lovingscience/chembl-v37-raw-activities huggingface dataset:
Compound (SMILES): CCOC(=O)c1cnn(-c2ccc(Nc3nn([C@@H](CC#N)C4CC4)c4cc[nH]c(=O)c34)cc2C)c1
InChIKey: ZNHJTMAKBXDJIV-FQEVSTJZSA-N
Target: Tyrosine-protein kinase JAK1 (SINGLE PROTEIN), Homo sapiens
UniProt: P23458
Assay: Jak Biochemical HTRF Assay Protocol: The ability of compounds to inhibit the activity of JAK1, JAK2, JAK3, and Tyk2 was measured using a recombinant purified GST-tagged catalytic domain for each enzyme (Invitrogen JAK1 #M4290, JAK2 #M4290, JAK3 #M4290, Tyk2 #M4290) in an HTRF format biochemical assay. The reactions employed a common peptide substrate, LCB-EQEDEPEGDYFEWLW-NH2 (in-house). The basic assay protocol is as follows: First, 250 nL of diluted compounds in DMSO were dispensed into the wells of a dry 384-well Black plate (Greiner #781076) using a Labcyte Echo 555 acoustic dispenser. Subsequent reagent additions employed an Agilent Bravo. Next, 18 μL of 1.11× enzyme and 1.11× substrate in 1× assay buffer (Invitrogen kinase buffer #PV3189, 2 mM DTT, 0.05% BSA) were added to the wells and shaken and then preincubated for 30 minutes at room temperature to allow compound binding to equilibrate. After equilibration, 2 μL of 10×ATP in 1× assay buffer was added to initiate the kinase reaction and the plates were shaken and then incubated at room temperature for 120 minutes. At the end of the incubation, 20 μL of 2× stop buffer (streptavidin-Dylight 650 (Thermo #84547B/100 mL), Eu-tagged pY20 antibody (Perkin Elmer #AD0067), EDTA, HEPES, and Triton) was added to quench the reaction. Plates were shaken and centrifuged and then incubated 60 minutes at room temperature and then read on a Perkin Elmer Envision (λex=337 nm, λem=665 and 615 nm, TRF delay time=20 μs). HTRF signal=10,000*665 nm reading/615 nm reading. After normalization to untreated controls, the percent inhibition of the HTRF signal at each compound concentration was calculated.
Measurement: IC50 = 700 nM (pChEMBL 6.159999847412109)
Descriptors: MW 471.52, ALogP 4, HBA 9, HBD 2, PSA 130.62, QED 0.37
Compound (SMILES): CCCCc1nc(O)c(-c2nnc(Cc3cnn(-c4ccccc4)c3)o2)c(O)c1-c1c(OC)cccc1OC
InChIKey: JDUDGMHVSXVGEZ-UHFFFAOYSA-N
Target: Apelin receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P35414
Assay: Intracellular cAMP Accumulation Assay: HEK293 cells stably expressing human APJ receptor were used to assess the activity of compounds. Cultured cells were detached and resuspended in the cAMP Homogeneous Time-Resolved Fluorescence (HTRF) assay buffer (Cisbio cat; #62AM4PEJ). The assay was performed in 384-well assay plates (Perkin-Elmer; cat #6008289) according to assay protocol provided by the manufacturer. Serial dilutions of a compound together with assay buffer containing 0.2 nM IBMX and 2 μM forskolin were added to each well containing 5,000 cells and incubated for 30 minutes at room temperature. Subsequently, cAMP D2 reagent was added in the lysis buffer followed by the EuK antibody (Cisbio; cat #62AM4PEJ) and incubated for 60 min. The fluorescence emission ratio was measured using fluorometer. The intracellular cAMP concentrations (compound-stimulated inhibition of forskolin-mediated cAMP production) were calculated by extrapolation from a standard curve using known cAMP concentrations. The EC50 values were obtained by fitting the data to a sigmoidal concentration-response curve with variable slope. The maximal achievable inhibition of forskolin-induced cAMP levels (Ymax) for each compound was expressed as relative percentage of inhibition attained using pyroglutamated apelin-13 ((Pyr1)apelin-13) peptide, which was set to 100%.The examples disclosed below were tested in the APJ in vitro assays described above and were found having human APJ cyclic AMP (hcAMP) activity. The EC50 value of each compound is presented at the end of the example description.
Measurement: EC50 = 55 nM (pChEMBL 7.260000228881836)
Descriptors: MW 527.58, ALogP 5.35, HBA 10, HBD 2, PSA 128.55, QED 0.25
Compound (SMILES): COc1ccc(N2CCN(C3CCN(c4ccc(-c5cn[nH]c5)c(OC)n4)C3=O)CC2)cc1
InChIKey: NDMOJAGIULBVAJ-UHFFFAOYSA-N
Target: Rho-associated protein kinase 2 (SINGLE PROTEIN), Homo sapiens
UniProt: O75116
Assay: In Vitro Assays: 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 (SEQ ID NO: 1)). 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 = 1150 nM (pChEMBL 5.940000057220459)
Descriptors: MW 448.53, ALogP 2.42, HBA 7, HBD 1, PSA 86.82, QED 0.62
Compound (SMILES): CC(C)(C)c1ccc2c(=O)c3ccc(Cl)c(Cl)c3n(CC(=O)O)c2c1
InChIKey: FCLILAVMASBPEC-UHFFFAOYSA-N
Target: Stimulator of interferon genes protein (SINGLE PROTEIN), Homo sapiens
UniProt: Q86WV6
Assay: Radiometric Filtration Binding Competition Assay: Briefly, the recombinant C-terminal ligand binding domain of the human STING protein (140-379) or mouse STING protein (139-328) [0.5 μM] was incubated in the presence of a compound of the present application (8 point, 2 fold dilution starting at 300 μM) for 30 min or under control conditions. H3 labeled 2′3′cGAMP [25 nM] was then added and allowed to reach binding equilibrium (1 hour). The resulting complexes were then filtered, dried, and scintillation fluid was added and the remaining radio signal was measured to determine the degree of compound ligand inhibition. This assay format was optimized for both the WT and HAQ isoforms of the human protein and the mouse orthologue protein. Assay DevelopmentParameters Optimized:a. Form of protein: 6×HIS-SUMO tag or untaggedb. Concentration of target protein: Minimum concentration that achieved >80 max signalc. Assay DMSO concentration: 10%-0.1%d. Assay Buffer: Base (Tris or phosphate), Salt concentration, +/−Tween (0.1-2%)e. Synthesis of Probe: (1) Enzymatic incorporation of a S35-ATP and cold GTP into a 2′3′cGAMP product using a mouse cGAS enzyme with subsequent purification. (2) Tritium incorporation into 2′3′cGAMPf. Probe Concentration: Minimum concentration that achieved max assay windowg. Assay Format: Scintillation Proximity Bead or Filtration Binding Plateh. Assay Plate: 384 vs 96 well formati. Assay incubation times for successive incubation steps
Measurement: IC50 < 100000 nM (pChEMBL 4)
Descriptors: MW 378.26, ALogP 4.84, HBA 3, HBD 1, PSA 59.3, QED 0.65
Compound (SMILES): Cc1[nH]c(-c2ccc(C(F)(F)F)cc2)nc1CN1CCC(n2c(=O)[nH]c3ccc(N4CCCC4)cc32)CC1
InChIKey: GQPLVMOARWOTCY-UHFFFAOYSA-N
Target: Cathepsin S (SINGLE PROTEIN), Homo sapiens
UniProt: P25774
Assay: Cathepsin Enzyme Inhibition Assay: Enzyme activity is measured by observing the increase in fluorescence intensity caused by cleavage of a peptide substrate containing a fluorophore whose emission is quenched in the intact peptide.Assay buffer: 100 mM potassium phosphate pH 6.5, EDTA-Na 5 mM, Triton X-100 0.001%, DTT 5 mM.Enzymes (all at 1 nM): human and mouse Cathepsin S, Cat K, Cat B, Cat L.Substrate (20 Z-Val-Val-Arg-AMC, except for Cat K which uses Z-Leu-Arg-AMC (both from Bachem).Z=Benzyloxycarbonyl.AMC=7-Amino-4-Methyl-Coumarin.DTT=dithiothreitol.Final volume: 100 μL.Excitation 360 nm, Emission 465 nm.Enzyme is added to the substance dilutions in 96-well microtitre plates and the reaction is started with substrate. Fluorescence emission is measured over 20 minutes, during which time a linear increase is observed in the absence of inhibitor.
Measurement: IC50 = 190 nM (pChEMBL 6.71999979019165)
Descriptors: MW 524.59, ALogP 5.48, HBA 5, HBD 2, PSA 72.95, QED 0.36
Compound (SMILES): CN(C)C/C=C/C(=O)Nc1ccc(C(=O)Nc2cccc(Nc3ncc(Cl)c(-c4ccc5ccn(C)c5n4)n3)c2)cc1
InChIKey: KWRZXFVKKDOYHF-VMPITWQZSA-N
Target: Cyclin-dependent kinase 7/ cyclin H (PROTEIN COMPLEX), Homo sapiens
UniProt: P51946
Assay: In Vitro Kinase Assay: In vitro kinase assays with immunoprecipitated proteins (or recombinant CAK complexes) were performed as follows. Kinase reactions were performed in 25 μL of final volume of reaction buffer (50 mM Hepes, pH 7.5, 150 mM NaCl, 10 mM MgCl2, 5% glycerol, 25 μM ATP, and 10 μCi of [γ-32P]ATP). Each reaction contained 1 μg of RNAP II. Incubation was at 30° C. for 30-45 min. Reactions were terminated by addition of 5 μL of 5×SDS-PAGE sample buffer and incubated at 95° C. for 5 min. Reactions were analyzed by SDS-PAGE, and the dried gel was exposed to film or PhosphorImager plate.
Measurement: IC50 = 550 nM (pChEMBL 6.260000228881836)
Descriptors: MW 581.08, ALogP 5.74, HBA 8, HBD 3, PSA 117.07, QED 0.19
Compound (SMILES): C=CC(=O)Nc1cccc(C(=O)Nc2n[nH]c3c2CN(C(=O)N[C@H](CN(C)C)c2ccccc2)C3(C)C)c1
InChIKey: STSHHEUAGAQVAK-JOCHJYFZSA-N
Target: CDK7/Cyclin H/MNAT1 (PROTEIN COMPLEX), Homo sapiens
UniProt: P51946
Assay: Inhibition of Kinase Activity: Compounds of the invention were assayed for activity against a variety of different kinases.
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 515.62, ALogP 3.85, HBA 5, HBD 4, PSA 122.46, QED 0.34
Compound (SMILES): CS(=O)(=O)N1CCC(Nc2ncc(C(F)(F)F)c(-c3cn(-c4ccc(Cn5cnnn5)cc4Cl)cn3)n2)CC1
InChIKey: AJCQGTTZWCJOHV-UHFFFAOYSA-N
Target: Cyclin-dependent kinase 2/cyclin E1 (PROTEIN COMPLEX), Homo sapiens
UniProt: P24941
Assay: 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 were conducted in white 384-well polystyrene plates in a final reaction volume of 8 μL. CDK2/Cyclin E1 (0.25 nM) was incubated with the compounds of the Examples (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 were 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 were read after 1 hour at room temperature on a PHERAstar FS plate reader (BMG Labtech). Data was 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 583, ALogP 2.87, HBA 11, HBD 1, PSA 136.61, QED 0.35
Compound (SMILES): CCc1nc2c(n1CC1CC1)-c1nc(Nc3ccc(N4CCNCC4)cn3)ncc1C2
InChIKey: DPEQDVGECWOZRS-UHFFFAOYSA-N
Target: Cyclin-dependent kinase 4/cyclin D1 (PROTEIN COMPLEX), Homo sapiens
UniProt: P11802
Assay: In Vitro Kinase Assay: In the kinase test, a compound to be tested was 1:3 diluted for 8 gradient points, added into a reaction plate, and then added an appropriate amount of recoinbinase. A buffer [50 mM HEPES pH17.5, 10 mM MgCl2, 3 mM MnCl2, 1 mM EGTA, 0.01% Tween-20, 1 mM TCEP] containing ATP/Ulight-4EBP1 premix with predetermined concentration was subsequently added, and the kinase reaction was started at room temperature. A test solution pre-mixed with 10 mM EDTA and detection antibody was added after a suitable reaction time, and then the fluorescence value was read on Tecan infinite pro after reacting for 1 hour at room temperature. The IC50 was calculated using a four-factor model fitting in a XLfit software.
Measurement: IC50 = 59 nM (pChEMBL 7.230000019073486)
Descriptors: MW 416.53, ALogP 2.76, HBA 8, HBD 2, PSA 83.79, QED 0.5
Compound (SMILES): COc1ccc(C(=O)CCC(=O)c2cccnc2)cc1OC
InChIKey: HETKXPIAVFELSW-UHFFFAOYSA-N
Target: Catenin beta-1/Protein Wnt-3a (PROTEIN COMPLEX), Homo sapiens
UniProt: P35222
Assay: Evaluation of Biological Activity: Compounds that enhance the Wnt activity, or Activators, were assayed as follows. Reporter cell lines were generated by stably transducing cells of colon cancer cell lines (with a lentiviral construct that included a Wnt-responsive promoter driving expression of the firefly luciferase gene).Lentiviral constructs were made in which the SP5 promoter, a promoter having eight TCF/LEF binding sites derived from the SP5 promoter, was linked upstream of the firefly luciferase gene. The lentiviral constructs also included a hygromycin resistance gene as a selectable marker. The SP5 promoter construct was used to transduce SW480 cells, a colon cancer cell line having a mutated APC gene that generates a truncated APC protein, leading to de-regulated accumulation of β-catenin.Cultured SW480 cells bearing a reporter construct were distributed at approximately 10,000 cells per well into 384- or 96-well multiwell plates. Compounds from a small molecule compound library were then added to the wells in half-log dilutions using a maximum concentration of three or ten micromolar. A series of control wells for each cell type received only buffer and compound solvent DMSO. Twenty-four hours after the addition of compound, reporter activity for luciferase was assayed, for example, by addition of the BrightGlo luminescence reagent (Promega) and the Victor3 plate reader (Perkin Elmer). Readings were normalized to DMSO only treated cells, and any activities above DMSO were considered activation. Compounds were considered activators if reporter activities were 2× fold or greater than DMSO. EC50 is the concentration at half maximal activation.
Measurement: EC50 > 10000 nM (pChEMBL 5)
Descriptors: MW 299.33, ALogP 2.94, HBA 5, HBD 0, PSA 65.49, QED 0.74
Compound (SMILES): CC(C)n1c([C@H](C)O)nc2c(F)cc(-c3cc(N[C@@H]4CCOC[C@H]4O)ncc3Cl)cc21
InChIKey: LFGOPXAPKINMIT-OEPMTFCVSA-N
Target: Cyclin-dependent kinase 4/cyclin D1 (PROTEIN COMPLEX), Homo sapiens
UniProt: P11802
Assay: Biological Assay: CDK4/Cyclin D1: The purpose CDK4/Cyclin D1 assay is to evaluate the inhibition (% inhibition, Kiapp and Ki values) in the presence of small molecule inhibitors by using a fluorescence based microfluidic mobility shift assay. CDK4/Cyclin D1 catalyzes the production of ADP from ATP that accompanies the phosphoryl transfer to the substrate peptide 5-FAM-Dyrktide (5-FAM-RRRFRPASPLRGPPK) (SEQ ID NO:1). The mobility shift assay electrophoretically separates the fluorescently labeled peptides (substrate and phosphorylated product) following the kinase reaction. Both substrate and product are measured and the ratio of these values is used to generate % Conversion of substrate to product by the LabChip EZ Reader. Typical reaction solutions contained 2% DMSO (±inhibitor), 10 mM MgCl2, 1 mM DTT, 3.5 mM ATP, 0.005% TW-20, 3 μM 5-FAM-Dyrktide, 3 nM (active sites) activated CDK4/Cyclin D1 in 40 mM HEPES buffer at pH 7.5.
Measurement: Ki = 1 nM (pChEMBL 9)
Descriptors: MW 448.93, ALogP 4.09, HBA 7, HBD 3, PSA 92.43, QED 0.54
Compound (SMILES): CNC1CCN(c2ccc(-n3cc(-c4nc(NC5CCN(S(C)(=O)=O)CC5)ncc4C(F)(F)F)nc3C)c(F)c2)CC1
InChIKey: AVJYXYAGWRVZDD-UHFFFAOYSA-N
Target: Cyclin-dependent kinase 2/cyclin E1 (PROTEIN COMPLEX), Homo sapiens
UniProt: P24941
Assay: 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 were conducted in white 384-well polystyrene plates in a final reaction volume of 8 μL. CDK2/Cyclin E1 (0.25 nM) was incubated with the compounds of the Examples (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 were 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 were read after 1 hour at room temperature on a PHERAstar FS plate reader (BMG Labtech). Data was 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 610.68, ALogP 3.82, HBA 9, HBD 2, PSA 108.28, QED 0.39
Compound (SMILES): CCS(=O)(=O)N1CCC(Nc2ncc3ccc(=O)n(C4CCCC4)c3n2)CC1
InChIKey: RCOWWHMFBKCUJC-UHFFFAOYSA-N
Target: Cyclin-dependent kinase 2/cyclin E1 (PROTEIN COMPLEX), Homo sapiens
UniProt: P24941
Assay: CDK2/Cyclin E1 Mobility Shift Assay: The purpose of the CDK2/Cyclin E1 assay is to evaluate the inhibition (% inhibition, Kiapp and Ki values) of small molecule inhibitors by using a fluorescence-based microfluidic mobility shift assay. CDK2/Cyclin E1 catalyzes the production of ADP from ATP that accompanies the phosphoryl transfer to the substrate peptide FL-Peptide-18 (5-FAM-QSPKKG-CONH2) (SEQ ID NO:1). (CPC Scientific, Sunnyvale, Calif.). The mobility shift assay electrophoretically separates the fluorescently labeled peptides (substrate and phosphorylated product) following the kinase reaction. Both substrate and product are measured and the ratio of these values is used to generate % conversion of substrate to product by the LabChip EZ Reader. Wild-type full length CDK2/wild-type full length Cyclin E1 enzyme complex was produced in-house (baculoviral expression, LJIC-2080/LJIC-2103) and phosphorylated by CDK7/Cyclin H1/Mat1 enzyme complex with CDK2:CDK7 ratio of 50:1 (concentration mg/mL) in the presence of 10 mM MgCl2 and 5 mM ATP at room temperature for one hour. Typical reaction solutions (50 μL final reaction volume) contained 2% DMSO (± inhibitor), 4 mM MgCl2, 1 mM DTT, 150 μM ATP (ATP Km=67.4 μM), 0.005% Tween-20, 3 μM FL-Peptide-18, and 0.36 nM (catalytically competent active site) phosphorylated wild-type full length CDK2/Cyclin E1 enzyme complex in 25 mM HEPES buffer at pH 7.15. The assay was initiated with the addition of ATP, following a fifteen minutes pre-incubation of enzyme and inhibitor at room temperature in the reaction mixture. The reaction was stopped after 45 minutes at room temperature by the addition of 50 μL of 80 mM EDTA, pH 7.5. The Ki value was determined from the fit of the data to the Morrison tight-binding competitive inhibition equation with the enzyme concentration as a variable.
Measurement: Ki = 0.48 nM (pChEMBL 9.319999694824219)
Descriptors: MW 405.52, ALogP 2.13, HBA 7, HBD 1, PSA 97.19, QED 0.82
Compound (SMILES): Cc1nc(N[C@@H](CCN(CCCCc2ccc3c(n2)NCCC3)CCOC2CC2(F)F)C(=O)O)c2ccc(F)cc2n1
InChIKey: TVGIBZQQUWICLR-QSAPEBAKSA-N
Target: Integrin alpha-5/beta-6 (PROTEIN COMPLEX), Homo sapiens
UniProt: P08648
Assay: Binding Assay: Microplates were coated with recombinant human integrin αVβ6 (2 μg/mL) in PBS (100 μL/well 25° C., overnight). The coating solution was removed, washed with wash buffer (0.05% Tween 20; 0.5 mM MnCl2; in 1×TBS). The plate was blocked with 200 μL/well of Block Buffer (1% BSA; 5% sucrose; 0.5 mM MnCl2; in 1×TBS) at 37° C. for 2 h. Dilutions of testing compounds and recombinant TGFβ1 LAP (0.67 μg/mL) in binding buffer (0.05% BSA; 2.5% sucrose; 0.5 mM MnCl2; in 1×TBS) were added. The plate was incubated for 2 hours at 25° C., washed, and incubated for 1 hour with Biotin-Anti-hLAP. Bound antibody was detected by peroxidase-conjugated streptavidin. The IC50 values for testing compounds were calculated by a four-parameter logistic regression.
Measurement: IC50 < 50 nM (pChEMBL 7.301000118255615)
Descriptors: MW 586.66, ALogP 4.83, HBA 8, HBD 3, PSA 112.5, QED 0.22
Compound (SMILES): C[C@]1(O)CCC[C@@H]1n1c(=O)ccc2cnc(NC3CCN(S(C)(=O)=O)CC3)nc21
InChIKey: DVAPMYJVUZJNDK-KXBFYZLASA-N
Target: Cyclin-dependent kinase 2/cyclin E1 (PROTEIN COMPLEX), Homo sapiens
UniProt: P24941
Assay: CDK2/Cyclin E1 Mobility Shift Assay: The purpose of the CDK2/Cyclin E1 assay is to evaluate the inhibition (% inhibition, Kiapp and Ki values) of small molecule inhibitors by using a fluorescence-based microfluidic mobility shift assay. CDK2/Cyclin E1 catalyzes the production of ADP from ATP that accompanies the phosphoryl transfer to the substrate peptide FL-Peptide-18 (5-FAM-QSPKKG-CONH2) (SEQ ID NO:1). (CPC Scientific, Sunnyvale, Calif.). The mobility shift assay electrophoretically separates the fluorescently labeled peptides (substrate and phosphorylated product) following the kinase reaction. Both substrate and product are measured and the ratio of these values is used to generate % conversion of substrate to product by the LabChip EZ Reader. Wild-type full length CDK2/wild-type full length Cyclin E1 enzyme complex was produced in-house (baculoviral expression, LJIC-2080/LJIC-2103) and phosphorylated by CDK7/Cyclin H1/Mat1 enzyme complex with CDK2:CDK7 ratio of 50:1 (concentration mg/mL) in the presence of 10 mM MgCl2 and 5 mM ATP at room temperature for one hour. Typical reaction solutions (50 μL final reaction volume) contained 2% DMSO (± inhibitor), 4 mM MgCl2, 1 mM DTT, 150 μM ATP (ATP Km=67.4 μM), 0.005% Tween-20, 3 μM FL-Peptide-18, and 0.36 nM (catalytically competent active site) phosphorylated wild-type full length CDK2/Cyclin E1 enzyme complex in 25 mM HEPES buffer at pH 7.15. The assay was initiated with the addition of ATP, following a fifteen minutes pre-incubation of enzyme and inhibitor at room temperature in the reaction mixture. The reaction was stopped after 45 minutes at room temperature by the addition of 50 μL of 80 mM EDTA, pH 7.5. The Ki value was determined from the fit of the data to the Morrison tight-binding competitive inhibition equation with the enzyme concentration as a variable.
Measurement: Ki = 3.07 nM (pChEMBL 8.510000228881836)
Descriptors: MW 421.52, ALogP 1.1, HBA 8, HBD 2, PSA 117.42, QED 0.76
Compound (SMILES): CS(=O)(=O)N1CCC(Nc2ncc(C(F)(F)F)c(-c3cn(-c4ccc(CN5CCN6CCCC6C5)cc4Cl)cn3)n2)CC1
InChIKey: GZSJZUTVJQFFSP-UHFFFAOYSA-N
Target: Cyclin-dependent kinase 2/cyclin E1 (PROTEIN COMPLEX), Homo sapiens
UniProt: P24941
Assay: 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 were conducted in white 384-well polystyrene plates in a final reaction volume of 8 μL. CDK2/Cyclin E1 (0.25 nM) was incubated with the compounds of the Examples (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 were 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 were read after 1 hour at room temperature on a PHERAstar FS plate reader (BMG Labtech). Data was 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 639.15, ALogP 4.12, HBA 9, HBD 1, PSA 99.49, QED 0.41
Compound (SMILES): CN1CCN(C(=O)c2ccc(Nc3ncc4cc5n(c4n3)C3(CCCCC3)C(=O)NC5O)cc2)CC1
InChIKey: VASFNXBXECJRSY-UHFFFAOYSA-N
Target: CDK2/Cyclin A2 (PROTEIN COMPLEX), Homo sapiens
UniProt: P24941
Assay: CDK Inhibition In Vitro Assay: Selected compounds disclosed herein were tested in kinase assays by Nanosyn (Santa Clara, Calif.) to determine their inhibitory effect on these CDKs. The assays were performed using microfluidic kinase detection technology (Caliper Assay Platform). The compounds were tested in 12-point dose-response format in singlicate at Km for ATP. Specifics of each assay are as described below:CDK1/Cyclin B1: Enzyme concentration: 0.08 nM; ATP concentration: 40 μM; Incubation time: 3 hr. CDK2/Cyclin A: Enzyme concentration: 0.1 nM; ATP concentration: 50 μM; Incubation time: 3 hr. CDK2/Cyclin E: Enzyme concentration: 0.15 nM; ATP concentration: 100 μM; Incubation time: 3 hr. CDK4/Cyclin D1: Enzyme concentration: 1 nM; ATP concentration: 200 μM; Incubation time: 3 hr. CDK6/Cyclin D3: Enzyme concentration: 2 nM; ATP concentration: 300 μM; Incubation time: 3 hr. CDK9/Cyclin T1: Enzyme concentration: 5 nM; ATP concentration: 10 μM; Incubation time: 17 hr.
Measurement: IC50 = 2 nM (pChEMBL 8.699999809265137)
Descriptors: MW 489.58, ALogP 2.34, HBA 8, HBD 3, PSA 115.62, QED 0.52
Compound (SMILES): COc1ccc(F)cc1-c1ccnc2[nH]c(C3=CC4CCC(C3)N4CC(O)CO)cc12
InChIKey: OYKMGKHMOLIWKB-UHFFFAOYSA-N
Target: CDK9/cyclin T1 (PROTEIN COMPLEX), Homo sapiens
UniProt: O60563
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 = 41 nM (pChEMBL 7.389999866485596)
Descriptors: MW 423.49, ALogP 3.35, HBA 5, HBD 3, PSA 81.61, QED 0.57
Compound (SMILES): Cc1cccc(OCc2cn(-c3ccc(F)c(C(F)F)c3)nn2)n1
InChIKey: RQFODQBXSXAFLK-UHFFFAOYSA-N
Target: Glutamate NMDA receptor; GRIN1/GRIN2B (PROTEIN COMPLEX), Homo sapiens
UniProt: Q13224
Assay: NMDA receptor FLIPR assay: NMDA receptors are ion channels that are highly permeable to Ca2+ ions, rendering it possible to monitor NMDA receptor function using cell-based calcium flux assay. In this assay, co-agonists glutamate and glycine are added to cells heterologously expressing human GluN1/GluN2B NMDA receptors to initiate cellular Ca2+ influx. The time course of the changes in intracellular calcium is measured using a fluorescent dye and a FLIPR (Fluorometric Imaging Plate Reader) device.Twenty four hours before measurements, the expression of the NMDA receptors in the stable cell line is induced with Tet-On inducible system in the presence of a non-selective NMDA receptor blocker. On the day of the experiment, cell culture media is carefully washed and the cells are loaded with Calcium 5 Dye Kit (Molecular Devices) in dye loading buffer containing 149 mM NaCl, 4 mM KCl, 2 mM CaCl2, and 1.5 mM MgCl2, 10 mM HEPES and 5 mM D-glucose; pH 7.4. After 1 h incubation at the room temperature, the dye is washed away with the assay buffer (149 mM NaCl (standard assay) or 150 mM (HTS assay), 4 mM KCl (standard assay) or 3 mM (HTS assay), 2 mM CaCl2, 0.01 mM EDTA, 10 mM HEPES and 5 mM D-glucose; pH 7.4) In the FLIPR TETRA reader, various concentrations of the test compounds are added to the cells for 5 min while fluorescence is monitored to detect potential agonist activity. Next, co-agonists, glutamate and glycine are added for another 5 minutes. The concentration of glutamate corresponding to EC80 (standard assay) or EC40 (HTS assay) is used to maximize the assay's signal window or the ability to detect NMDA receptor antagonists and negative allosteric modulators, respectively. A saturating concentration (10 μM) of glycine is also present in the assay. A non-selective NMDA receptor antagonist, (+)MK-801 is used as a positive control for antagonist activity. The fluorescent signal in the presence of test compounds is quantified and normalized to the signal defined by the appropriate control wells.
Measurement: IC50 = 38 nM (pChEMBL 7.420000076293945)
Descriptors: MW 334.3, ALogP 3.63, HBA 5, HBD 0, PSA 52.83, QED 0.71
Compound (SMILES): O=C(N[C@@H](CCN1CC[C@@H](OCCc2ccc3c(n2)NCCC3)C1)C(=O)O)c1c(Cl)cccc1Cl
InChIKey: GNRYKTGJZWOZEG-NQIIRXRSSA-N
Target: Integrin alpha-V/beta-6 (PROTEIN COMPLEX), Homo sapiens
UniProt: P18564
Assay: Binding Assay: Microplates were coated with recombinant human integrin αvβ6 (2 ug/ml) in PBS (100 ul/well 25° C., overnight). The coating solution was removed, washed with wash buffer (0.05% Tween 20; 0.5 mM MnCl2; in 1×TBS). Plate was blocked with 200 ul/well of Block Buffer (1% BSA; 5% sucrose; 0.5 mM MnCl2; in 1×TBS) at 37° C. for 2 h. Dilutions of testing compounds and recombinant TGFβ1 LAP (0.67 ug/ml) in binding buffer (0.05% BSA; 2.5% sucrose; 0.5 mM MnCl2; in 1×TBS) were added. The plate was incubated for 2 hours at 25° C., washed, and incubated for 1 hour with Biotin-Anti-hLAP. Bound antibody was detected by peroxidase-conjugated streptavidin. The IC50 values for testing compounds were calculated by a four-parameter logistic regression.
Measurement: IC50 = 625 nM (pChEMBL 6.199999809265137)
Descriptors: MW 521.45, ALogP 3.65, HBA 6, HBD 3, PSA 103.79, QED 0.44
Compound (SMILES): Cc1nc(C#N)ccc1-n1cnc(-c2nc(NC3CCN(S(C)(=O)=O)CC3)ncc2C(F)(F)F)c1
InChIKey: LCGREZIWDBFPAV-UHFFFAOYSA-N
Target: Cyclin-dependent kinase 2/cyclin E1 (PROTEIN COMPLEX), Homo sapiens
UniProt: P24941
Assay: 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 were conducted in white 384-well polystyrene plates in a final reaction volume of 8 μL. CDK2/Cyclin E1 (0.25 nM) was incubated with the compounds of the Examples (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 were 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 were read after 1 hour at room temperature on a PHERAstar FS plate reader (BMG Labtech). Data was 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 506.51, ALogP 2.76, HBA 9, HBD 1, PSA 129.69, QED 0.56
Compound (SMILES): Cc1ncc(-c2ccc(F)c(OC(F)F)c2)nc1CN1CC2(CC2)OC1=O
InChIKey: LVNLQZDHINQWHE-UHFFFAOYSA-N
Target: Glutamate NMDA receptor; GRIN1/GRIN2B (PROTEIN COMPLEX), Homo sapiens
UniProt: Q13224
Assay: Effect of Compounds of Formula (I) on Cloned Human GluN1/GluN2B Ion Channels Expressed in Mammalian Cells: NMDA receptors are ion channels that are highly permeable to Ca2+ ions, rendering it possible to monitor NMDA receptor function using cell-based calcium flux assay. In this assay, co-agonists glutamate and glycine are added to cells heterologously expressing human GluN1/GluN2B NMDA receptors to initiate cellular Ca2+ influx. The time course of the changes in intracellular calcium is measured using a fluorescent dye and a FLIPR (Fluorometric Imaging Plate Reader) device.Twenty four hours before measurements, the expression of the NMDA receptors in the stable cell line is induced with Tet-On inducible system in the presence of a non-selective NMDA receptor blocker. On the day of the experiment, cell culture media is carefully washed and the cells are loaded with Calcium 5 Dye Kit (Molecular Devices) in dye loading buffer containing 137 mM NaCl, 4 mM KCl, 2 mM CaCl2, 0.5 mM MgCl2 (standard assay) or 1.5 mM MgCl2 (HTS assay), 10 mM HEPES and 5 mM D-glucose; pH 7.4. After 1 h incubation at the room temperature, the dye is washed away with the assay buffer (137 mM NaCl (standard assay) or 150 mM (HTS assay), 4 mM KCl (standard assay) or 3 mM (HTS assay), 2 mM CaCl2, 0.01 mM EDTA, 10 mM HEPES and 5 mM D-glucose; pH 7.4) In the FLIPR TETRA reader, various concentrations of the test compounds are added to the cells for 5 min while fluorescence is monitored to detect potential agonist activity. Next, co-agonists, glutamate and glycine are added for another 5 minutes. The concentration of glutamate corresponding to EC40 (standard assay) or EC40 (HTS assay) is used to maximize the assay's signal window and ability to detect NMDA receptor antagonists and negative allosteric modulators. A saturating concentration (10 μM) of glycine is also present in the assay. A non-selective NMDA receptor antagonist, (+)MK-801 is used as a positive control for antagonist activity. The fluorescent signal in the presence of test compounds is quantified and normalized to the signal defined by the appropriate control wells.
Measurement: IC50 = 66 nM (pChEMBL 7.179999828338623)
Descriptors: MW 379.34, ALogP 3.68, HBA 5, HBD 0, PSA 64.55, QED 0.79
Compound (SMILES): CN(C)S(=O)(=O)N1CCC(Nc2ncc3ccc(=O)n(C4CCCC4)c3n2)CC1
InChIKey: NZJDXYOHDCAUSE-UHFFFAOYSA-N
Target: Cyclin-dependent kinase 2/cyclin E1 (PROTEIN COMPLEX), Homo sapiens
UniProt: P24941
Assay: CDK2/Cyclin E1 Mobility Shift Assay: The purpose of the CDK2/Cyclin E1 assay is to evaluate the inhibition (% inhibition, Kiapp and Ki values) of small molecule inhibitors by using a fluorescence-based microfluidic mobility shift assay. CDK2/Cyclin E1 catalyzes the production of ADP from ATP that accompanies the phosphoryl transfer to the substrate peptide FL-Peptide-18 (5-FAM-QSPKKG-CONH2) (SEQ ID NO:1). (CPC Scientific, Sunnyvale, Calif.). The mobility shift assay electrophoretically separates the fluorescently labeled peptides (substrate and phosphorylated product) following the kinase reaction. Both substrate and product are measured and the ratio of these values is used to generate % conversion of substrate to product by the LabChip EZ Reader. Wild-type full length CDK2/wild-type full length Cyclin E1 enzyme complex was produced in-house (baculoviral expression, LJIC-2080/LJIC-2103) and phosphorylated by CDK7/Cyclin H1/Mat1 enzyme complex with CDK2:CDK7 ratio of 50:1 (concentration mg/mL) in the presence of 10 mM MgCl2 and 5 mM ATP at room temperature for one hour. Typical reaction solutions (50 μL final reaction volume) contained 2% DMSO (± inhibitor), 4 mM MgCl2, 1 mM DTT, 150 μM ATP (ATP Km=67.4 μM), 0.005% Tween-20, 3 μM FL-Peptide-18, and 0.36 nM (catalytically competent active site) phosphorylated wild-type full length CDK2/Cyclin E1 enzyme complex in 25 mM HEPES buffer at pH 7.15. The assay was initiated with the addition of ATP, following a fifteen minutes pre-incubation of enzyme and inhibitor at room temperature in the reaction mixture. The reaction was stopped after 45 minutes at room temperature by the addition of 50 μL of 80 mM EDTA, pH 7.5. The Ki value was determined from the fit of the data to the Morrison tight-binding competitive inhibition equation with the enzyme concentration as a variable.
Measurement: Ki = 1.12 nM (pChEMBL 8.949999809265137)
Descriptors: MW 420.54, ALogP 1.59, HBA 7, HBD 1, PSA 100.43, QED 0.79
Compound (SMILES): C[C@H](COCc1nnn(C)n1)N[C@H]1CC[C@H](Nc2cc(-c3cccc(NCC4(C#N)CCOCC4)n3)c(Cl)cn2)CC1
InChIKey: BGJDLKTYVVXXRM-YPAWHYETSA-N
Target: CDK9/cyclin T1 (PROTEIN COMPLEX), Homo sapiens
UniProt: O60563
Assay: CDK9 Biochemical Assay: To each well of a 96-well plate was added 5× kinase assay buffer with 10 mM DTT (6 μL), 500 μM ATP (1 μL), 5×CDK substrate (10 μL), and water (8 μL). 5 μL of compound were added to the test and positive control groups, while 5 μL of solvent was added to the blank groups. 100 ng of CDK9/CyclinT in 20 μL water was added to the test and positive control groups, while 20 μL of 1× kinase assay buffer was added to the blank groups. The reaction mixtures were incubated at 30° C. for 45 minutes, and 50 μL of Kinase-Glo® Max was added to each well and the plates were shielded from light and incubated for 15 minutes at room temperature. Luminescence was measured on a microplate reader and IC50values were calculated using Prism 9 software.
Measurement: IC50 = 7.5 nM (pChEMBL 8.119999885559082)
Descriptors: MW 595.15, ALogP 3.97, HBA 12, HBD 3, PSA 147.72, QED 0.28
Compound (SMILES): Cc1nc2cc(-c3nc(Nc4ccc(C5CN(C)CCO5)cn4)ncc3F)sc2n1C(C)C
InChIKey: JXJFLPJLSCQWPW-UHFFFAOYSA-N
Target: Cyclin-dependent kinase 4/cyclin D1 (PROTEIN COMPLEX), Homo sapiens
UniProt: P11802
Assay: Enzymatic Activity Assay: The inhibitory activity of compounds was evaluated in vitro using TR-FRET assay with white 384-well low volume microplate (Greiner Bio-One). CDK4/Cyclin D1 catalyzed phosphorylation of peptide in the presence and absence of compounds was measured. IC50 determination. Recombinant protein complex CDK4/Cyclin D1, expressed from insect cell, was purchased from ProQinase. Testing compounds were dissolved in DMSO at 0.1 mM and tested in 9-dose IC50 mode. The reaction mixture was prepared by mixing CDK4/CyclinD1 (1 nM final), ULight-4E-BP1 (100 nM final, Perkinelmer, TRF0128-D), and ATP (2 mM final) in assay buffer (20 mM of HEPES pH 7.4, 1 mM of EGTA, 0.05% BSA, 0.005% Tween 20, and 1 mM TCEP). The compound of interest in DMSO was added to each well in 3-fold serial dilution by dispenser (TECAN D300E). After 20 minutes preincubation at room temperature, MgCl2(10 mM final) was added to initiate the reaction. Following a 60 minutes incubation at 37° C., the reaction was stopped by addition of 2 μL of quenching buffer consisting of Lance detection buffer (Perkinelmer CR97-100C), 2 nM LANCE Ultra Europium-anti-P-4E-BP1 (Perkinelmer, TRF0216-D), 10 mM EDTA, and incubate at room temperature for additional 60 minutes in dark. The reaction signal was measured by Envision multimode plate reader (PerkinElmer, 2102-0010).
Measurement: IC50 < 20 nM (pChEMBL 7.698999881744385)
Descriptors: MW 467.57, ALogP 4.72, HBA 9, HBD 1, PSA 80.99, QED 0.45
Compound (SMILES): O=C(CCc1ccccc1)Nc1ccccc1C(=O)NCc1ccccc1
InChIKey: UVGIBYJYUGXORP-UHFFFAOYSA-N
Target: Integrin alpha-4/beta-7 (PROTEIN COMPLEX), Homo sapiens
UniProt: P13612
Assay: ELISA assay : The studies began by screening for the optimal protein concentrations for the method. The studies were conducted in goat anti-mouse IgG coated black React-Bind 96 welled-plates (R&D Biosystems), referred to herein as the anti-mouse IgG plate. Twelve stock solutions were prepared containing a 1:1 stoichiometric mixture of gp120 and α4β7 integrin in PBS at pH 7.2 as given by 0 μM or control, 0.001 μM, 0.01 μM, 0.01 μM, 0.05 μM, 0.1 μM, 0.5 μM. 1 μM, 2.5 μM, 5 μM, 10 μM and 25 μM in protein (step 1, FIG. 1). A 200 μL aliquot of each stock solutions was then loaded across a 96 welled plate and treated either with 20 μL of PBS pH 7.2 (control) or 20 μL of a 100 μM stock solution of repandusinic acid (RA: compound whose anti-HIV activity is to be tested) in PBS pH 7.2 containing 1% DMSO. Three repetitions were run for both the control and positive or repandusinic acid treated experiments. The final concentration of repandusinic acid in each positive well was 10 μM. The plate was incubated for 4 h at 4° C. on a plate mixer at a speed that created a vortex in each well.
Measurement: IC50 = 0.33 nM (pChEMBL 9.479999542236328)
Descriptors: MW 358.44, ALogP 4.19, HBA 2, HBD 2, PSA 58.2, QED 0.67
Compound (SMILES): CNC(=O)N1CC=C(c2cc3c(-c4ccccc4OC)ccnc3[nH]2)CC1
InChIKey: JPVGLBMPNLVXTF-UHFFFAOYSA-N
Target: CDK9/cyclin T1 (PROTEIN COMPLEX), Homo sapiens
UniProt: O60563
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 = 19 nM (pChEMBL 7.71999979019165)
Descriptors: MW 362.43, ALogP 3.67, HBA 3, HBD 2, PSA 70.25, QED 0.75
Compound (SMILES): COc1ccc(F)cc1-c1c(OC)cnc2[nH]c(C3=CC4CN(CC(=O)N(C)C)CC4C3)cc12
InChIKey: SVSDPSOSGWKVGH-UHFFFAOYSA-N
Target: CDK9/cyclin T1 (PROTEIN COMPLEX), Homo sapiens
UniProt: O60563
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 = 1150 nM (pChEMBL 5.940000057220459)
Descriptors: MW 464.54, ALogP 3.81, HBA 5, HBD 1, PSA 70.69, QED 0.6
Compound (SMILES): O=C(NC(CCO[C@H]1C[C@H](CCc2ccc3c(n2)NCCC3)C1)C(=O)O)c1ccccc1-c1ccccn1
InChIKey: NLRRBRMXDCHLJK-WKHLBODFSA-N
Target: Integrin alpha-V/beta-6 (PROTEIN COMPLEX), Homo sapiens
UniProt: P18564
Assay: Binding Assay: The biochemical potency of compounds was determined using a proximity-based assay (ALPHASCREEN®, Perkin Elmer, Waltham, Mass.) as described previously (Ullman E F et al., Luminescent oxygen channeling immunoassay: Measurement of particle binding kinetics by chemiluminescence. Proc. Natl. Acad. Sci. USA, Vol. 91, pp. 5426-5430, June 1994). To gauge the potency of inhibitors of binding to human integrin αvβ6, inhibitor compounds and integrin were incubated together with TGF-b1 LAP and biotinylated anti-LAP antibody plus acceptor and donor beads, following the manufacture's recommendations. The donor beads were coated with streptavidin. The acceptor beads had a nitrilotriacetic acid Ni chelator, for binding to a 6×His Tag on human integrin αvβ6. All incubations occurred at room temperatures in 50 mM Tris-HCl, pH 7.5, 0.1% BSA supplemented with 1 mM each CaCl2 and MgCl2.The order of reagent addition was as follows:1. Alpha-v-beta-6 integrin, test inhibitor compound, LAP, biotinylated anti-LAP antibody and acceptor beads were all added together.2. After 2 hours, donor beads were added. After another 30 minute incubation, samples were then read.Integrin binding was evaluated by exciting donor beads at 680 nm, and measuring the fluorescent signal produced, between 520-620 nm, using a Biotek Instruments (Winooski, Vt., USA) SynergyNeo2 multimode plate reader.
Measurement: IC50 = 150 nM (pChEMBL 6.820000171661377)
Descriptors: MW 514.63, ALogP 4.5, HBA 6, HBD 3, PSA 113.44, QED 0.35
Compound (SMILES): CC1(NC(=O)O[C@@H]2CC[C@H](c3cc(NC(=O)Cc4cn(CC(F)(F)F)nn4)n[nH]3)C2)CC1
InChIKey: XTCJOPTWZANNEO-WCQYABFASA-N
Target: Cyclin-dependent kinase 2/cyclin E1 (PROTEIN COMPLEX), Homo sapiens
UniProt: P24941
Assay: Biochemical Assay: The purpose of CDK2/Cyclin E1 assay is to evaluate the inhibition (% inhibition, Kiapp and Ki values) of small molecule inhibitors by using a fluorescence-based microfluidic mobility shift assay. CDK2/Cyclin E1 full length catalyzes the production of ADP from ATP that accompanies the phosphoryl transfer to the substrate peptide FL-Peptide-18 (5-FAM-QSPKKG-CONH2, CPC Scientific, Sunnyvale, Calif.) (SEQ ID NO:1). The mobility shift assay electrophoretically separates the fluorescently labeled peptides (substrate and phosphorylated product) following the kinase reaction. Both substrate and product are measured and the ratio of these values is used to generate % conversion of substrate to product by the LabChip EZ Reader. Wild-type CDK2/wild-type full length Cyclin E1 enzyme complex was produced in-house (baculoviral expression, LJIC-2080/LJIC-2103) and phosphorylated by CDK7/Cyclin H1/Matl enzyme complex with CDK2:CDK7 ratio of 50:1 (concentration mg/mL) in the presence of 10 mM MgCl2 and 5 mM ATP at room temperature for one hour. Typical reaction solutions (50 μL final reaction volume) contained 2% DMSO (±inhibitor), 4 mM MgCl2, 1 mM DTT, 150 μM ATP (ATP Km=67.4 μM), 0.005% Tween-20, 3 μM FL-Peptide-18, and 0.36 nM (catalytically competent active site) phosphorylated wild-type full length CDK2/Cyclin E1 enzyme complex in 25 mM HEPES buffer at pH 7.15. The assay was initiated with the addition of ATP, following a fifteen minutes pre-incubation of enzyme and inhibitor at room temperature in the reaction mixture. The reaction was stopped after 45 minutes at room temperature by the addition of 50 μL of 80 mM EDTA.
Measurement: Ki = 1.7 nM (pChEMBL 8.770000457763672)
Descriptors: MW 455.44, ALogP 2.66, HBA 7, HBD 3, PSA 126.82, QED 0.59
Compound (SMILES): CCc1cccc([C@@H](C)[C@H](NS(=O)(=O)c2ccc(Cl)cc2OC)c2n[nH]c(=O)o2)c1CC
InChIKey: AJJCSPFCKDBZBH-XCLFUZPHSA-N
Target: Ribonucleotide reductase (PROTEIN COMPLEX), Homo sapiens
UniProt: Q7LG56
Assay: Human RNR Inhibition Effect: The inhibitory activity against the ribonucleotide reduction reaction (hereinafter referred to as RNR reaction) of the test compound was determined by measuring the formation of deoxycytidine diphosphate (hereinafter referred to as dCDP) from cytidine diphosphate (hereinafter referred to as CDP) by the following method.Human M1 subunit and human M2 subunit (mutant lacking amino terminal 59 amino acids), which are fused a histidine tag at the amino terminus, are overexpressed in Escherichia coli and are solubilized after collection, and histidine tagged human M1 and M2 proteins were purified on a nickel chelate column.For measuring the inhibitory activity of the test compound against the RNR reaction, the method described in the document [CANCER RESEARCH 64, 1-6, 2004] was referred to.
Measurement: IC50 = 840 nM (pChEMBL 6.079999923706055)
Descriptors: MW 479.99, ALogP 3.97, HBA 6, HBD 2, PSA 114.29, QED 0.48