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. 2025 Apr 17;64(17):e202408701.
doi: 10.1002/anie.202408701. Epub 2025 Feb 25.

Target Agnostic Photoaffinity Labelling by Sulfonylhydrazones

Affiliations

Target Agnostic Photoaffinity Labelling by Sulfonylhydrazones

Kristóf Garami et al. Angew Chem Int Ed Engl. .

Abstract

Photoaffinity labeling is a widely used methodology for interrogating small molecule-protein interactions. However, these applications are limited by the few photo-crosslinkers that typically modify the affinity and the binding mode of the original ligand. Here, we report the development of new target agnostic photoaffinity warheads, sulfohydrazones that form a reactive carbene upon UV irradiation. Careful optimization of the reaction conditions allowed us to effectively label five different amino acid residues in proteins. Our approach turned biologically relevant hydrazones and sulfohydrazones to intrinsically irreversible covalent binders without structural modifications by photoactivation as demonstrated on monoamine oxidase A (MAO-A) enzyme and STAT5b (Signal transducer and activator of transcription 5b) transcription factor. Sulfohydrazones are readily accessible by transforming the corresponding carbonyl group of a ligand or a suitable tag that extends the application domain of the method for any ligands exemplified by conditional labelling of the acetylcholine esterase enzyme and the oncogenic mutant of GTP-ase KRasG12D.

Keywords: KRas; STAT; acetylcholine esterase; photoaffinity; sulfohydrazone.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Scheme 1
Scheme 1
A) Collection of the most frequently used and the most recent photoaffinity tags and photo‐crosslinkers and their reaction upon photoactivation highlighting the activating wavelength by different colors. B) Protein labeling strategies with sulfonyl hydrazones: natively embedded sulfohydrazone moiety in the ligand (yellow), oxo group transformed to sulfohydrazone (cyan) and sulfohydrazone tag resulting in fully functionalized ligands (green).
Figure 1
Figure 1
A) General reaction scheme from the base and photocatalyzed Bamford‐Stevens reaction. Nu: nucleophile e.g thiol/thiolate, alcohol, water, amine, carboxylate. B) Conversion of tosylhydrazone 1a in time at 380 nm irradiation in PBS pH 7.4. C) Scope of acetophenone hydrazones. Blue percentage indicate conversion of the hydrazone after photoactivation. Irradiations were performed at 380 nm in PBS pH 7.4 for 10 min. D) Scope of ketohydrazones and aldohydrazones after 10 min irradiation at 380 nm in PBS pH 7.4. Blue percentage indicate conversion of the hydrazone after photoactivation. E. Conversion of the tosylhydrazone tags after 10 min irradiation at 380 nm in PBS pH 7.4. Blue percentage indicate conversion of the hydrazone after photoactivation. * During the full conversion of 4d rapid substitution of the Br to OH was also detected. F. Conversion of benzophenone and derivatives compared to the corresponding tosylhydrazone after 10 min irradiation at 380 nm in PBS pH 7.4. Blue percentage indicate conversion of the probe after photoactivation.
Figure 2
Figure 2
A) Bioactive sulfonyl hydrazone inhibitors against AChE (6), urease (7) and MAOs (8). MSMS spectrum proving the labelling of compound 8 on D123. Crystal structure of MAO‐A (PBD: 1O5 W) highlighting cofactor FAD (flavin adenine dinucleotide) by cyan, G110‐loop by green and D123 by dark blue. Blue percentage values refer for the conversion of the tosylhydrazones after 10 min irradiation in PBS pH 7.4. B) Sulfohydrazone derivatives (9 and 10) of bioactive carbohydrazones. Blue percentage values refer for the conversion of the tosylhydrazones after 10 min irradiation PBS pH 7.4. STAT5b inhibition assay curve (n=3) showing IC50 with irradiation. MSMS spectrum proving the labeling of compound 10 on STAT5b C688. C) KRasG12C fragment and the tosylhydrazone derivative 11. Modelling of binding sites based on tryptic digestion and MSMS (PDB: 7RPZ). Blue percentage values refer for the conversion of the tosylhydrazone after 10 min irradiation in PBS pH 7.4. D) Donepezil and donepezil‐tosylhydrazone (12). Results of the biochemical enzyme inhibition assay (n=3) with control experiments. Blue percentage values refer for the conversion of the tosylhydrazone after 10 min irradiation in PBS pH 7.4. Crystal structure of AChE with donepezil (PDB: 4EY7) highlighting the labelled amino acid sequences and binding site surfaces by blue and donepezil by cyan.
Figure 3
Figure 3
A) Chemical structure of the probe applied in proteomic labeling experiments; B) Dot blot analysis of biotinylation rate in HEK293 lysates treated with generic fragment probe (4b). The blot confirms the increased biotinylation rate after 4b probe treatment; C) Volcano plot depiction of MS‐identified protein labelling effectiveness in HEK293 cell lysates achieved by probe 4b compared to the DMSO‐treated control.

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