lovingscience/chembl-v37-raw-activities huggingface dataset:
Compound (SMILES): CC(C)(Sc1ccc2cncc(-c3ccc(C#N)c(Cl)c3)c2c1)C(=O)O
InChIKey: PIYIYFUVTJWCHX-UHFFFAOYSA-N
Target: Solute carrier family 22 member 12 (SINGLE PROTEIN), Homo sapiens
UniProt: Q96S37
Assay: hURAT1 Inhibition Assay: Human embryonic kidney cells (HEK293) was incubated in DMEM tissue culture medium, at 37° C., under 5% CO2 and 95% air atmosphere. TransIT-293 transfection agent (MIRUS BIO, Cat. No. MIR2706) and model URAT1 were used to construct transfected HEK293 cells. Transfected HEK293/hURAT1 cells were used to the test for 14C-uric acid transport activity. HEK293/hURAT1 cells were seeded in a 96-well plate (BD, Cat. No. 356461) fully coating with poly-D-lysine at a density of 6×104 cells per well. Cells were incubated at 37° C. for at least 12 hrs in the calorstat, and then washed with pre-heated washing buffer (125 mM sodium gluconate, 10 mM HEPES pH=7.4) at an amount of 200 μL per well to wash out the culture medium. The uric acid [8-14C] (ARC, Cat. No. ARC0513-250UCI) containing or not containing the compound was added to 50 μL HBSS buffer which was free of chloric ion each well (HBSS buffer: 125 mM sodium gluconate, 4.8 mM potassium gluconate, 1.3 mM calcium gluconate, 1.2 mM potassium dihydrogen phosphate, 1.2 mM magnesium sulfate, 5.6 mM glucose, 25 mM HEPES pH=7.4) to make the specific concentration of the uric acid 1 μCi per well. The incubating solution was removed after 10 mins incubation, followed by adding 100 μL cold washing buffer, after washing with this buffer for 3 times, the buffer was completely removed from the well. 50 μL Lysis buffer (0.1 mM NaOH) was added to each well, and transferred to a 96-well plate (PERKIN ELMER, Cat. No. 6005040) containing scintillation fluid after 5 mins, and counted by MicroBeta Trilux (PerkinElmer) to give IC50 value eventually.
Measurement: IC50 = 55 nM (pChEMBL 7.260000228881836)
Descriptors: MW 382.87, ALogP 5.38, HBA 4, HBD 1, PSA 73.98, QED 0.61
Compound (SMILES): C=CC(=O)Nc1cccc(Sc2cnc(N3CCC4(CCC[C@H]4N)CC3)n3cnnc23)c1Cl
InChIKey: JMKPROPBCWXYRX-GOSISDBHSA-N
Target: Tyrosine-protein phosphatase non-receptor type 11 (SINGLE PROTEIN), Homo sapiens
UniProt: Q06124
Assay: SHP2 Enzyme Activity Inhibition Assay: Compound powders were dissolved in DMSO to prepare mother liquor.During the experiment, the stock solutions of the compounds were subjected to 3-fold gradient dilution with DMSO, and 10 different test concentrations of each compound were set. 1 μL of each concentration of the compounds was transferred into the wells of detection plates (Corning, Costar 3915), setting two duplicates for each concentration. The protein used was the activated protein SHP2E76A with amino acid mutation at position 76 of the protein, and the substrate used was DiFMUP (Invitrogen, E12020). SHP2E76A protein and substrate were diluted with buffer (0.1 M NaAc (pH 7.2), 0.02% Tween 20, 0.1% BSA, 1 mM EDTA, 5 mM DTT) to a concentration of 1.2 nM and 20 M, respectively. 50 L of enzyme solution was added to the detection wells, followed by 50 L of substrate. On the Spectra max i3 (Molecular Devices) instrument, the fluorescence signal was recorded (Ex 358 nm/Em 455 nm) every minute, from which the product accumulation rate was calculated to characterize the enzyme activity. GraphPad Prism 5 was used for nonlinear regression analysis, and the curve of enzyme activity versus compound concentration was fitted by the equation Y=Bottom+(Top-Bottom)/(1+10{circumflex over ( )}((Log IC50−X)*HillSlope)) to calculate the IC50 value of each compound.
Measurement: IC50 = 550 nM (pChEMBL 6.260000228881836)
Descriptors: MW 484.03, ALogP 4.15, HBA 8, HBD 2, PSA 101.44, QED 0.53
Compound (SMILES): COc1cc(C#N)ccc1C(=O)Nc1ccc2[nH]nc(-c3cnn(C(F)F)c3)c2c1
InChIKey: GVNMOTZDXUQDTF-UHFFFAOYSA-N
Target: Leucine-rich repeat serine/threonine-protein kinase 2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q5S007
Assay: LanthaScreen Kinase Activity Assay: The assay was carried out under the following protocol conditions: 1 mM compound in DMSO was serially diluted 1:3, 11 points in DMSO with a Biomek FX and 0.1 μL of the diluted compound was subsequently stamped into the assay plate (384-well format Lumitrac 200, Greiner, 781075) with an Echo Labcyte such that the final compound concentration in the assay was 10 μM to 169 μM. Subsequently, 5 μL of 2× kinase solution (2.9 nM final concentration) was added to the assay plate in assay buffer composed of 50 mM Tris pH 8.5 (Sigma, T6791), 5 mM MgCl2 (Fluka, 63020), 1 mM EGTA (Sigma, E3889), 0.01% BRIJ-35 (Sigma, P1254) and 2 mM DTT. The reaction was started by addition of 2×ATP/LRRKtide solution in assay buffer such that the final concentration was 400 nM LRRKtide and 25 μM ATP. After 60 min incubation at room temperature, the reaction was stopped by addition of 10 μL of 2× stop solution containing a final concentration of 2 nM anti-pERM antibody and 10 mM EDTA. After a 30 min incubation at RT, the TR-FRET signal was measured on a Wallac 2104 EnVision multilabel reader at an excitation wavelength of 340 nm and reading emission at 520 nm and 495 nm. The ratio of the 520 nm and 495 nm emission was used to analyze the data.
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 408.37, ALogP 3.95, HBA 6, HBD 2, PSA 108.62, QED 0.52
Compound (SMILES): CN1Cc2cccc(c2)-c2cccc3[nH]c(nc23)-c2n[nH]c3ccc(cc23)-c2cnn(C)c2C1
InChIKey: MURDNBFVOLGBQI-UHFFFAOYSA-N
Target: Dual specificity tyrosine-phosphorylation-regulated kinase 1A (SINGLE PROTEIN), Homo sapiens
UniProt: Q13627
Assay: Kinase Assay: The DYRK1A kinase assay was run using the Ser/Thr 18 peptide Z-lyte assay kit according to manufacturer's instructions (Life Technologiesa Division of Thermo-Fisher). This is a non-radioactive assay using fluorescence resonance energy transfer (FRET) between coumarin and fluorescein to detect kinase activity which is represented as a ratio of coumarin emission/fluorescein emission.Briefly, recombinant DYRK1A kinase, ATP and Ser/Thr peptide 18 were prepared in 1× Kinase buffer to final concentrations of 0.19 μg/mL, 30 μM, and 4 μM respectively. The mixture was allowed to incubate with the representative compounds for one hour at room temperature. All reactions were performed in duplicate. Unphosphorylated (0% Control) and phosphorylated (100% control) forms of Ser/Thr 18 served as control reactions. Additionally, an 11-point dose-response curve of Staurosporine (1 uM top) was run to serve as a positive compound control.
Measurement: EC50 = 412 nM (pChEMBL 6.380000114440918)
Descriptors: MW 445.53, ALogP 5.12, HBA 5, HBD 2, PSA 78.42, QED 0.34
Compound (SMILES): Cc1cc(-c2cc3ncccc3c(-c3ccn(C4(CC#N)CC(C#N)C4)n3)n2)n[nH]1
InChIKey: PWIBKSIVHXHSJZ-UHFFFAOYSA-N
Target: Non-receptor tyrosine-protein kinase TYK2 (SINGLE PROTEIN), Homo sapiens
UniProt: P29597
Assay: JAK Caliper Enzyme Assay at 1mM ATP: Compounds were added to a 384-well plate. Reaction mixtures contained 10 mM HEPES, pH 7.4, 10 mM MgCl2, 0.01% BSA, 0.0005% Tween 20, 1 mM ATP and 1 μM peptide substrate. The JAK1 and TYK2 assays contained 1 μM of the IRStide peptide (5FAM-KKSRGDYMTMQID) and the JAK2 and JAK3 assays contained 1 μM of the JAKtide peptide (FITC-KGGEEEEYFELVKK). The assays were initiated by the addition of 20 nM JAK1, 1 nM JAK2, 1 nM JAK or 1 nM TYK2 enzyme and were incubated at room temperature for three hours for JAK1, 60 minutes for JAK2, 75 minutes for JAK3 or 135 minutes for TYK2. Enzyme concentrations and incubation times were optimized for each new enzyme preps and were modified slightly over time to ensure 20% to 30% phosphorylation. The assays were stopped with 15 μL of 180 mM HEPES, pH 7.4, 20 mM EDTA, and 0.2% Coating Reagent 3. The assay plates were placed on a Caliper Life Science LC3000 instrument, and each well was sampled using appropriate separation conditions to measure the unphosphorylated and phosphorylated peptide.
Measurement: IC50 = 14 nM (pChEMBL 7.849999904632568)
Descriptors: MW 394.44, ALogP 3.73, HBA 7, HBD 1, PSA 119.86, QED 0.56
Compound (SMILES): Cc1cccc(C(=O)N[C@@H](c2ccc(Cl)c(Cl)c2)c2ccc(Cl)cn2)c1
InChIKey: BRAIJBNSAYJEEB-IBGZPJMESA-N
Target: Sphingosine 1-phosphate receptor 2 (SINGLE PROTEIN), Homo sapiens
UniProt: O95136
Assay: TBD: TBD
Measurement: IC50 = 24600 nM (pChEMBL 4.610000133514404)
Descriptors: MW 405.71, ALogP 5.87, HBA 2, HBD 1, PSA 41.99, QED 0.59
Compound (SMILES): CN(C)C(=O)N1Cc2ccc(-c3nc(Nc4ccc(-c5cn[nH]c5)cc4)c4occc4n3)cc2C1
InChIKey: QUJFNTBYCDSIDC-UHFFFAOYSA-N
Target: Rho-associated protein kinase 2 (SINGLE PROTEIN), Homo sapiens
UniProt: O75116
Assay: Biological Assay: The kinase IC50 was determined by a commercialized CISBIO kinase detection kit, HTRF KinEASE-STK S2 kit (62ST2PEC). ROCK2 (01-119) employed in the reaction was purchased from Carna Biosciences.Before the assay, the following working solutions as needed were formulated with corresponding reagents according to the instruction of the kinase detection kit: 1×kinase buffer, 5×STK-52 substrate working solution (1.5 μM) and 5×ATP working solution (1.5 μM), 5×ROCK2 kinase working solution, 4×Streptavidin-XL665 working solution, and 4×STK-Ab-Cryptate 2 detection solution. Then the assay was performed according to the following procedure.A solution of a compound at a concentration of 10000 nM was prepared with the 1×kinase buffer containing 2.5% DMSO. Gradient dilution of the solution of the compound was performed with the kinase buffer containing DMSO, so as to obtain solutions of a test compound at 9 different concentrations. In addition to wells of test compounds, a positive well (containing all the reagents except the compound) and a negative well (containing all the reagents except the test compound and kinase) were set. Except for the control wells (positive and negative wells), a solution of a test compound (4 μL) was added to each of the reaction wells, and a solution of 2.5% DMSO was added to the control wells. Then the substrate (2 μM, i.e., 2 μL. 5×STK-S2 substrate working solution) was added to each of the reaction wells. The 5×ROCK2 kinase working solution (2 μL, containing 1.4 ng ROCK2 kinase) was added to each of the reaction wells except for the negative well, the volume of which was made up with the 1×kinase buffer (2 μL). The 5×ATP working solution (2 μL) was added to each of the reaction wells, and the mixtures were incubated at room temperature for 2 hours. After the kinase reaction was complete, the 4×Streptavidin-XL665 working solution was added to each of the reaction wells, the solutions were mixed, followed by immediate addition of the 4×STK-Ab-Cryptate 2 detection solution (5 μL), and the mixtures were incubated at room temperature for 1 hour. The fluorescence signal was read on ENVISION (Perkinelmer) (excitation wavelength: 320 nm, and emission wavelength: 665 nm and 615 nm). The inhibitory rate in each well was calculated based on the fluorescence intensity value: ER (Emission Ratio)=(fluorescence intensity at 665 nm/fluorescence intensity at 615 nm); inhibitory rate=(ERpositive−ERtest compound)/(ERpositive−ERnegative)*100%. Curves were plotted and fitted to obtain the median inhibitory concentration (IC50) of each teat compound with the PRISM 5.0 software.
Measurement: IC50 = 186 nM (pChEMBL 6.730000019073486)
Descriptors: MW 465.52, ALogP 5.02, HBA 6, HBD 2, PSA 103.18, QED 0.39
Compound (SMILES): CC1=C(N2CCC3(CCN(C[C@@H](O)c4ccc5c(c4C)CCc4nnnn4-5)CC3)C2=O)COC1=O
InChIKey: VISUOJIFTHZXFP-OAQYLSRUSA-N
Target: ATP-sensitive inward rectifier potassium channel 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P48048
Assay: Thallium Flux Assay: A Thallium Flux Assay was performed on the compounds of the Examples. This assay has been described previously; see, e.g., PCT Published Application WO 2013/062900.Data collected for compounds in the Examples of the present invention using the Thallium Flux Assay are shown in Table 3 below. All of the tested final product compounds in the Examples (diastereomeric mixtures and individual diastereomers) had IC50 potencies less than 1 μM in the Thallium Flux Assay.
Measurement: IC50 = 47 nM (pChEMBL 7.329999923706055)
Descriptors: MW 478.55, ALogP 1.25, HBA 9, HBD 1, PSA 113.68, QED 0.65
Compound (SMILES): Cc1cc(-c2n[nH]c3cc4c(cc23)CN(Cc2ccc(C(F)(F)F)cc2)C(=O)N4)ccn1
InChIKey: HUJMNXSPGOVGJT-UHFFFAOYSA-N
Target: Mitogen-activated protein kinase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P28482
Assay: Z′-LYTE Kinase Assay: The inhibition of ERK activity by the compounds disclosed herein was determined using the Z′-LYTE kinase assay kit (Life Technologies) with a Ser/Thr 3 peptide substrate (Life Technologies) according to manufacturers' instructions. The assay was run with an ERK2 enzyme (Life Technologies) concentration of 0.47 ng/μL at 100 μM ATP (approximately the ATP Km for ERK2). The IC50 values for the compounds were determined with 3-fold serial dilutions in duplicate. The compounds were first diluted in 1:3 dilutions in 100% DMSO at 100× the desired concentration, and then further diluted (1:25) in 20 mM HEPES buffer (Invitrogen) to make 4× solutions prior to adding to the enzyme solution. The final DMSO concentration in the assay was 1%. Final reaction volume was 20 μL/well in 384-well plates. Kinase reactions were conducted or 1 hour followed by the assay development reaction (1 hour) in a 20 ul/well in a 384 well plate format.
Measurement: IC50 = 150 nM (pChEMBL 6.820000171661377)
Descriptors: MW 437.43, ALogP 5.5, HBA 3, HBD 2, PSA 73.91, QED 0.44
Compound (SMILES): CC(C)CC(=O)NC1(C)CCN(c2ccc(-c3cc(OCCO)cn4ncc(C#N)c34)cn2)CC1
InChIKey: BLFBSWZIJWRDGL-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 = 15.5 nM (pChEMBL 7.809999942779541)
Descriptors: MW 476.58, ALogP 3.16, HBA 8, HBD 2, PSA 115.78, QED 0.51
Compound (SMILES): O=C(NCC1CCC(F)(F)CC1)c1cn(C2CCNC2)c2cccc(Cl)c12
InChIKey: ZYNCIRVQJPNTOS-UHFFFAOYSA-N
Target: P2X purinoceptor 7 (SINGLE PROTEIN), Homo sapiens
UniProt: Q99572
Assay: Pore Permeation Assay: Agonist-induced pore formation was determined by measuring cellular uptake of YO PRO fluorescence dye in HEK293 transfected with human P2X7 receptor. A HEK293 cell over expressing human P2X7 was harvested using HQTase reagent to detach the cells from T75 cm flask. The harvested cells are centrifuged @ 1200 rpm for 5 min at room temperature. The viability of cells was determined by Trypan blue dye and the cells are plated @ 10,000 cell/well in 50 ul volume in a 384W BD Poly lysine coated plate and incubated overnight at 37 C. After overnight incubation, the culture medium was replaced with 35 ul/well assay buffer (5 mM KCl, 0.1 mM CaCl2, 5 mM Glucose, 10 mM HEPES buffer pH7.4 containing 125 mM NaCl. The serial dilution of compounds was performed using Bravo liquid handling instrument and the compounds were added using Bravo to the cell assay plate starting at 2.5 uM with three dilutions for 10 points. The positive control inhibitor compound was added to column 23. The plate was shaken slowly on a plate shaker for 10 seconds. The cells were incubated with the compound for 20 minutes at room temperature. After the incubation period, YO PRO dye (1 uM) along with BzATP (10 uM) were added to cells at 10 ul/well. The plate was centrifuged at 1000 rpm for 5 seconds and incubated at room temperature for 30 minutes. The uptake of YO PRO dye into the cells was measured using Envision Fluorescence plate reader instrument (Perkin Elmer).
Measurement: IC50 = 55 nM (pChEMBL 7.260000228881836)
Descriptors: MW 395.88, ALogP 4.38, HBA 3, HBD 2, PSA 46.06, QED 0.81
Compound (SMILES): N#Cc1cccc(F)c1-c1cc(-n2ccc(CCO)n2)c2c(n1)CNC2=O
InChIKey: SMCIQHAIMKAVBS-UHFFFAOYSA-N
Target: Non-receptor tyrosine-protein kinase TYK2 (SINGLE PROTEIN), Homo sapiens
UniProt: P29597
Assay: Caliper Assay: The caliper machine employs an off chip mobility shift assay to detect phosphorylated peptide substrates from kinase assays, using microfluidics technology. The assays are carried out at ATP concentration equivalent to the ATP Km, and at 1 mM ATP. Compounds are serially diluted in DMSO then further diluted in assay buffer (25 mM HEPES, pH 7.5, 0.01% Brij-35, 0.01% Triton, 0.5 mM EGTA). 5 ul of diluted compound was added into wells first, then 10 ul of enzyme mix was added into wells, followed by 10 uL of substrate mix (peptide and ATP in 10 mM MgCl2) to start reaction. Reaction was incubated at 28° C. for 25 min and then added 25 ul stop buffer (100 mM HEPES, 0.015% Brij-35, 50 mM EDTA), followed by reading with Caliper. JAK2 at 1 nM final concentration and TYK2 at 9.75 nM are from Carna, and substrates used are ATP at 20 and 16 uM, respectively. JAK2 assay uses peptide 22 and TYK2 uses peptide 30 (Caliper), each at 3 uM.
Measurement: Ki = 55 nM (pChEMBL 7.260000228881836)
Descriptors: MW 363.35, ALogP 1.72, HBA 6, HBD 2, PSA 103.83, QED 0.73
Compound (SMILES): COCc1nn(Cc2ccc(Cn3ccc(C)cc3=O)cc2)cc1C(=O)NCCOc1cccc(Cl)c1
InChIKey: FTMLEBBQRKVAFL-UHFFFAOYSA-N
Target: Plasma kallikrein (SINGLE PROTEIN), Homo sapiens
UniProt: P03952
Assay: Inhibitory Activity Assay: Plasma kallikrein inhibitory activity in vitro was determined using standard published methods. Human plasma kallikrein (Protogen) was incubated at 25° C. with the fluorogenic substrate H-DPro-Phe-Arg-AFC and various concentrations of the test compound. Residual enzyme activity (initial rate of reaction) was determined by measuring the change in optical absorbance at 410 nm and the IC50 value for the test compound was determined.
Measurement: IC50 = 413 nM (pChEMBL 6.380000114440918)
Descriptors: MW 521.02, ALogP 4.06, HBA 7, HBD 1, PSA 87.38, QED 0.3
Compound (SMILES): O=C(Nc1cn2nc(-c3c(-c4ccc(F)cc4)ncn3C3CC3)ccc2n1)c1ccncc1
InChIKey: BTUKALJLOOIFHL-UHFFFAOYSA-N
Target: Casein kinase I isoform epsilon (SINGLE PROTEIN), Homo sapiens
UniProt: P49674
Assay: Kinase Assay: The kinase assay was performed in V-bottom 384-well plates. The final assay volume was 30 μl prepared from 15 μl additions of enzyme, substrates (fluoresceinated peptide FL-AHA-KRRRAL-PSER-VASLPGL-OH and ATP) and test compounds in assay buffer (20 mM HEPES pH 7.4, 30 mM MgCl2, 0.015% Brij35 and 4 mM DTT). The reaction was incubated at room temperature for 22 hours and terminated by adding 45 μl of 35 mM EDTA to each sample. The reaction mixture was analyzed on the Caliper LABCHIP®3000 (Caliper, Hopkinton, Mass.) by electrophoretic separation of the unphosphorylated substrate and phosphorylated product. Inhibition data were calculated by comparison of the no enzyme control reactions for 100% inhibition and vehicle-only reactions for 0% inhibition. The final concentration of reagents in the assay were 200 pM CK1ε or CK1δ, 50 μM ATP, 1.5 μM FL-AHA-KRRRAL-PSER-VASLPGL-OH, and 1.6% DMSO. Dose response curves were generated to determine the concentration required to inhibit 50% of the kinase activity (IC50). Compounds were dissolved at 10 mM in dimethylsulfoxide (DMSO) and evaluated at eleven concentrations.
Measurement: IC50 = 6.5 nM (pChEMBL 8.1899995803833)
Descriptors: MW 439.45, ALogP 4.38, HBA 7, HBD 1, PSA 90, QED 0.44
Compound (SMILES): Cc1c(OCCN(C)CCCF)ccc(F)c1[C@@H]1c2[nH]c3ccccc3c2C[C@H](C)N1C[C@@H](C)C(=O)O
InChIKey: OUWDZJIZWQEYKX-FSQFJQELSA-N
Target: Estrogen receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P03372
Assay: Binding Assay: ERα: The ability of compounds to bind to isolated Estrogen Receptor Alpha Ligand binding domain (ER alpha-LBD (GST)) was assessed in competition assays using a LanthaScreen is Time-Resolved Fluorescence Resonance Energy Transfer (TR-FRET) detection end-point. For the LanthaScreen TR-FRET endpoint, a suitable fluorophore (Fluormone ES2, ThermoFisher, Product code P2645) and recombinant human Estrogen Receptor alpha ligand binding domain, residues 307-554 (expressed and purified in-house) were used to measure compound binding. The assay principle is that ER alpha-LBD (GST) is added to a fluorescent ligand to form a receptor/fluorophore complex. A terbium-labelled anti-GST antibody (Product code PV3551) is used to indirectly label the receptor by binding to its GST tag, and competitive binding is detected by a test compound's ability to displace the fluorescent ligand, resulting in a loss of TR-FRET signal between the Tb-anti-GST antibody and the tracer. The assay was performed as follows with all reagent additions carried out using the Beckman Coulter BioRAPTR FRD microfluidic workstation:    1. Acoustic dispense 120 nL of the test compound into a black low volume 384 well assay plates.    2. Prepare 1×ER alpha-LBD/Tb-antiGST Ab in ES2 screening buffer and incubate for 15 minutes.    3. Dispense 6 μL of the 1×AR-LBD/Tb-anti-GST Ab reagent into each well of the assay plate followed by 6 μL of Fluorophore reagent into each well of the assay plate    4. Cover the assay plate to protect the reagents from light and evaporation, and incubate at room temperature for 4 hours.    5. Excite at 337 nm and measure the fluorescent emission signal of each well at 490 nm and 520 nm using the BMG PheraSTAR.Compounds were dosed directly from a compound source microplate containing serially diluted compound (4 wells containing 10 mM, 0.1 mM, 1 μM and 10 nM final compound respectively) to an assay microplate using the Labcyte Echo 550. The Echo 550 is a liquid handler that uses acoustic technology to perform direct microplate-to-microplate transfers of DMSO compound solutions and the system can be programmed to transfer multiple small nL volumes of compound from the different source plate wells to give the desired serial dilution of compound in the assay which is then back-filled to normalise the DMSO concentration across the dilution range.
Measurement: IC50 = 8.2 nM (pChEMBL 8.09000015258789)
Descriptors: MW 513.63, ALogP 5.34, HBA 4, HBD 2, PSA 68.8, QED 0.36
Compound (SMILES): Cn1cc(-c2cnn(-c3cccc(F)c3C#N)c2)c(-c2ccc(NCC3CC3)nc2)cc1=O
InChIKey: ZCYMIVMSGILCCD-UHFFFAOYSA-N
Target: CREB-binding protein (SINGLE PROTEIN), Homo sapiens
UniProt: Q92793
Assay: In Vitro CBP Inhibition: The CBP-inhibitory activity of the compounds described herein was determined by calculating the IC50. More specifically, CBP inhibitor activity was assayed as follows: CBP was cloned and expressed in E. coli as His-tag protein and purified by Nickel affinity and gel-filtration chromatography. The protein was further characterized as a single band with the correct molecular weight by SDS-PAGE. CBP binding and inhibition is assessed by monitoring the interaction of biotinylated H4-tetraacetyl peptide (AnaSpec, H4K5/8/12/16(Ac), biotin-labeled) with the target using the AlphaScreen technology (Perkin Elmer). In a 384-well ProxiPlate CBP (50 nM final) was combined with peptide (20 nM final) in 50 mM HEPES (pH 7.3), 10 mM NaCl, 0.25 mM TCEP, 0.1% (w/v) BSA, and 0.005% (w/v) Brij-35 either in the presence of DMSO (final 0.4% DMSO) or compound dilution series in DMSO. After 20 min incubation at room temp, Alpha-streptavidin donor beads and Nickel Chelate acceptor beads were added to a final concentration of 5 μg/mL. After 2 hr of equilibration, plates were read on an Envision instrument and the IC50 calculated using a four parameter non-linear curve fit.
Measurement: IC50 < 500 nM (pChEMBL 6.301000118255615)
Descriptors: MW 440.48, ALogP 4.13, HBA 7, HBD 1, PSA 88.53, QED 0.49
Compound (SMILES): CCNCc1cc(F)c(-c2ncc3[nH]nc(-c4cnn(C)c4)c3n2)c(OC)c1
InChIKey: LBCVZDGRKSAKAK-UHFFFAOYSA-N
Target: Mitogen-activated protein kinase kinase kinase kinase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q92918
Assay: Kinase Binding Assay: A stock solution of 1 mM test compound was prepared in DMSO. The compound plate was prepared by 3-fold and 11-point serial dilutions. 0.1 μL of the compound in DMSO was transferred from the compound plate to the white 384 well polystyrene plates. The assay buffer contained 50 mM HEPES, pH 7.5, 0.01% Tween-20, 5 mM MgCl2, 0.01% BSA, and 5 mM DTT. 5 μl of 4 nM active HPK1 (SignalChem M23-11G) prepared in the buffer was added to the plate. The enzyme concentration given was based on the given stock concentration reported by the vender. 5 μl of 18 nM tracer 222 (ThermoFisher PV6121) and 4 nM LanthaScreen Eu-Anti GST antibody (ThermoFisher PV5595) were added. After one hour incubation at 25° C., the plates were read on a PHERAstar FS plate reader (BMG Labtech).
Measurement: Ki < 50 nM (pChEMBL 7.301000118255615)
Descriptors: MW 381.42, ALogP 2.68, HBA 7, HBD 2, PSA 93.54, QED 0.53
Compound (SMILES): CC(c1cnc(C(F)(F)F)cn1)n1nc(C#N)c2c(=O)[nH]c(C3CCC3c3ncccn3)nc21
InChIKey: SESUHSGADRECQZ-UHFFFAOYSA-N
Target: High affinity cGMP-specific 3',5'-cyclic phosphodiesterase 9A (SINGLE PROTEIN), Homo sapiens
UniProt: O76083
Assay: Fluorescence Polarization Assay: Human PDE9 (PDE9A2, GenBank Accession No. NM_001001567), full length with N-terminal GST tag, was purchased from BPS Bioscience. The fluorescence polarization assay for cyclic nucleotide phosphodiesterases was performed using an IMAP® FP kit supplied by Molecular Devices, Sunnyvale, Calif. (product #R8139). IMAP® technology has been applied previously to phosphodiesterase assays. Assays were performed at room temperature in 384-well microtiter plates with an incubation volume of 20.2 μL. Solutions of test compounds were prepared in DMSO and serially diluted with DMSO to yield 8 μL of each of 10 solutions differing by 3-fold in concentration, at 32 serial dilutions per plate.
Measurement: Ki = 19.6 nM (pChEMBL 7.710000038146973)
Descriptors: MW 467.42, ALogP 2.86, HBA 9, HBD 1, PSA 138.92, QED 0.48
Compound (SMILES): CC(C)C[C@H](N)[C@](O)(Cc1nccc2c1C[C@H](CCC1CC1)C2)C(=O)O
InChIKey: PXESZBHBYSOLEN-MFMILTCTSA-N
Target: Leucyl-cystinyl aminopeptidase (SINGLE PROTEIN), Rattus norvegicus
UniProt: P97629
Assay: Inhibition Assay: The inventors have assumed that inhibition of nocturnal activity of placental leucine aminopeptidase (P-LAP), i.e. aminopeptidase that cleaves AVP, would maintain and/or increase an endogenous AVP level to enhance the antidiuretic effect, which would contribute to a decreased number of nocturnal voids, and have extensively studied compounds which inhibit P-LAP.
Measurement: IC50 = 5.9 nM (pChEMBL 8.229999542236328)
Descriptors: MW 360.5, ALogP 2.72, HBA 4, HBD 3, PSA 96.44, QED 0.63
Compound (SMILES): CC(C)n1nc(-c2ccccc2)c2c1CN(C#N)C2
InChIKey: GHUWLPWYLALVPP-UHFFFAOYSA-N
Target: Ubiquitin carboxyl-terminal hydrolase 7 (SINGLE PROTEIN), Homo sapiens
UniProt: Q93009
Assay: Biochemical Assay: Dilution plates were prepared at 21 times the final concentration (2100 μM for a final concentration of 100 μM) in 50% DMSO in a 96-well polypropylene V-bottom plate (Greiner #651201). A typical 8-point dilution series would be 100, 30, 10, 3, 1, 0.3, 0.1, 0.03 μM final. Reactions were performed in duplicate in black 384 well plates (small volume, Greiner 784076) in a final reaction volume of 21 μl. Either 1 μl of 50% DMSO or diluted compound was added to the plate. USP7 was diluted in reaction buffer (20 mM Tris, pH 7.5, 100 mM NaCl, 0.05% Tween 20, 0.5 mg/ml BSA, 5 mMbeta mercaptoethanol) to the equivalent of 0.0025 μl/well and 10 μl of diluted USP7 was added to the compound. Enzyme and compound were incubated for 30 min at room temp. Reactions were initiated by the addition of 50 nM of TAMRA labelled peptide linked to ubiquitin via an iso-peptide bond as fluorescence polarisation substrate. Reactions were read immediately after addition of substrate and following a 2 hr incubation at room temperature. Readings were performed on a Pherastar Plus (BMG Labtech). λ Excitation 540 nm; λ Emission 590 nm.
Measurement: IC50 < 10000 nM (pChEMBL 5)
Descriptors: MW 252.32, ALogP 2.93, HBA 4, HBD 0, PSA 44.85, QED 0.77
Compound (SMILES): OCCc1ccc(Cc2cc(C(F)(F)F)nc(-c3cccc(Cl)c3)n2)cc1
InChIKey: NCPPCVZMWDRHFG-UHFFFAOYSA-N
Target: 3',5'-cyclic-AMP phosphodiesterase 4B (SINGLE PROTEIN), Homo sapiens
UniProt: Q07343
Assay: Kinetic Assay: Assays are performed in 96-well plates in a total volume of 200 μl/well. Compounds are dissolved in dimethylsulfoxide (DMSO) and added to plates in a volume of 10 μl followed by addition of 165 μl of assay mix. Plates are pre-incubated at 25° C. for 15 min and the reactions are initiated by the addition of 25 μl of cAMP followed by thorough mixing. Reaction rates are measured by monitoring the decrease in fluorescence using excitation at 355 nm and emission at 460 nm for a period of 10 min in a fluorescence plate reader. Initial rates (slopes) are determined from linear portions of the progress curves. Final concentrations of assay components are as follows: 50 mM Tris, pH 8, 10 mM MgCl2, 50 mM KCl, 2% DMSO, 5 mM tris(2-carboxyethyl)phosphine (TCEP), 0.4 mM phosphenolpyruvate (PEP), 0.01 mM NADH, 0.04 mM adenosine triphosphate (ATP), 0.004 mM cAMP, 7.5 units myokinase from yeast, 1.6 units pyruvate kinase, 2 units lactate dehydrogenase, and either 0.5 nM human PDE4D7 or 10 nM human PDE4B1. All data are percent normalized relative to controls and are presented as percent inhibition. An inhibitory concentration 50% (IC50) value is calculated by fitting of a sigmoidal dose response curve. Human PDE4D7 contained a mutation of serine 54 to aspartic acid to mimic activation by cAMP-dependent protein kinase A (PKA). Human PDE4B1 contained a corresponding mutation of serine 133 to aspartic acid to also mimic PKA activation.
Measurement: IC50 > 1000 nM (pChEMBL 6)
Descriptors: MW 392.81, ALogP 4.94, HBA 3, HBD 1, PSA 46.01, QED 0.67
Compound (SMILES): CN1C(=O)C(NC(=O)c2n[nH]c(Cc3ccccc3)n2)COc2ccc(C#CC(C)(C)O)cc21
InChIKey: SQBZJKIINNRUFC-UHFFFAOYSA-N
Target: Receptor-interacting serine/threonine-protein kinase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q13546
Assay: 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 column1) add 2× rxn 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. The plate was quickly centrifuged and the reaction was incubated at 30° C. for 30 minutes. Adding 5 μl of ADP-Glo terminated the reaction. The plate was quickly centrifuged and the reaction was incubated at room temperature for 40 minutes. Kinase Detection Reagent was then added and incubated at room temperature for 30 minutes. The relative light unit (RLU) of kinase reaction was determined by luminescent (Luminescence 0.1 s) using a Wallac Victor2 Luminometer (PerkinElmer, Waltham, Mass., USA).
Measurement: IC50 = 28.6 nM (pChEMBL 7.539999961853027)
Descriptors: MW 459.51, ALogP 1.67, HBA 6, HBD 3, PSA 120.44, QED 0.51
Compound (SMILES): Cn1cc(-c2cc(-c3ccc(N4CCC(Nc5ccccc5)CC4)nc3)c3c(C#N)cnn3c2)cn1
InChIKey: QDMRTNJSXBZEQK-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 = 6.2 nM (pChEMBL 8.210000038146973)
Descriptors: MW 474.57, ALogP 4.75, HBA 8, HBD 1, PSA 87.07, QED 0.4
Compound (SMILES): CN1Cc2cc(Nc3ncc4c(=O)n5n(c4n3)-c3cccc(n3)OCC/C=C\C5)ccc2C2(CC2)C1
InChIKey: YCDCSAXFBHLEGT-IHWYPQMZSA-N
Target: Wee1-like protein kinase (SINGLE PROTEIN), Homo sapiens
UniProt: P30291
Assay: Enzymatic Activity of Wee1: In the present disclosure, Wee1 enzyme catalysis assay was carried out using the ATP-Glo Max kinase luminescence detection kit (Promega). Kinase activity was assessed by quantitative detection of the amount of ATP retained in the solution following the kinase reaction. The luminescent signal in the assay is proportional to the amount of ATP and inversely proportional to the kinase activity. The concentration of the compound in the assay ranged from 0.5 nM to 30 μM. The compound was dissolved in 10% DMSO, and 5 μL of the solution was added to 50 μL of the reaction, and the concentration of DMSO in the final reaction was 1%. The reaction was carried out at 30° C. for 50 minutes. 40 mM trishydroxymetyl aminomethane, pH 7.4, 10 mM MgCl2, 0.1 mg/ml BSA, 2 mM DTT, 0.1 mg/ml Poly (Lys, Tyr) substrate, 10 μM ATP and Wee1 were contained in the reaction mixture. After the enzymatic reaction, 50 μL of ATP-Glo Max kinase luminescence detection assay solution (Promega) was added and incubated for 15 minutes at room temperature. The luminescent signal was measured using a microplate reader. In some assays, a known Wee1 inhibitor was added as a positive control. Luminance data was analyzed using Graphpad software.
Measurement: IC50 = 5.4 nM (pChEMBL 8.270000457763672)
Descriptors: MW 481.56, ALogP 3.54, HBA 9, HBD 1, PSA 90.1, QED 0.44
Compound (SMILES): COc1cccc2c(C(=O)NCc3cccc(CSc4nc5ccccc5[nH]4)n3)nc(N)nc12
InChIKey: XPLKNJUPWPHNLI-UHFFFAOYSA-N
Target: Adenosine receptor A2a (SINGLE PROTEIN), Homo sapiens
UniProt: P29274
Assay: A2a Ki assay: Binding affinities of compounds of the invention for the human A2a receptor were determined in a competition binding assay using Scintillation Proximity technology. Thus, 1* μg, or preferably 0.25 μg of membranes from HEK293 cells expressing the human A2a receptor were incubated with a compound of the invention at concentrations ranging from 3000 nM to 0.15 nM in a reaction mixture containing also 0.5 nM of a tritiated form of 5-amino-7-[2-phenethyl]-2-(furan-2-yl)-7H-pyrazolo[4,3-e][1,2,4]triazolo[1,5-c]pyrimidine (the tritiated compound) and 25 μg of wheat germ agglutin-coated yttrium silicate SPA beads for one hour at room temperature with agitation. The beads were then allowed to settle to the bottom of the wells for 1 hr, after which the membrane-associated radioactivity was determined by scintillation counting in a TopCount microplate reader. Ki values were determined using the Cheng-Prusoff equation.
Measurement: Ki = 6.3 nM (pChEMBL 8.199999809265137)
Descriptors: MW 471.55, ALogP 3.71, HBA 8, HBD 3, PSA 131.7, QED 0.31
Compound (SMILES): COC[C@@H](C)Nc1n[nH]c2nccc(Oc3ccc(NC(=O)c4cc(C)cn(-c5ccc(F)cc5)c4=O)nc3)c12
InChIKey: RKCVCQRGHMAAHS-QGZVFWFLSA-N
Target: Tyrosine-protein kinase receptor UFO (SINGLE PROTEIN), Homo sapiens
UniProt: P30530
Assay: Enzyme Assay: The affinity of compound binding to wild type and mutant human MET kinases is measured using Invitrogen's LanthaScreen Eu Kinase Binding technology. Briefly, GST-tagged recombinant human MET kinase domain from Signal Chem (see Table 8 below for concentration in assay) is incubated with 50 nM Alexa-Fluor® Tracer 236 (Invitrogen Cat No. PR9078A) and 2 nM Europium-anti-GST (Invitrogen Cat. No. A151 6) along with test compound in a buffer consisting of 25 mM HEPES, pH 7.4, 10 mM MgCl2, 0.01% Triton X-100, 1 mM DTT, and 2% DMSO. Compounds are typically prepared in a three-fold serial dilution in DMSO and added to the assay to give the appropriate final concentration. After a 60-minute incubation at 22° C., the reaction is measured using a PerkinElmer EnVision multimode plate reader via TR-FRET dual wavelength detection, and the percent of control (POC) calculated using a ratiometric emission factor. 100 POC is determined using no test compounds and 0 POC is determined using a concentration of control compound that completely inhibits the enzyme
Measurement: IC50 = 3600 nM (pChEMBL 5.440000057220459)
Descriptors: MW 543.56, ALogP 4.44, HBA 9, HBD 3, PSA 136.05, QED 0.25
Compound (SMILES): COc1cc(N2C(=O)c3nc(C4CC4)n(C)c3C2c2ccc(Cl)cc2)cn2c(C)nnc12
InChIKey: SGQSLPKSTYGKNO-UHFFFAOYSA-N
Target: Bromodomain-containing protein 4 (SINGLE PROTEIN), Homo sapiens
UniProt: O60885
Assay: In-Vitro Binding Assay: All assays were performed in 384 well microtiter plates. Each assay plate contained 8-point serial dilutions for 40 test compounds, plus 16 high- and 16 low controls. Liquid handling and incubation steps were done on an Innovadyne Nanodrop Express equipped with a robotic arm (Thermo CatX, Perkin Elmer/Caliper Twister II) and an incubator (Liconic STX40, Thermo Cytomat 2C450). The assay plates were prepared by addition of 50 nl per well of compound solution in 90% DMSO HummingBird nanodispenser (Zinsser Analytic). The assay was started by stepwise addition of 4.5 ul per well of bromo domain protein (50 mM HEPES, pH 7.5, 0.005% Tween20, 0.1% BSA, 50 mM NaCl, 45 nM His-Brd2(60-472) or 45 nM His-Brd3(20-477) or 45 nM His-Brd4(44-477) all proteins produced in-house) and 4.5ul per well of peptide solution (50 mM HEPES, pH 7.5, 0.005% Tween20, 0.1% BSA, 50 mM NaCl, 60 nM acetyl-histone H4 (AcK 5, 8, 12, 16) (Biosyntan GmbH)). Reactions were incubated at 30° C. for 35 minutes. Subsequently 4.5 ul per well detection mix (50 mM HEPES, pH 7.5, 0.005% Tween20, 0.1% BSA, 50 mM NaCl, 3 nM Eu-labeled anti-His6 antibody, 21 nM streptavidin-allophycocyanin) were added. After 35 minutes incubation at 30° C., plates were measured in a Perkin Elmer EnVision multilabel reader. Concentrations causing 50% inhibition (IC50 values) were determined from percent inhibition values at different compound concentrations by non-linear regression analysis.
Measurement: IC50 = 130 nM (pChEMBL 6.889999866485596)
Descriptors: MW 448.91, ALogP 4.06, HBA 7, HBD 0, PSA 77.55, QED 0.47
Compound (SMILES): CCc1cc(N2CCN(C(=O)CCC3(CC(=O)O)NC(=O)NC3=O)C[C@@H]2C)ccc1Cl
InChIKey: GSHKFLCNIDFHJZ-JRTLGTJJSA-N
Target: A disintegrin and metalloproteinase with thrombospondin motifs 5 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9UNA0
Assay: In Vitro Assay: ADAMTS-5:Protocol 2: The basis for the assay is the cleavage of the substrate TBIS-1 (5 FAM-TEGEARGSVILLK (5TAMRA)K-NH2) (SEQ ID No 2) by human ADAMTS-5.For the dose response (10 point), 4 μL of a dilution series of compound (2 mM highest concentration, 1/5 dilution in DMSO further diluted 1 in 10 in water, corresponding to a final highest concentration of 20 μM), is transferred to 384 well Fluotrac 200 plate (Greiner, cat #781076) and incubated at room temperature for 30 min with a 26 μL buffer solution (50 mM Hepes pH7.5, 100 mM NaCl, 5 mM CaCl2, 0.1% CHAPS 1) containing hADAMTS-5 (1 ng/μL, affinity purified, followed by overnight digestion of 6His tag by thrombin and dialysis) (it will be appreciated by the skilled person that the potency read out is independent of the enzyme concentration).The reaction is initiated by adding to the assay plate TBIS-1 (10 μL, 4.5 μM, Anaspec) in the same buffer.Finally, the fluorescence is read on the Envision (Perkin Elmer) after an incubation of 45 min at 37° C. (Excitation 485 nm, emission 530).
Measurement: IC50 = 852 nM (pChEMBL 6.070000171661377)
Descriptors: MW 450.92, ALogP 1.77, HBA 5, HBD 3, PSA 119.05, QED 0.54
Compound (SMILES): Cc1ccncc1-c1cc2cc(Nc3ccn(C)c(=O)c3)ncc2c(N)n1
InChIKey: SJNGDNCXABAXAN-UHFFFAOYSA-N
Target: Mitogen-activated protein kinase kinase kinase kinase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q92918
Assay: Binding Assay: Lanth: For the binding assay, 4 ul 2×HPK1 and Eu-anti-GST antibody were added to each well of the assay plate using a Multidrop reagent dispenser. The solutions were incubated in a 23 C incubator for 1 h. To each well of the assay plate was added 4 ul 2× Tracer-222 using a Multidrop reagent dispenser. The solutions were again incubated in a 23 C incubator for 1 h. The results of the assay were read using an Envision plate reader with the following parameters: TR_FRET, 340ex/615 and 665em; 100 usec Delay; and 200 usec integration.
Measurement: Ki = 3 nM (pChEMBL 8.520000457763672)
Descriptors: MW 358.41, ALogP 3.02, HBA 7, HBD 2, PSA 98.72, QED 0.58
Compound (SMILES): Cc1nnc2c(NCCCCNC(=O)[C@@H](NC(=O)C(C)O)C(C)C)nc3cc(N4CCOCC4)ccc3n12
InChIKey: LNPRWVBRXZTVDW-UGNFMNBCSA-N
Target: Bromodomain-containing protein 4 (SINGLE PROTEIN), Homo sapiens
UniProt: O60885
Assay: Evaluation of BRD4 binding inhibition ability: The following experiment was performed to evaluate the ability of [1,2,4]triazolo[4,3-a]quinoxaline derivative of the present invention to inhibit the interaction between BRD4 (BD1+BD2) bromodomain, one of BET protein family, and tetraacetylated histone H4 peptide.The compound was serially diluted at the ratio of 1:5 in assay buffer from 10 mM stock in DMSO (initial concentration: 100 μM) on white OptiPlate-384 (PerkinElmer). A mixture comprising 100 nM GST-BRD4 (BD1+BD2) and 100 nM biotinylated acetyl-histone H4 (Lys5,8,12,16) peptide was prepared in assay buffer (50 mM HEPES pH 7.4; 25 mM NaCl; 0.05% Tween 20; 0.1% bovine serum albumin (BSA); 10 mM dithiothreitol (DTT)). After adding 6 μl of the mixture to the diluent, 6 μl of the pre-mixed AlphaLISA Glutathione Acceptor Beads and AlphaScreen Streptavidin Donor Beads (PerkinElmer, 10 μg/ml in assay buffer, respectively) was added thereto. The samples were incubated in the dark at room temperature for 30 minutes (shaking at 300 rpm). Then, the signals were measured with PerkinElmer Envision HTS Multilabel Reader using an alpha screen protocol of PerkinElmer. Each plate contained the negative control in which biotinylated acetyl-histone H4 peptide and GST-BRD4 (BD1+BD2) were replaced by assay buffer. In the case of using the software GraphPad Prism for calculation, the negative control point was input as a low standard value. The positive control (probe molecule l-BET762 containing protein/peptide mixture) proceeded to pipetting. IC50 value was determined by using GraphPad Prism 3.03 software (or an updated version thereof).
Measurement: IC50 = 34 nM (pChEMBL 7.46999979019165)
Descriptors: MW 526.64, ALogP 1.25, HBA 10, HBD 4, PSA 146.01, QED 0.27
Compound (SMILES): CC(C)C[C@@H](C)n1cc(-c2nc(-c3cnn(C[C@H](O)CO)c3)cn3nccc23)cn1
InChIKey: XOLGTWQACRYHDY-QAPCUYQASA-N
Target: Non-receptor tyrosine-protein kinase TYK2 (SINGLE PROTEIN), Homo sapiens
UniProt: P29597
Assay: Tyk2 Inhibition Assay: Compounds of Formula I were screened for their ability to inhibit Tyk2 using the general enzyme inhibition assay method, in which the assay mixture contained 1 mM ATP, 8 μM Omnia Y12 peptide (Catalog # IVGN KPZ3121C; Invitrogen Corporation, Carlsbad, Calif.) and 1 nM Tyk2 in a total volume of 25 μL. Human Tyk2 kinase domain, comprising amino acids 886 to 1187 with 10 additional histidine residues (histidine tag) on the carboxy terminus, was expressed and purified from bacculovirus in-house at Array BioPharma Inc. (Boulder, Colo.). The histidine tag was cleaved after purification using standard conditions.
Measurement: IC50 = 55 nM (pChEMBL 7.260000228881836)
Descriptors: MW 409.49, ALogP 2.42, HBA 9, HBD 2, PSA 106.29, QED 0.46
Compound (SMILES): Cc1cc2sc(N3CCC[C@@H]3C(=O)NCc3ccccc3)nc2nc1C
InChIKey: DYYBOOHCPODCNG-MRXNPFEDSA-N
Target: Kynurenine/alpha-aminoadipate aminotransferase, mitochondrial (SINGLE PROTEIN), Homo sapiens
UniProt: Q8N5Z0
Assay: Inhibitory Assay: To a reaction mixture (45 μL) containing 3.0 μmol/L kynurenine, 10 μmol/L pyridoxal phosphate, 2.0 ng/μL human recombinant KAT-II, and 150 mmol/L tris(hydroxymethyl)aminomethane-acetate buffer (pH 8.0) was added a 10% dimethyl sulfoxide solution (5 μL) of each test compound prepared, and the mixture was reacted at 37° C. for 1 hr. After the reaction, 50% trichloroacetic acid (5 μL) was added to terminate the reaction.The resultant kynurenic acid was quantified as follows by high performance liquid chromatography. An enzyme reaction mixture was separated by an octadecylsilane reversed-phase column (SC-50DS, Eicom Corporation; mobile phase: 250 mmol/L zinc acetate, 50 mmol/L sodium acetate, and 5.0% acetonitrile (pH 6.2)) incubated at 30° C., and kynurenic acid was quantified using a fluorescence detector (RF-20Axs, Shimadzu Corporation) at excitation wavelength 354 nm, detection wavelength 460 nm. The analytical curve was drawn every time by an external standard method. Each test compound was tested by dual measurement at each concentration. The kynurenic acid level in the presence of a test compound at each concentration was converted into % relative to kynurenic acid resulting from a reaction with an enzyme alone as 100%, and the obtained values were fitted to S-curve to determine IC50.
Measurement: IC50 = 900 nM (pChEMBL 6.050000190734863)
Descriptors: MW 366.49, ALogP 3.59, HBA 5, HBD 1, PSA 58.12, QED 0.77