lovingscience/chembl-v37-raw-activities huggingface dataset:
Compound (SMILES): CN[C@@H](C)C(=O)N[C@@H]1C(=O)N(Cc2cc(-c3ccccc3F)ccc2OC)c2ccc(C#N)cc2N(C(C)=O)[C@H]1C
InChIKey: LBFDNWJWBWGNLJ-GWZHAIQQSA-N
Target: E3 ubiquitin-protein ligase XIAP (SINGLE PROTEIN), Homo sapiens
UniProt: P98170
Assay: TR-FRET Assay: The peptide AVPIAQKSEK-(ε-biotin)-OH 1:2 TFA ("Peptide A") was identified as a substrate for the TR-FRET assay by screening the 6× Histidine-tagged BIR2 domain and BIR3 domain of XIAP against a set of 29 peptides synthesized based on sequences reported by Sweeny et al. (Biochemistry, 2006, 45, 14740 14748). The peptides were labeled with the fluorescent tags FITC or TAMRA and Kd values were determined by fluorescence polarization assay. The sequence AVPIAQKSEK was identified as optimal for using in an assay. The peptide sequence was derivatized with biotin to provide AVPIAQKSEK-(ε-biotin)-OH 1:2 TFA as the substrate for the TR-FRET assay. Ten nanomolar of 6× Histidine-tagged BIR2 domain, corresponding to amino acids 124-240 of XIAP, or BIR3 domain, corresponding to amino acids 241-356 of XIAP, was mixed with 20 nM of the peptide AVPIAQKSEK-(ε-biotin)-OH 1:2 TFA, in the presence of 50 mM Tris-C1, pH 7.5, 100 mM NaCl, 1 mM dithiothreitol (DTT) and 0.1 mg/mL bovine serum albumin (BSA). Following a 45 mM incubation at 37° C., Europium-Streptavidin and Allophycocyanin conjugated anti-Histidine antibody were added to a final concentration of 1.5 nM and 15 nM, respectively. Time-resolved fluorescence resonance energy transfer (TR-FRET) signals were measured 1 hour later at room temperature. Test compound potency was assessed at 10 serially diluted concentrations.
Measurement: IC50 = 16380 nM (pChEMBL 4.789999961853027)
Descriptors: MW 557.63, ALogP 3.75, HBA 6, HBD 2, PSA 114.77, QED 0.46
Compound (SMILES): O=C1CNc2ccc(C3(O)c4ccccc4C(=O)N3Cc3ccccc3)cc2N1
InChIKey: ICONQZBKGMYHET-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 = 1000 nM (pChEMBL 6)
Descriptors: MW 385.42, ALogP 2.9, HBA 4, HBD 3, PSA 81.67, QED 0.65
Compound (SMILES): c1cnc2cc(CN3CCC(c4nnn5cnc6[nH]ccc6c45)CC3)ccc2n1
InChIKey: JNDZDAVBXAXRLV-UHFFFAOYSA-N
Target: Tyrosine-protein kinase JAK2 (SINGLE PROTEIN), Homo sapiens
UniProt: O60674
Assay: Enzyme Assay: JAK1, JAK2, JAK3 and Tyk2 were purchased from Carna Biosciences, Inc. As the substrate, LANCE Ultra ULight-JAK1 Peptide (manufactured by PerkinElmer Co., Ltd. (PE)) was used. Dilute solutions of compounds and enzymes in assay buffer (50 mM HEPES pH7.5, 1 mM EGTA, 1 mM MgCl2, 2 mM DTT, 0.01% Tween20) were dispensed into wells of a 384-well black plate. After 5 minutes of preincubation, dilute solutions of the substrate and ATP (adenosine triphosphate) were added at a final concentration of 100 μM, and the plate was incubated at room temperature for 2 hours. After addition of a termination reagent containing EDTA (ethylenediamine tetraacetic acid) at a final concentration of 10 mM, LANCE Eu-W1024 Anti-phosphotyrosine (PT66) (manufactured by PE) was added, and after 1 hour of incubation, the fluorescences were measured with ARVO-HTS.
Measurement: IC50 = 140 nM (pChEMBL 6.849999904632568)
Descriptors: MW 384.45, ALogP 2.93, HBA 7, HBD 1, PSA 87.89, QED 0.51
Compound (SMILES): COc1ccc(CCN2CCC(Cc3cc(OC)ccc3Br)CC2)cc1
InChIKey: JZMJEIJWBRTMLR-UHFFFAOYSA-N
Target: 5-hydroxytryptamine receptor 1A (SINGLE PROTEIN), Homo sapiens
UniProt: P08908
Assay: Displacement of [3H]-8-OH-DPAT from human 5-HT1A receptor expressed in CHOK1 cells after 30 mins by liquid scintillation counting analysis
Measurement: Ki = 0.81 nM (pChEMBL 9.09000015258789)
Descriptors: MW 418.38, ALogP 4.96, HBA 3, HBD 0, PSA 21.7, QED 0.63
Compound (SMILES): CN1CC2(C1)C(=N)N[C@](C)(c1cc(NC(=O)c3ccc(OC(F)(F)F)cn3)ccc1F)CS2(=O)=O
InChIKey: HMAVKEZTRORLPM-IBGZPJMESA-N
Target: Beta-secretase 2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9Y5Z0
Assay: Time-Resolved Endpoint Proteolysis Assay: Inhibitor IC50s at purified human autoBACE-2 are determined in a time-resolved endpoint proteolysis assay that measures hydrolysis of the QSY7-EISEVNLDAEFC-Eu-amide FRET peptide substrate (BACE-HTRF assay). BACE-mediated hydrolysis of this peptide results in an increase in relative fluorescence (RFU) at 620 nm after excitation with 320 nm light. Inhibitor compounds, prepared at 3x the desired final concentration in 1Ã¿BACE assay buffer (20 mM sodium acetate pH 5.0, 10% glycerol, 0.1% Brij-35) supplemented with 7.5% DMSO are pre-incubated with an equal volume of autoBACE-2 enzyme diluted in 1xBACE assay buffer (final enzyme concentration 1 nM) in black 384-well NUNC plates for 30 minutes at 30° C. The assay is initiated by addition of an equal volume of the QSY7-EISEVNLDAEFC-Eu-amide substrate (200 nM final concentration, Km=8 uM for 4 uM for autoBACE-2) prepared in 1xBACE assay buffer supplemented with 7.5% DMSO and incubated for 90 minutes at 30° C. DMSO is present at 5% final concentration in the assay. Following laser excitation of sample wells at 320 nm, the fluorescence signal at 620 nm is collected for 400 ms following a 50 us delay on a RUBYstar HTRF plate reader (BMG Labtechnologies). Raw RFU data is normalized to maximum (1.0 nM BACE/DMSO) and minimum (no enzyme/DMSO) RFU values. IC50s are determined by nonlinear regression analysis (sigmoidal dose response, variable slope) of percent inhibition data with minimum and maximum values set to 0 and 100 percent respectively. Similar IC50s are obtained when using raw RFU data. The Ki values are calculated from the IC50 using the Cheng-Prusoff equation.
Measurement: Ki = 10.3 nM (pChEMBL 7.989999771118164)
Descriptors: MW 515.49, ALogP 2.27, HBA 7, HBD 3, PSA 124.48, QED 0.53
Compound (SMILES): COc1cc2c(Nc3ccc(Cl)c(Cl)c3F)ncnc2cc1OC[C@@H]1CN2CCCC[C@H]2C(=O)N1
InChIKey: ZDTMGSSRQLKYTK-UGSOOPFHSA-N
Target: Ephrin type-A receptor 2 (SINGLE PROTEIN), Homo sapiens
UniProt: P29317
Assay: AlphaScreen Assay: In AlphaScreen, when the donor and acceptor beads are close in proximity, a series of photochemical events will give rise to a fluorescent signal upon light activation. Here we use biotinylated poly-(Glu, Tyr) 4:1 as the kinase substrate, streptavidin-coated donor beads and anti-phosphortyrosine antibody PY100-coated acceptor beads. Upon phosphorylation, the peptide substrate can bind to both donor and acceptor beads, thus gives rise to fluorescence. Compounds, ATP, biotinylated poly-(Glu, Tyr) and kinases are mixed in a volume of 20 μL for 1 hr at ambient temperature using Greiner 384-well white clear bottom medium binding plates. Then 10 uL solution containing 15-30 mg/mL AlphaScreen beads, 75 mM Hepes, pH 7.4, 300 mM NaCl, 120 mM EDTA, 0.3% BSA and 0.03% Tween-20 is added to each well. After 2-16 hr incubation of the beads, plates are read in a Perkin Elmer AlphaQuest reader (available from PerkinElmer Life and Analytical Sciences, Inc., of Boston Mass.).
Measurement: IC50 = 525 nM (pChEMBL 6.28000020980835)
Descriptors: MW 520.39, ALogP 4.56, HBA 7, HBD 2, PSA 88.61, QED 0.46
Compound (SMILES): COc1cc(-c2cn(CC(=O)Nc3cccc(F)c3F)nn2)ccc1-n1cnc(C)c1
InChIKey: QXKSETBKJXFOOV-UHFFFAOYSA-N
Target: Probable G-protein coupled receptor 142 (SINGLE PROTEIN), Homo sapiens
UniProt: Q7Z601
Assay: Agonist activity at human GPR142 expressed in CHO cells measured for 3 to 5 mins by Fluo-4AM dye-based FLIPR assay
Measurement: EC50 = 634 nM (pChEMBL 6.199999809265137)
Descriptors: MW 424.41, ALogP 3.36, HBA 7, HBD 1, PSA 86.86, QED 0.51
Compound (SMILES): O=C(O)CCCOc1ccc(-c2ccccc2Oc2ccccc2)cc1
InChIKey: RRKJCHFRHOQLNR-UHFFFAOYSA-N
Target: Free fatty acid receptor 4 (SINGLE PROTEIN), Mus musculus
UniProt: Q7TMA4
Assay: Agonist activity at mouse GPR120 expressed in CHOK1 cells measured after 60 mins by IP1-HTRF assay
Measurement: EC50 = 418 nM (pChEMBL 6.380000114440918)
Descriptors: MW 348.4, ALogP 5.39, HBA 3, HBD 1, PSA 55.76, QED 0.55
Compound (SMILES): CC(C)Oc1ccc(Nc2nc(=O)n(CC(CO)CO)c(=O)n2Cc2ccc(Cl)cc2)cc1Cl
InChIKey: PLKBMAZPUXNVOD-UHFFFAOYSA-N
Target: P2X purinoceptor 3 (SINGLE PROTEIN), Homo sapiens
UniProt: P56373
Assay: Antagonist activity at P2X3 receptor (unknown origin)
Measurement: IC50 = 6.7 nM (pChEMBL 8.170000076293945)
Descriptors: MW 509.39, ALogP 2.89, HBA 9, HBD 3, PSA 118.61, QED 0.38
Compound (SMILES): CCNC(CC)C(=O)Nc1cc(-c2ncnc3c(C)nn(C)c23)cc(C#Cc2ccc3c(C)nccc3c2)n1
InChIKey: GEJGWYOHCBQTRW-UHFFFAOYSA-N
Target: Baculoviral IAP repeat-containing protein 2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q13490
Assay: XIAP BIR3 & cIAP1 BIR3 Binding Assays (DELFIA): BIR3 domains (10 nM) were incubated with SMAC peptide (10 nM) in assay buffer (50 mM Tris, 120 mM NaCl, 0.1% BSA, 1 mM DTT, 0.05% TritonX100) for one hour at room temperature in the presence of inhibitory compounds. The assay mixture was transferred to a strepatvidin coated plate and incubated for one hour at room temperature to allow binding of the biotinylated peptide and associated BIR3 domains to the plate. After several washing steps Eu labeled anti-GST antibody (e.g. Perkin Elmer DELFIA Eu-N1-antiGST AD0250) was added to detect BIR3 domain-SMAC peptide interactions according to Perkin Elmer's instructions. Briefly, the antibody was added (dilution 1:5000 in Perkin Elmer DELFIA Assay Buffer 2013-01) and incubated for one hour. After 3 washing steps using Delfia Washing Buffer (Perkin Elmer DELFIA Wash 2013-05), Enhancement Solution (Perkin Elmer Enhancement Asolution 2013-02) was added and incubation continued for 10 minutes. Time resolved Europium fluorescence was measured in a Wallac Victor using Standard assay settings.
Measurement: IC50 = 6 nM (pChEMBL 8.220000267028809)
Descriptors: MW 518.63, ALogP 4.32, HBA 8, HBD 2, PSA 110.51, QED 0.32
Compound (SMILES): Cc1cc(N=S(C)(C)=O)cc2ncnc(Nc3ccc(F)cc3OCCC(F)(F)F)c12
InChIKey: FKXRAFMZZZCZIC-UHFFFAOYSA-N
Target: MAP kinase-interacting serine/threonine-protein kinase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9BUB5
Assay: In vitro Kinase Assay: The Z-LYTE biochemical assay employs a fluorescence-based, coupled-enzyme format and is based on the differential sensitivity of phosphorylated and non-phosphorylated peptides to proteolytic cleavage. The peptide substrate is labeled with two fluorophores one at each end that make up a FRET pair.In the primary reaction, the kinase transfers the gamma-phosphate of ATP to a single tyrosine, serine or threonine residue in a synthetic FRET-peptide. In the secondary reaction, a site-specific protease recognizes and cleaves non-phosphorylated FRET-peptides. Phosphorylation of FRET-peptides suppresses cleavage by the Development Reagent. Cleavage disrupts FRET between the donor (i.e., coumarin) and acceptor (i.e., fluorescein) fluorophores on the FRET-peptide, whereas uncleaved, phosphorylated FRET-peptides maintain FRET.
Measurement: IC50 = 75 nM (pChEMBL 7.119999885559082)
Descriptors: MW 456.47, ALogP 5.51, HBA 6, HBD 1, PSA 76.47, QED 0.5
Compound (SMILES): CC(C)C[C@H](NC(=O)C(=O)Nc1cccc2ccccc12)C(=O)N[C@@H](CC(=O)O)C(=O)O
InChIKey: SGVNFLQYMIJSSK-IRXDYDNUSA-N
Target: Caspase-8 (SINGLE PROTEIN), Homo sapiens
UniProt: Q14790
Assay: Inhibition of recombinant MCH5 (unknown origin) pre-incubated for 30 mins before Acetyl-Asp-Glu-Val-Asp-amino-4-methylcoumarin substrate addition measured 1 hr after substrate addition by fluorescence based assay
Measurement: IC50 = 179 nM (pChEMBL 6.75)
Descriptors: MW 443.46, ALogP 1.35, HBA 5, HBD 5, PSA 161.9, QED 0.37
Compound (SMILES): C[C@H](Nc1cc(C(N)=O)nc(-c2ccc(Oc3cc(C(F)(F)F)ccn3)cc2)n1)C(N)=O
InChIKey: DVTYMPZMXZNCFG-JTQLQIEISA-N
Target: N-formyl peptide receptor 2 (SINGLE PROTEIN), Homo sapiens
UniProt: P25090
Assay: FLIPR Assay: Assay Buffer: The assay buffer was formulated by removing 120 mL from a 1 L bottle of fresh, sterile dH2O (Mediatech, Herndon, Va.) and adding 100 mL of 10×HBSS that does not contain Ca++ or Mg++ (Gibco, Invitrogen, Grand Island, N.Y.) followed by 20 mL of 1.0 M Hepes, pH 7.3 (Fisher Scientific, BP299-100). The final buffer consisted of 20 mM Hepes, pH 7.3, 1.261 mM CaCl2, 0.493 mM MgCl2, 0.407 mM Mg(SO)4, 5.33 mM KCl, 0.441 mM KH2PO4, 137 mM NaCl, 0.336 mM Na2HPO4 and 0.556 mM D-glucose (Hanks et al., Proc. Soc. Exp. Biol. Med. 71:196 (1949)), and the simple formulation was typically the basic buffer throughout the assay (i.e., all wash and addition steps).
Measurement: IC50 = 3250 nM (pChEMBL 5.489999771118164)
Descriptors: MW 446.39, ALogP 2.73, HBA 7, HBD 3, PSA 146.11, QED 0.51
Compound (SMILES): COc1cc(N2Cc3cn(-c4cccc(S(C)(=O)=O)c4)nc3[C@H]2C(C)(C)C)ccn1
InChIKey: XOPXTKOQXWMGEF-NRFANRHFSA-N
Target: Oxysterols receptor LXR-beta (SINGLE PROTEIN), Homo sapiens
UniProt: P55055
Assay: Displacement of radiolabeled T0901317 from LXRbeta LBD (unknown origin)
Measurement: Ki = 5 nM (pChEMBL 8.300000190734863)
Descriptors: MW 426.54, ALogP 3.79, HBA 7, HBD 0, PSA 77.32, QED 0.63
Compound (SMILES): Cc1ccc(C(=O)Nc2cccc(C(F)(F)F)c2)cc1-c1cc(N2CCS(=O)(=O)CC2)nc(N2CCOCC2)n1
InChIKey: CJQHHPNKIQNPAX-UHFFFAOYSA-N
Target: Serine/threonine-protein kinase B-raf (SINGLE PROTEIN), Homo sapiens
UniProt: P15056
Assay: In Vitro Enzyme Assay: Final assay conditions were 4 pM cRaf, 3 uM ATP, 10 nM biotin tagged MEK1 kinase dead protein substrate. Reactions were performed in Greiner384 well plates, catalog #784075. Reaction buffer was 50 mM Tris, pH 7.5, 50 mM NaCl, 10 mM MgCl2, 0.01% Tween-20, 0.05% BSA and 1 mM DTT. 5 ul of 2×cRaf plus MEK1 were dispensed into plates containing 0.25 ul of compound in 100% DMSO and incubated at room temperature for 30 minutes. Reactions were started by addition of 5 ul of 2×ATP. Reactions were run for two hours and terminated by addition of 10 ul of 2× antibody and Alphascreen bead mixture in Stop Buffer. Stop Buffer was 50 mM Tris, pH 7.5, 0.005% Tween-20, 25 mM EDTA.
Measurement: IC50 = 3.146 nM (pChEMBL 8.5)
Descriptors: MW 575.61, ALogP 3.79, HBA 8, HBD 1, PSA 104.73, QED 0.49
Compound (SMILES): COCCOCCSc1ccc(S(=O)(=O)NC(=O)c2ccc(N3CCN(CC4=C(c5ccc(Cl)cc5)CC(C)(C)CC4)CC3)cc2Oc2ccc3[nH]ccc3c2)cc1[N+](=O)[O-]
InChIKey: YLAKCIHBXDGLNK-UHFFFAOYSA-N
Target: Bcl-2-like protein 1 (SINGLE PROTEIN), Mus musculus
UniProt: Q64373
Assay: Time Resolved-Fluorescence Resonance Energy Transfer (TR-FRET) Assay: The inhibition constant (Ki) is the dissociation constant of an enzyme-inhibitor complex or a protein/small molecule complex, wherein the small molecule is inhibiting binding of one protein to another protein or peptide. Where the Ki for a compound is represented as > (greater than) a certain numerical value, it is intended to mean that the binding affinity value (e.g., for Bcl-XL) is greater than the limits of detection of the assay used. Where the binding selectivity ratio for a compound is represented as > (greater than) a certain numerical value, it is intended to mean that the selectivity of a particular compound for Bcl-2 over Bcl-XL is at least as great as the number indicated. Where the Ki for a compound is represented as < (less than) a certain numerical value, it is intended to mean that the binding affinity value (e.g., for Bcl-2) is lower than the limit of detection of the assay used. Inhibition constants were determined using Wang's equation (Wang Z-X).
Measurement: Ki = 174.91 nM (pChEMBL 6.760000228881836)
Descriptors: MW 888.51, ALogP 9.18, HBA 11, HBD 2, PSA 156.34, QED 0.04
Compound (SMILES): O=c1ccc2c(OCCCCOc3ccc(Cl)cc3)c3ccoc3cc2o1
InChIKey: FVZIWWDONGSZLH-UHFFFAOYSA-N
Target: Potassium voltage-gated channel subfamily A member 5 (SINGLE PROTEIN), Homo sapiens
UniProt: P22460
Assay: Inhibition of Kv1.5 (unknown origin)
Measurement: EC50 = 10 nM (pChEMBL 8)
Descriptors: MW 384.82, ALogP 5.43, HBA 5, HBD 0, PSA 61.81, QED 0.31
Compound (SMILES): Fc1ccc(-c2n[nH]c3ccc(-c4n[nH]c(CN5CCCC5)n4)cc23)cc1
InChIKey: GDRSAYYDIMBYNZ-UHFFFAOYSA-N
Target: Casein kinase I isoform delta (SINGLE PROTEIN), Homo sapiens
UniProt: P48730
Assay: Inhibition of CSNK1D (unknown origin) using CSNK1D/Ser/Thr 11 peptide incubated for 1 hr by FRET based Z'-LYTE assay
Measurement: IC50 = 848 nM (pChEMBL 6.070000171661377)
Descriptors: MW 362.41, ALogP 3.75, HBA 4, HBD 2, PSA 73.49, QED 0.58
Compound (SMILES): Cc1ccc(-c2c(N)ncnc2N2CCC(c3nc(-c4ccc(F)c(C(F)(F)F)c4)cn3C)CC2)s1
InChIKey: VEHOASQICBVOCK-UHFFFAOYSA-N
Target: RAC-alpha serine/threonine-protein kinase (SINGLE PROTEIN), Homo sapiens
UniProt: P31749
Assay: AKT Enzyme Assay: A TTP Mosquito liquid handling instrument is used to place 125 nl of the appropriate concentration of inhibitor in 100% DMSO (for a dose response curve calculation) into each well of a 384-well plate. To this reaction components are added to a final volume of 12.5 .mu.l: 0.1 ng/.mu.l His-AKT (Full Length), (Invitrogen, Part # P2999, Lot #641228C). 160 uM ATP (Fluka, 02055) 1 mM DTT (Sigma, D0632) 1 mM MgCl2 (Sigma, M1028) 1 .mu.M substrate peptide (sequence FITC-AHA-GRPRTSSFAEG-NH2), synthesized by Tufts Peptide Synthesis service. 100 mM HEPES pH 7.5 (Calbiochem, 391338)0.015% Brij-35 (Sigma, B4184)The reaction is incubated for 90 min at 25 C, and then stopped by the addition of 70 .mu.l of Stop buffer (100 mM HEPES pH 7.5, 0.015% Brij-35, 10 mM EDTA (Sigma, E7889)).The plate is read on a Caliper LC 3000 in an Off-Chip mobility shift assay format, using the following parameters for a 12-sipper chip: screening pressure -2.3 psi, upstream voltage -500, and downstream voltage -3000.
Measurement: IC50 = 2700 nM (pChEMBL 5.570000171661377)
Descriptors: MW 516.57, ALogP 6.04, HBA 7, HBD 1, PSA 72.86, QED 0.34
Compound (SMILES): Cc1cnc(Nc2ccc(N3CCOCC3)cc2)nc1-c1cc2c(o1)CCN(S(C)(=O)=O)C2
InChIKey: YDZHOVOPBMIATL-UHFFFAOYSA-N
Target: Tyrosine-protein kinase JAK3 (SINGLE PROTEIN), Homo sapiens
UniProt: P52333
Assay: Inhibition of GST-tagged recombinant human cytoplasmic JAK3 expressed in baculovirus using TK-substrate-biotin as substrate preincubated for 5 mins followed by substrate addition after 30 mins by HTRF assay
Measurement: IC50 = 23.2 nM (pChEMBL 7.630000114440918)
Descriptors: MW 469.57, ALogP 2.94, HBA 8, HBD 1, PSA 100.8, QED 0.61
Compound (SMILES): CC(C)S(=O)(=O)c1ccc(Oc2ccc(F)cc2F)c(-c2cn(C)c(=O)c3[nH]ccc23)c1
InChIKey: IWFKOKYYFVBLCY-UHFFFAOYSA-N
Target: Bromodomain-containing protein 4 (SINGLE PROTEIN), Homo sapiens
UniProt: O60885
Assay: Bromodomain Domain Binding Assay: A time-resolved fluorescence resonance energy transfer (TR-FRET) assay was used to determine the affinities of compounds of the Examples listed in Table 1 for each bromodomain of human BRD4. His-tagged first (BD1: amino acids K57-E168) and second (BD2: amino acids E352-E168) bromodomains of human BRD4 were expressed and purified. An Alexa647-labeled BET-inhibitor was used as the fluorescent probe in the assay. Compound dilution series were prepared in DMSO via a 3-fold serial dilution from 2.5 mM to 42 nM. Compounds were then diluted 6:100 in 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) to yield 3× working solutions. Six microliters (L) of the working solution was then transferred to white, low-volume assay plates (Costar #3673). A 1.5× assay mixture containing His-tagged bromodomain, Europium-conjugated anti-His antibody (Invitrogen PV5596) and the Alexa-647-conjugated probe molecule was also prepared. Twelve L of this solution were added to the assay plate to reach a final volume of 18 L. The final concentration of 1× assay buffer contains 2% DMSO, 50 M-0.85 nM compound, 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). After a one-hour incubation at room temperature, TR-FRET ratios were determined using an Envision multilabel plate reader (Ex 340, Em 495/520).
Measurement: Ki = 1.9 nM (pChEMBL 8.720000267028809)
Descriptors: MW 458.49, ALogP 4.79, HBA 5, HBD 1, PSA 81.16, QED 0.47
Compound (SMILES): O=C(Nc1ccc(N2CCN(C(=O)c3cccc(F)c3)CC2)cc1)N1Cc2ccccc2C1
InChIKey: ZHSDKXOLIROYPY-UHFFFAOYSA-N
Target: Nicotinamide phosphoribosyltransferase (SINGLE PROTEIN), Homo sapiens
UniProt: P43490
Assay: Time-Resolved Fluorescence Resonance Energy Transfer (TR-FRET) Binding Assay : Probe 2:The assay was carried out in 18 uL low volume plates (Owens Corning) in reaction buffer (50 mM HEPES (NaOH), pH 7.5, 100 mM NaCl, 10 mM MgCl2, 1 mM DTT, 1% Glycerol) using 6.8 nM recombinant, human, C-terminally-His tagged NAMPT, 1 nM Tb-anti-His antibody (Invitrogen, Cat #PV5895), and 200 nM probe (Oregon Green 488-conjugated APO0866; A-1287128.0. Plates were covered, and reactions were carried out for 2-3 hours. Plates were read with Envision (Laser Lantha low volume protocol) after 2 to 3 hours. Excitation was carried out at 337 nm, and the ratio of emission of Oregon Green (520 nm) to terbium (492 nm) was determined and used to calculate IC50 values of test compounds.
Measurement: IC50 = 66.6 nM (pChEMBL 7.179999828338623)
Descriptors: MW 444.51, ALogP 4.34, HBA 3, HBD 1, PSA 55.89, QED 0.65
Compound (SMILES): Cc1nc(C)n(CC(=O)N2CCN(c3sc(C(F)(F)F)nc3-c3nc4ccc(-n5cncn5)cc4[nH]3)C[C@H]2C)n1
InChIKey: OEOHNTRTGVOMDH-CYBMUJFWSA-N
Target: C-X-C chemokine receptor type 3 (SINGLE PROTEIN), Homo sapiens
UniProt: P49682
Assay: Antagonist activity at human recombinant CXCR3 in human venous blood assessed as receptor internalization measured for 30 mins in presence of CXCL10 by flow cytometric analysis
Measurement: IC50 = 1135 nM (pChEMBL 5.949999809265137)
Descriptors: MW 571.59, ALogP 3.23, HBA 11, HBD 1, PSA 126.54, QED 0.34
Compound (SMILES): Cn1cc(-c2ccc3nc(NC(=O)c4cccc(-c5cccnc5)c4)cn3n2)c(-c2ccc(F)cc2)n1
InChIKey: NADLMTUCSMOWHY-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 = 1.7 nM (pChEMBL 8.770000457763672)
Descriptors: MW 489.51, ALogP 5.25, HBA 7, HBD 1, PSA 90, QED 0.36
Compound (SMILES): CN1CCN(c2ccc(C(=O)NCCCCCC(=O)Nc3ccccc3N)cc2)CC1
InChIKey: QHJWAXOBPZOUFE-UHFFFAOYSA-N
Target: Histone deacetylase 3 (SINGLE PROTEIN), Homo sapiens
UniProt: O15379
Assay: HDAC enzyme inhibitionAssay: HDAC enzyme inhibition assays were performed using purified HDACs 1-10 essentially as described in Beckers et al., 2007, Int. J. Cancer., 121:1138-48 and Perez-Balado et al., 2007, J. Med. Chem., 50:2497-2505. Inhibition assays using nuclear extract were performed essentially as described in Herman et al., 2006, Nat. Chem. Biol., 2:551-558. Briefly, the purified HDACs or nuclear extract were incubated with an acetylated substrate in the absence of the compound to be assayed and with increasing concentrations of the compound. The rate of substrate deacetylation was measured under each condition, and half-maximal inhibitory concentration with regard to each HDAC was determined by standard means.
Measurement: IC50 = 94 nM (pChEMBL 7.03000020980835)
Descriptors: MW 423.56, ALogP 2.95, HBA 5, HBD 3, PSA 90.7, QED 0.43
Compound (SMILES): COc1cc2c(N)nccc2cc1C[C@H](C(=O)OCC1CC1)N1CC[C@H](NS(=O)(=O)c2cnc(N3CCC(F)(F)C3)s2)C1=O
InChIKey: MEJMIZVFTUQQKE-LEWJYISDSA-N
Target: Coagulation factor X (SINGLE PROTEIN), Homo sapiens
UniProt: P00742
Assay: Inhibition Assay: The compounds according to the invention are tested in a range of concentrations (10pM to 10 µM final) in assay buffer (50 mM TRIS, 100 mM NaCl, 0.1 % BSA, pH 7,5) with 0.1% maximal final DMSO concentration and deposited in a proportion of 25 µl per well, on 25 µL enzyme (human coagulation factor Xa: Enzyme Research Laboratories HFXa, final concentration 0.003Ul/ml). The reactive were mixed, centrifuged and incubated 10 minutes at 37°C in a 96 well microtiter plate. The enzyme reaction was started with 50 µL substrate (S-2765, Biogenic ref 821413 in a final concentration of 62.5µM final). The time course of the reaction was monitored at 405 nm in a microtiter plate reader (Tecan M200) for 20 minutes at 37°C. The percentage of inhibition of enzymatic activity (expressed as maximum rate of cleavage of the substrate) is calculated with respect to the enzymatic activity in the absence of inhibitor.
Measurement: IC50 = 1000 nM (pChEMBL 6)
Descriptors: MW 650.73, ALogP 2.57, HBA 11, HBD 2, PSA 157.05, QED 0.3
Compound (SMILES): CC1(C)CC2=NNC(=O)c3cccc4c3c2c(n4CC2CO2)C1
InChIKey: CHUANYXVPMSWFM-UHFFFAOYSA-N
Target: Poly [ADP-ribose] polymerase 2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9UGN5
Assay: PARP-2 and PARP-3 Enzymatic Assay: PARP-2 and PARP-3 enzymatic assays were conducted using commercial PARP-2/PARP-3 Chemiluminescent Assay Kit (BPS Biosciences) and the protocols with the kits. Briefly, histones were coated in a high binding plate first, and incubated with PARP-2 or PARP-3, and increasing concentrations of compounds for 0.5 h. Then, biotinylated NAD and activated DNA were added to the wells. The biotinylated PARylation product was measured by adding streptavidin-HRP and HRP substrates which produce chemiluminescence.
Measurement: IC50 = 21 nM (pChEMBL 7.679999828338623)
Descriptors: MW 309.37, ALogP 2.46, HBA 4, HBD 1, PSA 58.92, QED 0.87
Compound (SMILES): CC1(O)CCN(c2cc(NC(=O)N3CCCc4ccc(C=O)nc43)ncc2C#N)CC1
InChIKey: RDOLOIPFRWACGW-UHFFFAOYSA-N
Target: Fibroblast growth factor receptor 4 (SINGLE PROTEIN), Homo sapiens
UniProt: P22455
Assay: Inhibition of TEL-fused FGFR4 cytoplasmic/juxta membrane domain (unknown origin) expressed in mouse Ba/F3 cells assessed as reduction in FGFR4 autophosphorylation after 1 hr by ELISA
Measurement: IC50 = 24 nM (pChEMBL 7.619999885559082)
Descriptors: MW 420.47, ALogP 2.5, HBA 7, HBD 2, PSA 122.45, QED 0.73
Compound (SMILES): CN1Cc2c(Cl)cc(Cl)cc2[C@H](c2cccc(S(=O)(=O)NCCOCCOCCNC(=O)CCC(CCC(=O)NCCOCCOCCNS(=O)(=O)c3cccc([C@@H]4CN(C)Cc5c(Cl)cc(Cl)cc54)c3)(CCC(=O)NCCOCCOCCNS(=O)(=O)c3cccc([C@@H]4CN(C)Cc5c(Cl)cc(Cl)cc54)c3)NC(=O)N[C@@H](CO)C(=O)O)c2)C1
InChIKey: LRDXMYUMSHCCGW-MUOCXZOZSA-N
Target: Sodium/hydrogen exchanger 3 (SINGLE PROTEIN), Homo sapiens
UniProt: P48764
Assay: Inhibition of human NHE3-mediated sodium-dependant hydrogen ion antiport in Opossum kidney cells preincubated in NH4Cl-HEPES buffer containing BCECF-AM dye followed by washout with ammonium free HEPES and subsequent addition of compound in presence of NHE1 antagonist EIPA by fluorescence assay
Measurement: IC50 = 25.12 nM (pChEMBL 7.599999904632568)
Descriptors: MW 1831.68
Compound (SMILES): N#CCC1(n2cc(C(N)=O)c(Nc3ccccc3)n2)CCN(C(=O)c2cccs2)CC1
InChIKey: ODFGAAWXPAYLML-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 = 8 nM (pChEMBL 8.100000381469727)
Descriptors: MW 434.53, ALogP 3.33, HBA 7, HBD 2, PSA 117.04, QED 0.62
Compound (SMILES): Cc1cccc(-c2ocnc2C(=O)Nc2cnn(CCOc3nc4ccccc4o3)c2)c1
InChIKey: ICJXFNRWHWUDQF-UHFFFAOYSA-N
Target: Orexin receptor type 2 (SINGLE PROTEIN), Homo sapiens
UniProt: O43614
Assay: Antagonist activity at human OX2R expressed in CHO cells assessed as orexin A-induced intracellular Ca2+ mobilization preincubated for 120 mins followed by orexin A addition by FLIPR assay
Measurement: IC50 = 2545 nM (pChEMBL 5.590000152587891)
Descriptors: MW 429.44, ALogP 4.32, HBA 8, HBD 1, PSA 108.21, QED 0.41
Compound (SMILES): CCOc1ccc(CC(=O)CNN(C(=O)CC)[C@@H](Cc2ccccc2)C(=O)NCc2ccc(CN)c(F)c2)cc1
InChIKey: QGGICRDCUBZHLM-LJAQVGFWSA-N
Target: Kallikrein-1 (SINGLE PROTEIN), Homo sapiens
UniProt: P06870
Assay: In Vitro Inhibition Assay: KLK1 inhibitory activity in vitro was determined using standard published methods (see e.g. Johansen et al., Int. J. Tiss. Reac. 1986, 8, 185; Shori et al., Biochem. Pharmacol., 1992, 43, 1209; Stürzebecher et al., Biol. Chem. Hoppe-Seyler, 1992, 373, 1025). Human KLK1 (Callbiochem) was incubated at 37° C. with the fluorogenic substrate H-DVal-Leu-Arg-AFC and various concentrations of the test compound. Residual enzyme activity (initial rate of reaction) was determined by measuring the change in optical absorbance at 410 nm and the IC50 value for the test compound was determined.
Measurement: IC50 > 1000 nM (pChEMBL 6)
Descriptors: MW 548.66, ALogP 3.47, HBA 6, HBD 3, PSA 113.76, QED 0.25
Compound (SMILES): CN1c2nc(C3=CC4CN(C(=O)C(C)(C)O)CC4C3)ccc2N(CC2CC2(F)F)S1(=O)=O
InChIKey: ZLXJBYSKCONUDP-UHFFFAOYSA-N
Target: Metabotropic glutamate receptor 2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q14416
Assay: [35S]-GTPγS Assay: The stimulation of [35S]-GTPγS binding is a common functional assay to monitor Gαi-coupled receptor in native and recombinant receptor membrane preparation. Membrane from cells stably expressing hmGlu2 CHO-K1 (50 μg) are incubated in a 96 well plate for 1 hour in the presence of GTPγS35 (0.05 nM), GDP (5 μM) and compounds. The reaction is stopped by rapid filtration over Unifilter GF/B plate (Packard, Bioscience, Meriden Conn.) using a 96-well cell harvester (Brandel Gaithersburg, Md.). The filter plates are counted using Topcount counter (Packard, Bioscience, Meriden Conn., USA). When compounds are evaluated as potentiators they are tested in the presence of glutamate (1 μM).
Measurement: EC50 = 88.63 nM (pChEMBL 7.050000190734863)
Descriptors: MW 468.53, ALogP 1.87, HBA 5, HBD 1, PSA 94.05, QED 0.73
Compound (SMILES): CN1Cc2c(Cl)cc(Cl)cc2[C@H](c2cccc(S(=O)(=O)NCCOCCOCCNC(=O)NCCN(CCNC(=O)NCCOCCOCCNS(=O)(=O)c3cccc([C@@H]4CN(C)Cc5c(Cl)cc(Cl)cc54)c3)C(=O)CCOCCOCCOCCOCCOCCC(=O)N(CCNC(=O)NCCOCCOCCNS(=O)(=O)c3cccc([C@@H]4CN(C)Cc5c(Cl)cc(Cl)cc54)c3)CCNC(=O)NCCOCCOCCNS(=O)(=O)c3cccc([C@@H]4CN(C)Cc5c(Cl)cc(Cl)cc54)c3)c2)C1
InChIKey: WQUKYQUIZBXSJY-ZTYOEKFNSA-N
Target: Sodium/hydrogen exchanger 3 (SINGLE PROTEIN), Homo sapiens
UniProt: P48764
Assay: Inhibition of human NHE3-mediated sodium-dependant hydrogen ion antiport in Opossum kidney cells preincubated in NH4Cl-HEPES buffer containing BCECF-AM dye followed by washout with ammonium free HEPES and subsequent addition of compound in presence of NHE1 antagonist EIPA by fluorescence assay
Measurement: IC50 = 79.43 nM (pChEMBL 7.099999904632568)
Descriptors: MW 2622.49
Compound (SMILES): CC(C)(CC(N)=O)NC(=O)c1cnc(C2CC2)c(OCC2CC2)n1
InChIKey: SQEJNRMIGWJALG-UHFFFAOYSA-N
Target: Cannabinoid receptor 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P21554
Assay: cAMP Assay: CHO cells expressing human CB1 or CB2 receptors are seeded 17-24 hours prior to the experiment 50.000 cells per well in a black 96 well plate with flat clear bottom (Corning Costar #3904) in DMEM (Invitrogen No. 31331), 1×HT supplement, with 10% fetal calf serum and incubated at 5% CO2 and 37° C. in a humidified incubator. The growth medium was exchanged with Krebs Ringer Bicarbonate buffer with 1 mM IBMX and incubated at 30° C. for 30 min. Compounds were added to a final assay volume of 100 μl and incubated for 30 min at 30° C. Using the cAMP-Nano-TRF detection kit the assay (Roche Diagnostics) was stopped by the addition of 50 μl lysis reagent (Tris, NaCl, 1.5% Triton X100, 2.5% NP40, 10% NaN3) and 50 μl detection solutions (20 μM mAb Alexa700-cAMP 1:1, and 48 μM Ruthenium-2-AHA-cAMP) and shaken for 2 h at room temperature. The time-resolved energy transfer is measured by a TRF reader (Evotec Technologies GmbH), equipped with a ND:YAG laser as excitation source. The plate is measured twice with the excitation at 355 nm and at the emission with a delay of 100 ns and a gate of 100 ns, total exposure time 10s at 730 (bandwidth 30 nm) or 645 nm (bandwidth 75 nm), respectively.
Measurement: EC50 > 10000 nM (pChEMBL 5)
Descriptors: MW 332.4, ALogP 1.53, HBA 5, HBD 2, PSA 107.2, QED 0.75
Compound (SMILES): CNC(=O)c1c(-c2ccc(F)cc2)oc2cc(N(C)S(C)(=O)=O)c(-c3ccc4c(c3)-c3cc5ccccc5n3CC(O)(C(F)(F)F)CO4)cc12
InChIKey: YZUWVJNRQMENRD-UHFFFAOYSA-N
Target: Genome polyprotein (SINGLE PROTEIN), Hepatitis C virus genotype 1b (isolate Japanese) (HCV)
UniProt: P26662
Assay: ELISA-Based Assay: Measurement of inhibition by compounds was performed using the HCV replicon system. Several different replicons encoding different HCV genotypes or mutations were used. In addition, potency measurements were made using different formats of the replicon assay, including different ways of measurements and different plating formats. See Jan M. Vrolijk et al., A replicons-based bioassay for the measurement of interferons in patients with chronic hepatitis C, 110 J. VIROLOGICAL METHODS 201 (2003); Steven S. Carroll et al., Inhibition of Hepatitis C Virus RNA Replication by 2'-Modified Nucleoside Analogs, 278(14) J. BIOLOGICAL CHEMISTRY 11979 (2003). However, the underlying principles are common to all of these determinations, and are outlined below.Stable neomycin phosphotransferase encoding replicons-harboring cell lines were used, so all cell lines were maintained under G418 selection prior to the assay. Potency was determined using a cell ELISA assay with an antibody to the replicons encoded NS3/4a protease. See Caterina Trozzi et al., In Vitro Selection and Characterization of Hepatitis C Virus Serine Protease Variants Resistant to an Active-Site Peptide Inhibitor, 77(6) J. Virol. 3669 (2003).
Measurement: IC50 = 11 nM (pChEMBL 7.960000038146973)
Descriptors: MW 707.7, ALogP 6.97, HBA 7, HBD 2, PSA 114.01, QED 0.19
Compound (SMILES): Nc1ncnc(N2CCC(c3nc(-c4ccc(F)c(F)c4)cn3CCN3CCCC3)CC2)c1Cl
InChIKey: AJHYNXPIENOPCI-UHFFFAOYSA-N
Target: RAC-alpha serine/threonine-protein kinase (SINGLE PROTEIN), Homo sapiens
UniProt: P31749
Assay: AKT Enzyme Assay: A TTP Mosquito liquid handling instrument is used to place 125 nl of the appropriate concentration of inhibitor in 100% DMSO (for a dose response curve calculation) into each well of a 384-well plate. To this reaction components are added to a final volume of 12.5 .mu.l: 0.1 ng/.mu.l His-AKT (Full Length), (Invitrogen, Part # P2999, Lot #641228C). 160 uM ATP (Fluka, 02055) 1 mM DTT (Sigma, D0632) 1 mM MgCl2 (Sigma, M1028) 1 .mu.M substrate peptide (sequence FITC-AHA-GRPRTSSFAEG-NH2), synthesized by Tufts Peptide Synthesis service. 100 mM HEPES pH 7.5 (Calbiochem, 391338)0.015% Brij-35 (Sigma, B4184)The reaction is incubated for 90 min at 25 C, and then stopped by the addition of 70 .mu.l of Stop buffer (100 mM HEPES pH 7.5, 0.015% Brij-35, 10 mM EDTA (Sigma, E7889)).The plate is read on a Caliper LC 3000 in an Off-Chip mobility shift assay format, using the following parameters for a 12-sipper chip: screening pressure -2.3 psi, upstream voltage -500, and downstream voltage -3000.
Measurement: IC50 = 3.2 nM (pChEMBL 8.489999771118164)
Descriptors: MW 487.99, ALogP 4.33, HBA 7, HBD 1, PSA 76.1, QED 0.56
Compound (SMILES): COc1ccc(O)c(/C=C/C(=O)CC(=O)/C=C/c2ccc(O)c(OC)c2)c1
InChIKey: KNFRXGSLSBTHLJ-SNCPXTGUSA-N
Target: Amyloid-beta precursor protein (SINGLE PROTEIN), Homo sapiens
UniProt: P05067
Assay: Inhibition of amyloid beta (1 to 42 residues) (unknown origin) aggregation after 24 hrs by thioflavin T fluorescence assay
Measurement: IC50 = 1800 nM (pChEMBL 5.75)
Descriptors: MW 368.39, ALogP 3.37, HBA 6, HBD 2, PSA 93.06, QED 0.55
Compound (SMILES): CC1=C(N2C(=O)C3(CCN(C[C@H](O)c4ccc5c(c4C)COC5=O)CC3)CC2C)COC1=O
InChIKey: GZQBZFYKKVFELZ-YNNZGITBSA-N
Target: ATP-sensitive inward rectifier potassium channel 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P48048
Assay: Inhibition of human ROMK expressed in CHO cells by whole cell voltage clamp electrophysiology assay
Measurement: IC50 = 18 nM (pChEMBL 7.75)
Descriptors: MW 454.52, ALogP 2.23, HBA 7, HBD 1, PSA 96.38, QED 0.7
Compound (SMILES): CNC(=O)C(C)(C)NC(=O)c1ccc(C2CC2)c(-c2cccc(Cl)c2)n1
InChIKey: NUDFOKPZCULQMP-UHFFFAOYSA-N
Target: Cannabinoid receptor 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P21554
Assay: cAMP Assay: CHO cells expressing human CB1 or CB2 receptors are seeded 17-24 hours prior to the experiment 50.000 cells per well in a black 96 well plate with flat clear bottom (Corning Costar #3904) in DMEM (Invitrogen No. 31331), 1.times.HT supplement, with 10% fetal calf serum and incubated at 5% CO2 and 37° C. in a humidified incubator. The growth medium was exchanged with Krebs Ringer Bicarbonate buffer with 1 mM IBMX and incubated at 30.degree. C. for 30 min Compounds were added to a final assay volume of 100 ul and incubated for 30 min at 30° C. Using the cAMP-Nano-TRF detection kit the assay (Roche Diagnostics) was stopped by the addition of 50 ul lysis reagent (Tris, NaCl, 1.5% Triton X100, 2.5% NP40, 10% NaN.sub.3) and 50 ul detection solutions (20 uM mAb Alexa700-cAMP 1:1, and 48 uM Ruthenium-2-AHA-cAMP) and shaken for 2 h at room temperature. The time-resolved energy transfer is measured by a TRF reader (Evotec Technologies GmbH), equipped with a ND:YAG laser as excitation source. The plate is measured twice with the excitation at 355 nm and at the emission with a delay of 100 ns and a gate of 100 ns, total exposure time 10 s at 730 (bandwidth 30 nm) or 645 nm (bandwidth 75 nm), respectively. The FRET signal is calculated.
Measurement: EC50 > 10000 nM (pChEMBL 5)
Descriptors: MW 371.87, ALogP 3.53, HBA 3, HBD 2, PSA 71.09, QED 0.84
Compound (SMILES): O=C1NCCc2[nH]c(-c3cccc4ccc(O[C@@H]5CCCNC5)nc34)cc21
InChIKey: NTBQVSFBQPCPSN-CQSZACIVSA-N
Target: Serine/threonine-protein kinase pim-2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9P1W9
Assay: Homogeneous Time Resolved Fluorescence Assay (HTRF): The assay for the determination of Pim activity is based on the formation of phosphorylated biotinylated-BAD peptide at the Serine 112 residue (S112) and employs HTRF® (homogeneous time resolved fluorescence) technology to detect the product in a 96-well plate format. The phosphorylation of biotinylated-BAD (S112) peptide by full length recombinant Pim-1, Pim-2, or Pim-3 protein was detected with streptavidin:Allophycocyanin (APC) conjugate and a europium (Eu) labeled antibody directed against phosphorylated-BAD (S112). Excitation of Eu by a high energy laser light (337 nm) leads to a transfer of energy to the APC molecule, and results in an emission at 665 nm. The fluorescence is directly proportional to the amount of phosphorylated BAD peptide present in the reaction. Compounds were prepared in DMSO by conducting 3-fold serial dilutions to give a 10-point dosing curve having a high dose of 1 μM. A reference compound was included on each assay plate in order to validate that plate; on one plate of every assay run, two additional reference compounds were included. The final buffer conditions were as follows: 60 mM Hepes, pH 7.0, 0.05% BSA, 2 mM DTT. Incubations were carried out at RT (22° C.) for 2 h for Pim-1, 1 hour and 30 min for Pim-3, and 45 min for Pim-2. The reaction was stopped by the addition of 3 mM EDTA, and fluorescence was measured by an HTRF® Rubystar microplate reader.
Measurement: IC50 = 340 nM (pChEMBL 6.46999979019165)
Descriptors: MW 362.43, ALogP 2.65, HBA 4, HBD 3, PSA 79.04, QED 0.67
Compound (SMILES): FC(F)(F)C1CCN(C[C@H]2CC[C@H](c3nnn4cnc5[nH]ccc5c34)CC2)CC1
InChIKey: CFYKFXXPQVORBG-HDJSIYSDSA-N
Target: Tyrosine-protein kinase JAK2 (SINGLE PROTEIN), Homo sapiens
UniProt: O60674
Assay: Enzyme Assay: JAK1, JAK2, JAK3 and Tyk2 were purchased from Carna Biosciences, Inc. As the substrate, LANCE Ultra ULight-JAK1 Peptide (manufactured by PerkinElmer Co., Ltd. (PE)) was used. Dilute solutions of compounds and enzymes in assay buffer (50 mM HEPES pH7.5, 1 mM EGTA, 1 mM MgCl2, 2 mM DTT, 0.01% Tween20) were dispensed into wells of a 384-well black plate. After 5 minutes of preincubation, dilute solutions of the substrate and ATP (adenosine triphosphate) were added at a final concentration of 100 μM, and the plate was incubated at room temperature for 2 hours. After addition of a termination reagent containing EDTA (ethylenediamine tetraacetic acid) at a final concentration of 10 mM, LANCE Eu-W1024 Anti-phosphotyrosine (PT66) (manufactured by PE) was added, and after 1 hour of incubation, the fluorescences were measured with ARVO-HTS.
Measurement: IC50 = 81 nM (pChEMBL 7.090000152587891)
Descriptors: MW 406.46, ALogP 4.15, HBA 5, HBD 1, PSA 62.11, QED 0.71
Compound (SMILES): CC(C)c1cc(CNc2nccc(Nc3cc(C4CC4)[nH]n3)n2)on1
InChIKey: YHLMCLCAANHRIL-UHFFFAOYSA-N
Target: Insulin-like growth factor 1 receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P08069
Assay: Inhibition of human IGF1R kinase domain (M954 to C1367 residues) tri-phosphorylated enzyme form expressed in baculovirus based expression system using poly-EY peptide incubated for 2 hrs by luciferase-luciferin coupled chemiluminescent assay
Measurement: IC50 = 50 nM (pChEMBL 7.300000190734863)
Descriptors: MW 339.4, ALogP 3.54, HBA 7, HBD 3, PSA 104.55, QED 0.6
Compound (SMILES): CC(C)C[C@@H]1NCCOc2ccccc2CCCNC(=O)[C@H](Cc2ccccc2)NC(=O)C2(CCCC2)NC1=O
InChIKey: CLSFYQHIABHVHP-SVBPBHIXSA-N
Target: Growth hormone secretagogue receptor type 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q92847
Assay: Agonist activity at human GSHR1a transfected in CHOK1 cells co-expressing Galpha16 assessed as increase in Ca2+ release by aequorin assay
Measurement: EC50 = 100 nM (pChEMBL 7)
Descriptors: MW 548.73, ALogP 3.29, HBA 5, HBD 4, PSA 108.56, QED 0.47
Compound (SMILES): Cc1noc(C)c1-c1cc(C(C)c2ccc(F)cc2)c2[nH]c(C3CC3)nc2c1
InChIKey: UTXGSIWMYUWWGY-UHFFFAOYSA-N
Target: Bromodomain-containing protein 4 (SINGLE PROTEIN), Homo sapiens
UniProt: O60885
Assay: Homogeneous Time Resolved Fluorescence Resonance Energy Transfer (TR-FRET) Assay: Binding of the two tandem bromodomains, BRD4-1 and BRD4-2, to an acetylated histone H4 peptide was measured using a homogeneous time resolved fluorescence resonance energy transfer (TR-FRET) assay. The synthetic peptide containing amino acids 1-18 of histone H4 was acetylated at lysine 5, 8, 12, 16 and conjugated to biotin (SGRGACKGGACKGLGACKGGAACKRH-GSGSK-biotin[SEQ ID NO:1]) was purchased from Millipore. BRD4-1 and BRD4-2 were expressed and purified from Escherichia coli as N-terminal His6-tagged proteins. An XL665 labeled anti-His antibody (Cisbio) was used to specifically bind BRD4 and a cryptate labeled streptavidin protein was used because it specifically recognized the biotinylated H4 peptide. Binding of BRD4 to the peptide resulted in an increase in FRET signal whereas disruption of this protein-peptide interaction with a small molecule inhibitor resulted in a decrease in FRET signal. Assays were performed in 50 mM Hepes (pH 7.5), 150 mM NaCl, 0.1 mg/ml BSA, 0.01% (v/v) Brij, 0.5% (v/v) DMSO and 200 nM H4 peptide at the following concentrations for each BRD4 isoform: 60 nM BRD4-1 and 120 nM BRD4-2. After an assay reaction time of 60 minutes at 25° C., binding was measured with 2 nM crytptate labeled streptavidin and 10 nM anti-His-XL665 antibody. TR-FRET signal was detected on an Envision plate reader (Ex: 320 nm; Em: 615/665 nm; 100 As delay and 200 Î¼s read window). Data were normalized based on a positive (2 Î¼M I-BET) and negative (DMSO) controls and IC50values were calculated from the fit of the dose-response curves to a four-parameter equation. All IC50 values represent geometric mean values of a minimum of four determinations.
Measurement: IC50 = 110.9 nM (pChEMBL 6.960000038146973)
Descriptors: MW 375.45, ALogP 6, HBA 3, HBD 1, PSA 54.71, QED 0.48
Compound (SMILES): CS(=N)(=O)c1ccc(Nc2ncc(Br)c(Nc3ccccc3)n2)cc1
InChIKey: UOXXHPOQLJPWNQ-UHFFFAOYSA-N
Target: Cyclin-dependent kinase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P06493
Assay: Kinase Assay: Kinase assay using either CDK1, CDK2 or VEGFR-2.
Measurement: IC50 = 160 nM (pChEMBL 6.800000190734863)
Descriptors: MW 418.32, ALogP 4.76, HBA 6, HBD 3, PSA 90.76, QED 0.56
Compound (SMILES): O=S(=O)(Nc1cccnn1)c1ccc2c(c1)OCCN2c1ccc(C(F)(F)F)cc1C1=CCNCC1
InChIKey: MDUMLNKWTWMUSR-UHFFFAOYSA-N
Target: Sodium channel protein type 9 subunit alpha (SINGLE PROTEIN), Homo sapiens
UniProt: Q15858
Assay: 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 = 381 nM (pChEMBL 6.420000076293945)
Descriptors: MW 517.53, ALogP 4.2, HBA 7, HBD 2, PSA 96.45, QED 0.52
Compound (SMILES): COc1ccc(-c2cc(C(N)=O)nc3cc(C(C)Cc4cccnc4N)ccc23)c(F)c1
InChIKey: HRAMGXORLGQLPY-UHFFFAOYSA-N
Target: Metabotropic glutamate receptor 2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q14416
Assay: Biological Assay: The utility of the compounds in accordance with the present invention as antagonists of metabotropic glutamate receptor activity, in particular mGluR2 activity, may be demonstrated by methodology known in the art. Antagonist constants are determined as follows. The compounds of the present invention were tested in a fluorescence laser imaging plate reader based assay. This assay is a common functional assay to monitor Ca2+ mobilization in whole cells expressing recombinant receptor coupled with a promiscuous G-protein. CHO dhfr- cells stably expressing recombinant human mGluR2 and Gα16 loaded with Fluo-4 AM (Invitrogen, Carlsbad Calif.) are treated with various concentrations of antagonists of compounds and the Ca2+ response is monitored on a FLIPR384 (Molecular Devices, Sunnydale Calif.) for agonist activity.
Measurement: IC50 = 14 nM (pChEMBL 7.849999904632568)
Descriptors: MW 430.48, ALogP 4.47, HBA 5, HBD 2, PSA 104.12, QED 0.47