lovingscience/chembl-v37-raw-activities huggingface dataset:
Compound (SMILES): C=C1CN(C)C(=O)Cn2nc(Nc3cc4cc(-c5cnccc5C)c(F)c(N)c4cn3)cc21
InChIKey: MGXWVFDZJJYEBG-UHFFFAOYSA-N
Target: Mitogen-activated protein kinase kinase kinase kinase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q92918
Assay: HPK1-FL HTRF Enzymatic Assay: To a 384 well Proxiplate with 80 nL compound or DMSO spotted on was added 4 μl/well kinase mix. The mixture was preincubated for 30 minutes and then 4 μl/well substrate mix was added. The solution was incubated for 60 min and then 4 μl/well detection mix was added. The solution was incubated for another 60 min. The plates were then loaded onto a Perkin Elmer Envision and the TR-FRET signal was measured at 615 and 665 nm. A ratio of 665/620 was used to calculate the % activity at each concentration of compound.
Measurement: Ki = 0.043 nM (pChEMBL 10.369999885559082)
Descriptors: MW 443.49, ALogP 3.75, HBA 7, HBD 2, PSA 101.96, QED 0.47
Compound (SMILES): CCC(CC)n1cc(C(=O)NC2CCc3ccc(C)cc32)c2cc(OC)c(OC)cc2c1=O
InChIKey: NKSDYAQSTNXSMY-UHFFFAOYSA-N
Target: cAMP and cAMP-inhibited cGMP 3',5'-cyclic phosphodiesterase 10A (SINGLE PROTEIN), Homo sapiens
UniProt: Q9Y233
Assay: Enzymatic Assay: The PDE enzymatic reaction was carried out in assay buffer (20 mM Tris-HCl pH7.5, 10 mM MgCl2, 0.1% bovine serum albumin) containing enzyme and substrate. The PDE enzymes concentration ranged from 10 pM 250 pM, depending on each enzyme's specific activity. The substrate cyclic nucleotide (cAMP or cGMP) concentration used in the assay was 20 nM for PDE10, and 100 nM for other PDEs. The inhibitory effect of compound was determined by incubating various concentration of inhibitor in the enzymatic assay. Typically, compound was serial diluted in DMSO then further diluted in assay buffer. Next, the compound at varying concentration was mixed with PDE enzyme. The reaction was initiated by addition of cyclic nucleotide substrate, and incubated for 60 minutes at 29 C. The reaction was stopped by addition of lysis buffer from assay kit. The cAMP-d2 and anti-cAMP cryptate in the lysis buffer detected the level of cAMP left from the PDE hydrolysis reaction. The PDE activity is reversely correlated with the amount of cAMP left in the reaction and can be converted to the percent activity of an uninhibited control (100%).
Measurement: IC50 = 150 nM (pChEMBL 6.820000171661377)
Descriptors: MW 448.56, ALogP 5.11, HBA 5, HBD 1, PSA 69.56, QED 0.55
Compound (SMILES): N#Cc1ccc(C(=O)N[C@@H](CCCN[C@@H]2C[C@H]2c2ccc(F)cc2)C(=O)N2CCS(=O)(=O)CC2)cc1
InChIKey: ZLYXPWDMIDPPKL-KMDXXIMOSA-N
Target: Lysine-specific histone demethylase 1A (SINGLE PROTEIN), Homo sapiens
UniProt: O60341
Assay: In Vitro Assay: Briefly, bulk histones, histone peptides or nucleosomes are incubated with purified human recombinant KDM1A, in the histone demethylase activity (HDM) assay buffer 1 (50 mM Tris pH 8.5, 50 mM KCl, 5 mM MgCl, 0.5% BSA, and 5% glycerol) from 30 minutes to 4 hours at 37° C. A typical reaction is conducted in 100 microliters in which either 20 micrograms of purified bulk histones or 3 micrograms of modified histone peptides are used as substrates. Different amounts of KDM1A ranging from 1-20 micrograms are used in the reaction along with, as necessary, other co-factors such as FAD or CoREST, depending on the chosen substrate. The reaction mixture is analyzed by SDS-PAGE and Western blotting using histone methyl-specific antibodies or by formaldehyde formation assay to examine the removal and conversion of the methyl group to formaldehyde, or by mass spectrometry in the case of peptide substrates to identify the demethylated histone peptide.
Measurement: IC50 = 3000 nM (pChEMBL 5.519999980926514)
Descriptors: MW 512.61, ALogP 1.98, HBA 6, HBD 2, PSA 119.37, QED 0.5
Compound (SMILES): C=CC(=O)N1CC[C@]2(C1)C[C@H](n1nc(-c3ccc(C(F)(F)F)cc3)c(C(N)=O)c1N)C2
InChIKey: UNFXTUAGYXFPMI-OYOVHJISSA-N
Target: Tyrosine-protein kinase BTK (SINGLE PROTEIN), Homo sapiens
UniProt: Q06187
Assay: BTK Assay: TBDAn HTRF assay (Cisbio KinEASE-TK cat #62TKOPEC) was performed to quantitate the ability of test compounds to inhibit BTK mediated phosphorylation of substrate. Assays were assembled in 384 well plates where 6 nM of full-length human His-tagged BTK (Life Technologies cat # PV3587) and test compound at varying concentrations were preincubated for 15 minutes at 28° C. Then, 1 uM of TK substrate-biotin and 30 uM ATP were added and incubated for an additional 30 minutes at 28° C. Phospohrylation was detected by adding 62.5 nM Streptavidin-XL665 and TK-Antibody Cryptate diluted 1:100 in HTRF detection buffer (Cisbio cat #62SDBRDF) and incubated for 60 minutes at RT. The plate was read on an Envision plate reader and the fluorescence is measured at 620 nm (cryptate) and 665 nm (XL665). A ratio is calculated (665/620) and converted to POC relative to control and blank wells.
Measurement: IC50 = 6.4 nM (pChEMBL 8.1899995803833)
Descriptors: MW 433.43, ALogP 2.99, HBA 5, HBD 2, PSA 107.24, QED 0.72
Compound (SMILES): O=C(NC(CCOCCCCc1ccc2c(n1)NCCC2)C(=O)O)c1c(Cl)cccc1Cl
InChIKey: BMYVUXVAXJDFAV-UHFFFAOYSA-N
Target: Integrin alpha-V/beta-6 (PROTEIN COMPLEX), Homo sapiens
UniProt: P06756
Assay: Binding Assay: The biochemical potency of compounds was determined using a proximity-based assay (ALPHASCREEN®, Perkin Elmer, Waltham, Mass.) as described previously (Ullman E F et al., Luminescent oxygen channeling immunoassay: Measurement of particle binding kinetics by chemiluminescence. Proc. Natl. Acad. Sci. USA, Vol. 91, pp. 5426-5430, June 1994). To gauge the potency of inhibitors of binding to human integrin αvβ6, inhibitor compounds and integrin were incubated together with TGF-b1 LAP and biotinylated anti-LAP antibody plus acceptor and donor beads, following the manufacture's recommendations. The donor beads were coated with streptavidin. The acceptor beads had a nitrilotriacetic acid Ni chelator, for binding to a 6×His Tag on human integrin αvβ6. All incubations occurred at room temperatures in 50 mM Tris-HCl, pH 7.5, 0.1% BSA supplemented with 1 mM each CaCl2 and MgCl2.The order of reagent addition was as follows:1. Alpha-v-beta-6 integrin, test inhibitor compound, LAP, biotinylated anti-LAP antibody and acceptor beads were all added together.2. After 2 hours, donor beads were added. After another 30 minute incubation, samples were then read.Integrin binding was evaluated by exciting donor beads at 680 nm, and measuring the fluorescent signal produced, between 520-620 nm, using a Biotek Instruments (Winooski, Vt., USA) SynergyNeo2 multimode plate reader.
Measurement: IC50 = 150 nM (pChEMBL 6.820000171661377)
Descriptors: MW 480.39, ALogP 4.36, HBA 5, HBD 3, PSA 100.55, QED 0.41
Compound (SMILES): N#Cc1cc(-c2ncnc3[nH]c(-c4ccc(N5CCN(CC(F)F)CC5)cc4)nc23)ccc1OC1CCNCC1
InChIKey: GEMZAQKPULZSJL-UHFFFAOYSA-N
Target: Serine/threonine-protein kinase TBK1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9UHD2
Assay: Biological Assay for TBK1 and IKKepsilon: Enzymatic activity of IKKε and TBK1 was measured using a homogeneous time resolved fluorescence resonance energy transfer (TR-FRET) assay that monitors enzyme dependent phosphorylation of a biotinylated serine/threonine peptide substrate. An increase in the amount of phopshorylated peptide results in an increase in TR-FRET signal. TBK1 and IKKε were expressed and purified as full length recombinant proteins. Detection reagents for the assay were purchased from Cisbio. TBK1 and IKKε enzymes were assayed under initial rate conditions in the presence of 2×Km ATP (40-80 μM) and 1 μM peptide, hepes (pH 7), 0.1 mM orthovanadate, 0.02% NaN3, 0.01% BSA, 10 mM MgCl2, 0.01% (v/v) tritonX, 1 mM dithiothreitol, 0.5% (v/v) DMSO at the following concentrations for each enzyme: TBK1 at 2.5 mM and IKKε at 0.3 nM. After an assay reaction time of 240 minutes at 25° C., reactions were terminated with EDTA.Amount of phosphorylated peptide was determined by the addition of 125 nM streptavidin XL665 and europium cryptate labeled anti-phospho monoclonal antibody and the resulting TR-FRET signal was recorded on an Envision plate reader (Ex: 340 nm; Em: 615/665 nm; 100 μs delay and 200 μs read window). Data was normalized based on a positive (1 μM Staurosporine) and negative (DMSO) controls and IC50 values calculated from the fit of the dose-response curves to a four-parameter equation. All IC50 values represent geometric mean values of a minimum of four determinations. These assays generally produced results within 3-fold of the reported mean.
Measurement: IC50 = 16.2 nM (pChEMBL 7.789999961853027)
Descriptors: MW 544.61, ALogP 4.08, HBA 8, HBD 2, PSA 105.99, QED 0.36
Compound (SMILES): CCn1nc(CCCOC(=O)c2ccc(C(=O)N3CCOCC3)cc2)c2c1C(=O)NCC1(CCOCC1)C2
InChIKey: DGSOYRANHJANOQ-UHFFFAOYSA-N
Target: 3',5'-cyclic-AMP phosphodiesterase 4D (SINGLE PROTEIN), Homo sapiens
UniProt: Q08499
Assay: PDE4 Assay: The human PDE4D catalytic domain (UniProt no. Q08499 [S380-L740]) was incubated with a mixture of non-labelled cAMP (cyclic adenosine monophosphate) and fluorescein amidite (FAM) conjugated cAMP and titrated test or reference Compound. Following brief incubation the enzymatic reaction was stopped by addition of binding buffer containing nanoparticles with immobilized trivalent metal ions capable of binding 1) AMP phospho groups and 2) terbium (Tb) donor fluorophores. Subsequent excitation of the Tb donor triggers time-resolved FRET to adjacent FAM acceptor molecules resulting in light emission. In the presence of a PDE4 inhibitor, AMP generation was reduced resulting in a lower fluorescence signal. The cAMP phosphodiester is not bound by the detection system.The results were calculated as the molar concentrations resulting in 50% inhibition of the substrate cleavage compared to controls samples, and are expressed as a range of IC50 (nM).
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 524.62, ALogP 2.25, HBA 8, HBD 1, PSA 111.99, QED 0.44
Compound (SMILES): CC(=O)n1cc(NC(=O)Nc2cn(C(C)=O)c3ccccc23)c2ccccc21
InChIKey: XIAJBEMWPKMHPB-UHFFFAOYSA-N
Target: Complement factor D (SINGLE PROTEIN), Homo sapiens
UniProt: P00746
Assay: Inhibition 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 10 μM each. The increase in color is recorded at OD405 nm in a microplate in kinetic mode over 30 minutes with 30 second time points in a spectrofluorimeter. IC50 values are calculated by non-linear regression from the percentage of inhibition of complement factor D activity as a function of test compound concentration.
Measurement: IC50 > 10000 nM (pChEMBL 5)
Descriptors: MW 374.4, ALogP 4.56, HBA 5, HBD 2, PSA 85.13, QED 0.54
Compound (SMILES): CN(c1ccccc1CNc1nc(-c2cc(F)c(O)cc2CC(F)(F)F)cc(N)c1C=N)S(C)(=O)=O
InChIKey: IBZKOYSOUCDPJP-UHFFFAOYSA-N
Target: Tyrosine-protein kinase JAK1 (SINGLE PROTEIN), Homo sapiens
UniProt: P23458
Assay: JAK Caliper Enzyme Assay at 1 mM ATP: Test article was solubilized in dimethyl sulfoxide (DMSO) to a stock concentration of 30 mM. An 11-point half log dilution series was created in DMSO with a top concentration of 600 μM. The test compound plate also contained positive control wells containing a known inhibitor to define 100% inhibition and negative control wells containing DMSO to define no inhibition. The compound plates were diluted 1 to 60 resulting in a top final assay compound concentration of 10 μM and a 2% DMSO concentration.Test article and assay controls were added to a 384-well plate. Reaction mixtures contained 20 mM HEPES, pH 7.4, 10 mM magnesium chloride, 0.01% bovine serum albumin (BSA), 0.0005% Tween 20, 1 mM ATP and 1 μM peptide substrate. The JAK1 and TYK2 assays contained 1 μM of the IRStide peptide (5FAM-KKSRGDYMTMQID) and the JAK2 and JAK3 assays contained 1 μM of the JAKtide peptide (FITC-KGGEEEEYFELVKK). The assays were initiated by the addition of 20 nM JAK1, 1 nM JAK2, 1 nM JAK3 or 1 nM TYK2 enzyme and were incubated at room temperature for three hours for JAK1, 60 minutes for JAK2, 75 minutes for JAK3 or 135 minutes for TYK2. Enzyme concentrations and incubation times were optimized for each new enzyme preps and were modified slightly over time to ensure 20%-30% phosphorylation. The assays were stopped with a final concentration of 10 mM EDTA, 0.1% Coating Reagent and 100 mM HEPES, pH=7.4. The assay plates were placed on a Caliper Life Science Lab Chip 3000 (LC3000) instrument, and each well was sampled using appropriate separation conditions to measure the unphosphorylated and phosphorylated peptide.
Measurement: IC50 = 1.8 nM (pChEMBL 8.739999771118164)
Descriptors: MW 525.53, ALogP 4.29, HBA 7, HBD 4, PSA 132.4, QED 0.26
Compound (SMILES): CC1=C(N2CC[C@]3(CC[C@H](N(CCO)Cc4ccc(-n5cnnn5)nc4)CC3)C2=O)COC1=O
InChIKey: YHNQCIRYSHDPBD-QRTMHTFLSA-N
Target: ATP-sensitive inward rectifier potassium channel 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P48048
Assay: Thallium Flux Assay: A Thallium Flux Assay was performed on each of the final product compounds of the Examples. This assay has been described previously; see, e.g., PCT Published Application WO 2013/062900.
Measurement: IC50 = 160 nM (pChEMBL 6.800000190734863)
Descriptors: MW 467.53, ALogP 0.84, HBA 10, HBD 1, PSA 126.57, QED 0.59
Compound (SMILES): CNCC[C@@H](OCc1cccc(N2CCN(C)c3nc(SC)ncc3C2=O)c1)c1cccs1
InChIKey: QZTSBVNAYFBWIC-HXUWFJFHSA-N
Target: Voltage-dependent calcium channel subunit alpha-2/delta-1 (SINGLE PROTEIN), Homo sapiens
UniProt: P54289
Assay: Binding Assay: α2δ-1: Binding Assay to Human α2δ-1 Subunit of Cav2.2 Calcium Channel.Human α2δ-1 enriched membranes (2.5 μg) were incubated with 15 nM of radiolabeled [3H]-Gabapentin in assay buffer containing Hepes-KOH 10 mM, pH 7.4.NSB (non specific binding) was measured by adding 10 μM pregabalin. After 60 min incubation at 27° C., binding reaction was terminated by filtering through Multiscreen GF/C (Millipore) presoaked in 0.5% polyethyleneimine in Vacuum Manifold Station, followed by 3 washes with ice-cold filtration buffer containing 50 mM Tris-HCl, pH 7.4.Filter plates were dried at 60° C. for 1 hour and 30 μl of scintillation cocktail were added to each well before radioactivity reading.Readings were performed in a Trilux 1450 Microbeta radioactive counter (Perkin Elmer).
Measurement: Ki = 300 nM (pChEMBL 6.519999980926514)
Descriptors: MW 483.66, ALogP 4.22, HBA 8, HBD 1, PSA 70.59, QED 0.36
Compound (SMILES): CN1CCCC1CCNC(=O)c1ccc(Nc2nc3c(N4CCC(O)(c5ccc(Cl)cc5)CC4)cccn3n2)cc1
InChIKey: WZNRQBDJUAKYLO-UHFFFAOYSA-N
Target: Tyrosine-protein kinase JAK1 (SINGLE PROTEIN), Homo sapiens
UniProt: P23458
Assay: Enzyme Assay: The activity of the isolated recombinant JAK1 and JAK2 kinase domain was measured by monitoring phosphorylation of a peptide derived from JAK3 (Val-Ala-Leu-Val-Asp-Gly-Tyr-Phe-Arg-Leu-Thr-Thr, fluorescently labeled on the N-terminus with 5-carboxyfluorescein) using the Caliper LabChip® technology (Caliper Life Sciences, Hopkinton, Mass.). To determine inhibition constants (Ki), compounds were diluted serially in DMSO and added to 50 μL kinase reactions containing purified enzyme (1.5 nM JAK1, or 0.2 nM JAK2), 100 mM HEPES buffer (pH 7.2), 0.015% Brij-35, 1.5 μM peptide substrate, ATP (25 μM), 10 mM MgCl2, 4 mM DTT at a final DMSO concentration of 2%. Reactions were incubated at 22° C. in 384-well polypropylene microtiter plates for 30 minutes and then stopped by addition of 25 μL of an EDTA containing solution (100 mM HEPES buffer (pH 7.2), 0.015% Brij-35, 150 mM EDTA), resulting in a final EDTA concentration of 50 mM. After termination of the kinase reaction, the proportion of phosphorylated product was determined as a fraction of total peptide substrate using the Caliper LabChip® 3000 according to the manufacturer's specifications.
Measurement: Ki = 1.73 nM (pChEMBL 8.760000228881836)
Descriptors: MW 574.13, ALogP 4.83, HBA 8, HBD 3, PSA 98.03, QED 0.28
Compound (SMILES): CCOc1cc(F)c(Cn2nc(-c3ncc(OC)c(Nc4ccncc4)n3)c(Br)c2C)c(F)c1
InChIKey: QKYAVYXVDRYVBQ-UHFFFAOYSA-N
Target: Mitotic checkpoint serine/threonine-protein kinase BUB1 (SINGLE PROTEIN), Homo sapiens
UniProt: O43683
Assay: Time-Resolved Fluorescence Energy Transfer (TR-FRET) Kinase Assay: In a typical assay 11 different concentrations of each compound (0.1 nM, 0.33 nM, 1.1 nM, 3.8 nM, 13 nM, 44 nM, 0.15 μM, 0.51 μM, 1.7 μM, 5.9 μM and 20 μM) were tested in duplicate within the same microtiter plate. To this end, 100-fold concentrated compound solutions (in DMSO) were previously prepared by serial dilution (1:3.4) of 2 mM stocks in a clear low volume 384-well source microtiter plate (Greiner Bio-One, Frickenhausen, Germany), from which 50 nL of compounds were transferred into a black low volume test microtiter plate from the same supplier. Subsequently, 2 μL of Bub1 (the final concentration of Bub1 was adjusted depending on the activity of the enzyme lot in order to be within the linear dynamic range of the assay: typically 200 ng/mL were used) in aqueous assay buffer [50 mM Tris/HCl pH 7.5, 10 mM magnesium chloride (MgCl2), 200 mM potassium chloride (KCl), 1.0 mM dithiothreitol (DTT), 0.1 mM sodium ortho-vanadate, 1% (v/v) glycerol, 0.01% (w/v) bovine serum albumine (BSA), 0.005% (v/v) Trition X-100 (Sigma), 1× Complete EDTA-free protease inhibitor mixture (Roche)] were added to the compounds in the test plate and the mixture was incubated for 15 min at 22° C. to allow pre-equilibration of the putative enzyme-inhibitor complexes before the start of the kinase reaction, which was initiated by the addition of 3 μL 1.67-fold concentrated solution (in assay buffer) of adenosine-tri-phosphate (ATP, 10 μM final concentration) and peptide substrate (1 μM final concentration). The resulting mixture (5 μL final volume) was incubated at 22° C. during 60 min., and the reaction was stopped by the addition of 5 μL of an aqueous EDTA-solution (50 mM EDTA, in 100 mM HEPES pH 7.5 and 0.2% (w/v) bovine serum albumin) which also contained the TR-FRET detection reagents (0.2 μM streptavidin-XL665 [Cisbio Bioassays, Codolet, France] and 1 nM anti-phosho-Serine antibody [Merck Millipore, cat. #35-001] and 0.4 nM LANCE EU-W1024 labeled anti-mouse IgG antibody [Perkin-Elmer, product no.
Measurement: IC50 = 24 nM (pChEMBL 7.619999885559082)
Descriptors: MW 531.36, ALogP 5.28, HBA 8, HBD 1, PSA 86.98, QED 0.33
Compound (SMILES): Cc1nn(C(C)c2cc(Cl)c(C#N)c3c2O[C@H](C)CN(Cc2cccnc2)C3)c2ncnc(N)c12
InChIKey: OKQDZXAQQNFYAC-IURRXHLWSA-N
Target: Phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit delta isoform (SINGLE PROTEIN), Homo sapiens
UniProt: O00329
Assay: Scintillation Proximity Assay: The kinase reaction was conducted in polystyrene 384-well matrix white plate from Thermo Fisher Scientific in a final volume of 25 μL. Inhibitors were first diluted serially in DMSO and added to the plate wells before the addition of other reaction components. The final concentration of DMSO in the assay was 0.5%. The PI3K assays were carried out at room temperature in 20 mM MOPS, pH 6.7, 10 mM MgCl2, 5 mM DTT and CHAPS 0.03%. Reactions were initiated by the addition of ATP, the final reaction mixture consisted of 20 M PIP2, 20 M ATP, 0.2 μCi [γ-33P]ATP, 4 nM PI3Kδ. Reactions were incubated for 210 min and terminated by the addition of 40 μL SPA beads suspended in quench buffer: 150 mM potassium phosphate pH 8.0, 20% glycerol. 25 mM EDTA, 400 M ATP. The final concentration of SPA beads was 1.0 mg/mL. After the plate sealing, plates were shaken overnight at room temperature and centrifuged at 1800 rpm for 10 minutes, the radioactivity of the product was determined by scintillation counting on Topcount (Perkin-Elmer). IC50 determination was performed by fitting the curve of percent control activity versus the log of the inhibitor concentration using the GraphPad Prism 3.0 software.
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 488.98, ALogP 4.03, HBA 9, HBD 1, PSA 118.77, QED 0.46
Compound (SMILES): CCCCn1c(C)nc2c(NS(C)(=O)=O)cc(-c3cc(OC)c(=O)n(C)c3)cc21
InChIKey: HBCJKSTXPDACPP-UHFFFAOYSA-N
Target: Bromodomain-containing protein 4 (SINGLE PROTEIN), Homo sapiens
UniProt: O60885
Assay: In Vitro Enzyme Inhibition Assay: Determination of the IC50 for the heterocyclic derivative BRD4 inhibitors disclosed herein was performed as follows. His-tagged BREM was cloned, expressed and purified to homogeneity. Filipakopoulos et al., 468 Nature 1067 (2010). BRD4 binding and inhibition was assessed by monitoring the interaction of biotinylated H4-tetraacetyl peptide (AnaSpec, H4K5/8/12/16(Ac), biotinlabeled) with the target using the AlphaScreen technology (Life Technologies). In a 384-well ProxiPlate BRD4(BD1) (2 nM final) was combined with peptide (15 nM final) in 50 mM HEPES (pH 7.3), 10 mM NaCl, 0.25 mM TCEP, 0.1% (w/v) BSA, and 0.005% (w/v) Brij-35 either in the presence of DMSO (final 0.4% DMSO) or compound dilution series in DMSO. After 20 min incubation at RT, Alpha streptavidin donor beads and Nickel Chelate acceptor beads were added to a final concentration of 5 μg/mL. After 2 hr of equilibration, plates were read on an Envision instrument and the IC50 was calculated using a four parameter non-linear curve fit.
Measurement: IC50 < 500 nM (pChEMBL 6.301000118255615)
Descriptors: MW 418.52, ALogP 2.89, HBA 7, HBD 1, PSA 95.22, QED 0.64
Compound (SMILES): CC(=O)Nc1cc2c(-c3ccc(C(=O)O)cc3F)cn(C(=O)c3c(Cl)cccc3C(F)(F)F)c2cn1
InChIKey: RPWHNSYDDRMDCE-UHFFFAOYSA-N
Target: Nuclear receptor ROR-gamma (SINGLE PROTEIN), Homo sapiens
UniProt: P51449
Assay: Biological Assays: The compounds of the invention inhibit RORgammaT activity. Activation of RORgammaT activity can be measured using, e.g., biochemical TR-FRET assay. In such an assay, interaction of cofactor-derived peptides with human RORgammaT-Ligand Binding Domain (LBD) can be measured. The TR-FRET technique is a sensitive biochemical proximity assay that will give information concerning the interaction of a ligand with the LBD, in the presence of cofactor-derived peptides (Zhou et al., Methods 25:54-61, 2001).To identify novel antagonists of RORgammaT, an assay was developed which employs the interaction of RORgammaT with its co-activator peptide SRC1_2. This peptide mimics the recruitment of co-activators to RORgammaT through its interaction with the LXXLL (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 expressed in SF9 cells using a baculovirus expression system. The RORγ-LBD protein was purified by glutathione sepharose chromatography. Separately, SF9 cells not expressing any recombinant protein were lysed and the lysate was added to the purified RORγ-LBD at 0.25 μl lysate (from 10,000 SF9 cells)/nM purified protein. The mixture was then diluted in assay buffer (50 mM Tris pH 7.0, 50 mM KCl, 1 mM EDTA, 0.1 mM DTT) to obtain RORγ-LBD final concentration of 3 nM in 384-well assay plate.Compounds to be tested were injected to the assay plate using Acoustic Droplet Ejection technology by Echo 550 liquid handler (Labcyte, Calif.).A stock of biotinylated-LXXLL peptide from coactivator SRC1 (Biotin-CPSSHSSLTERHKILHRLLQEGSPS) (SEQ ID NO:2) was prepared in assay buffer and added to each well (100 nM final concentration). A solution of Europium tagged anti-HIS antibody (1.25 nM final concentration) and APC conjugated streptavidin (8 nM final concentration) were also added to each well.The final assay mixture was incubated overnight at 4° C., and the fluorescence signal was measured on an Envision plate reader: (Excitation filter=340 nm; APC emission=665 nm; Europium emission=615 nm; dichroic mirror=D400/D630; delay time=100 μs, integration time=200 μs). IC50 values for test compounds were calculated from the quotient of the fluorescence signal at 665 nm divided by the fluorescence signal at 615 nm.
Measurement: IC50 = 6 nM (pChEMBL 8.220000267028809)
Descriptors: MW 519.84, ALogP 5.86, HBA 5, HBD 2, PSA 101.29, QED 0.33
Compound (SMILES): N#Cc1ncc(N2CCC3(CC2)COC(=O)N3CC2CC2)nc1-c1cnn(C(F)F)c1
InChIKey: BXXRGVMJHSTUBZ-UHFFFAOYSA-N
Target: Phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit delta isoform (SINGLE PROTEIN), Homo sapiens
UniProt: O00329
Assay: Biochemical Assay: The PI3-Kinase biochemical assay was optimized using the HTRF kit provided by Upstate (Millipore). The assay kit contains six reagents: 1) 4×Reaction Buffer; 2) native PIP2 (substrate); 3) Stop (EDTA); 4) Detection Mix A (Streptavidin-APC); 5) Detection Mix B (Eu-labeled Anti-GST plus GST-tagged PH-domain): 6) Detection Mix C. In addition, the following items were obtained or purchased; PI3Kinase (catalog #'s alpha 14-602, beta 14-603, gamma 14-558 and delta 14-604 from Upstate Biotechnology now EMD Millipore, Mahopac, N.Y., USA), dithiothreitol (Sigma-Aldrich Corp. (St. Louis, Mo., USA) Catalog #D-5545), Adenosine-5′ triphosphate (InVitrogen (Waltham, Mass., USA), Cat#AS001 Å), native PIP3 (PI(3,4,5)P3, diC8, H+, CELLSIGNALS, INC., (Columbus, Ohio, USA) Cat #907) DMSO ((Sigma-Aldrich Corp. (St. Louis. Mo., USA) Catalog #472301).PI3Kinase Reaction Buffer is prepared by dilution the stock 1:4 with de-ionized water. DTT (DL-Dithiothreitol), phosphatidylinositol 4,5-bisphosphate (PIP2) and Biotin-PIP3 were added to 1536 assay plate at a final concentration of 5 mM, 5 mM and 25 nM on the day of use. Enzyme addition and compound pre-incubation are initiated by the addition of 1.25 ul of PI3K (at twice its final concentration) in the 1× reaction buffer to all wells using a BioRaptor. Plates are incubated at room temperature for 15 minutes. Reactions are initiated by addition of 1.25 ul of 2× substrate solution (PIP2 and ATP in 1× reaction buffer) using BioRaptor. Plates are incubated in humidified chamber at room temperature for one hour. Reactions are quenched by addition of 0.625 uL of stop solution to all wells using the BioRaptor. The quenched reactions are then processed to detect product formation by adding 0.625 uL of Detection Solution to all wells using the BioRaptor (Detection mix C, Detection Mix A and Detection Mix B combined together in an 18:1:1 ratio prepared 2 hours prior to use). Following a one hour incubation in the dark, the HTRF signal is measured on the Envision plate reader set for 330 nm excitation and dual emission detection at 620 nM (Eu) and 665 nM (APC).
Measurement: IC50 = 54 nM (pChEMBL 7.269999980926514)
Descriptors: MW 429.43, ALogP 2.81, HBA 8, HBD 0, PSA 100.17, QED 0.72
Compound (SMILES): CCOc1c(C(C)n2nc(C)c3c(N)ncnc32)cc(C)c(C#N)c1C1CN(CCO)C1
InChIKey: GRMHNISBQAWILC-UHFFFAOYSA-N
Target: Phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit delta isoform (SINGLE PROTEIN), Homo sapiens
UniProt: O00329
Assay: Scintillation Proximity Assay: [γ-33P]ATP (10 mCi/mL) was purchased from PerkinElmer (Waltham, Mass.). Lipid kinase substrate, D-myo-Phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2)D (+)-sn-1,2-di-O-octanoylglyceryl, 3-O-phospho linked (PIP2), CAS 204858-53-7, was purchased from Echelon Biosciences (Salt Lake City, Utah). PI3Kδ (p110δ/p85α) was purchased from Millipore (Bedford, Mass.). ATP, MgCl2, DTT, EDTA, MOPS and CHAPS were purchased from SigmaAldrich (St. Louis, Mo.). Wheat Germ Agglutinin (WGA) YSi SPA Scintillation Beads was purchased from GE healthcare life sciences (Piscataway, N.J.).The kinase reaction was conducted in polystyrene 384-well matrix white plate from Thermo Fisher Scientific in a final volume of 25 μL Inhibitors were first diluted serially in DMSO and added to the plate wells before the addition of other reaction components. The final concentration of DMSO in the assay was 0.5%. The PI3K assays were carried out at room temperature in 20 mM MOPS, pH 6.7, 10 mM MgCl2, 5 mM DTT and CHAPS 0.03%. Reactions were initiated by the addition of ATP, the final reaction mixture consisted of 20 μM PIP2, 20 μM ATP, 0.2 μCi [γ-33P] ATP, 4 nM PI31(6. Reactions were incubated for 210 min and terminated by the addition of 40 μL SPA beads suspended in quench buffer: 150 mM potassium phosphate pH 8.0, 20% glycerol. 25 mM EDTA, 400 μM ATP. The final concentration of SPA beads was 1.0 mg/mL. After the plate sealing, plates were shaken overnight at room temperature and centrifuged at 1800 rpm for 10 minutes, the radioactivity of the product was determined by scintillation counting on Topcount (PerkinElmer).
Measurement: IC50 < 10 nM (pChEMBL 8)
Descriptors: MW 435.53, ALogP 2.3, HBA 9, HBD 2, PSA 126.11, QED 0.58
Compound (SMILES): Cc1cc(Oc2ccccc2)ccc1N1C(=O)Nc2c(C(=O)N[C@H]3CC[C@@H](NC#N)CC3)sc3nccc1c23
InChIKey: CNAXPCKRKWDXJD-KDURUIRLSA-N
Target: Tyrosine-protein kinase BTK (SINGLE PROTEIN), Homo sapiens
UniProt: Q06187
Assay: Kinase Lanthascreen Binding Assay: A BTK kinase lanthascreen binding assay monitors compound binding to unphosphorylated-BTK kinase domain (UP-BTK), by competing with a fluorescent labeled tracer. UP-BTK, consisting of the kinase domain of non-phosphorylated BTK protein (389-659aa), was produced in a Baculovirus/insect cell expression system. Into a 384-well plate, 2 ng of GST-tagged human BTK (389-659aa) was incubated with compound, 50 nM of Tracer 236 and 2 nM anti-GST antibody for 60 minutes using an optimized Lanthascreen assay. After 60 minutes, plates were read at 340 nM and 615/665 nM in an Infinite F500 (Tecan).
Measurement: IC50 = 170 nM (pChEMBL 6.769999980926514)
Descriptors: MW 538.63, ALogP 6.19, HBA 7, HBD 3, PSA 119.38, QED 0.2
Compound (SMILES): C[C@H]1CC[C@H](c2ccccc2)S(=O)(=O)N1Cc1ccc([N+]2([O-])CCC(n3cnnc3)CC2)cc1F
InChIKey: HPNPPXMTFCYSMO-YHMCQLTHSA-N
Target: Nuclear receptor ROR-gamma (SINGLE PROTEIN), Homo sapiens
UniProt: P51449
Assay: Binding Assay: Assays were carried out in 16-microL reaction volumes in black 384 Plus F Proxiplates (Perkin-Elmer 6008269). All assay components except test ligand were mixed in coregulator buffer D (Invitrogen PV4420) containing 5 mM DTT and added to the plate at twice their final concentrations in a volume of 8 microL. Test ligands at 2× the final concentration were then added to the wells in 8 □L of coregulator buffer D containing 5 mM DTT and 4% DMSO. Final incubations contained 1× coregulator buffer D, 5 mM DTT, test ligand, 2% DMSO, 50 nM biotinyl-CPSSHSSLTERKHKILHRLLQEGSPS (American Peptide Company; Vista, Calif.), 2 nM Europium anti-GST (Cisbio 61GSTKLB), 12.5 nM streptavidin-D2 (Cisbio 610SADAB), 50 mM KF, and 10 nM of bacterially-expressed human RORc ligand binding domain protein containing an N-terminal 6×His-GST-tag and residues 262-507 of Accession NP_005051. Ten test ligand concentrations were tested in duplicate. After the reaction plates were incubated for 3 h in the dark at room temperature (22-23° C.), the plate was read on an EnVision plate reader (PerkinElmer) following the Europium/D2 HTRF protocol (ex 320, em 615 and 665, 100 Os lag time, 100 flashes, 500 μs window). The time-resolved FRET signal at 665 nm was divided by that at 615 nm to generate the signal ratio of each well.
Measurement: IC50 = 680 nM (pChEMBL 6.170000076293945)
Descriptors: MW 499.61, ALogP 4.31, HBA 6, HBD 0, PSA 91.15, QED 0.39
Compound (SMILES): CN1CCN(c2ccc(Nc3ncc4c(n3)N3C(CC4=O)C(=O)NCC3(C)C)nc2)CC1
InChIKey: UNHLUCCQTMZQPA-UHFFFAOYSA-N
Target: Cyclin-dependent kinase 4/cyclin D1 (PROTEIN COMPLEX), Homo sapiens
UniProt: P24385
Assay: Inhibition In Vitro Assay: Selected compounds disclosed herein were tested in CDK4/cyclinD1, CDK2/CycA and CDK2/cyclinE kinase assays by Nanosyn (Santa Clara, Calif.) to determine their inhibitory effect on these CDKs. The assays were performed using microfluidic kinase detection technology (Caliper Assay Platform). The compounds were tested in 12-point dose-response format in singlicate at Km for ATP. Phosphoacceptor substrate peptide concentration used was 1 μM for all assays and Staurosporine was used as the reference compound for all assays. Specifics of each assay are as described below:CDK2/CyclinA: Enzyme concentration: 0.2 nM; ATP concentration: 50 μM; Incubation time: 3 hr.CDK2/CyclinE: Enzyme concentration: 0.28 nM; ATP concentration: 100 μM; Incubation time: 1 hr.CDK4/CyclinD1: Enzyme concentration: 1 nM; ATP concentration: 200 μM; Incubation time: 10 hr.
Measurement: IC50 = 8060 nM (pChEMBL 5.090000152587891)
Descriptors: MW 436.52, ALogP 1.04, HBA 9, HBD 2, PSA 106.59, QED 0.73
Compound (SMILES): C=CC(=O)N1CC(CCn2c(=O)ccc3cnc(Nc4ccc(N5CCN(C(C)=O)C[C@@H]5C)cc4)nc32)C1
InChIKey: CJLZIKXVQFSTSK-IBGZPJMESA-N
Target: Tyrosine-protein kinase JAK1 (SINGLE PROTEIN), Homo sapiens
UniProt: P23458
Assay: Biochemical JAK and Tyk2 Kinase Assays: A panel of four LanthaScreen JAK biochemical assays (JAK1, 2, 3 and Tyk2) were carried in a common kinase reaction buffer (50 mM HEPES, pH 7.5, 0.01% Brij-35, 10 mM MgCl2, and 1 mM EGTA). Recombinant GST-tagged JAK enzymes and a GFP-tagged STAT1 peptide substrate were obtained from Life Technologies. Serially or discretely diluted compounds were pre-incubated with each of the four JAK enzymes and the substrate in white 384-well microplates (Corning) at ambient temperature for 1 h. ATP was subsequently added to initiate the kinase reactions in 10 μL total volume, with 1% DMSO. The final enzyme concentrations for JAK1, 2, 3 and Tyk2 are 4.2 nM, 0.1 nM, 1 nM, and 0.25 nM respectively; the corresponding Km ATP concentrations used are 25 μM, 3 μM, 1.6 μM, and 10 μM; while the substrate concentration is 200 nM for all four assays. Kinase reactions were allowed to proceed for 1 hour at ambient temperature before a 10 μL preparation of EDTA (10 mM final concentration) and Tb-anti-pSTAT1 (pTyr701) antibody (Life Technologies, 2 nM final concentration) in TR-FRET dilution buffer (Life Technologies) was added. The plates were allowed to incubate at ambient temperature for 1 h before being read on the EnVision reader (Perkin Elmer). Emission ratio signals (520 nm/495 nm) were recorded and utilized to calculate the percent inhibition values based on DMSO and background controls.For dose-response analysis, percent inhibition data were plotted vs. compound concentrations, and IC50 values were determined from a 4-parameter robust fit model with the Prism software (GraphPad Software). Results were expressed as pIC50 (negative logarithm of IC50) and subsequently converted to pKi (negative logarithm of dissociation constant, Ki) using the Cheng-Prusoff equation.
Measurement: Ki > 100 nM (pChEMBL 7)
Descriptors: MW 515.62, ALogP 2.63, HBA 8, HBD 1, PSA 103.67, QED 0.48
Compound (SMILES): CC(C)C[C@@H](CN)C(=O)N1CCN(c2ccc(-c3nc(-c4cnn(C)c4)cn4ncc(C#N)c34)cn2)CC1
InChIKey: FIDLSJCDNCPBIF-FQEVSTJZSA-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. 62TKOPEC) 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. 62TKOPEC). 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 = 59.7 nM (pChEMBL 7.21999979019165)
Descriptors: MW 512.62, ALogP 2.33, HBA 10, HBD 1, PSA 134.26, QED 0.4
Compound (SMILES): CCS(=O)(=O)c1ccc(Cl)cc1Cn1c(=O)[nH]c2c(Cl)c(CN3CC[C@@H](O)C3)c(C(F)(F)F)cc2c1=O
InChIKey: UMCXMINCIYEHKL-CQSZACIVSA-N
Target: Epithelial discoidin domain-containing receptor 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q08345
Assay: Binding Activity: DDR1 binding activity was measured using the LanthaScreen(Registered trademark) Eu Kinase Binding Assay (manufactured by Life Technologies Corporation). The test compound and the Alexa Fluor® 647-labeled Kinase Tracer 178 (manufactured by Life Technologies Corporation) were added to a mixture of DDR1 and the LanthaScreen(Registered trademark) Eu-anti-GST antibody. After one-hour reaction at room temperature, the fluorescence resonance energy transfer was measured. The 50% inhibitory concentration (IC50) was calculated from the inhibition rate relative to the test compound-free control.
Measurement: IC50 = 49 nM (pChEMBL 7.309999942779541)
Descriptors: MW 580.41, ALogP 3.42, HBA 7, HBD 2, PSA 112.47, QED 0.46
Compound (SMILES): CC1(C)CN(c2ccc(F)cc2NC(=O)Nc2ccc(F)cc2F)c2c(O)ccc(C(F)(F)F)c21
InChIKey: AJTSSGGHKSSYPE-UHFFFAOYSA-N
Target: P2Y purinoceptor 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P47900
Assay: Scintillation Proximity Assay (SPA): A SPA membrane binding assay was used to identify inhibitors of [33P] 2MeS-ADP binding to cloned human P2Y1 receptors (The P2Y1 receptor membranes were provided by Biology and the cloning of the receptor and P2Y1 receptor membrane preparation is same as described by Biology). Binding reactions were performed in 384-well OptiPlates (PerkinElmer Life Sciences, Cat #6007299) in a volume of 50 μL containing 15 fmol of P2Y1 receptor (1.7 μg of total protein), 0.3 nM [33P] 2MeS-ADP (PerkinElmer; 2,000 Ci/mmol), various concentrations of the test compound (usually between 10 μM and 160 pM) in Buffer B containing 1% DMSO in assay buffer (15 mM, HEPES, 145 mM potassium chloride, 5 mM sodium chloride, 5 mM EDTA, 0.1 mM MgCl2, pH 7.4) and 100 μg of SPA bead (WGA polystyrene Image beads, #RPNQ 0260V, Amersham). Reactions were allowed to proceed to completion at room temperature for 1 hour followed by centrifugation of the plate for 5 min. About 40 μL of the aqueous solution was aspirated. Plates were sealed and the [33P] 2MeS-ADP bound to the P2Y1 receptor membranes that were bound to the SPA bead were determined in a Gen 4 LEADSEEKER (Amersham) Image Reader.
Measurement: Ki = 11 nM (pChEMBL 7.960000038146973)
Descriptors: MW 495.42, ALogP 6.9, HBA 3, HBD 3, PSA 64.6, QED 0.35
Compound (SMILES): Cc1cc(C(=O)NS(C)(=O)=O)c(F)cc1-c1cnc(OCCC2CC2)c(Cl)c1
InChIKey: JBRSXDCXOIIEHU-UHFFFAOYSA-N
Target: Sodium channel protein type 9 subunit alpha (SINGLE PROTEIN), Homo sapiens
UniProt: Q15858
Assay: Nav 1.7 In Vitro Patch Express (PX) Assay: HEK 293 cells stably transfected with human Nav1.7 were recorded in whole cell voltage clamp mode with the PatchXpress automated electrophysiology system (Molecular Devices, LLC, Sunnyvale, Calif.). Compound effects were measured on a partially inactivated state of the sodium channel. Cells were clamped to a holding potential yielding 20 to 50% inactivation. To elicit sodium current, channels were activated by pulsing to −10 mV for 20 msec. This voltage protocol was repeated at a rate of 0.1 Hz throughout the experiment. A single concentration of test compound was applied to cells for a duration of 3 minutes. Peak sodium current was measured at the end of the compound addition period to determine percent inhibition. Three to five cells were tested per concentration, and IC50 curves were fitted to percent inhibition as a function of concentration.
Measurement: IC50 = 111 nM (pChEMBL 6.960000038146973)
Descriptors: MW 426.9, ALogP 3.72, HBA 5, HBD 1, PSA 85.36, QED 0.73
Compound (SMILES): Cc1cc(=O)cc(C)n1/C=C/c1ccc(OC(F)F)c(OCC[S+](C)[O-])c1
InChIKey: SRHAGQZXDLAGGK-VOTSOKGWSA-N
Target: 3',5'-cyclic-AMP phosphodiesterase 4B (SINGLE PROTEIN), Homo sapiens
UniProt: Q07343
Assay: Determination of the inhibition of the compounds on PDE4B1 activity by using cAMP HTRF assay: The inhibitory effect of the compounds on human PDE4B1 enzyme activity was determined by quantifying the 5′-adenosine monophosphate (5′-AMP) formed from 3′,5′-cyclic adenosine monophosphate (cAMP).The test compound or water (control) and human recombinant PDE4B1 enzyme (4.8 U) were mixed in a buffer solution (pH 7.4) consisting of 1× Hanks' balanced salt solution (HBSS), 5 mM HEPES, 3 mM MgCl2, and 0.1% BSA and incubated for 10 min. A cAMP enzyme substrate (final concentration 40 nM) was added, and the mixture was incubated at room temperature for 60 min. Then a fluorescent acceptor (Dye2 labeled with cAMP), a fluorescent donor (anti-cAMP antibody labeled with europium cryptate) and a non-specific phosphodiesterase inhibitor IBMX (3-isobutyl-1-methyl xanthine; final concentration 1 mM) were added. After 60 min, the fluorescence transfer related to the amount of remaining cAMP was measured on a microplate reader (Rubystar, BMG) at λex=337 nm, λem=620 nm and λem=665 nm. The enzyme activity is calculated from the ratio of the signals measured at 665 nm and 620 nm. The results are expressed as percent inhibition of the enzyme activity of the control (without PDE4 inhibitor). The enzyme was omitted for measurement of the basic control. The IC50 value (IC50=the concentration that caused the half-maximum inhibition of the specific activity of the control) is derived from dose-response measurements with eight different concentrations (n=2; repeated 2 times).
Measurement: IC50 = 137 nM (pChEMBL 6.860000133514404)
Descriptors: MW 397.44, ALogP 3.45, HBA 5, HBD 0, PSA 63.52, QED 0.64
Compound (SMILES): C[C@H](C(=O)O)[C@H](c1cccc(OCC2CCN(Cc3cc(C(F)(F)F)ccc3C(F)(F)F)CC2)c1)C1CC1
InChIKey: HLBUJSIBJDQPHO-GKVSMKOHSA-N
Target: Free fatty acid receptor 1 (SINGLE PROTEIN), Homo sapiens
UniProt: O14842
Assay: GRP40 FLIPR Assays: FLIPR (Fluorimetric Imaging Plate Reader, Molecular Devices) assays were performed to measure agonist-induced calcium mobilization of the stable clones. For the FLIPR assay, one day before assay, GPR40/CHO NFAT BLA cells were seeded into black-wall-clear-bottom 384-well plates (Costar) at 1.4×10e4 cells/20 μL medium/well. The cells were incubated with 20 μl/well of the assay buffer (HBSS, 0.1% BSA, 20 mM HEPES, 2.5 mM probenecid, pH 7.4) containing 8 μM fluo-4, AM, 0.08% pluronic acid at room temperature for 100 minutes. Fluorescence output was measured using FLIPR. Compounds were dissolved in DMSO and diluted to desired concentrations with assay buffer. 13.3 μL/well of compound solution was added.The compounds of the present invention, including the compounds in Examples 1-159, have EC50 values less than 2 micromolar (μM) in the FLIPR assay described above
Measurement: EC50 = 1 nM (pChEMBL 9)
Descriptors: MW 543.55, ALogP 7.23, HBA 3, HBD 1, PSA 49.77, QED 0.34
Compound (SMILES): CC(=O)c1nn2c3c(cc(-c4cnc(C)nc4)cc13)CCCCCCCCCC[C@@]13C[C@@H](C(=O)Nc4nc(Br)ccc4C)N(C(=O)C2)[C@@H]1C3
InChIKey: DZZZYHFKDGVGIB-QWTIHQBASA-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 min 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 M each. Absorbance at 405 nm (A405) is recorded at 30 second intervals for 30 minutes using a microplate spectrophotometer. IC50 values are calculated by nonlinear regression of Complement Factor D reaction rates as a function of test compound concentration.
Measurement: IC50 < 1000 nM (pChEMBL 6)
Descriptors: MW 726.72, ALogP 7.54, HBA 8, HBD 1, PSA 122.97, QED 0.17
Compound (SMILES): CC(C)(OC(=O)Nc1ccc2c(c1)N(S(=O)(=O)c1cccc(C(F)(F)F)c1)C[C@H](C[C@H](C(=O)O)C1CC1)O2)C(F)(F)F
InChIKey: FWKLERUTVLQROJ-HKUYNNGSSA-N
Target: Nuclear receptor ROR-gamma (SINGLE PROTEIN), Homo sapiens
UniProt: P51449
Assay: Biochemical TR-FRET Assay: To identify novel antagonists of RORgammaT, an assay was developed which employs the interaction of RORgammaT with its co-activator peptide SRC1_2. This peptide mimics the recruitment of co-activators to RORgammaT through its interaction with the LXXLL (SEQ ID NO:1) (e.g., NR box) motifs (Xie et al., J. Immunol. 175: 3800-09, 2005; Kurebayashi et al., Biochem. Biophys. Res. Commun. 315: 919-27, 2004; Jin et al., Mol. Endocrinology 24:923-29, 2010). The RORγ-Ligand Binding Domain TR-FRET Assay was run according to the following protocol.HIS-tagged RORγ-LBD protein was recombinantly expressed in Escherichia coli. The RORγ-LBD protein was purified by Ni2+-affinity resin. Purified protein was then diluted in assay buffer (50 mM Tris pH 7.0, 50 mM KCl, 1 mM EDTA, 0.1 mM DTT, 100 μg/mL bovine serum albumin, delipidated) to obtain a RORγ-LBD final concentration of 3 nM. Europium tagged anti-HIS antibody was also added to this solution (1.25 nM). Separately, SF9 cells not expressing any recombinant protein were lysed (32,000 cells per μl in 25 mM Tris, 50 mM NaCl) and the previously frozen lysate was added to the diluted RORγ-LBD solution at a ratio of 0.75 μl SF9 lysate per 15 μl of diluted RORγ-LBD.
Measurement: IC50 = 60.3 nM (pChEMBL 7.21999979019165)
Descriptors: MW 624.56, ALogP 6.05, HBA 6, HBD 2, PSA 122.24, QED 0.34
Compound (SMILES): CCCc1cnc2ccc(-c3cnn4c3C(=O)N(c3ccc(C(F)(F)F)cc3)C[C@@H]4C)cn12
InChIKey: RIVZHOKNBIRZCR-HNNXBMFYSA-N
Target: Metabotropic glutamate receptor 2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q14416
Assay: Assessment of mGlu2 Receptor Negative Allosteric Modulator Activity: Human mGlu2 receptor stable expressing cells were prepared and incubated. Luminescence signals by mGlu2 receptor stimulation were detected with FDSS7000 (Hamamatsu Photonics). The compound solution prepared above was added to a plate where cells and luminescent substrates were added (10 μl/well). After 120 seconds of the addition, EC80 Glutamate-containing solution was added thereto (10 μl/well), and luminescence signals were measured for 300 seconds after the addition (center wavelength: 465 nm) for calculation of RLU (Integration). mGlu2 receptor negative allosteric modulator activity of a compound was calculated by (100-100×(RLU of each compound and concentration)/(RLU of DMSO group)).The present compound was assessed with the above biological test, and the compounds with mGlu2 receptor negative allosteric modulator activity were found out.
Measurement: IC50 = 13 nM (pChEMBL 7.889999866485596)
Descriptors: MW 453.47, ALogP 5.39, HBA 5, HBD 0, PSA 55.43, QED 0.42