lovingscience/chembl-v37-raw-activities huggingface dataset:
Compound (SMILES): CCc1nc2ccc(N3CCN(S(C)(=O)=O)[C@H](COC)C3)nn2c1N(C)c1nc(-c2ccc(F)cc2)cs1
InChIKey: WJHFDBUSZZQJBB-IBGZPJMESA-N
Target: Autotaxin (SINGLE PROTEIN), Homo sapiens
UniProt: Q13822
Assay: Biochemical Assay: Compound IC50 values are determined in a fluorescent hENPP2 (UniProtKB/SwissProt Sequence ref Q13822) biochemical assay using the fluorogenic autotaxin substrate FS-3 as substrate. FS-3 is a doubly labeled analog of LPC wherein the fluorophore is quenched through intramolecular energy transfer. Without hENPP2, the emission of the probe is quenched. If the substrate is hydrolyzed by hENPP2, the emission of the probe is not quenched anymore resulting in a fluorescence increase. Inhibition of hENPP2 by compounds will result in a decrease of the signal. 10 uL of a dilution series of compound, starting from 20 uM highest concentration, dilution, is added to the wells. hENPP2 is used at a final concentration of 0.4 ug/mL or 0.64 ug/mL (it will be appreciated by the skilled person that the potency read out is independent of the enzyme concentration). The enzyme is diluted in 50 mM Tris-HCl pH 8.0, 250 mM NaCl, 5 mM KCl, 1 mM MgCl2, 1 mM CaCl2, 0.1% fatty acid free BSA in a total volume of 20 uL. Enzyme mixture is added to compounds and the resulting mixture is incubated for 30 min at room temperature under shaking. The reaction is started by the addition of 20 uL of 0.75 uM FS-3 diluted in the same buffer as described above and the mixture is incubated at 30° C. for 30 min. Fluorescence is read on the Envision (Excitation 485 nm, emission 520 nM).
Measurement: IC50 > 10000 nM (pChEMBL 5)
Descriptors: MW 559.69, ALogP 3.42, HBA 10, HBD 0, PSA 96.17, QED 0.32
Compound (SMILES): CCc1cc(Cl)c(OC)c(N2CCN(CCC(O)CCNC(=O)c3cc4ccccc4o3)CC2)c1
InChIKey: ZZDBZNUPBFQLAW-UHFFFAOYSA-N
Target: D(2) dopamine receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P14416
Assay: Radioligand Binding Assay: Binding at dopamine D2-like receptors was determined using previously described methods. Membranes were prepared from HEK293 cells expressing human D2R, D3R or D4R, grown in a 50:50 mix of DMEM and Ham's F12 culture media, supplemented with 20 mM HEPES, 2 mM L-glutamine, 0.1 mM non-essential amino acids, 1× antibiotic/antimycotic, 10% heat-inactivated fetal bovine serum, and 200 μg/mL hygromycin (Life Technologies, Grand Island, N.Y.) and kept in an incubator at 37° C. and 5% CO2. Upon reaching 80-90% confluence, cells were harvested using pre-mixed Earle's Balanced Salt Solution (EBSS) with 5 μM EDTA (Life Technologies) and centrifuged at 3000 rpm for 10 min at 21° C. The supernatant was removed and the pellet was resuspended in 10 mL hypotonic lysis buffer (5 mM MgCl2.6H2O, 5 mM Tris, pH 7.4 at 4° C.) and centrifuged at 20,000 rpm for 30 min at 4° C. The pellet was then resuspended in fresh EBSS buffer made from 8.7 g/L Earle's Balanced Salts without phenol red (US Biological, Salem, Mass.), 2.2 g/L sodium bicarbonate, pH to 7.4. A Bradford protein assay (Bio-Rad, Hercules, Calif.) was used to determine the protein concentration and membranes were diluted to 500 μg/mL and stored in a −80° C. freezer for later use.
Measurement: Ki = 1619 nM (pChEMBL 5.789999961853027)
Descriptors: MW 500.04, ALogP 4.35, HBA 6, HBD 2, PSA 78.18, QED 0.43
Compound (SMILES): COc1cc2nc3c(c(NC4CCN(C(C)C)CC4)c2cc1OC)CCCC3
InChIKey: UUFKGZFOFONACR-UHFFFAOYSA-N
Target: Histone-lysine N-methyltransferase EHMT2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q96KQ7
Assay: Enzymatic Activity Assay: For the assay, the following buffer was used to set up reactions: 50 mM Tris-HCl pH9, 50 mM NaCl, 0.01% Tween-20 and 1 mM DTT (added fresh prior to starting the reactions). The assay is set up by adding a final concentration of 0.15 nM G9a, 15 uM S-adenosyl-methionine and 100 nM biotinylated histone 3 peptide (1-21). The reaction is incubated at rt for 1 hour and subsequently quenched with the acceptor beads (anti-H3k9me2 AlphaLISA acceptor beads, Perkin Elmer #AL117) at a final concentration of 20 ug/mL. The acceptor beads are incubated for 1 hour. After 1 hour, the donor beads are added at a final concentration of 20 ug/mL (Alpha Streptavidin donor beads, PerkinElmer #6760002). Donor beads are incubated for 0.5 hours. Both donor and acceptor beads are resuspended in AlphaLISA 5X Epigenetics Buffer 1 Kit (PerkinElmer #AL008) prior to addition to the reaction. All manipulations and incubations with the donor and acceptor beads are done in subdued light. Signal is detected in an EnVision plate reader in Alpha mode.
Measurement: IC50 = 10000 nM (pChEMBL 5)
Descriptors: MW 383.54, ALogP 4.42, HBA 5, HBD 1, PSA 46.62, QED 0.83
Compound (SMILES): CCOc1nc(N)c2nc(-c3cncc(F)c3)n(Cc3cccnc3)c2n1
InChIKey: ZWTYAWNGWXXTOA-UHFFFAOYSA-N
Target: Toll-like receptor 7 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9NYK1
Assay: Inhibition of the Activation Assay: As human TLR7 expressing cell line, HEK293 cell line was bought from IMGENEX Corporation (TLR7/NF-ηB/SEAPorter HEK293 cell), which is human embryonic kidney cell line and stably expresses full-length human TLR7 gene and secreted alkaline phosphatase (SEAP) reporter gene under the transcriptional control of an NE-ηB response element. The TLR7/NF-ηB/SEAPorter HEK293 cell was cultivated with DMEM containing 10% fetal bovine serum (FBS) and 10 μg/mL blasticidin S at 37° C. in the presence of 5% CO2. The TLR7/NF-ηB/SEAPorter HEK293 cell was seeded into 96-well microtiter plate at 5×104 cell/90 μL/well, and the place was cultivated at 37° C. in a CO2 incubator overnight. Each test compound that was diluted with the medium was added to the wells (10 μL/well), wherein each final concentration was adjusted to 0.001, 0.003, 0.01, 0.03, 0.1, 0.3, 1, 3, and 10 μmol/L, or 0.01, 0.03, 0.1, 0.3, 1, 3, 10, 30, and 100 μmol/L. After 0.5 hour, R-848 that is TLR7/8 ligand was added to each well (10 μL/well), wherein each final concentration was adjusted to 200 nmol/L. The total volume was adjusted to 110 μL/well, the test samples were incubated in CO2 incubator for 20±1 hours, and then the SEAP activity was measured as activation of TLR7. The SEAP activity was evaluated as follows: p-nitro-phenyl phosphate (pNPP) (Invitrogen) was added to the incubated sample (50 μL/well); after 15 minutes, 4 mol/L sodium hydroxide solution (nacalai tesque) was added thereto (50 μL/well) to quench the reaction; and the absorbance of each sample was measured at 405 nm with microplate reader Elx808 (BioTek). The 50% inhibitory concentration (IC50 value) of each sample compound was calculated based on 100% of the SEAP activity wherein the test sample comprises no sample compound.
Measurement: IC50 = 2000 nM (pChEMBL 5.699999809265137)
Descriptors: MW 365.37, ALogP 2.45, HBA 8, HBD 1, PSA 104.63, QED 0.58
Compound (SMILES): Cn1cc(C(C)(O)c2cc(F)c3c(c2)C(=O)N(Cc2ccc(C#N)cn2)[C@@]3(OCC2(C#N)CC2)c2ccc(Cl)cc2)cn1
InChIKey: RKSMIALTWWALNY-BDJZEIKTSA-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 = 1 nM (pChEMBL 9)
Descriptors: MW 597.05, ALogP 4.91, HBA 8, HBD 1, PSA 128.06, QED 0.31
Compound (SMILES): CS(=O)(=O)N(C[C@H](Nc1ncc(-c2nnc(C(F)F)o2)cn1)c1ccc(Cl)cc1)CC(F)(F)F
InChIKey: COHTVTBHWDLUQD-ZDUSSCGKSA-N
Target: Protein deacetylase HDAC6 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9UBN7
Assay: Enzymatic Activity Assay: All enzymatic reactions were conducted in duplicate at room temperature for 17 hours in a 50 μl mixture containing HDAC assay buffer (50 mM Tris-HCl, pH 7.4, 137 mM NaCl, 2.7 mM KCl, 0.05% Tween 20, 5 μg BSA), an HDAC substrate, an HDAC enzyme, and a test compound. Compound dilution was prepared ten-fold higher than the final concentration of the compounds with 10% DMSO in HDAC assay buffer and 5 μl of the dilution was added to a 50 μl reaction so that the final concentration of DMSO is 1% in all of reactions. After enzymatic reactions, 50 μl of HDAC Developer was added to each well and the plate was incubated at room temperature for an additional 20 minutes. Fluorescence intensity was measured at an excitation of 360 nm and an emission of 460 nm using a Tecan Infinite M1000 or Biotek Synergy microplate reader.The fluorescent intensity data were analyzed using the computer software, Graphpad Prism. In the absence of the compound, the fluorescent intensity (Ft) in each data set was defined as 100% activity. In the absence of HDAC, the fluorescent intensity (Fb) in each data set was defined as 0% activity. The percent activity in the presence of each compound was calculated according to the following equation: % activity=(F−Fb)/(Ft−Fb), where F=the fluorescent intensity in the presence of the compound.
Measurement: IC50 = 110 nM (pChEMBL 6.960000038146973)
Descriptors: MW 526.88, ALogP 4.09, HBA 8, HBD 1, PSA 114.11, QED 0.41
Compound (SMILES): CCN(CC)[C@H]1CC[C@@H](Nc2nc(Nc3cc(C)ns3)nc3ccsc23)CC1
InChIKey: LGUBLRVOWRGXPD-GASCZTMLSA-N
Target: Interleukin-1 receptor-associated kinase 4 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9NWZ3
Assay: Kinase Activity Inhibition Assay: IRAK4 kinase (purchased from Life Technologies, Cat. No.: PR5612U) was diluted to 2 folds of the final concentration (the final concentration is 0.76 ng/μL) with reaction buffer (40 mM Tris-HCl, pH 7.5; 20 mM MgCl2; 0.1 mg/mL BSA; 1 mM DTT), and added to a 384-well plate at 5 μL/well. The test drugs were 10-fold serial diluted from 10 μM to set six concentration points and added to the test wells of the 384-well plate at 2.5 μL/well. After incubation for 10 min at 25° C., 10 μM of ATP and 0.1 μg/L of an enzyme reaction substrate, myelin basic protein MBP (purchased from SignalChem, Cat. No.: M42-51N) were added at 2.5 L/well, and reacted at 25° C. for 60 minutes. After the reaction was completed, the kinase activity assay was performed using the ADP-Glo kinase assay kit (purchased from Promega, Cat. No.: V9102) according to the manufacture's instruction, i.e., 10 μL of ADP-Glo reaction reagent was first added and reacted at 25° C. for 40 minutes, 10 μL of the solution to be tested was taken and mixed with 10 μL of ADP-Glo detection reagent, and reacted at 25° C. for 30 minutes.
Measurement: IC50 = 550 nM (pChEMBL 6.260000228881836)
Descriptors: MW 416.62, ALogP 5.26, HBA 8, HBD 2, PSA 65.97, QED 0.55
Compound (SMILES): C[C@H]1CN(c2cc(F)c(-c3cnc(N4CCOCC4)nc3)cc2NC(=O)c2cn(C)c(=O)cc2C(F)(F)F)CCN1C
InChIKey: CGLBRJYXTDVSFE-KRWDZBQOSA-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 = 6.36 nM (pChEMBL 8.199999809265137)
Descriptors: MW 589.59, ALogP 3.23, HBA 9, HBD 1, PSA 95.83, QED 0.45
Compound (SMILES): CCn1nc(C)c(Br)c1C(=O)N1CCN(Cc2ccc(C(=O)NCCC(C)C)s2)CC1
InChIKey: CIJNWWHGISIHFV-UHFFFAOYSA-N
Target: Bifunctional epoxide hydrolase 2 (SINGLE PROTEIN), Homo sapiens
UniProt: P34913
Assay: Biochemical Screening Assay: Compounds were tested in a biochemical screening assay using recombinant sEH purified from Sf9 insect cells and an artificial substrate, (3-phenyl-oxiranyl)-acetic acid cyano-(6-methoxy-naphtalen-2-yl)-methyl ester, Phome. The biochemical assay was performed in analogy to a fluorometric described in the literature (P. D. Jones et al., Anal. Biochem. 2005, 343, 66-75). The assay principle bases on the sEH-catalyzed hydrolysis of an artificial α-cyano-ester substrate. O-deacetylation liberates a cyanohydrin intermediate that rapidly decomposes to the highly fluorescent 6-methoxy-2-naphtaldehyde. To discriminate sample autofluorescence, the assay was carried out as a kinetic measurement with two time points. The first measurement is performed immediately before addition of the substrate and the 15 second measurement is done after completion of the assay. The assay format is either in 96- or in 384-well format.Details of the assay using a 96-well plate format are described below. 40 μl recombinant sEH enzyme and 5 μl test compound are pre-incubated for 15 minutes at 30° C. Following pre-incubation, the reaction is started by addition of 5 μl Phome. The assay mixture containing 2 nM final sEH concentration, test compound ranging in a concentration from 0.0001-10 μM and 5 μM Phome is incubated for 60 minutes at 30° C.
Measurement: IC50 = 1.53 nM (pChEMBL 8.8100004196167)
Descriptors: MW 510.5, ALogP 3.77, HBA 6, HBD 1, PSA 70.47, QED 0.59
Compound (SMILES): CC1CN(c2c(C(NC(=O)c3c(N)nn4cccnc34)C3CC3)cc(Cl)c3c(C#N)ncn23)CCS1(=O)=O
InChIKey: SOPKGNSGEFHQOL-UHFFFAOYSA-N
Target: Phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit gamma isoform (SINGLE PROTEIN), Homo sapiens
UniProt: P48736
Assay: PI3K-gamma Scintillation Proximity Assay: [γ-33P]ATP (10 mCi/mL) and Wheat Germ Agglutinin (WGA) YSi SPA Scintillation Beads was purchased from Perkin-Elmer (Waltham, Mass.). Lipid kinase substrate, D-myo-Phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2)D (+)-sn-1,2-di-O-octanoylglyceryl, 3-O-phospho linked (PIP2), CAS 204858-53-7, was purchased from Echelon Biosciences (Salt Lake City, Utah). PI3Kγ (p110γ) Recombinant Human Protein was purchased from Life technology (Grand Island, N.Y.). ATP, MgCl2, DTT, EDTA, MOPS and CHAPS were purchased from Sigma-Aldrich (St. Louis, Mo.).The kinase reaction was conducted in polystyrene 384-well Greiner Bio-one white plate from Thermo Fisher Scientific in a final volume of 25 μL. Inhibitors were first diluted serially in DMSO and added to the plate wells before the addition of other reaction components. The final concentration of DMSO in the assay was 2%. The PI3Kγ assay was carried out at room temperature in 20 mM MOPS, pH 6.7, 10 mM MgCl2, 5 mM DTT and CHAPS 0.03%. Reactions were initiated by the addition of ATP, the final reaction mixture consisted of 20 μM PIP2, 2 μM ATP, 0.5 μCi [γ-33P] ATP, 13 nM PI3Kγ. Reactions were incubated for 120 min and terminated by the addition of 40 μL SPA beads suspended in quench buffer: 163 mM potassium phosphate pH 7.8, 20% glycerol, 25 mM EDTA. The final concentration of SPA beads is 1.0 mg/mL. After the plate sealing, plates were shaken overnight at room temperature and centrifuged at 1500 rpm for 10 min, the radioactivity of the product was determined by scintillation counting on Topcount (Perkin-Elmer). IC50 determination was performed by fitting the curve of percent of the solvent control activity versus the log of the inhibitor concentration using the GraphPad Prism 6.0 software.
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 554.04, ALogP 1.99, HBA 11, HBD 2, PSA 163.78, QED 0.38
Compound (SMILES): Cc1nc2cnc(N(C)C3CCN(CC4(C(F)(F)F)CC4)CC3)c(C#N)c2c(=O)n1CC#Cc1ccc(Cl)cc1-c1ccnc2c(C(=O)O)csc12
InChIKey: WFPDDAZJFUQNMK-UHFFFAOYSA-N
Target: Eukaryotic translation initiation factor 4E-binding protein 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q13541
Assay: Fluorescence Polarization Assay: eIF4E protein was pre-incubated with EDA-m7GDP-ATTO-550 in FPB at 2xconcentrations for 5 minutes prior to the addition of compounds. Compounds (100x) were prepared using 3-fold serial dilutions in 100% DMSO, and subsequently diluted 1:50 in FPB to produce 2x stocks. 50 ul of 2x eIF4E/EDA-m7GDP-ATTO-550 were transferred to the wells of a 96-well half-area black flat bottom polystyrene plate. 50 ul of 2x test compound were added, and binding reactions were allowed to equilibrate for 30 minutes at room temperature while protected from light. The fluorescent polarization signal was detected using a Victor 2 multi-label counter (Perkin Elmer) and the concentration necessary to achieve inhibition of eIF4E/EDA-m7GDP-ATTO-550 binding by 50% (IC50) was calculated using data from an 8-point compound dilution series.
Measurement: IC50 = 39.7 nM (pChEMBL 7.400000095367432)
Descriptors: MW 746.22, ALogP 6.89, HBA 10, HBD 1, PSA 128.24, QED 0.18
Compound (SMILES): C[C@H](CO)N(CC(=O)NCc1cccc(Cl)c1F)C(=O)Cn1nc(C(N)=O)c2cc(C(N)=O)ccc21
InChIKey: PZOVMKLQWPWMPQ-GFCCVEGCSA-N
Target: Complement factor D (SINGLE PROTEIN), Homo sapiens
UniProt: P00746
Assay: Inhibition Assay: An established esterolytic assay for the measurement of Factor D activity and inhibition of Factor D activity was used (Kam, C. M.; McRae, B. J.; Harper, J. W.; Niemann, M. A.; Volanakis, J. E.; Powers, J. C. Human complement proteins D, C2, and B Active site mapping with peptide thioester substrates. J Biol. Chem. 1987, 262, 3444-3451). For this assay Z-Lys-SBzl, 1.29 mM (Kim, S.; Narayana, S. V. L; Volanakis, J. E. Mutational analysis of the substrate binding site of human complement Factor D. Biochemistry. 1994, 33, 14393-14399) was used as the substrate for Factor D (104 mM). Hydrolysis of this compound by Factor D liberated a free sulfhydryl group which is then reacted with 5,5′-dithiobis(2nitrobenzoic acid) producing an intense yellow color (Habeeb, A. F. S. A. Reaction of protein sulfhydryl groups with Ellman's Reagent. Methods in Enzymol. 1976, 25, 457-464.). The assays were performed in 96 well microtiter plates and rates of hydrolysis were monitored at 405 nm on a Biotek Synergy H1 plate reader. Hydrolysis rates were reported as change in mOD/min. The assay was conducted in 100 mM HEPES, 500 mM NaCl, pH 7.5 containing 10% DMSO in a final volume of 50 μL per well.
Measurement: IC50 < 1000 nM (pChEMBL 6)
Descriptors: MW 518.93, ALogP 0.55, HBA 7, HBD 4, PSA 173.64, QED 0.3
Compound (SMILES): COc1nc2ccc([C@](O)(c3ccc(Cl)cc3)c3cncn3C)cc2c(Cl)c1OCC(F)(F)F
InChIKey: ANQBFYZUQVGBKP-HSZRJFAPSA-N
Target: Nuclear receptor ROR-gamma (SINGLE PROTEIN), Homo sapiens
UniProt: P51449
Assay: ThermoFluor Assay: ThermoFluor is a fluorescence based assay that estimates ligand binding affinities by measuring the effect of a ligand on protein thermal stability (Pantoliano, M. W., Petrella, E. C., Kwasnoski, J. D., Lobanov, V. S., Myslik, J., Graf, E., Carver, T., Asel, E., Springer, B. A., Lane, P., and Salemme, F. R. (2001) High-density miniaturized thermal shift assays as a general strategy for drug discovery. J Biomol Screen 6, 429-40, and Matulis, D., Kranz, J. K., Salemme, F. R., and Todd, M. J. (2005) Thermodynamic stability of carbonic anhydrase: measurements of binding affinity and stoichiometry using ThermoFluor. Biochemistry 44, 5258-66). This approach is applicable to a wide variety of systems, and rigorous in theoretical interpretation through quantitation of equilibrium binding constants (KD).In a ThermoFluor experiment where protein stability is monitored as the temperature is steadily increased, an equilibrium binding ligand causes the midpoint of an unfolding transition (Tm) to occur at a higher temperature. The shift in the melting point described as a ΔTm is proportional to the concentration and affinity of the ligand. The compound potency may be compared as a rank order of either ΔTm values at a single compound concentration or in terms of KD values, estimated from concentration response curves.
Measurement: Kd = 25 nM (pChEMBL 7.599999904632568)
Descriptors: MW 512.32, ALogP 5.51, HBA 6, HBD 1, PSA 69.4, QED 0.37
Compound (SMILES): CC(C)(C)n1nc(-c2ccc3cc(C(=O)NCc4ccccc4)[nH]c3c2)c2c(N)ncnc21
InChIKey: QUQMCVRDSYYOPV-UHFFFAOYSA-N
Target: Proto-oncogene tyrosine-protein kinase receptor Ret (SINGLE PROTEIN), Homo sapiens
UniProt: P07949
Assay: RET and KDR Enzyme Assay: Kinase activity was detected using CisBio HTRF kinEASE kit based on time-resolved fluorescence transfer (FRET). The assay was performed in 384-well white plates (Corning #3574) in a reaction volume of 10 μL containing 1× CisBio enzymatic buffer supplemented with a final concentration of 5 mM MgCl2, 1 mM DTT, 10 nM SEB and 0.01% Triton X100 for RET. The same buffer was used for the KDR biochemical assay with the addition of 2 mM MnCl2.Inhibitors were pre-incubated in the plate for 15 mins with 5 μL kinase and assay buffer at the following concentrations; 13 pM RET (Carna Biosciences; 08-159) and 150 pM KDR (Millipore; 14-630). The reaction was initiated by the addition of 5 μL ATP and substrate at 2× final reaction concentrations. For RET, this was 18 μM and 2 μM; for KDR, this was 16 μM and 1 μM, respectively. Reactions were performed at ATP Km for each target. The assay was allowed to proceed at room temperature for 20 mins before terminating with the addition of 10 μL HTRF detection buffer containing EDTA supplemented with TK-antibody labelled with Eu3+-Cryptate (1:100 dilution) and streptavidin-XL665 (128 nM). Following incubation at room temperature for 1 hour, FRET signal was measured using the Pherastar FS Microplate Reader.
Measurement: IC50 = 933 nM (pChEMBL 6.03000020980835)
Descriptors: MW 439.52, ALogP 4.24, HBA 6, HBD 3, PSA 114.51, QED 0.39
Compound (SMILES): CC[C@H](NS(=O)(=O)c1ccc(-c2sc(-c3nnc(CC(C)(C)C(=O)O)o3)nc2CN2CCCC(F)(F)C2)c2ccccc12)C(F)(F)F
InChIKey: CCOMNIREOMNHMF-QFIPXVFZSA-N
Target: Nuclear receptor ROR-gamma (SINGLE PROTEIN), Homo sapiens
UniProt: P51449
Assay: ThermoFluor Assay: For the RORγt construct used in the ThermoFluor assay, numbering for the nucleotide sequences was based on the reference sequence for human RORγt, transcript variant 2, NCBI Accession: NM_001001523.1 (SEQ ID NO:1). Nucleotides 850-1635 (SEQ ID NO:2) coding for the wild type human RORγt ligand binding domain (RORγt LBD) were cloned into the pHIS1 vector, a modified pET E. coli expression vector (Accelagen, San Diego), containing an in-frame N-terminal His-tag and a TurboTEV protease cleavage site (ENLYFQG, SEQ ID NO:3) upstream of the cloned insert sequence. The amino acid sequence for the RORγt construct used in the Thermofluor assay is shown as SEQ ID NO:4. ThermoFluor experiments were carried out using instruments owned by Janssen Research and Discovery, L.L.C. through its acquisition of 3-Dimensional Pharmaceuticals, Inc. 1,8-ANS (Invitrogen) was used as a fluorescent dye. Protein and compound solutions are dispensed into black 384-well polypropylene PCR microplates (Abgene) and overlayed with silicone oil (1 μL, Fluka, type DC 200) to prevent evaporation. Bar-coded assay plates are robotically loaded onto a thermostatically controlled PCR-type thermal block and then heated at a typical ramp-rate of 1° C./min for all experiments. Fluorescence was measured by continuous illumination with UV light (Hamamatsu LC6) supplied via fiber optic and filtered through a band-pass filter (380-400 nm; >6 OD cutoff). Fluorescence emission of the entire 384-well plate was detected by measuring light intensity using a CCD camera (Sensys, Roper Scientific) filtered to detect 500±25 nm, resulting in simultaneous and independent readings of all 384 wells. Images were collected at each temperature, and the sum of the pixel intensity in a given area of the assay plate was recorded versus temperature.
Measurement: Kd = 0.12 nM (pChEMBL 9.920000076293945)
Descriptors: MW 701.74, ALogP 6.52, HBA 9, HBD 2, PSA 138.52, QED 0.17
Compound (SMILES): Cc1ccc(N2C(=O)c3c(Cl)ccc(NS(=O)(=O)c4ccc(C(C)(C)C)cc4)c3C2=O)nn1
InChIKey: ZSBHKBLVXGKBDB-UHFFFAOYSA-N
Target: C-C chemokine receptor type 9 (SINGLE PROTEIN), Homo sapiens
UniProt: P51686
Assay: FLIPR Assay: A calcium flux assay was used to determine the ability of the compounds to interfere with the binding between CCR9 and its chemokine ligand (TECK) in Chem1-hCCR9 overexpressing cells. hCCR9 overexpressing cells were seeded (25,000 cells/well) into black Poly-D-Lysine coated clear bottom 96-well plates (BD Biosciences, Cat #356640) and incubated overnight at 37° C./5% CO2 in a humidified incubator. Media was aspirated and cells washed twice with 100 μL assay buffer (1×HBSS, 20 mM HEPES) containing 2.5 mM Probenecid. A 0.3× Fluo-4 NW calcium dye was prepared in assay buffer containing 5 mM Probenecid and stored in the dark. Each well was loaded with 100 μL of 0.3× Fluo-4 NW calcium dye and incubated at 37° C./5% CO2 for 60 minutes and then at room temperature for 30 minutes. A half-log serially diluted concentration response curve was prepared at a 3× final assay concentration for each compound (10 μM-0.1 nM final assay concentration) and 50 μL of the compound then transferred to the cells (150 μL final volume) for 60 minutes prior to stimulation (30 minutes at 37° C./5% CO2 and 30 minutes at room temperature). TECK was diluted to 4× its EC80 in assay buffer (containing 0.1% [w/v] bovine serum albumin [BSA]) and 50 μL dispensed through the fluorometric imaging plate reader (FLIPR) instrument to stimulate the cells (200 μL final volume). The increase in intracellular calcium levels was measured with the FLIPR instrument.
Measurement: Ki = 32 nM (pChEMBL 7.5)
Descriptors: MW 484.97, ALogP 4.34, HBA 6, HBD 1, PSA 109.33, QED 0.55
Compound (SMILES): O=C(O)c1ccc2c(c1)CCCN2S(=O)(=O)c1ccc2ccccc2c1
InChIKey: GNGLYELNNHHYQP-UHFFFAOYSA-N
Target: Induced myeloid leukemia cell differentiation protein Mcl-1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q07820
Assay: fluorescence polarization competition assay (FPCA): Molecular modeling and SILCS functional group affinity mapping (FragMaps) of the Mcl-1 binding site indicated that the carboxylic acid of designed molecule 3a (FIG. 1C) would occupy an energetically favorable region, associated with a salt bridge interaction with R263 of the Mcl-1 binding site, while the ring of the naphthyl core would bind in the p3 pocket demarcated by a favorable non-polar FragMap. The aniline was directed into the hydrophobic p2 pocket, which is also demarcated by a nonpolar FragMap. With the molecular modeling data in hand, compound 3a was then synthesized according to Scheme 1-2.Briefly, commercially available 1-hydroxy-2-naphthoic acid (4) was regioselectively 4-chlorosulfonylated to yield 5, which was isolated by pouring over ice and used without further purification. Sulfonyl chloride 5 was next reacted with 4-bromoaniline to furnish the target molecule 3a in excellent overall yield (83%). Evaluation of 3a in a fluorescence polarization competition assay (FPCA) indicated that it disrupted the Mcl-1-Bak-BH3 PPI with an IC50 of 10.9 μM, corresponding to a Ki of 2.76 μM. Given the ability of 3a to inhibit Mcl-1, a structure-activity relationship (SAR) study was developed, the results of which are presented in the tables below.
Measurement: Ki = 22000 nM (pChEMBL 4.659999847412109)
Descriptors: MW 367.43, ALogP 3.68, HBA 3, HBD 1, PSA 74.68, QED 0.77
Compound (SMILES): O=C(O)c1ccc(-n2cc(C(=O)c3c(F)cccc3C(F)(F)F)c3cccc(F)c32)c(F)c1
InChIKey: KCBQCNNVYCKGLK-UHFFFAOYSA-N
Target: Nuclear receptor ROR-gamma (SINGLE PROTEIN), Homo sapiens
UniProt: P51449
Assay: FRET Assay: The assay was run in black 384 well plates (Greiner cat no: 784900). Various concentrations of test ligands in 0.1 microlitres DMSO were dispensed to assay plates using an Labcyte Echo acoustic dispenser. Two pre-mixes were prepared and incubated for 1 hr at room temp in the dark. Pre-mix 1 comprised 100 nM Protein (Biotinylated HN-Avi-MBP-TCS-hRORg (258-518)) and 60 nM Streptavidin APC in assay buffer, 50 mM MOPS pH7.4, 50 mM KF, 0.003% (w/v) CHAPS, 10 mM DTT and 0.01% (w/v) BSA and pre-mix 2 comprised 160 nM biotinylated SRC-1 peptide (NCOA1-677-700) and 20 nM Europium-W8044 labelled Streptavidin in assay buffer. Five microlitres of pre-mix 2 was dispensed to assay plates containing test compound and incubated for 15 minutes prior to adding five microlitres of pre-mix 1. Plates were incubated at room temperature for 1 hour in the dark, prior to reading in a Pherastar multi-mode plate reader using HTRF filter set (ex 320, em 612 and 665). The FRET signal at 665 nM was divided by the signal at 612 nM and multiplied by 10,000 to generate a signal ratio value for each well. The raw data was transformed to % effect using the equation:Compound % effect=100*[(X−min)/(max−min)],where X represents the normalized value for the compound based on the Min (vehicle) and Max (reference compound) inhibition control.
Measurement: IC50 = 111 nM (pChEMBL 6.960000038146973)
Descriptors: MW 463.33, ALogP 6, HBA 3, HBD 1, PSA 59.3, QED 0.3
Compound (SMILES): Nc1ccc(Oc2ncnc(N)c2-c2ccc(Oc3ccccc3)cc2)cc1
InChIKey: BMLGAFSAYAYZBA-UHFFFAOYSA-N
Target: Tyrosine-protein kinase BTK (SINGLE PROTEIN), Homo sapiens
UniProt: Q06187
Assay: Enzyme Assay: The following describes a microfluidic, off-chip mobility shift kinase assay used to measure inherent potency of compounds against BTK enzyme. Compounds described by embodiments of the present invention were assayed using this protocol and the data from the same is recorded in Table 2 within the column labeled: Time Dependent BTK Enzyme Assay IC50. These IC50 values are reported in ranges wherein: A<100 nM, B<1 uM, and C>1 uM.2.5× stocks of full-length human BTK (08-080) from CarnaBio USA, Inc., Natick, Mass., 1.6×ATP and appropriate kinKDR peptide substrate (FITC-AHA-EEPLYWSFPAKKK-NH2) were prepared in kinase reaction buffer consisting of 25 mM MgCl2, 0.015% Brij-35 (30%), 100 mM Hepes, pH 7.5, and 10 mM DTT.5 uL of enzyme buffer and 7.5 uL of ATP/kinKDR peptide substrate mix were added to Matrix (#115304) 384-well, sterile, polypropylene plates (Thermo Fisher Scientific, Hudson, N.H.) with 125 nL of serially diluted compounds prepared in 100% DMSO, and incubated for 90 min. at 27 C. Following the incubation period, reactions were stopped by adding 60 uL stop buffer consisting of 100 mM Hepes, pH 7.5, 0.015% Brij-35 (30%), 0.277% Coating Reagent #3 (Caliper Life Sciences, Mountain View, Calif.), 5% DMSO. Stopped reactions were monitored at −2 PSI, −3000 V/−700 V in a LabChip 3000 plate reader from Caliper Life Sciences, a PerkinElmer Company (Hopkinton, Mass.), and the activity was measured by off-chip mobility shift assay measuring the charge/mass difference between substrate and product resulting from peptide phosphorylation. IC50 and efficacy were determined by plotting log [Inhibitor] vs. % Activity in GeneData Screener (Basel, Switzerland). Compounds described by embodiments of the present invention were assayed using this protocol and the data from the same is recorded in Table 2 within the column labeled: Time Dependent PBMC BTK Enzyme Assay IC50.
Measurement: IC50 > 1000 nM (pChEMBL 6)
Descriptors: MW 370.41, ALogP 4.89, HBA 6, HBD 2, PSA 96.28, QED 0.49
Compound (SMILES): COc1cccc(O[C@H]2C[C@@H]3CN(CC(O)c4ccc(O)cn4)C[C@@H]3C2)c1
InChIKey: NWYWCLDKDLBUND-BWRJHLJPSA-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 = 70.7 nM (pChEMBL 7.150000095367432)
Descriptors: MW 370.45, ALogP 2.62, HBA 6, HBD 2, PSA 75.05, QED 0.81
Compound (SMILES): N#Cc1cnc(OCc2cccnc2)nc1-n1cnc2ccccc21
InChIKey: LSGQAHMOYABZKO-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 < 1000 nM (pChEMBL 6)
Descriptors: MW 328.34, ALogP 2.66, HBA 7, HBD 0, PSA 89.51, QED 0.57
Compound (SMILES): O=C(O)C1CN(c2cccc(-c3noc(-c4ccc(OCCF)c(C(F)(F)F)c4)n3)c2)C1
InChIKey: UARWOWOEUTYJCR-UHFFFAOYSA-N
Target: Sphingosine 1-phosphate receptor 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P21453
Assay: In Vitro Binding Assay: Newly synthesized compounds were first evaluated for binding potency toward S1P1 by a [32P]S1P competitive binding assay following published procedure (J. Rosenberg, H. Liu, Z. Tu, A practical process for the preparation of [32P]S1P and binding assay for S1P receptor ligands, Appl. Radiat. Isot. 102 (2015) 5-9), and using S1P as a reference compound. [32P]S1P was first prepared by incubating sphingosine and [γ-32P]ATP with sphingosine kinase 1 as previously reported (J. Rosenberg, H. Liu, Z. Tu, A practical process for the preparation of [32P]S1P and binding assay for S1P receptor ligands, Appl. Radiat. Isot. 102 (2015) 5-9). [32P]S1P was dissolved in DMSO, and then diluted in the assay buffer (50 mM HEPES-Na with 5 mM MgCl2, 1 mM CaCl2, and 0.5% fatty acid-free bovine serum albumin, pH=7.5). Compounds were dissolved in DMSO and diluted to different concentrations with assay buffer, followed by adding commercial cell membranes expressing recombinant human S1P receptors (1, 2, 3, 4, and 5) in the assay buffer at room temperature in 96-well plate. [32P]S1P solution was then added to give a final volume of 150 μL containing 0.1 nM of [32P]S1P and 1 μg of membrane protein per well. Competitive binding was performed for 60 min at room temperature and terminated by collecting the membranes onto 96-well glass fiber (GF/B) filtration plates (Millipore, Billerica, Mass.). Each filter was washed with 200 μL of assay buffer for five times. The filter bound radionuclide was measured by a Beckman LS3801 scintillation counter using Cherenkov counting. The reported IC50 values were calculated using the 4 parameter equation, least-square non-linear regression curve-fit, with GraphPad Prism software (GraphPad Software, Inc). Each assay was repeated at least three times with duplicate wells for each compound; the reported values (mean±SD, nM) are calculated from the average of all assays. Assays for compounds which showed no activity (IC50>1000 nM) were only repeated twice.
Measurement: IC50 > 1000 nM (pChEMBL 6)
Descriptors: MW 451.38, ALogP 4.29, HBA 6, HBD 1, PSA 88.69, QED 0.54
Compound (SMILES): COc1cc(OC)c(Cl)c(-c2ccc3c(NC(=O)c4ccc(N5CCC(N(C)C)CC5)cc4)n[nH]c3c2)c1Cl
InChIKey: WLIUWVQPEGEUJK-UHFFFAOYSA-N
Target: Fibroblast growth factor receptor 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P11362
Assay: Enzyme Kinase Activity Assay: The enzyme reaction substrate Poly(Glu,Tyr) 4:1 was diluted with PBS without potassium ion (10 mM sodium phosphate buffer, 150 mM NaCl, pH7.2-7.4) to 20 μg/mL, 125 μL/well to coat the enzyme plate, and reacted at 37° C. for 12-16 hours. After the liquid was removed from the wells, the plate was washed three times with 200 μL/well of T-PBS (PBS containing 0.1% Tween-20) for 5 minutes each. The enzyme plate was dried in a 37° C. oven for 1-2 hours.50 μL of the ATP solution diluted with the reaction buffer (50 mM HEPES pH 7.4, 50 mM MgCl2, 0.5 mM MnCl2, 0.2 mM Na3VO4, 1 mM DTT) was added into each well at a 5 μM final concentration. The compounds were diluted to the appropriate concentration in DMSO, 1 μL/well or containing the corresponding concentrations of DMSO (negative control wells). The reaction was initiated by addition of each kinase domain recombinant protein diluted with 49 μL of reaction buffer. Two control wells without ATP were set in each experiment. The reaction mixtures were placed on a shaker (100 rpm) to react at 37° C. for 1 hour. The plates were washed with T-PBS for three times. 100 μL/well of the primary antibody PY99 dilution was added, and reacted on a shaker (100 rpm) at 37° C. for 0.5 hour. The plates were washed with T-PBS for three times. 100 μL/well of the secondary anti-horseradish peroxidase-labeled goat anti-mouse IgG dilution was added, and reacted on a shaker at 37° C. for 0.5 hour. The plates were washed with T-PBS for three times. 100 μL/well of 2 mg/mL OPD developing solution (diluted with 0.1M citric acid-sodium citrate buffer containing 0.03% H2O2 (pH=5.4)), and reacted in dark for 1-10 minutes at 25° C. (Ultrasound is needed in OPD dissolution, and the developing solution should be prepared on the site). The reaction was quenched with 50 μL/well of 2M H2SO4, and read out at 490 nm using a tunable microplate microplate reader SPECTRA MAX 190.
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 568.51, ALogP 6.34, HBA 6, HBD 2, PSA 82.72, QED 0.27
Compound (SMILES): Cc1cccnc1C1CCC(CCNC2(C)CCOCC2)CC1
InChIKey: VBPBLEMXXIWBSQ-UHFFFAOYSA-N
Target: Nociceptin receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P41146
Assay: Cell-Based NOP Assay: Formula (I) compounds were tested for antagonism of the nociceptin receptor (NOP) in a cell-based cAMP assay, using the LANCE cAMP Detection Kit, Perkin Elmer (Waltham, Mass.). This assay (LANCE cAMP) is a homogeneous, time-resolved fluorescence resonance energy transfer (TR-FRET) immunoassay designed to measure cAMP produced upon modulation of adenylyl cyclase activity by G protein-coupled receptors (GPCRs).ProtocolCells expressing human opioid NOP (ValiScreen cell line, cat #ES-230-C Perkin Elmer (Waltham, Mass.) were dispensed in simulation buffer (5 mM Hepes, 0.1% BSA, 0.5 mM IBMX in Hanks Buffered Saline Solution) at 2.5 μL cells per well (400 cells/well) into a 1536 white TC-treated assay plate. Compound was then added and plates were incubated for 15 minutes at room temperature. To each well was added 2.5 μL of a solution containing forskolin (3 μM final) and nociceptin peptide (390 pM final) diluted in stimulation buffer, and the plates were incubated for 30 minutes at room temperature. To each well was dispensed 5 μL of LANCE Ultra detection (1:300 dilution of anti-cAMP ULight with a 1:100 dilution of Eu-cAMP tracer diluted in cAMP Detection Buffer) reagent, and the plates were incubated for 1 hr at room temperature. The plate was then read on the ViewLux microplate reader (Perkin Elmer, Waltham, Mass.) using the LANCE 1536 protocol with 60 second exposure times and 50 μs delay. This protocol has an excitation wavelength of 340 nm (DUG11 filter) and emission wavelengths of 615 nm (Eu-Donor) and 665 nm (Alexa 647 cAMP antibody acceptor).Data AnalysisData was analyzed, normalized, and visualized using ACAS (John McNeil & Co.). Efficacy was calculated using activity of the vehicle (DMSO) as 0% efficacy and the activity of the corresponding reference positive control (SCH 221510, Tocris Bioscience (Pittsburgh, Pa.), Catalog number 3240) as 100% efficacy. Activity was calculated as the ratio of donor and acceptor emissions. All curve fits and IC50 determinations were performed using a non-linear regression model. The IC50 value was measured as the concentration at which half-maximal response was calculated.
Measurement: IC50 = 550 nM (pChEMBL 6.260000228881836)
Descriptors: MW 316.49, ALogP 4.21, HBA 3, HBD 1, PSA 34.15, QED 0.88
Compound (SMILES): CCN(c1nccnc1CNc1nc(-c2cc(F)c(O)cc2CC(F)(F)F)cc2[nH]nc(C(N)=O)c12)S(C)(=O)=O
InChIKey: YFOBCCOOEQSQOX-UHFFFAOYSA-N
Target: Tyrosine-protein kinase JAK1 (SINGLE PROTEIN), Homo sapiens
UniProt: P23458
Assay: JAK Caliper Enzyme Assay at 1 mM ATP: Test article was solubilized in dimethyl sulfoxide (DMSO) to a stock concentration of 30 mM. An 11-point half log dilution series was created in DMSO with a top concentration of 600 μM. The test compound plate also contained positive control wells containing a known inhibitor to define 100% inhibition and negative control wells containing DMSO to define no inhibition. The compound plates were diluted 1 to 60 resulting in a top final assay compound concentration of 10 μM and a 2% DMSO concentration.Test article and assay controls were added to a 384-well plate. Reaction mixtures contained 20 mM HEPES, pH 7.4, 10 mM magnesium chloride, 0.01% bovine serum albumin (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 JAK3 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%-30% phosphorylation. The assays were stopped with a final concentration of 10 mM EDTA, 0.1% Coating Reagent and 100 mM HEPES, pH=7.4. The assay plates were placed on a Caliper Life Science Lab Chip 3000 (LC3000) instrument, and each well was sampled using appropriate separation conditions to measure the unphosphorylated and phosphorylated peptide.
Measurement: IC50 = 2.9 nM (pChEMBL 8.539999961853027)
Descriptors: MW 582.54, ALogP 2.86, HBA 9, HBD 4, PSA 180.08, QED 0.22
Compound (SMILES): O=C(Nc1ccc(Cl)cc1)N[C@@H]1CCN(c2ccc(-c3ccccc3F)c(F)c2F)C1=O
InChIKey: IQFNGNSHVVYUNT-GOSISDBHSA-N
Target: N-formyl peptide receptor 2 (SINGLE PROTEIN), Homo sapiens
UniProt: P25090
Assay: FPR2 and FPR1 Cyclic Adenosine Monophosphate (cAMP) Assays: A mixture of forskolin (5 μM final for FPR2 or 10 μM final for FPR1) and IBMX (200 μM final) were added to 384-well Proxiplates (Perkin-Elmer) pre-dotted with test compounds in DMSO (1% final) at final concentrations in the range of 0.020 nM to 100 μM. Chinese Hamster Ovary cells (CHO) overexpressing human FPR1 or human FPR2 receptors were cultured in F-12 (Ham's) medium supplemented with 10% qualified FBS, 250 μg/ml zeocin and 300 μg/ml hygromycin (Life Technologies). Reactions were initiated by adding 2,000 human FPR2 cells per well or 4,000 human FPR1 cells per well in Dulbecco's PBS (with calcium and magnesium) (Life Technologies) supplemented with 0.1% BSA (Perkin-Elmer). The reaction mixtures were incubated for 30 min at room temperature. The level of intracellular cAMP was determined using the HTRF HiRange cAMP assay reagent kit (Cisbio) according to manufacturer's instruction. Solutions of cryptate conjugated anti-cAMP and d2 fluorophore-labelled cAMP were made in a supplied lysis buffer separately. Upon completion of the reaction, the cells were lysed with equal volume of the d2-cAMP solution and anti-cAMP solution. After a 1-h room temperature incubation, time-resolved fluorescence intensity was measured using the Envision (Perkin-Elmer) at 400 nm excitation and dual emission at 590 nm and 665 nm. A calibration curve was constructed with an external cAMP standard at concentrations ranging from 1 μM to 0.1 pM by plotting the fluorescent intensity ratio from 665 nm emission to the intensity from the 590 nm emission against cAMP concentrations. The potency and activity of a compound to inhibit cAMP production was then determined by fitting to a 4-parametric logistic equation from a plot of cAMP level versus compound concentrations.
Measurement: EC50 = 12 nM (pChEMBL 7.920000076293945)
Descriptors: MW 459.86, ALogP 5.35, HBA 2, HBD 2, PSA 61.44, QED 0.55
Compound (SMILES): O=C(CCC(F)(F)F)Nc1ccc([C@H]2O[C@@H]3CN[C@H]2C3)cc1
InChIKey: KXSALGASUZXSCF-SGMGOOAPSA-N
Target: Trace amine-associated receptor 1 (SINGLE PROTEIN), Mus musculus
UniProt: Q923Y8
Assay: Radioligand Binding Assay: HEK-293 cells stably expressing rat TAAR1 were maintained at 37° C. and 5% CO2 in DMEM high glucose medium, containing fetal calf serum (10%, heat inactivated for 30 min at 56° C.), penicillin/streptomycin (1%), and 375 μg/ml geneticin (Gibco). Cells were released from culture flasks using trypsin/EDTA, harvested, washed twice with ice-cold PBS (without Ca2+ and Mg2+), pelleted at 1'000 rpm for 5 min at 4° C., frozen and stored at −80° C. Frozen pellets were suspended in 20 ml HEPES-NaOH (20 mM, pH 7.4) containing 10 mM EDTA and homogenized with a Polytron (PT 6000, Kinematica) at 14′000 rpm for 20 s. The homogenate was centrifuged at 48'000×g for 30 min at 4° C. Subsequently, the supernatant was removed and discarded, and the pellet resuspended in 20 ml HEPES-NaOH (20 mM, pH 7.4) containing 0.1 mM EDTA using the Polytron (20 s at 14'000 rpm). This procedure was repeated and the final pellet resuspended in HEPES-NaOH containing 0.1 mM EDTA and homogenized using the Polytron.
Measurement: IC50 = 17.2 nM (pChEMBL 7.760000228881836)
Descriptors: MW 314.31, ALogP 2.77, HBA 3, HBD 2, PSA 50.36, QED 0.9