lovingscience/chembl-v37-raw-activities huggingface dataset:
Compound (SMILES): O=C(O)c1ccc(CCc2cccc3c2N(Cc2ccc4c(c2)OCO4)CC3)cc1
InChIKey: CWZNOMPZVARKDQ-UHFFFAOYSA-N
Target: Prostaglandin E2 receptor EP4 subtype (SINGLE PROTEIN), Homo sapiens
UniProt: P35408
Assay: Radioligand Binding Assay: HEK-293 cells stably expressing the human or feline FP receptor, or EP1, EP2, or EP4 receptors were washed with TME buffer, scraped from the bottom of the flasks, and homogenized for 30 sec using a Brinkman PT 10/35 polytron. TME buffer was added to achieve a final 40 ml volume in the centrifuge tubes (the composition of TME is 100 mM TRIS base, 20 mM MgCl2, 2M EDTA; 10N HCl is added to achieve a pH of 7.4).The cell homogenate was centrifuged at 19000 r.p.m. for 20 min at 4° C. using a Beckman Ti-60 rotor. The resultant pellet was resuspended in TME buffer to give a final 1 mg/ml protein concentration, as determined by Biorad assay. Radioligand binding competition assays vs. [3H-]17-phenyl PGF2a (5 nM) were performed in a 100 μl volume for 60 min. Binding reactions were started by adding plasma membrane fraction. The reaction was terminated by the addition of 4 ml ice-cold TRIS-HCl buffer and rapid filtration through glass fiber GF/B filters using a Brandel cell harvester. The filters were washed 3 times with ice-cold buffer and oven dried for one hour.[3H] PGE2 (specific activity 180 Ci mmol) was used as the radioligand for EP receptors. [3H] 17-phenyl PGF2a was employed for FP receptor binding studies. Binding studies employing EP1, EP2, EP4 and FP receptors were performed in duplicate in at least three separate experiments. A 200 μl assay volume was used. Incubations were for 60 min at 25° C. and were terminated by the addition of 4 ml of ice-cold 50 mM TRIS-HCl, followed by rapid filtration through Whatman GF/B filters and three additional 4 ml washes in a cell harvester (Brandel).
Measurement: Ki = 71 nM (pChEMBL 7.150000095367432)
Descriptors: MW 401.46, ALogP 4.46, HBA 4, HBD 1, PSA 59, QED 0.66
Compound (SMILES): CN(C)C(CNC(=O)NC(C)(C)Cc1ccsc1)Cc1ccccc1
InChIKey: NSAOLNUDHLFABH-UHFFFAOYSA-N
Target: Mu-type opioid receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P35372
Assay: Radioligand Binding Assay: Each compound was initially tested at 20 μM and was incubated with 3H-DPN at a concentration equal to the Kd (0.4 nM) of the radioligand in μOR containing Sf9 insect cell membranes. The reaction contained 40 fmol of μOR and was incubated in a buffer of 20 mM HEPES pH 7.5, 100 mM sodium chloride, and 0.1% bovine serum albumin for 1 hour at 25° C. To separate free from bound radioligand, reactions were rapidly filtered over Whatman GF/B filters with the aid of a Brandel harvester and 3H-DPN counts were measured by liquid scintillation. Compounds with more than 25% of 3H-DPN radioactivity were further tested in full dose-response to determine the affinity (Ki) in HEK293 membranes. Subsequently, the 15 analogs were tested in full dose-response for affinity at the μOR and the κOR by the National Institutes of Mental Health Psychoactive Drug Screen Program (PDSP), as were the affinities of compounds 12, PZM21, and their stereoisomers at the μOR, δOR, κOR and nociception receptor.
Measurement: Ki = 210 nM (pChEMBL 6.679999828338623)
Descriptors: MW 359.54, ALogP 3.54, HBA 3, HBD 2, PSA 44.37, QED 0.76
Compound (SMILES): CC[C@H]1CN(C(c2ccc(F)cc2)c2ccc(C(F)(F)F)cn2)[C@H](C)CN1c1nc(=O)n(C)c2ccc(C#N)nc12
InChIKey: KCXOIXFXILJQSD-CXVSQYHOSA-N
Target: Diacylglycerol kinase zeta (SINGLE PROTEIN), Homo sapiens
UniProt: Q13574
Assay: In Vitro DGK Inhibition Assay: The reactions were carried out in 50 mM MOPS pH 7.5, 100 mM NaCl, 10 mM MgCl2, 1 μM CaCl2, and 1 mM DTT (assay buffer). The reactions using a detergent/lipid micelle substrate also contained 50 mM octyl B-D-glucopyranoside. The lipid substrate concentrations were 11 mM PS and 1 mM DAG for the detergent/lipid micelle reactions. The lipid substrate concentrations were 2 mM PS, 0.25 mM DAG, and 2.75 mM PC for the extruded liposome reactions (5 mM total lipid). The reactions were carried out in 150 μM ATP. The enzyme concentrations for the DGKα and DGKζ were 5 nM. The compound inhibition studies were carried out as follows: 25 nL (ADPGLO assay) or 50 nL (LIPGLO assay) droplets of each test compound (top concentration 10 mM with 11 point, 3-fold dilution series for each compound) solubilized in DMSO were transferred to wells of a white 1536 well plate (Corning 3725). A 5 mL enzyme/lipid substrate solution at 2× final reaction concentration was prepared by combining 2.5 mL 4× enzyme solution (20 nM DGKα or DGKζ (prepared as described below) in assay buffer) and 2.5 mL of either 4× liposome or 4× detergent/lipid micelle solution (compositions described below) and incubated at room temperature for 10 minutes. Next, 1 μL 2× enzyme/lipid substrate solution was added to wells containing the test compound and reactions were initiated with the addition of 1 μL 300 uM ATP. The reactions were allowed to proceed for 2 hr (ADPGLO assay) or 1 hr (LIPGLO assay), after which 2 μL Glo Reagent (Promega V9101) was added and incubated for 40 minutes. Next, 4 μL Kinase Detection Reagent was added and incubated for 30 minutes. Luminescence was recorded using an EnVision microplate reader. The percent inhibition was calculated from the ATP conversion generated by no enzyme control reactions for 100% inhibition and vehicle-only reactions for 0% inhibition.
Measurement: IC50 = 2470 nM (pChEMBL 5.610000133514404)
Descriptors: MW 565.58, ALogP 4.83, HBA 8, HBD 0, PSA 90.94, QED 0.32
Compound (SMILES): Cn1cc(-c2cc3ncccc3c(-c3ccn(C4(CC#N)CC(C#N)C4)n3)n2)cn1
InChIKey: CQRLVCKYPXPONF-UHFFFAOYSA-N
Target: Tyrosine-protein kinase JAK3 (SINGLE PROTEIN), Homo sapiens
UniProt: P52333
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 = 9608 nM (pChEMBL 5.019999980926514)
Descriptors: MW 394.44, ALogP 3.44, HBA 8, HBD 0, PSA 109, QED 0.52
Compound (SMILES): Fc1ccc2nc(-c3cnnc(NC4CCOCC4)c3)n(C3CC3)c2c1
InChIKey: AHAJBVXKUBAKEF-UHFFFAOYSA-N
Target: Cytochrome P450 11B1, mitochondrial (SINGLE PROTEIN), Homo sapiens
UniProt: P15538
Assay: Enzyme Assay: Enzyme assays for inhibitor screening employed CYP11B2 and CYP11B1 enzyme enriched microsomes and were run at the Km of the respective substrates. Products of the enzyme reactions, aldosterone for CYP11B2 or cortisol for CYP11B1, were measured by LC-MS. Assays were run under conditions of less than 20% substrate turnover. Inhibitor IC50s were generated by determining the product formation in the absence or presence of inhibitor at various concentrations. In the absence of the test compound, the product formed (Pt) in each data set was defined as 100% activity. In the absence of enzyme, the product formed (Pb) in each data set was defined as 0% activity.
Measurement: IC50 > 10000 nM (pChEMBL 5)
Descriptors: MW 353.4, ALogP 3.56, HBA 6, HBD 1, PSA 64.86, QED 0.78
Compound (SMILES): C=CC(=O)N1CC[C@@H](Oc2cc(-c3ccc(O)cc3)cn3cncc23)C[C@H]1C
InChIKey: CFIRTRVEXIZQLT-DNVCBOLYSA-N
Target: Non-receptor tyrosine-protein kinase TYK2 (SINGLE PROTEIN), Homo sapiens
UniProt: P29597
Assay: Biochemical JAK and Tyk2 Kinase Assay: A panel of four LanthaScreen JAK biochemical assays (JAK1, 2, 3 and Tyk2) were carried in a common kinase reaction buffer (50 mM HEPES, pH 7.5, 0.01% Brij-35, 10 mM MgCl2, and 1 mM EGTA). Recombinant GST-tagged JAK enzymes and a GFP-tagged STAT1 peptide substrate were obtained from Life Technologies.Serially or discretely diluted compounds were pre-incubated with each of the four JAK enzymes and the substrate in white 384-well microplates (Corning) at ambient temperature for 1 h. ATP was subsequently added to initiate the kinase reactions in 10 μL total volume, with 1% DMSO. The final enzyme concentrations for JAK1, 2, 3 and Tyk2 are 4.2 nM, 0.1 nM, 1 nM, and 0.25 nM respectively; the corresponding Km ATP concentrations used are 25 μM, 3 μM, 1.6 μM, and 10 μM; while the substrate concentration is 200 nM for all four assays. Kinase reactions were allowed to proceed for 1 hour at ambient temperature before a 10 μL preparation of EDTA (10 mM final concentration) and Tb-anti-pSTAT1 (pTyr701) antibody (Life Technologies, 2 nM final concentration) in TR-FRET dilution buffer (Life Technologies) was added. The plates were allowed to incubate at ambient temperature for 1 h before being read on the EnVision reader (Perkin Elmer). Emission ratio signals (520 nm/495 nm) were recorded and utilized to calculate the percent inhibition values based on DMSO and background controls.
Measurement: Ki > 100 nM (pChEMBL 7)
Descriptors: MW 377.44, ALogP 3.65, HBA 5, HBD 1, PSA 67.07, QED 0.7
Compound (SMILES): CC1CN(c2cnc3ccc(-c4ccc5oc(N)nc5c4)cc3n2)CCO1
InChIKey: AZMRQKADLAPQSR-UHFFFAOYSA-N
Target: Phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit alpha isoform (SINGLE PROTEIN), Homo sapiens
UniProt: P42336
Assay: Inhibition Assay of PI3K: An exemplary system is PI 3-Kinase (human) HTRF Assay from Upstate. The assay can be carried out according to the procedures suggested by the manufacturer. Briefly, the assay is a time resolved FRET assay that indirectly measures PIP3 product formed by the activity of a PI3-K. The kinase reaction is performed in a microtitre plate (e.g., a 384 well microtitre plate). The total reaction volume is approximately 20 ul per well. In the first step, each well receives 2 ul of test compound in 20% dimethylsulphoxide resulting in a 2% DMSO final concentration. Next, approximately 14.5 ul of a kinase/PIP2 mixture (diluted in 1× reaction buffer) is added per well for a final concentration of 0.25-0.3 ug/ml kinase and 10 μM PIP2. The plate is sealed and incubated for 15 minutes at room temperature. To start the reaction, 3.5 ul of ATP (diluted in 1× reaction buffer) is added per well for a final concentration of 10 μM ATP. The plate is sealed and incubated for 1 hour at room temperature. The reaction is stopped by adding 5 ul of Stop Solution per well and then 5 ul of Detection Mix is added per well. The plate is sealed, incubated for 1 hour at room temperature, and then read on an appropriate plate reader.
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 361.41, ALogP 3.25, HBA 7, HBD 1, PSA 90.3, QED 0.59
Compound (SMILES): CCC(CC)n1cc(-c2nc(-c3cnn(C[C@@H]4CCCN4)c3)cn3nccc23)cn1
InChIKey: PEALVEWPZOQWRK-SFHVURJKSA-N
Target: Tyrosine-protein kinase JAK3 (SINGLE PROTEIN), Homo sapiens
UniProt: P52333
Assay: JAK3 Inhibition Assay: Compounds of Formula I were screened for their ability to inhibit JAK3 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 2 nM JAK3 in a total volume of 25 μL. JAK3 was purchased from Invitrogen Corporation, Carlsbad, Calif. (catalog # IVGN PV4080).
Measurement: IC50 = 5500 nM (pChEMBL 5.260000228881836)
Descriptors: MW 404.52, ALogP 3.57, HBA 8, HBD 1, PSA 77.86, QED 0.51
Compound (SMILES): COc1cc(Cn2cccn2)cc2onc(NS(=O)(=O)c3cccc(C)c3C#N)c12
InChIKey: UDQLVPWPXOQLGE-UHFFFAOYSA-N
Target: Histone acetyltransferase KAT5 (SINGLE PROTEIN), Homo sapiens
UniProt: Q92993
Assay: Kat5 Assay: A. Compound preparation    1. Prepare 10 mM stock solutions in 100% DMSO from solid material    2. Serial dilute 10 mM, 1 mM or 0.1 mM compound stocks 3-fold in 100% DMSO for 11-point dose responseB. Reagent preparation    1. Prepare 1× assay buffer containing 10 mM Tris HCL pH 8.0, 2.5 mM NaCl, 0.5 mM EDTA, 0.005% BSG and 0.02% Tween-20    2. Dilute Histone peptide (CPC Scientific) and AcCoA (Sigma) together in assay buffer to 2×.    3. Dilute KAT enzyme in assay buffer to 2×.C. Enzyme reaction    1. Final reaction conditions for each KAT assay in a 20 ul assay reaction volume:  KAT5 25 nM, 1 uM AcCoA, 2 uM H4 1-21 peptide, 30-minute reaction     2. Add 0.5 ul of diluted compound to the assay plate (384-well V-bottom polypropylene plates) or 0.5 ul of DMSO for control wells.    3. Add 10 ul of 2× Histone peptide/2×AcCoA mix to the assay plate.    4. Add 10 ul of 2× enzyme to the assay plate.    5. Stop the reaction after the indicated time with the addition of 2 ul of 5% formic acid    6. Each reaction was analyzed using self-assembled monolayer desorption/ionization time-of-flight mass spectrometry (Mrksich, Milan (2008) Mass Spectrometry of Self-Assembled Monolayers: A New Tool for Molecular Surface Science ACS Nano 2008 2 (1), 7-18; SAMDI Tech, Inc. (Chicago, Ill.)).    7. Area under the curve (AUC) for both substrate and product peaks was determined for KAT5 at M.W. 2561 [Substrate+H]+ and 2603 [Product+H]+ with a +/−1 Da tolerance, respectively    8. Area under the curve (AUC) for both substrate and product peaks was determined for KAT6a, KAT6B, KAT7 and KAT8 at M.W. 2723 [Substrate+H]+ and 2765 [Product+H]+ with a +/−1 Da tolerance, respectively. 9. Percent conversion to product was calculated by: AUCProduct/(AUCsubstrate+AUCProduct).D. Data analysis    1. IC50 values were determined by fitting the % conversion at each inhibitor concentration to the 4-parameter IC50 equation using Pfizer proprietary curve fitting software.    2. Ki values were determined by fitting the % conversion at each inhibitor concentration to the Morrison equation for tightbinding competitive inhibitors using Pfizer proprietary curve fitting software.
Measurement: Ki = 4380 nM (pChEMBL 5.360000133514404)
Descriptors: MW 423.45, ALogP 3.06, HBA 8, HBD 1, PSA 123.04, QED 0.51
Compound (SMILES): COc1cc(OC)c(F)c(N2Cc3cnc(-c4ccc(C#N)nc4)cc3C3(CC3)C2=O)c1F
InChIKey: XHOBSPUMHKAEIP-UHFFFAOYSA-N
Target: Fibroblast growth factor receptor 4 (SINGLE PROTEIN), Homo sapiens
UniProt: P22455
Assay: FGFR Enzymatic Assay 1: FGFR1, FGFR2, and FGFR4 are measured under equivalent conditions with the following changes in enzyme and ATP concentrations: FGFR1, 0.02 nM and 210 uM respectively, FGFR2, 0.01 nM and 100 uM, respectively, and FGFR4, 0.04 nM and 600 uM respectively. The enzymes were purchased from Millipore or Invitrogen. GraphPad prism3 was used to analyze the data. The IC50 values were derived by fitting the data to the equation for a sigmoidal dose-response with a variable slope. Y=Bottom+(Top−Bottom)/(1+10^((Log IC50−X)*HillSlope)) where X is the logarithm of concentration and Y is the response. Compounds having an IC50 of 1 μM or less are considered active. The compounds of the invention were found to be selective inhibitors of FGFR3 and/or FGFR4 according to the FGFR Enzymatic Assays. Compounds of Formula (I′) and (I) and all the compounds as described herein have been tested and exhibit an IC50 of less than 1 μM. Table 1 provides IC50 data for compounds of the invention assayed in the FGFR Enzymatic Assay after dilution in assay buffer, added to the plate and pre-incubated for 4 hours.
Measurement: IC50 = 20 nM (pChEMBL 7.699999809265137)
Descriptors: MW 448.43, ALogP 3.89, HBA 6, HBD 0, PSA 88.34, QED 0.6
Compound (SMILES): O=C1CCC(N2C(=O)c3cccc(NCCOCCOCCOCCOCCN4CCN(Cc5ccc(-c6cc7c(NCc8ccccc8Cl)ncnc7[nH]6)cc5)CC4)c3C2=O)C(=O)N1
InChIKey: AZYKVBYABJWTNN-UHFFFAOYSA-N
Target: Epidermal growth factor receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P00533
Assay: TR-FRET Assay for ERBB Kinases: All compounds and PROTACs were serially diluted in three-fold increments using 100% DMSO, followed by an intermediate 10-fold dilution using Buffer A (50 mM HEPES, pH 7.5, 50 mM NaCl, 10 mM MgCl2, 1 mM DTT, and 0.1% Pluronic F-68). Two microliters of serially diluted compound or PROTAC were then transferred to black 384-well Proxiplates (PerkinElmer, #6008260) using an Integra Viaflo96. Next, 10 uL of protein kinase in Buffer A was added to each well of the assay plate and pre-incubated with compound for 10 minutes. Kinase reactions were then initiated by addition of 5 uL substrate mix containing 3 mM ATP and 30 uM fluorescein-labeled Poly-GluTyr (Thermo Fisher, #PV3610) in Buffer A and allowed to proceed for 10 minutes at room temperature. Reactions were quenched by addition of a 5 uL mixture containing 5 nM LanthaScreen® Tb-pY20 Antibody (Thermo Fisher, #PV3552) and 40 mM EDTA in Buffer A. Assay plates were then read using a Synergy2 (Biotek Instruments, Winooski, Vt.) via excitation thru a 340/20 nm bandpass filter and emission collected thru 490/10 nm (donor) and 520/25 nm (acceptor) bandpass filters. The final kinase concentrations used for each 15 uL reaction were as follows: 0.2 nM EGFR Exon20NPG (SignalChem, #E10-132GG), 0.1 nM wild type EGFR (BPS Bioscience, #40187), 0.3 nM EGFR L858R/T790M/C797S (BPS Bioscience, #40351), 0.1 nM EGFR L858R (BPS Bioscience, #40189), 0.4 nM L858R/T790M (BPS Bioscience, #40350), InM EGFR Dell9 (SignalChem, #E10-122JG), 10 nM EGFR Dell9 T790M (SignalChem, #E10-122KG), 0.3 nM Her2 (BPS Bioscience, #40230), 15 nM Her2 InsYVMA (SignalChem, #E27-13BG).
Measurement: IC50 = 110 nM (pChEMBL 6.960000038146973)
Descriptors: MW 908.46, ALogP 4.59, HBA 14, HBD 4, PSA 192.58, QED 0.05
Compound (SMILES): O=C(c1cnc2ccc(-c3cncc(NS(=O)(=O)c4ccccc4)c3)cn12)N1CCOCC1
InChIKey: FMSKSPNHFCXGOC-UHFFFAOYSA-N
Target: Phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit alpha isoform (SINGLE PROTEIN), Homo sapiens
UniProt: P42336
Assay: Inhibition Assay of PI3K: An exemplary system is PI 3-Kinase (human) HTRF Assay from Upstate. The assay can be carried out according to the procedures suggested by the manufacturer. Briefly, the assay is a time resolved FRET assay that indirectly measures PIP3 product formed by the activity of a PI3-K. The kinase reaction is performed in a microtitre plate (e.g., a 384 well microtitre plate). The total reaction volume is approximately 20 ul per well. In the first step, each well receives 2 ul of test compound in 20% dimethylsulphoxide resulting in a 2% DMSO final concentration. Next, approximately 14.5 ul of a kinase/PIP2 mixture (diluted in 1× reaction buffer) is added per well for a final concentration of 0.25-0.3 ug/ml kinase and 10 μM PIP2. The plate is sealed and incubated for 15 minutes at room temperature. To start the reaction, 3.5 ul of ATP (diluted in 1× reaction buffer) is added per well for a final concentration of 10 μM ATP. The plate is sealed and incubated for 1 hour at room temperature. The reaction is stopped by adding 5 ul of Stop Solution per well and then 5 ul of Detection Mix is added per well. The plate is sealed, incubated for 1 hour at room temperature, and then read on an appropriate plate reader.
Measurement: IC50 < 1000 nM (pChEMBL 6)
Descriptors: MW 463.52, ALogP 2.67, HBA 7, HBD 1, PSA 105.9, QED 0.49
Compound (SMILES): COc1ccc(-c2nc3c(n2C(C)C)C(c2ccc(Cl)cc2)N(c2cc(OC)c4nnn(C)c4c2)C3=O)cn1
InChIKey: JUNKHWUBWMACDY-UHFFFAOYSA-N
Target: CREB-binding protein (SINGLE PROTEIN), Homo sapiens
UniProt: Q92793
Assay: AlphaScreen In-Vitro Binding Assay : In order to assess bromodomain selectivity, we set up a binding assay using the bromodomain encoded by the CREBBP gene. Compounds were tested in the CREBBP assay with a similar protocol, however using AlphaScreen (Amplified Luminescent Proximity Homogeneous Assay, Perkin Elmer) as detection readout instead of TR-FRET. The assay was started by stepwise addition of 4.5μl per well of bromo domain protein (50 mM HEPES, pH 7.5, 0.005% Tween20, 0.02% BSA, 150 mM NaCl, 324 nM His-CREBBP(1081-1197) (custom production at Viva Biotech Ltd.)) and 4.5μl per well of peptide solution (50 mM HEPES, pH 7.5, 0.005% Tween20, 0.02% BSA, 150 mM NaCl, 120 nM acetyl-histone H4 (AcK 5, 8, 12) (Biosyntan GmbH)). Reactions were incubated at 30° C. for 35 minutes. Subsequently 4.5 μl per well detection mix (50 mM HEPES, pH 7.5, 0.005% Tween20, 0.02% BSA, 150 mM NaCl, 45 μg/ml Ni-chelate acceptor beads, 45 μg/mIstreptavidin-donor beads) (Perkin Elmer)) were added. After 60 minutes incubation at room temperature, plates were measured in a Perkin Elmer EnVision multilabel reader. IC50 values were determined from percent inhibition values at different compound concentrations by non-linear regression analysis.
Measurement: IC50 > 37000 nM (pChEMBL 4.432000160217285)
Descriptors: MW 544.02, ALogP 5.23, HBA 9, HBD 0, PSA 100.19, QED 0.29
Compound (SMILES): CNC(=O)c1n[nH]c2nc(-c3cc(F)c(O)cc3CC(F)(F)F)nc(NCc3nccnc3N(C)S(C)(=O)=O)c12
InChIKey: CNLKMAASBAEKQE-UHFFFAOYSA-N
Target: Non-receptor tyrosine-protein kinase TYK2 (SINGLE PROTEIN), Homo sapiens
UniProt: P29597
Assay: JAK Caliper Enzyme Assay at 1 mM ATP: Test article was solubilized in dimethyl sulfoxide (DMSO) to a stock concentration of 30 mM. An 11-point half log dilution series was created in DMSO with a top concentration of 600 μM. The test compound plate also contained positive control wells containing a known inhibitor to define 100% inhibition and negative control wells containing DMSO to define no inhibition. The compound plates were diluted 1 to 60 resulting in a top final assay compound concentration of 10 μM and a 2% DMSO concentration.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 = 6112 nM (pChEMBL 5.210000038146973)
Descriptors: MW 583.53, ALogP 2.13, HBA 10, HBD 4, PSA 178.98, QED 0.22
Compound (SMILES): N[C@@H]1CN(c2ccncc2NC(=O)c2ccc3ncc(N4CCC5(CCCO5)CC4)cc3n2)C[C@H](C2CC2)[C@H]1O
InChIKey: ZGCYGVBXRSPXOD-DQCZWYHMSA-N
Target: Serine/threonine-protein kinase pim-2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9P1W9
Assay: Enzyme Assay: Pim-1 and Pim-3 kinase assays-20 uL reactions were run in white 384 well polystyrene plates dotted with 0.8 uL compound/DMSO in the assay buffer (50 mM Tris, pH 7.5, 0.01% Tween-20, 5 mM mgCl2, 0.01% BSA, 5 mM DTT), containing 0.05 uM Biotin-labeled BAD peptide substrate (AnaSpec 62269), 1 mM ATP, and 2.5 pM (Pim-1, Invitrogen PV3503) or 1.25 pM (Pim-3, Millipore 14-738) enzyme for 1 h at 25° C. Reactions were stopped by addition of 10 uL STOP Buffer (150 mM Tris, pH=7.5, 150 mM NaCl, 75 mM EDTA, 0.01% Tween-20, 0.3% BSA), supplemented with Phospho-Bad (Ser112) Antibody (Cell Signaling 9291) diluted 666-fold, and Streptavidin donor beads (PerkinElmer 6760002) along with Protein-A acceptor beads (PerkinElmer 6760137) at 15 ug/mL each. Supplementation of the STOP buffer with beads and stopping the reactions were done under reduced light. Prior to the stopping reactions STOP buffer with beads was preincubated for 1 h in the dark at room temperature. After stopping the reactions, plates were incubated for 1 h in the dark at room temperature before reading on a PHERAstar FS plate reader (BMG Labtech) under reduced light. Pim-2 kinase assay-20 uL reactions were run in white 384 well polystyrene plates dotted with 0.8 uL compound/DMSO in the assay buffer (50 mM Tris, pH 7.5, 0.01% Tween-20, 5 mM mgCl2, 0.01% BSA, 5 mM DTT), containing 0.05 uM Fluorescein-labeled CREBtide peptide substrate (Invitrogen PV3508), 1 mM ATP, and 1 nM enzyme (Invitrogen PV3649) for 2 h at 25° C. Reactions were stopped by addition of 10 uL TR-FRET Dilution Buffer (Invitrogen PV3574) with 30 mM EDTA and 1.5 nM LanthaScreen Tb-CREB pSer133 antibody (Invitrogen PV3566). After 30 min. incubation at room temperature, plates were read on a PHERAstar FS plate reader (BMG Labtech).
Measurement: IC50 = 550 nM (pChEMBL 6.260000228881836)
Descriptors: MW 543.67, ALogP 2.96, HBA 9, HBD 3, PSA 129.73, QED 0.44
Compound (SMILES): N#Cc1cccc2c1OCC(c1cncc(CC(=O)N3CCOCC3)c1)O2
InChIKey: VTVATTVNDOGQPR-UHFFFAOYSA-N
Target: Cytochrome P450 11B1, mitochondrial (SINGLE PROTEIN), Homo sapiens
UniProt: P15538
Assay: Inhibition Assay: Assays are performed in 96-well format in a final volume of 60 μL/well, containing 100 mM potassium phosphate, pH 7.4, 1% (v/v) DMSO, and additionally, 2 μM of corticosterone and 50 units of CYP11B2 activity. Reactions are started by the addition of NADPH to 1 mM and allowed to proceed for 90 minutes at 37° C. Reactions are terminated by the addition of 60 μL of MeCN containing an internal standard for mass spectrometry. One hundred microliters are then transferred to a glass filter plate and centrifuged at 570×g for 5 minutes and the filtrate is collected. Reaction product aldosterone is quantified by mass spectrometry. To determine the assay blank value (0% activity), NADPH is omitted from some reactions.
Measurement: IC50 > 30000 nM (pChEMBL 4.5229997634887695)
Descriptors: MW 365.39, ALogP 1.87, HBA 6, HBD 0, PSA 84.68, QED 0.83
Compound (SMILES): CCC(CC)n1cc(-c2nc(-c3cnn(C[C@@H](O)COC)c3)cn3nccc23)cn1
InChIKey: ISWBZDSAQHLONO-GOSISDBHSA-N
Target: Tyrosine-protein kinase JAK3 (SINGLE PROTEIN), Homo sapiens
UniProt: P52333
Assay: JAK3 Inhibition Assay: Compounds of Formula I were screened for their ability to inhibit JAK3 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 2 nM JAK3 in a total volume of 25 μL. JAK3 was purchased from Invitrogen Corporation, Carlsbad, Calif. (catalog # IVGN PV4080).
Measurement: IC50 = 5500 nM (pChEMBL 5.260000228881836)
Descriptors: MW 409.49, ALogP 2.82, HBA 9, HBD 1, PSA 95.29, QED 0.46
Compound (SMILES): CCC(CC)n1cc(-c2nc(-c3cnn(C[C@H](CN)OC)c3)cn3nccc23)cn1
InChIKey: OIAGAWSCJMASIN-SFHVURJKSA-N
Target: Tyrosine-protein kinase JAK3 (SINGLE PROTEIN), Homo sapiens
UniProt: P52333
Assay: JAK3 Inhibition Assay: Compounds of Formula I were screened for their ability to inhibit JAK3 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 2 nM JAK3 in a total volume of 25 μL. JAK3 was purchased from Invitrogen Corporation, Carlsbad, Calif. (catalog # IVGN PV4080).
Measurement: IC50 = 5500 nM (pChEMBL 5.260000228881836)
Descriptors: MW 408.51, ALogP 2.79, HBA 9, HBD 1, PSA 101.08, QED 0.46
Compound (SMILES): Cn1cc(-c2cc(Oc3ccc(NC(=O)N4CCC5(CN(S(C)(=O)=O)C5)C4=O)nc3)ccn2)cn1
InChIKey: KRCPKGPVOUOHOW-UHFFFAOYSA-N
Target: Platelet-derived growth factor receptor beta (SINGLE PROTEIN), Homo sapiens
UniProt: P09619
Assay: Kinase Activity Assay: c-Kit, PDGFRα, PDGFRβ, FLT-3:Add 5 μL of compound (10% DMSO) at 5 times the final concentration of the reaction to a 384-well plate. Add 10 μL of 2.5×enzyme solution and incubate for 10 minutes at room temperature, then add 10 μL of 2.5×substrate (ATP and peptide substrate, P2 or P22). After incubating at 28° C. for 60 minutes, add 25 μL stop solution to stop the reaction. Read conversion rate data on Caliper EZ Reader II (Caliper Life Sciences). Convert the conversion rate into inhibition rate data (% inhibition rate=(max−sample conversion rate)/(max−min)×100). Where max refers to the conversion rate of the DMSO control, and min refers to the conversion rate of the control without enzyme activity. Draw a curve with compound concentration and inhibition rate on the abscissa and ordinate, use XLFit excel add-in version 4.3.1 software to fit the curve and calculate IC50.
Measurement: IC50 = 2808 nM (pChEMBL 5.550000190734863)
Descriptors: MW 497.54, ALogP 1.7, HBA 9, HBD 1, PSA 139.62, QED 0.56
Compound (SMILES): CCCCCC[C@@H]1Cc2ccc([C@@H]3CC[C@](N)(COP(=O)(O)O)C3)cc2CO1
InChIKey: MJKUOBXNYRXRCS-HMXCVIKNSA-N
Target: Sphingosine 1-phosphate receptor 3 (SINGLE PROTEIN), Homo sapiens
UniProt: Q99500
Assay: [35S] GTPgammaS Binding Assays: Compounds were loaded in a 384 Falcon v-bottom plate (0.5 ul/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 ul/well, final protein 3 ug/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 uM GDP, 0.1% fatty acid free BSA, and 10 ug/ml Saponin to 0.4 nM. 40 ul 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 ul) was added to each well for counting on the Packard TOPCOUNT. EC50 is defined as the agonist concentration that corresponds to 50% of the Ymax (maximal response) obtained for each individual compound tested.
Measurement: EC50 > 625 nM (pChEMBL 6.203999996185303)
Descriptors: MW 411.48, ALogP 4.17, HBA 4, HBD 3, PSA 102.01, QED 0.42
Compound (SMILES): Cn1cc(-c2n[nH]c3cnc(N4C5CCC4C(=O)N(CCF)C5)nc23)cn1
InChIKey: PGRJFPIBHFUXKF-UHFFFAOYSA-N
Target: Fibroblast growth factor receptor 2 (SINGLE PROTEIN), Homo sapiens
UniProt: P21802
Assay: Enzymatic Assay: Phosphorylated (P): The inhibitor potency of the exemplified compounds was determined in an enzyme discontinuous assay that measures peptide phosphorylation using FRET measurements to detect product formation. Inhibitors were serially diluted in DMSO and a volume of 0.2 μL was transferred to the wells of a 384-well plate. For the isoforms of FGFR (−1, −2, −3 wild-type and mutant isoforms, −4) including phosphorylated (P)proteins, a 5 μL/well volume of enzyme 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 with inhibitor for 5 to 15 minutes at ambient temperature. Appropriate controls (enzyme blank and enzyme with no inhibitor) were included on the plate. The reaction was initiated by the addition of a 5 μL/well volume containing both biotinylated EQEDEPEGDYFEWLE (SEQ ID: 1) peptide substrate and ATP in assay buffer. The 10 μL/well reaction concentration of the peptide substrate was 500 nM whereas the ATP concentration was maintained near or below the ATP Km for each FGFR isoform. The ATP Km values were pre-determined for each FGFR isoform in a separate series of experiments. The reaction plate was incubated at 25° C. for 1 hr and the reactions were ended with the addition of 5 μL/well of quench solution (50 mM Tris, 150 mM NaCl, 0.5 mg/mL BSA, pH 7.8; 45 mM EDTA, 600 nM staurosporin, with Perkin Elmer Lance Reagents at 3.75 nM Eu-antibody PY20 and 180 nM APC-Streptavidin). The plate was allowed to equilibrate for −10 minutes at ambient temperature before scanning on a PheraStar plate reader (BMG Labtech) instrument.
Measurement: IC50 = 300 nM (pChEMBL 6.519999980926514)
Descriptors: MW 370.39, ALogP 0.9, HBA 7, HBD 1, PSA 95.83, QED 0.73
Compound (SMILES): CC(C)CN1CCOc2c(F)cc(-c3nc(Nc4ccc(C5CCN(C)CC5)cn4)ncc3F)cc21
InChIKey: LEKYSYXNVDOBCA-UHFFFAOYSA-N
Target: Macrophage colony-stimulating factor 1 receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P07333
Assay: LanthaScreen Eu Kinase Binding Assay: FMS: IC50 values of compounds against FMS kinase were determined by LanthaScreen™ Eu Kinase Binding Assay at Invitrogen (Life Technologies Grand Island, N.Y.) where total reaction volume was 10 μL in low-volume 384-well plates (#4511, Corning). Serially diluted compounds (3-fold) were incubated with kinase (1.25 nM) for 10 min, following which a mixture of ATP (10 μM) (#A1852, Sigma, St-Louis, Mo.) and fluorescent-PolyGT substrate (200 nM) (#PV3610, Invitrogen, Life Technologies Grand Island, N.Y.) was added and incubated in dark at room temperature for 1 h. After 1 h, 10 μL stop solution containing Terbium labeled antibody (4 nM) (#PV3529, Invitrogen, Life Technologies Grand Island, N.Y.) and EDTA (#E5134, Sigma, St-Louis, Mo.) (20 mM) in TR-FRET dilution buffer (#PV3574, Invitrogen, Life Technologies Grand Island, N.Y.) was added. Readings were taken in a Synergy Neo Plate reader (BioTek, Winooski) at single excitation of 340 nm and Dual emission at 495 nm and 520 nm respectively.
Measurement: IC50 = 2597 nM (pChEMBL 5.590000152587891)
Descriptors: MW 494.59, ALogP 5.22, HBA 7, HBD 1, PSA 66.41, QED 0.5
Compound (SMILES): CCN(CCCN1CCN(c2noc3cc(F)ccc23)CC1)S(=O)(=O)C(C)CO
InChIKey: UCUKBRIYXZZGPO-UHFFFAOYSA-N
Target: Voltage-gated inwardly rectifying potassium channel KCNH2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q12809
Assay: Inhibitory Activity for hERG Channel: Extracellular and intracellular fluids used in the measurement were prepared as follows.Extracellular fluid: 2 mmol/L CaCl2, 1 mmol/L MgCl2, 10 mmol/L HEPES, 4 mmol/L KCl, 145 mmol/L NaCl, 10 mmol/L GlucoseIntracellular fluid: 10 mmol/L HEPES, 10 mmol/L EGTA, 20 mmol/L KCl, 130 mmol/L KFTest compound solution: A test compound was dissolved in DMSO to reach the concentration of 2 mmol/L or 20 mmol/L in order to prepare a test compound solution. The test compound solution was further diluted 200 times with the extracellular fluid, and serial-diluted with the extracellular fluid to prepare a test compound solution in each concentration which is necessary to calculate IC50 value of hERG inhibition.
Measurement: IC50 = 7400 nM (pChEMBL 5.130000114440918)
Descriptors: MW 428.53, ALogP 1.51, HBA 7, HBD 1, PSA 90.12, QED 0.65
Compound (SMILES): Cc1cc(N2C(=O)c3ccn(C)c3C2c2ccc(C#N)cc2)cn(C)c1=O
InChIKey: VBWJYWJSBHIBOW-UHFFFAOYSA-N
Target: CREB-binding protein (SINGLE PROTEIN), Homo sapiens
UniProt: Q92793
Assay: Assay for CREB protein: All assays were performed in 384-well microtiter plates. Each assay plate contained 8-point serial dilutions for 40 test compounds, plus 16 high- and 16 low controls. Liquid handling and incubation steps were done on an Innovadyne Nanodrop Express equipped with a robotic arm (Thermo CatX, Perkin Elmer/Caliper Twister II) and an incubator (Liconic STX40, Thermo Cytomat 2C450). The assay plates were prepared by addition of 50 nl per well of compound solution in 90% DMSO HummingBird nanodispenser (Zinsser Analytic). The assay was started by stepwise addition of 4.5 uL per well of bromo domain protein (50 mM HEPES, pH 7.5, 0.005% Tween20, 0.1% BSA, 50 mM NaCl, 45 nM His-Brd2(60-472) or 45 nM His-Brd3(20-477) or 45 nM His-Brd4(44-477) all proteins produced in-house) and 4.5 uL per well of peptide solution (50 mM HEPES, pH 7.5, 0.005% Tween20, 0.1% BSA, 50 mM NaCl, 60 nM acetyl-histone H4 (AcK 5, 8, 12, 16) (Biosyntan GmbH)). Reactions were incubated at 30° C. for 35 minutes. Subsequently 4.5 uL per well detection mix (50 mM HEPES, pH 7.5, 0.005% Tween20, 0.1% BSA, 50 mM NaCl, 3 nM Eu-labeled anti-His6 antibody, 21 nM streptavidin-allophycocyanin) were added. After 35 minutes incubation at 30° C., plates were measured in a Perkin Elmer EnVision multilabel reader.
Measurement: IC50 = 7150 nM (pChEMBL 5.150000095367432)
Descriptors: MW 358.4, ALogP 2.65, HBA 5, HBD 0, PSA 71.03, QED 0.71
Compound (SMILES): N#CC[C@H]1CC[C@H](n2c(COC(=O)Nc3ccccc3)nc3cnc4[nH]ccc4c32)CC1
InChIKey: FXBRFZSKYKGXOD-SAABIXHNSA-N
Target: Non-receptor tyrosine-protein kinase TYK2 (SINGLE PROTEIN), Homo sapiens
UniProt: P29597
Assay: Enzymatic Inhibition Assay: 11-point dosing series were made for each compound by serially diluting 1:3 or 1:4 in DMSO, with point 12 being a DMSO control. From the serial dilution plates, sample was transferred to a 384 wells assay plate (#781280, Greiner, Monroe, N.C.) using Labcyte Echo (Sunnyvale, Calif.), or Biosero ATS (San Diego, Calif.). The compounds were tested in duplicate. Column 12 was used for positive controls, and column 24 contained negative controls with no enzyme added. A compound from our internal collection, with inhibitory activity for JAK isoforms, was used as a reference compound. The final concentration of DMSO was ≤0.25% in a 20 μL reaction. Assay conditions for each of the proteins are summarized in Table 3. The enzyme reaction was initiated by the addition of 10 μL of 2× enzyme and ATP mixture to 10 μL of 2× substrate solution prepared in reaction buffer (50 mM MOPS pH 7.5, 10 mM MgCl2, 1 mM EDTA, 2 mM DTT, 0.002% Tween-20). The Tyk2 enzyme was pre-incubated with 2 mM ATP for 30 min prior to the reaction initiation. Immediately after the addition of the enzyme to the reaction mixture, the plate was centrifuged at 1000 rpm for 1 minute and incubated at 25° C. for 45 minutes for JAK 3 and 90 minutes for JAK1, JAK2 and Tyk2. The reaction was quenched by the addition of 20 μL of 0.5% TFA containing 0.15 μM of internal standard peptide using Multidrop Combi reagent dispenser (Thermo Scientific, Waltham, Mass.). Several wells in column 24 were typically used for the product standard curve. After the quench, the assay plate was centrifuged at 3000 rpm for 3 minutes and sealed with pierceable aluminum foil (Cat #06644-001, Agilent) using a PlateLoc (Agilent Technologies, Santa Clara, Calif.). The plates then were transferred on to the RapidFire for the MS analysis. Compound inhibition was assessed by a decrease of the phosphorylated product levels in sample wells compared to the non-inhibited enzyme reaction. The assay conditions for the above assays are shown in Table 3 and the results of Ex. 1-209 as tested in these assays are shown in Table 4.TABLE 3Assay conditions for JAK family enzyme assays* [p], [ATP], [Substrate], [IS],Enzyme nM μM μM nMJAK1-JH1JH2 8.0 12.5 200 100JAK2-JH1JH2 7.0 or 3.6 30 40 100JAK3-JH1JH2 2.0 150 40 100Tyk2-JH1JH2   25 or 14.7 50 200 100*Reaction buffer: 50 mM MOPS, pH 7.5 10 mM MgCl2, 1 mM EDTA, 2 mM DTT, 0.002% Tween-20; IS stands for internal standard peptide; p stands for phospho-peptide (product); Substrate stands for peptide.
Measurement: IC50 = 11.3 nM (pChEMBL 7.949999809265137)
Descriptors: MW 428.5, ALogP 5.31, HBA 6, HBD 2, PSA 108.62, QED 0.45
Compound (SMILES): O=C(NC1(C(=O)O)CC1)c1cnn2ccc(N3CCC[C@@H]3c3cc(F)ccc3F)nc12
InChIKey: HUMIANOGLZOVBP-MRXNPFEDSA-N
Target: High affinity nerve growth factor receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P04629
Assay: TrkA ELISA Assay: An enzyme-linked immunosorbant assay (ELISA) was used to assess TrkA kinase activity in the presence of inhibitors. Immulon 4HBX 384-well microtiter plates (Thermo part #8755) were coated with a 0.025 mg/mL solution of poly (Glu, Ala, Tyr; 6:3:1; Sigma P3899). Various concentrations of test compound, 2.5 nM TrkA (Invitrogen Corp., histidine-tagged recombinant human TrkA, cytoplasmic domain), and 500 μM ATP were incubated for 25 minutes at ambient temperature in the coated plates while shaking. The assay buffer consisted of 25 mM MOPS pH 7.5, 0.005% (v/v) Triton X-100 and 5 mM MgCl2. The reaction mixture was removed from the plate by washing with PBS containing 0.1% (v/v) Tween 20. The phosphorylated reaction product was detected using 0.2 μg/mL of a phosphotyrosine specific monoclonal antibody (clone PY20) conjugated to horseradish peroxidase in conjunction with the TMB Peroxidase Substrate System (KPL). After the addition of 1M phosphoric acid, the chromogenic substrate color intensity was quantitated via absorbance at 450 nm. IC50 values were calculated using either a 4 or 5-parameter logistic curve fit.
Measurement: IC50 > 1000 nM (pChEMBL 6)
Descriptors: MW 427.41, ALogP 2.7, HBA 6, HBD 2, PSA 99.83, QED 0.65
Compound (SMILES): C=CC(=O)N1CCC[C@H]1c1nc(-c2ccc(C(=O)Nc3cc(C#N)ccn3)cc2)c2c(N)nccn12
InChIKey: QXUSAIWDESXIER-IBGZPJMESA-N
Target: Tyrosine-protein kinase Fyn (SINGLE PROTEIN), Homo sapiens
UniProt: P06241
Assay: FynT Enzyme Activity : FynT enzyme activity is measured using the IMAP (immobilized metal ion affinity-based fluorescence polarization) assay as outlined below.FynT enzyme (Biomol catalog # SE-287), is diluted to 0.5 μg/mL in KR buffer (10 mM Tris-HCl, 10 mM MgCl2, 0.01% Tween-20, 0.05% NaN3, 1 mM DTT, 2 mM MnCl2, pH 7.2).Serial dilution log 10 from 2 mM to 63.2 nM of test compounds are made in 100% DMSO. The dilutions in DMSO are then diluted 50-fold in KR-buffer of which 5 μl is used in the assay, leading to a final compound concentration range in the assay from 10 μM to 0.316 nM.5 μL/well of test compound in KR buffer (final DMSO concentration in the assay is 1%) is mixed with 5 μl/well of 0.5 μg/mL FynT enzyme (final concentration in the assay is 125 ng/mL). Test compounds and FynT enzyme are pre-incubated 60 minutes at room temperature, before adding 5 μL/well of 400 nM Fluorescin labeled substrate peptide (p34cdc2 substrate peptide, e.g. #R7157/#R7172, Molecular Devices) in KR-buffer. Final peptide substrate concentration in assay is 100 nM. The kinase assay is started by adding 5 μL/well of 0.8 μM ATP in KR-buffer (final ATP concentration is 0.2 μM ATP, Km ATP in FynT IMAP assay). Following incubation for 2 h at room temperature the enzyme reaction is stopped by adding 40 μL/well IMAP Progressive Binding Solution (according to suppliers (Molecular Devices) protocol using 75% 1× buffer A and 25% 1× buffer B with 1:600 Progressive Binding Solution). After 60 min incubation at room temperature in the dark the FP signal is read. Fluorescence at 535 nm is measured using parallel and perpendicular filters to determine differences in rotation due to binding of the phosphorylated substrate peptide to the beads. Values are calculated as percentage of the difference in readout (ΔmPi) of the controls with and without ATP. EC50 values are determined by curve fitting of the experimental results using Activity Base.
Measurement: EC50 > 1000 nM (pChEMBL 6)
Descriptors: MW 478.52, ALogP 3.35, HBA 8, HBD 2, PSA 142.3, QED 0.42
Compound (SMILES): C[C@H](NC(=O)[C@H]1CCN(c2ncnc3[nH]ccc23)CC12CC2)[C@@H](O)c1ccc(C#N)cc1
InChIKey: HMXBTXMYJLIBLR-CWFSZBLJSA-N
Target: Non-receptor tyrosine-protein kinase TYK2 (SINGLE PROTEIN), Homo sapiens
UniProt: P29597
Assay: JAK Kinase Assays: Human baculovirus-expressed JAK1, 2, 3 and TYK2 were purchased from Carna Biosciences, Inc. All four purified enzymes contain only the catalytic domain. JAK1 (aa 850-1154) and TYK2 (aa 871-1187) are expressed with an N-terminally fused GST-tag, and JAK2 and JAK3 with an N-terminally fused His-tag.Inhibition of phosphorylation of a synthetic peptide was measured in an HTRF-based assay using the TK substrate-Biotin from the Cisbio HTRFKinEASE TK kit. First, 2 μl of TK solution (TK substrate-biotin in kinase buffer [1× enzymatic buffer from HTRFKinEASE TK kit, 1 mM DTT]) is added to a plate containing 1 μl prediluted compound (final assay concentration DMSO: 0.75%). Then, 5 μl kinase-ATP mix (prepared in kinase buffer) is added to the wells and the plates are incubated at RT for 20-30 min. For all four kinases a concentration of ATP that corresponded to the Km for ATP was used. The final concentrations of buffers, substrate, kinase and ATP were: JAK1: 50 mM Hepes buffer pH 7.0, 0.01% BSA, 10 mM MgCl2, 1 mM DTT, 7 μM ATP, 50 nM SEB, 1 μM TK Substrate-Biotin and 5 ng JAK1; JAK2: 50 mM Hepes buffer pH 7.0, 0.01% BSA, 5 mM MgCl2, 1 mM DTT, 4 μM ATP, 1 μM TK Substrate-Biotin and 0.1 ng JAK2; JAK3: 50 mM Hepes buffer pH 7.0, 0.01% BSA, 5 mM MgCl2, 1 mM DTT, 2 μM ATP, 1 μM TK Substrate-Biotin and 0.3 ng JAK3; TYK2: 50 mM Hepes buffer pH 7.0, 0.01% BSA, 5 mM MgCl2, 1 mM DTT, 13 μM ATP, 50 nM SEB, 1 μM TK Substrate-Biotin and 0.8 ng TYK2. Thereafter, the kinase reaction is stopped by adding 4 μl detection mix (final concentrations: 50 mM Hepes buffer pH 7.0, 0.01% BSA, 0.8 M KF, 20 mM EDTA, 42 nM Streptavidin-XL665 and 1:400 STK Ab Cryptate) and the plates are incubated overnight in the dark. The HTRF signal is read using an Envision plate reader.
Measurement: EC50 = 131 nM (pChEMBL 6.880000114440918)
Descriptors: MW 430.51, ALogP 2.67, HBA 6, HBD 3, PSA 117.93, QED 0.57
Compound (SMILES): Cc1ccc(-c2cc3ncccc3c(-c3ccn(C4(CC#N)CN(S(C)(=O)=O)C4)n3)n2)c(N)n1
InChIKey: AQOQJHILJVKXRA-UHFFFAOYSA-N
Target: Tyrosine-protein kinase JAK3 (SINGLE PROTEIN), Homo sapiens
UniProt: P52333
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 > 10000 nM (pChEMBL 5)
Descriptors: MW 474.55, ALogP 2.33, HBA 9, HBD 1, PSA 143.68, QED 0.46
Compound (SMILES): CC[C@H](Nc1ccc2c(c1)OCCn1cc(N3C(=O)OC[C@H]3C(F)F)nc1-2)C(N)=O
InChIKey: ZWEJBXXYQYTACR-STQMWFEESA-N
Target: Phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit alpha isoform (SINGLE PROTEIN), Homo sapiens
UniProt: P42336
Assay: Biochemical Inhibition Assay: The biochemical inhibition of four PI3K isoforms by the Formula I compounds of Table 1. In addition, two clinically tested PI3K compounds, taselisib and pictilisib are included as comparators. The representative compounds of the invention exhibit strong activity against PI3Kα, and exhibit significantly enhanced selectivity relative to the other isoforms PI3Kβ, PI3Kδ, and PI3Kγ when compared to taselisib (GDC-0032) and pictilisib (GDC-0941). In particular, the selectivity ratios in the second from the right column of Table 2A show that each Formula I compounds 101-107 has a PI3K alpha to delta selectivity ratio far higher than taselisib or pictilisib. In fact, both taselisib and pictilisib have stronger activity against PI3K delta than against PI3K alpha, i.e. their selectivity ratios are less than 1. The selectivity ratios of Formula I compound 101-107 range from 301-fold to 634-fold.
Measurement: Ki = 0.048 nM (pChEMBL 10.319999694824219)
Descriptors: MW 421.4, ALogP 2.21, HBA 7, HBD 2, PSA 111.71, QED 0.74