Assay dependent activity of the sodium channel gating modifier protoxin‐I: implications for sodium channel drug discovery

Abstract

Sodium channels (NaV1.7) are extremely attractive analgesic drug targets. The Holy Grail in NaV1.7 drug discovery (in‐vitro) is to discover selective NaV1.7 antagonists. Most compounds, if not all bind to a highly conserved, "local anesthetic binding site". Gating modifiers may exert selectivity due to interaction at less conserved allosteric sites. Therefore, we were interested to determine if several of the most commonly used sodium channel screening assays can identify such allosteric hits. Using protoxin‐I (ProTx‐I) we show the ability to detect gating modifiers is assay dependent. Using a voltage‐sensitive dye in FLIPR, ProTX‐I was unable to inhibit veratridine activation of NaV1.7 at concentrations up to 1 μM. Widely used pore blockers (amitriptyline, tetracaine) were effective in this assay format with a pIC50 of 6. Likewise, ProTX‐I was inactive in a radioligand‐binding assay using [3H]‐batrachotoxin as a tracer. In a VIPR FRET assay, ProTX‐1 caused only a small 'delay' in veratridine activation. In contrast , protoxin‐I was a potent blocker (pIC50 of 6.4) of the channel in two automated patch‐clamp assays (IonWorks Quattro and PatchXpress). These findings suggest that gating modulators may be missed in commonly used assays such as FLIPR that use an end‐point read. Carefully optimized VIPR assays or HTEP assays may be the preferred platform to identify sub‐type selective sodium channel blockers.