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.

Figure 1.

IsoTOP-ABPP of NHS-ester-alkyne reactivity in mouse liver proteome. (A) Structure of NHS-ester-alkyne (NHSyne) and distribution of probe-modified peptides in mouse liver proteome assessed by isoTOP-ABPP. Mouse liver proteomes were labeled with NHS-ester-alkyne (500 or 100 μM), followed by copper-catalyzed azide–alkyne cycloaddition (CuAAC) conjugation of a biotin-azide tag bearing an isotopically light (for 500 μM) or heavy (100 μM) mass tag to probe-labeled proteins. Probe-labeled proteins were subsequently avidin-enriched and digested with trypsin, and probe-modified tryptic peptides were isolated and eluted by TEV protease for subsequent LC-LC/MS/MS proteomic analysis. Raw data and ratiometric analysis of heavy to light peptides can be found in Figure S1 and Table S1. NHS-ester-alkyne predominantly reacted with lysines, showed significant reactivity with threonines and serines, and showed minor reactivity with tyrosines, arginines, and cysteines. (B) Breakdown of probe-labeled sites compared against annotated UniProt functional sites. While the majority of labeled residues are unannotated, the annotated sites were predominantly post-translationally modified or involved in ligand binding. Data shown in A and B are representative of probe-modified peptides found in two out of four biological replicates.

Figure 2.

Examples of various types of nucleophilic residues targeted by NHS-ester-alkyne. (A) K230 on Aldob, a catalytic residue within the active site. (B) Y55 on Akr1c6, the catalytic proton donor within the active site. (C) K12 and R105 on Nme1, two positively charged residues involved in binding nucleoside diphosphates. (D) K46 on Gnmt, a surface lysine and known succinylation site. (E) K91 on Pfn1, a surface lysine which forms two salt bridges with aspartate residues at the actin–Pfn1 interface. (F) T31 on Anxa5, a surface threonine near the calcium binding site. PDB file designations are listed in the text. Specific structures are all from human counterparts.

Figure 3.

NHS-ester-based covalent ligands conferring selectivity for lysines on specific protein targets. (A) Structure of NHS-ester fragment CW 1–26. (B) Competitive isoTOP-ABPP analysis of CW 1–26 lysine reactivity in mouse liver proteomes. DMSO vehicle or CW 1–26 (100 μM) was preincubated with mouse liver proteomes for 30 min prior to labeling with NHS-ester-alkyne (500 μM) for 1 h. Probe-labeled samples were then taken through the isoTOP-ABPP procedure. Shown are individual isotopically light (vehicle-treated) to heavy (CW-1–26-treated) probe-labeled peptides, showing K497 as the primary target. (C) Competition of CW1–26 against NHS-ester alkyne labeling of recombinant pure human DPYD using gel-based in-gel fluorescence ABPP methods. (D) K497 is a surface lysine on DPYD which forms a salt bridge with D119 and resides at an interface between DPYD dimeric partners. (E) A second NHS-ester fragment CW1–33. (F) Competitive isoTOP-ABPP analysis of CW1–33 using the same approach described in B yielded three targets, K639 on Zfp318, K211 on Aldh2, and K71 Gstt1. (G–J) Validation of CW 1–33 competition against NHS-ester-alkyne labeling of pure human ALDH2 (G) and GSTT1 (I) protein and locations of lysines targeted by CW 1–33 on ALDH2 (H) and GSTT1 (J) structures. Data in B and D show average ratios for probe-labeled peptides identified in at least two out of three biological replicates. Gels shown in C, G, and I are representative gels from n = 3.