Table 4.
Compound activity outcome assignments based on multi-channel assay readouts (a) multi-channel fluorescence agonist mode assay (b) multi-channel fluorescence antagonist mode assay (c) luminescence antagonist mode assay with cytotoxicity counter screen
| (a) | ||||
| *Ratio outcome | Signal channel outcome | Same reporter assay promiscuity | Auto fluorescence outcome | Activity outcome |
| inactive | N/A | N/A | N/A | inactive |
| inconclusive | N/A | N/A | N/A | inconclusive |
| active agonist | agonist | average curve rank ≤4 | inactive or AC50 fluor/AC50 signal ≥3 | active agonist |
| inconclusive agonist | agonist | average curve rank ≤4 | inactive or AC50 fluor/AC50 signal ≥3 | inconclusive agonist |
| agonist | agonist | average curve rank >4 | agonist and AC50 fluor/AC50 signal <3 | inconclusive agonist (fluorescent) |
| active antagonist | antagonist | N/A | N/A | active antagonist |
| inconclusive antagonist | antagonist | N/A | N/A | inconclusive antagonist |
| (b) | ||||
| *Ratio outcome | Signal channel outcome | Cell viability outcome | Other conditions | Activity outcome |
| inactive | N/A | N/A | N/A | inactive |
| inconclusive | N/A | N/A | N/A | inconclusive |
| active agonist | agonist | inactive or agonist | N/A | active agonist |
| active agonist | agonist | antagonist | AC50 viability/AC50 signal ≥3 (p<0.05) | active agonist |
| inconclusive agonist | agonist | N/A | N/A | inconclusive agonist |
| agonist | agonist | antagonist | AC50 viability/AC50 signal <3 or p≥0.05 | inconclusive agonist (cytotoxic) |
| active antagonist | antagonist | inactive or agonist | N/A | active antagonist |
| active antagonist | antagonist | antagonist | AC50 viability/AC50 signal ≥3 (p<0.05) | active antagonist |
| inconclusive antagonist | antagonist | N/A | N/A | inconclusive antagonist |
| antagonist | antagonist | antagonist | AC50 viability/AC50 signal <3 or p≥0.05 | inconclusive antagonist (cytotoxic) |
| (c) | ||||
| Signal channel outcome | Cell viability outcome | Other conditions | Activity outcome | |
| inactive | N/A | N/A | inactive | |
| inconclusive | N/A | N/A | inconclusive | |
| active agonist | inactive or agonist | N/A | active agonist | |
| active agonist | antagonist | AC50 viability/AC50 signal ≥3 (p<0.05) | active agonist | |
| inconclusive agonist | N/A | N/A | inconclusive agonist | |
| agonist | antagonist | AC50 viability/AC50 signal <3 or p≥0.05 | inconclusive agonist (cytotoxic) | |
| active antagonist | inactive or agonist | N/A | active antagonist | |
| active antagonist | antagonist | AC50 viability/AC50 signal ≥3 (p<0.05) | active antagonist | |
| inconclusive antagonist | N/A | N/A | inconclusive antagonist | |
| antagonist | antagonist | AC50 viability/AC50 signal <3 or p≥0.05 | inconclusive antagonist (cytotoxic) | |
*
Ratio = signal channel/control channel
Abbreviations: AC50 fluor = AC50 in the auto fluorescence assay, AC50 signal = AC50 in the ratio channel
*
Ratio = signal channel/control channel
Abbreviations: AC50 viability = AC50 in the cell viability assay, AC50 signal = AC50 in the ratio channel
Abbreviations: AC50 viability = AC50 in the cell viability assay, AC50 signal = AC50 in the antagonist mode assay