lovingscience/chembl-v37-raw-activities huggingface dataset:
Compound (SMILES): CN(C)C1CCN(c2nc(-c3ccc(C#N)cc3)c(-c3cnc4c(c3)oc(=O)n4C)n3ccnc23)CC1
InChIKey: JOKHSKBKZNCIPJ-UHFFFAOYSA-N
Target: Lysine-specific histone demethylase 1A (SINGLE PROTEIN), Homo sapiens
UniProt: O60341
Assay: LANCE LSD1/KDM1A Demethylase Assay: 10 μL of 1 nM LSD-1 enzyme (ENZO BML-SE544-0050) in the assay buffer (50 mM Tris, pH 7.5, 0.01% Tween-20, 25 mM NaCl, 5 mM DTT) were preincubated for 1 hour at 25° C. with 0.8 μL compound/DMSO dotted in black 384 well polystyrene plates. Reactions were started by addition of 10 μL of assay buffer containing 0.4 M Biotin-labeled Histone H3 peptide substrate: ART-K(Me1)-QTARKSTGGKAPRKQLA-GGK(Biotin) SEQ ID NO:1 (AnaSpec 64355) and incubated for 1 hour at 25° C. Reactions were stopped by addition of 10 μL 1× LANCE Detection Buffer (PerkinElmer CR97-100) supplemented with 1.5 nM Eu-anti-unmodified H3K4 Antibody (PerkinElmer TRF0404), and 225 nM LANCE Ultra Streptavidin (PerkinElmer TRF102) along with 0.9 mM Tranylcypromine-HCl (Millipore 616431). After stopping the reactions plates were incubated for 30 minutes and read on a PHERAstar FS plate reader (BMG Labtech).
Measurement: IC50 < 50 nM (pChEMBL 7.301000118255615)
Descriptors: MW 494.56, ALogP 3.31, HBA 10, HBD 0, PSA 108.49, QED 0.37
Compound (SMILES): C[C@@H]1C[C@H]2[C@@H]3C[C@H](F)C4=CC(=O)C=C[C@]4(C)[C@@]3(F)[C@@H](O)C[C@]2(C)[C@@]1(O)C(=O)SCCCCP(=O)(O)O
InChIKey: BEQGDWGTHMWLCN-VAOFZIKSSA-N
Target: Glucocorticoid receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P04150
Assay: Glucocorticoid Receptor Competitor Assay: The potency of binding to gucocorticoid receptor by small molecule compounds was measured with the PolarScreen Glucocorticoid Receptor Competitor Assay Kit, Red (Life Technology, Catalog #A15898) according to the procedure in the kit. In brief, after compounds were diluted and transferred into assay plates, the fluormone GS red and GR full length protein were subsequently added into the assay plates, along with the positive and negative controls. The samples were mixed on a shaker for 1 minute and then the plates were incubated at room temperature for 2-4 hours with minimal light exposure. The plates were read with an Envision and the relative fluorescent signals were calculated. The data were plotted in GraphPad Prism and the EC50 values were calculated with non-linear regression curve fit of the data in GraphPad Prism.
Measurement: IC50 = 160 nM (pChEMBL 6.800000190734863)
Descriptors: MW 548.59, ALogP 3.5, HBA 6, HBD 4, PSA 132.13, QED 0.29
Compound (SMILES): CNCc1ccc(-n2cc(-c3nc(N[C@H]4CCN(S(=O)(=O)CCCC#N)C[C@H]4F)ncc3C#N)cn2)c(Cl)c1
InChIKey: UYSBHQALTCMJGZ-YADHBBJMSA-N
Target: Cyclin-dependent kinase 2/cyclin E1 (PROTEIN COMPLEX), Homo sapiens
UniProt: P24864
Assay: CDK2/Cyclin E1 HERE Enzyme Activity Assay: CDK2/Cyclin E1 enzyme activity assays utilize full-length human CDK2 co-expressed as N-terminal GST-tagged protein with FLAG-Cyclin E1 in a baculovirus expression system (Cama Product Number 04-165). Assays are conducted in white 384-well polystyrene plates in a final reaction volume of 8 μL. CDK2/Cyclin E1 (0.25 nM) is incubated with compounds (40 nL serially diluted in DMSO) in the presence of ATP (50 μM or 1 mM) and 50 nM ULight-labeled eIF4E-binding protein 1 (THR37/46) peptide (PerkinElmer) in assay buffer (containing 50 mM HEPES pH 7.5, 1 mM EGTA, 10 mM MgCl2, 2 mM DTT, 0.05 mg/ml BSA, and 0.01% Tween 20) for 60 minutes at room temperature. The reactions are stopped by the addition of EDTA and Europium-labeled anti-phospho-4E-BP1 antibody (PerkinElmer), for a final concentration of 15 mM and 1.5 nM, respectively. HTRF signals are read after 1 hour at room temperature on a PHERAstar FS plate reader (BMG Labtech). Data is analyzed with IDBS XLFit and GraphPad Prism 5.0 software using a three or four parameter dose response curve to determine IC50 for each compound.
Measurement: IC50 < 50 nM (pChEMBL 7.301000118255615)
Descriptors: MW 572.07, ALogP 3.03, HBA 10, HBD 2, PSA 152.62, QED 0.35
Compound (SMILES): C[C@@H]1CN(c2cc(F)c(C3=CCCN(c4ncc(F)cn4)C3)cc2NC(=O)c2c[nH]c(=O)cc2C(F)(F)F)C[C@H](C)N1C
InChIKey: GYNGZAVRQSHHMV-CALCHBBNSA-N
Target: WD repeat-containing protein 5 (SINGLE PROTEIN), Homo sapiens
UniProt: P61964
Assay: Surface Plasmon Resonance (SPR) Assay: Exemplary compounds of the application were dissolved in 100% DMSO at 10 mM, assayed fresh, and then stored at −20° C. for repeat studies and other experiments. Full length WDR5 with an N-terminal His tag and C-terminal AviTag (Avidity Inc.) was expressed in E. coli with coexpression of BirA to biotin labelled protein in vivo. Purification of the protein was performed using Ni-NTA. The purified WDR5 protein has a molecular weight of 41976 Da.SPR studies were performed using a Biacore T200 instrument (GE Health Sciences Inc.). Biotinylated WDR5 protein (approximately 3000 RU) was stably captured to streptavidin coupled SA chips according to the manufacture's protocol (GE Health Sciences Inc.). The running buffer used was HBS-EP (20 mM Hepes pH 7.4, 150 mM NaCl, 3 mM EDTA, 0.05% P-20) plus 5% DMSO with a flow rate of 40 μl/min. For SPR analysis, 5 different concentrations of each exemplary compound of the application were sprayed into 96 or 384 well plates using an HP D300 digital dispenser. The concentration ranged from about 195 nM to about 12 nM in a two-fold series. Concentration ranges were adjusted higher or lower for weaker or more potent compounds respectively when necessary. For the KD determinations, single cycle kinetic analysis was performed with an on time of 60 seconds, and an off time of 300 or 600 seconds. Curve fitting and KD calculations were performed with the Biacore T200 Evaluation software (GE Health Sciences Inc).
Measurement: Kd = 1.3 nM (pChEMBL 8.890000343322754)
Descriptors: MW 603.6, ALogP 4.54, HBA 7, HBD 2, PSA 97.46, QED 0.41
Compound (SMILES): CC(C)(C)CNc1c(C#N)cnc2c(C#N)cc(N[C@H](C3=CN(C4(C#N)CC4)NN3)c3cccc4ncsc34)cc12
InChIKey: KHNRORJMYRKXQJ-MHZLTWQESA-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 560.69, ALogP 5.42, HBA 11, HBD 4, PSA 148.51, QED 0.23
Compound (SMILES): COC(=O)[C@@H](C)Cn1c(=O)[nH]/c(=N\c2ccc(CC(C)C)c(F)c2)n(Cc2ccc(Cl)cc2)c1=O
InChIKey: AFQDQWQVLUAASG-INIZCTEOSA-N
Target: P2X purinoceptor 3 (SINGLE PROTEIN), Homo sapiens
UniProt: P56373
Assay: Human P2X3 Receptor Inhibition Assay: Stably expressing cell line (C6BU-1 transfected with human P2X3 receptor gene (GenBank accession number Y07683) was used. The cells were seeded in a 96-well microtiter plate at a concentration of 8000 cells/well and cultured in the medium (8.3% fetal bovine serum, 8.3% horse serum, 1% antibiotic and antifungal in DMEM) for one day at 37° C. under 5% carbon dioxide atmosphere. The medium was replaced with 4 μM Fluo-3-AM solution (20 mM HEPES, 137 mM NaCl, 2.7 mM KCl, 0.9 mM MgCl2, 5.0 mM CaCl2, 5.6 mM D-glucose, 2.5 mM probenecid, 10% BSA, and 0.08% Pluronic F-127, pH 7.5) and incubated at 37° C. under 5% dioxide carbon atmosphere for one hour. The plate was washed with washing buffer (20 mM HEPES, 137 mM NaCl, 2.7 KCl, 0.9 mM MgCl2, 5.0 mM CaCl2, 5.6 mM D-glucose, 2.5 mM probenecid, pH 7.5), each well was added with 40 μL of this buffer. The plate was placed in High-Throughput Screening System FDSS 3000 (Hamamatsu Photonics K.K.). Measurement of fluorescence intensity by FDSS 3000 was started, and 40 μl, of DMSO solutions containing different concentrations of the test, compound as prepared by dilution with dilution buffer (20 mM HEPES, 137 mM NaCl, 2.7 mM KCl, 0.9 mM MgCl2, 5.0 mM CaCl2, 5.6 mM D-glucose, 2.5 mM probenecid, 0.1.% Pluronic F-127, pH 7.5) were dispensed to each well through the built-in automatic dispenser. Five minutes after, 50 nM ATP solution (50 μL) prepared by dilution with the dilution buffer was dispensed through the built-in automatic dispenser, and the measurement of fluorescence intensity was continued for 3 min. For each well, the specific maximum fluorescence intensity was calculated as the ratio of the maximum fluorescence intensity after addition of the ATP solution to the fluorescence intensity at the starting of the measurement. The 50% inhibitory concentration (IC50) was calculated under the assumption that the specific maximum fluorescence intensity without test compound is 0% inhibition and that the specific maximum fluorescence intensity when the dilution buffer was added in place of ATP solution is 100% inhibition, to evaluate the inhibitory activity of the test compound. FDSS software (Hamamatsu Photonics K.K.) was used for calculation of the specific maximum fluorescence intensity. IC50 was calculated using Microsoft Excel (Microsoft Corporation) and Ltd.).
Measurement: IC50 = 45 nM (pChEMBL 7.349999904632568)
Descriptors: MW 502.97, ALogP 3.42, HBA 7, HBD 1, PSA 98.45, QED 0.48
Compound (SMILES): COc1ccc(CCC(=O)N[C@@H](CC2COc3c2ccc(C)c3C)B(O)O)nc1
InChIKey: JNTYBSNMNIHDRG-FUBQLUNQSA-N
Target: Proteasome Macropain subunit MB1 (SINGLE PROTEIN), Homo sapiens
UniProt: P28074
Assay: Determination of Beta5 Activity: Measurement of Beta5 inhibition is performed in 384 well format based on fluorescence intensity assay.Purified human constitutive proteasome (1.25 nM) and serial diluted compounds in DMSO (range of concentrations from 30 μM to 15 μM) or controls are incubated for 20 minutes at 25° C. in assay buffer containing 50 mM Tris pH 7.4, 0.03% SDS, 1 mM EDTA and 1% DMSO. The reaction is initiated by the addition of the fluorogenic peptide substrate, Suc-LLVY-AMC (Bachem 1-1395), at a concentration of 40 μM. After 60 minutes of incubation at 37° C., fluorescence intensity is measured at λex=350 nm and λem=450 nm with a fluorescence reader (Perkin Elmer Envision reader or equivalent).
Measurement: IC50 > 5000 nM (pChEMBL 5.301000118255615)
Descriptors: MW 398.27
Compound (SMILES): C/C=C/Cn1cc(-c2ccc(C(N)=O)c(Cl)c2)c2cc[nH]c2c1=O
InChIKey: HVGPJXMOBVSNGI-NSCUHMNNSA-N
Target: Chromatin remodeling regulator CECR2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9BXF3
Assay: IC50 Measurements for Inhibitors Using TAF1-BD2 TR-FRET Binding Assay: His/Flag epitope tagged TAF1-BD21504-1635 was cloned, expressed, and purified to homogeneity. TAF1-BD2 binding and inhibition was assessed by monitoring the engagement of a biotinylated small molecule compound 1000 (Example 124) with the target using the TR-FRET assay technology (Perkin-Elmer). Specifically, in a 384 well ProxiPlate TAF1-BD2 (6 nM final) was combined with biotin-ligand (50 nM final) in 50 mM HEPES (pH 7.5), 50 mM NaCl, 1 mM TCEP, 0.01% (w/v) BSA, and 0.008% (w/v) Brij-35 either in the presence of DMSO (final 0.2% DMSO) or compound dilution series in DMSO. After 10 minutes incubation at room temperature, a mixture Eu-W1024 Anti-6×His antibody (Perkin Elmer AD0110) and SureLight¿ Allophycocyanin-Streptavidin (APC-SA, Perkin Elmer CR130-100) were added to a final concentrations of 0.2 nMolar antibody and 25 nMolar APC-SA, respectively. After twenty minutes of equilibration, the plates were read on an Envision instrument and IC50s calculated using a four parameter non-linear curve fit. Novel compound 1000 and the TAF1-BD2 TR-FRET Binding Assay described above represent additional embodiments of the invention.
Measurement: IC50 = 1600 nM (pChEMBL 5.800000190734863)
Descriptors: MW 341.8, ALogP 3.33, HBA 3, HBD 2, PSA 80.88, QED 0.71
Compound (SMILES): C=CCn1c(=O)c2cnc(Nc3ccc(N4CCC5(CCN(C(C)=O)CC5)CC4)cc3)nc2n1-c1cccc(C(C)(C)O)n1
InChIKey: WHAFWTPMGRCXQX-UHFFFAOYSA-N
Target: Wee1-like protein kinase (SINGLE PROTEIN), Homo sapiens
UniProt: P30291
Assay: In Vitro Enzymatic Inhibitory Activity Assay: Experimental Reagents:Basic reaction buffer: 20 mM hydroxyethylpiperazine ethanesulfuric acid (pH 7.5), 10 mM magnesium chloride, 1 mM EGTA, 0.02% Brij35, 0.02 mg/mL bovine serum albumin, 0.1 mM Na3VO4, 2 mM DTT, 1% DMSOEnzyme: Wee-1 concentration is 150 nMMatrix: MBP concentration is 20 μMCompound Treatment:The tested compound was formulated into a 5 mM solution with 100% DMSO, and a 10 points 3-fold gradient dilution was performed with DMSO through an automated pipetting station epMotion 5070.Experiment Procedure:1. Fresh matrix configuration reaction buffer was prepared;2. The Wee-1 was added into the matrix solution and shaked gently;3. DMSO solution of the compound were added into the kinase reaction mixture using an acoustic technique (Echo 550; nanoliter range) and incubated for 20 minutes at room temperature;4. 33P-ATP (specific activity, 10 μCi/μL) was added into the reaction mixture to stimulate the reaction;5. Incubated for 2 hours at room temperature;6. Kinase activity was detected by filter-binding method.
Measurement: IC50 = 58.8 nM (pChEMBL 7.230000019073486)
Descriptors: MW 596.74, ALogP 4.36, HBA 10, HBD 2, PSA 121.41, QED 0.3
Compound (SMILES): COc1cccc(F)c1-c1nccc(C(=O)Nc2ccc3c(nc(-c4ccnn4C)n3C)c2N2CCC[C@H]2CN)n1
InChIKey: BZYDVOZYBUNHOK-KRWDZBQOSA-N
Target: Mitogen-activated protein kinase kinase kinase kinase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q92918
Assay: Kinase Binding Assay: A stock solution of 1 mM test compound was prepared in DMSO. The compound plate was prepared by 3-fold and 11-point serial dilutions. 0.1 μL of the compound in DMSO was transferred from the compound plate to the white 384 well polystyrene plates. The assay buffer contained 50 mM HEPES, pH 7.5, 0.01% Tween-20, 5 mM MgCl2, 0.01% BSA, and 5 mM DTT. 5 μL of 4 nM active HPK1 (SignalChem M23-11G) prepared in the buffer was added to the plate. The enzyme concentration given was based on the given stock concentration reported by the vender. 5 μl of 18 nM tracer 222 (ThermoFisher PV6121) and 4 nM LanthaScreen Eu-Anti GST antibody (ThermoFisher PV5595) were added. After one hour incubation at 25° C., the plates were read on a PHERAstar FS plate reader (BMG Labtech). Ki values were determined.
Measurement: Ki < 100 nM (pChEMBL 7)
Descriptors: MW 555.62, ALogP 3.76, HBA 10, HBD 2, PSA 129.01, QED 0.31
Compound (SMILES): CN(C)CCC(c1ccccc1)n1cc(NC(=O)c2cc(-c3ccccc3)on2)cn1
InChIKey: JPRMTWIEQXNKAZ-UHFFFAOYSA-N
Target: Stearoyl-CoA desaturase (SINGLE PROTEIN), Homo sapiens
UniProt: O00767
Assay: Compound Profiling Methods: Strains expressing SCD1 or SCD5 as the sole desaturase, the human SCD1 and SCD5 genes were used to evaluate inhibition of SCD1/SCD5 using reduced growth as a surrogate for SCD inhibition. These yeast strains express human SCD1 or SCD5 from a plasmid harbored in a strain in which the yeast OLE1 gene is deleted.All compound profiling experiments were performed using the same basic protocol. Yeast were cultured using standard techniques in complete synthetic media lacking uracil and containing yeast nitrogen base supplemented with 2% (w/v) glucose (SD-Ura) Starter cultures were inoculated in 3 mL SD-Ura media containing 0.01% tween and 0.2 mM palmitoleic and oleic acid. Cultures were incubated overnight in a 30° C. shaker incubator (225 rpm). Saturated morning cultures were centrifuged, washed in SD-Ura media lacking TWEEN-20 and fatty acids, and then diluted 1:20 in fresh SD-Ura media also lacking TWEEN-20 and fatty acids. Cells were grown for 6 h to an  (optical density) of 0.4-0.8 at 30° C. with shaking.Compound stocks (10 mM in 100% DMSO) were arrayed into 384-round well, v-bottom polypropylene plates and diluted according to indicated dilution factors. Compound administration was performed in two separate steps. First, 15 μL of SD-Ura was dispensed into clear 384-well assay plates using a MULTIDROP Combi reagent dispenser. The diluted compound stock plates were then applied to the assay plates using an automated workstation (Perkin Elmer JANUS) outfitted with a 384-pin tool containing slotted pins that deliver 100 nL of compound. The cultures described above were centrifuged and washed with media lacking TWEEN-20 or oleic and palmitoleic acids. Cultures were then resuspended at a 2-fold concentrated  of 0.02 (final  of 0.0.01) in SD-Ura. 15 μL of diluted culture was then dispensed into the pinned assay plate to achieve 30 μL of the 1× culture (0.01) and a top drug concentration of 33.3 μM.After yeast delivery, assay plates were incubated under humidified conditions at 30° C. for 40 h. Yeast growth was monitored by reading the  of each well using a microplate reader (Perkin Elmer EnVision). Data were analyzed as follows. Raw data were processed by background subtracting and converting values to a percent of the nontreated condition for that strain [(EXP-0.035)/(DMSO-0.035)×100%].
Measurement: IC50 > 45000 nM (pChEMBL 4.3470001220703125)
Descriptors: MW 415.5, ALogP 4.33, HBA 6, HBD 1, PSA 76.19, QED 0.46
Compound (SMILES): CN(C)C(=O)c1cccc(-c2cnc3sc(NC4CCC(O)CC4)nn23)c1
InChIKey: XKYXBELZDYKZJC-UHFFFAOYSA-N
Target: Tyrosine-protein kinase Fyn (SINGLE PROTEIN), Homo sapiens
UniProt: P06241
Assay: ADP-Glo Kinase Assay: The assay procedure determines the IC50 of each potential FYN kinase inhibitor by measuring the enzyme catalyzed ATP-dependent phosphorylation of the FYN substrate peptide. The ADP-Glo Kinase Assay is specifically designed to quantify kinase activity by measuring the ADP produced in the reaction The reaction buffer comprises 40 mM Tris.HCl (pH 7.5), 20 mM MgCl2, 0.1 mg/ml BSA and 5 microM DTT. Individual compounds within the chemical library were dissolved in DMSO at known concentrations. Briefly, 2.5 microliters of reaction buffer containing 3 picograms of FYN kinase and serial dilutions of each potential FYN kinase inhibitor (9 3-fold dilutions of a 10 micromolar top sample) are placed into each well of a 384 well microtiter plate and incubated at room temperature for 30 minutes. An additional 2.5 microliters of reaction buffer containing 100 picograms of the FYN substrate and 12.5 picomoles of ATP are then added to each well and the reaction carried out for 90 minutes at room temperature.
Measurement: IC50 = 533 nM (pChEMBL 6.269999980926514)
Descriptors: MW 385.49, ALogP 2.88, HBA 7, HBD 2, PSA 82.76, QED 0.72
Compound (SMILES): CC(C)C[C@@H](C(=O)N1C[C@]2(CC1C#N)C(=O)Nc1ccccc12)N(C)C(=O)c1ccc(F)cc1Cl
InChIKey: KLZWGLTULASLNG-UTMPKSGFSA-N
Target: Replicase polyprotein 1ab (SINGLE PROTEIN), Severe acute respiratory syndrome coronavirus 2
UniProt: P0DTD1
Assay: Fluorescence Assay (FRET): SARS-CoV-2 3C-like (3CL) protease fluorescence assay (FRET): Recombinant SARS-CoV-2 3CL-protease was expressed and purified. TAMRA-SITSAVLQSGFRKMK-Dabcyl-OH peptide 3CLpro substrate was synthesized. Black, low volume, round-bottom, 384 well microplates were used. In a typical assay, 0.85 μL of test compound was dissolved in DMSO then incubated with SARS-CoV-2 3CL-protease (10 nM) in 10 μL assay buffer (50 mM HEPES [pH 7.5], 1 mM DTT, 0.01% BSA, 0.01% Triton-X 100) for 30 min at RT. Next, 10 μL of 3CL-protease substrate (40 μM) in assay buffer was added and the assays were monitored continuously for 1 h in an Envision multimode plate reader operating in fluorescence kinetics mode with excitation at 540 nm and emission at 580 nm at RT. No compound (DMSO only) and no enzyme controls were routinely included in each plate. All experiments were run in duplicate. Data Analysis: SARS-CoV-2 3CL-protease enzyme activity was measured as initial velocity of the linear phase (RFU/s) and normalized to controlled samples DMSO (100% activity) and no enzyme (0% activity) to determine percent residual activity at various concentrations of test compounds (0-10 μM). Data were fitted to normalized activity (variable slope) versus concentration fit in GraphPad Prism 7 to determine IC50.
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 496.97, ALogP 3.98, HBA 4, HBD 1, PSA 93.51, QED 0.68
Compound (SMILES): Cc1ccc2c(n1)c(Cc1ccc(S(C)(=O)=O)cn1)c(C)n2C/C(F)=C/CN
InChIKey: PDLZPUKGZZHLND-NVNXTCNLSA-N
Target: Amine oxidase [copper-containing] 3 (SINGLE PROTEIN), Homo sapiens
UniProt: Q16853
Assay: Inhibit Human Recombinant Assay: SSAO/VAP-1: Human recombinant SSAO/VAP-1 amine oxidase activity was determined using the coupled colorimetric method as described for monoamine oxidase, copper-containing amine oxidases and related enzymes (Holt A. and Palcic M., A peroxidase-coupled continuous absorbance plate-reader assay for flavin monoamine oxidases, copper-containing amine oxidases and related enzymes. Nat Protoc 2006; 1: 2498-2505). Briefly, a cloned cDNA template corresponding to residues 34-763 of human SSAO/VAP-1, and incorporating a mouse Ig kappa (κ) signal sequence, N-terminal flag epitope tag and tobacco etch virus (TEV) cleavage site, was assembled in a mammalian expression vector (pLO-CMV) by Geneart AG. This vector containing human SSAO/VAP-1 residues was transfected into CHO-K1 glycosylation mutant cell line, Lec 8. A clone stably expressing human SSAO/VAP-1 was isolated and cultured in large scale. Active human SSAO/VAP-1 was purified and recovered using immunoaffinity chromatography. This was used as the source for SSAO/VAP-1 activity. A high-throughput colorimetric assay was developed using either 96 or 384 well format. Briefly, in a standard 96 well plate assay 50 μL of purified human SSAO/VAP-1 (0.25 μg/mL) in 0.1 M sodium phosphate buffer (pH 7.4) was added into each well. Test compounds were dissolved in DMSO and tested in a Concentration Response Curve (CRC) with 4-11 data points, typically in the micromolar or nanomolar range after incubation with human SSAO/VAP-1 for 30 min at 37° C.
Measurement: IC50 > 3000 nM (pChEMBL 5.5229997634887695)
Descriptors: MW 402.5, ALogP 2.85, HBA 6, HBD 1, PSA 90.87, QED 0.68
Compound (SMILES): CN1CCCN(c2cnc3ccc(-c4ccc5oc(N)nc5c4)cc3n2)CC1
InChIKey: NWHOAJOKVRQOOA-UHFFFAOYSA-N
Target: Phosphatidylinositol 3-kinase regulatory subunit beta (SINGLE PROTEIN), Homo sapiens
UniProt: O00459
Assay: PI 3-Kinase HTRF Assay: Briefly, the assay is a time resolved FRET assay that indirectly measures PIP3 product formed by the activity of a PI3-K. The kinase reaction is performed in a microtitre plate (e.g., a 384 well microtitre plate). The total reaction volume is approximately 20 ul per well. In the first step, each well receives 2 ul of test compound in 20% dimethylsulphoxide resulting in a 2% DMSO final concentration. Next, approximately 14.5 ul of a kinase/PIP2 mixture (diluted in 1x reaction buffer) is added per well for a final concentration of 0.25-0.3 ug/ml kinase and 10 uM PIP2. The plate is sealed and incubated for 15 minutes at room temperature. To start the reaction, 3.5 ul of ATP (diluted in 1x reaction buffer) is added per well for a final concentration of 10 uM ATP. The plate is sealed and incubated for 1 hour at room temperature. The reaction is stopped by adding 5 ul of Stop Solution per well and then 5 ul of Detection Mix is added per well. The plate is sealed, incubated for 1 hour at room temperature, and then read on an appropriate plate reader.
Measurement: IC50 < 1000 nM (pChEMBL 6)
Descriptors: MW 374.45, ALogP 3.16, HBA 7, HBD 1, PSA 84.31, QED 0.58
Compound (SMILES): Cc1cc(-c2cc(-c3cc(S(=O)(=O)NCC(C)(C)O)ccc3C)cnc2N)sn1
InChIKey: AZVJHJZDMFRHDB-UHFFFAOYSA-N
Target: Phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit beta isoform (SINGLE PROTEIN), Homo sapiens
UniProt: P42338
Assay: Kinase Glo Luminescent Kinase Assay (Kglo) for PI 3-Kinase Alpha (A), PI 3-Kinase Beta (B), Vps34 (C), PI 4-Kinase Beta (D): The luminescence-based ATP detection reagent KinaseGlo was obtained from Promega, (Cat. No. V6714, Lot No. 236161) through Catalys, Wallisellen, Switzerland. L-alpha-phosphatidylinositol (PI, liver, bovine) was obtained from Avanti Polar Lipid (Cat. No. 840042C, Lot#LPI-274), phosphatidylinositol-4,5-bisphosphate (PIP(4,5)2) was obtained from Avanti Polar Lipid (Cat. No. 840046X). L-α-phosphatidylserine (PS) was obtained from Avanti Polar Lipid (Cat. No. 840032C), n-octylglucoside from Avanti Polar Lipid (Cat. No. 10634425001). Luminescence is a well established readout to determine ATP concentrations and can thus be used to follow the activity of many kinases regardless of their substrate. The Kinase Glo Luminescent Kinase Assay (Promega, Madison, Wis., USA) is a homogeneous HTS method of measuring kinase activity by quantifying the amount of ATP remaining in solution following a kinase reaction.50 nL of compound dilutions were dispensed onto black 384-well low volume Non Binding Styrene (NBS) plates (Costar Cat. No. NBS#3676). L-α-phosphatidylinositol (PI), provided as 10 mg/ml solution in methanol, was transferred into a glass tube and dried under a nitrogen beam. It was then resuspended in 3% OctylGlucoside (1-O-n-octyl-beta-D-glucopyranoside) by vortexing and stored at 4° C. 5 μl of a mix of PI/OctylGlucoside with the PI 3-kinase alpha and PI 3-kinase beta subtypes, or Vps34 or PI 4-kinase beta were added. Kinase reactions were started by the addition of 5 μl of an ATP-mix containing in a final volume 10 μl 10 mM TRIS-HCl pH 7.5, 3 mM MgCl2, 50 mM NaCl, 0.05% CHAPS, 1 mM DTT and 1 μM ATP at room temperature. Reactions were stopped with 10 μl of KinaseGlo and plates were read 10 mins later in a Synergy2 reader using an integration time of 0.1 seconds per well. 2.5 μM of NVP-BGT226 (1-(3-(trifluoromethyl)-4-(piperazin-1-yl)phenyl)-8-(6-methoxypyridin-3-yl)-3-methyl-1H-imidazo[4,5-c]quinolin-2(3H)-one) was added to the assay plates to generate the 100% inhibition of the kinase reaction, and the 0% inhibition was given by the solvent vehicle (90% DMSO in water). (1-(3-(trifluoromethyl)-4-(piperazin-1-yl)phenyl)-8-(6-methoxypyridin-3-yl)-3-methyl-1H-imidazo[4,5-c]quinolin-2(3H)-one) was used as a reference compound and included in all assay plates in the form of 16 dilution points in duplicate.IC50 values of the percentage inhibition of each compound at 8 concentrations (10, 3.0, 1.0, 0.3, 0.1, 0.030, 0.010 and 0.003 μM) n=2 were derived by fitting a sigmoidal dose-response curve to a plot of assay readout over inhibitor concentration as described. All fits were performed with the program XLfit4 (ID Business Solutions, Guildford, UK).
Measurement: IC50 = 4650 nM (pChEMBL 5.329999923706055)
Descriptors: MW 432.57, ALogP 3.12, HBA 7, HBD 3, PSA 118.2, QED 0.55
Compound (SMILES): COc1cc(-n2cnn(Cc3cccc(F)c3)c2=O)ccc1-c1cn[nH]c1
InChIKey: OXQBBISLPHNAQJ-UHFFFAOYSA-N
Target: Rho-associated protein kinase 2 (SINGLE PROTEIN), Homo sapiens
UniProt: O75116
Assay: In Vitro Assay: The effectiveness of compounds of the present invention as ROCK inhibitors can be determined in a 30 μL, assay containing 20 mM HEPES, pH 7.5, 20 mM MgCl2, 0.015% Brij-35, 4 mM DTT, 5 μM ATP and 1.5 μM peptide substrate (FITC-AHA-AKRRRLSSLRA-OH). Compounds were dissolved in DMSO so that the final concentration of DMSO was <2%, and the reaction was initiated with Rho kinase variants. After incubation, the reaction was terminated by the addition of EDTA and the phosphorylated and non-phosphorylated peptides separated using a LABCHIP® 3000 Reader (Caliper Life Sciences). Controls consisted of assays that did not contain compound, and backgrounds consisted of assays that contained enzyme and substrate but had EDTA from the beginning of the reaction to inhibit kinase activity. Compounds were tested in dose-response format, and the inhibition of kinase activity was calculated at each concentration of compound. The inhibition data were fit using a curve-fitting program to determine the IC50; i.e., the concentration of compound required to inhibit 50% of kinase activity.
Measurement: IC50 = 150 nM (pChEMBL 6.820000171661377)
Descriptors: MW 365.37, ALogP 2.62, HBA 6, HBD 1, PSA 77.73, QED 0.59
Compound (SMILES): COc1ccc(-c2[nH]c3ccc(-c4ccc(N5CCCN(CCO)CC5)cc4)cc3c2C(C)C)cc1OC
InChIKey: MUONFJYETHSMQQ-UHFFFAOYSA-N
Target: Toll-like receptor 7 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9NYK1
Assay: TLR7/8/9 Inhibition Reporter Assays: HEK-Blue-cells (Invivogen) overexpressing human TLR7, TLR8 or TLR9 receptors were used for screening inhibitors of these receptors using an inducible SEAP (secreted embryonic alkaline phosphatase) reporter gene under the control of the IFN-β minimal promoter fused to five NF-κc Band AP-1-binding sites. Briefly, cells are seeded into Greiner 384 well plates (15000 cells per well for TLR7, 20,000 for TLR8 and 25,000 for TLR9) and then treated with test compounds in DMSO to yield a final dose response concentration range of 0.05 nM-50 μM. After a 30 minute compound pre-treatment at room temperature, the cells are then stimulated with a TLR7 ligand (gardiquimod at a final concentration of 7.5 μM), TLR8 ligand (R848 at a final concentration of 15.9 μM) or TLR9 ligand (ODN2006 at final concentration of 5 nM) to activate NF-κB and AP-1 which induce the production of SEAP. After a 22 hour incubation at 37° C., 5% CO2, SEAP levels are determined with the addition of HEK-Bue Detection reagent (Invivogen), a cell culture medium that allows for detection of SEAP, according to manufacturer's specifications. The percent inhibition is determined as the % reduction in the HEK-Blue signal present in wells treated with agonist plus DMSO alone compared to wells treated with a known inhibitor.
Measurement: IC50 = 692 nM (pChEMBL 6.159999847412109)
Descriptors: MW 513.68, ALogP 6.15, HBA 5, HBD 2, PSA 60.96, QED 0.3
Compound (SMILES): O=C(N[C@H]1CCCC[C@@H]1O)C1=CN(Cc2cccc(F)c2)c2ccccc2O1
InChIKey: PNNLLCXKXKLVGJ-HKUYNNGSSA-N
Target: Muscarinic acetylcholine receptor M1 (SINGLE PROTEIN), Homo sapiens
UniProt: P11229
Assay: Determination of Allosteric Potency EC50 Values for Muscarinic M1 Receptor: A stable CHO cell line expressing recombinant human Muscarinic M1 receptor and pCRE-Luc reporter system was used for cell-based assay. The assay offers a non-radioactive based approach to determine binding of a compound to GPCRs. In this specific assay, the level of intracellular cyclic AMP which is modulated by activation or inhibition of the receptor is measured. The recombinant cells harbor luciferase reporter gene under the control of cAMP response element.The above cells were grown in 96 well clear bottom white plates in Hams F12 medium containing 10% fetal bovine serum (FBS). Prior to the addition of compounds or standard agonist, cells were serum starved overnight. Increasing concentrations of test compounds were added along with EC20 of acetylcholine in OptiMEM medium to the cells. The incubation was continued at 37° C. in CO2 incubator for 4 hours. Medium was removed and cells were washed with phosphate buffered saline. The cells were lysed and luciferase activity was measured in a Luminometer. Luminescence units were plotted against the compound concentrations using Graphpad software. EC50 values of the compounds were defined as the concentration required in stimulating the luciferase activity by 50% in presence of EC20 of acetylcholine.
Measurement: EC50 = 891 nM (pChEMBL 6.050000190734863)
Descriptors: MW 382.44, ALogP 3.49, HBA 4, HBD 2, PSA 61.8, QED 0.85
Compound (SMILES): Cc1ncc(C)n2cc(CCc3nc4c5cccnc5ccc4n3C)nc12
InChIKey: LONDHTKWZYHACJ-UHFFFAOYSA-N
Target: cAMP and cAMP-inhibited cGMP 3',5'-cyclic phosphodiesterase 10A (SINGLE PROTEIN), Homo sapiens
UniProt: Q9Y233
Assay: In Vitro Pharmacology Assay: In one embodiment, the compounds provided herein were assayed for their ability to inhibit human PDE-10A. In one embodiment, the activities of the compounds were determined using the Molecular Devices IMAP PDE Fluorescence Polarization assay using recombinant human PDE-10 enzyme expressed in a baculoviral system. Briefly, 10 μL of a compound (0.2 nM-20 μM) was added to either a 96-well half area black plate or a 384-well black plate along with 10 μL of Fluorescein-labeled cAMP/cGMP substrate as per manufacturer's instructions and 10 μL of PDE enzyme (activity 0.1 U). Following a 40-minute incubation at 37° C., 60 μL of IMAP binding reagent was added. The plate was then read on a Perkin Elmer Victor (480-535 nm). The data was analyzed using Prism Software (GraphPad Inc, San Diego, Calif.).
Measurement: IC50 = 55 nM (pChEMBL 7.260000228881836)
Descriptors: MW 356.43, ALogP 3.57, HBA 6, HBD 0, PSA 60.9, QED 0.5
Compound (SMILES): COC(=O)N1c2ccc3c(nc(C[C@@H](C(=O)O)c4ccccc4)n3-c3ccccc3)c2CC[C@@H]1C
InChIKey: BFUQNUSFLXJFBN-PGRDOPGGSA-N
Target: CREB-binding protein (SINGLE PROTEIN), Homo sapiens
UniProt: Q92793
Assay: HTRF Biochemical Assay for CBP and BRD4 Activity: The assay was performed in a final volume of 6 μL in assay buffer containing 50 mM Hepes (pH 7.5, (0.5M Hepes, pH 7.5 solution; Teknova H1575)), 0.5 mM GSH, 0.01% BGG (0.22 μM filtered, Sigma, G7516-25G), 0.005% BSA (0.22 μM filtered, EMD Millipore Corporation, 126575) and 0.01% Triton X-100 (Sigma, T9284-10L). Nanoliter quantities of 10-point, 3-fold serial dilution in DMSO were pre-dispensed into 1536 assay plates (Corning, #3724BC) for a final test concentration of 33 μM to 1.7 nM, top to lowest dose, respectively. 3 of 2× Protein and 3 μL of 2× Peptide Ligand were added to assay plates (pre-stamped with compound). Plates were incubated for varying times at room temperature prior to measuring the signal. TR-FRET (Time-Resolved Fluorescence Resonance Energy Transfer) was measured on the PHERAstar (BMG, equipped with HTRF optic module [337/520/490]) or on the Envision (PerkinElmer, equipped with the TRF Laser unit, TRF dual mirror D400/D505 and emission filters M520 and M495). Data were reported as percent inhibition compared with control wells based on the following equation: % inh=1−((TR-FRET ratio−AveLow)/(AveHigh−AveLow)) where TR-FRET ratio=(Fluorescence at 520 nm/Fluorescence at 490 nm)*10000), AveLow=average TR-FRET ratio of no enzyme control (n=32), and AveHigh=average TR-FRET ratio of DMSO control (n=32). IC50 values were determined by curve fitting of the standard 4 parameter logistic fitting algorithm included in the Activity Base software package: IDBS XE Designer Model 205. Data is fitted using the Levenburg Marquardt algorithm. For all assay formats data were reported as percent inhibition compared with control wells based on the following equation: % inh=100*((FLU−AveLow)/(AveHigh−AveLow)) where FLU=measured Fluorescence, AveLow=average Fluorescence of no enzyme control (n=32), and AveHigh=average Fluorescence of DMSO control (n=32). IC50 values were determined by curve fitting of the standard 4 parameter logistic fitting algorithm included in the Activity Base software package: IDBS XE Designer Model 205. Data is fitted using the Levenburg Marquardt algorithm.
Measurement: IC50 = 550 nM (pChEMBL 6.260000228881836)
Descriptors: MW 469.54, ALogP 5.34, HBA 5, HBD 1, PSA 84.66, QED 0.43
Compound (SMILES): CCOc1cc(F)ccc1N(Cc1cn[nH]n1)c1ccnc(N)n1
InChIKey: KWBVVEQMYHOLGJ-UHFFFAOYSA-N
Target: Histamine H4 receptor (SINGLE PROTEIN), Homo sapiens
UniProt: Q9H3N8
Assay: Binding Assay on Recombinant Human Histamine H4 Receptor: Receptor binding was assessed using isolated plasma membranes from SKNMC neuroblastoma cell lines stably expressing recombinant human H4 receptor. To isolate plasma membrane enriched in recombinant human H4 receptor, cells were collected from 100 culture dishes (150 mm) in PBS-EDTA. The cells were pellet by centrifugation (800 g) and resuspended in 5×TE buffer (50 mM Tris/5 mM EDTA). The cells were then homogenized and centrifuged at 1000 g for 10 min. The supernatant was collected and centrifuged for an additional 26000 g for 30 min. The supernatant was removed and the pellet was gently resuspended in 30 ml of 5×TE buffer. Protein concentration was then determined using Bradford protein assay (Bradford, M. M., Anal. Biochem., 1976, 72: 248-254). Isolated cell membranes were incubated with 2×KD (10 nM), [3H] histamine (Specific activity: 23 Ci/mmol), with or without test compounds for 45 min at 25° C. Nonspecific binding was defined with 100 μM cold histamine. Ki values were calculated based on an experimentally determined appropriate KD values according to Cheng and Prusoff (Biochem. Pharmacol. 1973, 22(23):3099-3108). Membranes were harvested by rapid filtration using the 96 well Brandel system or a cell harvester using a Whatman GF/C filter or filter plates treated with 0.5% polyethylenimine (PEI), and washed 4 times with ice-cold 50 mM Tris/5 mM EDTA buffer. Filters were then dried, mixed with scintillant and radioactive counts were determined.
Measurement: Ki = 294 nM (pChEMBL 6.53000020980835)
Descriptors: MW 329.34, ALogP 2.05, HBA 7, HBD 2, PSA 105.84, QED 0.71
Compound (SMILES): Cc1cccc(OCC#Cc2cnc(C(=O)NCC(=O)O)c(O)c2)c1C
InChIKey: OAQNHOGJOBLVRP-UHFFFAOYSA-N
Target: Egl nine homolog 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9GZT9
Assay: EGLN-1 Activity Assay : The EGLN-1 (or EGLN-3) enzyme activity is determined using mass spectrometry (matrix-assisted laser desorption ionization, time-of-flight MS, MALDI-TOF MS for assay details, see reference (Greis et al., 2006). Recombinant human EGLN-1-179/426 is prepared as described above and in the Supplemental Data. Full-length recombinant human EGLN-3 is prepared in a similar way, however it is necessary to use the His-MBP-TVMV EGLN-3 fusion for the assay due to the instability of the cleaved protein. For both enzymes, the HIF-1C. peptide corresponding to residues 556-574 (DLDLEALAPYIPAD DDFQL) is used as substrate. The reaction is conducted in a total volume of 50 uL containing TrisCl (5 mM, pH 7.5), ascorbate (120 uM), 2-oxoglutarate (3.2 uM), HIF-1C. (8.6 uM), and bovine serum albumin (0.01%). The enzyme, quantity predetermined to hydroxylate 20% of substrate in 20 minutes, is added to start the reaction. Where inhibitors are used, compounds are prepared in dimethyl Sulfoxide at 10-fold final assay concentration. After 20 minutes at room temperature, the reaction is stopped by transferring 10 LIL of reaction mixture to 50 LL of a mass spectrometry matrix Solution (C-cyano-4-hydroxycinnamic acid, 5 mg/mL in 50% acetonitrile/0.1% TFA, 5 mM NHPO). Two micro liters of the mixture is spotted onto a MALDI-TOF MS target plate for analysis with an Applied Biosystems (Foster City, Calif.) 4700 Proteomics Analyzer MALDI-TOF MS equipped with a Nd:YAG laser (355 nm, 3 ns pulse width, 200 HZ repetition rate). Hydroxylated peptide product is identified from substrate by the gain of 16 Da. Data defined as percent conversion of Substrate to product is analyzed in GraphPad Prism 4 to calculate ICso values.
Measurement: IC50 = 79.2 nM (pChEMBL 7.099999904632568)
Descriptors: MW 354.36, ALogP 1.65, HBA 5, HBD 3, PSA 108.75, QED 0.7
Compound (SMILES): C[C@@H]1CN(c2cc(F)c(-c3cnc(N4CCOCC4)nc3)cc2NC(=O)c2c(F)cccc2C(F)(F)F)C[C@H](C)N1C
InChIKey: GSGOUECYJUQOBF-HDICACEKSA-N
Target: WD repeat-containing protein 5 (SINGLE PROTEIN), Homo sapiens
UniProt: P61964
Assay: Surface Plasmon Resonance (SPR) Assay: Exemplary compounds of the application were dissolved in 100% DMSO at 10 mM, assayed fresh, and then stored at −20° C. for repeat studies and other experiments. Full length WDR5 with an N-terminal His tag and C-terminal AviTag (Avidity Inc.) was expressed in E. coli with coexpression of BirA to biotin labelled protein in vivo. Purification of the protein was performed using Ni-NTA. The purified WDR5 protein has a molecular weight of 41976 Da.SPR studies were performed using a Biacore T200 instrument (GE Health Sciences Inc.). Biotinylated WDR5 protein (approximately 3000 RU) was stably captured to streptavidin coupled SA chips according to the manufacture's protocol (GE Health Sciences Inc.). The running buffer used was HBS-EP (20 mM Hepes pH 7.4, 150 mM NaCl, 3 mM EDTA, 0.05% P-20) plus 5% DMSO with a flow rate of 40 μl/min. For SPR analysis, 5 different concentrations of each exemplary compound of the application were sprayed into 96 or 384 well plates using an HP D300 digital dispenser. The concentration ranged from about 195 nM to about 12 nM in a two-fold series. Concentration ranges were adjusted higher or lower for weaker or more potent compounds respectively when necessary. For the KD determinations, single cycle kinetic analysis was performed with an on time of 60 seconds, and an off time of 300 or 600 seconds. Curve fitting and KD calculations were performed with the Biacore T200 Evaluation software (GE Health Sciences Inc).
Measurement: Kd = 22.9 nM (pChEMBL 7.639999866485596)
Descriptors: MW 590.6, ALogP 5.06, HBA 7, HBD 1, PSA 73.83, QED 0.42
Compound (SMILES): C[C@H]1CN(c2cc(F)c(C3=CCN(c4ncc(C#N)s4)CC3)cc2NC(=O)c2c[nH]c(=O)cc2C(F)(F)F)CCN1C
InChIKey: OACAGJNTMHTIAT-INIZCTEOSA-N
Target: WD repeat-containing protein 5 (SINGLE PROTEIN), Homo sapiens
UniProt: P61964
Assay: Surface Plasmon Resonance (SPR) Assay: Exemplary compounds of the application were dissolved in 100% DMSO at 10 mM, assayed fresh, and then stored at −20° C. for repeat studies and other experiments. Full length WDR5 with an N-terminal His tag and C-terminal AviTag (Avidity Inc.) was expressed in E. coli with coexpression of BirA to biotin labelled protein in vivo. Purification of the protein was performed using Ni-NTA. The purified WDR5 protein has a molecular weight of 41976 Da.SPR studies were performed using a Biacore T200 instrument (GE Health Sciences Inc.). Biotinylated WDR5 protein (approximately 3000 RU) was stably captured to streptavidin coupled SA chips according to the manufacture's protocol (GE Health Sciences Inc.). The running buffer used was HBS-EP (20 mM Hepes pH 7.4, 150 mM NaCl, 3 mM EDTA, 0.05% P-20) plus 5% DMSO with a flow rate of 40 μl/min. For SPR analysis, 5 different concentrations of each exemplary compound of the application were sprayed into 96 or 384 well plates using an HP D300 digital dispenser. The concentration ranged from about 195 nM to about 12 nM in a two-fold series. Concentration ranges were adjusted higher or lower for weaker or more potent compounds respectively when necessary. For the KD determinations, single cycle kinetic analysis was performed with an on time of 60 seconds, and an off time of 300 or 600 seconds. Curve fitting and KD calculations were performed with the Biacore T200 Evaluation software (GE Health Sciences Inc).
Measurement: Kd = 4.17 nM (pChEMBL 8.380000114440918)
Descriptors: MW 601.63, ALogP 4.55, HBA 8, HBD 2, PSA 108.36, QED 0.41
Compound (SMILES): C[C@@H]1CN(C(=O)[C@@](C)(O)C(F)(F)F)CC[C@@H]1N(C)C(=O)c1ccccc1
InChIKey: TUKRVNCBIZEUNL-HACGYAERSA-N
Target: [Pyruvate dehydrogenase (acetyl-transferring)] kinase isozyme 2, mitochondrial (SINGLE PROTEIN), Homo sapiens
UniProt: Q15119
Assay: Biochemical Activity Assay: The PDC inactivation assay is performed in Greiner 384-well microtiter plates and is used for high throughput screen. 4 μl of PDHK2 (human, rec, Carna Bioscience, 10 ng/μl-137 nM final concentration) and PDC (isolated from porcine heart, Sigma-Aldrich, 20 mU/ml final concentration) are incubated in the absence or presence of the test compound (10 dilution concentrations) for 30 min at room temperature in kinase buffer (15 mM potassium phosphate buffer, pH 7.0, 60 mM KCl, 1.5 mM DTT, 2.5 mM MgCl2, 0.0125% (w/v) BSA, 0.125% Pluronic F-68). The kinase reaction is started by the addition of 4 μl ATP substrate solution (fc 5 μM in kinase buffer). After 30 min incubation at 37° C. 40 μl of PDC reaction solution (100 mM Tris/HCl, pH 7.8, 0.5 mM EDTA, 1 mM MgCl2, 50 mM NaF, 0.25 mM Coenzyme A, 5 mM pyruvate, 1 mM NAD, 5 mM DTT, 1 mM thiamine pyrophosphate) is added. The first fluorescence measurement is performed on a Perkin Elmer Envision (Exc 340 nm, Em 450 nm). The reaction is incubated for 45 min at room temperature. Afterwards a second fluorescence measurement is performed and the PDC activity is calculated by the difference between both measurements. As full value for the PDHK2 assay the inhibitor-free PDHK2 reaction is used.
Measurement: IC50 = 710 nM (pChEMBL 6.150000095367432)
Descriptors: MW 372.39, ALogP 2.31, HBA 3, HBD 1, PSA 60.85, QED 0.89
Compound (SMILES): CCC[C@H](C)Nc1ncc2c(C3CCN(C(=O)C4CCN(CCF)CC4)CC3)cn([C@H]3CC[C@H](O)CC3)c2n1
InChIKey: XIYMFRUJZHQSEM-TUSQITKMSA-N
Target: Tyrosine-protein kinase Mer (SINGLE PROTEIN), Homo sapiens
UniProt: Q12866
Assay: Activity Assay: Activity Against Mer Tyrosine Kinase.
Measurement: IC50 < 3 nM (pChEMBL 8.52299976348877)
Descriptors: MW 542.74, ALogP 4.9, HBA 7, HBD 2, PSA 86.52, QED 0.47
Compound (SMILES): Cc1nc2ccc(-n3ncnc3[C@@H]3O[C@H](CO)[C@H](O)[C@H](n4cc(-c5ccc(Cl)c(F)c5)nn4)[C@H]3O)cc2s1
InChIKey: ZOZITOVEVOGMJP-YDXQKAQTSA-N
Target: Galectin-3 (SINGLE PROTEIN), Homo sapiens
UniProt: P17931
Assay: Biological Assay: The Gal-3 assays were performed in 384 white Opti plates in three replicates at room temperature with gentle shaking at 250-300 rpmFrom the original stocks, 2.525× working stock concentrations of His-tagged recombinant human Gal-3 (hGal-3) and that of B-ASF were prepared. From the working stock, 20 μl of hGal-3 (15 nM) and 20 μl B-ASF (15 nM) were added to the plates. In Negative Control, only hGal-3 was added. A concentration range of 50× working stocks were prepared for the compounds in 100% DMSO. Aliquots of 1 uL of the compounds were added to the wells and pre-incubated with 20 μl hGal-3 per well for 30 minutes Then 20 μl B-ASF wereadded and incubated for another 1 hour. To detect the signal, 5 μL (final conc. of 1.0 nM) terbium labelled Anti-His antibody was added and incubated for 30 min followed by adding 5 μL (final conc. of 20 nM) Streptavidin d2 and incubation for another 1 hour. The assay signal was detected using HTRF screen protocol (Excitation wavelength=340 nm, emission wavelength=615 nm/665 nm) on Envision 2104 Multilabel Reader. Data analysed using Toolset and Curve Master. Results are reported in the experimental section (IC50 in μM).
Measurement: IC50 = 70 nM (pChEMBL 7.159999847412109)
Descriptors: MW 558, ALogP 2.63, HBA 12, HBD 3, PSA 144.23, QED 0.3