lovingscience/chembl-v37-raw-activities huggingface dataset:
Compound (SMILES): Cc1nnc2c(NC(C)C)nc(-c3nc4cnc(N5CCOCC5)cc4n3Cc3ccc(F)cn3)cn12
InChIKey: KAYFVHVSQSDACO-UHFFFAOYSA-N
Target: Histone H4 (SINGLE PROTEIN), Homo sapiens
UniProt: P62805
Assay: AlphaScreen Assay: Compounds are diluted in serial dilution 1:5 in assay buffer from 10 mM stock in DMSO (100 uM start concentration) in white OptiPlate-384 (PerkinElmer). A mix consisting of 15 nM GST-BRD4-BD1 protein (aa 44-168) or 150 nM GST-BRD4-BD2 (aa 333-460) and 15 nM biotinylated Acetyl-Histone H4 (Lys5, 8, 12, 16) peptide is prepared in assay buffer (50 mM HEPES pH=7.4; 25 mM NaCl; 0.05% Tween 20; 0.1% bovine serum albumin (BSA); 10 mM dithiothreitol (DTT)). 6 ul of the mix is added to the compound dilutions. Subsequently, 6 ul of premixed AlphaLISA Glutathione Acceptor Beads and AlphaScreen Streptavidin Donor Beads from PerkinElmer (in assay buffer at a concentration of 10 ug/ml each) are added and the samples are incubated for 30 min at RT in the dark (shaking 300 rpm). Afterwards, the signal is measured in a PerkinElmer Envision HTS Multilabel Reader using the AlphaScreen protocol from PerkinElmer. Each plate contains negative controls where biotinylated Acetyl-Histone H4 peptide and GST-BRD4-BD1 or GST-BRD4-BD2 are left out and replaced by assay buffer.
Measurement: IC50 = 1 nM (pChEMBL 9)
Descriptors: MW 502.56, ALogP 3.08, HBA 11, HBD 1, PSA 111.18, QED 0.37
Compound (SMILES): COc1ccc(C2(c3ccc(OC)cc3)NC(=N)N(CC34CC5CC(CC(C5)C3)C4)C2=O)cc1
InChIKey: AWHOQSPPRQUTAN-UHFFFAOYSA-N
Target: Beta-secretase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P56817
Assay: Fluorescence Resonance Energy Transfer (FRET) Assay : The recombinant human BACE1, Cathepsin D, and Cathepsin E enzymes were purchased from R&D Systems (catalog numbers are 931-AS, 1014AS and 1294AS, respectively). The enzymatic inhibition activity assays were determined using the fluorescence resonance energy transfer (FRET) assay. The assays were performed in a 384-well plate format. The recombinant human BACE-1 enzyme (R&D Systems, catalog#931-AS) was diluted to 20 ng/μL in assay buffer (100 mM sodium acetate pH 4.0), 10 point 1:3 serial dilutions of compound in DMSO were preincubated with the enzyme for 15 min at room temperature. The concentration of Cathepsin D was 20 ng/μL, and 1 ng/μL of Cathepsin E was used. CatD and CatE were activated by incubation in assay buffer (0.1 M NaOAc, 0.2 M NaCl, pH 3.5) at room temperature for 30 min. Subsequently, the rhBACE-1 substrate (R&D Systems, catalog# ES004), the Cathepsin D and Cathepsin E substrate (R&D Systems, Catalog # ES001) were added accordingly to final concentration
Measurement: IC50 = 7850 nM (pChEMBL 5.110000133514404)
Descriptors: MW 459.59, ALogP 4.53, HBA 4, HBD 2, PSA 74.65, QED 0.67
Compound (SMILES): COc1cc2c(cc1OC)C(=O)/C(=C/c1ccc(N3CCN(c4ccc(C)cc4)CC3)cc1)C2
InChIKey: GYJIYEYMALSDQT-XQNSMLJCSA-N
Target: Cholinesterase (SINGLE PROTEIN), Equus caballus
UniProt: P81908
Assay: Inhibition of equine serum BuChE using butyrylthiocholine iodide as substrate preincubated for 5 mins followed by substrate addition measured after 2 mins by Ellman's method
Measurement: IC50 = 1849 nM (pChEMBL 5.730000019073486)
Descriptors: MW 454.57, ALogP 5.16, HBA 5, HBD 0, PSA 42.01, QED 0.5
Compound (SMILES): CC(=O)N1CCC(C(=O)Nc2nc3cc(C4(O)c5ccccc5C(=O)N4c4cccc(Cl)c4F)ccc3[nH]2)CC1
InChIKey: UDFJOUVBMPMUBP-UHFFFAOYSA-N
Target: RAF proto-oncogene serine/threonine-protein kinase (SINGLE PROTEIN), Homo sapiens
UniProt: P04049
Assay: Inhibition of c-Raf1 (unknown origin) pre-incubated for 30 mins by luciferase-luciferin-coupled chemiluminescence assay
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 562, ALogP 4.41, HBA 5, HBD 3, PSA 118.63, QED 0.34
Compound (SMILES): CN1CCC[C@H]1COc1cc(-c2ccc(O)cc2)cc([N+](=O)[O-])c1
InChIKey: XGWHXWNGYFVPQY-HNNXBMFYSA-N
Target: Neuronal acetylcholine receptor; alpha3/beta4 (PROTEIN COMPLEX), Homo sapiens
UniProt: P32297
Assay: Displacement of [3H]-epibatine from human alpha3beta4-nACHR expressed in HEK243 cell membranes preincubated for 5 mins followed by overnight incubation with [3H]-epibatine by liquid scintillation counting analysis
Measurement: Ki = 122 nM (pChEMBL 6.909999847412109)
Descriptors: MW 328.37, ALogP 3.44, HBA 5, HBD 1, PSA 75.84, QED 0.67
Compound (SMILES): O=C(N[C@H]1CCC[C@@H]1Nc1cnc(C(F)(F)F)cn1)c1ccccc1-n1ccnn1
InChIKey: LPEKTSGWBHVMRT-KBPBESRZSA-N
Target: Orexin receptor type 2 (SINGLE PROTEIN), Homo sapiens
UniProt: O43614
Assay: Fluorescent Imaging Plate Reader (FLIPR) Assay: Orexin antagonist activity was determined by measuring changes in intracellular calcium levels using a Ca2+ sensitive fluorescent dye. The changes in fluorescent signal were monitored by Fluorescent Imaging Plate Reader (FLIPR®) technology available from Molecular Devices, LLC, U.S.A. Orexin mediated increases in intracellular Ca2+ concentration were readily detected upon activation with orexin-A. Twenty-four hours prior to the assay, RBL-2H3 cells stably expressing either human orexin receptor 1 or human orexin receptor 2 were seeded in cell culture medium in black, clear-bottom 384-well plates (commercially available from Corning Inc., U.S.A.) and grown overnight at 37 °C., 5% CO2. On the day of the assay, cell culture media was removed and cells were loaded with Calcium 5 Dye (commercially sold by Molecular Devices, LLC, U.S.A.) for 1 hour at 37 °C., 5% CO2.
Measurement: IC50 > 10000 nM (pChEMBL 5)
Descriptors: MW 417.4, ALogP 2.84, HBA 7, HBD 2, PSA 97.62, QED 0.66
Compound (SMILES): C=CC(=O)Nc1cccc(Cc2nn(C(C)C)c3ncnc(N)c23)c1
InChIKey: DIULQFHOBGDIKM-UHFFFAOYSA-N
Target: Proto-oncogene tyrosine-protein kinase Src (SINGLE PROTEIN), Homo sapiens
UniProt: P12931
Assay: In vitro Kinase Assay: In vitro kinase assays for c-Src variants were performed in 50 mM Tris (pH 8.0), 10 mM MgCl2 and 1 mg/mL BSA. When obtaining kinetic parameters (kcat, Km) kinase and peptide substrate (IYGEFKKK) (SEQ ID NO:51) concentrations were 2 nM and 500 μM, respectively, while ATP concentrations ranged from 2000-0.655 μM. Addition of nonradioactive ATP supplemented with 32P ATP (3,000 Ci/mmol, NEN) was used to initiate kinase reactions. Time points were selected such that product formation never exceeded 10%. Reactions were quenched by spotting 3 μL quantities onto phosphocellulose sheets (P81, Whatman). Afterwards, the sheets were washed 3×5 minutes in 0.5% phosphoric acid and dried. Radioactivity was measured by phosphorimaging and recorded on a Typhoon fluorescence imager (Molecular Dynamics).
Measurement: IC50 = 2762 nM (pChEMBL 5.559999942779541)
Descriptors: MW 336.4, ALogP 2.7, HBA 6, HBD 2, PSA 98.72, QED 0.7
Compound (SMILES): Cl.O=C(O)[C@]1(Cc2cccc(Nc3nccs3)n2)CC[C@@H](Oc2cccc(C(F)(F)F)c2)CC1
InChIKey: CUZPKOJQNROUQM-UBTQVQQGSA-N
Target: Aurora kinase A (SINGLE PROTEIN), Homo sapiens
UniProt: O14965
Assay: Inhibition of human N-terminal His-tagged Aurora A expressed in Escherichia coli BL21-Codon-Plus(DE3)-RIL cells using 5-carboxy-fluorescein-y-aminobutyric acid-Ala-Leu-Arg-Arg-Ala-Ser-Leu-Gly-NH2 as substrate after 120 mins by fluorescence polarization assay
Measurement: IC50 = 0.17 nM (pChEMBL 9.770000457763672)
Descriptors: MW 477.51, ALogP 5.94, HBA 6, HBD 2, PSA 84.34, QED 0.44
Compound (SMILES): Cn1c(NCCc2ccc(-c3ncc(Cl)cn3)cc2)nc2cc(Cl)ccc2c1=O
InChIKey: ASEAOQHLLWXLNA-UHFFFAOYSA-N
Target: Cytochrome P450 3A4 (SINGLE PROTEIN), Homo sapiens
UniProt: P08684
Assay: Inhibition of CYP3A4 in human liver microsomes assessed as 1'-hydroxymidazolam metabolite formation using midazolam as substrate incubated for 10 mins by HPLC-MS method
Measurement: IC50 > 30000 nM (pChEMBL 4.5229997634887695)
Descriptors: MW 426.31, ALogP 4.35, HBA 6, HBD 1, PSA 72.7, QED 0.51
Compound (SMILES): N#CN1CCc2c(C(=O)N3CCC(c4cc(F)cc(F)c4C(F)(F)F)CC3)n[nH]c2C1
InChIKey: VGWBIQACZZJMGX-UHFFFAOYSA-N
Target: Retinol-binding protein 4 (SINGLE PROTEIN), Homo sapiens
UniProt: P02753
Assay: TR-FRET Assay: TR-FRET assay for retinol-induced RBP4-TTR interaction Binding of a desired RBP4 antagonist displaces retinol and induces hindrance for RBP4-TTR interaction resulting in the decreased FRET signal (FIG. 7). Bacterially expressed MBP-RBP4 and untagged TTR were used in this assay. For the use in the TR-FRET assay the maltose binding protein (MBP)-tagged human RBP4 fragment (amino acids 19-201) was expressed in the Gold(DE3)pLysS E. coli strain (Stratagene) using the pMAL-c4x vector. Following cell lysis, recombinant RBP4 was purified from the soluble fraction using the ACTA FPLC system (GE Healthcare) equipped with the 5-ml the MBP Trap HP column. Human untagged TTR was purchased from Calbiochem. Untagged TTR was labeled directly with Eu3+ Cryptate-NHS using the HTRF Cryptate Labeling kit from CisBio following the manufacturer's recommendations. HTRF assay was performed in white low volume 384 well plates (Greiner-Bio) in a final assay volume of 16 ul per well. The reaction buffer contained 10 mM Tris-HCl pH 7.5, 1 mM DTT, 0.05% NP-40, 0.05% Prionex, 6% glycerol, and 400 mM KF. Each reaction contained 60 nM MBP-RBP4 and 2 nM TTR-Eu along with 26.7 nM of anti-MBP antibody conjugated with d2 (Cisbio). Titration of test compounds in this assay was conducted in the presence of 1 uM retinol. All reactions were assembled in the dark under dim red light and incubated overnight at +4° C. wrapped in aluminum foil. TR-FRET signal was measured in the SpectraMax M5e Multimode Plate Reader (Molecular Device). Fluorescence was excited at 337 nm and two readings per well were taken: Reading 1 for time-gated energy transfer from Eu(K) to d2 (337 nm excitation, 668 nm emission, counting delay 75 microseconds, counting window 100 microseconds) and Reading 2 for Eu(K) time-gated fluorescence (337 nm excitation, 620 nm emission, counting delay 400 microseconds, counting window 400 microseconds).
Measurement: IC50 = 12 nM (pChEMBL 7.920000076293945)
Descriptors: MW 439.39, ALogP 3.57, HBA 4, HBD 1, PSA 76.02, QED 0.57
Compound (SMILES): CC(=O)N[C@@H](Cc1ccccc1)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CO)C(=O)N[C@@H](CCCCN)C(=O)NCC(=O)N[C@@H](CC(N)=O)C(=O)N[C@H](C(=O)N[C@@H](Cc1ccccc1)C(=O)N[C@H](C(=O)N[C@@H](CCCCN)C(=O)NCC(=O)N[C@@H](Cc1ccc(O)cc1)C(=O)N[C@@H](Cc1ccccc1)C(=O)N[C@@H](CC(=O)O)C(=O)N[C@@H](CCCNC(=N)N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCNC(=N)N)C(=O)N[C@@H](C)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC(C)C)C(N)=O)C(C)C)C(C)C
InChIKey: UHVQTPLSPRSFMD-GOYLXPRVSA-N
Target: Tissue factor pathway inhibitor (SINGLE PROTEIN), Homo sapiens
UniProt: P10646
Assay: Binding affinity to TFPI (unknown origin) incubated for 1.5 hrs using tracer peptide JBT0131 by screening ELISA
Measurement: IC50 = 66 nM (pChEMBL 7.179999828338623)
Descriptors: MW 2414.85
Compound (SMILES): CCOc1cc(=O)n(C)cc1-c1cc(N(Cc2cncnc2)S(=O)(=O)Cc2ccccc2)ccc1Oc1ccc(F)cc1F
InChIKey: ISJULVVCCSFJRX-UHFFFAOYSA-N
Target: Bromodomain-containing protein 4 (SINGLE PROTEIN), Homo sapiens
UniProt: O60885
Assay: Time-Resolved Fluorescence Resonance Energy Transfer (TR-FRET) Assay: Assay method C: Compound dilution series were prepared in DMSO via an approximately 3-fold serial dilution from one of the following: 0.047 mM to 0.78 nM or 5-fold serial dilution from Assay method A. Compound dilutions were added directly into white, low-volume assay plates (Perkin Elmer Proxiplate 384 Plus# 6008280) using a Labcyte Echo in conjunction with Labcyte Access and Thermo Multidrop CombinL robotics. Compounds were then suspended in eight microliters (μL) of assay buffer (20 mM Sodium Phosphate, pH 6.0, 50 mM NaCl, 1 mM Ethylenediaminetetraacetic acid disodium salt dihydrate, 0.01% Triton X-100, 1 mM DL-Dithiothreitol) containing His-tagged bromodomain, Europium-conjugated anti-His antibody (Invitrogen PV5596) and Alexa-647-conjugated probe. The final concentration of 1X assay mixture for assay methods A, B and C contains 2% DMSO, 8 nM His-tagged bromodomain, 1 nM Europium-conjugated anti-His-tag antibody and 100 nM or 30 nM probe (for BDI or BDII, respectively) and compound concentration in the range of: 49.02 μM-15.63 nM for method A, 9.19 μM 150 pM for method B, and 0.92 μM 15 pM for method C. After a one-hour equilibration at room temperature, TR-FRET ratios were determined using an Envision multilabel plate reader (Ex 340, Em 495/520).
Measurement: Ki = 266 nM (pChEMBL 6.579999923706055)
Descriptors: MW 618.66, ALogP 5.85, HBA 8, HBD 0, PSA 103.62, QED 0.18
Compound (SMILES): FC(F)(F)c1ccc(Nc2nc(-c3ccncc3)nc3c2CCN(c2ncccc2C(F)(F)F)CC3)cc1Cl
InChIKey: YKZGDXYKEQTAFA-UHFFFAOYSA-N
Target: Transient receptor potential cation channel subfamily V member 1 (SINGLE PROTEIN), Rattus norvegicus
UniProt: O35433
Assay: Functional Assay: The assay was performed as described below, using HEK293 cells transfected with rat TRPV1 (rTRPV1/HEK293). These cells had a geneticin selection marker and were grown in Dulbecco's Modified Eagle Medium (DMEM, Gibco BRL) supplemented with 10% fetal bovine serum, penicillin/streptomycin (50 units/mL), and 500 μg/mL geneticin in 5% CO2 at 37° C. Cells were passaged frequently, every 3-5 days, to avoid overgrowth, depletion of essential medium components, or acidic medium exposure. Cells were passaged using a brief wash in 0.05% trypsin with 1 mM EDTA, followed by dissociation in divalent-free phosphate-buffered saline (Hyclone #SH30028.02). Dissociated cells were seeded onto poly-D-lysine coated black-walled 96-well plates (Biocoat; Becton Dickinson #354640) at about 40,000 cells per well and grown for approximately 1 day in culture medium to near confluency. The assay buffer was composed of 130 mM NaCl, 2 mM KCl, 2 mM MgCl2, 10 mM HEPES, 5 mM glucose, and either 2 mM or 20 μM CaCl2. On the day of the experiment, the culture medium was replaced with 2 mM calcium assay buffer using an automated plate washer (ELx405; Biotek, VT). The cells were incubated in 100 μL/well Fluo-3/AM (2 μM; TEFLabs #0116) with Pluronic F127 (100 μg/mL; Sigma #P2443) for 1 h at rt in the dark. After loading the cells, the dye solution was replaced with 50 μL/well of 20 μM calcium assay buffer using the ELx405 plate washer. Test compounds (50 μL/well) were added to the plate and incubated for 30 min. Intracellular Ca2+ levels were subsequently assayed using a Fluorometric Imaging Plate Reader (FLIPR™ instrument, Molecular Devices, CA) to simultaneously monitor Fluo-3 fluorescence in all wells (λexcitation=488 nm, λemission=540 nm) during challenge with agonist (capsaicin). The IC50 values were determined. Cells were challenged with 150 nM capsaicin and the fluorescence counts were captured following agonist addition at a sampling rate of 0.33 Hz. The contents of the wells were mixed 3 times (40 μL mix volume) immediately after the additions were made. Concentration dependence of block was determined by exposing each well of cells in duplicate rows of a 96 well plate to a serial dilution of test compound. The concentration series usually started at 10 Î¼M with a three-fold serial decrement in concentration. The magnitude of the capsaicin response was determined by measuring the change in fluo3 fluorescence before and 100 seconds after the addition of the agonist.
Measurement: IC50 = 1010 nM (pChEMBL 6)
Descriptors: MW 564.92, ALogP 6.97, HBA 6, HBD 1, PSA 66.83, QED 0.27
Compound (SMILES): CC(C)C[C@H](NC(=O)[C@H](CO)NC(=O)[C@H](C/C=C/c1ccccc1)NC(=O)[C@@H]1CCCN1C(=O)[C@@H](N)CCCCN)C(=O)N[C@@H](CO)C(=O)N[C@@H](Cc1ccc(O)cc1)C(=O)N[C@@H](CCCNC(=N)N)C(=O)N[C@@H](CO)C(=O)N[C@@H](C)C(=O)NCCCC[C@H](NC(=O)[C@@H]1CSSC[C@H](NC(=O)[C@H](Cc2ccc3ccccc3c2)NC(=O)[C@H](CCCNC(=N)N)NC(=O)[C@@H](N)CCCNC(=N)N)C(=O)N[C@@H](Cc2ccc(O)cc2)C(=O)N[C@@H](CCCNC(=N)N)C(=O)N[C@@H](CCCCN)C(=O)N[C@H](CCCCN)C(=O)N2CCC[C@H]2C(=O)N[C@@H](Cc2ccc(O)cc2)C(=O)N[C@@H](CCCNC(=N)N)C(=O)N[C@@H](CCCNC(=N)N)C(=O)N1)C(=O)O
InChIKey: GDCBENLGSFGYIL-ZNWVDKDVSA-N
Target: C-X-C chemokine receptor type 4 (SINGLE PROTEIN), Homo sapiens
UniProt: P61073
Assay: Agonist activity at HA-tagged human CXCR4 receptor expressed in HEK293 cells co-transfected with Galphai1-91-RlucII, Gbeta1, and GFP10-G-gamma1 incubated for 7 mins by Galpha1 protein activation-based BRET assay
Measurement: EC50 = 50 nM (pChEMBL 7.300000190734863)
Descriptors: MW 3171.81
Compound (SMILES): CNC(=O)c1c(-c2ccc(F)cc2)oc2cc(N(C)S(C)(=O)=O)c(-c3ccc4c(n3)-c3cc5c(F)cccc5n3C(CO)C4)cc12
InChIKey: NWORADADRBBBRF-UHFFFAOYSA-N
Target: Genome polyprotein (SINGLE PROTEIN), Hepatitis C virus genotype 1a (isolate H) (HCV)
UniProt: P27958
Assay: Inhibition Assay: To initiate an assay, replicon cells were plated in the presence of a dilution series of test compound in the absence of G418. Typically, the assays were performed in a 96-well plate formate for manual operation, or a 384-well plate format for automated assay. Replicon cells and compound were incubated for 96 hours. At the end of the assay, cells were washed free of media and compound, and the cells were then lysed. RNA was quantified indirectly through detection of replicon-encoded NS3/4A protein levels, through an ELISA-based assay with an antibody specific for NS3/4A.
Measurement: IC50 = 8.069 nM (pChEMBL 8.09000015258789)
Descriptors: MW 642.68, ALogP 5.91, HBA 7, HBD 2, PSA 117.67, QED 0.23
Compound (SMILES): CCOC(=O)c1c(NC(=O)c2cccc3ccccc23)sc2c1CCCC2
InChIKey: SHEYBSXUFWAXMD-UHFFFAOYSA-N
Target: Cannabinoid receptor 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P21554
Assay: Displacement of [3H]-CP-55940 from human CB1 receptor expressed in HEK293 cell membranes after 90 mins by radioligand binding assay
Measurement: Ki = 1373.03 nM (pChEMBL 5.860000133514404)
Descriptors: MW 379.48, ALogP 5.21, HBA 4, HBD 1, PSA 55.4, QED 0.63
Compound (SMILES): CCCCC(O)(c1ccc2nc(Cl)c(-c3ccccc3)c(Cl)c2c1)c1cncn1C
InChIKey: GAMJCHAOPZZQJQ-UHFFFAOYSA-N
Target: Nuclear receptor ROR-gamma (SINGLE PROTEIN), Homo sapiens
UniProt: P51449
Assay: Reporter Assay: The reporter assay was performed by transiently transfecting HEK293T cells with 5 μg of pBIND-RORγt LBD or pBIND-RORγt LBD-AF2 and 5 μg pGL4.31 (Promega Cat no. C935A) using Fugene 6 (Invitrogen Cat no. E2691) at a 1:6 ratio of DNA:Fugene 6 in a T-75 flask in which cells were at least 80% confluent. Twenty four hours after bulk transfection, cells were plated into 96-well plates at 50,000 cells/well in phenol-red free DMEM containing 5% Lipid Reduced FCS and Pen/Strep. Six hours after plating, cells were treated with compounds for 24 hours. Media was removed and cells were lysed with 50 μL 1× Glo Lysis Buffer (Promega). Dual Glo Luciferase Reagent (50 μL/well) was then added and firefly luciferase luminescence was read on an Envision after a ten minute incubation. Finally, Stop and Glo reagent (50 μL/well) was added and renilla luciferase luminescence was read on an Envision after a ten minute incubation.
Measurement: IC50 = 1500 nM (pChEMBL 5.820000171661377)
Descriptors: MW 440.37, ALogP 6.37, HBA 4, HBD 1, PSA 50.94, QED 0.35
Compound (SMILES): N=C(N)NCCCC[C@H]1C(=O)N(CCc2ccc3ccccc3c2)C[C@@H]2SC[C@H](CCNC(=N)N)C(=O)N21
InChIKey: FPAHXUWMNPQZDD-VABKMULXSA-N
Target: C-X-C chemokine receptor type 4 (SINGLE PROTEIN), Homo sapiens
UniProt: P61073
Assay: Antagonist activity at wild-type human CXCR4 expressed in COS7 cells co-expressing chimeric galphai assessed as inhibition of CXCL12-induced [3H]inositol-phosphate accumulation measured after 90 mins in presence of Myo-[2-3H]-inositol by scintillation proximity assay
Measurement: EC50 > 100000 nM (pChEMBL 4)
Descriptors: MW 538.72, ALogP 1.64, HBA 5, HBD 6, PSA 164.42, QED 0.14
Compound (SMILES): C[C@@H](N)C(=O)NCc1nnc(-c2ccccc2)o1
InChIKey: CLUVLSXNTNWGTA-MRVPVSSYSA-N
Target: Histone deacetylase 2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q92769
Assay: Inhibition of HDAC2 in human MDA-MB-231 cells assessed as inhibition of deacetylation activity using Fluor-de-Lys as substrate measured after 15 mins by fluorimetric assay
Measurement: IC50 = 135 nM (pChEMBL 6.869999885559082)
Descriptors: MW 246.27, ALogP 0.7, HBA 5, HBD 2, PSA 94.04, QED 0.83
Compound (SMILES): COc1cn(C(C[C@H](C)OC)C(=O)Nc2ccc(C(=O)O)cc2)c(=O)cc1-c1cc(Cl)ccc1C#N
InChIKey: DYNSAJCVHKUDFF-UEDXYCIISA-N
Target: Coagulation factor XI (SINGLE PROTEIN), Homo sapiens
UniProt: P03951
Assay: Inhibition Assay: Test substances are dissolved in dimethyl sulphoxide and serially diluted in dimethyl sulphoxide (3000 μM to 0.0078 μM; resulting final concentrations in the test: 50 μM to 0.00013 μM). In each case 1 μl of the diluted substance solutions are placed into the wells of white microtitre plates from Greiner (384 wells). 20 μl of assay buffer (50 mM of Tris/HCl pH 7.4; 100 mM of sodium chloride solution; 5 mM of calcium chloride solution; 0.1% of bovine serum albumin) and 20 μl of factor XIa from Kordia (0.45 nM in assay buffer) are then added successively. After 15 min of incubation, the enzyme reaction is started by addition of 20 μl of the factor XIa substrate Boc-Glu(OBzl)-Ala-Arg-AMC dissolved in assay buffer (10 μM in assay buffer) from Bachem, the mixture is incubated at room temperature (22° C.) for 30 min and fluorescence is then measured (excitation: 360 nm, emission: 460 nm).
Measurement: IC50 = 0.7 nM (pChEMBL 9.149999618530273)
Descriptors: MW 509.95, ALogP 4.35, HBA 7, HBD 2, PSA 130.65, QED 0.44
Compound (SMILES): Cn1ccnc1C(=O)Nc1ccc(-c2n[nH]c(=O)c3ccccc23)cc1
InChIKey: SZJSNCJNTTVKBE-UHFFFAOYSA-N
Target: Rho-associated protein kinase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q13464
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). 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.
Measurement: IC50 = 544 nM (pChEMBL 6.260000228881836)
Descriptors: MW 345.36, ALogP 2.58, HBA 5, HBD 2, PSA 92.67, QED 0.6
Compound (SMILES): CC(=O)c1ccc(OCc2ccc(F)cc2)cc1F
InChIKey: GISSMJZWKYCPSI-UHFFFAOYSA-N
Target: Amine oxidase [flavin-containing] A (SINGLE PROTEIN), Homo sapiens
UniProt: P21397
Assay: Inhibition of recombinant human MAO-A using p-tyramine as substrate assessed as decrease in H2O2 production incubated for 15 mins by fluorimetric method
Measurement: IC50 > 100000 nM (pChEMBL 4)
Descriptors: MW 262.25, ALogP 3.75, HBA 2, HBD 0, PSA 26.3, QED 0.78
Compound (SMILES): Cn1cc(-c2ccc3nc(NC(=O)C4CC5CC46CC56)cn3n2)c(-c2ccc(F)cc2)n1
InChIKey: OHSJWPUMSBSYCT-UHFFFAOYSA-N
Target: Casein kinase I isoform delta (SINGLE PROTEIN), Homo sapiens
UniProt: P48730
Assay: Kinase Assay: The kinase assay was performed in V-bottom 384-well plates. The final assay volume was 30 ul prepared from 15 ul additions of enzyme, substrates (fluoresceinated peptide FL-AHA-KRRRAL-PSER-VASLPGL-OH and ATP) and test compounds in assay buffer (20 mM HEPES pH 7.4, 30 mM MgCl2, 0.015% Brij35 and 4 mM DTT). The reaction was incubated at room temperature for 22 hours and terminated by adding 45 ul of 35 mM EDTA to each sample. The reaction mixture was analyzed on the Caliper LABCHIPÂ®3000 (Caliper, Hopkinton, Mass.) by electrophoretic separation of the unphosphorylated substrate and phosphorylated product Inhibition data were calculated by comparison of the no enzyme control reactions for 100% inhibition and vehicle-only reactions for 0% inhibition. The final concentration of reagents in the assay were 200 pM CK1ε or CK1δ, 50 uM ATP, 1.5 uM FL-AHA-KRRRAL-PSER-VASLPGL-OH, and 1.6% DMSO. Dose response curves were generated to determine the concentration required to inhibit 50% of the 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 = 7.4 nM (pChEMBL 8.130000114440918)
Descriptors: MW 428.47, ALogP 3.92, HBA 6, HBD 1, PSA 77.11, QED 0.53
Compound (SMILES): CC(C)(C)OC(=O)N1CCC(CC#N)(n2cc(C(N)=O)c(Nc3ccc(F)c(F)c3)n2)CC1
InChIKey: VJBPDUSOWSUMHX-UHFFFAOYSA-N
Target: Tyrosine-protein kinase JAK2 (SINGLE PROTEIN), Homo sapiens
UniProt: O60674
Assay: HTRF Biochemical Assay: The reactions employed a common peptide substrate, LCB-EQEDEPEGDYFEWLW-NH2 (in-house). The basic assay protocol is as follows: First, 250 nL of diluted compounds in DMSO were dispensed into the wells of a dry 384-well Black plate (Greiner #781076) using a Labcyte Echo 555 acoustic dispenser. Subsequent reagent additions employed an Agilent Bravo. Next, 18 uL of 1.11x enzyme and 1.11x substrate in 1x assay buffer (Invitrogen kinase buffer # PV3189, 2 mM DTT, 0.05% BSA) were added to the wells and shaken and then preincubated for 30 minutes at room temperature to allow compound binding to equilibrate. After equilibration, 2 uL of 10xATP in 1x assay buffer was added to initiate the kinase reaction and the plates were shaken and then incubated at room temperature for 120 minutes. At the end of the incubation, 20 uL of 2x stop buffer (streptavidin-Dylight 650 (Thermo #84547B/100 mL), Eu-tagged pY20 antibody (Perkin Elmer #AD0067), EDTA, HEPES, and Triton) was added to quench the reaction.
Measurement: IC50 = 24 nM (pChEMBL 7.619999885559082)
Descriptors: MW 460.49, ALogP 3.64, HBA 7, HBD 2, PSA 126.27, QED 0.7
Compound (SMILES): CCC(C)NC(=O)N(CCN1[C@@H]2CC[C@H]1C[C@@H](c1cccc(C(N)=O)c1)C2)CC1CCCCC1
InChIKey: BMKPXMBYJOSBBK-KJASELGPSA-N
Target: Delta-type opioid receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P41143
Assay: Displacement of [3H]-diprenorphine from human delta opioid receptor expressed in cell membranes
Measurement: Ki = 100 nM (pChEMBL 7)
Descriptors: MW 468.69, ALogP 4.89, HBA 3, HBD 2, PSA 78.67, QED 0.54
Compound (SMILES): O=C(CCl)OCCn1c(=O)oc2cc3ncnc(Nc4ccc(Oc5cccc(C(F)(F)F)c5)c(Cl)c4)c3cc21
InChIKey: OSYFQPJNLCUQEU-UHFFFAOYSA-N
Target: Epidermal growth factor receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P00533
Assay: Inhibition of GST-tagged wild type recombinant human EGFR (668 to 1210 residues) expressed in baculovirus after 1 hr by Z-lyte assay
Measurement: IC50 = 510 nM (pChEMBL 6.289999961853027)
Descriptors: MW 593.35, ALogP 6.53, HBA 9, HBD 1, PSA 108.48, QED 0.16
Compound (SMILES): CN(CO)Cc1csc2c(-c3cnc(N)nc3)nc(N3CCOCC3)nc12
InChIKey: FYZFCLCVNWDVMC-UHFFFAOYSA-N
Target: Phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit delta isoform (SINGLE PROTEIN), Homo sapiens
UniProt: O00329
Assay: Enzymatic Inhibitory Activity Assay:   1. Buffer preparation: 50 mM HEPES, pH 7.5, 3 mM MgCl2, 1 mM EGTA, 100 mM NaCl, 0.03% CHAPS.  2. Compound was formulated in 100% DMSO in a concentration gradient, deposited to a 384-well plate to make final DMSO concentration of 1%.  3. PI3Kα and PI3Kδ enzymes were diluted to be the optimum concentration with the following buffer: 50 mM HEPES, pH 7.5, 3 mM MgCl2, 1 mM EGTA, 100 mM NaCl, 0.03% CHAPS, 2 mM DTT. Transferred to a 384-well plate and incubated with the compound for a certain time.  4. Substrate was diluted to an optimum concentration with following buffer: 50 mM HEPES, pH 7.5, 3 mM MgCl2, 1 mM EGTA, 100 mM NaCl, 0.03% CHAPS, 2 mM DTT, 50 μM PIP2, Km ATP. The reaction was performed in a 384-well plate for 1 h at room temperature for PI3Kα and 2 hrs at room temperature for PI3Kδ.  5. Read the conversion rate using Caliper Reader, and calculate the inhibition rate as the average of two tests.
Measurement: IC50 = 137 nM (pChEMBL 6.860000133514404)
Descriptors: MW 387.47, ALogP 0.95, HBA 10, HBD 2, PSA 113.52, QED 0.62
Compound (SMILES): CCOCc1c(-c2cc3cc(C)cc(OC)c3s2)c2c(N)ncnn2c1CNCC(N)=O
InChIKey: PIQRHUGIGRKALL-UHFFFAOYSA-N
Target: Fibroblast growth factor receptor 3 (SINGLE PROTEIN), Homo sapiens
UniProt: P22607
Assay: Kinase Assay: For the assay, 50 nl of each stock solution of the test compound in DMSO was pipetted into a black, low-volume 384-well microtiter plate (Greiner Bio-One, Frickenhausen, Germany) 2 ul of a solution of the above FGFR-3 fusion protein in aqueous assay buffer [8 mM MOPS pH 7.0, 10 mM magnesium acetate, 1.0 mM dithiothreitol, 0.05% (w/v) bovine serum albumin (BSA), 0.07% (v/v) Tween-20, 0.2 mM EDTA] was added, and the mixture was incubated for 15 min at 22° C. to allow pre-binding of the test compound to the enzyme. Then, the kinase reaction was started by the addition of 3 ul of a solution of adenosine triphosphate (ATP, 16.7 uM; final concentration in the 5 ul assay volume=10 uM) and substrate (0.8 uM; final concentration in the 5 ul assay volume=0.5 uM) in assay buffer, and the resulting mixture was incubated for a reaction time of 60 min at 22° C. The concentration of FGFR-3 fusion protein was adjusted depending on the activity of the enzyme lot.
Measurement: IC50 = 63.9 nM (pChEMBL 7.190000057220459)
Descriptors: MW 454.56, ALogP 2.62, HBA 9, HBD 3, PSA 129.79, QED 0.35
Compound (SMILES): COc1cc(/C=C2\SC(=S)NC2=O)cc(OC)c1O
InChIKey: PCIQZJDFKLRXIO-UITAMQMPSA-N
Target: CDGSH iron-sulfur domain-containing protein 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9NZ45
Assay: Displacement of [3H]rosiglitazone from recombinant human C-terminal His-tagged MitoNEET cytosolic domain (32 to 108 residues) expressed in Escherichia coli BL21 by Cheng-Prusoff analysis
Measurement: Ki = 191 nM (pChEMBL 6.71999979019165)
Descriptors: MW 297.36, ALogP 1.9, HBA 6, HBD 2, PSA 67.79, QED 0.66
Compound (SMILES): COCC(COC1(Cn2ncc(-c3ccc(N4CCc5cccc(C(=O)Nc6nc7ccccc7s6)c5C4)nc3C(=O)O)c2C)CCCCCC1)OC
InChIKey: CGTIQFGPEXLTCC-UHFFFAOYSA-N
Target: Bcl-2-like protein 1 (SINGLE PROTEIN), Mus musculus
UniProt: Q64373
Assay: FL5.12 Cellular Assay: The efficacy of the compounds of Formula (I) can also be determined in cell-based killing assays using a variety of cell lines and mouse tumor models. For example, their activity on cell viability can be assessed on a panel of cultured tumorigenic and non-tumorigenic cell lines, as well as primary mouse or human cell populations. In one exemplary set of conditions, mouse FL5.12 cells transfected with Bcl-XL were cultured under standard conditions in RPMI with 2 mM glutamine, 1% 100 mM sodium pyruvate, 2% 1 M HEPES, 4 uL/L of beta;-mercaptoethanol, 1% penicillin-streptomycin, 10% FBS, and 10% WEHI-3B conditioned media (for IL-3). For assaying the compound activity, the cells were exchanged into an IL-3-depleted deprivation media, which was identical to the growth media except for the absence of FBS and WEHI-3B conditional media, for 2 days. Then the cells were exchanged to 3% FBS assay media (RPMI with 2 mM glutamine, 1% 100 mM sodium pyruvate, 2% 1 M HEPES, 4 uL/L of beta;-mercaptoethanol, 1% penicillin-streptomycin, 10% FBS, and 10% WEHI-3B conditioned media (for IL-3).
Measurement: EC50 = 0.5 nM (pChEMBL 9.300000190734863)
Descriptors: MW 738.91, ALogP 7.15, HBA 11, HBD 2, PSA 140.93, QED 0.12
Compound (SMILES): COc1ccc(S(=O)(=O)Nc2ccc(F)c(Nc3ncccc3-c3ncnc4[nH]cnc34)c2F)cc1OC
InChIKey: KTMMOPBQRGMNBC-UHFFFAOYSA-N
Target: Serine/threonine-protein kinase B-raf (SINGLE PROTEIN), Homo sapiens
UniProt: P15056
Assay: Cell Activity Inhibition Assay: To check the B-Raf cell activity inhibitory capability of the compounds of the present invention, the following experiments were conducted in A375P cell line (ATCC). The A375P cell line (ATCC) was derived from a patient with human melanoma, and had V600E mutant of B-Raf gene. A375 cell was maintained in DMEM supplemented with 10% fetal bovine serum, glutamine (2 mM), penicillin (100 U/mL), and streptomycin (100 ug/mL). The cell was maintained at 37° C., and in 5% CO2 and 100% humidity. To conduct the growth inhibition test, the cell was plated by using a white 384-well microplate, with 1000 cells/20 Î¼l plated per 1 well. After 24 hours, 5 ul of 5x stock solution of drug was added. All the drugs were initially prepared with 200x stock solution in DMSO, and the final concentration of DMSO was 0.5%. The cell was incubated at 37° C. for 72 hours. For MTT assay, 'CellTiter 96 (R) Non-Radioactive Cell Proliferation Assay (G4100)â¿¿ kit by Promega Corp. was used.
Measurement: EC50 = 10200 nM (pChEMBL 4.989999771118164)
Descriptors: MW 539.52, ALogP 4.25, HBA 9, HBD 3, PSA 144.01, QED 0.26
Compound (SMILES): COc1cccc([C@@H]2C[C@H]2C(=O)N/N=C/c2cc(C)c3ncccc3c2)n1
InChIKey: UEAGEEKWWUCZEW-RFTFHOITSA-N
Target: Transient receptor potential cation channel subfamily M member 5 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9NZQ8
Assay: Inhibition of human TRPM5 transfected in HEK293 cells assessed as inhibition of KCl-stimulated membrane potential by FLIPR assay
Measurement: IC50 = 410 nM (pChEMBL 6.389999866485596)
Descriptors: MW 360.42, ALogP 3.2, HBA 5, HBD 1, PSA 76.47, QED 0.56
Compound (SMILES): CNS(=O)(=O)c1ccc(OC/C(=C\F)CN)cc1
InChIKey: RVCAAPOJOTYELI-TWGQIWQCSA-N
Target: Amine oxidase [copper-containing] 3 (SINGLE PROTEIN), Homo sapiens
UniProt: Q16853
Assay: Recombinant SSAO/VAP-1 Inhibtion Assay: Briefly, HMEC cell expressing human SSAO/VAP-1 were grown in several 10 cm petri dishes, once the cells reached 100% confluency, cells were harvested and homogenates were prepared. Cells were washed twice with 5 mL of chilled HES buffer (20 mM HEPES, 1 mM EDTA, 250 mM sucrose, pH 7.4). HES buffer containing 1× protease inhibitor (Sigma Aldrich) and added and cells were incubated on ice for 3 min. Buffer was removed and cells were scraped and transferred to a centrifuge tube. Cell lysates were prepared by passing through 23 G needle for, 10 times and followed by 27 G needle for 10 times. Alternatively the cell lysates were prepared by using IKA Ultra-Turrax T 10 homogenizer for 3 min for every 10 mL of cell suspensions. Cells were then spun for 5 min at 300×g. The clear supernatant was transferred to new centrifuge tube and stored at −80 °C. until colorimetric assay was performed. Prior to the assay, 0.5 mM pargyline was added in order to inhibit any residue MAO activities. Briefly, 50 μL of cell lysate was incubated with test compounds for 30 min at 37 °C. Reaction mixtures were added and kinetic was read as described in detail in Example 5: Briefly, in a standard 96 well plate assay 50 μL of purified human SSAO/VAP-1 (0.25 μg/mL) in 0.1 M NaPO4 buffer (pH 7.4) was added into each well. Test compounds were dissolved in DMSO and tested in a Concentration Response Curve (CRC) with 4-9 data points, typically in the micromolar or nanomolar range after incubation with human SSAO/VAP-1 for 30 min at 37 °C. After 30 min incubation, 50 μL of the reaction mixture containing 600 μM benzylamine (Sigma Aldrich), 120 μM Amplex Red (Sigma Aldrich) and 1.5 U/mL horseradish peroxidase (Sigma Aldrich) prepared in 0.1 M NaPO4 buffer (pH 7.4) were added to the corresponding well. The fluorescence unit (RFU) was read every 2.5 min for 30 min at 37 °C. excitation 565 nm and emission 590 (Optima; BMG labtech). The slope of the kinetics for each well was calculated using MARS data analysis software (BMG labtech) and this value was used to deduce the IC50 value (Dotmatics).
Measurement: IC50 < 100000 nM (pChEMBL 4)
Descriptors: MW 274.32, ALogP 0.79, HBA 4, HBD 2, PSA 81.42, QED 0.8
Compound (SMILES): COc1nc(C#CC2CCCC2)c(Cl)cc1-n1c(=O)ccc2cc(S(=O)(=O)Nc3cccnn3)ccc21
InChIKey: URWQYWNFWZQLIV-UHFFFAOYSA-N
Target: Sodium channel protein type 9 subunit alpha (SINGLE PROTEIN), Homo sapiens
UniProt: Q15858
Assay: Nav1.7 In Vitro PX Assay: HEK 293 cells stably transfected with human Nav1.7 were recorded in whole cell voltage clamp mode with the PatchXpress automated electrophysiology system (Molecular Devices, LLC, Sunnyvale, Calif.). Compound effects were measured on a partially inactivated state of the sodium channel. Cells were clamped to a holding potential yielding 20 to 50% inactivation. To elicit sodium current, channels were activated by pulsing to -10 mV for 20 msec. This voltage protocol was repeated at a rate of 0.1 Hz throughout the experiment. A single concentration of test compound was applied to cells for a duration of 3 minutes.
Measurement: IC50 = 124 nM (pChEMBL 6.909999847412109)
Descriptors: MW 536.01, ALogP 4.18, HBA 8, HBD 1, PSA 116.07, QED 0.38
Compound (SMILES): C#CCN(c1c(F)cc(C#N)cc1F)[C@@H]1CCN(c2ncnc3[nH]ccc23)C1
InChIKey: JKPVBGCJUHOUKU-CQSZACIVSA-N
Target: Tyrosine-protein kinase JAK3 (SINGLE PROTEIN), Homo sapiens
UniProt: P52333
Assay: Enzymatic Assay: JAK1/2/3 kinase assay are performed in vitro using Kit-Tyr 6 Peptide (Invitrogen, Cat. No. PV4122). TYK2 kinase assay are performed in vitro using Z'-LYTE™ Kinase Assay Kit-Tyr 3 Peptide (Invitrogen, Cat. No. PV3192). Recombinant human JAK1/2/3 or TYK2 catalytic domains are from Invitrogen (Cat No. PV4774/PV4210/PV3855/PV4790); All reactions (20 μL) are started by adding 2.5 μL of the testing compound in 4% DMSO solution, 5 μL of Kinase/Peptide substrate Mixture (3.2, 0.04, 0.2 or 8 μg/mL for Recombinant human JAK1/2/3 catalytic domains, 4 μM for Z-LYTE™ Tyr 6 peptide or Z-LYTE™ Tyr 3 peptide) or Phospho-Peptide solution (Invitrogen, Cat. No. PV3192, diluted with 1.33× Kinase Buffer), 2.5 μL ATP Solution (300/100/40/100M, JAK1/JAK2/JAK3/TYK2) or 1.33× Kinase Buffer (Invitrogen, Cat. No. PV3189, 5× diluted with distilled water). The 384-well assay plate (Corning, Cat. No. 3575) is mixed and incubated at room temperature for 1 hour. 5 μL of the Development Solution (Dilute Development Reagent A (Cat. No. PV3297) is diluted to 1/64 with Development Buffer (Cat. No. PV3127) for JAK1, JAK2 and JAK3 assay; Development Reagent A (Cat. No. PV3297) is diluted to 1/2048 with Development Buffer (Cat. No. PV3127) for TYK2 assay. The diluted Development Solution is then added to each well, mixed and incubated at room temperature for another 1 hour. The kinase reaction is then stopped by adding 5 μL of the Stop Reagent (Invitrogen, Cat. No. PV3094), and the plate is read with Wallac 1420 VICTOR3 Multilabel Counter (PerkinElmer™) at 445 nm and 520 nm fluorescence. All compounds are initially tested at 8 different concentrations (1 μM down to 0.0003 μM) using a 1:3 serial dilution scheme.
Measurement: IC50 = 0.055 nM (pChEMBL 10.260000228881836)
Descriptors: MW 378.39, ALogP 2.83, HBA 5, HBD 1, PSA 71.84, QED 0.71
Compound (SMILES): O=C(OCCOC(=O)[C@@H]1CCCCN1S(=O)(=O)Cc1ccccc1)c1cccnc1
InChIKey: KUWLHSSIHRTCQU-IBGZPJMESA-N
Target: Outer membrane protein MIP (SINGLE PROTEIN), Legionella pneumophila
UniProt: Q70YI1
Assay: Inhibition of Mip PPIase activity in Legionella pneumophila
Measurement: IC50 = 2570.4 nM (pChEMBL 5.590000152587891)
Descriptors: MW 432.5, ALogP 2.17, HBA 7, HBD 0, PSA 102.87, QED 0.47
Compound (SMILES): C(=C/c1n[nH]c2cc(-c3ccncc3)ccc12)\c1ccc2c(c1)OCO2
InChIKey: GBBRQLSNSLPPIQ-ORCRQEGFSA-N
Target: Serine/threonine-protein kinase Chk1 (SINGLE PROTEIN), Homo sapiens
UniProt: O14757
Assay: Inhibition of full length human C-terminal 6His-tagged CHK1 expressed in baculovirus/insect expression system assessed as reduction in NADH oxidation using Syntide2 as substrate by pyruvate kinase/lactate dehydrogenase coupled assay
Measurement: Ki = 72 nM (pChEMBL 7.139999866485596)
Descriptors: MW 341.37, ALogP 4.52, HBA 4, HBD 1, PSA 60.03, QED 0.59
Compound (SMILES): COc1ccc(OC)c(CCc2nnc(-c3ccc4[nH]cnc4c3)s2)c1
InChIKey: MNFOQDSKCWTBBI-UHFFFAOYSA-N
Target: Glutaminyl-peptide cyclotransferase-like protein (SINGLE PROTEIN), Homo sapiens
UniProt: Q9NXS2
Assay: Spectrophotometric Assay: This novel assay was used to determine the kinetic parameters for most of the QC substrates. QC activity was analyzed spectrophotometrically using a continuous method, that was derived by adapting a previous discontinuous assay (Bateman, R. C. J. 1989 J Neurosci Methods 30, 23-28) utilizing glutamate dehydrogenase as auxiliary enzyme. Samples consisted of the respective QC substrate, 0.3 mM NADH, 14 mM α-Ketoglutaric acid and 30 U/ml glutamate dehydrogenase in a final volume of 250 ul. Reactions were started by addition of QC and perused by monitoring of the decrease in absorbance at 340 nm for 8-15 min.The initial velocities were evaluated and the enzymatic activity was determined from a standard curve of ammonia under assay conditions. All samples were measured at 30° C., using either the SPECTRAFluor Plus or the Sunrise (both from TECAN) reader for microplates. Kinetic data was evaluated using GraFit software.
Measurement: IC50 = 480 nM (pChEMBL 6.320000171661377)
Descriptors: MW 366.45, ALogP 3.88, HBA 6, HBD 1, PSA 72.92, QED 0.56
Compound (SMILES): O=S(=O)(NCC1CC1)c1ccc(NCc2ccncc2)cc1C(F)(F)F
InChIKey: YSJPDKNWAXNLER-UHFFFAOYSA-N
Target: Cannabinoid receptor 2 (SINGLE PROTEIN), Homo sapiens
UniProt: P34972
Assay: Binding Assay: The binding assays were performed by testing the ability of the new compounds to displace the radiolabeled synthetic non-selective cannabinoid agonist [3H]CP55940 (168 Ci/mmol; PerkinElmer) from the human CB1 (hCB1) or human CB2 (hCB2) receptors on membranes derived from stably transfected HEK-293 cells (PerkinElmer). Membranes were diluted in assay buffer (50 mM Tris-HCl, 1 mM EDTA, 1 mM MgCl2, 1 mg/ml BSA, pH=7.4). The amount of membrane was determined for each batch of membranes according to protein binding assay. The minimum amount of membrane that gave 50% specific binding was used for the binding assay. In most assays, binding was tested using 8 μg and 4 μg protein of hCB1 and hCB2 membranes, respectively. The tested compounds were dissolved in DMSO and diluted in assay buffer to a final concentration of 0.1% solvent. Total binding of [3H]CP55940 was evaluated with 1.5 nM to hCB1 and with 0.5 nM to hCB2, according to Kd affinity of [3H]CP55940 for the respective membranes. The ability of the tested compounds to displace [3H]CP55940 was evaluated first at a single concentration point of 100 nM for binding toward hCB2 or hCB1. In certain cases, the displacement was tested at compound concentrations ranging from 0.03 nM to 10 μM. Non-specific binding was measured by the addition of 1 μM of unlabelled CP55940 to the tubes. Binding assays were performed in triplicate in a total volume of 500 μl for 60 minutes at 30° C., in a shaking bath. Free and bound radioligands were separated by rapid filtration through GF/C filter plates (PerkinElmer) that had been presoaked with 0.1% Polyethylenimine (Sigma). Filters were shaken for 1 hour in 7 ml scintillation fluid (PerkinElmer) and radioactivity was determined by liquid scintillation counter (Wallac; PerkinElmer).
Measurement: IC50 = 3167 nM (pChEMBL 5.5)
Descriptors: MW 385.41, ALogP 3.4, HBA 4, HBD 2, PSA 71.09, QED 0.77
Compound (SMILES): CNC(=N)NCCC[C@H](NC(=O)[C@H](CC(C)C)NC(=O)NNC(=O)[C@H](Cc1cccc(F)c1)NC(=O)[C@@H](NC(=O)[C@H](CC(N)=O)NC(=O)[C@@H](Cc1c[nH]c2ccccc12)NC(=O)[C@@H](Cc1ccc(O)cc1)NC(C)=O)[C@@H](C)O)C(=O)N[C@@H](Cc1c[nH]c2ccccc12)C(N)=O
InChIKey: RMJPJFQIOLLQHN-LYEJOWPUSA-N
Target: KiSS-1 receptor (SINGLE PROTEIN), Rattus norvegicus
UniProt: Q924U1
Assay: Agonist activity at rat KISS1R expressed in CHO/dhfr cells assessed as increase in intracellular Ca2+ levels measured for 180 secs by Fluo 3-AM dye based FLIPR assay
Measurement: EC50 = 4.8 nM (pChEMBL 8.319999694824219)
Descriptors: MW 1316.46
Compound (SMILES): COc1cc(OC)cc(N(CC2(C)COC2)c2ccc3ncc(-c4cnn(C)c4)nc3c2)c1
InChIKey: WLMOBZCQXJLKAR-UHFFFAOYSA-N
Target: Fibroblast growth factor receptor 3 (SINGLE PROTEIN), Homo sapiens
UniProt: P22607
Assay: Inhibition of FGFR3 (unknown origin) transfected in mouse BA/F3 cells assessed as decrease in cell proliferation in presence of mouse IL3 after 24 hrs by Alamar blue assay
Measurement: IC50 > 10000 nM (pChEMBL 5)
Descriptors: MW 445.52, ALogP 4.22, HBA 8, HBD 0, PSA 74.53, QED 0.42
Compound (SMILES): Cc1ccc(C2=C(c3nn[nH]n3)C(=O)NC(c3ccc(Br)cc3)(C(F)(F)F)C2)cc1
InChIKey: IZCXIAMLSGVDFU-UHFFFAOYSA-N
Target: Alpha-1,6-mannosyl-glycoprotein 2-beta-N-acetylglucosaminyltransferase (SINGLE PROTEIN), Homo sapiens
UniProt: Q10469
Assay: MGAT LCMS Assay: The MGAT enzyme reactions were performed in CORNING Falcon 96-well Polypropylene plates, in a total volume of 60 uL of 50 mM Potassium Phosphate buffer pH 7.4, containing a final concentration of 100 uM 2-oleoylglycerol, 15 uM oleoyl-Coenzyme A and 0.0013 ug/uL Human or Mouse MGAT-2 or 0.0026 ug/uL Rat recombinant MGAT-2 membranes expressed in Sf9 cells. Assay plates were run through a fully automated robotics system and shaken for 5 seconds every minute for a total 10 minutes. The reactions were then quenched with 120 uL of ice cold methanol containing 1 ug/mL 1,2-distearoyl-rac-glycerol as the internal standard. Plates were shaken for 2 minutes and spun down to remove protein precipitation. After the spin, samples were transferred to LC/MS compatible PCR plates. For LC/MS analysis, a ThermoFisher Surveyor pump, utilizing a Waters Symmetry C8, 50x2.1 mm column, was used for the chromatography of enzyme products.
Measurement: IC50 = 639 nM (pChEMBL 6.190000057220459)
Descriptors: MW 478.27, ALogP 4.16, HBA 4, HBD 2, PSA 83.56, QED 0.59
Compound (SMILES): O=C(Nc1cn(CC(=O)N2CCC3(CNC3)C2)nc1-c1cc(Cl)ccc1OC(F)F)c1cnn2cccnc12
InChIKey: KXFWROWENYUILI-UHFFFAOYSA-N
Target: Tyrosine-protein kinase JAK2 (SINGLE PROTEIN), Homo sapiens
UniProt: O60674
Assay: JAK Enzyme Assay: The activity of the isolated recombinant JAK1 and JAK2 kinase domain was measured by monitoring phosphorylation of a peptide derived from JAK3 (Val-Ala-Leu-Val-Asp-Gly-Tyr-Phe-Arg-Leu-Thr-Thr, fluorescently labeled on the N-terminus with 5-carboxyfluorescein) using the Caliper LabChip® technology (Caliper Life Sciences, Hopkinton, Mass.). To determine inhibition constants (Ki), compounds were diluted serially in DMSO and added to 50 μL kinase reactions containing purified enzyme (1.5 nM JAK1, or 0.2 nM JAK2), 100 mM HEPES buffer (pH 7.2), 0.015% Brij-35, 1.5 μM peptide substrate, ATP (25 μM), 10 mM MgCl2, 4 mM DTT at a final DMSO concentration of 2%. Reactions were incubated at 22° C. in 384-well polypropylene microtiter plates for 30 minutes and then stopped by addition of 25 μL of an EDTA containing solution (100 mM HEPES buffer (pH 7.2), 0.015% Brij-35, 150 mM EDTA), resulting in a final EDTA concentration of 50 mM. After termination of the kinase reaction, the proportion of phosphorylated product was determined as a fraction of total peptide substrate using the Caliper LabChip® 3000 according to the manufacturer's specifications.
Measurement: Ki = 1.28 nM (pChEMBL 8.890000343322754)
Descriptors: MW 556.96, ALogP 2.92, HBA 9, HBD 2, PSA 118.68, QED 0.36
Compound (SMILES): CN(c1ccc(S(=O)(=O)NC2COC2)cn1)[C@@H]1CCN(c2ncnc3[nH]ccc23)C1
InChIKey: SSQRGQRRPUZAFS-CQSZACIVSA-N
Target: Tyrosine-protein kinase JAK2 (SINGLE PROTEIN), Homo sapiens
UniProt: O60674
Assay: Enzymatic Assay: JAK1/2/3 kinase assay are performed in vitro using Kit-Tyr 6 Peptide (Invitrogen, Cat. No. PV4122). TYK2 kinase assay are performed in vitro using Z'-LYTE™ Kinase Assay Kit-Tyr 3 Peptide (Invitrogen, Cat. No. PV3192). Recombinant human JAK1/2/3 or TYK2 catalytic domains are from Invitrogen (Cat No. PV4774/PV4210/PV3855/PV4790); All reactions (20 μL) are started by adding 2.5 μL of the testing compound in 4% DMSO solution, 5 μL of Kinase/Peptide substrate Mixture (3.2, 0.04, 0.2 or 8 μg/mL for Recombinant human JAK1/2/3 catalytic domains, 4 μM for Z-LYTE™ Tyr 6 peptide or Z-LYTE™ Tyr 3 peptide) or Phospho-Peptide solution (Invitrogen, Cat. No. PV3192, diluted with 1.33× Kinase Buffer), 2.5 μL ATP Solution (300/100/40/100M, JAK1/JAK2/JAK3/TYK2) or 1.33× Kinase Buffer (Invitrogen, Cat. No. PV3189, 5× diluted with distilled water). The 384-well assay plate (Corning, Cat. No. 3575) is mixed and incubated at room temperature for 1 hour. 5 μL of the Development Solution (Dilute Development Reagent A (Cat. No. PV3297) is diluted to 1/64 with Development Buffer (Cat. No. PV3127) for JAK1, JAK2 and JAK3 assay; Development Reagent A (Cat. No. PV3297) is diluted to 1/2048 with Development Buffer (Cat. No. PV3127) for TYK2 assay. The diluted Development Solution is then added to each well, mixed and incubated at room temperature for another 1 hour. The kinase reaction is then stopped by adding 5 μL of the Stop Reagent (Invitrogen, Cat. No. PV3094), and the plate is read with Wallac 1420 VICTOR3 Multilabel Counter (PerkinElmer™) at 445 nm and 520 nm fluorescence. All compounds are initially tested at 8 different concentrations (1 μM down to 0.0003 μM) using a 1:3 serial dilution scheme.
Measurement: IC50 = 0.055 nM (pChEMBL 10.260000228881836)
Descriptors: MW 429.51, ALogP 0.75, HBA 8, HBD 2, PSA 116.34, QED 0.59