lovingscience/chembl-v37-raw-activities huggingface dataset:
Compound (SMILES): COC(=O)C1(c2cn(C3CCCCC3)cn2)CC1CCCN(C(=O)OC(C)(C)C)C(=O)OC(C)(C)C
InChIKey: QEDCJYLUJWBGTA-UHFFFAOYSA-N
Target: Carboxypeptidase B2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q96IY4
Assay: Enzyme Inhibitory Assay: HEPES buffered saline (20 mM HEPES, 150 mM NaCl, pH 7.4; hereinafter, referred to as HBS) was used in the preparation of a reaction solution. To 12 μL of a 250 μg/mL TAFI solution, 30 μL of an HBS solution containing 4 U/mL human thrombin, 12 U/mL rabbit lung thrombomodulin, and 12 mM CaCl2 was added, and the mixture was gently stirred. Then, TAFI was activated at room temperature. Ten minutes later, thrombin was neutralized by the addition of 10 μL of 100 μM PPACK (thrombin inhibitor) to terminate the activation of TAFI. The formed TAFIa was stored in ice and diluted immediately before use in determination with 2050 μL of an HBS solution containing BSA (bovine serum albumin) adjusted to 0.1% in terms of the final concentration. A test substance was dissolved in HBS to prepare a 10-fold dilution series of evaluation concentrations. 80 μL of the TAFIa solution and 10 μL of the test substance were added to each well of a 96-well plate and mixed by shaking for 10 minutes. 10 μL of furylacryloyl-alanyl-lysine (FAAK) adjusted to 5 mg/mL was added to each well, and the change in the absorbance of this mixed solution at 330 nm was read for 30 minutes to determine the degradation rate of the substrate.
Measurement: IC50 = 29 nM (pChEMBL 7.539999961853027)
Descriptors: MW 505.66, ALogP 5.77, HBA 8, HBD 0, PSA 99.96, QED 0.34
Compound (SMILES): COc1cccc(-n2cc(Cn3c(=O)c(O)c(C(=O)O)c4sccc43)cn2)c1
InChIKey: PKRQEKXXTOBANY-UHFFFAOYSA-N
Target: Flap endonuclease 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P39748
Assay: Enzymatic IC50 Assays: FEN1, EXO1 or XPG enzyme was incubated with compound or vehicle (DMSO) and the FAM-labeled DNA oligomer substrate in a microtiter plate. The stop buffer contains EDTA to stop the enzymatic reaction. The plate is read for fluorescence intensity. The high control (DMSO) with high fluorescence intensity represents no inhibition of enzymatic reaction while the low control (10 μM) with low fluorescence intensity represents full inhibition of enzymatic reaction.
Measurement: IC50 < 200 nM (pChEMBL 6.698999881744385)
Descriptors: MW 397.41, ALogP 2.71, HBA 8, HBD 2, PSA 106.58, QED 0.54
Compound (SMILES): CN1CCC(N(C(=O)c2cccs2)c2ccn(-c3cccnc3)n2)CC1
InChIKey: QDYDLZNPLZJWOY-UHFFFAOYSA-N
Target: Mu-type opioid receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P35372
Assay: Human mu-Opioid Receptor Radioligand Assay: To investigate binding properties of test compounds to human μ-opioid receptor, transfected CHO-K1 cell membranes and [3H]-DAMGO (Perkin Elmer, ES-542-C), as the radioligand, were used. The assay was carried out with 20 μg of membrane suspension, 1 nM of [3H]-DAMGO in either absence or presence of either buffer or 10 μM Naloxone for total and non-specific binding, respectively. Binding buffer contained Tris-HCl 50 mM, MgCl2 5 mM at pH 7.4. Plates were incubated at 27° C. for 60 minutes. After the incubation period, the reaction mix was then transferred to MultiScreen HTS, FC plates (Millipore), filtered and plates were washed 3 times with ice-cold 10 mM Tris-HCL (pH 7.4). Filters were dried and counted at approximately 40% efficiency in a MicroBeta scintillation counter (Perkin-Elmer) using EcoScint liquid scintillation cocktail.
Measurement: Ki > 500 nM (pChEMBL 6.301000118255615)
Descriptors: MW 367.48, ALogP 3.07, HBA 6, HBD 0, PSA 54.26, QED 0.71
Compound (SMILES): COCCC(=O)N1CCN(c2nc(C3CC3)c3c(c2C#N)CC(C)(C)OC3)C[C@H]1C
InChIKey: PQXMHIOYPWGJDU-OAHLLOKOSA-N
Target: Isocitrate dehydrogenase [NADP] cytoplasmic (SINGLE PROTEIN), Homo sapiens
UniProt: O75874
Assay: Inhibition Assay: Assays were conducted in a volume of 76 μl assay buffer (150 mM NaCl, 10 mM MgCl2, 20 mM Tris pH 7.5, 0.03% bovine serum albumin) as follows in a standard 384-well plate: To 25 ul of substrate mix (8 uM NADPH, 2 mM aKG), 1 μl of test compound was added in DMSO. The plate was centrifuged briefly, and then 25 μl of enzyme mix was added (0.2 μg/ml IDH1 R132H) followed by a brief centrifugation and shake at 100 RPM. The reaction was incubated for 50 minutes at room temperature, then 25 μl of detection mix (30 μM resazurin, 36 μg/ml) was added and the mixture further incubated for 5 minutes at room temperature. The conversion of resazurin to resorufin was detected by fluorescent spectroscopy at Ex544 Em590 c/o 590.
Measurement: IC50 = 750 nM (pChEMBL 6.119999885559082)
Descriptors: MW 412.53, ALogP 2.76, HBA 6, HBD 0, PSA 78.69, QED 0.74
Compound (SMILES): CN1C(=O)/C(=C\Nc2cccc3c2CN(C2CCC(=O)NC2=O)C3=O)C(=O)c2ccc(C(F)(F)F)cc21
InChIKey: ZNTSVCOHIRFREE-GDNBJRDFSA-N
Target: Microtubule-associated protein 1 light chain 3 beta (SINGLE PROTEIN), Homo sapiens
UniProt: Q9GZQ8
Assay: Assay Targeting LC3B: By constructing a prokaryotic expression system, the LC3B protein was successfully expressed and purified, and a preliminary screening and verification platform was established using fluorescence polarization experiments to determine the activity of purchased and synthesized small compound libraries. The recombinant protein GST-LC3B (final concentration 180 nM) (SEQ ID NO: 1) and N-terminal FITC-labeled peptide (SEQ ID NO: 2, final concentration 18 nM) were placed in the FP buffer (50 mM HEPES pH 7.5, 0.1 mg/ml BSA and 1 mM DTT), to which a compound serially diluted with the FP buffer was added. Then the mixture was incubated at 25° C. in the dark. The fluorescence polarization value (PerkinElmer Envision, emission wavelength 480 nm; absorption wavelength 535 nm) was monitored, and the IC50 value was calculated using the GraphPad Prism 6.0 program.
Measurement: IC50 = 750 nM (pChEMBL 6.119999885559082)
Descriptors: MW 512.44, ALogP 2.62, HBA 6, HBD 2, PSA 115.89, QED 0.37
Compound (SMILES): CC(Nc1cc2cc(C#N)ccc2cn1)C1CCN(C#N)C1
InChIKey: YBGZQHXEUGOVCC-UHFFFAOYSA-N
Target: Ubiquitin carboxyl-terminal hydrolase 30 (SINGLE PROTEIN), Homo sapiens
UniProt: Q70CQ3
Assay: Biochemical Assay: Dilution plates were prepared at 21 times the final concentration (2100 μM for a final concentration of 100 μM) in 50% DMSO in a 96-well polypropylene V-bottom plate (Greiner #651201). A typical 8-point dilution series to be 100, 30, 10, 3, 1, 0.3, 0.1, 0.03 μM final. Reactions were performed in duplicate in black 384 well plates (small volume, Greiner 784076) in a final reaction volume of 21 μl. Either 1 μl of 50% DMSO or diluted compound was added to the plate. USP30 was diluted in reaction buffer (40 mM Tris, pH 7.5, 0.005% Tween 20, 0.5 mg/ml BSA, 5 mM beta-mercaptoethanol) to the equivalent of 0.05 μl/well and 10 μl of diluted USP30 was added to the compound. Enzyme and compound were incubated for 30 min at room temp. Reactions were initiated by the addition of 50 nM of TAMRA labelled peptide linked to ubiquitin via an iso-peptide bond as fluorescence polarisation substrate. Reactions were read immediately after addition of substrate and following a 2 hr incubation at room temperature. Readings were performed on a Pherastar Plus (BMG Labtech). λ Excitation 540 nm; λ Emission 590 nm.
Measurement: IC50 = 5500 nM (pChEMBL 5.260000228881836)
Descriptors: MW 291.36, ALogP 2.71, HBA 5, HBD 1, PSA 75.74, QED 0.88
Compound (SMILES): CCOc1nc(N)c2nc(-c3cncc(F)c3)n(Cc3cccc(OCCN4CCCC4)c3)c2n1
InChIKey: DOUMIVMNFQNAKM-UHFFFAOYSA-N
Target: Toll-like receptor 7 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9NYK1
Assay: Inhibition of the Activation Assay: As human TLR7 expressing cell line, HEK293 cell line was bought from IMGENEX Corporation (TLR7/NF-ηB/SEAPorter HEK293 cell), which is human embryonic kidney cell line and stably expresses full-length human TLR7 gene and secreted alkaline phosphatase (SEAP) reporter gene under the transcriptional control of an NE-ηB response element. The TLR7/NF-ηB/SEAPorter HEK293 cell was cultivated with DMEM containing 10% fetal bovine serum (FBS) and 10 μg/mL blasticidin S at 37° C. in the presence of 5% CO2. The TLR7/NF-ηB/SEAPorter HEK293 cell was seeded into 96-well microtiter plate at 5×104 cell/90 μL/well, and the place was cultivated at 37° C. in a CO2 incubator overnight. Each test compound that was diluted with the medium was added to the wells (10 μL/well), wherein each final concentration was adjusted to 0.001, 0.003, 0.01, 0.03, 0.1, 0.3, 1, 3, and 10 μmol/L, or 0.01, 0.03, 0.1, 0.3, 1, 3, 10, 30, and 100 μmol/L. After 0.5 hour, R-848 that is TLR7/8 ligand was added to each well (10 μL/well), wherein each final concentration was adjusted to 200 nmol/L. The total volume was adjusted to 110 μL/well, the test samples were incubated in CO2 incubator for 20±1 hours, and then the SEAP activity was measured as activation of TLR7. The SEAP activity was evaluated as follows: p-nitro-phenyl phosphate (pNPP) (Invitrogen) was added to the incubated sample (50 μL/well); after 15 minutes, 4 mol/L sodium hydroxide solution (nacalai tesque) was added thereto (50 μL/well) to quench the reaction; and the absorbance of each sample was measured at 405 nm with microplate reader Elx808 (BioTek). The 50% inhibitory concentration (IC50 value) of each sample compound was calculated based on 100% of the SEAP activity wherein the test sample comprises no sample compound.
Measurement: IC50 = 59 nM (pChEMBL 7.230000019073486)
Descriptors: MW 477.54, ALogP 3.53, HBA 9, HBD 1, PSA 104.21, QED 0.39
Compound (SMILES): Cc1noc(C)c1-c1ccc2nc(NC(C)C)n3c2c1OCC3c1ccccc1
InChIKey: QJJHFRGVSUVABK-UHFFFAOYSA-N
Target: Bromodomain-containing protein 4 (SINGLE PROTEIN), Homo sapiens
UniProt: O60885
Assay: AlphaScreen Assay: BRD4-BD1 and BRD4-BD2 assays were conducted in white 384-well polystyrene plate in a final volume of 20 μL for BD1 and 40 μL for BD2. Inhibitors were first serially diluted in DMSO and added to the plate wells before the addition of other reaction components. The final concentration of DMSO in the assay was 1.25% (BD1) and 0.83% (BD2). The assays were carried out at room temperature for 75 min. in the assay buffer (50 mM HEPES, pH 7.4, 100 mM NaCl, 0.05% CHAPS, 0.01% BSA), containing 50 nM Biotin-labeled tetra-acetylated histone H4 peptide (H4Ac4), 3.8 nM (BRD4-BD1, BPS Bioscience #31040) or 20 nM (BRD4-BD2, BPS Bioscience #31041). The reaction followed by the addition of 20 μL of assay buffer supplemented with Streptavidin donor beads (PerkinElmer 6760002) and GSH Acceptor beads (PerkinElmer-AL109C) at 4 μg/mL under reduced light. After plate sealing, the plate was incubated in the dark at room temperature for 75 min. before reading on a PHERAstar FS plate reader (BMG Labtech).
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 388.47, ALogP 5.11, HBA 6, HBD 1, PSA 65.11, QED 0.53
Compound (SMILES): C[C@@H]1OC(=O)NN=C1c1ccc(-c2ccc(F)c(F)c2F)c(C(F)(F)F)c1
InChIKey: KQIHCUMWNXFYMS-ZETCQYMHSA-N
Target: cGMP-inhibited 3',5'-cyclic phosphodiesterase 3A (SINGLE PROTEIN), Homo sapiens
UniProt: Q14432
Assay: PDE3A Enzyme Inhibition: The commercially available 3H-cAMP Scintillation Proximity Assay (SPA, Perkin Elmer) system was used for enzyme inhibition studies. For the determination of the in vitro effect of example compounds on the PDE3A reactions 2 μl of the respective example compound solution in DMSO (serial dilutions) were placed in wells of microtiter plates (Isoplate-96/200W; Perkin Elmer). 50 μl of a dilution of PDE3A cell extract from Sf9 cells overexpressing human full length PDE3A (SB Drug Discovery, UK) in buffer A (50 mM Tris/HCl pH 7.5, 8.3 mM MgCl2, 1.7 mM EDTA, 0.2% BSA) was added. The dilution of the PDE3A cell extract was chosen such that the reaction kinetics was linear and less than 70% of the substrate was consumed (typical dilution 1:5000). The reaction was started by addition of 50 μl (0.025 μCi) of 1:2000 in buffer A w/o BSA diluted substrate [8-3H]adenosine 3′, 5′-cyclic phosphate (1 μCi/μl; Perkin Elmer). After incubation at room temperature for 60 min, the reaction was stopped by addition of 25 μl of a suspension containing 18 mg/ml yttrium scintillation proximity beads (Perkin Elmer) in water. The microtiter plates were sealed and measured in a Microbeta scintillation counter (PerkinElmer Wallac). IC50 values were determined from sigmoidal curves by plotting percentage PDE3A activity vs log compound concentration.
Measurement: IC50 = 190 nM (pChEMBL 6.71999979019165)
Descriptors: MW 388.27, ALogP 4.62, HBA 3, HBD 1, PSA 50.69, QED 0.6
Compound (SMILES): C=CC(=O)N1C[C@H](C)N(c2nc(=O)n(-c3c(C)ccnc3C(C)C)c3nc(-c4c(O)cccc4F)c(F)cc23)C[C@H]1C
InChIKey: FWLXQPHYLWQAMQ-MOPGFXCFSA-N
Target: GTPase KRas (SINGLE PROTEIN), Homo sapiens
UniProt: P01116
Assay: Biochemical Assay: The following assay conditions were employed: Coupled Nucleotide Exchange Assay: Purified GDP-bound KRAS protein (aa 1-169), containing both G12C and C118A amino acid substitutions and an N-terminal His-tag, was pre-incubated with a compound dose-response titration for 5 min in assay buffer (25 mM HEPES pH 7.4, 10 mM MgCl2, and 0.01% Triton X-100). Following compound pre-incubation, purified SOS protein (aa 564-1049) and GTP (Roche 10106399001) were added to the assay wells and incubated for an additional 30 min. To determine the extent of inhibition of SOS-mediated nucleotide exchange, purified GST-tagged cRAF (aa 1-149), nickel chelate AlphaLISA acceptor beads (PerkinElmer AL108R), and AlphaScreen glutathione donor beads (PerkinElmer 6765302) were added to the assay wells and incubated for 5 minutes. The assay plates were then read on a PerkinElmer EnVision Multilabel Reader, using AlphaScreen® technology, and data were analyzed using a 4-parameter logistic model to calculate IC50 values.
Measurement: IC50 = 79 nM (pChEMBL 7.099999904632568)
Descriptors: MW 574.63, ALogP 4.87, HBA 8, HBD 1, PSA 104.45, QED 0.34
Compound (SMILES): Cc1ccc2cc(-c3nc(C)no3)c(N3CC[C@@H](NC4CCCCC4)[C@H](O)C3)nc2c1
InChIKey: IADRKEOEFZWGJA-IFMALSPDSA-N
Target: Kappa-type opioid receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P41145
Assay: OPRK1 antagonist assay: The cell line for the OPRK1 antagonist assay stably expresses the following elements. The carboxy terminus of the OPRK1 receptor has a 7 amino acid linker, followed by the TEV protease cleavage site and a GAL4-VP16 fusion protein. The cell line also expresses a b-arrestin-2-TEV protease fusion protein and contains a reporter construct consisting of the UAS response element and the b-lactamase (bla) reporter gene. Upon activation of the receptor, g-protein receptor kinase (GRK) phosphorylates specific intracellular residues of OPKR1 and this induces recruitment of B-arrestin2-TEV protease fusion protein. The TEV protease recognizes and cleaves the TEV site, releasing the GAL4-VP16 fusion protein, which then translocates to the nucleus. The GAL4-V16 binds to the UAS element, driving expressing of the b-lactamase gene. B-lactamase expression is detected with the cell permeable, fluorescent substrate, CCF4-AM. This substrate consists of coumarin tethered to fluoroscein via a b-lactam ring. In the absence of b-lactamase, excitation of the dye with 405 nm light results in FRET from the coumarin to fluoroscein and emission of green (525 nm maximum) light. B-lactamase cleavage of the substrate separates the courmarin fluorophore from the fluorscein, and 405 nm excitation results in blue (460 nm maximum) emission. The assay is monitored by the blue/green emission ratio. The antagonist assay is performed by seeding the cells into 384 well plates and incubating them 16-24 hours at 37° C. Test antagonist compounds are added and incubated for 30 minutes at 37° C. Next an EC80 challenge of U-50488 (OKRK1 agonist) is added and the samples are incubated for 4 hours at 37° C., followed by addition of CCF4-AM substrate. The plates are then incubated 2 hours at room temperature in the dark and the blue/green ratio determined on a fluorescent plate reader. See J Biomol Screen April 2009, vol. 14 no. 4, pp 381-394.
Measurement: IC50 = 7 nM (pChEMBL 8.149999618530273)
Descriptors: MW 421.55, ALogP 3.76, HBA 7, HBD 2, PSA 87.31, QED 0.66
Compound (SMILES): CC(C)(Cn1c(=O)[nH]/c(=N\c2ccc(Oc3cc(F)nc(F)c3)cc2)n(Cc2ccc(Cl)cc2)c1=O)C(=O)O
InChIKey: BDVTWUFYGYQEGN-UHFFFAOYSA-N
Target: P2X purinoceptor 3 (SINGLE PROTEIN), Homo sapiens
UniProt: P56373
Assay: Receptor Inhibition Assay: Stably expressing cell line (C6BU-1 cell transfected with human P2X3 receptor gene (GenBank accession number Y07683) was used. The cells were seeded in a 384-well PDL-coated microtiter plate at a concentration of 3000 cells/well and cultured in the medium (8.3% fetal bovine serum, 8.3% horse serum, and 1% antibiotic and antifungal in DMEM) for one day at 37° C. under 5% carbon dioxide atmosphere. The medium was replaced with 4 μM Fluo-3-AM solution (20 mM HEPES, 137 mM NaCl, 2.7 mM KCl, 0.9 mM MgCl2, 5.0 mM CaCl2, 5.6 mM D-glucose, 2.5 mM probenecid, 0.5% BSA, and 0.04% Pluronic P-127, pH 7.5) and incubated at 37° C. under 5% dioxide carbon atmosphere for one hour. The plate was washed with washing buffer (20 mM HEPES, 137 mM NaCl, 2.7 mM KCl, 0.9 mM MgCl2, 5.0 mM CaCl2, 5.6 mM D-glucose, 2.5 mM probenecid, pH 7.5), and each well was added with 20 μL of the washing buffer. The plate was placed in High-Throughput Screening System FLIPR 384 (Molecular Device Co.). Measurement of fluorescence intensity by FLIPR 884 was started, and 20 μL of DMSO solutions containing different concentrations of the test compound as prepared by dilution with dilution buffer (20 mM HEPES, 137 mM NaCl, 2.7 mM KCl, 0.9 mM MgCl2, 5.0 mM CaCl2, 5.6 mM D-glucose, 2.5 mM probenecid, 0.1% Pluronic F-127, pH 7.5) were dispensed to each well through the built-in automatic dispenser. Five minutes after, 150 nM ATP solution (25 μL) prepared by dilution with the dilution buffer was dispensed through the built-in automatic dispenser, and the measurement of fluorescence intensity was continued for 4 min. For each well, the specific maximum fluorescence intensity was calculated as the ratio of the maximum fluorescence intensity after addition of the ATP solution to the fluorescence intensity at the starting of the measurement.
Measurement: IC50 = 7 nM (pChEMBL 8.149999618530273)
Descriptors: MW 557.94, ALogP 3.85, HBA 8, HBD 2, PSA 131.57, QED 0.32
Compound (SMILES): CCc1cccc(C(=O)NC2(C)CCN(c3ccc(-c4cc(OCC(C)(C)O)cn5ncc(C#N)c45)cn3)CC2)n1
InChIKey: ZAWMCTQAZKCKMV-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 = 61.3 nM (pChEMBL 7.210000038146973)
Descriptors: MW 553.67, ALogP 4.16, HBA 9, HBD 2, PSA 128.67, QED 0.33
Compound (SMILES): CCC(c1ccc2c(c1)OC(F)(F)O2)N1C[C@H](C)N(c2nc(=O)n(C)c3ccc(C#N)nc23)C[C@H]1CC
InChIKey: CALZFCOZSYEZAX-APBAKZIRSA-N
Target: Diacylglycerol kinase alpha (SINGLE PROTEIN), Homo sapiens
UniProt: P23743
Assay: In Vitro DGK Inhibition Assay: The reactions were carried out in 50 mM MOPS pH 7.5, 100 mM NaCl, 10 mM MgCl2, 1 μM CaCl2, and 1 mM DTT (assay buffer). The reactions using a detergent/lipid micelle substrate also contained 50 mM octyl B-D-glucopyranoside. The lipid substrate concentrations were 11 mM PS and 1 mM DAG for the detergent/lipid micelle reactions. The lipid substrate concentrations were 2 mM PS, 0.25 mM DAG, and 2.75 mM PC for the extruded liposome reactions (5 mM total lipid). The reactions were carried out in 150 μM ATP. The enzyme concentrations for the DGKα and DGKζ were 5 nM. The compound inhibition studies were carried out as follows: 25 nL (ADPGLO assay) or 50 nL (LIPGLO assay) droplets of each test compound (top concentration 10 mM with 11 point, 3-fold dilution series for each compound) solubilized in DMSO were transferred to wells of a white 1536 well plate (Corning 3725). A 5 mL enzyme/lipid substrate solution at 2× final reaction concentration was prepared by combining 2.5 mL 4× enzyme solution (20 nM DGKα or DGKζ (prepared as described below) in assay buffer) and 2.5 mL of either 4× liposome or 4× detergent/lipid micelle solution (compositions described below) and incubated at room temperature for 10 minutes. Next, 1 μL 2× enzyme/lipid substrate solution was added to wells containing the test compound and reactions were initiated with the addition of 1 μL 300 uM ATP. The reactions were allowed to proceed for 2 hr (ADPGLO assay) or 1 hr (LIPGLO assay), after which 2 μL Glo Reagent (Promega V9101) was added and incubated for 40 minutes. Next, 4 μL Kinase Detection Reagent was added and incubated for 30 minutes. Luminescence was recorded using an EnVision microplate reader. The percent inhibition was calculated from the ATP conversion generated by no enzyme control reactions for 100% inhibition and vehicle-only reactions for 0% inhibition.
Measurement: IC50 = 300 nM (pChEMBL 6.519999980926514)
Descriptors: MW 510.55, ALogP 3.96, HBA 9, HBD 0, PSA 96.51, QED 0.51
Compound (SMILES): Cc1ccc(OCc2ccc(C(=O)N3[C@@H](c4ccccc4Cl)CC[C@H]3C(=O)O)cc2)cc1
InChIKey: DGFGZMOZZVAKSP-RPWUZVMVSA-N
Target: Free fatty acid receptor 2 (SINGLE PROTEIN), Homo sapiens
UniProt: O15552
Assay: BINDING ASSAY: The following assay can be used for determination of GPR43 activation. When a GPCR is in its active state, either as a result of ligand binding or constitutive activation, the receptor couples to a G protein and stimulates the release of GDP and subsequent binding of GTP to the G protein. The alpha subunit of the G protein-receptor complex acts as a GTPase and slowly hydrolyses the GTP to GDP, at which point the receptor normally is deactivated. Activated receptors continue to exchange GDP for GTP. The non-hydrolysable GTP analog, [35S]GTPγS, was used to demonstrate enhance binding of [35S]GTPγS to membranes expressing receptors. The assay uses the ability of GPCR to stimulate [35S]GTPγS binding to membranes expressing the relevant receptors. The assay can, therefore, be used in the direct identification method to screen candidate compounds to endogenous or not endogenous GPCR.
Measurement: EC50 = 600 nM (pChEMBL 6.21999979019165)
Descriptors: MW 449.93, ALogP 5.66, HBA 3, HBD 1, PSA 66.84, QED 0.53
Compound (SMILES): Cn1nc2c(c1-c1cc(F)c(F)c(F)c1)C[C@@H]1CCC[C@H]2N1C(=O)c1ccnnc1
InChIKey: BKVSYYAESRWHBT-SUMWQHHRSA-N
Target: Monoglyceride lipase (SINGLE PROTEIN), Homo sapiens
UniProt: Q99685
Assay: In Vitro Activity Assay: The assay used to measure the in vitro activity of MGL is adapted from the assay used for another serine hydrolase (FAAH) described in Wilson et al., 2003 (A high-throughput-compatible assay for determining the activity of fatty acid amide hydrolase. Wilson S J, Lovenberg T W, Barbier A J. Anal Biochem. 2003 Jul. 15; 318(2):270-5). The assay consists of combining endogenously expressed MGL from HeLa cells with test compounds, adding [glycerol-1,3-3H]-oleoyl glycerol, incubating for one hour, and then measuring the amount of cleaved [1,3-3H]-glycerol that passes through an activated carbon filter. The amount of cleaved, tritiated glycerol passing through the carbon filter is proportional to the activity of the MGL enzyme in a particular well/test condition. Standard conditions for this assay combine 300 nM [Glycerol-1,3-3H]-oleoyl glycerol with human MGL from HeLa cells and test compounds for one hour, after which the reaction is filtered through activated carbon and tritium is measured in the flow through. The test compound concentration in screening mode is 10 uM, while the highest compound concentration in IC50 assays is determined empirically.
Measurement: IC50 = 3200 nM (pChEMBL 5.5)
Descriptors: MW 413.4, ALogP 3.59, HBA 5, HBD 0, PSA 63.91, QED 0.6
Compound (SMILES): CC1(C)Oc2ccc(Nc3ncc(F)c(N[C@@H]4C[C@@H]5CCCN5C(C)(C)C4)n3)cc2-n2c1nn(CCO)c2=O
InChIKey: LVUDWQSAIHLXRB-MSOLQXFVSA-N
Target: Interleukin-2 (SINGLE PROTEIN), Homo sapiens
UniProt: P60568
Assay: IL-2 ELISA Assay: Primary T cell stimulation and IL2 ELISA: Human primary T cells (100,000 cells per well) were pre-incubated with or without test compound in Yssel's medium for 1 hour at 37° C. Cells were then stimulated by transferring them to round-bottom 96-well plates pre-coated with 1 μm/ml αCD3 and 5 μg/ml αCD28. For counter assay, cells were instead stimulated by adding 8× stock solutions of PMA and ionomycin in Yssels (for final concentrations of 0.5 ng/ml PMA and 0.1 uM ionomycin, both from Calbiochem). Cells were incubated at 37° C. for 24 hours before 100 μL supernatants were harvested for quantification of IL-2 by ELISA using Human IL-2 Duoset ELISA Kit from R and D Systems, Cat. # DY202E.
Measurement: EC50 < 10000 nM (pChEMBL 5)
Descriptors: MW 538.63, ALogP 3.14, HBA 11, HBD 3, PSA 122.36, QED 0.43
Compound (SMILES): COC(=O)N1Cc2cc(Cl)ccc2-n2c(nnc2C2CC3(C2)CN(c2cccnn2)C3)C1
InChIKey: VDVOGYKCOWEEGH-UHFFFAOYSA-N
Target: Vasopressin V1a receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P37288
Assay: Vasopressin V1A Receptor Antagonist Assay: The purpose of this assay was to determine the inhibitory effect of synthesized compounds on the Vasopressin V1a receptor. The assay as performed in Chinese Hamster Ovary (CHO) cells expressing the human Arginine Vasopressin Receptor 1a (AVPR1a). Arginine Vasopressin (AVP) evokes an increase in intracellular calcium in CHO-AVPR1a cells which is measured in a fluorescence assay on the FLIPRTETRA using calcium sensitive dyes. Test compounds were assessed for their ability to affect the magnitude of the response to AVP, with antagonists showing a concentration-dependent reduction in the AVP-mediated fluorescence. Compounds were tested in duplicate in a 10-point, 1:3 dilution series starting at a nominal concentration of 3 μM in the assay.CHO-AVPR1a cells were maintained in routine culture in T175 Flasks at 37° C., 5% CO2. The growth medium consists of Ham's F12 media supplemented with 10% v/v fetal bovine serum, 1× non-essential amino acids, and 0.4 mg/ml Geneticin G418.
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 451.92, ALogP 3.18, HBA 8, HBD 0, PSA 89.27, QED 0.59
Compound (SMILES): O=C(Cc1cc(Cl)ccc1O)Nc1ccnc(C(=O)N[C@@H]2CCC[C@@H]2O)c1
InChIKey: GAIOCIMFJMOPET-PBHICJAKSA-N
Target: Anoctamin-1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q5XXA6
Assay: Automated Whole-Cell Patch Clamp Assay to Detect TMEM16A Activity in Recombinant Cells: Compound activity was quantified by measuring the increase in current upon compound addition and expressing this as a percentage increase of baseline TMEM16A current level. Percentage increases in current were determined for each concentration and the data plotted as a function of concentration using either the Qpatch software or Graphpad Prism v6.05 providing the concentration which gave 50% of its maximal effect (EC50) and maximum efficacy (percentage of baseline increase). The method of calculating the results is illustrated in FIG. 1, which shows an example trace from the Qpatch TMEM16A assay. In FIG. 1, IBL equals baseline current, I[#1] equals the peak current during test compound concentration 1 incubation period and so on.
Measurement: EC50 = 430 nM (pChEMBL 6.369999885559082)
Descriptors: MW 389.84, ALogP 2.27, HBA 5, HBD 4, PSA 111.55, QED 0.63
Compound (SMILES): Cc1cnc([C@@H](C)[C@H](C)S(=O)(=O)Nc2nnc([C@@H]3CCCO3)n2-c2c(F)cccc2F)nc1
InChIKey: DODGIMOEWGLMRR-ZQIUZPCESA-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 = 120 nM (pChEMBL 6.920000076293945)
Descriptors: MW 478.53, ALogP 3.43, HBA 8, HBD 1, PSA 111.89, QED 0.55
Compound (SMILES): COC(=O)N1CCCc2cccc(CN3CCC4(CCN(C(=O)OC(C(F)(F)F)C(F)(F)F)CC4)C3)c21
InChIKey: VSQAFBUWSHMADR-UHFFFAOYSA-N
Target: Monoglyceride lipase (SINGLE PROTEIN), Mus musculus
UniProt: O35678
Assay: In Vitro Competitive Activity-Based Protein Profiling: Proteomes (mouse brain membrane fraction or cell lysates) (50 μL, 1.0 mg/mL total protein concentration) were preincubated with varying concentrations of inhibitors at 37° C. After 30 min, FP¿Rh (1.0 μL, 50 μM in DMSO) was added and the mixture was incubated for another 30 min at 37° C. Reactions were quenched with SDS loading buffer (50 μL-4×) and run on SDS-PAGE. Following gel imaging, serine hydrolase activity was determined by measuring fluorescent intensity of gel bands corresponding to MAGL, ABHD6 and FAAH using ImageJ 1.43u software. Data from this assay is shown in Table 2 (% inhibition at 1 μM).
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 537.5, ALogP 5.12, HBA 5, HBD 0, PSA 62.32, QED 0.5
Compound (SMILES): CCc1[nH]c(-c2cc(-c3cncc(N4CCOCC4)c3)ccn2)nc1C
InChIKey: ZROMYDRTXVSXQU-UHFFFAOYSA-N
Target: Phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit gamma isoform (SINGLE PROTEIN), Homo sapiens
UniProt: P48736
Assay: PI3K-γ Scintillation Proximity Assay: The kinase reaction was conducted in polystyrene 384-well Greiner Bio-one white plate from Thermo Fisher Scientific in a final volume of 25 μL. Inhibitors were first diluted serially in DMSO and added to the plate wells before the addition of other reaction components. The final concentration of DMSO in the assay was 2%. The PI3Kγ assay was carried out at room temperature in 20 mM MOPS, pH 6.7, 10 mM MgCl2, 5 mM DTT and CHAPS 0.03%. Reactions were initiated by the addition of ATP, the final reaction mixture consisted of 20 μM PIP2, 2 μM ATP, 0.5 μCi [γ-33P] ATP, 13 nM PI3Kγ. Reactions were incubated for 120 min and terminated by the addition of 40 μL SPA beads suspended in quench buffer: 163 mM potassium phosphate pH 7.8, 20% glycerol, 25 mM EDTA. The final concentration of SPA beads is 1.0 mg/mL. After the plate sealing, plates were shaken overnight at room temperature and centrifuged at 1500 rpm for 10 min, the radioactivity of the product was determined by scintillation counting on Topcount (Perkin-Elmer). IC50 determination was performed by fitting the curve of percent of the solvent control activity versus the log of the inhibitor concentration using the GraphPad Prism 6.0 software.
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 349.44, ALogP 3.24, HBA 5, HBD 1, PSA 66.93, QED 0.78
Compound (SMILES): COC(=O)C(C)(C)Cn1c(=O)[nH]/c(=N\c2ccc(O[C@H](C)C(F)(F)F)c(F)c2)n(Cc2ccc(Cl)cc2)c1=O
InChIKey: NGZKOTWCBLAIHM-CQSZACIVSA-N
Target: P2X purinoceptor 3 (SINGLE PROTEIN), Homo sapiens
UniProt: P56373
Assay: Receptor Inhibition Assay: Stably expressing cell line (C6BU-1 cell transfected with human P2X3 receptor gene (GenBank accession number Y07683) was used. The cells were seeded in a 384-well PDL-coated microtiter plate at a concentration of 3000 cells/well and cultured in the medium (8.3% fetal bovine serum, 8.3% horse serum, and 1% antibiotic and antifungal in DMEM) for one day at 37° C. under 5% carbon dioxide atmosphere. The medium was replaced with 4 μM Fluo-3-AM solution (20 mM HEPES, 137 mM NaCl, 2.7 mM KCl, 0.9 mM MgCl2, 5.0 mM CaCl2, 5.6 mM D-glucose, 2.5 mM probenecid, 0.5% BSA, and 0.04% Pluronic P-127, pH 7.5) and incubated at 37° C. under 5% dioxide carbon atmosphere for one hour. The plate was washed with washing buffer (20 mM HEPES, 137 mM NaCl, 2.7 mM KCl, 0.9 mM MgCl2, 5.0 mM CaCl2, 5.6 mM D-glucose, 2.5 mM probenecid, pH 7.5), and each well was added with 20 μL of the washing buffer. The plate was placed in High-Throughput Screening System FLIPR 384 (Molecular Device Co.). Measurement of fluorescence intensity by FLIPR 884 was started, and 20 μL of DMSO solutions containing different concentrations of the test compound as prepared by dilution with dilution buffer (20 mM HEPES, 137 mM NaCl, 2.7 mM KCl, 0.9 mM MgCl2, 5.0 mM CaCl2, 5.6 mM D-glucose, 2.5 mM probenecid, 0.1% Pluronic F-127, pH 7.5) were dispensed to each well through the built-in automatic dispenser. Five minutes after, 150 nM ATP solution (25 μL) prepared by dilution with the dilution buffer was dispensed through the built-in automatic dispenser, and the measurement of fluorescence intensity was continued for 4 min. For each well, the specific maximum fluorescence intensity was calculated as the ratio of the maximum fluorescence intensity after addition of the ATP solution to the fluorescence intensity at the starting of the measurement.
Measurement: IC50 = 108 nM (pChEMBL 6.96999979019165)
Descriptors: MW 572.94, ALogP 3.94, HBA 8, HBD 1, PSA 107.68, QED 0.32
Compound (SMILES): O=C(O)CCc1ccc(Nc2nc(N3CCCCCC3)nc3cn[nH]c(=O)c23)cc1
InChIKey: XZJCWAZZWYBTOV-UHFFFAOYSA-N
Target: Tyrosine-protein kinase SYK (SINGLE PROTEIN), Homo sapiens
UniProt: P43405
Assay: Inhibition of Enzymatic Syk Kinase Activity: 2.5 μL of 10% DMSO or compound in 10% DMSO was added to the wells in a 96 well V-bottom plate. Optimized concentration of in-house Syk enzyme (different batches of Syk (356-635) kinase domain) were used at optimized concentrations) ranging from 0.035 ng to 7.5 ng/reaction diluted in assay buffer was added to a total volume of 12.5 μL). Compound and protein were incubated for 30 minutes at room temperature on a plate shaker. Ten μL of a substrate mix containing 100 μM ATP (0.25 μL), γ-P32-ATP (0.1 μL; 10 μCi/μL), pG4T (0.25 μL; 10 mg/mL) and 1× assay buffer (9.4 μL) was added to all the wells. Samples were incubated at 30° C. for 10 minutes after mixing. The reaction was stopped by the addition of 8N HCl (13 μL) containing 100 mM ATP. Thirty μL of sample was transferred to the center of a 2×2 cm2 Whatman P81 chromatography paper. After allowing the sample to dry for one minute, the assay squares were washed 3 times for 5 minutes each in ortho-phosphoric acid (0.5%) and once in acetone. Assay squares were dried for 15 minutes in a 30° C. oven and transferred to 96 well optiplate. Microscint-O reagent (100 μL, Perkin Elmer) was added to each well, the plate was sealed with Topseal-A microplates and incubated for 10 minutes at room temperature at very low speed on rocker and the plate was read in the Topcount NXL instrument.
Measurement: IC50 < 10 nM (pChEMBL 8)
Descriptors: MW 408.46, ALogP 2.85, HBA 7, HBD 3, PSA 124.1, QED 0.57
Compound (SMILES): C[C@@H]1N=C(N2CCN(C(=O)[C@@H](CCCCNS(C)(=O)=O)NC3CCCCC3)[C@H](C(=O)NCc3cccs3)C2)O[C@H]1c1ccc(C#N)cc1
InChIKey: ABCHEZNYPFCFNO-QIXIFPRPSA-N
Target: Coagulation factor XII (SINGLE PROTEIN), Homo sapiens
UniProt: P00748
Assay: Factor XIIa Assay: The effectiveness of a compound of the present invention as an inhibitor of Coagulation Factor XIIa 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 room temperature 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 IC50, the inhibitor concentration causing a 50% decrease in Factor XIIa protease activity.Factor XIIa activity determinations were made in 50 mM HEPES buffer containing 150 mM NaCl, 5 mM CaCl2, and 0.1% PEG 8000 (polyethylene glycol; JT Baker or Fisher Scientific) at pH 7.4. Determinations were made using purified human Factor XIIa at a final concentration of 500 pM (Sekisui Diagnostics) and the synthetic substrate, n-Acetyl-Lys-Pro-Arg-AFC, TFA salt (Sigma #C6608) at a concentration of 100 μM.Activity assays were performed by diluting a stock solution of substrate at least tenfold to a final concentration ≤0.1 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. Initial velocities of product formation in the absence (Vo) or presence of inhibitor (Vi) were measured. IC50 was determined as the concentration of I yielding Vi=Vo/2.
Measurement: IC50 = 52.6 nM (pChEMBL 7.28000020980835)
Descriptors: MW 697.93, ALogP 3.28, HBA 10, HBD 3, PSA 156.23, QED 0.27
Compound (SMILES): O=C(NCCCN1CCS(=O)(=O)CC1)c1cnccc1Nc1nc(-c2cccc(C(F)F)n2)nn2cccc12
InChIKey: MZCGAQAWJQRWGU-UHFFFAOYSA-N
Target: TGF-beta receptor type-1 (SINGLE PROTEIN), Homo sapiens
UniProt: P36897
Assay: Biological Assays: Assays are conducted in 1536-well plates and 2 mL reactions are prepared from addition of HIS-TGFβR1 T204D or HIS-TGFβR2 WT, anti-HIS detection antibody, a labeled small molecule probe (Kd=<100 nM; koff=<0.001 s−1.) and test compounds in assay buffer (20 mM HEPES pH 7.4, 10 mM MgCl2, 0.015% Brij35, 4 mM DTT, and 0.05 mg/ml BSA). The reaction is incubated for 1 hour at room temperature and the HTRF signal was measured on an Envision plate reader (Ex: 340 nm; Em: 520 nm/495 nm). Inhibition data were calculated by comparison to no enzyme control reactions for 100% inhibition and vehicle-only reactions for 0% inhibition. The final concentration of reagents in the assay are 1 nM HIS-TGFβR1 T204D or HIS-TGFβR2 WT, 0.2 nM anti-HIS detection antibody, labeled small molecule probe (at Kd) and 0.5% DMSO. Dose response curves were generated to determine the concentration required inhibiting 50% of kinase activity (IC50). Compounds were dissolved at 10 mM in dimethylsulfoxide (DMSO) and evaluated at eleven concentrations. IC50 values were derived by non-linear regression analysis.
Measurement: IC50 = 8.6 nM (pChEMBL 8.069999694824219)
Descriptors: MW 556.6, ALogP 2.72, HBA 10, HBD 2, PSA 134.48, QED 0.3
Compound (SMILES): Cc1ncc(CC(=O)Nc2cc([C@H]3CC[C@@H](OC(=O)NC(C)(C)C)C3)[nH]n2)s1
InChIKey: CSLLQHXRVVZIDB-QWHCGFSZSA-N
Target: Cyclin-dependent kinase 2/cyclin E1 (PROTEIN COMPLEX), Homo sapiens
UniProt: P24864
Assay: Biochemical Assay: The purpose of CDK2/Cyclin E1 assay is to evaluate the inhibition (% inhibition, Kiapp and Ki values) of small molecule inhibitors by using a fluorescence-based microfluidic mobility shift assay. CDK2/Cyclin E1 full length catalyzes the production of ADP from ATP that accompanies the phosphoryl transfer to the substrate peptide FL-Peptide-18 (5-FAM-QSPKKG-CONH2, CPC Scientific, Sunnyvale, Calif.) (SEQ ID NO:1). 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. Wild-type CDK2/wild-type full length Cyclin E1 enzyme complex was produced in-house (baculoviral expression, LJIC-2080/LJIC-2103) and phosphorylated by CDK7/Cyclin H1/Matl enzyme complex with CDK2:CDK7 ratio of 50:1 (concentration mg/mL) in the presence of 10 mM MgCl2 and 5 mM ATP at room temperature for one hour. Typical reaction solutions (50 μL final reaction volume) contained 2% DMSO (±inhibitor), 4 mM MgCl2, 1 mM DTT, 150 μM ATP (ATP Km=67.4 μM), 0.005% Tween-20, 3 μM FL-Peptide-18, and 0.36 nM (catalytically competent active site) phosphorylated wild-type full length CDK2/Cyclin E1 enzyme complex in 25 mM HEPES buffer at pH 7.15. The assay was initiated with the addition of ATP, following a fifteen minutes pre-incubation of enzyme and inhibitor at room temperature in the reaction mixture. The reaction was stopped after 45 minutes at room temperature by the addition of 50 μL of 80 mM EDTA.
Measurement: Ki = 0.16 nM (pChEMBL 9.800000190734863)
Descriptors: MW 405.52, ALogP 3.52, HBA 6, HBD 3, PSA 109, QED 0.71
Compound (SMILES): CC(C)(C)C(=O)Nc1cc(Nc2nc(-c3cccc(C(F)(F)F)n3)nn3cccc23)ccn1
InChIKey: FGLAOSSPEGQSIA-UHFFFAOYSA-N
Target: TGF-beta receptor type-1 (SINGLE PROTEIN), Homo sapiens
UniProt: P36897
Assay: Biological Assays: Assays are conducted in 1536-well plates and 2 mL reactions are prepared from addition of HIS-TGFβR1 T204D or HIS-TGFβR2 WT, anti-HIS detection antibody, a labeled small molecule probe (Kd=<100 nM; koff=<0.001 s−1.) and test compounds in assay buffer (20 mM HEPES pH 7.4, 10 mM MgCl2, 0.015% Brij35, 4 mM DTT, and 0.05 mg/ml BSA). The reaction is incubated for 1 hour at room temperature and the HTRF signal was measured on an Envision plate reader (Ex: 340 nm; Em: 520 nm/495 nm). Inhibition data were calculated by comparison to no enzyme control reactions for 100% inhibition and vehicle-only reactions for 0% inhibition. The final concentration of reagents in the assay are 1 nM HIS-TGFβR1 T204D or HIS-TGFβR2 WT, 0.2 nM anti-HIS detection antibody, labeled small molecule probe (at Kd) and 0.5% DMSO. Dose response curves were generated to determine the concentration required inhibiting 50% of kinase activity (IC50). Compounds were dissolved at 10 mM in dimethylsulfoxide (DMSO) and evaluated at eleven concentrations. IC50 values were derived by non-linear regression analysis.
Measurement: IC50 = 160 nM (pChEMBL 6.800000190734863)
Descriptors: MW 455.44, ALogP 4.93, HBA 7, HBD 2, PSA 97.1, QED 0.45
Compound (SMILES): CC(C)COc1cccc(F)c1-n1nc2c(c1-c1cc(F)c(NC(N)=O)cc1F)CN(C(C)(C)c1ccccc1)CC2
InChIKey: GWRDZILKOHTGAP-UHFFFAOYSA-N
Target: C5a anaphylatoxin chemotactic receptor 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P21730
Assay: chemotaxis assay: Optionally, compounds may be further assayed for their ability to inhibit chemotaxis in cells. Chemotaxis assays are performed using 5 μm pore polycarbonate, polyvinylpyrrolidone-coated filters in 96-well chemotaxis chambers (Neuroprobe; Gaithersburg, Md.) using chemotaxis buffer (Hank's balanced salt solution (HBSS) and 1% FBS). C5aR ligands (i.e., C5a, R&D Systems; Minneapolis, Minn.) are use to evaluate compound mediated inhibition of C5aR mediated migration. Other chemokines (i.e., SDF-1α; R&D Systems; Minneapolis, Minn.) are used as specificity controls. The lower chamber is loaded with 29 μl of chemokine (i.e., 0.03 nM C5a) and varying amounts of compound; the top chamber contains 100,000 U937 or neutrophil cells in 20 μl. The chambers are incubated 1.5 hours at 37° C., and the number of cells in the lower chamber quantified either by direct cell counts in five high powered fields per well or by the CyQuant assay (Molecular Probes), a fluorescent dye method that measures nucleic acid content and microscopic observation.
Measurement: IC50 < 5 nM (pChEMBL 8.300999641418457)
Descriptors: MW 577.65, ALogP 6.78, HBA 5, HBD 2, PSA 85.41, QED 0.24
Compound (SMILES): CN1C(=N)N[C@@]2(CCOc3ccc(NC(=O)c4cnc(OCC(F)(F)F)cn4)cc32)CS1(=O)=O
InChIKey: FNQYVKIMJXZYOM-SFHVURJKSA-N
Target: Beta-secretase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P56817
Assay: HTRF FRET Assay: A homogeneous time-resolved FRET assay can be used to determine IC50 values for inhibitors of the soluble human BACE1 catalytic domain. This assay monitors the increase of 620 nm fluorescence that resulted from BACE1 cleavage of an APPswedish APPswe mutant peptide FRET substrate (QSY7-EISEVNLDAEFC-Europium-amide). This substrate contains an N-terminal QSY7 moiety that serves as a quencher of the C-terminal Europium fluorophore (620 nm Em). In the absence of enzyme activity, 620 nm fluorescence is low in the assay and increased linearly over 3 hours in the presence of uninhibited BACE1 enzyme Inhibition of BACE1 cleavage of the QSY7-APPswe-Eu substrate by inhibitors is manifested as a suppression of 620 nm fluorescence.Varying concentrations of inhibitors at 3× the final desired concentration in a volume of 10 ul are preincubated with purified human BACE1 catalytic domain (3 nM in 10 μl) for 30 minutes at 30° C. in reaction buffer containing 20 mM Na-Acetate pH 5.0, 10% glycerol, 0.1% Brij-35 and 7.5% DSMO. Reactions are initiated by addition of 10 μl of 600 nM QSY7-APPswe-Eu substrate (200 nM final) to give a final reaction volume of 30 μl in a 384 well Nunc HTRF plate. The reactions are incubated at 30° C. for 1.5 hours. The 620 nm fluorescence is then read on a Rubystar HTRF plate reader (BMG Labtechnologies) using a 50 millisecond delay followed by a 400 millisecond acquisition time window Inhibitor IC50 values are derived from non-linear regression analysis of concentration response curves.
Measurement: Ki = 70 nM (pChEMBL 7.159999847412109)
Descriptors: MW 500.46, ALogP 1.45, HBA 8, HBD 3, PSA 146.6, QED 0.57
Compound (SMILES): CC(C)Oc1nc2cc(F)c(C(=O)Nc3cccc(-c4nnnn4C(C)C)n3)cc2nc1OC(C)C
InChIKey: VJOAGJMDKDVZSY-UHFFFAOYSA-N
Target: Mitogen-activated protein kinase kinase kinase 5 (SINGLE PROTEIN), Homo sapiens
UniProt: Q99683
Assay: HTRF KinEASE Assay : ASK1 was purchased from Thermofisher (Catalogue # PV4011), ATP was purchased from Sigma (Catalogue # A7699), HTRF® KinEASE Assay System was obtained from Cisbio (Bedford, Mass.). ½ Area plate was purchased from Perkin Elmer (Catalogue # #6005560). HTRF® KinEASE-STK is a generic method for measuring serine/threonine kinase activities using a time-resolved fluorescence resonance energy transfer (TR-FRET) immunoassay. The IC50 value for each compound was determined in the presence of compound (various concentration from 0 to 10 μM) and a fixed amount of ATP and peptide substrates. The test compound, 1 uM STK3 peptide substrate, and 5 nM of ASK1 kinase are incubated with kinase reaction buffer containing 50 mM HEPES pH 7.5, 0.01% BRIJ-35, 10 mM MgCl2, and 1 mM EGTA for 30 minutes. 100 uM ATP is added to start kinase reaction and incubated for 3 hours. The STK3-antibody labeled with Eu3+-Cryptate and 125 nM streptavidin-XL665 are mixed in a single addition with stop reagents provided by the Cisbio kit used to stop the kinase reaction. Fluorescence is detected using an Envision Multilabeled 2014 reader from PerkinElmer. The Fluorescence is measured at 615 nm (Cryptate) and 665 nm (XL665) and a ratio of 665 nm/615 nm is calculated for each well. The resulting TR-FRET is proportional to the phosphorylation level. Staurosporine was used as the positive control. IC50 was determined by XLfit 5.3.
Measurement: IC50 > 1000 nM (pChEMBL 6)
Descriptors: MW 494.53, ALogP 4.23, HBA 10, HBD 1, PSA 129.83, QED 0.38