lovingscience/chembl-v37-raw-activities huggingface dataset:
Compound (SMILES): Cc1ncoc1-c1nnc(SCCCCN2CCC3CCN(c4ccc(C(F)(F)F)cc4)C3C2)n1C
InChIKey: XPEWLIYLRNDQAK-UHFFFAOYSA-N
Target: D(2) dopamine receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P14416
Assay: Functional Calcium Assay: Functional Calcium Assay at hD2 recombinant receptor. CHO cells stably expressing human dopamine receptor type 2, long variant (hD2L), coupled to Gα16 protein (CHO-Gα16-hD2L) were seeded into black walled clear-base 384-well plates at a density of 8,000 cells per well and grown overnight at 37° C. After washing with the assay buffer (20 mM HEPES, 145 mM NaCl, 5 mM KCl, 5.5 mM glucose, 1 mM MgCl2 and 2 mM CaCl2, pH 7.4) containing 2.5 mM Probenecid, cells were incubated with the cytoplasmic Ca2+ probe Fluo-4 AM at 1 μM (final concentration), 37° C. for 60 min. Plates were washed three times as above and placed into a Fluorometric Imaging Plate Reader (FLIPR Tetra, Molecular Devices) to monitor cell fluorescence (ex=470-495 nm, em=515-575 nm) before and after the addition of different concentrations of test compounds. Compounds of invention were dissolved in DMSO and 200-fold diluted with assay buffer plus 0.01% Pluronic F-127. Cells were exposed first to test compounds for 10 min, then to a submaximal concentration of the hD2 receptor agonist dopamine (EC80, 50-140 nM). The fluorescence before compound addition (baseline) and before and after addition of agonist challenge was monitored. The peak of Ca2+ stimulation (baseline subtracted) was plotted versus the concentration of test compound and the curve fitted using a four-parameter logistic equation (XLfit) to assess the agonist/antagonist potency and maximal response.
Measurement: Ki = 347 nM (pChEMBL 6.460000038146973)
Descriptors: MW 520.63, ALogP 5.27, HBA 8, HBD 0, PSA 63.22, QED 0.3
Compound (SMILES): CC(=O)N[C@@H]1CCN(C(=O)c2nc(N[C@@H](C)c3cccnc3)nc3ccsc23)C1
InChIKey: HAAIWERVSNJXHE-SWLSCSKDSA-N
Target: Adenosine receptor A2a (SINGLE PROTEIN), Homo sapiens
UniProt: P29274
Assay: Activity Assay: Compounds (Adenosine Receptor modulators) and activity assay.
Measurement: IC50 > 500 nM (pChEMBL 6.301000118255615)
Descriptors: MW 410.5, ALogP 2.61, HBA 7, HBD 2, PSA 100.11, QED 0.67
Compound (SMILES): Cc1cc(C(=O)N2CCC[C@H]2c2nc(-c3cccc(Cl)c3C)no2)c(-n2cccn2)cc1C
InChIKey: YMCIMSDKANXDNQ-NRFANRHFSA-N
Target: Orexin receptor type 2 (SINGLE PROTEIN), Homo sapiens
UniProt: O43614
Assay: In Vitro Assay: Human orexin-A as an agonist is prepared as 1 mM stock solution in MeOH: water (1:1), diluted in HBSS containing 0.1% bovine serum albumin (BSA), NaHCO3: 0.375 g/L and 20 mM HEPES for use in the assay at a final concentration of 3 nM.Antagonists are prepared as 10 mM stock solution in DMSO, then diluted in 384-well plates using DMSO followed by a transfer of the dilutions into in HBSS containing 0.1% bovine serum albumin (BSA), NaHCO3: 0.375 g/L and 20 mM HEPES. On the day of the assay, 50 uL of staining buffer (HBSS containing 1% FCS, 20 mM HEPES, NaHCO3: 0.375 g/L, 5 mM probenecid (Sigma) and 3 uM of the fluorescent calcium indicator fluo-4 AM (1 mM stock solution in DMSO, containing 10% pluronic) is added to each well. The 384-well cell-plates are incubated for 50 min at 37° C. in 5% CO2 followed by equilibration at rt for 30 min before measurement.Within the Fluorescent Imaging Plate Reader (FLIPR Tetra, Molecular Devices), antagonists are added to the plate in a volume of 10 uL/well, incubated for 120 min and finally 10 uL/well of agonist is added. Fluorescence is measured for each well at 1 second intervals, and the height of each fluorescence peak is compared to the height of the fluorescence peak induced by 3 nM orexin-A with vehicle in place of antagonist. The IC50 value (the concentration of compound needed to inhibit 50% of the agonistic response) is determined and may be normalized using the obtained IC50 value of an on-plate reference compound. Optimized conditions were achieved by adjustment of pipetting speed and cell splitting regime. The calculated IC50 values may fluctuate depending on the daily cellular assay performance. Fluctuations of this kind are known to those skilled in the art. In the case where IC50 values have been determined several times for the same compound, the geometric mean has been given.
Measurement: IC50 = 6 nM (pChEMBL 8.220000267028809)
Descriptors: MW 461.95, ALogP 5.48, HBA 6, HBD 0, PSA 77.05, QED 0.4
Compound (SMILES): N#CCC1(n2cc(-c3nc(-c4cn[nH]c4)cc4ncccc34)cn2)CN(CC(F)(F)F)C1
InChIKey: CFARZEYJXRLTIQ-UHFFFAOYSA-N
Target: Tyrosine-protein kinase JAK1 (SINGLE PROTEIN), Homo sapiens
UniProt: P23458
Assay: JAK Caliper Enzyme Assay at 1mM ATP: Compounds were added to a 384-well plate. Reaction mixtures contained 10 mM HEPES, pH 7.4, 10 mM MgCl2, 0.01% BSA, 0.0005% Tween 20, 1 mM ATP and 1 μM peptide substrate. The JAK1 and TYK2 assays contained 1 μM of the IRStide peptide (5FAM-KKSRGDYMTMQID) and the JAK2 and JAK3 assays contained 1 μM of the JAKtide peptide (FITC-KGGEEEEYFELVKK). The assays were initiated by the addition of 20 nM JAK1, 1 nM JAK2, 1 nM JAK or 1 nM TYK2 enzyme and were incubated at room temperature for three hours for JAK1, 60 minutes for JAK2, 75 minutes for JAK3 or 135 minutes for TYK2. Enzyme concentrations and incubation times were optimized for each new enzyme preps and were modified slightly over time to ensure 20% to 30% phosphorylation. The assays were stopped with 15 μL of 180 mM HEPES, pH 7.4, 20 mM EDTA, and 0.2% Coating Reagent 3. The assay plates were placed on a Caliper Life Science LC3000 instrument, and each well was sampled using appropriate separation conditions to measure the unphosphorylated and phosphorylated peptide.
Measurement: IC50 = 35 nM (pChEMBL 7.460000038146973)
Descriptors: MW 438.42, ALogP 3.37, HBA 7, HBD 1, PSA 99.31, QED 0.51
Compound (SMILES): O=C1CCc2c(Oc3ccc4c(c3)[C@H]3[C@H](NC(=O)Nc5ccc(F)c(F)c5F)[C@H]3O4)ccnc2N1
InChIKey: UVFPAUPHTITLLA-NYVOZVTQSA-N
Target: RAF proto-oncogene serine/threonine-protein kinase (SINGLE PROTEIN), Homo sapiens
UniProt: P04049
Assay: Raf IC50 Assay: Compounds disclosed herein were tested against B-Raf (V600E) (PV3849, from Invitrogen) or C-Raf (Y340D/Y341D) (PV3805, from Invitrogen) in a time-resolved fluorescence energy transfer assay. The assay was carried out in reactions (10 μL) containing 0.0625 nM B-Raf or 0.5 nM C-Raf, 25 mM Tris pH7.4, 10 mM MgCl2, 0.5 mM EGTA, 0.5 mM Na3VO4, 5 mM beta-glycerophosphate, 0.01% Triton X-100, 2.5 mM DTT, 0.1% BSA, 0.1 mM ATP, 13.7 nM GST-tagged MEK1 (Full-length protein with K97R mutation, recombinant protein purified from bacterial expression system) and 0-5 μM compounds disclosed herein (final concentration of 1% DMSO). The enzyme was incubated with the compounds at room temperature for 60 minutes and the reactions were initiated by the addition of ATP and GST-MEK1. After reaction at room temperature for 60 minutes, an equal volume of stop/detection solution was added according to the manufacture's instruction (CisBio Bioassays). The stop/detection solution contained Eu3+ cryptate-conjugated anti-phospho MEK1/2 (Ser217/221) rabbit polyclonal antibody and d2-conjugated anti-GST mouse monoclonal antibody in buffer containing 25 mM Tris pH7.4, 400 mM KF, 50 mM EDTA, 0.01% BSA and 0.01% Triton X-100. Plates were sealed and incubated at room temperature for 2 hours, and the TR-FRET signals (ratio of fluorescence emission at 665 nm over emission at 620 nm with excitation at 337 nm wavelength) were recorded on a PHERAstar FS plate reader (BMG Labtech). Phosphorylation of MEK1 led to the binding of anti-phospho-MEK1/2 antibody to GST-MEK1 protein that place fluorescent donor (Eu3+ crypate) in close proximity to the accepter d2 on the anti-GST antibody, thus resulting in a high degree of fluorescence resonance energy transfer from the donor fluorophore (at 620 nm) to the acceptor fluorophore (at 665 nm). Inhibition of RAF kinase activity resulted in decrease of the TR-FRET signal. The IC50 for each compound was derived from fitting the dose-response % inhibition data to the four-parameter logistic model by Graphpad Prism software.
Measurement: IC50 = 220 nM (pChEMBL 6.659999847412109)
Descriptors: MW 482.42, ALogP 4.22, HBA 5, HBD 3, PSA 101.58, QED 0.48
Compound (SMILES): COc1cc(/C=C(\C#N)C(N)=O)cc(-c2cnc3[nH]cc(-c4ccnc(OC)c4)c3c2)c1
InChIKey: OEFSVAYBAKZLHC-UBKPWBPPSA-N
Target: 1-phosphatidylinositol 4,5-bisphosphate phosphodiesterase gamma-1 (SINGLE PROTEIN), Homo sapiens
UniProt: P19174
Assay: ITK Target Modulation PLC-gamma Assay: Compounds were serially diluted into DMSO, and then into media to concentrations 10× that of the final assay concentration. Compounds were then added to the cells with the final assay DMSO concentration being 0.1%. Cells were incubated for 1 hour at 37° C. Following compound incubation, αCD3 Dynabeads were added at 1 bead/cell to each assay well and incubated at 37° C. for 7 minutes. Unstimulated control wells received media only. The plate was then placed on a 96-well magnetic plate for an additional 60 seconds (total assay stimulation time equals 8 minutes). The plate was then dumped and gently patted dry on a paper towel. 50 ul of complete Meso Scale Discovery (MSD) Lysis buffer was then added to each well. Samples were assayed for phospho-PLCγ1 using Meso Scale Discovery technology.
Measurement: IC50 > 5000 nM (pChEMBL 5.301000118255615)
Descriptors: MW 425.45, ALogP 3.7, HBA 6, HBD 2, PSA 126.91, QED 0.36
Compound (SMILES): Cc1cc(C)c(-c2ccc(NCCc3ccc(S(N)(=O)=O)cc3)nn2)c(O)c1
InChIKey: LMGYQUMHLYXREQ-UHFFFAOYSA-N
Target: Tumor necrosis factor (SINGLE PROTEIN), Homo sapiens
UniProt: P01375
Assay: TNF-alpha Secretion Assay: Monocytic THP-1 cells were maintained according to providers' instructions in RPMI media as described above. Undifferentiated cells were plated at 50,000 cells per well in a 384-well flat-bottom cell culture plates (Greiner, #781986), and allowed to rest overnight. Experimental compounds were prepared and added as described above. TNF-α secretion was triggered by the addition of either 1 μg/mL LPS (Sigma, #L4391) or 100 ng/mL Pam3CSK4 (Invivogen, #tlrl-pms) depending on the experiment, and cells were incubated for 3 hours. 10 μL supernatant was removed, and TNF-α levels were monitored using an HTRF assay (CisBio, #62TNFPEC) according to manufacturers' instructions.
Measurement: IC50 = 16000 nM (pChEMBL 4.800000190734863)
Descriptors: MW 398.49, ALogP 2.77, HBA 6, HBD 3, PSA 118.2, QED 0.59
Compound (SMILES): N#CCC1CCN(c2nc(Nc3ccc(OCCN4CCNCC4)cc3)c3c(=O)[nH]ncc3n2)CC1
InChIKey: RYGOOQDQGBYSJO-UHFFFAOYSA-N
Target: Tyrosine-protein kinase JAK2 (SINGLE PROTEIN), Homo sapiens
UniProt: O60674
Assay: Inhibition of Enzymatic JAK2 Kinase Activity: Two μL of 10% DMSO in blank, substrate control and positive control wells and 2 μL of test compound in test wells was added. Thirteen μL of assay buffer in blank and substrate control wells and 13 μL of Enzyme buffer mix in positive and test wells was added. The reaction mixture was incubated for 30 minutes at RT on a plate shaker. Ultra Light-pGT substrate (5 μL) [poly Glu-Tyr (4:1) labeled with U Light TM dye, a tyrosine kinase substrate] and ATP mix was added to all wells. The reaction plate was incubated for 60 minutes at RT on a plate shaker. The reaction was stopped by adding 40 mM EDTA (10 μL) in buffer. Ten μL of antibody was added to all the wells. The plate was read in a Wallac 1420 Multilabel Counter Victor 3 instrument (Ex: 340 nm Em: 615 & 665 nm).
Measurement: IC50 > 100 nM (pChEMBL 7)
Descriptors: MW 489.58, ALogP 1.87, HBA 10, HBD 3, PSA 135.09, QED 0.43
Compound (SMILES): N#Cc1cnn(-c2ccc(Cl)cc2-c2ccc([C@H]3CCCCC(=O)Nc4cnn(C(F)F)c4-c4cccc3c4)[n+]([O-])c2)c1
InChIKey: YNDRQXGXYGJMNC-DEOSSOPVSA-N
Target: Kallikrein-1 (SINGLE PROTEIN), Homo sapiens
UniProt: P06870
Assay: Kallikrein Assay: The effectiveness of a compound of the present invention as an inhibitor of Kallikrein can be determined using a relevant purified serine protease, and an appropriate synthetic substrate. The rate of hydrolysis of the chromogenic or fluorogenic substrate by the relevant serine protease was measured both in the absence and presence of compounds of the present invention. Assays were conducted at rt or at 37° C. Hydrolysis of the substrate resulted in release of amino trifluoromethylcoumarin (AFC), which was monitored spectrofluorometrically by measuring the increase in emission at 510 nm with excitation at 405 nm. A decrease in the rate of fluorescence change in the presence of inhibitor is indicative of enzyme inhibition. Such methods are known to one skilled in the art. The results of this assay are expressed as the inhibitory constant, Ki.Kallikrein determinations were made in 50 mM HEPES buffer at pH 7.4 containing 150 mM NaCl, 5 mM CaCl2, and 0.1% PEG 8000 (polyethylene glycol; Fisher Scientific). Determinations were made using purified Human plasma Kallikrein at a final concentration of 0.5 nM (Enzyme Research Laboratories) and the synthetic substrate, Acetyl-K-P-R-AFC (Sigma # C6608) at a concentration of 100 mM.Activity assays were performed by diluting a stock solution of substrate at least tenfold to a final concentration ≤0.2 Km into a solution containing enzyme or enzyme equilibrated with inhibitor. Times required to achieve equilibration between enzyme and inhibitor were determined in control experiments. The reactions were performed under linear progress curve conditions and fluorescence increase measured at 405 Ex/510 Em nm. Values were converted to percent inhibition of the control reaction (after subtracting 100% Inhibition value). IC50 was determined by inflection point from a four parameter logistic curve fit. Ki was calculated using the Cheng Prusoff equation, Ki=IC50/(1+([S]/Km)).
Measurement: Ki = 94.2 nM (pChEMBL 7.03000020980835)
Descriptors: MW 600.03, ALogP 6.6, HBA 7, HBD 1, PSA 115.47, QED 0.18
Compound (SMILES): Cc1nc(C(=O)N[C@@H](c2ccc(C(F)(F)F)cc2)C2(C)CC2)cc(=O)[nH]1
InChIKey: AHAWZPGCCFRNHZ-HNNXBMFYSA-N
Target: cGMP-dependent 3',5'-cyclic phosphodiesterase (SINGLE PROTEIN), Homo sapiens
UniProt: O00408
Assay: Fluorescence Polarization (FP) Assay: In a typical experiment the PDE2 inhibitory activity of the compounds of the present invention was determined in accordance with the following experimental method. Rhesus PDE2A3 was amplified from rhesus macaque brain cDNA (Biochain Institute, Hayward, Calif.) using primers based on human PDE2A sequence (accession NM_002599.3) where the forward primer containing a Kozak consensus was 5′-gccaccatggggcaggcatgtggc-3′ and the reverse primer was 5′-tcactcagcatcaaggctgca-3′. Amplification with Easy-A High-Fidelity PCR cloning enzyme (Stratagene, La Jolla, Calif.) was 95° C. for 2 minutes followed by thirty three cycles of 95° C. for 40 seconds, 52° C. for 30 seconds, and 72° C. for 2 minutes 48 seconds. Final extension was 72° C. for 7 minutes. The PCR product was TA cloned into pcDNA3.3-TOPO (Invitrogen, Carlsbad, Calif.) according to standard protocol. A consensus sequence was developed from multiple clones and then deposited into GenBank (EU812167). AD293 cells (Stratagene, La Jolla, Calif.) with 70-80% confluency were transiently transfected with rhesus PDE2A3/pcDNA3.3-TOPO using Lipofectamine 2000 according to manufacturer specifications (Invitrogen, Carlsbad, Calif.).
Measurement: Ki = 11 nM (pChEMBL 7.960000038146973)
Descriptors: MW 365.36, ALogP 3.37, HBA 3, HBD 2, PSA 74.85, QED 0.87
Compound (SMILES): C=CC(=O)N1CCO[C@H]2CN(C(=O)N(C)[C@H](C(=O)N[C@H]3Cc4cccc(n4)-c4ccc5c(c4)c(c(-c4cccnc4[C@H](C)OC)n5CC(F)(F)F)CC(C)(C)COC(=O)[C@@H]4CCCN(N4)C3=O)C(C)C)C[C@H]21
InChIKey: ISDVCOAZTZDRGZ-NPWNKKAYSA-N
Target: GTPase KRas (SINGLE PROTEIN), Homo sapiens
UniProt: P01116
Assay: Biochemical Assay: A variety of Ras proteins may be inhibited by compounds of the present invention (e.g., K-Ras, N-Ras, H-Ras, and mutants thereof at positions 12, 13 and 61, such as G12C, G12D, G12V, G12S, G13C, G13D, and Q61L, and others described herein). The purpose of this biochemical assay is to measure the ability of test compounds to covalently label nucleotide-loaded K-Ras isoforms. In assay buffer containing 12.5 mM HEPES pH 7.4, 75 mM NaCl, 1 mM MgCl2, 1 mM BME, 5 uM Cyclophilin A and 2 uM test compound, a 5 uM stock of GMP-PNP-loaded K-Ras (1-169) G12C is diluted 10-fold to yield a final concentration of 0.5 uM; with final sample volume being 100 uL. The sample is incubated at 25�� C. for a time period of up to 24 hours prior to quenching by the addition of 10 uL of 5% Formic Acid. Quenched samples are centrifuged at 15000 rpm for 15 minutes in a benchtop centrifuge before injecting a 10 uL aliquot onto a reverse phase C4 column and eluting into the mass spectrometer with an increasing acetonitrile gradient in the mobile phase. Analysis of raw data may be carried out using Waters MassLynx MS software, with % bound calculated from the deconvoluted protein peaks for labeled and unlabeled K-Ras.
Measurement: IC50 = 550 nM (pChEMBL 6.260000228881836)
Descriptors: MW 1000.13
Compound (SMILES): COc1c(F)cc(CN2C3CC2CN(c2ccc(-c4cc(OCC(C)(C)O)cn5ncc(C#N)c45)cn2)C3)cc1F
InChIKey: PTHLLEHOQTYYDS-UHFFFAOYSA-N
Target: Proto-oncogene tyrosine-protein kinase receptor Ret (SINGLE PROTEIN), Homo sapiens
UniProt: P07949
Assay: RET Enzyme Assay: Compounds of Formula I were screened for their ability to inhibit wildtype and V804M mutant RET kinase using CisBio's HTRF® KinEASE¿-TK assay technology. Briefly, N-terminal GST tagged recombinant human RET cytoplasmic domain (aa 658-end) from Eurofins (0.25 nM RET; Catalog No. 14-570M) or N-terminal GST tagged recombinant human V804M mutant RET cytoplasmic domain (aa 658-end) from Millipore (0.25 nM enzyme; Catalog No. 14-760) was incubated with 250 nM TK-substrate biotin (CisBio, part of Catalog No. 62TK0PEC) and 1 mM ATP along with test compound in a buffer consisting of 25 mM HEPES pH 7.4, 10 mM MgCl2, 0.01% Triton X-100, and 2% DMSO in a volume of 8 μL. Compounds were typically prepared in a threefold serial dilution in DMSO and added to the assay to give the appropriate final concentration. After a 30-minute incubation at 22° C., the reaction was quenched by adding 8 μL of quench solution containing 31.25 nM Sa-XL665 and 1×TK-ab-Cryptate in HTRF detection buffer (all from CisBio, part of Cat. No. 62TK0PEC). After a 1 hour incubation at 22° C., the extent of reaction was determined using a PerkinElmer EnVision multimode plate reader via HTRF dual wavelength detection, and the percent of control (POC) was calculated using a ratiometric emission factor. 100 POC was determined using no test compounds and 0 POC was determined using pre-quenched control reactions. The POC values were fit to a 4 parameter logistic curve, and the IC50 is defined as the concentration of inhibitor at which the POC equals 50 for the fitted curve.
Measurement: IC50 = 23.2 nM (pChEMBL 7.630000114440918)
Descriptors: MW 560.61, ALogP 4.17, HBA 9, HBD 1, PSA 99.15, QED 0.34
Compound (SMILES): O=C(c1cccs1)N(c1cn(-c2ccccc2)nn1)C1CCN(Cc2ccccc2)CC1
InChIKey: MKXVLTLTANCGBG-UHFFFAOYSA-N
Target: Voltage-dependent calcium channel subunit alpha-2/delta-1 (SINGLE PROTEIN), Homo sapiens
UniProt: P54289
Assay: Human alpha2delta-1 Subunit of Cav2.2 Calcium Channel 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. 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 > 5000 nM (pChEMBL 5.301000118255615)
Descriptors: MW 443.58, ALogP 4.64, HBA 6, HBD 0, PSA 54.26, QED 0.44
Compound (SMILES): COC(=O)c1cc2c(Oc3ccc(NC(=O)C4(C(=O)Nc5ccc(F)cc5)CC4)cc3F)ccnc2cc1OC
InChIKey: XKZPGAOTMYFHJK-UHFFFAOYSA-N
Target: Tyrosine-protein kinase Mer (SINGLE PROTEIN), Homo sapiens
UniProt: Q12866
Assay: Mer Kinase Assay: Human Mer (residues R557-E882 with H628Q and R794A, 0.7 nM) was incubated with 8 mM MOPS pH 7.0, 0.2 mM EDTA, 30 mM NaCl, 250 μM GGMEDIYFEFMGGKKK (SEO ID NO. 44). 10 mM magnesium acetate, and 10 μM [γ-33P-ATP]. The reaction was initiated by the addition of the Mg/ATP mix. After incubation for 40 minutes at room temperature, the reaction was stopped by the addition of phosphoric acid to a concentration of 0.5%. A reaction aliquot of 10 μL was then spotted onto a P30 filtermat and washed four times for 4 minutes in 0.425% phosphoric acid and once in methanol prior to drying and scintillation counting. Incorporated 33P was measured using the Wallac Microbeta scintillation counter (Perkin Elmer).
Measurement: IC50 < 10 nM (pChEMBL 8)
Descriptors: MW 547.51, ALogP 5.46, HBA 7, HBD 2, PSA 115.85, QED 0.22
Compound (SMILES): O=C(NC1CC2(CCC2)C1)c1cc2cc(-c3ccc(OC4CC4)cc3)cnc2n(Cc2ccc(F)cc2)c1=O
InChIKey: ZMSNAVHGDPFSAO-UHFFFAOYSA-N
Target: Cannabinoid receptor 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P21554
Assay: CB1 and CB2 Receptor Binding Assay: CB1R binding protocol involves the use of the same solution buffer used for both incubation and washing reaction (Tris-HCl, 50 mM; EDTA, 2.5 mM; MgCl2, 2.5 mM; BSA, 0.5 mg/mL at pH 7.4), 0.4 nM for [3H]CP-55,940, test compounds (concentrations from 0.001 to 10 μM), and finally 8 μg/sample membrane in a total volume of 200 μL. CB2R binding assays were carried out with two different buffers: incubation buffer (Tris-HCl, 50 mM; MgCl2, 5 mM; CaCl2 1 mM; BSA, 0.2% at pH 7.4) and washing buffer (Tris-HCl, 50 mM; NaCl 500 mM; BSA, 0.1% at pH 7.4). The assay mixture contained incubation buffer, 0.4 nM [3H]CP-55,940, test substances (concentrations from 0.001 to 10 μM), and 4 μg/sample membrane in a total assay volume of 200 μL. Assays were performed in duplicate and incubated for 120 mM at 37° C. After the incubation, the assay mixture is filtered through 96 GF/C filter plates (Perkin Elmer #6005174) using Perkin Elmer Filtermate Harvester, and then washed four times with ice-cold washing buffer.
Measurement: IC50 > 1000 nM (pChEMBL 6)
Descriptors: MW 523.61, ALogP 5.85, HBA 5, HBD 1, PSA 73.22, QED 0.33
Compound (SMILES): Cc1nc2ncc(-c3cn(C)c(=O)c4[nH]ccc34)nc2n1Cc1ccc(C(F)(F)F)cc1
InChIKey: SONHSXTYAWZZFU-UHFFFAOYSA-N
Target: Bromodomain-containing protein 4 (SINGLE PROTEIN), Homo sapiens
UniProt: O60885
Assay: Inhibitory Activity Assay: The assays of BRD4 (D1) and BRD4 (D2) were conducted in a 384-well polystyrene plate. The test compounds were first serially diluted in DMSO and the test compound/DMSO were transferred to 384-well plates. The final concentration of DMSO in the assay was 0.1%. 2 volumes of protein/peptide mixture were added into 384-well plates, then 2 volumes of assay mixture were added, and shake for 30 s. The plate was incubated at room temperature for 2 h. Then the HTRF signal on EnVision was readed.
Measurement: IC50 = 1.6 nM (pChEMBL 8.800000190734863)
Descriptors: MW 438.41, ALogP 4.05, HBA 6, HBD 1, PSA 81.39, QED 0.46
Compound (SMILES): CC1(c2onc(-c3c(Cl)cc(Cl)cc3Cl)c2C(=O)n2cc3c4c(cccc42)/C(=C/C(=O)O)CC3)CC(F)(F)C1
InChIKey: RXFMFDIZFGQVGG-NTUHNPAUSA-N
Target: Isocitrate dehydrogenase [NADP] cytoplasmic (SINGLE PROTEIN), Homo sapiens
UniProt: O75874
Assay: Inhibitory Activity Against IDH1R132H and IDH1R132C Enzymes: The IDH1R132H protein and the IDH1R132C protein were prepared as follows: the IDH1R132H or IDH1R132C gene was integrated into a pET24b(+) vector (Novagen) to prepare a construct for C-terminal fusion of 6× histidine tag. After transformation of Rosetta 2 (DE3) E. coli, the expression of the protein was induced with IPTG. The E. coli was collected and homogenized, followed by the affinity purification of the 6× histidine fusion protein using HisTrap HP columns (GE Healthcare Japan Corp.) and gel filtration purification using Superdex 200 columns (GE Healthcare Japan Corp.) to obtain the IDH1R132H or IDH1R132C protein of interest.IDH1R132H and IDH1R132C each convert 2-oxoglutarate and NADPH to D-2-hydroxyglutarate (2-HG) and NADP+. Therefore, the activity of the IDH1R132H and IDH1R132C enzymes can be measured by detecting NADPH levels.The enzyme inhibitory activity evaluation was carried out as follows: 40 μL each of reaction solutions containing different concentrations of each compound (100 mM Tris-HCl (pH 7.5), 150 mM NaCl, 20 mM MgCl2, 0.5 mg/mL bovine serum albumin, 1 mM reduced glutathione, 40 μM NADPH, 0.5 mM 2-oxoglutarate, 0.5% dimethyl sulfoxide, 50000 to 0.128 nM compound, and 12 nM IDH1R132H or 10 nM IDH1R132C as the enzyme) was added to each well of 384-well plates (Greiner Bio One International GmbH, #781096) and incubated at room temperature. While NADPH-derived fluorescence was occasionally monitored, the reaction was terminated by the addition of 5 μL of 0.5 M EDTA before consumption of NADPH. 5 μL of WST-8 reagent (Dojindo Laboratories, #CK04) was further added and mixed therewith. 15 minutes later, the absorbance at 450 nm was measured using a plate reader (PerkinElmer, Inc., EnVision). The observed absorbance value reflects the amount of residual NADPH. From the absorbance data, the enzyme inhibitory activity of each compound at each concentration was calculated and analyzed using medical statistical analysis software GraphPad Prism to calculate an IC50 value.
Measurement: IC50 = 169 nM (pChEMBL 6.769999980926514)
Descriptors: MW 591.83, ALogP 8.05, HBA 5, HBD 1, PSA 85.33, QED 0.24
Compound (SMILES): Cc1ncsc1-c1ccc(CNC(=O)[C@@H]2C[C@@H](O)CN2C(=O)[C@@H](NC(=O)c2cccc(C#N)c2)C(C)(C)C)cc1
InChIKey: DMYFTMDRRIIONV-RMTZWNOUSA-N
Target: von Hippel-Lindau disease tumor suppressor (SINGLE PROTEIN), Homo sapiens
UniProt: P40337
Assay: Fluorescence Polarization Assay (10% DMSO): Ability of VHL ligands to compete for the HIF 1 a binding site on VCB was determined through a fluorescence polarization competition assay as described in Buckley et al. JACS, 2012, 134, 4465-4468, WO 2013/106643, and US 2014-0356322, which are incorporated herein by reference in their entirety for all purposes (10% DMSO).
Measurement: IC50 = 1380 nM (pChEMBL 5.860000133514404)
Descriptors: MW 559.69, ALogP 3.41, HBA 7, HBD 3, PSA 135.42, QED 0.41
Compound (SMILES): COC(c1ccc(F)c(-c2ncnc3cc(N4CCOCC4)ccc23)c1)c1nccnc1C
InChIKey: VJZQQSPYGNVLNF-UHFFFAOYSA-N
Target: Voltage-gated inwardly rectifying potassium channel KCNH2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q12809
Assay: Patch Clamp Assay: The patch-clamp measurement was carried out at room temperature in whole-cell configuration on human embryonic kidney cells (HEK293) which have been transfected in a stable manner with the hERG gene.The whole-cell configurations were carried out using an automated patch clamp device (Patchliner, Nanion Technologies, Munich). This is a glass chip-based system with which automated whole-cell measurements on up to 8 cells simultaneously are possible. The glass chip has a hole of defined size to which the cell is transferred into the Gigaseal by application of a reduced pressure and brought into the whole-cell configuration. Buffer, cell suspension and test substances were added to microchannels of the chip using a Teflon-coated pipette. The cells were clamped to a holding potential of -80 mV. For measurement of substance-promoted inhibition of the Kv11.1 channel, the following voltage protocol was applied at 10-second intervals: 51 ms/-80 mV, 500 ms/+40 mV, 500 ms/-40 mV, 200 ms/-80 mV. The leakage current is subtracted by means of the P4 method. The cells were resuspended in extracellular buffer (EC) and applied to the chip. After the cell had been collected, the seal was improved by addition of a seal enhancer buffer. As soon as the whole-cell configuration had been reached, the seal enhancer buffer was washed out and replaced by extracellular buffer. The measurement started in EC for 1.5 min. DMSO (vehicle control, 0.1% of DMSO) was then applied, and the control current was recorded for 3 min. The test substance was subsequently added twice in the same concentration, and the potassium current was measured for 3.5 min in each case.
Measurement: Ki > 25000 nM (pChEMBL 4.6020002365112305)
Descriptors: MW 445.5, ALogP 4.11, HBA 7, HBD 0, PSA 73.26, QED 0.46
Compound (SMILES): N[C@H](CNC(=O)c1cc2nccc(C(F)F)c2s1)C(=O)O
InChIKey: ASWNPURWTQJVQD-ZCFIWIBFSA-N
Target: Glutamate receptor ionotropic, NMDA 1/2C/3B (PROTEIN COMPLEX), Homo sapiens
UniProt: O60391
Assay: Scintillation Proximity Assay: To determine the affinity of the compounds of the present invention a SPA is used. The assay is run in a 384-plate format (OptiPlate-384) where each well contains a mix of 5 μL of test compound, 5 μL NR1s1s2 (ligand binding domains of the NMDA receptor, MW 35.6 kDa, 0.075 ug/well final), 5 μL [3H]-MDL-105,519 (radiolabelled, high affinity N-methyl-D-aspartate (NMDA) glutamate receptor antagonist at the glycine site, final concentration 5 nM, Kd=1.3 nM), 5 μL streptavidin coated imaging beads (Perkin Elmer cat. No.: RPNQ0273, 8 ug/well). The assay buffer contains 100 mM HEPES-NaOH, 150 mM NaCl, 1 mM EDTA, 10% glycerol at pH 7.4 in ultra-pure water. Non-specific binding is defined by inclusion of 10 μM L-689,560 (highly potent NMDA antagonist) and total binding by 1% DMSO. Following 30 minutes incubation in the dark (shaker, Multi-microplate Genie), the SPA beads are allowed to settle for 3 hours after which the signal is read on a Viewlux instrument (Perkin Elmer). Normalized data are used to calculate Ki values.
Measurement: Ki = 170 nM (pChEMBL 6.769999980926514)
Descriptors: MW 315.3, ALogP 1.38, HBA 5, HBD 3, PSA 105.31, QED 0.77
Compound (SMILES): CC(C)(C)[C@@H](NC(=O)c1cc(=O)[nH]c(-c2ncccn2)n1)c1ccc(C2CC2)c(F)c1
InChIKey: KZWMNKPMTHIBPM-IBGZPJMESA-N
Target: cGMP-dependent 3',5'-cyclic phosphodiesterase (SINGLE PROTEIN), Homo sapiens
UniProt: O00408
Assay: Fluorescence Polarization (FP) Assay: In a typical experiment the PDE2 inhibitory activity of the compounds of the present invention was determined in accordance with the following experimental method. Rhesus PDE2A3 was amplified from rhesus macaque brain cDNA (Biochain Institute, Hayward, Calif.) using primers based on human PDE2A sequence (accession NM_002599.3) where the forward primer containing a Kozak consensus was 5′-gccaccatggggcaggcatgtggc-3′ and the reverse primer was 5′-tcactcagcatcaaggctgca-3′. Amplification with Easy-A High-Fidelity PCR cloning enzyme (Stratagene, La Jolla, Calif.) was 95° C. for 2 minutes followed by thirty three cycles of 95° C. for 40 seconds, 52° C. for 30 seconds, and 72° C. for 2 minutes 48 seconds. Final extension was 72° C. for 7 minutes. The PCR product was TA cloned into pcDNA3.3-TOPO (Invitrogen, Carlsbad, Calif.) according to standard protocol. A consensus sequence was developed from multiple clones and then deposited into GenBank (EU812167). AD293 cells (Stratagene, La Jolla, Calif.) with 70-80% confluency were transiently transfected with rhesus PDE2A3/pcDNA3.3-TOPO using Lipofectamine 2000 according to manufacturer specifications (Invitrogen, Carlsbad, Calif.).
Measurement: Ki = 4.8 nM (pChEMBL 8.319999694824219)
Descriptors: MW 421.48, ALogP 3.76, HBA 5, HBD 2, PSA 100.63, QED 0.65
Compound (SMILES): COc1ccc(CC(=O)Nc2sccc2-c2noc(C)n2)cc1
InChIKey: JWKYWPAXWSIVQX-UHFFFAOYSA-N
Target: Mitogen-activated protein kinase 8 (SINGLE PROTEIN), Homo sapiens
UniProt: P45983
Assay: HTRF Assay: (1) Assay buffer #1 with 0.0025% Tween and 1 mM DTT was prepared using JNK buffer stock solution.(2) Assay buffer #2 with 0.025% BSA was prepared using the assay buffer #1.(3) Test compound solution preparation: 5× compound solution was prepared using assay buffer #1 with 5% DMSO. The compound solution (10 μl/well) was added to a 384-well plate (Corning, Cat No. 3654).(4) aJNK1, 2 or 3 preparation: aJNK stock (100 μg/ml) at −80° C. was thawed on ice and an aJNK (1.6 ng/ml) solution was prepared using assay buffer #2. The aJNK solution (20 μl/well) was added to the plate and the plate was shaken. The enzyme was incubated with the compound at RT for 10 min.(5) ATP/substrate solution preparation: ATP and substrate stocks were thawed on ice. 2.5×ATP/substrate (e.g., 25 μM or 2.5 mM ATP/50 nM EPIW-1) was prepared using assay buffer #1. The ATP/substrate (20 μl/well) was added to the plate and the plate was shaken. The plate was incubated at RT for 15 min. Note: In an exemplary assay, the final ATP concentration was about 1 mM.(6) EDTA preparation: 30 mM EDTA was prepared using 0.5M EDTA stock and assay buffer #1. The EDTA (10 μl/well) was added to the plate to quench the enzyme reaction and the plate was shaken well.(7) Detection reagent preparation: Eu-63 and SA-XL stocks were thawed on ice. 4× Eu-63/SA-XL (2 nM Eu-63/9.4 nM SA-XL) was prepared using JNK detection buffer. The Eu-63/SA-XL solution (20 μl/well) was added to the plate and the plate was shaken. The plate was incubated at RT for 1 hr before reading the plate on LJL using ratiometric method named HTRF.
Measurement: IC50 = 5050 nM (pChEMBL 5.300000190734863)
Descriptors: MW 329.38, ALogP 3.3, HBA 6, HBD 1, PSA 77.25, QED 0.78
Compound (SMILES): CCN(CCN(C)c1nccc(C(C)(C)O)n1)C(=O)c1cccnc1OC(C)C
InChIKey: IBJOSCHWGCACFL-UHFFFAOYSA-N
Target: Orexin/Hypocretin receptor type 1 (SINGLE PROTEIN), Homo sapiens
UniProt: O43613
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 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.
Measurement: IC50 = 10000 nM (pChEMBL 5)
Descriptors: MW 401.51, ALogP 2.48, HBA 7, HBD 1, PSA 91.68, QED 0.69
Compound (SMILES): CCN1CCN(Cc2ccc(C(=O)N[C@@H]3[C@H]4Oc5ccc(Oc6ccnc7c6CCC(=O)N7)cc5[C@@H]34)cc2Br)CC1
InChIKey: VFRPNIRNEYDCSJ-AWCRTANDSA-N
Target: Epidermal growth factor receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P00533
Assay: TBD: TBD
Measurement: IC50 = 0.55 nM (pChEMBL 9.260000228881836)
Descriptors: MW 618.53, ALogP 4.32, HBA 7, HBD 2, PSA 96.03, QED 0.41
Compound (SMILES): Nc1ncnc2c1c(-c1cc3ccccc3[nH]1)nn2C1CCCCC1
InChIKey: PNZXFLWANJRDEH-UHFFFAOYSA-N
Target: Vascular endothelial growth factor receptor 2 (SINGLE PROTEIN), Homo sapiens
UniProt: P35968
Assay: Time-Resolved Fluorescence Transfer (FRET) Assay: RET and KDR: Kinase activity was detected using CisBio HTRF kinEASE kit based on time-resolved fluorescence transfer (FRET). The assay was performed in 384-well white plates (Corning #3574) in a reaction volume of 10 μL containing 1× CisBio enzymatic buffer supplemented with a final concentration of 5 mM MgCl2, 1 mM DTT, 10 nM SEB and 0.01% Triton X100 for RET. The same buffer was used for the KDR biochemical assay with the addition of 2 mM MnCl2.Inhibitors were pre-incubated in the plate for 15 mins with 5 μL kinase and assay buffer at the following concentrations; 13 pM RET (Carna Biosciences; 08-159) and 150 pM KDR (Millipore; 14-630). The reaction was initiated by the addition of 5 μL ATP and substrate at 2× final reaction concentrations. For RET, this was 18 pM and 2 μM; for KDR, this was 16 μM and 1 μM, respectively. Reactions were performed at ATP Km for each target. The assay was allowed to proceed at room temperature for 20 mins before terminating with the addition of 10 μL HTRF detection buffer containing EDTA supplemented with TK-antibody labelled with Eu3+-Cryptate (1:100 dilution) and streptavidin-XL665 (128 nM). Following incubation at room temperature for 1 hour, FRET signal was measured using the Pherastar FS Microplate Reader.
Measurement: IC50 = 21000 nM (pChEMBL 4.679999828338623)
Descriptors: MW 332.41, ALogP 4.06, HBA 5, HBD 2, PSA 85.41, QED 0.58
Compound (SMILES): COCCOc1nc(Nc2ccc(CN3CCN(C)CC3)cc2)nc2[nH]cc(-c3ccc(C#N)cc3)c12
InChIKey: LVMCQNRIJXVCOS-UHFFFAOYSA-N
Target: Tyrosine-protein kinase Mer (SINGLE PROTEIN), Homo sapiens
UniProt: Q12866
Assay: Mer Kinase Assay: Certain compounds of formula (I) were tested for their potency in inhibiting Mer kinase. Inhibition of Mer kinase activity by a test compound was calculated by AlphaScreen (PerkinElmer 6760620M). Standard assay conditions included 2 ng of recombinant Mer kinase (SignalChem) with 5 ng Poly-(GT)-Biotin (Cisbio) in the assay buffer, i.e., 10 μM ATP (Sigma), 8 mM MOPs, pH7.0 (Sigma M-1254), 0.2 mM EDTA (Sigma E-6758), 0.5% glycerol (J. T Baker 2136-1), 0.1 mg/ml BSA (Bio-Rad 500-0007), 0.01% 2-mercaptoethanol (BDH 441433A), 0.001% Brij35 (Sigma P1254), 10 mM MgCl2, and 10 mM MnCl2 in a final volume of 25 μL. Reactions were incubated at 30° C. for 30 minutes and stopped by adding 5 μL of 50 mM EDTA. Product analysis was performed using the AlphaScreen kit and counted with Enspire Alpha (PerkinElmer). Screen values of inhibitors were determined after carrying out assays with each compound at serially diluted concentrations in duplication. Results were analyzed using linear regression software (GraphPad Prism 4; GraphPad Software Inc.).
Measurement: IC50 = 700 nM (pChEMBL 6.159999847412109)
Descriptors: MW 497.6, ALogP 4.01, HBA 8, HBD 2, PSA 102.33, QED 0.33
Compound (SMILES): CCSc1nc(O)c(C(=O)Nc2ccc(-c3nn[nH]n3)cc2)c(=O)[nH]1
InChIKey: KCMFZUUGRZMYJC-UHFFFAOYSA-N
Target: Xanthine dehydrogenase/oxidase (SINGLE PROTEIN), Homo sapiens
UniProt: P47989
Assay: xanthine oxidase activity assay: Xanthine oxidase inhibition was determined using a standard fluorescence-based assay for xanthine oxidase activity (McHale A, Grimes H, Coughlan M P: Int J Biochem. 10:317-9, 1979) with minor variations. The procedure was internally standardized using allopurinol and DPI as controls for all experiments after determination of their optimal inhibitory concentrations. Experiments on test compounds were performed in triplicate in multi-well plates using 10 concentrations of each compound that ranged over a 3-fold dilution.
Measurement: IC50 = 0.2 nM (pChEMBL 9.699999809265137)
Descriptors: MW 359.37, ALogP 1.02, HBA 8, HBD 4, PSA 149.54, QED 0.39
Compound (SMILES): CN(C)S(=O)(=O)c1ccc(Nc2nn(C3(CC#N)CCN(S(=O)(=O)C4CC4)CC3)c3cc[nH]c(=O)c23)cc1
InChIKey: LEPDKKSIRCQPDJ-UHFFFAOYSA-N
Target: Tyrosine-protein kinase JAK2 (SINGLE PROTEIN), Homo sapiens
UniProt: O60674
Assay: Biochemical HTRF Assay : The ability of compounds to inhibit the activity of JAK1, JAK2, JAK3, and Tyk2 was measured using a recombinant purified GST-tagged catalytic domain for each enzyme (Invitrogen JAK1 #M4290, JAK2 #M4290, JAK3 #M4290, Tyk2 #M4290) in an HTRF format biochemical assay. The reactions employed a common peptide substrate, LCB-EQEDEPEGDYFEWLW-NH2 (in-house). The basic assay protocol is as follows: First, 250 nL of diluted compounds in DMSO were dispensed into the wells of a dry 384-well Black plate (Greiner #781076) using a Labcyte Echo 555 acoustic dispenser. Subsequent reagent additions employed an Agilent Bravo. Next, 18 μL of 1.11× enzyme and 1.11× substrate in 1× assay buffer (Invitrogen kinase buffer #PV3189, 2 mM DTT, 0.05% BSA) were added to the wells and shaken and then preincubated for 30 minutes at room temperature to allow compound binding to equilibrate. After equilibration, 2 μL of 10×ATP in 1× assay buffer was added to initiate the kinase reaction and the plates were shaken and then incubated at room temperature for 120 minutes. At the end of the incubation, 20 μL of 2× stop buffer (streptavidin-Dylight 650 (Thermo #84547B/100 mL), Eu-tagged pY20 antibody (Perkin Elmer #AD0067), EDTA, HEPES, and Triton) was added to quench the reaction. Plates were shaken and centrifuged and then incubated 60 minutes at room temperature and then read on a Perkin Elmer Envision (λex=337 nm, λem=665 and 615 nm, TRF delay time=20 μs). HTRF signal=10,000*665 nm reading/615 nm reading.
Measurement: IC50 = 0.12 nM (pChEMBL 9.920000076293945)
Descriptors: MW 559.67, ALogP 1.92, HBA 9, HBD 2, PSA 161.26, QED 0.42
Compound (SMILES): Cc1ncc(CC(=O)Nc2cc([C@@H]3CC[C@H](OC(=O)N[C@@H]4CCOC[C@H]4C)C3)[nH]n2)s1
InChIKey: SNGPLWPDIQUJME-JJAZEVLHSA-N
Target: Glycogen synthase kinase-3 beta (SINGLE PROTEIN), Homo sapiens
UniProt: P49841
Assay: Biochemical Assay: The purpose of GSK3β assay is to evaluate the inhibition (% inhibition, Kiapp and Ki values) of small molecule inhibitors by using a fluorescence-based microfluidic mobility shift assay. GSK3β catalyzes the production of ADP from ATP that accompanies the phosphoryl transfer to the substrate peptide FL-Peptide-15 (5-FAM-KRREILSRRPpSYR-COOH, CPC Scientific, Sunnyvale, Calif.) (SEQ ID NO:2). The mobility shift assay electrophoretically separates the fluorescently labeled peptides (substrate and phosphorylated product) following the kinase reaction. Both substrate and product are measured and the ratio of these values is used to generate % conversion of substrate to product by the LabChip EZ Reader. Active GSK3β (H350L) was purchased from Upstate/Millipore. Typical reaction solutions (50 μL final reaction volume) contained 2% DMSO (±inhibitor), 4 mM MgCl2, 1 mM DTT, 40 μM ATP (ATP Km=9.43 μM), 0.005% Tween-20, 2 μM FL-Peptide-15, and 0.6 nM GSK3β in 25 mM HEPES buffer at pH 7.5. The assay was initiated with the addition of ATP, following 15 minutes pre-incubation of enzyme and inhibitor at room temperature in the reaction mixture. The reaction was stopped after 30 minutes at room temperature by the addition of 50 μL of 80 mM EDTA. The Ki value was determined from the fit of the data to the Morrison tight-binding competitive inhibition equation with the enzyme concentration as a variable.
Measurement: Ki = 14 nM (pChEMBL 7.849999904632568)
Descriptors: MW 447.56, ALogP 3.14, HBA 7, HBD 3, PSA 118.23, QED 0.63
Compound (SMILES): Nc1ccc(-c2ccc(F)cc2)nc1NC(=O)c1ccc(N2CCOCC2)nc1
InChIKey: GFIVYKYFGAHRTQ-UHFFFAOYSA-N
Target: Histone deacetylase 2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q92769
Assay: Histone Deacetylase Assay: The inhibitory activities of compounds of present invention were determined using biochemical HDAC assays (Reaction Biology Corp. biochemical assay services). Compound with indicated doses was tested in the biochemical assays of HDAC 1, HDAC2, HDAC3, HDAC4, HDAC5, HDAC6, HDAC7, HDAC 8, HDAC9, HDAC10, and HDAC11 enzyme.Compounds were tested in singlicate 10-dose IC50 mode with 3-fold serial dilution starting at 10 μM against 11 HDACs. HDAC reference compounds Trichostatin A (TSA) and TMP269 were tested in a 10-dose IC50 with 3-fold serial dilution starting at 10 μM.Substrate for HDAC1,2,3,6,10: Fluorogenic peptide from p53 residues 379-382 (RHKK(Ac)AMC). Substrate for HDAC4,5,7,9, and 11: Fluorogenic HDAC Class2a Substrate (Trifluoroacetyl Lysine). Substrate for HDAC 8: Fluorogenic peptide from p53 residues 379-382 (RHK(Ac)K(Ac)AMC).General Reaction Procedure: (Standard IC50 determination)    a. 2× enzyme was added to wells of reaction plate except to No Enzyme (No En) control wells. Add buffer in No En wells.    b. Compounds to be tested in 100% DMSO were added to the enzyme mixture by Acoustic technology (Echo550; nanoliter range). The mixture was spinned down and preincubated.    c. 2× Substrate Mixture (Fluorogenic HDAC Substrate and co-factor (500 μM of Nicotinamide adenine dinucleotide (NAD<+>) in all Sirt assay) were added to all reaction wells to initiate the reaction. The plates were spinned and shaken.    d. The plates were incubated for 1-2 hr. at 30° C. with seal.    e. Developer with Trichostatin A (or TMP269 or NAD<+>) was added to stop the reaction and to generate fluorescent color.    f. Fluorescence was read (excitatory, 360; emission, 460) using the EnVision Multilabel Plate Reader (Perkin Elmer)    g. Endpoint reading was taken for analysis after the development reaches plateau.Data Analysis: The percentages of enzyme activity (relative to DMSO controls) and IC50 values were calculated using the GraphPad Prism 4 program based on a sigmoidal dose-response equation. The blank (DMSO) value was entered as 1.00E-12 of concentration for curve fitting.
Measurement: IC50 < 200 nM (pChEMBL 6.698999881744385)
Descriptors: MW 393.42, ALogP 2.95, HBA 6, HBD 2, PSA 93.37, QED 0.71
Compound (SMILES): COc1ccc(CN2C[C@]3(CC[C@](c4ccccc4)(N(C)C)CC3)N(Cc3ccncn3)C2=O)cc1
InChIKey: MMQCILBZQQKEQC-ISILISOKSA-N
Target: Nociceptin receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P41146
Assay: Receptor Binding Assay: The hNOP receptor binding assay was performed as homogeneous SPA-assay (scintillation proximity assay) using the assay buffer 50 mM TRIS-HCl. 10 mM MgCl2. 1 mM EDTA (pH 7.4). The final assay volume (250 ul/well) included 0.5 nM of [leucyl-3H]nociceptin as ligand (PerkinElmer Life Sciences. Inc. Boston. Mass. USA). and either test compound in dilution series or 1 uM unlabelled nociceptin for determination of unspecific binding. The test compound was diluted with 25% DMSO in H2O to yield a final 0.5% DMSO concentration. which also served as a respective vehicle control. The assay was started by adding wheat germ agglutinin coated SPA beads (GE Healthcare UK Ltd. Buckinghamshire. UK) which had been preloaded with hMOP receptor membranes (PerkinElmer Life Sciences. Inc. Boston. Mass. USA). After incubation for 60 minutes at RT and centrifugation for 20 minutes at 500 rpm the signal rate was measured by means of a 1450 Microbeta Trilux beta-counter.
Measurement: Ki = 5135 nM (pChEMBL 5.289999961853027)
Descriptors: MW 485.63, ALogP 4.69, HBA 5, HBD 0, PSA 61.8, QED 0.49
Compound (SMILES): Cn1cc2c(Cl)c(-c3n[nH]c4nc(N5[C@H]6CC[C@@H]5[C@@H](F)[C@@H](N)C6)c(CO)nc34)ccc2n1
InChIKey: OYCTYGRYBSMUGZ-LKBDIMQLSA-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 = 9.1 nM (pChEMBL 8.039999961853027)
Descriptors: MW 456.91, ALogP 2.46, HBA 8, HBD 3, PSA 121.77, QED 0.43