lovingscience/chembl-v37-raw-activities huggingface dataset:
Compound (SMILES): Cc1cc(Nc2ccc3[nH]ncc3c2)nc(N2CCCNCC2)n1
InChIKey: DLSQLPZMMHMIBW-UHFFFAOYSA-N
Target: Rho-associated protein kinase 2 (SINGLE PROTEIN), Homo sapiens
UniProt: O75116
Assay: Determination of Compound's ROCK Inhibitory Activity In Vitro (Z'Lyte Assay): Recombinant ROCK1 (amino acids 1-535) and ROCK2 (amino acids 1-552) proteins were purchased from ThermoFisher Scientific. Compound's activities were measured by Z′-lyte kinase kit (ThermoFisher Scientific) and IC50 was calculated
Measurement: IC50 = 54 nM (pChEMBL 7.269999980926514)
Descriptors: MW 323.4, ALogP 2.2, HBA 6, HBD 3, PSA 81.76, QED 0.69
Compound (SMILES): Cc1noc(Cn2cc3c(Cl)c(-c4c[nH]c5nc(N6C7CC[C@H]6C[C@@H](N)C7)cnc45)ccc3n2)n1
InChIKey: CUQMJXHOOIWYEJ-GNXJLENFSA-N
Target: Tyrosine-protein phosphatase non-receptor type 11 (SINGLE PROTEIN), Homo sapiens
UniProt: Q06124
Assay: Biochemical Assay: SHP2 activity was monitored by measuring the conversion of the surrogate substrate 6,8-difluoromethylumbelliferyl phosphate (DiFMUP) to the fluorescent product, 6,8-difluoromethylumbelliferone (DiFMU).SHP2 was pre-incubated with test compounds and the activating peptide pIRS1 (H2N-LN(pY)IDLDLV-(PEG)8-LST(pY)ASINFQK-amide) for 30 min, prior to addition of the 6,8-difluoromethylumbelliferyl phosphate (DiFMUP), (Thermo Fisher D6567). Final assay concentrations were 10 μM SHP2, 0.25 μM pIRS1 peptide, 50 μM DiFMUP, 25 mM Bis-Tris propane, pH 7.0, 150 mM NaCl, 0.05% (v/v) Tween-20, 0.5 mM TCEP and 5% (v/v) DMSO. Rates of reaction were then measured over 30 min by monitoring fluorescence on a BMG Pherastar reader at excitation 360 nm/emission 450 nm. IC50 values were calculated from the normalized dose-response plots using four parameter logistic curve fit.
Measurement: IC50 = 6.8 nM (pChEMBL 8.170000076293945)
Descriptors: MW 489.97, ALogP 3.83, HBA 9, HBD 2, PSA 127.57, QED 0.39
Compound (SMILES): CCN(CCc1nc(C(C)(C)O)c(C)o1)C(=O)c1cccnc1OCC(F)F
InChIKey: ZPEWZAGBKKTSKC-UHFFFAOYSA-N
Target: Orexin receptor type 2 (SINGLE PROTEIN), Homo sapiens
UniProt: O43614
Assay: FLIPR Ca2+ Flux Assay: In a typical experiment the OX1 and OX2 receptor antagonistic activity of the compounds of the present invention was determined in accordance with the following experimental method. For intracellular calcium measurements, Chinese hamster ovary (CHO) cells expressing the rat or human orexin-1 receptor or the human orexin-2 receptor, are grown in Iscove's modified DMEM containing 2 mM L-glutamine, 0.5 g/ml G418, 1% hypoxanthine-thymidine supplement, 100 U/ml penicillin, 100 μg/ml streptomycin and 10% heat-inactivated fetal calf serum (FCS). The cells are seeded at 20,000 cells/well into Becton-Dickinson black 384-well clear bottom sterile plates coated with poly-D-lysine. All reagents were from GIBCO-Invitrogen Corp. The seeded plates are incubated overnight at 37° C. and 5% CO2. Ala-6,12 human orexin-A as the agonist is prepared as a 1 mM stock solution in 1% bovine serum albumin (BSA) and diluted in assay buffer (HBSS containing 20 mM HEPES, 0.1% BSA and 2.5 mM probenecid, pH7.4) for use in the assay at a final concentration of 70 pM. Test compounds are prepared as 10 mM stock solution in DMSO, then diluted in 384-well plates, first in DMSO, then assay buffer. On the day of the assay, cells are washed 3 times with 100 μl assay buffer and then incubated for 60 min (37° C., 5% CO2) in 60 μl assay buffer containing 1 μM Fluo-4AM ester, 0.02% pluronic acid, and 1% BSA. The dye loading solution is then aspirated and cells are washed 3 times with 100 μl assay buffer. 30 μl of that same buffer is left in each well. Within the Fluorescent Imaging Plate Reader (FLIPR, Molecular Devices), test compounds are added to the plate in a volume of 25 μl, incubated for 5 min and finally 25 μl of agonist is added. Fluorescence is measured for each well at 1 second intervals for 5 minutes and the height of each fluorescence peak is compared to the height of the fluorescence peak induced by 70 pM Ala-6,12 orexin-A with buffer in place of antagonist. For each antagonist, IC50 value (the concentration of compound needed to inhibit 50% of the agonist response) is determined. Alternatively, compound potency can be assessed by a radioligand binding assay (described in Bergman et. al. Bioorg. Med. Chem. Lett. 2008, 18, 1425-1430) in which the inhibition constant (Ki) is determined in membranes prepared from CHO cells expressing either the OX1 or OX2 receptor. The intrinsic orexin receptor antagonist activity of a compound which may be used in the present invention may be determined by these assays.
Measurement: IC50 = 224 nM (pChEMBL 6.650000095367432)
Descriptors: MW 397.42, ALogP 2.95, HBA 6, HBD 1, PSA 88.69, QED 0.7
Compound (SMILES): CC1(N)CCN(c2cnc(C3=CCCCC3)c(N)n2)CC1
InChIKey: HGRPCZNNVIXVHE-UHFFFAOYSA-N
Target: Tyrosine-protein phosphatase non-receptor type 11 (SINGLE PROTEIN), Homo sapiens
UniProt: Q06124
Assay: SHP2 Allosteric Inhibition Assay: More specifically, the phosphatase reactions were performed at room temperature in 384-well black polystyrene plate, flat bottom, low flange, non-binding surface (Corning, Cat#3575) using a final reaction volume of 25 μL and the following assay buffer conditions: 60 mM HEPES, pH 7.2, 75 mM NaCl, 75 mM KCl, 1 mM EDTA, 0.05% P-20, 5 mM DTT. The inhibition of SHP2 by compounds of the invention (concentrations varying from 0.003-100 μM) was monitored using an assay in which 0.5 nM of SHP2 was incubated with of 0.5 μM of peptide IRS1_pY1172(dPEG8)pY1222 (sequence: H2N-LN(pY)IDLDLV(dPEG8)LST(pY)ASINFQK-amide) (SEQ ID NO:1). After 30-60 minutes incubation at 25° C., the surrogate substrate DiFMUP (Invitrogen, cat# D6567) was added to the reaction and incubated at 25° C. for 30 minutes. The reaction was then quenched by the addition of 5 μl of a 160 μM solution of bpV(Phen) (Enzo Life Sciences cat# ALX-270-204). The fluorescence signal was monitored using a microplate reader (Envision, Perki-Elmer) using excitation and emission wavelengths of 340 nm and 450 nm, respectively. The inhibitor dose response curves were analyzed using normalized IC50 regression curve fitting with control based normalization.
Measurement: IC50 = 2585 nM (pChEMBL 5.590000152587891)
Descriptors: MW 287.41, ALogP 2.33, HBA 5, HBD 2, PSA 81.06, QED 0.87
Compound (SMILES): Cn1ncnc1CN1CC[C@@]2(S(=O)(=O)c3ccc(F)cc3)c3ccc(C(F)(C(F)(F)F)C(F)(F)F)cc3CC[C@@H]12
InChIKey: IBODDIAQDDHORD-IFMALSPDSA-N
Target: Nuclear receptor ROR-gamma (SINGLE PROTEIN), Mus musculus
UniProt: P51450
Assay: General RORgamma 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). The ligand binding domain of human RORγ (A262-S507) with an N-terminal His tag is expressed in E. coli and purified using nickel affinity chromatography. 15 μg/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 rt in PBS buffer (Invitrogen #14190-144) containing 0.5% fatty acid free BSA (Gemini Bio-Products, Cat. #700-107P) and 0.1% glycerol (Sigma Cat#G5516). 10 nM of [3H] 25-hydroxycholesterol is then added, and the reaction is incubated for 10 min. 10 mg/mL of Copper-His Tag-PVT beads (Perkin Elmer cat # RPNQ0095) are added, and the mixture is incubated for 60 min. The reaction is read on a TopCount Microplate scintillation plate reader (Perkin Elmer). The competition data of the test compound over a range of concentrations was plotted as percentage inhibition of radioligand specifically bound in the absence of test compound (percent of total signal). After correcting for non-specific binding, IC50 values were determined. The IC50 value is defined as the concentration of test compound needed to reduce [3H] 25-hydroxycholesterol specific binding by 50% and is calculated using the four parameter logistic equation to fit the normalized data.
Measurement: IC50 = 112 nM (pChEMBL 6.949999809265137)
Descriptors: MW 594.53, ALogP 5.13, HBA 6, HBD 0, PSA 68.09, QED 0.3
Compound (SMILES): CCCOCCOc1ccc(-c2cc(=O)[nH]c(-c3cc(CNC(=O)CCC)ccc3Cl)n2)cn1
InChIKey: XEBINXDZAQTRJQ-UHFFFAOYSA-N
Target: Prostaglandin E synthase (SINGLE PROTEIN), Homo sapiens
UniProt: O14684
Assay: Inhibitory Activity Assay: Microsomes were prepared from COS-1 cells transiently transfected with a plasmid containing human mPGES-1 cDNA, and used as mPGES-1 enzyme. The mPGES-1 enzyme was diluted with a sodium phosphate buffer (pH 7.2) containing reduced glutathione (2.5 mM) and EDTA (1 mM), DMSO or a DMSO solution of a test compound (final concentration of DMSO was 1%) was added to the enzyme, and the mixture was preincubated at 4° C. for 15 minutes. Then, PGH2 as the substrate was added at a final concentration of 1 μM to start the enzymatic reaction, and after incubation at 4° C. for 4 minutes, a solution of ferric chloride (25 mM) and citric acid (50 mM) was added to terminate the enzymatic reaction. Generated PGE2 was measured by using Prostaglandin E2 Express EIA Kit (Cayman Chemical).
Measurement: IC50 = 23 nM (pChEMBL 7.639999866485596)
Descriptors: MW 484.98, ALogP 4.37, HBA 6, HBD 2, PSA 106.2, QED 0.37
Compound (SMILES): CC(N)CCc1nc(-c2ccc(CNc3ncc(C#N)cc3C(=O)N[C@@H](C)c3ccc(F)cc3)cc2)cnc1N
InChIKey: AGTRPDJFYIHCHD-GGYWPGCISA-N
Target: 3-phosphoinositide-dependent protein kinase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: O15530
Assay: PDK1 kinase assay: PDK1 (amino acids 51-359) and AKT2 (amino acids 140-467 fused to PIFtide, amino acids EEQEMFRDFDYIADW) were expressed as N-terminally tagged GST fusion proteins in insect cells and purified to homogeneity. Enzyme activity was determined in a coupled PDK1/AKT/FAM-crosstide assay and phosphorylation of FAM-crosstide was determined by standard IMAP protocol (Molecular Devices). For inhibition studies, compounds were titrated 4-fold in DMSO and diluted 40-fold into assay buffer (10 mM Tris HCl pH7.2; 10 mM MgCl2; 0.01% Triton X-100; 1 mM DTT) containing PDK1, AKT2, and FAM-crosstide (final concentrations: 0.75 nM PDK1, 10 nM unphosphorylated AKT2, and 100 nM crosstide substrate). The kinase reaction was initiated by adding ATP to a final concentration of 40 μM of PDK1 and incubated at 25° C. for 30 min. To detect assay product, the kinase reaction was combined with Progressive Binding Solution (1:600 Progressive Binding Reagent, 50% Buffer A, 50% Buffer B, Molecular Devices) in a 1:3 ratio. The mixture was incubated for 2 hours at 25° C. and the plate was scanned on an Analyst AD with excitation at 485 nm and emission at 530 nm. The fluorescence polarization value P is defined by the equation below. The value mP is generated by multiplying the P value for each reaction well by a factor of 1000. The value ΔmP for each well is the mP value for that well minus the mP value for the average negative control. P=(Fpar−Fperp)/(Fpar+Fperp)  Eq.:Where par is fluorescence intensity parallel to the excitation plane; and perp is fluorescence intensity perpendicular to the excitation plane. ΔmP values were plotted as a function of compound concentration and the data were analyzed with a 4-parameter fit using GraphPad Prism software.
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 538.63, ALogP 4.51, HBA 8, HBD 4, PSA 155.63, QED 0.23
Compound (SMILES): C[C@H](Nc1nc(C(=O)N2CC(C(=O)NCCO)C2)c2sccc2n1)c1cncc(F)c1
InChIKey: NQZNCYRVWFNYJL-NSHDSACASA-N
Target: Adenosine receptor A2b (SINGLE PROTEIN), Homo sapiens
UniProt: P29275
Assay: Activity Assay: Compounds (Adenosine Receptor modulators) and activity assay.
Measurement: IC50 = 75 nM (pChEMBL 7.119999885559082)
Descriptors: MW 444.49, ALogP 1.58, HBA 8, HBD 3, PSA 120.34, QED 0.51
Compound (SMILES): COc1nc(-c2ccc(Cl)cc2)nc2cc(C(=O)NCCCc3ncc[nH]3)ccc12
InChIKey: MMPQEWMMWHQTSY-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 < 10 nM (pChEMBL 8)
Descriptors: MW 421.89, ALogP 4.04, HBA 5, HBD 2, PSA 92.79, QED 0.44
Compound (SMILES): COc1ccc(C(=O)N2Cc3ccccc3C[C@H]2CN)c(-n2nc(C(=O)N(c3ccccc3)c3ccccc3)c3ccccc32)c1
InChIKey: RAJCVYCWSNEKPB-PMERELPUSA-N
Target: Apoptosis regulator Bcl-2 (SINGLE PROTEIN), Homo sapiens
UniProt: P10415
Assay: Fluorescence Polarisation Assay: Method A:The fluorescence polarisation tests were carried out on microplates (384 wells). The Bcl-2 protein, labelled (histag-Bcl-2 such that Bcl-2 corresponds to the UniProtKB® primary accession number: P10415), at a final concentration of 2.50×10−8 M, is mixed with a fluorescent peptide (Fluorescein-REIGAQLRRMADDLNAQY), at a final concentration of 1.50×10−8M in a buffer solution (NaPO4 20 mM, NaCl 50 mM, EDTA 1 mM, pH 7.4), in the presence or absence of increasing concentrations of test compounds. After incubation for 2 hours, the fluorescence polarisation is measured.Method B:The fluorescence polarisation tests were carried out on microplates (384 wells). The Bcl-2 protein, labelled (histag-Bcl-2 such that Bcl-2 corresponds to the UniProtKB® primary accession number: P10415), at a final concentration of 2.50×10−8 M, is mixed with a fluorescent peptide (Fluorescein-REIGAQLRRMADDLNAQY), at a final concentration of 1.00×10−8M in a buffer solution (Hepes 10 mM, NaCl 150 mM, Tween20 0.05%, pH 7.4), in the presence or absence of increasing concentrations of test compounds. After incubation for 2 hours, the fluorescence polarisation is measured.
Measurement: IC50 = 51 nM (pChEMBL 7.289999961853027)
Descriptors: MW 607.71, ALogP 6.54, HBA 6, HBD 1, PSA 93.69, QED 0.22
Compound (SMILES): CCOC(=O)NC12CCC(Nc3cc(Nc4ccc5ncsc5c4)ncc3C(=O)NC[C@@H](F)C(C)(C)O)(CC1)C2
InChIKey: VVFRMUWIHWDMDW-BQEUZBSESA-N
Target: Interleukin-1 receptor-associated kinase 4 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9NWZ3
Assay: Inhibition Assay: The assays were performed in U-bottom 384-well plates. The final assay volume was 30 μL prepared from 15 μL additions of enzyme and substrates (fluoresceinated peptide and ATP) and test compounds in assay buffer (20 mM HEPES pH 7.2, 10 mM MgCl2, 0.015% Brij35 and 4 mM DTT). The reaction was initiated by the combination of IRAK4 with substrates and test compounds. The reaction was incubated at room temperature for 60 min. and terminated by adding 45 μL of 35 mM EDTA to each sample. The reaction mixture was analyzed on the Caliper LABCHIP® 3000 (Caliper, Hopkinton, Mass.) by electrophoretic separation of the fluorescent substrate and phosphorylated product. Inhibition data were calculated by comparison to no enzyme control reactions for 100% inhibition and vehicle-only reactions for 0% inhibition. The final concentrations of reagents in the assays are ATP, 500 μM; FL-IPTSPITTTYFFFKKK peptide 1.5 μM; IRAK4, 0.6 nM; and DMSO, 1.6%.
Measurement: IC50 = 4.9 nM (pChEMBL 8.3100004196167)
Descriptors: MW 570.69, ALogP 4.89, HBA 9, HBD 5, PSA 137.5, QED 0.23
Compound (SMILES): CO[C@](Cc1ccccc1)(C(=O)Nc1cccc(-c2nc(NC(P(=O)(O)O)P(=O)(O)O)c3ccsc3n2)c1)C(F)(F)F
InChIKey: HENJGFLGRBJMCC-HSZRJFAPSA-N
Target: Geranylgeranyl pyrophosphate synthase (SINGLE PROTEIN), Homo sapiens
UniProt: O95749
Assay: In Vitro hGGPPS Inhibition Assay: The assay was based on a literature procedure (Kavanagh, et al. J. Biol. Chem., 2006, 281, 22004-22012) with minor modifications. All assays were run in triplicate using recombinant human GGPPS (80 ng), FPP (10 μM), IPP (8.3 μM; 3H-IPP, 40 mCi/mmoL) in a final volume of 100 μL buffer containing 50 mM Tris pH 7.7, 2 mM MgCl2, 1 mM TCEP, 5 μg/mL BSA and 0.2% (w/v) Tween 20. The enzyme and test compound were pre-incubated in the assay buffer in a volume of 80 μL at 37° C. for 10 mins. Afterwards, the substrates (FPP, IPP) were added to start the reaction, which also bring the compound, substrate, and buffer contents to the desired final concentrations as indicated above. The assay mixture was then incubated at 37° C. for 15 mins (Note: the incubation time is based from the curve determined each time a new batch of enzyme is produced). Assays were terminated by the addition of 200 μL of HCl/MeOH (1:4) and incubated for 10 min at 37° C. The mixture was then extracted with 700 μL of petroleum ether, dried through a plug of anhydrous Mg2SO4 and 300 μL of the dried ligroin phase was combined with 8 mL of scintillation cocktail. Finally, the radioactivity was counted using a Beckman Coulter LS6500 liquid scintillation counter.
Measurement: IC50 = 2550 nM (pChEMBL 5.590000152587891)
Descriptors: MW 646.48, ALogP 4.54, HBA 8, HBD 6, PSA 191.2, QED 0.13
Compound (SMILES): C=CC(=O)N1CC[C@@H](n2nc(C(=O)Nc3nc4cc(Cl)ccc4o3)c3c(N)ncnc32)C1
InChIKey: OXZZHHRPRXUIEV-LLVKDONJSA-N
Target: Tyrosine-protein kinase BTK (SINGLE PROTEIN), Homo sapiens
UniProt: Q06187
Assay: BTK Inhibitory Activity: With regard to the setting of the conditions for a method for measuring the inhibitory activity of a compound against BTK kinase activity in vitro, it is described in the consumable reagent supplies price list for LabChip (registered trademark) series of PerkinElmer, Inc. that FL-PEPTIDE 2 corresponds to a substrate peptide for the measurement of BTK kinase activity. Therefore, FL-PEPTIDE 2 was used as a substrate. The purified recombinant human BTK protein used in the test was purchased from Carna Biosciences, Inc.With regard to the measurement of the inhibitory activity of the compounds, firstly, the compounds of the present invention were diluted stepwise with dimethyl sulfoxide (DMSO). Subsequently, BTK protein, a substrate peptide (final concentration was 1 μM), magnesium chloride (final concentration was 10 mM), ATP (final concentration was 45 μM), and a DMSO solution of the compounds of the present invention (final concentration of DMSO was 5%) were added to a buffer solution for kinase reaction (20 mM HEPES (pH 7.5), 2 mM dithiotheitol, 0.01% Triton X-100), and after the solution was incubated for 40 minutes at 25° C., a kinase reaction was carried out. The reaction was terminated by adding EDTA thereto in order to obtain a final concentration of 30 mM. Finally, a substrate peptide that was not phosphorylated (S) and a phosphorylated peptide (P) were separated and detected by microchannel capillary electrophoresis using a LabChip EZ Reader II (PerkinElmer, Inc.). The amounts of phosphorylation reaction were determined from the respective peak heights of S and P, and the compound concentration at which the phosphorylation reaction could be suppressed in 50% was defined as the IC50 value (nM).
Measurement: IC50 = 0.591 nM (pChEMBL 9.229999542236328)
Descriptors: MW 452.86, ALogP 2.41, HBA 9, HBD 2, PSA 145.06, QED 0.45
Compound (SMILES): CNCC[C@H](Oc1ccc(CN2CCN(C)c3nc(C)ccc3C2=O)cc1)c1cccs1
InChIKey: PVIBEZAPXVLWAG-QFIPXVFZSA-N
Target: Voltage-dependent calcium channel subunit alpha-2/delta-1 (SINGLE PROTEIN), Homo sapiens
UniProt: P54289
Assay: Binding Assay : Human α2δ-1 enriched membranes (2.5 μg) were incubated with 15 nM of radiolabeled [3H]-Gabapentin in assay buffer containing Hepes-KOH 10 mM, pH 7.4.NSB (non specific binding) was measured by adding 10 μM pregabalin. The binding of the test compound was measured in five different concentrations. After 60 min incubation at 27° C., binding reaction was terminated by filtering through Multiscreen GF/C (Millipore) presoaked in 0.5% polyethyleneimine in Vacuum Manifold Station, followed by 3 washes with ice-cold filtration buffer containing 50 mM Tris-HCl, pH 7.4.Filter plates were dried at 60° C. for 1 hour and 30 μl of scintillation cocktail were added to each well before radioactivity reading.Readings were performed in a Trilux 1450 Microbeta radioactive counter (Perkin Elmer).
Measurement: Ki = 300 nM (pChEMBL 6.519999980926514)
Descriptors: MW 450.61, ALogP 4.27, HBA 6, HBD 1, PSA 57.7, QED 0.56
Compound (SMILES): Cc1oc([C@@H]2CC[C@@H](C)N(C(=O)c3cscc3-n3nccn3)C2)nc1C(C)(C)O
InChIKey: OPDYYAHTBQVIPS-TZMCWYRMSA-N
Target: Orexin receptor type 2 (SINGLE PROTEIN), Homo sapiens
UniProt: O43614
Assay: FLIPR Ca2+ Flux Assay: For intracellular calcium measurements, Chinese hamster ovary (CHO) cells expressing the rat orexin-1 receptor or the human orexin-2 receptor, are grown in Iscove's modified DMEM containing 2 mM L-glutamine, 0.5 g/ml G418, 1% hypoxanthine-thymidine supplement, 100 U/ml penicillin, 100 μg/ml streptomycin and 10% heat-inactivated fetal calf serum (FCS). The cells are seeded at 20,000 cells/well into Becton-Dickinson black 384-well clear bottom sterile plates coated with poly-D-lysine. All reagents were from GIBCO-Invitrogen Corp. The seeded plates are incubated overnight at 37° C. and 5% CO2. Ala-6,12 human orexin-A as the agonist is prepared as a 1 mM stock solution in 1% bovine serum albumin (BSA) and diluted in assay buffer (HBSS containing 20 mM HEPES, 0.1% BSA and 2.5 mM probenecid, pH7.4) for use in the assay at a final concentration of 70 pM. Test compounds are prepared as 10 mM stock solution in DMSO, then diluted in 384-well plates, first in DMSO, then assay buffer. On the day of the assay, cells are washed 3 times with 100 μl assay buffer and then incubated for 60 min (37° C., 5% CO2) in 60 μl assay buffer containing 1 μM Fluo-4AM ester, 0.02% pluronic acid, and 1% BSA. The dye loading solution is then aspirated and cells are washed 3 times with 100 μl assay buffer. 30 μl of that same buffer is left in each well. Within the Fluorescent Imaging Plate Reader (FLIPR, Molecular Devices), test compounds are added to the plate in a volume of 25 μl, incubated for 5 min and finally 25 μl of agonist is added.
Measurement: IC50 = 11.7 nM (pChEMBL 7.929999828338623)
Descriptors: MW 415.52, ALogP 3.26, HBA 8, HBD 1, PSA 97.28, QED 0.7
Compound (SMILES): C=CC(=O)Nc1cc(Nc2ncc(C(=O)OC(C)C)c(-c3nn(C)c4ccccc34)n2)c(OC)cc1N(C)CCN(C)C
InChIKey: BDKAGMNAOQQPRC-UHFFFAOYSA-N
Target: Epidermal growth factor receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P00533
Assay: ASV & NPG EGFR Exon 20 Insertion Mutations: A compound's ability in selectively inhibiting EGFR exon 20 insertion mutations can be assessed using Ba/F3 cells, a murine pro-B cell line, which have been transduced with EGFR exon 20 insertions. An expression vector, pLVX-IRES puro (Clontech) coding for human EGFR exon 20 insertions NPG (H773_V774insNPG) or ASV (V769_D770insASV), was transfected into HEK293 cells by the Trans-Lentiviral ORF Packaging System (Thermo Scientific), to produce virus encoding EGFR exon 20 insertions. Ba/F3 (DSMZ) cells maintained in RPMI 1640 medium supplemented with 10% fetal bovine serum, 200 μM L-glutamine/200 μg/mL penicillin/200 μg/mL streptomycin (Life Technology) and 10 ng/mL IL-3 (R&D system), were infected by EGFR Exon20 virus and subsequently selected by puromycin (Life Technology) selection and IL-3 depletion. Ba/F3 cells expressing EGFR exon 20 insertions (named Ba/F3-EGFR-Exon20-NPG or Ba/F3-EGFR-Exon20-ASV) can proliferate in the absence of IL-3. The anti-proliferative activity of compounds were determined as follows: BaF3-EGFR-Exon20 cells (NPG or ASV) seeded in 96 well plates (2500 cells/well) were treated with test compound (dissolved in DMSO) at a series of concentrations (4-fold dilution, top concentration: 10,000 nM). The plates were incubated for 72 h in a 37° C. incubator with 5% CO2, and the number of viable cells in each well were measured indirectly by CellTiter 96® Aqueous One Solution Cell Proliferation Assay (Promega) This assay is a colorimetric method for determining the number of viable cells through measurement of their metabolic activity by detection of enzymatic conversion of tetrazolium salts into blue formazan derivatives. Reagent (20 μL) was added into each well, and the plates were returned to the incubator for 2 h. The absorbance in each well was then measured at 490 nm using an Envision plate reader (Perkin Elmer). IC50 values were calculated by determining the concentration of compound required to decrease the MTS signal by 50% comparing to the DMSO control in best-fit curves using Microsoft XLfit software or Accelrys Pipeline Pilot.
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 586.7, ALogP 4.47, HBA 11, HBD 2, PSA 126.74, QED 0.18
Compound (SMILES): O=C(O)c1ccncc1NC[C@@H]1CCOc2cc(C3CC3)ccc21
InChIKey: RCLHALAJVFJTSA-AWEZNQCLSA-N
Target: Lysine-specific demethylase 4C (SINGLE PROTEIN), Homo sapiens
UniProt: Q9H3R0
Assay: JMJD2C Assay: The ability of test compounds to inhibit the activity of JMJD2C was determined in 384-well plate format under the following reaction conditions: 0.3 nM JMJD2C, 300 nM H3K9me3-biotin labeled peptide (Anaspec cat #64360), 2 μM alpha-ketoglutaric acid in assay buffer of 50 mM HEPES, pH7.3, 0.005% Brij35, 0.5 mM TCEP, 0.2 mg/ml BSA, 50 μM sodium L-ascorbate, and 2 μM ammonium iron(II) sulfate. Reaction product was determined quantitatively by TR-FRET after the addition of detection reagent Phycolink Streptavidin-allophycocyanin (Prozyme) and Europium-anti-di-methylated histone H3 lysine 9 (H3K9me2) antibody (PerkinElmer) in the presence of 5 mM EDTA in LANCE detection buffer (PerkinElmer) at a final concentration of 50 nM and 1 nM, respectively.The assay reaction was initiated by the following: 2 μl of the mixture of 900 nM H3K9me3-biotin labeled peptide and 6 μM alpha-ketoglutaric acid with 2 μl of 11-point serial diluted inhibitor in 3% DMSO were added to each well of the plate, followed by the addition of 2 μl of 0.9 nM JMJD2C to initiate the reaction. The reaction mixture was incubated at room temperature for 30 min, and terminated by the addition of 6 μl of 5 mM EDTA in LANCE detection buffer containing 100 nM Phycolink Streptavidin-allophycocyanin and 2 nM Europium-anti-H3K9me2 antibody. Plates were read by EnVisionMultilabel Reader in TR-FRET mode (excitation at 320 nm, emission at 615 nm and 665 nm) after 1 hr incubation at rt. A ratio was calculated (665/615) for each well and fitted to determine inhibition constant (IC50).
Measurement: IC50 = 550 nM (pChEMBL 6.260000228881836)
Descriptors: MW 324.38, ALogP 3.64, HBA 4, HBD 2, PSA 71.45, QED 0.88
Compound (SMILES): CN1CC[C@H](COc2cc(F)c3c(=O)[nH]c(CS[C@H]4CC[C@H](O)CC4)nc3c2)[C@@H](F)C1
InChIKey: GFFHNERPJQOLNW-SZXPJGKVSA-N
Target: Protein mono-ADP-ribosyltransferase PARP14 (SINGLE PROTEIN), Homo sapiens
UniProt: Q460N5
Assay: Enzymatic Inhibition Assay: Enzymatic inhibition of PARP14 was measured using a dissociation-enhanced lanthanide fluorescence immunoassay (DELFIA) monitoring the auto-modification of PARP14 by biotinylated nicotinamide adenine dinucleotide (biotin-NAD). 1 μL of a dose response curve of each test compound was spotted in 384-well nickel-coated white microplates (Thermo) using a Mosquito (TTP Labtech). Reactions were performed in a 50 μL volume by adding 40 μL of PARP14 in assay buffer (20 mM HEPES pH==8, 100 mM NaCl, 0.1% bovine serum albumin, 2 mM DTT and 0.002%0 Tween20), incubating with test compound at 25° C. for 30 min, then adding 10 μL of biotin-NAD (Biolog). The final concentrations of PARP14 and biotin-NAD are 50 nM and 3 μM, respectively. Reactions proceeded at 25° C. for 3 h, then were quenched with 5 μL of 10 mM unmodified nicotinamide adenine dinucleotide (Sigma-Aldrich). The quenched reactions were washed 3 times with 100 μL of TBST wash buffer (50 mM Tris-HCl, 150 mM NaCl and 0.1% Tween20). Next, to the washed and dried plate was added 25 μL of DELFIA Europium-N1 streptavidin (Perkin Elmer) diluted in DELFIA assay buffer (Perkin Elmer). After a 30 min incubation at 25° C., the plate was washed 5 times with TBST wash buffer. Finally, 25 μL of DELFIA enhancement solution was added. After a 5 min incubation the plate was read on an Envision platereader equipped with a LANCE/DELFIA top mirror (Perkin Elmer) using excitation of 340 nm and emission of 615 nm to measure the amount of Europium present in each well, informing on the amount of biotin-NAD that was transferred in the automodification reaction.
Measurement: IC50 < 1000 nM (pChEMBL 6)
Descriptors: MW 453.56, ALogP 3.27, HBA 6, HBD 2, PSA 78.45, QED 0.7
Compound (SMILES): Cc1nc(N2CCOC(C)(C)C2)c2[nH]c(N)c(S(=O)(=O)c3cc(F)ccc3F)c2n1
InChIKey: JLXMNAHHADOMNV-UHFFFAOYSA-N
Target: Free fatty acid receptor 2 (SINGLE PROTEIN), Homo sapiens
UniProt: O15552
Assay: Ca-Flux Functional Assay: GPR43 agonist/PAM activity was determined by measuring changes in intracellular calcium levels using a Ca2+ sensitive fluorescent dye. The changes in fluorescent signal were monitored by FLIPR (manufactured by Molecular Devices). GPR43 mediated increases in intracellular Ca2+ concentration were readily detected upon activation with sodium acetate. Prior to the assay (24 hours), CHO-K1 Gα16 cells stably expressing human GPR43 were-seeded in cell culture medium in black, clear-bottom 384-well plates (Corning Inc) and grown overnight at 37° C., 5% CO2. On the day of the assay, cell culture media was removed and cells were loaded with Calcium 5 Dye (Molecular Devices) diluted in HBSS containing 25 mM HEPES, 2.5 mM Probenecid, 0.1% BSA for 1 hour at 37° C., 5% CO2. 10 point half log concentration response curves of sodium acetate from 10 mM were conducted prior to the testing of compounds to calculate the sodium acetate concentration that produces 20% of the maximal response (EC20). Test compounds (at 10 point half log concentration response curves from 10 μM) were added in the presence of sodium acetate to achieve a final concentration that produces approximately 20% maximal response as calculated from the previous experiment. The changes in fluorescent signal were monitored by FLIPR upon addition of the compound/EC20 sodium acetate mix. The EC50 values were determined from ten point concentration response curves. Curves were generated using the average of two wells for each data point.
Measurement: EC50 = 8 nM (pChEMBL 8.100000381469727)
Descriptors: MW 437.47, ALogP 2.57, HBA 7, HBD 2, PSA 114.2, QED 0.65
Compound (SMILES): O=C(NC1CCN(c2ccccc2)C1)c1cc(-c2ccco2)on1
InChIKey: NQSOKEGVCPFHBV-UHFFFAOYSA-N
Target: Stearoyl-CoA desaturase (SINGLE PROTEIN), Homo sapiens
UniProt: O00767
Assay: Compound Profiling Methods: Strains expressing SCD1 or SCD5 as the sole desaturase, the human SCD1 and SCD5 genes were used to evaluate inhibition of SCD1/SCD5 using reduced growth as a surrogate for SCD inhibition. These yeast strains express human SCD1 or SCD5 from a plasmid harbored in a strain in which the yeast OLE1 gene is deleted.All compound profiling experiments were performed using the same basic protocol. Yeast were cultured using standard techniques in complete synthetic media lacking uracil and containing yeast nitrogen base supplemented with 2% (w/v) glucose (SD-Ura) Starter cultures were inoculated in 3 mL SD-Ura media containing 0.01% tween and 0.2 mM palmitoleic and oleic acid. Cultures were incubated overnight in a 30° C. shaker incubator (225 rpm). Saturated morning cultures were centrifuged, washed in SD-Ura media lacking TWEEN-20 and fatty acids, and then diluted 1:20 in fresh SD-Ura media also lacking TWEEN-20 and fatty acids. Cells were grown for 6 h to an  (optical density) of 0.4-0.8 at 30° C. with shaking.Compound stocks (10 mM in 100% DMSO) were arrayed into 384-round well, v-bottom polypropylene plates and diluted according to indicated dilution factors. Compound administration was performed in two separate steps. First, 15 μL of SD-Ura was dispensed into clear 384-well assay plates using a MULTIDROP Combi reagent dispenser. The diluted compound stock plates were then applied to the assay plates using an automated workstation (Perkin Elmer JANUS) outfitted with a 384-pin tool containing slotted pins that deliver 100 nL of compound. The cultures described above were centrifuged and washed with media lacking TWEEN-20 or oleic and palmitoleic acids. Cultures were then resuspended at a 2-fold concentrated  of 0.02 (final  of 0.0.01) in SD-Ura. 15 μL of diluted culture was then dispensed into the pinned assay plate to achieve 30 μL of the 1× culture (0.01) and a top drug concentration of 33.3 μM.After yeast delivery, assay plates were incubated under humidified conditions at 30° C. for 40 h. Yeast growth was monitored by reading the  of each well using a microplate reader (Perkin Elmer EnVision). Data were analyzed as follows. Raw data were processed by background subtracting and converting values to a percent of the nontreated condition for that strain [(EXP-0.035)/(DMSO-0.035)×100%].
Measurement: IC50 > 45000 nM (pChEMBL 4.3470001220703125)
Descriptors: MW 323.35, ALogP 2.94, HBA 5, HBD 1, PSA 71.51, QED 0.8
Compound (SMILES): Cn1nc(C(C)(C)C)cc1NC(=O)c1cccc(C2CC2NCC2CC2)c1
InChIKey: WNQCSOBDFJDQDH-UHFFFAOYSA-N
Target: Lysine-specific histone demethylase 1A (SINGLE PROTEIN), Homo sapiens
UniProt: O60341
Assay: LSD1 Inhibition Assay: A test compound dissolved in 2.5% DMSO was added by 4 μL to 3 μL reaction solution (50 mM Tris-HCl (pH 8.0), 0.1% BSA, 1 mM DTT) containing 7.5 ng of LSD1, and the mixture was reacted at room temperature for 60 min. Biotin-histone H3 mono methylated K4 peptide solution (NH2-ART(me-K)QTARKSTGGKAPRKQLAGGK(Biotin)-CONH2) (3.3 μM) was added by 3 μL to start the reaction. After reaction at room temperature for 5 min, 1 mM 2-PCPA solution was added 5 μL to terminate the reaction. A detection solution (BOO mM potassium fluoride, 0.1% BSA) containing europium-labeled anti-histone H3 antibody (Wako Pure Chemical Industries, Ltd.) and Streptavidin-XL665 (Cisbio) was further added by 5 μL, and the mixture was left standing for 60 min. A time-resolved fluorescence (excitation 320 nm, emission 615 nm, 665 nm) was measured by Envision (PerkinElmer). The LSD1 inhibitory rate (%) of the test compound was calculated by the following formula.inhibitory rate(%)=(1−(test compound count−blank)÷(control−blank))×100
Measurement: IC50 = 380 nM (pChEMBL 6.420000076293945)
Descriptors: MW 366.51, ALogP 3.83, HBA 4, HBD 2, PSA 58.95, QED 0.82
Compound (SMILES): Oc1c(F)cc(-n2cc(-c3ccc4cc(OCCOCCN5CCOCC5)ccc4n3)nn2)cc1F
InChIKey: RABVLQFPBZIZKT-UHFFFAOYSA-N
Target: Macrophage migration inhibitory factor (SINGLE PROTEIN), Homo sapiens
UniProt: P14174
Assay: Inhibition of Tautomerase Activity of Human MIF: Inhibition of the tautomerase activity of MIF was measured using 4-hydroxyphenyl pyruvic acid (HPP) as substrate, largely following previously reported protocols (Taylor et al., Biochemistry, 1999, 38, 7444-7452). HPP was dissolved in 0.5 M acetate buffer, pH 6.0 to a final concentration of 10 mM and incubated overnight at room temperature to allow equilibration of the keto and enol forms. MIF (6 μL) was premixed in 500 mM boric acid, pH 6.2 (142 μL) and transferred to a transparent U bottom 96-well plate to a final concentration of 50 nM MIF. At this concentration high signal-to-noise and linearity were observed after analysis of progress curves for enol production at different protein concentrations. Inhibitors were dissolved in DMSO to 10 mM and an initial screen was performed. For compounds that showed ca. 25% or greater inhibition at 10 μM, an inhibition constant, was measured.Compounds were placed into wells (2 μL) at 6 different concentrations and incubated for 30 minutes until the assay was started by addition of HPP (504) at two concentrations (1.0 and 2.5 mM). The negative control was MIF incubated with DMSO vehicle, which in all assays was 1% and did not influence tautomerase activity. MIF activity was monitored at 305 nm for formation of the borate-enol complex using an Infinite F500 plate reader (TECAN, Morrisville, N.C.) for 175 seconds. Calculation of initial velocities and the nonlinear regression analyses for the enzyme kinetics were repeated three times with the program Prism6 (GraphPad, La Jolla, Calif.).Data obtained for the Example compounds, obtained using the methods described in Example B, are provided in Table 3. Results are also included for a reference compound ISO-1 (Chang, K. F.; Al-Abed, Y. Bioorg. Med. Chem. Lett. 2006, 16, 3376-3379; Balachandran, S. et al. Bioorg. Med. Chem. Lett. 2009, 19, 4773-4776). X-ray crystal structures were obtained for complexes of 3a, 3b, and 3v (Examples 1, 2, and 21) with human MIF as described below in Example C, which confirm the binding of these inhibitors in the tautomerase active site.
Measurement: Ki = 3746 nM (pChEMBL 5.429999828338623)
Descriptors: MW 497.5, ALogP 3.19, HBA 9, HBD 1, PSA 94.76, QED 0.35
Compound (SMILES): CC(C)(O)c1cnnn1[C@H]1C[C@@H](C(=O)NC2(C(=O)C(N)=O)CCSCC2)N(C(=O)[C@@H](CC2CCCCC2)NC(=O)c2ccc(C(=O)N3CCCC3)cc2)C1
InChIKey: GRMJBOCKDPOJPP-NHKHRBQYSA-N
Target: Serine protease HTRA1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q92743
Assay: HTRA1 Assay: Serial dilutions (1/3) from 1000 μM down to 0.051 μM of test compounds were prepared in dimethyl sulfoxide (DMSO). Then 2 μL of solution from each dilution were added to 100 μL of 4 nM full-length human His-HTRA1 in assay buffer (50 mM Tris, pH 7.5, 200 mM NaCl and 0.25% 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate or CHAPS) in white non-binding 96-well plates. The assay solutions were mixed for 5 seconds on a shaker plate and incubated for 10 minutes at room temperature. Mca-H2OPT (Mca-Ile-Arg-Arg-Val-Ser-Tyr-Ser-Phe-Lys(Dnp)-Lys-OH trifluoroacetate salt) (Mca=7-methoxycoumarin-4-acetic acid; Dnp=dinitrophenyl) (5 μM) in 100 μL of assay buffer was added to the assay solutions. The reaction mixture was shaken for 5 seconds on a shaker plate and cleavage of Mca-H2OPT was monitored by spectrofluorometry (SpectraMax M3 by Molecular Devices, CA) for 10 minutes (Exk=330 nm; Emk=420 nm).
Measurement: IC50 = 4 nM (pChEMBL 8.399999618530273)
Descriptors: MW 764.95, ALogP 2.09, HBA 11, HBD 4, PSA 209.92, QED 0.23
Compound (SMILES): O=C(NC1(c2nnc(-c3c[nH]c4ncccc34)o2)CC1)c1cccn(Cc2ccc(F)c(F)c2)c1=O
InChIKey: UWDOSWNLBZWUTQ-UHFFFAOYSA-N
Target: 3-phosphoinositide-dependent protein kinase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: O15530
Assay: Kinase Assay: A PDK1 kinase assay was performed as follows. PDK1 (amino acids 51-360) and AKT2 (amino acids 140-467 fused to PIFtide, amino acids EEQEMFRDFDYIADW) were expressed as N-terminally tagged GST fusion proteins in insect cells and purified to greater than 90% homogeneity. PDK1 protein was divided into two fractions, one of which was subsequently dephosphorylated. To generate dephosphorylated PDK1, the PDK1 was reacted with GST-tagged lambda-phosphatase in vitro. GST was subsequently cleaved proteolytically for both phosphorylated and dephosphorylated PDK1. Protein preparations were run on glutathione Sepharose columns to remove GST and GST-tagged lambda-phosphatase, if present. Phosphorylated PDK1 and dephosphorylated PDK1 were verified by mass-spectrometry. Enzyme activity was determined in a coupled PDK1/AKT/FAM-crosstide assay using either phosphorylated or unphosphorylated PDK1 and phosphorylation of FAM-crosstide was determined by standard IMAP protocol (Molecular Devices). For inhibition studies, compounds were titrated 3-fold in DMSO and diluted 40-fold into assay buffer (10 mM Tris HCl pH7.2; 10 mM MgCl2; 0.01% Triton X-100; 1 mM DTT) containing PDK1, AKT2, and FAM-crosstide. (final concentrations: 25 nM un-phosphorylated PDK1 or 0.5 nM phosphorylated PDK1, 30 nM unphosphorylated AKT2, and 100 nM crosstide substrate). The kinase reaction was initiated by adding ATP to a final concentration of 24 μM for both forms of PDK1 and incubated at 25° C. for 30 min. To detect assay product, the kinase reaction was combined with Progressive Binding Solution (1:600 Progressive Binding Reagent, 50% Buffer A, 50% Buffer B, Molecular Devices) in a 1:3 ratio. The mixture was incubated for 2 hours at 25° C. and the plate was scanned on an Analyst AD with excitation at 485 nm and emission at 530 nm.
Measurement: IC50 < 500 nM (pChEMBL 6.301000118255615)
Descriptors: MW 488.45, ALogP 3.52, HBA 7, HBD 2, PSA 118.7, QED 0.38
Compound (SMILES): COc1cccc(OC)c1-n1c(NS(=O)(=O)[C@@H](C)[C@H](OC)c2ncc(Cl)cn2)nnc1[C@H]1CCCOC1
InChIKey: KDYKCQNMJYOJOY-AVYPCKFXSA-N
Target: Apelin receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P35414
Assay: [35S]GTPgammaS Binding: The human APJ receptor was cloned by polymerase chain reaction and the gene encoding the receptor was subcloned in pFLAG-CMV-3 expression vector (Sigma, Saint Louis, Mo. USA) in-house at Amgen. A GTPγS binding assay was performed on membranes prepared from CHO cells stably expressing human APJ receptor. The optimum experimental conditions for the concentrations of GDP, MgCl2, and NaCl in the assay buffer were initially determined. The assay was performed in assay buffer [20 mM HEPES, pH 7.5, 5 mM MgCl2, and 0.1% (w/v) BSA with 200 mM NaCl, 3 μM GDP] and membranes expressing human APJ receptor/well along with WGA PS beads. The reaction was initiated by addition of 0.2 nM [35S]GTPγS (Perkin Elmer Life and Analytical Sciences, Waltham USA) in the absence or presence of various ligands and incubated at RT for 90 min. Nonspecific binding was determined in the presence of 100 μM GTPγS and was always less than 0.2% of total binding. All the results presented are means of several independent experiments and analyzed by non-linear regression methods using commercially available program Prism (GraphPad, San Diego, Calif.).
Measurement: EC50 = 29 nM (pChEMBL 7.539999961853027)
Descriptors: MW 553.04, ALogP 3.14, HBA 11, HBD 1, PSA 139.58, QED 0.4
Compound (SMILES): Nc1ncc(-c2cccc(C(F)(F)F)c2)nc1-c1nnc(-c2ccccc2)o1
InChIKey: TZPAQLLFUZSKMO-UHFFFAOYSA-N
Target: Serine/threonine-protein kinase ATR (SINGLE PROTEIN), Homo sapiens
UniProt: Q13535
Assay: Inhibition Assay: Compounds were screened for their ability to inhibit ATR kinase using a radioactive-phosphate incorporation assay. Assays were carried out in a mixture of 50 mM Tris/HCl (pH 7.5), 10 mM MgCl2 and 1 mM DTT. Final substrate concentrations were 10 M [γ-33P]ATP (3mCi 33P ATP/mmol ATP, Perkin Elmer) and 800 μM target peptide (ASELPASQPQPFSAKKK).Assays were carried out at 25° C. in the presence of 5 nM full-length ATR. An assay stock buffer solution was prepared containing all of the reagents listed above, with the exception of ATP and the test compound of interest. 13.5 μL of the stock solution was placed in a 96 well plate followed by addition of 2 μL of DMSO stock containing serial dilutions of the test compound (typically starting from a final concentration of 15 μM with 3-fold serial dilutions) in duplicate (final DMSO concentration 7%). The plate was pre-incubated for 10 minutes at 25° C. and the reaction initiated by addition of 15 μL [γ-33P]ATP (final concentration 10 μM).The reaction was stopped after 24 hours by the addition of 30 μL 0.1M phosphoric acid containing 2 mM ATP. A multiscreen phosphocellulose filter 96-well plate (Millipore, Cat no. MAPHN0B50) was pretreated with 100 μL 0.2M phosphoric acid prior to the addition of 45 μL of the stopped assay mixture. The plate was washed with 5×200 μL 0.2M phosphoric acid. After drying, 100 μL Optiphase SuperMix liquid scintillation cocktail (Perkin Elmer) was added to the well prior to scintillation counting (1450 Microbeta Liquid Scintillation Counter, Wallac).
Measurement: Ki = 5050 nM (pChEMBL 5.300000190734863)
Descriptors: MW 383.33, ALogP 4.46, HBA 6, HBD 1, PSA 90.72, QED 0.56
Compound (SMILES): Cc1ccc(S(=O)(=O)NC23CCC(CN4CCOCC4)(CC2)OC3)cc1-c1cnc2c(N)ncnn12
InChIKey: ZUIDQDCZMYWGCB-UHFFFAOYSA-N
Target: Phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit gamma isoform (SINGLE PROTEIN), Homo sapiens
UniProt: P48736
Assay: Scintillation Proximity Assay: PI3K-γ:[γ-33P]ATP (10 mCi/mL) and Wheat Germ Agglutinin (WGA) YSi SPA Scintillation Beads was purchased from Perkin-Elmer (Waltham, Mass.). Lipid kinase substrate, D-myo-Phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2)D (+)-sn-1,2-di-O-octanoylglyceryl, 3-O-phospho linked (PIP2), CAS 204858-53-7, was purchased from Echelon Biosciences (Salt Lake City, Utah). PI3Kγ (p110γ) Recombinant Human Protein was purchased from Life technology (Grand Island, N.Y.). ATP, MgCl2, DTT, EDTA, MOPS and CHAPS were purchased from Sigma-Aldrich (St. Louis, Mo.).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).
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 513.62, ALogP 1.37, HBA 10, HBD 2, PSA 136.97, QED 0.5
Compound (SMILES): Nc1nc(N)c(NC(=O)Nc2ccc(C(=O)N[C@@H](CCc3nnn[nH]3)C(=O)O)nc2)c(=O)[nH]1
InChIKey: PMGGZFQJUNWXHA-QMMMGPOBSA-N
Target: N-formyl peptide receptor 2 (SINGLE PROTEIN), Homo sapiens
UniProt: P25090
Assay: Inhibition of MTHFD2: To determine the IC50 value of a compound, an 11-concentration dose-response curve with 3-fold difference in concentration between assay points was generated by using an acoustic dispenser (Echo 550 Liquid handler, Labcyte). Each assay point was run in duplicate and the assay was performed in a white 384-well ProxiPlate Plus (6008280, PerkinElmer). DMSO was used as negative control. The serial dilution in DMSO, from compound DMSO stock solution, was created by dispensing from a 384-well low dead volume microplate (LP-0200, Labcyte) and a 384-well polypropylene microplate 2.0 (PP-0200, Labcyte). A total of 2.5 μL MTHFD2 was preincubated with compound or DMSO for 10 min. The enzymatic reaction was initiated by adding 2.5 μL folitixorin (F680350, Toronto Research Chemicals). For background control, 5 μL buffer was added to the well. Final concentrations of the components in the assay were 3.4 nmol/L MTHFD2, 5 μmol/L folitixorin and 250 μmol/L NAD+. The final concentrations of all reagents in a total assay volume of 5 μL per well were 50 mmol/L Tris-HCl at pH 8.0, 100 mmol/L NaCl, 5 mmol/L MgCl2, 25 mmol/L Na3PO4 at pH 8.0, 0.005% (v/v) Tween-20, and 2 mmol/L 2-mercaptoethanol. After 15 min reaction time, 5 μL NAD(P)H-Glo detection reagent (G9061 or G9062, Promega) was dispensed in all wells and the plate was incubated for 60 min. Luminescence was measured on a plate reader (Envision, PerkinElmer or Sense, Hidex). The light signal produced is proportional to the amount of NAD(P)H in the sample.
Measurement: IC50 = 12 nM (pChEMBL 7.920000076293945)
Descriptors: MW 458.4, ALogP -1.7, HBA 11, HBD 8, PSA 272.67, QED 0.19