lovingscience/chembl-v37-raw-activities huggingface dataset:
Compound (SMILES): O=C(O)c1cnn(-c2nc(NC3CC3)c3cc(OC4CCCCC4)c(Cl)cc3n2)c1
InChIKey: UPTIVJANSHTGOY-UHFFFAOYSA-N
Target: Egl nine homolog 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9GZT9
Assay: PHD enzymatic assay: The PHD enzymatic assay was performed in 0.5 ml of reaction mixture containing the following: purified PHD2181-417 polypeptide (3 μg), synthetic HIF-1α peptide comprising residues [KNPFSTGDTDLDLEMLAPYIPMDDDFQLRSFDQLS] (10 μM, California Peptide Research Inc., Napa, Calif.), and [5-14C]-2-oxoglutaric acid (50 mCi/mmol, Moravek Chemicals, Brea, Calif.) in reaction buffer (40 mM Tris-HCl, pH 7.5, 0.4 mg/ml catalase, 0.5 mM DTT, 1 mM ascorbate) for 10 minutes. Compounds were pre-incubated for 30 min before starting the reaction (all test compounds were dissolved at 10 mM in 100% DMSO (w/v) and were tested with final compound concentrations at 100 μM in 1% DMSO (w/v)). The reaction was stopped by addition of 50 μl of 70 mM H3PO4 and 50 μl of 500 mM NaH2PO4, pH 3.2. Detection of [14C]-succinic acid was achieved by separating from [5-14C]-2-oxoglutaric acid by incubating the reaction mixture with 100 μl of 0.16 M DNP prepared in 30% perchloric acid. Next, 50 μl of unlabeled 20 mM 2-oxoglutaric acid/20 mM succinic acid, serving as carrier for the radioactivity, was added to the mixture, and was allowed to proceed for 30 minutes at room temperature. The reaction was then incubated with 50 μl of 1 M 2-oxoglutaric acid for 30 additional minutes at room temperature to precipitate the excess DNP. The reaction was then centrifuged at 2800×g for 10 minutes at room temperature to separate [14C]-succinic acid in the supernatant from the precipitated [14C]-dinitrophenylhydrazone. Fractions of the supernatant (400 μI) were counted using a beta counter (Beckman Coulter, Fullerton, Calif.). Inhibition of PHD2181-417 activity was measured as a decrease in succinic acid production. The IC50 values were estimated by fitting the data to a three-parameter logistic function using GraphPad Prism, version 4.02 (Graph Pad Software, San Diego, Calif.).
Measurement: IC50 = 100 nM (pChEMBL 7)
Descriptors: MW 427.89, ALogP 4.45, HBA 7, HBD 2, PSA 102.16, QED 0.6
Compound (SMILES): CNc1cc2nc(N[C@@H](C)c3cncc(F)c3)nc(C(=O)N3CC(OC)C3)c2s1
InChIKey: RVSPZKPVLQDVPP-JTQLQIEISA-N
Target: Adenosine receptor A2b (SINGLE PROTEIN), Homo sapiens
UniProt: P29275
Assay: Activity Assay: Compounds (Adenosine Receptor modulators) and activity assay.
Measurement: IC50 < 10 nM (pChEMBL 8)
Descriptors: MW 416.48, ALogP 2.91, HBA 8, HBD 2, PSA 92.27, QED 0.64
Compound (SMILES): Cc1noc(C)c1-c1cnc2c(-c3cnn(CC(F)(F)F)c3)cn(S(=O)(=O)C3CCCC3)c2c1
InChIKey: YFDCOAKGGUGZTB-UHFFFAOYSA-N
Target: Bromodomain-containing protein 4 (SINGLE PROTEIN), Homo sapiens
UniProt: O60885
Assay: Alphascreen Binding Assay: Binding of compounds of formula (I) with bromodomain 2, 3 4 was assessed using Alphascreen binding assay. The inhibition of the interaction between bromodomain and its acetylated target protein (Filippakopoulos P et al. 2012) was measured quantitatively using recombinant BRD proteins, an acetylated Histone 4 peptide and AlphaScreen technology. In absence of inhibition the BRD protein bound to AlphaScreen nickel chelate acceptor beads can interact with the acetylated Histone 4 peptide which is immobilized by the AlphaScreen Streptavidin coated beads. This interaction brings donor and acceptor beads in proximity. The close proximity allows the singlet oxygen produced by laser excitation of the donor beads to reach the acceptor beads and generate a luminescence signal. BRD inhibitors result in a decrease in the proximity signal through an inhibition of the BRD-acetylated peptide interaction.Recombinant human bromodomains containing the N-terminal bromodomain (BRD2-BD1 (71-194), BRD3-BD1 (24-144) and BRD4-BD1 (44-164)) or dual bromodomains (BRD4-BD12 (1-477), BRD4-BD12 (1-472)) were prepared and purified as described in protein expression and purification session.
Measurement: IC50 < 1000 nM (pChEMBL 6)
Descriptors: MW 493.51, ALogP 4.85, HBA 8, HBD 0, PSA 95.81, QED 0.39
Compound (SMILES): O=C(NCC1(CO)CC(F)(F)C1)[C@H]1CCN(c2ncnc3[nH]ccc23)CC12CC2
InChIKey: DLUOYGKIUAKHJC-CQSZACIVSA-N
Target: Tyrosine-protein kinase JAK1 (SINGLE PROTEIN), Homo sapiens
UniProt: P23458
Assay: JAK Kinase Assays: Human baculovirus-expressed JAK1, 2, 3 and TYK2 were purchased from Carna Biosciences, Inc. All four purified enzymes contain only the catalytic domain. JAK1 (aa 850-1154) and TYK2 (aa 871-1187) are expressed with an N-terminally fused GST-tag, and JAK2 and JAK3 with an N-terminally fused His-tag.Inhibition of phosphorylation of a synthetic peptide was measured in an HTRF-based assay using the TK substrate-Biotin from the Cisbio HTRFKinEASE TK kit. First, 2 μl of TK solution (TK substrate-biotin in kinase buffer [1× enzymatic buffer from HTRFKinEASE TK kit, 1 mM DTT]) is added to a plate containing 1 μl prediluted compound (final assay concentration DMSO: 0.75%). Then, 5 μl kinase-ATP mix (prepared in kinase buffer) is added to the wells and the plates are incubated at RT for 20-30 min. For all four kinases a concentration of ATP that corresponded to the Km for ATP was used. The final concentrations of buffers, substrate, kinase and ATP were: JAK1: 50 mM Hepes buffer pH 7.0, 0.01% BSA, 10 mM MgCl2, 1 mM DTT, 7 μM ATP, 50 nM SEB, 1 μM TK Substrate-Biotin and 5 ng JAK1; JAK2: 50 mM Hepes buffer pH 7.0, 0.01% BSA, 5 mM MgCl2, 1 mM DTT, 4 μM ATP, 1 μM TK Substrate-Biotin and 0.1 ng JAK2; JAK3: 50 mM Hepes buffer pH 7.0, 0.01% BSA, 5 mM MgCl2, 1 mM DTT, 2 μM ATP, 1 μM TK Substrate-Biotin and 0.3 ng JAK3; TYK2: 50 mM Hepes buffer pH 7.0, 0.01% BSA, 5 mM MgCl2, 1 mM DTT, 13 μM ATP, 50 nM SEB, 1 μM TK Substrate-Biotin and 0.8 ng TYK2. Thereafter, the kinase reaction is stopped by adding 4 μl detection mix (final concentrations: 50 mM Hepes buffer pH 7.0, 0.01% BSA, 0.8 M KF, 20 mM EDTA, 42 nM Streptavidin-XL665 and 1:400 STK Ab Cryptate) and the plates are incubated overnight in the dark. The HTRF signal is read using an Envision plate reader.
Measurement: EC50 = 1.3 nM (pChEMBL 8.890000343322754)
Descriptors: MW 405.45, ALogP 2.09, HBA 5, HBD 3, PSA 94.14, QED 0.71
Compound (SMILES): CCOc1cc(Nc2nc(=O)c(OC)cn2Cc2cccc(Cl)c2)c(C)cc1C
InChIKey: VBELLBSDVQHJGL-UHFFFAOYSA-N
Target: P2X purinoceptor 7 (SINGLE PROTEIN), Homo sapiens
UniProt: Q99572
Assay: Evaluation of a Human P2X7 Receptor Inhibitory Activity: Stably expressing cell line (1321N1 cell transfected with the human P2X7 receptor gene (GenBank accession number NM_002562.5 including T606C and G952A SNP)) was used. The cells were seeded in a 384-well microtiter plate at a concentration of 8000 cells/well and cultured in the medium (10% fetal bovine serum, 25 mM HEPES, 1% penicillin and streptomycin in DMEM) for one day at 37° C. under 5% carbon dioxide atmosphere. After replacing with 20 μL of the HBSS buffer (20 mM HEPES, 55.6 mM D-glucose, 1×HBSS(−), pH7.4-7.5), 15 μL of 17.3 μM Yo-Pro solution in the HBSS buffer was added. The plate was placed in high-throughput cellular screening system FLIPR TETRA (Molecullar Devices, LLC.) and 15 μL of 130 μM BzATP solution in the HBSS buffer was added. Measurement of fluorescence intensity by FLIPR TETRA was started. After eight minutes, 15 μL of DMSO solutions containing different concentrations of the compound of the present invention as prepared by dilution with the HBSS buffer were dispensed to each well through the built-in automatic dispenser, and the measurement of fluorescence intensity was continued for 20 minutes. The maximum fluorescence intensity without the compound of the present invention is calculated as 0% inhibition and the maximum fluorescence intensity when the reference compound was added is calculated as 100% inhibition. Changing values of fluorescence intensity by the compound of the present invention were calculated by difference between maximum and minimum fluorescence intensity for 20 minutes. IC50 was calculated using logistic approximation.
Measurement: IC50 < 10 nM (pChEMBL 8)
Descriptors: MW 413.91, ALogP 4.71, HBA 6, HBD 1, PSA 65.38, QED 0.61
Compound (SMILES): O=S1(=Nc2cc(C(F)(F)F)c3c(Nc4ccc(F)cc4O[C@H]4COC[C@@H]4O)ncnc3c2)CCCC1
InChIKey: WGLSBCXBBQYTPL-ICSRJNTNSA-N
Target: MAP kinase-interacting serine/threonine-protein kinase 2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9HBH9
Assay: MNK2a In Vitro Kinase Assay (Assay 2): ASSAY SETUP: The inhibition of kinase activity of MNK2a was assessed using pre-activated GST-MNK2a. The white, 384-well OptiPlate F plates were purchased from PerkinElmer. The ADP-Glo Kinase Assay (including ultra pure ATP) was purchased from Promega (V9103). Activated MNK2a was obtained as described in WO2011/104340. The unlabeled eIF4E peptide (NH2-TATKSGSTTKNR-CONH2), differing from Seq. ID No. 5 of WO 2011/104340 by the C-terminal CONH2 group, was purchased from Thermo Fisher Scientific. All other materials were of highest grade commercially available. Compounds are tested in either serial dilutions or single dose concentrations. The compound stock solutions are 10 mM in 100% DMSO The serial compound dilutions are prepared in 100% DMSO followed by 1:27.3 intermediate dilution in assay buffer. The final DMSO concentration in assay will be <3%.In the 384-well plates 3 μl of test compound from the intermediate dilutionis mixed with 4 μl of the activated MNK2 enzyme (final concentration of 10 nM) and 4 μl of the peptide (final concentration of 25 μM)/ultra pure ATP (final concentration of 20 μM), all dissolved in assay buffer. This step is followed by an incubation time of 90 min, then 10 μl of ADP Glo reagent are added, followed by 40 min of incubation. Then 20 μl of kinase detection reagent are admixed. The plates are sealed and after an incubation period of 30 min, the luminescence signal is measured in an Envision reader to determine the amount of produced ADP. All incubation steps are performed at room temperature.The assay buffer consists of 20 mM HEPES, 2 mM DTT, 0.01% BSA, 20 mM MgCl2 and 0.1% Pluronic F-127.Each assay microtiter plate contains wells with vehicle controls instead of compound (1% DMSO in water) as reference for the high signal (100% CTL, high signal), and wells containing a potent MNK2 inhibitor (final 20 μM, 1% DMSO) as reference for low signal (0% CTL, low signal).The luminescent signal generated is proportional to the ADP concentration produced and is correlated with activated MNK2 activity. The analysis of the data is performed by the calculation of the percentage of ATP consumption of activated MNK2 in the presence of the test compound compared to the consumption of ATP in the presence of activated MNK2 without compound. RLU(sample)−RLU(low control))*100/(RLU(high value)−RLU(low control)) [RLU=relative luminescence units]An inhibitor of the MNK2 enzyme will give values between 100% CTL (no inhibition) and 0% CTL (complete inhibition). Values of more than 100% CTL are normally related to compound/sample specific physico-chemical properties (e.g. solubility, light absorbance, fluorescence).IC50 values based on dose response curves are calculated with the AssayExplorer software.
Measurement: IC50 = 4 nM (pChEMBL 8.399999618530273)
Descriptors: MW 526.51, ALogP 4.56, HBA 8, HBD 2, PSA 105.93, QED 0.47
Compound (SMILES): CN(C)Cc1nc(C2CC2)c2sc(N3CCC[C@@H]3C(=O)NCc3ccccc3)nc2n1
InChIKey: AZSFMVPSDHTDLK-QGZVFWFLSA-N
Target: Kynurenine/alpha-aminoadipate aminotransferase, mitochondrial (SINGLE PROTEIN), Homo sapiens
UniProt: Q8N5Z0
Assay: Inhibitory Assay: To a reaction mixture (45 μL) containing 3.0 μmol/L kynurenine, 10 μmol/L pyridoxal phosphate, 2.0 ng/μL human recombinant KAT-II, and 150 mmol/L tris(hydroxymethyl)aminomethane-acetate buffer (pH 8.0) was added a 10% dimethyl sulfoxide solution (5 μL) of each test compound prepared, and the mixture was reacted at 37° C. for 1 hr. After the reaction, 50% trichloroacetic acid (5 μL) was added to terminate the reaction.The resultant kynurenic acid was quantified as follows by high performance liquid chromatography. An enzyme reaction mixture was separated by an octadecylsilane reversed-phase column (SC-50DS, Eicom Corporation; mobile phase: 250 mmol/L zinc acetate, 50 mmol/L sodium acetate, and 5.0% acetonitrile (pH 6.2)) incubated at 30° C., and kynurenic acid was quantified using a fluorescence detector (RF-20Axs, Shimadzu Corporation) at excitation wavelength 354 nm, detection wavelength 460 nm. The analytical curve was drawn every time by an external standard method. Each test compound was tested by dual measurement at each concentration. The kynurenic acid level in the presence of a test compound at each concentration was converted into % relative to kynurenic acid resulting from a reaction with an enzyme alone as 100%, and the obtained values were fitted to S-curve to determine IC50.
Measurement: IC50 = 640 nM (pChEMBL 6.190000057220459)
Descriptors: MW 436.59, ALogP 3.31, HBA 7, HBD 1, PSA 74.25, QED 0.61
Compound (SMILES): Cc1cc(N)nc(C)c1CNC(=O)c1ccc2c(c1)[C@@H]1O[C@H]2c2ccc(NC(=O)c3ccccc3)cc21
InChIKey: BKQDSZUUZPRYAK-IZLXSDGUSA-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 = 10000 nM (pChEMBL 5)
Descriptors: MW 490.56, ALogP 4.99, HBA 5, HBD 3, PSA 106.34, QED 0.37
Compound (SMILES): CCn1c(=O)[nH]/c(=N\c2ccc(Oc3ccnc(C(=O)O)c3)c(F)c2)n(Cc2ccc(Cl)cc2)c1=O
InChIKey: IEUFDBKLBLTXAS-UHFFFAOYSA-N
Target: P2X purinoceptor 3 (SINGLE PROTEIN), Homo sapiens
UniProt: P56373
Assay: Human P2X3 Receptor Inhibition Assay: Stably expressing cell line (C6BU-1 transfected with human P2X3 receptor gene (GenBank accession number Y07683) was used. The cells were seeded in a 96-well microtiter plate at a concentration of 8000 cells/well and cultured in the medium (8.3% fetal bovine serum, 8.3% horse serum, 1% antibiotic and antifungal in DMEM) for one day at 37° C. under 5% carbon dioxide atmosphere. The medium was replaced with 4 μM Fluo-3-AM solution (20 mM HEPES, 137 mM NaCl, 2.7 mM KCl, 0.9 mM MgCl2, 5.0 mM CaCl2, 5.6 mM D-glucose, 2.5 mM probenecid, 10% BSA, and 0.08% Pluronic F-127, pH 7.5) and incubated at 37° C. under 5% dioxide carbon atmosphere for one hour. The plate was washed with washing buffer (20 mM HEPES, 137 mM NaCl, 2.7 KCl, 0.9 mM MgCl2, 5.0 mM CaCl2, 5.6 mM D-glucose, 2.5 mM probenecid, pH 7.5), each well was added with 40 μL of this buffer. The plate was placed in High-Throughput Screening System FDSS 3000 (Hamamatsu Photonics K.K.). Measurement of fluorescence intensity by FDSS 3000 was started, and 40 μl, of DMSO solutions containing different concentrations of the test, compound as prepared by dilution with dilution buffer (20 mM HEPES, 137 mM NaCl, 2.7 mM KCl, 0.9 mM MgCl2, 5.0 mM CaCl2, 5.6 mM D-glucose, 2.5 mM probenecid, 0.1.% Pluronic F-127, pH 7.5) were dispensed to each well through the built-in automatic dispenser. Five minutes after, 50 nM ATP solution (50 μL) prepared by dilution with the dilution buffer was dispensed through the built-in automatic dispenser, and the measurement of fluorescence intensity was continued for 3 min. For each well, the specific maximum fluorescence intensity was calculated as the ratio of the maximum fluorescence intensity after addition of the ATP solution to the fluorescence intensity at the starting of the measurement. The 50% inhibitory concentration (IC50) was calculated under the assumption that the specific maximum fluorescence intensity without test compound is 0% inhibition and that the specific maximum fluorescence intensity when the dilution buffer was added in place of ATP solution is 100% inhibition, to evaluate the inhibitory activity of the test compound. FDSS software (Hamamatsu Photonics K.K.) was used for calculation of the specific maximum fluorescence intensity. IC50 was calculated using Microsoft Excel (Microsoft Corporation) and Ltd.).
Measurement: IC50 = 7 nM (pChEMBL 8.149999618530273)
Descriptors: MW 511.9, ALogP 3.32, HBA 8, HBD 2, PSA 131.57, QED 0.39
Compound (SMILES): CC(C)c1cc2nc(C(=O)Nc3cnccc3N3C[C@@H](N)[C@H](O)[C@@H](C)C3)ccc2o1
InChIKey: GLYWPANTIWPDCD-PXIJXODZSA-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 = 30 nM (pChEMBL 7.519999980926514)
Descriptors: MW 409.49, ALogP 2.74, HBA 7, HBD 3, PSA 117.51, QED 0.61
Compound (SMILES): COc1cc(C(=O)N2CC3CCC2[C@@H]3N)cc2nc(-c3cc4ccc(-c5ccc(C(=O)NCCC(N)=O)cc5)nc4n3CC3CC3)n(C)c12
InChIKey: VOBZGOGESGOLJC-PWJCZFGMSA-N
Target: Protein-arginine deiminase type-4 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9UM07
Assay: Rapid-Fire Mass Spectrum (RFMS) Assay: Compounds were solubilized in 100% DMSO to achieve a 10 mM compound concentration. Compound stock solutions were stored at RT. A series of dilutions were prepared in DMSO and mixed 8 times with 20 μL mixing volume. Final top concentration of compound in the assay is 50 μM. Final assay conditions were as follows:Reaction volume: 26 μlAssay buffer: 25 mM hepes, pH 7.5, 5 mM NaCl, 1 mM DTT, 0.2 mg/ml BSA, 0.01% CHAPS, 50 μM Calcium, and 5 μM TPENFinal concentrations: 5 nM hPAD4 enzyme, 250 μM BAEE, and 0.5% DMSOTotal incubation time: 30 mins compound and enzyme preincubation at 37° C., 90 min enzyme/substrate reaction, 30 min reaction with phenyl glyoxal at 37° C.Stop solution: 40 μl 5% TCA in ACN
Measurement: IC50 = 658 nM (pChEMBL 6.179999828338623)
Descriptors: MW 660.78, ALogP 3.84, HBA 9, HBD 3, PSA 163.39, QED 0.2
Compound (SMILES): CC(=O)OCCn1c(=O)n(C)c2cc(S(=O)(=O)NC3(C)CC3)ccc21
InChIKey: OQLVWAPWEJEYAP-UHFFFAOYSA-N
Target: Poly(ADP-ribose) glycohydrolase (SINGLE PROTEIN), Homo sapiens
UniProt: Q86W56
Assay: PARG Assay: PARG In vitro assays were conducted in a total volume of 15 ul in a standard 384 well format. 5 ul of Human Full Length PARG (Produced internally by Astra Zeneca), used at a final reaction concentration of 80 μM, was added to 5 ul of Ribosylated PARP substrate (also produced internally by Astra Zeneca) at final reaction concentration of 4.5 nM in assay buffer (50 mM Tris pH7.4, 0.1 mg/ml BSA, 3 mM EDTA, 0.4 mM EGTA, 1 mM DTT, 0.01% Tween 20, 50 mM KCl). The reaction was incubated at room temperature for 10 minutes and then 5 ul detection reagent was added. Detection Reagent consists of 42 nM MAb Anti-6HIS XL665 (CisBio: 61 HISXLB) and 2.25 nM Streptavidin Europium Cryptate (CisBio: 610SAKLB), both at 3× working stock concentrations (final concentrations of 14 nM and 0.75 nM respectively), in a detection buffer of 50 mM Tris pH7.4, BSA at 0.1 mg/ml and KF at 100 mM. Following incubation at room temperature for 60 minutes in the dark, TR-FRET signal was measured at Ex 340 and Em 665 and Em 620. A ratio was calculated as Em665/EM620×104 for each well and used to calculate percent inhibition for test compounds.
Measurement: EC50 = 9181 nM (pChEMBL 5.039999961853027)
Descriptors: MW 367.43, ALogP 0.73, HBA 7, HBD 1, PSA 99.4, QED 0.76
Compound (SMILES): Cc1c(-c2cc3cc(NC(=O)O[C@H]4COC[C@@H]4C)ncc3c(N)c2F)cnc2c1NC[C@@H](F)O2
InChIKey: VDBSAPWJFNZJIJ-POJUEXTFSA-N
Target: Mitogen-activated protein kinase kinase kinase kinase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q92918
Assay: TBD: TBD
Measurement: Ki = 0.119 nM (pChEMBL 9.920000076293945)
Descriptors: MW 471.46, ALogP 4.01, HBA 8, HBD 3, PSA 120.62, QED 0.49
Compound (SMILES): C[C@@H]1CN(c2ncc(C(=O)NC3CC3)cn2)C[C@H](C)N1C(=O)NCCC1CCN(Cc2ccccc2)CC1
InChIKey: WQUOADCQKNSJAM-SZPZYZBQSA-N
Target: Muscarinic acetylcholine receptor M4 (SINGLE PROTEIN), Homo sapiens
UniProt: P08173
Assay: Binding Assay: Binding affinity (Ki) of compounds was measured by inhibition of radioligand binding to membranes from CHO cells expressing human M1, M2, M3, M4 and M5 receptors. Membranes were prepared by nitrogen cavitation and differential centrifugation as previously described (Hoare et al., Mol. Pharmacol. 2003 March; 63(3): 751-65). The radioligand employed was tritiated N-methylscopolamine, used at a concentration of 1.5 nM. A dose-response of twelve concentrations of compound was used, ranging from 10 Î¼M to 32 Î¼M. The assay buffer was 50 mM HEPES, 100 mM NaCl, 5 mM MgCl2, 1 mM ethylenediaminetetraacetic acid, pH-adjusted to pH 7.4.
Measurement: Ki = 55 nM (pChEMBL 7.260000228881836)
Descriptors: MW 519.69, ALogP 3.28, HBA 6, HBD 2, PSA 93.7, QED 0.56
Compound (SMILES): Cn1cnc2ccc(-c3cc(-c4ccnn4C4CCCCC4)c4c(N)ncnn34)cc21
InChIKey: IGDRSWAIEJAIOH-UHFFFAOYSA-N
Target: Phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit delta isoform (SINGLE PROTEIN), Homo sapiens
UniProt: O00329
Assay: ADP-Glo Format PI3K Assays: The ADP-Glo format PI3K assays were performed in Proxiplate 384-well plates (Perkin Elmer #6008280). The final assay volume was 2 μl prepared from 1 μl additions of enzyme/PIP2:PS lipid (Invitrogen #PV5100) mixture and 1 μl ATP (provided in kit, Promega #V9101) and test compounds in assay buffer (50 mM HEPES pH 7.5, 3 mM MgCl2, 100 mM NaCl, 0.5 mM EGTA, 2 mM DTT, 0.03% CHAPS). The reaction was initiated by the combination of enzyme/lipid, ATP, and test compounds. The reaction mixture was incubated at room temperature for 30 minutes (PI3K Alpha, Beta, Gamma) or 3 hours for PI3K Delta. ADP-Glo (2 μl), followed by Kinase Detection reagent (4 μl), were added to reactions following the initial incubation and allowed to incubate 40 minutes at room temperature. The reaction mixture was analyzed on the TOPCOUNT (Perkin Elmer). Inhibition data were calculated by comparison to no enzyme control reactions for 100% inhibition and vehicle-only reactions for 0% inhibition. The final concentration of enzyme in the assays are PI3K Alpha [0.5 nM], PI3K Beta [2 nM], PI3K Gamma [20 nM], PI3K Delta [0.5 nM]. ATP final concentrations are as follows: for Alpha [10 μM], for Beta [12.5 μM], for Gamma [6.5 μM], for Delta [100 μM]. Lipid final concentration was the same for all enzymes, [25 μM]. Dose response curves were generated to determine the concentration required to inhibit 50% of activity. Compounds were dissolved at 0.12 mM in dimethylsulfoxide (DMSO) and evaluated at eleven concentrations.
Measurement: IC50 = 9 nM (pChEMBL 8.050000190734863)
Descriptors: MW 412.5, ALogP 4.23, HBA 8, HBD 1, PSA 91.85, QED 0.48
Compound (SMILES): C[C@@H]1CN(CCS(=O)(=O)N(Cc2ncc(-c3nnc(C(F)F)o3)s2)c2cncc(F)c2)CCO1
InChIKey: YRKWWNMUMPQFRA-GFCCVEGCSA-N
Target: Protein deacetylase HDAC6 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9UBN7
Assay: Biochemical Assay: The compounds disclosed herein were tested for potency against HDAC6 and selectivity against HDAC1 in a biochemical assay. A biochemical assay was adopted using a luminescent HDAC-Glo I/II assay (Promega) and measured the relative activity of HDAC6 and HDAC1 recombinant proteins. Compounds were first incubated in the presence of HDAC6 or HDAC1 separately, followed by addition of the luminescent substrate. The data was acquired using a plate reader and the biochemical IC50 were calculated from the data accordingly.
Measurement: IC50 = 33.6 nM (pChEMBL 7.46999979019165)
Descriptors: MW 518.54, ALogP 2.72, HBA 10, HBD 0, PSA 114.55, QED 0.42
Compound (SMILES): Cn1nc2c(c1-c1cc(Cl)cc(Cl)c1)C[C@@H]1CCC[C@H]2N1C(=O)c1cccn2cncc12
InChIKey: ACNBBUMQYICGGR-FXAWDEMLSA-N
Target: Monoglyceride lipase (SINGLE PROTEIN), Homo sapiens
UniProt: Q99685
Assay: In Vitro Activity Assay: The assay used to measure the in vitro activity of MGL is adapted from the assay used for another serine hydrolase (FAAH) described in Wilson et al., 2003 (A high-throughput-compatible assay for determining the activity of fatty acid amide hydrolase. Wilson S J, Lovenberg T W, Barbier A J. Anal Biochem. 2003 Jul. 15; 318(2):270-5). The assay consists of combining endogenously expressed MGL from HeLa cells with test compounds, adding [glycerol-1,3-3H]-oleoyl glycerol, incubating for one hour, and then measuring the amount of cleaved [1,3-3H]-glycerol that passes through an activated carbon filter. The amount of cleaved, tritiated glycerol passing through the carbon filter is proportional to the activity of the MGL enzyme in a particular well/test condition. Standard conditions for this assay combine 300 nM [Glycerol-1,3-3H]-oleoyl glycerol with human MGL from HeLa cells and test compounds for one hour, after which the reaction is filtered through activated carbon and tritium is measured in the flow through. The test compound concentration in screening mode is 10 uM, while the highest compound concentration in IC50 assays is determined empirically.
Measurement: IC50 = 0.72 nM (pChEMBL 9.140000343322754)
Descriptors: MW 466.37, ALogP 5.33, HBA 5, HBD 0, PSA 55.43, QED 0.4
Compound (SMILES): C[C@@H]1CC(=O)Nc2cccc(-c3cccc(N(C)C)c3)c2N1
InChIKey: ZIHLWRPTXLDVDL-GFCCVEGCSA-N
Target: CREB-binding protein (SINGLE PROTEIN), Homo sapiens
UniProt: Q92793
Assay: CBP TR-FRET Binding Assay: His/Flag epitope tagged CBP was cloned, expressed, and purified to homogeneity. CBP binding and inhibition was assessed by monitoring the engagement of a biotinylated small molecule compound with the target using the TR-FRET assay technology (Perkin-Elmer). Specifically, in a 384 well ProxiPlate CBP (4 nM final) was combined with biotin-ligand (60 nM final) in 50 mM HEPES (pH 7.5), 50 mM NaCl, 1 mM TCEP, 0.01% (w/v) BSA, and 0.008% (w/v) Brij-35 either in the presence of DMSO (final 0.2% DMSO) or compound dilution series in DMSO. After 10 minutes incubation at room temperature, a mixture Eu-W1024 Anti-6×His antibody (Perkin Elmer ADO 110) and SureLight Allophycocyanin-Streptavidin (APC-SA, Perkin Elmer CR130-100) were added to a final concentrations of 0.2 nMolar antibody and 50 nMolar APC-SA, respectively. After twenty minutes of equilibration, the plates were read on an Envision instrument and IC50s calculated using a four parameter non-linear curve fit.
Measurement: IC50 = 210 nM (pChEMBL 6.679999828338623)
Descriptors: MW 295.39, ALogP 3.56, HBA 3, HBD 2, PSA 44.37, QED 0.89
Compound (SMILES): CCOC(=O)[C@H]1C[C@@H](n2c(=O)[nH]/c(=N\c3ccc(Oc4ccc(Cl)cn4)cc3)n(Cc3ccc(C)cc3)c2=O)C1
InChIKey: JXZZQSYZDBZVKO-UHGJSFDGSA-N
Target: P2X purinoceptor 3 (SINGLE PROTEIN), Homo sapiens
UniProt: P56373
Assay: Receptor Inhibition Assay: Stably expressing cell line (C6BU-1 cell transfected with human P2X3 receptor gene (GenBank accession number Y07683) was used. The cells were seeded in a 384-well PDL-coated microtiter plate at a concentration of 3000 cells/well and cultured in the medium (8.3% fetal bovine serum, 8.3% horse serum, and 1% antibiotic and antifungal in DMEM) for one day at 37° C. under 5% carbon dioxide atmosphere. The medium was replaced with 4 μM Fluo-3-AM solution (20 mM HEPES, 137 mM NaCl, 2.7 mM KCl, 0.9 mM MgCl2, 5.0 mM CaCl2, 5.6 mM D-glucose, 2.5 mM probenecid, 0.5% BSA, and 0.04% Pluronic P-127, pH 7.5) and incubated at 37° C. under 5% dioxide carbon atmosphere for one hour. The plate was washed with washing buffer (20 mM HEPES, 137 mM NaCl, 2.7 mM KCl, 0.9 mM MgCl2, 5.0 mM CaCl2, 5.6 mM D-glucose, 2.5 mM probenecid, pH 7.5), and each well was added with 20 μL of the washing buffer. The plate was placed in High-Throughput Screening System FLIPR 384 (Molecular Device Co.). Measurement of fluorescence intensity by FLIPR 884 was started, and 20 μL of DMSO solutions containing different concentrations of the test compound as prepared by dilution with dilution buffer (20 mM HEPES, 137 mM NaCl, 2.7 mM KCl, 0.9 mM MgCl2, 5.0 mM CaCl2, 5.6 mM D-glucose, 2.5 mM probenecid, 0.1% Pluronic F-127, pH 7.5) were dispensed to each well through the built-in automatic dispenser. Five minutes after, 150 nM ATP solution (25 μL) prepared by dilution with the dilution buffer was dispensed through the built-in automatic dispenser, and the measurement of fluorescence intensity was continued for 4 min. For each well, the specific maximum fluorescence intensity was calculated as the ratio of the maximum fluorescence intensity after addition of the ATP solution to the fluorescence intensity at the starting of the measurement.
Measurement: IC50 = 8 nM (pChEMBL 8.100000381469727)
Descriptors: MW 562.03, ALogP 4.28, HBA 9, HBD 1, PSA 120.57, QED 0.32
Compound (SMILES): NCCCNC(=O)c1ccc(-c2cc(NCCCC3CCCNC3)c3ccccc3n2)cc1
InChIKey: SDFCKQNSMPHIHS-UHFFFAOYSA-N
Target: Low molecular weight phosphotyrosine protein phosphatase (SINGLE PROTEIN), Homo sapiens
UniProt: P24666
Assay: LMPTP Primary Screening Protocol: A listing of materials is provided:    Item, source, catalog no.    LMPTP-A Enzyme Stock Solution (4.22 mg/ml or 206.8 μM), SBMRI Protein Facility,    N/A    OMFP, Sigma, M2629-100 MG    Bis-Tris pH 6.0, Fisher Sci, BP301-100    Triton-X 100, Sigma, T9284    DTT, Sigma, D9779    Mol. Grade Water, Mediatech, Inc., 46-000-CM    1536-well black High base opaque bottom plate, Nexus Biosystems, 00019120Final assay conditions are:    0.625 nM LMPTP-A Enzyme    40 μM OMFP    50 mM Bis-Tris pH 6.0    1 mM DTT    0.01% Triton-X 100    20 μM test compound    3% DMSO (2% from substrate and 1% from compounds)    6 μL reaction volume    50 minutes incubation at room tempAssay Procedure    1. Prepare Reagents as described in section F. Recipe.    2. Using LabCyte Echo, transfer 60 nL from 2 mM test compound source plate into assay plate Col. 5-48 (final concentration of test compounds is 20 μM). 60 nL of DMSO should be transferred to col. 1-4 for positive and negative control wells.    3. Spin plates at 1000 rpm for 1 minute in centrifuge.    4. Using the Beckman Coulter Bioraptr, add 3 μL/well of control buffer to columns 1 and 2.    5. Using the Bioraptr, add 3 μL/well of enzyme solution to col. 3-48.    6. Using the Bioraptr, add 3 μL/well of substrate solution to col. 1-48.    7. Spin plates at 1000 rpm for 1 minute in centrifuge.    8. Incubate plates in the dark at room temperature for 50 minutes.    9. Read plates on PerkinElmer Viewlux using a FI protocol.
Measurement: IC50 = 500 nM (pChEMBL 6.300000190734863)
Descriptors: MW 445.61, ALogP 4.17, HBA 5, HBD 4, PSA 92.07, QED 0.35
Compound (SMILES): CC(C)(C)[C@H]1CC[C@H](Oc2cccc3cc(CN4C5CCC4CC(C(=O)O)C5)ccc23)CC1
InChIKey: QGBJOPOOEGSIMA-ILDSLZLGSA-N
Target: Autotaxin (SINGLE PROTEIN), Homo sapiens
UniProt: Q13822
Assay: ATX biochemical assay: ATX (Autotaxin) is a 125 KDa glycoprotein with lysophospholipase D (LPLD) activity that generates the bioactive lipid lysophosphatidic acid (LPA) from lysophosphatidylcholine (LPC). The ATX biochemical assay utilizes a FRET (fluorescence resonance energy transfer) technology platform. The fluorescence signal of FRET substrate FS-3 is quenched due to intra-molecular FRET of a fluorophore to a non-fluorescing quencher (Ferguson, C. G., et al., Org Lett. 2006 May 11; 8(10): 2023-2026, which is incorporated by reference in its entirety). ATX catalyzes the hydrolysis of the substrate which separates the dabsyl quencher from the fluorescein reporter, which becomes fluorescent. The reaction is monitored by a SpectraMax M5 (Molecular Devices, Sunnyvale, Calif.) with at excitation wavelength 485 nm and emission wavelength 535 nm.ReagentsFatty acid free-BSA (Sigma A8806): 10 mg/mL in H2O, stored at 4° C.2×ATX assay buffer: 100 mM Tris, 280 mM NaCl, 10 mM KCl, 2 mM CaCl2, 2 mM MgCl2, pH 7.4.Human ATX protein: expressed and purified in house. Stored at −80° C.Substrate FS-3 (Echelon, L-2000): 100 μg in 77.74 μL H2O (1 mM stock), stored at −20° C.384-well flat bottom platesCorning #3575.AssayCompound dilutionAll compounds were provided at 10 mM in 100% DMSO. In the first well, 2 μL of 10 mM compound was added to 78 μL of DMSO (1:40 dilution). In subsequent wells 3-fold dilution (total 10 dilutions) were performed.1×ATX assay buffer was made up with a final concentration of 1 mg/mL fatty acid free-BSA using 2×ATX assay buffer, 10 mg/ml fatty acid free-BSA and ddH2O.ATX protein was diluted with 1×ATX assay buffer to a concentration of 1.32 μg/mL (1.32×). 38 μL was added per well to the assay plate. The final concentration of ATX in the reaction as 1.0 μg/mL.2 μL per well of compounds was transferred to provide the desired concentration. The plate was centrifuged, then incubated at room temperature for 30 minutes on the shaker.FS-3 was diluted with 1×ATX assay buffer to a concentration of FS-3 of 10 μM (5×). Then, 10 μL was added per well to the assay plate. The final concentration of FS-3 in the reaction was 2 μM. The plate was centrifuged. The plate was kept shaking at room temperature for 2 hours. Because FS-3 substrate is light sensitive, plates were kept covered and protected from light.Fluorescence was measured using SpectraMax M5 (excitation at 485 nm/emission at 538 nm, top read).
Measurement: IC50 = 2750 nM (pChEMBL 5.559999942779541)
Descriptors: MW 449.64, ALogP 6.65, HBA 3, HBD 1, PSA 49.77, QED 0.56
Compound (SMILES): Cc1ccc(/C=C/c2n[nH]c3cc(C4C[C@@]45C(=O)Nc4ccccc45)ccc23)c(C)n1
InChIKey: HPJPZVODQVTPBB-ONDVHJRDSA-N
Target: Serine/threonine-protein kinase PLK4 (SINGLE PROTEIN), Homo sapiens
UniProt: O00444
Assay: Inhibition Assay: PLK4 activity was measured using an indirect ELISA detection system. Dephosphorylated GST-PLK4 (4 nM) was incubated in the presence of 15 μM ATP (Sigma cat#A7699), 50 mM HEPES-Na2+ pH 7.4, 10 mM MgCl2, 0.01% Brij 35 (Sigma cat#03-3170), in a 96 well microtitre plate pre-coated with MBP (Millipore cat#30-011). The reaction was allowed to proceed for 30 minutes, followed by 5 washes of the plate with Wash Buffer (50 mM TRIS-C1 pH 7.4 and 0.2% Tween 20), and incubation for 30 minutes with a 1:3000 dilution of primary antibody (Cell Signaling cat#9381). The plate was washed 5 times with Wash Buffer, incubated for 30 minutes in the presence of secondary antibody coupled to horse radish peroxidase (BioRad cat#1721019, 1:3000 concentration), washed an additional 5 times with Wash Buffer, and incubated in the presence of TMB substrate (Sigma cat#T0440). The colourimetric reaction was allowed to continue for 5 minutes, followed by addition of stop solution (0.5 N sulphuric acid), and quantified by detection at 450 nm with either a monochromatic or filter based plate reader (Molecular Devices M5 or Beckman DTX880, respectively).
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 406.49, ALogP 5.12, HBA 3, HBD 2, PSA 70.67, QED 0.5
Compound (SMILES): C=CC(=O)N1CC[C@@H](Nc2cnc3[nH]ccc3n2)C1
InChIKey: JYUPDYULQGZBHP-SECBINFHSA-N
Target: Tyrosine-protein kinase JAK3 (SINGLE PROTEIN), Homo sapiens
UniProt: P52333
Assay: JAK Caliper Enzyme Assay: Test article was solubilized in dimethyl sulfoxide (DMSO) to a stock concentration of 30 mM. An 11-point half log dilution series was created in DMSO with a top concentration of 600 μM. The test compound plate also contained positive control wells containing a known inhibitor to define 100% inhibition and negative control wells containing DMSO to define no inhibition. The compound plates were diluted 1 to 60 resulting in a top final assay compound concentration of 10 μM and a 2% DMSO concentration.Test article and assay controls were added to a 384-well plate. Reaction mixtures contained 20 mM HEPES, pH 7.4, 10 mM magnesium chloride, 0.01% bovine serum albumin (BSA), 0.0005% Tween 20, 4 μM ATP and 1 μM peptide substrate. The JAK3 assays contained 1 μM of the JAKtide peptide (FITC-KGGEEEEYFELVKK). The assays were initiated by the addition 1 nM JAK3 enzyme and were incubated at room temperature 75 minutes for JAK3. Enzyme concentrations and incubation times were optimized for each new enzyme preps and were modified slightly over time to ensure 20%-30% phosphorylation. The assays were stopped with a final concentration of 10 mM EDTA, 0.1% Coating Reagent and 100 mM HEPES, pH=7.4. The assay plates were placed on a Caliper Life Science Lab Chip 3000 (LC3000) instrument, and each well was sampled using appropriate separation conditions to measure the unphosphorylated and phosphorylated peptide.
Measurement: IC50 = 622 nM (pChEMBL 6.210000038146973)
Descriptors: MW 257.3, ALogP 1.16, HBA 4, HBD 2, PSA 73.91, QED 0.81
Compound (SMILES): NC(=O)Cc1ccc(Nc2nc(-c3cccc(Cl)c3)nc3c2S(=O)(=O)CC3)cc1
InChIKey: ZARAMEQAEZGXSO-UHFFFAOYSA-N
Target: 3',5'-cyclic-AMP phosphodiesterase 4B (SINGLE PROTEIN), Homo sapiens
UniProt: Q07343
Assay: Kinetic Assay: Assays are performed in 96-well plates in a total volume of 200 μl/well. Compounds are dissolved in dimethylsulfoxide (DMSO) and added to plates in a volume of 10 μl followed by addition of 165 μl of assay mix. Plates are pre-incubated at 25° C. for 15 min and the reactions are initiated by the addition of 25 μl of cAMP followed by thorough mixing. Reaction rates are measured by monitoring the decrease in fluorescence using excitation at 355 nm and emission at 460 nm for a period of 10 min in a fluorescence plate reader. Initial rates (slopes) are determined from linear portions of the progress curves. Final concentrations of assay components are as follows: 50 mM Tris, pH 8, 10 mM MgCl2, 50 mM KCl, 2% DMSO, 5 mM tris(2-carboxyethyl)phosphine (TCEP), 0.4 mM phosphenolpyruvate (PEP), 0.01 mM NADH, 0.04 mM adenosine triphosphate (ATP), 0.004 mM cAMP, 7.5 units myokinase from yeast, 1.6 units pyruvate kinase, 2 units lactate dehydrogenase, and either 0.5 nM human PDE4D7 or 10 nM human PDE4B1. All data are percent normalized relative to controls and are presented as percent inhibition. An inhibitory concentration 50% (IC50) value is calculated by fitting of a sigmoidal dose response curve. Human PDE4D7 contained a mutation of serine 54 to aspartic acid to mimic activation by cAMP-dependent protein kinase A (PKA). Human PDE4B1 contained a corresponding mutation of serine 133 to aspartic acid to also mimic PKA activation.
Measurement: IC50 < 1000 nM (pChEMBL 6)
Descriptors: MW 428.9, ALogP 2.9, HBA 6, HBD 2, PSA 115.04, QED 0.65
Compound (SMILES): COC1CCN(Cc2c(C)cc(C)c3[nH]ccc23)C(c2ccc(F)cc2)C1
InChIKey: BWVLOTHJSYXNJA-UHFFFAOYSA-N
Target: Complement factor B (SINGLE PROTEIN), Homo sapiens
UniProt: P00751
Assay: Human Complement Factor B TR-FRET Assay: CVF-Bb complex prepared from purified cobra venom factor (1 uM), recombinant human complement factor B (expressed in drosophila cells and purified using standard methods) and human complement factor D (expressed in E. Coli, refolded and purified using standard methods). CVF-Bb complex at 3 nM concentration was incubated with test compound at various concentrations for 1 hour at room temperature in PBS pH 7.4 containing 10 mM MgCl2 and 0.05% (w/v) CHAPS. Human complement C3 substrate purified from plasma was added to a final concentration of 1 uM. After 1 hour incubation at room temperature, the enzyme reaction was stopped by addition of a cocktail of concentrated pan-protease inhibitors. The product of the reaction, C3a, was quantified by means of an enzyme-linked-immunosorbent assay. IC50 values were calculated from percentage of inhibition of CVF-Bb activity as a function of test compound concentration.
Measurement: IC50 = 24 nM (pChEMBL 7.619999885559082)
Descriptors: MW 366.48, ALogP 5.28, HBA 2, HBD 1, PSA 28.26, QED 0.68
Compound (SMILES): CC(C)Oc1ccc(S(=O)(=O)N2C[C@@H](n3nc(CC(=O)O)c4ccc(F)cc43)C[C@H]2C)cc1
InChIKey: APXJUHKUJIXCRS-WBVHZDCISA-N
Target: Prostaglandin D2 receptor 2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9Y5Y4
Assay: Receptor Binding Inhibition Assay: Cell membrane was prepared from CRTH2-expressing K562 cell. The cell membrane (20 μg/well) was mixed with reaction buffer (50 mM Tris/HCl, pH 7.4, 10 mM MgCl2). Then, [3H]-PGD2 was added to the mixture and incubated for 60 min at room temperature. The concentration of [3H]-PGD2 was determined as equal to Kd calculated from Scatchard plot. Then, the mixture was filtrated with glass paper immediately. The filter paper was washed several times and radio activity on the filter was measured. Specific binding was defined as difference between total and non-specific binding (radio activity with 10 μM PGD2). Ki values for each compound were calculated from displacement curve.
Measurement: Ki < 100 nM (pChEMBL 7)
Descriptors: MW 475.54, ALogP 3.61, HBA 6, HBD 1, PSA 101.73, QED 0.56
Compound (SMILES): CCOc1cc(-c2cc(OC)ncc2F)nc2cc(C(=O)NCCCc3nnc[nH]3)ccc12
InChIKey: UYQGXKQTCWJSQL-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 450.47, ALogP 3.32, HBA 7, HBD 2, PSA 114.91, QED 0.38
Compound (SMILES): O=C(Nc1cc2cn(CC(=O)N3CCOCC3)nc2cc1Cl)c1cccc(C(F)(F)F)n1
InChIKey: RDTHSAJLTGYFFE-UHFFFAOYSA-N
Target: Interleukin-1 receptor-associated kinase 4 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9NWZ3
Assay: Kinase Assay: For the assay, 11 different concentrations in the range from 20 μM to 0.073 nM were prepared from a 2 mM solution of the test substance in DMSO. For the assay, 50 nl of the respective solution were pipetted into a black low-volume 384-well microtitre plate (Greiner Bio-One, Frickenhausen, Germany), 2 μl of a solution of Irak4 in assay buffer [50 mM HEPES pH 7.5, 5 mM MgCl2, 1.0 mM dithiothreitol, 30 μM activated sodium orthovanadate, 0.1% (w/v) of bovine gamma-globulin (BGG) 0.04% (v/v) nonidet-P40 (Sigma)] were added and the mixture was incubated for 15 min to allow prebinding of the substances to the enzyme prior to the kinase reaction. The kinase reaction was then started by addition of 3 μl of a solution of adenosine triphosphate (ATP, 1.67 mM=final concentration in 5 μl of assay volume: 1 μM) and peptide substrate (0.83 μM=final concentration in 5 μl assay volume: 0.5 μM) in assay buffer, and the resulting mixture was incubated at 22° C. for the reaction time of 45 min. The concentration of the Irak4 was adjusted to the respective activity of the enzyme and set such that the assay was carried out in the linear range. Typical concentrations were in the order of about 0.2 nM. The reaction was stopped by addition of 5 μl of a solution of TR-FRET detection reagents [0.1 μM streptavidin-XL665 (Cisbio Bioassays; France, catalogue No. 610SAXLG) and 1.5 nM anti-phosphoserin antibody [Merck Millipore, STK Antibody, catalogue No. 35-002] and 0.6 nM LANCE EU-W1024-labelled anti-mouse-IgG antibody (Perkin-Elmer, product No. AD0077, alternatively it is possible to use a terbium cryptate-labelled anti-mouse-IgG antibody from Cisbio Bioassays) in aqueous EDTA solution (100 mM EDTA, 0.4% [w/v] bovine serum albumin [BSA] in 25 mM HEPES pH 7.5).
Measurement: IC50 = 72 nM (pChEMBL 7.139999866485596)
Descriptors: MW 467.84, ALogP 3.21, HBA 6, HBD 1, PSA 89.35, QED 0.64
Compound (SMILES): O=C(Cc1cncnc1)N[C@H]1C[C@@](c2ccc(C(F)(C(F)(F)F)C(F)(F)F)cc2)(S(=O)(=O)c2ccc(F)cc2)C1
InChIKey: JHPNXJOQDNXRTA-IQGASKDCSA-N
Target: Nuclear receptor ROR-gamma (SINGLE PROTEIN), Homo sapiens
UniProt: P51449
Assay: SPA Binding Assay: The binding of potential ligands to RORγ is measured by competition with [3H] 25-hydroxycholesterol (Perkin Elmer NET674250UC) using a scintillation proximity assay (SPA) binding assay. The ligand binding domain of human RORγ (A262-S507) with an N-terminal His tag is expressed in E. coli and purified using nickel affinity chromotography. 15 ug/well RORγ (A262-S507) is incubated with test compound at varying concentrations in 3-fold serial dilution, with final concentrations ranging from 16.6 μM to 0.28 nM for 10 min at room temperature in PBS buffer (Invitrogen #14190-144) containing 0.5% fatty acid free BSA (Gemini Bio-Products, Cat. #700-107P) and 0.1% Glycerol (Sigma Cat# G5516). 10 nM of [3H] 25-hydroxycholesterol is then added, and the reaction is incubated for 10 min. 10 mg/mL of Copper-His Tag-PVT beads (Perkin Elmer cat # RPNQ0095) are added, and the mixture is incubated for 60 min. The reaction is read on a TopCount Microplate scintillation plate reader (Perkin Elmer).
Measurement: IC50 = 67 nM (pChEMBL 7.170000076293945)
Descriptors: MW 593.5, ALogP 5.1, HBA 5, HBD 1, PSA 89.02, QED 0.3