lovingscience/chembl-v37-raw-activities huggingface dataset:
Compound (SMILES): CCn1cc(C(=O)NCc2ccccc2F)c2cc(OC)c(OC)cc2c1=O
InChIKey: DLIHKGQSCLQQNF-UHFFFAOYSA-N
Target: cAMP and cAMP-inhibited cGMP 3',5'-cyclic phosphodiesterase 10A (SINGLE PROTEIN), Homo sapiens
UniProt: Q9Y233
Assay: Enzymatic Assay: The PDE enzymatic reaction was carried out in assay buffer (20 mM Tris-HCl pH7.5, 10 mM MgCl2, 0.1% bovine serum albumin) containing enzyme and substrate. The PDE enzymes concentration ranged from 10 pM 250 pM, depending on each enzyme's specific activity. The substrate cyclic nucleotide (cAMP or cGMP) concentration used in the assay was 20 nM for PDE10, and 100 nM for other PDEs. The inhibitory effect of compound was determined by incubating various concentration of inhibitor in the enzymatic assay. Typically, compound was serial diluted in DMSO then further diluted in assay buffer. Next, the compound at varying concentration was mixed with PDE enzyme. The reaction was initiated by addition of cyclic nucleotide substrate, and incubated for 60 minutes at 29 C. The reaction was stopped by addition of lysis buffer from assay kit. The cAMP-d2 and anti-cAMP cryptate in the lysis buffer detected the level of cAMP left from the PDE hydrolysis reaction. The PDE activity is reversely correlated with the amount of cAMP left in the reaction and can be converted to the percent activity of an uninhibited control (100%).
Measurement: IC50 = 350 nM (pChEMBL 6.460000038146973)
Descriptors: MW 384.41, ALogP 3.11, HBA 5, HBD 1, PSA 69.56, QED 0.71
Compound (SMILES): COc1c(N=S2(=O)CCCC2)cc2ncnc(Nc3ccc(F)cc3O[C@H](C)C(=O)N[C@@H](C)C(F)(F)F)c2c1C
InChIKey: RNSPDPHEMGBBDH-CVEARBPZSA-N
Target: MAP kinase-interacting serine/threonine-protein kinase 2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9HBH9
Assay: MNK2a In Vitro Kinase Assay (Assay 1): The inhibition of kinase activity of MNK2a was assessed using pre-activated GST-MNK2a. The white, 384-well OptiPlate F plates were purchased from PerkinElmer. The ADP-Glo Kinase Assay (including ultra pure ATP) was purchased from Promega (V9103). Activated MNK2a was obtained as described in WO2011/104340. The unlabeled elF4E peptide (NH2-TATKSGSTTKNR-CONH2), differing from Seq. ID No. 5 of WO 2011/104340 by the C-terminal CONH2 group, was purchased from Thermo Fisher Scientific. All other materials were of highest grade commercially available. Compounds are tested in either serial dilutions or single dose concentrations. The compound stock solutions are 10 mM in 100% DMSO The serial compound dilutions are prepared in 100% DMSO followed by 1:27.3 intermediate dilution in assay buffer. The final DMSO concentration in assay will be <3%.In the 384-well plates 3 μl of test compound from the intermediate dilution is mixed with 4 μl of the activated MNK2 enzyme (final concentration of 10 nM) and 4 μl of the peptide (final concentration of 25 μM)/ultra pure ATP (final concentration of 20 μM), all dissolved in assay buffer. This step is followed by an incubation time of 90 min, then 10 μl of ADP Glo reagent are added, followed by 40 min of incubation. Then 20 μl of kinase detection reagent are admixed. The plates are sealed and after an incubation period of 30 min, the luminescence signal is measured in an Envision reader to determine the amount of produced ADP. All incubation steps are performed at room temperature.The assay buffer consists of 20 mM HEPES, 2 mM DTT, 0.01% BSA, 20 mM MgCl2 and 0.1% Pluronic F-127.
Measurement: IC50 = 2.5 nM (pChEMBL 8.600000381469727)
Descriptors: MW 583.61, ALogP 5.56, HBA 8, HBD 2, PSA 114.8, QED 0.33
Compound (SMILES): N#Cc1ccc(N2CCN(C(=O)c3cccc(NCC4CCCN4c4cn[nH]c(=O)c4C(F)(F)F)c3)CC2)nc1
InChIKey: AAADAGSXBHFTSM-UHFFFAOYSA-N
Target: Protein mono-ADP-ribosyltransferase TIPARP (SINGLE PROTEIN), Homo sapiens
UniProt: Q7Z3E1
Assay: Enzymatic Assay: Displacement of Probe A, a biotinylated probe binding to the TIPARP active site, was measured using a time-resolved fluorescence energy transfer (TR-FRET) assay. 20 nL of a dose response curve of each test compound was spotted in black 384-well polystyrene proxiplates (Perkin Elmer) using a Mosquito (TTP Labtech). Reactions were performed in a 8 μL volume by adding 6 JAL of TIPARP and Probe A in assay buffer (20 mM HEPES pH=8, 100 mM NaCl, 0.1% bovine serum albumin, 2 mM DTT and 0.002% Tween20), incubating with test compound at 25° C. for 30 min, then adding 2 μL of ULight-anti 6×His and LANCE Eu-W1024 labeled streptavidin (Perkin Elmer). The final concentrations of TIPARP and Probe A were 6 nM and 2 nM, respectively. The final concentration of ULight-anti 6×His and LANCE Eu-W1024 labeled streptavidin were 4 nM and 0.25 nM, respectively. Reactions were incubated at 25° C. for an additional 30 min, then read on an Envision plate reader equipped with a LANCE/DELFIA top mirror (Perkin Elmer) using excitation of 320 nm and emission of 615 nm and 665 nM with a 90 μs delay. The ratio of the 665/615 nm emission were calculated for each well to determine the amount of complex of TIPARP and Probe A in each well.
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 552.56, ALogP 3.1, HBA 8, HBD 2, PSA 121.25, QED 0.48
Compound (SMILES): CNC(=O)c1cc(-c2cc(N)c3cnc(NC(=O)C4CC4C#N)cc3c2)n(C)n1
InChIKey: LFHVTFMUMYKOIV-UHFFFAOYSA-N
Target: Mitogen-activated protein kinase kinase kinase kinase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q92918
Assay: HPK Lantha Binding Assay: For the binding assay, 4 ul 2×HPK1 and Eu-anti-GST antibody were added to each well of the assay plate using a Multidrop reagent dispenser. The solutions were incubated in a 23 C incubator for 1 h. To each well of the assay plate was added 4 ul 2× Tracer-222 using a Multidrop reagent dispenser. The solutions were again incubated in a 23 C incubator for 1 h. The results of the assay were read using an Envision plate reader with the following parameters:TR_FRET, 340ex/615 and 665em; 100 usec Delay; and 200 usec integration.
Measurement: Ki = 24.1 nM (pChEMBL 7.619999885559082)
Descriptors: MW 389.42, ALogP 1.68, HBA 7, HBD 3, PSA 138.72, QED 0.58
Compound (SMILES): Oc1ccc(C(O)CN2C[C@H]3C[C@H](Oc4cc(F)cc(F)c4)C[C@H]3C2)nc1
InChIKey: HJDCPTLBSVVLBI-SWBKGBCFSA-N
Target: Glutamate NMDA receptor; GRIN1/GRIN2B (PROTEIN COMPLEX), Homo sapiens
UniProt: Q05586
Assay: NR2B NAM Assay: Cell Culture and plating: HEK293 cells expressing NR1/NR2B (Chantest, Cleveland, Ohio) were grown to 70-80% confluency as an adherent monolayer in standard tissue culture flasks at 37° C., 5% CO2 per supplier's instructions. NR2B expression was induced by incubation with 0.3-0.4 μg/ml tetracycline in the presence of 4 mM ARL-15896 for 18-24 hours under the same growth conditions, then transferred to 30° C. for another 3-5 hours.After induction, cell culture medium was removed and cells were rinsed once with Ca2+ and Mg2+-free Dulbecco's phosphate buffered saline. Cells were then removed from the flask using TrypLE Express (Life Technologies) according to the manufacturer's instructions and collected to 50 ml centrifuge tubes. Following two washes in Ca2+/Mg2+-free HBSS with 20 mM HEPES (HHnoCa), cells were counted and viability assessed using trypan blue. To load cells with Ca2+-sensitive dye, they were resuspended in fluo-8 plus Component B (AAT Bioquest Products) diluted in HHnoCa and incubated 15 minutes at 37° C., followed by 30 minutes at room temp (in dark). Cells were then washed and resuspended in HHnoCa to remove extracellular dye and plated in 384-well plates (Falcon, uncoated) at 20,000-30,000 cells/well in a final volume of 25 μl/well.
Measurement: IC50 = 58.3 nM (pChEMBL 7.230000019073486)
Descriptors: MW 376.4, ALogP 2.89, HBA 5, HBD 2, PSA 65.82, QED 0.84
Compound (SMILES): O=C(NCc1cn[nH]c1)Nc1ccc(S(=O)(=O)c2ccccc2-c2cccnc2)cc1
InChIKey: HIEHZIVLSSOKLN-UHFFFAOYSA-N
Target: Nicotinamide phosphoribosyltransferase (SINGLE PROTEIN), Homo sapiens
UniProt: P43490
Assay: Enzyme Assay and NMN Detection: NAMPT reaction was performed in 50 mM HEPES, pH 7.5, containing 5 mM MgCl2, 0.1% Prionex, 0.005% Tween 20, and 1 mM TCEP at room temperature in Greiner 384-well black polypropylene plates. The concentration of substrates and enzymes in the final assay were as follows: ATP (120 uM), nicotinamide (5 uM), phosphoribosylpyrophosphate (6.25 uM), NAMPT enzyme (16 nM), pyrophosphatase (0.04 U/ml) in the final reaction volume 6 uL. Following 2 h incubation the reaction was stopped by addition 2.5 ul 20% acetophenone in dimethyl sulfoxide (DMSO) and 2.5 ul 2 MKOH. Plates were centrifuges and added with 10.5 ul 88% formic acid. The fluorescence (ex380/em460) was measured following another short incubation and 30 min incubation. The increased concentration of NMN was determined from a calibration curve obtained at the time of experiment.
Measurement: EC50 = 1050 nM (pChEMBL 5.980000019073486)
Descriptors: MW 433.49, ALogP 3.63, HBA 5, HBD 3, PSA 116.84, QED 0.43
Compound (SMILES): CS(=O)(=O)C(C(=O)NCC(=O)NC1CC1)c1nc2cc(F)c(-c3ccc(NC(=O)OCc4ccccc4)nc3)cc2s1
InChIKey: UCTXRIJOEMMAKQ-UHFFFAOYSA-N
Target: Endothelial lipase (SINGLE PROTEIN), Homo sapiens
UniProt: Q9Y5X9
Assay: Biological Activity: Endothelial lipase (EL) and hepatic lipase (HL) activities were measured using a fluorescent substrate, A10070, (Invitrogen, CA) doped into an artificial vesicle containing DMPG (Avanti Polar Lipids) as the excipient. Vesicles were prepared by combining 571 μL of 29 mM DMPG in a 1:1 mixture of MeOH and CHCl3 with 2000 μL of 1 mM A10070 in a 1:1 mixture of MeOH and CHCl3. The mixture was dried under nitrogen in multiple vials then resuspended in 20 mL total volume of 50 mM HEPES pH 8.0 buffer containing 50 mM NaCl and 0.2 mM EDTA. The sample was allowed to sit at room temperature for 15 min and then was sonicated 3×4 mins on ice with a Branson Sonicator using duty cycle 1. This preparation provides vesicles with a mole fraction of 0.11 for the FRET substrate.The enzymatic assay was measured using 384-well white Optiplates. Each well contained 20 μL of assay buffer (50 mM HEPES pH 8.0, 50 mM NaCl and 1 mM CaCl2) and 0.25 μL of a DMSO solution containing a compound of interest. EL or HL (10 μL) was added and allowed to incubate with the compound for 30 min at 37° C. The source of EL was conditioned media obtained from HT-1080 cells that were transformed using RAGE technology (Athersys) to overexpress endogenous EL, and HL was partially purified from conditioned media obtained from COS cells overexpressing HL. The reaction was started by the addition of 10 μL of a 1:10 dilution of vesicles. The final total reaction volume was 20.25 μL. The reaction rates were measured on a Gemini plate reader with an excitation wavelength of 490 nm and an emission wavelength of 530 nm. Readings were taken over a period of 60 minutes, and the slope between 300 and 900 secs of the readout was used to calculate the rate of the reaction.
Measurement: IC50 = 1 nM (pChEMBL 9)
Descriptors: MW 611.68, ALogP 3.73, HBA 9, HBD 3, PSA 156.45, QED 0.24
Compound (SMILES): Cc1nnn(Cc2cc(C(F)(F)F)ccc2/C=C/C(=O)N2C[C@@H](F)C[C@H]2CO)n1
InChIKey: UFZHSBSMLKMRBZ-TVKHYIBRSA-N
Target: Autotaxin (SINGLE PROTEIN), Homo sapiens
UniProt: Q13822
Assay: ATX Assay: Reagents LPC (oleoyl (18:1)) was purchased from Avanti Polar Lipids (Alabaster, Ala.) and solubilized in methanol to 20 mM. Amplex Red was obtained from Invitrogen Life Technologies (Paisley, UK) and dissolved in DMSO to 10 mM. Choline oxidase and horseradish peroxidase (HRP) were obtained from Sigma Aldrich (Dorset, UK) and dissolved in HBSS to 20 U/ml and 200 U/ml respectively. All reagents were stored at −20° C. in single use aliquots. All experimental measurements were performed in assay buffer made up immediately prior to use (HBSS, 0.01% BSA essentially fatty acid free).Protein Recombinant human ATX was prepared at Novartis (Basel, CH) in a human embryonic kidney (HEK) cell preparation, and stored in single use aliquots of 26 mg/ml (26 μM) stocks stored at −80° C.Method All experimental measurements were performed in black 384 well polystyrene (low volume, round bottom, Corning (3676)) plates. PerkinElmer EnVision (Fluorescence Intensity/Absorbance Monochromator) or Tecan Infinite 200 PRO series plate reader was used to detect change in fluorescent intensity.Assessing ATX inhibition ATX activity was determined by measurement of released choline in reactions containing ATX (10 nM), choline oxidase (0.1 U/ml), HRP (100 U/ml), amplex red (50 μM) and LPC 18:1 (10 μM). Compounds of the invention were prepared as 10 point serial dilutions from 1 μM in duplicate and pre-incubated with ATX at 37° C. for 20 minutes prior to the addition of remaining reagents. The liberated choline was measured from changes in fluorescence intensity (λex 530 nm, λem 590 nm) of the product resurofin at 37° C. every 2 minutes over a 40-minute period. ATX activity was measured as a slope of the linear portion of the progress curve, typically between 14 to 24 minutes.Data analysis Slope data was exported to Graphpad prism (Graphpad software, San Diego, Calif.)
Measurement: IC50 = 23 nM (pChEMBL 7.639999866485596)
Descriptors: MW 413.38, ALogP 1.99, HBA 6, HBD 1, PSA 84.14, QED 0.6
Compound (SMILES): COc1cc(Nc2ncc(F)c(N[C@@H]3C[C@@H]4CCCN4C(C)(C)C3)n2)ccc1O
InChIKey: PYLCVIJJKWPMAD-CABCVRRESA-N
Target: Interleukin-2 (SINGLE PROTEIN), Homo sapiens
UniProt: P60568
Assay: IL-2 ELISA Assay: Primary T cell stimulation and IL2 ELISA: Human primary T cells (100,000 cells per well) were pre-incubated with or without test compound in Yssel's medium for 1 hour at 37° C. Cells were then stimulated by transferring them to round-bottom 96-well plates pre-coated with 1 μm/ml αCD3 and 5 μg/ml αCD28. For counter assay, cells were instead stimulated by adding 8× stock solutions of PMA and ionomycin in Yssels (for final concentrations of 0.5 ng/ml PMA and 0.1 uM ionomycin, both from Calbiochem). Cells were incubated at 37° C. for 24 hours before 100 μL supernatants were harvested for quantification of IL-2 by ELISA using Human IL-2 Duoset ELISA Kit from R and D Systems, Cat. # DY202E.
Measurement: EC50 < 10000 nM (pChEMBL 5)
Descriptors: MW 401.49, ALogP 3.89, HBA 7, HBD 3, PSA 82.54, QED 0.66
Compound (SMILES): CC(C)COc1ccc2c(c1)O[C@H](C)CC(=O)N2
InChIKey: IJIRWLCAGWNHBJ-SNVBAGLBSA-N
Target: CREB-binding protein (SINGLE PROTEIN), Homo sapiens
UniProt: Q92793
Assay: CBP TR-FRET Binding Assay: His/Flag epitope tagged CBP was cloned, expressed, and purified to homogeneity. CBP binding and inhibition was assessed by monitoring the engagement of a biotinylated small molecule compound with the target using the TR-FRET assay technology (Perkin-Elmer). Specifically, in a 384 well ProxiPlate CBP (4 nM final) was combined with biotin-ligand (60 nM final) in 50 mM HEPES (pH 7.5), 50 mM NaCl, 1 mM TCEP, 0.01% (w/v) BSA, and 0.008% (w/v) Brij-35 either in the presence of DMSO (final 0.2% DMSO) or compound dilution series in DMSO. After 10 minutes incubation at room temperature, a mixture Eu-W1024 Anti-6×His antibody (Perkin Elmer ADO 110) and SureLight Allophycocyanin-Streptavidin (APC-SA, Perkin Elmer CR130-100) were added to a final concentrations of 0.2 nMolar antibody and 50 nMolar APC-SA, respectively. After twenty minutes of equilibration, the plates were read on an Envision instrument and IC50s calculated using a four parameter non-linear curve fit.
Measurement: IC50 = 538 nM (pChEMBL 6.269999980926514)
Descriptors: MW 249.31, ALogP 2.83, HBA 3, HBD 1, PSA 47.56, QED 0.9
Compound (SMILES): C[C@@H](COc1cn[nH]c(=O)c1C(F)(F)F)OCCC(=O)N1CCN(c2ccc(C#N)cn2)CC1
InChIKey: BBVRKVPXLZGXKX-AWEZNQCLSA-N
Target: Protein mono-ADP-ribosyltransferase TIPARP (SINGLE PROTEIN), Homo sapiens
UniProt: Q7Z3E1
Assay: Enzymatic Assay: Displacement of Probe A, a biotinylated probe binding to the TIPARP active site, was measured using a time-resolved fluorescence energy transfer (TR-FRET) assay. 20 nL of a dose response curve of each test compound was spotted in black 384-well polystyrene proxiplates (Perkin Elmer) using a Mosquito (TTP Labtech). Reactions were performed in a 8 μL volume by adding 6 JAL of TIPARP and Probe A in assay buffer (20 mM HEPES pH=8, 100 mM NaCl, 0.1% bovine serum albumin, 2 mM DTT and 0.002% Tween20), incubating with test compound at 25° C. for 30 min, then adding 2 μL of ULight-anti 6×His and LANCE Eu-W1024 labeled streptavidin (Perkin Elmer). The final concentrations of TIPARP and Probe A were 6 nM and 2 nM, respectively. The final concentration of ULight-anti 6×His and LANCE Eu-W1024 labeled streptavidin were 4 nM and 0.25 nM, respectively. Reactions were incubated at 25° C. for an additional 30 min, then read on an Envision plate reader equipped with a LANCE/DELFIA top mirror (Perkin Elmer) using excitation of 320 nm and emission of 615 nm and 665 nM with a 90 μs delay. The ratio of the 665/615 nm emission were calculated for each well to determine the amount of complex of TIPARP and Probe A in each well.
Measurement: IC50 = 550 nM (pChEMBL 6.260000228881836)
Descriptors: MW 480.45, ALogP 1.58, HBA 8, HBD 1, PSA 124.44, QED 0.6
Compound (SMILES): O=C(c1cc(-c2ccc3c(c2)OCO3)on1)N1CCC[C@H]1c1ccon1
InChIKey: VQFMCVSSAIXQEV-AWEZNQCLSA-N
Target: Stearoyl-CoA desaturase (SINGLE PROTEIN), Homo sapiens
UniProt: O00767
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 > 45000 nM (pChEMBL 4.3470001220703125)
Descriptors: MW 353.33, ALogP 3.04, HBA 7, HBD 0, PSA 90.83, QED 0.71
Compound (SMILES): O=C(O)CC1CCN(c2c(F)cc(-c3nccc(Oc4ccccc4)n3)cc2F)CC1
InChIKey: KXCKLTQYNIHNHU-UHFFFAOYSA-N
Target: Free fatty acid receptor 4 (SINGLE PROTEIN), Homo sapiens
UniProt: Q5NUL3
Assay: Measurement of Activity of GPR120 Agonist (Cell-Based Assay): CHO-K1 cells expressing Ga16 and hGPR120 were dispensed into each well of a 96-well plate (3×104 cells/100 μl/well) and then incubated in 5% CO2, 37° C. incubator for 18 hours. Each well was treated with 100 μl of Calcium 5 dye (Molecular Devices) solution including 2% DMSO and then incubated in 5% CO2, 37° C. incubator for 1 hour. Serially diluted GPR120 agonists were prepared to a final concentration of 0.5% DMSO in a 96-well plate. Each well was treated with 50 μl of the agonist compounds using Plexstation II, and then fluorescence was measured at Ex 485 nm and Em 525 nm.Fluorescence increased by the serially diluted GPR120 agonists is calculated as a relative percent (%) value based on the fluorescence represented by the treatment of 1% DMSO only. EC50 refers to the concentration of agonist which shows 50% of maximum fluorescence increased by the treatment of agonist. The calculation of measurement was carried out by using statistical software (Prizm).
Measurement: EC50 < 200 nM (pChEMBL 6.698999881744385)
Descriptors: MW 425.44, ALogP 4.91, HBA 5, HBD 1, PSA 75.55, QED 0.61
Compound (SMILES): COc1cn(Cc2cc(F)cc(F)c2)c(Nc2cc3c(cc2C)OCCC3)nc1=O
InChIKey: SLFGSWNTEIZKMA-UHFFFAOYSA-N
Target: P2X purinoceptor 7 (SINGLE PROTEIN), Homo sapiens
UniProt: Q99572
Assay: Evaluation of a Human P2X7 Receptor Inhibitory Activity: Stably expressing cell line (1321N1 cell transfected with the human P2X7 receptor gene (GenBank accession number NM_002562.5 including T606C and G952A SNP)) was used. The cells were seeded in a 384-well microtiter plate at a concentration of 8000 cells/well and cultured in the medium (10% fetal bovine serum, 25 mM HEPES, 1% penicillin and streptomycin in DMEM) for one day at 37° C. under 5% carbon dioxide atmosphere. After replacing with 20 μL of the HBSS buffer (20 mM HEPES, 55.6 mM D-glucose, 1×HBSS(−), pH7.4-7.5), 15 μL of 17.3 μM Yo-Pro solution in the HBSS buffer was added. The plate was placed in high-throughput cellular screening system FLIPR TETRA (Molecullar Devices, LLC.) and 15 μL of 130 μM BzATP solution in the HBSS buffer was added. Measurement of fluorescence intensity by FLIPR TETRA was started. After eight minutes, 15 μL of DMSO solutions containing different concentrations of the compound of the present invention as prepared by dilution with the HBSS buffer were dispensed to each well through the built-in automatic dispenser, and the measurement of fluorescence intensity was continued for 20 minutes. The maximum fluorescence intensity without the compound of the present invention is calculated as 0% inhibition and the maximum fluorescence intensity when the reference compound was added is calculated as 100% inhibition. Changing values of fluorescence intensity by the compound of the present invention were calculated by difference between maximum and minimum fluorescence intensity for 20 minutes. IC50 was calculated using logistic approximation.
Measurement: IC50 = 55 nM (pChEMBL 7.260000228881836)
Descriptors: MW 413.42, ALogP 3.96, HBA 6, HBD 1, PSA 65.38, QED 0.69
Compound (SMILES): CNC(=O)c1nnc(Nc2cc(C)nc(C)n2)cc1Nc1cccc(-c2ncn(C)n2)c1OC
InChIKey: PTIQFVKQCBWNJS-UHFFFAOYSA-N
Target: Non-receptor tyrosine-protein kinase TYK2 (SINGLE PROTEIN), Homo sapiens
UniProt: P29597
Assay: Probe Displacement Assay: The probe displacement assay is conducted as follows: In a 385 well plate, test compounds along with recombinantly expressed His-tagged protein corresponding to amino acids 575-869 of human Tyk2 (sequence shown below) at 2.5 nM, 40 nM ((R)N-(1-(3-(8-methyl-5-(methylamino)-8H-imidazo[4,5-d]thiazolo[5,4-b]pyridin-2-yl)phenyl)ethyl)-2-([3H]methylsulfonyl)benzamide) (preparation described below) and 80 μg/mL Copper His-Tag scintillation proximity assay beads (Perkin Elmer, Catalog #RPNQ0095) in 50 mM HEPES, pH 7.5, containing 100 μg/mL bovine serum albumin and 5% DMSO were incubated for 30 minutes at room temperature. The amount of radiolabeled probe (preparation described below) bound to Tyk2 was then quantified by scintillation counting, and the inhibition by the test compound calculated by comparison to wells either with no inhibitor (0% inhibition) or without Tyk2 (100% inhibition). The IC50 value is defined as the concentration of test compound required to inhibit radiolabeled probe binding by 50%.
Measurement: EC50 = 1.46 nM (pChEMBL 8.84000015258789)
Descriptors: MW 460.5, ALogP 2.53, HBA 11, HBD 3, PSA 144.66, QED 0.38
Compound (SMILES): Cc1c(Cc2ccc3c(n2)C(F)(F)CCS3(=O)=O)c2cc(F)ccc2n1CC(=O)O
InChIKey: GIXYEVAZXTXWGK-UHFFFAOYSA-N
Target: Prostaglandin D2 receptor 2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9Y5Y4
Assay: Biological Activity Assay: Experimental Method:PathHunter CHO-K1 CRTH2 β-arrestin cells (DiscoverX, catalogue number 93-0291C2) grew under standard conditions, and were inoculated into a white-wall 384-well microplate at a density of 5,000 cells/well. 20 μL of Cell Plating Reagent 1 was used in each well. Before the test, the cells were incubated overnight at 37° C./5% CO2. A test compound was serially diluted in DMSO with a dilution factor of 3-fold to give 8 concentrations of the test compound. Shortly before the test, the serially diluted test compound was further diluted with the test buffer to 5 times of the test concentration. 5 μL of the further diluted test compound was added to the cells, and the cells were incubated for 30 min at 37° C. The concentration of the solvent was 1%. 5 μL of 6×EC80 agonist (PGD2) buffer was added to the cells, and the cells were incubated for 90 min at 37° C. Measured signals were generated by one-time addition of 15 μL (50% v/v) of PathHunter detection mixture reagent and subsequent one-hour incubation. The microplate was read through the chemiluminescent signals of PerkinElmer Envision reader. Biological activity of the test compound was analyzed by CBIS data analysis suite (ChemInnovation, CA), and was denoted as IC50 value.
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 438.43, ALogP 3.43, HBA 5, HBD 1, PSA 89.26, QED 0.67
Compound (SMILES): COC[C@H](C(=O)Nc1nnc(N[C@@H]2CCN(c3cccnn3)C2)s1)c1cccc(OC)c1
InChIKey: SQQHQXXXLATMLV-WBVHZDCISA-N
Target: Glutaminase kidney isoform, mitochondrial (SINGLE PROTEIN), Homo sapiens
UniProt: O94925
Assay: GLS Enzyme Potency Assay: A Glutamate Oxidase/AmplexRed coupled assay was used to measure the ability of compounds to bind to and inhibit the activity of GLS1 in vitro. 6His tagged GLS protein (amino acids 63-669) expressed in E. Coli was purified and stored at −80° C. in aliquots. GLS1 was diluted to 2× working concentration and incubated at room temperature to allow the tetrameric/dimeric forms to reach steady state. Assay measurements were performed in buffer comprising 50 mM TRIS pH 7.8, 100 mM NaPO4, pH 7.8, 0.001% v/v Tween20. Purified recombinant GLS1 protein was diluted in assay buffer to 12 nM and pre-incubated at room temperature for 30 minutes. Test compounds were prepared by dilution in 100% DMSO to give the correct dose range for 12 point concentration response and an appropriate volume (2.5-60 nl) dispensed into 384 well micro assay plates (Greiner product code 784900) using a Labcyte Echo 555 acoustic dispenser. DMSO concentration was maintained at 2% by back filling with DMSO solution. 3 μL of diluted GLS1 protein (12 nM) was then dispensed into each well using a BioRaptr automated dispenser (Beckman-Coulter) and incubated for 15 minutes at room temperature. 3 μL of 100 mM glutamine diluted in assay buffer was then added and the reaction incubated at room temperature for 60 minutes. The reaction was then stopped by addition of 45 μM 6-(2-bromoethynyl)-2,3-dimethyl-quinazolin-4-one, 75 μM Amplex Red, 0.375 units/mL Horseradish Peroxidase, 0.12 units/mL Glutamate Oxidase in 100 mM TRIS pH7.5. After 30 minutes at room temp in the dark, plates were read on a Perkin Elmer EnVision using 535/590 nm optic filters and raw data analysed using Genedata to generate IC50 values. An artefact version of the assay where the 6His tagged GLS protein and glutamine were replaced with assay buffer was also used to rule out non specific effects on the assay components.
Measurement: IC50 = 28.8 nM (pChEMBL 7.539999961853027)
Descriptors: MW 455.54, ALogP 2.4, HBA 10, HBD 2, PSA 114.39, QED 0.5
Compound (SMILES): C=CC(=O)Nc1ccc(C(=O)N2CC[C@@H](Nc3cnc(Cl)cn3)C2)cc1
InChIKey: JFSZDNXDUFONBS-CQSZACIVSA-N
Target: Cyclin-dependent kinase 12 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9NYV4
Assay: Protein kinase assay (Condition A: Thiol Containing Conditions): For the assays, 5 μl solution from each well of the compound dilution plates/10% DMSO were transferred into the assay plates. The final volume of the assay was 50 μl. All compounds were tested at 10 final assay concentrations in the range from 1×10−04M to 3×10−09M, in singlicate. The final DMSO concentration in the reaction cocktails was 1% in all cases.Recombinant protein kinases: All protein kinases were expressed in Sf9 insect cells or in E. coli as recombinant GST-fusion proteins or His-tagged proteins, either as full-length or enzymatically active fragments. All kinases were produced from human cDNAs and purified by either GSH-affinity chromatography or immobilized metal. Affinity tags were removed from a number of kinases during purification. The purity of the protein kinases was examined by SDS-PAGE/Coomassie staining, the identity was checked by mass spectroscopy.Protein kinase assay: A radiometric protein kinase assay (33PanQinase® Activity Assay) was used for measuring the kinase activity of the three protein kinases. All kinase assays were performed in 96-well FlashPlates from PerkinElmer (Boston, Mass., USA) in a 50 μl reaction volume. The reaction cocktail was pipetted in four steps in the following order:    20 μl of assay buffer (standard buffer)    5 μl of ATP solution (in H2O)    5 μl of test compound (in 10% DMSO)    20 μl enzyme/substrate mixThe assay for all protein kinases contained 70 mM HEPES-NaOH pH 7.5, 3 mM MgCl2, 3 mM MnCl2, 3 μM Na-orthovanadate, 1.2 mM DTT, 50 μg/ml PEG20000, ATP (variable concentrations, corresponding to the apparent ATP-Km of the respective kinase), [γ-33P]-ATP (approx. 9×1005 cpm per well), protein kinase (variable amounts), and substrate (variable amounts).
Measurement: IC50 = 550 nM (pChEMBL 6.260000228881836)
Descriptors: MW 371.83, ALogP 2.58, HBA 5, HBD 2, PSA 87.22, QED 0.79
Compound (SMILES): O=C(NN1CCC(NCCOc2ccc(Cl)c(F)c2)CC1)c1ccc2cc(Cl)ccc2n1
InChIKey: GJMIVKQVWAFEJP-UHFFFAOYSA-N
Target: Cyclic AMP-dependent transcription factor ATF-4 (SINGLE PROTEIN), Homo sapiens
UniProt: P18848
Assay: Expression Inhibition Assay: The ATF4 reporter was prepared by fusing the human full length 5′UTR of ATF4 (NCBI Accession No. BC022088.2) upstream of the firefly luciferase coding sequence lacking the initiator methionine. The fused sequence was cloned into pLenti-EF1a-C-Myc-DDK-IRES-Puro cloning vector (Origen #PS100085) using standard methods. Virus production was carried out by using Lenti-X Packaging Single Shots Protocol (Clonetech #631276). Viral particles were used to transduce HEK293T cells (ATCC #CRL-3216, ATCC Manassas, Va.), which were subsequently selected with puromycin to generate stable cell line. Cells were maintained at 37° C. and 5% CO2 in DMEM-F12 (Hyclone #SH30023.02) supplemented with 10% heat-inactivated fetal bovine serum (Gibco #16000-044), 2 mM L-glutamine (Gibco #25030-081), 100 U/ml penicillin, and 100 μg/ml streptomycin (Gibco #15140-122).
Measurement: IC50 = 55.6 nM (pChEMBL 7.25)
Descriptors: MW 477.37, ALogP 4.46, HBA 5, HBD 2, PSA 66.49, QED 0.49
Compound (SMILES): C[C@@H]1CC[C@@H](C(=O)NC(c2ccccc2)C(O)c2ccccc2)CN1C(=O)c1ccccc1-n1nccn1
InChIKey: WQLMDLUHZCAZAA-YTQBYCBUSA-N
Target: Orexin receptor type 2 (SINGLE PROTEIN), Homo sapiens
UniProt: O43614
Assay: FLIPR Ca2+ Flux Assay: For intracellular calcium measurements, Chinese hamster ovary (CHO) cells expressing the rat orexin-1 receptor or the human orexin-2 receptor, are grown in Iscove's modified DMEM containing 2 mM L-glutamine, 0.5 g/ml G418, 1% hypoxanthine-thymidine supplement, 100 U/ml penicillin, 100 μg/ml streptomycin and 10% heat-inactivated fetal calf serum (FCS). The cells are seeded at 20,000 cells/well into Becton-Dickinson black 384-well clear bottom sterile plates coated with poly-D-lysine. All reagents were from GIBCO-Invitrogen Corp. The seeded plates are incubated overnight at 37° C. and 5% CO2. Ala-6,12 human orexin-A as the agonist is prepared as a 1 mM stock solution in 1% bovine serum albumin (BSA) and diluted in assay buffer (HBSS containing 20 mM HEPES, 0.1% BSA and 2.5 mM probenecid, pH7.4) for use in the assay at a final concentration of 70 pM. Test compounds are prepared as 10 mM stock solution in DMSO, then diluted in 384-well plates, first in DMSO, then assay buffer. On the day of the assay, cells are washed 3 times with 100 μl assay buffer and then incubated for 60 min (37° C., 5% CO2) in 60 μl assay buffer containing 1 μM Fluo-4AM ester, 0.02% pluronic acid, and 1% BSA. The dye loading solution is then aspirated and cells are washed 3 times with 100 μl assay buffer. 30 μl of that same buffer is left in each well. Within the Fluorescent Imaging Plate Reader (FLIPR, Molecular Devices), test compounds are added to the plate in a volume of 25 μl, incubated for 5 min and finally 25 μl of agonist is added.
Measurement: IC50 = 515 nM (pChEMBL 6.289999961853027)
Descriptors: MW 509.61, ALogP 4.1, HBA 6, HBD 2, PSA 100.35, QED 0.39
Compound (SMILES): Fc1ccc(-c2cnc3nc(COc4ccccc4)cn3n2)c(F)c1
InChIKey: UQZIDUTYHFQTND-UHFFFAOYSA-N
Target: Metabotropic glutamate receptor 5 (SINGLE PROTEIN), Homo sapiens
UniProt: P41594
Assay: Calcium Influx Assay Based on Fluorescence: Ca2+ (calcium) influx assay is an experiment for measuring the activity of a positive allosteric modulator of mGluR5 receptor in which human mGluR5 receptor-overexpressed HEK293 cell line is used. The day before the experiment, cells were prepared in a cell culture medium (DMEM, 5% FBS) with the density of 80,000/well, and 100 μl of cells were dispensed into each well of a poly-D-lysine-coated 96-well plate. Cells were incubated in a 5% CO2, 37° C. incubator. The next day, the cell culture medium was removed from the plate, and 100 μl of 1× Fluo-4 calcium indicator diluted with a buffer (1× Hank's balanced salt solution, 20 mM HEPES, 2.5 mM probenecid) were added to each well and incubated at 37° C. for 1 hour. The compound stock solutions were prepared in 100% DMSO, and the compounds were serially diluted with a ¼ dilution to 6 or 7 concentrations (final concentration was 10 μM to 10 nM). The diluted compound solutions were added to the plate with 0.1 to 0.2% of final DMSO concentration. 1 hour after the addition of Fluo-4 calcium indicator, L-glutamate (EC20 concentration) and the test compound solutions were added to the plate, and Ca2+ reaction was then measured by FLIPR at room temperature. The activity of the compounds was standardized on the basis of the results of maximum value-minimum value of fluorescent reaction, and the activity value was calculated on the basis that no activity on glutamate EC20 is 0% and the reaction to glutamate maximum value is 100%.
Measurement: EC50 = 300 nM (pChEMBL 6.519999980926514)
Descriptors: MW 338.32, ALogP 3.65, HBA 5, HBD 0, PSA 52.31, QED 0.57
Compound (SMILES): CC(C)Nc1nc(=O)n(-c2ccccc2Cl)c2cc(C3CC3)ccc12
InChIKey: VXJOETNMUBNHDH-UHFFFAOYSA-N
Target: S-adenosylmethionine synthase isoform type-2 (SINGLE PROTEIN), Homo sapiens
UniProt: P31153
Assay: Phosphate Sensor Fluorescence Assay : MAT2A enzyme is incubated with a test compound in DMSO or DMSO and its substrates (L-methionine and ATP) in a microtiter plate. The enzymatic reaction is stopped by the addition of Working Phosphate Sensor Mixture. The plate is analyzed for fluorescence at 450 nm. The high control (DMSO with enzyme and its substrates) gives high fluorescence which represents no inhibition of enzymatic activity while the low control (DMSO with MAT2A substrates and no enzyme) gives low fluorescence which represents full inhibition of enzymatic activity.Materials:Human MAT2A: Cepter, amino acids 1-395Tris, pH 7.5: Invitrogen cat #15567-027KCl: Ambion cat #AM9640GMgCl2: Ambion cat #AM9530GBrij-35: Sigma cat B4184-10MLDTT: Goldbio cat #DTT100BGG: Sigma cat #G5009-25GPNP: Novus Biologicals cat #NBP1-508727-MEG: Cayman Chemical cat #15988L-Methionine: Alfa Aesar cat #J61904ATP: Alfa Aesar cat #J60336Phosphate Sensor: Thermo Fisher cat #PV4407EDTA: Life Tech cat #15575-038Assay plate: 384-well black polypropylene plate: Thomas Scientific cat #1149Q35Final Assay Conditions:Assay Buffer: 50 mM Tris, pH 7.5/50 mM KCl/10 mM MgCl2/0.01% Brij-35/1 mM DTT/0.1% BGG/40 nM PNP/6 uM 7-MEGMAT2A: 10 nM for Cepter clone ID 329, lot 00023-123 before the addition of Working Phosphate Sensor Mixture    5 nM for Cepter clone ID 334, lot 00023-148 before the addition of Working Phosphate Sensor MixtureL-methionine: 500 uM before the addition of Working Phosphate Sensor MixtureATP: 500 uM before the addition of Working Phosphate Sensor MixtureProcedure:For the assay, a mixture of 1 mM L-methionine/1 mM ATP (2× final pre-stopped concentration) in assay buffer; MAT2A (2× final pre-stopped concentration) in Assay Buffer and Working Phosphate Sensor Mixture (1.5 uM Phosphate Sensor/30 mM EDTA in Assay Buffer, which is 3× final concentrations) were prepared. Test compounds or DMSO were added to the appropriate well suing D300e digital dispenser. 5 μl/well of Assay Buffer was added to the wells corresponding to the negative control and 5 μl/well of MAT2A was added to all the wells except for those corresponding to the negative control. After incubating the plate at room temperature for 15 minutes, 5 μl/well of the 1 mM L-methionine/1 mM ATP mixture was added to all wells. The plate was centrifuged at 1000 rpm for 1 minute and then incubated at room temperature for 1 hour. 5 μl of the Working Phosphate Sensor Mixture was added to all wells and the plate was centrifuged at 1000 rpm for 1 minute. The plate was read for fluorescence at 450 nm after exciting at 430 nm.
Measurement: IC50 < 200 nM (pChEMBL 6.698999881744385)
Descriptors: MW 353.85, ALogP 4.74, HBA 4, HBD 1, PSA 46.92, QED 0.74
Compound (SMILES): CCc1cccc(CC)c1-n1nc2c(c1-c1cc(F)c(NC(N)=O)cc1F)CN(Cc1ccccc1)C(C)C2
InChIKey: HIFZCBJWFSLVIA-UHFFFAOYSA-N
Target: C5a anaphylatoxin chemotactic receptor 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P21730
Assay: chemotaxis assay: Optionally, compounds may be further assayed for their ability to inhibit chemotaxis in cells. Chemotaxis assays are performed using 5 μm pore polycarbonate, polyvinylpyrrolidone-coated filters in 96-well chemotaxis chambers (Neuroprobe; Gaithersburg, Md.) using chemotaxis buffer (Hank's balanced salt solution (HBSS) and 1% FBS). C5aR ligands (i.e., C5a, R&D Systems; Minneapolis, Minn.) are use to evaluate compound mediated inhibition of C5aR mediated migration. Other chemokines (i.e., SDF-1α; R&D Systems; Minneapolis, Minn.) are used as specificity controls. The lower chamber is loaded with 29 μl of chemokine (i.e., 0.03 nM C5a) and varying amounts of compound; the top chamber contains 100,000 U937 or neutrophil cells in 20 μl. The chambers are incubated 1.5 hours at 37° C., and the number of cells in the lower chamber quantified either by direct cell counts in five high powered fields per well or by the CyQuant assay (Molecular Probes), a fluorescent dye method that measures nucleic acid content and microscopic observation.
Measurement: IC50 < 5 nM (pChEMBL 8.300999641418457)
Descriptors: MW 529.64, ALogP 6.38, HBA 4, HBD 2, PSA 76.18, QED 0.29
Compound (SMILES): CC(O)(c1cc(F)c2c(c1)C(=O)N(Cc1ccc(Cl)cn1)[C@@]2(OCCO)c1ccc(Cl)cc1)C1CCS(=O)(=O)CC1
InChIKey: LNHWDLFOWCRHJS-YPJJGMIRSA-N
Target: E3 ubiquitin-protein ligase Mdm2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q00987
Assay: Binding Assay (ELISA): The ELISA assay was performed in streptavidin coated plates which were preincubated with 200 ul per well of 1 μg ml−1 biotinylated IP3 peptide. The plates were ready to use for MDM2 binding after washing the plate with PBS.Compounds and control solutions in DMSO aliquoted in 96-well plates were pre-incubated in a final 2.5-5% (v/v) DMSO concentration at room temperature (for example 20C.) for 20 min with 190 ul aliquots of optimized concentrations of in vitro translated MDM2, before transfer of the MDM2-compound mixture to the b-IP3 streptavidin plates, and incubation at 4C. for 90 min. After washing three times with PBS to remove unbound MDM2, each well was incubated at 20C. for 1 hour with a TBS-Tween (50 mM Tris pH7.5; 150 mM NaCl; 0.05% Tween 20 nonionic detergent) buffered solution of primary mouse monoclonal anti-MDM2 antibody (Ab-5, Calbiochem, used at a 1/10000 or 1/200 dilution depending on the antibody stock solution used), then washed three times with TBS-Tween before incubation for 45 mins at 20C. with a TBS-Tween buffered solution of a goat-anti-mouse horseradish peroxidase (HRP) conjugated secondary antibody (used at 1/20000 or 1/2000 depending on the antibody stock solution). The unbound secondary antibody was removed by washing three times with TBS-Tween. The bound HRP activity was measured by enhanced chemiluminesence (ECL, Amersham Biosciences) using the oxidation of the diacylhydrazide substrate, luminol, to generate a quantifiable light signal. The percentage of MDM2 inhibition at a given concentration is calculated as the [1−(RLU detected in the compound treated sample−RLU negative DMSO control)÷(RLU of DMSO positive and negative controls)]x100 or as the (RLU detected in the compound treated sample ÷ RLU of DMSO controls)x100.
Measurement: IC50 = 2.8 nM (pChEMBL 8.550000190734863)
Descriptors: MW 623.53, ALogP 4.43, HBA 7, HBD 2, PSA 117.03, QED 0.38
Compound (SMILES): C[C@H]1CN(c2cc(F)c(-c3cnc4c(c3)OCCO4)cc2NC(=O)c2c[nH]c(=O)cc2C(F)(F)F)CCN1C
InChIKey: TUBYFMDAMSFKHJ-AWEZNQCLSA-N
Target: WD repeat-containing protein 5 (SINGLE PROTEIN), Homo sapiens
UniProt: P61964
Assay: Surface Plasmon Resonance (SPR) Assay: Exemplary compounds of the application were dissolved in 100% DMSO at 10 mM, assayed fresh, and then stored at −20° C. for repeat studies and other experiments. Full length WDR5 with an N-terminal His tag and C-terminal AviTag (Avidity Inc.) was expressed in E. coli with coexpression of BirA to biotin labelled protein in vivo. Purification of the protein was performed using Ni-NTA. The purified WDR5 protein has a molecular weight of 41976 Da.SPR studies were performed using a Biacore T200 instrument (GE Health Sciences Inc.). Biotinylated WDR5 protein (approximately 3000 RU) was stably captured to streptavidin coupled SA chips according to the manufacture's protocol (GE Health Sciences Inc.). The running buffer used was HBS-EP (20 mM Hepes pH 7.4, 150 mM NaCl, 3 mM EDTA, 0.05% P-20) plus 5% DMSO with a flow rate of 40 μl/min. For SPR analysis, 5 different concentrations of each exemplary compound of the application were sprayed into 96 or 384 well plates using an HP D300 digital dispenser. The concentration ranged from about 195 nM to about 12 nM in a two-fold series. Concentration ranges were adjusted higher or lower for weaker or more potent compounds respectively when necessary. For the KD determinations, single cycle kinetic analysis was performed with an on time of 60 seconds, and an off time of 300 or 600 seconds. Curve fitting and KD calculations were performed with the Biacore T200 Evaluation software (GE Health Sciences Inc).
Measurement: Kd = 2.82 nM (pChEMBL 8.550000190734863)
Descriptors: MW 547.51, ALogP 3.76, HBA 7, HBD 2, PSA 99.79, QED 0.48
Compound (SMILES): CC1(N)CCN(c2cnc3c(OCc4cccc5[nH]ccc45)n[nH]c3n2)CC1
InChIKey: INQQCPQLXQXPSJ-UHFFFAOYSA-N
Target: Tyrosine-protein phosphatase non-receptor type 11 (SINGLE PROTEIN), Homo sapiens
UniProt: Q06124
Assay: SHP2 Allosteric Inhibition Assay: SHP2 is allosterically activated through binding of bis-tyrosyl-phorphorylated peptides to its Src Homology 2 (SH2) domains. The latter activation step leads to the release of the auto-inhibitory interface of SHP2, which in turn renders the SHP2 protein tyrosine phosphatase (PIT) active and available for substrate recognition and reaction catalysis. The catalytic activity of SHP2 was monitored using the surrogate substrate DiFMUP in a prompt fluorescence assay format.</p><p id="p-0381" num="0414">More specifically, the phosphatase reactions were performed at room temperature in 96-well black polystyrene plate, flat bottom, low flange, non-binding surface (Corning, Cat #3575) using a final reaction volume of 50 μl and the following assay buffer conditions: 60 mM HEPES, pH 7.2, 75 mM NaCl, 75 mM KCl, 1 mM EDTA 0.005% Brij-35, 5 mM DTT. The inhibition of SHP2 by compounds of the invention (concentrations varying from 0.003-100 μM) was monitored using an assay in which 0.25 nM of SHP2 was incubated with of 0.5 μM of peptide IRSI_psf 1172(dPEG8)pY1222 (sequence: H2N LN(pY)IDLDLV(dPEG8)LST(pY)ASINFQK-amide). After 30-60 minutes incubation at 25° C., the surrogate substrate DiFMUP (Invitrogen, cat #D6567, 100 μM final) was added to the reaction and the conversion of DiFMUP to 6,8-difluoro-7-hydroxyl-4-methylcoumarin (DiFMU) was monitored continuously for 10 minutes with excitation at 355 nm and emission at 460 nm using a microplate reader (PolarStar, BMG). The inhibitor dose response curves were analyzed using normalized IC50 regression curve fitting with control based normalization.
Measurement: IC50 < 1000 nM (pChEMBL 6)
Descriptors: MW 377.45, ALogP 2.73, HBA 6, HBD 3, PSA 108.74, QED 0.5
Compound (SMILES): CS(=N)(=O)c1nc(N)c2[nH]c(=O)n(Cc3ccncc3)c2n1
InChIKey: GMEXKXFTVMLTOL-UHFFFAOYSA-N
Target: Toll-like receptor 7 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9NYK1
Assay: HEK-Blue-hTLR7 assay: HEK-Blue-hTLR7 cells were incubated at a density of 250,000˜450,000 cells/mL in a volume of 180 μL in a 96-well plate in Dulbecco's Modified Eagle's medium (DMEM) containing 4.5 g/L glucose, 50 U/mL penicillin, 50 mg/mL streptomycin, 100 mg/mL Normocin, 2 mM L-glutamine, 10% (V/V) heat-inactivated fetal bovine serum for 24 hrs. Then the HEK-Blue-hTLR-7 cells were incubated with addition of 20 μL test compound in a serial dilution in the presence of final DMSO at 1% and perform incubation under 37° C. in a CO2 incubator for 20 hrs. Then 20 μL of the supernatant from each well was incubated with 180 μL Quanti-blue substrate solution at 37° C. for 2 hrs and the absorbance was read at 620˜655 nm using a spectrophotometer. The signalling pathway that TLR7 activation leads to downstream NF-κB activation has been widely accepted, and therefore similar reporter assay was also widely used for evaluating TLR7 agonist (Tsuneyasu Kaisho and Takashi Tanaka, Trends in Immunology, Volume 29, Issue 7, July 2008, Pages 329.sci; Hiroaki Hemmi et al, Nature Immunology 3, 196-200 (2002)).
Measurement: EC50 = 260 nM (pChEMBL 6.579999923706055)
Descriptors: MW 319.35, ALogP 0.18, HBA 8, HBD 3, PSA 143.4, QED 0.59
Compound (SMILES): c1ccc2c(c1)ncn2-c1ccnc(NC2COC2)n1
InChIKey: FYKZPCLKTZZIBA-UHFFFAOYSA-N
Target: MAP kinase-interacting serine/threonine-protein kinase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9BUB5
Assay: Inhibition Assay: The synthesized compounds then were tested for inhibition of the kinase activity (IC50) of Mnk1 and/or Mnk2 using the ADP monitoring assay for kinases described in Zegzouti, H.; Zdanovskaia, M.; Hsiao, K.; Goueli, S. A., ADP-Glo: A Bioluminescent and homogeneous ADP monitoring assay for kinases.
Measurement: IC50 = 420 nM (pChEMBL 6.380000114440918)
Descriptors: MW 267.29, ALogP 1.63, HBA 6, HBD 1, PSA 64.86, QED 0.78