lovingscience/chembl-v37-raw-activities huggingface dataset:
Compound (SMILES): Cc1cccc(-c2nn3c(c2-c2ccnc4ccc(C(N)=O)cc24)C2CCC3CC2)n1
InChIKey: WXDFWGNBHNFNIF-UHFFFAOYSA-N
Target: Vascular endothelial growth factor receptor 2 (SINGLE PROTEIN), Homo sapiens
UniProt: P35968
Assay: Kinase Activity Assay: VEGFR2: Z′-LYTE® Kinase Assay KitTyrosine 1 Peptide (PV3190, Invitrogen) was used to assay enzyme activity. 5 μl of recombinant human VEGFR2 enzyme (PV3660, Invitrogen) and VEGFR2 substrate polypeptide (in the reaction system, the final concentration of enzyme was 0.14 ng/μL, and the final concentration of substrate was 2 NM) formulated with reaction buffer (50 mM HEPES pH7.5, 10 mM MgCl2, 1 mM EGTA, 0.05% BRIJ-35), 2.5 μl of a 2-fold gradient dilution of the compounds dissolved in 5% DMSO, and 2.5 μL of ATP solution (the final concentration of ATP was 50 μM) were added successively to a 384-well plate (4513, Corning). After reaction at 25° C. for 2 hours, 5 μL of detection reagent was added to each well. After the plate was placed at 25° C. for 1 hour, the fluorescence signal values at emission wavelengths of 445 nm and 520 nm were measured with a NOVOstar (BMG) multi-function microplate reader. The IC50 values of the compounds for enzyme inhibition were calculated using Graphpad prism software based on each concentration of the compound and the corresponding signal value thereof.
Measurement: IC50 = 1243 nM (pChEMBL 5.909999847412109)
Descriptors: MW 409.49, ALogP 4.78, HBA 5, HBD 1, PSA 86.69, QED 0.53
Compound (SMILES): CCc1nc2c(F)cc(-c3nc(N[C@@H]4CCN(S(C)(=O)=O)C[C@H]4O)ncc3Cl)cc2n1C1COC1
InChIKey: OKENGSCNEWBERF-SJLPKXTDSA-N
Target: CDK6/cyclin D3 (PROTEIN COMPLEX), Homo sapiens
UniProt: P30281
Assay: Biological Assay: CDK6/Cyclin D3: The purpose of the CDK6/Cyclin D3 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. CDK6/Cyclin D3 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), 2% glycerol, 10 mM MgCl2, 1 mM DTT, 3.5 mM ATP, 0.005% Tween 20 (TW-20), 3 μM 5-FAM-Dyrktide, 4 nM (active sites) activated CDK6/Cyclin D3 in 40 mM HEPES buffer at pH 7.5.
Measurement: Ki = 11.4 nM (pChEMBL 7.940000057220459)
Descriptors: MW 525.01, ALogP 2.23, HBA 9, HBD 2, PSA 122.47, QED 0.5
Compound (SMILES): COc1ncnc(OC)c1-n1c(NS(=O)(=O)[C@@H]2C[C@H](OC(C)C)CN(c3ncc(F)cn3)C2)nnc1[C@@H]1COCCO1
InChIKey: FJJPEMOTKSRQSO-KSZLIROESA-N
Target: Apelin receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P35414
Assay: [35S]GTPgammaS Binding: The human APJ receptor was cloned by polymerase chain reaction and the gene encoding the receptor was subcloned in pFLAG-CMV-3 expression vector (Sigma, Saint Louis, Mo. USA) in-house at Amgen. A GTPγS binding assay was performed on membranes prepared from CHO cells stably expressing human APJ receptor. The optimum experimental conditions for the concentrations of GDP, MgCl2, and NaCl in the assay buffer were initially determined. The assay was performed in assay buffer [20 mM HEPES, pH 7.5, 5 mM MgCl2, and 0.1% (w/v) BSA with 200 mM NaCl, 3 μM GDP] and membranes expressing human APJ receptor/well along with WGA PS beads. The reaction was initiated by addition of 0.2 nM [35S]GTPγS (Perkin Elmer Life and Analytical Sciences, Waltham USA) in the absence or presence of various ligands and incubated at RT for 90 min. Nonspecific binding was determined in the presence of 100 μM GTPγS and was always less than 0.2% of total binding. All the results presented are means of several independent experiments and analyzed by non-linear regression methods using commercially available program Prism (GraphPad, San Diego, Calif.).
Measurement: EC50 = 240 nM (pChEMBL 6.619999885559082)
Descriptors: MW 609.64, ALogP 0.91, HBA 15, HBD 1, PSA 177.83, QED 0.34
Compound (SMILES): NC1CCCC(Nc2ccc(C3=NNC(=O)OC3)cc2C(F)(F)F)C1
InChIKey: RCDJAPNFKIPWFJ-UHFFFAOYSA-N
Target: cGMP-inhibited 3',5'-cyclic phosphodiesterase 3B (SINGLE PROTEIN), Homo sapiens
UniProt: Q13370
Assay: PDE3B Enzyme Inhibition: The commercially available 3H-cAMP Scintillation Proximity Assay (SPA, Perkin Elmer) system was used for enzyme inhibition studies. For the determination of the in vitro effect of example compounds on the PDE3B reactions 2 μl of the respective example compound solution in DMSO (serial dilutions) were placed in wells of microtiter plates (Isoplate-96/200W; Perkin Elmer). 50 μl of a dilution of PDE3B cell extract from Sf9 cells overexpressing human full length PDE3B (SB Drug Discovery, UK) in buffer A (50 mM Tris/HCl pH 7.5, 8.3 mM MgCl2, 1.7 mM EDTA, 0.2% BSA) was added. The dilution of the PDE3B cell extract was chosen such that the reaction kinetics was linear and less than 70% of the substrate was consumed (typical dilution 1:6000). The reaction was started by addition of 50 μl (0.025 μCi) of 1:2000 in buffer A w/o BSA diluted substrate [8-3H]adenosine 3′, 5′-cyclic phosphate (1 μCi/μl; Perkin Elmer). After incubation at room temperature for 60 min, the reaction was stopped by addition of 25 μl of a suspension containing 18 mg/ml yttrium scintillation proximity beads (Perkin Elmer) in water. The microtiter plates were sealed and measured in a Microbeta scintillation counter (PerkinElmer Wallac). IC50 values were determined from sigmoidal curves by plotting percentage PDE3B activity vs log compound concentration.
Measurement: IC50 = 780 nM (pChEMBL 6.110000133514404)
Descriptors: MW 356.35, ALogP 2.83, HBA 5, HBD 3, PSA 88.74, QED 0.78
Compound (SMILES): COc1cn(CC(=O)Nc2ccc3nccnc3c2)c(=O)cc1-c1cc(Cl)ccc1-n1cnnn1
InChIKey: GOCDCIBQPRFDPL-UHFFFAOYSA-N
Target: Plasma kallikrein (SINGLE PROTEIN), Homo sapiens
UniProt: P03952
Assay: Activity Assay: To determine the plasma kallikrein inhibition of the substances according to the invention, a biochemical test system is used which utilizes the reaction of a peptidic plasma kallikrein substrate to determine the enzymatic activity of human plasma kallikrein. Here, plasma kallikrein cleaves from the peptic plasma kallikrein substrate the C-terminal aminomethylcoumarin (AMC), the fluorescence of which is measured. The determinations are carried out in microtitre plates.Test substances are dissolved in dimethyl sulphoxide and serially diluted in dimethyl sulphoxide (3000 μM to 0.0078 μM; resulting final concentrations in the test: 50 μM to 0.00013 μM). In each case 1 μl of the diluted substance solutions is placed into the wells of white microtitre plates from Greiner (384 wells). 20 μl of assay buffer (50 mM Tris/HCl pH 7.4; 100 mM sodium chloride solution; 5 mM of calcium chloride solution; 0.1% of bovine serum albumin) and 20 μl of plasma kallikrein from Kordia (0.6 nM in assay buffer) are then added successively. After 15 min of incubation, the enzyme reaction is started by addition of 20 μl of the substrate H-Pro-Phe-Arg-AMC dissolved in assay buffer (10 μM in assay buffer) from Bachem, the mixture is incubated at room temperature (22° C.) for 30 min and fluorescence is then measured (excitation: 360 nm, emission: 460 nm).
Measurement: IC50 = 1 nM (pChEMBL 9)
Descriptors: MW 488.9, ALogP 2.73, HBA 10, HBD 1, PSA 129.71, QED 0.39
Compound (SMILES): CCCCCNc1cc(-c2cnc(NCCCC)nc2NC2CCC(O)CC2)ncn1
InChIKey: MIOQFVQXGRHCSR-UHFFFAOYSA-N
Target: Tyrosine-protein kinase receptor TYRO3 (SINGLE PROTEIN), Homo sapiens
UniProt: Q06418
Assay: Activity Assay: Briefly, activity assays were performed in a 384 well, polypropylene microplate in a final volume of 50 μL of 50 mM Hepes, Ph 7.4 containing 10 mM MgCl2, 1.0 mM DTT, 0.01% Triton X-100, 0.1% Bovine Serum Albumin (BSA), containing 1.0 μM fluorescent substrate and ATP at the Km for each enzyme. All reactions were terminated by addition of 20 μL of 70 mM EDTA. After a 180 min incubation, phosphorylated and unphosphorylated substrate peptides were separated in buffer supplemented with 1×CR-8 on a LabChip EZ Reader equipped with a 12-sipper chip. Data were analyzed using EZ Reader software. Assay conditions for MCE assays
Measurement: IC50 = 112 nM (pChEMBL 6.949999809265137)
Descriptors: MW 427.6, ALogP 4.46, HBA 8, HBD 4, PSA 107.88, QED 0.37
Compound (SMILES): CN(C)S(=O)(=O)c1ccc(Nc2nn(C3(CC#N)CCN(C(=O)C4Cc5ccccc5C4)CC3)c3cc[nH]c(=O)c23)cc1
InChIKey: BJNXUMVTXUMLQP-UHFFFAOYSA-N
Target: Tyrosine-protein kinase JAK2 (SINGLE PROTEIN), Homo sapiens
UniProt: O60674
Assay: Biochemical HTRF Assay : The ability of compounds to inhibit the activity of JAK1, JAK2, JAK3, and Tyk2 was measured using a recombinant purified GST-tagged catalytic domain for each enzyme (Invitrogen JAK1 #M4290, JAK2 #M4290, JAK3 #M4290, Tyk2 #M4290) in an HTRF format biochemical assay. The reactions employed a common peptide substrate, LCB-EQEDEPEGDYFEWLW-NH2 (in-house). The basic assay protocol is as follows: First, 250 nL of diluted compounds in DMSO were dispensed into the wells of a dry 384-well Black plate (Greiner #781076) using a Labcyte Echo 555 acoustic dispenser. Subsequent reagent additions employed an Agilent Bravo. Next, 18 μL of 1.11× enzyme and 1.11× substrate in 1× assay buffer (Invitrogen kinase buffer #PV3189, 2 mM DTT, 0.05% BSA) were added to the wells and shaken and then preincubated for 30 minutes at room temperature to allow compound binding to equilibrate. After equilibration, 2 μL of 10×ATP in 1× assay buffer was added to initiate the kinase reaction and the plates were shaken and then incubated at room temperature for 120 minutes. At the end of the incubation, 20 μL of 2× stop buffer (streptavidin-Dylight 650 (Thermo #84547B/100 mL), Eu-tagged pY20 antibody (Perkin Elmer #AD0067), EDTA, HEPES, and Triton) was added to quench the reaction. Plates were shaken and centrifuged and then incubated 60 minutes at room temperature and then read on a Perkin Elmer Envision (λex=337 nm, λem=665 and 615 nm, TRF delay time=20 μs). HTRF signal=10,000*665 nm reading/615 nm reading.
Measurement: IC50 = 0.39 nM (pChEMBL 9.40999984741211)
Descriptors: MW 599.72, ALogP 3.36, HBA 8, HBD 2, PSA 144.19, QED 0.33
Compound (SMILES): CC(C)c1cnc2ccc3cnc(Nc4ccc(N5CCC6(CC5)CNC6)cn4)nc3n12
InChIKey: GITJQMOEQGGXTM-UHFFFAOYSA-N
Target: CDK6/cyclin D1 (PROTEIN COMPLEX), Homo sapiens
UniProt: P24385
Assay: Biochemical Kinase Inhibition Assay: Potency determination of biochemical kinase inhibition test. Kinase activity test and IC50 determination. Firstly, 10 ng of recombinant CDK4/Cyclin D1 (Life Technologies PV4204) was diluted in kinase buffer (20 mM Tris pH 7.5, 10 mM MgCl2, 0.01% NP-40, 2 mM DTT), and was incubated with inhibitors at the indicated concentration at room temperature for 30 minutes. The kinase reaction was initiated by adding 1 μg (1.5 μM) of recombinant retinoblastoma protein, 5 μM ATP and 10μ Ci γ-32P-ATP. The reaction was incubated at 30° C. for 20 minutes, and was terminated by adding 2×Laemmli sample buffer, heated at 95° C. for three minutes, and the resulting solution was dissolved with 12% acrylamide SDS-PAGE and autoradiographed. A densitometer (Bio-Rad) was used to quantify the corresponding phosphorylated substrate protein bands. The resulting density values were plotted as a function of the log drug concentration using Prism 4 Graphpad software, IC50 values were determined by plotting a non-linear regression curve with a variable slope.
Measurement: IC50 = 35 nM (pChEMBL 7.460000038146973)
Descriptors: MW 428.54, ALogP 3.73, HBA 8, HBD 2, PSA 83.27, QED 0.51
Compound (SMILES): CCOc1cc2ncc(C=O)c(Nc3ccc(OCc4ncccc4C)cc3Cl)c2cc1NC(=O)CCCN(C)C
InChIKey: XEEIXGBLWIQZDU-UHFFFAOYSA-N
Target: Serine/threonine-protein kinase 3 (SINGLE PROTEIN), Homo sapiens
UniProt: Q13188
Assay: MST1/MST2 Biochemical LanthaScreen Eu Kinase Binding Assay: MST1 and MST2 biochemical LanthaScreen Eu Kinase Binding Assay was based on the binding and displacement of kinase tracer to the kinase of interest. Compounds in 1000×DMSO stock solution was dispensed using automated dispensing system (Labcyte) to 384 well Corning Microplate at 15 nL, 5 uL of Kinase buffer A was added to each well. Plates were shaken and incubated for 1 min to ensure well dissolution of compounds. Kinase/Antibody mixture was added at a final concentration of 5 nM and 2 nM and kinase tracer 222 solution at a final concentration of 100 nM in a total volume of 20 uL. Plates were incubated for 1.5 hrs in dark at room temperature and assay plates were scanned on Envision plate reader with excitation: 340 nM and kinase Tracer Emission at 665 nM.
Measurement: IC50 = 5500 nM (pChEMBL 5.260000228881836)
Descriptors: MW 576.1, ALogP 6.41, HBA 8, HBD 2, PSA 105.68, QED 0.18
Compound (SMILES): Cc1ccc(Cn2nc3n(c2=O)C(C(=O)N2CCCC2)CN(C)C3)cc1
InChIKey: QBWRGTWFIDSGTJ-UHFFFAOYSA-N
Target: Prolyl endopeptidase (SINGLE PROTEIN), Homo sapiens
UniProt: P48147
Assay: Pharmacological Activity Assay: To recombinant full-length human prolyl endopeptidase (PREP, R&D-Systems, 4308-SE; final concentration, for example, 0.4 nM, volume: 25 al) in reaction buffer (50 mM Tris-HI, pH 7.5; 150 mM NaCl; 0.13% BSA, 5 mM EDTA, 3 mM GSH, 0.005% Brij-35) was added a test compound (in DMSO, in an appropriate final concentration range from 1 nM to 30 μM, volume: 1 μl) in a well of a 384-well microtiter plate. The reaction was started by adding the substrate Z-GlyPro-AMC (final concentration 50 μl; Z=carboxybenzyl; AMC=7-amino-4-methylcoumarin, volume: 25 μl). The progress of the PREP reaction was observed by measuring the fluorescence intensity in a Tecan SAFIRE II plate spectrophotometer at 3200 over a period of 60 min (excitation wavelength: 360 nm, emission wavelength: 465 nm).
Measurement: IC50 = 146 nM (pChEMBL 6.840000152587891)
Descriptors: MW 355.44, ALogP 1.01, HBA 6, HBD 0, PSA 63.37, QED 0.82
Compound (SMILES): O=C(c1cc(F)ccc1-n1nccn1)N1CC2CC(Nc3cnc(C(F)(F)F)cn3)C1C2
InChIKey: SLRLNNYNKDLOIV-UHFFFAOYSA-N
Target: Orexin/Hypocretin receptor type 1 (SINGLE PROTEIN), Rattus norvegicus
UniProt: P56718
Assay: Radioligand Binding Assay: Human Embryonic Kidney 293 cells (HEK293) stably expressing rat orexin 1 receptor (Genebank accession number NM_001525) or Chinese ovary cells (CHO) stably expressing human orexin 1 receptor (Genebank accession number NM_001526) were grown to confluency in DMEM (Hyclone, cat # SH30022), 10% FBS, 1× Pen/Strep, 1× sodium pyruvate, 10 mM HEPES, 600 μg/mL G418 and DMEM/F12 (Gibco, Cat #11039), 10% FBS, 1× Pen/Strep, 600 μg/mL G418 media, respectively on 150 cm2 tissue culture plates, washed with 5 mM EDTA in PBS (HyClone Dulbecco's Phosphate Buffered Saline 1× with Calcium and Magnesium, Cat # SH30264.01, hereafter referred to simply as PBS) and scraped into 50 ml tubes. After centrifugation (2K×G, 5 min at 4° C.), the supernatant was aspirated and the pellets frozen and stored at −80° C. Cells were resuspended in PBS in the presence of 1 tablet of protease inhibitor cocktail (Roche, Cat. #11836145001) per 50 mL. Each cell pellet from a 15 cm plate was resuspended in 10 mL, stored on ice, and homogenized for 45 sec prior to addition to the reactions. Competition binding experiments in 96 well polypropylene plates were performed using [3H]-(1-(5-(2-fluoro-phenyl)-2-methyl-thiazol-4-yl)-1-((S)-2-(5-phenyl-(1,3,4)oxadiazol-2-ylmethyl)-pyrrolidin-1-yl)-methanone) (Moraveck Corporation, specific activity=35.3 Ci/mmol), diluted to a 10 nM concentration in PBS (4 nM final). Compounds were solubilized in 100% DMSO (Acros Organics, Cat. #61042-1000) and tested over a range of 7 concentrations (from 0.1 nM to 10 μM). The final concentration of DMSO in the reactions is equal to or less than 0.1%. Total and nonspecific binding was determined in the absence and presence of 10 μM almorexant. The total volume of each reaction is 200 μL (20 μL of diluted compounds, 80 μL of [3H]-(1-(5-(2-fluoro-phenyl)-2-methyl-thiazol-4-yl)-1-((S)-2-(5-phenyl-(1,3,4)oxadiazol-2-ylmethyl)-pyrrolidin-1-yl)-methanone) diluted in PBS and 100 μL of the cell suspension). Reactions were run for 60 min at room temperature and terminated by filtration through GF/C filter plates (PerkinElmer, Cat. #6005174) presoaked in 0.3% polyethylenimine using the cell harvester (PerkinElmer Filtermate). The plates were washed 3 times by aspirating 30 ml PBS through the plates. Plates were dried in 55° C. oven for 60 min, scintillation fluid was added, and the radioactivity was counted on a Topcount (Packard).
Measurement: Ki = 8 nM (pChEMBL 8.100000381469727)
Descriptors: MW 447.4, ALogP 2.93, HBA 7, HBD 1, PSA 88.83, QED 0.62
Compound (SMILES): CC(C)(C)OC(=O)Nc1nc(N=[N+]=[N-])nc2c1ncn2[C@@H]1O[C@H](COC(Cc2cccc(C#N)c2)(C(=O)O)C(=O)O)[C@@H](O)[C@@H]1F
InChIKey: LARDGROTCOYXQT-DOHHPOSVSA-N
Target: 5'-nucleotidase (SINGLE PROTEIN), Homo sapiens
UniProt: P21589
Assay: In Vitro Enzyme Assay: For measurements of soluble CD73 enzyme activity, recombinant CD73 was obtained from R&D Systems, Cat. No. 5795-EN-010. Serial dilutions of test compounds were incubated with recombinant CD73 and AMP in reaction buffer (25 mM Tris HCl pH7.5, 5 mM MgCl2, 50 mM NaCl, 0.25 mM DTT, 0.005% Triton X-100). The final reaction volume was 25 μL and the final concentrations of recombinant CD73 and AMP were 0.5 nM and 50 μM, respectively. Reactions were allowed to proceed for 30 minutes at room temperature before the addition of 100 μL Malachite Green. After 5 minutes at room temperature, absorbance at 630 nm was determined on a microplate spectrophotometer. The concentration of inorganic phosphate was determined using a phosphate standard curve.
Measurement: IC50 = 1790 nM (pChEMBL 5.75)
Descriptors: MW 627.55, ALogP 2.75, HBA 13, HBD 4, PSA 267.77, QED 0.11
Compound (SMILES): Cc1nnc(N[C@H](C)c2cccc(F)c2F)c2cc(N3CCN4CCOC[C@@H]4C3)ncc12
InChIKey: KRCKDQBHWSIAQE-ZBFHGGJFSA-N
Target: Son of sevenless homolog 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q07889
Assay: Inhibiting SOS1-Mediated GTP Nucleotide Exchange by Blocking Mant-GDP Exchange: Table 24: This Example illustrates that exemplary compounds of the present invention prevent KRas-mediated GTP nucleotide exchange mediated by SOS1 to inhibit KRas activity thereby inhibiting the generation of the downstream effector pERK. MKN1 cells (15,000/w) or H358 (30,000/w) were seeded in a black clear flat bottom 96-well cell culture plate (Corning, #3904) and incubated at 37° C. overnight. Assay day 1, cells were dosed with compounds of Formula (I) with a 10 μm starting concentration and serially diluted 3× for a total of 9 concentrations. The cells were incubated for approximately 0.5-1 hour with the compounds solubilized in DMSO at 37° C. Cells were immediately fixed by adding 50 μL of 4% formaldehyde to all wells in a fume hood and the plates were incubated for 20 minutes at room temperature. The formaldehyde was discarded from the plates and 150 μL of ice-cold methanol was added to permeabilize the cells for 10 minutes at −20° C. The methanol was discarded from each of the plates and any liquid remaining in the plate by tapping the plate against paper towels. Cells were then blocked with 150 μL of Odyssey blocking buffer (LI-COR Biosciences #927-50010) using 0.05% Tween for 1 hour at room temperature on a shaker. The blocking buffer was discarded and 50 μL of primary antibodies pERK (cell signaling Technology #9101L; Rabbit, 1:500) and GapDH (Millipore #MAB34; Mouse, 1:5000) diluted in Odyssey blocking buffer was added. The plates were incubated overnight at 4° C. on a shaker. On Assay Day 2, the primary antibody solution was removed. Each plate was washed 3× times with 150 μL of 1×PBST (PBS+0.1% Tween 20) and incubated with 50 μL of secondary antibodies: Anti-Rabbit (LI-COR Biosciences #926-32211) and Anti-Mouse (LI-COR Biosciences #68070) at 1:800 dilution in Odyssey blocking buffer with Tween at room temperature on a shaker for 2 hours (protected from light). The secondary antibody solution as removed and each plate was washed with PBST 3× times. Any liquid remaining was discarded and the plate was imaged using the Licor Odyssey machine according to the manufacturer's instruction, using a set focus length at 3 mm and both 800 nm and 700 nm filters. The GAPDH normalized scan values for each well were divided by the average of vehicle wells to get the % of pERK inhibition. The IC50 values were then calculated with the Graph pad Prism software.
Measurement: IC50 = 87 nM (pChEMBL 7.059999942779541)
Descriptors: MW 440.5, ALogP 3.31, HBA 7, HBD 1, PSA 66.41, QED 0.67
Compound (SMILES): CN1CCN(c2ccc(Cc3cncc(C(=O)NCc4ccc5c(N)nccc5c4)c3)cn2)CC1
InChIKey: AYIZOVVOLGYDQH-UHFFFAOYSA-N
Target: Kallikrein-1 (SINGLE PROTEIN), Homo sapiens
UniProt: P06870
Assay: KLK1 inhibitory activity: KLK1 inhibitory activity in vitro was determined using standard published methods (see e.g. Johansen et al., Int. J. Tiss. Reac. 1986, 8, 185; Shori et al., Biochem. Pharmacol., 1992, 43, 1209; Stürzebecher et al., Biol. Chem. Hoppe-Seyler, 1992, 373, 1025). Human KLK1 (Callbiochem) was incubated at 37° C. with the fluorogenic substrate H-DVal-Leu-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 = 5340 nM (pChEMBL 5.269999980926514)
Descriptors: MW 467.58, ALogP 2.88, HBA 7, HBD 2, PSA 100.27, QED 0.45
Compound (SMILES): COc1ccc(S(=O)(=O)C[C@H]2C[C@@H](N(C)c3ncnc4[nH]ccc34)C2)c(F)c1
InChIKey: WCQVVINBTJFFGB-BETUJISGSA-N
Target: Tyrosine-protein kinase JAK2 (SINGLE PROTEIN), Homo sapiens
UniProt: O60674
Assay: Caliper Enzyme Assay: Test article and assay controls were added to a 384-well plate. Reaction mixtures contained 20 mM HEPES, pH 7.4, 10 mM magnesium chloride, 0.01% bovine serum albumin (BSA), 0.0005% Tween 20, 1 mM ATP and 1 μM peptide substrate. The JAK1 and TYK2 assays contained 1 μM of the IRStide peptide (5FAM-KKSRGDYMTMQID) and the JAK2 and JAK3 assays contained 1 μM of the JAKtide peptide (FITC-KGGEEEEYFELVKK). The assays were initiated by the addition of 20 nM JAK1, 1 nM JAK2, 1 nM JAK3 or 1 nM TYK2 enzyme and were incubated at room temperature for three hours for JAK1, 60 minutes for JAK2, 75 minutes for JAK3 or 135 minutes for TYK2. Enzyme concentrations and incubation times were optimized for each new enzyme preps and were modified slightly over time to ensure 20%-30% phosphorylation. The assays were stopped with a final concentration of 10 mM EDTA, 0.1% Coating Reagent and 100 mM HEPES, pH=7.4. The assay plates were placed on a Caliper Life Science Lab Chip 3000 (LC3000) instrument, and each well was sampled using appropriate separation conditions to measure the unphosphorylated and phosphorylated peptide.
Measurement: IC50 = 0.398 nM (pChEMBL 9.399999618530273)
Descriptors: MW 404.47, ALogP 2.79, HBA 6, HBD 1, PSA 88.18, QED 0.68
Compound (SMILES): CC(NC(=O)c1cc2cc(Br)cnc2n(Cc2ncccn2)c1=O)c1ccc(F)cc1
InChIKey: JPMSRYJKTVQZLP-UHFFFAOYSA-N
Target: Cannabinoid receptor 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P21554
Assay: CB1 and CB2 Receptor Binding Assay: CB1R binding protocol involves the use of the same solution buffer used for both incubation and washing reaction (Tris-HCl, 50 mM; EDTA, 2.5 mM; MgCl2, 2.5 mM; BSA, 0.5 mg/mL at pH 7.4), 0.4 nM for [3H]CP-55,940, test compounds (concentrations from 0.001 to 10 μM), and finally 8 μg/sample membrane in a total volume of 200 μL. CB2R binding assays were carried out with two different buffers: incubation buffer (Tris-HCl, 50 mM; MgCl2, 5 mM; CaCl2 1 mM; BSA, 0.2% at pH 7.4) and washing buffer (Tris-HCl, 50 mM; NaCl 500 mM; BSA, 0.1% at pH 7.4). The assay mixture contained incubation buffer, 0.4 nM [3H]CP-55,940, test substances (concentrations from 0.001 to 10 μM), and 4 μg/sample membrane in a total assay volume of 200 μL. Assays were performed in duplicate and incubated for 120 mM at 37° C. After the incubation, the assay mixture is filtered through 96 GF/C filter plates (Perkin Elmer #6005174) using Perkin Elmer Filtermate Harvester, and then washed four times with ice-cold washing buffer.
Measurement: IC50 > 1000 nM (pChEMBL 6)
Descriptors: MW 482.31, ALogP 3.63, HBA 6, HBD 1, PSA 89.77, QED 0.47
Compound (SMILES): O=C1Nc2cn(C3CCOCC3)nc2C(=O)NCCSCCCn2cc(cn2)-c2cccc1n2
InChIKey: WMIWTQABSHYHQO-UHFFFAOYSA-N
Target: Interleukin-1 receptor-associated kinase 4 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9NWZ3
Assay: Enzymatic Assay: IRAK4 is a human purified recombinant enzyme (His-TEV-IRAK4 (1-460)).In this assay, IRAK4 hydrolyses ATP, autophosphorylates and phosphorylates a Serine/Threonine generic peptidic substrate (STK: 61ST1BLC from CisBio International). Measurement of IRAK-4 inhibition is performed in 384-well format based on a luminescence assay (ADP-Glo Kinase Assay from Promega). Purified human recombinant IRAK4 (0.3 μg/ml) and serial diluted compounds in DMSO (range of concentration from 10 μM to 0.5 nM) or controls (1% DMSO) are incubated for 15 minutes at RT in assay buffer containing 50 mM Hepes pH 7.0, Fatty acid-free BSA 0.1%, Dithiothreitol (DTT) 2 mM, MgCl2 10 mM, EGTA 0.5 mM, Triton X-100 0.01%, MnCl2 5 mM. The kinase reaction is then initiated by the addition of ATP (2 μM) and the peptidic substrate STK1-biotin peptide (300 nM). After 2 hours of incubation at RT, the reaction is stopped and the unconsumed ATP depleted by the addition of ADP-Glo Reagent according to supplier instructions. After 40 minutes of incubation at RT, the Kinase Detection Reagent is then added to the assay plate according to supplier instructions. After 20 minutes of incubation at RT, the luminescence signal is measured with a plate-reading luminometer (PerkinElmer Envision or equivalent reader).
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 481.58, ALogP 2.61, HBA 9, HBD 2, PSA 115.96, QED 0.55
Compound (SMILES): C[C@@H](Cc1ccsc1)NC(=O)NC[C@]1(N(C)C)CCc2ccccc2C1
InChIKey: DUWAGTHSPLQZPC-KKSFZXQISA-N
Target: Nociceptin receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P41146
Assay: Inhibitory Activity Assay: This study supports a structure-based approach for GPCR ligand discovery. These new chemotypes may stabilize receptor conformations not explored previously and thereby generate novel biological effects. With a novel chemotype in hand, the docked structure provides a straight-forward strategy for optimization.
Measurement: Ki = 1500 nM (pChEMBL 5.820000171661377)
Descriptors: MW 371.55, ALogP 3.47, HBA 3, HBD 2, PSA 44.37, QED 0.82
Compound (SMILES): Cc1c(-c2[nH]c3ccc(N4CCN(CCC(=O)N(C)C)C[C@@H]4C)nc3c2C(C)C)cn2ncnc2c1C
InChIKey: UHAPAFRVPHTNEG-SFHVURJKSA-N
Target: Toll-like receptor 8 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9NR97
Assay: TLR7/8/9 Inhibition Reporter Assays: HEK-Blue-cells (Invivogen) overexpressing human TLR7, TLR8 or TLR9 receptors were used for screening inhibitors of these receptors using an inducible SEAP (secreted embryonic alkaline phosphatase) reporter gene under the control of the IFN-β minimal promoter fused to five NF-κB and AP-1-binding sites. Briefly, cells are seeded into Greiner 384 well plates (15000 cells per well for TLR7, 20,000 for TLR8 and 25,000 for TLR9) and then treated with test compounds in DMSO to yield a final dose response concentration range of 0.05 nM-50 μM. After a 30 minute compound pre-treatment at room temperature, the cells are then stimulated with a TLR7 ligand (gardiquimod at a final concentration of 7.5 μM), TLR8 ligand (R848 at a final concentration of 15.9 μM) or TLR9 ligand (ODN2006 at a final concentration of 5 nM) to activate NF-κB and AP-1 which induce the production of SEAP. After a 22 hour incubation at 37° C., 5% CO2, SEAP levels are determined with the addition of HEK-Blue Detection reagent (Invivogen), a cell culture medium that allows for detection of SEAP, according to manufacturer's specifications. The percent inhibition is determined as the % reduction in the HEK-Blue signal present in wells treated with agonist plus DMSO alone compared to wells treated with a known inhibitor.
Measurement: IC50 = 0.58 nM (pChEMBL 9.239999771118164)
Descriptors: MW 502.67, ALogP 4, HBA 7, HBD 1, PSA 85.66, QED 0.43
Compound (SMILES): COc1c(N=S2(=O)CCC2)cc2ncnc(Nc3ccc(Cl)cc3O[C@H](C)C(=O)NCC(F)(F)F)c2c1C
InChIKey: MWCAWBUYZQRAJP-CQSZACIVSA-N
Target: MAP kinase-interacting serine/threonine-protein kinase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9BUB5
Assay: MNK1 In Vitro Kinase Assay (Assay 2): The Z′-LYTE® biochemical assay employs a fluorescence-based, coupled-enzyme format and is based on the differential sensitivity of phosphorylated and non-phosphorylated peptides to proteolytic cleavage. The peptide substrate is labeled with two fluorophores¿one at each end¿that make up a FRET pair.In the primary reaction, the kinase transfers the gamma-phosphate of ATP to a single tyrosine, serine or threonine residue in a synthetic FRET-peptide. In the secondary reaction, a site-specific protease recognizes and cleaves non-phosphorylated FRET-peptides. Phosphorylation of FRET-peptides suppresses cleavage by the Development Reagent. Cleavage disrupts FRET between the donor (i.e., coumarin) and acceptor (i.e., fluorescein) fluorophores on the FRET-peptide, whereas uncleaved, phosphorylated FRET-peptides maintain FRET. A ratiometric method, which calculates the ratio (the Emission Ratio) of donor emission to acceptor emission after excitation of the donor fluorophore at 400 nm, is used to quantitate reaction progress, as shown in the equation below. Emission Ratio=Coumarin Emission(445 nm)/Fluorescein Emission(520 nm).
Measurement: IC50 = 66 nM (pChEMBL 7.179999828338623)
Descriptors: MW 572.01, ALogP 5.29, HBA 8, HBD 2, PSA 114.8, QED 0.38
Compound (SMILES): Cc1ccc(NC(=O)c2cnc3ccc(-c4ccc5oc(N)nc5c4)cn23)cn1
InChIKey: WAFDOIKRHACIEF-UHFFFAOYSA-N
Target: Phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit delta isoform (SINGLE PROTEIN), Homo sapiens
UniProt: O00329
Assay: PI 3-Kinase HTRF Assay: Briefly, the assay is a time resolved FRET assay that indirectly measures PIP3 product formed by the activity of a PI3-K. The kinase reaction is performed in a microtitre plate (e.g., a 384 well microtitre plate). The total reaction volume is approximately 20 ul per well. In the first step, each well receives 2 ul of test compound in 20% dimethylsulphoxide resulting in a 2% DMSO final concentration. Next, approximately 14.5 ul of a kinase/PIP2 mixture (diluted in 1x reaction buffer) is added per well for a final concentration of 0.25-0.3 ug/ml kinase and 10 uM PIP2. The plate is sealed and incubated for 15 minutes at room temperature. To start the reaction, 3.5 ul of ATP (diluted in 1x reaction buffer) is added per well for a final concentration of 10 uM ATP. The plate is sealed and incubated for 1 hour at room temperature. The reaction is stopped by adding 5 ul of Stop Solution per well and then 5 ul of Detection Mix is added per well. The plate is sealed, incubated for 1 hour at room temperature, and then read on an appropriate plate reader.
Measurement: IC50 < 10000 nM (pChEMBL 5)
Descriptors: MW 384.4, ALogP 3.68, HBA 7, HBD 2, PSA 111.34, QED 0.49
Compound (SMILES): C[C@H](NC(=O)[C@@H](CCc1ccccc1)NCc1ccncc1)C(=O)NCc1ccc(C(=N)N)cc1
InChIKey: PJJBKHOCJZLARK-YADARESESA-N
Target: Prothrombin (SINGLE PROTEIN), Homo sapiens
UniProt: P00734
Assay: Enzymatic Assay for Thrombin: The thrombin assay utilizes a fluorogenic peptide substrate (Boc-VPR-AMC (R&D Systems) and was run at room temperature in an assay buffer containing 20 mM Hepes, pH 7.4, 140 mM NaCl and 0.1% Tween 20. Assay parameters were adjusted such that the assay was linear with respect to time, enzyme and substrate concentrations. Under these optimized assays conditions, IC50values were equivalent to Ki values, except in a few cases of tight binding inhibitors. Cases of tight binding or possible slow binding inhibitors were handled by the methods described in Copeland R. A. (2013) Evaluation of Enzyme Inhibitors in Drug Discovery. 2nd Ed. John Wiley and Sons, Inc., Chapters 5-7.
Measurement: Ki = 1500 nM (pChEMBL 5.820000171661377)
Descriptors: MW 472.59, ALogP 2.28, HBA 5, HBD 5, PSA 132.99, QED 0.2
Compound (SMILES): COCCS(=O)(=O)NC(=O)c1cc(N2CCC(COC)CC2)c2c(C(C)C)nn(-c3ccc(F)cc3)c2n1
InChIKey: DPGMCWVUTKWPOQ-UHFFFAOYSA-N
Target: Cystic fibrosis transmembrane conductance regulator (SINGLE PROTEIN), Homo sapiens
UniProt: P13569
Assay: YFP-Halide Influx Assay for the CFTR-WT: For this purpose, HEK293 cells are transfected with plasmid DNA containing WT CFTR and seeded in 96 well plates (70,000 HEK cells/well). Two days after transfection, cells are treated with test compounds.The CFTR channels are activated by treatment with the cAMP inducer forskolin (10.67 μM) and a dose response of test compounds in 1×D-PBS (from Gibco, Cat n#14090-091) for 20 minutes prior to addition of an F solution (137 mM NaI, 2.7 mM KI, 1.76 mM KH2PO4, 10.1 mM Na2HPO4, 5 mM glucose). The I− induced quenching of fluorescence is recorded immediately after injection of for 7 seconds. The impact of a compound on the channel functionality can be measured by looking at the effect on fluorescence, and is expressed as (1-(fluorescence after 7 seconds (F)/fluorescence before injection (F0))) and an EC50 can be derived from a (1−F/F0) vs compound concentration plot.
Measurement: IC50 = 40 nM (pChEMBL 7.400000095367432)
Descriptors: MW 547.65, ALogP 3.25, HBA 9, HBD 1, PSA 115.65, QED 0.41
Compound (SMILES): C=CC(=O)N1CC[C@H](C(=O)N(C)[C@H](C(=O)N[C@H]2Cc3cc(O)cc(c3)-c3ccc4c(c3)c(c(-c3c(F)cccc3CCOC)n4CC)CC(C)(C)COC(=O)[C@@H]3CCCN(N3)C2=O)C(C)C)C1
InChIKey: VTNLYAFWNNMVSY-FMGRFAOQSA-N
Target: GTPase KRas (SINGLE PROTEIN), Homo sapiens
UniProt: P01116
Assay: Biochemical Assay: A variety of Ras proteins may be inhibited by compounds of the present invention (e.g., K-Ras, N-Ras, H-Ras, and mutants thereof at positions 12, 13 and 61, such as G12C, G12D, G12V, G12S, G13C, G13D, and Q61L, and others described herein). The purpose of this biochemical assay is to measure the ability of test compounds to covalently label nucleotide-loaded K-Ras isoforms. In assay buffer containing 12.5 mM HEPES pH 7.4, 75 mM NaCl, 1 mM MgCl2, 1 mM BME, 5 uM Cyclophilin A and 2 uM test compound, a 5 uM stock of GMP-PNP-loaded K-Ras (1-169) G12C is diluted 10-fold to yield a final concentration of 0.5 uM; with final sample volume being 100 uL. The sample is incubated at 25�� C. for a time period of up to 24 hours prior to quenching by the addition of 10 uL of 5% Formic Acid. Quenched samples are centrifuged at 15000 rpm for 15 minutes in a benchtop centrifuge before injecting a 10 uL aliquot onto a reverse phase C4 column and eluting into the mass spectrometer with an increasing acetonitrile gradient in the mobile phase. Analysis of raw data may be carried out using Waters MassLynx MS software, with % bound calculated from the deconvoluted protein peaks for labeled and unlabeled K-Ras.
Measurement: IC50 = 5500 nM (pChEMBL 5.260000228881836)
Descriptors: MW 921.12, ALogP 6.19, HBA 10, HBD 3, PSA 162.75, QED 0.12
Compound (SMILES): COCCn1cnc2c(N3CC[C@H](O)C3)nc(C(C)(C)C)nc21
InChIKey: ZCZFHQPVVYWVPE-NSHDSACASA-N
Target: Cannabinoid receptor 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P21554
Assay: cAMP Assay: CHO cells expressing human CB1 or CB2 receptors are seeded 17-24 hours prior to the experiment 50.000 cells per well in a black 96 well plate with flat clear bottom (Corning Costar #3904) in DMEM (Invitrogen No. 31331), 1x HT supplement, with 10% fetal calf serum and incubated at 5% CO2 and 37° C. in a humidified incubator. The growth medium was exchanged with Krebs Ringer Bicarbonate buffer with 1 mM IBMX and incubated at 30° C. for 30 min. Compounds were added to a final assay volume of 100 ul and incubated for 30 min at 30° C. Using the cAMP-Nano-TRF detection kit the assay (Roche Diagnostics) was stopped by the addition of 50 ul lysis reagent (Tris, NaCl, 1.5% Triton X100, 2.5% NP40, 10% NaN3) and 50 ul detection solutions (20 uM mAb Alexa700-cAMP 1:1, and 48 uM Ruthenium-2-AHA-cAMP) and shaken for 2 h at room temperature. The time-resolved energy transfer is measured by a TRF reader (Evotec Technologies GmbH), equipped with a ND:YAG laser as excitation source. The plate is measured twice with the excitation at 355 nm and at the emission with a delay of 100 ns and a gate of 100 ns, total exposure time 10 s at 730 (bandwidth 30 nm) or 645 nm (bandwidth 75 nm), respectively. The FRET signal is calculated as follows: FRET=T730-Alexa730-P(T645-B645) with P=Ru730-B730/Ru645-B645, where T730 is the test well measured at 730 nM, T645 is the test well measured at 645 nm, B730 and B645 are the buffer controls at 730 nm and 645 nm, respectively. cAMP content is determined from the function of a standard curve spanning from 10 uM to 0.13 nM cAMP. EC50 values were determined using Activity Base analysis (ID Business Solution, Limited). The EC50 values for a wide range of cannabinoid agonists generated from this assay were in agreement with the values published in the scientific literature. The compounds of the invention are CB2 receptor agonists with EC50 below 1 uM and selectivity versus CB1 in the corresponding assay of at least 10 fold. Particular compound of the invention are CB2 receptor agonists with EC50 below 0.05 uM and selectivity versus CB1 in the corresponding assay of at least 500 fold.
Measurement: EC50 > 10000 nM (pChEMBL 5)
Descriptors: MW 319.41, ALogP 1.34, HBA 7, HBD 1, PSA 76.3, QED 0.92
Compound (SMILES): CNC(=O)[C@@H](NC(=O)c1nn(-c2c[n+]([O-])ccn2)c2c1C[C@@H]1C[C@H]21)C(C)(C)C
InChIKey: IRXDIFILQJBGOI-XMZIXOGTSA-N
Target: Cannabinoid receptor 2 (SINGLE PROTEIN), Homo sapiens
UniProt: P34972
Assay: Radioligand Binding Assay: Preparation of Membranes: HEK293 cells stably expressing human CB2 receptor were collected, washed in ice cold PBS, and centrifuged at 48,000×g for 20 min at 4° C. The cell pellet was then collected, resuspended in wash buffer (20 mM HEPES, pH 7.4 and 1 mM EDTA), homogenized on ice using a Brinkman Polytron, and centrifuged at 48,000×g for 20 min at 4° C. The resultant pellet was resuspended in ice cold 20 mM HEPES, pH 7.4, homogenized again on ice, recentrifuged for 20 min at 4° C., and membrane pellets were then stored at −80° C. until needed.[3H]P55,940 and [3H]WIN55, 212-2 Radioligand Binding Assays: Radioligand binding assays for human CB2 receptors were performed using two different agonist radioligands, [3H]CP55,940 and [3H]WIN55, 212-2 and similar assay conditions. For both assays, nonspecific binding was determined in the presence of 10 μM unlabeled compound. Competition experiments consisted of addition of 20 μL of assay buffer (50 mM Tris, pH 7.4, 2.5 mM EDTA, 5 mM MgCl2, and 0.5 mg/mL of fatty acid free BSA) containing test compound (concentrations ranging from 1 pM to 100 μM), 25 μL of radioligand (1 nM final assay concentration for [3H]CP55,904 and [3H]WIN55, 212-2), and 50 μL of membranes (20 μg/mL final protein for both assays). Incubations were conducted for 1 h at room temperature, assay plates were filtered under reduced pressure over GF/B filters, washed with assay buffer and dried overnight in a 50° C. oven. Then, 25 μL of BetaScint scintillation cocktail was added to each well, and plates were read in a Packard TopCount scintillation counter.
Measurement: Ki = 1.45 nM (pChEMBL 8.84000015258789)
Descriptors: MW 384.44, ALogP 0.45, HBA 6, HBD 2, PSA 115.85, QED 0.59
Compound (SMILES): Cc1cnc(-c2cc(OC[C@@H]3CCCO3)cc(C(=O)N[C@H](C)c3cnc(C(F)(F)F)nc3)c2)s1
InChIKey: MXZPUDRHAAYJOR-KDOFPFPSSA-N
Target: P2X purinoceptor 3 (SINGLE PROTEIN), Homo sapiens
UniProt: P56373
Assay: Intracellular Calcium Measurement to Assess Antagonist Activity : A fluorescent imaging plate reader (FLEX/FLIPR station; Molecular Devices) was used to monitor intracellular calcium levels using the calcium-chelating dye Fluo-4 (Molecular Probes). The excitation and emission wavelengths used to monitor fluorescence were 470-495 nm and 515-575 nm, respectively. Cells expressing purinergic receptors P2X3 (human) or P2X2/3 (human) were plated at a density of 15,000 cells/well in collagen-coated 384-well plates approximately 20 hours before beginning the assay. On the day of the assay, 20 μl of loading buffer (Hank's balanced salt solution, 20 mM HEPES, 0.5 mM CaCl2, 0.5 mM MgCl2, 0.1% BSA, 5 mM probenecid, 10 mM D-glucose monohydrate, 2 μM Fluo-4, and 5 units/mL, hexokinase, pH=7.4) was added and cells dye-loaded for 90 min at 37° C. The dye supernatant was removed and replaced with 45 μl probenecid buffer (Hank's balanced salt solution, 20 mM HEPES, 0.5 mM CaCl2, 0.5 mM MgCl2, 0.1% BSA, 5 mM probenecid, 10 mM D-glucose monohydrate, pH=7.4). The test compound was added in a volume of 5 μl and allowed to incubate for 30 min at 37° C. The final assay DMSO concentration is 1%.The agonist, α,β-Me-ATP, was added in a volume of 20 μl at a concentration representing the EC80 value. The fluorescence was measured for an interval of 90 sec at 2 sec intervals and analyzed based on the increase in peak relative fluorescence units (RFU) compared to the basal fluorescence. Peak fluorescence was used to determine the response to agonist obtained at each concentration of test compound by the following equation: % Response=100*(RFU(test compound)−RFU(control))/(RFU(DMSO)−RFU(control)).
Measurement: IC50 = 4 nM (pChEMBL 8.399999618530273)
Descriptors: MW 492.52, ALogP 4.98, HBA 7, HBD 1, PSA 86.23, QED 0.5
Compound (SMILES): Cc1ncc(NC(=O)c2cc(C(C)(C)O)ccn2)cc1-c1cnc(OC2CC3(COC3)C2)c(N2CCOCC2)c1
InChIKey: CGPFHJZAGKIEFK-UHFFFAOYSA-N
Target: RAF proto-oncogene serine/threonine-protein kinase (SINGLE PROTEIN), Homo sapiens
UniProt: P04049
Assay: In Vitro Raf Activity: 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, then 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 = 4.98 nM (pChEMBL 8.300000190734863)
Descriptors: MW 545.64, ALogP 3.72, HBA 9, HBD 2, PSA 118.93, QED 0.46
Compound (SMILES): Fc1cnccc1Nc1nc(-c2cccc(C(F)(F)F)n2)nn2ccc(CN3CCC(F)(F)CC3)c12
InChIKey: SOEKIDXGHGPSDG-UHFFFAOYSA-N
Target: TGF-beta receptor type-2 (SINGLE PROTEIN), Homo sapiens
UniProt: P37173
Assay: Biological Assays: Assays are conducted in 1536-well plates and 2 mL reactions are prepared from addition of HIS-TGFβR1 T204D or HIS-TGFβR2 WT, anti-HIS detection antibody, a labeled small molecule probe (Kd=<100 nM; koff=<0.001 s−1.) and test compounds in assay buffer (20 mM HEPES pH 7.4, 10 mM MgCl2, 0.015% Brij35, 4 mM DTT, and 0.05 mg/ml BSA). The reaction is incubated for 1 hour at room temperature and the HTRF signal was measured on an Envision plate reader (Ex: 340 nm; Em: 520 nm/495 nm). 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 reagents in the assay are 1 nM HIS-TGFβR1 T204D or HIS-TGFβR2 WT, 0.2 nM anti-HIS detection antibody, labeled small molecule probe (at Kd) and 0.5% DMSO. Dose response curves were generated to determine the concentration required inhibiting 50% of kinase activity (IC50). Compounds were dissolved at 10 mM in dimethylsulfoxide (DMSO) and evaluated at eleven concentrations. IC50 values were derived by non-linear regression analysis.
Measurement: IC50 > 15000 nM (pChEMBL 4.823999881744385)
Descriptors: MW 507.44, ALogP 5.32, HBA 7, HBD 1, PSA 71.24, QED 0.37
Compound (SMILES): CNC(=O)c1ccc(C(=O)OC2CN(c3nc4c(c(N(C)C5CCOCC5)n3)[S+]([O-])CC4)C2)cc1
InChIKey: VWLOTQNDVDAKKK-UHFFFAOYSA-N
Target: Tumor necrosis factor (SINGLE PROTEIN), Homo sapiens
UniProt: P01375
Assay: TNF-alpha RELEASE: Human peripheral blood mononuclear cells (PBMC) were isolated from fresh buffy coats by density centrifugation using lymphoprep tubes (Medinor). Frozen PBMC's were washed in serum free assay buffer (RPMI1640 with 25 mM HEPES, 1% pen/strep, 200 mM L glutamine, 0.5% human serum albumin) and living cells counted. Lipopolysaccharide (1 microg/ml; SIGMA) was added to the cells which were then transferred to 384 well tissue culture plates (5×105 c/ml) containing titrated test compounds. The cells were incubated for 18 hours at 37° C. in serum free assay buffer and the level of TNFalpha in the supernatant was quantitated by AlphaLISA (PerkinElmer) by measuring fluorescence intensity at 615 nm.Results were expressed as IC50 values calculated from inhibition curves using as controls the secretion in LPS stimulated wells and the secretion in cells incubated with 10 microM of a PDE4 inhibitor reference compound.
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 499.59, ALogP 1.16, HBA 9, HBD 1, PSA 119.95, QED 0.46
Compound (SMILES): Cc1cc(C(C)Nc2ccccc2C(=O)O)c2oc(N3CCc4ccccc4C3)cc(=O)c2c1
InChIKey: XMNRTCXETMPSBT-UHFFFAOYSA-N
Target: Phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit alpha isoform (SINGLE PROTEIN), Homo sapiens
UniProt: P42336
Assay: PI3K-Alpha kinase (PIK3CA) activity, wild-type and H1047R mutant and determining IC50 values for inhibitors: Recombinant, catalytically active human full length PIK3KA Wild-type and H1047R mutant were purchased as 1:1 complex of N-terminal 6×his tagged p110<(catalytic) and untagged p85<(regulatory subunit) from EMD Millipore Sigma (cat. no. 14-602M and 14-792M, respectively). The enzyme stocks were diluted to 5× stocks in buffer (20 mM HEPES pH 7.4, 100 mM NaCl, 0.5 mM EGTA, 0.01% triton-x-100) just before use. PIP2diC8 (Avanti Polar Lipids Inc., cat. no. 850185) or phosphoinositol-4,5-bisphosphate with phosphoserine (PIP2:PS) membrane (Thermo Fisher Scientific, cat. no. PV5100) was used as lipid substrates. PIP2diC8 lyophilized powder and PIP2:PS (1:19) membrane stock (1 mM in PIP2) were separately dissolved in milliQ water to a concentration of 250 uM and stored in −20° C. 10 mM stocks of compounds were serially diluted (3×) in neat DMSO and stored in a dessicator at room temperature. 5× compound stocks in 25% DMSO were prepared fresh from neat DMSO stocks. Wild-type (WT) and H1047R mutant protein, along with buffer components (except ATP), were incubated with or without compound at 27° C. for 1h. After incubation, the reaction was initiated by the addition of 5 uL of 125 uM ATP. A typical assay mixture (25 uL) comprised 40 mM HEPES buffer, pH 7.4, 25 mM MgCl2, 0.01% v/v triton-X-100, 5% v/v DMSO, 20 mM NaCl, 1-5 nM wt or H1047R, 25 uM ATP, and 50 uM PTP2diC8 or PTP2 in membrane. The reaction was allowed to proceed until about 1000 conversion (2.5 uM ADP) after which time, 10 uL of reaction mixture was quenched with 25 uL of transcreener reagent (Transcreener ADP2 FI assay kit, BellBrook labs, Cat. No. 3013). The contents were incubated at rt for 1h and fluorescence was measured using a plate reader (Paradigm, Molecular Devices). The same assay was also run at pH 6.0 or 6.4 using MOPS buffer (Fisher BioReagents, CAS 1132-61-2). A calibration curve was generated under identical buffer conditions with varying ADP amounts. Using that, the observed fluorescence was converted to uM ADP.
Measurement: IC50 < 500 nM (pChEMBL 6.301000118255615)
Descriptors: MW 454.53, ALogP 5.54, HBA 5, HBD 2, PSA 82.78, QED 0.41