lovingscience/chembl-v37-raw-activities huggingface dataset:
Compound (SMILES): C=CC(=O)N1CC2(CC(n3nc(-c4cnc5c(c4)ncn5C)c(-c4c(Cl)c(C)cc5[nH]ncc45)c3C)C2)C1
InChIKey: AYORTEJLITUNTG-UHFFFAOYSA-N
Target: GTPase KRas (SINGLE PROTEIN), Homo sapiens
UniProt: P01116
Assay: Scintillation Proximity Assay (SPA): Assays were run using 384-well plates (781207/Greiner) in which one column was designated as the high signal (no inhibition) control, and contained DMSO with no test compound, and another column was designated as the low signal control (maximal inhibition), and contained no protein. Serial dilutions of compounds to be tested were added to the assay plate (resulting in duplicate 11-point dose response with semi-log compound dilutions from 50 μM to 0.5 nM or from 5 μM to 0.05 nM for the most potent compounds). A 1/20 isotopic dilution of labeled (Compound B) and non-labeled covalent (Compound A) probe was prepared and added to all wells on the plate. The reaction was started by addition of KRasG12C (M1-K169, biotinylated on the N-terminus) to the compounds and incubated for 2 hours with continuous agitation allowing for full modification of KRasG12C with probe or test compounds. Final concentrations in an assay volume of 40 μL were 10 nM KRasG12C, 25 nM radio-ligand and 475 nM unlabelled ligand. The assay buffer contained 20 mM Tris-HCl pH 7.5 (Invitrogen), 150 mM NaCl (Sigma Aldrich), 0.1 mM MgCl2 (Sigma Aldrich), and 0.01% Tween-20 (Sigma Aldrich). Following addition of 50 μL of a 400 μg/ml. suspension of streptavidin-coated YSi beads (Perkin Elmer), plates were incubated for a further 30 min with continuous agitation before reading the plates on a scintillation counter (Topcount NXT 384 (Packard).
Measurement: IC50 > 50000 nM (pChEMBL 4.301000118255615)
Descriptors: MW 527.03, ALogP 4.99, HBA 7, HBD 1, PSA 97.52, QED 0.33
Compound (SMILES): CCOc1cc2nccc(Oc3ccc(NC(=O)NC4CC4)c(Cl)c3)c2c2c1OCCO2
InChIKey: QDZAYZVEGUECMK-UHFFFAOYSA-N
Target: Vascular endothelial growth factor receptor 2 (SINGLE PROTEIN), Homo sapiens
UniProt: P35968
Assay: Assay of Small Molecular Compounds for Inhibiting the Activity of VEGFR-2 Kinase: The assay is based on the LANCE TR-FRET technology of Perkin Elmer Inc., and the assay method is as follows:1. Dilution of compounds: a total of 11 concentrations were obtained using a 3-fold gradient dilution from the highest concentration of 2500 nM (the maximum final concentration of the drug used in this assay was 2500 nM, and the minimum final concentration was 0.042 nM).2. 2.5 μL of the gradient-diluted compounds were taken with a transfer pipette to a 384-well plate.3. Addition of enzyme: 5 μL of 2× VEGFR2 kinase solution (concentration was 0.5 nM) was taken with a transfer pipette to the corresponding reaction well of the 384-well plate, mixed well and pre-reacted at room temperature for 30 minutes.4. 2.5 μL 4× Ultra ULight-JAK-1 (Tyr1023) Peptide (concentration was 200 nM)/ATP (concentration was 40 μMM) mixture was taken with a transfer pipette to the corresponding reaction well of the 384-well plate.5. Negative control: 2.5 μL/well 4× substrate/ATP mixture and 7.5 μL 1× Kinase Assay Buffer were added to the wells of the 384-well plate.6. Positive control: 2.5 μL/well 4× substrate/ATP mixture, 2.5 μL/well 1× Kinase Assay Buffer containing 16% DMSO, and 5 μL/well 2× VEGFR2 kinase solution were added to the 384-well plate. The final concentration of DMSO in the reaction system was 4%.7. The mixture was mixed well and then centrifuged and reacted at room temperature in dark for 60 min.8. Termination of the enzymatic reaction: 5 μL of 4× stop solution was taken with a transfer pipette to the wells of the 384-well plate, mixed and then centrifuged, and reacted at room temperature for 5 min.9. Development of the reaction: 5 μL of 4× detection solution was taken with a transfer pipette to the wells of the 384-well plate for color development, and the mixture was mixed and then centrifuged and reacted at room temperature for 60 min.10. The 384-well plate was placed into the Envision plate reader and the signal was detected using the appropriate program.11. Analysis and processing of the raw data:The drug concentrations and the corresponding inhibition rates were input into GraphPad Prism5 for calculation, and the inhibition rate of the compounds were calculated as follows: inhibition rate (%)=(reading of positive well−reading of experimental well)/(reading of positive control well−reading of negative control well)×100%. Processing with GraphPad Prism5 software yielded the corresponding IC50 values (the concentration of the compound at which 50% of the highest inhibition of the enzyme is achieved).
Measurement: IC50 < 50 nM (pChEMBL 7.301000118255615)
Descriptors: MW 455.9, ALogP 5.13, HBA 6, HBD 2, PSA 90.94, QED 0.53
Compound (SMILES): Cc1ncc(CC(=O)N[C@H]2CC[C@H](CCN3CCc4ccc(OC5CCC5)nc4CC3)CC2)o1
InChIKey: NNCCKSDTBPBUII-AQYVVDRMSA-N
Target: D(2) dopamine receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P14416
Assay: Binding Inhibition for Dopamine D2 Receptor: Buffer solution: 50 mM Tris-HCl (35409-45, Nacalai Tesque) (pH 7.4) containing 120 mM NaCl (31320-05, Nacalai Tesque), 1 mM MgCl.06HO (20909-55, Nacalai Tesque), 5 mM KCl (28514-75, Nacalai Tesque) and 2 mM CaCl) (067-31, NAKARAI CHEMICALS, LTD.)Radioligand: (final concentration) 1.2 nM [H]-Methylspiperone ([HN-methyl-]-Methylspiperone, NET-856, 83.8 Ci/mmol, PerkinElmer) Non-specific ligand: (final concentration) 10 μM Butaclamol [(+)-Butaclamol Hydrochloride, D033, Sigma] SPA beads solution: SPA beads [WGA PVT SPA Scintillation Beads, RPNQ0001 (500 mg), RPNQ0060 (2 g), PerkinElmer] (0.2 mg/well)Incubation time and temperature: 120 min at 25° C.Kd: 0.272 nM(Preparation of Solutions of Non-Specific Ligand or the Compounds of the Present Invention)Butaclamol or the compounds of the present invention were weighed and DMSO was added to make a 10 mM solution. This solution was diluted to each concentration.(Preparation of Radioligand Solution),[H]-Methylspiperone was weighed and the buffer solution was added to make a 3.6 nM solution.(Preparation of SPA Beads Solution) SPA beads were weighed and stirred in water to make a 50 mg/mL solution. Using this solution, a mixture with the cell membranes was prepared.
Measurement: Ki > 2200 nM (pChEMBL 5.6579999923706055)
Descriptors: MW 466.63, ALogP 4.02, HBA 6, HBD 1, PSA 80.49, QED 0.63
Compound (SMILES): Nc1ncc(-c2nn[nH]n2)cc1-c1cc2cc(NC(=O)Nc3ccc(Cl)c(C(F)(F)F)c3)ccc2s1
InChIKey: LHUVPYZTQHNXRU-UHFFFAOYSA-N
Target: Interferon-induced, double-stranded RNA-activated protein kinase (SINGLE PROTEIN), Homo sapiens
UniProt: P19525
Assay: KinaseGlo Assay: PKR: Commercially available recombinant human GST-PKR (SignalChem, Canada; 1.5 uM-2 uM stock) is diluted to 500 nM in assay buffer (20 mM Tris-HCl, pH 7.2, 10 mM KCl, 10 mM MgCl2, 10% glycerol). Preactivated PKR is dispensed to 384/96-well black plates at 3.125/12.5 uls/well using the liquid handler Janus. Appropriate dilutions of inhibitors are added to 384/96-well plate followed by 6.6 uM ATP (final) and incubated for 10 minutes at room temperature. The remaining ATP/well is determined by adding 6.25/25 uls/well Kinase-Glo assay mix (Promega) and luminescence is measured on EnVision luminescence plate reader (integration time, 0.2 sec; Perkin-Elmer, Mass., USA). The % inhibition for the compounds is calculated using ATP only (100% inhibition) and PKR+ATP (0% inhibition).
Measurement: IC50 = 140 nM (pChEMBL 6.849999904632568)
Descriptors: MW 530.92, ALogP 6.04, HBA 7, HBD 4, PSA 134.5, QED 0.22
Compound (SMILES): Cc1nccc(-c2[nH]c3ccc(C4CCNCC4)cc3c2C(C)C)c1Cl
InChIKey: DDRQEGKSAFCYRC-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 = 14.4 nM (pChEMBL 7.840000152587891)
Descriptors: MW 367.92, ALogP 5.78, HBA 2, HBD 2, PSA 40.71, QED 0.62
Compound (SMILES): Cc1cc(Nc2cc3ncccc3c(N[C@@H]3C[C@H]4CC[C@@H](C3)N4CC(F)(F)F)n2)n[nH]1
InChIKey: ZHEHTFPUWPZJEG-QKDCVEJESA-N
Target: Tyrosine-protein kinase JAK2 (SINGLE PROTEIN), Homo sapiens
UniProt: O60674
Assay: Biochemical JAK Assay: A panel of four LanthaScreen JAK biochemical assays (JAK1, 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 lh. ATP was subsequently added to initiate the kinase reactions in 10 μL total volume, with 1% DMSO. The final enzyme concentrations for JAK1, 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 lh 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.For dose-response analysis, percent inhibition data were plotted vs. compound concentrations, and IC50 values were determined from a 4-parameter robust fit model with the Prism software (GraphPad Software). Results were expressed as pIC50 (negative logarithm of IC50) and subsequently converted to pKi (negative logarithm of dissociation constant, Ki) using the Cheng-Prusoff equation.Test compounds having a higher pKi value in each of the four JAK assays show greater inhibition of JAK activity. Compounds of the invention tested in this assay typically exhibited pKi values between about 7 and about 10.3
Measurement: Ki = 0.55 nM (pChEMBL 9.260000228881836)
Descriptors: MW 431.47, ALogP 4.37, HBA 6, HBD 3, PSA 81.76, QED 0.56
Compound (SMILES): CCC[C@@H](C(=O)Nc1ccc2nccnc2c1)n1cc(OC)c(-c2cc(Cl)ccc2-n2cnnn2)cc1=O
InChIKey: BSNRZZUTVDCZAA-QHCPKHFHSA-N
Target: Plasma kallikrein (SINGLE PROTEIN), Homo sapiens
UniProt: P03952
Assay: Activity Assay: To determine the plasma kallikrein inhibition of the substances according to the invention, a biochemical test system is used which utilizes the reaction of a peptidic plasma kallikrein substrate to determine the enzymatic activity of human plasma kallikrein. Here, plasma kallikrein cleaves from the peptic plasma kallikrein substrate the C-terminal aminomethylcoumarin (AMC), the fluorescence of which is measured. The determinations are carried out in microtitre plates.Test substances are dissolved in dimethyl sulphoxide and serially diluted in dimethyl sulphoxide (3000 μM to 0.0078 μM; resulting final concentrations in the test: 50 μM to 0.00013 μM). In each case 1 μl of the diluted substance solutions is placed into the wells of white microtitre plates from Greiner (384 wells). 20 μl of assay buffer (50 mM Tris/HCl pH 7.4; 100 mM sodium chloride solution; 5 mM of calcium chloride solution; 0.1% of bovine serum albumin) and 20 μl of plasma kallikrein from Kordia (0.6 nM in assay buffer) are then added successively. After 15 min of incubation, the enzyme reaction is started by addition of 20 μl of the substrate H-Pro-Phe-Arg-AMC dissolved in assay buffer (10 μM in assay buffer) from Bachem, the mixture is incubated at room temperature (22° C.) for 30 min and fluorescence is then measured (excitation: 360 nm, emission: 460 nm).
Measurement: IC50 = 0.57 nM (pChEMBL 9.239999771118164)
Descriptors: MW 530.98, ALogP 4.08, HBA 10, HBD 1, PSA 129.71, QED 0.32
Compound (SMILES): COCC(C)Oc1cc(-c2noc(C(C)(C)C)n2)ncc1C1CC1
InChIKey: PEYIQKRZOWIHGU-UHFFFAOYSA-N
Target: Cannabinoid receptor 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P21554
Assay: Radioligand Binding Assay: The affinity of the compounds of the invention for cannabinoid CB1 receptors was determined using recommended amounts of membrane preparations (PerkinElmer) of human embryonic kidney (HEK) cells expressing the human CNR1 or CNR2 receptors in conjunction with 1.5 or 2.6 nM [3H]-CP-55,940 (Perkin Elmer) as radioligand, respectively. Binding was performed in binding buffer (50 mM Tris, 5 mM MgCl2, 2.5 mM EDTA, and 0.5% (wt/vol) fatty acid free BSA, pH 7.4 for CB1 receptor and 50 mM Tris, 5 mM MgCl2, 2.5 mM EGTA, and 0.1% (wt/vol) fatty acid free BSA, pH 7.4 for CB2 receptor) in a total volume of 0.2 ml for 1 h at 30° C. shaking. The reaction was terminated by rapid filtration through microfiltration plates coated with 0.5% polyethylenimine (UniFilter GF/B filter plate; Packard). Bound radioactivity was analyzed for Ki using nonlinear regression analysis (Activity Base, ID Business Solution, Limited), with the Kd values for [3H]CP55,940 determined from saturation experiments. The compounds of formula (I) show an excellent affinity for the CB2 receptor with affinities below 10 μM, more particularly of 1 nM to 3 μM and most particularly of 1 nM to 100 nM.
Measurement: EC50 > 10000 nM (pChEMBL 5)
Descriptors: MW 331.42, ALogP 3.72, HBA 6, HBD 0, PSA 70.27, QED 0.8
Compound (SMILES): CN(C)C(=O)c1ccc(-c2cnc(N)c(-c3nnc(N4CCC4)o3)n2)cc1
InChIKey: RWWRZPXAOFAGKY-UHFFFAOYSA-N
Target: Serine/threonine-protein kinase ATR (SINGLE PROTEIN), Homo sapiens
UniProt: Q13535
Assay: Inhibition Assay: Compounds were screened for their ability to inhibit ATR kinase using a radioactive-phosphate incorporation assay. Assays were carried out in a mixture of 50 mM Tris/HCl (pH 7.5), 10 mM MgCl2 and 1 mM DTT. Final substrate concentrations were 10 μM [γ-33P]ATP (3 mCi 33P ATP/mmol ATP, Perkin Elmer) and 800 μM target peptide (ASELPASQPQPFSAKKK) (SEQ ID NO:1)).Assays were carried out at 25° C. in the presence of 5 nM full-length ATR. An assay stock buffer solution was prepared containing all of the reagents listed above, with the exception of ATP and the test compound of interest. 13.5 μL of the stock solution was placed in a 96 well plate followed by addition of 2 μL of DMSO stock containing serial dilutions of the test compound (typically starting from a final concentration of 15 μM with 3-fold serial dilutions) in duplicate (final DMSO concentration 7%). The plate was pre-incubated for 10 minutes at 25° C. and the reaction initiated by addition of 15 μL [γ-33P]ATP (final concentration 10 μM).
Measurement: Ki = 2550 nM (pChEMBL 5.590000152587891)
Descriptors: MW 365.4, ALogP 1.69, HBA 8, HBD 1, PSA 114.27, QED 0.74
Compound (SMILES): COc1cc2nc3n(c2cc1OC)CCn1cc(cn1)-c1ccc(=O)n(n1)Cc1cccc(c1)C(=O)N3
InChIKey: JEKNTDXKUAKXQX-UHFFFAOYSA-N
Target: Interleukin-1 receptor-associated kinase 4 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9NWZ3
Assay: IRAK4 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 is performed in streptavidin coated 384 well FlashPlate (PerkinElmer #SMP410A). His-TEV-IRAK4 (20 ng/well), ATP (2 μM, [33P]ATP 0.25 μCi/well), STK1-biotin peptide (300 nM) and compounds in DMSO (range of concentrations from 20 μM to 1 nM) or controls (2% DMSO) are incubated for 3 hours at 30° C. in assay buffer: Hepes pH 7.0 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.Kinase reaction is stopped by addition of EDTA. Supernatant is discarded, plates are washed three times with 150 mM NaCl and radioactivity is then measured in a Microbeta Trilux reader.
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 497.52, ALogP 2.79, HBA 10, HBD 1, PSA 118.09, QED 0.4
Compound (SMILES): COc1cc(F)cc2c1nc(N)n1nc([C@@H]3CCCN(c4cnn(C(C)C(C)O)c4)C3)nc21
InChIKey: KXQJKYKALIPNNM-JXQTWKCFSA-N
Target: Adenosine receptor A2b (SINGLE PROTEIN), Homo sapiens
UniProt: P29275
Assay: Measurement of A2A and A2B Antagonism in cAMP Cell-Based Assay : The ability of compounds to antagonize human A2A and A2B adenosine receptors was determined using a kit to measure changes in intracellular cyclic AMP levels (LANCE cAMP 384 Kit, Perkin Elmer, Cat. No. AD0264). HEK293 cells recombinantly expressing either human A2A or A2B receptors, previously frozen in Recovery Medium (Life Technologies, Cat. No. 12648-010) were thawed and diluted into stimulation buffer (HBSS (Hyclone SH 30268.01), 5 mM HEPES (Gibco 15630-106), 200 nM rolipram (Tocris, Cat. No. 0905), and 1.5% (V/v) BSA stabilizer (kit component). The cell suspension was centrifuged at 200×g for 10 min and then resuspended in stimulation buffer, supplemented with a 1:10 000 dilution of Alexa Fluor 647 anti-cAMP antibody, to a density of 6.0×105 cells/mL. A Labcyte Echo 550 acoustic dispenser was used to transfer up to 25 nL of test compound dissolved in DMSO into the wells of a dry Optiplate-384 plate (Perkin Elmer, Cat. No. 6008289). All subsequent liquid additions were performed using a multichannel pipettor. Next, 5 μL of the cell suspension was added to the wells of the Optiplate-384 and incubated for 30 min. at 37° C. and 5% CO2 in a humidified environment. After this time 5 μL of either 300 nM or 600 nM adenosine (Sigma Cat. No. A9251) for A2A and A2B respectively was added and incubated for 30 minutes at 37° C. and 5% CO2 in a humidified environment. At this time detection mix was prepared by combining the LANCE Eu-W8044 labeled streptavidin and Biotin-cAMP in detection buffer according to the manufacturers protocol. 10 μL of the detection mix was added to each well of the Optiplate-384 which was covered with a plate seal and incubated under ambient conditions for 2 hours prior to reading the plate using an Envision (Perkin Elmer, Waltham, Mass.) multimode plate reader. Data was normalized by defining minimal effect as stimulation in the presence of 0.25% (v/v) DMSO and maximal effect as stimulation in the presence of 1 μM ZM241385 (Cayman, Cat. No. 1036). Curve fitting of the percent effect data versus the log of compound concentration used a 4-parameter concentration response curve fitting algorithm to calculate IC50 values.
Measurement: IC50 = 49.8 nM (pChEMBL 7.300000190734863)
Descriptors: MW 454.51, ALogP 2.53, HBA 10, HBD 2, PSA 119.62, QED 0.47
Compound (SMILES): CC(F)(F)c1cc(-n2cc(COc3ncc(CO)cn3)nn2)ccc1F
InChIKey: GXWUNZYRWMEXFQ-UHFFFAOYSA-N
Target: Glutamate NMDA receptor; GRIN1/GRIN2B (PROTEIN COMPLEX), Homo sapiens
UniProt: Q05586
Assay: NMDA receptor FLIPR assay: NMDA receptors are ion channels that are highly permeable to Ca2+ ions, rendering it possible to monitor NMDA receptor function using cell-based calcium flux assay. In this assay, co-agonists glutamate and glycine are added to cells heterologously expressing human GluN1/GluN2B NMDA receptors to initiate cellular Ca2+ influx. The time course of the changes in intracellular calcium is measured using a fluorescent dye and a FLIPR (Fluorometric Imaging Plate Reader) device.Twenty four hours before measurements, the expression of the NMDA receptors in the stable cell line is induced with Tet-On inducible system in the presence of a non-selective NMDA receptor blocker. On the day of the experiment, cell culture media is carefully washed and the cells are loaded with Calcium 5 Dye Kit (Molecular Devices) in dye loading buffer containing 149 mM NaCl, 4 mM KCl, 2 mM CaCl2, and 1.5 mM MgCl2, 10 mM HEPES and 5 mM D-glucose; pH 7.4. After 1 h incubation at the room temperature, the dye is washed away with the assay buffer (149 mM NaCl (standard assay) or 150 mM (HTS assay), 4 mM KCl (standard assay) or 3 mM (HTS assay), 2 mM CaCl2, 0.01 mM EDTA, 10 mM HEPES and 5 mM D-glucose; pH 7.4) In the FLIPR TETRA reader, various concentrations of the test compounds are added to the cells for 5 min while fluorescence is monitored to detect potential agonist activity. Next, co-agonists, glutamate and glycine are added for another 5 minutes. The concentration of glutamate corresponding to EC80 (standard assay) or EC40 (HTS assay) is used to maximize the assay's signal window or the ability to detect NMDA receptor antagonists and negative allosteric modulators, respectively. A saturating concentration (10 μM) of glycine is also present in the assay. A non-selective NMDA receptor antagonist, (+)MK-801 is used as a positive control for antagonist activity. The fluorescent signal in the presence of test compounds is quantified and normalized to the signal defined by the appropriate control wells.
Measurement: IC50 = 21 nM (pChEMBL 7.679999828338623)
Descriptors: MW 365.32, ALogP 2.38, HBA 7, HBD 1, PSA 85.95, QED 0.72
Compound (SMILES): C=CC(=O)N1CCC[C@@H](Nc2nc(Nc3cnn(C4CC4)c3)nc3[nH]nc(C)c23)C1
InChIKey: CTLAGFKOZPODTI-CYBMUJFWSA-N
Target: Tyrosine-protein kinase BTK (SINGLE PROTEIN), Homo sapiens
UniProt: Q06187
Assay: Enzyme Inhibitory Activity: JAK3 and BTK kinase inhibitory activities were measured for the compounds prepared in the above Examples through in vitro analysis on the ADP Glow (Glo) platform.Specifically, the inhibitory activities of JAK3 and BTK kinase were measured using a JAK3 kinase assay kit (Promega, V9441) and a BTK kinase assay kit (Promega, V9071) which were purchased from Promega. Recombinant purified human JAK3 and BTK were diluted with 1×kinase reaction buffer (JAK3: 40 mM Tris-Cl, pH 7.5, 20 mM MgCl2, 0.1 mg/mL BSA and 50 uM DTT/BTK: 40 mM Tris-Cl, pH 7.5, 20 mM MgCl2, 0.1 mg/mL BSA, 2 mM MnCl2 and 50 uM DTT) and added to a 96 well plate (JAK3: final concentration of 4 ng per reaction/BTK: final concentration of 8 ng per reaction). The compounds were treated so as to be finally a 1% DMSO aqueous solution, and a substrate cocktail containing ATP (JAK3: final concentration of 5 uM/BTK: final concentration of 10 uM) and 0.2 μg/μL of Poly(Glu4, Tyr1) peptide (JAK3 and BTK final concentration) in the total 254 reactants was added to a 96-well plate to initiate enzymatic reaction. After incubation (30° C.) for 1 hour, equivalent volume (254 per reaction) of ADP Glo was added and incubated (30° C.) for 40 minutes at room temperature. Then, a kinase detection reagent (504 per reaction) was added and incubated (30° C.) for 30 minutes at room temperature. The kinase activity was measured by chemiluminescence according to the instructions of ADP Glo kinase assay kit, and the inhibitory activity of the compounds according to the present invention was calculated.
Measurement: IC50 = 2.9 nM (pChEMBL 8.539999961853027)
Descriptors: MW 407.48, ALogP 2.53, HBA 8, HBD 3, PSA 116.65, QED 0.54
Compound (SMILES): O=C(NCC1(O)CCC(F)(F)CC1)c1cn(CC(F)(F)F)c2cccc(Cl)c12
InChIKey: HKIPQWVBOHKCFR-UHFFFAOYSA-N
Target: Interleukin-1 beta (SINGLE PROTEIN), Homo sapiens
UniProt: P01584
Assay: IL-1beta release assay: The activation of P2X7 by ATP leads to a fast transient activation of cells resulting in influx of Ca2+ followed by conversion of pro-IL-1β to active IL-1β. The functional activity of P2X7 compounds was measured by the release of mature IL-13 in the culture medium of THP-1 cells, detected by sandwich ELISA. Cells were maintained in complete growth medium (RPMI 1640+10% HI-FCS+2 mM L-glutamine+1×PS). Every 3 days, the medium was renewed by diluting the cells 1/3 to 1/4 as cell density did not exceed 0.5 million cells per ml (seeding cell density @1×105/ml). THP-1 cells were harvested from the flask in 50 ml by centrifugation for 3 min at 100 g. The cells were resuspended to 2×105 cells/ml in medium supplemented with 0.5 μM PMA and incubated. The cells were washed and resuspended to 1.5×105 cells/ml in medium complemented with 10 ng/ml LPS, and the cells were primed for 4 h at 37° C., 5% CO2. After addition of 20 μL of prediluted test compounds, blank, standard and control reagents, cells were incubated for a further 20 min at 37° C. and stimulated with 0.8 mM BzATP for 30 minutes. The cells were centrifuged, supernatant was collected and the presence of mature IL-1β was detected using Dual human IL-1b kit following manufacturer's instruction. The tetrahydrobenzodiazepine analogs effectively modulated the activity of P2X7 in the cells as measured by the levels of pro-inflammatory cytokine IL-1β, which is released by the activation of P2X7 receptor.
Measurement: IC50 = 300 nM (pChEMBL 6.519999980926514)
Descriptors: MW 424.8, ALogP 4.53, HBA 3, HBD 2, PSA 54.26, QED 0.71
Compound (SMILES): COc1ccc(-n2cc(C(=O)Nc3ccc([C@H]4CNCCO4)cc3)cn2)cc1
InChIKey: ICERLSSUACJGGO-HXUWFJFHSA-N
Target: Trace amine-associated receptor 1 (SINGLE PROTEIN), Rattus norvegicus
UniProt: Q923Y9
Assay: Radioligand Binding Assay: HEK-293 cells stably expressing rat TAAR1 were maintained at 37° C. and 5% CO2 in DMEM high glucose medium, containing fetal calf serum (10%, heat inactivated for 30 min at 56° C.), penicillin/streptomycin (1%), and 375 ug/ml geneticin (Gibco). Cells were released from culture flasks using trypsin/EDTA, harvested, washed twice with ice-cold PBS (without Ca2+ and Mg2+), pelleted at 1,000 rpm for 5 min at 4° C., frozen and stored at -80° C. Frozen pellets were suspended in 20 ml HEPES-NaOH (20 mM, pH 7.4) containing 10 mM EDTA and homogenized with a Polytron (PT 6000, Kinematica) at 14,000 rpm for 20 s. The homogenate was centrifuged at 48,000xg for 30 min at 4° C. Subsequently, the supernatant was removed and discarded, and the pellet resuspended in 20 ml HEPES-NaOH (20 mM, pH 7.4) containing 0.1 mM EDTA using the Polytron (20 s at 14,000 rpm). This procedure was repeated and the final pellet resuspended in HEPES-NaOH containing 0.1 mM EDTA and homogenized using the Polytron. Typically, aliquots of 2 ml membrane portions were stored at -80° C. With each new membrane batch the dissociation constant (Kd) was determined via a saturation curve. The TAAR1 radioligand 3[H]-(S)-4-[(ethyl-phenyl-amino)-methyl]-4,5-dihydro-oxazol-2-ylamine (described in WO 2008/098857) was used at a concentration equal to the calculated Kd value, that was usually around 0.7 nM, resulting in the binding of approximately 0.5% of the radioligand and a specific binding representing approximately 70% of the total binding. Nonspecific binding was defined as the amount of 3[H]-(S)-4-[(ethyl-phenyl-amino)-methyl]-4,5-dihydro-oxazol-2-ylamine bound in the presence of 10 uM unlabeled ligand. All compounds were tested at a broad range of concentrations (10 pM to 10 uM) in duplicates. The test compounds (20 ul/well) were transferred into a 96 deep well plate (TreffLab), and 180 ul of HEPES-NaOH (20 mM, pH 7.4) containing MgCl2 (10 mM) and CaCl2 (2 mM) (binding buffer), 300 ul of the radioligand 3[H]-(S)-4-[(ethyl-phenyl-amino)-methyl]-4,5-dihydro-oxazol-2-ylamine at a concentration of 3.3xKd in nM and 500 ul of the membranes (resuspended at 60 ug protein per ml) added. The 96 deep well plates were incubated for 1 hr at 4° C. Incubations were terminated by rapid filtration through Unifilter-96 plates (Packard Instrument Company) and glass filters GF/C (Perkin Elmer) presoaked for 1 hr in polyethylenimine (0.3%) and washed 3 times with 1 ml of cold binding buffer. After addition of 45 ul of Microscint 40 (PerkinElmer) the Unifilter-96 plate was sealed and after 1 hr the radioactivity counted using a TopCount Microplate Scintillation Counter (Packard Instrument Company).
Measurement: Ki = 16.5 nM (pChEMBL 7.78000020980835)
Descriptors: MW 378.43, ALogP 2.79, HBA 6, HBD 2, PSA 77.41, QED 0.71
Compound (SMILES): Cc1nc(C(=O)N2CC(F)CCC2CNC(=O)c2cccc3cccnc23)c(-c2ccccc2)s1
InChIKey: BSPIQLLFYKKPND-UHFFFAOYSA-N
Target: Orexin receptor type 2 (SINGLE PROTEIN), Homo sapiens
UniProt: O43614
Assay: Cell-based Functional Assay: Measurement of [Ca2+]i using a FLIPR: CHO-OX1 or CHO-OX2 cells are seeded into black-walled clear-base 384-well plates (Costar) at a density of 20,000 cells per well in F12-K medium supplemented with 10% FBS and selection antibiotic (500 ug/ml G418 or 15 ug/ml Blasticidin) and cultured overnight. The cells are incubated with equal volume of calcium4 loading buffer (Molecular Devices, Inc.) containing 2.5 mM probenecid at 37° C. for 30 min, followed by putative OX1 or OX2 receptor antagonists (dose-range 0.1 nM-10 μM) for another 30 min.
Measurement: IC50 = 8 nM (pChEMBL 8.100000381469727)
Descriptors: MW 488.59, ALogP 5.04, HBA 5, HBD 1, PSA 75.19, QED 0.43
Compound (SMILES): Cn1cc(-c2cn3nccc3c(-c3cnn([C@]4(CC#N)C[C@@H](c5ccn[nH]5)C4)c3)n2)cn1
InChIKey: DVOYZGKQNUPXBI-VVONHTQRSA-N
Target: Tyrosine-protein kinase JAK1 (SINGLE PROTEIN), Homo sapiens
UniProt: P23458
Assay: JAK Caliper Enzyme Assay at 1 mM ATP: Compounds were added to a 384-well plate. Reaction mixtures contained 10 mM HEPES, pH 7.4, 10 mM MgCl2, 0.01% BSA, 0.0005% Tween 20, 1 mM ATP and 1 μM peptide substrate. The JAK1 and TYK2 assays contained 1 μM of the IRStide peptide (5FAM-KKSRGDYMTMQID) and the JAK2 and JAK3 assays contained 1 μM of the JAKtide peptide (FITC-KGGEEEEYFELVKK). The assays were initiated by the addition of 20 nM JAK1, 1 nM JAK2, 1 nM JAK or 1 nM TYK2 enzyme and were incubated at room temperature for three hours for JAK1, 60 minutes for JAK2, 75 minutes for JAK3 or 135 minutes for TYK2. Enzyme concentrations and incubation times were optimized for each new enzyme preps and were modified slightly over time to ensure 20% to 30% phosphorylation. The assays were stopped with 15 μL of 180 mM HEPES, pH 7.4, 20 mM EDTA, and 0.2% Coating Reagent 3. The assay plates were placed on a Caliper Life Science LC3000 instrument, and each well was sampled using appropriate separation conditions to measure the unphosphorylated and phosphorylated peptide.
Measurement: IC50 = 1079 nM (pChEMBL 5.96999979019165)
Descriptors: MW 424.47, ALogP 2.9, HBA 9, HBD 1, PSA 118.3, QED 0.46
Compound (SMILES): N#CN1CCC(NS(=O)(=O)c2ccc(Oc3ccc(C(F)(F)F)cn3)cc2)C1
InChIKey: KWAQJZBRETZPOX-UHFFFAOYSA-N
Target: Ubiquitin carboxyl-terminal hydrolase 30 (SINGLE PROTEIN), Homo sapiens
UniProt: Q70CQ3
Assay: USP30 Biochemical IC50 Assay: Dilution plates were prepared at 21 times the final concentration (2100 μM for a final concentration of 100 μM) in 50% DMSO in a 96-well polypropylene V-bottom plate (Greiner #651201). A typical 8-point dilution series would be 100, 30, 10, 3, 1, 0.3, 0.1, 0.03 μM final. Reactions were performed in duplicate in black 384 well plates (small volume, Greiner 784076) in a final reaction volume of 21 μl. Either 1 μl of 50% DMSO or diluted compound was added to the plate. USP30 was diluted in reaction buffer (40 mM Tris, pH 7.5, 0.005% Tween 20, 0.5 mg/ml BSA, 5 mM beta-mercaptoethanol) to the equivalent of 0.05 μl/well and 10 μl of diluted USP30 was added to the compound. Enzyme and compound were incubated for 30 min at room temp. Reactions were initiated by the addition of 50 nM of TAMRA labelled peptide linked to ubiquitin via an iso-peptide bond as fluorescence polarisation substrate. Reactions were read immediately after addition of substrate and following a 2-hour incubation at room temperature. Readings were performed on a Pherastar Plus (BMG Labtech). λ Excitation 540 nm; λ Emission 590 nm.
Measurement: IC50 = 550 nM (pChEMBL 6.260000228881836)
Descriptors: MW 412.39, ALogP 2.73, HBA 6, HBD 1, PSA 95.32, QED 0.76
Compound (SMILES): CNc1cc(Nc2cccn([C@H]3CC[C@H](OC)CC3)c2=O)nc2c(C(=O)N[C@@H]3CC[C@H]3OC)cnn12
InChIKey: RWTRNTPHYFRFAB-QQTQMKHKSA-N
Target: Non-receptor tyrosine-protein kinase TYK2 (SINGLE PROTEIN), Homo sapiens
UniProt: P29597
Assay: TYK2 JH2 Domain Binding Assay: The binding reaction was assembled by combining 16 μl of DNA-tagged kinase extract, 3.8 μl liganded affinity beads, and 0.18 μl test compound (PBS/0.05% Tween 20/10 mM DTT/0.1% BSA/2 μg/ml sonicated salmon sperm DNA)]. Extracts were used directly in binding assays without any enzyme purification steps at a ≥10,000-fold overall stock dilution (final DNA-tagged enzyme concentration <0.1 nM). Extracts were loaded with DNA-tag and diluted into the binding reaction in a two step process. First extracts were diluted 1:100 in 1× binding buffer (PBS/0.05% Tween 20/10 mM DTT/0.1% BSA/2 μg/ml sonicated salmon sperm DNA) containing 10 nM DNA-tag. This dilution was allowed to equilibrate at room temperature for 15 minutes and then subsequently diluted 1:100 in 1× binding buffer. Test compounds were prepared as 111× stocks in 100% DMSO. Kds were determined using an 11-point 3-fold compound dilution series with three DMSO control points. All compounds for Kd measurements are distributed by acoustic transfer (non-contact dispensing) in 100% DMSO
Measurement: Kd < 0.2 nM (pChEMBL 9.699000358581543)
Descriptors: MW 495.58, ALogP 2.71, HBA 10, HBD 3, PSA 123.81, QED 0.44
Compound (SMILES): Cc1cc(C(F)(F)F)cc(O)c1-c1ccc(NC2CCCC2O)nn1
InChIKey: UAHABEKKQMVOGP-UHFFFAOYSA-N
Target: Tumor necrosis factor (SINGLE PROTEIN), Homo sapiens
UniProt: P01375
Assay: TNF-alpha Secretion Assay: Monocytic THP-1 cells were maintained according to providers' instructions in RPMI media as described above. Undifferentiated cells were plated at 50,000 cells per well in a 384-well flat-bottom cell culture plates (Greiner, #781986), and allowed to rest overnight. Experimental compounds were prepared and added as described above. TNF-α secretion was triggered by the addition of either 1 μg/mL LPS (Sigma, #L4391) or 100 ng/mL Pam3CSK4 (Invivogen, #tlrl-pms) depending on the experiment, and cells were incubated for 3 hours. 10 μL supernatant was removed, and TNF-α levels were monitored using an HTRF assay (CisBio, #62TNFPEC) according to manufacturers' instructions.
Measurement: IC50 = 37000 nM (pChEMBL 4.429999828338623)
Descriptors: MW 353.34, ALogP 3.5, HBA 5, HBD 3, PSA 78.27, QED 0.79
Compound (SMILES): O=C(CCC(F)(F)F)N1CC=C(c2cccn3nc(Nc4ccc(C(=O)N5CCC(Cc6cccnc6)C5)cc4)nc23)CC1
InChIKey: OJJWEMDXVVBFRR-UHFFFAOYSA-N
Target: Tyrosine-protein kinase JAK2 (SINGLE PROTEIN), Homo sapiens
UniProt: O60674
Assay: Enzyme Assay: The activity of the isolated recombinant JAK1 and JAK2 kinase domain was measured by monitoring phosphorylation of a peptide derived from JAK3 (Val-Ala-Leu-Val-Asp-Gly-Tyr-Phe-Arg-Leu-Thr-Thr, fluorescently labeled on the N-terminus with 5-carboxyfluorescein) using the Caliper LabChip® technology (Caliper Life Sciences, Hopkinton, Mass.). To determine inhibition constants (Ki), compounds were diluted serially in DMSO and added to 50 μL kinase reactions containing purified enzyme (1.5 nM JAK1, or 0.2 nM JAK2), 100 mM HEPES buffer (pH 7.2), 0.015% Brij-35, 1.5 μM peptide substrate, ATP (25 μM), 10 mM MgCl2, 4 mM DTT at a final DMSO concentration of 2%. Reactions were incubated at 22° C. in 384-well polypropylene microtiter plates for 30 minutes and then stopped by addition of 25 μL of an EDTA containing solution (100 mM HEPES buffer (pH 7.2), 0.015% Brij-35, 150 mM EDTA), resulting in a final EDTA concentration of 50 mM. After termination of the kinase reaction, the proportion of phosphorylated product was determined as a fraction of total peptide substrate using the Caliper LabChip® 3000 according to the manufacturer's specifications.
Measurement: Ki = 0.27 nM (pChEMBL 9.569999694824219)
Descriptors: MW 603.65, ALogP 5.53, HBA 7, HBD 1, PSA 95.73, QED 0.28
Compound (SMILES): O=C(Nc1ccc(F)cc1)c1cccc(C2CC2NC2CCOCC2)c1
InChIKey: OFLGAFHBRCHRMU-UHFFFAOYSA-N
Target: Amine oxidase [flavin-containing] A (SINGLE PROTEIN), Homo sapiens
UniProt: P21397
Assay: MAO-A Inhibition Assay: A test compound dissolved in 4% DMSO was added by 12.5 μL to 25 μL reaction solution (100 mM HEPES (pH 7.5), 5% glycerol) containing 400 ng of MAO-A enzyme (Sigma-Aldrich Co. LLC.), and the mixture was reacted at room temperature for 10 min. MAO substrate (Promega KK) (160 μM) was added by 12.5 μL to start the reaction. After reaction at room temperature for 60 min, Luciferine detection reagent (Promega KK) (50 μL) was added to terminate the reaction. After reaction at room temperature for min with stirring, the luminescence was measured by Envision (PerkinElmer). The MAO-A inhibitory rate (%) of the test compound was calculated by the following formula.inhibitory rate(%)=(1−(test compound count−blank)÷(control−blank))×100
Measurement: IC50 > 10000 nM (pChEMBL 5)
Descriptors: MW 354.43, ALogP 3.7, HBA 3, HBD 2, PSA 50.36, QED 0.86
Compound (SMILES): C=C(C)C(=O)c1cccc(Nc2nc(Nc3cccc(OC(F)(F)F)c3)ncc2F)c1
InChIKey: YQEJTFBEHMVKDF-UHFFFAOYSA-N
Target: Tyrosine-protein kinase BTK (SINGLE PROTEIN), Homo sapiens
UniProt: Q06187
Assay: Omnia Assay: Briefly, 10x stocks of EGFR-WT (PV3872) from Invitrogen and EGFR-T790M/L858R (40350) from BPS Bioscience, San Diego, Calif., 1.13×ATP (AS001A) and appropriate Tyr-Sox conjugated peptide substrates (KCZ1001) were prepared in 1× kinase reaction buffer consisting of 20 mM Tris, pH 7.5, 5 mM MgCl2, 1 mM EGTA, 5 mM β-glycerophosphate, 5% glycerol (10× stock, KB002A) and 0.2 mM DTT (DS001A). 5 μL of each enzyme were pre-incubated in a Corning (#3574) 384-well, white, non-binding surface microtiter plate (Corning, N.Y.) for 30 min. at 27° C. with a 0.5 μL volume of 50% DMSO and serially diluted compounds prepared in 50% DMSO. Kinase reactions were started with the addition of 45 μL of the ATP/Tyr-Sox peptide substrate mix and monitored every 30-90 seconds for 60 minutes at λex360/λem485 in a Synergy4 plate reader from BioTek (Winooski, Vt.). At the conclusion of each assay, progress curves from each well were examined for linear reaction kinetics and fit statistics (R2, 95% confidence interval, absolute sum of squares). Initial velocity (0 minutes to 30 minutes) from each reaction was determined from the slope of a plot of relative fluorescence units vs time (minutes) and then plotted against inhibitor concentration to estimate IC50 from log[Inhibitor] vs Response, Variable Slope model in GraphPad Prism from GraphPad Software (San Diego, Calif.).
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 432.38, ALogP 5.76, HBA 6, HBD 2, PSA 76.14, QED 0.28
Compound (SMILES): CN[C@@H](C)C(=O)N[C@H]1CN(C(=O)Cc2cccnc2)c2ccccc2N(Cc2c(OC)ccc3c(Br)cccc23)C1=O
InChIKey: MGGITSBKIMQADG-FNZWTVRRSA-N
Target: E3 ubiquitin-protein ligase XIAP (SINGLE PROTEIN), Homo sapiens
UniProt: P98170
Assay: TR-FRET Assay for BIR2 and BIR3: Ten nanomolar of 6× Histidine-tagged BIR2 domain, corresponding to amino acids 124-240 of XIAP, or BIR3 domain, corresponding to amino acids 241-356 of XIAP, was mixed with 20 nM of the peptide AVPIAQKSEK-(c-biotin)-OH 1:2 TFA, in the presence of 50 mM Tris-Cl, pH 7.5, 100 mM NaCl, 1 mM dithiothreitol (DTT) and 0.1 mg/mL bovine serum albumin (BSA). Following a 45 min. incubation at 37° C., Europium-Streptavidin and Allophycocyanin conjugated anti-Histidine antibody were added to a final concentration of 1.5 nM and 15 nM, respectively. Time-resolved fluorescence resonance energy transfer (TR-FRET) signals were measured 1 hour later at room temperature. Test compound potency was assessed at 10 serially diluted concentrations. Percentage of inhibition at each concentration was determined to generate an IC50 value for each test compound.
Measurement: IC50 = 39890 nM (pChEMBL 4.400000095367432)
Descriptors: MW 630.54, ALogP 4.22, HBA 6, HBD 2, PSA 103.87, QED 0.3
Compound (SMILES): Cc1nc(C(=O)N[C@H](C)c2ccc(OC(F)(F)F)cc2)cc(=O)[nH]1
InChIKey: FIFOTGXFSFMDHB-MRVPVSSYSA-N
Target: cGMP-dependent 3',5'-cyclic phosphodiesterase (SINGLE PROTEIN), Homo sapiens
UniProt: O00408
Assay: Fluorescence Polarization (FP) Assay: In a typical experiment the PDE2 inhibitory activity of the compounds of the present invention was determined in accordance with the following experimental method. Rhesus PDE2A3 was amplified from rhesus macaque brain cDNA (Biochain Institute, Hayward, Calif.) using primers based on human PDE2A sequence (accession NM_002599.3) where the forward primer containing a Kozak consensus was 5′-gccaccatggggcaggcatgtggc-3′ and the reverse primer was 5′-tcactcagcatcaaggctgca-3′. Amplification with Easy-A High-Fidelity PCR cloning enzyme (Stratagene, La Jolla, Calif.) was 95° C. for 2 minutes followed by thirty three cycles of 95° C. for 40 seconds, 52° 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 pcDNA3.3-TOPO (Invitrogen, Carlsbad, Calif.) according to standard protocol. A consensus sequence was developed from multiple clones and then deposited into GenBank (EU812167). AD293 cells (Stratagene, La Jolla, Calif.) with 70-80% confluency were transiently transfected with rhesus PDE2A3/pcDNA3.3-TOPO using Lipofectamine 2000 according to manufacturer specifications (Invitrogen, Carlsbad, Calif.).
Measurement: Ki = 16 nM (pChEMBL 7.800000190734863)
Descriptors: MW 341.29, ALogP 2.47, HBA 4, HBD 2, PSA 84.08, QED 0.89
Compound (SMILES): Cn1cc(C(=O)Nc2cccc(-c3nnc(Nc4ccc5[nH]nc(F)c5c4)s3)c2)cn1
InChIKey: GADAFEOPHFNEBV-UHFFFAOYSA-N
Target: Rho-associated protein kinase 2 (SINGLE PROTEIN), Homo sapiens
UniProt: O75116
Assay: Biological Assays for Inhibition of Rho-Associated Protein Kinase: Assays for ROCK inhibition were performed using the following protein constructs: glutathione S-transferase (GST)-tagged human ROCK1 catalytic domain 1-477 from Carna Biosciences (cat #01-109; apparent Km value for ATP is 10 μM) and GST-tagged human ROCK2 catalytic domain 1-553 from Carna Biosciences (Cat#01-110; apparent Km value for ATP is 15 μM). Protein constructs were purified from a baculovirus expression system. The peptide substrate was fluorescent LANCE® Ultra ULight-CREBtide: CKRREILSRRPSYRK (PerkinElmer, # TRF0107-D). Kinase reactions were carried out in in a 10 μL volume in 384-well plates: 50 nM ULight-CREBtide substrate, 2 nM constitutively active ROCK1 or ROCK2 kinase, and test compound in DMSO (or DMSO only for controls) were diluted into assay buffer containing 50 mM Tris-HCl (pH=7.5), 10 mM MgCl2, 1 mM EGTA, 0.01% Tween-20, and 2 mM DTT such that the final concentration of DMSO was 0.5%. After a 90 minute incubation at room temperature on a shaker table, the kinase reaction was stopped by addition of 10 mM EDTA, and phosphorylation of the substrate was detected by adding 1 nM LANCE® Ultra Europium-anti-phospho-CREB (ser133) antibody (PerkinElmer, # TRF0200-D) and incubating for 60 minutes on a shaker at room temperature. The flurorescence resonance energy transfer (FRET) signals were read and analyzed on an Envision¿ 2103 Multilabel Reader (Perkin Elmer). The concentration of test compound required to inhibit substrate phosphorylation by 50% (the IC50) was calculated by non-linear regression using GraphPad PRIZM.
Measurement: IC50 > 30000 nM (pChEMBL 4.5229997634887695)
Descriptors: MW 434.46, ALogP 3.95, HBA 8, HBD 3, PSA 113.41, QED 0.39
Compound (SMILES): CNc1ccc(-c2cccc3c2C[C@@H](C)CC(=O)N3)cc1C(=N)C1CC1
InChIKey: ANIBATUKYBCBNA-CYBMUJFWSA-N
Target: Bromodomain-containing protein 4 (SINGLE PROTEIN), Homo sapiens
UniProt: O60885
Assay: BRD4 AlphaLisa Binding Assay: His/Flag epitope tagged BRD4 BD142-168 was cloned, expressed, and purified to. BRD4 binding and inhibition was assessed by monitoring the engagement of biotinylated H4-tetraacetyl peptide (New England Peptide, NEP2069-1/13) with the target using the AlphaLisa technology (Perkin-Elmer). Specifically, in a 384 well ProxiPlate BRD4(BD1) (30 nM final) was combined with peptide (200 nM final) in 40 mM HEPES (pH 7.0), 40 mM NaCl, 1 mM DTT, 0.01% (w/v) BSA, and 0.008% (w/v) Brij-35 either in the presence of DMSO (final 1.2% DMSO) or compound dilution series in DMSO. After 20 minutes incubation at room temperature Alpha streptavidin donor beads and AlphaLisa anti-Flag acceptor beads were added to a final concentration of 10 ug/mL each. After three hours equilibration plates were read on an Envision instrument and IC50s calculated using a four parameter non-linear curve fit.
Measurement: IC50 = 894 nM (pChEMBL 6.050000190734863)
Descriptors: MW 347.46, ALogP 4.69, HBA 3, HBD 3, PSA 64.98, QED 0.7
Compound (SMILES): CCCCOc1nc(N)c2nc(O)n(Cc3ccc(Oc4ccc(CNCCOC)cc4)cc3)c2n1
InChIKey: ORCANGUVKGRRQG-UHFFFAOYSA-N
Target: Interleukin-6 (SINGLE PROTEIN), Homo sapiens
UniProt: P05231
Assay: Interleukin 6 Induction Assay: Compounds diluted in DMSO were transferred to individual wells of a Matrix Technologies clear, V-bottom 384-well plate using ECHO acoustic liquid handling technology (25 nL per well). Human whole-blood samples (25 uL) were added to each well using a CyBio FeliX liquid handling instrument. The plate was shaken on a plate shaker for three min before incubating the reaction mixtures at 37° C. for 20 h. Basel RPMI 1640 medium (supplemented with L-glutamine) was then added to each well (25 uL per well) prior to liberating plasma from each sample by centrifugation (450×g, 5 min, ambient temperature). Treated plasma samples (3 uL) were subsequently transferred to individual wells of a white, shallow, 384-well ProxiPlate (Perkin Elmer) using the FeliX liquid handling instrument and their interleukin 6 levels were measured using AlphaLISA technology as described by the manufacturer, PerkinElmer. Data analyses software was used to determine compound EC50 values where the baseline was established using average DMSO values and 100% induction established using reference compound values at the highest concentration tested. EC50's can be determined with software such as Graphpad Prism.
Measurement: EC50 = 6.62 nM (pChEMBL 8.180000305175781)
Descriptors: MW 492.58, ALogP 3.87, HBA 10, HBD 3, PSA 129.57, QED 0.24
Compound (SMILES): COC1(C)CN(c2cnc3c(C(=O)Nc4cnccc4[C@@H]4C[C@H](C)C[C@H](N)C4)ccc(F)c3c2)C1
InChIKey: RQYVFQCCZWEMJR-KSZLIROESA-N
Target: Serine/threonine-protein kinase pim-2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9P1W9
Assay: Kinase Assay: Pim-2 kinase assay20 μL reactions were run in white 384 well polystyrene plates dotted with 0.8 μL compound/DMSO in the assay buffer (50 mM Tris, pH 7.5, 0.01% Tween-20, 5 mM mgCl2, 0.01% BSA, 5 mM DTT), containing 0.05 μM Fluorescein-labeled CREBtide peptide substrate (Invitrogen PV3508), 1 mM ATP, and 1 nM enzyme (Invitrogen PV3649) for 2 h at 25° C. Reactions were stopped by addition of 10 μL TR-FRET Dilution Buffer (Invitrogen PV3574) with 30 mM EDTA and 1.5 nM LanthaScreen Tb-CREB pSer133 antibody (Invitrogen PV3566). After 30 min. incubation at room temperature, plates were read on a PHERAstar FS plate reader (BMG Labtech).
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 477.58, ALogP 4.48, HBA 6, HBD 2, PSA 93.37, QED 0.56