| # | Aladdin ID | Assay Type | Description | Organism | Compounds | Reference | BAO Format | Source | |
|---|---|---|---|---|---|---|---|---|---|
| 1. | ALA2320318 | B | Binding affinity to menin (unknown origin) after 60 mins by fluorescence polarization assay | Homo sapiens | 37 | ALA2311406 | single protein format | Scientific Literature | |
| 2. | ALA3130537 | B | Inhibition of menin-FLAG-tagged MLL-AF9 interaction in HEK293 cells at 3 to 12 uM after 12 hrs by co-immunoprecipitation assay | Homo sapiens | 2 | ALA3124794 | cell-based format | Scientific Literature | |
| 3. | ALA3130538 | B | Binding affinity to human menin by isothermal titration calorimetric analysis | Homo sapiens | 3 | ALA3124794 | single protein format | Scientific Literature | |
| 4. | ALA3130539 | B | Competitive binding affinity to human menin at 100 uM by STD-NMR spectroscopic analysis in presence of MLL (1 to 46) | Homo sapiens | 1 | ALA3124794 | single protein format | Scientific Literature | |
| 5. | ALA3130540 | B | Binding affinity to human menin at 100 uM by STD-NMR spectroscopic analysis | Homo sapiens | 1 | ALA3124794 | single protein format | Scientific Literature | |
| 6. | ALA3130541 | B | Inhibition of human His-tagged menin-biotinylated MLL (4 to 15) interaction after 1 hr by HTRF assay | Homo sapiens | 29 | ALA3124794 | single protein format | Scientific Literature | |
| 7. | ALA3266203 | B | Binding affinity to menin (unknown origin) assessed as thermal stability at 50 uM by differential scanning fluorimetry (Rvb = 40.46 degC) | Homo sapiens | 2 | ALA3259787 | single protein format | Scientific Literature | |
| 8. | ALA3266204 | B | Binding affinity to menin (unknown origin) assessed as thermal stability at 200 uM by differential scanning fluorimetry (Rvb = 40.46 degC) | Homo sapiens | 2 | ALA3259787 | single protein format | Scientific Literature | |
| 9. | ALA3266205 | B | Binding affinity to menin (unknown origin) assessed as thermal stability at 400 uM by differential scanning fluorimetry (Rvb = 40.46 degC) | Homo sapiens | 2 | ALA3259787 | single protein format | Scientific Literature | |
| 10. | ALA3266206 | B | Binding affinity to menin (unknown origin) by isothermal titration calorimetry analysis | Homo sapiens | 2 | ALA3259787 | single protein format | Scientific Literature | |
| 11. | ALA3614558 | B | Inhibition of N-terminal thioredoxin His6-tagged full-length human menin expressed in Escherichia coli Rosetta (DE3) cells assessed as reduction in menin interaction with FITC-MBM1 peptide of MLL (4 to 15 residues) by fluorescence polarization assay | Homo sapiens | 9 | ALA3611991 | cell-based format | Scientific Literature | |
| 12. | ALA3705757 | B | Fluorescence Polarization Assay: Assays effective in monitoring the inhibition of the MLL binding to menin were developed during experiments performed during the development of embodiments of the present invention. A fluorescein-labeled 12-amino acid peptide derived from MLL containing the high affinity menin binding motif was produced (Yokoyama et al., Cell., 2005. 123(2): p. 207-18., herein incorporated by reference in its entirety). Upon binding of the peptide (1.7 kDa) to the much larger menin (-67 kDa), the rotational correlation time of the fluorophore (peptide labeled with fluorescein at N-terminus) changes significantly, resulting in a substantial increase in the measured fluorescence polarization and fluorescence anisotropy (excitation at 500 nm, emission at 525 nm). The fluorescence polarization (FP) assay was utilized to determine the Kd for the binding of menin and the MLL peptide using a serial dilution of menin and 50 nM fluorescein-labeled MLL peptide. | 41 | ALA3638703 | single protein format | BindingDB Database | ||
| 13. | ALA3705758 | B | Homogeneous Time-Resolve Fluorescence (HTRF) Assay: One potential limitation of the above FP assay is the risk of selection of compounds that may interfere with the FP assays and produce so called false-positives. Therefore, during development of embodiments of the present invention, a time-resolved fluorescence resonance energy transfer (TR-FRET) assay was utilized as a secondary, assay (e.g. for confirmation of results), commercialized by CIS-BIO as homogeneous time-resolve fluorescence (HTRF) assay. In some embodiments, the HTRF assay may be the primary assay and the FP assay is used as a secondary assay to confirm results. HTRF is based on the non-radiative energy transfer of the long-lived emission from the Europium cryptate (Eu3+-cryptate) donor to the allophycocyanin (XL665) acceptor, combined with time-resolved detection. When these two fluorophores are brought together by a biomolecular interaction, a portion of the energy captured by the Eu3+-cryptate during excitation at 337 nm is released through fluorescence emission. | 7 | ALA3638703 | single protein format | BindingDB Database | ||
| 14. | ALA3705759 | B | NMR Spectroscopy Assay: NMR spectroscopy validation of lead compounds. In embodiments of the present invention, and during development thereof, NMR spectroscopy: saturation transfer difference (STD), competition STD, and WaterLOGSY experiments to validate binding of compounds to menin. STD provides a reliable method, based on principles vastly different form fluorescence that is commonly used for drug screening (e.g. by pharmaceutical companies). The principle of the STD experiment is based on the transfer of magnetization from a protein to a small molecule. Such a transfer occurs only when the ligand-protein contact is direct, and can be detected when the ligand is in fast exchange between bound and unbound state (Mayer & Meyer. J Am Chem Soc., 2001. 123(25): p. 6108-17., herein incorporated by reference in its entirety). The difference spectrum of the ligand recorded with and without protein saturation is analyzed. | 24 | ALA3638703 | single protein format | BindingDB Database | ||
| 15. | ALA3782255 | B | Binding affinity to menin (unknown origin) | Homo sapiens | 2 | ALA3779881 | single protein format | Scientific Literature | |
| 16. | ALA4149600 | B | Inhibition of FITC-labelled MLL1 binding to menin (unknown origin) at 20 uM after 120 mins by fluorescence polarization method relative to control | Homo sapiens | 13 | ALA4145581 | single protein format | Scientific Literature | |
| 17. | ALA4149601 | B | Inhibition of FITC-labelled MLL1 binding to menin (unknown origin) after 120 mins by fluorescence polarization method | Homo sapiens | 1 | ALA4145581 | single protein format | Scientific Literature | |
| 18. | ALA4149708 | B | Inhibition of biotinylated MLL-1 derived biotinylated Btn-PEG2- PEG2-S-Nva-RWRFPARPGTT-Amide binding to recombinant full length N-terminal His-tagged menin (unknown origin) expressed in Escherichia coli incubated for 10 mins by TR-FRET assay | Homo sapiens | 9 | ALA4145588 | assay format | Scientific Literature | |
| 19. | ALA4186850 | B | Displacement of FITC-MBM1 from menin (unknown origin) measured after 1 hr by fluorescence polarization assay | Homo sapiens | 3 | ALA4184186 | single protein format | Scientific Literature | |
| 20. | ALA4186851 | B | Binding affinity to menin (unknown origin) by isothermal titration calorimetry | Homo sapiens | 3 | ALA4184186 | single protein format | Scientific Literature |
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