Profiling the proteome-wide selectivity of diverse electrophiles

Abstract

Targeted covalent inhibitors are powerful entities in drug discovery, but their application has so far mainly been limited to addressing cysteine residues. The development of cysteine-directed covalent inhibitors has largely profited from determining their proteome-wide selectivity using competitive residue-specific proteomics. Several probes have recently been described to monitor other amino acids using this technology and many more electrophiles exist to modify proteins. Nevertheless, a direct, proteome-wide comparison of the selectivity of diverse probes is still entirely missing. Here, we developed a completely unbiased workflow to analyse electrophile selectivity proteome-wide and applied it to directly compare 54 alkyne probes containing diverse reactive groups. In this way, we verified and newly identified probes to monitor a total of nine different amino acids as well as the N-terminus proteome-wide. This selection includes the first probes to globally monitor tryptophans, histidines and arginines as well as novel tailored probes for methionines, aspartates and glutamates.

The covalent reactivity of functionalized 5-hydroxy-butyrolactams is the basis for targeting of fatty acid binding protein 5 (FABP5) by the neurotrophic agent MT-21

“…For the direct substitution product we observed instability of the "R"-product S9), indicating that these are either not cysteine targets (see Fig. 3c) or, alternatively, cysteine targets that are not efficiently engaged by iodoacetamide 20,27 . To more directly probe the apparant loss in FABP5-reactivity in lysates, we first attempted pre-treatment with dithiotreitol (DTT) or TCEP, to reduce the potential C120-C127 disulphide bond but generally observed nearly complete loss of labelling (Fig.…”

“…These discoveries are enabled in large part by a combination of advances in chemical proteomics, [14][15][16] and an increasing knowledge of the biological performance and characteristics of different classes of reactive groups. [17][18][19][20] Structural motifs which are non-conventional, or which possess unusual selectivity, are particularly interesting as entry points for new covalent modulator design. Towards that end, natural products provide a valuable resource for continued discovery of novel electrophilic motifs [21][22][23][24][25][26][27] , but synthetic compounds [28][29][30][31][32][33] , designed or serendipitously discovered 30 , are also advancing this area.…”

Targeting the ubiquitin system by fragment-based drug discovery

“…A balance between reactivity and specificity is required, and acrylamides and chloroacetamides are often chosen for this reason. More recently, novel electrophiles have been developed for targeting residues beyond cysteine, including lysines, tyrosines and histidines ( Hacker et al, 2017 ; Ward et al, 2017 ; Abbasov et al, 2021 ; Zanon et al, 2021 ). The main advantage of covalent fragment screening over non-covalent screening is the increased simplicity of hit detection.…”

“…Profiling these residues with fragments could provide interesting insights into biology and highlight new drug targets. Despite their lower intrinsic reactivity, advances have been made in targeting lysine residues with sulfotetrafluorophenyl ester electrophiles ( Hacker et al, 2017 ; Abbasov et al, 2021 ), and serine, threonine, tyrosine and histidine residues with N-hydroxysuccinimide esters ( Ward et al, 2017 ; Zanon et al, 2021 ). In addition, profiling of cysteines, lysines, tyrosines and histidines has been characterised with sulfonyl fluorides ( Gilbert et al, 2022 ), and tyrosines with sulfur-triazole exchange chemistry ( Brulet et al, 2020 ).…”

The covalent reactivity of functionalized 5-hydroxy-butyrolactams is the basis for targeting of fatty acid binding protein 5 (FABP5) by the neurotrophic agent MT-21

“…These discoveries are enabled in large part by a combination of advances in chemical proteomics, [14][15][16] and an increasing knowledge of the biological performance and characteristics of different classes of reactive groups. [17][18][19][20] Structural motifs which are non-conventional, or which possess unusual selectivity, are particularly interesting as entry points for new covalent modulator design. Towards that end, natural products provide a lasting, valuable resource for continued discovery of novel electrophilic motifs [21][22][23][24][25][26][27] , but synthetic compounds [28][29][30][31][32][33] , designed or serendipitously discovered 30 , are also advancing this area.…”