lovingscience/chembl-v37-raw-activities huggingface dataset:
Compound (SMILES): CC(C)N(C)C(=O)N1C2CCC1CN(Cc1c(-c3ccc(Cl)cc3)nc3ncccn13)C2
InChIKey: ABCBAFNDQVXVCQ-UHFFFAOYSA-N
Target: Potassium channel subfamily K member 3 (SINGLE PROTEIN), Homo sapiens
UniProt: O14649
Assay: In Vitro Electrophysiological Assay: Table 1: In Vitro Electrophysiological Analysis of the Human TASK-1 and TASK-3 Channels Via Two-Electrode Voltage Clamp Technique in Xenopus laevis Oocytes. Xenopus laevis oocytes were selected as described elsewhere by way of illustration [Decher et al., FEBS Lett. 492, 84-89 (2001)]. Subsequently, the oocytes were injected with 0.5-5 ng of a cRNA solution coding for TASK-1 or TASK-3. For the electrophysiological analysis of the channel proteins expressed in the oocytes, the two-electrode voltage clamp technique [Stühmer, Methods Enzymol. 207, 319-339 (1992)] was used. The measurements were conducted as described [Decher et al., FEBS Lett. 492, 84-89 (2001)] at room temperature (21-22° C.) using a Turbo TEC 10CD amplifier (NPI), recorded at 2 kHz and filtered with 0.4 kHz. Substance administration was performed using a gravitation-driven perfusion system. Here, the oocyte is located in a measuring chamber and exposed to the solution stream of 10 ml/min. The level in the measuring chamber is monitored and regulated by sucking off the solution using a peristaltic pump.
Measurement: IC50 = 7400 nM (pChEMBL 5.130000114440918)
Descriptors: MW 452.99, ALogP 4.16, HBA 5, HBD 0, PSA 56.98, QED 0.6
Compound (SMILES): O=C(Nc1cc(-c2ccc3ncn(CC4CCOCC4)c3c2)c(Cl)cn1)C1CCS(=O)(=O)C12CCNC2
InChIKey: CCKOYRIGIRVEII-UHFFFAOYSA-N
Target: Cyclin-dependent kinase 9 (SINGLE PROTEIN), Homo sapiens
UniProt: P50750
Assay: TR-FRET Kinase Assay: CDK9 enzyme activities were measured using LANCE ULight TR-FRET kinase assay reagents (PerkinElmer, Waltham, Mass.). Compounds were directly added in 100% DMSO to white low volume assay plates (Perkin Elmer Proxiplate 6008289) using a Labcyte Echo acoustic dispenser. Assay reagents in serine/threonine kinase assay buffer containing 20 mM HEPES, 10 mM MgCl2, 100 mM Na3VO4, and 0.0075% Triton X-100. were added for final reaction mixture concentrations of 1000 μM ATP, 100 nM U-light MBP peptide (Perkin Elmer TRF0109M) and reaction initiated with 4 nM CDK9/Cyclin T1 (Carna Biosciences 04-110). The kinase reaction was carried out for 30 minutes before addition of stopping buffer to a final of 20 mM EDTA and 0.5 nM of LANCE Ultra Europium anti-phospho-MBP antibody (PerkinElmer TRF0201M) in LANCE detection buffer (PerkinElmer CR97-100). The reaction was equilibrated for 1 hour and the signal read in the Perkin Elmer Envision in TR-FRET mode (excitation at 320 nm and emission at 615665 nm).
Measurement: IC50 = 1500 nM (pChEMBL 5.820000171661377)
Descriptors: MW 544.08, ALogP 3.28, HBA 8, HBD 2, PSA 115.21, QED 0.51
Compound (SMILES): CC(C)Oc1ccc(-c2cc(COC(C)(C)C(=O)O)nn2Cc2ccccc2Cl)s1
InChIKey: FMUWIAZXIXXUOT-UHFFFAOYSA-N
Target: Monocarboxylate transporter 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P53985
Assay: Biological Activity Assay 4: MCT1 activity may be measured using BT-20 breast cancer cells that express high native levels of MCT1, but do not express MCT4 and are known to those with skill in the art. Preparation of BCECF loaded cells are as described for Assay 1. Lactate transport assay is as described for Assay 1, except 10 mM L-lactate (rather than 50 mM) is added.
Measurement: IC50 > 33000 nM (pChEMBL 4.480999946594238)
Descriptors: MW 448.97, ALogP 5.48, HBA 6, HBD 1, PSA 73.58, QED 0.47
Compound (SMILES): CC1(C)CN(c2ccccc2NC(=O)Nc2nc3ccc(Cl)cc3s2)c2c(O)ccc(-c3nc4cc(F)ccc4s3)c21
InChIKey: MQZFLMOMVCAEMJ-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 μL containing 15 fmol of P2Y1 receptor (1.7 μg of total protein), 0.3 nM [33P] 2MeS-ADP (PerkinElmer; 2,000 Ci/mmol), various concentrations of the test compound (usually between 10 μM 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 μg 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 μL of the aqueous solution 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 LEADSEEKER (Amersham) Image Reader.
Measurement: Ki = 17 nM (pChEMBL 7.769999980926514)
Descriptors: MW 616.14, ALogP 9.14, HBA 7, HBD 3, PSA 90.38, QED 0.18
Compound (SMILES): COCc1ccc(NC(=O)c2c(C#CC3(O)CCOC3)n(C3(C)CC3)c3ncnc(N)c23)cc1
InChIKey: DPCJGKJHILGTNQ-UHFFFAOYSA-N
Target: Proto-oncogene tyrosine-protein kinase receptor Ret (SINGLE PROTEIN), Homo sapiens
UniProt: P07949
Assay: Measurement of Inhibitory Activity Against RET (In Vitro): Regarding the conditions for measurement of in vitro inhibitory activity of compounds against RET kinase activity, the website of AnaSpec states that Srctide (GEEPLYWSFPAKKK) corresponds to the substrate peptide for reaction to measure RET kinase activity. Thus, the amino acid sequence was partly modified and biotinylated to prepare biotinylated peptides (biotin-EEPLYWSFPAKKK). The purified recombinant human RET protein used in the test was purchased from Carna Biosciences, Inc.To measure the inhibitory activity, first, the compounds of the present invention were individually diluted with dimethyl sulfoxide (DMSO) stepwise. Subsequently, RET protein, the substrate peptide (the final concentration: 250 nM), magnesium chloride (the final concentration: 10 mM), ATP (the final concentration: 10 μM), and a solution of the compound of the present invention in DMSO (the final concentration of DMSO: 2.5%) were added to a buffer for kinase reaction (13.5 mM Tris, pH of 7.5, 2 mM dithiothreitol, 0.009% Tween-20). Each of the mixtures was incubated at 25° C. for 100 minutes to perform a kinase reaction. EDTA was then added thereto to give a final concentration of 24 mM so that the reaction was terminated. A detection solution containing Eu-labeled antiphosphotyrosine antibody PT66 (PerkinElmer) and SureLight APC-SA (PerkinElmer) was added thereto, and each mixture was allowed to stand at room temperature for 2 hours or more. Finally, the intensity of fluorescence under the excitation light with a wavelength of 337 nm was measured with a PHERAstar FS (BMG Labtech) at the two wavelengths of 620 nm and 665 nm. The phosphorylation level was calculated from the ratio of the fluorescence intensity at the two wavelengths, and the compound concentration at which phosphorylation was inhibited by 50% was defined as the IC50 value (nM).
Measurement: IC50 = 0.8 nM (pChEMBL 9.100000381469727)
Descriptors: MW 461.52, ALogP 2.42, HBA 8, HBD 3, PSA 124.52, QED 0.5
Compound (SMILES): CC[C@@H](C)n1c(=O)c(NCc2ccc(S(C)(=O)=O)cc2)nc2cnc(-c3c(C)ncnc3C3CC3)nc21
InChIKey: ZHCKNBQBVJSNBZ-OAHLLOKOSA-N
Target: Nuclear receptor ROR-gamma (SINGLE PROTEIN), Homo sapiens
UniProt: P51449
Assay: Reporter Gene Assay: A nuclear receptor transactivation assay is performed to quantitate the ability of test compounds to inhibit RORγ transactivation of a luciferase reporter. A similar assay is described in: Khan et al., Bioorganic & Medicinal Chemistry Letters 23 (2013), 532-536. The system uses transiently transfected HEK 293 cells cotransfected with two plasmids (pGL4.3, luc2P/GAL4UAS/Hygro, and pBIND, Gal4DBD hRORC LBD1-3). The positive control is co-transiently transfected with both plasmids, and the negative control contains the pGL4.3 promoter sequence. Assays are assembled in 384 well plates where transiently transfected cells and test compound at varying concentrations are incubated for 20-24 h.The next day, assays plates are taken out and equilibrated at RT for 20-30 minutes. Bright-Glo Luciferase Assay System is used to detect Luciferase production. After addition of Bright GLO detection reagent, the plates are incubated at RT for 20 minutes. The plates are read on an Envision plate reader to measure luminescence signal. The RLU signal is converted to POC relative to control and blank wells.
Measurement: IC50 = 120 nM (pChEMBL 6.920000076293945)
Descriptors: MW 519.63, ALogP 3.82, HBA 10, HBD 1, PSA 132.62, QED 0.37
Compound (SMILES): CCS(=O)(=O)c1ccccc1-c1ncc2cnn(Cc3ccc(-c4nc(C(F)(F)F)cn4C)cc3)c2n1
InChIKey: NKSBTMFDBKJPOS-UHFFFAOYSA-N
Target: Ubiquitin carboxyl-terminal hydrolase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: O94782
Assay: Inhibitor Assay: Deubiquitinase activity was measured using ubiquitin-rhodamine 110 as a substrate. Cleavage of the amide bond between rhodamine and the c-terminal glycine of ubiquitin yields an increase in fluorescence signal. The assay was conducted in 20 ul total volume of assay buffer (50 mM Tris-HCl, pH 7.8, 0.5 mM EDTA, 0.01% Bovine Serum Albumin, 1 mM DTT, 0.01% Tween-20), and 0.05 nM USP1/UAF1 enzyme. Reaction was initiated by addition of 150 nM Ubiquitin-rhodamine (Boston Biochem) substrate.Compounds, dissolved in DMSO were tested in dose response format, beginning at 10 uM.Compounds depicted below were added to enzyme/assay buffer mix and incubated 10 min. Substrate mix was added, and reaction mix was read in kinetic mode for 30 min at Ex480/Em540 and IC50 response curves were plotted.
Measurement: IC50 = 150 nM (pChEMBL 6.820000171661377)
Descriptors: MW 526.54, ALogP 4.75, HBA 8, HBD 0, PSA 95.56, QED 0.32
Compound (SMILES): O=C(NCc1ccc(F)c(F)c1)c1cccnc1NCc1cc(-c2c[nH]c3ncc(-c4ccn[nH]4)cc23)ccn1
InChIKey: SYDKKYZFZMZLRT-UHFFFAOYSA-N
Target: 3-phosphoinositide-dependent protein kinase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: O15530
Assay: PDK1 kinase assay: PDK1 (amino acids 51-359) and AKT2 (amino acids 140-467 fused to PIFtide, amino acids EEQEMFRDFDYIADW) were expressed as N-terminally tagged GST fusion proteins in insect cells and purified to homogeneity. Enzyme activity was determined in a coupled PDK1/AKT/FAM-crosstide assay and phosphorylation of FAM-crosstide was determined by standard IMAP protocol (Molecular Devices). For inhibition studies, compounds were titrated 4-fold in DMSO and diluted 40-fold into assay buffer (10 mM Tris HCl pH7.2; 10 mM MgCl2; 0.01% Triton X-100; 1 mM DTT) containing PDK1, AKT2, and FAM-crosstide (final concentrations: 0.75 nM PDK1, 10 nM unphosphorylated AKT2, and 100 nM crosstide substrate). The kinase reaction was initiated by adding ATP to a final concentration of 40 μM of PDK1 and incubated at 25° C. for 30 min. To detect assay product, the kinase reaction was combined with Progressive Binding Solution (1:600 Progressive Binding Reagent, 50% Buffer A, 50% Buffer B, Molecular Devices) in a 1:3 ratio. The mixture was incubated for 2 hours at 25° C. and the plate was scanned on an Analyst AD with excitation at 485 nm and emission at 530 nm. The fluorescence polarization value P is defined by the equation below. The value mP is generated by multiplying the P value for each reaction well by a factor of 1000. The value ΔmP for each well is the mP value for that well minus the mP value for the average negative control. P=(Fpar−Fperp)/(Fpar+Fperp)  Eq.:Where par is fluorescence intensity parallel to the excitation plane; and perp is fluorescence intensity perpendicular to the excitation plane. ΔmP values were plotted as a function of compound concentration and the data were analyzed with a 4-parameter fit using GraphPad Prism software.
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 536.55, ALogP 5.23, HBA 6, HBD 4, PSA 124.27, QED 0.21
Compound (SMILES): Cc1nc2c(o1)C(=O)N(CCN1CCN(c3noc4ccccc34)CC1)CC2
InChIKey: RHACSLLPPLAATI-UHFFFAOYSA-N
Target: 5-hydroxytryptamine receptor 1A (SINGLE PROTEIN), Homo sapiens
UniProt: P08908
Assay: Binding Activity Assay: Binding affinity of the present compound for human 5-HT1A receptor, human 5-HT2A receptor, and human D2 receptor was measured by the following procedures.CHO cell membrane fraction in which human 5-HT1A receptor, human 5-HT2A receptor, and human D2 receptor were expressed was purchased from PerkinElmer, Inc. In a test for evaluating binding affinity, a test compound dissolved in dimethylsulfoxide (DMSO) and each receptor membrane sample diluted in buffer were mixed with [3H]8-OH-DPAT, [3H]Ketanserin, or [3H]Spiperone (all purchased from PerkinElmer, Inc.) for 5-HT1A receptor, 5-HT2A receptor, and D2 receptor, respectively. Each mixture was incubated at room temperature for 60 minutes. Then, the mixture was added quickly on a glassfiber filter plate (Multiscreen FB, Millipore, Inc.) coated with 0.3% aqueous polyethylenimine, and vacuum-filtered. Radioactivity remaining on the filter was measured with a liquid scintillation counter (PerkinElmer, Inc.). Binding inhibition rate was calculated from the following formula.
Measurement: Ki = 530 nM (pChEMBL 6.28000020980835)
Descriptors: MW 381.44, ALogP 1.94, HBA 7, HBD 0, PSA 78.85, QED 0.68
Compound (SMILES): Cc1ccc(N2CCC(NC3CCN(C(C)C)CC3)CC2)cc1-c1cc2ccccc2[nH]1
InChIKey: MREPSBCVPYNWMF-UHFFFAOYSA-N
Target: Transitional endoplasmic reticulum ATPase (SINGLE PROTEIN), Homo sapiens
UniProt: P55072
Assay: Biological Assay: To optimize p97 inhibitors, the C-5 trifluoromethylated trifluoromethylated indole 12 was generated as a promising lead structure. In the ADP-Glo assay, it was determined that this compound exhibits a 5.96±1.66 μM IC50 value (Table 1), after which the effect of replacing the CF3 with an SF5-group at the C-5 position as shown for compound 13 was explored. The ADP-Glo assay can be conducted as described in Chou, T.-F.; Bulfer, S. L.; Weihl, C. C.; Li, K.; Lis, L. G.; Walters, M. A.; Schoenen, F. J.; Lin, H. J.; Deshaies, R. J.; Arkin, M. R., Specific inhibition of p97/VCP atpase and kinetic analysis demonstrate interaction between D1 and D2 ATPase domains. J. Mol. Biol. 2014, 426, 2886-2899.
Measurement: IC50 = 500 nM (pChEMBL 6.300000190734863)
Descriptors: MW 430.64, ALogP 5.57, HBA 3, HBD 2, PSA 34.3, QED 0.55
Compound (SMILES): N#CCC(=O)N1CC[C@H](Oc2ccc(-c3ncnc(Nc4ccc(N5CCN(C6COC6)CC5)cc4)n3)cc2C#N)[C@H](F)C1
InChIKey: NKOOCLRGRSRSGW-IAPPQJPRSA-N
Target: Serine/threonine-protein kinase TBK1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9UHD2
Assay: TBK1 and IKKE Biological Assays: Enzymatic activity of IKKε and TBK1 was measured using a homogeneous time resolved fluorescence resonance energy transfer (TR-FRET) assay that monitors enzyme dependent phosphorylation of a biotinylated serine/threonine peptide substrate. An increase in the amount of phosphorylated peptide results in an increase in TR-FRET signal. TBK1 and IKKε were expressed and purified as full length recombinant proteins. Detection reagents for the assay were purchased from Cisbio. TBK1 and IKKε enzymes were assayed under initial rate conditions in the presence of 2×Km ATP (40-80 μM) and 1 μM peptide, hepes (pH 7), 0.1 mM orthovanadate, 0.02% NaN3, 0.01% BSA, 10 mM MgCl2, 0.01% (v/v) tritonX, 1 mM dithiothreitol, 0.5% (v/v) DMSO at the following concentrations for each enzyme: TBK1 at 2.5 nM and IKKε at 0.3 nM. After an assay reaction time of 240 minutes at 25° C., reactions were terminated with EDTA.Amount of phosphorylated peptide was determined by the addition of 125 nM streptavidin XL665 and europium cryptate labeled anti-phospho monoclonal antibody and the resulting TR-FRET signal was recorded on an Envision plate reader (Ex: 340 nm; Em: 615/665 nm; 100 μs delay and 200 μs read window). Data was normalized based on a positive (1 μM Staurosporine) and negative (DMSO) controls and IC50 values calculated from the fit of the dose-response curves to a four-parameter equation. All IC50 values represent geometric mean values of a minimum of four determinations.
Measurement: IC50 = 2000 nM (pChEMBL 5.699999809265137)
Descriptors: MW 597.66, ALogP 2.91, HBA 11, HBD 1, PSA 143.53, QED 0.41
Compound (SMILES): COc1cc(-n2cnc(Nc3nc(N4CCC[C@@H]4CO)nn4cccc34)c2)cc(OC)c1OC
InChIKey: LKAMFCLNKUITLK-OAHLLOKOSA-N
Target: Activin receptor type-1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q04771
Assay: Biochemical assay: Biochemical assays to measure the inhibitory effects of the compounds were performed by ThermoFisher Scientific (Life Technologies). ALK2 inhibition was tested using LanthaScreen Eu Kinase Binding Assay screening protocol.
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 465.51, ALogP 2.65, HBA 11, HBD 2, PSA 111.2, QED 0.41
Compound (SMILES): Cc1coc2c(C)c3oc(=O)cc(-c4ccccc4Cl)c3cc12
InChIKey: GMGQZUVWVFKXEP-UHFFFAOYSA-N
Target: Potassium voltage-gated channel subfamily A member 3 (SINGLE PROTEIN), Homo sapiens
UniProt: P22001
Assay: Patch-Clamp Assay for Inhibitory Activity on Kv1.3: For a description of a Kv1.3 patch-clamp assay, see Grissmer et al., Mol. Pharmacol. 1994, 45, 1227; whole cell patch-clamp recording was performed with n≥2 individual experiments at each compound concentration using different cells. Three or more different concentrations were determined per dose response curve.
Measurement: IC50 = 2250 nM (pChEMBL 5.650000095367432)
Descriptors: MW 324.76, ALogP 5.48, HBA 3, HBD 0, PSA 43.35, QED 0.44
Compound (SMILES): Cc1cc(N)nc(C)c1CNC(=O)c1ccc2c(c1)C1OC2c2cc(Nc3ccnn3C)ccc21
InChIKey: MOBICCQSALLBBS-UHFFFAOYSA-N
Target: Plasma kallikrein (SINGLE PROTEIN), Homo sapiens
UniProt: P03952
Assay: In Vitro Plasma Kallikrein Inhibition Assay: The chromogenic substrate D-Pro-Phe-Arg-pNa, 2HCl (BIOPHEN CS-31(02) from Hyphen BioMed, Neuville-Sur-oise, France) was dissolved in 5 mL deionized water and stored at 4° C. Concentration was determined in the spectrophotometer at 342 nm using an extinction coefficient of 8270. All other chemicals were of analytical grade.Human plasma kallikrein was purchased from Enzyme Research Labs (South Bend, Ind., USA, batch HPKa 2830). A stock solution of 7 μM in 50% glycerol was stored at −20° C.Enzyme reactions were conducted in assay buffer comprised of 20 mM HEPES at pH 7.4, 150 mM NaCl, 0.1% PEG-8000 and 0.01% Triton X-100.Both enzyme and substrate were diluted in assay buffer.The compound solutions as well as the enzyme and the substrate solutions were transferred to 96-well plates (Clear, UV-Star, Flat-bottom, Half-Area plates; cat. No. 675801 Greiner Bio-one, purchased from VWR International, Arlington Heights, Ill., USA) using a Rainin LTS 96-channel pipettor (Rainin, Columbus, Ohio, USA). Plate measurements were conducted using a SPECTROStar Nano reader (BMG Labtech, San Francisco, Calif., USA). The SPECTROStar Nano is a spectrophotometer and absorbance was measured at 405 nm. We used discrete wavelength, precise, kinetic reads of 15 cycles with a 60 sec cycle time.Determination of IC50 valuesFor the determination of IC50 values, the assays were performed at room temperature in 96-well plates with a total assay volume of 85 μL per well. The test compound was dissolved in 100% DMSO. The compounds were serially diluted in DMSO in a 7 point dose response. For the assays, 66.5 μL of protease solution (protease in assay buffer) was added per well followed by the addition of 8.5 μL of compound in 100% DMSO. The final assay concentration of the human plasma kallikrein was 250 μM. After 30 min. incubation at room temperature on an orbital shaker, the reactions were started by the addition of 10 μL substrate solution (in assay buffer, final assay concentration was 600 uM). After the addition of the substrate solution the final DMSO concentration was 10%. The plate was placed again on the shaker for 5 sec, spun at 2000 rpm for 5 sec and read on the spectrophotometer. The effect of the compound on the enzymatic activity was obtained from the linear part of the progress curves and determined after 15 minutes.
Measurement: IC50 = 457 nM (pChEMBL 6.340000152587891)
Descriptors: MW 466.55, ALogP 4.21, HBA 7, HBD 3, PSA 107.09, QED 0.41
Compound (SMILES): COc1ncc(-c2cc(=O)[nH]c(-c3cc(CNC(=O)C(C)C)ccc3Cl)n2)cn1
InChIKey: NQIOBXSFUGAJME-UHFFFAOYSA-N
Target: Prostaglandin E synthase (SINGLE PROTEIN), Homo sapiens
UniProt: O14684
Assay: Inhibitory Activity Assay: Microsomes were prepared from COS-1 cells transiently transfected with a plasmid containing human mPGES-1 cDNA, and used as mPGES-1 enzyme. The mPGES-1 enzyme was diluted with a sodium phosphate buffer (pH 7.2) containing reduced glutathione (2.5 mM) and EDTA (1 mM), DMSO or a DMSO solution of a test compound (final concentration of DMSO was 1%) was added to the enzyme, and the mixture was preincubated at 4° C. for 15 minutes. Then, PGH2 as the substrate was added at a final concentration of 1 μM to start the enzymatic reaction, and after incubation at 4° C. for 4 minutes, a solution of ferric chloride (25 mM) and citric acid (50 mM) was added to terminate the enzymatic reaction. Generated PGE2 was measured by using Prostaglandin E2 Express EIA Kit (Cayman Chemical).
Measurement: IC50 = 16 nM (pChEMBL 7.800000190734863)
Descriptors: MW 413.87, ALogP 2.83, HBA 6, HBD 2, PSA 109.86, QED 0.64
Compound (SMILES): C#Cc1cncc(-c2cc(=O)[nH]c(-c3cc(CNC(=O)C(C)C)ccc3Cl)n2)c1
InChIKey: GVBDBTRDKYCGTG-UHFFFAOYSA-N
Target: Prostaglandin E synthase (SINGLE PROTEIN), Homo sapiens
UniProt: O14684
Assay: Inhibitory Activity Assay: Microsomes were prepared from COS-1 cells transiently transfected with a plasmid containing human mPGES-1 cDNA, and used as mPGES-1 enzyme. The mPGES-1 enzyme was diluted with a sodium phosphate buffer (pH 7.2) containing reduced glutathione (2.5 mM) and EDTA (1 mM), DMSO or a DMSO solution of a test compound (final concentration of DMSO was 1%) was added to the enzyme, and the mixture was preincubated at 4° C. for 15 minutes. Then, PGH2 as the substrate was added at a final concentration of 1 μM to start the enzymatic reaction, and after incubation at 4° C. for 4 minutes, a solution of ferric chloride (25 mM) and citric acid (50 mM) was added to terminate the enzymatic reaction. Generated PGE2 was measured by using Prostaglandin E2 Express EIA Kit (Cayman Chemical).
Measurement: IC50 < 1 nM (pChEMBL 9)
Descriptors: MW 406.87, ALogP 3.41, HBA 4, HBD 2, PSA 87.74, QED 0.64
Compound (SMILES): O=CN1CCN(CCc2ccc(-c3cnc(NCc4cccc5c4CCO5)n4cnnc34)cn2)CC1
InChIKey: ILAGMDVVNQKKBN-UHFFFAOYSA-N
Target: Polycomb protein EED (SINGLE PROTEIN), Homo sapiens
UniProt: O75530
Assay: EED-H3K27Me3 Peptide Competition Binding Assay by AlphaScreen: To assess the compounds potency in the EED-H3K27Me3 competition binding assay, compounds were serially diluted 3-fold in DMSO to obtain a total of twelve concentrations. Then compounds at each concentration (75 nL of each) were transferred by Mosquito into a 384-well Perkin Elmer ProxiPlate 384 plus plates. 8 uL of solutions containing 30 nM EED (1-441)-His protein and 15 nM biotin-H3K27Me3 (19-33) peptide in the buffer (25 mM HEPES, pH 8, 0.02% Tween-20, 0.5% BSA) were added to the wells and then incubated with compound for 20 min. AlphaScreen detection beads mix was prepared immediately before use by mixing nickel chelate acceptor beads and streptavidin donor beads in a 1:1 ratio (Perkin Elmer, Product No. 6760619C/M/R) into the buffer described above. Then 4 μL of detection beads mix was added to the plate and incubate in the dark at the rt for 1 h. The final concentration of donor and acceptor beads was 10 μg/mL for each. Plates were read on EnVision (PerkinElmer) using the AlphaScreen setting adapted for optimal signal detection with a 615 nm filter, after sample excitation at 680 nm. The emission signal at 615 nm was used to quantify compounds inhibition. AlphaScreen signals were normalized based on the reading coming from the positive (maximum signal control) and negative controls (minimum signal control) to give percentage of activities left. The data were then fit to a dose response equation using the program Helios (Novartis) to get the IC50 values. Helios is a Novartis in-house assay data analysis software using the methods described by Normolle, D. P., Statistics in Medicine, 12:2025-2042 (1993); Formenko, I. et al, Computer Methods and Programs in Biomedicine, 82, 31-37 (2006); Sebaugh, J. L., Pharmaceutical Statistics, 10:128-134 (2011); Kelly, C. et al., Biometrics, 46(4):1071-1085 (1990); and Kahm, M. et al., Journal of Statistical Software, 33(7): (2010) (grofit: Fitting Biological Growth Curves with R, pages 1-21, available at http://www.jstatsoft.org/).Each compound was counterscreened to determine if it interfered with the AlphaScreen beads. Compounds were diluted as described in the preceding section, and the assay was performed by adding 12 μL of 10 nM biotin-miniPEG-His6 peptide in the above buffer and incubating for 20 min at rt prior to addition of the beads to 10 μg/mL each. The plates were then incubated for 1 h at rt in dark before being read on EnVison.
Measurement: IC50 = 12.4 nM (pChEMBL 7.909999847412109)
Descriptors: MW 484.56, ALogP 2.05, HBA 9, HBD 1, PSA 100.78, QED 0.38
Compound (SMILES): CNc1ncc2c(-c3ccccc3)nn(CC3CCC(N)CC3)c2n1
InChIKey: RENRJGXFTHYPPG-UHFFFAOYSA-N
Target: Tyrosine-protein kinase Mer (SINGLE PROTEIN), Homo sapiens
UniProt: Q12866
Assay: Inhibition Assay: The ectopic expression of Mer receptor tyrosine kinase (Mer) has been identified as a tumor cell survival gene product in Acute Lymphoblastic Leukemia (ALL) cells and a potential cause of ALL chemoresistance. Hence, we investigated whether the development of small molecule Mer inhibitors was possible.
Measurement: IC50 = 550 nM (pChEMBL 6.260000228881836)
Descriptors: MW 336.44, ALogP 3.05, HBA 6, HBD 2, PSA 81.65, QED 0.77
Compound (SMILES): N#Cc1cc(C(F)(F)F)cc2nc(C#N)n(CCOc3ccc(Cl)cc3-c3cccc(C(=O)O)c3)c(=O)c12
InChIKey: BUXPHDKHVYGBHZ-UHFFFAOYSA-N
Target: Eukaryotic translation initiation factor 4E-binding protein 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q13541
Assay: Fluorescence Polarization Assay: eIF4E protein was pre-incubated with EDA-m7GDP-ATTO-550 in FPB at 2xconcentrations for 5 minutes prior to the addition of compounds. Compounds (100x) were prepared using 3-fold serial dilutions in 100% DMSO, and subsequently diluted 1:50 in FPB to produce 2x stocks. 50 ul of 2x eIF4E/EDA-m7GDP-ATTO-550 were transferred to the wells of a 96-well half-area black flat bottom polystyrene plate. 50 ul of 2x test compound were added, and binding reactions were allowed to equilibrate for 30 minutes at room temperature while protected from light. The fluorescent polarization signal was detected using a Victor 2 multi-label counter (Perkin Elmer) and the concentration necessary to achieve inhibition of eIF4E/EDA-m7GDP-ATTO-550 binding by 50% (IC50) was calculated using data from an 8-point compound dilution series.
Measurement: IC50 = 94.9 nM (pChEMBL 7.019999980926514)
Descriptors: MW 538.87, ALogP 5.26, HBA 7, HBD 1, PSA 129, QED 0.35
Compound (SMILES): CNC[C@H](c1ccc(Cl)c(Cl)c1)[C@H](O)c1cncc(OC)c1
InChIKey: XJMDOSGWUHOTCM-CZUORRHYSA-N
Target: Sodium-dependent noradrenaline transporter (SINGLE PROTEIN), Homo sapiens
UniProt: P23975
Assay: Uptake Assay Protocol: NET: This protocol was designed to measure inhibition of uptake by the human norepinephrine transporter. The reagents were human NET (HEK293F) cells, desipramine (Sigma), nomifensine, neurotransmitter transporter uptake assay kit (Molecular Devices), freestyle 293 expression medium (Invitrogen), 10× Hank's Balanced Salt Solution (HBSS; Invitrogen), 1 M HEPES (Mediatech), Biocoat poly-D-lysine 96-well, black, clear plates (Becton, Dickinson), and 500 μL polypropylene U-bottom 96-well plates (Fisher). The Assay Buffer (AB) was 1×HBSS and 0.02 M HEPES.The HEK293F cells were transfected with the human norepinephrine transporter and frozen in 1 mL aliquots at about 1E+07 cells/mL. On the day of the experiment, the cells were removed from −80° C. or liquid nitrogen and thawed in a room temperature water bath. The cells were dilute to about 1-2E+06 with Freestyle medium. A 1 mL sample (1:2 dilution) was prepared, and the cells were counted. The cells were spun at 1100 rpm for 5 minutes, and the medium was aspirated off. The cells were resuspend in medium at 1.5E+06 cells/mL for about 60,000 cells per well. 40 μL of cells were dispensed per well in the Biocoat plates. The plates were spun at 1100 rpm for 1 minute to improve homogeneity of the cell layer and were incubated at 37° C. for a minimum of 3 hours.
Measurement: Ki = 92.4 nM (pChEMBL 7.03000020980835)
Descriptors: MW 341.24, ALogP 3.43, HBA 4, HBD 2, PSA 54.38, QED 0.85
Compound (SMILES): C[C@]12C=CC(=O)C=C1[C@@H](F)C[C@H]1[C@@H]3C[C@H]4O[C@@H](c5ccc(Sc6cccc(N)c6)cc5)O[C@@]4(C(=O)CO)[C@@]3(C)C[C@H](O)[C@@]12F
InChIKey: STJWTSUWXXKJNZ-APTODLHGSA-N
Target: Glucocorticoid receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P04150
Assay: Glucocorticoid Receptor Binding Assay: Small molecules were tested for glucocorticoid receptor (GR) binding using the Polarscreen Glucocorticoid Receptor Assay Kit, Red (ThermoFisher A 15898) according to the manufacturer's protocol. Briefly, compounds were serially diluted in DMSO then transferred into assay kit buffer at a 1:10 dilution. Compounds were further diluted 1:5 in assay kit buffer, and 10 μl was transferred to a 384 well low volume black walled plate (Corning 4514). 5 μl of 4×Fluormone GS Red stock solution and 5 ul of 4×GR full length stock solution were added to each well containing test compound, and plates were incubated protected from light at room temperature for 4 hours. Fluorescence Polarization (mP) was measured for each plate using an EnVision Multilabel Plate Reader (Perkinelmer #2104-0010), and data were analyzed using a four parameter curve fit to generate EC50 values.
Measurement: IC50 = 15.6 nM (pChEMBL 7.809999942779541)
Descriptors: MW 623.72, ALogP 5.06, HBA 8, HBD 3, PSA 119.08, QED 0.4
Compound (SMILES): O[C@H]1CCCN[C@@H]1CCCn1cnc2c(N3CCCC3)ncnc21
InChIKey: TVXZRPLDNNIFNN-KGLIPLIRSA-N
Target: Ribose-phosphate pyrophosphokinase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P60891
Assay: PRS Enzyme Activity Inhibition Experiment: In order to confirm the biological activities of the compounds prepared in Examples, % inhibition or IC50 values of PRS enzyme (phosphoribosylpyrophosphate synthetase enzyme) activities were calculated.Specifically, the portion corresponding to PRS in cDNA of EPRS was subcloned, and the obtained high-purity PRS protein was purified and used in the experiment. The compounds (1 μM) prepared in Examples were added into the reaction buffer (20 mM KPO4 (pH 7.4), 6 mM MgAc, 5 mM ATP, 400 mg/mL tRNA, 0.5 mM DTT, 20 mCi[3H]proline (1 mCi/mL)) and allowed to react at 37° C. for 5 to 10 minutes. The reaction was terminated with 3M paper that was in advance dried by addition of 5% TCA. The radioactivity was measured using a liquid scintillation counter.% Inhibition and IC50 values of the respective compounds were calculated and analyzed using Microsoft Excel or Sigma Plot 8.0.
Measurement: IC50 > 500 nM (pChEMBL 6.301000118255615)
Descriptors: MW 330.44, ALogP 1.32, HBA 7, HBD 2, PSA 79.1, QED 0.86
Compound (SMILES): O=C(Nc1cn2cc(-c3c(Br)ccc4[nH]ncc34)ccc2n1)[C@@H]1C[C@@H]1F
InChIKey: KCRZXZUYLYEKQU-MFKMUULPSA-N
Target: Mitogen-activated protein kinase kinase kinase kinase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q92918
Assay: ADP-Glo kinase assay: HPK1 kinase activity was measured by Promega's ADP-Glo kinase assay. In this assay, 5 ng of recombinant human HPK1 (signalchem) is incubated with 5 μL of compounds (0.5% DMSO), 5 μL of MBP (0.5 μg/μl) and 5 μL of ATP (25 μM) in buffer (40 mM Tris, 7.5; 20 mM MgCl2; 0.1 mg/ml BSA; 5 μM DTT). The assay was started by incubating the reaction mixture in a 96-well plate at 30′ C for 40 minutes. After the incubation, 25 uL ADP-Glo reagent was added and the reaction was incubated at room temperature for 40-min to stop the reaction and degrade residual ATP. The ADP product was then converted to ATP by adding 50 uL per well of detection reagent. Luminescence was detected after 30-min room temperature incubation with the Molecular device I3X plate reader. The IC50 values were calculated from a series of percent inhibition values determined at a range of inhibitor concentration using software routines as implemented in the GraphPad Prism 7 software and SigmaPlot13.0.
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 414.24, ALogP 3.94, HBA 4, HBD 2, PSA 75.08, QED 0.53
Compound (SMILES): Cc1cc(C2CCCN(Cc3cc(F)cc(F)c3)C2)n2ncnc2n1
InChIKey: MQDOOQOPTYCBBS-UHFFFAOYSA-N
Target: cGMP-dependent 3',5'-cyclic phosphodiesterase (SINGLE PROTEIN), Homo sapiens
UniProt: O00408
Assay: Enzymatic Assay: An IMAP TR-FRET-based phosphodiesterase assay was developed using the PDE2A isoform. IMAP technology is based on high-affinity binding of phosphate by immobilized metal (MIII) coordination complexes on nanoparticles. The IMAP binding reagent recognizes phosphate groups on AMP or GMP generated from cAMP or cGMP in a PDE reaction. Cyclic nucleotides that carry a phosphodiester bond and not a free phosphate are not recognized by the binding reagent. The time resolved fluorescence resonance energy transfer (TR-FRET) is afforded by a Terbium (Tb)-Donor pre-bound to the nanoparticles. FRET occurs when the fluorescent-labeled AMP or GMP product of a PDE reaction binds and comes into close proximity to the Tb-Donor complex. Due to the long lifetime of Tb fluorescence, detection can be run in time-resolved mode to reduce or eliminate interference from auto-fluorescent compounds.The IMAP TR-FRET PDE2A assay was performed in 1536-well white plates. A total of 250 pg per well of FLAG-tagged PDE2A1 (amino acids 2-941) was dispensed in 2.5 μL IMAP assay buffer consisting of 10 mM Tris pH 7.2, 10 mM MgCl2, 1 mM DTT, and 0.1% fatty acid free BSA. 30 nL of compound was then added from 1 mM stocks in DMSO using a Kalypsys Pintool. Plates were incubated for 5 min at room temperature before dispensing 1.5 μL of 533 nM FAM-cAMP substrate for a final concentration of 200 nM. Following a brief centrifugation, plates were incubated for 30 min at room temperature. The assay was terminated by adding 5 μL IMAP binding reagent Tb complex to each well which was prepared according to manufacturer's recommendations (Molecular Devices). Plates were incubated an additional 90 minutes at room temperature and read on a Viewlux plate reader. All compounds were solvated at a concentration of 10 mM in DMSO and tested in 11-point half-log dose-response. Curve fitting and IC50 values were determined using a standard four parameter fit.PDE2
Measurement: IC50 = 550 nM (pChEMBL 6.260000228881836)
Descriptors: MW 343.38, ALogP 3.09, HBA 5, HBD 0, PSA 46.32, QED 0.73
Compound (SMILES): Nc1[nH]c2c(N3CCOC(C4CC4)C3)ncnc2c1S(=O)(=O)c1ccccc1
InChIKey: IOXXOCGVKLEZAU-UHFFFAOYSA-N
Target: Free fatty acid receptor 2 (SINGLE PROTEIN), Homo sapiens
UniProt: O15552
Assay: Ca-Flux Functional Assay: GPR43 agonist/PAM activity was determined by measuring changes in intracellular calcium levels using a Ca2+ sensitive fluorescent dye. The changes in fluorescent signal were monitored by FLIPR (manufactured by Molecular Devices). GPR43 mediated increases in intracellular Ca2+ concentration were readily detected upon activation with sodium acetate. Prior to the assay (24 hours), CHO-K1 Gα16 cells stably expressing human GPR43 were-seeded in cell culture medium in black, clear-bottom 384-well plates (Corning Inc) and grown overnight at 37° C., 5% CO2. On the day of the assay, cell culture media was removed and cells were loaded with Calcium 5 Dye (Molecular Devices) diluted in HBSS containing 25 mM HEPES, 2.5 mM Probenecid, 0.1% BSA for 1 hour at 37° C., 5% CO2. 10 point half log concentration response curves of sodium acetate from 10 mM were conducted prior to the testing of compounds to calculate the sodium acetate concentration that produces 20% of the maximal response (EC20). Test compounds (at 10 point half log concentration response curves from 10 μM) were added in the presence of sodium acetate to achieve a final concentration that produces approximately 20% maximal response as calculated from the previous experiment. The changes in fluorescent signal were monitored by FLIPR upon addition of the compound/EC20 sodium acetate mix. The EC50 values were determined from ten point concentration response curves. Curves were generated using the average of two wells for each data point.
Measurement: EC50 = 40 nM (pChEMBL 7.400000095367432)
Descriptors: MW 399.48, ALogP 1.99, HBA 7, HBD 2, PSA 114.2, QED 0.69
Compound (SMILES): CCOc1cc(S(C)(=O)=O)ccc1Nc1nc2ccc(-c3ccc(NC(=O)[C@H](C)c4ccc(F)cc4)cc3)cn2n1
InChIKey: GIJBGFMZYQQSPA-LJQANCHMSA-N
Target: Dual specificity protein kinase TTK (SINGLE PROTEIN), Homo sapiens
UniProt: P33981
Assay: Mps-1 Kinase Assay : For the assay 50 nl of a 100-fold concentrated solution of the test compound in DMSO was pipetted into a black low volume 384 well microtiter plate (Greiner Bio-One, Frickenhausen, Germany), 2 μl of a solution of Mps-1 in assay buffer [0.1 mM sodium-ortho-vanadate, 10 mM MgCl2, 2 mM DTT, 25 mM Hepes pH 7.7, 0.05% BSA, 0.001% Pluronic F-127] were added and the mixture was incubated for 15 min at 22° C. to allow pre-binding of the test compounds to Mps-1 before the start of the kinase reaction. Then the kinase reaction was started by the addition of 3 μl of a solution of 16.7 adenosine-tri-phosphate (ATP, 16.7 μM=>final conc. in the 5 μl assay volume is 10 μM) and peptide substrate (1.67 μM=>final conc. in the 5 μl assay volume is 1 μM) in assay buffer and the resulting mixture was incubated for a reaction time of 60 min at 22° C. The concentration of Mps-1 in the assay was adjusted to the activity of the enzyme lot and was chosen appropriate to have the assay in the linear range, typical enzyme concentrations were in the range of about 1 nM (final conc. in the 5 μl assay volume). The reaction was stopped by the addition of 3 μl of a solution of HTRF detection reagents (100 mM Hepes pH 7.4, 0.1% BSA, 40 mM EDTA, 140 nM Streptavidin-XLent [#61GSTXLB, Fa. Cis Biointernational, Marcoule, France], 1.5 nM anti-phospho(Ser/Thr)-Europium-antibody [#AD0180, PerkinElmer LAS, Rodgau-Jügesheim, Germany].
Measurement: IC50 < 1 nM (pChEMBL 9)
Descriptors: MW 573.65, ALogP 5.82, HBA 8, HBD 2, PSA 114.69, QED 0.23
Compound (SMILES): COc1cc(C)c(NC(=O)c2cc(Cl)c(-n3nc4n(c3=O)CCCC4)cc2O[C@@H](C)C(F)(F)F)cn1
InChIKey: IKYAQHNFZXWBFW-ZDUSSCGKSA-N
Target: Dihydroorotate dehydrogenase (quinone), mitochondrial (SINGLE PROTEIN), Homo sapiens
UniProt: Q02127
Assay: Enzymatic Assay: Assay1: The enzymatic assay couples DHODH activity with bleaching of the dye 2,6-dichlorophenolindophenol (DCIP) (Knecht and Loffler, 1998; Miller et al., 1968). The assay was conducted in buffer containing 50 mM Tris, 0.1% Triton X-100, 150 mM potassium chloride, 2 nM DHODH, 1 mM dihydroorotate, 0.1 mM decylubiquinone, 0.06 mM DCIP, and 2% DMSO at pH 8.0 at 32 degree Celsius. The reaction was initiated by addition of substrates. Enzyme activity was monitored kinetically by the reduction in DCIP absorbance at 600 nm. Purified recombinant human DHODH enzyme was purchased from Novus (cat. no. NBP1-98916). Other chemicals were purchased from Sigma-Aldrich. Absorbance measurements were obtained using a BMG clarion star plate-reading spectrophotometer.
Measurement: IC50 = 700 nM (pChEMBL 6.159999847412109)
Descriptors: MW 525.92, ALogP 4.32, HBA 8, HBD 1, PSA 100.27, QED 0.51
Compound (SMILES): Cc1cnc(OCc2cn(-c3cc(F)ccc3F)nn2)nc1
InChIKey: GBHNIHCIAKIREI-UHFFFAOYSA-N
Target: Glutamate NMDA receptor; GRIN1/GRIN2B (PROTEIN COMPLEX), Homo sapiens
UniProt: Q05586
Assay: NMDA receptor FLIPR assay: NMDA receptors are ion channels that are highly permeable to Ca2+ ions, rendering it possible to monitor NMDA receptor function using cell-based calcium flux assay. In this assay, co-agonists glutamate and glycine are added to cells heterologously expressing human GluN1/GluN2B NMDA receptors to initiate cellular Ca2+ influx. The time course of the changes in intracellular calcium is measured using a fluorescent dye and a FLIPR (Fluorometric Imaging Plate Reader) device.Twenty four hours before measurements, the expression of the NMDA receptors in the stable cell line is induced with Tet-On inducible system in the presence of a non-selective NMDA receptor blocker. On the day of the experiment, cell culture media is carefully washed and the cells are loaded with Calcium 5 Dye Kit (Molecular Devices) in dye loading buffer containing 149 mM NaCl, 4 mM KCl, 2 mM CaCl2, and 1.5 mM MgCl2, 10 mM HEPES and 5 mM D-glucose; pH 7.4. After 1 h incubation at the room temperature, the dye is washed away with the assay buffer (149 mM NaCl (standard assay) or 150 mM (HTS assay), 4 mM KCl (standard assay) or 3 mM (HTS assay), 2 mM CaCl2, 0.01 mM EDTA, 10 mM HEPES and 5 mM D-glucose; pH 7.4) In the FLIPR TETRA reader, various concentrations of the test compounds are added to the cells for 5 min while fluorescence is monitored to detect potential agonist activity. Next, co-agonists, glutamate and glycine are added for another 5 minutes. The concentration of glutamate corresponding to EC80 (standard assay) or EC40 (HTS assay) is used to maximize the assay's signal window or the ability to detect NMDA receptor antagonists and negative allosteric modulators, respectively. A saturating concentration (10 μM) of glycine is also present in the assay. A non-selective NMDA receptor antagonist, (+)MK-801 is used as a positive control for antagonist activity. The fluorescent signal in the presence of test compounds is quantified and normalized to the signal defined by the appropriate control wells.
Measurement: IC50 = 10430 nM (pChEMBL 4.980000019073486)
Descriptors: MW 303.27, ALogP 2.22, HBA 6, HBD 0, PSA 65.72, QED 0.74
Compound (SMILES): CNc1cc(C(=O)N2CCCC(c3ccc(Cl)cc3C(F)(F)F)C2)ccn1
InChIKey: UMYLOSNSUMKTRX-UHFFFAOYSA-N
Target: Prokineticin receptor 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q8TCW9
Assay: Biological Assay: Prokineticin receptor 1 (PKR1) antagonists may be functionally assessed by measurement of change in intracellular calcium levels induced by Gq mediated increase in inositol triphosphate (IP3) levels. The ability of a compound to block the intracellular release of calcium mediated by PK1 in RBL2H3 cells expressing human PKR1 receptors is determined as a measure of the compound's antagonist activity in vitro.Approximately 10,000 cells per assay well are seeded in normal culture medium in a 384 well plate (Corning). Twenty-four hours after seeding, the cells are loaded with a calcium sensitive fluorescent dye by replacing the culture medium with assay buffer (1× Hanks buffered saline, 25 mM HEPES, 0.1% w/v fatty acid free BSA (bovine serum albumin), pH 7.4) containing 1 mM probenecid and 1× Calcium 5 Reagent (Molecular Devices). Cells are incubated at 37° C. for 1 hour to allow for dye uptake.To test for antagonist activity, test compounds at a final concentration range between 0.32 nM-10 μM (diluted in assay buffer) are added to the assay wells and allowed to incubate for 10 minutes prior to stimulation with PK1. After incubation with test compounds the assay plate is placed in a FLIPR Tetra (Molecular Devices) and PK1 (diluted in assay buffer) is added at the determined EC80 concentration (final). Ligand-dependent changes in intracellular calcium levels are determined by measuring changes in fluorescence of the dye at 525 nM following excitation at 485 nM.
Measurement: IC50 = 50 nM (pChEMBL 7.300000190734863)
Descriptors: MW 397.83, ALogP 4.82, HBA 3, HBD 1, PSA 45.23, QED 0.81
Compound (SMILES): N#CCC(=O)N1CCC[C@@H](NC(=O)c2sc3nccc4c3c2NC(=O)N4c2ccc(Oc3ccccc3)cc2)C1
InChIKey: KGPGJOUVUSTNLA-GOSISDBHSA-N
Target: Tyrosine-protein kinase BTK (SINGLE PROTEIN), Homo sapiens
UniProt: Q06187
Assay: TBD: TBD
Measurement: IC50 = 79.4 nM (pChEMBL 7.099999904632568)
Descriptors: MW 552.62, ALogP 5.41, HBA 7, HBD 2, PSA 127.66, QED 0.33
Compound (SMILES): Nc1noc2c(N3CC[C@@H](NC(=O)c4ccc(-n5cnc(CO)n5)cc4Cl)C3)cc(Cl)cc12
InChIKey: AZTAGHOIITWDTD-GFCCVEGCSA-N
Target: Coagulation factor IX (SINGLE PROTEIN), Homo sapiens
UniProt: P00740
Assay: Inhibitory Activity Assay: Inhibitory activity against factor IXa was tested using the substrate SPECTROFLUOR FIXa (american diagnostica inc.; 500 West Avenue, Stamford, Conn. 06902 USA; Pr. No. 299F) and human factor IXa (american diagnostica inc.; Pr. No. 449b). Test substances were dissolved in buffer A (50 mM α,α,α-tris(hydroxymethyl) methylamine (Tris), 100 mM NaCl, 5 mM CaCl2, 15% (v/v) ethylene glycol, pH 8.0) were mixed with factor IXa (0.1 μg/ml final concentration). The enzyme reaction was started by addition of SPECTROFLUOR FIXa (100 μM final concentration). After incubation for 60 minutes at room temperature, the reaction was stopped by the addition of 20% (v/v) acetic acid solution, and then the fluorescence value was measured (Excitation Wavelength: 355 nm, Emission Wavelength; 460 nm) in a microtiter plate reader (ARVO 1420 Multilabel Counter; PerkinElmer).The IC50 was calculated from a dilution series of the test substance with the aid of the software, Symyx Assay Explorer (Symyx Technologies, Inc.).
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 488.34, ALogP 2.8, HBA 9, HBD 3, PSA 135.33, QED 0.39
Compound (SMILES): [C-]#[N+]c1ccc(-c2nc(CN3CC[C@H](N(C)C)C3)c3nccn3c2-c2ccc(C)cc2)cc1
InChIKey: KCFQXFNBMBVJLF-QHCPKHFHSA-N
Target: Lysine-specific histone demethylase 1A (SINGLE PROTEIN), Homo sapiens
UniProt: O60341
Assay: LSD1 Histone Demethylase Biochemical Assay: LANCE LSD1/KDM1A demethylase assay-10 μL of 1 nM LSD-1 enzyme (ENZO BML-SE544-0050) in the assay buffer (50 mM Tris, pH 7.5, 0.01% Tween-20, 25 mM NaCl, 5 mM DTT) were preincubated for 1 hour at 25° C. with 0.8 μL compound/DMSO dotted in black 384 well polystyrene plates. Reactions were started by addition of 10 μL of assay buffer containing 0.4 μM Biotin-labeled Histone H3 peptide substrate: ART-K(Me1)-QTARKSTGGKAPRKQLA-GGK(Biotin) SEQ ID NO:1 (AnaSpec 64355) and incubated for 1 hour at 25° C. Reactions were stopped by addition of 10 μL 1× LANCE Detection Buffer (PerkinElmer CR97-100) supplemented with 1.5 nM Eu-anti-unmodified H3K4 Antibody (PerkinElmer TRF0404), and 225 nM LANCE Ultra Streptavidin (PerkinElmer TRF102) along with 0.9 mM Tranylcypromine-HCl (Millipore 616431). After stopping the reactions plates were incubated for 30 minutes and read on a PHERAstar FS plate reader (BMG Labtech).
Measurement: IC50 < 50 nM (pChEMBL 7.301000118255615)
Descriptors: MW 436.56, ALogP 5.06, HBA 5, HBD 0, PSA 41.03, QED 0.41
Compound (SMILES): N[C@]1(COP(=O)(O)O)CC[C@H](c2ccc3c(c2)CC[C@@H](CSCCc2ccncc2)C3)C1
InChIKey: RRMDNESQPXJWEG-VEXUSMLFSA-N
Target: Sphingosine 1-phosphate receptor 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P21453
Assay: Binding Assay: Membranes were prepared from CHO cells expressing human S1P1. Cells pellets (1x10^9 cells/pellet) were suspended in buffer containing 20 mM HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid), pH 7.5, 50 mM NaCl, 2 mM EDTA (Ethylenediaminetetraacetic acid) and Protease Inhibitor cocktail (Roche), and disrupted on ice using the Polytron homogenizer. The homogenate was centrifuged at 20,000 rpm (48,000 g) and the supernatant was discarded. The membrane pellets were resuspended in buffer containing 50 mM HEPES, pH 7.5, 100 mM NaCl, 1 mM MgCl2, 2 mM EDTA and stored in aliquots at -80Â° C. after protein concentration determination.Membranes (2 ug/well) and 0.03 nM final concentration of 33P-S1P ligand (1 mCi/ml, Perkin elmer or American Radiolabeled Chemicals) diluted in assay buffer (50 mM HEPES, pH7.4, 5 mM MgCl2, 1 mM CaCl2, 0.5% fatty acid free BSA (bovine serum albumin), 1 mM NaF) were added to the compound plates (384 Falcon v-bottom plate (0.5 ul/well in a 11 point, 3-fold dilution). Binding was performed for 45 minutes at room temperature, terminated by collecting the membranes onto 384-well Millipore FB filter plates, and radioactivity was measured by TOPCOUNT. The competition data of the test compounds over a range of concentrations was plotted as percentage inhibition of radioligand specific binding. The IC50 is defined as the concentration of competing ligand needed to reduce specific binding by 50%.
Measurement: IC50 < 10 nM (pChEMBL 8)
Descriptors: MW 476.58, ALogP 4.24, HBA 5, HBD 3, PSA 105.67, QED 0.37