lovingscience/chembl-v37-raw-activities huggingface dataset:
Compound (SMILES): COC(=O)Nc1ccc2c(c1)NC(=O)[C@H](C)CCC[C@H](N1CCC(c3c(C(C)=O)ccc(Cl)c3F)=CC1=O)c1cc(F)cc-2c1
InChIKey: WKGAFUMVYFTGCQ-LBEKAKSKSA-N
Target: Coagulation factor XI (SINGLE PROTEIN), Homo sapiens
UniProt: P03951
Assay: Inhibition Assay: Factor XIa determinations were made in 50 mM HEPES buffer at pH 7.4 containing 145 mM NaCl, 5 mM KCl, and 0.1% PEG 8000 (polyethylene glycol; JT Baker or Fisher Scientific). Determinations were made using purified human Factor XIa at a final concentration of 25-200 pM (Haematologic Technologies) and the synthetic substrate S-2366 (pyroGlu-Pro-Arg-pNA; CHROMOGENIX® or AnaSpec) at a concentration of 0.0002-0.001 M.
Measurement: Ki = 0.23 nM (pChEMBL 9.640000343322754)
Descriptors: MW 636.1, ALogP 7.78, HBA 5, HBD 2, PSA 104.81, QED 0.28
Compound (SMILES): COCCOCCOCCO[C@H]1CC[C@@]2(O)C3Cc4ccc(O)c5c4[C@@]2(CCN3C)[C@H]1O5
InChIKey: ONUDGLVFWSENGG-FVDYRSBZSA-N
Target: Mu-type opioid receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P35372
Assay: HiRange Homogenous Time-Resolved Fluorescence (HTRF) Assay: Briefly, suspensions of cells expressing either the mu, kappa or delta opioid receptors were prepared in buffer containing 0.5 mM isobutyl-methyl xanthine (IBMX). Cells were incubated with varying concentrations of PEG-opioid conjugates and 3 uM forskolin for 30 minutes at room temperature. cAMP was detected following a two-step assay protocol per the manufacturer's instructions and time resolved fluorescence was measured with the following settings: 330 nm excitation; 620 nm and 665 nm emission; 380 nm dichroic mirror. The 665 nm/620 nm ratio is expressed as Delta F % and test compound-related data is expressed as a percentage of average maximum response in wells without forskolin. EC50 values were calculated for each compound from a sigmoidal dose-response plot of concentrations versus maximum response.
Measurement: EC50 = 270 nM (pChEMBL 6.570000171661377)
Descriptors: MW 449.54, ALogP 1.24, HBA 8, HBD 2, PSA 89.85, QED 0.52
Compound (SMILES): CN(c1ccc(C#N)c(F)c1)[C@@H]1CCN(c2ncnc3[nH]ccc23)C1
InChIKey: UIBBXLYNSIGGKF-CQSZACIVSA-N
Target: Tyrosine-protein kinase JAK1 (SINGLE PROTEIN), Homo sapiens
UniProt: P23458
Assay: Enzymatic Assay: JAK1/2/3 kinase assay are performed in vitro using Kit-Tyr 6 Peptide (Invitrogen, Cat. No. PV4122). TYK2 kinase assay are performed in vitro using Z'-LYTE™ Kinase Assay Kit-Tyr 3 Peptide (Invitrogen, Cat. No. PV3192). Recombinant human JAK1/2/3 or TYK2 catalytic domains are from Invitrogen (Cat No. PV4774/PV4210/PV3855/PV4790); All reactions (20 μL) are started by adding 2.5 μL of the testing compound in 4% DMSO solution, 5 μL of Kinase/Peptide substrate Mixture (3.2, 0.04, 0.2 or 8 μg/mL for Recombinant human JAK1/2/3 catalytic domains, 4 μM for Z-LYTE™ Tyr 6 peptide or Z-LYTE™ Tyr 3 peptide) or Phospho-Peptide solution (Invitrogen, Cat. No. PV3192, diluted with 1.33× Kinase Buffer), 2.5 μL ATP Solution (300/100/40/100M, JAK1/JAK2/JAK3/TYK2) or 1.33× Kinase Buffer (Invitrogen, Cat. No. PV3189, 5× diluted with distilled water). The 384-well assay plate (Corning, Cat. No. 3575) is mixed and incubated at room temperature for 1 hour. 5 μL of the Development Solution (Dilute Development Reagent A (Cat. No. PV3297) is diluted to 1/64 with Development Buffer (Cat. No. PV3127) for JAK1, JAK2 and JAK3 assay; Development Reagent A (Cat. No. PV3297) is diluted to 1/2048 with Development Buffer (Cat. No. PV3127) for TYK2 assay. The diluted Development Solution is then added to each well, mixed and incubated at room temperature for another 1 hour. The kinase reaction is then stopped by adding 5 μL of the Stop Reagent (Invitrogen, Cat. No. PV3094), and the plate is read with Wallac 1420 VICTOR3 Multilabel Counter (PerkinElmer™) at 445 nm and 520 nm fluorescence. All compounds are initially tested at 8 different concentrations (1 μM down to 0.0003 μM) using a 1:3 serial dilution scheme.
Measurement: IC50 = 0.055 nM (pChEMBL 10.260000228881836)
Descriptors: MW 336.37, ALogP 2.68, HBA 5, HBD 1, PSA 71.84, QED 0.8
Compound (SMILES): COc1ccc(C(=O)Nc2ccc(F)c([C@]34COC(CF)[C@H]3S(=O)(=O)C(C)(C)C(=N)N4)c2)nc1
InChIKey: POWOFNJUGKWUOO-WMISUKIASA-N
Target: Beta-secretase 2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9Y5Z0
Assay: Time-Resolved Endpoint Proteolysis Assay: Inhibitor IC50s at purified human autoBACE-2 are determined in a time-resolved endpoint proteolysis assay that measures hydrolysis of the QSY7-EISEVNLDAEFC-Eu-amide FRET peptide substrate (BACE-HTRF assay). BACE-mediated hydrolysis of this peptide results in an increase in relative fluorescence (RFU) at 620 nm after excitation with 320 nm light. 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% fin
Measurement: Ki = 96 nM (pChEMBL 7.019999980926514)
Descriptors: MW 494.52, ALogP 2.19, HBA 7, HBD 3, PSA 130.47, QED 0.58
Compound (SMILES): C[C@@H]1CCC[C@H](N2CCC(c3c(F)ccc(Cl)c3F)=CC2=O)c2cc(ccn2)-c2c(C#N)cccc2NC1=O
InChIKey: VPKWFQMGARBSGB-NSYGIPOTSA-N
Target: Coagulation factor XI (SINGLE PROTEIN), Homo sapiens
UniProt: P03951
Assay: Inhibition Assay: Factor XIa determinations were made in 50 mM HEPES buffer at pH 7.4 containing 145 mM NaCl, 5 mM KCl, and 0.1% PEG 8000 (polyethylene glycol; JT Baker or Fisher Scientific). Determinations were made using purified human Factor XIa at a final concentration of 25-200 pM (Haematologic Technologies) and the synthetic substrate S-2366 (pyroGlu-Pro-Arg-pNA; CHROMOGENIX® or AnaSpec) at a concentration of 0.0002-0.001 M.
Measurement: Ki = 98.6 nM (pChEMBL 7.010000228881836)
Descriptors: MW 547.01, ALogP 6.67, HBA 4, HBD 1, PSA 86.09, QED 0.36
Compound (SMILES): Cc1nsc(-c2nnc3n2CCN(C(=O)c2cc(F)cc(F)c2)[C@@H]3C)n1
InChIKey: IQKKFYUURGEICP-MRVPVSSYSA-N
Target: Substance-K receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P21452
Assay: Binding Assay: The affinity of compounds of the invention for the NK-2 receptor was evaluated in CHO recombinant cells which express the human NK-2 receptor. Membrane suspensions were prepared from these cells. The following radioligand [125I]-Neurokinin A (PerkinElmer Cat#NEX252) was used in this assay. Binding assays were performed in a 25 mM HEPES/1 mM CaCl2/5 mM MgCl2/0.5% BSA/10 μg/ml saponin, at pH 7.4. Binding assays consisted of 25 μl of membrane suspension (approximately 3.75 μg of protein/well in a 96 well plate), 50 μl of compound or reference ligand (Neurokinin A) at increasing concentrations (diluted in assay buffer) and 0.1 nM [125I]-Neurokinin A. The plate was incubated 60 min at 25° C. in a water bath and then filtered over GF/C filters (Perkin Elmer, 6005174, presoaked in assay buffer without saponine for 2 h at room temperature) with a Filtration unit (Perkin Elmer). The radioactivity retained on the filters was measured by using the TopCount-NXT reader (Packard).
Measurement: Ki > 30000 nM (pChEMBL 4.5229997634887695)
Descriptors: MW 376.39, ALogP 2.6, HBA 7, HBD 0, PSA 76.8, QED 0.69
Compound (SMILES): Cc1nnc(Nc2ccc3c(c2)NC(=O)[C@H](C)CCC[C@H](N2CCC(c4c(F)ccc(Cl)c4F)=CC2=O)c2cc-3ccn2)o1
InChIKey: WNNVCOPVUBHDRW-CRYYWNKWSA-N
Target: Coagulation factor XI (SINGLE PROTEIN), Homo sapiens
UniProt: P03951
Assay: Inhibition Assay: Factor XIa determinations were made in 50 mM HEPES buffer at pH 7.4 containing 145 mM NaCl, 5 mM KCl, and 0.1% PEG 8000 (polyethylene glycol; JT Baker or Fisher Scientific). Determinations were made using purified human Factor XIa at a final concentration of 25-200 pM (Haematologic Technologies) and the synthetic substrate S-2366 (pyroGlu-Pro-Arg-pNA; CHROMOGENIX® or AnaSpec) at a concentration of 0.0002-0.001 M.
Measurement: Ki = 0.46 nM (pChEMBL 9.34000015258789)
Descriptors: MW 619.07, ALogP 7.23, HBA 7, HBD 2, PSA 113.25, QED 0.23
Compound (SMILES): CC[C@@H]1c2nnc(-c3nc(C)ns3)n2CCN1C(=O)c1ccc(F)cc1
InChIKey: OZKSTTVUQRWBIO-CYBMUJFWSA-N
Target: Substance-K receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P21452
Assay: Binding Assay: The affinity of compounds of the invention for the NK-2 receptor was evaluated in CHO recombinant cells which express the human NK-2 receptor. Membrane suspensions were prepared from these cells. The following radioligand [125I]-Neurokinin A (PerkinElmer Cat#NEX252) was used in this assay. Binding assays were performed in a 25 mM HEPES/1 mM CaCl2/5 mM MgCl2/0.5% BSA/10 μg/ml saponin, at pH 7.4. Binding assays consisted of 25 μl of membrane suspension (approximately 3.75 μg of protein/well in a 96 well plate), 50 μl of compound or reference ligand (Neurokinin A) at increasing concentrations (diluted in assay buffer) and 0.1 nM [125I]-Neurokinin A. The plate was incubated 60 min at 25° C. in a water bath and then filtered over GF/C filters (Perkin Elmer, 6005174, presoaked in assay buffer without saponine for 2 h at room temperature) with a Filtration unit (Perkin Elmer). The radioactivity retained on the filters was measured by using the TopCount-NXT reader (Packard).
Measurement: Ki > 30000 nM (pChEMBL 4.5229997634887695)
Descriptors: MW 372.43, ALogP 2.85, HBA 7, HBD 0, PSA 76.8, QED 0.71
Compound (SMILES): Cc1nc(-c2nnc3n2CCN(C(=O)c2ccc(-c4cccs4)cc2)[C@@H]3C)sc1C
InChIKey: KSIGHAFTVLYICN-CQSZACIVSA-N
Target: Transcriptional regulator ERG (SINGLE PROTEIN), Homo sapiens
UniProt: P11308
Assay: hERG Inhibition Assay: The human Ether-a-go-go Related Gene (hERG) encodes the inward rectifying voltage gated potassium channel in the heart (IKr) which is involved in cardiac repolarisation. IKr current inhibition has been shown to elongate the cardiac action potential, a phenomenon associated with increased risk of arrhythmia. IKr current inhibition accounts for the vast majority of known cases of drug-induced QT-prolongation. A number of drugs have been withdrawn from late stage clinical trials due to these cardiotoxic effects, therefore it is important to identify inhibitors early in drug discovery.The hERG inhibition study aims at quantifying the in vitro effects of compounds of the invention on the potassium-selective IKr current generated in normoxic conditions in stably transfected HEK 293 cells with the human ether-a-go-go-related gene (hERG).Whole-cell currents (acquisition by manual patch-clamp) elicited during a voltage pulse were recorded in baseline conditions and following application of tested compounds (5 minutes of exposure). The concentrations of tested compounds (0.3 μM; 3 μM; 10 μM; 30 μM) reflect a range believed to exceed the concentrations at expected efficacy doses in preclinical models.The pulses protocol applied is described as follow: the holding potential (every 3 seconds) was stepped from −80 mV to a maximum value of +40 mV, starting with −40 mV, in eight increments of +10 mV, for a period of 1 second. The membrane potential was then returned to −55 mV, after each of these incremented steps, for 1 second and finally repolarized to −80 mV for 1 second.The current density recorded were normalized against the baseline conditions and corrected for solvent effect and time-dependent current run-down using experimental design in test compound free conditions.Inhibition curves were obtained for compounds and the concentrations which decreased 50% of the current density determined in the baseline conditions (IC50) were determined. All compounds for which the IC50 value is above 10 M are not considered to be potent inhibitors of the hERG channel whereas compounds with IC50 values below 1 μM are considered potent hERG channel inhibitors.
Measurement: IC50 = 12000 nM (pChEMBL 4.920000076293945)
Descriptors: MW 435.58, ALogP 4.96, HBA 7, HBD 0, PSA 63.91, QED 0.46
Compound (SMILES): COC(=O)Nc1ccc2c(c1)NC(=O)[C@H](C)CCC[C@H](N1CCC(c3c(F)ccc(Cl)c3F)=CC1=O)c1nc-2c(C)[nH]1
InChIKey: UWCHLKPJXDNUJJ-CMJOXMDJSA-N
Target: Coagulation factor XI (SINGLE PROTEIN), Homo sapiens
UniProt: P03951
Assay: Inhibition Assay: Factor XIa determinations were made in 50 mM HEPES buffer at pH 7.4 containing 145 mM NaCl, 5 mM KCl, and 0.1% PEG 8000 (polyethylene glycol; JT Baker or Fisher Scientific). Determinations were made using purified human Factor XIa at a final concentration of 25-200 pM (Haematologic Technologies) and the synthetic substrate S-2366 (pyroGlu-Pro-Arg-pNA; CHROMOGENIX® or AnaSpec) at a concentration of 0.0002-0.001 M.
Measurement: Ki = 0.2 nM (pChEMBL 9.699999809265137)
Descriptors: MW 598.05, ALogP 6.61, HBA 5, HBD 3, PSA 116.42, QED 0.29
Compound (SMILES): COc1ccc2c3c1O[C@H]1[C@@]4(OC)CC[C@@]5(C[C@@H]4COCc4ccccc4)[C@@H](C2)NCC[C@]315
InChIKey: FDYCYWQDIVTVDR-RHRQCQTHSA-N
Target: Delta-type opioid receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P41143
Assay: Radioligand Dose-Displacement Binding Assay: Delta-opioid Receptor Binding Assay Procedures were conducted as follows. Radioligand dose-displacement assays used 0.3 nM [3H]-Naltrindole (Perkin Elmer, Shelton, Conn.; 33.0 Ci/mmole) with 5 ug membrane protein (Perkin Elmer, Shelton, Conn.) in a final volume of 500 ul binding buffer (5 mM MgCl2, 5% DMSO, 50 mM Trizma base, pH 7.4). Non-specific binding was determined in the presence of 25 uM unlabeled naloxone. All reactions were performed in 96-deep well polypropylene plates for 1 hr at a temperature of about 25° C. Binding reactions were terminated by rapid filtration onto 96-well Unifilter GF/C filter plates (Perkin Elmer, Shelton, Conn.) presoaked in 0.5% polyethylenimine (Sigma). Harvesting was performed using a 96-well tissue harvester (Perkin Elmer, Shelton, Conn.) followed by five filtration washes with 500 ul ice-cold binding buffer. Filter plates were subsequently dried at 50° C. for 1-2 hours.
Measurement: Ki = 341.52 nM (pChEMBL 6.46999979019165)
Descriptors: MW 447.58, ALogP 4.01, HBA 5, HBD 1, PSA 48.95, QED 0.73
Compound (SMILES): C[C@]1(c2nc(NC(=O)c3ncc(C(F)F)nc3N)ccc2F)N=C(N)OCC1(F)F
InChIKey: VJQYWRACFCYUPR-OAHLLOKOSA-N
Target: Amyloid-beta precursor protein (SINGLE PROTEIN), Homo sapiens
UniProt: P05067
Assay: Inhibition Assay: Chinese hamster ovary cells are transfected with the human gene for amyloid precursor protein. The cells are plated at a density of 8000 cells/well into 96-well microtiter plates and cultivated for 24 hours in DMEM cell culture medium containing 10% FCS. The test compound is added to the cells at various concentrations, and the cells are cultivated for 24 hours in the presence of the test compound. The supernatants are collected, and the concentration of amyloid peptide 1-40 is determined using state of the art immunoassay techniques, for example sandwich ELISA, homogenous time-resolved fluorescence (HTRF) immunoassay, or electro-chemiluminescence immunoassay. The potency of the compound is calculated from the percentage of inhibition of amyloid peptide release as a function of the test compound concentration.
Measurement: IC50 = 60 nM (pChEMBL 7.21999979019165)
Descriptors: MW 431.33, ALogP 1.98, HBA 8, HBD 3, PSA 141.4, QED 0.63
Compound (SMILES): O=C(N[C@@H]1CCN(C[C@H]2CC[C@H](c3nnn4cnc5[nH]ccc5c34)CC2)C1)C(F)(F)F
InChIKey: KEVBCHWWBKDFLB-MGPQQGTHSA-N
Target: Non-receptor tyrosine-protein kinase TYK2 (SINGLE PROTEIN), Homo sapiens
UniProt: P29597
Assay: Enzyme Assay: JAK1, JAK2, JAK3 and Tyk2 were purchased from Carna Biosciences, Inc. As the substrate, LANCE Ultra ULight-JAK1 Peptide (manufactured by PerkinElmer Co., Ltd. (PE)) was used. Dilute solutions of compounds and enzymes in assay buffer (50 mM HEPES pH7.5, 1 mM EGTA, 1 mM MgCl2, 2 mM DTT, 0.01% Tween20) were dispensed into wells of a 384-well black plate. After 5 minutes of preincubation, dilute solutions of the substrate and ATP (adenosine triphosphate) were added at a final concentration of 100 μM, and the plate was incubated at room temperature for 2 hours. After addition of a termination reagent containing EDTA (ethylenediamine tetraacetic acid) at a final concentration of 10 mM, LANCE Eu-W1024 Anti-phosphotyrosine (PT66) (manufactured by PE) was added, and after 1 hour of incubation, the fluorescences were measured with ARVO-HTS.
Measurement: IC50 = 860 nM (pChEMBL 6.070000171661377)
Descriptors: MW 435.45, ALogP 2.63, HBA 6, HBD 2, PSA 91.21, QED 0.66
Compound (SMILES): COCCOCCOCCOCCOCCOCCO[C@H]1CC[C@@]2(O)C3Cc4ccc(O)c5c4[C@@]2(CCN3C)[C@H]1O5
InChIKey: OULSSTIBLLAHID-FYJXLGDTSA-N
Target: Mu-type opioid receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P35372
Assay: HiRange Homogenous Time-Resolved Fluorescence (HTRF) Assay: Briefly, suspensions of cells expressing either the mu, kappa or delta opioid receptors were prepared in buffer containing 0.5 mM isobutyl-methyl xanthine (IBMX). Cells were incubated with varying concentrations of PEG-opioid conjugates and 3 uM forskolin for 30 minutes at room temperature. cAMP was detected following a two-step assay protocol per the manufacturer's instructions and time resolved fluorescence was measured with the following settings: 330 nm excitation; 620 nm and 665 nm emission; 380 nm dichroic mirror. The 665 nm/620 nm ratio is expressed as Delta F % and test compound-related data is expressed as a percentage of average maximum response in wells without forskolin. EC50 values were calculated for each compound from a sigmoidal dose-response plot of concentrations versus maximum response.
Measurement: EC50 = 415 nM (pChEMBL 6.380000114440918)
Descriptors: MW 581.7, ALogP 1.29, HBA 11, HBD 2, PSA 117.54, QED 0.23
Compound (SMILES): C[C@H](C1CC1)N(Cc1nc2c(c(=O)[nH]1)COCC2)C(=O)CN1CCC(C(=O)c2ccc(F)cc2)CC1
InChIKey: KGBRRRXNQADTEZ-QGZVFWFLSA-N
Target: Poly [ADP-ribose] polymerase 2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9UGN5
Assay: Biochemical Assay: The autoparsylation activity of the TNKS 1/2 or PARP1/2 enzymes was measured by the liquid chromatography-mass spectrometry (LC/MS) detection of nicotinamide as readout. Compound activity in inhibiting the TNKS and PARP autoparsylation was evaluated by IC50 measurements. In the compound screening assays, the reaction is composed of 5 μL of compound in 8-point serial dilutions with concentrations ranging from 0.0086 to 18.75 μM, 20 nM of purified enzyme, and 250 μM of β-NAD+ in the 1× Assay Buffer. After 60 min incubation at room temperature, the reactions were quenched by the addition of 10 μL of 5× quenching solution (20% formic acid and 500 nM [d]-nicotinamide in water). For the background control wells, 10 μL of the 5× quenching solution per well was added prior to the addition of β-NAD+.
Measurement: IC50 > 19000 nM (pChEMBL 4.7210001945495605)
Descriptors: MW 496.58, ALogP 2.7, HBA 6, HBD 1, PSA 95.6, QED 0.56
Compound (SMILES): COc1ccc2c3c1O[C@H]1[C@@]4(OC)CC[C@@]5(C[C@@H]4CN(Cc4ccccc4)C(C)=O)[C@@H](C2)N(CC2CC2)CC[C@]315
InChIKey: QERDWLFJAPHTPD-DPODCYPTSA-N
Target: Delta-type opioid receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P41143
Assay: Radioligand Dose-Displacement Binding Assay: Delta-opioid Receptor Binding Assay Procedures were conducted as follows. Radioligand dose-displacement assays used 0.3 nM [3H]-Naltrindole (Perkin Elmer, Shelton, Conn.; 33.0 Ci/mmole) with 5 ug membrane protein (Perkin Elmer, Shelton, Conn.) in a final volume of 500 ul binding buffer (5 mM MgCl2, 5% DMSO, 50 mM Trizma base, pH 7.4). Non-specific binding was determined in the presence of 25 uM unlabeled naloxone. All reactions were performed in 96-deep well polypropylene plates for 1 hr at a temperature of about 25° C. Binding reactions were terminated by rapid filtration onto 96-well Unifilter GF/C filter plates (Perkin Elmer, Shelton, Conn.) presoaked in 0.5% polyethylenimine (Sigma). Harvesting was performed using a 96-well tissue harvester (Perkin Elmer, Shelton, Conn.) followed by five filtration washes with 500 ul ice-cold binding buffer. Filter plates were subsequently dried at 50° C. for 1-2 hours.
Measurement: Ki = 387.09 nM (pChEMBL 6.409999847412109)
Descriptors: MW 542.72, ALogP 4.97, HBA 5, HBD 0, PSA 51.24, QED 0.47
Compound (SMILES): C[C@@H]1CCC[C@H](N2CCC(c3c(-n4cnnn4)ccc(Cl)c3F)=CC2=O)c2cc(ccn2)-c2ccccc2NC1=O
InChIKey: JSZAYFVPBHXVCY-CJAUYULYSA-N
Target: Coagulation factor XI (SINGLE PROTEIN), Homo sapiens
UniProt: P03951
Assay: Inhibition Assay: Factor XIa determinations were made in 50 mM HEPES buffer at pH 7.4 containing 145 mM NaCl, 5 mM KCl, and 0.1% PEG 8000 (polyethylene glycol; JT Baker or Fisher Scientific). Determinations were made using purified human Factor XIa at a final concentration of 25-200 pM (Haematologic Technologies) and the synthetic substrate S-2366 (pyroGlu-Pro-Arg-pNA; CHROMOGENIX® or AnaSpec) at a concentration of 0.0002-0.001 M.
Measurement: Ki = 0.05 nM (pChEMBL 10.300000190734863)
Descriptors: MW 572.04, ALogP 5.63, HBA 7, HBD 1, PSA 105.9, QED 0.34
Compound (SMILES): C[C@@H]1c2nnc(-c3nc(C(F)F)ns3)n2CCN1C(=O)c1ccc(F)cc1
InChIKey: NJRIZUYLVJHUMO-MRVPVSSYSA-N
Target: Substance-K receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P21452
Assay: Binding Assay: The affinity of compounds of the invention for the NK-2 receptor was evaluated in CHO recombinant cells which express the human NK-2 receptor. Membrane suspensions were prepared from these cells. The following radioligand [125I]-Neurokinin A (PerkinElmer Cat#NEX252) was used in this assay. Binding assays were performed in a 25 mM HEPES/1 mM CaCl2/5 mM MgCl2/0.5% BSA/10 μg/ml saponin, at pH 7.4. Binding assays consisted of 25 μl of membrane suspension (approximately 3.75 μg of protein/well in a 96 well plate), 50 μl of compound or reference ligand (Neurokinin A) at increasing concentrations (diluted in assay buffer) and 0.1 nM [125I]-Neurokinin A. The plate was incubated 60 min at 25° C. in a water bath and then filtered over GF/C filters (Perkin Elmer, 6005174, presoaked in assay buffer without saponine for 2 h at room temperature) with a Filtration unit (Perkin Elmer). The radioactivity retained on the filters was measured by using the TopCount-NXT reader (Packard).
Measurement: Ki > 30000 nM (pChEMBL 4.5229997634887695)
Descriptors: MW 394.38, ALogP 3.09, HBA 7, HBD 0, PSA 76.8, QED 0.68
Compound (SMILES): Cc1cncc(-c2cc3c(cn2)cnn3-c2cccc(N3CCNC[C@H]3C)n2)n1
InChIKey: KTRUDSZOTYPXRK-OAHLLOKOSA-N
Target: Serine/threonine-protein kinase pim-1 (SINGLE PROTEIN), Homo sapiens
UniProt: P11309
Assay: In Vitro Cell Proliferation Potency Assay: BaF3, a murine interleukin-3 dependent pro-B cell line, parental cells, BaF3 PIM1 cells, BaF3 PIM2 cells, and MM1.S (multiple myeloma) cells were seeded at 2 k/well, 5 k/well, 5 k/well, and 10 k/well respectively, in a 384-well plate, at 45 μL/well. Test compound was added at 5 μL/well. BaF3 cells (parental and transfected) were incubated overnight, while MM1.S cells were incubated for 72 hours at 37° C., 5% CO2. CELL TITER GLO® Reagent (Promega) was added at 50 μL/well, the plates were incubated for 30 minutes, and their luminescence read on an HT Analyst.
Measurement: IC50 = 919 nM (pChEMBL 6.039999961853027)
Descriptors: MW 386.46, ALogP 2.38, HBA 8, HBD 1, PSA 84.65, QED 0.58
Compound (SMILES): CCOC(=O)OC(CC)n1nnc(-c2c(-c3ccc(C(=O)N(c4ncccc4C)[C@@H]4CCCNC4)cc3)cnn2C)n1
InChIKey: XTKBKDFZXZYCQE-LETIRJCYSA-N
Target: Proprotein convertase subtilisin/kexin type 9 (SINGLE PROTEIN), Homo sapiens
UniProt: Q8NBP7
Assay: Cell Free Assay: In order to eliminate the permeability barrier inherent to the WT7 and SCHH cell-based assays a cell-free system was also established to access compound activity. A sequence containing the full length human PCSK9 (NCBI reference identifier, NM-174936.3, where coding sequence start annotated at position 363) along with 84 additional 3â¿² nucleotides, comprising a V5 tag and polylinkinker followed by an in frame modified firefly luciferase reporter (corresponding to nucleotide positions 283-1929 of pGL3, Gen Bank reference identifier JN542721.1) was cloned into the pT7CFE1 expression vector (ThermoScientific). The construct was then in-vitro transcribed using the MEGAscript T7 Kit (Life Technologies) and RNA subsequently purified incorporating the MEGAclear Kit (Life Technologies) according to manufacturer's protocols. HeLa cell lysates were prepared following the protocols described by Mikami (reference is Cell-Free Protein Synthesis Systems with Extracts from Cultured Human Cells, S. Mikami, T. Kobayashi and H. Imataka; from Methods in Molecular Biology, vol. 607, pages 43-52, Y. Endo et al. (eds.), Humana Press, 2010) with the following modifications.
Measurement: IC50 = 23700 nM (pChEMBL 4.619999885559082)
Descriptors: MW 573.66, ALogP 3.92, HBA 12, HBD 1, PSA 142.18, QED 0.29
Compound (SMILES): COC(=O)CCCC[S@@](C)(=O)=NC(=O)c1cnc(N)c(C#Cc2cccc(NC(=O)c3occc3C)c2)c1
InChIKey: SXGZAYZKUGJQPT-KXQOOQHDSA-N
Target: Platelet-derived growth factor receptor beta (SINGLE PROTEIN), Homo sapiens
UniProt: P09619
Assay: PDGFRbeta : Automated FLIPR (Fluorometric Imaging Plate Reader) technology was used to screen for inhibitors of PDGF-induced increases in intracellular calcium levels in fluorescent dye loaded endothelial cells. NHDF-Ad (Normal Human Dermal Fibroblasts, Adult; Lonza) were seeded in 384-well fibronectin coated black-walled plates overnight @ 37° C./5% CO2. Cells were loaded with calcium indicator Fluo-4 for 45 minutes at 37° C. Cells were washed 2 times (Elx405, Biotek Instruments) to remove extracellular dye. For screening, cells were pre-incubated with test agents for 30 minutes, at a single concentration (10 uM) or at concentrations ranging from 0.0001 to 10.0 uM followed by PDGF-BB stimulation (30 ng/mL). Changes in fluorescence at 516 nm were measured simultaneously in all 384 wells using a cooled CCD camera. Data were generated by determining max-min fluorescence levels for unstimulated, stimulated, and drug treated samples.
Measurement: IC50 = 86 nM (pChEMBL 7.070000171661377)
Descriptors: MW 536.61, ALogP 3.8, HBA 8, HBD 2, PSA 153.95, QED 0.25
Compound (SMILES): CO[C@@H]1CC[C@@H](n2c(=O)n(C)c3cnc4ccc(-c5ccc(OCCCN(C)C)nc5)cc4c32)C1
InChIKey: ZPOCWOPPQUMNCW-NHCUHLMSSA-N
Target: Serine/threonine-protein kinase mTOR (SINGLE PROTEIN), Homo sapiens
UniProt: P42345
Assay: Cellular Assay: Assay d) MDA-MB-468 cells (human breast adenocarcinoma ##ATCC HTB 132) were seeded at 1500 cells/well in 40 μl of DMEM containing 10% FBS and 1% glutamine into Greiner 384 well black flat-bottomed plates. Cell plates were incubated for 18 h in a 37° C. incubator before dosing with compounds of formula (I) in 100% DMSO using acoustic dispensing. Compounds were dosed in a 12 point concentration range into a randomised plate map. Control wells were generated either by dosing of 100% DMSO (max signal) or addition of a reference compound (a PI3K-β inhibitor) that completely eliminated the pAKT signal (min control). Plates were incubated, at 37° C. for 2 h, cells were then fixed by the addition of 10 μl of a 3.7% formaldehyde solution. After 30 minutes the plates were washed with PBS using a Tecan PW384 plate washer. Wells were blocked and cells permeabilised with the addition of 40 μl of PBS containing 0.5% Tween20 and 1% Marvel™ (dried milk powder) and incubated for 60 minutes at r.t. The plates were washed with PBS containing 0.5% (v/v) Tween20 and 20 μl rabbit anti-phospho AKT Ser473 (Cell Signalling Technologies, #3787) in same PBS-Tween+1% Marvel™ was added and incubated overnight at 4° C. Plates were washed 3 times with PBS 0.05% Tween 20 using a Tecan PW384. 20 μl of secondary antibody Alexa Fluor 488 anti-Rabbit (Molecular Probes, #A11008) diluted in PBS+0.05% Tween20 containing 1% Marvel™ was added to each well and incubated for 1 h at r.t. Plates were washed three times as before then 20 μl PBS added to each well and plates sealed with a black plate sealer. The plates were read on an Acumen plate reader as soon as possible, measuring green fluorescence after excitation with 488 nm laser.
Measurement: IC50 = 1190 nM (pChEMBL 5.920000076293945)
Descriptors: MW 475.59, ALogP 4.02, HBA 8, HBD 0, PSA 74.41, QED 0.36
Compound (SMILES): COC(=O)Nc1ccc2c(c1)NC(=O)[C@H](C)CCC[C@H](N1CCC(c3c(C#N)ccc4c(N)noc34)=CC1=O)c1cc-2cc(OC)n1
InChIKey: ZWUUYRIAOVUGDT-CLYVBNDRSA-N
Target: Coagulation factor XI (SINGLE PROTEIN), Homo sapiens
UniProt: P03951
Assay: Inhibition Assay: Factor XIa determinations were made in 50 mM HEPES buffer at pH 7.4 containing 145 mM NaCl, 5 mM KCl, and 0.1% PEG 8000 (polyethylene glycol; JT Baker or Fisher Scientific). Determinations were made using purified human Factor XIa at a final concentration of 25-200 pM (Haematologic Technologies) and the synthetic substrate S-2366 (pyroGlu-Pro-Arg-pNA; CHROMOGENIX® or AnaSpec) at a concentration of 0.0002-0.001 M.
Measurement: Ki = 33.2 nM (pChEMBL 7.480000019073486)
Descriptors: MW 635.68, ALogP 5.65, HBA 10, HBD 3, PSA 185.7, QED 0.25
Compound (SMILES): O[C@H](CNC[C@H]1CC[C@H](c2nnn3cnc4[nH]ccc4c23)CC1)c1ccccc1
InChIKey: FYGIJAJABTZJIG-SZVBFZGTSA-N
Target: Non-receptor tyrosine-protein kinase TYK2 (SINGLE PROTEIN), Homo sapiens
UniProt: P29597
Assay: Enzyme Assay: JAK1, JAK2, JAK3 and Tyk2 were purchased from Carna Biosciences, Inc. As the substrate, LANCE Ultra ULight-JAK1 Peptide (manufactured by PerkinElmer Co., Ltd. (PE)) was used. Dilute solutions of compounds and enzymes in assay buffer (50 mM HEPES pH7.5, 1 mM EGTA, 1 mM MgCl2, 2 mM DTT, 0.01% Tween20) were dispensed into wells of a 384-well black plate. After 5 minutes of preincubation, dilute solutions of the substrate and ATP (adenosine triphosphate) were added at a final concentration of 100 μM, and the plate was incubated at room temperature for 2 hours. After addition of a termination reagent containing EDTA (ethylenediamine tetraacetic acid) at a final concentration of 10 mM, LANCE Eu-W1024 Anti-phosphotyrosine (PT66) (manufactured by PE) was added, and after 1 hour of incubation, the fluorescences were measured with ARVO-HTS.
Measurement: IC50 = 50 nM (pChEMBL 7.300000190734863)
Descriptors: MW 390.49, ALogP 3.2, HBA 6, HBD 3, PSA 91.13, QED 0.47
Compound (SMILES): COC(=O)Nc1ccc2c(c1)NC(=O)[C@H](C)CCC[C@H](N1CC(C)C(c3c(F)ccc(Cl)c3F)=CC1=O)c1cc-2ccn1
InChIKey: NPKCSHLGQLVUNO-ILPYMAIGSA-N
Target: Plasma kallikrein (SINGLE PROTEIN), Homo sapiens
UniProt: P03952
Assay: In Vitro Assay: Plasma kallikrein determinations were made in 0.1 M sodium phosphate buffer at a pH of 7.5 containing 0.1-0.2 M sodium chloride and 0.5% PEG 8000. Determinations were made using purified human plasma kallikrein (Enzyme Research Laboratories) at a final assay concentration of 200 pM and the synthetic substrate S-2302 (H-(D)-Pro-Phe-Arg-pNA; CHROMOGENIX®) at a concentration of 0.00008-0.0004 M.
Measurement: Ki = 6 nM (pChEMBL 8.220000267028809)
Descriptors: MW 609.07, ALogP 7.22, HBA 5, HBD 2, PSA 100.63, QED 0.31
Compound (SMILES): CC(=O)N[C@@H]1CCN(C[C@H]2CC[C@H](c3nnn4cnc5[nH]ccc5c34)CC2)C1
InChIKey: VAMYQOUPUFFEEZ-BZUAXINKSA-N
Target: Non-receptor tyrosine-protein kinase TYK2 (SINGLE PROTEIN), Homo sapiens
UniProt: P29597
Assay: Enzyme Assay: JAK1, JAK2, JAK3 and Tyk2 were purchased from Carna Biosciences, Inc. As the substrate, LANCE Ultra ULight-JAK1 Peptide (manufactured by PerkinElmer Co., Ltd. (PE)) was used. Dilute solutions of compounds and enzymes in assay buffer (50 mM HEPES pH7.5, 1 mM EGTA, 1 mM MgCl2, 2 mM DTT, 0.01% Tween20) were dispensed into wells of a 384-well black plate. After 5 minutes of preincubation, dilute solutions of the substrate and ATP (adenosine triphosphate) were added at a final concentration of 100 μM, and the plate was incubated at room temperature for 2 hours. After addition of a termination reagent containing EDTA (ethylenediamine tetraacetic acid) at a final concentration of 10 mM, LANCE Eu-W1024 Anti-phosphotyrosine (PT66) (manufactured by PE) was added, and after 1 hour of incubation, the fluorescences were measured with ARVO-HTS.
Measurement: IC50 = 610 nM (pChEMBL 6.210000038146973)
Descriptors: MW 381.48, ALogP 2.09, HBA 6, HBD 2, PSA 91.21, QED 0.72
Compound (SMILES): C[C@@H]1c2nnc(-c3nc(C(C)(F)F)no3)n2CCN1C(=O)c1ccc(F)cc1
InChIKey: UZZXVHHPLPQWGG-SECBINFHSA-N
Target: Substance-K receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P21452
Assay: Binding Assay: The affinity of compounds of the invention for the NK-2 receptor was evaluated in CHO recombinant cells which express the human NK-2 receptor. Membrane suspensions were prepared from these cells. The following radioligand [125I]-Neurokinin A (PerkinElmer Cat#NEX252) was used in this assay. Binding assays were performed in a 25 mM HEPES/1 mM CaCl2/5 mM MgCl2/0.5% BSA/10 μg/ml saponin, at pH 7.4. Binding assays consisted of 25 μl of membrane suspension (approximately 3.75 μg of protein/well in a 96 well plate), 50 μl of compound or reference ligand (Neurokinin A) at increasing concentrations (diluted in assay buffer) and 0.1 nM [125I]-Neurokinin A. The plate was incubated 60 min at 25° C. in a water bath and then filtered over GF/C filters (Perkin Elmer, 6005174, presoaked in assay buffer without saponine for 2 h at room temperature) with a Filtration unit (Perkin Elmer). The radioactivity retained on the filters was measured by using the TopCount-NXT reader (Packard).
Measurement: Ki > 30000 nM (pChEMBL 4.5229997634887695)
Descriptors: MW 392.34, ALogP 2.8, HBA 7, HBD 0, PSA 89.94, QED 0.68
Compound (SMILES): C[C@@H]1CCC[C@H](n2cnc(-c3cc(Cl)ccc3-n3cc(Cl)nn3)cc2=O)c2cc(ccn2)-c2c(cnn2C(F)F)NC1=O
InChIKey: FSWFYCYPTDLKON-CMJOXMDJSA-N
Target: Coagulation factor VII (SINGLE PROTEIN), Homo sapiens
UniProt: P08709
Assay: Binding affinity to recombinant human coagulation factor 7a assessed as inhibition constant using N-alpha-Benzyloxycarbonyl-D-arginyl-glycyl-Larginine p-Nitroaniline as substrate measured upto 120 mins by coupled enzyme assay
Measurement: Ki = 1200 nM (pChEMBL 5.920000076293945)
Descriptors: MW 626.46, ALogP 5.8, HBA 10, HBD 1, PSA 125.41, QED 0.27
Compound (SMILES): C[C@]12CCCN1CC1=NNC(=O)c3cc(F)cc4[nH]c2c1c34
InChIKey: DENYZIUJOTUUNY-MRXNPFEDSA-N
Target: Poly [ADP-ribose] polymerase tankyrase-2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9H2K2
Assay: Tankyrase-2 Enzymatic Assay: Tankyrase-2 enzymatic assay was conducted using commercial Tankyrase-2 Chemiluminescent Assay Kit (BPS Biosciences) and the protocol with the kit. GST-fused tankyrase-2 (recombinant protein expressed and purified from Bacluovirus) were coated on a GSH-precoated plate first, and incubated with increasing concentrations of compounds for 0.5 h. Then, biotinylated NAD was added to the wells. The biotinylated auto-PARylation product was measured by adding streptavidin-HRP and HRP substrates which produce chemiluminescence.
Measurement: IC50 = 766 nM (pChEMBL 6.119999885559082)
Descriptors: MW 298.32, ALogP 2.08, HBA 3, HBD 2, PSA 60.49, QED 0.78
Compound (SMILES): CC(Nc1ncc(F)c(N2C(=O)OC[C@@H]2[C@@H](C)O)n1)c1noc(Cc2ccccc2)n1
InChIKey: RUTLACTVCZTWSF-AADWAWOOSA-N
Target: Isocitrate dehydrogenase [NADP] cytoplasmic (SINGLE PROTEIN), Homo sapiens
UniProt: O75874
Assay: Mutant IDH1 Biochemical Assay: LC-MS Detection of 2-HG: Mutant IDH1 R132H catalytic activity was monitored using the quantitative liquid chromatography/mass spectrometry (LC-MS) detection of 2-HG, a product of the NADPH-dependent alpha-KG reduction reaction.More specifically, the biochemical reactions were performed at room temperature in 384-well Greiner flat-bottom plates (Costar, Cat. No. 781201) using a final reaction volume of 30 μL and the following assay buffer conditions: 50 mM HEPES pH 7.4, 10 mM MgCl2, 50 mM KCl, 1 mM DTT, 0.02% BSA, 5 uM NADPH and 100 uM alpha-KG.The final reaction mixture contained 3.3% DMSO and inhibitors with concentrations ranging 0.02-50 μM. The IDH1 enzyme was used at a final concentration of 0.25 nM. Following 45 minutes incubation, the reaction mixtures were quenched by the addition of 10 μL of 16% formic acid containing 800 nM of 5-carbon labeled 13C-2-HG). The protein was then precipitated by the addition of 2.5 volumes of acetonitrile followed by centrifugation (3000×g, 20 minutes). The concentration of 2-HG in the resulting supernatants was measured by LC-MS (see below).LC-MS method. Reaction mixture supernatants were submitted to chromatographic separation on a BiobasicAX column (2.1 mm×20 mm, 5 μm particle, Thermo Scientific Inc.). The chromatographic mobile phases were A) 25 mM ammonium biocarbonate and B) acetonitrile (0.1% ammonium hydroxide). Nicotinamide was eluted at 1 ml/min using a 85-5% B gradient over 0.9 minutes (Agilent 1200SL LC system, Thermofisher LX-4 autosampler) and analyzed by multiple reaction monitoring (MRM) on a API4000 QTrap mass spectrometer (ABSciex, Framingham, Mass.) in the positive electrospray ionization (ESI+) mode. The mass transition for 2-HG and 13C-2-HG were 147→129 and 152→134, respectively. The relative responses (2-HG/13C-2-HG) were measured at varied inhibitor concentrations and used to calculate inhibitory IC50 values (normalized IC50 regression curves).
Measurement: IC50 > 50000 nM (pChEMBL 4.301000118255615)
Descriptors: MW 428.42, ALogP 2.47, HBA 9, HBD 2, PSA 126.5, QED 0.58
Compound (SMILES): Cc1ccc(-c2noc(C(C)Nc3ncc(F)c(N4C(=O)OC[C@@H]4[C@@H](C)O)n3)n2)cc1F
InChIKey: SNSAPFWSGVJLEA-HUFXEGEASA-N
Target: Isocitrate dehydrogenase [NADP] cytoplasmic (SINGLE PROTEIN), Homo sapiens
UniProt: O75874
Assay: Mutant IDH1 Biochemical Assay: LC-MS Detection of 2-HG: Mutant IDH1 R132H catalytic activity was monitored using the quantitative liquid chromatography/mass spectrometry (LC-MS) detection of 2-HG, a product of the NADPH-dependent alpha-KG reduction reaction.More specifically, the biochemical reactions were performed at room temperature in 384-well Greiner flat-bottom plates (Costar, Cat. No. 781201) using a final reaction volume of 30 μL and the following assay buffer conditions: 50 mM HEPES pH 7.4, 10 mM MgCl2, 50 mM KCl, 1 mM DTT, 0.02% BSA, 5 uM NADPH and 100 uM alpha-KG.The final reaction mixture contained 3.3% DMSO and inhibitors with concentrations ranging 0.02-50 μM. The IDH1 enzyme was used at a final concentration of 0.25 nM. Following 45 minutes incubation, the reaction mixtures were quenched by the addition of 10 μL of 16% formic acid containing 800 nM of 5-carbon labeled 13C-2-HG). The protein was then precipitated by the addition of 2.5 volumes of acetonitrile followed by centrifugation (3000×g, 20 minutes). The concentration of 2-HG in the resulting supernatants was measured by LC-MS (see below).LC-MS method. Reaction mixture supernatants were submitted to chromatographic separation on a BiobasicAX column (2.1 mm×20 mm, 5 μm particle, Thermo Scientific Inc.). The chromatographic mobile phases were A) 25 mM ammonium biocarbonate and B) acetonitrile (0.1% ammonium hydroxide). Nicotinamide was eluted at 1 ml/min using a 85-5% B gradient over 0.9 minutes (Agilent 1200SL LC system, Thermofisher LX-4 autosampler) and analyzed by multiple reaction monitoring (MRM) on a API4000 QTrap mass spectrometer (ABSciex, Framingham, Mass.) in the positive electrospray ionization (ESI+) mode. The mass transition for 2-HG and 13C-2-HG were 147→129 and 152→134, respectively. The relative responses (2-HG/13C-2-HG) were measured at varied inhibitor concentrations and used to calculate inhibitory IC50 values (normalized IC50 regression curves).
Measurement: IC50 = 268 nM (pChEMBL 6.570000171661377)
Descriptors: MW 446.41, ALogP 2.99, HBA 9, HBD 2, PSA 126.5, QED 0.59
Compound (SMILES): CN1C[C@@H]2CN(C(=O)Cn3cc(NC(=O)c4cnn5cccnc45)c(-c4cc(Cl)ccc4OC(F)F)n3)C[C@]2(c2ccccc2)C1
InChIKey: KCUIVGZJJQBSCF-KTBRYMIGSA-N
Target: Tyrosine-protein kinase JAK2 (SINGLE PROTEIN), Homo sapiens
UniProt: O60674
Assay: JAK Enzyme Assay: The activity of the isolated recombinant JAK1 and JAK2 kinase domain was measured by monitoring phosphorylation of a peptide derived from JAK3 (Val-Ala-Leu-Val-Asp-Gly-Tyr-Phe-Arg-Leu-Thr-Thr, fluorescently labeled on the N-terminus with 5-carboxyfluorescein) using the Caliper LabChip® technology (Caliper Life Sciences, Hopkinton, Mass.). To determine inhibition constants (Ki), compounds were diluted serially in DMSO and added to 50 μL kinase reactions containing purified enzyme (1.5 nM JAK1, or 0.2 nM JAK2), 100 mM HEPES buffer (pH 7.2), 0.015% Brij-35, 1.5 μM peptide substrate, ATP (25 μM), 10 mM MgCl2, 4 mM DTT at a final DMSO concentration of 2%. Reactions were incubated at 22° C. in 384-well polypropylene microtiter plates for 30 minutes and then stopped by addition of 25 μL of an EDTA containing solution (100 mM HEPES buffer (pH 7.2), 0.015% Brij-35, 150 mM EDTA), resulting in a final EDTA concentration of 50 mM. After termination of the kinase reaction, the proportion of phosphorylated product was determined as a fraction of total peptide substrate using the Caliper LabChip® 3000 according to the manufacturer's specifications.
Measurement: Ki = 0.22 nM (pChEMBL 9.65999984741211)
Descriptors: MW 647.09, ALogP 4.44, HBA 9, HBD 1, PSA 109.89, QED 0.27
Compound (SMILES): Cc1ccc(-c2noc(C(C)Nc3nccc(N4C(=O)OC[C@@H]4[C@@H](C)O)n3)n2)cc1F
InChIKey: CQNIUVQCNAZHDZ-AADWAWOOSA-N
Target: Isocitrate dehydrogenase [NADP] cytoplasmic (SINGLE PROTEIN), Homo sapiens
UniProt: O75874
Assay: Mutant IDH1 Biochemical Assay: LC-MS Detection of 2-HG: Mutant IDH1 R132H catalytic activity was monitored using the quantitative liquid chromatography/mass spectrometry (LC-MS) detection of 2-HG, a product of the NADPH-dependent alpha-KG reduction reaction.More specifically, the biochemical reactions were performed at room temperature in 384-well Greiner flat-bottom plates (Costar, Cat. No. 781201) using a final reaction volume of 30 μL and the following assay buffer conditions: 50 mM HEPES pH 7.4, 10 mM MgCl2, 50 mM KCl, 1 mM DTT, 0.02% BSA, 5 uM NADPH and 100 uM alpha-KG.The final reaction mixture contained 3.3% DMSO and inhibitors with concentrations ranging 0.02-50 μM. The IDH1 enzyme was used at a final concentration of 0.25 nM. Following 45 minutes incubation, the reaction mixtures were quenched by the addition of 10 μL of 16% formic acid containing 800 nM of 5-carbon labeled 13C-2-HG). The protein was then precipitated by the addition of 2.5 volumes of acetonitrile followed by centrifugation (3000×g, 20 minutes). The concentration of 2-HG in the resulting supernatants was measured by LC-MS (see below).LC-MS method. Reaction mixture supernatants were submitted to chromatographic separation on a BiobasicAX column (2.1 mm×20 mm, 5 μm particle, Thermo Scientific Inc.). The chromatographic mobile phases were A) 25 mM ammonium biocarbonate and B) acetonitrile (0.1% ammonium hydroxide). Nicotinamide was eluted at 1 ml/min using a 85-5% B gradient over 0.9 minutes (Agilent 1200SL LC system, Thermofisher LX-4 autosampler) and analyzed by multiple reaction monitoring (MRM) on a API4000 QTrap mass spectrometer (ABSciex, Framingham, Mass.) in the positive electrospray ionization (ESI+) mode. The mass transition for 2-HG and 13C-2-HG were 147→129 and 152→134, respectively. The relative responses (2-HG/13C-2-HG) were measured at varied inhibitor concentrations and used to calculate inhibitory IC50 values (normalized IC50 regression curves).
Measurement: IC50 = 45 nM (pChEMBL 7.349999904632568)
Descriptors: MW 428.42, ALogP 2.85, HBA 9, HBD 2, PSA 126.5, QED 0.61
Compound (SMILES): C[C@@H]1CCC[C@H](n2cnc(-c3cc(Cl)ccc3-n3cc(C(F)(F)F)nn3)cc2=O)c2ccnc(c2)-c2c(cnn2C)NC1=O
InChIKey: FQRJWJILLRZDIQ-MWTRTKDXSA-N
Target: Plasma kallikrein (SINGLE PROTEIN), Homo sapiens
UniProt: P03952
Assay: In Vitro Assay: Plasma kallikrein determinations were made in 0.1 M sodium phosphate buffer at a pH of 7.5 containing 0.1-0.2 M sodium chloride and 0.5% PEG 8000. Determinations were made using purified human plasma kallikrein (Enzyme Research Laboratories) at a final assay concentration of 200 pM and the synthetic substrate S-2302 (H-(D)-Pro-Phe-Arg-pNA; CHROMOGENIX®) at a concentration of 0.00008-0.0004 M.
Measurement: Ki = 19 nM (pChEMBL 7.71999979019165)
Descriptors: MW 624.03, ALogP 5.31, HBA 10, HBD 1, PSA 125.41, QED 0.29
Compound (SMILES): CC12C[C@H]3C[C@@H](N4[C@@H]5CCC[C@H]4C[C@@H](n4c(=O)c(N6CC(C(=O)O)C6)nc6ccccc64)C5)C[C@@H]1C[C@@H]2C3
InChIKey: CRCHFTNTZRGKSN-GWVJWZHSSA-N
Target: Delta-type opioid receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P41143
Assay: In Vitro Delta-Opioid Receptor Binding Assay: Radioligand dose-displacement assays used 0.2 nM [3H]-Naltrindole (NEN; 33.0 Ci/mmole) with 10-20 μg membrane protein (recombinant delta opioid receptor expressed in CHO-K1 cells; Perkin Elmer) in a final volume of 500 μL binding buffer (5 mM MgCl2, 5% DMSO, 50 mM Trizma base, pH 7.4). Non-specific binding was determined in the presence of 25 μM unlabeled naloxone. All reactions were performed in 96-deep well polypropylene plates for 1 h at a temperature of about 25 °C. Binding reactions were determined by rapid filtration onto 96-well Unifilter GF/C filter plates (Packard) presoaked in 0.5% polyethylenimine (Sigma-Aldrich). Harvesting was performed using a 96-well tissue harvester (Packard) followed by five filtration washes with 500 μL ice-cold binding buffer. Filter plates were subsequently dried at 50 °C. for 1-2 hours. Fifty μL/well scintillation cocktail (MicroScint20, Packard) was added and plates were counted in a Packard Top-Count for 1 min/well.
Measurement: Ki = 1516 nM (pChEMBL 5.820000171661377)
Descriptors: MW 516.69, ALogP 4.69, HBA 6, HBD 1, PSA 78.67, QED 0.64
Compound (SMILES): CC(Nc1nccc(N2C(=O)OC[C@@H]2[C@@H](C)O)n1)c1nc(-c2ccc3c(c2)CCC3)no1
InChIKey: BOZGVLGFGSEXBU-QQGYXAEISA-N
Target: Isocitrate dehydrogenase [NADP] cytoplasmic (SINGLE PROTEIN), Homo sapiens
UniProt: O75874
Assay: Fluorescence Biochemical Assay: The IDH1 (R132H) mutant catalyzes the reduced form of NADP+ (NADPH) and α-ketoglutarate (α-KG) to form nicotinamide adenine dinucleotide phosphate (NADP+) and R(−)-2-hydroxyglutarate (2HG). The reaction can be monitored kinetically by following the oxidation of NADPH to NADP+ which is measured using fluorescence, excitation at 355 nm and emission at 530 nm. Reactions were monitored using the Perkin-Elmer Envision, Model 2101. More specifically, the biochemical reactions were performed at room temperature in 384-well Greiner flat-bottom plates (Cat. No. 781076) using a final reaction volume of 20 μL and the following assay buffer conditions: 50 mM HEPES pH 7.5, 10 mM MgCl2, 1 mM DTT, 0.02% BSA, 0.02% Tween-20, 10 μM NADPH and 100 μM α-KG. The final reaction mixture contained 2.5% DMSO and test compounds with concentrations ranging 0.0000008-25 μM. The IDH1 (R132H) enzyme was used at a final concentration of 10 nM. Curve fitting for dose response IC50 determinations was done using a 4-parameter logistic model: y=min+((max−min)/1+(x/IC50)slope).
Measurement: IC50 = 36 nM (pChEMBL 7.440000057220459)
Descriptors: MW 436.47, ALogP 2.89, HBA 9, HBD 2, PSA 126.5, QED 0.6
Compound (SMILES): COC(=O)Nc1ccc2c(c1)NC(=O)[C@H](C)CCC[C@H](N1CCC(c3c(C#N)ccc(C#N)c3F)=CC1=O)c1cc-2cc(OC)n1
InChIKey: FJMCAFXMYKYZAO-GDJIYFAZSA-N
Target: Coagulation factor XI (SINGLE PROTEIN), Homo sapiens
UniProt: P03951
Assay: Inhibition Assay: Factor XIa determinations were made in 50 mM HEPES buffer at pH 7.4 containing 145 mM NaCl, 5 mM KCl, and 0.1% PEG 8000 (polyethylene glycol; JT Baker or Fisher Scientific). Determinations were made using purified human Factor XIa at a final concentration of 25-200 pM (Haematologic Technologies) and the synthetic substrate S-2366 (pyroGlu-Pro-Arg-pNA; CHROMOGENIX® or AnaSpec) at a concentration of 0.0002-0.001 M.
Measurement: Ki = 5.42 nM (pChEMBL 8.270000457763672)
Descriptors: MW 622.66, ALogP 5.93, HBA 8, HBD 2, PSA 157.44, QED 0.36
Compound (SMILES): C[C@@H]1CCC[C@H](N2CCC(c3c(F)ccc(Cl)c3F)=CC2=O)c2cccc(c2)-c2cc(C(=O)O)ccc2NC1=O
InChIKey: VNKIWRSSMQDOEY-QUGAMOGWSA-N
Target: Coagulation factor XI (SINGLE PROTEIN), Homo sapiens
UniProt: P03951
Assay: Inhibition Assay: Factor XIa determinations were made in 50 mM HEPES buffer at pH 7.4 containing 145 mM NaCl, 5 mM KCl, and 0.1% PEG 8000 (polyethylene glycol; JT Baker or Fisher Scientific). Determinations were made using purified human Factor XIa at a final concentration of 25-200 pM (Haematologic Technologies) and the synthetic substrate S-2366 (pyroGlu-Pro-Arg-pNA; CHROMOGENIX® or AnaSpec) at a concentration of 0.0002-0.001 M.
Measurement: Ki = 6.38 nM (pChEMBL 8.199999809265137)
Descriptors: MW 565.02, ALogP 7.1, HBA 3, HBD 2, PSA 86.71, QED 0.33