lovingscience/chembl-v37-raw-activities huggingface dataset:
Compound (SMILES): C[C@@H](O)Cc1coc(-c2cncc([C@@]34CC[C@@H](c5cc(-c6c(F)cccc6F)nnc53)C4(C)C)n2)n1
InChIKey: ISVPFCDTVVTNCI-YKSJCTFJSA-N
Target: Nuclear receptor ROR-gamma (SINGLE PROTEIN), Homo sapiens
UniProt: P51449
Assay: In Vitro Ligand Binding Assay: 50 mM HEPES buffer (pH 7.4); 150 mM NaCl; 1 mM DTT; 5 mM MgCl2; 0.01% BSA; 5% DMSO; 0.6 ug/mL RORc receptor; 6 nM 25-[3H]hydroxycholesterol. For NSB wells, 1 uM 25-hydroxycholesterol was also present. Assay plates were 96-well polypropylene V-bottom plates. 10 uL of 5× compound in 25% DMSO/75% Assay Buffer was added to Test wells. 10 uL of 25% DMSO/75% Assay Buffer was added to Total Binding or No Receptor wells. 10 uL of 5 uM 25-hydroxycholesterol in 25% DMSO/75% Assay Buffer was added to NSB wells. 20 uL of 15 nM 25-[3H]hydroxycholesterol prepared in Assay Buffer was added to all wells. 20 uL of 1.5 ug/mL RORc receptor was added to wells (or 40 uL Assay Buffer to No R wells). Following addition to the wells, the plates were incubated 3 h at 25° C. Filtration
Measurement: IC50 = 7.6 nM (pChEMBL 8.119999885559082)
Descriptors: MW 489.53, ALogP 4.99, HBA 7, HBD 1, PSA 97.82, QED 0.42
Compound (SMILES): C[C@H]1CCCN1C(=O)c1nc(C(=O)NCC(C)(C)O)sc1-c1cnc(N[C@H]2CCCCC2(F)F)cc1C(F)(F)F
InChIKey: UNIONGBHINDNNE-YOEHRIQHSA-N
Target: Nuclear receptor ROR-gamma (SINGLE PROTEIN), Homo sapiens
UniProt: P51449
Assay: ThermoFluor Assay: 1,8-ANS (Invitrogen) was used as a fluorescent dye. Protein and compound solutions are dispensed into black 384-well polypropylene PCR microplates (Abgene) and overlayed with silicone oil (1 μL, Fluka, type DC 200) to prevent evaporation.Bar-coded assay plates are robotically loaded onto a thermostatically controlled PCR-type thermal block and then heated at a typical ramp-rate of 1° C./min for all experiments. Fluorescence was measured by continuous illumination with UV light (Hamamatsu LC6) supplied via fiber optic and filtered through a band-pass filter (380-400 nm; >6 OD cutoff). Fluorescence emission of the entire 384-well plate was detected by measuring light intensity using a CCD camera (Sensys, Roper Scientific) filtered to detect 500±25 nm, resulting in simultaneous and independent readings of all 384 wells. Images were collected at each temperature, and the sum of the pixel intensity in a given area of the assay plate was recorded versus temperature. Reference wells contained RORγt without compounds, and the assay conditions were as follows:    0.065 mg/mL RORγt    60 μM 1,8-ANS    100 mM Hepes, pH 7.0    10 mM NaCl    2.5 mM GSH    0.002% Tween-20
Measurement: Kd = 1.7 nM (pChEMBL 8.770000457763672)
Descriptors: MW 589.63, ALogP 5.34, HBA 7, HBD 3, PSA 107.45, QED 0.38
Compound (SMILES): CCCCn1cc(-c2cccc(C(=O)N(C)C)c2)c2cc(C)[nH]c2c1=O
InChIKey: KZIMSPZDXKGJPU-UHFFFAOYSA-N
Target: Bromodomain-containing protein 4 (SINGLE PROTEIN), Homo sapiens
UniProt: O60885
Assay: IC50 Measurements for Inhibitors Using BRD4 AlphaLisa Binding Assay: His/Flag epitope tagged BRD4 BD142-168 was cloned, expressed, and purified to homogeneity. BRD4 binding and inhibition was assessed by monitoring the engagement of biotinylated H4-tetraacetyl peptide (New England Peptide, NEP2069-1/13) with the target using the AlphaLisa technology (Perkin-Elmer). Specifically, in a 384 well ProxiPlate BRD4(BD1) (30 nM final) was combined with peptide (200 nM final) in 40 mM HEPES (pH 7.0), 40 mM NaCl, 1 mM DTT, 0.01% (w/v) BSA, and 0.008% (w/v) Brij-35 either in the presence of DMSO (final 1.2% DMSO) or compound dilution series in DMSO. After 20 minutes incubation at room temperature Alpha streptavidin donor beads and AlphaLisa anti-Flag acceptor beads were added to a final concentration of 10 ug/mL each. After three hours equilibration plates were read on an Envision instrument and IC50s calculated using a four parameter non-linear curve fit.
Measurement: IC50 = 2300 nM (pChEMBL 5.639999866485596)
Descriptors: MW 351.45, ALogP 3.81, HBA 3, HBD 1, PSA 58.1, QED 0.76
Compound (SMILES): CCc1nc2cc(C(=O)NC)ccc2n1-c1ccc2c(c1)CCC(=O)N2
InChIKey: ODJADIRVHYABHQ-UHFFFAOYSA-N
Target: Protein cereblon (SINGLE PROTEIN), Homo sapiens
UniProt: Q96SW2
Assay: Â Fluorescence Direct Binding Protocol: Process 1) Optimization of measurement parameters to minimize protein consumption and to minimize the dilution effect and the DMSO content 2) Titration measurements of the protein against ligand by at least 12 titration steps to obtain a good s-curve fit 3) Repeat the same titration measurements with the ligand alone to enable correction 4) Check the stability of the protein once by titration against DMSO alone 5) Determination of the molar extinction coefficients of the ligand at 280 and 340 nm with help of a UV-spectrophotometer 6) Use Excel template for the correction of the measured raw data 7) Use GraphPad Prism software for the quadratic binding fit and the Kd evaluation.
Measurement: Kd = 190 nM (pChEMBL 6.71999979019165)
Descriptors: MW 348.41, ALogP 2.83, HBA 4, HBD 2, PSA 76.02, QED 0.76
Compound (SMILES): c1cc(-c2cnn3cc(-c4ccc(OCCN5CCNCC5)cn4)cnc23)c2cccnc2c1
InChIKey: ULQWBLKUEAKIFZ-UHFFFAOYSA-N
Target: Activin receptor type-1-like (SINGLE PROTEIN), Homo sapiens
UniProt: P37023
Assay: Enzyme Assay: Compounds were assessed in ALK1-6 enzymatic assays. Specifically, compounds were assayed using LANCE® Ultra ULight technology (Perkin Elmer) against human ALK1-6 enzymes (ALK1: Life Technologies, ALK2-6: Carna Biosciences). Briefly, ALK enzyme (10 nM) was prepared in kinase buffer (50 mM HEPES, pH 7.5, 10 mM MgCl2, 3 mM MgCl2, 0.005% Tween-20 and 2 mM DTT) and dispensed at 2.5 μL/well into a 1536 well, white, solid bottom, microtiter plate (Greiner, 789175-F). Negative controls for the assay were generated by adding one column containing kinase buffer only. Compounds in DMSO solution were transferred to the assay plates at 23 nL/well via a NX-TR pin tool workstation (WAKO, San Diego, Calif.) and incubated with enzyme for 10 minutes at ambient temperature. ULight Topo IIa Substrate (50 nM) was prepared in kinase buffer containing either 10 μM or 1000 μM ATP and dispensed at 2.5 μL/well into the assay plate. Following a 1 hour incubation at ambient temperature, Europium anti-phospho DNA Topoisomerase 2-alpha antibody (4 nM) was prepared in 1× LANCE detection buffer containing 12 mM EDTA and dispensed at 5 μL/well into the assay plate. Plates were measured using the EnVision plate reader (Perkin Elmer), with excitation 320 nm and emissions of 615 nm and 665 nm.
Measurement: IC50 = 341 nM (pChEMBL 6.46999979019165)
Descriptors: MW 451.53, ALogP 3.29, HBA 8, HBD 1, PSA 80.47, QED 0.42
Compound (SMILES): O=C(Nc1cc(S(=O)(=O)NCC(F)(F)F)ccc1-c1ccccc1)NC1CCCCC1
InChIKey: BMIZABGBVMXEEG-UHFFFAOYSA-N
Target: Isocitrate dehydrogenase [NADP], mitochondrial (SINGLE PROTEIN), Homo sapiens
UniProt: P48735
Assay: IDH2 Enzymatic Assay: Compounds were assayed for IDH2 R140Q inhibitory activity through a cofactor depletion assay. Compounds were preincubated with enzyme, then the reaction was started by the addition of NADPH and α-KG, and allowed to proceed for 60 minutes under conditions previously demonstrated to be linear with respect for time for consumption of both cofactor and substrate. The reaction was terminated by the addition of a second enzyme, diaphorase, and a corresponding substrate, resazurin. Diaphorase reduces resazurin to the highly fluorescent resorufin with the concomitant oxidation of NADPH to NADP, both halting the IDH2 reaction by depleting the available cofactor pool and facilitating quantitation of the amount of cofactor remaining after a specific time period through quantitative production of an easily detected fluorophore.Specifically, into each of 12 wells of a 384-well plate, 1 μl of compound dilution series was placed, followed by the addition of 40 μl of buffer (50 mM potassium phosphate, pH 7.5; 150 mM NaCl; 10 mM MgCl2, 10% glycerol, 0.05% bovine serum albumin, 2 mM beta-mercaptoethanol) containing 1.25 μg/ml IDH2 R140Q. The compound was then incubated for one hour at room temperature with the enzyme; before starting the IDH2 reaction with the addition of 10 μl of substrate mix containing 50 μM NADPH and 6.3 mM α-KG in the buffer described above. After a further one hour of incubation at room temperature, the reaction was halted and the remaining NADPH measured through conversion of resazurin to resorufin by the addition of 25 μl Stop Mix (36 μg/ml diaphorase enzyme and 60 μM resazurin; in buffer). After one minute of incubation the plate was read on a plate reader at Ex544/Em590.
Measurement: IC50 = 550 nM (pChEMBL 6.260000228881836)
Descriptors: MW 455.5, ALogP 4.65, HBA 3, HBD 3, PSA 87.3, QED 0.59
Compound (SMILES): CC1CN(c2ncnc3c(Sc4ccccc4)c(N)[nH]c23)CCO1
InChIKey: QHBLTWKEMHEMGT-UHFFFAOYSA-N
Target: Free fatty acid receptor 2 (SINGLE PROTEIN), Homo sapiens
UniProt: O15552
Assay: Ca-Flux Functional Assay: GPR43 agonist/PAM activity was determined by measuring changes in intracellular calcium levels using a Ca2+ sensitive fluorescent dye. The changes in fluorescent signal were monitored by FLIPR (manufactured by Molecular Devices). GPR43 mediated increases in intracellular Ca2+ concentration were readily detected upon activation with sodium acetate. Prior to the assay (24 hours), CHO-K1 Gα16 cells stably expressing human GPR43 were-seeded in cell culture medium in black, clear-bottom 384-well plates (Corning Inc) and grown overnight at 37° C., 5% CO2. On the day of the assay, cell culture media was removed and cells were loaded with Calcium 5 Dye (Molecular Devices) diluted in HBSS containing 25 mM HEPES, 2.5 mM Probenecid, 0.1% BSA for 1 hour at 37° C., 5% CO2. 10 point half log concentration response curves of sodium acetate from 10 mM were conducted prior to the testing of compounds to calculate the sodium acetate concentration that produces 20% of the maximal response (EC20). Test compounds (at 10 point half log concentration response curves from 10 μM) were added in the presence of sodium acetate to achieve a final concentration that produces approximately 20% maximal response as calculated from the previous experiment. The changes in fluorescent signal were monitored by FLIPR upon addition of the compound/EC20 sodium acetate mix. The EC50 values were determined from ten point concentration response curves. Curves were generated using the average of two wells for each data point.
Measurement: EC50 = 2 nM (pChEMBL 8.699999809265137)
Descriptors: MW 341.44, ALogP 2.92, HBA 6, HBD 2, PSA 80.06, QED 0.76
Compound (SMILES): CCN1CCN(c2ccc(Nc3ncc(F)c(-c4sc5c(C6CCCC6)ccnc5c4Cl)n3)nc2)CC1
InChIKey: FITODFIOLMCESC-UHFFFAOYSA-N
Target: Cyclin-dependent kinase 4/cyclin D1 (PROTEIN COMPLEX), Homo sapiens
UniProt: P24385
Assay: Enzymatic Activity Assay: The inhibitory activity of compounds was evaluated in vitro using TR-FRET assay with white 384-well low volume microplate (Greiner Bio-One). CDK4/Cyclin D1 catalyzed phosphorylation of peptide in the presence and absence of compounds was measured. IC50 determination. Recombinant protein complex CDK4/Cyclin D1, expressed from insect cell, was purchased from ProQinase. Testing compounds were dissolved in DMSO at 0.1 mM and tested in 9-dose IC50 mode. The reaction mixture was prepared by mixing CDK4/CyclinD1 (1 nM final), ULight-4E-BP1 (100 nM final, Perkinelmer, TRF0128-D), and ATP (2 mM final) in assay buffer (20 mM of HEPES pH 7.4, 1 mM of EGTA, 0.05% BSA, 0.005% Tween 20, and 1 mM TCEP). The compound of interest in DMSO was added to each well in 3-fold serial dilution by dispenser (TECAN D300E). After 20 minutes preincubation at room temperature, MgCl2(10 mM final) was added to initiate the reaction. Following a 60 minutes incubation at 37° C., the reaction was stopped by addition of 2 μL of quenching buffer consisting of Lance detection buffer (Perkinelmer CR97-100C), 2 nM LANCE Ultra Europium-anti-P-4E-BP1 (Perkinelmer, TRF0216-D), 10 mM EDTA, and incubate at room temperature for additional 60 minutes in dark. The reaction signal was measured by Envision multimode plate reader (PerkinElmer, 2102-0010).
Measurement: IC50 < 20 nM (pChEMBL 7.698999881744385)
Descriptors: MW 538.1, ALogP 6.48, HBA 8, HBD 1, PSA 70.07, QED 0.3
Compound (SMILES): O=C(c1cc(Cl)cc(-c2ccc(CO)cc2)c1)N1CCN(c2nc3ccccc3c(=O)[nH]2)CC1
InChIKey: UWNGWKVJQFQGOP-UHFFFAOYSA-N
Target: Monoglyceride lipase (SINGLE PROTEIN), Homo sapiens
UniProt: Q99685
Assay: MAGL Inhibitory Activity: Compounds of the present invention are MAGL inhibitors. Thus, in one aspect, the present invention provides the use of compounds of Formula (I) or a pharmaceutically acceptable salt thereof as described herein for inhibiting MAGL in a mammal.In a further aspect, the present invention provides compounds of Formula (I) or a pharmaceutically acceptable salt thereof as described herein for use in a method of inhibiting MAGL in a mammal.In a further aspect, the present invention provides the use of compounds of Formula (I) or a pharmaceutically acceptable salt thereof as described herein for the preparation of a medicament for inhibiting MAGL in a mammal.In a further aspect, the present invention provides a method for inhibiting MAGL in a mammal, which method comprises administering an effective amount of a compound or a pharmaceutically acceptable salt thereof of Formula (I) as described herein to the mammal.Compounds were profiled for MAGL inhibitory activity by measuring the enzymatic activity of MAGL by following the hydrolysis of 4-nitorphenylacetate resulting in 4-nitrophenol, which absorbs at 405-412 nm (G. G. Muccioli, G. Labar, D. M. Lambert, Chem. Bio. Chem. 2008, 9, 2704-2710).The assay was carried out in 384 well assay plates (black with clear bottom, non-binding surface treated, Corning Ref. 3655) in a total volume of 40 μL. Compound dilutions were made in 100% DMSO (VWR Chemicals 23500.297) in a polypropylene plate in 3-fold dilution steps to give a final concentration range in the assay from 25 μM to 1.7 nM. 1 μL compound dilutions (100% DMSO) were added to 19 μL MAGL (recombinant wild-type) in assay buffer (50 mM TRIS (GIBCO, 15567-027), 1 mM EDTA (Fluka, 03690-100 ml)). The plate was shaked for 1 min at 2000 rpm (Variomag Teleshake) and then incubated for 15 min at RT. To start the reaction, 20 μL 4-Nitrophenlyacetate (Sigma N-8130) in assay buffer with 6% EtOH was added. The final concentrations in the assay were 1 nM MAGL and 300 μM 4-Nitrophenylacetate. After shaking (1 min, 2000 rpm) and 5 min incubation at RT, the absorbance at 405 nm was measured for a fist time (Molecular Devices, SpectraMax Paradigm). A second measurement was then done after incubation for 80 min at RT. From the two measurements, the slope was calculated by subtracting the first from the second measurement.
Measurement: IC50 = 140 nM (pChEMBL 6.849999904632568)
Descriptors: MW 474.95, ALogP 3.7, HBA 5, HBD 2, PSA 89.53, QED 0.47
Compound (SMILES): O=C(CNC(=O)c1ccc2c(c1)OCc1cnccc1-2)c1cccc([N+](=O)[O-])c1
InChIKey: ARJVNTVHKCOBBR-UHFFFAOYSA-N
Target: Rho-associated protein kinase 2 (SINGLE PROTEIN), Homo sapiens
UniProt: O75116
Assay: In Vitro Assay: The effectiveness of compounds of the present invention as ROCK inhibitors can be determined in a 30 μL assay containing 20 mM HEPES, pH 7.5, 20 mM MgCl2, 0.015% Brij-35, 4 mM DTT, 5 μM ATP and 1.5 μM peptide substrate (FITC-AHA-AKRRRLSSLRA-OH). Compounds were dissolved in DMSO so that the final concentration of DMSO was <2%, and the reaction was initiated with Rho kinase variants. After incubation, the reaction was terminated by the addition of EDTA and the phosphorylated and non-phosphorylated peptides separated using a LABCHIP® 3000 Reader (Caliper Life Sciences). Controls consisted of assays that did not contain compound, and backgrounds consisted of assays that contained enzyme and substrate but had EDTA from the beginning of the reaction to inhibit kinase activity. Compounds were tested in dose-response format, and the inhibition of kinase activity was calculated at each concentration of compound. The inhibition data were fit using a curve-fitting program to determine the IC50; i.e., the concentration of compound required to inhibit 50% of kinase activity.
Measurement: IC50 = 1200 nM (pChEMBL 5.920000076293945)
Descriptors: MW 389.37, ALogP 3.16, HBA 6, HBD 1, PSA 111.43, QED 0.41
Compound (SMILES): CC(=O)c1cn(CC(=O)N2C[C@H](F)C[C@H]2C(=O)NC2CCCc3nc(-c4ccc(F)cc4)sc32)c2ccc(Nc3cncnc3)cc12
InChIKey: VZFSQLHHDAVXMK-YHRVPXSNSA-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-SBz1 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 > 10000 nM (pChEMBL 5)
Descriptors: MW 655.73, ALogP 5.77, HBA 9, HBD 2, PSA 122.11, QED 0.2
Compound (SMILES): CS(=O)(=O)c1ccc(-c2[nH]cc3c2CCCC3=O)cc1
InChIKey: DRAZPLJDOFGOIK-UHFFFAOYSA-N
Target: Bromodomain-containing protein 4 (SINGLE PROTEIN), Homo sapiens
UniProt: O60885
Assay: TR-FRET Assay: Compound dilution series were prepared in DMSO via a 3-fold serial dilution from 2.5 mM to 42 nM. Compounds were then diluted 6:100 in assay buffer (20 mM Sodium Phosphate, pH 6.0, 50 mM NaCl, 1 mM EDTA, 0.01% Triton X-100, 1 mM DTT) to yield 3× working solutions. Six microliters (μL) of the working solution was then transferred to white, low-volume assay plates (Costar #3673). A 1.5× assay mixture containing His-tagged bromodomain, Europium-conjugated anti-His antibody (Invitrogen PV5596) and the Alexa-647-conjugated probe molecule was also prepared. Twelve μL of this solution were added to the assay plate to reach a final volume of 18 μL. The final concentration of 1× assay buffer contains 2% DMSO, 50 μM−0.85 nM compound, 8 nM bromodomain, 1 nM antibody and 100 or 30 nM probe (for BDI or BDII, respectively). After a one-hour incubation at room temperature, TR-FRET ratios were determined using an Envision multilabel plate reader (Ex 340, Em 495/520).TR-FRET data were normalized to the means of 24 no-compound controls (high) and 8 controls containing 1 μM un-labeled probe (low). Percent inhibition was plotted as a function of compound concentration and the data were fit with the 4 parameter logistic equation to obtain IC50s Inhibition constants (Ki) were calculated from the IC50s, probe Kd and probe concentration. Typical Z′ values were between 0.65 and 0.75. The minimum significant ratio was determined to evaluate assay reproducibility (Eastwood et al., (2006) J Biomol Screen, 11: 253-261).
Measurement: Ki = 1430 nM (pChEMBL 5.840000152587891)
Descriptors: MW 289.36, ALogP 2.6, HBA 3, HBD 1, PSA 67, QED 0.92
Compound (SMILES): CC(C)Nc1cc(-n2ncc3cc(C#N)cnc32)ncc1-c1cn(C2CN(CC(F)F)C2)nn1
InChIKey: MKNBSVXIVJQKEE-UHFFFAOYSA-N
Target: Interleukin-1 receptor-associated kinase 4 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9NWZ3
Assay: IRAK4 Inhibition Assay: The assays were performed in U-bottom 384-well plates. The final assay volume was 30 μL prepared from 15 μL additions of enzyme and substrates (fluoresceinated peptide and ATP) and test compounds in assay buffer (20 mM HEPES pH 7.2, 10 mM MgCl2, 0.015% Brij 35 and 4 mM DTT). The reaction was initiated by the combination of IRAK4 with substrates and test compounds. The reaction mixture was incubated at room temperature for 60 min. and terminated by adding 45 μL of 35 mM EDTA to each sample. The reaction mixture was analyzed on the Caliper LABCHIP® 3000 (Caliper, Hopkinton, Mass.) by electrophoretic separation of the fluorescent substrate and phosphorylated product. Inhibition data were calculated by comparison to no enzyme control reactions for 100% inhibition and vehicle-only reactions for 0% inhibition. The final concentrations of reagents in the assays are ATP, 500 μM; FL-IPTSPITTTYFFFKKK peptide 1.5 μM; IRAK4, 0.6 nM; and DMSO, 1.6%.
Measurement: IC50 = 2 nM (pChEMBL 8.699999809265137)
Descriptors: MW 464.48, ALogP 2.89, HBA 10, HBD 1, PSA 113.37, QED 0.44
Compound (SMILES): O=C(O)c1ccc(-c2nn(C(=O)c3c(Cl)cccc3C3(C(F)(F)F)CC3)c3c2CCC(C(=O)N2CCOC4(CC4)C2)C3)c(F)c1
InChIKey: LSZKJEPPFPLUQW-UHFFFAOYSA-N
Target: Nuclear receptor ROR-gamma (SINGLE PROTEIN), Homo sapiens
UniProt: P51449
Assay: Biological Assay: The compounds of the invention inhibit RORgammaT activity. Activation of RORgammaT activity can be measured using, e.g., biochemical TR-FRET assay. In such an assay, interaction of cofactor-derived peptides with human RORgammaT-Ligand Binding Domain (LBD) can be measured. The TR-FRET technique is a sensitive biochemical proximity assay that will give information concerning the interaction of a ligand with the LBD, in the presence of cofactor-derived peptides (Zhou et al., Methods 25:54-61, 2001).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 (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 mg/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 ml 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 ml SF9 lysate per 15 ml of diluted RORγ-LBD.Compounds to be tested were injected to the 384-well assay plate using Acoustic Droplet Ejection technology by Echo 550 liquid handler (Labcyte, CA).A stock of biotinylated-LXXLL peptide from coactivator SRC1 (Biotin-SPSSHSSLTERHKILHRLLQEGSP) (SEQ ID NO:1) and APC-conjugated streptavidin (final concentrations 100 nM and 8 nM respectively) were also added to each well.
Measurement: IC50 = 1.2 nM (pChEMBL 8.920000076293945)
Descriptors: MW 646.04, ALogP 5.82, HBA 6, HBD 1, PSA 101.73, QED 0.35
Compound (SMILES): NC(=O)c1cnc(N[C@@H]2CCCC[C@@H]2N)nc1Nc1cccc(Sc2ccc(O)cc2)c1
InChIKey: LOQMHCLXTRAJKX-VQTJNVASSA-N
Target: Tyrosine-protein kinase SYK (SINGLE PROTEIN), Homo sapiens
UniProt: P43405
Assay: ADP-Glo Kinase Assay: The Promega ADP-Glo Kinase Assay is a luminescent ADP detection assay that provides a universal, homogeneous, high-throughput screening method to measure kinase activity by quantifying the amount of ADP produced during a kinase reaction.
Measurement: IC50 = 74.5 nM (pChEMBL 7.130000114440918)
Descriptors: MW 450.57, ALogP 3.86, HBA 8, HBD 5, PSA 139.18, QED 0.37
Compound (SMILES): Cc1ccc(-n2nccn2)c(C(=O)N2CCC[C@@]2(C)c2nc3cc(C(F)(F)F)c(Cl)cc3[nH]2)c1
InChIKey: JVWVAASSQXQBNF-QFIPXVFZSA-N
Target: Orexin/Hypocretin receptor type 1 (SINGLE PROTEIN), Homo sapiens
UniProt: O43613
Assay: Inhibition Assay: Chinese hamster ovary (CHO) cells expressing the human orexin-1 receptor and the human orexin-2 receptor, respectively, are grown in culture medium (Ham F-12 with L-Glutamine) containing 300 μg/ml G418, 100 U/ml penicillin, 100 μg/ml streptomycin and 10% heat inactivated fetal calf serum (FCS). The cells are seeded at 20,000 cells/well into 384-well black clear bottom sterile plates (Greiner). The seeded plates are incubated overnight at 37° C. in 5% CO2.Human orexin-A as an agonist is prepared as 1 mM stock solution in MeOH: water (1:1), diluted in HBSS containing 0.1% bovine serum albumin (BSA), NaHCO3: 0.375 g/l and 20 mM HEPES for use in the assay at a final concentration of 3 nM.Antagonists 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 in HBSS containing 0.1% bovine serum albumin (BSA), NaHCO3: 0.375 g/l and 20 mM HEPES. On the day of the assay, 50 μl of staining buffer (HBSS containing 1% FCS, 20 mM HEPES, NaHCO3: 0.375 g/l, 5 mM probenecid (Sigma) and 3 M of the fluorescent calcium indicator fluo-4 AM (1 mM stock solution in DMSO, containing 10% pluronic) is added to each well. The 384-well cell-plates are incubated for 50 min at 37° C. in 5% CO2 followed by equilibration at RT for 30 min before measurement.
Measurement: IC50 = 2 nM (pChEMBL 8.699999809265137)
Descriptors: MW 488.9, ALogP 5.28, HBA 5, HBD 1, PSA 79.7, QED 0.43
Compound (SMILES): Cc1ccc(-n2nccn2)c(C(=O)N2CC3CC(Oc4ccc(C(F)F)cn4)C2C3)n1
InChIKey: FPVWQZKZLYHJBA-UHFFFAOYSA-N
Target: Orexin/Hypocretin receptor type 1 (SINGLE PROTEIN), Homo sapiens
UniProt: O43613
Assay: Human Orexin 1 Receptor Ca2+ Mobilization Assay: CHO cells stably transfected with the human orexin 1 receptor (Genebank accession number NM_001526) were grown to confluency in DMEM/F12, 10% FBS, 1×pen-strep, 400 μg/ml G418. Cells were seeded on to 384-well Packard viewplates at a density of 10,000 cells/well and incubated overnight at 37° C., 5% CO2. The cells were dye-loaded with BD Calcium Assay kit (BD, cat #640178) in HBSS (Gibco, cat #14025-092) with 2.5 mM probenecid and incubated at 37° C., 5% CO2 for 45 min. Cells were pre-incubated with compounds (diluted in DMEM/F-12) for 15-30 minutes before agonist (orexin A, 10 nM) stimulation. Ligand-induced Ca2+ release was measured using a Fluorometric Imaging Plate Reader (FLIPR, Molecular Devices, Sunnyvale, Calif.). Functional responses were measured as peak fluorescence intensity minus basal. The concentration of agonist that produced a half-maximal response is represented by the EC50 value. Antagonistic potency values were converted to apparent pKB values using a modified Cheng-Prusoff correction. Apparent pKB=−log IC50/1+[conc agonist/EC50].
Measurement: Ki = 30 nM (pChEMBL 7.519999980926514)
Descriptors: MW 426.43, ALogP 2.99, HBA 7, HBD 0, PSA 86.03, QED 0.62
Compound (SMILES): O=C(c1cccc(Oc2cc(F)cc(F)c2)c1Cl)N1CCN2C[C@@H](c3ccc(Cl)cc3)CC[C@@H]2C1
InChIKey: CXDHTFUSVWYGGB-PGRDOPGGSA-N
Target: Monoglyceride lipase (SINGLE PROTEIN), Homo sapiens
UniProt: Q99685
Assay: Inhibition Assay: In one embodiment, the present invention provides compounds of formula (Ie) and their pharmaceutically acceptable salts as described herein, wherein said compounds of formula (Ie) and their pharmaceutically acceptable salts have an IC50 for MAGL below 25 μM, preferably below 10 μM, more preferably below 5 μM as measured in an assay comprising the steps of: list-style a) providing a solution of a compound formula (e), or a pharmaceutically acceptable salt thereof, in DMSO; b) providing a solution of MAGL (recombinant wild-type) in assay buffer (50 mM tris(hydroxymethyl)aminomethane; 1 mM ethylenediaminetetraacetic acid); c) adding 1 μL of compound solution from step a) to 19 μL of MAGL solution from step b); d) shaking the mixture for 1 min at 2000 rpm; e) incubating for 15 min at RT; f) adding 20 μL of a solution of 4-nitrophenlyacetate in assay buffer (50 mM tris(hydroxymethyl)aminomethane; 1 mM ethylenediaminetetraacetic acid, 6% EtOH); g) shaking the mixture for 1 min at 2000 rpm;h) incubating for 5 min at RT; i) measuring the absorbance of the mixture at 405 nm a first time; j) incubating a further 80 min at RT;k) measuring the absorbance of the mixture at 405 nm a second time; l) substracting the absorbance measured under i) from the absorbance measured under k) and calculating the slope of absorbance; wherein: i) the concentration of the compound of formula (Ie), or the pharmaceutically acceptable salt thereof in the assay after step f) is in the range of 25 μM to 1.7 nM; ii) the concentration of MAGL in the assay after step f) is 1 nM; >iii) the concentration of 4-nitrophenylacetate in the assay after step f) is 300 μM; and iv) steps a) to 1) are repeated for at least 3 times, each time with a different concentration of the compound of formula (Ie), or the pharmaceutically acceptable salt thereof.
Measurement: IC50 = 3960 nM (pChEMBL 5.400000095367432)
Descriptors: MW 517.4, ALogP 6.77, HBA 3, HBD 0, PSA 32.78, QED 0.38
Compound (SMILES): CN(C)[C@H]1CC[C@H](Oc2ccn(-c3cc(-c4c(F)cccc4C#N)nc4c3C(=O)NC4)n2)CC1
InChIKey: YHLGKVZOIAECOR-QAQDUYKDSA-N
Target: Non-receptor tyrosine-protein kinase TYK2 (SINGLE PROTEIN), Homo sapiens
UniProt: P29597
Assay: Caliper Assay: The caliper machine employs an off chip mobility shift assay to detect phosphorylated peptide substrates from kinase assays, using microfluidics technology. The assays are carried out at ATP concentration equivalent to the ATP Km, and at 1 mM ATP. Compounds are serially diluted in DMSO then further diluted in assay buffer (25 mM HEPES, pH 7.5, 0.01% Brij-35, 0.01% Triton, 0.5 mM EGTA). 5 ul of diluted compound was added into wells first, then 10 ul of enzyme mix was added into wells, followed by 10 uL of substrate mix (peptide and ATP in 10 mM MgCl2) to start reaction. Reaction was incubated at 28° C. for 25 min and then added 25 ul stop buffer (100 mM HEPES, 0.015% Brij-35, 50 mM EDTA), followed by reading with Caliper. JAK2 at 1 nM final concentration and TYK2 at 9.75 nM are from Carna, and substrates used are ATP at 20 and 16 uM, respectively. JAK2 assay uses peptide 22 and TYK2 uses peptide 30 (Caliper), each at 3 uM.
Measurement: Ki = 5 nM (pChEMBL 8.300000190734863)
Descriptors: MW 460.51, ALogP 3.44, HBA 7, HBD 1, PSA 96.07, QED 0.63
Compound (SMILES): N#Cc1ccc2ncc(-c3nccc(N4CCOC(c5cn[nH]c5C(F)F)C4)n3)n2c1
InChIKey: NRLMXTOUZLCRJG-UHFFFAOYSA-N
Target: eIF-2-alpha kinase GCN2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9P2K8
Assay: Enzyme Inhibition Assay: An assay buffer containing GCN2 kinase and (RS)7 was prepared. 4.7 μL of this stock solution was placed per well of a black, low volume, 384-well microtitre plate (e.g. catalogue number 3676, Corning Inc., NY). To this was added 0.65 μM of DMSO containing serial dilutions of the test compound (typical final concentrations of test compound were 0 to 8 μM). The plate was incubated for 10 minutes at 25° C. prior to the addition of 4.7 μL of ATP stock buffer to initiate the enzyme reaction. The reaction was allowed to proceed for 1 hour at 25° C., prior to the addition of 10 μL detection buffer (consisting of appropriate concentrations of ADP2 antibody and ADP Alexa633 tracer in 1× stop and detect buffer as supplied by BellBrook Labs). The reaction was left to incubate for 1 hour at 25° C., prior to measuring the fluorescence polarisation signal (mP) in each well using a PHERAstar FS reader (BMG Labtech, Germany).
Measurement: IC50 < 10 nM (pChEMBL 8)
Descriptors: MW 422.4, ALogP 2.9, HBA 8, HBD 1, PSA 108.02, QED 0.54
Compound (SMILES): Cc1noc(C)c1-c1cnc2c3ccc(C(=O)O)cc3n(C(c3ccccc3)c3ccccc3)c2c1
InChIKey: BKKAWPITIHYVHK-UHFFFAOYSA-N
Target: Bromodomain-containing protein 4 (SINGLE PROTEIN), Homo sapiens
UniProt: O60885
Assay: TR-FRET Assay: All assay components were dissolved in buffer composition 20 mM Hepes pH 7.5, 150 mM NaCl, 5 mM DTT, 0.005% Tween 20, and 100 ug/ml BSA for BRD4 (1-477 and 44-460). The final concentrations of the bromodomain proteins are 1.6 nM BRD4(44-168), 1 nM BRD4(333-460), and 1 nM BRD4(1-477 or 44-460), and the fluorescent probe molecule is 100 nM, 50 nM, and 7.5 nM respectively. All proteins were biotinylated. A streptavidin labeled with terbium cryptate (Cisbio SA-Tb) was used as detection, and pre-mixed with the bromodomain protein at a final concentration of 0.2 nM. In some instances for BRD4 (44-460), anti-His terbium cryptate was used as a detection. 7.5 nl of dose-responsed test compound or dmso vehicle (0.0375%) was pre-spotted in a black Corning 384 well plate and 10 ul each of bromodomain/detection reagent and fluorescent small molecule solution were added to the plate, and the reaction incubated for 60 min at room temperature. Plates were then read on EnVision plate reader, (λex=340 nm, acceptor λEm=520 nm, and donor λEm=615 nm, LANCE D400 mirror). Time resolved fluorescence intensity measurements were made at both emissions, and the ratio of acceptor/donor was calculated and used for data analysis.
Measurement: IC50 < 5 nM (pChEMBL 8.300999641418457)
Descriptors: MW 473.53, ALogP 6.8, HBA 5, HBD 1, PSA 81.15, QED 0.3
Compound (SMILES): Cn1ccc2c([C@H](Nc3cc(C#N)c4ncc(C#N)c(NCC(C)(C)C)c4c3)C3=CN(C4(C)CC4)NN3)cccc2c1=O
InChIKey: VREQCXFFVBBTLM-PMERELPUSA-N
Target: Epidermal growth factor receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P00533
Assay: High Throughput Biochemical Assay: EGFR activity was measured using KinEASE (Cisbio), a time-resolved fluorescence resonance energy transfer (TR-FRET) immunoassay. In this assay, EGFR-catalyzes the phosphorylation of a universal Tyrosine kinase peptide substrate labeled with XL665. Europium conjugated phosphor-tyrosine specific antibody binds the resulting phosphorylated peptide. Formation of phosphorylated peptide is quantified by TR-FRET with Europium as the donor and XL665 the acceptor. The assay was performed in two main steps. The first step is the kinase reaction step and the second step is the detection step with TR-FRET reagents. In brief, test compounds 1:3 serially diluted in DMSO were delivered into Corning white, low volume, non-binding 384 well plates using the Echo 550 acoustic liquid dispenser (Labcyte®). EGFR enzyme (Human EGFR, cytoplasmic domain [669-1210] from Carna Biosciences Cat. No. 08-115) and substrates TK substrate-biotin (included in Cisbio HTRF KinEASE-TK kit Cat. No. 62TK0PEJ) were dispensed into assay plates using a Multi-Flo (Bio-Tek Instruments). The standard 10 μL reaction mixture contained 6 μM ATP (1×Km) or 12 μM ATP (2×Km), 1 μM biotinylated peptide, 0.3 nM EGFR (for 1×Km ATP) or 0.1 nM EGFR (for 2×Km ATP) in reaction buffer (10 mM MOPs, pH 7.0, 1.5% Glycerol, 0.5 mg/ml BSA, 10 mM Mg-Acetate, 1 mM DTT, 0.025% NP-40). After 60 min of incubation at room temperature, 10 μL of Stop and Detect Solution (1:400 Europium Cryptate labeled anti-phosphorylated peptide antibody solution and 125 nM strepavidin-XL665 Tracer in a 50 mM Hepes pH 7.0 detection buffer containing sufficient EDTA) was added. The plate was then further incubated for over 60 minutes at room temperature and read using an Envision 2103 Multilabeled reader (PerkinElmer) with excitation/emission/FRET emission at 340 nm/615 nm/665 nm, respectively.
Measurement: IC50 = 1 nM (pChEMBL 9)
Descriptors: MW 573.71, ALogP 5.16, HBA 10, HBD 4, PSA 133.83, QED 0.24
Compound (SMILES): CC(C)Oc1cc2c(OC[C@@H]3C[C@](F)(CO)C(=O)N3)nccc2cc1C(N)=O
InChIKey: WEXWXNYZUDCSRT-BUXKBTBVSA-N
Target: Interleukin-1 receptor-associated kinase 4 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9NWZ3
Assay: IRAK4 Enzymatic DELFIA Assay, Protocol A: This is an in vitro assay to measure IRAK4 enzymatic activity utilizing the DELFIA (Dissociation-Enhanced Lanthanide Fluorescent Immunoassay, Perkin-Elmer) platform, with the human IRAK4 FL (Full Length) construct to characterize IRAK4 inhibitor and control compounds at 0.6 mM ATP (KM). The final amount of enzyme in the assay is 0.1 nM IRAK4 FL, final concentration of substrate is 50 nM, and final concentration of DMSO is 2.5%.The test compound was solubilized in DMSO to a stock concentration of 30 mM. The dose response plates were prepared with a 4 mM primary compound concentration, and then diluted in DMSO in a four-fold series for a total of 11 data points. Compounds were prepared as a 40-fold multiple of the final in-assay concentration.To begin the assay, 19 μL of reaction mixture containing 20 mM HEPES pH=7.5, 5 mM MgCl2, 0.0025% Brij-35, 600 μM ATP, 0.21 nM Full-length phosphorylated recombinant human IRAK4 (GenBank ID AF445802) were aliquoted into Ultra-Clear Polypropylene, 384-well, U-Bottom Plates (Corning Life Sciences). 1 μL of test compound from the dose-response plate was added to the reaction mixture and incubated for 20 minutes at room temperature. Then 20 μL of 20 mM HEPES pH=7.5, 5 mM MgCl2, 0.0025% Brij-35, 600 μM ATP, and 100 nM ERM-biotinylated peptide (AGAGRDKYKTLRQIR) was added to start the reaction. The reaction was incubated for 60 minutes at room temperature and stopped by the addition of 20 μL 0.3M EDTA.50 μL of the reaction mixture was transferred to a streptavidin coated detection plate (DELFIA streptavidin coated plates, 384-well, white plates, Perkin-Elmer Life Sciences) and incubated for 30 minutes at room temperature. The plates were washed 4× with 75 μL per well of PBS containing 0.05% Tween-20. Plates were then incubated with 50 μL per well of antibody cocktail of Anti-pERM antibody at 0.125 μg/mL (Cell Signaling Technology), plus Anti-Rabbit IgG EuN1 at 0.25 ug/ml (Perkin-Elmer Life Sciences) in a solution of 10 mM MOPS pH=7.5, 150 mM NaCl, 0.05% Tween-20, 0.02% NaN3, 1% BSA, 0.1% Gelatin for 45 minutes. The plates were washed 4× with 50 μL per well of PBS containing 0.05% Tween-20. Then 50 μL per well of DELFIA Enhancement Solution (Perkin-Elmer Life Sciences) were added to the plate and then read on an EnVision Model 2103 using a 340 nm excitation wavelength and a 665 nm emission wavelength for detection.
Measurement: IC50 = 1396 nM (pChEMBL 5.860000133514404)
Descriptors: MW 391.4, ALogP 1.09, HBA 6, HBD 3, PSA 123.77, QED 0.65
Compound (SMILES): CCOc1ncc(-c2cccc(N(CC34CCC(c5ccc(C6(C#N)CC6)cc5)(CC3)OC4)C(=O)C34CC(F)(C3)C4)c2)cn1
InChIKey: JGSMOICZRNLRAY-UHFFFAOYSA-N
Target: Bile acid receptor (SINGLE PROTEIN), Homo sapiens
UniProt: Q96RI1
Assay: transient human FXR/Gal4-luciferase reporter assay: A Gal4-hFXR fusion construct reporter system was used as the primary assay to characterize compound activity. A construct including 5 copies of the Gal4 promoter response element upstream of a firefly luciferase reporter cDNA was stably expressed in HEK293 cells. This reporter cell line was maintained in Dulbecco's Modified Eagle's medium (DMEM; Gibco) supplemented with 1% penicillin-streptomycin (P/S) solution, 500 μg/mL Zeocin and 10% charcoal/dextran-treated fetal bovine serum (cs-FBS) at 37° C. in a humidified 5% CO2 atmosphere. Another plasmid was constructed in which the human cytomegalovirus promoter in the pcDNA3.1 vector directs the expression of the cDNA encoding a fusion protein comprised of the DNA binding domain from the Gal4 transcription factor fused to the ligand binding domain from human FXR.The day prior to transfection, the reporter cells in culture are detached from the plate with trypsin and plated into a T75 flask at a sufficient density to achieve approximately 90% confluence the next morning. The transfection reagents are prepared by separately diluting 25 μg of the pcDNA3.1-Gal4-FXR plasmid into 1.87 mL of Opti-MEM (Thermo-Fisher), and 40 μL of Lipofectamine 2000 (Thermo-Fisher) into 1.87 mL of Opti-MEM, and then adding the diluted DNA solution into the diluted Lipofectamine 2000 solution and incubating at room temperature for 15-20 minutes. The mixture is further diluted with 10 mL of a solution comprised of DMEM, 10% cs-FBS, and 1% P/S immediately prior to transferring to the cells. The maintenance culture media is aspirated from the cells and the final transfection mixture is added before the cells are incubated overnight at 37° C. in a humidified 5% CO2 atmosphere. This protocol can be scaled up, and the transiently transfected cells can be cryopreserved in an assay-ready format.For compound testing, 100 nL of the compounds (serial dilutions in DMSO) are dispensed with an Echo acoustic dispenser (Labcyte) into the wells of a Corning/Costar clear bottom 384-well white plate. The transfected cells are harvested, counted, and diluted such that 10-25,000 cells in 25 μL are plated into each well of the 384-well compound assay plate. The compound-treated cells are incubated overnight at 37° C. in a humidified 5% CO2 atmosphere. The next morning 25 μL of Steady-Glo (Promega) are added to each well of the plate, the mixture is incubated for 15 min. with shaking, and luminescence is measured on an Envision (Perkin Elmer) plate reader. Background counts from cells treated with DMSO alone are subtracted from all raw counts, and the corrected values are converted to a percentage of the control response attained with 8 μM GW-4064. These data are fit to a 4-parameter log agonist-response equation to calculate an EC50 value.
Measurement: EC50 = 167 nM (pChEMBL 6.78000020980835)
Descriptors: MW 592.72, ALogP 6.81, HBA 6, HBD 0, PSA 88.34, QED 0.27
Compound (SMILES): O=C1Nc2c(Cl)cc3cc(C(=O)N(CCO)Cc4ccccc4)oc3c2C2(CCCCC2)N1
InChIKey: NSLGWJQMYSRMLG-UHFFFAOYSA-N
Target: 3',5'-cyclic-AMP phosphodiesterase 7B (SINGLE PROTEIN), Homo sapiens
UniProt: Q9NP56
Assay: Enzymatic Assay: PDE7 inhibition was determined by an IMAP TR-FRET assay using PDE7B. The IMAP TR-FRET PDE assay was optimized for concentration of enzyme, FAM-cAMP substrate, reducing agent, DMSO tolerance, and incubation time. First, PDE7 inhibitor compounds in 10 mM DMSO stock were serially diluted in 100% DMSO. Next, into each well of a solid white 1536 well plate (Corning) was dispensed 12.8 pg of N-terminal truncated recombinant human PDE7B enzyme (91-450aa, prep #DBVC-D04614, made in-house by structural biology) in 2.5 μL IMAP BSA reaction buffer (Molecular Devices, Sunnyvale, Calif.) containing 1 mM DTT (Sigma Aldrich.) After a brief centrifugation, 30 nL of serially diluted compounds in DMSO were added by transfer from 1 mM stock using a Kalypsys 1536 Pintool. Plates were incubated for 5 minutes at room temperature before dispensing 1.5 μL of 134 nM 5-carboxy fluorescein (FAM)-labeled cAMP (Molecular Devices, Sunnyvale, Calif.) for a final concentration of 50 nM. After a brief centrifugation, the plates were incubated for 15 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).
Measurement: IC50 = 55 nM (pChEMBL 7.260000228881836)
Descriptors: MW 467.95, ALogP 5.02, HBA 4, HBD 3, PSA 94.81, QED 0.49
Compound (SMILES): CC(C)[C@@H](NC(=O)c1cc(C2CC2)ccc1F)C(=O)N1CCC2(CC1)C(=O)N(C)C(=O)N2c1ccccc1
InChIKey: MZTDGOBVIVLPLI-XMMPIXPASA-N
Target: Autotaxin (SINGLE PROTEIN), Homo sapiens
UniProt: Q13822
Assay: Autotaxin LPC Cascade Assay: The efficacy of the Examples described herein, as inhibitors of ATX are demonstrated and confirmed by pharmacological in vitro assays. The following assays and their respective methods are carried out with the compounds according to the present invention. Activity possessed by the compounds may be demonstrated in vivo. Those skilled in the art will appreciate that a variety of assay formats may be used to determine the activity of the compounds of this invention.Autotaxin LPC Cascade Assay:Assay Buffer:100 mM Tris-HCl, pH=9500 mM NaCl5 mM MgCl25 mM CaCl20.05% Triton X 100Reagents:Enzyme Source Stock conc Working conc. Final conc. Autotaxin X-Chem 4.6 μM 20 nM 5 nM LPC Sigma L5254 8 mM in assay buffer 400 μM 100 μM Choline Oxidase Sigma C5896 50 U/mL in water 0.8 U/mL 0.1 U/mL HRP Sigma P8375 1000 U/mL in water 8 U/mL 1 U/mL Ampliflu Red Sigma 90101 100 mM in DMSO 400 μM 50 μM.
Measurement: IC50 < 500 nM (pChEMBL 6.301000118255615)
Descriptors: MW 520.61, ALogP 3.92, HBA 4, HBD 1, PSA 90.03, QED 0.59
Compound (SMILES): CC(=O)Nc1cc(-c2cncc(N3CCOc4cc(F)ccc4S3(=O)=O)c2)ccn1
InChIKey: WMCCWHWGHMIDFA-UHFFFAOYSA-N
Target: Phosphatidylinositol 3-kinase catalytic subunit type 3 (SINGLE PROTEIN), Homo sapiens
UniProt: Q8NEB9
Assay: VPS34 Enzyme Assays: 100 nL compounds in DMSO are added to wells of a 384 well microtitre plate (Greiner 780076). At room temperature: 5 ul VPS34 reaction buffer (Invitrogen Assay Buffer Q (diluted 1 in 5 with nanopure water) plus 2 mM DTT and 2 mM MnCl2) containing ATP (20 uM, Promega) and 200 uM PI-PS substrate (Invitrogen PV5122) is added followed immediately by 5 ul VPS34 reaction buffer (as above) containing VPS34 (5 nM, Millennium Protein Sciences Group) and the mixture is incubated with shaking at room temperature for 1 hour. Then 5 ul VPS34 stop-detect mix (as per Invitrogen Adapta Assay kit (PV5009) instructions (contains kinase quench buffer, TR-FRET buffer, Adapta Eu anti-ADP antibody and Alexa Fluor 647 ADP tracer)) is added to quench the reaction. The plates are then incubated for 30 minutes at room temperature with shaking and then read on a BMG PheraStar Plus reader.
Measurement: IC50 = 30 nM (pChEMBL 7.519999980926514)
Descriptors: MW 428.45, ALogP 2.83, HBA 6, HBD 1, PSA 101.49, QED 0.69
Compound (SMILES): C[C@H](CCc1ccc(OC(=O)N(C)C)c(OC(F)(F)F)c1)N1Cc2ccc(F)cc2C1
InChIKey: JWOJDKMHSKNBEA-CQSZACIVSA-N
Target: Sigma non-opioid intracellular receptor 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q99720
Assay: Radioligand Binding Assay: Radioligand binding assays for Sigma-1 receptors and Sigma-2 receptors were carried out by a commercial contract research organization. For Sigma-1 binding, various concentrations of test compounds from 100 μM to 1 nM were used to displace 8 nM [3H](+)pentazocine from endogenous receptors on Jurkat cell membranes (Ganapathy M E et al. 1991, J Pharmacol. Exp. Ther. 289:251-260). 10 μM Haloperidol was used to define non-specific binding. For Sigma-2 receptors various concentrations of test compounds from 100 μM to 1 nM were used to displace 5 nM [3H] 1,3-Di-(2-tolyl)guanidine from endogenous receptors on membranes from rat cerebral cortex in the presence of 300 nM (+)pentazocine to mask Sigma-1 receptors. (Bowen W D, et al. 1993, Mol. Neuropharmcol 3:117-126). 10 μM Haloperidol was used to define non-specific binding. Reactions were terminated by rapid filtration through Whatman GF/C filters using a Brandel 12R cell harvester followed by two washes with ice-cold buffer. Radioactivity on the dried filter discs was measured using a liquid scintillation analyzer (Tri-Carb 2900TR; PerkinElmer Life and Analytical Sciences). The displacement curves were plotted and the Ki values of the test ligands for the receptor subtypes were determined using GraphPad Prism (GraphPad Software Inc., San Diego, Calif.). The percentage specific binding was determined by dividing the difference between total bound (disintegrations per minute) and nonspecific bound (disintegrations per minute) by the total bound (disintegrations per minute).
Measurement: Ki = 380 nM (pChEMBL 6.420000076293945)
Descriptors: MW 440.44, ALogP 5.12, HBA 4, HBD 0, PSA 42.01, QED 0.58
Compound (SMILES): N[C@H]1c2cccnc2CC12CCN(c1ncc(Sc3cccc(Cl)c3Cl)c3nccn13)CC2
InChIKey: JVKMRWOADDUHNG-NRFANRHFSA-N
Target: Tyrosine-protein phosphatase non-receptor type 11 (SINGLE PROTEIN), Homo sapiens
UniProt: Q06124
Assay: Biochemical Assay: Inhibition of SHP-2 activity by small molecule inhibitors was monitored by a biochemical DiFMUP pseudosubstrate-fluorogenic assay. SHP-2 enzyme at final concentration of 0.2 nM was incubated with an IRS-1 activator peptide at a final concentration of 1 μM in SHP-2 assay buffer (60 mM HEPES, pH 7.2-7.5, 0.05% Tween-20, 75 mM NaCl, 75 mM KCl, 1 mM EDTA, and 5 mM DTT) for 75 min at room temperature. The reaction mix was added to the 384-well non-binding plate which had been pre-spotted with 1:3 serial diluted compounds from 10,000-0.005 nM, and incubated at room temperature for 90 min. DiFMUP substrate at final concentration of 50 μM was then added to the wells and incubated for additional 30 min at room temperature. The final reaction volume was 20 μl/well. The fluorescence intensity of the reaction product was measured by Envision at excitation and emission wavelengths of 340 nm and 450 nm, respectively. The inhibitor dose response curves were generated using normalized IC50 regression curve fitting by in house dose response analysis tools. The wells with DMSO vehicle were used as controls (100% signal). The percent (%) control of SHP-2 compound was calculated as: (Compound well fluorescence counts/DMSO fluorescence counts)×100%.
Measurement: IC50 = 41 nM (pChEMBL 7.389999866485596)
Descriptors: MW 497.46, ALogP 5.43, HBA 7, HBD 1, PSA 72.34, QED 0.41