lovingscience/chembl-v37-raw-activities huggingface dataset:
Compound (SMILES): Cc1nc(Nc2ncc(C(=O)Nc3c(C)cccc3Cl)s2)cc(N2CCN(CCO)CC2)n1.O
InChIKey: XHXFZZNHDVTMLI-UHFFFAOYSA-N
Target: Bcr/Abl fusion protein (CHIMERIC PROTEIN), Homo sapiens
UniProt: P00519
Assay: Inhibition of Bcr-Abl in presence of ATP
Measurement: IC50 < 1 nM (pChEMBL 9)
Descriptors: MW 488.02, ALogP 3.31, HBA 9, HBD 3, PSA 106.51, QED 0.47
Compound (SMILES): Cc1nc(Nc2ncc(C(=O)Nc3c(C)cccc3Cl)s2)cc(N2CCN(CCO)CC2)n1.O
InChIKey: XHXFZZNHDVTMLI-UHFFFAOYSA-N
Target: Bcr/Abl fusion protein (CHIMERIC PROTEIN), Homo sapiens
UniProt: P00519
Assay: Inhibition of Bcr-Abl kinase
Measurement: IC50 < 1 nM (pChEMBL 9)
Descriptors: MW 488.02, ALogP 3.31, HBA 9, HBD 3, PSA 106.51, QED 0.47
Compound (SMILES): Cc1nc(Nc2ncc(C(=O)Nc3c(C)cccc3Cl)s2)cc(N2CCN(CCO)CC2)n1.O
InChIKey: XHXFZZNHDVTMLI-UHFFFAOYSA-N
Target: Inhibitor of NF-kappa-B kinase (IKK) (PROTEIN COMPLEX), Homo sapiens
UniProt: O14920
Assay: Inhibition of IKK
Measurement: IC50 > 10000 nM (pChEMBL 5)
Descriptors: MW 488.02, ALogP 3.31, HBA 9, HBD 3, PSA 106.51, QED 0.47
Compound (SMILES): Cc1nc(Nc2ncc(C(=O)Nc3c(C)cccc3Cl)s2)cc(N2CCN(CCO)CC2)n1.O
InChIKey: XHXFZZNHDVTMLI-UHFFFAOYSA-N
Target: Bcr/Abl fusion protein (CHIMERIC PROTEIN), Homo sapiens
UniProt: P00519
Assay: Inhibition of wild type Bcr-Abl (unknown origin) using Tyr2 peptide substrate after 2 hrs by FRET-based Z'-lyte assay
Measurement: IC50 = 3 nM (pChEMBL 8.520000457763672)
Descriptors: MW 488.02, ALogP 3.31, HBA 9, HBD 3, PSA 106.51, QED 0.47
Compound (SMILES): Cc1nc(Nc2ncc(C(=O)Nc3c(C)cccc3Cl)s2)cc(N2CCN(CCO)CC2)n1.O
InChIKey: XHXFZZNHDVTMLI-UHFFFAOYSA-N
Target: Bcr/Abl fusion protein (CHIMERIC PROTEIN), Homo sapiens
UniProt: P00519
Assay: Inhibition of Bcr-Abl T315I mutant (unknown origin) using Tyr2 peptide substrate after 2 hrs by FRET-based Z'-lyte assay
Measurement: IC50 > 1000 nM (pChEMBL 6)
Descriptors: MW 488.02, ALogP 3.31, HBA 9, HBD 3, PSA 106.51, QED 0.47
Compound (SMILES): Cc1nc(Nc2ncc(C(=O)Nc3c(C)cccc3Cl)s2)cc(N2CCN(CCO)CC2)n1.O
InChIKey: XHXFZZNHDVTMLI-UHFFFAOYSA-N
Target: Bcr/Abl fusion protein (CHIMERIC PROTEIN), Homo sapiens
UniProt: P00519
Assay: Inhibition of wild type Bcr-Abl (unknown origin) by ADP-Glo assay
Measurement: IC50 = 7.6 nM (pChEMBL 8.119999885559082)
Descriptors: MW 488.02, ALogP 3.31, HBA 9, HBD 3, PSA 106.51, QED 0.47
Compound (SMILES): Cc1nc(Nc2ncc(C(=O)Nc3c(C)cccc3Cl)s2)cc(N2CCN(CCO)CC2)n1.O
InChIKey: XHXFZZNHDVTMLI-UHFFFAOYSA-N
Target: Bcr/Abl fusion protein (CHIMERIC PROTEIN), Homo sapiens
UniProt: P00519
Assay: Inhibition of Bcr-Abl T315I mutant (unknown origin) by ADP-Glo assay
Measurement: IC50 = 220 nM (pChEMBL 6.659999847412109)
Descriptors: MW 488.02, ALogP 3.31, HBA 9, HBD 3, PSA 106.51, QED 0.47
Compound (SMILES): Cc1nc(Nc2ncc(C(=O)Nc3c(C)cccc3Cl)s2)cc(N2CCN(CCO)CC2)n1.O
InChIKey: XHXFZZNHDVTMLI-UHFFFAOYSA-N
Target: Bcr/Abl fusion protein (CHIMERIC PROTEIN), Homo sapiens
UniProt: P00519
Assay: Inhibition of BCR-ABL phosphorylation in human K562 cells
Measurement: IC50 < 1 nM (pChEMBL 9)
Descriptors: MW 488.02, ALogP 3.31, HBA 9, HBD 3, PSA 106.51, QED 0.47
Compound (SMILES): Cc1nc(Nc2ncc(C(=O)Nc3c(C)cccc3Cl)s2)cc(N2CCN(CCO)CC2)n1.O
InChIKey: XHXFZZNHDVTMLI-UHFFFAOYSA-N
Target: Bcr/Abl fusion protein (CHIMERIC PROTEIN), Homo sapiens
UniProt: P00519
Assay: Inhibition of Bcr-Abl (unknown origin)
Measurement: IC50 = 4200 nM (pChEMBL 5.380000114440918)
Descriptors: MW 488.02, ALogP 3.31, HBA 9, HBD 3, PSA 106.51, QED 0.47
Compound (SMILES): Cc1nc(Nc2ncc(C(=O)Nc3c(C)cccc3Cl)s2)cc(N2CCN(CCO)CC2)n1.O
InChIKey: XHXFZZNHDVTMLI-UHFFFAOYSA-N
Target: Bcr/Abl fusion protein (CHIMERIC PROTEIN), Homo sapiens
UniProt: P00519
Assay: Inhibition of BCR-Abl (unknown origin)
Measurement: IC50 = 0.016 nM (pChEMBL 10.800000190734863)
Descriptors: MW 488.02, ALogP 3.31, HBA 9, HBD 3, PSA 106.51, QED 0.47
Compound (SMILES): Cc1nc(Nc2ncc(C(=O)Nc3c(C)cccc3Cl)s2)cc(N2CCN(CCO)CC2)n1.O
InChIKey: XHXFZZNHDVTMLI-UHFFFAOYSA-N
Target: Bcr/Abl fusion protein (CHIMERIC PROTEIN), Homo sapiens
UniProt: P00519
Assay: Binding affinity to Bcr-Abl (unknown origin) assessed as inhibition constant in presence of ATP by competitive binding assay
Measurement: Ki = 0.03 nM (pChEMBL 10.520000457763672)
Descriptors: MW 488.02, ALogP 3.31, HBA 9, HBD 3, PSA 106.51, QED 0.47
Compound (SMILES): Cc1nc(Nc2ncc(C(=O)Nc3c(C)cccc3Cl)s2)cc(N2CCN(CCO)CC2)n1.O
InChIKey: XHXFZZNHDVTMLI-UHFFFAOYSA-N
Target: Inhibitor of NF-kappa-B kinase (IKK) (PROTEIN COMPLEX), Homo sapiens
UniProt: O15111
Assay: Inhibition of IKK
Measurement: IC50 > 10000 nM (pChEMBL 5)
Descriptors: MW 488.02, ALogP 3.31, HBA 9, HBD 3, PSA 106.51, QED 0.47
Compound (SMILES): COc1cc(-c2cccc(-c3cccc(NC(=O)c4cccnc4O)c3C)c2C)cc(F)c1CN
InChIKey: OCAPFQCINOEZPE-UHFFFAOYSA-N
Target: Programmed cell death 1 ligand 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9NZQ7
Assay: Enzyme-Linked Immunosorbent AssayELISA: 96 Well plates were coated with 111 g/mL of human PD-L1 (obtained from R&D) in PBS overnight at 4° C. The wells were then blocked with 2% BSA in PBS (W/V) with 0.05% TWEEN-20 for 1 hour at 37° C. The plates were washed 3 times with PBS/0.05% TWEEN-20 and the compounds were serial diluted (1:5) in dilution medium and added to the ELISA plates. Human PD-1 and biotin 0.31 μg/mL (ACRO Biosystems) were added and incubated for 1 hour at 37° C. then washed 3 times with PBS/0.05% TWEEN-20. A second block was performed with 2% BSA in PBS (W/V)/0.05% TWEEN-20 for 10 min at 37° C. and the plates were washed 3 times with PBS/0.05% TWEEN-20. Streptavidin-HRP was added for 1 hour at 37° C. then the plates were washed 3 times with PBS/0.05% TWEEN-20. TMB substrate was added and reacted for 20 min at 37° C. A stop solution (2 N aqueous H2SO4) was added. The absorbance was read at 450 nm using a micro-plate spectrophotometer.
Measurement: IC50 = 505 nM (pChEMBL 6.300000190734863)
Descriptors: MW 471.53, ALogP 5.6, HBA 5, HBD 3, PSA 97.47, QED 0.34
Compound (SMILES): CCCNC(=O)Nc1ncnc2c1cnn2CCCCN1CCC(C(C)(C)C)CC1
InChIKey: SCYQBCPJAGSJNO-UHFFFAOYSA-N
Target: Sigma non-opioid intracellular receptor 1 (SINGLE PROTEIN), Cavia porcellus
UniProt: Q60492
Assay: Binding Assay: Brain membrane preparation and binding assays for the σ1-receptor were performed as described (DeHaven-Hudkins, D. L., L. C. Fleissner, and F. Y. Ford-Rice, 1992, Characterization of the binding of [3H]-(+)-pentazocine to 6 recognition sites in guinea pig brain, Eur. J. Pharmacol. 227, 371-378) with some modifications. Guinea pig brains were homogenized in 10 vols. (w/v) of Tris-HCl 50 mM 0.32 M sucrose, pH 7.4, with a Kinematica Polytron PT 3000 at 15000 r.p.m. for 30 s. The homogenate was centrifuged at 1000 g for 10 min at 4° C. and the supernatants collected and centrifuged again at 48000 g for 15 min at 4° C. The pellet was resuspended in 10 volumes of Tris-HCl buffer (50 mM, pH 7.4), incubated at 37° C. for 30 min, and centrifuged at 48000 g for 20 min at 4° C. Following this, the pellet was resuspended in fresh Tris-HCl buffer (50 mM, pH 7.4) and stored on ice until use.The radioligand used was [3H]-(+)-pentazocine at 5.0 nM and the final volume was 200 μl. The incubation was initiated with the addition of 100 μA of membrane at a final tissue concentration of approximately 5 mg tissue net weight/mL and the incubation time was 150 m. at 37° C. After incubation, the membranes were collected onto pretreated glass fiber filterplate (MultiScreen-FC, Millipore), with polyethylenimine 0.1%. The filters were washed two times with 200 μA of washing buffer (50 mM Tris CI, pH=7.4) and then 25 μl of Ecoscint H liquid scintillation cocktail were added. Microplates were allowed to set for several hours and then quantified by liquid scintillation spectrophotometry (1450 Microbeta, Wallac).
Measurement: Ki = 280 nM (pChEMBL 6.550000190734863)
Descriptors: MW 415.59, ALogP 3.9, HBA 6, HBD 2, PSA 87.97, QED 0.64
Compound (SMILES): N#Cc1ccc2c(ccn2-c2cc(F)c(OCCCC(=O)O)c(F)c2)c1
InChIKey: QMAUESTWQQEXGO-UHFFFAOYSA-N
Target: Free fatty acid receptor 4 (SINGLE PROTEIN), Homo sapiens
UniProt: Q5NUL3
Assay: Measurement of Activity of GPR120 Agonist (Cell-Based Assay): CHO-K1 cells expressing Ga16 and hGPR120 were dispensed into each well of a 96-well plate (3×104 cells/100 μl/well) and then incubated in 5% CO2, 37° C. incubator for 18 hours. Each well was treated with 100 μl of Calcium 5 dye (Molecular Devices) solution including 2% DMSO and then incubated in 5% CO2, 37° C. incubator for 1 hour. Serially diluted GPR120 agonists were prepared to a final concentration of 0.5% DMSO in a 96-well plate. Each well was treated with 50 μl of the agonist compounds using Plexstation II, and then fluorescence was measured at Ex 485 nm and Em 525 nm.Fluorescence increased by the serially diluted GPR120 agonists is calculated as a relative percent (%) value based on the fluorescence represented by the treatment of 1% DMSO only. EC50 refers to the concentration of agonist which shows 50% of maximum fluorescence increased by the treatment of agonist. The calculation of measurement was carried out by using statistical software (Prizm).
Measurement: EC50 = 11000 nM (pChEMBL 4.960000038146973)
Descriptors: MW 356.33, ALogP 4.02, HBA 4, HBD 1, PSA 75.25, QED 0.68
Compound (SMILES): CC1(C)C(N)=N[C@](C)(c2cc(/C=C(\F)c3cc4c(cn3)OCCO4)ccc2F)C(F)(F)S1(=O)=O
InChIKey: ISINGNQBVRWZCL-WHIQYKQFSA-N
Target: Beta-secretase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P56817
Assay: In Vitro Enzymatic BACE1 and BACE2 FRET (Fluorescence Resonance Energy Transfer) Assays: The cDNAs for both human recombinant BACE1 and 2 with C-terminal 6-His Tags were cloned into transient protein expression vectors, which were subsequently transfected into mammalian cell lines. These recombinant proteins were further purified using Ni-NTA affinity chromatography (Qiagen). The assay buffer used in these screens was 0.05 M acetate, pH 4.5, 8% DMSO final, 100 μM genapol (which is a nonionic detergent, below its Critical Micelle Concentration). The β-secretase enzyme (0.02 nM for BACE1 and 0.64 nM for BACE2), which was pre-incubated for one hour with the test compound, typically in about 1 uL of DMSO according to a serial dilution, was added thereto. The assay was effectively started by the addition of FRET substrate (50 nM) and the combination was incubated for one hour. The FRET assay was terminated by the addition of tris buffer, which raised the pH to neutrality, and the fluorescence was determined. The FRET substrate was a peptide with commercially available fluorophore and quencher, on opposite sides of the BACE cleavage site. The specific FRET substrate used in this assay was made by Amgen in-house. Commercially available FRET substrates, for example, the FRET substrate offered with the BACE1 FRET Assay Kit sold by ThermoFisher Scientific (Catalog Number P2985), may be used in this assay with the appropriate modifications, which are within the purview of the ability of a person with ordinary skill in the art. Proteolytic cleavage of the FRET substrate released quenching of fluorescence (excitation 488 nm and emission 590 nm).
Measurement: IC50 = 3135 nM (pChEMBL 5.5)
Descriptors: MW 499.49, ALogP 3.83, HBA 7, HBD 1, PSA 103.87, QED 0.64
Compound (SMILES): C[C@H](Nc1nc(C(=O)NC2CC[C@@H](O)C2)c2sccc2n1)c1ccccn1
InChIKey: YJKYOVAWMNRRSS-IAMFDIQRSA-N
Target: Adenosine receptor A2b (SINGLE PROTEIN), Homo sapiens
UniProt: P29275
Assay: Activity Assay: Compounds (Adenosine Receptor modulators) and activity assay.
Measurement: IC50 < 10 nM (pChEMBL 8)
Descriptors: MW 383.48, ALogP 2.9, HBA 7, HBD 3, PSA 100.03, QED 0.63
Compound (SMILES): C[C@@H]1CC[C@@H](c2nsc(C(C)(C)O)n2)CN1C(=O)c1ccccc1CS(C)(=O)=O
InChIKey: AJJUVSQDFMETJV-ZIAGYGMSSA-N
Target: Orexin receptor type 2 (SINGLE PROTEIN), Homo sapiens
UniProt: O43614
Assay: FLIPR assay: The utility of the compounds in accordance with the present invention as orexin receptor OX1R and/or OX2R antagonists may be readily determined without undue experimentation by methodology well known in the art, including the FLIPR Ca2+ Flux Assay (Okumura et al., Biochem. Biophys. Res. Comm. 280:976-981, 2001). In a typical experiment the OX1 and OX2 receptor antagonistic activity of the compounds of the present invention was determined in accordance with the following experimental method. For intracellular calcium measurements, Chinese hamster ovary (CHO) cells expressing the rat orexin-1 receptor or the human orexin-2 receptor, are grown in Iscove's modified DMEM containing 2 mM L-glutamine, 0.5 g/ml G418, 1% hypoxanthine-thymidine supplement, 100 U/ml penicillin, 100 μg/ml streptomycin and 10% heat-inactivated fetal calf serum (FCS). The cells are seeded at 20,000 cells/well into Becton-Dickinson black 384-well clear bottom sterile plates coated with poly-D-lysine. All reagents were from GIBCO-Invitrogen Corp. The seeded plates are incubated overnight at 37° C. and 5% CO2. Ala-6,12 human orexin-A as the agonist is prepared as a 1 mM stock solution in 1% bovine serum albumin (BSA) and diluted in assay buffer (HBSS containing 20 mM HEPES, 0.1% BSA and 2.5 mM probenecid, pH7.4) for use in the assay at a final concentration of 70 pM. Test compounds are prepared as 10 mM stock solution in DMSO, then diluted in 384-well plates, first in DMSO, then assay buffer. On the day of the assay, cells are washed 3 times with 100 μl assay buffer and then incubated for 60 min (37° C., 5% CO2) in 60 μl assay buffer containing 1 μM Fluo-4AM ester, 0.02% pluronic acid, and 1% BSA. The dye loading solution is then aspirated and cells are washed 3 times with 100 μl assay buffer. 30 μl of that same buffer is left in each well. Within the Fluorescent Imaging Plate Reader (FLIPR, Molecular Devices), test compounds are added to the plate in a volume of 25 μl, incubated for 5 min and finally 25 μl of agonist is added. Fluorescence is measured for each well at 1 second intervals for 5 minutes and the height of each fluorescence peak is compared to the height of the fluorescence peak induced by 70 μM Ala-6,12 orexin-A with buffer in place of antagonist. For each antagonist, IC50 value (the concentration of compound needed to inhibit 50% of the agonist response) is determined. Alternatively, compound potency can be assessed by a radioligand binding assay (described in Bergman et. al. Bioorg. Med. Chem. Lett. 2008, 18, 1425-1430) in which the inhibition constant (Ki) is determined in membranes prepared from CHO cells expressing either the OX1 or OX2 receptor. The intrinsic orexin receptor antagonist activity of a compound which may be used in the present invention may be determined by these assays.
Measurement: IC50 = 194 nM (pChEMBL 6.710000038146973)
Descriptors: MW 437.59, ALogP 2.72, HBA 7, HBD 1, PSA 100.46, QED 0.77
Compound (SMILES): C[C@H](CCc1ccc(OC(=O)N(C)C)c(OC(F)(F)F)c1)N1Cc2cccc(F)c2C1
InChIKey: ZGNPRTXHIWGVPG-CQSZACIVSA-N
Target: Voltage-gated inwardly rectifying potassium channel KCNH2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q12809
Assay: In Vitro Toxicity: Representative sigma-2 antagonists test compounds do not induce neuronal or glial toxicity with acute or chronic dosing in vitro. The sigma-2 receptor antagonists eliminate or reduce Abeta oligomer-induced changes in membrane trafficking. No significant effect of compounds on membrane trafficking occurs when dosed without oligomers. There is no toxicity relative to neuron number, glial number, nuclear size, nuclear morphology, neurite length, cytoskeletal morphology when tested up to 10 times the EC50 concentration for three days. See, e.g., WO2013/029060, Table 12, which is incorporated herein by reference.In vitro toxicity for Test Compounds is tested in a number of standard assays. Preferrably, testing in vitro tox studies reveals there is no genotoxicity at 10 μM (AMES, micronucleus, bacterial cytotox); HepG2 toxicity at 100-fold above affinity at sigma-2 receptor, in HepG2 tumor cell line; inhibition of CYP 450 enzymes 2D6, 3A4, and 2C19 at 10 μM; and hERG inhibition.
Measurement: IC50 = 2800 nM (pChEMBL 5.550000190734863)
Descriptors: MW 440.44, ALogP 5.12, HBA 4, HBD 0, PSA 42.01, QED 0.58
Compound (SMILES): O=C(O)c1cnn(-c2nc3cc(C(=O)Nc4ccccc4)ccc3[nH]2)c1
InChIKey: GZICKMLJCRIRKE-UHFFFAOYSA-N
Target: Egl nine homolog 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9GZT9
Assay: Enzyme Activity Assay: The PHD2181-417 polypeptide (3 μg) was pre-incubated for 30 minutes with test compound prior to assessing the enzymatic activity of the polypeptide. The PHD enzymatic assay was then performed by transferring the purified PHD2181-417 polypeptide (3 μg) mixture with compound to 0.5 ml of reaction mixture containing the following: synthetic HIF-1α peptide comprising residues [KNPFSTGDTDLDLEMLAPYIPMDDDFQLRSFDQLS] (10 μM, California Peptide Research Inc., Napa, Calif.), and [5-14C]-2-oxoglutaric acid (50 mCi/mmol, Moravek Chemicals, Brea, Calif.) in reaction buffer (40 mM Tris-HCl, pH 7.5, 0.4 mg/ml catalase, 0.5 mM DTT, 1 mM ascorbate) for 10 minutes in the presence of compound. The reaction was stopped by addition of 50 μl of 70 mM H3PO4 and 50 μl of 500 mM NaH2PO4, pH 3.2. Detection of [14C]-succinic acid was achieved by separating from [5-14C]-2-oxoglutaric acid by incubating the reaction mixture with 100 μl of 0.16 M DNP prepared in 30% perchloric acid. Next, 50 μl of unlabeled 20 mM 2-oxoglutaric acid/20 mM succinic acid, serving as carrier for the radioactivity, was added to the mixture, and was allowed to proceed for 30 minutes at room temperature. The reaction was then incubated with 50 μl of 1 M 2-oxoglutaric acid for 30 additional minutes at room temperature to precipitate the excess DNP. The reaction was then centrifuged at 2800×g for 10 minutes at room temperature to separate [14C]-succinic acid in the supernatant from the precipitated [14C]-dinitrophenylhydrazone. Fractions of the supernatant (400 μl) were counted using a beta counter (Beckman Coulter, Fullerton, Calif.). Inhibition of PHD2181-417 activity was measured as a decrease in [14C]-succinic acid production. The IC50 values were estimated by fitting the data to a three-parameter logistic function using GraphPad Prism, version 4.02 (Graph Pad Software, San Diego, Calif.). IC50 values up to 10 μM were quantified otherwise were noted as >10 μM. All compounds were diluted at 10 mM in 100% DMSO (w/v) and tested from 10 μM to 3 nM at half-log serial dilutions, with a final concentration of 2% DMSO (w/v) in the assay.
Measurement: IC50 = 126 nM (pChEMBL 6.900000095367432)
Descriptors: MW 347.33, ALogP 2.7, HBA 5, HBD 3, PSA 112.9, QED 0.53
Compound (SMILES): O=C1CCc2c(Oc3ccc4c(c3)CC(c3nc(-c5cccs5)c[nH]3)CO4)ccnc2N1
InChIKey: WCSQDCPCGRYUND-UHFFFAOYSA-N
Target: Serine/threonine-protein kinase B-raf (SINGLE PROTEIN), Homo sapiens
UniProt: P15056
Assay: LanthaScreen Eu kinase binding assay: To determine whether compounds bind to RAF kinases, they are tested in a competition binding assay. The Invitrogen LanthaScreen Eu binding assay involves the binding of an Alexa-Fluor® 647-labelled, ATP-competitive kinase tracer to the kinase of interest. A Europium-labelled anti-tag antibody also binds to the kinase of interest. Simultaneous binding of the tracer and the antibody brings them into close proximity and upon excitation at 340 nm, triggers fluorescence resonance energy transfer (FRET) between the Europium donor fluorophore on the antibody and the Alexa Fluor® 647 acceptor on the tracer. The dual emission signal produced can be measured at 665 and 615 nm.Compounds at a concentration of 3 mM are serially diluted (e.g. 10 μl 90 μl of 100% dimethyl sulfoxide (DMSO)) seven times in 96-well plates for a total of 8 dilution points. Each DMSO dilution is further diluted 1:100 in kinase buffer (e.g. 5 μl into 495 μl kinase buffer) containing 50 mM HEPES pH 7.5, 10 mM MgCl2, 1 mM EGTA, 0.01% Brij-35.Each well in a 96-well Optiplate (Perkin Elmer 6005569) contains 30 μl of final volume per sample, including 10 μl compound at 3× the desired concentration, providing 10 μM at the final maximum concentration, 10 μl of kinase/antibody mixture at 3× the desired concentration of RAF recombinant kinases and antibody providing 5 nM final concentration of B-RAFV600E kinase (Invitrogen PV3849) and 3 nM final concentration of C-RAF Y340D/Y341D kinase (Invitrogen PV3805) and 2 nM final concentration of Eu-anti-GST antibody (PV5594), and 10 μl of 3× the desired concentration of kinase tracer 178 (Invitrogen PV5593) providing final concentrations of 20 nM tracer for B-RAFV600E kinase and 6 nM tracer for C-RAF kinase. The plates are incubated for 5 hours at room temperature and read on an EnVision plate reader (Perkin Elmer). All data are analysed using the GraphPad Prism software package. Inhibition of tracer binding to the kinase of interest is assessed by determination of IC50 value, which is defined as the concentration of compound which decreased the level of FRET signal measured at 665 nm by 50%.
Measurement: IC50 < 4 nM (pChEMBL 8.39799976348877)
Descriptors: MW 444.52, ALogP 4.93, HBA 6, HBD 2, PSA 89.13, QED 0.47
Compound (SMILES): O=C(NCC1CCNCC1)c1ccc(-c2cc(NCCCC3CCCNC3)c3ccccc3n2)cc1
InChIKey: UCEJBFUALIFQKN-UHFFFAOYSA-N
Target: Low molecular weight phosphotyrosine protein phosphatase (SINGLE PROTEIN), Homo sapiens
UniProt: P24666
Assay: LMPTP Primary Screening Protocol: A listing of materials is provided:    Item, source, catalog no.    LMPTP-A Enzyme Stock Solution (4.22 mg/ml or 206.8 μM), SBMRI Protein Facility,    N/A    OMFP, Sigma, M2629-100 MG    Bis-Tris pH 6.0, Fisher Sci, BP301-100    Triton-X 100, Sigma, T9284    DTT, Sigma, D9779    Mol. Grade Water, Mediatech, Inc., 46-000-CM    1536-well black High base opaque bottom plate, Nexus Biosystems, 00019120Final assay conditions are:    0.625 nM LMPTP-A Enzyme    40 μM OMFP    50 mM Bis-Tris pH 6.0    1 mM DTT    0.01% Triton-X 100    20 μM test compound    3% DMSO (2% from substrate and 1% from compounds)    6 μL reaction volume    50 minutes incubation at room tempAssay Procedure    1. Prepare Reagents as described in section F. Recipe.    2. Using LabCyte Echo, transfer 60 nL from 2 mM test compound source plate into assay plate Col. 5-48 (final concentration of test compounds is 20 μM). 60 nL of DMSO should be transferred to col. 1-4 for positive and negative control wells.    3. Spin plates at 1000 rpm for 1 minute in centrifuge.    4. Using the Beckman Coulter Bioraptr, add 3 μL/well of control buffer to columns 1 and 2.    5. Using the Bioraptr, add 3 μL/well of enzyme solution to col. 3-48.    6. Using the Bioraptr, add 3 μL/well of substrate solution to col. 1-48.    7. Spin plates at 1000 rpm for 1 minute in centrifuge.    8. Incubate plates in the dark at room temperature for 50 minutes.    9. Read plates on PerkinElmer Viewlux using a FI protocol.
Measurement: IC50 = 500 nM (pChEMBL 6.300000190734863)
Descriptors: MW 485.68, ALogP 4.82, HBA 5, HBD 4, PSA 78.08, QED 0.33
Compound (SMILES): Cc1cc(CC(C#N)C(=O)Nc2nc(-c3ccccc3)cs2)cc(Cl)c1O
InChIKey: UUGDXHOTOITNFZ-UHFFFAOYSA-N
Target: Double-strand break repair protein MRE11 (SINGLE PROTEIN), Homo sapiens
UniProt: P49959
Assay: HTS Assay: The conditions of the assay are as follows:Microplate type: 1536 Well Black Round Bottom Polystyrene Not Treated (Corning cat no. 3936)Total reaction volume: 5 μLMRE11 concentration in the reaction: 18 nMDNA substrate concentration in the reaction: 40 nMNumber of compounds to test: 257Inhibitor concentration range tested: 7 nM-50 μMMultiplicates: 3Concentration points: 13Dilution step: 2.1Each plate contains a series of high and low signal control wells, where no compound is added:High signal: MRE11+DNA substrateLow signal: DNA substrate onlyThese are used during data evaluation.Two Extra Assay Controls:1) To check whether unwinding of DNA by the compounds alone occurs.The DNA substrate [CY5+BBQ(650)] is mixed with the compounds with no protein present.This is done as a single measurement at 25 μM inhibitor concentration.2) To check whether the compounds are able to quench the CY5.The CY5 single stranded oligo is mixed with the compounds with no protein present. This is done as a single measurement at 25 μM inhibitor concentration.The layout of the plates is created by the in-house software (CZ-Openscreen Prague) and this information is transferred to the robotic HTS station.Assay Steps1) Prepare 50 mL of master mix:16.7 mL 5× reaction buffer (150 mM Bis Tris pH 7; 5 mM DTT)1042 μL 400 mM MnCl232.3 mL H2O2) Fill the plates with 3 μL of master mix per well using MultiDrop (Thermo Scientific)3) Transfer of compounds to the plates at the robotic station with the contactless Echo dispenser (Labcyte)4) Measurement of autofluorescence with the EnVision reader (PerkinElmer)5) Prepare 20 mL of 90 nM MRE11 in T+50 buffer (25 mM Tris-HCl pH7.5, 50 mM KCl 8.7% glycerol, 0.5 mM EDTA)6) Add 1 μL of 90 nM MRE11 to the corresponding wells using MultiDrop7) Preincubation at RT for 30 min8) Prepare 20 mL of a 200 nM solution of 5′ overhang DNA substrate: 480 μL 6 μM DNA+19.52 mL H2O9) Prepare 4 mL of a 200 nM solution of the single stranded DNA (oligo 1): 8 μL 100 μM DNA+4 mL H2O10) Add 1 μL of each 200 nM DNA solution to the corresponding wells with the MultiDrop11) Fluorescence measurement with the EnVision reader every 45 minutesFluorescence readout: CY5λex/em=620/665 nmAnalysis
Measurement: IC50 = 50000 nM (pChEMBL 4.300000190734863)
Descriptors: MW 397.89, ALogP 4.8, HBA 5, HBD 2, PSA 86.01, QED 0.65
Compound (SMILES): COC(=O)C(C)(C)n1cc([C@@H]2CC[C@@H](C)N(C(=O)c3ccccc3-n3nccn3)C2)cn1
InChIKey: JKHUPHRIVNHDEC-IAGOWNOFSA-N
Target: Orexin receptor type 2 (SINGLE PROTEIN), Homo sapiens
UniProt: O43614
Assay: FLIPR Ca2+ Flux Assay: In a typical experiment the OX1 and OX2 receptor antagonistic activity of the compounds of the present invention was determined in accordance with the following experimental method. For intracellular calcium measurements, Chinese hamster ovary (CHO) cells expressing the rat orexin-1 receptor or the human orexin-2 receptor, are grown in Iscove's modified DMEM containing 2 mM L-glutamine, 0.5 g/ml G418, 1% hypoxanthine-thymidine supplement, 100 U/ml penicillin, 100 μg/ml streptomycin and 10% heat-inactivated fetal calf serum (FCS). The cells are seeded at 20,000 cells/well into Becton-Dickinson black 384-well clear bottom sterile plates coated with poly-D-lysine. All reagents were from GIBCO-Invitrogen Corp. The seeded plates are incubated overnight at 37° C. and 5% CO2. Ala-6,12 human orexin-A as the agonist is prepared as a 1 mM stock solution in 1% bovine serum albumin (BSA) and diluted in assay buffer (HBSS containing 20 mM HEPES, 0.1% BSA and 2.5 mM probenecid, pH7.4) for use in the assay at a final concentration of 70 pM. Test compounds are prepared as 10 mM stock solution in DMSO, then diluted in 384-well plates, first in DMSO, then assay buffer. On the day of the assay, cells are washed 3 times with 100 μl assay buffer and then incubated for 60 min (37° C., 5% CO2) in 60 μl assay buffer containing 1 μM Fluo-4AM ester, 0.02% pluronic acid, and 1% BSA. The dye loading solution is then aspirated and cells are washed 3 times with 100 μl assay buffer. 30 μl of that same buffer is left in each well. Within the Fluorescent Imaging Plate Reader (FLIPR, Molecular Devices), test compounds are added to the plate in a volume of 25 μl, incubated for 5 min and finally 25 μl of agonist is added. Fluorescence is measured for each well at 1 second intervals for 5 minutes and the height of each fluorescence peak is compared to the height of the fluorescence peak induced by 70 pM Ala-6,12 orexin-A with buffer in place of antagonist. For each antagonist, IC50 value (the concentration of compound needed to inhibit 50% of the agonist response) is determined. Alternatively, compound potency can be assessed by a radioligand binding assay (described in Bergman et. al. Bioorg. Med. Chem. Lett. 2008, 18, 1425-1430) in which the inhibition constant (Ki) is determined in membranes prepared from CHO cells expressing either the OX1 or OX2 receptor. The intrinsic orexin receptor antagonist activity of a compound which may be used in the present invention may be determined by these assays.
Measurement: IC50 = 46 nM (pChEMBL 7.340000152587891)
Descriptors: MW 436.52, ALogP 2.78, HBA 8, HBD 0, PSA 95.14, QED 0.57
Compound (SMILES): C[C@@H]1COCCN1c1cc(-c2cccnc2O)c2ccnc(-c3ccnn3C3CCCCO3)c2n1
InChIKey: WDORVQBRIYQTFB-LIXIDFRTSA-N
Target: Serine/threonine-protein kinase ATR (SINGLE PROTEIN), Homo sapiens
UniProt: Q13535
Assay: Time-Resolved Fluorescence Energy Transfer (TR-FRET Tracer A): To determine of binding activity of the test compounds, full-length human ATR protein was expressed and purified together with ATRIP as described above. Furthermore, a fluorescently labelled compound (tracer A) was used as a tracer molecule. Detection of the binding event of the tracer was achieved by time-resolved fluorescence energy transfer (TR-FRET). We used an anti-GST-Terbium antibody (CisBio) that binds to the GST-tag at the N-terminus of ATR-kinase. Excitation of Terbium with 337 nm light results in emission of fluorescent light with 545 nm. In case a tetrameric complex has formed (antiGST-Tb+GST-ATR+Strp2-ATRIP+tracer), part of the energy will be transferred from the Terbium to the fluorophore that itself emits light of 570 nm. Displacement of the fluorescent tracer by a test compound leads to a reduction of the TR-FRET-signal.For the assay 50 nl of a 100-fold concentrated solution of the test compound in DMSO was pipetted into a black low volume 384well microtiter plate (MTP, Greiner Bio-One, Frickenhausen, Germany). To prepare the ATR-working solution, ATR/ATRIP stock solution was diluted in assay buffer [50 mM HEPES (pH 7.0), 10 mM MgCl2, 1 mM DTT, 0.01% (w/v) Igepal, 0.01% (w/v) BSA] to 4.2 nM protein concentration (concentration may vary from lot to lot of protein preparation). AntiGST-Tb antibody was diluted to 4.2 nM. The ATR-working solution was incubated for 30 min at 22° C. prior to dispensing to pre-form the complex of antiGST-Tb+GST-ATR+ATRIP. Then, 3 μl of the ATR-working solution were added to the test compound and the mixture was incubated for 10 min at 22° C. to allow pre-binding of the test compounds to ATR/ATRIP. Then, 2 μl of a 100 nM solution of either tracer A or B in assay buffer were added to the ATR-working solution. The resulting mixture was incubated for 30 min at 22° C. The measurement of the TR-FRET signal was performed in a standard HTRF-compatible MTP reader instrument (e.g. BMG Pherastar) by recording the fluorescence emissions at 545 nm and 570 nm after excitation at 337-350 nm. The ratio between emission at 570 nm divided by emission at 545 nm was calculated to give the well ratio. The experimental data (well ratios) were normalised by the following way: positive control contained ATR-working solution plus either tracer A or B solution (=0% inhibition), the negative control contained all components except GST-ATR/ATRIP (=100% inhibition).
Measurement: IC50 = 408 nM (pChEMBL 6.389999866485596)
Descriptors: MW 472.55, ALogP 4.19, HBA 9, HBD 1, PSA 98.42, QED 0.47
Compound (SMILES): COc1cn(Cc2cc(F)cc(F)c2)c(Nc2cc(-c3ccccc3)ccc2C)nc1=O
InChIKey: WQKUVQCRCZIMMP-UHFFFAOYSA-N
Target: P2X purinoceptor 7 (SINGLE PROTEIN), Homo sapiens
UniProt: Q99572
Assay: Evaluation of a Human P2X7 Receptor Inhibitory Activity: Stably expressing cell line (1321N1 cell transfected with the human P2X7 receptor gene (GenBank accession number NM_002562.5 including T606C and G952A SNP)) was used. The cells were seeded in a 384-well microtiter plate at a concentration of 8000 cells/well and cultured in the medium (10% fetal bovine serum, 25 mM HEPES, 1% penicillin and streptomycin in DMEM) for one day at 37° C. under 5% carbon dioxide atmosphere. After replacing with 20 μL of the HBSS buffer (20 mM HEPES, 55.6 mM D-glucose, 1×HBSS(−), pH7.4-7.5), 15 μL of 17.3 μM Yo-Pro solution in the HBSS buffer was added. The plate was placed in high-throughput cellular screening system FLIPR TETRA (Molecullar Devices, LLC.) and 15 μL of 130 μM BzATP solution in the HBSS buffer was added. Measurement of fluorescence intensity by FLIPR TETRA was started. After eight minutes, 15 μL of DMSO solutions containing different concentrations of the compound of the present invention as prepared by dilution with the HBSS buffer were dispensed to each well through the built-in automatic dispenser, and the measurement of fluorescence intensity was continued for 20 minutes. The maximum fluorescence intensity without the compound of the present invention is calculated as 0% inhibition and the maximum fluorescence intensity when the reference compound was added is calculated as 100% inhibition. Changing values of fluorescence intensity by the compound of the present invention were calculated by difference between maximum and minimum fluorescence intensity for 20 minutes. IC50 was calculated using logistic approximation.
Measurement: IC50 = 55 nM (pChEMBL 7.260000228881836)
Descriptors: MW 433.46, ALogP 5.3, HBA 5, HBD 1, PSA 56.15, QED 0.45
Compound (SMILES): Cc1c([C@H](OC(C)(C)C)C(=O)O)c(-c2ccc(Cl)cc2)c2cc(C)n3c2c1N(S(C)(=O)=O)[C@@H](CC(F)F)C3
InChIKey: RHFOXZIZRGFTMT-BVZFJXPGSA-N
Target: Cytochrome P450 2C9 (SINGLE PROTEIN), Homo sapiens
UniProt: P11712
Assay: Inhibition Assay: Each five kinds of substrates, human hepatic microsome, and a test drug in 50 mmol/L Hepes buffer as a reaction solution was added to a 96-well plate as the composition ad described above, NADPH, as a coenzyme was added to initiate metabolism reactions as markers and, after the incubation at 37° C. for 15 minutes, a methanol/acetonitrile=1/1 (V/V) solution was added to stop the reaction. After the centrifugation at 3000 rpm for 15 minutes, resorufin (CYP1A2 metabolite) in the supernatant was quantified by a fluorescent multilabel counter and tolbutamide hydroxide (CYP2C9 metabolite), mephenytoin 4′ hydroxide (CYP2C19 metabolite), dextrorphan (CYP2D6 metabolite), and terfenadine alcohol (CYP3A4 metabolite) were quantified by LC/MS/MS.
Measurement: IC50 > 20000 nM (pChEMBL 4.698999881744385)
Descriptors: MW 569.07, ALogP 6.32, HBA 5, HBD 1, PSA 88.84, QED 0.36
Compound (SMILES): COC(=O)c1cc(C)c(N2C[C@]3(CC[C@](c4ccccc4)(N(C)C)CC3)N(CC3CCC3)C2=O)cn1
InChIKey: NZJQVCPBDKKIBX-ISILISOKSA-N
Target: Nociceptin receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P41146
Assay: Human Nociceptin/Orphanin FQ Peptide (hNOP) Receptor Binding Assay: The hNOP receptor binding assay was performed as homogeneous SPA-assay (scintillation proximity assay) using the assay buffer 50 mM TRIS-HCl. 10 mM MgCl2. 1 mM EDTA (pH 7.4). The final assay volume (250 μl/well) included 0.5 nM of [leucyl-3H]nociceptin as ligand (PerkinElmer Life Sciences. Inc. Boston. Mass. USA). and either test compound in dilution series or 1 μM unlabelled nociceptin for determination of unspecific binding. The test compound was diluted with 25% DMSO in H2O to yield a final 0.5% DMSO concentration. which also served as a respective vehicle control. The assay was started by adding wheat germ agglutinin coated SPA beads (GE Healthcare UK Ltd. Buckinghamshire. UK) which had been preloaded with hMOP receptor membranes (PerkinElmer Life Sciences. Inc. Boston. Mass. USA). After incubation for 60 minutes at RT and centrifugation for 20 minutes at 500 rpm the signal rate was measured by means of a 1450 Microbeta Trilux β-counter (PerkinElmer Life Sciences/Wallac. Turku. Finland). Half-maximal inhibitory concentration (IC50) values reflecting 50% displacement of [3H]nociceptin-specific receptor binding were calculated by nonlinear regression analysis and Ki values were calculated by using the Cheng-Prusoff equation. (Cheng and Prusoff. 1973).
Measurement: Ki = 1.6 nM (pChEMBL 8.800000190734863)
Descriptors: MW 490.65, ALogP 4.99, HBA 5, HBD 0, PSA 65.98, QED 0.53
Compound (SMILES): CN1CC[C@@](O)(C#Cc2ccnc(-n3nc(C(N)=O)c4c3CC(C)(C)[C@H](O)C4)c2)C1=O
InChIKey: NNSYVCGIXIMRBO-ZHRRBRCNSA-N
Target: Mitogen-activated protein kinase kinase kinase 14 (SINGLE PROTEIN), Homo sapiens
UniProt: Q99558
Assay: NIK Enzyme Inhibition Assay: The ability of the nuclear factor-kappa B (NF-kB)-inducing kinase (NIK) to catalyze the hydrolysis of adenosine-5′-triphosphate (ATP) was monitored using the Transcreener ADP (adenosine-5′-diphosphate) assay (BellBrook Labs). Purified NIK (0.5 nM) derived from a baculovirus-infected insect cell expression system was incubated with test compounds for 1-3.5 hours in 50 mM 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid buffer (pH 7.2) containing 10 mM MgCl2, 2 mM dithiothreitol, 10 μM ATP, 0.01% Triton X-100, 0.1% gamma-globulins from bovine blood, 1% dimethylsulfoxide (DMSO), 7 μg/mLADP antibody and 5 nM ADP-MR121 633 tracer. Reactions were quenched by the addition of 20 mM 2,2′,2″,2′″-(ethane-1,2-diyldinitrilo)tetraacetic acid and 0.01% Brij 35. The tracer bound to the antibody was displaced by the ADP generated during the NIK reaction, which causes a decrease in fluorescence polarization that was measured by laser excitation at 633 nm with a Fluorescence Correlation Spectroscopy Plus reader (Evotec AG). Equilibrium dissociation constant (Ki) values for NIK inhibitors are calculated from plots of activity vs. inhibitor concentration using Morrison's quadratic equation that accounts for the potential of tight binding, and by also applying the conversion factor that accounted for competitive inhibition and the concentration of substrate used in the assay relative to its Michaelis constant (Km).
Measurement: Ki = 33.2 nM (pChEMBL 7.480000019073486)
Descriptors: MW 423.47, ALogP -0.2, HBA 7, HBD 3, PSA 134.57, QED 0.57
Compound (SMILES): O=S(=O)(c1nc2n(n1)[C@H](c1ccccc1)C[C@@H]2F)C1(F)CC1
InChIKey: UWTUBMJYUVVFGR-QWRGUYRKSA-N
Target: Receptor-interacting serine/threonine-protein kinase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q13546
Assay: Biochemical Assay: The ability of the receptor interacting protein kinase (RIPK1) to catalyze the hydrolysis of adenosine-5′-triphosphate (ATP) is monitored using the Transcreener ADP (adenosine-5′-diphosphate) assay (BellBrook Labs). Purified human RIP1 kinase domain (2-375) (50 nM) derived from a baculovirus-infected insect cell expression system is incubated with test compounds for 2 hours in 50 mM Hepes buffer (pH 7.5) containing 30 mM MgCl2, 1 mM dithiothreitol, 50 uM ATP, 0.002% Brij-35, and 0.5% dimethyl sulfoxide (DMSO). Reactions are quenched by the addition of 1× Bell Brooks Stop buffer B (20 mM Hepes (ph7.5), 40 mM ethylenediaminetetraacetic acid and 0.02% Brij-35) containing an additional 12 mM EDTA and 55 ug/mL ADP2 antibody and 4 nM ADP-AlexaFluor® 633 tracer. The tracer bound to the antibody is displaced by the ADP generated during the RIP1K reaction, which causes a decrease in fluorescence polarization that is measured by laser excitation at 633 nm with a FP microplate reader M1000. Fractional activity was plotted against test article concentration. Using Genedata Screener software (Genedata; Basel, Switzerland), the data were fit to the tight-binding apparent inhibition constant (Ki app) Morrison equation [Williams, J. W. and Morrison, J. F. (1979) The kinetics of reversible tight-binding inhibition. Methods Enzymol 63: 437-67]. The following equation was used to calculate fractional activity and Ki app:Fractional ⁢ ⁢ activity = V i V o = 1 - ( [ E ] T + [ I ] T + K i app ) - ( [ E ] T + [ I ] T + K i app ) 2 - 4 ⁡ [ E ] T ⁡ [ I ] T 2 ⁡ [ E ] Twhere [E]T and [I]T are the total concentrations of active enzyme and test article, respectively.
Measurement: Ki = 6 nM (pChEMBL 8.220000267028809)
Descriptors: MW 325.34, ALogP 2.52, HBA 5, HBD 0, PSA 64.85, QED 0.87
Compound (SMILES): O=C(NCCCc1ncc[nH]1)c1ccc2nc(-c3ccc(F)cc3)c(-c3ccc4c(c3)OCCO4)nc2c1
InChIKey: WTWQHGGDQFFQHU-UHFFFAOYSA-N
Target: Nicotinamide phosphoribosyltransferase (SINGLE PROTEIN), Homo sapiens
UniProt: P43490
Assay: NAMPT Enzyme Assay: To measure the inhibition of NAMPT activity hNAMPT protein stock and anti 6His-Tb (Cisbio; Cat. No. 61HISTLB) is diluted to 3× final concentration with assay buffer (50 mM Tris-HCl (pH7.5), 1 mM DTT, 100 mM NaCl, 10 mM MgCl2, 0.005% Tween 20). To this solution is added test compounds or vehicle control (DMSO) and BodiPY ligand (structure below). The plate is shaken for 1-2 min, sealed and incubated for 1 hour at rt in the dark. The TR-FRET signal is measured using BMG Pherastar (Lanthascreen protocol on BMG Pherastar). Excitation was carried out at 320 nm, and the ratio of emission of BodiPY (520 nm) to terbium (486 nm) was determined. Concentration response curves are generated by calculating the excitation increase in test compound-treated samples relative to DMSO-treated controls. For the assay method described above, test compound percent inhibition values at a single concentration are calculated relative to control (DMSO) treated samples. Compound concentration response curves are fitted to generate IC50 values from those curves. One skilled in the art will appreciate that these values generated either as percentage inhibition at a single concentration or IC50 values are subject to experimental variation.
Measurement: IC50 = 750 nM (pChEMBL 6.119999885559082)
Descriptors: MW 509.54, ALogP 4.96, HBA 6, HBD 2, PSA 102.02, QED 0.3
Compound (SMILES): CC(=O)OC[C@@H](C)n1nnnc1-c1cccc(NC(=O)c2cc3c(cc2F)CCNC3)n1
InChIKey: DMYASARKOKXVJF-GFCCVEGCSA-N
Target: Mitogen-activated protein kinase kinase kinase 5 (SINGLE PROTEIN), Homo sapiens
UniProt: Q99683
Assay: HTRF KinEASE Assay : HTRF KinEASE-STK is a generic method for measuring serine/threonine kinase activities using a time-resolved fluorescence resonance energy transfer (TR-FRET) immunoassay. The IC50 value for each compound was determined in the presence of compound (various concentration from 0 to 10 μM) and a fixed amount of ATP and peptide substrates. The test compound, 1 Um STK3 peptide substrate, and 5 Nm of ASK1 kinase are incubated with kinase reaction buffer containing 50 Mm HEPES Ph 7.5, 0.01% BRIJ-35, 10 Mm MgCl2, and 1 Mm EGTA for 30 minutes. 100 Um ATP is added to start kinase reaction and incubated for 3 hours. The STK3-antibody labeled with Eu3+-Cryptate and 125 Nm streptavidin-XL665 are mixed in a single addition with stop reagents provided by the Cisbio kit used to stop the kinase reaction. Fluorescence is detected using an Envision Multilabeled 2014 reader from PerkinElmer. The Fluorescence is measured at 615 nm (Cryptate) and 665 nm (XL665) and a ratio of 665 nm/615 nm is calculated for each well.
Measurement: IC50 > 1000 nM (pChEMBL 6)
Descriptors: MW 439.45, ALogP 1.9, HBA 9, HBD 2, PSA 123.92, QED 0.56
Compound (SMILES): CCn1c(-c2cccc(C(F)(F)F)c2)nc2cc(Nc3nc(-c4ccncc4)cs3)c(C)cc21
InChIKey: WZPVDURRZYKUAS-UHFFFAOYSA-N
Target: Mast/stem cell growth factor receptor Kit (SINGLE PROTEIN), Homo sapiens
UniProt: P10721
Assay: In Vitro TK Inhibition Assay: Colorimetric cell proliferation and viability assay (reagent CellTiter-Blue purchased from Promega cat N°G8081) was performed on BaF3 Kit WT cell line.A total of 2.104 cells/50 μl were seeded per well of a 96-wells plate. Treatment was initiated by addition of a 2× drug solution of ½ serial dilutions ranging from 0 to 10 μM. After incubating for 48 hours at 37° C., 10 μl of a ½ dilution of CellTiter-Blue reagent was added to each well and the plates were returned to the incubator for an additional 4 hours. The fluorescence intensity from the CellTiter-Blue reagent is proportional to the number of viable cells and data were recorded (544Ex/590Em) using a POLARstar OMEGA microplate reader (BMG LabteckSarl). A background control without cells was used as a blank. The positive control of the assay corresponds to the cell proliferation obtained in the absence of drug treatment (100% proliferation). Each sample was done in triplicate. The results were expressed as a percentage of the proliferation obtained in absence of treatment. All drugs were prepared as 20 mM stock solutions in DMSO and conserved at −80° C. Drug dilutions were made fresh in medium before each experiment. A DMSO control was included in each experiment.
Measurement: EC50 < 1000 nM (pChEMBL 6)
Descriptors: MW 479.53, ALogP 7.31, HBA 6, HBD 1, PSA 55.63, QED 0.29
Compound (SMILES): O=C(NCCc1ccccc1F)Nc1cc2cnn(-c3ccncc3)c2cn1
InChIKey: BPKOOQJLPFNRKT-UHFFFAOYSA-N
Target: Mitogen-activated protein kinase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P28482
Assay: IMAP-FP Assay: Activated ERK2 activity was determined in an IMAP-FP assay (Molecular Devices). Using this assay format, the potency (IC50) of each compound was determined from a 10 point (1:3 serial dilution, 3 μM starting compound concentration) titration curve using the following outlined procedure. To each well of a black Corning 384-well plate (Corning Catalog #3575), 7.5 nL of compound (3333 fold dilution in final assay volume of 25 μL) was dispensed, followed by the addition of 15 μL of kinase buffer (tween containing kinase buffer, Molecular Devices) containing 0.0364 ng/mL (0.833 nM) of phosphorylated active hERK2 enzyme. Following a 15 minute compound & enzyme incubation, each reaction was initiated by the addition of 10 μL kinase buffer containing 2.45 μM ERK2 IMAP substrate peptides and 75 μM ATP. The final reaction in each well of 25 μL consists of 0.5 nM hERK2, 900 nM unlabeled peptide, 80 nM labeled-peptide, and 30 μM ATP. Phosphorylation reactions were allowed to proceed for 60 minutes and were immediately quenched by the addition of 60 μL IMAP detection beads (1:1000 dilutions) in IMAP binding buffer (Molecular Devices) with 24 mM NaCl. Plates were read on EnVision reader after 60 minutes binding equilibration using Fluorescence Polarization protocol (Perkin Elmer).
Measurement: IC50 = 9.2 nM (pChEMBL 8.039999961853027)
Descriptors: MW 376.4, ALogP 3.32, HBA 5, HBD 2, PSA 84.73, QED 0.56
Compound (SMILES): CC(=O)N1CCC/C=C\Cn2c(=O)c3cnc(Nc4ccc5c(c4)CN(C)CC54CC4)nc3n2-c2cccc1n2
InChIKey: XJMXYZQFAVITFS-XQRVVYSFSA-N
Target: Wee1-like protein kinase (SINGLE PROTEIN), Homo sapiens
UniProt: P30291
Assay: Enzymatic Activity of Wee1: In the present disclosure, Wee1 enzyme catalysis assay was carried out using the ATP-Glo Max kinase luminescence detection kit (Promega). Kinase activity was assessed by quantitative detection of the amount of ATP retained in the solution following the kinase reaction. The luminescent signal in the assay is proportional to the amount of ATP and inversely proportional to the kinase activity. The concentration of the compound in the assay ranged from 0.5 nM to 30 μM. The compound was dissolved in 10% DMSO, and 5 μL of the solution was added to 50 μL of the reaction, and the concentration of DMSO in the final reaction was 1%. The reaction was carried out at 30° C. for 50 minutes. 40 mM trishydroxymetyl aminomethane, pH 7.4, 10 mM MgCl2, 0.1 mg/ml BSA, 2 mM DTT, 0.1 mg/ml Poly (Lys, Tyr) substrate, 10 μM ATP and Wee1 were contained in the reaction mixture. After the enzymatic reaction, 50 μL of ATP-Glo Max kinase luminescence detection assay solution (Promega) was added and incubated for 15 minutes at room temperature. The luminescent signal was measured using a microplate reader. In some assays, a known Wee1 inhibitor was added as a positive control. Luminance data was analyzed using Graphpad software.
Measurement: IC50 = 3.7 nM (pChEMBL 8.430000305175781)
Descriptors: MW 536.64, ALogP 3.9, HBA 9, HBD 1, PSA 101.18, QED 0.39