lovingscience/chembl-v37-raw-activities huggingface dataset:
Compound (SMILES): COc1cc2c(Nc3ccc(Br)c(Cl)c3F)ncnc2cc1OC[C@H]1CN2CCOCC2CO1
InChIKey: CMTRUERFSJKMJQ-ARLHGKGLSA-N
Target: Receptor tyrosine-protein kinase erbB-2 (SINGLE PROTEIN), Homo sapiens
UniProt: P04626
Assay: AlphaScreen Assay: In AlphaScreen, when the donor and acceptor beads are close in proximity, a series of photochemical events will give rise to a fluorescent signal upon light activation. Here we use biotinylated poly-(Glu, Tyr) 4:1 as the kinase substrate, streptavidin-coated donor beads and anti-phosphortyrosine antibody PY100-coated acceptor beads. Upon phosphorylation, the peptide substrate can bind to both donor and acceptor beads, thus gives rise to fluorescence. Compounds, ATP, biotinylated poly-(Glu, Tyr) and kinases are mixed in a volume of 20 μL for 1 hr at ambient temperature using Greiner 384-well white clear bottom medium binding plates. Then 10 uL solution containing 15-30 mg/mL AlphaScreen beads, 75 mM Hepes, pH 7.4, 300 mM NaCl, 120 mM EDTA, 0.3% BSA and 0.03% Tween-20 is added to each well. After 2-16 hr incubation of the beads, plates are read in a Perkin Elmer AlphaQuest reader (available from PerkinElmer Life and Analytical Sciences, Inc., of Boston Mass.).
Measurement: IC50 = 525 nM (pChEMBL 6.28000020980835)
Descriptors: MW 553.82, ALogP 4.42, HBA 8, HBD 1, PSA 77.97, QED 0.45
Compound (SMILES): Cc1cnc(CNC2CCC(c3ccc4[nH]c(-c5cn6ncnc6c(C)c5C)c(C(C)C)c4n3)CC2)cn1
InChIKey: PGPBYQIYURVNGO-UHFFFAOYSA-N
Target: Toll-like receptor 8 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9NR97
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 = 4.4 nM (pChEMBL 8.359999656677246)
Descriptors: MW 508.67, ALogP 5.93, HBA 7, HBD 2, PSA 96.68, QED 0.3
Compound (SMILES): Cn1cc(-c2cn3nccc3c(-c3cnn(C4CCCC4O)c3)n2)cn1
InChIKey: VHBISBSNGUVGTR-UHFFFAOYSA-N
Target: Tyrosine-protein kinase JAK1 (SINGLE PROTEIN), Homo sapiens
UniProt: P23458
Assay: JAK1 Inhibition Assay: Compounds of Formula I were screened for their ability to inhibit JAK1 using the general enzyme inhibition assay method, in which the assay mixture contained 1 mM ATP, 8 μM Omnia Y12 peptide (Catalog # IVGN KPZ3121C; Invitrogen Corporation, Carlsbad, Calif.) and 12.5 nM JAK1 in a total volume of 25 μL. JAK1 was purchased from Invitrogen Corporation, Carlsbad, Calif. (catalog # IVGN PV4775).
Measurement: IC50 = 5500 nM (pChEMBL 5.260000228881836)
Descriptors: MW 349.4, ALogP 2.08, HBA 8, HBD 1, PSA 86.06, QED 0.61
Compound (SMILES): CN(c1ncnc2[nH]ccc12)[C@H]1C[C@@H](CS(=O)(=O)c2ccc(C(F)(F)F)nc2)C1
InChIKey: PYTYUNMFVXMLHB-TXEJJXNPSA-N
Target: Tyrosine-protein kinase JAK2 (SINGLE PROTEIN), Homo sapiens
UniProt: O60674
Assay: Caliper Enzyme Assay: Test article and assay controls were added to a 384-well plate. Reaction mixtures contained 20 mM HEPES, pH 7.4, 10 mM magnesium chloride, 0.01% bovine serum albumin (BSA), 0.0005% Tween 20, 1 mM ATP and 1 μM peptide substrate. The JAK1 and TYK2 assays contained 1 μM of the IRStide peptide (5FAM-KKSRGDYMTMQID) and the JAK2 and JAK3 assays contained 1 μM of the JAKtide peptide (FITC-KGGEEEEYFELVKK). The assays were initiated by the addition of 20 nM JAK1, 1 nM JAK2, 1 nM JAK3 or 1 nM TYK2 enzyme and were incubated at room temperature for three hours for JAK1, 60 minutes for JAK2, 75 minutes for JAK3 or 135 minutes for TYK2. Enzyme concentrations and incubation times were optimized for each new enzyme preps and were modified slightly over time to ensure 20%-30% phosphorylation. The assays were stopped with a final concentration of 10 mM EDTA, 0.1% Coating Reagent and 100 mM HEPES, pH=7.4. The assay plates were placed on a Caliper Life Science Lab Chip 3000 (LC3000) instrument, and each well was sampled using appropriate separation conditions to measure the unphosphorylated and phosphorylated peptide.
Measurement: IC50 = 0.561 nM (pChEMBL 9.25)
Descriptors: MW 425.44, ALogP 3.06, HBA 6, HBD 1, PSA 91.84, QED 0.68
Compound (SMILES): COc1cc(C(F)(F)F)c(OC)cc1C1CCCNC1
InChIKey: NZKYTYHIERLZBG-UHFFFAOYSA-N
Target: 5-hydroxytryptamine receptor 2C (SINGLE PROTEIN), Homo sapiens
UniProt: P28335
Assay: Radioligand Binding Assay: The binding affinities shown in table 5 of psilocin and compound 8 at various monoaminergic receptors were determined in radioligand competition binding assays by Psychiatric Drug Screening Program according to the experimental described in Assay Protocol Book, Version III 11, 16. As shown compound 8 shows less affinity for various monoaminergic receptors compared to Psilocin.
Measurement: Ki = 42 nM (pChEMBL 7.380000114440918)
Descriptors: MW 289.3, ALogP 3.19, HBA 3, HBD 1, PSA 30.49, QED 0.93
Compound (SMILES): Cc1cc(-c2c(-c3ccccc3F)nc(N)n3cnnc23)cc(C)n1
InChIKey: WBEQFIXFBVCQJG-UHFFFAOYSA-N
Target: Adenosine receptor A2b (SINGLE PROTEIN), Homo sapiens
UniProt: P29275
Assay: Adenosine A2b receptor (A2bR): CHO-K1/A2bR cells were cultured in DMEM/F12 medium containing 10% fetal bovine serum and 1 mg/ml G418. The cells were digested with the cell separation buffer during the experiment. The cells were resuspended in the balanced salt buffer containing 20 mM HEPES and 0.1% bovine serum albumin and counted, and the cell density was adjusted to 106 cells/ml. In the 384-well plate, each well was added with 5 μl of cell suspension, and 2.5 μl of test compound (4× concentration) formulated with the balanced salt buffer containing 20 mM HEPES, 0.1% bovine serum albumin, 54 μM rolipram and 2.7 U/ml adenosine deaminase, and the plate was incubated at room temperature for 30 minutes. Each well was then added with 2.5 μl of ethyl carbazole (4× concentration) (Torcis, 1691/10) formulated with the balanced salt buffer containing 20 mM HEPES, 0.1% bovine serum albumin, 54 μM rolipram and 2.7 U/ml adenosine deaminase, and the plate was incubated at room temperature for 30 minutes. The final concentrations of the compounds were: 100000, 10000, 1000, 100, 10, 1, 0.1 and 0 nM. The final concentration of ethyl carbazole was 1 μM. Intracellular cAMP concentration was detected with the cAMP dynamic 2 kit. cAMP-d2 and Anti-cAMP-Eu-Cryptate were diluted respectively with the cAMP lysis buffer at a ratio of 1:4. Each well was added with 5 μl of diluted cAMP-d2, followed by addition of 5 μl of diluted Anti-cAMP-Eu-Cryptate, and the plate was incubated at room temperature in the dark for 1 hour. The HTRF signal values were read by the PHERAstar multi-function microplate reader. IC50 values of inhibition activity of the compounds were calculated by Graphpad Prism software.
Measurement: IC50 = 85.4 nM (pChEMBL 7.070000171661377)
Descriptors: MW 334.36, ALogP 3.19, HBA 6, HBD 1, PSA 81.99, QED 0.61
Compound (SMILES): CNCc1nc2cc(C(F)(F)F)cc(C(F)(F)F)c2[nH]1
InChIKey: PEJUVFLXTQGLKO-UHFFFAOYSA-N
Target: Voltage-dependent L-type calcium channel subunit alpha-1C (SINGLE PROTEIN), Homo sapiens
UniProt: Q13936
Assay: Nav1.5 Assay: To assess the potential cardiac liability of compounds at an early stage in the drug discovery process, a Nav1.5 sodium channel screening assay was be performed on Molecular Device's PatchXpress™ automated electrophysiology platform. Under voltage-clamp conditions, Nav1.5 currents were recorded from HEK cells expressing the human Nav1.5 channel in the absence and presence of increasing concentrations of the test compound to obtain an IC50 value. The external recording solution contained (in mM): 90 TEACl, 50 NaCl, 1.8 CaCl, 1 MgCl2, 10 HEPES, 10 glucose, adjusted to pH 7.4 with TEA-OH and to 300 mOsm with sucrose (if necessary), while the internal patch pipette solution contained (in mM): 129 CsF, 2 MgCl2, 11 EGTA, 10 HEPES, 3 Na2ATP adjusted to pH 7.2 with CsOH and to 290 mOsm with sucrose (if necessary). Nav1.5 channel currents were evoked using a cardiac action potential waveform at 1 Hz, digitized at 31.25 kHz and low-pass filtered at 12 kHz.
Measurement: IC50 > 10000 nM (pChEMBL 5)
Descriptors: MW 297.2, ALogP 3.32, HBA 2, HBD 2, PSA 40.71, QED 0.83
Compound (SMILES): Cc1cccn2c(C(C)(C)NC(=O)[C@H]3[C@@H]4CNC[C@@H]43)ncc12
InChIKey: LWBGHNNRSMCRTE-IMRBUKKESA-N
Target: Somatostatin receptor type 5 (SINGLE PROTEIN), Homo sapiens
UniProt: P35346
Assay: Radioligand Binding Assay: Receptor binding assays refer to a technique in which labeled receptor ligands are used to detect binding to a receptor. In competition experiments test compounds, which are not labeled, compete with the binding side of a labeled ligand. The displacement of the labeled ligand by the test compound leads to a decreased signal.Procedure:For the binding experiments 200 μL of membrane homogenate from one of the following protein amounts is used: hSSTR1 (40 μg/well); hSSTR2 (25 μg/well); hSSTR3 (1.5 μg/well); hSSTR4 (0.5 μg/well); hSSTR5 (25 μg/well). The homogenate is incubated with 0.05 nM of radioligand ([3-125I-Tyr]-Somatostatin-(1-14)) in addition to increasing concentrations of a test compound or vehicle (100% binding) in a total volume of 250 μL using a Hepes buffer (10 mM, EDTA 1 mM, MgCl2 5 mM, pH7.6, BSA 0.5%, Bacitracin 0.003%, DMSO 1%) for 180 min at room temperature. The incubation is terminated by filtration with ice cold NaCl 0.9% through polyethyleneimine treated (0.3%) GF/B glass fiber filters using a cell harvester. The protein-bound radioactivity is measured in a suitable reader. The non-specific binding is defined as radioactivity bound in the presence of 1 μM Somatostatin-14 during the incubation period.
Measurement: Ki > 9860 nM (pChEMBL 5.00600004196167)
Descriptors: MW 298.39, ALogP 1.46, HBA 4, HBD 2, PSA 58.43, QED 0.9
Compound (SMILES): Nc1ccc(S(=O)(=O)Nc2ncc3cc4n(c3n2)C2(CCCCC2)C(=O)NN4)cc1
InChIKey: QGIFQLWCNWTFNA-UHFFFAOYSA-N
Target: Cyclin-dependent kinase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P06493
Assay: CDK Inhibition In Vitro Assay: Selected compounds disclosed herein were tested in kinase assays by Nanosyn (Santa Clara, Calif.) to determine their inhibitory effect on these CDKs. The assays were performed using microfluidic kinase detection technology (Caliper Assay Platform). The compounds were tested in 12-point dose-response format in singlicate at Km for ATP. Specifics of each assay are as described below:CDK1/Cyclin B1: Enzyme concentration: 0.08 nM; ATP concentration: 40 μM; Incubation time: 3 hr. CDK2/Cyclin A: Enzyme concentration: 0.1 nM; ATP concentration: 50 μM; Incubation time: 3 hr. CDK2/Cyclin E: Enzyme concentration: 0.15 nM; ATP concentration: 100 μM; Incubation time: 3 hr. CDK4/Cyclin D1: Enzyme concentration: 1 nM; ATP concentration: 200 μM; Incubation time: 3 hr. CDK6/Cyclin D3: Enzyme concentration: 2 nM; ATP concentration: 300 μM; Incubation time: 3 hr. CDK9/Cyclin T1: Enzyme concentration: 5 nM; ATP concentration: 10 μM; Incubation time: 17 hr.
Measurement: IC50 > 1000 nM (pChEMBL 6)
Descriptors: MW 427.49, ALogP 1.93, HBA 8, HBD 4, PSA 144.03, QED 0.47
Compound (SMILES): CCc1nc2ccc(CN3CCN(c4ccc(C(=O)NC)nc4F)CC3)cc2[nH]c1=O
InChIKey: IEYVHGZKGZORPQ-UHFFFAOYSA-N
Target: Voltage-gated inwardly rectifying potassium channel KCNH2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q12809
Assay: hERG Electrophysiological Assay: Electrophysiological recordings (all performed at RT) from stably transfected CHO hKv11.1 cells were obtained using the Nanion Syncropatch 768PE. Test compounds, vehicle or positive controls were added with 6 compound plates each at a different concentration to allow cumulative dosing onto cells (10 mM, 3.167 mM, 1 mM, 0.3167 mM, 0.1 mM, 0.03167 mM). 600 Figure US11325906-20220510-P00001of compound is resuspended into 90 μl of reference buffer (in mM, NaCl 80, KCL 4, CaCl 5, MgCl 1, NMDG Cl 60, D-Glucose monohydrate 5, HEPES 10 (pH7.4 HCL, 298 mOsm) for a final compound concentration of 39.6 μM, 13.2 μM, 4.4 μM, 1.46 μM, 0.48 μM, 0.16 μM. For each Nanion Syncropatch 768PE run, the current amplitude in each cell in the presence of extracellular solution (in mM, NaCl 80, KCL 4, CaCl 5, MgCl 1, NMDG Cl 60, D-Glucose monohydrate 5, HEPES 10 (pH7.4 HCL, 298 mOsm) is measured with all liquid additions performed using the Syncropatch liquid handling system. Add 40 μL external solution (in mM, HBPS, CaCl2 2, MgCl2 1 (pH7.4, NaOH) to 384 well multihole medium resistance recording chip and perfuse internal buffer (in mM, KF 130, KCl 20, MgCl2 1, EGTA 10, HEPES 10, Escin 25 (all Sigma-Aldrich; pH 7.2-7.30 using 10 M KOH, 320 mOsm) to the underside of plate. Dispense 20 μL of cells at a density of 1e6 cells/ml maintained at 9° C. into each well of the chip followed by 20 μL of seal enhancer (in mM, NaCl 80, KCl 3, CaCl 10, HEPES 10, MgCl 1 (pH7.4 NaOH). Perform wash step leaving a residual volume of 40 μL. Dispense 40 μL of reference buffer to establish a stable baseline prior to the addition of test compounds, with a removal step of 40 μL after 3 min, repeat this step. Dispense 40 μL of compound concentration 1 (0.16 μM), real time recordings for 3 min exposure prior to removal of 40 μL. This step is repeated for 5 further subsequent compound plates to generate cumulative curve analysis. All data is leak subtracted, 2 pulses to −80 mV 100 ms with 100 ms delay. Outward K+ currents are then evoked by voltage step to +60 mV from a holding potential of −90 mV, Each pulse is delivered at a frequency of 2 Hz with a 15 s pulse interval.
Measurement: IC50 > 40000 nM (pChEMBL 4.3979997634887695)
Descriptors: MW 424.48, ALogP 1.7, HBA 6, HBD 2, PSA 94.22, QED 0.61
Compound (SMILES): CN(c1ncc(C(=O)NO)cn1)C12CC3CC(CC(C3)C1)C2
InChIKey: IXDSXOZCSDIMHI-UHFFFAOYSA-N
Target: Histone deacetylase 5 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9UQL6
Assay: IC50 Assay: IC50 measurements were conducted by BPS Biosciences (Table 1) or by Nanosyn (Table 1A) with an established fluorescence assay.
Measurement: IC50 = 121 nM (pChEMBL 6.920000076293945)
Descriptors: MW 302.38, ALogP 2, HBA 5, HBD 2, PSA 78.35, QED 0.66
Compound (SMILES): O=C(NO)c1ccc2oc(-c3ccccc3Cl)nc2c1
InChIKey: SOQHGPLOGBSSPZ-UHFFFAOYSA-N
Target: Histone deacetylase 8 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9BY41
Assay: In Vitro Enzymatic Assay: The assay principle is well known and all necessary reagents like enzymes, substrates, developer and reference compounds are commercially available. Stock solutions (10 mM in DMSO) of compounds were serially diluted 1:3 in 11 concentrations with a top concentration of 200 μM for HDAC1,2,3 and 2 μM for HDAC6 and HDAC8. The enzymatic reactions were conducted in a mixture containing assay buffer, bovine serum albumin, HDAC substrate, and a test compound. After enzymatic reaction, developer was added and after an additional incubation time, fluorescence intensity was measured at an excitation wavelength of 360 nm and an emission wavelength of 460 nm. All experiments were performed in duplicate.
Measurement: IC50 = 880 nM (pChEMBL 6.059999942779541)
Descriptors: MW 288.69, ALogP 3.27, HBA 4, HBD 2, PSA 75.36, QED 0.56
Compound (SMILES): Cc1cccc(CNCC[C@@]2(c3ccc(F)cn3)CCO[C@]3(CCOC3)C2)c1
InChIKey: KDBYCIGWZXGKPW-DHIUTWEWSA-N
Target: Cytochrome P450 3A4 (SINGLE PROTEIN), Homo sapiens
UniProt: P08684
Assay: Inhibition for Cytochrome P450 Isoenzymes: To test the inhibitory effects of the compounds of the present disclosure on different isoforms of human cytochrome P450 isoenzymes
Measurement: IC50 > 50000 nM (pChEMBL 4.301000118255615)
Descriptors: MW 384.5, ALogP 3.92, HBA 4, HBD 1, PSA 43.38, QED 0.77
Compound (SMILES): Cc1c(-c2[nH]c3ccc(C4CCC(N(C)CC(N)=O)CC4)nc3c2C(C)C)cn2ncnc2c1C
InChIKey: GUEANUQRFJJSOW-UHFFFAOYSA-N
Target: Toll-like receptor 8 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9NR97
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 = 3 nM (pChEMBL 8.520000457763672)
Descriptors: MW 473.63, ALogP 4.46, HBA 6, HBD 2, PSA 105.2, QED 0.43
Compound (SMILES): CC(C)(C)CSC[C@@H]1CCc2cc([C@H]3CC[C@](N)(COP(=O)(O)O)C3)ccc2C1
InChIKey: TZEMKQKSWRONQO-QGCDCVKKSA-N
Target: Sphingosine-1-phosphate lyase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: O95470
Assay: hS1P1 ERK Phosphorylation (S1P1 pERK): hS1P1/CHO cells were plated into BD Amine 384-well plates the day before the assay. On the day of the assay, growth medium was removed and replaced with serum-free medium (Ham's F-12 Invitrogen) and incubated for 2 hours. Test compounds pre-diluted in HBSS (Gibco)/20 mM HEPES (Gibco) were transferred to the cell plates and incubated for 7 minutes at 37° C. Cells were lysed in lysis buffer (Perkin Elmer) and phospho-ERK was measured using the SureFire pERK kit (Perkin Elmer) as described by the manufacturer. Data was plotted as percentage activation of the test compound relative to the efficacy of 10 μM S1P. The EC50 is defined as the concentration of test compound which produces 50% of the maximal response and was quantified using the 4 parameter logistic equation to fit the data.
Measurement: EC50 < 10 nM (pChEMBL 8)
Descriptors: MW 441.57, ALogP 4.65, HBA 4, HBD 3, PSA 92.78, QED 0.53
Compound (SMILES): Cc1nc2c(F)cc(-c3nc(Nc4ccc5c(n4)CCN(C(=O)CN(C)C)C5)ncc3F)cc2n1C1CC1
InChIKey: OIQZLVBXHSEFQP-UHFFFAOYSA-N
Target: Cyclin-dependent kinase 4 (SINGLE PROTEIN), Homo sapiens
UniProt: P11802
Assay: LANCE Ultra ULight Assay: 1. Take 10 mM stock solution of the test compound, in 96-well compound plate, DMSO was used to dilute the compound to an initial concentration of 100x, then this concentration was used as the first concentration, 3-fold diluted to make 10 serial concentrations; 1 μL each serial dilution was then added to 19 μL 1x reaction buffer to prepare 5x compound for use; 2 μL 5x compound was transferred from 96-well plate to 384-well plate; compound-free control well was added with 2 μL the following liquid: 1x reaction buffer with the addition of 1 μL DMSO; 2 μL 250 mM EDTA was added to the Min control well.2.1x reaction buffer was used to formulate the kinase, substrate and ATP into a 2.5xenzyme/substrate mixture and 2.5xATP solution respectively. In the experiment, the final concentration of CDK4/CycD3 kinase is 0.76 ng/μL, the final concentration of ATP is 8 μM; the final concentration of CDK6/CycD3 kinase is 0.5 ng/μL, the final concentration of ATP is 50M; the final concentration of CDK2/CycA2 kinase is 0.86 ng/μL, the final concentration of ATP is 1 μM; the final concentration of CDK2/CycE1 kinase is 1.016 ng/μL, the final concentration of ATP is 20M; 2.5xenzyme/substrate mixture was added to a 384-well plate, incubated at room temperature for 5 minutes; then added with 2.5xATP solution, reacted at room temperature for 30 minutes.3. LANCE Detection Buffer was used, 1xto prepare 2xLANCE Ultra Europium-anti-phospho-eIF4E-binding protein 1 (Thr37/46) for use. After the enzymatic reaction was continued for 30 minutes, 10 mM EDTA was added to 384-well plate and the mixture was reacted at room temperature for 5 minutes. Then LANCE Ultra Europium-anti-phospho-eIF4E-binding protein 1 (Thr37/46) was added, reacted at room temperature for 1 hour.4. The 384-well plate was placed in HERAEUS Multifuge X1R centrifuge, centrifuged at 2000 rpm for 2 minutes; data were measured on EnVision, 337 nM wavelength laser was used as the excitation light, test at RFU665 nM and RFU615 nM, and RFU665 nM/RFU615 nM x10000 was used as the final data for analysis.
Measurement: IC50 = 3.26 nM (pChEMBL 8.489999771118164)
Descriptors: MW 518.57, ALogP 4, HBA 8, HBD 1, PSA 92.07, QED 0.41
Compound (SMILES): CCCCCOC[C@@H]1COc2cc([C@H]3CC[C@](N)(COP(=O)(O)O)C3)ccc2O1
InChIKey: XFNWLBLKUWRFPE-SQGPQFPESA-N
Target: Sphingosine-1-phosphate lyase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: O95470
Assay: hS1P1 ERK Phosphorylation (S1P1 pERK): hS1P1/CHO cells were plated into BD Amine 384-well plates the day before the assay. On the day of the assay, growth medium was removed and replaced with serum-free medium (Ham's F-12 Invitrogen) and incubated for 2 hours. Test compounds pre-diluted in HBSS (Gibco)/20 mM HEPES (Gibco) were transferred to the cell plates and incubated for 7 minutes at 37° C. Cells were lysed in lysis buffer (Perkin Elmer) and phospho-ERK was measured using the SureFire pERK kit (Perkin Elmer) as described by the manufacturer. Data was plotted as percentage activation of the test compound relative to the efficacy of 10 μM S1P. The EC50 is defined as the concentration of test compound which produces 50% of the maximal response and was quantified using the 4 parameter logistic equation to fit the data.
Measurement: EC50 < 10 nM (pChEMBL 8)
Descriptors: MW 429.45, ALogP 3.11, HBA 6, HBD 3, PSA 120.47, QED 0.38
Compound (SMILES): CC1C(=O)N(C)c2cc3cnc(Nc4ccc(S(N)(=O)=O)cc4)nc3n2C12CCCCC2
InChIKey: FLIVKVQMXOAQAK-UHFFFAOYSA-N
Target: Cyclin-dependent kinase 5 activator 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q15078
Assay: CDK Inhibition In Vitro Assay: Selected compounds disclosed herein were tested in kinase assays by Nanosyn (Santa Clara, Calif.) to determine their inhibitory effect on these CDKs. The assays were performed using microfluidic kinase detection technology (Caliper Assay Platform). The compounds were tested in 12-point dose-response format in singlicate at Km for ATP. Specifics of each assay are as described below:CDK1/Cyclin B1: Enzyme concentration: 0.08 nM; ATP concentration: 40 μM; Incubation time: 3 hr. CDK2/Cyclin A: Enzyme concentration: 0.1 nM; ATP concentration: 50 μM; Incubation time: 3 hr. CDK2/Cyclin E: Enzyme concentration: 0.15 nM; ATP concentration: 100 μM; Incubation time: 3 hr. CDK4/Cyclin D1: Enzyme concentration: 1 nM; ATP concentration: 200 μM; Incubation time: 3 hr. CDK6/Cyclin D3: Enzyme concentration: 2 nM; ATP concentration: 300 μM; Incubation time: 3 hr. CDK9/Cyclin T1: Enzyme concentration: 5 nM; ATP concentration: 10 μM; Incubation time: 17 hr.
Measurement: IC50 = 15 nM (pChEMBL 7.820000171661377)
Descriptors: MW 454.56, ALogP 3.09, HBA 7, HBD 2, PSA 123.21, QED 0.63
Compound (SMILES): Cc1cnc(C(=O)N[C@@H](C)c2cc3cccc(C#Cc4cnn(C)c4)c3c(=O)n2-c2ccccc2)c(N)n1
InChIKey: KRQKZGMXITZFSA-IBGZPJMESA-N
Target: Phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit delta isoform (SINGLE PROTEIN), Homo sapiens
UniProt: O00329
Assay: PI3K-delta Selective Assay: A compound's ability in selectively inhibiting PI3K-δ can be assessed using RAJI cells, i.e., B lymphocyte cells derived from lymphoma patients. Briefly, serum-starved RAJI cells are stimulated with anti-human IgM, thereby causing signaling through the B-cell receptors, as described in, for example, He et al., Leukemia Research (2009) 33: 798-802. B-cell receptor signaling is important for the activation, differentiation, and survival of B cells and certain B-cell derived cancers. Reduction of phospho-AKT is indicative of compounds that may inhibit B-cell proliferation and function in certain diseases. By monitoring the reduction of phospho-AKT in stimulated RAJI cells (using for example, phospho-AKT antibodies), a compound's potential efficacy in selectively inhibiting PI3Kδ can be assessed.
Measurement: IC50 = 4000 nM (pChEMBL 5.400000095367432)
Descriptors: MW 503.57, ALogP 3.3, HBA 8, HBD 2, PSA 120.72, QED 0.36
Compound (SMILES): N#CCC1(n2cc(-c3cc(-c4cc[nH]n4)cc4nccn34)cn2)CC(C#N)C1
InChIKey: UXYXSKPNTUCZFD-UHFFFAOYSA-N
Target: Tyrosine-protein kinase JAK2 (SINGLE PROTEIN), Homo sapiens
UniProt: O60674
Assay: JAK Caliper Enzyme Assay at 1mM ATP: Compounds were added to a 384-well plate. Reaction mixtures contained 10 mM HEPES, pH 7.4, 10 mM MgCl2, 0.01% BSA, 0.0005% Tween 20, 1 mM ATP and 1 μM peptide substrate. The JAK1 and TYK2 assays contained 1 μM of the IRStide peptide (5FAM-KKSRGDYMTMQID) and the JAK2 and JAK3 assays contained 1 μM of the JAKtide peptide (FITC-KGGEEEEYFELVKK). The assays were initiated by the addition of 20 nM JAK1, 1 nM JAK2, 1 nM JAK or 1 nM TYK2 enzyme and were incubated at room temperature for three hours for JAK1, 60 minutes for JAK2, 75 minutes for JAK3 or 135 minutes for TYK2. Enzyme concentrations and incubation times were optimized for each new enzyme preps and were modified slightly over time to ensure 20% to 30% phosphorylation. The assays were stopped with 15 μL of 180 mM HEPES, pH 7.4, 20 mM EDTA, and 0.2% Coating Reagent 3. The assay plates were placed on a Caliper Life Science LC3000 instrument, and each well was sampled using appropriate separation conditions to measure the unphosphorylated and phosphorylated peptide.
Measurement: IC50 = 5676 nM (pChEMBL 5.25)
Descriptors: MW 368.4, ALogP 3.13, HBA 7, HBD 1, PSA 111.38, QED 0.59
Compound (SMILES): COCCCN1C(=O)Cc2cc(-c3cncc(-c4ccc(=O)n(C(C)C)c4)c3)ccc21
InChIKey: ICKRRSVRNGVVPJ-UHFFFAOYSA-N
Target: Bone morphogenetic protein receptor type-1B (SINGLE PROTEIN), Homo sapiens
UniProt: O00238
Assay: In Vitro Enzyme Inhibition Using a Biochemical Peptide Phosphorylation Assay- Caliper Assay: Example 54:The assays were performed in 384-well, low volume microtiter assay plates in a final reaction volume of 9 ul. Dose-response curves were generated by incubating 10 nM of each kinase together with 2 uM of the fluorescently labeled substrate peptide 5-Fluo-Ahx-KKYQAEEN-T-YDEYENKK-amid (10 mM stock solution in DMSO) in 50 mM Hepes pH 7.5, 0.02% Tween 20, 0.02% BSA, 1 mM DTT, 10 uM Na3VO4, 10 mM ß-Glycerolphosphate, 1 mM MgCl2, 12 mM MnCl2 (ALK1 and ALK6 7 mM) and 15 uM ATP for 60 min at 30° C. in the presence or absence of compound diluted in DMSO.Kinase reactions were terminated by adding 15 ul STOP buffer (100 mM HEPES pH 7.5, 5% DMSO, 0.1% Caliper coating reagent, 10 mM EDTA, and 0.015% Brij35.
Measurement: IC50 = 2600 nM (pChEMBL 5.579999923706055)
Descriptors: MW 417.51, ALogP 4.08, HBA 5, HBD 0, PSA 64.43, QED 0.54
Compound (SMILES): NS(=O)(=O)c1ccc(C(=O)N2C[C@H]3CN(C(=O)c4cnc(OC5CCOCC5)c(C5CC5)c4)C[C@@H]3C2)cn1
InChIKey: YLIKCNACGAJJCL-PMACEKPBSA-N
Target: Ataxin-2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q99700
Assay: Enzyme Inhibition Assay : ATX inhibition was measured by a fluorescence quenching assay using a specifically labeled substrate analogue (MR121 substrate). To obtain this MR121 substrate, BOC and TBS protected 6-amino-hexanoic acid (R)-3-({2-[3-(2-{2-[2-(2-amino-ethoxy)-ethoxy]-ethoxy}-ethoxy)-propionylamino]-ethoxy}-hydroxy-phosphoryloxy)-2-hydroxy-propyl ester (Ferguson et al., Org Lett 2006, 8 (10), 2023) was labeled with MR121 fluorophore (CAS 185308-24-1, 1-(3-carboxypropyl)-11-ethyl-1,2,3,4,8,9,10,11-octahydro-dipyrido[3,2-b:2′,3′-i]phenoxazin-13-ium) on the free amine of the ethanolamine side and then, after deprotection, subsequently with tryptophan on the side of the aminohexanoic acid.Assay working solutions were made as follows:Assay buffer (50 mM Tris-HCl, 140 mM NaCl, 5 mM KCl, 1 mM CaCl2, 1 mM MgCl2, 0.01% Triton-X-100, pH 8.0; ATX solution: ATX (human His-tagged) stock solution (1.08 mg/mL in 20 mM bicine, pH 8.5, 0.15 M NaCl, 10% glycerol, 0.3% CHAPS, 0.02% NaN3), diluted to 1.4-2.5× final concentration in assay buffer; MR121 substrate solution: MR121 substrate stock solution (800 μM MR121 substrate in DMSO), diluted to 2-5× final concentration in assay buffer.Test compounds (10 mM stock in DMSO, 8 μL) were obtained in 384 well sample plates (Corning Costar #3655) and diluted with 8 μL DMSO. Row-wise serial dilutions were made by transferring 8 μL cpd solution to the next row up to row O. The compound and control solutions were mixed five times and 2 L were transferred to 384 well assay plates (Corning Costar #3702). Then, 15 μL of 41.7 nM ATX solution was added (30 nM final concentration), mixed five times and then incubated for 15 minutes at 30° C. 10 μL of MR121 substrate solution was added (1 μM final concentration), mixed 30 times and then incubated for 15 minutes at 30° C. Fluorescence was then measured every 2 minutes for 1 hour (Perkin Elmer plate: vision multimode reader); light intensity: 2.5%; exp. time: 1.4 sec, Filter: Fluo_630/690 nm).
Measurement: IC50 = 7 nM (pChEMBL 8.149999618530273)
Descriptors: MW 541.63, ALogP 1.4, HBA 8, HBD 1, PSA 145.02, QED 0.58
Compound (SMILES): CNCCN1CCc2nc(Nc3ncc(F)c(-c4cc(F)c5nc(C)n(C6(C)CC6)c5c4)n3)ccc2C1
InChIKey: SAKHMWHBWMDUMO-UHFFFAOYSA-N
Target: Cyclin-dependent kinase 9 (SINGLE PROTEIN), Homo sapiens
UniProt: P50750
Assay: LANCE Ultra ULight Assay: 1. Take 10 mM stock solution of the test compound, in 96-well compound plate, DMSO was used to dilute the compound to an initial concentration of 100x, then this concentration was used as the first concentration, 3-fold diluted to make 10 serial concentrations; 1 μL each serial dilution was then added to 19 μL 1x reaction buffer to prepare 5x compound for use; 2 μL 5x compound was transferred from 96-well plate to 384-well plate; compound-free control well was added with 2 μL the following liquid: 1x reaction buffer with the addition of 1 μL DMSO; 2 μL 250 mM EDTA was added to the Min control well.2.1x reaction buffer was used to formulate the kinase, substrate and ATP into a 2.5xenzyme/substrate mixture and 2.5xATP solution respectively. In the experiment, the final concentration of CDK4/CycD3 kinase is 0.76 ng/μL, the final concentration of ATP is 8 μM; the final concentration of CDK6/CycD3 kinase is 0.5 ng/μL, the final concentration of ATP is 50M; the final concentration of CDK2/CycA2 kinase is 0.86 ng/μL, the final concentration of ATP is 1 μM; the final concentration of CDK2/CycE1 kinase is 1.016 ng/μL, the final concentration of ATP is 20M; 2.5xenzyme/substrate mixture was added to a 384-well plate, incubated at room temperature for 5 minutes; then added with 2.5xATP solution, reacted at room temperature for 30 minutes.3. LANCE Detection Buffer was used, 1xto prepare 2xLANCE Ultra Europium-anti-phospho-eIF4E-binding protein 1 (Thr37/46) for use. After the enzymatic reaction was continued for 30 minutes, 10 mM EDTA was added to 384-well plate and the mixture was reacted at room temperature for 5 minutes. Then LANCE Ultra Europium-anti-phospho-eIF4E-binding protein 1 (Thr37/46) was added, reacted at room temperature for 1 hour.4. The 384-well plate was placed in HERAEUS Multifuge X1R centrifuge, centrifuged at 2000 rpm for 2 minutes; data were measured on EnVision, 337 nM wavelength laser was used as the excitation light, test at RFU665 nM and RFU615 nM, and RFU665 nM/RFU615 nM x10000 was used as the final data for analysis.
Measurement: IC50 = 42.3 nM (pChEMBL 7.369999885559082)
Descriptors: MW 504.59, ALogP 4.31, HBA 8, HBD 2, PSA 83.79, QED 0.39
Compound (SMILES): COc1cc2c(Nc3ccc(Br)c(Cl)c3F)ncnc2cc1OC[C@H]1CN2CCOCC2CO1
InChIKey: CMTRUERFSJKMJQ-ARLHGKGLSA-N
Target: Ephrin type-B receptor 4 (SINGLE PROTEIN), Homo sapiens
UniProt: P54760
Assay: AlphaScreen Assay: In AlphaScreen, when the donor and acceptor beads are close in proximity, a series of photochemical events will give rise to a fluorescent signal upon light activation. Here we use biotinylated poly-(Glu, Tyr) 4:1 as the kinase substrate, streptavidin-coated donor beads and anti-phosphortyrosine antibody PY100-coated acceptor beads. Upon phosphorylation, the peptide substrate can bind to both donor and acceptor beads, thus gives rise to fluorescence. Compounds, ATP, biotinylated poly-(Glu, Tyr) and kinases are mixed in a volume of 20 μL for 1 hr at ambient temperature using Greiner 384-well white clear bottom medium binding plates. Then 10 uL solution containing 15-30 mg/mL AlphaScreen beads, 75 mM Hepes, pH 7.4, 300 mM NaCl, 120 mM EDTA, 0.3% BSA and 0.03% Tween-20 is added to each well. After 2-16 hr incubation of the beads, plates are read in a Perkin Elmer AlphaQuest reader (available from PerkinElmer Life and Analytical Sciences, Inc., of Boston Mass.).
Measurement: IC50 = 525 nM (pChEMBL 6.28000020980835)
Descriptors: MW 553.82, ALogP 4.42, HBA 8, HBD 1, PSA 77.97, QED 0.45
Compound (SMILES): Cn1cc(-c2cn3nccc3c(-c3cnn(C4CCCC4O)c3)n2)cn1
InChIKey: VHBISBSNGUVGTR-UHFFFAOYSA-N
Target: Tyrosine-protein kinase JAK2 (SINGLE PROTEIN), Homo sapiens
UniProt: O60674
Assay: JAK2 Inhibition Assay: Compounds of Formula I were screened for their ability to inhibit JAK2 using the general enzyme inhibition assay method, in which the assay mixture contained 1 mM ATP, 10 μM Omnia Y7 peptide (Catalog # IVGN KNZ3071C, Invitrogen Corporation, Carlsbad, Calif.) and 4 nM JAK2 in a total volume of 25 μL. JAK2 was purchased from Invitrogen Corporation, Carlsbad, Calif. (catalog # IVGN PV4288).
Measurement: IC50 = 550 nM (pChEMBL 6.260000228881836)
Descriptors: MW 349.4, ALogP 2.08, HBA 8, HBD 1, PSA 86.06, QED 0.61
Compound (SMILES): CN(c1ncnc2[nH]ccc12)[C@H]1C[C@@H](CS(=O)(=O)c2ccc(C(F)(F)F)nc2)C1
InChIKey: PYTYUNMFVXMLHB-TXEJJXNPSA-N
Target: Tyrosine-protein kinase JAK3 (SINGLE PROTEIN), Homo sapiens
UniProt: P52333
Assay: Caliper Enzyme Assay: Test article and assay controls were added to a 384-well plate. Reaction mixtures contained 20 mM HEPES, pH 7.4, 10 mM magnesium chloride, 0.01% bovine serum albumin (BSA), 0.0005% Tween 20, 1 mM ATP and 1 μM peptide substrate. The JAK1 and TYK2 assays contained 1 μM of the IRStide peptide (5FAM-KKSRGDYMTMQID) and the JAK2 and JAK3 assays contained 1 μM of the JAKtide peptide (FITC-KGGEEEEYFELVKK). The assays were initiated by the addition of 20 nM JAK1, 1 nM JAK2, 1 nM JAK3 or 1 nM TYK2 enzyme and were incubated at room temperature for three hours for JAK1, 60 minutes for JAK2, 75 minutes for JAK3 or 135 minutes for TYK2. Enzyme concentrations and incubation times were optimized for each new enzyme preps and were modified slightly over time to ensure 20%-30% phosphorylation. The assays were stopped with a final concentration of 10 mM EDTA, 0.1% Coating Reagent and 100 mM HEPES, pH=7.4. The assay plates were placed on a Caliper Life Science Lab Chip 3000 (LC3000) instrument, and each well was sampled using appropriate separation conditions to measure the unphosphorylated and phosphorylated peptide.
Measurement: IC50 = 8.62 nM (pChEMBL 8.0600004196167)
Descriptors: MW 425.44, ALogP 3.06, HBA 6, HBD 1, PSA 91.84, QED 0.68
Compound (SMILES): COc1cc(C(F)(F)F)c(OC)cc1C1CCCNC1
InChIKey: NZKYTYHIERLZBG-UHFFFAOYSA-N
Target: 5-hydroxytryptamine receptor 3A (SINGLE PROTEIN), Homo sapiens
UniProt: P46098
Assay: Radioligand Binding Assay: The binding affinities shown in table 5 of psilocin and compound 8 at various monoaminergic receptors were determined in radioligand competition binding assays by Psychiatric Drug Screening Program according to the experimental described in Assay Protocol Book, Version III 11, 16. As shown compound 8 shows less affinity for various monoaminergic receptors compared to Psilocin.
Measurement: Ki > 10000 nM (pChEMBL 5)
Descriptors: MW 289.3, ALogP 3.19, HBA 3, HBD 1, PSA 30.49, QED 0.93
Compound (SMILES): CN(c1ncc(C(=O)NO)cn1)C12CC3CC(CC(C3)C1)C2
InChIKey: IXDSXOZCSDIMHI-UHFFFAOYSA-N
Target: Histone deacetylase 7 (SINGLE PROTEIN), Homo sapiens
UniProt: Q8WUI4
Assay: IC50 Assay: IC50 measurements were conducted by BPS Biosciences (Table 1) or by Nanosyn (Table 1A) with an established fluorescence assay.
Measurement: IC50 = 36 nM (pChEMBL 7.440000057220459)
Descriptors: MW 302.38, ALogP 2, HBA 5, HBD 2, PSA 78.35, QED 0.66
Compound (SMILES): Cc1cccc(CNCC[C@@]2(c3ccc(F)cn3)CCO[C@]3(CCOC3)C2)c1
InChIKey: KDBYCIGWZXGKPW-DHIUTWEWSA-N
Target: Voltage-gated inwardly rectifying potassium channel KCNH2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q12809
Assay: Testing of Effects on hERG Potassium Channels: To test the blocking effects of the compounds of the present disclosure on hERG potassium currents.
Measurement: IC50 = 18800 nM (pChEMBL 4.730000019073486)
Descriptors: MW 384.5, ALogP 3.92, HBA 4, HBD 1, PSA 43.38, QED 0.77
Compound (SMILES): Cc1c(-c2[nH]c3ccc(C4CCC(N(C)CC(N)=O)CC4)nc3c2C(C)C)cn2ncnc2c1C
InChIKey: GUEANUQRFJJSOW-UHFFFAOYSA-N
Target: Toll-like receptor 9 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9NR96
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 = 1466 nM (pChEMBL 5.829999923706055)
Descriptors: MW 473.63, ALogP 4.46, HBA 6, HBD 2, PSA 105.2, QED 0.43
Compound (SMILES): Cc1nc2c(F)cc(-c3nc(Nc4ccc5c(n4)CCN(C(=O)CN(C)C)C5)ncc3F)cc2n1C1CC1
InChIKey: OIQZLVBXHSEFQP-UHFFFAOYSA-N
Target: Cyclin-dependent kinase 6 (SINGLE PROTEIN), Homo sapiens
UniProt: Q00534
Assay: LANCE Ultra ULight Assay: 1. Take 10 mM stock solution of the test compound, in 96-well compound plate, DMSO was used to dilute the compound to an initial concentration of 100x, then this concentration was used as the first concentration, 3-fold diluted to make 10 serial concentrations; 1 μL each serial dilution was then added to 19 μL 1x reaction buffer to prepare 5x compound for use; 2 μL 5x compound was transferred from 96-well plate to 384-well plate; compound-free control well was added with 2 μL the following liquid: 1x reaction buffer with the addition of 1 μL DMSO; 2 μL 250 mM EDTA was added to the Min control well.2.1x reaction buffer was used to formulate the kinase, substrate and ATP into a 2.5xenzyme/substrate mixture and 2.5xATP solution respectively. In the experiment, the final concentration of CDK4/CycD3 kinase is 0.76 ng/μL, the final concentration of ATP is 8 μM; the final concentration of CDK6/CycD3 kinase is 0.5 ng/μL, the final concentration of ATP is 50M; the final concentration of CDK2/CycA2 kinase is 0.86 ng/μL, the final concentration of ATP is 1 μM; the final concentration of CDK2/CycE1 kinase is 1.016 ng/μL, the final concentration of ATP is 20M; 2.5xenzyme/substrate mixture was added to a 384-well plate, incubated at room temperature for 5 minutes; then added with 2.5xATP solution, reacted at room temperature for 30 minutes.3. LANCE Detection Buffer was used, 1xto prepare 2xLANCE Ultra Europium-anti-phospho-eIF4E-binding protein 1 (Thr37/46) for use. After the enzymatic reaction was continued for 30 minutes, 10 mM EDTA was added to 384-well plate and the mixture was reacted at room temperature for 5 minutes. Then LANCE Ultra Europium-anti-phospho-eIF4E-binding protein 1 (Thr37/46) was added, reacted at room temperature for 1 hour.4. The 384-well plate was placed in HERAEUS Multifuge X1R centrifuge, centrifuged at 2000 rpm for 2 minutes; data were measured on EnVision, 337 nM wavelength laser was used as the excitation light, test at RFU665 nM and RFU615 nM, and RFU665 nM/RFU615 nM x10000 was used as the final data for analysis.
Measurement: IC50 = 8.47 nM (pChEMBL 8.069999694824219)
Descriptors: MW 518.57, ALogP 4, HBA 8, HBD 1, PSA 92.07, QED 0.41
Compound (SMILES): CCCCCOC[C@@H]1COc2cc([C@H]3CC[C@](N)(COP(=O)(O)O)C3)ccc2O1
InChIKey: XFNWLBLKUWRFPE-SQGPQFPESA-N
Target: Sphingosine 1-phosphate receptor 3 (SINGLE PROTEIN), Homo sapiens
UniProt: Q99500
Assay: Binding Assay: GTPγS:Compounds were loaded in a 384 Falcon v-bottom plate (0.5 μl/well in a 11 point, 3-fold dilution). Membranes prepared from S1P1/CHO cells or EDG3-Gal5-bla HEK293T cells (EDG3 equivalent S1P3) were added to the compound plate (40 μl/well, final protein 3 μg/well) with MULTIDROP®. [35S]GTP (1250 Ci/mmol, Perkin Elmer) was diluted in assay buffer: 20 mM HEPES, pH7.5, 10 mM MgCl2, 150 mM NaCl, 1 mM EGTA(ethylene glycol tetraacetic acid), 1 mM DTT (Dithiothreitol), 10 μM GDP, 0.1% fatty acid free BSA, and 10 μg/ml Saponin to 0.4 nM. 40 μl of the [35S] GTP solution was added to the compound plate with a final concentration of 0.2 nM. The reaction was kept at room temperature for 45 min. At the end of incubation, all the mixtures in the compound plate were transferred to Millipore 384-well FB filter plates via the VELOCITY11® Vprep liquid handler. The filter plate was washed with water 4 times by using the manifold Embla plate washer and dried at 60° C. for 45 min. MicroScint 20 scintillation fluid (30 μl) was added to each well for counting on the Packard TOPCOUNT®.
Measurement: IC50 > 10000 nM (pChEMBL 5)
Descriptors: MW 429.45, ALogP 3.11, HBA 6, HBD 3, PSA 120.47, QED 0.38
Compound (SMILES): CC1C(=O)N(C)c2cc3cnc(Nc4ccc(S(N)(=O)=O)cc4)nc3n2C12CCCCC2
InChIKey: FLIVKVQMXOAQAK-UHFFFAOYSA-N
Target: Cyclin-dependent kinase 7 (SINGLE PROTEIN), Homo sapiens
UniProt: P50613
Assay: CDK Inhibition In Vitro Assay: Selected compounds disclosed herein were tested in kinase assays by Nanosyn (Santa Clara, Calif.) to determine their inhibitory effect on these CDKs. The assays were performed using microfluidic kinase detection technology (Caliper Assay Platform). The compounds were tested in 12-point dose-response format in singlicate at Km for ATP. Specifics of each assay are as described below:CDK1/Cyclin B1: Enzyme concentration: 0.08 nM; ATP concentration: 40 μM; Incubation time: 3 hr. CDK2/Cyclin A: Enzyme concentration: 0.1 nM; ATP concentration: 50 μM; Incubation time: 3 hr. CDK2/Cyclin E: Enzyme concentration: 0.15 nM; ATP concentration: 100 μM; Incubation time: 3 hr. CDK4/Cyclin D1: Enzyme concentration: 1 nM; ATP concentration: 200 μM; Incubation time: 3 hr. CDK6/Cyclin D3: Enzyme concentration: 2 nM; ATP concentration: 300 μM; Incubation time: 3 hr. CDK9/Cyclin T1: Enzyme concentration: 5 nM; ATP concentration: 10 μM; Incubation time: 17 hr.
Measurement: IC50 = 250 nM (pChEMBL 6.599999904632568)
Descriptors: MW 454.56, ALogP 3.09, HBA 7, HBD 2, PSA 123.21, QED 0.63
Compound (SMILES): Cc1cnc(C(=O)N[C@@H](C)c2cc3cccc(C#Cc4cnn(C)c4)c3c(=O)n2-c2ccccc2)c(N)n1
InChIKey: KRQKZGMXITZFSA-IBGZPJMESA-N
Target: Phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit gamma isoform (SINGLE PROTEIN), Homo sapiens
UniProt: P48736
Assay: PI3K-gamma Selective Assay: A compound's ability in selectively inhibiting PI3K-γ can be assessed using RAW264.7 macrophages. Briefly, serum-starved RAW264.7 cells are stimulated with a known GPCR agonist C5a. See, e.g., Camps et al., Nature Medicine (2005) 11(9):936-943. Cells can be treated with test compounds prior to, simultaneously with, or subsequent to the stimulation by C5a. RAW 264.7 cells respond to the complement component fragment C5a through activation of the C5a receptor, and the C5a receptor activates macrophages and induces cell migration. Test compounds' ability to inhibit C5a-mediated AKT phosphorylation is indicative of selective inhibition of PI3K-γ. Thus, by monitoring the reduction of phospho-AKT in stimulated RAW 264.7 cells (using for example, phospho-AKT antibodies), a compound's potential efficacy in selectively inhibiting PI3Kγ can be assessed.
Measurement: IC50 = 5000 nM (pChEMBL 5.300000190734863)
Descriptors: MW 503.57, ALogP 3.3, HBA 8, HBD 2, PSA 120.72, QED 0.36
Compound (SMILES): COc1cc2c(Nc3ccc(Br)c(Cl)c3F)ncnc2cc1OC[C@H]1CN2CCOCC2CO1
InChIKey: CMTRUERFSJKMJQ-ARLHGKGLSA-N
Target: Ephrin type-A receptor 2 (SINGLE PROTEIN), Homo sapiens
UniProt: P29317
Assay: AlphaScreen Assay: In AlphaScreen, when the donor and acceptor beads are close in proximity, a series of photochemical events will give rise to a fluorescent signal upon light activation. Here we use biotinylated poly-(Glu, Tyr) 4:1 as the kinase substrate, streptavidin-coated donor beads and anti-phosphortyrosine antibody PY100-coated acceptor beads. Upon phosphorylation, the peptide substrate can bind to both donor and acceptor beads, thus gives rise to fluorescence. Compounds, ATP, biotinylated poly-(Glu, Tyr) and kinases are mixed in a volume of 20 μL for 1 hr at ambient temperature using Greiner 384-well white clear bottom medium binding plates. Then 10 uL solution containing 15-30 mg/mL AlphaScreen beads, 75 mM Hepes, pH 7.4, 300 mM NaCl, 120 mM EDTA, 0.3% BSA and 0.03% Tween-20 is added to each well. After 2-16 hr incubation of the beads, plates are read in a Perkin Elmer AlphaQuest reader (available from PerkinElmer Life and Analytical Sciences, Inc., of Boston Mass.).
Measurement: IC50 = 525 nM (pChEMBL 6.28000020980835)
Descriptors: MW 553.82, ALogP 4.42, HBA 8, HBD 1, PSA 77.97, QED 0.45
Compound (SMILES): CN(c1ncnc2[nH]ccc12)[C@H]1C[C@@H](CS(=O)(=O)c2ccc(C(F)(F)F)nc2)C1
InChIKey: PYTYUNMFVXMLHB-TXEJJXNPSA-N
Target: Non-receptor tyrosine-protein kinase TYK2 (SINGLE PROTEIN), Homo sapiens
UniProt: P29597
Assay: Caliper Enzyme Assay: Test article and assay controls were added to a 384-well plate. Reaction mixtures contained 20 mM HEPES, pH 7.4, 10 mM magnesium chloride, 0.01% bovine serum albumin (BSA), 0.0005% Tween 20, 1 mM ATP and 1 μM peptide substrate. The JAK1 and TYK2 assays contained 1 μM of the IRStide peptide (5FAM-KKSRGDYMTMQID) and the JAK2 and JAK3 assays contained 1 μM of the JAKtide peptide (FITC-KGGEEEEYFELVKK). The assays were initiated by the addition of 20 nM JAK1, 1 nM JAK2, 1 nM JAK3 or 1 nM TYK2 enzyme and were incubated at room temperature for three hours for JAK1, 60 minutes for JAK2, 75 minutes for JAK3 or 135 minutes for TYK2. Enzyme concentrations and incubation times were optimized for each new enzyme preps and were modified slightly over time to ensure 20%-30% phosphorylation. The assays were stopped with a final concentration of 10 mM EDTA, 0.1% Coating Reagent and 100 mM HEPES, pH=7.4. The assay plates were placed on a Caliper Life Science Lab Chip 3000 (LC3000) instrument, and each well was sampled using appropriate separation conditions to measure the unphosphorylated and phosphorylated peptide.
Measurement: IC50 = 2.98 nM (pChEMBL 8.529999732971191)
Descriptors: MW 425.44, ALogP 3.06, HBA 6, HBD 1, PSA 91.84, QED 0.68