A High-Throughput Screening Assay for NKCC1 Cotransporter Using Nonradioactive Rubidium Flux Technology

Abstract

A high-throughput screening (HTS) assay was developed for cotransporter, NKCC1, which is a potential target for the treatment of diverse disorders. This nonradioactive rubidium flux assay coupled with ion channel reader series provides a working screen for this target expressed in human embryonic kidney (HEK) cell line. An eightfold window of detection was achieved with the optimized assay. This new functional assay offered a robust working model for NKCC1 in determining reliable and concordant rank orders of the test compounds supporting its sensitivity and specificity. The robustness of manual assay indicated by Z' of 0.9 qualified its amenability to automation. The Z' of 0.7 was displayed by automated assay employed in high-throughput screening of compound libraries against this target. Being electrically neutral, the NKCC1 screening is difficult to achieve by both manual and automated electrophysiological techniques. These techniques, although considered gold standard, suffer from their inherent problems of being too slow to be in high-throughput format and with high running costs. In addition to being a functional assay for NKCC1, it is nontoxic as compared with thallium flux assay, which is prone to generate high number of false-positive/false-negative rates because of its innate fluorescence issues.

Azosemide is more potent than bumetanide and various other loop diuretics to inhibit the sodium-potassium-chloride-cotransporter human variants hNKCC1A and hNKCC1B

“…IC 50 for bumetanide (1.16 µM) determined by the latter technology was similar to the bumetanide IC 50 s reported here, but none of the blind compounds reported by Gill et al . 47 reached the potency of azosemide determined in the present study.…”

“…However, it may be difficult to develop NKCC1B-selective inhibitors because the only difference between NKCC1A and NKCC1B is the lack of the small exon 21, a region that is very poorly conserved, predicted to be unstructured, and seems unlikely to be involved in an important way with either ion translocation or diuretic inhibition and probably not to a significant extent to the phosphorylation-activation mechanism 38 . One feasible plan to tackle the goal would be a high-throughput screening (HTS) assay for the splice variants of the hNKCC1 cotransporter similar to the HTS approach recently described for hNKCC1 expressed in human embryonic kidney (HEK) cell line 47 . The latter cell-based Rb + flux assay has been used to screen for NKCC1 inhibitors in a focused library of 1450 compounds, followed by a full HTS of 1.2 million compounds, using the Ion Channel Reader (ICR) technology for detecting intracellular concentration of Rb + in cell lysates 47 .…”

“…One feasible plan to tackle the goal would be a high-throughput screening (HTS) assay for the splice variants of the hNKCC1 cotransporter similar to the HTS approach recently described for hNKCC1 expressed in human embryonic kidney (HEK) cell line 47 . The latter cell-based Rb + flux assay has been used to screen for NKCC1 inhibitors in a focused library of 1450 compounds, followed by a full HTS of 1.2 million compounds, using the Ion Channel Reader (ICR) technology for detecting intracellular concentration of Rb + in cell lysates 47 . IC 50 for bumetanide (1.16 µM) determined by the latter technology was similar to the bumetanide IC 50 s reported here, but none of the blind compounds reported by Gill et al .…”

Screening Strategies for the Discovery of Ion Channel Monoclonal Antibodies

“…AAS screens for ion flux have been described for small molecules (Karczewski et al., ; Qi et al., ) but to our knowledge have not been described for ion channel antibody discovery. AAS‐based flux assays offer a robust alternative (Z’ > 0.9) compatible with automation, miniaturization, and the potential to avoid the toxicity artefacts associated with fluorescent assays (Gill et al., ). AAS requires cell washing, separation from supernatants, and lysis steps, thus having a poorer temporal resolution relative to fluorescence‐based assays.…”

“…Current Protocols in Pharmacology automation, miniaturization, and the potential to avoid the toxicity artefacts associated with fluorescent assays (Gill et al, 2017). AAS requires cell washing, separation from supernatants, and lysis steps, thus having a poorer temporal resolution relative to fluorescence-based assays.…”