NHS-Esters As Versatile Reactivity-Based Probes for Mapping Proteome-Wide Ligandable Hotspots

Abstract

Most of the proteome is considered undruggable, oftentimes hindering translational efforts for drug discovery. Identifying previously unknown druggable hotspots in proteins would enable strategies for pharmacologically interrogating these sites with small molecules. Activity-based protein profiling (ABPP) has arisen as a powerful chemoproteomic strategy that uses reactivity-based chemical probes to map reactive, functional, and ligandable hotspots in complex proteomes, which has enabled inhibitor discovery against various therapeutic protein targets. Here, we report an alkyne-functionalized N-hydroxysuccinimide-ester (NHS-ester) as a versatile reactivity-based probe for mapping the reactivity of a wide range of nucleophilic ligandable hotspots, including lysines, serines, threonines, and tyrosines, encompassing active sites, allosteric sites, post-translational modification sites, protein interaction sites, and previously uncharacterized potential binding sites. Surprisingly, we also show that fragment-based NHS-ester ligands can be made to confer selectivity for specific lysine hotspots on specific targets including Dpyd, Aldh2, and Gstt1. We thus put forth NHS-esters as promising reactivity-based probes and chemical scaffolds for covalent ligand discovery.

Chemoproteomics-Enabled Covalent Ligand Screening Reveals a Thioredoxin-Caspase 3 Interaction Disruptor That Impairs Breast Cancer Pathogenicity

“…In addition, to further test the proteome-wide selectivity for KEA1–97, we also performed isoTOP-ABPP studies competing KEA1–97 against our recently disclosed NHS-ester-alkyne reactivity-based probe. 10 Interestingly, while this probe identified >1000 probe-modified lysines in 231MFP cells, compared to the 80 lysines profiled by the DCT-alkyne probe, we did not identify K72 of TXN. Rather, the NHS-ester-alkyne probe labeled K94 of TXN which showed an isotopically light to heavy ratio of 1.0, indicating that this site was not a target of KEA1–97.…”

Global profiling of lysine reactivity and ligandability in the human proteome

“…Alternative electrophiles, when used as broad profiling probes, may also provide access to additional lysine residues in the proteome, although the chemoselectivity of such probes could present a challenge. While our manuscript was under review, for instance, Ward and colleagues characterized the proteomic reactivity of an NHS-ester probe and found that, while this activated ester labeled lysines, it also showed substantive reactivity with several other amino acid residues (serine, threonine, tyrosine, arginine, cysteine) across the mouse liver proteome 61 . These results are consistent with our initial gel-based profiling experiments studies of a similar NHS ester probe ( 8 ), which showed substantially higher overall proteomic reactivity compared to the STP probe 1 (Supplementary Fig.…”

Discovery and elucidation of unknown protein modifications : brominated coumarin azide as a probe for alkynes

“…32 The mAb-alkyne was used to demonstrate this analytical approach, illustrated in Figure 1. Lysine or primary amine conjugation in a mAb is highly heterogeneous due to the more than eighty lysine residues, making detection of the conjugation site quite challenging.…”

“…[31][32][33][34][35][36][37][38][39] Detection of alkynes is of great significance due to its broader applications and coupling with click chemistry. However, since the 1.2.3-triazole product often lacks specific properties, the detection becomes challenging especially with heterogeneous protein modifications, in vivo sample matrices and low abundant protein samples.…”

“…32,34 The Ɛ-amine in lysine residues and at the N-terminus are expected to be acylated. Once the reactions were confirmed, the mAb-trizole was analyzed by tryptic mapping on a UPLC with both fluorescence and mass spectrometry detectors to elucidate the alkyne modification sites and chemistry.…”