Assay Detail
Binding
CHEMBL5732230
Review assay metadata, readout intent, target linkage, and publication context from the same page.
ROCK1 and ROCK2 Compound Selectivity: Dose response curves for Rho-kinase inhibition were derived from a Millipore immuno-based 96 well plate assay (Millipore catalog number CSA001). Purified active ROCK1 and ROCK2 were obtained from Invitrogen (catalog numbers ROCK1, PV3691 and ROCK2, PV3759). The kit components include assay plates, which are pre-coated with recombinant MYPT1, which contains a specifically phosphorylatable Thr696. The inhibitory activities of compounds are measured according to the manufactures protocol. Briefly, decreasing concentrations of test compounds or the known ROCK inhibitor Y-27963, are added, from 50 uM to 0.003 uM to reaction buffer containing 5 mM MgCl2, and 10 mUnits of ROCK1 or ROCK2 in assay dilution buffer. This mixture is overlayed into the 96 well plate and the reaction is initiated with the addition of 2.5 uM ATP. The assay proceeds at 30° Celsius for 30 minutes with gentle shaking at 120 rpm. The assay is terminated by washing of the plate 3 times with Tris-buffered saline and tween wash buffer. Anti-phospho-MYPT1 (Thr696) antibody is added to each well to detect the phosphorylated substrate and incubated for 1 hour at room temperature after which HRP conjugated anti-rabbit IgG secondary is added for 1 hour at room temperature. After washing the assay is developed using a substrate reagent and the absorbance is read at 450 nm on a Tecan Infinite M1000 reflecting the relative remaining ROCK phosphorylation activity.
141
Total Activities
36
Compounds Tested
4
Activity Types
0
Assay Parameters
Assay Information
| Assay Type | Binding |
| Organism | Homo sapiens |
| Confidence | 9 — Direct single protein target |
| Curated By | Autocuration |
Publication
Rho kinase inhibitors
Activity Statistics
| Type | Count | Avg pChEMBL | Best pChEMBL |
|---|---|---|---|
| kon | 47 | - | - |
| k_off | 47 | - | - |
| IC50 | 39 | 6.65 | 7.89 |
| Ki | 8 | 7.27 | 7.70 |
Compounds Tested
| Compound | Name | Phase | Activities | Best pChEMBL |
|---|---|---|---|---|
| CHEMBL5992096 | — | — | 3 | 7.89 |
| CHEMBL5858473 | — | — | 6 | 7.70 |
| CHEMBL5819970 | — | — | 3 | 7.70 |
| CHEMBL5768727 | — | — | 9 | 7.70 |
| CHEMBL5749663 | — | — | 3 | 7.52 |
| CHEMBL5815614 | — | — | 3 | 7.52 |
| CHEMBL5936293 | — | — | 3 | 7.52 |
| CHEMBL5850835 | — | — | 9 | 7.52 |
| CHEMBL5898334 | — | — | 3 | 7.40 |
| CHEMBL2005186 | BELUMOSUDIL | 4.0 | 6 | 7.40 |
| CHEMBL5882290 | — | — | 3 | 7.40 |
| CHEMBL5763746 | — | — | 3 | 7.30 |
| CHEMBL6021969 | — | — | 3 | 7.22 |
| CHEMBL559147 | Y-27632 | — | 6 | 7.22 |
| CHEMBL5896038 | — | — | 3 | 7.22 |
| CHEMBL5751696 | — | — | 3 | 7.16 |
| CHEMBL5790296 | — | — | 3 | 7.16 |
| CHEMBL6016872 | — | — | 9 | 7.16 |
| CHEMBL5819432 | — | — | 3 | 7.10 |
| CHEMBL5744109 | — | — | 3 | 7.10 |
| CHEMBL5828705 | — | — | 3 | 6.92 |
| CHEMBL5986438 | — | — | 6 | 6.77 |
| CHEMBL5841511 | — | — | 6 | 6.75 |
| CHEMBL5817799 | — | — | 3 | 6.72 |
| CHEMBL5794019 | — | — | 3 | 6.70 |
| CHEMBL6003524 | — | — | 3 | 6.51 |
| CHEMBL5989311 | — | — | 3 | 6.47 |
| CHEMBL5898268 | — | — | 3 | 6.42 |
| CHEMBL5958076 | — | — | 3 | 6.40 |
| CHEMBL5953458 | — | — | 3 | 6.30 |
Activity Data
| Compound | Name | Type | Rel. | Value | Units | pChEMBL |
|---|---|---|---|---|---|---|
| CHEMBL5992096 | — | IC50 | = | 13.0 | nM | 7.89 |
| CHEMBL5858473 | — | Ki | = | 20.0 | nM | 7.70 |
| CHEMBL5768727 | — | Ki | = | 20.0 | nM | 7.70 |
| CHEMBL5819970 | — | IC50 | = | 20.0 | nM | 7.70 |
| CHEMBL5936293 | — | IC50 | = | 30.0 | nM | 7.52 |
| CHEMBL5850835 | — | Ki | = | 30.0 | nM | 7.52 |
| CHEMBL5815614 | — | IC50 | = | 30.0 | nM | 7.52 |
| CHEMBL5749663 | — | IC50 | = | 30.0 | nM | 7.52 |
| CHEMBL5850835 | — | IC50 | = | 30.0 | nM | 7.52 |
| CHEMBL2005186 | BELUMOSUDIL | Ki | = | 40.0 | nM | 7.40 |
| CHEMBL5898334 | — | IC50 | = | 40.0 | nM | 7.40 |
| CHEMBL5882290 | — | IC50 | = | 40.0 | nM | 7.40 |
| CHEMBL5763746 | — | IC50 | = | 50.0 | nM | 7.30 |
| CHEMBL6021969 | — | IC50 | = | 60.0 | nM | 7.22 |
| CHEMBL559147 | Y-27632 | Ki | = | 60.0 | nM | 7.22 |
| CHEMBL5896038 | — | IC50 | = | 60.0 | nM | 7.22 |
| CHEMBL5790296 | — | IC50 | = | 70.0 | nM | 7.16 |
| CHEMBL6016872 | — | IC50 | = | 70.0 | nM | 7.16 |
| CHEMBL5751696 | — | IC50 | = | 70.0 | nM | 7.16 |
| CHEMBL5819432 | — | IC50 | = | 80.0 | nM | 7.10 |
| CHEMBL6016872 | — | Ki | = | 80.0 | nM | 7.10 |
| CHEMBL5744109 | — | IC50 | = | 80.0 | nM | 7.10 |
| CHEMBL5828705 | — | IC50 | = | 120.0 | nM | 6.92 |
| CHEMBL5986438 | — | Ki | = | 170.0 | nM | 6.77 |
| CHEMBL5841511 | — | Ki | = | 180.0 | nM | 6.75 |
| CHEMBL5817799 | — | IC50 | = | 191.0 | nM | 6.72 |
| CHEMBL5794019 | — | IC50 | = | 200.0 | nM | 6.70 |
| CHEMBL6003524 | — | IC50 | = | 310.0 | nM | 6.51 |
| CHEMBL5989311 | — | IC50 | = | 340.0 | nM | 6.47 |
| CHEMBL5898268 | — | IC50 | = | 380.0 | nM | 6.42 |
| CHEMBL5958076 | — | IC50 | = | 400.0 | nM | 6.40 |
| CHEMBL5768727 | — | IC50 | = | 470.0 | nM | 6.33 |
| CHEMBL5953458 | — | IC50 | = | 500.0 | nM | 6.30 |
| CHEMBL5858473 | — | IC50 | = | 540.0 | nM | 6.27 |
| CHEMBL6056601 | — | IC50 | = | 700.0 | nM | 6.16 |
| CHEMBL5850835 | — | IC50 | = | 720.0 | nM | 6.14 |
| CHEMBL5899291 | — | IC50 | = | 1000.0 | nM | 6.00 |
| CHEMBL5768727 | — | IC50 | = | 1000.0 | nM | 6.00 |
| CHEMBL2005186 | BELUMOSUDIL | IC50 | = | 1020.0 | nM | 5.99 |
| CHEMBL559147 | Y-27632 | IC50 | = | 1630.0 | nM | 5.79 |
| CHEMBL6016872 | — | IC50 | = | 1960.0 | nM | 5.71 |
| CHEMBL5991297 | — | IC50 | = | 3200.0 | nM | 5.50 |
| CHEMBL5986438 | — | IC50 | = | 4250.0 | nM | 5.37 |
| CHEMBL5841511 | — | IC50 | = | 4640.0 | nM | 5.33 |
| CHEMBL5967940 | — | IC50 | = | 4900.0 | nM | 5.31 |
| CHEMBL5794019 | — | k_off | = | - | s-1 | - |
| CHEMBL5815614 | — | kon | = | - | — | - |
| CHEMBL5828705 | — | k_off | = | - | s-1 | - |
| CHEMBL5898268 | — | kon | = | - | — | - |
| CHEMBL5828705 | — | kon | = | - | — | - |