Fast and Site-Specific Covalent Targeting of Proteins by Arylfluorosulfate-Modified Aptamers

Sulfur fluoride exchange (SuFEx) is a versatile click chemistry platform with emerging applications in covalent inhibitor development, among which arylfluorosulfate (AFS) derivatives have shown great promise because of their exclusive selectivity to target proteins. Nevertheless, due to the inherently mild electrophilicity of AFS, the high target specificity often comes at the expense of target reactivity. Here, we challenge this conventional view and show that in vitro selected AFS-modified covalent aptamers (AFS-Aps) can be both highly target selective and reactive. One such AFS-Ap is able to site-specifically modify the extracellular domain (ECD) of human epidermal growth factor receptor 3 (HER3) with a "click" reaction half-life (<i>t</i>_1/2) as short as 1 min and covalently inhibit the protein-protein interaction (PPI) of HER3/EGFRs. Another AFS-Ap targeting human vascular endothelial growth factor (VEGF) can also rapidly cross-link VEGF165 and block the PPI of VEGF/VEGFR. The inactivation rate constants (<i>k</i>_inact) of these AFS-Aps are one to two orders of magnitude higher than many reported AFS-based covalent molecules. This unexpectedly fast and site-specific covalent targeting requires a precisely organized aptamer-protein binding interface, where the AFS structure, rather than electrophilicity, is the decisive factor. We believe the target reactivity potential of AFS derivatives may have long been underestimated in covalent inhibitor development. AFS-adapted combinatorial techniques are therefore invaluable for discovering covalent inhibitors with both high target reactivity and selectivity.