lovingscience/chembl-v37-raw-activities huggingface dataset:
Compound (SMILES): CCO[C@H]1CC[C@]2(CC1)Cc1ccc(Br)cc1C21N=C(C)C(N)=N1
InChIKey: BGVASHDMRXAQIW-DUWYVTJWSA-N
Target: Beta-secretase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P56817
Assay: TR-FRET Assay: The beta-secretase enzyme used in the TR-FRET is prepared as follows: The cDNA for the soluble part of the human beta-Secretase (AA 1-AA 460) was cloned using the ASP2-Fc10-1-IRES-GFP-neoK mammalian expression vector. The gene was fused to the Fc domain of IgG1 (affinity tag) and stably cloned into HEK 293 cells. Purified sBACE-Fc was stored in -80 C. in Tris buffer, pH 9.2 and had a purity of 40%.The enzyme (truncated form) was diluted to 6 ug/mL (stock 1.3 mg/mL) and the substrate (Europium)CEVNLDAEFK(Qsy7) to 200 nM (stock 120 uM) in reaction buffer (NaAcetate, chaps, triton x-100, EDTA pH4.5). The robotic systems Biomek FX and Velocity 11 were used for all liquid handling and the enzyme and substrate solutions were kept on ice until they were placed in the robotic system. Enzyme (9 ul) was added to the plate then 1 ul of compound in dimethylsulphoxide was added, mixed and pre-incubated for 10 minutes. Substrate (10 ul) was then added, mixed.
Measurement: IC50 = 842 nM (pChEMBL 6.079999923706055)
Descriptors: MW 390.33, ALogP 3.96, HBA 4, HBD 1, PSA 59.97, QED 0.83
Compound (SMILES): O=C1OCCN1c1cc(Nc2nccc(C(F)(F)F)n2)cc(-c2ccc([C@@H](O)C(F)F)cc2)c1
InChIKey: XUGAGRWXIPMYGA-GOSISDBHSA-N
Target: Tyrosine-protein kinase SYK (SINGLE PROTEIN), Homo sapiens
UniProt: P43405
Assay: Homogeneous Time-Resolved Fluorescence (HTRF) Assay: A recombinant GST-hSYK fusion protein was used to measure potency of compounds to inhibit human Syk activity. The recombinant human GST-SYK (Carna Biosciences #08-176) (5 μM final concentration) was incubated with various concentrations of the inhibitor diluted in DMSO (0.1% final concentration) for 10 minutes at room temperature in 15 mM Tris-HCl (pH 7.5), 0.01% tween 20, 2 mM DTT in 384 well plate format. To initiate the reaction the biotinylated substrate peptide (250 nM final concentration) that contains the phosphorylation site for Syk was added with magnesium (5 mM final concentration) and ATP (25 μM final concentration). Final volume of the reaction was 10 μL. Phosphorylation of the peptide was allowed to proceed for 45' at room temperature. To quench the reaction and detect the phosphorylated product, 2 nM of a Europium-anti-phosphotyrosine antibody (Perkin Elmer #AD0161) and 70 nM SA-APC (Perkin-Elmer #CR130-100) were added together in 15 mM Tris pH 7.5, 40 mM EDTA, 0.01% tween 20. Final volume of the quenching solution was 10 μL. The resulting HTRF signal was measured after 30 minutes on an EnVision (Perkin-Elmer) reader using a time-resolved fluorescence protocol.
Measurement: IC50 = 43.7 nM (pChEMBL 7.360000133514404)
Descriptors: MW 480.39, ALogP 5.16, HBA 6, HBD 2, PSA 87.58, QED 0.47
Compound (SMILES): NC1=N[C@@]2(c3ccc(F)cc3F)CO[C@@H](c3ccccc3F)C[C@H]2CS1
InChIKey: GNYNEASZTBNKNJ-JVMOKEISSA-N
Target: Beta-secretase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P56817
Assay: BACE1 Cell-Free Assay: A synthetic APP substrate that can be cleaved by beta-secretase having N-terminal biotin and made fluorescent by the covalent attachment of Oregon Green at the Cys residue is used to assay beta-secretase activity in the presence or absence of the inhibitory compounds. The substrate is Biotin-GLTNIKTEEISEISY^EVEFR-C[Oregon Green]KK-OH. The BACE1 enzyme is affinity purified material from conditioned media of CHO-K1 cells that have been transfected with a soluble BACE construct (BACE1deltaTM96His). Compounds are incubated in a log dose response curve from a top concentration of 100 uM with BACE1 enzyme and the biotinylated fluorescent peptide in 384-well black plates (Thermo Scientific #4318). BACE1 is at a final concentration of 0.1 nM with a final concentration of peptide substrate of 150 nM in a reaction volume of 30 uL assay buffer [100 mM sodium acetate, pH 4.5 (brought to pH with acetic acid), and 0.001% Tween-20]. Plates are covered and incubated for 3 hours at 37° C. The reaction is stopped with the addition of 30 uL of 1.5 uM Streptavidin (Pierce, #21125). After a 10 minute incubation at room temperature, plates are read on a PerkinElmer EnVision for fluorescence polarization (Ex485 nm/Em530 nm). The activity of the beta-secretase enzyme is detected by changes in the fluorescence polarization that occur when the substrate is cleaved by the enzyme. Incubation in the presence of compound inhibitor demonstrates specific inhibition of beta-secretase enzymatic cleavage of the synthetic APP substrate.
Measurement: IC50 = 86 nM (pChEMBL 7.070000171661377)
Descriptors: MW 378.42, ALogP 4.14, HBA 4, HBD 1, PSA 47.61, QED 0.85
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](Cc1ccccc1)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)N[C@@H](CCCNC(=N)N)C(=O)N[C@@H](C)C(=O)N[C@@H](CCCCN)C(=O)N(C)[C@@H](CC(C)C)C(N)=O)C(C)C)C(C)C
InChIKey: CDNHUWREEODNFN-FLYJGWEFSA-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 = 891 nM (pChEMBL 6.050000190734863)
Descriptors: MW 2461.9
Compound (SMILES): CNS1(O)C[C@@](C)(c2cc(NC(=O)c3ccc(F)cn3)ccc2F)NC(=N)C1(C)C
InChIKey: RXLGQBLPZKLQKA-FQEVSTJZSA-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 = 10 nM (pChEMBL 8)
Descriptors: MW 437.52, ALogP 3.6, HBA 5, HBD 5, PSA 110.13, QED 0.5
Compound (SMILES): C[C@H]1C[C@H]2CSC(N)=NC2(c2nc(NC(=O)c3ccn(C(F)F)n3)cs2)CO1
InChIKey: RODMHPIORBBNPR-IJLCECLYSA-N
Target: Beta-secretase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P56817
Assay: BACE1 Cell-Free Assay: The substrate is Biotin-GLTNIKTEEISEISY^EVEFR-C[Oregon Green]KK-OH. The BACE1 enzyme is affinity purified material from conditioned media of CHO-K1 cells that have been transfected with a soluble BACE construct (BACE1deltaTM96His). Compounds are incubated in a ½ log dose response curve from a top concentration of 100 μM with BACE1 enzyme and the biotinylated fluorescent peptide in 384-well black plates (Thermo Scientific #4318). BACE1 is at a final concentration of 0.1 nM with a final concentration of peptide substrate of 150 nM in a reaction volume of 30 μL assay buffer [100 mM sodium acetate, pH 4.5 (brought to pH with acetic acid), and 0.001% Tween-20]. Plates are covered and incubated for 3 hours at 37 °C. The reaction is stopped with the addition of 30 μL of 1.5 μM Streptavidin (Pierce, #21125). After a 10 minute incubation at room temperature, plates are read on a PerkinElmer EnVision for fluorescence polarization (Ex485 nm/Em530 nm).
Measurement: IC50 = 19 nM (pChEMBL 7.71999979019165)
Descriptors: MW 428.49, ALogP 2.67, HBA 9, HBD 2, PSA 107.42, QED 0.78
Compound (SMILES): CCCCN(Cc1ccc(OCCn2c(=O)ccn(C)c2=O)c(OC)c1)C(CC(=O)O)c1ccc2c(c1)CCO2
InChIKey: IKSIQQMWYSWFTD-UHFFFAOYSA-N
Target: C-X-C chemokine receptor type 3 (SINGLE PROTEIN), Homo sapiens
UniProt: P49682
Assay: Displacement of [125I]-CXCL10 from human recombinant CXCR3 transfected in Flp-In-CHO cell membranes after 60 mins by gamma counting method
Measurement: IC50 = 122 nM (pChEMBL 6.909999847412109)
Descriptors: MW 551.64, ALogP 3.39, HBA 9, HBD 1, PSA 112.23, QED 0.32
Compound (SMILES): Cn1cc(-c2cc(NS(C)(=O)=O)ccc2OC2CCCCC2)c2cc[nH]c2c1=O
InChIKey: ZKMZBCLSLVYACG-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 = 0.88 nM (pChEMBL 9.0600004196167)
Descriptors: MW 415.52, ALogP 3.62, HBA 5, HBD 2, PSA 93.19, QED 0.67
Compound (SMILES): CC1CCCCN1CCCNC(=O)c1ccc(-c2cc(NC3CC3)n3ncc(/C=C4\NC(=O)NC4=O)c3n2)s1
InChIKey: ZSAQPHNDHIVGPZ-MOSHPQCFSA-N
Target: Casein kinase II subunit beta (SINGLE PROTEIN), Homo sapiens
UniProt: P67870
Assay: Inhibition Assay: 5 uM ATP: Test compounds dissolved and diluted in DMSO (2 μl) were added to a reaction mixture comprising 10 μl of 5× Reaction Buffer (40 mM MOPS pH 7.0, 5 mM EDTA), 10 μl of recombinant human PIM2 solution (4 ng PIM-2 dissolved in dilution buffer (20 mM MOPS pH 7.0; EDTA 1 mM; 5% Glycerol; 0.01% Brij 35; 0.1%; 0.1% 2-mercaptoethanol; 1 mg/ml BSA)) and 8 ul of water. Reactions were initiated by the addition of 10 ul of ATP Solution (49% (15 mM MgCl2; 5 uM ATP) 1% ([γ-33P]ATP: Stock 1 mCi/100 μl; 3000 Ci/mmol (Perkin Elmer)) and 10 ul of substrate peptide solution (RSRSSYPAGT (SEQ ID NO.: 6), dissolved in water at a concentration of 1 mM), Reactions were maintained for 10 min at 30° C. The reactions were quenched with 100 ul of 0.75% Phosphoric acid, then transferred to and filtered through a Phosphocellulose filter plate (Millipore, MSPH-N6B-50). After washing each well 4 times with 0.75% Phosphoric acid, scintillation fluid (20 uL) was added to each well and
Measurement: IC50 > 2500 nM (pChEMBL 5.6020002365112305)
Descriptors: MW 548.67, ALogP 3.21, HBA 9, HBD 4, PSA 132.76, QED 0.18
Compound (SMILES): Cc1nc(NC(=O)N2CCC(N3CC(CC#N)(n4cc(-c5ncnc6[nH]ccc56)cn4)C3)CC2)sc1C
InChIKey: FHAOWDSJKGGBCZ-UHFFFAOYSA-N
Target: Tyrosine-protein kinase JAK1 (SINGLE PROTEIN), Homo sapiens
UniProt: P23458
Assay: Inhibition of human N-terminal His-tagged JAK1 catalytic domain (837 to 1142 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 > 5 nM (pChEMBL 8.300999641418457)
Descriptors: MW 516.64, ALogP 3.52, HBA 9, HBD 2, PSA 131.65, QED 0.41
Compound (SMILES): NC(=O)N1CCC(C(=O)N2CC[C@](c3ccc(C(OCc4c(F)cccc4F)(C(F)(F)F)C(F)(F)F)cc3)(S(=O)(=O)c3ccc(F)cc3)C2)CC1
InChIKey: ITFHMWNKBWVAFU-PMERELPUSA-N
Target: Nuclear receptor ROR-gamma (SINGLE PROTEIN), Homo sapiens
UniProt: P51449
Assay: Scintillation Proximity Assay (SPA): The binding of potential ligands to RORγ is measured by competition with [3H] 25-hydroxycholesterol (Perkin Elmer NET674250UC) using a scintillation proximity assay (SPA) binding assay. The ligand binding domain of human RORγ (A262-S507) with an N-terminal His tag is expressed in E. coli and purified using nickel affinity chromatography. 15 ug/well RORγ (A262-S507) is incubated with test compound at varying concentrations in 3-fold serial dilution, with final concentrations ranging from 16.6 uM to 0.28 nM for 10 min at room temperature in PBS buffer (Invitrogen #14190-144) containing 0.5% fatty acid free BSA (Gemini Bio-Products, Cat. #700-107P) and 0.1% Glycerol (Sigma Cat#G5516). 10 nM of [3H] 25-hydroxycholesterol is then added, and the reaction is incubated for 10 min. 10 mg/mL of Copper-His Tag-PVT beads (Perkin Elmer cat #RPNQ0095) are added, and the mixture is incubated for 60 min. The reaction is read on a TopCount Microplate scintillation plate reader (Perkin Elmer). The competition data of the test compound over a range of concentrations was plotted as percentage inhibition of radioligand specifically bound in the absence of test compound (percent of total signal). After correcting for non-specific binding, IC50 values were determined. The IC50 value is defined as the concentration of test compound needed to reduce [3H] 25-hydroxycholesterol specific binding by 50% and is calculated using the four parameter logistic equation to fit the normalized data.
Measurement: IC50 = 79 nM (pChEMBL 7.099999904632568)
Descriptors: MW 751.67, ALogP 6.33, HBA 5, HBD 1, PSA 110.01, QED 0.21
Compound (SMILES): CN1CCN(c2cc(NCCN3CCCCC3)c3c4c(onc24)-c2ccccc2C3=O)CC1
InChIKey: HQXMLEOWBROEKL-UHFFFAOYSA-N
Target: Histone-lysine N-methyltransferase EHMT2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q96KQ7
Assay: Inhibition of G9a (unknown origin) using biotinylated-histone H3 (1 to 21 residues)/S-adenosyl-methionine as substrate/methyl donor after 3 hrs by AlphaScreen assay
Measurement: IC50 = 1870 nM (pChEMBL 5.730000019073486)
Descriptors: MW 445.57, ALogP 3.69, HBA 7, HBD 1, PSA 64.85, QED 0.5
Compound (SMILES): Cn1c(=O)c2c(CC(=O)Nc3nc(-c4ccc(I)cc4)cs3)cccc2n(C)c1=O
InChIKey: GBWICTTWLYIICR-UHFFFAOYSA-N
Target: Transient receptor potential cation channel subfamily A member 1 (SINGLE PROTEIN), Homo sapiens
UniProt: O75762
Assay: Radiometric Calcium Assay: The inhibition of TRPA1 receptor activation was measured as inhibition of allylisothiocyanate (AITC) induced cellular uptake of radioactive calcium. Test compounds were dissolved in DMSO to prepare 10 mM stock solution and then diluted using plain medium with 0.1% BSA and 1.8 mM CaCl2 to get desired concentration. Final concentration of DMSO in the reaction was 0.5% (v/v). Human TRPA1 expressing CHO cells were grown in F-12 DMEM medium with 10% FBS, 1% penicillin-streptomycin solution, and 400 ug/ml of G-418. Cells were seeded 24 h prior to the assay in 96 well plates so as to get 50,000 cells per well on the day of experiment. Cells were treated with test compounds for 10 min followed by addition of AITC at a final concentration of 30 micromolar and 5 uCi/ml 45Ca+2 for 3 min at 25° C. Cells were washed and lysed using buffer containing 1% Triton X-100, 0.1% deoxycholate and 0.1% SDS. Radioactivity in the lysate was measured in Packard Top count.
Measurement: IC50 = 66.28 nM (pChEMBL 7.179999828338623)
Descriptors: MW 532.36, ALogP 3.15, HBA 7, HBD 1, PSA 85.99, QED 0.41
Compound (SMILES): CC(C)C[C@H](NC(=O)c1cccc(-c2cccc(Cl)c2)n1)c1nccs1
InChIKey: VOWWDTZOXKOIRE-SFHVURJKSA-N
Target: Cannabinoid receptor 2 (SINGLE PROTEIN), Homo sapiens
UniProt: P34972
Assay: cAMP Assay: CHO cells expressing human CB1 or CB2 receptors are seeded 17-24 hours prior to the experiment 50.000 cells per well in a black 96 well plate with flat clear bottom (Corning Costar #3904) in DMEM (Invitrogen No. 31331), 1.times.HT supplement, with 10% fetal calf serum and incubated at 5% CO2 and 37° C. in a humidified incubator. The growth medium was exchanged with Krebs Ringer Bicarbonate buffer with 1 mM IBMX and incubated at 30.degree. C. for 30 min Compounds were added to a final assay volume of 100 ul and incubated for 30 min at 30° C. Using the cAMP-Nano-TRF detection kit the assay (Roche Diagnostics) was stopped by the addition of 50 ul lysis reagent (Tris, NaCl, 1.5% Triton X100, 2.5% NP40, 10% NaN.sub.3) and 50 ul detection solutions (20 uM mAb Alexa700-cAMP 1:1, and 48 uM Ruthenium-2-AHA-cAMP) and shaken for 2 h at room temperature. The time-resolved energy transfer is measured by a TRF reader (Evotec Technologies GmbH), equipped with a ND:YAG laser as excitation source. The plate is measured twice with the excitation at 355 nm and at the emission with a delay of 100 ns and a gate of 100 ns, total exposure time 10 s at 730 (bandwidth 30 nm) or 645 nm (bandwidth 75 nm), respectively. The FRET signal is calculated.
Measurement: EC50 = 173.3 nM (pChEMBL 6.760000228881836)
Descriptors: MW 385.92, ALogP 5.38, HBA 4, HBD 1, PSA 54.88, QED 0.62
Compound (SMILES): Cc1cc(CNc2nc(Nc3cc(C4CC4)[nH]n3)cc(N3CC4CC3CN4C)n2)on1
InChIKey: GXKBWVNTEUMNOU-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) un-phosphorylated apo-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 421.51, ALogP 2.62, HBA 9, HBD 3, PSA 111.03, QED 0.53
Compound (SMILES): COc1ccc(C(=O)Nc2ccc3c(c2)[C@@]2(CCOC(N)=N2)c2cc(C4=CCOCC4)nc(F)c2O3)nc1
InChIKey: ZXVUTPMCWBMAHM-MHZLTWQESA-N
Target: Beta-secretase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P56817
Assay: Fluorescence Resonance Energy Transfer (FRET) Assay: The assay buffer used in this screen is 0.05 M acetate, pH 4.2, 10% DMSO final, 100 uM genapol (which is a nonionic detergent, below its Critical Micelle Concentration). The beta secretase enzyme (0.2 nM) is pre-incubated for one hour with inhibitors, typically in about 1 uL of DMSO according to a serial dilution, are added thereto. This assay is effectively started by the addition of FRET substrate (50 nM) and the combination is incubated for one hour. The FRET assay is terminated with by addition of Tris buffer, which raises the pH to neutrality, and the fluorescence is determined. The FRET substrate is a peptide with commercially available fluorophore and quencher, on opposite sides of the BACE cleavage site. Proteolytic cleavage of the FRET substrate releases quenching of fluorescence (excitation 488 nm and emission 425 nm).
Measurement: IC50 = 1.5 nM (pChEMBL 8.819999694824219)
Descriptors: MW 517.52, ALogP 3.76, HBA 9, HBD 2, PSA 130.18, QED 0.5
Compound (SMILES): O=S(=O)(Nc1nccs1)c1ccc2c(c1)OCCN2c1ccc(C(F)(F)F)cc1-c1cncnc1
InChIKey: BDPIGJCLXMOTDZ-UHFFFAOYSA-N
Target: Sodium channel protein type 9 subunit alpha (SINGLE PROTEIN), Homo sapiens
UniProt: Q15858
Assay: In Vitro Assay: HEK 293 cells stably transfected with either Nav 1.7 or with Nav 1.5 were recorded in population patch-clamp mode with the IonWorks® Quattro automated electrophysiology system in accordance with the manufacturer's specifications (Molecular Devices, LLC, Sunnyvale, Calif.). Sodium channel currents were measured in response to a train of depolarizations that induced successively greater inactivation. Cells were held at −110 mV for three seconds (Nav 1.7) or half a second (Nav 1.5) from a holding voltage of −15 mV, then put through a series of 26 pulses of 150 msec duration to −20 mV at a frequency of 5 Hz. Cells were then left unclamped for a period of 3 to 8 minutes while a single concentration of test compound was added. Cells were then reclamped and put through the same voltage protocol. Current at the end of the 26th pulse to −20 mV was subtracted from the peak current evoked by the 26th pulse to −20 mV to correct for leak current.
Measurement: IC50 = 11.5 nM (pChEMBL 7.940000057220459)
Descriptors: MW 519.53, ALogP 4.95, HBA 8, HBD 1, PSA 97.31, QED 0.4
Compound (SMILES): CCN(CC)CCNC(=O)CCc1ccc(Nc2nc(C)cc(Nc3cc(C4CC4)n[nH]3)n2)cc1
InChIKey: XMAHDEYITIVOHY-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) un-phosphorylated apo-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 476.63, ALogP 4.26, HBA 7, HBD 4, PSA 110.86, QED 0.29
Compound (SMILES): NCCCC[C@@H]1N[C@H](CNC(=O)Cc2ccc3ccccc3c2)CCN(Cc2ccc3ccccc3c2)C1=O
InChIKey: KABTYBLCGLFRIC-CONSDPRKSA-N
Target: Melanocortin receptor 5 (SINGLE PROTEIN), Homo sapiens
UniProt: P33032
Assay: Radioligand Binding Assay: Assessments of compound binding to human MC5R (hMC5R)) by displacement of an 125I-labeled NDP-MSH receptor ligand peptide were performed essentially as described in the data sheets produced by Perkin Elmer to accompany their frozen hMC5R membranes (Perkin Elmer catalog number RBXMC5M400UA). [125I] NDP-MSH: Radiolabeled in House and Purified by HPLC: Na125I (0.5 mCi, 17.4 Ci/mg) was added to 50 μL sodium phosphate (50 mM, pH 7.4) in an eppendorf tube precoated with IODOGEN. After incubation for 10 mins the phosphate buffer containing the iodine was added to NDP-MSH (10 μl at 1 mg/mL) in a separate eppendorf tube. This was incubated for a further 10 mins. The iodinated NDP-MSH was purified by HPLC on a Zorbax SB 300 column using solvent A: 0.05% TFA and solvent B: 90% acetonitrile 0.045% TFA with a linear gradient, 0-67% B over 60 mins. The 125I NDP-MSH eluted at 52 mins after the unlabeled starting material (48 min) and was counted and stored in the freezer. It was used within 48 hrs, as radioactive decay and ligand decomposition resulted in greatly reduced specific binding observed after 72 hrs. Incubation buffer: 25 mM HEPES-KOH (pH 7.0), 1.5 mM CaCl2, 1 mM MgSO4, 0.1 M NaCl, 1 mM 1,10-phenanthroline, and 1 Complete™ protease inhibitor tablet/100 mL (Roche, catalog number 1873580). Perkin Elmer frozen hMC5 membranes: catalog number RBXMC5M400UA, 0.4 mL/vial; 400 microassays/vial, 0.78 mg/mL protein concentration. Vials of frozen membranes were thawed rapidly immediately before use, diluted with binding buffer and vortexed. Resuspended membranes were kept on ice until they were added to the wells of the plate. Assays were performed in 96 well polypropylene plates. Membranes (0.78 μg 40 μL of a 1:40 dilution in incubation buffer) were added to [125I] NDP-MSH (0.84 nM; 2200 Ci/mmol) and test compounds in a total volume of 140 μL. This was incubated for 1 hr at 37° C. Non-specific binding was determined with 3 mM NDP-MSH. Plates were filtered using a Tomtec cell harvester with GF/A filters (Wallac) (presoaked in 0.6% polyethylenimine) and washed three times with 1.0 mL ice-cold wash buffer (the above incubation buffer without 1,10-phenanthroline and Complete™ protease inhibitor tablet). The filters were dried in a 37° C. oven, placed in a sample bag and 5 mL Betaplatescint (Wallac) was added. Prepared filters were counted in cassettes in a Microbeta Trilux (Wallac) for 1 min.
Measurement: IC50 < 10000 nM (pChEMBL 5)
Descriptors: MW 522.69, ALogP 4.54, HBA 4, HBD 3, PSA 87.46, QED 0.27
Compound (SMILES): CCn1c(=O)n(-c2cnn(Cc3c(C)noc3C)c2)c(=O)n1Cc1ccccc1
InChIKey: BTLCMQNNBQBXLV-UHFFFAOYSA-N
Target: Taste receptor type 2 member 8 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9NYW2
Assay: Fluorescence Polarization Assays: In another embodiment, Fluorescence Polarization ("FP") based assays may be used to detect and monitor ligand binding. Fluorescence polarization is a versatile laboratory technique for measuring equilibrium binding, nucleic acid hybridization, and enzymatic activity. Fluorescence polarization assays are homogeneous in that they do not require a separation step such as centrifugation, filtration, chromatography, precipitation, or electrophoresis. These assays are done in real time, directly in solution and do not require an immobilized phase. Polarization values can be measured repeatedly and after the addition of reagents since measuring the polarization is rapid and does not destroy the sample. Generally, this technique can be used to measure polarization values of fluorophores from low picomolar to micromolar levels.
Measurement: IC50 = 40 nM (pChEMBL 7.400000095367432)
Descriptors: MW 394.44, ALogP 1.72, HBA 9, HBD 0, PSA 92.78, QED 0.5
Compound (SMILES): CCOc1cc(C(=O)NC2CC2)cc(-c2cccc(-c3cc(NC(=O)Nc4c(Cl)cncc4Cl)c(=O)n(CC)n3)c2)c1
InChIKey: JFHRDTICJOZJOB-UHFFFAOYSA-N
Target: Adenosine receptor A2b (SINGLE PROTEIN), Homo sapiens
UniProt: P29275
Assay: Antagonist activity at adenosine A2b receptor (unknown origin)
Measurement: IC50 > 1000 nM (pChEMBL 6)
Descriptors: MW 607.5, ALogP 6.23, HBA 7, HBD 3, PSA 127.24, QED 0.21
Compound (SMILES): CCc1cc(CNc2cc(Cl)nn3c(-c4ccc(OC)c(S(=O)(=O)n5cccn5)c4)c(C)nc23)ccn1
InChIKey: LHVUCJFYZUOADA-UHFFFAOYSA-N
Target: Phosphatidylinositol 4-kinase beta (SINGLE PROTEIN), Homo sapiens
UniProt: Q9UBF8
Assay: Inhibition of PI4KB (unknown origin) by ADP-Glo kinase assay
Measurement: IC50 = 19.1 nM (pChEMBL 7.71999979019165)
Descriptors: MW 538.03, ALogP 4.37, HBA 10, HBD 1, PSA 116.3, QED 0.31
Compound (SMILES): CCCN1CCN(c2ccc(Nc3nccc(-c4c(-c5ccc(OCC)c(C(=O)Nc6c(F)cccc6F)c5)nc5ccccn45)n3)c(OCC)c2)CC1
InChIKey: UDKAIUKQYUAKGM-UHFFFAOYSA-N
Target: Insulin-like growth factor 1 receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P08069
Assay: Inhibition of human GST-rTEV-tagged IGF1R (957 to 1367 residues) expressed in baculovirus infected insect Sf9 cells assessed as decrease in biotin-aminohexyl-AEEEEYMMMMAKKKK-NH2 phosphorylation after 1 hr by fluorometer
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 732.84, ALogP 8.06, HBA 10, HBD 2, PSA 109.15, QED 0.12
Compound (SMILES): OCCNc1cc(-c2cnc(N3CCOCC3)c(NCc3cccc(F)c3)c2)c(Cl)cn1
InChIKey: JWMNGINJCKZUHW-UHFFFAOYSA-N
Target: CDK9/cyclin T1 (PROTEIN COMPLEX), Homo sapiens
UniProt: P50750
Assay: Inhibition of recombinant human full length C-terminal His6-tagged CDK9/cyclin T1 expressed in baculovirus infected Sf21 insect cells using 5TAMRA-cdk7tide as substrate after 4.5 hrs by IMAP assay
Measurement: IC50 = 6917 nM (pChEMBL 5.159999847412109)
Descriptors: MW 457.94, ALogP 3.79, HBA 7, HBD 3, PSA 82.54, QED 0.47
Compound (SMILES): CC1(C)C(=O)N(c2ccc(C#N)c(C(F)(F)F)c2)C(=S)N1c1ccc(/C=C/C(=O)Nc2ccccc2N)c(F)c1
InChIKey: LPHGULGLUJFAAG-FMIVXFBMSA-N
Target: Histone deacetylase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q13547
Assay: Inhibition of HDAC1 (unknown origin) assessed as inhibition of fluorogenic peptide deacetylation
Measurement: IC50 > 100000 nM (pChEMBL 4)
Descriptors: MW 567.57, ALogP 5.87, HBA 5, HBD 2, PSA 102.46, QED 0.17
Compound (SMILES): CNCCn1cc(-c2ccnc(C(F)(F)F)c2)nc1C1CCN(c2ncnc(N)c2Cl)CC1
InChIKey: UOFRCDSWOWULBQ-UHFFFAOYSA-N
Target: Ribosomal protein S6 kinase beta-1 (SINGLE PROTEIN), Homo sapiens
UniProt: P23443
Assay: P70S6K Enzyme Assay: P70S6K inhibitor compounds are diluted and plated in 96 well plates. A reaction mixture including the following components is then added to the compound plate to initiate the enzyme reaction; P70S6K (3 nM, T412E mutant, Millipore) is mixed with 24 uM ATP in an assay buffer containing 100 mM Hepes (pH 7.5), 5 mM MgCl2, 1 mM DTT, 0.015% Brij and 1 uM of the substrate peptide FITC-AHA-AKRRRLSSLRA-OH (derived from the S6 ribosomal protein sequence, FITC=fluorescein isothiocyanate, AHA=6-aminohexanoic acid). The reaction is incubated for 90 min at 25.degree. C., before the addition of 10 mM EDTA to stop the reaction. The proportion of substrate and product (phosphorylated) peptide is analysed on a Caliper Life Sciences Lab Chip 3000, using a pressure of -1.4 psi, and upstream and downstream voltages of -3000 and -700 respectively. Product peaks are resolved before substrate peaks on the resulting chromatograms.
Measurement: IC50 = 2 nM (pChEMBL 8.699999809265137)
Descriptors: MW 480.93, ALogP 3.59, HBA 8, HBD 2, PSA 97.78, QED 0.56
Compound (SMILES): Cc1nc(C2CC2c2nnc(C)o2)cc(-n2nc(C)nc2C)n1
InChIKey: ZKTOHHKAYFDNFM-UHFFFAOYSA-N
Target: cAMP and cAMP-inhibited cGMP 3',5'-cyclic phosphodiesterase 10A (SINGLE PROTEIN), Homo sapiens
UniProt: Q9Y233
Assay: Fluorescence Polarization Assay: In a typical experiment the PDE10 inhibitory activity of the compounds of the present invention was determined in accordance with the following experimental method. PDE10A2 was amplified from human fetal brain cDNA (Clontech, Mountain View, Calif.) using a forward primer corresponding to nucleotides 56-77 of human PDE10A2 (Accession No. AF127480, Genbank Identifier 4894716), containing a Kozak consensus sequence, and a reverse primer corresponding to nucleotides 2406-2413 of human PDE10A2 (Accession No. AF127480, Genbank Identifier 4894716). Amplification with Easy-A polymerase (Stratagene, La Jolla, Calif.) was 95° C. for 2 minutes followed by thirty three cycles of 95° C. for 40 seconds, 55° C. for 30 seconds, and 72° C. for 2 minutes 48 seconds. Final extension was 72° C. for 7 minutes. The PCR product was TA cloned into pcDNA3.2-TOPO (Invitrogen, Carlsbad, Calif.) according to standard protocol. AD293 cells with 70-80% confluency were transiently transfected with human PDE10A2.
Measurement: Ki = 918 nM (pChEMBL 6.039999961853027)
Descriptors: MW 311.35, ALogP 1.95, HBA 8, HBD 0, PSA 95.41, QED 0.73
Compound (SMILES): CC(C)CN(Cc1ccc(C(=O)O)cc1)C(=O)c1ccc(Oc2ccccc2F)cc1
InChIKey: CMQWTHVXHRXUNC-UHFFFAOYSA-N
Target: Lysophosphatidic acid receptor 5 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9H1C0
Assay: Receptor Assay: Engagement of the LPA5 receptor by its ligand, oleoyl-L-Î±-lysophosphatidic acid (LPA), leads to the release of intracellular stores of calcium into the cytoplasm mediated through a Gq-driven increase in inositol triphosphate (IP3) levels. Gq is a heterotrimeric G protein subunit that activates phospholipase C.The ability of test compounds to prevent LPA-driven intracellular release of stored calcium from RH7777 cells expressing human LPA5 receptors was assayed by stimulating the cells with LPA in the presence of various test compounds to measure the test compounds' antagonism, i.e. activity in vitro. Approximately 10,000 cells in normal culture medium (Minimal Essential Media, 10% Foetal Calf Serum) were dispensed per assay well in a black clear bottom poly-D-lysine coated 384 well plate (Corning). Cells were allowed to settle for thirty minutes at room temperature before being incubated overnight at 37° C., 5% CO2. Sixteen to twenty hours after dispensing, the cells were loaded with a calcium-sensitive fluorescent dye by replacing the culture medium with assay buffer (1x Hanks buffered saline, 25 mM HEPES, 0.1% w/v fatty acid free BSA (bovine serum albumin), pH7.4) containing 1.25 mM probenecid and 1x Calcium 5 Reagent (Molecular Devices). Cells were incubated at 37° C. for one hour to allow for dye uptake.
Measurement: IC50 = 63 nM (pChEMBL 7.199999809265137)
Descriptors: MW 421.47, ALogP 5.61, HBA 3, HBD 1, PSA 66.84, QED 0.51
Compound (SMILES): Cc1cc(-c2ccc(CNC(=O)c3ccc4c(c3)C(F)(F)c3ccccc3-4)cc2)ccn1
InChIKey: SREWAVPCIPWWPP-UHFFFAOYSA-N
Target: Protein-serine O-palmitoleoyltransferase porcupine (SINGLE PROTEIN), Mus musculus
UniProt: Q9JJJ7
Assay: Inhibition of porcupine in mouse L Wnt3A cells co-cultured with HEK293 cells after 48 hrs by Super-top flash reporter gene assay
Measurement: IC50 = 60 nM (pChEMBL 7.21999979019165)
Descriptors: MW 426.47, ALogP 6.11, HBA 2, HBD 1, PSA 41.99, QED 0.43
Compound (SMILES): CN(C)C(=O)c1ccc2c(c1)c(Sc1ccccc1)nn2-c1ccc(C(=O)O)cc1
InChIKey: ZGDDULRRLLCPBU-UHFFFAOYSA-N
Target: Nuclear receptor ROR-gamma (SINGLE PROTEIN), Homo sapiens
UniProt: P51449
Assay: Inverse agonist activity at GAL4 DBD-fused RORgammat LBD (unknown origin) expressed in HG5LN cells after 18 hrs by luciferase reporter gene assay
Measurement: IC50 > 10000 nM (pChEMBL 5)
Descriptors: MW 417.49, ALogP 4.58, HBA 5, HBD 1, PSA 75.43, QED 0.51
Compound (SMILES): O=C(O)[C@H]1CC[C@H](C(=O)N2CCC(c3ccc(C(F)(C(F)(F)F)C(F)(F)F)cc3)(S(=O)(=O)c3cccc(CC(F)(F)F)c3)C2)CC1
InChIKey: SWOBOUPOGIPMKL-DFPMAXHOSA-N
Target: Nuclear receptor ROR-gamma (SINGLE PROTEIN), Homo sapiens
UniProt: P51449
Assay: Scintillation Proximity Assay (SPA): The binding of potential ligands to RORγ is measured by competition with [3H] 25-hydroxycholesterol (Perkin Elmer NET674250UC) using a scintillation proximity assay (SPA) binding assay. The ligand binding domain of human RORγ (A262-S507) with an N-terminal His tag is expressed in E. coli and purified using nickel affinity chromatography. 15 ug/well RORγ (A262-S507) is incubated with test compound at varying concentrations in 3-fold serial dilution, with final concentrations ranging from 16.6 uM to 0.28 nM for 10 min at room temperature in PBS buffer (Invitrogen #14190-144) containing 0.5% fatty acid free BSA (Gemini Bio-Products, Cat. #700-107P) and 0.1% Glycerol (Sigma Cat#G5516). 10 nM of [3H] 25-hydroxycholesterol is then added, and the reaction is incubated for 10 min. 10 mg/mL of Copper-His Tag-PVT beads (Perkin Elmer cat #RPNQ0095) are added, and the mixture is incubated for 60 min. The reaction is read on a TopCount Microplate scintillation plate reader (Perkin Elmer). The competition data of the test compound over a range of concentrations was plotted as percentage inhibition of radioligand specifically bound in the absence of test compound (percent of total signal). After correcting for non-specific binding, IC50 values were determined. The IC50 value is defined as the concentration of test compound needed to reduce [3H] 25-hydroxycholesterol specific binding by 50% and is calculated using the four parameter logistic equation to fit the normalized data.
Measurement: IC50 = 72 nM (pChEMBL 7.139999866485596)
Descriptors: MW 691.58, ALogP 6.87, HBA 4, HBD 1, PSA 91.75, QED 0.32
Compound (SMILES): COCCNc1ccc(C(c2ccc(Cl)cc2)c2cc(NCCOC)c3[nH]c(=O)cc(NC4CCN(S(=O)(=O)C(F)(F)F)CC4)c3c2)cc1
InChIKey: MCIVUQCGJIAZEA-UHFFFAOYSA-N
Target: Cannabinoid receptor 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P21554
Assay: cAMP Assay: The following mixtures and buffer solutions were prepared: (a) Buffer 1: HBSS (Mediatech Cat#21-023-CV) with 5 mM HEPES (1 mM stock, Gibco BRL Cat#15630-056) and 0.1% BSA (7.5% stock, Invitrogen Cat#15260-037); (b) Buffer 2: 0.5 mM IBMX (200 mM stock in DMSO, Sigma 15879) in Buffer 1; (c) 1 uM cAMP Standard (50 uM stock, Perkin Elmer Cat# AD0262) diluted in Buffer 2 and serially diluted in Buffer 2, 12 doses at 1/2 dilutions resulting in a dose range of 1 uM to 0.5 nM; (d) d2 labelled cAMP (CisBio HTRF Detection Kit Cat #62AM4PEB reconstituted with 6 ml dH2O) diluted 1/20 with lysis buffer (CisBio HTRF Detection Kit Cat #62AM4PEB); (e) anti-cAMP (CisBio HTRF Detection Kit Cat #62AM4PEB reconstituted with 5 ml dH2O) diluted 1/20 with lysis buffer (CisBio HTRF Detection Kit Cat #62AM4PEB); and (f) Forskolin (Sigma Cat# F6886, 10 mM in DMSO) diluted first in DMSO to 1 mM and then to 1.5 uM in Buffer 2.
Measurement: EC50 = 15 nM (pChEMBL 7.820000171661377)
Descriptors: MW 722.23, ALogP 6.2, HBA 8, HBD 4, PSA 124.79, QED 0.09
Compound (SMILES): CN1CCC(c2cnc(N)c(-c3ccc(C(=O)N[C@H](CO)c4cccc(Cl)c4)c(F)c3)c2)CC1=O
InChIKey: QPFNXFHQWKPHCC-RYDFDWSBSA-N
Target: Mitogen-activated protein kinase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P28482
Assay: Kinase Assay (New): Compound potency against activated ERK2 is determined using a kinase assay that measures ERK2-catalyzed phosphorylation of biotinylated ERKtide peptide substrate ([Biotin]-AHA-K-R-E-L-V-E-P-L-T-P-S-G-E-A-P-N-Q-A-L-L-R-[NH2], the peptide sequence derived from EGF receptor: SEQ ID NO:1). The assay is carried out in 50 mM HEPES [pH 7.5], 5 mM MgCl2, 1 mM DTT, 0.01% Tween-20, 0.05% BSA using 0.25 nM ERK2, 200 nM ERKtide peptide and 35 uM ATP (all concentrations are final in the reaction) in a total volume of 10.25 uL. A 16-point, half-log dilution series of compounds at 41x final concentration is used for generating IC50 curves. Compound dilution series are prepared in 100% DMSO. ERK2 is preincubated with compounds for 30 minutes at ambient temperature. Reaction is initiated by addition of a substrate cocktail of ERKtide peptide and ATP and is allowed to proceed for 2-3 hours at ambient temperature. Reaction is terminated by addition of 10 uL of a 2x stop buffer consisting of 100 mM Tris-Cl [pH 7.5], 25 mM EDTA, 0.01% Tween 20, 10 ug/mL of AlphaScreen Protein A Acceptor Beads, 10 ug/mL of Streptavidin Donor Beads (PerkinElmer, Waltham, Mass.), and 1.4 ug/mL phospho-EGF Receptor (Thr669) antibody (Cat #3056, Cell Signaling Technology, Danvers, Mass.). Terminated reactions are read, after overnight incubation in the dark, on an EnVision Multilabel Plate Reader (PerkinElmer, Waltham, Mass.), with excitation and emission wavelengths set to 680 nm and 570 nm, respectively. IC50 values are determined using a four-parameter fit.
Measurement: IC50 = 3.27 nM (pChEMBL 8.479999542236328)
Descriptors: MW 496.97, ALogP 3.92, HBA 5, HBD 3, PSA 108.55, QED 0.48
Compound (SMILES): COC(=O)N[C@@H](Cc1ccccc1)C(=O)N[C@@H](Cc1ccc(NS(=O)(=O)O)cc1)c1nc(-c2ccc(Cl)s2)cs1
InChIKey: HWJBEXMPVQKCEM-PMACEKPBSA-N
Target: Receptor-type tyrosine-protein phosphatase beta (SINGLE PROTEIN), Homo sapiens
UniProt: P23467
Assay: Inhibitory Activity Assay: Non-limiting examples of the HPTPβ IC50 (μM) activity.
Measurement: IC50 < 9000 nM (pChEMBL 5.046000003814697)
Descriptors: MW 621.16, ALogP 5.11, HBA 8, HBD 4, PSA 146.72, QED 0.17
Compound (SMILES): C[C@@H]1CN(CC2CCCCO2)c2ccccc2[C@H](C)N1C(=O)Nc1cccnc1
InChIKey: OUIXCBSYDNQJPG-ZOVQDZKKSA-N
Target: Voltage-dependent T-type calcium channel subunit alpha-1G (SINGLE PROTEIN), Homo sapiens
UniProt: O43497
Assay: Channel blocking activity at recombinant human CaV 3.1 channel expressed in HEK293 cells assessed as inhibition of Ca2+ flux preincubated for 3 mins followed by CaCl2 addition by fluo-4 dye-based FLIPR assay
Measurement: IC50 = 875 nM (pChEMBL 6.059999942779541)
Descriptors: MW 394.52, ALogP 4.45, HBA 4, HBD 1, PSA 57.7, QED 0.84
Compound (SMILES): COCCOCCOCC(=O)N1CCC(c2nc(-c3ccc([S+](C)[O-])cc3)c(Sc3ccc(Cl)cn3)o2)CC1
InChIKey: NLLJTWLSCZKSBJ-UHFFFAOYSA-N
Target: Fatty-acid amide hydrolase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: O00519
Assay: FAAH Assay: Enzyme activity was demonstrated in a radioenzymatic test based on measuring the product of hydrolysis (ethanolamine [3]H) of anandamide [ethanolamine 1-.sup.3H] (American Radiolabeled Chemicals; 1 mCi/ml) with FAAH (Life Sciences (1995), 56, 1999-2005 and Journal of Pharmacology and Experimented Therapeutics (1997), 283, 729-734), Analytical. Biochemistry (2003), 318, 270-5. In addition, routine assays were performed monitoring hydrolysis of arachidonyl-7-amino-4-methylcoumarin amide (AAMCA) by following increase in fluorescence upon release of 7-amino 4-methyl coumarin (λEX=355 nm, (λEM=460 nm). Analytical. Biochemistry (2005), 343, 143-51. Assays are performed on either cell lysate fractions prepared as described or in whole cell format employing either the fluorescent substrate AAMCA (Cayman chemical, Ann Arbor, Mich.,) or 3H-anandamide ([ETHANOLAMINE-1-3H] American Radiolabeled Chemicals; 1 mCi/ml). The cell lysate assay is performed in black PerkinElmer OptiPlates-384F.
Measurement: IC50 = 747.7 nM (pChEMBL 6.130000114440918)
Descriptors: MW 594.16, ALogP 4.66, HBA 9, HBD 0, PSA 109.98, QED 0.21
Compound (SMILES): NS(=O)(=O)c1ccccc1NS(=O)(=O)c1cccc(-c2ccc3[nH]ccc3c2)c1
InChIKey: HOTXPWMEDFTQKG-UHFFFAOYSA-N
Target: Prostaglandin E synthase 2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9H7Z7
Assay: Biochemical Assay: A solution of test compound was added to a diluted microsome preparation containing human mPGES-1 and pre-incubated for 15 minutes in potassium phosphate buffer pH 6.8 with cofactor glutathione (GSH). Corresponding solutions without test compound were used as positive controls, and corresponding solutions without test compound and without microsomes were used as negative controls. The enzymatic reaction was then started by addition of the substrate PGH2 in an organic solution (dry acetonitrile).The typical reaction conditions of the enzymatic reaction were thus: Test compound: ranging from 60 uM to 0.002 uM, or zero in positive and negative controls; potassium phosphate buffer pH 6.8: 50 mM; GSH: 2.5 mM; mPGES-1-containing microsomes: 2 ug/mL (sample and positive controls) or 0 ug/mL (negative control); PGH2: 10.8 uM; Acetonitrile: 7.7% (v/v); DMSO: 0.6% (v/v). The reaction was stopped after one minute by adding an acidic solution (pH 1.9) of ferric chloride.
Measurement: IC50 = 3300 nM (pChEMBL 5.480000019073486)
Descriptors: MW 427.51, ALogP 3.28, HBA 4, HBD 3, PSA 122.12, QED 0.45
Compound (SMILES): CC(=O)N1CCC(C(=O)N2CC[C@](c3ccc(C(OCc4c(F)cccc4F)(C(F)(F)F)C(F)(F)F)cc3)(S(=O)(=O)c3ccc(F)cc3)C2)(c2ccccc2)CC1
InChIKey: XJDZJCKJDMBKSY-QNGWXLTQSA-N
Target: Nuclear receptor ROR-gamma (SINGLE PROTEIN), Homo sapiens
UniProt: P51449
Assay: Scintillation Proximity Assay (SPA): The binding of potential ligands to RORγ is measured by competition with [3H] 25-hydroxycholesterol (Perkin Elmer NET674250UC) using a scintillation proximity assay (SPA) binding assay. The ligand binding domain of human RORγ (A262-S507) with an N-terminal His tag is expressed in E. coli and purified using nickel affinity chromatography. 15 ug/well RORγ (A262-S507) is incubated with test compound at varying concentrations in 3-fold serial dilution, with final concentrations ranging from 16.6 uM to 0.28 nM for 10 min at room temperature in PBS buffer (Invitrogen #14190-144) containing 0.5% fatty acid free BSA (Gemini Bio-Products, Cat. #700-107P) and 0.1% Glycerol (Sigma Cat#G5516). 10 nM of [3H] 25-hydroxycholesterol is then added, and the reaction is incubated for 10 min. 10 mg/mL of Copper-His Tag-PVT beads (Perkin Elmer cat #RPNQ0095) are added, and the mixture is incubated for 60 min. The reaction is read on a TopCount Microplate scintillation plate reader (Perkin Elmer). The competition data of the test compound over a range of concentrations was plotted as percentage inhibition of radioligand specifically bound in the absence of test compound (percent of total signal). After correcting for non-specific binding, IC50 values were determined. The IC50 value is defined as the concentration of test compound needed to reduce [3H] 25-hydroxycholesterol specific binding by 50% and is calculated using the four parameter logistic equation to fit the normalized data.
Measurement: IC50 = 471 nM (pChEMBL 6.329999923706055)
Descriptors: MW 826.78, ALogP 8.12, HBA 5, HBD 0, PSA 83.99, QED 0.13
Compound (SMILES): CC[C@H](C)[C@@H]1NCCOc2ccccc2/C=C\CNC(=O)[C@H](Cc2ccccc2)NC(=O)C2(CCCC2)NC1=O
InChIKey: NTJPDCDVACGKEF-HJFOHLNVSA-N
Target: Growth hormone secretagogue receptor type 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q92847
Assay: Displacement of [125I]-Ghrelin from human GSHR1a transfected in HEK293 cell membranes after 30 mins by microplate scintillation and luminescence counting method
Measurement: Ki = 10 nM (pChEMBL 8)
Descriptors: MW 546.71, ALogP 3.37, HBA 5, HBD 4, PSA 108.56, QED 0.47
Compound (SMILES): NC1CCN(c2cccc(-c3n[nH]c4cnc(-c5cccnc5)cc34)n2)CC1
InChIKey: UJHVWBSOPCVARA-UHFFFAOYSA-N
Target: Serine/threonine-protein kinase pim-1 (SINGLE PROTEIN), Homo sapiens
UniProt: P11309
Assay: PIM Kinase Binding Activity Assay: PIM-1, PIM-2, and PIM-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.
Measurement: Ki = 0.0839 nM (pChEMBL 10.079999923706055)
Descriptors: MW 371.45, ALogP 3.01, HBA 6, HBD 2, PSA 96.61, QED 0.57
Compound (SMILES): O=S1(=O)Nc2c(cc(F)c3cccnc23)-c2cc(C(F)(F)F)ccc21
InChIKey: WHBQSQZGHXRDDK-UHFFFAOYSA-N
Target: Ferroportin (SINGLE PROTEIN), Homo sapiens
UniProt: Q9NP59
Assay: Antagonist activity at C-terminal halo-tag fused human ferroportin expressed in MDCK cells assessed as inhibition of hepcidin-induced ferroportin internalization after overnight incubation by Draq5 staining-based fluorescence assay
Measurement: IC50 = 4190 nM (pChEMBL 5.380000114440918)
Descriptors: MW 368.31, ALogP 4.17, HBA 3, HBD 1, PSA 59.06, QED 0.61
Compound (SMILES): Cc1cccc2cc(Cn3nc(-c4cc(O)cc(F)c4)c4c(N)ncnc43)n(C3CCOCC3)c(=O)c12
InChIKey: IVMXUWQNWPMPGL-UHFFFAOYSA-N
Target: PI3-kinase p110-delta/p85-alpha (PROTEIN COMPLEX), Homo sapiens
UniProt: O00329
Assay: Inhibition of N-terminal His6-tagged recombinant full-length human p110delta/untagged recombinant full length p85alpha expressed in baculovirus infected Sf21 cells using phosphatidylinositol as substrate in presence of gamma-32P-ATP after 5 mins by thin layer chromatographic method
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 500.53, ALogP 3.94, HBA 9, HBD 2, PSA 121.08, QED 0.38
Compound (SMILES): Cc1ccn2nc([C@H](C)Nc3ncnc4[nH]cc(-c5cnn(CCN(C)C)c5)c34)n(-c3ccccc3)c(=O)c12
InChIKey: FSXBPLVRNPPEMD-IBGZPJMESA-N
Target: Phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit delta isoform (SINGLE PROTEIN), Homo sapiens
UniProt: O00329
Assay: HTRF Assay : PI-3 Kinase HTRF kit (ref. #33-037) and the different PI3K recombinant isoforms (ref. #14-602, ref. #14-603, ref. #14-604, ref. #15-558 for Alpha, Beta, Delta and Gamma respectively) were purchased at Millipore (expressed in insect cells). ATP was purchased at Sigma Aldrich (ref. #A7699).The compounds were pre-incubated with the enzyme for 30 min before starting of the catalytic reaction. [PIP2] was used at its Km. [ATP] was used at 15 μM for all isoforms for technical reasons (Km values varied between 10 and 20 μM depending on the isoform). Time of assay and [Enzyme] were optimized to work in the linear range. Stop and Detection mixtures were used as specified in the Millipore PI-3 Kinase kit.
Measurement: IC50 = 2 nM (pChEMBL 8.699999809265137)
Descriptors: MW 522.62, ALogP 3.66, HBA 10, HBD 2, PSA 113.96, QED 0.31
Compound (SMILES): COc1cccc(F)c1C1CC(C)C(=O)N1Cc1cnc2ccccc2c1
InChIKey: JMFKYGMKQKXEDK-UHFFFAOYSA-N
Target: Orexin/Hypocretin receptor type 1 (SINGLE PROTEIN), Homo sapiens
UniProt: O43613
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 = 959 nM (pChEMBL 6.019999980926514)
Descriptors: MW 364.42, ALogP 4.49, HBA 3, HBD 0, PSA 42.43, QED 0.68