lovingscience/chembl-v37-raw-activities huggingface dataset:
Compound (SMILES): COC(=O)N1c2ccc3c(nc(Cc4ccccc4)n3C[C@@H]3CCCCN3)c2CC[C@@H]1C
InChIKey: XMLLVRNVCHKWAX-ICSRJNTNSA-N
Target: Bromodomain-containing protein 4 (SINGLE PROTEIN), Homo sapiens
UniProt: O60885
Assay: HTRF Biochemical Assay for CBP and BRD4 Activity: The assay was performed in a final volume of 6 μL in assay buffer containing 50 mM Hepes (pH 7.5, (0.5M Hepes, pH 7.5 solution; Teknova H1575)), 0.5 mM GSH, 0.01% BGG (0.22 μM filtered, Sigma, G7516-25G), 0.005% BSA (0.22 μM filtered, EMD Millipore Corporation, 126575) and 0.01% Triton X-100 (Sigma, T9284-10L). Nanoliter quantities of 10-point, 3-fold serial dilution in DMSO were pre-dispensed into 1536 assay plates (Corning, #3724BC) for a final test concentration of 33 μM to 1.7 nM, top to lowest dose, respectively. 3 of 2× Protein and 3 μL of 2× Peptide Ligand were added to assay plates (pre-stamped with compound). Plates were incubated for varying times at room temperature prior to measuring the signal. TR-FRET (Time-Resolved Fluorescence Resonance Energy Transfer) was measured on the PHERAstar (BMG, equipped with HTRF optic module [337/520/490]) or on the Envision (PerkinElmer, equipped with the TRF Laser unit, TRF dual mirror D400/D505 and emission filters M520 and M495). Data were reported as percent inhibition compared with control wells based on the following equation: % inh=1−((TR-FRET ratio−AveLow)/(AveHigh−AveLow)) where TR-FRET ratio=(Fluorescence at 520 nm/Fluorescence at 490 nm)*10000), AveLow=average TR-FRET ratio of no enzyme control (n=32), and AveHigh=average TR-FRET ratio of DMSO control (n=32). IC50 values were determined by curve fitting of the standard 4 parameter logistic fitting algorithm included in the Activity Base software package: IDBS XE Designer Model 205. Data is fitted using the Levenburg Marquardt algorithm. For all assay formats data were reported as percent inhibition compared with control wells based on the following equation: % inh=100*((FLU−AveLow)/(AveHigh−AveLow)) where FLU=measured Fluorescence, AveLow=average Fluorescence of no enzyme control (n=32), and AveHigh=average Fluorescence of DMSO control (n=32). IC50 values were determined by curve fitting of the standard 4 parameter logistic fitting algorithm included in the Activity Base software package: IDBS XE Designer Model 205. Data is fitted using the Levenburg Marquardt algorithm.
Measurement: IC50 = 50500 nM (pChEMBL 4.300000190734863)
Descriptors: MW 432.57, ALogP 4.68, HBA 5, HBD 1, PSA 59.39, QED 0.65
Compound (SMILES): CC(C)(C)c1nc(N2CCC(F)(F)C2)c2cnn(Cc3ccc(F)cc3Cl)c2n1
InChIKey: WKKWCYSKPGGNJJ-UHFFFAOYSA-N
Target: Cannabinoid receptor 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P21554
Assay: cAMP Assay: CHO cells expressing human CB1 or CB2 receptors are seeded 17-24 hours prior to the experiment 50.000 cells per well in a black 96 well plate with flat clear bottom (Corning Costar #3904) in DMEM (Invitrogen No. 31331), lx HT supplement, with 10% fetal calf serum and incubated at 5% CO2 and 37° C. in a humidified incubator. The growth medium was exchanged with Krebs Ringer Bicarbonate buffer with 1 mM IBMX and incubated at 30° C. for 30 min. Compounds were added to a final assay volume of 100 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% NaN3) 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 as follows: FRET=T730-Alexa730-P(T645-B645) with P=Ru730-B730/Ru645-B645, where T730 is the test well measured at 730 nM, T645 is the test well measured at 645 nm, B730 and B645 are the buffer controls at 730 nm and 645 nm, respectively. cAMP content is determined from the function of a standard curve spanning from 10 uM to 0.13 nM cAMP. EC50 values were determined using Activity Base analysis (ID Business Solution, Limited). The EC50 values for a wide range of cannabinoid agonists generated from this assay were in agreement with the values published in the scientific literature.
Measurement: EC50 > 10000 nM (pChEMBL 5)
Descriptors: MW 423.87, ALogP 4.81, HBA 5, HBD 0, PSA 46.84, QED 0.61
Compound (SMILES): c1ccc(C2C[C@@H]2NCCC2CCCCCC2)cc1
InChIKey: BGTYDVHTSKDFPF-ZVAWYAOSSA-N
Target: Amine oxidase [flavin-containing] B (SINGLE PROTEIN), Homo sapiens
UniProt: P27338
Assay: Monoamine Oxidase Assays : Briefly, a fixed amount of MAO (0.25 μg for MAO-A and 0.5 μg for MAO-B) was incubated on ice for 15 minutes in the reaction buffer, in the absence and/or in the presence of various concentrations of inhibitor (e.g., from 0 to 50 μM, depending on the inhibitor strength). Tranylcypromine (Biomol International) was used as a control for inhibition.After leaving the enzyme(s) interacting with the inhibitor, 60 to 90 μM of kynuramine was added to each reaction for MAO-B and MAO-A assay respectively, and the reaction was left for 1 hour at 37° C. in the dark. The oxidative deamination of the substrate was stopped by adding 50 μL (v/v) of NaOH 2N. The conversion of kynuramine to 4-hydroxyquinoline, was monitored by fluorescence (excitation at 320 nm, emission at 360 nm) using a microplate reader (Infinite 200, Tecan). Arbitrary units were used to measure levels of fluorescence produced in the absence and/or in the presence of inhibitor.The maximum of oxidative deamination activity was obtained by measuring the amount of 4-hydroxyquinoline formed from kynuramine deamination in the absence of inhibitor and corrected for background fluorescence in the absence of MAO enzymes.
Measurement: Ki = 20500 nM (pChEMBL 4.690000057220459)
Descriptors: MW 257.42, ALogP 4.49, HBA 1, HBD 1, PSA 12.03, QED 0.77
Compound (SMILES): O=C(CCO)N1CCC(c2nn(C3CC3)c3ccc(C(c4ccc(Cl)cc4)c4ccc(Cl)cc4)cc23)CC1
InChIKey: NQVCAKNWBKOEJZ-UHFFFAOYSA-N
Target: Cannabinoid receptor 2 (SINGLE PROTEIN), Homo sapiens
UniProt: P34972
Assay: Biological Assay: CB1 and CB2 receptors are Gi-coupled GPCR. Activation of CB1 and CB2 receptors results in a decrease in cAMP production. An inverse agonist of the CB1 or CB2 receptor results in the opposite effect, an increase of cAMP production. The principle of this assay is based on HTRF® technology (Homogeneous Time-Resolved Fluorescence). The method is a competitive immunoassay between native cAMP produced by cells and the cAMP labeled with the fluorophore d2. The tracer binding is quantified by a Mab anti-cAMP labeled with Eu3+TBP-NHS Cryptate (supplied as part of the assay kit). The specific signal (i.e. energy transfer) is inversely proportional to the concentration of cAMP in the standard or sample.
Measurement: EC50 > 10000 nM (pChEMBL 5)
Descriptors: MW 548.51, ALogP 6.95, HBA 4, HBD 1, PSA 58.36, QED 0.25
Compound (SMILES): Cc1ccnc2cc(C(=O)NC[C@@H](N)C(=O)O)sc12
InChIKey: QRAZNSMFVGTRFM-SSDOTTSWSA-N
Target: Glutamate NMDA receptor; GRIN1/GRIN2A (PROTEIN COMPLEX), Homo sapiens
UniProt: Q12879
Assay: Agonist activity at recombinant human GluN1/2A expressed in Xenopus laevis oocytes by TEVC recording method
Measurement: EC50 = 470 nM (pChEMBL 6.329999923706055)
Descriptors: MW 279.32, ALogP 0.75, HBA 5, HBD 3, PSA 105.31, QED 0.77
Compound (SMILES): CC1(c2ccccc2)CCN(C(=O)c2cc(-c3ccc4c(c3)OCO4)on2)CC1
InChIKey: GQANMRBECUPGLA-UHFFFAOYSA-N
Target: Stearoyl-CoA desaturase 5 (SINGLE PROTEIN), Homo sapiens
UniProt: Q86SK9
Assay: Compound Profiling Methods: Strains expressing SCD1 or SCD5 as the sole desaturase, the human SCD1 and SCD5 genes were used to evaluate inhibition of SCD1/SCD5 using reduced growth as a surrogate for SCD inhibition. These yeast strains express human SCD1 or SCD5 from a plasmid harbored in a strain in which the yeast OLE1 gene is deleted.All compound profiling experiments were performed using the same basic protocol. Yeast were cultured using standard techniques in complete synthetic media lacking uracil and containing yeast nitrogen base supplemented with 2% (w/v) glucose (SD-Ura) Starter cultures were inoculated in 3 mL SD-Ura media containing 0.01% tween and 0.2 mM palmitoleic and oleic acid. Cultures were incubated overnight in a 30° C. shaker incubator (225 rpm). Saturated morning cultures were centrifuged, washed in SD-Ura media lacking TWEEN-20 and fatty acids, and then diluted 1:20 in fresh SD-Ura media also lacking TWEEN-20 and fatty acids. Cells were grown for 6 h to an  (optical density) of 0.4-0.8 at 30° C. with shaking.Compound stocks (10 mM in 100% DMSO) were arrayed into 384-round well, v-bottom polypropylene plates and diluted according to indicated dilution factors. Compound administration was performed in two separate steps. First, 15 μL of SD-Ura was dispensed into clear 384-well assay plates using a MULTIDROP Combi reagent dispenser. The diluted compound stock plates were then applied to the assay plates using an automated workstation (Perkin Elmer JANUS) outfitted with a 384-pin tool containing slotted pins that deliver 100 nL of compound. The cultures described above were centrifuged and washed with media lacking TWEEN-20 or oleic and palmitoleic acids. Cultures were then resuspended at a 2-fold concentrated  of 0.02 (final  of 0.0.01) in SD-Ura. 15 μL of diluted culture was then dispensed into the pinned assay plate to achieve 30 μL of the 1× culture (0.01) and a top drug concentration of 33.3 μM.After yeast delivery, assay plates were incubated under humidified conditions at 30° C. for 40 h. Yeast growth was monitored by reading the  of each well using a microplate reader (Perkin Elmer EnVision). Data were analyzed as follows. Raw data were processed by background subtracting and converting values to a percent of the nontreated condition for that strain [(EXP-0.035)/(DMSO-0.035)×100%].
Measurement: IC50 = 2940 nM (pChEMBL 5.53000020980835)
Descriptors: MW 390.44, ALogP 4.26, HBA 5, HBD 0, PSA 64.8, QED 0.67
Compound (SMILES): COCCO[C@@H]1CC[C@@H](C[C@@H](C)C2CC(=O)C(C)/C=C(/C)C(O)C(OC)C(=O)C(C)CC/C=C\C=C/C=C(/C)C(NS(C)(=O)=O)C[C@H]3CC[C@H](C)[C@](O)(O3)C(=O)C(=O)N(C)C(C)C(=O)O2)C[C@H]1OC
InChIKey: JXJBFDQGPVGFNA-YAQIDPNYSA-N
Target: Peptidyl-prolyl cis-trans isomerase FKBP5 (SINGLE PROTEIN), Homo sapiens
UniProt: Q13451
Assay: SPR Assay to Determine Binding Affinity to FKBP51: Biotinylated avi-FKBP51 was immobilized on a streptavidin chip (Cytiva Series S SA) using a Biacore 8K or 8 k+ (Cytiva). To achieve an immobilization level of 2000 RU, 3 μg/ml biotinylated avi-FKBP51 were injected for 360 sec at a flow rate of 10 μl/min. Rapalogs were diluted in DMSO to 100× working concentration. Each Rapalog was 100-fold diluted in 50 mM HEPES pH 7.5, 150 mM NaCl, 2 mM MgCl2, 1 mM DTT, 0.05% Tween-20 and a serial dilution prepared (8 concentrations, 3-fold dilutions, 0.5-1000 nM). Rapamycin was used as reference sample (8 concentrations, 3-fold dilutions, 0.5-1000 nM). The compound dilutions were then injected at 100 uL/min for 120 seconds contact time and with 3600 seconds dissociation time with increasing concentrations. 50 mM HEPES pH 7.5, 150 mM NaCl, 2 mM MgCl2, 1 mM DTT, 0.05% Tween-20, 1% DMSO was used as running buffer. Multi-cycle kinetics data were fit to a 1:1 binding model to measure the association rate ka (1/Ms), the dissociation rate kd (1/s) and the affinity Kd (M).
Measurement: Kd = 55 nM (pChEMBL 7.260000228881836)
Descriptors: MW 995.28, ALogP 4.97, HBA 15, HBD 3, PSA 230.6, QED 0.11
Compound (SMILES): COc1ncc2c(n1)CCN(CCN1CCN(c3noc4ccccc34)CC1)C2=O
InChIKey: WVTIRDAEXLIBJI-UHFFFAOYSA-N
Target: 5-hydroxytryptamine receptor 2A (SINGLE PROTEIN), Homo sapiens
UniProt: P28223
Assay: Binding Affinity Assay: Present compound for human 5-HT1A receptor, human 5-HT2A receptor, and human D2 receptor was measured by the following procedures. CHO cell membrane fraction in which human 5-HT1A receptor, human 5-HT2A receptor, and human D2 receptor were expressed was purchased from PerkinElmer, Inc. In a test for evaluating binding affinity, a test compound dissolved in dimethylsulfoxide (DMSO) and each receptor membrane sample diluted in buffer were mixed with [3H]8-OH-DPAT, [3H]Ketanserin, or [3H]Spiperone (all purchased from PerkinElmer, Inc.) for 5-HT1A receptor, 5-HT2A receptor, and D2 receptor, respectively. Each mixture was incubated at room temperature for 60 minutes. Then, the mixture was added quickly on a glassfiber filter plate (Multiscreen FB, Millipore, Inc.) coated with 0.3% aqueous polyethylenimine, and vacuum-filtered. Radioactivity remaining on the filter was measured with a liquid scintillation counter (PerkinElmer, Inc.). Binding inhibition rate was calculated from the following formula.
Measurement: Ki = 22.9 nM (pChEMBL 7.639999866485596)
Descriptors: MW 408.46, ALogP 1.45, HBA 8, HBD 0, PSA 87.83, QED 0.63
Compound (SMILES): COc1cccc(NCc2ccc(-c3noc(C(F)(F)F)n3)cc2)c1
InChIKey: FGRHBOCVOGOKKY-UHFFFAOYSA-N
Target: Histone deacetylase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q13547
Assay: Enzymatic Assay: HDAC1 and 6 reagents: FLAG-tagged HDACs 1 and 6 were prepared in-house by protein expression in HEK293F cells followed by anti-FLAG affinity purification. Assays were performed with buffer containing 20 mM HEPES, pH 8.0 [Boston BioProducts, catalog #BB-104, 1M stock], 137 mM NaCl [Sigma, catalog #S5150, 5M stock], 2.7 mM KCl [BioChemika, catalog #87526, 4M stock], 1 mM MgCl2 [Fluka, catalog #63020, 1M stock], and 0.05% BSA (Fraction V) [Invitrogen, catalog #15260, 7.5% stock]. In addition to the above buffer ingredients, TCEP [CalBiochem, catalog #580561, 500 mM stock] was added at a final concentration of 0.5 mM to the buffer for the HDAC6 assays. HDAC1, 2, 3, and 6 enzymes were run at the final concentrations of 0.3 nM, 1.5 nM, 0.3 nM, and 1.333 nM, respectively. Fluor-de-Lys substrate [BioMol Research Laboratories, catalog #KI-104], used to evaluate enzyme activity, was added at the final concentrations of 20 uM, 40 uM, 20 uM, and 2.5 uM for HDACs 1, 2, 3, and 6. To enable detection of the signal, Developer [BioMol Research Laboratories, catalog #KI-105] was added at a 1:250 dilution to the stop solution, which also included 10 uM SAHA [Sigma, catalog #SML0061] to ensure complete termination of the reaction.
Measurement: IC50 = 1700 nM (pChEMBL 5.769999980926514)
Descriptors: MW 349.31, ALogP 4.38, HBA 5, HBD 1, PSA 60.18, QED 0.74
Compound (SMILES): Cc1ccc(S(=O)(=O)N2CCN(C)C(=O)C2)cc1-c1cnc2c(N)nc(C(F)(F)F)cn12
InChIKey: YIMXJADGBZLWDY-UHFFFAOYSA-N
Target: Phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit delta isoform (SINGLE PROTEIN), Homo sapiens
UniProt: O00329
Assay: Scintillation Proximity Assay: PI3Kδ:[γ-33P]ATP (10 mCi/mL) and Wheat Germ Agglutinin (WGA) YSi SPA Scintillation Beads was purchased from Perkin-Elmer (Waltham, Mass.). Lipid kinase substrate, D-myo-Phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2)D (+)-sn-1,2-di-O-octanoylglyceryl, 3-O-phospho linked (PIP2), CAS 204858-53-7, was purchased from Echelon Biosciences (Salt Lake City, Utah). PI3Kδ (p110δ/p85α) Recombinant Human Protein was purchased from Eurofins (St Charles, Mo.). ATP, MgCl2, DTT, EDTA, MOPS and CHAPS were purchased from Sigma-Aldrich (St. Louis, Mo.).The kinase reaction was conducted in polystyrene 384-well Greiner Bio-one white plate from Thermo Fisher Scientific in a final volume of 25 μL. Inhibitors were first diluted serially in DMSO and added to the plate wells before the addition of other reaction components. The final concentration of DMSO in the assay was 2%. The PI3Kδ assay was carried out at room temperature in 20 mM MOPS, pH 6.7, 10 mM MgCl2, 5 mM DTT and CHAPS 0.03%. Reactions were initiated by the addition of ATP, the final reaction mixture consisted of 20 μM PIP2, 2 μM ATP, 0.5 μCi [γ-33P] ATP, 3.4 nM PI3Kδ. Reactions were incubated for 120 min and terminated by the addition of 40 μL SPA beads suspended in quench buffer: 163 mM potassium phosphate pH 7.8, 20% glycerol, 25 mM EDTA. The final concentration of SPA beads is 1.0 mg/mL. After the plate sealing, plates were shaken overnight at room temperature and centrifuged at 1500 rpm for 10 min, the radioactivity of the product was determined by scintillation counting on Topcount (Perkin-Elmer).
Measurement: IC50 < 2000 nM (pChEMBL 5.698999881744385)
Descriptors: MW 468.46, ALogP 1.77, HBA 7, HBD 1, PSA 113.9, QED 0.63
Compound (SMILES): C[C@@]1(c2cc(NC(=O)c3ccc(F)cn3)cc(F)c2F)Cn2nncc2C(N)=N1
InChIKey: DEALMQADSASPQK-SFHVURJKSA-N
Target: Beta-secretase 2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9Y5Z0
Assay: Biochemical FRET Based Assay : This assay is a Fluorescence Resonance Energy Transfer Assay (FRET) based assay. The substrate for this assay contains the ¿Swedish¿ Lys-Met/Asn-Leu mutation of the amyloid precursor protein (APP) beta-secretase cleavage site. This substrate also contains two fluorophores: (7-methoxycoumarin-4-yl) acetic acid (Mca) is a fluorescent donor with excitation wavelength at 320 nm and emission at 405 nm and 2,4-Dinitrophenyl (Dnp) is a proprietary quencher acceptor. The distance between those two groups has been selected so that upon light excitation, the donor fluorescence energy is significantly quenched by the acceptor, through resonance energy transfer. Upon cleavage by the beta-secretase, the fluorophore Mca is separated from the quenching group Dnp, restoring the full fluorescence yield of the donor. The increase in fluorescence is linearly related to the rate of proteolysis.Briefly in a 384-well format recombinant BACE2 protein in a final concentration of 0.4 μg/ml is incubated for 450 minutes at room temperature with 10 μM substrate in incubation buffer (50 mM Citrate buffer pH 5.0, 0.05% PEG, no DMSO) in the absence or presence of compound. Next the amount of proteolysis is directly measured by fluorescence measurement at T=0 and T=450 (excitation at 320 nm and emission at 405 nm). Results are expressed in RFU (Relative Fluorescence Units), as difference between T450 and TO.
Measurement: IC50 = 3.02 nM (pChEMBL 8.520000457763672)
Descriptors: MW 401.35, ALogP 1.98, HBA 7, HBD 2, PSA 111.08, QED 0.7
Compound (SMILES): COc1cccc(C(=O)N2CC3CN(Cc4c(-c5ccc(Cl)cc5)nc5ccccn45)CC3C2)c1
InChIKey: JQQNQHGNKFGHED-UHFFFAOYSA-N
Target: Potassium channel subfamily K member 9 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9NPC2
Assay: In Vitro Electrophysiological Assay: In Vitro Electrophysiological Analysis of the Human TASK-1 and TASK-3 Channels Via Two-Electrode Voltage Clamp Technique in Xenopus laevis OocytesSubsequently, the oocytes were injected with 0.5-5 ng of a cRNA solution coding for TASK-1 or TASK-3. For the electrophysiological analysis of the channel proteins expressed in the oocytes, the two-electrode voltage clamp technique was used. The measurements were conducted at room temperature (21-22° C.) using a Turbo TEC 10CD amplifier (NPI), recorded at 2 kHz and filtered with 0.4 kHz. Substance administration was performed using a gravitation-driven perfusion system. Here, the oocyte is located in a measuring chamber and exposed to the solution stream of 10 ml/min. The level in the measuring chamber is monitored and regulated by sucking off the solution using a peristaltic pump.
Measurement: IC50 = 21.8 nM (pChEMBL 7.659999847412109)
Descriptors: MW 487, ALogP 4.87, HBA 5, HBD 0, PSA 50.08, QED 0.4
Compound (SMILES): CN(CB(O)O)C(=O)[C@@H](N)CCC(=O)O
InChIKey: FNHCVVVEFVZKKN-YFKPBYRVSA-N
Target: Dipeptidyl peptidase 9 (SINGLE PROTEIN), Homo sapiens
UniProt: Q86TI2
Assay: EnPlex (purified enzyme activity assay): This assay is described in Bachovchin et al. Nature Chemical Biology 10, 656-663 (2014). Briefly, purified enzymes are coupled to Luminex microspheres, with a different bead color for each enzyme. Multiplexed bead complexes are incubated with a compound before being treated with a biotinylated activity-based probe and a streptavidin R-phycoerythrin conjugate (SAPE). The mixtures are scanned on a Luminex flow cytometer, where one laser detects the bead color (enzyme identity) and a second laser detects the R-phycoerythrin signal (enzyme activity). The enzyme concentration is calculated assuming 100% of the protein was coupled to the beads.
Measurement: IC50 = 8400 nM (pChEMBL 5.079999923706055)
Descriptors: MW 218.02
Compound (SMILES): CCn1c(-c2cccc(Cl)c2)cc2cc(NC(=O)c3cc(CNC(=O)C(C)(C)F)cnc3C(F)F)ccc21
InChIKey: YTEHFCYGLNBMHW-UHFFFAOYSA-N
Target: Prostaglandin E synthase (SINGLE PROTEIN), Homo sapiens
UniProt: O14684
Assay: mPGES1 enzyme assay: mPGES1 was diluted with assay buffer and added into plate at 49 μL/well supplemented with 1 μL of compound (the final concentration of each compound at seven gradient concentrations was 10000 nM, 1000 nM, 100 nM, 10 nM, 1 nM, 0.1 nM and 0.01 nM). Each well was added with 3.8 μL of 20 μg/mL PGH precooled on ice after shaking for 30 seconds and incubated for 7 minutes on ice. Then, each well was added with 53.8 μL of 6 mg/mL SnCl2 to quench the reaction. The sample was diluted with dilute buffer at 1:400. 10 μl of diluted sample, 5 μL PGE2-d2 and 5 μL anti-PGE2 Cryptate were added to a black 384-well plate, and incubated at 4° C. overnight. HTRF was determined using Flexstation, and the IC50 of the compounds was obtained by data processing software.
Measurement: IC50 = 12.6 nM (pChEMBL 7.900000095367432)
Descriptors: MW 542.99, ALogP 6.93, HBA 4, HBD 2, PSA 76.02, QED 0.25
Compound (SMILES): CCc1cc(O)c(F)cc1-c1ccc2c(-c3nc4c([nH]3)CCN(CCN3CCC[C@@H]3C)C4)n[nH]c2c1
InChIKey: ZFFSYYHNBNAIRW-KRWDZBQOSA-N
Target: Tyrosine-protein kinase JAK2 (SINGLE PROTEIN), Homo sapiens
UniProt: O60674
Assay: Biochemical JAK and Off-Target Kinase Assays: A panel of four LanthaScreen JAK biochemical assays (JAKL 2, 3 and Tyk2) were carried in a common kinase reaction buffer (50 mM HEPES, pH 7.5, 0.01% Brij-35, 10 mM MgCl2, and 1 mM EGTA). Recombinant GST-tagged JAK enzymes and a GFP-tagged STAT1 peptide substrate were obtained from Life Technologies. Serially diluted compounds were pre-incubated with each of the four JAK enzymes and the substrate in white 384-well microplates (Corning) at ambient temperature for 1 h. ATP was subsequently added to initiate the kinase reactions in 10 μL total volume, with 1% DMSO. The final enzyme concentrations for JAKL 2, 3 and Tyk2 are 4.2 nM, 0.1 nM, 1 nM, and 0.25 nM respectively; the corresponding Km ATP concentrations used are 25 μM, 3 μM, 1.6 μM, and 10 μM; while the substrate concentration is 200 nM for all four assays. Kinase reactions were allowed to proceed for 1 hour at ambient temperature before a 10 μL preparation of EDTA (10 mM final concentration) and Tb-anti-pSTAT1 (pTyr701) antibody (Life Technologies, 2 nM final concentration) in TR-FRET dilution buffer (Life Technologies) was added. The plates were allowed to incubate at ambient temperature for 1 h before being read on the EnVision reader (Perkin Elmer). Emission ratio signals (520 nm/495 nm) were recorded and utilized to calculate the percent inhibition values based on DMSO and background controls.
Measurement: Ki < 0.1 nM (pChEMBL 10)
Descriptors: MW 488.61, ALogP 4.87, HBA 5, HBD 3, PSA 84.07, QED 0.36
Compound (SMILES): Cn1cc(-c2cccc(-c3ncc(-c4cnn(C)c4)c(NC4CC5CC4N(S(C)(=O)=O)C5)n3)c2)cn1
InChIKey: LRCCRTWLBUDDTG-UHFFFAOYSA-N
Target: Interleukin-1 receptor-associated kinase 4 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9NWZ3
Assay: Enzymatic Assay: IRAK4 is a human purified recombinant enzyme (His-TEV-IRAK1 (194-712). IRAK4 hydrolyses ATP, autophosphorylates and phosphorylates a Serine/Threonine generic peptidic substrate (STK: 61ST1BLC from CisBio International based in Bagnols/Cèze FR).Measurement of IRAK-4 inhibition was performed in streptavidin coated 384well FlashPlate (PerkinElmer #SMP410A). His-TEV-IRAK4 (20 ng/well), ATP (2 μM, [33P]ATP 0.25 Ci/well), STK1-biotin peptide (300 nM) and compounds in DMSO (range of concentrations from 20 M to 1 nM) or controls (2% DMSO) were incubated for 3 hours at 30° C. in assay buffer: Hepes pH7.0 50 mM, Fatty acid-free BSA 0.1%, Dithiothreitol DTT 2 mM, MgCl2 10 mM, EGTA 0.5 mM, Tween-20 0.01%, MnCl2 5 mM.
Measurement: IC50 > 5000 nM (pChEMBL 5.301000118255615)
Descriptors: MW 504.62, ALogP 2.78, HBA 9, HBD 1, PSA 110.83, QED 0.43
Compound (SMILES): CCC(CC)n1cc(-c2nc(-c3cnn(CCN4CC[C@H](OC)C4)c3)cn3nccc23)cn1
InChIKey: KEBMLZWSFJAEJQ-NRFANRHFSA-N
Target: Tyrosine-protein kinase JAK1 (SINGLE PROTEIN), Homo sapiens
UniProt: P23458
Assay: JAK1 Inhibition Assay: Compounds of Formula I were screened for their ability to inhibit JAK1 using the general enzyme inhibition assay method, in which the assay mixture contained 1 mM ATP, 8 μM Omnia Y12 peptide (Catalog # IVGN KPZ3121C; Invitrogen Corporation, Carlsbad, Calif.) and 12.5 nM JAK1 in a total volume of 25 μL. JAK1 was purchased from Invitrogen Corporation, Carlsbad, Calif. (catalog # IVGN PV4775).
Measurement: IC50 = 5500 nM (pChEMBL 5.260000228881836)
Descriptors: MW 448.58, ALogP 3.54, HBA 9, HBD 0, PSA 78.3, QED 0.39
Compound (SMILES): Cc1cc(-c2[nH]c3ccc(C4CCN(CCC#N)CC4)cc3c2C(C)C)c(C)cn1
InChIKey: UJXSZAADJUJQHH-UHFFFAOYSA-N
Target: Toll-like receptor 8 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9NR97
Assay: Inhibition Reporter Assay: HEK-Blue-cells (Invivogen) overexpressing human TLR7, TLR8 or TLR9 receptors were used for screening inhibitors of these receptors using an inducible SEAP (secreted embryonic alkaline phosphatase) reporter gene under the control of the IFN-β minimal promoter fused to five NF-κB and AP-1-binding sites. Briefly, cells are seeded into Greiner 384 well plates (15000 cells per well for TLR7, 20,000 for TLR8 and 25,000 for TLR9) and then treated with test compounds in DMSO to yield a final dose response concentration range of 0.05 nM-50 μM. After a 30 minute compound pre-treatment at room temperature, the cells are then stimulated with a TLR7 ligand (gardiquimod at a final concentration of 7.5 μM), TLR8 ligand (R848 at a final concentration of 15.9 μM) or TLR9 ligand (ODN2006 at a final concentration of 5 nM) to activate NF-κB and AP-1 which induce the production of SEAP. After a 22 hour incubation at 37° C., 5% CO2, SEAP levels are determined with the addition of HEK-Blue Detection reagent (Invivogen), a cell culture medium that allows for detection of SEAP, according to manufacturer's specifications.
Measurement: IC50 = 2.2 nM (pChEMBL 8.65999984741211)
Descriptors: MW 400.57, ALogP 6.06, HBA 3, HBD 1, PSA 55.71, QED 0.57
Compound (SMILES): CCNC(=O)c1cc2c(-c3cc(C(C)(C)O)ccc3Oc3c(C)cc(F)cc3C)cn(C)c(=O)c2s1
InChIKey: XHZXLIMJVCTFEQ-UHFFFAOYSA-N
Target: Bromodomain-containing protein 4 (SINGLE PROTEIN), Homo sapiens
UniProt: O60885
Assay: Binding Assay (BRD4-2): The bromodomain binding assays were performed by Reaction Biology Corp., Malvern, Pa., USA (www.reactionbiology.com). The BET binding assays were conducted in 384 well microplates in assay buffer (50 mM HEPES-HCl, pH 7.5, 100 mM NaCl, 1 mg/ml BSA, 0.05% CHAPS, and 0.5% DMSO) with compounds added as DMSO stocks at a single concentration or with 10-point dose response titrations. BET protein (130 nM for BRD4) or assay buffer were delivered to the appropriate wells of the microplate. Test compound (70 nM) was then delivered by acoustic technology via a Labcyte Echo550 liquid handler. The microplate was centrifuged for 5 min and pre-incubated for 30 min at RT with gentle shaking. The ligand (histone H4 peptide (1-21) K5/8/12/16Ac-biotin) was delivered and the microplate was again centrifuged for 5 min and allowed to incubate for 30 min at RT with gentle shaking. Donor beads were then added in the absence of light and the microplate was centrifuged and gently shaken. After 5 min, acceptor beads were added in the absence of light and the microplate was centrifuged and gently shaken in the dark for 60 min. The microplate was read using a Perkin Elmer EnSpire Alpha plate reader (λ Ex/λ Em=680/520-620 nm). Percent inhibition was calculated relative to positive and negative controls on a per plate basis.
Measurement: IC50 = 2 nM (pChEMBL 8.699999809265137)
Descriptors: MW 508.62, ALogP 5.79, HBA 6, HBD 2, PSA 80.56, QED 0.35
Compound (SMILES): O=C(O)CCCc1cccc2c1CCN(c1ccc3ccccc3c1)C2
InChIKey: ATHPUMZOPARQFZ-UHFFFAOYSA-N
Target: Sphingosine 1-phosphate receptor 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P21453
Assay: Measurement of the Inhibitory Action of the Compound of the Present Invention on Binding of [33P]-S1P to S1P5 (EDG-8): Compound of the Present Invention on Binding of [33P]-S1P to S1P5 (EDG-8)A reaction was carried out in a 96-well microplate by using membrane fractions of Chinese hamster ovary (CHO) cells each of which was made to overexpress human S1P1 (EDG-1) or human S1P5 gene respectively, in an amount of the membrane fraction of 1 mg protein/mL. To each of the wells, 100 μL of a vehicle (DMSO) solution or a two-fold concentration-ligand solution each of which was diluted with Binding Buffer (50 mmol/L, Tris pH 7.5, 5 mmol/L, MgCl2, 0.5% BSA and Complete EDTA free (1 tablet/50 mL)), and 50 μL of 0.16 nmol/L [33P]-S1P (manufactured by American Radiolabeled Chemicals, Inc.) diluted with Binding Buffer were added. Thereafter, the membrane fraction solutions (50 μL) was added to the wells and the reaction was carried out at room temperature for 60 minutes. After the reaction, suction filtration was carried out by using a 96-well UNIFILTER, and the 96-well microplate was washed with Wash Buffer (50 mmol/L, Tris pH 7.5, 0.5% BSA) (150 mL), and thereafter, was dried at 60° C. for 45 minutes. MicroScint (trade name) 20 (50 μL/well) was added and the plate was covered with TopSeal-A, and thereafter, the radioactivity was measured by using TopCount (manufactured by PerkinElmer Inc.).
Measurement: IC50 > 30000 nM (pChEMBL 4.5229997634887695)
Descriptors: MW 345.44, ALogP 4.81, HBA 2, HBD 1, PSA 40.54, QED 0.72
Compound (SMILES): O=C(Nc1ccc(Oc2ncnc3cc(OCCCN4CCCC4)c4c(c23)OCCO4)cc1Cl)NC1CC1
InChIKey: QDGKOJXPQOIIRJ-UHFFFAOYSA-N
Target: RAF proto-oncogene serine/threonine-protein kinase (SINGLE PROTEIN), Homo sapiens
UniProt: P04049
Assay: Test of Small Molecule Compounds for Inhibiting the Activity of C-RAF and B-RAF Kinases: 1. Preparation of test compounds: according to the molecular weight of the compounds, an appropriate volume of DMSO was directly added to dissolve the test compounds. For storing the compound, the concentration of DMSO is 100%, and the final concentration of DMSO in the experimental system is 1%. The compounds were 3-fold serially diluted with DMSO to obtain a total of 8 dilutions, with a maximum concentration of 1000 nM and a minimum concentration of 0.46 nM.2. Preparation of the sorafenib positive control: sorafenib, a selective inhibitor of BRAF and RAF1, was used as the positive control of this experiment, and the dilution method thereof was the same as that of the above test compounds.3. Test Conditions:Enzyme: B-RAF: 0.1 ng/l (the final concentration in the reaction system); C-RAF: 0.1 ng/μl (the final concentration in the reaction system)Substrate and ATP: inactive MEKI: 2 ng/μl (the final concentration in the reaction system); ATP: 35 μM (the final concentration in the reaction system)HPE: the reaction without enzyme (1% DMSO)ZPE: the reaction with enzyme but without compound (1% DMSO)4. Test procedure:a) 1 ul of 10-fold diluted compound or 10% DMSO was added to a 384-well assay plate,b) 4 ul enzyme solution or assay buffer was added to the wells of the assay plate,c) the plate was centrifuged at 1000 rpm for 1 minute to homogeneous,d) 5 ul of ATP-substrate mixture was added to the wells of the assay plate,e) the plate was shaked for mixing for 2 minutes,f) the plate was incubated at 30° C. for 1 hour,g) 10 ul of ADP-Glo reagent was added to the wells of the assay plate, and the plate was incubated for 40 minutes at 27° C.,h) 20 ul of kinase assay solution was added to the wells of the plate, and the plate was incubated for 30 minutes at 27° C.,i) the chemiluminescent signal was read with Envision.5. Analysis of the results: calculation of the compound inhibition rate:Inhibition rate (%)=(control measurement without compound−sample measurement)/(control measurement without compound−control measurement without enzyme)*100%The IC50 values of the positive control compound and the test compounds were calculated using the Prism software according to the variable slope of the curve.
Measurement: IC50 > 5000 nM (pChEMBL 5.301000118255615)
Descriptors: MW 540.02, ALogP 5, HBA 8, HBD 2, PSA 107.07, QED 0.37
Compound (SMILES): COc1cccc(O)c1S(=O)(=O)Nc1noc2cc(Cn3cccn3)cc(OC)c12
InChIKey: DOIQAHRSIIRMIB-UHFFFAOYSA-N
Target: Histone acetyltransferase KAT6A (SINGLE PROTEIN), Homo sapiens
UniProt: Q92794
Assay: Kat6A Assay 2: A. Compound preparation    1. Prepare 10 mM stock solutions in 100% DMSO from solid material    2. Serial dilute 10 mM, 1 mM or 0.1 mM compound stocks 3-fold in 100% DMSO for 11-point dose responseB. Reagent preparation    1. Prepare 1× assay buffer containing 10 mM Tris HCL pH 8.0, 2.5 mM NaCl, 0.5 mM EDTA, 0.005% BSG and 0.02% Tween-20    2. Dilute Histone peptide (CPC Scientific) and AcCoA (Sigma) together in assay buffer to 2×.    3. Dilute KAT enzyme in assay buffer to 2×.C. Enzyme reaction    1. Final reaction conditions for each KAT assay in a 20 ul assay reaction volume:  KAT6A 15 nM, 10 uM AcCoA, 2 uM H3 1-21 peptide, 45-minute reaction   2. Add 0.5 ul of diluted compound to the assay plate (384-well V-bottom polypropylene plates) or 0.5 ul of DMSO for control wells.    3. Add 10 ul of 2× Histone peptide/2×AcCoA mix to the assay plate.    4. Add 10 ul of 2× enzyme to the assay plate.    5. Stop the reaction after the indicated time with the addition of 2 ul of 5% formic acid    6. Each reaction was analyzed using self-assembled monolayer desorption/ionization time-of-flight mass spectrometry (Mrksich, Milan (2008) Mass Spectrometry of Self-Assembled Monolayers: A New Tool for Molecular Surface Science ACS Nano 2008 2 (1), 7-18; SAMDI Tech, Inc. (Chicago, Ill.)).    7. Area under the curve (AUC) for both substrate and product peaks was determined for KAT5 at M.W. 2561 [Substrate+H]+ and 2603 [Product+H]+ with a +/−1 Da tolerance, respectively    8. Area under the curve (AUC) for both substrate and product peaks was determined for KAT6a, KAT6B, KAT7 and KAT8 at M.W. 2723 [Substrate+H]+ and 2765 [Product+H]+ with a +/−1 Da tolerance, respectively. 9. Percent conversion to product was calculated by: AUCProduct/(AUCsubstrate+AUCProduct).D. Data analysis    1. IC50 values were determined by fitting the % conversion at each inhibitor concentration to the 4-parameter IC50 equation using Pfizer proprietary curve fitting software.    2. Ki values were determined by fitting the % conversion at each inhibitor concentration to the Morrison equation for tightbinding competitive inhibitors using Pfizer proprietary curve fitting software.
Measurement: Ki = 3.89 nM (pChEMBL 8.40999984741211)
Descriptors: MW 430.44, ALogP 2.6, HBA 9, HBD 2, PSA 128.71, QED 0.46
Compound (SMILES): COCCN1CCC(COc2cc(-c3ccc(N4CC5CC(C4)N5Cc4ccc(OC)nc4)nc3)c3c(C#N)cnn3c2)CC1
InChIKey: WOLZFTHPBAAXCN-UHFFFAOYSA-N
Target: Proto-oncogene tyrosine-protein kinase receptor Ret (SINGLE PROTEIN), Homo sapiens
UniProt: P07949
Assay: RET Enzyme Assay: Compounds of Formula I were screened for their ability to inhibit wildtype and V804M mutant RET kinase using CisBio's HTRF® KinEASE¿-TK assay technology. Briefly, N-terminal GST tagged recombinant human RET cytoplasmic domain (aa 658-end) from Eurofins (0.25 nM RET; Catalog No. 14-570M) or N-terminal GST tagged recombinant human V804M mutant RET cytoplasmic domain (aa 658-end) from Millipore (0.25 nM enzyme; Catalog No. 14-760) was incubated with 250 nM TK-substrate biotin (CisBio, part of Catalog No. 62TK0PEC) and 1 mM ATP along with test compound in a buffer consisting of 25 mM HEPES pH 7.4, 10 mM MgCl2, 0.01% Triton X-100, and 2% DMSO in a volume of 8 μL. Compounds were typically prepared in a threefold serial dilution in DMSO and added to the assay to give the appropriate final concentration. After a 30-minute incubation at 22° C., the reaction was quenched by adding 8 μL of quench solution containing 31.25 nM Sa-XL665 and 1×TK-ab-Cryptate in HTRF detection buffer (all from CisBio, part of Cat. No. 62TK0PEC). After a 1 hour incubation at 22° C., the extent of reaction was determined using a PerkinElmer EnVision multimode plate reader via HTRF dual wavelength detection, and the percent of control (POC) was calculated using a ratiometric emission factor. 100 POC was determined using no test compounds and 0 POC was determined using pre-quenched control reactions. The POC values were fit to a 4 parameter logistic curve, and the IC50 is defined as the concentration of inhibitor at which the POC equals 50 for the fitted curve.
Measurement: IC50 = 11.9 nM (pChEMBL 7.920000076293945)
Descriptors: MW 608.75, ALogP 3.87, HBA 11, HBD 0, PSA 104.28, QED 0.25
Compound (SMILES): COC(=O)N1c2ccc3c(nc(Cc4cc(F)cc(OC)c4)n3[C@H]3CC[C@H](C(=O)O)CC3)c2CC[C@@H]1C
InChIKey: OPLLJRZYXODUEK-QRFRQXIXSA-N
Target: Bromodomain-containing protein 4 (SINGLE PROTEIN), Homo sapiens
UniProt: O60885
Assay: HTRF Biochemical Assay for CBP and BRD4 Activity: The assay was performed in a final volume of 6 μL in assay buffer containing 50 mM Hepes (pH 7.5, (0.5M Hepes, pH 7.5 solution; Teknova H1575)), 0.5 mM GSH, 0.01% BGG (0.22 μM filtered, Sigma, G7516-25G), 0.005% BSA (0.22 μM filtered, EMD Millipore Corporation, 126575) and 0.01% Triton X-100 (Sigma, T9284-10L). Nanoliter quantities of 10-point, 3-fold serial dilution in DMSO were pre-dispensed into 1536 assay plates (Corning, #3724BC) for a final test concentration of 33 μM to 1.7 nM, top to lowest dose, respectively. 3 of 2× Protein and 3 μL of 2× Peptide Ligand were added to assay plates (pre-stamped with compound). Plates were incubated for varying times at room temperature prior to measuring the signal. TR-FRET (Time-Resolved Fluorescence Resonance Energy Transfer) was measured on the PHERAstar (BMG, equipped with HTRF optic module [337/520/490]) or on the Envision (PerkinElmer, equipped with the TRF Laser unit, TRF dual mirror D400/D505 and emission filters M520 and M495). Data were reported as percent inhibition compared with control wells based on the following equation: % inh=1−((TR-FRET ratio−AveLow)/(AveHigh−AveLow)) where TR-FRET ratio=(Fluorescence at 520 nm/Fluorescence at 490 nm)*10000), AveLow=average TR-FRET ratio of no enzyme control (n=32), and AveHigh=average TR-FRET ratio of DMSO control (n=32). IC50 values were determined by curve fitting of the standard 4 parameter logistic fitting algorithm included in the Activity Base software package: IDBS XE Designer Model 205. Data is fitted using the Levenburg Marquardt algorithm. For all assay formats data were reported as percent inhibition compared with control wells based on the following equation: % inh=100*((FLU−AveLow)/(AveHigh−AveLow)) where FLU=measured Fluorescence, AveLow=average Fluorescence of no enzyme control (n=32), and AveHigh=average Fluorescence of DMSO control (n=32). IC50 values were determined by curve fitting of the standard 4 parameter logistic fitting algorithm included in the Activity Base software package: IDBS XE Designer Model 205. Data is fitted using the Levenburg Marquardt algorithm.
Measurement: IC50 = 550 nM (pChEMBL 6.260000228881836)
Descriptors: MW 509.58, ALogP 5.5, HBA 6, HBD 1, PSA 93.89, QED 0.49
Compound (SMILES): CCc1nc2ccc(C3=NC(CO)CO3)cn2c1N(C)c1nc(-c2ccc(F)cc2)cs1
InChIKey: LZLYKNYLKYQYJN-UHFFFAOYSA-N
Target: Autotaxin (SINGLE PROTEIN), Homo sapiens
UniProt: Q13822
Assay: Biochemical Assay: TABLE VI: (UniProtKB/SwissProt Sequence ref Q13822) biochemical assay using the fluorogenic autotaxin substrate FS-3 as substrate. FS-3 is a doubly labeled analog of LPC wherein the fluorophore is quenched through intramolecular energy transfer. Without hENPP2, the emission of the probe is quenched. If the substrate is hydrolyzed by hENPP2, the emission of the probe is not quenched anymore resulting in a fluorescence increase. Inhibition of hENPP2 by compounds will result in a decrease of the signal.10 μL of a dilution series of compound, starting from 20 μM highest concentration, 1/5 dilution, is added to the wells. hENPP2 is used at a final concentration of 0.4 μg/mL or 0.64 μg/mL (it will be appreciated by the skilled person that the potency read out is independent of the enzyme concentration). The enzyme is diluted in 50 mM Tris-HCl pH 8.0, 250 mM NaCl, 5 mM KCl, 1 mM MgCl2, 1 mM CaCl2, 0.1% fatty acid free BSA in a total volume of 20 μL. Enzyme mixture is added to compounds and the resulting mixture is incubated for 30 min at room temperature under shaking. The reaction is started by the addition of 20 μL of 0.75 μM FS-3 diluted in the same buffer as described above and the mixture is incubated at 30° C. for 30 min. Fluorescence is read on the Envision (Excitation 485 nm, emission 520 nM).
Measurement: IC50 = 50 nM (pChEMBL 7.300000190734863)
Descriptors: MW 451.53, ALogP 4.06, HBA 8, HBD 1, PSA 75.25, QED 0.48
Compound (SMILES): O=c1n(Cc2ccc(Cl)c(F)c2)c2sc3c(c2c2ncnn12)CCOC3
InChIKey: DGAOAGBVDNUYSF-UHFFFAOYSA-N
Target: Dual specificity calcium/calmodulin-dependent 3',5'-cyclic nucleotide phosphodiesterase 1B (SINGLE PROTEIN), Homo sapiens
UniProt: Q01064
Assay: PDE1B Inhibitory Assay: PDE1B inhibition was determined by an IMAP TR-FRET assay. The IMAP TR-FRET PDE assay was optimized for concentration of enzyme, Calmodulin, cAMP or cGMP substrate, DMSO tolerance, and incubation time. Into each well of a solid white 1536 well plate (Corning) was dispensed 250 pg full-length recombinant NH-terminal GST tagged human PDE1B enzyme (BPS Bioscience Cat #60011, San Diego, Calif.) in 2.5 μL IMAP BSA reaction buffer (Molecular Devices, Sunnyvale, Calif.) containing 10 U/ml Calmodulin and 2.5 mM CaCl2 (Sigma Aldrich.) After a brief centrifugation, 30 nl compound was added by transfer from 1 mM stock in DMSO using a Kalypsys 1536 Pintool. Plates were incubated for 5 minutes at room temperature before dispensing 1.5 μL of 533 nM 5-carboxy fluorescein (FAM)-labeled cAMP (Molecular Devices, Sunnyvale, Calif.) for a final concentration of 200 nM. After a brief centrifugation, the plates were incubated for 30 minutes at room temperature. The assay was terminated by adding 5 μL IMAP binding reagent/Tb complex (Molecular Devices, Sunnyvale, Calif.) to each well. Plates were incubated 1 hour at room temperature and read on a Viewlux multimode plate reader (Perkin Elmer). The instrument was set to excite using the DUG11 filter and measure using 490/10 nm and 520/10 nm filters. Ratios of acceptor and donor were then calculated.
Measurement: IC50 = 550 nM (pChEMBL 6.260000228881836)
Descriptors: MW 390.83, ALogP 3.02, HBA 7, HBD 0, PSA 61.42, QED 0.53
Compound (SMILES): Nc1nccn2c([C@@H]3CCCCN3C(=O)c3ccnc(Cl)n3)nc(-c3ccc(C(=O)Nc4cccnn4)cc3)c12
InChIKey: RVSFDJGGLLQAOC-IBGZPJMESA-N
Target: Tyrosine-protein kinase BTK (SINGLE PROTEIN), Homo sapiens
UniProt: Q06187
Assay: Btk Enzyme Activity: Btk enzyme (His-Btk (Millipore catalog #14-552), is diluted to 0.4 U/mL in KR buffer (10 mM Tris-HCl, 10 mM MgCl2, 0.01% Tween-20, 0.05% NaN3, 1 mM DTT, 2 mM MnCl2, pH 7.2).Serial dilution log 10 from 2 mM to 63.2 nM of test compounds are made in 100% DMSO. The dilutions in DMSO are then diluted 50-fold in KR-buffer. Final compound concentration range in the assay from 10 μM to 0.316 nM.5 μL/well of test compound in KR buffer (final DMSO concentration in the assay is 1%) is mixed with 5 μl/well of 0.4 U/mL Btk enzyme (final concentration in the assay is 0.1 U/mL). Test compounds and Btk enzyme are pre-incubated 60 minutes at room temperature, before adding 5 μL/well of 200 nM Fluorescin labeled substrate peptide (Blk/Lyntide substrate, e.g. #R7188/#R7233, Molecular Devices) in KR-buffer. Final peptide substrate concentration in assay is 50 nM. The kinase assay is started by adding 5 μL/well of 20 μM ATP in KR-buffer (final ATP concentration is 5 μM ATP, Km ATP in Btk IMAP assay). Following incubation for 2 h at room temperature the enzyme reaction is stopped by adding 40 μL/well IMAP Progressive Binding Solution (according to suppliers (Molecular Devices) protocol using 75% 1× buffer A and 25% 1× buffer B with 1:600 Progressive Binding Solution). After 60 min incubation at room temperature in the dark the FP signal is read. Fluorescence at 535 nm is measured using parallel and perpendicular filters to determine differences in rotation due to binding of the phosphorylated substrate peptide to the beads. Values are calculated as percentage of the difference in readout (ΔmPi) of the controls with and without ATP. EC50 values are determined by curve fitting of the experimental results using Activity Base.
Measurement: EC50 > 100 nM (pChEMBL 7)
Descriptors: MW 555, ALogP 3.83, HBA 10, HBD 2, PSA 157.18, QED 0.31
Compound (SMILES): O=C(N[C@@H]1CCNC1)c1sc2nccc3c2c1NC(=O)N3c1ccc(Oc2ccccc2)cc1
InChIKey: WUCKMTZUSNOPJL-OAHLLOKOSA-N
Target: Tyrosine-protein kinase BTK (SINGLE PROTEIN), Homo sapiens
UniProt: Q06187
Assay: TBD: TBD
Measurement: IC50 = 251 nM (pChEMBL 6.599999904632568)
Descriptors: MW 471.54, ALogP 4.86, HBA 6, HBD 3, PSA 95.59, QED 0.39
Compound (SMILES): COc1cccc(-c2nc(-c3ccc(CCN4C5CCC4COC5)cn3)nn2CC(=O)NC(C)(C)C)c1
InChIKey: PMQMWNGJQFXWLW-UHFFFAOYSA-N
Target: Vasopressin V1b receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P47901
Assay: Binding Assay: Human V1b receptor was transiently expressed in 293FT cells (Invitrogen). The cells were collected and then homogenated in a 15 mmol/L tris-hydrochloric acid buffer (pH 7.4 and containing 2 mmol/L magnesium chloride, 0.3 mmol/L ethylenediaminetetracetic acid, and 1 mmol/L glycol ether diaminetetraacetic acid). The resulting homogenate was centrifuged at 50,000xg at 4° C. for 20 minutes. The precipitate was resuspended in a 75 mmol/L tris-hydrochloric acid buffer (pH 7.4 and containing 12.5 mmol/L magnesium chloride, 0.3 mmol/L ethylenediaminetetracetic acid, 1 mmol/L glycol ether diaminetetraacetic acid, and 250 mmol/L sucrose) to give a crude membrane preparation, which was stored at â¿¿80° C. until the binding test was initiated. In the binding test, the crude membrane preparation was diluted with a 50 mmol/L tris-hydrochloric acid buffer (pH 7.4 and containing 10 mmol/L magnesium chloride and 0.1% bovine serum albumin) and mixed with each test compound and [3H]AVP (final concentration: 0.4 to 1 nmol/L), followed by incubation at room temperature for 60 minutes. The test compound was serially diluted with DMSO so that it would have final concentrations of 0.01 nmol/L to 1 umol/L at the time of mixing. After the incubation, the mixture was suction filtered through a GF/C filter that was preliminarily impregnated with 0.3% polyethyleneimine. The GF/C filter was dried and after adding a scintillator, the residual radioactivity on the filter was measured using TopCount (PerkinElmer Inc.). The radioactivity in the presence of unlabeled AVP at 10 mmol/L was defined as 0%, and the radioactivity in the absence of unlabeled AVP was defined as 100%. A dose-response curve was plotted from radio activities in the presence of a test compound at various concentrations, and the 50% inhibitory concentration (IC50 value) of the test compound was calculated. The IC50 values of the compounds of the present invention were in the range of 0.1 to 1000 nM.
Measurement: IC50 = 4.3 nM (pChEMBL 8.369999885559082)
Descriptors: MW 504.64, ALogP 3.34, HBA 8, HBD 1, PSA 94.4, QED 0.5