lovingscience/chembl-v37-raw-activities huggingface dataset:
Compound (SMILES): CC1CN(c2ccc(NC3=NC4C(C=N3)C=C3C(=O)NCC5(CCCC5)N34)nc2)CC(C)O1
InChIKey: LUNNUDCFOOEYJM-UHFFFAOYSA-N
Target: Cyclin-dependent kinase 2/cyclin E1 (PROTEIN COMPLEX), Homo sapiens
UniProt: P24864
Assay: Biological Activity Assay: Kinase enzymatic reactions were performed in 384-well microplates using a 12-channel Caliper LabChip instrument as a detection device. The enzymatic phosphorylation of a peptide results in a change in net charge, enabling electrophoretic separation of product from substrate. As substrate and product are separated, two peaks of fluorescence are observed. Change in the relative fluorescence intensity of the substrate and product peaks is the parameter measured, reflecting enzyme activity. In the presence of an inhibitor, the ratio between product and substrate is altered. The signal of the product decreases, while the signal of the substrate increases.For the measurement of CDK2/cyclinE activity, enzyme (0.22 nM) was incubated with 100 mM ATP and the phosphoacceptor substrate peptide (1 mM) for one hour. For the measurement of CDK4/CyclinD activity, enzyme (0.85 nM) was incubated with 200 mM ATP and the phosphoacceptor substrate peptide (1 mM) for three hours. Potential inhibitor compounds (as HCl salts) were tested using 12-point dose response curves in single point at the Km for ATP.
Measurement: IC50 = 50500 nM (pChEMBL 4.300000190734863)
Descriptors: MW 449.56, ALogP 2.13, HBA 8, HBD 2, PSA 94.45, QED 0.72
Compound (SMILES): COCCNC(=O)CN1CCN(C(=O)c2ccc(N/C(=C3\C(=O)Nc4cc(C(=O)OC)c(C)cc43)c3ccccc3)cc2)CC1
InChIKey: OLBVLPUBUBRBGW-KTMFPKCZSA-N
Target: Fibroblast growth factor receptor 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P11362
Assay: FGFR1 Enzyme Inhibition: The inhibitory activities of compounds of the invention against FGFR1 (FGFR1 Kinase Enzyme System: Promega), were evaluated by mixing the FGFR1 protein (3.12 ng/mL, 2 μL), substrate (Poly (4:1 Glu4, Tyr1), 100 ng/mL, 2 μL) with the test compound (2 μL at either 3 μM, 0.67 μM, 0.15 μM, 0.033 μM, 0.0073 μM, 0.0016 μM, 0.0036 μM or 0.00008 μM) for 90 min at 25° C. The kinase reaction was then initiated by adding ATP (50 μM, 2 μL) and the mixture was incubated for 1 hr at 25° C. ADP-Glo reagent was added for 40 min (8 μL) then development reagent (16 μL) was added for 40 min prior to detection in a microplate reader (EnVision Multilabel Reader, Perkin Elmer).
Measurement: Ki = 47.4 nM (pChEMBL 7.320000171661377)
Descriptors: MW 611.7, ALogP 3.23, HBA 8, HBD 3, PSA 129.31, QED 0.18
Compound (SMILES): COc1cc(C(=O)N2CC3CCC2[C@@H]3N)cc2nc(-c3cc4ccc(-c5cnc6[nH]c(=O)oc6c5)nc4n3CC3CC3)n(C)c12
InChIKey: ZLXKVDDXBJNFCQ-IBYOELKQSA-N
Target: Protein-arginine deiminase type-4 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9UM07
Assay: Rapid-Fire Mass Spectrum (RFMS) Assay: Compounds were solubilized in 100% DMSO to achieve a 10 mM compound concentration. Compound stock solutions were stored at RT. A series of dilutions were prepared in DMSO and mixed 8 times with 20 μL mixing volume. Final top concentration of compound in the assay is 50 μM. Final assay conditions were as follows:Reaction volume: 26 μlAssay buffer: 25 mM hepes, pH 7.5, 5 mM NaCl, 1 mM DTT, 0.2 mg/ml BSA, 0.01% CHAPS, 50 μM Calcium, and 5 μM TPENFinal concentrations: 5 nM hPAD4 enzyme, 250 μM BAEE, and 0.5% DMSOTotal incubation time: 30 mins compound and enzyme preincubation at 37° C., 90 min enzyme/substrate reaction, 30 min reaction with phenyl glyoxal at 37° C.Stop solution: 40 μl 5% TCA in ACN
Measurement: IC50 = 71 nM (pChEMBL 7.150000095367432)
Descriptors: MW 604.67, ALogP 4.07, HBA 10, HBD 2, PSA 150.09, QED 0.29
Compound (SMILES): CC[C@H]1CC[C@@H](c2ccccc2)S(=O)(=O)N1Cc1ccc(N2C[C@@H]3[C@H](C2)[C@H]3n2cnnc2)cc1F
InChIKey: NLZOBQVDLKJKTG-OECXIXJESA-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 = 51 nM (pChEMBL 7.289999961853027)
Descriptors: MW 495.62, ALogP 4.17, HBA 6, HBD 0, PSA 71.33, QED 0.51
Compound (SMILES): Cn1cc(-c2cc(-c3ccc(N4CCN(Cc5cc(F)cc(F)c5)CC4)nc3)c3c(C#N)cnn3c2)cn1
InChIKey: BCORKFVUMGRFED-UHFFFAOYSA-N
Target: Proto-oncogene tyrosine-protein kinase receptor Ret (SINGLE PROTEIN), Homo sapiens
UniProt: P07949
Assay: RET Enzyme Assay: Compounds of General Formula I were screened for their ability to inhibit wild type and V804M mutant RET kinase using CisBio's HTRF® KinEASE -TK assay technology. Briefly, N-terminal GST tagged recombinant human RET cytoplasmic domain (aa 658-end) from Eurofins (0.25 nM RET; Cat. No. 14-570M) or N-terminal GST tagged recombinant human V804M mutant RET cytoplasmic domain (aa 658-end) from Millipore (0.25 nM enzyme; Cat. No. 14-760) was incubated with 250 nM TK-substrate biotin (CisBio, part of Cat. No. 62TK0PEC) and 1 mM ATP along with test compound in a buffer consisting of 25 mM HEPES pH 7.4, 10 mM MgCl2, 0.01% Triton X-100, and 2% DMSO in a volume of 8 μL. Compounds were typically prepared in a threefold serial dilution in DMSO and added to the assay to give the appropriate final concentration. After a 30-minute incubation at 22° C., the reaction was quenched by adding 8 μL of quench solution containing 31.25 nM Sa-XL665 and 1×TK-ab-Cryptate in HTRF detection buffer (all from CisBio, part of Cat. No. 62TK0PEC). After a 1 hour incubation at 22° C., the extent of reaction was determined using a PerkinElmer EnVision multimode plate reader via HTRF dual wavelength detection, and the percent of control (POC) was calculated using a ratiometric emission factor. 100 POC was determined using no test compounds and 0 POC was determined using pre-quenched control reactions. The POC values were fit to a 4 parameter logistic curve, and the IC50 is defined as the concentration of inhibitor at which the POC equals 50 for the fitted curve.
Measurement: IC50 = 9 nM (pChEMBL 8.050000190734863)
Descriptors: MW 510.55, ALogP 4.27, HBA 8, HBD 0, PSA 78.28, QED 0.35
Compound (SMILES): CN1C(=O)[C@@H](NC(=O)c2cc(Oc3ccccc3)ccn2)COc2ccc(C#CC(C)(C)O)nc21
InChIKey: KOUHIWPWBGFRSA-NRFANRHFSA-N
Target: Receptor-interacting serine/threonine-protein kinase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q13546
Assay: Kinase Inhibition Assay: In this example, compounds of the disclosure were evaluated using a biochemical assay using the ADP-Glo technology. ADP-Glo (Promega, Madison, Wis., USA) reagents were thawed at ambient temperature. Kinase Detection Reagent was prepared by mixing kinase detection buffer with the lyophilized kinase detection substrate. A 500 ml stock volume of 5× Reaction Kinase Buffer was made by mixing 1000 μl of 1M MgCl2, 500 μl of 1M Tris-HCL pH7.4, 0.5 mg/ml (25 mg) of BSA, and 3475 μl of distilled H2O. A 3 ml 2× working stock volume of Reaction Kinase Buffer was made containing a final concentration of 100 μM DTT and 4 mM MnCl2.Components of RIPK1 enzyme (Rigel Pharmaceuticals, South San Francisco, Calif., USA) were thawed on ice. Diluted RIPK1 was prepared in 1× Kinase Reaction Buffer (diluted from 2× buffer) to 31 ng/well. A 166 M working stock ATP assay solution was prepared in 1× Kinase Reaction Buffer (diluted from 2× buffer).Compounds were serially diluted in DMSO from 250 uM in 4-fold dilutions then diluted 1:5 in 2× Reaction Buffer in a 96 well plate. 1.0 ul of diluted compound was added to a 384 well plate in duplicate. 2 μl of diluted Active RIPK1 was added to 384 well plate (do not add to column 1) add 2× r×n buffer to column 1. AKT (Anaspec, Fremont, Calif., USA) at 150 nM was combined with ATP working stock at equal volume and 2 ul/well were added to the 384 well plate. The final reaction volume was 5.0 μl.
Measurement: IC50 = 22.7 nM (pChEMBL 7.639999866485596)
Descriptors: MW 472.5, ALogP 2.55, HBA 7, HBD 2, PSA 113.88, QED 0.56
Compound (SMILES): CCOc1cc(-c2ccc(N3CCC(C)(NC(=O)OCC4CC4)CC3)nc2)c2c(C#N)cnn2c1
InChIKey: HCXGWEIZPFERRU-UHFFFAOYSA-N
Target: Proto-oncogene tyrosine-protein kinase receptor Ret (SINGLE PROTEIN), Homo sapiens
UniProt: P07949
Assay: RET Enzyme Assay: Compounds of Formula I were screened for their ability to inhibit wildtype and V804M mutant RET kinase using CisBio's HTRF KinEASE-TK assay technology. Briefly, N-terminal GST tagged recombinant human RET cytoplasmic domain (aa 658-end) from Eurofins (0.25 nM RET; Catalog No. 14-570M) or N-terminal GST tagged recombinant human V804M mutant RET cytoplasmic domain (aa 658-end) from Millipore (0.25 nM enzyme; Catalog No. 14-760) was incubated with 250 nM TK-substrate biotin (CisBio, part of Catalog No. 62TK0PEC) and 1 mM ATP along with test compound in a buffer consisting of 25 mM HEPES pH 7.4, 10 mM MgCl2, 0.01% Triton X-100, and 2% DMSO in a volume of 8 μL. Compounds were typically prepared in a threefold serial dilution in DMSO and added to the assay to give the appropriate final concentration. After a 30-minute incubation at 22° C., the reaction was quenched by adding 8 μL of quench solution containing 31.25 nM Sa-XL665 and 1×TK-ab-Cryptate in HTRF detection buffer (all from CisBio, part of Cat. No. 62TK0PEC). After a 1 hour incubation at 22° C., the extent of reaction was determined using a PerkinElmer EnVision multimode plate reader via HTRF dual wavelength detection, and the percent of control (POC) was calculated using a ratiometric emission factor. 100 POC was determined using no test compounds and 0 POC was determined using pre-quenched control reactions. The POC values were fit to a 4 parameter logistic curve, and the IC50 is defined as the concentration of inhibitor at which the POC equals 50 for the fitted curve.
Measurement: IC50 = 19.9 nM (pChEMBL 7.699999809265137)
Descriptors: MW 474.57, ALogP 4.16, HBA 8, HBD 1, PSA 104.78, QED 0.55
Compound (SMILES): COc1cc(CNC(=O)c2cc(Cn3ccn(C)c3=N)cc(-c3c(C)cc(F)cc3C)c2)cc(OC)c1
InChIKey: XJZNKYPGIBYVEP-UHFFFAOYSA-N
Target: WD repeat-containing protein 5 (SINGLE PROTEIN), Homo sapiens
UniProt: P61964
Assay: TR-FRET Binding Assay : A Time-Resolved Fluorescence Resonance Energy Transfer (TR-FRET) assay that measures the displacement of the 10mer-Thr-FAM probe in response to compound treatment was performed for compounds wherein the IC50 from FPA assay using 10mer-Thr-FAM was below the lower assay IC50 limit 1 nM. Excess 10mer-Thr-FAM probe was utilized with His-tagged WDR5 in conjunction with a commercial anti-His antibody containing a Terbium label. The LanthaScreen Elite Tb-anti-HIS Antibody from ThermoFisher Scientific was used for this purpose. This Th-anti-HIS has an excitation/emission of 340 nm and 490 nm, respectively. The 10mer-Thr-FAM probe when bound to WDR5 will undergo a FRET interaction with the Tb-anti-HIS and emit at 520 nm. The ratio of the 520 and 495 signals are then utilized to generate a dose-response curve to calculate an IC50 value. By virtue of FRET there is little to no background fluorescence interference from 10mer-Thr-FAM probe allowing an excess of the probe to be used permitting an increase in the lower limit of the calculated Ki when testing against highly potent inhibitors with Ki<<1 nM. WDR5-His Tag (A23, residues 24-334) is expressed and purified in our lab in sufficient quantities for screening. 10mer-Thr-FAM peptide is used anywhere from 15 to 150 nM depending on the window of sensitivity required. WDR5-His tag protein is used at 2 nM. A source plate is prepared using an Echo Liquid Handler, which distributes the compounds to the assay plate (white, flat-bottom; OptiPlate) in a 10-point, 3-fold dilution schemes with a top concentration of either 5 or 20 μM depending on the anticipated potency of the compounds, in a final volume of 20 μL. A final target (WDR5)/Tb-Ab concentration of 2 nM/1 nM is dispensed from appropriate stock solutions, respectively. The final DMSO concentration in each well of the assay plate is 1% or lower. As before the plate is covered, shielded from light, and incubated for 60 minutes at room temperature with rocking. Anisotropy is then measured on a Biotek Cytation 3 at excitation wavelength of 340 nm, and emission wavelengths of 495 nm and 520 nm. Working buffer conditions (pH 7.0) are similar to that in FPA above. TR-FRET signal is plotted and IC50 and Ki values are calculated in the same manner as the fluorescence polarization anisotropy based competition assays.
Measurement: Ki = 0.63 nM (pChEMBL 9.199999809265137)
Descriptors: MW 502.59, ALogP 4.72, HBA 6, HBD 2, PSA 81.27, QED 0.37
Compound (SMILES): CCC(O)(CC)Cn1ncc2cc(-c3ccc(C(=O)N4[C@@H]5CC[C@H]4[C@@H](N)C5)cc3-c3ccc(C#N)c(F)c3)c(F)cc21
InChIKey: OTQUVMAHLBKNQO-STQWGSIPSA-N
Target: Lysine-specific histone demethylase 1A (SINGLE PROTEIN), Homo sapiens
UniProt: O60341
Assay: Inhibitory Activity Assay: The conditions for measuring inhibitory activity of compounds against LSD1 activity were determined with reference to a document available from the website of PerkinElmer (U-TRF #38) and a patent of GlaxoSmithKline (WO2012135113).To measure the inhibitory activity, first, the compound of the present invention was serially diluted in dimethylsulfoxide (DMSO). Sequentially, the solution of the compound of the present invention in DMSO (final concentration of DMSO: 5%) and human LSD1 protein (Abcam, ab80379) were added to a reaction buffer (25 mM Tris-HCl (pH 7.5), 50 mM KCl, 2 mM CHAPS, 1 mM DTT, 0.02% BSA). The mixture was preincubated at 25° C. for 30 minutes. Thereafter, a H3K4 (Me1)-biotin-labeled peptide (Anaspec #64355) (final concentration: 200 nM) was added thereto and reacted for 60 minutes. Tranylcypromine (final concentration: 3 mM) was then added thereto to terminate the reaction. Thereafter, a detection solution containing an Eu-labeled anti-H3K4 antibody (PerkinElmer, TRF0404) and Streptavidin Alexa Fluor 647 (Thermo Fisher Scientific, 521374) was added thereto, and the mixture was allowed to stand at room temperature for 1 hour. Finally, the intensity of fluorescence under the excitation light with a wavelength of 337 nm was measured with a PHERAstar FS (BMG Labtech) at two wavelengths: 620 nm and 665 nm. The demethylation level was calculated from the ratio of the fluorescence intensity at the two wavelengths, and the compound concentration at which demethylation was inhibited by 50% was defined as IC50 (nM).
Measurement: IC50 = 0.3 nM (pChEMBL 9.520000457763672)
Descriptors: MW 569.66, ALogP 5.78, HBA 6, HBD 2, PSA 108.17, QED 0.29
Compound (SMILES): COc1cc(-c2cccc(-c3cccc(NC(=O)c4c[nH]c(=O)n(C)c4=O)c3C)c2C)cc(F)c1CN[C@H]1COC[C@@H]1O
InChIKey: UCMOYJWVBCVLIL-NSOVKSMOSA-N
Target: Programmed cell death 1 ligand 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9NZQ7
Assay: Enzyme-Linked Immunosorbent AssayELISA: 96 Well plates were coated with 111 g/mL of human PD-L1 (obtained from R&D) in PBS overnight at 4° C. The wells were then blocked with 2% BSA in PBS (W/V) with 0.05% TWEEN-20 for 1 hour at 37° C. The plates were washed 3 times with PBS/0.05% TWEEN-20 and the compounds were serial diluted (1:5) in dilution medium and added to the ELISA plates. Human PD-1 and biotin 0.31 μg/mL (ACRO Biosystems) were added and incubated for 1 hour at 37° C. then washed 3 times with PBS/0.05% TWEEN-20. A second block was performed with 2% BSA in PBS (W/V)/0.05% TWEEN-20 for 10 min at 37° C. and the plates were washed 3 times with PBS/0.05% TWEEN-20. Streptavidin-HRP was added for 1 hour at 37° C. then the plates were washed 3 times with PBS/0.05% TWEEN-20. TMB substrate was added and reacted for 20 min at 37° C. A stop solution (2 N aqueous H2SO4) was added. The absorbance was read at 450 nm using a micro-plate spectrophotometer.
Measurement: IC50 < 10 nM (pChEMBL 8)
Descriptors: MW 588.64, ALogP 3.27, HBA 8, HBD 4, PSA 134.68, QED 0.25
Compound (SMILES): Cc1cc2c(cc1C#N)c(-c1cncc(N)c1Cl)cn2C1CCCC(C(N)=O)C1
InChIKey: FWYSGLDOSVIVAZ-UHFFFAOYSA-N
Target: Lysine-specific histone demethylase 1A (SINGLE PROTEIN), Homo sapiens
UniProt: O60341
Assay: FL-LSD1 LC-MS Assay: Representative compounds of the present invention were serially and separately diluted 3-fold in DMSO to obtain a total of twelve concentrations. Then the test compounds at each concentration (100 nL of each) were transferred into a 384-well Perkin Elmer ProxiPlate 384 plus plates by Mosquito Solutions (5 μL) of 0.8 nM, the full-length LSD1 and 0.5 μM FAD in reaction buffer (40 mM Tris-HCl, 0.01% Triton-×100.10 mM KCl, 1 mM DTT) were added into the wells and then incubated with the test compound for 30 min. A 5 μL solution of 1 μM of the peptide substrate H3K4me1 (histone H3[1-21]-biotin) in reaction buffer was added to each initiate reaction. The final components in the reaction solution include 0.4 nM FL-LSD1, 0.25 μM FAD, and 0.5 μM H3K4me1 peptide with varying concentration of the compounds. A positive control consisted of the enzyme, 0.25 μM FAD and 0.5 μM substrate in the absence of the test compound, and a negative control consisted of 0.5 μM substrate only. Each reaction was incubated at room temperature for 60 min, and then stopped by the addition of 3 μL quench solution (2.5% TFA with 320 nM d4-SAH). The reaction mixture was centrifuged (Eppendorf centrifuge 5810, Rotor A-4-62) for 1 min at 2000 rpm and read on an API 4000 triple quadrupole mass spec with Turbulon Spray (Applied Biosystem) coupled with Prominence UFLC (Shimadzu).
Measurement: IC50 = 34 nM (pChEMBL 7.46999979019165)
Descriptors: MW 407.91, ALogP 4.34, HBA 5, HBD 2, PSA 110.72, QED 0.67
Compound (SMILES): CN1CCN(c2ccc(NCc3ccc(-c4cccc5nc(NC(=O)C6CC6)nn45)cc3)cn2)CC1
InChIKey: MFOGBHHPNSJUBL-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 < 100 nM (pChEMBL 7)
Descriptors: MW 482.59, ALogP 3.5, HBA 8, HBD 2, PSA 90.69, QED 0.42
Compound (SMILES): O=C(OC(C(F)(F)F)C(F)(F)F)N1CCC2(CCCN2C(=O)c2cncnc2C(F)(F)F)CC1
InChIKey: NUGHZFYNULUAET-UHFFFAOYSA-N
Target: Monoglyceride lipase (SINGLE PROTEIN), Homo sapiens
UniProt: Q99685
Assay: In Vitro Competitive Activity-Based Protein Profiling: Proteomes (human prefrontal cortex or cell membrane fractions) (50 μL, 1.0 mg/mL total protein concentration) were preincubated with varying concentrations of inhibitors at 37° C. After 30 min, FP-Rh or HT-01 (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4×) 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 = 550 nM (pChEMBL 6.260000228881836)
Descriptors: MW 508.34, ALogP 4.2, HBA 5, HBD 0, PSA 75.63, QED 0.56
Compound (SMILES): COc1ccc(N2CCN(C(C)c3ccc4nccnc4c3)CC2)cn1
InChIKey: GKFBRHZDDKRUDR-UHFFFAOYSA-N
Target: Protein O-GlcNAcase (SINGLE PROTEIN), Homo sapiens
UniProt: O60502
Assay: Inhibition Assay: 5 μl of the appropriate concentration of a solution of inhibitor in McIlvaine's Buffer (pH 6.5) in 2% DMSO (for a dose response curve calculation) is added into each well of a 384-well plate (Greiner, 781900). Then, 20 nM of His-Tagged hOGA and 10 μM of FL-GlcNAc (Fluorescein mono-beta-D-(2-deoxy-2-N-acetyl) glucopyranoside; Marker Gene Technologies Inc, M1485) were added to the 384-well plate for a final volume of 20 μl. After incubation for 60 min at room temperature, the reaction was terminated by the addition of 10 μL of stop buffer (200 mM glycine, pH 10.75). The level of fluorescence (λexc 485 nm; (λemm 520 nm) was read on a PHERAstar machine. The amount of fluorescence measured was plotted against the concentration of inhibitor to produce a sigmoidal dose response curve to calculate an IC50. All individual data was corrected by subtraction of the background (Thiamet 3 uM=100% inhibition) whilst 0.5% DMSO was considered as the control value (no inhibition).
Measurement: IC50 = 600 nM (pChEMBL 6.21999979019165)
Descriptors: MW 349.44, ALogP 2.92, HBA 6, HBD 0, PSA 54.38, QED 0.72
Compound (SMILES): Cc1cc(N(Cc2cccc(CO)c2)CC(F)(F)F)ccc1Cl
InChIKey: XTPDNFOSJZYCKC-UHFFFAOYSA-N
Target: Sphingosine 1-phosphate receptor 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P21453
Assay: BCA Protein Assay: Inhibitory activity evaluation was performed by multi-screen (registered trademark) HTS 96-well plate (Millipore) using the cell membrane fraction stably expressing S1P1 obtained by the above operation. 25 μL of assay solution (50 mM Tris, 100 mM sodium chloride, 5 mM magnesium chloride, 1 mM EDTA, 1 mM DTT (dithiothreitol), 10 μM GDP (guanosine diphosphate) and 0.5% BSA (bovine serum albumin), pH 7.4), 25 μL of test compounds solution diluted by assay solution and 25 μL of the membrane fraction (0.2 μg protein/μL) were added to each well, and the mixture was gently shaken at room temperature: for 30 minutes. Then, 25 μL of 35S-GTP, and 25 μL of 50 nM S1P1 receptor selective agonist (1-[4-(4-phenyl-5-trifluoromethylthiophen-2-ylmethoxy)benzyl]azetidine-3-carboxylic acid, ¿J. Med. Chem.¿, 2004, vol. 47, pp. 6662-6665, compound 18) were added to each well, respectively, and it was reacted at room temperature for 60 minutes. After the reaction, a reaction solution of each well was suction-filtered. After suction filtration, ice-colded wash solution (50 mM Tris, 100 mM sodium chloride, 5 mL magnesium chloride and 1 mM EDTA, pH 7.4) was added to each well, and moreover filters were washed by suction filtration. This wash procedure was performed three times. A bottom of the plate containing the filters was dried at 60° C. After drying, 30 μL of MicroScinti-40 (PerkinElmer) was added to each well, and the radioactivity adsorbed on the filter was determined by TopCount NXT (registered trademark) (PerkinElmer) after shaking for 30 minutes at room temperature.
Measurement: IC50 = 2.5 nM (pChEMBL 8.600000381469727)
Descriptors: MW 343.78, ALogP 4.71, HBA 2, HBD 1, PSA 23.47, QED 0.85
Compound (SMILES): COCC1CCCCN1C(=O)c1ccc(CN2C(=O)Cc3cnc(-c4ccccc4C(C)C)nc32)cc1
InChIKey: LMMXUGAHOHFSLH-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 = 125 nM (pChEMBL 6.900000095367432)
Descriptors: MW 498.63, ALogP 5, HBA 5, HBD 0, PSA 75.63, QED 0.46
Compound (SMILES): Cc1cc2c(cc1C#N)c(-c1cncc(N)c1Cl)cn2[C@@H]1CC[C@H](NS(C)(=O)=O)C1
InChIKey: BRYVAFMZKHMXQO-LSDHHAIUSA-N
Target: Lysine-specific histone demethylase 1A (SINGLE PROTEIN), Homo sapiens
UniProt: O60341
Assay: FL-LSD1 LC-MS Assay: Representative compounds of the present invention were serially and separately diluted 3-fold in DMSO to obtain a total of twelve concentrations. Then the test compounds at each concentration (100 nL of each) were transferred into a 384-well Perkin Elmer ProxiPlate 384 plus plates by Mosquito Solutions (5 μL) of 0.8 nM, the full-length LSD1 and 0.5 μM FAD in reaction buffer (40 mM Tris-HCl, 0.01% Triton-×100.10 mM KCl, 1 mM DTT) were added into the wells and then incubated with the test compound for 30 min. A 5 μL solution of 1 μM of the peptide substrate H3K4me1 (histone H3[1-21]-biotin) in reaction buffer was added to each initiate reaction. The final components in the reaction solution include 0.4 nM FL-LSD1, 0.25 μM FAD, and 0.5 μM H3K4me1 peptide with varying concentration of the compounds. A positive control consisted of the enzyme, 0.25 μM FAD and 0.5 μM substrate in the absence of the test compound, and a negative control consisted of 0.5 μM substrate only. Each reaction was incubated at room temperature for 60 min, and then stopped by the addition of 3 μL quench solution (2.5% TFA with 320 nM d4-SAH). The reaction mixture was centrifuged (Eppendorf centrifuge 5810, Rotor A-4-62) for 1 min at 2000 rpm and read on an API 4000 triple quadrupole mass spec with Turbulon Spray (Applied Biosystem) coupled with Prominence UFLC (Shimadzu).
Measurement: IC50 = 77 nM (pChEMBL 7.110000133514404)
Descriptors: MW 443.96, ALogP 3.76, HBA 6, HBD 2, PSA 113.8, QED 0.64
Compound (SMILES): Cn1c(SCCCN2C[C@@H]3CCN(c4ccccc4C(F)(F)F)[C@@H]3C2)nnc1-c1cnccn1
InChIKey: MGPBHFVVGJDFMY-OXJNMPFZSA-N
Target: D(3) dopamine receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P35462
Assay: Inhibition Assay: 3H]-Spiperone Binding Assay at hD3 and hD4 recombinant receptors CHO cells transiently transfected with human dopamine type 3 or 4 receptors (CHO-hD3 or CHO-hD4, respectively), were re-suspended in 20 mM HEPES, 2 mM EDTA (pH 7.4), homogenised and centrifuged at 40,000 g (20 min, 4° C.). After re-suspension, homogenization and centrifugation as above, the final pellet was re-suspended in 20 mM HEPES, 100 mM NaCl, 10 mM MgCl2, 1 mM EDTA (pH 7.4) and aliquots were kept at −80° C. [3H]-Spiperone Binding experiments were performed in 96 deep-well polypropylene plates in 50 mM Tris/HCl, 120 mM NaCl, 5 mM KCl, 5 mM MgCl2 (pH 7.4). Compounds of invention were serially diluted in DMSO at 100 fold final concentrations in the assay (1% DMSO final in the assay). Displacement was performed in the presence of 0.3 nM [3H]-Spiperone. The reaction was initiated by the addition of membrane suspension (4 μg and 12 μg of protein for CHO-hD3- and CHO-hD4 membranes, respectively) and lasted for 90 or 100 min (for hD3 or hD4 membranes, respectively) at 23° C. in a final volume of 500 μl. Non specific binding (NSB) was determined in the presence of 1 μM Spiperone. The binding reaction was stopped by rapid filtration through GF/B filterplates pre-soaked in 0.5% polyetylenimmine (PEI) using a Packard cell harvester. After washing with ice-cold 0.9% NaCl, the plate was left to dry before the addition of Microscint 20 (50 μl/well, PerkinElmer). Radioactivity was counted with a TopCount (PerkinElmer). Data were analysed by non-linear regression analysis using GraphPad Prism 5.0 (GraphPad Software). Saturation binding experiments were performed similar to the competition binding experiments using a radioligand concentrations ranging from 0.015 to 4.0 nM. Ref: Mackenzie R. G. et al. (1994). Characterization of the human dopamine D3 receptor expressed in transfected cell lines. Eur. J. Pharmacol. 266: 79-85.
Measurement: Ki = 2.51 nM (pChEMBL 8.600000381469727)
Descriptors: MW 489.57, ALogP 3.98, HBA 8, HBD 0, PSA 62.97, QED 0.37
Compound (SMILES): Cc1ccc(Sc2cc(C(=O)N3Cc4ccccc4C3)c(O)cc2CO)cc1C
InChIKey: PAPOFWBFRFFDLW-UHFFFAOYSA-N
Target: Heat shock protein HSP 90-beta (SINGLE PROTEIN), Homo sapiens
UniProt: P08238
Assay: Florescence Polarization Assay: Assay was performed in 96-well format in black, flat bottom plates (Santa Cruz Biotechnology) with a final volume of 100 μL. 25 μL of assay buffer (20 mM HEPES, pH 7.3, 50 mM KCl, 5 mM MgCl2, 20 mM Na2MoO4, 2 mM DTT, 0.1 mg/mL BGG, and 0.01% NP-40) containing 6 nM FITC-GDA (fluorescent tracer, stock in DMSO and diluted in assay buffer) and 50 μL of assay buffer containing 10 nM of either Grp94 or Hsp90α were added to each well. Compounds were tested in triplicate wells (1% DMSO final concentration). For each plate, wells containing buffer only (background), tracer in buffer only (low polarization control), and protein and tracer in buffer with 1% DMSO (highpolarization control) were included. Plates were incubated at 4° C. with rocking for 24 h. Polarization values (in mP units) was measured at 37° C. with an excitation filter at 485 nm and an emission filter at 528 nm. Polarization values were correlated to % tracer bound and compound concentrations. The concentration at which the tracer was 50% displaced by the inhibitor was determined using Graphpad Prism.
Measurement: Kd = 2860 nM (pChEMBL 5.539999961853027)
Descriptors: MW 405.52, ALogP 4.81, HBA 4, HBD 2, PSA 60.77, QED 0.65
Compound (SMILES): CC(C)C1CCCCN1c1nc2nc(C(=O)O)nc(N[C@H](C)C3CCC3)c2n1C[C@H]1CC[C@H](C)CC1
InChIKey: KIDCBBFKBGIRJO-ILTMHZNQSA-N
Target: E3 ubiquitin-protein ligase Mdm2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q00987
Assay: FRET Assay: An HDM2 FRET assay was developed to assess the compounds' inhibitory activity towards binding of p53 protein. A truncated version of HDM2 with residues 17 to 125 (containing p53 binding surface, Science (1994) 265, 346-355), with N-terminal His and Thioredoxin tag was generated in pET32a expression vector and expressed in E. coli strain BL21(DE3)Rosetta. Protein was purified using Ni-affinity chromatography, followed by size exclusion chromatography using Superdex 75 26/60 column. To assess inhibition of p53 binding to HDM2, a FITC labeled 8-mer peptide (sequence: Ac-Phe-Arg-Dpr-Ac6c-(6-Br)Trp-Glu-Glu-Leu-NH2; Anal Biochem. 2004 Aug. 1; 331(1):138-46) with strong affinity towards p53 binding pocket of HDM2 was used. The HDM2 assay buffer contained 1× Phosphate Buffered Saline (Invitrogen, Cat#14190), 0.01% BSA (Jackson ImmunoResearch, Cat#001-000-162), 0.01% Tween-20. In the 1× assay buffer recombinant HDM2 protein, peptide and Lumi4-Tb Cryptate-conjugate mouse anti-6×His antibody (cisbio, Cat# Tb61HISTLB) were added and transferred to ProxiPlate PLUS (PerkinElmer, Cat#6008269), containing compounds so that final DMSO concentration is 0.1%. Final concentrations of reagents in the assay wells are 0.5 nM HDM2, 0.25 nM anti HIS (Tb label) antibody and 3 nM peptide. After two hour incubation at room temperature in a humidified chamber plates were read on EnVision plate reader with the following settings: excitation: UV, 340 nM, two emission filters: 520 nm and 495 nm respectively. Ratio of em520/em495 was used to calculate % inhibition and to obtain IC50 with 4-parameter logistic equation.
Measurement: IC50 = 3.309 nM (pChEMBL 8.479999542236328)
Descriptors: MW 496.7, ALogP 5.97, HBA 7, HBD 2, PSA 96.17, QED 0.46
Compound (SMILES): CCOc1ncc(-c2cc(NC(=O)c3ccnc(C(F)(F)F)c3)cnc2C)cc1C1(C)COC1
InChIKey: MLGNXKIPPIXQET-UHFFFAOYSA-N
Target: RAF proto-oncogene serine/threonine-protein kinase (SINGLE PROTEIN), Homo sapiens
UniProt: P04049
Assay: In Vitro Raf Activity Determination: Assay Materials: Assay buffer is 50 mM Tris, pH 7.5, 15 mM MgCl2, 0.01% Bovine Serum Albumin (BSA) and 1 mM dithiothreitol (DTT); Stop buffer is 60 mM ethylenediaminetetraacetic acid (EDTA) and 0.01% Tween® 20: b-Raf (V600E), active; biotinylated Mek, kinase dead; Alpha Screen detection kit (available from PerkinElmer, #6760617R); Anti phospho-MEK1/2 (available from Cell Signaling Technology, Inc. #9121); 384 well low volume assay plates (White Greiner® plates).Assay conditions: b-Raf (V600E) approximately 4 μM; c-Raf approximately 4 nM; biotinylated Mek, kinase dead approximately 10 nM; ATP 10 μM for BRAF (V600E) and 1 μM for CRAF; Pre-incubation time with compounds 60 minutes at room temperature; Reaction time 1 or 3 hours at room temperature.Assay protocol: Raf and biotinylated Mek (kinase dead) were combined at 2× final concentrations in assay buffer (50 mM Tris, pH 7.5, 15 mM MgCl2, 0.01% BSA and 1 mM DTT) and dispensed 5 ml per well in assay plates (Greiner white 384 well assay plates #781207) containing 0.25 ml of 40× of a Raf kinase inhibitor test compound diluted in 100% DMSO. The plate was incubated for 60 minutes at room temperature. The Raf kinase activity reaction was started by the addition of 5 mL per well of 2×ATP diluted in assay buffer. After 3 hours (b-Raf(V600E)) or 1 hour (c-Raf). The reactions were stopped and the phosphorylated product was measured using a rabbit anti-p-MEK (Cell Signaling, #9121) antibody and the Alpha Screen IgG (ProteinA) detection Kit (PerkinElmer #6760617R), by the addition of 10 mL to the well of a mixture of the antibody (1:2000 dilution) and detection beads (1:2000 dilution of both beads) in Stop/bead buffer (25 mM EDTA, 50 mM Tris, pH 7.5, 0.01% Tween20). The additions were carried out under dark conditions to protect the detection beads from light. A lid was placed on top of the plate and incubated for 1 hour at room temperature, after which the luminescence was read on a PerkinElmer Envision instrument. The concentration of each compound for 50% inhibition (IC50) was calculated by non-linear regression using XL Fit data analysis software.
Measurement: IC50 = 183 nM (pChEMBL 6.739999771118164)
Descriptors: MW 472.47, ALogP 4.8, HBA 6, HBD 1, PSA 86.23, QED 0.55
Compound (SMILES): C[C@H](NC(=O)c1cn2c(c(O)c1=O)C(=O)N1[C@@H]3CC[C@@H](C3)O[C@H]1C2)c1ccc(F)cc1F
InChIKey: HZQGBUQIMZZLDE-IMODNSLOSA-N
Target: Solute carrier family 22 member 2 (SINGLE PROTEIN), Homo sapiens
UniProt: O15244
Assay: Inhibition Assay: Test compounds were serially diluted in DMSO and then spiked (2 μL) into in 0.4 mL KHB buffer containing wild-type or OCT2-transfected cells and incubated for 10 minutes. Assay was initiated with the addition of 0.1 mL of 100 μM 14C-TEA in KHB buffer (20 μM final concentration after mixing). The concentration of TEA is based on the Km. After 10 minutes of incubation, the assay mixture was quenched with addition of 0.5 mL of ice-cold IX PBS buffer. Samples were then centrifuged at 1000 rpm for 5 min and supernatants were removed. Wash steps were repeated four times with ice-cold PBS.
Measurement: IC50 = 610 nM (pChEMBL 6.210000038146973)
Descriptors: MW 445.42, ALogP 2.06, HBA 6, HBD 2, PSA 100.87, QED 0.75
Compound (SMILES): CS(=O)(=O)N1CCC[C@@H](C(=O)N[C@@H](CCCCCCCc2ccc3c(n2)NCCC3)C(=O)O)C1
InChIKey: CWZXCJPGXXAJEK-CTNGQTDRSA-N
Target: Integrin alpha-V/beta-6 (PROTEIN COMPLEX), Homo sapiens
UniProt: P06756
Assay: Solid Phase Integrin αVβ6 Binding Assay: Microplates were coated with recombinant human integrin αVβ6 (2 μg/mL) in PBS (100 μL/well 25 OC, overnight). The coating solution was removed, washed with wash buffer (0.05% Tween 20; 0.5 mM MnCl2; in 1×TBS). Plate was blocked with 200 μL/well of Block Buffer (1% BSA; 5% sucrose; 0.5 mM MnCl2; in 1×TBS) at 37° C. for 2 h. Dilutions of testing compounds and recombinant TGFβ1 LAP (0.67 μg/mL) in binding buffer (0.05% BSA; 2.5% sucrose; 0.5 mM MnCl2; in 1×TBS) were added. The plate was incubated for 2 hours at 25° C., washed, and incubated for 1 hour with Biotin-Anti-hLAP. Bound antibody was detected by peroxidase-conjugated streptavidin. The IC50 values for testing compounds were calculated by a four-parameter logistic regression.
Measurement: IC50 < 50 nM (pChEMBL 7.301000118255615)
Descriptors: MW 494.66, ALogP 2.56, HBA 6, HBD 3, PSA 128.7, QED 0.38
Compound (SMILES): CN(CCCc1ccc(F)cc1)[C@H]1C[C@@H](C(=O)NS(=O)(=O)N(C)C)N(C(=O)C(c2ccccc2)c2ccccc2)C1
InChIKey: UDXHQRMQQJQHMN-NSOVKSMOSA-N
Target: Type-2 angiotensin II receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P50052
Assay: Receptor Binding Assay:     120 μL membrane (5 mg protein/well) was incubated with 15 μL of [125I]-CGP42112A and 15 μL of compound at RT for 1.5 hrs.    The binding reaction was stopped by rapid filtration through Unifilter GF/C plates (presoaked in 0.3% (v:v) BSA).    Plate was washed three times with ice cold wash buffer.    The filtration plates were dried at 37° C. overnight.    50 μL of scintillation cocktail was added to each well.    Radioactivity was determined using MicroBetaTriluxmicroplate scintillation counter.
Measurement: IC50 = 3604 nM (pChEMBL 5.440000057220459)
Descriptors: MW 580.73, ALogP 3.41, HBA 5, HBD 1, PSA 90.03, QED 0.38
Compound (SMILES): CC(=O)N1CCC(NC(=O)c2cccc(-c3cc(-c4ccnn4C4CCCCC4)c4c(N)ncnn34)c2)CC1
InChIKey: RPUPYIFIPXQDAV-UHFFFAOYSA-N
Target: Phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit delta isoform (SINGLE PROTEIN), Homo sapiens
UniProt: O00329
Assay: ADP-Glo Format PI3K Assay: The ADP-Glo format PI3K assays were performed in Proxiplate 384-well plates (Perkin Elmer #6008280). The final assay volume was 2 μl prepared from 1 μl additions of enzyme/PIP2:PS lipid (Invitrogen #PV5100) mixture and 1 μl ATP (provided in kit, Promega #V9101) and test compounds in assay buffer (50 mM HEPES pH 7.5, 3 mM MgCl2, 100 mM NaCl, 0.5 mM EGTA, 2 mM DTT, 0.03% CHAPS). The reaction was initiated by the combination of enzyme/lipid, ATP, and test compounds. The reaction mixture was incubated at room temperature for 30 minutes (PI3K Alpha, Beta, Gamma) or 3 hours for PI3K Delta. ADP-Glo (2 μl), followed by Kinase Detection reagent (4 μl), were added to reactions following the initial incubation and allowed to incubate 40 minutes at room temperature. The reaction mixture was analyzed on the TOPCOUNT (Perkin Elmer). Inhibition data were calculated by comparison to no enzyme control reactions for 100% inhibition and vehicle-only reactions for 0% inhibition. The final concentration of enzyme in the assays are PI3K Alpha [0.5 nM], PI3K Beta [2 nM], PI3K Gamma [20 nM], PI3K Delta [0.5 nM]. ATP final concentrations are as follows: for Alpha [10 μM], for Beta [12.5 μM], for Gamma [6.5 μM], for Delta [100 μM]. Lipid final concentration was the same for all enzymes, [25 μM]. Dose response curves were generated to determine the concentration required to inhibit 50% of activity. Compounds were dissolved at 0.12 mM in dimethylsulfoxide (DMSO) and evaluated at eleven concentrations. The IC50 values were derived by non-linear regression analysis.
Measurement: IC50 = 2 nM (pChEMBL 8.699999809265137)
Descriptors: MW 526.65, ALogP 4.09, HBA 8, HBD 2, PSA 123.44, QED 0.4
Compound (SMILES): CC(C)n1c(=O)n(-c2ccc(Cl)c(O)c2)c2c(N)nc(Nc3ccc(C(=O)N4C=CC(O)C=C4)cc3F)nc21
InChIKey: NMFVXNXKOGFOQY-UHFFFAOYSA-N
Target: Protein-tyrosine kinase 6 (SINGLE PROTEIN), Homo sapiens
UniProt: Q13882
Assay: Measurement of Brk Inhibitory Activity: Measurement of an inhibitory activity on Brk enzyme was performed by using LanthaScreen (registered trademark) system (Invitrogen) in accordance with the attached manual. Reagents used are described below.Reaction Buffer: A solution containing 50 mmol/L HEPES (pH 7.5), 0.01% Brij 35, 10 mmol/L MgCl2 and 1 mmol/L EGTA was prepared by using purified water.A solution of a test substance (the compound of the present invention): A solution of each concentration of a test compound in DMSO was diluted 20-fold with Reaction Buffer, and a solution containing a test compound at a concentration 5 times a final concentration was prepared.An enzyme solution: A solution containing 480 ng/mL of Brk enzyme was prepared by using Reaction Buffer.A substrate solution: A solution containing 57 μmol/L of ATP and 500 nmol/L of Fluorescein-Poly GT (Invitrogen) was prepared by using Reaction Buffer.A detection solution: A solution containing 20 mmol/L of EDTA and 4 nmol/L of PY20 (Invitrogen) was prepared by using Dilution B (Invitrogen).To a 96-well plate (Nunc), a solution of 10 mmol/L of a test compound in DMSO was dispensed, and further, a dilution series at a common ratio of three was prepared by using DMSO. To each of wells of the 96-well plate for the measurement, 5 μL of Reaction Buffer containing DMSO was added for a blank group and a vehicle group and 5 μL of a test substance solution was added for a test substance group. Next, 10 μL per well of Reaction Buffer was added for the blank group, and 10 μL per well of the enzyme solution was added for the vehicle group and the test compound group, and thereafter, the mixture was stirred at room temperature for 10 minutes. After completion of stirring, 10 μL of the substrate solution was added to each of wells, and the mixture was stirred at room temperature under a shading condition for 1 hour. After completion of the reaction, 25 μL of the detection solution was added to each well, and the mixture was left to stand at room temperature under a shading condition for 30 minutes. After being left standing, fluorescence intensities at 520 nm and 495 nm were measured by using Analyst GT (Molecular Devices, LLC) when being irradiated with an excitation light at 340 nm. The phosphorylation of the artificial substrate was quantified by Time-Resolved Fluorescence Resonance Energy Transfer (TR-FRET). With regard to each well, the TR-FRET ratio was calculated by divining the fluorescence signal at 520 run by the fluorescence signal at 495 nm, and the inhibition rate (%) in the test compound group was calculated according to the following Numerical Formula 1.Inhibition rate (%)={1−(TR-FRET ratio of test compound group−A)/(B−A)}×100  [Numerical Formula 1].
Measurement: IC50 = 14 nM (pChEMBL 7.849999904632568)
Descriptors: MW 551.97, ALogP 3.83, HBA 10, HBD 4, PSA 151.53, QED 0.29
Compound (SMILES): Cc1nc2ccc(-c3nc(N[C@@H]4CCN(S(C)(=O)=O)C[C@H]4O)ncc3F)cc2n1C(C)(C)C
InChIKey: XGPZVHJSIGQLBU-IEBWSBKVSA-N
Target: Cyclin-dependent kinase 4/cyclin D1 (PROTEIN COMPLEX), Homo sapiens
UniProt: P24385
Assay: Biological Assay: CDK4/Cyclin D1: The purpose CDK4/Cyclin D1 assay is to evaluate the inhibition (% inhibition, Kiapp and Ki values) in the presence of small molecule inhibitors by using a fluorescence based microfluidic mobility shift assay. CDK4/Cyclin D1 catalyzes the production of ADP from ATP that accompanies the phosphoryl transfer to the substrate peptide 5-FAM-Dyrktide (5-FAM-RRRFRPASPLRGPPK) (SEQ ID NO:1). 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. Typical reaction solutions contained 2% DMSO (±inhibitor), 10 mM MgCl2, 1 mM DTT, 3.5 mM ATP, 0.005% TW-20, 3 μM 5-FAM-Dyrktide, 3 nM (active sites) activated CDK4/Cyclin D1 in 40 mM HEPES buffer at pH 7.5.
Measurement: Ki = 0.1 nM (pChEMBL 10)
Descriptors: MW 476.58, ALogP 2.5, HBA 8, HBD 2, PSA 113.24, QED 0.59
Compound (SMILES): CS(=O)(=O)CC(=O)Nc1c2c(nn1CC1CC(F)(F)C1)C[C@]1(CCc3cc(OCC(F)(F)F)ccc31)NC2=O
InChIKey: QRMZAMQZSGUGDM-QFIPXVFZSA-N
Target: Alpha-1,6-mannosyl-glycoprotein 2-beta-N-acetylglucosaminyltransferase (SINGLE PROTEIN), Homo sapiens
UniProt: Q10469
Assay: Inhibitory Activity: Solutions of the compounds of the present invention in DMSO were each aliquoted into 0.2-μL portions in a 384-well polystyrene microplate produced by Corning Incorporated, and 5 μL of an enzyme solution prepared with an assay buffer (100 mmol/L phosphate buffer (pH 7.4) containing 2 mmol/L DTT) and 5 μL of a substrate solution (100 mmol/L phosphate buffer (pH 7.4), 30 μmol/L 2-Oleoylglycerol, 10 μmol/L Oleoyl-CoA) were added thereto, and the resultant was stirred and centrifuged, and incubated in a moist chamber at room temperature for 1 hour. After enzymatic reaction, 50 μL of a quenching solution (containing 0.2 μmol/L Diolein-d5, 0.4% formic acid, and 50% isopropanol) containing Internal Standard (IS) was added to terminate the reaction, and the resultant was sealed in a plate produced by Shimadzu GLC Ltd., and then stirred and centrifuged, and measurement was performed by using an electrospray ionization method with a RapidFire360 and Agilent 6550 Q-TOF mass spectrometer. Diolein as a reaction product (P) of 2-Oleoylglycerol as the substrate and an ammonium adduct ion of the IS were detected, and the peak intensity ratio, P/IS, was calculated from the peak heights to evaluate the inhibitory activity. Inhibitory activities with/without addition of enzyme were defined as Control (+)/Control (−), respectively, and the respective % inhibitions were defined as 0% inhibition and 100% inhibition. The inhibitory activity was calculated from formula below with TIBCO Spotfire (produced by TIBCO Software Inc.):Inhibitory activity (%)=[1−{Sample−Control(−)}/{Control(+)−Control(−)}]*100where Sample indicates a peak intensity ratio: P/IS, when the compound of the present invention was added.
Measurement: IC50 = 35 nM (pChEMBL 7.460000038146973)
Descriptors: MW 576.54, ALogP 2.98, HBA 7, HBD 2, PSA 119.39, QED 0.49