lovingscience/chembl-v37-raw-activities huggingface dataset:
Compound (SMILES): C[C@@H](NC[C@H](O)c1ccc(O)c2[nH]c(=O)ccc12)c1cccc(CCNC(=O)c2ccc(N(C)C(=O)CCN3CCC(OC(=O)Nc4ccccc4-c4ccccc4)CC3)cc2)c1
InChIKey: KOHQSQYVBTZTTO-PWOKGZDBSA-N
Target: Beta-2 adrenergic receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P07550
Assay: cAMP Assay: cAMP assays were performed in a radioimmunoassay format using the Flashplate Adenylyl Cyclase Activation Assay System with [125I]-cAMP (NEN SMP004, PerkinElmer Life Sciences Inc., Boston, Mass.), according to the manufacturers instructions. For this assay, HEK-293 cell lines stably expressing cloned human β1 or β2 receptors were grown to near confluency in DMEM supplemented with 10% FBS and Geneticin (500 μg/mL); or CHO-K1 cell lines stably expressing cloned human β3 adrenergic receptors were grown to near confluency in Hams F-12 media supplemented with 10% FBS and Geneticin (250 μg/mL). Cells were rinsed with PBS and detached in dPBS (Dulbecco's Phosphate Buffered Saline, without CaCl2 and MgCl2) containing 2 mM EDTA or Trypsin-EDTA solution (0.05% trypsin/0.53 mM EDTA). After counting cells in Coulter cell counter, cells were pelleted by centrifugation at 1,000 rpm and re-suspended in stimulation buffer containing TBMX (PerkinElmer Kit) pre-warmed to room temperature to a concentration of 1.6×106 to 2.8×106 cells/mL. About 40,000 to 80,000 cells per well were used in this assay. Test compounds (10 mM in DMSO) were diluted into PBS containing 0.1% BSA in Beckman Biomek-2000 and tested at 11 different concentrations ranging from 100 μM to 1 pM. Reactions were incubated for 10 min at 37° C. and stopped by adding 100 μL of cold detection buffer containing [125I]-cAMP (NEN SMP004, PerkinElmer Life Sciences, Boston, Mass.).
Measurement: EC50 = 1 nM (pChEMBL 9)
Descriptors: MW 851.02, ALogP 7.32, HBA 9, HBD 6, PSA 176.33, QED 0.06
Compound (SMILES): Nc1ccc(S(=O)(=O)Nc2cccc(-c3ccc([C@H]4C[C@@H]4NC4CCC(N)CC4)cc3)c2)cn1
InChIKey: PWPKPNWKBRXCMZ-GBBDAIBUSA-N
Target: Amine oxidase [flavin-containing] B (SINGLE PROTEIN), Homo sapiens
UniProt: P27338
Assay: Fluorescence-Based (Inhibitor)-Screening Assay: In order to monitor MAO enzymatic activities and/or their inhibition rate by inhibitor(s) of interest, a fluorescence-based (inhibitor)-screening assay was set up. 3-(2-Aminophenyl)-3-oxopropanamine (kynuramine dihydrobromide, Sigma Aldrich), a non fluorescent compound was chosen as a substrate. Kynuramine is a non-specific substrate for both MAO-A and MAO-B activities. While undergoing oxidative deamination by MAO activities, kynuramine is converted into 4-hydroxyquinoline (4-HQ), a resulting fluorescent product.The monoamine oxidase activity was estimated by measuring the conversion of kynuramine into 4-hydroxyquinoline. Assays were conducted in 96-well black plates with clear bottom (Corning) in a final volume of 100 uL. The assay buffer was 100 mM HEPES, pH 7.5. Each experiment was performed in duplicate within the same experiment.Briefly, a fixed amount of MAO (0.25 ug for MAO-A and 0.5 ug for MAO-B) was incubated on ice for 15 minutes in the reaction buffer, in the absence and/or in the presence of at least eight 3-fold serial dilutions each. Clorgyline and Deprenyl (Sigma Aldrich) was used as a control for specific inhibition of MAO-A and MAO-B respectively. After leaving the enzyme(s) interacting with the inhibitor, KM of kynuramine was added to each reaction for MAO-B and MAO-A assay respectively, and the reaction was left for 1 hour at 37° C. in the dark. The oxidative deamination of the substrate was stopped by adding 50 uL of NaOH 2N. The conversion of kynuramine to 4-hydroxyquinoline, was monitored by fluorescence (excitation at 320 nm, emission at 360 nm) using a microplate reader (Infinite 200, Tecan). Arbitrary units were used to measure levels of fluorescence produced in the absence and/or in the presence of inhibitor. The maximum of oxidative deamination activity was obtained by measuring the amount of 4-hydroxyquinoline formed from kynuramine deamination in the absence of inhibitor and corrected for background fluorescence in the absence of MAO enzymes. The IC50 values of each inhibitor were calculated with GraphPad Prism Software.
Measurement: IC50 = 100000 nM (pChEMBL 4)
Descriptors: MW 477.63, ALogP 3.85, HBA 6, HBD 4, PSA 123.13, QED 0.41
Compound (SMILES): CC(C)(C)OC(=O)[C@H](C1CC1)N1C(=O)[C@@H](CC(=O)O)C[C@H](c2cccc(Cl)c2)[C@H]1c1ccc(Cl)cc1
InChIKey: KJMQFBPHXPWLEW-MPMKUJPUSA-N
Target: E3 ubiquitin-protein ligase Mdm2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q00987
Assay: Homogenous Time-Resolved Fluorescence Assay (HTRF2 Assay): The standard assay conditions for the in vitro HTRF assay consisted of a 50 ul total reaction volume in black 384-well Costar polypropylene plates in 1×PBS buffer pH 7.4, 1 mM DTT, 0.1% BSA, 2.5 nM GST-hMDM2 (aa 1-188), 5 nM biotinylated-p53 (aa 1-83), 1.8 nM SA-XLent (Cisbio; Bedford, Mass.), 0.6 nM anti-GST cryptate monoclonal antibody (Cisbio; Bedford, Mass.) and 200 mM KF. Amino acid residues 1-188 of human MDM2 were expressed as an amino-terminal glutathione-S-transferase (GST) fusion protein (GST-hMDM2) in Escherichia coli. Residues 1-83 of human p53 were expressed as an amino-terminal AviTag™-TrxA-6×His fusion protein (biotinylated p53) in E. coli. Each protein was purified from cell paste by affinity chromatography.Specifically, 10 uL of GST-hMDM2 was incubated with 10 ul of diluted compound (various concentrations, serially diluted) in 10% DMSO for 20 minutes at room temperature. 20 uL of biotinylated-p53 was added to the GST-hMDM2+compound mixture, and then incubated at room temperature for 60 min. 10 uL of detection buffer consisting of SA-XLent, anti-GST cryptate antibody and KF was added to GST-hMDM2, biotinylated-p53 and compound reaction and left at room temperature to reach equilibrium for >4 hrs. The final concentration of DMSO in the reaction was 2%. Time-resolved fluorescence readings were measured on a microplate multilabel reader. Percentage of inhibition was calculated relative to nutlin-3. All assay conditions remained the same as described above, with the exception of the following changes in reagent concentrations: 0.2 nM GST-hMDM2 (1-188), 0.5 nM biotinylated-p53 (1-83), 0.18 nM SA-XLent, and 100 mM KF.
Measurement: IC50 = 6 nM (pChEMBL 8.220000267028809)
Descriptors: MW 532.46, ALogP 6.26, HBA 4, HBD 1, PSA 83.91, QED 0.43
Compound (SMILES): CC(C)C[C@@H](NC(=O)c1ccc(N2CC(F)(F)C2)c(OCC2CC2)n1)c1cccnn1
InChIKey: JNWQBXIJVLIUBQ-GOSISDBHSA-N
Target: Cannabinoid receptor 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P21554
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), 1.times.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.degree. 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% NaN.sub.3) 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.
Measurement: EC50 > 10000 nM (pChEMBL 5)
Descriptors: MW 431.49, ALogP 3.63, HBA 6, HBD 1, PSA 80.24, QED 0.65
Compound (SMILES): Cn1cc2c(n1)CCC[C@H]2n1nc(-c2ccc(C(=O)Nc3cc(C(F)(F)F)ccn3)cc2)c2c(N)ncnc21
InChIKey: PMTZBZSNTTWACN-LJQANCHMSA-N
Target: Tyrosine-protein kinase BTK (SINGLE PROTEIN), Homo sapiens
UniProt: Q06187
Assay: Btk Enzyme Activity Assay C: BTK enzymatic activity was determined with the LANCE (Lanthanide Chelate Excite) TR-FRET (Time-resolved fluorescence resonance energy transfer) assay. In this assay, the potency (IC50) of each compound was determined from an eleven point (1:3 serial dilution; final compound concentration range in assay from 1 μM to 0.017 nM) titration curve using the following outlined procedure. To each well of a black non-binding surface Corning 384-well microplate (Corning Catalog #3820), 5 nL of compound (2000 fold dilution in final assay volume of 10 μL) was dispensed, followed by the addition of 7.5 μL of 1× kinase buffer (50 mM Hepes 7.5, 10 mM MgCl2, 0.01% Brij-35, 1 mM EGTA, 0.05% BSA & 1 mM DTT) containing 5.09 pg/μL (66.67 pM) of BTK enzyme (recombinant protein from baculovirus-transfected Sf9 cells: full-length BTK, 6HIS-tag cleaved). Following a 60 minute compound & enzyme incubation, each reaction was initiated by the addition of 2.5 μL 1× kinase buffer containing 8 μM biotinylated A5 peptide (Biotin-EQEDEPEGDYFEWLE-NH2), and 100 μM ATP. The final reaction in each well of 10 μL consists of 50 pM hBTK, 2 μM biotin-A5-peptide, and 25 μM ATP. Phosphorylation reactions were allowed to proceed for 120 minutes. Reactions were immediately quenched by the addition of 20 uL of 1× quench buffer (15 mM EDTA, 25 mM Hepes 7.3, and 0.1% Triton X-100) containing detection reagents (0.626 nM of LANCE-Eu-W1024-anti-phospho Tyrosine antibody, PerkinElmer and 86.8 nM of Streptavidin-conjugated Dylight 650, Dyomics/ThermoFisher Scientific). After 60 minutes incubation with detection reagents, reaction plates were read on a PerkinElmer EnVision plate reader using standard TR-FRET protocol. Briefly, excitation of donor molecules (Eu-chelate:anti-phospho-antibody) with a laser light source at 337 nm produces energy that can be transferred to Dylight-650 acceptor molecules if this donor:acceptor pair is within close proximity. Fluorescence intensity at both 665 nm (acceptor) and 615 nm (donor) are measured and a TR-FRET ratio calculated for each well (acceptor intensity/donor intensity). IC50 values were determined by 4 parameter robust fit of TR-FRET ratio values vs. (Log10) compound concentrations.
Measurement: IC50 = 550 nM (pChEMBL 6.260000228881836)
Descriptors: MW 533.52, ALogP 4.4, HBA 9, HBD 2, PSA 129.43, QED 0.35
Compound (SMILES): Cc1ccc(Cc2cc([C@@H]3O[C@H](CO)[C@@H](O)[C@H](O)[C@H]3O)c3c(c2C)OCO3)cc1
InChIKey: UPXRNMVSQVCWSW-YRIDSSQKSA-N
Target: Sodium/glucose cotransporter 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P13866
Assay: SGLT1 Inhibition Assay: For sodium-dependent glucose transport assay, cells expressing hSGLT1 were seeded into a 96-well culture plate at a density of 5×104 cells/well in RPMI medium 1640 containing 10% fetal bovine serum. The cells were used 1 day after plating. They were incubated in pretreatment buffer (10 mM HEPES, 5 mM Tris, 140 mM choline chloride, 2 mM KCl, 1 mM CaCl2, and 1 mM MgCl2, pH 7.4) at 37 °C. for 10 min. They were then incubated in uptake buffer (10 mM HEPES, 5 mM Tris, 140 mM NaCl, 2 mM KCl, 1 mM CaCl2, 1 mM MgCl2, and 1 mM 14C-nonlabeled AMG pH 7.4) containing 14C-labeled (8 μM) and inhibitor or dimethyl sulfoxide (DMSO) vehicle at 37 °C. for 2 h. Cells were washed twice with washing buffer (pretreatment buffer containing 10 mM AMG at room temperature) and then the radioactivity was measured using a liquid scintillation counter.
Measurement: IC50 = 27 nM (pChEMBL 7.570000171661377)
Descriptors: MW 402.44, ALogP 1.14, HBA 7, HBD 4, PSA 108.61, QED 0.61
Compound (SMILES): C[C@@H](NC(=O)c1ccc(-c2cn[nH]c2)c(OC(F)F)c1)c1ccc(F)cc1
InChIKey: MRSCRUMYAVOXFF-LLVKDONJSA-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.
Measurement: IC50 = 7.49 nM (pChEMBL 8.130000114440918)
Descriptors: MW 375.35, ALogP 4.31, HBA 3, HBD 2, PSA 67.01, QED 0.67
Compound (SMILES): CN1CCC(COC(=O)N2CC[C@@H](n3cc(C(N)=O)c(Nc4ccc(F)cc4)n3)[C@H](C#N)C2)CC1
InChIKey: JFLXPNCMPSZGDJ-DYESRHJHSA-N
Target: Tyrosine-protein kinase JAK2 (SINGLE PROTEIN), Homo sapiens
UniProt: O60674
Assay: Biochemical HTRF Assay: The ability of compounds to inhibit the activity of JAK1, JAK2, JAK3, and Tyk2 was measured using a recombinant purified GST-tagged catalytic domain for each enzyme (Invitrogen JAK1 #M4290, JAK2 #M4290, JAK3 #M4290, Tyk2 #M4290) in an HTRF format biochemical assay. The reactions employed a common peptide substrate, LCB-EQEDEPEGDYFEWLW-NH2 (in-house). The basic assay protocol is as follows: First, 250 nL of diluted compounds in DMSO were dispensed into the wells of a dry 384-well Black plate (Greiner #781076) using a Labcyte Echo 555 acoustic dispenser. Subsequent reagent additions employed an Agilent Bravo. Next, 18 μL of 1.11× enzyme and 1.11× substrate in 1× assay buffer (Invitrogen kinase buffer # PV3189, 2 mM DTT, 0.05% BSA) were added to the wells and shaken and then preincubated for 30 minutes at ambient temperature to allow compound binding to equilibrate. After equilibration, 2 μL of 10×ATP in 1× assay buffer was added to initiate the kinase reaction and the plates were shaken and then incubated at ambient temperature for 120 minutes. At the end of the incubation, 20 μL of 2× stop buffer (streptavidin-Dylight 650 (Thermo #84547B/100 mL), Eu-tagged pY20 antibody (Perkin Elmer #AD0067), EDTA, HEPES, and Triton) was added to quench the reaction. Plates were shaken and centrifuged and then incubated 60 minutes at ambient temperature and then read on a Perkin Elmer Envision (λex=337 nm, λem=665 and 615 nm, TRF delay time=20 μs). HTRF signal=10,000*665 nm reading/615 nm reading.
Measurement: IC50 = 1089 nM (pChEMBL 5.960000038146973)
Descriptors: MW 483.55, ALogP 2.73, HBA 8, HBD 2, PSA 129.51, QED 0.65
Compound (SMILES): COc1cccc(C(=O)N2CCn3c(-c4nc(C)ns4)nnc3[C@H]2C)c1
InChIKey: YKIGSSIUJRGFEY-SNVBAGLBSA-N
Target: Neuromedin-K receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P29371
Assay: Competitive Binding Assay: The affinity of compounds of the invention for the human NK-3 receptor was determined by measuring the ability of compounds of the invention to competitively and reversibly displace a well-characterized NK-3 radioligand in a concentration-dependent manner.3H-SB222200 Binding Competition Assay with Human NK-3 ReceptorThe ability of compounds of the invention to inhibit the binding of the NK-3 receptor selective antagonist 3H-SB222200 was assessed by an in vitro radioligand binding assay. Membranes were prepared from Chinese hamster ovary recombinant cells stably expressing the human NK-3 receptor. The membranes were incubated with 5 nM 3H-SB222200 (ARC) in a HEPES 25 mM/NaCl 0.1M/CaCl2 1 mM/MgCl2 5 mM/BSA 0.5%/Saponin 10 μg/ml buffer at pH 7.4 and various concentrations of compounds of the invention. The amount of 3H-SB222200 bound to the receptor was determined after filtration by the quantification of membrane associated radioactivity using the TopCount-NXT reader (Packard).
Measurement: Ki = 100 nM (pChEMBL 7)
Descriptors: MW 370.44, ALogP 2.33, HBA 8, HBD 0, PSA 86.03, QED 0.7
Compound (SMILES): Cc1cc(C#N)cnc1C(=O)Nc1ccc(F)c([C@@]2(C)N=C(N)OCC2(F)F)n1
InChIKey: ISXCJKRNXUQSFB-QGZVFWFLSA-N
Target: Beta-secretase 2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9Y5Z0
Assay: Inhibition Assay: Recombinant BACE-2 (extracellular domain, expressed in baculovirus and purified using standard methods) at 0.1 to 1 nM concentrations is incubated with the test compound at various concentrations for 1 hour at room temperature in 100 mM acetate buffer, pH 4.5, containing 0.1% CHAPS. Activity was measured using a final concentration of 3 μM of the fluorescence-quenched substrate Q-C(HSO3)-Ile-Asp-Leu-Ala-Val-Leu-Asp-HN-CH2-CH2-Mca, where Q=2-nitro-5-amino benzoic acid and Mca=7-methoxy-4-coumarinyl acetic acid. Catalytic turnover was monitored in a Spectramax Gemini fluorescence plate reader (Molecular Devices) in black 96-well microplates using excitation/emission wavelength of 325 nm and 400 nm, respectively. Fluorescence increase was followed for 15 min, in 1 minute's intervals. The fluorescence/time slopes were calculated from duplicate wells and from wells without inhibitor and the IC50 values were calculated using a logistic 4-parameter model.
Measurement: IC50 = 66 nM (pChEMBL 7.179999828338623)
Descriptors: MW 404.35, ALogP 2.24, HBA 7, HBD 2, PSA 126.28, QED 0.81
Compound (SMILES): CC(O)c1cc(C(=O)N[C@H](C)c2ccc(F)cc2)ccc1-c1cn[nH]c1
InChIKey: XRCQVMGYIXAQNO-PZORYLMUSA-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.
Measurement: IC50 = 48.1 nM (pChEMBL 7.320000171661377)
Descriptors: MW 353.4, ALogP 3.76, HBA 3, HBD 3, PSA 78.01, QED 0.65
Compound (SMILES): C[C@@H]1CCC[C@H](N2CCC(c3cc(Cl)ccc3-n3cncn3)=CC2=O)c2cc(ccn2)-c2ccccc2NC1=O
InChIKey: WOYLVPNPEGMMRB-OLILMLBXSA-N
Target: Plasma kallikrein (SINGLE PROTEIN), Homo sapiens
UniProt: P03952
Assay: In Vitro Assay: Plasma kallikrein determinations were made in 0.1 M sodium phosphate buffer at a pH of 7.5 containing 0.1-0.2 M sodium chloride and 0.5% PEG 8000. Determinations were made using purified human plasma kallikrein (Enzyme Research Laboratories) at a final assay concentration of 200 pM and the synthetic substrate S-2302 (H-(D)-Pro-Phe-Arg-pNA; CHROMOGENIX®) at a concentration of 0.00008-0.0004 M.
Measurement: Ki = 60 nM (pChEMBL 7.21999979019165)
Descriptors: MW 553.07, ALogP 6.1, HBA 6, HBD 1, PSA 93.01, QED 0.33
Compound (SMILES): CCc1ccc(Cc2cc([C@@H]3O[C@H](CO)[C@@H](O)[C@H](O)[C@H]3O)c3c(c2C)CCCC3)cc1
InChIKey: RFBRRXRFMKJCAK-RTJMFUJLSA-N
Target: Sodium/glucose cotransporter 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P13866
Assay: SGLT1 Inhibition Assay: For sodium-dependent glucose transport assay, cells expressing hSGLT1 were seeded into a 96-well culture plate at a density of 5×104 cells/well in RPMI medium 1640 containing 10% fetal bovine serum. The cells were used 1 day after plating. They were incubated in pretreatment buffer (10 mM HEPES, 5 mM Tris, 140 mM choline chloride, 2 mM KCl, 1 mM CaCl2, and 1 mM MgCl2, pH 7.4) at 37 °C. for 10 min. They were then incubated in uptake buffer (10 mM HEPES, 5 mM Tris, 140 mM NaCl, 2 mM KCl, 1 mM CaCl2, 1 mM MgCl2, and 1 mM 14C-nonlabeled AMG pH 7.4) containing 14C-labeled (8 μM) and inhibitor or dimethyl sulfoxide (DMSO) vehicle at 37 °C. for 2 h. Cells were washed twice with washing buffer (pretreatment buffer containing 10 mM AMG at room temperature) and then the radioactivity was measured using a liquid scintillation counter.
Measurement: IC50 = 10.4 nM (pChEMBL 7.980000019073486)
Descriptors: MW 426.55, ALogP 2.54, HBA 5, HBD 4, PSA 90.15, QED 0.59
Compound (SMILES): CC(Nc1nccc(N2C(=O)OC[C@@H]2[C@@H](C)O)n1)c1nc(-c2ccc(F)c(OC(F)(F)F)c2)no1
InChIKey: NVQKTZLJBNQZPU-SVLLLCHKSA-N
Target: Isocitrate dehydrogenase [NADP] cytoplasmic (SINGLE PROTEIN), Homo sapiens
UniProt: O75874
Assay: LC-MS Detection of 2-HG Assay: The biochemical reactions were performed at room temperature in 384-well Greiner flat-bottom plates (Costar, Cat. No. 781201) using a final reaction volume of 30 μL and the following assay buffer conditions: 50 mM HEPES pH 7.4, 10 mM MgCl2, 50 mM KCl, 1 mM DTT, 0.02% BSA, 5 uM NADPH and 100 uM alpha-KG. The final reaction mixture contained 3.3% DMSO and inhibitors with concentrations ranging 0.02-50 μM. The IDH1 enzyme was used at a final concentration of 0.25 nM. Following 45 minutes incubation, the reaction mixtures were quenched by the addition of 10 μL of 16% formic acid containing 800 nM of 5-carbon labeled 13C-2-HG). The protein was then precipitated by the addition of 2.5 volumes of acetonitrile followed by centrifugation (3000×g, minutes). The concentration of 2-HG in the resulting supernatants was measured by LC-MS (see below). LC-MS method. Reaction mixture supernatants were submitted to chromatographic separation on a BiobasicAX column (2.1 mm×20 mm, 5 μm particle, Thermo Scientific Inc.). The chromatographic mobile phases were A) 25 mM ammonium biocarbonate and B) acetonitrile (0.1% ammonium hydroxide). Nicotinamide was eluted at 1 ml/min using a 85-5% B gradient over 0.9 minutes (Agilent 1200SL LC system, Thermofisher LX-4 autosampler) and analyzed by multiple reaction monitoring (MRM) on a API4000 QTrap mass spectrometer (ABSciex, Framingham, Mass.) in the positive electrospray ionization (ESI+) mode. The mass transition for 2-HG and 13C-2-HG were 147→129 and 152→134, respectively.
Measurement: IC50 = 152 nM (pChEMBL 6.820000171661377)
Descriptors: MW 498.39, ALogP 3.44, HBA 10, HBD 2, PSA 135.73, QED 0.47
Compound (SMILES): Cc1nsc(-c2nnc3n2CCN(C(=O)c2ccc(F)c(Cl)c2)[C@@H]3C)n1
InChIKey: DMCGPNZWXATPEH-MRVPVSSYSA-N
Target: Neuromedin-K receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P29371
Assay: Competitive Binding Assay: The affinity of compounds of the invention for the human NK-3 receptor was determined by measuring the ability of compounds of the invention to competitively and reversibly displace a well-characterized NK-3 radioligand in a concentration-dependent manner.3H-SB222200 Binding Competition Assay with Human NK-3 ReceptorThe ability of compounds of the invention to inhibit the binding of the NK-3 receptor selective antagonist 3H-SB222200 was assessed by an in vitro radioligand binding assay. Membranes were prepared from Chinese hamster ovary recombinant cells stably expressing the human NK-3 receptor. The membranes were incubated with 5 nM 3H-SB222200 (ARC) in a HEPES 25 mM/NaCl 0.1M/CaCl2 1 mM/MgCl2 5 mM/BSA 0.5%/Saponin 10 μg/ml buffer at pH 7.4 and various concentrations of compounds of the invention. The amount of 3H-SB222200 bound to the receptor was determined after filtration by the quantification of membrane associated radioactivity using the TopCount-NXT reader (Packard).
Measurement: Ki = 24 nM (pChEMBL 7.619999885559082)
Descriptors: MW 392.85, ALogP 3.11, HBA 7, HBD 0, PSA 76.8, QED 0.67
Compound (SMILES): C[C@H](C1CC1)N1CC[C@]23CCCC[C@H]2[C@H]1Cc1ccc(C(N)=O)cc13
InChIKey: WKSWYCJGHDWJFK-PDPGBCDDSA-N
Target: Mu-type opioid receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P35372
Assay: null: The Ki (binding affinity) for u opioid receptors was determined using a competitive displacement assay as previously described in Neumeyer (Journal of Med. Chem. 2012, p 3878), which is incorporated herein in its entirety. Briefly, membrane protein from CHO (Chinese Hamster Ovarian) cells that stably expressed the cloned human u opioid receptor were incubated with 12 different concentrations of the compound set forth herein in the presence of 0.25 nM [3H]DAMGO (see Tiberi et al., Can. J. Physiol. Pharmacol. 1988, Vol. 66, p 1368, which is incorporated by reference herein in its entirety) in a final volume of 1 mL of 50 mM Tris-HCl, pH 7.5 at 25° C. Incubation times of 60 min were used for [3H]DAMGO (see Gulati et al., Life Sci. 1990, Vol. 47, p 159, which is incorporated by reference herein in its entirety). Nonspecific binding was measured by inclusion of 10 uM naloxone. The binding was terminated by filtering the samples through Schleicher & Schuell No. 32 glass fiber.
Measurement: IC50 = 1.7 nM (pChEMBL 8.770000457763672)
Descriptors: MW 338.5, ALogP 3.64, HBA 2, HBD 1, PSA 46.33, QED 0.91
Compound (SMILES): C[C@@H]1CCC[C@H](N2CCC(c3c(F)ccc(Cl)c3F)=CC2=O)c2cc(ccn2)-c2cc(C#N)ccc2NC1=O
InChIKey: OQVHPCHZBPUBCJ-QUGAMOGWSA-N
Target: Plasma kallikrein (SINGLE PROTEIN), Homo sapiens
UniProt: P03952
Assay: In Vitro Assay: Plasma kallikrein determinations were made in 0.1 M sodium phosphate buffer at a pH of 7.5 containing 0.1-0.2 M sodium chloride and 0.5% PEG 8000. Determinations were made using purified human plasma kallikrein (Enzyme Research Laboratories) at a final assay concentration of 200 pM and the synthetic substrate S-2302 (H-(D)-Pro-Phe-Arg-pNA; CHROMOGENIX®) at a concentration of 0.00008-0.0004 M.
Measurement: Ki = 31.7 nM (pChEMBL 7.5)
Descriptors: MW 547.01, ALogP 6.67, HBA 4, HBD 1, PSA 86.09, QED 0.36
Compound (SMILES): CNCCCC[C@@H](NC(=O)[C@@H](CC(C)C)NC(=O)[C@@H](Cc1ccccc1)NC(=O)[C@H](N)Cc1ccccc1)C(=O)N1CCCN(C(=N)N)CC1
InChIKey: BWOLOWBBCGTZIF-SEVDZJIVSA-N
Target: Kappa-type opioid receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P41145
Assay: Time Resolved Fluorescence Resonance Energy Transfer (TR-FRET)-Based cAMP Immunoassay: Mouse R1.G1 cells (ATCC, Manassas, Va.) were grown in suspension in high glucose-DMEM (Dulbecco's Modified Eagle's Medium, Cellgro, Herndon, Va.) containing 10% horse serum and 2% glutaMax (Invitrogen, Carlsbad. Calif.) without added antibiotics. On the day of the experiment, cells were spun at 1,000 rpm for 5 minutes at room temperature and then washed once with HBSS (HEPES Buffered Saline Solution, Invitrogen, Carlsbad, Calif.). Cells were then spun again and resuspended in stimulation buffer (HBSS with 0.05% FAF-BSA (Fatty acid-free bovine serum albumin, Roche Applied Science, Indianapolis, Ind.), 5 mM HEPES) to 2 million cells per ml. Antibody supplied with the LANCE™ cAMP immunoassay kit was then added to the cells according to the manufacturer's instructions, and 12,000 cells per well were then added to the wells containing forskolin to a predetermined fixed final concentration (typically about 2.5 μM) and the previously determined amount of the synthetic peptide amide to be tested. The synthetic peptide amides were tested in a range of concentrations to determine potency. Cells were incubated with the synthetic peptide amide plus forskolin for about 20 minutes at room temperature. After incubation, cells are lysed by adding 12 μl of detection mix as supplied with the LANCE™ kit, followed by incubation for one hour at room temperature. Time resolved fluorescence was read using a 330-380 nm excitation filter, a 665 nm emission filter, dichroic mirror 380, and Z=1 mm.
Measurement: EC50 = 0.15 nM (pChEMBL 9.819999694824219)
Descriptors: MW 691.92, ALogP 1.12, HBA 7, HBD 7, PSA 198.77, QED 0.07
Compound (SMILES): Cn1ncc(NC(=O)c2ccc(F)c(-c3c(F)cccc3F)n2)c1[C@@H]1CC[C@@H](N)[C@H](F)CO1
InChIKey: RTIBEHQDRMSSBP-RKVPGOIHSA-N
Target: Serine/threonine-protein kinase pim-1 (SINGLE PROTEIN), Homo sapiens
UniProt: P11309
Assay: PIM Kinase Binding Assay: LC3K assay: PIM-1, -2, and -3 enzymes were generated as fusion proteins expressed in bacteria and purified by IMAC column chromatography (Sun, X., Chiu, J. F., and He, Q. Y. (2005) Expert Rev. Proteomics, 2:649-657). A fluorescent-labeled Pim-specific peptide substrate, was custom synthesized by American Peptide Company (Sunnyvale, Calif.). Reaction Buffer contained 10 mM HEPES, pH 7.2, 10 mM MgCl2, 0.01% Tween 20, 2 mM DTT. Termination Buffer contained 190 mM HEPES, pH 7.2, 0.015% Brij-35, 0.2% Coating Reagent 3 (Caliper Life Sciences, Hopkinton, Mass.), 20 mM EDTA. Separation Buffer contained 100 mM HEPES, pH 7.2, 0.015% Brij-35, 0.1% Coating Reagent 3, 1:200 Coating Reagent 8 (Caliper Life Sciences, Hopkinton, Mass.), 10 mM EDTA and 5% DMSO. PIM reactions were carried out in a final volume of 10 μL per well in a 384-well plate. A standard enzymatic reaction, initiated by the addition of 5 μL 2×ATP and test compound to 5 μL of 2× enzyme and FAM-peptide, contained 20 pM PIM1, 50 pM PIM2, or 55 pM PIM3, 1 μM FAM-peptide, and 10 μM ATP, in Reaction Buffer. After 90 minutes of incubation at room temperature, the phosphorylation reaction was stopped by the addition of 10 μL Termination Buffer. The product and substrate in each independent reaction were separated on a 12-sipper microfluidic chip (Caliper Life Sciences, Hopkinton, Mass.) run on a Caliper LC3000® (Caliper Life Sciences, Hopkinton, Mass.). The separation of product and substrate was optimized by choosing voltages and pressure using Caliper's Optimizer software (Hopkinton, Mass.). The separation conditions used a downstream voltage of −500V, an upstream voltage of −2150V, and a screening pressure of −1.2 psi. The product and substrate fluorophore were excited at 488 nm and detected at 530 nm.
Measurement: Ki = 0.016 nM (pChEMBL 10.800000190734863)
Descriptors: MW 463.44, ALogP 3.67, HBA 6, HBD 2, PSA 95.06, QED 0.58
Compound (SMILES): COC(=O)NCCCn1nc([C@@H](C)N(C(=O)[C@H]2CNCCO2)C2CC2)c2cccnc21
InChIKey: IGXZOTGJSKGMAP-RHSMWYFYSA-N
Target: Renin (SINGLE PROTEIN), Homo sapiens
UniProt: P00797
Assay: Inhibitory Assay: A substrate of synthetic peptide (Nma-KHPFHLVIHK(Dnp)-NH2) and test compound were mixed, and fluorescence intensity was assayed using a fluorophotometer before staring an enzymatic reaction (exciting wavelength: 340 nm, measuring wavelength: 460 nm). Recombinant human renin was added and the mixture was incubated at 37° C. for 1 hour, and the fluorescence intensity was measured after the reaction using a fluorophotometer (exciting wavelength: 340 nm, measuring wavelength: 460 nm). Renin activity was evaluated on the ground of fluorescence intensity which was obtained by deduction of the intensity before the reaction from the intensity after the reaction, and 50% inhibitory concentration (IC50) was calculated from renin activities under the existence of various concentration of the test compound.
Measurement: IC50 = 3.6 nM (pChEMBL 8.4399995803833)
Descriptors: MW 430.51, ALogP 1.22, HBA 8, HBD 2, PSA 110.61, QED 0.61
Compound (SMILES): Cc1ccc(-c2nnc(C(C)Nc3nccc(N4C(=O)OC[C@@H]4[C@@H](C)O)n3)o2)cc1
InChIKey: FXPWKPAUTKSPOQ-JYRZLJSNSA-N
Target: Isocitrate dehydrogenase [NADP] cytoplasmic (SINGLE PROTEIN), Homo sapiens
UniProt: O75874
Assay: LC-MS Detection of 2-HG Assay: The biochemical reactions were performed at room temperature in 384-well Greiner flat-bottom plates (Costar, Cat. No. 781201) using a final reaction volume of 30 μL and the following assay buffer conditions: 50 mM HEPES pH 7.4, 10 mM MgCl2, 50 mM KCl, 1 mM DTT, 0.02% BSA, 5 uM NADPH and 100 uM alpha-KG. The final reaction mixture contained 3.3% DMSO and inhibitors with concentrations ranging 0.02-50 μM. The IDH1 enzyme was used at a final concentration of 0.25 nM. Following 45 minutes incubation, the reaction mixtures were quenched by the addition of 10 μL of 16% formic acid containing 800 nM of 5-carbon labeled 13C-2-HG). The protein was then precipitated by the addition of 2.5 volumes of acetonitrile followed by centrifugation (3000×g, minutes). The concentration of 2-HG in the resulting supernatants was measured by LC-MS (see below). LC-MS method. Reaction mixture supernatants were submitted to chromatographic separation on a BiobasicAX column (2.1 mm×20 mm, 5 μm particle, Thermo Scientific Inc.). The chromatographic mobile phases were A) 25 mM ammonium biocarbonate and B) acetonitrile (0.1% ammonium hydroxide). Nicotinamide was eluted at 1 ml/min using a 85-5% B gradient over 0.9 minutes (Agilent 1200SL LC system, Thermofisher LX-4 autosampler) and analyzed by multiple reaction monitoring (MRM) on a API4000 QTrap mass spectrometer (ABSciex, Framingham, Mass.) in the positive electrospray ionization (ESI+) mode. The mass transition for 2-HG and 13C-2-HG were 147→129 and 152→134, respectively.
Measurement: IC50 = 3057 nM (pChEMBL 5.510000228881836)
Descriptors: MW 410.43, ALogP 2.71, HBA 9, HBD 2, PSA 126.5, QED 0.63
Compound (SMILES): O=C(Nc1ccc([C@H]2CNCCO2)cc1)c1cnn(-c2ccc(C(F)(F)F)cc2)n1
InChIKey: IBYMAJRGHIHKQY-GOSISDBHSA-N
Target: Trace amine-associated receptor 1 (SINGLE PROTEIN), Rattus norvegicus
UniProt: Q923Y9
Assay: Radioligand Binding (rat TAAR1): HEK-293 cells stably expressing rat TAAR1 were maintained at 37Â° C. and 5% CO2 in DMEM high glucose medium, containing fetal calf serum (10%, heat inactivated for 30 min at 56Â° C.), penicillin/streptomycin (1%), and 375 Î¼g/ml geneticin (Gibco). Cells were released from culture flasks using trypsin/EDTA, harvested, washed twice with ice-cold PBS (without Ca2+ and Mg2+), pelleted at 1'000 rpm for 5 min at 4Â° C., frozen and stored at â¿¿80Â° C. Frozen pellets were suspended in 20 ml HEPES-NaOH (20 mM, pH 7.4) containing 10 mM EDTA and homogenized with a Polytron (PT 6000, Kinematica) at 14'000 rpm for 20 s. The homogenate was centrifuged at 48'000Ã¿g for 30 min at 4Â° C. Subsequently, the supernatant was removed and discarded, and the pellet resuspended in 20 ml HEPES-NaOH (20 mM, pH 7.4) containing 0.1 mM EDTA using the Polytron (20 s at 14'000 rpm). This procedure was repeated and the final pellet resuspended in HEPES-NaOH containing 0.1 mM EDTA and homogenized using the Polytron. Typically, aliquots of 2 ml membrane portions were stored at â¿¿80Â° C. With each new membrane batch the dissociation constant (Kd) was determined via a saturation curve. The TAAR1 radioligand 3[H]â¿¿(S)-4-[(ethyl-phenyl-amino)-methyl]-4,5-dihydro-oxazol-2-ylamine (described in WO 2008/098857) was used at a concentration equal to the calculated Kd value, that was usually around 2.3 nM, resulting in the binding of approximately 0.2% of the radioligand and a specific binding representing approximately 85% of the total binding. Nonspecific binding was defined as the amount of 3[H]â¿¿(S)-4-[(ethyl-phenyl-amino)-methyl]-4,5-dihydro-oxazol-2-ylamine bound in the presence of 10 Î¼M unlabeled ligand. All compounds were tested at a broad range of concentrations (10 Î¼M to 10 Î¼M) in duplicates. The test compounds (20 Î¼l/well) were transferred into a 96 deep well plate (TreffLab), and 180 Î¼l of HEPES-NaOH (20 mM, pH 7.4) containing MgCl2 (10 mM) and CaCl2 (2 mM) (binding buffer), 300 Î¼l of the radioligand 3[H]â¿¿(S)-4-[(ethyl-phenyl-amino)-methyl]-4,5-dihydro-oxazol-2-ylamine at a concentration of 3.3Ã¿Kd in nM and 500 Î¼l of the membranes (resuspended at 50 Î¼g protein per ml) added. The 96 deep well plates were incubated for 1 hr at 4Â° C. Incubations were terminated by rapid filtration through Unifilter-96 plates (Packard Instrument Company) and glass filters GF/C (Perkin Elmer) presoaked for 1 hr in polyethylenimine (0.3%) and washed 3 times with 1 ml of cold binding buffer. After addition of 45 Î¼l of Microscint 40 (PerkinElmer) the Unifilter-96 plate was sealed and after 1 hr the ratioactivity counted using a TopCount Microplate Scintillation Counter (Packard Instrument Company).
Measurement: Ki = 0.2 nM (pChEMBL 9.699999809265137)
Descriptors: MW 417.39, ALogP 3.2, HBA 6, HBD 2, PSA 81.07, QED 0.68
Compound (SMILES): CC(C)C[C@@H](NC(=O)[C@@H](Cc1ccccc1)NC(=O)[C@H](N)Cc1ccccc1)C(=O)N[C@H](CCCCNC(=N)N)C(=O)N1CCC(N2CCC[C@H]2C(=O)O)CC1
InChIKey: NLHUFIKAQYWPME-JSMUHSIHSA-N
Target: Kappa-type opioid receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P41145
Assay: Time Resolved Fluorescence Resonance Energy Transfer (TR-FRET)-Based cAMP Immunoassay: Mouse R1.G1 cells (ATCC, Manassas, Va.) were grown in suspension in high glucose-DMEM (Dulbecco's Modified Eagle's Medium, Cellgro, Herndon, Va.) containing 10% horse serum and 2% glutaMax (Invitrogen, Carlsbad. Calif.) without added antibiotics. On the day of the experiment, cells were spun at 1,000 rpm for 5 minutes at room temperature and then washed once with HBSS (HEPES Buffered Saline Solution, Invitrogen, Carlsbad, Calif.). Cells were then spun again and resuspended in stimulation buffer (HBSS with 0.05% FAF-BSA (Fatty acid-free bovine serum albumin, Roche Applied Science, Indianapolis, Ind.), 5 mM HEPES) to 2 million cells per ml. Antibody supplied with the LANCE™ cAMP immunoassay kit was then added to the cells according to the manufacturer's instructions, and 12,000 cells per well were then added to the wells containing forskolin to a predetermined fixed final concentration (typically about 2.5 μM) and the previously determined amount of the synthetic peptide amide to be tested. The synthetic peptide amides were tested in a range of concentrations to determine potency. Cells were incubated with the synthetic peptide amide plus forskolin for about 20 minutes at room temperature. After incubation, cells are lysed by adding 12 μl of detection mix as supplied with the LANCE™ kit, followed by incubation for one hour at room temperature. Time resolved fluorescence was read using a 330-380 nm excitation filter, a 665 nm emission filter, dichroic mirror 380, and Z=1 mm.
Measurement: EC50 = 0.17 nM (pChEMBL 9.770000457763672)
Descriptors: MW 776, ALogP 1.49, HBA 8, HBD 8, PSA 236.07, QED 0.05
Compound (SMILES): C[C@@H](Oc1cc(-c2cnn(C3CC4(CCCNC4)C3)c2)cnc1N)c1c(Cl)ccc(F)c1Cl
InChIKey: WUIOBNGTWSVFMF-FLORGGSBSA-N
Target: Hepatocyte growth factor receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P08581
Assay: Inhibition Assay: The inhibitory activities of compounds of Formula I were assay at Reaction Biology Corporation, One Great Valley Parkway, Malvern, Pa., USA. Human ALK and cMet enzymes were used and the substrate was a peptide substrate, poly[Glu:Tyr] (4:1) at a concentration of 0.2 mg/ml. The ATP concentration for the assay was 10 uM and Staurosporine was used as a standardwith an IC50 of 2.3 nM, and 75 nM, respectively for ALK and cMet.
Measurement: IC50 = 2.09 nM (pChEMBL 8.680000305175781)
Descriptors: MW 490.41, ALogP 5.82, HBA 6, HBD 2, PSA 77.99, QED 0.44
Compound (SMILES): CC(Nc1nccc(N2C(=O)OC[C@@H]2[C@@H](C)O)n1)c1cc(-c2ccc(Cl)cc2)no1
InChIKey: QCIXJXCZOHQOBC-ZLXOECBPSA-N
Target: Isocitrate dehydrogenase [NADP] cytoplasmic (SINGLE PROTEIN), Homo sapiens
UniProt: O75874
Assay: LC-MS Detection of 2-HG Assay: The biochemical reactions were performed at room temperature in 384-well Greiner flat-bottom plates (Costar, Cat. No. 781201) using a final reaction volume of 30 μL and the following assay buffer conditions: 50 mM HEPES pH 7.4, 10 mM MgCl2, 50 mM KCl, 1 mM DTT, 0.02% BSA, 5 uM NADPH and 100 uM alpha-KG. The final reaction mixture contained 3.3% DMSO and inhibitors with concentrations ranging 0.02-50 μM. The IDH1 enzyme was used at a final concentration of 0.25 nM. Following 45 minutes incubation, the reaction mixtures were quenched by the addition of 10 μL of 16% formic acid containing 800 nM of 5-carbon labeled 13C-2-HG). The protein was then precipitated by the addition of 2.5 volumes of acetonitrile followed by centrifugation (3000×g, minutes). The concentration of 2-HG in the resulting supernatants was measured by LC-MS (see below). LC-MS method. Reaction mixture supernatants were submitted to chromatographic separation on a BiobasicAX column (2.1 mm×20 mm, 5 μm particle, Thermo Scientific Inc.). The chromatographic mobile phases were A) 25 mM ammonium biocarbonate and B) acetonitrile (0.1% ammonium hydroxide). Nicotinamide was eluted at 1 ml/min using a 85-5% B gradient over 0.9 minutes (Agilent 1200SL LC system, Thermofisher LX-4 autosampler) and analyzed by multiple reaction monitoring (MRM) on a API4000 QTrap mass spectrometer (ABSciex, Framingham, Mass.) in the positive electrospray ionization (ESI+) mode. The mass transition for 2-HG and 13C-2-HG were 147→129 and 152→134, respectively.
Measurement: IC50 = 2052 nM (pChEMBL 5.690000057220459)
Descriptors: MW 429.86, ALogP 3.66, HBA 8, HBD 2, PSA 113.61, QED 0.61
Compound (SMILES): O=C(Nc1ccc(OC(F)(F)F)cc1)c1cnc(N2CC[C@@H](O)C2)c(-c2ccccc2)c1
InChIKey: ATWBZFNCHJUFLK-GOSISDBHSA-N
Target: Tyrosine-protein kinase ABL1 (SINGLE PROTEIN), Homo sapiens
UniProt: P00519
Assay: Cellular Proliferation Assay: Test compounds were resuspended in DMSO at a concentration of 10 mM. A serial three-fold dilution of each compound with DMSO was performed in 384-well plates using the Janus Liquid Dispenser (PerkinElmer). Compound was delivered to the assay plates containing 2500 cells in a 50 μL volume via Acoustic delivery from an ATS-100 (EDC). For Ba/F3-BCR-ABL1-WT cell assays, 2 nL of each compound dilution was transferred to the assay plate for final assay concentrations of 0.4 μM, 0.13 μM, 0.044 μM, 0.015 μM, 0.005 μM, 0.001 μM, 0.00033 μM, 0.00011 μM, 0.000037 μM, 0.000012 μM. For Ba/F3-WT and Ba/F3-BCR-ABL1-T315I cell assays, 50 nL of each compound dilution was transferred to the assay plate for final assay concentrations of 10 μM, 3.33 μM, 1.11 μM, 0.37 μM, 0.12 μM, 0.041 μM, 0.014 μM, 0.0046 μM, 0.0015 μM, 0.00051 μM.Cells were incubated at 37° C. in a humidified environment with 5% car
Measurement: IC50 = 6.6 nM (pChEMBL 8.180000305175781)
Descriptors: MW 443.43, ALogP 4.47, HBA 5, HBD 2, PSA 74.69, QED 0.61
Compound (SMILES): Cc1nn(C)c(C)c1NS(=O)(=O)c1ccc(-c2ccnc(N3CCNC(C(C)C)C3)c2)cc1
InChIKey: NKPDOAKEBATUJP-UHFFFAOYSA-N
Target: Glycylpeptide N-tetradecanoyltransferase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P30419
Assay: Enzyme Inhibition Assay: Measurements were performed using a modification of the scintillation proximity assay platform described previously by Georgopapadakou, N. H. et al. (22nd International Congress on Chemotherapy, 2001, Abstract P16.001). Compounds were solubilised in DMSO at a top concentration of 10 mM and serially diluted in half log steps to achieve a range of final assay concentrations of 100 uM to 1 nM. Compound at each concentration (100-fold final) was added to white 384 well plates in a volume of 0.5 ml. Human, A. fumigatus, T. brucei or L. major N-myristoyl transferase enzyme, dissolved to a working concentration of 10 nM in assay buffer (30 mM Tris/HCl pH 7.4, 0.5 mM EGTA, 0.5 mM EDTA, 1.25 mM DTT, 0.1% Triton X-100), was then added to columns 1 to 11 and 13 to 23 of the plates in a volume of 20 ml. To columns 12 and 24, 20 ml assay buffer was added to provide a no enzyme control. Following a 5 minute incubation at room temperature the substrates (GCGGSKVKPQPPQAK(Biotin)-Amide and myristoyl coenzyme A), dissolved in assay buffer, were added to all wells in a volume of 20 ml to start the reaction. The final concentrations of peptide and 3H-myristoyl coenzyme A were 0.5 mM and 125 nM respectively and the specific activity of the radiolabel was 8 Ci/mmol. Plates were then incubated at room temperature for up to 50 minutes (dependant upon the period of linearity for the different enzyme species) before SPA beads, suspended to 1 mg/ml in a stop solution (200 mM Phosphoric Acid/NaOH pH 4, 750 mM MgCl2), were added in a volume of 40 ml.
Measurement: IC50 = 240 nM (pChEMBL 6.619999885559082)
Descriptors: MW 468.63, ALogP 3.33, HBA 7, HBD 2, PSA 92.15, QED 0.58
Compound (SMILES): C[C@@H]1CCC[C@H](N2CCC(c3c(F)ccc(Cl)c3F)=CC2=O)c2cc(ccn2)-c2ccc(Nc3nnc(C4CC4)o3)cc2NC1=O
InChIKey: XRRSNWWFDKBFCV-WRHNGFHOSA-N
Target: Plasma kallikrein (SINGLE PROTEIN), Homo sapiens
UniProt: P03952
Assay: In Vitro Assay: Plasma kallikrein determinations were made in 0.1 M sodium phosphate buffer at a pH of 7.5 containing 0.1-0.2 M sodium chloride and 0.5% PEG 8000. Determinations were made using purified human plasma kallikrein (Enzyme Research Laboratories) at a final assay concentration of 200 pM and the synthetic substrate S-2302 (H-(D)-Pro-Phe-Arg-pNA; CHROMOGENIX®) at a concentration of 0.00008-0.0004 M.
Measurement: Ki = 2 nM (pChEMBL 8.699999809265137)
Descriptors: MW 645.11, ALogP 7.8, HBA 7, HBD 2, PSA 113.25, QED 0.21
Compound (SMILES): COc1cccc([C@@H](C)NC(=O)c2ccc(-c3c[nH]nc3C)cc2OCCN(C)C)c1
InChIKey: PUGDUBQQBUJNPW-MRXNPFEDSA-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.
Measurement: IC50 = 34.79 nM (pChEMBL 7.460000038146973)
Descriptors: MW 422.53, ALogP 3.83, HBA 5, HBD 2, PSA 79.48, QED 0.55
Compound (SMILES): CC(C)(c1nc(-[n+]2c(N3CCOCC3)[nH]c3ccccc32)nc2c1OC[C@@H]1COCCN21)S(C)(=O)=O
InChIKey: BYNUQELMPZFSBA-INIZCTEOSA-O
Target: DNA-dependent protein kinase catalytic subunit (SINGLE PROTEIN), Homo sapiens
UniProt: P78527
Assay: ADP Hunter Plus Assay: The kinase activity was measured by using the commercial ADP Hunter Plus assay available from DiscoveRx (#90-0083), which is a homogeneous assay to measure the accumulation of ADP, a universal product of kinase activity. The enzyme, DNA-PK was purchased from Promega (#V5811). The assay was done following the manufacturer recommendations with slight modifications: Mainly the kinase buffer was replace by 15 mM HEPES, pH 7.5, 10 mM MgCl2, 20 mM NaCl, 1 mM EGTA, 0.1 mg/mL BGG, 0.02% Tween 20. The DNA-PK was assayed in a titration experiment to determine the optimal protein concentration for the inhibition assay. To calculate the 1050 of the Compounds, serial 1:3 dilutions of the compounds were added to the enzyme at a fixed concentration (2 U/uL). The enzyme was preincubated with the inhibitor and 200 uM DNA substrate and then ATP was added to a final 75 uM concentration. Reaction was carried out for 1 hour at 37° C. Reagent A and B were sequentially added to the wells and plates were incubated for 30 min at RT. Fluorescence counts were read in a EnVision instrument (Perkin Elmer) with the recommended settings (550 and 590 nm as excitation and emission wavelengths, respectively). Values were normalized against the control activity included for each enzyme (i.e., 100% DNA_PK kinase activity, without compound). These values were plotted against the inhibitor concentration and were fit to a sigmoid dose-response curve by using the model sigmoidal Four-Parameter Logistc implement for Activity base - software.
Measurement: IC50 = 10000 nM (pChEMBL 5)
Descriptors: MW 515.62, ALogP 0.95, HBA 9, HBD 1, PSA 113.76, QED 0.51