lovingscience/chembl-v37-raw-activities huggingface dataset:
Compound (SMILES): CCC1c2ncn(-c3cnccn3)c2CCN1C(=O)c1cccc(C(F)(F)F)c1Cl
InChIKey: RHSLGGDFAHTXIY-UHFFFAOYSA-N
Target: P2X purinoceptor 7 (SINGLE PROTEIN), Homo sapiens
UniProt: Q99572
Assay: FLIPR Assay: 1321 N1 cells expressing the recombinant human, rat or mouse P2X7 channel was cultured in HyQ DME/(HyClone/Dulbecco's Modified Eagle Medium) high glucose supplemented with 10% Fetal Bovine Serum (FBS) and appropriate selection marker. Cells were seeded at a density of 25000 cells/well (96-well clear bottom black walled plates) in 100 ul volume/well. On the day of the experiment, cell plates were washed with assay buffer, containing (in mM): 130 NaCl, 2 KCl, 1 CaCl2, 1 MgCl2, 10 HEPES, 5 glucose; pH 7.40 and 300 mOs. After the wash, cells were loaded with the Calcium-4 dye (Molecular Device) and incubated in the dark for 60 minutes. Test compounds were prepared at 250Ã¿ the test concentration in neat DMSO. Intermediate 96-well compound plates were prepared by transferring 1.2 uL of the compound into 300 uL of assay buffer. A further 3x dilution occurred when transferring 50 uL/well of the compound plate to 100 uL/well in the cell plate. Cells were incubateÂ  with test compounds and dye for 30 minutes. Calcium dye fluorescence was monitored in FLIPR as the cells were challenged by adding 50 uL/well of BzATP (final concentration is 250 uM BzATP (human and rat) or 600 (mouse)). The fluorescence change was measured 180 seconds after adding the agonist.
Measurement: IC50 = 87.5 nM (pChEMBL 7.059999942779541)
Descriptors: MW 435.84, ALogP 4.48, HBA 5, HBD 0, PSA 63.91, QED 0.61
Compound (SMILES): Cc1cc(Oc2ccccc2OCCn2ccc(=O)[nH]c2=O)cc2cc(C#N)ccc12
InChIKey: XZUICUJGMQEYTJ-UHFFFAOYSA-N
Target: Human immunodeficiency virus type 1 reverse transcriptase (SINGLE PROTEIN), Human immunodeficiency virus 1
UniProt: Q72547
Assay: Inhibition of HIV1 reverse transcriptase Y181C mutant infected in human MT2 cells assessed as protection against viral infection by MTT assay
Measurement: EC50 = 800 nM (pChEMBL 6.099999904632568)
Descriptors: MW 413.43, ALogP 3.74, HBA 6, HBD 1, PSA 97.11, QED 0.52
Compound (SMILES): Nc1ccccc1NC(=O)CCCCCNC(=O)c1cc2cccc(F)c2[nH]1
InChIKey: SRJBNYGUCHLOJS-UHFFFAOYSA-N
Target: Histone deacetylase 3 (SINGLE PROTEIN), Homo sapiens
UniProt: O15379
Assay: HDAC enzyme inhibitionAssay: HDAC enzyme inhibition assays were performed using purified HDACs 1-10 essentially as described in Beckers et al., 2007, Int. J. Cancer., 121:1138-48 and Perez-Balado et al., 2007, J. Med. Chem., 50:2497-2505. Inhibition assays using nuclear extract were performed essentially as described in Herman et al., 2006, Nat. Chem. Biol., 2:551-558. Briefly, the purified HDACs or nuclear extract were incubated with an acetylated substrate in the absence of the compound to be assayed and with increasing concentrations of the compound. The rate of substrate deacetylation was measured under each condition, and half-maximal inhibitory concentration with regard to each HDAC was determined by standard means.
Measurement: IC50 = 41 nM (pChEMBL 7.389999866485596)
Descriptors: MW 382.44, ALogP 3.82, HBA 3, HBD 4, PSA 100.01, QED 0.35
Compound (SMILES): CC(C(N)=O)c1cccc(Nc2nccc(Nc3cccc(C(F)(F)F)c3)n2)c1
InChIKey: KXPATQPDYGJJOM-UHFFFAOYSA-N
Target: Aurora kinase A (SINGLE PROTEIN), Homo sapiens
UniProt: O14965
Assay: Enzyme Inhibition Assay: The phosphorylation of L-Hse was monitored by coupling the formation of ADP with pyruvate kinase and lactate dehydrogenase (PK/LDH). The resulting oxidation of NADH was monitored at 340 nm by using a SpectraMax plate reader in a 96-well format.
Measurement: IC50 = 1400 nM (pChEMBL 5.849999904632568)
Descriptors: MW 401.39, ALogP 4.57, HBA 5, HBD 3, PSA 92.93, QED 0.56
Compound (SMILES): CCC(C)C(=O)N1CCC(Oc2ccc(NC(=O)N3Cc4ccccc4C3)cc2)CC1
InChIKey: ISGVOILXCPQWBH-UHFFFAOYSA-N
Target: Nicotinamide phosphoribosyltransferase (SINGLE PROTEIN), Homo sapiens
UniProt: P43490
Assay: Time-Resolved Fluorescence Resonance Energy Transfer (TR-FRET) Binding Assay : Probe 2:The assay was carried out in 18 uL low volume plates (Owens Corning) in reaction buffer (50 mM HEPES (NaOH), pH 7.5, 100 mM NaCl, 10 mM MgCl2, 1 mM DTT, 1% Glycerol) using 6.8 nM recombinant, human, C-terminally-His tagged NAMPT, 1 nM Tb-anti-His antibody (Invitrogen, Cat #PV5895), and 200 nM probe (Oregon Green 488-conjugated APO0866; A-1287128.0. Plates were covered, and reactions were carried out for 2-3 hours. Plates were read with Envision (Laser Lantha low volume protocol) after 2 to 3 hours. Excitation was carried out at 337 nm, and the ratio of emission of Oregon Green (520 nm) to terbium (492 nm) was determined and used to calculate IC50 values of test compounds.
Measurement: IC50 = 51.9 nM (pChEMBL 7.289999961853027)
Descriptors: MW 421.54, ALogP 4.65, HBA 3, HBD 1, PSA 61.88, QED 0.76
Compound (SMILES): O=C1COc2ccc(C3(O)c4ccccc4C(=O)N3c3cccc(CO)c3)cc2N1
InChIKey: GQBDSNNFWUMJQQ-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 = 1000 nM (pChEMBL 6)
Descriptors: MW 402.41, ALogP 2.36, HBA 5, HBD 3, PSA 99.1, QED 0.62
Compound (SMILES): O=C(Nc1cccc(C2CNCCO2)c1)c1ccc(Cl)cc1
InChIKey: HLJFMYBGKKFDOK-UHFFFAOYSA-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 = 107.2 nM (pChEMBL 6.96999979019165)
Descriptors: MW 316.79, ALogP 3.25, HBA 3, HBD 2, PSA 50.36, QED 0.91
Compound (SMILES): Cc1ccc2nc(N3CCN(c4ncccn4)CC3)nc(NCc3ccc(NC(=O)C4CCN(Cc5ccccc5)CC4)cc3)c2c1
InChIKey: XHLPHDKEOZRIGH-UHFFFAOYSA-N
Target: TCF4/beta-catenin (PROTEIN COMPLEX), Homo sapiens
UniProt: P35222
Assay: Inhibition of TCF-4/beta catenin in human Tcf22C11 cells expressing Tcf-4/SV40 binding sites driving firefly luciferase reporter gene after 24 hrs by luminometer
Measurement: IC50 = 872 nM (pChEMBL 6.059999942779541)
Descriptors: MW 627.8, ALogP 5.52, HBA 9, HBD 2, PSA 102.41, QED 0.22
Compound (SMILES): CC(C1=C(c2ccc(Cl)cc2)CC(C)(C)CC1)N1CCN(c2ccc(C(=O)NS(=O)(=O)c3ccc(NCCCN4CCOCC4)c([N+](=O)[O-])c3)c(Oc3ccc4[nH]ccc4c3)c2)CC1
InChIKey: BUZLZTNLOGFYNY-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 = 17.13 nM (pChEMBL 7.769999980926514)
Descriptors: MW 910.54, ALogP 8.95, HBA 11, HBD 3, PSA 162.38, QED 0.05
Compound (SMILES): C[C@@]1(c2cc(Nc3ccon3)ccc2F)COCC(N)=N1
InChIKey: QRXTWNWAFMAMIP-AWEZNQCLSA-N
Target: Beta-secretase 2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9Y5Z0
Assay: Cellular Assay: The assay uses the principle of inhibition of human TMEM27 cleavage by endogenous cellular BACE2 in the Ins1e rat cell line and shedding from the cell surface into the culture medium, followed by detection in an ELISA assay. Inhibition of BACE2 prevents the cleavage and shedding in a dose-dependent mannerThe stable cell line "INS-TMEM27" represents an INS1e-derived cell line with inducible expression (using the TetOn system) of full-length hTMEM27 in a doxycycline-dependent manner. The cells are cultured throughout the experiment in RPMI1640 +Glutamax (Invitrogen) Penicillin/Streptomycin, 10% Fetal bovine serum, 100 mM pyruvate, 5 mM beta-mercatptoethanol, 100 micrograms/ml G418 and 100 microgram/ml hygromycin and are grown inadherent culture at 37° C. in a standard CO2 cell culture incubator.INS-TMEM27 cells are seeded in 96-well plates. After 2 days in culture, BACE2 inhibitor is added in a range of concentrations as required by the assay and after a further two hours, doxycycline i
Measurement: IC50 = 1210 nM (pChEMBL 5.920000076293945)
Descriptors: MW 290.3, ALogP 2.16, HBA 6, HBD 2, PSA 85.67, QED 0.9
Compound (SMILES): NC(=O)c1ccc(N2CCC(OC(=O)N3CCN(C4CCC4)CC3)CC2)nc1
InChIKey: PESWRCJNZDSPCQ-UHFFFAOYSA-N
Target: Histamine H3 receptor (SINGLE PROTEIN), Homo sapiens
UniProt: Q9Y5N1
Assay: Radioligand Binding Assay: The affinity of compounds of the invention to the H3 receptor can be assessed by measuring displacement of binding of the radioligand [3H]-N-Î±-Methylhistamine (PerkinElmer, #NET1027250UC) to membranes containing human H3 receptors (PerkinElmer, #ES-392-M400UA; GenBank: NM-007232.2; Hill S J et al, International Union of Pharmacology XIII. Classification of histamine receptors, Pharmacol Rev, 49(3), 253-278, 1997).Protocol: 1. Preparation of binding assay buffer (500 ml): 25 ml 1 M Tris-HCl pH 7.5 (Invitrogen, #15567-027), 2.5 ml 1 M MgCl2 (Sigma, #M1028-100ML), 472.5 ml ddH2O. 2. Compound serial dilution: Dilution was performed by BioTek Precision on compound dilution plate. Compound concentrations start at 5 or 10 uM, 10 point dose titrations with 3- or 5-fold serial dilutions. 3. Preparation of 2x membrane solution (25 ml): 1.25 ml human Histamine H3 receptor stock, 23.75 ml assay buffer. 4. Preparation of 2x solution of [3H]-N-Î±-methylhistamine (25 ml).
Measurement: Ki = 33 nM (pChEMBL 7.480000019073486)
Descriptors: MW 387.48, ALogP 1.46, HBA 6, HBD 1, PSA 92, QED 0.84
Compound (SMILES): C/C(=C\c1cc(F)c(Oc2ccc(S(=O)(=O)NCCOCCOCCOCCNCc3ccc(CNCCOCCOCCOCCNS(=O)(=O)c4ccc(Oc5c(F)cc(/C=C(\C)C(=O)N=C(N)N)cc5F)cc4)cc3)cc2)c(F)c1)C(=O)N=C(N)N
InChIKey: QCOMQSLWHIBJPY-UTKMUYHOSA-N
Target: Sodium/hydrogen exchanger 3 (SINGLE PROTEIN), Homo sapiens
UniProt: P48764
Assay: Inhibition of human NHE3-mediated sodium-dependant hydrogen ion antiport in Opossum kidney cells preincubated in NH4Cl-HEPES buffer containing BCECF-AM dye followed by washout with ammonium free HEPES and subsequent addition of compound in presence of NHE1 antagonist EIPA by fluorescence assay
Measurement: IC50 = 39.81 nM (pChEMBL 7.400000095367432)
Descriptors: MW 1273.4
Compound (SMILES): CO[C@H]1CC[C@H](CC(=O)N[C@H]2CC[C@H](CCN3CCC(c4noc5ccc(Cl)cc45)CC3)CC2)CC1
InChIKey: CMOUHGOJSYWQFR-LRHALGNLSA-N
Target: D(3) dopamine receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P35462
Assay: Radioligand Binding Assay: Aliquots of membrane preparations were thawed at RT, resuspended in assay buffer (D2, D3: 50 mM Tris-HCl, 120 mM NaCl, 5 mM MgCl2, 1 mM EDTA, 5 mM KCl, 1.5 mM CaCl2, pH=7.4; 5-HT2A: 50 mM Tris-HCl, 10 mM MgCl2, 1 mM EGTA, pH=7.4), homogenized with a Polytron for 20-30 sec at 12,000 rpm and adjusted to a final concentration of approximately 7.5 ug protein/well (D2, D3) and 15 ug protein/well (5-HT2A), respectively.The binding affinity (Ki) of the compounds was determined using radioligand binding. Membranes were incubated in a total volume of 200 ul with a fixed concentration of radioligand (final concentration approximately 0.7 nM [3H]-spiperone for D2, 0.5 nM [3H]-spiperone for D3, and 1.1 nM [3H]-ketanserin for 5-HT2A) and ten concentrations of test compound in ranging between 10 uM-0.1 nM for 1 h at RT. At the end of the incubation, the reaction mixtures were filtered on to unifilter 96-well white microplates with bonded GF/C filters.
Measurement: Ki = 8.381 nM (pChEMBL 8.079999923706055)
Descriptors: MW 516.13, ALogP 6.32, HBA 5, HBD 1, PSA 67.6, QED 0.45
Compound (SMILES): CCCC[C@H](NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CCCNC(=N)N)NC(=O)[C@H](C)NC(=O)[C@H](Cc1ccccc1)NC(=O)[C@H](Cc1ccc(O)cc1)NC(=O)CNC(=O)[C@H](C)NC(=O)[C@@H](NC(=O)[C@H](Cc1ccccc1)NC(=O)[C@@H](NC(=O)[C@H](C)NC(=O)[C@@H](C)NC(=O)[C@H](CCCCN)NC(=O)[C@H](CO)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@H](Cc1ccccc1)NC(C)=O)C(C)C)C(C)C)C(=O)N[C@@H](C)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC(C)C)C(N)=O
InChIKey: QYGDZSKAWLACHO-JDJSXECJSA-N
Target: Tissue factor pathway inhibitor (SINGLE PROTEIN), Homo sapiens
UniProt: P10646
Assay: Inhibition of C-terminus 10x-His-taged human TFPI-1 (29 to 282 residues) assessed as inhibition of factor 10a using S-2222 substrate incubated for 115 mins by micro-plate reader based assay
Measurement: EC50 = 290 nM (pChEMBL 6.539999961853027)
Descriptors: MW 2241.72
Compound (SMILES): CN1C(=N)N[C@](C)(c2sc(-c3nnc(-c4ccc(F)cc4)o3)cc2Cl)CS1(=O)=O
InChIKey: GHVBKTVASKIYHA-KRWDZBQOSA-N
Target: Beta-secretase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P56817
Assay: HTRF FRET Assay: Varying concentrations of inhibitors at 3× the final desired concentration in a volume of 10 ul are preincubated with purified human BACE1 catalytic domain (3 nM in 10 μl) for 30 minutes at 30° C. in reaction buffer containing 20 mM Na-Acetate pH 5.0, 10% glycerol, 0.1% Brij-35 and 7.5% DSMO. Reactions are initiated by addition of 10 μl of 600 nM QSY7-APPswe-Eu substrate (200 nM final) to give a final reaction volume of 30 μl in a 384 well Nunc HTRF plate. The reactions are incubated at 30° C. for 1.5 hours. The 620 nm fluorescence is then read on a Rubystar HTRF plate reader (BMG Labtechnologies) using a 50 millisecond delay followed by a 400 millisecond acquisition time window.
Measurement: Ki = 102 nM (pChEMBL 6.989999771118164)
Descriptors: MW 455.92, ALogP 3.27, HBA 7, HBD 2, PSA 112.18, QED 0.63
Compound (SMILES): Cc1cc(-c2cn(CCNC(C)C)c(C3CCN(c4ncnc(N)c4C(N)=O)CC3)n2)ccc1F
InChIKey: GEZVKRSPTDGJTN-UHFFFAOYSA-N
Target: RAC-alpha serine/threonine-protein kinase (SINGLE PROTEIN), Homo sapiens
UniProt: P31749
Assay: AKT Enzyme Assay: A TTP Mosquito liquid handling instrument is used to place 125 nl of the appropriate concentration of inhibitor in 100% DMSO (for a dose response curve calculation) into each well of a 384-well plate. To this reaction components are added to a final volume of 12.5 .mu.l: 0.1 ng/.mu.l His-AKT (Full Length), (Invitrogen, Part # P2999, Lot #641228C). 160 uM ATP (Fluka, 02055) 1 mM DTT (Sigma, D0632) 1 mM MgCl2 (Sigma, M1028) 1 .mu.M substrate peptide (sequence FITC-AHA-GRPRTSSFAEG-NH2), synthesized by Tufts Peptide Synthesis service. 100 mM HEPES pH 7.5 (Calbiochem, 391338)0.015% Brij-35 (Sigma, B4184)The reaction is incubated for 90 min at 25 C, and then stopped by the addition of 70 .mu.l of Stop buffer (100 mM HEPES pH 7.5, 0.015% Brij-35, 10 mM EDTA (Sigma, E7889)).The plate is read on a Caliper LC 3000 in an Off-Chip mobility shift assay format, using the following parameters for a 12-sipper chip: screening pressure -2.3 psi, upstream voltage -500, and downstream voltage -3000.
Measurement: IC50 = 15 nM (pChEMBL 7.820000171661377)
Descriptors: MW 480.59, ALogP 2.85, HBA 8, HBD 3, PSA 127.98, QED 0.45
Compound (SMILES): O=S(=O)(Nc1ccc(-c2nc3cc(NS(=O)(=O)c4ccc(Cl)cc4)ccc3o2)cc1)c1ccc(Cl)cc1
InChIKey: JUWFOWVHTSWLCC-UHFFFAOYSA-N
Target: GroEL/GroES (PROTEIN COMPLEX), Escherichia coli
UniProt: Q7BGE6
Assay: Inhibition of Escherichia coli GroEL/GroES-ATPase activity expressed in Escherichia coli DH5alpha/BL21 (DE3) assessed as inhibition of denatured MDH refolding preincubated for 10 mins followed by ATP addition measured after 20 to 40 mins by malachite green dye based fluorescence assay
Measurement: IC50 = 35000 nM (pChEMBL 4.460000038146973)
Descriptors: MW 574.47, ALogP 6.4, HBA 6, HBD 2, PSA 118.37, QED 0.23
Compound (SMILES): N[C@H](C(=O)O)C(NS(=O)(=O)c1ccc(Br)cc1)C(=O)O
InChIKey: NPZGPHKBQHVBNJ-JAMMHHFISA-N
Target: Excitatory amino acid transporter 3 (SINGLE PROTEIN), Homo sapiens
UniProt: P43005
Assay: Inhibition of [3H]-D-Asp uptake at human EAAT3 expressed in HEK293 cells measured after 3 mins by TopCount method
Measurement: IC50 = 2600 nM (pChEMBL 5.579999923706055)
Descriptors: MW 367.18, ALogP -0.41, HBA 5, HBD 4, PSA 146.79, QED 0.53
Compound (SMILES): N#CCC1(n2cc(-c3ncnc4[nH]ccc34)cn2)CN(C2CCN(C(=O)c3ccc(-c4ccc5cnccc5c4)c(F)c3)CC2)C1
InChIKey: XCNBPIQLDHQRRB-UHFFFAOYSA-N
Target: Tyrosine-protein kinase JAK2 (SINGLE PROTEIN), Homo sapiens
UniProt: O60674
Assay: In Vitro JAK Kinase Assay: Compounds herein were tested for inhibitory activity of JAK targets according to the following in vitro assay described in Park et al., Analytical Biochemistry 1999, 269, 94-104. The catalytic domains of human JAK1 (a.a. 837-1142), Jak2 (a.a. 828-1132) and Jak3 (a.a. 781-1124) with an N-terminal His tag were expressed using baculovirus in insect cells and purified. The catalytic activity of JAK1, JAK2 or JAK3 was assayed by measuring the phosphorylation of a biotinylated peptide. The phosphorylated peptide was detected by homogenous time resolved fluorescence (HTRF). IC50s of compounds were measured for each kinase in the 40 microL reactions that contain the enzyme, ATP and 500 nM peptide in 50 mM Tris (pH 7.8) buffer with 100 mM NaCl, 5 mM DTT, and 0.1 mg/mL (0.01%) BSA. For the 1 mM IC50 measurements, ATP concentration in the reactions was 1 mM. Reactions were carried out at room temperature for 1 hr and then stopped with 20 μL 45 mM EDTA, 300 nM SA-APC, 6 nM Eu-Py20 in assay buffer (Perkin Elmer, Boston, Mass.). Binding to the Europium labeled antibody took place for 40 minutes and HTRF signal was measured on a Fusion plate reader (Perkin Elmer, Boston, Mass.).
Measurement: IC50 = 12.5 nM (pChEMBL 7.900000095367432)
Descriptors: MW 611.69, ALogP 5.41, HBA 8, HBD 1, PSA 119.62, QED 0.27
Compound (SMILES): CS(=O)(=O)NC[C@H]1CC[C@H](N2CN(C3CC3)C(=O)c3cnc4[nH]ccc4c32)CC1
InChIKey: RXTFZIUWWZLOEV-HDJSIYSDSA-N
Target: Tyrosine-protein kinase JAK2 (SINGLE PROTEIN), Homo sapiens
UniProt: O60674
Assay: Enzyme Assay: The inhibitory activity of the compounds of the present invention against JAK was measured.The respective enzymes (JAK1, JAK2, JAK3 and Tyk2) were purchased from Carna Biosciences, Inc.As a substrate of the enzymes (hereinafter referred to as substrate), LANCE Ultra ULight-JAK-1 (Tyr1023) Peptide (manufactured by PerkinElmer Inc.) was used.As an antibody for detecting phosphorylation of the substrate, LANCE Ultra Europium-anti-phospho tyrosine antibody (PT66) (manufactured by PerkinElmer Inc.) was used.The other reagents were purchased from the following.Adenosine triphosphate (ATP): Sigma-Aldrich4-(2-Hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES): DOJINDO LABORATORIESGlycol ether diamine tetraacetic acid (EGTA): DOJINDO LABORATORIES Magnesium chloride (MgCl2): Wako Pure Chemical Industries, Ltd.Dithiothreitol (DTT): Wako Pure Chemical Industries, Ltd.Tween 20: Sigma-AldrichEthylenediaminetetraacetic acid (EDTA): DOJINDO LABORATORIESThe compounds of the present invention, the enzymes (JAK1, JAK2, JAK3 and Tyk2), the substrate and ATP were used for assay as diluted with assay buffer.Â  As the assay buffer, one having the following composition was used.Â  HEPES (pH7.5): 50 mM EGTA: 1 mM MgCl2: 10 mM DTT: 2 mM Tween 20: 0.01% (wt/wt). The dilute concentration and the addition amount on a well plate as described hereinafter were adjusted so that the following final concentrations were achieved on the well plate.
Measurement: IC50 = 4.9 nM (pChEMBL 8.3100004196167)
Descriptors: MW 417.54, ALogP 2.05, HBA 5, HBD 2, PSA 98.4, QED 0.78
Compound (SMILES): CC1=C(N2CCC3(CCN(C(CO)Cc4ccc5c(c4C)COC5=O)CC3)C2=O)COC1
InChIKey: WDIVWQJNSTZVSP-UHFFFAOYSA-N
Target: ATP-sensitive inward rectifier potassium channel 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P48048
Assay: Thallium Flux Assay: The ROMK channel functional thallium flux assay is performed in 384 wells, using the FLIPR-Tetra instrument. HEK-hKir1.1 cells are seeded in Poly-D-Lysine microplates and kept in a 37° C.-10% CO2 incubator overnight. On the day of the experiment, the growth media is replaced with the FluxOR reagent loading buffer and incubated, protected from light, at ambient temperature (23-25° C.) for 90 min. The loading buffer is replaced with assay buffer+/-test compound followed by 30 min incubation at ambient temperature, where the Thallium/Potassium stimulant is added to the microplate.Step Protocol 1. Seed HEK-hKir1.1 cells (50 uA at 20,000 cells/well) in 384-well PDL coated Microplates 2. Allow cells to adhere overnight in humidified 37° C./10% CO2 incubator 3. Completely remove cell growth media from microplate and replace with 25 ul loading buffer 4. Incubate Microplate at room temperature, protected form light, for 90 min.
Measurement: IC50 = 70 nM (pChEMBL 7.159999847412109)
Descriptors: MW 440.54, ALogP 2.19, HBA 6, HBD 1, PSA 79.31, QED 0.71
Compound (SMILES): Nc1cccc(Nc2ncc(Br)c(Nc3cc(C4CC4)n[nH]3)n2)c1
InChIKey: LNMZHHWSSYEJRW-UHFFFAOYSA-N
Target: Insulin-like growth factor 1 receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P08069
Assay: Inhibition of human IGF1R kinase domain (M954 to C1367 residues) tri-phosphorylated enzyme form expressed in baculovirus based expression system using poly-EY peptide incubated for 2 hrs by luciferase-luciferin coupled chemiluminescent assay
Measurement: IC50 = 50 nM (pChEMBL 7.300000190734863)
Descriptors: MW 386.26, ALogP 3.91, HBA 6, HBD 4, PSA 104.54, QED 0.5
Compound (SMILES): COCCOCCOCc1cn(CCCC[C@H](NC(=O)N[C@@H](CCC(=O)O)C(=O)O)C(=O)O)nn1
InChIKey: BRDYNJDVRFBXJS-HOTGVXAUSA-N
Target: Diacylglycerol O-acyltransferase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: O75907
Assay: PMSA Inhibition Assay: For IC50 measurements, inhibitors were dissolved in Reaction Buffer containing 40 μM NAAG to a final volume of 100 μL. Then, 25 μL of this solution was transferred to each of three wells in a microtiter plate, and 5 μL aliquots were serially diluted into 20 μL of solution containing 40 μM NAAG over 10 wells (5-fold dilutions). Inhibitor concentration therefore ranged over 6 orders of magnitude in these experiments. rhPSMA (20 ng/mL in Reaction Buffer, 20 μL, R&D Research) was then added to each well. The plate was incubated at room temperature for 15 min, and then heated to 95 °C. for 3 minutes. The plate was allowed to cool to room temperature, and glutamic acid levels were measured using a commercially available Amplex® − Red Glutamic Acid/Glutamate Oxidase Assay Kit (Invitrogen). Fluorescence intensities were measured using a Synergy 2 multiwell plate reader (Biotek), fitted with excitation and emission filters of 545 nm and 590 nm, respectively. Ki values were obtained from IC50 values using the Cheng-Prusoff equation, a substrate concentration of 20 μM, and a Km value of 0.925 μM.
Measurement: IC50 = 40.8 nM (pChEMBL 7.389999866485596)
Descriptors: MW 503.51, ALogP -0.3, HBA 10, HBD 5, PSA 211.43, QED 0.15
Compound (SMILES): CC(C)Sc1ccc(S(=O)(=O)N2CCN(c3ccc(Cl)c(OCC(=O)O)c3)CC2)cc1
InChIKey: JDDNRTYTJOLZBD-UHFFFAOYSA-N
Target: Prostaglandin D2 receptor (SINGLE PROTEIN), Homo sapiens
UniProt: Q13258
Assay: Displacement of [3H]-PGD2 from PGD2 receptor in human platelet membranes after 2 hrs by micro beta scintillation counting analysis
Measurement: Ki = 3100 nM (pChEMBL 5.510000228881836)
Descriptors: MW 485.03, ALogP 3.81, HBA 6, HBD 1, PSA 87.15, QED 0.57
Compound (SMILES): O=C(c1ccccc1)c1ccc2c(/C=C/c3ccccc3)n[nH]c2c1
InChIKey: YPBBBWMWVRXIJU-SDNWHVSQSA-N
Target: Serine/threonine-protein kinase Chk1 (SINGLE PROTEIN), Homo sapiens
UniProt: O14757
Assay: Inhibition of full length human C-terminal 6His-tagged CHK1 expressed in baculovirus/insect expression system assessed as reduction in NADH oxidation using Syntide2 as substrate by pyruvate kinase/lactate dehydrogenase coupled assay
Measurement: Ki = 532 nM (pChEMBL 6.269999980926514)
Descriptors: MW 324.38, ALogP 4.96, HBA 2, HBD 1, PSA 45.75, QED 0.54
Compound (SMILES): COc1cc2c(cc1OC)C(=O)/C(=C/c1ccc(CN3CCN(C)CC3)c(O)c1)CO2
InChIKey: HVVULCYWWXFSFM-VCHYOVAHSA-N
Target: Amine oxidase [flavin-containing] B (SINGLE PROTEIN), Homo sapiens
UniProt: P27338
Assay: Inhibition of human recombinant MAO-B using kynuramine as substrate measured after 30 mins by fluorescence assay
Measurement: IC50 = 915 nM (pChEMBL 6.039999961853027)
Descriptors: MW 424.5, ALogP 2.82, HBA 7, HBD 1, PSA 71.47, QED 0.74
Compound (SMILES): CCOc1cc(=O)n(C)cc1-c1cc(NC(=O)Nc2ccc(C)cc2C)ccc1Oc1ccc(F)cc1F
InChIKey: HUFOXXDKNBRRJB-UHFFFAOYSA-N
Target: Bromodomain-containing protein 4 (SINGLE PROTEIN), Homo sapiens
UniProt: O60885
Assay: Time-Resolved Fluorescence Resonance Energy Transfer (TR-FRET) Assay: Assay method B: Compound dilution series were prepared in DMSO via an approximately 3-fold serial dilution from one of the following: 0.47 mM to 7.8 nM. Compound dilutions were added directly into white, low-volume assay plates (Perkin Elmer Proxiplate 384 Plus# 6008280) using a Labcyte Echo in conjunction with Labcyte Access and Thermo Multidrop CombinL robotics. Compounds were then suspended in eight microliters (μL) of 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) containing His-tagged bromodomain, Europium-conjugated anti-His antibody (Invitrogen PV5596) and Alexa-647-conjugated probe. The final concentration of 1X assay mixture for assay methods A, B and C contains 2% DMSO, 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) and compound concentration in the range of: 49.02 μM-15.63 nM for method A, 9.19 μM 150 pM for method B, and 0.92 μM 15 pM for method C. After a one-hour equilibration at room temperature, TR-FRET ratios were determined using an Envision multilabel plate reader (Ex 340, Em 495/520).
Measurement: Ki = 28.2 nM (pChEMBL 7.550000190734863)
Descriptors: MW 519.55, ALogP 6.78, HBA 5, HBD 2, PSA 81.59, QED 0.28
Compound (SMILES): COc1cc(F)cc(N(CCN)c2ccc3ncc(-c4cnn(C)c4)nc3c2)c1
InChIKey: AJGWZFROWJUKON-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 392.44, ALogP 3.27, HBA 7, HBD 1, PSA 82.09, QED 0.54
Compound (SMILES): N#Cc1cccc(-c2c[nH]c3ncnc(N4CCOC(c5nccs5)C4)c23)c1
InChIKey: TUPFGHYCHSDHDW-UHFFFAOYSA-N
Target: Leucine-rich repeat serine/threonine-protein kinase 2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q5S007
Assay: Lantha Screen Technology Assay: LRRK2 kinase activity was measured using Lantha Screen technology from Invitrogen. GST-tagged truncated LRRK2 from Invitrogen (Cat # PV4874) was incubated with a fluorescein-labeled peptide substrate based upon ezrin/radixin/moesin (ERM), also known as LRRKtide (Invitrogen cat #PR8976A), in the presence of a dose response of compound. Upon completion, the assay was stopped and detected with a terbium labeled anti-phospho-ERM antibody (Invitrogen, cat #PR8975A). The assay was carried out under the following protocol: 3 uL of a working solution of substrate (233 nM LRRKtide, 117 uM ATP) prepared in assay buffer (50 mM HEEPES, pH 7.5, 3 mM MgCl2, with 2 mM DTT and 0.01% Brij35 added fresh) was added to a low volume Greiner 384-well plate. The compound dose response was prepared by diluting compound to a top concentration of 3.16 mM in 100% DMSO and serial diluted by half-log in DMSO 11 times. Aliquots (3.5 uL) of the 100% DMSO dose response were mixed with 46.5 uL water.
Measurement: IC50 = 181 nM (pChEMBL 6.739999771118164)
Descriptors: MW 388.46, ALogP 3.53, HBA 7, HBD 1, PSA 90.72, QED 0.58
Compound (SMILES): Cc1cccc(C)c1NC(=O)c1cn2cc(-c3ccc(C4CCC(CC(=O)O)CC4)cc3)ccc2n1
InChIKey: HSXUOTMVAPMQKN-UHFFFAOYSA-N
Target: Diacylglycerol O-acyltransferase 1 (SINGLE PROTEIN), Mus musculus
UniProt: Q9Z2A7
Assay: Inhibition Assay: Â Assay buffer [20 mM HEPES (pH 7.4), 100 mM MgCl2 and 0.04% bovine serum albumin (BSA)] containing 500 uM of enzyme substrate (didecanoyl glycerol) and 7.5 uM radiolabeled acyl-CoA substrate ([14C]decanoyl-CoA) was added to each well of a phospholipid-coated radiometric assay plate. A small aliquot of cell lysate having human DGAT1 activity (5 ug/well) obtained in above, of human liver microsomes having human DGAT1 activity (5 ug/well), or of mouse liver microsomes having mouse DGAT1 activity (5 ug/well) was added to the assay plate to start the reaction, and the reaction was allowed to proceed for 60 minutes at 25° C. The reaction was terminated upon the addition of an equal volume (100 uL) of isopropanol. The plates were sealed, incubated overnight and counted the next morning on a liquid scintillation counter and luminometer.
Measurement: IC50 < 1 nM (pChEMBL 9)
Descriptors: MW 481.6, ALogP 6.62, HBA 4, HBD 2, PSA 83.7, QED 0.32
Compound (SMILES): O=C(Nc1ccc([C@H]2CCCNC2)cc1)c1ccc(Cl)cn1
InChIKey: FUSOYBOVDDVOIU-ZDUSSCGKSA-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 = 4.8 nM (pChEMBL 8.319999694824219)
Descriptors: MW 315.8, ALogP 3.45, HBA 3, HBD 2, PSA 54.02, QED 0.91
Compound (SMILES): C[C@@H](CCCc1ccccc1)[C@H](O)/C=C/[C@H]1CC(F)(F)C(=O)N1CCCc1ccc(C(=O)O)s1
InChIKey: UXQWNSWYEJSWBM-GJWBGHMGSA-N
Target: Prostaglandin E2 receptor EP4 subtype (SINGLE PROTEIN), Homo sapiens
UniProt: P35408
Assay: Radioligand Binding Assay: Assay Volume and Format:200 μl in 96-well plateCell membrane homogenates (20 μg protein) are incubated for 120 min at 22° C. with 0.5 nM [3H]PGE2 in the absence or presence of the test compound in a buffer containing 10 mM MES/KOH (pH 6.0), 10 mM MgCl2 and 1 mM EDTA.Nonspecific binding is determined in the presence of 10 M PGE2.Following incubation, the samples are filtered rapidly under vacuum through glass fiber filters (GF/B, Packard) presoaked with 0.3% PEI and rinsed several times with ice-cold 50 mM Tris-HCl using a 96-sample cell harvester (Unifilter, Packard). The filters are dried then counted for radioactivity in a scintillation counter (Topcount, Packard) using a scintillation cocktail (Microscint 0, Packard).
Measurement: IC50 = 0.74 nM (pChEMBL 9.130000114440918)
Descriptors: MW 491.6, ALogP 5.19, HBA 4, HBD 2, PSA 77.84, QED 0.4
Compound (SMILES): Cc1nc2cc(C3(O)c4ccccc4C(=O)N3Cc3ccccc3)ccc2[nH]1
InChIKey: VMVSXMARTHVYRB-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 >= 10000 nM (pChEMBL 5)
Descriptors: MW 369.42, ALogP 3.72, HBA 3, HBD 2, PSA 69.22, QED 0.58
Compound (SMILES): O=C(Nc1cc2cccnc2cc1O[C@@H]1CC[C@H](O)C1)c1csc2cncnc12
InChIKey: XPMRQKGHVCXNDM-UONOGXRCSA-N
Target: Tyrosine-protein kinase SYK (SINGLE PROTEIN), Homo sapiens
UniProt: P43405
Assay: In vitro SYK Kinase Assay: SYK activity is measured by phosphorylation of a peptide substrate (Biotin-EPEGDYEEVLE) with [gamma-33P] ATP. The enzyme reaction was conducted at 20 uM ATP with 0.05 uCi [gamma-33P]ATP (2 uCi for 40 ul assay) and 10 uM peptide substrate at final volume of 40 ul in buffer containing 50 mM Hepes, pH 7.2, 1 mM dithiothreitol, 10 mM MgCl2, 100 uM Na3VO4, 0.1% BSA and 10% DMSO. The enzyme assay was carried out with human full length SYK in the presence or absence of ten compound concentrations. SYK and compound were pre-incubated for 10 minutes. Then, the enzymatic reaction was initiated by addition of ATP and peptide substrate. The reaction mixture was incubated at room temperature for 30 minutes. At the end of incubation, the reaction was terminated by transferring 25 ul of the reaction mixture to 100 ul of 10% streptavidin slurry containing 100 mM EDTA. The reaction product was captured on the affinity resin and sequentially washed on a filtration plate (Millipore, MABVNOB50) with 2M NaCl, 2M M NaCl in 1% phosphoric acid and water to remove free radio nucleotide. Then the incorporation of 33P into peptide substrate was quantified on a microplate scintillation counter.
Measurement: IC50 = 500 nM (pChEMBL 6.300000190734863)
Descriptors: MW 406.47, ALogP 3.78, HBA 7, HBD 2, PSA 97.23, QED 0.54
Compound (SMILES): CC(=O)N[C@@H](Cc1ccccc1)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CO)C(=O)N[C@@H](CCCCN)C(=O)NCC(=O)N[C@@H](CC(N)=O)C(=O)N[C@H](C(=O)N[C@@H](Cc1ccccc1)C(=O)N[C@H](C(=O)N[C@@H](CC(=O)O)C(=O)NCC(=O)N[C@@H](Cc1ccc(O)cc1)C(=O)N[C@@H](Cc1ccccc1)C(=O)N[C@@H](CCC(=O)O)C(=O)N[C@@H](CCCNC(=N)N)C(=O)N[C@@H](CC(C)C)C(=O)NC(C)(C)C(=O)N[C@@H](C)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CS)C(N)=O)C(C)C)C(C)C
InChIKey: HBMZMDUWQNIFTD-URURHIFTSA-N
Target: Tissue factor pathway inhibitor (SINGLE PROTEIN), Homo sapiens
UniProt: P10646
Assay: Binding affinity to TFPI (unknown origin) incubated for 1.5 hrs using tracer peptide JBT0131 by screening ELISA
Measurement: IC50 = 45 nM (pChEMBL 7.349999904632568)
Descriptors: MW 2447.85
Compound (SMILES): OC1CN(c2cncc(-c3cc4c(cn3)cnn4-c3cccc(N4CCNCC4)n3)n2)C1
InChIKey: LVCFJRVHJZAYQL-UHFFFAOYSA-N
Target: Serine/threonine-protein kinase pim-2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9P1W9
Assay: Kinase Binding Assay: PIM-1, -2, and -3 enzymes were generated as fusion proteins expressed in bacteria and purified by IMAC column chromatography (Sun, X., Chiu, J. F., and He, Q. Y. (2005) Expert Rev. Proteomics, 2:649-657). A fluorescent-labeled Pim-specific peptide substrate, was custom synthesized by American Peptide Company (Sunnyvale, Calif.). Reaction Buffer contained 10 mM HEPES, pH 7.2, 10 mM MgCl2, 0.01% Tween 20, 2 mM DTT. Termination Buffer contained 190 mM HEPES, pH 7.2, 0.015% Brij-35, 0.2% Coating Reagent 3 (Caliper Life Sciences, Hopkinton, Mass.), 20 mM EDTA. Separation Buffer contained 100 mM HEPES, pH 7.2, 0.015% Brij-35, 0.1% Coating Reagent 3, 1:200 Coating Reagent 8 (Caliper Life Sciences, Hopkinton, Mass.), 10 mM EDTA and 5% DMSO.PIM reactions were carried out in a final volume of 10 uL per well in a 384-well plate. A standard enzymatic reaction, initiated by the addition of 5 uL 2Ã¿Â¿ATP and test compound to 5 uL of 2x enzyme and FAM-peptide, contained 20 uM PIM1, 50 uM PIM2, or 55 uM PIM3, 1 uM FAM-peptide, and 10 uM ATP, in Reaction Buffer. After 90 minutes of incubation at room temperature, the phosphorylation reaction was stopped by the addition of 10 uL Termination Buffer. The product and substrate in each independent reaction were separated on a 12-sipper microfluidic chip (Caliper Life Sciences, Hopkinton, Mass.) run on a Caliper LC3000 (Caliper Life Sciences, Hopkinton, Mass.). The separation of product and substrate was optimized by choosing voltages and pressure using Caliper's Optimizer software (Hopkinton, Mass.). The separation conditions used a downstream voltage of -500V, an upstream voltage of -2150V, and a screening pressure of -1.2 psi. The product and substrate fluorophore were excited at 488 nm and detected at 530 nm. Substrate conversion was calculated from the electropherogram using HTS Well Analyzer software (Caliper Life Sciences, Hopkinton, Mass.). Ki values for the test compound were calculated.
Measurement: Ki = 3.37 nM (pChEMBL 8.470000267028809)
Descriptors: MW 429.49, ALogP 0.86, HBA 10, HBD 2, PSA 108.12, QED 0.49
Compound (SMILES): CCS(=O)(=O)c1ccc(Oc2cccc(CO)c2)c(-c2cn(C)c(=O)c3[nH]ccc23)c1
InChIKey: UAHRULIEGJEKKJ-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 = 3.6 nM (pChEMBL 8.4399995803833)
Descriptors: MW 438.51, ALogP 3.61, HBA 6, HBD 2, PSA 101.39, QED 0.48
Compound (SMILES): Cc1ccc(NC(=O)c2cc(C(C)(C)C)no2)cc1-c1cc(N2CCOCC2)nc(N2CCOCC2)n1
InChIKey: PNGGYMKHJMOIPI-UHFFFAOYSA-N
Target: Serine/threonine-protein kinase B-raf (SINGLE PROTEIN), Homo sapiens
UniProt: P15056
Assay: In Vitro Enzyme Assay: Final assay conditions were 4 pM cRaf, 3 uM ATP, 10 nM biotin tagged MEK1 kinase dead protein substrate. Reactions were performed in Greiner384 well plates, catalog #784075. Reaction buffer was 50 mM Tris, pH 7.5, 50 mM NaCl, 10 mM MgCl2, 0.01% Tween-20, 0.05% BSA and 1 mM DTT. 5 ul of 2×cRaf plus MEK1 were dispensed into plates containing 0.25 ul of compound in 100% DMSO and incubated at room temperature for 30 minutes. Reactions were started by addition of 5 ul of 2×ATP. Reactions were run for two hours and terminated by addition of 10 ul of 2× antibody and Alphascreen bead mixture in Stop Buffer. Stop Buffer was 50 mM Tris, pH 7.5, 0.005% Tween-20, 25 mM EDTA.
Measurement: IC50 = 1.356 nM (pChEMBL 8.869999885559082)
Descriptors: MW 506.61, ALogP 3.66, HBA 9, HBD 1, PSA 105.85, QED 0.55
Compound (SMILES): CC(C)c1cncc(-c2cc3c(cn2)cnn3-c2cccc(N3CCCNCC3)n2)c1
InChIKey: BKZIXMFZEWLONB-UHFFFAOYSA-N
Target: Serine/threonine-protein kinase pim-2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9P1W9
Assay: Kinase Binding Assay: PIM-1, -2, and -3 enzymes were generated as fusion proteins expressed in bacteria and purified by IMAC column chromatography (Sun, X., Chiu, J. F., and He, Q. Y. (2005) Expert Rev. Proteomics, 2:649-657). A fluorescent-labeled Pim-specific peptide substrate, was custom synthesized by American Peptide Company (Sunnyvale, Calif.). Reaction Buffer contained 10 mM HEPES, pH 7.2, 10 mM MgCl2, 0.01% Tween 20, 2 mM DTT. Termination Buffer contained 190 mM HEPES, pH 7.2, 0.015% Brij-35, 0.2% Coating Reagent 3 (Caliper Life Sciences, Hopkinton, Mass.), 20 mM EDTA. Separation Buffer contained 100 mM HEPES, pH 7.2, 0.015% Brij-35, 0.1% Coating Reagent 3, 1:200 Coating Reagent 8 (Caliper Life Sciences, Hopkinton, Mass.), 10 mM EDTA and 5% DMSO.PIM reactions were carried out in a final volume of 10 uL per well in a 384-well plate. A standard enzymatic reaction, initiated by the addition of 5 uL 2Ã¿Â¿ATP and test compound to 5 uL of 2x enzyme and FAM-peptide, contained 20 uM PIM1, 50 uM PIM2, or 55 uM PIM3, 1 uM FAM-peptide, and 10 uM ATP, in Reaction Buffer. After 90 minutes of incubation at room temperature, the phosphorylation reaction was stopped by the addition of 10 uL Termination Buffer. The product and substrate in each independent reaction were separated on a 12-sipper microfluidic chip (Caliper Life Sciences, Hopkinton, Mass.) run on a Caliper LC3000 (Caliper Life Sciences, Hopkinton, Mass.). The separation of product and substrate was optimized by choosing voltages and pressure using Caliper's Optimizer software (Hopkinton, Mass.). The separation conditions used a downstream voltage of -500V, an upstream voltage of -2150V, and a screening pressure of -1.2 psi. The product and substrate fluorophore were excited at 488 nm and detected at 530 nm. Substrate conversion was calculated from the electropherogram using HTS Well Analyzer software (Caliper Life Sciences, Hopkinton, Mass.). Ki values for the test compound were calculated.
Measurement: Ki = 2.76 nM (pChEMBL 8.5600004196167)
Descriptors: MW 413.53, ALogP 3.8, HBA 7, HBD 1, PSA 71.76, QED 0.55
Compound (SMILES): Cn1ccc2c(-c3cnc(OCCC4CCNCC4)c(C(F)(F)F)c3)nc(C#N)nc21
InChIKey: FPHDGXFXSNDLKM-UHFFFAOYSA-N
Target: Cathepsin S (SINGLE PROTEIN), Mus musculus
UniProt: O70370
Assay: Inhibition Assay: First, spleen cells collected from C57BL/6 mice were seeded in a 96-well plate so as to be a 1× 105 cells/well, then, 5-fold dilution of 10 mM DMSO solution of the test compound was serially performed with RPMI 1640 medium (including 10% fetal bovine serum (FCS), 5×10^−5 M 2-mercaptoethanol, 50 IU/mL penicillin and 50 μg/mL streptomycin) nine times such that the final concentration becomes 0.026 nM to 10 μM, and the resultant products were added thereto (the final DMSO concentration was 0.1%). At the same time, LPS (Sigma L4005) was added to the wells such that the final concentration becomes 2 μg/mL, and culture was performed at 37° C. for 48 hours in 5% CO2. After culturing, the cells were stained at 4° C. for 20 minutes with PE-labeled streptavidin (BD BIOSCIENCE 554061) and biotin-labeled YAe antibody (EBIOSCIENCE 13-5741-85) together with FITC-labeled anti-mouse B220 antibody (BD BIOSCIENCE 553088), and an expression level (fluorescence intensity of YAe-bioti
Measurement: IC50 = 1.8 nM (pChEMBL 8.739999771118164)
Descriptors: MW 430.43, ALogP 3.69, HBA 7, HBD 1, PSA 88.65, QED 0.66