| # | Aladdin ID | Assay Type | Description | Organism | Compounds | Reference | BAO Format | Source | |
|---|---|---|---|---|---|---|---|---|---|
| 1. | ALA3888984 | B | ELISA-Based In Vitro Kinase Assay: GST-tagged recombinant wild-type or L1196M mutated ALK kinase (rALK) was expressed in Sf9 insect cells using the pBacPAK baculovirus vector system (Clontech) and purified using Glutathione Sepharose 4B affinity beads (GE Healthcare). Recombinant 3C protease was used to remove the GST tag. Purified ALK was used to screen inhibitors in the ELISA-based kinase assay, as follows: Nunc-Immuno 96-well plates were incubated overnight at 30° C. with coating solution containing 2 μg of a specific ALK peptide substrate (ARDIYRASFFRKGGCAMLPVK) in PBS. Wells were then washed with 200 μL of wash buffer (PBS-Tween 0.05%) and incubated with 4% BSA in PBS for at least 2 h at 30° C. The kinase reaction was performed in the presence of 50 mM Tris pH 7.5, 5 mM MnCl2, 5 mM MgCl2, 0.3 mM ATP and purified rALK in a total volume of 100 μL/well at 30° C. for 15 min. For inhibitor testing the reaction mix was preincubated with inhibitor or vehicle for 10 min at room temperature before transferring to the ELISA plate. After the reaction, the wells were washed 5 times with 200 μL of wash buffer. Phosphorylated peptide was detected using 100 μL/well of a mouse monoclonal anti-phosphotyrosine antibody (clone 4G10 UpstateBiotech Ltd) diluted 1:2000 in PBS+4% BSA. After 30 min incubation at room temperature the antibody was removed and wells were washed as described above. 100 μL of a secondary antibody (anti-mouse IgG, Horseradish Peroxidase linked whole antibody, Amersham Pharmacia Biotech) diluted 1:1000 in PBS+4% BSA was added to each well and the plate was incubated again for 30 min at room temperature before washing as above. The plate was developed using 100 μL/well TMB Substrate Solution (Pierce) and the reaction was stopped by adding an equal volume of 1M H2SO4. Finally, the absorbance was read at 450 nm using an ELISA plate reader (Bio-Rad). | Homo sapiens | 57 | ALA3886908 | cell-based format | BindingDB Database | |
| 2. | ALA3888985 | B | ELISA-Based In Vitro Kinase Assay: GST-tagged recombinant wild-type or L1196M mutated ALK kinase (rALK) was expressed in Sf9 insect cells using the pBacPAK baculovirus vector system (Clontech) and purified using Glutathione Sepharose 4B affinity beads (GE Healthcare). Recombinant 3C protease was used to remove the GST tag. Purified ALK was used to screen inhibitors in the ELISA-based kinase assay, as follows: Nunc-Immuno 96-well plates were incubated overnight at 30° C. with coating solution containing 2 μg of a specific ALK peptide substrate (ARDIYRASFFRKGGCAMLPVK) in PBS. Wells were then washed with 200 μL of wash buffer (PBS-Tween 0.05%) and incubated with 4% BSA in PBS for at least 2 h at 30° C. The kinase reaction was performed in the presence of 50 mM Tris pH 7.5, 5 mM MnCl2, 5 mM MgCl2, 0.3 mM ATP and purified rALK in a total volume of 100 μL/well at 30° C. for 15 min. For inhibitor testing the reaction mix was preincubated with inhibitor or vehicle for 10 min at room temperature before transferring to the ELISA plate. After the reaction, the wells were washed 5 times with 200 μL of wash buffer. Phosphorylated peptide was detected using 100 μL/well of a mouse monoclonal anti-phosphotyrosine antibody (clone 4G10 UpstateBiotech Ltd) diluted 1:2000 in PBS+4% BSA. After 30 min incubation at room temperature the antibody was removed and wells were washed as described above. 100 μL of a secondary antibody (anti-mouse IgG, Horseradish Peroxidase linked whole antibody, Amersham Pharmacia Biotech) diluted 1:1000 in PBS+4% BSA was added to each well and the plate was incubated again for 30 min at room temperature before washing as above. The plate was developed using 100 μL/well TMB Substrate Solution (Pierce) and the reaction was stopped by adding an equal volume of 1M H2SO4. Finally, the absorbance was read at 450 nm using an ELISA plate reader (Bio-Rad). | 56 | ALA3886908 | cell-based format | BindingDB Database | ||
| 3. | ALA5140257 | B | Inhibition of recombinant GST-tagged ALK L1196M mutant (1064 to 1427 residues) (unknown origin) expressed in Sf9 cells using TMB peptide as substrate preincubated for 10 mins followed by ATP addition and measured after 15 mins by ELISA analysis | Homo sapiens | 71 | ALA5137040 | cell-based format | Scientific Literature | |
| 4. | ALA5140258 | B | Inhibition of recombinant GST-tagged wild type ALK (1064 to 1427 residues) (unknown origin) expressed in Sf9 cells using TMB peptide as substrate preincubated for 10 mins followed by ATP addition and measured after 15 mins by ELISA analysis | Homo sapiens | 71 | ALA5137040 | cell-based format | Scientific Literature | |
| 5. | ALA5140259 | F | Cytotoxicity against mouse BaF3 cells assessed as inhibition of cell proliferation incubated for 72 hrs by MTS assay | Mus musculus | 15 | ALA5137040 | cell-based format | Scientific Literature | |
| 6. | ALA5140260 | F | Cytotoxicity against mouse BaF3 cells harboring crizotinib resistant wild type NPM/ALK assessed as inhibition of cell proliferation incubated for 72 hrs by MTS assay | Mus musculus | 15 | ALA5137040 | cell-based format | Scientific Literature | |
| 7. | ALA5140261 | F | Cytotoxicity against mouse BaF3 cells harboring crizotinib resistant NPM/ALK L1196M mutant assessed as inhibition of cell proliferation incubated for 72 hrs by MTS assay | Mus musculus | 15 | ALA5137040 | cell-based format | Scientific Literature | |
| 8. | ALA5140262 | F | Cytotoxicity against mouse BaF3 cells harboring crizotinib resistant NPM/ALK L1152R mutant assessed as inhibition of cell proliferation incubated for 72 hrs by MTS assay | Mus musculus | 1 | ALA5137040 | cell-based format | Scientific Literature | |
| 9. | ALA5140263 | F | Cytotoxicity against mouse BaF3 cells harboring crizotinib resistant NPM/ALK C1156Y mutant assessed as inhibition of cell proliferation incubated for 72 hrs by MTS assay | Mus musculus | 1 | ALA5137040 | cell-based format | Scientific Literature | |
| 10. | ALA5140264 | F | Cytotoxicity against mouse BaF3 cells harboring crizotinib resistant NPM/ALK I1171N mutant assessed as inhibition of cell proliferation incubated for 72 hrs by MTS assay | Mus musculus | 1 | ALA5137040 | cell-based format | Scientific Literature | |
| 11. | ALA5140265 | F | Cytotoxicity against mouse BaF3 cells harboring crizotinib resistant NPM/ALK F1174L mutant assessed as inhibition of cell proliferation incubated for 72 hrs by MTS assay | Mus musculus | 1 | ALA5137040 | cell-based format | Scientific Literature | |
| 12. | ALA5140266 | F | Cytotoxicity against mouse BaF3 cells harboring crizotinib resistant NPM/ALK G1202R mutant assessed as inhibition of cell proliferation incubated for 72 hrs by MTS assay | Mus musculus | 1 | ALA5137040 | cell-based format | Scientific Literature | |
| 13. | ALA5140267 | F | Resistance index, ratio of IC50 for cytotoxicity against mouse BaF3 cells harboring crizotinib resistant NPM/ALK L1152R mutant to IC50 for cytotoxicity against mouse BaF3 cells harboring crizotinib resistant wild type NPM/ALK assessed as inhibition of cell proliferation incubated for 72 hrs by MTS assay | Mus musculus | 1 | ALA5137040 | cell-based format | Scientific Literature | |
| 14. | ALA5140268 | F | Resistance index, ratio of IC50 for cytotoxicity against mouse BaF3 cells harboring crizotinib resistant NPM/ALK C1156Y mutant to IC50 for cytotoxicity against mouse BaF3 cells harboring crizotinib resistant wild type NPM/ALK assessed as inhibition of cell proliferation incubated for 72 hrs by MTS assay | Mus musculus | 1 | ALA5137040 | cell-based format | Scientific Literature | |
| 15. | ALA5140269 | F | Resistance index, ratio of IC50 for cytotoxicity against mouse BaF3 cells harboring crizotinib resistant NPM/ALK I1171N mutant to IC50 for cytotoxicity against mouse BaF3 cells harboring crizotinib resistant wild type NPM/ALK assessed as inhibition of cell proliferation incubated for 72 hrs by MTS assay | Mus musculus | 1 | ALA5137040 | cell-based format | Scientific Literature | |
| 16. | ALA5140270 | F | Resistance index, ratio of IC50 for cytotoxicity against mouse BaF3 cells harboring crizotinib resistant NPM/ALK F1174L mutant to IC50 for cytotoxicity against mouse BaF3 cells harboring crizotinib resistant wild type NPM/ALK assessed as inhibition of cell proliferation incubated for 72 hrs by MTS assay | Mus musculus | 1 | ALA5137040 | cell-based format | Scientific Literature | |
| 17. | ALA5140271 | F | Resistance index, ratio of IC50 for cytotoxicity against mouse BaF3 cells harboring crizotinib resistant NPM/ALK L1196M mutant to IC50 for cytotoxicity against mouse BaF3 cells harboring crizotinib resistant wild type NPM/ALK assessed as inhibition of cell proliferation incubated for 72 hrs by MTS assay | Mus musculus | 1 | ALA5137040 | cell-based format | Scientific Literature | |
| 18. | ALA5140272 | F | Resistance index, ratio of IC50 for cytotoxicity against mouse BaF3 cells harboring crizotinib resistant NPM/ALK G1202R mutant to IC50 for cytotoxicity against mouse BaF3 cells harboring crizotinib resistant wild type NPM/ALK assessed as inhibition of cell proliferation incubated for 72 hrs by MTS assay | Mus musculus | 1 | ALA5137040 | cell-based format | Scientific Literature | |
| 19. | ALA5140273 | F | Antiproliferative activity against human KARPAS-299 cells harboring crizotinib resistant ALK L1196Q mutant assessed as inhibition of cell growth at 0.1 to 10 uM incubated for 72 hrs by MTS assay | Homo sapiens | 1 | ALA5137040 | cell-based format | Scientific Literature | |
| 20. | ALA5140274 | F | Antiproliferative activity against human SUPM2 cells harboring crizotinib resistant ALK L1171N mutant assessed as inhibition of cell growth at 0.1 to 10 uM incubated for 72 hrs by MTS assay | Homo sapiens | 1 | ALA5137040 | cell-based format | Scientific Literature |
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