lovingscience/chembl-v37-raw-activities huggingface dataset:
Compound (SMILES): COc1cccc2c1/C(=C/c1c(Cl)cccc1C1CC1)C(=O)N2c1ccc(C(=O)O)c(O)c1
InChIKey: RHEHHKQGUZOMKE-UYRXBGFRSA-N
Target: Nuclear receptor ROR-gamma (SINGLE PROTEIN), Homo sapiens
UniProt: P51449
Assay: TR-FRET Screening: The compounds of the present application can regulate (inhibit) the biological activity of the nuclear receptor RORγ, and the intensity of such a regulation (inhibition) can be evaluated by TR-FRET (Time-Resolved Fluorescence Resonance Energy Transfer) screening system. Nuclear receptor cofactors (co-activators and co-repressors) can regulate the transcription of target genes through interactions with nuclear receptors. If a ligand (test compound) interferes with the interaction between a nuclear receptor and a cofactor, such a ligand (test compound) can regulate the transcription of the corresponding gene.The method employs LanthaScreen TR-FRET (Time-Resolved Fluorescence Resonance Energy Transfer) technique of Life Technologies Co. to test the compounds for their abilities to regulate (agonize or inversely agonize) the interaction between RORγ and its co-activator. RORγ-LBD is indirectly labeled by binding a terbium(Tb)-labeled anti-GST antibody (Life Technologies #PV3550) onto the GST tag of RORγ-LBD (Life Technologies #PV5887). When no ligand is present, RORγ can continuously bind to a fluorescein-labeled co-activator, and the binding of an agonist to RORγ can enhance the interaction between RORγ and the fluorescein-labeled co-activator; while the binding of an inverse agonist to RORγ can inhibit the interaction between RORγ and the fluorescein-labeled co-activator. When the fluorescein-labeled co-activator and the terbium-labeled anti-GST antibody-RORγ-LBD composite are close to each other to a certain distance, an energy transfer can occur, generating a TR-FRET signal. The co-activators employed in the method are not limited to D22 (Life Technologies #PV4386).(1) The final concentrations in the reaction system and the reaction conditions in the method are shown in the table below:TABLE 2 Concentration of Total Concentration of Fluorescein-labeled Concentration of Tb-labeled volume Reaction ROR γ-LBD co-activator activator peptide anti-GST antibody (μL) conditions 2 nM D22 150 nM 2 nM 20 22° C., 5 hr Fluorescein-D22
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 461.9, ALogP 5.85, HBA 4, HBD 2, PSA 87.07, QED 0.47
Compound (SMILES): COc1cc(OCc2cccc(-c3ccccc3)c2C)cc(OC)c1CN[C@H]1COC[C@@H]1O
InChIKey: OPKHCRVDVMEODN-DQEYMECFSA-N
Target: Programmed cell death 1 ligand 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9NZQ7
Assay: Homogenous Time-Resolved Fluorescence (HTRF) Binding Assay: All binding studies were performed in an HTRF assay buffer consisting of dPBS supplemented with 0.1% (withv) bovine serum albumin and 0.05% (v/v) Tween-20. For the PD-1-Ig/PD-L1-His binding assay, inhibitors were pre-incubated with PD-L1-His (10 nM final) for 15 m in 4 μl of assay buffer, followed by addition of PD-1-Ig (20 nM final) in 1 μl of assay buffer and further incubation for 15 m. PD-L1 from either human, cyno, or mouse were used. HTRF detection was achieved using europium crypate-labeled anti-Ig (1 nM final) and allophycocyanin (APC) labeled anti-His (20 nM final). Antibodies were diluted in HTRF detection buffer and 5 μl was dispensed on top of binding reaction. The reaction mixture was allowed to equilibrate for 30 minutes and signal (665 nm/620 nm ratio) was obtained using an EnVision fluorometer. Additional binding assays were established between PD-1-Ig/PD-L2-His (20 & 5 nM, respectively), CD80-His/PD-L1-Ig (100 & 10 nM, respectively) and CD80-His/CTLA4-Ig (10 & 5 nM, respectively). Competition studies between biotinylated SEQ ID NO:71 and human PD-L1-His were performed as follows. Inhibitors were pre-incubated with PD-L1-His (10 nM final) for 60 m in 4 μl of assay buffer followed by addition of biotinylated SEQ ID NO:71 (0.5 nM final) in 1 μl of assay buffer. Binding was allowed to equilibrate for 30 m followed by addition of europium crypated labeled Strepatavidin (2.5 pM final) and APC-labeled anti-His (20 nM final) in 5 μl of HTRF buffer. The reaction was allowed to equilibrate for 30 m and signal (665 nm/620 nm ratio) was obtained using an EnVision fluorometer.
Measurement: IC50 = 53 nM (pChEMBL 7.28000020980835)
Descriptors: MW 449.55, ALogP 4.11, HBA 6, HBD 2, PSA 69.18, QED 0.51
Compound (SMILES): C=CC(=O)N1CCC[C@@H](Nc2nc(Nc3cnn(CC(=O)NC)c3)nc3[nH]cc(Cl)c23)C1
InChIKey: MAISEFJAAIILEY-GFCCVEGCSA-N
Target: Tyrosine-protein kinase JAK3 (SINGLE PROTEIN), Homo sapiens
UniProt: P52333
Assay: Enzyme Inhibitory Activity: JAK3 and BTK kinase inhibitory activities were measured for the compounds prepared in the above Examples through in vitro analysis on the ADP Glow (Glo) platform.Specifically, the inhibitory activities of JAK3 and BTK kinase were measured using a JAK3 kinase assay kit (Promega, V9441) and a BTK kinase assay kit (Promega, V9071) which were purchased from Promega. Recombinant purified human JAK3 and BTK were diluted with 1×kinase reaction buffer (JAK3: 40 mM Tris-Cl, pH 7.5, 20 mM MgCl2, 0.1 mg/mL BSA and 50 uM DTT/BTK: 40 mM Tris-Cl, pH 7.5, 20 mM MgCl2, 0.1 mg/mL BSA, 2 mM MnCl2 and 50 uM DTT) and added to a 96 well plate (JAK3: final concentration of 4 ng per reaction/BTK: final concentration of 8 ng per reaction). The compounds were treated so as to be finally a 1% DMSO aqueous solution, and a substrate cocktail containing ATP (JAK3: final concentration of 5 uM/BTK: final concentration of 10 uM) and 0.2 μg/μL of Poly(Glu4, Tyr1) peptide (JAK3 and BTK final concentration) in the total 254 reactants was added to a 96-well plate to initiate enzymatic reaction. After incubation (30° C.) for 1 hour, equivalent volume (254 per reaction) of ADP Glo was added and incubated (30° C.) for 40 minutes at room temperature. Then, a kinase detection reagent (504 per reaction) was added and incubated (30° C.) for 30 minutes at room temperature. The kinase activity was measured by chemiluminescence according to the instructions of ADP Glo kinase assay kit, and the inhibitory activity of the compounds according to the present invention was calculated.
Measurement: IC50 = 0.2 nM (pChEMBL 9.699999809265137)
Descriptors: MW 457.93, ALogP 1.89, HBA 8, HBD 4, PSA 132.86, QED 0.4
Compound (SMILES): CC(C)C(=O)NCc1ccc(Cl)c(-c2nc(-c3cccc4ccsc34)cc(=O)[nH]2)c1
InChIKey: PCMUADGTVZEYAB-UHFFFAOYSA-N
Target: Prostaglandin E synthase (SINGLE PROTEIN), Homo sapiens
UniProt: O14684
Assay: Inhibitory Activity Assay: Microsomes were prepared from COS-1 cells transiently transfected with a plasmid containing human mPGES-1 cDNA, and used as mPGES-1 enzyme. The mPGES-1 enzyme was diluted with a sodium phosphate buffer (pH 7.2) containing reduced glutathione (2.5 mM) and EDTA (1 mM), DMSO or a DMSO solution of a test compound (final concentration of DMSO was 1%) was added to the enzyme, and the mixture was preincubated at 4° C. for 15 minutes. Then, PGH2 as the substrate was added at a final concentration of 1 μM to start the enzymatic reaction, and after incubation at 4° C. for 4 minutes, a solution of ferric chloride (25 mM) and citric acid (50 mM) was added to terminate the enzymatic reaction. Generated PGE2 was measured by using Prostaglandin E2 Express EIA Kit (Cayman Chemical).
Measurement: IC50 = 25 nM (pChEMBL 7.599999904632568)
Descriptors: MW 437.95, ALogP 5.24, HBA 4, HBD 2, PSA 74.85, QED 0.45
Compound (SMILES): COc1ccc(Br)c(C(=O)N2CCN(Cc3c(-c4ccc(Cl)cc4)nc4ccccn34)CC2)n1
InChIKey: BZPMLHWCBMBLHH-UHFFFAOYSA-N
Target: Potassium channel subfamily K member 3 (SINGLE PROTEIN), Homo sapiens
UniProt: O14649
Assay: Inhibition In Vito Assay: The investigations on the inhibition of the recombinant TASK-1 and TASK-3 channels were carried out using stably transfected CHO cells. Here, the compounds according to the invention were tested with administration of 40 mM of potassium chloride in the presence of a voltage-sensitive dye for high-throughput screening of potassium channel from Molecular Devices FLIPR Application Note: Measuring membrane potential using the FLIPR membrane potential assay kit on Fluorometric Imaging Plate Reader (FLIPR) systems. The activity of the test substances was determined as their ability to inhibit a depolarization induced in the recombinant cells by 40 mM potassium chloride.
Measurement: IC50 = 310 nM (pChEMBL 6.510000228881836)
Descriptors: MW 540.85, ALogP 4.78, HBA 6, HBD 0, PSA 62.97, QED 0.36
Compound (SMILES): COc1ccc(-c2ccc3nc4c(n3n2)[C@@H](c2ccccc2OC(F)F)NC[C@@H]4O)cn1
InChIKey: BKTVDSFXXWQGOH-HNAYVOBHSA-N
Target: Tumor necrosis factor (SINGLE PROTEIN), Homo sapiens
UniProt: P01375
Assay: TNF induced HEK-Blue assay: Test compounds serially diluted in DMSO were plated in an assay plate (Labcyte, Cat. #LP-0200) at final concentrations ranging from 0.004 μM to 25μM. TNFα (final concentration 0.5 ng/ml) or CD4OL (final concentration 30 ng/ml) in assay buffer [DMEM, 4.5 g/l glucose (Gibco, Cat. 21063-029), 10% FBS (Sigma, F4135), 1% Penicillin-Streptomycin (Gibco, Cat. 15140-122), 1% Anti-Anti (Gibco, Cat. 15240-112) and 2 mM L-glutamine (Gibco, Cat. 25030-081)] was then added to the assay plate. After a 30 minute pre-incubation at 37° C. and 5% CO2, HEK-Blue-CD40L cells (InvivoGen, Cat. Code hkb-cd40) containing a NF-κB-driven secreted alkaline phosphatase reporter gene were seeded into the assay plate at a density of 20,000 cells per well. This plate was then incubated for 18 h at 37° C. and 5% CO2. Secreted alkaline phosphatase expression was measured using QUANTI-Blue (InvivoGen, Cat. Code rep-qb1) according to manufacturer's specifications and the assay plate was read on a PerkinElmer Envision at 620 nm.
Measurement: IC50 = 17500 nM (pChEMBL 4.760000228881836)
Descriptors: MW 439.42, ALogP 3.13, HBA 8, HBD 2, PSA 93.8, QED 0.49
Compound (SMILES): Cc1cc(Nc2nccc(-c3cc(C#N)c4c(c3)[C@@](C)(CO)CN4)n2)n(CCCO)n1
InChIKey: HXNDUJQNAWLKBP-JOCHJYFZSA-N
Target: Mitogen-activated protein kinase kinase kinase 14 (SINGLE PROTEIN), Homo sapiens
UniProt: Q99558
Assay: Inhibition of auto-phosphorylation of recombinant human NF-kappaB-inducing kinase (NIK/MAP3K14) activity: NIK/MAP3K14 auto-phosphorylation activity was measured using the AlphaScreen® (αscreen) format (Perkin Elmer). All compounds tested were dissolved in dimethyl sulfoxide (DMSO) and further dilutions were made in assay buffer. Final DMSO concentration was 1% (v/v) in assays. Assay buffer was 50 mM Tris pH 7.5 containing 1 mM EGTA (ethylene glycol tetraacetic acid), 1 mM DTT (dithiothreitol), 0.1 mM Na3VO4, 5 mM MgCl2, 0.01% Tween® 20. Assays were carried out in 384 well Alphaplates (Perkin Elmer). Incubations consisted of compound, 25 microM Adenosine-5′-triphosphate (ATP), and 0.2 nM NIK/MAP3K14. Incubations were initiated by addition of GST-tagged NIK/MAP3K14 enzyme, carried out for 1 h at 25° C. and terminated by addition of stop buffer containing anti-phospho-IKK Ser176/180 antibody. Protein A Acceptor and Glutathione-Donor beads were added before reading using an EnVision® Multilabel Plate Reader (Perkin Elmer). Signal obtained in the wells containing blank samples was subtracted from all other wells and IC50's were determined by fitting a sigmoidal curve to % inhibition of control versus Log10 compound concentration.
Measurement: IC50 = 3.5 nM (pChEMBL 8.460000038146973)
Descriptors: MW 419.49, ALogP 2.32, HBA 9, HBD 4, PSA 131.91, QED 0.46
Compound (SMILES): [C-]#[N+]c1ccc(NC(=O)N2Cc3c(C(N)=O)c(-c4cccc(Cl)c4)nn3[C@@H](CF)C2)cc1Cl
InChIKey: BMVCWLIUGFYWMO-HNNXBMFYSA-N
Target: Casein kinase I isoform delta (SINGLE PROTEIN), Homo sapiens
UniProt: P48730
Assay: Kinase Assay: The kinase assay was performed in V-bottom 384-well plates. The final assay volume was 30 μl prepared from 15 μl additions of enzyme, substrates (fluoresceinated peptide FL-AHA-KRRRAL-PSER-VASLPGL-OH and ATP) and test compounds in assay buffer (20 mM HEPES pH 7.4, 30 mM MgCl2, 0.015% Brij35 and 4 mM DTT). The reaction was incubated at room temperature for 22 hours and terminated by adding 45 μl of 35 mM EDTA to each sample. The reaction mixture was analyzed on the Caliper LABCHIP®3000 (Caliper, Hopkinton, Mass.) by electrophoretic separation of the unphosphorylated substrate and phosphorylated product. Inhibition data were calculated by comparison of the no enzyme control reactions for 100% inhibition and vehicle-only reactions for 0% inhibition. The final concentration of reagents in the assay were 200 pM CK1ε or CK1δ, 50 μM ATP, 1.5 μM FL-AHA-KRRRAL-PSER-VASLPGL-OH, and 1.6% DMSO. Dose response curves were generated to determine the concentration required to inhibit 50% of the kinase activity (IC50). Compounds were dissolved at 10 mM in dimethylsulfoxide (DMSO) and evaluated at eleven concentrations. IC50 values were derived by non-linear regression analysis.
Measurement: IC50 < 10 nM (pChEMBL 8)
Descriptors: MW 487.32, ALogP 5.06, HBA 4, HBD 2, PSA 97.61, QED 0.5
Compound (SMILES): CCOc1ccc2nccc(-c3cnn4cc(N5CCC(N6CCN(C)CC6)CC5)cnc34)c2c1
InChIKey: MHKQEZQAUKOFSS-UHFFFAOYSA-N
Target: Activin receptor type-1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q04771
Assay: Luciferase Reporter Gene Assay for ALK2 and TGFbeta Activity: In this reporter gene cell based ALK2 and TGFβ assay, the C2Cl2 cell line was employed for the measurement of ALK2 activity, using a BRE-Luc SMAD1/5/8 reporter and BMP6 as the agonist. The HEK293T cell line was employed to measure TGFβ activity, using a SBE-Luc SMAD2/3 reporter and TGFβ as the agonist. Luciferase reporter assays were read using Promega Steady-Glo Luciferase Assay System.Cells were plated in 96 well white clear bottom assay plates at 10 k cells/well for C2Cl2-BRE, 15 k cells/well for HEK293-SBE in DMEM containing 2% FBS 1% P/S. Cells were given a minimum of 4 hours in incubator at 37° C./5% CO2 to adhere prior to further treatment. Compounds were diluted in DMSO to a 10-point dilution curve and added to the plate to reach the following final concentrations: 10000, 3000, 1000, 300, 100, and 30 nM in the HEK293-SBE assay and 1000, 300, 100, 30, 10, 3, 1, 0.3 nM in the C2Cl2-BRE assay. Negative and positive control wells received 2 μL DMSO as vehicle treatment. Plates were returned to incubator for 45 minutes and then BMP6 and TGFB were added to a final concentration of 50 ng/mL and 5 ng/mL, respectively. Plates were returned to the incubator and left overnight. After a minimum of 18 hours post BMP6/TGFβ addition, the plates were read using Promega Steady-Glo Luciferase Assay System. A 1:1 mixture of prepared steady-glo and phenol free DMEM was prepared, and 50 μL/well was added to the assay plates whose media had been flicked off. Plates were given 10 minutes post steady-glo addition before luminescence was read on a Spectramax M5e microplate reader. Negative control wells were averaged and subtracted from all other wells on the plate. Inhibition was calculated as the percent of signal loss compared to the averaged positive control wells.
Measurement: IC50 = 27 nM (pChEMBL 7.570000171661377)
Descriptors: MW 471.61, ALogP 3.56, HBA 8, HBD 0, PSA 62.03, QED 0.44
Compound (SMILES): COc1cncc(NCc2cc3cc(COc4ccccc4CC(=O)O)cc(I)c3o2)c1
InChIKey: ITCALPJUMROMRJ-UHFFFAOYSA-N
Target: Complement factor D (SINGLE PROTEIN), Homo sapiens
UniProt: P00746
Assay: Factor D Esterolytic Assay: An established esterolytic assay for the measurement of Factor D activity and inhibition of Factor D activity was used (Kam, C. M.; McRae, B. J.; Harper, J. W.; Niemann, M. A.; Volanakis, J. E.; Powers, J. C. Human complement proteins D, C2, and B Active site mapping with peptide thioester substrates. J Biol. Chem. 1987, 262, 3444-3451). For this assay Z-Lys-SBzl, 1.29 mM (Kim, S.; Narayana, S. V. L; Volanakis, J. E. Mutational analysis of the substrate binding site of human complement Factor D. Biochemistry. 1994, 33, 14393-14399.) was used as the substrate for Factor D (104 mM). Hydrolysis of this compound by Factor D liberated a free sulfhydryl group which is then reacted with 5,5′-dithiobis(2nitrobenzoic acid) producing an intense yellow color (Habeeb, A. F. S. A. Reaction of protein sulfhydryl groups with Ellman's Reagent. Methods in Enzymol. 1976, 25, 457-464). The assays were performed in 96 well microtiter plates and rates of hydrolysis were monitored at 405 nm on a Biotek Synergy H1 plate reader. Hydrolysis rates were reported as change in mOD/min. The assay was conducted in 100 mM HEPES, 500 mM NaCl, pH 7.5 containing 10% DMSO in a final volume of 50 μL per well.
Measurement: IC50 < 1000 nM (pChEMBL 6)
Descriptors: MW 544.35, ALogP 5.26, HBA 6, HBD 2, PSA 93.82, QED 0.28
Compound (SMILES): CS(=O)(=O)c1nc2n(n1)[C@H](c1ccccc1F)CC2
InChIKey: KNHQFFKMMQEAQC-JTQLQIEISA-N
Target: Receptor-interacting serine/threonine-protein kinase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q13546
Assay: Biochemical Assay: The ability of the receptor interacting protein kinase (RIPK1) to catalyze the hydrolysis of adenosine-5′-triphosphate (ATP) is monitored using the Transcreener ADP (adenosine-5′-diphosphate) assay (BellBrook Labs). Purified human RIP1 kinase domain (2-375) (50 nM) derived from a baculovirus-infected insect cell expression system is incubated with test compounds for 2 hours in 50 mM Hepes buffer (pH 7.5) containing 30 mM MgCl2, 1 mM dithiothreitol, 50 uM ATP, 0.002% Brij-35, and 0.5% dimethyl sulfoxide (DMSO). Reactions are quenched by the addition of 1× Bell Brooks Stop buffer B (20 mM Hepes (ph7.5), 40 mM ethylenediaminetetraacetic acid and 0.02% Brij-35) containing an additional 12 mM EDTA and 55 ug/mL ADP2 antibody and 4 nM ADP-AlexaFluor® 633 tracer. The tracer bound to the antibody is displaced by the ADP generated during the RIP1K reaction, which causes a decrease in fluorescence polarization that is measured by laser excitation at 633 nm with a FP microplate reader M1000. Fractional activity was plotted against test article concentration. Using Genedata Screener software (Genedata; Basel, Switzerland), the data were fit to the tight-binding apparent inhibition constant (Ki app) Morrison equation [Williams, J. W. and Morrison, J. F. (1979) The kinetics of reversible tight-binding inhibition. Methods Enzymol 63: 437-67]. The following equation was used to calculate fractional activity and Ki app:Fractional ⁢ ⁢ activity = V i V o = 1 - ( [ E ] T + [ I ] T + K i app ) - ( [ E ] T + [ I ] T + K i app ) 2 - 4 ⁡ [ E ] T ⁡ [ I ] T 2 ⁡ [ E ] Twhere [E]T and [I]T are the total concentrations of active enzyme and test article, respectively.
Measurement: Ki = 250 nM (pChEMBL 6.599999904632568)
Descriptors: MW 281.31, ALogP 1.36, HBA 5, HBD 0, PSA 64.85, QED 0.83
Compound (SMILES): CCc1cc(CN(C)C(=O)c2cc3cc(Cl)c4c(c3o2)C2(CCCCC2)NC(=O)N4)on1
InChIKey: VBWAZHGTMYMLOV-UHFFFAOYSA-N
Target: 3',5'-cyclic-AMP phosphodiesterase 7B (SINGLE PROTEIN), Homo sapiens
UniProt: Q9NP56
Assay: Enzymatic Assay: PDE7 inhibition was determined by an IMAP TR-FRET assay using PDE7B. The IMAP TR-FRET PDE assay was optimized for concentration of enzyme, FAM-cAMP substrate, reducing agent, DMSO tolerance, and incubation time. First, PDE7 inhibitor compounds in 10 mM DMSO stock were serially diluted in 100% DMSO. Next, into each well of a solid white 1536 well plate (Corning) was dispensed 12.8 pg of N-terminal truncated recombinant human PDE7B enzyme (91-450aa, prep #DBVC-D04614, made in-house by structural biology) in 2.5 μL IMAP BSA reaction buffer (Molecular Devices, Sunnyvale, Calif.) containing 1 mM DTT (Sigma Aldrich.) After a brief centrifugation, 30 nL of serially diluted compounds in DMSO were added by transfer from 1 mM stock using a Kalypsys 1536 Pintool. Plates were incubated for 5 minutes at room temperature before dispensing 1.5 μL of 134 nM 5-carboxy fluorescein (FAM)-labeled cAMP (Molecular Devices, Sunnyvale, Calif.) for a final concentration of 50 nM. After a brief centrifugation, the plates were incubated for 15 minutes at room temperature. The assay was terminated by adding 5 μL IMAP binding reagent/Tb complex (Molecular Devices, Sunnyvale, Calif.) to each well. Plates were incubated 1 hour at room temperature and read on a Viewlux multimode plate reader (Perkin Elmer).
Measurement: IC50 < 10 nM (pChEMBL 8)
Descriptors: MW 456.93, ALogP 5.2, HBA 5, HBD 2, PSA 100.61, QED 0.56
Compound (SMILES): Cc1cc(S(=O)(=O)Nc2cscn2)c(F)cc1O[C@@]1(C)CCN(Cc2ccccc2)C1
InChIKey: PGHBQSPVIDPUTJ-QFIPXVFZSA-N
Target: Sodium channel protein type 8 subunit alpha (SINGLE PROTEIN), Homo sapiens
UniProt: Q9UQD0
Assay: Sodium Influx Assay (In Vitro Assay): This sodium influx assay employs the use of the cell permeable, sodium sensitive dye ANG2 to quantify sodium ion influx through sodium channels which are maintained in an open state by use of sodium channel modulators. This high throughput sodium influx assay allows for rapid profiling and characterization of sodium channel blockers.In general, Trex HEK293 cells were stably transfected with an inducible expression vector containing the full-length cDNA coding for the desired human sodium channel α-subunit and with an expression vector containing full length cDNA coding for the β1-subunit. Sodium channel expressing cell lines were induced with tetracycline (1 μg/mL) and plated on 384-well PDL-coated plates at a density of 25K-30K cells/well in culture media (DMEM, containing 10% FBS and 1% L-glutamine). After overnight incubation (37° C., 5% CO2), culture media was removed and cells were loaded with 5 uM ANG2 dye for 1-1.5 h in Buffer 1 (155 mM NMDG, 5 mM KCl, 2 mM CaCl2, 1 mM MgCl2, 10 mM HEPES, 10 mM glucose, adjusted with Tris to pH 7.4). Access dye was removed and cells were incubated with test compounds for 1 hr in buffer 1 containing sodium channel modulator(s) at room temperature. Hamamatsu FDSS ρCell was used to perform a 1:1 addition of Na/K challenge buffer (140 mM NaCl, 20 mM HEPES, 1 mM CaCl2, 15 mM KCl, 1 mM MgCl2, 10 mM glucose, adjusted with Tris to pH 7.4) and simultaneously read plates at excitation wavelength of 530 nm and emission wavelength set at 558 nm. Percent inhibition of sodium ion influx was calculated for each test compound at each test concentration to determine the IC50 values.
Measurement: IC50 = 178 nM (pChEMBL 6.75)
Descriptors: MW 461.58, ALogP 4.43, HBA 6, HBD 1, PSA 71.53, QED 0.56
Compound (SMILES): Nc1cc(C2CCN3C[C@H](Cc4ccccc4)[C@@H](C3)Oc3cccc(c3)Cn3cc2cn3)c2nn[nH]c2n1
InChIKey: WHOPNONEBDNGIG-MVASODGFSA-N
Target: Myeloperoxidase (SINGLE PROTEIN), Homo sapiens
UniProt: P05164
Assay: MPO Peroxidation Assay (Amplex Red Assay): MPO peroxidation activity was measured in 100 mM KPi (pH 7.4) by utilizing the non-fluorescent reagent Amplex Red (Invitrogen Cat. #A12222) which can be oxidized to the highly fluorescent resorufin. Amplex Red is oxidized by the peroxidase action of MPO to resorufin. Reactions were carried out in 50 μL total volume by adding a 25 μL mixture of 200 pM myeloperoxidase and 40 nM H2O2 (Sigma #349887) to 100 nL inhibitor in 100% DMSO in a 384 well Perkin Elmer Optiplate. Enzyme and compound were preincubated for ten minutes at room temperature.After the ten minute preincubation, 25 μL of an Amplex Red mixture containing 200 μM Amplex Red and 10 mM H2O2 was added to the plate. Kinetic determinations were carried out immediately on a Perkin Elmer Envision (15 minute kinetic read, Ex: 535 nm, Em: 590 nm).
Measurement: IC50 = 550 nM (pChEMBL 6.260000228881836)
Descriptors: MW 506.61, ALogP 3.64, HBA 8, HBD 2, PSA 110.77, QED 0.38
Compound (SMILES): CCCCn1cc(-c2cc(C(=O)N(C)C)ccc2OC)c2cc(C)[nH]c2c1=O
InChIKey: VTYUQHJERJKSHW-UHFFFAOYSA-N
Target: Bromodomain-containing protein 9 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9H8M2
Assay: IC50 measurements for inhibitors using BRD9 AlphaLisa Binding Assay: His/Flag epitope tagged BRD9134-239 was cloned, expressed, and purified to homogeneity. BRD9 binding and inhibition was assessed by monitoring the engagement of biotinylated H4-tetraacetyl peptide (New England Peptide, NEP2069-11/13) with the target using the AlphaLisa technology (Perkin-Elmer). Specifically, in a 384 well ProxiPlate BRD9 (50 nM final) was combined with peptide (3 nM final) in 50 mM HEPES (pH 7.5), 150 mM NaCl, 1 mM TCEP, 0.01% (w/v) BSA, and 0.008% (w/v) Brij-35 either in the presence of DMSO (final 0.8% DMSO) or compound dilution series in DMSO. After 20 minutes incubation at room temperature AlphaLisa Streptavidin Acceptor Beads (Perkin-AL125C) and AlphaLisa Nickel donor beads (Perkin AS 10 ID) were added to a final concentration of 15 ug/mL each. After ninety minutes of equilibration in the dark, the plates were read on an Envision instrument and IC50s calculated using a four parameter non-linear curve fit.
Measurement: IC50 = 66 nM (pChEMBL 7.179999828338623)
Descriptors: MW 381.48, ALogP 3.82, HBA 4, HBD 1, PSA 67.33, QED 0.71
Compound (SMILES): CCOc1ccc(CN2CCn3nnnc3C2c2cn(-c3ccccc3)nc2C(C)C)cc1
InChIKey: ZFRGMPIYWJVILV-UHFFFAOYSA-N
Target: Nuclear receptor ROR-gamma (SINGLE PROTEIN), Homo sapiens
UniProt: P51449
Assay: TR-FRET Assay: In this assay, a GST-tagged human RORγ ligand binding domain (GST-hRORγ-LBD) interacts with a synthetic biotinylated TRAP220 cofactor peptide containing an LXXLL motif (amino acids 631-655 from NP_004765). The ligand-binding domain (LBD) of RORγ is expressed as fusion protein with GST in BL-21 (BL3) cells using the vector pDEST15. Cells are lysed by lysozyme-treatment and sonication, and the fusion proteins purified over glutathione sepharose (Pharmacia) according to the manufacturers instructions. The strong constitutive interaction is disrupted upon binding of functional antagonists. The strength of the interaction is monitored by TR-FRET between streptavidine APC interacting with the biotinylated peptide and Europium-labelled Anti-GST. 6 μl RORγ-LBD-GST solution (final 8.7 nM) in assay buffer (20 mM Tris-HCl, pH 6.8, 5 mM MgCl2, 60 mM KCl, 0.1% delipidated BSA, 1 mM DTT) are dispensed into black 384 well small volume plates (Greiner #784076). 6 μl TRAP220 peptide (final 400 nM), SA-APC (final 1.6 ng/μl), Eu-anti-GST (final 0.125 ng/μl) in assay buffer and 2 μl test compounds (DMSO stocks prediluted in 20 mM Tris-HCl, pH 6.8, 5 mM MgCl2, 60 mM KCl) are added and the mixture incubated 60 min at RT in the dark before measuring FRET (excitation 337 nm, emission at 615 and 665 nm).
Measurement: IC50 = 24950 nM (pChEMBL 4.599999904632568)
Descriptors: MW 443.56, ALogP 3.99, HBA 8, HBD 0, PSA 73.89, QED 0.43
Compound (SMILES): CCN(CCN(C)c1cccc(C(C)(C)O)n1)C(=O)c1cccnc1CC(F)(F)F
InChIKey: IYJFCMIDHUENES-UHFFFAOYSA-N
Target: Orexin receptor type 2 (SINGLE PROTEIN), Homo sapiens
UniProt: O43614
Assay: FLIPR Ca2+ Flux Assay: In a typical experiment the OX1 and OX2 receptor antagonistic activity of the compounds of the present invention was determined in accordance with the following experimental method. For intracellular calcium measurements, Chinese hamster ovary (CHO) cells expressing the rat orexin-1 receptor or the human orexin-2 receptor, are grown in Iscove's modified DMEM containing 2 mM L-glutamine, 0.5 g/ml G418, 1% hypoxanthine-thymidine supplement, 100 U/ml penicillin, 100 μg/ml streptomycin and 10% heat-inactivated fetal calf serum (FCS). The cells are seeded at 20,000 cells/well into Becton-Dickinson black 384-well clear bottom sterile plates coated with poly-D-lysine. All reagents were from GIBCO-Invitrogen Corp. The seeded plates are incubated overnight at 37° C. and 5% CO2. Ala-6,12 human orexin-A as the agonist is prepared as a 1 mM stock solution in 1% bovine serum albumin (BSA) and diluted in assay buffer (HBSS containing 20 mM HEPES, 0.1% BSA and 2.5 mM probenecid, pH7.4) for use in the assay at a final concentration of 70 pM. Test compounds are prepared as 10 mM stock solution in DMSO, then diluted in 384-well plates, first in DMSO, then assay buffer. On the day of the assay, cells are washed 3 times with 100 μl assay buffer and then incubated for 60 min (37° C., 5% CO2) in 60 μl assay buffer containing 1 μM Fluo-4AM ester, 0.02% pluronic acid, and 1% BSA. The dye loading solution is then aspirated and cells are washed 3 times with 100 μl assay buffer. 30 μl of that same buffer is left in each well. Within the Fluorescent Imaging Plate Reader (FLIPR, Molecular Devices), test compounds are added to the plate in a volume of 25 μl, incubated for 5 min and finally 25 μl of agonist is added. Fluorescence is measured for each well at 1 second intervals for 5 minutes and the height of each fluorescence peak is compared to the height of the fluorescence peak induced by 70 pM Ala-6,12 orexin-A with buffer in place of antagonist.
Measurement: IC50 = 2266 nM (pChEMBL 5.639999866485596)
Descriptors: MW 424.47, ALogP 3.41, HBA 5, HBD 1, PSA 69.56, QED 0.7
Compound (SMILES): CC[C@H](O)[C@@H]1C[C@@H](C(C)(C)OC(C)=O)[C@H](n2c(=O)sc3c(=O)[nH]c(N)nc32)O1
InChIKey: PIZRDTIURRKKIK-MAGOMRGPSA-N
Target: Toll-like receptor 7 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9NYK1
Assay: HEK293-hTLR-7 assay: HEK293-Blue-hTLR7 cells were incubated at a density of 250,000-450,000 cells/mL in a volume of 180 μL in a 96-well plate in Dulbecco's Modified Eagle's medium (DMEM) containing 4.5 g/l glucose, 50 U/ml penicillin, 50 mg/ml streptomycin, 100 mg/ml Normocin, 2 mM L-glutamine, 10% (v/v) heat-inactivated fetal bovine serum for 24 h. Then the HEK293-Blue-hTLR-7 cells were incubated with addition of 20 μL test compound in a serial dilution in the presence of final DMSO at 1% and perform incubation under 37° C. in a CO2 incubator for 20 hours. Then 20 μL of the supernatant from each well was incubated with 180 μL Quanti-blue substrate solution at 37° C. for 1-3 hours and the absorbance was read at 620-655 nm using a spectrophotometer. The signalling pathway that TLR7 activation leads to downstream NF-κB activation has been widely accepted, and therefore similar reporter assay was also widely used for evaluating TLR7 agonist (Tsuneyasu Kaisho and Takashi Tanaka, Trends in Immunology, Volume 29, Issue 7, July 2008, Pages 329.sci; Hiroaki Hemmi et al, Nature Immunology 3, 196-200 (2002).The compounds of the present invention were tested in the above assay for their TLR7 agonism activity as described herein and results are listed in Table 1. The Examples were found to have EC50 of about 3 μM to about 500 μM. Particular compounds of formula (I) or (Ia) were found to have EC50 of about 3 μM to about 200 μM.
Measurement: EC50 = 19000 nM (pChEMBL 4.71999979019165)
Descriptors: MW 412.47, ALogP 0.74, HBA 10, HBD 3, PSA 149.53, QED 0.61
Compound (SMILES): CCC[C@H](N)c1cccc(-c2cc(Cl)c3cnn(-c4cccc(CO)n4)c3c2)n1
InChIKey: RSRNMFXWODENAT-SFHVURJKSA-N
Target: Interleukin-1 receptor-associated kinase 4 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9NWZ3
Assay: Biochemical Assay: The biochemical assay is in a AlphaScreen format. The kinase reaction is based on the IRAK-4 phosphorylation of a biotin labeled peptide. The phosphopeptide is incubated with anti-phosphothreonine antibody as well as streptavidin- and protein A-coated beads. Binding of the protein-A coated beads to the antibody and the streptavidin beads to the peptide, leads to an energy transfer from one bead to the other, ultimately producing a luminescent/fluorescent signal.Generally, the kinase reaction is carried out at 1 nM IRAK4, 1.6 μM peptide, 1 mM ATP in reaction buffer 50 mM Hepes, 60 mM NaCl, 5 mM MgCl2, 0.25 mM MnCl2, 2 mM DTT, 0.01% BSA, 0.01% Tween-20) for 3.5 h at RT.
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 407.91, ALogP 4.43, HBA 6, HBD 2, PSA 89.85, QED 0.49
Compound (SMILES): O=C(Nc1ccc(-c2n[nH]c(=O)c3ccccc23)cc1)c1ccn(C(F)F)n1
InChIKey: XXRUTFCEEGCCQC-UHFFFAOYSA-N
Target: Rho-associated protein kinase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q13464
Assay: In Vitro Assay: The effectiveness of compounds of the present invention as ROCK inhibitors can be determined in a 30 μL assay containing 20 mM HEPES, pH 7.5, 20 mM MgCl2, 0.015% Brij-35, 4 mM DTT, 5 μM ATP and 1.5 μM peptide substrate (FITC-AHA-AKRRRLSSLRA-OH). Compounds were dissolved in DMSO so that the final concentration of DMSO was <2%, and the reaction was initiated with Rho kinase variants. After incubation, the reaction was terminated by the addition of EDTA and the phosphorylated and non-phosphorylated peptides separated using a LABCHIP® 3000 Reader (Caliper Life Sciences). Controls consisted of assays that did not contain compound, and backgrounds consisted of assays that contained enzyme and substrate but had EDTA from the beginning of the reaction to inhibit kinase activity. Compounds were tested in dose-response format, and the inhibition of kinase activity was calculated at each concentration of compound. The inhibition data were fit using a curve-fitting program to determine the IC50; i.e., the concentration of compound required to inhibit 50% of kinase activity.
Measurement: IC50 > 1000 nM (pChEMBL 6)
Descriptors: MW 381.34, ALogP 3.43, HBA 5, HBD 2, PSA 92.67, QED 0.57
Compound (SMILES): Cc1nc(C(=O)NCC(=O)O)c(O)c2ccc(Oc3ccc4c(c3)OCCO4)nc12
InChIKey: DLGCRYLHUYIBNN-UHFFFAOYSA-N
Target: Egl nine homolog 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9GZT9
Assay: Fluorescence polarization (FP) Assay: To a NETN (20 mM Tris.HCl, 100 mM NaCl, 1 mM EDTA, 0.5% NP-40, 1 mM PMSF) buffer containing 200 μM ascorbic acid, 20 μM α-ketoglutaric acid, 100 μM FeCl2 was added FAM-HIF (556-575) at a final concentration of 1 μM in the dark. Subsequently, the desired concentration of the test compound or the positive compound was added (the compound was replaced by the buffer in the negative control and the positive control). Finally, PHD2 was added at a final concentration of 0.5 μg/μl (PHD2 was replaced by the buffer in the negative control). They were mixed well and allowed to stand at room temperature for 30 minutes in the dark followed by 95° C. water bath for 1 minute, and then the reaction was terminated. After the temperature drops to room temperature, the sample was prepared well for use. EBC buffer (50 mM Tris.HCl, 120 mM NaCl, 0.5% NP-40) was added to the corresponding wells of a black 96-well test plate. A GST-VBC complex was added to the corresponding test wells at a final concentration of 300 nM (using the wells containing only EBC buffer as blank wells). Subsequently, the corresponding PHD2 prolyl hydroxylation reaction sample was added in the dark as a substrate with a final concentration of 100 nM. After mixing well, the lateral and longitudinal fluorescence intensity values were measured using a full-wavelength multifunctional microplate reader (TECAN infinite M1000) at an excitation wavelength of 407 nm and an emission wavelength of 518 nm.
Measurement: IC50 = 210 nM (pChEMBL 6.679999828338623)
Descriptors: MW 411.37, ALogP 2.02, HBA 8, HBD 3, PSA 140.1, QED 0.57
Compound (SMILES): COc1cc(OC)c(Cl)c(-c2cc3cnc(Nc4ccc(N5CCC(N(C)C)CC5)cc4)nc3n3cnnc23)c1
InChIKey: KBMMQFUIHXLUTH-UHFFFAOYSA-N
Target: Fibroblast growth factor receptor 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P11362
Assay: Kinase Assay: The inhibitory activity of some compounds of the present disclosure against FGFR1, FGFR2, FGFR3, FGFR4, KDR was tested by mobility shift assay (concentration of ATP is Km value).Test method:Reagent: basic kinase buffer: 50 mM HEPES (pH 7.5); 0.0015% Brij-35Stop buffer: 100 mM HEPES (pH 7.5); 0.015% Brij-35; 0.2% Coating Reagent #3; 50 mM EDTAPreparing of compounds: dilute test compounds to specific concentration using 100% DMSOReaction process:    1) preparing of 2.5× enzyme solution    adding kinase to 1× basic kinase buffer    2) preparing of 2.5× peptide solution    adding FAM-labeled peptide and ATP to 1× basic kinase buffer  3) preparing of analysis board    transferring 10 μL of test compound to 384-well plate, adding 904, of 1× basic kinase buffer    4) adding 104, of 2.5× enzyme solution to each well of the assay plate and incubating for 10 min at room temperature    5) adding 104, of 2.5× enzyme solution to each well of the assay plate and incubating for specific time at 28° C. 6) stop the reaction by adding 254, of stop solution to each well    7) reading data with Caliper and calculating IC50 value.
Measurement: IC50 < 1 nM (pChEMBL 9)
Descriptors: MW 559.07, ALogP 5.28, HBA 10, HBD 1, PSA 92.94, QED 0.29
Compound (SMILES): CN1CCN(CCC#Cc2ccc(CO[C@H]3CCC[C@@H]3NC(=O)c3cc(-c4cnn(C)c4)cnc3N)cc2)CC1
InChIKey: IXEXFVBVSLCJNA-VMPREFPWSA-N
Target: Tyrosine-protein kinase Mer (SINGLE PROTEIN), Homo sapiens
UniProt: Q12866
Assay: TBD: TBD
Measurement: IC50 = 55 nM (pChEMBL 7.260000228881836)
Descriptors: MW 541.7, ALogP 2.92, HBA 8, HBD 2, PSA 101.54, QED 0.42
Compound (SMILES): Cc1cc2nc(C(CNC(=O)c3ccc(-n4cnnc4)cc3Cl)c3cccc(F)c3)oc2cc1C
InChIKey: MNYBRERVVDKSQG-UHFFFAOYSA-N
Target: Coagulation factor IX (SINGLE PROTEIN), Homo sapiens
UniProt: P00740
Assay: Determination of Inhibitory Activity Against Factor IXa: Formation of a clot to stem bleeding at a site of blood vessel injury involves the coordinated activity of a group of plasma proteins that initiate and propagate fibrin formation and subsequently protect fibrin from premature degradation. Factor IX is a key component of the plasma system that forms a fibrin clot at a site of vascular injury. The activity of Factor IXa is measured by monitoring the cleavage of the fluorescent peptide, CH3SO2-D-CHG-Gly-Arg-AFC.AcOH (CHG is cyclohexyl-glycine and AFC is trifluoro aminomethyl coumarin). Factor IXa cleaves the amide bond between Arg and AFC, thereby releasing the AFC fluorophore. The free AFC can be detected with a fluorescence detector at an excitation wavelength of 405 nM and emission wavelength of 510 nM.
Measurement: IC50 = 71.9 nM (pChEMBL 7.139999866485596)
Descriptors: MW 489.94, ALogP 5.38, HBA 6, HBD 1, PSA 85.84, QED 0.35
Compound (SMILES): Cc1nc(N(C)C(=O)Cc2ccc(-c3ccccn3)cc2)sc1[S@@](C)(=N)=O
InChIKey: ZDGRIBNQGIXLIO-MHZLTWQESA-N
Target: Carbonic anhydrase 2 (SINGLE PROTEIN), Homo sapiens
UniProt: P00918
Assay: Enzymatic Activity Assay: A protocol for determination of the carbonic anhydrase enzymatic activity at rt using the pH indicator method is described below:1 μL inhibitor (50 mM stock solution in DMSO) is diluted to a final test concentration ranging from 100 μM down to 1 nM (or 1 μL water in controls) and incubated for 2 min with 0.5 to 2 EU human Carboanhydrase I (180 U/mg) in 400 μL water and 200 μL phenol red indicator solution (20 mg/L). An enzymactic unit (EU) is defined as an amount which doubles the non catalyzied rate. The hydration reaction is initiated by adding 100 μL 0.5M bicarbonate buffer (0.3M Na2CO3; 0.2M NaHCO3) and subsequent dumping of CO2 through a needle (0.7×30 mm; 22 G×1.25) into the assay solution at a rate of 10 mL gas/minute. The time to colour change (pH 7.2) is determined with a microchronometer or stop watch.
Measurement: IC50 > 10000 nM (pChEMBL 5)
Descriptors: MW 400.53, ALogP 3.75, HBA 6, HBD 1, PSA 87.01, QED 0.71
Compound (SMILES): Cc1cc(-c2cnc3cnn(Cc4cccnn4)c3c2)ccc1Cl
InChIKey: CUPKBSXUQDXOKC-UHFFFAOYSA-N
Target: Glutamate NMDA receptor; GRIN1/GRIN2B (PROTEIN COMPLEX), Homo sapiens
UniProt: Q05586
Assay: Effects of Test Articles on Cloned Human NR1/GuN2B Ion Channels Expressed in Mammalian Cells: NMDA receptors are ion channels that are highly permeable to Ca2+ ions, rendering it possible to monitor NMDA receptor function using cell-based calcium flux assay. In this assay, co-agonists glutamate and glycine are added to cells heterologously expressing human GluN1/GluN2B NMDA receptors to initiate cellular Ca2+ influx. The time course of the changes in intracellular calcium is measured using a fluorescent dye and a FLIPR (Fluorometric Imaging Plate Reader) device.Twenty-four hours before measurements, the expression of the NMDA receptors in the stable cell line is induced with Tet-On inducible system in the presence of a non-selective NMDA receptor blocker. On the day of the experiment, cell culture media is carefully washed, and the cells are loaded with Calcium 5 Dye Kit (Molecular Devices) in dye loading buffer containing 137 mM NaCl, 4 mM KCl, 2 mM CaCl2, 0.5 mM MgCl2 (standard assay) or 1.5 mM MgCl2 (HTS assay), 10 mM HEPES and 5 mM D-glucose; pH 7.4. After 1 h incubation at the room temperature, the dye is washed away with the assay buffer (137 mM NaCl (standard assay) or 150 mM (HTS assay), 4 mM KCl (standard assay) or 3 mM (HTS assay), 2 mM CaCl2), 0.01 mM EDTA, 10 mM HEPES and 5 mM D-glucose; pH 7.4) In the FLIPR TETRA reader, various concentrations of the test compounds are added to the cells for 5 min while fluorescence is monitored to detect potential agonist activity. Next, co-agonists, glutamate and glycine are added for another 5 minutes. The concentration of glutamate corresponding to EC40 (standard assay) or EC40 (HTS assay) is used to maximize the assay's signal window and ability to detect NMDA receptor antagonists and negative allosteric modulators. A saturating concentration (10 μM) of glycine is also present in the assay. A non-selective NMDA receptor antagonist, (+)MK-801 is used as a positive control for antagonist activity. The fluorescent signal in the presence of test compounds is quantified and normalized to the signal defined by the appropriate control wells.
Measurement: IC50 = 404 nM (pChEMBL 6.389999866485596)
Descriptors: MW 335.8, ALogP 3.9, HBA 5, HBD 0, PSA 56.49, QED 0.57
Compound (SMILES): CC(C)(C)CNc1c(C#N)cnc2c(C#N)cc(N[C@H](C3=CN(C4C[C@@H]4F)NN3)c3cccc4cnccc34)cc12
InChIKey: VVWRBRKNGYZLSA-PHSGMAHSSA-N
Target: Epidermal growth factor receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P00533
Assay: High Throughput Biochemical Assay: EGFR activity was measured using KinEASE (Cisbio), a time-resolved fluorescence resonance energy transfer (TR-FRET) immunoassay. In this assay, EGFR-catalyzes the phosphorylation of a universal Tyrosine kinase peptide substrate labeled with XL665. Europium conjugated phosphor-tyrosine specific antibody binds the resulting phosphorylated peptide. Formation of phosphorylated peptide is quantified by TR-FRET with Europium as the donor and XL665 the acceptor. The assay was performed in two main steps. The first step is the kinase reaction step and the second step is the detection step with TR-FRET reagents. In brief, test compounds 1:3 serially diluted in DMSO were delivered into Corning white, low volume, non-binding 384 well plates using the Echo 550 acoustic liquid dispenser (Labcyte®). EGFR enzyme (Human EGFR, cytoplasmic domain [669-1210] from Carna Biosciences Cat. No. 08-115) and substrates TK substrate-biotin (included in Cisbio HTRF KinEASE-TK kit Cat. No. 62TK0PEJ) were dispensed into assay plates using a Multi-Flo (Bio-Tek Instruments). The standard 10 μL reaction mixture contained 6 μM ATP (1×Km) or 12 μM ATP (2×Km), 1 μM biotinylated peptide, 0.3 nM EGFR (for 1×Km ATP) or 0.1 nM EGFR (for 2×Km ATP) in reaction buffer (10 mM MOPs, pH 7.0, 1.5% Glycerol, 0.5 mg/ml BSA, 10 mM Mg-Acetate, 1 mM DTT, 0.025% NP-40). After 60 min of incubation at room temperature, 10 μL of Stop and Detect Solution (1:400 Europium Cryptate labeled anti-phosphorylated peptide antibody solution and 125 nM strepavidin-XL665 Tracer in a 50 mM Hepes pH 7.0 detection buffer containing sufficient EDTA) was added. The plate was then further incubated for over 60 minutes at room temperature and read using an Envision 2103 Multilabeled reader (PerkinElmer) with excitation/emission/FRET emission at 340 nm/615 nm/665 nm, respectively.
Measurement: IC50 = 1 nM (pChEMBL 9)
Descriptors: MW 547.64, ALogP 5.41, HBA 9, HBD 4, PSA 124.72, QED 0.24
Compound (SMILES): O=C(Cc1ccnnc1)N1CC[C@@]2(S(=O)(=O)c3ccc(F)cc3)c3ccc(C(F)(C(F)(F)F)C(F)(F)F)cc3CC[C@@H]12
InChIKey: PTUBSSJVFTVOLU-ISKFKSNPSA-N
Target: Nuclear receptor ROR-gamma (SINGLE PROTEIN), Mus musculus
UniProt: P51450
Assay: General RORgamma SPA binding assay : The binding of potential ligands to RORγ is measured by competition with [3H]25-hydroxycholesterol (Perkin Elmer NET674250UC) using a scintillation proximity assay (SPA). 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 chromatography. 15 μg/well RORγ (A262-S507) is incubated with test compound at varying concentrations in 3-fold serial dilution, with final concentrations ranging from 16.6 μM to 0.28 nM, for 10 min at rt in PBS buffer (Invitrogen #14190-144) containing 0.5% fatty acid free BSA (Gemini Bio-Products, Cat. #700-107P) and 0.1% glycerol (Sigma Cat#G5516). 10 nM of [3H] 25-hydroxycholesterol is then added, and the reaction is incubated for 10 min. 10 mg/mL of Copper-His Tag-PVT beads (Perkin Elmer cat # RPNQ0095) are added, and the mixture is incubated for 60 min. The reaction is read on a TopCount Microplate scintillation plate reader (Perkin Elmer). The competition data of the test compound over a range of concentrations was plotted as percentage inhibition of radioligand specifically bound in the absence of test compound (percent of total signal). After correcting for non-specific binding, IC50 values were determined. The IC50 value is defined as the concentration of test compound needed to reduce [3H] 25-hydroxycholesterol specific binding by 50% and is calculated using the four parameter logistic equation to fit the normalized data.
Measurement: IC50 = 114 nM (pChEMBL 6.940000057220459)
Descriptors: MW 619.53, ALogP 5.36, HBA 5, HBD 0, PSA 80.23, QED 0.28
Compound (SMILES): CC(=O)O[C@@H]1CCC[C@H]1C(C#N)(c1ccccc1F)C1CCN(CC2CN(c3ccc(S(=O)(=O)C4CC4)cc3)C2)CC1
InChIKey: GKIFDNSCASLJLJ-RDGBXCDUSA-N
Target: Menin (SINGLE PROTEIN), Homo sapiens
UniProt: O00255
Assay: Fluorescence Polarization (FP) Competitive Binding Assay: A fluorescence polarization (FP) competitive binding assay was used to determine the binding affinities of representative menin inhibitors. A FAM labeled fluorescent probe was designed and synthesized based on a MLL1 peptide (FAM-MM2). Equilibrium dissociation constant (Kd) value of FAM-MM2 to menin protein was determined from protein saturation experiments by monitoring the total fluorescence polarization of mixtures composed with the fluorescent probe at a fixed concentration and the protein with increasing concentrations up to full saturation. Serial dilutions of the protein were mixed with FAM-MM2 to a final volume of 200 μl in the assay buffer (PBS with 0.02% Bovine γ-Globulin and 4% DMSO. 0.01% Triton X-100 was added right before assays). Final FAM-MM2 concentration was 2 nM. Plates were incubated at room temperature for 30 minutes with gentle shaking to assure equilibrium. FP values in millipolarization units (mP) were measured using the Infinite M-1000 plate reader (Tecan U.S., Research Triangle Park, N.C.) in Microfluor 1 96-well, black, v-bottom plates (Thermo Scientific, Waltham, Mash.) at an excitation wavelength of 485 nm and an emission wavelength of 530 nm. Kd value of FAM-MM2, which was calculated by fitting the sigmoidal dose-dependent FP increases as a function of protein concentrations using Graphpad Prism 6.0 software (Graphpad Software, San Diego, Calif.), is determined as 1.4 nM.
Measurement: IC50 = 5700 nM (pChEMBL 5.239999771118164)
Descriptors: MW 593.77, ALogP 5.1, HBA 7, HBD 0, PSA 90.71, QED 0.37
Compound (SMILES): COc1cc(C(=O)N2CC3CCC2[C@@H]3N)cc2nc(-c3cc4ccc(-c5ccc6nc(C(=O)O)cc(O)c6c5)nc4n3CC3CC3)n(C)c12
InChIKey: VGSLOUFPKUYVTO-DOZIQCTLSA-N
Target: Protein-arginine deiminase type-4 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9UM07
Assay: Rapid-Fire Mass Spectrum (RFMS) Assay: Compounds were solubilized in 100% DMSO to achieve a 10 mM compound concentration. Compound stock solutions were stored at RT. A series of dilutions were prepared in DMSO and mixed 8 times with 20 μL mixing volume. Final top concentration of compound in the assay is 50 μM. Final assay conditions were as follows:Reaction volume: 26 μlAssay buffer: 25 mM hepes, pH 7.5, 5 mM NaCl, 1 mM DTT, 0.2 mg/ml BSA, 0.01% CHAPS, 50 μM Calcium, and 5 μM TPENFinal concentrations: 5 nM hPAD4 enzyme, 250 μM BAEE, and 0.5% DMSOTotal incubation time: 30 mins compound and enzyme preincubation at 37° C., 90 min enzyme/substrate reaction, 30 min reaction with phenyl glyoxal at 37° C.Stop solution: 40 μl 5% TCA in ACN
Measurement: IC50 = 116 nM (pChEMBL 6.940000057220459)
Descriptors: MW 657.73, ALogP 5.19, HBA 10, HBD 3, PSA 161.62, QED 0.21