lovingscience/chembl-v37-raw-activities huggingface dataset:
Compound (SMILES): CN(C1COC2(CCN(C(=O)ON3C(=O)CCC3=O)CC2)C1)S(=O)(=O)Cc1ccccc1
InChIKey: GTTDKAZTTJGHIJ-UHFFFAOYSA-N
Target: Monoglyceride lipase (SINGLE PROTEIN), Homo sapiens
UniProt: Q99685
Assay: MAGL Enzymatic Assay (0 minute): Assessment of MAGL inhibition utilizes human recombinant Monoacylglycerol Lipase and the fluorogenic substrate 7-hydroxycoumarinyl arachidonate (7-HCA, Biomol ST-502). 400 nL of a test compound at decreasing concentration (ranging from 150 μM down to 1.5 nM) was spotted into a 384-well back plate (PerkinElmer, 6007279) using a Labcyte Echo, followed by addition of 10 μL of MAGL enzyme in assay buffer (50 mM HEPES, pH 7.4, 100 mM NaCl, 5 mM MgCl2, 0.1% Triton X-100 and 25% glycerin). An equal volume of 7-HCA in assay buffer with 10% DMSO was added either immediately (T=0 min) or after a 30 minute incubation (T=30 min) to initiate the reaction. The final concentration of MAGL enzyme was 88 μM and 7-HCA substrate was 5 μM. After these dilutions, the final concentration of the test compound ranged from 3 μM to 0.03 nM. The reaction was allowed to progress for 60 minutes, after which the plate was read at an Ex/Em of 340/465. Percent inhibitions were calculated based on control wells containing no compound (0% inhibition) and a control compound (e.g., a MAGL inhibitor whose activity is known or was previously reported in the literature, such as one with about 100% inhibition). IC50 values were generated based on a four parameter fit model using ABASE software from IDBS. See e.g., Wang, Y. et al., A Fluorescence-Based Assay for Monoacylglycerol Lipase Compatible with Inhibitor Screening, Assay and Drug Development Technologies, 2008, Vol. 6 (3) pp 387-393 (reporting an assay for measuring MAGL activity).To measure MAGL inactivation, the same protocol for the (T=0 min) MAGL inhibition IC50 assay was performed with data collected every minute to acquire enzyme progress curves at decreasing concentrations of compound. Kobs values were calculated from this data and kinact/Kl ratios were determined from a plot of Kobs values vs. compound concentrations.
Measurement: IC50 = 192 nM (pChEMBL 6.71999979019165)
Descriptors: MW 465.53, ALogP 1.27, HBA 7, HBD 0, PSA 113.53, QED 0.6
Compound (SMILES): O=C(NCc1cc(F)cc(Cl)c1)[C@]1(F)CC[C@H](O)c2ncccc21
InChIKey: NSNRRXKVRXXPNA-YOEHRIQHSA-N
Target: P2X purinoceptor 7 (SINGLE PROTEIN), Homo sapiens
UniProt: Q99572
Assay: Pharmacological Assay: 1. Wash each well with 80 microL of assay buffer (20 mM HEPES, 1×HBSS, pH 7.4 adjusted with NaOH) three times and leave 20 microL using plate washer, ELx-405 Select CW (BIO-TEK).2. Add 20 microL of assay buffer containing 2.5 mM probenecid, 0.5 microM Fluo-4-AM (Molecular Probes) and 0.1% Pluronic F-127 to each well.3. Incubate the plate at 37° C. in 5% CO2 for 1 h.4. Wash each well with 80 microL of assay buffer (see below) three times and leave 20 microL using plate washer, ELx-405 Select CW (BIO-TEK).5. Test compounds were prepared at 100× the test concentration in DMSO by serial dilution with Biomek-FX liquid handling instrument. 33× diluted compound solutions in assay buffer were prepared in intermediate compound plate with Biomek-NX liquid handling instrument. A further 3× dilution occurred in below steps 6 and 7.6. Add 20 microL of 33× diluted compound solutions into each well and leave the plate for 10 min under the dark at room temperature.7. Measure activity by FDSS as follows:Set the assay plate on the stacker of FDSS.Start the detection of fluorescence intensity at 540 nm by 480 nm exicitation.After 30 seconds, add 20 microL of assay buffer containing 240 microM BzATP (final concentration 80 microM).
Measurement: IC50 = 75 nM (pChEMBL 7.119999885559082)
Descriptors: MW 352.77, ALogP 3.18, HBA 3, HBD 2, PSA 62.22, QED 0.89
Compound (SMILES): CC1(C)CNCc2cc(Nc3ncc4c(n3)OCN(c3c(Cl)cccc3Cl)C4=O)ccc21
InChIKey: OSOBUUOUCAFBBY-UHFFFAOYSA-N
Target: Wee1-like protein kinase (SINGLE PROTEIN), Homo sapiens
UniProt: P30291
Assay: WEE1 IC50 Determination: IC50 values of compounds against WEE1 kinase enzyme were determined by LanthaScreen Terbium Labeled TR-FRET assay. Kinase assays were performed in 1× kinase buffer (# PV6135, 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 WEE1 Enzyme (1 nM) (# PR7373A, Invitrogen, Life Technologies Grand Island, N.Y.) for 10 min, following which a mixture of ATP (10 M) (# 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 = 11 nM (pChEMBL 7.960000038146973)
Descriptors: MW 470.36, ALogP 4.9, HBA 6, HBD 2, PSA 79.38, QED 0.57
Compound (SMILES): CN[C@H]1CC[C@@H](S(=O)(=O)N2CC[C@@H](NC(=O)c3cc(C4CC4)on3)[C@@H](C)C2)CC1
InChIKey: CLYANLXDANAIKQ-QMCVQRASSA-N
Target: Histone-lysine N-methyltransferase SMYD3 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9H7B4
Assay: SMYD3 Biochemical Assay: The assays were all performed in a buffer consisting of 25 mM Tris-Cl pH 8.0, 1 mM TCEP, 0.005% BSG, and 0.005% Tween 20, prepared on the day of use. Compounds in 100% DMSO (1 ul) were spotted into a 384-well white opaque OptiPlate using a Bravo automated liquid handling platform outfitted with a 384-channel head (Agilent Technologies). DMSO (1 ul) was added to Columns 11, 12, 23, 24, rows A-H for the maximum signal control and 1 ul of SAH, a known product and inhibitor of SMYD3, was added to columns 11, 12, 23, 24, rows I-P for the minimum signal control. A cocktail (40 ul) containing the SMYD3 enzyme was added by Multidrop Combi (Thermo-Fisher). The compounds were allowed to incubate with SMYD3 for 30 min at room temperature, then a cocktail (10 ul) containing SAM and MEKK2 was added to initiate the reaction (final volume=51 ul). The final concentrations of the components were as follows: SMYD3 was 0.4 nM, 3H-SAM was 8 nM, MEKK2 was 12 nM, SAH in the minimum signal control wells was 1 mM, and the DMSO concentration was 2%. The assays were stopped by the addition of non-radiolabeled SAM (10 ul) to a final concentration of 100 uM, which dilutes the 3H-SAM to a level where its incorporation into MEKK2 is no longer detectable. Radiolabeled MEKK2 was detected using a scintillation proximity assay (SPA). 10 uL of a 10 mg/mL solution of SPA beads in 0.5 M citric acid was added and the plates centrifuged at 600 rpm for 1 min to precipitate the radiolabeled MEKK2 onto the SPA beads. The plates were then read in a PerkinElmer TopCount plate reader to measure the quantity of 3H-labeled MEKK2 as disintegrations per minute (dpm) or alternatively, referred to as counts per minute (cpm).% Inhibition Calculation% ⁢ ⁢ inh = 100 - ( dpm cmpd - dpm min dpm max - dpm min ) × 100Where dpm=disintegrations per minute, cmpd=signal in assay well, and min and max are the respective minimum and maximum signal controls.Four-Parameter IC50 FitY = Bottom + ( Top - Bottom ) ( 1 + ( X IC 50 ) Hill ⁢ ⁢ CoefficientWhere top and bottom are the normally allowed to float, but may be fixed at 100 or 0 respectively in a 3-parameter fit. The Hill Coefficient normally allowed to float but may also be fixed at 1 in a 3-parameter fit. Y is the % inhibition and X is the compound concentration.
Measurement: IC50 = 14.6 nM (pChEMBL 7.840000152587891)
Descriptors: MW 424.57, ALogP 1.85, HBA 6, HBD 2, PSA 104.54, QED 0.72
Compound (SMILES): Cc1nc2ccc(-c3ccnc(N(CC45CCC(c6cc(C7CC7)nn6C)(CC4)CC5)C(=O)C45CC(F)(C4)C5)c3)cc2s1
InChIKey: YQOKYBVPOKOCRH-UHFFFAOYSA-N
Target: Bile acid receptor (SINGLE PROTEIN), Homo sapiens
UniProt: Q96RI1
Assay: transient human FXR/Gal4-luciferase reporter assay: A Gal4-hFXR fusion construct reporter system was used as the primary assay to characterize compound activity. A construct including 5 copies of the Gal4 promoter response element upstream of a firefly luciferase reporter cDNA was stably expressed in HEK293 cells. This reporter cell line was maintained in Dulbecco's Modified Eagle's medium (DMEM; Gibco) supplemented with 1% penicillin-streptomycin (P/S) solution, 500 μg/mL Zeocin and 10% charcoal/dextran-treated fetal bovine serum (cs-FBS) at 37° C. in a humidified 5% CO2 atmosphere. Another plasmid was constructed in which the human cytomegalovirus promoter in the pcDNA3.1 vector directs the expression of the cDNA encoding a fusion protein comprised of the DNA binding domain from the Gal4 transcription factor fused to the ligand binding domain from human FXR.The day prior to transfection, the reporter cells in culture are detached from the plate with trypsin and plated into a T75 flask at a sufficient density to achieve approximately 90% confluence the next morning. The transfection reagents are prepared by separately diluting 25 μg of the pcDNA3.1-Gal4-FXR plasmid into 1.87 mL of Opti-MEM (Thermo-Fisher), and 40 μL of Lipofectamine 2000 (Thermo-Fisher) into 1.87 mL of Opti-MEM, and then adding the diluted DNA solution into the diluted Lipofectamine 2000 solution and incubating at room temperature for 15-20 minutes. The mixture is further diluted with 10 mL of a solution comprised of DMEM, 10% cs-FBS, and 1% P/S immediately prior to transferring to the cells. The maintenance culture media is aspirated from the cells and the final transfection mixture is added before the cells are incubated overnight at 37° C. in a humidified 5% CO2 atmosphere. This protocol can be scaled up, and the transiently transfected cells can be cryopreserved in an assay-ready format.For compound testing, 100 nL of the compounds (serial dilutions in DMSO) are dispensed with an Echo acoustic dispenser (Labcyte) into the wells of a Corning/Costar clear bottom 384-well white plate. The transfected cells are harvested, counted, and diluted such that 10-25,000 cells in 25 μL are plated into each well of the 384-well compound assay plate. The compound-treated cells are incubated overnight at 37° C. in a humidified 5% CO2 atmosphere. The next morning 25 μL of Steady-Glo (Promega) are added to each well of the plate, the mixture is incubated for 15 min. with shaking, and luminescence is measured on an Envision (Perkin Elmer) plate reader. Background counts from cells treated with DMSO alone are subtracted from all raw counts, and the corrected values are converted to a percentage of the control response attained with 8 μM GW-4064. These data are fit to a 4-parameter log agonist-response equation to calculate an EC50 value.
Measurement: EC50 = 48 nM (pChEMBL 7.320000171661377)
Descriptors: MW 595.79, ALogP 7.8, HBA 6, HBD 0, PSA 63.91, QED 0.22
Compound (SMILES): COCCN(C)C(=O)c1ccc(-c2cnc(N)c(-c3nnc(-c4ccccc4)o3)n2)cc1
InChIKey: JFASWDFQIWAYJL-UHFFFAOYSA-N
Target: Serine/threonine-protein kinase ATR (SINGLE PROTEIN), Homo sapiens
UniProt: Q13535
Assay: Inhibition Assay: Compounds were screened for their ability to inhibit ATR kinase using a radioactive-phosphate incorporation assay. Assays were carried out in a mixture of 50 mM Tris/HCl (pH 7.5), 10 mM MgCl2 and 1 mM DTT. Final substrate concentrations were 10 M [γ-33P]ATP (3mCi 33P ATP/mmol ATP, Perkin Elmer) and 800 μM target peptide (ASELPASQPQPFSAKKK).Assays were carried out at 25° C. in the presence of 5 nM full-length ATR. An assay stock buffer solution was prepared containing all of the reagents listed above, with the exception of ATP and the test compound of interest. 13.5 μL of the stock solution was placed in a 96 well plate followed by addition of 2 μL of DMSO stock containing serial dilutions of the test compound (typically starting from a final concentration of 15 μM with 3-fold serial dilutions) in duplicate (final DMSO concentration 7%). The plate was pre-incubated for 10 minutes at 25° C. and the reaction initiated by addition of 15 μL [γ-33P]ATP (final concentration 10 μM).The reaction was stopped after 24 hours by the addition of 30 μL 0.1M phosphoric acid containing 2 mM ATP. A multiscreen phosphocellulose filter 96-well plate (Millipore, Cat no. MAPHN0B50) was pretreated with 100 μL 0.2M phosphoric acid prior to the addition of 45 μL of the stopped assay mixture. The plate was washed with 5×200 μL 0.2M phosphoric acid. After drying, 100 μL Optiphase SuperMix liquid scintillation cocktail (Perkin Elmer) was added to the well prior to scintillation counting (1450 Microbeta Liquid Scintillation Counter, Wallac).
Measurement: Ki < 10 nM (pChEMBL 8)
Descriptors: MW 430.47, ALogP 3.16, HBA 8, HBD 1, PSA 120.26, QED 0.47
Compound (SMILES): CCOC(=O)Cc1ccc(CC(=O)Nc2ccc3c(c2)nc(C(F)(F)F)n3Cc2cccc3c2OC(F)(F)O3)cc1
InChIKey: JAFWCLYNXWKTTD-UHFFFAOYSA-N
Target: Nuclear receptor ROR-gamma (SINGLE PROTEIN), Homo sapiens
UniProt: P51449
Assay: Binding Assay : Compounds described herein were tested for ability to bind to RORγ in a cell-free competition assay with commercially available radio-ligand (RL), 25-hydroxy [26,27-3H]-cholesterol (PerkinElmer, Cat. #NET674250UC), for a ligand binding site on a recombinant RORγ Ligand Binding Domain (LBD) protein expressed as a 6×His-Glutathione-S-Transferase (GST) fusion. The assay was performed in 96-well SPA plates (PerkinElmer, Cat. #1450-401) in 50 mM HEPES buffer, pH 7.4, containing 150 mM NaCl, 5 mM MgCl2, 10% (v/v) glycerol, 2 mM CHAPS, 0.5 mM β-octylglucopyranoside and 5 mM DTT. Tested compounds were dissolved in DMSO, and semi-log (3.162×) serial dilutions of the compounds were prepared in the same solvent. Two μL of the DMSO solutions were mixed with 28 μL of 8.6 nM 25-hydroxy [26,27-3H]-cholesterol and 50 μL of 24 nM RORγ LBD. The plate was shaken at 700 rpm for 20 min and incubated for 10 min at rt, after which 40 μL of poly-Lys YSi SPA beads (PerkinElmer, Cat. #RPNQ0010) were added to achieve 50 ag of the beads per well. The plate was incubated on an orbital shaker for 20 min and then for 10 min without agitation at rt. SPA signal for tritium beta radiation was registered on PerkinElmer Microbeta plate reader. Percent inhibition values were calculated based on the high signal obtained with DMSO control and the low signal observed with 10 μM standard RORγ inverse agonist T0901317 (SigmaAldrich, Cat. #T2320). The percent inhibition vs. concentration data were fit into a four-parameter model, and IC50 values were calculated from the fit as the concentrations corresponding to the inflection points on the dose-response curves.
Measurement: Ki = 550 nM (pChEMBL 6.260000228881836)
Descriptors: MW 575.49, ALogP 5.71, HBA 7, HBD 1, PSA 91.68, QED 0.21
Compound (SMILES): CONC(=O)c1ccccc1NC(C)c1cc(C)cc2c(=O)cc(N3CCC(C)(C)CC3)oc12
InChIKey: HPMBVISMWXQGJV-UHFFFAOYSA-N
Target: Phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit alpha isoform (SINGLE PROTEIN), Homo sapiens
UniProt: P42336
Assay: PI3K-Alpha kinase (PIK3CA) activity, wild-type and H1047R mutant and determining IC50 values for inhibitors: Recombinant, catalytically active human full length PIK3KA Wild-type and H1047R mutant were purchased as 1:1 complex of N-terminal 6×his tagged p110<(catalytic) and untagged p85<(regulatory subunit) from EMD Millipore Sigma (cat. no. 14-602M and 14-792M, respectively). The enzyme stocks were diluted to 5× stocks in buffer (20 mM HEPES pH 7.4, 100 mM NaCl, 0.5 mM EGTA, 0.01% triton-x-100) just before use. PIP2diC8 (Avanti Polar Lipids Inc., cat. no. 850185) or phosphoinositol-4,5-bisphosphate with phosphoserine (PIP2:PS) membrane (Thermo Fisher Scientific, cat. no. PV5100) was used as lipid substrates. PIP2diC8 lyophilized powder and PIP2:PS (1:19) membrane stock (1 mM in PIP2) were separately dissolved in milliQ water to a concentration of 250 uM and stored in −20° C. 10 mM stocks of compounds were serially diluted (3×) in neat DMSO and stored in a dessicator at room temperature. 5× compound stocks in 25% DMSO were prepared fresh from neat DMSO stocks. Wild-type (WT) and H1047R mutant protein, along with buffer components (except ATP), were incubated with or without compound at 27° C. for 1h. After incubation, the reaction was initiated by the addition of 5 uL of 125 uM ATP. A typical assay mixture (25 uL) comprised 40 mM HEPES buffer, pH 7.4, 25 mM MgCl2, 0.01% v/v triton-X-100, 5% v/v DMSO, 20 mM NaCl, 1-5 nM wt or H1047R, 25 uM ATP, and 50 uM PTP2diC8 or PTP2 in membrane. The reaction was allowed to proceed until about 1000 conversion (2.5 uM ADP) after which time, 10 uL of reaction mixture was quenched with 25 uL of transcreener reagent (Transcreener ADP2 FI assay kit, BellBrook labs, Cat. No. 3013). The contents were incubated at rt for 1h and fluorescence was measured using a plate reader (Paradigm, Molecular Devices). The same assay was also run at pH 6.0 or 6.4 using MOPS buffer (Fisher BioReagents, CAS 1132-61-2). A calibration curve was generated under identical buffer conditions with varying ADP amounts. Using that, the observed fluorescence was converted to uM ADP.
Measurement: IC50 = 750 nM (pChEMBL 6.119999885559082)
Descriptors: MW 463.58, ALogP 5.19, HBA 6, HBD 2, PSA 83.81, QED 0.49
Compound (SMILES): C[C@H]1CC[C@H](c2ccccc2)S(=O)(=O)N1Cc1cc(F)c(C(CO)CCC(N)=O)cc1F
InChIKey: YKVZSSIEFHOJON-SSMNUEENSA-N
Target: Nuclear receptor ROR-gamma (SINGLE PROTEIN), Homo sapiens
UniProt: P51449
Assay: Binding Assay: Assays were carried out in 16-microL reaction volumes in black 384 Plus F Proxiplates (Perkin-Elmer 6008269). All assay components except test ligand were mixed in coregulator buffer D (Invitrogen PV4420) containing 5 mM DTT and added to the plate at twice their final concentrations in a volume of 8 microL. Test ligands at 2× the final concentration were then added to the wells in 8 □L of coregulator buffer D containing 5 mM DTT and 4% DMSO. Final incubations contained 1× coregulator buffer D, 5 mM DTT, test ligand, 2% DMSO, 50 nM biotinyl-CPSSHSSLTERKHKILHRLLQEGSPS (American Peptide Company; Vista, Calif.), 2 nM Europium anti-GST (Cisbio 61GSTKLB), 12.5 nM streptavidin-D2 (Cisbio 610SADAB), 50 mM KF, and 10 nM of bacterially-expressed human RORc ligand binding domain protein containing an N-terminal 6×His-GST-tag and residues 262-507 of Accession NP_005051. Ten test ligand concentrations were tested in duplicate. After the reaction plates were incubated for 3 h in the dark at room temperature (22-23° C.), the plate was read on an EnVision plate reader (PerkinElmer) following the Europium/D2 HTRF protocol (ex 320, em 615 and 665, 100 Os lag time, 100 flashes, 500 μs window). The time-resolved FRET signal at 665 nm was divided by that at 615 nm to generate the signal ratio of each well.
Measurement: IC50 = 84 nM (pChEMBL 7.079999923706055)
Descriptors: MW 466.55, ALogP 3.36, HBA 4, HBD 2, PSA 100.7, QED 0.62
Compound (SMILES): COc1cc(C)cc(C)c1C(O)c1nc2cc(C#N)ccc2[nH]1
InChIKey: ULGNGBHYQFNATA-UHFFFAOYSA-N
Target: Complement factor B (SINGLE PROTEIN), Homo sapiens
UniProt: P00751
Assay: ELISA Assay: CVF-Bb complex prepared from purified cobra venom factor (1 μM), recombinant human complement factor B (expressed in drosophila cells and purified using standard methods) and human complement factor D (expressed in E. Coli, refolded and purified using standard methods). CVF-Bb complex at 3 nM concentration was incubated with test compound at various concentrations for 1 hour at room temperature in PBS pH 7.4 containing 10 mM MgCl2 and 0.05% (w/v) CHAPS. Human complement C3 substrate purified from plasma was added to a final concentration of 1 μM. After 1 hour incubation at room temperature, the enzyme reaction was stopped by addition of a cocktail of concentrated pan-protease inhibitors. The product of the reaction, C3a, was quantified by means of an enzyme-linked-immunosorbent assay. IC50 values were calculated from percentage of inhibition of CVF-Bb activity as a function of test compound concentration.
Measurement: IC50 = 5750 nM (pChEMBL 5.239999771118164)
Descriptors: MW 307.35, ALogP 3.14, HBA 4, HBD 2, PSA 81.93, QED 0.78
Compound (SMILES): NC(=O)c1nn(CC(=O)N2[C@@H]3C[C@@H]3C[C@H]2C(=O)N[C@H]2CCCC3(CC3)C2)c2cnccc12
InChIKey: QGGPWWBKUIIOTO-WTTBNOFXSA-N
Target: Complement factor D (SINGLE PROTEIN), Homo sapiens
UniProt: P00746
Assay: Human Complement Factor D Assay: Method 1: Recombinant human factor D (expressed in E. coli and purified using standard methods) at 10 nM concentration is incubated with test compound at various concentrations for 1 hour at room temperature in 0.1 M Hepes buffer, pH 7.5, containing 1 mM MgCl2, 1 M NaCl and 0.05% CHAPS. A synthetic substrate Z-Lys-thiobenzyl and 2,4-dinitrobenzenesulfonyl-fluoresceine are added to final concentrations of 200 μM and 25 μM, respectively. The increase in fluorescence is recorded at excitation of 485 nm and emission at 535 nm in a microplate spectrofluorimeter. IC50 values are calculated from percentage of inhibition of complement factor D-activity as a function of test compound concentration.
Measurement: IC50 = 156 nM (pChEMBL 6.809999942779541)
Descriptors: MW 436.52, ALogP 1.36, HBA 6, HBD 2, PSA 123.21, QED 0.73
Compound (SMILES): O=C1NCCCOc2ncc(F)cc2[C@H]2COC(=O)N2c2ccn3ncc1c3n2
InChIKey: HODOGORARXFPKU-CYBMUJFWSA-N
Target: High affinity nerve growth factor receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P04629
Assay: TrkA Elisa Enzyme 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 = 55 nM (pChEMBL 7.260000228881836)
Descriptors: MW 398.35, ALogP 1.47, HBA 8, HBD 1, PSA 110.95, QED 0.61
Compound (SMILES): CN(CCO)C(=O)[C@@H]1CN(C2CCCCC2)CC[C@H]1NC(=O)c1cc(-c2ccc(F)cc2F)on1
InChIKey: BPMPVIYPBYIYQA-TZIWHRDSSA-N
Target: Atypical chemokine receptor 3 (SINGLE PROTEIN), Homo sapiens
UniProt: P25106
Assay: In Vitro Assay: Tango CXCR7-bla U2OS cells are detached from culture dishes with 0.05% trypsin-EDTA and collected in growing medium (McCoy's 5A 90% (v/v), dialyzed FCS 10% (v/v), 0.1 mM NEAA, 25 mM HEPES (pH7.3), 1 mM sodium pyruvate, P/S 1% (v/v) 50 μg/ml Hygromycin, 100 μg/ml Geneticin, 200 μg/ml Zeocin), spinned down and resuspended in assay medium (McCoy's 5A 90% (v/v), dialyzed FCS 1% (v/v), 0.1 mM NEAA, 25 mM HEPES (pH7.3), P/S 1% (v/v)). 10′000 cells per well (in 30 μl) are seeded in a 384 well plate (black-walled, clear bottom). The plate is incubated at 37° C./5% CO2 for 24 hours. Test compounds are dissolved to 10 mM in DMSO and serially diluted in DMSO to 500× of the final concentration for dose response curves. Compounds are then diluted 1:100 in assay medium to 5× of the final concentration. 10 μl/well of diluted compounds are added to the assay plate and incubated for 15 minutes at 37° C. Thereafter CXCL12/SDF1-α is diluted in assay medium to 5× of the final concentration (its EC80 value for receptor activation) and 10 μl/well are added to the assay plate. The agonist leads to activation of the receptor and therefore to b-arrestin recruitment. Compounds acting as antagonists reduce this activation. The plate is incubated for 22 hrs at 37° C. 10 μl/well of detection reagent (LiveBLAzer-FRET BIG (CCF4-AM) substrate) is transferred to the assay plate and the plate is incubated for 2 hours at room temperature protected from light. Fluorescent counts are determined (Scan1: Ex 409/20 nm, Em 460/30 nm, Scan 2: Ex 409/20 nm, Em 530/30 nm).
Measurement: IC50 = 198 nM (pChEMBL 6.699999809265137)
Descriptors: MW 490.55, ALogP 2.82, HBA 6, HBD 2, PSA 98.91, QED 0.62
Compound (SMILES): CC(C)N1CCC[C@H]1COC(=O)Nc1ccccc1-c1ccccc1
InChIKey: ADDLJMRWTOPDCH-SFHVURJKSA-N
Target: Muscarinic acetylcholine receptor M3 (SINGLE PROTEIN), Homo sapiens
UniProt: P20309
Assay: Binding Assay: Cell membrane proteins (Perkin Elmer) wherein human muscarinic M3 receptor was overexpressed, [3H]-methyl scopolamine and test compounds in various concentration were cultured in 0.2 ml of Tris-HCl buffer at 25° C. for 120 minutes. The same was filtered under suction through glass fiber filter (Whatman GF/B), and then the filter was washed 5 times with 1 ml of Tris-HCl buffer. The radioactivity of [3H]-methyl scopolamine adsorbed on the filter was measured by a liquid scintillation counter. Non-specific binding was evaluated under existence of 5 μM of atropine. Affinity of the compound of the present invention to muscarinic M3 receptor was calculated as the dissociation constant (Ki), which can be calculated from concentration (IC50) of test compounds inhibiting 50% of binding of [3H]-methyl scopolamine (i.e. labeled ligand) according to Cheng and Prusoff [Cheng and Prusoff, Biochem. Pharmacol., 22, 3099, 1973]. In following Table, compounds having stronger binding affinity to human muscarinic M3 receptor have lower dissociation constant (Ki).
Measurement: Ki = 3.77 nM (pChEMBL 8.420000076293945)
Descriptors: MW 338.45, ALogP 4.77, HBA 3, HBD 1, PSA 41.57, QED 0.85
Compound (SMILES): CC(C)c1ccccc1-c1ncc2c(n1)N(Cc1ccc(C(=O)CCc3nccn3C)cc1)C(=O)C2
InChIKey: SNTAAMBFSNUVJI-UHFFFAOYSA-N
Target: Ubiquitin carboxyl-terminal hydrolase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: O94782
Assay: HTS Assay: The HTS assay was performed in a final volume of 20 μL in assay buffer containing 20 mM Tris-HCl (pH 8.0, (1M Tris-HCl, pH 8.0 solution; Corning 46-031-CM)), 2 mM CaCl2) (1M Calcium Chloride solution; Sigma #21114) 1 mM GSH (L-Glutathione reduced; Sigma #G4251), 0.01% Prionex (0.22 μM filtered, Sigma #G-0411), and 0.01% Triton X-100. Stock compound solutions were stored at −20° C. as 10 mM in DMSO. Up to 1 month prior to the assay, 2 mM test compounds were pre-dispensed into assay plates (Black, low volume; Corning #3820) and frozen at −20° C. Prestamped assay plates were allowed to come to room temperature on the day of the assay. For the screen, 100 nL of 2 mM was pre-dispensed for a final screening concentration of 10 μM (DMSO(fc)=0.5%). The final concentration of the enzyme (USP1, construct USP1 (I-785, GG670, 671AA)/UAF1 (I-677)-Flag; Viva) in the assay was 100 μM. Final substrate (Ub-Rh110; Ubiquitin-Rhodamine 110, R&D Systems #U-555) concentration was 25 nM with [Ub-Rh110]<<Km. 10 μL of 2× enzyme was added to assay plates (pre-stamped with compound) either simultaneously with 2×Ub-Rh110 or preincubated with USP1 40 minutes prior to the addition of 10 μL of 2×Ub-Rh110 to compound plates. Plates were incubated stacked for 45 minutes at room temperature before fluorescence was read on the Envision (Excitation at 485 nm and Emission at 535 nm; Perkin Elmer) or on the PheraSTAR (Excitation at 485 nm and Emission at 535 nm; BMG Labtech).
Measurement: IC50 = 1100 nM (pChEMBL 5.960000038146973)
Descriptors: MW 479.58, ALogP 4.91, HBA 6, HBD 0, PSA 80.98, QED 0.34
Compound (SMILES): N#CN1CCCC(c2nc(-c3cccc(-c4ccccc4)c3)co2)C1
InChIKey: GCXMPVYWTNQRMX-UHFFFAOYSA-N
Target: Ubiquitin carboxyl-terminal hydrolase 30 (SINGLE PROTEIN), Homo sapiens
UniProt: Q70CQ3
Assay: Biochemical Assay: Dilution plates were prepared at 21 times the final concentration (2100 μM for a final concentration of 100 μM) in 50% DMSO in a 96-well polypropylene V-bottom plate (Greiner #651201). A typical 8-point dilution series would be 100, 30, 10, 3, 1, 0.3, 0.1, 0.03 M final. Reactions were performed in duplicate in black 384 well plates (small volume, Greiner 784076) in a final reaction volume of 21 μl. Either 1 μl of 50% DMSO or diluted compound was added to the plate. USP30 was diluted in reaction buffer (40 mM Tris, pH 7.5, 0.005% Tween 20, 0.5 mg/ml BSA, 5 mM beta-mercaptoethanol) to the equivalent of 0.05 μl/well and 10 μl of diluted USP30 was added to the compound. Enzyme and compound were incubated for 30 min at room temp. Reactions were initiated by the addition of 50 nM of TAMRA labelled peptide linked to ubiquitin via an iso-peptide bond as fluorescence polarisation substrate. Reactions were read immediately after addition of substrate and following a 2 h incubation at room temperature. Readings were performed on a Pherastar Plus (BMG Labtech). λ Excitation 540 nm; λ Emission 590 nm.
Measurement: IC50 > 10000 nM (pChEMBL 5)
Descriptors: MW 329.4, ALogP 4.67, HBA 4, HBD 0, PSA 53.06, QED 0.66
Compound (SMILES): N#CC(Cc1ccc(O)c(O)c1)C(=O)Nc1nc(-c2cc(Cl)sc2Cl)cs1
InChIKey: NFWSQOGPIWMFDH-UHFFFAOYSA-N
Target: Double-strand break repair protein MRE11 (SINGLE PROTEIN), Homo sapiens
UniProt: P49959
Assay: HTS Assay: The conditions of the assay are as follows:Microplate type: 1536 Well Black Round Bottom Polystyrene Not Treated (Corning cat no. 3936)Total reaction volume: 5 μLMRE11 concentration in the reaction: 18 nMDNA substrate concentration in the reaction: 40 nMNumber of compounds to test: 257Inhibitor concentration range tested: 7 nM-50 μMMultiplicates: 3Concentration points: 13Dilution step: 2.1Each plate contains a series of high and low signal control wells, where no compound is added:High signal: MRE11+DNA substrateLow signal: DNA substrate onlyThese are used during data evaluation.Two Extra Assay Controls:1) To check whether unwinding of DNA by the compounds alone occurs.The DNA substrate [CY5+BBQ(650)] is mixed with the compounds with no protein present.This is done as a single measurement at 25 μM inhibitor concentration.2) To check whether the compounds are able to quench the CY5.The CY5 single stranded oligo is mixed with the compounds with no protein present. This is done as a single measurement at 25 μM inhibitor concentration.The layout of the plates is created by the in-house software (CZ-Openscreen Prague) and this information is transferred to the robotic HTS station.Assay Steps1) Prepare 50 mL of master mix:16.7 mL 5× reaction buffer (150 mM Bis Tris pH 7; 5 mM DTT)1042 μL 400 mM MnCl232.3 mL H2O2) Fill the plates with 3 μL of master mix per well using MultiDrop (Thermo Scientific)3) Transfer of compounds to the plates at the robotic station with the contactless Echo dispenser (Labcyte)4) Measurement of autofluorescence with the EnVision reader (PerkinElmer)5) Prepare 20 mL of 90 nM MRE11 in T+50 buffer (25 mM Tris-HCl pH7.5, 50 mM KCl 8.7% glycerol, 0.5 mM EDTA)6) Add 1 μL of 90 nM MRE11 to the corresponding wells using MultiDrop7) Preincubation at RT for 30 min8) Prepare 20 mL of a 200 nM solution of 5′ overhang DNA substrate: 480 μL 6 μM DNA+19.52 mL H2O9) Prepare 4 mL of a 200 nM solution of the single stranded DNA (oligo 1): 8 μL 100 μM DNA+4 mL H2O10) Add 1 μL of each 200 nM DNA solution to the corresponding wells with the MultiDrop11) Fluorescence measurement with the EnVision reader every 45 minutesFluorescence readout: CY5λex/em=620/665 nmAnalysis
Measurement: IC50 = 6000 nM (pChEMBL 5.21999979019165)
Descriptors: MW 440.33, ALogP 4.91, HBA 7, HBD 3, PSA 106.24, QED 0.49
Compound (SMILES): O=C(OC(C(F)(F)F)C(F)(F)F)N1CCC2(CCN(Cc3ccc(C(F)(F)F)c(N4CCCCC4)c3)C2)CC1
InChIKey: KPYZAKPNXWDXIG-UHFFFAOYSA-N
Target: Monoglyceride lipase (SINGLE PROTEIN), Homo sapiens
UniProt: Q99685
Assay: In Vitro Competitive Activity-Based Protein Profiling Assay: Human: Proteomes (human prefrontal cortex or cell membrane fractions) (50 μL, 1.0-2.0 mg/mL total protein concentration) were preincubated with varying concentrations of inhibitors at 37° C. After 30 min, FP-Rh or JW912 (1.0 μL, 50 μM in DMSO) was added and the mixture was incubated for another 30 min at room temperature. Reactions were quenched with SDS loading buffer (15 μL-4X) and run on SDS-PAGE. Following gel imaging, serine hydrolase activity was determined by measuring fluorescent intensity of gel bands corresponding to MAGL using ImageJ 1.49 k software.
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 575.52, ALogP 6.61, HBA 4, HBD 0, PSA 36.02, QED 0.38
Compound (SMILES): Cc1c(-c2[nH]c3ccc(N4C[C@@H](C)N(CCCS(C)(=O)=O)C[C@@H]4C)nc3c2C(C)C)cn2ncnc2c1C
InChIKey: VBCCGMZLTJNHAB-MOPGFXCFSA-N
Target: Toll-like receptor 7 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9NYK1
Assay: TLR7/8/9 Inhibition Reporter Assays: HEK-Blue-cells (Invivogen) overexpressing human TLR7, TLR8 or TLR9 receptors were used for screening inhibitors of these receptors using an inducible SEAP (secreted embryonic alkaline phosphatase) reporter gene under the control of the IFN-β minimal promoter fused to five NF-κB and AP-1-binding sites. Briefly, cells are seeded into Greiner 384 well plates (15000 cells per well for TLR7, 20,000 for TLR8 and 25,000 for TLR9) and then treated with test compounds in DMSO to yield a final dose response concentration range of 0.05 nM-50 μM. After a 30 minute compound pre-treatment at room temperature, the cells are then stimulated with a TLR7 ligand (gardiquimod at a final concentration of 7.5 μM), TLR8 ligand (R848 at a final concentration of 15.9 μM) or TLR9 ligand (ODN2006 at a final concentration of 5 nM) to activate NF-κB and AP-1 which induce the production of SEAP. After a 22 hour incubation at 37° C., 5% CO2, SEAP levels are determined with the addition of HEK-Blue Detection reagent (Invivogen), a cell culture medium that allows for detection of SEAP, according to manufacturer's specifications. The percent inhibition is determined as the % reduction in the HEK-Blue signal present in wells treated with agonist plus DMSO alone compared to wells treated with a known inhibitor.
Measurement: IC50 = 0.55 nM (pChEMBL 9.260000228881836)
Descriptors: MW 537.73, ALogP 4.35, HBA 8, HBD 1, PSA 99.49, QED 0.37
Compound (SMILES): CCCCNc1ncc(C(=O)NCc2ccc(-c3cccnc3)nc2)c(N[C@H]2CC[C@H](O)CC2)n1
InChIKey: MKUAZUIGYWTRKA-MEMLXQNLSA-N
Target: Tyrosine-protein kinase Mer (SINGLE PROTEIN), Homo sapiens
UniProt: Q12866
Assay: Activity Assay: Briefly, activity assays were performed in a 384 well, polypropylene microplate in a final volume of 50 μL of 50 mM Hepes, Ph 7.4 containing 10 mM MgCl2, 1.0 mM DTT, 0.01% Triton X-100, 0.1% Bovine Serum Albumin (BSA), containing 1.0 μM fluorescent substrate and ATP at the Km for each enzyme. All reactions were terminated by addition of 20 μL of 70 mM EDTA. After a 180 min incubation, phosphorylated and unphosphorylated substrate peptides were separated in buffer supplemented with 1×CR-8 on a LabChip EZ Reader equipped with a 12-sipper chip. Data were analyzed using EZ Reader software. Assay conditions for MCE assays
Measurement: IC50 = 3.8 nM (pChEMBL 8.420000076293945)
Descriptors: MW 475.6, ALogP 3.79, HBA 8, HBD 4, PSA 124.95, QED 0.33
Compound (SMILES): O=C1NCCN(CC(F)(F)C2CCC(O)CC2)c2c1oc1ccc(C(F)(F)F)cc21
InChIKey: NXSRTOYFJHZVEC-UHFFFAOYSA-N
Target: Serine/threonine-protein kinase pim-1 (SINGLE PROTEIN), Homo sapiens
UniProt: P11309
Assay: Pim-1 Inhibitory Activity Assay: The calculation of the Pim-1 inhibitory activity of the compound was performed using the following solution according to the protocol attached to ADP-Glo Kinase Assay (cat. V9102, Promega). The purified enzyme of human Pim-1 described above was used. (i) Preparation of Solution. The kinase buffer solution (50 mmol/L HEPES (pH 7.5), 5 mmol/L MgCl2, 1 mmol/L DTT, 0.05% BSA) was prepared by dissolving HEPES (Jena Bioscience), MgCl2 (Sigma-Aldrich), DTT (Sigma-Aldrich) and BSA (Sigma-Aldrich) in purified water. The ATP solution (288 μmol/L) was prepared by dissolving 100 mmol/L ATP (Promega) in the kinase buffer solution. The enzyme/substrate solution (0.2 mmol/L Pim-1, 30 μmol/L Pim2tide) was prepared by dissolving Pim-1 (described above) and Pim2tide (custom synthetic product by GenScript USA, the same product as PIM2tide cat. 12-542 by Millipore) in the kinase buffer solution. The test compound solution (containing 12.5% DMSO) was prepared by dissolving the DMSO solution of the test compound in the kinase buffer solution. The vehicle solution (containing 12.5% DMSO) was prepared by dissolving DMSO in the kinase buffer solution. (ii) Method. To a 384-well assay plate (Corning, 4513), the test compound solution or vehicle solution (control) was added by 1 μL/well, the enzyme/substrate solution or kinase buffer solution (blank) was added by 2 μL/well, and the ATP solution was added by 2 μL/well, and they were mixed. After enzymatically reacting at room temperature for 45 minutes, ADP-Glo Reagent (Promega) was added by 5 μL/well, and they were mixed. After reacting at room temperature for 60 minutes, Kinase Detection Reagent (Promega) was added by 10 μL/well, and they were mixed. After reacting at room temperature for 30 minutes, the luminescence amount of each well was measured for 10 msec with a multi-label plate reader EnVision (PerkinElmer).
Measurement: Ki = 0.086 nM (pChEMBL 10.069999694824219)
Descriptors: MW 432.39, ALogP 4.19, HBA 4, HBD 2, PSA 65.71, QED 0.71
Compound (SMILES): Cc1cc(C2CCOCC2)n2nc(NCc3cc(-c4ccccc4)nn3C)nc2n1
InChIKey: ZXDYQMWZUOASKB-UHFFFAOYSA-N
Target: cGMP-dependent 3',5'-cyclic phosphodiesterase (SINGLE PROTEIN), Homo sapiens
UniProt: O00408
Assay: IMAP TR-FRET-based phosphodiesterase assay: An IMAP TR-FRET-based phosphodiesterase assay was developed using the PDE2A isoform. IMAP technology is based on high-affinity binding of phosphate by immobilized metal (MIII) coordination complexes on nanoparticles. The IMAP binding reagent recognizes phosphate groups on AMP or GMP generated from cAMP or cGMP in a PDE reaction. Cyclic nucleotides that carry a phosphodiester bond and not a free phosphate are not recognized by the binding reagent. The time resolved fluorescence resonance energy transfer (TR-FRET) is afforded by a Terbium (Tb)-Donor pre-bound to the nanoparticles. FRET occurs when the fluorescent-labeled AMP or GMP product of a PDE reaction binds and comes into close proximity to the Tb-Donor complex. Due to the long lifetime of Tb fluorescence, detection can be run in time-resolved mode to reduce or eliminate interference from auto-fluorescent compounds.The IMAP TR-FRET PDE2A assay was performed in 1536-well white plates. A total of 250 pg per well of FLAG-tagged PDE2A1 (amino acids 2-941) was dispensed in 2.5 μL IMAP assay buffer consisting of 10 mM Tris pH 7.2, 10 mM MgCl2, 1 mM DTT, and 0.1% fatty acid free BSA. 30 nL of compound was then added from 1 mM stocks in DMSO using a Kalypsys Pintool. Plates were incubated for 5 min at room temperature before dispensing 1.5 μL of 533 nM FAM-cAMP substrate for a final concentration of 200 nM. Following a brief centrifugation, plates were incubated for 30 min at room temperature. The assay was terminated by adding 5 μL IMAP binding reagent Tb complex to each well which was prepared according to manufacturer's recommendations (Molecular Devices). Plates were incubated an additional 90 minutes at room temperature and read on a Viewlux plate reader. All compounds were solvated at a concentration of 10 mM in DMSO and tested in 11-point half-log dose-response. Curve fitting and IC50 values were determined using a standard four parameter fit.
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 403.49, ALogP 3.34, HBA 8, HBD 1, PSA 82.16, QED 0.55
Compound (SMILES): Fc1cccc2c1CCN1CCNC[C@@H]21
InChIKey: WKACMCSEGGICSS-LBPRGKRZSA-N
Target: Trace amine-associated receptor 1 (SINGLE PROTEIN), Rattus norvegicus
UniProt: Q923Y9
Assay: Radioligand Binding Assay: The TAAR1 radioligand 3[H]-(S)-4-[(ethyl-phenyl-amino)-methyl]-4,5-dihydro-oxazol-2-ylamine (described in WO 2008/098857) was used at a concentration equal to the calculated Kd value, that was usually around 2.3 nM, resulting in the binding of approximately 0.2% of the radioligand and a specific binding representing approximately 85% of the total binding. Nonspecific binding was defined as the amount of [H]-(S)-4-[(ethyl-phenyl-amino)-methyl]-4,5-dihydro-oxazol-2-ylamine bound in the presence of 10 μM unlabeled ligand. All compounds were tested at a broad range of concentrations (10 μM to 10 μM) in duplicates. The test compounds (20□ μl/well) were transferred into a 96 deep well plate (TreffLab), and 180 μl of HEPES-NaOH (20 mM, pH 7.4) containing MgCl2 (10 mM) and CaCl2 (2 mM) (binding buffer), 300 μl of the radioligand 3[H]-(S)-4-[(ethyl-phenyl-amino)-methyl]-4,5-dihydro-oxazol-2-ylamine at a concentration of 3.3×Kd in nM and 500 μl of the membranes (resuspended at 50 μg protein per ml) added.
Measurement: Ki = 176 nM (pChEMBL 6.75)
Descriptors: MW 206.26, ALogP 1.33, HBA 2, HBD 1, PSA 15.27, QED 0.69
Compound (SMILES): CCc1nc2ccc(N3CCN(S(C)(=O)=O)CC3)cn2c1N(C)c1nc(-c2ccc(Cl)cc2)ns1
InChIKey: WZBGCAPUOXTTLF-UHFFFAOYSA-N
Target: Autotaxin (SINGLE PROTEIN), Homo sapiens
UniProt: Q13822
Assay: Biochemical Assay: TABLE VI: (UniProtKB/SwissProt Sequence ref Q13822) biochemical assay using the fluorogenic autotaxin substrate FS-3 as substrate. FS-3 is a doubly labeled analog of LPC wherein the fluorophore is quenched through intramolecular energy transfer. Without hENPP2, the emission of the probe is quenched. If the substrate is hydrolyzed by hENPP2, the emission of the probe is not quenched anymore resulting in a fluorescence increase. Inhibition of hENPP2 by compounds will result in a decrease of the signal.10 μL of a dilution series of compound, starting from 20 μM highest concentration, 1/5 dilution, is added to the wells. hENPP2 is used at a final concentration of 0.4 μg/mL or 0.64 μg/mL (it will be appreciated by the skilled person that the potency read out is independent of the enzyme concentration). The enzyme is diluted in 50 mM Tris-HCl pH 8.0, 250 mM NaCl, 5 mM KCl, 1 mM MgCl2, 1 mM CaCl2, 0.1% fatty acid free BSA in a total volume of 20 μL. Enzyme mixture is added to compounds and the resulting mixture is incubated for 30 min at room temperature under shaking. The reaction is started by the addition of 20 μL of 0.75 μM FS-3 diluted in the same buffer as described above and the mixture is incubated at 30° C. for 30 min. Fluorescence is read on the Envision (Excitation 485 nm, emission 520 nM).
Measurement: IC50 = 50 nM (pChEMBL 7.300000190734863)
Descriptors: MW 532.1, ALogP 3.92, HBA 9, HBD 0, PSA 86.94, QED 0.37
Compound (SMILES): CCOC(=O)O[C@@H](C)n1nnc(COC[C@H](C)N[C@H]2CC[C@H](Nc3cc(-c4cccc(NCC5(C#N)CCOCC5)n4)c(Cl)cn3)CC2)n1
InChIKey: HQVRFXBZJOXOKH-QORCZRPOSA-N
Target: CDK9/cyclin T1 (PROTEIN COMPLEX), Homo sapiens
UniProt: P50750
Assay: CDK9 Biochemical Assay: To each well of a 96-well plate was added 5× kinase assay buffer with 10 mM DTT (6 μL), 500 μM ATP (1 μL), 5×CDK substrate (10 μL), and water (8 μL). 5 μL of compound were added to the test and positive control groups, while 5 μL of solvent was added to the blank groups. 100 ng of CDK9/CyclinT in 20 μL water was added to the test and positive control groups, while 20 μL of 1× kinase assay buffer was added to the blank groups. The reaction mixtures were incubated at 30° C. for 45 minutes, and 50 μL of Kinase-Glo® Max was added to each well and the plates were shielded from light and incubated for 15 minutes at room temperature. Luminescence was measured on a microplate reader and IC50values were calculated using Prism 9 software.
Measurement: IC50 > 10 nM (pChEMBL 8)
Descriptors: MW 697.24, ALogP 5.13, HBA 15, HBD 3, PSA 183.25, QED 0.18
Compound (SMILES): O=C(NS(=O)(=O)c1ccc(NCC2CCOCC2)c([N+](=O)[O-])c1)c1ccc(-c2ccc(CN3CCCC3c3ccc(Cl)cc3)cc2)cc1Oc1cnc2[nH]ccc2c1
InChIKey: UUJMVHLFKALLPR-UHFFFAOYSA-N
Target: Apoptosis regulator Bcl-2 (SINGLE PROTEIN), Homo sapiens
UniProt: P10415
Assay: Bcl-2/Bcl-X Fluorescence Polarization Assay: Compounds disclosed herein were tested for blocking of Bcl-2/Bcl-xl protein with its ligand in an assay based on fluorescence polarization methodology. Recombinant human 2.7 nM Bcl-2/1.3 nM Bcl-xl protein was pre-incubated with a serial dilution of compounds disclosed herein (maximum concentration is 1 μM for Bcl-2 assay, and 10 μM or 1 μM for Bcl-xl assay, 3-fold serially diluted, 10 points) at room temperature for 0.5 hour in an assay buffer containing 20 ml potassium phosphate buffer, pH 7.5, 50 mM NaCl, 1 mM EDTA, 0.05% Tween-20, 0.01% BSA. Then the FITC labeled Bak peptide Ac-GQVGRQLAIIGDK (FITC)INR-amide (1 nM for Bcl-2, 0.82 nM for Bcl-xl) was added to plate and further incubated at room temperature for 0.5 h. The FP signals (485 nm-520 nm-520 nm) were read on BMG PHERAstar FS or BMG PHERAstar FSX instalment. The inhibition percentage of Bcl-2/Bcl-xl interaction with its ligand in presence of increasing concentrations of compounds was calculated based on the FP signals. The IC50 for each compound was derived from fitting the data to the four-parameter logistic equation by Graphpad Prism software.
Measurement: IC50 = 141 nM (pChEMBL 6.849999904632568)
Descriptors: MW 821.36, ALogP 8.88, HBA 10, HBD 3, PSA 168.79, QED 0.08
Compound (SMILES): CS(=O)(=O)N1CCC(Nc2ncc3cc(C(F)(F)F)c(=O)n(C4CCCC4)c3n2)CC1
InChIKey: ONIGOMQTGTVZJA-UHFFFAOYSA-N
Target: Cyclin-dependent kinase 2/cyclin E1 (PROTEIN COMPLEX), Homo sapiens
UniProt: P24864
Assay: CDK2/Cyclin E1 Mobility Shift Assay: The purpose of the CDK2/Cyclin E1 assay is to evaluate the inhibition (% inhibition, Kiapp and Ki values) of small molecule inhibitors by using a fluorescence-based microfluidic mobility shift assay. CDK2/Cyclin E1 catalyzes the production of ADP from ATP that accompanies the phosphoryl transfer to the substrate peptide FL-Peptide-18 (5-FAM-QSPKKG-CONH2) (SEQ ID NO:1). (CPC Scientific, Sunnyvale, Calif.). The mobility shift assay electrophoretically separates the fluorescently labeled peptides (substrate and phosphorylated product) following the kinase reaction. Both substrate and product are measured and the ratio of these values is used to generate % conversion of substrate to product by the LabChip EZ Reader. Wild-type full length CDK2/wild-type full length Cyclin E1 enzyme complex was produced in-house (baculoviral expression, LJIC-2080/LJIC-2103) and phosphorylated by CDK7/Cyclin H1/Mat1 enzyme complex with CDK2:CDK7 ratio of 50:1 (concentration mg/mL) in the presence of 10 mM MgCl2 and 5 mM ATP at room temperature for one hour. Typical reaction solutions (50 μL final reaction volume) contained 2% DMSO (± inhibitor), 4 mM MgCl2, 1 mM DTT, 150 μM ATP (ATP Km=67.4 μM), 0.005% Tween-20, 3 μM FL-Peptide-18, and 0.36 nM (catalytically competent active site) phosphorylated wild-type full length CDK2/Cyclin E1 enzyme complex in 25 mM HEPES buffer at pH 7.15. The assay was initiated with the addition of ATP, following a fifteen minutes pre-incubation of enzyme and inhibitor at room temperature in the reaction mixture. The reaction was stopped after 45 minutes at room temperature by the addition of 50 μL of 80 mM EDTA, pH 7.5. The Ki value was determined from the fit of the data to the Morrison tight-binding competitive inhibition equation with the enzyme concentration as a variable.
Measurement: Ki = 4.18 nM (pChEMBL 8.380000114440918)
Descriptors: MW 459.49, ALogP 2.76, HBA 7, HBD 1, PSA 97.19, QED 0.76