lovingscience/chembl-v37-raw-activities huggingface dataset:
Compound (SMILES): CCCn1cnnc1-c1cccc(N2Cc3c(cc(N(C)CC)nc3CNC)C2=O)n1
InChIKey: RIQLJJYZCGIHPB-UHFFFAOYSA-N
Target: Mitogen-activated protein kinase kinase kinase kinase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q92918
Assay: HPK1 Biochemical Enzyme Assay: HPK1 enzyme inhibition was measured using a microfluidic mobility shift assay (MSA). The reactions were conducted in 50 μL volumes in 96-well plates, and contained 0.5 nM human full-length recombinant HPK1, 3 μM phosphoacceptor peptide, 5FAM-AKRRRLSSLRA-COOH (CPC Scientific, Sunnyvale, Calif.), test compound (11-dose 3-fold serial dilutions, 2% DMSO final) or DMSO only, 0.002% Tween-20, 1 mM DTT and 2.5 mM MgCl2 in 50 mM MOPS (3-(N-morpholino)propanesulfonic acid), pH 7.8, buffer and were initiated by addition of 75 μM ATP, following a 20-min preincubation. The reactions were conducted for 60 min at 37° C., stopped by the addition of 50 μL of 0.015 M EDTA, pH 8, and the extent of reactions (15-20% conversion with no inhibitor) was determined after electrophoretic separation of the fluorescently labeled peptide substrate and phosphorylated product on an LabChip EZ Reader II (PerkinElmer, Inc., Waltham, Mass.).Inhibition of HPK1 was also measured using the fluorescence based chelation-enhanced fluorescence (CHEF) method (1), using a proprietary fluorescent peptide substrate, in which a cysteine residue is alkylated with a sulfonamido-oxine based derivative to afford an amino acid termed C-Sox (CSx). The assay was conducted similarly as described for the MSA method above, but using 3 μM Ac-[CSx]HSLPRFNR-amide peptide substrate (also known as AQT0178 when purchased from AssayQuant Technologies Inc., Hopkinton, Mass.) and 45 μM ATP. Initial reaction velocities were determined by following the peptide fluorescence (λex=360 nm, λem=500 nm) at 30° C. for 15 min in a Tecan M1000 plate reader (Tecan Group Ltd., Männedorf, Zürich, Switzerland). The inhibition constant (Ki) values were calculated by fitting the % conversion based (MSA method) or fluorescence based initial velocities (CHEF method) to the Morrison equation (2) for tight-binding competitive inhibition using non-linear regression method and an experimentally measured ATP Km (29 μM by MSA and 19 μM by CHEF, respectively). The inhibitors were shown to be ATP-competitive from kinetic and crystallographic studies. HPK1 protein was produced in-house and preactivated by autophosphorylation of enzyme with MgATP as described in the section Production of recombinant autophosphorylated full-length HPK1.
Measurement: Ki = 0.19 nM (pChEMBL 9.720000267028809)
Descriptors: MW 420.52, ALogP 2.48, HBA 8, HBD 1, PSA 92.07, QED 0.6
Compound (SMILES): CCOc1ccc(N2CCN(C)CC2)cc1N(C)c1ccc(Oc2nc(O)c3ccncc3n2)cc1C
InChIKey: AJAWOCBVVWSTSL-UHFFFAOYSA-N
Target: Lysine-specific demethylase 4C (SINGLE PROTEIN), Homo sapiens
UniProt: Q9H3R0
Assay: JMJD2C Assay: The ability of test compounds to inhibit the activity of JMJD2C was determined in 384-well plate-format under the following reaction conditions: 0.3 nM JMJD2C, 300 nM H3K9me3-biotin labeled peptide (Anaspec cat #64360), 2 μM alpha-ketoglutaric acid in assay buffer of 50 mM HEPES, pH 7.3, 0.005% Brij35, 0.5 mM TCEP, 0.2 mg/ml BSA, 50 μM sodium L-ascorbate, and 2 μM ammonium iron(II) sulfate. Reaction product was determined quantitatively by TR-FRET after the addition of detection reagent Phycolink Streptavidin-allophycocyanin (Prozyme) and Europium-anti-di-methylated histone H3 lysine 9 (H3K9me2) antibody (PerkinElmer) in the presence of 5 mM EDTA in LANCE detection buffer (PerkinElmer) at a final concentration of 50 nM and 1 nM, respectively.The assay reaction was initiated by the following: 2 μl of the mixture of 900 nM H3K9me3-biotin labeled peptide and 6 μM alpha-ketoglutaric acid with 2 μl of 11-point serial diluted inhibitor in 3% DMSO were added to each well of the plate, followed by the addition of 2 μl of 0.9 nM JMJD2C to initiate the reaction. The reaction mixture was incubated at room temperature for 30 min, and terminated by the addition of 6 μl of 5 mM EDTA in LANCE detection buffer containing 100 nM Phycolink Streptavidin-allophycocyanin and 2 nM Europium-anti-H3K9me2 antibody. Plates were read by EnVisionMultilabel Reader in TR-FRET mode (excitation at 320 nm, emission at 615 nm and 665 nm) after 1 hr incubation at room temperature. A ratio was calculated (665/615) for each well and fitted to determine inhibition constant (IC50).
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 500.6, ALogP 4.75, HBA 9, HBD 1, PSA 87.08, QED 0.39
Compound (SMILES): Cc1nc(N)nc2ccc(Nc3cc(F)c(F)c(F)c3)nc12
InChIKey: RCIPLCUNHJEGOY-UHFFFAOYSA-N
Target: Dihydrofolate reductase (SINGLE PROTEIN), Homo sapiens
UniProt: P00374
Assay: Inhibitory Activity Assay: These assays were carried out at 37° C. under 18 μM dihydrofolic acid concentration.
Measurement: IC50 = 80 nM (pChEMBL 7.099999904632568)
Descriptors: MW 305.26, ALogP 3.08, HBA 5, HBD 2, PSA 76.72, QED 0.71
Compound (SMILES): N#Cc1cccc(-c2nc(N)c3cc(C(O)c4ccccc4F)nn3c2Br)c1
InChIKey: UNPZMEKPIMYZQV-UHFFFAOYSA-N
Target: Adenosine receptor A2a (SINGLE PROTEIN), Homo sapiens
UniProt: P29274
Assay: HTRF Assay: Assays were conducted in black low volume 384-well polystyrene plates (Greiner 784076-25) in a final volume of 10 μL. Test compounds were first serially diluted in DMSO and 100 nl added to the plate wells before the addition of other reaction components. The final concentration of DMSO was 1%. Tag-Lite® Adenosine A2A labeled cells (CisBio C1TT1A2A) were diluted 1:5 into Tag-lite buffer (CisBio LABMED) and spun 1200 g for 5 mins. The pellet was resuspended at a volume 10.4× the initial cell suspension volume in Tag-lite buffer, and Adenosine A2A Receptor Red antagonist fluorescent ligand (CisBio L0058RED) added at 12.5 nM final concentration. 10 ul of the cell and ligand mix was added to the assay wells and incubated at room temperature for 45 minutes before reading on a PHERAstar FS plate reader (BMG Labtech) with HTRF 337/620/665 optical module. Percent binding of the fluorescent ligand was calculated; where 100 nM of A2A antagonist control ZM 241385 (Tocris 1036) displaces the ligand 100% and 1% DMSO has 0% displacement. The % binding data versus the log of the inhibitor concentration was fitted to a one-site competitive binding model (GraphPad Prism version 7.02) where the ligand constant=12.5 nM and the ligand Kd=1.85 nM.
Measurement: Ki < 10 nM (pChEMBL 8)
Descriptors: MW 438.26, ALogP 3.83, HBA 6, HBD 2, PSA 100.23, QED 0.51
Compound (SMILES): COc1ccc(Cn2c(=O)n3ncnc3c3cc(N[C@H]4CCCC[C@@H]4N)cnc32)cc1
InChIKey: IQXGHTMTUMPVTA-OALUTQOASA-N
Target: Dual specificity calcium/calmodulin-dependent 3',5'-cyclic nucleotide phosphodiesterase 1B (SINGLE PROTEIN), Homo sapiens
UniProt: Q01064
Assay: PDE1B Inhibitory Assay: PDE1B inhibition was determined by an IMAP TR-FRET assay. The IMAP TR-FRET PDE assay was optimized for concentration of enzyme, Calmodulin, cAMP or cGMP substrate, DMSO tolerance, and incubation time. Into each well of a solid white 1536 well plate (Corning) was dispensed 250 pg full-length recombinant NH-terminal GST tagged human PDE1B enzyme (BPS Bioscience Cat #60011, San Diego, Calif.) in 2.5 μL IMAP BSA reaction buffer (Molecular Devices, Sunnyvale, Calif.) containing 10 U/ml Calmodulin and 2.5 mM CaCl2 (Sigma Aldrich.) After a brief centrifugation, 30 nl compound was added by transfer from 1 mM stock in DMSO using a Kalypsys 1536 Pintool. Plates were incubated for 5 minutes at room temperature before dispensing 1.5 μL of 533 nM 5-carboxy fluorescein (FAM)-labeled cAMP (Molecular Devices, Sunnyvale, Calif.) for a final concentration of 200 nM. After a brief centrifugation, the plates were incubated for 30 minutes at room temperature. The assay was terminated by adding 5 μL IMAP binding reagent/Tb complex (Molecular Devices, Sunnyvale, Calif.) to each well. Plates were incubated 1 hour at room temperature and read on a Viewlux multimode plate reader (Perkin Elmer). The instrument was set to excite using the DUG11 filter and measure using 490/10 nm and 520/10 nm filters. Ratios of acceptor and donor were then calculated.
Measurement: IC50 = 5500 nM (pChEMBL 5.260000228881836)
Descriptors: MW 419.49, ALogP 2.18, HBA 9, HBD 2, PSA 112.36, QED 0.51
Compound (SMILES): COC(C)(C)COc1cc(C(=O)N[C@H](C)c2cnc(C)cn2)cc(-c2ncc(Cl)s2)c1
InChIKey: IVUVTQZOIPAQFU-CQSZACIVSA-N
Target: P2X purinoceptor 3 (SINGLE PROTEIN), Homo sapiens
UniProt: P56373
Assay: Intracellular Calcium Measurement to Assess Antagonist Activity at Human P2X3 and Human P2X2/3 Receptors: 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 = 36 nM (pChEMBL 7.440000057220459)
Descriptors: MW 460.99, ALogP 4.86, HBA 7, HBD 1, PSA 86.23, QED 0.52
Compound (SMILES): COc1cc(OC)c(F)c(-c2ccc3c(NC(=O)c4ccc(CN5CCN(C)CC5)cc4)n[nH]c3c2)c1F
InChIKey: YDRALAXHOSQZQX-UHFFFAOYSA-N
Target: Fibroblast growth factor receptor 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P11362
Assay: Enzyme Kinase Activity Assay: The enzyme reaction substrate Poly(Glu,Tyr) 4:1 was diluted with PBS without potassium ion (10 mM sodium phosphate buffer, 150 mM NaCl, pH7.2-7.4) to 20 μg/mL, 125 μL/well to coat the enzyme plate, and reacted at 37° C. for 12-16 hours. After the liquid was removed from the wells, the plate was washed three times with 200 μL/well of T-PBS (PBS containing 0.1% Tween-20) for 5 minutes each. The enzyme plate was dried in a 37° C. oven for 1-2 hours.50 μL of the ATP solution diluted with the reaction buffer (50 mM HEPES pH 7.4, 50 mM MgCl2, 0.5 mM MnCl2, 0.2 mM Na3VO4, 1 mM DTT) was added into each well at a 5 μM final concentration. The compounds were diluted to the appropriate concentration in DMSO, 1 μL/well or containing the corresponding concentrations of DMSO (negative control wells). The reaction was initiated by addition of each kinase domain recombinant protein diluted with 49 μL of reaction buffer. Two control wells without ATP were set in each experiment. The reaction mixtures were placed on a shaker (100 rpm) to react at 37° C. for 1 hour. The plates were washed with T-PBS for three times. 100 μL/well of the primary antibody PY99 dilution was added, and reacted on a shaker (100 rpm) at 37° C. for 0.5 hour. The plates were washed with T-PBS for three times. 100 μL/well of the secondary anti-horseradish peroxidase-labeled goat anti-mouse IgG dilution was added, and reacted on a shaker at 37° C. for 0.5 hour. The plates were washed with T-PBS for three times. 100 μL/well of 2 mg/mL OPD developing solution (diluted with 0.1M citric acid-sodium citrate buffer containing 0.03% H2O2 (pH=5.4)), and reacted in dark for 1-10 minutes at 25° C. (Ultrasound is needed in OPD dissolution, and the developing solution should be prepared on the site). The reaction was quenched with 50 μL/well of 2M H2SO4, and read out at 490 nm using a tunable microplate microplate reader SPECTRA MAX 190.
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 521.57, ALogP 4.53, HBA 6, HBD 2, PSA 82.72, QED 0.37
Compound (SMILES): CC(=O)c1cn(CC(=O)N2C[C@H](F)C[C@H]2C(=O)Nc2cccc(Br)n2)c2ccc(-c3cnc(F)nc3)cc12
InChIKey: ODUZXFLSXMMLEF-UTKZUKDTSA-N
Target: Complement factor D (SINGLE PROTEIN), Homo sapiens
UniProt: P00746
Assay: Factor D Assay: Human factor D (purified from human serum, Complement Technology, Inc.) at 80 nM final concentration is incubated with test compound at various concentrations for 5 minutes at room temperature in 50 mM Tris, 1M NaCl, pH 7.5. A synthetic substrate Z-L-Lys-SBzl and DTNB (Ellman's reagent) are added to final concentrations of 100 μM each. The increase in color is recorded at OD405 nm in a microplate in kinetic mode over 30 minutes with 30 second time points in a spectrofluorimeter. IC50 values are calculated by non-linear regression from the percentage of inhibition of complement factor D activity as a function of test compound concentration.
Measurement: IC50 < 1000 nM (pChEMBL 6)
Descriptors: MW 583.39, ALogP 4.18, HBA 7, HBD 1, PSA 110.08, QED 0.21
Compound (SMILES): Cc1cc(-c2cnn3cc(N4CC(N5CCN(C)CC5)C4)cnc23)c2cccc(F)c2n1
InChIKey: LBCISVVKWNMZCI-UHFFFAOYSA-N
Target: Activin receptor type-1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q04771
Assay: Luciferase Reporter Gene Assay for ALK2 and TGFbeta Activity: In this reporter gene cell based ALK2 and TGFβ assay, the C2Cl2 cell line was employed for the measurement of ALK2 activity, using a BRE-Luc SMAD1/5/8 reporter and BMP6 as the agonist. The HEK293T cell line was employed to measure TGFβ activity, using a SBE-Luc SMAD2/3 reporter and TGFβ as the agonist. Luciferase reporter assays were read using Promega Steady-Glo Luciferase Assay System.Cells were plated in 96 well white clear bottom assay plates at 10 k cells/well for C2Cl2-BRE, 15 k cells/well for HEK293-SBE in DMEM containing 2% FBS 1% P/S. Cells were given a minimum of 4 hours in incubator at 37° C./5% CO2 to adhere prior to further treatment. Compounds were diluted in DMSO to a 10-point dilution curve and added to the plate to reach the following final concentrations: 10000, 3000, 1000, 300, 100, and 30 nM in the HEK293-SBE assay and 1000, 300, 100, 30, 10, 3, 1, 0.3 nM in the C2Cl2-BRE assay. Negative and positive control wells received 2 μL DMSO as vehicle treatment. Plates were returned to incubator for 45 minutes and then BMP6 and TGFB were added to a final concentration of 50 ng/mL and 5 ng/mL, respectively. Plates were returned to the incubator and left overnight. After a minimum of 18 hours post BMP6/TGFβ addition, the plates were read using Promega Steady-Glo Luciferase Assay System. A 1:1 mixture of prepared steady-glo and phenol free DMEM was prepared, and 50 μL/well was added to the assay plates whose media had been flicked off. Plates were given 10 minutes post steady-glo addition before luminescence was read on a Spectramax M5e microplate reader. Negative control wells were averaged and subtracted from all other wells on the plate. Inhibition was calculated as the percent of signal loss compared to the averaged positive control wells.
Measurement: IC50 = 22 nM (pChEMBL 7.659999847412109)
Descriptors: MW 431.52, ALogP 2.83, HBA 7, HBD 0, PSA 52.8, QED 0.5
Compound (SMILES): O=C(NC1CCS(=O)(=O)CC1)c1ccc2c(-c3ccccc3Cl)nn(-c3ccc(F)cc3)c2c1
InChIKey: XSLKOTQFOUADLX-UHFFFAOYSA-N
Target: Diacylglycerol O-acyltransferase 2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q96PD7
Assay: Enzymatic Activity Assay: DGAT2 activity was determined by measuring the amount of enzymatic product triolein (1,2,3-Tri(cis-9-octadecenoyl)glycerol) using the membrane prep mentioned above. The assay was carried out in deep well 384 plates in a final volume of 40 μL at room temperature. The assay mixture contained the following: assay buffer (100 mM Tris.Cl, pH 7.0, 20 mM MgCl2, 5% ethanol), 25 μM of diolein, 10 μM of oleoyl-CoA and 10 ng/μL of DGAT2 membrane.
Measurement: IC50 = 61 nM (pChEMBL 7.210000038146973)
Descriptors: MW 497.98, ALogP 4.79, HBA 5, HBD 1, PSA 81.06, QED 0.44
Compound (SMILES): COC(=O)Nc1ccc2c(c1)NC(=O)CCCC[C@H](c1ccc(-c3c(F)cccc3OC(F)F)c[n+]1[O-])c1cccc-2c1
InChIKey: FYWBHLIHHLFVDV-DEOSSOPVSA-N
Target: Coagulation factor XI (SINGLE PROTEIN), Homo sapiens
UniProt: P03951
Assay: Factor XIa Assay: The effectiveness of a compound of the present invention as an inhibitor of Coagulation Factor XIa can be determined using a relevant purified serine protease, and an appropriate synthetic substrate. The rate of hydrolysis of the chromogenic or fluorogenic substrate by the relevant serine protease was measured both in the absence and presence of compounds of the present invention. Assays were conducted at rt or at 37° C. Hydrolysis of the substrate resulted in release of amino trifluoromethylcoumarin (AFC), which was monitored spectrofluorometrically by measuring the increase in emission at 510 nm with excitation at 405 nm. A decrease in the rate of fluorescence change in the presence of inhibitor is indicative of enzyme inhibition. Such methods are known to one skilled in the art. The results of this assay are expressed as the inhibitory constant, Ki.Factor XIa determinations were made in 50 mM HEPES buffer at pH 7.4 containing 150 mM NaCl, 5 mM CaCl2, and 0.1% PEG 8000 (polyethylene glycol; JT Baker or Fisher Scientific). Determinations were made using purified human Factor XIa at a final concentration of 40 pM (Sekisui Diagnostics) and he synthetic substrate, Z-Gly-Pro-Arg-AFC, TFA salt (Sigma #C0980) at a concentration of 100 μM.Activity assays were performed by diluting a stock solution of substrate at least tenfold to a final concentration ≤0.1 Km into a solution containing enzyme or enzyme equilibrated with inhibitor. Times required to achieve equilibration between enzyme and inhibitor were determined in control experiments. Initial velocities of product formation in the absence (Vo) or presence of inhibitor (Vi) were measured. Assuming competitive inhibition, and that unity is negligible compared Km/[S], [I]/e, and [I]/e (where [S], [I], and e respectively represent the total concentrations, of substrate, inhibitor and enzyme), the equilibrium constant (Ki) for dissociation of the inhibitor from the enzyme can be obtained from the dependence of Vo/Vi on [I] shown in the following equation.
Measurement: Ki = 1.52 nM (pChEMBL 8.819999694824219)
Descriptors: MW 591.59, ALogP 7.22, HBA 5, HBD 2, PSA 103.6, QED 0.19
Compound (SMILES): CC(=O)N1CCc2cc(-c3cncc(C(F)(F)F)c3)cc(Cl)c21
InChIKey: WFPKESACWWRPPG-UHFFFAOYSA-N
Target: Cytochrome P450 11B2, mitochondrial (SINGLE PROTEIN), Homo sapiens
UniProt: P19099
Assay: HTRF-Based Assay : V79 cell lines stably expressing the either the human CYP11B2 or the human CYP11B1 enzyme were generated using a standard transfection protocol. V79 cells were transfected with plasmids pTriEx3-Hygro-hCyp11B2 or pTriEx3-Hygro-hCyp11B1 using Lipofectamine2000 reagent. V79 cells that stably express the human CYP11B2 or human CYP11B1 enzyme were selected for and maintained in DMEM supplemented with 10% FBS and 400 μg/mL hygromycin for 2 weeks. Single cell clones were generated by infinite dilution in DMEM supplemented with 10% FBS and 400 μg/mL hygromycin until single colonies were obtained. Clones V79-hCYP11B2-CLE9 and V79-hCYP11B1-CL8C7, were determined to produce the most aldosterone and cortisol, respectively, and were selected for inhibitor screening. For testing of inhibitors, cells were harvested at 80% confluency with 0.05% Trypsan-EDTA, washed once in PBS, and reconstituted in DMEM+0.1% BSA media at a cell concentration of 600,000 cells/mL for the CYP11B2 assay and 280,000 cells/mL for the CYP11B1 assay. 25 μL of cells were added to a 384 well tissue culture treated plate and mixed with 0.3 μL of inhibitor or DMSO (1% final DMSO concentration) for 1 hour at 37° C., 5% CO2. After pre-incubation with inhibitor, the reaction was initiated by adding 5 μL of substrate (final concentration of 125 nM 11-deoxycorticosterone for the CYP11B2 assay or 250 nM 11-deoxycortisol for the CYP11B1 assay). The reaction was carried out for 3 hours at 37° C., 5% CO2 and was stopped by harvesting the supernatants. The amount of product in the supernatant (aldosterone for CYP11B2 assay and cortisol for the CYP11B1 assay) was measured using HTRF-based assay kit (Aldosterone HTRF-CisBio#64ALDPEB, Cortisol HTRF-CisBio #63IDC002-CORT).
Measurement: IC50 = 7.5 nM (pChEMBL 8.119999885559082)
Descriptors: MW 340.73, ALogP 4.33, HBA 2, HBD 0, PSA 33.2, QED 0.77
Compound (SMILES): Cn1cc(-c2cc(-c3ccc(N4CCC5(CC4)CN(C(=O)C4CC4)C5)nc3)c3c(C#N)cnn3c2)cn1
InChIKey: VJZXWOMNLUZTQM-UHFFFAOYSA-N
Target: Proto-oncogene tyrosine-protein kinase receptor Ret (SINGLE PROTEIN), Homo sapiens
UniProt: P07949
Assay: RET Enzyme Assay: Compounds of General Formula I were screened for their ability to inhibit wild type and V804M mutant RET kinase using CisBio's HTRF® KinEASE-TK assay technology. Briefly, N-terminal GST tagged recombinant human RET cytoplasmic domain (aa 658-end) from Eurofins (0.25 nM RET; Cat. No. 14-570M) or N-terminal GST tagged recombinant human V804M mutant RET cytoplasmic domain (aa 658-end) from Millipore (0.25 nM enzyme; Cat. No. 14-760) was incubated with 250 nM TK-substrate biotin (CisBio, part of Cat. No. 62TK0PEC) and 1 mM ATP along with test compound in a buffer consisting of 25 mM HEPES pH 7.4, 10 mM MgCl2, 0.01% Triton X-100, and 2% DMSO in a volume of 8 μL. Compounds were typically prepared in a threefold serial dilution in DMSO and added to the assay to give the appropriate final concentration. After a 30-minute incubation at 22° C., the reaction was quenched by adding 8 μL of quench solution containing 31.25 nM Sa-XL665 and 1× TK-ab-Cryptate in HTRF detection buffer (all from CisBio, part of Cat. No. 62TK0PEC). After a 1 hour incubation at 22° C., the extent of reaction was determined using a PerkinElmer EnVision multimode plate reader via HTRF dual wavelength detection, and the percent of control (POC) was calculated using a ratiometric emission factor. 100 POC was determined using no test compounds and 0 POC was determined using pre-quenched control reactions. The POC values were fit to a 4 parameter logistic curve, and the IC50 is defined as the concentration of inhibitor at which the POC equals 50 for the fitted curve.
Measurement: IC50 = 9 nM (pChEMBL 8.050000190734863)
Descriptors: MW 492.59, ALogP 3.51, HBA 8, HBD 0, PSA 95.35, QED 0.43
Compound (SMILES): CC(C)N1CCN(c2cccc(N(C)c3ccc(Oc4nc(O)c5ccncc5n4)cc3)c2)CC1
InChIKey: PJQNDBNJFCXLPU-UHFFFAOYSA-N
Target: Lysine-specific demethylase 4C (SINGLE PROTEIN), Homo sapiens
UniProt: Q9H3R0
Assay: JMJD2C Assay: The ability of test compounds to inhibit the activity of JMJD2C was determined in 384-well plate-format under the following reaction conditions: 0.3 nM JMJD2C, 300 nM H3K9me3-biotin labeled peptide (Anaspec cat #64360), 2 μM alpha-ketoglutaric acid in assay buffer of 50 mM HEPES, pH7.3, 0.005% Brij35, 0.5 mM TCEP, 0.2 mg/ml BSA, 50 μM sodium L-ascorbate, and 2 μM ammonium iron(II) sulfate. Reaction product was determined quantitatively by TR-FRET after the addition of detection reagent Phycolink Streptavidin-allophycocyanin (Prozyme) and Europium-anti-di-methylated histone H3 lysine 9 (H3K9me2) antibody (PerkinElmer) in the presence of 5 mM EDTA in LANCE detection buffer (PerkinElmer) at a final concentration of 50 nM and 1 nM, respectively.The assay reaction was initiated by the following: 2 μl of the mixture of 900 nM H3K9me3-biotin labeled peptide and 6 μM alpha-ketoglutaric acid with 2 μl of 11-point serial diluted inhibitor in 3% DMSO were added to each well of the plate, followed by the addition of 2 μl of 0.9 nM JMJD2C to initiate the reaction. The reaction mixture was incubated at room temperature for 30 min, and terminated by the addition of 6 μl of 5 mM EDTA in LANCE detection buffer containing 100 nM Phycolink Streptavidin-allophycocyanin and 2 nM Europium-anti-H3K9me2 antibody. Plates were read by EnVisionMultilabel Reader in TR-FRET mode (excitation at 320 nm, emission at 615 nm and 665 nm) after 1 hr incubation at room temperature. A ratio was calculated (665/615) for each well and fitted to determine inhibition constant (IC50).
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 470.58, ALogP 4.82, HBA 8, HBD 1, PSA 77.85, QED 0.43
Compound (SMILES): CC(C)(OC(=O)Nc1ccc2c(c1)N(S(=O)(=O)c1ccc(F)c(C(F)(F)F)c1)C[C@H]([C@H]1C[C@H]1C(=O)O)O2)C(F)(F)F
InChIKey: NATQKFCKEVJSOH-IQUTYRLHSA-N
Target: Nuclear receptor ROR-gamma (SINGLE PROTEIN), Homo sapiens
UniProt: P51449
Assay: Biochemical TR-FRET Assay: To identify novel antagonists of RORgammaT, an assay was developed which employs the interaction of RORgammaT with its co-activator peptide SRC1_2. This peptide mimics the recruitment of co-activators to RORgammaT through its interaction with the LXXLL (SEQ ID NO:1) (e.g., NR box) motifs (Xie et al., J. Immunol. 175: 3800-09, 2005; Kurebayashi et al., Biochem. Biophys. Res. Commun. 315: 919-27, 2004; Jin et al., Mol. Endocrinology 24:923-29, 2010). The RORγ-Ligand Binding Domain TR-FRET Assay was run according to the following protocol.HIS-tagged RORγ-LBD protein was recombinantly expressed in Escherichia coli. The RORγ-LBD protein was purified by Ni2+-affinity resin. Purified protein was then diluted in assay buffer (50 mM Tris pH 7.0, 50 mM KCl, 1 mM EDTA, 0.1 mM DTT, 100 μg/mL bovine serum albumin, delipidated) to obtain a RORγ-LBD final concentration of 3 nM. Europium tagged anti-HIS antibody was also added to this solution (1.25 nM). Separately, SF9 cells not expressing any recombinant protein were lysed (32,000 cells per μl in 25 mM Tris, 50 mM NaCl) and the previously frozen lysate was added to the diluted RORγ-LBD solution at a ratio of 0.75 μl SF9 lysate per 15 μl of diluted RORγ-LBD.
Measurement: IC50 = 22.4 nM (pChEMBL 7.650000095367432)
Descriptors: MW 614.49, ALogP 5.41, HBA 6, HBD 2, PSA 122.24, QED 0.42
Compound (SMILES): Cc1cc(OCCCc2c(C)n(Cc3cccc(C(=O)O)c3)c3c(-c4c(C)n[nH]c4C)cccc23)cc(C)c1Cl
InChIKey: XNUGGGNUKPIEGI-UHFFFAOYSA-N
Target: Induced myeloid leukemia cell differentiation protein Mcl-1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q07820
Assay: Fluorescence Polarization Anisotropy Competition Assay: Compound affinity was measured using a fluorescence polarization anisotropy competition assay. Anisotropy measurements were carried out in 384-well, black, flat-bottom plates (Greiner Bio-one, Monroe, N.C., USA). The assay was run using a fluorescein isothiocyanate-labeled BH3 peptide derived from Bak (FITC-AHx-GQVGRQLAIIGDDINR-NH2) that was purchased from GenScript (Piscataway, N.J.) at >95% purity and used without further purification. 10 nM FITC-Bak peptide and 14 nM recombinant Mcl-1 (residues 172-327) were added to assay buffer (3 mM dithiothreitol, 50 mM NaCl, 20 mM Tris, pH 7.5). For selectivity assays, 40 nM Bcl-2 (residues 1-207A96T,G110R, Δ35-91, replaced with Bcl-xL35-50) or 4 nM Bcl-xL (residues 1-209, loop 45-86 deleted) were incubated with 10 nM FITC-Bak in assay buffer.Compounds are diluted in DMSO in a 10-point, 3-fold serial dilution scheme. 2.5 uL compound is added to 47.5 uL of assay buffer containing FITC-Bak and protein, for a final DMSO concentration of 5% and a top concentration of 20 uM. A FITC-Bak peptide alone (100% inhibition) and peptide plus protein (0% inhibition) control is included on each assay plate. The plate was mixed and incubated for 90 minutes at room temperature. Anisotropy is measured at excitation wavelength 480 nm and emission wavelength 535 nm using an EnVision Multi-label plate reader (PerkinElmer, Wellesley, Mass., USA). Fluorescence anisotropy is plotted against compound concentration to generate an IC50 (inhibitor concentration at which 50% of bound peptide is displaced) by fitting the data to a 4-parameter logistic model using XLFit software (Guildford, Surrey, UK). IC50 is converted to a binding dissociation constant (Ki value)
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 556.11, ALogP 7.99, HBA 4, HBD 2, PSA 80.14, QED 0.18
Compound (SMILES): C[C@@H](C[C@@]1(C)NC(=O)NC1=O)C(=O)N1CCC(c2ccc(F)cc2)CC1
InChIKey: RPDOQOLWCQNQJE-HXPMCKFVSA-N
Target: A disintegrin and metalloproteinase with thrombospondin motifs 5 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9UNA0
Assay: hADAMTS-5 assay: 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, 5% glycerol) containing hADAMTS-5 (0.5 ng/μL) (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 60 min at Room Temperature (Excitation 485 nm, emission 530).
Measurement: IC50 = 75 nM (pChEMBL 7.119999885559082)
Descriptors: MW 361.42, ALogP 2.16, HBA 3, HBD 2, PSA 78.51, QED 0.81
Compound (SMILES): FC(F)(F)c1ccc(Nc2ccc([C@H]3CNCCO3)nc2)cc1
InChIKey: CWRDKDSHVOKMNB-OAHLLOKOSA-N
Target: Trace amine-associated receptor 1 (SINGLE PROTEIN), Mus musculus
UniProt: Q923Y8
Assay: Radioligand Binding Assay : HEK-293 cells stably expressing mouse TAAR1 were maintained at 37° C. and 5% CO2 in DMEM high glucose medium, containing fetal calf serum (10%, heat inactivated for 30 min at 56° C.), penicillin/streptomycin (1%), and 375 μg/ml geneticin (Gibco). Cells were released from culture flasks using trypsin/EDTA, harvested, washed twice with ice-cold PBS (without Ca2+ and Mg2+), pelleted at 1,000 rpm for 5 min at 4° C., frozen and stored at −80° C. Frozen pellets were suspended in 20 ml HEPES-NaOH (20 mM, pH 7.4) containing 10 mM EDTA and homogenized with a Polytron (PT 6000, Kinematica) at 14′000 rpm for 20 s. The homogenate was centrifuged at 48,000×g for 30 min at 4° C. Subsequently, the supernatant was removed and discarded, and the pellet resuspended in 20 ml HEPES-NaOH (20 mM, pH 7.4) containing 0.1 mM EDTA using the Polytron (20 s at 14′000 rpm). This procedure was repeated and the final pellet resuspended in HEPES-NaOH containing 0.1 mM EDTA and homogenized using the Polytron. Typically, aliquots of 2 ml membrane portions were stored at −80° C. With each new membrane batch the dissociation constant (Kd) was determined via a saturation curve. The TAAR1 radioligand 3[H](S)-4-[(ethyl-phenyl-amino)-methyl]-4,5-dihydro-oxazol-2-ylamine (described in WO 2008/098857) was used at a concentration equal to the calculated Kd value, that was usually around 0.7 nM, resulting in the binding of approximately 0.5% of the radioligand and a specific binding representing approximately 70% of the total binding.
Measurement: Ki = 4.8 nM (pChEMBL 8.319999694824219)
Descriptors: MW 323.32, ALogP 3.5, HBA 4, HBD 2, PSA 46.18, QED 0.91
Compound (SMILES): C[C@@](C(=O)N[C@@H]1CCCC[C@@H]1O)(c1ccccc1)n1ncc2c1nc(N)n1nc(-c3ccco3)nc21
InChIKey: PJJGCZPQOJDWDD-UZEJHQAJSA-N
Target: Adenosine deaminase (SINGLE PROTEIN), Homo sapiens
UniProt: P00813
Assay: Luciferase Reporter Assay: HEK293-A2AR-luc2p/CRE/Hygro cells were seeded at a density of 5,000 cells/well in DMEM with 1% FBS and 1 U/mL adenosine deaminase (ADA) (Sigma). After 18 h, the cells were treated with 3 nM CGS21680 plus a series dilution of A2AR antagonist, the compounds disclosed herein at the concentration of 0.110000 nM, prepared in DMEM with 1% FBS. After 5 h incubation, the luciferase activity in cells were measured using the Bright-Glo Luciferase Assay System (Promega) according to manufacturer's instructions. The luminescence signal was measured using a PHERAstar FS plate reader (BMG Labtech). Luminescence intensity from 10 μM preladenant treatment was set as 0%. Maximal luminescence intensity was determined in the presence of 3 nM CGS21680 and was set as 100%. The IC50 value was calculated from a dose dependent inhibition curve across the range of compound concentrations.
Measurement: IC50 = 14.2 nM (pChEMBL 7.849999904632568)
Descriptors: MW 486.54, ALogP 2.5, HBA 10, HBD 3, PSA 149.39, QED 0.34
Compound (SMILES): COc1cc(-c2cnn(-c3ccc(C(=O)NCCCN(C)C)cn3)c2O)ccn1
InChIKey: PVYUGIQQQFZPBD-UHFFFAOYSA-N
Target: Egl nine homolog 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9GZT9
Assay: Inhibition Assay: The IC50 values for the PHD2 enzyme (residues 181-417) were determined by mixing increasing amounts of inhibitor with a fixed amount of enzyme (5 nM, final concentration) and Biotin labeled peptide (Biotin-Asp-Leu-Glu-Met-Leu-Ala-Pro-Tyr-Ile-Pro-Met-Asp-Asp-Asp-Phe-Gln-Leu, 1 uM final concentration) and 2-Oxyglutarate (2 uM final concentration) in 50 mM HEPES, 50 mM KCl, 0.5 mM TCEP, 2 uM FeCl2, 0.1 mg/ml BSA, at pH 7.3. The reaction was conducted by pre-incubating the enzyme in the presence of inhibitor for 60 min at room temperature. The activity of the free enzyme was measured by adding the peptide, the 2-Oxoglutarate (see above for final concentrations), and Ascorbic Acid (1 mM final concentration). The enzymatic activity was quenched after 60 min by adding an excess of a tight binding inhibitor to the assay mixture. The amount of product released was measured by using a LC/MS system (Agilent HPLC with Applied Biosystems API3000 Mass Spectrometer).
Measurement: IC50 = 50.1 nM (pChEMBL 7.300000190734863)
Descriptors: MW 396.45, ALogP 1.73, HBA 8, HBD 2, PSA 105.4, QED 0.56
Compound (SMILES): Fc1ncccc1-c1cnc2nc(COc3ccccc3)cn2c1
InChIKey: CEVNKNJDOGSMEY-UHFFFAOYSA-N
Target: Metabotropic glutamate receptor 5 (SINGLE PROTEIN), Homo sapiens
UniProt: P41594
Assay: Calcium Influx Assay Based on Fluorescence: Ca2+ (calcium) influx assay is an experiment for measuring the activity of a positive allosteric modulator of mGluR5 receptor in which human mGluR5 receptor-overexpressed HEK293 cell line is used. The day before the experiment, cells were prepared in a cell culture medium (DMEM, 5% FBS) with the density of 80,000/well, and 100 μl of cells were dispensed into each well of a poly-D-lysine-coated 96-well plate. Cells were incubated in a 5% CO2, 37° C. incubator. The next day, the cell culture medium was removed from the plate, and 100 μl of 1× Fluo-4 calcium indicator diluted with a buffer (1× Hank's balanced salt solution, 20 mM HEPES, 2.5 mM probenecid) were added to each well and incubated at 37° C. for 1 hour. The compound stock solutions were prepared in 100% DMSO, and the compounds were serially diluted with a ¼ dilution to 6 or 7 concentrations (final concentration was 10 μM to 10 nM). The diluted compound solutions were added to the plate with 0.1 to 0.2% of final DMSO concentration. 1 hour after the addition of Fluo-4 calcium indicator, L-glutamate (EC20 concentration) and the test compound solutions were added to the plate, and Ca2+ reaction was then measured by FLIPR at room temperature. The activity of the compounds was standardized on the basis of the results of maximum value-minimum value of fluorescent reaction, and the activity value was calculated on the basis that no activity on glutamate EC20 is 0% and the reaction to glutamate maximum value is 100%.
Measurement: EC50 = 300 nM (pChEMBL 6.519999980926514)
Descriptors: MW 320.33, ALogP 3.51, HBA 5, HBD 0, PSA 52.31, QED 0.54
Compound (SMILES): C[S@@+]([O-])c1cc(Cl)ccc1CN1C(=O)c2cc(C(C)(C)O)cc(F)c2[C@]1(OCC1(CO)CC1)c1ccc(Cl)cc1
InChIKey: PVRYETGYPNIACM-HFOCTHEHSA-N
Target: E3 ubiquitin-protein ligase Mdm2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q00987
Assay: MDM2-p53 Interaction Using a 96-Well Plate Binding Assay (ELISA): The ELISA assay was performed in streptavidin coated plates which were preincubated with 200 μl per well of 1 μg ml−1 biotinylated IP3 peptide. The plates were ready to use for MDM2 binding after washing the plate with PBS.Compounds and control solutions in DMSO aliquoted in 96-well plates were pre-incubated in a final 2.5-5% (v/v) DMSO concentration at room temperature (for example 20° C.) for 20 min with 190 μl aliquots of optimized concentrations of in vitro translated MDM2, before transfer of the MDM2-compound mixture to the b-IP3 streptavidin plates, and incubation at 4° C. for 90 min. After washing three times with PBS to remove unbound MDM2, each well was incubated at 20° C. for 1 hour with a TBS-Tween (50 mM Tris pH7.5; 150 mM NaCl; 0.05% Tween 20 nonionic detergent) buffered solution of primary mouse monoclonal anti-MDM2 antibody (Ab-5, Calbiochem, used at a 1/10000 or 1/200 dilution depending on the antibody stock solution used), then washed three times with TBS-Tween before incubation for 45 mins at 20° C. with a TBS-Tween buffered solution of a goat-anti-mouse horseradish peroxidase (HRP) conjugated secondary antibody (used at 1/20000 or 1/2000 depending on the antibody stock solution). The unbound secondary antibody was removed by washing three times with TBS-Tween. The bound HRP activity was measured by enhanced chemiluminescence (ECL, Amersham Biosciences) using the oxidation of the diacylhydrazide substrate, luminol, to generate a quantifiable light signal. The percentage of MDM2 inhibition at a given concentration is calculated as the [1−(RLU detected in the compound treated sampleRLU negative DMSO control)÷(RLU of DMSO positive and negative controls)]×100 or as the (RLU detected in the compound treated sample÷RLU of DMSO controls)×100. The IC50 was calculated using a plot of % MDM2 inhibition vs concentration and is the average of two or three independent experiments.
Measurement: IC50 = 2.3 nM (pChEMBL 8.640000343322754)
Descriptors: MW 606.54, ALogP 5.74, HBA 5, HBD 2, PSA 93.06, QED 0.3
Compound (SMILES): COc1nc2nc(C)nc(N[C@H](C)c3cccc(C(F)F)c3F)c2cc1C1(C#N)CC1
InChIKey: ITZSTKVARHEHBZ-LLVKDONJSA-N
Target: Son of sevenless homolog 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q07889
Assay: Ras-SOS Interaction Assay: The ability of any compound of the present disclosure to reduce a Ras protein signaling output by, e.g., interfering or disrupting interaction (or binding) between SOS1 and a Ras protein can be assessed in vitro. For example, the equilibrium interaction of human SOS1 (hSOS1) with human wildtype Kras or K-Ras mutant (e.g., hK-Ras G12C mutant, or hK-RasG12C) can be assessed as a proxy or an indication for a subject compound's ability to inhibit SOS. Detection of such interaction is achieved by measuring homogenous time-resolved fluorescence resonance energy transfer (HTRF) from (i) a fluorescence resonance energy transfer (FRET) donor (e.g., antiGST-Europium) that is bound to GST-tagged K-RasG12C to (ii) a FRET acceptor (e.g., anti-6His-XL665) bound to a His-tagged hSOS1. The assay buffer can contain 5 mM HEPES pH 7.4, 150 mM NaCl, 10 mM EDTA, 1 mM DTT, 0.05% BSA, 0.0025% (v/v) Igepal and 100 mM KF. A Ras working solution is prepared in assay buffer containing typically 10 nM of the protein construct (e.g., GST-tagged hK-RasG12C) and 2 nM of the FRET donor (e.g., antiGST-Eu(K) from Cisbio, France). A SOS1 working solution is prepared in assay buffer containing typically 10 nM of the protein construct (e.g., His-hSOS1) and 10 nM of the FRET acceptor (e.g., anti-6His-XL665 from Cisbio, France). An inhibitor control solution is prepared in assay buffer containing 10 nM of the FRET acceptor without the SOS1 protein. A fixed reaction mixture with or without test compound is transferred into a 384-well plate. Ras working solution is added to all wells of the test plate. SOS1 working solution is added to all wells except for those that are subsequently filled the inhibitor control solution. After approximately 60 min incubation, the fluorescence is measured with a M1000Pro plate reader (Tecan) using HTRF detection (excitation 337 nm, emission 1: 620 nm, emission 2: 665 nm).
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 427.43, ALogP 5.15, HBA 6, HBD 1, PSA 83.72, QED 0.59
Compound (SMILES): CN1CCCc2cc(ccn2)-c2nc(cs2)C(=O)Nc2cn(C3CCOCC3)nc2C(=O)NCC1
InChIKey: QSXIGAYIJFICPE-UHFFFAOYSA-N
Target: Interleukin-1 receptor-associated kinase 4 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9NWZ3
Assay: Biochemical Assay: The biochemical assay is in a AlphaScreen format. The kinase reaction is based on the IRAK-4 phosphorylation of a biotin labeled peptide. The phosphopeptide is incubated with anti-phosphothreonine antibody as well as streptavidin- and protein A-coated beads. Binding of the protein-A coated beads to the antibody and the streptavidin beads to the peptide, leads to an energy transfer from one bead to the other, ultimately producing a luminescent/fluorescent signal.Generally, the kinase reaction is carried out at 1 nM IRAK4, 1.6 μM peptide, 10 μM ATP in reaction buffer 50 mM Hepes, 60 mM NaCl, 5 mM MgCl2, 0.25 mM MnCl2, 2 mM DTT, 0.01% BSA, 0.01% Tween-20) for 3.5 h at RT.
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 495.61, ALogP 2.61, HBA 9, HBD 2, PSA 114.27, QED 0.53
Compound (SMILES): O=C(Cc1csc(-c2ccc(Cl)cc2Cl)n1)N[C@@H](Cc1ccccc1)C(=O)O.S
InChIKey: BCEUZRYRQHHNEA-LMOVPXPDSA-N
Target: Serine hydroxymethyltransferase, cytosolic (SINGLE PROTEIN), Homo sapiens
UniProt: P34896
Assay: SHMT1 and SHMT2 Activity Assays: Full length human cytosolic Serine Hydroxymethyl transferase 1 (SHMT1, residues 1-483 in Uniport ID P34896) was expressed as an N-terminal His6 tagged protein and purified in E. coli using nickel capture followed by size-exclusion chromatography. Human mitochondrial SHMT2 (residues 30-504 in Uniprot ID P34897) with mitochondrial leader sequence deleted was expressed as an N-terminal His6 tagged protein and purified in E. coli using nickel capture followed by size-exclusion chromatography.Serine hydroxymethyltransferases catalyze the reversible hydroxymethylation of glycine to serine, with methylene tetrahydrofolate (CH2-THF) providing the additional carbon. SHMT1 and SHMT2 activity was determined by measuring serine production from glycine and CH2-THF using mass spectroscopy. Briefly, 15 ul of 0.5 mM glycine and 0.2 mM CH2-THF in 20 mM TEA, pH8.0, 0.2 mM NADPH were added to 384 well plate containing 0, 0.05 mM, 0.005 mM or 0.0005 mM of inhibitor. 15 ul of SHMT1 or 2 were added to initiate the reaction. The plate was incubated for 60 minutes at room temperature and the reaction was quenched by the addition of 30 ul of 10% trichloroacetic acid. Serine produced was analyzed using Rapidfire 360 and API4000+ in the positive ion mode.
Measurement: IC50 = 55000 nM (pChEMBL 4.260000228881836)
Descriptors: MW 469.42
Compound (SMILES): Cc1ccc([C@@H](C)NC(=O)c2cc(OC3COC3)cc(-c3ncc(C)s3)c2)cn1
InChIKey: MHQNYPHPPSGTBD-OAHLLOKOSA-N
Target: P2X purinoceptor 3 (SINGLE PROTEIN), Homo sapiens
UniProt: P56373
Assay: Intracellular Calcium Measurement to Assess Antagonist Activity at Human P2X3 and Human P2X2/3 Receptors: 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 = 74 nM (pChEMBL 7.130000114440918)
Descriptors: MW 409.51, ALogP 4.09, HBA 6, HBD 1, PSA 73.34, QED 0.66
Compound (SMILES): Cc1nn2c3c(nc2s1)CCN(C(=O)COc1ccc(N2CCOCC2)nc1Cl)C3c1cc(-c2nn[nH]n2)cs1
InChIKey: KPJSDGHTZGSETK-UHFFFAOYSA-N
Target: Tryptophan 5-hydroxylase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P17752
Assay: Biochemical In Vitro Assay: To generate the enzyme, full length human TPH1 is cloned into the plasmid pET20b(+) (Novagen) and expressed in E. coli. The bacterial cells are ruptured by sonication on ice and the lysate is cleared by centrifugation. The resulting protein in the pellet is re-extracted and TPH1 is purified from the obtained lysate by affinity chromatography using a pterin cosubstrate analog immobilized to the resin of the column. The protein is further purified by size exclusion chromatography to remove protein aggregates. The activity of TPH1 is determined by using a fluorescence assay. The enzyme activity assay is carried out at 15° C. with atmosphere oxygen for the duration of 60 minutes in a volume of 64 μl. The reaction is carried out in a 0.1 M Tris-HCl buffer, adjusted to pH 7.6, containing 1 mM DTT, 0.2 mg/mL catalase, 100 μM (±)-6-methyl-5,6,7,8-tetrahydropterine dihydrochloride, 40 μM L-tryptophan, and 40-80 nM of TPH1. The reaction is started by bringing together L-tryptophan with all the other reaction substituents and stopped by quenching with perchloric acid (HClO4). The amount of 5-hydroxy-L-tryptophan produced during the enzymatic reaction is determined by fluorescence readout. Fluorescence, as determined at 540 nm when excited at 300 nm wavelength, increases proportionally to the 5-hydroxy-L-tryptophan formed. Compounds are prepared as 10 mM stock solution in DMSO, then diluted in 384-well plates using DMSO followed by a transfer of the dilutions into the assay plate. Fluorescence is measured for each well and the fluorescence at 540 nm wavelength is compared to the fluorescence of the vehicle in place of compound. Inhibitory activities of example compounds with respect to the TPH1 protein are determined by calculating the IC50 value (the concentration of compound needed to inhibit 50% of the enzyme activity).
Measurement: IC50 = 23 nM (pChEMBL 7.639999866485596)
Descriptors: MW 599.1, ALogP 2.78, HBA 13, HBD 1, PSA 139.55, QED 0.29
Compound (SMILES): Cc1cc(Cn2nc(C)c3c(Cl)c(C(=O)Nc4ccc(C(C)C)cc4)cnc32)ncn1
InChIKey: BYRXXZPAMHDICB-UHFFFAOYSA-N
Target: Glucocorticoid receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P04150
Assay: Biological Evaluation: The assay was performed using H4 neuroglioma cells and using indirect immunofluorescence detection of the endogenous GR receptor. Fixative: 4% PFA. Permeabilization buffer: 0.3% Triton X-100 in PBS. Primary antibody: 1:200 in 5% BSA block. Secondary antibody: 1:800 in 5% BSA block. HCS Cell Mask: 1:6000 in PBS. DAPI: 100 ng/ml.
Measurement: IC50 < 50 nM (pChEMBL 7.301000118255615)
Descriptors: MW 434.93, ALogP 4.92, HBA 6, HBD 1, PSA 85.59, QED 0.48