lovingscience/chembl-v37-raw-activities huggingface dataset:
Compound (SMILES): CN1CCC(N(C)Cc2cc(F)cc(-c3ccc4ncnc(N)c4n3)c2)C1
InChIKey: YKOJCVHJEYEHQT-UHFFFAOYSA-N
Target: Mitogen-activated protein kinase kinase kinase kinase 4 (SINGLE PROTEIN), Homo sapiens
UniProt: O95819
Assay: Inhibition Assay: The kinase activity of purified human MAP4K4 kinase domain was measured by monitoring the phosphorylation of a peptide substrate derived from moesin protein (Leu-Gly-Arg-Asp-Lys-Tyr-Lys-Thr-Leu-Arg-Gln-Ile-Arg-Gln) fluorescently labeled on the N-terminus with 5-carboxyfluorescein using the Caliper LabChip technology (Caliper Life Sciences, Hopkinton, Mass.). To determine inhibition constants (IC50), compounds were serially diluted in DMSO and added to 10 uL kinase reactions containing 1 nM purified MAP4K4 enzyme, 1 uM peptide substrate, 10 uM ATP, 10 mM MgCl2, 1 mM EGTA, 50 mM Hepes pH 7.2, 1 mM DTT, 0.01% Triton X-100, and 2% DMSO. Reactions were incubated at room temperature in Perkin Elmer Proxiplates for 45 minutes and stopped by the addition of 10 uL of an EDTA-containing solution (50 mM Hepes pH 7.2, 40 mM EDTA, 0.02% Triton X-100). The fraction of phosphorylated peptide was determined as a fraction of total peptide substrate using the Caliper Lab Chip 3000 according to the manufacturer's instructions. IC50 values were determined using the four-parameter non-linear fit model.
Measurement: IC50 = 271 nM (pChEMBL 6.570000171661377)
Descriptors: MW 366.44, ALogP 2.55, HBA 6, HBD 1, PSA 71.17, QED 0.77
Compound (SMILES): COC[C@H]1CCCN1c1nc2n(n1)CCC(NC(=O)c1c(-c3nc(C(F)F)cs3)cnn1C)C2
InChIKey: JNGLWZBNZXTYKE-ZGTCLIOFSA-N
Target: cAMP and cAMP-inhibited cGMP 3',5'-cyclic phosphodiesterase 10A (SINGLE PROTEIN), Homo sapiens
UniProt: Q9Y233
Assay: Phosphodiersterase Evaluation Assay: IMAP TR-FRET Phosphodiersterase Evaluation Assay Kit (Molecular Device) was used for the measurement. Ten μL of a diluted test compound having each concentration and 5 μL of the enzyme PDE10 (BPA Bioscience) that had been diluted to 2 ng/mL with 1×IMAP Reaction Buffer (prepared with 5×included with the kit, 10 mM Tris-HCl, pH=7.2, 10 mM MgCl2, 0.05% NaN3, and 0.1% BSA) containing 0.1% BSA were added to a 384-well plate (Corning). The obtained mixture was then pre-incubated at room temperature for 5 minutes. Thereafter, 5 μL of cAMP substrate solution which was included with the kit and diluted to 400 nM with 1×IMAP Reaction Buffer containing 0.1% BSA, was added to the reaction solution, and the obtained mixture was reacted at room temperature for 60 minutes. Thereafter, 60 μL of an IMAP TR-FRET Binding solution included with the kit was further added to the reaction solution, and the obtained mixture was then left for 3 hours or more. Subsequently, using ARVO SX (PerkinElmer), the fluorescent intensity of Terbium (Emission=490 nm) and TR-FRET (Emission=520 nm) in the reaction solution were measured at an excitation wavelength of 340 nm, so as to calculate the amount of the generated 5′-AMP. The count of a well to which a solvent had been added instead of the test compound was set at 0%, and the count of a well to which the enzyme PDE10 had not been added was set at 100%. Thus, the inhibitory activity of each test compound was calculated.
Measurement: IC50 = 52 nM (pChEMBL 7.28000020980835)
Descriptors: MW 492.56, ALogP 2.43, HBA 10, HBD 1, PSA 102.99, QED 0.54
Compound (SMILES): Cc1cc(-c2cc(Cl)ccc2OCCn2c(C)nc3cc(C(F)F)c(C)c(C#N)c3c2=O)c2scc(C(=O)O)c2n1
InChIKey: CKYJELJQEOEWNF-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 = 6.72 nM (pChEMBL 8.170000076293945)
Descriptors: MW 595.03, ALogP 6.84, HBA 8, HBD 1, PSA 118.1, QED 0.22
Compound (SMILES): Fc1cnccc1Nc1nc(-c2ccccn2)nc2[nH]ccc12
InChIKey: OFFLEANUVDDEBL-UHFFFAOYSA-N
Target: TGF-beta receptor type-1 (SINGLE PROTEIN), Homo sapiens
UniProt: P36897
Assay: Biological Assays: Assays are conducted in 1536-well plates and 2 mL reactions are prepared from addition of HIS-TGFβR1 T204D or HIS-TGFβR2 WT, anti-HIS detection antibody, a labeled small molecule probe (Kd=<100 nM; koff=<0.001 s−1.) and test compounds in assay buffer (20 mM HEPES pH 7.4, 10 mM MgCl2, 0.015% Brij35, 4 mM DTT, and 0.05 mg/ml BSA). The reaction is incubated for 1 hour at room temperature and the HTRF signal was measured on an Envision plate reader (Ex: 340 nm; Em: 520 nm/495 nm). Inhibition data were calculated by comparison to no enzyme control reactions for 100% inhibition and vehicle-only reactions for 0% inhibition. The final concentration of reagents in the assay are 1 nM HIS-TGFβR1 T204D or HIS-TGFβR2 WT, 0.2 nM anti-HIS detection antibody, labeled small molecule probe (at Kd) and 0.5% DMSO. Dose response curves were generated to determine the concentration required inhibiting 50% of kinase activity (IC50). Compounds were dissolved at 10 mM in dimethylsulfoxide (DMSO) and evaluated at eleven concentrations. IC50 values were derived by non-linear regression analysis.
Measurement: IC50 = 0.26 nM (pChEMBL 9.59000015258789)
Descriptors: MW 306.3, ALogP 3.3, HBA 5, HBD 2, PSA 79.38, QED 0.61
Compound (SMILES): CCc1ccn(Cc2ccccc2Cl)c(=O)c1
InChIKey: ZYLSQWYAPKFESZ-UHFFFAOYSA-N
Target: Enoyl-[acyl-carrier-protein] reductase [NADPH] FabI (SINGLE PROTEIN), Staphylococcus aureus
UniProt: Q6GI75
Assay: Tight-binding assay: Kinetics were performed on a Cary 100 spectrophotometer (Varian) at 20° C. Reaction velocities were measured by monitoring the oxidation of NAD(P)H to NAD(P)+ at 340 nm (Figure US10071965-20180911-P00001=6220 M−1 cm−1). For saFabI, the reaction mixture was identical to that described previously for progress curve experiments (Schiebel et al. (2012). Structure 20:802-13). For ecFabI, the final reaction mixture contained ecFabI (75 nM), trans-2-butenoyl-CoA (800 μM; Sigma and Advent Bio), NADH (300 μM; Sigma), NAD+ (400 μM; Sigma), and inhibitor (2 v/v % DMSO) in 50 mM potassium phosphate, pH 7.5, 150 mM NaCl, 8 v/v % glycerol. For InhA, the final reaction mixture contained InhA (100 nM), trans-2-octenoyl-CoA (200 μM), NADH (250 μM), NAD+ (200 μM), and inhibitor (2 v/v % DMSO) in 30 mM PIPES, pH 6.8, 150 mM NaCl, 1 mM EDTA, 8 v/v % glycerol. The resulting curves were fit to the Morrison and Walsh integrated rate equation (Equation 1) (Morrison et al.
Measurement: Ki = 40.7 nM (pChEMBL 7.389999866485596)
Descriptors: MW 247.72, ALogP 3.11, HBA 2, HBD 0, PSA 22, QED 0.82
Compound (SMILES): C[C@@H]1COCCN1c1cc(N2CCN(S(=O)(=O)N3CCOCC3)CC2C(F)(F)F)[nH]c(=O)c1
InChIKey: GEQMLAFCKWRJLD-IURRXHLWSA-N
Target: Phosphatidylinositol 3-kinase catalytic subunit type 3 (SINGLE PROTEIN), Homo sapiens
UniProt: Q8NEB9
Assay: Vps34 Biochemical Assay: Dilution series of compounds of the invention were prepared in DMSO at 100 times the final assay concentration (n1=n0/3 in 10 points). The compounds were further diluted to 4 times the assay concentration in assay buffer (Life technologies buffer Q, PV5125, diluted 5 times supplemented with 2 mM DTT and 2 mM MnCl2). 2.5 μl of the diluted compounds were added to a 384 well assay plate followed by 2.5 μl of 16.5 nM Vps34 enzyme (Life technologies, PV5126). Enzyme and compounds were pre-incubated at rt for 15 min. Then 5 μl of substrate mix containing 20 μM ATP (Life technologies, PV3227) and 200 μM PI:PS substrate (Life technologies, PV5122) in assay buffer was added to the wells containing compound and enzyme. Mixing was performed by pipetting several times. The reaction was incubated at room temperature for 1 h. Then 5 μl stop-detection mix, prepared as described in the Adapta kinase assay kit instructions (Life technologies, PV5099) containing Adapta Eu-anti-ADP antibody (2.3 nM), Alexa Fluor 647 ADP tracer (9 nM) and EDTA (30 mM) in TR-FRET buffer, was added to quench the reaction. Mixing was performed by pipetting several times. The assay plate was then incubated at room temperature for 30 min and read with Artemis micro plate reader. Percent inhibition of the compounds as compared to DMSO treated control samples was calculated. By the use of Dotmatics software compound concentration versus percent inhibition was fitted to generate IC50 values.
Measurement: IC50 < 5 nM (pChEMBL 8.300999641418457)
Descriptors: MW 495.52, ALogP 0.23, HBA 7, HBD 1, PSA 98.42, QED 0.64
Compound (SMILES): C[C@@H]1OC(=O)NN=C1c1ccc(N2CCN(C)CC2)c(C(F)(F)F)c1
InChIKey: MOMGZCKRXOIAQX-JTQLQIEISA-N
Target: cGMP-inhibited 3',5'-cyclic phosphodiesterase 3A (SINGLE PROTEIN), Homo sapiens
UniProt: Q14432
Assay: PDE3A Enzyme Inhibition: The commercially available 3H-cAMP Scintillation Proximity Assay (SPA, Perkin Elmer) system was used for enzyme inhibition studies. For the determination of the in vitro effect of example compounds on the PDE3A reactions 2 μl of the respective example compound solution in DMSO (serial dilutions) were placed in wells of microtiter plates (Isoplate-96/200W; Perkin Elmer). 50 μl of a dilution of PDE3A cell extract from Sf9 cells overexpressing human full length PDE3A (SB Drug Discovery, UK) in buffer A (50 mM Tris/HCl pH 7.5, 8.3 mM MgCl2, 1.7 mM EDTA, 0.2% BSA) was added. The dilution of the PDE3A cell extract was chosen such that the reaction kinetics was linear and less than 70% of the substrate was consumed (typical dilution 1:5000). The reaction was started by addition of 50 μl (0.025 μCi) of 1:2000 in buffer A w/o BSA diluted substrate [8-3H]adenosine 3′, 5′-cyclic phosphate (1 μCi/μl; Perkin Elmer). After incubation at room temperature for 60 min, the reaction was stopped by addition of 25 μl of a suspension containing 18 mg/ml yttrium scintillation proximity beads (Perkin Elmer) in water. The microtiter plates were sealed and measured in a Microbeta scintillation counter (PerkinElmer Wallac). IC50 values were determined from sigmoidal curves by plotting percentage PDE3A activity vs log compound concentration.
Measurement: IC50 = 100 nM (pChEMBL 7)
Descriptors: MW 356.35, ALogP 2.29, HBA 5, HBD 1, PSA 57.17, QED 0.88
Compound (SMILES): CS(=O)(=O)N1CCC(CC#N)(N2CC(CNC3CC3c3ccccc3)C2)CC1
InChIKey: NCGLLDRHTVMYPJ-UHFFFAOYSA-N
Target: Lysine-specific histone demethylase 1A (SINGLE PROTEIN), Homo sapiens
UniProt: O60341
Assay: LSD1 histone demethylase biochemical assay: LANCE LSD1/KDM1A demethylase assay10 μL of 1 nM LSD-1 enzyme (ENZO BML-SE544-0050) in the assay buffer (50 mM Tris, pH 7.5, 0.01% Tween-20, 25 mM NaCl, 5 mM DTT) were preincubated for 1 hour at 25° C. with 0.8 μL compound/DMSO dotted in black 384 well polystyrene plates. Reactions were started by addition of 10 μL of assay buffer containing 0.4 μM Biotin-labeled Histone H3 peptide substrate: ART-K(Me1)-QTARKSTGGKAPRKQLA-GGK(Biotin) SEQ ID NO: 1 (AnaSpec 64355) and incubated for 1 hour at 25° C. Reactions were stopped by addition of 10 μL 1× LANCE Detection Buffer (PerkinElmer CR97-100) supplemented with 1.5 nM Eu-anti-unmodified H3K4 Antibody (PerkinElmer TRF0404), and 225 nM LANCE Ultra Streptavidin (PerkinElmer TRF102) along with 0.9 mM Tranylcypromine-HCl (Millipore 616431). After stopping the reactions plates were incubated for 30 minutes and read on a PHERAstar FS plate reader (BMG Labtech). Compounds having an IC50 of 1 μM or less were considered active.
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 402.56, ALogP 1.77, HBA 5, HBD 1, PSA 76.44, QED 0.75
Compound (SMILES): C[C@@H]1C[C@H](N)C[C@H](c2ccncc2NC(=O)c2ccc(F)c3cc(N4C[C@H]5C[C@@H]4CN5S(C)(=O)=O)cnc23)C1
InChIKey: GYJDXXMDLHOBRX-ARXXEPJESA-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 pM (Pim-1, Invitrogen PV3503) or 1.25 pM (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 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 552.68, ALogP 3.47, HBA 7, HBD 2, PSA 121.52, QED 0.5
Compound (SMILES): NC1=N[C@@]2(c3cc(/C=C(\F)c4ncc(C(F)(F)F)cc4Cl)ccc3F)CO[C@H](C(F)(F)F)[C@H]2CS1
InChIKey: NVNVYXHMZVSSEQ-WUXFZBHOSA-N
Target: Beta-secretase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P56817
Assay: n Vitro Enzymatic BACE1 and BACE2 FRET (Fluorescence Resonance Energy Transfer) Assay: The cDNAs for both human recombinant BACE1 and 2 with C-terminal 6-His Tags were cloned into transient protein expression vectors, which were subsequently transfected into mammalian cell lines. These recombinant proteins were further purified using Ni-NTA affinity chromatography (Qiagen). The assay buffer used in these screens was 0.05 M acetate, pH 4.5, 8% DMSO final, 100 μM genapol (which is a nonionic detergent, below its Critical Micelle Concentration). The β-secretase enzyme (0.02 nM for BACE1 and 0.64 nM for BACE2), which was pre-incubated for one hour with the test compound, typically in about luL of DMSO according to a serial dilution, was added thereto. The assay was effectively started by the addition of FRET substrate (50 nM) and the combination was incubated for one hour. The FRET assay was terminated by the addition of tris buffer, which raised the pH to neutrality, and the fluorescence was determined. The FRET substrate was a peptide with commercially available fluorophore and quencher, on opposite sides of the BACE cleavage site. The specific FRET substrate used in this assay was made by Amgen in-house. Commercially available FRET substrates, for example, the FRET substrate offered with the BACE1 FRET Assay Kit sold by ThermoFisher Scientific (Catalog Number P2985), may be used in this assay with the appropriate modifications, which are within the purview of the ability of a person with ordinary skill in the art. Proteolytic cleavage of the FRET substrate released quenching of fluorescence (excitation 488 nm and emission 590 nm).
Measurement: IC50 = 15 nM (pChEMBL 7.820000171661377)
Descriptors: MW 543.87, ALogP 6.19, HBA 5, HBD 1, PSA 60.5, QED 0.47
Compound (SMILES): C[C@@H](NC(=O)c1ccc2c(c1)[nH]c1cnccc12)c1ccccc1
InChIKey: ZUPXTGFATWSXGC-CYBMUJFWSA-N
Target: Rho-associated protein kinase 2 (SINGLE PROTEIN), Homo sapiens
UniProt: O75116
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 = 16.3 nM (pChEMBL 7.789999961853027)
Descriptors: MW 315.38, ALogP 4.21, HBA 2, HBD 2, PSA 57.78, QED 0.59
Compound (SMILES): COc1ccc(-c2nc(-c3ccc(CN4CC(C(=O)O)C4)cc3)no2)cc1C(F)(F)F
InChIKey: FWUHHFDNBPBTII-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 = 103 nM (pChEMBL 6.989999771118164)
Descriptors: MW 433.39, ALogP 3.95, HBA 6, HBD 1, PSA 88.69, QED 0.63
Compound (SMILES): COc1cc(-n2cnc(Nc3nc(N4CCC[C@@H]4CO)nc4cc(Br)ccc34)c2)cc(OC)c1OC
InChIKey: PVIWFSBAJLIEGH-MRXNPFEDSA-N
Target: Activin receptor type-1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q04771
Assay: Biochemical assay: Biochemical assays to measure the inhibitory effects of the compounds were performed by ThermoFisher Scientific (Life Technologies). ALK2 inhibition was tested using LanthaScreen Eu Kinase Binding Assay screening protocol.
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 555.43, ALogP 4.31, HBA 10, HBD 2, PSA 106.79, QED 0.33
Compound (SMILES): CCOc1ncccc1-c1cc(N[C@@H]2CCC[C@H]2OC)c2c(cnn2C)n1
InChIKey: NJSQUDFJMLLGLH-RDTXWAMCSA-N
Target: Dual specificity calcium/calmodulin-dependent 3',5'-cyclic nucleotide phosphodiesterase 1B (SINGLE PROTEIN), Homo sapiens
UniProt: Q01064
Assay: Inhibition Assay: PDE1A, PDE1B and PDE1C assays were performed as follows: the assays were performed in 60 μL samples containing a fixed amount of the PDE1 enzyme (sufficient to convert 20-25% of the cyclic nucleotide substrate), a buffer (50 mM HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) pH 7.6; 10 mM MgCl2; 0.02% Tween20), 0.1 mg/ml BSA (bovine serum albumin), 15 nM tritium labelled cAMP and varying amounts of inhibitors. Reactions were initiated by addition of the cyclic nucleotide substrate, and reactions were allowed to proceed for 1 hr at room temperature before being terminated through mixing with 20 μL (0.2 mg) yttrium silicate SPA beads (PerkinElmer). The beads were allowed to settle for 1 hour in the dark before the plates were counted in a Wallac 1450 Microbeta counter. The measured signals were converted to activity relative to an uninhibited control (100%) and IC50 values were calculated using XIFit (model 205, IDBS).
Measurement: IC50 = 536 nM (pChEMBL 6.269999980926514)
Descriptors: MW 367.45, ALogP 3.41, HBA 7, HBD 1, PSA 74.09, QED 0.72
Compound (SMILES): Cc1cc(C)c(CN2CCc3c(Cl)c4c(c(C)c3C2=O)OC(C)(C23CCC(N(C)C)(CC2)CC3)O4)c(=O)[nH]1
InChIKey: HSPAORFDQDFABD-UHFFFAOYSA-N
Target: Histone-lysine N-methyltransferase EZH1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q92800
Assay: Inhibitory Activity Assay: The inhibitory activity of the synthesized compounds above against EZH1 or EZH2 methyltransferase was measured. The experiments were consigned to Reaction Biology, and EZH1 or EZH2 activity was measured using a radiometric scintillation proximity assay. To measure the IC50 of the synthesized compounds for EZH1 or EZH2, 2.3 nmol/L EZH1 or EZH2, 1 μmol/L histone H3 (21-44)-lys(biotin), 1.5 μmol/L S-adenyl methionine (SAM), and 500 nmol/L 3H-SAM were added to the compounds or DMSO with a buffer solution and reacted for 90 minutes at room temperature. The buffer solution was comprised of 50 mmol/L Tris-HCl pH 8.0, 50 mmol/L NaCl, 1 mmol/L EDTA, 1 mmoL/L DTT, 1 mmol/L PMSF and 1% DMSO. Trichloroacetic acid was added to end the reaction, and SPA beads coated with PVT streptavidin were added followed by 1 hour reaction at room temperature. A TopCount NXT plate reader was used to measure the methylation value of the substrate peptide. The measured values were converted to percent activity, setting the mean value for wells treated with DMSO as 100% and the background mean value as 0%.
Measurement: IC50 = 15 nM (pChEMBL 7.820000171661377)
Descriptors: MW 540.1, ALogP 5.29, HBA 5, HBD 1, PSA 74.87, QED 0.57
Compound (SMILES): Cn1cncc1COc1cc(F)cc(Oc2ccc(-c3nn(CC(C)(C)O)c4ncnc(N)c34)cc2)c1
InChIKey: DBFIVGZVRSEGSB-UHFFFAOYSA-N
Target: Tyrosine-protein kinase BTK (SINGLE PROTEIN), Homo sapiens
UniProt: Q06187
Assay: Fluorescence Polarization-Based Kinase Assay: Fluorescence polarization-based kinase assays were performed in 384 well-plate format using histidine tagged recombinant human full-length Bruton Agammaglobulinemia Tyrosine Kinase (Btk) and a modified protocol of the KinEASE FP Fluorescein Green Assay supplied from Millipore. Kinase reaction were performed at room temperature for 60 minutes in presence of 250 μM substrate, 10 μM ATP and variable test article concentrations. The reaction was stopped with EDTA/kinase detection reagents and the polarization measured on a Tecan 500 instrument. From the dose-response curve obtained, the IC50 was calculated using Graph Pad Prisms® using a non linear fit curve.
Measurement: Ki < 100 nM (pChEMBL 7)
Descriptors: MW 503.54, ALogP 4.09, HBA 10, HBD 2, PSA 126.13, QED 0.32
Compound (SMILES): CCOc1ccc(COc2ccc(Nc3c(C#N)cnc4cc(OCC)c(NC(=O)CCCN(C)C)cc34)cc2Cl)nc1
InChIKey: LHKYBHFMIMOWNS-UHFFFAOYSA-N
Target: Serine/threonine-protein kinase 4 (SINGLE PROTEIN), Homo sapiens
UniProt: Q13043
Assay: MST1/MST2 Biochemical LanthaScreen Eu Kinase Binding Assay: MST1 and MST2 biochemical LanthaScreen Eu Kinase Binding Assay was based on the binding and displacement of kinase tracer to the kinase of interest. Compounds in 1000×DMSO stock solution was dispensed using automated dispensing system (Labcyte) to 384 well Corning Microplate at 15 nL, 5 uL of Kinase buffer A was added to each well. Plates were shaken and incubated for 1 min to ensure well dissolution of compounds. Kinase/Antibody mixture was added at a final concentration of 5 nM and 2 nM and kinase tracer 222 solution at a final concentration of 100 nM in a total volume of 20 uL. Plates were incubated for 1.5 hrs in dark at room temperature and assay plates were scanned on Envision plate reader with excitation: 340 nM and kinase Tracer Emission at 665 nM.
Measurement: IC50 = 550 nM (pChEMBL 6.260000228881836)
Descriptors: MW 603.12, ALogP 6.56, HBA 9, HBD 2, PSA 121.63, QED 0.17
Compound (SMILES): Cc1cc(Cn2c(=O)c3cc(S(=O)(=O)NC4(C)CC4)ccc3n(CC3(F)COC3)c2=O)on1
InChIKey: YPEABEZHKZSAOP-UHFFFAOYSA-N
Target: Poly(ADP-ribose) glycohydrolase (SINGLE PROTEIN), Homo sapiens
UniProt: Q86W56
Assay: PARG Assay: PARG In vitro assays were conducted in a total volume of 15 ul in a standard 384 well format. 5 ul of Human Full Length PARG (Produced internally by Astra Zeneca), used at a final reaction concentration of 80 pM, was added to 5 ul of Ribosylated PARP substrate (also produced internally by Astra Zeneca) at final reaction concentration of 4.5 nM in assay buffer (50 mM Tris pH7.4, 0.1 mg/ml BSA, 3 mM EDTA, 0.4 mM EGTA, 1 mM DTT, 0.01% Tween 20, 50 mM KCl). The reaction was incubated at room temperature for 10 minutes and then 5 ul detection reagent was added. Detection Reagent consists of 42 nM MAb Anti-6HIS XL665 (CisBio: 61HISXLB) and 2.25 nM Streptavidin Europium Cryptate (CisBio: 610SAKLB), both at 3× working stock concentrations (final concentrations of 14 nM and 0.75 nM respectively), in a detection buffer of 50 mM Tris pH7.4, BSA at 0.1 mg/ml and KF at 100 mM. Following incubation at room temperature for 60 minutes in the dark, TR-FRET signal was measured at Ex 340 and Em 665 and Em 620. A ratio was calculated as Em665/EM620×104 for each well and used to calculate percent inhibition for test compounds.
Measurement: IC50 = 320 nM (pChEMBL 6.5)
Descriptors: MW 478.5, ALogP 1.08, HBA 9, HBD 1, PSA 125.43, QED 0.54
Compound (SMILES): CCCCOc1nc(N)c2nc(O)n(Cc3ccc(CNCCCOCCOCCOCCCNC(=O)OC(C)(C)C)cn3)c2n1
InChIKey: BRQKNUSXTOXEOB-UHFFFAOYSA-N
Target: Toll-like receptor 7 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9NYK1
Assay: HEK-Blue TLR7 reporter assay: Engineered human embryonic kidney blue cells (HEK-Blue TLR cells; Invivogen) possessing a human TLR7-secreted embryonic alkaline phosphatase (SEAP) reporter transgene were suspended in a non-selective, culture medium (DMEM high-glucose (Invitrogen), supplemented with 10% fetal bovine serum (Sigma)). HEK-Blue TLR7 cells were added to each well of a 384-well tissue-culture plate (15,000 cells per well) and incubated 16-18 h at 37° C., 5% CO2. Compounds (100 nl) were dispensed into wells containing the HEK-Blue TLR cells and the treated cells were incubated at 37° C., 5% CO2. After 18 h treatment ten microliters of freshly-prepared Quanti-Blue reagent (Invivogen) was added to each well, incubated for 30 min (37° C., 5% CO2) and SEAP levels measured using an Envision plate reader (OD=620 nm). The half maximal effective concentration values (EC50; compound concentration which induced a response halfway between the assay baseline and maximum) were calculated.
Measurement: EC50 = 47 nM (pChEMBL 7.329999923706055)
Descriptors: MW 646.79, ALogP 3.18, HBA 14, HBD 4, PSA 190.02, QED 0.12
Compound (SMILES): CCn1c(CO)nn(-c2cc(O[C@@H](C)C(F)(F)F)c(C(=O)Nc3c(F)ccc(OC)c3Cl)cc2F)c1=O
InChIKey: UIPCXTGICSQKQG-JTQLQIEISA-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 = 5.2 nM (pChEMBL 8.279999732971191)
Descriptors: MW 550.87, ALogP 4.07, HBA 8, HBD 2, PSA 107.61, QED 0.41
Compound (SMILES): CCn1cc(Nc2nc(S[C@@H]3CCCN(C(=O)/C(C#N)=C/C4CC4)C3)c3cc[nH]c3n2)cn1
InChIKey: PCUJMLFUSIIHIF-GANRHZMWSA-N
Target: Tyrosine-protein kinase JAK3 (SINGLE PROTEIN), Homo sapiens
UniProt: P52333
Assay: Enzyme Inhibitory Activity: JAK3 and BTK kinase inhibitory activities were measured for the compounds prepared in the above Examples through in vitro analysis on the ADP Glow (Glo) platform.Specifically, the inhibitory activities of JAK3 and BTK kinase were measured using a JAK3 kinase assay kit (Promega, V9441) and a BTK kinase assay kit (Promega, V9071) which were purchased from Promega. Recombinant purified human JAK3 and BTK were diluted with 1×kinase reaction buffer (JAK3: 40 mM Tris-Cl, pH 7.5, 20 mM MgCl2, 0.1 mg/mL BSA and 50 uM DTT/BTK: 40 mM Tris-Cl, pH 7.5, 20 mM MgCl2, 0.1 mg/mL BSA, 2 mM MnCl2 and 50 uM DTT) and added to a 96 well plate (JAK3: final concentration of 4 ng per reaction/BTK: final concentration of 8 ng per reaction). The compounds were treated so as to be finally a 1% DMSO aqueous solution, and a substrate cocktail containing ATP (JAK3: final concentration of 5 uM/BTK: final concentration of 10 uM) and 0.2 μg/μL of Poly(Glu4, Tyr1) peptide (JAK3 and BTK final concentration) in the total 254 reactants was added to a 96-well plate to initiate enzymatic reaction. After incubation (30° C.) for 1 hour, equivalent volume (254 per reaction) of ADP Glo was added and incubated (30° C.) for 40 minutes at room temperature. Then, a kinase detection reagent (504 per reaction) was added and incubated (30° C.) for 30 minutes at room temperature. The kinase activity was measured by chemiluminescence according to the instructions of ADP Glo kinase assay kit, and the inhibitory activity of the compounds according to the present invention was calculated.
Measurement: IC50 = 1 nM (pChEMBL 9)
Descriptors: MW 462.58, ALogP 3.86, HBA 8, HBD 2, PSA 115.52, QED 0.31
Compound (SMILES): c1ccc(CCN2CCCC3(CC2)CN(c2ccccc2)CC2(CC2)O3)cc1
InChIKey: CQDJVNJOMNGSPB-UHFFFAOYSA-N
Target: Sigma non-opioid intracellular receptor 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q99720
Assay: Radioligand Assay: To investigate binding properties of test compounds to human σ1 receptor, transfected HEK-293 membranes and [3H](+)-pentazocine (Perkin Elmer, NET-1056), as the radioligand, were used. The assay was carried out with 7 μg of membrane suspension, 5 nM of [3H](+)-pentazocine in either absence or presence of either buffer or 10 μM Haloperidol for total and non-specific binding, respectively. Binding buffer contained Tris-HCl 50 mM at pH 8. Plates were incubated at 37° C. for 120 minutes. After the incubation period, the reaction mix was then transferred to MultiScreen HTS, FC plates (Millipore), filtered and plates were washed 3 times with ice-cold 10 mM Tris-HCL (pH7.4). Filters were dried and counted at approximately 40% efficiency in a MicroBeta scintillation counter (Perkin-Elmer) using EcoScint liquid scintillation cocktail.
Measurement: Ki < 100 nM (pChEMBL 7)
Descriptors: MW 376.54, ALogP 4.52, HBA 3, HBD 0, PSA 15.71, QED 0.78
Compound (SMILES): COC(=O)c1cccc(-c2ccc(CNC(=O)C3CCN(c4ccc(C(F)(F)F)cc4)CC3)c3c2CCN(C(=O)NC(C)(C)C)C3)c1
InChIKey: GQMWGXUCLAWMPB-UHFFFAOYSA-N
Target: Diacylglycerol O-acyltransferase 2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q96PD7
Assay: Enzymatic Activity Assay: DGAT2 activity was determined by measuring the amount of enzymatic product triolein (1,2,3-Tri(cis-9-octadecenoyl)glycerol) using the membrane prep mentioned above. The assay was carried out in deep well 384 plates in a final volume of 40 μL at room temperature. The assay mixture contained the following: assay buffer (100 mM Tris·Cl, pH 7.0, 20 mM MgCl2, 5% ethanol), 25 μM of diolein, 10 μM of oleoyl-CoA and 10 ng/μL of DGAT2 membrane.
Measurement: IC50 = 1.75 nM (pChEMBL 8.760000228881836)
Descriptors: MW 650.74, ALogP 6.56, HBA 5, HBD 2, PSA 90.98, QED 0.3
Compound (SMILES): COc1cccc(OC)c1-n1c(NS(=O)(=O)[C@@H](C)[C@H](OC)c2ncc(C)cn2)nnc1-c1cccc(S(=O)(=O)N(C)C)c1
InChIKey: STPRPAJVYDUYHF-UUOWRZLLSA-N
Target: Apelin receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P35414
Assay: [35S]GTPγS Binding Assay: 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 9 μL assay buffer [20 mM HEPES, pH 7.5, 5 mM MgCl2, 100 mM NaCl and 0.1% (w/v) BSA], 1 μL of diluted test compound (starting with 0.75 mM, 2-fold serial dilution with DMSO, total 22 points), 10 μL of 18 μM GDP (final concentration of 3 μM GDP), 20 μL of 0.25 μg/mL membrane protein expressing human APJ receptor captured with WGA PS beads (final concentration of 5 μg per well), and 20 μL of 0.3 nM [35S]GTPγS (final concentration is 0.1 nM [35S]GTPγS)(Perkin Elmer Life and Analytical Sciences, Waltham USA). One column of the plate was 1 μL of DMSO as background and another column of the plate was 1 μL of 180 μM Pyr-Apelin-13 which was used as control at a final concentration of 3 μM. Incubation was at RT for 90 min and the microplate was read using a ViewLux ultra HTS Microplate Imager (PerkinElmer, Inc.).
Measurement: EC50 = 31 nM (pChEMBL 7.510000228881836)
Descriptors: MW 631.74, ALogP 2.82, HBA 12, HBD 1, PSA 167.73, QED 0.24
Compound (SMILES): FC(F)(F)c1cccc(-c2nc(Nc3ccnc4cccnc34)c3cccn3n2)n1
InChIKey: BKIMZLSUOAVQNQ-UHFFFAOYSA-N
Target: TGF-beta receptor type-1 (SINGLE PROTEIN), Homo sapiens
UniProt: P36897
Assay: Biological Assays: Assays are conducted in 1536-well plates and 2 mL reactions are prepared from addition of HIS-TGFβR1 T204D or HIS-TGFβR2 WT, anti-HIS detection antibody, a labeled small molecule probe (Kd=<100 nM; koff=<0.001 s−1.) and test compounds in assay buffer (20 mM HEPES pH 7.4, 10 mM MgCl2, 0.015% Brij35, 4 mM DTT, and 0.05 mg/ml BSA). The reaction is incubated for 1 hour at room temperature and the HTRF signal was measured on an Envision plate reader (Ex: 340 nm; Em: 520 nm/495 nm). Inhibition data were calculated by comparison to no enzyme control reactions for 100% inhibition and vehicle-only reactions for 0% inhibition. The final concentration of reagents in the assay are 1 nM HIS-TGFβR1 T204D or HIS-TGFβR2 WT, 0.2 nM anti-HIS detection antibody, labeled small molecule prode (at Kd) and 0.5% DMSO. Dose response curves were generated to determine the concentration required inhibiting 50% of kinase activity (IC50). Compounds were dissolved at 10 mM in dimethylsulfoxide (DMSO) and evaluated at eleven concentrations. IC50 values were derived by non-linear regression analysis.
Measurement: IC50 = 20 nM (pChEMBL 7.699999809265137)
Descriptors: MW 407.36, ALogP 4.5, HBA 7, HBD 1, PSA 80.89, QED 0.47
Compound (SMILES): NC(=O)c1cc(-c2nc3cc(C4CCNCC4)cnc3[nH]2)ccc1OCc1ccccc1
InChIKey: XJADZQGEQBONFQ-UHFFFAOYSA-N
Target: Protein arginine N-methyltransferase 5 (SINGLE PROTEIN), Homo sapiens
UniProt: O14744
Assay: Biochemical Assay : The 6 μL assay reactions are run in Greiner brand black 384-well low volume plates. All reactions contained assay buffer (phosphate buffered saline, 0.01% (v/v) Tween-20, 0.01% (w/v) albumin from chicken egg white, 1 mM dithiothreitol, 200 nM peptide substrate, 1 μM S-Adenosyl methionine, and 15 ng/reaction enzyme, with the enzyme being omitted from negative control reactions. Compounds were added in a volume of 100 nL from dilution series prepared in DMSO, positive and negative control reactions receiving the same volume DMSO without compound. The plates were sealed with adhesive seals and incubated for 4 hours at 37° C. Reaction progress was measured using the Transcreener EPIGEN methyltransferase assay (BellBrook Labs, Madison, Wis.) as recommended by the manufacturer. To each reaction 2 μL detection mix were added, containing coupling enzymes, fluorescence polarisation tracer, and AMP antibody. Plates were incubated for 90 minutes before being read on a PerkinElmer EnVision plate reader in fluorescence polarisation mode.
Measurement: IC50 = 1285 nM (pChEMBL 5.889999866485596)
Descriptors: MW 427.51, ALogP 3.77, HBA 5, HBD 3, PSA 105.92, QED 0.43
Compound (SMILES): Cn1nnc(-c2ccc(Oc3ccc(F)c4c3CC[C@H]4Oc3ccc4c(c3)OC[C@H]4CC(=O)O)cc2)n1
InChIKey: PVERUTLCDVOZFZ-WAIKUNEKSA-N
Target: Free fatty acid receptor 1 (SINGLE PROTEIN), Homo sapiens
UniProt: O14842
Assay: Pharmacological Assay: IP1 accumulation measurements using the IPOne assay system 1321N1 cells stably expressing human GPR40 receptor (Euroscreen, Belgium) are seeded 24 h before the assay in white 384-well plates in culture medium containing 10% FCS, 1% Na-Pyruvate and 400 μg/mL G418. IP1 is assayed according to the manufacturer's description (Cisbio Bioassays, France). In brief, the assay is started by substitution of the culture medium by stimulation buffer (Hepes 10 mM, CaCl2 1 mM, MgCl2 0.5 mM, KCl 4.2 mM, NaCl 146 mM, glucose 5.5 mM and LiCl 50 mM, pH 7.4). Cells are stimulated for 1 h at 37° C., 5% CO2 by addition of the compounds that are diluted in stimulation buffer containing LiCl. Assays are stopped by adding HTRF-conjugates (IP1-d2 and Anti-IP1 cryptate Tb) and lysis buffer, provided by the manufacturer. After an incubation time of 1 h at room temperature plates are measured using an EnVision, Perkin Elmer. The obtained fluorescence ratios at 665/615 nM are then used to calculate the pEC50 values using Assay Explorer 3.3 Software (Accelrys, Inc.) by interpolation using an IP1 reference curve and subsequent sigmoidal curve fitting allowing for a variable hill slope.
Measurement: EC50 = 3 nM (pChEMBL 8.520000457763672)
Descriptors: MW 502.5, ALogP 4.83, HBA 8, HBD 1, PSA 108.59, QED 0.38
Compound (SMILES): Nc1cc(C[S+]([O-])c2ccc(Cl)cc2NS(=O)(=O)c2cc3ccccc3o2)n[nH]1
InChIKey: OYRJSMWOKYNBLF-UHFFFAOYSA-N
Target: C-C chemokine receptor type 2 (SINGLE PROTEIN), Homo sapiens
UniProt: P41597
Assay: Biological Assay: HEK-Gqi5 cells stably expressing CCR2 were cultured in (DMEM high glucose, 10% FBS, 1% PSA, 400 μg/ml geneticin and 50 μg/ml hygromycin. Appropriate positive control chemokines (MCP-1, MIP1A or RANTES) was used as the positive control agonist for screening compound-induced calcium activity assayed on the FLIPRTetra. The drug plates were prepared in 384-well microplates using the EP3 and the MuItiPROBE robotic liquid handling systems.
Measurement: IC50 = 1381 nM (pChEMBL 5.860000133514404)
Descriptors: MW 450.93, ALogP 3.5, HBA 6, HBD 3, PSA 137.07, QED 0.38
Compound (SMILES): NCC(c1ccc(C(F)(F)F)cc1)N1CCN(c2ncnc3c2CCC3)CC1
InChIKey: URQPFVVLTWIGDH-UHFFFAOYSA-N
Target: Proline-rich AKT1 substrate 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q96B36
Assay: AKT Enzyme Assay: A TTP Mosquito liquid handling instrument was used to place 125 nl of the appropriate concentration of inhibitor in 100% DMSO (for a dose response curve calculation) into each well of a 384-well plate. To this reaction components were added to a final volume of 12.5 μl:0.1 ng/μl His-AKT (Full Length), (Invitrogen, Part # P2999, Lot #641228C).160 uM ATP (Fluka, 02055)1 mM DTT (Sigma, D0632)1 mM MgCl2 (Sigma, M1028)1 μM substrate peptide (sequence FITC-AHA-GRPRTSSFAEG-NH2),synthesized by Tufts Peptide Synthesis service.100 mM HEPES pH 7.5 (Calbiochem, 391338)0.015% Brij-35 (Sigma, B4184)The reaction was incubated for 90 min at 25° C., and then stopped by the addition of 70 μl of Stop buffer (100 mM HEPES pH 7.5, 0.015% Brij-35, 10 mM EDTA (Sigma, E7889)).The plate was read on a Caliper LC 3000 in an Off-Chip mobility shift assay format, using the following parameters for a 12-sipper chip: screening pressure −2.3 psi, upstream voltage −500, and downstream voltage −3000. These conditions caused unphosphorylated substrate and phosphorylated product peptide to resolve as separate peaks allowing direct measurement of percentage of conversion of substrate to product. The percent conversion was plotted against concentration of inhibitor to produce a sigmoidal dose response curve, from which an IC50 was calculated.
Measurement: IC50 > 1000 nM (pChEMBL 6)
Descriptors: MW 391.44, ALogP 2.81, HBA 5, HBD 1, PSA 58.28, QED 0.87
Compound (SMILES): N#Cc1ccc(CCCC(=O)O)c(NC(=O)[C@@H]2C[C@]23CCOc2ccc(C(=O)Nc4cccnn4)cc23)c1
InChIKey: ZUTDVAODKSCPLI-KMRXNPHXSA-N
Target: Prostaglandin E2 receptor EP4 subtype (SINGLE PROTEIN), Homo sapiens
UniProt: P35408
Assay: EP4 Antagonistic Activity Measurement: EP4 Antagonistic Activity Measurement Experiment Using Prostanoid Receptor Subtype Expressing CellsCHO cells expressing rat EP4 receptor subtypes were prepared according to the methods of Nishigaki et al. (FEBS Letters, Vol. 364, p. 339-341, 1995), and used for experiment. Cultured subconfluent cells were detached, and suspended in an assay medium (MEM containing 1 mmol/L IBMX, 1% HSA) in a concentration of 1×106 cells/mL. For reaction, PGE2 was added to the cell suspension (25 μL) in a final concentration of 10 nmol/L, either alone or as a 25-μL PGE2 solution containing the test compound. After 30 minutes of reaction at room temperature, the amount of cAMP in the cells was quantified according to the method in the descriptions of the cAMP assay kit (CISBIO).The antagonistic effect (IC50 value) of the test compound was calculated as a value that represents an inhibition rate against a reaction with PGE2 alone at 10 nM, a concentration that produces a submaximal cAMP producing effect.
Measurement: IC50 = 2.5 nM (pChEMBL 8.600000381469727)
Descriptors: MW 511.54, ALogP 3.69, HBA 7, HBD 3, PSA 154.3, QED 0.41
Compound (SMILES): CCN1C(=O)N(c2c(F)c(OC)cc(OC)c2F)Cc2cnc3[nH]c(CN4CCN(C)CC4)cc3c21
InChIKey: KYOFPLBLRISEMM-UHFFFAOYSA-N
Target: Fibroblast growth factor receptor 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P11362
Assay: Enzymatic Assay: The inhibitor potency of the exemplified compounds was measured in an enzyme assay that measures peptide phosphorylation using FRET measurements to detect product formation. Inhibitors were serially diluted in DMSO and a volume of 0.5 μL was transferred to the wells of a 384-well plate. For FGFR3, a 10 μL volume of FGFR3 enzyme (Millipore) diluted in assay buffer (50 mM HEPES, 10 mM MgCl2, 1 mM EGTA, 0.01% Tween-20, 5 mM DTT, pH 7.5) was added to the plate and pre-incubated for 5-10 minutes. Appropriate controls (enzyme blank and enzyme with no inhibitor) were included on the plate. The assay was initiated by the addition of a 10 μL solution containing biotinylated EQEDEPEGDYFEWLE peptide substrate (SEQ ID NO: 1) and ATP (final concentrations of 500 nM and 140 μM respectively) in assay buffer to the wells. The plate was incubated at 25° C. for 1 hr. The reactions were ended with the addition of 10 μL/well of quench solution (50 mM Tris, 150 mM NaCl, 0.5 mg/mL BSA, pH 7.8; 30 mM EDTA with Perkin Elmer Lance Reagents at 3.75 nM Eu-antibody PY20 and 180 nM APC-Streptavidin). The plate was allowed to equilibrate for 1 hr before scanning the wells on a PheraStar plate reader (BMG Labtech).
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 500.55, ALogP 3.57, HBA 6, HBD 1, PSA 77.17, QED 0.56
Compound (SMILES): COCc1cccc2c1CCCN2c1n[nH]c2nc(N3CCC4(CC3)CO[C@@H](C)[C@H]4N)cnc12
InChIKey: ZBDVHVLMTQPZFK-KSFYIVLOSA-N
Target: Tyrosine-protein phosphatase non-receptor type 11 (SINGLE PROTEIN), Homo sapiens
UniProt: Q06124
Assay: Inhibition Assay: SHP2 is allosterically activated through binding of bis-tyrosyl-phorphorylated peptides to its Src Homology 2 (SH2) domains. The latter activation step leads to the release of the auto-inhibitory interface of SHP2, which in turn renders the SHP2 protein tyrosine phosphatase (PTP) active and available for substrate recognition and reaction catalysis. The catalytic activity of SHP2 was monitored using the surrogate substrate DiFMUP in a prompt fluorescence assay format.The inhibition of SHP2 by compounds of the disclosure (concentrations varying from 0.003-100 μM) was monitored using an assay in which 0.25 nM of SHP2 was incubated with of 0.5 μM of peptide IRS1_pY1172(dPEG8)pY1222 (sequence: H2N-LN(pY)IDLDLV(dPEG8)LST(pY)ASINFQK-amide) After 30-60 minutes incubation at 25° C., the surrogate substrate DiFMUP (Invitrogen, cat #D6567, 100 μM final) was added to the reaction and the conversion of DiFMUP to 6,8-difluoro-7-hydroxyl-4-methyl coumarin (DiFMU) was monitored continuously for 10 minutes with excitation at 355 nm and emission at 460 nm using a microplate reader (PolarStar, BMG).
Measurement: IC50 < 1000 nM (pChEMBL 6)
Descriptors: MW 463.59, ALogP 2.92, HBA 8, HBD 2, PSA 105.42, QED 0.61
Compound (SMILES): C[C@H](CN[C@H](C(=O)Nc1ccc(-c2cnn(C)n2)cn1)c1ccccc1)c1ccc(C(F)(F)F)cc1
InChIKey: HGEYJGGOUSTJML-OSPHWJPCSA-N
Target: Histone acetyltransferase p300 (SINGLE PROTEIN), Homo sapiens
UniProt: Q09472
Assay: Biochemical Assay: The full length p300 SPA assay was run following the same protocol as p300 HAT SPA assay, but used 6 nM purified full length p300 (purchased from Active Motif) in place of the purified p300-HAT domain.Select compounds were also evaluated in a H3K18Ac MSD cellular assay that measures the ability of compounds to inhibit the the acetylation of chromatin at H3K18, a process catalyzed by p300 and CBP. In a typical experiment the p300 HAT inhibitory activity inside cells of the compounds described herein was determined in accordance with the following experimental method. 20 k HCT-116 cells per well are plated in 75 μL RPMI+10% FBS media the night before treatment. Compounds plated in DMSO at 4× final concentration are resuspended in 30 μL RPMI+10% FBS, then 25 μL is combined with corresponding wells containing cells. Treated cells are incubated for 2 hr at 37° C., then lysed in 500 μL final volume and frozen at −80° C. MSD plates (Meso Scale Discovery) are coated overnight at 4° C. with 60 μL 1:500 α-total histone antibody (Millipore MAB3422) in PBS. Plates are then blocked with 5% BSA in TBST shaking at RT for 1 hr, washed, and 30 μL lysate added to each well for 2 hr shaking at RT. Plates are washed and 25 μL 1:216 α-H3K18ac antibody (CST 9675) in PBS added, then incubated for 1 hr shaking at RT. Plates are washed again, then 25 μL 1:1000 Sulfo-Tag goat α-rabbit antibody (Meso Scale Discovery R32Ab-1) in PBS is added for 1 hr shaking at RT. Plates are washed once more, then 150 μL 1× Read Buffer (MSD #R92TD-3) is added to all wells and read on MSD SECTOR Imager 2400 using the conventional read setup.
Measurement: IC50 = 28.3 nM (pChEMBL 7.550000190734863)
Descriptors: MW 494.52, ALogP 4.97, HBA 6, HBD 2, PSA 84.73, QED 0.36