lovingscience/chembl-v37-raw-activities huggingface dataset:
Compound (SMILES): O=C(Nc1ccc(C2CCCNC2)cc1)c1cnc(Cl)cn1
InChIKey: VCQRGMMHDZMTLR-UHFFFAOYSA-N
Target: Trace amine-associated receptor 1 (SINGLE PROTEIN), Rattus norvegicus
UniProt: Q923Y9
Assay: Radioligand Binding Assay: HEK-293 cells stably expressing rat TAAR1 were maintained at 37° C. and 5% CO2 in DMEM high glucose medium, containing fetal calf serum (10%, heat inactivated for 30 min at 56° C.), penicillin/streptomycin (1%), and 375 ug/ml geneticin (Gibco). Cells were released from culture flasks using trypsin/EDTA, harvested, washed twice with ice-cold PBS (without Ca2+ and Mg2+), pelleted at 1,000 rpm for 5 min at 4° C., frozen and stored at -80° C. Frozen pellets were suspended in 20 ml HEPES-NaOH (20 mM, pH 7.4) containing 10 mM EDTA and homogenized with a Polytron (PT 6000, Kinematica) at 14,000 rpm for 20 s. The homogenate was centrifuged at 48,000Ã¿g for 30 min at 4° C. Subsequently, the supernatant was removed and discarded, and the pellet resuspended in 20 ml HEPES-NaOH (20 mM, pH 7.4) containing 0.1 mM EDTA using the Polytron (20 s at 14,000 rpm). This procedure was repeated and the final pellet resuspended in HEPES-NaOH containing 0.1 mM EDTA and homogenized using the Polytron. Typically, aliquots of 2 ml membrane portions were stored at -80° C. With each new membrane batch the dissociation constant (Kd) was determined via a saturation curve. The TAAR1 radioligand 3[H]â¿¿(S)-4-[(ethyl-phenyl-amino)-methyl]-4,5-dihydro-oxazol-2-ylamine (described in WO 2008/098857) was used at a concentration equal to the calculated Kd value, that was usually around 2.3 nM, resulting in the binding of approximately 0.2% of the radioligand and a specific binding representing approximately 85% of the total binding. Nonspecific binding was defined as the amount of 3[H]â¿¿(S)-4-[(ethyl-phenyl-amino)-methyl]-4,5-dihydro-oxazol-2-ylamine bound in the presence of 10 uM unlabeled ligand. All compounds were tested at a broad range of concentrations (10 pM to 10 uM) in duplicates. The test compounds (20 ul/well) were transferred into a 96 deep well plate (TreffLab), and 180 ul of HEPES-NaOH (20 mM, pH 7.4) containing MgCl2 (10 mM) and CaCl2 (2 mM) (binding buffer), 300 ul of the radioligand 3[H]â¿¿(S)-4-[(ethyl-phenyl-amino)-methyl]-4,5-dihydro-oxazol-2-ylamine at a concentration of 3.3xKd in nM and 500 ul of the membranes (resuspended at 50 ug protein per ml) added. The 96 deep well plates were incubated for 1 hr at 4° C. Incubations were terminated by rapid filtration through Unifilter-96 plates (Packard Instrument Company) and glass filters GF/C (Perkin Elmer) presoaked for 1 hr in polyethylenimine (0.3%) and washed 3 times with 1 ml of cold binding buffer. After addition of 45 ul of Microscint 40 (PerkinElmer) the Unifilter-96 plate was sealed and after 1 hr the ratioactivity counted using a TopCount Microplate Scintillation Counter (Packard Instrument Company).
Measurement: Ki = 4.6 nM (pChEMBL 8.34000015258789)
Descriptors: MW 316.79, ALogP 2.85, HBA 4, HBD 2, PSA 66.91, QED 0.91
Compound (SMILES): Cc1nnc(C(C)(CC2COC2)NC(=O)c2cc(OCC(F)(F)F)c(C3CC3)cn2)o1
InChIKey: GRCCRCIRSYGQEF-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 10 s at 730 (bandwidth 30 nm) or 645 nm (bandwidth 75 nm), respectively. The FRET signal is calculated as follows: FRET=T730-Alexa730-P(T645-B645) with P=Ru730-B730/Ru645-B645, where T730 is the test well measured at 730 nM, T645 is the test well measured at 645 nm, B730 and B645 are the buffer controls at 730 nm and 645 nm, respectively. cAMP content is determined from the function of a standard curve spanning from 10 μM to 0.13 nM cAMP.
Measurement: EC50 > 10000 nM (pChEMBL 5)
Descriptors: MW 440.42, ALogP 3.27, HBA 7, HBD 1, PSA 99.37, QED 0.67
Compound (SMILES): CC1(C)[C@@H]2CC[C@@]1(C)[C@@H](NC(=O)[C@H](CC1CCCCC1)NS(=O)(=O)N[C@@H](CCCCN)C(=O)O)C2.Cl
InChIKey: SXAUCWSUBICAAS-LZZNFXAASA-N
Target: Cytochrome P450 3A4 (SINGLE PROTEIN), Homo sapiens
UniProt: P08684
Assay: Inhibition of CYP3A4 in human liver microsomes using testosterone as substrate assessed as suppression of 6beta-hydroxytestosterone formation after 30 mins by LC-MS/MS analysis
Measurement: IC50 > 30000 nM (pChEMBL 4.5229997634887695)
Descriptors: MW 514.73, ALogP 2.66, HBA 5, HBD 5, PSA 150.62, QED 0.24
Compound (SMILES): CC1(C)C(=O)N(c2ccc(C#N)c(C(F)(F)F)c2)C(=S)N1c1ccc(C(=O)NCCCn2cccc(O)c2=O)c(F)c1
InChIKey: FHOQVZJRWRBYOV-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 > 100000 nM (pChEMBL 4)
Descriptors: MW 601.58, ALogP 4.32, HBA 7, HBD 2, PSA 118.67, QED 0.24
Compound (SMILES): CN(C)C1(c2ccccc2)CCC(c2ccccc2)(N(C)C(=O)c2ccn(C)n2)CC1
InChIKey: IPUHZZJQIGQBOQ-UHFFFAOYSA-N
Target: Nociceptin receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P41146
Assay: Binding Assay: The compounds were examined with membranes of recombinant CHO-ORL 1 cells in a receptor binding assay with 3H-nociceptin/orphanin FQ. This test system was conducted in accordance with the method outlined by Ardati et al. (Mol. Pharmacol., 51, 1997, pp. 816-824). The concentration of 3H-nociceptin/orphanin FQ amounted to 0.5 nM in these tests. The binding assays were conducted in each case on 20 ug of membrane protein per 200ul of preparation in 50 mM of HEPES, pH 7.4, 10 nM of MgCl2 and 1 mM of EDTA. The binding to the ORL 1-receptor was determined using 1 mg of WGA-SPA beads (Amersham-Pharmacia, Freiburg) in each case by incubating the preparation for one hour at RT and then conducting measurements in the Trilux scintillation counter (Wallac, Finland).
Measurement: Ki = 235 nM (pChEMBL 6.630000114440918)
Descriptors: MW 416.57, ALogP 4.42, HBA 4, HBD 0, PSA 41.37, QED 0.62
Compound (SMILES): C=CC(=O)Nc1c(C)cccc1Nc1cc(N(C)C(=O)Nc2c(Cl)c(OC)cc(OC)c2Cl)ncn1
InChIKey: WNUARQDAEXHHFR-UHFFFAOYSA-N
Target: Fibroblast growth factor receptor 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P11362
Assay: Kinase HotSpotâ¿¢ Assay: Compounds were profiled for FGFR inhibition activity at Reaction Biology Corporation (Malvern, Pa.) with their Kinase HotSpot assay. See, Anastassiadis et al., 2011, Comprehensive assay of kinase catalytic activity reveals features of kinase inhibitor selectivity. Nat Biotechnol 29, 1039-1045.Recombinant FGFR1 (2.5 nM), FGFR2 (1 nM), FGFR3 (5 nM), or FGFR4 (12 nM) (Invitrogen) was prepared as a mixture with substrate KKKSPGEYVNIEFG (SEQ ID NO:1) (20 uM, FGFR1 substrate); and Poly [E,Y]4:1 (0.2 mg/ml, FGFR2,3,4 substrate)] in kinase reaction buffer (20 mM HEPES-HCl, pH 7.5, 10 mM MgCl2, 2 mM MnCl2, 1 mM EGTA, 0.02% Brij35, 0.1 mM Na3VO4, 0.02 mg/ml BSA, 2 mM DTT, and 1% DMSO). Compound was added to the enzyme/substrate mixture using acoustic technology (Labcyte Echo 550, Sunnyvale, Calif.) (see, Olechno et al., 2006, Improving IC50 results with acoustic droplet ejection. JALA 11, 240-246) and pre-incubated for 0, 15, or 60 minutes at room temperature. After compound pre-incubation, a mixture of ATP (Sigma-Aldrich) and 33P-γ-ATP (PerkinElmer) was added to a final concentration of 10 uM to initiate kinase reactions. Reactions were incubated for 120 minutes at room temperature and then spotted onto Whatman P81 ion exchange filter paper. Unbound phosphate was removed by extensively washing filters in 0.75% phosphoric acid. See, Anastassiadis et al., 2011, Comprehensive assay of kinase catalytic activity reveals features of kinase inhibitor selectivity. Nat Biotechnol 29, 1039-1045.
Measurement: IC50 = 6475 nM (pChEMBL 5.190000057220459)
Descriptors: MW 531.4, ALogP 5.65, HBA 7, HBD 3, PSA 117.71, QED 0.32
Compound (SMILES): Nc1ccccc1NC(=O)CCCCCNC(=O)c1cccc(Cl)c1
InChIKey: DVCBTEJLIHVUGC-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 = 400 nM (pChEMBL 6.400000095367432)
Descriptors: MW 359.86, ALogP 3.85, HBA 3, HBD 3, PSA 84.22, QED 0.49
Compound (SMILES): COc1cc2c(Nc3ccc(Cl)c(Cl)c3)ncnc2cc1OCC1CN(C(=O)c2cc(F)ccc2F)CCO1
InChIKey: QPCXTIGWORLPNS-UHFFFAOYSA-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 575.4, ALogP 5.89, HBA 7, HBD 1, PSA 85.81, QED 0.29
Compound (SMILES): COc1cc(OC)cc(N(CCN2CCOCC2)c2ccc3ncc(-c4cnn(C)c4)nc3c2)c1.Cl
InChIKey: IOZOSZRZSAEJGK-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 > 10000 nM (pChEMBL 5)
Descriptors: MW 474.57, ALogP 3.52, HBA 9, HBD 0, PSA 77.77, QED 0.38
Compound (SMILES): CSCC[C@H](NC(=O)[C@H](CC(C)C)NC(=O)[C@H](Cc1cnc[nH]1)NC(=O)CNC(=O)[C@@H](NC(=O)[C@H](C)NC(=O)[C@H](Cc1c[nH]c2ccccc12)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@H](Cc1cccc2ccccc12)NC(=O)[C@H](CC1CCCCC1)NC(=O)CN1CCN(CC(=O)O)CCN(CC(=O)O)CCN(CC(=O)O)CC1)C(C)C)C(N)=O
InChIKey: PTOWMYSEGATBAG-NNRBTSICSA-N
Target: Neuromedin-B receptor (SINGLE PROTEIN), Rattus norvegicus
UniProt: P24053
Assay: Displacement of [125I]-NMB from NMB receptor in rat C6 cells
Measurement: IC50 = 66.5 nM (pChEMBL 7.179999828338623)
Descriptors: MW 1677.01
Compound (SMILES): CCNC(=O)N(C)C1(c2ccccc2)CCC(c2ccccc2)(N(C)C)CC1
InChIKey: ULIIEFZWMMPAHZ-UHFFFAOYSA-N
Target: Nociceptin receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P41146
Assay: Binding Assay: The compounds were examined with membranes of recombinant CHO-ORL 1 cells in a receptor binding assay with 3H-nociceptin/orphanin FQ. This test system was conducted in accordance with the method outlined by Ardati et al. (Mol. Pharmacol., 51, 1997, pp. 816-824). The concentration of 3H-nociceptin/orphanin FQ amounted to 0.5 nM in these tests. The binding assays were conducted in each case on 20 ug of membrane protein per 200ul of preparation in 50 mM of HEPES, pH 7.4, 10 nM of MgCl2 and 1 mM of EDTA. The binding to the ORL 1-receptor was determined using 1 mg of WGA-SPA beads (Amersham-Pharmacia, Freiburg) in each case by incubating the preparation for one hour at RT and then conducting measurements in the Trilux scintillation counter (Wallac, Finland).
Measurement: Ki = 51 nM (pChEMBL 7.289999961853027)
Descriptors: MW 379.55, ALogP 4.57, HBA 2, HBD 1, PSA 35.58, QED 0.83
Compound (SMILES): CNC(=N)NCCC[C@H](NC(=O)[C@H](CC(C)C)NC(=O)NNC(=O)[C@H](Cc1ccccc1)NC(=O)[C@@H](NC(=O)[C@H](CC(N)=O)NC(=O)[C@H]1CCCN(C(=O)[C@@H](Cc2ccc(O)cc2)NC(C)=O)C1)[C@@H](C)O)C(=O)N[C@@H](Cc1c[nH]c2ccccc12)C(N)=O
InChIKey: AQWHKHMLNBUPJS-NZAHRQIBSA-N
Target: KiSS-1 receptor (SINGLE PROTEIN), Rattus norvegicus
UniProt: Q924U1
Assay: Agonist activity at rat KISS1R expressed in CHO/dhfr cells assessed as increase in intracellular Ca2+ levels measured for 180 secs by Fluo 3-AM dye based FLIPR assay
Measurement: EC50 = 45 nM (pChEMBL 7.349999904632568)
Descriptors: MW 1223.4
Compound (SMILES): CC#CCCN
InChIKey: IFGQNSAUMOFJNS-UHFFFAOYSA-N
Target: Amine oxidase [copper-containing] 3 (SINGLE PROTEIN), Bos taurus
UniProt: Q9TTK6
Assay: Inhibition Assay: In the following Examples, 0.9 mL aliquots of a solution of candidate inhibitor in 100 mM potassium phosphate buffer, pH 7.2 was mixed bovine plasma amine oxidase (0.1 mL, about 8 AM) and incubated at 30° C. aerobically. Aliquots (0.1 mL) were periodically withdrawn using disposable calibrated Drummond micropipettes and diluted with 1.0 mL of benzylamine (5 mM in 50 mM sodium phosphate buffer, pH 7.2) in a 1 cm quartz cuvette (1.5 mL volume). The rate of oxidation of benzylamine to benzaldehyde was measured by recording the increase in absorbance at 250 nm for 1 minute and compared to the rate of benzylamine oxidation in a companion control solution of enzyme without inhibitor. The concentration of active BPAO was estimated from the rate of benzylamine oxidation. Inhibitory potency values (IC50 values) and partition values for inactivation of BPAO were measured at various times as in indicated in the Tables.
Measurement: IC50 = 10000 nM (pChEMBL 5)
Descriptors: MW 83.13, ALogP 0.36, HBA 1, HBD 1, PSA 26.02, QED 0.45
Compound (SMILES): Cl.O=C(O)C1CN(C(c2ccccc2)c2ccccc2)CCN1C(=O)CC(c1ccccc1)c1ccccc1
InChIKey: HYQGWLLAKFRPTI-UHFFFAOYSA-N
Target: Voltage-dependent L-type calcium channel alpha1C/alpha2delta/beta1b (PROTEIN COMPLEX), Homo sapiens
UniProt: Q02641
Assay: Antagonist activity at alpha1C/alpha2delta/beta1b L-type calcium channel (unknown origin) expressed in HEK293 cells assessed as 0.1 Hz current frequency dependent channel blocking for 2 to 3 mins in presence of Ba2+ by whole-cell patch clamp assay
Measurement: IC50 = 37140 nM (pChEMBL 4.429999828338623)
Descriptors: MW 504.63, ALogP 5.6, HBA 3, HBD 1, PSA 60.85, QED 0.34
Compound (SMILES): C[C@@H](CO)Nc1cc(C(N)=O)nc(-c2ccc(Oc3ccc(F)cc3)cc2)n1
InChIKey: ATRLVPLKGMTSHY-LBPRGKRZSA-N
Target: Sodium channel protein type 9 subunit alpha (SINGLE PROTEIN), Homo sapiens
UniProt: Q15858
Assay: Electrophysiology Assay: For electrophysiological recordings the external solution was either standard, DMEM supplemented with 10 mM HEPES (pH adjusted to 7.34 with NaOH and the osmolarity adjusted to 320) or Tyrodes salt solution (Sigma, USA) supplemented with 10 mM HEPES (pH adjusted to 7.4 with NaOH; osmolarity=320). The internal pipette solution contained (in mM): NaCl (10), CsF (140), CaCl2 (1), MgCl2 (5), EGTA (11), HEPES (10: pH 7.4, 305 mOsm). Compounds were prepared first as a series of stock solutions in DMSO and then dissolved in external solution; DMSO content in final dilutions did not exceed 0.3%. At this concentration, DMSO did not affect sodium currents. Vehicle solution used to establish base line was also contacting 0.3% DMSO.
Measurement: Ki = 24 nM (pChEMBL 7.619999885559082)
Descriptors: MW 382.4, ALogP 2.97, HBA 6, HBD 3, PSA 110.36, QED 0.58
Compound (SMILES): CCc1ccc(OCCCCOc2c3ccoc3cc3oc(=O)ccc23)cc1
InChIKey: AYMOUKKEXWPUMU-UHFFFAOYSA-N
Target: Potassium voltage-gated channel subfamily A member 5 (SINGLE PROTEIN), Homo sapiens
UniProt: P22460
Assay: Inhibition of Kv1.5 (unknown origin)
Measurement: EC50 = 52 nM (pChEMBL 7.28000020980835)
Descriptors: MW 378.42, ALogP 5.34, HBA 5, HBD 0, PSA 61.81, QED 0.31
Compound (SMILES): Cc1noc(C)c1-c1cc(C(O)(c2ccccn2)c2ccccn2)c2nc(C3CC3)[nH]c2c1
InChIKey: CMSUJGUHYXQSOK-UHFFFAOYSA-N
Target: Bromodomain-containing protein 4 (SINGLE PROTEIN), Homo sapiens
UniProt: O60885
Assay: Ligand KI Assay: The labeled ligand specifically binds BRD4-1 and BRD4-2 and can be displaced by a small molecule inhibitor that shares a similar or overlapping binding site. BRD4-I and BRD4-2 were expressed and purified from Escherichia coli as N-terminal His6, tagged proteins. A Eu-cryptate labeled anti-His antibody (Perkin Elmer) was used to specifically bind BRD4. Binding of BRD4 to the labeled probe/ligand resulted in an increase in FRET signal whereas displacement of this labeled ligand from BRD4 with a small molecule inhibitor resulted in a decrease in FRET signal. Assays were performed in 50 mM Hepes (pH 7.5), 150 mM NaCl, 0.1 mg/ml BSA, 0.01% (v/v) Brij, 0.5% (v/v) DMSO and 10 nM labeled ligand at the following concentrations for each BRD4 isoform: 2 nM BRD4-1 and 0.5 nM BRD4-2. After an assay reaction time of 60 minutes at 25° C., binding was measured with 2 nM Eu-cryptate labeled anti-His antibody. TR-FRET signal was detected on an Envision plate reader (Ex: 320 nm; Em: 615/665 nm; 100 us delay and 200 us read window). Data were normalized based on a positive (2 uM I-BET) and negative (DMSO) controls and IC50 values were calculated from the fit of the dose-response curves to a four-parameter equation. All IC50 values represent geometric mean values of a minimum of four determinations. The IC50 values were converted to Ki values (dissociation constant for BRD4-inhibitor complex) using the Cheng and Prusoff equation for a competitive inhibitor mode of action.
Measurement: IC50 = 11.7 nM (pChEMBL 7.929999828338623)
Descriptors: MW 437.5, ALogP 4.79, HBA 6, HBD 2, PSA 100.72, QED 0.41
Compound (SMILES): CN1C(=O)N(c2cccc(C(F)(F)F)c2)C2=C(C(=O)CC2)C1c1ccc(C#N)cn1
InChIKey: YNXCSNKOJBRMIV-UHFFFAOYSA-N
Target: Neutrophil elastase (SINGLE PROTEIN), Homo sapiens
UniProt: P08246
Assay: Neutrophil Elastase Assay: Materials: Human neutrophil elastase was purchased from Calbiochem (Cat. No.: 324681) and the elastase substrate MeOSuc-Ala-Ala-Pro-Val-AMC from Bachem (Cat. No.: I-1270). All other materials were of the highest grade commercially available. 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 uL of these compound dilutions were mixed with 10 ul 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 uL substrate solution in assay buffer were added (250 uM 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. Each plate contains wells with a high value control (DMSO+enzyme+substrate) and wells with a low value control (DMSO+inactivated enzyme+substrate). IC50 values were estimated using a sigmoidal concentration response curve with variable slope.
Measurement: IC50 = 26.8 nM (pChEMBL 7.570000171661377)
Descriptors: MW 412.37, ALogP 4.2, HBA 4, HBD 0, PSA 77.3, QED 0.74
Compound (SMILES): CSc1ccc(CNC(=O)c2sc3nnc(C)c(C)c3c2N)cc1
InChIKey: RGOMECWMAXEGDR-UHFFFAOYSA-N
Target: Muscarinic acetylcholine receptor M4 (SINGLE PROTEIN), Homo sapiens
UniProt: P08173
Assay: Positive allosteric modulation of human M4 receptor expressed in CHO cells co-expressing Gqui5 by calcium mobilization assay
Measurement: EC50 = 60.2 nM (pChEMBL 7.21999979019165)
Descriptors: MW 358.49, ALogP 3.54, HBA 6, HBD 2, PSA 80.9, QED 0.7
Compound (SMILES): Cl.NC(N)=N/N=C/c1ccc(Cl)c(Cl)c1
InChIKey: XWTMACVBCUKXFY-GAYQJXMFSA-N
Target: Neuropeptide FF receptor 2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9Y5X5
Assay: Agonist activity at NPFFR2 (unknown origin) expressed in mouse NIH3T3 cells by receptor selection and amplification technology assay
Measurement: EC50 = 794.33 nM (pChEMBL 6.099999904632568)
Descriptors: MW 231.09, ALogP 1.81, HBA 2, HBD 3, PSA 74.26, QED 0.41
Compound (SMILES): N#CCC1(n2cc(-c3c(F)cnc4[nH]ccc34)cn2)CN(C2CCN(C(=O)c3ccc(C#N)cc3F)CC2)C1
InChIKey: WIQUSYCVUZBNNO-UHFFFAOYSA-N
Target: Tyrosine-protein kinase JAK2 (SINGLE PROTEIN), Homo sapiens
UniProt: O60674
Assay: In Vitro JAK Kinase Assay: Compounds herein were tested for inhibitory activity of JAK targets according to the following in vitro assay described in Park et al., Analytical Biochemistry 1999, 269, 94-104. The catalytic domains of human JAK1 (a.a. 837-1142), Jak2 (a.a. 828-1132) and Jak3 (a.a. 781-1124) with an N-terminal His tag were expressed using baculovirus in insect cells and purified. The catalytic activity of JAK1, JAK2 or JAK3 was assayed by measuring the phosphorylation of a biotinylated peptide. The phosphorylated peptide was detected by homogenous time resolved fluorescence (HTRF). IC50s of compounds were measured for each kinase in the 40 microL reactions that contain the enzyme, ATP and 500 nM peptide in 50 mM Tris (pH 7.8) buffer with 100 mM NaCl, 5 mM DTT, and 0.1 mg/mL (0.01%) BSA. For the 1 mM IC50 measurements, ATP concentration in the reactions was 1 mM. Reactions were carried out at room temperature for 1 hr and then stopped with 20 μL 45 mM EDTA, 300 nM SA-APC, 6 nM Eu-Py20 in assay buffer (Perkin Elmer, Boston, Mass.). Binding to the Europium labeled antibody took place for 40 minutes and HTRF signal was measured on a Fusion plate reader (Perkin Elmer, Boston, Mass.).
Measurement: IC50 = 12.5 nM (pChEMBL 7.900000095367432)
Descriptors: MW 526.55, ALogP 3.81, HBA 7, HBD 1, PSA 117.63, QED 0.42
Compound (SMILES): CCc1c(N)ncnc1N1CCC(c2nc(-c3cccc(C(F)(F)F)c3)cn2CCN2CCCC2)CC1
InChIKey: KAWNIHAFIANXQO-UHFFFAOYSA-N
Target: RAC-alpha serine/threonine-protein kinase (SINGLE PROTEIN), Homo sapiens
UniProt: P31749
Assay: AKT Enzyme Assay: A TTP Mosquito liquid handling instrument is used to place 125 nl of the appropriate concentration of inhibitor in 100% DMSO (for a dose response curve calculation) into each well of a 384-well plate. To this reaction components are added to a final volume of 12.5 .mu.l: 0.1 ng/.mu.l His-AKT (Full Length), (Invitrogen, Part # P2999, Lot #641228C). 160 uM ATP (Fluka, 02055) 1 mM DTT (Sigma, D0632) 1 mM MgCl2 (Sigma, M1028) 1 .mu.M substrate peptide (sequence FITC-AHA-GRPRTSSFAEG-NH2), synthesized by Tufts Peptide Synthesis service. 100 mM HEPES pH 7.5 (Calbiochem, 391338)0.015% Brij-35 (Sigma, B4184)The reaction is incubated for 90 min at 25 C, and then stopped by the addition of 70 .mu.l of Stop buffer (100 mM HEPES pH 7.5, 0.015% Brij-35, 10 mM EDTA (Sigma, E7889)).The plate is read on a Caliper LC 3000 in an Off-Chip mobility shift assay format, using the following parameters for a 12-sipper chip: screening pressure -2.3 psi, upstream voltage -500, and downstream voltage -3000.
Measurement: IC50 = 9.6 nM (pChEMBL 8.020000457763672)
Descriptors: MW 513.61, ALogP 4.98, HBA 7, HBD 1, PSA 76.1, QED 0.48
Compound (SMILES): COc1cc(Nc2ncnc3ccc(-c4cn(CCCCCCC(=O)NO)nn4)cc23)ccc1Oc1ccccc1
InChIKey: NCUZLDZWYWPMHU-UHFFFAOYSA-N
Target: Histone deacetylase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q13547
Assay: Inhibition of HDAC1 (unknown origin) using acetylated peptide substrate preincubated for 15 mins followed by substrate addition measured after 60 mins by fluorescence analysis
Measurement: IC50 = 12.8 nM (pChEMBL 7.889999866485596)
Descriptors: MW 553.62, ALogP 5.89, HBA 10, HBD 3, PSA 136.31, QED 0.09
Compound (SMILES): O=C(Nc1ccc(OC(F)(F)F)cc1)c1cnc(N2CCOC(CO)C2)c(-c2ccn[nH]2)c1
InChIKey: CGBWPZREDCDGKW-UHFFFAOYSA-N
Target: Tyrosine-protein kinase ABL1 (SINGLE PROTEIN), Homo sapiens
UniProt: P00519
Assay: Caliper Assay: The assay plates were prepared by addition of 50 nL per well of compound solution in 90% DMSO. The kinase reactions were started by stepwise addition of 4.5 μL per well of peptide/ATP-solution (50 mM HEPES, pH 7.5, 1 mM DTT, 0.02% BSA, 0.6% DMSO, 10 mM beta-glycerophosphate, and 10 μM sodium orthovanadate, 20 mM MgCl2, 2 mM MnCl2, 4 μM ATP, 4 μM peptide (FITC-Ahx-EAIYAAPFAKKK-NH2)) and 4.5 μL per well of enzyme solution (50 mM HEPES, pH 7.5, 1 mM DTT, 0.02% BSA, 0.6% DMSO, 10 mM beta-glycerophosphate, and 10 μM sodium orthovanadate, 20 mM MgCl2, 2 mM MnCl2, 3.5 nM ABL (ABL(64-515), produced in-house from E. coli)). Kinase reactions were incubated at 30° C. for 60 minutes and subsequently terminated by addition of 16 μL per well of stop solution (100 mM HEPES pH 7.5, 5% DMSO, 0.1% Caliper coating reagent, 10 mM EDTA, and 0.015% Brij35). Plates with terminated kinase reactions were transferred to the Caliper LC3000 workstations for reading. Phosphorylated and unphosphorylated peptides were separated using the Caliper microfluidic mobility shift technology. Briefly, samples from terminated kinase reactions were applied to the chip. Analytes are transported through the chip by constant buffer flow and the migration of the substrate peptide is monitored by the fluorescence signal of its label. Phosphorylated peptide (product) and unphosphorylated peptide (substrate) are separated in an electric field by their charge/mass ratio.
Measurement: IC50 = 8.1 nM (pChEMBL 8.09000015258789)
Descriptors: MW 463.42, ALogP 2.82, HBA 7, HBD 3, PSA 112.6, QED 0.52
Compound (SMILES): CN1CCC(n2cc(Nc3ncc(Cl)c(CCc4ccccc4C4(C(N)=O)CC4)n3)cn2)CC1
InChIKey: GZQZGOLXVRSKEE-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 = 5 nM (pChEMBL 8.300000190734863)
Descriptors: MW 480.02, ALogP 3.64, HBA 7, HBD 2, PSA 101.96, QED 0.51
Compound (SMILES): COc1cc(OC)cc(N(CC#Cc2nccc(OC(C)C)n2)c2ccc3ncc(-c4cnn(C)c4)nc3c2)c1
InChIKey: QHWNOTNFJRKXDT-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 > 10000 nM (pChEMBL 5)
Descriptors: MW 535.61, ALogP 4.81, HBA 10, HBD 0, PSA 100.31, QED 0.26
Compound (SMILES): CC1(N)CCN(c2cccnc2NC(=O)c2nc(-c3ncccc3C(F)(F)F)cnc2N)CC1
InChIKey: XXJXHXJWQSCNPX-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 = 0.4 nM (pChEMBL 9.399999618530273)
Descriptors: MW 472.48, ALogP 3.1, HBA 8, HBD 3, PSA 135.94, QED 0.53
Compound (SMILES): CN(C)[C@H]1Cc2c(Cl)cc(Cl)cc2[C@@H]1Oc1ccc(S(=O)(=O)NCCOCCOCCNC(=O)C(C)(C)C(=O)NCCOCCOCCNS(=O)(=O)c2ccc(O[C@H]3c4cc(Cl)cc(Cl)c4C[C@@H]3N(C)C)cc2)cc1
InChIKey: VYNJQGSVUMGYJX-KVXOYYPDSA-N
Target: Sodium/hydrogen exchanger 3 (SINGLE PROTEIN), Homo sapiens
UniProt: P48764
Assay: Inhibition of human NHE3-mediated sodium-dependant hydrogen ion antiport in Opossum kidney cells preincubated in NH4Cl-HEPES buffer containing BCECF-AM dye followed by washout with ammonium free HEPES and subsequent addition of compound in presence of NHE1 antagonist EIPA by fluorescence assay
Measurement: IC50 = 31.62 nM (pChEMBL 7.5)
Descriptors: MW 1161.06
Compound (SMILES): O=C(O)CCC[C@@H]1CC[C@@H]2[C@@H](/C=C/[C@@H](O)COc3ccccc3)[C@H](O)C[C@@H]2O1
InChIKey: FTBNLUSUCXCIGD-OVCPVMRKSA-N
Target: Prostaglandin E2 receptor EP3 subtype (SINGLE PROTEIN), Homo sapiens
UniProt: P43115
Assay: Agonist activity at human EP3 receptor expressed in CHO cells assessed as increase in intracellular calcium level by Fura 2-AM dye based fluorescence assay
Measurement: EC50 = 4.1 nM (pChEMBL 8.390000343322754)
Descriptors: MW 390.48, ALogP 2.78, HBA 5, HBD 3, PSA 96.22, QED 0.56
Compound (SMILES): COc1cc(N2CCN(C(C)=O)CC2)ccc1Nc1ncc(Br)c(Nc2ccc(N3CCN(C(C)=O)CC3)cc2OC)n1
InChIKey: QFCVPFUQKUFBSV-UHFFFAOYSA-N
Target: ALK tyrosine kinase receptor (SINGLE PROTEIN), Homo sapiens
UniProt: Q9UM73
Assay: Homogeneous Time-resolved fluorescence (HTRF) Assay: In order to measure the inhibitory activity against ALK, a Grainer 96-well round type bottom plate was added with the compounds (2 μl) prepared in Examples 1 to 89, and mixed with ALK enzyme (1 ul) and a peptide substrate (2 μl) with biotin attached thereto for 15 minutes and cultured thereafter. Here, ATP solution (5 μl) was added thereto and kinase reaction was performed at room temperature for 30 minutes. XL 665 (5 μl), to which Streptavidin dissolved in ethylenediaminetetraacetic acid solution was attached, anti-phosphotyrosine antibody (5 μl), to which europium (Eu3+) was attached, were added to a reaction solution to stop the reaction, cultured for 1 hour, and analyzed using Homogeneous Time-resolved fluorescence (HTRF, Cisbio). The result was read by a Wallac Envision 2103 device at the wavelength range of 615/665 nm.
Measurement: IC50 = 86 nM (pChEMBL 7.070000171661377)
Descriptors: MW 653.58, ALogP 4.08, HBA 10, HBD 2, PSA 115.4, QED 0.37
Compound (SMILES): COc1cc(OC)cc(N(CCCN2CCC[C@H]2CO)c2ccc3ncc(-c4cnn(C)c4)nc3c2)c1.Cl
InChIKey: QVPOZCFIENYNSR-FTBISJDPSA-N
Target: Vascular endothelial growth factor receptor 2 (SINGLE PROTEIN), Homo sapiens
UniProt: P35968
Assay: Inhibition of recombinant human N-terminal His6-tagged VEGFR2 (790 end residues) expressed in baculovirus infected insect Sf21 cells using Btn-Flt3 as substrate after 1 hr by time resolved fluorescence assay
Measurement: IC50 = 30.2 nM (pChEMBL 7.519999980926514)
Descriptors: MW 502.62, ALogP 4.03, HBA 9, HBD 1, PSA 88.77, QED 0.35
Compound (SMILES): COc1cccc2c1ncc1c(=O)n(-c3cccc(SC)c3)c(=O)n(C3CCCC3)c12
InChIKey: ZWHJGDGKNAKKAK-UHFFFAOYSA-N
Target: Lipopolysaccharide-induced tumor necrosis factor-alpha factor (SINGLE PROTEIN), Homo sapiens
UniProt: Q99732
Assay: TNF inhibition assay: Samples were tested for TNF as per the manufacturer's instructions (TNF ELISA; R&D Systems, Cat #DY210). The samples were serially diluted with PBS+1% BSA to the appropriate dilution condition for the cell prep, typically a 1:4 or 1:6 dilution.
Measurement: IC50 = 74 nM (pChEMBL 7.130000114440918)
Descriptors: MW 433.53, ALogP 4.55, HBA 7, HBD 0, PSA 66.12, QED 0.35
Compound (SMILES): CN1C(=N)N[C@](C)(c2sc(-c3nnc(-c4ncccn4)o3)cc2Cl)CS1(=O)=O
InChIKey: YVXLHYGRGOHSRH-HNNXBMFYSA-N
Target: Beta-secretase 2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9Y5Z0
Assay: Time-Resolved Endpoint Proteolysis Assay: Inhibitor compounds, prepared at 3× the desired final concentration in 1×BACE assay buffer (20 mM sodium acetate pH 5.0, 10% glycerol, 0.1% Brij-35) supplemented with 7.5% DMSO are pre-incubated with an equal volume of autoBACE-2 enzyme diluted in 1×BACE assay buffer (final enzyme concentration 1 nM) in black 384-well NUNC plates for 30 minutes at 30° C. The assay is initiated by addition of an equal volume of the QSY7-EISEVNLDAEFC-Eu-amide substrate (200 nM final concentration, Km=8 μM for 4 μM for autoBACE-2) prepared in 1×BACE assay buffer supplemented with 7.5% DMSO and incubated for 90 minutes at 30° C. DMSO is present at 5% final concentration in the assay. Following laser excitation of sample wells at 320 nm, the fluorescence signal at 620 nm is collected for 400 ms following a 50 μs delay on a RUBYstar HTRF plate reader (BMG Labtechnologies).
Measurement: Ki = 104.5 nM (pChEMBL 6.980000019073486)
Descriptors: MW 439.91, ALogP 1.92, HBA 9, HBD 2, PSA 137.96, QED 0.63
Compound (SMILES): CC(C)(C)Cn1cc(-c2ccc(N3CCc4cccc(C(=O)Nc5nc6ccccc6s5)c4C3)nc2C(=O)O)cn1
InChIKey: LCCKVNXYMGVDQL-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 566.69, ALogP 6.11, HBA 8, HBD 2, PSA 113.24, QED 0.25
Compound (SMILES): Cc1nc(-c2cccnc2)sc1C(=O)Nc1cn2nc(-c3c(-c4ccc(F)cc4)nn(C)c3C3CCN(C)CC3)ccc2n1
InChIKey: CKGPDAVXBRCHHB-UHFFFAOYSA-N
Target: Casein kinase I isoform delta (SINGLE PROTEIN), Homo sapiens
UniProt: P48730
Assay: Kinase Assay: The kinase assay was performed in V-bottom 384-well plates. The final assay volume was 30 ul prepared from 15 ul additions of enzyme, substrates (fluoresceinated peptide FL-AHA-KRRRAL-PSER-VASLPGL-OH and ATP) and test compounds in assay buffer (20 mM HEPES pH 7.4, 30 mM MgCl2, 0.015% Brij35 and 4 mM DTT). The reaction was incubated at room temperature for 22 hours and terminated by adding 45 ul of 35 mM EDTA to each sample. The reaction mixture was analyzed on the Caliper LABCHIPÂ®3000 (Caliper, Hopkinton, Mass.) by electrophoretic separation of the unphosphorylated substrate and phosphorylated product Inhibition data were calculated by comparison of the no enzyme control reactions for 100% inhibition and vehicle-only reactions for 0% inhibition. The final concentration of reagents in the assay were 200 pM CK1ε or CK1δ, 50 uM ATP, 1.5 uM FL-AHA-KRRRAL-PSER-VASLPGL-OH, and 1.6% DMSO. Dose response curves were generated to determine the concentration required to inhibit 50% of the kinase activity (IC50). Compounds were dissolved at 10 mM in dimethylsulfoxide (DMSO) and evaluated at eleven concentrations. IC50 values were derived by non-linear regression analysis.
Measurement: IC50 = 3 nM (pChEMBL 8.520000457763672)
Descriptors: MW 607.72, ALogP 5.82, HBA 10, HBD 1, PSA 106.13, QED 0.25
Compound (SMILES): CC(OC(=O)N[C@H]1C(=O)O[C@H]1C)c1ccc(-c2ccccc2)cc1
InChIKey: IVXYXOKUGHRSBD-PMBLOPFUSA-N
Target: N-acylethanolamine-hydrolyzing acid amidase (SINGLE PROTEIN), Homo sapiens
UniProt: Q02083
Assay: UPLC/MS Assay: NAAA protein preparation (10 ug) was pre-incubated with various concentrations of test compound or vehicle control in 100 mM NaH2PO4, 100 mM Tri Sodium Citrate Dehydrate, 0.1% Triton-X 100, 3 mM DTT, pH 4.5 for 30 min at 37° C. Duplicate samples were then incubated with 50 uM C17:1 10-cis-heptadecenoylethanolamide (Avanti Polar Lipids, Alabaster, Ala. USA) at 37° C. for 30 minutes. The reaction was terminated by the addition of 0.2 mL of cold methanol containing 1 nmol of heptadecanoic acid (NuChek Prep, Elysian, Minn. USA) as internal standard. Samples were then analyzed by UPLC/MS. Heptadecenoic and heptadecanoic acids were eluted on an Acquity UPLC BEH C18 column (50 mm length, 2.1 mm i.d., 1.7 um pore size, Waters) isocratically at 0.5 mL/min for 1.5 min with a solvent mixture of 95% methanol and 5% water, both containing 0.25% Acetic Acid and 5 mM Ammonium Acetate. The column temperature was 40° C. Electrospray ionization was in the negative mode, capillary voltage was 0.5 kV, cone voltage was 25 kV, desolvation temperature was 500° C. N2 was used as drying gas at a flow rate of 1000 L/hour and a temperature of 500° C. The [M-H]- ion was monitored in the selected-ion monitoring mode (m/z values: heptadecenoic acid 267.37, heptadecanoic acid 269.37). Calibration curves were generated using commercial heptadecenoic acid (NuCheck Prep). Inhibition of NAAA activity was calculated as reduction of heptadecenoic acid in the samples compared to vehicle controls. IC50 values were calculated by non-linear regression analysis of log [concentration]/inhibition curves using GraphPad Prism 5 (GraphPad Software Inc., CA-USA) applying a standard slope curve fitting.
Measurement: IC50 = 10 nM (pChEMBL 8)
Descriptors: MW 325.36, ALogP 3.45, HBA 4, HBD 1, PSA 64.63, QED 0.87
Compound (SMILES): Cc1cc(N=S2(=O)CCCC2)cc2ncnc(Nc3ccc(F)cc3OC(CF)CF)c12
InChIKey: MOUJAAFOCZZGAI-UHFFFAOYSA-N
Target: MAP kinase-interacting serine/threonine-protein kinase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9BUB5
Assay: In vitro Kinase Assay: The Z-LYTE biochemical assay employs a fluorescence-based, coupled-enzyme format and is based on the differential sensitivity of phosphorylated and non-phosphorylated peptides to proteolytic cleavage. The peptide substrate is labeled with two fluorophores one at each end that make up a FRET pair.In the primary reaction, the kinase transfers the gamma-phosphate of ATP to a single tyrosine, serine or threonine residue in a synthetic FRET-peptide. In the secondary reaction, a site-specific protease recognizes and cleaves non-phosphorylated FRET-peptides. Phosphorylation of FRET-peptides suppresses cleavage by the Development Reagent. Cleavage disrupts FRET between the donor (i.e., coumarin) and acceptor (i.e., fluorescein) fluorophores on the FRET-peptide, whereas uncleaved, phosphorylated FRET-peptides maintain FRET.
Measurement: IC50 = 34 nM (pChEMBL 7.46999979019165)
Descriptors: MW 464.51, ALogP 5.4, HBA 6, HBD 1, PSA 76.47, QED 0.51
Compound (SMILES): CN1CCN(CC(=O)OCc2ccc(CC(SCCc3ccc(F)cc3)C(=O)O)cc2)CC1
InChIKey: UEMBYQGWCHULKT-UHFFFAOYSA-N
Target: Peroxisome proliferator-activated receptor gamma (SINGLE PROTEIN), Homo sapiens
UniProt: P37231
Assay: Reporter Gene Assay: Transactivation Assay Expression vectors were prepared by inserting the ligand binding domain cDNA (complementary DNA) of human PPARalpha (amino acid 168-468) and human PPARgamma (amino acid 205-505), 3′ to and in frame with, the yeast GAL4 transcription factor DNA binding domain and the nuclear localization signal from the T-antigen of Polyoma Virus into the mammalian expression vector pSGS (Stratagene). The resulting expression vectors pSGGAL-PPARalpha and pSGGAL-PPARgamma were used in co-transfection experiments together with a modified pGL3 promoter plasmid (Promega) containing five copies of the UAS GAL4 recognition site. 2.5 μg pSGGAL-PPARalpha or pSGGAL-PPARgamma were mixed with 25 μg pGL3p 1×UAS and 22.5 μg pBluescript (Stratagene) in 0.95 mL ice cold PBS containing between 9-12 million U-2 OS (human osteosarcoma) cells. The cell/DNA mixture was incubated on ice for 5 minutes and then divided between two 0.4 cm cuvettes and electroporated at 960 μF, 2
Measurement: EC50 = 810 nM (pChEMBL 6.090000152587891)
Descriptors: MW 474.6, ALogP 3.09, HBA 6, HBD 1, PSA 70.08, QED 0.5
Compound (SMILES): O=C1COc2ccc(C3(O)c4ccccc4C(=O)N3c3cc(F)cc(F)c3)cc2N1
InChIKey: YMLLEMATODMZJH-UHFFFAOYSA-N
Target: Serine/threonine-protein kinase B-raf (SINGLE PROTEIN), Homo sapiens
UniProt: P15056
Assay: Inhibition of B-Raf (unknown origin) pre-incubated for 30 mins by luciferase-luciferin-coupled chemiluminescence assay
Measurement: IC50 = 1000 nM (pChEMBL 6)
Descriptors: MW 408.36, ALogP 3.15, HBA 4, HBD 2, PSA 78.87, QED 0.68
Compound (SMILES): CCCCc1c(-c2ccc(C(=O)NS(=O)(=O)c3ccc4ccccc4c3)cc2C(=O)N2CCc3ccccc3C2)c(CO)nn1-c1ccc(OCC(O)CO)cc1
InChIKey: IWBPSTXSCXLBAV-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 = 4.96 nM (pChEMBL 8.300000190734863)
Descriptors: MW 788.92, ALogP 5.58, HBA 10, HBD 4, PSA 171.29, QED 0.11
Compound (SMILES): O=S(=O)(Nc1nccs1)c1ccc2c(c1)OCCN2c1ccccc1N1CCNCC1
InChIKey: DTSNBUNQRWNESH-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 = 252 nM (pChEMBL 6.599999904632568)
Descriptors: MW 457.58, ALogP 2.88, HBA 8, HBD 2, PSA 86.8, QED 0.61
Compound (SMILES): CC(C)C(Nc1ccc(F)c([C@]2(C)COCC(N)=N2)c1)c1ccc(Cl)cn1
InChIKey: XUDAQSPLHDYMTP-ANYOKISRSA-N
Target: Beta-secretase 2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9Y5Z0
Assay: Cellular Assay: The assay uses the principle of inhibition of human TMEM27 cleavage by endogenous cellular BACE2 in the Ins1e rat cell line and shedding from the cell surface into the culture medium, followed by detection in an ELISA assay. Inhibition of BACE2 prevents the cleavage and shedding in a dose-dependent mannerThe stable cell line "INS-TMEM27" represents an INS1e-derived cell line with inducible expression (using the TetOn system) of full-length hTMEM27 in a doxycycline-dependent manner. The cells are cultured throughout the experiment in RPMI1640 +Glutamax (Invitrogen) Penicillin/Streptomycin, 10% Fetal bovine serum, 100 mM pyruvate, 5 mM beta-mercatptoethanol, 100 micrograms/ml G418 and 100 microgram/ml hygromycin and are grown inadherent culture at 37° C. in a standard CO2 cell culture incubator.INS-TMEM27 cells are seeded in 96-well plates. After 2 days in culture, BACE2 inhibitor is added in a range of concentrations as required by the assay and after a further two hours, doxycycline i
Measurement: IC50 = 1662 nM (pChEMBL 5.78000020980835)
Descriptors: MW 390.89, ALogP 4.29, HBA 5, HBD 2, PSA 72.53, QED 0.8
Compound (SMILES): COc1cc(N)cnc1Nc1ccc(F)c([C@]2(C(F)F)COCC(N)=N2)c1
InChIKey: BULJFAPFXZXMNJ-KRWDZBQOSA-N
Target: Beta-secretase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P56817
Assay: BACE-1 Inhibition Assay: Recombinant BACE-1 (extracellular domain, expressed in baculovirus and purified using standard methods) at 0.1 to 1 nM concentrations is incubated with the test compound at various concentrations for 1 hour at room temperature in 100 mM acetate buffer, pH 4.5, containing 0.1% CHAPS. Activity was measured using a final concentration of 3 uM of the fluorescence-quenched substrate Q-C(HSO3)-Ile-Asp-Leu-Ala-Val-Leu-Asp-HN-CH2-CH2-Mca, where Q=2-nitro-5-amino benzoic acid and Mca=7-methoxy-4-coumarinyl acetic acid. Catalytic turnover was monitored in a Spectramax Gemini fluorescence plate reader (Molecular Devices) in black 96-well microplates using excitation/emission wavelength of 325 nm and 400 nm, respectively. Fluorescence increase was followed for 15 min, in 1 minute's intervals.
Measurement: IC50 = 3000 nM (pChEMBL 5.519999980926514)
Descriptors: MW 381.36, ALogP 2.4, HBA 7, HBD 3, PSA 107.78, QED 0.73
Compound (SMILES): C[C@@H]1CN(c2sc(C(F)(F)F)nc2-c2nc3ccccc3[nH]2)CCN1C(=O)Cn1cccn1
InChIKey: CRYXEDQPNCNOKN-CYBMUJFWSA-N
Target: C-X-C chemokine receptor type 3 (SINGLE PROTEIN), Homo sapiens
UniProt: P49682
Assay: Antagonist activity at human recombinant CXCR3 expressed in CHO-K1 cells measured after 10 mins in presence of CXCL10 by FLIPR analysis
Measurement: IC50 = 13 nM (pChEMBL 7.889999866485596)
Descriptors: MW 475.5, ALogP 3.64, HBA 7, HBD 1, PSA 82.94, QED 0.49