lovingscience/chembl-v37-raw-activities huggingface dataset:
Compound (SMILES): CC(C)(Nc1ccc(Cl)c(C(=O)NC2CC2)c1)C(=O)Nc1nc2c(C(F)(F)F)cc3[nH]ncc3c2s1
InChIKey: WOBKYDKXSVLQGJ-UHFFFAOYSA-N
Target: CDK3/Cyclin E (PROTEIN COMPLEX), Homo sapiens
UniProt: Q00526
Assay: Inhibition of C-terminal His6-tagged human full length Cdk3/N-terminal GST-tagged human full length Cyclin E expressed in Sf21 cells
Measurement: IC50 = 41 nM (pChEMBL 7.389999866485596)
Descriptors: MW 536.97, ALogP 5.57, HBA 6, HBD 4, PSA 111.8, QED 0.26
Compound (SMILES): CC(=O)Nc1cc(NC(C)(C)C(=O)Nc2nc3c(C(F)(F)F)cc4[nH]ncc4c3s2)ccc1F
InChIKey: ICVWYIZCPUIPOY-UHFFFAOYSA-N
Target: CDK3/Cyclin E (PROTEIN COMPLEX), Homo sapiens
UniProt: Q00526
Assay: Inhibition of C-terminal His6-tagged human full length Cdk3/N-terminal GST-tagged human full length Cyclin E expressed in Sf21 cells
Measurement: IC50 = 55 nM (pChEMBL 7.260000228881836)
Descriptors: MW 494.47, ALogP 5.12, HBA 6, HBD 4, PSA 111.8, QED 0.29
Compound (SMILES): CC(C)(Nc1cccc(S(N)(=O)=O)c1)C(=O)Nc1nc2c(C(F)(F)F)cc3[nH]ncc3c2s1
InChIKey: FMUUGBFOPCVEBP-UHFFFAOYSA-N
Target: CDK3/Cyclin E (PROTEIN COMPLEX), Homo sapiens
UniProt: Q00526
Assay: Inhibition of C-terminal His6-tagged human full length Cdk3/N-terminal GST-tagged human full length Cyclin E expressed in Sf21 cells
Measurement: IC50 = 5.9 nM (pChEMBL 8.229999542236328)
Descriptors: MW 498.51, ALogP 3.67, HBA 7, HBD 4, PSA 142.86, QED 0.33
Compound (SMILES): CC(C)(Nc1ccc(Cl)c(C(=O)NCC(N)=O)c1)C(=O)Nc1nc2c(C(F)(F)F)cc3[nH]ncc3c2s1
InChIKey: UQHPGIWKYQJUIE-UHFFFAOYSA-N
Target: CDK3/Cyclin E (PROTEIN COMPLEX), Homo sapiens
UniProt: Q00526
Assay: Inhibition of C-terminal His6-tagged human full length Cdk3/N-terminal GST-tagged human full length Cyclin E expressed in Sf21 cells
Measurement: IC50 = 0.6 nM (pChEMBL 9.220000267028809)
Descriptors: MW 553.95, ALogP 3.89, HBA 7, HBD 5, PSA 154.89, QED 0.23
Compound (SMILES): CC(C)(Nc1ccc(Cl)c(O)c1)C(=O)Nc1nc2c(C(F)(F)F)cc3[nH]ncc3c2s1
InChIKey: FNAWEKRTBLXUJI-UHFFFAOYSA-N
Target: CDK3/Cyclin E (PROTEIN COMPLEX), Homo sapiens
UniProt: Q00526
Assay: Inhibition of C-terminal His6-tagged human full length Cdk3/N-terminal GST-tagged human full length Cyclin E expressed in Sf21 cells
Measurement: IC50 = 22 nM (pChEMBL 7.659999847412109)
Descriptors: MW 469.88, ALogP 5.38, HBA 6, HBD 4, PSA 102.93, QED 0.32
Compound (SMILES): O=C(Nc1nc2ccc3[nH]ncc3c2s1)C1(Nc2ccc(Cl)cc2)CC1
InChIKey: ZODPLLYTMWNTCA-UHFFFAOYSA-N
Target: CDK3/Cyclin E (PROTEIN COMPLEX), Homo sapiens
UniProt: Q00526
Assay: Inhibition of C-terminal His6-tagged human full length Cdk3/N-terminal GST-tagged human full length Cyclin E expressed in Sf21 cells
Measurement: IC50 = 44 nM (pChEMBL 7.360000133514404)
Descriptors: MW 383.86, ALogP 4.41, HBA 5, HBD 3, PSA 82.7, QED 0.49
Compound (SMILES): CNC(=O)c1cc(NC2(C(=O)Nc3nc4ccc5[nH]ncc5c4s3)CC2)ccc1Cl
InChIKey: FVEIPMUKOTZXBA-UHFFFAOYSA-N
Target: CDK3/Cyclin E (PROTEIN COMPLEX), Homo sapiens
UniProt: Q00526
Assay: Inhibition of C-terminal His6-tagged human full length Cdk3/N-terminal GST-tagged human full length Cyclin E expressed in Sf21 cells
Measurement: IC50 = 38 nM (pChEMBL 7.420000076293945)
Descriptors: MW 440.92, ALogP 3.77, HBA 6, HBD 4, PSA 111.8, QED 0.38
Compound (SMILES): COc1cc(C(C)C)c(Oc2cnc(N)nc2N)cc1S(N)(=O)=O
InChIKey: HLWURFKMDLAKOD-UHFFFAOYSA-N
Target: P2X2/P2X3 heterotrimeric receptor (PROTEIN COMPLEX), Homo sapiens
UniProt: P56373
Assay: Antagonist activity at human P2X2/3 receptor
Measurement: IC50 = 520 nM (pChEMBL 6.28000020980835)
Descriptors: MW 353.4, ALogP 1.21, HBA 8, HBD 3, PSA 156.44, QED 0.72
Compound (SMILES): COc1ccc(OC(C)C(=O)[O-])cc1.[Na+]
InChIKey: SKORRGYRKQDXRS-UHFFFAOYSA-M
Target: T1R2/T1R3 (PROTEIN COMPLEX), Homo sapiens
UniProt: Q8TE23
Assay: Inhibition of human T1R2/T1R3 expressed in HEK293T cells co-expressing Galpha15/i1 assessed as reduction in 50 mM sucrose-induced response
Measurement: IC50 = 19600 nM (pChEMBL 4.710000038146973)
Descriptors: MW 196.2, ALogP 1.55, HBA 3, HBD 1, PSA 55.76, QED 0.79
Compound (SMILES): CC(C)(Nc1ccc(Cl)c(C(=O)NCCCC(=O)O)c1)C(=O)Nc1nc2c(C(F)(F)F)cc3[nH]ncc3c2s1
InChIKey: YWKCCOXVWMNFSF-UHFFFAOYSA-N
Target: CDK3/Cyclin E (PROTEIN COMPLEX), Homo sapiens
UniProt: Q00526
Assay: Inhibition of C-terminal His6-tagged human full length Cdk3/N-terminal GST-tagged human full length Cyclin E expressed in Sf21 cells
Measurement: IC50 = 180 nM (pChEMBL 6.75)
Descriptors: MW 582.99, ALogP 5.27, HBA 7, HBD 5, PSA 149.1, QED 0.17
Compound (SMILES): CC(C)(Nc1ccc(Cl)c(C(=O)NCC(=O)O)c1)C(=O)Nc1nc2c(C(F)(F)F)cc3[nH]ncc3c2s1
InChIKey: ULMMSSXMAQOVME-UHFFFAOYSA-N
Target: CDK3/Cyclin E (PROTEIN COMPLEX), Homo sapiens
UniProt: Q00526
Assay: Inhibition of C-terminal His6-tagged human full length Cdk3/N-terminal GST-tagged human full length Cyclin E expressed in Sf21 cells
Measurement: IC50 = 134 nM (pChEMBL 6.869999885559082)
Descriptors: MW 554.94, ALogP 4.49, HBA 7, HBD 5, PSA 149.1, QED 0.23
Compound (SMILES): CC(C)(Nc1ccc(Cl)c(C(N)=O)c1)C(=O)Nc1nc2c(C(F)(F)F)cc3[nH]ncc3c2s1
InChIKey: ITHPWIHYZQLUIG-UHFFFAOYSA-N
Target: CDK3/Cyclin E (PROTEIN COMPLEX), Homo sapiens
UniProt: Q00526
Assay: Inhibition of C-terminal His6-tagged human full length Cdk3/N-terminal GST-tagged human full length Cyclin E expressed in Sf21 cells
Measurement: IC50 = 23 nM (pChEMBL 7.639999866485596)
Descriptors: MW 496.9, ALogP 4.77, HBA 6, HBD 4, PSA 125.79, QED 0.32
Compound (SMILES): CC(C)(Nc1ccc(Cl)c(C(=O)NCCC(=O)O)c1)C(=O)Nc1nc2c(C(F)(F)F)cc3[nH]ncc3c2s1
InChIKey: OVBAFUGPHNUQFH-UHFFFAOYSA-N
Target: CDK3/Cyclin E (PROTEIN COMPLEX), Homo sapiens
UniProt: Q00526
Assay: Inhibition of C-terminal His6-tagged human full length Cdk3/N-terminal GST-tagged human full length Cyclin E expressed in Sf21 cells
Measurement: IC50 = 70 nM (pChEMBL 7.159999847412109)
Descriptors: MW 568.97, ALogP 4.88, HBA 7, HBD 5, PSA 149.1, QED 0.2
Compound (SMILES): COc1ccc(OC(C)C(=O)[O-])cc1.[Na+]
InChIKey: SKORRGYRKQDXRS-UHFFFAOYSA-M
Target: T1R1/T1R3 (PROTEIN COMPLEX), Homo sapiens
UniProt: Q7RTX1
Assay: Inhibition of human T1R1/T1R3 expressed in HEK293T cells co-expressing Galpha15 assessed as reduction in L-glutamate/IMP-induced response in presence of 80 mM L-glutamate
Measurement: IC50 = 820 nM (pChEMBL 6.090000152587891)
Descriptors: MW 196.2, ALogP 1.55, HBA 3, HBD 1, PSA 55.76, QED 0.79
Compound (SMILES): COc1ccc(OC(C)C(=O)[O-])cc1.[Na+]
InChIKey: SKORRGYRKQDXRS-UHFFFAOYSA-M
Target: T1R1/T1R3 (PROTEIN COMPLEX), Homo sapiens
UniProt: Q7RTX1
Assay: Inhibition of human T1R1/T1R3 expressed in HEK293T cells co-expressing Galpha15 assessed as reduction in L-glutamate/IMP-induced response in presence of 0.8 mM L-glutamate
Measurement: IC50 = 350 nM (pChEMBL 6.460000038146973)
Descriptors: MW 196.2, ALogP 1.55, HBA 3, HBD 1, PSA 55.76, QED 0.79
Compound (SMILES): COc1ccc(OC(C)C(=O)[O-])cc1.[Na+]
InChIKey: SKORRGYRKQDXRS-UHFFFAOYSA-M
Target: T1R1/T1R3 (PROTEIN COMPLEX), Homo sapiens
UniProt: Q7RTX1
Assay: Inhibition of human T1R1/T1R3 expressed in HEK293T cells co-expressing Galpha15 assessed as reduction in 80 mM L-glutamate-induced response
Measurement: IC50 = 210 nM (pChEMBL 6.679999828338623)
Descriptors: MW 196.2, ALogP 1.55, HBA 3, HBD 1, PSA 55.76, QED 0.79
Compound (SMILES): COc1ccc(OC(C)C(=O)[O-])cc1.[Na+]
InChIKey: SKORRGYRKQDXRS-UHFFFAOYSA-M
Target: T1R1/T1R3 (PROTEIN COMPLEX), Homo sapiens
UniProt: Q7RTX1
Assay: Inhibition of human T1R1/T1R3 expressed in HEK293T cells co-expressing Galpha15 assessed as reduction in 8 mM L-glutamate-induced response
Measurement: IC50 = 190 nM (pChEMBL 6.71999979019165)
Descriptors: MW 196.2, ALogP 1.55, HBA 3, HBD 1, PSA 55.76, QED 0.79
Compound (SMILES): COc1ccc(OC(C)C(=O)[O-])cc1.[Na+]
InChIKey: SKORRGYRKQDXRS-UHFFFAOYSA-M
Target: T1R2/T1R3 (PROTEIN COMPLEX), Homo sapiens
UniProt: Q8TE23
Assay: Inhibition of human T1R2/T1R3 expressed in HEK293T cells co-expressing Galpha15/i1 assessed as reduction in 150 mM sucrose-induced response
Measurement: IC50 = 24000 nM (pChEMBL 4.619999885559082)
Descriptors: MW 196.2, ALogP 1.55, HBA 3, HBD 1, PSA 55.76, QED 0.79
Compound (SMILES): C[C@@]1(c2cc(NC(=O)N3CC=CCC3)ccc2F)C[C@@H](C(F)(F)F)OC(N)=N1
InChIKey: FUSXNZWCAWAAIL-YOEHRIQHSA-N
Target: Beta-secretase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P56817
Assay: In Vitro BACE Cell-Based Assay: The cell-based assay measures inhibition or reduction of Aβ40 in conditioned medium of test compound treated cells expressing amyloid precursor protein. Cells stably expressing Amyloid Precursor Protein (APP) were plated at a density of 40K cells/well in 96 well plates (Costar). The cells were cultivated for 24 hours at 37° C. and 5% CO2 in DMEM supplemented with 10% FBS. The test compounds were then added to cells in 10-point dose response concentrations with the starting concentration being either 100 uM or 10 uM. The compounds were diluted from stock solutions in DMSO and the final DMSO concentration of the test compounds on cells was 0.1%. After 24 h of incubation with the test compounds the supernatant conditioned media was collected and the Aβ 40 levels were determined using a sandwich ELISA.
Measurement: IC50 = 908 nM (pChEMBL 6.039999961853027)
Descriptors: MW 400.38, ALogP 3.5, HBA 4, HBD 2, PSA 79.95, QED 0.59
Compound (SMILES): Cc1cccc(-c2cc(N)nc3[nH]cc(C#N)c23)c1
InChIKey: KWTGXMXCSXFSJX-UHFFFAOYSA-N
Target: Leucine-rich repeat serine/threonine-protein kinase 2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q5S007
Assay: Homogeneous Time Resolved-fluorescence Resonance Energy Transfer (TR-FRET) Assay: LRRK2 kinase activity is measured using a LanthaScreen kinase activity assay available from Invitrogen (Life Technologies Corporation). The assay is a homogeneous time resolved-fluorescence resonance energy transfer (TR-FRET) assay that measures phosphorylation of a fluorescein-labelled peptide substrate (fluorescein-LRRKtide, fluorescein-GAGRLGRDKYKTLRQIRQ) (SEQ ID NO 1) as a result of LRRK2 kinase activity. The phosphorylated peptide is recognized by a terbium-labelled phospho-specific anti-LRRKtide antibody and, subsequently, the phosphorylated LRRKtide can be quantified by the extent of TR-FRET between the terbium donor and fluorescein acceptor.The LRRK2 kinase is obtained from Invitrogen (Life Technologies Corporation) and comprises residues 970 to 2527 of the full length human wild-type LRRK2 kinase or a similar sequence with the G2019S mutation. As discussed above, this mutation increases the kinase activity relative to the wild-type. The kinase reactions are performed in a 20 uL volume in 384-well plates. The kinase reaction buffer consists of 50 mM Tris pH 8.5, 0.01% BRIJ-35, 10 mM MgCl2, 1 mM EGTA, and 2 mM DTT.Â  In the assay, 1 nM LRRK2 WT or 250 pM LRRK2 G2019S kinase is incubated with the test compound (typically at 0 to 30 uM) for 30 minutes before the kinase reaction is initiated by addition of 1.3 mM ATP and 0.4 uM fluorescein-LRRKtide. The reaction mixture (20 ul total volume) is incubated for 2 hours at 30° C. before the reaction is terminated by addition of 10 mM EDTA and 1 nM terbium-labelled anti-phospho-LRRKtide antibody (final volume 20 ul). The mixture is further incubated for 30 minutes at RT. TR-FRET is measured by excitation of the terbium-donor with 340 nm light and subsequent (delay time 100 us) measurement of terbium and fluorescein emission at 495 nm and 520 nm, respectively, over a time window of 1000 us. The measurement is repeated 10 times for fluorescein and 10 times for terbium emission with a 2000 us time window between repeats. TR-FRET measurements are performed on a Biomek Synergy plate. The TR-FRET signal is calculated as the emission-ratio at 520 nm over 495 nm.
Measurement: IC50 = 15 nM (pChEMBL 7.820000171661377)
Descriptors: MW 248.29, ALogP 2.99, HBA 3, HBD 2, PSA 78.49, QED 0.69
Compound (SMILES): O=C(N/N=C/c1cc(C(F)(F)F)c2ncccc2c1)[C@@H]1C[C@H]1c1ccccc1
InChIKey: KDWQHEPFWKJVQJ-RYZSJLKPSA-N
Target: Transient receptor potential cation channel subfamily M member 5 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9NZQ8
Assay: Inhibition of human TRPM5 transfected in HEK293 cells assessed as inhibition of KCl-stimulated membrane potential by FLIPR assay
Measurement: IC50 = 10000 nM (pChEMBL 5)
Descriptors: MW 383.37, ALogP 4.51, HBA 3, HBD 1, PSA 54.35, QED 0.53
Compound (SMILES): CCCCN(CCOc1ccc(C)cc1)C1CCN(C(=O)Cn2cc(NC(=O)c3cnn4cccnc34)c(-c3cc(Cl)ccc3OC(F)F)n2)CC1
InChIKey: UOLLHOKAUPKEPG-UHFFFAOYSA-N
Target: Tyrosine-protein kinase JAK2 (SINGLE PROTEIN), Homo sapiens
UniProt: O60674
Assay: JAK Enzyme Assay: The activity of the isolated recombinant JAK1 and JAK2 kinase domain was measured by monitoring phosphorylation of a peptide derived from JAK3 (Val-Ala-Leu-Val-Asp-Gly-Tyr-Phe-Arg-Leu-Thr-Thr, fluorescently labeled on the N-terminus with 5-carboxyfluorescein) using the Caliper LabChip® technology (Caliper Life Sciences, Hopkinton, Mass.). To determine inhibition constants (Ki), compounds were diluted serially in DMSO and added to 50 μL kinase reactions containing purified enzyme (1.5 nM JAK1, or 0.2 nM JAK2), 100 mM HEPES buffer (pH 7.2), 0.015% Brij-35, 1.5 μM peptide substrate, ATP (25 μM), 10 mM MgCl2, 4 mM DTT at a final DMSO concentration of 2%. Reactions were incubated at 22° C. in 384-well polypropylene microtiter plates for 30 minutes and then stopped by addition of 25 μL of an EDTA containing solution (100 mM HEPES buffer (pH 7.2), 0.015% Brij-35, 150 mM EDTA), resulting in a final EDTA concentration of 50 mM. After termination of the kinase reaction, the proportion of phosphorylated product was determined as a fraction of total peptide substrate using the Caliper LabChip® 3000 according to the manufacturer's specifications.
Measurement: Ki = 3.66 nM (pChEMBL 8.4399995803833)
Descriptors: MW 735.24, ALogP 6.58, HBA 10, HBD 1, PSA 119.12, QED 0.13
Compound (SMILES): COc1cc2c(cc1OC)C(=O)/C(=C/c1ccc(N3CCN(Cc4ccc5c(c4)OCO5)CC3)cc1)C2
InChIKey: SRRFJOCTTBPYJP-YDZHTSKRSA-N
Target: Cholinesterase (SINGLE PROTEIN), Equus caballus
UniProt: P81908
Assay: Inhibition of equine serum BuChE using butyrylthiocholine iodide as substrate preincubated for 5 mins followed by substrate addition measured after 2 mins by Ellman's method
Measurement: IC50 = 12220 nM (pChEMBL 4.909999847412109)
Descriptors: MW 498.58, ALogP 4.58, HBA 7, HBD 0, PSA 60.47, QED 0.46
Compound (SMILES): O=c1[nH]c(C2CCC2c2ncccn2)nc2c1cnn2C1CCC(F)(F)CC1
InChIKey: LRRFQGYMGYXVHJ-UHFFFAOYSA-N
Target: High affinity cGMP-specific 3',5'-cyclic phosphodiesterase 9A (SINGLE PROTEIN), Homo sapiens
UniProt: O76083
Assay: Scintillation Proximity Assay: The PDE9A2 enzymatic activity assay was run as scintillation proximity assay (SPA), in general according to the protocol of the manufacturer (GE Healthcare, former Amersham Biosciences, product number: TRKQ 7100). As enzyme source, lysate (PBS with 1% Triton X-100 supplemented with protease inhibitors, cell debris removed by centrifugation at 13.000 rpm for 30 min) of SF 9 cell expressing the human PDE9A2 was used. The total protein amount included in the assay varied upon infection and production efficacy of the SF9 cells and lay in the range of 0.1-100 ng. In general, the assay conditions were as follows: total assay volume: 40 microlitre; protein amount: 0.1-50 ng; substrate concentration (cGMP): 20 nanomolar; ~1 mCi/l; incubation time: 60 min at room temperature; final DMSO concentration: 0.2-1%. The assays were run in 384-well format. The test reagents as well as the enzyme and the substrate were diluted in assay buffer. The assay buffer contained 50 mM Tris, 8.3 mM MgCl2, 1.7 mM EGTA, 0.1% BSA, 0.05% Tween 20; the pH of assay buffer was adjusted to 7.5. The reaction was stopped by applying a PDE9 specific inhibitor (e.g. compounds according to WO 2004/099210 or WO 2004/099211, like one of the enantiomers of example 37, e.g. 1-(2-Chlorophenyl)-6-[2R)-3,3,3-trifluoro-2-methyl-propyl]-1,5-dihydro-4H-pyrazolo[3,4-d]pyrimidine-4-one) in excess.
Measurement: IC50 = 4 nM (pChEMBL 8.399999618530273)
Descriptors: MW 386.41, ALogP 3.32, HBA 6, HBD 1, PSA 89.35, QED 0.75
Compound (SMILES): CC(C)(C)CN1CCc2c(C(=O)N3CCC(c4ccc(F)c(F)c4C(F)(F)F)CC3)n[nH]c2C1
InChIKey: RMKSAIFOYKMQFY-UHFFFAOYSA-N
Target: Retinol-binding protein 4 (SINGLE PROTEIN), Homo sapiens
UniProt: P02753
Assay: TR-FRET Assay: TR-FRET assay for retinol-induced RBP4-TTR interaction Binding of a desired RBP4 antagonist displaces retinol and induces hindrance for RBP4-TTR interaction resulting in the decreased FRET signal (FIG. 7). Bacterially expressed MBP-RBP4 and untagged TTR were used in this assay. For the use in the TR-FRET assay the maltose binding protein (MBP)-tagged human RBP4 fragment (amino acids 19-201) was expressed in the Gold(DE3)pLysS E. coli strain (Stratagene) using the pMAL-c4x vector. Following cell lysis, recombinant RBP4 was purified from the soluble fraction using the ACTA FPLC system (GE Healthcare) equipped with the 5-ml the MBP Trap HP column. Human untagged TTR was purchased from Calbiochem. Untagged TTR was labeled directly with Eu3+ Cryptate-NHS using the HTRF Cryptate Labeling kit from CisBio following the manufacturer's recommendations. HTRF assay was performed in white low volume 384 well plates (Greiner-Bio) in a final assay volume of 16 ul per well. The reaction buffer contained 10 mM Tris-HCl pH 7.5, 1 mM DTT, 0.05% NP-40, 0.05% Prionex, 6% glycerol, and 400 mM KF. Each reaction contained 60 nM MBP-RBP4 and 2 nM TTR-Eu along with 26.7 nM of anti-MBP antibody conjugated with d2 (Cisbio). Titration of test compounds in this assay was conducted in the presence of 1 uM retinol. All reactions were assembled in the dark under dim red light and incubated overnight at +4° C. wrapped in aluminum foil. TR-FRET signal was measured in the SpectraMax M5e Multimode Plate Reader (Molecular Device). Fluorescence was excited at 337 nm and two readings per well were taken: Reading 1 for time-gated energy transfer from Eu(K) to d2 (337 nm excitation, 668 nm emission, counting delay 75 microseconds, counting window 100 microseconds) and Reading 2 for Eu(K) time-gated fluorescence (337 nm excitation, 620 nm emission, counting delay 400 microseconds, counting window 400 microseconds).
Measurement: IC50 = 169.72 nM (pChEMBL 6.769999980926514)
Descriptors: MW 484.51, ALogP 5.13, HBA 3, HBD 1, PSA 52.23, QED 0.61
Compound (SMILES): Oc1ncn(-c2ccc(-c3c(/C=C/c4ccc5ccccc5n4)nc4c(N5CCOCC5)ccnn34)cn2)n1
InChIKey: OUJVXBOIHRBQEX-CMDGGOBGSA-N
Target: cAMP and cAMP-inhibited cGMP 3',5'-cyclic phosphodiesterase 10A (SINGLE PROTEIN), Homo sapiens
UniProt: Q9Y233
Assay: In Vitro Assay: Human recombinant PDE10A2 was expressed in Sf9 cells, using a recombinant baculovirus construct containing the full length sequence containing a 6Ã¿His sequence following the start Met to allow metal affinity purification of the recombinant protein. Cells were harvested and the phosphodiesterase protein was purified by metal chelate chromatography on Ni-sepharose 6FF.The affinity of the compounds of Formula (I) for phophodiesterases (PDE) was measured by a scintillation proximity assay (SPA). PDE Yttrium Silicate SPA beads allow PDE activity to be measured by direct binding of the primary phosphate groups of non-cyclic AMP or GMP to the beads via a complex iron chelation mechanism. The amount of bound tritiated product ([3H]-AMP) is measured by liquid scintillation counting.The compounds were dissolved and diluted in 100% DMSO in polystyrene plates to a concentration of 100-fold the final concentration in the assay. Human PDE10A enzyme solution (10 uL) was added.
Measurement: IC50 = 4 nM (pChEMBL 8.399999618530273)
Descriptors: MW 517.55, ALogP 3.63, HBA 11, HBD 1, PSA 119.38, QED 0.37
Compound (SMILES): CC(C)(C)CCN1CCN(c2ccc(Nc3ncc4cc5n(c4n3)C3(CCCC3)CNC5=O)nc2)CC1(C)C
InChIKey: QTTLRBURMUNSGV-UHFFFAOYSA-N
Target: Cyclin-dependent kinase 2 (SINGLE PROTEIN), Homo sapiens
UniProt: P24941
Assay: Caliper Assay: Selected compounds disclosed herein were tested in CDK4/cyclinD1, CDK6/CycD3 CDK2/CycA and CDK2/cyclinE 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. Phosphoacceptor substrate peptide concentration used was 1 μM for all assays and Staurosporine was used as the reference compound for all assays. Specifics of each assay are as described below:CDK2/CyclinA: Enzyme concentration: 0.2 nM; ATP concentration: 50 μM; Incubation time: 3 hr.CDK2/CyclinE: Enzyme concentration: 0.28 nM; ATP concentration: 100 μM; Incubation time: 1 hr.CDK4/CyclinD1: Enzyme concentration: 1 nM; ATP concentration: 200 μM; Incubation time: 10 hr.CDK6/CyclinD3: Enzyme concentration: 1 nM; ATP concentration: 300 μM; Incubation time: 3 hr.
Measurement: IC50 = 457 nM (pChEMBL 6.340000152587891)
Descriptors: MW 530.72, ALogP 4.92, HBA 8, HBD 2, PSA 91.21, QED 0.48
Compound (SMILES): O=C(Nc1nccs1)[C@@H]1CC[C@@H]2CN1C(=O)N2OS(=O)(=O)O
InChIKey: COSQMHAARQJIKM-RQJHMYQMSA-N
Target: Beta-lactamase (SINGLE PROTEIN), Klebsiella pneumoniae
UniProt: Q93LQ9
Assay: Spectrophotometric Assay: Enzyme activities were measured in the presence of the test inhibitor in spectrophotometric assay.
Measurement: IC50 = 36 nM (pChEMBL 7.440000057220459)
Descriptors: MW 348.36, ALogP 0.08, HBA 7, HBD 2, PSA 129.14, QED 0.74
Compound (SMILES): CNS(=O)(=O)c1ccccc1Nc1nc(Nc2ccc(N3CCOCC3)cc2OC)nc2c1C1(CC1)CN2
InChIKey: IJNKMKPYXXIHRM-UHFFFAOYSA-N
Target: Protein-tyrosine kinase 2-beta (SINGLE PROTEIN), Homo sapiens
UniProt: Q14289
Assay: Biochemical Assay: To measure inhibitory activity of FAK/PYK2 inhibitors, compounds were first prepare as a 1 mM stock in 100% DMSO and 3-fold serial dilution was performed in a 96-well plate (Corning, 3897) to generate 12 different concentration of 100× stock. A 5 μl of 100× stock of each concentration was added to wells containing 95 μl of 1× reaction buffer (40 mM Tris, pH7.5, mM MgCl2, 1 mM DTT and 1 mM CHAPS) to generate 5× stock. Then 2 μl of 5× stock of each concentration was added to a 384 wells-OptiPlate (PerkinElmer, 6007299). For FAK, 4 μl of 2.5 nM FAK, 1 μl of 8 μg/μl BSA and 1.5 μl of 666 nM ULight-poly Glu, Ala, Tyr (1:1:1) peptide substrate, prepared in above reaction buffer, were added to each well. The reaction was initiated by adding 1.5 μl of 33.3 μM ATP. The reaction was allowed to proceed for 120 min before being quenched with 5 μl of 40 mM EDTA stop buffer prepared in 1×LANCE detection buffer (PerkinElmer, CR97-100). For PYK2, 5 μl of 2 nM PYK2 and 1.5 μl of 666 nM ULight-poly Glu, Ala, Tyr (1:1:1) peptide substrate, prepared in above reaction buffer, were added to each well. The reaction was initiated by adding 1.5 μl of 46.6 μM ATP. The reaction was allowed to proceed for 40 min before being quenched with 5 μl of 40 mM EDTA stop buffer prepared in 1×LANCE detection buffer (PerkinElmer, CR97-100). Upon quenching of the reaction, 5 μl of 8 nM anti-phosphotyrosine antibody was added to each well and incubated for 60 minutes.
Measurement: IC50 < 20 nM (pChEMBL 7.698999881744385)
Descriptors: MW 537.65, ALogP 3.17, HBA 10, HBD 4, PSA 129.74, QED 0.34
Compound (SMILES): O=S(=O)(Nc1nccs1)c1ccc2c(c1)OCCN2c1ccccc1Cl
InChIKey: JIUSFZSCMYJTBW-UHFFFAOYSA-N
Target: Sodium channel protein type 9 subunit alpha (SINGLE PROTEIN), Homo sapiens
UniProt: Q15858
Assay: In Vitro PX Assay: HEK 293 cells stably transfected with human Nav1.7 were recorded in whole cell voltage clamp mode with the PatchXpress automated electrophysiology system (Molecular Devices, LLC, Sunnyvale, Calif.). Compound effects were measured on a partially inactivated state of the sodium channel. Cells were clamped to a holding potential yielding 20 to 50% inactivation. To elicit sodium current, channels were activated by pulsing to −10 mV for 20 msec. This voltage protocol was repeated at a rate of 0.1 Hz throughout the experiment. A single concentration of test compound was applied to cells for a duration of 3 minutes.
Measurement: IC50 = 1080 nM (pChEMBL 5.96999979019165)
Descriptors: MW 407.9, ALogP 4.13, HBA 6, HBD 1, PSA 71.53, QED 0.7
Compound (SMILES): CN(c1ccncn1)[C@H]1CCN(c2ncnc3[nH]ccc23)C1
InChIKey: KZBFERTYFCJTEX-NSHDSACASA-N
Target: Tyrosine-protein kinase JAK2 (SINGLE PROTEIN), Homo sapiens
UniProt: O60674
Assay: Enzymatic Assay: JAK1/2/3 kinase assay are performed in vitro using Kit-Tyr 6 Peptide (Invitrogen, Cat. No. PV4122). TYK2 kinase assay are performed in vitro using Z'-LYTE™ Kinase Assay Kit-Tyr 3 Peptide (Invitrogen, Cat. No. PV3192). Recombinant human JAK1/2/3 or TYK2 catalytic domains are from Invitrogen (Cat No. PV4774/PV4210/PV3855/PV4790); All reactions (20 μL) are started by adding 2.5 μL of the testing compound in 4% DMSO solution, 5 μL of Kinase/Peptide substrate Mixture (3.2, 0.04, 0.2 or 8 μg/mL for Recombinant human JAK1/2/3 catalytic domains, 4 μM for Z-LYTE™ Tyr 6 peptide or Z-LYTE™ Tyr 3 peptide) or Phospho-Peptide solution (Invitrogen, Cat. No. PV3192, diluted with 1.33× Kinase Buffer), 2.5 μL ATP Solution (300/100/40/100M, JAK1/JAK2/JAK3/TYK2) or 1.33× Kinase Buffer (Invitrogen, Cat. No. PV3189, 5× diluted with distilled water). The 384-well assay plate (Corning, Cat. No. 3575) is mixed and incubated at room temperature for 1 hour. 5 μL of the Development Solution (Dilute Development Reagent A (Cat. No. PV3297) is diluted to 1/64 with Development Buffer (Cat. No. PV3127) for JAK1, JAK2 and JAK3 assay; Development Reagent A (Cat. No. PV3297) is diluted to 1/2048 with Development Buffer (Cat. No. PV3127) for TYK2 assay. The diluted Development Solution is then added to each well, mixed and incubated at room temperature for another 1 hour. The kinase reaction is then stopped by adding 5 μL of the Stop Reagent (Invitrogen, Cat. No. PV3094), and the plate is read with Wallac 1420 VICTOR3 Multilabel Counter (PerkinElmer™) at 445 nm and 520 nm fluorescence. All compounds are initially tested at 8 different concentrations (1 μM down to 0.0003 μM) using a 1:3 serial dilution scheme.
Measurement: IC50 = 0.55 nM (pChEMBL 9.260000228881836)
Descriptors: MW 295.35, ALogP 1.46, HBA 6, HBD 1, PSA 73.83, QED 0.79
Compound (SMILES): CCCS(=O)(=O)Nc1ccc(F)c(C(=O)c2c[nH]c3ncc(-c4ccc(O)nc4)cc23)c1F
InChIKey: MKVMMAUBKKDDSE-UHFFFAOYSA-N
Target: Serine/threonine-protein kinase B-raf (SINGLE PROTEIN), Homo sapiens
UniProt: P15056
Assay: Inhibition of B-Raf (unknown origin) assessed as decrease in biotin-tagged MEK1 phosphorylation after 45 mins
Measurement: IC50 < 10 nM (pChEMBL 8)
Descriptors: MW 472.47, ALogP 3.99, HBA 6, HBD 3, PSA 125.04, QED 0.35
Compound (SMILES): C[C@@]1(c2nc(NC(=O)c3ccc(Cl)cn3)ccc2F)C[C@@H](C(F)(F)F)OC(N)=N1
InChIKey: FWHRMLYVMNQBGS-ZBEGNZNMSA-N
Target: Beta-secretase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P56817
Assay: In Vitro BACE Cell-Based Assay: The cell-based assay measures inhibition or reduction of Aβ40 in conditioned medium of test compound treated cells expressing amyloid precursor protein. Cells stably expressing Amyloid Precursor Protein (APP) were plated at a density of 40K cells/well in 96 well plates (Costar). The cells were cultivated for 24 hours at 37° C. and 5% CO2 in DMEM supplemented with 10% FBS. The test compounds were then added to cells in 10-point dose response concentrations with the starting concentration being either 100 uM or 10 uM. The compounds were diluted from stock solutions in DMSO and the final DMSO concentration of the test compounds on cells was 0.1%. After 24 h of incubation with the test compounds the supernatant conditioned media was collected and the Aβ 40 levels were determined using a sandwich ELISA.
Measurement: IC50 = 38.9 nM (pChEMBL 7.409999847412109)
Descriptors: MW 431.78, ALogP 3.4, HBA 6, HBD 2, PSA 102.49, QED 0.73
Compound (SMILES): Cc1cc(Nc2nccc(Oc3ccc(NC(=O)Nc4cc(C(C)(C)C)nn4-c4ccc(=O)n(CCN5CCOCC5)c4)c4ccccc34)n2)cc2cn[nH]c12
InChIKey: XIFFATZVUNRCKL-UHFFFAOYSA-N
Target: Proto-oncogene tyrosine-protein kinase Src (SINGLE PROTEIN), Homo sapiens
UniProt: P12931
Assay: c-Src and Syk Enzyme Inhibition Assay: The inhibitory activities of compounds of the invention against c-Src and Syk enzymes (Invitrogen), are evaluated in a similar fashion to that described hereinabove. The relevant enzyme (3000 ng/mL or 2000 ng/mL respectively, 2.5 uL) is incubated with the test compound (either 4 ug/mL, 0.4 ug/mL, 0.04 ug/mL, or 0.004 ug/mL, 2.5 uL each) for 2 hr at RT. The FRET peptides (8 uM, 2.5 uL), and appropriate ATP solutions (2.5 uL, 800 uM for c-Src, and 60 uM ATP for Syk) are then added to the enzymes/compound mixtures and incubated for 1 hr. Development reagent (protease, 5 uL) is added for 1 hr prior to detection in a fluorescence microplate reader (Varioskan Flash, ThermoFisher Scientific).
Measurement: IC50 < 1 nM (pChEMBL 9)
Descriptors: MW 753.87, ALogP 6.97, HBA 12, HBD 4, PSA 169.14, QED 0.11
Compound (SMILES): CN(C)CCOc1ccc(/C(=C(/CCCF)c2ccccc2)c2ccc(O)cc2)cc1.Cl
InChIKey: KXTIGEUSLGAAHZ-JTHROIFXSA-N
Target: Steroid hormone receptor ERR1 (SINGLE PROTEIN), Homo sapiens
UniProt: P11474
Assay: Inhibition of fluorescien-conjugated coactivator PGC1a binding to ERRalpha (unknown origin) after 1 hr by TR-FRET assay
Measurement: IC50 = 8010 nM (pChEMBL 5.099999904632568)
Descriptors: MW 419.54, ALogP 6.04, HBA 3, HBD 1, PSA 32.7, QED 0.41
Compound (SMILES): N#C[C@@H]1CCN(C(=O)OC2CCOCC2)C[C@H]1n1cc(C(N)=O)c(Nc2ccc(F)cc2)n1
InChIKey: DIOZMTCEMXTFKU-IFXJQAMLSA-N
Target: Tyrosine-protein kinase JAK2 (SINGLE PROTEIN), Homo sapiens
UniProt: O60674
Assay: Biochemical HTRF Assay: 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 ambient 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 ambient 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 ambient 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.
Measurement: IC50 = 87 nM (pChEMBL 7.059999942779541)
Descriptors: MW 456.48, ALogP 2.57, HBA 8, HBD 2, PSA 135.5, QED 0.71
Compound (SMILES): NC1(C(=O)O)CN(c2nc3ccccc3n([C@H]3C[C@H]4CCC[C@@H](C3)N4[C@@H]3C[C@@H]4CCCC[C@@H](C4)C3)c2=O)C1
InChIKey: IIQBWWKHQCWLSI-SSIAJPCRSA-N
Target: Opioid growth factor receptor-like protein 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q5TC84
Assay: In Vitro ORL-1 Receptor Functional Assay: ORL-1 membrane solution was prepared by sequentially adding final concentrations of 0.066 μg/μL ORL-1 membrane protein, 10 μg/mL saponin, 3 μM GDP and 0.20 nM [35S]GTPγS to binding buffer (100 mM NaCl, 10 mM MgCl2, 20 mM HEPES, pH 7.4) on ice. The prepared membrane solution (190 μL/well) was transferred to 96-shallow well polypropylene plates containing 10 μL of 20× concentrated stock solutions of agonist/nociceptin prepared in DMSO. Plates were incubated for 30 min at about 25 °C. with shaking. Reactions were terminated by rapid filtration onto 96-well Unifilter GF/B filter plates (Packard) using a 96-well tissue harvester (Packard) and followed by three filtration washes with 200 μL ice-cold binding buffer (10 mM NaH2PO4, 10 mM Na2HPO4, pH 7.4). Filter plates were subsequently dried at 50 °C. for 2-3 hours. Fifty μL/well scintillation cocktail (BetaScint; Wallac) was added and plates were counted in Packard Top-Count for 1 min/well.
Measurement: EC50 > 20000 nM (pChEMBL 4.698999881744385)
Descriptors: MW 519.69, ALogP 3.92, HBA 7, HBD 2, PSA 104.69, QED 0.63
Compound (SMILES): CC1(C)CC2=NNC(=O)c3cc(F)cc4[nH]c(c2c34)C1=O
InChIKey: YADMFPWTDIQDHU-UHFFFAOYSA-N
Target: Poly [ADP-ribose] polymerase 2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9UGN5
Assay: PARP-2 and PARP-3 Enzymatic Assay: PARP-2 and PARP-3 enzymatic assays were conducted using commercial PARP-2/PARP-3 Chemiluminescent Assay Kit (BPS Biosciences) and the protocols with the kits. Briefly, histones were coated in a high binding plate first, and incubated with PARP-2 or PARP-3, and increasing concentrations of compounds for 0.5 h. Then, biotinylated NAD and activated DNA were added to the wells. The biotinylated PARylation product was measured by adding streptavidin-HRP and HRP substrates which produce chemiluminescence.
Measurement: IC50 = 0.9 nM (pChEMBL 9.050000190734863)
Descriptors: MW 285.28, ALogP 2.37, HBA 3, HBD 2, PSA 74.32, QED 0.78
Compound (SMILES): CN(C)CCCOc1ccc(-c2ccc3ncc4c(c3c2)n(C2(C)CCOCC2)c(=O)n4C)cn1
InChIKey: SIKMYPMDQGCEPL-UHFFFAOYSA-N
Target: Serine/threonine-protein kinase ATR (SINGLE PROTEIN), Homo sapiens
UniProt: Q13535
Assay: Cellular Assay: Assay b) HT29 cells (ECACC #85061109) were seeded into 384 well assay plates (Costar #3712) at a density of 6000 cells/well in 40 μl EMEM medium containing 1% L glutamine and 10% FBS and allowed to adhere overnight. The following morning compound of Formula (I) in 100% DMSO was added to assay plates by acoustic dispensing. After 1 h incubation at 37° C. and 5% CO2, 40 nl of 3 mM 4NQO in 100% DMSO was added to all wells by acoustic dispensing, except minimum control wells which were left untreated with 4NQO to generate a null response control. Plates were returned to the incubator for a further 1 h. Then cells were fixed by adding 20 μl of 3.7% formaldehyde in PBS solution and incubating for 20 mins at r.t. Then 20 μl of 0.1% Triton X100 in PBS was added and incubated for 10 minutes at r.t., to permeabalise cells. Then the plates were washed once with 50 μl/well PBS, using a Biotek EL405 plate washer. Phospho-Chk1 Ser 345 antibody (Cell Signalling Technology #2348) was diluted 150 fold in PBS containing 0.05% polysorbate/Tween and 15 μl was added to each well and incubated over night at r.t. The next morning plates were washed three times with 50 μl/well PBS, using a Biotek EL405 plate washer, and then 20 μl of secondary Ab solution, containing 500 fold diluted Alexa Fluor 488 Goat anti-rabbit IgG (Molecular Probes #A-11008) and 0.002 mg/ml Hoeschst dye (Molecular Probes #H-3570), in PBST, was added. After 2 h incubation at r.t., the plates were washed three times with 50 μl/well PBS, using a Biotek EL405 plate washer, and plates were then sealed with black plate seals until read. Plates were read using an ArrayScan VTI instrument, using an XF53 filter with 10× objective. A two laser set up was used to analyse nuclear staining with Hoeschst (405 nm) and secondary antibody staining of pChk1 (488 nm).
Measurement: IC50 = 10800 nM (pChEMBL 4.96999979019165)
Descriptors: MW 475.59, ALogP 3.81, HBA 8, HBD 0, PSA 74.41, QED 0.38
Compound (SMILES): CN[C@@H](C)C(=O)N[C@H](C(=O)N1CCc2ccc(F)cc2[C@@H]1C(=O)Nc1c(F)cccc1F)C(C)C
InChIKey: RRQHSAJDWNSTCR-PRPYNDMISA-N
Target: E3 ubiquitin-protein ligase XIAP (SINGLE PROTEIN), Homo sapiens
UniProt: P98170
Assay: TR-FRET Assay: The peptide AVPIAQKSEK-(ε-biotin)-OH 1:2 TFA ("Peptide A") was identified as a substrate for the TR-FRET assay by screening the 6× Histidine-tagged BIR2 domain and BIR3 domain of XIAP against a set of 29 peptides synthesized based on sequences reported by Sweeny et al. (Biochemistry, 2006, 45, 14740 14748). The peptides were labeled with the fluorescent tags FITC or TAMRA and Kd values were determined by fluorescence polarization assay. The sequence AVPIAQKSEK was identified as optimal for using in an assay. The peptide sequence was derivatized with biotin to provide AVPIAQKSEK-(Îµ-biotin)-OH 1:2 TFA as the substrate for the TR-FRET assay. Ten nanomolar of 6× Histidine-tagged BIR2 domain, corresponding to amino acids 124-240 of XIAP, or BIR3 domain, corresponding to amino acids 241-356 of XIAP, was mixed with 20 nM of the peptide AVPIAQKSEK-(ε-biotin)-OH 1:2 TFA, in the presence of 50 mM Tris-Cl, pH 7.5, 100 mM NaCl, 1 mM dithiothreitol (DTT) and 0.1 mg/mL bovine serum albumin (BSA). Following a 45 min. incubation at 37° C., Europium-Streptavidin and Allophycocyanin conjugated anti-Histidine antibody were added to a final concentration of 1.5 nM and 15 nM, respectively. Time-resolved fluorescence resonance energy transfer (TR-FRET) signals were measured 1 hour later at room temperature. Test compound potency was assessed at 10 serially diluted concentrations.
Measurement: IC50 > 54800 nM (pChEMBL 4.261000156402588)
Descriptors: MW 490.53, ALogP 2.92, HBA 4, HBD 3, PSA 90.54, QED 0.56
Compound (SMILES): Nc1cncc(-c2cc3c(cn2)cnn3-c2cccc(N3CCCNCC3)n2)n1
InChIKey: IWSDHPXZBHZPMC-UHFFFAOYSA-N
Target: Serine/threonine-protein kinase pim-2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9P1W9
Assay: Kinase Binding Assay: PIM-1, -2, and -3 enzymes were generated as fusion proteins expressed in bacteria and purified by IMAC column chromatography (Sun, X., Chiu, J. F., and He, Q. Y. (2005) Expert Rev. Proteomics, 2:649-657). A fluorescent-labeled Pim-specific peptide substrate, was custom synthesized by American Peptide Company (Sunnyvale, Calif.). Reaction Buffer contained 10 mM HEPES, pH 7.2, 10 mM MgCl2, 0.01% Tween 20, 2 mM DTT. Termination Buffer contained 190 mM HEPES, pH 7.2, 0.015% Brij-35, 0.2% Coating Reagent 3 (Caliper Life Sciences, Hopkinton, Mass.), 20 mM EDTA. Separation Buffer contained 100 mM HEPES, pH 7.2, 0.015% Brij-35, 0.1% Coating Reagent 3, 1:200 Coating Reagent 8 (Caliper Life Sciences, Hopkinton, Mass.), 10 mM EDTA and 5% DMSO.PIM reactions were carried out in a final volume of 10 uL per well in a 384-well plate. A standard enzymatic reaction, initiated by the addition of 5 uL 2Ã¿Â¿ATP and test compound to 5 uL of 2x enzyme and FAM-peptide, contained 20 uM PIM1, 50 uM PIM2, or 55 uM PIM3, 1 uM FAM-peptide, and 10 uM ATP, in Reaction Buffer. After 90 minutes of incubation at room temperature, the phosphorylation reaction was stopped by the addition of 10 uL Termination Buffer. The product and substrate in each independent reaction were separated on a 12-sipper microfluidic chip (Caliper Life Sciences, Hopkinton, Mass.) run on a Caliper LC3000 (Caliper Life Sciences, Hopkinton, Mass.). The separation of product and substrate was optimized by choosing voltages and pressure using Caliper's Optimizer software (Hopkinton, Mass.). The separation conditions used a downstream voltage of -500V, an upstream voltage of -2150V, and a screening pressure of -1.2 psi. The product and substrate fluorophore were excited at 488 nm and detected at 530 nm. Substrate conversion was calculated from the electropherogram using HTS Well Analyzer software (Caliper Life Sciences, Hopkinton, Mass.). Ki values for the test compound were calculated.
Measurement: Ki = 0.664 nM (pChEMBL 9.180000305175781)
Descriptors: MW 387.45, ALogP 1.65, HBA 9, HBD 2, PSA 110.67, QED 0.55
Compound (SMILES): O=C(OC(C(F)(F)F)C(F)(F)F)N1CCN(C(c2ccc(Cl)cc2)c2ccc(Cl)cc2)CC1
InChIKey: UPIHQFKOCOKGEO-UHFFFAOYSA-N
Target: Platelet-activating factor acetylhydrolase (SINGLE PROTEIN), Homo sapiens
UniProt: Q13093
Assay: Inhibition of PLA2G7 derived from human PC3 cell lysates preincubated for 30 mins followed by JW912 addition after 30 mins by gel-based ABPP assay
Measurement: IC50 > 1000 nM (pChEMBL 6)
Descriptors: MW 515.28, ALogP 6.33, HBA 3, HBD 0, PSA 32.78, QED 0.45
Compound (SMILES): CC(C)C[C@@H]1NCCOc2ccccc2/C=C\CNC(=O)C2(CCCC2)NC(=O)C2(CCCC2)NC1=O
InChIKey: FFCXJXXNEYZJSH-IDWGIVCRSA-N
Target: Growth hormone secretagogue receptor type 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q92847
Assay: Agonist activity at human GSHR1a transfected in CHOK1 cells co-expressing Galpha16 assessed as increase in Ca2+ release by aequorin assay
Measurement: EC50 > 1000 nM (pChEMBL 6)
Descriptors: MW 510.68, ALogP 3.07, HBA 5, HBD 4, PSA 108.56, QED 0.49
Compound (SMILES): CN(C)c1cccc(CNC(=O)c2ccc(-c3cc(C4CCOCC4)cnc3N)cc2)c1
InChIKey: QMPRUVNSZISTDL-UHFFFAOYSA-N
Target: Mitogen-activated protein kinase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P28482
Assay: AlphaScreen Assay: Compound potency against activated ERK2 is determined using a kinase assay that measures ERK2-catalyzed phosphorylation of biotinylated ERKtide peptide substrate ([Biotin]-AHA-K-R-E-L-V-E-P-L-T-P-S-G-E-A-P-N-Q-A-L-L-R-[NH2], the peptide sequence derived from EGF receptor: SEQ ID NO:1). The assay is carried out in 50 mM HEPES [pH 7.5], 5 mM MgCl2, 1 mM DTT, 0.01% Tween-20, 0.05% BSA using 0.25 nM ERK2, 200 nM ERKtide peptide and 35 uM ATP (all concentrations are final in the reaction) in a total volume of 10.25 uL. A 16-point, half-log dilution series of compounds at 41x final concentration is used for generating IC50 curves. Compound dilution series are prepared in 100% DMSO. ERK2 is preincubated with compounds for 30 minutes at ambient temperature. Reaction is initiated by addition of a substrate cocktail of ERKtide peptide and ATP and is allowed to proceed for 2-3 hours at ambient temperature. Reaction is terminated by addition of 10 uL of a 2x stop buffer consisting of 100 mM Tris-Cl [pH 7.5], 25 mM EDTA, 0.01% Tween 20, 10 ug/mL of AlphaScreen Protein A Acceptor Beads, 10 ug/mL of Streptavidin Donor Beads (PerkinElmer, Waltham, Mass.), and 1.4 ug/mL phospho-EGF Receptor (Thr669) antibody (Cat #3056, Cell Signaling Technology, Danvers, Mass.).
Measurement: IC50 = 85.5 nM (pChEMBL 7.070000171661377)
Descriptors: MW 430.55, ALogP 4.22, HBA 5, HBD 2, PSA 80.48, QED 0.61
Compound (SMILES): CNCCN1CCC(OCc2ccccc2)CC1
InChIKey: HBOJWAYLSJLULG-UHFFFAOYSA-N
Target: Histone-arginine methyltransferase CARM1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q86X55
Assay: Inhibition of PRMT4 in HEK293 cells assessed as reduction in Med12-Rme2a level measured after 72 hrs by western blot analysis
Measurement: IC50 = 1400 nM (pChEMBL 5.849999904632568)
Descriptors: MW 248.37, ALogP 1.89, HBA 3, HBD 1, PSA 24.5, QED 0.83
Compound (SMILES): CCN1CC(C)(C)Oc2nc(N3CCOC[C@@H]3C)nc(-c3ccc(NC(=O)Nc4cnccn4)c(F)c3)c2C1=O
InChIKey: WOCZOFWHYNKJCD-INIZCTEOSA-N
Target: Phosphatidylinositol 3-kinase regulatory subunit alpha (SINGLE PROTEIN), Homo sapiens
UniProt: P27986
Assay: Enzyme Assay: The test uses a lucifeurerin/luciferase system to measure the concentration of ATP and its consumption during the enzymatic reaction. The test is performed in 96-well format (Corning/Costar 96 black flat-bottomed half-wells plate, ref. 3694) in a total volume of 30 μl. To 1 μl of inhibitor in 100% DMSO are added (final concentrations) 50 μM of the substrate PIP2 ((L-α-phosphatidyl-D-myoinositol 4,5-bisphosphate, Echelon 117P-4516-0500), 2 μM of ATP and 1.7 μg/ml of PI3Kα, (p110α/p85α, Invitrogen PV4788) in a buffer of Tris/HCl 50 mM pH 7.5, EGTA 1 mM, MgCl2 10 mM, Chaps 0.03%, 1 mM DTT). After 90 minutes, the reaction is quenched by adding 20 μl/well of KinaseGlo reagent (Promega V6713). After 10 minutes in the dark, the luminescence is read on the PHERAStar microplate reader (reading at 0.8 sec/well).
Measurement: IC50 = 4900 nM (pChEMBL 5.309999942779541)
Descriptors: MW 550.6, ALogP 3.57, HBA 9, HBD 2, PSA 134.7, QED 0.49
Compound (SMILES): COC(=O)Nc1ccc(-c2[nH]c([C@@H]3C[C@H](N4CCN(S(C)(=O)=O)CC4)CN3C(=O)C3CCC(C(=N)N)CC3)nc2Cl)cc1
InChIKey: NUIFYMFMVFUBSY-NZUHHQEFSA-N
Target: Prothrombin (SINGLE PROTEIN), Homo sapiens
UniProt: P00734
Assay: In Vitro Assay: Human Factor Xa (American Diagnostica Inc.) and human thrombin (Sigma) activities were measured at the enzyme concentrations of 0.18 U/mL and 0.12 U/mL, respectively in the same buffer containing 150 mM NaCl, 2 mg/mL PEG6000, 50 mM Tris-HCl (pH7.4), except that the reactions were started with 300 μM S-2222 (phenyl-Ile-Glu-Gly-Arg-pNA, Chromogenix) and 300 μM S-2366, respectively.
Measurement: IC50 = 84000 nM (pChEMBL 4.079999923706055)
Descriptors: MW 635.19, ALogP 2.87, HBA 8, HBD 4, PSA 177.81, QED 0.27
Compound (SMILES): O=S(=O)(c1ccc(Nc2nc(OC3CCCC3)nc3c2CCN(c2ncccc2C(F)(F)F)CC3)cc1)N1CCCC1
InChIKey: AMBOIFXWSJYRKD-UHFFFAOYSA-N
Target: Transient receptor potential cation channel subfamily V member 1 (SINGLE PROTEIN), Rattus norvegicus
UniProt: O35433
Assay: Functional Assay: The assay was performed as described below, using HEK293 cells transfected with rat TRPV1 (rTRPV1/HEK293). These cells had a geneticin selection marker and were grown in Dulbecco's Modified Eagle Medium (DMEM, Gibco BRL) supplemented with 10% fetal bovine serum, penicillin/streptomycin (50 units/mL), and 500 μg/mL geneticin in 5% CO2 at 37° C. Cells were passaged frequently, every 3-5 days, to avoid overgrowth, depletion of essential medium components, or acidic medium exposure. Cells were passaged using a brief wash in 0.05% trypsin with 1 mM EDTA, followed by dissociation in divalent-free phosphate-buffered saline (Hyclone #SH30028.02). Dissociated cells were seeded onto poly-D-lysine coated black-walled 96-well plates (Biocoat; Becton Dickinson #354640) at about 40,000 cells per well and grown for approximately 1 day in culture medium to near confluency. The assay buffer was composed of 130 mM NaCl, 2 mM KCl, 2 mM MgCl2, 10 mM HEPES, 5 mM glucose, and either 2 mM or 20 μM CaCl2. On the day of the experiment, the culture medium was replaced with 2 mM calcium assay buffer using an automated plate washer (ELx405; Biotek, VT). The cells were incubated in 100 μL/well Fluo-3/AM (2 μM; TEFLabs #0116) with Pluronic F127 (100 μg/mL; Sigma #P2443) for 1 h at rt in the dark. After loading the cells, the dye solution was replaced with 50 μL/well of 20 μM calcium assay buffer using the ELx405 plate washer. Test compounds (50 μL/well) were added to the plate and incubated for 30 min. Intracellular Ca2+ levels were subsequently assayed using a Fluorometric Imaging Plate Reader (FLIPR™ instrument, Molecular Devices, CA) to simultaneously monitor Fluo-3 fluorescence in all wells (λexcitation=488 nm, λemission=540 nm) during challenge with agonist (capsaicin). The IC50 values were determined. Cells were challenged with 150 nM capsaicin and the fluorescence counts were captured following agonist addition at a sampling rate of 0.33 Hz. The contents of the wells were mixed 3 times (40 μL mix volume) immediately after the additions were made. Concentration dependence of block was determined by exposing each well of cells in duplicate rows of a 96 well plate to a serial dilution of test compound. The concentration series usually started at 10 Î¼M with a three-fold serial decrement in concentration. The magnitude of the capsaicin response was determined by measuring the change in fluo3 fluorescence before and 100 seconds after the addition of the agonist.
Measurement: IC50 = 40 nM (pChEMBL 7.400000095367432)
Descriptors: MW 602.68, ALogP 5.35, HBA 8, HBD 1, PSA 100.55, QED 0.39
Compound (SMILES): O=c1ccc2cc(OCCCN3CCN(c4cccc5sccc45)CC3)cc(F)c2[nH]1
InChIKey: ZFZUJZBNGFMIEE-UHFFFAOYSA-N
Target: 5-hydroxytryptamine receptor 2A (SINGLE PROTEIN), Rattus norvegicus
UniProt: P14842
Assay: Serotonin 5-HT2A Receptor Binding Assay: The assay was performed according to the method by Leysen J E et al. (Leysen J E, Niemegeers C J E, Van Nueten J M and Laduron P M. [3H] Ketanserin (R 41 468), a selective 3H-ligand for serotonin 2 receptor binding sites. Mol. Pharmacol., 1982, 21: 301-314). The binding assay was performed using 40 ul of the membrane specimen, 20 ul of [3H]-Ketanserin (final concentration 1 to 3 nM), 20 ul of a test drug and 50 mM Tris-hydrochloric acid buffer (pH 7.4) so that the total amount was 200 ul (final dimethylsulfoxide concentration 1%). The reaction was performed at 37° C. for 20 minutes and terminated by conducting suction filtration with a cell harvester on a glass fiber filter plate.The filter plate made of glass fiber was washed with 50 mM Tris-hydrochloric acid buffer (pH 7.4), and after dried, a microplate liquid scintillation cocktail was added and the radioactivity was measured with a microplate scintillation counter.
Measurement: Ki = 3.4 nM (pChEMBL 8.470000267028809)
Descriptors: MW 437.54, ALogP 4.47, HBA 5, HBD 1, PSA 48.57, QED 0.45