lovingscience/chembl-v37-raw-activities huggingface dataset:
Compound (SMILES): NC(=O)c1ccc2c(c1O)[C@]13CCN(CC4CCC4)[C@H](C2)[C@@H]1CCC(=O)C3
InChIKey: WXCUTWYVZVSZGP-SVMVAKDDSA-N
Target: Mu-type opioid receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P35372
Assay: null: The Ki (binding affinity) for u opioid receptors was determined using a competitive displacement assay as previously described in Neumeyer (Journal of Med. Chem. 2012, p 3878), which is incorporated herein in its entirety. Briefly, membrane protein from CHO (Chinese Hamster Ovarian) cells that stably expressed the cloned human u opioid receptor were incubated with 12 different concentrations of the compound set forth herein in the presence of 0.25 nM [3H]DAMGO (see Tiberi et al., Can. J. Physiol. Pharmacol. 1988, Vol. 66, p 1368, which is incorporated by reference herein in its entirety) in a final volume of 1 mL of 50 mM Tris-HCl, pH 7.5 at 25° C. Incubation times of 60 min were used for [3H]DAMGO (see Gulati et al., Life Sci. 1990, Vol. 47, p 159, which is incorporated by reference herein in its entirety). Nonspecific binding was measured by inclusion of 10 uM naloxone. The binding was terminated by filtering the samples through Schleicher & Schuell No. 32 glass fiber.
Measurement: IC50 = 1.1 nM (pChEMBL 8.960000038146973)
Descriptors: MW 368.48, ALogP 2.53, HBA 4, HBD 2, PSA 83.63, QED 0.86
Compound (SMILES): c1ccc2sc(NC3=NCC4(CN5CCC4CC5)O3)nc2c1
InChIKey: VUSUESCSIUWQMT-UHFFFAOYSA-N
Target: 5-hydroxytryptamine receptor 3A (SINGLE PROTEIN), Homo sapiens
UniProt: P46098
Assay: Antagonist activity at human 5-HT3A expressed in HEK293 cells assessed as reduction in acetylcholine-induced activity preincubated for 30 mins followed by addition of acetylcholine measured for 2 mins by Fluo-4AM dye based FLIPR assay
Measurement: IC50 = 21 nM (pChEMBL 7.679999828338623)
Descriptors: MW 314.41, ALogP 2.56, HBA 6, HBD 1, PSA 49.75, QED 0.88
Compound (SMILES): O=C(Nc1ccc(N2CCN(C(=O)c3ccc[nH]3)CC2)cc1)N1Cc2ccccc2C1
InChIKey: NRTOKRLWCYRPAS-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 = 24.3 nM (pChEMBL 7.610000133514404)
Descriptors: MW 415.5, ALogP 3.52, HBA 3, HBD 2, PSA 71.68, QED 0.69
Compound (SMILES): O=C(O)c1ccc(NCCOc2ccc3cc2COC/C=C/COCc2cccc(c2)-c2ccnc(n2)N3)cc1
InChIKey: HVGFTTQGMMUWGW-OWOJBTEDSA-N
Target: Protein deacetylase HDAC6 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9UBN7
Assay: Inhibition of full length human recombinant N-terminal GST-tagged HDAC6 (1 to 1215 residues) expressed in sf9 cells preincubated with enzyme followed by fluorogenic Arg-His-Lys-Lys(Ac)-AMC substrate addition measured after 2 hrs by fluorescence assay
Measurement: IC50 = 64000 nM (pChEMBL 4.190000057220459)
Descriptors: MW 538.6, ALogP 5.68, HBA 8, HBD 3, PSA 114.83, QED 0.21
Compound (SMILES): CC1(C)CCC(CN2CCN(c3ccc(C(=O)NS(=O)(=O)c4ccc(N[C@@H]5CCN(CC(F)F)C5)c([N+](=O)[O-])c4)c(Oc4cccc5[nH]cnc45)c3)CC2)=C(c2ccc(Cl)cc2)C1
InChIKey: FOTGOGLEQJRYAN-JGCGQSQUSA-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 = 74.56 nM (pChEMBL 7.130000114440918)
Descriptors: MW 903.45, ALogP 8.57, HBA 11, HBD 3, PSA 166.04, QED 0.07
Compound (SMILES): CCCCN(CCCC)C(=O)c1nn(-c2ccc(C(=O)NS(=O)(=O)c3ccc4c(c3)c(Cl)cn4CC)cc2C(=O)N2CCc3ccccc3C2)c(C)c1Cl
InChIKey: WUSYFHJUCZLPFK-UHFFFAOYSA-N
Target: Bcl-2-like protein 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q07817
Assay: Fluorescence Resonance Energy Transfer Assay (FRET): The assays were performed in black flat-bottom 384-well plates. The final assay volume was 55 μl prepared from additions of Biotin-Bcl-xL (Bcl-xL: GENBANK® Accession No. Q07817), fluoresceinated 18-mer BIM peptide (NH2-YYANFEDGIRRLEQAIWI-[FAM]) (SEQ ID NO: 1), Streptavidin-Terbium FRET detection reagent, and test compounds in assay buffer consisting of 20 mM Sodium Phosphate, 1 mM EDTA, 50 mM NaCl, and 0.05% Pluronic Acid. The reaction was incubated at room temperature for 60 minutes. After 60 minutes, 5 μL of Streptavidin-Terbium FRET detection reagent (Perkin Elmer) was added to the reaction mixture and incubated at room temperature in the dark for 30 mins. The FRET signal generated by the reaction was detected on the Envision Plate Reader Inhibition data were calculated from FRET values generated by the no protein control reactions for 100% inhibition and vehicle-only reactions for 0% inhibition. The final concentration of reagents in the assay was 10 nM Bcl-xL, 5 nM fluo
Measurement: IC50 = 40 nM (pChEMBL 7.400000095367432)
Descriptors: MW 791.8, ALogP 7.82, HBA 8, HBD 1, PSA 126.61, QED 0.13
Compound (SMILES): NCCOc1c(-c2ccc(N3CCOCC3)cc2)[nH]c(=O)c2cc(F)ccc12
InChIKey: KSTGCHRYORWWOO-UHFFFAOYSA-N
Target: Poly [ADP-ribose] polymerase 2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9UGN5
Assay: Biochemical Assay: The identification of compounds capable of binding several PARP proteins is carried out through a screening method including the steps of  a) providing a reaction mixture containing:  b) comparing the polarization signal generated in the absence of the test compound with the one generated in the presence of different concentrations of the test compound, and  c) evaluating the ability of the test compound to displace the compound of formula (IP) as defined above indicated from a decreased fluorescence polarization level.The polarization signal can be measured, e.g., by a plate reader such as the Saphire2 (Tecan).
Measurement: Kd = 7200 nM (pChEMBL 5.139999866485596)
Descriptors: MW 383.42, ALogP 2.51, HBA 5, HBD 2, PSA 80.58, QED 0.71
Compound (SMILES): CC(C)Oc1ccc(S(=O)(=O)N2CCN(c3cc(Cl)cc(OCC(=O)O)c3)CC2)cc1
InChIKey: MAIBWIPFPQMJRH-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 = 630 nM (pChEMBL 6.199999809265137)
Descriptors: MW 468.96, ALogP 3.1, HBA 6, HBD 1, PSA 96.38, QED 0.64
Compound (SMILES): N#CCC1(n2ccc(-c3ncnc4[nH]ccc34)c2)CN(C2CCN(C(=O)c3cccc(C#N)c3)CC2)C1
InChIKey: FDEXCMVSAHLSDJ-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 > 50 nM (pChEMBL 7.301000118255615)
Descriptors: MW 490.57, ALogP 3.53, HBA 7, HBD 1, PSA 117.63, QED 0.46
Compound (SMILES): CCOc1n[nH]c2cc(NC(=O)N[C@@H](c3ccc(F)cc3)C(C)(C)O)ncc12
InChIKey: DVTYGBADWQQMFP-INIZCTEOSA-N
Target: Mitogen-activated protein kinase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P28482
Assay: IMAP-FP Assay: Activated ERK2 activity was determined in an IMAP-FP assay (Molecular Devices). Using this assay format, the potency (IC50) of each compound was determined from a 10 point (1:3 serial dilution, 3 uM starting compound concentration) titration curve using the following outlined procedure. To each well of a black Corning 384-well plate (Corning Catalog #3575), 7.5 nL of compound (3333 fold dilution in final assay volume of 25 uL) was dispensed, followed by the addition of 15 uL of kinase buffer (tween containing kinase buffer, Molecular Devices) containing 0.0364 ng/mL (0.833 nM) of phosphorylated active hERK2 enzyme. Following a 15 minute compound & enzyme incubation, each reaction was initiated by the addition of 10 uL kinase buffer containing 2.45 uM ERK2 IMAP substrate peptides (2.25 uM-unlabeled IPTTPITTTYFFFK-COOH and 200 nM-labeled IPTTPITTTYFFFK-SFAM (5-carboxyfluorescein)-COOH), and 75 uM ATP.
Measurement: IC50 = 0.7 nM (pChEMBL 9.149999618530273)
Descriptors: MW 387.42, ALogP 3.13, HBA 5, HBD 4, PSA 112.16, QED 0.52
Compound (SMILES): Cc1c(C(=O)Nc2cccc(C(F)(F)F)c2)nc(-c2ccccc2)n1CCCN1CCOCC1
InChIKey: UMWLUXMSHVOKGA-UHFFFAOYSA-N
Target: Transient receptor potential cation channel subfamily V member 4 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9HBA0
Assay: Inhibition Assay: Test Example 1: (1) The TRPV4 inhibitory activity was measured using CHO-K1 cells stably expressing human TRPV4 (hTRPV4/CHO cells). (2) In the day before the experimental day, frozen cell were thawed and washed with the culture medium (MEM-α, 10% FBS, 2 mmol/L GlutaMax, 50 unit Penicillin, 50 μg/mL Streptomycin). Then cells were suspended in the culture medium. (3) The 384-well plates which hTRPV4/CHO cells were seeded at densities of 4000 cells/well in the culture medium and cultured in a CO2 incubator in the presence of 5% CO2 at 37° C. for overnight was used as assay plates. (4) The assay plate was washed with assay buffer (Hanks, 20 mmol/L HEPES, 2.5 mmol/L probenecid, pH7.4), and the buffer were remained at 20 μL/well. (5) 10 μl of dye loading buffer (9 μmol/L Fluo 4-AM, 0.09% Pluronic F-127/assay buffer) was added to each well, then the assay plate was incubated in a CO2 incubator in the presence of 5% CO2 at 37° C. for one hour. (final 3 μmol/L Fluo4-AM). (6) The assay plate was washed with the assay buffer, and the buffers were remained at 20 μL/well. Then the assay plate was incubated for 10 min at room temperature. (7) Diluted compound solution was dispensed to each well in the compound plate, and mixed with built-in Pipette and Mixer of the Fluorescence analysis system FLIPR TETRA (Molecular devices). (8) After incubation for 5 min, 20 μL of 4α-PDD solution were applied to each well of assay plate and mixed with the FLIPR TETRA. (final 1 Î¼mol/L 4α-PDD). (9) The fluorescent intensity was measured with FLIPR TETRA system for 10 min from the point of time addition the compound solution, at Ex 470-495 nm, Em 515-575 nm wavelength.
Measurement: IC50 = 710 nM (pChEMBL 6.150000095367432)
Descriptors: MW 472.51, ALogP 4.85, HBA 5, HBD 1, PSA 59.39, QED 0.53
Compound (SMILES): O=C(Nc1ccc(OC(F)(F)F)cc1)c1cnc(N2CC[C@H](O)C2)c(-c2cncnc2)c1
InChIKey: LARFZNXVNANWFD-INIZCTEOSA-N
Target: Tyrosine-protein kinase ABL1 (SINGLE PROTEIN), Homo sapiens
UniProt: P00519
Assay: Caliper ABL : The assay plates were prepared by addition of 50 nL per well of compound solution in 90% DMSO. The kinase reactions were started by stepwise addition of 4.5 uL per well of peptide/ATP-solution (50 mM HEPES, pH 7.5, 1 mM DTT, 0.02% BSA, 0.6% DMSO, 10 mM beta-glycerophosphate, and 10 uM sodium orthovanadate, 20 mM MgCl2, 2 mM MnCl2, 4 uM ATP, 4 uM peptide (FITC-Ahx-EAIYAAPFAKKK-NH2)) and 4.5 uL per well of enzyme solution (50 mM HEPES, pH 7.5, 1 mM DTT, 0.02% BSA, 0.6% DMSO, 10 mM beta-glycerophosphate, and 10 uM sodium orthovanadate, 20 mM MgCl2, 2 mM MnCl2, 3.5 nM ABL (ABL(64-515), produced in-house from E. coli)). Kinase reactions were incubated at 30° C. for 60 minutes and subsequently terminated by addition of 16 uL per well of stop solution (100 mM HEPES pH 7.5, 5% DMSO, 0.1% Caliper coating reagent, 10 mM EDTA, and 0.015% Brij35). Plates with terminated kinase reactions were transferred to the Caliper LC3000 workstations for reading. Phosphorylated and unphosphorylated peptides were separated using the Caliper microfluidic mobility shift technology.
Measurement: IC50 = 4.8 nM (pChEMBL 8.319999694824219)
Descriptors: MW 445.4, ALogP 3.26, HBA 7, HBD 2, PSA 100.47, QED 0.62
Compound (SMILES): COC(=O)[C@@H](Cc1cc2ccnc(N)c2cc1OC(F)(F)F)N1CC[C@H](NS(=O)(=O)c2cnc(N3CCCC3)s2)C1=O
InChIKey: LTEGXTZXBWOHGG-ZWKOTPCHSA-N
Target: Coagulation factor X (SINGLE PROTEIN), Homo sapiens
UniProt: P00742
Assay: Inhibition Assay: The compounds according to the invention are tested in a range of concentrations (10pM to 10 µM final) in assay buffer (50 mM TRIS, 100 mM NaCl, 0.1 % BSA, pH 7,5) with 0.1% maximal final DMSO concentration and deposited in a proportion of 25 µl per well, on 25 µL enzyme (human coagulation factor Xa: Enzyme Research Laboratories HFXa, final concentration 0.003Ul/ml). The reactive were mixed, centrifuged and incubated 10 minutes at 37°C in a 96 well microtiter plate. The enzyme reaction was started with 50 µL substrate (S-2765, Biogenic ref 821413 in a final concentration of 62.5µM final). The time course of the reaction was monitored at 405 nm in a microtiter plate reader (Tecan M200) for 20 minutes at 37°C. The percentage of inhibition of enzymatic activity (expressed as maximum rate of cleavage of the substrate) is calculated with respect to the enzymatic activity in the absence of inhibitor.
Measurement: IC50 = 1120 nM (pChEMBL 5.949999809265137)
Descriptors: MW 628.65, ALogP 2.44, HBA 11, HBD 2, PSA 157.05, QED 0.34
Compound (SMILES): COC(=O)Nc1nc2cc(C3(O)c4ccccc4C(=O)N3c3cccc(C4CCNCC4)c3)ccc2[nH]1
InChIKey: NQIDKYDDEPOOGC-UHFFFAOYSA-N
Target: RAF proto-oncogene serine/threonine-protein kinase (SINGLE PROTEIN), Homo sapiens
UniProt: P04049
Assay: Inhibition of c-Raf1 (unknown origin) pre-incubated for 30 mins by luciferase-luciferin-coupled chemiluminescence assay
Measurement: IC50 = 100 nM (pChEMBL 7)
Descriptors: MW 497.56, ALogP 4.06, HBA 6, HBD 4, PSA 119.58, QED 0.34
Compound (SMILES): COc1cccc(OC)c1C1CC(C)C(=O)N1Cc1cccc(-c2noc(C)n2)c1
InChIKey: LQQRWVVSBRNSJK-UHFFFAOYSA-N
Target: Orexin receptor type 2 (SINGLE PROTEIN), Homo sapiens
UniProt: O43614
Assay: In Vitro Assay: Intracellular Calcium Measurements : In Vitro Assay: Intracellular Calcium Measurements:Chinese hamster ovary (CHO) cells expressing the human orexin-1 receptor and the human orexin-2 receptor, respectively, are grown in culture medium (Ham F-12 with L-Glutamine) containing 300 g/ml G418, 100 U/ml penicillin, 100 g/ml streptomycin and 10% heat inactivated fetal calf serum (FCS). The cells are seeded at 20,000 cells/well into 384-well black clear bottom sterile plates (Greiner). The seeded plates are incubated overnight at 37° C. in 5% CO2. Human orexin-A as an agonist is prepared as 1 mM stock solution in MeOH:water (1:1), diluted in HBSS containing 0.1% bovine serum albumin (BSA), NaHCO3: 0.375 g/l and 20 mM HEPES for use in the assay at a final concentration of 3 nM.Antagonists are prepared as 10 mM stock solution in DMSO, then diluted in 384-well plates using DMSO followed by a transfer of the dilutions into in HBSS containing 0.1% bovine serum albumin (BSA), NaHCO3: 0.375 g/l and 20 mM HEPES. On the day of the assay, 50 μl of staining buffer (HBSS containing 1% FCS, 20 mM HEPES, NaHCO3: 0.375 g/l, 5 mM probenecid (Sigma) and 3 M of the fluorescent calcium indicator fluo-4 AM (1 mM stock solution in DMSO, containing 10% pluronic) is added to each well. The 384-well cell-plates are incubated for 50 min at 37° C. in 5% CO2 followed by equilibration at RT for 30 min before measurement.
Measurement: IC50 = 46 nM (pChEMBL 7.340000152587891)
Descriptors: MW 407.47, ALogP 4.17, HBA 6, HBD 0, PSA 77.69, QED 0.61
Compound (SMILES): NC(=O)c1ccc(NCc2cccc(Cl)c2)c2cncnc12
InChIKey: WSEAJWAXWQGKPA-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 ul 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 12.50 0.1 ng/uL 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 uM 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 ul 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 = 12000 nM (pChEMBL 4.920000076293945)
Descriptors: MW 312.76, ALogP 2.99, HBA 4, HBD 2, PSA 80.9, QED 0.78
Compound (SMILES): Cc1cc(NC(=O)Nc2ccc(Cl)c(C(C)(F)F)c2)ccc1Oc1ccnc(NC(=O)C2CC2)c1
InChIKey: CFMORAKHQKGHJV-UHFFFAOYSA-N
Target: Vascular endothelial growth factor receptor 2 (SINGLE PROTEIN), Homo sapiens
UniProt: P35968
Assay: Inhibition Assay: [Experimental method]: Use Lance@Ultra Ulight™-TK assay kit from PerKinElmer to evaluate the inhibitory activity of compounds at KDR kinase. [Instrument]: PerKinElmer's ENVISION plate reader. [Materials]: Optiplate-384 well plate (PerKinElmer), kinase buffer (50 mM Hepes pH7.5, 0.25 mM EGTA, 2 mM DTT, 0.01% Tween 20, 10 mM Mg2+, 0.5 mM Mn2+), KDR kinase (790-1356 AA, Crown Bioscience), KDR kinase substrate (PerKinElmer catalogue # TRF0127-M), Lance@Eu-W1024-anti-phosphotyrosine (PT66) (PerKinElmer, catalogue #AD0068), ATP (Invitrogen), DMSO (Sigma, catalogue #34869), purified water (Millipore, type: Milli-Q). [Study conditions]: Mix KDR (final concentration: 20 nM) and compound (final DMSO: 0.5%), pre-incubate for 20 min at 30° C.; then add ATP (final concentration: 90 μM) and the substrate (final concentration 50 μM). React for 2 hrs at 30° C. After the reaction, add antibody and reaction for 60 min at 30° C. Read the plate (615 nm, 665 nm), calculate the ratio of the value at 665 nm vs 615 nm and analyze the data.
Measurement: IC50 = 9.5 nM (pChEMBL 8.020000457763672)
Descriptors: MW 500.93, ALogP 6.94, HBA 4, HBD 3, PSA 92.35, QED 0.33
Compound (SMILES): CN[C@@H](C)Cn1cc(NC(=O)c2cnn3cccnc23)c(-c2cc(Cl)ccc2OC(F)F)n1
InChIKey: VPXLZWSMTPLGSL-LBPRGKRZSA-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 = 1.25 nM (pChEMBL 8.899999618530273)
Descriptors: MW 475.89, ALogP 3.71, HBA 8, HBD 2, PSA 98.37, QED 0.4
Compound (SMILES): CN1CCc2cc(N(C(=O)c3cc(-c4cc5c(cc4C(=O)N4Cc6ccccc6C[C@H]4CN4CCOCC4)OCO5)n4ccccc34)c3ccc(O)cc3)ccc21
InChIKey: SIJWBAYDEANRLU-DHUJRADRSA-N
Target: Apoptosis regulator Bcl-2 (SINGLE PROTEIN), Homo sapiens
UniProt: P10415
Assay: Fluorescence Polarisation Assay: The fluorescence polarisation tests were carried out on microplates (384 wells). The Bcl-2 protein, at a final concentration of 2.50×10−8 M, is mixed with a fluorescent peptide (Fluorescein-REIGAQLRRMADDLNAQY), at a final concentration of 1.00×10−8 M in a buffer solution (Hepes 10 mM, NaCl 150 mM, Tween20 0.05%, pH 7.4), in the presence or in the absence of increasing concentrations of test compounds. After incubation for 2 hours, the fluorescence polarisation is measured.
Measurement: IC50 = 22 nM (pChEMBL 7.659999847412109)
Descriptors: MW 761.88, ALogP 6.91, HBA 9, HBD 1, PSA 99.43, QED 0.19
Compound (SMILES): CC1(C)CN(c2cncc(N3CCC4(CC3)CNC(=O)C4)n2)C1
InChIKey: XQBNNOFAFYZWBB-UHFFFAOYSA-N
Target: Cytochrome P450 11B1, mitochondrial (SINGLE PROTEIN), Homo sapiens
UniProt: P15538
Assay: Inhibition of human CYP11B1-8C7 expressed in chinese hamster V79 cells using 11-deoxycortisol as substrate preincubated for 1 hr followed by substrate addition measured for 3 hrs by HTRF assay
Measurement: IC50 = 1739 nM (pChEMBL 5.760000228881836)
Descriptors: MW 315.42, ALogP 1.43, HBA 5, HBD 1, PSA 61.36, QED 0.89
Compound (SMILES): CCS(=O)(=O)c1cc(C#N)ccc1C1C2=C(CCC2=O)N(c2ccnc(C(F)(F)F)c2)C(=O)N1C
InChIKey: ATCYXNWGEBMATL-UHFFFAOYSA-N
Target: Neutrophil elastase (SINGLE PROTEIN), Homo sapiens
UniProt: P08246
Assay: Human Neutrophil Elastase Assay: The following buffers were used: Compound buffer: 100 mM Tris, 500 mM NaCl, adjusted to pH 7.5; Assay buffer: 100 mM Tris, 500 mM NaCl, adjusted to pH 7.5, containing 0.01% BSA.Assay conditions: Test compounds were prediluted in DMSO and subsequently in compound buffer (5% DMSO final). 5 μL of these compound dilutions were mixed with 10 μl Neutrophil elastase (9 ng/ml in assay buffer) in a black 384 well OptiPlate (Perkin Elmer, Cat No.: 6007270) and incubated for 15 min at room temperature. Subsequently 10 μL substrate solution in assay buffer were added (250 μM final concentration) and the plates were incubated for 60 min at room temperature. After inactivation of the enzyme, fluorescence intensities were measured at 380 nm excitation and 460 nm emission wavelengths.
Measurement: IC50 = 1.4 nM (pChEMBL 8.850000381469727)
Descriptors: MW 504.49, ALogP 4, HBA 6, HBD 0, PSA 111.44, QED 0.62
Compound (SMILES): N#Cc1ccc(CN2CCC(c3nnn4cnc5[nH]cc(Cl)c5c34)CC2)cc1
InChIKey: LWXYBHBSDREWSX-UHFFFAOYSA-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 = 17 nM (pChEMBL 7.769999980926514)
Descriptors: MW 391.87, ALogP 3.51, HBA 6, HBD 1, PSA 85.9, QED 0.58
Compound (SMILES): Cc1ccc2nc(NC(=O)N3CCN(c4ncc([C@H](O)[C@H](C)O)cc4F)CC3)sc2c1
InChIKey: WLFKITVCNICDEW-SCLBCKFNSA-N
Target: Transient receptor potential cation channel subfamily V member 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q8NER1
Assay: pH-based Assay: pH dependent Ca2+ responses in TRPV1/CHO cells cultured in a 96-well plate were determined (see, e.g., FIG. 2 of U.S. Patent Application Publication No. US 2009/0170868 A1). In particular, Ca2+ influx into TRPV1/CHO cells in response to low pH as measured by Fura-2 AM fluorescence was determined. The cells were stimulated using 3.0 mM (well numbers B1-B6), 3.1 mM (C1-C6), 3.2 mM (D1-D6), 3.3 mM (E1-E6), 3.4 mM (F1-F6), 3.5 mM (G1-G6), or 3.6 mM (H1-H6) H2SO4 or pH 7.2 measuring buffer without H2SO4 (A1-A6). Culture medium was removed using an 8-channel-pipette (Rainin, USA) from the 96-well plate and the wells were refilled with 100 uL of loading buffer (20 mM HEPES, 115 mM NaCl, 5.4 mM KCl, 0.8 mM MgCl2, 1.8 mM CaCl2, 13.8 mM D-glucose, 2.5 mM probenecid, pH 7.4) containing 5 uM Fura-2 AM (Dojin, Japan). The 96-well plate was incubated at 37° C. for 45 min. The loading buffer was removed from each well. The cells were subsequently washed twice with 150 uL of measuring buffer (20 mM HEPES, 115 mM NaCl, 5.4 mM KCl, 0.8 mM MgCl2, 5.0 mM CaCl2, 13.8 mM D-glucose, 0.1% BSA, pH 7.4) (no probenecid). The wells were then refilled with 80 uL of measuring buffer. After an incubation at 4° C. for 15 min, the 96-well plate was transferred to a model FDSS-3000 plate reader apparatus (Hamamatsu Photonics K.K., Japan). The Fura-2 fluorescent intensity was monitored at a wavelength of 340 nm and at 380 nm, respectively, at a rate of 0.5 Hz for a total of 240 seconds. After 16 time points (32 sec) of baseline detection, 20 uL of agonist solution was added to each well. The final volume was 100 uL/well. Fluorescence intensity ratio refers to the fluorescence intensity at 340 nm over the fluorescence intensity at 380 nm at a particular time point. The baseline was set as the average of the fluorescent intensity ratios for the first 16 time points before the addition of agonist solution. The maximum response was the highest fluorescent intensity ratio during the 60 time points following addition of agonist solution.
Measurement: IC50 = 67 nM (pChEMBL 7.170000076293945)
Descriptors: MW 445.52, ALogP 2.91, HBA 7, HBD 3, PSA 101.82, QED 0.57
Compound (SMILES): CCCN(CCC)C(=O)C1=Cc2ccc(C(=O)Nc3ccc(CCCCN)nc3)cc2N=C(N)C1
InChIKey: HKPNKAYHHNWTBL-UHFFFAOYSA-N
Target: Toll-like receptor 7 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9NYK1
Assay: Pharmacological Assay: For TLR8 and TLR7 activity testing, HEK-Blue human TLR8 or TLR7 cells (Invivogen, San Diego, Calif., USA) are used, respectively. These cells are designed for studying the stimulation of human TLR8 or TLR7 by monitoring the activation of NF-κB. A SEAP (secreted embryonic alkaline phosphatase) reporter gene is placed under the control of the IFN-b minimal promoter fused to five NF-κB and AP-1-binding sites. Therefore the reporter expression is regulated by the NF-κB promoter upon stimulation of human TLR8 or TLR7 for 20 hours. The cell culture supernatant SEAP reporter activity was determined using Quanti Blue kit (Invivogen, San Diego, Ca, USA) at a wavelength of 640 nm, a detection medium that turns purple/blue in the presence of alkaline phosphatase.
Measurement: EC50 > 100000 nM (pChEMBL 4)
Descriptors: MW 476.63, ALogP 4.04, HBA 6, HBD 3, PSA 126.7, QED 0.42
Compound (SMILES): OCc1cccc(-c2c[nH]c3ncnc(N4CCOCC4)c23)c1
InChIKey: WSFKANMHWWIKSD-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 = 19 nM (pChEMBL 7.71999979019165)
Descriptors: MW 310.36, ALogP 1.95, HBA 5, HBD 2, PSA 74.27, QED 0.77
Compound (SMILES): CCCc1nn(C)c2c(=O)[nH]c(-c3cc(N4CCCC5(CCC(C(=O)NO)CC5)C4)ccc3OCC)nc12
InChIKey: LECHQHPOTZRDEW-UHFFFAOYSA-N
Target: Histone deacetylase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q13547
Assay: Inhibition of full length recombinant human C-terminal His/Flag-tagged HDAC1 expressed in baculovirus infected Sf9 cells using fluorogenic HDAC substrate after 30 mins by fluorescence analysis
Measurement: IC50 = 9270 nM (pChEMBL 5.03000020980835)
Descriptors: MW 522.65, ALogP 3.96, HBA 8, HBD 3, PSA 125.37, QED 0.32
Compound (SMILES): O=C(N[C@H]1CC[C@H](CCN2CCC(c3noc4cc(F)ccc34)CC2)CC1)c1ccc(N2CCOCC2)nn1
InChIKey: RJFINFRRQCTSTE-JKIUYZKVSA-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 = 25.33 nM (pChEMBL 7.599999904632568)
Descriptors: MW 536.65, ALogP 4.15, HBA 8, HBD 1, PSA 96.62, QED 0.48
Compound (SMILES): Cc1nc(-c2cccnc2)sc1C(=O)Nc1cn2nc(-c3cn(C)nc3-c3ccc(F)cc3)ccc2n1
InChIKey: ZSQXBJVHGCHBDR-UHFFFAOYSA-N
Target: Casein kinase I isoform delta (SINGLE PROTEIN), Homo sapiens
UniProt: P48730
Assay: Kinase Assay: The kinase assay was performed in V-bottom 384-well plates. The final assay volume was 30 ul prepared from 15 ul additions of enzyme, substrates (fluoresceinated peptide FL-AHA-KRRRAL-PSER-VASLPGL-OH and ATP) and test compounds in assay buffer (20 mM HEPES pH 7.4, 30 mM MgCl2, 0.015% Brij35 and 4 mM DTT). The reaction was incubated at room temperature for 22 hours and terminated by adding 45 ul of 35 mM EDTA to each sample. The reaction mixture was analyzed on the Caliper LABCHIPÂ®3000 (Caliper, Hopkinton, Mass.) by electrophoretic separation of the unphosphorylated substrate and phosphorylated product Inhibition data were calculated by comparison of the no enzyme control reactions for 100% inhibition and vehicle-only reactions for 0% inhibition. The final concentration of reagents in the assay were 200 pM CK1ε or CK1δ, 50 uM ATP, 1.5 uM FL-AHA-KRRRAL-PSER-VASLPGL-OH, and 1.6% DMSO. Dose response curves were generated to determine the concentration required to inhibit 50% of the kinase activity (IC50). Compounds were dissolved at 10 mM in dimethylsulfoxide (DMSO) and evaluated at eleven concentrations. IC50 values were derived by non-linear regression analysis.
Measurement: IC50 = 1.2 nM (pChEMBL 8.920000076293945)
Descriptors: MW 510.56, ALogP 5.02, HBA 9, HBD 1, PSA 102.89, QED 0.35
Compound (SMILES): CCCc1n[nH]c2c1C(c1ccc(OC)c(OC)c1)c1c(nc3c(c1N)CCCC3)O2
InChIKey: KVQYRPWLNMXJJF-UHFFFAOYSA-N
Target: Cholinesterase (SINGLE PROTEIN), Homo sapiens
UniProt: P06276
Assay: Inhibition of human recombinant BChE using butyrylthiocholine iodide as substrate incubated for 5 mins followed by substrate addition measured for 1 min by Ellman's method
Measurement: IC50 = 365 nM (pChEMBL 6.440000057220459)
Descriptors: MW 420.51, ALogP 4.52, HBA 6, HBD 2, PSA 95.28, QED 0.49
Compound (SMILES): CC(C)C1c2ncn(-c3cnccn3)c2CCN1C(=O)c1cccc(C(F)(F)F)c1Cl
InChIKey: RHCOFPRUWCRZDY-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 = 103.8 nM (pChEMBL 6.980000019073486)
Descriptors: MW 449.86, ALogP 4.73, HBA 5, HBD 0, PSA 63.91, QED 0.58
Compound (SMILES): CN1CCC(Nc2ccc(S(=O)(=O)NC(=O)c3ccc(N4CCN(CC5=C(c6ccc(Cl)cc6)CCC5)CC4)cc3Oc3cccc4[nH]ccc34)cc2[N+](=O)[O-])CC1
InChIKey: WXJAPUWTZHRPLV-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 824.4, ALogP 7.91, HBA 10, HBD 3, PSA 153.15, QED 0.08
Compound (SMILES): CC(C)Nc1cncc(-c2c[nH]c3ccc(-c4nc(N)no4)cc23)n1
InChIKey: ROJNKYJPUZLUTF-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 = 58.88 nM (pChEMBL 7.230000019073486)
Descriptors: MW 335.37, ALogP 3.08, HBA 7, HBD 3, PSA 118.54, QED 0.52
Compound (SMILES): CCN1CC(C)(C)Oc2nc(N3CC4CCC(C3)O4)nc(-c3ccc(NC(=O)Nc4ccc(OC5CCN(C)CC5)nc4)c(F)c3)c2C1=O
InChIKey: FNLHVEWGLIZNDP-UHFFFAOYSA-N
Target: Phosphatidylinositol 3-kinase regulatory subunit alpha (SINGLE PROTEIN), Homo sapiens
UniProt: P27986
Assay: Enzyme Assay: The test uses a lucifeurerin/luciferase system to measure the concentration of ATP and its consumption during the enzymatic reaction. The test is performed in 96-well format (Corning/Costar 96 black flat-bottomed half-wells plate, ref. 3694) in a total volume of 30 μl. To 1 μl of inhibitor in 100% DMSO are added (final concentrations) 50 μM of the substrate PIP2 ((L-α-phosphatidyl-D-myoinositol 4,5-bisphosphate, Echelon 117P-4516-0500), 2 μM of ATP and 1.7 μg/ml of PI3Kα, (p110α/p85α, Invitrogen PV4788) in a buffer of Tris/HCl 50 mM pH 7.5, EGTA 1 mM, MgCl2 10 mM, Chaps 0.03%, 1 mM DTT). After 90 minutes, the reaction is quenched by adding 20 μl/well of KinaseGlo reagent (Promega V6713). After 10 minutes in the dark, the luminescence is read on the PHERAStar microplate reader (reading at 0.8 sec/well).
Measurement: IC50 = 3070 nM (pChEMBL 5.510000228881836)
Descriptors: MW 674.78, ALogP 4.8, HBA 10, HBD 2, PSA 134.28, QED 0.36
Compound (SMILES): C=CC(=O)Nc1cccc(Nc2nc(Nc3ccc(OC)c(OC)c3OC)ncc2Br)c1
InChIKey: UKFLBAQMJNODAB-UHFFFAOYSA-N
Target: Epidermal growth factor receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P00533
Assay: Omnia Assay: Briefly, 10x stocks of EGFR-WT (PV3872) from Invitrogen and EGFR-T790M/L858R (40350) from BPS Bioscience, San Diego, Calif., 1.13x ATP (AS001A) and appropriate Tyr-Sox conjugated peptide substrates (KCZ1001) were prepared in 1x kinase reaction buffer consisting of 20 mM Tris, pH 7.5, 5 mM MgCl2, 1 mM EGTA, 5 mM β-glycerophosphate, 5% glycerol (10x stock, KB002A) and 0.2 mM DTT (DS001A). 5 uL of each enzyme were pre-incubated in a Corning (#3574) 384-well, white, non-binding surface microtiter plate (Corning, N.Y.) for 30 min. at 25° C. with a 0.5 uL volume of 50% DMSO and serially diluted compounds prepared in 50% DMSO. Kinase reactions were started with the addition of 45 uL of the ATP/Tyr-Sox peptide substrate mix and monitored every 71 seconds for 60 minutes at λex360/λem485 in a Synergy4 plate reader from BioTek (Winooski, Vt.). At the conclusion of each assay, progress curves from each well were examined for linear reaction kinetics and fit statistics.
Measurement: IC50 = 65 nM (pChEMBL 7.190000057220459)
Descriptors: MW 500.35, ALogP 4.88, HBA 8, HBD 3, PSA 106.63, QED 0.36
Compound (SMILES): NC(=O)C[C@H](NC(=O)[C@@H]1CCC(=O)N1)C(=O)N1CCC[C@H]1C(N)=O
InChIKey: LLRIQFIHWHSMMB-CIUDSAMLSA-N
Target: Thyrotropin-releasing hormone receptor (SINGLE PROTEIN), Rattus norvegicus
UniProt: Q01717
Assay: Displacement of [3H]-3MeHis-TRH from TRH receptor in Sprague-Dawley rat cortical membranes
Measurement: Ki = 7079.46 nM (pChEMBL 5.150000095367432)
Descriptors: MW 339.35, ALogP -2.9, HBA 5, HBD 4, PSA 164.69, QED 0.41
Compound (SMILES): COC(=O)CSCCCS[C@H]1C(=O)C(C)(C)[C@@H](O)[C@@H]1/C=C/C(O)CCc1sc2ccccc2c1Cl
InChIKey: LFQLFJPHMATZPF-IACHTARMSA-N
Target: Prostaglandin E2 receptor EP4 subtype (SINGLE PROTEIN), Homo sapiens
UniProt: P35408
Assay: Radioligand Binding: HEK-293 cells stably expressing the human or feline FP receptor, or EP1, EP2, EP3, or EP4 receptors were washed with TME buffer, scraped from the bottom of the flasks, and homogenized for 30 sec using a Brinkman PT 10/35 polytron. TME buffer was added to achieve a final 40 ml volume in the centrifuge tubes (the composition of TME is 100 mM TRIS base, 20 mM MgCl2, 2M EDTA; 10 N HCl is added to achieve a pH of 7.4).The cell homogenate was centrifuged at 19000 r.p.m. for 20 min at 4 C. using a Beckman Ti-60 rotor. The resultant pellet was resuspended in TME buffer to give a final 1 mg/ml protein concentration, as determined by Biorad assay. Radioligand binding competition assays vs. [3H-]17-phenyl PGF2 (5 nM) were performed in a 100 ul volume for 60 min. Binding reactions were started by adding plasma membrane fraction. The reaction was terminated by the addition of 4 ml ice-cold TRIS-HCl buffer and rapid filtration through glass fiber GF/B filters using a Brandel cell.
Measurement: IC50 = 98 nM (pChEMBL 7.010000228881836)
Descriptors: MW 557.2, ALogP 5.39, HBA 8, HBD 2, PSA 83.83, QED 0.21
Compound (SMILES): CCCCN(C)C(=O)c1cc(-c2n[nH]c(=O)n2-c2ccccc2)c(O)cc1O
InChIKey: PHYBXAQDXCJFDV-UHFFFAOYSA-N
Target: Heat shock protein HSP 90-alpha (SINGLE PROTEIN), Homo sapiens
UniProt: P07900
Assay: Recombinant Hsp90 [His-Tev-huHsp90-alpha (9-236)] was immobilized on a Biacore CM5 chip at 25 degrees C and a flow rate of 30 uL/min using amine coupling at pH 4.50 according to Biacore standard protocol. Hsp90 was applied at a concentration of 20 ug/mL with 50uM 17-DMAG.Data sets were processed and analyzed using the software Biacore 4000 Evaluation. Solvent corrected and double-referenced association and dissociation phase data were fitted to a simple 1:1 interaction model with mass transport limitations
Measurement: Kd = 8.801 nM (pChEMBL 8.050000190734863)
Descriptors: MW 382.42, ALogP 2.51, HBA 6, HBD 3, PSA 111.45, QED 0.61
Compound (SMILES): Cc1ccc(C2=C(C#N)C(=O)NC(c3ccc(OCCCC(F)(F)F)cc3)(C(F)(F)F)C2)cc1F
InChIKey: FGFXVWOTSZPHLE-UHFFFAOYSA-N
Target: Alpha-1,6-mannosyl-glycoprotein 2-beta-N-acetylglucosaminyltransferase (SINGLE PROTEIN), Homo sapiens
UniProt: Q10469
Assay: MGAT LCMS Assay: The MGAT enzyme reactions were performed in CORNING Falcon 96-well Polypropylene plates, in a total volume of 60 uL of 50 mM Potassium Phosphate buffer pH 7.4, containing a final concentration of 100 uM 2-oleoylglycerol, 15 uM oleoyl-Coenzyme A and 0.0013 ug/uL Human or Mouse MGAT-2 or 0.0026 ug/uL Rat recombinant MGAT-2 membranes expressed in Sf9 cells. Assay plates were run through a fully automated robotics system and shaken for 5 seconds every minute for a total 10 minutes. The reactions were then quenched with 120 uL of ice cold methanol containing 1 ug/mL 1,2-distearoyl-rac-glycerol as the internal standard. Plates were shaken for 2 minutes and spun down to remove protein precipitation. After the spin, samples were transferred to LC/MS compatible PCR plates. For LC/MS analysis, a ThermoFisher Surveyor pump, utilizing a Waters Symmetry C8, 50x2.1 mm column, was used for the chromatography of enzyme products.
Measurement: IC50 = 22 nM (pChEMBL 7.659999847412109)
Descriptors: MW 500.41, ALogP 6.11, HBA 3, HBD 1, PSA 62.12, QED 0.38
Compound (SMILES): O=C(Cc1ccccc1)N1CCN(c2ccc(NC(=O)N3Cc4ccccc4C3)cc2)CC1
InChIKey: LHLGIZOHWVIKBW-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 = 41.7 nM (pChEMBL 7.380000114440918)
Descriptors: MW 440.55, ALogP 4.13, HBA 3, HBD 1, PSA 55.89, QED 0.66
Compound (SMILES): CNC(=O)Cn1cc(-c2cnc3ccc(N(CCCO)c4cc(OC)cc(OC)c4)cc3n2)cn1
InChIKey: FRFIDSQIINSGEA-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 476.54, ALogP 2.78, HBA 9, HBD 2, PSA 114.63, QED 0.36
Compound (SMILES): Brc1cnc(NCc2cc(-c3ccccc3)no2)nc1Nc1cc(C2CCCC2)n[nH]1
InChIKey: YYKGOTKXCAOTNQ-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 = 500 nM (pChEMBL 6.300000190734863)
Descriptors: MW 480.37, ALogP 5.63, HBA 7, HBD 3, PSA 104.55, QED 0.31
Compound (SMILES): Cc1cc(Nc2nn(C3(CC#N)CCN(C(=O)OC(C)(C)C)CC3)cc2C(N)=O)ncn1
InChIKey: XITGQWNXBUDAGU-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 = 126 nM (pChEMBL 6.900000095367432)
Descriptors: MW 440.51, ALogP 2.46, HBA 9, HBD 2, PSA 152.05, QED 0.72
Compound (SMILES): NC(=O)c1cccc([C@H]2C[C@H]3CC[C@@H](C2)N3CCN(Cc2cccc(F)c2)C(=O)CO)c1.O=C(O)C(F)(F)F
InChIKey: WFRXRRDAYNWHBH-XWFSNYJISA-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 439.53, ALogP 2.66, HBA 4, HBD 2, PSA 86.87, QED 0.66