A cell-based high-throughput screen identifies novel residual transport inhibitors.

(A) Kinetics of osmotic lysis in indicated solutes with 0, 200, or 2000 μM furosemide (black, red, and green traces where present in each panel, respectively). Notice that 200 μM furosemide abolishes lysis due to sorbitol or alanine uptake, but yields residual uptake of PhTMA+ and proline (indicated with arrow and “R”). (B) All points histogram of compound activities against osmotic lysis in PhTMA+ + 200 μM furosemide, as measured at 3 h and normalized to 0% block in DMSO-only wells and to 100% block in positive controls (HBS+1600 μM furosemide). Ordinate shows the number of compounds at each block level on a logarithmic scale, revealing that most compounds fell within 3*SD of negative control wells (grey bars). A small number of hits inhibited or accentuated lysis (red and blue bars, respectively). Two of the three compounds producing ≥ 50% block were unavailable for downstream experiments; the third did not reproduce in secondary studies. (C) Histogram comparing compound activities against primary and residual transport (x and y axes reflecting inhibition in parallel sorbitol and PhTMA+ + furosemide screens, respectively). Notice that mean ± SEM % activity against residual PhTMA+ transport gradually increases with activity in the sorbitol screen (grey histogram bars). Key hits reported previously in the sorbitol screen (compounds 1–8 from ref. [10]) exhibited weak activity in the PhTMA+ screen (black circles). Novel hits from the present screen are superimposed as red circles. (D) Structures and names of key hits from the PhTMA+ screen, with the prefix PRT referring to action against the PSAC residual transport.