lovingscience/chembl-v37-raw-activities huggingface dataset:
Compound (SMILES): CC(CC(F)F)Nc1n[nH]c2nccc(Oc3ccc(NC(=O)c4cn(C(C)C)c(=O)n(-c5ccc(F)cc5)c4=O)cc3F)c12
InChIKey: GSJTVHKWTWIIFJ-UHFFFAOYSA-N
Target: Hepatocyte growth factor receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P08581
Assay: Enzyme Assay: The affinity of compound binding to wild type and mutant human MET kinases is measured using Invitrogen's LanthaScreen Eu Kinase Binding technology. Briefly, GST-tagged recombinant human MET kinase domain from Signal Chem (see Table 8 below for concentration in assay) is incubated with 50 nM Alexa-Fluor® Tracer 236 (Invitrogen Cat No. PR9078A) and 2 nM Europium-anti-GST (Invitrogen Cat. No. A151 6) along with test compound in a buffer consisting of 25 mM HEPES, pH 7.4, 10 mM MgCl2, 0.01% Triton X-100, 1 mM DTT, and 2% DMSO. Compounds are typically prepared in a three-fold serial dilution in DMSO and added to the assay to give the appropriate final concentration. After a 60-minute incubation at 22° C., the reaction is measured using a PerkinElmer EnVision multimode plate reader via TR-FRET dual wavelength detection, and the percent of control (POC) calculated using a ratiometric emission factor. 100 POC is determined using no test compounds and 0 POC is determined using a concentration of control compound that completely inhibits the enzyme
Measurement: IC50 = 6.2 nM (pChEMBL 8.210000038146973)
Descriptors: MW 625.58, ALogP 5.63, HBA 9, HBD 3, PSA 135.93, QED 0.17
Compound (SMILES): COc1ccc2c(c1)[C@]1(CC1c1ccc3c(/C=C/c4ccc(CN(C)C)cc4)n[nH]c3c1)C(=O)N2
InChIKey: DKVKUPRCFKTRIY-OWJSMZNMSA-N
Target: Aurora kinase B (SINGLE PROTEIN), Homo sapiens
UniProt: Q96GD4
Assay: Inhibition Assay: Aurora B inhibition was determined using the Z-Lyte assay kit from Invitrogen. The assay was performed using the recommended manufacturer's instructions with 128 μM ATP and 28 nM Aurora B (Invitrogen cat #PV3970). The % inhibition values were determined according to the manufacturer's directions and IC50 values were obtained using a non-linear 4 point logistic curve fit (XLfit4, IDBS)
Measurement: IC50 = 550 nM (pChEMBL 6.260000228881836)
Descriptors: MW 464.57, ALogP 5.18, HBA 4, HBD 2, PSA 70.25, QED 0.41
Compound (SMILES): C[C@@H]1Cc2c(ccc3[nH]ncc23)[C@@H](c2c(F)cc(NC3CN(CCCF)C3)cc2F)N1CC(F)(F)CO
InChIKey: PBTFDTKJUCHOCI-BZQUYTCOSA-N
Target: Estrogen receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P03372
Assay: Binding Assay: ER: The ability of compounds to bind to isolated Estrogen Receptor Alpha Ligand binding domain (ER alpha-LBD (GST)) was assessed in competition assays using a LanthaScreen Time-Resolved Fluorescence Resonance Energy Transfer (TR-FRET) detection end-point. For the LanthaScreen TR-FRET endpoint, a suitable fluorophore (Fluormone ES2, ThermoFisher, Product code P2645) and recombinant human Estrogen Receptor alpha ligand binding domain, residues 307-554 (expressed and purified in-house) were used to measure compound binding. The assay principle is that ER alpha-LBD (GST) is added to a fluorescent ligand to form a receptor/fluorophore complex. A terbium-labelled anti-GST antibody (Product code PV3551) is used to indirectly label the receptor by binding to its GST tag, and competitive binding is detected by a test compound's ability to displace the fluorescent ligand, resulting in a loss of TR-FRET signal between the Tb-anti-GST antibody and the tracer. The assay was performed as follows with all reagent additions carried out using the Beckman Coulter BioRAPTR FRD microfluidic workstation:    1. Acoustic dispense 120 nL of the test compound into a black low volume 384 well assay plates.    2. Prepare 1×ER alpha-LBD/Tb-antiGST Ab in ES2 screening buffer and incubate for 15 minutes.    3. Dispense 6 μL of the 1×AR-LBD/Tb-anti-GST Ab reagent into each well of the assay plate followed by 6 μL of Fluorophore reagent into each well of the assay plate    4. Cover the assay plate to protect the reagents from light and evaporation, and incubate at room temperature for 4 hours.    5. Excite at 337 nm and measure the fluorescent emission signal of each well at 490 nm and 520 nm using the BMG PheraSTAR.Compounds were dosed directly from a compound source microplate containing serially diluted compound (4 wells containing 10 mM, 0.1 mM, 1 mM and 10 nM final compound respectively) to an assay microplate using the Labcyte Echo 550. The Echo 550 is a liquid handler that uses acoustic technology to perform direct microplate-to-microplate transfers of DMSO compound solutions and the system can be programmed to transfer multiple small nL volumes of compound from the different source plate wells to give the desired serial dilution of compound in the assay which is then back-filled to normalise the DMSO concentration across the dilution range.
Measurement: IC50 = 13000 nM (pChEMBL 4.889999866485596)
Descriptors: MW 523.55, ALogP 4.26, HBA 5, HBD 3, PSA 67.42, QED 0.37
Compound (SMILES): Cc1cc(Nc2nc(N3CCC[C@@H](NC(=O)N(C)C)[C@H]3C)cnc2C(N)=O)sn1
InChIKey: IQBNYZLVGAFZIF-VXGBXAGGSA-N
Target: Tyrosine-protein kinase JAK3 (SINGLE PROTEIN), Homo sapiens
UniProt: P52333
Assay: HotSpot kinase Assay: The degree of inhibition of a panel of kinases is determined using the in vitro HotSpot kinase assay (purified enzymes, 33P-ATP, an appropriate substrate and 1 uM ATP).
Measurement: IC50 = 550 nM (pChEMBL 6.260000228881836)
Descriptors: MW 418.53, ALogP 1.71, HBA 8, HBD 3, PSA 129.37, QED 0.67
Compound (SMILES): CCS(=O)(=O)c1ccc(CNC(=O)c2cc3c(s2)C2(CCN(C(C)c4cnc(C(F)(F)F)nc4)CC2)O[C@H](C)C3)nc1
InChIKey: ILXLMNLDODDHOQ-QNSVNVJESA-N
Target: Nuclear receptor ROR-beta (SINGLE PROTEIN), Homo sapiens
UniProt: Q92753
Assay: Biological Assay: Receptor competition binding assays are run in a buffer made up of DPBS (1 L) (Hyclone #SH30028.03), 2.2 g BSA Fraction v (Roche #9048-46-8), 100 mL glycerol (Fischer #56-81-5) and 40 mL DMSO (reagent grade). The final wells contain 20 μg/mL aprotinin and 20 μg/mL leupeptin and 10 μM Pefabloc. Typically, receptor binding assays include radio-labeled ligands, such as 7 nM [3H]-25-hydroxycholesterol for alpha binding, 20 nM [3H]-3-[[4-[[3-(2,6-dichlorophenyl)-5-isopropyl-isoxazol-4-yl]methoxy]-N,2-dimethyl-anilino]methyl]benzoic acid for beta binding, and 6 nM [3H]-25-hydroxycholesterol for gamma binding, and 0.5 μg RORa receptor, 0.03 μg RORb receptor, or 0.13 μg RORg receptor per well. Assays are typically run in 96-well format. Competing test compounds are added at various concentrations ranging from about 0.4 nM to 25 μM. Non-specific binding is determined in the presence of 250 nM 25-hydroxycholesterol for RORa and RORg binding, 250 nM 3-[[4-[[3-(2,6-dichlorophenyl)-5-isopropyl-isoxazol-4-yl]methoxy]-N,2-dimethyl-anilino]methyl]benzoic acid for RORb binding. The sample, label and receptor solutions are combined in a 96 well assay plate (Costar 3632) and incubated overnight at room temperature, then 25 μl beads (Amersham YSi (2-5 micron) copper His-tag Spa Beads, #RPNQ0096) for a final bead concentration of 1 mg/well is added to each reaction. Plates are mixed for 30 minutes on an orbital shaker at room temperature. After an incubation of 4 hours, plates are read in a Wallac MICROBETA® counter.
Measurement: Ki = 407 nM (pChEMBL 6.389999866485596)
Descriptors: MW 623.72, ALogP 4.69, HBA 9, HBD 1, PSA 114.38, QED 0.4
Compound (SMILES): N#Cc1cc(N2CCC(C(=O)NCCC3CCN(Cc4ccccc4)CC3)CC2)ccc1F
InChIKey: BLQUDHHDWHNLFJ-UHFFFAOYSA-N
Target: Muscarinic acetylcholine receptor M5 (SINGLE PROTEIN), Homo sapiens
UniProt: P08912
Assay: Binding Assay: Binding affinity (Ki) of compounds was measured by inhibition of radioligand binding to membranes from CHO cells expressing human M1, M2, M3, M4 and M5 receptors. Membranes were prepared by nitrogen cavitation and differential centrifugation as previously described (Hoare et al., Mol. Pharmacol. 2003 March; 63(3): 751-65). The radioligand employed was tritiated N-methylscopolamine, used at a concentration of 1.5 nM. A dose-response of twelve concentrations of compound was used, ranging from 10 Î¼M to 32 Î¼M. The assay buffer was 50 mM HEPES, 100 mM NaCl, 5 mM MgCl2, 1 mM ethylenediaminetetraacetic acid, pH-adjusted to pH 7.4.
Measurement: Ki > 100 nM (pChEMBL 7)
Descriptors: MW 448.59, ALogP 4.33, HBA 4, HBD 1, PSA 59.37, QED 0.68
Compound (SMILES): CC(C)N1C[C@H]2C[C@@]2(c2ccc(-c3cnc(N)c(C(=O)N[C@H]4CC[C@H](O)CC4)c3)cc2)C1
InChIKey: HZFKGEGMOFIQKL-BILGYAHESA-N
Target: Bone morphogenetic protein receptor type-1B (SINGLE PROTEIN), Homo sapiens
UniProt: O00238
Assay: Caliper Assay: In the applied method, this separation takes place inside a chip that contains a complex capillary system for simultaneous analysis of 12 samples (12-sipper chip, Caliper Technologies Corp., Mountain View, USA). In order to allow the detection and quantification of the peptides in the capillary system, the peptides carry a fluorescent label (fluorescein). With this label the peptides can be quantified by fluorescence intensity through the instruments laser and detection system (LC3000, Caliper Life Sciences).The assays were performed in 384-well, low volume microtiter assay plates in a final reaction volume of 9 ul. Dose-response curves were generated by incubating 10 nM of each kinase together with 2 μm of the fluorescently labeled substrate peptide 5-Fluo-Ahx-KKYQAEEN-T-YDEYENKK-amid (10 mM stock solution in DMSO) in 50 mM Hepes pH 7.5, 0.02% Tween 20, 0.02% BSA, 1 mM DTT, 10 μm Na3VO4, 10 mM β-Glycerolphosphate, 1 mM MgCl2, 12 mM MnCl2 (ALK1 and ALK6 7 mM) and 15 μm ATP for 60 min at 30° C. in the presence or absence of compound diluted in DMSO.Kinase reaction were terminated by adding 15 ul STOP buffer (100 mM HEPES pH 7.5, 5% DMSO, 0.1% Caliper coating reagent, 10 mM EDTA, and 0.015% Brij35.Plates with terminated kinase reactions were transferred to the Caliper LC3000 workstation (Caliper Technologies Corp., Mountain View, USA) for reading. The relative amount of phosphorylated peptide r, was calculated using the heights of the substrate peak, s, and the product peak, p: r=p/(p+s).
Measurement: IC50 = 2500 nM (pChEMBL 5.599999904632568)
Descriptors: MW 434.58, ALogP 3.35, HBA 5, HBD 3, PSA 91.48, QED 0.67
Compound (SMILES): CC(CO)(CO)Nc1n[nH]c2nccc(Oc3ccc(NC(=O)c4ccnn(-c5ccc(F)cc5)c4=O)cc3F)c12
InChIKey: CTPHZJUGWFVHAY-UHFFFAOYSA-N
Target: Hepatocyte growth factor receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P08581
Assay: Enzyme Assay: The affinity of compound binding to wild type and mutant human MET kinases is measured using Invitrogen's LanthaScreen Eu Kinase Binding technology. Briefly, GST-tagged recombinant human MET kinase domain from Signal Chem (see Table 8 below for concentration in assay) is incubated with 50 nM Alexa-Fluor® Tracer 236 (Invitrogen Cat No. PR9078A) and 2 nM Europium-anti-GST (Invitrogen Cat. No. A151 6) along with test compound in a buffer consisting of 25 mM HEPES, pH 7.4, 10 mM MgCl2, 0.01% Triton X-100, 1 mM DTT, and 2% DMSO. Compounds are typically prepared in a three-fold serial dilution in DMSO and added to the assay to give the appropriate final concentration. After a 60-minute incubation at 22° C., the reaction is measured using a PerkinElmer EnVision multimode plate reader via TR-FRET dual wavelength detection, and the percent of control (POC) calculated using a ratiometric emission factor. 100 POC is determined using no test compounds and 0 POC is determined using a concentration of control compound that completely inhibits the enzyme
Measurement: IC50 = 7.2 nM (pChEMBL 8.140000343322754)
Descriptors: MW 563.52, ALogP 2.98, HBA 10, HBD 5, PSA 167.28, QED 0.18
Compound (SMILES): Cc1ccc(COC(=O)N2CC[C@H](CNc3nccn4cnnc34)C(F)(F)C2)cc1
InChIKey: VUFCSTHNGFATSK-MRXNPFEDSA-N
Target: Glutamate receptor ionotropic, NMDA 2A (SINGLE PROTEIN), Homo sapiens
UniProt: Q12879
Assay: NR2B Antagonist Activity: HEK293 cell lines stably expressing cloned human NR1/NR2B and NR1/NR2A, respectively, were established according to standard previously described methods (Hansen et al., Comb. Chem High Throughput Screen. 11:304, 2008). Activation of the NR2A or NR2B subtype of NMDA receptor with glutamate as an agonist and glycine co-agonist on these cells results in calcium influx, which can be monitored with fluorescent indicator Fluo-4. A cell based assay has been implemented to evaluate the effect of a compound on NR2A and NR2B receptors by measuring the fluorescent changes (Hansen et al., Comb. Chem High Throughput Screen. 11:304, 2008).HEK293 cells stably expressing NR2A or NR2B receptors were cultured at 37° C. in a humidified CO2 incubator in DMEM supplemented with 10% fetal bovine serum (FBS) (Hyclone), 10 μM MK801 (Sigma-Aldrich) and 50 μM AP-5 (Tocris). For experiments, the cells were seeded onto poly-D-lysine-coated 96-well black plates with clear bottom (Corning) at a density of 50,000 cells/well. After overnight culture, the growth medium was removed from the wells and the cells were incubated at 37° C. for 60 min in Hanks buffer containing 4 μM fluo-4-AM (Invitrogen) and 0.1% bovine serum albumin (BSA). After dye-loading, the cells were washed three times with Hanks buffer and incubated for 10 min at room temperature with various concentrations of test compounds prepared in Hanks buffer with 0.1% BSA. The cell plates were placed onto FDSS μCell fluorescence reader (Hamamatsu). After 20 sec reading of background fluorescence, agonist glutamate at final 100 μM and co-agonist glycine at final 50 μM were added to the cells to activate the receptor, and the resulting fluorescence changes were recorded and quantified. Based on the changes in fluorescence intensity, the pharmacological effect of test compounds were analyzed and the IC50 values derived from a non-linear least squares fitting of the concentration-dependent response to a standard logistic equation using Prism (Graphpad, Inc):Amplitude=Max Amplitude/(1+(IC 50/[antagonist])n).
Measurement: IC50 > 10000 nM (pChEMBL 5)
Descriptors: MW 416.43, ALogP 3.14, HBA 7, HBD 1, PSA 84.65, QED 0.69
Compound (SMILES): Cc1cc(CN2CCC[C@H]2C)ccc1-c1cnc(N)c(C(=O)N[C@H]2CC[C@](C)(O)CC2)c1
InChIKey: DYPKOAOCIPJRSB-KAWPREARSA-N
Target: Activin receptor type-1-like (SINGLE PROTEIN), Homo sapiens
UniProt: P37023
Assay: Caliper Assay: In the applied method, this separation takes place inside a chip that contains a complex capillary system for simultaneous analysis of 12 samples (12-sipper chip, Caliper Technologies Corp., Mountain View, USA). In order to allow the detection and quantification of the peptides in the capillary system, the peptides carry a fluorescent label (fluorescein). With this label the peptides can be quantified by fluorescence intensity through the instruments laser and detection system (LC3000, Caliper Life Sciences).The assays were performed in 384-well, low volume microtiter assay plates in a final reaction volume of 9 ul. Dose-response curves were generated by incubating 10 nM of each kinase together with 2 μm of the fluorescently labeled substrate peptide 5-Fluo-Ahx-KKYQAEEN-T-YDEYENKK-amid (10 mM stock solution in DMSO) in 50 mM Hepes pH 7.5, 0.02% Tween 20, 0.02% BSA, 1 mM DTT, 10 μm Na3VO4, 10 mM β-Glycerolphosphate, 1 mM MgCl2, 12 mM MnCl2 (ALK1 and ALK6 7 mM) and 15 μm ATP for 60 min at 30° C. in the presence or absence of compound diluted in DMSO.Kinase reaction were terminated by adding 15 ul STOP buffer (100 mM HEPES pH 7.5, 5% DMSO, 0.1% Caliper coating reagent, 10 mM EDTA, and 0.015% Brij35.Plates with terminated kinase reactions were transferred to the Caliper LC3000 workstation (Caliper Technologies Corp., Mountain View, USA) for reading. The relative amount of phosphorylated peptide r, was calculated using the heights of the substrate peak, s, and the product peak, p: r=p/(p+s).
Measurement: IC50 = 10000 nM (pChEMBL 5)
Descriptors: MW 436.6, ALogP 4.05, HBA 5, HBD 3, PSA 91.48, QED 0.66
Compound (SMILES): C[C@H](O)C(=O)N1C[C@H]2C[C@H](N(C)c3ncnc4[nH]ccc34)C[C@H]2C1
InChIKey: IJKKBQHULDYNJO-WUHRBBMRSA-N
Target: Tyrosine-protein kinase JAK2 (SINGLE PROTEIN), Homo sapiens
UniProt: O60674
Assay: In Vitro Kinase Assay: In vitro kinase assays described below can be used to determine the activity of a test compound for inhibiting the activity of JAK2 kinase. The test compounds were dissolved in dimethyl sulfoxide and diluted with water to a serial concentration gradient as required in the experiment. JAK2 substrates (Cell Signaling Technology, Catalog Number: 1305s) and ATP (2 mM) solution were diluted with water to obtain a final concentration of 20 uM ATP and 1.2 uM substrate solution. The appropriate amount of JAK2 kinase (Invitrogen, Catalog Number: pv4210) was mixed with 4x buffer (prepared by user, and comprising 50 mM HEPES, pH 7.3, 125 mM NaCl, 24 mM MgCl2, 1.25 mM DTT) to a final concentration of 8 ng/uL. To each well of a microplate [DELFIA Streptavidin-coated clear plate (Perkin Elmer, Catalog Number: AAAND-0005)]17.5 uL of ATP/substrate mixture, 5 uL of aqueous solution of a test compound (5 uL of pure water only were added to control and blank), and 7.5 uL of the kinase solution prepared above (4x buffer only was added to control) were added. Each well was mixed sufficiently, then incubated at room temperature (27° C.) for 50 minutes, washed with wash buffer, and dried three times, then HRP conjugated antibody [Phospho-Tyrosine Mouse mAb (P-Tyr-100) (HRP Conjugate, Cell signaling Technology, Catalog Number: 5465)] was added, and incubated for 1 hour. The microplate was washed with wash buffer and dried three times, and then TMB (Sigma, Catalog Number: T4444) was added and incubated for 5 to 15 minutes to allow for color change. Stop solution (1 N sulfuric acid solution) was added to stop the reaction. Absorbance was measured on a Novostar microplate reader at a wavelength of 450 nm. IC50 values of test compounds were calculated from the data of the test compounds for inhibiting the activity of JAK2 kinase at different concentrations.
Measurement: IC50 = 3 nM (pChEMBL 8.520000457763672)
Descriptors: MW 329.4, ALogP 1.01, HBA 5, HBD 2, PSA 85.35, QED 0.88
Compound (SMILES): COc1cc(OC)c(F)c(N(C[C@H]2CCC(=O)N2)c2ncc3ncc(-c4cnn(C)c4)nc3n2)c1
InChIKey: LUJNIROONWQIJG-CQSZACIVSA-N
Target: VEGFR1-VEGA (PROTEIN COMPLEX), Homo sapiens
UniProt: P17948
Assay: KDR (VEGFR2) (Enzymatic Assay): In a final reaction volume of 30 μL, KDR (h) (150 ng/ml) was incubated with 50 mM HEPES pH 7.5, 6 mM MnCl2, 1 mM DTT, 0.1 mM Na3VO4, 0.01% Triton-X-100, 500 nM Btn-Flt3 and 3 μM ATP in the presence of compound (1% DMSO final). After incubation for 120 minutes at room temperature the reaction was stopped with 2.27 nM EU-anti P-Tyr, 7 mM EDTA, 31.25 nM SA-XL-665 and 0.02% BSA which was present for 60 minutes at room temperature. Time-Resolved Fluorescence Resonance Energy Transfer (TR-FRET) signal (ex340 nm. Em 620 nm, em 655 nm) was measured afterwards and results are expressed in RFU (Relative Fluorescence Units). In this assay, the inhibitory effect of different compound concentrations (range 10 μM to 0.1 nM) was determined and used to calculate an IC50 (M) and pIC50 (−log IC50) value.
Measurement: IC50 = 1000 nM (pChEMBL 6)
Descriptors: MW 478.49, ALogP 2.39, HBA 10, HBD 1, PSA 120.18, QED 0.43
Compound (SMILES): CCc1cncc2ccc(NC(=O)[C@@H]3CC3c3ccc(S(=O)(=O)Nc4ccccn4)cc3)cc12
InChIKey: ZPRHGJOOCBVZCP-XMMISQBUSA-N
Target: Tyrosine-protein kinase JAK3 (SINGLE PROTEIN), Homo sapiens
UniProt: P52333
Assay: JAK Kinase Assays: Compounds were prepared in the exact same manner as described in the ROCK Kinase Assay with the exception to the substrate and enzyme. The JAK 2× substrate/enzyme solution consisted of acceptor substrate (800 nM Abl peptide EAIYAAPFAKKK (SEQ ID NO: 2)), JAK2 or JAK3 enzyme (10 nM) and DTT (2 μM). All other steps and solutions remain identical to the ROCK Kinase Assay above.
Measurement: IC50 = 4.2 nM (pChEMBL 8.380000114440918)
Descriptors: MW 472.57, ALogP 4.74, HBA 5, HBD 2, PSA 101.05, QED 0.4
Compound (SMILES): O=C1c2cc(-c3cccnc3)c(OC3CCCC3)cc2CN1c1cccc(-c2nncn2C2CC2)n1
InChIKey: QXEUJUYSECCFGD-UHFFFAOYSA-N
Target: Tumor necrosis factor (SINGLE PROTEIN), Homo sapiens
UniProt: P01375
Assay: Inhibition of LPS-induced TNFalpha in Human PBMCs: Cryopreserved human PBMCs were obtained from AllCells (cat# PB003F). After thawing/dilution protocols using RPMI medium supplemented with 5% FBS (heat inactivated), 100 ul/well of 1×106 cells/ml were plated into 96 well tissue culture plates (Corning). Cells were then pre-incubated for 1 hr at 37° C. in humidified 5% CO2 and 95% air with test compounds diluted in DMSO (final DMSO concentration 0.3%). Each compound was tested at 10 concentrations in duplicate wells. After the pre-incubation, 100 ng/ml LPS (E. Coli; Sigma) in RPMI media with 5% FBS was added for a 6 hr incubation at 37° C. in humidified 5% CO2 and 95% air. Controls on each plate included cells and LPS only, cells and media only (no LPS), and media only. After the 6 hr incubation, plates were centrifuged and the supernatants transferred to a new plate and frozen for subsequent TNFalpha analysis. Human TNFalpha was analyzed by ELISA according to the manufacturer's instructions (BD Sciences, BD OptEIA Cat#550610) and analyzed on a SpectraMax M series (Molecular Devices) microplate reader at OD 450 nm. IC50s were calculated using XLFit4 curve fitting software (IDBS) and a 4-parameter one-site sigmoid dose response fit.
Measurement: IC50 < 200 nM (pChEMBL 6.698999881744385)
Descriptors: MW 478.56, ALogP 5.22, HBA 7, HBD 0, PSA 86.03, QED 0.38
Compound (SMILES): C=CC(=O)N1CCc2c(cccc2-c2c(F)cc(C(N)=O)c3[nH]c(C)c(C)c23)C1
InChIKey: JAVLNNZFNZNYEY-UHFFFAOYSA-N
Target: Tyrosine-protein kinase BTK (SINGLE PROTEIN), Homo sapiens
UniProt: Q06187
Assay: Enzyme Assay: To V-bottom 384-well plates were added test compounds, human recombinant Btk (1 nM, Invitrogen Corporation), fluoresceinated peptide (1.5 μM), ATP (20 μM), and assay buffer (20 mM HEPES pH 7.4, 10 mM MgCl2, 0.015% Brij 35 surfactant and 4 mM DTT in 1.6% DMSO), with a final volume of 30 μL. After incubating at room temperature for 60 min., the reaction was terminated by adding 45 μL of 35 mM EDTA to each sample. The reaction mixture was analyzed on the Caliper LABCHIP® 3000 (Caliper, Hopkinton, Mass.) by electrophoretic separation of the fluorescent substrate and phosphorylated product. Inhibition data were calculated by comparison to control reactions with no enzyme (for 100% inhibition) and controls with no inhibitor (for 0% inhibition). Dose response curves were generated to determine the concentration required for inhibiting 50% of Btk activity (IC50). Compounds were dissolved at 10 mM in DMSO and evaluated at eleven concentrations.
Measurement: IC50 = 150 nM (pChEMBL 6.820000171661377)
Descriptors: MW 391.45, ALogP 3.76, HBA 2, HBD 2, PSA 79.19, QED 0.67
Compound (SMILES): CCn1cc(Nc2ncnc3[nH]cc(C(=O)c4c(F)ccc(NS(=O)(=O)c5cc(F)ccc5F)c4F)c23)cn1
InChIKey: VVARIPLFBATYLY-UHFFFAOYSA-N
Target: Cytochrome P450 3A4 (SINGLE PROTEIN), Homo sapiens
UniProt: P08684
Assay: Inhibition Assay: The inhibition of CYP enzyme activity (IC50) for each of CYP1A2, CYP2C19, CYP2C9, CYP2D6, CYP3A4(BFC) and CYP3A4(BQ) is determined for compounds, where inhibition of metabolism of a known substrate leads to a decrease in the fluorescence of the metabolized product. The fluorescence of the product is monitored as a function of compound concentration. Compounds are dissolved in dimethyl sulfoxide to a concentration of 100 mM. These are diluted 1 μL into 82 μL of acetonitrile. An 11 μL aliquot of this solution is then added to 204 μL of cofactor mix (1.3% NADPH Regeneration system Solution A, 1.04% NADPH Regeneration system Solution B from BD Biosciences, 5% acetonitrile and 0.05% dimethyl sulfoxide). These are then serially diluted 1:1 (160 μL to 160 μL co-factor mix) for a total of 10 points. A 10 μL aliquot of this final mixture is dispensed into 384 well assay plates and incubated for 10 minutes at 37° C. Enzyme and substrate mix (10 μL; 0.5 pmol CYP1A2/5 μM CEC; 1.0 pmol CYP2C9/75 μM MFC; 0.5 pmol CYP2C19/25 μM CEC; 1.5 pmol CYP2D6/1.5 μM AMMC; 1.0 pmol CYP3A4/50 μM BFC; or 1.0 pmol CYP3A4/40 μM BQ) is added to these assay plates. Assay plates are incubated at 37° C. (CYP1A2-15 min; CYP2C9-45 min; CYP2C19, 2D6 and 3A4-30 min) and read in a Tecan Safire 2 plate reader (CYP1A2, 2C19 and 3A4 409 ex/460 em; CYP2C9 and 2D6 409 ex/530 em).
Measurement: IC50 > 10000 nM (pChEMBL 5)
Descriptors: MW 559.51, ALogP 4.51, HBA 8, HBD 3, PSA 134.66, QED 0.19
Compound (SMILES): CC(C)N1CCC(n2nc(-c3cc4cccc(O)c4[nH]3)c3c(N)ncnc32)CC1
InChIKey: XAWSEOWTVPLGSW-UHFFFAOYSA-N
Target: Phosphatidylinositol 3-kinase regulatory subunit beta (SINGLE PROTEIN), Homo sapiens
UniProt: O00459
Assay: In vitro kinase assay: This assay is relatively simple, reasonably sensitive, and the peptide substrate can be adjusted both in terms of sequence and concentration to meet the assay requirements. Other exemplary kinase assays are detailed in U.S. Pat. No. 5,759,787 and U.S. application Ser. No. 12/728,926, both of which are incorporated herein by reference.
Measurement: IC50 > 500 nM (pChEMBL 6.301000118255615)
Descriptors: MW 391.48, ALogP 3.31, HBA 7, HBD 3, PSA 108.88, QED 0.49
Compound (SMILES): C=CC(=O)N1CC(Oc2cc3c(Nc4ccc(Cl)cc4Cl)ncnc3cc2OC)C1
InChIKey: VURUUKGYNYHYRK-UHFFFAOYSA-N
Target: Epidermal growth factor receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P00533
Assay: Cell Viability Assay and IC50 Estimation: Cell viability was determined using the Cell Titer Glo assay (Promega) as previously described. Briefly, 2000-3000 cells per well were plated in 384-well plates (Greiner Bio-One) in technical triplicate. Cells were treated with seven different concentrations of tyrosine kinase inhibitors or vehicle alone at a final volume of 40 μL per well. After 3 days hours, 11 μL of Cell Titer Glo was added to each well. Plates were shaken for 15 minutes, and bioluminescence was determined using a FLUOstar OPTIMA multi-mode micro-plate reader (BMG LABTECH). Bioluminescence values were normalized to DMSO treated cells, and normalized values were plotted in GraphPad Prism using non-linear regression fit to normalized data with a variable slope. IC50 values were calculated by GraphPad Prism at 50% inhibition.
Measurement: IC50 = 43 nM (pChEMBL 7.369999885559082)
Descriptors: MW 445.31, ALogP 4.46, HBA 6, HBD 1, PSA 76.58, QED 0.56
Compound (SMILES): CC(C)(N)CC(=O)N1CC(Cc2nc3c(C(F)(F)F)cc(C(F)(F)F)cc3[nH]2)C1
InChIKey: RJOKSRUIVHIBFY-UHFFFAOYSA-N
Target: Voltage-dependent L-type calcium channel subunit alpha-1C (SINGLE PROTEIN), Homo sapiens
UniProt: Q13936
Assay: Nav1.5 Assay: To assess the potential cardiac liability of compounds at an early stage in the drug discovery process, a Nav1.5 sodium channel screening assay was be performed on Molecular Device's PatchXpress™ automated electrophysiology platform. Under voltage-clamp conditions, Nav1.5 currents were recorded from HEK cells expressing the human Nav1.5 channel in the absence and presence of increasing concentrations of the test compound to obtain an IC50 value. The external recording solution contained (in mM): 90 TEACl, 50 NaCl, 1.8 CaCl, 1 MgCl2, 10 HEPES, 10 glucose, adjusted to pH 7.4 with TEA-OH and to 300 mOsm with sucrose (if necessary), while the internal patch pipette solution contained (in mM): 129 CsF, 2 MgCl2, 11 EGTA, 10 HEPES, 3 Na2ATP adjusted to pH 7.2 with CsOH and to 290 mOsm with sucrose (if necessary). Nav1.5 channel currents were evoked using a cardiac action potential waveform at 1 Hz, digitized at 31.25 kHz and low-pass filtered at 12 kHz.
Measurement: IC50 > 10000 nM (pChEMBL 5)
Descriptors: MW 422.37, ALogP 3.73, HBA 3, HBD 2, PSA 75.01, QED 0.74
Compound (SMILES): CNc1ncc2cc(-c3cc(NC(=O)c4ccnc(C(F)(F)F)c4)ccc3C)c(C)nc2n1
InChIKey: YTVCZPUNBLVANT-UHFFFAOYSA-N
Target: RAF proto-oncogene serine/threonine-protein kinase (SINGLE PROTEIN), Homo sapiens
UniProt: P04049
Assay: Kinase Assay: Protein kinase assay: Assay platform was used to measure kinase/inhibitor interactions as described previously (Anastassiadis et al., 2011). In brief, for each reaction, kinase and substrate were mixed in a buffer containing 20 mM HEPES (pH 7.5), 10 mM MgCl2, 1 mM EGTA, 0.02% Brij35, 0.02 mg/mL BSA, 0.1 mM Na3VO4, 2 mM DTT, and 1% DMSO. All compounds were solubilized in DMSO. Compounds were then added to each reaction mixture via acoustic dispense using an ECHO 550 nanoliter dispenser. For human RAF1 testing, human MEK1 (K97R) was used as a substrate at a concentration of 3 micromolar, with a final ATP concentration of 10 micromolar. For human BRAF testing, human MEK1 (K97R) was used as a substrate at 1 micromolar concentration, with a final ATP concentration of 25 micromolar. Compounds were tested in 10-dose IC50 mode with a 3-fold serial dilution starting at 10 micromolar. After a 20-min incubation, ATP (Sigma-Aldrich, St. Louis, Mo. 63178) and [g33P] ATP (specific activity 10 microCi/microliter) purchased at PerkinElmer (Boston, Mass., 02118 Cat #BLU 003H250UC) were added at a final total concentration of 10 mM. Reactions were carried out at room temperature for 2 hr and spotted onto P81 ion exchange cellulose chromatography paper (Reaction Biology). Filter paper was washed in 0.75% phosphoric acid to remove unincorporated ATP. The percent remaining kinase activity relative to a vehicle-containing (DMSO) kinase reaction was calculated for each kinase/inhibitor pair.
Measurement: IC50 < 10 nM (pChEMBL 8)
Descriptors: MW 452.44, ALogP 5.02, HBA 6, HBD 2, PSA 92.69, QED 0.45
Compound (SMILES): COc1cc2c(Nc3cc(Cl)c(Br)cc3F)ncnc2cc1OC[C@H]1CN2CCC[C@@H]2CO1
InChIKey: NGRFKPHUUUCTAT-ZIAGYGMSSA-N
Target: Receptor tyrosine-protein kinase erbB-2 (SINGLE PROTEIN), Homo sapiens
UniProt: P04626
Assay: AlphaScreen Assay: In AlphaScreen, when the donor and acceptor beads are close in proximity, a series of photochemical events will give rise to a fluorescent signal upon light activation. Here we use biotinylated poly-(Glu, Tyr) 4:1 as the kinase substrate, streptavidin-coated donor beads and anti-phosphortyrosine antibody PY100-coated acceptor beads. Upon phosphorylation, the peptide substrate can bind to both donor and acceptor beads, thus gives rise to fluorescence. Compounds, ATP, biotinylated poly-(Glu, Tyr) and kinases are mixed in a volume of 20 μL for 1 hr at ambient temperature using Greiner 384-well white clear bottom medium binding plates. Then 10 uL solution containing 15-30 mg/mL AlphaScreen beads, 75 mM Hepes, pH 7.4, 300 mM NaCl, 120 mM EDTA, 0.3% BSA and 0.03% Tween-20 is added to each well. After 2-16 hr incubation of the beads, plates are read in a Perkin Elmer AlphaQuest reader (available from PerkinElmer Life and Analytical Sciences, Inc., of Boston Mass.).
Measurement: IC50 = 525 nM (pChEMBL 6.28000020980835)
Descriptors: MW 537.82, ALogP 5.18, HBA 7, HBD 1, PSA 68.74, QED 0.43
Compound (SMILES): COc1cc(-c2[nH]c3ccc(C4CCC(NC(C)C)CC4)nc3c2C(C)C)cn2ncnc12
InChIKey: KWMAAQNRLXTVKG-UHFFFAOYSA-N
Target: Toll-like receptor 9 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9NR96
Assay: TLR7/8/9 Inhibition Reporter Assays: 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. The percent inhibition is determined as the % reduction in the HEK-Blue signal present in wells treated with agonist plus DMSO alone compared to wells treated with a known inhibitor.
Measurement: IC50 = 648 nM (pChEMBL 6.190000057220459)
Descriptors: MW 446.6, ALogP 5.43, HBA 6, HBD 2, PSA 80.13, QED 0.41
Compound (SMILES): O=C(O)c1ccncc1Nc1nn(C2CCC(F)(F)CC2)c2ccc(F)cc12
InChIKey: NGZUHYKCIFSOED-UHFFFAOYSA-N
Target: Lysine-specific demethylase 4C (SINGLE PROTEIN), Homo sapiens
UniProt: Q9H3R0
Assay: Enzymatic Assay: The ability of test compounds to inhibit the activity of JMJD2C was determined in 384-well plate format under the following reaction conditions: 0.3 nM JMJD2C, 300 nM H3K9me3-biotin labeled peptide (Anaspec cat #64360), 2 μM alpha-ketoglutaric acid in assay buffer of 50 mM HEPES, pH7.3, 0.005% Brij35, 0.5 mM TCEP, 0.2 mg/ml BSA, 50 μM sodium L-ascorbate, and 2 μM ammonium iron(II) sulfate. Reaction product was determined quantitatively by TR-FRET after the addition of detection reagent Phycolink Streptavidin-allophycocyanin (Prozyme) and Europium-anti-di-methylated histone H3 lysine 9 (H3K9me2) antibody (PerkinElmer) in the presence of 5 mM EDTA in LANCE detection buffer (PerkinElmer) at a final concentration of 50 nM and 1 nM, respectively.The assay reaction was initiated by the following: 2 μl of the mixture of 900 nM H3K9me3-biotin labeled peptide and 6 μM alpha-ketoglutaric acid with 2 μl of 11-point serial diluted inhibitor in 3% DMSO were added to each well of the plate, followed by the addition of 2 μl of 0.9 nM JMJD2C to initiate the reaction. The reaction mixture was incubated at room temperature for 30 minutes, and terminated by the addition of 6 μl of 5 mM EDTA in LANCE detection buffer containing 100 nM Phycolink Streptavidin-allophycocyanin and 2 nM Europium-anti-H3K9me2 antibody. Plates were read by EnVisionMultilabel Reader in TR-FRET mode (excitation at 320 nm, emission at 615 nm and 665 nm) after 1 hour incubation at room temperature. A ratio was calculated (665/615) for each well and fitted to determine inhibition constant (IC50).
Measurement: IC50 = 5500 nM (pChEMBL 5.260000228881836)
Descriptors: MW 390.37, ALogP 4.76, HBA 5, HBD 2, PSA 80.04, QED 0.67
Compound (SMILES): N#CCC1CC(CC#N)(n2ccc(-c3nc(-c4cn[nH]c4)cc4ncccc34)n2)C1
InChIKey: OGDHHJRTWUJUDG-UHFFFAOYSA-N
Target: Tyrosine-protein kinase JAK1 (SINGLE PROTEIN), Homo sapiens
UniProt: P23458
Assay: JAK Caliper Enzyme Assay at 1mM 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 = 1049 nM (pChEMBL 5.980000019073486)
Descriptors: MW 394.44, ALogP 3.82, HBA 7, HBD 1, PSA 119.86, QED 0.55
Compound (SMILES): Cc1c(Br)cc(C(=O)Nc2ccc(-c3cc(C4CCN(C(=O)C(C)C)CC4)n4ncnc(N)c34)cc2)c(=O)n1-c1cncc(F)c1
InChIKey: YKAKDBAZAFVYTA-UHFFFAOYSA-N
Target: Tyrosine-protein kinase receptor TYRO3 (SINGLE PROTEIN), Homo sapiens
UniProt: Q06418
Assay: TAM Enzymatic Assay: The kinase assay buffer contained 50 mM HEPES, pH7.5, 10 mM MgCl2, 1 mM EGTA, 0.01% NP-40 and 2 mM DTT. 0.1 ul test compounds dissolved in DMSO were transferred from compound plates to white 384-well assay plates (Greiner LLIMITRAC plates). The final concentration of DMSO was 1.25%. Enzyme solutions of 5.1 nM phosphor-Axl, or 0.0625 nM c-Mer (Carna Biosciences, 08-108), or 0.366 nM Tyro3 (Life Technologies, PR7480A) were prepared in assay buffer. A 1 mM stock solution of peptide substrate Biotin-EQEDEPEGDYFEWLE-amide SEQ ID NO: 1 (Quality Controlled Biochemicals, MA) dissolved in DMSO was diluted to 1 uM in assay buffer containing 2000 uM ATP. 4 ul enzyme solution (or assay buffer for the enzyme blank) was added to the appropriate wells in each plate, and then 4 ul/well substrate solution was added to initiate the reaction. The plate was protected from light and incubated at room temperature for 60 min. The reaction was stopped by adding 4 ul detection solution containing 50 mM Tris-HCl, pH7.8, 150 mM NaCl, 0.05% BSA, 45 mM EDTA, 180 nM SA-APC (Perkin Elmer, CR130-100) and 3 nM Eu-W1024 anti-phosphotyrosine PY20 (Perkin Elmer, AD0067). The plate was incubated for 1 h at room temperature, and HTRF (homogenous time resolved fluorescence) signal was measured on a PHERAstar FS plate reader (BMG labtech). Percentage of inhibition was calculated for each concentration and IC50 value was generated from curve fitting with GraphPad Prism software.
Measurement: IC50 < 5 nM (pChEMBL 8.300999641418457)
Descriptors: MW 687.57, ALogP 5.35, HBA 9, HBD 2, PSA 140.51, QED 0.25
Compound (SMILES): Nc1ncc(-c2ccc([C@]34C[C@H]3CN(C3CCOCC3)C4)cc2)cc1C(=O)NC12CCC(O)(CC1)CC2
InChIKey: KPRPFTOLWQQUAV-GXLPGVSKSA-N
Target: Activin receptor type-1-like (SINGLE PROTEIN), Homo sapiens
UniProt: P37023
Assay: Caliper Assay: In the applied method, this separation takes place inside a chip that contains a complex capillary system for simultaneous analysis of 12 samples (12-sipper chip, Caliper Technologies Corp., Mountain View, USA). In order to allow the detection and quantification of the peptides in the capillary system, the peptides carry a fluorescent label (fluorescein). With this label the peptides can be quantified by fluorescence intensity through the instruments laser and detection system (LC3000, Caliper Life Sciences).The assays were performed in 384-well, low volume microtiter assay plates in a final reaction volume of 9 ul. Dose-response curves were generated by incubating 10 nM of each kinase together with 2 μm of the fluorescently labeled substrate peptide 5-Fluo-Ahx-KKYQAEEN-T-YDEYENKK-amid (10 mM stock solution in DMSO) in 50 mM Hepes pH 7.5, 0.02% Tween 20, 0.02% BSA, 1 mM DTT, 10 μm Na3VO4, 10 mM β-Glycerolphosphate, 1 mM MgCl2, 12 mM MnCl2 (ALK1 and ALK6 7 mM) and 15 μm ATP for 60 min at 30° C. in the presence or absence of compound diluted in DMSO.Kinase reaction were terminated by adding 15 ul STOP buffer (100 mM HEPES pH 7.5, 5% DMSO, 0.1% Caliper coating reagent, 10 mM EDTA, and 0.015% Brij35.Plates with terminated kinase reactions were transferred to the Caliper LC3000 workstation (Caliper Technologies Corp., Mountain View, USA) for reading. The relative amount of phosphorylated peptide r, was calculated using the heights of the substrate peak, s, and the product peak, p: r=p/(p+s).
Measurement: IC50 = 540 nM (pChEMBL 6.269999980926514)
Descriptors: MW 502.66, ALogP 3.65, HBA 6, HBD 3, PSA 100.71, QED 0.58
Compound (SMILES): Cc1cccn2c(C(C)(C)NC(=O)[C@@H]3CNCCO3)ncc12
InChIKey: MNHAYZLLHUEGPR-ZDUSSCGKSA-N
Target: Somatostatin receptor type 5 (SINGLE PROTEIN), Homo sapiens
UniProt: P35346
Assay: Radioligand Binding Assays: For the binding experiments 200 μL of membrane homogenate from one of the following protein amounts is used: hSSTR1 (40 μg/well); hSSTR2 (25 μg/well); hSSTR3 (1.5 μg/well); hSSTR4 (0.5 μg/well); hSSTR5 (25 μg/well). The homogenate is incubated with 0.05 nM of radioligand ([3-125I-Tyr]-Somatostatin-(1-14)) in addition to increasing concentrations of a test compound or vehicle (100% binding) in a total volume of 250 μL using a Hepes buffer (10 mM, EDTA 1 mM, MgCl2 5 mM, pH7.6, BSA 0.5%, Bacitracin 0.003%, DMSO 1%) for 180 min at room temperature. The incubation is terminated by filtration with ice cold NaCl 0.9% through polyethyleneimine treated (0.3%) GF/B glass fiber filters using a cell harvester. The protein-bound radioactivity is measured in a suitable reader. The non-specific binding is defined as radioactivity bound in the presence of 1 μM Somatostatin-14 during the incubation period.
Measurement: Ki = 9770 nM (pChEMBL 5.010000228881836)
Descriptors: MW 302.38, ALogP 0.98, HBA 5, HBD 2, PSA 67.66, QED 0.89
Compound (SMILES): CC(C)c1c(-c2cn(C)c3ncccc23)[nH]c2ccc(C3CCC(NCC(=O)N(C)C)CC3)nc12
InChIKey: WMQSQIWBKXSCTC-UHFFFAOYSA-N
Target: Toll-like receptor 9 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9NR96
Assay: TLR7/8/9 Inhibition Reporter Assays: 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. The percent inhibition is determined as the % reduction in the HEK-Blue signal present in wells treated with agonist plus DMSO alone compared to wells treated with a known inhibitor.
Measurement: IC50 = 1031 nM (pChEMBL 5.989999771118164)
Descriptors: MW 472.64, ALogP 4.94, HBA 5, HBD 2, PSA 78.84, QED 0.42
Compound (SMILES): Cc1ccc(-n2nccn2)c(C(=O)N2C3CC(C3)[C@H](C)C2COc2ccc(Cl)cc2)n1
InChIKey: DCNHWCVUWVDUDQ-ZQWKGRKISA-N
Target: Orexin receptor type 2 (SINGLE PROTEIN), Homo sapiens
UniProt: O43614
Assay: Scintillation Proximity Assay (SPA) Binding Assay: CHO cells stably transfected with human Orexin type 1 receptors (CHO-hOX1) or HEK-293 cells transiently transfected with human Orexin type 2 receptors (HEK-hOX2) were collected after 16 h induction with 5 mM sodium butyrate. The cell pellets were re-suspended, homogenized in 15 mM Tris/HCl pH=7.5, 1 mM EGTA, 0.3 mM EDTA, 2 mM MgCl2, protease inhibitors and centrifuged at 40,000 g (20 min, 4° C.). After re-suspension, homogenization and centrifugation as above, the final pellets were re-suspended in 75 mM Tris/HCl pH=7.5, 1 mM EGTA, 0.3 mM EDTA, 12.5 mM MgCl2, 250 mM Sucrose, protease inhibitors, divided into aliquots and frozen down at −80° C.Compounds of invention were serially diluted in neat DMSO at 100-fold concentrations (1% DMSO final in the assay) and 2 μl/well were plated into 96-well Isoplates (Perkin Elmer).
Measurement: Ki = 1047 nM (pChEMBL 5.980000019073486)
Descriptors: MW 437.93, ALogP 3.94, HBA 6, HBD 0, PSA 73.14, QED 0.6
Compound (SMILES): O=C(c1ccccn1)C1CCCN(Cc2ccccc2)C1
InChIKey: XKHWOQWKHIBRFE-UHFFFAOYSA-N
Target: Cytochrome P450 3A4 (SINGLE PROTEIN), Homo sapiens
UniProt: P08684
Assay: CYP Inhibition Assay: The effect of compounds IV-6, IV-7, and V-4 on Cytochromes P450 (CYP) inhibition was evaluated. For each isozyme, microsomes-buffer-substrate mixture (MBS mix) is prepared by premixing appropriate volumes of buffer, microsomes and substrate. MBS mixture (179 μL) is transferred to a 96-well reaction plate. An aliquot (1 μL) is spiked from corresponding wells of Test Item stock solution plate to reaction plate. The reaction plate is pre-incubated for 5 minutes at 37° C. Reaction is initiated by the addition of 20 μL of NADPH solution. Each experiment is performed in duplicate. Reaction plate is incubated for predetermined time at 37° C. and quenched using either 200 μL of acetonitrile (for CYP2C9, CYP2D6, CYP2C19 and CYP3A4) or 200 μL of a mixture of 70:30 1% formic acid:acetonitrile (for CYP1A2).
Measurement: IC50 = 35000 nM (pChEMBL 4.460000038146973)
Descriptors: MW 280.37, ALogP 3.18, HBA 3, HBD 0, PSA 33.2, QED 0.81
Compound (SMILES): CC(CC(F)F)Nc1n[nH]c2nccc(Oc3ccc(NC(=O)c4cn(C(C)C)c(=O)n(-c5ccc(F)cc5)c4=O)cc3F)c12
InChIKey: GSJTVHKWTWIIFJ-UHFFFAOYSA-N
Target: Hepatocyte growth factor receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P08581
Assay: Enzyme Assay: The affinity of compound binding to wild type and mutant human MET kinases is measured using Invitrogen's LanthaScreen Eu Kinase Binding technology. Briefly, GST-tagged recombinant human MET kinase domain from Signal Chem (see Table 8 below for concentration in assay) is incubated with 50 nM Alexa-Fluor® Tracer 236 (Invitrogen Cat No. PR9078A) and 2 nM Europium-anti-GST (Invitrogen Cat. No. A151 6) along with test compound in a buffer consisting of 25 mM HEPES, pH 7.4, 10 mM MgCl2, 0.01% Triton X-100, 1 mM DTT, and 2% DMSO. Compounds are typically prepared in a three-fold serial dilution in DMSO and added to the assay to give the appropriate final concentration. After a 60-minute incubation at 22° C., the reaction is measured using a PerkinElmer EnVision multimode plate reader via TR-FRET dual wavelength detection, and the percent of control (POC) calculated using a ratiometric emission factor. 100 POC is determined using no test compounds and 0 POC is determined using a concentration of control compound that completely inhibits the enzyme
Measurement: IC50 = 7.3 nM (pChEMBL 8.140000343322754)
Descriptors: MW 625.58, ALogP 5.63, HBA 9, HBD 3, PSA 135.93, QED 0.17
Compound (SMILES): CCS(=O)(=O)c1ccc(CNC(=O)c2cc3c(s2)C2(CCN(C(C)c4cnc(C(F)(F)F)nc4)CC2)O[C@H](C)C3)nc1
InChIKey: ILXLMNLDODDHOQ-QNSVNVJESA-N
Target: Nuclear receptor ROR-gamma (SINGLE PROTEIN), Homo sapiens
UniProt: P51449
Assay: Biological Assay: Receptor competition binding assays are run in a buffer made up of DPBS (1 L) (Hyclone #SH30028.03), 2.2 g BSA Fraction v (Roche #9048-46-8), 100 mL glycerol (Fischer #56-81-5) and 40 mL DMSO (reagent grade). The final wells contain 20 μg/mL aprotinin and 20 μg/mL leupeptin and 10 μM Pefabloc. Typically, receptor binding assays include radio-labeled ligands, such as 7 nM [3H]-25-hydroxycholesterol for alpha binding, 20 nM [3H]-3-[[4-[[3-(2,6-dichlorophenyl)-5-isopropyl-isoxazol-4-yl]methoxy]-N,2-dimethyl-anilino]methyl]benzoic acid for beta binding, and 6 nM [3H]-25-hydroxycholesterol for gamma binding, and 0.5 μg RORa receptor, 0.03 μg RORb receptor, or 0.13 μg RORg receptor per well. Assays are typically run in 96-well format. Competing test compounds are added at various concentrations ranging from about 0.4 nM to 25 μM. Non-specific binding is determined in the presence of 250 nM 25-hydroxycholesterol for RORa and RORg binding, 250 nM 3-[[4-[[3-(2,6-dichlorophenyl)-5-isopropyl-isoxazol-4-yl]methoxy]-N,2-dimethyl-anilino]methyl]benzoic acid for RORb binding. The sample, label and receptor solutions are combined in a 96 well assay plate (Costar 3632) and incubated overnight at room temperature, then 25 μl beads (Amersham YSi (2-5 micron) copper His-tag Spa Beads, #RPNQ0096) for a final bead concentration of 1 mg/well is added to each reaction. Plates are mixed for 30 minutes on an orbital shaker at room temperature. After an incubation of 4 hours, plates are read in a Wallac MICROBETA® counter.
Measurement: Ki = 153 nM (pChEMBL 6.820000171661377)
Descriptors: MW 623.72, ALogP 4.69, HBA 9, HBD 1, PSA 114.38, QED 0.4