lovingscience/chembl-v37-raw-activities huggingface dataset:
Compound (SMILES): COc1cn(C(CCOC2CCC2)C(=O)Nc2ccc(C(=O)O)cc2)c(=O)cc1-c1cc(Cl)ccc1C(C)=O
InChIKey: OMRPNYPLMAJARJ-UHFFFAOYSA-N
Target: Coagulation factor XI (SINGLE PROTEIN), Homo sapiens
UniProt: P03951
Assay: Biological Assay: Test Example 1: Biological Activity of the Compounds of the Present Invention onThe Inhibition of Factor XIa Detected by Absorption Photometry1. Experimental MaterialsEnzyme: Coagulation Factor XIa protease (Abcam, Art. No ab62411)Substrate: Coagulation Factor XIa specific substrate (HYPHEN1310 med, Art. No. Biophen cs-21(66))Buffer: 100 mM tris-HCl, 200 mM NaCl, 0.02% Tween20, pH 7.42. Experimental Procedure20 mM of test compound dissolved in 100% DMSO was diluted to 200, 20, 2, 0.2, 0.02, 0.002 μM with 100% DMSO; 1 μl of the compound was added to each well in a 384-well plate, blank and control wells were replaced with DMSO. The plate was centrifuged to remove the compound to the bottom. 10 μl (2.5 μg/ml) of FXIa enzyme solution was added to each well, and 10 μl of buffer was added to the blank well. The plate was centrifuged to remove the enzyme solution to the bottom.Finally, 10 μl of 2 mM substrate were added to each well, and the plate was centrifuged to remove the substrate solution to the bottom.The plate was incubated for 10 minutes at 37° C.; and then the absorbance was measured at 405 nm. The absorbance was curve-fitted by graphpad
Measurement: IC50 = 35 nM (pChEMBL 7.460000038146973)
Descriptors: MW 553.01, ALogP 5.22, HBA 7, HBD 2, PSA 123.93, QED 0.31
Compound (SMILES): Cc1ncsc1-c1ccc(CNC(=O)[C@@H]2C[C@@H](O)CN2C(=O)[C@H](C(C)C)N2Cc3cccc(F)c3C2=O)cc1
InChIKey: HWJIMIISFDRVKH-VTZPFEBOSA-N
Target: von Hippel-Lindau disease tumor suppressor (SINGLE PROTEIN), Homo sapiens
UniProt: P40337
Assay: Fluorescence Polarization Assay (0.25% DMSO): Ability of VHL ligands to compete for the HIF 1 a binding site on VCB was determined through a fluorescence polarization competition assay as described in Buckley et al. JACS, 2012, 134, 4465-4468, WO 2013/106643, and US 2014-0356322, which are incorporated herein by reference in their entirety for all purposes (0.25% DMSO).
Measurement: IC50 = 3040 nM (pChEMBL 5.519999980926514)
Descriptors: MW 550.66, ALogP 3.52, HBA 6, HBD 2, PSA 102.84, QED 0.47
Compound (SMILES): O=c1[nH]c(SCc2cccnc2)nc2c(Cl)cccc12
InChIKey: SPZBRBFXOHJWFJ-UHFFFAOYSA-N
Target: High affinity 3',5'-cyclic-AMP phosphodiesterase 7A (SINGLE PROTEIN), Homo sapiens
UniProt: Q13946
Assay: Inhibition Assay: Measurement of PDE7 inhibition was carried out using a commercial phosphodiesterase activity measurement kit.Some of the compounds of the present invention (including those synthesised in Example 1) were evaluated. For this purpose, said compounds were incubated (in a range of concentrations from 0.1 nM to 100 μM) in the presence of 0.02 U/well of PDE7A1 and 0.05 μCi of [3H] cAMP, for 20 minutes at 30° C. in the assay buffer supplied with the kit (total volume per well=100 μL). Once this time had elapsed, 50 μL of a 20 mg/mL suspension of SPA glass silicate microspheres were added and kept in agitation at ambient temperature for 60 minutes. After allowing the plate to rest for 20 minutes, the radioactivity was tested in a scintillation detector. All the assays included two points in the absence of PDE7A1 (blank/negative control) and two points with PDE7A1 in the absence of inhibitors (positive control).
Measurement: IC50 = 540 nM (pChEMBL 6.269999980926514)
Descriptors: MW 303.77, ALogP 3.26, HBA 4, HBD 1, PSA 58.64, QED 0.6
Compound (SMILES): Cc1ccc(-n2nc(C(C)(C)C)cc2NC(=O)Nc2ncc(CCc3ccnc(NC(=O)OCc4ccccc4)c3)s2)cc1
InChIKey: UKJKWKMMZJXNCO-UHFFFAOYSA-N
Target: Mitogen-activated protein kinase 13 (SINGLE PROTEIN), Homo sapiens
UniProt: O15264
Assay: MAPK13 and MAPK14 enzyme assays: ach of these analogs were checked for physical chemical characteristics based on molecular weight, Lipinski's Rule of 5, partition coefficient (log P), and topological polar surface area (tPSA). Acceptable analogs were assessed for IC50 in MAPK13 and MAPK14 enzyme assays, using inhibition of the closely related MAPK14 as a surrogate for specificity against the broader kinome.
Measurement: IC50 = 625 nM (pChEMBL 6.199999809265137)
Descriptors: MW 609.76, ALogP 7.51, HBA 8, HBD 3, PSA 123.06, QED 0.16
Compound (SMILES): Cc1cccc(-c2nc3cc(Nc4nc(-c5cccc(C#N)c5)cs4)c(C)cc3[nH]2)c1
InChIKey: WRMPLAAISSHIGI-UHFFFAOYSA-N
Target: Mast/stem cell growth factor receptor Kit (SINGLE PROTEIN), Homo sapiens
UniProt: P10721
Assay: In Vitro TK Inhibition Assay: Colorimetric cell proliferation and viability assay (reagent CellTiter-Blue purchased from Promega cat N°G8081) was performed on BaF3 Kit WT cell line.A total of 2.104 cells/50 μl were seeded per well of a 96-wells plate. Treatment was initiated by addition of a 2× drug solution of ½ serial dilutions ranging from 0 to 10 μM. After incubating for 48 hours at 37° C., 10 μl of a ½ dilution of CellTiter-Blue reagent was added to each well and the plates were returned to the incubator for an additional 4 hours. The fluorescence intensity from the CellTiter-Blue reagent is proportional to the number of viable cells and data were recorded (544Ex/590Em) using a POLARstar OMEGA microplate reader (BMG LabteckSarl). A background control without cells was used as a blank. The positive control of the assay corresponds to the cell proliferation obtained in the absence of drug treatment (100% proliferation). Each sample was done in triplicate. The results were expressed as a percentage of the proliferation obtained in absence of treatment. All drugs were prepared as 20 mM stock solutions in DMSO and conserved at −80° C. Drug dilutions were made fresh in medium before each experiment. A DMSO control was included in each experiment.
Measurement: EC50 < 1000 nM (pChEMBL 6)
Descriptors: MW 421.53, ALogP 6.59, HBA 5, HBD 2, PSA 77.39, QED 0.34
Compound (SMILES): CCc1oc2ccc(/N=c3\[nH]c(=O)n(C[C@@H](C)NC(C)=O)c(=O)n3Cc3ccc(Cl)cc3)cc2c1C
InChIKey: PKUKFRPSLHTKMI-OAHLLOKOSA-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 = 14 nM (pChEMBL 7.849999904632568)
Descriptors: MW 509.99, ALogP 3.41, HBA 7, HBD 2, PSA 114.39, QED 0.4
Compound (SMILES): CC(C)(O)c1cnc2c(NC(=O)c3cc(-c4cc[nH]n4)c(C(F)(F)F)cc3F)n(-c3ccccc3)nc2c1
InChIKey: ODJPBHRPHYLZGR-UHFFFAOYSA-N
Target: High affinity nerve growth factor receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P04629
Assay: Biological Assay: TrkA functional activity was measured using a DiscoverX PathHunter assay. In this assay, U2OS cells express the human TrkA receptor as a fusion with the weakly complementing fragment of B-galactosidase, which DiscoverX calls Prolink (PK); additionally, Shc1 is fused with a larger fragment, which is called Enzyme Acceptor (EA). Activation of the TrkA receptor, upon NGF addition, results in the kinase domain being phosphorylated, resulting in subsequent recruitment of Shc1-EA protein. That recruitment results in an active B-galactosidase enzyme that is detected by addition of a chemiluminescent substrate. The human p75NTR protein was also expressed as a co-receptor for NGF.
Measurement: IC50 = 2.6 nM (pChEMBL 8.59000015258789)
Descriptors: MW 524.48, ALogP 5.45, HBA 6, HBD 3, PSA 108.72, QED 0.27
Compound (SMILES): N[C@]1(NC2CC2)CCCN(c2ccnc3[nH]cc(-c4ccnnc4)c23)C1
InChIKey: GCHWCYMGCVYULP-IBGZPJMESA-N
Target: Serine/threonine-protein kinase MRCK alpha (SINGLE PROTEIN), Homo sapiens
UniProt: Q5VT25
Assay: Kinase Assay: MRCKα, MRCKβ, ROCK1 and ROCK2 assays were performed using an IMAP fluorescence polarization assay format (Molecular Devices Inc.). 8-12 nM of each kinase (Life Technologies) was incubated for 60 min at room temperature with 100 nM FAM-S6-ribosomal protein derived peptide (synthesized by Alta Biosciences, University of Birmingham UK) in the presence of 1 μM ATP and 0.5 mM MgCl2 in 20 mM Tris buffer (pH 7.4) containing 0.01% Tween-20 and 1 mM DTT (MRCKα and β); or 1 μM ATP, 10 mM MgCl2 in 20 mM Tris buffer (pH 7.5) containing 0.25 mM EGTA 0.01% Triton X-100 and 1 mM DTT (ROCK1 and ROCK2). Typically, dose response analyses were performed over concentration ranges from 0.005-100 μM. Reactions were stopped by adding 2 assay volumes of 0.25% (v/v) IMAP binding reagent in 1×IMAP binding buffer A (Molecular Devices). After 30 min incubation to allow binding reagent to bind phosphorylated peptide, fluorescence polarization was measured on a Tecan Saphire2 plate reader at excitation (470 nm) and emission (530 nm) wavelengths. Inhibition was calculated using no inhibitor and no enzyme controls as 0 and 100% inhibition, respectively.
Measurement: Ki = 0.322 nM (pChEMBL 9.489999771118164)
Descriptors: MW 349.44, ALogP 2.03, HBA 6, HBD 3, PSA 95.75, QED 0.62
Compound (SMILES): Cc1cc(N)nc(C)c1CNC(=O)c1cn(Cc2ccc3ncc(C4CC4)cc3c2)nc1C#N
InChIKey: NXWBCYNOFIKWBT-UHFFFAOYSA-N
Target: Plasma kallikrein (SINGLE PROTEIN), Homo sapiens
UniProt: P03952
Assay: In Vitro Enzyme Inhibition: A 10 mM solution of the test compound was made in DMSO. This solution was serially diluted 1:5 in DMSO to yield 2000, 400, 80, 16, 3.2, 0.64, 0.128, 0.0256 and 0.00512 μM compound test solutions. A control tube containing only DMSO is included. 16 μL of each compound test solution was combined with 384 μL of assay buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.01% Triton X-100) to yield a 4× test compound buffer stock.Separately, a 40 nM solution of human Plasma Kallikrein (Abcam) and a 93.6 μM solution Pro-Phe-Arg-AMC (Bachem) were made using assay buffer. These solutions are hereby referred to as 4×hPK and 2×PFR-AMC, respectively.60 μL of each 4× test compound buffer stock was combined with 60 μL of 4×hPK to yield 120 μL of 2× test compound buffer stock/2×hPK. 50 μL was removed from this mixture and placed into duplicate wells on a Microfluor 1Black U-bottom microtiter plate (Thermo Scientific). This plate was incubated for 5 minutes at 37° C. To each well, 50 μL of pre-warmed 2×PFR-AMC was added to start the enzymatic reaction. Cleavage of PFR-AMC was monitored in a Biotek Synergy H4 reader set at 37° C. Readings are taken every 43 seconds for 1 hour. The highest mean velocity over 20 reads (15 minutes) is used to calculate the IC50. The IC50 is calculated using the Gen5 (Biotek Instruments).
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 451.53, ALogP 3.75, HBA 7, HBD 2, PSA 122.51, QED 0.46
Compound (SMILES): CC1(C)CN(C(=O)c2cccc(Cl)c2)CC/C1=C\C#Cc1ccc(F)cc1
InChIKey: RRMDVZQUPRLYLK-FBCYGCLPSA-N
Target: Metabotropic glutamate receptor 5 (SINGLE PROTEIN), Homo sapiens
UniProt: P41594
Assay: Radioligand Binding Assay at Cloned Human mGluR5 Receptor Subtype: Affinity at transmembrane glutamate metabotropic cloned human mGluR5 receptor was evaluated according to the methods of Anderson (Anderson et al., J Pharmacol. Exp. Ther., (2002), Vol. 303(3), pp. 1044-51), with some modifications. Human cloned mGluR5 was obtained by re-suspending CHO T-REx h-mGluR5 cells (50 μg/well) in 20 mM HEPES, 2 mM MgCl2, 2 mM CaCl2, pH 7.4, that then were incubated in a final volume of 1 ml for 60 min at 25° C. with 4 nM [3H]MPEP in the absence or presence of competing drugs. Non-specific binding was determined in the presence of 10 μM MPEP. The incubation was stopped by addition of cold Tris buffer pH 7.4 and rapid filtration through 0.2% polyethyleneimine pretreated Filtermat 1204-401 (Perkin Elmer) filters. The filters were then washed with cold buffer and the radioactivity retained on the filters was counted by liquid scintillation spectrometry (Betaplate 1204 BS-Wallac).
Measurement: Ki = 4.02 nM (pChEMBL 8.399999618530273)
Descriptors: MW 381.88, ALogP 5.33, HBA 1, HBD 0, PSA 20.31, QED 0.61
Compound (SMILES): CC(C)[C@@H]1CN(c2nc(C3CC3)c3c(c2C=N)CC(C)(C)OC3)CCN1C(=O)Cn1cncn1
InChIKey: JPMUICRQOFAYOX-NRFANRHFSA-N
Target: Isocitrate dehydrogenase [NADP] cytoplasmic (SINGLE PROTEIN), Homo sapiens
UniProt: O75874
Assay: Inhibition Assay: Assays were conducted in a volume of 76 μl assay buffer (150 mM NaCl, 10 mM MgCl2, 20 mM Tris pH 7.5, 0.03% bovine serum albumin) as follows in a standard 384-well plate: To 25 ul of substrate mix (8 uM NADPH, 2 mM aKG), 1 μl of test compound was added in DMSO. The plate was centrifuged briefly, and then 25 μl of enzyme mix was added (0.2 μg/ml IDH1 R132H) followed by a brief centrifugation and shake at 100 RPM. The reaction was incubated for 50 minutes at room temperature, then 25 μl of detection mix (30 μM resazurin, 36 μg/ml) was added and the mixture further incubated for 5 minutes at room temperature. The conversion of resazurin to resorufin was detected by fluorescent spectroscopy at Ex544 Em590 c/o 590.
Measurement: IC50 = 5500 nM (pChEMBL 5.260000228881836)
Descriptors: MW 465.6, ALogP 2.77, HBA 8, HBD 1, PSA 100.23, QED 0.66
Compound (SMILES): CCC(C)c1nc(-c2cnc(N)c(OC(F)(F)F)c2)cn1C12CC(N3CCOCC3)(C1)C2
InChIKey: BUVPPKZCXFYHEG-UHFFFAOYSA-N
Target: Mitogen-activated protein kinase kinase kinase 12 (SINGLE PROTEIN), Homo sapiens
UniProt: Q12852
Assay: Biological Activity Assay: The DLK dissociation constants (Kd) have been determined in the KINOMEscan KdELECT Service at DiscoveRx. A fusion protein of full length of human DLK (amino acids 1-859) and the DNA binding domain of NFkB was expressed in transiently transfected HEK293 cells. From these HEK 293 cells, extracts were prepared in M-PER extraction buffer (Pierce) in the presence of Protease Inhibitor Cocktail Complete (Roche) and Phosphatase Inhibitor Cocktail Set II (Merck) per manufacturers' instructions. The DLK fusion protein was labeled with a chimeric double-stranded DNA tag containing the NFkB binding site (5′-GGGAATTCCC-3′) fused to an amplicon for qPCR readout, which was added directly to the expression extract (the final concentration of DNA-tag in the binding reaction is 0.1 nM).
Measurement: Kd = 8.6 nM (pChEMBL 8.069999694824219)
Descriptors: MW 451.49, ALogP 3.9, HBA 7, HBD 1, PSA 78.43, QED 0.72
Compound (SMILES): CN[C@@H](C)C(=O)N[C@H]1CCc2ccccc2N(Cc2cccc3c(CN4CCNCC4)cccc23)C1=O
InChIKey: BHYOCBKZUPMHEF-IDISGSTGSA-N
Target: E3 ubiquitin-protein ligase XIAP (SINGLE PROTEIN), Homo sapiens
UniProt: P98170
Assay: TR-FRET Assay: The peptide AVPIAQKSEK-(ε-biotin)-OH (SEQ ID NO: 1) 1:2 TFA (Peptide A) was identified as a substrate for the TR-FRET assay by screening the 6× Histidine-tagged (SEQ ID NO: 2) BIR2 domain and BIR3 domain of XIAP against a set of 29 peptides synthesized based on sequences reported by Sweeny et al. (Biochemistry, 2006, 45, 14740 14748). The peptides were labeled with the fluorescent tags FITC or TAMRA and Kd values were determined by fluorescence polarization assay. The sequence AVPIAQKSEK (SEQ ID NO: 3) was identified as optimal for using in an assay. The peptide sequence was derivatized with biotin to provide AVPIAQKSEK-(ε-biotin)-OH (SEQ ID NO: 1) 1:2 TFA as the substrate for the TR-FRET assay.The XIAP protein sequence was obtained from the SWISS-PROT protein sequence database and the BIR2 and BIR3 domains were derived from that. The sequence of the BIR2 domain used for the TR-FRET assay is MRHHHHHHRDHFALDRPSETHADYLLRTGQVVDISDTIYPRNPAMYSEEARLKSFQNW PDYAHLTPRELASAGLYYTGIGDQVQCFACGGKLKNWEPGDRAWSEHRRHFPNCFFVL GRNLNIRSE (SEQ ID NO: 4).The sequence of the BIR3 domain used for the TR-FRET assay is MRHHHHHHRSDAVSSDRNFPNSTNLPRNPSMADYEARIFTFGTWIYSVNKEQLARAGF YALGEGDKVKCFHCGGGLTDWKPSEDPWEQHAKWYPGCKYLLEQKGQEYINNIHLTH SLEECLVRTT (SEQ ID NO: 5).Ten nanomolar of 6× Histidine-tagged (SEQ ID NO: 2) BIR2 domain, corresponding to amino acids 124-240 of XIAP, or BIR3 domain, corresponding to amino acids 241-356 of XIAP, was mixed with 20 nM of the peptide AVPIAQKSEK-(ε-biotin)-OH (SEQ ID NO: 1) 1:2 TFA, in the presence of 50 mM Tris-Cl, pH 7.5, 100 mM NaCl, 1 mM dithiothreitol (DTT) and 0.1 mg/niL bovine serum albumin (BSA). Following a 45 min. incubation at 37° C., Europium-Streptavidin and Allophycocyanin conjugated anti-Histidine antibody were added to a final concentration of 1.5 nM and 15 nM, respectively. Time-resolved fluorescence resonance energy transfer (TR-FRET) signals were measured 1 hour later at room temperature. Test compound potency was assessed at 10 serially diluted concentrations.
Measurement: IC50 = 2236 nM (pChEMBL 5.650000095367432)
Descriptors: MW 499.66, ALogP 2.82, HBA 5, HBD 3, PSA 76.71, QED 0.47
Compound (SMILES): Clc1ccc2[nH]c(-c3cnco3)cc2c1Oc1ccccc1
InChIKey: AHIQMHNAJPAEAY-UHFFFAOYSA-N
Target: GTP-binding protein Rheb (SINGLE PROTEIN), Homo sapiens
UniProt: Q15382
Assay: In Vitro Kinase Assay: For the Rheb-dependent in vitro kinase assay, HA-GST-Rheb was first loaded with GTP-γ-S (Millipore #20-176) by incubating 600 ng/μL Rheb with 10 mM EDTA and 0.4 mM GTP-γ-S at 30° C. for 10 minutes. MgCl2 was added to a final concentration of 20 mM to stop the reaction. This mixture was used at 20× in the kinase assay.Loaded HA-GST-Rheb was then incubated with compound (final DMSO concentration 1%) for 30 minutes at room temperature. mTORC1, GFP-4E-BP1 substrate (Life Technologies #PV4759), and kinase assay buffer (HEPES pH 7.4, KCl, MgCl2) were added to the Rheb-compound mixture and incubated at room temperature for 20 minutes. ATP was then added, and the kinase reaction proceeded at 30° C. for 45 min before being stopped by the addition of SDS-PAGE loading buffer for Westerns or 10 mM EDTA for the LanthaScreen readout. During the kinase reaction, the final concentration of reagents is as follows: Rheb30 ng/μL, compoundvariable, GTPγS0.02 mM, EDTA0.5 mM, MgCl21 mM, mTORC1fixed volume of prep (molarity unknown), GFP-4E-BP1 substrate15 ng/μL, ATP0.5 mM. For LanthaScreen analysis (Thermo Fisher #PV4757), the manufacturer's instructions were followed and conditions were optimized for the amount of Rheb and mTORC1. The final concentration of reagents was as follows: 100 nM loaded HA-GST-Rheb, compound (variable), 0.5 mM EDTA, 10 mM MgCl2, 25 mM HEPES pH 7.4, 50 mM KCl, mTORC1 (fixed volume0.1 μL in 10 μL total volume), 0.4 μM GFP-4E-BP1 substrate, 0.5 mM ATP. Optimization is summarized in FIG. 1. Results were read on an EnVision reader (PerkinElmer) using 495ex/520em filters (Life Technologies, #PV00315).
Measurement: IC50 = 55000 nM (pChEMBL 4.260000228881836)
Descriptors: MW 310.74, ALogP 5.27, HBA 3, HBD 1, PSA 51.05, QED 0.56
Compound (SMILES): O=C(C[C@@H]1CCc2cc(C(O)(C(F)(F)F)C(F)(F)F)ccc2N1S(=O)(=O)c1ccc(F)cc1)N[C@@H](CO)Cc1c[nH]cn1
InChIKey: BGVXOKUVBXSBBS-UXHICEINSA-N
Target: Nuclear receptor ROR-gamma (SINGLE PROTEIN), Homo sapiens
UniProt: P51449
Assay: Spa Binding Assay: The binding of potential ligands to RORγ is measured by competition with [3H] 25-hydroxycholesterol using a scintillation proximity assay (SPA) binding assay. The ligand binding domain of human RORγ (A262-S507) with an N-terminal His tag is expressed in E. coli and purified using nickel affinity chromotography. 50 nM RORγ (A262-S507) is incubated with test compound at varying concentrations for 15 min at room temperature in PBS buffer containing 0.5% fatty acid free BSA. 10 nM of [3H] 25-hydroxycholesterol is then added, and the reaction is incubated for 15 min. 4 mg/mL of Ysi Copper HIS-TAG SPA Beads (Perkin Elmer) are added, and the mixture is incubated for 30 min. The reaction is read on a MicroBeta Trilux scintillation plate reader (Perkin Elmer).
Measurement: IC50 = 95.3 nM (pChEMBL 7.019999980926514)
Descriptors: MW 638.56, ALogP 3.48, HBA 6, HBD 4, PSA 135.62, QED 0.27
Compound (SMILES): COc1cc(N2CCN(C)CC2)ccc1Nc1nccc(-c2cc(F)c3nc(C)n(C(C)C)c3c2)n1
InChIKey: VYGCBAWMDOWVPT-UHFFFAOYSA-N
Target: Epidermal growth factor receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P00533
Assay: Kinase Inhibition Assay: Reagents and Materials:WT EGFR (Carna, Cat. No. 08-115), EGFR [L858R] (Carna, Cat. No. 08-502), EGFR [L858R/T790M] (Carna, Cat. No. 08-510), ATP (Sigma, Cat. No. A7699-1G), DMSO (Sigma, Cat. No. D2650), 96-well plate (Corning, Cat. No. 3365), 384-well plate (Greiner, Cat. No. 784076), HTRF Kinase TK Kit (Cisbio, Cat. No. 62TK0PEJ), Erlotinib (Selleckchem, Cat. No. S7787), EGFR [d746-750] (Life Technologies, Cat. No. PV6178), 5× Kinase Buffer A (Life Technologies, Cat. No. PV3186), Kinase Tracer 199 (Life Technologies, Cat. No. PV5830), LanthaScreen® Eu-anti-GST antibody (Life Technologies, Cat. No. PV5594).Specific Experimental Protocol:Compound preparation: the test compound was dissolved in DMSO to make a 20 mM stock solution. Then, it was diluted in DMSO with a 3-fold series gradient dilution for 10 times. The dilutions were diluted 10 fold with buffer when dosing.WT EGFR and EGFR [L858R/T790M] kinase assay: WT EGFR or EGFR [L858R/T790M] kinase was mixed with different concentrations of pre-diluted compounds for 10 minutes in 5× Kinase Buffer A in duplicate. The corresponding substrate and ATP were added and reacted at room temperature for 20 minutes (in which a negative and a positive control were set: the negative control is blank and the positive control is erlotinib). After the reaction, the detection reagent (the reagent in the HTRF Kinase TK kit) was added, and after incubation at room temperature for 30 minutes, the enzyme activity in the presence of the compounds of the present disclosure at each concentration was measured by an Evnvision microplate reader, and the inhibition of the enzyme by the compound at each concentrations were calculated. The inhibitions of the enzyme activity by the compounds at different concentrations were then fitted using Graphpad 5.0 software according to the four-parameter equation, and the IC50 values were calculated.
Measurement: IC50 = 25 nM (pChEMBL 7.599999904632568)
Descriptors: MW 489.6, ALogP 5.03, HBA 8, HBD 1, PSA 71.34, QED 0.41
Compound (SMILES): O=C(NC1CC1)c1nc2cccnc2n1-c1ccc(Cl)cc1F
InChIKey: JPYAMLLEZNRELS-UHFFFAOYSA-N
Target: 3',5'-cyclic-AMP phosphodiesterase 4B (SINGLE PROTEIN), Homo sapiens
UniProt: Q07343
Assay: Scintillation Proximity Assay (SPA): The PDE4B and 4D assays use scintillation proximity assay (SPA) technology to measure the inhibition of human recombinant PDE4B and PDE4D enzyme activity by compounds in vitro. The PDE4B and 4D assays are run in parallel using identical parameters, except for the concentration of enzyme (32 pM PDE4B and 16 pM PDE4D). The assays are performed in a 384-well format with 50 μL assay buffer (50 mM Tris pH 7.5; 1.3 mM MgCl2; 0.01% Brij) containing enough PDE4B and PDE4D to convert 20% of substrate (1 μM cAMP consisting of 20 nM 3H-cAMP+980 μM cold cAMP) and a range of inhibitors. Reactions are incubated for 30 min at 25° C. The addition of 20 μL of 8 mg/mL yitrium silicate SPA beads (PerkinElmer) stops the reaction. The plates are sealed (TopSeal, PerkinElmer) and the beads are allowed to settle for 8 hrs, after which they are read on the Trilux Microbeta overnight.
Measurement: IC50 = 632 nM (pChEMBL 6.199999809265137)
Descriptors: MW 330.75, ALogP 3.11, HBA 4, HBD 1, PSA 59.81, QED 0.8
Compound (SMILES): O=C(N[C@H](CO)C(F)(F)F)c1cc(N2CCC(c3n[nH]c4ncccc34)CC2)nc(OC[C@H]2CCCO2)n1
InChIKey: RXKXYXHACAMGAS-CRAIPNDOSA-N
Target: Tyrosine-protein kinase receptor UFO (SINGLE PROTEIN), Homo sapiens
UniProt: P30530
Assay: Enzyme Assay: AXL enzyme inhibition (% inhibition, Kiapp and Ki values) by small molecule inhibitors was evaluated using a fluorescence-based microfluidic mobility shift assay. AXL catalyzes the production of ADP from ATP that accompanies the phosphoryl transfer to the substrate peptide FL-Peptide-30 (5-FAM-KKKKEEIYFFF-CONH2, CPC Scientific, Sunnyvale, Calif.). The mobility shift assay electrophoretically separates the fluorescently labeled peptides (substrate and phosphorylated product) following the kinase reaction. Both substrate and product are measured and the ratio of these values is used to generate % conversion of substrate to product by the LabChip EZ Reader. Human wild-type receptor tyrosine kinase protein Axl comprising residues 505-811 was produced in-house using the baculoviral expression vector system that incorporated a hexahistidine affinity tag into the protein (LJIC-1916B1.1). The enzyme was preactivated by auto-phosphorylation of 34 uM non-activated enzyme in the presence of 2 mM ATP, 4 mM MgCl2, 50 mM NaCl and 1 mM TCEP in 20 mM HEPES, pH 7.3 at 4° C. for 30 minutes. Typical reaction solutions (50 μL final reaction volume) contained 2% DMSO (±inhibitor), 10 mM MgCl2, 1 mM DTT, 120 μM ATP (ATP Km=70.4 μM), 0.01% Tween-20, 3 μM FL-Peptide-30, and 0.5 nM phosphorylated AXL enzyme in 100 mM HEPES buffer at pH 7.3. The assay was initiated with the addition of ATP, following a fifteen minutes pre-incubation of enzyme and inhibitor at room temperature in the reaction mixture. The reaction was stopped after 30 minutes at 25° C. by the addition of 50 μL of 200 mM EDTA, pH 7.5. The Ki values were determined from the fit of the data to the Morrison tight-binding competitive inhibition equation with the enzyme concentration as a variable. (See, Morrison, J. F. (1969) Kinetics of the reversible inhibition of enzyme-catalysed reactions by tight-binding inhibitors, Biochimica et biophysica acta 185, 269-286; and Murphy, D. J. (2004) Determination of accurate KI values for tight-binding enzyme inhibitors: an in silico study of experimental error and assay design, Analytical biochemistry 327, 61-67.)
Measurement: Ki = 4.22 nM (pChEMBL 8.380000114440918)
Descriptors: MW 535.53, ALogP 2.34, HBA 9, HBD 3, PSA 138.38, QED 0.4
Compound (SMILES): Cc1csc(O[C@@H]2CCOc3cc(-c4ccnc(Nc5ccnn5C)n4)cc4nnc2n34)n1
InChIKey: ZWWWUZHELAGQTF-OAHLLOKOSA-N
Target: Mitogen-activated protein kinase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P28482
Assay: ERK-2 Enzymatic Assay: Compounds were tested in an enzymatic assay using human ERK-2 (Mitogen Activated Kinase 1), recombinantly expressed as an n-terminal 6-His fusion protein in E. coli and corresponding to aa 8-360. The substrate used was the fluorescent Omnia peptide S/T17 (Invitrogen of Carlsbad, Calif.; Cat. KNZ1171C). Test compounds were diluted in DMSO in 3-fold serial dilutions at 100× final concentrations. In addition to compound, the assay contained 50 mM HEPES [pH 7.3], 10 mM MgCl2, 2 mM DTT, 0.005% Triton-X100, 5 nM ERK-2 enzyme, 6.25 μM S/T17 peptide substrate and 25 μM ATP (corresponding to the observed Km) for a total reaction volume of 25 μL. The assay was run at ambient temperature in a white 384-well polypropylene plate (Nunc, Inc of Naperville, Ill.; Cat. 267462) collecting data every 50 seconds for approximately 30 minutes on an Envision plate reader (PerkinElmer, Inc. of Waltham, Mass.); Excitation 340 nm/Emission 495 nm. The data collected from each well was fit to a straight line, and the resulting rates were used to calculate percent of control. Percent of control was plotted against compound concentration, and IC50 values were determined using a four-parameter fit. Table 1 contains representative data for compounds disclosed herein.
Measurement: IC50 < 0.02 nM (pChEMBL 10.699000358581543)
Descriptors: MW 461.51, ALogP 3.33, HBA 12, HBD 1, PSA 117.17, QED 0.42
Compound (SMILES): COc1ccc(CN2C(=O)N(C)C(=O)C23CCN(C(=O)[C@H](NC(=O)c2cc(C(F)(F)F)ccc2F)C(C)C)CC3)c(F)c1
InChIKey: UQAFTGVVBVVLNA-HSZRJFAPSA-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 610.58, ALogP 4.2, HBA 5, HBD 1, PSA 99.26, QED 0.37
Compound (SMILES): NC(=O)c1cc(-c2ccc(F)cc2F)c2c(n1)[C@@H]1CC[C@H](C2)O1
InChIKey: MTMLJAALOFBSKB-PSLIRLAXSA-N
Target: Metabotropic glutamate receptor 2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q14416
Assay: Biological Assays: The utility of the compounds of the invention as an inhibitor of metabotropic glutamate receptor activity, in particular mGluR2 activity, may be demonstrated by methodology known in the art and as described as follows. Inhibition constants (IC50s; the concentration of compound required to provide 50% of maximal activity) are determined as follows. The compounds of the invention were tested in a fluorescence laser imaging plate reader based assay. This assay is a common functional assay to monitor Ca2+ mobilization in whole cells expressing recombinant receptor coupled with a promiscuous G-protein. CHO (Chinese hamster ovary) dhfr− cells stably expressing recombinant human mGluR2 and Gα16 loaded with Fluo-4 AM (Invitrogen, Carlsbad Calif., USA) were treated with various concentrations of each of the tested compounds of the invention and the Ca2+ response was monitored on a FLIPR384 instrument (Molecular Devices, Sunnydale Calif., USA). Maximal agonist activity was measured in the presence of 2,500 nM glutamate and the inhibition provided by a range of compound concentrations sufficient to minimally and maximally inhibit the glutamate-dependent response was monitored over time. The maximum calcium response at each concentration of compound for agonist or antagonist were plotted as dose responses and the curves were fitted with a four parameter logistic equation giving IC50 and Hill coefficient using the iterative non-linear curve fitting software ADA (Merck & Co., Inc.). Data in the following table lists the activity of each compound to inhibit glutamate-dependent mGluR2 activity in this cellular assay.
Measurement: IC50 = 21 nM (pChEMBL 7.679999828338623)
Descriptors: MW 316.31, ALogP 2.9, HBA 3, HBD 1, PSA 65.21, QED 0.93
Compound (SMILES): Cc1c(C(=O)C(=O)NC2(c3c[nH]nn3)CC2)c2n(c1C(=O)Nc1cccc(C(F)F)c1)CCC2
InChIKey: MADCJRUEHZZXGR-UHFFFAOYSA-N
Target: Hepatic sodium/bile acid cotransporter (SINGLE PROTEIN), Homo sapiens
UniProt: Q14973
Assay: HBV DNA Quantification Assay: A HepG2 cell line overexpressing the HBV virus attachment receptor sodium-taurocholate cotransporting polypeptide (NTCP) was grown to confluency in DMEM growth medium, Dulbecco's Modified Eagle Medium without sodium pyruvate (Life Technologies, Rockville, Md.) supplemented with 10% FBS (Thermo Scientific, Waltham, Md.), 1% penicillin/streptomycin (Life Technologies, Rockville, Md.) and 2 mM L-glutamine (Life Technologies, Rockville, Md.) in T175 flasks. Cells were infected with HBV AD38 viral particles (Texcell, Frederick, USA) at 4000 genome equivalents per cell. After allowing viral infection to take place for 4 days, the infected cells were harvested from the flasks by trypsinization, washed twice with OptiMEM (Life Technologies, Rockville, Md.) and re-suspended in DMEM containing 2% FBS and 1% DMSO at a density of 0.25E6 cells/ml. Infected cells were seeded on 384 well collagen coated plates (Greiner, Austria) at a density of 20,000 cells/well containing serially diluted compounds of the present disclosure or DMSO (0.5%) in a final volume of 80 μl. The assay plates were incubated for a period of 5 days and the antiviral activity of the test compounds were assayed by detecting the presence of HBV DNA in the culture supernatant using the QuantiGene 2.0 nucleic acid quantification kit (Affymetrix, Santa Clara, Calif.).The culture supernatant was harvested and treated with lysis buffer containing Proteinase K (Affymetrix, Santa Clara, Calif.). The supernatant was incubated with HBV viral DNA specific probes (Affymetrix, Santa Clara, Calif.) for 30 minutes at 55° C. This was followed by addition of 0.2M NaOH for 30 minutes at room temperature to denature the DNA, followed by addition of Neutralization buffer (Affymetrix, Santa Clara, Calif.). The resulting lysed and neutralized supernatant was then added to QuantiGene 2.0 384 well plates coated with capture oligonucleotides and incubated overnight at 55° C. The HBV specific probe set consists of Capture Extender oligonucleotides (CE's) and blocking probes. Following the overnight incubation, the wells were incubated for one hour sequentially with a Pre-Amplifier, Amplifier and Labeled probes conjugated to alkaline phosphatase with a wash step between incubations. After the final wash step, the alkaline phosphatase substrate (Luminol APS5) was added and the resulting luminescence signal was read in an EnVision Multilabel Plate Reader (PerkinElmer, Santa Clara, Calif.). The EC50 values were calculated from the fit of the doseresponse curves to a four-parameter equation. All EC50 values represent geometric mean values of a minimum of four determinations.
Measurement: EC50 = 40.8 nM (pChEMBL 7.389999866485596)
Descriptors: MW 468.46, ALogP 3.04, HBA 6, HBD 3, PSA 121.77, QED 0.36
Compound (SMILES): Cn1cc2c([C@H](Nc3cc(Cl)c4ncc(C#N)c(NCC(C)(C)C)c4c3)C3=CN(C4CC4)NN3)cccc2n1
InChIKey: UAQDZKILDBSBGI-NDEPHWFRSA-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 = 2 nM (pChEMBL 8.699999809265137)
Descriptors: MW 542.09, ALogP 5.59, HBA 9, HBD 4, PSA 105.86, QED 0.24
Compound (SMILES): CO[C@H](C(=O)N1CCN(c2ccc(-c3cc(OCCCO)cn4ncc(C#N)c34)cn2)CC1)c1ccccc1
InChIKey: XSFRMRKLGPGNSN-NDEPHWFRSA-N
Target: Proto-oncogene tyrosine-protein kinase receptor Ret (SINGLE PROTEIN), Homo sapiens
UniProt: P07949
Assay: RET Enzyme Assay: Compounds of Formula I were screened for their ability to inhibit wildtype and V804M mutant RET kinase using CisBio's HTRF® KinEASE¿-TK assay technology. Briefly, N-terminal GST tagged recombinant human RET cytoplasmic domain (aa 658-end) from Eurofins (0.25 nM RET; Catalog No. 14-570M) or N-terminal GST tagged recombinant human V804M mutant RET cytoplasmic domain (aa 658-end) from Millipore (0.25 nM enzyme; Catalog No. 14-760) was incubated with 250 nM TK-substrate biotin (CisBio, part of Catalog No. 62TK0PEC) and 1 mM ATP along with test compound in a buffer consisting of 25 mM HEPES pH 7.4, 10 mM MgCl2, 0.01% Triton X-100, and 2% DMSO in a volume of 8 μL. Compounds were typically prepared in a threefold serial dilution in DMSO and added to the assay to give the appropriate final concentration. After a 30-minute incubation at 22° C., the reaction was quenched by adding 8 μL of quench solution containing 31.25 nM Sa-XL665 and 1×TK-ab-Cryptate in HTRF detection buffer (all from CisBio, part of Cat. No. 62TK0PEC). After a 1 hour incubation at 22° C., the extent of reaction was determined using a PerkinElmer EnVision multimode plate reader via HTRF dual wavelength detection, and the percent of control (POC) was calculated using a ratiometric emission factor. 100 POC was determined using no test compounds and 0 POC was determined using pre-quenched control reactions. The POC values were fit to a 4 parameter logistic curve, and the IC50 is defined as the concentration of inhibitor at which the POC equals 50 for the fitted curve.
Measurement: IC50 = 69.2 nM (pChEMBL 7.159999847412109)
Descriptors: MW 526.6, ALogP 3.07, HBA 9, HBD 1, PSA 116.22, QED 0.33
Compound (SMILES): COc1cc(-c2cccc(-c3cccc(NC(=O)c4cn(C)c(=O)n(C)c4=O)c3C)c2C)cc(F)c1CNC[C@H]1CCC(=O)N1
InChIKey: HOEIQNQJXILPMO-JOCHJYFZSA-N
Target: Programmed cell death 1 ligand 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9NZQ7
Assay: Enzyme-Linked Immunosorbent AssayELISA: 96 Well plates were coated with 111 g/mL of human PD-L1 (obtained from R&D) in PBS overnight at 4° C. The wells were then blocked with 2% BSA in PBS (W/V) with 0.05% TWEEN-20 for 1 hour at 37° C. The plates were washed 3 times with PBS/0.05% TWEEN-20 and the compounds were serial diluted (1:5) in dilution medium and added to the ELISA plates. Human PD-1 and biotin 0.31 μg/mL (ACRO Biosystems) were added and incubated for 1 hour at 37° C. then washed 3 times with PBS/0.05% TWEEN-20. A second block was performed with 2% BSA in PBS (W/V)/0.05% TWEEN-20 for 10 min at 37° C. and the plates were washed 3 times with PBS/0.05% TWEEN-20. Streptavidin-HRP was added for 1 hour at 37° C. then the plates were washed 3 times with PBS/0.05% TWEEN-20. TMB substrate was added and reacted for 20 min at 37° C. A stop solution (2 N aqueous H2SO4) was added. The absorbance was read at 450 nm using a micro-plate spectrophotometer.
Measurement: IC50 < 10 nM (pChEMBL 8)
Descriptors: MW 613.69, ALogP 3.8, HBA 8, HBD 3, PSA 123.46, QED 0.26
Compound (SMILES): N#Cc1cnc(N2CCC[C@@H](Nc3nc(-c4cnn5ccccc45)nc(OCCO)c3F)C2)cn1
InChIKey: KNWWIXHOFCTMFC-OAHLLOKOSA-N
Target: Tyrosine-protein kinase JAK3 (SINGLE PROTEIN), Homo sapiens
UniProt: P52333
Assay: In Vitro JAK kinase Assays: The catalytic domains of human JAK1 (aa 850-1154), JAK2 (aa 826-1132), JAK3 (aa 795-1124) and Tyk2 (aa 871-1187) were expressed as N-terminal GST-fusion proteins using a baculovirus expression system and were purchased from Carna Biosciences. The enzymatic activity was assayed using as substrate a biotinylated peptide, poly (GT)-Biotin (CisBio). The peptide concentration in the reactions was 60 nM for JAK1, 20 nM for JAK2, 140 nM for JAK3 and 50 nM for Tyk2. The degree of phosphorylation was detected by TR-FRET (time-resolved fluorescence energy transfer).IC50s of compounds were measured for each kinase in a reaction mixture containing the enzyme, ATP and the peptide in 8 mM MOPS (pH 7.0), 10 mM MgCl2, 0.05% β-mercaptoethanol, 0.45 mg/ml BSA. The ATP concentration in the reactions was 3 μM for JAK1, 0.2 μM for JAK2, 0.6 μM for JAK3 and 1.8 μM for Tyk2. The enzymatic reactions took place for 30 minutes at room temperature. Then, the reactions were stopped with 20 μL of quench detection buffer (50 mM HEPES, 0.5 M KF, EDTA 0.25 M, 0.1% (w/v) BSA, pH 7.5) containing 0.115 μg/mL of anti-phosphoTyr (PT66)-Cryptate (CisBio) and a variable concentration of SA-XL665 (CisBio) to keep the SA-B ratio constant. Incubate for 3 h and read on Victor 2V spectrofluorometer (Perkin Elmer) set to read fluorescence resonance energy transfer.
Measurement: IC50 = 2 nM (pChEMBL 8.699999809265137)
Descriptors: MW 475.49, ALogP 2.04, HBA 11, HBD 2, PSA 137.38, QED 0.41
Compound (SMILES): Nc1nnc(N2CCC(N3CCS(=O)(=O)CC3Cc3ccc(Cl)cc3)CC2)[nH]1
InChIKey: CCZUIJXVLDHFRC-UHFFFAOYSA-N
Target: Acidic mammalian chitinase (SINGLE PROTEIN), Homo sapiens
UniProt: Q9BZP6
Assay: Human AMCase Activity Assay: An enzymatic assay with recombinant human AMCase was used in order to establish inhibitory activity of the compounds (Boot et al., 2001, JBC: 276). The assay was run in the 96-well plate format, each reaction in the total volume of 100 μL. 4-Methylumbelliferyl B-D-N,N′-diacetylchitobioside hydrate was used as a substrate for the enzyme. Upon hydrolysis by AMCase, the substrate releases 4-methylumbelliferyl (4MU) that, when ionized in basic pH, emits fluorescence at 460 nm.Briefly, 40 μL of a substrate was added to each well, followed by 10 μL of compound dilution and 50 μL of hAMCase recombinant enzyme solution. The reaction was carried out in citrate buffer, pH 5.2, in the dark, at 37° C. for 60 minutes with shaking. After that time the reaction was stopped by adding 195 μL of Stop Buffer (pH 10.5) to each well. The fluorescence of the reaction product was measured in Perkin Elmer Envision fluorescent plate reader at an excitation wavelength of 355 nm. The IC50 values were calculated using GraphPad Prism.
Measurement: IC50 = 550 nM (pChEMBL 6.260000228881836)
Descriptors: MW 424.96, ALogP 1.35, HBA 7, HBD 2, PSA 108.21, QED 0.76
Compound (SMILES): CC(C)(OC(=O)Nc1ccc2c(c1)N(S(=O)(=O)c1ccc(F)c(Cl)c1)C[C@H](CNC(=O)C13CC(C1)C3)O2)C(F)(F)F
InChIKey: CTWZOBITQRQWPT-JLBFDCLDSA-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 = 6.59 nM (pChEMBL 8.180000305175781)
Descriptors: MW 620.02, ALogP 5.24, HBA 6, HBD 2, PSA 114.04, QED 0.41
Compound (SMILES): OCCN1CCN(c2cccc(Nc3ncc4[nH]c(-c5ccccc5Cl)cc4n3)c2)CC1
InChIKey: DFYXCLCXXSZGCX-UHFFFAOYSA-N
Target: Receptor-interacting serine/threonine-protein kinase 2 (SINGLE PROTEIN), Homo sapiens
UniProt: O43353
Assay: RIPK2 Inhibition Assay: RIPK2 activity was measured using an indirect ELISA detection system. HisRIPK2 (0.6 nM) was incubated in the presence of 6 μM ATP (Sigma cat # A7699), 20 mM Hepes, pH 7.5, 1 mM EGTA, 2.5 mM MgCl2, 2.5 mM MnCl2 and 0.01% Triton X-100 in a 96 well microtitre plate pre-coated with amino terminal 6 histidine, sumo tagged TTK (amino acid residues 1-275). The reaction was allowed to proceed for 30 minutes, followed by 5 washes of the plate with Wash Buffer (phosphate buffered saline supplemented with 0.2% Tween 20), and incubation for 30 minutes with a 1:3000 dilution of primary antibody (Cell Signaling cat #9381). The plate was washed 5 times with Wash Buffer, incubated for 30 minutes in the presence of secondary antibody coupled to horse radish peroxidase (BioRad cat #1721019, 1:3000 concentration), washed an additional 5 times with Wash Buffer, and incubated in the presence of TMB substrate (Sigma cat # T0440). The colorimetric reaction was allowed to continue for 5 minutes, followed by addition of stop solution (0.5 N H2SO4), and quantified by detection at 450 nm with either a monoChromatic or filter based plate reader (Molecular Devices M5 or Beckman DTX880, respectively).
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 448.96, ALogP 4.14, HBA 6, HBD 3, PSA 80.31, QED 0.41