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. 2017 Mar 30;12(3):e0174742. doi: 10.1371/journal.pone.0174742

Fig 3. Workflow for identifying AMPAR-auxiliary subunit modulators.

Fig 3

(A) 39,202 compounds were initially screened using the VSD assay against A2R-stg cells. (B) 1,184 hits from (A) were counter-screened against A2R, TetON, and A2R-C3 cells. (C) 116 compounds were identified from counter-screening in (B) as being stargazin or auxiliary subunit specific (i.e. they did not hit on A2R or TetON cells). These were tested for full compound CRCs against A2R-stg and A2R-C3 cells using the VSD assay. These CRCs identified 90 hits that fit to sigmoidal dose response curves with potency under 10 μM. (D) We identified 39 stargazin specific PAMs, 2 CNIH3 specific PAMs, and 36 PAMs that had activity in both A2R-stg and A2R-C3 cells. We also found 1 stargazin specific NAM and 9 compounds with NAM activity on both cell lines. Three compounds gave opposite effects in the two cell lines. Hits were discarded for reorder if they showed activity in the compound only window. Hits with activity in the CMPD only windows were discarded. (E) 57 of the 90 compounds in (D) were re-screened with new batch samples as compound CRCs in the VSD assay. (F) 57 hits were tested in the glutamate potency fold-shift calcium flux assay and 28 were subjected to a full compound CRC calcium flux assay to study their effects using an orthogonal approach.