Physicochemical properties of probe BAY-707 and control BAY-604
| BAY-707 | |
|---|---|
| log D (pH 7.5) | 1.6 |
| cLog P | 0.6 |
| LE | 0.6 |
| BAY-604 | |
|---|---|
| log D (pH 7.5) | 1.9 |
Solubility
| BAY-707: |
aqueous, pH 6.5: 288 mg/L
|
| BAY-604: |
aqueous, pH 6.5: 2170 mg/L
|
Redox activity data
Redox activity of the probe/control was tested in order to find compounds that interfere with assays or exhibit unwanted mechanisms of bioreactivity leading to false positive inhibitions. The horseradish peroxidase-phenol red (HRP-PR) assay is used to detect H2O2 generated by redox cycling compounds in the presence or absence of the reducing agent dithiotreitol (DTT). The resazurin assay detects redox active compounds, that catalyze the conversion of resazurin to resorufin through the formation of free radicals in the presence of the reducing agent DTT. The fluorescent probe, 2',7'-dichlorodihydrofluorescein diacetate (H2DCFDA) is used to find reactive oxygen species-generating compounds in the presence or absence of the reducing agent tris(2-carboxyethyl)phosphine (TCEP). All assays are performed in a cell-free environment.
| Assay name | Cmp. conc. | Result | Activity |
|---|---|---|---|
| HRP-PR assay (redox-free) | 100 µM | A = 1 | No |
| HRP-PR assay (1 mM DTT) | 100 µM | A = 1 | No |
| Resazurin assay | 10 µM | F = 0.9 | No |
| Resazurin assay | 1 µM | F = 0.9 | No |
| Resazurin assay | 0.1 µM | F = 1 | No |
| H2DCFDA assay (redox-free) | 100 µM | F = 1.6 | Yes |
| H2DCFDA assay (100 µM TCEP) | 100 µM | F = 4.7 | Yes |
| Assay name | Cmp. conc. | Result | Activity |
|---|---|---|---|
| HRP-PR assay (redox-free) | 100 µM | A = 1.1 | No |
| HRP-PR assay (1 mM DTT) | 100 µM | A = 1 | No |
| Resazurin assay | 10 µM | F = 1 | No |
| Resazurin assay | 1 µM | F = 1 | No |
| Resazurin assay | 0.1 µM | F = 1 | No |
| H2DCFDA assay (redox-free) | 100 µM | F = 1 | No |
| H2DCFDA assay (100 µM TCEP) | 100 µM | F = 0.9 | No |
A: Absorbance; F: Fluorescence; PS: poor solubility. Final DMSO concentrations were 5% in the HRP-PR and the H2DCFDA assays and 1% in the resazurin assay. Absorbance or fluorescence relative to the DMSO blank is reported. Threshold for activity flag was greater than 10-fold of standard deviation relative to the mean of three duplicates of DMSO blank. Shown are data from PMID:35301150.