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Activity-based profiling of primary brain cells identifies covalent allosteric modulators of HCN channels

Ye, E.; Russo, A.; Castelli, R.; Westlake, G. T.; Jiang, X.; Spiro, D. A.; Quejido, S.; Henry, C. L.; Blankman, J. L.; Simon, G. M.; Melillo, B.; Santoro, B.; Moroni, A.; Cravatt, B. F.

2026-08-26 biochemistry
10.64898/2026.08.25.747126 bioRxiv
Show abstract

Chemical proteomics can provide global portraits of small molecule-protein interactions in native biological systems. Such ligandability maps have, however, been mostly restricted to readily accessible cell lines and primary immune cells. Here, we describe an activity-based protein profiling (ABPP) strategy for mapping the covalent ligandability of primary brain cells isolated from mice. By investigating sets of stereochemically defined electrophilic small molecules (stereoprobes), we identify liganding events for diverse brain cell proteins, including many with nervous system-enriched expression. In this category were multiple hyperpolarization-activated cyclic nucleotide-gated (HCN) ion channels, which we show are covalently liganded by tryptoline acrylamide stereoprobes at a conserved cysteine in their cyclic nucleotide-binding domain. The stereoprobes were found to block cAMP-dependent shifts in voltage dependence while sparing basal activity of HCN channels. We thus describe an advanced ABPP platform for identifying ligands targeting nervous system-enriched proteins, including chemical probes that modulate HCN channel function in cells.

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