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

Supplementary material

Supporting Information
Experimental details about chemical synthesis and characterization, supporting figures
Supplementary Data 1
Data analysis of MS-based ABPP experiments on HepG2 cell lysates using NAIA-5 and IAA as the cysteine-reactive probe
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
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
Supplementary Data 4
Protein targets of CL1 in HepG2 cell lysates identified by competitive MS-based ABPP experiment