lovingscience/chembl-v37-raw-activities huggingface dataset:
Compound (SMILES): CN1CCN(c2ccc(C(=O)N3CCC(CC#N)(n4cc(C(N)=O)c(Nc5ccccc5)n4)CC3)cc2)CC1
InChIKey: KMXZLJKMVZPEDE-UHFFFAOYSA-N
Target: Tyrosine-protein kinase JAK2 (SINGLE PROTEIN), Homo sapiens
UniProt: O60674
Assay: HTRF Biochemical Assay: The reactions employed a common peptide substrate, LCB-EQEDEPEGDYFEWLW-NH2 (in-house). The basic assay protocol is as follows: First, 250 nL of diluted compounds in DMSO were dispensed into the wells of a dry 384-well Black plate (Greiner #781076) using a Labcyte Echo 555 acoustic dispenser. Subsequent reagent additions employed an Agilent Bravo. Next, 18 uL of 1.11x enzyme and 1.11x substrate in 1x assay buffer (Invitrogen kinase buffer # PV3189, 2 mM DTT, 0.05% BSA) were added to the wells and shaken and then preincubated for 30 minutes at room temperature to allow compound binding to equilibrate. After equilibration, 2 uL of 10xATP in 1x assay buffer was added to initiate the kinase reaction and the plates were shaken and then incubated at room temperature for 120 minutes. At the end of the incubation, 20 uL of 2x stop buffer (streptavidin-Dylight 650 (Thermo #84547B/100 mL), Eu-tagged pY20 antibody (Perkin Elmer #AD0067), EDTA, HEPES, and Triton) was added to quench the reaction.
Measurement: IC50 = 830 nM (pChEMBL 6.079999923706055)
Descriptors: MW 526.65, ALogP 3.02, HBA 8, HBD 2, PSA 123.52, QED 0.49
Compound (SMILES): C#CCOC(=O)Nc1nc2cc(C3(O)c4ccccc4C(=O)N3Cc3ccccc3)ccc2[nH]1
InChIKey: YOESWAGAIFDFHR-UHFFFAOYSA-N
Target: Serine/threonine-protein kinase B-raf (SINGLE PROTEIN), Homo sapiens
UniProt: P15056
Assay: Inhibition of B-Raf (unknown origin) pre-incubated for 30 mins by luciferase-luciferin-coupled chemiluminescence assay
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 452.47, ALogP 3.59, HBA 5, HBD 3, PSA 107.55, QED 0.4
Compound (SMILES): Cc1cn(CCC(=O)N/N=C/c2ccc3nccnc3c2)c2ccccc12
InChIKey: UUBWJWIDPSAZHE-ZMOGYAJESA-N
Target: Transient receptor potential cation channel subfamily M member 5 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9NZQ8
Assay: Inhibition of human TRPM5 transfected in HEK293 cells assessed as inhibition of KCl-stimulated membrane potential by FLIPR assay
Measurement: IC50 = 2200 nM (pChEMBL 5.659999847412109)
Descriptors: MW 357.42, ALogP 3.43, HBA 5, HBD 1, PSA 72.17, QED 0.44
Compound (SMILES): CC(C)(NC(=O)c1ccc(N2CCCS2(=O)=O)c(OCC2CC2)n1)c1nccs1
InChIKey: DYELJUPTUVISDY-UHFFFAOYSA-N
Target: Cannabinoid receptor 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P21554
Assay: cAMP Assay: CHO cells expressing human CB1 or CB2 receptors are seeded 17-24 hours prior to the experiment 50.000 cells per well in a black 96 well plate with flat clear bottom (Corning Costar #3904) in DMEM (Invitrogen No. 31331), 1.times.HT supplement, with 10% fetal calf serum and incubated at 5% CO2 and 37° C. in a humidified incubator. The growth medium was exchanged with Krebs Ringer Bicarbonate buffer with 1 mM IBMX and incubated at 30.degree. C. for 30 min Compounds were added to a final assay volume of 100 ul and incubated for 30 min at 30° C. Using the cAMP-Nano-TRF detection kit the assay (Roche Diagnostics) was stopped by the addition of 50 ul lysis reagent (Tris, NaCl, 1.5% Triton X100, 2.5% NP40, 10% NaN.sub.3) and 50 ul detection solutions (20 uM mAb Alexa700-cAMP 1:1, and 48 uM Ruthenium-2-AHA-cAMP) and shaken for 2 h at room temperature. The time-resolved energy transfer is measured by a TRF reader (Evotec Technologies GmbH), equipped with a ND:YAG laser as excitation source. The plate is measured twice with the excitation at 355 nm and at the emission with a delay of 100 ns and a gate of 100 ns, total exposure time 10 s at 730 (bandwidth 30 nm) or 645 nm (bandwidth 75 nm), respectively. The FRET signal is calculated.
Measurement: EC50 > 10000 nM (pChEMBL 5)
Descriptors: MW 436.56, ALogP 2.53, HBA 7, HBD 1, PSA 101.49, QED 0.72
Compound (SMILES): COc1cc(OC)cc(N(CC2CC2)c2ccc3ncc(-c4cnn(CCCN)c4)nc3c2)c1
InChIKey: VCNJSFBAEBDCKK-UHFFFAOYSA-N
Target: Fibroblast growth factor receptor 3 (SINGLE PROTEIN), Homo sapiens
UniProt: P22607
Assay: Inhibition of FGFR3 (unknown origin) transfected in mouse BA/F3 cells assessed as decrease in cell proliferation in presence of mouse IL3 after 24 hrs by Alamar blue assay
Measurement: IC50 = 5754.4 nM (pChEMBL 5.239999771118164)
Descriptors: MW 458.57, ALogP 4.41, HBA 8, HBD 1, PSA 91.32, QED 0.38
Compound (SMILES): NC(=O)c1cc(-c2ccc(F)cc2)c2ccc(CN3CCOC(C(F)(F)F)C3)cc2n1
InChIKey: PQGBVTXVOBDHIA-UHFFFAOYSA-N
Target: Voltage-gated inwardly rectifying potassium channel KCNH2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q12809
Assay: Displacement of [35S]-MK-499 from hERG expressed in HEK293 cells by automated patch-clamp assay
Measurement: IC50 = 5500 nM (pChEMBL 5.260000228881836)
Descriptors: MW 433.41, ALogP 3.9, HBA 4, HBD 1, PSA 68.45, QED 0.63
Compound (SMILES): COc1cc(C(=O)N2CCC(N3CC(CC#N)(n4cc(-c5ncnc6[nH]ccc56)cn4)C3)CC2)ccc1F
InChIKey: GVQJMBULVQMONV-UHFFFAOYSA-N
Target: Tyrosine-protein kinase JAK2 (SINGLE PROTEIN), Homo sapiens
UniProt: O60674
Assay: Inhibition of human N-terminal His-tagged JAK2 catalytic domain (828 to 1132 residues) expressed in baculovirus infected insect cells assessed as decrease in phosphorylation of biotinylated peptide substrate after 1 hr in presence of 1 mM ATP by HTRF method
Measurement: IC50 <= 10 nM (pChEMBL 8)
Descriptors: MW 514.57, ALogP 3.2, HBA 8, HBD 1, PSA 115.96, QED 0.42
Compound (SMILES): Cn1c(Nc2c(F)cccc2Cl)nc2cc(C(=O)Nc3ccc(F)c(C(F)(F)F)c3)c3c(c21)OCCO3
InChIKey: MRUCMCCYXQYKGZ-UHFFFAOYSA-N
Target: Prostaglandin E synthase (SINGLE PROTEIN), Homo sapiens
UniProt: O14684
Assay: Inhibition of human recombinant mPGES-1 expressed in CHO cells assessed as reduction in conversion of PGH2 to PGE2 incubated for 10 mins followed by substrate addition measured after 1 min in presence of GSH
Measurement: IC50 = 7.99 nM (pChEMBL 8.100000381469727)
Descriptors: MW 538.86, ALogP 6.29, HBA 6, HBD 2, PSA 77.41, QED 0.3
Compound (SMILES): Cn1cc(-c2nc3c(N4CCN(Cc5cccnc5)CC4)c(Br)cnc3[nH]2)cn1
InChIKey: KLCAJPNNJWCSJD-UHFFFAOYSA-N
Target: Voltage-gated inwardly rectifying potassium channel KCNH2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q12809
Assay: Cell-Based Electrophysiology Assays: All hERG percentage inhibitions at 10 uM compound concentration were determined by Millipore in a high-throughput cell-based electrophysiology assay for inhibition of hERG tail current41, and values are reported as a mean of multiple determinations. 0.3% DMSO aqueous vehicle negative control gave 7-16% inhibition. Cisapride (1 uM) positive control gave 96-104% inhibition. All hERG IC50 values were determined by Millipore,41 and the hERG IC50 for Example 1 was also determined by Cyprotex plc measuring hERG tail-currents by whole-cell voltage-clamping.
Measurement: IC50 = 5500 nM (pChEMBL 5.260000228881836)
Descriptors: MW 453.35, ALogP 2.84, HBA 7, HBD 1, PSA 78.76, QED 0.51
Compound (SMILES): Cc1cc(C)c(CNC(=O)c2cc(-c3ccc(N4CCNCC4)nc3)cc3c2C(C)CN3C2CCCC2)c(=O)[nH]1
InChIKey: WZMNJYXLTFTXGO-UHFFFAOYSA-N
Target: Histone-lysine N-methyltransferase EZH2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q15910
Assay: Inhibition of human wild type EZH2 using histone H3 as substrate after 1 hr in presence of 3H-SAM by filter paper detection analysis
Measurement: IC50 = 21 nM (pChEMBL 7.679999828338623)
Descriptors: MW 540.71, ALogP 4.26, HBA 6, HBD 3, PSA 93.36, QED 0.43
Compound (SMILES): CCCCOc1cnc(-c2c[nH]c3ccc(-c4nnc(N)s4)cc23)cn1
InChIKey: WCRZDULJXSERSJ-UHFFFAOYSA-N
Target: Serine/threonine-protein kinase pim-2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9P1W9
Assay: Enzyme Assay: The assay for the determination of Pim activity is based on the formation of phosphorylated biotinylated-BAD peptide at the Serine 112 residue (S112) and employs HTRF® (homogeneous time resolved fluorescence) technology to detect the product in a 96-well plate format. The phosphorylation of biotinylated-BAD (S112) peptide by full length recombinant Pim-1, Pim-2, or Pim-3 protein was detected with streptavidin:Allophycocyanin (APC) conjugate and a europium (Eu) labeled antibody directed against phosphorylated-BAD (S112). Excitation of Eu by a high energy laser light (337 nm) leads to a transfer of energy to the APC molecule, and results in an emission at 665 nm. The fluorescence is directly proportional to the amount of phosphorylated BAD peptide present in the reaction.Compounds were prepared in DMSO by conducting 3-fold serial dilutions to give a 10-point dosing curve having a high dose of 1 uM. A reference compound was included on each assay plate in order to validate that plate; on
Measurement: IC50 = 1546.83 nM (pChEMBL 5.809999942779541)
Descriptors: MW 366.45, ALogP 3.9, HBA 7, HBD 2, PSA 102.6, QED 0.5
Compound (SMILES): COc1cc2c(Nc3ccc(Cl)c(Cl)c3)ncnc2cc1OCC1CN(C/C=C/Br)CCO1
InChIKey: IAULTELZJHNXGT-GORDUTHDSA-N
Target: Ephrin type-A receptor 2 (SINGLE PROTEIN), Homo sapiens
UniProt: P29317
Assay: Inhibition of recombinant human N-terminal His6-tagged EphA2 (N598 to R890 residues) expressed in baculovirus using poly-EY as substrate after 180 mins by luciferase coupled chemiluminescence assay
Measurement: IC50 = 50 nM (pChEMBL 7.300000190734863)
Descriptors: MW 554.27, ALogP 5.68, HBA 7, HBD 1, PSA 68.74, QED 0.38
Compound (SMILES): CC(O)(C(=O)N1CCC(CC#N)(n2cc(C(N)=O)c(Nc3ccnc(F)c3)n2)CC1)C(F)F
InChIKey: FGTMUTZTXAXMBI-UHFFFAOYSA-N
Target: Tyrosine-protein kinase JAK2 (SINGLE PROTEIN), Homo sapiens
UniProt: O60674
Assay: HTRF Biochemical Assay: The reactions employed a common peptide substrate, LCB-EQEDEPEGDYFEWLW-NH2 (in-house). The basic assay protocol is as follows: First, 250 nL of diluted compounds in DMSO were dispensed into the wells of a dry 384-well Black plate (Greiner #781076) using a Labcyte Echo 555 acoustic dispenser. Subsequent reagent additions employed an Agilent Bravo. Next, 18 uL of 1.11x enzyme and 1.11x substrate in 1x assay buffer (Invitrogen kinase buffer # PV3189, 2 mM DTT, 0.05% BSA) were added to the wells and shaken and then preincubated for 30 minutes at room temperature to allow compound binding to equilibrate. After equilibration, 2 uL of 10xATP in 1x assay buffer was added to initiate the kinase reaction and the plates were shaken and then incubated at room temperature for 120 minutes. At the end of the incubation, 20 uL of 2x stop buffer (streptavidin-Dylight 650 (Thermo #84547B/100 mL), Eu-tagged pY20 antibody (Perkin Elmer #AD0067), EDTA, HEPES, and Triton) was added to quench the reaction.
Measurement: IC50 = 38 nM (pChEMBL 7.420000076293945)
Descriptors: MW 465.44, ALogP 1.51, HBA 8, HBD 3, PSA 150.16, QED 0.52
Compound (SMILES): CCCCCOc1ccc(-c2nnc(-c3cc(Cl)c([C@]4(C)CS(=O)(=O)N(C)C(=N)N4)s3)o2)cc1
InChIKey: LDILEPQPHIRRKQ-QFIPXVFZSA-N
Target: Beta-secretase 2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9Y5Z0
Assay: Time-Resolved Endpoint Proteolysis Assay: Inhibitor compounds, prepared at 3× the desired final concentration in 1×BACE assay buffer (20 mM sodium acetate pH 5.0, 10% glycerol, 0.1% Brij-35) supplemented with 7.5% DMSO are pre-incubated with an equal volume of autoBACE-2 enzyme diluted in 1×BACE assay buffer (final enzyme concentration 1 nM) in black 384-well NUNC plates for 30 minutes at 30° C. The assay is initiated by addition of an equal volume of the QSY7-EISEVNLDAEFC-Eu-amide substrate (200 nM final concentration, Km=8 μM for 4 μM for autoBACE-2) prepared in 1×BACE assay buffer supplemented with 7.5% DMSO and incubated for 90 minutes at 30° C. DMSO is present at 5% final concentration in the assay. Following laser excitation of sample wells at 320 nm, the fluorescence signal at 620 nm is collected for 400 ms following a 50 μs delay on a RUBYstar HTRF plate reader (BMG Labtechnologies).
Measurement: Ki = 355.8 nM (pChEMBL 6.449999809265137)
Descriptors: MW 524.07, ALogP 4.7, HBA 8, HBD 2, PSA 121.41, QED 0.41
Compound (SMILES): C[C@H](CN1[C@@H]2CC[C@H]1C[C@@H](c1cccc(O)c1)C2)N(Cc1ccccc1)C(=O)Cc1ccccc1
InChIKey: AKXBEMDXMYDZEW-HYJYBNJOSA-N
Target: Delta-type opioid receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P41143
Assay: Displacement of [3H]-diprenorphine from human delta opioid receptor expressed in cell membranes
Measurement: Ki = 100 nM (pChEMBL 7)
Descriptors: MW 468.64, ALogP 5.76, HBA 3, HBD 1, PSA 43.78, QED 0.46
Compound (SMILES): Cc1ccc(F)c(NC(=O)Nc2ccc(Oc3ccnc4cc(C(=O)NCCCN)sc34)cc2F)c1
InChIKey: JHYGYHSMYDQRSI-UHFFFAOYSA-N
Target: Vascular endothelial growth factor receptor 2 (SINGLE PROTEIN), Homo sapiens
UniProt: P35968
Assay: Kinase Assay: Kinase assays were performed in 96 well microtiter plates that were coated overnight with 30 μg of poly-Glu-Tyr (4:1) in 10 mM Phosphate Buffered Saline (PBS), pH 7.2-7.4. The plates were incubated with 1% BSA and then washed four times with PBS prior to starting the reaction. Reactions were carried out in 120 μL reaction volumes containing 3.6 μM ATP in kinase buffer (50 mM Hepes buffer pH 7.4, 20 mM MgCl2, 0.1 mM MnCl2 and 0.2 mM Na3VO4). Test compounds were reconstituted in 100% DMSO and added to the reaction to give a final DMSO concentration of 5%. Reactions were initiated by the addition 0.5 ng of purified protein. Following a ten minute incubation at 25 °C., the reactions were washed four times with PBS containing 0.05% Tween-20. 100 μl of a monoclonal anti-phosphotyrosine antibody-peroxidase conjugate was diluted 1:10000 in PBS-Tween-20 and added to the wells for 30 minutes. Following four washes with PBS-Tween-20, 100 μl of 0-phenylenediamine Dihydrochloride in Phosphate-citrate buffer, containing urea hydrogen peroxide, was added to the wells for 7 minutes as a colorimetric substrate for the peroxidase. The reaction was terminated by the addition of 100 μl of 2.5N H2 SO4 to each well and read using a microplate ELISA reader set at 492 nm.
Measurement: IC50 = 7 nM (pChEMBL 8.149999618530273)
Descriptors: MW 511.55, ALogP 5.4, HBA 6, HBD 4, PSA 118.37, QED 0.24
Compound (SMILES): Cn1ccc2ccc(-c3cccc(OCC(O)CN4CCc5ccccc5C4)c3)nc21
InChIKey: LCAFUBIHLJBTGT-UHFFFAOYSA-N
Target: Protein arginine N-methyltransferase 5 (SINGLE PROTEIN), Homo sapiens
UniProt: O14744
Assay: Inhibition of PRMT5 in human Z138 cells assessed as decrease in di-methyl arginine levels after 96 hrs by Western blot method
Measurement: EC50 = 101 nM (pChEMBL 7)
Descriptors: MW 413.52, ALogP 4.04, HBA 5, HBD 1, PSA 50.52, QED 0.52
Compound (SMILES): O=C(NS(=O)(=O)c1ccc(NC2CCN(C3CCOCC3)CC2)c([N+](=O)[O-])c1)c1ccc(N2CCN(Cc3ccc(C(F)(F)F)cc3-c3ccc(Cl)cc3)CC2)cc1Oc1ccc2[nH]ccc2c1
InChIKey: RZFHYDPUKFLDDB-UHFFFAOYSA-N
Target: Bcl-2-like protein 1 (SINGLE PROTEIN), Mus musculus
UniProt: Q64373
Assay: Time Resolved-Fluorescence Resonance Energy Transfer (TR-FRET) Assay: The inhibition constant (Ki) is the dissociation constant of an enzyme-inhibitor complex or a protein/small molecule complex, wherein the small molecule is inhibiting binding of one protein to another protein or peptide. Where the Ki for a compound is represented as > (greater than) a certain numerical value, it is intended to mean that the binding affinity value (e.g., for Bcl-XL) is greater than the limits of detection of the assay used. Where the binding selectivity ratio for a compound is represented as > (greater than) a certain numerical value, it is intended to mean that the selectivity of a particular compound for Bcl-2 over Bcl-XL is at least as great as the number indicated. Where the Ki for a compound is represented as < (less than) a certain numerical value, it is intended to mean that the binding affinity value (e.g., for Bcl-2) is lower than the limit of detection of the assay used. Inhibition constants were determined using Wang's equation (Wang Z-X).
Measurement: Ki > 660 nM (pChEMBL 6.179999828338623)
Descriptors: MW 972.49, ALogP 9.7, HBA 11, HBD 3, PSA 162.38, QED 0.07
Compound (SMILES): CCOC(=O)C1(Nc2cc(C(N)=O)nc(-c3ccc(Oc4ccc(F)cc4)cc3)n2)CC1
InChIKey: ALQOLRCTDNIXDY-UHFFFAOYSA-N
Target: N-formyl peptide receptor 2 (SINGLE PROTEIN), Homo sapiens
UniProt: P25090
Assay: FLIPR Assay: Assay Buffer: The assay buffer was formulated by removing 120 mL from a 1 L bottle of fresh, sterile dH2O (Mediatech, Herndon, Va.) and adding 100 mL of 10×HBSS that does not contain Ca++ or Mg++ (Gibco, Invitrogen, Grand Island, N.Y.) followed by 20 mL of 1.0 M Hepes, pH 7.3 (Fisher Scientific, BP299-100). The final buffer consisted of 20 mM Hepes, pH 7.3, 1.261 mM CaCl2, 0.493 mM MgCl2, 0.407 mM Mg(SO)4, 5.33 mM KCl, 0.441 mM KH2PO4, 137 mM NaCl, 0.336 mM Na2HPO4 and 0.556 mM D-glucose (Hanks et al., Proc. Soc. Exp. Biol. Med. 71:196 (1949)), and the simple formulation was typically the basic buffer throughout the assay (i.e., all wash and addition steps).
Measurement: IC50 = 100 nM (pChEMBL 7)
Descriptors: MW 436.44, ALogP 3.68, HBA 7, HBD 2, PSA 116.43, QED 0.52
Compound (SMILES): O=C1NN=C2CCCc3c2c2c1cccc2n3CCN1CCCC1
InChIKey: ASIFNLNMOYRKGY-UHFFFAOYSA-N
Target: Poly [ADP-ribose] polymerase tankyrase-2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9H2K2
Assay: Tankyrase-2 Enzymatic Assay: Tankyrase-2 enzymatic assay was conducted using commercial Tankyrase-2 Chemiluminescent Assay Kit (BPS Biosciences) and the protocol with the kit. GST-fused tankyrase-2 (recombinant protein expressed and purified from Bacluovirus) were coated on a GSH-precoated plate first, and incubated with increasing concentrations of compounds for 0.5 h. Then, biotinylated NAD was added to the wells. The biotinylated auto-PARylation product was measured by adding streptavidin-HRP and HRP substrates which produce chemiluminescence.
Measurement: IC50 = 150 nM (pChEMBL 6.820000171661377)
Descriptors: MW 322.41, ALogP 2.52, HBA 4, HBD 1, PSA 49.63, QED 0.94
Compound (SMILES): Cn1c(=O)n([C@H]2C[C@@H](O)C2)c2c3cc(-c4ccc(OCCCN5CCCC5)nc4)ccc3ncc21
InChIKey: USWRLFCEADLRGY-OYRHEFFESA-N
Target: Serine/threonine-protein kinase ATR (SINGLE PROTEIN), Homo sapiens
UniProt: Q13535
Assay: Cellular Assay: Assay b) HT29 cells (ECACC #85061109) were seeded into 384 well assay plates (Costar #3712) at a density of 6000 cells/well in 40 μl EMEM medium containing 1% L glutamine and 10% FBS and allowed to adhere overnight. The following morning compound of Formula (I) in 100% DMSO was added to assay plates by acoustic dispensing. After 1 h incubation at 37° C. and 5% CO2, 40 nl of 3 mM 4NQO in 100% DMSO was added to all wells by acoustic dispensing, except minimum control wells which were left untreated with 4NQO to generate a null response control. Plates were returned to the incubator for a further 1 h. Then cells were fixed by adding 20 μl of 3.7% formaldehyde in PBS solution and incubating for 20 mins at r.t. Then 20 μl of 0.1% Triton X100 in PBS was added and incubated for 10 minutes at r.t., to permeabalise cells. Then the plates were washed once with 50 μl/well PBS, using a Biotek EL405 plate washer. Phospho-Chk1 Ser 345 antibody (Cell Signalling Technology #2348) was diluted 150 fold in PBS containing 0.05% polysorbate/Tween and 15 μl was added to each well and incubated over night at r.t. The next morning plates were washed three times with 50 μl/well PBS, using a Biotek EL405 plate washer, and then 20 μl of secondary Ab solution, containing 500 fold diluted Alexa Fluor 488 Goat anti-rabbit IgG (Molecular Probes #A-11008) and 0.002 mg/ml Hoeschst dye (Molecular Probes #H-3570), in PBST, was added. After 2 h incubation at r.t., the plates were washed three times with 50 μl/well PBS, using a Biotek EL405 plate washer, and plates were then sealed with black plate seals until read. Plates were read using an ArrayScan VTI instrument, using an XF53 filter with 10× objective. A two laser set up was used to analyse nuclear staining with Hoeschst (405 nm) and secondary antibody staining of pChk1 (488 nm).
Measurement: IC50 > 30000 nM (pChEMBL 4.5229997634887695)
Descriptors: MW 473.58, ALogP 3.51, HBA 8, HBD 1, PSA 85.41, QED 0.41
Compound (SMILES): CN[C@@H](C)C(=O)N[C@H](C(=O)N1CCc2ccccc2[C@H]1C(=O)Nc1c(F)cccc1F)C(C)C
InChIKey: PZXNEDCIDKOIEV-LVWPNOBMSA-N
Target: E3 ubiquitin-protein ligase XIAP (SINGLE PROTEIN), Homo sapiens
UniProt: P98170
Assay: TR-FRET Assay: The peptide AVPIAQKSEK-(ε-biotin)-OH 1:2 TFA ("Peptide A") was identified as a substrate for the TR-FRET assay by screening the 6× Histidine-tagged BIR2 domain and BIR3 domain of XIAP against a set of 29 peptides synthesized based on sequences reported by Sweeny et al. (Biochemistry, 2006, 45, 14740 14748). The peptides were labeled with the fluorescent tags FITC or TAMRA and Kd values were determined by fluorescence polarization assay. The sequence AVPIAQKSEK was identified as optimal for using in an assay. The peptide sequence was derivatized with biotin to provide AVPIAQKSEK-(Îµ-biotin)-OH 1:2 TFA as the substrate for the TR-FRET assay. Ten nanomolar of 6× Histidine-tagged BIR2 domain, corresponding to amino acids 124-240 of XIAP, or BIR3 domain, corresponding to amino acids 241-356 of XIAP, was mixed with 20 nM of the peptide AVPIAQKSEK-(ε-biotin)-OH 1:2 TFA, in the presence of 50 mM Tris-Cl, pH 7.5, 100 mM NaCl, 1 mM dithiothreitol (DTT) and 0.1 mg/mL bovine serum albumin (BSA). Following a 45 min. incubation at 37° C., Europium-Streptavidin and Allophycocyanin conjugated anti-Histidine antibody were added to a final concentration of 1.5 nM and 15 nM, respectively. Time-resolved fluorescence resonance energy transfer (TR-FRET) signals were measured 1 hour later at room temperature. Test compound potency was assessed at 10 serially diluted concentrations.
Measurement: IC50 > 54800 nM (pChEMBL 4.261000156402588)
Descriptors: MW 472.54, ALogP 2.78, HBA 4, HBD 3, PSA 90.54, QED 0.58
Compound (SMILES): CC(C)(C)Oc1ncnc2[nH]cc(/C=C/C(N)=O)c12
InChIKey: OIZYRHLPFJOIQM-SNAWJCMRSA-N
Target: TAK1/TAB1 (PROTEIN COMPLEX), Homo sapiens
UniProt: Q15750
Assay: Inhibition of kinase tracer-236 binding to full length recombinant human His-tagged TAK1-TAB1 expressed in baculovirus by FRET assay
Measurement: IC50 = 13 nM (pChEMBL 7.889999866485596)
Descriptors: MW 260.3, ALogP 1.63, HBA 4, HBD 2, PSA 93.89, QED 0.82
Compound (SMILES): Cc1nc(C2(NC(=O)c3cnc(N4CC(F)(F)C4)c(OCC4CC4)n3)CCC2)no1
InChIKey: BCQUNMHAZHYLTP-UHFFFAOYSA-N
Target: Cannabinoid receptor 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P21554
Assay: cAMP Assay: CHO cells expressing human CB1 or CB2 receptors are seeded 17-24 hours prior to the experiment 50.000 cells per well in a black 96 well plate with flat clear bottom (Corning Costar #3904) in DMEM (Invitrogen No. 31331), 1×HT supplement, with 10% fetal calf serum and incubated at 5% CO2 and 37° C. in a humidified incubator. The growth medium was exchanged with Krebs Ringer Bicarbonate buffer with 1 mM IBMX and incubated at 30° C. for 30 min. Compounds were added to a final assay volume of 100 μl and incubated for 30 min at 30° C. Using the cAMP-Nano-TRF detection kit the assay (Roche Diagnostics) was stopped by the addition of 50 μl lysis reagent (Tris, NaCl, 1.5% Triton X100, 2.5% NP40, 10% NaN3) and 50 μl detection solutions (20 μM mAb Alexa700-cAMP 1:1, and 48 μM Ruthenium-2-AHA-cAMP) and shaken for 2 h at room temperature. The time-resolved energy transfer is measured by a TRF reader (Evotec Technologies GmbH), equipped with a ND:YAG laser as excitation source. The plate is measured twice with the excitation at 355 nm and at the emission with a delay of 100 ns and a gate of 100 ns, total exposure time 10s at 730 (bandwidth 30 nm) or 645 nm (bandwidth 75 nm), respectively.
Measurement: EC50 = 1285.8 nM (pChEMBL 5.889999866485596)
Descriptors: MW 420.42, ALogP 2.22, HBA 8, HBD 1, PSA 106.27, QED 0.73
Compound (SMILES): O=C(Nc1ccc([C@@H]2CNCCO2)cc1)c1ccc(Cl)cc1
InChIKey: KGPOKGIHPOHHOL-INIZCTEOSA-N
Target: Trace amine-associated receptor 1 (SINGLE PROTEIN), Rattus norvegicus
UniProt: Q923Y9
Assay: Radioligand Binding Assay: HEK-293 cells stably expressing rat TAAR1 were maintained at 37° C. and 5% CO2 in DMEM high glucose medium, containing fetal calf serum (10%, heat inactivated for 30 min at 56° C.), penicillin/streptomycin (1%), and 375 ug/ml geneticin (Gibco). Cells were released from culture flasks using trypsin/EDTA, harvested, washed twice with ice-cold PBS (without Ca2+ and Mg2+), pelleted at 1,000 rpm for 5 min at 4° C., frozen and stored at -80° C. Frozen pellets were suspended in 20 ml HEPES-NaOH (20 mM, pH 7.4) containing 10 mM EDTA and homogenized with a Polytron (PT 6000, Kinematica) at 14,000 rpm for 20 s. The homogenate was centrifuged at 48,000Ã¿g for 30 min at 4° C. Subsequently, the supernatant was removed and discarded, and the pellet resuspended in 20 ml HEPES-NaOH (20 mM, pH 7.4) containing 0.1 mM EDTA using the Polytron (20 s at 14,000 rpm). This procedure was repeated and the final pellet resuspended in HEPES-NaOH containing 0.1 mM EDTA and homogenized using the Polytron. Typically, aliquots of 2 ml membrane portions were stored at -80° C. With each new membrane batch the dissociation constant (Kd) was determined via a saturation curve. The TAAR1 radioligand 3[H]â¿¿(S)-4-[(ethyl-phenyl-amino)-methyl]-4,5-dihydro-oxazol-2-ylamine (described in WO 2008/098857) was used at a concentration equal to the calculated Kd value, that was usually around 2.3 nM, resulting in the binding of approximately 0.2% of the radioligand and a specific binding representing approximately 85% of the total binding. Nonspecific binding was defined as the amount of 3[H]â¿¿(S)-4-[(ethyl-phenyl-amino)-methyl]-4,5-dihydro-oxazol-2-ylamine bound in the presence of 10 uM unlabeled ligand. All compounds were tested at a broad range of concentrations (10 pM to 10 uM) in duplicates. The test compounds (20 ul/well) were transferred into a 96 deep well plate (TreffLab), and 180 ul of HEPES-NaOH (20 mM, pH 7.4) containing MgCl2 (10 mM) and CaCl2 (2 mM) (binding buffer), 300 ul of the radioligand 3[H]â¿¿(S)-4-[(ethyl-phenyl-amino)-methyl]-4,5-dihydro-oxazol-2-ylamine at a concentration of 3.3xKd in nM and 500 ul of the membranes (resuspended at 50 ug protein per ml) added. The 96 deep well plates were incubated for 1 hr at 4° C. Incubations were terminated by rapid filtration through Unifilter-96 plates (Packard Instrument Company) and glass filters GF/C (Perkin Elmer) presoaked for 1 hr in polyethylenimine (0.3%) and washed 3 times with 1 ml of cold binding buffer. After addition of 45 ul of Microscint 40 (PerkinElmer) the Unifilter-96 plate was sealed and after 1 hr the ratioactivity counted using a TopCount Microplate Scintillation Counter (Packard Instrument Company).
Measurement: Ki = 0.6 nM (pChEMBL 9.220000267028809)
Descriptors: MW 316.79, ALogP 3.25, HBA 3, HBD 2, PSA 50.36, QED 0.91
Compound (SMILES): Cn1cc(-c2cc(C(=O)NC3CCS(=O)(=O)C3)ccc2Oc2ccccc2)c2cc[nH]c2c1=O
InChIKey: GDCAGGAWVJAPOC-UHFFFAOYSA-N
Target: Bromodomain-containing protein 4 (SINGLE PROTEIN), Homo sapiens
UniProt: O60885
Assay: Bromodomain Domain Binding Assay: A time-resolved fluorescence resonance energy transfer (TR-FRET) assay was used to determine the affinities of compounds of the Examples listed in Table 1 for each bromodomain of human BRD4. His-tagged first (BD1: amino acids K57-E168) and second (BD2: amino acids E352-E168) bromodomains of human BRD4 were expressed and purified. An Alexa647-labeled BET-inhibitor was used as the fluorescent probe in the assay. Compound dilution series were prepared in DMSO via a 3-fold serial dilution from 2.5 mM to 42 nM. Compounds were then diluted 6:100 in assay buffer (20 mM Sodium Phosphate, pH 6.0, 50 mM NaCl, 1 mM Ethylenediaminetetraacetic acid disodium salt dihydrate, 0.01% Triton X-100, 1 mM DL-Dithiothreitol) to yield 3× working solutions. Six microliters (L) of the working solution was then transferred to white, low-volume assay plates (Costar #3673). A 1.5× assay mixture containing His-tagged bromodomain, Europium-conjugated anti-His antibody (Invitrogen PV5596) and the Alexa-647-conjugated probe molecule was also prepared. Twelve L of this solution were added to the assay plate to reach a final volume of 18 L. The final concentration of 1× assay buffer contains 2% DMSO, 50 M-0.85 nM compound, 8 nM His-tagged bromodomain, 1 nM Europium-conjugated anti-His-tag antibody and 100 nM or 30 nM probe (for BDI or BDII, respectively). After a one-hour incubation at room temperature, TR-FRET ratios were determined using an Envision multilabel plate reader (Ex 340, Em 495/520).
Measurement: Ki = 10.3 nM (pChEMBL 7.989999771118164)
Descriptors: MW 477.54, ALogP 3.24, HBA 6, HBD 2, PSA 110.26, QED 0.46
Compound (SMILES): O=C(c1n[nH]c2c1CN(C1COC1)CC2)N1CCC(c2cccc(F)c2C(F)(F)F)CC1
InChIKey: JABDGKTYXKTOFM-UHFFFAOYSA-N
Target: Retinol-binding protein 4 (SINGLE PROTEIN), Homo sapiens
UniProt: P02753
Assay: 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).
Measurement: IC50 = 28.34 nM (pChEMBL 7.550000190734863)
Descriptors: MW 452.45, ALogP 3.34, HBA 4, HBD 1, PSA 61.46, QED 0.73
Compound (SMILES): O[C@@H](CN[C@H]1CC[C@H](c2nnn3cnc4[nH]ccc4c23)CC1)c1ccccc1
InChIKey: RJVYQTHPYZENAN-BQFCYCMXSA-N
Target: Tyrosine-protein kinase JAK2 (SINGLE PROTEIN), Homo sapiens
UniProt: O60674
Assay: Enzyme Assay: JAK1, JAK2, JAK3 and Tyk2 were purchased from Carna Biosciences, Inc. As the substrate, LANCE Ultra ULight-JAK1 Peptide (manufactured by PerkinElmer Co., Ltd. (PE)) was used. Dilute solutions of compounds and enzymes in assay buffer (50 mM HEPES pH7.5, 1 mM EGTA, 1 mM MgCl2, 2 mM DTT, 0.01% Tween20) were dispensed into wells of a 384-well black plate. After 5 minutes of preincubation, dilute solutions of the substrate and ATP (adenosine triphosphate) were added at a final concentration of 100 μM, and the plate was incubated at room temperature for 2 hours. After addition of a termination reagent containing EDTA (ethylenediamine tetraacetic acid) at a final concentration of 10 mM, LANCE Eu-W1024 Anti-phosphotyrosine (PT66) (manufactured by PE) was added, and after 1 hour of incubation, the fluorescences were measured with ARVO-HTS.
Measurement: IC50 = 190 nM (pChEMBL 6.71999979019165)
Descriptors: MW 376.46, ALogP 2.96, HBA 6, HBD 3, PSA 91.13, QED 0.5
Compound (SMILES): Cc1cc(Nc2nn(C3(CC#N)CCN(C(=O)OC(C)(C)C)CC3)cc2C(N)=O)ccn1
InChIKey: LPULVUIIYXMNDP-UHFFFAOYSA-N
Target: Tyrosine-protein kinase JAK2 (SINGLE PROTEIN), Homo sapiens
UniProt: O60674
Assay: HTRF Biochemical Assay: The reactions employed a common peptide substrate, LCB-EQEDEPEGDYFEWLW-NH2 (in-house). The basic assay protocol is as follows: First, 250 nL of diluted compounds in DMSO were dispensed into the wells of a dry 384-well Black plate (Greiner #781076) using a Labcyte Echo 555 acoustic dispenser. Subsequent reagent additions employed an Agilent Bravo. Next, 18 uL of 1.11x enzyme and 1.11x substrate in 1x assay buffer (Invitrogen kinase buffer # PV3189, 2 mM DTT, 0.05% BSA) were added to the wells and shaken and then preincubated for 30 minutes at room temperature to allow compound binding to equilibrate. After equilibration, 2 uL of 10xATP in 1x assay buffer was added to initiate the kinase reaction and the plates were shaken and then incubated at room temperature for 120 minutes. At the end of the incubation, 20 uL of 2x stop buffer (streptavidin-Dylight 650 (Thermo #84547B/100 mL), Eu-tagged pY20 antibody (Perkin Elmer #AD0067), EDTA, HEPES, and Triton) was added to quench the reaction.
Measurement: IC50 = 133 nM (pChEMBL 6.880000114440918)
Descriptors: MW 439.52, ALogP 3.07, HBA 8, HBD 2, PSA 139.16, QED 0.73
Compound (SMILES): C#Cc1cc(S(=O)(=O)NC(=O)c2ccc(N3CCN(CC4=C(c5ccc(Cl)cc5)CC(C)(C)CC4)CC3)cc2Oc2cccc3[nH]ccc23)cnc1OCC1CCOCC1
InChIKey: BJBNHPNEGVDAOF-UHFFFAOYSA-N
Target: Bcl-2-like protein 1 (SINGLE PROTEIN), Mus musculus
UniProt: Q64373
Assay: Time Resolved-Fluorescence Resonance Energy Transfer (TR-FRET) Assay: The inhibition constant (Ki) is the dissociation constant of an enzyme-inhibitor complex or a protein/small molecule complex, wherein the small molecule is inhibiting binding of one protein to another protein or peptide. Where the Ki for a compound is represented as > (greater than) a certain numerical value, it is intended to mean that the binding affinity value (e.g., for Bcl-XL) is greater than the limits of detection of the assay used. Where the binding selectivity ratio for a compound is represented as > (greater than) a certain numerical value, it is intended to mean that the selectivity of a particular compound for Bcl-2 over Bcl-XL is at least as great as the number indicated. Where the Ki for a compound is represented as < (less than) a certain numerical value, it is intended to mean that the binding affinity value (e.g., for Bcl-2) is lower than the limit of detection of the assay used. Inhibition constants were determined using Wang's equation (Wang Z-X).
Measurement: Ki > 660 nM (pChEMBL 6.179999828338623)
Descriptors: MW 848.47, ALogP 8.7, HBA 9, HBD 2, PSA 126.09, QED 0.12
Compound (SMILES): NCCC[C@H]1CNC(=O)c2cc(-c3cccc(OC(F)(F)F)c3)c(-c3ccc(O)cc3)n21
InChIKey: BFDYPWZGYIYZPW-INIZCTEOSA-N
Target: Serine/threonine-protein kinase pim-2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9P1W9
Assay: Trans-Phosphorylation Assay: The inhibitory activity of putative kinase inhibitors and the potency of selected compounds were determined using a trans-phosphorylation assay.Specific peptide or protein substrates were trans-phosphorylated by their specific ser-thr or tyr kinase in the presence of ATP traced with 33P-gamma-ATP, and in the presence of their own optimal buffer and cofactors. At the end of the phosphorylation reaction, more than 98% unlabeled ATP and radioactive ATP were captured by an excess of the ion exchange dowex resin; the resin then settled down to the bottom of the reaction plate by gravity. Supernatant was subsequently withdrawn and transferred into a counting plate, then evaluated by beta-counting. Kinase Buffer (KB):The buffer for PIM2 assay was composed of HEPES 50 mM, at pH 7.5, with 1 mM MgCl2, 1 mM DTT, 3 uM Na3VO4, and 0.2 mg/mL BSA.Â  Full-length human PIM2 was expressed and purified as described in Fedorov O, et al., PNAS 2007 104, 51, 20523-28.
Measurement: IC50 = 48 nM (pChEMBL 7.320000171661377)
Descriptors: MW 445.44, ALogP 4.45, HBA 5, HBD 3, PSA 89.51, QED 0.52
Compound (SMILES): O=C(CO)ONC(=O)c1cccc([C@H]2C[C@H]3CC[C@@H](C2)N3CCN(CC2CCCCC2)C(=O)[C@@H](O)CO)c1
InChIKey: YYGKFFUAZRPQFA-NDBXHCKUSA-N
Target: Delta-type opioid receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P41143
Assay: Displacement of [3H]-diprenorphine from human delta opioid receptor expressed in cell membranes
Measurement: Ki = 100 nM (pChEMBL 7)
Descriptors: MW 531.65, ALogP 1.34, HBA 8, HBD 4, PSA 139.64, QED 0.33
Compound (SMILES): CN1CCC2C(C1)OCCN2C(=O)Cn1cc(NC(=O)c2c(N)nn3cccnc23)c(-c2cc(Cl)ccc2OC(F)F)n1
InChIKey: AUTGBGJOJQVLAH-UHFFFAOYSA-N
Target: Tyrosine-protein kinase JAK2 (SINGLE PROTEIN), Homo sapiens
UniProt: O60674
Assay: JAK Enzyme Assay: The activity of the isolated recombinant JAK1 and JAK2 kinase domain was measured by monitoring phosphorylation of a peptide derived from JAK3 (Val-Ala-Leu-Val-Asp-Gly-Tyr-Phe-Arg-Leu-Thr-Thr, fluorescently labeled on the N-terminus with 5-carboxyfluorescein) using the Caliper LabChip® technology (Caliper Life Sciences, Hopkinton, Mass.). To determine inhibition constants (Ki), compounds were diluted serially in DMSO and added to 50 μL kinase reactions containing purified enzyme (1.5 nM JAK1, or 0.2 nM JAK2), 100 mM HEPES buffer (pH 7.2), 0.015% Brij-35, 1.5 μM peptide substrate, ATP (25 μM), 10 mM MgCl2, 4 mM DTT at a final DMSO concentration of 2%. Reactions were incubated at 22° C. in 384-well polypropylene microtiter plates for 30 minutes and then stopped by addition of 25 μL of an EDTA containing solution (100 mM HEPES buffer (pH 7.2), 0.015% Brij-35, 150 mM EDTA), resulting in a final EDTA concentration of 50 mM. After termination of the kinase reaction, the proportion of phosphorylated product was determined as a fraction of total peptide substrate using the Caliper LabChip® 3000 according to the manufacturer's specifications.
Measurement: Ki = 1.26 nM (pChEMBL 8.899999618530273)
Descriptors: MW 616.03, ALogP 2.61, HBA 11, HBD 2, PSA 145.14, QED 0.32
Compound (SMILES): COc1nc2ccc(C(C)(O)c3cnnn3C)cc2c(Cl)c1Cc1ccc(C(F)(F)F)cc1
InChIKey: KCEVZBCVYHCXBO-UHFFFAOYSA-N
Target: Nuclear receptor ROR-gamma (SINGLE PROTEIN), Homo sapiens
UniProt: P51449
Assay: ThermoFluor® Assay: ThermoFluor® experiments were carried out using instruments owned by Janssen Research and Discovery, L.L.C. through its acquisition of 3-Dimensional Pharmaceuticals, Inc. 1,8-ANS (Invitrogen) was used as a fluorescent dye. Protein and compound solutions are dispensed into black 384-well polypropylene PCR microplates (Abgene) and overlayed with silicone oil (1 μL, Fluka, type DC 200) to prevent evaporation. Bar-coded assay plates are robotically loaded onto a thermostatically controlled PCR-type thermal block and then heated at a typical ramp-rate of 1° C./min for all experiments. Fluorescence was measured by continuous illumination with UV light (Hamamatsu LC6) supplied via fiber optic and filtered through a band-pass filter (380-400 nm; >6 OD cutoff). Fluorescence emission of the entire 384-well plate was detected by measuring light intensity using a CCD camera (Sensys, Roper Scientific) filtered to detect 500±25 nm, resulting in simultaneous and independent readings of all 384 wells.Â  Reference wells contained RORγt without compounds, and the assay conditions were as follows: 0.065 mg/mL RORγt, 60 μM 1,8-ANS, 100 mM Hepes, pH 7.0, 10 mM NaCl, 2.5 mM GSH, 0.002% Tween-20.
Measurement: Kd = 290 nM (pChEMBL 6.539999961853027)
Descriptors: MW 476.89, ALogP 4.89, HBA 6, HBD 1, PSA 73.06, QED 0.44
Compound (SMILES): O=C(Nc1cn(CC(=O)N2CC[C@H]3CN(CC4CCOCC4)C[C@H]3C2)nc1-c1cc(Cl)ccc1OC(F)F)c1cnn2cccnc12
InChIKey: HZHXIWLMJLULHF-VXKWHMMOSA-N
Target: Tyrosine-protein kinase JAK2 (SINGLE PROTEIN), Homo sapiens
UniProt: O60674
Assay: JAK Enzyme Assay: The activity of the isolated recombinant JAK1 and JAK2 kinase domain was measured by monitoring phosphorylation of a peptide derived from JAK3 (Val-Ala-Leu-Val-Asp-Gly-Tyr-Phe-Arg-Leu-Thr-Thr, fluorescently labeled on the N-terminus with 5-carboxyfluorescein) using the Caliper LabChip® technology (Caliper Life Sciences, Hopkinton, Mass.). To determine inhibition constants (Ki), compounds were diluted serially in DMSO and added to 50 μL kinase reactions containing purified enzyme (1.5 nM JAK1, or 0.2 nM JAK2), 100 mM HEPES buffer (pH 7.2), 0.015% Brij-35, 1.5 μM peptide substrate, ATP (25 μM), 10 mM MgCl2, 4 mM DTT at a final DMSO concentration of 2%. Reactions were incubated at 22° C. in 384-well polypropylene microtiter plates for 30 minutes and then stopped by addition of 25 μL of an EDTA containing solution (100 mM HEPES buffer (pH 7.2), 0.015% Brij-35, 150 mM EDTA), resulting in a final EDTA concentration of 50 mM. After termination of the kinase reaction, the proportion of phosphorylated product was determined as a fraction of total peptide substrate using the Caliper LabChip® 3000 according to the manufacturer's specifications.
Measurement: Ki = 0.94 nM (pChEMBL 9.029999732971191)
Descriptors: MW 669.13, ALogP 4.31, HBA 10, HBD 1, PSA 119.12, QED 0.28
Compound (SMILES): COc1ccc(COCCOc2ccc3c(NC(=O)c4ccc(N5CCN(C)CC5)cc4)n[nH]c3c2)cc1
InChIKey: IIFDFWIYQQQBGS-UHFFFAOYSA-N
Target: Mast/stem cell growth factor receptor Kit (SINGLE PROTEIN), Homo sapiens
UniProt: P10721
Assay: Biochemical Assay: The assay has been performed using KIT cytoplasmic domain product and purified in house as GST fused protein. The KIT protein (4.5 microM) was pre activated with 300 microM ATP for 1 hour at 28*C in order to obtain a linear kinetic. Kinase buffer was composed of 50 mM HEPES pH 7.9 containing 5 mM MgCl2, 1 mM MnCl2, 10 mM DTT, 3 microM Na3VO4, and 0.2 mg/mL BSA. The KIT kinase assay was run with a final pre activated enzyme concentration of 4 nM, in the presence of 4.4 microM ATP (residual ATP from KIT pre activation step is negligible), 3.9 nM 33P-γ-ATP and 2.5 microM of substrate BioDB n*138 (Aminoacidic sequence: KWEEINGNNYVYIDPTQLPYDHKWEFPRNR- SEQ ID NO: 2).
Measurement: IC50 = 88 nM (pChEMBL 7.059999942779541)
Descriptors: MW 515.61, ALogP 4.17, HBA 7, HBD 2, PSA 91.95, QED 0.31
Compound (SMILES): Cc1oc2c(c1CC(=O)Nc1nc(-c3ccc(C(F)(F)F)c(F)c3F)cs1)c(=O)n(C)c(=O)n2C
InChIKey: MQPVCEGYFVTWDL-UHFFFAOYSA-N
Target: Transient receptor potential cation channel subfamily V member 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q8NER1
Assay: Radiometric Calcium Assay: The inhibition of TRPA1 receptor activation was measured as inhibition of allylisothiocyanate (AITC) induced cellular uptake of radioactive calcium. Test compounds were dissolved in DMSO to prepare 10 mM stock solution and then diluted using plain medium with 0.1% BSA and 1.8 mM CaCl2 to get desired concentration. Final concentration of DMSO in the reaction was 0.5% (v/v). Human TRPA1 expressing CHO cells were grown in F-12 DMEM medium with 10% FBS, 1% penicillin-streptomycin solution, and 400 ug/ml of G-418. Cells were seeded 24 h prior to the assay in 96 well plates so as to get 50,000 cells per well on the day of experiment. Cells were treated with test compounds for 10 min followed by addition of AITC at a final concentration of 30 micromolar and 5 uCi/ml 45Ca+2 for 3 min at 25° C. Cells were washed and lysed using buffer containing 1% Triton X-100, 0.1% deoxycholate and 0.1% SDS. Radioactivity in the lysate was measured in Packard Top count.
Measurement: IC50 = 8010 nM (pChEMBL 5.099999904632568)
Descriptors: MW 514.43, ALogP 3.74, HBA 8, HBD 1, PSA 99.13, QED 0.42
Compound (SMILES): NC(=O)CC1(NC(=O)c2cc(OCC3CC3)c(N3CC(F)(F)C3)cn2)COC1
InChIKey: FPBGJMMSUSSBTL-UHFFFAOYSA-N
Target: Cannabinoid receptor 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P21554
Assay: cAMP Assay: CHO cells expressing human CB1 or CB2 receptors are seeded 17-24 hours prior to the experiment 50.000 cells per well in a black 96 well plate with flat clear bottom (Corning Costar #3904) in DMEM (Invitrogen No. 31331), 1×HT supplement, with 10% fetal calf serum and incubated at 5% CO2 and 37° C. in a humidified incubator. The growth medium was exchanged with Krebs Ringer Bicarbonate buffer with 1 mM IBMX and incubated at 30° C. for 30 min. Compounds were added to a final assay volume of 100 μl and incubated for 30 min at 30° C. Using the cAMP-Nano-TRF detection kit the assay (Roche Diagnostics) was stopped by the addition of 50 μl lysis reagent (Tris, NaCl, 1.5% Triton X100, 2.5% NP40, 10% NaN3) and 50 μl detection solutions (20 μM mAb Alexa700-cAMP 1:1, and 48 μM Ruthenium-2-AHA-cAMP) and shaken for 2 h at room temperature. The time-resolved energy transfer is measured by a TRF reader (Evotec Technologies GmbH), equipped with a ND:YAG laser as excitation source. The plate is measured twice with the excitation at 355 nm and at the emission with a delay of 100 ns and a gate of 100 ns, total exposure time 10 s at 730 (bandwidth 30 nm) or 645 nm (bandwidth 75 nm), respectively. The FRET signal is calculated as follows: FRET=T730-Alexa730-P(T645-B645) with P=Ru730-B730/Ru645-B645, where T730 is the test well measured at 730 nM, T645 is the test well measured at 645 nm, B730 and B645 are the buffer controls at 730 nm and 645 nm, respectively. cAMP content is determined from the function of a standard curve spanning from 10 μM to 0.13 nM cAMP.
Measurement: EC50 > 10000 nM (pChEMBL 5)
Descriptors: MW 396.39, ALogP 0.7, HBA 6, HBD 2, PSA 106.78, QED 0.67
Compound (SMILES): Cc1cc(Nc2nccc(Oc3ccc(NC(=O)Nc4cc(C(C)(C)C)nn4-c4cccc(OCCN5CCOCC5)c4)c4ccccc34)n2)cc2cn[nH]c12
InChIKey: QJIXTISVQSBZTF-UHFFFAOYSA-N
Target: Proto-oncogene tyrosine-protein kinase Src (SINGLE PROTEIN), Homo sapiens
UniProt: P12931
Assay: c-Src and Syk Enzyme Inhibition Assay: The inhibitory activities of compounds of the invention against c-Src and Syk enzymes (Invitrogen), are evaluated in a similar fashion to that described hereinabove. The relevant enzyme (3000 ng/mL or 2000 ng/mL respectively, 2.5 uL) is incubated with the test compound (either 4 ug/mL, 0.4 ug/mL, 0.04 ug/mL, or 0.004 ug/mL, 2.5 uL each) for 2 hr at RT. The FRET peptides (8 uM, 2.5 uL), and appropriate ATP solutions (2.5 uL, 800 uM for c-Src, and 60 uM ATP for Syk) are then added to the enzymes/compound mixtures and incubated for 1 hr. Development reagent (protease, 5 uL) is added for 1 hr prior to detection in a fluorescence microplate reader (Varioskan Flash, ThermoFisher Scientific).
Measurement: IC50 = 2 nM (pChEMBL 8.699999809265137)
Descriptors: MW 752.88, ALogP 8.19, HBA 11, HBD 4, PSA 156.37, QED 0.1
Compound (SMILES): CO[C@H]1CC[C@]2(CC1)Cc1ccc(OCCC(F)(F)F)cc1[C@@]21N=C(C)C(N)=N1
InChIKey: JJEORDNWHQBLEW-ZRCAFCQKSA-N
Target: Amyloid-beta precursor protein (SINGLE PROTEIN), Homo sapiens
UniProt: P05067
Assay: Release Assay: SH-SY5Y cells were cultured in DMEM/F-12 with Glutamax, 10% FCS and 1% non-essential amino acids and cryopreserved and stored at -140 C. at a concentration of 7.5-9.5x106 cells per vial. Thaw cells and seed at a conc. of around 10000 cells/well in DMEM/F-12 with Glutamax, 10% FCS and 1% non-essential amino acids to a 384-well tissue culture treated plate, 100 uL cell susp/well. The cell plates were then incubated for 7-24 h at 37 C., 5% CO2. The cell medium was removed, followed by addition of 30 uL compound diluted in DMEM/F-12 with Glutamax, 10% FCS, 1% non-essential amino acids and 1% PeSt to a final conc. of 1% DMSO. The compounds were incubated with the cells for 17 h (overnight) at 37 C., 5% CO2. Meso Scale Discovery (MSD) plates were used for the detection of sAPPbeta release. MSD sAPPbeta plates were blocked in 1% BSA in Tris wash buffer (40 uL/well) for 1 h on shake at r.t. and washed 1 time in Tris wash buffer (40 uL/well).
Measurement: IC50 = 0.7 nM (pChEMBL 9.149999618530273)
Descriptors: MW 409.45, ALogP 4.13, HBA 5, HBD 1, PSA 69.2, QED 0.81
Compound (SMILES): Cl.O=C1c2ccc(Cl)cc2CC1CCN1CCc2ccccc2C1
InChIKey: CDKCTEJHUVKKNR-UHFFFAOYSA-N
Target: D(3) dopamine receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P35462
Assay: Displacement of [3H]-N-methylspiperone from human dopamine D3 receptor by liquid scintillation counting method
Measurement: Ki = 783 nM (pChEMBL 6.110000133514404)
Descriptors: MW 325.84, ALogP 4.14, HBA 2, HBD 0, PSA 20.31, QED 0.84
Compound (SMILES): COc1cc(OC)cc(N(CCCO)c2ccc3ncc(-c4c(C)nn(C)c4C)nc3c2)c1
InChIKey: WCHAWRNEALMUFZ-UHFFFAOYSA-N
Target: Fibroblast growth factor receptor 3 (SINGLE PROTEIN), Homo sapiens
UniProt: P22607
Assay: Inhibition of FGFR3 (unknown origin) transfected in mouse BA/F3 cells assessed as decrease in cell proliferation in presence of mouse IL3 after 24 hrs by Alamar blue assay
Measurement: IC50 > 10000 nM (pChEMBL 5)
Descriptors: MW 447.54, ALogP 4.18, HBA 8, HBD 1, PSA 85.53, QED 0.43