lovingscience/chembl-v37-raw-activities huggingface dataset:
Compound (SMILES): CC[C@H](NC(=O)[C@H](C)NC)C(=O)N1C[C@@H](F)C[C@H]1Cc1c(-c2nc3cc(F)ccc3n2C[C@@H]2C[C@H](F)CN2C(=O)[C@@H](NC(=O)[C@H](C)NC)C2CCCCC2)[nH]c2cc(F)ccc12
InChIKey: APTRBTUTSFUIJT-IAECAYPDSA-N
Target: Baculoviral IAP repeat-containing protein 2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q13490
Assay: Biological Assay 1: affinity of compounds to cIAP1-BIR3, cIAP2-BIR3, XIAP-BIR3: 1) Affinity of compounds listed in table to cIAP1-BIR3, Ciap2-BIR3, XIAP-BIR3 was tested referring to Nikolovska-Colesks, Z. et.Al. (Analytical Biochemistry,2004,232:261-273) to obtain IC50 value. Generally speaking, in 10-dose IC50 mode, IAP antagonists with different concentrations were diluted in series by 3-fold and fluorescence labelled ARPFAQ-K (5-FAM)-NH2 peptide was used as probe to test compounds binding to protein BIR3 domain.2) Test condition: cIAP1-BIR3: 20 nM; cIAP2-BIR3: 60 nM; XIAP-BIR3: 30 nM; 5 nM probe in 100 mM tripotassium phosphate, pH 7.5, 0.1 mg/mL BSA, 0.005% Triton X100 and 0.5% DMSO.3) Test procedure: compounds were added into protein with different concentrations by using ECHO (LabCyte), and then the mixture was preincubated for 15 min. The probe was added and the final concentration was 5 nM. After incubating for 60 min, FP was tested and mP was calculated.
Measurement: IC50 < 10 nM (pChEMBL 8)
Descriptors: MW 866.02, ALogP 5.06, HBA 8, HBD 5, PSA 156.49, QED 0.1
Compound (SMILES): COc1cccc(OC(F)F)c1-n1c(NS(=O)(=O)[C@@H](C)[C@H](OC)c2ncc(C)cn2)nnc1-c1cncc(C)c1
InChIKey: LHJNLYPTZJOLMB-KKSFZXQISA-N
Target: Apelin receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P35414
Assay: [35S]GTPγS Binding: The human APJ receptor was cloned by polymerase chain reaction and the gene encoding the receptor was subcloned in pFLAG-CMV-3 expression vector (Sigma, Saint Louis, Mo. USA) in-house at Amgen. A GTPγS binding assay was performed on membranes prepared from CHO cells stably expressing human APJ receptor. The optimum experimental conditions for the concentrations of GDP, MgCl2, and NaCl in the assay buffer were initially determined. The assay was performed in assay buffer [20 mM HEPES, pH 7.5, 5 mM MgCl2, and 0.1% (w/v) BSA with 200 mM NaCl, 3 M GDP] and membranes expressing human APJ receptor/well along with WGA PS beads. The reaction was initiated by addition of 0.2 nM [35S]GTPγS (Perkin Elmer Life and Analytical Sciences, Waltham USA) in the absence or presence of various ligands and incubated at RT for 90 min. Nonspecific binding was determined in the presence of 100 M GTPγS and was always less than 0.2% of total binding. All the results presented are means of several independent experiments and analyzed by non-linear regression methods using commercially available program Prism (GraphPad, San Diego, Calif.) to obtain EC50.
Measurement: EC50 = 0.76 nM (pChEMBL 9.119999885559082)
Descriptors: MW 575.6, ALogP 3.86, HBA 11, HBD 1, PSA 143.24, QED 0.28
Compound (SMILES): NCc1cccc(-c2cc(COc3ccccc3CC(=O)O)cc3cc(COCc4cccnc4)oc23)c1
InChIKey: KJCZSCKOFWNQLF-UHFFFAOYSA-N
Target: Complement factor D (SINGLE PROTEIN), Homo sapiens
UniProt: P00746
Assay: Factor D Esterolytic Assay: An established esterolytic assay for the measurement of Factor D activity and inhibition of Factor D activity was used (Kam, C. M.; McRae, B. J.; Harper, J. W.; Niemann, M. A.; Volanakis, J. E.; Powers, J. C. Human complement proteins D, C2, and B Active site mapping with peptide thioester substrates. J Biol. Chem. 1987, 262, 3444-3451). For this assay Z-Lys-SBzl, 1.29 mM (Kim, S.; Narayana, S. V. L; Volanakis, J. E. Mutational analysis of the substrate binding site of human complement Factor D. Biochemistry. 1994, 33, 14393-14399.) was used as the substrate for Factor D (104 mM). Hydrolysis of this compound by Factor D liberated a free sulfhydryl group which is then reacted with 5,5′-dithiobis(2nitrobenzoic acid) producing an intense yellow color (Habeeb, A. F. S. A. Reaction of protein sulfhydryl groups with Ellman's Reagent. Methods in Enzymol. 1976, 25, 457-464). The assays were performed in 96 well microtiter plates and rates of hydrolysis were monitored at 405 nm on a Biotek Synergy H1 plate reader. Hydrolysis rates were reported as change in mOD/min. The assay was conducted in 100 mM HEPES, 500 mM NaCl, pH 7.5 containing 10% DMSO in a final volume of 50 μL per well.
Measurement: IC50 < 1000 nM (pChEMBL 6)
Descriptors: MW 508.57, ALogP 5.88, HBA 6, HBD 2, PSA 107.81, QED 0.23
Compound (SMILES): Cc1c(N2CCC3(CC2)Cc2ccccc2[C@H]3N)nc(C)n(-c2ccc(F)c(F)c2Cl)c1=O
InChIKey: DTAMYNIRMKDWCL-JOCHJYFZSA-N
Target: Tyrosine-protein phosphatase non-receptor type 11 (SINGLE PROTEIN), Homo sapiens
UniProt: Q06124
Assay: Biochemical Assay: The inhibition of SHP2 by compounds of the invention was monitored using the surrogate substrate DiFMUP after protein activation by a peptide bearing two appropriately spaced phosphotyrosine. Full length SHP2 protein (Recombinant HumanSHP-2, E. coli derived Ser2Arg593, N-terminal 6His tag from R&D systems; 0.0.24 nM) was incubated with activating peptide, IRSI_2pY (New England Peptide, 140 nM) and DiFMUP (molecular probes, 80 uM) at RT in buffer (HEPES pH 7.2 60 mM, DDT 5 mM, KCl 75 mM, NaCl 75 mM, EDTA 1 mM, Tween 20 0.05%) in presence of compound (10 concentrations range, top concentration 50 μM) for 60 min. The generation of the DiFMU product by activated SHP2 was monitored through Fluorescence measurement with a PerkinElmer Envision reader. The inhibitor dose response curves were analyzed with Genedata Screener.
Measurement: IC50 = 6 nM (pChEMBL 8.220000267028809)
Descriptors: MW 470.95, ALogP 4.62, HBA 5, HBD 1, PSA 64.15, QED 0.55
Compound (SMILES): N#CC1(COc2nc(C(=O)NCC(F)(F)F)nc(N3CCC(c4c[nH]c5ncncc45)CC3)n2)CC1
InChIKey: XQUVNCURHOGKBP-UHFFFAOYSA-N
Target: Tyrosine-protein kinase receptor UFO (SINGLE PROTEIN), Homo sapiens
UniProt: P30530
Assay: Enzyme Assay: AXL enzyme inhibition (% inhibition, Kiapp and Ki values) by small molecule inhibitors was evaluated using a fluorescence-based microfluidic mobility shift assay. AXL catalyzes the production of ADP from ATP that accompanies the phosphoryl transfer to the substrate peptide FL-Peptide-30 (5-FAM-KKKKEEIYFFF-CONH2, 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. Human wild-type receptor tyrosine kinase protein Axl comprising residues 505-811 was produced in-house using the baculoviral expression vector system that incorporated a hexahistidine affinity tag into the protein (LJIC-1916B1.1). The enzyme was preactivated by auto-phosphorylation of 34 uM non-activated enzyme in the presence of 2 mM ATP, 4 mM MgCl2, 50 mM NaCl and 1 mM TCEP in 20 mM HEPES, pH 7.3 at 4° C. for 30 minutes. Typical reaction solutions (50 μL final reaction volume) contained 2% DMSO (±inhibitor), 10 mM MgCl2, 1 mM DTT, 120 μM ATP (ATP Km=70.4 μM), 0.01% Tween-20, 3 μM FL-Peptide-30, and 0.5 nM phosphorylated AXL enzyme in 100 mM HEPES buffer at pH 7.3. 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 30 minutes at 25° C. by the addition of 50 μL of 200 mM EDTA, pH 7.5. The Ki values were determined from the fit of the data to the Morrison tight-binding competitive inhibition equation with the enzyme concentration as a variable. (See, Morrison, J. F. (1969) Kinetics of the reversible inhibition of enzyme-catalysed reactions by tight-binding inhibitors, Biochimica et biophysica acta 185, 269-286; and Murphy, D. J. (2004) Determination of accurate KI values for tight-binding enzyme inhibitors: an in silico study of experimental error and assay design, Analytical biochemistry 327, 61-67.)
Measurement: Ki = 10.5 nM (pChEMBL 7.980000019073486)
Descriptors: MW 501.47, ALogP 2.5, HBA 9, HBD 2, PSA 145.6, QED 0.5
Compound (SMILES): N#CC1(CS(=O)(=O)N2C3CCC2CN(c2cc(NC(=O)C4CC4)nc4[nH]ccc24)C3)CC1
InChIKey: OOBUMDVTESCIBH-UHFFFAOYSA-N
Target: Tyrosine-protein kinase JAK1 (SINGLE PROTEIN), Homo sapiens
UniProt: P23458
Assay: ADP-Glo Kinase Assay: A control material and a test material were prepared for each concentration, in such a way that they were diluted by means of DMSO. At the same time, ATP (250 uM) and JAK substrate (JAK1, IRS-itide 40 ng/mL) were prepared, in such a way that they were diluted by means of kinase buffer (40 mM Tris-HCl pH 7.5, 20 mM MgCl2, 0.5 mg/mL BSA, 50 uM DTT).A test drug for each concentration, substrate, ATP and JAK enzyme were mixed in an eppendorf tube, and reacted in a 30° C. incubator for 40 minutes.ADP-Glo reagent contained in ADP-Glo Kinase Enzyme System (Promega, USA, V9571) was added into each eppendorf tube, and reacted in a 30° C. incubator for 40 minutes. A kinase detection reagent contained in ADP-Go Kinase Enzyme System was inserted into the eppendorf tube, then an integration time was set to 1 second by using Wallac Victor 2, then luminescence was measured, and then JAKs phosphorylation inhibition ability of the test material was analyzed. The concentration of the compound, which causes 50% of the JAK enzyme activity inhibition in comparison With the control group, was determined as IC50 (nM) of an inhibitor, wherein the results thereof are shown in a following table 1.
Measurement: IC50 < 20 nM (pChEMBL 7.698999881744385)
Descriptors: MW 454.56, ALogP 2.2, HBA 6, HBD 2, PSA 122.19, QED 0.69
Compound (SMILES): O=C(NS(=O)(=O)c1ccc(NCC2CCOCC2)c([N+](=O)[O-])c1)c1ccc(C2=CCC(N3CCCC3c3cccc(Cl)c3C3CC3)CC2)cc1Oc1cnc2[nH]ccc2c1
InChIKey: QUFFEBFYDGSUQY-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 = 14 nM (pChEMBL 7.849999904632568)
Descriptors: MW 851.43, ALogP 9.52, HBA 10, HBD 3, PSA 168.79, QED 0.08
Compound (SMILES): O=C(c1cc(Cl)c(O)cc1O)N1CCOCC1
InChIKey: OXQDHJGMTBNPMO-UHFFFAOYSA-N
Target: Heat shock protein HSP 90-alpha (SINGLE PROTEIN), Homo sapiens
UniProt: P07900
Assay: Endonuclease Activity Assay: Endonuclease activity assays were carried out in Black Costar 96-well plates. Each well contained a total volume of 100 μL comprised of: buffer (20 mM Tris, 150 mM NaCl, 2 mM MnCl2, 10 mM β-mercaptoethanol, 0.2% Triton-X100, pH=8.0), influenza PA endonuclease (25 nM) prepared from Example 1, inhibitor (various concentrations) in buffer, and fluorescent ssDNA-oligo substrate (200 nM). A single-stranded, 17-mer DNA substrate labeled with a 5′-FAM fluorophore and a 3′-TAMRA quencher ([6-FAM]AATCGCAGGCAGCACTC[TAM]) (SEQ ID NO:2) synthesized by Sigma-Aldrich was employed to measure endonucleic cleavage. Upon addition of the substrate, the change in fluorescence was measured over 45 minutes at 37° C. (excitation: 485 nm; emission 528 nm). The positive control wells contained no inhibitor for preliminary screens, and were set as an arbitrary 100% activity. Compounds that exhibited >80% inhibition at a concentration of 200 μM were re-evaluated at a concentration of 50 μM. Confirmation screens employed EGCG (Epigallocatechin 3-gallate), a previously validated inhibitor, as a positive control. The gain was set to 100 and the first 10 data points (the first 10 minutes) were excluded from the activity calculations. Dose-response curves were generated, fitted, and analyzed using Origin8 graphing software. Dose-response curves were compiled and IC50 values determined for compounds exhibiting >50% inhibition at 50 μM.
Measurement: IC50 = 383 nM (pChEMBL 6.420000076293945)
Descriptors: MW 257.67, ALogP 1.22, HBA 4, HBD 2, PSA 70, QED 0.79
Compound (SMILES): O=C(Nc1nc2cccc(-c3ccc(C(=O)N4CCC(n5nnc6ccccc65)CC4)cc3)n2n1)C1CC1
InChIKey: BXDHULAAYYZGOK-UHFFFAOYSA-N
Target: Tyrosine-protein kinase JAK1 (SINGLE PROTEIN), Homo sapiens
UniProt: P23458
Assay: JAK1 Inhibition Assay: Recombinant human JAK1 catalytic domain (amino acids 850-1154; catalog number 08-144) was purchased from Carna Biosciences. 10 ng of JAK1 was incubated with 12.5 μg polyGT substrate (Sigma catalog number P0275) in kinase reaction buffer (15 mM Tris-HCl pH 7.5, 1 mM DTT, 0.01% Tween-20, 10 mM MgCl2, 2 μM non-radioactive ATP, 0.25 μCi 33P-gamma-ATP (GE Healthcare, catalog number AH9968) final concentrations) with or without 5 μL containing test compound or vehicle (DMSO, 1% final concentration), in a total volume of 25 μL, in a polypropylene 96-well plate (Greiner, V-bottom). After 45 min at 30° C., reactions were stopped by adding of 25 μL/well of 150 mM phosphoric acid. All of the terminated kinase reaction was transferred to prewashed (75 mM phosphoric acid) 96 well filter plates (Perkin Elmer catalog number 6005177) using a cell harvester (Perkin Elmer). Plates were washed 6 times with 300 μL per well of a 75 mM phosphoric acid solution and the bottom of the plates was sealed. 40 μL/well of Microscint-20 was added, the top of the plates was sealed and readout was performed using the Topcount (Perkin Elmer). Kinase activity was calculated by subtracting counts per minute (cpm) obtained in the presence of a positive control inhibitor (10 μM staurosporine) from cpm obtained in the presence of vehicle. The ability of a test compound to inhibit this activity was determined as:Percentage inhibition=((cpm determined for sample with test compound present−cpm determined for sample with positive control inhibitor) divided by (cpm determined in the presence of vehicle−cpm determined for sample with positive control inhibitor))*100%.
Measurement: IC50 = 550 nM (pChEMBL 6.260000228881836)
Descriptors: MW 506.57, ALogP 3.97, HBA 8, HBD 1, PSA 110.31, QED 0.39
Compound (SMILES): COc1cc(CO)cc(S(=O)(=O)N2CCc3cccc(CCc4ccc(C(=O)O)cc4)c32)c1
InChIKey: GNQFVWHBHKWCJW-UHFFFAOYSA-N
Target: Prostaglandin E2 receptor EP2 subtype (SINGLE PROTEIN), Homo sapiens
UniProt: P43116
Assay: FLIPR Assay: Cells were seeded at a density of 5×104 cells per well in Biocoat® Poly-D-lysine-coated black-wall, clear-bottom 96-well plates (Becton-Dickinson) and allowed to attach overnight in an incubator at 37° C. Cells were then washed two times with HBSS-HEPES buffer (Hanks Balanced Salt Solution without bicarbonate and phenol red, 20 mM HEPES, pH 7.4) using a Denley Cellwash plate washer (Labsystems). After 45 minutes of dye-loading in the dark, using the calcium-sensitive dye Fluo-4 AM at a final concentration of 2 mM, plates were washed four times with HBSS-HEPES buffer to remove excess dye leaving 100 ml in each well. Plates were re-equilibrated to 37° C. for a few minutes.Cells were excited with an Argon laser at 488 nm, and emission was measured through a 510-570 nm bandwidth emission filter (FLIPR™, Molecular Devices, Sunnyvale, Calif.). Drug solution was added in a 50 μl volume to each well to give the desired final concentration. The peak increase in fluorescence intensity was recorded for each well. On each plate, four wells each served as negative (HBSS-HEPES buffer) and positive controls (standard agonists: BW245C (hDP); PGE2 (hEP1; hEP2/Gqs5; hEP3A/Gqi5; hEP4/Gqs5); PGF2a (hFP); carbacyclin (hIP); U-46619 (hTP), depending on receptor). The peak fluorescence change in each drug-containing well was then expressed relative to the controls.
Measurement: Ki = 776 nM (pChEMBL 6.110000133514404)
Descriptors: MW 467.54, ALogP 3.42, HBA 5, HBD 2, PSA 104.14, QED 0.53
Compound (SMILES): CCc1cccc(CC)c1-n1nc2c(c1-c1cc(F)c(NC(N)=O)cc1F)CN(Cc1cc(C(F)(F)F)ccc1C)CC2
InChIKey: DBDQQFQAVLKHIU-UHFFFAOYSA-N
Target: C5a anaphylatoxin chemotactic receptor 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P21730
Assay: chemotaxis assay: Optionally, compounds may be further assayed for their ability to inhibit chemotaxis in cells. Chemotaxis assays are performed using 5 μm pore polycarbonate, polyvinylpyrrolidone-coated filters in 96-well chemotaxis chambers (Neuroprobe; Gaithersburg, Md.) using chemotaxis buffer (Hank's balanced salt solution (HBSS) and 1% FBS). C5aR ligands (i.e., C5a, R&D Systems; Minneapolis, Minn.) are use to evaluate compound mediated inhibition of C5aR mediated migration. Other chemokines (i.e., SDF-1α; R&D Systems; Minneapolis, Minn.) are used as specificity controls. The lower chamber is loaded with 29 μl of chemokine (i.e., 0.03 nM C5a) and varying amounts of compound; the top chamber contains 100,000 U937 or neutrophil cells in 20 μl. The chambers are incubated 1.5 hours at 37° C., and the number of cells in the lower chamber quantified either by direct cell counts in five high powered fields per well or by the CyQuant assay (Molecular Probes), a fluorescent dye method that measures nucleic acid content and microscopic observation.
Measurement: IC50 < 5 nM (pChEMBL 8.300999641418457)
Descriptors: MW 597.63, ALogP 7.32, HBA 4, HBD 2, PSA 76.18, QED 0.22
Compound (SMILES): N#CC1(c2cnc3c(C(=O)Nc4cncc(F)c4N4CCN(C5COC5)CC4)c(N)nn3c2)CC1
InChIKey: CSQPCWDGHLQJSD-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 μM [γ-33P]ATP (3mCi 33P ATP/mmol ATP, Perkin Elmer) and 800 μM target peptide (ASELPASQPQPFSAKKK) (SEQ ID NO:1).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 454 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 = 505 nM (pChEMBL 6.300000190734863)
Descriptors: MW 477.5, ALogP 1.17, HBA 10, HBD 2, PSA 137.7, QED 0.55
Compound (SMILES): C[C@@H]1CN(c2ccc(CN3[C@@H](C)CC[C@H](c4ccccc4)S3(=O)=O)c(F)c2)CC[C@@H]1n1cnnc1
InChIKey: LSHQMJIVDVPWGV-XWJWGENMSA-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 = 15 nM (pChEMBL 7.820000171661377)
Descriptors: MW 497.64, ALogP 4.56, HBA 6, HBD 0, PSA 71.33, QED 0.52
Compound (SMILES): CCN1CCN(Cc2cc3c4c(cnc3[nH]2)CN(c2c(F)c(OC)cc(OC)c2F)C(=O)C4(C)C)CC1
InChIKey: CQZQLQVEMYIPJL-UHFFFAOYSA-N
Target: Fibroblast growth factor receptor 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P11362
Assay: Enzymatic Assay: The inhibitor potency of the exemplified compounds was measured in an enzyme assay that measures peptide phosphorylation using FRET measurements to detect product formation. Inhibitors were serially diluted in DMSO and a volume of 0.5 μL was transferred to the wells of a 384-well plate. For FGFR3, a 10 μL volume of FGFR3 enzyme (Millipore) diluted in assay buffer (50 mM HEPES, 10 mM MgCl2, 1 mM EGTA, 0.01% Tween-20, 5 mM DTT, pH 7.5) was added to the plate and pre-incubated for 5-10 minutes. Appropriate controls (enzyme blank and enzyme with no inhibitor) were included on the plate. The assay was initiated by the addition of a 10 μL solution containing biotinylated EQEDEPEGDYFEWLE peptide substrate (SEQ ID NO: 1) and ATP (final concentrations of 500 nM and 140 μM respectively) in assay buffer to the wells. The plate was incubated at 25° C. for 1 hr. The reactions were ended with the addition of 10 μL/well of quench solution (50 mM Tris, 150 mM NaCl, 0.5 mg/mL BSA, pH 7.8; 30 mM EDTA with Perkin Elmer Lance Reagents at 3.75 nM Eu-antibody PY20 and 180 nM APC-Streptavidin). The plate was allowed to equilibrate for 1 hr before scanning the wells on a PheraStar plate reader (BMG Labtech).
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 513.59, ALogP 3.82, HBA 6, HBD 1, PSA 73.93, QED 0.54
Compound (SMILES): O=c1c2cccnc2ncn1Cc1nc([C@H]2C[C@](O)(c3ccc(Cl)cc3)C2)no1
InChIKey: DLXGKTWQOPDGDA-KWUMSVIQSA-N
Target: Transient receptor potential cation channel subfamily A member 1 (SINGLE PROTEIN), Homo sapiens
UniProt: O75762
Assay: FLIPR Assay (15 minutes): The IC50 (effective concentration) of compounds on the human TRPA1 channel was determined using a FLIPR Tetra instrument. CHO cells expressing TRPA1 were plated into 384-well plates, incubated overnight at 37° C., and loaded with BD calcium indicator dye for 1 hr at 37° C. followed by 15 minutes at room temperature. The assay buffer was Hank's Balanced Salt Solution (HBSS) containing 20 mM HEPES (pH readjusted to 7.4) along with 0.02% BSA.Following dye load and plate cool down, compounds were added to the cells using FLIPR Tetra. Plates were then incubated with compounds for 10 minutes or 90 minutes at room temperature prior to adding agonist. Following this incubation, about an EC50 concentration of cinnamaldehyde (75) was added to active the channels and block of cinnamaldehyde induced calcium influx was measured.The IC50 results were fit with a standard Hill function, keeping the Hill coefficient (n) fixed to 1.5. Fixing the Hill coefficient will generally reduce variability of the IC50 determination. The IC50 results were individually examined to make sure the MIN and MAX points were set correctly prior to validation of the results.
Measurement: IC50 = 261 nM (pChEMBL 6.579999923706055)
Descriptors: MW 409.83, ALogP 2.64, HBA 8, HBD 1, PSA 106.93, QED 0.55
Compound (SMILES): Cn1cc(NC(=O)c2cnn3ccc(N4CCN5CCC4C5)nc23)c(C(F)(F)F)n1
InChIKey: QGWFKBZCSRDLJM-UHFFFAOYSA-N
Target: Interleukin-1 receptor-associated kinase 4 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9NWZ3
Assay: IRAK4 Kinase Assay: The kinase activity of IRAK4 is determined by its ability to catalyze the phosphorylation of a fluorescent polypeptide substrate. The extent of phosphorylation is measured using the IMAP technology (Molecular Devices) where the phosphorylated fluorescent substrate binds to the large M(III)-based nanoparticles which reduces the rotational speed of the substrate and thus increases its fluorescent polarization (FP).20 μL reaction mixture contains 10 mM TriHCl, pH 7.2, 0.5 nM GST tagged IRAK4 (SignalChem), 100 nM fluorescent peptide substrate (RP7030, Molecular Devices), 100 μM ATP, 1 mM DDT, 1 mM MgCl2, and 0.01% Tween 20. The reaction is initiated by the addition of ATP. After incubation for 30 minutes at 25° C., 60 μL of Progressive IMAP Reagent (Molecular Devices) is added to stop the reaction. Change in RP7030's FP is determined by a FP reader (Analyst HT, LJL BioSystems).
Measurement: IC50 = 45 nM (pChEMBL 7.349999904632568)
Descriptors: MW 420.4, ALogP 1.63, HBA 8, HBD 1, PSA 83.59, QED 0.69
Compound (SMILES): Cc1cc(Nc2cc3ncccc3c(N[C@@H]3C[C@H]4CCC[C@@H](C3)N4Cc3cccc(O)c3)n2)n[nH]1
InChIKey: WSFQMCDKLICTAR-MZADTFQBSA-N
Target: Tyrosine-protein kinase JAK1 (SINGLE PROTEIN), Homo sapiens
UniProt: P23458
Assay: Biochemical JAK 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 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 lh. 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 lh 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.For dose-response analysis, percent inhibition data were plotted vs. compound concentrations, and IC50 values were determined from a 4-parameter robust fit model with the Prism software (GraphPad Software). Results were expressed as pIC50 (negative logarithm of IC50) and subsequently converted to pKi (negative logarithm of dissociation constant, Ki) using the Cheng-Prusoff equation.Test compounds having a higher pKi value in each of the four JAK assays show greater inhibition of JAK activity. Compounds of the invention tested in this assay typically exhibited pKi values between about 7 and about 10.3
Measurement: Ki < 0.1 nM (pChEMBL 10)
Descriptors: MW 469.59, ALogP 5.11, HBA 7, HBD 4, PSA 101.99, QED 0.31
Compound (SMILES): COc1cc(-c2ccc(Cl)c(C)c2)c(F)cc1-c1ncnc2cc(S(=O)(=O)Nc3ccon3)ccc12
InChIKey: IBIWZZGLUXGVJX-UHFFFAOYSA-N
Target: Sodium channel protein type 9 subunit alpha (SINGLE PROTEIN), Homo sapiens
UniProt: Q15858
Assay: In Vitro PX Assay: HEK 293 cells stably transfected with human Nav1.7 were recorded in whole cell voltage clamp mode with the PatchXpress automated electrophysiology system (Molecular Devices, LLC, Sunnyvale, Calif.). Compound effects were measured on a partially inactivated state of the sodium channel. Cells were clamped to a holding potential yielding 20 to 50% inactivation. To elicit sodium current, channels were activated by pulsing to â¿¿10 mV for 20 msec. This voltage protocol was repeated at a rate of 0.1 Hz throughout the experiment. A single concentration of test compound was applied to cells for a duration of 3 minutes.
Measurement: IC50 = 24 nM (pChEMBL 7.619999885559082)
Descriptors: MW 524.96, ALogP 5.86, HBA 7, HBD 1, PSA 107.21, QED 0.3
Compound (SMILES): Cc1ccc(-c2ccc(NC(=O)[C@H](NC[C@@H](C)c3ccc(C#N)cc3)c3ccccc3)nc2)nn1
InChIKey: WEFNYOHMOPVIBQ-XHCCPWGMSA-N
Target: Histone acetyltransferase p300 (SINGLE PROTEIN), Homo sapiens
UniProt: Q09472
Assay: Biochemical Assay: The full length p300 SPA assay was run following the same protocol as p300 HAT SPA assay, but used 6 nM purified full length p300 (purchased from Active Motif) in place of the purified p300-HAT domain.Select compounds were also evaluated in a H3K18Ac MSD cellular assay that measures the ability of compounds to inhibit the the acetylation of chromatin at H3K18, a process catalyzed by p300 and CBP. In a typical experiment the p300 HAT inhibitory activity inside cells of the compounds described herein was determined in accordance with the following experimental method. 20 k HCT-116 cells per well are plated in 75 μL RPMI+10% FBS media the night before treatment. Compounds plated in DMSO at 4× final concentration are resuspended in 30 μL RPMI+10% FBS, then 25 μL is combined with corresponding wells containing cells. Treated cells are incubated for 2 hr at 37° C., then lysed in 500 μL final volume and frozen at −80° C. MSD plates (Meso Scale Discovery) are coated overnight at 4° C. with 60 μL 1:500 α-total histone antibody (Millipore MAB3422) in PBS. Plates are then blocked with 5% BSA in TBST shaking at RT for 1 hr, washed, and 30 μL lysate added to each well for 2 hr shaking at RT. Plates are washed and 25 μL 1:216 α-H3K18ac antibody (CST 9675) in PBS added, then incubated for 1 hr shaking at RT. Plates are washed again, then 25 μL 1:1000 Sulfo-Tag goat α-rabbit antibody (Meso Scale Discovery R32Ab-1) in PBS is added for 1 hr shaking at RT. Plates are washed once more, then 150 μL 1× Read Buffer (MSD #R92TD-3) is added to all wells and read on MSD SECTOR Imager 2400 using the conventional read setup.
Measurement: IC50 > 2000 nM (pChEMBL 5.698999881744385)
Descriptors: MW 462.56, ALogP 4.79, HBA 6, HBD 2, PSA 103.59, QED 0.39
Compound (SMILES): COc1c(F)cc(Cn2ncc(NC(=O)Cc3ccc(OCC4COC4)cc3)n2)cc1F
InChIKey: KGTZFYVRNHGHIN-UHFFFAOYSA-N
Target: Voltage-dependent T-type calcium channel subunit alpha-1H (SINGLE PROTEIN), Homo sapiens
UniProt: O95180
Assay: Measurement of Calcium Channel Flux by Means of FLIPR Assay: Stock solutions of test compounds are prepared to a concentration of 10 mM in DMSO. For the Cav3.2 assay, serial dilutions of the compounds are prepared in TEAC buffer (100 mM tetraethylammonium chloride; 20 mM Hepes; 2.5 mM CaCl2; 5 mM KCl; 1 mM MgCl2; 1% FBS; pH 7.2), for the Cav1.2 assay serial dilutions are prepared in assay buffer. Test compounds are added to the cells to give a 3-fold dilution range from 10 μM to 0.05 nM. The compounds are incubated with the cells for 3 minutes and Ca2+ entry is stimulated by adding either CaCl2 to a final concentration of 10 mM (Cav3.2 assay) or by adding KCl to a final concentration of 20 mM (Cav1.2 assay). The kinetics of fluorescence increase are recorded for every well and the area under the fluorescence trace for every compound concentration is used to generate inhibition curves using non-linear regression sigmoidal concentration-response curve analysis with in-house software. IC50 values are calculated and represent the compound concentration required to inhibit 50% of the signal that is obtained in the presence of vehicle instead of test compound. In analogy, antagonistic activities (IC50 values) of all exemplified compounds have been measured for the for the Cav3.1- and the Cav3.3-channel. Antagonistic activities (IC50 values) of all exemplified compounds are in the range of 1.7 to 970 nM with respect to Cav3.1; and in the range of 1.1 to 620 nM with respect to Cav3.3.
Measurement: IC50 = 86 nM (pChEMBL 7.070000171661377)
Descriptors: MW 444.44, ALogP 2.82, HBA 7, HBD 1, PSA 87.5, QED 0.55
Compound (SMILES): C[C@H](NC(=O)c1c(N)nn2cccnc12)c1cc2cccc(Cl)c2c(=O)n1-c1cccc(S(C)(=O)=O)c1
InChIKey: JFOUTONBSIXQAW-AWEZNQCLSA-N
Target: Phosphatidylinositol 3-kinase regulatory subunit beta (SINGLE PROTEIN), Homo sapiens
UniProt: O00459
Assay: Inhibition Assay: Class I PI3-Ks can be either purchased (p110α/p85α, p110β/p85α, p110δ/p85α from Upstate, and p110γ from Sigma) or expressed as previously described (Knight et al., 2004). IC50 values are measured using either a standard TLC assay for lipid kinase activity (described below) or a high-throughput membrane capture assay. Kinase reactions are performed by preparing a reaction mixture containing kinase, inhibitor (2% DMSO final concentration), buffer (25 mM HEPES, pH 7.4, 10 mM MgCl2), and freshly sonicated phosphatidylinositol (100 μg/ml). Reactions are initiated by the addition of ATP containing 10 μCi of γ-32P-ATP to a final concentration of 10 or 100 μM and allowed to proceed for 5 minutes at room temperature. For TLC analysis, reactions are then terminated by the addition of 105 μL 1N HCl followed by 160 μL CHCl3:MeOH (1:1). The biphasic mixture is vortexed, briefly centrifuged, and the organic phase is transferred to a new tube using a gel loading pipette tip precoated with CHCl3. This extract is spotted on TLC plates and developed for 3-4 hours in a 65:35 solution of n-propanol:1M acetic acid. The TLC plates are then dried, exposed to a phosphorimager screen (Storm, Amersham), and quantitated. For each compound, kinase activity is measured at 10-12 inhibitor concentrations representing two-fold dilutions from the highest concentration tested (typically, 200 μM). For compounds showing significant activity, IC50 determinations are repeated two to four times, and the reported value is the average of these independent measurements.
Measurement: IC50 = 750 nM (pChEMBL 6.119999885559082)
Descriptors: MW 537, ALogP 3.16, HBA 9, HBD 2, PSA 141.45, QED 0.35
Compound (SMILES): O=C(CN1CN(c2ccccc2)C2(CCN(C(=O)c3ccc4cn[nH]c4c3)CC2)C1=O)Nc1ccccc1
InChIKey: HPIYVMMUJURNEW-UHFFFAOYSA-N
Target: Epithelial discoidin domain-containing receptor 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q08345
Assay: In Vitro Binding Assay for DDR1: This assay is based on the intracellular domain of the DDR1 protein which contains the kinase active site. The recombinant protein additionally carries a GST-tag that can be recognized by an Eu-labeled anti-GST antibody. A tracer compound binding to the active site is labeled with a dye so that a FRET donor acceptor pair can be formed. Excitation energy absorbed by the Europium complex (350 nm flash light or pulsed laser) is transferred to a suitable fluorescent dye, if it is in close proximity. Compounds binding competitively with the tracer molecule will displace the bound tracer molecules and reduce the FRET signal in a dose dependent manner. Due to the long lifetime of the Eu excited state, the emission of the donor and the acceptor can be measured in time-gated mode such that most of the intrinsic fluorescence contributions have already decayed. This results in high sensitivity, excellent reproducibility and high data quality. This sensitive detection method enables protein concentrations below 20 nM.Protein, tracer and labeled antibody were obtained from commercial sources. The assay was performed in 384 low volume microtiter-plates with a final volume of 15 μl. Dose response curves were generated from 16 compound dilutions in DMSO as solvent compound dilutions, a solution containing protein and labeled antibody, and a solution containing the tracer which is added in the last step. The fluorescence of donor and acceptor were then measured after one hour incubation at room temperature. Every assay run was quality-controlled with dose response curves for two reference compounds.
Measurement: IC50 = 496 nM (pChEMBL 6.300000190734863)
Descriptors: MW 508.58, ALogP 3.48, HBA 5, HBD 2, PSA 101.64, QED 0.43
Compound (SMILES): COc1cc(C#Cc2cn(C3CN(C(=O)/C=C/CN4CCC4)C3)c3ncnc(N)c23)cc(OC)c1
InChIKey: LJEXNLQQUUZCQM-HWKANZROSA-N
Target: Fibroblast growth factor receptor 2 (SINGLE PROTEIN), Homo sapiens
UniProt: P21802
Assay: FGFR2 Kinase Activity: When setting conditions for the measurement of the inhibitory effect of the compounds on FGFR2 kinase activity, FL-Peptide 22 (Caliper Life Sciences, Inc.) was used as a substrate. The purified recombinant human FGFR2 protein used in the test was purchased from Carna Biosciences, Inc. In the measurement of the inhibitory effect of the compounds, first, a test compound was gradually diluted with dimethylsulfoxide (DMSO) to a concentration that was 20 times higher than the final concentration. Next, the purified human FGFR2 protein, FL-Peptide 22 (final concentration: 1.5 μM), magnesium chloride (final concentration: 5 mM), ATP (final concentration: 75 μM), and the test compound DMSO solution (final concentration of DMSO: 5%) were added to a reaction buffer (15 mM Tris-HCl pH 7.5, 0.01% Tween-20, 2 mM DTT), and the mixture was incubated at 25° C. for 120 minutes to perform a kinase reaction. EDTA (final concentration: 30 mM) diluted with a separation buffer (Caliper Life Sciences, Inc.) was added thereto to terminate the kinase reaction. Finally, using a LabChip (registered trademark) 3000 system (Caliper Life Sciences, Inc.; excitation wavelength: 488 nm, detection wavelength: 530 nm), phosphorylated peptides and non-phosphorylated peptides were separated, and the amount of each peptide was measured. The level of phosphorylation was determined from the quantitative ratio. The compound concentration at which phosphorylation was inhibited by 50% was defined as the IC50 value (nM).
Measurement: IC50 = 2.5 nM (pChEMBL 8.600000381469727)
Descriptors: MW 472.55, ALogP 2.08, HBA 8, HBD 1, PSA 98.74, QED 0.43
Compound (SMILES): c1ccc(Nc2ncc3[nH]c(-c4ccccc4)cc3n2)cc1
InChIKey: YHCGTIVCLOQBAU-UHFFFAOYSA-N
Target: Receptor-interacting serine/threonine-protein kinase 2 (SINGLE PROTEIN), Homo sapiens
UniProt: O43353
Assay: RIPK2 Inhibition Assay: RIPK2 activity was measured using an indirect ELISA detection system. HisRIPK2 (0.6 nM) was incubated in the presence of 6 μM ATP (Sigma cat # A7699), 20 mM Hepes, pH 7.5, 1 mM EGTA, 2.5 mM MgCl2, 2.5 mM MnCl2 and 0.01% Triton X-100 in a 96 well microtitre plate pre-coated with amino terminal 6 histidine, sumo tagged TTK (amino acid residues 1-275). The reaction was allowed to proceed for 30 minutes, followed by 5 washes of the plate with Wash Buffer (phosphate buffered saline supplemented with 0.2% Tween 20), and incubation for 30 minutes with a 1:3000 dilution of primary antibody (Cell Signaling cat #9381). The plate was washed 5 times with Wash Buffer, incubated for 30 minutes in the presence of secondary antibody coupled to horse radish peroxidase (BioRad cat #1721019, 1:3000 concentration), washed an additional 5 times with Wash Buffer, and incubated in the presence of TMB substrate (Sigma cat # T0440). The colorimetric reaction was allowed to continue for 5 minutes, followed by addition of stop solution (0.5 N H2SO4), and quantified by detection at 450 nm with either a monoChromatic or filter based plate reader (Molecular Devices M5 or Beckman DTX880, respectively).
Measurement: IC50 > 500 nM (pChEMBL 6.301000118255615)
Descriptors: MW 286.34, ALogP 4.37, HBA 3, HBD 2, PSA 53.6, QED 0.59
Compound (SMILES): CC(C(=O)Nc1cnc2ccccn12)[C@H]1[C@@H]2C[C@@H](n3cnc4cc(F)c(F)cc43)C[C@@H]21
InChIKey: LIKRLOAPJSTOMH-GZUFLWRWSA-N
Target: Indoleamine 2,3-dioxygenase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P14902
Assay: IDO1 Cell-Based Assay: HeLa cells were obtained from the American Type Culture Collection (ATCC) and maintained in DMEM media containing 10% FBS. Cells (7,000/well) were seeded onto a 384 well plate in 50 μL of media and incubated at 37° C., 5% CO2 overnight. Cell media was aspirated, fresh media containing 10 ng/mL IFNgamma was added, and cells were incubated in absence or presence of various concentrations of test compound (final 0.5% DMSO) for 24 hours. Aliquots of the cell conditioned media were removed from the cell plate, and mixed with an equal volume of 200 mM ZnSO4 to precipitate media containing proteins. Two volumes of acetonitrile were added, mixed, and samples were then centrifuged at 2250 G for 20 minutes at 4° C. Aliquots of the supernatant were diluted 1:10 in 0.1% formic acid containing 3 μM of deuterated Tryptophan as an internal standard.Samples were analyzed via RFMS to quantify N-Formyl Kynurenine (AUC) and L-tryptophan (AUC). A C18 cartridge was used with mobile phases of 0.1% Formic Acid and 80% ACN/0.1% Formic Acid under isocratic conditions. Dose-response curves were analyzed using IC50 regression curve fitting (GeneData Screener). Curves were plotted as percent of control and normalized by high controls without inhibitor (100%), and low controls (0%) containing 1 μM of epacadostat, a potent cell-permeable IDO1 inhibitor. Cell viability was also assessed using the Cell Titer Glo Kit (Promega) following manufacturer recommendation.
Measurement: IC50 = 495 nM (pChEMBL 6.300000190734863)
Descriptors: MW 421.45, ALogP 4.43, HBA 5, HBD 1, PSA 64.22, QED 0.53
Compound (SMILES): Cc1ccc(/C=C/CC(=O)Nc2ccc(C(=O)Nc3ccc(F)cc3N)cc2)cn1
InChIKey: FFYPWXUBGIMTJU-NSCUHMNNSA-N
Target: Histone deacetylase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q13547
Assay: HDACs 1, 2, and 3 Enzymatic Inhibitions: HDACs assay was performed according to standard Protocols (Fluorgenic HDACs 1, 2, and 3 assay kit, BPS Bioscience). All of the compounds, with Chidamide and Entinostat as positive control, at doses ranging from 20 μM to 1.28 nM, were mixed with kit buffer and incubated at 37° C. for 1 hour. After 1 h, assay developer was added to the samples and the absorbance was read at fluorogenic wavelength. The relative inhibition to HDACs 1, 2, and 3 activities in each sample was determined. Example 10 Enzyme Inhibition Kinetic of HDAC3 by GNTbm-02 was Determined HDAC3
Measurement: IC50 = 560 nM (pChEMBL 6.25)
Descriptors: MW 404.45, ALogP 4.41, HBA 4, HBD 3, PSA 97.11, QED 0.53
Compound (SMILES): COC(=O)c1cnc(N)c(-c2cc3cc(NC(=O)Nc4cc(C(F)(F)F)ccc4F)ccc3s2)c1
InChIKey: LSCATNNDFBVKMR-UHFFFAOYSA-N
Target: Platelet-derived growth factor receptor beta (SINGLE PROTEIN), Homo sapiens
UniProt: P09619
Assay: Biochemical Assay: Biochemical PDGFRβ kinase assays were performed in 96 well microtiter plates that were coated overnight with 75 μg of poly-Glu-Tyr (4:1) in 10 mM Phosphate Buffered Saline (PBS), pH 7.4. The coated plates were washed with 2 mls per well PBS+0.05% Tween-20 (PBS-T), blocked by incubation with PBS containing 1% BSA, then washed with 2 mls per well PBS-T prior to starting the reaction. Reactions were carried out in 100 μL reaction volumes containing 36 μM ATP in kinase buffer (50 mM Hepes buffer pH 7.4, 20 mM MgCl2, 0.1 mM MnCl2 and 0.2 mM Na3VO4). Test compounds were reconstituted in 100% DMSO and added to the reaction to give a final DMSO concentration of 5%. Reactions were initiated by the addition 20 μl per well of kinase buffer containing 200-300 ng purified cytoplasmic domain PDGFR-b protein (Millipore). Following a 60 minute incubation at 30° C., the reactions were washed 2 mls per well PBS-T. 100 μl of a monoclonal anti-phosphotyrosine antibody-peroxidase conjugate diluted 1:10,000 in PBS-T was added to the wells for 30 minutes. Following a 2 mls per well wash with PBS-Tween-20, 100 μl of O-Phenylenediamine Dihydrochloride in phosphate-citrate buffer, containing urea hydrogen peroxide, was added to the wells for 7-10 minutes as a colorimetric substrate for the peroxidase. The reaction was terminated by the addition of 100 μl of 2.5N H2SO4 to each well and read using a microplate ELISA reader set at 492 nm. IC50 values for compound inhibition were calculated directly from graphs of optical density (arbitrary units) versus compound concentration following subtraction of blank values.
Measurement: IC50 = 10 nM (pChEMBL 8)
Descriptors: MW 504.47, ALogP 6.13, HBA 6, HBD 3, PSA 106.34, QED 0.23
Compound (SMILES): Cc1nn(C)c2c(-n3nc(COC(C)(C)C(=O)O)cc3-c3cccc(OCC(C)(C)C)c3)cccc12
InChIKey: LONCIMGLEVRXRB-UHFFFAOYSA-N
Target: Monocarboxylate transporter 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P53985
Assay: Biological Activity Assay 4: MCT1 activity may be measured using BT-20 breast cancer cells that express high native levels of MCT1, but do not express MCT4 and are known to those with skill in the art. Preparation of BCECF loaded cells are as described for Assay 1. Lactate transport assay is as described for Assay 1, except 10 mM L-lactate (rather than 50 mM) is added.
Measurement: IC50 = 7300 nM (pChEMBL 5.139999866485596)
Descriptors: MW 490.6, ALogP 5.54, HBA 7, HBD 1, PSA 91.4, QED 0.35
Compound (SMILES): COc1ccc(-c2nc3ccn(C[C@@H]4CCNC4)c3cc2-c2ccc(C#N)c(F)c2)cc1F
InChIKey: WPVOPJWYYVEJHN-MRXNPFEDSA-N
Target: Lysine-specific histone demethylase 1A (SINGLE PROTEIN), Homo sapiens
UniProt: O60341
Assay: In Vitro Enzyme Inhibition Assay - LSD-1: The enzymatic assay of LSD1 activity is based on Time Resolved-Fluorescence Resonance Energy Transfer (TR-FRET) detection. The inhibitory properties of compounds to LSD1 were determined in 384-well plate format under the following reaction conditions: 0.1 nM LSD1, 50 nM H3K4mel-biotin labeled peptide (Anaspec cat #64355), 2 μM FAD in assay buffer of 50 mM HEPES, pH7.3, 10 mM NaCl, 0.005% Brij35, 0.5 mM TCEP, 0.2 mg/ml BSA. Reaction product was determined quantitatively by TR-FRET after the addition of detection reagent Phycolink Streptavidin-allophycocyanin (Prozyme) and Europium-anti-unmodified histone H3 lysine 4 (H3K4) antibody (PerkinElmer) in the presence of LSD1 inhibitor such as 1.8 mM of Tranylcypromine hydrochloride (2-PCPA) in LANCE detection buffer (PerkinElmer) to final concentration of 12.5 nM and 0.25 nM respectively.The assay reaction was performed according to the following procedure: 2 μL of the mixture of 150 nM H3K4mel-biotin labeled peptide with 2 μL of 11-point serial diluted test compound in 3% DMSO was added to each well of plate, followed by the addition of 2 μL of 0.3 nM LSD1 and 6 μM of FAD to initiate the reaction. The reaction mixture was then incubated at room temperature for one hour, and terminated by the addition of 6 μL of 1.8 mM 2-PCPA in LANCE detection buffer containing 25 nM Phycolink Streptavidin-allophycocyanin and 0.5 nM Europium-anti-unmodified H3K4 antibody. Plates were read by EnVision Multilabel Reader in TR-FRET mode (excitation at 320 nm, emission at 615 nm and 665 nm) after 1 hr incubation at room temperature. A ratio was calculated (665/615) for each well and fitted to determine inhibition constant (IC50).
Measurement: IC50 < 10 nM (pChEMBL 8)
Descriptors: MW 444.49, ALogP 5.14, HBA 5, HBD 1, PSA 62.87, QED 0.46