lovingscience/chembl-v37-raw-activities huggingface dataset:
Compound (SMILES): COCCC(=O)N1CCc2c(-c3cccc(C(=O)OC)c3)ccc(CNC(=O)C3CC3c3ccccc3)c2C1
InChIKey: HOIDEHIBJSZCEQ-UHFFFAOYSA-N
Target: Diacylglycerol O-acyltransferase 2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q96PD7
Assay: Enzymatic Activity Assay: DGAT2 activity was determined by measuring the amount of enzymatic product triolein (1,2,3-Tri(cis-9-octadecenoyl)glycerol) using the membrane prep mentioned above. The assay was carried out in deep well 384 plates in a final volume of 40 μL at room temperature. The assay mixture contained the following: assay buffer (100 mM Tris.Cl, pH 7.0, 20 mM MgCl2, 5% ethanol), 25 μM of diolein, 10 μM of oleoyl-CoA and 10 ng/μL of DGAT2 membrane.
Measurement: IC50 = 1772 nM (pChEMBL 5.75)
Descriptors: MW 526.63, ALogP 4.48, HBA 5, HBD 1, PSA 84.94, QED 0.42
Compound (SMILES): NC(=O)c1nn(CC(=O)N2C[C@H](F)C[C@H]2C(=O)N[C@@H]2CCC[C@H]2O)c2ccc(-c3ccnnc3)cc12
InChIKey: NTKMUBWYJKVCJW-WSTLGDPDSA-N
Target: Complement factor D (SINGLE PROTEIN), Homo sapiens
UniProt: P00746
Assay: Factor D Assay: Human factor D (purified from human serum, Complement Technology, Inc.) at 80 nM final concentration is incubated with test compound at various concentrations for 5 minutes 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. The increase in color is recorded at OD405 nm in a microplate in kinetic mode over 30 minutes with 30 second time points in a spectrofluorimeter. IC50 values are calculated by non-linear regression from the percentage of inhibition of complement factor D activity as a function of test compound concentration.
Measurement: IC50 > 10000 nM (pChEMBL 5)
Descriptors: MW 495.52, ALogP 0.56, HBA 8, HBD 3, PSA 156.33, QED 0.45
Compound (SMILES): COc1cc(F)c(Nc2cc(-c3ccc(F)cc3OC)ncn2)cc1C(N)=O
InChIKey: WUKHXYWKXVPSKC-UHFFFAOYSA-N
Target: Cyclin-dependent kinase 9 (SINGLE PROTEIN), Homo sapiens
UniProt: P50750
Assay: Fluorescent Polarization Assay: The activity of the compounds described in the present invention can be determined by measuring the phosphorylation of a fluorescently-labeled peptide by human CDK9/CyclinT kinase complex by fluorescent polarization using a commercially available IMAP Screening Express Assay Kit (Molecular devices).Test compounds were diluted in 100% DMSO to 5 mM stock concentration, then further dilutions were made in H2O or 100% DMSO to desirable concentrations. Each reaction consisted of 5 nM enzyme: CDK9/CyclinT (Proqinase cat#0371-0345-1), 400 nM TAMRA-Rbtide (synthetic 15-mer peptide derived from human retinoblastoma tumor suppressor protein labeled with TAMRA dye, Genecust Europe), 12 μM ATP (=Kmapp, Sigma-Aldrich) and kinase buffer: 20 mM MOPS pH 7 (Sigma-Aldrich), 1 mM DTT (Sigma-Aldrich), 10 mM MgCl2 (Sigma-Aldrich), 0.01% Tween 20 (Sigma-Aldrich).For each reaction, 4 or 6 μl containing TAMRA-Rbtide, ATP and kianse buffer were combined with 2 μl diluted compound in H2O or 0.028 μl compound in 100% DMSO. The kinase reaction was started by the addition of 2 μl diluted enzyme.
Measurement: IC50 < 1000 nM (pChEMBL 6)
Descriptors: MW 386.36, ALogP 3.28, HBA 6, HBD 2, PSA 99.36, QED 0.67
Compound (SMILES): O=C1CN(C(=O)c2ccc3c(c2)N(c2ncc(-c4cccc(F)c4)cn2)CC32CC2)CCN1
InChIKey: ISAXWUCMDQUNJC-UHFFFAOYSA-N
Target: 3',5'-cyclic-AMP phosphodiesterase 4B (SINGLE PROTEIN), Homo sapiens
UniProt: Q07343
Assay: cAMP HTRF Assay to Determine the Activity of hPDE4B1: The inhibiting effect of the compounds on the enzyme activity of human PDE4B1 was measured by the quantification of 5′-adenosine monophosphate (5′-AMP), which is formed from 3′,5′-cyclic adenosine monophosphate (cAMP). Human recombinant enzyme, expressed in Sf9 cells, and the HTRF (homogeneous time-resolved fluorescence) detection method were used in the assay.The test compound or water (control) was mixed with the human recombinant PDE4B1 enzyme (4.8 U) in a buffer consisting of 44.4 mM tris-HCl, 5.28 mM MgCl2, 2.64 mM DTT and 0.044% Tween 20 (pH 7.8). After adding the cAMP enzyme substrate (final concentration 40 nM), the mixture was incubated for 30 minutes at room temperature. Then a fluorescence acceptor (Dye2 marked with cAMP), a fluorescence donor (anti-cAMP antibody marked with a europium cryptate) and the non-specific phosphodiesterase inhibitor IBMX (3-isobutyl-1-methylxanthine; final concentration 1 mM) were added. After 60 min the fluorescence transfer, which correlates with the amount of remaining cAMP, was measured with a microplate reader (Rubystar, BMG) at λex=337 nm, λem=620 nm and λem=665 nm. The enzyme activity was calculated from the quotient formed from the measured signal at 665 nm and that at 620 nm. The result was expressed as the percentage inhibition of enzyme activity of the control (without PDE4 inhibitor). The enzyme was omitted for measurement of the basal control.[N. Saldou et al., Comparison of recombinant human PDE4 isoforms: interaction with substrate and inhibitors, Cell. Signal. Vol. 10, No. 6, 427-440, 1998].
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 443.48, ALogP 3.04, HBA 5, HBD 1, PSA 78.43, QED 0.67
Compound (SMILES): N#Cc1cc(C2CC2)cn2cc(Cc3nnc(C(=O)NCc4ncn5ccc(Br)c(F)c45)o3)nc12
InChIKey: BOEXVDRNOHZYSF-UHFFFAOYSA-N
Target: Plasma kallikrein (SINGLE PROTEIN), Homo sapiens
UniProt: P03952
Assay: Inhibitory Activity Assay: Example compounds were evaluated for inhibition of the human activated kallikrein enzyme in two formats of an assay employing a fluorogenic peptide substrate. In one assay format, the concentrations of reagents were as follows: 20 mM Tris pH 7.5, 1 mM EDTA, 150 mM sodium chloride, 0.1% PEG-400, 0.1% Triton X-100, 500 pM activated kallikrein enzyme, 300 uM Pro-Phe-Arg-7-amido-4-methylcoumarin substrate. Prior to reaction initiation with substrate, enzyme and inhibitors were preincubated for 30 min at RT. After initiation with substrate, reactions were incubated for 10 min at RT and fluorescence emission at 460 nm from 380 nm excitation measured with a microplate reader. In another assay format, the concentrations of reagents were as follows: 20 mM Tris pH 7.5, 1 mM EDTA, 150 mM sodium chloride, 0.1% PEG-400, 0.1% Triton X-100, 5 pM activated kallikrein enzyme, 300 uM Pro-Phe-Arg-7-amido-4-methylcoumarin substrate. Prior to reaction initiation with substrate, enzyme and inhibitors were preincubated for 30 min at RT. After initiation with substrate, reactions were incubated for 18 h at RT and fluorescence emission at 460 nm from 380 nm excitation measured with a microplate reader.
Measurement: EC50 < 1 nM (pChEMBL 9)
Descriptors: MW 535.34, ALogP 3.54, HBA 9, HBD 1, PSA 126.41, QED 0.35
Compound (SMILES): COc1ccc(C(=O)N2C3CC2CN(c2ccc(-c4cc(OCC5(C)COC5)cn5ncc(C#N)c45)cn2)C3)cn1
InChIKey: PMWIFLDYJYLIQH-UHFFFAOYSA-N
Target: Proto-oncogene tyrosine-protein kinase receptor Ret (SINGLE PROTEIN), Homo sapiens
UniProt: P07949
Assay: RET Enzyme Assay: Compounds of Formula I were screened for their ability to inhibit wildtype and V804M mutant RET kinase using CisBio's HTRF® KinEASE¿-TK assay technology. Briefly, N-terminal GST tagged recombinant human RET cytoplasmic domain (aa 658-end) from Eurofins (0.25 nM RET; Catalog No. 14-570M) or N-terminal GST tagged recombinant human V804M mutant RET cytoplasmic domain (aa 658-end) from Millipore (0.25 nM enzyme; Catalog No. 14-760) was incubated with 250 nM TK-substrate biotin (CisBio, part of Catalog No. 62TK0PEC) and 1 mM ATP 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 in a volume of 8 μL. Compounds were typically prepared in a threefold serial dilution in DMSO and added to the assay to give the appropriate final concentration. After a 30-minute incubation at 22° C., the reaction was quenched by adding 8 μL of quench solution containing 31.25 nM Sa-XL665 and 1×TK-ab-Cryptate in HTRF detection buffer (all from CisBio, part of Cat. No. 62TK0PEC). After a 1 hour incubation at 22° C., the extent of reaction was determined using a PerkinElmer EnVision multimode plate reader via HTRF 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 pre-quenched control reactions. The POC values were fit to a 4 parameter logistic curve, and the IC50 is defined as the concentration of inhibitor at which the POC equals 50 for the fitted curve.
Measurement: IC50 = 59 nM (pChEMBL 7.230000019073486)
Descriptors: MW 551.61, ALogP 3.19, HBA 10, HBD 0, PSA 118.11, QED 0.34
Compound (SMILES): O=C(NCc1ccncc1)Nc1ccc(C(=O)Nc2ccc(Cl)cc2)cc1
InChIKey: GBNIRKSYVYOHGD-UHFFFAOYSA-N
Target: Nicotinamide phosphoribosyltransferase (SINGLE PROTEIN), Homo sapiens
UniProt: P43490
Assay: Enzyme Assay and NMN Detection: NAMPT reaction was performed in 50 mM HEPES, pH 7.5, containing 5 mM MgCl2, 0.1% Prionex, 0.005% Tween 20, and 1 mM TCEP at room temperature in Greiner 384-well black polypropylene plates. The concentration of substrates and enzymes in the final assay were as follows: ATP (120 uM), nicotinamide (5 uM), phosphoribosylpyrophosphate (6.25 uM), NAMPT enzyme (16 nM), pyrophosphatase (0.04 U/ml) in the final reaction volume 6 uL. Following 2 h incubation the reaction was stopped by addition 2.5 ul 20% acetophenone in dimethyl sulfoxide (DMSO) and 2.5 ul 2 MKOH. Plates were centrifuges and added with 10.5 ul 88% formic acid. The fluorescence (ex380/em460) was measured following another short incubation and 30 min incubation. The increased concentration of NMN was determined from a calibration curve obtained at the time of experiment.
Measurement: EC50 = 1050 nM (pChEMBL 5.980000019073486)
Descriptors: MW 380.83, ALogP 4.31, HBA 3, HBD 3, PSA 83.12, QED 0.62
Compound (SMILES): FC(F)(F)c1cccc(CN2CCC[C@@H](C3CC(Br)=NO3)C2)c1
InChIKey: AEBCNMCBIWJGSW-PUODRLBUSA-N
Target: Transcriptional enhancer factor TEF-1 (SINGLE PROTEIN), Homo sapiens
UniProt: P28347
Assay: TEAD Inhibition Assay: TEAD inhibition can be assayed using Hippo Pathway TEAD ReporterMCF7 Cell Line (BPS Bioscience, Catalog #: 60618).
Measurement: EC50 > 500 nM (pChEMBL 6.301000118255615)
Descriptors: MW 391.23, ALogP 4.41, HBA 3, HBD 0, PSA 24.83, QED 0.76
Compound (SMILES): Cc1n[nH]c(C(=O)Nc2cnc3sc(-c4ccccc4)nc3c2)c1C
InChIKey: CKSNDOMNWPWMNX-UHFFFAOYSA-N
Target: Mast/stem cell growth factor receptor Kit (SINGLE PROTEIN), Homo sapiens
UniProt: P10721
Assay: Exemplary c-Kit Mutant Biochemical Assay: The c-kit mutant D816V (or kinase domain thereof) is an active kinase in AlphaScreen. IC50 values are determined with respect to inhibition of c-Kit mutant D816V kinase activity, where inhibition of phosphorylation of a peptide substrate is measured as a function of compound concentration. Compounds to be tested were dissolved in DMSO to a concentration of 20 mM. These were diluted 30 μl into 120 μl of DMSO (4 mM) and 1 μl was added to an assay plate. These were then serially diluted 1:3 (50 μl to 100 μl DMSO) for a total of 8 points. Plates were prepared such that each kinase reaction is 20 μl in 1× kinase buffer (25 mM HEPES, pH 7.2, 8 mM MgCl2, 2 mM MnCl2, 50 mM NaCl, 0.01% Brij, 1 mM DTT, 0.01% BSA), 5% DMSO and 10 μM ATP. Substrate was 30 nM biotin-(E4Y)10 (EMD Millipore, Cat#12-440). C-kit mutant D816V kinase was at 0.75 ng per sample. After incubation of the kinase reaction for 30 minutes at room temperature, 5 μl of donor beads (Streptavidin coated beads (Perkin Elmer Life Science) final concentration 7.5 μg/ml) in stop buffer (25 mM Hepes pH 7.5, 100 mM EDTA, 0.01% BSA) was added, the sample was mixed and incubated for 20 minutes at room temperature before adding 5 μl of acceptor beads (PY20 coated beads (Perkin Elmer Life Science) final concentration 7.5 ug/ml) in stop buffer. The samples were incubated for 60 minutes at room temperature and the signal per well was read on EnVision reader. Phosphorylated substrate results in binding of the PY20 antibody and association of the donor and acceptor beads such that signal correlates with kinase activity.
Measurement: Ki = 5500 nM (pChEMBL 5.260000228881836)
Descriptors: MW 349.42, ALogP 3.95, HBA 5, HBD 2, PSA 83.56, QED 0.59
Compound (SMILES): COc1cc(C(=O)N2CC3CCC2[C@@H]3N)cc2nc(-c3cc4ccc(-c5c(F)cc(O)cc5F)cc4n3CC3CC3)n(C)c12
InChIKey: RPYBUDYJXBOBAX-ABNDPDSASA-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 = 92 nM (pChEMBL 7.039999961853027)
Descriptors: MW 597.67, ALogP 5.83, HBA 7, HBD 2, PSA 98.54, QED 0.26
Compound (SMILES): CNc1nc(Nc2cnn(CC(C)OC(F)F)c2C)ncc1C(F)(F)F
InChIKey: RKFXATMQAIFGBD-UHFFFAOYSA-N
Target: Leucine-rich repeat serine/threonine-protein kinase 2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q5S007
Assay: Biochemical Assay : 50 mM Tris pH 7.5, 10 mM MgCl2, 1 mM EGTA, 0.01% Brij-35, 2 mM DTT, 5 nM LRRK2, 134 μM ATP, 60 minute reaction time, 23° C. reaction temperature, and 10 μL total reaction volume.Detection Reaction Conditions:1×TR-FRET dilution buffer, 10 mM EDTA, 2 nM antibody, 23° C. reaction temperature, and 10 μL total reaction volume.Compound solutions were prepared by initially diluting to 1 mM with DMSO. 35 μL of reference compound solution, 35 μL of test compound solution, and 35 μL HPE were successively added to the source plate (384-well assay plate, Labcyte). The plates were centrifuged at 2500 rpm for 1 minute and sealed in foil. POD was used to perform a 3.162 fold serial dilution and 100 nL of reference compound solution, test compound solution, HPE and ZPE were transferred to assay plates. The assay plate was centrifuged at 2500 rpm for 1 minute, and sealed with foil.
Measurement: IC50 > 60 nM (pChEMBL 7.2220001220703125)
Descriptors: MW 380.32, ALogP 3.41, HBA 7, HBD 2, PSA 76.89, QED 0.72
Compound (SMILES): CC[C@H]1[C@@H](O)[C@@H]2[C@H](CC[C@]3(C)[C@@H]([C@H](C)CCNC(=O)NS(=O)(=O)c4cccc(-c5nc(C)cs5)c4)CC[C@@H]23)[C@@]2(C)CC[C@@H](O)C[C@@H]12
InChIKey: KQCMXVPWDBRZFW-KSDNQMEYSA-N
Target: Bile acid receptor (SINGLE PROTEIN), Homo sapiens
UniProt: Q96RI1
Assay: Human FXR (NR1H4) Assay: The potency and efficacy of the compounds of the invention on TGR5 receptor was evaluated using in vitro assays which carried out using the express kit from DiscoverX (cAMP HUNTER eXpress GPBAR1 CHO-K1 GPCR Assay; Cataloguer number: 95-0049E2CP2S)GPBAR1 (G protein-coupled bile acid receptor 1) encodes a member of the G protein-coupled receptor (GPCR) superfamily. GPBAR1 activation following ligand binding initiates a series of second messenger cascades that result in a cellular response. Treatment of CHO cells expressing GPBAR1 with bile acids induces the production of intracellular cAMP and internalization of the receptor. The potency and efficacy of compound for GPBAR1 activation by measuring cyclic adenosine monophosphate (cyclic AMP or cAMP) levels in live cells using a competitive immunoassay based on Enzyme Fragment Complementation (EFC).In briefly, following seeding the cells into the white, 96 well microplate, place it in a 37° C., 5% CO2 in a humidified incubator for 18-24 hours prior to testing. On second day, proceed to the appropriate cAMP HUNTER eXpress Protocol according to the manufacturer's instructions. Dissolve agonist compound in DMSO at the desired stock concentration, and prepare 3-fold serial dilutions of agonist compound in Cell Assay Buffer. The concentration of each dilution should be prepared at 4× of the final screening concentration (i.e. 15 μL compound+45 μL Cell Assay Buffer/cAMP Antibody Reagent). For each dilution, the final concentration of solvent should remain constant. Transfer 15 μL diluted compound the assay plate and incubate the plate for 30 minutes at 37° C. Following agonist incubation, add 60 μL of working cAMP detection reagents/cAMP Solution mixture (cAMP Lysis Buffer, Substrate Reagent 1, cAMP Solution D) to the appropriate wells. Incubate for 1 hour at room temperature (23° C.), protected from light. Add 60 μl of cAMP Solution A to the appropriate wells. Incubate for 3 hours at room temperature (23° C.), protected from light. Read samples on Envision standard luminescence plate reader. Calculate of average EC50 after logarithm transformation.
Measurement: EC50 = 550 nM (pChEMBL 6.260000228881836)
Descriptors: MW 671.97, ALogP 6.76, HBA 7, HBD 4, PSA 128.62, QED 0.25
Compound (SMILES): Cc1nnc(-n2c(=O)n(CCN(C)C)c3ccc(S(=O)(=O)NC4(C)CC4)cc32)o1
InChIKey: BBZDXYRXDZKBMX-UHFFFAOYSA-N
Target: Poly(ADP-ribose) glycohydrolase (SINGLE PROTEIN), Homo sapiens
UniProt: Q86W56
Assay: PARG Assay: PARG In vitro assays were conducted in a total volume of 15 ul in a standard 384 well format. 5 ul of Human Full Length PARG (Produced internally by Astra Zeneca), used at a final reaction concentration of 80 μM, was added to 5 ul of Ribosylated PARP substrate (also produced internally by Astra Zeneca) at final reaction concentration of 4.5 nM in assay buffer (50 mM Tris pH7.4, 0.1 mg/ml BSA, 3 mM EDTA, 0.4 mM EGTA, 1 mM DTT, 0.01% Tween 20, 50 mM KCl). The reaction was incubated at room temperature for 10 minutes and then 5 ul detection reagent was added. Detection Reagent consists of 42 nM MAb Anti-6HIS XL665 (CisBio: 61 HISXLB) and 2.25 nM Streptavidin Europium Cryptate (CisBio: 610SAKLB), both at 3× working stock concentrations (final concentrations of 14 nM and 0.75 nM respectively), in a detection buffer of 50 mM Tris pH7.4, BSA at 0.1 mg/ml and KF at 100 mM. Following incubation at room temperature for 60 minutes in the dark, TR-FRET signal was measured at Ex 340 and Em 665 and Em 620. A ratio was calculated as Em665/EM620×104 for each well and used to calculate percent inhibition for test compounds.
Measurement: EC50 = 2284 nM (pChEMBL 5.639999866485596)
Descriptors: MW 420.5, ALogP 0.88, HBA 9, HBD 1, PSA 115.26, QED 0.6
Compound (SMILES): C=CC(=O)Nc1cc(Nc2ncc(C(=O)OC(C)C)c(-c3c[nH]c4ccccc34)n2)c(OC)cc1N(C)C[C@H]1CCCN1C
InChIKey: DQIRQCGGCGXFTQ-OAQYLSRUSA-N
Target: Epidermal growth factor receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P00533
Assay: ASV & NPG EGFR Exon 20 Insertion Mutations: A compound's ability in selectively inhibiting EGFR exon 20 insertion mutations can be assessed using Ba/F3 cells, a murine pro-B cell line, which have been transduced with EGFR exon 20 insertions. An expression vector, pLVX-IRES puro (Clontech) coding for human EGFR exon 20 insertions NPG (H773_V774insNPG) or ASV (V769_D770insASV), was transfected into HEK293 cells by the Trans-Lentiviral ORF Packaging System (Thermo Scientific), to produce virus encoding EGFR exon 20 insertions. Ba/F3 (DSMZ) cells maintained in RPMI 1640 medium supplemented with 10% fetal bovine serum, 200 μM L-glutamine/200 μg/mL penicillin/200 μg/mL streptomycin (Life Technology) and 10 ng/mL IL-3 (R&D system), were infected by EGFR Exon20 virus and subsequently selected by puromycin (Life Technology) selection and IL-3 depletion. Ba/F3 cells expressing EGFR exon 20 insertions (named Ba/F3-EGFR-Exon20-NPG or Ba/F3-EGFR-Exon20-ASV) can proliferate in the absence of IL-3. The anti-proliferative activity of compounds were determined as follows: BaF3-EGFR-Exon20 cells (NPG or ASV) seeded in 96 well plates (2500 cells/well) were treated with test compound (dissolved in DMSO) at a series of concentrations (4-fold dilution, top concentration: 10,000 nM). The plates were incubated for 72 h in a 37° C. incubator with 5% CO2, and the number of viable cells in each well were measured indirectly by CellTiter 96® Aqueous One Solution Cell Proliferation Assay (Promega) This assay is a colorimetric method for determining the number of viable cells through measurement of their metabolic activity by detection of enzymatic conversion of tetrazolium salts into blue formazan derivatives. Reagent (20 μL) was added into each well, and the plates were returned to the incubator for 2 h. The absorbance in each well was then measured at 490 nm using an Envision plate reader (Perkin Elmer). IC50 values were calculated by determining the concentration of compound required to decrease the MTS signal by 50% comparing to the DMSO control in best-fit curves using Microsoft XLfit software or Accelrys Pipeline Pilot.
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 597.72, ALogP 5.6, HBA 9, HBD 3, PSA 124.71, QED 0.15
Compound (SMILES): O=C1CS(=O)(=O)c2cc(Cl)cnc2N1Cc1ccc(F)c(OC(F)(F)F)c1
InChIKey: ODHIDXCWOJIAFN-UHFFFAOYSA-N
Target: Intermediate conductance calcium-activated potassium channel protein 4 (SINGLE PROTEIN), Homo sapiens
UniProt: O15554
Assay: KCa3.1 Assay: HEK293 cells over-expressing human KCa3.1 are used to measure the effects of compounds in inhibiting the KCa3.1 channel function. The assay is based on measuring the influx of Tl+ through KCa3.1 using a FLIPR dye assay (FLIPR Potassium Ion Channel Assay Kit #R8154 by Molecular Devices). The day before the assay HEK293/KCa3.1 cells are seeded on a 384 well plate. On the day of the assay, media is removed and replaced with Molecular Devices Potassium Ion Channel dye then incubated for 1 hour according to manufacturer's recommendations. The cells are then treated with compounds or DMSO control for 15 min, followed by addition of thallium (final concentration=1.5 mM) and ionomycin (final concentration=1 uM). The plate is read on HAMAMATSU 60000 measuring fluorescence signal for 50 seconds using (Em: 565-625 nm). Data analysis: HAMAMATSU 6000 data are exported using max-min value for frame 10 and 40 and corrected by spatial uniformity correction. Percent of control (POC) is calculated as 100×(max-minFlsample−max-minFllow)/(max-minFlhi−max-minFllow). Max-min Flsample is max-min value of each sample well containing test compound. Max-min Fllow is average max-min value of wells containing reference inhibitory compound.
Measurement: IC50 = 92 nM (pChEMBL 7.039999961853027)
Descriptors: MW 424.76, ALogP 3.09, HBA 5, HBD 0, PSA 76.57, QED 0.71
Compound (SMILES): COc1ncc(-c2cc(C(F)(F)F)c3c(N)ncnn23)cc1C(=O)N[C@@H]1CN(C(C)CC(O)C(F)(F)F)C[C@@H]1F
InChIKey: DREFOKCKQORHNO-MDMLBXLCSA-N
Target: Receptor-interacting serine/threonine-protein kinase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q13546
Assay: RIPK1 HTRF Binding Assay: A solution was prepared containing 0.2 nM Anti GST-Tb (Cisbio, 61GSTTLB), 90.6 nM probe and 1 nM His-GST-TVMV-hRIPK1(1-324) in FRET Buffer (20 mM HEPES, 10 mM MgCl2, 0.015% Brij-35, 4 mM DTT, 0.05 mg/mL BSA). Using Formulatrix Tempest, the detection antibody/enzyme/probe solution (2 mL) was dispensed into wells of a 1536 plate (Black Low Binding Polystyrene 1536 Plate (Corning, 3724)) containing 10 nL of compounds of interest at appropriate concentration in DMSO. The plate was incubated at rt for 1 h. FRET was measured using the EnVision plate reader (Excitation: 340 nM, Emission: 520 nM/495 nM). Total signal (0% inhibition) was calculated from wells containing 10 nL DMSO only. Blank signal (100% inhibition) calculated from wells containing 10 nL of 15 nM staurosporine and internal controls.
Measurement: IC50 = 87000 nM (pChEMBL 4.059999942779541)
Descriptors: MW 579.48, ALogP 2.85, HBA 9, HBD 3, PSA 130.9, QED 0.36
Compound (SMILES): Nc1c(-c2ccc3ncc(-c4cc(F)cc(F)c4)c(N4CCC5NCCO[C@@H]5C4)c3c2)cncc1C1CC1
InChIKey: WBDFPVARKUPMFX-PLYLYKGUSA-N
Target: Somatostatin receptor type 2 (SINGLE PROTEIN), Homo sapiens
UniProt: P30874
Assay: cAMP Assay: Four days prior to the assay, 5,000 Chinese hamster ovary cells (CHO-K1, ATCC # CCL-61) stably expressing the human SSTR2 are plated in each well of a 96-well tissue culture-treated plate in Ham's F12 growth media (ThermoFisher #10-080-CM) supplemented with 10% donor bovine serum (Gemini Bio-Products #100-506), 100 U/mL penicillin; 100 ug/mL streptomycin; 2 mM L-glutamine (Gemini Bio-Products #400-110) and 0.2 mg/mL hygromycin B (GoldBio #31282-04-9). The cells are cultured at 37° C., 5% CO2 and 95% humidity. On the day of the assay, the media is aspirated and the cells are treated with 50 μL of 1.6 μM NKH477 (Sigma # N3290) plus various dilutions of compounds of the invention in assay buffer [1× Hank's Balanced Salt Solution (ThermoFisher # SH3058802), 0.5 mM HEPES pH 7.4, 0.1% bovine serum albumin, 0.2 mM 3-Isobutyl-1-methylxanthine (IBMX, VWR #200002-790)]. The cells are incubated for 20 minutes at 37° C. (the final concentration of the compounds of the invention are typically 0-10,000 nM). The cells are treated with 50 μL of lysis buffer (HRTF cAMP kit, Cisbio). The lysate is transferred to 384-well plates and cAMP detection and visualization antibodies are added and incubated for 1-24 hours at room temperature. The time-resolved fluorescent signal is read with a Tecan M1000Pro multiplate reader.
Measurement: EC50 = 55 nM (pChEMBL 7.260000228881836)
Descriptors: MW 513.59, ALogP 5.27, HBA 6, HBD 2, PSA 76.3, QED 0.39
Compound (SMILES): CC(C)S(=O)(=O)c1ccc(Nc2nn([C@@H](CC#N)C3CC3)c3cc[nH]c(=O)c23)cc1
InChIKey: ORSAEBWZIDGJRU-KRWDZBQOSA-N
Target: Tyrosine-protein kinase JAK1 (SINGLE PROTEIN), Homo sapiens
UniProt: P23458
Assay: Jak Biochemical HTRF Assay Protocol: The ability of compounds to inhibit the activity of JAK1, JAK2, JAK3, and Tyk2 was measured using a recombinant purified GST-tagged catalytic domain for each enzyme (Invitrogen JAK1 #M4290, JAK2 #M4290, JAK3 #M4290, Tyk2 #M4290) in an HTRF format biochemical assay. The reactions employed a common peptide substrate, LCB-EQEDEPEGDYFEWLW-NH2 (in-house). The basic assay protocol is as follows: First, 250 nL of diluted compounds in DMSO were dispensed into the wells of a dry 384-well Black plate (Greiner #781076) using a Labcyte Echo 555 acoustic dispenser. Subsequent reagent additions employed an Agilent Bravo. Next, 18 μL of 1.11× enzyme and 1.11× substrate in 1× assay buffer (Invitrogen kinase buffer #PV3189, 2 mM DTT, 0.05% BSA) were added to the wells and shaken and then preincubated for 30 minutes at room temperature to allow compound binding to equilibrate. After equilibration, 2 μL of 10×ATP in 1× assay buffer was added to initiate the kinase reaction and the plates were shaken and then incubated at room temperature for 120 minutes. At the end of the incubation, 20 μL of 2× stop buffer (streptavidin-Dylight 650 (Thermo #84547B/100 mL), Eu-tagged pY20 antibody (Perkin Elmer #AD0067), EDTA, HEPES, and Triton) was added to quench the reaction. Plates were shaken and centrifuged and then incubated 60 minutes at room temperature and then read on a Perkin Elmer Envision (λex=337 nm, λem=665 and 615 nm, TRF delay time=20 μs). HTRF signal=10,000*665 nm reading/615 nm reading. After normalization to untreated controls, the percent inhibition of the HTRF signal at each compound concentration was calculated.
Measurement: IC50 = 120 nM (pChEMBL 6.920000076293945)
Descriptors: MW 425.51, ALogP 3.52, HBA 7, HBD 2, PSA 120.64, QED 0.6
Compound (SMILES): Cc1ncc(NC(=O)c2ccnc(C(F)(F)F)c2)cc1-c1cc(C2CCOCC2)nc(N2CCC(O)CC2)c1
InChIKey: RNCXZGGSSNOHCF-UHFFFAOYSA-N
Target: RAF proto-oncogene serine/threonine-protein kinase (SINGLE PROTEIN), Homo sapiens
UniProt: P04049
Assay: In Vitro Raf Activity Determination: Assay Materials: Assay buffer is 50 mM Tris, pH 7.5, 15 mM MgCl2, 0.01% Bovine Serum Albumin (BSA) and 1 mM dithiothreitol (DTT); Stop buffer is 60 mM ethylenediaminetetraacetic acid (EDTA) and 0.01% Tween® 20: b-Raf (V600E), active; biotinylated Mek, kinase dead; Alpha Screen detection kit (available from PerkinElmer, #6760617R); Anti phospho-MEK1/2 (available from Cell Signaling Technology, Inc. #9121); 384 well low volume assay plates (White Greiner® plates).Assay conditions: b-Raf (V600E) approximately 4 μM; c-Raf approximately 4 nM; biotinylated Mek, kinase dead approximately 10 nM; ATP 10 μM for BRAF (V600E) and 1 μM for CRAF; Pre-incubation time with compounds 60 minutes at room temperature; Reaction time 1 or 3 hours at room temperature.Assay protocol: Raf and biotinylated Mek (kinase dead) were combined at 2× final concentrations in assay buffer (50 mM Tris, pH 7.5, 15 mM MgCl2, 0.01% BSA and 1 mM DTT) and dispensed 5 ml per well in assay plates (Greiner white 384 well assay plates #781207) containing 0.25 ml of 40× of a Raf kinase inhibitor test compound diluted in 100% DMSO. The plate was incubated for 60 minutes at room temperature. The Raf kinase activity reaction was started by the addition of 5 mL per well of 2×ATP diluted in assay buffer. After 3 hours (b-Raf(V600E)) or 1 hour (c-Raf). The reactions were stopped and the phosphorylated product was measured using a rabbit anti-p-MEK (Cell Signaling, #9121) antibody and the Alpha Screen IgG (ProteinA) detection Kit (PerkinElmer #6760617R), by the addition of 10 mL to the well of a mixture of the antibody (1:2000 dilution) and detection beads (1:2000 dilution of both beads) in Stop/bead buffer (25 mM EDTA, 50 mM Tris, pH 7.5, 0.01% Tween20). The additions were carried out under dark conditions to protect the detection beads from light. A lid was placed on top of the plate and incubated for 1 hour at room temperature, after which the luminescence was read on a PerkinElmer Envision instrument. The concentration of each compound for 50% inhibition (IC50) was calculated by non-linear regression using XL Fit data analysis software.
Measurement: IC50 = 383 nM (pChEMBL 6.420000076293945)
Descriptors: MW 541.57, ALogP 4.97, HBA 7, HBD 2, PSA 100.47, QED 0.47
Compound (SMILES): COC(=O)N[C@@H](CC/C=C/C(=O)N(C)C)C(=O)Nc1cccn(Cc2nc3c(CCC(C)(C)C)ncnc3[nH]2)c1=O
InChIKey: NBYXOBMJBOIYAT-BEBFYNPSSA-N
Target: Mitogen-activated protein kinase kinase kinase kinase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q92918
Assay: HPK1 Biochemical Enzyme Assay: HPK1 biochemical enzyme assay: HPK1 enzyme inhibition was measured using a microfluidic mobility shift assay. Reactions were performed in a 384-well plate, containing 1.5 nM HPK1 (Invitrogen), in assay buffer (Carna Biosciences; pH 7.4). Test compounds were titrated in ten point curves (top final assay concentration 3 μM), and preincubated with enzyme/substrate mix for 30 min prior to initiation of the reaction by addition of ATP (1 mM final concentration) and substrate (1 μM final concentration; Carna Biosciences) diluted in assay buffer supplemented by MgCl2 (final assay concentration of 5 mM). Following 60 min incubation at RT, the reaction was terminated by addition of 60 μl/well termination buffer (Carna Biosciences) and signal determination using a Caliper EZ Reader (Perkin Elmer, UK).
Measurement: IC50 < 500 nM (pChEMBL 6.301000118255615)
Descriptors: MW 566.66, ALogP 2.63, HBA 9, HBD 3, PSA 164.2, QED 0.3
Compound (SMILES): N#Cc1ccc(-c2cc(OCc3ccccc3)cc3ncnn23)cc1
InChIKey: WRHATRFGTXCACG-UHFFFAOYSA-N
Target: Prolyl hydroxylase EGLN2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q96KS0
Assay: PHD1 Enzyme Assay: The IC50 values for the PHD1 enzyme (residues 1-407) were determined by mixing increasing amounts of a compound of the invention with a fixed amount of the enzyme (20 nM final concentration) and peptide substrate (Asp-Leu-Asp-Leu-Glu-Ala-Leu-Ala-Pro-Tyr-Ile-Pro-Ala-Asp-Asp-Asp-Phe-Gln-Leu, 1 μM final concentration) and 2-oxoglutarate (0.5 M final concentration) in an assay buffer comprising 30 mM 2-(N-morpholino)ethanesulfonic acid pH 6.0, 2 mM sodium ascorbate, 100 μM dithiothreitol, 2 mg/ml bovine serum albumin, 60 μg/ml catalase enzyme and 1 μM iron (II) sulphate (FeSO4). The reaction was conducted by pre-incubating the PHD1 enzyme in the presence of a compound of the invention for 60 minutes at room temperature. The activity of the free enzyme was measured by adding the peptide, the 2-oxoglutarate and sodium ascorbate (see above for final concentrations). The assay was quenched by the addition of 30% v/v trichloroacetic acid (final concentration 5%). The amount of product released was measured using a UPLC-MS (Agilent 1290 with an ABSciex 4000qTrap Mass Spectrometer). Data were analysed using the classical isotherm equation for the determination of IC50.
Measurement: IC50 = 49 nM (pChEMBL 7.309999942779541)
Descriptors: MW 326.36, ALogP 3.85, HBA 5, HBD 0, PSA 63.21, QED 0.57
Compound (SMILES): COC(=O)N1CCC[C@H](NS(C)(=O)=O)[C@@H]1CO[C@H]1CC[C@@H](c2cc(F)ccc2F)CC1
InChIKey: QDCQATOYWNLFAU-ZXUOCUECSA-N
Target: Orexin receptor type 2 (SINGLE PROTEIN), Homo sapiens
UniProt: O43614
Assay: Measurement of Orexin Type 2 Receptor Agonist Activity: Chinese hamster ovary (CHO) dhfr-cells forcibly expressing human orexin type 2 receptor (hOX2R) were seeded in each well of Black clear bottom plate (384 wells) (Becton, Dickinson and Company) by 10,000 cells, and cultured for 16 hr in an MEM-alpha (Nikken-Bio Co., Ltd.) medium containing 100 U/ml penicillin, 100 μg/ml streptomycin, 0.5 g/ml G418 (all above Invitrogen), and 10% fetal calf serum (Thermo), under the conditions of 37° C., 5% CO2. After removal of the medium, 30 μL of assay buffer 1 (0.1% bovine serum albumin (Wako Pure Chemical Industries, Ltd.), 1.25 mM probenecid, 10% B2-Quencher, 2.5 μg/mL Fluo-4AM, 10 mM HEPES (DOJINDO)) was added, and the cells were incubated for 60 min under the conditions of 37° C., 5% CO2. A test compound was dissolved in dimethyl sulfoxide to 10 mM, and then diluted with assay buffer 2 (20 mM HEPES, Hanks' balanced salt solution (Invitrogen), 0.1% bovine serum albumin). For the reaction, a test compound solution (10 μL) was added using Fluorescent Imaging Plate Reader TETRA (FLIPR TETRA; manufactured by Molecular Devices), a fluorescence value (excitation wavelength 488 nm, measurement wavelength 570 nm) of each well was measured every one second for 1 min, and the agonist activity was determined using the area of the fluorescence value as an indicator of intracellular Ca2+ concentration. The agonist activity of the test compound was calculated assuming that the fluorescence value of the well added with only the dilution buffer was 0% and the fluorescence value of the well added with 10 nM human orexin B (PEPTIDE INSTITUTE, INC.) buffer was 100%. The agonist activity values EC50 and Emax of each compound are shown below. As used herein, Emax indicates the value at 30 uM concentration when orexin B is converted to a full agonist (maximum value of agonist activity: 100%). As is clear from the results, the compound of the present invention was shown to have an agonist activity on hOX2R.
Measurement: EC50 = 2 nM (pChEMBL 8.699999809265137)
Descriptors: MW 460.54, ALogP 3.16, HBA 5, HBD 1, PSA 84.94, QED 0.71
Compound (SMILES): COC(=O)/C=C/CC[C@H](NC(=O)[C@H](CC(N)=O)NC(C)=O)C(=O)N[C@@H](CCC(=O)O)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@H](C(=O)N[C@@H](CO)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CC(C)C)C(=O)N[C@H](C(=O)O)C(C)O)C(C)C
InChIKey: SGNXSVCSTVRNNI-HGNXJLQFSA-N
Target: Coagulation factor XIII A chain (SINGLE PROTEIN), Homo sapiens
UniProt: P00488
Assay: Isopeptidase Assay for Estimating Inhibitor Potency: FXIIIa activity has been determined using substrate A101 (Zedira GmbH, Darmstadt, Germany), which is based on the N-terminal dodecapeptide of α2-antipiasmin. FXIIIa catalyzes by its isopeptidase activity the release of dark quencher dinitrophenyl at the original substrate glutamine position resulting in fluorescence increase (based on the N-terminal 2-aminobenzoyl fluorescent dye) (Oertel K, HunfekJ A, Specker E, Reiff C, Seitz R, Pasternack R, Dodt J. A highly sensitive fluorometric assay for determination of human coagulation factor XIII in plasma. Anal Biochem 2007; 367:152-8.)Briefly, 12 μL recombinant cFXIII-A2 (T027, Zedira GmbH, Darmstadt, Germany) or FXIII-A2B2 derived from human plasma (T007) (25 μg/mL) and 3 μL human α-thrombin (0.5 U/mL, T056, Zedira) were mixed with 270 μL assay buffer (50 mM Tris-HCl, 10 mM CaCl2), 150 mM NaCl, 5.56 mM glycine methyl ester, 5 mM DTT, pH 7.5) containing 55 μM A101 substrate. The mixture was incubated for 20 min at room temperature to activate FXIII. 15 μL of inhibitor solution (serial dilution from 1.25 μM to 1.25 nM) dissolved in DMSO/assay buffer were added, mixed and the kinetic measurement started after 3 min. Fluorescence emission was monitored at 418 nm (λex=313 nm) and 37° C. for 30 min using a CLARIOstar fluorescence micro plate reader (BMG Labtech, Ortenberg, Germany). For measurements without inhibitor, 15 μL of assay buffer/2% (v/v) DMSO were added. All measurements were performed in triplicate. The respective IC50 values were calculated by non-linear regression using the MARS software package (BMG Labtech).
Measurement: IC50 = 8475 nM (pChEMBL 5.070000171661377)
Descriptors: MW 1098.17
Compound (SMILES): CC[C@H](Nc1cc(C(F)(F)F)c(-c2sc(C(=O)NCC(C)(C)O)nc2C(=O)N2C3CCC2CC3)cn1)C(F)(F)F
InChIKey: OKBHNPDIMWPTQZ-ZUEPYMLJSA-N
Target: Nuclear receptor ROR-gamma (SINGLE PROTEIN), Homo sapiens
UniProt: P51449
Assay: ThermoFluor Assay: 1,8-ANS (Invitrogen) was used as a fluorescent dye. Protein and compound solutions are dispensed into black 384-well polypropylene PCR microplates (Abgene) and overlayed with silicone oil (1 μL, Fluka, type DC 200) to prevent evaporation.Bar-coded assay plates are robotically loaded onto a thermostatically controlled PCR-type thermal block and then heated at a typical ramp-rate of 1° C./min for all experiments. Fluorescence was measured by continuous illumination with UV light (Hamamatsu LC6) supplied via fiber optic and filtered through a band-pass filter (380-400 nm; >6 OD cutoff). Fluorescence emission of the entire 384-well plate was detected by measuring light intensity using a CCD camera (Sensys, Roper Scientific) filtered to detect 500±25 nm, resulting in simultaneous and independent readings of all 384 wells. Images were collected at each temperature, and the sum of the pixel intensity in a given area of the assay plate was recorded versus temperature. Reference wells contained RORγt without compounds, and the assay conditions were as follows:    0.065 mg/mL RORγt    60 μM 1,8-ANS    100 mM Hepes, pH 7.0    10 mM NaCl    2.5 mM GSH    0.002% Tween-20
Measurement: Kd = 3.2 nM (pChEMBL 8.489999771118164)
Descriptors: MW 593.59, ALogP 5.24, HBA 7, HBD 3, PSA 107.45, QED 0.36
Compound (SMILES): CCCCCn1c2cc[nH]c2c(=O)n2c(C)nnc12
InChIKey: UTULOYNJGOVYDE-UHFFFAOYSA-N
Target: Adenosine receptor A2a (SINGLE PROTEIN), Homo sapiens
UniProt: P29274
Assay: HTRF Assay: Assays were conducted in black low volume 384-well polystyrene plates (Greiner 784076-25) in a final volume of 10 μL. Test compounds were first serially diluted in DMSO and 100 nl added to the plate wells before the addition of other reaction components. The final concentration of DMSO was 1%. Tag-lite® Adenosine A2A labeled cells (CisBio C1TT1A2A) were diluted 1:5 into Tag-lite buffer (CisBio LABMED) and spun 1200 g for 5 mins. The pellet was resuspended at a volume 10.4× the initial cell suspension volume in Tag-lite buffer, and Adenosine A2A Receptor Red antagonist fluorescent ligand (CisBio L0058RED) added at 12.5 nM final concentration. 10 ul of the cell and ligand mix was added to the assay wells and incubated at room temperature for 45 minutes before reading on a PHERAstar FS plate reader (BMG Labtech) with HTRF 337/620/665 optical module. Percent binding of the fluorescent ligand was calculated; where 100 nM of A2A antagonist control ZM 241385 (Tocris 1036) displaces the ligand 100% and 1% DMSO has 0% displacement. The % binding data versus the log of the inhibitor concentration was fitted to a one-site competitive binding model (GraphPad Prism version 7.02) where the ligand constant=12.5 nM and the ligand Kd=1.85 nM.
Measurement: Ki = 550 nM (pChEMBL 6.260000228881836)
Descriptors: MW 259.31, ALogP 1.87, HBA 5, HBD 1, PSA 67.98, QED 0.73
Compound (SMILES): CC(C)C(=N)/C=C(\N)C(=O)Nc1ccc([C@@H]2O[C@H]3CN[C@@H]2C3)cc1
InChIKey: GJCQBOUAEJUWPW-WCTMDYHZSA-N
Target: Trace amine-associated receptor 1 (SINGLE PROTEIN), Mus musculus
UniProt: Q923Y8
Assay: Radioligand Binding Assay: HEK-293 cells stably expressing rat 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 μg/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 48'000×g 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.
Measurement: IC50 = 9.9 nM (pChEMBL 8)
Descriptors: MW 328.42, ALogP 1.95, HBA 5, HBD 4, PSA 100.23, QED 0.49
Compound (SMILES): CNC(=O)c1cc(C(=O)N2CCC(c3ccccc3)C2)no1
InChIKey: FBMGHVBJNLZMPL-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 = 4190 nM (pChEMBL 5.380000114440918)
Descriptors: MW 299.33, ALogP 1.66, HBA 4, HBD 1, PSA 75.44, QED 0.94
Compound (SMILES): COCCC(=O)N1CCC[C@@H](c2nc(-c3ccc(C(=O)Nc4ccccn4)nc3)c3c(N)nccn23)C1
InChIKey: UWTCDSRULALDQF-GOSISDBHSA-N
Target: Tyrosine-protein kinase BTK (SINGLE PROTEIN), Homo sapiens
UniProt: Q06187
Assay: Btk Enzyme Activity Assay A: Btk enzyme activity is measured using the IMAP (immobilized metal ion affinity-based fluorescence polarization) assay as outlined below. Btk enzyme (His-Btk (Millipore catalog #14-552), is diluted to 0.4 U/mL in KR buffer (10 mM Tris-HCl, 10 mM MgCl2, 0.01% Tween-20, 0.05% NaN3, 1 mM DTT, 2 mM MnCl2, pH 7.2).Serial dilution log 10 from 2 mM to 63.2 nM of test compounds are made in 100% DMSO. The dilutions in DMSO are then diluted 50-fold in KR-buffer. Final compound concentration range in the assay from 10 μM to 0.316 nM. 5 μL/well of test compound in KR buffer (final DMSO concentration in the assay is 1%) is mixed with 5 μl/well of 0.4 U/mL Btk enzyme (final concentration in the assay is 0.1 U/mL). Test compounds and Btk enzyme are pre-incubated 60 minutes at room temperature, before adding 5 μL/well of 200 nM Fluorescin labeled substrate peptide (Blk/Lyntide substrate, e.g. #R7188/#R7233, Molecular Devices) in KR-buffer. Final peptide substrate concentration in assay is 50 nM. The kinase assay is started by adding 5 μL/well of 20 μM ATP in KR-buffer (final ATP concentration is 5 μM ATP, Km ATP in Btk IMAP assay). Following incubation for 2 h at room temperature the enzyme reaction is stopped by adding 40 μL/well IMAP Progressive Binding Solution (according to suppliers (Molecular Devices) protocol using 75% 1× buffer A and 25% 1x buffer B with 1:600 Progressive Binding Solution). After 60 min incubation at room temperature in the dark the FP signal is read. Fluorescence at 535 nm is measured using parallel and perpendicular filters to determine differences in rotation due to binding of the phosphorylated substrate peptide to the beads. Values are calculated as percentage of the difference in readout (ΔmPi) of the controls with and without ATP. EC50 values are determined by curve fitting of the experimental results using Activity Base.
Measurement: EC50 = 55 nM (pChEMBL 7.260000228881836)
Descriptors: MW 500.56, ALogP 2.76, HBA 9, HBD 2, PSA 140.63, QED 0.39
Compound (SMILES): COc1cccc(-c2ccc(CNC(=O)[C@@H]3CC3c3ccccc3)c3c2CCN(C(=O)NC2(C)COC2)C3)c1
InChIKey: XGNJRNNQHRXMDE-PLYLYKGUSA-N
Target: Diacylglycerol O-acyltransferase 2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q96PD7
Assay: Enzymatic Activity Assay: DGAT2 activity was determined by measuring the amount of enzymatic product triolein (1,2,3-Tri(cis-9-octadecenoyl)glycerol) using the membrane prep mentioned above. The assay was carried out in deep well 384 plates in a final volume of 40 μL at room temperature. The assay mixture contained the following: assay buffer (100 mM Tris.Cl, pH 7.0, 20 mM MgCl2, 5% ethanol), 25 μM of diolein, 10 μM of oleoyl-CoA and 10 ng/μL of DGAT2 membrane.
Measurement: IC50 = 13.8 nM (pChEMBL 7.860000133514404)
Descriptors: MW 525.65, ALogP 4.64, HBA 4, HBD 2, PSA 79.9, QED 0.47