lovingscience/chembl-v37-raw-activities huggingface dataset:
Compound (SMILES): COc1ccc(COc2ccc(Nc3ccc4ncc(N5CCNCC5)nc4c3C#N)cc2OC)cn1
InChIKey: XLNPHXDPVQICCU-UHFFFAOYSA-N
Target: Macrophage colony-stimulating factor 1 receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P07333
Assay: Kinase Assay: CSF-1R: Inhibition of CSF-1R kinase activity by a test compound disclosed herein was estimated by AlphaScreen (PerkinElmer). Standard assay conditions were 1.25 ng of recombinant CSF-1R kinase (SignalChem) with 10 ng Biotin-conjugated Poly-(Glu 4:Tyr 1) (Cisbio) in the assay buffer (10 μM ATP, 10 mM MOPs, pH7.0, 0.21 mM EDTA, 0.5% glycerol, 1 mg/ml BSA, 0.01% 2-mercaptoethanol, 0.001% Brij35) in a final volume of 25 μL. Reactions were incubated at 30° C. for 45 min and stopped by adding 5 μL of 50 mM EDTA. Analysis of resulting product using an AlphaScreen kit, and counted with Enspire Alpha (PerkinElmer). The readout from control reaction (complete reaction mixture) was designated as 0% inhibition and the readout for the reaction without enzyme as 100% inhibition. The IC50 values of quinoxaline compounds of this invention against CSF-1R kinase were determined using the GraphPad Prism 5 software.
Measurement: IC50 = 27.5 nM (pChEMBL 7.559999942779541)
Descriptors: MW 497.56, ALogP 3.65, HBA 10, HBD 2, PSA 117.45, QED 0.37
Compound (SMILES): Nc1nn2cc(F)cnc2c1C(=O)Nc1cnccc1-c1ccc(S(=O)(=O)N2CCC2)cc1
InChIKey: AVDXLGUZSCJCTG-UHFFFAOYSA-N
Target: Serine/threonine-protein kinase ATR (SINGLE PROTEIN), Homo sapiens
UniProt: Q13535
Assay: Inhibition Assay: Compounds were screened for their ability to inhibit ATR kinase using a radioactive-phosphate incorporation assay. Assays were carried out in a mixture of 50 mM Tris/HCl (pH 7.5), 10 mM MgCl2 and 1 mM DTT. Final substrate concentrations were 10 μM [γ-33P]ATP (3mCi 33P ATP/mmol ATP, Perkin Elmer) and 800 μM target peptide (ASELPASQPQPFSAKKK) (SEQ ID NO:1).Assays were carried out at 25° C. in the presence of 5 nM full-length ATR. An assay stock buffer solution was prepared containing all of the reagents listed above, with the exception of ATP and the test compound of interest. 13.5 μL of the stock solution was placed in a 96 well plate followed by addition of 2 μL of DMSO stock containing serial dilutions of the test compound (typically starting from a final concentration of 15 μM with 3-fold serial dilutions) in duplicate (final DMSO concentration 7%). The plate was pre-incubated for 10 minutes at 25° C. and the reaction initiated by addition of 15 μL [γ-33P]ATP (final concentration 10 μM).The reaction was stopped after 24 hours by the addition of 30 μL 0.1M phosphoric acid containing 2 mM ATP. A multiscreen phosphocellulose filter 96-well plate (Millipore, Cat no. MAPHN0B50) was pretreated with 100 μL 0.2M phosphoric acid prior to the addition of 454 of the stopped assay mixture. The plate was washed with 5×200 μL 0.2M phosphoric acid. After drying, 100 μL Optiphase SuperMix liquid scintillation cocktail (Perkin Elmer) was added to the well prior to scintillation counting (1450 Microbeta Liquid Scintillation Counter, Wallac).
Measurement: Ki = 505 nM (pChEMBL 6.300000190734863)
Descriptors: MW 467.49, ALogP 2.16, HBA 8, HBD 2, PSA 135.58, QED 0.46
Compound (SMILES): NC(=O)c1ccc(N2CCC[C@@H](n3cnc4ccc(F)cc4c3=O)C2)c2cc(-c3ccc(F)cc3)[nH]c12
InChIKey: JEZAWSITKMVAGA-LJQANCHMSA-N
Target: Tyrosine-protein kinase BTK (SINGLE PROTEIN), Homo sapiens
UniProt: Q06187
Assay: Time-Resolved Fluorescence Resonance Energy Transfer (TR-FRET) : The in-house BTK corresponds to recombinant human catalytic domain (aa 393-659), which was expressed in SF9 cells with an N-terminal his tag and purified by immobilized metal affinity chromatography. BTK was mixed with peptide substrate (biotin-TYR1, Sequence: Biotin-(Ahx)-GAEEEIYAAFFA-COOH, 0.2 μM final) at varying inhibitor concentrations in reaction buffer: 50 mM MOPSO pH 6.5, 10 mM MgCl2, 2 mM MnCl2, 2.5 mM DTT, 0.01% BSA, 0.1 mM Na3VO4 and 0.001 mM ATP. After about 60 mM incubation at room temperature, the reaction was quenched by addition of EDTA (final concentration: 100 mM) and developed by addition of detection reagents (final approximate concentrations: 30 mM HEPES pH 7.0, 0.06% BSA, 0.006% Tween-20, 0.24 M KF, 80 ng/mL PT66K (europium labeled anti-phosphotyrosine antibody cat #61T66KLB Cisbio, Bedford, Mass.) and 0.6 μg/mL SAXL (Phycolink streptavidin-allophycocyanin acceptor, cat #PJ25S, Prozyme, San Leandro, Calif.). The developed reaction was incubated in the dark for about 60 mM at room temperature, then read via a time-resolved fluorescence detector (Rubystar, BMG) using a 337 nm laser for excitation and monitoring emission wavelength at 665 nm. Within the linear range of the assay, the observed signal at 665 nm was directly related to phosphorylated product and can be used to calculate the IC50 values.
Measurement: IC50 = 5500 nM (pChEMBL 5.260000228881836)
Descriptors: MW 499.52, ALogP 4.76, HBA 5, HBD 2, PSA 97.01, QED 0.37
Compound (SMILES): NCCN(Cc1ccc(F)cc1)C(=O)c1ccc(-c2cn[nH]c2)cc1
InChIKey: AVPPHRGAVXSSMY-UHFFFAOYSA-N
Target: Rho-associated protein kinase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q13464
Assay: In Vitro Effect Assay: Table 2: Inhibitory effect of each compound synthesized above on Rho-associated protein kinase (ROCK) was tested.Method 1. 10 mM test compound was diluted to 1 mM with DMSO, and then further diluted to 300 nM. Netarsudil (AR-13324) is a commercial drug for decreasing intraocular pressure, and AR-13503 is an active metabolite of AR-13324. 2. The 300 nM test compound mentioned above was serially diluted to obtain test compounds with concentrations of 100 nM, 33 nM, 11 nM, 3.7 nM, 1.2 nM and 0.4 nM, respectively. 3. 1 μL of the serially diluted test compound mentioned above was added to 49 μL of the modified ROCK reaction buffer (0.05 M Trizma® hydrochloride buffer (pH 7.5) containing 0.1 M KCl, 0.01 M MgCl2, 0.1 mM EGTA and 2.25 μg/mL ROCK1) to obtain an experimental group sample. 1 μL of DMSO was taken to add to 49 μL of an adjusted ROCK reaction buffer to obtain a positive control group sample (maximum value=100%). 1 μL of DMSO was taken to add to 49 μL of a buffer (0.05 M Trizma® hydrochloride buffer (pH 7.5) containing 0.1 M KCl, 0.01 M MgCl2 and 0.1 mM EGTA) to obtain a vehicle control group sample (minimum value=0%). 4. 20 μL of the above prepared sample was added to a flat-bottomed 96-well plate, and 20 μL ROCK ATP buffer was added to each well. 5. The 96-well plate mentioned above was placed on an orbital shaker and reacted at room temperature for 90 minutes.6. Next, 40 μL of Kinase-Glo luminescent kinase assay solution (Promega, RV6712) was added to the 96-well plate mentioned above, and the 96-well plate was placed on an orbital shaker and reacted at room temperature for 10 minutes. 7. The luminescence value of each well of the 96-well plate was determined by SpectraMax M5 microplate reader, and the ROCK inhibition rate (%) was calculated by the following formula.
Measurement: IC50 = 550 nM (pChEMBL 6.260000228881836)
Descriptors: MW 338.39, ALogP 2.82, HBA 3, HBD 2, PSA 75.01, QED 0.73
Compound (SMILES): COc1cccc(Nc2ncc(Cl)c(Nc3cccc(OC)c3)n2)c1
InChIKey: CUYHMISXEVDIMK-UHFFFAOYSA-N
Target: Serine/threonine-protein kinase ULK1 (SINGLE PROTEIN), Homo sapiens
UniProt: O75385
Assay: ULK1 inhibition assay: Gamma-32P assays to measure ULK1 kinase activity were performed as previously described. Briefly, Flag ULK1 was transfected into HEK293T cells and 20 hours later treated as indicated. The immmunoprecipitate was washed in IP buffer 3 times, and washed in kinase buffer (25 mM MOPS, pH 7.5, 1 mM EGTA, 0.1 mM Na3VO4, 15 mM MgCl2,). Hot and cold ATP were added at a 100 μM final concentration. As substrates, GST or the recombinant protein GST-Atg101 purified from E. coli were used at 1 μg for each reaction. Reactions were boiled, run out on SDS page gel. The gel was dried, and imaged using PhosphoImager software. For cold assays to asses ULK1, Flag ULK1 which was transiently overexpressed and immunoprecipited from HEK293T cells. Reactions were then run out on SDS page gel, transferred to PVDF membrane and blotted for total levelsFluorescence MicroscopyVps34flox/flox MEFs were reconstituted with Flag-VPS34 and either p40FX or GFP-DFCP1. 48 hours post infection with adenovirus expressing Cre recombinase (MOI of 100), cells were plated on glass coverslips at a density of 3×105 cells per well in 6-well tissue culture plates. 18 h later, cells were fixed in 4% PFA in PBS for 10 minutes and permeabilized in 0.2% Triton in PBS for 10 minutes. The following primary antibodies were used: mouse anti-Myc epitope and LC3B XP antibody (2276 and 3868 respectively, Cell Signaling Technologies). Secondary antibodies were anti-rabbit Alexa488 and anti-mouse Alexa594 (Molecular Probes, 1:1000. Cells were then fixed and counter stained with DAPI. Coverslips were mounted in FluoromountG (SouthernBiothech). Images were acquired on a Zeiss Axioplan2 epifluorescence microscope coupled to the Openlab software. Confocal images of mitotracker were taken on Zeiss LSM 710 laser scanning confocal microscope. 10 random fields per condition were acquired using the 100× objective and representative images shown. Glass coverslips were mounted directly on plate with FluoromountG and images taken on Zeiss Axioplan2 epifluorescence microscope.
Measurement: IC50 < 200 nM (pChEMBL 6.698999881744385)
Descriptors: MW 356.81, ALogP 4.63, HBA 6, HBD 2, PSA 68.3, QED 0.67
Compound (SMILES): CNC(=O)NC(=O)C(C)(C)[C@@H]1c2ccc(-c3ccc(OC(C)C)nc3)nc2Oc2c(F)cccc21
InChIKey: XCNNCZAWOFPSPF-NRFANRHFSA-N
Target: Glucocorticoid receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P04150
Assay: Receptor Binding Assay: Binding Assay (I):In order to assess the affinity of test compounds for the human glucocorticoid receptor, a commercially available kit was used (Glucocorticoid Receptor Competitor Assay Kit, Invitrogen Part #2893). Briefly, purified human recombinant full-length glucocorticoid receptor (2 nM) was mixed with fluorescently labeled glucocorticoid (1 nM Fluormone GS Red) in the presence or absence of test compound. After two hour incubation at room temperature in the dark, the fluorescence polarization (FP) of the samples was measured. The FP of a mixture of receptor, fluorescent probe (i.e., Fluormone GS Red) and 5 μM dexamethasone represented background fluorescence or 100% inhibition, whereas, the FP of the mixture without dexamethasone (but in the presence of vehicle) was taken to be 100% binding. The percentage inhibition of test compounds were then compared to the sample with 5 μM dexamethasone and expressed as % relative binding activity with dexamethasone being 100% and no inhibition is 0%. Test compounds were analyzed in the concentration range from 8.5E-05 μM to 5 μM. Binding Assay (II):In order to measure the binding of compounds on the glucocorticoid receptor a commercially available kit was used (Glucocorticoid receptor competitor assay kit, PanVera Co., Madison, Wis., P2816). Briefly, a cell lysate containing recombinantly expressed human full-length glucocorticoid receptor was mixed with a fluorescently labeled glucocorticoid (1 nM Fluormone GS1) in the presence or absence of test compound. After one hour at room temperature, the fluorescence polarization (FP) of the samples were measured. The FP of a mixture of receptor, fluorescent probe (i.e., Fluormone GS1) and 1 mM dexamethasone represented background fluorescence or 100% inhibition, whereas, the FP of the mixture without dexamethasone was taken to be 100% binding. The percentage inhibition of test molecules were then compared to the sample with 1 mM dexamethasone and expressed as % relative binding activity with dexamethasone being 100% and no inhibition is 0%. Test molecules were analyzed in the concentration range from 2.4 nM to 40 μM.Site I binding assays for any NHR (Nuclear Hormone Receptor) are conducted similarly to the above. An appropriate cell lysate or purified NHR is used as the source of the NHR. The fluorescent probe and unlabeled competitor are appropriate for the specific NHR, i.e., are ligands for the specific NHR.
Measurement: Ki = 9.68 nM (pChEMBL 8.010000228881836)
Descriptors: MW 478.52, ALogP 4.79, HBA 6, HBD 2, PSA 102.44, QED 0.55
Compound (SMILES): O=C(O)[C@@H]1c2ccccc2C(=O)N(c2ccc(Cl)c(F)c2)[C@H]1c1ccc2c(c1)OCCO2
InChIKey: DSWAKYKFWKGKFU-YADHBBJMSA-N
Target: Stimulator of interferon genes protein (SINGLE PROTEIN), Homo sapiens
UniProt: Q86WV6
Assay: H-cGAMP Filtration Binding Assay (HAQ STING): The ability of compounds to bind STING is quantified by their ability to compete with tritiated cGAMP ligand for human STING receptor membrane using a radioactive filter-binding assay. The binding assay employs STING receptor obtained from Trichoplusia ni (T. ni; Expression Systems, cat #94-002F, Expression Systems, Davis, Calif., USA) cell membranes overexpressing full-length HAQ STING prepared in-house and tritiated cGAMP ligand also purified in-house.
Measurement: IC50 = 3927 nM (pChEMBL 5.409999847412109)
Descriptors: MW 453.85, ALogP 4.82, HBA 4, HBD 1, PSA 76.07, QED 0.61
Compound (SMILES): CC(C)[S+]([O-])c1sc2nc(-c3nccs3)cc(-c3nccn3C)c2c1N
InChIKey: GKXXLEPMXUMXCY-UHFFFAOYSA-N
Target: 15-hydroxyprostaglandin dehydrogenase [NAD(+)] (SINGLE PROTEIN), Homo sapiens
UniProt: P15428
Assay: In Vitro Assay: Each compound for inhibiting enzymatic activity of recombinant 15-PGDH in an in vitro assay.
Measurement: IC50 = 4.18 nM (pChEMBL 8.380000114440918)
Descriptors: MW 403.56, ALogP 3.92, HBA 8, HBD 1, PSA 92.68, QED 0.52
Compound (SMILES): CCn1c(=O)n(-c2nnc(C)o2)c2cc(S(=O)(=O)NC3(C)CC3)ccc21
InChIKey: ZMNFRMAJNOQMQW-UHFFFAOYSA-N
Target: Poly(ADP-ribose) glycohydrolase (SINGLE PROTEIN), Homo sapiens
UniProt: Q86W56
Assay: PARG Assay: PARG In vitro assays were conducted in a total volume of 15 ul in a standard 384 well format. 5 ul of Human Full Length PARG (Produced internally by Astra Zeneca), used at a final reaction concentration of 80 μM, was added to 5 ul of Ribosylated PARP substrate (also produced internally by Astra Zeneca) at final reaction concentration of 4.5 nM in assay buffer (50 mM Tris pH7.4, 0.1 mg/ml BSA, 3 mM EDTA, 0.4 mM EGTA, 1 mM DTT, 0.01% Tween 20, 50 mM KCl). The reaction was incubated at room temperature for 10 minutes and then 5 ul detection reagent was added. Detection Reagent consists of 42 nM MAb Anti-6HIS XL665 (CisBio: 61 HISXLB) and 2.25 nM Streptavidin Europium Cryptate (CisBio: 610SAKLB), both at 3× working stock concentrations (final concentrations of 14 nM and 0.75 nM respectively), in a detection buffer of 50 mM Tris pH7.4, BSA at 0.1 mg/ml and KF at 100 mM. Following incubation at room temperature for 60 minutes in the dark, TR-FRET signal was measured at Ex 340 and Em 665 and Em 620. A ratio was calculated as Em665/EM620×104 for each well and used to calculate percent inhibition for test compounds.
Measurement: EC50 = 934 nM (pChEMBL 6.03000020980835)
Descriptors: MW 377.43, ALogP 1.33, HBA 8, HBD 1, PSA 112.02, QED 0.72
Compound (SMILES): NS(=O)(=O)c1cccc(-c2cn(S(=O)(=O)c3ccccc3)c3ncccc23)c1-c1nn[nH]n1
InChIKey: GKLQFJBSJRLNRT-UHFFFAOYSA-N
Target: Metallo-beta-lactamase type 2 (SINGLE PROTEIN), Bacteria
UniProt: C7C422
Assay: Class B Enzyme Assay: The Class B enzyme activities were measured in the presence of the test inhibitor in a fluorescence assay against a commercially available substrate consisting of a cephalosporin core linking 7-hydroxycoumarin to fluorescein (CCF2-FA). The enzyme (NDM-1, IMP-1 or VIM-1) and the substrate were diluted in 100 mM KH2PO4 buffer (pH 7) containing 0.005% Tween-20 and 10 μM ZnSO4. In the assay, the final concentration of enzyme was 1 pM, 2 pM and 30 pM for NDM-1, IMP-1 and VIM-1, respectively, and the final concentration of CCF2-FA was 1.25 μM. The test inhibitor was dissolved in dimethylsulfoxide and diluted 1:50 in the assay, resulting in a final concentration range of 20 uM to 0.00063 uM. In a 384-well microplate, the test inhibitor was incubated with the metallo-β-lactamase enzyme and the substrate for 2 hours at 25° C. Fluorescence at 460 nm following excitation at 405 nm was measured. The IC50 value was determined from semi-logarithmic plots of enzyme inhibition versus inhibitor concentration, with a curve generated using a 4-parameter fit.
Measurement: IC50 = 2.21 nM (pChEMBL 8.65999984741211)
Descriptors: MW 481.52, ALogP 1.77, HBA 9, HBD 2, PSA 166.58, QED 0.38
Compound (SMILES): Cc1c(-c2cc3cc(NC(=O)O[C@H]4CCC5(CCN(C6COC6)CC5)C4)ncc3c(N)c2F)cnc2c1NCCO2
InChIKey: DWKMDBVUSRQBJL-FQEVSTJZSA-N
Target: Mitogen-activated protein kinase kinase kinase kinase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q92918
Assay: TBD: TBD
Measurement: Ki = 0.017 nM (pChEMBL 10.770000457763672)
Descriptors: MW 562.65, ALogP 4.71, HBA 9, HBD 3, PSA 123.86, QED 0.39
Compound (SMILES): CC(C)(NC(=O)[C@H]1[C@@H]2CNC[C@@H]21)c1ncc2cccc(F)n12
InChIKey: AAUTVKKLKBMPKY-PJXYFTJBSA-N
Target: Somatostatin receptor type 4 (SINGLE PROTEIN), Homo sapiens
UniProt: P31391
Assay: AMP Assay with Human Somatostatin 4 Receptor: The activation of the SSTR4 receptor (Gi coupled) causes an inhibition of intracellular cAMP after stimulation with Forskolin, which can be quantifiable by use of a suitable assay Kit and an adequate plate reader. This technique is used to characterize pharmacological effects of the SSTR4 receptor agonists by use of hSSTR4 expressing H4 cells. Compounds are dissolved and diluted in DMSO. The final test solution contains 1% DMSO. The cAMP standard (Lance cAMP 384 Kit; PerkinElmer, Cat# AD0264) is prepared in assay buffer (HBSS with 0.1% BSA, 5 mM HEPES, 0.5 M IBMX, pH 7.4) containing 1% DMSO and the cAMP standard curve is included at least on one plate. Cells are centrifuged and suspended in assay buffer (incl. 1:100 diluted Alexa antibody).
Measurement: EC50 = 7.6 nM (pChEMBL 8.119999885559082)
Descriptors: MW 302.35, ALogP 1.29, HBA 4, HBD 2, PSA 58.43, QED 0.84
Compound (SMILES): COc1cc(-c2[nH]c3cc(C)c(C4CCC(N)CC4)nc3c2C(C)C)cn2ncnc12
InChIKey: HEHSWKIMUQMIQL-UHFFFAOYSA-N
Target: Toll-like receptor 8 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9NR97
Assay: TLR7/8/9 Inhibition Reporter Assays: HEK-Blue-cells (Invivogen) overexpressing human TLR7, TLR8 or TLR9 receptors were used for screening inhibitors of these receptors using an inducible SEAP (secreted embryonic alkaline phosphatase) reporter gene under the control of the IFN-β minimal promoter fused to five NF-κB and AP-1-binding sites. Briefly, cells are seeded into Greiner 384 well plates (15000 cells per well for TLR7, 20,000 for TLR8 and 25,000 for TLR9) and then treated with test compounds in DMSO to yield a final dose response concentration range of 0.05 nM-50 μM. After a 30 minute compound pre-treatment at room temperature, the cells are then stimulated with a TLR7 ligand (gardiquimod at a final concentration of 7.5 μM), TLR8 ligand (R848 at a final concentration of 15.9 μM) or TLR9 ligand (ODN2006 at a final concentration of 5 nM) to activate NF-κB and AP-1 which induce the production of SEAP. After a 22 hour incubation at 37° C., 5% CO2, SEAP levels are determined with the addition of HEK-Blue Detection reagent (Invivogen), a cell culture medium that allows for detection of SEAP, according to manufacturer's specifications. The percent inhibition is determined as the % reduction in the HEK-Blue signal present in wells treated with agonist plus DMSO alone compared to wells treated with a known inhibitor.
Measurement: IC50 = 2.1 nM (pChEMBL 8.680000305175781)
Descriptors: MW 418.55, ALogP 4.7, HBA 6, HBD 2, PSA 94.12, QED 0.5
Compound (SMILES): CC(=O)c1cn(CC(=O)N2C[C@H](F)C[C@H]2C(=O)NCc2cccc(Cl)c2F)c2cc(P(=O)(OCC(F)(F)F)OCC(F)(F)F)ccc12
InChIKey: AHNBSVMXCCNKSN-HXOBKFHXSA-N
Target: Complement factor D (SINGLE PROTEIN), Homo sapiens
UniProt: P00746
Assay: Human 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. IC5o 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 717.93, ALogP 5.87, HBA 7, HBD 1, PSA 106.94, QED 0.15
Compound (SMILES): CC(=O)NC[C@H]1CN(S(=O)(=O)c2cn(C(F)F)nc2C)c2cc(NC(=O)OCc3ccccc3)ccc2O1
InChIKey: NOFLWLBPNZYBCG-IBGZPJMESA-N
Target: Nuclear receptor ROR-gamma (SINGLE PROTEIN), Homo sapiens
UniProt: P51449
Assay: Biochemical TR-FRET assay: The compounds of the invention inhibit RORgammaT activity. Activation of RORgammaT activity can be measured using, e.g., a 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 (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 = 109 nM (pChEMBL 6.960000038146973)
Descriptors: MW 549.56, ALogP 3.43, HBA 8, HBD 2, PSA 131.86, QED 0.44
Compound (SMILES): CCC(=O)N1CC(Nc2cncc(N3C(=O)c4ccc(Cl)cc4C3C)c2)C1
InChIKey: ZTWWIDHBLIYTCM-UHFFFAOYSA-N
Target: Cytochrome P450 11B2, mitochondrial (SINGLE PROTEIN), Homo sapiens
UniProt: P19099
Assay: Cellular Enzyme Assay: G-402 cells expressing CYP11 constructs were established as described above and maintained in McCoy's 5a Medium Modified, ATCC Catalog No. 30-2007 containing 10% FCS and 400 ug/ml G418 (Geneticin) at 37° C. under an atmosphere of 5% CO2/95% air. Cellular enzyme assays were performed in DMEM/F12 medium containing 2.5% charcoal treated FCS and appropriate concentration of substrate (0.3-10 uM 11-Deoxycorticosterone, 11-Deoxycortisol or Corticosterone). For assaying enzymatic activity, cells were plated onto 96 well plates and incubated for 16 h. An aliquot of the supernatant is then transferred and analyzed for the concentration of the expected product (Aldosterone for CYP11B2; Cortisol for CYP11B1). The concentrations of these steroids can be determined using HTRF assays from CisBio analyzing either Aldosterone or Cortisol.
Measurement: EC50 = 65 nM (pChEMBL 7.190000057220459)
Descriptors: MW 384.87, ALogP 3.49, HBA 4, HBD 1, PSA 65.54, QED 0.88
Compound (SMILES): N#Cc1cc(-c2cc(-c3nnc[nH]3)c3ncn(-c4cc(N5CCNCC5)nc5ccccc45)c3c2)ccn1
InChIKey: LWSHSWWGADGWQS-UHFFFAOYSA-N
Target: Phosphatidylinositol 3-kinase regulatory subunit beta (SINGLE PROTEIN), Homo sapiens
UniProt: O00459
Assay: TR-FRET Assay: Class I PI3K isoforms were expressed and purified as heterodimeric recombinant proteins. All assay reagents and buffers for the TR-FRET assay were purchased from Millipore. PI3K isoforms were assayed under initial rate conditions in the presence of 25 mM Hepes (pH 7.4), and 2× Km ATP (75-500 μM), 2 μM PIP2, 5% glycerol, 5 mM MgCl2, 50 mM NaCl, 0.05% (v/v) Chaps, 1 mM dithiothreitol, and 1% (v/v) DMSO at the following concentrations for each isoform: PI3Kα, PI3Kβ, and PI3Kδ between 25 and 50 pM, and PI3Kγ at 2 nM. The compounds were added to the assay solution and incubated for 30 minutes at 25° C. The reactions were terminated with a final concentration of 10 mM EDTA, 10 nM labeled-PIPS, and 35 nM Europium labeled GRP-1 detector protein before reading TR-FRET on an Envision plate reader (Ex: 340 nm; Em: 615/665 nm; 100 μs delay and 500 μs read window).
Measurement: IC50 = 10 nM (pChEMBL 8)
Descriptors: MW 498.55, ALogP 3.7, HBA 9, HBD 2, PSA 124.23, QED 0.38
Compound (SMILES): Cc1ccc(OCCN2CCC(N[C@@H]3C[C@H]3c3ccccc3)CC2)cc1
InChIKey: PYAGOBRWOPLLCY-XZOQPEGZSA-N
Target: Lysine-specific histone demethylase 1A (SINGLE PROTEIN), Homo sapiens
UniProt: O60341
Assay: LSD1 enzymatic activity: Briefly, compounds of the present invention were solubilized in DMSO and a series of 10, three-fold serial dilutions were made for each compound in 15% DMSO. The initial starting concentration for the serial dilutions of each compound was 10 μM. Control samples lacking compound, LSD1 enzyme or various reaction components also were prepared and processed in parallel with compound test samples.An aliquot of each serial dilution of test compound was added to a 96 well plate containing 50 nM purified N-truncated LSD1 enzyme (RBC Cat# PDM-11-350), 50 mM Tris-HCl, pH 7.5, 0.05% CHAPS and 1% DMSO in a 10 microliter reaction volume. The plate was pre-incubated at room temperature for 30 min to which 10 M of histone 3.3 peptide (aa 1-21) was added to initiate the enzymatic reaction. The reaction mixture was incubated at room temperature for one hour. After one hour, 10 μl of a detection mixture of horseradish peroxidase (Sigma Cat #P8375) and Amplex Red (InVitrogen A36006) was added and kinetic measurements were read at 5 minute intervals for a period of 30 minutes using an Envision Multiplate Reader (PerkinElmer; excitation at 535 nM and emmission read at 590 nM). The IC50 value for each compound was determined from each 10-point dose-response curve using GraphPad Prism 4 software with a sigmodial dose response.
Measurement: IC50 = 5 nM (pChEMBL 8.300000190734863)
Descriptors: MW 350.51, ALogP 3.98, HBA 3, HBD 1, PSA 24.5, QED 0.82
Compound (SMILES): CC[C@H](C)COc1ccc(/N=c2\[nH]c(=O)n(C[C@H](C)C(=O)OC)c(=O)n2Cc2ccc(C)cc2)cc1
InChIKey: BBBYKFGODQVCIO-ICSRJNTNSA-N
Target: P2X purinoceptor 3 (SINGLE PROTEIN), Homo sapiens
UniProt: P56373
Assay: Human P2X3 Receptor Inhibition Assay: Stably expressing cell line (C6BU-1 transfected with human P2X3 receptor gene (GenBank accession number Y07683) was used. The cells were seeded in a 96-well microtiter plate at a concentration of 8000 cells/well and cultured in the medium (8.3% fetal bovine serum, 8.3% horse serum, 1% antibiotic and antifungal in DMEM) for one day at 37° C. under 5% carbon dioxide atmosphere. The medium was replaced with 4 μM Fluo-3-AM solution (20 mM HEPES, 137 mM NaCl, 2.7 mM KCl, 0.9 mM MgCl2, 5.0 mM CaCl2, 5.6 mM D-glucose, 2.5 mM probenecid, 10% BSA, and 0.08% Pluronic F-127, pH 7.5) and incubated at 37° C. under 5% dioxide carbon atmosphere for one hour. The plate was washed with washing buffer (20 mM HEPES, 137 mM NaCl, 2.7 KCl, 0.9 mM MgCl2, 5.0 mM CaCl2, 5.6 mM D-glucose, 2.5 mM probenecid, pH 7.5), each well was added with 40 μL of this buffer. The plate was placed in High-Throughput Screening System FDSS 3000 (Hamamatsu Photonics K.K.). Measurement of fluorescence intensity by FDSS 3000 was started, and 40 μl, of DMSO solutions containing different concentrations of the test, compound as prepared by dilution with dilution buffer (20 mM HEPES, 137 mM NaCl, 2.7 mM KCl, 0.9 mM MgCl2, 5.0 mM CaCl2, 5.6 mM D-glucose, 2.5 mM probenecid, 0.1.% Pluronic F-127, pH 7.5) were dispensed to each well through the built-in automatic dispenser. Five minutes after, 50 nM ATP solution (50 μL) prepared by dilution with the dilution buffer was dispensed through the built-in automatic dispenser, and the measurement of fluorescence intensity was continued for 3 min. For each well, the specific maximum fluorescence intensity was calculated as the ratio of the maximum fluorescence intensity after addition of the ATP solution to the fluorescence intensity at the starting of the measurement. The 50% inhibitory concentration (IC50) was calculated under the assumption that the specific maximum fluorescence intensity without test compound is 0% inhibition and that the specific maximum fluorescence intensity when the dilution buffer was added in place of ATP solution is 100% inhibition, to evaluate the inhibitory activity of the test compound. FDSS software (Hamamatsu Photonics K.K.) was used for calculation of the specific maximum fluorescence intensity. IC50 was calculated using Microsoft Excel (Microsoft Corporation) and Ltd.).
Measurement: IC50 = 159 nM (pChEMBL 6.800000190734863)
Descriptors: MW 494.59, ALogP 3.16, HBA 8, HBD 1, PSA 107.68, QED 0.44
Compound (SMILES): C[C@@H]1CC[C@H](c2ncc(C(C)(C)O)s2)CN1C(=O)c1ccccc1-n1nccn1
InChIKey: MFXDQNYSVOUGOX-CABCVRRESA-N
Target: Orexin receptor type 2 (SINGLE PROTEIN), Homo sapiens
UniProt: O43614
Assay: FLIPR Ca2+ Flux Assay: Briefly, for intracellular calcium measurements, Chinese hamster ovary (CHO) cells expressing the orexin-1 receptor (e.g., rat or human) or the orexin-2 receptor (e.g., rat or human), are grown in Iscove's modified DMEM containing 2 mM L-glutamine, 0.5 g/ml G418, 1% hypoxanthine-thymidine supplement, 100 U/ml penicillin, 100 μg/ml streptomycin and 10% heat-inactivated fetal calf serum (FCS). The cells are seeded at approximately 20,000 cells per well into 384-well clear bottom sterile plates coated with poly-D-lysine. The seeded plates are incubated overnight at 37° C. and 5% CO2. Human ala-6,12 orexin-A can be used as the agonist and prepared as a 1 mM stock solution in 1% bovine serum albumin (BSA) and diluted in assay buffer (HBSS containing 20 mM HEPES, 0.1% BSA and 2.5 mM probenecid, pH7.4) for use in the assay at a final concentration of 70 pM. Test compounds are prepared as 10 mM stock solution in DMSO, then diluted in 384-well plates, first in DMSO, then in assay buffer. On the day of the assay, cells are washed 3 times with 100 μl assay buffer and then incubated for 60 minutes (37° C., 5% CO2) in 60 μl assay buffer containing 1 μM Fluo-4 AM ester, 0.02% pluronic acid, and 1% BSA. The dye loading solution is then aspirated and cells are washed 3 times with 100 μl assay buffer. 30 μl of that same buffer is left in each well. Within the Fluorescent Imaging Plate Reader (FLIPR, Molecular Devices), test compounds are added to the plate in a volume of 25 μl, incubated for 5 minutes, and then 25 μl of agonist is added. Fluorescence is measured for each well at 1 second intervals for 5 minutes, and the height of each fluorescence peak is compared to the height of the fluorescence peak induced by 70 pM of agonist ala-6,12 orexin-A with buffer in place of test compound.
Measurement: IC50 = 2650 nM (pChEMBL 5.579999923706055)
Descriptors: MW 411.53, ALogP 3.36, HBA 7, HBD 1, PSA 84.14, QED 0.71
Compound (SMILES): O=c1[nH]c(CSC2CCS(=O)(=O)CC2)nc2cc(NC3CCCC3)cc(F)c12
InChIKey: UEIQAKQYFNHJDR-UHFFFAOYSA-N
Target: Protein mono-ADP-ribosyltransferase PARP14 (SINGLE PROTEIN), Homo sapiens
UniProt: Q460N5
Assay: Enzymatic Inhibition Assay: Enzymatic inhibition of PARP14 was measured using a dissociation-enhanced lanthanide fluorescence immunoassay (DELFIA) monitoring the auto-modification of PARP14 by biotinylated nicotinamide adenine dinucleotide (biotin-NAD). 1 μL of a dose response curve of each test compound was spotted in 384-well nickel-coated white microplates (Thermo) using a Mosquito (TTP Labtech). Reactions were performed in a 50 μL volume by adding 40 μL of PARP14 in assay buffer (20 mM HEPES pH==8, 100 mM NaCl, 0.1% bovine serum albumin, 2 mM DTT and 0.002%0 Tween20), incubating with test compound at 25° C. for 30 min, then adding 10 μL of biotin-NAD (Biolog). The final concentrations of PARP14 and biotin-NAD are 50 nM and 3 μM, respectively. Reactions proceeded at 25° C. for 3 h, then were quenched with 5 μL of 10 mM unmodified nicotinamide adenine dinucleotide (Sigma-Aldrich). The quenched reactions were washed 3 times with 100 μL of TBST wash buffer (50 mM Tris-HCl, 150 mM NaCl and 0.1% Tween20). Next, to the washed and dried plate was added 25 μL of DELFIA Europium-N1 streptavidin (Perkin Elmer) diluted in DELFIA assay buffer (Perkin Elmer). After a 30 min incubation at 25° C., the plate was washed 5 times with TBST wash buffer. Finally, 25 μL of DELFIA enhancement solution was added. After a 5 min incubation the plate was read on an Envision platereader equipped with a LANCE/DELFIA top mirror (Perkin Elmer) using excitation of 340 nm and emission of 615 nm to measure the amount of Europium present in each well, informing on the amount of biotin-NAD that was transferred in the automodification reaction.
Measurement: IC50 < 1000 nM (pChEMBL 6)
Descriptors: MW 425.55, ALogP 3.23, HBA 6, HBD 2, PSA 91.92, QED 0.76
Compound (SMILES): CN(C)[C@H]1C[C@@H](c2cccc(OC(F)F)c2)CC[C@@H]1Nc1cc(F)c(S(=O)(=O)Nc2ccncn2)cc1Cl
InChIKey: CZBRRFOWIVYWKE-BUKVSMQUSA-N
Target: Sodium channel protein type 9 subunit alpha (SINGLE PROTEIN), Homo sapiens
UniProt: Q15858
Assay: Electrophysiological Assay (EP) (In Vitro Assay): The following voltage clamp electrophysiology studies are performed on representative compounds using cells heterologously expressing Nav1.7 or Nav1.5 channels. cDNAsfor Nav1.7 (NM_002977) and Nav1.5 (AC137587) are stably expressed in Chinese Hamstr Ovary (CHO) cells and CHL (Chinese Hamster Lung) cells respectively. Sodium currents are measured in the whole-cell configuration using Syncropatch 384PE (Nanlon Technologies, Germany). 1NPC®-384 chips with custom medium resistance and single hole mode are used. Internal solution consists of (in mM): 110 CsCl, 10 CsCl, 20 EGTA, and 10 Hepes (pH adjusted to 7.2); and external solution contains (in mM): 60 NMDG, 80 NaCl, 4 KCl, 1 MgCl2, 2 CaCl2, 2 D-Glucose monohydrate, 10 Hepes (pH adjusted to 7.4 with NaOH).
Measurement: IC50 = 1.63 nM (pChEMBL 8.789999961853027)
Descriptors: MW 570.04, ALogP 5.35, HBA 7, HBD 2, PSA 96.45, QED 0.36
Compound (SMILES): O=C(COc1cc(Cl)cc(Cl)c1)N1CCC2(CC1)CN(C(=O)c1ccc3[nH]nnc3c1)C2
InChIKey: GEGDEOMKGFQOPG-UHFFFAOYSA-N
Target: Autotaxin (SINGLE PROTEIN), Homo sapiens
UniProt: Q13822
Assay: Enzyme Inhibition Assay: ATX inhibition was measured by a fluorescence quenching assay using a specifically labeled substrate analogue (MR121 substrate). To obtain this MR121 substrate, BOC and TBS protected 6-amino-hexanoic acid (R)-3-({2-[3-(2-{2-[2-(2-amino-ethoxy)-ethoxy]-ethoxy}-ethoxy)-propionylamino]-ethoxy}-hydroxy-phosphoryloxy)-2-hydroxy-propyl ester (Ferguson et al., Org Lett 2006, 8 (10), 2023) was labeled with MR121 fluorophore (CAS 185308-24-1, 143-carboxypropyl)-11-ethyl-1,2,3,4,8,9,10,11-octahydro-dipyrido[3,2-b:2',3'-i]phenoxazin-13-ium) on the free amine of the ethanolamine side and then, after deprotection, subsequently with tryptophan on the side of the aminohexanoic acid.Assay working solutions were made as follows:Assay buffer (50 mM Tris-HCl, 140 mM NaCl, 5 mM KCl, 1 mM CaCl2, 1 mM MgCl2, 0.01% Triton-X-100, pH 8.0;ATX solution: ATX (human His-tagged) stock solution (1.08 mg/mL in 20 mM bicine, pH 8.5, 0.15 M NaCl, 10% glycerol, 0.3% CHAPS, 0.02% NaN3), diluted to 1.4-2.5x final concentration in assay buffer. MR121 substrate solution: MR121 substrate stock solution (800 uM MR121 substrate in DMSO), diluted to 2-5x final concentration in assay buffer. Test compounds (10 mM stock in DMSO, 8 uL) were obtained in 384 well sample plates (Corning Costar #3655) and diluted with 8 uL DMSO. Row-wise serial dilutions were made by transferring 8 uL cpd solution to the next row up to row 0. The compound and control solutions were mixed five times and 2 uL were transferred to 384 well assay plates (Corning Costar #3702). Then, 15 uL of 41.7 nM ATX solution was added (30 nM final concentration), mixed five times and then incubated for 15 minutes at 30° C. 10 uL of MR121 substrate solution was added (1 uM final concentration), mixed 30 times and then incubated for 15 minutes at 30° C. Fluorescence was then measured every 2 minutes for 1 hour (Perkin Elmer plate: vision multimode reader); light intensity: 2.5%; exp. time: 1.4 sec, Filter: Fluo_630/690 nm) and IC50 values were calculated from these readouts.
Measurement: IC50 = 298 nM (pChEMBL 6.53000020980835)
Descriptors: MW 474.35, ALogP 3.41, HBA 5, HBD 1, PSA 91.42, QED 0.63
Compound (SMILES): CCOC(=O)c1cn(-c2ccc(-c3nnc(C)o3)cc2)c(=O)n(Cc2cccc(C(F)(F)F)c2Cl)c1=O
InChIKey: QRBGCQMNPSRRLP-UHFFFAOYSA-N
Target: Chymase (SINGLE PROTEIN), Homo sapiens
UniProt: P23946
Assay: Enzymatic Assay : The enzyme source used is recombinant human chymase (expressed in HEK293 cells) or chymase purified from hamsters' tongues. The substrate used for chymase is Abz-HPFHL-Lys(Dnp)-NH2. For the assay, 1 μl of a 50-fold concentrated solution of test substance in DMSO, 24 al of enzyme solution (dilution 1:80 000 human or 1:4000 hamster) and 25 al of substrate solution (final concentration 10 μM) in assay buffer (Tris 50 mM (pH 7.5), sodium chloride 150 mM, BSA 0.10%, Chaps 0.10%, glutathione 1 mM, EDTA 1 mM) are combined in a white 384-hole microtiter plate (Greiner Bio-One, Frickenhausen, Germany). The reaction is incubated at 32 degrees for 60 min and the fluorescence emission at 465 nm after excitation at 340 nm is measured in a fluorescence reader, for example Tecan Ultra (Tecan, Männedorf, Switzerland).
Measurement: IC50 = 2000 nM (pChEMBL 5.699999809265137)
Descriptors: MW 534.88, ALogP 4.25, HBA 9, HBD 0, PSA 109.22, QED 0.34
Compound (SMILES): C[C@@H]1CC(=O)Nc2cccc(-c3cc4ccc(Cl)cc4n3C)c2O1
InChIKey: PODGPNDFLZAAFP-LLVKDONJSA-N
Target: CREB-binding protein (SINGLE PROTEIN), Homo sapiens
UniProt: Q92793
Assay: CBP TR-FRET Binding Assay: His/Flag epitope tagged CBP was cloned, expressed, and purified to homogeneity. CBP binding and inhibition was assessed by monitoring the engagement of a biotinylated small molecule compound with the target using the TR-FRET assay technology (Perkin-Elmer). Specifically, in a 384 well ProxiPlate CBP (4 nM final) was combined with biotin-ligand (60 nM final) in 50 mM HEPES (pH 7.5), 50 mM NaCl, 1 mM TCEP, 0.01% (w/v) BSA, and 0.008% (w/v) Brij-35 either in the presence of DMSO (final 0.2% DMSO) or compound dilution series in DMSO. After 10 minutes incubation at room temperature, a mixture Eu-W1024 Anti-6×His antibody (Perkin Elmer ADO 110) and SureLight Allophycocyanin-Streptavidin (APC-SA, Perkin Elmer CR130-100) were added to a final concentrations of 0.2 nMolar antibody and 50 nMolar APC-SA, respectively. After twenty minutes of equilibration, the plates were read on an Envision instrument and IC50s calculated using a four parameter non-linear curve fit.
Measurement: IC50 = 182 nM (pChEMBL 6.739999771118164)
Descriptors: MW 340.81, ALogP 4.61, HBA 3, HBD 1, PSA 43.26, QED 0.7
Compound (SMILES): Cc1noc(C)c1-c1cnc2c3ccc(C(C)(C)O)cc3n(Cc3ccc(F)cc3)c2c1
InChIKey: FKJPEXOLMVUXRZ-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 < 25 nM (pChEMBL 7.6020002365112305)
Descriptors: MW 429.5, ALogP 5.88, HBA 5, HBD 1, PSA 64.08, QED 0.39
Compound (SMILES): C[C@H](Nc1cc(N2C(=O)OCC2(C)C)ccn1)c1cc2cc(Cl)ccc2[nH]c1=O
InChIKey: AJYIONBIQGQQEY-LBPRGKRZSA-N
Target: Isocitrate dehydrogenase [NADP] cytoplasmic (SINGLE PROTEIN), Homo sapiens
UniProt: O75874
Assay: IDH1-R132H and IDH1-R132C Enzymatic Assay: Assays were performed in a 384-well black plate. An aliquot of 250 nL of compound was incubated with 10 μL of 30 nM IDH1-R132H or 10 nM IDH1-R132C recombinant protein in assay buffer (50 mM Tris pH=7.5, 150 mM NaCl, 5 mM MgCl2, 0.1% (w/v) Bovine Serum Albumin, and 0.01% Triton X-100) in each well at 25° C. for 15 minutes. After the plate was centrifuged briefly, an aliquot of 10 μL of 2 mM α-ketoglutarate and 20 μM NADPH solution prepared in assay buffer was then added to each well and the reaction was maintained at 25° C. for 45 minutes. An aliquot of 10 μL of diaphorase solution (0.15 U/mL diaphorase and 30 μM Resazurin in assay buffer) was added to each well. The plate was maintained at 25° C. for 15 minutes and then read on a plate reader with excitation and emission wavelengths at 535 nm and 590 nm, respectively. The IC50 of a given compound was calculated by fitting the dose response curve of inhibition of NADPH consumption at a given concentration with the four parameter logistic equation.
Measurement: IC50 = 55 nM (pChEMBL 7.260000228881836)
Descriptors: MW 412.88, ALogP 4.48, HBA 5, HBD 2, PSA 87.32, QED 0.66
Compound (SMILES): NCc1cnnn1-c1ncc(NC(=O)c2cnn(-c3cccc4ncccc34)c2C(F)(F)F)cc1Cl
InChIKey: SKNZJAOTWQCQES-UHFFFAOYSA-N
Target: Mucosa-associated lymphoid tissue lymphoma translocation protein 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9UDY8
Assay: In Vitro Assay: MALT1 protease activity was assessed in an in vitro assay using a tetrapeptide as substrate and full-length MALT1 protein (Strep-MALT1(1-824)-His) purified from baculovirus-infected insect cells. The tetrapeptide LRSR is coupled to AMC (7-amino-4-methylcoumarin) and provides a quenched, fluorescent substrate for the MALT1 protease (SM Biochemicals). Cleavage of AMC from the Arginine residue results in an increase in coumarin fluorescence measured at 460 nm (excitation 355 nm). The final assay buffer consisted of 10 nM FL MALT1 protein, 200 μM Ac-LRSR-AMC, 50 mM Tris pH 7.5, 0.6 M Citrate, 1 mM DTT, 1 mM EDTA, 0.05% BSA and 1.5% DMSO. Test compounds were spotted at 50 nL in 100% DMSO per well of a black 384-Proxiplate (Perkin Elmer). Test compound concentrations ranged from 30 μM to 0.5 nM using 11 dilution steps (1:3). Background signal was measured from control wells containing assay buffer without enzyme which functions as low control (LC). High control (HC) values were generated using the reaction with enzyme but no compound treatment. Compounds were pre-incubated with MALT1 enzyme for 50 minutes at RT. Substrate was added subsequently and fluorescence was measured in Labsystems fluoroskan at excitation 355 nm and emission 460 nm to determine time 0. The reaction was subsequently incubated for 4 h at RT and fluorescence was measured.
Measurement: IC50 = 331 nM (pChEMBL 6.480000019073486)
Descriptors: MW 513.87, ALogP 3.78, HBA 9, HBD 2, PSA 129.43, QED 0.37
Compound (SMILES): COC(=O)c1cc2cc(-c3ccc4cc(-c5nc6cc(C(=O)N7CC8CCC7[C@@H]8N)cc(OC)c6n5C)n(CC5CC5)c4n3)ccc2[nH]1
InChIKey: QNDOICZZNOXIHY-OXZXHNBXSA-N
Target: Protein-arginine deiminase type-4 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9UM07
Assay: Rapid-Fire Mass Spectrum (RFMS) Assay: Compounds were solubilized in 100% DMSO to achieve a 10 mM compound concentration. Compound stock solutions were stored at RT. A series of dilutions were prepared in DMSO and mixed 8 times with 20 μL mixing volume. Final top concentration of compound in the assay is 50 μM. Final assay conditions were as follows:Reaction volume: 26 μlAssay buffer: 25 mM hepes, pH 7.5, 5 mM NaCl, 1 mM DTT, 0.2 mg/ml BSA, 0.01% CHAPS, 50 μM Calcium, and 5 μM TPENFinal concentrations: 5 nM hPAD4 enzyme, 250 μM BAEE, and 0.5% DMSOTotal incubation time: 30 mins compound and enzyme preincubation at 37° C., 90 min enzyme/substrate reaction, 30 min reaction with phenyl glyoxal at 37° C.Stop solution: 40 μl 5% TCA in ACN
Measurement: IC50 = 107 nM (pChEMBL 6.96999979019165)
Descriptors: MW 643.75, ALogP 5.51, HBA 9, HBD 2, PSA 133.29, QED 0.22
Compound (SMILES): CC(C)C[C@H](N)[C@](O)(Cc1cc(OC2CC3CCC2C3)ccn1)C(=O)O
InChIKey: IJZMAUGVQAHPHH-XSCIWRGYSA-N
Target: Leucyl-cystinyl aminopeptidase (SINGLE PROTEIN), Rattus norvegicus
UniProt: P97629
Assay: Inhibition of IRAP Assay: Rat epididymal fat pads were homogenized and subjected to ultracentrifugation at 100,000×g for 30 minutes to obtain microsomes containing IRAP. The microsomes (with a total protein content of 55 g/well) were mixed with a solvent (dimethyl sulfoxide; hereinafter, abbreviated as DMSO (final concentration: 0.1%)) or with each test compound (common ratio: 3; maximum concentration: 10 μM). AVP was then added to the solution to a final concentration of 25 μM, and the resulting solution was allowed to react for one hour at 37° C. An aqueous trifluoroacetic acid (hereinafter, abbreviated as TFA) solution was then added to the solution (final concentration: 1%) to stop the enzymatic reaction. Residual AVP was then determined by mass spectrometry (MALDI-MS). Based on the results, IC50 values (nM), i.e. concentrations required for 50% inhibition of decrease in AVP level in the solvent control group, of the individual test compounds were calculated by the logistic regression to evaluate inhibition of IRAP activity. As comparative examples, similar tests were performed with the compounds of the Reference Examples 1 and 2 described below: (2S,3S)-3-amino-2-hydroxy-5-methyl-2-({4-[(trans-4-methylcyclohexyl)oxy]pyridin-2-yl}methyl)hexanoic acid dihydrochloride; and (2S,3R)-3-amino-2-hydroxy-5-methyl-2-({4-[(trans-4-methylcyclohexyl)oxy]pyridin-2-yl}methyl)hexanoic acid hydrochloride, respectively.
Measurement: IC50 = 2 nM (pChEMBL 8.699999809265137)
Descriptors: MW 362.47, ALogP 2.38, HBA 5, HBD 3, PSA 105.67, QED 0.66
Compound (SMILES): CC(C)N1CCN(C(C)(C)C=C(C#N)C(=O)N2CCC[C@@H](n3nc(-c4ccc(Oc5ccccc5)cc4F)c4c(N)ncnc43)C2)CC1
InChIKey: BICRABDPLNFWOV-AREMUKBSSA-N
Target: Tyrosine-protein kinase BTK (SINGLE PROTEIN), Homo sapiens
UniProt: Q06187
Assay: Caliper-Based Kinase Assay: A Caliper-based kinase assay (Caliper Life Sciences, Hopkinton, Mass.) was used to measure inhibition of Btk kinase activity of a compound of the present disclosure. Serial dilutions of test compounds were incubated with human recombinant Btk (0.5 nM), ATP (16 μM) and a phosphoacceptor peptide substrate FAM-GEEPLYWSFPAKKK-NH2 (1 μM) at room temperature for 3 h. The reaction was then terminated with EDTA, final concentration 20 mM and the phosphorylated reaction product was quantified on a Caliper Desktop Profiler (Caliper LabChip 3000).
Measurement: IC50 = 0.7 nM (pChEMBL 9.149999618530273)
Descriptors: MW 651.79, ALogP 5.42, HBA 10, HBD 1, PSA 129.43, QED 0.2