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Assay dependent activity of the sodium channel gating modifier protoxin‐I: implications for sodium channel drug discovery
Author(s) -
Bhattacharya Anindya,
Wang Qi,
Wu Nancy,
Wickenden Alan D
Publication year - 2009
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.23.1_supplement.998.31
Subject(s) - sodium channel , chemistry , veratridine , allosteric regulation , gating , pharmacology , batrachotoxin , biophysics , sodium channel blocker , sodium , biochemistry , enzyme , biology , organic chemistry
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 pIC 50 of 6. Likewise, ProTX‐I was inactive in a radioligand‐binding assay using [ 3 H]‐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 (pIC 50 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.