Broad-Spectrum Kinase Profiling in Live Cells with Lysine-Targeted Sulfonyl Fluoride Probes

Abstract

Protein kinases comprise a large family of structurally related enzymes. A major goal in kinase-inhibitor development is to selectively engage the desired kinase while avoiding myriad off-target kinases. However, quantifying inhibitor interactions with multiple endogenous kinases in live cells remains an unmet challenge. Here, we report the design of sulfonyl fluoride probes that covalently label a broad swath of the intracellular kinome with high efficiency. Protein crystallography and mass spectrometry confirmed a chemoselective reaction between the sulfonyl fluoride and a conserved lysine in the ATP binding site. Optimized probe 2 (XO44) covalently modified up to 133 endogenous kinases, efficiently competing with high intracellular concentrations of ATP. We employed probe 2 and label-free mass spectrometry to quantify intracellular kinase engagement by the approved drug, dasatinib. The data revealed saturable dasatinib binding to a small subset of kinase targets at clinically relevant concentrations, highlighting the utility of lysine-targeted sulfonyl fluoride probes in demanding chemoproteomic applications.

Global profiling of lysine reactivity and ligandability in the human proteome

“…Additionally, because probe reactivity with lysines blocks tryptic cleavage sites, the use of alternative proteases may uncover additional probe-lysine reactivity events that evade detection in conventional tryptic digest protocols. Finally, subsets of lysines can be selectively targeted with greater sensitivity using tailored electrophilic probes that leverage recognition elements to favor binding to functional protein pockets, such as the ATP-binding sites of kinases 11,29 .…”

“…Success of such a program, however, may depend on identifying alternative amine-reactive chemotypes, as the activated esters tested herein are likely too prone to enzymatic and non-enzymatic hydrolysis for development into cellular or in vivo probes. Alternative amine-reactive electrophiles, such as sulfonyl fluorides 28,29 or the N , N ′-diacyl-pyrazolecarboxamidines explored herein may offer more suitable starting points for optimization of lysine-targeting covalent ligands that are more suitable for cell biological studies. 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.…”

“…Individual lysine residues within functional protein pockets are also susceptible to modification by electrophilic small molecules, including natural products, such as Wortmannin 18 , which targets a lysine in the active sites of PI3K kinases, activated esters that react with a lysine in transthyretin (TTR) 19 , and boronic acid carbonyl antagonists of the apoptosis regulatory protein MCL-1 13 . Additional electrophiles that have been shown to react with proteinaceous lysine residues include dichlorotriazines 20,21 , imidoesters 22 , 2-acetyl- or 2-formyl-benzeneboronic acids 13,23 , isothiocyanates 24,25 , pyrazolecarboxamidines 26,27 , sulfonyl fluorides 28,29 , and vinyl sulfonamides 30 .…”

Lysine‐Targeting Covalent Inhibitors

“…By comparing PP1 (24)t ot he substrate-bound protein crystal structure,t hey were able to exploit abulk-solvent-exposed vector,substituting the 2'-hydroxyl with an alkynyl moiety linker to develop the inhibitor towards an intracellular ABPP probe. [66] This chemotype has been shown to bind to the conserved hinge region of many kinases, [67] so it should keep selectivity within the kinase family whilst allowing the probe to covalently tag al arge proportion of the kinome. [66] This chemotype has been shown to bind to the conserved hinge region of many kinases, [67] so it should keep selectivity within the kinase family whilst allowing the probe to covalently tag al arge proportion of the kinome.…”

Activity‐Based Kinome Profiling Using Chemical Proteomics and ATP Acyl Phosphates

“…Examples of such methodologies include kinobeads, which utilize a pan-kinase inhibitor to enrich kinases from complex cell lysates (Bantscheff et al, 2007), and newer activity-based probes, such as XO44, which utilizes a reporter-tagged sulfonyl fluoride to covalently modify the catalytic lysine of kinases in lysates and live cells (Zhao et al, 2017). Examples of such methodologies include kinobeads, which utilize a pan-kinase inhibitor to enrich kinases from complex cell lysates (Bantscheff et al, 2007), and newer activity-based probes, such as XO44, which utilizes a reporter-tagged sulfonyl fluoride to covalently modify the catalytic lysine of kinases in lysates and live cells (Zhao et al, 2017).…”