lovingscience/chembl-v37-raw-activities huggingface dataset:
Compound (SMILES): COCC1CCN(C(=O)/C=C/c2ccc(C(F)(F)F)cc2Cn2nnc(C)n2)CC1
InChIKey: GHNLGCBEDHCYTG-GQCTYLIASA-N
Target: Autotaxin (SINGLE PROTEIN), Homo sapiens
UniProt: Q13822
Assay: ATX Assay: Reagents LPC (oleoyl (18:1)) was purchased from Avanti Polar Lipids (Alabaster, Ala.) and solubilized in methanol to 20 mM. Amplex Red was obtained from Invitrogen Life Technologies (Paisley, UK) and dissolved in DMSO to 10 mM. Choline oxidase and horseradish peroxidase (HRP) were obtained from Sigma Aldrich (Dorset, UK) and dissolved in HBSS to 20 U/ml and 200 U/ml respectively. All reagents were stored at −20° C. in single use aliquots. All experimental measurements were performed in assay buffer made up immediately prior to use (HBSS, 0.01% BSA essentially fatty acid free).Protein Recombinant human ATX was prepared at Novartis (Basel, CH) in a human embryonic kidney (HEK) cell preparation, and stored in single use aliquots of 26 mg/ml (26 μM) stocks stored at −80° C.Method All experimental measurements were performed in black 384 well polystyrene (low volume, round bottom, Corning (3676)) plates. PerkinElmer EnVision (Fluorescence Intensity/Absorbance Monochromator) or Tecan Infinite 200 PRO series plate reader was used to detect change in fluorescent intensity.Assessing ATX inhibition ATX activity was determined by measurement of released choline in reactions containing ATX (10 nM), choline oxidase (0.1 U/ml), HRP (100 U/ml), amplex red (50 μM) and LPC 18:1 (10 μM). Compounds of the invention were prepared as 10 point serial dilutions from 1 μM in duplicate and pre-incubated with ATX at 37° C. for 20 minutes prior to the addition of remaining reagents. The liberated choline was measured from changes in fluorescence intensity (λex 530 nm, λem 590 nm) of the product resurofin at 37° C. every 2 minutes over a 40-minute period. ATX activity was measured as a slope of the linear portion of the progress curve, typically between 14 to 24 minutes.Data analysis Slope data was exported to Graphpad prism (Graphpad software, San Diego, Calif.)
Measurement: IC50 = 25 nM (pChEMBL 7.599999904632568)
Descriptors: MW 423.44, ALogP 2.95, HBA 6, HBD 0, PSA 73.14, QED 0.67
Compound (SMILES): N#CC(NNc1cc(F)cc(Cl)c1)C(=O)c1noc2ccccc12
InChIKey: XMVPARVULIQZSI-UHFFFAOYSA-N
Target: Rap guanine nucleotide exchange factor 3 (SINGLE PROTEIN), Homo sapiens
UniProt: O95398
Assay: In Vitro Evaluation of EPAC1 Inhibition: To explore the SARs and examine how the modifications on the isoxazole ring affect biological activities of newly synthesized analogues, we first evaluated their ability to inhibit EPAC1-mediated Rap1b-bGDP exchange activity using purified recombinant full-length EPAC1 proteins. Previous hit 1 was used as the reference compound, with an IC50 value of 10.8 μM in inhibiting EPAC1 (Zhu et al., Sci. Rep. (2015) 5:9344).
Measurement: IC50 = 18900 nM (pChEMBL 4.71999979019165)
Descriptors: MW 344.73, ALogP 3.31, HBA 6, HBD 2, PSA 90.95, QED 0.54
Compound (SMILES): Cn1ccc(-c2nc(C(N)=O)[nH]c2-c2ccc3ncsc3c2)n1
InChIKey: OTSRYBQLSNIGHW-UHFFFAOYSA-N
Target: TGF-beta receptor type-1 (SINGLE PROTEIN), Homo sapiens
UniProt: P36897
Assay: LanthaScreenEu Kinase Binding Assay TGFBR1 (ALK5): Exemplified compounds were screened in 1% DMSO (final concentration) by 3-fold serial dilutions from 10,000 nM to 0.316 nM to produce an IC50 or at single concentrations of 10,000, 1000 and 100 nM according to the following protocol. To low volume, white 384-well plates (Greiner Cat #784207) add 160 nL 100× compound in 100% DMSO, 3.84 uL kinase buffer (50 mM HEPES, pH 7.5, 0.01% BRIJ-35, 10 mM MgCl2, 1 mM EGTA), 8 uL 2× kinase/antibody mixture in kinase buffer (final ALK5 concentration of 5 nM; final Eu-anti-GST antibody concentration of 2 nM), 4 uL 4× Tracer #178 in kinase buffer (final concentration of 5 nM). Gently shake the plates for 30 seconds and incubate at room temperature for 1 hour before reading fluorescence on a plate reader. Percent inhibition was determined compared to DMSO only control (maximal ALK5 tracer binding) and sigmoidal dose response curve fit yielded IC50 values as indicated in Table 2. For those compounds marked with an asterisk, activity was evaluated at three concentrations (10000, 1000, 10 nM) and the IC50 is depicted as less than the lowest concentration producing >50% inhibition.
Measurement: IC50 = 45.8 nM (pChEMBL 7.340000152587891)
Descriptors: MW 324.37, ALogP 2.19, HBA 6, HBD 2, PSA 102.48, QED 0.6
Compound (SMILES): Cn1cc(-c2nc3c(-c4ccc(CNC(=O)c5noc(C(C)(C)N)n5)c(F)c4)ccnc3[nH]2)cn1
InChIKey: QTKNXMMRIYLQTM-UHFFFAOYSA-N
Target: Tyrosine-protein kinase BTK (SINGLE PROTEIN), Homo sapiens
UniProt: Q06187
Assay: Microfluidic Off-Chip Mobility Shift Assay Protocol for Potency Assessment Against BTK Enzyme: Briefly, 2.5× stocks of full-length human BTK (08-080) from CarnaBio USA, Inc., Natick, Mass., 1.6×ATP and appropriate kinKDR peptide substrate (FITC-AHA-EEPLYWSFPAKKK-NH2; developed in-house) were prepared in kinase reaction buffer consisting of 25 mM MgCl2, 0.015% Brij-35 (30%), 100 mM HEPES, pH 7.5, and 10 mM DTT. 5 uL of enzyme buffer and 7.5 uL of ATP/kinKDR peptide substrate mix were added to Matrix (#115304) 384-well, sterile, polypropylene plates (Thermo Fisher Scientific, Hudson, N.H.) with 125 nL of serially diluted compounds prepared in 100% DMSO, and incubated for 90 min. at 27° C. Following the incubation period, reactions were stopped by adding 60 uL stop buffer consisting of 100 mM HEPES, pH 7.5, 0.015% Brij-35 (30%), 0.277% Coating Reagent #3 (Caliper Life Sciences, Mountain View, Calif.), 5% DMSO. Stopped reactions were monitored at −2 PSI, −3000 V/−700 V in a LabChip 3000 plate reader from Caliper Life Sciences, a PerkinElmer Company (Hopkinton, Mass.), and the activity was measured by off-chip mobility shift assay measuring the charge/mass difference between substrate and product resulting from peptide phosphorilation. IC50 and efficacy were determined by plotting log [Inhibitor] vs. % Activity in GeneData Screener (Basel. Switzerland).
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 475.49, ALogP 2.67, HBA 9, HBD 3, PSA 153.43, QED 0.34
Compound (SMILES): CCS(O)(O)c1ccc(OCC2CC2)c(-c2cn(C)c(=O)c3ccoc23)c1
InChIKey: SLTJQUOAFLMIRL-UHFFFAOYSA-N
Target: Bromodomain-containing protein 4 (SINGLE PROTEIN), Homo sapiens
UniProt: O60885
Assay: In Vitro Enzyme Inhibition Assay: Determination of the IC50 for the heterocyclic derivative BRD4 inhibitors disclosed herein was performed as follows. His-tagged BRD4 was cloned, expressed and purified to homogeneity. BRD4 binding and inhibition was assessed by monitoring the interaction of biotinylated H4-tetraacetyl peptide (AnaSpec, H4K5/8/12/16(Ac), biotin-labeled) with the target using the AlphaScreen technology (Life Technologies). In a 384-well ProxiPlate BRD4(BD1) (2 nM final) was combined with peptide (15 nM final) in 50 mM HEPES (pH 7.3), 10 mM NaCl, 0.25 mM TCEP, 0.1% (w/v) BSA, and 0.005% (w/v) Brij-35 either in the presence of DMSO (final 0.4% DMSO) or compound dilution series in DMSO. After 20 min incubation at room temp, Alpha streptavidin donor beads and Nickel Chelate acceptor beads were added to a final concentration of 5 μg/mL. After 2 hr of equilibration, plates were read on an Envision instrument and the IC50 was calculated using a four parameter non-linear curve fit.
Measurement: IC50 < 500 nM (pChEMBL 6.301000118255615)
Descriptors: MW 389.47, ALogP 4.72, HBA 6, HBD 2, PSA 84.83, QED 0.64
Compound (SMILES): Nc1nc2cc(CCN3CCN(c4cccc5sccc45)CC3)ccc2s1
InChIKey: AMCGNHPMLVBAQK-UHFFFAOYSA-N
Target: 5-hydroxytryptamine receptor 1A (SINGLE PROTEIN), Homo sapiens
UniProt: P08908
Assay: 5-HT1A Receptor Agonism Activity Test: The 5-HT1A receptor agonism activity test (The agonism activity of test compounds on 5-HT1A receptor expressing human recombinant 5-HT1A receptor in HEK293 cells) was performed using LANCE cAMP 384 Kit (Product of USA PerkinElmer Inc.). The 5-HT1A receptor agonism activity of test compounds was evaluated through their inhibition on cAMP production in HEK293 cells. cAMP concentration test was performed according to the method documented in the kit instructions. The concentration of the test compounds was 0.1 nM-10000 nM, 8-OH-DPAT was used as a positive control, EC50 value was calculated by the Excelfit software.
Measurement: EC50 = 0.379 nM (pChEMBL 9.420000076293945)
Descriptors: MW 394.57, ALogP 4.46, HBA 6, HBD 1, PSA 45.39, QED 0.56
Compound (SMILES): COc1cc(C(=O)N2C[C@H](N)C[C@@H](F)C2)cc2nc(-c3cc4ccc(-c5cnc6[nH]c(=O)[nH]c6c5)nc4n3CC3CC3)n(C)c12
InChIKey: UDLOKUPYPMDNIK-NHCUHLMSSA-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 = 18 nM (pChEMBL 7.75)
Descriptors: MW 609.67, ALogP 3.75, HBA 9, HBD 3, PSA 152.74, QED 0.26
Compound (SMILES): O=C(Nc1ccc(-c2cc[nH]n2)cc1)C1c2cccc(-c3cncnc3)c2CCN1C(=O)c1cn(-c2cccc(Cl)c2F)nn1
InChIKey: UKOSTGTZPKRMJF-UHFFFAOYSA-N
Target: Coagulation factor XI (SINGLE PROTEIN), Homo sapiens
UniProt: P03951
Assay: Enzyme Inhibition Assay: Factor XIa determinations were made in 50 mM HEPES buffer at pH 7.4 containing 145 mM NaCl, 5 mM KCl, and 0.1% PEG 8000 (polyethylene glycol; JT Baker or Fisher Scientific). Determinations were made using purified human Factor XIa at a final concentration of 25-200 pM (Haematologic Technologies) and the synthetic substrate S-2366 (pyroGlu-Pro-Arg-pNA; CHROMOGENIX or AnaSpec) at a concentration of 0.0002-0.001 M.
Measurement: Ki < 5 nM (pChEMBL 8.300999641418457)
Descriptors: MW 620.05, ALogP 5.29, HBA 8, HBD 2, PSA 134.58, QED 0.26
Compound (SMILES): Cc1nc(-c2ccc(Nc3cc(Nc4cnc(C)c(C)n4)nc4[nH]c(C(F)F)nc34)c(N(C)S(C)(=O)=O)c2)n(C)n1
InChIKey: YGWUMDOKPAMYSH-UHFFFAOYSA-N
Target: Non-receptor tyrosine-protein kinase TYK2 (SINGLE PROTEIN), Homo sapiens
UniProt: P29597
Assay: Binding Assay: Binding constants for compounds of the present invention against the JH2 domain were determined by the following protocol for a KINOMEscan® assay (DiscoveRx). A fusion protein of a partial length construct of human TYK2 (JH2domain-pseudokinase) (amino acids G556 to D888 based on reference sequence NP_003322.3) and the DNA binding domain of NFkB was expressed in transiently transfected HEK293 cells. From these HEK 293 cells, extracts were prepared in M-PER extraction buffer (Pierce) in the presence of Protease Inhibitor Cocktail Complete (Roche) and Phosphatase Inhibitor Cocktail Set II (Merck) per manufacturers' instructions. The TYK2 (JH2domain-pseudokinase) fusion protein was labeled with a chimeric double-stranded DNA tag containing the NFkB binding site (5′-GGGAATTCCC-3′) fused to an amplicon for qPCR readout, which was added directly to the expression extract (the final concentration of DNA-tag in the binding reaction is 0.1 nM).
Measurement: Kd = 0.3 nM (pChEMBL 9.520000457763672)
Descriptors: MW 583.63, ALogP 4.29, HBA 11, HBD 3, PSA 159.5, QED 0.24
Compound (SMILES): CCS(=O)(=O)c1ccc(Cl)cc1Cn1c(=O)[nH]c2cc(Br)c(OC(F)(F)F)cc2c1=O
InChIKey: QVOLEGNLUXOSJZ-UHFFFAOYSA-N
Target: Epithelial discoidin domain-containing receptor 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q08345
Assay: Binding Activity: DDR1 binding activity was measured using the LanthaScreen(Registered trademark) Eu Kinase Binding Assay (manufactured by Life Technologies Corporation). The test compound and the Alexa Fluor® 647-labeled Kinase Tracer 178 (manufactured by Life Technologies Corporation) were added to a mixture of DDR1 and the LanthaScreen(Registered trademark) Eu-anti-GST antibody. After one-hour reaction at room temperature, the fluorescence resonance energy transfer was measured. The 50% inhibitory concentration (IC50) was calculated from the inhibition rate relative to the test compound-free control.
Measurement: IC50 = 70 nM (pChEMBL 7.159999847412109)
Descriptors: MW 541.73, ALogP 3.85, HBA 6, HBD 1, PSA 98.23, QED 0.53
Compound (SMILES): CN1C(=O)CC[C@H]1C(=O)N1CCC2(S(=O)(=O)c3ccc(F)cc3)c3ccc(C(F)(C(F)(F)F)C(F)(F)F)cc3CC12
InChIKey: SLLNCMQNYXQVBT-UDTLNKQYSA-N
Target: Nuclear receptor ROR-gamma (SINGLE PROTEIN), Mus musculus
UniProt: P51450
Assay: General RORgamma SPA binding assay : The binding of potential ligands to RORγ is measured by competition with [3H]25-hydroxycholesterol (Perkin Elmer NET674250UC) using a scintillation proximity assay (SPA). The ligand binding domain of human RORγ (A262-S507) with an N-terminal His tag is expressed in E. coli and purified using nickel affinity chromatography. 15 μg/well RORγ (A262-S507) is incubated with test compound at varying concentrations in 3-fold serial dilution, with final concentrations ranging from 16.6 μM to 0.28 nM, for 10 min at rt in PBS buffer (Invitrogen #14190-144) containing 0.5% fatty acid free BSA (Gemini Bio-Products, Cat. #700-107P) and 0.1% glycerol (Sigma Cat#G5516). 10 nM of [3H] 25-hydroxycholesterol is then added, and the reaction is incubated for 10 min. 10 mg/mL of Copper-His Tag-PVT beads (Perkin Elmer cat # RPNQ0095) are added, and the mixture is incubated for 60 min. The reaction is read on a TopCount Microplate scintillation plate reader (Perkin Elmer). The competition data of the test compound over a range of concentrations was plotted as percentage inhibition of radioligand specifically bound in the absence of test compound (percent of total signal). After correcting for non-specific binding, IC50 values were determined. The IC50 value is defined as the concentration of test compound needed to reduce [3H] 25-hydroxycholesterol specific binding by 50% and is calculated using the four parameter logistic equation to fit the normalized data.
Measurement: IC50 = 336 nM (pChEMBL 6.46999979019165)
Descriptors: MW 610.52, ALogP 4.56, HBA 4, HBD 0, PSA 74.76, QED 0.38
Compound (SMILES): CC(C)(O)[C@H](F)CNC(=O)c1cnc(-c2ccc3cc(C#N)cnn23)cc1N[C@H]1CC[C@H](C2CCOCC2)CC1
InChIKey: WWIMEJDBWFXPOY-QJCIYXEWSA-N
Target: Interleukin-1 receptor-associated kinase 4 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9NWZ3
Assay: Biochemical Assay: The enzyme and peptide solution were incubated with compound for 15 minutes at room temp before the reaction was initiated by the addition of ATP. The standard 5ul reaction mixture contained 500uM ATP, 2uM peptide (STK1 Peptide), 0.75 nM of IRAK4 in reaction buffer (50 mM HEPES, pH 7.0 nM of IRAK4 in reaction buffer (50nM HEPES, pH 7.0, 0.02% NaN3, 0.01% BSA, 0.1mM Orthovanadate, 5nM MgCl2, 0.025% NP-40, 1mM DTT). After 120 min of incubation at room temperature, 5ul of Stop and Detect Solution *1:100 Cryptate labeled anti-phosphorylated peptide antibody solution and 125nM Tracer in a 50 nM HEPES pH 7.0 detection buffer containing sufficient EDTA) was added. The plate was then further incubated for 60 minutes at room temperature and read on Envision 2013 Multilabeled reader (PerkinElmer) with excitation/emission/FRET emission at 340nm/615nm/665nM, respectively.
Measurement: IC50 = 1 nM (pChEMBL 9)
Descriptors: MW 548.66, ALogP 4.5, HBA 8, HBD 3, PSA 124.57, QED 0.38
Compound (SMILES): CCC1CN(c2nnc(C)cc2-c2nc(C)no2)CCC1NC1CCCCC1
InChIKey: FVGZZFCTAQZKEG-UHFFFAOYSA-N
Target: Kappa-type opioid receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P41145
Assay: enzyme assay: Well-known assay for kappa opioid receptor
Measurement: IC50 = 24 nM (pChEMBL 7.619999885559082)
Descriptors: MW 384.53, ALogP 3.67, HBA 7, HBD 1, PSA 79.97, QED 0.84
Compound (SMILES): C=CC(=O)N1CCC(C(=O)Nc2cccc(CNc3nc(OC)nc4c(C(C)C)cnn34)c2)CC1
InChIKey: ZSEPOSLDBYTTOP-UHFFFAOYSA-N
Target: CDK7/Cyclin H/MNAT1 (PROTEIN COMPLEX), Homo sapiens
UniProt: P51948
Assay: Biochemical Assay for CDK7: The ability of compounds to inhibit CDK7 kinase activity was tested in a TR-FRET assay using 5 nM of CDK7/CycH/MNAT1 obtained from Invitrogen, USA. Test compounds were pre-incubated with the kinase at room temperature for 60 min. After incubation, substrate mix [100 nM Ultra-light MBP (Perkin Elmer, USA) and 1 mM ATP (Sigma)] was added. The above reaction was stopped by the addition of 40 mM EDTA after 60 minutes of kinase reaction. 1 nM Eu-labelled antiphospho-MBP antibody [Perkin Elmer, USA] was added, mixed well and the fluorescence emission at 615 nm and 665 nm [excitation at 340 nm] was measured. The final DMSO concentration in the assay was 1%. For IC50 determination, appropriate concentrations were made by ⅓rd serial dilutions of 10 mM DMSO stock solution of test compound. All the fluorescence measurements were made in a Victor 3 Multilabel Counter [Perkin Elmer, USA]. The IC50 was determined by fitting the dose response data to sigmoidal curve fitting equation using GraphPad Prism software V5. To identify compounds that inhibit CDK7 irreversibly, time depended inhibition studies were carried by pre-incubating compound with the enzyme at three time points (20, 60 and 180 min) and carrying out assay as described above.
Measurement: IC50 > 1000 nM (pChEMBL 6)
Descriptors: MW 477.57, ALogP 3.23, HBA 8, HBD 2, PSA 113.75, QED 0.48
Compound (SMILES): CC1(C)C(=O)Nc2ccc(CC(=O)Nc3cnn(Cc4ccc(F)c(F)c4)n3)cc21
InChIKey: VDASFGIPWWDHFA-UHFFFAOYSA-N
Target: Voltage-dependent T-type calcium channel subunit alpha-1H (SINGLE PROTEIN), Homo sapiens
UniProt: O95180
Assay: Measurement of Calcium Channel Flux by Means of FLIPR Assay: Stock solutions of test compounds are prepared to a concentration of 10 mM in DMSO. For the Cav3.2 assay, serial dilutions of the compounds are prepared in TEAC buffer (100 mM tetraethylammonium chloride; 20 mM Hepes; 2.5 mM CaCl2; 5 mM KCl; 1 mM MgCl2; 1% FBS; pH 7.2), for the Cav1.2 assay serial dilutions are prepared in assay buffer. Test compounds are added to the cells to give a 3-fold dilution range from 10 μM to 0.05 nM. The compounds are incubated with the cells for 3 minutes and Ca2+ entry is stimulated by adding either CaCl2 to a final concentration of 10 mM (Cav3.2 assay) or by adding KCl to a final concentration of 20 mM (Cav1.2 assay). The kinetics of fluorescence increase are recorded for every well and the area under the fluorescence trace for every compound concentration is used to generate inhibition curves using non-linear regression sigmoidal concentration-response curve analysis with in-house software. IC50 values are calculated and represent the compound concentration required to inhibit 50% of the signal that is obtained in the presence of vehicle instead of test compound. In analogy, antagonistic activities (IC50 values) of all exemplified compounds have been measured for the for the Cav3.1- and the Cav3.3-channel. Antagonistic activities (IC50 values) of all exemplified compounds are in the range of 1.7 to 970 nM with respect to Cav3.1; and in the range of 1.1 to 620 nM with respect to Cav3.3.
Measurement: IC50 = 180 nM (pChEMBL 6.75)
Descriptors: MW 411.41, ALogP 3.02, HBA 5, HBD 2, PSA 88.91, QED 0.68
Compound (SMILES): Cc1ccc(-n2nc3c(c2NC(=O)CS(C)(=O)=O)C(=O)N[C@@]2(CCOc4cc(OCC(F)(F)F)ccc42)C3)cc1F
InChIKey: XWYWICVMPWGTKL-VWLOTQADSA-N
Target: Alpha-1,6-mannosyl-glycoprotein 2-beta-N-acetylglucosaminyltransferase (SINGLE PROTEIN), Homo sapiens
UniProt: Q10469
Assay: Inhibitory Activity: Solutions of the compounds of the present invention in DMSO were each aliquoted into 0.2-μL portions in a 384-well polystyrene microplate produced by Corning Incorporated, and 5 μL of an enzyme solution prepared with an assay buffer (100 mmol/L phosphate buffer (pH 7.4) containing 2 mmol/L DTT) and 5 μL of a substrate solution (100 mmol/L phosphate buffer (pH 7.4), 30 μmol/L 2-Oleoylglycerol, 10 μmol/L Oleoyl-CoA) were added thereto, and the resultant was stirred and centrifuged, and incubated in a moist chamber at room temperature for 1 hour. After enzymatic reaction, 50 μL of a quenching solution (containing 0.2 μmol/L Diolein-d5, 0.4% formic acid, and 50% isopropanol) containing Internal Standard (IS) was added to terminate the reaction, and the resultant was sealed in a plate produced by Shimadzu GLC Ltd., and then stirred and centrifuged, and measurement was performed by using an electrospray ionization method with a RapidFire360 and Agilent 6550 Q-TOF mass spectrometer. Diolein as a reaction product (P) of 2-Oleoylglycerol as the substrate and an ammonium adduct ion of the IS were detected, and the peak intensity ratio, P/IS, was calculated from the peak heights to evaluate the inhibitory activity. Inhibitory activities with/without addition of enzyme were defined as Control (+)/Control (−), respectively, and the respective % inhibitions were defined as 0% inhibition and 100% inhibition. The inhibitory activity was calculated from formula below with TIBCO Spotfire (produced by TIBCO Software Inc.):Inhibitory activity (%)=[1−{Sample−Control(−)}/{Control(+)−Control(−)}]*100where Sample indicates a peak intensity ratio: P/IS, when the compound of the present invention was added.
Measurement: IC50 = 1.8 nM (pChEMBL 8.739999771118164)
Descriptors: MW 596.56, ALogP 3.21, HBA 8, HBD 2, PSA 128.62, QED 0.42
Compound (SMILES): COc1cc(OC)c(F)c(N2Cc3cnc4c(c3N(CC3CC3)C2=O)CC(=O)N4)c1F
InChIKey: YKVCPGLYXSLOHD-UHFFFAOYSA-N
Target: Fibroblast growth factor receptor 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P11362
Assay: Enzymatic Assay: The inhibitor potency of the exemplified compounds was measured in an enzyme assay that measures peptide phosphorylation using FRET measurements to detect product formation. Inhibitors were serially diluted in DMSO and a volume of 0.5 μL was transferred to the wells of a 384-well plate. For FGFR3, a 10 μL volume of FGFR3 enzyme (Millipore) diluted in assay buffer (50 mM HEPES, 10 mM MgCl2, 1 mM EGTA, 0.01% Tween-20, 5 mM DTT, pH 7.5) was added to the plate and pre-incubated for 5-10 minutes. Appropriate controls (enzyme blank and enzyme with no inhibitor) were included on the plate. The assay was initiated by the addition of a 10 μL solution containing biotinylated EQEDEPEGDYFEWLE peptide substrate (SEQ ID NO: 1) and ATP (final concentrations of 500 nM and 140 μM respectively) in assay buffer to the wells. The plate was incubated at 25° C. for 1 hr. The reactions were ended with the addition of 10 μL/well of quench solution (50 mM Tris, 150 mM NaCl, 0.5 mg/mL BSA, pH 7.8; 30 mM EDTA with Perkin Elmer Lance Reagents at 3.75 nM Eu-antibody PY20 and 180 nM APC-Streptavidin). The plate was allowed to equilibrate for 1 hr before scanning the wells on a PheraStar plate reader (BMG Labtech).
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 430.41, ALogP 3.23, HBA 5, HBD 1, PSA 84, QED 0.79
Compound (SMILES): O=C(N[C@@H]1CS(=O)(=O)C[C@H]1O)c1ccc2c(-c3ccccc3Cl)nn(-c3ccc(F)cc3)c2c1
InChIKey: WPLXDLHGBKXFLR-IFMALSPDSA-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 = 531 nM (pChEMBL 6.28000020980835)
Descriptors: MW 499.95, ALogP 3.37, HBA 6, HBD 2, PSA 101.29, QED 0.45
Compound (SMILES): Cc1nc2cnc(N3CCN(C)CC3)c(C#N)c2c(=O)n1CC#Cc1ccc(Cl)cc1-c1ccnc2c(C(=O)O)csc12
InChIKey: LCQVQDIBGZWVCY-UHFFFAOYSA-N
Target: Eukaryotic translation initiation factor 4E-binding protein 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q13541
Assay: Fluorescence Polarization Assay: eIF4E protein was pre-incubated with EDA-m7GDP-ATTO-550 in FPB at 2xconcentrations for 5 minutes prior to the addition of compounds. Compounds (100x) were prepared using 3-fold serial dilutions in 100% DMSO, and subsequently diluted 1:50 in FPB to produce 2x stocks. 50 ul of 2x eIF4E/EDA-m7GDP-ATTO-550 were transferred to the wells of a 96-well half-area black flat bottom polystyrene plate. 50 ul of 2x test compound were added, and binding reactions were allowed to equilibrate for 30 minutes at room temperature while protected from light. The fluorescent polarization signal was detected using a Victor 2 multi-label counter (Perkin Elmer) and the concentration necessary to achieve inhibition of eIF4E/EDA-m7GDP-ATTO-550 binding by 50% (IC50) was calculated using data from an 8-point compound dilution series.
Measurement: IC50 = 20.9 nM (pChEMBL 7.679999828338623)
Descriptors: MW 610.1, ALogP 4.4, HBA 10, HBD 1, PSA 128.24, QED 0.29
Compound (SMILES): CCNc1nc(Nc2cnn(C3CCC3)c2C#N)ncc1Br
InChIKey: AIEQUNWPQVTKPG-UHFFFAOYSA-N
Target: Leucine-rich repeat serine/threonine-protein kinase 2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q5S007
Assay: Biochemical Assay: Materials:    LRRK2 G2019S enzyme    Substrate (LRRKtide)    ATP    TR-FRET dilution buffer    pLRRKtide antibody    384-well assay plate    DMSOEnzyme Reaction Conditions    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    10 μL total reaction volumeDetection Reaction Conditions    1× TR-FRET dilution buffer    10 mM EDTA    2 nM antibody    23° C. reaction temperature    10 μL total reaction volumeCompounds 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.To perform the enzyme reaction, 5 μL of LRRKtide substrate and kinase mixture in assay buffer was added to all wells of the assay plate. The plate was centrifuged to concentrate the mixture at the bottom of the wells. The assay plate was incubated at 23° C. for 20 minutes. Following incubation, 5 μL of 2× ATP in assay buffer was added to each well, and plates were centrifuged to concentrate the mixture at the bottom of the wells. The plate was incubated at 23° C. for 60 minutes.To perform the detection of the reaction, EDTA completely mixed in TR-FRET dilution buffer was added to antibody reagent. 10 μL of detection reagent was added to all wells of each well of the assay plate and the plate was centrifuged to concentrate the mixture at the bottom of the wells. The plate was then incubated at 23° C. for 60 minutes. Plates were read on Perkin Elmer Envision 2104 instrument in TR-FRET mode using a 340 nm excitation filter, 520 nm fluorescence emission filter, and 490 or 495 nm terbium emission filter.
Measurement: IC50 < 30 nM (pChEMBL 7.5229997634887695)
Descriptors: MW 362.24, ALogP 3.21, HBA 7, HBD 2, PSA 91.45, QED 0.85
Compound (SMILES): O=C(NS(=O)(=O)c1ccc(NCC2CN(C3COC3)CCO2)c([N+](=O)[O-])c1)c1ccc(C2=CCC(N3CCCC3c3ccccc3C3CC3)CC2)cc1Oc1cnc2[nH]ccc2c1
InChIKey: WTGWSMXBZCHERZ-UHFFFAOYSA-N
Target: Apoptosis regulator Bcl-2 (SINGLE PROTEIN), Homo sapiens
UniProt: P10415
Assay: Bcl-2/Bcl-X Fluorescence Polarization Assay: Compounds disclosed herein were tested for blocking of Bcl-2/Bcl-xl protein with its ligand in an assay based on fluorescence polarization methodology. Recombinant human 2.7 nM Bcl-2/1.3 nM Bcl-xl protein was pre-incubated with a serial dilution of compounds disclosed herein (maximum concentration is 1 μM for Bcl-2 assay, and 10 μM or 1 μM for Bcl-xl assay, 3-fold serially diluted, 10 points) at room temperature for 0.5 hour in an assay buffer containing 20 ml potassium phosphate buffer, pH 7.5, 50 mM NaCl, 1 mM EDTA, 0.05% Tween-20, 0.01% BSA. Then the FITC labeled Bak peptide Ac-GQVGRQLAIIGDK (FITC)INR-amide (1 nM for Bcl-2, 0.82 nM for Bcl-xl) was added to plate and further incubated at room temperature for 0.5 h. The FP signals (485 nm-520 nm-520 nm) were read on BMG PHERAstar FS or BMG PHERAstar FSX instalment. The inhibition percentage of Bcl-2/Bcl-xl interaction with its ligand in presence of increasing concentrations of compounds was calculated based on the FP signals. The IC50 for each compound was derived from fitting the data to the four-parameter logistic equation by Graphpad Prism software.
Measurement: IC50 = 4.5 nM (pChEMBL 8.350000381469727)
Descriptors: MW 874.03, ALogP 7.54, HBA 12, HBD 3, PSA 181.26, QED 0.07
Compound (SMILES): CCN(C[C@@]12CC[C@@H](c3cc(-c4c(F)cccc4F)nnc31)C2(C)C)C(=O)c1cc(CO)on1
InChIKey: AIZQJFPLZHVHEH-LMKMVOKYSA-N
Target: Nuclear receptor ROR-gamma (SINGLE PROTEIN), Homo sapiens
UniProt: P51449
Assay: In Vitro RORc Ligand Binding Assay: Consumable Supplier and product code GFB Unifilter plates Perkin Elmer 6005177 3-[(3- Sigma C5070 Cholamidopropyl)dimethylammonio]- 1-propanesulfonate (CHAPS) 96-well polypropylene U-bottom Nunc 267245 assay plate HEPES buffer, 1M Sigma H3375 Magnesium chloride (MgCl2) Sigma M8266 D,L-Dithiothreitol (DTT) Sigma D0632 Sodium chloride (NaCl) Sigma 71382 Bovine serum albumin (BSA) Sigma A7030 [lyophilized powder, ≥98% (agarose gel electrophoresis), Essentially fatty acid free, essentially globulin free] 25-hydroxycholesterol Sigma H1015 25-[26,27-3H]hydroxycholesterol Perkin Elmer NET674250UC American Radiolabeled Chemicals ART0766 RORc ligand binding domain Genentech (e.g., PUR 28048), expressed in E. coli Plate seals Perkin Elmer 6005185 Microscint 0 Perkin Elmer 6013611. Final concentrations were as follows: 50 mM HEPES buffer (pH 7.4); 150 mM NaCl; 1 mM DTT; 5 mM MgCl2, 0.01% BSA; 5% DMSO; 0.6 ug/mL RORc receptor; 6 nM 25-[3H]hydroxycholesterol. For NSB wells, 1 uM 25-hydroxycholesterol was also present.
Measurement: IC50 = 6.9 nM (pChEMBL 8.15999984741211)
Descriptors: MW 468.5, ALogP 4.22, HBA 6, HBD 1, PSA 92.35, QED 0.58
Compound (SMILES): Nc1ncc(-c2ccc([C@@]34C[C@@H]3CN(CCN3CCOCC3)C4)cc2)cc1C(=O)NC12CCC(O)(CC1)CC2
InChIKey: GAIBPGQSYNAFNN-XSVJXTIASA-N
Target: Activin receptor type-1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q04771
Assay: ADPGlo Assay: The assays were performed in 384-well, low volume microtiter assay plates in a final reaction volume of 6 ul. Dose-response curves were generated by incubating 10 nM of each kinase in 50 mM Hepes pH 7.5, 0.02% Tween 20, 0.02% BSA, 1 mM DTT, 10 μm Na3VO4, 10 mM β-Glycerolphosphate, 1 mM MgCl2, 12 mM MnCl2 and 15 μm ATP for 60 min at 32° C. in the presence or absence of compound diluted in DMSO. The amount of generated ADP is a measure of kinase activity and is quantified using the ADP-Glo™ Kinase Assay (Promega) according to manufacturer's instructions. ADP is converted to ATP by adding 3 ul of ADP-Glo™ Reagent and incubation at 32° C. for 60 min. ATP is subsequently converted into a bioluminescent signal by adding 6 ul luciferase assay reagents (Kinase detection buffer+Kinase Detection Substrate (Promega)) and further incubation at 32° C. for 60 min. For the measurement of luminescence a PHERAstar™ Multilabel Reader was used at a measurement interval time of 0.1 second (optical module for luminescence measurements in the 230 nm to 750 nm wavelength range).
Measurement: IC50 = 29 nM (pChEMBL 7.539999961853027)
Descriptors: MW 531.7, ALogP 2.8, HBA 7, HBD 3, PSA 103.95, QED 0.5
Compound (SMILES): Cc1c(-c2ccc3c(c2)CCNC3)cccc1-c1cccc(OCCCN2[C@H](C)CC[C@H]2C)c1Cl
InChIKey: XMVFJOAUOUIIDX-FGZHOGPDSA-N
Target: Programmed cell death 1 ligand 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9NZQ7
Assay: Enzyme-Linked Immunosorbent AssayELISA: 96 Well plates were coated with 1 μg/mL of human PD-L1 (obtained from R&D) in PBS overnight at 4′° C. The wells were then blocked with 2% BSA in PBS (W/V) with 0.05% TWEEN-20 for 1 hour at 37′° C. The plates were washed 3 times with PBS/0.05% TWEEN-20 and the compounds were serial diluted (1:5) in dilution medium and added to the ELISA plates. Human PD-1 and biotin 0.3 μg/mL (ACRO Biosystems) were added and incubated for 1 hour at 37° C. then washed 3 times with PBS/0.05% TWEEN-20. A second block was performed with 2% BSA in PBS (WNV)/0.05% TWEEN-20 for 10 min at 37° C. and the plates were washed 3 times with PBS/0.05% TWEEN-20. Streptavidin-HIRP was added for 1 hour at 37° C. then the plates were washed 3 times with PBS/0.05% TWEEN-20. TMB substrate was added and reacted for 20 min at 37° C. A stop solution (2 N aqueous H2SO4) was added.
Measurement: IC50 < 1000 nM (pChEMBL 6)
Descriptors: MW 489.1, ALogP 7.27, HBA 3, HBD 1, PSA 24.5, QED 0.35
Compound (SMILES): COc1cc(-n2cnc(Nc3nc4c(c(N5CCC[C@H]5C(N)=O)n3)CCNC4)c2)cc(OC)c1OC
InChIKey: XXRRBWZZSGOQRC-KRWDZBQOSA-N
Target: Activin receptor type-1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q04771
Assay: Biochemical assay: Biochemical assays to measure the inhibitory effects of the compounds were performed by ThermoFisher Scientific (Life Technologies). ALK2 inhibition was tested using LanthaScreen Eu Kinase Binding Assay screening protocol.
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 494.56, ALogP 1.53, HBA 11, HBD 3, PSA 141.68, QED 0.42
Compound (SMILES): CC(=O)N[C@@H](C)CO[C@H]1CC[C@H](Oc2cc3sc(OC(C)CF)nc3cn2)CC1
InChIKey: PEFQJNWKDUUZGU-DXSPHCOUSA-N
Target: Acetyl-CoA carboxylase 2 (SINGLE PROTEIN), Homo sapiens
UniProt: O00763
Assay: Inhibitory Activity on Human ACC1 and the ACC2: Recombinant human ACC1 and recombinant human ACC2, which were prepared by the method mentioned above, were preincubated with assay buffer solution (50 mM HEPES-KOH (pH 7.4), 10 mM magnesium chloride, 6-10 mM potassium citrate, 4 mM reduced form of glutathione, 1.5 mg/ml bovine serum albumin) for one hour. Then, 0.2 μL of each this invention compound solution (in DMSO) were dispensed to 384-well microplate, 5 μL of the preincubated enzyme solution and 5 μL of substrate solution (50 mM HEPES-KOH (pH 7.4), 1 mM ATP, 0.8 mM acetyl CoA and 25-50 mM potassium bicarbonate) were added to microplate. After centrifugation and shaking, the reaction mixtures were incubated in a humidified box at room temperature for 1 to 3 hours. After the incubation, the enzyme reactions were stopped by the addition of EDTA. Then, after the samples were cocrystallized with CHCA (α-cyano-4-hydroxy cinnamic acid) matrices on MALDI target plate, by using the matrix assist laser deionization time-of-flight mass spectrometer (MALDI-TOF MS), samples were measured in reflector negative mode. Deprotonated ions of acetyl CoA (AcCoA) of substrate and malonyl CoA (MalCoA) of the reaction product were detected, then, the conversion rates of acetyl CoA to malonyl CoA was calculated by the intensity of [MalCoA-H]−/(Intensity of [MalCoA-H]−+Intensity of [AcCoA-H]−) using each signal strength. The 50% inhibitory concentration (IC50) was calculated from the inhibition rate of the enzymatic reaction at each concentration of the compounds. In addition, potassium citrate concentrations in assay buffer solution, potassium hydrogen carbonate concentrations in substrate solution and incubation time were adjusted by each lot of enzyme.
Measurement: IC50 = 48 nM (pChEMBL 7.320000171661377)
Descriptors: MW 425.53, ALogP 3.66, HBA 7, HBD 1, PSA 82.57, QED 0.66
Compound (SMILES): CC(=O)Nc1nc(C)c(-c2cc3c(c(NS(=O)(=O)CC4CC4)c2)C(=O)N([C@@H](C)C2CC2)C3)s1
InChIKey: ZKNGXRWHBHUFFL-ZDUSSCGKSA-N
Target: Phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit gamma isoform (SINGLE PROTEIN), Homo sapiens
UniProt: P48736
Assay: Enzymatic Activity Assay for Rec Human PI3Kgamma: The activity of recombinant human PI3Kγ (aa144-1102)-6His was determined by measuring the ADP level after phosphorylation of DiC8-PIP2 using a commercially available ADP-Glo kit from Promega. The assay was carried out in white low volume 384 well plates in a final volume of 14 μl at rt. The assay conditions contained the following: 50 mM Tris buffer pH 7.4, 2.1 mM DTT, 3 mM MgCl2, 0.05% CHAPS, 20 μM ATP, 80 μM DiC8-PIP2 and 1.2 nM PI3Kγ. Potential inhibitors were made up in DMSO and then diluted in the assay to give a final concentration of not exceeding 1% (v/v) DMSO. A 10-point half-log dilution series of the inhibitors (highest concentration typically 0.1 μM) was tested and the pIC50 determined using a 4-parameter logistic equation in a non-linear curve fitting routine. Routinely, inhibitors were pre-incubated with 3 μl of PI3Kγ for 15 min prior to the addition of 2 l substrate mixture for a further 60 min enzyme reaction. The phosphorylation was stopped with the addition of 3 μl ADP-Glo reagent (stop solution) followed by a 40 min incubation. Prior to detection 6 μl of ADP-Glo Kinase Detection Reagent was added and the plates were read in a micro plate reader using a Luminescence filter.
Measurement: IC50 = 0.794 nM (pChEMBL 9.100000381469727)
Descriptors: MW 488.64, ALogP 3.98, HBA 6, HBD 2, PSA 108.47, QED 0.58
Compound (SMILES): COc1cccc(C(=O)N2CC/C(=C\C#Cc3cccc(Cl)c3)C(C)(C)C2)c1
InChIKey: GYUDNFZVPHGXKV-KEBDBYFISA-N
Target: Metabotropic glutamate receptor 5 (SINGLE PROTEIN), Homo sapiens
UniProt: P41594
Assay: Fold Shift of Agonist Curve Determination: The PAM potency was evaluated by performing an agonist concentration response curve in the presence of the compounds. The compounds at fixed concentration (0.01 or 0.1 or 1 μM) were applied 10 minutes before the incubation of the agonist (quisqualate or glutamate) dose-response curve. The fold shift was determined as ratio of EC50 agonist curves in presence and in absence of test compound. Selected folds shift for some of the compounds of interest, prepared according to the invention, are shown in Table 5 below.
Measurement: EC50 = 268 nM (pChEMBL 6.570000171661377)
Descriptors: MW 393.91, ALogP 5.2, HBA 2, HBD 0, PSA 29.54, QED 0.66
Compound (SMILES): CC1(C)Oc2cnccc2-c2[nH]c(C(=O)O)c(Br)c21
InChIKey: ZHJUEEVXBXXRCT-UHFFFAOYSA-N
Target: Serine/threonine-protein kinase haspin (SINGLE PROTEIN), Homo sapiens
UniProt: Q8TF76
Assay: Haspin Kinase Assay: The kinase assay relies on ADP-GloKM biochemical kinase assay (Promega). This is a luminescent kinase assay that measures ADP formed from a kinase reaction. Then ADP is converted into ATP, which is transformed into a light signal by Ultra-Glo Luciferase. The luminescent signal positively correlates with kinase activity. The assay measures the intrinsic ATPase activity (in the absence of peptidic substrate as phosphate acceptor).The enzyme was purchase from Invitrogen (PV5708) as the kinase domain of GST tagged recombinant human proteins.Assay conditions were as indicated by the kit manufacturers with the following adaptations:Assay buffer: 15 mM HEPES pH 7.4, 20 mM NaCl, 1 mM EGTA, 0.02% Tween 20, 10 mM MgCl2, 0.1 mg/ml BGGAssay volume: 20 μlIncubation time and temperature: 60 min at 30° C.Haspin concentration: 20 nMATP concentration: 150 μMTested compound: Serial 1:3 dilutionsFinal DMSO concentration in the assay: 1%Assays were performed in 384-well plates. The final read out was generated using an EnVision plate reader (Perkin-Elmer). Luminescent relative units are normalized against the control activity included for each compound (i.e., 100% Haspin activity, without compound) and the percentage of inhibition is calculated.
Measurement: IC50 = 89.3 nM (pChEMBL 7.050000190734863)
Descriptors: MW 323.15, ALogP 3.16, HBA 3, HBD 2, PSA 75.21, QED 0.85
Compound (SMILES): CC(=O)N1CCC(c2cc(F)c3ncn(CC(=O)N(C)Cc4ccc(C#N)c(Cl)c4)c(=O)c3c2)CC1
InChIKey: QGEPSJRORVPVGA-UHFFFAOYSA-N
Target: Autotaxin (SINGLE PROTEIN), Homo sapiens
UniProt: Q13822
Assay: Human ATX Enzyme Inhibition Assay: ATX inhibition was measured by a fluorescence quenching assay using a specifically labeled substrate analogue (MR121 substrate). To obtain this MR121 substrate, BOC and TBS protected 6-amino-hexanoic acid (R)-3-({2-[3-(2-{2-[2-(2-amino-ethoxy)-ethoxy]-ethoxy}-ethoxy)-propionylamino]-ethoxy}-hydroxy-phosphoryloxy)-2-hydroxy-propyl ester (Ferguson et al., Org. Lett. 2006, 8, 2023) was labeled with MR121 fluorophore [CAS RN 185308-24-1], 1-(3-carboxypropyl)-11-ethyl-1,2,3,4,8,9,10,11-octahydro-dipyrido[3,2-b:2′,3′-i]phenoxazin-13-ium) on the free amine of the ethanolamine side and then, after deprotection, subsequently with tryptophan on the side of the aminohexanoic acid.Assay working solutions were made as follows:Assay buffer (50 mM Tris-HCl, 140 mM NaCl, 5 mM KCl, 1 mM CaCl2, 1 mM MgCl2, 0.01% Triton-X-100, pH 8.0;ATX solution: ATX (human His-tagged) stock solution (1.08 mg/mL in 20 mM bicine, pH 8.5, 0.15 M NaCl, 10% glycerol, 0.3% CHAPS, 0.02% NaN3), diluted to 1.4-2.5× final concentration in assay buffer;MR121 substrate solution: MR121 substrate stock solution (800 μM MR121 substrate in DMSO), diluted to 2-5× final concentration in assay buffer.Test compounds (10 mM stock in DMSO, 8 μL) were obtained in 384 well sample plates (Corning Costar #3655) and diluted with 8 μL DMSO. Row-wise serial dilutions were made by transferring 8 μL cpd solution to the next row up to row O. The compound and control solutions were mixed five times and 2 μL were transferred to 384 well assay plates (Corning Costar #3702). Then, 15 μL of 41.7 nM ATX solution was added (30 nM final concentration), mixed five times and then incubated for 15 minutes at 30° C. 10 μL of MR121 substrate solution was added (1 μM final concentration), mixed 30 times and then incubated for 15 minutes at 30° C. Fluorescence was then measured every 2 min for 1 h (Perkin Elmer plate: vision multimode reader); light intensity: 2.5%; exp. time: 1.4 s, Filter: Fluo_630/690 nm) and IC50 values were calculated from these readouts.
Measurement: IC50 = 1 nM (pChEMBL 9)
Descriptors: MW 509.97, ALogP 3.45, HBA 6, HBD 0, PSA 99.3, QED 0.52
Compound (SMILES): CC1(C)OC[C@H](COc2cc3ncc(C#N)c(Nc4ccc(OCc5cccc(F)c5)c(Cl)c4)c3cc2NC(=O)C[C@H]2CCSS2)O1
InChIKey: CZFLJSHHAMLIOE-LOSJGSFVSA-N
Target: Epidermal growth factor receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P00533
Assay: Kinase assay: KINOMEscan is based on a competition binding assay that quantitatively measures the ability of a compound to compete with an immobilized, active-site directed ligand. The assay is performed by combining three components: DNA-tagged kinase; immobilized ligand; and a test compound. The ability of the test compound to compete with the immobilized ligand is measured via quantitative PCR of the DNA tag.For most assays, kinase-tagged T7 phage strains were prepared in an E. coli host derived from the BL21 strain. E. coli were grown to log-phase and infected with T7 phage and incubated with shaking at 32° C. until lysis. The lysates were centrifuged and filtered to remove cell debris. The remaining kinases were produced in HEK-293 cells and subsequently tagged with DNA for qPCR detection. Streptavidin-coated magnetic beads were treated with biotinylated small molecule ligands for 30 minutes at room temperature to generate affinity resins for kinase assays. 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 nonspecific binding. Binding reactions were assembled by combining kinases, liganded affinity beads, and test compounds in 1× binding buffer (20% SeaBlock, 0.17×PBS, 0.05% Tween 20, 6 mM DTT). All reactions were performed in polystyrene 96-well plates in a final volume of 0.135 ml. The assay plates were incubated at room temperature with shaking for 1 hour and the affinity beads were washed with wash buffer (1×PBS, 0.05% Tween 20). The beads were then re-suspended in elution buffer (1×PBS, 0.05% Tween 20, 0.5 M non-biotinylated affinity ligand) and incubated at room temperature with shaking for 30 minutes. The kinase concentration in the eluates was measured by qPCR.
Measurement: Kd = 50 nM (pChEMBL 7.300000190734863)
Descriptors: MW 695.24, ALogP 8.23, HBA 10, HBD 2, PSA 114.73, QED 0.15
Compound (SMILES): c1ccc(C2CCCN2c2nc(Nc3cn(C4CC4)cn3)c3cccn3n2)cc1
InChIKey: SQNXDJJFTSITAI-UHFFFAOYSA-N
Target: Activin receptor type-1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q04771
Assay: Biochemical assay: Biochemical assays to measure the inhibitory effects of the compounds were performed by ThermoFisher Scientific (Life Technologies). ALK2 inhibition was tested using LanthaScreen Eu Kinase Binding Assay screening protocol.
Measurement: IC50 = 550 nM (pChEMBL 6.260000228881836)
Descriptors: MW 385.48, ALogP 4.35, HBA 7, HBD 1, PSA 63.28, QED 0.55
Compound (SMILES): Cc1cc(OCCCc2c3n(c4c(-c5c(C)nn(C)c5C)cccc24)CCCN(c2ccnc4c(C(=O)O)cccc24)C3=O)cc(C)c1Cl
InChIKey: DXTPYBOZKAFXDB-UHFFFAOYSA-N
Target: Induced myeloid leukemia cell differentiation protein Mcl-1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q07820
Assay: Fluorescence Polarization Anisotropy Competition Assay: Compound affinity was measured using a fluorescence polarization anisotropy competition assay. Anisotropy measurements were carried out in 384-well, black, flat-bottom plates (Greiner Bio-one, Monroe, N.C., USA). The assay was run using either a fluorescein isothiocyanate-labeled BH3 peptide derived from Bak (FITC-AHx-GQVGRQLAIIGDDINR-NH2) or a fluorescein isothiocyanate-labeled BH3 peptide derived from Bim (FITC-AHx-EARIAQELRRIGDEFNETYTR-NH2) that were purchased from GenScript (Piscataway, N.J.) at >95% purity and used without further purification. 10 nM FITC-Bak peptide and 15 nM recombinant Mcl-1 (residues 172-327) were added to assay buffer (3 mM dithiothreitol, 50 mM NaCl, 20 mM Tris, pH 7.5). The Bim based assay was run with 1 nM FITC-Bim peptide and 1.5 nM recombinant Mcl-1 (residues 172-327) added to assay buffer (20 mM TRIS pH 7.5, 50 mM NaCl, 3 mM DTT, 0.01% CHAPS). For selectivity assays, 40 nM Bcl-2 (residues 1-207A96T,G110R, Î35-91, replaced with Bcl-xL35-50) or 4 nM Bcl-xL (residues 1-209, loop 45-86 deleted) were incubated with 10 nM FITC-Bak in assay buffer.
Measurement: Ki < 50 nM (pChEMBL 7.301000118255615)
Descriptors: MW 676.22, ALogP 8.24, HBA 7, HBD 1, PSA 102.48, QED 0.16
Compound (SMILES): CS(=O)(=O)N1CCC(Nc2ncc3c(n2)-c2c(nc(Br)n2C2CC2)CC3)CC1
InChIKey: BBUORPXDQISNHL-UHFFFAOYSA-N
Target: Cyclin-dependent kinase 2/cyclin E1 (PROTEIN COMPLEX), Homo sapiens
UniProt: P24864
Assay: CDK2/Cyclin E1 HTRF Enzyme Activity Assay: CDK2/Cyclin E1 enzyme activity assays utilize full-length human CDK2 co-expressed as N-terminal GST-tagged protein with FLAG-Cyclin E1 in a baculovirus expression system (Carna Product Number 04-165). Assays are conducted in white 384-well polystyrene plates in a final reaction volume of 8 μL. CDK2/Cyclin E1 (0.25 nM) is incubated with compounds (40 nL serially diluted in DMSO) in the presence of ATP (50 μM or 1 mM) and 50 nM ULight-labeled eIF4E-binding protein 1 (THR37/46) peptide (PerkinElmer) in assay buffer (containing 50 mM HEPES pH 7.5, 1 mM EGTA, 10 mM MgCl2, 2 mM DTT, 0.05 mg/ml BSA, and 0.01% Tween 20) for 60 minutes at room temperature. The reactions are stopped by the addition of EDTA and Europium-labeled anti-phospho-4E-BP1 antibody (PerkinElmer), for a final concentration of 15 mM and 1.5 nM, respectively. HTRF signals are read after 1 hour at room temperature on a PHERAstar FS plate reader (BMG Labtech). Data is analyzed with IDBS XLFit and GraphPad Prism 5.0 software using a three or four parameter dose response curve to determine IC50 for each compound.
Measurement: IC50 < 50 nM (pChEMBL 7.301000118255615)
Descriptors: MW 467.39, ALogP 2.37, HBA 7, HBD 1, PSA 93.01, QED 0.74
Compound (SMILES): Cc1nnc2n1CCN(c1cccc3c1CCN(C(=O)[C@H]1CC(c4cccc(Cl)c4F)=NO1)[C@H]3C(=O)Nc1ccc(C(=O)O)cc1)C2
InChIKey: QNQFMFYHIZXXRE-POURPWNDSA-N
Target: Coagulation factor XI (SINGLE PROTEIN), Homo sapiens
UniProt: P03951
Assay: Enzyme Inhibition Assay: Factor XIa determinations were made in 50 mM HEPES buffer at pH 7.4 containing 145 mM NaCl, 5 mM KCl, and 0.1% PEG 8000 (polyethylene glycol; JT Baker or Fisher Scientific). Determinations were made using purified human Factor XIa at a final concentration of 25-200 pM (Haematologic Technologies) and the synthetic substrate S-2366 (pyroGlu-Pro-Arg-pNA; CHROMOGENIX or AnaSpec) at a concentration of 0.0002-0.001 M.
Measurement: Ki < 5 nM (pChEMBL 8.300999641418457)
Descriptors: MW 658.09, ALogP 4.36, HBA 9, HBD 2, PSA 142.25, QED 0.31