lovingscience/chembl-v37-raw-activities huggingface dataset:
Compound (SMILES): Cc1ccc(C(=O)Nc2cc(C(C)(C)C)on2)cc1-n1cc(-c2cncc(N(C)C3CCOC3)c2)nn1
InChIKey: VUVJZAGCIKDPQH-UHFFFAOYSA-N
Target: Serine/threonine-protein kinase B-raf (SINGLE PROTEIN), Homo sapiens
UniProt: P15056
Assay: B-RAF (V600E) Kinase Assay: In a dilution series 10 uL/well of test substance solution are placed in a multiwell plate. The dilution series is selected so that generally a range of concentrations of 2 uM to 0.119 nM or 0.017 nM is covered. If necessary the initial concentration of 2 uM is changed to 50 uM, 10 uM or 0.4 uM or 0.2857 uM and further dilution is carried out accordingly. The final concentration of DMSO is 5%. 10 uL/well of the B-Raf (V600E)-kinase solution are pipetted in (containing 0.5 ng B-Raf (V600E)-kinase (e.g. from Upstate) in 20 mM Tris-HCl pH 7.5, 0.1 mM EDTA, 0.1 mM EGTA, 0.286 mM sodium orthovanadate, 10% glycerol, 1 mg/mL bovine serum albumin, 1 mM dithiothreitol) and the mixture is incubated for 1 h at RT under with shaking. The kinase reaction is started by the addition of 20 uL/well ATP solution [final concentration: 250 uM ATP, 30 mM Tris-HCl pH 7.5, 0.02% Brij, 0.2 mM sodium orthovanadate, 10 mM magnesium acetate, 0.1 mM EGTA, phosphatase cocktail (Sigma, #P2850, dilution recommended by the manufacturer)] and 10 uL/well MEK1 solution [containing 50 ng biotinylated MEK1 (prepared from purified MEK1 according to standard procedure, e.g. with EZ-Link Sulpho-NHS-LC-Biotin reagent, Pierce, #21335)] and carried out for 60 min at RT with constant shaking. The reaction is stopped by the addition of 12 uL/well of a 100 mM EDTA solution and incubation is continued for a further 5 min. 55 uL/well of the reaction solution are transferred into a streptavidin-coated plate (e.g. Streptawell HighBond, Roche, #11989685001) and shaken gently for 1 h at RT, in order to bind biotinylated MEK1 to the plate. After elimination of the liquid the plate is washed five times with 200 uL/well of 1Ã¿PBS and 100 uL/well solution of primary antibody plus europium-labelled secondary antibody [Anti Phospho-MEK (Ser217/221), Cell Signaling, #9121 and Eu-N1 labelled goat-anti-rabbit antibody, Perkin Elmer, #AD0105] is added, the primary antibody is diluted 1:2000 and the secondary antibody is diluted to 0.4-0.5 ug/mL in Delfia Assay Buffer (Perkin Elmer, #1244-111). After 1 h shaking at RT the solution is poured away and washed five times with 200 uL/well Delfia Wash Buffer (Perkin Elmer, #4010-0010/#1244-114). After the addition of 200 uL/well Enhancement Solution (Perkin Elmer, #4001-0010/#1244-105) the mixture is shaken for 10 min at RT and then measured in a Wallac Victor using the program "Delfia Time Resolved Fluorescence (Europium)". IC50 values are obtained from these dosage-activity curves using a software program (GraphPadPrizm).
Measurement: IC50 = 8 nM (pChEMBL 8.100000381469727)
Descriptors: MW 501.59, ALogP 4.4, HBA 9, HBD 1, PSA 111.2, QED 0.41
Compound (SMILES): Cc1cc(F)ccc1NC(=O)c1cccc(Cc2n[nH]c(=O)c3c2CCCC3)c1
InChIKey: IEWWLTJWWFBYLR-UHFFFAOYSA-N
Target: Poly [ADP-ribose] polymerase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P09874
Assay: PARP Enzyme Inhibition Assay: PARP1 assay was conducted in PARP assay buffer containing 50 mM Tris pH 8.0, 1 mM DTT, 4 mM MgCl2. PARP reactions contained 1.5 uM [3H]-NAD+ (1.6 uCi/mmol), 200 nM biotinylated histone H1, 200 nM sIDNA, and 1 nM PARP enzyme. Auto reactions utilizing SPA bead-based detection were carried out in 100 uL volumes in white 96 well plates. Reactions were initiated by adding 50 ul of 2xNAD+ substrate mixture to 50 uL of 2x enzyme mixture containing PARP and DNA. These reactions were terminated by the addition of 150 uL of 1.5 mM benzamide (~1000-fold over its IC50). 170 uL of the stopped reaction mixtures were transferred to streptavidin Flash Plates, incubated for 1 hour, and counted using a TopCount microplate scintillation counter.
Measurement: Ki = 279 nM (pChEMBL 6.550000190734863)
Descriptors: MW 391.45, ALogP 3.94, HBA 3, HBD 2, PSA 74.85, QED 0.71
Compound (SMILES): CCN(CC)C(=O)CN[C@@H]1CCc2c(-c3noc(-c4ccc(OC(C)C)c(C#N)c4)n3)cccc21
InChIKey: GJWOVIPYQPVQMK-HSZRJFAPSA-N
Target: Sphingosine 1-phosphate receptor 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P21453
Assay: Agonist activity at human S1P1 expressed in CRE-bla CHOK1 cells assessed as inhibition of forskolin-induced cAMP formation preincubated for 4 hrs followed by FRET-based beta-lactamase fluorescent substrate addition measured after 2 hrs
Measurement: EC50 = 0.5 nM (pChEMBL 9.300000190734863)
Descriptors: MW 473.58, ALogP 4.51, HBA 7, HBD 1, PSA 104.28, QED 0.49
Compound (SMILES): C[C@@]1(c2cc(NC(=O)c3nn(C(F)F)cc3Cl)cnc2F)C[C@@H](C(F)(F)F)OC(N)=N1
InChIKey: VJJOURSDKWNMOD-VFZGTOFNSA-N
Target: Beta-secretase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P56817
Assay: In Vitro Enzymatic BACE FRET Assay: The assay buffer used in this screen is 0.05 M acetate, pH 4.2, 10% DMSO final, 100 uM genapol (which is a nonionic detergent, below its Critical Micelle Concentration). The Beta Secretase enzyme (0.2 nM) is pre-incubated for one hour with inhibitors, typically in about 1 uL of DMSO according to a serial dilution, are added thereto. This assay is effectively started by the addition of FRET substrate (50 nM) and the combination is incubated for one hour. The FRET assay is terminated with by addition of Tris buffer, which raises the pH to neutrality, and the fluorescence is determined. The FRET substrate is a peptide with commercially available fluorophore and quencher, on opposite sides of the BACE cleavage site. Proteolytic cleavage of the FRET substrate releases quenching of fluorescence (excitation 488 nm and emission 425 nm).
Measurement: IC50 = 24 nM (pChEMBL 7.619999885559082)
Descriptors: MW 470.76, ALogP 3.6, HBA 7, HBD 2, PSA 107.42, QED 0.52
Compound (SMILES): O=C1COc2ccc(C3(O)c4ccccc4C(=O)N3c3ccc(F)c(C(F)(F)F)c3)cc2N1
InChIKey: ODOWXUUIDMYMAO-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 >= 10000 nM (pChEMBL 5)
Descriptors: MW 458.37, ALogP 4.03, HBA 4, HBD 2, PSA 78.87, QED 0.57
Compound (SMILES): COc1cn(C(CC2CCC2)C(=O)Nc2ccc(-c3c[nH]c(=O)o3)cc2)c(=O)cc1-c1cc(Cl)ccc1C#N
InChIKey: LUITXNJTLSXQQE-UHFFFAOYSA-N
Target: Coagulation factor XI (SINGLE PROTEIN), Homo sapiens
UniProt: P03951
Assay: Inhibition Assay: Test substances are dissolved in dimethyl sulphoxide and serially diluted in dimethyl sulphoxide (3000 μM to 0.0078 μM; resulting final concentrations in the test: 50 μM to 0.00013 μM). In each case 1 μl of the diluted substance solutions are placed into the wells of white microtitre plates from Greiner (384 wells). 20 μl of assay buffer (50 mM of Tris/HCl pH 7.4; 100 mM of sodium chloride solution; 5 mM of calcium chloride solution; 0.1% of bovine serum albumin) and 20 μl of factor XIa from Kordia (0.45 nM in assay buffer) are then added successively. After 15 min of incubation, the enzyme reaction is started by addition of 20 μl of the factor XIa substrate Boc-Glu(OBzl)-Ala-Arg-AMC dissolved in assay buffer (10 μM in assay buffer) from Bachem, the mixture is incubated at room temperature (22° C.) for 30 min and fluorescence is then measured (excitation: 360 nm, emission: 460 nm).
Measurement: IC50 = 22 nM (pChEMBL 7.659999847412109)
Descriptors: MW 545, ALogP 5.37, HBA 7, HBD 2, PSA 130.12, QED 0.31
Compound (SMILES): O=C(c1cc(Cc2n[nH]c(=O)c3c2CCCC3)ccc1F)N1CCN(C(=O)[C@@H]2CSCN2)CC1
InChIKey: AQNOMPVQOHZJRS-NRFANRHFSA-N
Target: Poly [ADP-ribose] polymerase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P09874
Assay: PARP Enzyme Inhibition Assay: PARP1 assay was conducted in PARP assay buffer containing 50 mM Tris pH 8.0, 1 mM DTT, 4 mM MgCl2. PARP reactions contained 1.5 uM [3H]-NAD+ (1.6 uCi/mmol), 200 nM biotinylated histone H1, 200 nM sIDNA, and 1 nM PARP enzyme. Auto reactions utilizing SPA bead-based detection were carried out in 100 uL volumes in white 96 well plates. Reactions were initiated by adding 50 ul of 2xNAD+ substrate mixture to 50 uL of 2x enzyme mixture containing PARP and DNA. These reactions were terminated by the addition of 150 uL of 1.5 mM benzamide (~1000-fold over its IC50). 170 uL of the stopped reaction mixtures were transferred to streptavidin Flash Plates, incubated for 1 hour, and counted using a TopCount microplate scintillation counter.
Measurement: Ki = 1.4 nM (pChEMBL 8.850000381469727)
Descriptors: MW 485.59, ALogP 1.33, HBA 6, HBD 2, PSA 98.4, QED 0.68
Compound (SMILES): CCOC(=O)c1cc(-c2ccccc2)sc1NC(=O)c1ccc2ccccc2c1
InChIKey: BOXQWPWSSYVTDZ-UHFFFAOYSA-N
Target: Cannabinoid receptor 2 (SINGLE PROTEIN), Homo sapiens
UniProt: P34972
Assay: Displacement of [3H]-CP-55940 from human CB2 receptor expressed in HEK293 cell membranes after 90 mins by radioligand binding assay
Measurement: Ki > 10000 nM (pChEMBL 5)
Descriptors: MW 401.49, ALogP 6, HBA 4, HBD 1, PSA 55.4, QED 0.42
Compound (SMILES): C[C@@H](c1ccccc1)N1CCC(N2CC[C@@H](c3ccc(O)cc3)[C@H](F)C2)C1=O
InChIKey: CAYQYXDBKVLPMB-ONKNLRSLSA-N
Target: Glutamate receptor ionotropic, NMDA 2B (SINGLE PROTEIN), Rattus norvegicus
UniProt: Q00960
Assay: Radioligand Binding Assay: To perform the competition binding assay, thawed membrane homogenate was added to each well of a 96-well plate (20 ug/well). The experimental compounds were serially diluted in 100% DMSO and added to each row of the assay plate to achieve desired compound concentrations, keeping the DMSO concentration in the assay plate at 1.33% of the final reaction volume. Next, 3H Ro 25-6981 (4 nM) was added to the assay plate. After incubation for 1 hr at room temperature, the membrane bound radioligand was harvested on to GF/B filter plates (treated with 0.5% PEI for 1 hr at room temperature). The filter plates were dried at 50° C. for 20 mins, incubated with microscint 20 for 10 minutes and finally, the counts were read on TopCount (Perkin Elmer). Non-specific binding was determined using MK-0657 (the preparation of this compound is described as example 1 in WO 2004 108705 (40 uM).
Measurement: Ki = 710 nM (pChEMBL 6.150000095367432)
Descriptors: MW 382.48, ALogP 3.88, HBA 3, HBD 1, PSA 43.78, QED 0.87
Compound (SMILES): O=C(Nc1cnn2ccc(N3CCC[C@@H]3c3cc(F)ccc3F)nc12)[C@H]1CC[C@H](O)C1
InChIKey: FJCFOBAIIMESRH-ZUEVXXBESA-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 = 42.5 nM (pChEMBL 7.369999885559082)
Descriptors: MW 427.46, ALogP 3.45, HBA 6, HBD 2, PSA 82.76, QED 0.67
Compound (SMILES): Cc1cc(Nc2cc(C(F)(F)F)ccn2)nc(-c2ccc(C3(O)CCNCC3)nc2)c1
InChIKey: AYASSQPFHIGBOH-UHFFFAOYSA-N
Target: Tyrosine-protein kinase SYK (SINGLE PROTEIN), Homo sapiens
UniProt: P43405
Assay: Homogeneous Time-Resolved Fluorescence (HTRF) Assay: A recombinant GST-hSyk fusion protein was used to measure potency of compounds to inhibit human Syk activity. The recombinant human GST-Syk (Carna Biosciences #08-176) (5 pM final concentration) was incubated with various concentrations of the inhibitor diluted in DMSO (0.1% final concentration) for 10 minutes at room temperature in 15 mM Tris-HCl (pH 7.5), 0.01% tween 20, 2 mM DTT in 384 well plate format. To initiate the reaction the biotinylated substrate peptide (250 nM final concentration) that contains the phosphorylation site for Syk was added with magnesium (5 mM final concentration) and ATP (25 uM final concentration). Final volume of the reaction was 10 uL. Phosphorylation of the peptide was allowed to proceed for 45x at room temperature. To quench the reaction and detect the phosphorylated product, 2 nM of a Europium-anti-phosphotyrosine antibody (Perkin Elmer #AD0161) and 70 nM SA-APC (Perkin-Elmer #CR130-100) were added together in 15 mM Tris pH 7.5.
Measurement: IC50 = 9 nM (pChEMBL 8.050000190734863)
Descriptors: MW 429.45, ALogP 4.18, HBA 6, HBD 3, PSA 82.96, QED 0.58
Compound (SMILES): CC(C)c1ccc(S(=O)(=O)NC[C@@H](O)C[C@@H](Cc2ccccc2)C(=O)N[C@H]2c3ccccc3C[C@H]2O)cc1
InChIKey: YDNYPGVDVMVJBS-BVIJCPSYSA-N
Target: Vasoactive intestinal polypeptide receptor 2 (SINGLE PROTEIN), Homo sapiens
UniProt: P41587
Assay: Antagonist activity at recombinant human N-terminal signal peptide/FLAG epitope-tagged Rluc8-fused VIPR2 expressed in HEK293 cells assessed as inhibition of VIP-induced human mVenus-beta-arrestin2 binding preincubated for 5 mins with luciferin, coelenterazine H followed by VIP addition measured after 5 mins by BRET assay
Measurement: IC50 = 3440 nM (pChEMBL 5.460000038146973)
Descriptors: MW 536.69, ALogP 3.47, HBA 5, HBD 4, PSA 115.73, QED 0.3
Compound (SMILES): C[C@@]1(c2cc(Nc3cc(Cl)cc4cc(Cl)cnc34)ccc2F)COCC(N)=N1
InChIKey: LBDVFEXBWOROJV-FQEVSTJZSA-N
Target: Beta-secretase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P56817
Assay: Cellular Aβ-Lowering Assay: DNA of the human APP wt gene (APP695) were used to assess the potency of the compounds in a cellular assay. The cells were seeded in 96-well microtiter plates in cell culture medium (Iscove, plus 10% (v/v) fetal bovine serum, glutamine, penicillin/streptomycin) to about 80% confluence and the compounds were added at a 10× concentration in 1/10 volume of medium without FCS containing 8% DMSO (final concentration of DMSO was kept at 0.8% v/v). After 18-20 hrs incubation at 37° C. and 5% CO2 in a humidified incubator the culture supernatant was harvested for the determination of Aβ340 concentrations. 96well ELISA plates (e.g., Nunc MaxiSorb) were coated with monoclonal antibody which specifically recognize the C-terminal end of Aβ340 (Brockhaus et al., NeuroReport 9, 1481-1486; 1998). After blocking of non-specific binding sites with e.g. 1% BSA and washing, the culture supernatants were added in suitable dilutions together with a horseradish peroxidase-coupled Aβ detectio
Measurement: IC50 = 660 nM (pChEMBL 6.179999828338623)
Descriptors: MW 419.29, ALogP 5.03, HBA 5, HBD 2, PSA 72.53, QED 0.63
Compound (SMILES): NS(=O)(=O)OC[C@@H]1C[C@@H](Cc2cc(N[C@H]3CCc4ccccc43)ncn2)C[C@@H]1O
InChIKey: ZVNMCNUGHPOVTE-BMIPRAEKSA-N
Target: NEDD8 activating enzyme (PROTEIN COMPLEX), Homo sapiens
UniProt: Q13564
Assay: Inhibition of recombinant human NAEalpha/N-terminal His-tagged NAEbeta expressed in baculovirus infected insect Sf9 cells using N-terminal FLAG tagged Nedd8 as substrate incubated for 105 mins in presence of N-terminal GST-tagged Ubc12 by FRET assay
Measurement: IC50 = 500 nM (pChEMBL 6.300000190734863)
Descriptors: MW 418.52, ALogP 1.73, HBA 7, HBD 3, PSA 127.43, QED 0.62
Compound (SMILES): COc1cc(-c2ccncc2Cl)c(F)cc1-n1c(=O)ccc2cc(S(=O)(=O)Nc3ccon3)ccc21
InChIKey: JFPXXJUPWVYVFP-UHFFFAOYSA-N
Target: Sodium channel protein type 9 subunit alpha (SINGLE PROTEIN), Homo sapiens
UniProt: Q15858
Assay: Nav1.7 In Vitro PX Assay: HEK 293 cells stably transfected with human Nav1.7 were recorded in whole cell voltage clamp mode with the PatchXpress automated electrophysiology system (Molecular Devices, LLC, Sunnyvale, Calif.). Compound effects were measured on a partially inactivated state of the sodium channel. Cells were clamped to a holding potential yielding 20 to 50% inactivation. To elicit sodium current, channels were activated by pulsing to -10 mV for 20 msec. This voltage protocol was repeated at a rate of 0.1 Hz throughout the experiment. A single concentration of test compound was applied to cells for a duration of 3 minutes.
Measurement: IC50 = 573 nM (pChEMBL 6.239999771118164)
Descriptors: MW 526.93, ALogP 4.64, HBA 8, HBD 1, PSA 116.32, QED 0.34
Compound (SMILES): CC(C)C[C@@H]1NC(=O)[C@H](CCC(N)=O)NC(=O)[C@@H]2CCCN2C(=O)[C@@H](NC(=O)[C@H](C)N)CSCc2cc3cc(c2)CSC[C@H](C(=O)NCC(N)=O)NC(=O)[C@H](CO)NC(=O)[C@H](C(C)C)NC(=O)[C@H](CCCNC(=N)N)NC(=O)[C@H](CSC3)NC(=O)[C@@H]2CCCN2C(=O)[C@@H]2CCCN2C1=O
InChIKey: AREDASPITDNTEJ-CJUNNKGMSA-N
Target: Sortase A (SINGLE PROTEIN), Staphylococcus aureus
UniProt: Q9S446
Assay: Inhibition of Staphylococcus aureus His6-biotinylated SrtA expressed in Escherichia coli XL1-blue using Dabcyl-LPETG-Edans as substrate assessed as residual activity by fluorescence assay
Measurement: Ki = 4300 nM (pChEMBL 5.369999885559082)
Descriptors: MW 1443.79
Compound (SMILES): CC(=O)N1CCC(C(=O)N2CC[C@](c3ccc(C(OCc4c(C)cccc4C)(C(F)(F)F)C(F)(F)F)cc3)(S(=O)(=O)c3ccc(F)cc3)C2)CC1
InChIKey: HTTORLKYJHKSFA-XIFFEERXSA-N
Target: Nuclear receptor ROR-gamma (SINGLE PROTEIN), Homo sapiens
UniProt: P51449
Assay: Scintillation Proximity Assay (SPA): The binding of potential ligands to RORγ is measured by competition with [3H] 25-hydroxycholesterol (Perkin Elmer NET674250UC) using a scintillation proximity assay (SPA) binding assay. The ligand binding domain of human RORγ (A262-S507) with an N-terminal His tag is expressed in E. coli and purified using nickel affinity chromatography. 15 ug/well RORγ (A262-S507) is incubated with test compound at varying concentrations in 3-fold serial dilution, with final concentrations ranging from 16.6 uM to 0.28 nM for 10 min at room temperature in PBS buffer (Invitrogen #14190-144) containing 0.5% fatty acid free BSA (Gemini Bio-Products, Cat. #700-107P) and 0.1% Glycerol (Sigma Cat#G5516). 10 nM of [3H] 25-hydroxycholesterol is then added, and the reaction is incubated for 10 min. 10 mg/mL of Copper-His Tag-PVT beads (Perkin Elmer cat #RPNQ0095) are added, and the mixture is incubated for 60 min. The reaction is read on a TopCount Microplate scintillation plate reader (Perkin Elmer). The competition data of the test compound over a range of concentrations was plotted as percentage inhibition of radioligand specifically bound in the absence of test compound (percent of total signal). After correcting for non-specific binding, IC50 values were determined. The IC50 value is defined as the concentration of test compound needed to reduce [3H] 25-hydroxycholesterol specific binding by 50% and is calculated using the four parameter logistic equation to fit the normalized data.
Measurement: IC50 = 188 nM (pChEMBL 6.730000019073486)
Descriptors: MW 742.75, ALogP 7.14, HBA 5, HBD 0, PSA 83.99, QED 0.18
Compound (SMILES): N#Cc1ccc(CCc2cc(O)c(=O)[nH]n2)cc1
InChIKey: ODAJAZMNOCKMGG-UHFFFAOYSA-N
Target: D-amino-acid oxidase (SINGLE PROTEIN), Homo sapiens
UniProt: P14920
Assay: In Vitro DAAO Enzyme Assay: Human DAAO enzyme was supplied by the Takeda Pharmaceutical Company (Osaka) and each batch was tested and used at concentrations giving comparable levels of activity. The Km of D-Serine was measured for each enzyme batch to maintain consistency; this Km was used in subsequent assays.On the day of the assay compounds were serially diluted in DMSO before being diluted 1:20 with assay buffer (20 mM Tris ph 7.4). A 5 ul portion of assay buffer was added to the wells of a 384 clear base black-walled plate (Corning), 5 ul of diluted compound was then added via automated plate to plate transfer using the Bravo liquid handler (Agilent technologies) followed by 5 ul of human DAAO enzyme and then 5 ul D-Serine 50 mM was added to all but the negative control wells (final concentration of 10 mM). Finally 5 ul Amplex red reagent (Invitrogen) was added to all wells as per manufacturer's protocol. The plate was incubated for 60 minutes in the dark at 25° C., and the fluorescence in each well was measured in the Envision plate reader.
Measurement: IC50 = 13 nM (pChEMBL 7.889999866485596)
Descriptors: MW 241.25, ALogP 1.13, HBA 4, HBD 2, PSA 89.77, QED 0.84
Compound (SMILES): O=S(=O)(NCC1CCOCC1)c1ccc(Nc2ccc(Cl)cc2Cl)cc1C(F)(F)F
InChIKey: AVNHLPCVJAOWMG-UHFFFAOYSA-N
Target: Cannabinoid receptor 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P21554
Assay: Binding Assay: The binding assays were performed by testing the ability of the new compounds to displace the radiolabeled synthetic non-selective cannabinoid agonist [3H]CP55940 (168 Ci/mmol; PerkinElmer) from the human CB1 (hCB1) or human CB2 (hCB2) receptors on membranes derived from stably transfected HEK-293 cells (PerkinElmer). Membranes were diluted in assay buffer (50 mM Tris-HCl, 1 mM EDTA, 1 mM MgCl2, 1 mg/ml BSA, pH=7.4). The amount of membrane was determined for each batch of membranes according to protein binding assay. The minimum amount of membrane that gave 50% specific binding was used for the binding assay. In most assays, binding was tested using 8 μg and 4 μg protein of hCB1 and hCB2 membranes, respectively. The tested compounds were dissolved in DMSO and diluted in assay buffer to a final concentration of 0.1% solvent. Total binding of [3H]CP55940 was evaluated with 1.5 nM to hCB1 and with 0.5 nM to hCB2, according to Kd affinity of [3H]CP55940 for the respective membranes. The ability of the tested compounds to displace [3H]CP55940 was evaluated first at a single concentration point of 100 nM for binding toward hCB2 or hCB1. In certain cases, the displacement was tested at compound concentrations ranging from 0.03 nM to 10 μM. Non-specific binding was measured by the addition of 1 μM of unlabelled CP55940 to the tubes. Binding assays were performed in triplicate in a total volume of 500 μl for 60 minutes at 30° C., in a shaking bath. Free and bound radioligands were separated by rapid filtration through GF/C filter plates (PerkinElmer) that had been presoaked with 0.1% Polyethylenimine (Sigma). Filters were shaken for 1 hour in 7 ml scintillation fluid (PerkinElmer) and radioactivity was determined by liquid scintillation counter (Wallac; PerkinElmer).
Measurement: Ki = 5841 nM (pChEMBL 5.230000019073486)
Descriptors: MW 483.34, ALogP 5.46, HBA 4, HBD 2, PSA 67.43, QED 0.57
Compound (SMILES): N[C@H]1CCCN(c2ccncc2Nc2nnc3ccccn23)C1
InChIKey: XQXCEKIWYGDQJP-LBPRGKRZSA-N
Target: Serine/threonine-protein kinase pim-1 (SINGLE PROTEIN), Homo sapiens
UniProt: P11309
Assay: PIM Enzyme Assay: 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 uM. A reference compound was included on each assay plate in order to validate that plate.
Measurement: IC50 = 38.3 nM (pChEMBL 7.420000076293945)
Descriptors: MW 309.38, ALogP 1.8, HBA 7, HBD 2, PSA 84.37, QED 0.77
Compound (SMILES): CN(C)CCCCC(=O)Nc1ccc(NC(=S)NC(=O)c2cc(F)c(F)c(F)c2)cc1
InChIKey: SCYXENJCDZJASM-UHFFFAOYSA-N
Target: NAD-dependent protein deacetylase sirtuin-1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q96EB6
Assay: Inhibition Assay: All compounds were tested for inhibition of SIRT1 and SIRT2 using human recombinant enzymes available in the Fluor de Lys fluorescence-based assay kit (BIOMOL, AK555, AK556). All other required reagents were provided in the kit which was stored at −78° C. Before use, small aliquots of each enzyme (2-3 μL) were prepared, snap frozen in liquid nitrogen and stored at −78° C. Fresh dilutions of compounds were prepared in DMSO and further diluted in assay buffer. NAD+ (12.5 ΞL at 4 mM) and Fluor de Lys SIRT1 or SIRT2 (12.5 μL at 100 μM) in assay buffer were added to a white 96 well plate, followed by compound (10 μL) and lastly enzyme (15 μL, 0.07 U/μL or SIRT1 and 0.3 U/μL for SIRT2). After incubation for 1 h at 37° C. a developer solution (50 μL) was added to each reaction. The developer solution contained 38 μL buffer, 10 μL developer and 2 μL nicotinamide (50 mM) per reaction. The plate was then incubated for 45 min at rt and then read using a Spectra Max Gemini fluorimeter with an excitation wavelength of 355 nm and an emission wavelength of 460 nm.
Measurement: IC50 > 90000 nM (pChEMBL 4.046000003814697)
Descriptors: MW 452.5, ALogP 3.9, HBA 4, HBD 3, PSA 73.47, QED 0.32
Compound (SMILES): CC(C)Nc1cc(-n2ccc3cncnc32)ncc1C(=O)NCCC(C)(C)O
InChIKey: QXCJSXBXJGFACL-UHFFFAOYSA-N
Target: Interleukin-1 receptor-associated kinase 4 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9NWZ3
Assay: Inhibition Assay: The assays were performed in U-bottom 384-well plates. The final assay volume was 30 L prepared from 15 L additions of enzyme and substrates (fluoresceinated peptide and ATP) and test compounds in assay buffer (20 mM HEPES pH 7.2, 10 mM MgCl2, 0.015% Brij 35 and 4 mM DTT). The reaction was initiated by the combination of IRAK4 with substrates and test compounds. The reaction mixture was incubated at room temperature for 60 min. and terminated by adding 45 μL of 35 mM EDTA to each sample. The reaction mixture was analyzed on the Caliper LABCHIP® 3000 (Caliper, Hopkinton, Mass.) by electrophoretic separation of the fluorescent substrate and phosphorylated product. Inhibition data were calculated by comparison to no enzyme control reactions for 100% inhibition and vehicle-only reactions for 0% inhibition. The final concentrations of reagents in the assays are ATP, 500 μM; FL-IPTSPITTTYFFFKKK peptide 1.5 μM; IRAK4, 0.6 nM; and DMSO, 1.6%.
Measurement: IC50 = 4.2 nM (pChEMBL 8.380000114440918)
Descriptors: MW 382.47, ALogP 2.53, HBA 7, HBD 3, PSA 104.96, QED 0.58
Compound (SMILES): COc1ccc(C(=O)Nc2ccc(F)c([C@@]3(C(F)F)N=C(N)CCC3(F)F)c2)nc1
InChIKey: PEEPRUHZLOVBDY-IBGZPJMESA-N
Target: Beta-secretase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P56817
Assay: BACE1 Binding Assay: The binding assay was performed as SPA-based assay using a biotinylated form of human BACE1 recombinantly expressed and subsequently purified from Freestyle HEK293 cells. The binding assay was run in a 50 mM sodium acetate buffer, pH 4.5 containing 50 mM NaCl and 0.03% Tween-20 in white clear bottom 384 plates (Corning #3653). 10 nM (final concentration) radioligand ([3H]−N-((1S,2R)-1-benzyl-3-cyclopropylamino-2-hydroxy-propyl)-5-(methanesulfonyl-methyl-amino)-N−((R)-1-phenyl-ethyl)-isophthalamide) (TRQ11569 purchased from GE Healthcare) was mixed with test compound at a given concentration, 6 nM (final concentration) human BACE1 and 25 μg Streptavidin coated PVT core SPA beads (RPNQ0007, GE Healthcare Life Sciences) in a total volume of 40 μl. Several concentrations of each test compound were tested in the assay for IC50 determination. The plates were incubated for one hour at room temperature and counted in a Wallac Trilux counter. Total and non-specific binding were determined using buffer and 1 μM (final concentration) of the high affinity BACE1 reference inhibitor (S)-6-[3-chloro-5-(5-prop-1-ynyl-pyridin-3-yl)-thiophen-2-yl]-2-imino-3,6-dimethyl-tetrahydro-pyrimidin-4-one, respectively.
Measurement: Ki = 6.7 nM (pChEMBL 8.170000076293945)
Descriptors: MW 428.36, ALogP 3.73, HBA 5, HBD 2, PSA 89.6, QED 0.71
Compound (SMILES): O=C(/C=C/c1ccc(O)c(O)c1)NC(Cc1c[nH]c2ccccc12)C(=O)O
InChIKey: XITPERBRJNUFSB-SOFGYWHQSA-N
Target: NAD-dependent protein deacetylase sirtuin-1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q96EB6
Assay: Inhibition of recombinant human SIRT1 assessed as deacetylation activity using fluorescent substrate in presence of NAD+ by fluorescence analysis
Measurement: IC50 = 34000 nM (pChEMBL 4.46999979019165)
Descriptors: MW 366.37, ALogP 2.4, HBA 4, HBD 5, PSA 122.65, QED 0.34
Compound (SMILES): COc1cccc(OC)c1C1CCC(=O)N1Cc1ccc(Oc2ccccc2)cc1
InChIKey: QDIASNGSQGQKNP-UHFFFAOYSA-N
Target: Orexin/Hypocretin receptor type 1 (SINGLE PROTEIN), Homo sapiens
UniProt: O43613
Assay: In Vitro Assay: Intracellular Calcium Measurements : In Vitro Assay: Intracellular Calcium Measurements:Chinese hamster ovary (CHO) cells expressing the human orexin-1 receptor and the human orexin-2 receptor, respectively, are grown in culture medium (Ham F-12 with L-Glutamine) containing 300 g/ml G418, 100 U/ml penicillin, 100 g/ml streptomycin and 10% heat inactivated fetal calf serum (FCS). The cells are seeded at 20,000 cells/well into 384-well black clear bottom sterile plates (Greiner). The seeded plates are incubated overnight at 37° C. in 5% CO2. Human orexin-A as an agonist is prepared as 1 mM stock solution in MeOH:water (1:1), diluted in HBSS containing 0.1% bovine serum albumin (BSA), NaHCO3: 0.375 g/l and 20 mM HEPES for use in the assay at a final concentration of 3 nM.Antagonists are prepared as 10 mM stock solution in DMSO, then diluted in 384-well plates using DMSO followed by a transfer of the dilutions into in HBSS containing 0.1% bovine serum albumin (BSA), NaHCO3: 0.375 g/l and 20 mM HEPES. On the day of the assay, 50 μl of staining buffer (HBSS containing 1% FCS, 20 mM HEPES, NaHCO3: 0.375 g/l, 5 mM probenecid (Sigma) and 3 M of the fluorescent calcium indicator fluo-4 AM (1 mM stock solution in DMSO, containing 10% pluronic) is added to each well. The 384-well cell-plates are incubated for 50 min at 37° C. in 5% CO2 followed by equilibration at RT for 30 min before measurement.
Measurement: IC50 = 233 nM (pChEMBL 6.630000114440918)
Descriptors: MW 403.48, ALogP 5.36, HBA 4, HBD 0, PSA 48, QED 0.53
Compound (SMILES): O=C(CBr)OCCn1c(=O)oc2cc3ncnc(Nc4ccc(N5CCCCC5)c(Cl)c4)c3cc21
InChIKey: KRZUYDHNTSGCKX-UHFFFAOYSA-N
Target: Receptor tyrosine-protein kinase erbB-2 (SINGLE PROTEIN), Homo sapiens
UniProt: P04626
Assay: Inhibition of His-tagged human HER2 (676 to 1255 residues) expressed in baculovirus after 1 hr by Z-lyte assay
Measurement: IC50 > 10000 nM (pChEMBL 5)
Descriptors: MW 560.84, ALogP 4.86, HBA 9, HBD 1, PSA 102.49, QED 0.25
Compound (SMILES): N#Cc1ccc2cccc(Oc3nc(NC4CC4)n4ncc(/C=C5\NC(=O)NC5=O)c4n3)c2n1
InChIKey: WXODCENIESBQPD-NVNXTCNLSA-N
Target: Casein kinase II subunit beta (SINGLE PROTEIN), Homo sapiens
UniProt: P67870
Assay: Inhibition Assay: Test compounds dissolved and diluted in DMSO (2 μl) were added to a reaction mixture comprising 10 μl of 5× Reaction Buffer (40 mM MOPS pH 7.0, 5 mM EDTA), 10 μl of recombinant human PIM2 solution (4 ng PIM-2 dissolved in dilution buffer (20 mM MOPS pH 7.0; EDTA 1 mM; 5% Glycerol; 0.01% Brij 35; 0.1%; 0.1% 2-mercaptoethanol; 1 mg/ml BSA)) and 8 ul of water. Reactions were initiated by the addition of 10 ul of ATP Solution (49% (15 mM MgCl2; 75 uM ATP) 1% ([γ-33P]ATP: Stock 1 mCi/100 μl; 3000 Ci/mmol (Perkin Elmer)) and 10 ul of substrate peptide solution (RSRSSYPAGT (SEQ ID NO.: 6), dissolved in water at a concentration of 1 mM), Reactions were maintained for 10 min at 30° C. The reactions were quenched with 100 ul of 0.75% Phosphoric acid, then transferred to and filtered through a Phosphocellulose filter plate (Millipore, MSPH-N6B-50). After washing each well 4 times with 0.75% Phosphoric acid, scintillation fluid (20 uL) was added to each well and the resid
Measurement: IC50 > 1000 nM (pChEMBL 6)
Descriptors: MW 453.42, ALogP 2.09, HBA 10, HBD 3, PSA 159.22, QED 0.3
Compound (SMILES): COc1ccc(F)c(OC)c1[C@@H]1C[C@H](F)C(=O)N1Cc1ccc(OC(F)(F)F)cc1
InChIKey: QOSKJOLEGJZPDC-GJZGRUSLSA-N
Target: Orexin/Hypocretin receptor type 1 (SINGLE PROTEIN), Homo sapiens
UniProt: O43613
Assay: In Vitro Assay: Intracellular Calcium Measurements : In Vitro Assay: Intracellular Calcium Measurements:Chinese hamster ovary (CHO) cells expressing the human orexin-1 receptor and the human orexin-2 receptor, respectively, are grown in culture medium (Ham F-12 with L-Glutamine) containing 300 g/ml G418, 100 U/ml penicillin, 100 g/ml streptomycin and 10% heat inactivated fetal calf serum (FCS). The cells are seeded at 20,000 cells/well into 384-well black clear bottom sterile plates (Greiner). The seeded plates are incubated overnight at 37° C. in 5% CO2. Human orexin-A as an agonist is prepared as 1 mM stock solution in MeOH:water (1:1), diluted in HBSS containing 0.1% bovine serum albumin (BSA), NaHCO3: 0.375 g/l and 20 mM HEPES for use in the assay at a final concentration of 3 nM.Antagonists are prepared as 10 mM stock solution in DMSO, then diluted in 384-well plates using DMSO followed by a transfer of the dilutions into in HBSS containing 0.1% bovine serum albumin (BSA), NaHCO3: 0.375 g/l and 20 mM HEPES. On the day of the assay, 50 μl of staining buffer (HBSS containing 1% FCS, 20 mM HEPES, NaHCO3: 0.375 g/l, 5 mM probenecid (Sigma) and 3 M of the fluorescent calcium indicator fluo-4 AM (1 mM stock solution in DMSO, containing 10% pluronic) is added to each well. The 384-well cell-plates are incubated for 50 min at 37° C. in 5% CO2 followed by equilibration at RT for 30 min before measurement.
Measurement: IC50 = 1018 nM (pChEMBL 5.989999771118164)
Descriptors: MW 431.36, ALogP 4.55, HBA 4, HBD 0, PSA 48, QED 0.63
Compound (SMILES): CC1(C)CN(c2ncnc3[nH]cc(-c4cccc(C#N)c4)c23)CCO1
InChIKey: RFDOWEJYIPXXRM-UHFFFAOYSA-N
Target: Leucine-rich repeat serine/threonine-protein kinase 2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q5S007
Assay: Lantha Screen Technology Assay: LRRK2 kinase activity was measured using Lantha Screen technology from Invitrogen. GST-tagged truncated LRRK2 from Invitrogen (Cat # PV4874) was incubated with a fluorescein-labeled peptide substrate based upon ezrin/radixin/moesin (ERM), also known as LRRKtide (Invitrogen cat #PR8976A), in the presence of a dose response of compound. Upon completion, the assay was stopped and detected with a terbium labeled anti-phospho-ERM antibody (Invitrogen, cat #PR8975A). The assay was carried out under the following protocol: 3 uL of a working solution of substrate (233 nM LRRKtide, 117 uM ATP) prepared in assay buffer (50 mM HEEPES, pH 7.5, 3 mM MgCl2, with 2 mM DTT and 0.01% Brij35 added fresh) was added to a low volume Greiner 384-well plate. The compound dose response was prepared by diluting compound to a top concentration of 3.16 mM in 100% DMSO and serial diluted by half-log in DMSO 11 times. Aliquots (3.5 uL) of the 100% DMSO dose response were mixed with 46.5 uL water.
Measurement: IC50 = 35.2 nM (pChEMBL 7.449999809265137)
Descriptors: MW 333.4, ALogP 3.11, HBA 5, HBD 1, PSA 77.83, QED 0.78
Compound (SMILES): CC(=O)N(C)C1(c2ccccc2)CCC(c2ccccc2)(N(C)C)CC1
InChIKey: IAPPMVYMEDGDEU-UHFFFAOYSA-N
Target: Nociceptin receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P41146
Assay: Binding Assay: The compounds were examined with membranes of recombinant CHO-ORL 1 cells in a receptor binding assay with 3H-nociceptin/orphanin FQ. This test system was conducted in accordance with the method outlined by Ardati et al. (Mol. Pharmacol., 51, 1997, pp. 816-824). The concentration of 3H-nociceptin/orphanin FQ amounted to 0.5 nM in these tests. The binding assays were conducted in each case on 20 ug of membrane protein per 200ul of preparation in 50 mM of HEPES, pH 7.4, 10 nM of MgCl2 and 1 mM of EDTA. The binding to the ORL 1-receptor was determined using 1 mg of WGA-SPA beads (Amersham-Pharmacia, Freiburg) in each case by incubating the preparation for one hour at RT and then conducting measurements in the Trilux scintillation counter (Wallac, Finland).
Measurement: Ki = 190 nM (pChEMBL 6.71999979019165)
Descriptors: MW 350.51, ALogP 4.39, HBA 2, HBD 0, PSA 23.55, QED 0.82
Compound (SMILES): Cc1cc(N)nc(C)c1CNC(=O)c1cnn(Cc2ccc(Cn3ccccc3=O)cc2)c1
InChIKey: LAHWWHTYCCSIGH-UHFFFAOYSA-N
Target: Plasma kallikrein (SINGLE PROTEIN), Homo sapiens
UniProt: P03952
Assay: In-vitro Plasma Kallikrein Inhibition: Enzymatic reactions were conducted in â¿¿assay bufferâ¿¿, comprising 50 mM Hepes/NaOH at pH 7.8, 150 mM NaCl, 1 mM EDTA and 0.05% (w/v) CHAPS. For the determination of IC50 values, the assays were performed at room temperature in 384-well plates with a total assay volume of 25.25 ul per well. The test compound was dissolved in 90% (v/v) DMSO/water. For the assays, 250 nL of the 90% (v/v) DMSO/water solution or compound solution were added per well, followed by the addition of 12.5 ul protease solution (protease in assay buffer). The final assay concentration of the human plasma kallikrein was nominally 25 pM, the 11 compound concentrations in the dilution series were in the range form 1 nM to 100 uM. After 1 hour of pre-incubation at room temperature, the reactions were started by the addition of 12.5 ul substrate solution (in assay buffer, final assay concentration was 0.5 uM). After the addition of the substrate solution, the final DMSO concentration in the assay was 0.9% (v/v).
Measurement: IC50 = 0.0224 nM (pChEMBL 10.649999618530273)
Descriptors: MW 442.52, ALogP 2.67, HBA 7, HBD 2, PSA 107.83, QED 0.46
Compound (SMILES): CC(C)(C)C(=O)c1c[nH]c2ncc(Nc3ccc(/C=C4/SC(=O)NC4=O)cc3)nc12
InChIKey: ZEKMHJMIMRPSOF-RIYZIHGNSA-N
Target: Tyrosine-protein kinase JAK3 (SINGLE PROTEIN), Homo sapiens
UniProt: P52333
Assay: Phosphorylation Assay: Phosphorylation of the appropriate peptide substrate (fluorescently labeled Srctide) by recombinant JAK3 (catalytic domain, aa 781-1124) was measured in the presence and absence of different concentrations of test compounds. Test compound, enzyme, fluorescently labeled substrate, and cofactors (ATP and Mg2+) were combined in a well of a microtiter plate and incubated for 2 hours at 25° C. The composition of the reaction buffer was 100 mM HEPES pH 7.5, 5 mM MgCl2, 0.01% Triton-X 100, 0.1% Bovine Serum Albumin, and 1% DMSO. Enzyme concentration in the final reaction mixture was 0.5 nM while peptide substrate was at 1 uM. ATP was used at 1xKm concentration corresponding to 2 uM. Serial dilutions (3-fold steps) of compounds were prepared in DMSO. At the end of the incubation, the reaction was stopped by the addition of 20 mM EDTA. Substrate and product (i.e. phosphorylated substrate) were separated electrophoretically and both were quantified by fluorescence intensity.
Measurement: IC50 = 580 nM (pChEMBL 6.239999771118164)
Descriptors: MW 421.48, ALogP 4.25, HBA 7, HBD 3, PSA 116.84, QED 0.43
Compound (SMILES): COC(=O)N[C@H](C(=O)N1CCC[C@H]1C1=Nc2cc(-c3cnc(-c4ccc(-c5cnc([C@@H]6CCCN6C(=O)[C@@H](NC(=O)OC)C(C)C)[nH]5)cc4)c4c3CC3(CCCC3)C4)ccc2S(=O)(=O)N1)C(C)C
InChIKey: FNONDSVNKZWWOV-ZSMOYPKZSA-N
Target: Nonstructural protein 5A (SINGLE PROTEIN), Hepatitis C virus
UniProt: Q5L478
Assay: Luciferase Assay: The Huh-7 cells transfected with HCV replicons system were seeded into 96-well plates (8,000 cells in 125 μL/well) respectively; each test compound was diluted to desired concentration using 5-fold serial dilutions protocol, 10 doses in duplicate, and added to wells with POD™ 810 Plate Assembler. The plates were incubated in a CO2 incubator for 72 hours; after that, 40 μL of Luciferase assay substrate (Promega Bright-Glo) was added to each well, and detected by a chemiluminescence detection system (Topcount Microplate Scintillation and Luminescence Counter) 5 minutes later; the EC50 (half-maximal effective concentration, concentration for 50% of maximal effect) values of test compounds were analyzed by GraphPad Prism software, respectively. In this paper, experiments were repeated twice and set the holes without compounds as negative control.
Measurement: EC50 = 0.061 nM (pChEMBL 10.210000038146973)
Descriptors: MW 948.16, ALogP 7.2, HBA 11, HBD 4, PSA 217.38, QED 0.12
Compound (SMILES): O=S(=O)(c1ccccc1)N(Cc1ccc(F)c(C(F)(F)F)c1)c1sc2ncccc2c1Cl
InChIKey: DFNWXWZEMJIPCY-UHFFFAOYSA-N
Target: Transient receptor potential M8 protein (SINGLE PROTEIN), Canis lupus familiaris
UniProt: Q1A7N1
Assay: Functional Assay: The functional activity of compounds of the formula (I) was determined by measuring changes in intracellular calcium concentration using a Ca2+-sensitive fluorescent dye. The changes in fluorescent signal were monitored by a fluorescence plate reader, either a FLIPR (Molecular Devices) or FDSS (Hamamatsu). Increases in intracellular Ca2+ concentration were readily detected upon activation with icilin.At 24 hrs prior to assay, HEK293 cells stably expressing canine TRPM8 were seeded in culture medium in black wall, clear-base poly-D-lysine coated 384-well plates (BD Biosciences, NJ, USA) and grown overnight in 5% CO2 at 37° C. On assay day, growth media was removed and cells were loaded with Calcium 3 Dye (Molecular Devices) for 35 min at 37° C., under 5% CO2 and then for 25 min at room temperature and atmosphere. Subsequently, cells were tested for agonist-induced increases in intracellular Ca2+ levels using FLIPR or FDSS. Cells were challenged with a compound of the Formula (I) (at varying concentrations) and intracellular Ca2+ was measured for 5 min prior to the addition of icilin to all wells to achieve a final concentration that produces approximately an 80% maximal response. EC50 or IC50 values for compounds of the present invention were determined from eight-point dose-response studies.
Measurement: IC50 = 28 nM (pChEMBL 7.550000190734863)
Descriptors: MW 500.93, ALogP 6.5, HBA 4, HBD 0, PSA 50.27, QED 0.29
Compound (SMILES): CN1C(=N)N[C@](C)(c2sc(-c3nnc(-c4ccc(N5CCCCC5)cc4)o3)cc2Cl)CS1(=O)=O
InChIKey: AEYDNTDXIXLOBT-QFIPXVFZSA-N
Target: Beta-secretase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P56817
Assay: HTRF FRET Assay: Varying concentrations of inhibitors at 3× the final desired concentration in a volume of 10 ul are preincubated with purified human BACE1 catalytic domain (3 nM in 10 μl) for 30 minutes at 30° C. in reaction buffer containing 20 mM Na-Acetate pH 5.0, 10% glycerol, 0.1% Brij-35 and 7.5% DSMO. Reactions are initiated by addition of 10 μl of 600 nM QSY7-APPswe-Eu substrate (200 nM final) to give a final reaction volume of 30 μl in a 384 well Nunc HTRF plate. The reactions are incubated at 30° C. for 1.5 hours. The 620 nm fluorescence is then read on a Rubystar HTRF plate reader (BMG Labtechnologies) using a 50 millisecond delay followed by a 400 millisecond acquisition time window.
Measurement: Ki = 5362 nM (pChEMBL 5.269999980926514)
Descriptors: MW 521.07, ALogP 4.12, HBA 8, HBD 2, PSA 115.42, QED 0.53
Compound (SMILES): CCS(=O)(=O)N(Cc1ccc(F)c(F)c1)c1sc2ccccc2c1C
InChIKey: URVPJMQFXGBYSR-UHFFFAOYSA-N
Target: Transient receptor potential M8 protein (SINGLE PROTEIN), Canis lupus familiaris
UniProt: Q1A7N1
Assay: Functional Assay: The functional activity of compounds of the formula (I) was determined by measuring changes in intracellular calcium concentration using a Ca2+-sensitive fluorescent dye. The changes in fluorescent signal were monitored by a fluorescence plate reader, either a FLIPR (Molecular Devices) or FDSS (Hamamatsu). Increases in intracellular Ca2+ concentration were readily detected upon activation with icilin.At 24 hrs prior to assay, HEK293 cells stably expressing canine TRPM8 were seeded in culture medium in black wall, clear-base poly-D-lysine coated 384-well plates (BD Biosciences, NJ, USA) and grown overnight in 5% CO2 at 37° C. On assay day, growth media was removed and cells were loaded with Calcium 3 Dye (Molecular Devices) for 35 min at 37° C., under 5% CO2 and then for 25 min at room temperature and atmosphere. Subsequently, cells were tested for agonist-induced increases in intracellular Ca2+ levels using FLIPR or FDSS. Cells were challenged with a compound of the Formula (I) (at varying concentrations) and intracellular Ca2+ was measured for 5 min prior to the addition of icilin to all wells to achieve a final concentration that produces approximately an 80% maximal response. EC50 or IC50 values for compounds of the present invention were determined from eight-point dose-response studies.
Measurement: IC50 = 5.4 nM (pChEMBL 8.270000457763672)
Descriptors: MW 381.47, ALogP 4.84, HBA 3, HBD 0, PSA 37.38, QED 0.63
Compound (SMILES): CC(=O)N[C@@H](Cc1ccccc1)C(=O)N[C@@H](Cc1ccccc1)C(=O)N[C@@H](CO)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@H](C(=O)N[C@@H](Cc1ccccc1)C(=O)N[C@H](C(=O)N[C@@H](Cc1ccccc1)C(=O)NCC(=O)N[C@@H](Cc1ccc(O)cc1)C(=O)N[C@@H](Cc1ccccc1)C(=O)N[C@@H](CCC(=O)O)C(=O)N[C@@H](CCCNC(=N)N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCNC(=N)N)C(=O)N[C@@H](C)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC(C)C)C(N)=O)C(C)C)C(C)C
InChIKey: NMZLHGLGLMKOQW-YNGFWQFOSA-N
Target: Tissue factor pathway inhibitor (SINGLE PROTEIN), Homo sapiens
UniProt: P10646
Assay: Binding affinity to TFPI (unknown origin) incubated for 1.5 hrs using tracer peptide JBT0131 by screening ELISA
Measurement: IC50 > 5000 nM (pChEMBL 5.301000118255615)
Descriptors: MW 2538.05
Compound (SMILES): COC(=O)[C@@H](Cc1cc2ccnc(N)c2cc1Cl)N1CC[C@H](NS(=O)(=O)c2cnc(N3CCCC3)s2)C1=O
InChIKey: JACIRYISMLCOOZ-RBUKOAKNSA-N
Target: Coagulation factor IX (SINGLE PROTEIN), Homo sapiens
UniProt: P00740
Assay: Inhibition Assay: The compounds according to the invention are tested in a range of concentrations (4.9 nM to 5 µM final) and deposited in a proportion of 25 µl per well. After the deposition of 50 µl of Spectrozyme 229 substrate (American Diagnostica), at the final concentration of 625 µM, the reaction is triggered by the addition of 25 µl of FIXa (human factor IXa supplied by Enzyme Research Laboratories (ERL)), at the final concentration of 2.5 U/ml. Reading is carried out at 405 nm for 15 min 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 = 2080 nM (pChEMBL 5.679999828338623)
Descriptors: MW 579.1, ALogP 2.19, HBA 10, HBD 2, PSA 147.82, QED 0.38
Compound (SMILES): COc1ccc2c(c1)cc(C(=O)NCCCCCC(=O)Nc1ccccc1N)n2C
InChIKey: BFRHVORGADPJTP-UHFFFAOYSA-N
Target: Histone deacetylase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q13547
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 = 95 nM (pChEMBL 7.019999980926514)
Descriptors: MW 408.5, ALogP 3.7, HBA 5, HBD 3, PSA 98.38, QED 0.37
Compound (SMILES): CCOc1cc(CC(=O)O)ccc1-c1ccc(F)c2c1CN(C(=O)OCC1CCCC1)CC2
InChIKey: YNENPYQPBGZUSL-UHFFFAOYSA-N
Target: Prostaglandin D2 receptor 2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9Y5Y4
Assay: Radioligand Displacement Assay: Binding assay was performed in a final assay volume of 250 ul. First, 25 ul of test compound, previously diluted in Binding-Buffer (Binding-Buffer: 50 mM Tris-Base, 100 mM NaCl, 1 mM EDTA, 0.1% BSA (protease free), 0.01% NaN3, 10 mM MnCl2, pH 7.0) was placed into each well. After addition of 75 ul Binding-Buffer, 50 ul of the radioligand 3H-PGD2 (at 2.5 nM (220.000 dpm/well) from ANAWA ART0662) was added to each well. Binding assay was started by addition of 100 ul CRTH2 membrane fragments, reaching a final concentration of 20 ug/well. For non-specific binding, PGD2 was added to the reaction mixture to 10 mM final concentration. This assay mix was incubated for 90 minutes at room temperature and then filtered through a GF/C filter 96-well plate which was pre-soaked for 3 hours in 0.5% polyethyleneimine (PEI). The filter-wells were washed three times with ice cold Binding-Buffer. Then, 40 ul of Microscint-40 (Packard) was added to each well and the retained radioactivity quantified in a Topcount (Packard).
Measurement: IC50 = 5.9 nM (pChEMBL 8.229999542236328)
Descriptors: MW 455.53, ALogP 5.2, HBA 4, HBD 1, PSA 76.07, QED 0.62