Rational Targeting of Active-Site Tyrosine Residues Using Sulfonyl Fluoride Probes

Abstract

This work describes the first rational targeting of tyrosine residues in a protein binding site by small-molecule covalent probes. Specific tyrosine residues in the active site of the mRNA-decapping scavenger enzyme DcpS were modified using reactive sulfonyl fluoride covalent inhibitors. Structure-based molecular design was used to create an alkyne-tagged probe bearing the sulfonyl fluoride warhead, thus enabling the efficient capture of the protein from a complex proteome. Use of the probe in competition experiments with a diaminoquinazoline DcpS inhibitor permitted the quantification of intracellular target occupancy. As a result, diaminoquinazoline upregulators of survival motor neuron protein that are used for the treatment of spinal muscular atrophy were confirmed as inhibitors of DcpS in human primary cells. This work illustrates the utility of sulfonyl fluoride probes designed to react with specific tyrosine residues of a protein and augments the chemical biology toolkit by these probes uses in target validation and molecular pharmacology.

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

“…Previous studies have shown that sulfonyl fluorides can react with tyrosine as well as lysine residues. 14,21,22 Despite having 16 lysines and 13 tyrosines, most of which are solvent accessible, superstoichiometric labeling of SRC by the sulfonyl fluoride probes (3 equiv) was not observed. Specific modification of SRC at Lys295, corresponding to the catalytic lysine, was confirmed for probe 2 by trypsinization of the adduct, followed by LC–MS/MS analysis (Figure S2).…”

Arylfluorosulfates Inactivate Intracellular Lipid Binding Protein(s) through Chemoselective SuFEx Reaction with a Binding Site Tyr Residue

“…This comparison revealed that 1 labeled very few proteins, whereas S1 formed conjugates with numerous proteome members, reflecting the overall muted reactivity of arylfluorosulfates in comparison to aryl sulfonyl fluorides (Figure 1c). 2,3,14–16 Subjecting HEK293T cells to 1 for up to 48 h did not result in the substantial modification of the proteome relative to treatment by S1 , which is significantly more reactive over all time intervals examined (Figure S3). …”

SRPKIN-1: A Covalent SRPK1/2 Inhibitor that Potently Converts VEGF from Pro-angiogenic to Anti-angiogenic Isoform

“…Following a successful strategy for improving the selectivity in the cases of Ibrutinib (Davids and Brown, 2014), AZD9291 (Finlay et al, 2014), and THZ1 (Kwiatkowski et al, 2014), we considered targeting Tyr227, which is located proximal to the morpholine group of Alectinib (Figure 2D) with the known tyrosine-reactive sulfonyl fluoride substituent (Hett et al, 2015). Reasoning that a benzenesulfonyl fluoride would maintain the required size in order to fit in the solvent-exposed region, which could also orient the warhead in the required position to covalently modify Tyr227, we replaced the pyrazole ring in JH-VII-139-1 with a 3-benzene-sulfonyl fluoride to generate a new compound, which we named SRPKIN-1.…”

“…Medicinal chemists have been making great efforts to expand the scope of both the nucleophile in target protein as well as an electrophilic warhead in the small-molecule inhibitor. A recent report on the discovery of a covalent inhibitor of mRNA-decapping scavenger enzyme DcpS highlighted the phenol group of tyrosine as a suitable nucleophile for the sulfonyl fluoride warhead (Hett et al, 2015). …”