lovingscience/chembl-v37-raw-activities huggingface dataset:
Compound (SMILES): CCc1cc([C@]23C[C@H]2CN(CC[C@H]2CC[C@H](NC(=O)Cc4cnn(C)n4)CC2)C3)no1
InChIKey: ADCYPKNNPPDYMB-ORQYLMBXSA-N
Target: D(3) dopamine receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P35462
Assay: Binding Inhibition for Dopamine D3 Receptor: Buffer solution: 50 mM Tris-HCl (35409-45, Nacalai Tesque) (pH 7.4) containing 120 mM NaCl (31320-05, Nacalai Tesque), 1 mM MgCl2.6H2O (20909-55, Nacalai Tesque), 5 mM KCl (28514-75, Nacalai Tesque) and 2 mM CaCl2 (067-31, NAKARAI CHEMICALS, LTD.)Radioligand: (final concentration) 2 nM [3H]-Methylspiperone ([3HN-methyl-]-Methylspiperone, NET-856, 83.8 Ci/mmol, PerkinElmer)Non-specific ligand: (final concentration) 10 μM Butaclamol [(+)-Butaclamol Hydrochloride, D033, Sigma]SPA beads solution: SPA beads [WGA PVT SPA Scintillation Beads, RPNQ0001 (500 mg), RPNQ0060 (2 g), PerkinElmer] (0.2 mg/well)Incubation time and temperature: 120 min at 25° C.
Measurement: Ki = 0.48 nM (pChEMBL 9.319999694824219)
Descriptors: MW 426.57, ALogP 2.25, HBA 7, HBD 1, PSA 89.08, QED 0.7
Compound (SMILES): COc1nc(N[C@H]2CN(S(=O)(=O)c3cn(C)cn3)C[C@@H]2c2ccccc2)cc(C(F)(F)F)n1
InChIKey: HUTRNOQBLQCCPK-CABCVRRESA-N
Target: Alpha-1,3-mannosyl-glycoprotein 2-beta-N-acetylglucosaminyltransferase (SINGLE PROTEIN), Homo sapiens
UniProt: P26572
Assay: Radioligand Binding Assay: Radioligand binding assays using recombinant CHO cell membranes expressing human GlyT1: Radioligand binding to human GlyT1c transporter-expressing membranes was determined as described in Mezler et al., Molecular Pharmacology 74:1705-1715, 2008.
Measurement: Ki < 1000 nM (pChEMBL 6)
Descriptors: MW 482.49, ALogP 2.51, HBA 8, HBD 1, PSA 102.24, QED 0.58
Compound (SMILES): CC[C@H](O)[C@@H](NC(=O)[C@@H](C)N1Cc2ccc(-c3nc(NC4CCOCC4)ncc3Cl)cc2C1=O)c1ccnc(OC)c1
InChIKey: HVJXAGNSXVXYSL-MROGCYOMSA-N
Target: Mitogen-activated protein kinase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P28482
Assay: ERK2 In Vitro Inhibition Assay: Activity of ERK2 enzyme (Life Technologies) was determined using a time-resolved fluorescence format measuring the phosphorylation of a truncated version of Activating transcription factor 2 labelled with green fluorescent protein (ATF2-GFP) (Life Technologies). Assay reactions containing 50 mM Tris pH 7.5, 10 mM MgCl2, 1 mM EGTA, 0.01% Triton X-100, 1 mM DTT, 2.5% DMSO, 0.4 mM ATF2-GFP, 20 μM ATP and 0.25 nM ERK2 were set up in the presence of compound and allowed to proceed for 30 min at room temperature. Reactions were then stopped using TR-FRET dilution buffer (Life Technologies), 25 mM EDTA and 2 nM Tb-Anti-pATF2 (Thr71) (Life Technologies). After a further incubation period of at least 30 minutes, fluorescence was read on a Pherastar reader (Lanthascreen optic module; excitation 340 nm, emission 520 nm (channel A), 495 nm (channel B)). The ratio between A and B counts was used to calculate signal. IC50 values were calculated using a sigmoidal dose response equation (Prism GraphPad software, La Jolla, Calif., USA).
Measurement: IC50 = 2.1 nM (pChEMBL 8.680000305175781)
Descriptors: MW 595.1, ALogP 3.76, HBA 9, HBD 3, PSA 138.8, QED 0.32
Compound (SMILES): CC(CCN)n1c([C@H](C)Nc2ncnc(N)c2C#N)nc2cccc(Cl)c2c1=O
InChIKey: XKXDQJLRKPXIPL-DTIOYNMSSA-N
Target: Phosphatidylinositol 3-kinase regulatory subunit beta (SINGLE PROTEIN), Homo sapiens
UniProt: O00459
Assay: a time-resolved fluorescence resonance energy transfer (TR-FRET) assay: Class I PI3K isoforms were expressed and purified as heterodimeric recombinant proteins. All assay reagents and buffers for the TR-FRET assay were purchased from Millipore. PI3K isoforms were assayed under initial rate conditions in the presence of 25 mM Hepes (pH 7.4), and 2×Km ATP (75-500 μM), 2 μM PIP2, 5% glycerol, 5 mM MgCl2, 50 mM NaCl, 0.05% (v/v) Chaps, 1 mM dithiothreitol, and 1% (v/v) DMSO at the following concentrations for each isoform: PI3Kα, PI3Kβ, and PI3Kδ between 25 and 50 pM, and PI3Kγ at 2 nM. The compounds of Table 1 and Compound X, ((S)-2,4-diamino-6-((1-(5-chloro-4-oxo-3-phenyl-3,4-dihydroquinazolin-2-yl)ethyl)amino)pyrimidine-5-carbonitrile) were added to the assay solution and incubated for 30 minutes at 25° C. The reactions were terminated with a final concentration of 10 mM EDTA, 10 nM labeled-PIP3, and 35 nM Europium labeled GRP-1 detector protein before reading TR-FRET on an Envision plate reader (Ex: 340 nm; Em: 615/665 nm; 100 μs delay and 500 μs read window).The results were normalized based on positive (1 pM wortmanin) and negative (DMSO) controls, and the IC50 values for PI3K α, β, δ, and γ were calculated from the fit of the dose-response curves to a four-parameter equation. These assays generally produced results within 3-fold of the reported mean.
Measurement: IC50 = 51 nM (pChEMBL 7.289999961853027)
Descriptors: MW 412.89, ALogP 2.38, HBA 9, HBD 3, PSA 148.53, QED 0.56
Compound (SMILES): CN(C)C(=O)c1cc2ccc(-c3nccc(Nc4ccc(-c5cn[nH]c5)cc4)n3)cc2[nH]1
InChIKey: PQKNVEFRWWFVMD-UHFFFAOYSA-N
Target: Rho-associated protein kinase 2 (SINGLE PROTEIN), Homo sapiens
UniProt: O75116
Assay: Biological Assay: The kinase IC50 was determined by a commercialized CISBIO kinase detection kit, HTRF KinEASE-STK S2 kit (62ST2PEC). ROCK2 (01-119) employed in the reaction was purchased from Carna Biosciences.Before the assay, the following working solutions as needed were formulated with corresponding reagents according to the instruction of the kinase detection kit: 1×kinase buffer, 5×STK-52 substrate working solution (1.5 μM) and 5×ATP working solution (1.5 μM), 5×ROCK2 kinase working solution, 4×Streptavidin-XL665 working solution, and 4×STK-Ab-Cryptate 2 detection solution. Then the assay was performed according to the following procedure.A solution of a compound at a concentration of 10000 nM was prepared with the 1×kinase buffer containing 2.5% DMSO. Gradient dilution of the solution of the compound was performed with the kinase buffer containing DMSO, so as to obtain solutions of a test compound at 9 different concentrations. In addition to wells of test compounds, a positive well (containing all the reagents except the compound) and a negative well (containing all the reagents except the test compound and kinase) were set. Except for the control wells (positive and negative wells), a solution of a test compound (4 μL) was added to each of the reaction wells, and a solution of 2.5% DMSO was added to the control wells. Then the substrate (2 μM, i.e., 2 μL 5×STK-S2 substrate working solution) was added to each of the reaction wells. The 5×ROCK2 kinase working solution (2 μL, containing 1.4 ng ROCK2 kinase) was added to each of the reaction wells except for the negative well, the volume of which was made up with the 1×kinase buffer (2 μL). The 5×ATP working solution (2 μL) was added to each of the reaction wells, and the mixtures were incubated at room temperature for 2 hours. After the kinase reaction was complete, the 4×Streptavidin-XL665 working solution was added to each of the reaction wells, the solutions were mixed, followed by immediate addition of the 4×STK-Ab-Cryptate 2 detection solution (5 μL), and the mixtures were incubated at room temperature for 1 hour. The fluorescence signal was read on ENVISION (Perkinelmer) (excitation wavelength: 320 nm, and emission wavelength: 665 nm and 615 nm). The inhibitory rate in each well was calculated based on the fluorescence intensity value: ER (Emission Ratio)=(fluorescence intensity at 665 nm/fluorescence intensity at 615 nm); inhibitory rate=(ERpositive−ERtest compound) (ERpositive−ERnegative)*100%. Curves were plotted and fitted to obtain the median inhibitory concentration (IC50) of each teat compound with the PRISM 5.0 software.
Measurement: IC50 = 352 nM (pChEMBL 6.449999809265137)
Descriptors: MW 423.48, ALogP 4.46, HBA 5, HBD 3, PSA 102.59, QED 0.39
Compound (SMILES): Cc1cc(C2CCOCC2)n2nc(NCc3cc(F)cc(Br)c3)nc2n1
InChIKey: ADKQMSNFRIIQFT-UHFFFAOYSA-N
Target: cGMP-dependent 3',5'-cyclic phosphodiesterase (SINGLE PROTEIN), Homo sapiens
UniProt: O00408
Assay: IMAP TR-FRET-based phosphodiesterase assay: An IMAP TR-FRET-based phosphodiesterase assay was developed using the PDE2A isoform. IMAP technology is based on high-affinity binding of phosphate by immobilized metal (MIII) coordination complexes on nanoparticles. The IMAP binding reagent recognizes phosphate groups on AMP or GMP generated from cAMP or cGMP in a PDE reaction. Cyclic nucleotides that carry a phosphodiester bond and not a free phosphate are not recognized by the binding reagent. The time resolved fluorescence resonance energy transfer (TR-FRET) is afforded by a Terbium (Tb)-Donor pre-bound to the nanoparticles. FRET occurs when the fluorescent-labeled AMP or GMP product of a PDE reaction binds and comes into close proximity to the Tb-Donor complex. Due to the long lifetime of Tb fluorescence, detection can be run in time-resolved mode to reduce or eliminate interference from auto-fluorescent compounds.The IMAP TR-FRET PDE2A assay was performed in 1536-well white plates. A total of 250 pg per well of FLAG-tagged PDE2A1 (amino acids 2-941) was dispensed in 2.5 μL IMAP assay buffer consisting of 10 mM Tris pH 7.2, 10 mM MgCl2, 1 mM DTT, and 0.1% fatty acid free BSA. 30 nL of compound was then added from 1 mM stocks in DMSO using a Kalypsys Pintool. Plates were incubated for 5 min at room temperature before dispensing 1.5 μL of 533 nM FAM-cAMP substrate for a final concentration of 200 nM. Following a brief centrifugation, plates were incubated for 30 min at room temperature. The assay was terminated by adding 5 μL IMAP binding reagent Tb complex to each well which was prepared according to manufacturer's recommendations (Molecular Devices). Plates were incubated an additional 90 minutes at room temperature and read on a Viewlux plate reader. All compounds were solvated at a concentration of 10 mM in DMSO and tested in 11-point half-log dose-response. Curve fitting and IC50 values were determined using a standard four parameter fit.
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 420.29, ALogP 3.84, HBA 6, HBD 1, PSA 64.34, QED 0.7
Compound (SMILES): CC(=O)c1ccc(S(=O)(=O)c2ccc(NC(=O)NCc3cccnc3)cc2)cc1
InChIKey: VVIFRQHLJQNFPG-UHFFFAOYSA-N
Target: Nicotinamide phosphoribosyltransferase (SINGLE PROTEIN), Homo sapiens
UniProt: P43490
Assay: Biochemical Inhibition Assay: The NAMPT enzymatic reactions were carried out in Buffer A (50 mM Hepes pH 7.5, 50 mM NaCl, 5 mM MgCl2, and 1 mM THP) in 96-well V-bottom plates. The compound titrations were performed in a separate dilution plate by serially diluting the compounds in DMSO to make a 100× stock. Buffer A (89 μL) containing 33 nM of NAMPT protein was added to 1 μL of 100× compound plate containing controls (e.g. DMSO or blank). The compound and enzyme mix was incubated for 15 minutes at room temperature, then 10 μL of 10× substrate and co-factors in Buffer A were added to the test well to make a final concentration of 1 μM NAM, 100 μM 5-Phospho-D-ribose 1-diphosphate (PRPP), and 2.5 mM Adenosine 5′-triphosphate (ATP). The reaction was allowed to proceed for 30 minutes at room temperature, then was quenched with the addition of 11 μL of a solution of formic acid and L-Cystathionine to make a final concentration of 1% formic acid and 10 μM L-Cystathionine. Background and signal strength was determined by addition (or non-addition) of a serial dilution of NMN to a pre-quenched enzyme and cofactor mix.
Measurement: IC50 = 2 nM (pChEMBL 8.699999809265137)
Descriptors: MW 409.47, ALogP 3.44, HBA 5, HBD 2, PSA 105.23, QED 0.61
Compound (SMILES): CC[C@H]1NC(=O)[C@@H]([C@H](O)[C@H](C)CCCc2ccnn2C)N(C)C(=O)[C@@H](C(C)C)N(C)C(=O)[C@@H](CC(C)C)N(C)C(=O)[C@@H](CC(C)C)N(C)C(=O)[C@H](C)NC(=O)[C@@H](C)NC(=O)[C@@H](CC(C)C)N(C)C(=O)[C@@H](C(C)C)NC(=O)[C@H](CC(C)C)N(C)C(=O)[C@@H](C)N(C)C1=O
InChIKey: PJFCXNVGTPNOPO-LBRYMDJZSA-N
Target: Peptidyl-prolyl cis-trans isomerase A (SINGLE PROTEIN), Homo sapiens
UniProt: P62937
Assay: Protease-Free PPIase Assay: The protease-free PPIase assay measures the rate of cis to trans conversion of a peptide substrate catalyzed by the enzyme cyclophilin A. Addition of a cyclophilin A inhibitor (e.g., a test compound) slows the catalyzed rate and a Ki value is obtained. A Ki value of less than 10 nM demonstrates that the test compound is a potent inhibitor of cyclophilin A.MaterialsAssay Buffer:35 mM HEPES pH 7.8, filtered through a 0.2 μm filter. 50 μM DTT was added prior to use each day and then the buffer was stored on ice.Enzyme:Human recombinant cyclophilin A (Cyp A) (Sigma C3805) enzyme was diluted to 1 μM with enzyme dilution buffer (20 mM HEPES pH 7.8, 40% glycerol, 50 μM DTT and 1 μM BSA) and stored at −20° C.Substrate:Succinimide-Ala-Ala-Pro-Phe-p-nitroanilide (SUC-AAPF-pNA) (from Bachem AG, L-1400), 20 mg/ml prepared in 0.5 M LiCl in trifluoroethanol.MethodAll readings were taken with an Agilent 8453 Spectrophotometer which includes of a cuvette holder, stirrer and chiller to maintain a stirred cuvette temperature of 10.0±0.1° C. The temperature is monitored by the use of a temperature probe. To prevent UV degradation of test compounds, the light below 290 nm was blocked using a glass slide in the light path. 1.5 ml of the assay buffer was put into a 3 ml quartz cuvette and cooled to 10.0±0.1° C. while stirring (vigorous but not so fast as to produce cavitation). The inhibitor was diluted in 100% DMSO, and then added to the assay to a maximum final concentration of 0.5% DMSO in the assay. A blank spectrum was obtained, then 3 μL of enzyme was added (2 nM final concentration) and then 3 μL substrate (60 μM final concentration) added. The absorbance was measured at 330 nm for 300 s or 500 s for blank runs (NOTE: the substrate must be added in one quick injection and the measurements started immediately to minimize mixing errors).A first order rate equation was fitted to the absorbance data, for each concentration of inhibitor, to obtain the rate constant (the first 10 to 15 seconds were excluded as mixing causes errors in this portion of curve). The catalytic rate was calculated from the enzymatic rate constant minus the background rate constant. An exponential curve was generated using the catalytic rate constants versus the inhibitor concentration to obtain the Ki value for the inhibitor. The Ki value is indicative of the binding affinity between the test compound and cyclophilin A.
Measurement: Ki = 2.1 nM (pChEMBL 8.680000305175781)
Descriptors: MW 1284.74
Compound (SMILES): Cc1cc(F)ccc1-c1cc([C@H]2CC[C@H](CC(=O)O)CC2)ncc1N(C)C(=O)C(C)(C)c1cc(C(F)(F)F)cc(C(F)(F)F)c1
InChIKey: JTHFKRBAGMFRLP-MXVIHJGJSA-N
Target: Substance-P receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P25103
Assay: Receptor Binding Assay: The buffer solution for the receptor binding test was dispensed to the wells of a 96-well assay plate (Greiner) at 22.5 μL/well. DMSO solutions of a test compound, which were prepared at an 80-time higher concentration using 100% dimethyl sulfoxide (DMSO), were added to the wells at 2.5 μL/well (final concentrations of 1 nM to 100 nM), and the solutions were mixed. As a radiolabeled ligand, 125I-substance P (Substance P, [125I]Tyr8-, PerkinElmer) was used. 125I-substance P was diluted with the buffer solution for the receptor binding test to a concentration resulting in 125 pmol/25 μL/well and added to the 96-well assay plate, and the solutions were mixed. The membrane fraction prepared from the human NK1 receptor-expressing cells was diluted with the buffer solution for the receptor binding test to a concentration resulting in 8 to 10 μg/well, suspended until the suspension became in such a homogenous state that the suspension could flow through a 27 G injection needle smoothly and then added to the 96-well assay plate at 150 μL/well. Then, the plate was incubated at room temperature for 60 minutes while shaking the plate. The reaction solutions were suction-filtered through a multiscreen 96-well filter plate (Millipore) which had been pre-treated with 0.3% polyethyleneimine, and the reaction was terminated by washing with a washing solution (50 mM Tris and 0.02% bovine serum albumin, pH 7.4) four times. The bottom of the microplate was dried at 60° C., and then 100 μL/well of MicroScint 20 (PerkinElmer) was dispensed to the wells. The top of the plate was sealed with TopSeal A (PerkinElmer), and the plate was shaken for 5 to 10 minutes. Then, the radioactivities were measured with TopCount NXT (registered trademark) (PerkinElmer). The radioactivity of each well was calculated by subtracting the radioactivity of the well to which 10 μM aprepitant was added (non-specific binding). The binding rate (%) of 125I-substance P=(the radioactivity of the group to which the test compound was added)/(the radioactivity of the group to which the vehicle was added)×100 was calculated. Using analysis software, GraphPad Prism (GraphPad Software), the binding rate (%) was plotted against the concentration of the test compound and linearly approximated, and the concentration required for 50% inhibition, IC50, was calculated.
Measurement: IC50 = 1.62 nM (pChEMBL 8.789999961853027)
Descriptors: MW 638.62, ALogP 8.92, HBA 3, HBD 1, PSA 70.5, QED 0.26
Compound (SMILES): COc1ncc(-c2cc(CN3CCC(F)(F)CC3)c3c(N)ncnn23)cc1C(=O)N[C@@H]1CN(C(=O)c2cc(F)cc(F)c2)C[C@@H]1F
InChIKey: HCRVEXZRJMKIEV-XZOQPEGZSA-N
Target: Receptor-interacting serine/threonine-protein kinase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q13546
Assay: RIPK1 HTRF Binding Assay: A solution was prepared containing 0.2 nM Anti GST-Tb (Cisbio, 61GSTTLB), 90.6 nM probe and 1 nM His-GST-TVMV-hRIPK1(1-324) in FRET Buffer (20 mM HEPES, 10 mM MgCl2, 0.015% Brij-35, 4 mM DTT, 0.05 mg/mL BSA). Using Formulatrix Tempest, the detection antibody/enzyme/probe solution (2 mL) was dispensed into wells of a 1536 plate (Black Low Binding Polystyrene 1536 Plate (Corning, 3724)) containing 10 nL of compounds of interest at appropriate concentration in DMSO. The plate was incubated at rt for 1 h. FRET was measured using the EnVision plate reader (Excitation: 340 nM, Emission: 520 nM/495 nM). Total signal (0% inhibition) was calculated from wells containing 10 nL DMSO only. Blank signal (100% inhibition) calculated from wells containing 10 nL of 15 nM staurosporine and internal controls.
Measurement: IC50 = 2500 nM (pChEMBL 5.599999904632568)
Descriptors: MW 644.6, ALogP 3.48, HBA 9, HBD 2, PSA 130.98, QED 0.29
Compound (SMILES): COc1cc2c(Oc3ccc(NC(=O)Nc4cc(C)on4)c(Cl)c3)ncnc2cc1OCCCOC1COC1
InChIKey: STAIEXXCCDJVGT-UHFFFAOYSA-N
Target: Vascular endothelial growth factor receptor 2 (SINGLE PROTEIN), Homo sapiens
UniProt: P35968
Assay: VEGFR2 Binding Assay: A competition binding assay (DISCOVERX KINOMESCAN) was used to measure the ability of a compound to compete for binding of an immobilized adenosine triphosphosphate (ATP) site directed ligand using a DNA-tagged vascular endothelial growth receptor 2 (VEGFR2) as the target. The ability of the test compound to compete with the immobilized ligand was measured using quantitative polymerase chain reaction (qPCR) of the DNA tag (Fabian, M. A. et al., 23 Nature Biotechnology 329-336 (2005); Karaman, M. W. et al., 26 Nature Biotechnology 127-132 (2008)).A VEGFR2 tagged T7 phage strain was prepared in an Escherichia coli (E. coli) derived from the BL21 strain. The E. coli were grown to log-phase, infected with VEGFR2 tagged T7 phage and then incubated with shaking at 32° C. until lysis. The lysate containing the kinase was then centrifuged and filtered to remove cell debris. Affinity resin for the VEGFR2 assay was prepared by treating Streptavidin-coated magnetic beads with a biotinylated small molecule ligand for 30 minutes at room temperature. The beads were blocked with excess biotin and then washed with blocking buffer (SEABLOCK (PIERCE), 1% bovine serum albumin, 0.17% phosphate buffered saline, 0.05% TWEEN 20, 6 mM dithiothreitol). The binding reaction was initiated by combining in a well of a polystyrene 96-well plate, DNA tagged VEGFR2, liganded affinity beads and the serial diluted test compound in 1× binding buffer (20% SEABLOCK, 0.17× phosphate buffered saline, 0.05% TWEEN 20, 6 mM dithiothreitol) in a final volume of 0.135 ml. The assay plates were incubated at room temperature with shaking for 1 hour and then the beads were washed with wash buffer (1× phosphate buffered saline, 0.05% TWEEN 20). The beads were re-suspended in elution buffer (1× phosphate buffered saline, 0.05% TWEEN 20, 0.05 μM non-biotinylated affinity ligand) and incubated at room temperature with shaking for 30 minutes. The VEGFR2 concentration in the eluate was measured using qPCR.An 11-point dose response curve of 3-fold serial diluted test compound starting at 1 μM was used to determine the VEGFR2 binding constant (Kd). The compounds were prepared in 100% DMSO at 100× the final test concentration and the diluted to 1× in the assay for final DMSO concentration of 1%. Binding constants were calculated with standard dose-response curve using the Hill equation with Hill slope set to −1. Curves were fit using a non-linear least square fit with the Levenberg-Marquardt algorithm.
Measurement: Kd = 980 nM (pChEMBL 6.010000228881836)
Descriptors: MW 555.98, ALogP 5.21, HBA 10, HBD 2, PSA 139.09, QED 0.24
Compound (SMILES): C[C@@H]1CN(c2nccc(C#N)n2)CCN1C(=O)NCCC1CCN(Cc2ccccc2)CC1
InChIKey: XBONZKCZRZMMLR-HXUWFJFHSA-N
Target: Muscarinic acetylcholine receptor M4 (SINGLE PROTEIN), Homo sapiens
UniProt: P08173
Assay: Binding Assay: Binding affinity (Ki) of compounds was measured by inhibition of radioligand binding to membranes from CHO cells expressing human M1, M2, M3, M4 and M5 receptors. Membranes were prepared by nitrogen cavitation and differential centrifugation as previously described (Hoare et al., Mol. Pharmacol. 2003 March; 63(3): 751-65). The radioligand employed was tritiated N-methylscopolamine, used at a concentration of 1.5 nM. A dose-response of twelve concentrations of compound was used, ranging from 10 Î¼M to 32 Î¼M. The assay buffer was 50 mM HEPES, 100 mM NaCl, 5 mM MgCl2, 1 mM ethylenediaminetetraacetic acid, pH-adjusted to pH 7.4.
Measurement: Ki = 55 nM (pChEMBL 7.260000228881836)
Descriptors: MW 447.59, ALogP 2.87, HBA 6, HBD 1, PSA 88.39, QED 0.73
Compound (SMILES): Cc1cnc2ccc(-c3c(N4CCC5(COC5)C4)nc(N)n4nc(Cc5ncccc5F)nc34)cn12
InChIKey: KLNPLIKINOXCQC-UHFFFAOYSA-N
Target: Adenosine receptor A2a (SINGLE PROTEIN), Homo sapiens
UniProt: P29274
Assay: Binding Affinities to Different Adenosine Receptors: The compounds at different concentrations were incubate with hA1 membrane (from PerkinElmer) and [3H]-8-Cyclopentyl-1,3-dipropylxanthine (DPCPX) for 50 min at 25° C., meanwhile 100 μL 0.5% PEI solution was added into UNFILTER-96 GF/B filter plate for 60 min at 4° C., then UNIFILTER-96 GF/B filter plate was washed twice with 50 ml wash buffer, the membrane mix was transferred into UNIFILTER-96 GF/B filter plate, and the filter plate was washed 4 times before incubated at 55° C. for 10 min. At last, 40 L ULTIMA GOLD was added into each well, and CPM was read by TopCount.The compounds at different concentrations were incubate with hA2a membrane (from PerkinElmer) and [3H]-CGS21680 for 90 min at 25° C., meanwhile 100 μL 0.5% PEI solution was added into UNFILTER-96 GF/B filter plate for 60 min at 4° C., then UNIFILTER-96 GF/B filter plate was washed twice with 50 ml wash buffer, the membrane mix was transferred into UNIFILTER-96 GF/B filter plate, and the filter plate was washed 4 times before incubated at 55° C. for 10 min. At last, 40 L ULTIMA GOLD was added into each well, and CPM was read by TopCount.The compounds at different concentrations were incubate with hA2b membrane (from PerkinElmer) and [3H]-DPCPX for 60 min at 27° C., and the binding reactions were stopped by rapid filtration through 0.5% BSA coated UNIFILTER-96 GF/C plates using cell harvester. The filter plates were then washed three times with ice cold wash buffer, and dried at 37° C. for 120 min. At last, 50 L of scintillation cocktail was added into each well, and CPM was read by TopCount.The compounds at different concentrations were incubate with hA3 membrane (from PerkinElmer) and [1211]-AB-MECA for 60 min at 27° C., the binding reactions were stopped by rapid filtration through 0.50% BSA coated UNIFILTER-96 GF/C plates using cell harvester. The filter plates were then washed three times with ice cold wash buffer, and dried at 37° C. for 120 min. At last, 50 L of scintillation cocktail was added into each well, and CPM was read by TopCount.
Measurement: IC50 = 795 nM (pChEMBL 6.099999904632568)
Descriptors: MW 485.53, ALogP 2.68, HBA 10, HBD 1, PSA 111.76, QED 0.41
Compound (SMILES): CC(=O)c1cn(CC(=O)N2C[C@H](F)C[C@H]2C(=O)N[C@H]2CCCC[C@@H]2OCc2ccccc2)c2ccc(-c3ccnnc3)cc12
InChIKey: VDKAWHFQBJIXRL-PVKGFQCZSA-N
Target: Complement factor D (SINGLE PROTEIN), Homo sapiens
UniProt: P00746
Assay: Factor D Assay: Human factor D (purified from human serum, Complement Technology, Inc.) at 80 nM final concentration is incubated with test compound at various concentrations for 5 minutes at room temperature in 50 mM Tris, 1M NaCl, pH 7.5. A synthetic substrate Z-L-Lys-SBzl and DTNB (Ellman's reagent) are added to final concentrations of 100 μM each. The increase in color is recorded at OD405 nm in a microplate in kinetic mode over 30 minutes with 30 second time points in a spectrofluorimeter. IC50 values are calculated by non-linear regression from the percentage of inhibition of complement factor D activity as a function of test compound concentration.
Measurement: IC50 > 10000 nM (pChEMBL 5)
Descriptors: MW 597.69, ALogP 4.88, HBA 7, HBD 1, PSA 106.42, QED 0.28
Compound (SMILES): COc1cc(-c2cccc(-c3cccc(NC(=O)c4c[nH]c(=O)n(C)c4=O)c3C)c2C)cc(F)c1CNCCCC(N)=O
InChIKey: HEXDJBSPMSKGOK-UHFFFAOYSA-N
Target: Programmed cell death 1 ligand 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9NZQ7
Assay: Enzyme-Linked Immunosorbent AssayELISA: 96 Well plates were coated with 111 g/mL of human PD-L1 (obtained from R&D) in PBS overnight at 4° C. The wells were then blocked with 2% BSA in PBS (W/V) with 0.05% TWEEN-20 for 1 hour at 37° C. The plates were washed 3 times with PBS/0.05% TWEEN-20 and the compounds were serial diluted (1:5) in dilution medium and added to the ELISA plates. Human PD-1 and biotin 0.31 μg/mL (ACRO Biosystems) were added and incubated for 1 hour at 37° C. then washed 3 times with PBS/0.05% TWEEN-20. A second block was performed with 2% BSA in PBS (W/V)/0.05% TWEEN-20 for 10 min at 37° C. and the plates were washed 3 times with PBS/0.05% TWEEN-20. Streptavidin-HRP was added for 1 hour at 37° C. then the plates were washed 3 times with PBS/0.05% TWEEN-20. TMB substrate was added and reacted for 20 min at 37° C. A stop solution (2 N aqueous H2SO4) was added. The absorbance was read at 450 nm using a micro-plate spectrophotometer.
Measurement: IC50 < 10 nM (pChEMBL 8)
Descriptors: MW 587.65, ALogP 3.78, HBA 7, HBD 4, PSA 148.31, QED 0.2
Compound (SMILES): N#Cc1cc(C(=O)Nc2ccc3[nH]nc(-c4ccncc4)c3c2)ccn1
InChIKey: SARWLKOJEYYDRF-UHFFFAOYSA-N
Target: Leucine-rich repeat serine/threonine-protein kinase 2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q5S007
Assay: LanthaScreen Kinase Activity Assay: The assay was carried out under the following protocol conditions: 1 mM compound in DMSO was serially diluted 1:3, 11 points in DMSO with a Biomek FX and 0.1 μL of the diluted compound was subsequently stamped into the assay plate (384-well format Lumitrac 200, Greiner, 781075) with an Echo Labcyte such that the final compound concentration in the assay was 10 μM to 169 μM. Subsequently, 5 μL of 2× kinase solution (2.9 nM final concentration) was added to the assay plate in assay buffer composed of 50 mM Tris pH 8.5 (Sigma, T6791), 5 mM MgCl2 (Fluka, 63020), 1 mM EGTA (Sigma, E3889), 0.01% BRIJ-35 (Sigma, P1254) and 2 mM DTT. The reaction was started by addition of 2×ATP/LRRKtide solution in assay buffer such that the final concentration was 400 nM LRRKtide and 25 μM ATP. After 60 min incubation at room temperature, the reaction was stopped by addition of 10 μL of 2× stop solution containing a final concentration of 2 nM anti-pERM antibody and 10 mM EDTA. After a 30 min incubation at RT, the TR-FRET signal was measured on a Wallac 2104 EnVision multilabel reader at an excitation wavelength of 340 nm and reading emission at 520 nm and 495 nm. The ratio of the 520 nm and 495 nm emission was used to analyze the data.
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 340.35, ALogP 3.14, HBA 5, HBD 2, PSA 107.35, QED 0.6
Compound (SMILES): Cc1ccc(S(=O)(=O)N(CC(=O)N(Cc2ccc(C3CCCCC3)cc2)c2ccc(C(=O)O)c(O)c2)Cc2cccc(C(=O)CO)c2)cc1
InChIKey: SJQOVAAGRRQFKO-UHFFFAOYSA-N
Target: Signal transducer and activator of transcription 3 (SINGLE PROTEIN), Homo sapiens
UniProt: P40763
Assay: Chromatin Immunoprecipitation (CHIP): ChIP assay was performed using EZ ChIP Kit (Millipore, Billerica, Mass., USA) as per manufacturer's instruction. Briefly, after crosslinking with formaldehyde (1%), cell were lysed using SDS Lysis Buffer containing Protease Inhibitor Cocktail and nuclear extract were obtained. Then, crosslinked DNA was sheared using sonication; sheared DNA size was checked on agarose gel and ranged, in all cases, between 300-500 bp. An amount of 1/20 of the shared chromatin was kept as an input. Sheared chromatin was incubated with rabbit polyclonal Phospho-Stat5 (Tyr694) Antibody (Cell Signaling, Danvers, Mass., USA) at dilution 1:50 or rabbit polyclonal IgG at appropriate concentration. Incubation was performed at 4° C. overnight. The antibody/chromatin complex was precipitated using Protein G Agarose beads. After elution, the crosslink of protein/DNA was reversed, and DNA was purified using Spin Columns. PCR was performed using platinum Taq (Invitrogen Canada, Burlington, ON, Canada). Two sets of primers were designed to amplify two DNA segments that contain STAT5-binding sites located 1672 bp and 428 bp upstream the start sites of C-Myc and Cyclin-D1, respectively. The STAT5-binding site in the C-MYC promoter is characterized by a 4N spacer and has the sequence (ttcccccgaa), whereas the one in the Cyclin D1 promoter is characterized by a 3N spacer and has the sequence (ttcttggaa).
Measurement: Ki = 9200 nM (pChEMBL 5.039999961853027)
Descriptors: MW 684.81, ALogP 6.05, HBA 7, HBD 3, PSA 152.52, QED 0.14
Compound (SMILES): CC(C)n1ccc2c(-c3ccnc(CN)c3F)cc(COc3ccccc3CC(=O)O)cc21
InChIKey: USVHGBLLGRFRAK-UHFFFAOYSA-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 < 1000 nM (pChEMBL 6)
Descriptors: MW 447.51, ALogP 5.09, HBA 5, HBD 2, PSA 90.37, QED 0.39
Compound (SMILES): Cc1cc(C#N)ccc1-c1cnn(-c2ccc(C(=O)NC3CN(C)C3)cn2)c1O
InChIKey: JJKADPBVPYYJOU-UHFFFAOYSA-N
Target: Egl nine homolog 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9GZT9
Assay: Inhibition Assay: The IC50 values for the PHD2 enzyme (residues 181-417) were determined by mixing increasing amounts of inhibitor with a fixed amount of enzyme (5 nM, final concentration) and Biotin labeled peptide (Biotin-Asp-Leu-Glu-Met-Leu-Ala-Pro-Tyr-Ile-Pro-Met-Asp-Asp-Asp-Phe-Gln-Leu, 1 uM final concentration) and 2-Oxyglutarate (2 uM final concentration) in 50 mM HEPES, 50 mM KCl, 0.5 mM TCEP, 2 uM FeCl2, 0.1 mg/ml BSA, at pH 7.3. The reaction was conducted by pre-incubating the enzyme in the presence of inhibitor for 60 min at room temperature. The activity of the free enzyme was measured by adding the peptide, the 2-Oxoglutarate (see above for final concentrations), and Ascorbic Acid (1 mM final concentration). The enzymatic activity was quenched after 60 min by adding an excess of a tight binding inhibitor to the assay mixture. The amount of product released was measured by using a LC/MS system (Agilent HPLC with Applied Biosystems API3000 Mass Spectrometer).
Measurement: IC50 = 15.8 nM (pChEMBL 7.800000190734863)
Descriptors: MW 388.43, ALogP 1.86, HBA 7, HBD 2, PSA 107.07, QED 0.71
Compound (SMILES): Nc1ccc(NC(=O)c2ccc3c(c2)[C@@H]2O[C@H]3c3ccc(-c4ccc(F)cc4F)cc32)cn1
InChIKey: RDJJVSRRINGZCZ-LOSJGSFVSA-N
Target: Plasma kallikrein (SINGLE PROTEIN), Homo sapiens
UniProt: P03952
Assay: In Vitro Plasma Kallikrein Inhibition Assay: The chromogenic substrate D-Pro-Phe-Arg-pNa, 2HCl (BIOPHEN CS-31(02) from Hyphen BioMed, Neuville-Sur-oise, France) was dissolved in 5 mL deionized water and stored at 4° C. Concentration was determined in the spectrophotometer at 342 nm using an extinction coefficient of 8270. All other chemicals were of analytical grade.Human plasma kallikrein was purchased from Enzyme Research Labs (South Bend, Ind., USA, batch HPKa 2830). A stock solution of 7 μM in 50% glycerol was stored at −20° C.Enzyme reactions were conducted in assay buffer comprised of 20 mM HEPES at pH 7.4, 150 mM NaCl, 0.1% PEG-8000 and 0.01% Triton X-100.Both enzyme and substrate were diluted in assay buffer.The compound solutions as well as the enzyme and the substrate solutions were transferred to 96-well plates (Clear, UV-Star, Flat-bottom, Half-Area plates; cat. No. 675801 Greiner Bio-one, purchased from VWR International, Arlington Heights, Ill., USA) using a Rainin LTS 96-channel pipettor (Rainin, Columbus, Ohio, USA). Plate measurements were conducted using a SPECTROStar Nano reader (BMG Labtech, San Francisco, Calif., USA). The SPECTROStar Nano is a spectrophotometer and absorbance was measured at 405 nm. We used discrete wavelength, precise, kinetic reads of 15 cycles with a 60 sec cycle time.Determination of IC50 valuesFor the determination of IC50 values, the assays were performed at room temperature in 96-well plates with a total assay volume of 85 μL per well. The test compound was dissolved in 100% DMSO. The compounds were serially diluted in DMSO in a 7 point dose response. For the assays, 66.5 μL of protease solution (protease in assay buffer) was added per well followed by the addition of 8.5 μL of compound in 100% DMSO. The final assay concentration of the human plasma kallikrein was 250 μM. After 30 min. incubation at room temperature on an orbital shaker, the reactions were started by the addition of 10 μL substrate solution (in assay buffer, final assay concentration was 600 uM). After the addition of the substrate solution the final DMSO concentration was 10%. The plate was placed again on the shaker for 5 sec, spun at 2000 rpm for 5 sec and read on the spectrophotometer. The effect of the compound on the enzymatic activity was obtained from the linear part of the progress curves and determined after 15 minutes.
Measurement: IC50 = 10000 nM (pChEMBL 5)
Descriptors: MW 441.44, ALogP 5.38, HBA 4, HBD 2, PSA 77.24, QED 0.45
Compound (SMILES): Cc1cc(Cn2nc(NC3CCOCC3)c3ccc(S(=O)(=O)NC4(C)CC4)cc3c2=O)on1
InChIKey: PEZSAXNBIZNNKR-UHFFFAOYSA-N
Target: Poly(ADP-ribose) glycohydrolase (SINGLE PROTEIN), Homo sapiens
UniProt: Q86W56
Assay: PARG Assay: PARG In vitro assays were conducted in a total volume of 15 ul in a standard 384 well format. 5 ul of Human Full Length PARG (Produced internally by Astra Zeneca), used at a final reaction concentration of 80 pM, was added to 5 ul of Ribosylated PARP substrate (also produced internally by Astra Zeneca) at final reaction concentration of 4.5 nM in assay buffer (50 mM Tris pH7.4, 0.1 mg/ml BSA, 3 mM EDTA, 0.4 mM EGTA, 1 mM DTT, 0.01% Tween 20, 50 mM KCl). The reaction was incubated at room temperature for 10 minutes and then 5 ul detection reagent was added. Detection Reagent consists of 42 nM MAb Anti-6HIS XL665 (CisBio: 61HISXLB) and 2.25 nM Streptavidin Europium Cryptate (CisBio: 610SAKLB), both at 3× working stock concentrations (final concentrations of 14 nM and 0.75 nM respectively), in a detection buffer of 50 mM Tris pH7.4, BSA at 0.1 mg/ml and KF at 100 mM. Following incubation at room temperature for 60 minutes in the dark, TR-FRET signal was measured at Ex 340 and Em 665 and Em 620. A ratio was calculated as Em665/EM620×104 for each well and used to calculate percent inhibition for test compounds.
Measurement: IC50 = 367 nM (pChEMBL 6.429999828338623)
Descriptors: MW 473.56, ALogP 2.16, HBA 9, HBD 2, PSA 128.35, QED 0.53
Compound (SMILES): Cc1ccc(-c2nc(N)nc3c2[nH]c(=O)n3Cc2ccccc2C(F)(F)F)o1
InChIKey: AOWRBTDAPSAERW-UHFFFAOYSA-N
Target: Adenosine receptor A2a (SINGLE PROTEIN), Homo sapiens
UniProt: P29274
Assay: Binding Affinity Assay: 1. Preparation of the reagents1) Detection buffer: 50 mM Tris-HCl pH 7.4, 10 mM MgCl2, 1 mM EDTA, 1 μg/mL Adenosine Deaminase, and stored at 4° C. for use.2) Washing solution: 50 mM Tris-HCl pH 7.4, 154 mM NaCl, and stored at 4° C. for use.3) 0.5% PEI solution: 0.5 g PEI is dissolved in 100 mL ddH2O, and stored at 4° C. for use.2. Operation procedure1) Addition of compound: 250 nL of the compound was added to the Opti-plate with Echo 550, and sealed by sealing film.2) Membrane dilution: 1 mL assay buffer was added into 20 U A2A membrane, 0.75 uCi [3H]-CGS 21680 (final 25 nM), and 50 uL of which was added into the Opti-plate.3) Incubation: the above mixture was incubated for 90 minutes at 25° C.4) Preparation of the pre-filter plate: 100 uL of 0.5% PEI solution was added into UNIFILTER-96 GF/B filter plate, and soaked for 40 minutes at 4° C.5) Filtration:a. Cell Harvester transferred 500 uL of cleaning solution/empty cleaning UNIFILTER-96 GF/B filter plate 2 times.b. The mixed system in the Opti-plate was suspended and transfer to the UNIFILTER-96 GF/B filter plate.c. 500 uL cleaning solution/empty cleaning UNIFILTER-96 GF/B filter plate 9 times.d. Incubated for 10 minutes in a 55° C. incubator.3. Readings
Measurement: IC50 = 177 nM (pChEMBL 6.75)
Descriptors: MW 389.34, ALogP 3.34, HBA 6, HBD 2, PSA 102.73, QED 0.56
Compound (SMILES): N#Cc1sccc1NC(=O)c1csc2ncc(N[C@@H]3CCCC(F)(F)[C@@H]3N)nc12.O=C(O)C(F)(F)F
InChIKey: XHUYYWIPKZJGAV-GPKQSYPGSA-N
Target: Interleukin-1 receptor-associated kinase 4 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9NWZ3
Assay: IRAK4 Kinase Assay: The kinase activity of IRAK4 is determined by its ability to catalyze the phosphorylation of a fluorescent polypeptide substrate. The extent of phosphorylation is measured using the IMAP technology (Molecular Devices) where the phosphorylated fluorescent substrate binds to the large M(III)-based nanoparticles which reduces the rotational speed of the substrate and thus increases its fluorescent polarization (FP).20 μL reaction mixture contains 10 mM TriHCl, pH 7.2, 0.5 nM GST tagged IRAK4 (SignalChem), 100 nM fluorescent peptide substrate (RP7030, Molecular Devices), 100 μM ATP, 1 mM DDT, 1 mM MgCl2, and 0.01% Tween 20. The reaction is initiated by the addition of ATP. After incubation for 30 minutes at 25° C., 60 μL of Progressive IMAP Reagent (Molecular Devices) is added to stop the reaction. Change in RP7030's FP is determined by a FP reader (Analyst HT, LJL BioSystems).
Measurement: IC50 = 3659 nM (pChEMBL 5.440000057220459)
Descriptors: MW 434.5, ALogP 3.8, HBA 8, HBD 3, PSA 116.72, QED 0.57
Compound (SMILES): COCCN1C[C@@H](NC(=O)Nc2c3c(nn2-c2ccccc2)CCCC3)[C@H](c2ccccc2)C1
InChIKey: ZSASIQWGVLBATF-UKILVPOCSA-N
Target: High affinity nerve growth factor receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P04629
Assay: TrkA Omnia Assay: Trk enzymatic selectivity was assessed using Omnia Kinase Assay reagents from Invitrogen Corp. Enzyme (TrkA from Invitrogen Corp.) and test compound (various concentrations) were incubated for 10 minutes at ambient temperature in a 384-well white polypropylene plate (Nunc catalog#267462). Omnia Tyr Peptide #4, as well as ATP, were then added to the plate. Final concentrations were as follows: 20 nM enzyme, 500 μM of ATP, 10 μM peptide substrate. The assay buffer consisted of 25 mM MOPS pH 7.5, 0.005% (v/v) Triton X-100 and 5 mM MgCl2. The production of phosphorylated peptide was monitored continuously for 70 minutes using a Molecular Devices FlexStation II384 microplate reader (excitation=360 nm; emission=485 nm). Initial rates were calculated from the progress curves. IC50 values were calculated from these rates using either a 4 or 5-parameter logistic curve fit.
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 459.59, ALogP 3.99, HBA 5, HBD 2, PSA 71.42, QED 0.56
Compound (SMILES): CCC(CC)(Cc1ccc(C(=O)Oc2ccc(C(=N)N)cc2F)s1)C(=O)N[C@@H](CCC(=O)O)C(=O)O.Cl
InChIKey: ASCMVBGGEBCPSH-NTISSMGPSA-N
Target: Enteropeptidase (SINGLE PROTEIN), Homo sapiens
UniProt: P98073
Assay: Inhibitory Activity Assay: Using a 96 well plate (#3915, Costar), a test compound (25 μL) was mixed with 20 μM fluorescence enzyme substrate (Boc-Phe-Ser-Arg-AMC, 50 μL) mixed with 200 mM Tris-HCl buffer (pH 8.0), and human trypsin (Sigma, 25 μL) was added. Using a fluorescence plate reader fmax (Molecular Devices, Inc.), the reaction rate was measured from the time-course changes at excitation wavelength 355 nm and fluorescence wavelength 460 nm. The Ki value was calculated from the concentration of the test compound, reciprocal of reaction rate, and Km value of the enzyme substrate, and by using Dixon plot.
Measurement: Ki = 0.27 nM (pChEMBL 9.569999694824219)
Descriptors: MW 521.57, ALogP 3.17, HBA 7, HBD 5, PSA 179.87, QED 0.12
Compound (SMILES): COc1cc(CCc2cnc3[nH]c(C4CCN(C)CC4)cc3n2)cc(OC)c1
InChIKey: HSCXDCSZMPPBAZ-UHFFFAOYSA-N
Target: Fibroblast growth factor receptor 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P11362
Assay: Enzymatic Assay: The inhibitor potency of compounds was measured in an enzyme assay that measures peptide phosphorylation using FRET measurements to detect product formation. Inhibitors were serially diluted in DMSO and a volume of 0.5 μL was transferred to the wells of a 384-well plate. For FGFR3, a 10 μL volume of FGFR3 enzyme (Millipore) diluted in assay buffer (50 mM HEPES, 10 mM MgCl2, 1 mM EGTA, 0.01% Tween-20, 5 mM DTT, pH 7.5) was added to the plate and pre-incubated for 5-10 minutes. Appropriate controls (enzyme blank and enzyme with no inhibitor) were included on the plate. The assay was initiated by the addition of a 10 μL solution containing biotinylated EQEDEPEGDYFEWLE peptide substrate (SEQ ID NO: 1) and ATP (final concentrations of 500 nM and 140 μM respectively) in assay buffer to the wells. The plate was incubated at 25° C. for 1 hr. The reactions were ended with the addition of 10 μL/well of quench solution (50 mM Tris, 150 mM NaCl, 0.5 mg/mL BSA, pH 7.8; added fresh 30 mM EDTA and Perkin Elmer Lance Reagents for HTRF at 3.75 nM Eu-antibody PY20 and 180 nM APC-Streptavidin). The plate was allowed to equilibrate for 1 hr before scanning the wells on a PheraStar plate reader.FGFR1 and FGFR2 were measured under equivalent conditions with the following changes in enzyme and ATP concentrations: FGFR1, 0.02 nM and 210 μM, respectively and FGFR2, 0.01 nM and 100 μM, respectively.
Measurement: IC50 = 300 nM (pChEMBL 6.519999980926514)
Descriptors: MW 380.49, ALogP 3.57, HBA 5, HBD 1, PSA 63.27, QED 0.71
Compound (SMILES): COc1cc2ncnc(Sc3cccc(NC(=O)Nc4cc(C(C)(C)C)on4)c3)c2cc1OCCN1CCC(CO)CC1
InChIKey: TXSBXVVJFGIDET-UHFFFAOYSA-N
Target: Serine/threonine-protein kinase B-raf (SINGLE PROTEIN), Homo sapiens
UniProt: P15056
Assay: Competition Binding Assay: Competition binding assays used herein were developed, validated and performed as described in Fabian et al., Nature Biotechnology 2005, 23, 329-336. Kinases were produced as fusions to T7 phage (See, Fabian et al. or WO04/015142) or alternatively, the kinases were expressed in HEK-293 cells and subsequently tagged with DNA for PCR detection (See, WO08/005310). For the binding assays, streptavidin-coated magnetic beads were treated with biotinylated affinity ligands for 30 min at room temperature to generate affinity resins. The liganded beads were blocked with excess biotin and washed with blocking buffer (SeaBlock (Pierce), 1% BSA, 0.05% Tween 20, 1 mM DTT) to remove unbound ligand and to reduce non-specific binding. Binding reactions were assembled by combining kinase, liganded affinity beads, and test compounds in 1× binding buffer (20% SeaBlock, 0.17×PBS, 0.05% Tween 20, 6 mM DTT). Test compounds were prepared as 100× stocks in DMSO and rapidly diluted into the aqueous environment. DMSO was added to control assays lacking a test compound. Primary screen interactions were performed in polypropylene 384-well plates in a final volume of 34 μL, while Kd determinations were performed in polystyrene 96-well plates in a final volume of 135 μL. The assay plates were incubated at room temperature with shaking for 1 hour, long enough for binding reactions to reach equilibrium, and the affinity beads were washed extensively with wash buffer (1×PBS, 0.05% Tween 20) to remove unbound protein. The beads were then resuspended in elution buffer (1×PBS, 0.05% Tween 20, 2 μM non-biotinylated affinity ligand) and incubated at room temperature with shaking for 30 min. The kinase concentration in the eluates was measured by quantitative PCR. Each kinase was tested individually against each compound. KdS were determined using eleven serial threefold dilutions.
Measurement: Kd < 250 nM (pChEMBL 6.6020002365112305)
Descriptors: MW 606.75, ALogP 5.8, HBA 10, HBD 3, PSA 134.87, QED 0.19
Compound (SMILES): C[C@@H](COCCC(=O)N1CCN(c2ncc(C(F)(F)F)s2)CC1)Oc1cn[nH]c(=O)c1Br
InChIKey: FYFDKXGNXCRLGL-NSHDSACASA-N
Target: Protein mono-ADP-ribosyltransferase TIPARP (SINGLE PROTEIN), Homo sapiens
UniProt: Q7Z3E1
Assay: Enzymatic Assay for Inhibition of PARP7: Displacement of Probe A, a biotinylated probe binding to the TIPARP active site, was measured using a time-resolved fluorescence energy transfer (TR-FRET) assay. 20 nL of a dose response curve of each test compound was spotted in black 384-well polystyrene proxiplates (Perkin Elmer) using a Mosquito (TTP Labtech). Reactions were performed in a 8 μL volume by adding 6 μL of TIPARP and Probe A in assay buffer (20 mM HEPES pH=8, 100 mM NaCl, 0.1% bovine serum albumin, 2 mM DTT and 0.002% Tween20), incubating with test compound at 25° C. for 30 min, then adding 2 μL of ULight-anti 6×His and LANCE Eu-W1024 labeled streptavidin (Perkin Elmer). The final concentrations of TIPARP and Probe A were 6 nM and 2 nM, respectively. The final concentration of ULight-anti 6xHis and LANCE Eu-W1024 labeled streptavidin were 4 nM and 0.25 nM, respectively. Reactions were incubated at 25° C. for an additional 30 min, then read on an Envision platereader equipped with a LANCE/DELFIA top mirror (Perkin Elmer) using excitation of 320 nm and emission of 615 nm and 665 nM with a 90 μs delay. The ratio of the 665/615 nm emission were calculated for each well to determine the amount of complex of TIPARP and Probe A in each well. Control wells containing a negative control of 0.25% DMSO vehicle or a positive control of 100 μM Example 190 were used to calculate the % inhibition as described below:% ⁢ ⁢ inhibition = 100 × TRF cmpd - TRF min TRF max - TRF minwhere TRFcmpd is the TR-FRET ratio from the compound treated well, TRFmin is the TR-FRET ratio from the Example 190-treated positive control well and TRFmax is the TR-FRET ratio from the DMSO-treated negative control well.The % inhibition values were plotted as a function of compound concentration and the following 4-parameter fit was applied to derive the IC50 the values:Y = Bottom + ( Top - Bottom ) ( 1 + ( X IC 50 ) Hill ⁢ ⁢ Coefficientwhere top and bottom are normally allowed to float, but may be fixed at 100 or 0 respectively in a 3-parameter fit. The Hill Coefficient is normally allowed to float but may also be fixed at 1 in a 3-parameter fit. Y is the % inhibition and x is the compound concentration.
Measurement: IC50 = 550 nM (pChEMBL 6.260000228881836)
Descriptors: MW 540.36, ALogP 2.53, HBA 8, HBD 1, PSA 100.65, QED 0.51