lovingscience/chembl-v37-raw-activities huggingface dataset:
Compound (SMILES): C=CC(=O)Nc1ccccc1Nc1cc(N(C)C(=O)Nc2c(Cl)c(OC)cc(OC)c2Cl)ncn1
InChIKey: SNAPABPRVHZAGB-UHFFFAOYSA-N
Target: Fibroblast growth factor receptor 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P11362
Assay: Kinase HotSpotâ¿¢ Assay: Compounds were profiled for FGFR inhibition activity at Reaction Biology Corporation (Malvern, Pa.) with their Kinase HotSpot assay. See, Anastassiadis et al., 2011, Comprehensive assay of kinase catalytic activity reveals features of kinase inhibitor selectivity. Nat Biotechnol 29, 1039-1045.Recombinant FGFR1 (2.5 nM), FGFR2 (1 nM), FGFR3 (5 nM), or FGFR4 (12 nM) (Invitrogen) was prepared as a mixture with substrate KKKSPGEYVNIEFG (SEQ ID NO:1) (20 uM, FGFR1 substrate); and Poly [E,Y]4:1 (0.2 mg/ml, FGFR2,3,4 substrate)] in kinase reaction buffer (20 mM HEPES-HCl, pH 7.5, 10 mM MgCl2, 2 mM MnCl2, 1 mM EGTA, 0.02% Brij35, 0.1 mM Na3VO4, 0.02 mg/ml BSA, 2 mM DTT, and 1% DMSO). Compound was added to the enzyme/substrate mixture using acoustic technology (Labcyte Echo 550, Sunnyvale, Calif.) (see, Olechno et al., 2006, Improving IC50 results with acoustic droplet ejection. JALA 11, 240-246) and pre-incubated for 0, 15, or 60 minutes at room temperature. After compound pre-incubation, a mixture of ATP (Sigma-Aldrich) and 33P-γ-ATP (PerkinElmer) was added to a final concentration of 10 uM to initiate kinase reactions. Reactions were incubated for 120 minutes at room temperature and then spotted onto Whatman P81 ion exchange filter paper. Unbound phosphate was removed by extensively washing filters in 0.75% phosphoric acid. See, Anastassiadis et al., 2011, Comprehensive assay of kinase catalytic activity reveals features of kinase inhibitor selectivity. Nat Biotechnol 29, 1039-1045.
Measurement: IC50 > 10000 nM (pChEMBL 5)
Descriptors: MW 517.37, ALogP 5.34, HBA 7, HBD 3, PSA 117.71, QED 0.35
Compound (SMILES): N#CCC1(n2cc(-c3ncnc4[nH]ccc34)cn2)CN(C2CCN(Cc3cc(F)cc(F)c3)CC2)C1
InChIKey: SWQZXCPMESABJL-UHFFFAOYSA-N
Target: Tyrosine-protein kinase JAK2 (SINGLE PROTEIN), Homo sapiens
UniProt: O60674
Assay: In Vitro JAK Kinase Assay: Compounds herein were tested for inhibitory activity of JAK targets according to the following in vitro assay described in Park et al., Analytical Biochemistry 1999, 269, 94-104. The catalytic domains of human JAK1 (a.a. 837-1142), Jak2 (a.a. 828-1132) and Jak3 (a.a. 781-1124) with an N-terminal His tag were expressed using baculovirus in insect cells and purified. The catalytic activity of JAK1, JAK2 or JAK3 was assayed by measuring the phosphorylation of a biotinylated peptide. The phosphorylated peptide was detected by homogenous time resolved fluorescence (HTRF). IC50s of compounds were measured for each kinase in the 40 microL reactions that contain the enzyme, ATP and 500 nM peptide in 50 mM Tris (pH 7.8) buffer with 100 mM NaCl, 5 mM DTT, and 0.1 mg/mL (0.01%) BSA. For the 1 mM IC50 measurements, ATP concentration in the reactions was 1 mM. Reactions were carried out at room temperature for 1 hr and then stopped with 20 μL 45 mM EDTA, 300 nM SA-APC, 6 nM Eu-Py20 in assay buffer (Perkin Elmer, Boston, Mass.). Binding to the Europium labeled antibody took place for 40 minutes and HTRF signal was measured on a Fusion plate reader (Perkin Elmer, Boston, Mass.).
Measurement: IC50 = 12.5 nM (pChEMBL 7.900000095367432)
Descriptors: MW 488.55, ALogP 3.69, HBA 7, HBD 1, PSA 89.66, QED 0.45
Compound (SMILES): CCN(CC)c1ccc(N(C(=O)c2cc(-c3cc(Cl)ccc3C(=O)N3Cc4ccccc4C[C@H]3CN3CCOCC3)n3c2CCCC3)c2ccc(O)cc2)cc1
InChIKey: SKDKNEQHSQMYLA-LHEWISCISA-N
Target: Apoptosis regulator Bcl-2 (SINGLE PROTEIN), Homo sapiens
UniProt: P10415
Assay: Fluorescence Polarisation Assay: The fluorescence polarisation tests were carried out on microplates (384 wells). The Bcl-2 protein, at a final concentration of 2.50×10−8 M, is mixed with a fluorescent peptide (Fluorescein-REIGAQLRRMADDLNAQY), at a final concentration of 1.00×10−8 M in a buffer solution (Hepes 10 mM, NaCl 150 mM, Tween20 0.05%, pH 7.4), in the presence or in the absence of increasing concentrations of test compounds. After incubation for 2 hours, the fluorescence polarisation is measured.
Measurement: IC50 = 25 nM (pChEMBL 7.599999904632568)
Descriptors: MW 772.39, ALogP 8.57, HBA 7, HBD 1, PSA 81.49, QED 0.15
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)N(C)C)c2)C1
InChIKey: GSRIIQVWJDRVGA-NLXGDKCYSA-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 = 31.62 nM (pChEMBL 7.5)
Descriptors: MW 1728.65
Compound (SMILES): COc1ccc(OC(F)(F)F)cc1CN1C(=O)C(C)CC1c1c(OC)cccc1OC
InChIKey: DQPCWUWHHKIBQE-UHFFFAOYSA-N
Target: Orexin receptor type 2 (SINGLE PROTEIN), Homo sapiens
UniProt: O43614
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 = 449 nM (pChEMBL 6.349999904632568)
Descriptors: MW 439.43, ALogP 4.72, HBA 5, HBD 0, PSA 57.23, QED 0.62
Compound (SMILES): NC(=O)c1c(N)ncnc1N1CCC(c2nc(-c3ccc(F)c(Cl)c3)cn2CCN2CCC2)CC1
InChIKey: SKBGXGJACXJIHW-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 = 7.5 nM (pChEMBL 8.119999885559082)
Descriptors: MW 498.99, ALogP 2.9, HBA 8, HBD 2, PSA 119.19, QED 0.51
Compound (SMILES): COc1ccc(C(=O)Nc2cccc(CNc3ncnc4c(C(N)=O)cc(C(O)CO)cc34)c2)cc1
InChIKey: SVDPMUQDJQIHAL-UHFFFAOYSA-N
Target: Aurora kinase A (SINGLE PROTEIN), Homo sapiens
UniProt: O14965
Assay: Enzyme Assay: The Aurora assays described here are performed on two Caliper Life Sciences systems: the LC3000 and the Desktop Profiler. These provide data on enzyme activity via measurement of the relative amounts of phosphorylated or unphosphorylated fluorescently labelled substrate peptide at the end of an enzymatic reaction. These different states of peptide are resolved by applying a potential difference across the sample. The presence of the charged phosphate group on the product (as opposed to the substrate) causes a different peptide mobility between the two peptides. This is visualized by excitation of the fluorescent label on the substrate and product peptides and represented as peaks within the analysis software.
Measurement: IC50 = 1.2 nM (pChEMBL 8.920000076293945)
Descriptors: MW 487.52, ALogP 2.63, HBA 8, HBD 5, PSA 159.69, QED 0.24
Compound (SMILES): CCOc1ccc(CC2CC2C(=O)NO)cc1-c1nc2c(CC)nn(C)c2c(=O)[nH]1
InChIKey: HDBVIGOTKGSSOC-UHFFFAOYSA-N
Target: Histone deacetylase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q13547
Assay: Inhibition of full length recombinant human C-terminal His/Flag-tagged HDAC1 expressed in baculovirus infected Sf9 cells using fluorogenic HDAC substrate after 30 mins by fluorescence analysis
Measurement: IC50 = 2610 nM (pChEMBL 5.579999923706055)
Descriptors: MW 411.46, ALogP 1.97, HBA 7, HBD 3, PSA 122.13, QED 0.4
Compound (SMILES): COC(=O)Nc1nc2cc(C3(O)c4ccccc4C(=O)N3Cc3cccc(OC)c3)ccc2[nH]1
InChIKey: CSAJGJCTRLXUEU-UHFFFAOYSA-N
Target: RAF proto-oncogene serine/threonine-protein kinase (SINGLE PROTEIN), Homo sapiens
UniProt: P04049
Assay: Inhibition of c-Raf1 (unknown origin) pre-incubated for 30 mins by luciferase-luciferin-coupled chemiluminescence assay
Measurement: IC50 = 100 nM (pChEMBL 7)
Descriptors: MW 458.47, ALogP 3.6, HBA 6, HBD 3, PSA 116.78, QED 0.42
Compound (SMILES): CN(C)S(=O)(=O)c1cnc(OC2CCOC2)c(-c2cn(C)c(=O)c3[nH]ccc23)c1
InChIKey: BYVUXCOWLZMHNK-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 = 5.1 nM (pChEMBL 8.289999961853027)
Descriptors: MW 418.48, ALogP 1.35, HBA 7, HBD 1, PSA 106.52, QED 0.67
Compound (SMILES): CN1C(=N)N[C@](C)(c2sc(-c3nnc(-c4ccc(C5(C#N)COC5)cc4)o3)cc2Cl)CS1(=O)=O
InChIKey: DSOROAFTBZZSKC-FQEVSTJZSA-N
Target: Beta-secretase 2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9Y5Z0
Assay: Time-Resolved Endpoint Proteolysis Assay: Inhibitor compounds, prepared at 3× the desired final concentration in 1×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 1×BACE 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 μM for 4 μM for autoBACE-2) prepared in 1×BACE 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 μs delay on a RUBYstar HTRF plate reader (BMG Labtechnologies).
Measurement: Ki = 1070 nM (pChEMBL 5.96999979019165)
Descriptors: MW 519.01, ALogP 2.92, HBA 9, HBD 2, PSA 145.2, QED 0.54
Compound (SMILES): N#CC1CC12CCC(c1nnn3cnc4[nH]ccc4c13)CC2
InChIKey: NLCXLKGYQDXCMB-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 = 9 nM (pChEMBL 8.050000190734863)
Descriptors: MW 292.35, ALogP 2.79, HBA 5, HBD 1, PSA 82.66, QED 0.75
Compound (SMILES): COC1CCN(c2ccc(S(=O)(=O)NC(=O)c3ccc(N4CCN(CC5=C(c6ccc(Cl)cc6)CC(C)(C)CC5)CC4)cc3Oc3ccc4[nH]ccc4c3)cc2[N+](=O)[O-])CC1
InChIKey: CDVCPMHQQKVGNQ-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 > 660 nM (pChEMBL 6.179999828338623)
Descriptors: MW 867.47, ALogP 9.04, HBA 10, HBD 2, PSA 150.35, QED 0.09
Compound (SMILES): NC(=O)c1cccc([C@H]2C[C@H]3CC[C@@H](C2)N3CCN(CCCc2ccccc2)C(=O)CO)c1
InChIKey: RBEQOHMLGWWJOC-QEIWAGNOSA-N
Target: Delta-type opioid receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P41143
Assay: Displacement of [3H]-diprenorphine from human delta opioid receptor expressed in cell membranes
Measurement: Ki = 100 nM (pChEMBL 7)
Descriptors: MW 449.6, ALogP 2.95, HBA 4, HBD 2, PSA 86.87, QED 0.58
Compound (SMILES): O=C(c1ccccc1-n1nccn1)N1CCOC[C@H]1Cc1cccc(-c2ncccn2)c1
InChIKey: RHBCBAZYIYZIMR-HXUWFJFHSA-N
Target: Orexin receptor type 2 (SINGLE PROTEIN), Homo sapiens
UniProt: O43614
Assay: OX Inhibition Assay: Chinese hamster ovary (CHO) cells expressing the human orexin-1 receptor and the human orexin-2 receptor, respectively, are grown in culture medium (Ham F-12 with L-Glutamine) containing 300 ug/mL G418, 100 U/mL penicillin, 100 ug/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.
Measurement: IC50 = 1892 nM (pChEMBL 5.71999979019165)
Descriptors: MW 426.48, ALogP 2.81, HBA 7, HBD 0, PSA 86.03, QED 0.49
Compound (SMILES): CNC(=O)c1c(-c2ccc(F)cc2)oc2cc(C(=O)OC)c(-c3ccc4c(n3)-c3cc5c(F)cccc5n3CO4)cc12
InChIKey: SWPTUMKAJXHBIM-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 = 23 nM (pChEMBL 7.639999866485596)
Descriptors: MW 565.53, ALogP 6.56, HBA 7, HBD 1, PSA 95.59, QED 0.24
Compound (SMILES): C[C@H]1NC(=O)c2cc(-c3cccc4c(=O)[nH]c(N(C)C(C)(C)C)nc34)[nH]c21
InChIKey: XUPURJISFWXKAO-SNVBAGLBSA-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 384-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 22-point dosing curve having a high dose of 1 μM. A reference compound was included on each assay plate [Costar 3658] in order to validate that plate; on one plate of every assay run, two additional reference compounds were included. The Reaction Buffer consisted of 45 mM Hepes, pH 7.0, 15 mM NaCl, and 1 mM MgCl. The quench/detection buffer consisted of 50 mM Tris, 100 mM NaCl, 0.05% BSA, 0.1% Tween and 3 mM EDTA. Biotinylated BAD peptide (Biopeptide), 10 mM ATP (Sigma), Labeled p-BAD (S112) mAb (Cell Signalling and Perkin Elmer) [with 0.05% BSA and 2 mM DTT added] streptavidin:Allophycocyanin [Perkin Elmer]. Final concentrations either Pim-1 enzyme [5 pM], or Pim-2 enzyme [0.5 pM], DMSO [1%], BLC BAD (S112) [0.5 μM], ATP [1.5 μM], streptavidin:Allophycocyanin [0.002 mg/mL] and biotinylated-BAD (S112) mAb [100 pM]. Initial incubations were carried out at RT (22° C.) for 30 min for both Pim-1 and for Pim-2. Pim enzyme is added to compound in buffer, and plates are incubated of 30 min. Biotinylated BAD and ATP are added and plates are incubated for 1 h. A mixture of labeled p-BAD (S112) mAb and quench/detection buffer are added and incubated for 2 h. Fluorescence was measured by an HTRF® Envision microplate reader.
Measurement: IC50 = 0.4 nM (pChEMBL 9.399999618530273)
Descriptors: MW 365.44, ALogP 2.96, HBA 4, HBD 3, PSA 93.88, QED 0.65
Compound (SMILES): O=C(NCC1CCCCC1)c1cc2cnccc2[nH]1
InChIKey: WQAOIKREBALUNB-UHFFFAOYSA-N
Target: Nicotinamide phosphoribosyltransferase (SINGLE PROTEIN), Homo sapiens
UniProt: P43490
Assay: Inhibition of NAMPT in human A2780 cells assessed as decrease in cell viability after 72 hrs by SRB assay
Measurement: IC50 > 2000 nM (pChEMBL 5.698999881744385)
Descriptors: MW 257.34, ALogP 2.87, HBA 2, HBD 2, PSA 57.78, QED 0.89
Compound (SMILES): CCCCCCCCCCCc1ccc(C(=O)N2CCC[C@H]2/C(N)=N/O)cc1
InChIKey: DAKJVLCDXHVRPL-NRFANRHFSA-N
Target: Sphingosine kinase 2 (SINGLE PROTEIN), Mus musculus
UniProt: Q9JIA7
Assay: Kinase Assay: Sphingosine kinase activity was assessed as described previously (Kharel, Y., Lee, S., Snyder, A. H., Sheasley-O'Neill, S. L., Morris, M. A., Setiady, Y., Zhu, R., Zigler, M. A., Burcin, T. L., Ley, K., Tung, K. S. K., Engelhard, V. H., Macdonald, T. L. and Lynch, K. R. Sphingosine kinase 2 is required for modulation of lymphocyte traffic by FTY720. J Biological Chemistry 280: 36865-36872 (2005)). High levels of human sphingosine kinase type 1 (mSK1) and mouse sphingosine kinase type 2 (mSK2) were expressed in Sf9 insect cells by infection with cognate baculoviruses. Crude homogenates were incubated with γ-[32P]ATP and 10 micromolar D-erythro-sphingosine in the presence of 100 micromolar concentrations of test compounds for 20 minutes at 37 C. The product, radiolabeled S1P, was isolated by extraction into organic solvents after acidification and displaced by thin layer chromatography.
Measurement: Ki = 2000 nM (pChEMBL 5.699999809265137)
Descriptors: MW 387.57, ALogP 5.11, HBA 3, HBD 2, PSA 78.92, QED 0.17
Compound (SMILES): COc1cccc(OC)c1C1CCCC(=O)N1Cc1cccc(-c2cncs2)c1
InChIKey: ACRPTJULOPXGFI-UHFFFAOYSA-N
Target: Orexin receptor type 2 (SINGLE PROTEIN), Homo sapiens
UniProt: O43614
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 = 17 nM (pChEMBL 7.769999980926514)
Descriptors: MW 408.52, ALogP 5.08, HBA 5, HBD 0, PSA 51.66, QED 0.57
Compound (SMILES): CCNc1ncc(-c2ccc(Oc3ncc(NC(=O)Nc4cc(C(F)(F)F)cnc4-c4ccc(C)cc4)cn3)cc2)s1
InChIKey: UFEAYYJBORUWKT-UHFFFAOYSA-N
Target: High affinity nerve growth factor receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P04629
Assay: Inhibition Assay: TrkA kinase-inhibiting activity in cell systems was measured using CHO-K1 cells expressing human TrkA and NFAT-bla (CellSenser TrkA-NFAT-bla CHO-K1 cells, Invitrogen).On the day before the assay, CellSenser TrkA-NFAT-bla CHO-K1 cells were suspended in an assay medium (Opti-MEM1 Reduced Serum Medium (Invitrogen) containing 0.5% dialysed fetal bovine serum (Invitrogen), 0.1 mM nonessential amino acids (Invitrogen), 1 mM sodium pyruvate (Invitrogen) and antibiotics (100 U/mL penicillin and 100 ug/mL streptomycin (Invitrogen)) and plated at a density of 2.4x104 cells/40 uL/well in a 96-well clear bottom plate (Corning, Catalogue No.: 3882). In some wells were added only the assay medium at 40 uL/well (Cell-free). On the day of the assay, 10 mM of the present compound (DMSO solution) was distributed in a 96-well plate (Costar, Catalogue No.: 3363) and serially diluted with DMSO with the geometrical ratio of 3. The serial dilutions were diluted with the assay medium to 100-fold to prepare a solution of the present compound with a 10-times concentration (DMSO concentration: 1%).
Measurement: IC50 = 4 nM (pChEMBL 8.399999618530273)
Descriptors: MW 591.62, ALogP 7.86, HBA 8, HBD 3, PSA 113.95, QED 0.17
Compound (SMILES): CCc1c(C(=O)NC23CC4CC(CC(C4)C2)C3)cc(C)n1-c1ccccc1
InChIKey: OPJPPPHWOSHFCK-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 = 6.15 nM (pChEMBL 8.210000038146973)
Descriptors: MW 362.52, ALogP 5.05, HBA 2, HBD 1, PSA 34.03, QED 0.81
Compound (SMILES): COc1c(F)cccc1-c1noc([C@H]2CCN2C(=O)c2ccc(C)cc2-n2nccn2)n1
InChIKey: HJDKRWJGSBWZFK-QGZVFWFLSA-N
Target: Orexin receptor type 2 (SINGLE PROTEIN), Homo sapiens
UniProt: O43614
Assay: Intracellular Calcium Assay: Chinese hamster ovary (CHO) cells expressing the human orexin-1 receptor and the human orexin-2 receptor, respectively, are grown in culture medium (Ham F-12 with L-Glutamine) containing 300 μg/mL G418, 100 U/mL penicillin, 100 μg/mL streptomycin and 10% heat inactivated fetal calf serum (FCS). The cells are seeded at 20,000 cells/well into 384-well black clear bottom sterile plates (Greiner). The seeded plates are incubated overnight at 37° C. in 5% CO2. Human orexin-A as an agonist is prepared as 1 mM stock solution in MeOH:water (1:1), diluted in HBSS containing 0.1% bovine serum albumin (BSA), NaHCO3: 0.375 g/L and 20 mM HEPES for use in the assay at a final concentration of 3 nM. Antagonists are prepared as 10 mM stock solution in DMSO, then diluted in 384-well plates using DMSO followed by a transfer of the dilutions into in HBSS containing 0.1% bovine serum albumin (BSA), NaHCO3: 0.375 g/L and 20 mM HEPES. On the day of the assay, 50 μL of staining buffer (HBSS containing 1% FCS, 20 mM HEPES, NaHCO3: 0.375 g/L, 5 mM probenecid (Sigma) and 3 μM 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. Within the Fluorescent Imaging Plate Reader (FLIPR Tetra, Molecular Devices), antagonists are added to the plate in a volume of 10 μL/well, incubated for 120 min and finally 10 μL/well of agonist is added. Fluorescence is measured for each well at 1 second intervals, and the height of each fluorescence peak is compared to the height of the fluorescence peak induced by 3 nM orexin-A with vehicle in place of antagonist.
Measurement: IC50 = 33 nM (pChEMBL 7.480000019073486)
Descriptors: MW 434.43, ALogP 3.36, HBA 8, HBD 0, PSA 99.17, QED 0.47
Compound (SMILES): CN(CC1CCC(=O)O1)C1CCN(C(=O)Cn2cc(NC(=O)c3cnn4cccnc34)c(-c3cc(Cl)ccc3OC(F)F)n2)CC1
InChIKey: DUSGXGLCHHSZSB-UHFFFAOYSA-N
Target: Tyrosine-protein kinase JAK2 (SINGLE PROTEIN), Homo sapiens
UniProt: O60674
Assay: JAK Enzyme Assay: The activity of the isolated recombinant JAK1 and JAK2 kinase domain was measured by monitoring phosphorylation of a peptide derived from JAK3 (Val-Ala-Leu-Val-Asp-Gly-Tyr-Phe-Arg-Leu-Thr-Thr, fluorescently labeled on the N-terminus with 5-carboxyfluorescein) using the Caliper LabChip® technology (Caliper Life Sciences, Hopkinton, Mass.). To determine inhibition constants (Ki), compounds were diluted serially in DMSO and added to 50 μL kinase reactions containing purified enzyme (1.5 nM JAK1, or 0.2 nM JAK2), 100 mM HEPES buffer (pH 7.2), 0.015% Brij-35, 1.5 μM peptide substrate, ATP (25 μM), 10 mM MgCl2, 4 mM DTT at a final DMSO concentration of 2%. Reactions were incubated at 22° C. in 384-well polypropylene microtiter plates for 30 minutes and then stopped by addition of 25 μL of an EDTA containing solution (100 mM HEPES buffer (pH 7.2), 0.015% Brij-35, 150 mM EDTA), resulting in a final EDTA concentration of 50 mM. After termination of the kinase reaction, the proportion of phosphorylated product was determined as a fraction of total peptide substrate using the Caliper LabChip® 3000 according to the manufacturer's specifications.
Measurement: Ki = 1.9 nM (pChEMBL 8.720000267028809)
Descriptors: MW 657.08, ALogP 3.73, HBA 11, HBD 1, PSA 136.19, QED 0.25
Compound (SMILES): COc1cccc2c1ncc1c(=O)n(-c3ccc4c(c3)OCO4)c(=O)n(C3CCN(C(=O)OC(C)(C)C)CC3)c12
InChIKey: MYNFJASRUQCJTA-UHFFFAOYSA-N
Target: Lipopolysaccharide-induced tumor necrosis factor-alpha factor (SINGLE PROTEIN), Homo sapiens
UniProt: Q99732
Assay: TNF inhibition assay: Samples were tested for TNF as per the manufacturer's instructions (TNF ELISA; R&D Systems, Cat #DY210). The samples were serially diluted with PBS+1% BSA to the appropriate dilution condition for the cell prep, typically a 1:4 or 1:6 dilution.
Measurement: IC50 = 1 nM (pChEMBL 9)
Descriptors: MW 546.58, ALogP 4.01, HBA 10, HBD 0, PSA 114.12, QED 0.35
Compound (SMILES): On1nnc2cc(CCc3nn[nH]n3)ccc21
InChIKey: ILSPBLDTHMAASO-UHFFFAOYSA-N
Target: Catenin beta-1 (SINGLE PROTEIN), Homo sapiens
UniProt: P35222
Assay: Fluorescence Polarization (FP) Assay: Experiments were performed in 96-well Microfluor 2 black plates (Waltham, Mass.), and the samples were read by a Synergy 2 plate reader (Biotek, Winooski, Vt.). The fluorescence polarization (FP) was measured at room temperature with an excitation wavelength at 485 nm and an emission wavelength at 535 nm. The FP saturation experiment was performed in an assay buffer of 137 mM of NaCl, 2.7 mM of KCl, 10 mM of Na2HPO4, 2 mM of KH2PO4, 100 ug/mL of bovine gamma globulin, and 0.01% Triton-X 100. The final reaction volume is 100 uL. The competitive FP assays used 2.5 nM of Tcf4 fluorescence tracer, 10 nM of β-catenin, and different concentrations of the tested inhibitors in 100 uL of assay buffer. Each assay plate was covered black and gently mixed on an orbital shaker for 3 h to reach equilibrium before polarization values were read. For each inhibitor competition assay, the negative controls (equivalent to 0% inhibition) refer to 2.5 nM of Tcf4 fluorescence tracer and 10 nM of β-catenin, but no tested peptide inhibitor was presented. The positive controls (equivalent to 100% inhibition) refer to only 2.5 nM of Tcf4 fluorescence tracer in a final volume of 100 uL.
Measurement: Ki = 3140 nM (pChEMBL 5.5)
Descriptors: MW 231.22, ALogP -0.03, HBA 7, HBD 2, PSA 105.4, QED 0.61
Compound (SMILES): CCCCN(CCCC)C(=O)c1cc(C)n(-c2ccc(N[S+]([O-])c3ccc(CNC(=O)c4ccc(Cl)cc4)s3)cc2C(=O)N2Cc3ccccc3C[C@H]2CO)n1
InChIKey: RZPYLLUHUAWYPG-NAWFSSCASA-N
Target: Apoptosis regulator Bcl-2 (SINGLE PROTEIN), Homo sapiens
UniProt: P10415
Assay: Surface plasmon resonance (SPR): Methods for Determining IC50s.The present method includes utility of a Surface plasmon resonance (SPR)- based biosensor (Biacore GE Healthcare, Uppsala, Sweden) to characterize BCL-2 inhibitors.Biacore utilizes the phenomenon of surface plasmon resonance (SPR) to detect and measure binding interactions. In a typical Biacore experiment, one of the interacting molecules (ligand) is immobilized on a flexible dextran matrix while the interacting partner (analyte) is allowed to flow across that surface. A binding interaction results in an increase in mass on the sensor surface and a corresponding direct change in the refractive index of the medium in the vicinity of the sensor surface. Changes in refractive index or signal are recorded in resonance units (R.U.) Signal changes due to association and dissociation of complexes are monitored in a non-invasive manner, continuously and in real-time, the results of which are reported in the form of a sensorgram.
Measurement: IC50 = 2392.61 nM (pChEMBL 5.619999885559082)
Descriptors: MW 815.46, ALogP 7.56, HBA 9, HBD 3, PSA 142.86, QED 0.09
Compound (SMILES): Cc1nc(C2C[C@H]2c2nc3ccccc3n2C)cc(-n2nc(C)nc2[C@H](C)O)n1
InChIKey: KJNPQKXNYILKNS-AXWJTWPOSA-N
Target: Cytochrome P450 2C9 (SINGLE PROTEIN), Homo sapiens
UniProt: P11712
Assay: Inhibition Assay: Inhibition assay using CYP2C9
Measurement: IC50 > 50000 nM (pChEMBL 4.301000118255615)
Descriptors: MW 389.46, ALogP 2.89, HBA 8, HBD 1, PSA 94.54, QED 0.58
Compound (SMILES): CNC(=O)c1c(-c2ccc(F)cc2)oc2cc(N(C)S(C)(=O)=O)c(-c3ccc4oc(=O)n5c6cccc(F)c6cc5c4n3)cc12
InChIKey: KKKHKGARLYBVFL-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 = 39 nM (pChEMBL 7.409999847412109)
Descriptors: MW 628.61, ALogP 5.71, HBA 8, HBD 1, PSA 127.13, QED 0.26
Compound (SMILES): Cc1nccn1CC(=O)Nc1ccc(Nc2nccc(-c3cnc(N4CC[C@H](F)C4)c(C#N)c3)n2)cn1
InChIKey: RWGAMRFXAUSZLC-IBGZPJMESA-N
Target: Serine/threonine-protein kinase TBK1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9UHD2
Assay: Time-Resolved Fluorescence-based Lanthascreen Assay: Kinase inhibition assays (10 μL) were performed at 20° C. in 384-well plate format. Compound IC50 values were determined at the apparent Km for ATP (20 μM) based on a radiometric assay (Invitrogen) using 8 or 10 point curves in duplicate. The final reaction conditions contained 400 nM fluorescein-IkBα substrate (DRHDSGLDSMKDE), 20 μM ATP, 2 nM or 8 nM IKKε or TBK1 kinase respectively, and 3% DMSO in kinase assay buffer consisting of 50 mM HEPES (pH 7.5), 10 mM MgCl, 1 mM EGTA, 0.01% Brij-35.Compound dilutions were prepared from 10 mM DMSO stocks by dilution into DMSO. Compound dilution series were further diluted in kinase assay buffer to give a 12% DMSO stock, the final concentration in the assay being 3% DMSO.The kinase phosphorylation assay was initiated by the addition of the kinase and the reaction was allowed to proceed for 1 h or 2.5 h for IKKε and TBK-1 kinase respectively.
Measurement: IC50 < 30 nM (pChEMBL 7.5229997634887695)
Descriptors: MW 498.53, ALogP 3.24, HBA 10, HBD 2, PSA 137.54, QED 0.39
Compound (SMILES): O=C1COc2ccc(C3(O)c4ccccc4C(=O)N3Cc3cccc(F)c3)cc2N1
InChIKey: KDTAQSMAQLUPGB-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 404.4, ALogP 3.01, HBA 4, HBD 2, PSA 78.87, QED 0.7
Compound (SMILES): CC1(C)CC1NC[C@H]1CC[C@H](c2nnn3cnc4[nH]ccc4c23)CC1
InChIKey: YSNALLPGDSFESC-JOQLWKCKSA-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 = 39 nM (pChEMBL 7.409999847412109)
Descriptors: MW 338.46, ALogP 3.27, HBA 5, HBD 2, PSA 70.9, QED 0.77
Compound (SMILES): FC(F)(F)c1ccc(Nc2nc(NCCN3CCCCC3)nc3c2CCN(c2ncccc2C(F)(F)F)CC3)cc1
InChIKey: KAWLJCOVVJZFAO-UHFFFAOYSA-N
Target: Transient receptor potential cation channel subfamily V member 1 (SINGLE PROTEIN), Rattus norvegicus
UniProt: O35433
Assay: Functional Assay: The assay was performed as described below, using HEK293 cells transfected with rat TRPV1 (rTRPV1/HEK293). These cells had a geneticin selection marker and were grown in Dulbecco's Modified Eagle Medium (DMEM, Gibco BRL) supplemented with 10% fetal bovine serum, penicillin/streptomycin (50 units/mL), and 500 μg/mL geneticin in 5% CO2 at 37° C. Cells were passaged frequently, every 3-5 days, to avoid overgrowth, depletion of essential medium components, or acidic medium exposure. Cells were passaged using a brief wash in 0.05% trypsin with 1 mM EDTA, followed by dissociation in divalent-free phosphate-buffered saline (Hyclone #SH30028.02). Dissociated cells were seeded onto poly-D-lysine coated black-walled 96-well plates (Biocoat; Becton Dickinson #354640) at about 40,000 cells per well and grown for approximately 1 day in culture medium to near confluency. The assay buffer was composed of 130 mM NaCl, 2 mM KCl, 2 mM MgCl2, 10 mM HEPES, 5 mM glucose, and either 2 mM or 20 μM CaCl2. On the day of the experiment, the culture medium was replaced with 2 mM calcium assay buffer using an automated plate washer (ELx405; Biotek, VT). The cells were incubated in 100 μL/well Fluo-3/AM (2 μM; TEFLabs #0116) with Pluronic F127 (100 μg/mL; Sigma #P2443) for 1 h at rt in the dark. After loading the cells, the dye solution was replaced with 50 μL/well of 20 μM calcium assay buffer using the ELx405 plate washer. Test compounds (50 μL/well) were added to the plate and incubated for 30 min. Intracellular Ca2+ levels were subsequently assayed using a Fluorometric Imaging Plate Reader (FLIPR™ instrument, Molecular Devices, CA) to simultaneously monitor Fluo-3 fluorescence in all wells (λexcitation=488 nm, λemission=540 nm) during challenge with agonist (capsaicin). The IC50 values were determined. Cells were challenged with 150 nM capsaicin and the fluorescence counts were captured following agonist addition at a sampling rate of 0.33 Hz. The contents of the wells were mixed 3 times (40 μL mix volume) immediately after the additions were made. Concentration dependence of block was determined by exposing each well of cells in duplicate rows of a 96 well plate to a serial dilution of test compound. The concentration series usually started at 10 Î¼M with a three-fold serial decrement in concentration. The magnitude of the capsaicin response was determined by measuring the change in fluo3 fluorescence before and 100 seconds after the addition of the agonist.
Measurement: IC50 = 1500 nM (pChEMBL 5.820000171661377)
Descriptors: MW 579.59, ALogP 6.16, HBA 7, HBD 2, PSA 69.21, QED 0.33
Compound (SMILES): C=CC(=O)N1CCc2c([nH]c3c(C(N)=O)c(-c4ccc(Oc5ccccc5)cc4)nn23)C1
InChIKey: HCRQKHQHICVPMB-UHFFFAOYSA-N
Target: Tyrosine-protein kinase BTK (SINGLE PROTEIN), Homo sapiens
UniProt: Q06187
Assay: Inhibition of BTK in human Ramos cells assessed as reduction in BTK phosphorylation at Tyr223 residue preincubated for 1 hr followed by pervanadate or Na3VO4 stimulation for 20 mins by HTRF assay
Measurement: IC50 = 1.5 nM (pChEMBL 8.819999694824219)
Descriptors: MW 427.46, ALogP 3.29, HBA 5, HBD 2, PSA 105.72, QED 0.48
Compound (SMILES): NC(=O)c1c(N)ncnc1N1CCC(c2nc(-c3ccoc3)cn2CCN2CCC2)CC1
InChIKey: AQWOGQIBQPRDAX-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 = 3600 nM (pChEMBL 5.440000057220459)
Descriptors: MW 436.52, ALogP 1.7, HBA 9, HBD 2, PSA 132.33, QED 0.57
Compound (SMILES): CSc1ccc(C(O)(CN[C@H]2CC[C@H](c3nnn4cnc5[nH]ccc5c34)CC2)C(F)(F)F)cc1
InChIKey: UXHYCSPSXIPCCF-LOXQESOXSA-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 = 27 nM (pChEMBL 7.570000171661377)
Descriptors: MW 490.56, ALogP 4.39, HBA 7, HBD 3, PSA 91.13, QED 0.35
Compound (SMILES): Brc1cnc(NCCC(c2ccccc2)c2ccccc2)nc1Nc1cc(C2CC2)[nH]n1
InChIKey: VRVRRNNCXIOKST-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 >= 5000 nM (pChEMBL 5.301000118255615)
Descriptors: MW 489.42, ALogP 6.22, HBA 5, HBD 3, PSA 78.52, QED 0.26
Compound (SMILES): CN1CC(Oc2ccc(-c3cc4c(-c5cccc(N6CCOc7cc(C8CC8)ccc7C6=O)c5CO)ncnc4[nH]3)cc2)C1
InChIKey: PEASILRRNHAHPE-UHFFFAOYSA-N
Target: Tyrosine-protein kinase BTK (SINGLE PROTEIN), Homo sapiens
UniProt: Q06187
Assay: Recombinant Human BTK Inhibition Assay: The inhibition assays were performed using the generic tyrosine kinase assay kit from C is Bio (HTRF KinEASE-TK #62TK0PEB). The assay is based on the principle of fluorescence resonance energy transfer (FRET) between the Europium Cryptate labeled anti-phosphotyrosine antibody and streptavidin labeled fluorophore, XL-665. In an uninhibited kinase reaction the biotinylated peptide substrate (Tk-Biotin) gets phosphorylated at an intermediate tyrosine site which is captured by anti-phosphotyrosine antibody which eventually acts as a FRET donor. The streptavidin labeled XL-665 binds strongly to the Tk-Biotin substrate and act as a FRET acceptor. The resultant FRET signal is measured at 665 nm. A ratio of this specific fluorescence (665 nm) to the background fluorescence (620 nm), conferred by the free antibody, is directly proportional to the kinase activity. Inhibition of the kinase activity by a pharmacological intervention such as a Btk inhibitor of the present disclosure will result into decreased FRET signal.
Measurement: IC50 = 5 nM (pChEMBL 8.300000190734863)
Descriptors: MW 587.68, ALogP 5.39, HBA 7, HBD 2, PSA 103.81, QED 0.26
Compound (SMILES): O=C(Nc1ccncc1)c1cc(NC2CCCCC2)ncn1
InChIKey: ORDMRAVRIJZRRC-UHFFFAOYSA-N
Target: Sphingosine 1-phosphate receptor 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P21453
Assay: 35S-GTPgammaS Binding: Measurements of 35S-GTPgammaS Binding: Membranes (1 to 10 ug protein) prepared as described above, were incubated in 96-well Scintiplates (PerkinElmer) with test compounds diluted in DMSO, in 180 ul of 20 mM HEPES, pH 7.4, 10 mM MgCl2, 2 ug/well Saponin, 0.2% fatty acid free BSA (Assay buffer), 140 mM NaCl and 1.7 uM GDP. The assay was initiated with the addition of 20 ul of 1.5 nM [35S]-GTPgammaS (1100 Ci/mmol; GE Healthcare) in assay buffer. After 60 min incubation at 30° C. on a shaker, plates were centrifuged for 10 min at 2000 RPM. Supernatant was discarded and membrane bound radioactivity was measured on a PerkinElmer 1450 MicroBeta counter. Triplicate samples were averaged and expressed as % response relative to S1P activation in absence of compound (n=2).
Measurement: EC50 = 19500 nM (pChEMBL 4.710000038146973)
Descriptors: MW 297.36, ALogP 2.87, HBA 5, HBD 2, PSA 79.8, QED 0.91