lovingscience/chembl-v37-raw-activities huggingface dataset:
Compound (SMILES): Cc1ccc(-c2cc(C(=O)NCc3cnc(C)nc3)cc(-n3c(=O)oc4ccccc43)c2)nc1
InChIKey: BMCDSOJUZHNPHG-UHFFFAOYSA-N
Target: P2X2/P2X3 heterotrimeric receptor (PROTEIN COMPLEX), Homo sapiens
UniProt: P56373
Assay: Antagonist activity against human P2X2/3 receptor expressed in CHOK1 cells assessed as inhibition of alpha,beta-meATP-induced calcium response incubated for 20 mins by Fluo-3AM dye based FLIPR assay
Measurement: Ki = 13.8 nM (pChEMBL 7.860000133514404)
Descriptors: MW 451.49, ALogP 3.98, HBA 7, HBD 1, PSA 102.91, QED 0.43
Compound (SMILES): Cc1ccc(-c2cc(C(=O)N[C@@H](C)CO)cc(N3C(=O)OC[C@H]3C(C)C)c2)cc1
InChIKey: QRDUNINWOFCAMQ-KKSFZXQISA-N
Target: P2X2/P2X3 heterotrimeric receptor (PROTEIN COMPLEX), Homo sapiens
UniProt: P56373
Assay: Antagonist activity against human P2X2/3 receptor expressed in CHOK1 cells assessed as inhibition of alpha,beta-meATP-induced calcium response incubated for 20 mins by Fluo-3AM dye based FLIPR assay
Measurement: Ki = 79.43 nM (pChEMBL 7.099999904632568)
Descriptors: MW 396.49, ALogP 3.75, HBA 4, HBD 2, PSA 78.87, QED 0.78
Compound (SMILES): Cc1ccc(-c2cc(C(=O)NC(C)c3cnccn3)cc(N3C(=O)OC[C@H]3C(C)C)c2)cc1
InChIKey: PXQNUEJJIJCDAS-LUTIACGYSA-N
Target: P2X2/P2X3 heterotrimeric receptor (PROTEIN COMPLEX), Homo sapiens
UniProt: P56373
Assay: Antagonist activity against human P2X2/3 receptor expressed in CHOK1 cells assessed as inhibition of alpha,beta-meATP-induced calcium response incubated for 20 mins by Fluo-3AM dye based FLIPR assay
Measurement: Ki = 38.02 nM (pChEMBL 7.420000076293945)
Descriptors: MW 444.54, ALogP 4.92, HBA 5, HBD 1, PSA 84.42, QED 0.58
Compound (SMILES): Cc1ccc(-c2cc(C(=O)NCc3cnc(C)cn3)cc(N3C(=O)OC[C@H]3C(C)C)c2)cc1
InChIKey: LZHFVMNPYNSOFI-DEOSSOPVSA-N
Target: P2X2/P2X3 heterotrimeric receptor (PROTEIN COMPLEX), Homo sapiens
UniProt: P56373
Assay: Antagonist activity against human P2X2/3 receptor expressed in CHOK1 cells assessed as inhibition of alpha,beta-meATP-induced calcium response incubated for 20 mins by Fluo-3AM dye based FLIPR assay
Measurement: Ki = 38.02 nM (pChEMBL 7.420000076293945)
Descriptors: MW 444.54, ALogP 4.67, HBA 5, HBD 1, PSA 84.42, QED 0.6
Compound (SMILES): Cc1ccc(-c2cc(C(=O)NC3CC3)cc(N3C(=O)OC[C@H]3C(C)C)c2)cc1
InChIKey: FLIRILDGLQMFEZ-NRFANRHFSA-N
Target: P2X2/P2X3 heterotrimeric receptor (PROTEIN COMPLEX), Homo sapiens
UniProt: P56373
Assay: Antagonist activity against human P2X2/3 receptor expressed in CHOK1 cells assessed as inhibition of alpha,beta-meATP-induced calcium response incubated for 20 mins by Fluo-3AM dye based FLIPR assay
Measurement: Ki = 83.18 nM (pChEMBL 7.079999923706055)
Descriptors: MW 378.47, ALogP 4.54, HBA 3, HBD 1, PSA 58.64, QED 0.83
Compound (SMILES): Cc1ccc(-c2cc(C(=O)NC(C)c3cnccn3)cc(N3C(=O)OC[C@@H]3C(C)C)c2)cc1
InChIKey: PXQNUEJJIJCDAS-VCUSLETLSA-N
Target: P2X2/P2X3 heterotrimeric receptor (PROTEIN COMPLEX), Homo sapiens
UniProt: P56373
Assay: Antagonist activity against human P2X2/3 receptor expressed in CHOK1 cells assessed as inhibition of alpha,beta-meATP-induced calcium response incubated for 20 mins by Fluo-3AM dye based FLIPR assay
Measurement: Ki = 7585.78 nM (pChEMBL 5.119999885559082)
Descriptors: MW 444.54, ALogP 4.92, HBA 5, HBD 1, PSA 84.42, QED 0.58
Compound (SMILES): Cc1ccc(-c2cc(C(=O)N[C@@H](C)CO)cc(N3C(=O)OC[C@H]3C(C)C)c2)c(F)c1
InChIKey: DRAPDSWDHFUIHZ-BTYIYWSLSA-N
Target: P2X2/P2X3 heterotrimeric receptor (PROTEIN COMPLEX), Homo sapiens
UniProt: P56373
Assay: Antagonist activity against human P2X2/3 receptor expressed in CHOK1 cells assessed as inhibition of alpha,beta-meATP-induced calcium response incubated for 20 mins by Fluo-3AM dye based FLIPR assay
Measurement: Ki = 93.33 nM (pChEMBL 7.03000020980835)
Descriptors: MW 414.48, ALogP 3.89, HBA 4, HBD 2, PSA 78.87, QED 0.75
Compound (SMILES): Cc1ccc(-c2cc(C(=O)NC(C)c3cnccn3)cc(N3C(=O)OC[C@H]3C(C)C)c2)c(F)c1
InChIKey: OSQFXHWKOIFZOO-UCSBTNPJSA-N
Target: P2X2/P2X3 heterotrimeric receptor (PROTEIN COMPLEX), Homo sapiens
UniProt: P56373
Assay: Antagonist activity against human P2X2/3 receptor expressed in CHOK1 cells assessed as inhibition of alpha,beta-meATP-induced calcium response incubated for 20 mins by Fluo-3AM dye based FLIPR assay
Measurement: Ki = 44.67 nM (pChEMBL 7.349999904632568)
Descriptors: MW 462.53, ALogP 5.06, HBA 5, HBD 1, PSA 84.42, QED 0.55
Compound (SMILES): Cc1ccc(-c2cc(C(=O)NCc3cnc(C)cn3)cc(N3C(=O)OC[C@H]3C(C)C)c2)c(F)c1
InChIKey: NXXXMKXJEBTPJP-DEOSSOPVSA-N
Target: P2X2/P2X3 heterotrimeric receptor (PROTEIN COMPLEX), Homo sapiens
UniProt: P56373
Assay: Antagonist activity against human P2X2/3 receptor expressed in CHOK1 cells assessed as inhibition of alpha,beta-meATP-induced calcium response incubated for 20 mins by Fluo-3AM dye based FLIPR assay
Measurement: Ki = 38.02 nM (pChEMBL 7.420000076293945)
Descriptors: MW 462.53, ALogP 4.81, HBA 5, HBD 1, PSA 84.42, QED 0.57
Compound (SMILES): Cc1ccc(-c2cc(C(=O)NC3CC3)cc(N3C(=O)OC[C@H]3C(C)C)c2)c(F)c1
InChIKey: QAFMSMLCHQICCA-NRFANRHFSA-N
Target: P2X2/P2X3 heterotrimeric receptor (PROTEIN COMPLEX), Homo sapiens
UniProt: P56373
Assay: Antagonist activity against human P2X2/3 receptor expressed in CHOK1 cells assessed as inhibition of alpha,beta-meATP-induced calcium response incubated for 20 mins by Fluo-3AM dye based FLIPR assay
Measurement: Ki = 123.03 nM (pChEMBL 6.909999847412109)
Descriptors: MW 396.46, ALogP 4.67, HBA 3, HBD 1, PSA 58.64, QED 0.8
Compound (SMILES): Cc1ccc(-c2cc(C(=O)NC(C)c3cnccn3)cc(-n3c(=O)oc4ccccc43)c2)cc1
InChIKey: AEHQLZRGGMTEMV-UHFFFAOYSA-N
Target: P2X2/P2X3 heterotrimeric receptor (PROTEIN COMPLEX), Homo sapiens
UniProt: P56373
Assay: Antagonist activity against human P2X2/3 receptor expressed in CHOK1 cells assessed as inhibition of alpha,beta-meATP-induced calcium response incubated for 20 mins by Fluo-3AM dye based FLIPR assay
Measurement: Ki = 26.3 nM (pChEMBL 7.579999923706055)
Descriptors: MW 450.5, ALogP 4.84, HBA 6, HBD 1, PSA 90.02, QED 0.41
Compound (SMILES): Cc1ccc(-c2cc(C(=O)NCc3cnc(C)cn3)cc(-n3c(=O)oc4ccccc43)c2)cc1
InChIKey: RWHGQAGSVCDSDE-UHFFFAOYSA-N
Target: P2X2/P2X3 heterotrimeric receptor (PROTEIN COMPLEX), Homo sapiens
UniProt: P56373
Assay: Antagonist activity against human P2X2/3 receptor expressed in CHOK1 cells assessed as inhibition of alpha,beta-meATP-induced calcium response incubated for 20 mins by Fluo-3AM dye based FLIPR assay
Measurement: Ki = 31.62 nM (pChEMBL 7.5)
Descriptors: MW 450.5, ALogP 4.59, HBA 6, HBD 1, PSA 90.02, QED 0.42
Compound (SMILES): Cc1ccc(-c2cc(C(=O)NCc3cnc(C)cn3)cc(N3C(=O)Cc4ccccc43)c2)cc1
InChIKey: LILYTSLDQVOVAO-UHFFFAOYSA-N
Target: P2X2/P2X3 heterotrimeric receptor (PROTEIN COMPLEX), Homo sapiens
UniProt: P56373
Assay: Antagonist activity against human P2X2/3 receptor expressed in CHOK1 cells assessed as inhibition of alpha,beta-meATP-induced calcium response incubated for 20 mins by Fluo-3AM dye based FLIPR assay
Measurement: Ki = 123.03 nM (pChEMBL 6.909999847412109)
Descriptors: MW 448.53, ALogP 4.91, HBA 4, HBD 1, PSA 75.19, QED 0.47
Compound (SMILES): Cc1ccc(-c2cc(C(=O)NC(C)c3cnccn3)cc(N3C(=O)Cc4ccccc43)c2)cc1
InChIKey: XUTUDMYKUNRZSS-UHFFFAOYSA-N
Target: P2X2/P2X3 heterotrimeric receptor (PROTEIN COMPLEX), Homo sapiens
UniProt: P56373
Assay: Antagonist activity against human P2X2/3 receptor expressed in CHOK1 cells assessed as inhibition of alpha,beta-meATP-induced calcium response incubated for 20 mins by Fluo-3AM dye based FLIPR assay
Measurement: Ki = 112.2 nM (pChEMBL 6.949999809265137)
Descriptors: MW 448.53, ALogP 5.16, HBA 4, HBD 1, PSA 75.19, QED 0.46
Compound (SMILES): Cc1ccc(-c2cc(C(=O)NCc3cnc(C)cn3)cc(-c3snnc3C(C)C)c2)nc1
InChIKey: QGKWEGPZHJOINP-UHFFFAOYSA-N
Target: P2X2/P2X3 heterotrimeric receptor (PROTEIN COMPLEX), Homo sapiens
UniProt: P56373
Assay: Antagonist activity against human P2X2/3 receptor expressed in CHOK1 cells assessed as inhibition of alpha,beta-meATP-induced calcium response incubated for 20 mins by Fluo-3AM dye based FLIPR assay
Measurement: Ki = 37.15 nM (pChEMBL 7.429999828338623)
Descriptors: MW 444.56, ALogP 4.73, HBA 7, HBD 1, PSA 93.55, QED 0.46
Compound (SMILES): Cc1ccc(-c2cc(C(=O)N[C@H](C)CO)cc(-c3snnc3C(C)C)c2)nc1
InChIKey: ONAOVNAFHOSAPO-CQSZACIVSA-N
Target: P2X2/P2X3 heterotrimeric receptor (PROTEIN COMPLEX), Homo sapiens
UniProt: P56373
Assay: Antagonist activity against human P2X2/3 receptor expressed in CHOK1 cells assessed as inhibition of alpha,beta-meATP-induced calcium response incubated for 20 mins by Fluo-3AM dye based FLIPR assay
Measurement: Ki = 213.8 nM (pChEMBL 6.670000076293945)
Descriptors: MW 396.52, ALogP 3.81, HBA 6, HBD 2, PSA 88, QED 0.66
Compound (SMILES): Cc1ccc(-c2cc(C(=O)NCc3cnc(C)nc3)cc(-c3snnc3C(C)C)c2)nc1
InChIKey: DRWUOGAXURZYOQ-UHFFFAOYSA-N
Target: P2X2/P2X3 heterotrimeric receptor (PROTEIN COMPLEX), Homo sapiens
UniProt: P56373
Assay: Antagonist activity against human P2X2/3 receptor expressed in CHOK1 cells assessed as inhibition of alpha,beta-meATP-induced calcium response incubated for 20 mins by Fluo-3AM dye based FLIPR assay
Measurement: Ki = 33.88 nM (pChEMBL 7.46999979019165)
Descriptors: MW 444.56, ALogP 4.73, HBA 7, HBD 1, PSA 93.55, QED 0.46
Compound (SMILES): Cc1ccc(-c2cc(C(=O)NCc3ncc(C)cn3)cc(-c3snnc3C(C)C)c2)nc1
InChIKey: XDSMVWUQPSBETH-UHFFFAOYSA-N
Target: P2X2/P2X3 heterotrimeric receptor (PROTEIN COMPLEX), Homo sapiens
UniProt: P56373
Assay: Antagonist activity against human P2X2/3 receptor expressed in CHOK1 cells assessed as inhibition of alpha,beta-meATP-induced calcium response incubated for 20 mins by Fluo-3AM dye based FLIPR assay
Measurement: Ki = 104.71 nM (pChEMBL 6.980000019073486)
Descriptors: MW 444.56, ALogP 4.73, HBA 7, HBD 1, PSA 93.55, QED 0.46
Compound (SMILES): Cc1ccc(-c2cc(C(=O)NCc3cnc(C)cn3)cc(-c3nsnc3C(C)C)c2)nc1
InChIKey: GFPCDVNWJYDKRC-UHFFFAOYSA-N
Target: P2X2/P2X3 heterotrimeric receptor (PROTEIN COMPLEX), Homo sapiens
UniProt: P56373
Assay: Antagonist activity against human P2X2/3 receptor expressed in CHOK1 cells assessed as inhibition of alpha,beta-meATP-induced calcium response incubated for 20 mins by Fluo-3AM dye based FLIPR assay
Measurement: Ki = 43.65 nM (pChEMBL 7.360000133514404)
Descriptors: MW 444.56, ALogP 4.73, HBA 7, HBD 1, PSA 93.55, QED 0.46
Compound (SMILES): Cc1ccc(F)c(NC(=O)Nc2ccc(Oc3ccnc4cc(C(=O)NCCCN5CCOC(CO)C5)sc34)cc2F)c1
InChIKey: DNBSBOYLZWOFKX-UHFFFAOYSA-N
Target: Vascular endothelial growth factor receptor 2 (SINGLE PROTEIN), Homo sapiens
UniProt: P35968
Assay: Kinase Assay: Kinase assays were performed in 96 well microtiter plates that were coated overnight with 30 μg of poly-Glu-Tyr (4:1) in 10 mM Phosphate Buffered Saline (PBS), pH 7.2-7.4. The plates were incubated with 1% BSA and then washed four times with PBS prior to starting the reaction. Reactions were carried out in 120 μL reaction volumes containing 3.6 μM ATP in kinase buffer (50 mM Hepes buffer pH 7.4, 20 mM MgCl2, 0.1 mM MnCl2 and 0.2 mM Na3VO4). Test compounds were reconstituted in 100% DMSO and added to the reaction to give a final DMSO concentration of 5%. Reactions were initiated by the addition 0.5 ng of purified protein. Following a ten minute incubation at 25 °C., the reactions were washed four times with PBS containing 0.05% Tween-20. 100 μl of a monoclonal anti-phosphotyrosine antibody-peroxidase conjugate was diluted 1:10000 in PBS-Tween-20 and added to the wells for 30 minutes. Following four washes with PBS-Tween-20, 100 μl of 0-phenylenediamine Dihydrochloride in Phosphate-citrate buffer, containing urea hydrogen peroxide, was added to the wells for 7 minutes as a colorimetric substrate for the peroxidase. The reaction was terminated by the addition of 100 μl of 2.5N H2 SO4 to each well and read using a microplate ELISA reader set at 492 nm.
Measurement: IC50 = 7 nM (pChEMBL 8.149999618530273)
Descriptors: MW 611.67, ALogP 5.13, HBA 8, HBD 4, PSA 125.05, QED 0.18
Compound (SMILES): N#Cc1ccc(CN2CCC(c3nnn4cnc5[nH]ccc5c34)CC2)cc1C#N
InChIKey: MLVQDEQCRFDZOB-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 = 52 nM (pChEMBL 7.28000020980835)
Descriptors: MW 382.43, ALogP 2.73, HBA 7, HBD 1, PSA 109.69, QED 0.58
Compound (SMILES): NC(=O)OC(CNC(=O)Cn1nc(-c2ccc(Cl)cc2)n(C[C@H](O)C(F)(F)F)c1=O)c1ccccc1Cl
InChIKey: ZGMNJHJAVDYJPA-DJNXLDHESA-N
Target: Vasopressin V2 receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P30518
Assay: Cellular in Vitro Assay: On the day before the assay, the cells are plated out in culture medium (DMEM, 10% FCS, 2 mM glutamine, 10 mM HEPES) in 384-well microtitre plates and kept in a cell incubator (96% humidity, 5% v/v CO2, 37° C.). On the day of the assay, the culture medium is replaced by a Tyrode solution (140 mM NaCl, 5 mM KCl, 1 mM MgCl2, 2 mM CaCl2, 20 mM glucose, 20 mM HEPES), which additionally contains the cofactor coelenterazine (50 μM), and the microtitre plate is then incubated for a further 3-4 hours. The test substances in various concentrations are placed for 10 to 20 minutes in the wells of the microtitre plate before the agonist [Arg8]-vasopressin is added, and the resulting light signal is measured immediately in the luminometer.
Measurement: IC50 = 2 nM (pChEMBL 8.699999809265137)
Descriptors: MW 562.33, ALogP 2.89, HBA 8, HBD 3, PSA 141.47, QED 0.37
Compound (SMILES): O=C(Nc1ccc(N2CCOCC2)cn1)[C@@H]1CC[C@@H]2CN1C(=O)N2OS(=O)(=O)O
InChIKey: YYAOFRAEXXYSPX-OLZOCXBDSA-N
Target: Beta-lactamase (SINGLE PROTEIN), Pseudomonas aeruginosa
UniProt: Q8RL90
Assay: Spectrophotometric Assay: Enzyme activities were measured in the presence of the test inhibitor in spectrophotometric assay.
Measurement: IC50 = 5 nM (pChEMBL 8.300000190734863)
Descriptors: MW 427.44, ALogP -0.14, HBA 8, HBD 2, PSA 141.61, QED 0.61
Compound (SMILES): Cc1c(C#N)cccc1[C@@H]1CN2CCN(C(=O)Cc3ccc(-n4cnnn4)nc3)C[C@@H]2CO1
InChIKey: CHVOVRGBCPPLHQ-CTNGQTDRSA-N
Target: Voltage-gated inwardly rectifying potassium channel KCNH2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q12809
Assay: Inhibition of human ERG by electrophysiology method
Measurement: IC50 = 14800 nM (pChEMBL 4.829999923706055)
Descriptors: MW 444.5, ALogP 1.06, HBA 9, HBD 0, PSA 113.06, QED 0.59
Compound (SMILES): CN(C)c1ccc2cc(C(=O)NCCCCCC(=O)Nc3ccccc3N)[nH]c2c1
InChIKey: LJHGOODLFAKLNF-UHFFFAOYSA-N
Target: Histone deacetylase 3 (SINGLE PROTEIN), Homo sapiens
UniProt: O15379
Assay: HDAC enzyme inhibitionAssay: HDAC enzyme inhibition assays were performed using purified HDACs 1-10 essentially as described in Beckers et al., 2007, Int. J. Cancer., 121:1138-48 and Perez-Balado et al., 2007, J. Med. Chem., 50:2497-2505. Inhibition assays using nuclear extract were performed essentially as described in Herman et al., 2006, Nat. Chem. Biol., 2:551-558. Briefly, the purified HDACs or nuclear extract were incubated with an acetylated substrate in the absence of the compound to be assayed and with increasing concentrations of the compound. The rate of substrate deacetylation was measured under each condition, and half-maximal inhibitory concentration with regard to each HDAC was determined by standard means.
Measurement: IC50 = 115 nM (pChEMBL 6.940000057220459)
Descriptors: MW 407.52, ALogP 3.75, HBA 4, HBD 4, PSA 103.25, QED 0.32
Compound (SMILES): COC1(Cn2nnc(-c3ccc(N4CCc5cccc(C(=O)Nc6nc7ccccc7s6)c5C4)nc3C(=O)O)c2C)CCCC(C)(C)C1
InChIKey: HFGPGDMPFNZLFE-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 = 30 nM (pChEMBL 7.519999980926514)
Descriptors: MW 665.82, ALogP 6.76, HBA 10, HBD 2, PSA 135.36, QED 0.18
Compound (SMILES): [N-]=[N+]=Nc1ccc(N2CCOc3cc(S(=O)(=O)Nc4nccs4)ccc32)c(Br)c1
InChIKey: KSKVPCKKKJJWTF-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 = 1070 nM (pChEMBL 5.96999979019165)
Descriptors: MW 493.37, ALogP 5.18, HBA 7, HBD 1, PSA 120.29, QED 0.3
Compound (SMILES): CN1Cc2c(Cl)cc(Cl)cc2[C@H](c2cccc(S(=O)(=O)NCCOCCOCCOCCNC(=O)CN(CCOCCOCCN(CC(=O)NCCOCCOCCOCCNS(=O)(=O)c3cccc([C@@H]4CN(C)Cc5c(Cl)cc(Cl)cc54)c3)CC(=O)NCCOCCOCCOCCNS(=O)(=O)c3cccc([C@@H]4CN(C)Cc5c(Cl)cc(Cl)cc54)c3)CC(=O)NCCOCCOCCOCCNS(=O)(=O)c3cccc([C@@H]4CN(C)Cc5c(Cl)cc(Cl)cc54)c3)c2)C1
InChIKey: CHSODALJUANGIF-QZESFGMVSA-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 = 10 nM (pChEMBL 8)
Descriptors: MW 2494.36
Compound (SMILES): Cc1csc(NC(=O)C2=C(C(=O)O)CCC2)c1-c1nc(C2CC2)no1
InChIKey: OPAHDAOJXHDREH-UHFFFAOYSA-N
Target: Fatty acid-binding protein 5 (SINGLE PROTEIN), Homo sapiens
UniProt: Q01469
Assay: Time Resolved-Fluorescence Energy Transfer (TR-FRET) Assay: Stock DMSO solutions (1.8 mM) of compounds were serially diluted 3-fold for ten concentrations with 100% DMSO (50 μM to 0.003 μM final compound concentration). 1 μl of these compound dilutions and 1 μl of Bodipy labeled fatty acid 4.5 μM in 100% DMSO (Bodipy FL C11, cat. no. D3862, Invitrogen) were sequentially pipetted in wells of 384-well black polypropylene plates (Thermo Matrix cat. no. 4344). FABP4 or FABP5 protein was then added (28 μl of 64 nM protein in 25 mM Tris pH 7.5, 0.4 mg/ml α-globulin, 1 mM DTT, 0.012% NP40, final protein concentration: 50 nM). Assay blanks contained ligand, but no protein. Neutral controls contained ligand, but no compound. After adding the detection reagent (Tb antiHis6 antibody, Columbia Biosciences, TB-110, 6 μl of a 24 nM Ab solution in 25 mM Tris pH 7.5, 0.4 mg/ml γ-globulin, final Tb antiHis6 Ab concentration: 4 nM), plates were spun one minute at 1000 rpm.
Measurement: IC50 = 98.6 nM (pChEMBL 7.010000228881836)
Descriptors: MW 359.41, ALogP 3.49, HBA 6, HBD 2, PSA 105.32, QED 0.85
Compound (SMILES): Cc1ccc(-n2nccn2)c(C(=O)N2CC[C@H]2c2nc(-c3c(C)cccc3F)no2)c1
InChIKey: CAQSJYDHFANXMS-SFHVURJKSA-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 = 115 nM (pChEMBL 6.940000057220459)
Descriptors: MW 418.43, ALogP 3.66, HBA 7, HBD 0, PSA 89.94, QED 0.5
Compound (SMILES): O=C(Nc1nc2ccc(C3(O)c4ccccc4C(=O)N3c3cccc(Cl)c3F)cc2[nH]1)C1CC1(F)F
InChIKey: HHSVKZYLXCCZHB-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 512.88, ALogP 4.8, HBA 4, HBD 3, PSA 98.32, QED 0.37
Compound (SMILES): CCCCCCCCCCCCc1ccc(NC(=O)C2(/C(N)=N/O)CC2)cc1
InChIKey: OOIRIHAFMWHMRF-UHFFFAOYSA-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 = 6000 nM (pChEMBL 5.21999979019165)
Descriptors: MW 387.57, ALogP 5.62, HBA 3, HBD 3, PSA 87.71, QED 0.13
Compound (SMILES): COCCC1(N)CCN(c2cccnc2NC(=O)c2nc(-c3ncccc3C(F)(F)F)cnc2N)CC1
InChIKey: OCJFSDQZBJFGTN-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 = 29 nM (pChEMBL 7.539999961853027)
Descriptors: MW 516.53, ALogP 3.12, HBA 9, HBD 3, PSA 145.17, QED 0.43
Compound (SMILES): CC1CCN(c2cccc(-c3c[nH]c4ccc(-c5nnc(N)s5)cc34)n2)CC1
InChIKey: KIVJUGCLEZDIID-UHFFFAOYSA-N
Target: Serine/threonine-protein kinase pim-2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9P1W9
Assay: Enzyme Assay: The assay for the determination of Pim activity is based on the formation of phosphorylated biotinylated-BAD peptide at the Serine 112 residue (S112) and employs HTRF® (homogeneous time resolved fluorescence) technology to detect the product in a 96-well plate format. The phosphorylation of biotinylated-BAD (S112) peptide by full length recombinant Pim-1, Pim-2, or Pim-3 protein was detected with streptavidin:Allophycocyanin (APC) conjugate and a europium (Eu) labeled antibody directed against phosphorylated-BAD (S112). Excitation of Eu by a high energy laser light (337 nm) leads to a transfer of energy to the APC molecule, and results in an emission at 665 nm. The fluorescence is directly proportional to the amount of phosphorylated BAD peptide present in the reaction.Compounds were prepared in DMSO by conducting 3-fold serial dilutions to give a 10-point dosing curve having a high dose of 1 uM. A reference compound was included on each assay plate in order to validate that plate; on
Measurement: IC50 = 117.31 nM (pChEMBL 6.929999828338623)
Descriptors: MW 390.52, ALogP 4.57, HBA 6, HBD 2, PSA 83.72, QED 0.54
Compound (SMILES): CNC(=O)c1c(-c2ccc(F)cc2)oc2cc(N(C)S(C)(=O)=O)c(-c3ccc4c(n3)-c3cc5c(F)cccc5n3CC=C4)cc12
InChIKey: OLTHMJNUAUFBDS-UHFFFAOYSA-N
Target: Genome polyprotein (SINGLE PROTEIN), Hepatitis C virus genotype 1b (isolate Japanese) (HCV)
UniProt: P26662
Assay: Inhibition Assay: To initiate an assay, replicon cells were plated in the presence of a dilution series of test compound in the absence of G418. Typically, the assays were performed in a 96-well plate formate for manual operation, or a 384-well plate format for automated assay. Replicon cells and compound were incubated for 96 hours. At the end of the assay, cells were washed free of media and compound, and the cells were then lysed. RNA was quantified indirectly through detection of replicon-encoded NS3/4A protein levels, through an ELISA-based assay with an antibody specific for NS3/4A.
Measurement: IC50 = 8.646 nM (pChEMBL 8.0600004196167)
Descriptors: MW 624.67, ALogP 6.84, HBA 6, HBD 1, PSA 97.44, QED 0.23
Compound (SMILES): C[C@H](NC(=O)C[S+]([O-])c1nc2cccnc2n1Cc1ccc(OC(F)(F)F)cc1)c1ccc(F)cc1
InChIKey: DSBWSBQQGOKUGE-QJFJEURWSA-N
Target: Autotaxin (SINGLE PROTEIN), Homo sapiens
UniProt: Q13822
Assay: Inhibition of human recombinant ATX using Rac-1-Palmitoyl-glycero-3-phosphocholine as substrate incubated for 2 hrs using ADHP fluorogenic peroxidase substrate by horseradish peroxidase/choline oxidase-coupled assay
Measurement: IC50 = 178 nM (pChEMBL 6.75)
Descriptors: MW 520.51, ALogP 4.5, HBA 6, HBD 1, PSA 92.1, QED 0.27
Compound (SMILES): CNc1nc(NCc2ccc(NC(=O)c3ccc(Cl)cc3F)cc2)c2cc(C)cc(C)c2n1
InChIKey: PBVSVNBYMXGULL-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 > 21554 nM (pChEMBL 4.665999889373779)
Descriptors: MW 463.94, ALogP 5.95, HBA 5, HBD 3, PSA 78.94, QED 0.33
Compound (SMILES): CCCCN(CCCC)c1nn(-c2ccc(C(=O)NS(=O)(=O)c3ccc4ccccc4c3)cc2C(=O)N2CCc3ccccc3C2)c(C)c1Cl
InChIKey: HQANQJQFPFHZAZ-UHFFFAOYSA-N
Target: Bcl-2-like protein 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q07817
Assay: Fluorescence Resonance Energy Transfer Assay (FRET): The assays were performed in black flat-bottom 384-well plates. The final assay volume was 55 μl prepared from additions of Biotin-Bcl-xL (Bcl-xL: GENBANK® Accession No. Q07817), fluoresceinated 18-mer BIM peptide (NH2-YYANFEDGIRRLEQAIWI-[FAM]) (SEQ ID NO: 1), Streptavidin-Terbium FRET detection reagent, and test compounds in assay buffer consisting of 20 mM Sodium Phosphate, 1 mM EDTA, 50 mM NaCl, and 0.05% Pluronic Acid. The reaction was incubated at room temperature for 60 minutes. After 60 minutes, 5 μL of Streptavidin-Terbium FRET detection reagent (Perkin Elmer) was added to the reaction mixture and incubated at room temperature in the dark for 30 mins. The FRET signal generated by the reaction was detected on the Envision Plate Reader Inhibition data were calculated from FRET values generated by the no protein control reactions for 100% inhibition and vehicle-only reactions for 0% inhibition. The final concentration of reagents in the assay was 10 nM Bcl-xL, 5 nM fluo
Measurement: IC50 = 690 nM (pChEMBL 6.159999847412109)
Descriptors: MW 712.32, ALogP 7.71, HBA 7, HBD 1, PSA 104.61, QED 0.14
Compound (SMILES): Cc1cccc2cc(Cn3nc(-c4cccc(O)c4)c4c(N)ncnc43)n(-c3ccccc3C(C)C)c(=O)c12
InChIKey: RKLGNXMPYTTYRU-UHFFFAOYSA-N
Target: Phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit gamma isoform (SINGLE PROTEIN), Homo sapiens
UniProt: P48736
Assay: Inhibition of recombinant human C-terminal His6-tagged p110gamma expressed in insect cells using phosphatidylinositol as substrate in presence of gamma-32P-ATP after 5 mins by thin layer chromatographic method
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 516.61, ALogP 5.57, HBA 8, HBD 2, PSA 111.85, QED 0.31
Compound (SMILES): [C-]#[N+]c1ccc(OC)c(-c2c[nH]c3ncnc(N4CCOCC4)c23)c1
InChIKey: DUVOQILPRVJODM-UHFFFAOYSA-N
Target: Leucine-rich repeat serine/threonine-protein kinase 2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q5S007
Assay: Lantha Screen Technology Assay: LRRK2 kinase activity was measured using Lantha Screen technology from Invitrogen. GST-tagged truncated LRRK2 from Invitrogen (Cat # PV4874) was incubated with a fluorescein-labeled peptide substrate based upon ezrin/radixin/moesin (ERM), also known as LRRKtide (Invitrogen cat #PR8976A), in the presence of a dose response of compound. Upon completion, the assay was stopped and detected with a terbium labeled anti-phospho-ERM antibody (Invitrogen, cat #PR8975A). The assay was carried out under the following protocol: 3 uL of a working solution of substrate (233 nM LRRKtide, 117 uM ATP) prepared in assay buffer (50 mM HEEPES, pH 7.5, 3 mM MgCl2, with 2 mM DTT and 0.01% Brij35 added fresh) was added to a low volume Greiner 384-well plate. The compound dose response was prepared by diluting compound to a top concentration of 3.16 mM in 100% DMSO and serial diluted by half-log in DMSO 11 times. Aliquots (3.5 uL) of the 100% DMSO dose response were mixed with 46.5 uL water.
Measurement: IC50 = 121 nM (pChEMBL 6.920000076293945)
Descriptors: MW 335.37, ALogP 3.02, HBA 5, HBD 1, PSA 67.63, QED 0.75
Compound (SMILES): CN1C(=O)Cc2ccc(-c3ccc(C[C@@H](C#N)NC(=O)[C@@]45CCO[C@@H](CN4)C5)cc3)cc21
InChIKey: FNFKANIAANVVRI-BKSPAHHJSA-N
Target: Dipeptidyl peptidase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P53634
Assay: Inhbition Assay: The recombinant human DPPI was diluted in TAGZyme buffer to 1 U/ml (38.1 ug/ml, respectively), and then activated by mixing in a 1:2 ratio with a Cysteamine aqueous solution (2 mM) and incubating for 5 min at room temperature.Five uL test compound (final concentration 0.1 nM to 100 uM) in aqua bidest (containing 4% DMSO, final DMSO concentration 1%) were mixed with 10 uL of DPPI in MES buffer (final concentration 0.0125 ng/uL) and incubated for 10 min. Then, 5 uL of substrate in MES buffer (final concentration 50 uM) were added. The microtiter plates were then incubated at room temperature for 30 min. Then, the reaction was stopped by adding 10 uL of Gly-Phe-DMK in MES-buffer (final concentration 1 uM). The fluorescence in the wells was determined using a Molecular Devices SpectraMax M5 Fluorescence Reader (Ex 360 nm, Em 460 nm) or an Envision Fluorescence Reader (Ex 355 nm, Em 460 nm).
Measurement: IC50 = 50.3 nM (pChEMBL 7.300000190734863)
Descriptors: MW 430.51, ALogP 1.94, HBA 5, HBD 2, PSA 94.46, QED 0.76
Compound (SMILES): CNC(=O)c1c(C2CC2)oc2cc(N(C)S(C)(=O)=O)c(-c3ncc4c(n3)-c3cc5c(F)cccc5n3CO4)cc12
InChIKey: SULYLMQQDKEIDV-UHFFFAOYSA-N
Target: Genome polyprotein (SINGLE PROTEIN), Hepatitis C virus genotype 1b (isolate Japanese) (HCV)
UniProt: P26662
Assay: Inhbition Assay: To initiate an assay, replicon cells were plated in the presence of a dilution series of test compound in the absence of G418. Typically, the assays were performed in a 96-well plate formate for manual operation, or a 384-well plate format for automated assay. Replicon cells and compound were incubated for 96 hours. At the end of the assay, cells were washed free of media and compound, and the cells were then lysed. RNA was quantified indirectly through detection of replicon-encoded NS3/4A protein levels, through an ELISA-based assay with an antibody specific for NS3/4A.
Measurement: IC50 = 30 nM (pChEMBL 7.519999980926514)
Descriptors: MW 561.6, ALogP 4.63, HBA 8, HBD 1, PSA 119.56, QED 0.33
Compound (SMILES): Cc1nc([C@H](CC2CC2)NC(=O)c2ccc(C3CC3)c(OCC(F)(F)C(F)(F)F)n2)no1
InChIKey: VZFNTIBCUQHUTG-HNNXBMFYSA-N
Target: Cannabinoid receptor 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P21554
Assay: cAMP Assay: CHO cells expressing human CB1 or CB2 receptors are seeded 17-24 hours prior to the experiment 50.000 cells per well in a black 96 well plate with flat clear bottom (Corning Costar #3904) in DMEM (Invitrogen No. 31331), 1.times.HT supplement, with 10% fetal calf serum and incubated at 5% CO2 and 37° C. in a humidified incubator. The growth medium was exchanged with Krebs Ringer Bicarbonate buffer with 1 mM IBMX and incubated at 30.degree. C. for 30 min Compounds were added to a final assay volume of 100 ul and incubated for 30 min at 30° C. Using the cAMP-Nano-TRF detection kit the assay (Roche Diagnostics) was stopped by the addition of 50 ul lysis reagent (Tris, NaCl, 1.5% Triton X100, 2.5% NP40, 10% NaN.sub.3) and 50 ul detection solutions (20 uM mAb Alexa700-cAMP 1:1, and 48 uM Ruthenium-2-AHA-cAMP) and shaken for 2 h at room temperature. The time-resolved energy transfer is measured by a TRF reader (Evotec Technologies GmbH), equipped with a ND:YAG laser as excitation source. The plate is measured twice with the excitation at 355 nm and at the emission with a delay of 100 ns and a gate of 100 ns, total exposure time 10 s at 730 (bandwidth 30 nm) or 645 nm (bandwidth 75 nm), respectively. The FRET signal is calculated.
Measurement: EC50 > 10000 nM (pChEMBL 5)
Descriptors: MW 460.4, ALogP 4.5, HBA 6, HBD 1, PSA 90.14, QED 0.55
Compound (SMILES): CCOc1ccc(C[C@@H](NC(=O)c2scnc2C)C(=O)N[C@@H](Cc2ccccc2)C(=O)NCc2ccc(CN)cc2)cc1
InChIKey: NKONHEXDLBKQFF-URLMMPGGSA-N
Target: Kallikrein-1 (SINGLE PROTEIN), Homo sapiens
UniProt: P06870
Assay: In Vitro Inhibition Assay: KLK1 inhibitory activity in vitro was determined using standard published methods (see e.g. Johansen et al., Int. J. Tiss. Reac. 1986, 8, 185; Shori et al., Biochem. Pharmacol., 1992, 43, 1209; Stürzebecher et al., Biol. Chem. Hoppe-Seyler, 1992, 373, 1025). Human KLK1 (Callbiochem) was incubated at 37° C. with the fluorogenic substrate H-DVal-Leu-Arg-AFC and various concentrations of the test compound. Residual enzyme activity (initial rate of reaction) was determined by measuring the change in optical absorbance at 410 nm and the IC50 value for the test compound was determined.
Measurement: IC50 = 6100 nM (pChEMBL 5.210000038146973)
Descriptors: MW 599.76, ALogP 3.69, HBA 7, HBD 4, PSA 135.44, QED 0.17
Compound (SMILES): COc1cc(C(=O)NCc2ccc3sccc3c2)ccc1-c1cn[nH]c1
InChIKey: ADMZUAQDCHMTRI-UHFFFAOYSA-N
Target: Rho-associated protein kinase 2 (SINGLE PROTEIN), Homo sapiens
UniProt: O75116
Assay: In Vitro Assay: The effectiveness of compounds of the present invention as ROCK inhibitors can be determined in a 30 μL assay containing 20 mM HEPES, pH 7.5, 20 mM MgCl2, 0.015% Brij-35, 4 mM DTT, 5 μM ATP and 1.5 μM peptide substrate (FITC-AHA-AKRRRLSSLRA-OH). Compounds were dissolved in DMSO so that the final concentration of DMSO was <2%, and the reaction was initiated with Rho kinase variants. After incubation, the reaction was terminated by the addition of EDTA and the phosphorylated and non-phosphorylated peptides separated using a LABCHIP® 3000 Reader (Caliper Life Sciences). Controls consisted of assays that did not contain compound, and backgrounds consisted of assays that contained enzyme and substrate but had EDTA from the beginning of the reaction to inhibit kinase activity.
Measurement: IC50 = 9.44 nM (pChEMBL 8.029999732971191)
Descriptors: MW 363.44, ALogP 4.23, HBA 4, HBD 2, PSA 67.01, QED 0.56
Compound (SMILES): Cc1cc(Nc2cc(C3CC3)[nH]n2)nc(NCc2ncc[nH]2)n1
InChIKey: QLZDJSZWNLYOLI-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 = 500 nM (pChEMBL 6.300000190734863)
Descriptors: MW 310.37, ALogP 2.46, HBA 6, HBD 4, PSA 107.2, QED 0.56
Compound (SMILES): CN[C@@H](C)C(=O)Nc1ccc(-c2c(-c3ccc(OC)nc3)nc3cc(C)ccn23)c(C#Cc2ccc3c(C)nccc3c2)n1
InChIKey: WCEKPSGXNVKDPR-QHCPKHFHSA-N
Target: E3 ubiquitin-protein ligase XIAP (SINGLE PROTEIN), Homo sapiens
UniProt: P98170
Assay: Binding Assays (DELFIA): BIR3 domains (10 nM) were incubated with SMAC peptide (10 nM) in assay buffer (50 mM Tris, 120 mM NaCl, 0.1% BSA, 1 mM DTT, 0.05% Triton X100) for one hour at room temperature in the presence of inhibitory compounds. The assay mixture was transferred to a streptavidin coated plate and incubated for one hour at room temperature to allow binding of the biotinylated peptide and associated BIR3 domains to the plate. After several washing steps Eu labeled anti-GST antibody (e.g. Perkin Elmer DELFIA Eu-N1-antiGST AD0250) was added to detect BIR3 domain-SMAC peptide interactions according to Perkin Elmer's instructions. Briefly, the antibody was added (dilution 1:5000 in Perkin Elmer DELFIA Assay Buffer 2013-01) and incubated for one hour. After 3 washing steps using Delfia Washing Buffer (Perkin Elmer DELFIA Wash 2013-05), Enhancement Solution (Perkin Elmer Enhancement Asolution 2013-02) was added and incubation continued for 10 minutes.
Measurement: IC50 = 267 nM (pChEMBL 6.570000171661377)
Descriptors: MW 581.68, ALogP 5.58, HBA 8, HBD 2, PSA 106.33, QED 0.25
Compound (SMILES): N=C(NC(=N)N1CCN(C(=N)NC(=N)Nc2ccccc2F)CC1)Nc1ccccc1F
InChIKey: SVAUEUFROJRERJ-UHFFFAOYSA-N
Target: Acetylcholinesterase (SINGLE PROTEIN), Electrophorus electricus
UniProt: O42275
Assay: Competitive inhibition of Electrophorus electricus acetylcholinesterase in presence of varying acetylthiocholine iodide substrate level preincubated for 20 mins followed by substrate addition measured every 1 min for 30 mins
Measurement: Ki = 14920 nM (pChEMBL 4.829999923706055)
Descriptors: MW 442.48, ALogP 2.02, HBA 4, HBD 8, PSA 150, QED 0.26
Compound (SMILES): O=C(Nc1ccccn1)N1CCN(Cc2cnc3ccccc3c2)CC1
InChIKey: IITSALAZNAIVNX-UHFFFAOYSA-N
Target: Fatty-acid amide hydrolase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: O00519
Assay: FAAH Assay: T84 frozen cell pellets or transfected SK-N-MC cells (contents of 1x15 cm culture dishes) were homogenized in 50 mL of FAAH assay buffer (125 mM Tris, 1 mM EDTA, 0.2% Glycerol, 0.02% Triton X-100, 0.4 mM Hepes, pH 9). The assay mixture consisted of 50 uL of the cell homogenate, 10 uL of the test compound, and 40 uL of anandamide [1-3H-ethanolamine] (3H-AEA, Perkin-Elmer, 10.3 Ci/mmol), which was added last, for a final tracer concentration of 80 nM. The reaction mixture was incubated at rt for 1 h. During the incubation, 96-well Multiscreen filter plates (catalog number MAFCNOB50; Millipore, Bedford, Mass., USA) were loaded with 25 uL of activated charcoal (Multiscreen column loader, catalog number MACL09625, Millipore) and washed once with 100 uL of MeOH. Also during the incubation, 96-well DYNEX MicroLite plates (catalog number NL510410) were loaded with 100 uL of MicroScint40 (catalog number 6013641, Packard Bioscience, Meriden, Conn., USA).
Measurement: IC50 = 8000 nM (pChEMBL 5.099999904632568)
Descriptors: MW 347.42, ALogP 2.98, HBA 4, HBD 1, PSA 61.36, QED 0.79
Compound (SMILES): C=Cc1nc(-c2nnc3n2CCN(C(=O)c2ccc(-c4cccs4)cc2)C3)cs1
InChIKey: BGMHMCCXCXGAEX-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 = 19 nM (pChEMBL 7.71999979019165)
Descriptors: MW 419.54, ALogP 4.43, HBA 7, HBD 0, PSA 63.91, QED 0.49
Compound (SMILES): CN(CCCN1CCC(n2cc(NC(=O)c3c(N)nn4cccnc34)c(-c3cc(Cl)ccc3OC(F)F)n2)CC1)C(=O)c1ccccc1
InChIKey: SFBUNBMVYTYLGS-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 = 0.42 nM (pChEMBL 9.380000114440918)
Descriptors: MW 678.14, ALogP 5.48, HBA 10, HBD 2, PSA 135.91, QED 0.19
Compound (SMILES): Cn1cc(-c2cc(S(C)(=O)=O)ccc2Oc2ccc(F)cc2F)c2cc(C(N)=O)[nH]c2c1=O
InChIKey: PMFAAQHHEWEOPY-UHFFFAOYSA-N
Target: Bromodomain-containing protein 4 (SINGLE PROTEIN), Homo sapiens
UniProt: O60885
Assay: Bromodomain Domain Binding Assay: A time-resolved fluorescence resonance energy transfer (TR-FRET) assay was used to determine the affinities of compounds of the Examples listed in Table 1 for each bromodomain of human BRD4. His-tagged first (BD1: amino acids K57-E168) and second (BD2: amino acids E352-E168) bromodomains of human BRD4 were expressed and purified. An Alexa647-labeled BET-inhibitor was used as the fluorescent probe in the assay. Compound dilution series were prepared in DMSO via a 3-fold serial dilution from 2.5 mM to 42 nM. Compounds were then diluted 6:100 in assay buffer (20 mM Sodium Phosphate, pH 6.0, 50 mM NaCl, 1 mM Ethylenediaminetetraacetic acid disodium salt dihydrate, 0.01% Triton X-100, 1 mM DL-Dithiothreitol) to yield 3× working solutions. Six microliters (L) of the working solution was then transferred to white, low-volume assay plates (Costar #3673). A 1.5× assay mixture containing His-tagged bromodomain, Europium-conjugated anti-His antibody (Invitrogen PV5596) and the Alexa-647-conjugated probe molecule was also prepared. Twelve L of this solution were added to the assay plate to reach a final volume of 18 L. The final concentration of 1× assay buffer contains 2% DMSO, 50 M-0.85 nM compound, 8 nM His-tagged bromodomain, 1 nM Europium-conjugated anti-His-tag antibody and 100 nM or 30 nM probe (for BDI or BDII, respectively). After a one-hour incubation at room temperature, TR-FRET ratios were determined using an Envision multilabel plate reader (Ex 340, Em 495/520).
Measurement: Ki = 1.7 nM (pChEMBL 8.770000457763672)
Descriptors: MW 473.46, ALogP 3.11, HBA 6, HBD 2, PSA 124.25, QED 0.46
Compound (SMILES): COc1cc(C(=O)NCc2ccc(F)c(C(=O)O)c2)ccc1-c1cn[nH]c1
InChIKey: RJXSCRRYUBZADY-UHFFFAOYSA-N
Target: Rho-associated protein kinase 2 (SINGLE PROTEIN), Homo sapiens
UniProt: O75116
Assay: In Vitro Assay: The effectiveness of compounds of the present invention as ROCK inhibitors can be determined in a 30 μL assay containing 20 mM HEPES, pH 7.5, 20 mM MgCl2, 0.015% Brij-35, 4 mM DTT, 5 μM ATP and 1.5 μM peptide substrate (FITC-AHA-AKRRRLSSLRA-OH). Compounds were dissolved in DMSO so that the final concentration of DMSO was <2%, and the reaction was initiated with Rho kinase variants. After incubation, the reaction was terminated by the addition of EDTA and the phosphorylated and non-phosphorylated peptides separated using a LABCHIP® 3000 Reader (Caliper Life Sciences). Controls consisted of assays that did not contain compound, and backgrounds consisted of assays that contained enzyme and substrate but had EDTA from the beginning of the reaction to inhibit kinase activity.
Measurement: IC50 = 4.41 nM (pChEMBL 8.359999656677246)
Descriptors: MW 369.35, ALogP 2.85, HBA 4, HBD 3, PSA 104.31, QED 0.62
Compound (SMILES): COc1ccc(N)c(NC(=O)CCCCCNC(=O)c2ccc(C)cc2)c1
InChIKey: MWNRCVQDELNBDV-UHFFFAOYSA-N
Target: Histone deacetylase 3 (SINGLE PROTEIN), Homo sapiens
UniProt: O15379
Assay: HDAC enzyme inhibitionAssay: HDAC enzyme inhibition assays were performed using purified HDACs 1-10 essentially as described in Beckers et al., 2007, Int. J. Cancer., 121:1138-48 and Perez-Balado et al., 2007, J. Med. Chem., 50:2497-2505. Inhibition assays using nuclear extract were performed essentially as described in Herman et al., 2006, Nat. Chem. Biol., 2:551-558. Briefly, the purified HDACs or nuclear extract were incubated with an acetylated substrate in the absence of the compound to be assayed and with increasing concentrations of the compound. The rate of substrate deacetylation was measured under each condition, and half-maximal inhibitory concentration with regard to each HDAC was determined by standard means.
Measurement: IC50 = 1000 nM (pChEMBL 6)
Descriptors: MW 369.46, ALogP 3.51, HBA 4, HBD 3, PSA 93.45, QED 0.47