Oxidation of a protein cysteine thiols to sulfenic acid plays a crucial role in regulating protein function and is correlated with inflammation, ageing, and malignant transformation. Most popular reaction-based small molecule probes used for detecting sulfenic acids are based on the dimedone (5,5-dimethyl-1,3-cyclohexanedione) scaffold, which is selective, but displays poor reactivity. In addition, mechanistic details and features that ‘tune’ the reactivity of the nucleophile remain obscure.<br/><br/>Herein, we report a series of structure-guided development of cyclic/linear C-nucleophiles suitable for capturing sulfenic acid in proteins. First, we evaluated reactivity of dimedone probe with cysteine sulfenic acid in peptide model and critical role of solvent molecules in this reaction. Using a combination of all-atom MD simulations, QM calculations, molecular docking, lead optimisation, synthetic medicinal chemistry, and mass spectrometry, we have developed a series of probes capturing EGFR Cys797 sulfenic acid. Our MD simulations data show differences in intrinsic dynamics between “normal” and S-sulfenylated EGFR, which are likely to affect interactions with inhibitors. Developed compound showed affinities in the nanomolar range for the double mutant EFGR whilst being selective over the wild type. Next, using force-pulling and equilibrium MD simulations of human cellular senescence marker p16 at diverse redox states, which was followed by solvent-mapping, we identified a small yet ‘druggable’ pocket adjacent to S-sulfenylated Cys72, suitable for accommodating a small molecule probe. The “warheads” have been synthesised and validated by mass-spectrometry, but their selectivity and suitability for cellular studies remain to be tested. Finally, we designed, developed, and evaluated reactivity of novel probe based on 1,5-benzodiazepine scaffold, which can be used for S-sulfenylation studies in inflammatory conditions.

Structure-guided development of novel molecular probes for capturing protein sulfenic acid: a combined computational and experimental approach

ACS Spring Meeting 2021

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