lovingscience/chembl-v37-raw-activities huggingface dataset:
Compound (SMILES): CC(C)[C@H](Oc1ccc(-c2ncnc(Nc3ccc(N4CCN(C5COC5)CC4)cc3)n2)cc1C#N)C(N)=O
InChIKey: ZPXLMPJUMHHLKC-VWLOTQADSA-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 = 94000 nM (pChEMBL 4.03000020980835)
Descriptors: MW 528.62, ALogP 2.56, HBA 10, HBD 2, PSA 142.52, QED 0.43
Compound (SMILES): CC(C)(C)c1ccc(S(=O)(=O)Nc2ccc(Cl)c3c2C(=O)N(c2ccc[nH]c2=O)C3=O)cc1
InChIKey: IXDDRQLZQFTDEL-UHFFFAOYSA-N
Target: C-C chemokine receptor type 9 (SINGLE PROTEIN), Homo sapiens
UniProt: P51686
Assay: FLIPR Assay: A calcium flux assay was used to determine the ability of the compounds to interfere with the binding between CCR9 and its chemokine ligand (TECK) in Chem1-hCCR9 overexpressing cells. hCCR9 overexpressing cells were seeded (25,000 cells/well) into black Poly-D-Lysine coated clear bottom 96-well plates (BD Biosciences, Cat #356640) and incubated overnight at 37° C./5% CO2 in a humidified incubator. Media was aspirated and cells washed twice with 100 μL assay buffer (1×HBSS, 20 mM HEPES) containing 2.5 mM Probenecid. A 0.3× Fluo-4 NW calcium dye was prepared in assay buffer containing 5 mM Probenecid and stored in the dark. Each well was loaded with 100 μL of 0.3× Fluo-4 NW calcium dye and incubated at 37° C./5% CO2 for 60 minutes and then at room temperature for 30 minutes. A half-log serially diluted concentration response curve was prepared at a 3× final assay concentration for each compound (10 μM-0.1 nM final assay concentration) and 50 μL of the compound then transferred to the cells (150 μL final volume) for 60 minutes prior to stimulation (30 minutes at 37° C./5% CO2 and 30 minutes at room temperature). TECK was diluted to 4× its EC80 in assay buffer (containing 0.1% [w/v] bovine serum albumin [BSA]) and 50 μL dispensed through the fluorometric imaging plate reader (FLIPR) instrument to stimulate the cells (200 μL final volume). The increase in intracellular calcium levels was measured with the FLIPR instrument.
Measurement: Ki = 4 nM (pChEMBL 8.399999618530273)
Descriptors: MW 485.95, ALogP 3.93, HBA 5, HBD 2, PSA 116.41, QED 0.54
Compound (SMILES): CNC(=O)c1ccc(OC)cc1Oc1ncc(Br)c(Nc2cc(OC)c(OC)c(OC)c2)n1
InChIKey: JLNOVOOWIVTQGG-UHFFFAOYSA-N
Target: Serine/threonine-protein kinase ULK1 (SINGLE PROTEIN), Homo sapiens
UniProt: O75385
Assay: ULK1 inhibition assay: Gamma-32P assays to measure ULK1 kinase activity were performed as previously described. Briefly, Flag ULK1 was transfected into HEK293T cells and 20 hours later treated as indicated. The immmunoprecipitate was washed in IP buffer 3 times, and washed in kinase buffer (25 mM MOPS, pH 7.5, 1 mM EGTA, 0.1 mM Na3VO4, 15 mM MgCl2,). Hot and cold ATP were added at a 100 μM final concentration. As substrates, GST or the recombinant protein GST-Atg101 purified from E. coli were used at 1 μg for each reaction. Reactions were boiled, run out on SDS page gel. The gel was dried, and imaged using PhosphoImager software. For cold assays to asses ULK1, Flag ULK1 which was transiently overexpressed and immunoprecipited from HEK293T cells. Reactions were then run out on SDS page gel, transferred to PVDF membrane and blotted for total levelsFluorescence MicroscopyVps34flox/flox MEFs were reconstituted with Flag-VPS34 and either p40FX or GFP-DFCP1. 48 hours post infection with adenovirus expressing Cre recombinase (MOI of 100), cells were plated on glass coverslips at a density of 3×105 cells per well in 6-well tissue culture plates. 18 h later, cells were fixed in 4% PFA in PBS for 10 minutes and permeabilized in 0.2% Triton in PBS for 10 minutes. The following primary antibodies were used: mouse anti-Myc epitope and LC3B XP antibody (2276 and 3868 respectively, Cell Signaling Technologies). Secondary antibodies were anti-rabbit Alexa488 and anti-mouse Alexa594 (Molecular Probes, 1:1000. Cells were then fixed and counter stained with DAPI. Coverslips were mounted in FluoromountG (SouthernBiothech). Images were acquired on a Zeiss Axioplan2 epifluorescence microscope coupled to the Openlab software. Confocal images of mitotracker were taken on Zeiss LSM 710 laser scanning confocal microscope. 10 random fields per condition were acquired using the 100× objective and representative images shown. Glass coverslips were mounted directly on plate with FluoromountG and images taken on Zeiss Axioplan2 epifluorescence microscope.
Measurement: IC50 > 2000 nM (pChEMBL 5.698999881744385)
Descriptors: MW 519.35, ALogP 4.17, HBA 9, HBD 2, PSA 113.06, QED 0.43
Compound (SMILES): CC(=O)c1nn(CC(=O)N2C[C@H](F)C[C@H]2C(=O)Nc2nc(C(C)(F)F)ccc2C)c2ccc(-c3cnc(C)nc3)cc12
InChIKey: RNPKWZMEPDQQJV-OFNKIYASSA-N
Target: Complement factor D (SINGLE PROTEIN), Homo sapiens
UniProt: P00746
Assay: Human Factor D Assay: Human Factor D (purified from human serum, Complement Technology, Inc.) at 80 nM final concentration was incubated with test compound at various concentrations for 5 minutes at room temperature in 50 mM Tris, 1M NaCl, pH 7.5. A synthetic substrate Z-L-Lys-SBzl and DTNB (Ellman's reagent) are added to final concentrations of 100 μM each. Absorbance at 405 nm (A405) was recorded at 30 second intervals for 30 minutes using a microplate spectrophotometer. IC50 values are calculated by nonlinear regression of complement Factor D reaction rates as a function of test compound concentration.
Measurement: IC50 < 1000 nM (pChEMBL 6)
Descriptors: MW 579.58, ALogP 4.4, HBA 8, HBD 1, PSA 122.97, QED 0.32
Compound (SMILES): CN1CCSC(c2nc(-c3ccc(C(=O)Nc4cc(C(F)(F)F)ccn4)cc3)c3c(N)nccn23)C1
InChIKey: IICHIZQBMUSVAS-UHFFFAOYSA-N
Target: Tyrosine-protein kinase BTK (SINGLE PROTEIN), Homo sapiens
UniProt: Q06187
Assay: Btk Enzyme Activity Assay B: BTK enzymatic activity was determined in an IMAP-FP assay (Immobilized Metal ion Affinity-based fluorescence Polarization; Molecular Devices). Using this assay format, the potency (IC50) of each compound was determined from a 10 point (1:3 serial dilution, final compound concentration range in assay from 10 μM to 0.508 nM) titration curve using the following outlined procedure. To each well of a black Corning 384-well microplate (Corning Catalog #3575), 200 nL of compound (100 fold dilution in final assay volume of 20 μL) was dispensed, followed by the addition of 10 μL of 1× kinase buffer (10 mM Tris pH 7.2, 10 mM MgCl2, 0.01% Tween 20, 2 mM MnCl2, and 1 mM DTT) containing 0.16 ng/μL (2.1 nM) of BTK enzyme (recombinant protein from baculovirus-transfected Sf9 cells: full-length BTK, 6HIS-tag cleaved). Following a 60 minute compound & enzyme incubation, each reaction was initiated by the addition of 10 μL kinase buffer containing 100 nM Blk/Lyntide IMAP substrate peptide (5-carboxyfluorescein-EFPIYDFLPAKKK-NH2; Molecular Devices Catalog #R7188), and 10 μM ATP. The final reaction in each well of 20 μL consists of 1.05 nM hBTK, 50 nM Blk/Lyntide IMAP substrate, and 5 μM ATP. Phosphorylation reactions were allowed to proceed for 120 minutes and were immediately quenched by the addition of 40 μL IMAP detection beads resuspended in IMAP progressive binding buffer (beads were diluted 1:600 in 75% and 25% progressive binding buffer A and B, respectively; Molecular Devices). Plates were read on Packard microplate reader after 60 minutes binding equilibration using Fluorescence Polarization protocol. Specifically, fluorescence at 535 nm is measured using emission filters oriented both parallel and perpendicular to the polarized excitation filter. This allows detection of changes in Fluorescence Polarization due to binding of the phosphorylated substrate peptide to the beads. Values are calculated as percentage of the difference in readout (ΔmP) of the controls with and without hBTK enzyme. IC50 values are determined by curve fitting of the experimental results using Activity Base.
Measurement: IC50 = 550 nM (pChEMBL 6.260000228881836)
Descriptors: MW 513.55, ALogP 4.36, HBA 8, HBD 2, PSA 101.44, QED 0.42
Compound (SMILES): CS[C@H]1O[C@@H](c2ccc(C)c(Cc3ccc(OCCCC(=O)NC(C)(C)C(=O)NC4CCN(C)CC4)cc3)c2)[C@H](O)[C@@H](O)[C@@H]1O
InChIKey: HGEBSKMVZRCHTO-DSSMMGBBSA-N
Target: Sodium/glucose cotransporter 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P13866
Assay: In Vitro Human SGLT1 Inhibition Assay: Human sodium/glucose co-transporter type 1 (SGLT1; accession number NP_000334; GI: 4507031) was cloned into pIRESpuro2 vector for mammalian expression (construct: HA-SGLT1-pIRESpuro2). HEK293 cells were transfected with the human HA-SGLT1-pIRESpuro2 vector and the bulk stable cell line was selected in presence of 0.5 μg/mL of puromycin. Human HA-SGLT1 cells were maintained in DMEM media containing 10% FBS, 1% GPS and 0.5 μg/mL of puromycin. The HEK293 cells expressing the human HA-SGLT1 were seeded in 384 well plates (30,000 cells/well) in DMEM media containing 10% FBS, 1% GPS and 0.5 μg/mL of puromycin, then incubated overnight at 37 C, 5% CO2. Cells were then washed with uptake buffer (140 mM NaCl, 2 mM KCl, 1 mM CaCl2, 1 mM MgCl2, 10 mM HEPES, 5 mM Tris, 1 mg/mL bovine serum albumin (BSA), pH 7.3). Twenty microliters of uptake buffer with or without testing compounds were added to the cells. Then, 20 microliters of uptake buffer containing 14C-AMG (100 nCi) were also added to cells. The cell plates were incubated at 37° C., 5% CO2 for 1-2 hours. After washing the cells with uptake buffer, scintillation fluid was added (40 microliters/well) and 14C-AMG uptake was measured by counting radioactivity using a scintillation coulter (TopCoulter NXT; Packard Instruments).
Measurement: IC50 < 10 nM (pChEMBL 8)
Descriptors: MW 643.85, ALogP 2.69, HBA 9, HBD 5, PSA 140.59, QED 0.22
Compound (SMILES): CC1(C)CCN1Cc1cccc(Cl)c1CNc1cc(F)c(S(=O)(=O)Nc2cscn2)c(F)c1
InChIKey: GQHXKZRLHCSYDT-UHFFFAOYSA-N
Target: Sodium channel protein type 8 subunit alpha (SINGLE PROTEIN), Homo sapiens
UniProt: Q9UQD0
Assay: Sodium Influx Assay (In Vitro Assay): This sodium influx assay employs the use of the cell permeable, sodium sensitive dye ANG2 to quantify sodium ion influx through sodium channels which are maintained in an open state by use of sodium channel modulators. This high throughput sodium influx assay allows for rapid profiling and characterization of sodium channel blockers.In general, Trex HEK293 cells were stably transfected with an inducible expression vector containing the full-length cDNA coding for the desired human sodium channel α-subunit and with an expression vector containing full length cDNA coding for the β1-subunit. Sodium channel expressing cell lines were induced with tetracycline (1 μg/mL) and plated on 384-well PDL-coated plates at a density of 25K-30K cells/well in culture media (DMEM, containing 10% FBS and 1% L-glutamine). After overnight incubation (37° C., 5% CO2), culture media was removed and cells were loaded with 5 uM ANG2 dye for 1-1.5 h in Buffer 1 (155 mM NMDG, 5 mM KCl, 2 mM CaCl2, 1 mM MgCl2, 10 mM HEPES, 10 mM glucose, adjusted with Tris to pH 7.4). Access dye was removed and cells were incubated with test compounds for 1 hr in buffer 1 containing sodium channel modulator(s) at room temperature. Hamamatsu FDSS μCell was used to perform a 1:1 addition of Na/K challenge buffer (140 mM NaCl, 20 mM HEPES, 1 mM CaCl2, 15 mM KCl, 1 mM MgCl2, 10 mM glucose, adjusted with Tris to pH 7.4) and simultaneously read plates at excitation wavelength of 530 nm and emission wavelength set at 558 nm. Percent inhibition of sodium ion influx was calculated for each test compound at each test concentration to determine the IC50 values.
Measurement: IC50 = 213 nM (pChEMBL 6.670000076293945)
Descriptors: MW 513.04, ALogP 5.47, HBA 6, HBD 2, PSA 74.33, QED 0.42
Compound (SMILES): CCOc1cc(-c2ccc(C)nc2OC)nc2cc(C(=O)NCCc3c[nH]nc3C)ccc12
InChIKey: WVKIIQOEBHTLCA-UHFFFAOYSA-N
Target: Nicotinamide phosphoribosyltransferase (SINGLE PROTEIN), Homo sapiens
UniProt: P43490
Assay: NAMPT Enzyme Assay: To measure the inhibition of NAMPT activity hNAMPT protein stock and anti 6His-Tb (Cisbio; Cat. No. 61HISTLB) is diluted to 3× final concentration with assay buffer (50 mM Tris-HCl (pH 7.5), 1 mM DTT, 100 mM NaCl, 10 mM MgCl2, 0.005% Tween 20). To this solution is added a test compound or vehicle control (DMSO) and BodiPY ligand (structure below). The plate is shaken for 1-2 min sealed and incubated for 1 h at rt in the dark. The TR-FRET signal is measured using BMG Pherastar (Lanthascreen protocol on BMG Pherastar). Excitation was carried out at 320 nm, and the ratio of emission of BodiPY (520 nm) to terbium (486 nm) was determined. Concentration response curves are generated by calculating the excitation increase in test compound-treated samples relative to DMSO-treated controls.For the assay method described above, test compound percent inhibition values at a single concentration are calculated relative to control (DMSO) treated samples. Compound concentration response curves are fitted to generate IC50 values from those curves. One skilled in the art will appreciate that these values generated either as percentage inhibition at a single concentration or IC50 values are subject to experimental variation.
Measurement: IC50 = 30 nM (pChEMBL 7.519999980926514)
Descriptors: MW 445.52, ALogP 4.02, HBA 6, HBD 2, PSA 102.02, QED 0.42
Compound (SMILES): CCn1nc(C(=O)NC[C@H]2CC[C@H](C)CC2)c(Cl)c1-c1cnc(CC(C)(C)C(F)(F)F)cc1C(F)F
InChIKey: IPZYEPAKBMMIBI-SHTZXODSSA-N
Target: Nuclear receptor ROR-gamma (SINGLE PROTEIN), Homo sapiens
UniProt: P51449
Assay: Fluorescence Based Assay: ThermoFluor® is a fluorescence based assay that estimates ligand binding affinities by measuring the effect of a ligand on protein thermal stability (Pantoliano, M. W., Petrella, E. C., Kwasnoski, J. D., Lobanov, V. S., Myslik, J., Graf, E., Carver, T., Asel, E., Springer, B. A., Lane, P., and Salemme, F. R. (2001) High-density miniaturized thermal shift assays as a general strategy for drug discovery. J Biomol Screen 6, 429-40, and Matulis, D., Kranz, J. K., Salemme, F. R., and Todd, M. J. (2005) Thermodynamic stability of carbonic anhydrase: measurements of binding affinity and stoichiometry using ThermoFluor. Biochemistry 44, 5258-66). This approach is applicable to a wide variety of systems, and rigorous in theoretical interpretation through quantitation of equilibrium binding constants (KD).
Measurement: Kd = 17 nM (pChEMBL 7.769999980926514)
Descriptors: MW 535, ALogP 7.24, HBA 4, HBD 1, PSA 59.81, QED 0.36
Compound (SMILES): Cc1cncc2ccc(NC(=O)[C@@H]3C[C@H]3c3ccc(S(=O)(=O)NC4CCNC4)cc3)cc12
InChIKey: GYMRIGZBUYFIEU-NWZLQDFTSA-N
Target: Inhibitor of nuclear factor kappa-B kinase subunit beta (SINGLE PROTEIN), Homo sapiens
UniProt: O14920
Assay: ROCK Kinase Inhibition Assays: All compounds were initially prepared as 10 mM stocks in anhydrous dimethylsulfoxide (DMSO). A 20 μl aliquot of the 10 mM solutions was transferred to individual wells in column 1 of a 96-well polypropylene microtiter plate (Corning #3363) and diluted with DMSO to give a final compound concentration of 4 mM. Test compounds were then serially diluted 1:5 in DMSO for an 11-point concentration response and further diluted in the assay buffer bringing all compound concentrations to a final range of 100 μM to 10 pM in 2.5% DMSO. The assay was performed in white 96-well, flat-bottom, half-area, non-binding assay plate (Corning #3642) in assay buffer consisting of 20 mM HEPES (pH 7.5), 10 mM MgCl2*6H2O, 100 μM sodium orthovanadate, 0.05% CHAPS and 0.1% bovine serum albumin. A 10 μL aliquot of compound from each well of the intermediate dilution plate and 20 μL of a 2× substrate/enzyme solution containing acceptor substrate (800 nM RSK2 peptide KRRRLSSLRA (SEQ ID NO: 1)), ROCK2 enzyme (10 nM), or ROCK1 enzyme, and 1,4-Dithiothreitol (DTT, 2 μM) were added to all wells. The reaction was initiated by the addition of 10 μL of 4× stock solution ATP (2 ∥M). Reactions were thoroughly mixed manually, covered and allowed to incubate at room temperature for 75 min. Protein kinase activity was quantitated using Promega's KINASE-GLO luminescent Kinase Assay Kit according to the manufacturer's directions. ATP concentrations remaining in Test wells following the termination of the enzymatic reaction were compared against control wells containing equivalent amounts of DMSO containing no inhibitor (CTRL). ATP concentrations in both Test wells and CTRL wells were normalized against background (BKG) ATP concentrations in wells containing concentrations of inhibitor that completely inhibited the protein kinase under investigation (i.e. a concentration that prevented any consumption of ATP over the course of the incubation). Percent of Control (POC) values were determined for each concentration of compound tested according to the equation: POC=((Test well value−BKG)/(CTRL−BKG))*100IC50 values were calculated using the following 4-parameter logistic curve-fitting algorithm: f(x)=(A+((B−A)/(1+((x/C){circumflex over ( )}D))))IC50 values were converted to Ki values using the Cheng-Prusoff Equation: Ki=IC50/(1+([ATP]/Km ATP])).
Measurement: IC50 = 759 nM (pChEMBL 6.119999885559082)
Descriptors: MW 450.56, ALogP 2.93, HBA 5, HBD 3, PSA 100.19, QED 0.54
Compound (SMILES): Cc1cc(OC2(c3ccc(C(F)(F)F)c(F)c3)CC2)n2ccnc2n1
InChIKey: LYXOWFCHMGUTIW-UHFFFAOYSA-N
Target: cGMP-dependent 3',5'-cyclic phosphodiesterase (SINGLE PROTEIN), Homo sapiens
UniProt: O00408
Assay: Inhibitory Activity Assay (Lab B): In a typical experiment the PDE2 inhibitory activity of the compounds of the present invention was determined in accordance with the following experimental method. Rhesus PDE2A3 was amplified from rhesus macaque brain cDNA (Biochain Institute, Hayward, Calif.) using primers based on human PDE2A sequence (accession NM_002599.3) where the forward primer containing a Kozak consensus was 5′-gccaccatggggcaggcatgtggc-3′ and the reverse primer was 5′-tcactcagcatcaaggctgca-3′. Amplification with Easy-A High-Fidelity PCR cloning enzyme (Stratagene, La Jolla, Calif.) was 95° C. for 2 minutes followed by thirty three cycles of 95° C. for 40 seconds, 52° C. for 30 seconds, and 72° C. for 2 minutes 48 seconds. Final extension was 72° C. for 7 minutes. The PCR product was TA cloned into pcDNA3.3-TOPO (Invitrogen, Carlsbad, Calif.) according to standard protocol. A consensus sequence was developed from multiple clones and then deposited into GenBank (EU812167). AD293 cells (Stratagene, La Jolla, Calif.) with 70-80% confluency were transiently transfected with rhesus PDE2A3/pcDNA3.3-TOPO using Lipofectamine 2000 according to manufacturer specifications (Invitrogen, Carlsbad, Calif.). Cells were harvested 48 hours post-transfection and lysed by sonication (setting 3, 10×5 sec pulses) in a buffer containing 20 mM HEPES pH 7.4, 1 mM EDTA and Complete Protease Inhibitor Cocktail Tablets (Roche, Indianapolis, Ind.). Lysate was collected by centrifugation at 75,000×g for 20 minutes at 4° C. and supernatant utilized for evaluation of PDE2 activity.
Measurement: Ki = 55 nM (pChEMBL 7.260000228881836)
Descriptors: MW 351.3, ALogP 4.26, HBA 4, HBD 0, PSA 39.42, QED 0.66
Compound (SMILES): COc1c(F)cc(-c2c(C(=O)N3CCC3)nn3c2OCCC3)cc1F
InChIKey: ARADJHAPDWYDIE-UHFFFAOYSA-N
Target: 3',5'-cyclic-AMP phosphodiesterase 4A (SINGLE PROTEIN), Homo sapiens
UniProt: P27815
Assay: Scintillation Proximity Assay (SPA): Human PDE4A3 coding sequence (amino acids 2 to 825 from the sequence with accession number NP_001104779) was cloned into the baculovirus expression vector pFastBac (Invitrogen) engineered to include an N-terminal His6 affinity tag and a C-terminal FLAG affinity tag to aid in purification. The recombinant Bacmid was isolated and used to transfect insect cells to generate a viral stock. To generate cell paste for purification, insect cells were infected with the virus stock and cells were harvested 72 hours after infection. Insect cell paste was lysed and after centrifugation, the supernatant was batch bound to Ni-NTA agarose (GE Healthcare) and eluted with 250 mM imidazole. This eluate was diluted with FLAG buffer (50 mM Tris HCl pH 7.5, 100 mM NaCl, 5% glycerol, 1 mM TCEP with protease inhibitors) and batch bound to ant-FLAG M2 agarose (Sigma) overnight at 4° C. The agarose was packed into a column, washed with buffer and eluted with buffer containing elute using 250 μg/mL Flag-peptide. Fractions were analyzed using SDS-PAGE Coomassie blue staining and pooled based on purity. Pooled fractions were chromatographed on a S200 120 mL column (GE Healthcare) in 50 mM Tris HCl pH 7.5, 150 mM NaCl, 10% glycerol, 2 mM TCEP with protease inhibitors. PDE4A3 fractions were analyzed by SDS-PAGE Coomassie blue staining, pooled based on purity, dialyzed against 50 mM Tris HCl pH 7.5, 100 mM NaCl, 20% glycerol, 2 mM TCEP, frozen and stored at −80° C.
Measurement: IC50 = 681 nM (pChEMBL 6.170000076293945)
Descriptors: MW 349.34, ALogP 2.47, HBA 5, HBD 0, PSA 56.59, QED 0.85
Compound (SMILES): O=C1c2cccnc2CCN1CCN1CCC(c2noc3cc(F)ccc23)CC1
InChIKey: WXUKVMDDBRUFBU-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 = 60.9 nM (pChEMBL 7.210000038146973)
Descriptors: MW 394.45, ALogP 3.24, HBA 5, HBD 0, PSA 62.47, QED 0.68
Compound (SMILES): Nc1ccc(-c2cccs2)cc1NC(=O)c1ccc(NC2CCN(C(=O)C3(O)CCCC3)CC2)cc1
InChIKey: NXCPBNLDBYSAEM-UHFFFAOYSA-N
Target: Histone deacetylase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q13547
Assay: Enzyme Assay: Compounds for testing were diluted in DMSO to 50 fold the final concentration and a ten point three fold dilution series was made. The compounds were diluted in assay buffer (50 mM HEPES, pH 7.4, 100 mM KCl, 0.001% Tween-20, 0.05% BSA, 20 μM tris(2-carboxyethyl)phosphine) to 6 fold their final concentration. The HDAC enzymes (purchased from BPS Biosciences) were diluted to 1.5 fold their final concentration in assay buffer and pre-incubated with the compounds for 24 hours prior to addition of the substrate.
Measurement: IC50 = 7 nM (pChEMBL 8.149999618530273)
Descriptors: MW 504.66, ALogP 4.96, HBA 6, HBD 4, PSA 107.69, QED 0.36
Compound (SMILES): CCC(=O)N[C@H]1CC[C@@H](N(C)C(=O)c2ccc(-c3nc4ccc(Cl)cc4o3)nc2)C1
InChIKey: LUEHPTDHJCYTAS-JKSUJKDBSA-N
Target: Fatty acid synthase (SINGLE PROTEIN), Homo sapiens
UniProt: P49327
Assay: Biochemical Assay of FASN: The biochemical FASN activity testing was performed as 384 well two-time-point kinetic fluorescence intensity assay format in Greiner low volume medium binding 384-well black microtiter plates in a total assay volume of 8 μl and was used for high throughput screen. In each well 3 μl 40 nM human recombinant full-length fatty acid synthase (produced in-house in SF9 cells) were dispensed in the following assay buffer: 50 mM potassium phosphate buffer pH 7.0, 0.005% (w/v) BSA, 2 mM Glutathione, 0.02% Tween-20. Then 2 μl of 200 μM NADPH in assay buffer were added, followed by the addition of the test compounds in 10 dilution concentrations starting with 30 μM (final concentration) to get a final DMSO content of 1% (v/v). The mixture was incubated for at least 15 min at room temperature. After the pre-incubation the enzymatic reaction was started by the addition of 2 μl substrate solution (80 μM acetyl-CoA, 240 μM malonyl-CoA). A first fluorescence intensity measurement (time point one) was performed with an Envision multimode reader (Perkin Elmer LAS Germany GmbH) at excitation wavelength 340 nm (lamp mode) and emission wavelength 460 nm. The reaction was incubated for 30 minutes at room temperature. After this the fluorescence intensity was measured again in the Envision using the same parameters as described above (second time point measurement). The data were analysed by subtracting the first time point measurement value from the second time point measurement value (after the enzymatic reaction). The differences of the emission signals were determined. These reflect directly the conversion rate of NADPH. The full value used was the inhibitor-free reaction. A pharmacological zero value was used like GSK837149A (Sigma-Aldrich) in a final concentration of 5-10 μM. The inhibitory values (IC50) were determined using either the program Symyx Assay Explorer® or Condosseo® from GeneData.
Measurement: IC50 < 300 nM (pChEMBL 6.5229997634887695)
Descriptors: MW 426.9, ALogP 4.06, HBA 5, HBD 1, PSA 88.33, QED 0.66
Compound (SMILES): Cc1[nH]c(-c2cc(-c3cncc(S(C)(=O)=O)c3)ccn2)nc1C(=O)NC(C)C
InChIKey: YPSJGDFFLRZMDN-UHFFFAOYSA-N
Target: Phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit gamma isoform (SINGLE PROTEIN), Homo sapiens
UniProt: P48736
Assay: PI3K-γ Scintillation Proximity Assay: The kinase reaction was conducted in polystyrene 384-well Greiner Bio-one white plate from Thermo Fisher Scientific in a final volume of 25 μL. Inhibitors were first diluted serially in DMSO and added to the plate wells before the addition of other reaction components. The final concentration of DMSO in the assay was 2%. The PI3Kγ assay was carried out at room temperature in 20 mM MOPS, pH 6.7, 10 mM MgCl2, 5 mM DTT and CHAPS 0.03%. Reactions were initiated by the addition of ATP, the final reaction mixture consisted of 20 μM PIP2, 2 μM ATP, 0.5 μCi [γ-33P] ATP, 13 nM PI3Kγ. Reactions were incubated for 120 min and terminated by the addition of 40 μL SPA beads suspended in quench buffer: 163 mM potassium phosphate pH 7.8, 20% glycerol, 25 mM EDTA. The final concentration of SPA beads is 1.0 mg/mL. After the plate sealing, plates were shaken overnight at room temperature and centrifuged at 1500 rpm for 10 min, the radioactivity of the product was determined by scintillation counting on Topcount (Perkin-Elmer). IC50 determination was performed by fitting the curve of percent of the solvent control activity versus the log of the inhibitor concentration using the GraphPad Prism 6.0 software.
Measurement: IC50 < 50 nM (pChEMBL 7.301000118255615)
Descriptors: MW 399.48, ALogP 2.38, HBA 6, HBD 2, PSA 117.7, QED 0.68
Compound (SMILES): CC1(C)Cc2c(C3CC3)sc(-n3c(Br)nnc3SC3(C(=O)O)CCC3)c2C1
InChIKey: COLUZXDVTGQFKS-UHFFFAOYSA-N
Target: Solute carrier family 22 member 12 (SINGLE PROTEIN), Homo sapiens
UniProt: Q96S37
Assay: In Vitro Inhibitory Activity Assay: Experimental Purpose:Determination of the IC50 value of the inhibitory activity of the compound against uric acid reabsorption by the HEK293 cell line stably transfected with the URAT-1 (uric acid transporter) gene.Background Introduction:Gout is a progressive disease induced by abnormal elevation of the uric acid level in blood. The coding URAT-1 gene exists in uric acid transporter in renal tubules. Small molecule compounds can promote uric acid excretion by inhibiting the function of this protein, thereby preventing gout attacks.
Measurement: IC50 < 500 nM (pChEMBL 6.301000118255615)
Descriptors: MW 468.44, ALogP 5.19, HBA 6, HBD 1, PSA 68.01, QED 0.65
Compound (SMILES): COCCC(=O)OC1CN(c2nc3c(c(N(C)C4CCOCC4)n2)[S+]([O-])CC3)C1
InChIKey: HLUPPSXYWZJFNN-UHFFFAOYSA-N
Target: 3',5'-cyclic-AMP phosphodiesterase 4D (SINGLE PROTEIN), Homo sapiens
UniProt: Q08499
Assay: PDE4 Assay: The human PDE4D catalytic domain (UniProt no. Q08499 [S380-L740]) was incubated with a mixture of non-labelled cAMP (cyclic adenosine monophosphate) and fluorescein amidite (FAM) conjugated cAMP and titrated test or reference compound.Following brief incubation the enzymatic reaction was stopped by addition of binding buffer containing nanoparticles with immobilized trivalent metal ions capable of binding 1) AMP phospho groups and 2) terbium (Tb) donor fluorophores. Subsequent excitation of the Tb donor triggered time-resolved FRET to adjacent FAM acceptor molecules resulting in light emission. In the presence of a PDE4 inhibitor, AMP generation was reduced resulting in a lower fluorescence signal. The cAMP phosphodiester is not bound by the detection system.Results were expressed as IC50 values (nM) calculated from inhibition curves where the TR-FRET signal was normalized to Tb fluorescence intensity and the negative (DMSO vehicle) and positive (10 microM a PDE4 inhibitor reference compound) controls.
Measurement: IC50 = 300 nM (pChEMBL 6.519999980926514)
Descriptors: MW 424.52, ALogP 0.52, HBA 9, HBD 0, PSA 100.08, QED 0.46
Compound (SMILES): CC(C)(C)OC(=O)NCc1ccc(-c2nc3ccc(C(=O)NCCCn4ccnc4)cc3nc2-c2ccccc2)cc1
InChIKey: DAELNYPSIWNDBT-UHFFFAOYSA-N
Target: Nicotinamide phosphoribosyltransferase (SINGLE PROTEIN), Homo sapiens
UniProt: P43490
Assay: NAMPT Enzyme Assay: To measure the inhibition of NAMPT activity hNAMPT protein stock and anti 6His-Tb (Cisbio; Cat. No. 61HISTLB) is diluted to 3× final concentration with assay buffer (50 mM Tris-HCl (pH7.5), 1 mM DTT, 100 mM NaCl, 10 mM MgCl2, 0.005% Tween 20). To this solution is added test compounds or vehicle control (DMSO) and BodiPY ligand (structure below). The plate is shaken for 1-2 min, sealed and incubated for 1 hour at rt in the dark. The TR-FRET signal is measured using BMG Pherastar (Lanthascreen protocol on BMG Pherastar). Excitation was carried out at 320 nm, and the ratio of emission of BodiPY (520 nm) to terbium (486 nm) was determined. Concentration response curves are generated by calculating the excitation increase in test compound-treated samples relative to DMSO-treated controls. For the assay method described above, test compound percent inhibition values at a single concentration are calculated relative to control (DMSO) treated samples. Compound concentration response curves are fitted to generate IC50 values from those curves. One skilled in the art will appreciate that these values generated either as percentage inhibition at a single concentration or IC50 values are subject to experimental variation.
Measurement: IC50 = 300 nM (pChEMBL 6.519999980926514)
Descriptors: MW 562.67, ALogP 6.01, HBA 7, HBD 2, PSA 111.03, QED 0.21
Compound (SMILES): Cc1nn2c3c(nc2s1)CCN(C(=O)COc1ccc(N2CCOCC2)cc1Cl)C3c1ccc(Cl)cc1F
InChIKey: ADJMZANHOAZVCE-UHFFFAOYSA-N
Target: Tryptophan 5-hydroxylase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P17752
Assay: Biochemical In Vitro Assay: To generate the enzyme, full length human TPH1 is cloned into the plasmid pET20b(+) (Novagen) and expressed in E. coli. The bacterial cells are ruptured by sonication on ice and the lysate is cleared by centrifugation. The resulting protein in the pellet is re-extracted and TPH1 is purified from the obtained lysate by affinity chromatography using a pterin cosubstrate analog immobilized to the resin of the column. The protein is further purified by size exclusion chromatography to remove protein aggregates. The activity of TPH1 is determined by using a fluorescence assay. The enzyme activity assay is carried out at 15° C. with atmosphere oxygen for the duration of 60 minutes in a volume of 64 μl. The reaction is carried out in a 0.1 M Tris-HCl buffer, adjusted to pH 7.6, containing 1 mM DTT, 0.2 mg/mL catalase, 100 μM (±)-6-methyl-5,6,7,8-tetrahydropterine dihydrochloride, 40 μM L-tryptophan, and 40-80 nM of TPH1. The reaction is started by bringing together L-tryptophan with all the other reaction substituents and stopped by quenching with perchloric acid (HClO4). The amount of 5-hydroxy-L-tryptophan produced during the enzymatic reaction is determined by fluorescence readout. Fluorescence, as determined at 540 nm when excited at 300 nm wavelength, increases proportionally to the 5-hydroxy-L-tryptophan formed. Compounds 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 the assay plate. Fluorescence is measured for each well and the fluorescence at 540 nm wavelength is compared to the fluorescence of the vehicle in place of compound. Inhibitory activities of example compounds with respect to the TPH1 protein are determined by calculating the IC50 value (the concentration of compound needed to inhibit 50% of the enzyme activity).
Measurement: IC50 = 4 nM (pChEMBL 8.399999618530273)
Descriptors: MW 576.48, ALogP 4.93, HBA 8, HBD 0, PSA 72.2, QED 0.34
Compound (SMILES): CN(CCC(=O)N1CCN(c2ccc(C#N)cn2)CC1)C[C@H]1c2ccccc2CN1c1cn[nH]c(=O)c1C(F)(F)F
InChIKey: PQEREFCCJHRNSM-QHCPKHFHSA-N
Target: Protein mono-ADP-ribosyltransferase TIPARP (SINGLE PROTEIN), Homo sapiens
UniProt: Q7Z3E1
Assay: Enzymatic Assay: Displacement of Probe A, a biotinylated probe binding to the TIPARP active site, was measured using a time-resolved fluorescence energy transfer (TR-FRET) assay. 20 nL of a dose response curve of each test compound was spotted in black 384-well polystyrene proxiplates (Perkin Elmer) using a Mosquito (TTP Labtech). Reactions were performed in a 8 μL volume by adding 6 JAL of TIPARP and Probe A in assay buffer (20 mM HEPES pH=8, 100 mM NaCl, 0.1% bovine serum albumin, 2 mM DTT and 0.002% Tween20), incubating with test compound at 25° C. for 30 min, then adding 2 μL of ULight-anti 6×His and LANCE Eu-W1024 labeled streptavidin (Perkin Elmer). The final concentrations of TIPARP and Probe A were 6 nM and 2 nM, respectively. The final concentration of ULight-anti 6×His and LANCE Eu-W1024 labeled streptavidin were 4 nM and 0.25 nM, respectively. Reactions were incubated at 25° C. for an additional 30 min, then read on an Envision plate reader equipped with a LANCE/DELFIA top mirror (Perkin Elmer) using excitation of 320 nm and emission of 615 nm and 665 nM with a 90 μs delay. The ratio of the 665/615 nm emission were calculated for each well to determine the amount of complex of TIPARP and Probe A in each well.
Measurement: IC50 = 550 nM (pChEMBL 6.260000228881836)
Descriptors: MW 566.59, ALogP 2.79, HBA 8, HBD 1, PSA 112.46, QED 0.46
Compound (SMILES): CC(C)C[C@H]1COCCN1c1nc2nc(-c3noc(=O)[nH]3)nc(-c3cccc(Cl)c3)c2n1C[C@H]1CC[C@H](C)CC1
InChIKey: AUHBHTJJJWPBAU-IPJJNNNSSA-N
Target: E3 ubiquitin-protein ligase Mdm2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q00987
Assay: FRET Assay: An HDM2 FRET assay was developed to assess the compounds' inhibitory activity towards binding of p53 protein. A truncated version of HDM2 with residues 17 to 125 (containing p53 binding surface, Science (1994) 265, 346-355), with N-terminal His and Thioredoxin tag was generated in pET32a expression vector and expressed in E. coli strain BL21(DE3)Rosetta. Protein was purified using Ni-affinity chromatography, followed by size exclusion chromatography using Superdex 75 26/60 column. To assess inhibition of p53 binding to HDM2, a FITC labeled 8-mer peptide (sequence: Ac-Phe-Arg-Dpr-Ac6c-(6-Br)Trp-Glu-Glu-Leu-NH2; Anal Biochem. 2004 Aug. 1; 331(1):138-46) with strong affinity towards p53 binding pocket of HDM2 was used. The HDM2 assay buffer contained 1× Phosphate Buffered Saline (Invitrogen, Cat#14190), 0.01% BSA (Jackson ImmunoResearch, Cat#001-000-162), 0.01% Tween-20. In the 1× assay buffer recombinant HDM2 protein, peptide and Lumi4-Tb Cryptate-conjugate mouse anti-6×His antibody (cisbio, Cat# Tb61HISTLB) were added and transferred to ProxiPlate PLUS (PerkinElmer, Cat#6008269), containing compounds so that final DMSO concentration is 0.1%. Final concentrations of reagents in the assay wells are 0.5 nM HDM2, 0.25 nM anti HIS (Tb label) antibody and 3 nM peptide. After two hour incubation at room temperature in a humidified chamber plates were read on EnVision plate reader with the following settings: excitation: UV, 340 nM, two emission filters: 520 nm and 495 nm respectively. Ratio of em520/em495 was used to calculate % inhibition and to obtain IC50 with 4-parameter logistic equation.
Measurement: IC50 = 47.47 nM (pChEMBL 7.320000171661377)
Descriptors: MW 566.11, ALogP 5.57, HBA 9, HBD 1, PSA 114.96, QED 0.31
Compound (SMILES): COCCCOc1ccc(N2CCc3nc(-c4ccccn4)ncc3C2)cc1F
InChIKey: BGULWXDVRQNJDH-UHFFFAOYSA-N
Target: Genome polyprotein (SINGLE PROTEIN), GB virus-B
UniProt: Q69422
Assay: HBsAg Assay: HepG2.2.15 cells were seeded in duplicate into white, 96-well plates at 1.5×104 cells/well. The cells were treated with a three-fold serial dilution series of the compounds in DMSO. The final DMSO concentration in all wells was 1% and DMSO was used as no drug control.The HBsAg chemiluminescence immunoassay (CLIA) kit (Autobio Diagnostics Co., Zhengzhou, China, Catalog number: CL0310-2) was used to measure the levels of secreted HBV antigens semi-quantitatively. For the detection 50 μL/well culture supernatant was used and HBsAg was quantified using HBsAg chemiluminescence immunoassay (CLIA) kit (Autobio Diagnostics Co., Zhengzhou, China, Catalog number: CL0310-2), 50 μL of the supernatant was transferred to the CLIA assay plate and 50 μL of enzyme conjugate reagent was added into each well. The plates were sealed and gently agitated for 1 hour at room temperature. The supernatant-enzyme-mixture was discarded and wells were washed 6 times with 300 μL of PBS. The residual liquid was removed by plating the CLIA plate right side down on absorbent tissue paper. 25 μL of substrates A and B were added to each well. Luminance was measured using a luminometer (Mithras LB 940 Multimode Microplate Reader) after 10 minutes incubation. Dose-response curves were generated and the IC50 value was extrapolated by using the E-WorkBook Suite (ID Business Solutions Ltd., Guildford, UK). The IC50 was defined as the compound concentration (or conditioned media log dilution) at which HBsAg secretion was reduced by 50% compared to the no drug control.
Measurement: IC50 = 1837 nM (pChEMBL 5.739999771118164)
Descriptors: MW 394.45, ALogP 3.66, HBA 6, HBD 0, PSA 60.37, QED 0.57
Compound (SMILES): COc1cc(-c2cnc3[nH]cc(-c4ccc(NC(C)=O)cc4)c3c2)cc(OC)c1OC
InChIKey: WMRASQJTJHIKGG-UHFFFAOYSA-N
Target: Mitogen-activated protein kinase kinase kinase 11 (SINGLE PROTEIN), Homo sapiens
UniProt: Q16584
Assay: Radiometric Filter Plate Assay: 200 ng (130 nM) MLK3 (Dundee, DU8313) was incubated with 1 μM inactive MKK7b (Dundee, DU703) in the presence of 2 μM cold ATP (Km) and 0.5 μCi/assay 33P ATP and appropriate concentrations of compounds. After a twenty minute incubation, the reactions were washed through filter plates and read on a scintillation counter.
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 417.47, ALogP 4.88, HBA 5, HBD 2, PSA 85.47, QED 0.47
Compound (SMILES): O=C(O)CN1CCN(C(c2ccc(Cl)cc2)c2ccc(Cl)cc2)CC1
InChIKey: JXBXMYFRUKHWCQ-UHFFFAOYSA-N
Target: Sodium- and chloride-dependent glycine transporter 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P48067
Assay: Cell-Based Assay: This cell-based assay measures the ability of test compounds to inhibit the uptake of glycine by the glycine transporter type 1. Human placental choriocarcinoma (JAR) cells endogenously expressing human glycine transporter type 1 (Gly-T1) were used for this assay. For uptake assays, JAR cells were cultured in 96-well cytostar T scintillating microplates (Amersham Biosciences) in RPMI 1640 medium containing 10% fetal bovine serum in the presence of penicillin (100 μg/ml) and streptomycin (100 μg/ml). Cells were plated at a density of 4×104 cells/well and grown at 37° C. in a humidified atmosphere of 5% CO2 for 24 h.Culture medium was removed from Cytostar plate and JAR cells were incubated with 30 μl of Uptake buffer (120 mM NaCl, 2 mM KCl, 1 mM CaCl2, 1 mM MgCl2, 10 mM Hepes, 5 mM alanine, pH 7.5) with or without compound for 5 min. Then 30 μl of [14C] glycine (101 mCi/mmol, obtained from Perkin Elmer) diluted in Uptake buffer was added to each well to give a final concentration of 5 μM. After incubation at room temperature for the desired time usually 1-2 h, sealed 96-well Cytostar plates were counted on a TopCount (Packard). Nonspecific uptake of [14C] glycine was determined in the presence of 10 μM cold ALX-5407 (Sigma).
Measurement: IC50 = 79.1 nM (pChEMBL 7.099999904632568)
Descriptors: MW 379.29, ALogP 3.79, HBA 3, HBD 1, PSA 43.78, QED 0.86
Compound (SMILES): Cc1ccc(F)c(NC(=O)Nc2ccc(-c3cccn4nncc34)cc2)c1
InChIKey: NUEIRIWDUMCISX-UHFFFAOYSA-N
Target: Vascular endothelial growth factor receptor 2 (SINGLE PROTEIN), Homo sapiens
UniProt: P35968
Assay: VEGF-R2 Binding Assay: A competition binding assay (DiscoveRx KINOMEscan) was used to measure the ability of the compound to compete for binding of an immobilized adenosine triphosphosphate (ATP) site directed ligand using a DNA-tagged vascular endothelial growth receptor 2 (VEGFR2) as the target. The ability of the test compound to compete with the immobilized ligand was measured using quantitative polymerase chain reaction (qPCR) of the DNA tag (Fabian, et al, Nature Biotechnology (2005) 23, 329-336; Karaman, et al, Nature Biotechnology (2008) 26, 127-132).A VEGFR2 tagged T7 phage strain was prepared in an Escherichia coli (E. coli) derived from the BL21 strain. The E. coli were grown to log-phase, infected with VEGFR2 tagged T7 phage and then incubated with shaking at 32° C. until lysis. The lysate containing the kinase was then centrifuged and filtered to remove cell debris. Affinity resin for the VEGFR2 assay was prepared by treating Streptavidin-coated magnetic beads with a biotinylated small molecule ligand for 30 minutes at room temperature. The beads were blocked with excess biotin and then washed with blocking buffer (SeaBlock (Pierce), 1% bovine serum albumin, 0.17% phosphate buffered saline, 0.05% Tween 20, 6 mM dithiothreitol). The binding reaction was initiated by combining in a well of a polystyrene 96-well plate, DNA tagged VEGFR2, liganded affinity beads and the serial diluted test compound in 1× binding buffer (20% SeaBlock, 0.17× phosphate buffered saline, 0.05% Tween 20, 6 mM dithiothreitol) in a final volume of 0.135 ml. The assay plates were incubated at room temperature with shaking for 1 hour and then the beads were washed with wash buffer (1× phosphate buffered saline, 0.05% Tween 20). The beads were re-suspended in elution buffer (1× phosphate buffered saline, 0.05% Tween 20, 0.05 μM non-biotinylated affinity ligand) and incubated at room temperature with shaking for 30 minutes. The VEGFR2 concentration in the eluate was measured using qPCR.An 11-point dose response curve of 3-fold serial diluted test compound starting at 1 μM was used to determine the VEGFR2 binding constant (Kd). The compounds were prepared in 100% DMSO at 100× the final test concentration and then diluted to 1× in the assay for final DMSO concentration of 1%. Binding constants were calculated with standard dose-response curve using the Hill equation with Hill slope set to −1. Curves were fit using a non-linear least square fit with the Levenberg-Marquardt algorithm.
Measurement: Kd = 2300 nM (pChEMBL 5.639999866485596)
Descriptors: MW 361.38, ALogP 4.49, HBA 4, HBD 2, PSA 71.32, QED 0.56
Compound (SMILES): C[C@H]1CN(c2ccncc2NC(=O)c2ccc3cc([C@H]4C[C@@H]4c4ccccc4)oc3n2)C[C@@H](N)[C@@H]1O
InChIKey: LESRWQUHWMOYGM-WBRHXPQJSA-N
Target: Serine/threonine-protein kinase pim-1 (SINGLE PROTEIN), Homo sapiens
UniProt: P11309
Assay: Enzyme Assay: Pim-1 and Pim-3 kinase assays 20 μL reactions were run in white 384 well polystyrene plates dotted with 0.8 μL compound/DMSO in the assay buffer (50 mM Tris, pH 7.5, 0.01% Tween-20, 5 mM MgCl2, 0.01% BSA, 5 mM DTT), containing 0.05 μM Biotin-labeled BAD peptide substrate (AnaSpec 62269), 1 mM ATP, and 2.5 μM (Pim-1, Invitrogen PV3503) or 1.25 μM (Pim-3, Millipore 14-738) enzyme for 1 h at 25° C. Reactions were stopped by addition of 10 μL STOP Buffer (150 mM Tris, pH=7.5, 150 mM NaCl, 75 mM EDTA, 0.01% Tween-20, 0.3% BSA) supplemented with Phospho-Bad (Ser112) Antibody (Cell Signaling 9291) diluted 666-fold, and Streptavidin donor beads (PerkinElmer 6760002) along with Protein-A acceptor beads (PerkinElmer 6760137) at 15 μg/mL each. Supplementation of the STOP buffer with beads and stopping the reactions were done under reduced light. Prior to the stopping reactions STOP buffer with beads was preincubated for 1 h in the dark at room temperature. After stopping the reactions, plates were incubated for 1 h in the dark at room temperature before reading on a PHERAstar FS plate reader (BMG Labtech) under reduced light.
Measurement: IC50 < 10 nM (pChEMBL 8)
Descriptors: MW 483.57, ALogP 3.89, HBA 7, HBD 3, PSA 117.51, QED 0.39
Compound (SMILES): O=C(NC1(c2c[nH]nn2)CC(F)(F)C1)C(=O)N1CC[C@H](C(=O)Nc2cc(F)c(F)c(Cl)c2)C1
InChIKey: FAOAERWHKLCXNN-VIFPVBQESA-N
Target: Core antigen (SINGLE PROTEIN), Hepatitis B virus
UniProt: Q03732
Assay: Anti-HBV Activity: The anti HBV activity was measured using the HepG2.117 cell line, a stable, inducible HBV producing cell line, which replicates HBV in the absence of doxicycline (Tet-off system). The HepG2 cell line is available from ATCC®) under number HB-8065. Transfection of the HepG2 cell line can be as described in Sun and Nassal 2006 Journal of Hepatology 45 (2006) 636-645 Stable HepG2- and Huh7-based human hepatoma cell lines for efficient regulated expression of infectious hepatitis B virus.For the antiviral assay, HBV replication was induced, followed by a treatment with serially diluted compound in 96-well plates. After 3 days of treatment, the antiviral activity was determined by quantification of intracellular HBV DNA using real-time PCR and an HBV specific primer set and probe.Cytotoxicity of the compounds was tested using HepG2 or HepG2.117 cells, incubated for 3 days in the presence of compounds. The viability of the cells was assessed using the Perkin Elmer ATPlite Luminescence Assay System.
Measurement: EC50 = 45 nM (pChEMBL 7.349999904632568)
Descriptors: MW 488.83, ALogP 1.96, HBA 5, HBD 3, PSA 120.08, QED 0.35