lovingscience/chembl-v37-raw-activities huggingface dataset:
Compound (SMILES): COc1ccc(-n2c(-c3ccc(S(C)(=O)=O)cc3)cc(CC(=O)OCCCCO[N+](=O)[O-])c2C)cc1
InChIKey: UMPJRQFMZFJMCP-UHFFFAOYSA-N
Target: Prostaglandin G/H synthase 2 (SINGLE PROTEIN), Homo sapiens
UniProt: P35354
Assay: In Vitro Inhibition Assay: The murine monocyte/macrophage cell line J774 is grown in Dulbecco's modified Eagle's medium (DMEM), enriched with glutamine (2 mM), HEPES (25 mM), penicillin (100 ug/mL), streptomycin (100 ug/mL), 10% of fetal bovine serum (FBS) and 1.2% of sodium pyruvate. The cells are distributed in 24-well plates at a density of 2.5x105 cells/mL or in culture dishes with a diameter of 10 cm (1x107 cells/10 mL/dish) and kept for 2 hours at 37° C. in a CO2 (5%)/O2 (95%) atmosphere. Just before the experiments, the culture medium is replaced with fresh medium without FBS to avoid interference during the radio-immunological phase and the cells are stimulated as described below.Evaluation of COX-1 Activity.The cells are pretreated with the test compounds for 15 minutes and then incubated for 30 minutes with arachidonic acid (15x10-6 m). At the end of incubation the supernatants are collected for evaluating, by means of radio-immunological assays, the amount of PGE2 produced.
Measurement: IC50 = 550 nM (pChEMBL 6.260000228881836)
Descriptors: MW 516.57, ALogP 3.94, HBA 9, HBD 0, PSA 126.97, QED 0.15
Compound (SMILES): CCN1CC(C)(C)Oc2nc(N3CCOC[C@@H]3C)nc(-c3ccc(NC(=O)Nc4cnc(C)cn4)c(F)c3)c2C1=O
InChIKey: TVZVOFUSEIVRHM-KRWDZBQOSA-N
Target: Phosphatidylinositol 3-kinase regulatory subunit alpha (SINGLE PROTEIN), Homo sapiens
UniProt: P27986
Assay: Enzyme Assay: The test uses a lucifeurerin/luciferase system to measure the concentration of ATP and its consumption during the enzymatic reaction. The test is performed in 96-well format (Corning/Costar 96 black flat-bottomed half-wells plate, ref. 3694) in a total volume of 30 μl. To 1 μl of inhibitor in 100% DMSO are added (final concentrations) 50 μM of the substrate PIP2 ((L-α-phosphatidyl-D-myoinositol 4,5-bisphosphate, Echelon 117P-4516-0500), 2 μM of ATP and 1.7 μg/ml of PI3Kα, (p110α/p85α, Invitrogen PV4788) in a buffer of Tris/HCl 50 mM pH 7.5, EGTA 1 mM, MgCl2 10 mM, Chaps 0.03%, 1 mM DTT). After 90 minutes, the reaction is quenched by adding 20 μl/well of KinaseGlo reagent (Promega V6713). After 10 minutes in the dark, the luminescence is read on the PHERAStar microplate reader (reading at 0.8 sec/well).
Measurement: IC50 = 3520 nM (pChEMBL 5.449999809265137)
Descriptors: MW 564.62, ALogP 3.88, HBA 9, HBD 2, PSA 134.7, QED 0.47
Compound (SMILES): COc1nc2ccc(C(C)(O)c3oc(C)nc3C)cc2c(Cl)c1Cc1ccc(C(F)(F)F)cc1
InChIKey: GZHHSXYGQHWIGF-UHFFFAOYSA-N
Target: Nuclear receptor ROR-gamma (SINGLE PROTEIN), Homo sapiens
UniProt: P51449
Assay: ThermoFluor® Assay: ThermoFluor® experiments were carried out using instruments owned by Janssen Research and Discovery, L.L.C. through its acquisition of 3-Dimensional Pharmaceuticals, Inc. 1,8-ANS (Invitrogen) was used as a fluorescent dye. Protein and compound solutions are dispensed into black 384-well polypropylene PCR microplates (Abgene) and overlayed with silicone oil (1 μL, Fluka, type DC 200) to prevent evaporation. Bar-coded assay plates are robotically loaded onto a thermostatically controlled PCR-type thermal block and then heated at a typical ramp-rate of 1° C./min for all experiments. Fluorescence was measured by continuous illumination with UV light (Hamamatsu LC6) supplied via fiber optic and filtered through a band-pass filter (380-400 nm; >6 OD cutoff). Fluorescence emission of the entire 384-well plate was detected by measuring light intensity using a CCD camera (Sensys, Roper Scientific) filtered to detect 500±25 nm, resulting in simultaneous and independent readings of all 384 wells.Â  Reference wells contained RORγt without compounds, and the assay conditions were as follows: 0.065 mg/mL RORγt, 60 μM 1,8-ANS, 100 mM Hepes, pH 7.0, 10 mM NaCl, 2.5 mM GSH, 0.002% Tween-20.
Measurement: Kd = 140 nM (pChEMBL 6.849999904632568)
Descriptors: MW 490.91, ALogP 6.37, HBA 5, HBD 1, PSA 68.38, QED 0.35
Compound (SMILES): Cc1cncc(-c2nn3c(c2C2CC2)NC(=O)CC3)c1
InChIKey: FFQAARSNMOVRFM-UHFFFAOYSA-N
Target: Cytochrome P450 11B1, mitochondrial (SINGLE PROTEIN), Homo sapiens
UniProt: P15538
Assay: HTRF-Based Assay: Methods for V79-Human-CYP11B2 and V79-Human-CYP11B1 Assays: V79 cell lines stably expressing the either the human CYP11B2 or the human CYP11B1 enzyme were generated using a standard transfection protocol. V79 cells were transfected with plasmids pTriEx3-Hygro-hCyp11B2 or pTriEx3-Hygro-hCyp11B1 using Lipofectamine2000 reagent. V79 cells that stably express the human CYP11B2 or human CYP11B1 enzyme were selected for and maintained in DMEM supplemented with 10% FBS and 400 μg/mL hygromycin for 2 weeks. Single cell clones were generated by infinite dilution in DMEM supplemented with 10% FBS and 400 μg/mL hygromycin until single colonies were obtained. Clones V79-hCYP11B2-CLE9 and V79-hCYP11B1-8CLC7, were determined to produce the most aldosterone and cortisol, respectively, and were selected for inhibitor screening. For testing of inhibitors, cells were harvested at 80% confluency with 0.5% Trypsan-EDTA, washed once in PBS, and reconstituted in DMEM+0.1% BSA media at a cell concentration of 600,000 cells/mL for the CYP11B2 assay and 280,000 cells/mL for the CYP11B1 assay. 25 μL of cells were added to a 384 well tissue culture treated plate and mixed with 0.3 μL of inhibitor or DMSO (1% final DMSO concentration) for 1 hour at 37° C., 5% CO2. After pre-incubation with inhibitor, the reaction was initiated by adding 5 μL of substrate (final concentration of 125 nM 11-deoxycorticosterone for the CYP11B2 assay or 250 nM 11-deoxycortisol for the CYP11B1 assay). The reaction was carried out for 3 hours at 37° C., 5% CO2 and was stopped by harvesting the supernatants. The amount of product in the supernatant (aldosterone for CYP11B2 assay and cortisol for the CYP11B1 assay) was measured using HTRF-based assay kit (Aldosterone HTRF-CisBio#64ALDPEB, Cortisol HTRF-CisBio #63IDC002-CORT).
Measurement: IC50 = 71 nM (pChEMBL 7.150000095367432)
Descriptors: MW 268.32, ALogP 2.47, HBA 4, HBD 1, PSA 59.81, QED 0.91
Compound (SMILES): CN(C)Cc1cccc(Cn2cc(-c3ccc(N4CCc5cccc(C(=O)Nc6nc7ccccc7s6)c5C4)nc3C(=O)O)cn2)c1
InChIKey: BHCPDYBYBYGVKV-UHFFFAOYSA-N
Target: Bcl-2-like protein 1 (SINGLE PROTEIN), Mus musculus
UniProt: Q64373
Assay: FL5.12 Cellular Assay: The efficacy of the compounds of Formula (I) can also be determined in cell-based killing assays using a variety of cell lines and mouse tumor models. For example, their activity on cell viability can be assessed on a panel of cultured tumorigenic and non-tumorigenic cell lines, as well as primary mouse or human cell populations. In one exemplary set of conditions, mouse FL5.12 cells transfected with Bcl-XL were cultured under standard conditions in RPMI with 2 mM glutamine, 1% 100 mM sodium pyruvate, 2% 1 M HEPES, 4 uL/L of beta;-mercaptoethanol, 1% penicillin-streptomycin, 10% FBS, and 10% WEHI-3B conditioned media (for IL-3). For assaying the compound activity, the cells were exchanged into an IL-3-depleted deprivation media, which was identical to the growth media except for the absence of FBS and WEHI-3B conditional media, for 2 days. Then the cells were exchanged to 3% FBS assay media (RPMI with 2 mM glutamine, 1% 100 mM sodium pyruvate, 2% 1 M HEPES, 4 uL/L of beta;-mercaptoethanol, 1% penicillin-streptomycin, 10% FBS, and 10% WEHI-3B conditioned media (for IL-3).
Measurement: EC50 > 1000 nM (pChEMBL 6)
Descriptors: MW 643.77, ALogP 6.18, HBA 9, HBD 2, PSA 116.48, QED 0.19
Compound (SMILES): O=C(COc1ccc2cc(C(O)(C(F)(F)F)C(F)(F)F)c(O)cc2c1)NC1CC1
InChIKey: KRJZZXKNHIXOJI-UHFFFAOYSA-N
Target: Tyrosine-protein phosphatase non-receptor type 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P18031
Assay: Inhibition of recombinant PTP1B (unknown origin) using pNPP as substrate by spectrophotometric method
Measurement: IC50 > 20000 nM (pChEMBL 4.698999881744385)
Descriptors: MW 423.31, ALogP 3.52, HBA 4, HBD 3, PSA 78.79, QED 0.64
Compound (SMILES): CC1(C)C(=O)N(c2ccc(C#N)c(C(F)(F)F)c2)C(=S)N1c1ccc(C(=O)NCCCCCCc2ccc(C(=O)C(F)(F)F)s2)c(F)c1
InChIKey: GSSIWEALQCBBSM-UHFFFAOYSA-N
Target: Histone deacetylase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q13547
Assay: Inhibition of HDAC1 (unknown origin) assessed as inhibition of fluorogenic peptide deacetylation
Measurement: IC50 > 10000 nM (pChEMBL 5)
Descriptors: MW 712.71, ALogP 7.97, HBA 6, HBD 1, PSA 93.51, QED 0.09
Compound (SMILES): C[C@H](NC(=O)c1cnn2cccnc12)c1cc2cccc(Cl)c2c(=O)n1-c1ccccc1
InChIKey: HMXXEARIYBJYFT-HNNXBMFYSA-N
Target: Phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit gamma isoform (SINGLE PROTEIN), Homo sapiens
UniProt: P48736
Assay: Inhibition of human recombinant full length N-terminal His-tagged PI3Kgamma expressed in Sf9 insect cells using diC8PIP2 as substrate incubated for 15 mins followed by substrate addition measured after 2 hr by ADP-Glo luminescence assay
Measurement: IC50 = 400 nM (pChEMBL 6.400000095367432)
Descriptors: MW 443.89, ALogP 4.18, HBA 6, HBD 1, PSA 81.29, QED 0.45
Compound (SMILES): O=C(c1ccc(-c2cccs2)cc1)N1CCn2c(nnc2-c2csc(C(F)(F)F)n2)C1
InChIKey: UMVKCAIIJZHOQD-UHFFFAOYSA-N
Target: Neuromedin-K receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P29371
Assay: Binding Competition Assay: The ability of compounds of the invention to inhibit the binding of the NK-3 receptor selective antagonist 3H-SB222200 was assessed by an in vitro radioligand binding assay. Membranes were prepared from Chinese hamster ovary recombinant cells stably expressing the human NK3 receptor. The membranes were incubated with 5 nM 3H-SB222200(ARC) in a HEPES 25 mM/NaCl 0.1M/CaCl2 1 mM/MgCl2 5 Mm/BSA 0.5%/Saponin 10 μg/ml buffer at pH 7.4 and various concentrations of compounds of the invention. The amount of 3H-SB222200 bound to the receptor was determined after filtration by the quantification of membrane associated radioactivity using the TopCount-NXT reader (Packard).
Measurement: Ki = 56 nM (pChEMBL 7.25)
Descriptors: MW 461.49, ALogP 4.8, HBA 7, HBD 0, PSA 63.91, QED 0.44
Compound (SMILES): CC1CCN(C(=O)C(CCn2ccnc2[N+](=O)[O-])NS(=O)(=O)c2cc(Cl)c(N)c(Cl)c2)CC1
InChIKey: FVJCVTWWUJRNDE-UHFFFAOYSA-N
Target: C-C chemokine receptor type 10 (SINGLE PROTEIN), Homo sapiens
UniProt: P46092
Assay: Antagonist activity at human CCR10 expressed in mouse BA/F3 cells assessed as inhibition of human CCL27-dependent chemotaxis
Measurement: IC50 = 50.12 nM (pChEMBL 7.300000190734863)
Descriptors: MW 519.41, ALogP 2.68, HBA 8, HBD 2, PSA 153.46, QED 0.31
Compound (SMILES): Cn1cc(-c2cc(S(C)(=O)=O)ccc2OCC2CC2)c2cc[nH]c2c1=O
InChIKey: COSBTRPLBVPWCS-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 = 9.7 nM (pChEMBL 8.010000228881836)
Descriptors: MW 372.45, ALogP 2.73, HBA 5, HBD 1, PSA 81.16, QED 0.75
Compound (SMILES): O=C(Nc1cn(CC(=O)N2C[C@@H]3CCN[C@@H]3C2)nc1-c1cc(Cl)ccc1OC(F)F)c1cnn2cccnc12
InChIKey: PXAAENHUXRCKIS-KBXCAEBGSA-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 = 0.89 nM (pChEMBL 9.050000190734863)
Descriptors: MW 556.96, ALogP 2.92, HBA 9, HBD 2, PSA 118.68, QED 0.36
Compound (SMILES): CCOc1cccc(F)c1[C@@H]1C[C@H](Cl)C(=O)N1Cc1ccc(OC(F)(F)F)c(F)c1
InChIKey: PUSWJMUZMOHRFX-WFASDCNBSA-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 = 30 nM (pChEMBL 7.519999980926514)
Descriptors: MW 449.8, ALogP 5.34, HBA 3, HBD 0, PSA 38.77, QED 0.44
Compound (SMILES): CC(NC(=O)OC(C)(C)C)c1ccc(Nc2ncc(Cl)c(CCc3ccccc3C3(C(N)=O)CC3)n2)cc1
InChIKey: YOOZCWNOLYRGFW-UHFFFAOYSA-N
Target: Vascular endothelial growth factor receptor 3 (SINGLE PROTEIN), Homo sapiens
UniProt: P35916
Assay: VEGFR3 Phospho ELISA Assay: Adult human dermal lymphatic microvascular endothelial cells (HMVEC-dLyAD) (Cat# CC-2810, Lonza) were seeded into clear-bottom, TC treated 12 well plates (Cat #665180, Greiner Bio-One) in EGM-2MV (Cat# CC-3202, Lonza) at 180,000 cells/well (volume 1 mL), and the plates incubated at 37° C. and 5% CO2 for 6 hours. The media was replaced with EBM-2 (Cat # CC-3156, Lonza)+0.1% BSA (Cat# A8412, Sigma) and cells incubated for a further period (overnight at 37° C. and 5% CO2).96 well Maxisorp immuno plates (Cat #439454, Nunc) were coated with 100 uL of Total VEGFR3 capture antibody (Part #841888, Human Total VEGFR3/FLT4 ELISA Kit, Cat # DYC3491, R&D Systems), or Phospho VEGFR3 Capture antibody (Part #841885, Human Phospho VEGFR3/FLT4 ELISA Kit, Cat# DYC2724, R&D Systems). The plates were covered and incubated at room temperature overnight .The coating antibody was flicked out and the plates washed three times with Wash Buffer (Phosphate buffered saline (137 mM NaCl, 2.7 nM KCl, 8.1 nM Na2HPO4, 1.5 mL KH2PO4, pH 7.2-7.4), 0.05% Tween 20). 300 uL of blocking buffer (5% v/v Tween 20, 5% w/v sucrose in PBS) was then added to wells and plate incubated for 2 hours at room temperature.
Measurement: IC50 = 25 nM (pChEMBL 7.599999904632568)
Descriptors: MW 536.08, ALogP 5.76, HBA 6, HBD 3, PSA 119.23, QED 0.32
Compound (SMILES): CC(O)CN1CCN(C(=O)CSC(c2ccc(F)cc2)c2ccc(F)cc2)CC1
InChIKey: VMHOZLUMJYUETI-UHFFFAOYSA-N
Target: Sodium-dependent serotonin transporter (SINGLE PROTEIN), Rattus norvegicus
UniProt: P31652
Assay: Displacement of [3H]citalopram from SERT in Sprague-Dawley rat brain stem membranes incubated for 60 mins by radioligand binding assay
Measurement: Ki = 11400 nM (pChEMBL 4.940000057220459)
Descriptors: MW 420.52, ALogP 3.31, HBA 4, HBD 1, PSA 43.78, QED 0.75
Compound (SMILES): CN1C(=O)N(c2cccc(C(F)(F)F)c2)C2=C(C(=O)CC2)C1c1ccc(C#N)cc1C(=O)N1CCN(C2CC2)CC1
InChIKey: LNFYNZWJRSRZCE-UHFFFAOYSA-N
Target: Neutrophil elastase (SINGLE PROTEIN), Homo sapiens
UniProt: P08246
Assay: Human Neutrophil Elastase Assay: The following buffers were used: Compound buffer: 100 mM Tris, 500 mM NaCl, adjusted to pH 7.5; Assay buffer: 100 mM Tris, 500 mM NaCl, adjusted to pH 7.5, containing 0.01% BSA.Assay conditions: Test compounds were prediluted in DMSO and subsequently in compound buffer (5% DMSO final). 5 μL of these compound dilutions were mixed with 10 μl Neutrophil elastase (9 ng/ml in assay buffer) in a black 384 well OptiPlate (Perkin Elmer, Cat No.: 6007270) and incubated for 15 min at room temperature. Subsequently 10 μL substrate solution in assay buffer were added (250 μM final concentration) and the plates were incubated for 60 min at room temperature. After inactivation of the enzyme, fluorescence intensities were measured at 380 nm excitation and 460 nm emission wavelengths.
Measurement: IC50 = 15 nM (pChEMBL 7.820000171661377)
Descriptors: MW 563.58, ALogP 4.73, HBA 5, HBD 0, PSA 87.96, QED 0.54
Compound (SMILES): Cc1ccc2nc(NC(=O)N3CCN(c4ncc([C@H](O)CO)cc4F)C[C@@H]3C)sc2c1
InChIKey: MOYYIRTYGZTKTA-SUMWQHHRSA-N
Target: Transient receptor potential cation channel subfamily V member 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q8NER1
Assay: pH-based Assay: pH dependent Ca2+ responses in TRPV1/CHO cells cultured in a 96-well plate were determined (see, e.g., FIG. 2 of U.S. Patent Application Publication No. US 2009/0170868 A1). In particular, Ca2+ influx into TRPV1/CHO cells in response to low pH as measured by Fura-2 AM fluorescence was determined. The cells were stimulated using 3.0 mM (well numbers B1-B6), 3.1 mM (C1-C6), 3.2 mM (D1-D6), 3.3 mM (E1-E6), 3.4 mM (F1-F6), 3.5 mM (G1-G6), or 3.6 mM (H1-H6) H2SO4 or pH 7.2 measuring buffer without H2SO4 (A1-A6). Culture medium was removed using an 8-channel-pipette (Rainin, USA) from the 96-well plate and the wells were refilled with 100 uL of loading buffer (20 mM HEPES, 115 mM NaCl, 5.4 mM KCl, 0.8 mM MgCl2, 1.8 mM CaCl2, 13.8 mM D-glucose, 2.5 mM probenecid, pH 7.4) containing 5 uM Fura-2 AM (Dojin, Japan). The 96-well plate was incubated at 37° C. for 45 min. The loading buffer was removed from each well. The cells were subsequently washed twice with 150 uL of measuring buffer (20 mM HEPES, 115 mM NaCl, 5.4 mM KCl, 0.8 mM MgCl2, 5.0 mM CaCl2, 13.8 mM D-glucose, 0.1% BSA, pH 7.4) (no probenecid). The wells were then refilled with 80 uL of measuring buffer. After an incubation at 4° C. for 15 min, the 96-well plate was transferred to a model FDSS-3000 plate reader apparatus (Hamamatsu Photonics K.K., Japan). The Fura-2 fluorescent intensity was monitored at a wavelength of 340 nm and at 380 nm, respectively, at a rate of 0.5 Hz for a total of 240 seconds. After 16 time points (32 sec) of baseline detection, 20 uL of agonist solution was added to each well. The final volume was 100 uL/well. Fluorescence intensity ratio refers to the fluorescence intensity at 340 nm over the fluorescence intensity at 380 nm at a particular time point. The baseline was set as the average of the fluorescent intensity ratios for the first 16 time points before the addition of agonist solution. The maximum response was the highest fluorescent intensity ratio during the 60 time points following addition of agonist solution.
Measurement: IC50 = 12.2 nM (pChEMBL 7.909999847412109)
Descriptors: MW 445.52, ALogP 2.91, HBA 7, HBD 3, PSA 101.82, QED 0.57
Compound (SMILES): CC1(N)CCN(c2cccnc2NC(=O)c2nc(-c3nc(C4CC4)ccc3C(F)(F)F)cnc2N)CC1
InChIKey: UPSXKHFTVWTQRJ-UHFFFAOYSA-N
Target: Protein kinase C theta type (SINGLE PROTEIN), Homo sapiens
UniProt: Q04759
Assay: In Vitro Inhibition Activity Assay: The compounds of formula I were tested for their activity on different PKC isoforms according to a published method (D. Geiges et al. Biochem. Pharmacol. 1997; 53:865-875) The assay is performed in a 96-well polypropylene microtiterplate (Costar 3794) that has been previously siliconized with Sigmacote (Sigma SL-2). The reaction mixture (50 μL) contains 10 μL of the relevant PKC isozyme together with 25 μL of the PKC inhibitor compound and 15 μL of a mix solution that contains 200 μg/mL protamine sulfate, 10 mM Mg(NO3)2, 10 μM ATP (Boehringer 519987) and 3750 Bq of 33P-ATP (Hartmann Analytic SFC301, 110 TBq/mmol) in 20 mM Tris-buffer pH 7.4+0.1% BSA. Incubation was performed for 15 minutes at 32° C. in a microtiterplate shaking incubator (Biolabo Scientific Instruments). Reaction was stopped by adding 10 μl of 0.5 M Na2EDTA, pH 7.4. 50 μl of mixture are pipetted onto a pre-wetted phosphocellulose paper (Whatmann 3698-915) under gentle pressure.
Measurement: IC50 = 2.1 nM (pChEMBL 8.680000305175781)
Descriptors: MW 512.54, ALogP 3.98, HBA 8, HBD 3, PSA 135.94, QED 0.47
Compound (SMILES): C[C@H]1C(=O)N[C@@H](Cc2ccc(F)cc2)C(=O)NC[C@H]2C[C@@H]2c2ccccc2OCCN[C@H](C2CC2)C(=O)N1C
InChIKey: DNNIOJOUHTXROV-QVLUIDJUSA-N
Target: Growth hormone secretagogue receptor type 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q92847
Assay: Agonist activity at human GSHR1a transfected in CHOK1 cells co-expressing Galpha16 assessed as increase in Ca2+ release by aequorin assay
Measurement: EC50 = 100 nM (pChEMBL 7)
Descriptors: MW 536.65, ALogP 2.38, HBA 5, HBD 3, PSA 99.77, QED 0.56
Compound (SMILES): CN(C)S(=O)(=O)N(CCN1[C@@H]2CC[C@H]1C[C@@H](c1cccc(C(N)=O)c1)C2)CC1CCCCC1.O=C(O)C(F)(F)F
InChIKey: KCVJLNHJKMIUCV-MGJLCPRGSA-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 476.69, ALogP 3.18, HBA 4, HBD 1, PSA 86.95, QED 0.59
Compound (SMILES): Cc1cc(C(F)(F)F)ccc1Nc1nc(N2CCOC(CO)C2)nc2c1CCN(c1ncccc1C(F)(F)F)CC2
InChIKey: ZFHPXSRPNQRBCM-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 = 680 nM (pChEMBL 6.170000076293945)
Descriptors: MW 582.55, ALogP 4.76, HBA 8, HBD 2, PSA 86.64, QED 0.42
Compound (SMILES): O=S(=O)(Nc1ncccn1)c1ccc(Nc2nncc3ccccc23)cc1
InChIKey: AUAMVGQDEAESMO-UHFFFAOYSA-N
Target: Vascular endothelial growth factor receptor 2 (SINGLE PROTEIN), Homo sapiens
UniProt: P35968
Assay: Inhibition of VEGFR-2 (unknown origin) using poly (Glu, Tyr) 4:1 as substrate after 1 hr by ELISA
Measurement: IC50 = 8.5 nM (pChEMBL 8.069999694824219)
Descriptors: MW 378.42, ALogP 2.96, HBA 7, HBD 2, PSA 109.76, QED 0.55
Compound (SMILES): COc1ccc2c3c1O[C@H]1[C@@]4(OC)CC[C@@]5(C[C@@H]4COCC4CCCCC4)[C@@H](C2)N(C)CC[C@]315
InChIKey: CROXCUGUTMDKDP-ZYGOFEOWSA-N
Target: Opioid growth factor receptor-like protein 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q5TC84
Assay: Binding Assay: Binding assay of certain compounds of this invention to the opioid receptor was determine using radioligand binding assay (screening and dose-displacement).
Measurement: Ki = 1034.67 nM (pChEMBL 5.989999771118164)
Descriptors: MW 467.65, ALogP 4.74, HBA 5, HBD 0, PSA 40.16, QED 0.6
Compound (SMILES): COc1cn(C(CC(C)C)C(=O)Nc2ccc(C(=O)O)cc2)c(=O)cc1-c1cc(Cl)ccc1C#N
InChIKey: PDLAMRDOLIUATC-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 = 4.7 nM (pChEMBL 8.329999923706055)
Descriptors: MW 493.95, ALogP 4.97, HBA 6, HBD 2, PSA 121.42, QED 0.46
Compound (SMILES): CNc1nc(NCc2ccc(NC(=O)c3ccc(Cl)nc3)cc2)c2cc(OC)c(OC)cc2n1
InChIKey: DCANTODNDYGXLJ-UHFFFAOYSA-N
Target: TCF4/beta-catenin (PROTEIN COMPLEX), Homo sapiens
UniProt: P35222
Assay: Inhibition of TCF-4/beta catenin in human Tcf22C11 cells expressing Tcf-4/SV40 binding sites driving firefly luciferase reporter gene after 24 hrs by luminometer
Measurement: IC50 = 888 nM (pChEMBL 6.050000190734863)
Descriptors: MW 478.94, ALogP 4.6, HBA 8, HBD 3, PSA 110.29, QED 0.32
Compound (SMILES): COc1ncc(C(F)(F)F)cc1N1CCOc2cc(S(=O)(=O)Nc3nccs3)ccc21
InChIKey: OWRSUSYZFMGANM-UHFFFAOYSA-N
Target: Sodium channel protein type 9 subunit alpha (SINGLE PROTEIN), Homo sapiens
UniProt: Q15858
Assay: In Vitro PX Assay: HEK 293 cells stably transfected with human Nav1.7 were recorded in whole cell voltage clamp mode with the PatchXpress automated electrophysiology system (Molecular Devices, LLC, Sunnyvale, Calif.). Compound effects were measured on a partially inactivated state of the sodium channel. Cells were clamped to a holding potential yielding 20 to 50% inactivation. To elicit sodium current, channels were activated by pulsing to −10 mV for 20 msec. This voltage protocol was repeated at a rate of 0.1 Hz throughout the experiment. A single concentration of test compound was applied to cells for a duration of 3 minutes.
Measurement: IC50 = 11800 nM (pChEMBL 4.929999828338623)
Descriptors: MW 472.47, ALogP 3.9, HBA 8, HBD 1, PSA 93.65, QED 0.6
Compound (SMILES): Cc1nc(C2(NC(=O)c3cnc(C4CC4)c(OCC4CC4)n3)CCC2)no1
InChIKey: BLCQCFSLZDMXMV-UHFFFAOYSA-N
Target: Cannabinoid receptor 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P21554
Assay: cAMP Assay: CHO cells expressing human CB1 or CB2 receptors are seeded 17-24 hours prior to the experiment 50.000 cells per well in a black 96 well plate with flat clear bottom (Corning Costar #3904) in DMEM (Invitrogen No. 31331), 1×HT supplement, with 10% fetal calf serum and incubated at 5% CO2 and 37° C. in a humidified incubator. The growth medium was exchanged with Krebs Ringer Bicarbonate buffer with 1 mM IBMX and incubated at 30° C. for 30 min. Compounds were added to a final assay volume of 100 μl and incubated for 30 min at 30° C. Using the cAMP-Nano-TRF detection kit the assay (Roche Diagnostics) was stopped by the addition of 50 μl lysis reagent (Tris, NaCl, 1.5% Triton X100, 2.5% NP40, 10% NaN3) and 50 μl detection solutions (20 μM mAb Alexa700-cAMP 1:1, and 48 μM Ruthenium-2-AHA-cAMP) and shaken for 2 h at room temperature. The time-resolved energy transfer is measured by a TRF reader (Evotec Technologies GmbH), equipped with a ND:YAG laser as excitation source. The plate is measured twice with the excitation at 355 nm and at the emission with a delay of 100 ns and a gate of 100 ns, total exposure time 10s at 730 (bandwidth 30 nm) or 645 nm (bandwidth 75 nm), respectively.
Measurement: EC50 > 10000 nM (pChEMBL 5)
Descriptors: MW 369.43, ALogP 2.64, HBA 7, HBD 1, PSA 103.03, QED 0.8
Compound (SMILES): C#CCOc1cnc(C(=O)Nc2ccc(F)c([C@]3(C)C[C@@H](C(F)(F)F)OC(N)=N3)n2)cn1
InChIKey: IVYZSVSPKSDHAE-SGTLLEGYSA-N
Target: Beta-secretase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P56817
Assay: In Vitro BACE Cell-Based Assay: The cell-based assay measures inhibition or reduction of Aβ40 in conditioned medium of test compound treated cells expressing amyloid precursor protein. Cells stably expressing Amyloid Precursor Protein (APP) were plated at a density of 40K cells/well in 96 well plates (Costar). The cells were cultivated for 24 hours at 37° C. and 5% CO2 in DMEM supplemented with 10% FBS. The test compounds were then added to cells in 10-point dose response concentrations with the starting concentration being either 100 uM or 10 uM. The compounds were diluted from stock solutions in DMSO and the final DMSO concentration of the test compounds on cells was 0.1%. After 24 h of incubation with the test compounds the supernatant conditioned media was collected and the Aβ 40 levels were determined using a sandwich ELISA.
Measurement: IC50 = 8.4 nM (pChEMBL 8.079999923706055)
Descriptors: MW 452.37, ALogP 2.16, HBA 8, HBD 2, PSA 124.61, QED 0.53
Compound (SMILES): COc1cc(N2CCC(N3CCN(S(C)(=O)=O)CC3)CC2)ccc1Nc1nccc(-c2c(-c3cccc(C(=O)Nc4c(F)cccc4F)c3)nc3c(C)cccn23)n1
InChIKey: LAVUQVGESJYCJA-UHFFFAOYSA-N
Target: Insulin receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P06213
Assay: Inhibition of human GST-rTEV-tagged insulin receptor (979 to 1382 residues) expressed in baculovirus infected insect Sf9 cells assessed as decrease in biotin-aminohexyl-AEEEEYMMMMAKKKK-NH2 phosphorylation after 1 hr by fluorometer
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 807.93, ALogP 6.6, HBA 11, HBD 2, PSA 137.3, QED 0.16
Compound (SMILES): CC(C)n1cc(-c2cc(-c3ccc(CN4CCOCC4)c(F)c3)cnc2N)nn1
InChIKey: JATRXSPQYZZTFU-UHFFFAOYSA-N
Target: Leucine-rich repeat serine/threonine-protein kinase 2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q5S007
Assay: Kinase Activity Assay: The kinase reaction buffer consists of 50 mM Tris pH 8.5, 0.01% BRIJ-35, 10 mM MgCl2, 1 mM EGTA, and 2 mM DTT. In the assay, 1 nM LRRK2 WT or 250 pM LRRK2 G2019S kinase is incubated with the test compound (typically at 0 to 30 μM) for 30 minutes before the kinase reaction is initiated by addition of 1.3 mM ATP and 0.4 μM fluorescein-LRRKtide. The reaction mixture (20 μl total volume) is incubated for 2 hours at 30 °C. before the reaction is terminated by addition of 10 mM EDTA and 1 nM terbium-labelled anti-phospho-LRRKtide antibody (final volume 20 μl). The mixture is further incubated for 30 minutes at RT. TR-FRET is measured by excitation of the terbium-donor with 340 nm light and subsequent (delay time 100 μs) measurement of terbium and fluorescein emission at 495 nm and 520 nm, respectively, over a time window of 1000 μs. The measurement is repeated 10 times for fluorescein and 10 times for terbium emission with a 2000 μs time window between repeats. TR-FRET measurements are performed on a Biomek Synergy plate. The TR-FRET signal is calculated as the emission-ratio at 520 nm over 495 nm.
Measurement: IC50 = 200 nM (pChEMBL 6.699999809265137)
Descriptors: MW 396.47, ALogP 3.14, HBA 7, HBD 1, PSA 82.09, QED 0.71
Compound (SMILES): Cc1nn(C)c(C)c1N(C(F)F)S(=O)(=O)c1c(Cl)cc(CCCOC2CC(C)(C)N(C)C(C)(C)C2)cc1Cl
InChIKey: OTKMKBYGZQEUCC-UHFFFAOYSA-N
Target: Glycylpeptide N-tetradecanoyltransferase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P30419
Assay: Enzyme Inhibition Assay: Measurements were performed using a modification of the scintillation proximity assay platform described previously by Georgopapadakou, N. H. et al. (22nd International Congress on Chemotherapy, 2001, Abstract P16.001). Compounds were solubilised in DMSO at a top concentration of 10 mM and serially diluted in half log steps to achieve a range of final assay concentrations of 100 uM to 1 nM. Compound at each concentration (100-fold final) was added to white 384 well plates in a volume of 0.5 ml. Human, A. fumigatus, T. brucei or L. major N-myristoyl transferase enzyme, dissolved to a working concentration of 10 nM in assay buffer (30 mM Tris/HCl pH 7.4, 0.5 mM EGTA, 0.5 mM EDTA, 1.25 mM DTT, 0.1% Triton X-100), was then added to columns 1 to 11 and 13 to 23 of the plates in a volume of 20 ml. To columns 12 and 24, 20 ml assay buffer was added to provide a no enzyme control. Following a 5 minute incubation at room temperature the substrates (GCGGSKVKPQPPQAK(Biotin)-Amide and myristoyl coenzyme A), dissolved in assay buffer, were added to all wells in a volume of 20 ml to start the reaction. The final concentrations of peptide and 3H-myristoyl coenzyme A were 0.5 mM and 125 nM respectively and the specific activity of the radiolabel was 8 Ci/mmol. Plates were then incubated at room temperature for up to 50 minutes (dependant upon the period of linearity for the different enzyme species) before SPA beads, suspended to 1 mg/ml in a stop solution (200 mM Phosphoric Acid/NaOH pH 4, 750 mM MgCl2), were added in a volume of 40 ml.
Measurement: IC50 > 1000 nM (pChEMBL 6)
Descriptors: MW 595.58, ALogP 6.36, HBA 6, HBD 0, PSA 67.67, QED 0.25
Compound (SMILES): Cc1c(-c2ccc(C(N)=O)c3[nH]c4cc(C(C)(C)O)ccc4c23)cccc1-n1c(=O)c2cccc(F)c2n(C)c1=O
InChIKey: LEOYMYOSQPPAAS-UHFFFAOYSA-N
Target: Tyrosine-protein kinase BTK (SINGLE PROTEIN), Homo sapiens
UniProt: Q06187
Assay: Inhibition of full length recombinant human His-tagged BTK expressed in baculovirus using fluoresceinated peptide incubated for 60 mins by fluorescent assay
Measurement: IC50 = 4.6 nM (pChEMBL 8.34000015258789)
Descriptors: MW 550.59, ALogP 4.76, HBA 6, HBD 3, PSA 123.11, QED 0.29
Compound (SMILES): COc1cc2c(Nc3cc(Cl)c(Cl)cc3F)ncnc2cc1OC[C@@H]1CN2CCOC[C@H]2CO1
InChIKey: KHSJGHHCLKKKKU-KBPBESRZSA-N
Target: Ephrin type-A receptor 2 (SINGLE PROTEIN), Homo sapiens
UniProt: P29317
Assay: Inhibition of recombinant human N-terminal His6-tagged EphA2 (N598 to R890 residues) expressed in baculovirus using poly-EY as substrate after 180 mins by luciferase coupled chemiluminescence assay
Measurement: IC50 = 50 nM (pChEMBL 7.300000190734863)
Descriptors: MW 509.37, ALogP 4.31, HBA 8, HBD 1, PSA 77.97, QED 0.49
Compound (SMILES): CC(C)CCNC(=O)c1ccc(NC(=O)N2Cc3ccncc3C2)cc1
InChIKey: LMYQDAFZILWDHN-UHFFFAOYSA-N
Target: Nicotinamide phosphoribosyltransferase (SINGLE PROTEIN), Homo sapiens
UniProt: P43490
Assay: Inhibition of C-terminal His-tagged human recombinant NAMPT using FK866 or isoindoline urea-based Oregon green (488) probe incubated for 3 hrs by TR-FRET assay
Measurement: Ki = 7.5 nM (pChEMBL 8.119999885559082)
Descriptors: MW 352.44, ALogP 3.41, HBA 3, HBD 2, PSA 74.33, QED 0.87
Compound (SMILES): Cc1cc(O)cc(C)c1C[C@@H]1NC(=O)C(CCCN)NC(=O)[C@H](Cc2ccccc2)NC(=O)[C@@H](CCCNC(=N)N)NC1=O
InChIKey: JAUDAXVKEGMCPI-CSQIJOFWSA-N
Target: Delta-type opioid receptor (SINGLE PROTEIN), Rattus norvegicus
UniProt: P33533
Assay: Displacement of [3H]DSLET from DOR in rat brain membrane measured after 2 hrs
Measurement: Ki = 3.84 nM (pChEMBL 8.420000076293945)
Descriptors: MW 608.74, ALogP -0.25, HBA 7, HBD 9, PSA 224.55, QED 0.09
Compound (SMILES): O=C(NO)c1ccc(CN(c2ccccc2)c2ccccc2)cc1
InChIKey: IGWBWSHLQZLWRG-UHFFFAOYSA-N
Target: Protein deacetylase HDAC6 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9UBN7
Assay: HDAC Activity Assay: HDAC assay is performed using fluorescently-labeled acetylated substrate, which comprises an acetylated lysine side chain. After incubation with HDAC, deacetylation of the substrate sensitizes the substrate such that, in a second step, treatment with the detection enzyme produces a fluorophore. HDACs 1 and 6 were expressed as full length fusion proteins. Purified proteins were incubated with 50 μM fluorescently-labeled acetylated peptide substrate and test compound for 2 hours at RT in HDAC assay buffer containing 50 mM Tris-HCl (pH 8.0), 137 mM NaCl, 2.7 mM KCl, 1 mM MgCl2, 1% DMSO, and 1% BSA. Reactions were terminated by the addition of the Developer after 2 hours, and the development of fluorescence signal, which was relative to the amount of deacetylated peptide, was monitored by time-course measurement of EnVision (PerkinElmer). The HDAC activity was estimated from the slope of time-course measurement of the fluorescence intensity.
Measurement: IC50 = 4500 nM (pChEMBL 5.349999904632568)
Descriptors: MW 318.38, ALogP 4.14, HBA 3, HBD 2, PSA 52.57, QED 0.55
Compound (SMILES): N#Cc1c(NC(=O)c2ccc(Cl)c([N+](=O)[O-])c2)n(Cc2ccccc2)c2nc3ccccc3nc12
InChIKey: OFJFAJHQAVKRSQ-UHFFFAOYSA-N
Target: Dihydrolipoyllysine-residue acetyltransferase component of pyruvate dehydrogenase complex (SINGLE PROTEIN), Mycobacterium tuberculosis (strain ATCC 25618 / H37Rv)
UniProt: P9WIS7
Assay: Inhibition Assay: Mtb PDH (Lpd+DlaT+AceE) is provided by Dr. Bryk Ruslana. The assay was performed in a manner similar to that described in Bryk et al., Biochemistry (2010) 49:1616-1627 and modified for an online robotics screening system.A solution containing PDH (Lpd=15 μM; DlaT=30 μM; AceE=15 μM) was diluted 2-fold in 100 mM of potassium phosphate buffer (pH 7.0) for the assay. Another reaction solution containing 50 mM potassium phosphate buffer (pH 7.0), 200 μM TPP, 2 mM Pyruvate, 200 μM CoA, 1 mM MgCl2, 1 mM NAD+ was prepared. 5 μL/well of PDH solution was dispensed into 1536-well black plates. Next, 50 nL of each test compound (1 mM dissolved in DMSO) was added into each well. After 30 minutes of incubation, 5 μL/well of reaction solution was added. At the 30th minutes of the reaction, the florescence signal (excitation 360 nm and emission 460 nm) was obtained by Viewlux reader (PerkinElmer).
Measurement: IC50 > 5000 nM (pChEMBL 5.301000118255615)
Descriptors: MW 482.89, ALogP 5.32, HBA 7, HBD 1, PSA 126.74, QED 0.27
Compound (SMILES): Cc1noc(C)c1Cn1cc(NC(=O)Cc2ccccc2)cn1
InChIKey: VNWOEVYYILNNAE-UHFFFAOYSA-N
Target: Taste receptor type 2 member 8 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9NYW2
Assay: Antagonist activity at recombinant human TAS2R8 stably expressed in cells co-expressing Galpha16gust44 assessed as inhibition of andrographolide-induced intracellular calcium level measured for 100 secs by fluo-4 dye based FLIPR assay
Measurement: IC50 = 800 nM (pChEMBL 6.099999904632568)
Descriptors: MW 310.36, ALogP 2.72, HBA 5, HBD 1, PSA 72.95, QED 0.79
Compound (SMILES): CC(=O)N1CCN(C2CCC(CC#N)(n3cc(C(N)=O)c(Nc4ccc(F)cc4)n3)CC2)CC1
InChIKey: DUUQCXDMKROSBX-UHFFFAOYSA-N
Target: Tyrosine-protein kinase JAK2 (SINGLE PROTEIN), Homo sapiens
UniProt: O60674
Assay: HTRF Biochemical Assay: The reactions employed a common peptide substrate, LCB-EQEDEPEGDYFEWLW-NH2 (in-house). The basic assay protocol is as follows: First, 250 nL of diluted compounds in DMSO were dispensed into the wells of a dry 384-well Black plate (Greiner #781076) using a Labcyte Echo 555 acoustic dispenser. Subsequent reagent additions employed an Agilent Bravo. Next, 18 uL of 1.11x enzyme and 1.11x substrate in 1x assay buffer (Invitrogen kinase buffer # PV3189, 2 mM DTT, 0.05% BSA) were added to the wells and shaken and then preincubated for 30 minutes at room temperature to allow compound binding to equilibrate. After equilibration, 2 uL of 10xATP in 1x assay buffer was added to initiate the kinase reaction and the plates were shaken and then incubated at room temperature for 120 minutes. At the end of the incubation, 20 uL of 2x stop buffer (streptavidin-Dylight 650 (Thermo #84547B/100 mL), Eu-tagged pY20 antibody (Perkin Elmer #AD0067), EDTA, HEPES, and Triton) was added to quench the reaction.
Measurement: IC50 = 225 nM (pChEMBL 6.650000095367432)
Descriptors: MW 467.55, ALogP 2.58, HBA 7, HBD 2, PSA 120.28, QED 0.67
Compound (SMILES): COc1cc(OC)cc(N(CC2CC2)c2ccc3ncc(-c4cnn(CC5(OC)CCNCC5)c4)nc3c2)c1
InChIKey: IJNFMKJSNQLEOT-UHFFFAOYSA-N
Target: Fibroblast growth factor receptor 3 (SINGLE PROTEIN), Homo sapiens
UniProt: P22607
Assay: Inhibition of FGFR3 (unknown origin) transfected in mouse BA/F3 cells assessed as decrease in cell proliferation in presence of mouse IL3 after 24 hrs by Alamar blue assay
Measurement: IC50 = 5495.41 nM (pChEMBL 5.260000228881836)
Descriptors: MW 528.66, ALogP 4.83, HBA 9, HBD 1, PSA 86.56, QED 0.31
Compound (SMILES): O=C(O)CN1CCN(C(=O)Cn2cc(NC(=O)c3cnn4cccnc34)c(-c3cc(Cl)ccc3OC(F)F)n2)CC1
InChIKey: BADPAEQLSZZUJD-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 = 0.59 nM (pChEMBL 9.229999542236328)
Descriptors: MW 588.96, ALogP 2.33, HBA 10, HBD 2, PSA 147.19, QED 0.3
Compound (SMILES): CN1C(=N)N[C@](C)(c2sc(-c3cccc(-c4ncon4)c3)cc2F)CS1(=O)=O
InChIKey: GGCKFWXYKBJBSD-KRWDZBQOSA-N
Target: Beta-secretase 2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9Y5Z0
Assay: HTRF FRET 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 = 0.34 nM (pChEMBL 9.470000267028809)
Descriptors: MW 421.48, ALogP 2.62, HBA 7, HBD 2, PSA 112.18, QED 0.67
Compound (SMILES): CCN1CC(C)(C)Oc2nc(N3CCOC[C@@H]3C)nc(-c3ccc(NC(=O)NC)c(F)c3)c2C1=O
InChIKey: REMUXVMJAABOQT-AWEZNQCLSA-N
Target: Phosphatidylinositol 3-kinase regulatory subunit alpha (SINGLE PROTEIN), Homo sapiens
UniProt: P27986
Assay: Enzyme Assay: The test uses a lucifeurerin/luciferase system to measure the concentration of ATP and its consumption during the enzymatic reaction. The test is performed in 96-well format (Corning/Costar 96 black flat-bottomed half-wells plate, ref. 3694) in a total volume of 30 μl. To 1 μl of inhibitor in 100% DMSO are added (final concentrations) 50 μM of the substrate PIP2 ((L-α-phosphatidyl-D-myoinositol 4,5-bisphosphate, Echelon 117P-4516-0500), 2 μM of ATP and 1.7 μg/ml of PI3Kα, (p110α/p85α, Invitrogen PV4788) in a buffer of Tris/HCl 50 mM pH 7.5, EGTA 1 mM, MgCl2 10 mM, Chaps 0.03%, 1 mM DTT). After 90 minutes, the reaction is quenched by adding 20 μl/well of KinaseGlo reagent (Promega V6713). After 10 minutes in the dark, the luminescence is read on the PHERAStar microplate reader (reading at 0.8 sec/well).
Measurement: IC50 = 547 nM (pChEMBL 6.260000228881836)
Descriptors: MW 486.55, ALogP 2.89, HBA 7, HBD 2, PSA 108.92, QED 0.68