Subunit selective modulation of GABAA receptors using pharmacogenetically tethered neurosteroids

Neurosteroids are endogenous neuromodulators and emerging therapeutics, but understanding but understanding how these compounds modulate receptor signaling within defined neuronal populations and networks has been limited by an inability to deliver these molecules with receptor-level and cell-type specificity. Here, we developed a neurosteroid DART (Drug Acutely Restricted by Tethering) that combines the GABAA receptor subunit-selectivity of a neuroactive steroid (NAS) with the cell-type specificity of the DART platform. Screening of seventeen NAS analogs identified seven scaffolds suitable for further engineering, and structure-activity analysis revealed that DART linker attachment at the C11 position preserved NAS activity on GABAA receptors, whereas C2 and C17 attachment failed to exhibit activity. Functional profiling of C11-linked NAS-DARTs slowed IPSC decay kinetics and showed variable off-target modulation of NMDA and AMPA EPSCs. The most selective compound, YX85.1DART.2, potentiated GABA-evoked currents in neurons expressing pharmacogenetically isolated 4/{delta}-containing GABAA receptors but not in {gamma}2-expressing neurons. A previously validated BZP.1DART.2 produced complementary selectivity on the two receptor populations. Together, these findings establish new tools for interrogating subunit-specific NAS actions on inhibitory signaling in defined neuronal populations.