lovingscience/chembl-v37-raw-activities huggingface dataset:
Compound (SMILES): COC[C@H](C[C@@H](Cc1ccc(-c2cc(Cl)ccc2F)cc1)NC(=O)c1ccc(C(F)(F)F)cn1)C(=O)O
InChIKey: YVQDONHLOZXBSO-FXAWDEMLSA-N
Target: Neprilysin (SINGLE PROTEIN), Homo sapiens
UniProt: P08473
Assay: Enzyme Inhibition Assay: Recombinant human NEP and recombinant human ACE were obtained commercially (R&D Systems, Minneapolis, Minn., catalog numbers 1182-ZN and 929-ZN, respectively). The fluorogenic peptide substrate Mca-D-Arg-Arg-Leu-Dap-(Dnp)-OH (Medeiros et al. (1997) Braz. J. Med. Biol. Res. 30:1157-62; Anaspec, San Jose, Calif.) and Abz-Phe-Arg-Lys(Dnp)-Pro-OH (Araujo et al. (2000) Biochemistry 39:8519-8525; Bachem, Torrance, Calif.) were used in the NEP and ACE assays respectively.The assays were performed in 384-well white opaque plates at 37° C. using the fluorogenic peptide substrates at a concentration of 10 μM in Assay Buffer (NEP: 50 mM HEPES, pH 7.5, 100 mM NaCl, 0.01% polyethylene glycol sorbitan monolaurate (Tween-20), 10 μM ZnSO4; ACE: 50 mM HEPES, pH 7.5, 100 mM NaCl, 0.01% Tween-20, 1 μM ZnSO4). The respective enzymes were used at concentrations that resulted in quantitative proteolysis of 1 μM of substrate after 20 minutes at 37° C.Test compounds were assayed over the range of concentrations from 10 μM to 20 pM. Test compounds were added to the enzymes and incubated for 30 minute at 37° C. prior to initiating the reaction by the addition of substrate. Reactions were terminated after 20 minutes of incubation at 37° C. by the addition of glacial acetic acid to a final concentration of 3.6% (v/v).
Measurement: Ki < 1 nM (pChEMBL 9)
Descriptors: MW 538.93, ALogP 5.64, HBA 4, HBD 2, PSA 88.52, QED 0.33
Compound (SMILES): CNC(=O)N1CCc2c(N3CCOCC3)nn(-c3cccc4cc(-c5cnn(C)c5)ncc34)c2C1
InChIKey: VRVMHHGGFDAADO-UHFFFAOYSA-N
Target: CREB-binding protein (SINGLE PROTEIN), Homo sapiens
UniProt: Q92793
Assay: TR-FRET Binding Assay: His/Flag epitope tagged CBP was cloned, expressed, and purified to homogeneity. CBP binding and inhibition was assessed by monitoring the engagement of a biotinylated small molecule compound with the target using the TR-FRET assay technology (Perkin-Elmer). Specifically, in a 384 well ProxiPlate CBP (4 nM final) was combined with biotin-ligand (60 nM final) in 50 mM HEPES (pH 7.5), 50 mM NaCl, 1 mM TCEP, 0.01% (w/v) BSA, and 0.008% (w/v) Brij-35 either in the presence of DMSO (final 0.2% DMSO) or compound dilution series in DMSO. After 10 minutes incubation at room temperature, a mixture Eu-W1024 Anti-6xHis antibody (6xHis is disclosed as SEQ ID NO: 3) (Perkin Elmer ADO 110) and SureLight Allophycocyanin-Streptavidin (APC-SA, Perkin Elmer CR130-100) were added to a final concentrations of 0.2 nMolar antibody and 50 nMolar APC-SA, respectively. After twenty minutes of equilibration, the plates were read on an Envision instrument and IC50s calculated using a four parameter non-linear curve fit.
Measurement: IC50 = 9.61 nM (pChEMBL 8.020000457763672)
Descriptors: MW 472.55, ALogP 2.36, HBA 8, HBD 1, PSA 93.34, QED 0.49
Compound (SMILES): N#Cc1ccc(-n2nc3c(c2C(=O)NCC(N)=O)C(=O)N[C@]2(CCc4cc(OCC(F)(F)F)ccc42)C3)nc1
InChIKey: QZXGHBSTBVJVHU-XMMPIXPASA-N
Target: Alpha-1,6-mannosyl-glycoprotein 2-beta-N-acetylglucosaminyltransferase (SINGLE PROTEIN), Homo sapiens
UniProt: Q10469
Assay: Inhibitory Activity: Solutions of the compounds of the present invention in DMSO were each aliquoted into 0.2-μL portions in a 384-well polystyrene microplate produced by Corning Incorporated, and 5 μL of an enzyme solution prepared with an assay buffer (100 mmol/L phosphate buffer (pH 7.4) containing 2 mmol/L DTT) and 5 μL of a substrate solution (100 mmol/L phosphate buffer (pH 7.4), 30 μmol/L 2-Oleoylglycerol, 10 μmol/L Oleoyl-CoA) were added thereto, and the resultant was stirred and centrifuged, and incubated in a moist chamber at room temperature for 1 hour. After enzymatic reaction, 50 μL of a quenching solution (containing 0.2 μmol/L Diolein-d5, 0.4% formic acid, and 50% isopropanol) containing Internal Standard (IS) was added to terminate the reaction, and the resultant was sealed in a plate produced by Shimadzu GLC Ltd., and then stirred and centrifuged, and measurement was performed by using an electrospray ionization method with a RapidFire360 and Agilent 6550 Q-TOF mass spectrometer. Diolein as a reaction product (P) of 2-Oleoylglycerol as the substrate and an ammonium adduct ion of the IS were detected, and the peak intensity ratio, P/IS, was calculated from the peak heights to evaluate the inhibitory activity. Inhibitory activities with/without addition of enzyme were defined as Control (+)/Control (−), respectively, and the respective % inhibitions were defined as 0% inhibition and 100% inhibition. The inhibitory activity was calculated from formula below with TIBCO Spotfire (produced by TIBCO Software Inc.):Inhibitory activity (%)=[1−{Sample−Control(−)}/{Control(+)−Control(−)}]*100where Sample indicates a peak intensity ratio: P/IS, when the compound of the present invention was added.
Measurement: IC50 = 11.3 nM (pChEMBL 7.949999809265137)
Descriptors: MW 539.47, ALogP 1.42, HBA 8, HBD 3, PSA 165.02, QED 0.43
Compound (SMILES): CCOC(=O)N(C)C1CN(C(=O)c2nc(N[C@@H](C)c3cncc(F)c3)nc3cc(C)sc23)C1
InChIKey: WVEJSMJZKRZNHF-ZDUSSCGKSA-N
Target: Adenosine receptor A2b (SINGLE PROTEIN), Homo sapiens
UniProt: P29275
Assay: Activity Assay: Compounds (Adenosine Receptor modulators) and activity assay.
Measurement: IC50 = 30 nM (pChEMBL 7.519999980926514)
Descriptors: MW 472.55, ALogP 3.62, HBA 8, HBD 1, PSA 100.55, QED 0.58
Compound (SMILES): CNC(=O)N1CCn2c(C3CC3)nc(-c3c(F)ccc4cc(-c5cn(C)nc5C)ncc34)c2C1
InChIKey: ORUJYOLVIPHMHG-UHFFFAOYSA-N
Target: CREB-binding protein (SINGLE PROTEIN), Homo sapiens
UniProt: Q92793
Assay: CBP AlphaScreen Assay: 5 nM GST-CBP(1081-1197) and 20 nM biotin-H4(1-21) Ac-K5/8/12/16 (AnaSpec. 64989) were incubated with varying concentrations of CBP inhibitors in 15 μL of buffer containing 50 mM HEPES 7.5, 100 nM NaCl, 1 mM TCEP, and 0.003% Tween-20. After 30 minutes incubation at room temperature, 15 μL of detection buffer (BPS Bio. 33006) containing 7 μg/mL of Glutathione AlphaLisa acceptor beads (Perkin Elmer AL109) and 14 μg/mL of Streptavidin donor beads (Perkin Elmer 676002) was then added to the previous mixture. The reaction was incubated for an additional 2 hours at room temperature, and the AlphaScreen signal was quantified using the Envision Multilabel plate reader. As negative control, GST-CBP(1081-1197) was incubated with the non-acetylated biotin-H4(1-21) peptide(AnaSpec. 62555) and in presence of 0.25% of final DMSO concentration.
Measurement: IC50 = 54 nM (pChEMBL 7.269999980926514)
Descriptors: MW 459.53, ALogP 3.98, HBA 6, HBD 1, PSA 80.87, QED 0.5
Compound (SMILES): CCS(=O)(=O)c1ccc(Cl)cc1Nn1cnc2c(Br)c(CN3CCNCC3)c(Br)cc2c1=O
InChIKey: AAVYKKRDHNHICC-UHFFFAOYSA-N
Target: Epithelial discoidin domain-containing receptor 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q08345
Assay: LanthaScreen Assay: DDR1 binding activity was measured using the LanthaScreen (Registered trademark) Eu Kinase Binding Assay (manufactured by Life Technologies Corporation). The test compound and the Alexa Fluor® 647-labeled Kinase Tracer 178 (manufactured by Life Technologies Corporation) were added to a mixture of DDR1 and the LanthaScreen (Registered trademark) Eu-anti-GST antibody. After one-hour reaction at room temperature, the fluorescence resonance energy transfer was measured. The 50% inhibitory concentration (IC50) was calculated from the inhibition rate relative to the test compound-free control.
Measurement: IC50 = 82 nM (pChEMBL 7.090000152587891)
Descriptors: MW 619.77, ALogP 3.65, HBA 8, HBD 2, PSA 96.33, QED 0.44
Compound (SMILES): CCc1ccc(-c2c(-c3ccc(C#N)c(F)c3)cc(NC[C@H]3CCNC3)c(=O)n2C)cc1
InChIKey: NGHZQIQVMRSJOS-SFHVURJKSA-N
Target: Lysine-specific histone demethylase 1A (SINGLE PROTEIN), Homo sapiens
UniProt: O60341
Assay: In Vitro Enzyme Inhibition Assay: The enzymatic assay of LSD1 activity is based on Time Resolved-Fluorescence Resonance Energy Transfer (TR-FRET) detection. The inhibitory properties of compounds to LSD1 were determined in 384-well plate format under the following reaction conditions: 0.1-0.5 nM LSD1, 50 nM H3K4me1-biotin labeled peptide (Anaspec cat #64355), 2 uM FAD in assay buffer of 50 mM HEPES, pH7.3, 10 mM NaCl, 0.005% Brij35, 0.5 mM TCEP, 0.2 mg/ml BSA. Reaction product was determined quantitatively by TR-FRET after the addition of detection reagent Phycolink Streptavidin-allophycocyanin (Prozyme) and Europium-anti-unmodified histone H3 lysine 4 (H3K4) antibody (PerkinElmer) in the presence of LSD1 inhibitor such as 1.8 mM of Tranylcypromine hydrochloride (2-PCPA) in LANCE detection buffer (PerkinElmer) to final concentration of 12.5 nM and 0.25 nM respectively. The assay reaction was performed according to the following procedure: 2 uL of the mixture of 150 nM H3K4me1-biotin labeled peptide with 2 uL of 11-point serial diluted test compound in 3% DMSO were added to each well of plate, followed by the addition of 2 uL of 0.3 nM LSD1 and 6 LM of FAD to initiate the reaction. The reaction mixture was then incubated at room temperature for one hour, and terminated by the addition of 6 uL of 1.8 mM 2-PCPA in LANCE detection buffer containing 25 nM Phycolink Streptavidin-allophycocyanin and 0.5 nM Europium-anti-unmodified H3K4 antibody. Enzymatic reaction is terminated within 15 minutes if 0.5 LSD1 enzyme is used in the plate. Plates were read by EnVision Multilabel Reader in TR-FRET mode (excitation at 320 nm, emission at 615 nm and 665 nm) after 1 hour incubation at room temperature.
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 430.53, ALogP 4.31, HBA 5, HBD 2, PSA 69.85, QED 0.61
Compound (SMILES): Cc1nc(N2CC3C(C2)C3(F)F)ccc1Cn1cc(C(=O)N[C@@H]2CCc3c2ccnc3N)cn1
InChIKey: JECFULPDSRFDSJ-SOAGJPPSSA-N
Target: Plasma kallikrein (SINGLE PROTEIN), Homo sapiens
UniProt: P03952
Assay: Inhibition Assay: Human KLKB1 (1.78 nM or 0.025 U/mL; Enzyme Research Laboratories) was incubated at 24° C. with 0.25 mM fluorogenic substrate H-Pro-Phe-Arg-AMC (11295 from Bachem) and various concentrations of the test compound in assay buffer. Measurements were performed in a kinetic interval every 2nd minute for 16 min using a Spectramax M5 (Molecular Devices) with the following settings of the wavelength excitation of 350 nm and wavelength emission of 450 nm.
Measurement: Ki = 0.4 nM (pChEMBL 9.399999618530273)
Descriptors: MW 465.51, ALogP 2.73, HBA 7, HBD 2, PSA 101.96, QED 0.6
Compound (SMILES): Cc1c(Nc2cc(Oc3ccc(NC(=O)c4cccn(-c5ccc(F)cc5)c4=O)cc3F)c3ccnn3c2)cnn1C
InChIKey: KOPHHWPBBQLROP-UHFFFAOYSA-N
Target: Tyrosine-protein kinase receptor UFO (SINGLE PROTEIN), Homo sapiens
UniProt: P30530
Assay: AXL Kinase Assay: The ability of compounds to inhibit the kinase activity of recombinant human baculovirus-expressed AXL was measured by homogeneous TRF (HTRF) using Cisbio's KinEASE assay system in white 384-well Optiplates. Assay buffer contained 1 mM DTT, 2 mM MnCl2, 2% DMSO, 50 nM supplement enzymatic buffer, and 1× enzymatic buffer. A 2× concentration of tyrosine kinase (TK) substrate-biotin/ATP mixture made in assay buffer was added to plates at 10 μL/well using the Multidrop Combi (Thermo Fisher Scientific, Waltham, Mass.). The final concentrations were 0.3 μM TK substrate-biotin, and 1.3 μM ATP. Compounds (100 nL), diluted in 100% DMSO on the Biomek FX, (Beckman Coulter, Inc., Brea, Calif.), were transferred to the assay plates using the Biomek FX pintool (2.5% final DMSO in assay). A 2× concentration (final=12 ng/mL) of GST-AXL (diluted in assay buffer) was added to plates at 10 uL/well using the Multidrop Combi. Plates were sealed, briefly shaken and incubated at 25° C. for 30 minutes. A 4× stock of Streptavidin-XL665 (final=18.8 nM) and a 1:100 diluted stock of TK antibody-cryptate were made in HTRF detection buffer and mixed together just prior to adding 20 μL/well on the Multidrop Combi. Plates were sealed, briefly shaken and incubated at 25° C. for 1 hour. The fluorescence of the resulting solution was measured using the PerkinElmer EnVision 2102 multi-label plate reader (PerkinElmer, Waltham, Mass.) with an excitation wavelength of 337 nm (laser) and emission wavelengths of 590 and 665 nm. Raw data was expressed as the ratio of 665/590×10,000.
Measurement: IC50 = 55 nM (pChEMBL 7.260000228881836)
Descriptors: MW 567.56, ALogP 5.59, HBA 9, HBD 2, PSA 107.48, QED 0.26
Compound (SMILES): O=C(Nc1ccc(-c2n[nH]c(=O)c3ccccc23)cc1)C1CC(=O)N(c2ccccc2)C1
InChIKey: UEHNHNAPLAIXLI-UHFFFAOYSA-N
Target: Rho-associated protein kinase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q13464
Assay: In Vitro Assay: The effectiveness of compounds of the present invention as ROCK inhibitors can be determined in a 30 μL assay containing 20 mM HEPES, pH 7.5, 20 mM MgCl2, 0.015% Brij-35, 4 mM DTT, 5 μM ATP and 1.5 μM peptide substrate (FITC-AHA-AKRRRLSSLRA-OH). Compounds were dissolved in DMSO so that the final concentration of DMSO was <2%, and the reaction was initiated with Rho kinase variants. After incubation, the reaction was terminated by the addition of EDTA and the phosphorylated and non-phosphorylated peptides separated using a LABCHIP® 3000 Reader (Caliper Life Sciences). Controls consisted of assays that did not contain compound, and backgrounds consisted of assays that contained enzyme and substrate but had EDTA from the beginning of the reaction to inhibit kinase activity. Compounds were tested in dose-response format, and the inhibition of kinase activity was calculated at each concentration of compound. The inhibition data were fit using a curve-fitting program to determine the IC50; i.e., the concentration of compound required to inhibit 50% of kinase activity.
Measurement: IC50 = 25500 nM (pChEMBL 4.590000152587891)
Descriptors: MW 424.46, ALogP 3.58, HBA 4, HBD 2, PSA 95.16, QED 0.52
Compound (SMILES): CCC(C)[C@@H]1CN(c2cccc(S(C)(=O)=O)c2)CCN1Cc1ccccc1
InChIKey: RCUJWQHUVDWKFQ-YSYXNDDBSA-N
Target: Oxysterols receptor LXR-alpha (SINGLE PROTEIN), Homo sapiens
UniProt: Q13133
Assay: Human LXRalpha and LXRbeta Coactivator Recruitment FRET Assay: This assay is based on the ability of the LXR-LBDs (LXRα and LXRβ) to recruit and interact with a co-activator peptide. This assay was run in agonist mode, to characterize compounds of the invention that exhibit LXR agonist activity, and in antagonist mode to characterize compounds of the invention having LXR antagonist activity and which cause a concentration-dependent release of the co-activator peptide in the presence of the full LXR agonist T0901317. The interaction between glutathione S-transferase (GST)-tagged, recombinant human LXRα-LBD or LXRβ-LBD and fluorescein-conjugated co-activator peptide SRC2-3 (Cat# PV4588, Life Technologies, Grand Island, N.Y.) was measured via time resolved-fluorescence resonance energy transfer (TR-FRET). The assay was performed in 96-well half-area black Opti-plates (Cat# 3686, Corning, Lowell, Mass.) in 20 mM TRIS-HCl buffer, pH 8, containing 150 mM NaCl and 5 mM DDT in a total volume of 80 μL. Test compounds were dissolved in DMSO, and semi-log (3.162×) serial dilutions were also prepared in DMSO, as 40× solutions.
Measurement: EC50 = 586 nM (pChEMBL 6.230000019073486)
Descriptors: MW 386.56, ALogP 3.83, HBA 4, HBD 0, PSA 40.62, QED 0.76
Compound (SMILES): Cn1cnc2ncn(Cc3nc([C@@H]4[C@H]5C=C(c6ccc(F)cc6)C[C@H]54)no3)c(=O)c21
InChIKey: DHGRAFMYKRWRSJ-ZMSDIMECSA-N
Target: Transient receptor potential cation channel subfamily A member 1 (SINGLE PROTEIN), Homo sapiens
UniProt: O75762
Assay: FLIPR Assay (15 minutes): The IC50 (effective concentration) of compounds on the human TRPA1 channel was determined using a FLIPR Tetra instrument. CHO cells expressing TRPA1 were plated into 384-well plates, incubated overnight at 37° C., and loaded with BD calcium indicator dye for 1 hr at 37° C. followed by 15 minutes at room temperature. The assay buffer was Hank's Balanced Salt Solution (HBSS) containing 20 mM HEPES (pH readjusted to 7.4) along with 0.02% BSA.Following dye load and plate cool down, compounds were added to the cells using FLIPR Tetra. Plates were then incubated with compounds for 10 minutes or 90 minutes at room temperature prior to adding agonist. Following this incubation, about an EC50 concentration of cinnamaldehyde (75) was added to active the channels and block of cinnamaldehyde induced calcium influx was measured.The IC50 results were fit with a standard Hill function, keeping the Hill coefficient (n) fixed to 1.5. Fixing the Hill coefficient will generally reduce variability of the IC50 determination. The IC50 results were individually examined to make sure the MIN and MAX points were set correctly prior to validation of the results.
Measurement: IC50 = 10.9 nM (pChEMBL 7.960000038146973)
Descriptors: MW 404.41, ALogP 2.52, HBA 8, HBD 0, PSA 91.63, QED 0.52
Compound (SMILES): CCc1cc(CN2CC3(C2)CN(S(C)(=O)=O)C3)ccc1-c1ccc(C(O)(C(F)(F)F)C(F)(F)F)cc1Cl
InChIKey: WCWUUFCECJJLKD-UHFFFAOYSA-N
Target: Nuclear receptor ROR-gamma (SINGLE PROTEIN), Homo sapiens
UniProt: P51449
Assay: Gal4 Ligand Binding Assay: Compounds of the present invention were tested in a human RORγ ligand binding assay using a commercially available cell based ligand binding reporter assay in 96-well format (Cat #1304001, INDIGO Biosciences, State College, Pa.). The N-terminal DNA binding domains (DBD) of the native RORγ and RORγt receptors have been substituted with that of the yeast GAL4-DBD and stably transfected in HEK293T cells that also stably express luciferase under the regulation by upstream activation sequence of yeast Gal4. These cells constitutively express high level RORγ activity due to binding of endogenous co-factors. Both agonist and inverse agonist activity can be detected. The assay was performed according to kit manufacturer's instructions as follows. 10 mM compound stocks were diluted serially 1:3 with DMSO and further diluted with provided media to generate 10 titration points from 60 μM to 3 nM. These treatment conditions were added to the plates as 2× media in 100 μL volume. Each plate includes a positive control with 10 titration points as well as 6 negative control wells with vehicle only, with final DMSO concentration of 0.2%. RORγ reporter cells were rapidly thawed and added to the plates in 100 μL volume. The plates were incubated for 24 h in a 37° C. humidified 5% CO2 incubator. Media was removed before the addition of room temperature luciferous detection substrate. After 5 minute incubation, relative light units (RLUs) were quantified using a plate reading luminometer. Data was normalized to positive control wells with only 0.2%0DMSO. Before establishing internal controls, ursolic acid was used as control.
Measurement: IC50 < 50 nM (pChEMBL 7.301000118255615)
Descriptors: MW 570.98, ALogP 4.96, HBA 4, HBD 1, PSA 60.85, QED 0.5
Compound (SMILES): Cc1cncc(NC(=O)c2cccc3c2NC(C)CC(=O)N3)c1
InChIKey: NGQZYUFXSRCSFJ-UHFFFAOYSA-N
Target: CREB-binding protein (SINGLE PROTEIN), Homo sapiens
UniProt: Q92793
Assay: CBP TR-FRET Binding Assay: His/Flag epitope tagged CBP was cloned, expressed, and purified to homogeneity. CBP binding and inhibition was assessed by monitoring the engagement of a biotinylated small molecule compound with the target using the TR-FRET assay technology (Perkin-Elmer). Specifically, in a 384 well ProxiPlate CBP (4 nM final) was combined with biotin-ligand (60 nM final) in 50 mM HEPES (pH 7.5), 50 mM NaCl, 1 mM TCEP, 0.01% (w/v) BSA, and 0.008% (w/v) Brij-35 either in the presence of DMSO (final 0.2% DMSO) or compound dilution series in DMSO. After 10 minutes incubation at room temperature, a mixture Eu-W1024 Anti-6×His antibody (Perkin Elmer ADO 110) and SureLight Allophycocyanin-Streptavidin (APC-SA, Perkin Elmer CR130-100) were added to a final concentrations of 0.2 nMolar antibody and 50 nMolar APC-SA, respectively. After twenty minutes of equilibration, the plates were read on an Envision instrument and IC50s calculated using a four parameter non-linear curve fit.
Measurement: IC50 = 826 nM (pChEMBL 6.079999923706055)
Descriptors: MW 310.36, ALogP 2.78, HBA 4, HBD 3, PSA 83.12, QED 0.8
Compound (SMILES): Cc1ccc(S(=O)(=O)N(C)CCC#N)cc1-c1cnc2c(N)ncnn12
InChIKey: ALFZRHNURTYRHC-UHFFFAOYSA-N
Target: Phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit gamma isoform (SINGLE PROTEIN), Homo sapiens
UniProt: P48736
Assay: Scintillation Proximity Assay: PI3K-γ:[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).
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 371.43, ALogP 1.22, HBA 8, HBD 1, PSA 130.27, QED 0.71
Compound (SMILES): C[C@H]1CN(c2ccncc2NC(=O)c2ccc3oc(-c4c(F)cccc4F)cc3n2)C[C@@H](N)[C@@H]1O
InChIKey: MHWGZEKDCLWVNJ-CNAJZPKWSA-N
Target: Serine/threonine-protein kinase pim-1 (SINGLE PROTEIN), Homo sapiens
UniProt: P11309
Assay: Enzyme Assay: Pim-1 and Pim-3 kinase assays 20 μL reactions were run in white 384 well polystyrene plates dotted with 0.8 μL compound/DMSO in the assay buffer (50 mM Tris, pH 7.5, 0.01% Tween-20, 5 mM MgCl2, 0.01% BSA, 5 mM DTT), containing 0.05 μM Biotin-labeled BAD peptide substrate (AnaSpec 62269), 1 mM ATP, and 2.5 μM (Pim-1, Invitrogen PV3503) or 1.25 μM (Pim-3, Millipore 14-738) enzyme for 1 h at 25° C. Reactions were stopped by addition of 10 μL STOP Buffer (150 mM Tris, pH=7.5, 150 mM NaCl, 75 mM EDTA, 0.01% Tween-20, 0.3% BSA) supplemented with Phospho-Bad (Ser112) Antibody (Cell Signaling 9291) diluted 666-fold, and Streptavidin donor beads (PerkinElmer 6760002) along with Protein-A acceptor beads (PerkinElmer 6760137) at 15 μg/mL each. Supplementation of the STOP buffer with beads and stopping the reactions were done under reduced light. Prior to the stopping reactions STOP buffer with beads was preincubated for 1 h in the dark at room temperature. After stopping the reactions, plates were incubated for 1 h in the dark at room temperature before reading on a PHERAstar FS plate reader (BMG Labtech) under reduced light.
Measurement: IC50 < 10 nM (pChEMBL 8)
Descriptors: MW 479.49, ALogP 3.56, HBA 7, HBD 3, PSA 117.51, QED 0.41
Compound (SMILES): CCS(=O)(=O)c1ccc([C@@H](CO)NC(=O)c2ccc3c(c2)nc(CC2CCC(C(F)(F)F)CC2)n3C2CC2)nc1
InChIKey: WGVDGZZVENHSQZ-VWLBVBLESA-N
Target: Nuclear receptor ROR-gamma (SINGLE PROTEIN), Homo sapiens
UniProt: P51449
Assay: Radio-Ligand Binding Assay: Compounds described herein were tested for ability to bind to RORγ in a cell-free competition assay with commercially available radio-ligand (RL), 25-hydroxy [26,27-3H]-cholesterol (PerkinElmer, Cat. # NET674250UC), for a ligand binding site on a recombinant RORγ Ligand Binding Domain (LBD) protein expressed as a 6×His-Glutathione-S-Transferase (GST) fusion. The assay was performed in 96-well SPA plates (PerkinElmer, Cat. #1450-401) in 50 mM HEPES buffer, pH 7.4, containing 150 mM NaCl, 5 mM MgCl2, 10% (v/v) glycerol, 2 mM CHAPS, 0.5 mM β-octylglucopyranoside and 5 mM DTT. Tested compounds were dissolved in DMSO, and semi-log (3.162×) serial dilutions of the compounds were prepared in the same solvent. Two μL of the DMSO solutions were mixed with 28 μL of 8.6 nM 25-hydroxy [26,27-3H]-cholesterol and 50 μL of 24 nM RORγ LBD. The plate was shaken at 700 rpm for 20 min and incubated for 10 min at rt, after which 40 μL of poly-Lys YSi SPA beads (PerkinElmer, Cat. # RPNQ0010) were added to achieve 50 μg of the beads per well. The plate was incubated on an orbital shaker for 20 min and then for 10 min without agitation at rt. SPA signal for tritium beta radiation was registered on PerkinElmer Microbeta plate reader. Percent inhibition values were calculated based on the high signal obtained with DMSO control and the low signal observed with 10 μM standard RORγ inverse agonist T0901317 (SigmaAldrich, Cat. # T2320).
Measurement: Ki > 1000 nM (pChEMBL 6)
Descriptors: MW 578.66, ALogP 4.93, HBA 7, HBD 2, PSA 114.18, QED 0.37
Compound (SMILES): CC(C)Cc1nc(-c2cnn3c(C4CCNCC4)cc(=O)[nH]c23)no1
InChIKey: OYQJNGMXWYAENW-UHFFFAOYSA-N
Target: Tissue-type plasminogen activator (SINGLE PROTEIN), Homo sapiens
UniProt: P00750
Assay: Plasma-based Clot Lysis Assay: The clot-lysis test system configures the kinetics of clot formation and degradation in vitro and allows quantifying modulation of the process by selected test compounds.The test compounds were dissolved in 1% acetic acid and further complemented with an equal volume of DMSO. The resulting stock solutions were serially diluted in 0.5% acetic acid/50% DMSO. 1 μL aliquots of these solutions were placed into 384 well microplates (Greiner, black, transparent bottom), followed by 30 μL of diluted human citrated plasma (platelet-poor, final concentration: 5%; supplemented with fibrinogen, final concentration: 3 μM; dilution buffer: 20 mM HEPES, 150 mM NaCl, 0.01% Brij (pH 7)). The reactions were started by addition of 20 μL of CaCl2 (final concentration: 10 mM), and tPA (tissue plasminogen activator, final concentration: 0.2 nM) in dilution buffer, followed by an additional volume of 20 μL dilution buffer for improved mixing. The reactions were incubated at 37° C. Clot formation and degradation was monitored spectrophotometrically by kinetic optical density measurements at 405 nm. IC50 values were determined by comparing the resulting time courses with the time course of a blank control reaction.
Measurement: IC50 = 32 nM (pChEMBL 7.5)
Descriptors: MW 342.4, ALogP 1.74, HBA 7, HBD 2, PSA 101.11, QED 0.75
Compound (SMILES): Cn1cc(C2CCN(C(=O)[C@H]3CCCS3)CC2)c2c(C(F)(F)F)c(NC(=O)c3cccc(C#N)c3)cnc21
InChIKey: JUBPLDQCQLLZST-OAQYLSRUSA-N
Target: Nuclear receptor ROR-gamma (SINGLE PROTEIN), Homo sapiens
UniProt: P51449
Assay: Assay of Co-Activator Recruitment by TR-FRET: Specifically, in one embodiment the aforementioned assay was performed as outlined below. The assay was carried out in black polystyrene, 384-well plates in a total assay volume of 50.5 μL. The assay buffer contained 50 mM TRIS¿HCL pH 7.5, 1 mM NaCl, 2 mM MgCl2, 0.5 mg/mL bovine serum albumin, and 5 mM dithiothreitol. The final concentration of reagents was 6.3 nM RORC2 LBD, 200 nM SRC1-2, 50 nM streptavidin APC, 1 nM Europium-labeled anti-His antibody, and varying concentrations of compounds such that final concentration of DMSO is 1% (v/v). The assay steps were: (1) dispensing 500 μL compound at 100× final concentration in DMSO (test wells) or DMSO only (control wells for no inhibition); and (2) dispensing 50 μL mixture of the other assay components including receptor (test wells) or excluding receptor (control wells for maximal inhibition). Assay mixtures were incubated are room temperature for 3 hr and read in EnVision 2100 Multilabel Reader (PerkinElmer Life Sciences) at Excitation Filter 320, Emission Europium Filter 615, Emission APC Filter 665, Dichroic Mirror D400/D630. TR-FRET signal was determined by calculating the ratio of 665 nm by 615 nm and IC50 values of compounds of the invention (Table 1) were determined by the non-linear regression analysis of dose response curves.
Measurement: IC50 = 7.1 nM (pChEMBL 8.149999618530273)
Descriptors: MW 541.6, ALogP 5.32, HBA 6, HBD 1, PSA 91.02, QED 0.48
Compound (SMILES): CC(C)C[C@@H](N)[C@](O)(Cc1cc(O[C@H]2CC[C@H](C)CC2)ccn1)C(=O)O
InChIKey: GGIXEKOHFTUBMY-QSFMABPGSA-N
Target: Leucyl-cystinyl aminopeptidase (SINGLE PROTEIN), Rattus norvegicus
UniProt: P97629
Assay: Inhibition of IRAP Assay: Rat epididymal fat pads were homogenized and subjected to ultracentrifugation at 100,000×g for 30 minutes to obtain microsomes containing IRAP. The microsomes (with a total protein content of 55 g/well) were mixed with a solvent (dimethyl sulfoxide; hereinafter, abbreviated as DMSO (final concentration: 0.1%)) or with each test compound (common ratio: 3; maximum concentration: 10 μM). AVP was then added to the solution to a final concentration of 25 μM, and the resulting solution was allowed to react for one hour at 37° C. An aqueous trifluoroacetic acid (hereinafter, abbreviated as TFA) solution was then added to the solution (final concentration: 1%) to stop the enzymatic reaction. Residual AVP was then determined by mass spectrometry (MALDI-MS). Based on the results, IC50 values (nM), i.e. concentrations required for 50% inhibition of decrease in AVP level in the solvent control group, of the individual test compounds were calculated by the logistic regression to evaluate inhibition of IRAP activity. As comparative examples, similar tests were performed with the compounds of the Reference Examples 1 and 2 described below: (2S,3S)-3-amino-2-hydroxy-5-methyl-2-({4-[(trans-4-methylcyclohexyl)oxy]pyridin-2-yl}methyl)hexanoic acid dihydrochloride; and (2S,3R)-3-amino-2-hydroxy-5-methyl-2-({4-[(trans-4-methylcyclohexyl)oxy]pyridin-2-yl}methyl)hexanoic acid hydrochloride, respectively.
Measurement: IC50 = 34 nM (pChEMBL 7.46999979019165)
Descriptors: MW 364.49, ALogP 2.77, HBA 5, HBD 3, PSA 105.67, QED 0.65
Compound (SMILES): CCC(C)C1CN(c2cnc3ccccc3n2)CCN1/C(=N\C#N)Nc1ccccc1C
InChIKey: RXAJCLSFNZLFHO-UHFFFAOYSA-N
Target: Protein M2-1 (SINGLE PROTEIN), Human respiratory syncytial virus A (strain A2)
UniProt: P04545
Assay: Antiviral Assay: A cytopathetic effect (CPE) protection assay was performed to determine the ability of a compound to protect the cells from viral infection and thus the CPE induced by viral infection. 96-Well plates were first seeded with 3×10 HEp-2 cells per well in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum (FBS). One day after the cells were seeded, they were preincubated with serial dilutions of compounds prepared in 100 μL assay medium (DMEM mixed with F12 medium at a 1:1 ratio, supplemented with 2% FBS and 1 mM sodium pyruvate) for 1 hour at 37° C. 100 μL of assay medium containing 0.2 multiplicity of infection (MOI) of RSV was then added to each well of cells. In addition to wells containing infected cells incubated with compounds, each plate also contained replicates of two kinds of controls: (1) Virus control contained cells infected with 0.2 MOI of RSV in assay medium, (2) Uninfected cell control contained cells incubated with assay medium only. After 4 days of incubation at 37° C., the viability of cells was assessed using MTT (Thiazolyl blue tetrazolium bromide, Sigma). A stock solution of MTT, at a concentration of 4 mg/mL in phosphate-buffered saline, was added to all wells at 25 μL per well. Plates were further incubated for 4 hours, and each well was then treated with 50 μL of a solution containing 20% sodium dodecyl sulfate (SDS) and 0.02 N HCl. After an overnight incubation, the plates were measured on a BioTek® microtiter plate reader at wavelengths of 570 nm and 650 nm. The MTT detection is based on the fact that viable (uninfected) cells can reduce the tetrazolium salts into colored formazan products, which can then be quantitated by spectrometry. Based on the spectrometric absorbance of each sample, the percent of protection from CPE, which is an indicator of protection from viral infection, can be calculated for each compound and the 50% effective concentrations (EC50) can be calculated using a nonlinear regression curve fitting equation provided by the GraphPad Prism® 4 software. Using the above-described assay, compounds of the present disclosure showed obvious inhibitory activities against RSV replication.
Measurement: EC50 > 1380 nM (pChEMBL 5.860000133514404)
Descriptors: MW 427.56, ALogP 4.42, HBA 5, HBD 1, PSA 80.44, QED 0.38
Compound (SMILES): C[C@H](C(=O)N[C@H](C)c1cccc(N2CCN(CCO)CC2)n1)N1Cc2ccc(-c3nc(NC4CCOCC4)ncc3Cl)cc2C1=O
InChIKey: OKNACXYGJUSOFT-FGZHOGPDSA-N
Target: Mitogen-activated protein kinase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P28482
Assay: ERK2 In Vitro Inhibition Assay: Activity of ERK2 enzyme (Life Technologies) was determined using a time-resolved fluorescence format measuring the phosphorylation of a truncated version of Activating transcription factor 2 labelled with green fluorescent protein (ATF2-GFP) (Life Technologies). Assay reactions containing 50 mM Tris pH 7.5, 10 mM MgCl2, 1 mM EGTA, 0.01% Triton X-100, 1 mM DTT, 2.5% DMSO, 0.4 mM ATF2-GFP, 20 μM ATP and 0.25 nM ERK2 were set up in the presence of compound and allowed to proceed for 30 min at room temperature. Reactions were then stopped using TR-FRET dilution buffer (Life Technologies), 25 mM EDTA and 2 nM Tb-Anti-pATF2 (Thr71) (Life Technologies). After a further incubation period of at least 30 minutes, fluorescence was read on a Pherastar reader (Lanthascreen optic module; excitation 340 nm, emission 520 nm (channel A), 495 nm (channel B)). The ratio between A and B counts was used to calculate signal. IC50 values were calculated using a sigmoidal dose response equation (Prism GraphPad software, La Jolla, Calif., USA).
Measurement: IC50 = 3 nM (pChEMBL 8.520000457763672)
Descriptors: MW 649.2, ALogP 3.12, HBA 10, HBD 3, PSA 136.05, QED 0.3
Compound (SMILES): Cc1cc(-c2cc(-c3cnn(C)c3)cn3ncc(C#N)c23)cnc1N1CCN(C(=O)N2CCCC2)CC1
InChIKey: CUJDEBGUBOJLEH-UHFFFAOYSA-N
Target: Proto-oncogene tyrosine-protein kinase receptor Ret (SINGLE PROTEIN), Homo sapiens
UniProt: P07949
Assay: RET Enzyme Assay: Compounds of General Formula I were screened for their ability to inhibit wild type and V804M mutant RET kinase using CisBio's HTRF® KinEASE -TK assay technology. Briefly, N-terminal GST tagged recombinant human RET cytoplasmic domain (aa 658-end) from Eurofins (0.25 nM RET; Cat. No. 14-570M) or N-terminal GST tagged recombinant human V804M mutant RET cytoplasmic domain (aa 658-end) from Millipore (0.25 nM enzyme; Cat. No. 14-760) was incubated with 250 nM TK-substrate biotin (CisBio, part of Cat. No. 62TK0PEC) and 1 mM ATP along with test compound in a buffer consisting of 25 mM HEPES pH 7.4, 10 mM MgCl2, 0.01% Triton X-100, and 2% DMSO in a volume of 8 μL. Compounds were typically prepared in a threefold serial dilution in DMSO and added to the assay to give the appropriate final concentration. After a 30-minute incubation at 22° C., the reaction was quenched by adding 8 μL of quench solution containing 31.25 nM Sa-XL665 and 1×TK-ab-Cryptate in HTRF detection buffer (all from CisBio, part of Cat. No. 62TK0PEC). After a 1 hour incubation at 22° C., the extent of reaction was determined using a PerkinElmer EnVision multimode plate reader via HTRF dual wavelength detection, and the percent of control (POC) was calculated using a ratiometric emission factor. 100 POC was determined using no test compounds and 0 POC was determined using pre-quenched control reactions. The POC values were fit to a 4 parameter logistic curve, and the IC50 is defined as the concentration of inhibitor at which the POC equals 50 for the fitted curve.
Measurement: IC50 = 176 nM (pChEMBL 6.75)
Descriptors: MW 495.59, ALogP 3.31, HBA 8, HBD 0, PSA 98.59, QED 0.43
Compound (SMILES): CC(C)c1ccccc1C1CCCCN1c1ccc(-c2ccc(C(=O)NS(=O)(=O)c3ccc(NCC4CCOCC4)c([N+](=O)[O-])c3)c(Oc3cnc4[nH]ccc4c3)c2)cc1
InChIKey: QLOLQBCTRGJVMX-UHFFFAOYSA-N
Target: Apoptosis regulator Bcl-2 (SINGLE PROTEIN), Homo sapiens
UniProt: P10415
Assay: Bcl-2/Bcl-X Fluorescence Polarization Assay: Compounds disclosed herein were tested for blocking of Bcl-2/Bcl-xl protein with its ligand in an assay based on fluorescence polarization methodology. Recombinant human 2.7 nM Bcl-2/1.3 nM Bcl-xl protein was pre-incubated with a serial dilution of compounds disclosed herein (maximum concentration is 1 μM for Bcl-2 assay, and 10 μM or 1 μM for Bcl-xl assay, 3-fold serially diluted, 10 points) at room temperature for 0.5 hour in an assay buffer containing 20 ml potassium phosphate buffer, pH 7.5, 50 mM NaCl, 1 mM EDTA, 0.05% Tween-20, 0.01% BSA. Then the FITC labeled Bak peptide Ac-GQVGRQLAIIGDK (FITC)INR-amide (1 nM for Bcl-2, 0.82 nM for Bcl-xl) was added to plate and further incubated at room temperature for 0.5 h. The FP signals (485 nm-520 nm-520 nm) were read on BMG PHERAstar FS or BMG PHERAstar FSX instalment. The inhibition percentage of Bcl-2/Bcl-xl interaction with its ligand in presence of increasing concentrations of compounds was calculated based on the FP signals. The IC50 for each compound was derived from fitting the data to the four-parameter logistic equation by Graphpad Prism software.
Measurement: IC50 = 27 nM (pChEMBL 7.570000171661377)
Descriptors: MW 828.99, ALogP 9.74, HBA 10, HBD 3, PSA 168.79, QED 0.08
Compound (SMILES): CCn1c(C(N)=O)nn(-c2cc(O[C@@H](C)C(F)(F)F)c(C(=O)Nc3c(F)cccc3Cl)cc2F)c1=O
InChIKey: IPTGCOJTXSXNDO-VIFPVBQESA-N
Target: Dihydroorotate dehydrogenase (quinone), mitochondrial (SINGLE PROTEIN), Homo sapiens
UniProt: Q02127
Assay: Enzymatic Assay: Assay 1: The enzymatic assay couples DHODH activity with bleaching of the dye 2,6-dichlorophenolindophenol (DCIP) (Knecht and Loffler, 1998; Miller et al., 1968). The assay was conducted in buffer containing 50 mM Tris, 0.1% Triton X-100, 150 mM potassium chloride, 2 nM DHODH, 1 mM dihydroorotate, 0.1 mM decylubiquinone, 0.06 mM DCIP, and 2% DMSO at pH 8.0 at 32 degree Celsius. The reaction was initiated by addition of substrates. Enzyme activity was monitored kinetically by the reduction in DCIP absorbance at 600 nm. Purified recombinant human DHODH enzyme was purchased from Novus (cat. no. NBP1-98916). Other chemicals were purchased from Sigma-Aldrich. Absorbance measurements were obtained using a BMG clarion star plate-reading spectrophotometer.
Measurement: IC50 = 3 nM (pChEMBL 8.520000457763672)
Descriptors: MW 533.84, ALogP 3.67, HBA 7, HBD 2, PSA 121.24, QED 0.45
Compound (SMILES): NC(=O)CC1(NC(=O)c2ccc(N3CC(F)(F)C3)c(OCC3CC3)n2)CCOC1
InChIKey: VKZXWJSWOPPLFH-UHFFFAOYSA-N
Target: Cannabinoid receptor 2 (SINGLE PROTEIN), Homo sapiens
UniProt: P34972
Assay: cAMP Assay: CHO cells expressing human CB1 or CB2 receptors are seeded 17-24 hours prior to the experiment 50.000 cells per well in a black 96 well plate with flat clear bottom (Corning Costar #3904) in DMEM (Invitrogen No. 31331), 1xHT supplement, with 10% fetal calf serum and incubated at 5% CO2 and 37° C. in a humidified incubator. The growth medium was exchanged with Krebs Ringer Bicarbonate buffer with 1 mM IBMX and incubated at 30° C. for 30 min. Compounds were added to a final assay volume of 100 ul and incubated for 30 min at 30° C. Using the cAMP-Nano-TRF detection kit the assay (Roche Diagnostics) was stopped by the addition of 50 ul lysis reagent (Tris, NaCl, 1.5% Triton X100, 2.5% NP40, 10% NaN3) and 50 ul detection solutions (20 uM mAb Alexa700-cAMP 1:1, and 48 uM Ruthenium-2-AHA-cAMP) and shaken for 2 h at room temperature. The time-resolved energy transfer is measured by a TRF reader (Evotec Technologies GmbH), equipped with a ND:YAG laser as excitation source. The plate is measured twice with the excitation at 355 nm and at the emission with a delay of 100 ns and a gate of 100 ns, total exposure time 10 s at 730 (bandwidth 30 nm) or 645 nm (bandwidth 75 nm), respectively. The FRET signal is calculated as follows: FRET=T730-Alexa730-P(T645-B645) with P=Ru730-B730/Ru645-B645, where T730 is the test well measured at 730 nM, T645 is the test well measured at 645 nm, B730 and B645 are the buffer controls at 730 nm and 645 nm, respectively. cAMP content is determined from the function of a standard curve spanning from 10 uM to 0.13 nM cAMP. EC50 values were determined using Activity Base analysis (ID Business Solution, Limited). The EC50 values for a wide range of cannabinoid agonists generated from this assay were in agreement with the values published in the scientific literature. The compounds of the invention are CB2 receptor agonists with EC50 below 1 uM and selectivity versus CB1 in the corresponding assay of at least 10 fold.
Measurement: EC50 = 1.5 nM (pChEMBL 8.819999694824219)
Descriptors: MW 410.42, ALogP 1.09, HBA 6, HBD 2, PSA 106.78, QED 0.66
Compound (SMILES): COc1cccc(-c2ccncc2N(C)C(=O)c2cc(C(F)(F)F)cc(C(F)(F)F)c2)c1F
InChIKey: JAVHQCSOFPTTJZ-UHFFFAOYSA-N
Target: G-protein coupled bile acid receptor 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q8TDU6
Assay: cAMP Assay: CHO-dhfr(minus) cells expressing human GPBAR1 receptors are seeded 17-24 hours prior to the experiment 50.000 cells per well in a black 96 well plate with flat clear bottom (Corning Costar #3904) in DMEM (Invitrogen No. 31331), lx HT supplement, with 10% fetal calf serum and incubated at 5% CO2 and 37° C. in a humidified incubator. The growth medium was exchanged with Krebs Ringer Bicarbonate buffer with 1 mM IBMX and incubated at 30° C. for 30 min Compounds were added to a final assay volume of 100 μl and incubated for 30 min at 30° C. The assay was stopped by the addition of 50 μl lysis reagent (Tris, NaCl, 1.5% Triton X100, 2.5% NP40, 10% NaN3) and 50 detection solutions (20 μM mAb Alexa700-cAMP 1:1, and 48 μM Ruthenium-2-AHA-cAMP) and shaked for 2 h at room temperature. The time-resolved energy transfer is measured by a TRF reader (Evotec Technologies GmbH, Hamburg Germany), equipped with a ND:YAG laser as excitation source. The plate is measured twice with the excitation at 355 nm and at the emission with a delay of 100 ns and a gate of 100 ns, total exposure time 10 s at 730 (bandwidth 30 nm) or 645 nm (bandwidth 75 nm), respectively. The measured signal at 730 nm has to be corrected for the ruthenium background, the direct excitation of Alexa and the buffer control. The FRET signal is calculated as follows: FRET=T730−Alexa730−P(T645−B645) with P=Ru730−B730/Ru645−B645, where T730 is the test well measured at 730 nM, T645 is the test well measured at 645 nm, B730 and B645 are the buffer controls at 730 nm and 645 nm, respectively. cAMP content is determined from the function of a standard curve spanning from 10 μM to 0.13 nM cAMP.
Measurement: EC50 = 337 nM (pChEMBL 6.46999979019165)
Descriptors: MW 472.36, ALogP 6.21, HBA 3, HBD 0, PSA 42.43, QED 0.42
Compound (SMILES): Cc1cc(-c2nnc(N)nc2-c2ccccc2)cc(C)[n+]1[O-]
InChIKey: NKQOTFYKXPHZJD-UHFFFAOYSA-N
Target: Adenosine receptor A2a (SINGLE PROTEIN), Homo sapiens
UniProt: P29274
Assay: Binding Assay: All test compounds were prepared as a stock solution of 10 mM in 100% DMSO.Inhibition binding assays were performed using 2.5 μg of membranes prepared from HEK293 cells transiently transfected with human adenosine A2a receptor or 10 μg of membranes prepared from CHO cells stably transfected with human adenosine A1 receptor. Membranes were incubated in 50 mM Tris-HCl (HEK293-hA2a; pH 7.4) or 20 mM HEPES, 100 mM NaCl, 10 mM MgCl2 (CHO-hA1; pH 7.4) in the presence of varying concentrations of test compound and 1 nM [3H]ZM241385 (HEK293-hA2a) or [3H]DPCPX (CHO-hA1) at 25° C. for 1 h. The assay was then terminated by rapid filtration onto GF/B grade Unifilter plates using a TomTec cell harvester, followed by 5×0.5 ml washes with double distilled H2O. Nonspecific binding was defined in the presence of 1 μM CGS15943 (HEK293-hA2a) or 1 μM DPCPX (CHO-hA1). Bound radioactivity was determined by liquid scintillation counting (Trilux Microbeta® Counter) and inhibition curves were analysed using a four-parameter logistic equation.
Measurement: Ki = 30.9 nM (pChEMBL 7.510000228881836)
Descriptors: MW 293.33, ALogP 2.04, HBA 5, HBD 1, PSA 91.63, QED 0.58
Compound (SMILES): Cc1cnc(C[C@](O)(C(=O)O)[C@@H](N)CC(C)C)cc1C1CCCC1
InChIKey: QLOJGNSFHKYJON-PKOBYXMFSA-N
Target: Leucyl-cystinyl aminopeptidase (SINGLE PROTEIN), Rattus norvegicus
UniProt: P97629
Assay: Inhibition Assay: The inventors have assumed that inhibition of nocturnal activity of placental leucine aminopeptidase (P-LAP), i.e. aminopeptidase that cleaves AVP, would maintain and/or increase an endogenous AVP level to enhance the antidiuretic effect, which would contribute to a decreased number of nocturnal voids, and have extensively studied compounds which inhibit P-LAP.
Measurement: IC50 = 73 nM (pChEMBL 7.139999866485596)
Descriptors: MW 334.46, ALogP 2.78, HBA 4, HBD 3, PSA 96.44, QED 0.71
Compound (SMILES): N#Cc1ccc(CNc2ncc(C(=O)c3ccc(F)c(C(F)(F)F)c3)cn2)cc1
InChIKey: FDGVFOFDAOWJTD-UHFFFAOYSA-N
Target: Pantetheinase (SINGLE PROTEIN), Homo sapiens
UniProt: O95497
Assay: Human Vanin-1 Enzyme Assay 1: The vanin-1 protein was prepared in-house from a construct expressing the extracellular domain of human vanin-1 (GenBank ID NM_004666) preceded N-terminally by the honey bee melittin signal peptide, a GSG linker sequence, a His6X tag and a FLAG tag. The secreted, soluble enzyme was purified from the conditioned medium from a CHO cell line stably expressing the resulting protein. Enzyme purification was performed through sequential Ni NTA and size-exclusion chromatography steps.The test inhibitors were solubilized in DMSO to a stock concentration of 30 mM. On the day of the assay, dose response plates were prepared by diluting the inhibitors in DMSO at compound concentration 200-fold the final in-assay concentration. Intermediate concentrations were prepared by diluting in DMSO in a four-fold series for a total of 11 data points.To prepare a working solution of human vanin-1, the enzyme was diluted to 33.3 pM in the assay buffer consisting of 50 mM Tris-HCl pH=8.0, 50 mM KCl, 0.005% Brij-35 and 1.6 mM cysteamine. To begin the assay 100 nL was transferred from the compound plate to the assay plate. Next, 15 μL of the vanin-1 working solution were transferred to the assay plate. The inhibitor and enzyme were incubated at room temperature for 30 minutes. The enzyme reaction was then initiated by the addition of 5 μL of 200 μM pantetheine 7-amino-4-trifluoromethylcoumarin prepared in assay buffer. The final concentrations in the assay were 25 pM human vanin-1 and 50 uM substrate. The final concentration of DMSO was 0.5%. The assay plates were incubated for 60 minutes and before they were read on a Perkin Elmer EnVision Model 2103 using a 405 nm excitation wavelength and a 510 nm emission wavelength for detection.
Measurement: IC50 = 503 nM (pChEMBL 6.300000190734863)
Descriptors: MW 400.34, ALogP 4.35, HBA 5, HBD 1, PSA 78.67, QED 0.51
Compound (SMILES): COCC(COC)n1c(NS(=O)(=O)[C@@H](C)[C@H](C)c2ccc(Cl)cn2)nnc1[C@H]1CC[C@@H](C)O1
InChIKey: DIWOWOARXDSCSY-MMMWYMCRSA-N
Target: Apelin receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P35414
Assay: [35S]GTPgammaS Binding: The human APJ receptor was cloned by polymerase chain reaction and the gene encoding the receptor was subcloned in pFLAG-CMV-3 expression vector (Sigma, Saint Louis, Mo. USA) in-house at Amgen. A GTPγS binding assay was performed on membranes prepared from CHO cells stably expressing human APJ receptor. The optimum experimental conditions for the concentrations of GDP, MgCl2, and NaCl in the assay buffer were initially determined. The assay was performed in assay buffer [20 mM HEPES, pH 7.5, 5 mM MgCl2, and 0.1% (w/v) BSA with 200 mM NaCl, 3 μM GDP] and membranes expressing human APJ receptor/well along with WGA PS beads. The reaction was initiated by addition of 0.2 nM [35S]GTPγS (Perkin Elmer Life and Analytical Sciences, Waltham USA) in the absence or presence of various ligands and incubated at RT for 90 min. Nonspecific binding was determined in the presence of 100 μM GTPγS and was always less than 0.2% of total binding. All the results presented are means of several independent experiments and analyzed by non-linear regression methods using commercially available program Prism (GraphPad, San Diego, Calif.).
Measurement: EC50 = 240 nM (pChEMBL 6.619999885559082)
Descriptors: MW 502.04, ALogP 3.33, HBA 9, HBD 1, PSA 117.46, QED 0.5