Generation of a Nav1.9 overexpressing cell-line for screening compounds using automated patch-clamp

Na_V1.9, is a voltage-gated sodium channel involved in pain signaling in the primary afferent sensory pathway. Genetic and pharmacological studies highlight the central role of NaV1.9 in chronic and inflammatory pain, making it a promising non-opioid analgesic target. High-throughput screening (HTS) assays are critical for discovering potent inhibitors of sodium channels and developing robust HTS assays enables the rapid identification of desirable selective inhibitors, accelerating the path to novel pain therapeutics with fewer side effects.

Discovery efforts to screen against Na_V1.9 have been somewhat held back due to the unusually slow activation and inactivation kinetics of the channel, difficulties to establish robust overexpression in heterologous systems and the slightly more nuanced role of NaV1.9 in pain signaling compared to other sodium channels.

Here, we discuss the development of a HEK293 Na_V1.9 overexpressing cell line, its characterization and its validation for implementation in a drug discovery screening cascade. Electrophysiological data generated on the Sophion Qube 384 well automated system show that the biophysical properties and pharmacological profile of the recorded currents closely resemble those expected from Na_V1.9. Pilot study data shows that the assay is robust, stable and scalable to enable its use for HTS.

We also discuss the development of a secondary assay to examine Na_V1.9 state dependency. The pharmacological profile of selected compounds shows that the assay is suitable for examining compound potency, and can show preferential block of the inactivated state for compounds that show state-dependent pharmacology.

By providing the cellular reagents and methodology required for HTS we can begin to unlock the potential of selective NaV1.9 inhibitors and allow the development of next-generation analgesics, offering hope for patients suffering from debilitating pain conditions.