lovingscience/chembl-v37-raw-activities huggingface dataset:
Compound (SMILES): C=CC(=O)N1CC[C@@H](F)[C@@H](Nc2nc(Nc3cnoc3)nc3[nH]cc(C)c23)C1
InChIKey: HZFQEZSJFVDTDI-OLZOCXBDSA-N
Target: Tyrosine-protein kinase JAK3 (SINGLE PROTEIN), Homo sapiens
UniProt: P52333
Assay: Inhibitory Activity Assay: JAK3 and BTK kinases inhibitory activities were measured for the compounds prepared in the Examples through in vitro analysis on the ADP Glow (Glo) platform.Specifically, the inhibitory activities against JAK3 and BTK kinase were measured using a JAK3 kinase assay kit (Promega, V9441) and a BTK kinase assay kit (Promega, V9071) which were purchased from Promega. Recombinant purified human JAK3 and BTK were diluted with 1× kinase reaction buffer (JAK3: 40 mM Tris-Cl, pH 7.5, 20 mM MgCl2, 0.1 mg/mL BSA and 50 uM DTT/BTK: 40 mM Tris-Cl, pH 7.5, 20 mM MgCl2, 0.1 mg/mL BSA, 2 mM MnCl2 and 50 uM DTT) and added to 96 well plates (JAK3: final concentration of 4 ng per reaction/BTK: final concentration of 8 ng per reaction). The compounds prepared in the previous Examples were treated so as to be finally a 1% DMSO aqueous solution, and a substrate cocktail containing ATP (JAK3: final concentration of 5 uM/BTK: final concentration of 10 uM) and 0.2 ug/uL of Poly(Glu4, Tyr1)peptide (JAK3 and BTK final concentration) in the total 25 uL reactants was added to 96-well plates to initiate enzymatic reaction. After incubation (30° C.) for 1 hour, equivalent volume (25 uL per reaction) of ADP Glo was added and incubated (30° C.) for 40 minutes at room temperature. Then, a kinase detection reagent (50 uL per reaction) was added and incubated (30° C.) for 30 minutes at room temperature. The kinase activity was measured by chemiluminescence according to the instructions of ADP Glo kinase assay kit, and the inhibitory activity of the compounds according to the present invention was calculated.
Measurement: IC50 = 0.7 nM (pChEMBL 9.149999618530273)
Descriptors: MW 385.4, ALogP 2.53, HBA 7, HBD 3, PSA 111.97, QED 0.58
Compound (SMILES): N[C@H](c1cc(-c2cc(COc3ccccc3CC(=O)O)cc3ccoc23)ccn1)C(F)(F)F
InChIKey: RWGPLOYGFRZHIO-HSZRJFAPSA-N
Target: Complement factor D (SINGLE PROTEIN), Homo sapiens
UniProt: P00746
Assay: Factor D Esterolytic Assay: An established esterolytic assay for the measurement of Factor D activity and inhibition of Factor D activity was used (Kam, C. M.; McRae, B. J.; Harper, J. W.; Niemann, M. A.; Volanakis, J. E.; Powers, J. C. Human complement proteins D, C2, and B Active site mapping with peptide thioester substrates. J Biol. Chem. 1987, 262, 3444-3451). For this assay Z-Lys-SBzl, 1.29 mM (Kim, S.; Narayana, S. V. L; Volanakis, J. E. Mutational analysis of the substrate binding site of human complement Factor D. Biochemistry. 1994, 33, 14393-14399.) was used as the substrate for Factor D (104 mM). Hydrolysis of this compound by Factor D liberated a free sulfhydryl group which is then reacted with 5,5′-dithiobis(2nitrobenzoic acid) producing an intense yellow color (Habeeb, A. F. S. A. Reaction of protein sulfhydryl groups with Ellman's Reagent. Methods in Enzymol. 1976, 25, 457-464). The assays were performed in 96 well microtiter plates and rates of hydrolysis were monitored at 405 nm on a Biotek Synergy H1 plate reader. Hydrolysis rates were reported as change in mOD/min. The assay was conducted in 100 mM HEPES, 500 mM NaCl, pH 7.5 containing 10% DMSO in a final volume of 50 μL per well.
Measurement: IC50 > 10000 nM (pChEMBL 5)
Descriptors: MW 456.42, ALogP 5.26, HBA 5, HBD 2, PSA 98.58, QED 0.39
Compound (SMILES): C[C@@]1(O)CC[C@@]2(C)[C@H](CC[C@@H]3[C@@H]2CC[C@]2(C)[C@@H](C(=O)Cn4nnc5ccc(Cl)cc54)CC[C@@H]32)C1
InChIKey: RLAIWYIKPYXWQG-NLJQKHSCSA-N
Target: Gamma-aminobutyric acid type B receptor subunit 1 (SINGLE PROTEIN), Rattus norvegicus
UniProt: Q9Z0U4
Assay: TBD: TBD
Measurement: IC50 < 10 nM (pChEMBL 8)
Descriptors: MW 484.08, ALogP 6.06, HBA 5, HBD 1, PSA 68.01, QED 0.58
Compound (SMILES): CCSc1nc(N2CCC(F)(F)C2)c2nnn(Cc3ccccc3Cl)c2n1
InChIKey: RHXCJUZAMMHKJT-UHFFFAOYSA-N
Target: Cannabinoid receptor 2 (SINGLE PROTEIN), Homo sapiens
UniProt: P34972
Assay: cAMP Assay: CHO cells expressing human CB1 or CB2 receptors are seeded 17-24 hours prior to the experiment 50.000 cells per well in a black 96 well plate with flat clear bottom (Corning Costar #3904) in DMEM (Invitrogen No. 31331), 1×HT supplement, with 10% fetal calf serum and incubated at 5% CO2 and 37° C. in a humidified incubator. The growth medium was exchanged with Krebs Ringer Bicarbonate buffer with 1 mM IBMX and incubated at 30° C. for 30 min. Compounds were added to a final assay volume of 100 μl and incubated for 30 min at 30° C. Using the cAMP-Nano-TRF detection kit the assay (Roche Diagnostics) was stopped by the addition of 50 μl lysis reagent (Tris, NaCl, 1.5% Triton X100, 2.5% NP40, 10% NaN3) and 50 μl detection solutions (20 μM mAb Alexa700-cAMP 1:1, and 48 μM Ruthenium-2-AHA-cAMP) and shaken for 2 h at room temperature. The time-resolved energy transfer is measured by a TRF reader (Evotec Technologies GmbH), equipped with a ND:YAG laser as excitation source. The plate is measured twice with the excitation at 355 nm and at the emission with a delay of 100 ns and a gate of 100 ns, total exposure time 10 s at 730 (bandwidth 30 nm) or 645 nm (bandwidth 75 nm), respectively. The FRET signal is calculated as follows: FRET=T730-Alexa730-P(T645-B645) with P=Ru730-B730/Ru645-B645, where T730 is the test well measured at 730 nM, T645 is the test well measured at 645 nm, B730 and B645 are the buffer controls at 730 nm and 645 nm, respectively. cAMP content is determined from the function of a standard curve spanning from 10 μM to 0.13 nM cAMP.
Measurement: EC50 = 0.7 nM (pChEMBL 9.149999618530273)
Descriptors: MW 410.88, ALogP 3.88, HBA 7, HBD 0, PSA 59.73, QED 0.47
Compound (SMILES): Cc1cccc(-c2cn(C)nc2C(=O)N2CC(F)(F)C[C@@H](C)C2CNc2ncc(C(F)(F)F)cn2)c1
InChIKey: IZSZWEMDNFEKMB-NYRJJRHWSA-N
Target: Orexin receptor type 2 (SINGLE PROTEIN), Homo sapiens
UniProt: O43614
Assay: Orexin Receptor Cell-Based Functional Assay: Measurement of [Ca2+]i using a FLIPR: CHOOX1 or CHOOX2 cells were seeded into black-walled clear-base 384-well plates (Corning, catalog #3712) at a density of 20,000 cells per well in F12-K medium supplemented with 10% FBS and then incubated in a 5% CO2, 37° C. incubator overnight to reach 90% confluency. The cells were incubated with equal volume of calcium 6 loading buffer (Molecular Devices, Inc.) containing 2.5 mM probenecid at 37° C. for 2 h, followed by test compounds (dose-range 0.1 nM-10 μM) for another 30 min. The plates were then placed into a FLIPR (Molecular Devices, Inc.) to monitor fluorescence (λ excitation 488 nm, λ emission 540 nm) before and after the addition of EC90 of [OX].
Measurement: IC50 > 5000 nM (pChEMBL 5.301000118255615)
Descriptors: MW 508.5, ALogP 4.8, HBA 6, HBD 1, PSA 75.94, QED 0.5
Compound (SMILES): Cc1ccc(S(=O)(=O)C23CCN(C(=O)C4(O)CCS(=O)(=O)CC4)C2CCc2cc(C(F)(C(F)(F)F)C(F)(F)F)ccc23)cc1Cl
InChIKey: NHMKCWFDDYHIQL-UHFFFAOYSA-N
Target: Nuclear receptor ROR-gamma (SINGLE PROTEIN), Mus musculus
UniProt: P51450
Assay: General RORgamma SPA binding assay : The binding of potential ligands to RORγ is measured by competition with [3H]25-hydroxycholesterol (Perkin Elmer NET674250UC) using a scintillation proximity assay (SPA). The ligand binding domain of human RORγ (A262-S507) with an N-terminal His tag is expressed in E. coli and purified using nickel affinity chromatography. 15 μg/well RORγ (A262-S507) is incubated with test compound at varying concentrations in 3-fold serial dilution, with final concentrations ranging from 16.6 μM to 0.28 nM, for 10 min at rt in PBS buffer (Invitrogen #14190-144) containing 0.5% fatty acid free BSA (Gemini Bio-Products, Cat. #700-107P) and 0.1% glycerol (Sigma Cat#G5516). 10 nM of [3H] 25-hydroxycholesterol is then added, and the reaction is incubated for 10 min. 10 mg/mL of Copper-His Tag-PVT beads (Perkin Elmer cat # RPNQ0095) are added, and the mixture is incubated for 60 min. The reaction is read on a TopCount Microplate scintillation plate reader (Perkin Elmer). The competition data of the test compound over a range of concentrations was plotted as percentage inhibition of radioligand specifically bound in the absence of test compound (percent of total signal). After correcting for non-specific binding, IC50 values were determined. The IC50 value is defined as the concentration of test compound needed to reduce [3H] 25-hydroxycholesterol specific binding by 50% and is calculated using the four parameter logistic equation to fit the normalized data.
Measurement: IC50 = 175 nM (pChEMBL 6.760000228881836)
Descriptors: MW 706.1, ALogP 5.09, HBA 6, HBD 1, PSA 108.82, QED 0.44
Compound (SMILES): COc1ccc(C[C@H]2CC(=O)N(C)c3cc(-c4ccnc(Nc5ccnn5C)n4)cc4nnc2n34)c(F)c1
InChIKey: FOBHQXWBDGWDCB-KRWDZBQOSA-N
Target: Mitogen-activated protein kinase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P28482
Assay: ERK-2 Enzymatic Assay: Compounds were tested in an enzymatic assay using human ERK-2 (Mitogen Activated Kinase 1), recombinantly expressed as an n-terminal 6-His fusion protein in E. coli and corresponding to aa 8-360. The substrate used was the fluorescent Omnia peptide S/T17 (Invitrogen of Carlsbad, Calif.; Cat. KNZ1171C). Test compounds were diluted in DMSO in 3-fold serial dilutions at 100× final concentrations. In addition to compound, the assay contained 50 mM HEPES [pH 7.3], 10 mM MgCl2, 2 mM DTT, 0.005% Triton-X100, 5 nM ERK-2 enzyme, 6.25 μM S/T17 peptide substrate and 25 μM ATP (corresponding to the observed Km) for a total reaction volume of 25 μL. The assay was run at ambient temperature in a white 384-well polypropylene plate (Nunc, Inc of Naperville, Ill.; Cat. 267462) collecting data every 50 seconds for approximately 30 minutes on an Envision plate reader (PerkinElmer, Inc. of Waltham, Mass.); Excitation 340 nm/Emission 495 nm. The data collected from each well was fit to a straight line, and the resulting rates were used to calculate percent of control. Percent of control was plotted against compound concentration, and IC50 values were determined using a four-parameter fit. Table 1 contains representative data for compounds disclosed herein.
Measurement: IC50 < 0.02 nM (pChEMBL 10.699000358581543)
Descriptors: MW 513.54, ALogP 3.5, HBA 10, HBD 1, PSA 115.36, QED 0.37
Compound (SMILES): O=c1[nH]c2c(-c3ccccc3)cccc2c(=O)n1O
InChIKey: DOKPESCMELTHQQ-UHFFFAOYSA-N
Target: Flap endonuclease 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P39748
Assay: Enzymatic Assay: Fluorogenic biochemical assays used recombinant full-length FEN-1 or catalytic domains of Exo1, XPG or GEN1 and 200 nM of a DNA substrate (formed by annealing three oligonucleotidesquencher (5′-CAC GTT GAC TAC CGC TCA ATC CTG ACG AAC ACA TC-BHQ2), flap (5′-TAMRA-GA TGT CAA GCA GTC CTA ACT TTG AGG CAG AGT CCG C) and template (5′-GC GGA CTC TGC CTC AAG ACG GTA GTC AAC GTG-3′) (PMID: 21062821)) in 50 mM Tris pH 8.0, 10 mM MgCl2, 1 mM DTT, and 0.01% Tween-20. Inhibitors arrayed in dose response were added from DMSO stocks (using a V&P 384-pintool head) to the enzyme solution in low volume, black polystyrene 384-well plates (Corning #3677) 15 minutes prior to the addition of the DNA substrate. Reactions were allowed to proceed for 2-4 hours at 25° C. or 37° C. Fluorescence data (Excitation: 530 nm/10 nm bandwidth; Emission: 590 nm/20 nm bandwith) were measured with an Infinite M1000 plate reader (Tecan).
Measurement: IC50 = 113 nM (pChEMBL 6.949999809265137)
Descriptors: MW 254.25, ALogP 1.59, HBA 4, HBD 2, PSA 75.09, QED 0.65
Compound (SMILES): CC1(C)Cc2cc(NC(=O)c3cnn4cccnc34)c(N3CC4(CCC4(F)F)C3)cc2O1
InChIKey: NGIFYSALWVIOHD-UHFFFAOYSA-N
Target: Interleukin-1 receptor-associated kinase 4 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9NWZ3
Assay: Biological Assay: Kinase activities were assayed using the Transcreener-Fluorecescence polarization platform (BelBrook Labs, Madison, Wis., USA) that measures amounts of the reaction product, ADP. The IRAK4 reaction conditions were optimized using an IRAK1-derived peptide (sequence H-KKARFSRFAGSSPSQSSMVAR) to provide a linear reaction rate over the course of a 90 min incubation, which resulted in 10-12% conversion of the starting ATP to ADP. Final IRAK4 assay conditions were 1.25 nM IRAK4; 125 uM ATP; 10 uM MgCl2; 125 uM peptide in reaction buffer (25 mM HEPES (pH7.4); 2 mM Dithiothreitol; 0.015% Brij-35; and 0.5% dimethyl sulfoxide. The IRAK1 activity was optimized similarly, yielding final assay conditions of 3 mM IRAK1; 62.5 uM ATP; 5 uM MgCl2, and 62.5 uM IRAK1 peptide in reaction buffer for 60 min.Assays of compounds for kinase inhibition were performed using inhibitors serially-diluted in dimethyl sulfoxide, which was accomplished with a LabCyte Echo 555 liquid dispenser. 384 well assay plates spotted with compound received 4 ul of a 2× substrate (ATP+peptide) mix in reaction buffer, followed by 4 ul of 2× enzyme diluted in reaction buffer. Reactions were halted at 60 (IRAK1) or 90 (IRAK4) min by addition of 6 ul of detection buffer, containing EDTA (40 nM final concentration), 0.95 ug of the ADP-binding antibody ADP2, ADP tracer (3 nM final concentration), and 25 uM HEPES. Following a 1 hr incubation, fluorescence polarization of the ADP2-antibody TRACER complex was read on a Tecan M1000 plate reader using a 635/20 excitation filter in combination with a 670/20 emission filter.
Measurement: Ki = 730 nM (pChEMBL 6.139999866485596)
Descriptors: MW 439.47, ALogP 3.93, HBA 6, HBD 1, PSA 71.76, QED 0.67
Compound (SMILES): Cc1cc(C(F)(F)F)cc2cnn(C(=O)c3c(Cl)ccc(C(=O)N4CCC(CC(=O)O)CC4)c3Cl)c12
InChIKey: PQGVIMZCRYQSHB-UHFFFAOYSA-N
Target: Nuclear receptor ROR-gamma (SINGLE PROTEIN), Homo sapiens
UniProt: P51449
Assay: transient transactivation assay: The primary screen was performed by transient transactivation assays. These cell-based assays were carried out using Cos-7 cells transfected with a chimeric human RORγ-Gal4 receptor expression plasmid and a 5Gal4 pGL3 TK Luc reporter plasmid. Transfections were performed by a chemical agent (Jet PEI). Transfected cells were distributed in 384-wells plates and were allowed to recover for 24 h. The culture medium was then removed and fresh medium containing the compounds to be tested was added (final concentration ranging from 10−4 M to 3 10−10 M). After an overnight incubation, luciferase expression was measured by adding SteadyGlo according to the manufacturer's instructions (Promega). T0901317 at 10−5 M was used as reference. Results were expressed as fold induction compared to basal level or as percentage activity compared to references taken as 100% (basal) or 0% (T0901317). Dose-effect curves and IC50 were calculated using the software Assay Explorer (MDL).
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 542.34, ALogP 5.69, HBA 5, HBD 1, PSA 92.5, QED 0.46
Compound (SMILES): N#Cc1ccc(-n2cnc3c2C(=O)C[C@@]2(CCOC2)C3)cc1Cl
InChIKey: KDPULPOLUSRSSA-QGZVFWFLSA-N
Target: Cytochrome P450 11B2, mitochondrial (SINGLE PROTEIN), Homo sapiens
UniProt: P19099
Assay: Inhibition of Aldosterone Synthase: Assays are performed in 96-well format in a final volume of 60 microL/well, containing 100 mM potassium phosphate, pH 7.4, 1% (v/v) DMSO, and additionally, 2 μM of corticosterone and 50 units of CYP11B2 activity. Reactions are started by the addition of NADPH to 1 mM and allowed to proceed for 90 minutes at 37° C. Reactions are terminated by the addition of 60 μL of MeCN containing an internal standard for mass spectrometry. One hundred microliters are then transferred to a glass filter plate and centrifuged at 570×g for 5 minutes and the filtrate is collected. Reaction product aldosterone is quantified by mass spectrometry. To determine the assay blank value (0% activity), NADPH is omitted from some reactions.Dose dependent inhibition is quantified by the inclusion of compound at various concentrations. Maximum activity (100%) is defined by reactions containing NADPH, but without compound. Activities at each concentration are expressed as a percentage of the maximum activity (y-axis) and plotted against concentration of compound (x-axis) and the concentration corresponding to 50% activity (IC50) determined using the XLFit curve-fitting program using a 4-parameter logistic model.
Measurement: IC50 = 16 nM (pChEMBL 7.800000190734863)
Descriptors: MW 327.77, ALogP 2.93, HBA 5, HBD 0, PSA 67.91, QED 0.81
Compound (SMILES): O=C1N[C@H](c2cncc(C#CC34CCCCC3C4)c2)[C@@H](c2cccc(F)c2)O1
InChIKey: BMWHPTGGFJCRDA-QXMMQCAPSA-N
Target: Metabotropic glutamate receptor 5 (SINGLE PROTEIN), Homo sapiens
UniProt: P41594
Assay: mGluR5 FLIPR Assay: HEK293 (ZF) cells stably transfected with human mGluR5A (pIRES neo) and the rat glutamate-aspartate transporter (GLAST; pIRES puro) are grown in a monolayer culture at 37° C. in 5% CO2 and fed with Minimum Essential Medium (MEM) supplemented with 10% dialysed fetal bovine serum. 24 hours prior to assay, cells are enzymatically dissociated from the culture flask (Trypsin, 0.25%), spun down (1000 rpm, 3 min), resuspended, and plated on Greiner black clear bottomed PDL-coated 384-well plates at a density of 30 thousand cells/well. On the day of the experiment, media is removed from the cell plates and replaced with Molecular Devices Calcium 4 microfluorometric Ca++ sensitive dye in assay buffer (HBSS; Gibco #14025+20 mM HEPES and 250 uM probenacid). Plates are incubated in dye at 37° C. in 5% CO2 for 60 minutes prior to delivery of test compounds in assay buffer. Test compounds are incubated with cells in the presence of dye for 10 minutes prior to being read on the FLIPR platform (Molecular Devices). A Ca++ signal is induced in the assay plates via the delivery of an ¿EC10 concentration of the endogenous agonist 1-glutamate; images are acquired at 1 Hz for 100 seconds post-delivery of agonist stimulus. Positive modulator activity (i.e. the ability of test compounds to increase the Ca++ response to a sub-maximal concentration of agonist) is normalized to a saturating concentration of a known mGluR5 PAM run in each assay plate.
Measurement: EC50 = 377 nM (pChEMBL 6.420000076293945)
Descriptors: MW 376.43, ALogP 4.67, HBA 3, HBD 1, PSA 51.22, QED 0.77
Compound (SMILES): Cc1ccc(-n2ncc(C)n2)c(C(=O)N2C3CC(C3)[C@H](C)C2COc2ccc(F)cc2)n1
InChIKey: VIRNLKBWGGBTIE-QOYRLARYSA-N
Target: Orexin/Hypocretin receptor type 1 (SINGLE PROTEIN), Homo sapiens
UniProt: O43613
Assay: Scintillation Proximity Assay (SPA) Binding Assay: CHO cells stably transfected with human Orexin type 1 receptors (CHO-hOX1) or HEK-293 cells transiently transfected with human Orexin type 2 receptors (HEK-hOX2) were collected after 16 h induction with 5 mM sodium butyrate. The cell pellets were re-suspended, homogenized in 15 mM Tris/HCl pH=7.5, 1 mM EGTA, 0.3 mM EDTA, 2 mM MgCl2, protease inhibitors and centrifuged at 40,000 g (20 min, 4° C.). After re-suspension, homogenization and centrifugation as above, the final pellets were re-suspended in 75 mM Tris/HCl pH=7.5, 1 mM EGTA, 0.3 mM EDTA, 12.5 mM MgCl2, 250 mM Sucrose, protease inhibitors, divided into aliquots and frozen down at −80° C.Compounds of invention were serially diluted in neat DMSO at 100-fold concentrations (1% DMSO final in the assay) and 2 μl/well were plated into 96-well Isoplates (Perkin Elmer).
Measurement: Ki = 12.3 nM (pChEMBL 7.909999847412109)
Descriptors: MW 435.5, ALogP 3.74, HBA 6, HBD 0, PSA 73.14, QED 0.61
Compound (SMILES): Nc1nc2c3c(ccc2c2nc([C@H]4CCCN(c5cncnc5)C4)nn12)OC(F)(F)O3
InChIKey: JJESMNPJLANBMC-JTQLQIEISA-N
Target: Adenosine receptor A2a (SINGLE PROTEIN), Homo sapiens
UniProt: P29274
Assay: competition binding assay: The following table shows representative data for the compounds of the Examples as A2a receptor antagonists as determined by a competition binding assay using Scintillation Proximity technology. Thus, 1.0 μg of membranes from CHO-K1 cells expressing the human A2a receptor were incubated with a compound of the invention at concentrations ranging from 3000 nM to 0.15 nM in a reaction mixture also containing 2.0 nM of a tritiated form of 5-amino-7-[2-phenethyl]-2-(furan-2-yl)-7H-pyrazolo[4,3-e][1,2,4]triazolo[1,5-c]pyrimidine (the tritiated compound) and 100 μg of wheatgerm agglutin-coated yttrium silicate SPA beads for one hour at room temperature with agitation. The beads were then allowed to settle to the bottom of the wells for 1 hr, after which the membrane-associated radioactivity was determined by scintillation counting in a TopCount microplate reader. Ki values were determined using the Cheng-Prusoff equation.
Measurement: Ki = 44 nM (pChEMBL 7.360000133514404)
Descriptors: MW 426.39, ALogP 2.36, HBA 10, HBD 1, PSA 116.58, QED 0.52
Compound (SMILES): CN1CCN(c2ccc(NC(=O)c3n[nH]c4cc(F)c(-c5cnn(C)c5)cc34)cn2)CC1
InChIKey: HBAHDQPPMZAYNN-UHFFFAOYSA-N
Target: Dual specificity tyrosine-phosphorylation-regulated kinase 1A (SINGLE PROTEIN), Homo sapiens
UniProt: Q13627
Assay: Kinase Assay: DYRK1A: Each compound was dissolved in DMSO as a 10 mM stock and used to prepare compound source plates. Serial dilution (1:3, 11-point dose-response curves from 10 M to 0.00016 M) and compound transfer was performed using the ECHO 550 (Labcyte, Sunnyvale, Calif.) into 1536-well black-walled round bottom plates (Corning).The DYRK1A kinase assay was run using the Ser/Thr 18 peptide Z-lyte assay kit according to manufacturer's instructions (Life Technologiesa Division of Thermo-Fisher). This is a non-radioactive assay using fluorescence resonance energy transfer (FRET) between coumarin and fluorescein to detect kinase activity which is represented as a ratio of coumarin emission/fluorescein emission.Briefly, recombinant DYRK1A kinase, ATP and Ser/Thr peptide 18 were prepared in 1× Kinase buffer to final concentrations of 0.19 μg/mL, 30 μM, and 4 μM respectively. The mixture was allowed to incubate with the representative compounds for one hour at room temperature. All reactions were performed in duplicate. Unphosphorylated (0% Control) and phosphorylated (100% control) forms of Ser/Thr 18 served as control reactions. Additionally, an 11-point dose-response curve of Staurosporine (1 uM top) was run to serve as a positive compound control.After incubation, Development Reagent A was diluted in Development Buffer then added to the reaction and allowed to further incubate for one hour at room temperature. The plate was read at Ex 400 Em 455 to detect the coumarin signal and Ex 400 Em 520 to measure the signal (EnVision Multilabel Plate Reader, PerkinElmer).
Measurement: EC50 = 1 nM (pChEMBL 9)
Descriptors: MW 434.48, ALogP 2.5, HBA 7, HBD 2, PSA 94.97, QED 0.51
Compound (SMILES): CS(=O)(=O)NC(=O)CCc1ccc(N2C[C@@H]3C[C@H]2C[C@H]3OCc2c(-c3c(Cl)cccc3Cl)noc2C2CC2)c(F)c1
InChIKey: JNGDBOLLQYJFCT-XHNVNVPESA-N
Target: Bile acid receptor (SINGLE PROTEIN), Homo sapiens
UniProt: Q96RI1
Assay: Binding Assay: The affinity of FXR ligands for the ligand binding domain of FXR was determined using a commercially available human FXR ligand binding assay (LanthaScreen, Thermofisher Cat # PV4833). The purified ligand binding domain of human FXR tagged with GST (glutathiones-S-transferase) is incubated with a terbium labelled anti-GLT antibody and a fluorescein-labelled SRC2-2 peptide (LKEKHKILHRLLQDSSSPV (SEQ ID NO.: 1)). Binding of FXR ligands to the FXR ligand binding domain promotes binding of the fluorescein-labelled SRC2-2 peptide. This causes a FRET signal between the terbium-labelled anti-GST antibody and the fluorescein-labelled SRC peptide which are both bound to the FXR ligand binding domain.Test compounds are dissolved in DMSO and a 3-fold serial dilution series is generated, then further diluted into assay buffer. The compounds are mixed with 5 nM GST-tagged FXR ligand binding domain, 5 nM Tb-labelled anti-GST antibody and 500 nM fluorescein-labelled SRC2-2 peptide in a pH7.4 buffer. The reaction is incubated at room temperature for 1 hour, then the FRET signal is measured as the ratio of the 520 nm/495 nm emission following excitation at 340 nm. The change in FRET signal is plotted against the test article concentration and fit to a 3-parameter logistical equation.
Measurement: EC50 = 3.98 nM (pChEMBL 8.399999618530273)
Descriptors: MW 622.55, ALogP 5.86, HBA 7, HBD 1, PSA 101.74, QED 0.3
Compound (SMILES): COc1cc(-c2[nH]c3cc(C)c(C4CCN(C5COC5)CC4)nc3c2C(C)C)c[nH]/c1=N\C=N
InChIKey: ZGTCOWPDTMSISO-UHFFFAOYSA-N
Target: Toll-like receptor 7 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9NYK1
Assay: TLR7/8/9 Inhibition Reporter Assays: HEK-Blue-cells (Invivogen) overexpressing human TLR7, TLR8 or TLR9 receptors were used for screening inhibitors of these receptors using an inducible SEAP (secreted embryonic alkaline phosphatase) reporter gene under the control of the IFN-β minimal promoter fused to five NF-κB and AP-1-binding sites. Briefly, cells are seeded into Greiner 384 well plates (15000 cells per well for TLR7, 20,000 for TLR8 and 25,000 for TLR9) and then treated with test compounds in DMSO to yield a final dose response concentration range of 0.05 nM-50 μM. After a 30 minute compound pre-treatment at room temperature, the cells are then stimulated with a TLR7 ligand (gardiquimod at a final concentration of 7.5 μM), TLR8 ligand (R848 at a final concentration of 15.9 μM) or TLR9 ligand (ODN2006 at a final concentration of 5 nM) to activate NF-κB and AP-1 which induce the production of SEAP. After a 22 hour incubation at 37° C., 5% CO2, SEAP levels are determined with the addition of HEK-Blue Detection reagent (Invivogen), a cell culture medium that allows for detection of SEAP, according to manufacturer's specifications. The percent inhibition is determined as the % reduction in the HEK-Blue signal present in wells treated with agonist plus DMSO alone compared to wells treated with a known inhibitor.
Measurement: IC50 = 0.39 nM (pChEMBL 9.40999984741211)
Descriptors: MW 462.6, ALogP 4.08, HBA 5, HBD 3, PSA 102.38, QED 0.38
Compound (SMILES): CCNS(=O)(=O)c1cccc(CNC(=O)c2ccc3c(c2)OCc2cnccc2-3)c1
InChIKey: YXPBWTSEJDZUGR-UHFFFAOYSA-N
Target: Rho-associated protein kinase 2 (SINGLE PROTEIN), Homo sapiens
UniProt: O75116
Assay: In Vitro Assay: The effectiveness of compounds of the present invention as ROCK inhibitors can be determined in a 30 μL assay containing 20 mM HEPES, pH 7.5, 20 mM MgCl2, 0.015% Brij-35, 4 mM DTT, 5 μM ATP and 1.5 μM peptide substrate (FITC-AHA-AKRRRLSSLRA-OH). Compounds were dissolved in DMSO so that the final concentration of DMSO was <2%, and the reaction was initiated with Rho kinase variants. After incubation, the reaction was terminated by the addition of EDTA and the phosphorylated and non-phosphorylated peptides separated using a LABCHIP® 3000 Reader (Caliper Life Sciences). Controls consisted of assays that did not contain compound, and backgrounds consisted of assays that contained enzyme and substrate but had EDTA from the beginning of the reaction to inhibit kinase activity. Compounds were tested in dose-response format, and the inhibition of kinase activity was calculated at each concentration of compound. The inhibition data were fit using a curve-fitting program to determine the IC50; i.e., the concentration of compound required to inhibit 50% of kinase activity.
Measurement: IC50 = 25.2 nM (pChEMBL 7.599999904632568)
Descriptors: MW 423.49, ALogP 2.87, HBA 5, HBD 2, PSA 97.39, QED 0.64
Compound (SMILES): Cc1nc(C(=O)N2C(COc3ccc4ccccc4n3)CC3CC2C3)c(-c2ccccc2)s1
InChIKey: YMSYZOCTMYHHTM-UHFFFAOYSA-N
Target: Orexin/Hypocretin receptor type 1 (SINGLE PROTEIN), Homo sapiens
UniProt: O43613
Assay: Scintillation Proximity Assay (SPA) Binding Assay: CHO cells stably transfected with human Orexin type 1 receptors (CHO-hOX1) or HEK-293 cells transiently transfected with human Orexin type 2 receptors (HEK-hOX2) were collected after 16 h induction with 5 mM sodium butyrate. The cell pellets were re-suspended, homogenized in 15 mM Tris/HCl pH=7.5, 1 mM EGTA, 0.3 mM EDTA, 2 mM MgCl2, protease inhibitors and centrifuged at 40,000 g (20 min, 4° C.). After re-suspension, homogenization and centrifugation as above, the final pellets were re-suspended in 75 mM Tris/HCl pH=7.5, 1 mM EGTA, 0.3 mM EDTA, 12.5 mM MgCl2, 250 mM Sucrose, protease inhibitors, divided into aliquots and frozen down at −80° C.Compounds of invention were serially diluted in neat DMSO at 100-fold concentrations (1% DMSO final in the assay) and 2 μl/well were plated into 96-well Isoplates (Perkin Elmer).
Measurement: Ki = 10.5 nM (pChEMBL 7.980000019073486)
Descriptors: MW 455.58, ALogP 5.74, HBA 5, HBD 0, PSA 55.32, QED 0.39
Compound (SMILES): Cc1ccc(C(=O)O)cc1N1C(=O)CSC1c1ccc(F)cc1
InChIKey: DRUGRVTYZYPXMC-UHFFFAOYSA-N
Target: 5-hydroxytryptamine receptor 7 (SINGLE PROTEIN), Homo sapiens
UniProt: P34969
Assay: FLIPR Calcium Assay: One day before the assay, CORNING black with clear flat bottom 96-well assay plates were coated with a 0.1 mg/mL Poly-L-Lysine solution. CHO-K1/MOR/Gα15 cells were suspended in the F12 medium and plated at a density of 8×104 cells/well in 200 μL medium. Cells were incubated in a humidified atmosphere of 10% CO2 at 37° C. overnight so as to reach an 80-90% confluent cell monolayer before assay. At the day of assay, 150 μL medium/well was removed from plate. To each well, 50 μL FLIPR calcium assay reagent dissolved in 1× assay buffer (HBSS: KCl 5 mM, KH2PO4 0.3 mM, NaCl 138 mM, NaHCO3 4 mM, Na2HPO4 0.3 mM, d-glucose 5.6 mM, with additional 20 mM HEPES and 13 mM CaCl2, pH 7.4), with 2.5 mM probenecid was added and the plate was incubated at 37° C. for 1 h. Compounds and other reagents were dissolved in the assay buffer. Using a FlexStationIII (Molecular Devices Corp.), the [Ca2+]i fluorescence increases after robotic injections of compounds or other reagents were monitored every 1.52 s interval with excitation wavelength at 485 nm and with emission wavelength at 525 nm. The [Ca2+]i fluorescence was measured up to 90 s after agonist injection. The fluorescence intensity from 6 to 12 wells of cells were averaged and the relative amount of [Ca2+]i release was determined by integrating the AUC of the [Ca2+]i fluorescence averages.
Measurement: EC50 = 3300 nM (pChEMBL 5.480000019073486)
Descriptors: MW 331.37, ALogP 3.61, HBA 3, HBD 1, PSA 57.61, QED 0.93
Compound (SMILES): Cc1cc(-c2[nH]c3ccc(-c4nnc([C@H]5CSCN5)o4)cc3c2C(C)C)ccn1
InChIKey: CQZNWZZJPLULOM-GOSISDBHSA-N
Target: Toll-like receptor 7 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9NYK1
Assay: TLR7/8/9 Inhibition Reporter Assays: HEK-Blue-cells (Invivogen) overexpressing human TLR7, TLR8 or TLR9 receptors were used for screening inhibitors of these receptors using an inducible SEAP (secreted embryonic alkaline phosphatase) reporter gene under the control of the IFN-β minimal promoter fused to five NF-κc Band AP-1-binding sites. Briefly, cells are seeded into Greiner 384 well plates (15000 cells per well for TLR7, 20,000 for TLR8 and 25,000 for TLR9) and then treated with test compounds in DMSO to yield a final dose response concentration range of 0.05 nM-50 μM. After a 30 minute compound pre-treatment at room temperature, the cells are then stimulated with a TLR7 ligand (gardiquimod at a final concentration of 7.5 μM), TLR8 ligand (R848 at a final concentration of 15.9 μM) or TLR9 ligand (ODN2006 at final concentration of 5 nM) to activate NF-κB and AP-1 which induce the production of SEAP. After a 22 hour incubation at 37° C., 5% CO2, SEAP levels are determined with the addition of HEK-Bue Detection reagent (Invivogen), a cell culture medium that allows for detection of SEAP, according to manufacturer's specifications. The percent inhibition is determined as the % reduction in the HEK-Blue signal present in wells treated with agonist plus DMSO alone compared to wells treated with a known inhibitor.
Measurement: IC50 = 596 nM (pChEMBL 6.21999979019165)
Descriptors: MW 405.53, ALogP 5.05, HBA 6, HBD 2, PSA 79.63, QED 0.49
Compound (SMILES): Cc1cc(C(=O)OC(C)(C)C)ccc1NC(=O)c1ccc(-n2nc3n(c2=O)CCCC3)cc1O[C@@H](C)C1CCCCC1
InChIKey: KNBWYRSFPUWODT-QFIPXVFZSA-N
Target: Dihydroorotate dehydrogenase (quinone), mitochondrial (SINGLE PROTEIN), Homo sapiens
UniProt: Q02127
Assay: Enzymatic Assay: Assay1: The enzymatic assay couples DHODH activity with bleaching of the dye 2,6-dichlorophenolindophenol (DCIP) (Knecht and Loffler, 1998; Miller et al., 1968). The assay was conducted in buffer containing 50 mM Tris, 0.1% Triton X-100, 150 mM potassium chloride, 2 nM DHODH, 1 mM dihydroorotate, 0.1 mM decylubiquinone, 0.06 mM DCIP, and 2% DMSO at pH 8.0 at 32 degree Celsius. The reaction was initiated by addition of substrates. Enzyme activity was monitored kinetically by the reduction in DCIP absorbance at 600 nm. Purified recombinant human DHODH enzyme was purchased from Novus (cat. no. NBP1-98916). Other chemicals were purchased from Sigma-Aldrich. Absorbance measurements were obtained using a BMG clarion star plate-reading spectrophotometer.
Measurement: IC50 = 50 nM (pChEMBL 7.300000190734863)
Descriptors: MW 574.72, ALogP 6.23, HBA 8, HBD 1, PSA 104.45, QED 0.34
Compound (SMILES): COc1nn(-c2ccc(C(F)(F)F)nc2)cc1CNC(=O)[C@@H]1CCC[C@@H]1S(=O)(=O)c1ccc(F)cc1
InChIKey: ULYAWZCWOGWOHO-MOPGFXCFSA-N
Target: Transient receptor potential cation channel subfamily A member 1 (SINGLE PROTEIN), Homo sapiens
UniProt: O75762
Assay: Inhibition Assay: IC50 values (effective concentration) of compounds on the human and rat TRPA1 channels were determined using a FLIPR Tetra instrument. CHO cells expressing TRPA1 were plated into 384-well plates, incubated overnight at 37 C, and loaded with BD calcium indicator dye for 1 hr at 37° C. followed by 15 minutes. at room temperature. The assay buffer was Hank's Balanced Salt Solution (HBSS) containing 20 mM HEPES (pH readjusted to 7.4) along with 0.02% BSA.Following dye load and plate cool down, compounds were added to the cells using FLIPR Tetra. Plates were then incubated with compounds for 20 minutes at room temperature prior to adding agonist. Following this incubation, EC80 concentration of cinnamaldehyde (75 uM for human TRPA1 and 45 uM for rat TRPA1) was added to active the channels and block of cinnamaldehyde induced calcium influx was measured.
Measurement: IC50 = 159 nM (pChEMBL 6.800000190734863)
Descriptors: MW 526.51, ALogP 3.69, HBA 7, HBD 1, PSA 103.18, QED 0.37
Compound (SMILES): O=C(N[C@H](C(=O)N1C[C@H](Cl)[C@H]2OCC(=O)[C@H]21)C1CCCCC1)c1cnc(-c2ccnnc2)o1
InChIKey: WHGKLAHPYJXNIO-WQANXHIZSA-N
Target: Cathepsin S (SINGLE PROTEIN), Homo sapiens
UniProt: P25774
Assay: Enzyme Assay: Recombinant human cathepsins (CatS, CatK, CatL) were purchased from a Enzo Life Sciences. All assays were carried out in 96-well format using a buffer of 50 mM KH2PO4, 50 mM NaCl, 2 mM EDTA, 0.5 mM DTT and 1% Triton-X-100, pH 6.5 for Cathepsin S and a buffer of 50 mM NaOAc, 10 mM EDTA, 1 mM DTT and 0.01% Triton-X-100, pH 5.5 for CatK/L/B. For CatS, the enzyme (0.0007 mU/well) was incubated with fluorogeninc substrate (Z-VVR-AMC, 5 μM) at RT for 10 min. For CatK the enzyme (0.00175 mU/well) was incubated with fluorogeninc substrate (Z-FR-AMC, 40 μM) at RT for 10 min. For CatL, the enzyme (0.000874 mU/well) was incubated with fluorogeninc substrate (Z-VVR-AMC, 40 μM) at RT for 10 min. Flourogenic substrate turnover was detected using a microplate reader (Synergy H4, BioTek). Ki values were calculated using the Cheng Prusoff equation (Cheng & Prosoff 1973).
Measurement: Ki = 527 nM (pChEMBL 6.28000020980835)
Descriptors: MW 473.92, ALogP 1.6, HBA 8, HBD 1, PSA 127.52, QED 0.65
Compound (SMILES): CNC(=O)c1cn(C)c2c(C(C)C)cc(N3CCCc4cc(-c5cnn(C)c5)c(C(F)F)cc43)cc12
InChIKey: MFEDGSDMDRUZJQ-UHFFFAOYSA-N
Target: CREB-binding protein (SINGLE PROTEIN), Homo sapiens
UniProt: Q92793
Assay: TR FRET Binding Assay: His/Flag epitope tagged CBP was cloned, expressed, and purified to homogeneity. CBP binding and inhibition was assessed by monitoring the engagement of a biotinylated small molecule compound with the target using the TR-FRET assay technology (Perkin-Elmer). Specifically, in a 384 well ProxiPlate CBP (4 nM final) was combined with biotin-ligand (60 nM final) in 50 mM HEPES (pH 7.5), 50 mM NaCl, 1 mM TCEP, 0.01% (w/v) BSA, and 0.008% (w/v) Brij-35 either in the presence of DMSO (final 0.2% DMSO) or compound dilution series in DMSO. After 10 minutes incubation at room temperature, a mixture Eu-W1024 Anti-6×His antibody (6×His is disclosed as SEQ ID NO: 3) (Perkin Elmer AD0110) and SureLight Allophycocyanin-Streptavidin (APC-SA, Perkin Elmer CR130-100) were added to a final concentrations of 0.2 nMolar antibody and 50 nMolar APC-SA, respectively.
Measurement: IC50 = 31 nM (pChEMBL 7.510000228881836)
Descriptors: MW 491.59, ALogP 6.08, HBA 5, HBD 1, PSA 55.09, QED 0.37
Compound (SMILES): Cn1c(=O)n(C)c2cc(-n3cc(C(=O)O)c(=O)n(Cc4cc(F)cc(C(F)(F)F)c4)c3=O)ccc21
InChIKey: SAFPZCFBZFJZRU-UHFFFAOYSA-N
Target: Chymase (SINGLE PROTEIN), Homo sapiens
UniProt: P23946
Assay: Inhibition of human recombinant chymase using Abz-HPFHL-Lys(Dnp)-NH2 as substrate by fluorescence based analysis
Measurement: IC50 = 2300 nM (pChEMBL 5.639999866485596)
Descriptors: MW 492.39, ALogP 2.09, HBA 8, HBD 1, PSA 108.23, QED 0.44
Compound (SMILES): CN1C(=O)C(NC(=O)c2n[nH]c(Cc3ccccc3)n2)COc2ccc(CCC(=O)N3CCOCC3)cc21
InChIKey: NINRIIKRGLAIMX-UHFFFAOYSA-N
Target: Receptor-interacting serine/threonine-protein kinase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q13546
Assay: biochemical assay using the ADP-Glo technology: ADP-Glo (Promega, Madison, Wis., USA) reagents were thawed at ambient temperature. Kinase Detection Reagent was prepared by mixing kinase detection buffer with the lyophilized kinase detection substrate.A 500 ml stock volume of 5× Reaction Kinase Buffer was made by mixing 1000 μl of 1M MgCl2, 500 μl of 1M Tris-HCL pH7.4, 0.5 mg/ml (25 mg) of BSA, and 3475 μl of distilled H2O. A 3 ml 2× working stock volume of Reaction Kinase Buffer was made containing a final concentration of 100 μM DTT and 4 mM MnCl2.Components of RIPK1 enzyme (Rigel Pharmaceuticals, South San Francisco, Calif., USA) were thawed on ice. Diluted RIPK1 was prepared in 1× Kinase Reaction Buffer (diluted from 2× buffer) to 31 ng/well. A 166 M working stock ATP assay solution was prepared in 1× Kinase Reaction Buffer (diluted from 2× buffer).Compounds were serially diluted in DMSO from 250 uM in 4-fold dilutions then diluted 1:5 in 2× Reaction Buffer in a 96 well plate. 1.0 ul of diluted compound was added to a 384 well plate in duplicate. 2 μl of diluted Active RIPK1 was added to 384 well plate (do not add to column1) add 2× rxn buffer to column 1. AKT (Anaspec, Fremont, Calif., USA) at 150 nM was combined with ATP working stock at equal volume and 2 ul/well were added to the 384 well plate. The final reaction volume was 5.0 μl. The plate was quickly centrifuged and the reaction was incubated at 30° C. for 30 minutes. Adding 5 μl of ADP-Glo terminated the reaction. The plate was quickly centrifuged and the reaction was incubated at room temperature for 40 minutes. Kinase Detection Reagent was then added and incubated at room temperature for 30 minutes. The relative light unit (RLU) of kinase reaction was determined by luminescent (Luminescence 0.1 s) using a Wallac Victor2 Luminometer (PerkinElmer, Waltham, Mass., USA).
Measurement: IC50 = 57.1 nM (pChEMBL 7.239999771118164)
Descriptors: MW 518.57, ALogP 1.34, HBA 7, HBD 2, PSA 129.75, QED 0.48
Compound (SMILES): Cn1nc(N)c2cc(-c3cccc(-n4ncc5cc(C(C)(C)C)cc(F)c5c4=O)c3CO)cc(C(N)=O)c21
InChIKey: HDVYUHIZICKEMJ-UHFFFAOYSA-N
Target: Tyrosine-protein kinase BTK (SINGLE PROTEIN), Homo sapiens
UniProt: Q06187
Assay: 25P Btk Enzyme Activity Assay: Btk enzymatic activity was determined with the LANCE (Lanthanide Chelate Excite) TR-FRET (lime-resolved fluorescence resonance energy transfer) assay. In this assay, the potency (IC50) of each compound was determined from an eleven point (1:3 serial dilution; final compound concentration range in assay from 1000 nM to 0.017 nM) titration curve using the following outlined procedure. To each well of a black non-binding surface Corning 384-well microplate (Corning Catalog #3820), 5 nL of compound (2000 fold dilution in final assay volume of 10 μL) was dispensed, followed by the addition of 7.5 μL of 1× kinase buffer (50 mM Hepes 7.5, 10 mM MgCl2, 0.01% Brij-35, 1 mM EGTA, 0.05% BSA and 1 mM DTT) containing 26.67 pg/μL (266.7 pM) of 25P Btk enzyme (recombinant protein from baculovirus-transfected Sf9 cells: full-length Btk; MW=79378 Da). Following a 60 minute compound and enzyme incubation, each reaction was initiated by the addition of 2.5 μL 1× kinase buffer containing 8 μM biotinylated A5 peptide (Biotin-EQEDEPEGDYFEWLE-NH2), and 100 μM ATP. The final reaction in each well of 10 consists of 200 pM 25P Btk, 2 μM biotin-A5-peptide, and 25 μM ATP. Phosphorylation reactions were allowed to proceed for 120 minutes. Reactions were immediately quenched by the addition of 20 uL of 1× quench buffer (15 mM EDTA, 25 mM Hepes 7.3, and 0.1% Triton X-100) containing detection reagents (0.626 nM of LANCE-Eu-W1024-anti-phosphoTyrosine antibody, PerkinElmer and 86.8 nM of Streptavidin-conjugated Dylight 650, Dyomics/ThermoFisher Scientific). After 60 minutes incubation with detection reagents, reaction plates were read on a PerkinElmer EnVision plate reader using standard TR-FRET protocol. Briefly, excitation of donor molecules (Eu-chelate:anti-phospho-antibody) with a laser light source at 337 nm produces energy that can be transferred to Dylight-650 acceptor molecules if this donor:acceptor pair is within close proximity. Fluorescence intensity at both 665 nm (acceptor) and 615 nm (donor) are measured and a TR-FRET ratio calculated for each well (acceptor intensity/donor intensity). IC50 values were determined by 4 parameter robust fit of TR-FRET ratio values vs. (Log10) compound concentrations.
Measurement: IC50 = 47.3 nM (pChEMBL 7.329999923706055)
Descriptors: MW 514.56, ALogP 3.55, HBA 8, HBD 3, PSA 142.05, QED 0.33
Compound (SMILES): CC1(C)CC(=O)c2c([nH]c(-c3ccnc(NC(=O)c4cnn(Cc5ccccc5)c4)c3)c2Nc2ccccc2)C1
InChIKey: CZKLNTCZBDRDTE-UHFFFAOYSA-N
Target: Mitotic checkpoint serine/threonine-protein kinase BUB1 (SINGLE PROTEIN), Homo sapiens
UniProt: O43683
Assay: Kinase Assay: In a typical assay 11 different concentrations of each compound (0.1 nM, 0.33 nM, 1.1 nM, 3.8 nM, 13 nM, 44 nM, 0.15 μM, 0.51 μM, 1.7 μM, 5.9 μM and 20 μM) were tested in duplicate within the same microtiter plate. To this end, 100-fold concentrated compound solutions (in DMSO) were previously prepared by serial dilution (1:3.4) of 2 mM stocks in a clear low volume 384-well source microtiter plate (Greiner Bio-One, Frickenhausen, Germany), from which 50 nl of compounds were transferred into a black low volume test microtiter plate from the same supplier. Subsequently, 2 μL of Bub1 (the final concentration of Bub1 was adjusted depending on the activity of the enzyme lot in order to be within the linear dynamic range of the assay: typically 200 ng/mL were used) in aqueous assay buffer [50 mM Tris/HCl pH 7.5, 10 mM magnesium chloride (MgCl2), 200 mM potassium chloride (KCl), 1.0 mM dithiothreitol (DTT), 0.1 mM sodium ortho-vanadate, 1% (v/v) glycerol, 0.01% (w/v) bovine serum albumine (BSA), 0.005% (v/v) Trition X-100 (Sigma), 1× Complete EDTA-free protease inhibitor mixture (Roche)] were added to the compounds in the test plate and the mixture was incubated for 15 min at 22° C. to allow pre-equilibration of the putative enzyme-inhibitor complexes before the start of the kinase reaction, which was initiated by the addition of 3 μL 1.67-fold concentrated solution (in assay buffer) of adenosine-tri-phosphate (ATP, 10 μM final concentration) and peptide substrate (1 μM final concentration). The resulting mixture (5 μL final volume) was incubated at 22° C. during 60 min., and the reaction was stopped by the addition of 5 μL of an aqueous EDTA-solution (50 mM EDTA, in 100 mM HEPES pH 7.5 and 0.2% (w/v) bovine serum albumin) which also contained the TR-FRET detection reagents (0.2 μM streptavidin-XL665 [Cisbio Bioassays, Codolet, France] and 1 nM anti-phosho-Serine antibody [Merck Millipore, cat. #35-002] and 0.4 nM LANCE EU-W1024 labeled anti-mouse IgG antibody [Perkin-Elmer, product no. AD0077, alternatively a Terbium-cryptate-labeled anti-mouse IgG antibody from Cisbio Bioassays can be used]). The stopped reaction mixture was further incubated 1 h at 22° C. in order to allow the formation of complexes between peptides and detection reagents.
Measurement: IC50 = 4.23 nM (pChEMBL 8.369999885559082)
Descriptors: MW 530.63, ALogP 6.47, HBA 6, HBD 3, PSA 104.7, QED 0.22
Compound (SMILES): Cc1nnc(-c2cccc(N3CCN(C)c4nc(NCCc5ccccc5)ncc4C3=O)c2)o1
InChIKey: GBKBMKMHPMFPFR-UHFFFAOYSA-N
Target: Voltage-dependent calcium channel subunit alpha-2/delta-1 (SINGLE PROTEIN), Homo sapiens
UniProt: P54289
Assay: Binding Assay: α2δ-1: Binding Assay to Human α2δ-1 Subunit of Cav2.2 Calcium Channel.Human α2δ-1 enriched membranes (2.5 μg) were incubated with 15 nM of radiolabeled [3H]-Gabapentin in assay buffer containing Hepes-KOH 10 mM, pH 7.4.NSB (non specific binding) was measured by adding 10 μM pregabalin. After 60 min incubation at 27° C., binding reaction was terminated by filtering through Multiscreen GF/C (Millipore) presoaked in 0.5% polyethyleneimine in Vacuum Manifold Station, followed by 3 washes with ice-cold filtration buffer containing 50 mM Tris-HCl, pH 7.4.Filter plates were dried at 60° C. for 1 hour and 30 μl of scintillation cocktail were added to each well before radioactivity reading.Readings were performed in a Trilux 1450 Microbeta radioactive counter (Perkin Elmer).
Measurement: Ki > 3000 nM (pChEMBL 5.5229997634887695)
Descriptors: MW 455.52, ALogP 3.59, HBA 8, HBD 1, PSA 100.28, QED 0.47
Compound (SMILES): CC1(C)Oc2cc(Cl)ccc2C2OCC3(CCN(CCn4ccnc4CCN)CC3)CC21
InChIKey: GAHYWFXDIVLVNW-UHFFFAOYSA-N
Target: Voltage-gated inwardly rectifying potassium channel KCNH2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q12809
Assay: Test for Evaluating hERG Inhibition: To a cultured CHO cell strain which stably expresses hERG was added each test compound to adjust the final concentration of DMSO to 0.0135 to 0.5%. The hERG current was measured with QPatch HT (Sophion Inc.), and the concentration at which 50% of the hERG current was inhibited by each test compound (IC50 value; μM) was calculated.
Measurement: IC50 = 6300 nM (pChEMBL 5.199999809265137)
Descriptors: MW 459.03, ALogP 4.07, HBA 6, HBD 1, PSA 65.54, QED 0.73
Compound (SMILES): C[C@@]1(c2cc(NC(=O)c3ccc(Cl)cn3)ccc2F)Cn2nncc2C(N)=N1
InChIKey: FIUOQWQRVSRKHL-SFHVURJKSA-N
Target: Beta-secretase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P56817
Assay: FRET Based Assay: This assay is a Fluorescence Resonance Energy Transfer Assay (FRET) based assay. The substrate for this assay is an APP derived 13 amino acids peptide that contains the ‘Swedish’ Lys-Met/Asn-Leu mutation of the amyloid precursor protein (APP) beta-secretase cleavage site. This substrate also contains two fluorophores: (7-methoxycoumarin-4-yl) acetic acid (Mca) is a fluorescent donor with excitation wavelength at 320 nm and emission at 405 nm and 2,4-Dinitrophenyl (Dnp) is a proprietary quencher acceptor. The distance between those two groups has been selected so that upon light excitation, the donor fluorescence energy is significantly quenched by the acceptor, through resonance energy transfer. Upon cleavage by BACE1, the fluorophore Mca is separated from the quenching group Dnp, restoring the full fluorescence yield of the donor. The increase in fluorescence is linearly related to the rate of proteolysis.
Measurement: IC50 = 1.74 nM (pChEMBL 8.760000228881836)
Descriptors: MW 399.82, ALogP 2.35, HBA 7, HBD 2, PSA 111.08, QED 0.7