lovingscience/chembl-v37-raw-activities huggingface dataset:
Compound (SMILES): O=C(O)CCC1CC1c1c(F)cc(-c2cccnc2OC2CCCC2)cc1F
InChIKey: GHUYJMFDYCKUJX-UHFFFAOYSA-N
Target: Free fatty acid receptor 4 (SINGLE PROTEIN), Homo sapiens
UniProt: Q5NUL3
Assay: Measurement of Activity of GPR120 Agonist (Cell-Based Assay): CHO-K1 cells expressing Ga16 and hGPR120 were dispensed into each well of a 96-well plate (3×104 cells/100 μl/well) and then incubated in 5% CO2, 37° C. incubator for 18 hours. Each well was treated with 100 μl of Calcium 5 dye (Molecular Devices) solution including 2% DMSO and then incubated in 5% CO2, 37° C. incubator for 1 hour. Serially diluted GPR120 agonists were prepared to a final concentration of 0.5% DMSO in a 96-well plate. Each well was treated with 50 μl of the agonist compounds using Plexstation II, and then fluorescence was measured at Ex 485 nm and Em 525 nm.Fluorescence increased by the serially diluted GPR120 agonists is calculated as a relative percent (%) value based on the fluorescence represented by the treatment of 1% DMSO only. EC50 refers to the concentration of agonist which shows 50% of maximum fluorescence increased by the treatment of agonist. The calculation of measurement was carried out by using statistical software (Prizm).
Measurement: EC50 < 200 nM (pChEMBL 6.698999881744385)
Descriptors: MW 387.43, ALogP 5.32, HBA 3, HBD 1, PSA 59.42, QED 0.7
Compound (SMILES): CC(C)(C)NC(=O)N1CCc2c(-c3cccc(C(F)(F)F)c3)ccc(C(=O)NCc3cnn4ccccc34)c2C1
InChIKey: KVYLGHVYVPXSNB-UHFFFAOYSA-N
Target: Diacylglycerol O-acyltransferase 2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q96PD7
Assay: Enzymatic Activity Assay: DGAT2 activity was determined by measuring the amount of enzymatic product triolein (1,2,3-Tri(cis-9-octadecenoyl)glycerol) using the membrane prep mentioned above. The assay was carried out in deep well 384 plates in a final volume of 40 μL at room temperature. The assay mixture contained the following: assay buffer (100 mM Tris·Cl, pH 7.0, 20 mM MgCl2, 5% ethanol), 25 μM of diolein, 10 μM of oleoyl-CoA and 10 ng/μL of DGAT2 membrane.
Measurement: IC50 = 351 nM (pChEMBL 6.460000038146973)
Descriptors: MW 549.6, ALogP 5.82, HBA 4, HBD 2, PSA 78.74, QED 0.34
Compound (SMILES): CCCC[C@@H](CC(=O)NC[C@H](Cc1ccc2[nH]c(=O)oc2c1)N(C)C)c1ccccc1
InChIKey: XWMFWTVDIBANEE-SFTDATJTSA-N
Target: Mu-type opioid receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P35372
Assay: MOR cAMP Agonist and Antagonist Assays: CHO Tag-lite human mOR stable cell line from Cisbio (NCBI accession number: NM_000914.3, Bedford, Mass.) were seeded and grown to approximately 80% confluence in Ham F-12 with 10% FBS, 50 U/ml penicillin, 50 μg/ml streptomycin, 2 mM Hepes, and 1 mg/ml geneticin (Invitrogen, Carlsbad, Calif.). Cells were then harvested using accutase (Corning. Coming, N.Y.), centrifuged at 1300 RPM for 5 min, and plated at 5000 cells per 5 μL per well in a 5× dilution of stimulation buffer consisting of the HTRF cAMP Gi kit, water and IBMX at 0.5 mM (Cisbio, Bedford, Mass.) in white HTRF low volume 384 well plates (Cisbio, Bedford, Mass.). Plates were then incubated at 37° C. in 5% CO2 for 10 minutes. For the agonist assay, forskolin was added to a final concentration of 4 μM. For the antagonist assay, forskolin (4 μM) and DAMGO at EC90 final concentration were added. Test compounds were dissolved in DMSO and water, and then serially diluted to working concentrations such that the concentration of DMSO was less than 0.1%. Diluted test compounds were added at 2.5 μL per well, and plates were incubated at 37° C., and 5% CO2 for 15 minutes, and then at room temperature for 15 minutes. Next 5 μl/well of cAMP Eu-cryptate and 5 μl/well of anti-cAMP-d2 (both diluted 1:20 in lysis buffer) were added, and plates were incubated at room temperature for 1 hour. Following incubation, plates were read in a Synergy Neo2 multi-mode reader (Biotek, Winooski, Vt.). Plate reader settings were set to time resolved fluorescence with excitation at 330 nm and emissions of 620 nm and 665 nm. Emission fluorescence was normalized (665/620 nm signal×1000). For the agonist assay, data was normalized using the maximal DAMGO response. Measurements were performed in triplicate and the dose response was fit using nonlinear regression.
Measurement: IC50 > 3000 nM (pChEMBL 5.5229997634887695)
Descriptors: MW 423.56, ALogP 4.07, HBA 4, HBD 2, PSA 78.34, QED 0.49
Compound (SMILES): CCn1nc(CCCOC(=O)c2cccc(OC)c2Cl)c2c1C(=O)NCC1(CCOCC1)C2
InChIKey: TZSDUYLZZWTKHP-UHFFFAOYSA-N
Target: 3',5'-cyclic-AMP phosphodiesterase 4D (SINGLE PROTEIN), Homo sapiens
UniProt: Q08499
Assay: PDE4 Assay: The human PDE4D catalytic domain (UniProt no. Q08499 [S380-L740]) was incubated with a mixture of non-labelled cAMP (cyclic adenosine monophosphate) and fluorescein amidite (FAM) conjugated cAMP and titrated test or reference Compound. Following brief incubation the enzymatic reaction was stopped by addition of binding buffer containing nanoparticles with immobilized trivalent metal ions capable of binding 1) AMP phospho groups and 2) terbium (Tb) donor fluorophores. Subsequent excitation of the Tb donor triggers time-resolved FRET to adjacent FAM acceptor molecules resulting in light emission. In the presence of a PDE4 inhibitor, AMP generation was reduced resulting in a lower fluorescence signal. The cAMP phosphodiester is not bound by the detection system.The results were calculated as the molar concentrations resulting in 50% inhibition of the substrate cleavage compared to controls samples, and are expressed as a range of IC50 (nM).
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 475.97, ALogP 3.44, HBA 7, HBD 1, PSA 91.68, QED 0.49
Compound (SMILES): NCCC(=O)N[C@H]1CC[C@H](NC(=O)c2cncc(O)c2)CC1
InChIKey: NDGGGLBKRZWZJF-HAQNSBGRSA-N
Target: Histone-lysine N-methyltransferase SMYD3 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9H7B4
Assay: SMYD3 Biochemical Assay: The assays were all performed in a buffer consisting of 25 mM Tris-Cl pH 8.0, 1 mM TCEP, 0.005% BSG, and 0.005% Tween 20, prepared on the day of use. Compounds in 100% DMSO (1 ul) were spotted into a 384-well white opaque OptiPlate using a Bravo automated liquid handling platform outfitted with a 384-channel head (Agilent Technologies). DMSO (1 ul) was added to Columns 11, 12, 23, 24, rows A-H for the maximum signal control and 1 ul of SAH, a known product and inhibitor of SMYD3, was added to columns 11, 12, 23, 24, rows I-P for the minimum signal control. A cocktail (40 ul) containing the SMYD3 enzyme was added by Multidrop Combi (Thermo-Fisher). The compounds were allowed to incubate with SMYD3 for 30 min at room temperature, then a cocktail (10 ul) containing SAM and MEKK2 was added to initiate the reaction (final volume=51 ul). The final concentrations of the components were as follows: SMYD3 was 0.4 nM, 3H-SAM was 8 nM, MEKK2 was 12 nM, SAH in the minimum signal control wells was 1 mM, and the DMSO concentration was 2%. The assays were stopped by the addition of non-radiolabeled SAM (10 ul) to a final concentration of 100 uM, which dilutes the 3H-SAM to a level where its incorporation into MEKK2 is no longer detectable. Radiolabeled MEKK2 was detected using a scintillation proximity assay (SPA). 10 uL of a 10 mg/mL solution of SPA beads in 0.5 M citric acid was added and the plates centrifuged at 600 rpm for 1 min to precipitate the radiolabeled MEKK2 onto the SPA beads. The plates were then read in a PerkinElmer TopCount plate reader to measure the quantity of 3H-labeled MEKK2 as disintegrations per minute (dpm) or alternatively, referred to as counts per minute (cpm).
Measurement: IC50 > 50000 nM (pChEMBL 4.301000118255615)
Descriptors: MW 306.37, ALogP 0.29, HBA 5, HBD 4, PSA 117.34, QED 0.62
Compound (SMILES): CO[C@H]1CN(C(=O)c2c(F)cccc2-n2nccn2)C(C)(c2nc3c(C)c(Cl)ccc3[nH]2)C1
InChIKey: DVBLWCSCSNQUJD-PDNQZYNLSA-N
Target: Orexin/Hypocretin receptor type 1 (SINGLE PROTEIN), Homo sapiens
UniProt: O43613
Assay: Inhibition Assay: Chinese hamster ovary (CHO) cells expressing the human orexin-1 receptor and the human orexin-2 receptor, respectively, are grown in culture medium (Ham F-12 with L-Glutamine) containing 300 μg/ml G418, 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 384-well black clear bottom sterile plates (Greiner). The seeded plates are incubated overnight at 37° C. in 5% CO2.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 μl of staining buffer (HBSS containing 1% FCS, 20 mM HEPES, NaHCO3: 0.375 g/l, 5 mM probenecid (Sigma) and 3 M 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.
Measurement: IC50 = 193 nM (pChEMBL 6.710000038146973)
Descriptors: MW 468.92, ALogP 4.02, HBA 6, HBD 1, PSA 88.93, QED 0.49
Compound (SMILES): CN1CCNC(=O)c2nn(C)cc2NC(=O)c2cccc(n2)-c2cnn(c2)CC1
InChIKey: SRPZQITYKMNCOH-UHFFFAOYSA-N
Target: Interleukin-1 receptor-associated kinase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P51617
Assay: Enzymatic Assay: IRAK1 is a human purified recombinant enzyme (His-TEV-IRAK1 (194-712))In this assay, IRAK1 hydrolyses ATP and autophosphorylates. Measurement of IRAK-1 inhibition is performed in 384-well format based on luminescence assay (ADP-Glo Kinase Assay from Promega). Purified human recombinant IRAK1 (0.3 g/ml) and serial diluted compounds in DMSO (range of concentration from 10 μM to 0.5 nM) or controls (1% DMSO) are incubated for 15 minutes at RT in assay buffer containing 50 mM Hepes pH 7.0, Fatty acid-free BSA 0.1%, Dithiothreitol (DTT) 2 mM, MgCl2 10 mM, EGTA 0.5 mM, Triton X-100 0.01%. The kinase reaction is then initiated by the addition of ATP at a concentration of 1μM. After 2 hours of incubation at RT, the reaction is stopped and the unconsumed ATP depleted by the addition of ADP-Glo Reagent according to supplier instructions. After 40 minutes of incubation at RT, the Kinase Detection Reagent is then added to the assay plate according to supplier instructions. After 20 minutes of incubation at RT, the luminescence signal is measured with a luminometer (PerkinElmer Envision or equivalent reader).
Measurement: IC50 = 3000 nM (pChEMBL 5.519999980926514)
Descriptors: MW 394.44, ALogP 0.61, HBA 8, HBD 2, PSA 109.97, QED 0.58
Compound (SMILES): Cc1c([C@H](c2ccc3c(c2)[C@H](N2Cc4cnccc4OC(C)(C)C2)CC3)C(C)(C)C(=O)O)ccc2c1nnn2C
InChIKey: HELMUQRMQBFUKI-NAKRPHOHSA-N
Target: NAD(P)H dehydrogenase [quinone] 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P15559
Assay: BEAS-2B NQO1 MTT Assay: NAD(P)H:quinone oxidoreductase 1 (NQO1), also called DT diaphorase, is a homodimeric FAD-containing enzyme that catalyzes obligatory NAD(P)H-dependent two-electron reductions of quinones and protects cells against the toxic and neoplastic effects of free radicals and reactive oxygen species arising from one-electron reductions. The transcription of NQO1 is finely regulated by NRF2, and thus NQO1 activity is a good marker for NRF2 activation. On day one, frozen BEAS-2B cells (ATCC) are thawed in a water bath, counted, and re-suspended at a concentration of 250,000 cells/mL. Fifty microliters of cells are plated in 384 well black clear-bottomed plates. Plates are incubated at 37° C., 5% CO2 overnight. On day two, plates are centrifuged and 50 nL of compound or controls are added to the cells. Plates are then incubated at 37° C., 5% CO2 for 48 hours. On day four, medium is aspirated from the plate and crude cell lysates are made by adding 13 uL of 1× Cell Signaling Technologies lysis buffer with 1 Complete, Mini, EDTA-free Protease Inhibitor Tablet (Roche) for each 10 mL of lysis buffer. After lysis plates are incubated for 20 minutes at room temperature. Two microliters of lysate are removed for use in Cell Titer Glo assay (Promega) and MTT cocktail is prepared (Prochaska et. al. 1998) for measurement of NQO1 activity. Fifty microliters of MTT cocktail is added to each well, plate is centrifuged, and analyzed on an Envision plate reader (Perkin Elmer) using Absorbance 570 nm label for 30 minutes. Product formation is measured kinetically and the pEC50 of NQO1 specific activity induction is calculated by plotting the change in absorbance (Delta OD/min) versus the log of compound concentration followed by 3-parameter fitting.
Measurement: EC50 < 1 nM (pChEMBL 9)
Descriptors: MW 539.68, ALogP 5.57, HBA 7, HBD 1, PSA 93.37, QED 0.35
Compound (SMILES): Cc1cc(C(=O)Nc2cnc3[nH]c(C#CCF)cc3c2)[nH]n1
InChIKey: ZQESFHAJYXLRSF-UHFFFAOYSA-N
Target: Mast/stem cell growth factor receptor Kit (SINGLE PROTEIN), Homo sapiens
UniProt: P10721
Assay: Exemplary c-Kit Biochemical Assay: Assays for biochemical cell-based activity of c-kit kinase are known in the art, for example, as described in U.S. Pat. Nos. 7,498,342 and 7,846,941, the disclosures of which are hereby incorporated by reference as it relates to such assays. The c-kit (or kinase domain thereof) is an active kinase in AlphaScreen. IC50 values are determined with respect to inhibition of c-Kit kinase activity, where inhibition of phosphorylation of a peptide substrate is measured as a function of compound concentration. Compounds to be tested were dissolved in DMSO to a concentration of 20 mM. These were diluted 30 μl into 120 μl of DMSO (4 mM) and 1 μl was added to an assay plate. These were then serially diluted 1:3 (50 μl to 100 μl DMSO) for a total of 8 points. Plates were prepared such that each kinase reaction is 20 μl in 1× kinase buffer (50 mM HEPES, pH 7.2, 5 mM MgCl2, 5 mM MnCl2, 0.01% NP-40, 0.2% BSA), 5% DMSO and 10 gM ATP. Substrate was 100 nM biotin-(E4Y)3 (Open Source Biotech, Inc.). C-kit kinase was at 0.1 ng per sample. After incubation of the kinase reaction for 1 hour at room temperature, 5 μl of donor beads (Streptavidin coated beads (Perkin Elmer Life Science) final concentration 1 μg/ml) in stop buffer (50 mM EDTA in 1× kinase buffer) was added, the sample was mixed and incubated for 20 minutes at room temperature before adding 5 μl of acceptor beads (PY20 coated beads (Perkin Elmer Life Science) final concentration 1 μg/ml) in stop buffer. The samples were incubated for 60 minutes at room temperature and the signal per well was read on AlphaQuest reader. Phosphorylated substrate results in binding of the PY20 antibody and association of the donor and acceptor beads such that signal correlates with kinase activity. The signal vs. compound concentration was used to determine the IC50.
Measurement: Ki = 5500 nM (pChEMBL 5.260000228881836)
Descriptors: MW 297.29, ALogP 2.17, HBA 3, HBD 3, PSA 86.46, QED 0.63
Compound (SMILES): Cc1nc2cnc(N3CC4(C3)CN(C3(C)CC(F)(F)C3)C4)c(C#N)c2c(=O)n1CCOc1ccc(Cl)cc1-c1ccnc2c(C(=O)O)csc12
InChIKey: HRKAKEZHHLEMID-UHFFFAOYSA-N
Target: Eukaryotic translation initiation factor 4E-binding protein 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q13541
Assay: Fluorescence Polarization Assay: eIF4E protein was pre-incubated with EDA-m7GDP-ATTO-550 in FPB at 2xconcentrations for 5 minutes prior to the addition of compounds. Compounds (100x) were prepared using 3-fold serial dilutions in 100% DMSO, and subsequently diluted 1:50 in FPB to produce 2x stocks. 50 ul of 2x eIF4E/EDA-m7GDP-ATTO-550 were transferred to the wells of a 96-well half-area black flat bottom polystyrene plate. 50 ul of 2x test compound were added, and binding reactions were allowed to equilibrate for 30 minutes at room temperature while protected from light. The fluorescent polarization signal was detected using a Victor 2 multi-label counter (Perkin Elmer) and the concentration necessary to achieve inhibition of eIF4E/EDA-m7GDP-ATTO-550 binding by 50% (IC50) was calculated using data from an 8-point compound dilution series.
Measurement: IC50 = 26.4 nM (pChEMBL 7.579999923706055)
Descriptors: MW 718.19, ALogP 5.99, HBA 11, HBD 1, PSA 137.47, QED 0.2
Compound (SMILES): Cc1cc(C(C)C)cnc1N1CCc2nn(-c3c(C)cccc3OCC(C)C)c(-c3cc(F)c(NC(N)=O)cc3F)c2C1
InChIKey: YZLKXUGGOALXQC-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 588.7, ALogP 7.04, HBA 6, HBD 2, PSA 98.3, QED 0.23
Compound (SMILES): Cc1cccc(C)c1-n1nc2c(c1-c1ccc(CO)cc1)CN(c1ccccc1C(C)C)CC2
InChIKey: XFBQHQFGGPHONN-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 = 275 nM (pChEMBL 6.559999942779541)
Descriptors: MW 451.61, ALogP 6.33, HBA 4, HBD 1, PSA 41.29, QED 0.39
Compound (SMILES): COC[C@]12C[C@H]1[C@@](C)(c1cc(/C=C(\F)c3cnc(OCC4CC(F)(F)C4)cn3)ccc1F)N=C(N)S2
InChIKey: NWIYSYRPQNHSKZ-QKQHWZHFSA-N
Target: Beta-secretase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P56817
Assay: In Vitro Enzymatic BACE1 and BACE2 FRET (Fluorescence Resonance Energy Transfer) Assays: The cDNAs for both human recombinant BACE1 and 2 with C-terminal 6-His Tags were cloned into transient protein expression vectors, which were subsequently transfected into mammalian cell lines. These recombinant proteins were further purified using Ni-NTA affinity chromatography (Qiagen). The assay buffer used in these screens was 0.05M acetate, pH 4.5, 8% DMSO final, 100 uM genapol (which is a nonionic detergent, below its Critical Micelle Concentration). The Beta Secretase enzyme (0.02 nM for BACE1 and 0.64 nM for BACE2) is pre-incubated for one hour with inhibitors, typically in about 1 uL of DMSO according to a serial dilution, are added thereto. This assay is effectively started by the addition of FRET substrate (50 nM) and the combination is incubated for one hour. The FRET assay is terminated by addition of Tris buffer, which raises the pH to neutrality, and the fluorescence is determined. The FRET substrate is a peptide with commercially available fluorophore and quencher, on opposite sides of the BACE cleavage site. Proteolytic cleavage of the FRET substrate releases quenching of fluorescence (excitation 488 nm and emission 590 nm).
Measurement: IC50 = 2280 nM (pChEMBL 5.639999866485596)
Descriptors: MW 522.57, ALogP 5.19, HBA 7, HBD 1, PSA 82.62, QED 0.48
Compound (SMILES): CC(=O)c1nn(CC(=O)N2[C@H](C(=O)Nc3nc(Br)ccc3C(C)C)C[C@@]3(C)C[C@@H]23)c2ccc(-c3cnc(C)nc3)cc12
InChIKey: RDCKBZYLOJHTIS-NTWBYJAHSA-N
Target: Complement factor D (SINGLE PROTEIN), Homo sapiens
UniProt: P00746
Assay: Factor D Assay: Human Factor D (purified from human serum, Complement Technology, Inc.) at 80 nM final concentration is incubated with test compound at various concentrations for 5 min at room temperature in 50 mM Tris, 1M NaCl, pH 7.5. A synthetic substrate Z-L-Lys-SBz and DTNB (Ellman's reagent) are added to final concentrations of 100 μM each. Absorbance at 405 nm (A405) is recorded at 30 second intervals for 30 min using a microplate spectrophotometer. IC50 values are calculated by nonlinear regression of complement Factor D reaction rates as a function of test compound concentration.
Measurement: IC50 < 1000 nM (pChEMBL 6)
Descriptors: MW 630.55, ALogP 5.3, HBA 8, HBD 1, PSA 122.97, QED 0.22
Compound (SMILES): CNc1cc(Nc2cccn(-c3ocnc3C)c2=O)nc2c(C(=O)NC3CCC[C@@H](OC)C3)cnn12
InChIKey: ICHMLBPIJNNLJA-OEMAIJDKSA-N
Target: Non-receptor tyrosine-protein kinase TYK2 (SINGLE PROTEIN), Homo sapiens
UniProt: P29597
Assay: TYK2 JH2 Domain Binding Assay: The binding reaction was assembled by combining 16 μl of DNA-tagged kinase extract, 3.8 μl liganded affinity beads, and 0.18 μl test compound (PBS/0.05% Tween 20/10 mM DTT/0.1% BSA/2 μg/ml sonicated salmon sperm DNA)]. Extracts were used directly in binding assays without any enzyme purification steps at a ≥10,000-fold overall stock dilution (final DNA-tagged enzyme concentration <0.1 nM). Extracts were loaded with DNA-tag and diluted into the binding reaction in a two step process. First extracts were diluted 1:100 in 1× binding buffer (PBS/0.05% Tween 20/10 mM DTT/0.1% BSA/2 μg/ml sonicated salmon sperm DNA) containing 10 nM DNA-tag. This dilution was allowed to equilibrate at room temperature for 15 minutes and then subsequently diluted 1:100 in 1× binding buffer. Test compounds were prepared as 111× stocks in 100% DMSO. Kds were determined using an 11-point 3-fold compound dilution series with three DMSO control points. All compounds for Kd measurements are distributed by acoustic transfer (non-contact dispensing) in 100% DMSO
Measurement: Kd = 0.6 nM (pChEMBL 9.220000267028809)
Descriptors: MW 492.54, ALogP 2.65, HBA 11, HBD 3, PSA 140.61, QED 0.35
Compound (SMILES): CCc1c(-c2ccnc(O)c2)[nH]c2ccc(C3CCN(C4CCN(CC(C)C)CC4)CC3)cc12
InChIKey: JRGAXLGFQIZWHP-UHFFFAOYSA-N
Target: Toll-like receptor 7 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9NYK1
Assay: Inhibition Reporter Assay: HEK-Blue-cells (Invivogen) overexpressing human TLR7, TLR8 or TLR9 receptors were used for screening inhibitors of these receptors using an inducible SEAP (secreted embryonic alkaline phosphatase) reporter gene under the control of the IFN-β minimal promoter fused to five NF-κB and AP-1-binding sites. Briefly, cells are seeded into Greiner 384 well plates (15000 cells per well for TLR7, 20,000 for TLR8 and 25,000 for TLR9) and then treated with test compounds in DMSO to yield a final dose response concentration range of 0.05 nM-50 μM. After a 30 minute compound pre-treatment at room temperature, the cells are then stimulated with a TLR7 ligand (gardiquimod at a final concentration of 7.5 μM), TLR8 ligand (R848 at a final concentration of 15.9 μM) or TLR9 ligand (ODN2006 at a final concentration of 5 nM) to activate NF-κB and AP-1 which induce the production of SEAP. After a 22 hour incubation at 37° C., 5% CO2, SEAP levels are determined with the addition of HEK-Blue Detection reagent (Invivogen), a cell culture medium that allows for detection of SEAP, according to manufacturer's specifications.
Measurement: IC50 = 10978 nM (pChEMBL 4.960000038146973)
Descriptors: MW 460.67, ALogP 5.8, HBA 4, HBD 2, PSA 55.39, QED 0.49
Compound (SMILES): CC(C)[C@@H](C(=O)N1C[C@](C)(O)C[C@H]1C(=O)O)N1Cc2ccccc2C1=O
InChIKey: QUBUYLUWNCJKOM-YZVOILCLSA-N
Target: von Hippel-Lindau disease tumor suppressor (SINGLE PROTEIN), Homo sapiens
UniProt: P40337
Assay: Fluorescence Polarization Assay (1% 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 (1% DMSO).
Measurement: IC50 > 100000 nM (pChEMBL 4)
Descriptors: MW 360.41, ALogP 1.1, HBA 4, HBD 2, PSA 98.15, QED 0.84
Compound (SMILES): CCOC(=O)Cn1nc(C(C)(C)C)cc1NC(=O)Nc1cccc(Sc2ncnc3cc(OC)c(OC)cc23)c1
InChIKey: CVRZJGJLTURVDM-UHFFFAOYSA-N
Target: Serine/threonine-protein kinase B-raf (SINGLE PROTEIN), Homo sapiens
UniProt: P15056
Assay: Competition Binding Assay: Competition binding assays used herein were developed, validated and performed as described in Fabian et al., Nature Biotechnology 2005, 23, 329-336. Kinases were produced as fusions to T7 phage (See, Fabian et al. or WO04/015142) or alternatively, the kinases were expressed in HEK-293 cells and subsequently tagged with DNA for PCR detection (See, WO08/005310). For the binding assays, streptavidin-coated magnetic beads were treated with biotinylated affinity ligands for 30 min at room temperature to generate affinity resins. The liganded beads were blocked with excess biotin and washed with blocking buffer (SeaBlock (Pierce), 1% BSA, 0.05% Tween 20, 1 mM DTT) to remove unbound ligand and to reduce non-specific binding. Binding reactions were assembled by combining kinase, liganded affinity beads, and test compounds in 1× binding buffer (20% SeaBlock, 0.17×PBS, 0.05% Tween 20, 6 mM DTT). Test compounds were prepared as 100× stocks in DMSO and rapidly diluted into the aqueous environment. DMSO was added to control assays lacking a test compound. Primary screen interactions were performed in polypropylene 384-well plates in a final volume of 34 μL, while Kd determinations were performed in polystyrene 96-well plates in a final volume of 135 μL. The assay plates were incubated at room temperature with shaking for 1 hour, long enough for binding reactions to reach equilibrium, and the affinity beads were washed extensively with wash buffer (1×PBS, 0.05% Tween 20) to remove unbound protein. The beads were then resuspended in elution buffer (1×PBS, 0.05% Tween 20, 2 μM non-biotinylated affinity ligand) and incubated at room temperature with shaking for 30 min. The kinase concentration in the eluates was measured by quantitative PCR. Each kinase was tested individually against each compound. KdS were determined using eleven serial threefold dilutions.
Measurement: Kd = 375 nM (pChEMBL 6.429999828338623)
Descriptors: MW 564.67, ALogP 5.5, HBA 10, HBD 2, PSA 129.49, QED 0.2
Compound (SMILES): COC(=O)c1c(OC)ccnc1O[C@@H]1CC[C@@H](C)N(C(=O)c2ccccc2-n2ncnn2)C1
InChIKey: MNSIINCVACWHTA-HUUCEWRRSA-N
Target: Orexin receptor type 2 (SINGLE PROTEIN), Homo sapiens
UniProt: O43614
Assay: FLIPR Ca2+ Flux Assay: In a typical experiment the OX1 and OX2 receptor antagonistic activity of the compounds of the present invention was determined in accordance with the following experimental method. For intracellular calcium measurements, Chinese hamster ovary (CHO) cells expressing the rat orexin-1 receptor or the human orexin-2 receptor, are grown in Iscove's modified DMEM containing 2 mM L-glutamine, 0.5 g/ml G418, 1% hypoxanthine-thymidine supplement, 100 U/ml penicillin, 100 μg/ml streptomycin and 10% heat-inactivated fetal calf serum (FCS). The cells are seeded at 20,000 cells/well into Becton-Dickinson black 384-well clear bottom sterile plates coated with poly-D-lysine. All reagents were from GIBCO-Invitrogen Corp. The seeded plates are incubated overnight at 37° C. and 5% CO2. Ala-6,12 human orexin-A as the agonist is prepared as a 1 mM stock solution in 1% bovine serum albumin (BSA) and diluted in assay buffer (HBSS containing 20 mM HEPES, 0.1% BSA and 2.5 mM probenecid, pH7.4) for use in the assay at a final concentration of 70 pM. Test compounds are prepared as 10 mM stock solution in DMSO, then diluted in 384-well plates, first in DMSO, then assay buffer. On the day of the assay, cells are washed 3 times with 100 μl assay buffer and then incubated for 60 min (37° C., 5% CO2) in 60 μl assay buffer containing 1 μM Fluo-4AM ester, 0.02% pluronic acid, and 1% BSA. The dye loading solution is then aspirated and cells are washed 3 times with 100 μl assay buffer. 30 μl of that same buffer is left in each well. Within the Fluorescent Imaging Plate Reader (FLIPR, Molecular Devices), test compounds are added to the plate in a volume of 25 μl, incubated for 5 min and finally 25 μl of agonist is added.
Measurement: IC50 = 27.7 nM (pChEMBL 7.559999942779541)
Descriptors: MW 452.47, ALogP 1.92, HBA 10, HBD 0, PSA 121.56, QED 0.52
Compound (SMILES): O=C(C[C@@H]1CCc2cc(C(O)(C(F)(F)F)C(F)(F)F)ccc2N1S(=O)(=O)c1ccc(F)cc1)Nc1cccnc1CO
InChIKey: JQISLIRLFHEPBX-SFHVURJKSA-N
Target: Nuclear receptor ROR-gamma (SINGLE PROTEIN), Homo sapiens
UniProt: P51449
Assay: Spa Binding Assay: The binding of potential ligands to RORγ is measured by competition with [3H] 25-hydroxycholesterol using a scintillation proximity assay (SPA) binding assay. The ligand binding domain of human RORγ (A262-S507) with an N-terminal His tag is expressed in E. coli and purified using nickel affinity chromotography. 50 nM RORγ (A262-S507) is incubated with test compound at varying concentrations for 15 min at room temperature in PBS buffer containing 0.5% fatty acid free BSA. 10 nM of [3H] 25-hydroxycholesterol is then added, and the reaction is incubated for 15 min. 4 mg/mL of Ysi Copper HIS-TAG SPA Beads (Perkin Elmer) are added, and the mixture is incubated for 30 min. The reaction is read on a MicroBeta Trilux scintillation plate reader (Perkin Elmer).
Measurement: IC50 = 48.2 nM (pChEMBL 7.320000171661377)
Descriptors: MW 621.53, ALogP 4.56, HBA 6, HBD 3, PSA 119.83, QED 0.33
Compound (SMILES): O=C(NCCC1CCN(Cc2ccccc2)CC1)N1CCN(c2cc(F)c(F)c(F)c2)CC1
InChIKey: GKKNLDRLLJQNSE-UHFFFAOYSA-N
Target: Muscarinic acetylcholine receptor M4 (SINGLE PROTEIN), Homo sapiens
UniProt: P08173
Assay: Binding Assay: Binding affinity (Ki) of compounds was measured by inhibition of radioligand binding to membranes from CHO cells expressing human M1, M2, M3, M4 and M5 receptors. Membranes were prepared by nitrogen cavitation and differential centrifugation as previously described (Hoare et al., Mol. Pharmacol. 2003 March; 63(3): 751-65). The radioligand employed was tritiated N-methylscopolamine, used at a concentration of 1.5 nM. A dose-response of twelve concentrations of compound was used, ranging from 10 Î¼M to 32 Î¼M. The assay buffer was 50 mM HEPES, 100 mM NaCl, 5 mM MgCl2, 1 mM ethylenediaminetetraacetic acid, pH-adjusted to pH 7.4.
Measurement: Ki = 55 nM (pChEMBL 7.260000228881836)
Descriptors: MW 460.54, ALogP 4.24, HBA 3, HBD 1, PSA 38.82, QED 0.66
Compound (SMILES): Cc1cc(C(=O)NCc2ccc(C3(N)CC3)cc2)c(C)n1Cc1csc(-c2ccccc2)n1
InChIKey: JDDOXSIDIBNPSA-UHFFFAOYSA-N
Target: Plasma kallikrein (SINGLE PROTEIN), Homo sapiens
UniProt: P03952
Assay: Plasma kallikrein assay: Plasma kallikrein inhibitory activity in vitro was determined using standard published methods (see e.g. Johansen et al., Int. J. Tiss. Reac. 1986, 8, 185; Shori et al., Biochem. Pharmacol., 1992, 43, 1209; Stürzebecher et al., Biol. Chem. Hoppe-Seyler, 1992, 373, 1025). Human plasma kallikrein (Protogen) was incubated at 37° C. with the fluorogenic substrate H-DPro-Phe-Arg-AFC and various concentrations of the test compound. Residual enzyme activity (initial rate of reaction) was determined by measuring the change in optical absorbance at 410 nm and the IC50 value for the test compound was determined.
Measurement: IC50 = 121 nM (pChEMBL 6.920000076293945)
Descriptors: MW 456.62, ALogP 5.15, HBA 5, HBD 2, PSA 72.94, QED 0.4
Compound (SMILES): C=CC(=O)NC1CCN(c2ccc(C(N)=O)c(-c3ccc(Oc4ccccc4)cc3)n2)C1
InChIKey: MPKDWKUHJVCUNA-UHFFFAOYSA-N
Target: Tyrosine-protein kinase BTK (SINGLE PROTEIN), Homo sapiens
UniProt: Q06187
Assay: ADP-Glo Kinase Assay: 1× and 2× assay buffer were made at first. BTK kinase was diluted with 1× assay buffer but substrate was diluted with 2× assay buffer. 1 ul of diluted compound was transferred into 384-well assay plate, and then 2.0 ul of enzyme solution was added, and spun at 2000 rpm for 1 min. This mixture was incubated at 24° C. for 30 mins. 2 ul of peptide substrate/ATP mixture was added into the assay plate to start the reaction. The mixture was mixed thoroughly and then the 384-well plate was spun and incubated at 24° C. for 60 mins. 5.0 ul of ADP-Glo Reagent was added to stop the kinase activity and deplete the ATP unconsumed, and the plate was mixed thoroughly and incubated at 24° C. for 40 min. Then, 10.0 ul of Kinase Detection reagent was added, and the plate was centrifuged and then kept at 24° C. for 30 min. The luminescence signal was read on Envision.
Measurement: IC50 < 500 nM (pChEMBL 6.301000118255615)
Descriptors: MW 428.49, ALogP 3.52, HBA 5, HBD 2, PSA 97.55, QED 0.56
Compound (SMILES): Cn1nnn(-c2cc(Nc3ncc(C#N)c(N[C@@H]4C[C@@H]5CCCN5C(C)(C)C4)n3)c(F)cc2OC2COC2)c1=O
InChIKey: MKRIJPOTGYQXJX-SJORKVTESA-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 < 500 nM (pChEMBL 6.301000118255615)
Descriptors: MW 550.6, ALogP 2.11, HBA 13, HBD 2, PSA 148.04, QED 0.45
Compound (SMILES): O=C(NO)c1ccc2nc(-c3ccc4[nH]nnc4c3)oc2c1
InChIKey: PEJMEJGSUMWYKV-UHFFFAOYSA-N
Target: Protein deacetylase HDAC6 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9UBN7
Assay: In Vitro Enzymatic Assay: The assay principle is well known and all necessary reagents like enzymes, substrates, developer and reference compounds are commercially available. Stock solutions (10 mM in DMSO) of compounds were serially diluted 1:3 in 11 concentrations with a top concentration of 200 μM for HDAC1,2,3 and 2 μM for HDAC6 and HDAC8. The enzymatic reactions were conducted in a mixture containing assay buffer, bovine serum albumin, HDAC substrate, and a test compound. After enzymatic reaction, developer was added and after an additional incubation time, fluorescence intensity was measured at an excitation wavelength of 360 nm and an emission wavelength of 460 nm. All experiments were performed in duplicate.
Measurement: IC50 = 60 nM (pChEMBL 7.21999979019165)
Descriptors: MW 295.26, ALogP 1.89, HBA 6, HBD 3, PSA 116.93, QED 0.38
Compound (SMILES): CCC(C)n1cc(-c2cncc(-c3ccc4c(c3)CC(=O)N4C)c2)ccc1=O
InChIKey: ZBNPOKDTSKZDCA-UHFFFAOYSA-N
Target: Activin receptor type-1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q04771
Assay: Luminescence-Based ADPGlo Kinase Activity Assay: Example 53: The assays were performed in 384-well, low volume microtiter assay plates in a final reaction volume of 6 ul. Dose-response curves were generated by incubating 10 nM of each kinase in 50 mM Hepes pH 7.5, 0.02% Tween 20, 0.02% BSA, 1 mM DTT, 10 uM Na3VO4, 10 mM ß-Glycerolphosphate, 1 mM MgCl2, 12 mM MnCl2 and 15 uM ATP for 60 min at 32° C. in the presence or absence of compound diluted in DMS. The amount of generated ADP is a measure of kinase activity and is quantified using the ADP-Glo Kinase Assay (Promega) according to manufacturer's instructions. ADP is converted to ATP by adding 3 ul of ADP-Glo Reagent and incubation at 32° C. for 60 min. ATP is subsequently converted into a bioluminescent signal by adding 6 ul luciferase assay reagents (Kinase detection buffer+Kinase Detection Substrate (Promega)) and further incubation at 32° C. for 60 min. For the measurement of luminescence a PHERAstar Multilabel Reader was used at a measurement interval time of 0.1 second (optical module for luminescence measurements in the 230 nm to 750 nm wavelength range). The luminescent signal positively correlates with kinase activity.
Measurement: IC50 = 1300 nM (pChEMBL 5.889999866485596)
Descriptors: MW 373.46, ALogP 4.07, HBA 4, HBD 0, PSA 55.2, QED 0.69
Compound (SMILES): CC(C)[C@H](NC(=O)[C@@H](Cc1ccc2ccccc2n1)NC(=O)[C@H](CC(=O)O)NC(=O)OCc1ccccc1)C(=O)N[C@H](/C=C/S(=O)(=O)c1ccccc1)CC(=O)O
InChIKey: CPFXCSLAAJOPLW-IHJNOYNJSA-N
Target: Caspase-3 (SINGLE PROTEIN), Homo sapiens
UniProt: P42574
Assay: In Vitro Inhibitory Activity of Caspase: Selectivity of compound 55, compound 63, compound 48, compound 57, compound 88 toward caspase-1 (pro-inflammatory group), caspase-5 (group I), caspase-9 (group II) and caspase-3 and caspase-7 (group III) was evaluated by using fluorometric methods using the Caspase-1, -3, -5, -7, -9 Inhibitor Drug Screening Kit (Catalog #: K151-100, K153-100, K155-100, K157-100, K159-100 respectively, BioVision). Briefly, using instructions of the manufacturer, a wide range of different concentrations of the compound: 3333, 1000, 333, 100, 33, 10, 3, and 1 nM (final concentration) was added directly to the reaction mixtures containing the substrate and the enzyme in a final volume of 10 μl After a 30-minute incubation at 37° C., the liberation of AFC was measured as an endpoint assay using the Flexstation3 (Molecular Devices) with an excitation wavelength of 400 nm and an emission wavelength of 505 nm. The level of inhibition of caspase-1, -3, -5, -7, -9 activity was determined by comparison of the relative fluorescence intensity in samples with or without the compound.
Measurement: IC50 = 300 nM (pChEMBL 6.519999980926514)
Descriptors: MW 801.88, ALogP 3.12, HBA 10, HBD 6, PSA 247.26, QED 0.08
Compound (SMILES): CCOc1ccc(S(=O)(=O)N2C[C@H](n3nc(CC(=O)O)c4ccccc43)[C@H](C)[C@H]2C#N)cn1
InChIKey: YFGHBDWSHJFRHC-XMCHAPAWSA-N
Target: Prostaglandin D2 receptor 2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9Y5Y4
Assay: Receptor Binding Inhibition Assay: Cell membrane was prepared from CRTH2-expressing K562 cell. The cell membrane (20 μg/well) was mixed with reaction buffer (50 mM Tris/HCl, pH 7.4, 10 mM MgCl2). Then, [3H]-PGD2 was added to the mixture and incubated for 60 min at room temperature. The concentration of [3H]-PGD2 was determined as equal to Kd calculated from Scatchard plot. Then, the mixture was filtrated with glass paper immediately. The filter paper was washed several times and radio activity on the filter was measured. Specific binding was defined as difference between total and non-specific binding (radio activity with 10 μM PGD2). Ki values for each compound were calculated from displacement curve.
Measurement: Ki = 2505 nM (pChEMBL 5.599999904632568)
Descriptors: MW 469.52, ALogP 2.23, HBA 8, HBD 1, PSA 138.41, QED 0.56
Compound (SMILES): N#Cc1ccc(COc2cccc(-c3cc(F)c(Cc4nc5ccc(C(=O)O)cc5n4C[C@@H]4CCO4)c(F)c3)n2)c(F)c1
InChIKey: YHWOWBZYTVVUQA-QFIPXVFZSA-N
Target: cAMP and cAMP-inhibited cGMP 3',5'-cyclic phosphodiesterase 10A (SINGLE PROTEIN), Homo sapiens
UniProt: Q9Y233
Assay: Phosphodiesterase 10 (PDE10) Enzyme Activity Assay: PDE10A1 enzyme activities are measured with a yittrium silicate based scintillation proximity assay (SPA) that detects radioactive nucleotide monophosphates but not cyclic monophosphates. The assay buffer is composed of 50 mM Tris-HCl pH 7.5, 8 mM MgCl2, 3.4 mM EDTA, and 0.1% BSA (Sigma). Assays are conducted in 384 well plates (3706, Corning) in a total volume of 50 μl: comprised of 24 μl PDE10A1 enzyme, 1 μl test compound and 25 μl of cyclic nucleotide. Test compounds are diluted in pure DMSO using ten-point concentration response curves with a 3-fold dilution factor and 1 μl is acoustically dispensed into assay plates using the Echo555 (LabCyte). 24 μl PDE10A1 protein is incubated with 1 μl compound for 30 min before the reaction is started by the addition of [8-3H]-cGMP substrate (6.5 Ci/mmol, Perkin Elmer). Final concentration of components is 70 μM PDE10A1, 80 nM (3H-cGMP), and 2% DMSO in assay buffer. Maximal compound concentration in the reaction mixture is 10 μM. Reactions are incubated for 60 min at RT before quenching and the addition of 400 mg/per well SPA beads (RPNQ0150, Perkin Elmer). Bead bound radioactivity (product) is quantified 12 h later with a Microbeta counter (Perkin Elmer). Data is normalized to % inhibition and IC50 values are calculated using the 4 parameter logistic equation as described (Campbell R. M.; Dymshitz, J.; Eastwood, B. J.; et al. Data Standardization for Results Management. In: Sittampalam, G. S.; Grossman, A.; Brimacombe, K.; et al.; eds.<i>Assay Guidance Manual. Bethesda (Md.): Eli Lilly & Company and the National Center for Advancing Translational Sciences; 2004.).
Measurement: IC50 = 5410 nM (pChEMBL 5.269999980926514)
Descriptors: MW 584.55, ALogP 6.04, HBA 7, HBD 1, PSA 110.26, QED 0.22
Compound (SMILES): O=C(NO)c1cccc2c1CCC1(CCN(c3cccc(C(F)(F)F)c3)C1=O)C2
InChIKey: GAJLLVCGGCDVJK-UHFFFAOYSA-N
Target: Histone deacetylase 11 (SINGLE PROTEIN), Homo sapiens
UniProt: Q96DB2
Assay: HDAC Inhibition Assays: This example describes in vitro inhibition properties of exemplary HDAC11 inhibitors for various HDACs. HDAC inhibition assays were performed using an electrophoretic mobility shift assay at Nanosyn, Inc. (Santa Clara, Calif.). Full length human recombinant HDAC proteins were expressed in the baculoviral system and purified by affinity chromatography. A human recombinant HDAC3 was co-expressed with nuclear receptor corepressor (Ncor2). The following peptide substrates were used: FAM-RHKK(Ac)-NH2 for HDAC3, HDAC6 and HDAC8; FITC-H3K27(Ac)-NH2 for HDAC1, HDAC2 and HDAC10; FAM-RHKK(tri-fluor-Ac)-NH2 for HDAC4, HDAC5, HDAC7, HDAC9 and HDAC11. Reactions consisting of compound, enzyme, and substrate were performed in reaction buffer (comprised of 100 mM HEPES, pH7.5, 25 mM KCl, 0.1% bovine serum albumin, 0.01% Triton X-100) at 25° C. and quenched by the addition of termination buffer (100 mM HEPES, pH7.5, 0.01% Triton X-100, 0.05% SDS). The fluorescence intensity of the electrophoretically separated de-acetylated product and substrate peptide were measured and analyzed using the LabChip® 3000 microfluidic electrophoresis instrument (Perkin Elmer/Caliper Life Sciences). The IC50 values of inhibitors were determined by fitting the %-inhibition curves with 4 parameter dose-response model using XLfit 4 software (IDBS).
Measurement: IC50 = 750 nM (pChEMBL 6.119999885559082)
Descriptors: MW 404.39, ALogP 3.74, HBA 3, HBD 2, PSA 69.64, QED 0.59
Compound (SMILES): COc1ccc2c(c1)CCc1sc(NC(=O)Cc3ccc(Oc4ncccc4C(N)=O)cc3)nc1-2
InChIKey: UQKMLEXGSZTUHS-UHFFFAOYSA-N
Target: Rho-associated protein kinase 2 (SINGLE PROTEIN), Homo sapiens
UniProt: O75116
Assay: In Vitro Assay: The effectiveness of compounds of the present invention as ROCK inhibitors can be determined in a 30 μL assay containing 20 mM HEPES, pH 7.5, 20 mM MgCl2, 0.015% Brij-35, 4 mM DTT, 5 μM ATP and 1.5 μM peptide substrate (FITC-AHA-AKRRRLSSLRA-OH) (SEQ ID No. 1). Compounds were dissolved in DMSO so that the final concentration of DMSO was <2%, and the reaction was initiated with Rho kinase variants. After incubation, the reaction was terminated by the addition of EDTA and the phosphorylated and non-phosphorylated peptides separated using a LABCHIP® 3000 Reader (Caliper Life Sciences). Controls consisted of assays that did not contain compound, and backgrounds consisted of assays that contained enzyme and substrate but had EDTA from the beginning of the reaction to inhibit kinase activity. Compounds were tested in dose-response format, and the inhibition of kinase activity was calculated at each concentration of compound. The inhibition data were fit using a curve-fitting program to determine the IC50; i.e., the concentration of compound required to inhibit 50% of kinase activity.
Measurement: IC50 = 21 nM (pChEMBL 7.679999828338623)
Descriptors: MW 486.55, ALogP 4.38, HBA 7, HBD 2, PSA 116.43, QED 0.4
Compound (SMILES): Cn1nc(-c2ccccc2)c(F)c1CSc1cc(N)nc2[nH]nnc12
InChIKey: MTWQGJKSXGDJQH-UHFFFAOYSA-N
Target: Myeloperoxidase (SINGLE PROTEIN), Homo sapiens
UniProt: P05164
Assay: MPO Chlorination (APF) Assay : MPO chlorination activity was measured in 100 mM KPi (pH 7.4) by utilizing the non-fluorescent reagent Aminophenyl fluorescein (APF, Invitrogen catalog #A36003). APF is cleaved by (OCl) to yield the fluorescent compound fluorescein. Reactions were carried out in 50 μL total volume by adding a 25 μL mixture of 200 pM myeloperoxidase and 120 mM NaCl to 100 nL inhibitor in 100% DMSO in a 384 well, non-binding surface clear bottom plate (CORNING® #3655). Enzyme, inhibitor, and chloride were preincubated for ten minutes at rt.After the ten minute preincubation, 25 μL of an APF mixture containing 10 mM APF, 120 mM NaCl and 10 μM H2O2 was added to the plate using the internal dispensing system of a Hammatsu FDSS 6000. Kinetic determinations were carried out immediately on the FDSS 6000 (3 minute kinetic read, 1 read every second, ex: 485 nm, em: 535 nm). IC50 values for inhibitors were calculated by taking the slope of the linear portion of the kinetic measurement (20 seconds to 80-120 secs).IC50 values were calculated by determining the slope of the linear portion of the kinetic trace (180-540 secs), and using that calculated slope to determine % inhibition occurring at each concentration of inhibitor using the following equation:Y = A + B - A 1 + ( C / x ) Dwhere A=minimal Y value (activity level of inhibited sample), B=maximal Y value (activity level of uninhibited sample), C=Log IC50, D=Hill Slope, x=concentration of inhibitor.
Measurement: IC50 < 60 nM (pChEMBL 7.2220001220703125)
Descriptors: MW 355.4, ALogP 2.77, HBA 7, HBD 2, PSA 98.3, QED 0.55