| # | Aladdin ID | Assay Type | Description | Organism | Compounds | Reference | BAO Format | Source | |
|---|---|---|---|---|---|---|---|---|---|
| 1. | ALA1804183 | B | Binding affinity to His-tagged recombinant human sRBP expressed in Escherichia coli BL21(DE3) assessed as apparent dissociation constant after 5 mins by fluorescence spectrophotometric analysis | Homo sapiens | 8 | assay format | Scientific Literature | ||
| 2. | ALA1804184 | B | Binding affinity to His-tagged recombinant human sRBP expressed in Escherichia coli BL21(DE3) assessed as disruption of sRBP-TTR protein interaction at 1 to 100 uM after 1 hr by SDS-PAGE and silver staining method | Homo sapiens | 21 | assay format | Scientific Literature | ||
| 3. | ALA1804185 | B | Binding affinity to His-tagged recombinant human sRBP expressed in Escherichia coli BL21(DE3) assessed as disruption of sRBP-TTR protein interaction after 1 hr by SDS-PAGE and silver staining method | Homo sapiens | 47 | assay format | Scientific Literature | ||
| 4. | ALA1804186 | B | Binding affinity to His-tagged recombinant human sRBP expressed in Escherichia coli BL21(DE3) assessed as disruption of sRBP-TTR protein interaction at 100 uM after 1 hr by SDS-PAGE and silver staining method relative to 10 uM ROH | Homo sapiens | 1 | assay format | Scientific Literature | ||
| 5. | ALA1804187 | F | Antagonist activity at His-tagged recombinant human sRBP expressed in Escherichia coli BL21(DE3) assessed as disruption of ROH-sRBP-TTR protein interaction after 2 hr by TR-FRET assay | Homo sapiens | 7 | assay format | Scientific Literature | ||
| 6. | ALA1804571 | F | Antagonist activity at His-tagged recombinant human sRBP expressed in Escherichia coli BL21(DE3) assessed as disruption of ROH-sRBP-TTR protein interaction after 2 hr by TR-FRET assay relative to control | Homo sapiens | 7 | assay format | Scientific Literature | ||
| 7. | ALA1804572 | F | Antagonist activity at His-tagged recombinant human sRBP expressed in Escherichia coli BL21(DE3) assessed as disruption of sRBP-TTR protein interaction after 2 hr by surface plasmon resonance assay | Homo sapiens | 2 | assay format | Scientific Literature | ||
| 8. | ALA1804573 | B | Binding affinity to immobilized His-tagged recombinant human sRBP assessed as inhibition of sRBP and detergent treated HEK293 membrane interaction at 100 uM after 1 hr by surface plasmon resonance assay | Homo sapiens | 7 | cell-based format | Scientific Literature | ||
| 9. | ALA3291832 | B | Displacement of [3H]-retinol from human recombinant RBP4 by scintillation proximity assay | Homo sapiens | 33 | single protein format | Scientific Literature | ||
| 10. | ALA3291833 | B | Inhibition of human recombinant RBP4-transthyretin interaction by FRET analysis | Homo sapiens | 26 | single protein format | Scientific Literature | ||
| 11. | ALA3380009 | B | Displacement of [3H]retinol from biotinylated human RBP4 by scintillation proximity assay | Homo sapiens | 35 | single protein format | Scientific Literature | ||
| 12. | ALA3380010 | B | Antagonist activity at maltose binding protein-tagged RBP4 (unknown origin) expressed in Escherichia coli assessed as inhibition of retinol-induced protein/transthyretin interaction by HTRF assay | Homo sapiens | 35 | assay format | Scientific Literature | ||
| 13. | ALA3594967 | B | Displacement of [3H]-retinol from RBP4 (unknown origin) by scintillation proximity assay | Homo sapiens | 36 | single protein format | Scientific Literature | ||
| 14. | ALA3594968 | B | Inhibition of retinol-induced interaction of bacterially expressed MBP-tagged RBP4 (unknown origin) with Eu3+ cryptate labeled TTR by HTRF assay | Homo sapiens | 35 | single protein format | Scientific Literature | ||
| 15. | ALA3705134 | B | Binding Assay: The action of the compound of the present invention to inhibit binding of RBP4 and retinol and TTR was evaluated using the Retinol-RBP4-TTR ELISA (human-type ELISA) system. | Homo sapiens | 15 | single protein format | BindingDB Database | ||
| 16. | ALA3705730 | B | Binding Assay: Streptavidin (20 ul) (10 ug/ml Streptavidin type II (Wako Pure Chemical Industries, Ltd.), 10 mM Tris-HCl (pH 7.5), 10 mM NaCl) was added to a 384 well black plate (Nunc MaxiSorp, Thermo Fisher Scientific Inc.), and the plate was subjected to centrifugation (1000 rpm, 1 min) and coated overnight at 4 C. The plate was washed twice with PBST (PBS, 0.05% Tween 20, 100 ul/well) and blocked with 25% Block Ace (Snow Brand Milk Products Co., Ltd., PBS, 100 ul/well). The plate was subjected to centrifugation (1000 rpm, 1 min) and incubated at room temperature for 4 hr or overnight at 4 C. The plate was washed twice with PBST (PBS, 0.05% Tween 20, 100 ul/well), and biotinylate human TTR (stock solution concentration 1.0 mg/ml) diluted 750-fold with PBST was added at 20 ul/well. The plate was subjected to centrifugation (1000 rpm, 1 min) and stood still at room temperature for 1.5 hr or overnight at 4 C. The plate was washed 3 times with PBST (100 ul/well). | Homo sapiens | 17 | single protein format | BindingDB Database | ||
| 17. | ALA3889135 | B | Scintillation Proximity Binding Assay: Untagged human RBP4 purified from urine of tubular proteinuria patients was purchased from Fitzgerald Industries International. It was biotinylated using the EZ-Link Sulfo-NHS-LC-Biotinylation kit from Pierce following the manufacturer's recommendations. Binding experiments were performed in 96-well plates (OptiPlate, PerkinElmer) in a final assay volume of 100 ul per well in SPA buffer (IX PBS, pH 7.4, 1 mM EDTA, 0.1% BSA, 0.5% CHAPS). The reaction mix contained 10 nM 3H-Retinol (48.7 Ci/mmol; PerkinElmer), 0.3 mg/well Streptavidin-PVT beads, 50 nM biotinylated RBP4 and a test compound. Nonspecific binding was determined in the presence of 20 uM of unlabeled retinol. The reaction mix was assembled in the dark under dim red light. The plates were sealed with clear tape (TopSeal-A: 96-well microplate, PerkinElmer), wrapped in the aluminum foil, and allowed to equilibrate 6 hours at room temperature followed by overnight incubation at +4° C. Radiocounts were measured using a TopCount NXT counter (Packard Instrument Company). | Homo sapiens | 112 | single protein format | BindingDB Database | ||
| 18. | ALA3889136 | B | TR-FRET Assay: TR-FRET assay for retinol-induced RBP4-TTR interaction Binding of a desired RBP4 antagonist displaces retinol and induces hindrance for RBP4-TTR interaction resulting in the decreased FRET signal (FIG. 7). Bacterially expressed MBP-RBP4 and untagged TTR were used in this assay. For the use in the TR-FRET assay the maltose binding protein (MBP)-tagged human RBP4 fragment (amino acids 19-201) was expressed in the Gold(DE3)pLysS E. coli strain (Stratagene) using the pMAL-c4x vector. Following cell lysis, recombinant RBP4 was purified from the soluble fraction using the ACTA FPLC system (GE Healthcare) equipped with the 5-ml the MBP Trap HP column. Human untagged TTR was purchased from Calbiochem. Untagged TTR was labeled directly with Eu3+ Cryptate-NHS using the HTRF Cryptate Labeling kit from CisBio following the manufacturer's recommendations. HTRF assay was performed in white low volume 384 well plates (Greiner-Bio) in a final assay volume of 16 ul per well. The reaction buffer contained 10 mM Tris-HCl pH 7.5, 1 mM DTT, 0.05% NP-40, 0.05% Prionex, 6% glycerol, and 400 mM KF. Each reaction contained 60 nM MBP-RBP4 and 2 nM TTR-Eu along with 26.7 nM of anti-MBP antibody conjugated with d2 (Cisbio). Titration of test compounds in this assay was conducted in the presence of 1 uM retinol. All reactions were assembled in the dark under dim red light and incubated overnight at +4° C. wrapped in aluminum foil. TR-FRET signal was measured in the SpectraMax M5e Multimode Plate Reader (Molecular Device). Fluorescence was excited at 337 nm and two readings per well were taken: Reading 1 for time-gated energy transfer from Eu(K) to d2 (337 nm excitation, 668 nm emission, counting delay 75 microseconds, counting window 100 microseconds) and Reading 2 for Eu(K) time-gated fluorescence (337 nm excitation, 620 nm emission, counting delay 400 microseconds, counting window 400 microseconds). | Homo sapiens | 112 | assay format | BindingDB Database | ||
| 19. | ALA4305280 | B | Competitive displacement of 3[H]retinol from biotinylated RBP4 (unknown origin) by surface plasmon resonance analysis | Homo sapiens | 47 | single protein format | Scientific Literature | ||
| 20. | ALA4305353 | B | Displacement of 3[H]retinol from human biotinylated RBP4 after overnight incubation by surface plasmon resonance analysis | Homo sapiens | 4 | single protein format | Scientific Literature |
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