lovingscience/chembl-v37-raw-activities huggingface dataset:
Compound (SMILES): CNCCCC[C@@H](NC(=O)[C@@H](CC(C)C)NC(=O)[C@@H](Cc1ccccc1)NC(=O)[C@H](N)Cc1ccccc1)C(=O)N1CCC(N)(C(=O)O)CC1
InChIKey: DHKPJPUVNZWGFO-OMRVPHBLSA-N
Target: Kappa-type opioid receptor (SINGLE PROTEIN), Mus musculus
UniProt: P33534
Assay: Time Resolved Fluorescence Resonance Energy Transfer (TR-FRET)-Based cAMP Immunoassay: Mouse R1.G1 cells (ATCC, Manassas, Va.) were grown in suspension in high glucose-DMEM (Dulbecco's Modified Eagle's Medium, Cellgro, Herndon, Va.) containing 10% horse serum and 2% glutaMax (Invitrogen, Carlsbad. Calif.) without added antibiotics. On the day of the experiment, cells were spun at 1,000 rpm for 5 minutes at room temperature and then washed once with HBSS (HEPES Buffered Saline Solution, Invitrogen, Carlsbad, Calif.). Cells were then spun again and resuspended in stimulation buffer (HBSS with 0.05% FAF-BSA (Fatty acid-free bovine serum albumin, Roche Applied Science, Indianapolis, Ind.), 5 mM HEPES) to 2 million cells per ml. Antibody supplied with the LANCE™ cAMP immunoassay kit was then added to the cells according to the manufacturer's instructions, and 12,000 cells per well were then added to the wells containing forskolin to a predetermined fixed final concentration (typically about 2.5 μM) and the previously determined amount of the synthetic peptide amide to be tested. The synthetic peptide amides were tested in a range of concentrations to determine potency. Cells were incubated with the synthetic peptide amide plus forskolin for about 20 minutes at room temperature. After incubation, cells are lysed by adding 12 μl of detection mix as supplied with the LANCE™ kit, followed by incubation for one hour at room temperature. Time resolved fluorescence was read using a 330-380 nm excitation filter, a 665 nm emission filter, dichroic mirror 380, and Z=1 mm.
Measurement: EC50 = 0.052 nM (pChEMBL 10.279999732971191)
Descriptors: MW 693.89, ALogP 1.09, HBA 8, HBD 7, PSA 208.98, QED 0.1
Compound (SMILES): Cc1cc(-c2n[nH]c3ccc(C(=O)N[C@@H]4CCC[C@](O)(Cn5ncc6ccncc65)C4)cc23)ccn1
InChIKey: IWQJZQDKQPYMKC-JIPXPUAJSA-N
Target: Mitogen-activated protein kinase 1 (SINGLE PROTEIN), Mus musculus
UniProt: P63085
Assay: Active ERK2 (aERK) Assay: Activated ERK2 activity was also determined in the IMAP assay format using the procedure outlined above. 1 μl of 25× compound was added to 14 μl of kinase buffer containing 0.25 ng fully phosphorylated, active Mouse ERK2 protein. Following a 30 minute incubation, the reactions were initiated by addition of 10 μl of kinase buffer containing 1 μM ERK2 IMAP substrate peptide (0.9 μM unlabeled IPTTPITTTYFFFK-CONH2 and 100 nM IPTTPITTTYFFFK(5-carboxyfluorescein)-CONH2) and 30 μM ATP. Reactions proceeded for 30 minutes before termination by addition of 60 μl IMAP detection beads in binding buffer. Plates were read as above after 30 minute binding equilibration. Active compounds were reconfirmed in an independent assay.
Measurement: IC50 = 792.1 nM (pChEMBL 6.099999904632568)
Descriptors: MW 481.56, ALogP 3.78, HBA 7, HBD 3, PSA 121.61, QED 0.35
Compound (SMILES): N#CC1(NC(=O)[C@@H]2Cc3ccc(c(Cl)c3)OC/C=C/COc3cc4c(cc3C(=O)N2)CCCC4)CC1
InChIKey: AEMSQVNLKWJOIJ-CRTIWDKESA-N
Target: Procathepsin L (SINGLE PROTEIN), Homo sapiens
UniProt: P07711
Assay: Cathepsin Enzyme Inhibition Assay: Enzyme activity is measured by observing the increase in fluorescence intensity caused by cleavage of a peptide substrate containing a fluorophore whose emission is quenched in the intact peptide. Assay buffer: 100 mM potassium phosphate pH 6.5, EDTA-Na 5 mM, Triton X-100 0.001%, DDT 5 mM. Enzymes (all at 1 nM): human and mouse Cathepsin S, Cat K, Cat B, Cat LSubstrate (20 uM): Z-Val-Val-Arg-AMC, except for Cat K which uses Z-Leu-Arg-AMC (both from Bachem). Z=Benzyloxycarbonyl. AMC=7-Amino-4-Methyl-Coumarin. Final volume: 100 uL. Excitation 360 nm, Emission 465 nm. Enzyme is added to the substance dilutions in 96-well microtitre plates and the reaction is started with substrate. Fluorescence emission is measured over 20 minutes, during which time a linear increase is observed in the absence of inhibitor.
Measurement: IC50 = 16 nM (pChEMBL 7.800000190734863)
Descriptors: MW 506, ALogP 4.06, HBA 5, HBD 2, PSA 100.45, QED 0.6
Compound (SMILES): COC(=O)Nc1ccc2c(c1)NC(=O)[C@H](C)C(F)CC[C@H](N1CCC(c3c(F)ccc(Cl)c3F)=CC1=O)c1cc-2ccn1
InChIKey: QMJWWNVVTLEZIW-ALZQUROESA-N
Target: Coagulation factor IX (SINGLE PROTEIN), Homo sapiens
UniProt: P00740
Assay: In Vitro Assay: Factor XIa determinations were made in 50 mM HEPES buffer at pH 7.4 containing 145 mM NaCl, 5 mM KCl, and 0.1% PEG 8000 (polyethylene glycol; JT Baker or Fisher Scientific). Determinations were made using purified human Factor XIa at a final concentration of 25-200 pM (Haematologic Technologies) and the synthetic substrate S-2366 (pyroGlu-Pro-Arg-pNA; CHROMOGENIX® or AnaSpec) at a concentration of 0.0002-0.001 M.
Measurement: Ki = 0.18 nM (pChEMBL 9.739999771118164)
Descriptors: MW 613.04, ALogP 6.92, HBA 5, HBD 2, PSA 100.63, QED 0.31
Compound (SMILES): CC(C)(C)CC(C)(C)NC(=O)N1CCn2c(c(C(N)=O)c(-c3ccccc3)c2C2CC2)C1
InChIKey: AIEXWVUKUDSHCL-UHFFFAOYSA-N
Target: Casein kinase I isoform epsilon (SINGLE PROTEIN), Homo sapiens
UniProt: P49674
Assay: In Vitro ATP-33P Casein Assay: "U"-bottomed Falcon plates are prepared by placing 5 uL of solutions of the compounds according to the invention at concentrations of 10, 1, 0.1, 0.01 or 0.001 uM in different wells. The solutions of the compounds according to the invention at these various concentrations are prepared by diluting in a test buffer (Tris 50 mM pH 7.5, MgCl2 10 M, DTT 2 mM and EGTA 1 mM) a stock solution in DMSO at a concentration of 10 mM. Next, 5 uL of dephosphorylated casein are added to a final concentration of 0.2 ug/uL, 20 uL of CK1 epsilon to a final concentration of 3 ng/uL, and 20 uL of ATP-33P to a final concentration of 0.02 uCi/uL mixed with cold ATP (10 uM final approximately 2x106 CPM per well). The final total test volume per well is equal to 50 uL. The "U"-bottomed Falcon test plate mentioned above is vortexed, and then incubated at room temperature for 2 hours. After 2 hours the reaction is stopped by adding an ice-cold solution of 65 uL of cold ATP (2 mM) prepared in test buffer. 100 uL of the reaction mixture are then transferred from the "U"-bottomed Falcon plate into Millipore MAPH filter plates, preimpregnated with 25 uL of ice-cold 100% TCA. The Millipore MAPH filter plates are agitated gently and are left to stand at room temperature for at least 30 minutes to precipitate the proteins. After 30 minutes, the filter plates are sequentially washed and filtered with 2x150 uL of 20% TCA, 2x150 uL of 10% TCA and 2x150 uL of 5% TCA (6 washes in total per plate/900 uL per well). The plates are left to dry overnight at room temperature. Next, 40 uL of Microscint-20 Packard scintillation liquid are added per well and the plates are closed in a leaktight manner. The radiation emitted by each well is then measured for 2 minutes in a TopCount NXT Packard scintillation counter, in which the values of CPM/well are measured.
Measurement: IC50 = 45 nM (pChEMBL 7.349999904632568)
Descriptors: MW 436.6, ALogP 4.87, HBA 3, HBD 2, PSA 80.36, QED 0.7
Compound (SMILES): N#CCC1(COC(=O)N2CC[C@@H](n3cc(C(N)=O)c(Nc4ccc(F)cc4)n3)[C@H](C#N)C2)CC1
InChIKey: VSQCPYADUJGPPM-DNVCBOLYSA-N
Target: Tyrosine-protein kinase JAK1 (SINGLE PROTEIN), Homo sapiens
UniProt: P23458
Assay: Biochemical HTRF Assay: The ability of compounds to inhibit the activity of JAK1, JAK2, JAK3, and Tyk2 was measured using a recombinant purified GST-tagged catalytic domain for each enzyme (Invitrogen JAK1 #M4290, JAK2 #M4290, JAK3 #M4290, Tyk2 #M4290) in an HTRF format biochemical assay. The reactions employed a common peptide substrate, LCB-EQEDEPEGDYFEWLW-NH2 (in-house). The basic assay protocol is as follows: First, 250 nL of diluted compounds in DMSO were dispensed into the wells of a dry 384-well Black plate (Greiner #781076) using a Labcyte Echo 555 acoustic dispenser. Subsequent reagent additions employed an Agilent Bravo. Next, 18 μL of 1.11× enzyme and 1.11× substrate in 1× assay buffer (Invitrogen kinase buffer # PV3189, 2 mM DTT, 0.05% BSA) were added to the wells and shaken and then preincubated for 30 minutes at ambient temperature to allow compound binding to equilibrate. After equilibration, 2 μL of 10×ATP in 1× assay buffer was added to initiate the kinase reaction and the plates were shaken and then incubated at ambient temperature for 120 minutes. At the end of the incubation, 20 μL of 2× stop buffer (streptavidin-Dylight 650 (Thermo #84547B/100 mL), Eu-tagged pY20 antibody (Perkin Elmer #AD0067), EDTA, HEPES, and Triton) was added to quench the reaction. Plates were shaken and centrifuged and then incubated 60 minutes at ambient temperature and then read on a Perkin Elmer Envision (λex=337 nm, λem=665 and 615 nm, TRF delay time=20 μs). HTRF signal=10,000*665 nm reading/615 nm reading.
Measurement: IC50 = 25 nM (pChEMBL 7.599999904632568)
Descriptors: MW 465.49, ALogP 3.08, HBA 8, HBD 2, PSA 150.06, QED 0.64
Compound (SMILES): Cc1nn(C)c(C)c1NS(=O)(=O)c1ccc(Br)cc1F
InChIKey: LGQWPYLOBBSGDL-UHFFFAOYSA-N
Target: Glycylpeptide N-tetradecanoyltransferase (SINGLE PROTEIN), Trypanosoma brucei brucei (strain 927/4 GUTat10.1)
UniProt: Q388H8
Assay: Enzyme Inhibition Assay: Measurements were performed using a modification of the scintillation proximity assay platform described previously by Georgopapadakou, N. H. et al. (22nd International Congress on Chemotherapy, 2001, Abstract P16.001). Compounds were solubilised in DMSO at a top concentration of 10 mM and serially diluted in half log steps to achieve a range of final assay concentrations of 100 uM to 1 nM. Compound at each concentration (100-fold final) was added to white 384 well plates in a volume of 0.5 ml. Human, A. fumigatus, T. brucei or L. major N-myristoyl transferase enzyme, dissolved to a working concentration of 10 nM in assay buffer (30 mM Tris/HCl pH 7.4, 0.5 mM EGTA, 0.5 mM EDTA, 1.25 mM DTT, 0.1% Triton X-100), was then added to columns 1 to 11 and 13 to 23 of the plates in a volume of 20 ml. To columns 12 and 24, 20 ml assay buffer was added to provide a no enzyme control. Following a 5 minute incubation at room temperature the substrates (GCGGSKVKPQPPQAK(Biotin)-Amide and myristoyl coenzyme A), dissolved in assay buffer, were added to all wells in a volume of 20 ml to start the reaction. The final concentrations of peptide and 3H-myristoyl coenzyme A were 0.5 mM and 125 nM respectively and the specific activity of the radiolabel was 8 Ci/mmol. Plates were then incubated at room temperature for up to 50 minutes (dependant upon the period of linearity for the different enzyme species) before SPA beads, suspended to 1 mg/ml in a stop solution (200 mM Phosphoric Acid/NaOH pH 4, 750 mM MgCl2), were added in a volume of 40 ml.
Measurement: IC50 = 10000 nM (pChEMBL 5)
Descriptors: MW 362.22, ALogP 2.74, HBA 4, HBD 1, PSA 63.99, QED 0.91
Compound (SMILES): C[C@@H](NC(=O)Nc1cc2[nH]nc(OC3CCNC3)c2cn1)c1ccccc1
InChIKey: FEAPBSNWTMHACN-PUODRLBUSA-N
Target: Mitogen-activated protein kinase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P28482
Assay: IMAP-FP Assay: Activated ERK2 activity was determined in an IMAP-FP assay (Molecular Devices). Using this assay format, the potency (IC50) of each compound was determined from a 10 point (1:3 serial dilution, 3 uM starting compound concentration) titration curve using the following outlined procedure. To each well of a black Corning 384-well plate (Corning Catalog #3575), 7.5 nL of compound (3333 fold dilution in final assay volume of 25 uL) was dispensed, followed by the addition of 15 uL of kinase buffer (tween containing kinase buffer, Molecular Devices) containing 0.0364 ng/mL (0.833 nM) of phosphorylated active hERK2 enzyme. Following a 15 minute compound & enzyme incubation, each reaction was initiated by the addition of 10 uL kinase buffer containing 2.45 uM ERK2 IMAP substrate peptides (2.25 uM-unlabeled IPTTPITTTYFFFK-COOH and 200 nM-labeled IPTTPITTTYFFFK-SFAM (5-carboxyfluorescein)-COOH), and 75 uM ATP.
Measurement: IC50 = 109.5 nM (pChEMBL 6.960000038146973)
Descriptors: MW 366.43, ALogP 2.58, HBA 5, HBD 4, PSA 103.96, QED 0.56
Compound (SMILES): CC(Nc1nccc(N2C(=O)OC[C@@H]2[C@@H](C)O)n1)c1nc(-c2ccc3c(c2)OC(F)(F)O3)no1
InChIKey: RGPKYJCYYMOGBR-GTFYECCDSA-N
Target: Isocitrate dehydrogenase [NADP] cytoplasmic (SINGLE PROTEIN), Homo sapiens
UniProt: O75874
Assay: Fluorescence Biochemical Assay: The IDH1 (R132H) mutant catalyzes the reduced form of NADP+ (NADPH) and α-ketoglutarate (α-KG) to form nicotinamide adenine dinucleotide phosphate (NADP+) and R (−)-2-hydroxyglutarate (2HG). The reaction can be monitored kinetically by following the oxidation of NADPH to NADP+ which is measured using fluorescence, excitation at 355 nm and emission at 530 nm. Reactions were monitored using the Perkin-Elmer Envision, Model 2101. More specifically, the biochemical reactions were performed at room temperature in 384-well Greiner flat-bottom plates (Cat. No. 781076) using a final reaction volume of 20 μL and the following assay buffer conditions: 50 mM HEPES pH 7.5, 10 mM MgCl2, 1 mM DTT, 0.02% BSA, 0.02% Tween-20, 10 μM NADPH and 100 μM α-KG. The final reaction mixture contained 2.5% DMSO and test compounds with concentrations ranging 0.0000008-25 μM. The IDH1 (R132H) enzyme was used at a final concentration of 10 nM.
Measurement: IC50 = 38 nM (pChEMBL 7.420000076293945)
Descriptors: MW 476.4, ALogP 2.73, HBA 11, HBD 2, PSA 144.96, QED 0.54
Compound (SMILES): COCc1nc(NC(=O)N[C@H](COC(F)F)c2ccccc2)cc2[nH]nc(OC)c12
InChIKey: XHWSCVSQURGSHE-CYBMUJFWSA-N
Target: Mitogen-activated protein kinase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P28482
Assay: IMAP-FP Assay: Activated ERK2 activity was determined in an IMAP-FP assay (Molecular Devices). Using this assay format, the potency (IC50) of each compound was determined from a 10 point (1:3 serial dilution, 3 uM starting compound concentration) titration curve using the following outlined procedure. To each well of a black Corning 384-well plate (Corning Catalog #3575), 7.5 nL of compound (3333 fold dilution in final assay volume of 25 uL) was dispensed, followed by the addition of 15 uL of kinase buffer (tween containing kinase buffer, Molecular Devices) containing 0.0364 ng/mL (0.833 nM) of phosphorylated active hERK2 enzyme. Following a 15 minute compound & enzyme incubation, each reaction was initiated by the addition of 10 uL kinase buffer containing 2.45 uM ERK2 IMAP substrate peptides (2.25 uM-unlabeled IPTTPITTTYFFFK-COOH and 200 nM-labeled IPTTPITTTYFFFK-SFAM (5-carboxyfluorescein)-COOH), and 75 uM ATP.
Measurement: IC50 = 8.6 nM (pChEMBL 8.069999694824219)
Descriptors: MW 421.4, ALogP 3.21, HBA 6, HBD 3, PSA 110.39, QED 0.49
Compound (SMILES): Cn1ncc(-c2ccc(C(=O)N(c3ncccc3Cl)[C@@H]3CCCNC3)cc2)c1C(=O)NS(=O)(=O)C(F)(F)F
InChIKey: IZXPGSOJEDBUKP-MRXNPFEDSA-N
Target: Proprotein convertase subtilisin/kexin type 9 (SINGLE PROTEIN), Homo sapiens
UniProt: Q8NBP7
Assay: AlphaLISA Assay: An in-vitro AlphaLISA assay (Perkin Elmer) was developed in order to quantitate the level of PCSK9 secreted into the cell culture media following compound treatment. To detect and measure PCSK9 protein an in-house generated anti-PCSK9 antibody was coupled to AlphaLISA acceptor beads by an external vendor (PerkinElmer) and a second internally developed anti-PCSK9 antibody with an epiptope distinct from that of the acceptor beads was biotinylated in house. Streptavidin coated-donor beads (Perkin Elmer) are also included in the assay mixture which then binds the biotinylated anti-PCSK9 antibody and in the presence of PCSK9 this donor complex and acceptor beads are brought into close proximity. Upon excitation of the donor beads at 680 nm singlet oxygen molecules are released that trigger an energy transfer cascade within the acceptor beads resolving as a single peak of light emitted at 615 nm. The ability of compound to modulate PCSK9 protein levels in conditioned media by AlphaLISA was assessed in the human hepatocellular carcinoma cell line Huh7, stably over-expressing human PCSK9.
Measurement: IC50 > 20000 nM (pChEMBL 4.698999881744385)
Descriptors: MW 570.98, ALogP 3.11, HBA 8, HBD 2, PSA 126.29, QED 0.47
Compound (SMILES): O=C(N[C@H]1CN2CC[C@@H]1C2)c1cc(NC(=O)c2cc(-c3ncccc3F)ccc2Cl)n(-c2ccccc2)n1
InChIKey: MAVBNADNFASPAI-KOSHJBKYSA-N
Target: High affinity nerve growth factor receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P04629
Assay: Cell Based Assay: TRKA co-expressed with p75: The assays are based upon DiscoveRx's proprietary Enzyme Fragment Complementation (EFC) technology. In the case of the TRK cell lines, the enzyme acceptor (EA) protein is fused to a SH2 protein and the TRK receptor of interest has been tagged with a Prolink tag. Cryopreserved PathHunter cells are used from either in-house produced batches or bulk batches bought directly from DiscoveRx. Cryopreserved cells are resuscitated, spun 1000 rpm for 4 min to remove freezing media, and resuspended in MEM+0.5% horse serum (both Invitrogen) to 5e^5 cells/ml. The cells are then plated using a Multidrop into Greiner white tissue culture treated plates at 20 μl/well and incubated for 24 h at 37 °C., 5% CO2, high humidity. On the day of the assay, the cell plates are allowed to cool to room temperature for 30 min prior to the assay. 4 mM stock solutions of test compounds are prepared and serially diluted in 100% DMSO. A standard curve using the compound of Example 135, WO2005/116035 at a top concentration of 150 μM is also prepared on each test plate. High percentage effect (HPE) is defined by 150 μM of the compound of Example 135, WO2005/116035 and 0% effect (ZPE) is defined by 100% DMSO. Plates containing 1 μl of serially diluted compound, standard and HPE/ZPE are diluted 1/66 in assay buffer (PBS minus Ca2+, minus Mg2+ with 0.05% pluronic F127) using a Wellmate. Using a Platemate Plus, 5 μl of 1/66 diluted test compounds is then transferred to the cell plate and allowed to reach equilibrium by incubating for 30 min at room temperature before addition of agonist stimulus: 10 μl/well of 2 nM (0.571 nM FAC) of the cognate neurotrophin (Peprotech) diluted in agonist buffer (HBSS with 0.25% BSA). Final assay concentration of the test compounds is 8.66 μM, (the compound of Example 135, WO2005/116035 FAC is 0.325 μM). The plates are left at room temperature for a further 2 hours before addition of 10 μl of the DiscoveRx PathHunter detection reagent (made up by adding 1 part Galacton Star, 5 parts Emerald II and 19 parts Cell Assay Buffer as per the manufacturer's instructions). After reagent addition, plates are covered and incubated at room temperature for 60 minutes. Luminescence signal is read using an Envision.
Measurement: IC50 = 149 nM (pChEMBL 6.829999923706055)
Descriptors: MW 530.99, ALogP 4.41, HBA 6, HBD 2, PSA 92.15, QED 0.39
Compound (SMILES): CC[C@@H](NC(=O)c1cc(C(=O)N(CC)Cc2nnn(C)n2)n2c1COCC2)c1ccccc1
InChIKey: CISXVHHDZYCIAI-GOSISDBHSA-N
Target: Potassium channel subfamily K member 3 (SINGLE PROTEIN), Homo sapiens
UniProt: O14649
Assay: TASK-1 Inhibiition Assay: Human TASK-1 channels were expressed in Xenopus oocytes. For this purpose, oocytes were isolated from Xenopus laevis and defoliculated. Subsequently, TASK-1-encoding RNA synthesized in vitro was injected into the oocytes. After two days of TASK-1 protein expression, TASK-1 currents were measured by two-microelectrode voltage clamp. Data were acquired and analyzed using a TEC-10cx amplifier (NPI Electronic, Tamm, Germany) connected to an ITC-16 interface (Instrutech Corp., Long Island, USA) and Pulse software (HEKA Elektronik, Lambrecht, Germany). Oocytes were clamped to -90 mV and TASK-1 mediated currents were measured during 500 ms voltage pulses to 40 mV. Oocytes were continuously superfused with ND96 buffer containing 96 mM sodium chloride, 2 mM potassium chloride, 1.8 mM calcium chloride, 1 mM magnesium chloride, 5 mM 4-(2-hydroxyethyl)-piperazine-1-ethanesulfonic acid (HEPES; pH adjusted to 7.4 with sodium hydroxide). All experiments were performed at room temperature.
Measurement: IC50 = 450 nM (pChEMBL 6.349999904632568)
Descriptors: MW 451.53, ALogP 2.09, HBA 8, HBD 1, PSA 107.17, QED 0.56
Compound (SMILES): C#CCCOc1cc(C(=O)Nc2cc(C(F)(F)F)ccn2)ccc1-c1nc([C@@H]2CC[C@H]3CCC(=O)N3C2)n2ccnc(N)c12
InChIKey: NKBKOUANIHBRAL-CTNGQTDRSA-N
Target: Tyrosine-protein kinase BTK (SINGLE PROTEIN), Homo sapiens
UniProt: Q06187
Assay: LANCE (Lanthanide Chelate Excite) TR-FRET (Time-resolved fluorescence resonance energy transfer) Assay: BTK enzymatic activity was determined with the LANCE (Lanthanide Chelate Excite) TR-FRET (Time-resolved fluorescence resonance energy transfer) assay. In this assay, the potency (IC50) of each compound was determined from an eleven point (1:3 serial dilution; final compound concentration range in assay from 1 uM to 0.017 nM) titration curve using the following outlined procedure. To each well of a black non-binding surface Corning 384-well microplate (Corning Catalog #3820), 5 nL of compound (2000 fold dilution in final assay volume of 10 uL) was dispensed, followed by the addition of 7.5 uL of 1x kinase buffer (50 mM Hepes 7.5, 10 mM MgCl2, 0.01% Brij-35, 1 mM EGTA, 0.05% BSA & 1 mM DTT) containing 5.09 pg/uL (66.67 pM) of BTK enzyme (recombinant protein from baculovirus-transfected Sf9 cells: full-length BTK, 6HIS-tag cleaved). Following a 60 minute compound & enzyme incubation, each reaction was initiated by the addition of 2.5 uL 1x kinase buffer containing 8 uM biotinylated "A5" peptide (Biotin-EQEDEPEGDYFEWLE-NH2) (SEQ ID NO: 1), and 100 uM ATP. The final reaction in each well of 10 uL consists of 100 pM hBTK, 2 uM biotin-A5-peptide, and 25 uM ATP. Phosphorylation reactions were allowed to proceed for 120 minutes. Reactions were immediately quenched by the addition of 20 uL of 1x quench buffer (15 mM EDTA, 25 mM Hepes 7.3, and 0.1% Triton X-100) containing detection reagents (0.626 nM of LANCE-Eu-W1024-anti-phosphoTyrosine antibody, PerkinElmer and 86.8 nM of Streptavidin-conjugated Dylight 650, Dyomics/ThermoFisher Scientific). After 60 minutes incubation with detection reagents, reaction plates were read on a PerkinElmer EnVision plate reader using standard TR-FRET protocol. Briefly, excitation of donor molecules (Eu-chelate:anti-phospho-antibody) with a laser light source at 337 nm produces energy that can be transferred to Dylight-650 acceptor molecules if this donor:acceptor pair is within close proximity. Fluorescence intensity at both 665 nm (acceptor) and 615 nm (donor) are measured and a TR-FRET ratio calculated for each well (acceptor intensity/donor intensity). IC50 values were determined by 4 parameter robust fit of TR-FRET ratio values vs. (Log10) compound concentrations.
Measurement: IC50 = 0.75 nM (pChEMBL 9.119999885559082)
Descriptors: MW 603.6, ALogP 4.92, HBA 8, HBD 2, PSA 127.74, QED 0.23
Compound (SMILES): COC(=O)[C@H]1OC(Nc2ccc3c(c2)NC(=O)[C@H](C)CCC[C@H](N2CCC(c4c(F)ccc(Cl)c4F)=CC2=O)c2cc-3ccn2)[C@H](OC(C)=O)[C@@H](OC(C)=O)[C@@H]1OC(C)=O
InChIKey: NGYYEMLUUNCVGC-LDGUVKFUSA-N
Target: Coagulation factor IX (SINGLE PROTEIN), Homo sapiens
UniProt: P00740
Assay: In Vitro Assay: Factor XIa determinations were made in 50 mM HEPES buffer at pH 7.4 containing 145 mM NaCl, 5 mM KCl, and 0.1% PEG 8000 (polyethylene glycol; JT Baker or Fisher Scientific). Determinations were made using purified human Factor XIa at a final concentration of 25-200 pM (Haematologic Technologies) and the synthetic substrate S-2366 (pyroGlu-Pro-Arg-pNA; CHROMOGENIX® or AnaSpec) at a concentration of 0.0002-0.001 M.
Measurement: Ki = 33.56 nM (pChEMBL 7.46999979019165)
Descriptors: MW 853.27, ALogP 5.9, HBA 13, HBD 2, PSA 188.76, QED 0.16
Compound (SMILES): CC[C@H](NC(=O)c1cc(C(=O)N[C@H](C)c2ccccc2)n2c1COCC2)c1cnc(OC)cn1
InChIKey: WLCRBISUHGHDRK-APWZRJJASA-N
Target: Potassium channel subfamily K member 3 (SINGLE PROTEIN), Homo sapiens
UniProt: O14649
Assay: TASK-1 Inhibiition Assay: Human TASK-1 channels were expressed in Xenopus oocytes. For this purpose, oocytes were isolated from Xenopus laevis and defoliculated. Subsequently, TASK-1-encoding RNA synthesized in vitro was injected into the oocytes. After two days of TASK-1 protein expression, TASK-1 currents were measured by two-microelectrode voltage clamp. Data were acquired and analyzed using a TEC-10cx amplifier (NPI Electronic, Tamm, Germany) connected to an ITC-16 interface (Instrutech Corp., Long Island, USA) and Pulse software (HEKA Elektronik, Lambrecht, Germany). Oocytes were clamped to -90 mV and TASK-1 mediated currents were measured during 500 ms voltage pulses to 40 mV. Oocytes were continuously superfused with ND96 buffer containing 96 mM sodium chloride, 2 mM potassium chloride, 1.8 mM calcium chloride, 1 mM magnesium chloride, 5 mM 4-(2-hydroxyethyl)-piperazine-1-ethanesulfonic acid (HEPES; pH adjusted to 7.4 with sodium hydroxide). All experiments were performed at room temperature.
Measurement: IC50 = 190 nM (pChEMBL 6.71999979019165)
Descriptors: MW 463.54, ALogP 3.19, HBA 7, HBD 2, PSA 107.37, QED 0.53
Compound (SMILES): CCn1cc(C(=O)C2CC2)c2cc(Nc3nccc(-n4cc(CN5C[C@@H](O)CO5)c(C)n4)n3)ccc21
InChIKey: PWEKRGOZHHLNMO-HXUWFJFHSA-N
Target: Tyrosine-protein kinase SYK (SINGLE PROTEIN), Homo sapiens
UniProt: P43405
Assay: Inhibition Assay: Compounds of the invention were initially diluted to 10 mM in 100% DMSO (CALBIOCHEM) for storage and made into kinase buffer solution to create a compound concentration ranging from 1 uM and 10 uM. Serial dilutions of compounds of the invention were dispensed into a 96-well plate (GREINER BIOSCIENCES) at 6 uL each. Purified full-length human SYK (CARNA BIOSCIENCES) were diluted in kinase buffer and added to the compound solutions and pre-incubated for 30 minutes at room temperature. Next, ATP (TEKNOVA) of Km (15 uM) and substrate solution (suggested manufacture substrates of PerkinElmer, Ulight-TK peptide for SYK) was added (12 uL each) to the wells containing the compound solution and enzyme. The reaction mixture was incubated for 1 hour. Following the incubation, the stop solution made with EDTA, water, and Lance detection buffer (PERKINELMER) was added (12 uL each) to stop phosphorylation. Following the addition of the stop solution and 5 minutes of shaking.
Measurement: IC50 = 6.28 nM (pChEMBL 8.199999809265137)
Descriptors: MW 487.56, ALogP 3.39, HBA 10, HBD 2, PSA 110.33, QED 0.36
Compound (SMILES): Cc1ccc(CNc2cc(OC[C@H]3C[C@@H]3c3ccn(C)n3)nn(C)c2=O)c(C)n1
InChIKey: CCCDFHJNABCGQN-SJORKVTESA-N
Target: cAMP and cAMP-inhibited cGMP 3',5'-cyclic phosphodiesterase 10A (SINGLE PROTEIN), Homo sapiens
UniProt: Q9Y233
Assay: Fluorescence Polarization Assay: The fluorescence polarization assay for cyclic nucleotide phosphodiesterases was performed using an IMAP® FP kit supplied by Molecular Devices, Sunnyvale, Calif. (product #R8139). IMAP® technology has been applied previously to phosphodiesterase assays (Huang, W., et al., J. Biomol Screen, 2002, 7: 215).
Measurement: Ki = 0.023 nM (pChEMBL 10.640000343322754)
Descriptors: MW 394.48, ALogP 2.32, HBA 8, HBD 1, PSA 86.86, QED 0.66
Compound (SMILES): Cc1cc([C@](C)(O)C#Cc2cc3c(cc2F)C2CC(C2)n2c-3nc(C(N)=O)c2CNC(=O)C2CC2)no1
InChIKey: WZLDGTJHZVPFLA-IYIBJFCGSA-N
Target: Mitogen-activated protein kinase kinase kinase 14 (SINGLE PROTEIN), Homo sapiens
UniProt: Q99558
Assay: Inhibition Assay: The ability of the nuclear factor-kappa B (NF-kB)-inducing kinase (NIK) to catalyze the hydrolysis of adenosine-5'-triphosphate (ATP) was monitored using the Transcreener ADP (adenosine-5'-diphosphate) assay (BellBrook Labs). Purified NIK (0.2-1 nM) derived from a baculovirus-infected insect cell expression system was incubated with test compounds for 1-3.5 hours in 50 mM 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid buffer (pH 7.2) containing 10 mM MgCl.sub.2, 2 mM dithiothreitol, 10 uM ATP, 0.01% Triton X-100, 0.1% gamma-globulins from bovine blood, 1% dimethylsulfoxide (DMSO), 12 ug/mL ADP antibody and 4 nM ADP-AlexaFluor.RTM. 633 tracer. Reactions were quenched by the addition of 20 mM 2,2',2'',2'''-(ethane-1,2-diyldinitrilo)tetraacetic acid and 0.01% Brij 35. The tracer bound to the antibody was displaced by the ADP generated during the NIK reaction, which causes a decrease in fluorescence polarization that was measured by laser excitation.
Measurement: Ki = 3.3 nM (pChEMBL 8.479999542236328)
Descriptors: MW 503.53, ALogP 2.8, HBA 7, HBD 3, PSA 136.27, QED 0.46
Compound (SMILES): CC(C)c1cc(NC(=O)N2CC=C(c3ncc([C@H](O)CO)cc3Cl)CC2)ccc1OC(F)(F)F
InChIKey: ZAZDKMKNKZEHQF-LJQANCHMSA-N
Target: Transient receptor potential cation channel subfamily V member 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q8NER1
Assay: Capsaicin-Based Assay: Two days prior to performing this assay, cells are seeded in poly-D-lysine-coated 96-well clear-bottom black plates (50,000 cells/well) in growth media containing 5 μM PonA (commercially available from Invitrogen) to induce expression of TRPV1. On the day of the assay, the plates are washed with 0.2 mL 1× Hank's Balanced Salt Solution (commercially available from Life Technologies) containing 1 mM CaCl2 and 20 mM HEPES, pH 7.4, and cells are loaded using 0.1 mL of wash buffer containing Fluo-4 (3 μM final). After one hour, the cells are washed twice with 0.2 mL of wash buffer and resuspended in 0.1 mL of wash buffer. The plates are transferred to a FLIPR for assay. 50 μL of test compound diluted with assay buffer (1× Hank's Balanced Salt Solution containing 1 mM CaCl2 and 20 mM HEPES, pH 7.4) are added to the cell plates and incubated for 2 min. The final concentration of the compound is adjusted to range from about 50 picoM to about 3 μM. Human TRPV1 is activated by the addition of 50 μL of capsaicin (400 nM), and the plates are incubated for an additional 3 min.
Measurement: IC50 = 77.6 nM (pChEMBL 7.110000133514404)
Descriptors: MW 499.92, ALogP 5.1, HBA 5, HBD 3, PSA 94.92, QED 0.51
Compound (SMILES): C[C@@H]1CCC[C@H](N2CCC(c3c(F)ccc(Cl)c3F)=CC2=O)c2cc(ccn2)-c2cc(Cl)c(O)c(Cl)c2NC1=O
InChIKey: CCYMFXPYIKCNFP-PEBXRYMYSA-N
Target: Plasma kallikrein (SINGLE PROTEIN), Homo sapiens
UniProt: P03952
Assay: In Vitro Assay: Plasma kallikrein determinations were made in 0.1 M sodium phosphate buffer at a pH of 7.5 containing 0.1-0.2 M sodium chloride and 0.5% PEG 8000. Determinations were made using purified human plasma kallikrein (Enzyme Research Laboratories) at a final assay concentration of 200 pM and the synthetic substrate S-2302 (H-(D)-Pro-Phe-Arg-pNA; CHROMOGENIX®) at a concentration of 0.00008-0.0004 M.
Measurement: Ki = 973 nM (pChEMBL 6.010000228881836)
Descriptors: MW 606.88, ALogP 7.81, HBA 4, HBD 2, PSA 82.53, QED 0.29
Compound (SMILES): CNC[C@@H](NC(=O)c1ccc(-c2nc([C@@H]3CC[C@@H](O)[C@H](F)C3)cnc2N)cc1F)c1cc(F)cc(I)c1
InChIKey: LWXUIBDGJPDETH-XBWNDRIYSA-N
Target: Mitogen-activated protein kinase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P28482
Assay: ERK2 (20 pM) Kinase Assay: Compound potency against activated ERK2 was determined using a kinase assay that measures ERK2-catalyzed phosphorylation of biotinylated ERKtide peptide substrate ([Biotin]-AHA-K-R-E-L-V-E-P-L-T-P-S-G-E-A-P-N-Q-A-L-L-R-[NH2], the peptide sequence derived from EGF receptor: SEQ ID NO:1). The assay was carried out in 20 mM HEPES [pH 7.5], 5 mM MgCl2, 1 mM DTT, 0.01% Tween-20, 0.05% BSA using 0.02 nM ERK2, 400 nM ERKtide peptide and 35 uM ATP (all concentrations are final in the reaction) in a total volume of 10.25 uL. A 16-point, half-log dilution series of compounds at 41x final concentration was used for generating IC50 curves. Compound dilution series were prepared in 100% DMSO. ERK2 was preincubated with compounds for 30 minutes at ambient temperature. Reaction was initiated by addition of a substrate cocktail of ERKtide peptide and ATP and was allowed to proceed for 4 hours at ambient temperature. Reaction was terminated by addition of 10 uL of a 2x stop buffer consisting of 100 mM Tris-Cl [pH 7.5], 25 mM EDTA, 0.01% Tween 20, 20 ug/mL of AlphaScreen Protein A Acceptor Beads, 20 ug/mL of Streptavidin Donor Beads (PerkinElmer, Waltham, Mass.), and 1:1000 dilution phospho-EGF Receptor (Thr669) antibody (Cat #8808, Cell Signaling Technology, Danvers, Mass.). Terminated reactions were read, after overnight incubation in the dark, on an EnVision Multilabel Plate Reader (PerkinElmer, Waltham, Mass.), with excitation and emission wavelengths set to 680 nm and 570 nm, respectively. IC50 values were determined using a four-parameter fit.
Measurement: IC50 = 0.0205 nM (pChEMBL 10.6899995803833)
Descriptors: MW 625.43, ALogP 4.27, HBA 6, HBD 4, PSA 113.16, QED 0.29
Compound (SMILES): Cn1ncc(-c2ccc(C(=O)N(c3nccc4ccn(C)c34)[C@@H]3CCCNC3)cc2)c1C(=O)O
InChIKey: IOHAAGRRFZIREZ-LJQANCHMSA-N
Target: Proprotein convertase subtilisin/kexin type 9 (SINGLE PROTEIN), Homo sapiens
UniProt: Q8NBP7
Assay: AlphaLISA Assay: An in-vitro AlphaLISA assay (Perkin Elmer) was developed in order to quantitate the level of PCSK9 secreted into the cell culture media following compound treatment. To detect and measure PCSK9 protein an in-house generated anti-PCSK9 antibody was coupled to AlphaLISA acceptor beads by an external vendor (PerkinElmer) and a second internally developed anti-PCSK9 antibody with an epiptope distinct from that of the acceptor beads was biotinylated in house. Streptavidin coated-donor beads (Perkin Elmer) are also included in the assay mixture which then binds the biotinylated anti-PCSK9 antibody and in the presence of PCSK9 this donor complex and acceptor beads are brought into close proximity. Upon excitation of the donor beads at 680 nm singlet oxygen molecules are released that trigger an energy transfer cascade within the acceptor beads resolving as a single peak of light emitted at 615 nm. The ability of compound to modulate PCSK9 protein levels in conditioned media by AlphaLISA was assessed in the human hepatocellular carcinoma cell line Huh7, stably over-expressing human PCSK9.
Measurement: IC50 > 20000 nM (pChEMBL 4.698999881744385)
Descriptors: MW 458.52, ALogP 3.07, HBA 7, HBD 2, PSA 105.28, QED 0.48
Compound (SMILES): COc1ccc([C@H](NC(=O)c2ccc3cnc(N[C@H]4CCOC[C@H]4F)nc3c2)c2ccnn2C)cc1F
InChIKey: XGVGQGFUQGCUNQ-NMMYMHLASA-N
Target: Mitogen-activated protein kinase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P28482
Assay: Enzymatic Assay: Compounds were tested in an enzymatic assay using human ERK-2 (Mitogen Activated Kinase 1), recombinantly expressed as an N-terminal 6-His fusion protein in E. coli and corresponding to aa 8-360. The substrate used was the fluorescent Omnia peptide S/T17 (Invitrogen of Carlsbad, Calif.; Cat. KNZ1171C). Test compounds were diluted in DMSO in 3-fold serial dilutions at 100× final concentrations. In addition to compound, the assay contained 50 mM HEPES [pH 7.3], 10 mM MgCl2, 1 mM DTT, 0.005% Triton-X100, 5 nM ERK-2 enzyme, 6.25 μM S/T17 peptide substrate and 25 μM ATP (corresponding to the observed Km) for a total reaction volume of 25 μL. The assay was run at ambient temperature in a white 384-well polypropylene plate (Nunc, Inc of Naperville, Ill.; Cat. 267462) collecting data every 50 seconds for approximately 30 minutes on an Envision plate reader (PerkinElmer, Inc. of Waltham, Mass.); Excitation 340 nm/Emission 495 nm.
Measurement: IC50 = 3.8 nM (pChEMBL 8.420000076293945)
Descriptors: MW 508.53, ALogP 3.57, HBA 8, HBD 2, PSA 103.19, QED 0.39
Compound (SMILES): CC(C)C[C@@H](NC(=O)[C@@H](Cc1ccccc1)NC(=O)[C@H](N)Cc1ccccc1)C(=O)N[C@H](CCCNC(=N)N)C(=O)N1CCCNCC1
InChIKey: SAUBOLOHAOXQJP-SKKKGAJSSA-N
Target: Kappa-type opioid receptor (SINGLE PROTEIN), Mus musculus
UniProt: P33534
Assay: Time Resolved Fluorescence Resonance Energy Transfer (TR-FRET)-Based cAMP Immunoassay: Mouse R1.G1 cells (ATCC, Manassas, Va.) were grown in suspension in high glucose-DMEM (Dulbecco's Modified Eagle's Medium, Cellgro, Herndon, Va.) containing 10% horse serum and 2% glutaMax (Invitrogen, Carlsbad. Calif.) without added antibiotics. On the day of the experiment, cells were spun at 1,000 rpm for 5 minutes at room temperature and then washed once with HBSS (HEPES Buffered Saline Solution, Invitrogen, Carlsbad, Calif.). Cells were then spun again and resuspended in stimulation buffer (HBSS with 0.05% FAF-BSA (Fatty acid-free bovine serum albumin, Roche Applied Science, Indianapolis, Ind.), 5 mM HEPES) to 2 million cells per ml. Antibody supplied with the LANCE™ cAMP immunoassay kit was then added to the cells according to the manufacturer's instructions, and 12,000 cells per well were then added to the wells containing forskolin to a predetermined fixed final concentration (typically about 2.5 μM) and the previously determined amount of the synthetic peptide amide to be tested. The synthetic peptide amides were tested in a range of concentrations to determine potency. Cells were incubated with the synthetic peptide amide plus forskolin for about 20 minutes at room temperature. After incubation, cells are lysed by adding 12 μl of detection mix as supplied with the LANCE™ kit, followed by incubation for one hour at room temperature. Time resolved fluorescence was read using a 330-380 nm excitation filter, a 665 nm emission filter, dichroic mirror 380, and Z=1 mm.
Measurement: EC50 = 0.012 nM (pChEMBL 10.920000076293945)
Descriptors: MW 663.87, ALogP 0.38, HBA 7, HBD 8, PSA 207.56, QED 0.07
Compound (SMILES): N=C(Nc1cccc(OC[C@H]2NCC[C@@H]2Oc2cccc(NC(=N)c3cccs3)c2)c1)c1cccs1
InChIKey: XWGONJBPPMPHKV-PKTZIBPZSA-N
Target: Nitric oxide synthase 1 (SINGLE PROTEIN), Rattus norvegicus
UniProt: P29476
Assay: Inhibition Assay: The compounds were evaluated for in vitro inhibition against three NOS isozymes: rat nNOS, bovine eNOS and marine iNOS using known literature methods. (Hevel, J. M.; Marietta, M. A. Nitric-oxide synthase assays. Method Enzymol. 1994, 233, 250-258).
Measurement: Ki = 28.2 nM (pChEMBL 7.550000190734863)
Descriptors: MW 517.68, ALogP 5.87, HBA 7, HBD 5, PSA 102.25, QED 0.14
Compound (SMILES): O=C(Nc1cnn2ccc(N3CCC[C@@H]3c3cc(F)ccc3C(F)(F)F)nc12)N1CCC[C@@H](O)C1
InChIKey: SDQDEHYSFKIQQB-DNVCBOLYSA-N
Target: High affinity nerve growth factor receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P04629
Assay: ELISA Assay: An enzyme-linked immunosorbant assay (ELISA) was used to assess TrkA kinase activity in the presence of inhibitors. Immulon 4HBX 384-well microtiter plates (Thermo part #8755) were coated with a 0.025 mg/mL solution of poly (Glu, Ala, Tyr; 6:3:1; Sigma P3899). Various concentrations of test compound, 2.5 nM TrkA (Invitrogen Corp., histidine-tagged recombinant human TrkA, cytoplasmic domain), and 500 uM ATP were incubated for 25 minutes at ambient temperature in the coated plates while shaking. The assay buffer consisted of 25 mM MOPS pH 7.5, 0.005% (v/v) Triton X-100 and 5 mM MgCl2. The reaction mixture was removed from the plate by washing with PBS containing 0.1% (v/v) Tween 20. The phosphorylated reaction product was detected using 0.2 ug/mL of a phosphotyrosine specific monoclonal antibody (clone PY20) conjugated to horseradish peroxidase in conjunction with the TMB Peroxidase Substrate System (KPL).
Measurement: IC50 = 14 nM (pChEMBL 7.849999904632568)
Descriptors: MW 492.48, ALogP 4.22, HBA 6, HBD 2, PSA 86, QED 0.54
Compound (SMILES): O=C(Nc1cnc2ccc(N3CCC[C@@H]3c3cc(F)ccc3F)nn12)N1CCC(F)(F)C1
InChIKey: JBOCEQMXBSZMDR-MRXNPFEDSA-N
Target: High affinity nerve growth factor receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P04629
Assay: ELISA Assay: An enzyme-linked immunosorbant assay (ELISA) was used to assess TrkA kinase activity in the presence of inhibitors. Immulon 4HBX 384-well microtiter plates (Thermo part #8755) were coated with a 0.025 mg/mL solution of poly (Glu, Ala, Tyr; 6:3:1; Sigma P3899). Various concentrations of test compound, 2.5 nM TrkA (Invitrogen Corp., histidine-tagged recombinant human TrkA, cytoplasmic domain), and 500 uM ATP were incubated for 25 minutes at ambient temperature in the coated plates while shaking. The assay buffer consisted of 25 mM MOPS pH 7.5, 0.005% (v/v) Triton X-100 and 5 mM MgCl2. The reaction mixture was removed from the plate by washing with PBS containing 0.1% (v/v) Tween 20. The phosphorylated reaction product was detected using 0.2 ug/mL of a phosphotyrosine specific monoclonal antibody (clone PY20) conjugated to horseradish peroxidase in conjunction with the TMB Peroxidase Substrate System (KPL).
Measurement: IC50 = 22.7 nM (pChEMBL 7.639999866485596)
Descriptors: MW 448.42, ALogP 4.22, HBA 5, HBD 1, PSA 65.77, QED 0.61
Compound (SMILES): CN[C@H](C)[C@H]1COC(=O)N1c1ccnc(N[C@@H](C)c2cnc(-c3ccc(Cl)cc3)s2)n1
InChIKey: HSAYUWLDZHIRGI-DVOMOZLQSA-N
Target: Isocitrate dehydrogenase [NADP] cytoplasmic (SINGLE PROTEIN), Homo sapiens
UniProt: O75874
Assay: Fluorescence Biochemical Assay: The IDH1 (R132H) mutant catalyzes the reduced form of NADP+ (NADPH) and α-ketoglutarate (α-KG) to form nicotinamide adenine dinucleotide phosphate (NADP+) and R (−)-2-hydroxyglutarate (2HG). The reaction can be monitored kinetically by following the oxidation of NADPH to NADP+ which is measured using fluorescence, excitation at 355 nm and emission at 530 nm. Reactions were monitored using the Perkin-Elmer Envision, Model 2101. More specifically, the biochemical reactions were performed at room temperature in 384-well Greiner flat-bottom plates (Cat. No. 781076) using a final reaction volume of 20 μL and the following assay buffer conditions: 50 mM HEPES pH 7.5, 10 mM MgCl2, 1 mM DTT, 0.02% BSA, 0.02% Tween-20, 10 μM NADPH and 100 μM α-KG. The final reaction mixture contained 2.5% DMSO and test compounds with concentrations ranging 0.0000008-25 μM. The IDH1 (R132H) enzyme was used at a final concentration of 10 nM.
Measurement: IC50 = 17750 nM (pChEMBL 4.75)
Descriptors: MW 458.98, ALogP 4.36, HBA 8, HBD 2, PSA 92.27, QED 0.54
Compound (SMILES): COC[C@@H](NC(=O)Nc1cc2[nH]nc(NC(C)=O)c2cn1)c1ccc(F)cc1
InChIKey: KGAFBIKDOHNXCE-OAHLLOKOSA-N
Target: Mitogen-activated protein kinase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P28482
Assay: IMAP-FP Assay: Activated ERK2 activity was determined in an IMAP-FP assay (Molecular Devices). Using this assay format, the potency (IC50) of each compound was determined from a 10 point (1:3 serial dilution, 3 uM starting compound concentration) titration curve using the following outlined procedure. To each well of a black Corning 384-well plate (Corning Catalog #3575), 7.5 nL of compound (3333 fold dilution in final assay volume of 25 uL) was dispensed, followed by the addition of 15 uL of kinase buffer (tween containing kinase buffer, Molecular Devices) containing 0.0364 ng/mL (0.833 nM) of phosphorylated active hERK2 enzyme. Following a 15 minute compound & enzyme incubation, each reaction was initiated by the addition of 10 uL kinase buffer containing 2.45 uM ERK2 IMAP substrate peptides (2.25 uM-unlabeled IPTTPITTTYFFFK-COOH and 200 nM-labeled IPTTPITTTYFFFK-SFAM (5-carboxyfluorescein)-COOH), and 75 uM ATP. The final reaction in each well of 25 uL consists of 0.5 nM hERK2, 900 nM unlabeled peptide, 80 nM labeled-peptide, and 30 uM ATP.
Measurement: IC50 = 0.56 nM (pChEMBL 9.25)
Descriptors: MW 386.39, ALogP 2.56, HBA 5, HBD 4, PSA 121.03, QED 0.52
Compound (SMILES): C=C[C@@H]1C[C@]1(NC(=O)[C@@H]1C[C@@]2(CN1C(=O)[C@@H](NC(=O)C(NC(=O)[C@@H]1CCCCN1C(C)C)C1CCCC1)C(C)(C)C)C(C)(C)C21CCC1)C(=O)NS(=O)(=O)N(CC)CC
InChIKey: OJIHYRADUMQJCL-NYESPQOFSA-N
Target: Genome polyprotein (SINGLE PROTEIN), Hepatitis C virus genotype 1a (isolate H) (HCV)
UniProt: P27958
Assay: Protease Assay: The inhibitory activity of certain compounds of Table A against HCV NS3-4A serine protease is determined in a homogenous assay using the full-length NS3-4A protein (genotype 1a, strain HCV-1) and a commercially available internally-quenched fluorogenic peptide substrate as described by Taliani, M., et al. 1996 Anal. Biochem. 240:60-67, which is incorporated by reference in its entirety.
Measurement: IC50 = 2.5 nM (pChEMBL 8.600000381469727)
Descriptors: MW 844.18, ALogP 4.02, HBA 8, HBD 4, PSA 177.33, QED 0.18
Compound (SMILES): CC(C)(C)[C@@]1(C)OCO[C@]23CC[C@@]4(C[C@@H]21)[C@H]1Cc2ccc(OCc5ccccc5)c5c2[C@@]4(CCN1CC1CC1)[C@H]3O5
InChIKey: GMWKLQVLCZTICI-BHPNABNESA-N
Target: Mu-type opioid receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P35372
Assay: μ-Opioid Receptor Functional Assay: [35S]GTPγS functional assays were conducted using freshly thawed μ-receptor membranes prepared in-house from a cell line expressing recombinant μ opioid receptor in a HEK293 background or purchased from a commercial source (Perkin Elmer, Shelton, Conn.). Assay reactions were prepared by sequentially adding the following reagents to binding buffer (100 mM NaCl, 10 mM MgCl2, 20 mM HEPES, pH 7.4) on ice (final concentrations indicated): membrane protein (0.026 mg/mL), saponin (10 mg/mL), GDP (3 mM) and [35S]GTPγS (0.20 nM; Perkin Elmer, Shelton, Conn.). The prepared membrane solution (190 μl/well) was transferred to 96-shallow well polypropylene plates containing 10 μl of 20× concentrated stock solutions of the agonist [D-Ala2, N-methyl-Phe4 Gly-ol5]-enkephalin (DAMGO) prepared in dimethyl sulfoxide (DMSO). Plates were incubated for 30 min at about 25 °C. with shaking. Reactions were terminated by rapid filtration onto 96-well Unifilter GF/B filter plates (Perkin Elmer, Shelton, Conn.) using a 96-well tissue harvester (Perkin Elmer, Shelton, Conn.) followed by three filtration washes with 200 μl of ice-cold wash buffer (10 mM NaH2PO4, 10 mM Na2HPO4, pH 7.4). Filter plates were subsequently dried at 50 °C. for 2-3 hours. BetaScint scintillation cocktail (Perkin Elmer, Shelton, Conn.) was added (50 μl/well) and plates were counted using a Packard Top-Count for 1 min/well.
Measurement: EC50 = 950 nM (pChEMBL 6.019999980926514)
Descriptors: MW 555.76, ALogP 6.65, HBA 5, HBD 0, PSA 40.16, QED 0.41
Compound (SMILES): CCOc1cc(C(=O)Nc2cc(C(F)(F)F)n(C)n2)ccc1-c1nc([C@@H]2CC[C@H]3CCC(=O)N3C2)n2c(F)cnc(N)c12
InChIKey: GWLBSELKJQBHPD-CVEARBPZSA-N
Target: Tyrosine-protein kinase BTK (SINGLE PROTEIN), Homo sapiens
UniProt: Q06187
Assay: LANCE (Lanthanide Chelate Excite) TR-FRET (Time-resolved fluorescence resonance energy transfer) Assay: BTK enzymatic activity was determined with the LANCE (Lanthanide Chelate Excite) TR-FRET (Time-resolved fluorescence resonance energy transfer) assay. In this assay, the potency (IC50) of each compound was determined from an eleven point (1:3 serial dilution; final compound concentration range in assay from 1 uM to 0.017 nM) titration curve using the following outlined procedure. To each well of a black non-binding surface Corning 384-well microplate (Corning Catalog #3820), 5 nL of compound (2000 fold dilution in final assay volume of 10 uL) was dispensed, followed by the addition of 7.5 uL of 1x kinase buffer (50 mM Hepes 7.5, 10 mM MgCl2, 0.01% Brij-35, 1 mM EGTA, 0.05% BSA & 1 mM DTT) containing 5.09 pg/uL (66.67 pM) of BTK enzyme (recombinant protein from baculovirus-transfected Sf9 cells: full-length BTK, 6HIS-tag cleaved). Following a 60 minute compound & enzyme incubation, each reaction was initiated by the addition of 2.5 uL 1x kinase buffer containing 8 uM biotinylated "A5" peptide (Biotin-EQEDEPEGDYFEWLE-NH2) (SEQ ID NO: 1), and 100 uM ATP. The final reaction in each well of 10 uL consists of 100 pM hBTK, 2 uM biotin-A5-peptide, and 25 uM ATP. Phosphorylation reactions were allowed to proceed for 120 minutes. Reactions were immediately quenched by the addition of 20 uL of 1x quench buffer (15 mM EDTA, 25 mM Hepes 7.3, and 0.1% Triton X-100) containing detection reagents (0.626 nM of LANCE-Eu-W1024-anti-phosphoTyrosine antibody, PerkinElmer and 86.8 nM of Streptavidin-conjugated Dylight 650, Dyomics/ThermoFisher Scientific). After 60 minutes incubation with detection reagents, reaction plates were read on a PerkinElmer EnVision plate reader using standard TR-FRET protocol. Briefly, excitation of donor molecules (Eu-chelate:anti-phospho-antibody) with a laser light source at 337 nm produces energy that can be transferred to Dylight-650 acceptor molecules if this donor:acceptor pair is within close proximity. Fluorescence intensity at both 665 nm (acceptor) and 615 nm (donor) are measured and a TR-FRET ratio calculated for each well (acceptor intensity/donor intensity). IC50 values were determined by 4 parameter robust fit of TR-FRET ratio values vs. (Log10) compound concentrations.
Measurement: IC50 = 3.1 nM (pChEMBL 8.510000228881836)
Descriptors: MW 600.58, ALogP 4.39, HBA 9, HBD 2, PSA 132.67, QED 0.31