lovingscience/chembl-v37-raw-activities huggingface dataset:
Compound (SMILES): Cc1cc(Br)cc([C@H](CC(=O)O)NC(=O)CNC(=O)c2cc(O)cc(NC3=NCC(O)CN3)c2)c1
InChIKey: OBOJFQICXTYCRY-IBGZPJMESA-N
Target: Integrin alpha-V/beta-6 (PROTEIN COMPLEX), Homo sapiens
UniProt: P06756
Assay: Solid Phase Receptor Assay (SPRA) for alphaVbeta6: Recombinant human LAP (R&D Systems, 246-LP) diluted to 0.25 μg/mL in TBS+ buffer (25 mM Tris pH 7.4, 137 mM NaCl, 2.7 mM KCl, 1 mM CaCl2, 1 mM MgCl2, 1 mM MnCl2) was added to wells (50 μL/well) of a 96-well half-well transparent microtiter plate (Greiner 675061) and incubated overnight at 4° C. Wells were washed 3 times with 150 μL TBS+, and 150 μL of blocking buffer (TBS+ with 1% bovine serum albumin, Sigma A7906) were added. The plate was incubated for 1 hr at 37° C., and then washed 3× with TBS+ buffer. Recombinant human integrin αvβ6 (R&D Systems, 3817-AV) was diluted to 0.1 μg/mL in TBS+/0.1% bovine serum albumin. Compounds were diluted 1:100 into the integrin solution and then 50 μL added to empty wells of the washed LAP-coated plate according to a standard template with each sample repeated in triplicate. After incubation for two hours at room temperature, the plate was washed 3× with 150 μL of TBS+ buffer. To each well, 50 μL of biotinylated anti-αv antibody (R&D Systems, BAF1219) at 0.5 μg/mL in TBS+/0.1% BSA were added and the plate was covered and incubated for 1 hr at room temperature. After washing the plate 3× with 150 μL of TBS+ buffer, 50 μL of streptavidin-conjugated horseradish peroxidase (R&D Systems, DY998) diluted in TBS+ blocking buffer were added to the wells and the plate incubated for 20 min at room temperature. The plate was washed 3× with TBS+ buffer followed by 50 μL of TMB substrate (Sigma T4444) added to each well and the plate incubated for 20 min at room temperature. Plates were read by colorimetric detection at 650 nm wavelength using a Tecan Safire II plate reader. Concentration-response curves were constructed by non-linear regression (best fit) analysis, and IC50 values were calculated for each compound.
Measurement: IC50 = 0.5 nM (pChEMBL 9.300000190734863)
Descriptors: MW 548.39, ALogP 1.26, HBA 8, HBD 7, PSA 172.38, QED 0.26
Compound (SMILES): CCC([C@@H]1CC1(F)F)n1cc(-c2nc(-c3cnn(C[C@@H](O)CO)c3)cn3nccc23)cn1
InChIKey: HQYCIYSQINFXHM-LNKXUWQBSA-N
Target: Non-receptor tyrosine-protein kinase TYK2 (SINGLE PROTEIN), Homo sapiens
UniProt: P29597
Assay: Tyk2 Inhibition Assay: Compounds of Formula I were screened for their ability to inhibit Tyk2 using the general enzyme inhibition assay method, in which the assay mixture contained 1 mM ATP, 8 μM Omnia® Y12 peptide (Catalog # IVGN KPZ3121C; Invitrogen Corporation, Carlsbad, Calif.) and 1 nM Tyk2 in a total volume of 25 μL. Human Tyk2 kinase domain, comprising amino acids 886 to 1187 with 10 additional histidine residues (histidine tag) on the carboxy terminus, was expressed and purified from bacculovirus in-house at Array BioPharma Inc. (Boulder, Colo.). The histidine tag was cleaved after purification using standard conditions.
Measurement: IC50 = 550 nM (pChEMBL 6.260000228881836)
Descriptors: MW 443.46, ALogP 2.42, HBA 9, HBD 2, PSA 106.29, QED 0.43
Compound (SMILES): C=CC(=O)N1CCCC(c2cc3c(cn2)CN(c2c(F)c(OC)cc(OC)c2F)C(=O)C32CC2)C1
InChIKey: KLYNIAXDZOJTNU-UHFFFAOYSA-N
Target: Fibroblast growth factor receptor 2 (SINGLE PROTEIN), Homo sapiens
UniProt: P21802
Assay: Enzymatic Assay: The inhibitor potency of the exemplified compounds was measured in an enzyme assay that measures peptide phosphorylation using FRET measurements to detect product formation. Inhibitors were serially diluted in DMSO and a volume of 0.5 μL was transferred to the wells of a 384-well plate. For FGFR3, a 10 μL volume of FGFR3 enzyme (Millipore) diluted in assay buffer (50 mM HEPES, 10 mM MgCl2, 1 mM EGTA, 0.01% Tween-20, 5 mM DTT, pH 7.5) was added to the plate and pre-incubated for a time between 5-10 minutes and up to 4 hours. Appropriate controls (enzyme blank and enzyme with no inhibitor) were included on the plate. The assay was initiated by the addition of a 10 μL solution containing biotinylated EQEDEPEGDYFEWLE peptide substrate (SEQ ID NO: 1) and ATP (final concentrations of 500 nM and 140 μM respectively) in assay buffer to the wells. The plate was incubated at 25° C. for 1 hr. The reactions were ended with the addition of 10 μL/well of quench solution (50 mM Tris, 150 mM NaCl, 0.5 mg/mL BSA, pH 7.8; 30 mM EDTA with Perkin Elmer Lance Reagents at 3.75 nM Eu-antibody PY20 and 180 nM APC-Streptavidin). The plate was allowed to equilibrate for 1 hr before scanning the wells on a PheraStar plate reader (BMG Labtech).FGFR1, FGFR2, and FGFR4 are measured under equivalent conditions with the following changes in enzyme and ATP concentrations: FGFR1, 0.02 nM and 210 uM respectively, FGFR2, 0.01 nM and 100 uM, respectively, and FGFR4, 0.04 nM and 600 uM respectively. The enzymes were purchased from Millipore or Invitrogen.
Measurement: IC50 > 200 nM (pChEMBL 6.698999881744385)
Descriptors: MW 483.52, ALogP 3.85, HBA 5, HBD 0, PSA 71.97, QED 0.6
Compound (SMILES): O=C1CCc2cc(OCCCN3CCC(C4=NOC5C=C(F)C=CC45)CC3)ccc2N1
InChIKey: OCNUPGFDBUFRFR-UHFFFAOYSA-N
Target: Histamine H1 receptor (SINGLE PROTEIN), Rattus norvegicus
UniProt: P31390
Assay: Receptor Binding Assay: Histamine H1: (1) The prepared membrane was applied with appropriate amount of buffer, and homogenizer was used for evenly dispersing. 15 tubes were mixed into a 100 ml container, and appropriate amount of buffer (potassium dihydrogen phosphate 1.36 g, 0.1 mol/L sodium hydroxide 79 ml, metered to 200 ml with double-distilled water) was added to give 50 ml of membrane suspension, which was reserved for future use. (2) 100 uL of membrane preparation was added into each reaction tube. (3) 100 uL of buffer was added into the total binding tube (TB), 100 uL of promethazine (final concentration 10-5M) was added into the nonspecific binding tube (NB), 100 uL of the test compound (final concentration 10-5M) was added into the specific binding tube (SB) of each test compound. (4) 10 uL of radioactive ligand 3H-pyrilamine was respectively added into each reaction tube (2 parallel tubes were used for each reaction tube, and each of them was placed on ice when adding sample). (5) Each of the reaction tubes was incubated at 30° C. for 60 min. After the reaction was completed, the bound ligands were rapidly filtered under reduced pressure, and the ice-chilled assay buffer was used for adequate washing. The filter was taken out and put into a 3 ml scintillation vial, and 2 ml of toluene scintillation solution was added and blended. 2* Isotope ligand [3H]-5-CT (85.4 Ci/mmol) was purchased from PerkinElmer Company; (+-)-pindolol was purchased from RBI Company; GF/C glass fiber filter paper was purchased from Whatman Company; Tris was imported and divided into aliquots; PPO and POPOP were purchased from Shanghai No. 1 Reagent Factory; liposoluble scintillation cocktail was purchased from Shanghai Reagent Factory. Beckman LS-6500 Multi-function Liquid Scintillation Counter was used.
Measurement: Ki = 178 nM (pChEMBL 6.75)
Descriptors: MW 425.5, ALogP 3.85, HBA 5, HBD 1, PSA 63.16, QED 0.7
Compound (SMILES): Nc1nc2cc(-c3ccc4ncn(C5CC5)c(=O)c4c3)ccc2o1
InChIKey: WMZMCTLVCWIPLX-UHFFFAOYSA-N
Target: Phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit beta isoform (SINGLE PROTEIN), Homo sapiens
UniProt: P42338
Assay: Inhibition Assay of PI3K: An exemplary system is PI 3-Kinase (human) HTRF Assay from Upstate. The assay can be carried out according to the procedures suggested by the manufacturer. Briefly, the assay is a time resolved FRET assay that indirectly measures PIP3 product formed by the activity of a PI3-K. The kinase reaction is performed in a microtitre plate (e.g., a 384 well microtitre plate). The total reaction volume is approximately 20 ul per well. In the first step, each well receives 2 ul of test compound in 20% dimethylsulphoxide resulting in a 2% DMSO final concentration. Next, approximately 14.5 ul of a kinase/PIP2 mixture (diluted in 1× reaction buffer) is added per well for a final concentration of 0.25-0.3 ug/ml kinase and 10 μM PIP2. The plate is sealed and incubated for 15 minutes at room temperature. To start the reaction, 3.5 ul of ATP (diluted in 1× reaction buffer) is added per well for a final concentration of 10 μM ATP. The plate is sealed and incubated for 1 hour at room temperature. The reaction is stopped by adding 5 ul of Stop Solution per well and then 5 ul of Detection Mix is added per well. The plate is sealed, incubated for 1 hour at room temperature, and then read on an appropriate plate reader.
Measurement: IC50 > 10000 nM (pChEMBL 5)
Descriptors: MW 318.34, ALogP 3.12, HBA 6, HBD 1, PSA 86.94, QED 0.61
Compound (SMILES): Cc1ccc(S(=O)(=O)N[C@H]2CC[C@H](O)CC2)cc1-c1cncc(-c2cccnc2)n1
InChIKey: UOHNJRXFMONSKM-IYARVYRRSA-N
Target: Phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit delta isoform (SINGLE PROTEIN), Homo sapiens
UniProt: O00329
Assay: Kinase Glo Luminescent Kinase Assay: PI 3-Kinase Alpha (A), Pb 3-Kinase Beta (B), Vps34 (C), PI 4-Kinase Beta (D):The luminescence-based ATP detection reagent KinaseGlo was obtained from Promega, (Cat. No. V6714, Lot No. 236161) through Catalys, Wallisellen, Switzerland. L-alpha-phosphatidylinositol (PI, liver, bovine) was obtained from Avanti Polar Lipid (Cat. No. 840042C, Lot#LPI-274), Phosphatidylinositol-4,5-bisphosphate (PIP(4,5)2) was also obtained from Avanti Polar Lipid (Cat. No. 840046X). L-α-Phosphatidylserine (PS) was obtained from Avanti Polar Lipid (Cat. No. 840032C), n-Octylglucoside from Avanti Polar Lipid (Cat. No. 10634425001). Luminescence is a well established readout to determine ATP concentrations and can thus be used to follow the activity of many kinases regardless of their substrate. The Kinase Glo Luminescent Kinase Assay (Promega, Madison/WI, USA) is a homogeneous HTS method of measuring kinase activity by quantifying the amount of ATP remaining in solution following a kinase reaction.
Measurement: IC50 = 120 nM (pChEMBL 6.920000076293945)
Descriptors: MW 424.53, ALogP 3.1, HBA 6, HBD 2, PSA 105.07, QED 0.65
Compound (SMILES): N#CC[C@]1(n2cc(-c3ncnc4[nH]ccc34)cn2)C[C@H](N2CCC(Oc3cc(CN4CCC[C@@H]4CO)cc(C(F)(F)F)n3)CC2)C1
InChIKey: WQXUGBZSZWSUTM-OVMXQCPESA-N
Target: Tyrosine-protein kinase JAK1 (SINGLE PROTEIN), Homo sapiens
UniProt: P23458
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) 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 μL 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 hour 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 PHERA star plate reader (BMG, Cary, N.C.). The data for the JAK1 and/or JAK2 inhibitors were obtained by testing the compounds in the Example A assay at 1 mM ATP.
Measurement: IC50 < 50 nM (pChEMBL 7.301000118255615)
Descriptors: MW 635.7, ALogP 4.51, HBA 10, HBD 2, PSA 132.01, QED 0.27
Compound (SMILES): Cc1ccc(Cl)c2[nH]c3c(C(=O)O)ccc(F)c3c12
InChIKey: HPPILTSFNNPAGH-UHFFFAOYSA-N
Target: Metabotropic glutamate receptor 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q13255
Assay: New CNS Specificity Data for AK3-238 (FA44) and AK3-99B (FA26): Two exemplary compounds, FA44 and FA26, were screened against a panel of CNS receptors, transporters, and ion channels to examine specificity for CLC-2 in the brain. A comprehensive primary binding assay screen was performed through the NIH Psychoactive Drug Screening Program (PDSP) at UNC-Chapel Hill. Secondary or functional assays were performed for compounds showing >50% activity in the primary assay.
Measurement: Ki > 10000 nM (pChEMBL 5)
Descriptors: MW 277.68, ALogP 4.12, HBA 1, HBD 2, PSA 53.09, QED 0.7
Compound (SMILES): CC#CCN1C[C@@H]2C[C@]2(c2ccc(-c3cnc(N)c(C(=O)N[C@H]4CC[C@H](O)CC4)c3)cc2)C1
InChIKey: BBWFHMVFMVFXJP-RNLGNXJRSA-N
Target: Bone morphogenetic protein receptor type-1B (SINGLE PROTEIN), Homo sapiens
UniProt: O00238
Assay: Caliper Assay: In the applied method, this separation takes place inside a chip that contains a complex capillary system for simultaneous analysis of 12 samples (12-sipper chip, Caliper Technologies Corp., Mountain View, USA). In order to allow the detection and quantification of the peptides in the capillary system, the peptides carry a fluorescent label (fluorescein). With this label the peptides can be quantified by fluorescence intensity through the instruments laser and detection system (LC3000, Caliper Life Sciences).The assays were performed in 384-well, low volume microtiter assay plates in a final reaction volume of 9 ul. Dose-response curves were generated by incubating 10 nM of each kinase together with 2 μm of the fluorescently labeled substrate peptide 5-Fluo-Ahx-KKYQAEEN-T-YDEYENKK-amid (10 mM stock solution in DMSO) in 50 mM Hepes pH 7.5, 0.02% Tween 20, 0.02% BSA, 1 mM DTT, 10 μm Na3VO4, 10 mM β-Glycerolphosphate, 1 mM MgCl2, 12 mM MnCl2 (ALK1 and ALK6 7 mM) and 15 μm ATP for 60 min at 30° C. in the presence or absence of compound diluted in DMSO.Kinase reaction were terminated by adding 15 ul STOP buffer (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 workstation (Caliper Technologies Corp., Mountain View, USA) for reading. The relative amount of phosphorylated peptide r, was calculated using the heights of the substrate peak, s, and the product peak, p: r=p/(p+s).
Measurement: IC50 = 1700 nM (pChEMBL 5.769999980926514)
Descriptors: MW 444.58, ALogP 2.96, HBA 5, HBD 3, PSA 91.48, QED 0.62
Compound (SMILES): Nc1ncc(-c2ccc([C@]34C[C@H]3CN(CCCN3CCOCC3)C4)cc2)cc1C(=O)N[C@H]1CC[C@H](O)CC1
InChIKey: FCURQEMLQWEPFI-VWDBNSKXSA-N
Target: Activin receptor type-1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q04771
Assay: Caliper Assay: A kinase selectivity panel which measures substrate peptide phosphorylation was set-up for wild-type ALK2 (aa172-499), ALK2 FOP mutant (aa172-499 R206H), ALK1 (aa166-493), ALK5 (aa162-503) and ALK6 (aa168-495). The technology used for the described assay is based on the separation and quantification of substrate and product in an electrical field. In the course of the kinase reaction the peptide substrate is phosphorylated by a kinase. The transfer of a phosphate residue also causes the introduction of two additional negative charges and hence to a change in the net charge of the phospho-peptide compared to the unphosphorylated peptide. Due to this difference in charge the phosphorylated und unphosphorylated peptides migrate with different velocities in an electrical field.In the applied method, this separation takes place inside a chip that contains a complex capillary system for simultaneous analysis of 12 samples (12-sipper chip, Caliper Technologies Corp., Mountain View, USA). In order to allow the detection and quantification of the peptides in the capillary system, the peptides carry a fluorescent label (fluorescein). With this label the peptides can be quantified by fluorescence intensity through the instruments laser and detection system (LC3000, Caliper Life Sciences).The assays were performed in 384-well, low volume microtiter assay plates in a final reaction volume of 9 ul. Dose-response curves were generated by incubating 10 nM of each kinase together with 2 μm of the fluorescently labeled substrate peptide 5-Fluo-Ahx-KKYQAEEN-T-YDEYENKK-amid (10 mM stock solution in DMSO) in 50 mM Hepes pH 7.5, 0.02% Tween 20, 0.02% BSA, 1 mM DTT, 10 m Na3VO4, 10 mM If-Glycerolphosphate, 1 mM MgCl2, 12 mM MnCl2 (ALK1 and ALK6 7 mM) and 15 μm ATP for 60 min at 30° C. in the presence or absence of compound diluted in DMSO. Kinase reaction were terminated by adding 15 ul STOP buffer (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 workstation (Caliper Technologies Corp., Mountain View, USA) for reading. The relative amount of phosphorylated peptide r, was calculated using the heights of the substrate peak, s, and the product peak, p: r=p/(p+s).
Measurement: IC50 = 11 nM (pChEMBL 7.960000038146973)
Descriptors: MW 519.69, ALogP 2.66, HBA 7, HBD 3, PSA 103.95, QED 0.49
Compound (SMILES): CNC[C@H](c1ccc(Cl)c(Cl)c1)[C@H](O)c1cccc(N)c1
InChIKey: BQCHSJJIVZNPPW-CZUORRHYSA-N
Target: Cytochrome P450 2D6 (SINGLE PROTEIN), Homo sapiens
UniProt: P10635
Assay: Inhibition Assay: Cytochrome P450 3A4 and 2D6:Recombinant enzymes, 3A4 and 2D6, generated using the ABL yeast expression system were used. For CYP3A4, the enzyme amount was 2 pmol per well, and the substrate (7-benzyloxy-4-(trifluoromethyl) coumarin; BFC) was used at 15 μM. The assay mixture also included K2HPO4 (pH 7.4; conc. 0.1 M) and NADPH (conc. 1 mM). The incubation time was 30 minutes, and the reference inhibitor was A-naphthoflavone. For CYP2D6, the enzyme amount was 2 pmol per well, and the substrate (7-methoxy-4-(aminomethyl)-coumarin; MAMC) was used at 20 μM. The assay mixture also included K2HPO4 (pH 7.4; conc. 0.1 M) and NADPH (conc. 0.06 mM). The incubation time was 35 minutes, and the reference inhibitor was quinidine.Briefly, 0.6 μL of serially diluted compound was added to 75 μL water. 10 μL of diluted compound in water was then added to each assay plate. 20 μL of a K2HPO4, enzyme, and substrate mixture was added. After 10 minutes of room temperature pre-incubation, 10 μL of NADPH was added, and the plates were placed at 37° C. for 30 or 35 minutes depending on the enzyme being tested. After the incubation was complete, 20 μL of 0.1% Tris/ACN was added to the plate. The plate was read on a FluorStar fluorescence plate reader.
Measurement: IC50 = 1400 nM (pChEMBL 5.849999904632568)
Descriptors: MW 325.24, ALogP 3.61, HBA 3, HBD 3, PSA 58.28, QED 0.74
Compound (SMILES): C=C(C)c1cnc(-c2cc(Cl)ccc2F)cc1Nc1ccncc1C(=O)N1CCCC1
InChIKey: FHRZBVSUXBSDPP-UHFFFAOYSA-N
Target: Bone morphogenetic protein receptor type-1B (SINGLE PROTEIN), Homo sapiens
UniProt: O00238
Assay: In Vitro Kinase Assay: Compound of the invention were screened in an in vitro kinase assay against several members of the TGFβ family of Ser/Thr kinases. The kinases tested were ALK1 (ACVRL1), ALK2 (ACVR1), ALK3 (BMPR1A), ALK4 (ACVR1B), ALK5 (TGFBR1), and ALK6 (BMPR1B). Standard kinase testing conditions and techniques were employed. For each case, specific kinase/substrate pairs along with required cofactors were prepared in reaction buffer. Compound of the invention were delivered into the reaction, followed 15-20 min later by addition of a mixture of ATP and 33P ATP to a final concentration of 10 μM. Reactions were carried out at RT for 120 min, followed by spotting of the reactions onto P81 ion exchange filter paper. Unbound phosphate was removed by extensive washing of filters in 0.75% phosphoric acid. Kinase activity data was expressed as the percent of remaining kinase activity in test samples compared to vehicle.
Measurement: IC50 > 10000 nM (pChEMBL 5)
Descriptors: MW 436.92, ALogP 5.95, HBA 4, HBD 1, PSA 58.12, QED 0.54
Compound (SMILES): COc1cccc(-c2nnc(NS(=O)(=O)[C@@H](C)[C@H](OC)c3ncc(C)cn3)n2-c2c(OC)cccc2OC)n1
InChIKey: RZJCKIXEYMQFPY-AOMKIAJQSA-N
Target: Apelin receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P35414
Assay: [35S]GTPγS Binding: The human APJ receptor was cloned by polymerase chain reaction and the gene encoding the receptor was subcloned in pFLAG-CMV-3 expression vector (Sigma, Saint Louis, Mo. USA) in-house at Amgen. A GTPγS binding assay was performed on membranes prepared from CHO cells stably expressing human APJ receptor. The optimum experimental conditions for the concentrations of GDP, MgCl2, and NaCl in the assay buffer were initially determined. The assay was performed in assay buffer [20 mM HEPES, pH 7.5, 5 mM MgCl2, and 0.1% (w/v) BSA with 200 mM NaCl, 3 M GDP] and membranes expressing human APJ receptor/well along with WGA PS beads. The reaction was initiated by addition of 0.2 nM [35S]GTPγS (Perkin Elmer Life and Analytical Sciences, Waltham USA) in the absence or presence of various ligands and incubated at RT for 90 min. Nonspecific binding was determined in the presence of 100 M GTPγS and was always less than 0.2% of total binding. All the results presented are means of several independent experiments and analyzed by non-linear regression methods using commercially available program Prism (GraphPad, San Diego, Calif.) to obtain EC50.
Measurement: EC50 = 0.18 nM (pChEMBL 9.739999771118164)
Descriptors: MW 555.62, ALogP 2.97, HBA 12, HBD 1, PSA 152.47, QED 0.29
Compound (SMILES): CN(C)C(CNC(=O)NC(C)(C)Cc1ccsc1)Cc1ccccc1
InChIKey: NSAOLNUDHLFABH-UHFFFAOYSA-N
Target: Delta-type opioid receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P41143
Assay: Radioligand Binding Assay: Each compound was initially tested at 20 μM and was incubated with 3H-DPN at a concentration equal to the Kd (0.4 nM) of the radioligand in μOR containing Sf9 insect cell membranes. The reaction contained 40 fmol of μOR and was incubated in a buffer of 20 mM HEPES pH 7.5, 100 mM sodium chloride, and 0.1% bovine serum albumin for 1 hour at 25° C. To separate free from bound radioligand, reactions were rapidly filtered over Whatman GF/B filters with the aid of a Brandel harvester and 3H-DPN counts were measured by liquid scintillation. Compounds with more than 25% of 3H-DPN radioactivity were further tested in full dose-response to determine the affinity (Ki) in HEK293 membranes. Subsequently, the 15 analogs were tested in full dose-response for affinity at the μOR and the κOR by the National Institutes of Mental Health Psychoactive Drug Screen Program (PDSP), as were the affinities of compounds 12, PZM21, and their stereoisomers at the μOR, δOR, κOR and nociception receptor.
Measurement: Ki = 3000 nM (pChEMBL 5.519999980926514)
Descriptors: MW 359.54, ALogP 3.54, HBA 3, HBD 2, PSA 44.37, QED 0.76
Compound (SMILES): Cn1cc(-c2cc3ncccc3c(-c3ccn(C4(CC#N)CC(C#N)C4)n3)n2)cn1
InChIKey: CQRLVCKYPXPONF-UHFFFAOYSA-N
Target: Tyrosine-protein kinase JAK1 (SINGLE PROTEIN), Homo sapiens
UniProt: P23458
Assay: JAK Caliper Enzyme Assay at 1mM ATP: Compounds were added to a 384-well plate. Reaction mixtures contained 10 mM HEPES, pH 7.4, 10 mM MgCl2, 0.01% BSA, 0.0005% Tween 20, 1 mM ATP and 1 μM peptide substrate. The JAK1 and TYK2 assays contained 1 μM of the IRStide peptide (5FAM-KKSRGDYMTMQID) and the JAK2 and JAK3 assays contained 1 μM of the JAKtide peptide (FITC-KGGEEEEYFELVKK). The assays were initiated by the addition of 20 nM JAK1, 1 nM JAK2, 1 nM JAK or 1 nM TYK2 enzyme and were incubated at room temperature for three hours for JAK1, 60 minutes for JAK2, 75 minutes for JAK3 or 135 minutes for TYK2. Enzyme concentrations and incubation times were optimized for each new enzyme preps and were modified slightly over time to ensure 20% to 30% phosphorylation. The assays were stopped with 15 μL of 180 mM HEPES, pH 7.4, 20 mM EDTA, and 0.2% Coating Reagent 3. The assay plates were placed on a Caliper Life Science LC3000 instrument, and each well was sampled using appropriate separation conditions to measure the unphosphorylated and phosphorylated peptide.
Measurement: IC50 = 55 nM (pChEMBL 7.260000228881836)
Descriptors: MW 394.44, ALogP 3.44, HBA 8, HBD 0, PSA 109, QED 0.52
Compound (SMILES): COc1cc(Cn2cccn2)cc2onc(NS(=O)(=O)c3cccc(C)c3C#N)c12
InChIKey: UDQLVPWPXOQLGE-UHFFFAOYSA-N
Target: Histone acetyltransferase KAT8 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9H7Z6
Assay: Kat8 Assay: TBDA. Compound preparation    1. Prepare 10 mM stock solutions in 100% DMSO from solid material    2. Serial dilute 10 mM, 1 mM or 0.1 mM compound stocks 3-fold in 100% DMSO for 11-point dose responseB. Reagent preparation    1. Prepare 1× assay buffer containing 10 mM Tris HCL pH 8.0, 2.5 mM NaCl, 0.5 mM EDTA, 0.005% BSG and 0.02% Tween-20    2. Dilute Histone peptide (CPC Scientific) and AcCoA (Sigma) together in assay buffer to 2×.    3. Dilute KAT enzyme in assay buffer to 2×.C. Enzyme reaction    1. Final reaction conditions for each KAT assay in a 20 ul assay reaction volume:   KAT8 15 nM, 1 uM AcCoA, 2 uM H3 1-21 peptide, 45-minute reaction    2. Add 0.5 ul of diluted compound to the assay plate (384-well V-bottom polypropylene plates) or 0.5 ul of DMSO for control wells.    3. Add 10 ul of 2× Histone peptide/2×AcCoA mix to the assay plate.    4. Add 10 ul of 2× enzyme to the assay plate.    5. Stop the reaction after the indicated time with the addition of 2 ul of 5% formic acid    6. Each reaction was analyzed using self-assembled monolayer desorption/ionization time-of-flight mass spectrometry (Mrksich, Milan (2008) Mass Spectrometry of Self-Assembled Monolayers: A New Tool for Molecular Surface Science ACS Nano 2008 2 (1), 7-18; SAMDI Tech, Inc. (Chicago, Ill.)).    7. Area under the curve (AUC) for both substrate and product peaks was determined for KAT5 at M.W. 2561 [Substrate+H]+ and 2603 [Product+H]+ with a +/−1 Da tolerance, respectively    8. Area under the curve (AUC) for both substrate and product peaks was determined for KAT6a, KAT6B, KAT7 and KAT8 at M.W. 2723 [Substrate+H]+ and 2765 [Product+H]+ with a +/−1 Da tolerance, respectively. 9. Percent conversion to product was calculated by: AUCProduct/(AUCsubstrate+AUCProduct).D. Data analysis    1. IC50 values were determined by fitting the % conversion at each inhibitor concentration to the 4-parameter IC50 equation using Pfizer proprietary curve fitting software.    2. Ki values were determined by fitting the % conversion at each inhibitor concentration to the Morrison equation for tightbinding competitive inhibitors using Pfizer proprietary curve fitting software.
Measurement: Ki = 12500 nM (pChEMBL 4.900000095367432)
Descriptors: MW 423.45, ALogP 3.06, HBA 8, HBD 1, PSA 123.04, QED 0.51
Compound (SMILES): O=C1CCC(N2C(=O)c3cccc(NCCOCCOCCOCCOCCN4CCN(Cc5ccc(-c6cc7c(NCc8ccccc8Cl)ncnc7[nH]6)cc5)CC4)c3C2=O)C(=O)N1
InChIKey: AZYKVBYABJWTNN-UHFFFAOYSA-N
Target: Epidermal growth factor receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P00533
Assay: TR-FRET Assay for ERBB Kinases: All compounds and PROTACs were serially diluted in three-fold increments using 100% DMSO, followed by an intermediate 10-fold dilution using Buffer A (50 mM HEPES, pH 7.5, 50 mM NaCl, 10 mM MgCl2, 1 mM DTT, and 0.1% Pluronic F-68). Two microliters of serially diluted compound or PROTAC were then transferred to black 384-well Proxiplates (PerkinElmer, #6008260) using an Integra Viaflo96. Next, 10 uL of protein kinase in Buffer A was added to each well of the assay plate and pre-incubated with compound for 10 minutes. Kinase reactions were then initiated by addition of 5 uL substrate mix containing 3 mM ATP and 30 uM fluorescein-labeled Poly-GluTyr (Thermo Fisher, #PV3610) in Buffer A and allowed to proceed for 10 minutes at room temperature. Reactions were quenched by addition of a 5 uL mixture containing 5 nM LanthaScreen® Tb-pY20 Antibody (Thermo Fisher, #PV3552) and 40 mM EDTA in Buffer A. Assay plates were then read using a Synergy2 (Biotek Instruments, Winooski, Vt.) via excitation thru a 340/20 nm bandpass filter and emission collected thru 490/10 nm (donor) and 520/25 nm (acceptor) bandpass filters. The final kinase concentrations used for each 15 uL reaction were as follows: 0.2 nM EGFR Exon20NPG (SignalChem, #E10-132GG), 0.1 nM wild type EGFR (BPS Bioscience, #40187), 0.3 nM EGFR L858R/T790M/C797S (BPS Bioscience, #40351), 0.1 nM EGFR L858R (BPS Bioscience, #40189), 0.4 nM L858R/T790M (BPS Bioscience, #40350), InM EGFR Dell9 (SignalChem, #E10-122JG), 10 nM EGFR Dell9 T790M (SignalChem, #E10-122KG), 0.3 nM Her2 (BPS Bioscience, #40230), 15 nM Her2 InsYVMA (SignalChem, #E27-13BG).
Measurement: IC50 = 8500 nM (pChEMBL 5.070000171661377)
Descriptors: MW 908.46, ALogP 4.59, HBA 14, HBD 4, PSA 192.58, QED 0.05
Compound (SMILES): CC[C@H](Nc1ccc2c(c1)OCCn1cc(N3C(=O)OC[C@H]3C(F)F)nc1-2)C(N)=O
InChIKey: ZWEJBXXYQYTACR-STQMWFEESA-N
Target: Phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit gamma isoform (SINGLE PROTEIN), Homo sapiens
UniProt: P48736
Assay: Biochemical Inhibition Assay: The biochemical inhibition of four PI3K isoforms by the Formula I compounds of Table 1. In addition, two clinically tested PI3K compounds, taselisib and pictilisib are included as comparators. The representative compounds of the invention exhibit strong activity against PI3Kα, and exhibit significantly enhanced selectivity relative to the other isoforms PI3Kβ, PI3Kδ, and PI3Kγ when compared to taselisib (GDC-0032) and pictilisib (GDC-0941). In particular, the selectivity ratios in the second from the right column of Table 2A show that each Formula I compounds 101-107 has a PI3K alpha to delta selectivity ratio far higher than taselisib or pictilisib. In fact, both taselisib and pictilisib have stronger activity against PI3K delta than against PI3K alpha, i.e. their selectivity ratios are less than 1. The selectivity ratios of Formula I compound 101-107 range from 301-fold to 634-fold.
Measurement: Ki = 17.5 nM (pChEMBL 7.760000228881836)
Descriptors: MW 421.4, ALogP 2.21, HBA 7, HBD 2, PSA 111.71, QED 0.74
Compound (SMILES): C[C@H](CO)Nc1n[nH]c2nccc(Oc3ccc(NC(=O)c4cn(C)n(-c5ccc(F)cc5)c4=O)cc3F)c12
InChIKey: SLHFUYONPRUNQR-CQSZACIVSA-N
Target: Hepatocyte growth factor receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P08581
Assay: Enzyme Assay: The affinity of compound binding to wild type and mutant human MET kinases is measured using Invitrogen's LanthaScreen Eu Kinase Binding technology. Briefly, GST-tagged recombinant human MET kinase domain from Signal Chem (see Table 8 below for concentration in assay) is incubated with 50 nM Alexa-Fluor® Tracer 236 (Invitrogen Cat No. PR9078A) and 2 nM Europium-anti-GST (Invitrogen Cat. No. A151 6) along with test compound in a buffer consisting of 25 mM HEPES, pH 7.4, 10 mM MgCl2, 0.01% Triton X-100, 1 mM DTT, and 2% DMSO. Compounds are typically prepared in a three-fold serial dilution in DMSO and added to the assay to give the appropriate final concentration. After a 60-minute incubation at 22° C., the reaction is measured using a PerkinElmer EnVision multimode plate reader via TR-FRET dual wavelength detection, and the percent of control (POC) calculated using a ratiometric emission factor. 100 POC is determined using no test compounds and 0 POC is determined using a concentration of control compound that completely inhibits the enzyme
Measurement: IC50 = 21.3 nM (pChEMBL 7.670000076293945)
Descriptors: MW 535.51, ALogP 3.56, HBA 9, HBD 4, PSA 139.09, QED 0.24
Compound (SMILES): CN1CCN(c2cnc3ccc(-c4ccc5oc(N)nc5c4)nc3n2)CC1
InChIKey: SQYMECIRTPXKHH-UHFFFAOYSA-N
Target: Phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit gamma isoform (SINGLE PROTEIN), Homo sapiens
UniProt: P48736
Assay: PI 3-Kinase HTRF Assay: Briefly, the assay is a time resolved FRET assay that indirectly measures PIP3 product formed by the activity of a PI3-K. The kinase reaction is performed in a microtitre plate (e.g., a 384 well microtitre plate). The total reaction volume is approximately 20 ul per well. In the first step, each well receives 2 ul of test compound in 20% dimethylsulphoxide resulting in a 2% DMSO final concentration. Next, approximately 14.5 ul of a kinase/PIP2 mixture (diluted in 1x reaction buffer) is added per well for a final concentration of 0.25-0.3 ug/ml kinase and 10 uM PIP2. The plate is sealed and incubated for 15 minutes at room temperature. To start the reaction, 3.5 ul of ATP (diluted in 1x reaction buffer) is added per well for a final concentration of 10 uM ATP. The plate is sealed and incubated for 1 hour at room temperature. The reaction is stopped by adding 5 ul of Stop Solution per well and then 5 ul of Detection Mix is added per well. The plate is sealed, incubated for 1 hour at room temperature, and then read on an appropriate plate reader.
Measurement: IC50 < 1000 nM (pChEMBL 6)
Descriptors: MW 361.41, ALogP 2.17, HBA 8, HBD 1, PSA 97.2, QED 0.58
Compound (SMILES): CNC[C@H](c1ccc2ccccc2c1)[C@H](O)c1cc(F)cc(F)c1
InChIKey: OKQCBRXAICHRKL-WOJBJXKFSA-N
Target: Sodium-dependent serotonin transporter (SINGLE PROTEIN), Homo sapiens
UniProt: P31645
Assay: Uptake Assay Protocol: SERT: This protocol was designed to measure inhibition of uptake by the human serotonin transporter. The reagents were human SERT (HEK293F) cells, fluoxetine (Sigma), nomifensine, neurotransmitter transporter uptake assay kit (Molecular Devices), freestyle 293 expression medium (Invitrogen), 10× Hank's Balanced Salt Solution (HBSS; Invitrogen), 1 M HEPES (Mediatech), Biocoat poly-D-lysine 96-well, black, clear plates (Becton, Dickinson), and 500 μL polypropylene U-bottom 96-well plates (Fisher). The Assay Buffer (AB) was 1λ HBSS and 0.02 M HEPES.The HEK293F cells were transfected with the human serotonin transporter and frozen in 1 mL aliquots at about 1E+07 cells/mL. On the day of the experiment, the cells were removed from −80° C. or liquid nitrogen and thawed in a room temperature water bath. The cells were dilute to about 1-2E+06 with Freestyle medium. A 1 mL sample (1:2 dilution) was prepared, and the cells were counted. The cells were spun at 1100 rpm for 5 minutes, and the medium was aspirated off. The cells were resuspend in medium at 1.5E+06 cells/mL for about 60,000 cells per well. 40 μL of cells were dispensed per well in the Biocoat plates. The plates were spun at 1100 rpm for 1 minute to improve homogeneity of the cell layer and were incubated at 37° C. for a minimum of 3 hours.
Measurement: Ki = 52.5 nM (pChEMBL 7.28000020980835)
Descriptors: MW 327.37, ALogP 4.15, HBA 2, HBD 2, PSA 32.26, QED 0.74
Compound (SMILES): Cc1cccc(C2=CCN(CCNC(=O)[C@H]3C[C@@H]4CC[C@H]3O4)CC2)n1
InChIKey: WOHMWCGVHSYPFG-JENIJYKNSA-N
Target: Voltage-gated inwardly rectifying potassium channel KCNH2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q12809
Assay: Evaluation of Activity for Inhibiting hERG Channel : The activity of the present compound for inhibiting hERG channel was measured by whole-cell patch clamp method with auto patch clamp system, using CHO cell wherein hERG channel involved in human rapidly activating delayed rectifier potassium current (IKr) was forcibly expressed.(Preparation of Cell Suspension)hERG-CHO cell purchased from ChanTest was incubated at 37° C. in a CO2 incubator, and the cell was exfoliated from the flask with trypsin to prepare a cell suspension, shortly before the hERG current measurement.(Preparation of Solution)The extracellular fluid and intracellular fluid which were used in the measurement were prepared as follows.Extracellular fluid: 2 mmol/L CaCl2, 1 mmol/L MgCl2, 10 mmol/L HEPES, 4 mmol/L KCl, 145 mmol/L NaCl, 10 mmol/L glucoseIntracellular fluid: 5.4 mmol/L CaCl2, 1.8 mmol/L MgCl2, 10 mmol/L HEPES, 31 mmol/L KOH, 10 mmol/L EGTA, 120 mmol/L KCl, 4 mmol/L Na2-ATPTest compound solution: The test compound was dissolved in DMSO by adjusting the concentration to 2 mmol/L or 20 mmol/L to prepare each test compound solution. Further, the test compound solution was diluted with the extracellular fluid by 200-fold, which was serially diluted with the extracellular fluid to prepare each concentration of the test compound solution which is used to calculate IC50 value of hERG inhibition.
Measurement: IC50 > 10000 nM (pChEMBL 5)
Descriptors: MW 341.46, ALogP 2.16, HBA 4, HBD 1, PSA 54.46, QED 0.89
Compound (SMILES): C[C@H](NC(=O)c1c(N)nn2cccnc12)c1cc2cccc(C#Cc3ccc(=O)[nH]c3)c2c(=O)n1-c1ccccc1
InChIKey: OOPAAQGQPZGVGQ-IBGZPJMESA-N
Target: Phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit beta isoform (SINGLE PROTEIN), Homo sapiens
UniProt: P42338
Assay: Inhibition Assays: Class I PI3-Ks can be either purchased (p110α/p85α, p110β/p85α, p110δ/p85α from Upstate, and p110γ from Sigma) or expressed as previously described (Knight et al., 2004). IC50 values are measured using either a standard TLC assay for lipid kinase activity (described below) or a high-throughput membrane capture assay. Kinase reactions are performed by preparing a reaction mixture containing kinase, inhibitor (2% DMSO final concentration), buffer (25 mM HEPES, pH 7.4, 10 mM MgCl2), and freshly sonicated phosphatidylinositol (100 μg/mL). Reactions are initiated by the addition of ATP containing 10 μCi of γ-32P-ATP to a final concentration of 10 or 100 μM and allowed to proceed for 5 minutes at room temperature. For TLC analysis, reactions are then terminated by the addition of 105 μL 1N HCl followed by 160 μL CHCl3:MeOH (1:1). The biphasic mixture is vortexed, briefly centrifuged, and the organic phase is transferred to a new tube using a gel loading pipette tip precoated with CHCl3. This extract is spotted on TLC plates and developed for 3-4 hours in a 65:35 solution of n-propanol:1M acetic acid.
Measurement: IC50 = 2000 nM (pChEMBL 5.699999809265137)
Descriptors: MW 541.57, ALogP 3.19, HBA 8, HBD 3, PSA 140.17, QED 0.29
Compound (SMILES): C=CC(=O)N1CC[C@@H](n2nc(-c3ccc(Oc4ccccc4)cc3)c3cnccc32)C1
InChIKey: UZTXHEWMOHGSCD-LJQANCHMSA-N
Target: Epidermal growth factor receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P00533
Assay: Kinases Assay : Btk kinase activity was determined using a homogenous time resolved fluorescence (HTRF) methodology. Measurements were performed in a reaction volume of 15 μL using 384-well assay plates. Kinase enzyme, inhibitor, ATP and 1 μM peptide substrate were incubated in a reaction buffer compose of Hepes50 mM (pH7.0), NaN3 0.02%, BSA 0.01%, Orthocanadate 0.1 mM. After one hour, the kinase reaction was quenched by the addition of E t-labeled antibody and XL-665 in 1×Detection buffer containing 60 mM EDTA (Cisbio), and the mixture was allowed to incubate for one hour. The HTRF signal was measured on a multimode plate reader (EnVision Multilabel Reader, Perkin Elmer) with an excitation wavelength (λEx) of 330 nm and detection wavelengths (λEm) of 615 and 665 nm. Activity was determined by the ratio of the fluorescence at 665 nm to that at 615 nm. For each compound, enzyme activity as measured at various concentrations of compound, Negative control reactions were performed in the absence of inhibitor in two replicates and eight no enzyme controls were used to determine baseline fluorescence levels. IC50s were obtained according to the equation:Y=100/(1+10{circumflex over ( )}((Log IC50−X)*HillSlope)).For BTK assay, [ATP]=80 μM, BTK=3.4 nM.For LYN assay, [ATP]=20 μM, LYN=0.12 n M. For LCK assay, [ATP]=20 μM, LCK=0.2 nM. For BLK assay, [ATP]=20 μM, BLK=0.6 n M.
Measurement: IC50 = 550 nM (pChEMBL 6.260000228881836)
Descriptors: MW 410.48, ALogP 4.85, HBA 5, HBD 0, PSA 60.25, QED 0.44
Compound (SMILES): CNc1cc2c(cn1)c(-c1cnn(C)c1)nn2C1CCC(Oc2nccc(=O)n2C)CC1
InChIKey: HCNFOYIEZHCOME-UHFFFAOYSA-N
Target: Tyrosine-protein kinase receptor TYRO3 (SINGLE PROTEIN), Homo sapiens
UniProt: Q06418
Assay: Enzyme Assay: TYRO3: Compounds of Formula I were screened for their ability to inhibit TYRO3 kinase using Invitrogen's LanthaScreen Eu Kinase Binding technology. GST-tagged recombinant human TYRO3 kinase domain from Carna (5 nM; Cat. No. PR7480A) was incubated with 20 nM Alexa-Fluor® Tracer 236 (PR9078A) and 2 nM Europium-anti-GST (Cat. No. A15116) along with test compound in a buffer consisting of 25 mM HEPES, pH 7.4, 10 mM MgCl2, 0.01% Triton X-100, and 2% DMSO. Compounds are typically prepared in a threefold serial dilution in DMSO and added to the assay to give the appropriate final concentration. After a 60-minute incubation at 22° C., the reaction was measured using a PerkinElmer EnVision multimode plate reader via TR-FRET dual wavelength detection, and the percent of control (POC) calculated using a ratiometric emission factor. 100 POC was determined using no test compounds and 0 POC was determined using a concentration of control compound that completely inhibits the enzyme. The POC values were fit to a 4 parameter logistic curve and the IC50 value is point where the curve crosses 50 POC.
Measurement: IC50 > 500 nM (pChEMBL 6.301000118255615)
Descriptors: MW 434.5, ALogP 2.53, HBA 10, HBD 1, PSA 104.68, QED 0.51
Compound (SMILES): CNc1cc2c(cn1)c(-c1cnn(C)c1)nn2C1CCC(Oc2ccccn2)CC1
InChIKey: VGOUFJFPXAHHOY-UHFFFAOYSA-N
Target: Tyrosine-protein kinase receptor TYRO3 (SINGLE PROTEIN), Homo sapiens
UniProt: Q06418
Assay: Enzyme Assay: TYRO3: Compounds of Formula I were screened for their ability to inhibit TYRO3 kinase using Invitrogen's LanthaScreen Eu Kinase Binding technology. GST-tagged recombinant human TYRO3 kinase domain from Carna (5 nM; Cat. No. PR7480A) was incubated with 20 nM Alexa-Fluor® Tracer 236 (PR9078A) and 2 nM Europium-anti-GST (Cat. No. A15116) along with test compound in a buffer consisting of 25 mM HEPES, pH 7.4, 10 mM MgCl2, 0.01% Triton X-100, and 2% DMSO. Compounds are typically prepared in a threefold serial dilution in DMSO and added to the assay to give the appropriate final concentration. After a 60-minute incubation at 22° C., the reaction was measured using a PerkinElmer EnVision multimode plate reader via TR-FRET dual wavelength detection, and the percent of control (POC) calculated using a ratiometric emission factor. 100 POC was determined using no test compounds and 0 POC was determined using a concentration of control compound that completely inhibits the enzyme. The POC values were fit to a 4 parameter logistic curve and the IC50 value is point where the curve crosses 50 POC.
Measurement: IC50 = 350 nM (pChEMBL 6.460000038146973)
Descriptors: MW 403.49, ALogP 3.83, HBA 8, HBD 1, PSA 82.68, QED 0.55
Compound (SMILES): CCC[C@@H](CCO)Nc1nc(N)nc(C)c1Cc1ccc(CN(CC)CCC(=O)O)cc1OC
InChIKey: WKSQSVJHSIYNRQ-FQEVSTJZSA-N
Target: Voltage-gated inwardly rectifying potassium channel KCNH2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q12809
Assay: hERG Analysis-Method 2: The hERG potassium current was measured in a hERG-stably-expressing Chinese hamster ovary K1 (CHO) cells. The experiments were performed using an automated planar patch-clamp system QPATCH HT (Sophion Bioscience A/S). The application of pressure for forming gigaseals and whole-cell patch clamp configuration were established using the QPATCH assay software. Patch-clamp experiments were performed in voltage-clamp mode and whole-cell currents were recorded from individual cells. The following stimulation protocol was applied to investigate the effects of compounds on hERG potassium channel. The membrane potential was held at −80 mV and repetitively (every 15 s) depolarized to +20 mV for 5 s after the pulse to −50 mV for 20 ms served to define the baseline, followed by repolarizing step to −50 mV for 5 s to evaluate of the tail current amplitude. Experiments were conducted at room temperature (22±2° C.).Effects of compounds were determined from cumulative applications of increasing 4 concentrations and calculated as percent of blocked current. The data points were fitted with Hill equation to calculate half-maximal inhibition concentrations. The test solution includes:Extracellular solution (mM): 2 mM of CaCl2, 1 mM of MgCl2, 10 mM of HEPES, 4 mM of KCl, 145 mM of NaCl, and 10 mM of Glucose; andIntracellular solution (mM): 5.4 mM of CaCl2, 1.8 mM of MgCl2, 10 mM of HEPES, 31 mM of KOH, 10 mM of EGTA, 120 mM of KCl, and 4 mM of ATP.
Measurement: IC50 > 10000 nM (pChEMBL 5)
Descriptors: MW 473.62, ALogP 3.23, HBA 8, HBD 4, PSA 133.83, QED 0.31
Compound (SMILES): CNCC1(c2ccc(OC(F)(F)F)cc2)CCCCC1O
InChIKey: YDFJSUAVFRHHMT-UHFFFAOYSA-N
Target: Sodium-dependent dopamine transporter (SINGLE PROTEIN), Homo sapiens
UniProt: Q01959
Assay: Functional Uptake Assay (hDAT): Inhibition of human dopamine reuptake transporter was assayed using the recombinant human dopamine transporter expressed in either CHO-K1 or HEK293 cells using a published method (Pristupa, Z. B. et al., Mol. Pharmacol. 45: 125-135, 1994). Either CHO-K1 or HEK293 cells expressing human recombinant dopamine transporter were plated before the assay. Test compound and/or vehicle was preincubated with cells in modified HEPES buffer pH 7.1 or pH 7.4 for 20 minutes at 18 to 25° C. and 50 nM [3H]dopamine was then added for an additional timed incubation period (10 to 30 minutes). After washing the cells to remove [3H]dopamine not internalized, the cells were lysed, and the amount of tritium in the lysate was measured using a liquid scintillation counter to determine [3H]dopamine uptake. Non-specific binding of tritium was measured in a control reaction containing 10 uM nomifensine, and was subtracted from the counts for assays to correct for non-specific binding of tritium. Reduction of [3H]dopamine uptake by 50 percent or more (50%) relative to an uninhibited control reaction indicates significant inhibitory activity. Compounds were screened at 10, 1, 0.1, 0.01 and 0.001 uM. The reference compound for the assay was nomifensine, for which the IC50 value of 11 nM was obtained in a typical experiment.
Measurement: IC50 > 10000 nM (pChEMBL 5)
Descriptors: MW 303.32, ALogP 2.98, HBA 3, HBD 2, PSA 41.49, QED 0.9
Compound (SMILES): CCC(CC)n1cc(-c2nc(-c3cnn(C4CN(C(=O)C5(O)CC5)C4)c3)cn3nccc23)cn1
InChIKey: PFOVYMSLQMATJJ-UHFFFAOYSA-N
Target: Tyrosine-protein kinase JAK1 (SINGLE PROTEIN), Homo sapiens
UniProt: P23458
Assay: JAK1 Inhibition Assay: Compounds of Formula I were screened for their ability to inhibit JAK1 using the general enzyme inhibition assay method, in which the assay mixture contained 1 mM ATP, 8 μM Omnia Y12 peptide (Catalog # IVGN KPZ3121C; Invitrogen Corporation, Carlsbad, Calif.) and 12.5 nM JAK1 in a total volume of 25 μL. JAK1 was purchased from Invitrogen Corporation, Carlsbad, Calif. (catalog # IVGN PV4775).
Measurement: IC50 = 550 nM (pChEMBL 6.260000228881836)
Descriptors: MW 460.54, ALogP 2.73, HBA 9, HBD 1, PSA 106.37, QED 0.45
Compound (SMILES): Cc1cc(Br)cc([C@H](CC(=O)O)NC(=O)CNC(=O)c2cc(O)cc(NC3=NCC(O)CN3)c2)c1
InChIKey: OBOJFQICXTYCRY-IBGZPJMESA-N
Target: Integrin alpha-V/beta-8 (PROTEIN COMPLEX), Homo sapiens
UniProt: P26012
Assay: Solid Phase Receptor Assay (SPRA) for alphaVbeta8: Recombinant human LAP protein (R&D Systems, Inc, 246-LP) diluted to 0.5 μg/mL in TBS+ buffer (25 mM Tris pH 7.4, 137 mM NaCl, 2.7 mM KCl, 1 mM CaCl2, 1 mM MgCl2, 1 mM MnCl2) was added to wells (50 μl/well) of a 96-well half-well transparent microtiter plate (Greiner 675061), and incubated overnight at 4° C. Wells were washed 3 times with 150 μL TBS+ and 150 μL of blocking buffer (TBS+ with 1% bovine serum albumin, Sigma A7906) were added. The plate was incubated for 1 hr at 37° C. and then washed 3× with TBS+. Recombinant human integrin αvβ8 (R&D Systems, 4135-AV) was diluted to 0.1 μg/mL in TBS+/0.1% bovine serum albumin. Compounds were diluted 1:100 into the integrin solution and 50 μL added to empty wells of the washed LAP-coated plate according to a standard template with each sample repeated in triplicate. After incubation for two hours at room temperature, the plate was washed 3× with 150 μL of TBS+. To each well, 50 μL of biotinylated anti-αv antibody (R&D Systems, BAF1219) at 1 μg/mL in TBS+/0.1% BSA were added and the plate was covered and incubated for 1 hr at room temperature. After washing the plate 3× with 150 μL of TBS+ buffer, 50 μL of streptavidin-conjugated horseradish peroxidase (R&D Systems, DY998) diluted in TBS+ blocking buffer were added to the wells and the plate incubated for 20 min at room temperature. The plate was washed 3× with TBS+ followed by 50 μL of TMB substrate (Sigma T4444) added to each well and the plate incubated for 20 min at room temperature. Plates were read by colorimetric detection at 650 nm wavelength using a Tecan Safire II plate reader. Concentration-response curves were constructed by non-linear regression (best fit) analysis, and IC50 values were calculated for each compound.
Measurement: IC50 = 0.6 nM (pChEMBL 9.220000267028809)
Descriptors: MW 548.39, ALogP 1.26, HBA 8, HBD 7, PSA 172.38, QED 0.26