lovingscience/chembl-v37-raw-activities huggingface dataset:
Compound (SMILES): COC(=O)c1cc(Cl)c(OC)c(S(=O)(=O)Nc2cc(-c3ccccc3)c(F)cc2F)c1
InChIKey: FVIONZBRQCHWDO-UHFFFAOYSA-N
Target: ATP-citrate synthase (SINGLE PROTEIN), Homo sapiens
UniProt: P53396
Assay: ADP-Glo Assay: Materials and Instruments Used in the ADP-Glo AssayVendor Cat No.Materials ACL Sino Biological 11769-H07BCoANa2 Sigma C3144Potassium Citrate Sigma 89306ATP Promega V915BADP-GloTM Kinase Assay Promega V9102kitHEPES Life Technologies 15630080MgCl2 Sigma M1028Brij 35 detergent Merck 203728DTT Sigma 646563DMSO MP 196055Optiplate-384, White 384- Perkin Elmer 6007290well384 dilution plate Corning 3657Topseal A Perkin Elmer E534196-well plate Nunc 249944InstrumentPlate reader Perkin Elmer Envision 2104Centrifuge Eppendorf 5810RCompounds of the present invention were evaluated in an ADP-GLO assay as follows:a) Dilute cpd 1:3 in succession in DMSO by hand for each cpds for 12 ptsb) Add 0.1 μL diluted cpd solution to assay plate, each dose with 2 replicatesc) Centrifuge 1000 RPM for 1 mind) Add 5 μL ACL working solution to 384-well assay plate, centrifuge 1000 RPM for 1 mine) Incubate at 25° C. for 15 minf) Add 5 μL substrate working solution to initiate reactiong) Final ACL reaction concentrations: 3 nM ACL, 15 μM ATP, 3 μM CoA, 300 μM Citrate, 0.01% Brij35, 4 mM DTT, 1% DMSO;h) Reference final conc: 30 uM starting Conc, 3× dilution, 11+0 points. Test cpd conc: 30/100 uM starting Conc, 3× dilution, 11+0 points.i) Incubate at 25° C. for 60 minj) Add 10 μL ADP Glo reagent, centrifuge 1000 RPM for 1 mink) Incubate at 25° C. for 40 minl) Add 20 μL kinase detection reagent, centrifuge 1000 RPM for 1 minm) Incubate at 25° C. for 40 minn) Read on Envision for US LUM as RLU
Measurement: IC50 = 10250 nM (pChEMBL 4.989999771118164)
Descriptors: MW 467.88, ALogP 4.88, HBA 5, HBD 1, PSA 81.7, QED 0.52
Compound (SMILES): CC(=O)Nc1ccc(C(=O)Nc2c(N)cccc2F)cc1
InChIKey: TWPLTSGSUZFOBC-UHFFFAOYSA-N
Target: Histone deacetylase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q13547
Assay: Inhibition of Histone Deacetylase Enzymatic Activity: The following non-trypsin coupled in-vitro HDAC enzymatic endpoint assay was used to assay the compounds of the invention. Below is a standardized protocol for running HDAC selectivity panel on Caliper LabChip EZ-Reader Instrument.
Measurement: IC50 = 2180 nM (pChEMBL 5.659999847412109)
Descriptors: MW 287.29, ALogP 2.62, HBA 3, HBD 3, PSA 84.22, QED 0.76
Compound (SMILES): CC(=O)Nc1ccc(Cn2c(Sc3nc4ccccc4[nH]3)nc3c2c(=O)[nH]c(=O)n3C)cc1
InChIKey: VDGUSZDWMNEHNT-UHFFFAOYSA-N
Target: Tryptophan 5-hydroxylase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P17752
Assay: In Vitro Enzyme Activity Assay: These compounds according to the afore-mentioned examples were tested for tryptophan hydroxylase (TPH) inhibitory activity in a fluorescence-based in vitro assay, using recombinant human TPH1 (Swiss-Prot: P17752) and TPH2 (Swiss-Prot: Q8IWU9).The full-length coding sequences of human TPH1 and TPH2 were PCR amplified, ligated into a MBP fusion vector (pMalc2x, New England Biolabs, MA, USA) and transformed into SCS1 (Stratagene, CA, USA) to amplify plasmid DNA. For the overexpression of TPH proteins, the constructs were transformed into Rosetta (DE3) (Novagen®/EMD Millipore, MA, USA) and cultivated in terrific broth (TB) medium (AppliChem, Darmstadt, Germany) at 37° C. When the bacterial cultures reached an ≈2, expression was induced with 0.5 mM IPTG (AppliChem, Darmstadt, Germany) over night at 17° C. The purification of soluble proteins started with sonication-mediated cell disruption in lysis buffer (1×PBS pH 7.4, 0.5 M NaCl, 5% Glycerol+CHAPS, DTT, PMSF, benzonase), followed by affinity purification (MBPTrap, GE Healthcare, UK) and gel filtration (26/60 Superdex 200 prep grade, GE Healthcare, UK), according to the manufacturer's protocol. The quality of protein expression and solubility was controlled by SDS-PAGE and Coomassie blue staining.The enzymatic reaction was carried out in black 96-well flat bottom plates (Corning GmbH, Wiesbaden). TPH1 and TPH2 activities were measured in a reaction mixture containing 50 mM 4-Morpholineethanesulfonic acid (MES), pH 7.0, 40 μM tryptophan, 200 mM ammonium sulfate, 25 μM ferrous ammonium sulfate, 50 μM tetrahydrobiopterin, 25 μg/ml catalase, and 7 mM DTT. The reactions were initiated by adding TPH1 or TPH2 to a final concentration of 5 μg/ml. Initial velocity of the reactions was determined by following the change of fluorescence at 330 nm (excitation wavelength=300 nm) (Infinite M200, Tecan, Crailsheim).TPH1 and TPH2 inhibition was determined by measuring a compound dose response, using a serial dilution of a 5 mM DMSO stock solution. The potency of a given compound was calculated in GraphPad PRISM 6 software (San Diego, USA) with a Nonlinear Regression fit (log(inhibitor) vs. response-variable slope) using the relative fluorescence units (RFU) of the sample triplicates.
Measurement: IC50 = 33010 nM (pChEMBL 4.480000019073486)
Descriptors: MW 461.51, ALogP 2.46, HBA 8, HBD 3, PSA 130.46, QED 0.37
Compound (SMILES): CC(C)Nc1c(C(N)=O)nnc2cc(N3CCCC3=O)ccc12
InChIKey: KZZQENOVNVAQQZ-UHFFFAOYSA-N
Target: Leucine-rich repeat serine/threonine-protein kinase 2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q5S007
Assay: In Vitro Kinase Activities (LRRK2 TR-FRET Peptide Assay): Compounds as described herein (compounds of Formula I, e.g., compounds of the above Examples) are tested for their in vitro kinase activities using various LRRK2 (including LRRK2 G2019S mutant) assays. For example, assays were performed in a total volume of 20 μL using the same kinase reaction buffer (50 mM HEPES pH 7.5, 10 mM MgCl2, 1 mM EGTA, 2 mM DTT and 0.01% Tween-20) for wild-type or G2019S mutant LRRK2. Serially diluted compounds (1% DMSO as co-solvent) were pre-incubated with recombinant GST-LRRK2 (wild-type, or G2019S mutant, Invitrogen) for 15 minutes at room temperature in 384-well Corning black plates. Mixtures of ATP and biotin-LRRKtide substrate (biotin-RLGRDKYKTLRQIRQ) (SEQ ID NO: 1) were added to the wells at a final concentration of 100 μM ATP and 100 nM substrate, with final kinase concentration of 10 nM. The kinase reactions were carried out at room temperature for 60 minutes, then the reaction was stopped with the addition of 10 μL/well of stop/detection buffer (25 mM HEPES pH 7.5, 66 mM EDTA, 0.8 M KF and 0.1% BSA) that contains streptavidin-XL665 (12.5 nM) and europium-conjugated phospho-specific antibody (2nM). The plates were read 1 hour later and the time-resolved fluorescence (665 nm to 615 nm ratio) measured using an Envision reader. The inhibition of each well was calculated using the control and background readings for that plate. IC50 values were determined from dose-response curves using eight concentrations from the serial dilution of the test compounds.Many compounds of the Examples are demonstrated to be inhibitors of LRRK2, with most of the compounds tested typically measuring an IC50 below 1 μM for both LRRK2 and G2019S mutant LRRK2 kinase activity.
Measurement: IC50 = 170 nM (pChEMBL 6.769999980926514)
Descriptors: MW 313.36, ALogP 1.68, HBA 5, HBD 2, PSA 101.21, QED 0.89
Compound (SMILES): C=CC(=O)Nc1cccc(-c2cnc3[nH]cc(-c4cccc(C(N)=O)c4)c3c2)c1
InChIKey: IAXUBJQEBGMQQC-UHFFFAOYSA-N
Target: Tyrosine-protein kinase ITK/TSK (SINGLE PROTEIN), Homo sapiens
UniProt: Q08881
Assay: In Vitro Inhibition Assay: ITK and JAK3 Kinase assay procedures:Enzyme was incubated with substrate peptide in reaction buffer in the presence and absence of test compounds or Staurosporine. All additions were done on ice, followed by the addition of ATP mix. Wells were uniformly mixed using an Eppendorff plate shaker and incubated at 30° C. for 20 min, and stopped by the addition of 5 μL of 3% phosphoric acid. Volume was increased to 100 μL by adding 0.8% phosphoric acid which was then transferred to PC filter mats (Millipore), pre-equilibrated with 70% ethanol and water. Plates were washed thrice with 100 μL 0.8% phosphoric acid and dried for an hour at 60° C. 100 μL scintillation fluid was added into each well and reading taken in Perkin Elmer TOPCOUNT beta counter. The data analysis was performed by averaging the duplicate top count readings for each standard, negative, positive control (enzyme control) and samples and subtracting the average negative control from each reading which results in corrected values.
Measurement: IC50 > 100 nM (pChEMBL 7)
Descriptors: MW 382.42, ALogP 4.12, HBA 3, HBD 3, PSA 100.87, QED 0.45
Compound (SMILES): CN(c1cc(F)cc(-c2ccc3ncnc(N)c3n2)c1)C1CCC(O)CC1
InChIKey: GMBCJYHYZOKCPT-UHFFFAOYSA-N
Target: Mitogen-activated protein kinase kinase kinase kinase 4 (SINGLE PROTEIN), Homo sapiens
UniProt: O95819
Assay: Inhibition Assay: The kinase activity of purified human MAP4K4 kinase domain was measured by monitoring the phosphorylation of a peptide substrate derived from moesin protein (Leu-Gly-Arg-Asp-Lys-Tyr-Lys-Thr-Leu-Arg-Gln-Ile-Arg-Gln) fluorescently labeled on the N-terminus with 5-carboxyfluorescein using the Caliper LabChip technology (Caliper Life Sciences, Hopkinton, Mass.). To determine inhibition constants (IC50), compounds were serially diluted in DMSO and added to 10 uL kinase reactions containing 1 nM purified MAP4K4 enzyme, 1 uM peptide substrate, 10 uM ATP, 10 mM MgCl2, 1 mM EGTA, 50 mM Hepes pH 7.2, 1 mM DTT, 0.01% Triton X-100, and 2% DMSO. Reactions were incubated at room temperature in Perkin Elmer Proxiplates for 45 minutes and stopped by the addition of 10 uL of an EDTA-containing solution (50 mM Hepes pH 7.2, 40 mM EDTA, 0.02% Triton X-100). The fraction of phosphorylated peptide was determined as a fraction of total peptide substrate using the Caliper Lab Chip 3000 according to the manufacturer's instructions. IC50 values were determined using the four-parameter non-linear fit model.
Measurement: IC50 = 1600 nM (pChEMBL 5.800000190734863)
Descriptors: MW 367.43, ALogP 3.15, HBA 6, HBD 2, PSA 88.16, QED 0.74
Compound (SMILES): Cc1nc(C)n(Cc2cn(-c3ccccc3)nn2)n1
InChIKey: KTZHEMRJQDFTCV-UHFFFAOYSA-N
Target: Voltage-gated inwardly rectifying potassium channel KCNH2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q12809
Assay: Inhibition Assay: Cells were split 2-3 times weekly between 1:3 and 1:4. For binding assays and membrane preparations the cell culture medium was removed, cells were washed with PBS. Crude membranes for radioligand binding experiments were prepared by scraping the cells off the dishes in ice cold 20 mM HEPES/0.1 mM KCl/pH 7.2. The cell suspension was homogenized on ice (Ultra turrax, 3×20 sec.) and the homogenate was spun for 10 min (1° C., 1000 g, OPTIMA, SW28, 2800 U/min). The supernatant was than centrifuged for 40 min at 100000 g (1° C., OPTIMA, SW28, 23000 U/min). The membrane pellet was resuspended in 20 mM HEPES/0.1 mM KCl/pH 7.2, frozen and stored at −80° C.After thawing on the day of the assay, the membrane suspension was diluted further with 20 mM HEPES/0.1 mM KCl/pH 7.2.The incubation mixture of 200 μl contained 1.5 nmol/l 3H-Dofetilide, optimized amount of membrane preparation, 20 mM HEPES/0.1 mM KCl/(pH 7.2) and inhibitor in 1% DMSO. Nonspecific binding was estimated in the presence of 10 M Dofetilide. The samples were incubated for 90 min. at RT.Binding was terminated by filtration of the incubated membrane preparations using Filtermat B (Pharmacia, Uppsala Sweden) and a Micro Cell Harvester (Skatron, Lier, Norway). The Filtermat B had been presoaked with 1% polyethylenimine and carefully washed with 0.05 M Tris/HCl-buffer pH=7.7 after the filtration to separate free and bound radioactivity. The filters were counted in a scintillation counter (Betaplate 1205, Berthold, Wildbad, Germany) in order to determine the specific binding of [3H]-Dofetilide.
Measurement: IC50 > 10000 nM (pChEMBL 5)
Descriptors: MW 254.3, ALogP 1.52, HBA 6, HBD 0, PSA 61.42, QED 0.71
Compound (SMILES): Cc1c(F)cccc1[C@]1(C(=O)NO)CC[C@H](c2ccc3nc(C4CC4)sc3c2)C1
InChIKey: FVSHRAFFHIROGV-HJPURHCSSA-N
Target: Histone deacetylase 4 (SINGLE PROTEIN), Homo sapiens
UniProt: P56524
Assay: Inhibition Assay: The potency of Class IIa Histone Deacetylase (HDAC) inhibitors is quantified by measuring the Histone Deacetylase 4 (HDAC4) catalytic domain enzymatic activity using the fluorogenic substrate, Boc-Lys(Tfa)-AMC. The substrate is deacetylated to Boc-Lys-AMC by HDAC4. Cleavage by trypsin results in the release of the fluorophore AMC from the deacetylated substrate. The fluorescence of the sample is directly related to the histone deacetylase activity in the sample.Serially dilute HDAC inhibitor compounds. Serial dilutions of the HDAC inhibitors and control reference compound (1-(5-(3-((4-(1,3,4-oxadiazol-2-yl)phenoxy)methyl)-1,2,4-oxadiazol-5-yl)thiophen-2-yl)-2,2,2-trifluoroethanone) are made by first resuspending the lyophilized compound to a final concentration of 10 mM in 100% dimethyl sulfoxide (DMSO). Stocks of 6 μl aliquots of the 10 mM compound in DMSO are prepared and stored at −2° C.
Measurement: IC50 = 310 nM (pChEMBL 6.510000228881836)
Descriptors: MW 410.51, ALogP 5.33, HBA 4, HBD 2, PSA 62.22, QED 0.45
Compound (SMILES): Cc1ncc(C)n2nc(C#Cc3nc(N4CCCC4=O)cn3C)nc12
InChIKey: CKDRPPMIFRXUHG-UHFFFAOYSA-N
Target: cAMP and cAMP-inhibited cGMP 3',5'-cyclic phosphodiesterase 10A (SINGLE PROTEIN), Homo sapiens
UniProt: Q9Y233
Assay: Inhibition Assay: The human PDE10A full length assay was performed in 96-well micro titer plates. The reaction mixture of 50 μl contained 20 mM HEPES pH=7.5/10 mM MgCl2/0.05 mg/ml BSA (Sigma cat. #A-7906), 50 nM cGMP (Sigma, cat. #G6129) and 50 nM [3H]-cGMP (GE Healthcare, cat. #TRK392 S.A. 13.2 Ci/mmol), 3.75 ng/well PDE10A enzyme (Enzo Life Science, Lausen, Switzerland cat #SE-534) with or without a specific test compound. A range of concentrations of the potential inhibitor was used to generate data for calculating the concentration of inhibitor resulting in 50% of the effect (e.g. IC50, the concentration of the competitor inhibiting PDE10A activity by 50%). Non-specific activity was tested without the enzyme. The reaction was initiated by the addition of the substrate solution (cGMP and [3H]-cGMP) and allowed to progress for 20 minutes at room temperature. The reaction was terminated by adding 25 μl of YSi-SPA scintillation beads (GE Healthcare, cat. # RPNQ0150) in 18 mM zinc sulphate solution (stop reagent). After 1 h under shaking, the plate was centrifuged one minute at 170 g to allow beads to settle. Afterwards, radioactive counts were measured on a Perkin Elmer TopCount Scintillation plate reader.
Measurement: IC50 = 29 nM (pChEMBL 7.539999961853027)
Descriptors: MW 335.37, ALogP 1, HBA 7, HBD 0, PSA 81.21, QED 0.62
Compound (SMILES): C[C@@H](CO[C@@H]1CCN([C@@H]2CCN(c3ncc(C(F)F)cn3)C[C@H]2O)C1=O)Nc1cn[nH]c(=O)c1C(F)(F)F
InChIKey: SGQLOALKYYFPIR-AEAUEXCSSA-N
Target: Protein mono-ADP-ribosyltransferase TIPARP (SINGLE PROTEIN), Homo sapiens
UniProt: Q7Z3E1
Assay: Enzymatic Assay: Displacement of Probe A, a biotinylated probe binding to the TIPARP active site, was measured using a time-resolved fluorescence energy transfer (TR-FRET) assay. 20 nL of a dose response curve of each test compound was spotted in black 384-well polystyrene proxiplates (Perkin Elmer) using a Mosquito (TTP Labtech). Reactions were performed in a 8 μl volume by adding 6 μL of TIPARP and Probe A in assay buffer (20 mM HEPES pH=8, 100 mM NaCl, 0.1% bovine serum albumin, 2 mM DTT and 0.002% Tween20), incubating with test compound at 25° C. for 30 min, then adding 2 μL of ULight-anti 6×His and LANCE Eu-W1024 labeled streptavidin (Perkin Elmer). The final concentrations of TIPARP and Probe A were 6 nM and 2 nM, respectively. The final concentration of ULight-anti 6×His and LANCE Eu-W1024 labeled streptavidin were 4 nM and 0.25 nM, respectively. Reactions were incubated at 25° C. for an additional 30 min, then read on an Envision platereader equipped with a LANCE/DELFIA top mirror (Perkin Elmer) using excitation of 320 nm and emission of 615 nm and 665 nM with a 90 is delay. The ratio of the 665/615 nm emission were calculated for each well to determine the amount of complex of TIPARP and Probe Ain each well. Control wells containing a negative control of 0.25% DMSO vehicle or a positive control of 100 μM 5-(5-(piperidin-4-yloxy)isoindolin-2-yl)-4-(trifluoromethyl)pyridazin-3(2H)-one were used to calculate the % inhibition.
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 547.49, ALogP 1.57, HBA 9, HBD 3, PSA 136.57, QED 0.42
Compound (SMILES): CC(C)c1cnn2c(NCc3cccc(NC(=O)/C=C/CN4CCC(F)C4)c3)nc(NC3CCOCC3)nc12
InChIKey: LNUBFZYACACGRH-QPJJXVBHSA-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 < 300 nM (pChEMBL 6.5229997634887695)
Descriptors: MW 536.66, ALogP 3.99, HBA 9, HBD 3, PSA 108.71, QED 0.33
Compound (SMILES): O=C(c1cnn[nH]1)N1CC2CCC(C1)N2S(=O)(=O)c1ccc(Cl)cc1
InChIKey: OLKAPIXATOZPTM-UHFFFAOYSA-N
Target: Aldo-keto reductase family 1 member C3 (SINGLE PROTEIN), Homo sapiens
UniProt: P42330
Assay: AKR1C3-Inhibitory Activity Assay: The enzyme used was recombinant human AKR1C3 (Aldo-keto reductase family 1 member C3; GenBank Accession No. NM_003739). This was expressed in E. coli as GST (glutathione S transferase) fusion protein and purified by glutathione Sepharose affinity chromatography. The GST was removed by digestion with thrombin and subsequent size exclusion chromatography (Dufort, I., Rheault, P., Huang, X F., Soucy, P., and Luu-The, V., Endocrinology 140, 568-574 (1999)).For the assay, 50 nl of a 100-fold concentrated solution of the test substance in DMSO were pipetted into a black low-volume 384-well microtiter plate (Greiner Bio-One, Frickenhausen, Germany), 2.5 μl of a solution of AKR1C3 in assay buffer [50 mM potassium phosphate buffer pH 7, 1 mM DTT, 0.0022% (w/v) Pluronic F-127, 0.01% BSA (w/v) and protease inhibitor cocktail (Complete, EDTA-free Protease Inhibitor Cocktail from Roche)] were added and the mixture was incubated for 15 min to allow pre-binding of the substances to the enzyme prior to the enzyme reaction. The enzyme reaction was then started by addition of 2.5 μl of a solution of NADPH (20 μM→final concentration in 5 μl of assay volume is 10 μM) and Coumberone (0.6 μM→final concentration in 5 μl of assay volume is 0.3 μM) in assay buffer, and the resulting mixture was incubated at 22° C. for the reaction time of typically 90 min. The concentration of the AKR1C3 and the reaction time was adapted to the respective activity of the enzyme preparation and adjusted such that the assay was carried out in the linear range. Typical AKR1C3 concentrations were in the region of 1 nM. The reaction was stopped by addition of 2.5 μl of a stop solution consisting of 3 μM EM-1404 as inhibitor (U.S. Pat. No. 6,541,463) in 50 mM HEPES pH7.5 (3 μM EM-1404→final concentration in 7.5 μl of assay volume is 1 μM). The fluorescence of the Coumberole was then measured at 520 nm (excitation at 380 nm) using a suitable measuring instrument (Pherastar from BMG Labtechnologies). The intensity of the fluorescence was used as a measure of the amount of Coumberole formed and thus of the enzyme activity of AKR1C3. The data were normalized (enzyme reaction without inhibitor=0% inhibition; all other assay components, but no enzyme=100% inhibition). Usually, the test substances were tested on the same microtiter plate at 11 different concentrations in the range from 20 μM to 73 pM (20 μM, 5.7 μM, 1.6 μM, 0.47 μM, 0.13 μM, 38 nM, 10.9 nM, 3.1 nM, 0.9 nM, 0.25 nM and 73 pM, the dilution series were prepared prior to the assay on the level of the 100-fold concentrated solution by serial 1:3 dilutions with 100% DMSO) in duplicates for each concentration, and the IC50 values were calculated using a 4-parameter fit.
Measurement: IC50 = 1.5 nM (pChEMBL 8.819999694824219)
Descriptors: MW 381.85, ALogP 1.14, HBA 5, HBD 1, PSA 99.26, QED 0.86
Compound (SMILES): C=CC(=O)N1CC[C@@H](n2nc(-c3ccc(Oc4cccc(OC)c4F)cc3)c3cncc(C(F)F)c32)C1
InChIKey: AIJJFJWHVXFKJK-QGZVFWFLSA-N
Target: Tyrosine-protein kinase BTK (SINGLE PROTEIN), Homo sapiens
UniProt: Q06187
Assay: Kinase Assay: Measurements were performed in a reaction volume of 15 μL using 384-well assay plates. Kinase enzyme, inhibitor, ATP and 1 μM peptide substrate were incubated in a reaction buffer compose of Hepes 50 mM (pH7.0), NaN3 0.02%, BSA 0.01%, Orthocanadate 0.1 mM. After one hour, the kinase reaction was quenched by the addition of Eμ-labeled antibody and XL-665 in 1ÃDetection buffer containing 60 mM EDTA (Cisbio), and the mixture was allowed to incubate for one hour. The HTRF signal was measured on a multimode plate reader (EnVisionÂ® Multilabel Reader, Perkin Elmer) with an excitation wavelength (λEx) of 330 nm and detection wavelengths (λEm) of 615 and 665 nm. Activity was determined by the ratio of the fluorescence at 665 nm to that at 615 nm. For each compound, enzyme activity as measured at various concentrations of compound, Negative control reactions were performed in the absence of inhibitor in two replicates and eight no enzyme controls.
Measurement: IC50 < 10 nM (pChEMBL 8)
Descriptors: MW 508.5, ALogP 5.94, HBA 6, HBD 0, PSA 69.48, QED 0.29
Compound (SMILES): CN(c1cccc(C#N)c1)c1ccc2c(c1)OCC[C@H]2CNc1cnccc1C(=O)O
InChIKey: QYOWPBZARUNSRN-KRWDZBQOSA-N
Target: Lysine-specific demethylase 4C (SINGLE PROTEIN), Homo sapiens
UniProt: Q9H3R0
Assay: JMJD2C Assay: The ability of test compounds to inhibit the activity of JMJD2C was determined in 384-well plate format under the following reaction conditions: 0.3 nM JMJD2C, 300 nM H3K9me3-biotin labeled peptide (Anaspec cat #64360), 2 μM alpha-ketoglutaric acid in assay buffer of 50 mM HEPES, pH7.3, 0.005% Brij35, 0.5 mM TCEP, 0.2 mg/ml BSA, 50 μM sodium L-ascorbate, and 2 μM ammonium iron(II) sulfate. Reaction product was determined quantitatively by TR-FRET after the addition of detection reagent Phycolink Streptavidin-allophycocyanin (Prozyme) and Europium-anti-di-methylated histone H3 lysine 9 (H3K9me2) antibody (PerkinElmer) in the presence of 5 mM EDTA in LANCE detection buffer (PerkinElmer) at a final concentration of 50 nM and 1 nM, respectively.The assay reaction was initiated by the following: 2 μl of the mixture of 900 nM H3K9me3-biotin labeled peptide and 6 μM alpha-ketoglutaric acid with 2 μl of 11-point serial diluted inhibitor in 3% DMSO were added to each well of the plate, followed by the addition of 2 μl of 0.9 nM JMJD2C to initiate the reaction. The reaction mixture was incubated at room temperature for 30 minutes, and terminated by the addition of 6 μl of 5 mM EDTA in LANCE detection buffer containing 100 nM Phycolink Streptavidin-allophycocyanin and 2 nM Europium-anti-H3K9me2 antibody. Plates were read by EnVisionMultilabel Reader in TR-FRET mode (excitation at 320 nm, emission at 615 nm and 665 nm) after 1 hour incubation at room temperature. A ratio was calculated (665/615) for each well and fitted to determine inhibition constant (IC50).
Measurement: IC50 = 550 nM (pChEMBL 6.260000228881836)
Descriptors: MW 414.47, ALogP 4.4, HBA 6, HBD 2, PSA 98.48, QED 0.62
Compound (SMILES): COc1ccc(NS(C)(=O)=O)cc1-c1ccc([C@H]2C[C@@H]2NC2CCNCC2)cc1
InChIKey: FXKFQJGJMSTPFG-CTNGQTDRSA-N
Target: Amine oxidase [flavin-containing] A (SINGLE PROTEIN), Homo sapiens
UniProt: P21397
Assay: Biological Assay: Human recombinant monoamine oxidase proteins MAO-A and MAO-B were purchased from Sigma Aldrich. MAOs catalyze the oxidative deamination of primary, secondary and tertiary amines. In order to monitor MAO enzymatic activities and/or their inhibition rate by inhibitor(s) of interest, a fluorescence-based (inhibitor)-screening assay was set up. 3-(2-Aminophenyl)-3-oxopropanamine (kynuramine dihydrobromide, Sigma Aldrich), a non fluorescent compound was chosen as a substrate. Kynuramine is a non-specific substrate for both MAO-A and MAO-B activities. While undergoing oxidative deamination by MAO activities, kynuramine is converted into 4-hydroxyquinoline (4-HQ), a resulting fluorescent product.The monoamine oxidase activity was estimated by measuring the conversion of kynuramine into 4-hydroxyquinoline. Assays were conducted in 96-well black plates with clear bottom (Corning) in a final volume of 100 uL. The assay buffer was 100 mM HEPES, pH 7.5. Each experiment was performed in duplicate within the same experiment. Briefly, a fixed amount of MAO (0.25 ug for MAO-A and 0.5 ug for MAO-B) was incubated on ice for 15 minutes in the reaction buffer, in the absence and/or in the presence of at least eight 3-fold serial dilutions each. Clorgyline and Deprenyl (Sigma Aldrich) was used as a control for specific inhibition of MAO-A and MAO-B respectively. After leaving the enzyme(s) interacting with the inhibitor, KM of kynuramine was added to each reaction for MAO-B and MAO-A assay respectively, and the reaction was left for 1 hour at 37° C. In the dark. The oxidative deamination of the substrate was stopped by adding 50 uL of NaOH 2N. The conversion of kynuramine to 4-hydroxyquinoline, was monitored by fluorescence (excitation at 320 nm, emission at 360 nm) using a microplate reader (Infinite 200, Tecan). Arbitrary units were used to measure levels of fluorescence produced in the absence and/or in the presence of inhibitor. The maximum of oxidative deamination activity was obtained by measuring the amount of 4-hydroxyquinoline formed from kynuramine deamination in the absence of inhibitor and corrected for background fluorescence in the absence of MAO enzymes. The IC50 values of each inhibitor were calculated with GraphPad Prism Software.
Measurement: IC50 = 100000 nM (pChEMBL 4)
Descriptors: MW 415.56, ALogP 2.93, HBA 5, HBD 3, PSA 79.46, QED 0.65
Compound (SMILES): O=c1n(-c2ccc(-c3cn[nH]c3)cc2)nc(CO)n1Cc1ccccc1
InChIKey: OKCWVMKGPLPEIB-UHFFFAOYSA-N
Target: Rho-associated protein kinase 2 (SINGLE PROTEIN), Homo sapiens
UniProt: O75116
Assay: In Vitro Assay: The effectiveness of compounds of the present invention as ROCK inhibitors can be determined in a 30 μL, assay containing 20 mM HEPES, pH 7.5, 20 mM MgCl2, 0.015% Brij-35, 4 mM DTT, 5 μM ATP and 1.5 μM peptide substrate (FITC-AHA-AKRRRLSSLRA-OH). Compounds were dissolved in DMSO so that the final concentration of DMSO was <2%, and the reaction was initiated with Rho kinase variants. After incubation, the reaction was terminated by the addition of EDTA and the phosphorylated and non-phosphorylated peptides separated using a LABCHIP® 3000 Reader (Caliper Life Sciences). Controls consisted of assays that did not contain compound, and backgrounds consisted of assays that contained enzyme and substrate but had EDTA from the beginning of the reaction to inhibit kinase activity. Compounds were tested in dose-response format, and the inhibition of kinase activity was calculated at each concentration of compound. The inhibition data were fit using a curve-fitting program to determine the IC50; i.e., the concentration of compound required to inhibit 50% of kinase activity.
Measurement: IC50 = 27.5 nM (pChEMBL 7.559999942779541)
Descriptors: MW 347.38, ALogP 1.96, HBA 6, HBD 2, PSA 88.73, QED 0.58
Compound (SMILES): CO[C@@H](C)C(=O)N[C@H]1CC[C@@H](N(C)C(=O)c2ccc(-c3nc4ccccc4o3)cc2)C1
InChIKey: LFTLGMZNYBPOJL-ZYSHUDEJSA-N
Target: Fatty acid synthase (SINGLE PROTEIN), Homo sapiens
UniProt: P49327
Assay: Biochemical Assay of FASN: The biochemical FASN activity testing was performed as 384 well two-time-point kinetic fluorescence intensity assay format in Greiner low volume medium binding 384-well black microtiter plates in a total assay volume of 8 μl and was used for high throughput screen. In each well 3 μl 40 nM human recombinant full-length fatty acid synthase (produced in-house in SF9 cells) were dispensed in the following assay buffer: 50 mM potassium phosphate buffer pH 7.0, 0.005% (w/v) BSA, 2 mM Glutathione, 0.02% Tween-20. Then 2 μl of 200 μM NADPH in assay buffer were added, followed by the addition of the test compounds in 10 dilution concentrations starting with 30 μM (final concentration) to get a final DMSO content of 1% (v/v). The mixture was incubated for at least 15 min at room temperature. After the pre-incubation the enzymatic reaction was started by the addition of 2 μl substrate solution (80 μM acetyl-CoA, 240 μM malonyl-CoA). A first fluorescence intensity measurement (time point one) was performed with an Envision multimode reader (Perkin Elmer LAS Germany GmbH) at excitation wavelength 340 nm (lamp mode) and emission wavelength 460 nm. The reaction was incubated for 30 minutes at room temperature. After this the fluorescence intensity was measured again in the Envision using the same parameters as described above (second time point measurement). The data were analysed by subtracting the first time point measurement value from the second time point measurement value (after the enzymatic reaction). The differences of the emission signals were determined. These reflect directly the conversion rate of NADPH. The full value used was the inhibitor-free reaction. A pharmacological zero value was used like GSK837149A (Sigma-Aldrich) in a final concentration of 5-10 μM. The inhibitory values (IC50) were determined using either the program Symyx Assay Explorer® or Condosseo® from GeneData.
Measurement: IC50 < 300 nM (pChEMBL 6.5229997634887695)
Descriptors: MW 421.5, ALogP 3.64, HBA 5, HBD 1, PSA 84.67, QED 0.66
Compound (SMILES): COc1ccc2cc(C(=O)[C@H]3CCCN(C4CCCC4)C3)ccc2c1
InChIKey: JNSVKSRNLAOUJI-IBGZPJMESA-N
Target: Lysine-specific demethylase 2B (SINGLE PROTEIN), Homo sapiens
UniProt: Q8NHM5
Assay: KDM2B TR-FRET Assay: Full length KDM2B was cloned, expressed, and purified to homogeneity. Compound inhibition of KDM2B demethylase activity was assessed by monitoring the methylation status of a biotin-H3K36me2 peptide substrate (H2N-RKSAPATGGV(KMe2)KPHRYRPGTV-NTPEGBiot; New England Peptide) in the presence of α-keotglutarate (2-OG) and iron (Fe2+) using the TR-FRET assay technology (Cisbio). Specifically, in a 384 well ProxiPlate KDM2B (5 mM final), ascorbate (500 μM final) and DTT (2 mM final) were combined with the biotin-H3K36me2 peptide substrate (200 nM final), 2-OG (0.3 μM or 6 μM final; Sigma K2010) and Fe2+ (100 μM final; Sigma F1543) in 50 mM HEPES (pH 6.5) and 0.01% Triton-X 100 either in the presence of DMSO (final 0.25% DMSO) or compound dilution series in DMSO and mixed. After a two hour incubation at room temperature, a mixture of EU-anti-H3K36me1 antibody (2 nM final; Cisbio #64CUSKAZ), and Streptavidin-d2 (50 nM final; Cisbio #64CUS000) in 200 mM KF, 200 mM EDTA, 0.1% BSA and 50 mM HEPES (pH 6.5) was added. After 1 hour incubation, the plates were read on an Envision instrument, the readouts were transformed into % inhibition, and IC50 values were generated using a four parameter logistic model (XLFIT5).
Measurement: IC50 = 56.6 nM (pChEMBL 7.25)
Descriptors: MW 337.46, ALogP 4.69, HBA 3, HBD 0, PSA 29.54, QED 0.76
Compound (SMILES): CC(C)n1nc(-c2ccc(OC/C(=C/F)CN)cc2)nc1C1CC1
InChIKey: ANEXDGJMMZCGNQ-UKTHLTGXSA-N
Target: Amine oxidase [copper-containing] 3 (SINGLE PROTEIN), Homo sapiens
UniProt: Q16853
Assay: Enzyme Activity Assay:: The inhibition of the sample on VAP-1 enzyme activity was measured by using an Amplex® Red Monoamine Oxidase kit (Invitrogen #A12214). 100 nL of the gradiently-diluted to-be-tested compound (solvent DMSO) was added to a 384-well plate. 25 μL of 10 nM VAP-1 enzyme solution was added and incubated for 30 minutes at room temperature. A substrate mixture of VAP-1 enzyme (200 μM Amplex Red, 1 U/mL HRP, 1 mM Benzylamine) was added and incubated for 60 minutes at room temperature. After the incubation, the fluorescent signal was read with a microplate reader Envision (excitation light wavelength 530-560 nm, emission light wavelength 590 nm). The fluorescence signal value after the background signal was removed was analyzed by Prism software, and the IC50 of the sample against the VAP-1 enzyme was calculated.
Measurement: IC50 = 1.2 nM (pChEMBL 8.920000076293945)
Descriptors: MW 330.41, ALogP 3.59, HBA 5, HBD 1, PSA 65.96, QED 0.84
Compound (SMILES): COc1ncc(-c2c(-c3nnn[nH]3)cc3c(N)ncnn23)cc1C(=O)N[C@@H]1CN(C(=O)[C@@](C)(O)C(F)(F)F)C[C@@H]1F
InChIKey: MSEOURRMOMBKIG-OSRJUQGESA-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 = 25000 nM (pChEMBL 4.599999904632568)
Descriptors: MW 579.48, ALogP 0.15, HBA 12, HBD 4, PSA 202.43, QED 0.23
Compound (SMILES): C=CC(=O)N1CCC[C@@H](n2nc(-c3ccc(C(=O)NCc4cccc(OC)c4)cc3)c3cnccc32)C1
InChIKey: UQNIDVWIHRMXFQ-HSZRJFAPSA-N
Target: Tyrosine-protein kinase BTK (SINGLE PROTEIN), Homo sapiens
UniProt: Q06187
Assay: Kinase Assay: Measurements were performed in a reaction volume of 15 μL using 384-well assay plates. Kinase enzyme, inhibitor, ATP and 1 μM peptide substrate were incubated in a reaction buffer compose of Hepes 50 mM (pH7.0), NaN3 0.02%, BSA 0.01%, Orthocanadate 0.1 mM. After one hour, the kinase reaction was quenched by the addition of Eμ-labeled antibody and XL-665 in 1ÃDetection buffer containing 60 mM EDTA (Cisbio), and the mixture was allowed to incubate for one hour. The HTRF signal was measured on a multimode plate reader (EnVisionÂ® Multilabel Reader, Perkin Elmer) with an excitation wavelength (λEx) of 330 nm and detection wavelengths (λEm) of 615 and 665 nm. Activity was determined by the ratio of the fluorescence at 665 nm to that at 615 nm. For each compound, enzyme activity as measured at various concentrations of compound, Negative control reactions were performed in the absence of inhibitor in two replicates and eight no enzyme controls.
Measurement: IC50 = 550 nM (pChEMBL 6.260000228881836)
Descriptors: MW 495.58, ALogP 4.39, HBA 6, HBD 1, PSA 89.35, QED 0.38
Compound (SMILES): Cc1sc2c(c1C)C(c1ccc(Cl)cc1)=N[C@@H](CC(=O)NCCCCCCCC(=O)Nc1cccc(C(=O)N3CCC4(CCC(=O)NC4=O)C3)c1)c1nnc(C)n1-2
InChIKey: YDJFNASTIGGGBC-MUAQNSFOSA-N
Target: Protein cereblon (SINGLE PROTEIN), Homo sapiens
UniProt: Q96SW2
Assay: Fluorescence Direct Binding Asaay: Determination of the affinities of compounds to protein containing one or more tryptophan is measurable by monitoring the fluorescence emission in direct mode. The measurements, depending on the protein available amounts, were performed either manually in a cuvette on ISS-PCI photon counting spectrofluorometer or automatically in well plates on a fluorescence plate reader device. Fluorescence titrations were performed at 20° C. in the chosen binding assay buffer by using a defined constant protein concentration against ligand concentration variations. Small aliquots of known ligand concentration solubilized in DMSO were added and the fluorescence, excited at 280 nm, was recorded at 340 nm. The fluorescence intensity was corrected for protein dilution and for the filter effect (Birdsall, B., King, R. W., Wheeler, M. R., Lewis, C. A. Jr, Goode, S. R., Dunlap, R. B. & Roberts, G. C. (1983). Anal. Biochem.132, 353-361). The corrected fluorescence intensity was plotted against the ligand concentration and fitted using a four-parameter sigmoidal function from which the equilibrium dissociation constant Kd was computed using the law of mass action assuming a 1:1 proteinligand complex (Eftink, Methods Enzymol. 1997; 278:221-57). The process includes:Optimization of measurement parameters to minimize protein consumption and to minimize the dilution effect and the DMSO content. Titration measurements of the protein against ligand by at least 12 titration steps to obtain an s-curve fit. Repeat the same titration measurements with the ligand alone to enable correction. Check the stability of the protein once by titration against DMSO alone. Determination of the molar extinction coefficients of the ligand at 280 and 340 nm with help of an UV-spectrophotometer. Use Excel template for the correction of the measured raw data. Use GraphPad Prism software for the quadratic binding fit and the KD evaluation.
Measurement: Kd = 106 nM (pChEMBL 6.96999979019165)
Descriptors: MW 811.41, ALogP 6.55, HBA 10, HBD 3, PSA 167.75, QED 0.1
Compound (SMILES): C#C[C@@]1(O)[C@@H](COC(Cc2ccccc2)C(=O)O)O[C@@H](n2cnc3c(NC4CCOC4)nc(Cl)nc32)[C@@H]1O
InChIKey: OXWLZHISWPZQQP-RXSOQNLOSA-N
Target: 5'-nucleotidase (SINGLE PROTEIN), Homo sapiens
UniProt: P21589
Assay: Inhibition of the CD73 Enzyme In Vitro: For measurements of soluble CD73 enzyme activity, recombinant CD73 was obtained from R&D Systems, Cat. No. 5795-EN-010. Serial dilutions of test compounds were incubated with recombinant CD73 and AMP in reaction buffer (25 mM Tris HCl pH7.5, 5 mM MgCl2, 50 mM NaCl, 0.25 mM DTT, 0.005% Triton X-100). The final reaction volume was 25 μL and the final concentrations of recombinant CD73 and AMP were 0.05 nM and 50 μM, respectively. Reactions were allowed to proceed for 1 hour at 37° C. before the addition of 100 μL Malachite Green (Cell Signaling Technology, Cat. No. 12776). After 5 minutes at room temperature, absorbance at 630 nm was determined on a microplate spectrophotometer.
Measurement: IC50 = 3 nM (pChEMBL 8.520000457763672)
Descriptors: MW 543.96, ALogP 1.02, HBA 11, HBD 4, PSA 161.08, QED 0.23
Compound (SMILES): C=CC(=O)N1C[C@H](C)N(c2nc(=O)n(-c3c(C(C)C)nc(OCCN(C)C)nc3C(C)C)c3nc(-c4ccccc4F)c(Cl)cc23)C[C@H]1C
InChIKey: PMNIJSSSDVNWEA-PKTZIBPZSA-N
Target: GTPase KRas (SINGLE PROTEIN), Homo sapiens
UniProt: P01116
Assay: Coupled Nucleotide Exchange Assay: Purified GDP-bound KRAS protein (aa 1-169), containing both G12C and C118A amino acid substitutions and an N-terminal His-tag, was pre-incubated in assay buffer (25 mM HEPES pH 7.4, 10 mM MgCl2, and 0.01% Triton X-100) with a compound dose-response titration for either 5 min or 2 hours (see Table 15). Following compound pre-incubation, purified SOS protein (aa 564-1049) and GTP (Roche 10106399001) were added to the assay wells and incubated for an additional 30 min (for 5 min compound pre-incubation) or 1 hour (for 2 hour compound pre-incubation). To determine the extent of inhibition of SOS-mediated nucleotide exchange, purified GST-tagged cRAF (aa 1-149), nickel chelate AlphaLISA acceptor beads (PerkinElmer AL108R), and AlphaScreen glutathione donor beads (PerkinElmer 6765302) were added to the assay wells and incubated for 10 minutes. The assay plates were then read on a PerkinElmer EnVision Multilabel Reader, using AlphaScreen® technology, and data were analyzed using a 4-parameter logistic model to calculate IC50 values.
Measurement: IC50 = 64 nM (pChEMBL 7.190000057220459)
Descriptors: MW 691.25, ALogP 5.83, HBA 10, HBD 0, PSA 109.58, QED 0.19
Compound (SMILES): CCCc1cnc2c(C(=O)Nc3cnccc3N3C[C@@H](N)[C@H](O)[C@@H](C)C3)c(N)sc2c1
InChIKey: MQMQKXIYQAHIDI-KBARZSOFSA-N
Target: Serine/threonine-protein kinase pim-1 (SINGLE PROTEIN), Homo sapiens
UniProt: P11309
Assay: Enzyme Assay: Pim-1 and Pim-3 kinase assays-20 μL reactions were run in white 384 well polystyrene plates dotted with 0.8 μL compound/DMSO in the assay buffer (50 mM Tris, pH 7.5, 0.01% Tween-20, 5 mM MgCl2, 0.01% BSA, 5 mM DTT), containing 0.05 μm Biotin-labeled BAD peptide substrate (AnaSpec 62269), 1 mM ATP, and 2.5 pM (Pim-1, Invitrogen PV3503) or 1.25 pM (Pim-3, Millipore 14-738) enzyme for 1 h at 25° C. Reactions were stopped by addition of 10 μL STOP Buffer (150 mM Tris, pH=7.5, 150 mM NaCl, 75 mM EDTA, 0.01% Tween-20, 0.3% BSA) supplemented with Phospho-Bad (Ser112) Antibody (Cell Signaling 9291) diluted 666-fold, and Streptavidin donor beads (PerkinElmer 6760002) along with Protein-A acceptor beads (PerkinElmer 6760137) at 15 μg/mL each. Supplementation of the STOP buffer with beads and stopping the reactions were done under reduced light. Prior to the stopping reactions STOP buffer with beads was pre-incubated for 1 h in the dark at room temperature. After stopping the reactions, plates were incubated for 1 h in the dark at room temperature before reading on a PHERAstar FS plate reader (BMG Labtech) under reduced light.Pim-2 kinase assay-20 μL reactions were run in white 384 well polystyrene plates dotted with 0.8 μL compound/DMSO in the assay buffer (50 mM Tris, pH 7.5, 0.01% Tween-20, 5 mM MgCl2, 0.01% BSA, 5 mM DTT), containing 0.05 μm Fluorescein-labeled CREBtide peptide substrate (Invitrogen PV3508), 1 mM ATP, and 1 nM enzyme (Invitrogen PV3649) for 2 h at 25° C. Reactions were stopped by addition of 10 μL TR-FRET Dilution Buffer (Invitrogen PV3574) with 30 mM EDTA and 1.5 nM LanthaScreen Tb-CREB pSer133 antibody (Invitrogen PV3566). After 30 min. incubation at room temperature, plates were read on a PHERAstar FS plate reader (BMG Labtech).
Measurement: IC50 < 10 nM (pChEMBL 8)
Descriptors: MW 440.57, ALogP 2.62, HBA 8, HBD 4, PSA 130.39, QED 0.48
Compound (SMILES): CCOC(=O)NC(=O)NCCSc1nonc1C(=N)Nc1ccc(F)c(Br)c1
InChIKey: GEWKVVFMNHGPNI-UHFFFAOYSA-N
Target: Indoleamine 2,3-dioxygenase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P14902
Assay: Enzyme Assay: HIS-tagged IDO1 protein was recombinantly expressed in Escherichia coli using ZYP5052 autoinduction media supplemented with 500 uM delta aminolevulinic acid for 48 h at 16° C. IDO1 protein was purified using Ni2+-affinity resin and size exclusion chromatography. Purified protein was then diluted in assay buffer (50 mM Tris pH 7.0, 1% glycerol, 20 uM methylene blue, 0.05% Tween-20, 20 mM sodium ascorbate, 100 units/mL catalase to obtain a final IDO1 concentration of 40 nM. IDO1 solution (30 uM) or buffer alone (30 uM) were dispensed to wells of the assay plate using a BioRAPTR liquid dispenser (Beckman Coulter). Assay plates containing compound and IDO1 enzyme were incubated at RT for 30 min. Afterwards, 10 uL of 400 uM tryptophan in assay buffer were added to each well of the assay plate using a BioRAPTR liquid dispenser. Plates were incubated at RT for 60 min and reactions were quenched by addition of 10 uL of 0.5 M methyl isonipecotate in dimethyl sulfoxide. Plates were sealed and incubated at 37° C. for 4 h or 50° C. for 2 h. The plates are allowed to cool and then centrifuged for 1 min at 1000 ug. The resulting fluorescence was measured in an Envision plate reader (Perkin Elmer) with a 400/25 nm excitation filter and an 510/20 nm emission filter.
Measurement: IC50 = 110 nM (pChEMBL 6.960000038146973)
Descriptors: MW 475.3, ALogP 2.96, HBA 8, HBD 4, PSA 142.23, QED 0.21
Compound (SMILES): CN(C)C(=O)c1cc2cc(-c3nc(Nc4ccc(N5CCNCC5)cn4)ncc3F)ccc2n1C1CCCC1
InChIKey: CBYUJXBONNVTCO-UHFFFAOYSA-N
Target: Cyclin-dependent kinase 4 (SINGLE PROTEIN), Homo sapiens
UniProt: P11802
Assay: Caliper Mobility Shift Assay: CDK4: The test compound was dissolved in DMSO and diluted with a kinase buffer solution (20 mM HEPES-pH 7.5, 0.01% Triton X-100, 10 mM MgCl2, 2 mM DTT) and 5 μL of compound at 5-fold final concentration of reaction dissolved in 10% DMSO was added in a 384-well plate. The compound-free control well was 5 μL of 10% DMSO, and the no-activity control well was 5 μL of kinase buffer. 10 μL of a 2.5-fold diluted CDK4 enzyme solution (GST-CDK4(1-303 end)) was added and incubated at room temperature for 10 min, and then 10 μL of the 2.5-fold diluted substrate solution Peptide FAM-P8 was added. The reaction was stopped by adding 25 μL of stop solution after incubation at 28° C. for 3 h, and the conversion rate data was read on a Caliper EZ Reader II (Caliper Life Sciences) and the conversion rate was converted to inhibition rate data according to above method.
Measurement: IC50 < 20 nM (pChEMBL 7.698999881744385)
Descriptors: MW 528.64, ALogP 4.6, HBA 8, HBD 2, PSA 91.21, QED 0.38
Compound (SMILES): COc1ccc(F)cc1-c1ccnc2[nH]c(C3CCN(S(=O)(=O)NC[C@H](O)CO)CC3)cc12
InChIKey: BEBOFNISPDSMFG-INIZCTEOSA-N
Target: CDK9/cyclin T1 (PROTEIN COMPLEX), Homo sapiens
UniProt: P50750
Assay: LANCE ULight TR-FRET Kinase Assay: CDK9 enzyme activities were measured using LANCE ULight TR-FRET kinase assay reagents (PerkinElmer, Waltham, Mass.). Compounds were directly added in 100% DMSO to white low volume assay plates (Perkin Elmer Proxiplate 6008289) using a Labcyte Echo acoustic dispenser. Assay reagents in serine/threonine kinase assay buffer containing 20 mM HEPES, 10 mM MgCl2, 100 mM Na3VO4, and 0.0075% Triton X-100. were added for final reaction mixture concentrations of 1000 μM ATP, 100 nM U-light MBP peptide (Perkin Elmer TRF0109M) and reaction initiated with 4 nM CDK9/Cyclin T1 (Carna Biosciences 04-110). The kinase reaction was carried out for 30 minutes before addition of stopping buffer to a final of 20 mM EDTA and 0.5 nM of LANCE Ultra Europium anti-phospho-MBP antibody (PerkinElmer TRF0201M) in LANCE detection buffer (PerkinElmer CR97-100). The reaction was equilibrated for 1 hour and the signal read in the Perkin Elmer Envision in TR-FRET mode (excitation at 320 nm and emission at 615/665 nm).
Measurement: IC50 = 39 nM (pChEMBL 7.409999847412109)
Descriptors: MW 478.55, ALogP 1.74, HBA 6, HBD 4, PSA 127.78, QED 0.39
Compound (SMILES): CC(C)(C)[S@@+]([O-])NC(CCC1CC1)(c1cccc(C#N)c1)c1ccc(F)c(NC(=O)[C@H]2CCCN2C(=O)Nc2ccc(Cl)cc2)c1
InChIKey: QMMABOBRWWOXDM-BLQLWLDUSA-N
Target: Plasma kallikrein (SINGLE PROTEIN), Homo sapiens
UniProt: P03952
Assay: Activity Assay: The effect of compounds of the invention on human plasma kallikrein activity was determined using the chromogenic substrates. In these experiments, 2 nM kallikrein was incubated with 80 μM S2302 (H-D-Pro-Phe-Arg-p-nitroaniline) in the absence or presence of increasing concentrations of compounds of the invention in a final volume of 200 μL Tris-HCl buffer (200 mM NaCl; 2.5 mM CaCl2; 50 mM Tris-HCl, pH 7.8).After incubation at 30° C., the activity of kallikrein was measured as a change in absorbance at OD 405 nm using BioTek PowerWave X340 Microplate Reader.
Measurement: Ki > 100 nM (pChEMBL 7)
Descriptors: MW 664.25, ALogP 7.47, HBA 5, HBD 3, PSA 120.32, QED 0.19