lovingscience/chembl-v37-raw-activities huggingface dataset:
Compound (SMILES): COCc1ccc(NC(=O)c2c(C#Cc3ccc(OC)cc3)n(C3(C)CC3)c3ncnc(N)c23)cc1
InChIKey: CIJMJPGEMBPFFJ-UHFFFAOYSA-N
Target: Proto-oncogene tyrosine-protein kinase receptor Ret (SINGLE PROTEIN), Homo sapiens
UniProt: P07949
Assay: Measurement of Inhibitory Activity Against RET (In Vitro): Regarding the conditions for measurement of in vitro inhibitory activity of compounds against RET kinase activity, the website of AnaSpec states that Srctide (GEEPLYWSFPAKKK) corresponds to the substrate peptide for reaction to measure RET kinase activity. Thus, the amino acid sequence was partly modified and biotinylated to prepare biotinylated peptides (biotin-EEPLYWSFPAKKK). The purified recombinant human RET protein used in the test was purchased from Carna Biosciences, Inc.To measure the inhibitory activity, first, the compounds of the present invention were individually diluted with dimethyl sulfoxide (DMSO) stepwise. Subsequently, RET protein, the substrate peptide (the final concentration: 250 nM), magnesium chloride (the final concentration: 10 mM), ATP (the final concentration: 10 μM), and a solution of the compound of the present invention in DMSO (the final concentration of DMSO: 2.5%) were added to a buffer for kinase reaction (13.5 mM Tris, pH of 7.5, 2 mM dithiothreitol, 0.009% Tween-20). Each of the mixtures was incubated at 25° C. for 100 minutes to perform a kinase reaction. EDTA was then added thereto to give a final concentration of 24 mM so that the reaction was terminated. A detection solution containing Eu-labeled antiphosphotyrosine antibody PT66 (PerkinElmer) and SureLight APC-SA (PerkinElmer) was added thereto, and each mixture was allowed to stand at room temperature for 2 hours or more. Finally, the intensity of fluorescence under the excitation light with a wavelength of 337 nm was measured with a PHERAstar FS (BMG Labtech) at the two wavelengths of 620 nm and 665 nm. The phosphorylation level was calculated from the ratio of the fluorescence intensity at the two wavelengths, and the compound concentration at which phosphorylation was inhibited by 50% was defined as the IC50 value (nM).
Measurement: IC50 = 2.9 nM (pChEMBL 8.539999961853027)
Descriptors: MW 481.56, ALogP 4.33, HBA 7, HBD 2, PSA 104.29, QED 0.4
Compound (SMILES): O=C(O)c1ccc2c3n(nc2c1)[C@@H]1[C@H](CO3)N(C2CCCCC2)[C@@]2(C(=O)Nc3cc(Cl)ccc32)[C@H]1c1cccc(Cl)c1F
InChIKey: WYGAHHDMOLTFMI-CKHSQSCFSA-N
Target: E3 ubiquitin-protein ligase Mdm2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q00987
Assay: Mdm2-p53 Inhibition AlphaScreen: This assay is used to determine whether the compounds inhibit the p53-MDM2 interaction and thus restore p53 function.15 μL of compound in 20% DMSO (serial pre-dilutions of compound are done in 100% DMSO) is pipetted to the wells of a white OptiPlate-96 (PerkinElmer). A mix consisting of 20 nM GST-MDM2 protein (aa 23-117) and 20 nM biotinylated p53 wt peptide (encompassing aa 16-27 of wt human p53, amino acid sequence QETFSDLWKLLP-Ttds-Lys-Biotin, molecular weight 2132.56 g/mol) is prepared in assay buffer (50 mM Tris/HCl pH 7.2; 120 mM NaCl; 0.1% bovine serum albumin (BSA); 5 mM dithiothreitol (DTT); 1 mM ethylenediaminetetraacetic acid (EDTA); 0.01% Tween 20). 30 μL of the mix is added to the compound dilutions and incubated for 15 min at rt while gently shaking the plate at 300 rounds per minute (rpm). Subsequently, 15 μL of premixed AlphaLISA Glutathione Acceptor Beads and AlphaScreen Streptavidin Donor Beads from PerkinElmer (in assay buffer at a concentration of 10 μg/mL each) are added and the samples are incubated for 30 min at rt in the dark (shaking 300 rpm). Afterwards, the signal is measured in a PerkinElmer Envision HTS Multilabel Reader using the AlphaScreen protocol from PerkinElmer.Each plate contains negative controls where biotinylated p53-peptide and GST-MDM2 are left out and replaced by assay buffer. Negative control values are entered as low basis value when using the software GraphPad Prism for calculations. Furthermore, a positive control (5% DMSO instead of test compound; with protein/peptide mix) is pipetted. Determination of IC50 values are carried out using GraphPad Prism 3.03 software (or updates thereof).
Measurement: IC50 = 36 nM (pChEMBL 7.440000057220459)
Descriptors: MW 621.5, ALogP 6.76, HBA 6, HBD 2, PSA 96.69, QED 0.26
Compound (SMILES): COC1CCC(O)(CCNC(=O)c2cnc(-c3cnn4cc(Cl)cnc34)cc2NC2COC2)CC1
InChIKey: UMVAQBYUGJCZOT-UHFFFAOYSA-N
Target: Interleukin-1 receptor-associated kinase 4 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9NWZ3
Assay: IRAK4 Inhibition Assay: The assays were performed in U-bottom 384-well plates. The final assay volume was 30 μL prepared from 15 μL additions of enzyme and substrates (fluoresceinated peptide and ATP) and test compounds in assay buffer (20 mM HEPES pH 7.2, 10 mM MgCl2, 0.015% Brij 35 and 4 mM DTT). The reaction was initiated by the combination of IRAK4 with substrates and test compounds. The reaction mixture was incubated at room temperature for 60 min. and terminated by adding 45 μL of 35 mM EDTA to each sample. The reaction mixture was analyzed on the Caliper LABCHIP® 3000 (Caliper, Hopkinton, Mass.) by electrophoretic separation of the fluorescent substrate and phosphorylated product. Inhibition data were calculated by comparison to no enzyme control reactions for 100% inhibition and vehicle-only reactions for 0% inhibition. The final concentrations of reagents in the assays are ATP, 500 μM; FL-IPTSPITTTYFFFKKK peptide 1.5 μM; IRAK4, 0.6 nM; and DMSO, 1.6%.
Measurement: IC50 = 14 nM (pChEMBL 7.849999904632568)
Descriptors: MW 500.99, ALogP 2.7, HBA 9, HBD 3, PSA 122.9, QED 0.43
Compound (SMILES): CS(=O)(=O)N(c1ccccc1Nc1cc(Nc2cccc(C#N)n2)nc2[nH]c(C(F)F)nc12)C(F)F
InChIKey: OBHPWHWOTUSGAZ-UHFFFAOYSA-N
Target: Non-receptor tyrosine-protein kinase TYK2 (SINGLE PROTEIN), Homo sapiens
UniProt: P29597
Assay: Binding Assay: Binding constants for compounds of the present invention against the JH2 domain were determined by the following protocol for a KINOMEscan® assay (DiscoveRx). A fusion protein of a partial length construct of human TYK2 (JH2domain-pseudokinase) (amino acids G556 to D888 based on reference sequence NP_003322.3) and the DNA binding domain of NFkB was expressed in transiently transfected HEK293 cells. From these HEK 293 cells, extracts were prepared in M-PER extraction buffer (Pierce) in the presence of Protease Inhibitor Cocktail Complete (Roche) and Phosphatase Inhibitor Cocktail Set II (Merck) per manufacturers' instructions. The TYK2 (JH2domain-pseudokinase) fusion protein was labeled with a chimeric double-stranded DNA tag containing the NFkB binding site (5′-GGGAATTCCC-3′) fused to an amplicon for qPCR readout, which was added directly to the expression extract (the final concentration of DNA-tag in the binding reaction is 0.1 nM).
Measurement: Kd = 5.5 nM (pChEMBL 8.260000228881836)
Descriptors: MW 520.47, ALogP 4.64, HBA 8, HBD 3, PSA 139.69, QED 0.23
Compound (SMILES): C=CC(=O)N1CCC(c2cc3cn[nH]c3c(-c3ccc(Oc4cccc(F)c4)cc3)n2)CC1
InChIKey: BTKBCMJQFVOPEL-UHFFFAOYSA-N
Target: Tyrosine-protein kinase BTK (SINGLE PROTEIN), Homo sapiens
UniProt: Q06187
Assay: BTK Enzyme Activity Testing: In vitro activity of BTK is measured by detecting ADP produced in a kinase reaction with an ADP-Glo kinase assay kit from Promega Company. In the kinase assay, the kinase consumes ATP to phosphorylate the substrate, while producing ADP. ADP-Glo reagent then terminates the kinase reaction and completely consumes the remaining ATP. Finally, a kinase detection reagent is added to convert generated ADP into new ATP. Luciferase in the detection reagent is capable of catalyzing fluorescein with the participation of ATP and O2 to produce oxidized fluorescein, AMP, and generate light quantum, thereby converting a chemical signal into an optical signal (Luminecence). The intensity of optical signal is positively correlated with the production of ADP in the kinase reaction, so that the activity of kinase BTK can be thereby quantitatively determined.All assays are conducted at a constant temperature of 23° C., using a Corning 3674 Type white 384-well plate; the BTK kinase (full length with His-Tag) is expressed and purified internally by the company; the substrate of kinase is polypeptide (4:1 Glu,Tyr) (from SignalChem) and ATP (from Sigma); and a microplate reader EnVision (Perkin Elmer) is used for reading an optical signal. The assay buffer includes 40 mM Tris-HCl (pH 7.5), 10 mM MgCl2 (Sigma), 2 mM MnCl2 (Sigma), 0.05 mM DTT (Sigma), and 0.01% BSA (Sigma); the BTK kinase is diluted with the assay buffer to a concentration of 1.6 ng/uL as a kinase reaction solution; and the substrate reaction solution comprises 0.2 mg/mL polypeptide substrate and 50 uM ATP.Compound's IC50 is calculated from 10 concentration points by the following method. The compound is dissolved and diluted in 100% DMSO to a concentration of 1 mM, followed by a serial 3× dilution with DMSO to a minimum concentration of 0.05 uM. Each concentration stock is further diluted 40× with the assay buffer. To a 384-well assay plate are added 1 uL of a series of compound solutions and 2 uL of the kinase reaction solution, followed by mixing homogeneously, and incubating in the dark at room temperature. After in the dark for 30 min, 2 uL of the substrate reaction solution is added to allowed the total reaction volume to 5 uL. The reaction mixture is incubated in the dark at room temperature in the dark for another 60 min. An equal volume of 5 uL ADP-Glo reagent is then added to terminate the reaction. The resulting mixture is homogeneously mixed, and stands at room temperature for 40 min. Finally, 10 uL of the kinase detection reagent is added, stands at room temperature for 30 min, and then a value is read on Envision.
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 442.49, ALogP 5.45, HBA 4, HBD 1, PSA 71.11, QED 0.42
Compound (SMILES): C[C@@H]1CN(C2COC2)CCN1c1ccc(Nc2ncnc(-c3ccc(O[C@H]4CCN(C(=O)CO)C[C@H]4F)c(C#N)c3)n2)cc1
InChIKey: OYQSRSUSXBDIKK-LVPRMVSMSA-N
Target: Serine/threonine-protein kinase TBK1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9UHD2
Assay: TBK1 and IKKE Biological Assays: Enzymatic activity of IKKε and TBK1 was measured using a homogeneous time resolved fluorescence resonance energy transfer (TR-FRET) assay that monitors enzyme dependent phosphorylation of a biotinylated serine/threonine peptide substrate. An increase in the amount of phosphorylated peptide results in an increase in TR-FRET signal. TBK1 and IKKε were expressed and purified as full length recombinant proteins. Detection reagents for the assay were purchased from Cisbio. TBK1 and IKKε enzymes were assayed under initial rate conditions in the presence of 2×Km ATP (40-80 μM) and 1 μM peptide, hepes (pH 7), 0.1 mM orthovanadate, 0.02% NaN3, 0.01% BSA, 10 mM MgCl2, 0.01% (v/v) tritonX, 1 mM dithiothreitol, 0.5% (v/v) DMSO at the following concentrations for each enzyme: TBK1 at 2.5 nM and IKKε at 0.3 nM. After an assay reaction time of 240 minutes at 25° C., reactions were terminated with EDTA.Amount of phosphorylated peptide was determined by the addition of 125 nM streptavidin XL665 and europium cryptate labeled anti-phospho monoclonal antibody and the resulting TR-FRET signal was recorded on an Envision plate reader (Ex: 340 nm; Em: 615/665 nm; 100 μs delay and 200 μs read window). Data was normalized based on a positive (1 μM Staurosporine) and negative (DMSO) controls and IC50 values calculated from the fit of the dose-response curves to a four-parameter equation. All IC50 values represent geometric mean values of a minimum of four determinations.
Measurement: IC50 = 2000 nM (pChEMBL 5.699999809265137)
Descriptors: MW 602.67, ALogP 2.37, HBA 11, HBD 2, PSA 139.97, QED 0.39
Compound (SMILES): CC(C)c1cc(-c2nc3nc(C(=O)O)nc(N[C@H](C)C4CCC4)c3n2C[C@H]2CC[C@H](C)CC2)ncc1F
InChIKey: MOQAWKPXLOGNPA-KZNAEPCWSA-N
Target: E3 ubiquitin-protein ligase Mdm2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q00987
Assay: FRET Assay: An HDM2 FRET assay was developed to assess the compounds' inhibitory activity towards binding of p53 protein. A truncated version of HDM2 with residues 17 to 125 (containing p53 binding surface, Science (1994) 265, 346-355), with N-terminal His and Thioredoxin tag was generated in pET32a expression vector and expressed in E. coli strain BL21(DE3)Rosetta. Protein was purified using Ni-affinity chromatography, followed by size exclusion chromatography using Superdex 75 26/60 column. To assess inhibition of p53 binding to HDM2, a FITC labeled 8-mer peptide (sequence: Ac-Phe-Arg-Dpr-Ac6c-(6-Br)Trp-Glu-Glu-Leu-NH2; Anal Biochem. 2004 Aug. 1; 331(1):138-46) with strong affinity towards p53 binding pocket of HDM2 was used. The HDM2 assay buffer contained 1× Phosphate Buffered Saline (Invitrogen, Cat#14190), 0.01% BSA (Jackson ImmunoResearch, Cat#001-000-162), 0.01% Tween-20. In the 1× assay buffer recombinant HDM2 protein, peptide and Lumi4-Tb Cryptate-conjugate mouse anti-6×His antibody (cisbio, Cat# Tb61HISTLB) were added and transferred to ProxiPlate PLUS (PerkinElmer, Cat#6008269), containing compounds so that final DMSO concentration is 0.1%. Final concentrations of reagents in the assay wells are 0.5 nM HDM2, 0.25 nM anti HIS (Tb label) antibody and 3 nM peptide. After two hour incubation at room temperature in a humidified chamber plates were read on EnVision plate reader with the following settings: excitation: UV, 340 nM, two emission filters: 520 nm and 495 nm respectively. Ratio of em520/em495 was used to calculate % inhibition and to obtain IC50 with 4-parameter logistic equation.
Measurement: IC50 = 1.139 nM (pChEMBL 8.9399995803833)
Descriptors: MW 508.64, ALogP 6.28, HBA 7, HBD 2, PSA 105.82, QED 0.37
Compound (SMILES): CN1CCCc2c1nc(N1CCN(Cc3ncccc3O)CC1)[nH]c2=O
InChIKey: VFXBLJYICQUSGU-UHFFFAOYSA-N
Target: Poly [ADP-ribose] polymerase tankyrase-1 (SINGLE PROTEIN), Homo sapiens
UniProt: O95271
Assay: Biochemical Assay: The enzymatic activity of hTNKS1 and hTNKS2 is assessed using an enzyme-linked immunosorbent assay (ELISA) which detects poly ADP ribose incorporated into plate-bound Telomeric repeat binding factor 1 (TRF1) protein (NCBI, Accession number NP_059523.2 (SEQ ID NO: 1) in a 384-well format. Using recombinant hTNKS1 (NCBI, Accession number NP_003738.2 (SEQ ID NO: 2) or hTNKS2 (NCBI, Accession number NP_079511.1 (SEQ ID NO: 3) enzyme, this assay uses biotinylated NAD+ and measures its incorporation into recombinant hTRF1 using a streptavidin-horseradish peroxidase (HRP) conjugate and TMB peroxidase substrate to generate a colorimetric signal. Recombinant Flag-tagged hTRF1 protein is generated by expressing full length human TRF1 protein with an N-terminal Flag tag in E. coli. Recombinant Flag-hTNKS1 (with a change of Q83P) protein is generated by expressing full length human TNKS1 with an N-terminal Flag tag in Baculovirus according to the manufacturer's protocol of Bac-to-Bac Baculovirus Expression system (Invitrogen; See also Invitrogen User Manual, Version F, dated 4 Sep. 2010; and Invitrogen Instruction Manual dated 27 Feb. 2002). The Flag tags on hTRF1 and hTNK1 are used only for purification of the enzymes and are not otherwise involved in the ELISA assay.
Measurement: IC50 = 26.4 nM (pChEMBL 7.579999923706055)
Descriptors: MW 356.43, ALogP 0.58, HBA 7, HBD 2, PSA 88.59, QED 0.83
Compound (SMILES): Cn1cc(-c2cccc(C(=O)Nc3ccc4nn(CC(=O)N5CCC(N6CCCC6)CC5)cc4c3)n2)cn1
InChIKey: XKSIASNOIXBKCM-UHFFFAOYSA-N
Target: Interleukin-1 receptor-associated kinase 4 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9NWZ3
Assay: Kinase Assay: For the assay, 11 different concentrations in the range from 20 μM to 0.073 nM were prepared from a 2 mM solution of the test substance in DMSO. For the assay, 50 nl of the respective solution were pipetted into a black low-volume 384-well microtitre plate (Greiner Bio-One, Frickenhausen, Germany), 2 μl of a solution of Irak4 in assay buffer [50 mM HEPES pH 7.5, 5 mM MgCl2, 1.0 mM dithiothreitol, 30 μM activated sodium orthovanadate, 0.1% (w/v) of bovine gamma-globulin (BGG) 0.04% (v/v) nonidet-P40 (Sigma)] were added and the mixture was incubated for 15 min to allow prebinding of the substances to the enzyme prior to the kinase reaction. The kinase reaction was then started by addition of 3 μl of a solution of adenosine triphosphate (ATP, 1.67 mM=final concentration in 5 μl of assay volume: 1 μM) and peptide substrate (0.83 μM=final concentration in 5 μl assay volume: 0.5 μM) in assay buffer, and the resulting mixture was incubated at 22° C. for the reaction time of 45 min. The concentration of the Irak4 was adjusted to the respective activity of the enzyme and set such that the assay was carried out in the linear range. Typical concentrations were in the order of about 0.2 nM. The reaction was stopped by addition of 5 μl of a solution of TR-FRET detection reagents [0.1 μM streptavidin-XL665 (Cisbio Bioassays; France, catalogue No. 610SAXLG) and 1.5 nM anti-phosphoserin antibody [Merck Millipore, STK Antibody, catalogue No. 35-002] and 0.6 nM LANCE EU-W1024-labelled anti-mouse-IgG antibody (Perkin-Elmer, product No. AD0077, alternatively it is possible to use a terbium cryptate-labelled anti-mouse-IgG antibody from Cisbio Bioassays) in aqueous EDTA solution (100 mM EDTA, 0.4% [w/v] bovine serum albumin [BSA] in 25 mM HEPES pH 7.5).
Measurement: IC50 = 27 nM (pChEMBL 7.570000171661377)
Descriptors: MW 512.62, ALogP 3.17, HBA 8, HBD 1, PSA 101.18, QED 0.43
Compound (SMILES): CC(C)n1cnnc1-c1cccc(NC(=O)c2cc3cc(N4CCCCC4)ccc3cn2)n1
InChIKey: QENVECNTXGVDFD-UHFFFAOYSA-N
Target: Mitogen-activated protein kinase kinase kinase 5 (SINGLE PROTEIN), Homo sapiens
UniProt: Q99683
Assay: HTRF KinEASE Assay : ASK1 was purchased from Thermofisher (Catalogue # PV4011), ATP was purchased from Sigma (Catalogue # A7699), HTRF® KinEASE Assay System was obtained from Cisbio (Bedford, Mass.). ½ Area plate was purchased from Perkin Elmer (Catalogue # #6005560). HTRF® KinEASE-STK is a generic method for measuring serine/threonine kinase activities using a time-resolved fluorescence resonance energy transfer (TR-FRET) immunoassay. The IC50 value for each compound was determined in the presence of compound (various concentration from 0 to 10 μM) and a fixed amount of ATP and peptide substrates. The test compound, 1 uM STK3 peptide substrate, and 5 nM of ASK1 kinase are incubated with kinase reaction buffer containing 50 mM HEPES pH 7.5, 0.01% BRIJ-35, 10 mM MgCl2, and 1 mM EGTA for 30 minutes. 100 uM ATP is added to start kinase reaction and incubated for 3 hours. The STK3-antibody labeled with Eu3+-Cryptate and 125 nM streptavidin-XL665 are mixed in a single addition with stop reagents provided by the Cisbio kit used to stop the kinase reaction. Fluorescence is detected using an Envision Multilabeled 2014 reader from PerkinElmer. The Fluorescence is measured at 615 nm (Cryptate) and 665 nm (XL665) and a ratio of 665 nm/615 nm is calculated for each well. The resulting TR-FRET is proportional to the phosphorylation level. Staurosporine was used as the positive control. IC50 was determined by XLfit 5.3.
Measurement: IC50 = 5.5 nM (pChEMBL 8.260000228881836)
Descriptors: MW 441.54, ALogP 4.71, HBA 7, HBD 1, PSA 88.83, QED 0.48
Compound (SMILES): C=CC(=O)NC1CCC=C(c2ncnc3[nH]cc(-c4ccsc4)c23)C1
InChIKey: AXWBIEGMJCJHCS-UHFFFAOYSA-N
Target: Tyrosine-protein kinase JAK1 (SINGLE PROTEIN), Homo sapiens
UniProt: P23458
Assay: Kinase-Inhibiting Activity Assay: JAK 1: Among materials for the measurement of this inhibiting activity, a substrate peptide and a kinase protein were acquired as follows. As such a substrate peptide, a substrate peptide for QSS Assist JAK1-MSA assay kit (Carna Biosciences, Inc.) was purchased. As such a kinase protein, a purified recombinant human JAK1 protein (Carna Biosciences, Inc.) was purchased.The method for measuring the inhibiting activity is as follows. First, the compounds of the present invention were each dissolved in dimethyl sulfoxide (DMSO), and a serial dilution was then prepared using DMSO. Subsequently, a serial dilution solution of the compound (the final concentration of DMSO upon a kinase reaction: 5.0%) or DMSO (final concentration: 5.0%) was mixed with a solution comprising the substrate peptide (final concentration: 1 μM), magnesium chloride (final concentration: 5 mM) and ATP (final concentration: 75 μM) in a buffer for kinase reaction (20 mM HEPES (pH 7.5), 2 mM dithiothreitol and 0.01% Triton X-100). Thereafter, a JAK1 protein was further added to the mixed solution, and the obtained mixture was then incubated at 25° C. for 120 minutes to carry out a kinase reaction. To the reaction solution, EDTA was added to a final concentration of 30 mM, so as to terminate the reaction. Finally, using LabChip EZ Reader II (Perkin Elmer Corp.), an unphosphorylated substrate peptide (S) and a phosphorylated peptide (P) were subjected to microchannel capillary electrophoresis, so that the two peptides were separated from each other and were then detected.
Measurement: IC50 = 1500 nM (pChEMBL 5.820000171661377)
Descriptors: MW 350.45, ALogP 3.92, HBA 4, HBD 2, PSA 70.67, QED 0.7
Compound (SMILES): Fc1ccc(-c2nc(Nc3cc(Cl)ncc3F)c3cccn3n2)nc1
InChIKey: NDCKNIOZEAUIGO-UHFFFAOYSA-N
Target: TGF-beta receptor type-1 (SINGLE PROTEIN), Homo sapiens
UniProt: P36897
Assay: Biological Assays: Assays are conducted in 1536-well plates and 2 mL reactions are prepared from addition of HIS-TGFβR1 T204D or HIS-TGFβR2 WT, anti-HIS detection antibody, a labeled small molecule probe (Kd=<100 nM; koff=<0.001 s−1.) and test compounds in assay buffer (20 mM HEPES pH 7.4, 10 mM MgCl2, 0.015% Brij35, 4 mM DTT, and 0.05 mg/ml BSA). The reaction is incubated for 1 hour at room temperature and the HTRF signal was measured on an Envision plate reader (Ex: 340 nm; Em: 520 nm/495 nm). Inhibition data were calculated by comparison to no enzyme control reactions for 100% inhibition and vehicle-only reactions for 0% inhibition. The final concentration of reagents in the assay are 1 nM HIS-TGFβR1 T204D or HIS-TGFβR2 WT, 0.2 nM anti-HIS detection antibody, labeled small molecule probe (at Kd) and 0.5% DMSO. Dose response curves were generated to determine the concentration required inhibiting 50% of kinase activity (IC50). Compounds were dissolved at 10 mM in dimethylsulfoxide (DMSO) and evaluated at eleven concentrations. IC50 values were derived by non-linear regression analysis.
Measurement: IC50 = 3000 nM (pChEMBL 5.519999980926514)
Descriptors: MW 358.74, ALogP 3.86, HBA 6, HBD 1, PSA 68, QED 0.56
Compound (SMILES): Cc1cc(F)ccc1CC(=O)Nc1ccc2c(c1)OCc1cnccc1-2
InChIKey: ORXMBZPQYXDQQX-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 = 11.1 nM (pChEMBL 7.960000038146973)
Descriptors: MW 348.38, ALogP 4.27, HBA 3, HBD 1, PSA 51.22, QED 0.77
Compound (SMILES): Cc1nc2cc(C(F)(F)F)cc(C#N)c2c(=O)n1CCOc1ccc(Cl)cc1-c1ccnc2c1CCC2C(=O)O
InChIKey: NCXOWLKMPXJCJB-UHFFFAOYSA-N
Target: Eukaryotic translation initiation factor 4E-binding protein 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q13541
Assay: Fluorescence Polarization Assay: eIF4E protein was pre-incubated with EDA-m7GDP-ATTO-550 in FPB at 2xconcentrations for 5 minutes prior to the addition of compounds. Compounds (100x) were prepared using 3-fold serial dilutions in 100% DMSO, and subsequently diluted 1:50 in FPB to produce 2x stocks. 50 ul of 2x eIF4E/EDA-m7GDP-ATTO-550 were transferred to the wells of a 96-well half-area black flat bottom polystyrene plate. 50 ul of 2x test compound were added, and binding reactions were allowed to equilibrate for 30 minutes at room temperature while protected from light. The fluorescent polarization signal was detected using a Victor 2 multi-label counter (Perkin Elmer) and the concentration necessary to achieve inhibition of eIF4E/EDA-m7GDP-ATTO-550 binding by 50% (IC50) was calculated using data from an 8-point compound dilution series.
Measurement: IC50 = 1060 nM (pChEMBL 5.96999979019165)
Descriptors: MW 568.94, ALogP 5.5, HBA 7, HBD 1, PSA 118.1, QED 0.33
Compound (SMILES): CCn1c(C(F)F)nc2sc(N3CCC[C@@H]3C(=O)NCc3cc(C)nn3C)nc2c1=O
InChIKey: ZXXUKVVHCGWSQT-GFCCVEGCSA-N
Target: Kynurenine/alpha-aminoadipate aminotransferase, mitochondrial (SINGLE PROTEIN), Homo sapiens
UniProt: Q8N5Z0
Assay: KAT-II inhibitory assay: The inhibitory action of the test compound on human recombinant KAT-II was determined by the following method.To a reaction mixture (45 μL) containing 3.0 μmol/L kynurenine, 10 μmol/L pyridoxal phosphate, 2.0 ng/μL human recombinant KAT-II, and 150 mmol/L tris(hydroxymethyl)aminomethane-acetate buffer (pH 8.0) was added a 10% dimethyl sulfoxide solution (5 μL) of each test compound prepared, and the mixture was reacted at 37° C. for 1 hr. After the reaction, 50% trichloroacetic acid (5 μL) was added to terminate the reaction.The resultant kynurenic acid was quantified as follows by high performance liquid chromatography. An enzyme reaction mixture was separated by an octadecylsilane reversed-phase column (SC-50DS, Eicom Corporation; mobile phase: 250 mmol/L zinc acetate, 50 mmol/L sodium acetate, and 5.0% acetonitrile (pH 6.2)) incubated at 30° C., and kynurenic acid was quantified using a fluorescence detector (RF-20Axs, Shimadzu Corporation) at excitation wavelength 354 nm, detection wavelength 460 nm. The analytical curve was drawn every time by an external standard method. Each test compound was tested by dual measurement at each concentration. The kynurenic acid level in the presence of a test compound at each concentration was converted into % relative to kynurenic acid resulting from a reaction with an enzyme alone as 100%, and the obtained values were fitted to S-curve to determine IC50.
Measurement: IC50 = 1100 nM (pChEMBL 5.960000038146973)
Descriptors: MW 451.5, ALogP 2.14, HBA 9, HBD 1, PSA 97.94, QED 0.62
Compound (SMILES): COc1cccc(F)c1-c1cc2c(N3CCc4ccc(C#N)cc4C3)n[nH]c2cn1
InChIKey: SVESIFPTQILSCN-UHFFFAOYSA-N
Target: Mitogen-activated protein kinase kinase kinase kinase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q92918
Assay: HPK1 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).
Measurement: Ki < 100 nM (pChEMBL 7)
Descriptors: MW 399.43, ALogP 4.21, HBA 5, HBD 1, PSA 77.83, QED 0.56
Compound (SMILES): Cc1noc(C)c1-c1cnc2c(c1)c(Sc1ccccc1)cn2C1COC1
InChIKey: FEGDOHGXEAVQFV-UHFFFAOYSA-N
Target: Bromodomain-containing protein 4 (SINGLE PROTEIN), Homo sapiens
UniProt: O60885
Assay: Alphascreen Binding Assay: Binding of compounds of formula (I) with bromodomain 2, 3 4 was assessed using Alphascreen binding assay. The inhibition of the interaction between bromodomain and its acetylated target protein (Filippakopoulos P et al. 2012) was measured quantitatively using recombinant BRD proteins, an acetylated Histone 4 peptide and AlphaScreen technology. In absence of inhibition the BRD protein bound to AlphaScreen nickel chelate acceptor beads can interact with the acetylated Histone 4 peptide which is immobilized by the AlphaScreen Streptavidin coated beads. This interaction brings donor and acceptor beads in proximity. The close proximity allows the singlet oxygen produced by laser excitation of the donor beads to reach the acceptor beads and generate a luminescence signal. BRD inhibitors result in a decrease in the proximity signal through an inhibition of the BRD-acetylated peptide interaction.Recombinant human bromodomains containing the N-terminal bromodomain (BRD2-BD1 (71-194), BRD3-BD1 (24-144) and BRD4-BD1 (44-164)) or dual bromodomains (BRD4-BD12 (1-477), BRD4-BD12 (1-472)) were prepared and purified as described in protein expression and purification session.
Measurement: IC50 = 5500 nM (pChEMBL 5.260000228881836)
Descriptors: MW 377.47, ALogP 5.03, HBA 6, HBD 0, PSA 53.08, QED 0.5
Compound (SMILES): N#Cc1ccc(CN2CC[C@@H](NC(=O)c3ccc(C(=O)N4CCN(Cc5ccc(F)cc5)CC4)cn3)[C@@H](F)C2)cc1
InChIKey: GGMOGBNZFSCMDK-WUFINQPMSA-N
Target: 5'-AMP-activated protein kinase catalytic subunit alpha-2 (SINGLE PROTEIN), Homo sapiens
UniProt: P54646
Assay: Enzyme-Linked Immunosorbent Assay: Compounds were assayed for their ability to activate AMPK using an enzyme-linked immunosorbent assay. Reagents and procedures for measuring AMPK activation are well known and kits for AMPK activation assays are commercially available.
Measurement: EC50 < 500 nM (pChEMBL 6.301000118255615)
Descriptors: MW 558.63, ALogP 3.39, HBA 6, HBD 1, PSA 92.57, QED 0.48
Compound (SMILES): Cc1c(C(=O)C(=O)NC2(c3nncs3)CC2)c2n(c1C(=O)Nc1ccc(F)c(F)c1)[C@@H]1C[C@@H]1C2
InChIKey: VSYNYKWQZPYCSY-IAQYHMDHSA-N
Target: Hepatic sodium/bile acid cotransporter (SINGLE PROTEIN), Homo sapiens
UniProt: Q14973
Assay: HBV DNA Quantification Assay: A HepG2 cell line overexpressing the HBV virus attachment receptor sodium-taurocholate cotransporting polypeptide (NTCP) was grown to confluency in DMEM growth medium, Dulbecco's Modified Eagle Medium without sodium pyruvate (Life Technologies, Rockville, Md.) supplemented with 10% FBS (Thermo Scientific, Waltham, Md.), 1% penicillin/streptomycin (Life Technologies, Rockville, Md.) and 2 mM L-glutamine (Life Technologies, Rockville, Md.) in T175 flasks. Cells were infected with HBV AD38 viral particles (Texcell, Frederick, USA) at 4000 genome equivalents per cell. After allowing viral infection to take place for 4 days, the infected cells were harvested from the flasks by trypsinization, washed twice with OptiMEM (Life Technologies, Rockville, Md.) and re-suspended in DMEM containing 2% FBS and 1% DMSO at a density of 0.25E6 cells/ml. Infected cells were seeded on 384 well collagen coated plates (Greiner, Austria) at a density of 20,000 cells/well containing serially diluted compounds of the present disclosure or DMSO (0.5%) in a final volume of 80 μl. The assay plates were incubated for a period of 5 days and the antiviral activity of the test compounds were assayed by detecting the presence of HBV DNA in the culture supernatant using the QuantiGene 2.0 nucleic acid quantification kit (Affymetrix, Santa Clara, Calif.).The culture supernatant was harvested and treated with lysis buffer containing Proteinase K (Affymetrix, Santa Clara, Calif.). The supernatant was incubated with HBV viral DNA specific probes (Affymetrix, Santa Clara, Calif.) for 30 minutes at 55° C. This was followed by addition of 0.2M NaOH for 30 minutes at room temperature to denature the DNA, followed by addition of Neutralization buffer (Affymetrix, Santa Clara, Calif.). The resulting lysed and neutralized supernatant was then added to QuantiGene 2.0 384 well plates coated with capture oligonucleotides and incubated overnight at 55° C. The HBV specific probe set consists of Capture Extender oligonucleotides (CE's) and blocking probes. Following the overnight incubation, the wells were incubated for one hour sequentially with a Pre-Amplifier, Amplifier and Labeled probes conjugated to alkaline phosphatase with a wash step between incubations. After the final wash step, the alkaline phosphatase substrate (Luminol APS5) was added and the resulting luminescence signal was read in an EnVision Multilabel Plate Reader (PerkinElmer, Santa Clara, Calif.). The EC50 values were calculated from the fit of the doseresponse curves to a four-parameter equation. All EC50 values represent geometric mean values of a minimum of four determinations.
Measurement: EC50 = 12.3 nM (pChEMBL 7.909999847412109)
Descriptors: MW 483.5, ALogP 3.28, HBA 7, HBD 2, PSA 105.98, QED 0.41
Compound (SMILES): O=C(NC(CCO[C@H]1C[C@H](CCc2ccc3c(n2)NCCC3)C1)C(=O)O)c1cccc(-c2cncs2)c1
InChIKey: JKOWFVGPFYHJES-SCRAKJMHSA-N
Target: Integrin alpha-V/beta-6 (PROTEIN COMPLEX), Homo sapiens
UniProt: P06756
Assay: Binding Assay: The biochemical potency of compounds was determined using a proximity-based assay (ALPHASCREEN®, Perkin Elmer, Waltham, Mass.) as described previously (Ullman E F et al., Luminescent oxygen channeling immunoassay: Measurement of particle binding kinetics by chemiluminescence. Proc. Natl. Acad. Sci. USA, Vol. 91, pp. 5426-5430, June 1994). To gauge the potency of inhibitors of binding to human integrin αvβ6, inhibitor compounds and integrin were incubated together with TGF-b1 LAP and biotinylated anti-LAP antibody plus acceptor and donor beads, following the manufacture's recommendations. The donor beads were coated with streptavidin. The acceptor beads had a nitrilotriacetic acid Ni chelator, for binding to a 6×His Tag on human integrin αvβ6. All incubations occurred at room temperatures in 50 mM Tris-HCl, pH 7.5, 0.1% BSA supplemented with 1 mM each CaCl2 and MgCl2.The order of reagent addition was as follows:1. Alpha-v-beta-6 integrin, test inhibitor compound, LAP, biotinylated anti-LAP antibody and acceptor beads were all added together.2. After 2 hours, donor beads were added. After another 30 minute incubation, samples were then read.Integrin binding was evaluated by exciting donor beads at 680 nm, and measuring the fluorescent signal produced, between 520-620 nm, using a Biotek Instruments (Winooski, Vt., USA) SynergyNeo2 multimode plate reader.
Measurement: IC50 = 150 nM (pChEMBL 6.820000171661377)
Descriptors: MW 520.66, ALogP 4.56, HBA 7, HBD 3, PSA 113.44, QED 0.34
Compound (SMILES): COc1cc(OC)cc(N2Cc3cnc4[nH]c(C(=O)N5CCN(C)CC5)cc4c3N(C)C2=O)c1
InChIKey: KVWDJGIRKNYBJM-UHFFFAOYSA-N
Target: Fibroblast growth factor receptor 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P11362
Assay: Enzymatic Assay: The inhibitor potency of the exemplified compounds was measured in an enzyme assay that measures peptide phosphorylation using FRET measurements to detect product formation. Inhibitors were serially diluted in DMSO and a volume of 0.5 μL was transferred to the wells of a 384-well plate. For FGFR3, a 10 μL volume of FGFR3 enzyme (Millipore) diluted in assay buffer (50 mM HEPES, 10 mM MgCl2, 1 mM EGTA, 0.01% Tween-20, 5 mM DTT, pH 7.5) was added to the plate and pre-incubated for 5-10 minutes. Appropriate controls (enzyme blank and enzyme with no inhibitor) were included on the plate. The assay was initiated by the addition of a 10 μL solution containing biotinylated EQEDEPEGDYFEWLE peptide substrate (SEQ ID NO: 1) and ATP (final concentrations of 500 nM and 140 μM respectively) in assay buffer to the wells. The plate was incubated at 25° C. for 1 hr. The reactions were ended with the addition of 10 μL/well of quench solution (50 mM Tris, 150 mM NaCl, 0.5 mg/mL BSA, pH 7.8; 30 mM EDTA with Perkin Elmer Lance Reagents at 3.75 nM Eu-antibody PY20 and 180 nM APC-Streptavidin). The plate was allowed to equilibrate for 1 hr before scanning the wells on a PheraStar plate reader (BMG Labtech).
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 464.53, ALogP 2.54, HBA 6, HBD 1, PSA 94.24, QED 0.64
Compound (SMILES): Cc1cccc2c(=O)n(C)nc(N[C@H](C)[C@@H](c3ccccc3)N(C)C)c12
InChIKey: QTMVVXTXPFBUQU-BEFAXECRSA-N
Target: Histone acetyltransferase KAT2B (SINGLE PROTEIN), Homo sapiens
UniProt: Q92831
Assay: PCAF AlphaLisa Binding Assay: His/Flag epitope tagged PCAF719-832 bromodomain was cloned, expressed and purified to homogeneity in-house. PCAF bromodomain binding and inhibition of the compounds disclosed herein was assessed by monitoring the engagement of biotinylated small molecule ligand (known to bind to the PCAF bromodomain) with the target using the AlphaLisa technology (Perkin-Elmer). Specifically, in a 384 well ProxiPlate PCAF bromodomain (225 nM final) was combined with the biotinylated small molecule ligand (6 nM final) in 50 mM HEPES (pH 7.5), 75 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 15 minute incubation at room temperature AlphaLisa streptavidin acceptor beads and AlphaLisa anti-histidine donor beads were added to a final concentration of 12.5 μg/mL each. After 90 minutes of equilibration plates were read on an Envision instrument and IC50s calculated using a four parameter non-linear curve fit.
Measurement: IC50 = 1800 nM (pChEMBL 5.75)
Descriptors: MW 350.47, ALogP 3.35, HBA 5, HBD 1, PSA 50.16, QED 0.77
Compound (SMILES): CN1CCN(C(=O)[C@H]2CC[C@H](C(=O)Nc3cc4cc(-c5cnn(C)c5)ccc4cn3)CC2)CC1
InChIKey: LYTBFSXOMIMCTF-WGSAOQKQSA-N
Target: Proto-oncogene Wnt-1 (SINGLE PROTEIN), Homo sapiens
UniProt: P04628
Assay: The screening assay for Wnt activity: The screening assay for Wnt activity is described as follows. Reporter cell lines can be generated by stably transducing cancer cell lines (e.g., colon cancer) or primary cells (e.g., IEC-6 intestinal cells) with a lentiviral construct that includes a Wnt-responsive promoter driving expression of the firefly luciferase gene.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 = 2949 nM (pChEMBL 5.53000020980835)
Descriptors: MW 460.58, ALogP 3.15, HBA 6, HBD 1, PSA 83.36, QED 0.65
Compound (SMILES): CC(=O)c1nn(CC(=O)N2[C@H](C(=O)Nc3nc(Br)ccc3C)C[C@@]3(C)C[C@@H]23)c2cc(S(C)(=N)=O)ccc12
InChIKey: ZXUKQDKIZFDZNO-HTGVHPAMSA-N
Target: Complement factor D (SINGLE PROTEIN), Homo sapiens
UniProt: P00746
Assay: Human Factor D Assay: Human Factor D (purified from human serum, Complement Technology, Inc.) at 80 nM final concentration is incubated with test compound at various concentrations for 5 min at room temperature in 50 mM Tris, 1M NaCl, pH 7.5. A synthetic substrate Z-L-Lys-SBzl and DTNB (Ellman's reagent) are added to final concentrations of 100 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 < 1000 nM (pChEMBL 6)
Descriptors: MW 587.5, ALogP 3.76, HBA 8, HBD 2, PSA 138.11, QED 0.33
Compound (SMILES): COc1cn(Cc2ccccc2)c(Nc2ccccc2Cc2ccccc2)nc1=O
InChIKey: MBRXHQRELPSWKY-UHFFFAOYSA-N
Target: P2X purinoceptor 7 (SINGLE PROTEIN), Homo sapiens
UniProt: Q99572
Assay: Evaluation of a Human P2X7 Receptor Inhibitory Activity: Stably expressing cell line (1321N1 cell transfected with the human P2X7 receptor gene (GenBank accession number NM_002562.5 including T606C and G952A SNP)) was used. The cells were seeded in a 384-well microtiter plate at a concentration of 8000 cells/well and cultured in the medium (10% fetal bovine serum, 25 mM HEPES, 1% penicillin and streptomycin in DMEM) for one day at 37° C. under 5% carbon dioxide atmosphere. After replacing with 20 μL of the HBSS buffer (20 mM HEPES, 55.6 mM D-glucose, 1×HBSS(−), pH7.4-7.5), 15 μL of 17.3 μM Yo-Pro solution in the HBSS buffer was added. The plate was placed in high-throughput cellular screening system FLIPR TETRA (Molecullar Devices, LLC.) and 15 μL of 130 μM BzATP solution in the HBSS buffer was added. Measurement of fluorescence intensity by FLIPR TETRA was started. After eight minutes, 15 μL of DMSO solutions containing different concentrations of the compound of the present invention as prepared by dilution with the HBSS buffer were dispensed to each well through the built-in automatic dispenser, and the measurement of fluorescence intensity was continued for 20 minutes. The maximum fluorescence intensity without the compound of the present invention is calculated as 0% inhibition and the maximum fluorescence intensity when the reference compound was added is calculated as 100% inhibition. Changing values of fluorescence intensity by the compound of the present invention were calculated by difference between maximum and minimum fluorescence intensity for 20 minutes. IC50 was calculated using logistic approximation.
Measurement: IC50 = 55 nM (pChEMBL 7.260000228881836)
Descriptors: MW 397.48, ALogP 4.63, HBA 5, HBD 1, PSA 56.15, QED 0.49
Compound (SMILES): COC(=O)N[C@H]1CCC[C@@H]1C(C#N)(c1ccccc1)C1CCN(CC2(C)CN(c3ccc(S(=O)(=O)c4cnn(C)c4)cc3)C2)CC1
InChIKey: AEGAGTFLYPDDPK-SVYHFPLZSA-N
Target: Menin (SINGLE PROTEIN), Homo sapiens
UniProt: O00255
Assay: Fluorescence Polarization (FP) Competitive Binding Assay: A fluorescence polarization (FP) competitive binding assay was used to determine the binding affinities of representative menin inhibitors. A FAM labeled fluorescent probe was designed and synthesized based on a MLL1 peptide (FAM-MM2). Equilibrium dissociation constant (Kd) value of FAM-MM2 to menin protein was determined from protein saturation experiments by monitoring the total fluorescence polarization of mixtures composed with the fluorescent probe at a fixed concentration and the protein with increasing concentrations up to full saturation. Serial dilutions of the protein were mixed with FAM-MM2 to a final volume of 200 μl in the assay buffer (PBS with 0.02% Bovine γ-Globulin and 4% DMSO. 0.01% Triton X-100 was added right before assays). Final FAM-MM2 concentration was 2 nM. Plates were incubated at room temperature for 30 minutes with gentle shaking to assure equilibrium. FP values in millipolarization units (mP) were measured using the Infinite M-1000 plate reader (Tecan U.S., Research Triangle Park, N.C.) in Microfluor 1 96-well, black, v-bottom plates (Thermo Scientific, Waltham, Mash.) at an excitation wavelength of 485 nm and an emission wavelength of 530 nm. Kd value of FAM-MM2, which was calculated by fitting the sigmoidal dose-dependent FP increases as a function of protein concentrations using Graphpad Prism 6.0 software (Graphpad Software, San Diego, Calif.), is determined as 1.4 nM.
Measurement: IC50 = 167 nM (pChEMBL 6.78000020980835)
Descriptors: MW 644.84, ALogP 4.78, HBA 9, HBD 1, PSA 120.56, QED 0.35
Compound (SMILES): Cc1ccc(CO[C@H]2CCC[C@@H]2NC(=O)c2cc(-c3ccc(C=O)cc3)cnc2N)cc1C
InChIKey: ZOHCQNHQJRAISM-DQEYMECFSA-N
Target: Tyrosine-protein kinase Mer (SINGLE PROTEIN), Homo sapiens
UniProt: Q12866
Assay: TBD: TBD
Measurement: IC50 = 55 nM (pChEMBL 7.260000228881836)
Descriptors: MW 443.55, ALogP 4.63, HBA 5, HBD 2, PSA 94.31, QED 0.52
Compound (SMILES): C[C@@H]1CN(c2ccc(F)cc2)C(=O)c2cc(CNc3c(F)cc(F)cc3F)nn21
InChIKey: TYQYNDLQAXSLCR-LLVKDONJSA-N
Target: Metabotropic glutamate receptor 5 (SINGLE PROTEIN), Rattus norvegicus
UniProt: P31424
Assay: mGlu3 Ca2+ Flux Assay: Gα15/TREx cells stably expressing rat mGlu3 were plated in black-walled, clear-bottomed, poly-D-lysine coated 384-well plates in 20 μL of assay medium (DMEM containing 10% dialyzed FBS, 20 mM HEPES, 25 ng/mL tetracycline, 100 units/mL penicillin/streptomycin plus 250 ng/mL Fungizone, and 1 mM sodium pyruvate) at a density of 15K cells/well. The cells were grown overnight at 37° C. in the presence of 5% CO2. The next day, medium was removed and the cells incubated with 20 μL of 2.3 μM Fluo-4, AM prepared as a 2.3 mM stock in DMSO and mixed in a 1:1 ratio with 10% (w/v) pluronic acid F-127 and diluted in assay buffer (Hank's balanced salt solution, 20 mM HEPES, and 2.5 mM probenecid) for 60 minutes at room temperature. Dye was removed, 20 μL of assay buffer was added, and the plate was incubated for 10 minutes at room temperature.
Measurement: IC50 = 10000 nM (pChEMBL 5)
Descriptors: MW 404.37, ALogP 4.27, HBA 4, HBD 1, PSA 50.16, QED 0.66
Compound (SMILES): CCc1cccc(CC)c1-n1nc2c(c1-c1cc(F)c(NC(N)=O)cc1F)CN(C(c1ccccc1)C(C)(C)O)CC2
InChIKey: MNHYPNSQVAVTDG-UHFFFAOYSA-N
Target: C5a anaphylatoxin chemotactic receptor 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P21730
Assay: chemotaxis assay: Optionally, compounds may be further assayed for their ability to inhibit chemotaxis in cells. Chemotaxis assays are performed using 5 μm pore polycarbonate, polyvinylpyrrolidone-coated filters in 96-well chemotaxis chambers (Neuroprobe; Gaithersburg, Md.) using chemotaxis buffer (Hank's balanced salt solution (HBSS) and 1% FBS). C5aR ligands (i.e., C5a, R&D Systems; Minneapolis, Minn.) are use to evaluate compound mediated inhibition of C5aR mediated migration. Other chemokines (i.e., SDF-1α; R&D Systems; Minneapolis, Minn.) are used as specificity controls. The lower chamber is loaded with 29 μl of chemokine (i.e., 0.03 nM C5a) and varying amounts of compound; the top chamber contains 100,000 U937 or neutrophil cells in 20 μl. The chambers are incubated 1.5 hours at 37° C., and the number of cells in the lower chamber quantified either by direct cell counts in five high powered fields per well or by the CyQuant assay (Molecular Probes), a fluorescent dye method that measures nucleic acid content and microscopic observation.
Measurement: IC50 < 5 nM (pChEMBL 8.300999641418457)
Descriptors: MW 573.69, ALogP 6.3, HBA 5, HBD 3, PSA 96.41, QED 0.23