lovingscience/chembl-v37-raw-activities huggingface dataset:
Compound (SMILES): CCn1cnc(-c2ccc(F)cc2)c1-c1ccc2ncc(C(N)=O)n2n1
InChIKey: FOWRMRUKQMVYKL-UHFFFAOYSA-N
Target: TGF-beta receptor type-1 (SINGLE PROTEIN), Homo sapiens
UniProt: P36897
Assay: Biological Assay: Assays for the compounds reported below were conducted in 1536-well plates and 2 mL reactions are prepared from addition of HIS-TGF-βR1 T204D or HIS-TGF-βR2 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-TGF-βR1 T204D or HIS-TGF-βR2 WT, 0.2 nM anti-HIS detection antibody, labeled small molecule prode (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.
Measurement: IC50 = 0.7 nM (pChEMBL 9.149999618530273)
Descriptors: MW 350.36, ALogP 2.52, HBA 6, HBD 1, PSA 91.1, QED 0.61
Compound (SMILES): COc1cc2nn(CC(=O)N3CCOCC3)cc2cc1NC(=O)c1cccc(C(O)C(F)(F)F)n1
InChIKey: UZYWWOWQGPMEIC-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 = 9 nM (pChEMBL 8.050000190734863)
Descriptors: MW 493.44, ALogP 2.15, HBA 8, HBD 2, PSA 118.81, QED 0.54
Compound (SMILES): O=C(NCCN1CCCC1)c1cnccc1Nc1nc(-c2cccc(C(F)(F)F)n2)nn2cccc12
InChIKey: UUGFFSMCHXZYGT-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 = 17 nM (pChEMBL 7.769999980926514)
Descriptors: MW 496.5, ALogP 3.77, HBA 8, HBD 2, PSA 100.34, QED 0.4
Compound (SMILES): O=C(Cc1ccc(-c2cnc3cc4c(cc3n2)OCCO4)cc1F)Nc1cc(C2(C(F)(F)F)CC2)on1
InChIKey: YQHICSNVABYXJV-UHFFFAOYSA-N
Target: Receptor-type tyrosine-protein kinase FLT3 (SINGLE PROTEIN), Homo sapiens
UniProt: P36888
Assay: Competition Binding Assay: Competition binding assays used herein were developed, validated and performed as described in Fabian et al., Nature Biotechnology 2005, 23, 329-336. Kinases were produced as fusions to T7 phage (See, Fabian et al. or WO04/015142) or alternatively, the kinases were expressed in HEK-293 cells and subsequently tagged with DNA for PCR detection (See, WO08/005,310). For the binding assays, streptavidin-coated magnetic beads were treated with biotinylated affinity ligands for 30 min at room temperature to generate affinity resins. The liganded beads were blocked with excess biotin and washed with blocking buffer (SeaBlock (Pierce), 1% BSA, 0.05% Tween 20, 1 mM DTT) to remove unbound ligand and to reduce non-specific binding. Binding reactions were assembled by combining kinase, liganded affinity beads, and test compounds in 1× binding buffer (20% SeaBlock, 0.17×PBS, 0.05% Tween 20, 6 mM DTT). Test compounds were prepared as 100× stocks in DMSO and diluted into the aqueous environment. Kds were determined using an eleven point threefold serial dilutions. DMSO or control compounds were was added to control assays lacking a test compound.
Measurement: Kd < 5 nM (pChEMBL 8.300999641418457)
Descriptors: MW 514.44, ALogP 4.97, HBA 7, HBD 1, PSA 99.37, QED 0.38
Compound (SMILES): CC(C)NC(=O)C1C2CN(C(=O)c3cc(C(C)C)[nH]n3)CC21
InChIKey: IXZBELFRWCXTJD-UHFFFAOYSA-N
Target: Lysine-specific demethylase 5A (SINGLE PROTEIN), Homo sapiens
UniProt: P29375
Assay: Biochemical KDM5A Inhibition Assay: Using 384-well white Greiner784075 (Greiner), representative compounds were characterized for their inhibition of KDM5A using HTRF® technology (Cisbio Bioassays). Briefly, the test compounds, reference compounds, and DMSO control (typical compound concentration range 1 μM-10 M, final assay concentration (FAC*) of DMSO 0.5%) were added to 384-well plate by the Echo® acoustic dispensing platform (Labcyte). Five (5) 1l of KDM5A protein (produced by the method described in Nat Chem Biol 12, 531-538 (2016)). (5-20 nM FAC*) in assay buffer (50 mM MES, 50 mM NaCl, 1 mM TCEP, 0.01% v/v Tween 20, 0.03% BSA, pH 6.5) was added to wells and plates were incubated for 10-20 minutes at 25 degrees celsius. Then, 5 μl of alpha-ketoglutaric acid (100 μM FAC*), biotin-labelled H3K4-Me3 substrate (Anaspec Cat #AS-64357-1; 200 nM FAC*), Fe(II)SO4 (100 μM FAC*) and ascorbic acid (2 mM FAC*) in assay buffer was added to wells and plates were incubated for 20 minutes at room temperature. The reaction was stopped with the addition of 10 μl of anti-H3K4-Me2-Eu(K) (Cisbio Bioassays Cat #61KA2KAH; 0.75 nM FAC**)+Streptavidin XL665 (Cisbio Bioassays Cat #610SAXLB; 25 nM FAC**) in HTRF detection buffer (Cisbio Bioassays Cat #62SDBRDF). The mixture was incubated for 30 minutes at room temperature before 340 nm excitation and measurement of dual emission at 620 nm and 665 run. *FAC: final assay concentration calculated based on 10 μl of the assay buffer**FAC: final assay concentration calculated based on 20 μl of the detection buffer.
Measurement: IC50 = 50 nM (pChEMBL 7.300000190734863)
Descriptors: MW 304.39, ALogP 1.38, HBA 3, HBD 2, PSA 78.09, QED 0.88
Compound (SMILES): Cc1[nH]c(=O)c(CN2CCc3c(Cl)cc(N4CC5(CCOCC5)C4)c(Cl)c3C2=O)c2occc12
InChIKey: XLWCANCNVVSLHY-UHFFFAOYSA-N
Target: Histone-lysine N-methyltransferase EZH2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q15910
Assay: EZH2 enzymatic activity: Ten-point dose-response curves for a subset of compounds shown in Table 1 were determined using a Transcreener® EPIGEN Methyltransferase HTS assay using purified EZH2 enzyme (BellBrook Labs). In this assay, the S-adenosylhomocysteine (SAH) generated by the transfer of the methyl group from S-adenosyl methionine (SAM) to purified histone 3.3 protein by EZH2 is enzymatically converted to AMP, which is detected using a fluorescence polarization readout.Briefly, compounds of the present invention were solubilized in DMSO and a series of 10, three-fold serial dilutions were made for each compound in 15% DMSO. The initial starting concentration for the serial dilutions of each compound was 1.0 μM. Control samples lacking compound, EZH2 enzyme or various reaction components also were prepared and processed in parallel with compound test samples. A panel of dilutions of SAH/SAM also was prepared as a standard curve to ensure the linearity of the assay.An aliquot of each serial dilution of test compound was added to deep 384 well plate containing 5 mg/ml purified EZH2 enzyme (Reaction Biology) in a 15 microliter reaction volume. The plate was pre-incubated at room temperature for 15 min to which 4 μM SAM and 6 mg/ml of full length histone 3.3 protein were added to initiate the enzymatic reaction. The reaction mixture was incubated at 30° C. for three hours during which the SAH produced from the methylation reaction is enzymatically converted to AMP. The reaction was stopped by quenching and a detector buffer containing the coupling enzymes, fluorescent indicator and AMP antibody was added to each well. After 1 hr, the resulting fluorescent output of the assay was read on Tecan Safire2 instrument according to the manufacturer's instructions.
Measurement: IC50 = 5 nM (pChEMBL 8.300000190734863)
Descriptors: MW 502.4, ALogP 4.55, HBA 5, HBD 1, PSA 78.78, QED 0.57
Compound (SMILES): CN(C)C(=O)N1CCC(SCc2nc3cc(NC4CCCC4)cc(F)c3c(=O)[nH]2)CC1
InChIKey: HAXLREFLMNQZCT-UHFFFAOYSA-N
Target: Protein mono-ADP-ribosyltransferase PARP14 (SINGLE PROTEIN), Homo sapiens
UniProt: Q460N5
Assay: Enzymatic Inhibition Assay: Enzymatic inhibition of PARP14 was measured using a dissociation-enhanced lanthanide fluorescence immunoassay (DELFIA) monitoring the auto-modification of PARP14 by biotinylated nicotinamide adenine dinucleotide (biotin-NAD). 1 μL of a dose response curve of each test compound was spotted in 384-well nickel-coated white microplates (Thermo) using a Mosquito (TTP Labtech). Reactions were performed in a 50 μL volume by adding 40 μL of PARP14 in assay buffer (20 mM HEPES pH==8, 100 mM NaCl, 0.1% bovine serum albumin, 2 mM DTT and 0.002%0 Tween20), incubating with test compound at 25° C. for 30 min, then adding 10 μL of biotin-NAD (Biolog). The final concentrations of PARP14 and biotin-NAD are 50 nM and 3 μM, respectively. Reactions proceeded at 25° C. for 3 h, then were quenched with 5 μL of 10 mM unmodified nicotinamide adenine dinucleotide (Sigma-Aldrich). The quenched reactions were washed 3 times with 100 μL of TBST wash buffer (50 mM Tris-HCl, 150 mM NaCl and 0.1% Tween20). Next, to the washed and dried plate was added 25 μL of DELFIA Europium-N1 streptavidin (Perkin Elmer) diluted in DELFIA assay buffer (Perkin Elmer). After a 30 min incubation at 25° C., the plate was washed 5 times with TBST wash buffer. Finally, 25 μL of DELFIA enhancement solution was added. After a 5 min incubation the plate was read on an Envision platereader equipped with a LANCE/DELFIA top mirror (Perkin Elmer) using excitation of 340 nm and emission of 615 nm to measure the amount of Europium present in each well, informing on the amount of biotin-NAD that was transferred in the automodification reaction.
Measurement: IC50 < 1000 nM (pChEMBL 6)
Descriptors: MW 447.58, ALogP 3.8, HBA 5, HBD 2, PSA 81.33, QED 0.73
Compound (SMILES): CC1CC(O)(CNC(=O)c2cn(C3CCOC3)c3cccc(Cl)c23)CCC1(F)F
InChIKey: SPDJGGSBVPJLBF-UHFFFAOYSA-N
Target: P2X purinoceptor 7 (SINGLE PROTEIN), Homo sapiens
UniProt: Q99572
Assay: Pore Permeation Assay: Agonist-induced pore formation was determined by measuring cellular uptake of YO PRO fluorescence dye in HEK293 transfected with human P2X7 receptor. A HEK293 cell over expressing human P2X7 was harvested using HQTase reagent to detach the cells from T75 cm flask. The harvested cells are centrifuged @1200 rpm for 5 min at room temperature. The viability of cells was determined by Trypan blue dye and the cells are plated @10,000 cell/well in 50 ul volume in a 384W BD Poly lysine coated plate and incubated overnight at 37 C. After overnight incubation, the culture medium was replaced with 35 ul/well assay buffer (5 mM KCl, 0.1 mM CaCl2, 5 mM Glucose, 10 mM HEPES buffer pH7.4 containing 125 mM NaCl. The serial dilution of compounds was performed using Bravo liquid handling instrument and the compounds were added using Bravo to the cell assay plate starting at 2.5 uM with three dilutions for 10 points. The positive control inhibitor compound was added to column 23. The plate was shaken slowly on a plate shaker for 10 seconds. The cells were incubated with the compound for 20 minutes at room temperature. After the incubation period, YO PRO dye (1 uM) along with BzATP (10 uM) were added to cells at 10 ul/well. The plate was centrifuged at 1000 rpm for 5 seconds and incubated at room temperature for 30 minutes. The uptake of YO PRO dye into the cells was measured using Envision Fluorescence plate reader instrument (Perkin Elmer).
Measurement: IC50 = 750 nM (pChEMBL 6.119999885559082)
Descriptors: MW 426.89, ALogP 4.17, HBA 4, HBD 2, PSA 63.49, QED 0.77
Compound (SMILES): CCCC(N)c1cccc(-c2ccc3cnc(-c4cccc(C)n4)n3c2)n1
InChIKey: VLHNOFFYZKBBIP-UHFFFAOYSA-N
Target: Interleukin-1 receptor-associated kinase 4 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9NWZ3
Assay: Biochemical Assay: The biochemical assay is in a AlphaScreen format. The kinase reaction is based on the IRAK-4 phosphorylation of a biotin labeled peptide. The phosphopeptide is incubated with anti-phosphothreonine antibody as well as streptavidin- and protein A-coated beads. Binding of the protein-A coated beads to the antibody and the streptavidin beads to the peptide, leads to an energy transfer from one bead to the other, ultimately producing a luminescent/fluorescent signal.Generally, the kinase reaction is carried out at 1 nM IRAK4, 1.6 μM peptide, 1 mM ATP in reaction buffer 50 mM Hepes, 60 mM NaCl, 5 mM MgCl2, 0.25 mM MnCl2, 2 mM DTT, 0.01% BSA, 0.01% Tween-20) for 3.5 h at RT.
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 357.46, ALogP 4.57, HBA 5, HBD 1, PSA 69.1, QED 0.57
Compound (SMILES): COc1cccc(F)c1C1CC1CNc1cnn2c(CC3CC3)nnc2c1Cl
InChIKey: ICTLLKUCPXFAGI-UHFFFAOYSA-N
Target: Metabotropic glutamate receptor 2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q14416
Assay: MGluR2 cAMP Assay: Clonally expressed human mGluR2 in HEK293 background was glutamate starved overnight in media without glutamine (GIBCO MEM 12360), containing 10% FBS, 1% penicillin streptomycin. Compounds were added to white, standard volume 384 non-binding surface plates (Corning 3574). Cells were resuspended in stimulation buffer consisting of Hanks Balanced Salt Solution (14175-095) pH 7.0, 20 mM HEPES, 2.0 mM CaCl2, 5.0 mM MgCl2, and 1 mM IBMX (Sigma 15879), 1 Î¼M forskolin, and 1 Î¼M LY-341495, for 30 minutes. Buffer without forskolin was used as a negative control. Solutions of D2 and cryptate detection reagents from the CISBIO dynamic cAMP kit (62AM4PEJ) were diluted 1:40 in lysis buffer. Lysis buffer consisted of 50 mM Phosphate Buffer pH 7.0, 800 mM Potassium Fluoride, 0.2% BSA, and 1.0% Triton. Assay reaction was terminated by addition of detection reagents in lysis buffer. One hour later, plates were read on a PE Viewlux.
Measurement: EC50 = 28 nM (pChEMBL 7.550000190734863)
Descriptors: MW 401.87, ALogP 4.09, HBA 6, HBD 1, PSA 64.34, QED 0.65
Compound (SMILES): CO[C@H]1[C@H](N)CC[C@@H](c2c(NC(=O)c3csc(-c4c(F)cccc4F)n3)cnn2C)O[C@H]1C
InChIKey: ZNMZBZGLWLFBPB-GSRMHOFYSA-N
Target: Serine/threonine-protein kinase pim-1 (SINGLE PROTEIN), Homo sapiens
UniProt: P11309
Assay: Biological Assay: Determination of the Pim kinase activity of a Formula I compound is possible by a number of direct and indirect detection methods. Certain exemplary compounds described herein were assayed for their Pim kinase binding activity, including isoforms Pim-1, Pim-2, andPim-3, (Example 901) and in vitro activity against tumor cells (Example 902). Certain exemplary compounds of the invention had Pim binding activity IC50 values less than about 1 micromolar (μM). Certain compounds of the invention had tumor cell-based activity EC50 values less than about 1 micromolar (μM).
Measurement: Ki = 0.032 nM (pChEMBL 10.489999771118164)
Descriptors: MW 477.54, ALogP 3.66, HBA 8, HBD 2, PSA 104.29, QED 0.58
Compound (SMILES): C[C@H](Nc1nc(N)nc(N)c1Cl)c1nc2cccc(Cl)c2c(=O)n1N1CCNCC1
InChIKey: CFHUDEJAZUMWJT-VIFPVBQESA-N
Target: Phosphatidylinositol 3-kinase regulatory subunit beta (SINGLE PROTEIN), Homo sapiens
UniProt: O00459
Assay: TR-FRET PIK3 Assay: TR-FRET monitored the formation of 3,4,5-inositol triphosphate molecule that competed with fluorescently labeled PIPS for binding to the GRP-1 pleckstrin homology domain protein. An increase in phosphatidylinositide 3-phosphate product resulted in a decrease in TR-FRET signal as the labeled fluorophore was displaced from the GRP-1 protein binding site.Class I PI3K isoforms were expressed and purified as heterodimeric recombinant proteins. All assay reagents and buffers for the TR-FRET assay were purchased from Millipore. PI3K isoforms were assayed under initial rate conditions in the presence of 25 mM Hepes (pH 7.4), and 2× Km ATP (75-500 μM), 2 μM PIP2, 5% glycerol, 5 mM MgCl2, 50 mM NaCl, 0.05% (v/v) Chaps, 1 mM dithiothreitol, and 1% (v/v) DMSO at the following concentrations for each isoform: PI3Kα, PI3Kβ, and PI3Kδ between 25 and 50 pM, and PI3Kγ at 2 nM. The compounds of Table 1 and Compound X ((S)-2,4-diamino-6-((1-(5-chloro-4-oxo-3-phenyl-3,4-dihydroquinazolin-2-yl)ethyl)amino)pyrimidine-5-carbonitrile) were added to the assay solution and incubated for 30 minutes at 25° C. The reactions were terminated with a final concentration of 10 mM EDTA, 10 nM labeled-PIPS, and 35 nM Europium labeled GRP-1 detector protein before reading TR-FRET on an Envision plate reader (Ex: 340 nm; Em: 615/665 nm; 100 is delay and 500 is read window).The results were normalized based on positive (1 μM wortmanin) and negative (DMSO) controls, and the IC50 values for PI3Kα, β, δ, and γ were calculated from the fit of the dose-response curves to a four-parameter equation. These assays generally produced results within 3-fold of the reported mean.
Measurement: IC50 = 28000 nM (pChEMBL 4.550000190734863)
Descriptors: MW 450.33, ALogP 1.37, HBA 10, HBD 4, PSA 140.01, QED 0.46
Compound (SMILES): COc1cccc(OC)c1-n1c(NS(=O)(=O)[C@@H]2C[C@@H](c3ccc(F)cc3)CN(C(C)=O)C2)nnc1-c1ccc(C)o1
InChIKey: IQOSFWBTMGNOEC-IFMALSPDSA-N
Target: Apelin receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P35414
Assay: GTPγS Binding: The human APJ receptor was cloned by polymerase chain reaction and the gene encoding the receptor was subcloned in pFLAG-CMV-3 expression vector (Sigma, Saint Louis, Mo. USA) in-house at Amgen. A GTPγS binding assay was performed on membranes prepared from CHO cells stably expressing human APJ receptor. The optimum experimental conditions for the concentrations of GDP, MgCl2, and NaCl in the assay buffer were initially determined. The assay was performed in assay buffer [20 mM HEPES, pH 7.5, 5 mM MgCl2, and 0.1% (w/v) BSA with 200 mM NaCl, 3 μM GDP] and membranes expressing human APJ receptor/well along with WGA PS beads. The reaction was initiated by addition of 0.2 nM [35S]GTPγS in the absence or presence of various ligands and incubated at RT for 90 min. Nonspecific binding was determined in the presence of 100 μM GTPγS and was always less than 0.2% of total binding.
Measurement: EC50 = 0.75 nM (pChEMBL 9.119999885559082)
Descriptors: MW 583.64, ALogP 4.14, HBA 9, HBD 1, PSA 128.79, QED 0.33
Compound (SMILES): CCNc1cc2c(cn1)c(-c1cnn(C(C)(C)C#N)c1)nn2C1CCC(Oc2ccc(=O)n(C)n2)CC1
InChIKey: IIILCAYWUVJASR-UHFFFAOYSA-N
Target: Tyrosine-protein kinase receptor UFO (SINGLE PROTEIN), Homo sapiens
UniProt: P30530
Assay: Enzyme Assay: AXL: Compounds of Formula I were screened for their ability to inhibit AXL kinase using Invitrogen's LanthaScreen Eu Kinase Binding technology. His-tagged recombinant human AXL cytoplasmic domain was incubated with 20 nM Alexa-Fluor® Tracer 236 (PR9078A), 2 nM biotinylated anti-His (Cat. No. M4408), and 2 nM europium-labeled Streptavidin (Cat. No. PV5899) along with test compound in a buffer consisting of 25 mM HEPES, pH 7.4, 10 mM MgCl2, 0.01% Triton X-100, and 2% DMSO. Compounds were typically prepared in a threefold serial dilution in DMSO and added to the assay to give the appropriate final concentration. After a 60-minute incubation at 22° C., the reaction was measured using a PerkinElmer EnVision multimode plate reader via TR-FRET dual wavelength detection, and the percent of control (POC) was calculated using a ratiometric emission factor. 100 POC was determined using no test compounds and 0 POC was determined using a concentration of control compound that completely inhibits the enzyme. The POC values are fit to a 4 parameter logistic curve and the IC50 value is point where the curve crosses 50 POC.
Measurement: IC50 = 350 nM (pChEMBL 6.460000038146973)
Descriptors: MW 501.6, ALogP 3.64, HBA 11, HBD 1, PSA 128.47, QED 0.41
Compound (SMILES): COc1ccc([C@H](Cc2c(Cl)c[n+]([O-])cc2Cl)OC(=O)c2ccc(CNC(C(=O)OCC3CCCCN3C)c3ccccc3)s2)cc1OC
InChIKey: TZYIKZPGFYGKKX-FEACODLBSA-N
Target: Muscarinic acetylcholine receptor M3 (SINGLE PROTEIN), Homo sapiens
UniProt: P20309
Assay: M3 Binding Assay: Human M3 receptor membranes (15 μg/well) from Perkin Elmer are incubated with 0.52 nM Scopolamine Methyl Chloride, [N-methyl-3H] with or without test compounds, or a saturating concentration of Atropine (5 μM) for the determination of non-specific binding. The assay is carried out in 96-well polypropylene plates in a volume of 250 μl. The assay buffer used is 50 mM Tris-HCl, 154 mM NaCl (pH 7.4). The final assay concentration of DMSO is 0.5% (v/v). The plates are sealed and incubated for 2 h at room temperature on an orbital shaker (slow speed). Membranes are harvested onto 96-well unifilter GF/C filter plates pre-treated with 0.5% polyethyleneimine (v/v), using a filter manifold, washed four times with 200 μl of assay buffer. The plates are dried before addition of 50 μl of microscint-0, sealed then read in a Trilux Microbeta scintillation counter. IC50 values are determined from competition curves using a non-linear curve fitting program. Ki values are calculated from IC50 values by the Cheng and Prusoff equation.
Measurement: IC50 = 55 nM (pChEMBL 7.260000228881836)
Descriptors: MW 728.69, ALogP 6.7, HBA 10, HBD 1, PSA 113.27, QED 0.09
Compound (SMILES): Cc1cc(C(=O)N2CCC(N)CC2)ccc1N1C(=O)CSC1c1ccc(F)cc1
InChIKey: MMDJWZDICQJJJK-UHFFFAOYSA-N
Target: 5-hydroxytryptamine receptor 7 (SINGLE PROTEIN), Homo sapiens
UniProt: P34969
Assay: FLIPR Calcium Assay: One day before the assay, CORNING black with clear flat bottom 96-well assay plates were coated with a 0.1 mg/mL Poly-L-Lysine solution. CHO-K1/MOR/Gα15 cells were suspended in the F12 medium and plated at a density of 8×104 cells/well in 200 μL medium. Cells were incubated in a humidified atmosphere of 10% CO2 at 37° C. overnight so as to reach an 80-90% confluent cell monolayer before assay. At the day of assay, 150 μL medium/well was removed from plate. To each well, 50 μL FLIPR calcium assay reagent dissolved in 1× assay buffer (HBSS: KCl 5 mM, KH2PO4 0.3 mM, NaCl 138 mM, NaHCO3 4 mM, Na2HPO4 0.3 mM, d-glucose 5.6 mM, with additional 20 mM HEPES and 13 mM CaCl2, pH 7.4), with 2.5 mM probenecid was added and the plate was incubated at 37° C. for 1 h. Compounds and other reagents were dissolved in the assay buffer. Using a FlexStationIII (Molecular Devices Corp.), the [Ca2+]i fluorescence increases after robotic injections of compounds or other reagents were monitored every 1.52 s interval with excitation wavelength at 485 nm and with emission wavelength at 525 nm. The [Ca2+]i fluorescence was measured up to 90 s after agonist injection. The fluorescence intensity from 6 to 12 wells of cells were averaged and the relative amount of [Ca2+]i release was determined by integrating the AUC of the [Ca2+]i fluorescence averages.
Measurement: EC50 = 10000 nM (pChEMBL 5)
Descriptors: MW 413.52, ALogP 3.48, HBA 4, HBD 1, PSA 66.64, QED 0.84
Compound (SMILES): COC(=O)c1c(F)cc(-c2ccc3cc(-c4nc5cc(C(=O)N6CC7CCC6[C@@H]7N)cc(OC)c5n4C)n(CC4CC4)c3c2)cc1F
InChIKey: NEEVNRSYPWVLMF-KUVLNDGKSA-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 = 112 nM (pChEMBL 6.949999809265137)
Descriptors: MW 639.7, ALogP 5.91, HBA 8, HBD 1, PSA 104.61, QED 0.22
Compound (SMILES): CCN(CC)C1CCN(S(=O)(=O)c2ccc(NC(=O)NCc3cccnc3)cc2)CC1
InChIKey: RUAXGENFCPAVCB-UHFFFAOYSA-N
Target: Nicotinamide phosphoribosyltransferase (SINGLE PROTEIN), Homo sapiens
UniProt: P43490
Assay: Biochemical Inhibition Assay: The NAMPT enzymatic reactions were carried out in Buffer A (50 mM Hepes pH 7.5, 50 mM NaCl, 5 mM MgCl2, and 1 mM THP) in 96-well V-bottom plates. The compound titrations were performed in a separate dilution plate by serially diluting the compounds in DMSO to make a 100× stock. Buffer A (89 μL) containing 33 nM of NAMPT protein was added to 1 μL of 100× compound plate containing controls (e.g. DMSO or blank). The compound and enzyme mix was incubated for 15 minutes at room temperature, then 10 μL of 10× substrate and co-factors in Buffer A were added to the test well to make a final concentration of 1 μM NAM, 100 μM 5-Phospho-D-ribose 1-diphosphate (PRPP), and 2.5 mM Adenosine 5′-triphosphate (ATP). The reaction was allowed to proceed for 30 minutes at room temperature, then was quenched with the addition of 11 μL of a solution of formic acid and L-Cystathionine to make a final concentration of 1% formic acid and 10 μM L-Cystathionine. Background and signal strength was determined by addition (or non-addition) of a serial dilution of NMN to a pre-quenched enzyme and cofactor mix.
Measurement: IC50 = 5 nM (pChEMBL 8.300000190734863)
Descriptors: MW 445.59, ALogP 2.9, HBA 5, HBD 2, PSA 94.64, QED 0.65
Compound (SMILES): O=C(Nc1ccc([C@H]2CNCCO2)cc1)c1cnn(-c2ccnc(C(F)(F)F)c2)c1
InChIKey: ZJOOIYPGMOMTSH-QGZVFWFLSA-N
Target: Trace amine-associated receptor 1 (SINGLE PROTEIN), Mus musculus
UniProt: Q923Y8
Assay: Radioligand Binding Assay: HEK-293 cells stably expressing mouse TAAR1 were maintained at 37° C. and 5% CO2 in DMEM high glucose medium, containing fetal calf serum (10%, heat inactivated for 30 min at 56° C.), penicillin/streptomycin (1%), and 375 ug/ml geneticin (Gibco). Cells were released from culture flasks using trypsin/EDTA, harvested, washed twice with ice-cold PBS (without Ca2+ and Mg2+), pelleted at 1,000 rpm for 5 min at 4° C., frozen and stored at -80° C. Frozen pellets were suspended in 20 ml HEPES-NaOH (20 mM, pH 7.4) containing 10 mM EDTA and homogenized with a Polytron (PT 6000, Kinematica) at 14,000 rpm for 20 s. The homogenate was centrifuged at 48,000xg for 30 min at 4° C. Subsequently, the supernatant was removed and discarded, and the pellet resuspended in 20 ml HEPES-NaOH (20 mM, pH 7.4) containing 0.1 mM EDTA using the Polytron (20 s at 14,000 rpm). This procedure was repeated and the final pellet resuspended in HEPES-NaOH containing 0.1 mM EDTA and homogenized using the Polytron. Typically, aliquots of 2 ml membrane portions were stored at -80° C. With each new membrane batch the dissociation constant (Kd) was determined via a saturation curve. The TAAR1 radioligand 3[H]-(S)-4-[(ethyl-phenyl-amino)-methyl]-4,5-dihydro-oxazol-2-ylamine (described in WO 2008/098857) was used at a concentration equal to the calculated Kd value, that was usually around 2.3 nM, resulting in the binding of approximately 0.2% of the radioligand and a specific binding representing approximately 85% of the total binding. Nonspecific binding was defined as the amount of 3[H]-(S)-4-[(ethyl-phenyl-amino)-methyl]-4,5-dihydro-oxazol-2-ylamine bound in the presence of 10 uM unlabeled ligand. All compounds were tested at a broad range of concentrations (10 pM to 10 uM) in duplicates. The test compounds (20 ul/well) were transferred into a 96 deep well plate (TreffLab), and 180 ul of HEPES-NaOH (20 mM, pH 7.4) containing MgCl2 (10 mM) and CaCl2 (2 mM) (binding buffer), 300 ul of the radioligand 3[H]-(S)-4-[(ethyl-phenyl-amino)-methyl]-4,5-dihydro-oxazol-2-ylamine at a concentration of 3.3xKd in nM and 500 ul of the membranes (resuspended at 50 ug protein per ml) added. The 96 deep well plates were incubated for 1 hr at 4° C. Incubations were terminated by rapid filtration through Unifilter-96 plates (Packard Instrument Company) and glass filters GF/C (Perkin Elmer) presoaked for 1 hr in polyethylenimine (0.3%) and washed 3 times with 1 ml of cold binding buffer. After addition of 45 ul of Microscint 40 (PerkinElmer) the Unifilter-96 plate was sealed and after 1 hr the radioactivity counted using a TopCount Microplate Scintillation Counter (Packard Instrument Company).
Measurement: Ki = 7.7 nM (pChEMBL 8.109999656677246)
Descriptors: MW 417.39, ALogP 3.2, HBA 6, HBD 2, PSA 81.07, QED 0.68
Compound (SMILES): C=CC(=O)NC1COCC1Nc1ncc2c(n1)N(C)C(=O)N(c1ccc(OC)cc1)C2
InChIKey: NJDHFNVZECIIBG-UHFFFAOYSA-N
Target: Fibroblast growth factor receptor 4 (SINGLE PROTEIN), Homo sapiens
UniProt: P22455
Assay: Omnia Assay: Omnia Assay Protocol for Potency Assessment Against FGFR 4 Enzyme. a) [FGFR4-WT]=10 nM, [ATP]=300 uM, [Y10-Sox]=10 uM (ATP KMapp 300 uM): A 10x stock solution of FGFR4-WT (PR4380C), (from Invitrogen, Carlsbad, Calif.) corresponding to method a in Table 7, was prepared as described below. Alternatively, a 10x stock solution of FGFR4-WT (F01-11G), (SignalChem, Richmond, BC) corresponding to method b in Table 7, was prepared as described below. A solution of 1.4xATP (AS001A) and 5x Tyr-Sox conjugated peptide substrate (KNZ3101) were prepared in 1x kinase reaction buffer consisting of 20 mM Tris, pH 7.5, 5 mM MgCl2, 1 mM EGTA, 5 mM β-glycerophosphate, 5% glycerol (10x stock, KB002A) and 0.2 mM DTT (DS001A). 5 uL of FGFR4 was pipetted into a Corning (#3574) 384-well, white, non-binding surface microtiter plate (Corning, N.Y.), containing a 0.5 uL volume of 100% DMSO. The serially diluted compounds were prepared on a Tecan EVO100. A second addition of 10 ul of Tyr-Sox FGFR4 substrate was added to each well and the kinase reactions were started with the addition of 35 uL of 1.4xATP. The reactions were monitored every 71 seconds for 240 minutes at λex360/λem485 in a Synergy plate reader from BioTek (Winooski, Vt.). At the conclusion of each assay, progress curves from each well were examined for linear reaction kinetics and fit statistics (R2, 95% confidence interval, absolute sum of squares). Initial velocity (0 minutes to ~60 minutes) from each reaction was determined from the slope of a plot of relative fluorescence units vs time (seconds) and then plotted against inhibitor concentration to estimate IC50 from log [Inhibitor] vs Response (Variable Slope model in GraphPad Prism from GraphPad Software (San Diego, Calif.)).
Measurement: IC50 > 1000 nM (pChEMBL 6)
Descriptors: MW 424.46, ALogP 1.54, HBA 7, HBD 2, PSA 108.92, QED 0.68
Compound (SMILES): CNC(=O)O[C@H]1CCC[C@@H]1C(C#N)(c1ccccc1)C1CCN(CC2CN(c3ccc(S(=O)(=O)c4ccn(C)n4)cc3)C2)CC1
InChIKey: XGDPJMZKJSYGJF-FVVNGJPMSA-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 = 7 nM (pChEMBL 8.149999618530273)
Descriptors: MW 630.82, ALogP 4.39, HBA 9, HBD 1, PSA 120.56, QED 0.37
Compound (SMILES): CCc1cc(C(=O)NCC2(O)CCCCC2)c(Cl)c2cc[nH]c12
InChIKey: ZCIHDOGZIWYRRH-UHFFFAOYSA-N
Target: P2X purinoceptor 7 (SINGLE PROTEIN), Homo sapiens
UniProt: Q99572
Assay: In Vitro Assay: The amplified sequence was subsequently ligated into a pcDNA3.1 (+) NotI, NheI digested plasmid. Human embryonic kidney (HEK) cells (ATCC CRL-1573, Manassas, Va., USA) were transfected with the pcDNA3.1 (+).hP2X7 plasmid using lipofectamine 2000 (Invitrogen AG, CH) according to the manufacturer's instructions. Following a 24 h exposure to DNA, cells were trypsinized and re-seeded at low density in the presence of 250 μg Geneticin. Geneticin resistant cells were then selected during two consecutive rounds of cloning by serial limiting dilution with visual inspection. Individual clones were screened for P2X7 expression by applying ATP and recording the resultant uptake of YO-PRO1. Specific cell clones were chosen based on RNA and protein expression. HEK cells stably expressing P2X7 were used to screen drugs using the YO-PRO1 assay. Cells were grown to confluency in adherent culture at 37° C. in a humidified 5% CO2 incubator (split 1/5 every 3-4 days with DMEM, 10% FCS, 1% Penicillin/Streptomycin, 250 μg/ml Geneticin). Adherent cells were detached by incubation with Trypsine (1 ml per 165 cm2 dish) for 2 minutes, then washed off with 10 ml PBS (without Mg2+ and Ca2+), and resuspended in DMEM, 10% FCS, 1% Penicillin/Streptomycin, no Geneticin. 10′000 cells per well (48 hours before the assay) or 25′000 cells per well (Vi-cell XR (Beckman Coulter) (24 hours before the assay) in 50 μl full medium were seeded on 384-well black-wall, clear bottom plates, that were coated before with 10 μl per well Poly-L-Lysine, incubated for 30-60 minutes at 37° C. and washed once with PBS. Medium was removed from cells and 50 μl of assay buffer containing 0.5 μM YO-PRO-1 was added into the wells. Solutions of antagonist compounds were prepared by serial dilutions of a 10 mM DMSO solution of the antagonist into PBS using a BioMek (Beckman Coulter). Each concentration was performed in duplicate. For IC50 measurements 10 concentration points were measured (10 μM being the highest concentration followed by 9 serial dilution steps 1/3). The cells were incubated with the antagonists of the present invention together with ATP at a final concentration of 250 μM for 90 minutes. During this time period, four time points were taken. Each time point comprised the average of several measurements made within a few seconds. Fluorescence was measured in the FLIPR tetra (Molecular Devices) using the filters appropriate for YO-PRO-1 fluorescence (excitation485/20, emission 530/25).
Measurement: IC50 = 4.1 nM (pChEMBL 8.390000343322754)
Descriptors: MW 334.85, ALogP 3.81, HBA 2, HBD 3, PSA 65.12, QED 0.8
Compound (SMILES): CNC(=O)c1ccc(C=C2CCN(Cc3cnc(NC(C)=O)s3)CC2)cc1
InChIKey: MUBGWHRAAGNGIP-UHFFFAOYSA-N
Target: Protein O-GlcNAcase (SINGLE PROTEIN), Homo sapiens
UniProt: O60502
Assay: Assay for Determination of KI Values for Inhibition of O-GlcNAcase Activity : Experimental procedure for kinetic analyses: Enzymatic reactions were carried out in a reaction containing 50 mM NaH2PO4, 100 mM NaCl and 0.1% BSA (pH 7.0) using 2 mM 4-Methylumbelliferyl N-acetyl-β-D-glucosaminide dihydrate (Sigma M2133) dissolved in ddH2O, as a substrate. The amount of purified human O-GlcNAcase enzyme used in the reaction was 0.7 nM. Test compound of varying concentrations was added to the enzyme prior to initiation of the reaction. The reaction was performed at room temperature in a 96-well plate and was initiated with the addition of substrate. The production of fluorescent product was measured every 60 sec for 45 min with a Tecan Infinite M200 plate-reader with excitation at 355 nM and emission detected at 460 nM, with 4-Methylumbelliferone (Sigma M1381) used to produce a standard curve. The slope of product production was determined for each concentration of compound tested and plotted, using standard curve fitting algorithms for sigmoidal dose response curves. The values for a four parameter logistic curve fit of the data were determined. KI values were determined using the Cheng-Prusoff equation; the Km of O-GlcNAcase for substrate was 0.2 mM. Many compounds of the invention exhibit KI values for inhibition of O-GlcNAcase in the range 0.1 nM-10 μM. The KI values for the compounds of the examples are shown in the table below. The following table shows representative data for the compounds of the Examples as determined by the assay described herein.
Measurement: Ki = 0.43 nM (pChEMBL 9.369999885559082)
Descriptors: MW 384.51, ALogP 3.14, HBA 5, HBD 2, PSA 74.33, QED 0.83
Compound (SMILES): O=C(c1cc(-c2ccncn2)on1)N1CC(c2ccccc2)C1
InChIKey: UOWHAXONWVKJBJ-UHFFFAOYSA-N
Target: Stearoyl-CoA desaturase (SINGLE PROTEIN), Homo sapiens
UniProt: O00767
Assay: Compound Profiling Methods: Strains expressing SCD1 or SCD5 as the sole desaturase, the human SCD1 and SCD5 genes were used to evaluate inhibition of SCD1/SCD5 using reduced growth as a surrogate for SCD inhibition. These yeast strains express human SCD1 or SCD5 from a plasmid harbored in a strain in which the yeast OLE1 gene is deleted.All compound profiling experiments were performed using the same basic protocol. Yeast were cultured using standard techniques in complete synthetic media lacking uracil and containing yeast nitrogen base supplemented with 2% (w/v) glucose (SD-Ura) Starter cultures were inoculated in 3 mL SD-Ura media containing 0.01% tween and 0.2 mM palmitoleic and oleic acid. Cultures were incubated overnight in a 30° C. shaker incubator (225 rpm). Saturated morning cultures were centrifuged, washed in SD-Ura media lacking TWEEN-20 and fatty acids, and then diluted 1:20 in fresh SD-Ura media also lacking TWEEN-20 and fatty acids. Cells were grown for 6 h to an  (optical density) of 0.4-0.8 at 30° C. with shaking.Compound stocks (10 mM in 100% DMSO) were arrayed into 384-round well, v-bottom polypropylene plates and diluted according to indicated dilution factors. Compound administration was performed in two separate steps. First, 15 μL of SD-Ura was dispensed into clear 384-well assay plates using a MULTIDROP Combi reagent dispenser. The diluted compound stock plates were then applied to the assay plates using an automated workstation (Perkin Elmer JANUS) outfitted with a 384-pin tool containing slotted pins that deliver 100 nL of compound. The cultures described above were centrifuged and washed with media lacking TWEEN-20 or oleic and palmitoleic acids. Cultures were then resuspended at a 2-fold concentrated  of 0.02 (final  of 0.0.01) in SD-Ura. 15 μL of diluted culture was then dispensed into the pinned assay plate to achieve 30 μL of the 1× culture (0.01) and a top drug concentration of 33.3 μM.After yeast delivery, assay plates were incubated under humidified conditions at 30° C. for 40 h. Yeast growth was monitored by reading the  of each well using a microplate reader (Perkin Elmer EnVision). Data were analyzed as follows. Raw data were processed by background subtracting and converting values to a percent of the nontreated condition for that strain [(EXP-0.035)/(DMSO-0.035)×100%].
Measurement: IC50 = 321 nM (pChEMBL 6.489999771118164)
Descriptors: MW 306.32, ALogP 2.37, HBA 5, HBD 0, PSA 72.12, QED 0.74
Compound (SMILES): Cn1ccc(-c2cccc(S(N)(=O)=O)c2-c2nn[nH]n2)n1
InChIKey: PLKVAQWXYWOENE-UHFFFAOYSA-N
Target: Metallo-beta-lactamase type 2 (SINGLE PROTEIN), Bacteria
UniProt: C7C422
Assay: Class B Enzyme Assay: The Class B enzyme activities were measured in the presence of the test inhibitor in a fluorescence assay against a commercially available substrate consisting of a cephalosporin core linking 7-hydroxycoumarin to fluorescein (CCF2-FA). The enzyme (NDM-1, IMP-1 or VIM-1) and the substrate were diluted in 100 mM KH2PO4 buffer (pH 7) containing 0.005% Tween-20 and 10 μM ZnSO4. In the assay, the final concentration of enzyme was 1 pM, 2 pM and 30 pM for NDM-1, IMP-1 and VIM-1, respectively, and the final concentration of CCF2-FA was 1.25 μM. The test inhibitor was dissolved in dimethylsulfoxide and diluted 1:50 in the assay, resulting in a final concentration range of 20 uM to 0.00063 uM. In a 384-well microplate, the test inhibitor was incubated with the metallo-β-lactamase enzyme and the substrate for 2 hours at 25° C. Fluorescence at 460 nm following excitation at 405 nm was measured. The IC50 value was determined from semi-logarithmic plots of enzyme inhibition versus inhibitor concentration, with a curve generated using a 4-parameter fit.
Measurement: IC50 = 71.1 nM (pChEMBL 7.150000095367432)
Descriptors: MW 305.32, ALogP -0.09, HBA 7, HBD 2, PSA 132.44, QED 0.69
Compound (SMILES): N#Cc1ccc(Oc2ccc(/N=c3\[nH]c(=O)n(CC(=O)O)c(=O)n3Cc3ccc(Cl)cc3)cc2)nc1
InChIKey: DPLICNAZDYZBIZ-UHFFFAOYSA-N
Target: P2X purinoceptor 3 (SINGLE PROTEIN), Homo sapiens
UniProt: P56373
Assay: Receptor Inhibition Assay: Stably expressing cell line (C6BU-1 cell transfected with human P2X3 receptor gene (GenBank accession number Y07683) was used. The cells were seeded in a 384-well PDL-coated microtiter plate at a concentration of 3000 cells/well and cultured in the medium (8.3% fetal bovine serum, 8.3% horse serum, and 1% antibiotic and antifungal in DMEM) for one day at 37° C. under 5% carbon dioxide atmosphere. The medium was replaced with 4 μM Fluo-3-AM solution (20 mM HEPES, 137 mM NaCl, 2.7 mM KCl, 0.9 mM MgCl2, 5.0 mM CaCl2, 5.6 mM D-glucose, 2.5 mM probenecid, 0.5% BSA, and 0.04% Pluronic P-127, pH 7.5) and incubated at 37° C. under 5% dioxide carbon atmosphere for one hour. The plate was washed with washing buffer (20 mM HEPES, 137 mM NaCl, 2.7 mM KCl, 0.9 mM MgCl2, 5.0 mM CaCl2, 5.6 mM D-glucose, 2.5 mM probenecid, pH 7.5), and each well was added with 20 μL of the washing buffer. The plate was placed in High-Throughput Screening System FLIPR 384 (Molecular Device Co.). Measurement of fluorescence intensity by FLIPR 884 was started, and 20 μL of DMSO solutions containing different concentrations of the test compound as prepared by dilution with dilution buffer (20 mM HEPES, 137 mM NaCl, 2.7 mM KCl, 0.9 mM MgCl2, 5.0 mM CaCl2, 5.6 mM D-glucose, 2.5 mM probenecid, 0.1% Pluronic F-127, pH 7.5) were dispensed to each well through the built-in automatic dispenser. Five minutes after, 150 nM ATP solution (25 μL) prepared by dilution with the dilution buffer was dispensed through the built-in automatic dispenser, and the measurement of fluorescence intensity was continued for 4 min. For each well, the specific maximum fluorescence intensity was calculated as the ratio of the maximum fluorescence intensity after addition of the ATP solution to the fluorescence intensity at the starting of the measurement.
Measurement: IC50 = 48 nM (pChEMBL 7.320000171661377)
Descriptors: MW 504.89, ALogP 2.42, HBA 9, HBD 2, PSA 155.36, QED 0.39
Compound (SMILES): Cc1nnn(C)c1-c1cnc2c3ccc(C(C)(C)O)c(F)c3n([C@H](c3ccc(F)cc3)C3CCOCC3)c2c1
InChIKey: JDZYRNKZAUHPTC-MUUNZHRXSA-N
Target: Bromodomain-containing protein 4 (SINGLE PROTEIN), Homo sapiens
UniProt: O60885
Assay: TR-FRET Assay: All assay components were dissolved in buffer composition 20 mM Hepes pH 7.5, 150 mM NaCl, 5 mM DTT, 0.005% Tween 20, and 100 ug/ml BSA for BRD4 (1-477 and 44-460). The final concentrations of the bromodomain proteins are 1.6 nM BRD4(44-168), 1 nM BRD4(333-460), and 1 nM BRD4(1-477 or 44-460), and the fluorescent probe molecule is 100 nM, 50 nM, and 7.5 nM respectively. All proteins were biotinylated. A streptavidin labeled with terbium cryptate (Cisbio SA-Tb) was used as detection, and pre-mixed with the bromodomain protein at a final concentration of 0.2 nM. In some instances for BRD4 (44-460), anti-His terbium cryptate was used as a detection. 7.5 nl of dose-responsed test compound or dmso vehicle (0.0375%) was pre-spotted in a black Corning 384 well plate and 10 ul each of bromodomain/detection reagent and fluorescent small molecule solution were added to the plate, and the reaction incubated for 60 min at room temperature. Plates were then read on EnVision plate reader, (λex=340 nm, acceptor λEm=520 nm, and donor λEm=615 nm, LANCE D400 mirror). Time resolved fluorescence intensity measurements were made at both emissions, and the ratio of acceptor/donor was calculated and used for data analysis.
Measurement: IC50 < 5 nM (pChEMBL 8.300999641418457)
Descriptors: MW 531.61, ALogP 5.82, HBA 7, HBD 1, PSA 77.99, QED 0.31
Compound (SMILES): O=C(NO)c1ccc(NC(=O)c2[nH]c(-c3ccoc3)cc2-c2ccc(O)cc2)cc1
InChIKey: PPBYALDLCGQTJN-UHFFFAOYSA-N
Target: Protein deacetylase HDAC6 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9UBN7
Assay: Enzyme Assay: The reactions were carried out in a 96-well microplate for fluorometry in a 50 μl reaction volume. After the deacetylation reaction, Fluor-de-Lys-Developer (BioMol Cat. # KI-105) was added to each well to digest the deacetylated substrate, thus producing the fluorescent signal. The reaction was allowed to develop for 45 minutes at 30° C. with 5% CO2; then the fluorescent signal was measured with an excitation wavelength at 360 nm and an emission wavelength at 460 nm in a microplate-reading fluorometer (GeminiXS; Molecular Devices, Sunnyvale, Calif.). A curve of Deacetylated Standard (Biomol, Cat. # KI-142; made from 100 μM with 1:2 dilution and 10-doses, 6 μl) allowed the conversion of fluorescent signal into micromoles of deacetylated product.
Measurement: IC50 = 6.8 nM (pChEMBL 8.170000076293945)
Descriptors: MW 403.39, ALogP 4.02, HBA 5, HBD 5, PSA 127.59, QED 0.25