The authors implemented an activity-based protein profiling (ABPP) approach to chart covalent ligandability in primary brain cells isolated from mice. This strategy extends chemical proteomics beyond commonly used cell lines and peripheral primary immune cells to native neural cell types, enabling direct assessment of small molecule–protein interactions in brain-derived proteomes. The platform was used to detect proteins susceptible to covalent engagement by electrophilic compounds under biologically relevant conditions.
To probe ligandable sites in the brain proteome, the study employed sets of stereochemically defined electrophilic small molecules, termed stereoprobes. These compounds are stereochemically distinct electrophiles designed to form covalent adducts with nucleophilic residues on proteins. By applying stereoprobes to primary brain cells and profiling resultant protein modifications, the investigators generated ligandability maps that reveal stereoselective covalent interactions across diverse neural proteins.
ABPP with stereoprobes identified numerous liganding events on proteins that show enriched expression in the nervous system. The profiling thus captured interactions relevant to brain biology rather than being limited to broadly expressed or easily cultured cell line proteins. The breadth of identified targets included ion channels and other proteins with functions pertinent to neuronal physiology.
Among the proteins identified were multiple members of the hyperpolarization-activated cyclic nucleotide-gated (HCN) channel family. HCN channels, which contribute to pacemaking and excitability in many neurons, were detected as direct covalent targets of the tryptoline acrylamide class of stereoprobes used in the screen. This result highlights the capacity of the ABPP platform to uncover ligandable sites on integral membrane ion channels in primary brain cells.
The stereoprobes were found to covalently modify a conserved cysteine residue located in the HCN channels’ cyclic nucleotide-binding domain (CNBD). The CNBD is the molecular module that mediates regulation of HCN channels by cyclic nucleotides such as cAMP. Covalent liganding at this conserved cysteine provides a plausible structural basis for altering CNBD-mediated modulation of channel gating.
Functional characterization reported in the source indicates that covalent modification of the CNBD by the tryptoline acrylamide stereoprobes blocked cAMP-dependent shifts in the voltage dependence of HCN channels. Importantly, these covalent allosteric modulators did not abolish the channels’ basal activity; rather, they selectively interfered with cAMP-driven modulation while sparing intrinsic channel function in the absence of cAMP shifts. This profile is consistent with an allosteric mechanism in which the probe perturbs ligand-dependent gating without preventing constitutive channel opening.
The study presents an advanced ABPP platform capable of mapping covalent ligandability in primary neural cells and of identifying small molecules that modulate the function of nervous system–enriched proteins in cells. The identification of covalent allosteric modulators of HCN channels demonstrates that electrophilic stereoprobes can yield chemical tools to selectively alter signaling-dependent channel regulation. Such probes could be useful for dissecting CNBD-dependent modulation of excitability in cellular models.
The supplied source text summarizes the main findings but does not report several experimental details in this excerpt. Specific information not provided here includes quantitative data, detailed experimental methods, the identities and stereochemical configurations of all stereoprobes screened, the exact biochemical assays and electrophysiological protocols used, sample sizes, and any in vivo or behavioral validation. Where those details are required for interpretation or translation, they were not available in the supplied source excerpt.
Using ABPP on primary mouse brain cells, the authors identified covalent liganding events by stereoprobes across nervous system–enriched proteins and discovered that tryptoline acrylamide stereoprobes covalently modify a conserved cysteine in the HCN channel CNBD. This covalent engagement selectively blocks cAMP-dependent modulation of HCN gating while preserving basal channel activity, and the work establishes an expanded chemical proteomics workflow for identifying modulators of neural proteins in their native cellular context.