lovingscience/chembl-v37-raw-activities huggingface dataset:
Compound (SMILES): CCc1cc(C)nc(C2CCN(C(=O)c3cc(C(C)C)n[nH]3)C2)c1
InChIKey: LJIWBTHSAZIRGT-UHFFFAOYSA-N
Target: Lysine-specific demethylase 5A (SINGLE PROTEIN), Homo sapiens
UniProt: P29375
Assay: KDM5 Enzyme Assay: Full length KDM5A enzyme was expressed and purified inhouse. Biotin-H3K4me3 peptide was purchased from New England Biolabs. HTRF reagents (containing Eu-labeled H3K4me1-2 antibody, and streptavidin-XL665) were purchased from Cis-Bio International. Plates were read on an Envision multi-label plate reader (Perkin Elmer).The HTRF assay mixture contained 2 nM full length KDM5A enzyme, 100 nM H3K4Me3 peptide substrate, 1 uM 2-OG, 100 uM Fe2+, 500 uM ascorbate. 50 mM HEPES pH 7.0 buffer, 0.01% Triton-X 100, 2 mM DTT, 0.25% DMSO at a final volume of 10 uL.The enzyme reaction was carried out at room temperature in black Proxiplate 384-Plus plate (Corning. Costar) within 30 minutes, in the presence of varying concentration of a test compound. At the end of enzyme reaction, 5 uL of 1 mM EDTA were added to quench the reaction and then the detection reagents (5 uL) were added to give final concentrations of 0.5 nM Eu-labeled H3K4me1-2 antibody, and 50 nM streptavidin-XL665. The plates were incubated at room temperature for 60 minutes and then read in the Envision plate reader. The readouts were transformed into % inhibition, and IC50 value of a test compounds was generated by using four parameters curve fitting (Model 205 in XLFIT5, iDBS).
Measurement: IC50 = 1.4 nM (pChEMBL 8.850000381469727)
Descriptors: MW 326.44, ALogP 3.43, HBA 3, HBD 1, PSA 61.88, QED 0.94
Compound (SMILES): FC(F)(F)c1cccc(N2CCN(C(=S)Nc3cc[nH]n3)CC2)c1
InChIKey: DDWVRSDHWVHFAL-UHFFFAOYSA-N
Target: D-3-phosphoglycerate dehydrogenase (SINGLE PROTEIN), Homo sapiens
UniProt: O43175
Assay: Enzyme Assay: PHGDH assay buffer contained 50 mM TEA pH 8.0, 10 mM MgCl2, 0.05% BSA, and 0.01% Tween-20. PHGDH enzyme buffer consisted of assay buffer with 20 nM PHGDH and 0.2 mg/mL diaphorase. PHGDH substrate buffer contained 0.3 mM NAD 1.25 mM glutamate, 0.1 mM 3-phosphoglycerate, 0.2 mM resazurin, 1 μM PSAT1, and 1 μM PSPH. qHTS was performed in 1536-well plates dispensed with a BioRAPTR FRD. Each well contained equal volumes of substrate buffer and assay buffer. Plates were read at 0 minutes and 20 minutes at room temperature with a ViewLux uHTS Microplate Imager (PerkinElmer). Follow-up assays were performed in black 384-well plates (Greiner) in 20 μL of enzyme buffer to which compounds were added in dose-response with an HP D300 digital dispenser (Hewlett-Packard), followed by addition of 20 μL of substrate buffer. Plates were incubated at room temperature (25° C.) and read at 0 and 20 minutes with a Spectramax M5 plate reader (Molecular Devices) in fluorescence intensity mode with a λex=550 nm and λem=600 nm (emission cutoff=590 nm).
Measurement: IC50 = 7500 nM (pChEMBL 5.119999885559082)
Descriptors: MW 355.39, ALogP 2.95, HBA 3, HBD 2, PSA 47.19, QED 0.81
Compound (SMILES): CC(C1CC1)N1CC[C@@H](NC(=O)c2cc(-c3ccc(F)cc3F)on2)[C@H](C(=O)N(C)C)C1
InChIKey: JOHHCDHGTVUTHN-BSTPMOINSA-N
Target: Atypical chemokine receptor 3 (SINGLE PROTEIN), Homo sapiens
UniProt: P25106
Assay: In Vitro Assay: Tango CXCR7-bla U2OS cells are detached from culture dishes with 0.05% trypsin-EDTA and collected in growing medium (McCoy's 5A 90% (v/v), dialyzed FCS 10% (v/v), 0.1 mM NEAA, 25 mM HEPES (pH7.3), 1 mM sodium pyruvate, P/S 1% (v/v) 50 μg/ml Hygromycin, 100 μg/ml Geneticin, 200 μg/ml Zeocin), spinned down and resuspended in assay medium (McCoy's 5A 90% (v/v), dialyzed FCS 1% (v/v), 0.1 mM NEAA, 25 mM HEPES (pH7.3), P/S 1% (v/v)). 10′000 cells per well (in 30 μl) are seeded in a 384 well plate (black-walled, clear bottom). The plate is incubated at 37° C./5% CO2 for 24 hours. Test compounds are dissolved to 10 mM in DMSO and serially diluted in DMSO to 500× of the final concentration for dose response curves. Compounds are then diluted 1:100 in assay medium to 5× of the final concentration. 10 μl/well of diluted compounds are added to the assay plate and incubated for 15 minutes at 37° C. Thereafter CXCL12/SDF1-α is diluted in assay medium to 5× of the final concentration (its EC80 value for receptor activation) and 10 μl/well are added to the assay plate. The agonist leads to activation of the receptor and therefore to b-arrestin recruitment. Compounds acting as antagonists reduce this activation. The plate is incubated for 22 hrs at 37° C. 10 μl/well of detection reagent (LiveBLAzer-FRET BIG (CCF4-AM) substrate) is transferred to the assay plate and the plate is incubated for 2 hours at room temperature protected from light. Fluorescent counts are determined (Scan1: Ex 409/20 nm, Em 460/30 nm, Scan 2: Ex 409/20 nm, Em 530/30 nm).
Measurement: IC50 = 28 nM (pChEMBL 7.550000190734863)
Descriptors: MW 446.5, ALogP 2.93, HBA 5, HBD 1, PSA 78.68, QED 0.74
Compound (SMILES): N[C@]1(COP(=O)(O)O)CC[C@H](c2ccc3c(c2)CC[C@@H](CCC2CCOCC2)C3)C1
InChIKey: HEKLITRSEQYQLU-YFXJRYMSSA-N
Target: Sphingosine 1-phosphate receptor 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P21453
Assay: Binding Assay: Membranes were prepared from CHO cells expressing human S1P1. Cells pellets (1x10^9 cells/pellet) were suspended in buffer containing 20 mM HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid), pH 7.5, 50 mM NaCl, 2 mM EDTA (Ethylenediaminetetraacetic acid) and Protease Inhibitor cocktail (Roche), and disrupted on ice using the Polytron homogenizer. The homogenate was centrifuged at 20,000 rpm (48,000 g) and the supernatant was discarded. The membrane pellets were resuspended in buffer containing 50 mM HEPES, pH 7.5, 100 mM NaCl, 1 mM MgCl2, 2 mM EDTA and stored in aliquots at -80Â° C. after protein concentration determination.Membranes (2 ug/well) and 0.03 nM final concentration of 33P-S1P ligand (1 mCi/ml, Perkin elmer or American Radiolabeled Chemicals) diluted in assay buffer (50 mM HEPES, pH7.4, 5 mM MgCl2, 1 mM CaCl2, 0.5% fatty acid free BSA (bovine serum albumin), 1 mM NaF) were added to the compound plates (384 Falcon v-bottom plate (0.5 ul/well in a 11 point, 3-fold dilution). Binding was performed for 45 minutes at room temperature, terminated by collecting the membranes onto 384-well Millipore FB filter plates, and radioactivity was measured by TOPCOUNT. The competition data of the test compounds over a range of concentrations was plotted as percentage inhibition of radioligand specific binding. The IC50 is defined as the concentration of competing ligand needed to reduce specific binding by 50%.
Measurement: IC50 < 10 nM (pChEMBL 8)
Descriptors: MW 437.52, ALogP 4.07, HBA 4, HBD 3, PSA 102.01, QED 0.56
Compound (SMILES): COc1cc(C(=O)N2CC3CCC2[C@@H]3N)cc2nc(-c3cc4ccc(-c5ccc(NC(C)=O)cc5C)nc4n3CC3CC3)n(C)c12
InChIKey: NRMQVMXLJIZNLP-KFHGAJFOSA-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 = 52 nM (pChEMBL 7.28000020980835)
Descriptors: MW 617.75, ALogP 5.5, HBA 8, HBD 2, PSA 120.3, QED 0.25
Compound (SMILES): CC(=O)Nc1ccc(S[C@H]2OC(CO)[C@H](O)[C@H](n3cc(-c4cc(F)c(F)c(F)c4)nn3)C2O)cc1
InChIKey: IOAIBUSJDFUFDE-YSZROVPYSA-N
Target: Galectin-3 (SINGLE PROTEIN), Homo sapiens
UniProt: P17931
Assay: fluorescence anisotropy assay : The affinity of Example 1-106 for galectins were determined by a fluorescence anisotropy assay where the compound was used as an inhibitor of the interaction between galectin and a fluorescein tagged saccharide probe as described Sörme, P., Kahl-Knutsson, B., Huflejt, M., Nilsson, U. J., and Leffler H. (2004) Fluorescence polarization as an analytical tool to evaluate galectin-ligand interactions. Anal. Biochem. 334: 36-47, (Sörme et al., 2004) and Monovalent interactions of Galectin-1 By Salomonsson, Emma; Larumbe, Amaia; Tejler, Johan; Tullberg, Erik; Rydberg, Hanna; Sundin, Anders; Khabut, Areej; Frejd, Torbjorn; Lobsanov, Yuri D.; Rini, James M.; et al, From Biochemistry (2010), 49(44), 9518-9532, (Salomonsson et al., 2010). The assay was also adapted to be able to measure the high affinity of compounds for galectin-3 by using the below probe constructed to have high affinity for galectin-3 which made it possible to use a low concentration of galectin-3 (50 nM). 100 nM albumin was included as a carrier to prevent protein loss at such low concentration of galectin.
Measurement: Kd = 313 nM (pChEMBL 6.5)
Descriptors: MW 510.49, ALogP 2.09, HBA 9, HBD 4, PSA 129.73, QED 0.37
Compound (SMILES): COc1nc2ccc([C@@](O)(c3ccc(C)nc3C)c3cnnn3C)cc2c(Cl)c1CC(F)(F)F
InChIKey: PYLOODPJFPYIJW-HSZRJFAPSA-N
Target: Nuclear receptor ROR-gamma (SINGLE PROTEIN), Homo sapiens
UniProt: P51449
Assay: ThermoFluor Assay: ThermoFluor is a fluorescence based assay that estimates ligand binding affinities by measuring the effect of a ligand on protein thermal stability (Pantoliano, M. W., Petrella, E. C., Kwasnoski, J. D., Lobanov, V. S., Myslik, J., Graf, E., Carver, T., Asel, E., Springer, B. A., Lane, P., and Salemme, F. R. (2001) High-density miniaturized thermal shift assays as a general strategy for drug discovery. J Biomol Screen 6, 429-40, and Matulis, D., Kranz, J. K., Salemme, F. R., and Todd, M. J. (2005) Thermodynamic stability of carbonic anhydrase: measurements of binding affinity and stoichiometry using ThermoFluor. Biochemistry 44, 5258-66). This approach is applicable to a wide variety of systems, and rigorous in theoretical interpretation through quantitation of equilibrium binding constants (KD).In a ThermoFluor experiment where protein stability is monitored as the temperature is steadily increased, an equilibrium binding ligand causes the midpoint of an unfolding transition (Tm) to occur at a higher temperature. The shift in the melting point described as a ΔTm is proportional to the concentration and affinity of the ligand. The compound potency may be compared as a rank order of either ΔTm values at a single compound concentration or in terms of KD values, estimated from concentration response curves.
Measurement: Kd = 1200 nM (pChEMBL 5.920000076293945)
Descriptors: MW 491.9, ALogP 4.43, HBA 7, HBD 1, PSA 85.95, QED 0.44
Compound (SMILES): COc1ccc(F)cc1-c1ccnc2[nH]c(C3=CCN(C4CC(C(=O)O)C4)CC3)cc12
InChIKey: JJXGECIPCDHZFQ-UHFFFAOYSA-N
Target: CDK9/cyclin T1 (PROTEIN COMPLEX), Homo sapiens
UniProt: P50750
Assay: LANCE ULight TR-FRET Kinase Assay: CDK9 enzyme activities were measured using LANCE ULight TR-FRET kinase assay reagents (PerkinElmer, Waltham, Mass.). Compounds were directly added in 100% DMSO to white low volume assay plates (Perkin Elmer Proxiplate 6008289) using a Labcyte Echo acoustic dispenser. Assay reagents in serine/threonine kinase assay buffer containing 20 mM HEPES, 10 mM MgCl2, 100 mM Na3VO4, and 0.0075% Triton X-100. were added for final reaction mixture concentrations of 1000 μM ATP, 100 nM U-light MBP peptide (Perkin Elmer TRF0109M) and reaction initiated with 4 nM CDK9/Cyclin T1 (Carna Biosciences 04-110). The kinase reaction was carried out for 30 minutes before addition of stopping buffer to a final of 20 mM EDTA and 0.5 nM of LANCE Ultra Europium anti-phospho-MBP antibody (PerkinElmer TRF0201M) in LANCE detection buffer (PerkinElmer CR97-100). The reaction was equilibrated for 1 hour and the signal read in the Perkin Elmer Envision in TR-FRET mode (excitation at 320 nm and emission at 615/665 nm).
Measurement: IC50 = 79 nM (pChEMBL 7.099999904632568)
Descriptors: MW 421.47, ALogP 4.33, HBA 4, HBD 2, PSA 78.45, QED 0.64
Compound (SMILES): CC1C[C@@]2(CCN(C#N)C2)C(=O)N1
InChIKey: QJSGPSYFYHOZNH-NHSZFOGYSA-N
Target: OTU domain-containing protein 7B (SINGLE PROTEIN), Homo sapiens
UniProt: Q6GQQ9
Assay: Biochemical Assay: Dilution plates were prepared at 21 times the final concentration (2100 μM for a final concentration of 100 μM) in 50% DMSO in a 96-well polypropylene V-bottom plate (Greiner #651201). A typical 8-point dilution series to be 100, 30, 10, 3, 1, 0.3, 0.1, 0.03 μM final. Reactions were performed in duplicate in black 384 well plates (small volume, Greiner 784076) in a final reaction volume of 21 μl. Either 1 μl of 50% DMSO or diluted compound was added to the plate. Cezanne 1 was diluted in reaction buffer (40 mM Tris, pH 7.5, 0.005% Tween 20, 0.5 mg/ml BSA, 5 mM beta-mercaptoethanol) to the equivalent of 0.005 μl/well and 10 μl of diluted Cezanne 1 was added to the compound. Enzyme and compound were incubated for 30 min at room temp. Reactions were initiated by the addition of 50 nM of TAMRA labelled peptide linked to ubiquitin via an iso-peptide bond as fluorescence polarisation substrate. Reactions were read immediately after addition of substrate and following a 2 hr incubation at room temperature.
Measurement: IC50 > 10000 nM (pChEMBL 5)
Descriptors: MW 179.22, ALogP 0.07, HBA 3, HBD 1, PSA 56.13, QED 0.54
Compound (SMILES): C[C@@H]1C[C@H](Nc2c(NC(=O)C3CC(C)(F)C3)cnc3ccc(Cl)cc23)CCO1
InChIKey: WNBLEXLGPNQPDM-QRJTUMAOSA-N
Target: Leucine-rich repeat serine/threonine-protein kinase 2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q5S007
Assay: LRRK2 Assay: LRRK2 kinase activity was measured using Lantha Screen technology from Invitrogen. GST-tagged truncated LRRK2 from Invitrogen (Cat #PV4874) was incubated with a fluorescein-labeled peptide substrate based upon ezrin/radixin/moesin (ERM), also known as LRRKtide (Invitrogen cat #PR8976A), in the presence of a dose response of compound. Upon completion, the assay was stopped and detected with a terbium labeled anti-phospho-ERM antibody (Invitrogen, cat #PR8975A). The assay was carried out under the following protocol: The compound dose response was prepared by diluting compound to a top concentration of 0.3 mM in 100% DMSO and serial diluted by half-log in DMSO to give an 11 point curve, 100× final assay concentration. Using Echo acoustic dispensing, 60 nL of compound was transferred to a low volume Corning 384-well assay plate. 3 μL of a working solution of substrate (200 nM LRRKtide, 2 mM ATP) prepared in assay buffer (50 mM HEPES, pH 7.5, 3 mM MgCl2, with 2 mM DTT and 0.01% Brij35 added fresh) was added to the 60 nL compound assay plate. The kinase reaction was started with 3 μL of a working solution of LRRK2 enzyme at a concentration of 4 μg/mL. The final reaction concentrations were 100 nM LRRKtide, 1 mM ATP, 2 μg/mL LRRK2 enzyme and a compound dose response with a top dose of 3 μM. The reaction was allowed to progress at room temperature for 30 minutes and then stopped with the addition of 6 μL of detection buffer (20 mM Tris pH 7.6, 0.01% NP-40, 6 mM EDTA with 2 nM terbium labeled anti-phospho-ERM). After an incubation of 1 hour at room temperature, the plate was read on an Envision with an excitation wavelength of 340 nm and a reading emission at both 520 nm and 495 nm. The ratio of the 520 nm and 495 nm emission was used to analyze the data. Inhibition of mutant G2019S LRRK2 (Invitrogen cat #PV4881) was measured in the exact same method. All final concentrations of substrate ATP and enzyme were the same.
Measurement: IC50 = 6.81 nM (pChEMBL 8.170000076293945)
Descriptors: MW 405.9, ALogP 4.94, HBA 4, HBD 2, PSA 63.25, QED 0.76
Compound (SMILES): Cc1ncc(NC(=O)c2ccnc(C(F)(F)F)c2)cc1-c1cc(N2CCOCC2)nc(N2CC(C)(O)C2)c1
InChIKey: NMDPUDHWDDVMOD-UHFFFAOYSA-N
Target: Serine/threonine-protein kinase B-raf (SINGLE PROTEIN), Homo sapiens
UniProt: P15056
Assay: Raf Activity Assay: Raf and biotinylated Mek, kinase dead, were combined at 2x final concentrations in assay buffer (50 mM Tris, pH 7.5, 15 mM MgCl2, 0.01% BSA and 1 mM DTT) and dispensed 5 ml per well in assay plates (Greiner white 384 well assay plates #781207) containing 0.25 ml of 40x of a Raf kinase inhibitor test compound diluted in 100% DMSO. The plate was incubated for 60 minutes at room temperature. The Raf kinase activity reaction was started by the addition of 5 mL per well of 2xATP diluted in assay buffer. After 3 hours (b-Raf(V600E)) or 1 hour (c-Raf). The reactions were stopped and the phosphorylated product was measured using a rabbit anti-p-MEK (Cell Signaling, #9121) antibody and the Alpha Screen IgG (ProteinA) detection Kit (PerkinElmer #6760617R), by the addition of 10 mL to the well of a mixture of the antibody (1:2000 dilution) and detection beads (1:2000 dilution of both beads) in Stop/bead buffer (25 mM EDTA, 50 mM Tris, pH 7.5, 0.01% Tween20). The additions were carried out under dark conditions to protect the detection beads from light. A lid was placed on top of the plate and incubated for 1 hour at room temperature, then the luminescence was read on a PerkinElmer Envision instrument. The concentration of each compound for 50% inhibition (IC50) was calculated by non-linear regression using XL Fit data analysis software.
Measurement: IC50 = 7.64 nM (pChEMBL 8.119999885559082)
Descriptors: MW 528.54, ALogP 3.53, HBA 8, HBD 2, PSA 103.71, QED 0.52
Compound (SMILES): CN1C(=O)[C@@H](NC(=O)c2ncc3cnn(C4CC4)c3n2)COc2ccccc21
InChIKey: XTANDMILNAKFQJ-ZDUSSCGKSA-N
Target: Receptor-interacting serine/threonine-protein kinase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q13546
Assay: Enzyme 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 (ph 7.5), 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.
Measurement: Ki = 22 nM (pChEMBL 7.659999847412109)
Descriptors: MW 378.39, ALogP 1.31, HBA 7, HBD 1, PSA 102.24, QED 0.74
Compound (SMILES): N/C(=N\S(=O)(=O)c1ccccc1)NCc1cnc(-c2ccc(Cl)cc2)c(-c2ccc(F)cc2)c1
InChIKey: GSOCNSPHTZTSAV-UHFFFAOYSA-N
Target: Mucosa-associated lymphoid tissue lymphoma translocation protein 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9UDY8
Assay: Inhibition of Protease Activity Assay: Full-length cDNA of human MALT1 gene (GenBank accession No: AB026118.1) amplified by PCR was inserted in flame to a SalI site located downstream of GST gene in a pGEX6P3 vector (GE Healthcare Japan Corp.) to prepare a vector (hereinafter, referred to as a pGEX6P3-MALT1 vector). Subsequently, E. coli for protein expression (BL21-RIL-codon plus-DE3, Agilent Technologies, Inc.) was transformed with the pGEX6P3-MALT1 vector and then analyzed by ampicillin resistance screening and colony PCR to obtain an E. coli strain expressing recombinant GST fusion MALT1. Protein expression was induced with isopropyl-(3-thiogalactopyranoside. After the expression induction, E. coli precipitates were recovered by centrifugation from the E. coli culture solution, and the E. coli precipitates were homogenized and then centrifuged to obtain a supernatant. The supernatant was purified using GSTrap FF column (GE Healthcare Japan Corp.) to obtain recombinant GST fusion MALT1.B) Evaluation of Inhibition of Protease Activity of MALT1:To 89 μL of an enzyme solution (4.8 g/mL GST fusion MALT1, 50 mmol/L MES, 150 mmol/L NaCl, 10% sucrose, 0.1% CHAPS, 10 mmol/L dithiothreitol, and 1 mol/L tri-ammonium citrate) per specimen, 1 μL of a test compound (DMSO-diluted solution) of each concentration was added to prepare a mixed solution. The mixed solution was incubated at room temperature for 30 minutes, followed by the measurement of the fluorescence value of the mixed solution (fluorescence value of the first measurement) (Ex: 380 nm, Em: 460 nm; Envision (Perkin Elmer Inc.)). Next, 10 μL of 200 μmol/L substrate (Ac-LRSR-AMC, SM Biochemicals LLC) was added (final concentration: 20 μmol/L) to the mixed solution, and the mixture was reacted by incubation at 30° C. for 80 minutes, followed by the measurement of the fluorescence value of the reaction solution (fluorescence value of the second measurement) (Ex: 380 nm, Em: 460 nm; Envision (Perkin Elmer Inc.)).
Measurement: IC50 = 1114 nM (pChEMBL 5.949999809265137)
Descriptors: MW 494.98, ALogP 5, HBA 3, HBD 2, PSA 97.44, QED 0.29
Compound (SMILES): NC(=O)c1cccc(Oc2ccc(-n3ncc(C(=O)c4cc5cc(C(=O)N6CCOCC6)ccc5[nH]4)c3N)c(Cl)c2)c1
InChIKey: QARVIPGXIAPCCM-UHFFFAOYSA-N
Target: Tyrosine-protein kinase BTK (SINGLE PROTEIN), Homo sapiens
UniProt: Q06187
Assay: TR-FRET (Time Resolved FRET) Assay: This BTK competition assay measures compound potency (IC50) for the inactivated state of Bruton's Tyrosine Kinase using FRET (Förster/Fluorescence Resonance Energy Transfer) technology. The BTK-Eu complex was incubated on ice one hour prior to use at a starting concentration of 50 nM BTK-Bioease: 10 nM Eu-streptavidin (Perkin-Elmer Catalog#AD0062). The assay buffer consisted of 20 mM HEPES (pH 7.15), 0.1 mM DTT, 10 mM MgCl2, 0.5 mg/ml BSA with 3% Kinase Stabilizer (Fremont Biosolutions, Catalog #STB-K02). After 1 h, the reaction mixture from above was diluted 10 fold in assay buffer to make 5 nM BTK: 1 nM Eu-Streptavidin complex (donor fluorophore). 18 μl of a mixture of 0.11 nM BTK-Eu and 0.11 nM Kinase Tracer 178 (Invitrogen, Catalog #PV5593,) with BTK-Eu alone as no negative control, was then dispensed into 384-well flat bottom plates (Greiner, 784076). Compounds to be tested in assay were prepared as 10× concentrations and serial dilution in half-log increments was performed in DMSO so as to generate 10 point curves. To initiate the FRET reaction, compounds prepared as 10× stock in DMSO was added to the plates and the plates were incubated 18-24 h at 14° C.After the incubation the plates were read on a BMG Pherastar Fluorescent plate reader (or equivalent) and used to measure the emission energy from the europium donor fluorophore (620 nm emission) and the FRET (665 nm emission).
Measurement: IC50 = 50 nM (pChEMBL 7.300000190734863)
Descriptors: MW 585.02, ALogP 4.18, HBA 8, HBD 3, PSA 158.56, QED 0.24
Compound (SMILES): Cc1ncc(-c2ccc3c(c2)c(C(=O)NCC(C)(C)C)nn3CC(=O)N2[C@H](C(=O)Nc3nc(Br)ccc3C)C[C@@]3(C)C[C@@H]23)cn1
InChIKey: LQKNNXWNCJVAIO-DVIHYUSWSA-N
Target: Complement factor D (SINGLE PROTEIN), Homo sapiens
UniProt: P00746
Assay: Factor D Assay: Human Factor D (purified from human serum, Complement Technology, Inc.) at 80 nM final concentration is incubated with test compound at various concentrations for 5 min at room temperature in 50 mM Tris, 1M NaCl, pH 7.5. A synthetic substrate Z-L-Lys-SBz and DTNB (Ellman's reagent) are added to final concentrations of 100 μM each. Absorbance at 405 nm (A405) is recorded at 30 second intervals for 30 min using a microplate spectrophotometer. IC50 values are calculated by nonlinear regression of complement Factor D reaction rates as a function of test compound concentration.
Measurement: IC50 > 10000 nM (pChEMBL 5)
Descriptors: MW 673.62, ALogP 5.06, HBA 8, HBD 2, PSA 135, QED 0.26
Compound (SMILES): CC(O)c1c(Cc2ccc(S(=O)(=O)N(C)C)cc2)c2ncccc2n1C/C(F)=C/CN
InChIKey: QZJVGNWPJBOTRW-YVLHZVERSA-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 446.55, ALogP 2.74, HBA 6, HBD 2, PSA 101.45, QED 0.55
Compound (SMILES): O=C(Nc1cc2cc(-c3cnco3)ccc2cn1)[C@H]1CCCO1
InChIKey: HCPWCMYSBPNFMN-CQSZACIVSA-N
Target: Proto-oncogene Wnt-1 (SINGLE PROTEIN), Homo sapiens
UniProt: P04628
Assay: The screening assay for Wnt activity: SW480 colon carcinoma cells were transduced with a lentiviral vector expressing luciferase with a human Sp5 promoter consisting of a sequence of eight TCF/LEF binding sites. SW480 cells stably expressing the Sp5-Luc reporter gene and a hygromycin resistance gene were selected by treatment with 150 μg/mL of hygromycin for 7 days. These stably transduced SW480 cells were expanded in cell culture and used for all further screening activities. Each compound was dissolved in DMSO as a 10 mM stock and used to prepare compound source plates. Serial dilution (1:3, 10-point dose-response curves starting from 10 μM) and compound transfer was performed using the ECHO 550 (Labcyte, Sunnyvale, Calif.) into 384-well white solid bottom assay plates (Greiner Bio-One) with appropriate DMSO backfill for a final DMSO concentration of 0.1%. For Sp5-Luc reporter gene assays, the cells were plated at 4,000 cells/well in 384-well plates with a DMEM medium containing 1% fetal bovine serum, and 1% Penicillin-Streptomycin and incubated for 36 to 48 hours at 37° C. and 5% CO2. Following incubation, 15 μl of BriteLite Plus luminescence reagent (Perkin Elmer) was added to each well of the 384-well assay plates. The plates were placed on an orbital shaker for 2 min and then luminescence was quantified using the Envision (Perkin Elmer) plate reader. Readings were normalized to DMSO only treated cells, and normalized activities were utilized for EC50 calculations using the dose-response log (inhibitor) vs. response-variable slope (four parameters) nonlinear regression feature available in GraphPad Prism 5.0 (or Dotmatics).
Measurement: EC50 = 3742 nM (pChEMBL 5.429999828338623)
Descriptors: MW 309.33, ALogP 3.01, HBA 5, HBD 1, PSA 77.25, QED 0.8
Compound (SMILES): O=COc1ccc(N2CC(O)(c3ccc(OCc4c(-c5c(Cl)cccc5Cl)noc4C4CC4)cc3Cl)C2)nc1C(F)(F)F
InChIKey: XEYUCFTWINTVFY-UHFFFAOYSA-N
Target: Bile acid receptor (SINGLE PROTEIN), Homo sapiens
UniProt: Q96RI1
Assay: FRET Activity Assay: Expression and purification of the FXR-LBD: An overnight preculture of a transformed E. coli strain was diluted 1:20 in LB-Ampicillin medium and grown at 30° C. to an optical density of =0.4-0.6. Gene expression was then induced by addition of 0.5 mM IPTG. Cells were incubated an additional 6 h at 30° C., 180 rpm. Cells were collected by centrifugation (7000×g, 7 min, rt). Per liter of original cell culture, cells were resuspended in 10 mL lysis buffer (50 mM Glucose, 50 mM Tris pH 7.9, 1 mM EDTA and 4 mg/mL lysozyme) and left on ice for 30 min. Cells were then subjected to sonication and cell debris removed via centrifugation (22000×g, 30 min, 4° C.). Per 10 mL of supernatant 0.5 mL prewashed Glutathione 4B sepharose slurry (Qiagen) was added and the suspension kept slowly rotating for 1 h at 4° C. Glutathione 4B sepharose beads were pelleted by centrifugation (2000×g, 15 sec, 4° C.) and washed twice in wash buffer (25 mM Tris, 50 mM KCl, 4 mM MgCl2 and 1M NaCl). The pellet was resuspended in 3 mL elution buffer per liter of original culture (elution buffer: 20 mM Tris, 60 mM KCl, 5 mM MgCl2 and 80 mM glutathione added immediately prior to use as powder). The suspension was left rotating for 15 min at 4° C., the beads pelleted and eluted again with half the volume of elution buffer than the first time. The eluates were pooled and dialysed overnight in 20 mM Hepes buffer (pH 7.5) containing 60 mM KCl, 5 mM MgCl2 as well as 1 mM dithiothreitol and 10% (v/v) glycerol. The protein was analysed by SDS-Page.
Measurement: EC50 = 3.1 nM (pChEMBL 8.510000228881836)
Descriptors: MW 654.86, ALogP 7.42, HBA 8, HBD 1, PSA 97.92, QED 0.19
Compound (SMILES): COC[C@]12CC[C@@](C)(O)C[C@H]1CC[C@H]1[C@@H]3CC[C@H](C(=O)Cn4nnc5cc(OC)ccc54)[C@@]3(C)CC[C@@H]12
InChIKey: DRMFZNTUFRPVCP-CWKDJKSQSA-N
Target: Gamma-aminobutyric acid type B receptor subunit 1 (SINGLE PROTEIN), Rattus norvegicus
UniProt: Q9Z0U4
Assay: Steroid Inhibition of TBPS Binding: TBPS binding assays using rat brain cortical membranes in the presence of 5 μM GABA has been described (Gee et al, J. Pharmacol. Exp. Ther. 1987, 241, 346-353; Hawkinson et al, Mol. Pharmacol. 1994, 46, 977-985; Lewin, A. H et al., Mol. Pharmacol. 1989, 35, 189-194).Briefly, cortices are rapidly removed following decapitation of carbon dioxide-anesthetized Sprague-Dawley rats (200-250 g). The cortices are homogenized in 10 volumes of ice-cold 0.32 M sucrose using a glass/teflon homogenizer and centrifuged at 1500×g for 10 min at 4° C. The resultant supernatants are centrifuged at 10,000×g for 20 min at 4° C. to obtain the P2 pellets. The P2 pellets are resuspended in 200 mM NaCl/50 mM NaK phosphate pH 7.4 buffer and centrifuged at 10,000×g for 10 min at 4° C. This washing procedure is repeated twice and the pellets are resuspended in 10 volumes of buffer. Aliquots (100 μL) of the membrane suspensions are incubated with 3 nM [35S]-TBPS and 5 μL aliquots of test drug dissolved in dimethyl sulfoxide (DMSO) (final 0.5%) in the presence of 5 μM GABA. The incubation is brought to a final volume of 1.0 mL with buffer. Nonspecific binding is determined in the presence of 2 μM unlabeled TBPS and ranged from 15 to 25%. Following a 90 min incubation at room temp, the assays are terminated by filtration through glass fiber filters (Schleicher and Schuell No. 32) using a cell harvester (Brandel) and rinsed three times with ice-cold buffer. Filter bound radioactivity is measured by liquid scintillation spectrometry. Non-linear curve fitting of the overall data for each drug averaged for each concentration is done using Prism (GraphPad). The data are fit to a partial instead of a full inhibition model if the sum of squares is significantly lower by F-test. Similarly, the data are fit to a two component instead of a one component inhibition model if the sum of squares is significantly lower by F-test. The concentration of test compound producing 50% inhibition (IC50) of specific binding and the maximal extent of inhibition (Imax) are determined for the individual experiments with the same model used for the overall data and then the means ±SEM.s of the individual experiments are calculated. Picrotoxin serves as the positive control for these studies as it has been demonstrated to robustly inhibit TBPS binding.
Measurement: IC50 < 10 nM (pChEMBL 8)
Descriptors: MW 509.69, ALogP 5.05, HBA 7, HBD 1, PSA 86.47, QED 0.59
Compound (SMILES): Cc1cccnc1C(O)c1ccc(F)c(-c2ncnc3cc(N4CCOCC4)ccc23)c1
InChIKey: GBYLJBVPBPYLRC-UHFFFAOYSA-N
Target: DNA-dependent protein kinase catalytic subunit (SINGLE PROTEIN), Homo sapiens
UniProt: P78527
Assay: Biochemical Assay: The kinase assay was carried out in streptavidin-coated 348-well microtitre flashplates. To this end, 1.5 ug of DNA-PK/protein complex and 100 ng of biotinylated substrate, such as, for example, PESQEAFADLWKK-biotin-NH2 (biotin-DNA-PK peptide), were incubated for 90 min at room temperature in a total volume of 36.5 ul (34.25 mM HEPES/KOH; 7.85 mM Tris HCl; 68.5 mM KCl; 5 uM ATP; 6.85 mM MgCl2; 0.5 mM EDTA; 0.14 mM EGTA; 0.69 mM DTT; pH 7.4) with 500 ng of DNA from calf thymus, 0.1 uCi of 33P-ATP and 1.8% of DMSO per well with and without the test compound. The reaction was stopped using 50 ul/well of 200 mM EDTA. After incubation for a further 30 min at room temperature, the liquid was removed. Each well was washed three times with 100 ul of 0.9% saline solution. A nonspecific reaction (blank value) was determined using 10 uM of an innate kinase inhibitor. The radioactivity measurement was carried out using a TopCount. IC50 values were calculated in RS1.
Measurement: IC50 = 5 nM (pChEMBL 8.300000190734863)
Descriptors: MW 430.48, ALogP 4.06, HBA 6, HBD 1, PSA 71.37, QED 0.53
Compound (SMILES): COCCC(=O)N1C[C@@H](F)[C@@H](NC(=O)c2sc3nccc4c3c2NC(=O)N4c2ccc(Oc3ccccc3)cc2C)C1
InChIKey: CPUUOVQQRGELBT-RTWAWAEBSA-N
Target: Tyrosine-protein kinase BTK (SINGLE PROTEIN), Homo sapiens
UniProt: Q06187
Assay: Kinase Lanthascreen Binding Assay: A BTK kinase lanthascreen binding assay monitors compound binding to unphosphorylated-BTK kinase domain (UP-BTK), by competing with a fluorescent labeled tracer. UP-BTK, consisting of the kinase domain of non-phosphorylated BTK protein (389-659aa), was produced in a Baculovirus/insect cell expression system. Into a 384-well plate, 2 ng of GST-tagged human BTK (389-659aa) was incubated with compound, 50 nM of Tracer 236 and 2 nM anti-GST antibody for 60 minutes using an optimized Lanthascreen assay. After 60 minutes, plates were read at 340 nM and 615/665 nM in an Infinite F500 (Tecan).
Measurement: IC50 = 25.1 nM (pChEMBL 7.599999904632568)
Descriptors: MW 589.65, ALogP 5.4, HBA 7, HBD 2, PSA 113.1, QED 0.28
Compound (SMILES): NCCCNc1nc(Nc2ccc(Cl)c(Cl)c2)ncc1Cl
InChIKey: OJBZKMRHGQTLHT-UHFFFAOYSA-N
Target: Serine/threonine-protein kinase 17B (SINGLE PROTEIN), Homo sapiens
UniProt: O94768
Assay: Activity Inhibition Assay: To measure the DRAK1 and DRAK2 activity, the substrate MRCL3 peptide and ATP were mixed with the enzymes. After an appropriate period of time, the reaction product ADP was quantitatively analyzed. The buffer solution A was used to make a 3x substrate solution of 3x concentration so that the final concentrations of ATP and MRCL3 peptides were 1 uM, respectively, and 2.5 ul of each solution was dispensed into Optiplate 384 (perkin-elmer) microplate. The final concentrations of ATP and MRCL3 peptide were made to be 1 uM respectively by using buffer A, followed by preparing a 3x substrate solution. The prepared substrate solution was distributed in Optiplate 384 (Perkin-Elmer) microplate (2.5 ul/well). Then, the compounds of Examples 1~39 and the compounds of Comparative Examples 1-6, whose concentrations were tripled by the final concentration were distributed thereto (2.5 ul/well). Lastly, DRAK1 or DRAK2 enzyme solution, properly diluted by using buffer A, was added thereto (2.5 ul/well). Centrifugation was performed at 800 rpm for 1 minute, and then enzyme reaction was induced at 30 C.
Measurement: IC50 = 310 nM (pChEMBL 6.510000228881836)
Descriptors: MW 346.65, ALogP 3.94, HBA 5, HBD 3, PSA 75.86, QED 0.69
Compound (SMILES): NS(=O)(=O)NCCNc1nonc1/C(=N/O)NC1Cc2ccc(F)cc21
InChIKey: MSCIHUFSFVFHHV-UHFFFAOYSA-N
Target: Indoleamine 2,3-dioxygenase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P14902
Assay: Enzyme Assay: HIS-tagged IDO1 protein was recombinantly expressed in Escherichia coli using ZYP5052 autoinduction media supplemented with 500 μM delta aminolevulinic acid for 48 hours at 16 degrees Celsius. IDO1 protein was purified using Ni2+-affinity resin and size exclusion chromatography. Purified protein was then diluted in assay buffer (50 mM Tris pH 7.0, 1% glycerol, 20 μM methylene blue, 0.05% Tween-20, 20 mM sodium ascorbate, 100 units/mL catalase to obtain a final IDO1 concentration of 40 nM. IDO1 solution (30 μM) or buffer alone (30 μM) were dispensed to wells of the assay plate using a BioRAPTR liquid dispenser (Beckman Coulter). Assay plates containing compound and IDO1 enzyme were incubated at room temperature for 30 minutes. Afterwards, 10 μL of 400 μM tryptophan in assay buffer were added to each well of the assay plate using a BioRAPTR liquid dispenser. Plates were incubated at room temperature for 60 minutes and reactions were quenched by addition of 10 μL of 0.5 M methyl isonipecotate in dimethyl sulfoxide. Plates were sealed and incubated at 37° C. for 4 hours or 50° C. for 2 h. The plates are allowed to cool and then centrifuged for 1 minute at 1000×g. The resulting fluoresence was measured in an Envision plate reader (Perkin Elmer) with a 400/25 nm excitation filter and an 510/20 nm emission filter.
Measurement: IC50 = 50.3 nM (pChEMBL 7.300000190734863)
Descriptors: MW 385.38, ALogP -0.56, HBA 8, HBD 5, PSA 167.76, QED 0.14
Compound (SMILES): Cc1cc(OCCCc2c3n(c4c(-c5c(C)nn(C)c5C)c(Cl)ccc24)CCCN(c2cc(F)cc(C(=O)O)c2)C3=O)cc(C)c1Cl
InChIKey: ZNIFEEFQQLQVAS-UHFFFAOYSA-N
Target: Induced myeloid leukemia cell differentiation protein Mcl-1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q07820
Assay: Fluorescence Polarization Anisotropy Competition Assay: Compound affinity was measured using a fluorescence polarization anisotropy competition assay. Anisotropy measurements were carried out in 384-well, black, flat-bottom plates (Greiner Bio-one, Monroe, N.C., USA). The assay was run using either a fluorescein isothiocyanate-labeled BH3 peptide derived from Bak (FITC-AHx-GQVGRQLAIIGDDINR-NH2) or a fluorescein isothiocyanate-labeled BH3 peptide derived from Bim (FITC-AHx-EARIAQELRRIGDEFNETYTR-NH2) that were purchased from GenScript (Piscataway, N.J.) at >95% purity and used without further purification. 10 nM FITC-Bak peptide and 15 nM recombinant Mcl-1 (residues 172-327) were added to assay buffer (3 mM dithiothreitol, 50 mM NaCl, 20 mM Tris, pH 7.5). The Bim based assay was run with 1 nM FITC-Bim peptide and 1.5 nM recombinant Mcl-1 (residues 172-327) added to assay buffer (20 mM TRIS pH 7.5, 50 mM NaCl, 3 mM DTT, 0.01% CHAPS). For selectivity assays, 40 nM Bcl-2 (residues 1-207A96T,G110R, Î35-91, replaced with Bcl-xL35-50) or 4 nM Bcl-xL (residues 1-209, loop 45-86 deleted) were incubated with 10 nM FITC-Bak in assay buffer.
Measurement: Ki < 50 nM (pChEMBL 7.301000118255615)
Descriptors: MW 677.6, ALogP 8.48, HBA 6, HBD 1, PSA 89.59, QED 0.17
Compound (SMILES): O=C(O)c1ccc(CNc2nc(-c3ccc(Cl)cc3)nc(C3CC3)n2)cc1
InChIKey: QIEPKBLBWKKCSD-UHFFFAOYSA-N
Target: 3',5'-cyclic-AMP phosphodiesterase 4B (SINGLE PROTEIN), Homo sapiens
UniProt: Q07343
Assay: Kinetic Assay: Assays are performed in 96-well plates in a total volume of 200 μl/well. Compounds are dissolved in dimethylsulfoxide (DMSO) and added to plates in a volume of 10 μl followed by addition of 165 μl of assay mix. Plates are pre-incubated at 25° C. for 15 min and the reactions are initiated by the addition of 25 μl of cAMP followed by thorough mixing. Reaction rates are measured by monitoring the decrease in fluorescence using excitation at 355 nm and emission at 460 nm for a period of 10 min in a fluorescence plate reader. Initial rates (slopes) are determined from linear portions of the progress curves. Final concentrations of assay components are as follows: 50 mM Tris, pH 8, 10 mM MgCl2, 50 mM KCl, 2% DMSO, 5 mM tris(2-carboxyethyl)phosphine (TCEP), 0.4 mM phosphenolpyruvate (PEP), 0.01 mM NADH, 0.04 mM adenosine triphosphate (ATP), 0.004 mM cAMP, 7.5 units myokinase from yeast, 1.6 units pyruvate kinase, 2 units lactate dehydrogenase, and either 0.5 nM human PDE4D7 or 10 nM human PDE4B1. All data are percent normalized relative to controls and are presented as percent inhibition. An inhibitory concentration 50% (IC50) value is calculated by fitting of a sigmoidal dose response curve. Human PDE4D7 contained a mutation of serine 54 to aspartic acid to mimic activation by cAMP-dependent protein kinase A (PKA). Human PDE4B1 contained a corresponding mutation of serine 133 to aspartic acid to also mimic PKA activation.
Measurement: IC50 > 1000 nM (pChEMBL 6)
Descriptors: MW 380.84, ALogP 4.38, HBA 5, HBD 2, PSA 88, QED 0.66
Compound (SMILES): Cc1cc(-n2ccc3cncnc32)cc2c1C(=O)NC2C
InChIKey: OPZFLEPCEIJFSE-UHFFFAOYSA-N
Target: MAP kinase-interacting serine/threonine-protein kinase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9BUB5
Assay: Biochemical Enzymatic Assay: Compounds are screened for MNK inhibition using the ADP-Glo kinase assay kit (Promega, catalogue No. V9101). All kinase reactions are performed in Reaction Buffer E (15 mM HEPES pH7.4, 20 mM NaCl, 1 mM EGTA, 10 mM MgCl2, 0.1 mg/ml BGG, and 0.02% Tween-20). Final MNK 1 reactions contained 10 nM recombinant MNK1 (Life Technologies, PR9138A), 100 μM MNK substrate peptide Ac-TATKSGSTTKNR-NH2 (American Peptide Company), 300 μM ATP, and varying concentrations of the inhibitory compound of interest. Final MNK2 reactions contained 3 nM recombinant MNK2 (Life Technologies, PV5607), 50 μM MNK substrate peptide Ac-TATKSGSTTKNR-NH2 (American Peptide Company), 10 μM ATP, and varying concentrations of the inhibitory compound of interest. Final DMSO concentration in each reaction is 1%.
Measurement: IC50 = 55 nM (pChEMBL 7.260000228881836)
Descriptors: MW 278.31, ALogP 2.53, HBA 4, HBD 1, PSA 59.81, QED 0.74
Compound (SMILES): Cc1nc2c(n1C1CC3CCC(C1)N3CC[C@H](NC(=O)C1CCC(F)(F)CC1)c1ccsc1)CCN(C(=O)C(C)C)C2
InChIKey: XADGEXPZZCPUIH-GVHAAITQSA-N
Target: C-C chemokine receptor type 5 (SINGLE PROTEIN), Homo sapiens
UniProt: P51681
Assay: Calcium Flux Inhibition Experiment: 1. HEK293 cells which can stably express CCR5 were inoculated in a 96-well plate and incubated overnight.2. The medium in each well into which cells were innoculated was removed and 40 μl/well of freshly prepared dye was added. The plate was placed in a 37° C. incubator and incubated for 40 minutes at constant temperature.3. The medicament to be determined was diluted with calcium buffer to eight concentration gradients, which is 1×10−4 M, 1×10−5 M, 1×10−6 M, 1×10−7 M, 1×10−8 M, 1×10−9 M, 1×10−10 M, and 1×10−11 M, respectively, and homogeneously mixed.4. The dye was removed. Freshly prepared calcium buffer was used to wash for one time, 50 μl of calcium buffer was added.5. FlexStation II was used for detection. 25 μl of calcium buffer containing the medicament to be determined was added automatically from the 15th second. The fluorescence value at 525 nm was read ultimately.
Measurement: IC50 = 8.34 nM (pChEMBL 8.079999923706055)
Descriptors: MW 601.81, ALogP 6.03, HBA 6, HBD 1, PSA 70.47, QED 0.4
Compound (SMILES): CC(C)c1ncccc1-c1ncc2c(n1)N(Cc1c(F)cc(-c3nc(C(F)(F)F)cn3C)cc1F)C(=O)C2
InChIKey: SYURTLAZDCOLFT-UHFFFAOYSA-N
Target: Ubiquitin carboxyl-terminal hydrolase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: O94782
Assay: HTS Assay: The HTS assay was performed in a final volume of 20 μL in assay buffer containing 20 mM Tris-HCl (pH 8.0, (1M Tris-HCl, pH 8.0 solution; Corning 46-031-CM)), 2 mM CaCl2) (1M Calcium Chloride solution; Sigma #21114) 1 mM GSH (L-Glutathione reduced; Sigma #G4251), 0.01% Prionex (0.22 μM filtered, Sigma #G-0411), and 0.01% Triton X-100. Stock compound solutions were stored at −20° C. as 10 mM in DMSO. Up to 1 month prior to the assay, 2 mM test compounds were pre-dispensed into assay plates (Black, low volume; Corning #3820) and frozen at −20° C. Prestamped assay plates were allowed to come to room temperature on the day of the assay. For the screen, 100 nL of 2 mM was pre-dispensed for a final screening concentration of 10 μM (DMSO(fc)=0.5%). The final concentration of the enzyme (USP1, construct USP1 (I-785, GG670, 671AA)/UAF1 (I-677)-Flag; Viva) in the assay was 100 μM. Final substrate (Ub-Rh110; Ubiquitin-Rhodamine 110, R&D Systems #U-555) concentration was 25 nM with [Ub-Rh110]<<Km. 10 μL of 2× enzyme was added to assay plates (pre-stamped with compound) either simultaneously with 2×Ub-Rh110 or preincubated with USP1 40 minutes prior to the addition of 10 μL of 2×Ub-Rh110 to compound plates. Plates were incubated stacked for 45 minutes at room temperature before fluorescence was read on the Envision (Excitation at 485 nm and Emission at 535 nm; Perkin Elmer) or on the PheraSTAR (Excitation at 485 nm and Emission at 535 nm; BMG Labtech).
Measurement: IC50 = 125 nM (pChEMBL 6.900000095367432)
Descriptors: MW 528.49, ALogP 5.45, HBA 6, HBD 0, PSA 76.8, QED 0.32
Compound (SMILES): CS(=O)(=O)Nc1cccc(C(=O)NC[C@@H](O)CNC2Cc3ccccc3C2)c1
InChIKey: CWPWRLACNLMJOS-IBGZPJMESA-N
Target: Protein arginine N-methyltransferase 5 (SINGLE PROTEIN), Homo sapiens
UniProt: O14744
Assay: iochemical Assay: Compounds of the invention may be tested for in vitro activity in the following assay: A histone H4 derived peptide is used as substrate (amino acid sequence: Ser-Gly-Arg-Gly-Lys-Gly-Gly-Lys-Gly-Leu-Gly-Lys-Gly-Gly-Ala-Lys-Arg-His-Arg-Lys-Val-NH2). Full-length PRMT5 enzyme (NCBI Reference sequence NP-006100.2) was co-expressed with Hiss-MEP50 in insect cells and purified via Nickel immobilized metal affinity and gel filtration chromatograph.The 6 μL assay reactions are run in Greiner brand black 384-well low volume plates. All reactions contained assay buffer (phosphate buffered saline, 0.01% (v/v) Tween-20, 0.01% (w/v) albumin from chicken egg white, 1 mM dithiothreitol, 200 nM peptide substrate, 1 μM S-Adenosyl methionine, and 15 ng/reaction enzyme, with the enzyme being omitted from negative control reactions. Compounds were added in a volume of 100 nL from dilution series prepared in DMSO, positive and negative control reactions receiving the same volume DMSO without compound. The plates were sealed with adhesive seals and incubated for 4 hours at 37° C. Reaction progress was measured using the Transcreener™ EPIGEN methyltransferase assay (BellBrook Labs, Madison, Wis.) as recommended by the manufacturer. To each reaction 2 μL detection mix were added, containing coupling enzymes, fluorescence polarisation tracer, and AMP antibody. Plates were incubated for 90 min before being read on a PerkinElmer EnVision™ plate reader in fluorescence polarisation mode. IC50 values were obtained from the raw readings by calculating percent inhibition (% I) for each reaction relative to controls on the same plate (% I=(I−CN)/(CP−CN) where CN/CP are the averages of the negative/positive reactions, respectively), then fitting the % I data vs. compound concentration [I] to % I=(A+((B−A)/(1+((C/[I])^D)))) where A is the lower asymptote, B is the upper asymptote, C is the IC50 value, and D is the slope.
Measurement: IC50 = 4679 nM (pChEMBL 5.329999923706055)
Descriptors: MW 403.5, ALogP 0.91, HBA 5, HBD 4, PSA 107.53, QED 0.52