Light-Activatable, 2,5-Disubstituted Tetrazoles for the Proteome-Wide Profiling of Aspartates and Glutamates in Living Bacteria

Abstract

Covalent inhibitors have recently seen a resurgence of interest in drug development. Nevertheless, compounds, that do not rely on an enzymatic activity, have almost exclusively been developed to target cysteines. Expanding the scope to other amino acids would be largely facilitated by the ability to globally monitor their engagement by covalent inhibitors. Here, we present the use of light-activatable 2,5-disubstituted tetrazoles that allow quantifying 8971 aspartates and glutamates in the bacterial proteome with excellent selectivity. Using these probes, we competitively map the binding sites of two isoxazolium salts and introduce hydrazonyl chlorides as a new class of carboxylic acid-directed covalent protein ligands. As the probes are unreactive prior to activation, they allow global profiling even in living Gram-positive and Gram-negative bacteria. Taken together, this method to monitor aspartates and glutamates proteome-wide will lay the foundation to efficiently develop covalent inhibitors targeting these amino acids

Labeling Preferences of Diazirines with Protein Biomolecules

“…14,48 Conversely, products resulting from labeling through the diazo are more-likely to represent weaker binding interactions, since the lifetime of the diazo is above the rate of diffusion. 49 There are relatively few chemistries that have been used to selectively label carboxylic acids in whole cells, [50][51][52] and fewer photoactivatable chemistries, 27 suggesting that this chemistry could be intentionally utilized to profile carboxylic acids in cells as a method that senses the protonation state of the acids.…”

Profiling the Proteome-Wide Selectivity of Diverse Electrophiles

“…In this way, we verified and newly identified tailored probes to study nine different amino acids as well as the N-terminus proteome-wide. Within our set of probes, we verify the selectivity of IA-and EBX2-alkyne for cysteines, 2,3 STP-alkyne for lysines, 18 SuTEx2-alkyne for tyrosines 23 as well as MeTet- 10 and Az-alkyne 20 for aspartates and glutamates. For several of these amino acids, we identify additional probes based on complementary chemistries like ArSq-and EBA-alkyne for lysines, HC-alkyne for aspartates and glutamates and PTAD-alkyne for tyrosines, which increase the proteome-wide coverage.…”

“…Light-activated probes hold the potential to monitor covalent target engagement in living cells as the probe itself is unreactive before activation and therefore has the potential to be non-toxic. 10 Proteome-wide, residue-specific monitoring of lysines in a lightdependent fashion would therefore be highly promising to study effects on lysines with temporal and spatial resolution in living cells. Nevertheless, no probe had been previously shown to allow this application.…”

“…9 However, as cysteine is a very rare amino acid, many interesting binding pockets contain no suitable cysteine for covalent engagement. 10 Recently, many reactive groups have been developed that selectively target other residues in proteins and that have promise for TCI development against a much broader set of target proteins. 11 For these compounds, one key challenge is to globally investigate their target and amino acid selectivity.…”

Prioritization of antimicrobial targets by CRISPR-based oligo recombineering

“…In recent years the scope, scale, and speed with which chemically reactive amino acids can be profiled has accelerated dramatically; chemoselective probes are now available for cysteine 4 , serine 39 , lysine 5 , methionine 6 , tyrosine 7 , aspartate/glutamate 9,40 , tryptophan 41 , and histidine 42 . Supporting this, advances in mass-spectrometry have significantly expanded the number and rate at which individual reactive sites can be profiled 43,44 , and subsequently exploited by electrophilic drug hunters.…”