 1
Data analysis of MS-based ABPP experiments on HepG2 cell lysates using NAIA-5 and IAA as the cysteine-reactive probe
Thumbnail image of NAIA_Supplementary Data 2_Comparison to state of the art paper in cysteine profiling.xlsx
Supplementary Data 2
Comparison of ligandable Cys identified by NAIA-5 with those identified by DBIA in the current state-of-the-art cysteine profiling experiment
Thumbnail image of NAIA_Supplementary Data 3_231MFP cell lysates.xlsx
Supplementary Data 3
Data analysis of MS-based ABPP experiments on MFP231cell lysates with MudPIT using NAIA-4 and IAA as the cysteine-reactive probe
Thumbnail image of NAIA_Supplementary Data 4_Competitive gel-based ABPP experiment.xlsx
Supplementary Data 4
Protein targets of CL1 in HepG2 cell lysates identified by competitive MS-based ABPP experiment