lovingscience/chembl-v37-raw-activities huggingface dataset:
Compound (SMILES): COc1cccc(OC)c1C1CCCC(=O)N1Cc1cccc(N2CCCCC2)c1
InChIKey: FSJIJHVMTMAXFG-UHFFFAOYSA-N
Target: Orexin/Hypocretin receptor type 1 (SINGLE PROTEIN), Homo sapiens
UniProt: O43613
Assay: In Vitro Assay: Intracellular Calcium Measurements : In Vitro Assay: Intracellular Calcium Measurements:Chinese hamster ovary (CHO) cells expressing the human orexin-1 receptor and the human orexin-2 receptor, respectively, are grown in culture medium (Ham F-12 with L-Glutamine) containing 300 g/ml G418, 100 U/ml penicillin, 100 g/ml streptomycin and 10% heat inactivated fetal calf serum (FCS). The cells are seeded at 20,000 cells/well into 384-well black clear bottom sterile plates (Greiner). The seeded plates are incubated overnight at 37° C. in 5% CO2. Human orexin-A as an agonist is prepared as 1 mM stock solution in MeOH:water (1:1), diluted in HBSS containing 0.1% bovine serum albumin (BSA), NaHCO3: 0.375 g/l and 20 mM HEPES for use in the assay at a final concentration of 3 nM.Antagonists are prepared as 10 mM stock solution in DMSO, then diluted in 384-well plates using DMSO followed by a transfer of the dilutions into in HBSS containing 0.1% bovine serum albumin (BSA), NaHCO3: 0.375 g/l and 20 mM HEPES. On the day of the assay, 50 μl of staining buffer (HBSS containing 1% FCS, 20 mM HEPES, NaHCO3: 0.375 g/l, 5 mM probenecid (Sigma) and 3 M of the fluorescent calcium indicator fluo-4 AM (1 mM stock solution in DMSO, containing 10% pluronic) is added to each well. The 384-well cell-plates are incubated for 50 min at 37° C. in 5% CO2 followed by equilibration at RT for 30 min before measurement.
Measurement: IC50 = 22 nM (pChEMBL 7.659999847412109)
Descriptors: MW 408.54, ALogP 4.95, HBA 4, HBD 0, PSA 42.01, QED 0.68
Compound (SMILES): CS(=O)(=O)N[C@H]1CC[C@H](Nc2cc(NC3CC3)n3ncc(/C=C4\NC(=O)NC4=O)c3n2)CC1
InChIKey: YQNATXAICQCDQY-WLOSUKBJSA-N
Target: Casein kinase II subunit beta (SINGLE PROTEIN), Homo sapiens
UniProt: P67870
Assay: Inhibition Assay: Test compounds dissolved and diluted in DMSO (2 μl) were added to a reaction mixture comprising 10 μl of 5× Reaction Buffer (40 mM MOPS pH 7.0, 5 mM EDTA), 10 μl of recombinant human PIM2 solution (4 ng PIM-2 dissolved in dilution buffer (20 mM MOPS pH 7.0; EDTA 1 mM; 5% Glycerol; 0.01% Brij 35; 0.1%; 0.1% 2-mercaptoethanol; 1 mg/ml BSA)) and 8 ul of water. Reactions were initiated by the addition of 10 ul of ATP Solution (49% (15 mM MgCl2; 75 uM ATP) 1% ([γ-33P]ATP: Stock 1 mCi/100 μl; 3000 Ci/mmol (Perkin Elmer)) and 10 ul of substrate peptide solution (RSRSSYPAGT (SEQ ID NO.: 6), dissolved in water at a concentration of 1 mM), Reactions were maintained for 10 min at 30° C. The reactions were quenched with 100 ul of 0.75% Phosphoric acid, then transferred to and filtered through a Phosphocellulose filter plate (Millipore, MSPH-N6B-50). After washing each well 4 times with 0.75% Phosphoric acid, scintillation fluid (20 uL) was added to each well and the resid
Measurement: IC50 = 476 nM (pChEMBL 6.320000171661377)
Descriptors: MW 474.55, ALogP 0.76, HBA 9, HBD 5, PSA 158.62, QED 0.29
Compound (SMILES): N#CCC1(n2cc(C(N)=O)c(Nc3ccc(F)cc3)n2)CCN(Cc2cccc(F)n2)CC1
InChIKey: CRWJPXGQHQUKGX-UHFFFAOYSA-N
Target: Tyrosine-protein kinase JAK1 (SINGLE PROTEIN), Homo sapiens
UniProt: P23458
Assay: HTRF 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 uL of 1.11x enzyme and 1.11x substrate in 1x 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 room temperature to allow compound binding to equilibrate. After equilibration, 2 uL of 10xATP in 1x assay buffer was added to initiate the kinase reaction and the plates were shaken and then incubated at room temperature for 120 minutes. At the end of the incubation, 20 uL of 2x 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.
Measurement: IC50 = 3 nM (pChEMBL 8.520000457763672)
Descriptors: MW 451.48, ALogP 3.3, HBA 7, HBD 2, PSA 112.86, QED 0.53
Compound (SMILES): COC(=O)NC(C(=O)N1CCC[C@H]1c1ncc(-c2ccc3cc(-c4ccc5nc([C@@H]6CCCN6C(=O)C(NC(=O)OC)c6ccccc6)[nH]c5c4)ccc3c2)[nH]1)c1ccccc1
InChIKey: OLWUAUYJBBJFTO-YZNAZWRISA-N
Target: Nonstructural protein 5A (SINGLE PROTEIN), Hepatitis C virus
UniProt: Q5L478
Assay: Transient Transfection Assay: In a transient set-up, a Huh-7 lunet hepatoma cell line was transiently transfected with an autonomously replicating RNA encoding a bi-cistronic expression construct. This construct comprises a firefly luciferase reporter gene preceding the NS3-NS5B subgenomic region of HCV (genotype 1a H77 or 1b Con1). Translation of the HCV subgenomic region is mediated by an internal ribosome entry site of encephalomyocarditis virus. The construct is furthermore flanked by 5' and 3' untranslated regions of HCV (genotype 1a H77 or 1b Con 1, respectively), which allow for replication of the RNA. In addition to the wild-type constructs, site-directed mutations were introduced into the transient HCV genotype 1b replicon in the gene encoding for the non-structural protein 5A (NS5A). More precisely, amino acid residues 28, 30, 31 and 93 in NS5A were independently altered. Cells were plated in 384 well plates in the presence of test and control compounds, which were added in various concentrations. Following an incubation of two days, replication of the HCV subgenomic replicon RNA was measured by assaying luciferase activity (using standard luciferase assay substrates and reagents and a Perkin Elmer ViewLux™ ultraHTS microplate imager).
Measurement: EC50 = 0.093 nM (pChEMBL 10.029999732971191)
Descriptors: MW 830.95, ALogP 8.29, HBA 8, HBD 4, PSA 174.64, QED 0.11
Compound (SMILES): CC(C)OC(=O)[C@H](C)NP(=O)(OC[C@H]1O[C@@H](c2cnn3c(N)ncnc23)[C@](C)(N)[C@@H]1O)Oc1ccccc1
InChIKey: PMCRYGFUVWXAHD-GHRVZJKLSA-N
Target: RNA-directed RNA polymerase (SINGLE PROTEIN), Hepacivirus hominis
UniProt: O39930
Assay: Cell-Based HCV Replicon Assay: To measure cell-based anti-HCV activity of selected compounds of the present invention, replicon cells were seeded at 5000 cells/well in 96-well collagen I-coated Nunc plates in the presence of the test compound. Various concentrations of test compound, typically in 10 serial 2-fold dilutions, were added to the assay mixture, with the starting concentration ranging from 250 uM to 1 uM. The final concentration of dimethylsulfoxide was 0.5% fetal bovine serum was 5%, in the assay media. Cells were harvested on day 3 by the addition of 1x cell lysis buffer (Ambion cat #8721).The replicon RNA level was measured using real time PCR (Taqman assay). The amplicon was located in 5B. The PCR primers were: 5B.2F, ATGGACAGGCGCCCTGA (SEQ ID. NO. 1); 5B.2R, TTGATGGGCAGCTTGGTTTC (SEQ ID. NO. 2); the probe sequence was FAM-labeled CACGCCATGCGCTGCGG (SEQ ID. NO. 3). GAPDH RNA was used as endogenous control and was amplified in the same reaction as NS5B (multiplex PCR) using primers and VIC-labeled probe recommended by the manufacturer (PE Applied Biosystem). The real-time RT-PCR reactions were run on ABI PRISM 7900HT Sequence Detection System using the following program: 48° C. for 30 minutes, 95° C. for 10 minutes, 40 cycles of 95° C. for 15 sec, 60° C. for 1 minute. The ΔCT values (CT5B-CTGAPDH) were plotted against the concentration of test compound and fitted to the sigmoid dose-response model using XLfit4 (MDL). EC50 was defined as the concentration of inhibitor necessary to achieve ΔCT=1 over the projected baseline; EC90 the concentration necessary to achieve ΔCT=3.2 over the baseline. Alternatively, to quantitate the absolute amount of replicon RNA, a standard curve was established by including serially diluted T7 transcripts of replicon RNA in the Taqman assay. All Taqman reagents were from petroleum ether Applied Biosystems. Such an assay procedure was described in detail in e.g. Malcolm et al., Antimicrobial Agents and Chemotherapy 50: 1013-1020 (2006).
Measurement: EC50 > 100000 nM (pChEMBL 4)
Descriptors: MW 549.53, ALogP 1.36, HBA 13, HBD 4, PSA 198.44, QED 0.21
Compound (SMILES): CC(=O)N1CCc2ccc(Nc3ccc(S(=O)(=O)NCC4CC4)c(C(F)(F)F)c3)cc21
InChIKey: MJZCCERKIDMRDR-UHFFFAOYSA-N
Target: Cannabinoid receptor 2 (SINGLE PROTEIN), Homo sapiens
UniProt: P34972
Assay: Binding Assay: The binding assays were performed by testing the ability of the new compounds to displace the radiolabeled synthetic non-selective cannabinoid agonist [3H]CP55940 (168 Ci/mmol; PerkinElmer) from the human CB1 (hCB1) or human CB2 (hCB2) receptors on membranes derived from stably transfected HEK-293 cells (PerkinElmer). Membranes were diluted in assay buffer (50 mM Tris-HCl, 1 mM EDTA, 1 mM MgCl2, 1 mg/ml BSA, pH=7.4). The amount of membrane was determined for each batch of membranes according to protein binding assay. The minimum amount of membrane that gave 50% specific binding was used for the binding assay. In most assays, binding was tested using 8 μg and 4 μg protein of hCB1 and hCB2 membranes, respectively. The tested compounds were dissolved in DMSO and diluted in assay buffer to a final concentration of 0.1% solvent. Total binding of [3H]CP55940 was evaluated with 1.5 nM to hCB1 and with 0.5 nM to hCB2, according to Kd affinity of [3H]CP55940 for the respective membranes. The ability of the tested compounds to displace [3H]CP55940 was evaluated first at a single concentration point of 100 nM for binding toward hCB2 or hCB1. In certain cases, the displacement was tested at compound concentrations ranging from 0.03 nM to 10 μM. Non-specific binding was measured by the addition of 1 μM of unlabelled CP55940 to the tubes. Binding assays were performed in triplicate in a total volume of 500 μl for 60 minutes at 30° C., in a shaking bath. Free and bound radioligands were separated by rapid filtration through GF/C filter plates (PerkinElmer) that had been presoaked with 0.1% Polyethylenimine (Sigma). Filters were shaken for 1 hour in 7 ml scintillation fluid (PerkinElmer) and radioactivity was determined by liquid scintillation counter (Wallac; PerkinElmer).
Measurement: Ki = 644 nM (pChEMBL 6.190000057220459)
Descriptors: MW 453.49, ALogP 4.05, HBA 4, HBD 2, PSA 78.51, QED 0.69
Compound (SMILES): CC(C)n1cnc(S(=O)(=O)N2CCC(C(=O)NC3CC3)CC2)c1
InChIKey: QEVQCAOOAVTZME-UHFFFAOYSA-N
Target: Dihydrolipoyl dehydrogenase (SINGLE PROTEIN), Mycobacterium tuberculosis (strain ATCC 25618 / H37Rv)
UniProt: P9WHH9
Assay: Inhibition Assay: The assay was performed in a manner similar to that described in Bryk et al., Biochemistry (2010) 49:1616-1627 and modified for an online robotics screening system.A solution containing Lpd (100 nM) and 100 mM sodium phosphate buffer (pH 7.0) was prepared. Another solution containing the substrate lipoamide (2 mM), 100 mM sodium phosphate buffer (pH 7.0), EDTA (4 mM), and NADH (200 uM) was also prepared. The detection reagent, DTNB was dissolved in DMSO and diluted to a concentration of 375 uM with a 100 mM sodium phosphate buffer (pH 7.0).4 uL/well of Lpd solution was dispensed into 1536-well clear bottom plates. Next, 50 nL of each test compounds (1 mM dissolved in DMSO) was added into each well. After one hour of incubation, 4 uL/well of lipoamide (LPA) solution was added, so that in each well, the concentrations of Lpd, LPA, and the test compound were 50 nM, 1 mM, and 6.25 uM, respectively.
Measurement: IC50 = 2990 nM (pChEMBL 5.519999980926514)
Descriptors: MW 340.45, ALogP 1.14, HBA 5, HBD 1, PSA 84.3, QED 0.87
Compound (SMILES): C#CCNC(=O)[C@H](CC(C)C)NC(=O)C1OC1c1ccccc1
InChIKey: YBOZSTKVIUDZCF-FHERZECASA-N
Target: Calpain-1 catalytic subunit (SINGLE PROTEIN), Homo sapiens
UniProt: P07384
Assay: Inhibition FRET Assay: The Calbiochem InnoZyme activity kit was used for measuring the inhibition effect inhibitors on human erythrocyte calpain 1 activity. A calpain FRET substrate, (DABCYL)-TPLKSPPPSPR-(EDANS) (SEQ ID NO: 9), was used to detect the activity of Cal1. 20 μM of the FRET substrate, 10 nM of native Cal1, and 20 μM TCEP (reducing agent) were added to the reaction mixture containing assay buffer (Tris [10 mM], NaCl [100 mM], pH 7.4). Cal1 was activated by the addition of 10 μM calcium. Cleavage of the scissile bond amide bond, K-S, releases the fluorophore (EDANS) from the internal quenching molecule (DABCYL), resulting in an increase in fluorescence measured at 320 nm excitation and 480 nm emission wave lengths for 30 min. Each inhibitor was added to the reaction mixture at varying concentrations (10 nM, 100 nM, 1 μM, and 10 μM) to detect inhibition of Cal1 and reduction in fluorescence was measured by 96 well-plate reader.
Measurement: IC50 > 1000 nM (pChEMBL 6)
Descriptors: MW 314.39, ALogP 1.41, HBA 3, HBD 2, PSA 70.73, QED 0.59
Compound (SMILES): COC(=O)C(C)C(C)(C)NC(=O)c1cc(OCC2CC2)c(Cl)cn1
InChIKey: CLVRLEZFLMKDMP-UHFFFAOYSA-N
Target: Cannabinoid receptor 2 (SINGLE PROTEIN), Homo sapiens
UniProt: P34972
Assay: cAMP Assay: CHO cells expressing human CB1 or CB2 receptors are seeded 17-24 hours prior to the experiment 50.000 cells per well in a black 96 well plate with flat clear bottom (Corning Costar #3904) in DMEM (Invitrogen No. 31331), 1×HT supplement, with 10% fetal calf serum and incubated at 5% CO2 and 37° C. in a humidified incubator. The growth medium was exchanged with Krebs Ringer Bicarbonate buffer with 1 mM IBMX and incubated at 30° C. for 30 min. Compounds were added to a final assay volume of 100 μl and incubated for 30 min at 30° C. Using the cAMP-Nano-TRF detection kit the assay (Roche Diagnostics) was stopped by the addition of 50 μl lysis reagent (Tris, NaCl, 1.5% Triton X100, 2.5% NP40, 10% NaN3) and 50 μl detection solutions (20 μM mAb Alexa700-cAMP 1:1, and 48 μM Ruthenium-2-AHA-cAMP) and shaken for 2 h at room temperature. The time-resolved energy transfer is measured by a TRF reader (Evotec Technologies GmbH), equipped with a ND:YAG laser as excitation source. The plate is measured twice with the excitation at 355 nm and at the emission with a delay of 100 ns and a gate of 100 ns, total exposure time 10 s at 730 (bandwidth 30 nm) or 645 nm (bandwidth 75 nm), respectively. The FRET signal is calculated as follows: FRET=T730-Alexa730-P(T645-B645) with P=Ru730-B730/Ru645-B645, where T730 is the test well measured at 730 nM, T645 is the test well measured at 645 nm, B730 and B645 are the buffer controls at 730 nm and 645 nm, respectively. cAMP content is determined from the function of a standard curve spanning from 10 μM to 0.13 nM cAMP.
Measurement: EC50 = 9.5 nM (pChEMBL 8.020000457763672)
Descriptors: MW 354.83, ALogP 2.84, HBA 5, HBD 1, PSA 77.52, QED 0.76
Compound (SMILES): COc1ccc(-c2cc(C)ccc2F)cc1C(CC(=O)O)c1ncc(C)s1
InChIKey: JJULKYKSRIXPSC-UHFFFAOYSA-N
Target: Lysosomal protective protein (SINGLE PROTEIN), Homo sapiens
UniProt: P10619
Assay: Inhibition Assay: The activated cathepsin A was diluted in assay buffer (25 mM MES, pH 5.5, containing 5 mM DTT) and mixed with the test compound (dissolved in assay buffer containing (v/v) 3% DMSO) or, in the control experiments, with the vehicle in a multiple assay plate. After incubation for 15 min at room temperature, as substrate then bradykinin carrying an N-terminal® Bodipy FL (4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-propionyl) label (JPT Peptide Technologies GmbH; dissolved in assay buffer) was added to the mixture. The final concentration of cathepsin A was 833 ng/ml and the final concentration of labeled bradykinin 2 μM. After incubation for 15 min at room temperature the reaction was stopped by the addition of stop buffer (130 mM 2-(4-(2-hydroxy-ethyl)-piperazin-1-yl)-ethanesulfonic acid, pH 7.4, containing (v/v) 0.013%° Triton X-100, 0.13% Coating Reagent 3 (Caliper Life Sciences), 6.5% DMSO and 20 μM ebelactone B (Sigma, #E0886)).
Measurement: IC50 = 5680 nM (pChEMBL 5.25)
Descriptors: MW 385.46, ALogP 5.18, HBA 4, HBD 1, PSA 59.42, QED 0.63
Compound (SMILES): CCCc1ncc(Cc2cnc3ccccc3c2)c2cc(OC)c(OC)cc12
InChIKey: HUZZQEPXBQCKKU-UHFFFAOYSA-N
Target: cAMP and cAMP-inhibited cGMP 3',5'-cyclic phosphodiesterase 10A (SINGLE PROTEIN), Homo sapiens
UniProt: Q9Y233
Assay: Diaxonhit Phosphodiesterase Assay: The phosphodiesterase assay was developed using the LANCE cAMP kit (PerkinElmer). The assay buffer contained HBSS with 5 mM HEPES, 0.1% BSA, and 1.5 mM MgCl2, pH 7.4. PDE10A (BPS Bioscience) was used at 200 pg/well (with a specific activity of 3200 pmole/min/ug with assay conditions: 10 mM Tris-HCl, pH 7.4, 10 mM MgCl2, 1 mM MnCl2, 200 uM cAMP, 2.5 kU 5' nucleotidase, 37° C., 20 min) and PDE4D3 (BPS Bioscience) was used at 100 pg/well (with a specific activity of 32713 pmole/min/ug with assay conditions: 10 mM Tris-HCl, pH 7.4, 10 mM MgCl2, 1 mM MnCl2, 200 uM cAMP, 2.5 kU 5' nucleotidase, 37° C., 20 min). The Biotin-cAMP tracer, supplied in 10 mmol/L Tris-HCl buffered (pH 8.0) salt solution with 1 mmol/L ethylenediaminetetraacetic acid (EDTA), 0.1% bovine serum albumin (BSA), and 0.05% sodium azide, is used at a dilution of 1/375. The assay detection mixture contained the LANCE Eu-W8044 labeled streptavidin 1/2250 (supplied in 50 mmol/L Tris-HCl buffer).
Measurement: IC50 = 11 nM (pChEMBL 7.960000038146973)
Descriptors: MW 372.47, ALogP 5.34, HBA 4, HBD 0, PSA 44.24, QED 0.46
Compound (SMILES): CCOC(c1ccc([C@]2(S(=O)(=O)c3ccc(F)cc3)CCN(C(=O)N3CCN(C(C)=O)CC3)C2)cc1)(C(F)(F)F)C(F)(F)F
InChIKey: XAYTUNWJIVMROS-VWLOTQADSA-N
Target: Nuclear receptor ROR-gamma (SINGLE PROTEIN), Homo sapiens
UniProt: P51449
Assay: Scintillation Proximity Assay (SPA): The binding of potential ligands to RORγ is measured by competition with [3H] 25-hydroxycholesterol (Perkin Elmer NET674250UC) using a scintillation proximity assay (SPA) binding assay. The ligand binding domain of human RORγ (A262-S507) with an N-terminal His tag is expressed in E. coli and purified using nickel affinity chromatography. 15 ug/well RORγ (A262-S507) is incubated with test compound at varying concentrations in 3-fold serial dilution, with final concentrations ranging from 16.6 uM to 0.28 nM for 10 min at room temperature in PBS buffer (Invitrogen #14190-144) containing 0.5% fatty acid free BSA (Gemini Bio-Products, Cat. #700-107P) and 0.1% Glycerol (Sigma Cat#G5516). 10 nM of [3H] 25-hydroxycholesterol is then added, and the reaction is incubated for 10 min. 10 mg/mL of Copper-His Tag-PVT beads (Perkin Elmer cat #RPNQ0095) are added, and the mixture is incubated for 60 min. The reaction is read on a TopCount Microplate scintillation plate reader (Perkin Elmer). The competition data of the test compound over a range of concentrations was plotted as percentage inhibition of radioligand specifically bound in the absence of test compound (percent of total signal). After correcting for non-specific binding, IC50 values were determined. The IC50 value is defined as the concentration of test compound needed to reduce [3H] 25-hydroxycholesterol specific binding by 50% and is calculated using the four parameter logistic equation to fit the normalized data.
Measurement: IC50 = 47 nM (pChEMBL 7.329999923706055)
Descriptors: MW 653.62, ALogP 4.84, HBA 5, HBD 0, PSA 87.23, QED 0.33
Compound (SMILES): CC(C)(C)OC(=O)N1CCC(CC#N)(n2cc(C(N)=O)c(Nc3cccc(Cn4ccnc4)c3)n2)CC1
InChIKey: UEOFWGMPBJMCRS-UHFFFAOYSA-N
Target: Tyrosine-protein kinase JAK1 (SINGLE PROTEIN), Homo sapiens
UniProt: P23458
Assay: HTRF 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 uL of 1.11x enzyme and 1.11x substrate in 1x 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 room temperature to allow compound binding to equilibrate. After equilibration, 2 uL of 10xATP in 1x assay buffer was added to initiate the kinase reaction and the plates were shaken and then incubated at room temperature for 120 minutes. At the end of the incubation, 20 uL of 2x 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.
Measurement: IC50 = 0.6 nM (pChEMBL 9.220000267028809)
Descriptors: MW 504.6, ALogP 3.61, HBA 9, HBD 2, PSA 144.09, QED 0.5
Compound (SMILES): CC(C)(C)CN1CCC2(CC1)CN(c1ccccc1NC(=O)Nc1ccc(OC(F)(F)F)cc1)c1c(O)ccc(-c3nc4c(Cl)cccc4s3)c12
InChIKey: DIDVGGOWANYFFD-UHFFFAOYSA-N
Target: P2Y purinoceptor 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P47900
Assay: Scintillation Proximity Assay (SPA): A SPA membrane binding assay was used to identify inhibitors of [33P] 2MeS-ADP binding to cloned human P2Y1 receptors (The P2Y1 receptor membranes were provided by Biology and the cloning of the receptor and P2Y1 receptor membrane preparation is same as described by Biology). Binding reactions were performed in 384-well OptiPlates (PerkinElmer Life Sciences, Cat #6007299) in a volume of 50 uL containing 15 fmol of P2Y1 receptor (1.7 ug of total protein), 0.3 nM [33P] 2MeS-ADP (PerkinElmer; 2,000 Ci/mmol), various concentrations of the test compound (usually between 10 uM and 160 pM) in Buffer B containing 1% DMSO in assay buffer (15 mM, HEPES, 145 mM potassium chloride, 5 mM sodium Chloride, 5 mM EDTA, 0.1 mM MgCl2, pH 7.4) and 100 ug of SPA bead (WGA polystyrene Image beads, #RPNQ 0260V, Amersham). Reactions were allowed to proceed to completion at room temperature for 1 hour followed by centrifugation of the plate for 5 min. About 40 uL of the aqueous soluttion was aspirated. Plates were sealed and the [33P] 2MeS-ADP bound to the P2Y1 receptor membranes that were bound to the SPA bead were determined in a Gen 4 LEADSEEKERSM (Amersham) Image Reader. Dose-response curves (IC50) were fit by non-linear regression (Toolset an in house data processing program) and binding constants (Ki) calculated using the Cheng-Prusoff relationship (Ki=IC50/(1+L/Kd) in which a Kd for 2MeS-ADP to the P2Y1 receptor was determined to be 14 nM.
Measurement: Ki = 22.8 nM (pChEMBL 7.639999866485596)
Descriptors: MW 736.26, ALogP 10.4, HBA 7, HBD 3, PSA 89.96, QED 0.16
Compound (SMILES): NS(=O)(=O)c1cccc(NC(=O)c2cc3ccccc3nc2Oc2ccccc2)c1
InChIKey: LBDGGIUDYRLMHK-UHFFFAOYSA-N
Target: Sodium channel protein type 10 subunit alpha (SINGLE PROTEIN), Rattus norvegicus
UniProt: Q62968
Assay: Electrophysiology Assay: Patch clamp electrophysiology was used to assess the efficacy and selectivity of sodium channel blockers in dorsal root ganglion neurons. Rat neurons were isolated from the dorsal root ganglions and maintained in culture for 2 to 10 days in the presence of NGF (50 ng/ml) (culture media consisted of NeurobasalA supplemented with B27, glutamine and antibiotics). Small diameter neurons (nociceptors, 8-12 um in diameter) were visually identified and probed with fine tip glass electrodes connected to an amplifier (Axon Instruments). The "voltage clamp" mode was used to assess the compound's IC50 holding the cells at -60 mV. In addition, the "current clamp" mode was employed to test the efficacy of the compounds in blocking action potential generation in response to current injections.
Measurement: IC50 = 81 nM (pChEMBL 7.090000152587891)
Descriptors: MW 419.46, ALogP 3.93, HBA 5, HBD 2, PSA 111.38, QED 0.51
Compound (SMILES): COC1(C(F)(F)F)CC(c2nc(-c3ccc(C)c(N(C)C(=O)c4cnc5ccccn45)c3)no2)C1
InChIKey: VLIRSVBOVAFGHU-UHFFFAOYSA-N
Target: Mast/stem cell growth factor receptor Kit (SINGLE PROTEIN), Homo sapiens
UniProt: P10721
Assay: Cell Proliferation Assay: The compounds was tested for inhibition of SCF dependent proliferation using human Mo7e cells which endogenously express c-kit in a 384 well format. Three-fold serially diluted test compounds (Cmax=10 mM) were evaluated for their antiproliferative activity of Mo7e cells stimulated with human recombinant SCF. After 48 hours of incubation at 37° C., cell viability was measured by adding 25 uL of CellTiter Glo (Promega) to the cells and the luminescence was measured by a CLIPR CCD camera (Molecular Devices).
Measurement: IC50 = 999 nM (pChEMBL 6)
Descriptors: MW 485.47, ALogP 4.79, HBA 7, HBD 0, PSA 85.76, QED 0.4
Compound (SMILES): COCc1ccc(C(C)=O)c2sc(C(=O)Nc3ccc4ccccc4n3)c(C)c12
InChIKey: IDQWJMSEANBNGL-UHFFFAOYSA-N
Target: cAMP and cAMP-inhibited cGMP 3',5'-cyclic phosphodiesterase 10A (SINGLE PROTEIN), Homo sapiens
UniProt: Q9Y233
Assay: Activity Assay: A human acute lymphoblastic lymphoma T cell line MOLT-4 (which can be purchased under ATCC No. CRL-1582 from ATCC) was cultured in an RPMI1640 medium containing 10% fetal bovine serum to obtain 5108 MOLT-4 cells. The cells were recovered by centrifugation and suspended in 10 ml of buffer solution A (25 mM Tris-HCl, 5 mM 2-mercaptoethanol, 2 mM benzamidine, 2 mM EDTA, and 0.1 mM 4-(2-aminoethyl)benzenesulfonyl hydrochloride, pH 7.5). The cells were homogenized using a Polytron homogenizer and centrifuged (4° C., 25,000 g, 10 minutes). Then, the supernatant was further ultracentrifuged (4° C., 100,000 g, 60 minutes). The obtained supernatant was filtered through a 0.2um filter to obtain a soluble fraction.HiTrap Q HP column (5 ml2, GE Healthcare Japan Corp.) equilibrated with buffer solution A was charged with the obtained soluble fraction. Phosphodiesterase was eluted with 300 ml of buffer solution A containing a linear gradient solution of 0 to 0.8 M NaCl to recover sixty 5-ml fractions. Each fraction was tested for cAMP-metabolizing phosphodiesterase activity. In each fraction, a fraction eluted as an active peak centered around 250 mM NaCl was collected from fractions that had cAMP metabolic activity and did not lose the metabolic activity by 10uM rolipram (PDE4 selective inhibitor) and 10uM milrinone (PDE3 selective inhibitor). In order to confirm whether or not PDE10A mRNA was expressed in the MOLT-4 cells, total RNA was prepared from the MOLT-4 cells according to a standard method and analyzed by RT-PCR using PDE10A gene-specific primers (PDE10A sense primer: 5'-TGCTCCATGGTGGAAGTGGA-3' (SEQ ID NO: 1) and PDE10A antisense primer: 5'-CAACTGGAAGCATGCGGTCA-3' (SEQ ID NO: 2)). As a result, PDE10A mRNA was detected from the total RNA of the MOLT-4 cells. On the other hand, total RNA was prepared from Jurkat cells, one type of human T cell, and similarly subjected to RT-PCR. As a result, PDE10A mRNA was rarely detected. An active peak centered around 250 mM NaCl was not observed in fractions obtained by the treatment of Jurkat cells in the same way as above. Eur J Biochem. 1999, 266 (3), 1118-2 has reported that PDE10A from the rat striatum and testis is eluted as an active peak centered around 250 mM NaCl.
Measurement: IC50 = 1.1 nM (pChEMBL 8.960000038146973)
Descriptors: MW 404.49, ALogP 5.36, HBA 5, HBD 1, PSA 68.29, QED 0.45
Compound (SMILES): Cc1c(N(Cc2ccccn2)S(=O)(=O)c2ccc(C(=O)O)cc2)sc2ccccc12
InChIKey: UFPGRAYJWQFBKN-UHFFFAOYSA-N
Target: Transient receptor potential M8 protein (SINGLE PROTEIN), Canis lupus familiaris
UniProt: Q1A7N1
Assay: Functional Assay: The functional activity of compounds of the formula (I) was determined by measuring changes in intracellular calcium concentration using a Ca2+-sensitive fluorescent dye. The changes in fluorescent signal were monitored by a fluorescence plate reader, either a FLIPR (Molecular Devices) or FDSS (Hamamatsu). Increases in intracellular Ca2+ concentration were readily detected upon activation with icilin.At 24 hrs prior to assay, HEK293 cells stably expressing canine TRPM8 were seeded in culture medium in black wall, clear-base poly-D-lysine coated 384-well plates (BD Biosciences, NJ, USA) and grown overnight in 5% CO2 at 37° C. On assay day, growth media was removed and cells were loaded with Calcium 3 Dye (Molecular Devices) for 35 min at 37° C., under 5% CO2 and then for 25 min at room temperature and atmosphere. Subsequently, cells were tested for agonist-induced increases in intracellular Ca2+ levels using FLIPR or FDSS. Cells were challenged with a compound of the Formula (I) (at varying concentrations) and intracellular Ca2+ was measured for 5 min prior to the addition of icilin to all wells to achieve a final concentration that produces approximately an 80% maximal response. EC50 or IC50 values for compounds of the present invention were determined from eight-point dose-response studies.
Measurement: IC50 = 55 nM (pChEMBL 7.260000228881836)
Descriptors: MW 438.53, ALogP 4.7, HBA 5, HBD 1, PSA 87.57, QED 0.47
Compound (SMILES): CC[C@H](Nc1nc(N)nc(N)c1C#N)c1nc2c(F)ccc(C#N)c2c(=O)n1-c1cccnc1
InChIKey: WHLUYTXJVKSHRJ-HNNXBMFYSA-N
Target: Phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit delta isoform (SINGLE PROTEIN), Homo sapiens
UniProt: O00329
Assay: Time-Resolved Fluorescence Resonance Energy transfer (TR-FRET) Assay: Class I PI3K isoforms were expressed and purified as heterodimeric recombinant proteins. All assay reagents and buffers for the TR-FRET assay were purchased from Millipore. PI3K isoforms were assayed under initial rate conditions in the presence of 25 mM Hepes (pH 7.4), and 2× Km ATP (75-500 μM), 2 μM PIP2, 5% glycerol, 5 mM MgCl2, 50 mM NaCl, 0.05% (v/v) Chaps, 1 mM dithiothreitol, 1% (v/v) DMSO at the following concentrations for each isoforms: PI3Kα, PI3Kβ, and PI3Kδ between 25 and 50 pM, and PI3Kγ at 2 nM. After an assay reaction time of 30 minutes at 25° C., reactions were terminated with a final concentration of 10 mM EDTA, 10 nM labeled-PIP3, and 35 nM Europium labeled GRP-1 detector protein before reading TR-FRET on an Envision plate reader (Ex: 340 nm; Em: 615/665 nm; 10lps delay and 500 μs read window).
Measurement: IC50 = 35 nM (pChEMBL 7.460000038146973)
Descriptors: MW 456.45, ALogP 2.18, HBA 11, HBD 3, PSA 185.21, QED 0.4
Compound (SMILES): CN1CCN(CCC(=O)Nc2ccc(NC(=O)N3Cc4ccccc4C3)cc2)CC1
InChIKey: DHTVRHWDRCZRBG-UHFFFAOYSA-N
Target: Nicotinamide phosphoribosyltransferase (SINGLE PROTEIN), Homo sapiens
UniProt: P43490
Assay: Time-Resolved Fluorescence Resonance Energy Transfer (TR-FRET) Binding Assay : Probe 1:The assay was carried out in 18 uL low volume plates (Owens Corning) in reaction buffer (50 mM HEPES (NaOH), pH 7.5, 100 mM NaCl, 10 mM MgCl2, 1 mM DTT, 1% Glycerol) using 6.8 nM recombinant, human, C-terminally-His tagged NAMPT, 1 nM Tb-anti-His antibody (Invitrogen, Cat #PV5895), and 200 nM probe (Oregon Green 488-conjugated APO0866; A-1251667.0. Plates were covered, and reactions were carried out for 2-3 hours. Plates were read with Envision (Laser Lantha low volume protocol) after 2 to 3 hours. Excitation was carried out at 337 nm, and the ratio of emission of Oregon Green (520 nm) to terbium (492 nm) was determined and used to calculate IC50 values of test compounds.
Measurement: IC50 = 114 nM (pChEMBL 6.940000057220459)
Descriptors: MW 407.52, ALogP 2.81, HBA 4, HBD 2, PSA 67.92, QED 0.8
Compound (SMILES): CN(c1ccc(Cl)cc1)c1ccc(C(=O)c2ccc(C(=O)C(C)(C)c3ccccc3)c(C(=O)O)c2)nc1
InChIKey: CZZBHEACPMFSDJ-UHFFFAOYSA-N
Target: Leukotriene C4 synthase (SINGLE PROTEIN), Homo sapiens
UniProt: Q16873
Assay: Inhibition of recombinant human LTC4 synthase expressed in Pichia pastoris using LTA4 as substrate preincubated for 10 mins followed by substrate addition measured after 5 mins by HTRF method
Measurement: IC50 = 55 nM (pChEMBL 7.260000228881836)
Descriptors: MW 512.99, ALogP 6.59, HBA 5, HBD 1, PSA 87.57, QED 0.27
Compound (SMILES): O=c1[nH]c(-c2ccc(S(=O)(=O)N(CCC(F)(F)F)c3cc4ccccc4s3)cc2)ns1
InChIKey: UTCUDJBSIMPCLK-UHFFFAOYSA-N
Target: Transient receptor potential M8 protein (SINGLE PROTEIN), Canis lupus familiaris
UniProt: Q1A7N1
Assay: Functional Assay: The functional activity of compounds of the formula (I) was determined by measuring changes in intracellular calcium concentration using a Ca2+-sensitive fluorescent dye. The changes in fluorescent signal were monitored by a fluorescence plate reader, either a FLIPR (Molecular Devices) or FDSS (Hamamatsu). Increases in intracellular Ca2+ concentration were readily detected upon activation with icilin.At 24 hrs prior to assay, HEK293 cells stably expressing canine TRPM8 were seeded in culture medium in black wall, clear-base poly-D-lysine coated 384-well plates (BD Biosciences, NJ, USA) and grown overnight in 5% CO2 at 37° C. On assay day, growth media was removed and cells were loaded with Calcium 3 Dye (Molecular Devices) for 35 min at 37° C., under 5% CO2 and then for 25 min at room temperature and atmosphere. Subsequently, cells were tested for agonist-induced increases in intracellular Ca2+ levels using FLIPR or FDSS. Cells were challenged with a compound of the Formula (I) (at varying concentrations) and intracellular Ca2+ was measured for 5 min prior to the addition of icilin to all wells to achieve a final concentration that produces approximately an 80% maximal response. EC50 or IC50 values for compounds of the present invention were determined from eight-point dose-response studies.
Measurement: IC50 = 144.8 nM (pChEMBL 6.840000152587891)
Descriptors: MW 485.53, ALogP 4.86, HBA 6, HBD 1, PSA 83.13, QED 0.42
Compound (SMILES): COc1c(OCC2CC(F)(F)C2)ccc2c1CCN(Cc1ccc([C@H](C)NC(C)=O)cc1)C2C
InChIKey: DIVQBPMASBFEFI-ZENAZSQFSA-N
Target: Acetyl-CoA carboxylase 2 (SINGLE PROTEIN), Homo sapiens
UniProt: O00763
Assay: Spectrophotometric 384 Well Assay: Assay reactions are then carried out in 384-well plates, with hACC2 in an appropriate dilution and at final assay concentrations (f.c.) of 100 mM Tris (pH 7.5), 10 mM trisodium citrate, 25 mM KHCO3, 10 mM MgCl2, 0.5 mg/ml BSA, 3.75 mM reduced L-glutathione, 15 U/ml lactate dehydrogenase, 0.5 mM phosphoenolpyruvate, 15 U/ml pyruvate kinase, compounds at different concentrations at final DMSO concentrations of 1%.The enzymatic reaction is then started by addition of a mixture of NADH, acetyl Coenzyme A (both 2000 f.c.) and ATP (500 uM f.c.). The decrease of the optical density (slope S) is then determined at 25° C. at a wavelength of 340 nm over 15 minutes in a spectrophotometric reader.Each assay microtiter plate contains wells with vehicle instead of compound as controls for the non-inhibited enzyme (100% CTL; HIGH) and wells without acetyl-CoA as controls for non-specific NADH degradation (0% CTL; LOW).
Measurement: IC50 = 189 nM (pChEMBL 6.71999979019165)
Descriptors: MW 472.58, ALogP 5.44, HBA 4, HBD 1, PSA 50.8, QED 0.56
Compound (SMILES): N#Cc1ccc(C2C3=C(CCC3=O)N(c3cccc(C(F)(F)F)c3)C(=O)N2C(=O)N[C@H]2CCCC[C@@H]2O)cc1
InChIKey: VTLLQJBZQLMZMJ-IIUCQXFBSA-N
Target: Neutrophil elastase (SINGLE PROTEIN), Homo sapiens
UniProt: P08246
Assay: Neutrophil Elastase Assay: Materials: Human neutrophil elastase was purchased from Calbiochem (Cat. No.: 324681) and the elastase substrate MeOSuc-Ala-Ala-Pro-Val-AMC from Bachem (Cat. No.: I-1270). All other materials were of the highest grade commercially available. The following buffers were used: Compound buffer: 100 mM Tris, 500 mM NaCl, adjusted to pH 7.5; Assay buffer: 100 mM Tris, 500 mM NaCl, adjusted to pH 7.5, containing 0.01% BSA. Assay conditions: Test compounds were prediluted in DMSO and subsequently in compound buffer (5% DMSO final). 5 uL of these compound dilutions were mixed with 10 ul Neutrophil elastase (9 ng/ml in assay buffer) in a black 384 well OptiPlate (Perkin Elmer, Cat No.: 6007270) and incubated for 15 min at room temperature. Subsequently 10 uL substrate solution in assay buffer were added (250 uM final concentration) and the plates were incubated for 60 min at room temperature. After inactivation of the enzyme, fluorescence intensities were measured at 380 nm excitation and 460 nm emission wavelengths. Each plate contains wells with a high value control (DMSO+enzyme+substrate) and wells with a low value control (DMSO+inactivated enzyme+substrate). IC50 values were estimated using a sigmoidal concentration response curve with variable slope.
Measurement: IC50 < 1 nM (pChEMBL 9)
Descriptors: MW 538.53, ALogP 5.19, HBA 5, HBD 2, PSA 113.74, QED 0.56
Compound (SMILES): Brc1cnc(NCCc2ccccn2)nc1Nc1cc(C2CC2)n[nH]1
InChIKey: MRKOFNHZJMWMPX-UHFFFAOYSA-N
Target: Insulin-like growth factor 1 receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P08069
Assay: Inhibition of human IGF1R kinase domain (M954 to C1367 residues) un-phosphorylated apo-enzyme form expressed in baculovirus based expression system using poly-EY peptide incubated for 2 hrs by luciferase-luciferin coupled chemiluminescent assay
Measurement: IC50 >= 5000 nM (pChEMBL 5.301000118255615)
Descriptors: MW 400.28, ALogP 3.63, HBA 6, HBD 3, PSA 91.41, QED 0.56
Compound (SMILES): CC(C)C[C@@H]1NCCOc2ccccc2CCCNC(=O)[C@H](Cc2ccccc2)NC(=O)[C@H](C)NC1=O
InChIKey: SXNDYZAPVYDWEK-TUSQITKMSA-N
Target: Growth hormone secretagogue receptor type 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q92847
Assay: Displacement of [125I]-Ghrelin from human GSHR1a transfected in HEK293 cell membranes after 30 mins by microplate scintillation and luminescence counting method
Measurement: Ki = 1000 nM (pChEMBL 6)
Descriptors: MW 508.66, ALogP 2.36, HBA 5, HBD 4, PSA 108.56, QED 0.51
Compound (SMILES): CC[C@]1(CC(=O)O)C[C@H](c2cccc(Cl)c2)[C@@H](c2ccc(Cl)cc2)N([C@H](CS(=O)(=O)c2cccc(F)c2)C2CC2)C1=O
InChIKey: FMTBMXZIUHWMDV-XDFGCPGZSA-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 = 1.4 nM (pChEMBL 8.850000381469727)
Descriptors: MW 632.58, ALogP 7.31, HBA 4, HBD 1, PSA 91.75, QED 0.25
Compound (SMILES): CCOc1ccc(C[C@@H](NC(=O)c2cccnc2C)C(=O)N[C@@H](Cc2ccccc2)C(=O)NCc2ccc(CN)cc2)cc1
InChIKey: ALBLURFVONFERJ-AJQTZOPKSA-N
Target: Plasma kallikrein (SINGLE PROTEIN), Homo sapiens
UniProt: P03952
Assay: In Vitro Inhibition Assay: Plasma kallikrein inhibitory activity in vitro was determined using standard published methods (see e.g. Johansen et al., Int. J. Tiss. Reac. 1986, 8, 185; Shori et al., Biochem. Pharmacol., 1992, 43, 1209; Stiirzebecher et al., Biol. Chem. Hoppe-Seyler, 1992, 373, 1025). Human plasma kallikrein (Protogen) was incubated at 37° C. with the fluorogenic substrate H-DPro-Phe-Arg-AFC and various concentrations of the test compound. Residual enzyme activity (initial rate of reaction) was determined by measuring the change in optical absorbance at 410 nm and the IC50 value for the test compound was determined.
Measurement: IC50 = 185 nM (pChEMBL 6.730000019073486)
Descriptors: MW 593.73, ALogP 3.63, HBA 6, HBD 4, PSA 135.44, QED 0.18
Compound (SMILES): N#Cc1cccc2c(O[C@H]3CC[C@H](NC(=O)c4cccc(F)c4)CC3)ccnc12
InChIKey: YPARZKBHGSVSFI-WGSAOQKQSA-N
Target: Androgen receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P10275
Assay: Transactivation Assay: For determining the androgen receptor-dependent transcription, a cellular assay system was used, consisting of PC-3 cells (Kaighn et al., Invest. Urol. 17: 16-23, 1979), which express the human androgen receptor stably and recombinantly (full length, wild-type form, see Swiss-Prot Acc. No. P10275, Entry Version 159, Sequence Version 2). In addition, these PC3 cells contain a stably integrated reporter gene plasmid, which is based on the commercially available plasmid pGL4.14 (#E6691, Promega Corporation, Madison, Wis., USA) and contains the luciferase gene from the American firefly (Photinus pyralis) under the control of the MMTV promoter (Cato et al., EMBO J. 6: 363-368, 1987). These cells were propagated in routine cell culture at 37° C. and 5% CO2 in a medium containing 90% RPMI 1640 (Invitrogen GmbH, Darmstadt, Germany), 100 U penicillin, 100 μg/ml streptomycin (Invitrogen), 4 mM L-glutamine (Invitrogen), 10% fetal calf serum (FCS Serum Gold, PAA Laboratories GmbH, Cölbe, Germany), 600 μg/ml Geneticin (G418-sulphate, Invitrogen) and 10 μg/ml puromycin (Sigma Aldrich GmbH, Germany). For carrying out the transactivation assays, approx. 1000 cells per well were plated out in a 384-well cell culture plate in a medium that contained activated charcoal-treated calf serum (FCS Serum Gold, PAA Laboratories) at a concentration of 5% (v/v). The test substances were added in a concentration series from 5.12×10^−12 to 1×10^−5 M in the presence of 1×10^−1° R1881 (methyltrienolone). The test plates were incubated overnight at 37° C. and 5% CO2. After 16 hours, 15 μl of Steady Glo Lysis and Detection reagent (E2550, Promega Corporation, Madison, Wis., USA) was added per well and the luminescence was read in a Topcount Luminometer (PerkinElmer, Waltham, Mass., USA) for 4 seconds per well. The luminescence values obtained were normalized, wherein 100% corresponded to the effect of the unstimulated control (without R1881), and 0% corresponded to the effect of the stimulated control (R1881 plus DMSO instead of test substance).
Measurement: IC50 = 52 nM (pChEMBL 7.28000020980835)
Descriptors: MW 389.43, ALogP 4.37, HBA 4, HBD 1, PSA 75.01, QED 0.72
Compound (SMILES): CC1(C)CCC(CN2CCN(c3ccc(C(=O)NS(=O)(=O)c4cnc(OCC5CCOCC5)c(C#N)c4)c(Oc4cnc5[nH]ccc5c4)c3)CC2)=C(c2ccc(Cl)cc2)C1
InChIKey: IYJJTGJTNSOTQA-UHFFFAOYSA-N
Target: Apoptosis regulator Bcl-2 (SINGLE PROTEIN), Homo sapiens
UniProt: P10415
Assay: Time Resolved-Fluorescence Resonance Energy Transfer (TR-FRET) Assay: Representative compounds were serially diluted in dimethyl sulfoxide (DMSO) starting at 50 uM (2x starting concentration; 10% DMSO) and 10 uL were transferred into a 384-well plate. Then 10 uL of a protein/probe/antibody mix was added to each well at final concentrations listed in TABLE 1. Protein:GST-Bcl-2, Probe: F-Bak Peptide Probe Acetyl-GQVGRQLAIIGDK(6-FAM)INR-amide(SEQ ID NO: 1), Protein(nM): 1, Probe (nM): 100, Antibody: Tb-anit-GST, Antibody (nm): 1.  The samples are then mixed on a shaker for 1 minute and incubated for an additional 3 hours at room temperature. For each assay, the probe/antibody and protein/probe/antibody were included on each assay plate as negative and positive controls, respectively. Fluorescence was measured on the ENVISION plate reader (Perkin Elmer) using a 340/35 nm excitation filter and 520/525 (F-Bak peptide) and 495/510 nm (Tb-labeled anti-Histidine antibody) emission filters.
Measurement: Ki = 0.017 nM (pChEMBL 10.770000457763672)
Descriptors: MW 850.44, ALogP 7.99, HBA 11, HBD 2, PSA 162.77, QED 0.13
Compound (SMILES): CC#CCn1c(N2CCC[C@@H](N)C2)nc2c1c(=O)n(CCCCN1CCC(C(=O)OC(C)C)CC1)c(=O)n2C
InChIKey: XPNLCZIPHRBXMF-JOCHJYFZSA-N
Target: Dipeptidyl peptidase 4 (SINGLE PROTEIN), Homo sapiens
UniProt: P27487
Assay: Inhibition of DPP4 (unknown origin) using Gly-Pro-p-nitroanilide as substrate incubated for 1 hr
Measurement: IC50 = 400 nM (pChEMBL 6.400000095367432)
Descriptors: MW 541.7, ALogP 1.29, HBA 11, HBD 1, PSA 120.62, QED 0.29
Compound (SMILES): Cn1cc(-c2cc3c(-c4cccc(N5CCC[C@H](O)C5)n4)n[nH]c3cn2)cn1
InChIKey: SCICSUQDLRSZHW-AWEZNQCLSA-N
Target: Serine/threonine-protein kinase pim-1 (SINGLE PROTEIN), Homo sapiens
UniProt: P11309
Assay: PIM Kinase Binding Activity Assay: PIM-1, PIM-2, and PIM-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.
Measurement: Ki = 0.154 nM (pChEMBL 9.8100004196167)
Descriptors: MW 375.44, ALogP 2.38, HBA 7, HBD 2, PSA 95.75, QED 0.57
Compound (SMILES): CC(=O)N1CCC(C(=O)N2CC[C@](c3ccc(C(F)(C(F)(F)F)C(F)(F)F)cc3)(S(=O)(=O)c3ccc(F)cc3)C2)CC1
InChIKey: ZCVYLZVBYDUMQY-DEOSSOPVSA-N
Target: Nuclear receptor ROR-gamma (SINGLE PROTEIN), Homo sapiens
UniProt: P51449
Assay: Scintillation Proximity Assay (SPA): The binding of potential ligands to RORγ is measured by competition with [3H] 25-hydroxycholesterol (Perkin Elmer NET674250UC) using a scintillation proximity assay (SPA) binding assay. The ligand binding domain of human RORγ (A262-S507) with an N-terminal His tag is expressed in E. coli and purified using nickel affinity chromatography. 15 ug/well RORγ (A262-S507) is incubated with test compound at varying concentrations in 3-fold serial dilution, with final concentrations ranging from 16.6 uM to 0.28 nM for 10 min at room temperature in PBS buffer (Invitrogen #14190-144) containing 0.5% fatty acid free BSA (Gemini Bio-Products, Cat. #700-107P) and 0.1% Glycerol (Sigma Cat#G5516). 10 nM of [3H] 25-hydroxycholesterol is then added, and the reaction is incubated for 10 min. 10 mg/mL of Copper-His Tag-PVT beads (Perkin Elmer cat #RPNQ0095) are added, and the mixture is incubated for 60 min. The reaction is read on a TopCount Microplate scintillation plate reader (Perkin Elmer). The competition data of the test compound over a range of concentrations was plotted as percentage inhibition of radioligand specifically bound in the absence of test compound (percent of total signal). After correcting for non-specific binding, IC50 values were determined. The IC50 value is defined as the concentration of test compound needed to reduce [3H] 25-hydroxycholesterol specific binding by 50% and is calculated using the four parameter logistic equation to fit the normalized data.
Measurement: IC50 = 48 nM (pChEMBL 7.320000171661377)
Descriptors: MW 626.57, ALogP 5.28, HBA 4, HBD 0, PSA 74.76, QED 0.33