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

Chemical proteomics offers a comprehensive view of small molecule-protein interactions within natural biological environments. Typically, these ligandability maps have primarily focused on readily available cell lines and primary immune cells. In this study, researchers detail a new activity-based protein profiling (ABPP) method to map the covalent ligandability of primary brain cells derived from mice.

By testing a variety of stereochemically defined electrophilic small molecules, or "stereoprobes," the team discovered ligands for a wide range of proteins found in various brain cells, with a particular emphasis on those enriched in nervous system expression. Among the proteins identified were multiple hyperpolarization-activated cyclic nucleotide-gated (HCN) ion channels, which were found to be covalently modified by tryptoline acrylamide stereoprobes at a conserved cysteine within their cyclic nucleotide-binding domain.

These stereoprobes were observed to inhibit cAMP-induced changes in voltage sensitivity without affecting the basal activity of HCN channels. The researchers demonstrate an enhanced ABPP platform capable of identifying ligands that target proteins enriched in the nervous system, including compounds that can modulate HCN channel function in cells.