lovingscience/chembl-v37-raw-activities huggingface dataset:
Compound (SMILES): COc1ccc(CC(=O)O)cc1-c1ccc(F)c2c1CN(C(=O)CN(C)C(c1ccccc1)c1ccc(Cl)cc1)CC2
InChIKey: VFYFWBYHSVPKAR-UHFFFAOYSA-N
Target: Prostaglandin D2 receptor 2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9Y5Y4
Assay: Radioligand Displacement Assay: Binding assay was performed in a final assay volume of 250 ul. First, 25 ul of test compound, previously diluted in Binding-Buffer (Binding-Buffer: 50 mM Tris-Base, 100 mM NaCl, 1 mM EDTA, 0.1% BSA (protease free), 0.01% NaN3, 10 mM MnCl2, pH 7.0) was placed into each well. After addition of 75 ul Binding-Buffer, 50 ul of the radioligand 3H-PGD2 (at 2.5 nM (220.000 dpm/well) from ANAWA ART0662) was added to each well. Binding assay was started by addition of 100 ul CRTH2 membrane fragments, reaching a final concentration of 20 ug/well. For non-specific binding, PGD2 was added to the reaction mixture to 10 mM final concentration. This assay mix was incubated for 90 minutes at room temperature and then filtered through a GF/C filter 96-well plate which was pre-soaked for 3 hours in 0.5% polyethyleneimine (PEI). The filter-wells were washed three times with ice cold Binding-Buffer. Then, 40 ul of Microscint-40 (Packard) was added to each well and the retained radioactivity quantified in a Topcount (Packard).
Measurement: IC50 = 10.6 nM (pChEMBL 7.96999979019165)
Descriptors: MW 587.09, ALogP 6.39, HBA 4, HBD 1, PSA 70.08, QED 0.25
Compound (SMILES): CN1CCC(Nc2ccc(S(=O)(=O)NC(=O)c3ccc(N4CCN(C/C5=C(\c6ccc(Cl)cc6)CCCCCC5)CC4)cc3Oc3cccc4[nH]ccc34)cc2[N+](=O)[O-])CC1
InChIKey: QTIWKELYIHAPRB-GUPKEBGXSA-N
Target: Apoptosis regulator Bcl-2 (SINGLE PROTEIN), Mus musculus
UniProt: P10417
Assay: Time Resolved-Fluorescence Resonance Energy Transfer (TR-FRET) Assay: The inhibition constant (Ki) is the dissociation constant of an enzyme-inhibitor complex or a protein/small molecule complex, wherein the small molecule is inhibiting binding of one protein to another protein or peptide. Where the Ki for a compound is represented as > (greater than) a certain numerical value, it is intended to mean that the binding affinity value (e.g., for Bcl-XL) is greater than the limits of detection of the assay used. Where the binding selectivity ratio for a compound is represented as > (greater than) a certain numerical value, it is intended to mean that the selectivity of a particular compound for Bcl-2 over Bcl-XL is at least as great as the number indicated. Where the Ki for a compound is represented as < (less than) a certain numerical value, it is intended to mean that the binding affinity value (e.g., for Bcl-2) is lower than the limit of detection of the assay used. Inhibition constants were determined using Wang's equation (Wang Z-X).
Measurement: Ki = 44.18 nM (pChEMBL 7.360000133514404)
Descriptors: MW 866.48, ALogP 9.08, HBA 10, HBD 3, PSA 153.15, QED 0.08
Compound (SMILES): O=C(Nc1ccn(COP(=O)(O)O)c(=O)c1)c1cc(C(F)(F)F)ccc1Oc1ccc(F)cc1
InChIKey: XWWGACAASLFUDK-UHFFFAOYSA-N
Target: Sodium channel protein type 10 subunit alpha (SINGLE PROTEIN), Rattus norvegicus
UniProt: Q62968
Assay: Electrophysiology Assay: Patch clamp electrophysiology was used to assess the efficacy and selectivity of sodium channel blockers in dorsal root ganglion neurons. Rat neurons were isolated from the dorsal root ganglions and maintained in culture for 2 to 10 days in the presence of NGF (50 ng/ml) (culture media consisted of NeurobasalA supplemented with B27, glutamine and antibiotics). Small diameter neurons (nociceptors, 8-12 um in diameter) were visually identified and probed with fine tip glass electrodes connected to an amplifier (Axon Instruments). The voltage clamp mode was used to assess the compound's IC50 holding the cells at -60 mV. In addition, the current clamp mode was employed to test the efficacy of the compounds in blocking action potential generation in response to current injections.
Measurement: IC50 = 457 nM (pChEMBL 6.340000152587891)
Descriptors: MW 502.31, ALogP 4.12, HBA 6, HBD 3, PSA 127.09, QED 0.33
Compound (SMILES): C=C1C[C@@](C)(c2cc(NC(=O)c3ccc(C#N)cn3)ccc2F)N=C(N)S1
InChIKey: QQOKHWYWSYNBSI-IBGZPJMESA-N
Target: Beta-secretase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P56817
Assay: Inhibition of recombinant human BACE1 (43 to 454 residues) expressed in Escherichia coli BL21 (DE3) using Biotin-XSEVNLDAEFRHDSGC-Eu fluorogenic peptide as substrate incubated for 3 hrs by HTRF assay
Measurement: IC50 = 9.1 nM (pChEMBL 8.039999961853027)
Descriptors: MW 381.44, ALogP 3.53, HBA 6, HBD 2, PSA 104.16, QED 0.85
Compound (SMILES): O=C(O)Cn1nnc(-c2nnc(N3CCC4(CC3)CC(=O)c3c(Cl)cccc3O4)s2)n1
InChIKey: NWNLGPIKBJCOEL-UHFFFAOYSA-N
Target: Stearoyl-CoA desaturase (SINGLE PROTEIN), Homo sapiens
UniProt: O00767
Assay: Enzyme Activity Assay: The potency of compounds of formula I against the stearoyl-CoA desaturase was determined by measuring the conversion of radiolabeled stearoyl-CoA to oleoyl-CoA using rat liver microsome or human SCD1 following previously published procedures with some modifications (Joshi, et al., J. Lipid Res., 18: 32-36 (1977); Talamo, et al., Anal. Biochem, 29: 300-304 (1969)). Liver microsome was prepared from male Wistar or Sprague Dawley rats on a high carbohydrate diet for 3 days (LabDiet #5803, Purina). The livers were homogenized (1:10 w/v) in a buffer containing 250 mM sucrose, 1 mM EDTA, 5 mM DTT and 50 mM Tris-HCl (pH 7.5). After a 100,000xg centrifugation for 60 min, the liver microsome pellet was suspended in a buffer containing 100 mM sodium phosphate, 20% glycerol, 2 mM DTT, and stored at -78° C. Human SCD1 desaturase system was reconstituted using human SCD1 from a baculovirus/Sf9 expression system, cytochrome B5 and cytochrome B5 reductase.
Measurement: IC50 = 24 nM (pChEMBL 7.619999885559082)
Descriptors: MW 461.89, ALogP 1.93, HBA 11, HBD 1, PSA 136.22, QED 0.61
Compound (SMILES): CC(=C(c1ccc(N)cc1)c1ccc(NCC(N)=O)cc1)c1ccc([N+](=O)[O-])cc1
InChIKey: VJRVGNMDZPKEME-UHFFFAOYSA-N
Target: Estrogen receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P03372
Assay: Displacement of fluorescent ES2 from recombinant human ERalpha after 2 hrs in absence of light by fluorometric analysis
Measurement: EC50 = 164.1 nM (pChEMBL 6.789999961853027)
Descriptors: MW 402.45, ALogP 4.05, HBA 5, HBD 3, PSA 124.28, QED 0.24
Compound (SMILES): CC(=O)N[C@@H](Cc1ccccc1)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CO)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CO)C(=O)N[C@@H](Cc1ccccc1)C(=O)N[C@H](C(=O)N[C@@H](Cc1ccccc1)C(=O)NCC(=O)N[C@@H](Cc1ccc(O)cc1)C(=O)N[C@@H](Cc1ccccc1)C(=O)N[C@@H](CCC(=O)O)C(=O)N[C@@H](CCCNC(=N)N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCNC(=N)N)C(=O)N[C@@H](C)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC(C)C)C(N)=O)C(C)C
InChIKey: IUWLKAYXOVFGAG-NXKXUXLOSA-N
Target: Tissue factor pathway inhibitor (SINGLE PROTEIN), Homo sapiens
UniProt: P10646
Assay: Binding affinity to TFPI (unknown origin) incubated for 1.5 hrs using tracer peptide JBT0131 by screening ELISA
Measurement: IC50 = 5000 nM (pChEMBL 5.300000190734863)
Descriptors: MW 2506.94
Compound (SMILES): C[C@@H]1OC(=O)N(c2ccc3[nH]cnc3c2)[C@H]1c1ccccc1
InChIKey: CVAPQNMJDHCBSC-MEDUHNTESA-N
Target: Glutaminyl-peptide cyclotransferase (SINGLE PROTEIN), Homo sapiens
UniProt: Q16769
Assay: Spectrophotometic Assay: Spectrophotometic Assay: This novel assay was used to determine the kinetic parameters for most of the QC substrates. QC activity was analyzed spectrophotometrically using a continuous method, that was derived by adapting a previous discontinuous assay (Bateman, R.C.J. 1989 J Neurosci Methods 30, 23-28).
Measurement: IC50 < 10000 nM (pChEMBL 5)
Descriptors: MW 293.33, ALogP 3.65, HBA 3, HBD 1, PSA 58.22, QED 0.78
Compound (SMILES): COc1cc(-c2cn(C)nc2C)ccc1Nc1ncc2ccnc(NCC(C)(C)C)c2n1
InChIKey: LEVJITMIUUMAFY-UHFFFAOYSA-N
Target: Dual specificity protein kinase TTK (SINGLE PROTEIN), Homo sapiens
UniProt: P33981
Assay: Enzyme Inhibition Assay: The enzyme reaction (total volume 10 μl) was carried out in black 384-well low volume plates containing full length MPS1 (12.5 nM or 3 nM), fluorescent labelled peptide [known as H236, which has the sequence: 5FAM-DHTGFLTEYVATR-CONH2] (5 μM), ATP(10 μM), either DMSO (1% v/v) or the test compound (in the range 0.25 nM-100 μM in 1% DMSO) and assay buffer (50 mM HEPES (pH 7.0), 0.02% NaN3, 0.01% BSA, 0.1 mM Orthovandate, 10 μM MgCl2, 1 μM DTT, Roche protease inhibitor). The reaction was carried out for 60 min at room temperature and stopped by the addition of buffer (10 μl) containing 20 mM EDTA, 0.05% (v/v) Brij-35, in 0.1M HEPES-buffered saline (Free acid, Sigma, UK). The plate was read on a Caliper EZ reader II (Caliper Life Sciences).
Measurement: IC50 = 5 nM (pChEMBL 8.300000190734863)
Descriptors: MW 431.54, ALogP 4.94, HBA 8, HBD 2, PSA 89.78, QED 0.45
Compound (SMILES): CC(c1ccccc1)c1ccc(O)cc1O
InChIKey: PQSXNIMHIHYFEE-UHFFFAOYSA-N
Target: Polyphenol oxidase 1 (SINGLE PROTEIN), Agaricus bisporus
UniProt: Q00024
Assay: Inhibition Assay: Reader: Synergy HT program: tyrosinase 280-490 kinetics: kinetics over 45 minutes, reading at t=10 minutes, Tests in transparent 96-well plates, Phosphate buffer (pH 6.8), Enzyme: mushroom tyrosinase (T-3824, Sigma), Substrate: L-tyrosine (T-3754, Sigma), Positive control: Kojic acid (KA) (60890, Fluka) (reference inhibitor). Reference Molecules for the Test: Kojic acid: 9 μM<IC50<20 μM (PPO), 3 &muM<IC50<7 μM (MPMO); Vitamin C: 20 μM<IC50<40 μM (PPO); Reduced glutathione: 55% inhibition at 25 μM (PPO), IC50=1-2 μM (MPMO); Hydroquinone: IC50=3-4 μM (MPMO); Arbutin: 57% inhibition at 88 μM (MPMO).
Measurement: IC50 = 100 nM (pChEMBL 7)
Descriptors: MW 214.26, ALogP 3.25, HBA 2, HBD 2, PSA 40.46, QED 0.81
Compound (SMILES): C.CCC1NC(C(=O)Nc2n[nH]c3c2CN(C(=O)N2CCN(CC4CCOCC4)C[C@@H]2C)C3(C)C)=CO1
InChIKey: HJHWICUBTRRZSF-JNTYTKDLSA-N
Target: Protein kinase C beta type (SINGLE PROTEIN), Homo sapiens
UniProt: P05771
Assay: Inhibition Assay: Protein Kinase C beta 2 (PKC beta II) catalyzes the production of ADP from ATP that accompanies the phosphoryl transfer to the PKC Pseudosubstrate peptide (A->S, RFARKGSLRQKNV). This transfer is coupled to the oxidation of p-NADH through the activities of Pyruvate Kinase (PK) and Lactate Dehydrogenase (LDH). (3-NADH conversion to NAD+ is monitored by the decrease in absorbance at 340 nm (e=6.22 cm-1 mM-1) using a Molecular Devices SPECTRA max PLUS spectrophotometer.A typical assay was carried out on a 96-well, clear microtiter plate in a Molecular Devices spectrophotometer for 20 minutes at 30 C. in 0.1 mL of assay buffer containing 50 mM HEPES, pH 7.4, 5 nM PKC, 23 units of pyruvate kinase, 33 units of lactate dehydrogenase, 0.15 mM peptide, 0.1 mM ATP, 1 mM DTT, 4 mM PEP, 8 mM MgCl2, 0.3 mM NADH, 60 mM CaCl2, 10 mg/mL PS, 50 ng/mL PMA, 7.5% DMSO and from about 10,000 nM to 0.169 nM compound inhibitor.
Measurement: Ki = 14.9 nM (pChEMBL 7.829999923706055)
Descriptors: MW 517.68
Compound (SMILES): CCOC(=O)O[C@@H](C)OC(=O)c1c(-c2ccc(C(=O)N(c3ncccc3Cl)[C@@H]3CCCNC3)cc2)cnn1C
InChIKey: DUHCQXCKCLIECZ-FXAWDEMLSA-N
Target: Proprotein convertase subtilisin/kexin type 9 (SINGLE PROTEIN), Homo sapiens
UniProt: Q8NBP7
Assay: AlphaLISA Assay: An in-vitro AlphaLISA assay (Perkin Elmer) was developed in order to quantitate the level of PCSK9 secreted into the cell culture media following compound treatment. To detect and measure PCSK9 protein an in-house generated anti-PCSK9 antibody was coupled to AlphaLISA acceptor beads by an external vendor (PerkinElmer) and a second internally developed anti-PCSK9 antibody with an epiptope distinct from that of the acceptor beads was biotinylated in house. Streptavidin coated-donor beads (Perkin Elmer) are also included in the assay mixture which then binds the biotinylated anti-PCSK9 antibody and in the presence of PCSK9 this donor complex and acceptor beads are brought into close proximity. Upon excitation of the donor beads at 680 nm singlet oxygen molecules are released that trigger an energy transfer cascade within the acceptor beads resolving as a single peak of light emitted at 615 nm. The ability of compound to modulate PCSK9 protein levels in conditioned media by AlphaLISA was assessed in the human hepatocellular carcinoma cell line Huh7, stably over-expressing human PCSK9.
Measurement: IC50 = 7300 nM (pChEMBL 5.139999866485596)
Descriptors: MW 556.02, ALogP 4.21, HBA 10, HBD 1, PSA 124.88, QED 0.32
Compound (SMILES): Cc1cccc2cc(Cn3nc(-c4cn[nH]c4)c4c(N)ncnc43)n(-c3cccc(F)c3)c(=O)c12
InChIKey: ZCGSFTXYADUZTH-UHFFFAOYSA-N
Target: Phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit gamma isoform (SINGLE PROTEIN), Homo sapiens
UniProt: P48736
Assay: Inhibition of recombinant human C-terminal His6-tagged p110gamma expressed in insect cells using phosphatidylinositol as substrate in presence of gamma-32P-ATP after 5 mins by thin layer chromatographic method
Measurement: IC50 < 1000 nM (pChEMBL 6)
Descriptors: MW 466.48, ALogP 3.6, HBA 8, HBD 2, PSA 120.3, QED 0.41
Compound (SMILES): CC(C)(C)CN1CCC2(CC1)CN(c1ccccc1NC(=O)Nc1nc3ccc(F)nc3s1)c1c(O)ccc(F)c12
InChIKey: FRRQCEZVXTUSMX-UHFFFAOYSA-N
Target: P2Y purinoceptor 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P47900
Assay: Scintillation Proximity Assay (SPA): A SPA membrane binding assay was used to identify inhibitors of [33P] 2MeS-ADP binding to cloned human P2Y1 receptors (The P2Y1 receptor membranes were provided by Biology and the cloning of the receptor and P2Y1 receptor membrane preparation is same as described by Biology). Binding reactions were performed in 384-well OptiPlates (PerkinElmer Life Sciences, Cat #6007299) in a volume of 50 uL containing 15 fmol of P2Y1 receptor (1.7 ug of total protein), 0.3 nM [33P] 2MeS-ADP (PerkinElmer; 2,000 Ci/mmol), various concentrations of the test compound (usually between 10 uM and 160 pM) in Buffer B containing 1% DMSO in assay buffer (15 mM, HEPES, 145 mM potassium chloride, 5 mM sodium Chloride, 5 mM EDTA, 0.1 mM MgCl2, pH 7.4) and 100 ug of SPA bead (WGA polystyrene Image beads, #RPNQ 0260V, Amersham). Reactions were allowed to proceed to completion at room temperature for 1 hour followed by centrifugation of the plate for 5 min. About 40 uL of the aqueous soluttion was aspirated. Plates were sealed and the [33P] 2MeS-ADP bound to the P2Y1 receptor membranes that were bound to the SPA bead were determined in a Gen 4 LEADSEEKERSM (Amersham) Image Reader. Dose-response curves (IC50) were fit by non-linear regression (Toolset an in house data processing program) and binding constants (Ki) calculated using the Cheng-Prusoff relationship (Ki=IC50/(1+L/Kd) in which a Kd for 2MeS-ADP to the P2Y1 receptor was determined to be 14 nM.
Measurement: Ki = 7 nM (pChEMBL 8.149999618530273)
Descriptors: MW 578.69, ALogP 6.85, HBA 7, HBD 3, PSA 93.62, QED 0.23
Compound (SMILES): CCC[C@H](NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CCCNC(=N)N)NC(=O)[C@H](CCC(=O)O)NC(=O)[C@H](Cc1ccccc1)NC(=O)[C@H](Cc1ccc(O)cc1)NC(=O)CNC(=O)[C@H](CC(=O)O)NC(=O)[C@@H](NC(=O)[C@H](Cc1ccccc1)NC(=O)[C@@H](NC(=O)[C@H](CC(N)=O)NC(=O)CNC(=O)[C@H](CCCCN)NC(=O)[C@H](CO)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@H](Cc1ccccc1)NC(C)=O)C(C)C)C(C)C)C(=O)N[C@@H](C)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC(C)C)C(N)=O
InChIKey: DRBBMCJNAIAQLF-URURHIFTSA-N
Target: Tissue factor pathway inhibitor (SINGLE PROTEIN), Homo sapiens
UniProt: P10646
Assay: Binding affinity to TFPI (unknown origin) incubated for 1.5 hrs using tracer peptide JBT0131 by screening ELISA
Measurement: IC50 = 26 nM (pChEMBL 7.579999923706055)
Descriptors: MW 2358.73
Compound (SMILES): C[C@@]1(c2cc(Nc3nccc4cc(C#N)cnc34)cc(F)c2F)C[C@@H](C(F)(F)F)OC(N)=N1
InChIKey: SWFXRUVBVUKMPN-YWZLYKJASA-N
Target: Beta-secretase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P56817
Assay: In Vitro Enzymatic BACE FRET Assay: The assay buffer used in this screen is 0.05 M acetate, pH 4.2, 10% DMSO final, 100 uM genapol (which is a nonionic detergent, below its Critical Micelle Concentration). The Beta Secretase enzyme (0.2 nM) is pre-incubated for one hour with inhibitors, typically in about 1 uL of DMSO according to a serial dilution, are added thereto. This assay is effectively started by the addition of FRET substrate (50 nM) and the combination is incubated for one hour. The FRET assay is terminated with by addition of Tris buffer, which raises the pH to neutrality, and the fluorescence is determined. The FRET substrate is a peptide with commercially available fluorophore and quencher, on opposite sides of the BACE cleavage site. Proteolytic cleavage of the FRET substrate releases quenching of fluorescence (excitation 488 nm and emission 425 nm).
Measurement: IC50 = 14 nM (pChEMBL 7.849999904632568)
Descriptors: MW 462.38, ALogP 4.4, HBA 7, HBD 2, PSA 109.21, QED 0.56
Compound (SMILES): Cc1cc(C)c(-c2[nH]c(C(C)O)nc2C)cc1C(=O)N1CC(c2ccc(C#N)cc2)C1
InChIKey: VINHYLOQNZOEFD-UHFFFAOYSA-N
Target: Fatty acid synthase (SINGLE PROTEIN), Homo sapiens
UniProt: P49327
Assay: Biochemical Assay: The FASN enzyme was isolated from SKBr3 cells. SKBr3 is a human breast cancer cell-line with high levels of FASN expression. It is estimated that FASN comprises about 25% of the cytosolic proteins in this cell line. SKBr3 cells were homogenized in a dounce homogenizer then centrifuged for 15 minutes at 4° C. to remove particulate matter. The supernatant was then analyzed for protein content, diluted to the appropriate concentration, and used to measure FASN activity. The presence of FASN was confirmed by western blot analysis. A similar method for isolation of FASN from SKBr3 cells is described in Teresa, P. et al. (Clin. Cancer Res. 2009; 15(24), 7608-7615). FASN activity of the SKBr3 cell extract was determined by measuring either NADPH oxidation or the amount of thiol-containing coenzyme A (CoA) released during the fatty acid synthase reaction. The dye CPM (7-diethylamino-3-(4'-maleimidyl-phenyl)-4-methylcoumarin) contains a thiol reactive group that increases its fluorescence emission on reaction with the sulfhydryl group of CoA.
Measurement: IC50 = 54 nM (pChEMBL 7.269999980926514)
Descriptors: MW 414.51, ALogP 4.17, HBA 4, HBD 2, PSA 93.01, QED 0.67
Compound (SMILES): Cc1noc(C)c1Cn1cc(N2C(=O)N(CCOc3ccccc3)C(C)(C)C2=O)cn1
InChIKey: DXVLDEYHGXZPNM-UHFFFAOYSA-N
Target: Taste receptor type 2 member 8 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9NYW2
Assay: Fluorescence Polarization Assays: In another embodiment, Fluorescence Polarization ("FP") based assays may be used to detect and monitor ligand binding. Fluorescence polarization is a versatile laboratory technique for measuring equilibrium binding, nucleic acid hybridization, and enzymatic activity. Fluorescence polarization assays are homogeneous in that they do not require a separation step such as centrifugation, filtration, chromatography, precipitation, or electrophoresis. These assays are done in real time, directly in solution and do not require an immobilized phase. Polarization values can be measured repeatedly and after the addition of reagents since measuring the polarization is rapid and does not destroy the sample. Generally, this technique can be used to measure polarization values of fluorophores from low picomolar to micromolar levels.
Measurement: IC50 = 350 nM (pChEMBL 6.460000038146973)
Descriptors: MW 423.47, ALogP 3.16, HBA 7, HBD 0, PSA 93.7, QED 0.54
Compound (SMILES): Cc1nnc(C(CC(=O)O)c2cc(Cl)cc(-c3ccccc3OC(F)(F)F)c2)o1
InChIKey: GNAMUJMORPJOQF-UHFFFAOYSA-N
Target: Lysosomal protective protein (SINGLE PROTEIN), Homo sapiens
UniProt: P10619
Assay: Inhibition Assay: Recombinant human cathepsin A (residues 29-480, with a C-terminal 10-His tag; R&D Systems, #1049-SE) was proteolytically activated with recombinant human cathepsin L (R&D Systems, #952-CY). Briefly, cathepsin A was incubated at 10 ug/ml with cathepsin L at 1 ug/ml in activation buffer (25 mM 2-(morpholin-4-yl)-ethanesulfonic acid (MES), pH 6.0, containing 5 mM dithiothreitol (DTT)) for 15 min at 37° C. Cathepsin L activity was then stopped by the addition of the cysteine protease inhibitor E-64 (N-(trans-epoxysuccinyl)-L-leucine-4-guanidinobutylamide; Sigma-Aldrich, # E3132; dissolved in activation buffer/DMSO) to a final concentration of 10 uM.The activated cathepsin A was diluted in assay buffer (25 mM MES, pH 5.5, containing 5 mM DTT) and mixed with the test compound (dissolved in assay buffer containing (v/v) 3% DMSO) or, in the control experiments, with the vehicle in a multiple assay plate. After incubation for 15 min at room temperature.
Measurement: IC50 = 89 nM (pChEMBL 7.050000190734863)
Descriptors: MW 426.78, ALogP 5.2, HBA 5, HBD 1, PSA 85.45, QED 0.58
Compound (SMILES): O=C(NO)C1CCN(CCOc2ccc3cc2COC/C=C/COCc2cccc(c2)-c2ccnc(n2)N3)CC1
InChIKey: HFQVERAALUPSOG-OWOJBTEDSA-N
Target: Tyrosine-protein kinase JAK2 (SINGLE PROTEIN), Homo sapiens
UniProt: O60674
Assay: Inhibition of JAK2 (unknown origin) preincubated for 20 mins followed by [33P]ATP addition measured after 120 mins by Hotspot assay
Measurement: IC50 = 0.97 nM (pChEMBL 9.010000228881836)
Descriptors: MW 545.64, ALogP 4.09, HBA 9, HBD 3, PSA 118.07, QED 0.25
Compound (SMILES): Cc1cc(-c2cn(CCN3CCC3)c(C3CCN(c4ncnc(N)c4C#N)CC3)n2)ccc1F
InChIKey: GIMRLCRHTAIQJM-UHFFFAOYSA-N
Target: Ribosomal protein S6 kinase beta-1 (SINGLE PROTEIN), Homo sapiens
UniProt: P23443
Assay: P70S6K Enzyme Assay: P70S6K inhibitor compounds are diluted and plated in 96 well plates. A reaction mixture including the following components is then added to the compound plate to initiate the enzyme reaction; P70S6K (3 nM, T412E mutant, Millipore) is mixed with 24 uM ATP in an assay buffer containing 100 mM Hepes (pH 7.5), 5 mM MgCl2, 1 mM DTT, 0.015% Brij and 1 uM of the substrate peptide FITC-AHA-AKRRRLSSLRA-OH (derived from the S6 ribosomal protein sequence, FITC=fluorescein isothiocyanate, AHA=6-aminohexanoic acid). The reaction is incubated for 90 min at 25.degree. C., before the addition of 10 mM EDTA to stop the reaction. The proportion of substrate and product (phosphorylated) peptide is analysed on a Caliper Life Sciences Lab Chip 3000, using a pressure of -1.4 psi, and upstream and downstream voltages of -3000 and -700 respectively. Product peaks are resolved before substrate peaks on the resulting chromatograms.
Measurement: IC50 = 0.7 nM (pChEMBL 9.149999618530273)
Descriptors: MW 460.56, ALogP 3.33, HBA 8, HBD 1, PSA 99.89, QED 0.6
Compound (SMILES): COc1ccc(OC(F)(F)F)cc1-c1cncc(C(CC(=O)O)c2nnc(C)o2)c1
InChIKey: RDLFDASXDQYGEN-UHFFFAOYSA-N
Target: Lysosomal protective protein (SINGLE PROTEIN), Homo sapiens
UniProt: P10619
Assay: Inhibition Assay: Recombinant human cathepsin A (residues 29-480, with a C-terminal 10-His tag; R&D Systems, #1049-SE) was proteolytically activated with recombinant human cathepsin L (R&D Systems, #952-CY). Briefly, cathepsin A was incubated at 10 ug/ml with cathepsin L at 1 ug/ml in activation buffer (25 mM 2-(morpholin-4-yl)-ethanesulfonic acid (MES), pH 6.0, containing 5 mM dithiothreitol (DTT)) for 15 min at 37° C. Cathepsin L activity was then stopped by the addition of the cysteine protease inhibitor E-64 (N-(trans-epoxysuccinyl)-L-leucine-4-guanidinobutylamide; Sigma-Aldrich, # E3132; dissolved in activation buffer/DMSO) to a final concentration of 10 uM.The activated cathepsin A was diluted in assay buffer (25 mM MES, pH 5.5, containing 5 mM DTT) and mixed with the test compound (dissolved in assay buffer containing (v/v) 3% DMSO) or, in the control experiments, with the vehicle in a multiple assay plate. After incubation for 15 min at room temperature.
Measurement: IC50 = 4760 nM (pChEMBL 5.320000171661377)
Descriptors: MW 423.35, ALogP 3.95, HBA 7, HBD 1, PSA 107.57, QED 0.61
Compound (SMILES): CNCc1ccc(Cl)c(-c2ccccc2OC)c1
InChIKey: NSASZAUFRUOUIN-UHFFFAOYSA-N
Target: 5-hydroxytryptamine receptor 7 (SINGLE PROTEIN), Homo sapiens
UniProt: P34969
Assay: Agonist activity at human 5-HT7R expressed in HEK293 cells assessed as induction of cAMP levels treated for 10 mins measured after 30 mins by D2-dye based fluorescence assay
Measurement: EC50 = 549.54 nM (pChEMBL 6.260000228881836)
Descriptors: MW 261.75, ALogP 3.74, HBA 2, HBD 1, PSA 21.26, QED 0.91
Compound (SMILES): N#CCC1(n2cc(C(N)=O)c(Nc3ccnc(F)c3)n2)CCC(NCc2ccncc2)CC1
InChIKey: ABAWAKYNDLSEJP-UHFFFAOYSA-N
Target: Tyrosine-protein kinase JAK1 (SINGLE PROTEIN), Homo sapiens
UniProt: P23458
Assay: HTRF 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 uL of 1.11x enzyme and 1.11x substrate in 1x 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 uL of 10xATP in 1x 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 uL of 2x 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.
Measurement: IC50 = 86 nM (pChEMBL 7.070000171661377)
Descriptors: MW 448.51, ALogP 3, HBA 8, HBD 3, PSA 134.54, QED 0.45
Compound (SMILES): O=C(Nc1ccc(C(=O)N2CCc3c([nH]c4ccccc34)C2)cc1)N1Cc2ccccc2C1
InChIKey: XSSYSCISAPCOGC-UHFFFAOYSA-N
Target: Nicotinamide phosphoribosyltransferase (SINGLE PROTEIN), Homo sapiens
UniProt: P43490
Assay: Time-Resolved Fluorescence Resonance Energy Transfer (TR-FRET) Binding Assay : Probe 1:The assay was carried out in 18 uL low volume plates (Owens Corning) in reaction buffer (50 mM HEPES (NaOH), pH 7.5, 100 mM NaCl, 10 mM MgCl2, 1 mM DTT, 1% Glycerol) using 6.8 nM recombinant, human, C-terminally-His tagged NAMPT, 1 nM Tb-anti-His antibody (Invitrogen, Cat #PV5895), and 200 nM probe (Oregon Green 488-conjugated APO0866; A-1251667.0. Plates were covered, and reactions were carried out for 2-3 hours. Plates were read with Envision (Laser Lantha low volume protocol) after 2 to 3 hours. Excitation was carried out at 337 nm, and the ratio of emission of Oregon Green (520 nm) to terbium (492 nm) was determined and used to calculate IC50 values of test compounds.
Measurement: IC50 = 265 nM (pChEMBL 6.579999923706055)
Descriptors: MW 436.52, ALogP 4.91, HBA 2, HBD 2, PSA 68.44, QED 0.47
Compound (SMILES): CC1(C)CCC(CN2CCN(c3ccc(C(=O)NS(=O)(=O)c4ccc(NC5CCN(CC(F)(F)F)CC5)c([N+](=O)[O-])c4)c(Oc4cnc5[nH]ccc5c4)c3)CC2)=C(c2ccc(Cl)cc2)C1
InChIKey: VYMWQJPSEXCBIC-UHFFFAOYSA-N
Target: Apoptosis regulator Bcl-2 (SINGLE PROTEIN), Homo sapiens
UniProt: P10415
Assay: Time Resolved-Fluorescence Resonance Energy Transfer (TR-FRET) Assay: The inhibition constant (Ki) is the dissociation constant of an enzyme-inhibitor complex or a protein/small molecule complex, wherein the small molecule is inhibiting binding of one protein to another protein or peptide. Where the Ki for a compound is represented as > (greater than) a certain numerical value, it is intended to mean that the binding affinity value (e.g., for Bcl-XL) is greater than the limits of detection of the assay used. Where the binding selectivity ratio for a compound is represented as > (greater than) a certain numerical value, it is intended to mean that the selectivity of a particular compound for Bcl-2 over Bcl-XL is at least as great as the number indicated. Where the Ki for a compound is represented as < (less than) a certain numerical value, it is intended to mean that the binding affinity value (e.g., for Bcl-2) is lower than the limit of detection of the assay used. Inhibition constants were determined using Wang's equation (Wang Z-X).
Measurement: Ki = 0.071 nM (pChEMBL 10.149999618530273)
Descriptors: MW 935.47, ALogP 9.26, HBA 11, HBD 3, PSA 166.04, QED 0.08
Compound (SMILES): O=C(NCCCO)c1cc(NC(=O)c2cc(-c3ncccc3F)ccc2Cl)n(-c2ccccc2)n1
InChIKey: WIHRBMADIQSVNT-UHFFFAOYSA-N
Target: High affinity nerve growth factor receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P04629
Assay: Cell Based Assay: TRKA co-expressed with p75: The assays are based upon DiscoveRx's proprietary Enzyme Fragment Complementation (EFC) technology. In the case of the TRK cell lines, the enzyme acceptor (EA) protein is fused to a SH2 protein and the TRK receptor of interest has been tagged with a Prolink tag. Cryopreserved PathHunter cells are used from either in-house produced batches or bulk batches bought directly from DiscoveRx. Cryopreserved cells are resuscitated, spun 1000 rpm for 4 min to remove freezing media, and resuspended in MEM+0.5% horse serum (both Invitrogen) to 5e^5 cells/ml. The cells are then plated using a Multidrop into Greiner white tissue culture treated plates at 20 μl/well and incubated for 24 h at 37 °C., 5% CO2, high humidity. On the day of the assay, the cell plates are allowed to cool to room temperature for 30 min prior to the assay. 4 mM stock solutions of test compounds are prepared and serially diluted in 100% DMSO. A standard curve using the compound of Example 135, WO2005/116035 at a top concentration of 150 μM is also prepared on each test plate. High percentage effect (HPE) is defined by 150 μM of the compound of Example 135, WO2005/116035 and 0% effect (ZPE) is defined by 100% DMSO. Plates containing 1 μl of serially diluted compound, standard and HPE/ZPE are diluted 1/66 in assay buffer (PBS minus Ca2+, minus Mg2+ with 0.05% pluronic F127) using a Wellmate. Using a Platemate Plus, 5 μl of 1/66 diluted test compounds is then transferred to the cell plate and allowed to reach equilibrium by incubating for 30 min at room temperature before addition of agonist stimulus: 10 μl/well of 2 nM (0.571 nM FAC) of the cognate neurotrophin (Peprotech) diluted in agonist buffer (HBSS with 0.25% BSA). Final assay concentration of the test compounds is 8.66 μM, (the compound of Example 135, WO2005/116035 FAC is 0.325 μM). The plates are left at room temperature for a further 2 hours before addition of 10 μl of the DiscoveRx PathHunter detection reagent (made up by adding 1 part Galacton Star, 5 parts Emerald II and 19 parts Cell Assay Buffer as per the manufacturer's instructions). After reagent addition, plates are covered and incubated at room temperature for 60 minutes. Luminescence signal is read using an Envision.
Measurement: IC50 = 61.4 nM (pChEMBL 7.210000038146973)
Descriptors: MW 493.93, ALogP 4.09, HBA 6, HBD 3, PSA 109.14, QED 0.32
Compound (SMILES): CCS(=O)(=O)NCc1cncc(-c2cnc3c(c2)CCCN3C(N)=O)c1
InChIKey: IBQGMGNYUQJBBT-UHFFFAOYSA-N
Target: Cytochrome P450 11B2, mitochondrial (SINGLE PROTEIN), Homo sapiens
UniProt: P19099
Assay: Aldosterone Synthase Inhibition Assay: The aldosterone synthase inhibition assay employs cynomolgus adrenal gland mitochondria as the source of aldosterone synthase (CYP11B2). Mitochondria are prepared from frozen cynomolgus monkey adrenal glands according to Method A described in by J. D. McGarry et al. (Biochem. J., 1983, 214, 21-28), with a final resuspension in the AT buffer described in R. Yamaguchi et al. (Cell Death and Differentiation, 2007, 14, 616-624), frozen as aliquots in liquid nitrogen and stored at -80° C. until use. Assays are performed in 96-well format in a final volume of 60 uL/well, containing 100 mM potassium phosphate, pH 7.4, 1% (v/v) DMSO, and additionally, 2 uM of corticosterone and 6 mg of mitochondrial protein. Reactions are started by the addition of NADPH to 1 mM and allowed to proceed for 60-90 minutes at 37° C. Reactions are terminated by the addition of 60 uL of acetonitrile. One hundred microliters are then transferred to a glass filter plate and centrifuged at 570xg.
Measurement: IC50 = 22.6 nM (pChEMBL 7.650000095367432)
Descriptors: MW 375.45, ALogP 1.41, HBA 5, HBD 2, PSA 118.28, QED 0.82
Compound (SMILES): CCNc1c(-c2cccc(Cl)c2)nc2ccc(C(F)(F)F)cn12
InChIKey: SKTMPVUPTJNYLK-UHFFFAOYSA-N
Target: Transient receptor potential cation channel subfamily A member 1 (SINGLE PROTEIN), Homo sapiens
UniProt: O75762
Assay: Dose Response Assay: Harvested cells with 0.025% trypsin/EDTA. Resuspended cells in growth media without selection antibiotics. Measured cell density and diluted to 2.4×10^5 cells/ml in media containing 1 μg/ml Doxycycline Plate 25 μl/well into 384 well black/clear tissue culture-treated plates. Incubated overnight at 37° C. Growth media was removed from the cell plates (20 μl) and 20 μl of the Replacement Buffer was added followed by addition of 25 μl of diluted dye. All three steps were performed using a Plate Washer BioTek 407. The plates were then incubated for 30' at RT. After incubation, both the cell and compound plates were brought to the FLIPR and 20 μl of the diluted compounds/antagonist/bk were transferred to the cell plates by the FLIPR. Plates were then incubated for 30' at room temperature. After 30' incubation, plates were returned to the FLIPR and 20 ul of 4.5× Cinnamaldehyde was added to the cell plates. During the compound addition as well as agonist addition, fluorescence readings were taken simultaneously from all 384 wells of the cell plate every 1.5 seconds. Five readings were taken to establish a stable baseline, then 20 μl of sample was rapidly (30 μl/sec) and simultaneously added to each well of the cell plate. The fluorescence was continuously monitored before, during and after sample/agonist addition for a total elapsed time of 100 seconds (compound addition) and 120 seconds (agonist addition).
Measurement: IC50 = 2067 nM (pChEMBL 5.679999828338623)
Descriptors: MW 339.75, ALogP 5.11, HBA 3, HBD 1, PSA 29.33, QED 0.72
Compound (SMILES): O=C(N[C@H]1CCC[C@@H]1Nc1ccc(C(F)(F)F)cn1)c1ccccc1Cl
InChIKey: GWLZLMIQIQBKLX-GJZGRUSLSA-N
Target: Orexin/Hypocretin receptor type 1 (SINGLE PROTEIN), Homo sapiens
UniProt: O43613
Assay: Fluorescent Imaging Plate Reader (FLIPR) Assay: Orexin antagonist activity was determined by measuring changes in intracellular calcium levels using a Ca2+ sensitive fluorescent dye. The changes in fluorescent signal were monitored by Fluorescent Imaging Plate Reader (FLIPR®) technology available from Molecular Devices, LLC, U.S.A. Orexin mediated increases in intracellular Ca2+ concentration were readily detected upon activation with orexin-A. Twenty-four hours prior to the assay, RBL-2H3 cells stably expressing either human orexin receptor 1 or human orexin receptor 2 were seeded in cell culture medium in black, clear-bottom 384-well plates (commercially available from Corning Inc., U.S.A.) and grown overnight at 37 °C., 5% CO2. On the day of the assay, cell culture media was removed and cells were loaded with Calcium 5 Dye (commercially sold by Molecular Devices, LLC, U.S.A.) for 1 hour at 37 °C., 5% CO2.
Measurement: IC50 = 321 nM (pChEMBL 6.489999771118164)
Descriptors: MW 383.8, ALogP 4.52, HBA 3, HBD 2, PSA 54.02, QED 0.82
Compound (SMILES): CNCCN1CCC(NC(=O)c2ccccc2)CC1
InChIKey: GHEBNMWUAGLPPM-UHFFFAOYSA-N
Target: Histone-arginine methyltransferase CARM1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q86X55
Assay: Inhibition of human full length PRMT4 (1 to 608 residues) expressed in 293F cells assessed as inhibition of methylation activity preincubated with protein followed by addition of biotin labeled histone H3 (1 to 25) peptide as substrate and [3H]-SAM measured after 3 hrs by scintillation proximity assay
Measurement: IC50 = 231 nM (pChEMBL 6.639999866485596)
Descriptors: MW 261.37, ALogP 1.1, HBA 3, HBD 2, PSA 44.37, QED 0.83
Compound (SMILES): COCCCNC(=O)c1cc(C#N)ccc1C1C2=C(CCC2=O)N(c2cccc(C(F)(F)F)c2)C(=O)N1C
InChIKey: WTWJFJWBIBKKIT-UHFFFAOYSA-N
Target: Neutrophil elastase (SINGLE PROTEIN), Homo sapiens
UniProt: P08246
Assay: Human Neutrophil Elastase Assay: The following buffers were used: Compound buffer: 100 mM Tris, 500 mM NaCl, adjusted to pH 7.5; Assay buffer: 100 mM Tris, 500 mM NaCl, adjusted to pH 7.5, containing 0.01% BSA.Assay conditions: Test compounds were prediluted in DMSO and subsequently in compound buffer (5% DMSO final). 5 μL of these compound dilutions were mixed with 10 μl Neutrophil elastase (9 ng/ml in assay buffer) in a black 384 well OptiPlate (Perkin Elmer, Cat No.: 6007270) and incubated for 15 min at room temperature. Subsequently 10 μL substrate solution in assay buffer were added (250 μM final concentration) and the plates were incubated for 60 min at room temperature. After inactivation of the enzyme, fluorescence intensities were measured at 380 nm excitation and 460 nm emission wavelengths.
Measurement: IC50 = 5.7 nM (pChEMBL 8.239999771118164)
Descriptors: MW 526.52, ALogP 4.57, HBA 5, HBD 1, PSA 102.74, QED 0.53
Compound (SMILES): N#CCC1(n2cc(-c3ncnc4[nH]ccc34)cn2)CN(C2CCN(C(=O)Nc3cc(F)ccc3F)CC2)C1
InChIKey: CIVCERVVQBMFGU-UHFFFAOYSA-N
Target: Tyrosine-protein kinase JAK1 (SINGLE PROTEIN), Homo sapiens
UniProt: P23458
Assay: In Vitro JAK Kinase Assay: Compounds herein were tested for inhibitory activity of JAK targets according to the following in vitro assay described in Park et al., Analytical Biochemistry 1999, 269, 94-104. The catalytic domains of human JAK1 (a.a. 837-1142), Jak2 (a.a. 828-1132) and Jak3 (a.a. 781-1124) with an N-terminal His tag were expressed using baculovirus in insect cells and purified. The catalytic activity of JAK1, JAK2 or JAK3 was assayed by measuring the phosphorylation of a biotinylated peptide. The phosphorylated peptide was detected by homogenous time resolved fluorescence (HTRF). IC50s of compounds were measured for each kinase in the 40 microL reactions that contain the enzyme, ATP and 500 nM peptide in 50 mM Tris (pH 7.8) buffer with 100 mM NaCl, 5 mM DTT, and 0.1 mg/mL (0.01%) BSA. For the 1 mM IC50 measurements, ATP concentration in the reactions was 1 mM. Reactions were carried out at room temperature for 1 hr and then stopped with 20 μL 45 mM EDTA, 300 nM SA-APC, 6 nM Eu-Py20 in assay buffer (Perkin Elmer, Boston, Mass.). Binding to the Europium labeled antibody took place for 40 minutes and HTRF signal was measured on a Fusion plate reader (Perkin Elmer, Boston, Mass.).
Measurement: IC50 < 10 nM (pChEMBL 8)
Descriptors: MW 517.54, ALogP 3.72, HBA 7, HBD 2, PSA 118.76, QED 0.42
Compound (SMILES): COC(CN1CC[C@H](CNC(=O)c2ccc3ccccc3c2)N[C@H](CCCNC(=N)N)C1=O)c1ccccc1
InChIKey: CBJRTXOFLNIBPS-QMMNWOAZSA-N
Target: Melanocortin receptor 5 (SINGLE PROTEIN), Homo sapiens
UniProt: P33032
Assay: Radioligand Binding Assay: Assessments of compound binding to human MC5R (hMC5R)) by displacement of an 125I-labeled NDP-MSH receptor ligand peptide were performed essentially as described in the data sheets produced by Perkin Elmer to accompany their frozen hMC5R membranes (Perkin Elmer catalog number RBXMC5M400UA). [125I] NDP-MSH: Radiolabeled in House and Purified by HPLC: Na125I (0.5 mCi, 17.4 Ci/mg) was added to 50 μL sodium phosphate (50 mM, pH 7.4) in an eppendorf tube precoated with IODOGEN. After incubation for 10 mins the phosphate buffer containing the iodine was added to NDP-MSH (10 μl at 1 mg/mL) in a separate eppendorf tube. This was incubated for a further 10 mins. The iodinated NDP-MSH was purified by HPLC on a Zorbax SB 300 column using solvent A: 0.05% TFA and solvent B: 90% acetonitrile 0.045% TFA with a linear gradient, 0-67% B over 60 mins. The 125I NDP-MSH eluted at 52 mins after the unlabeled starting material (48 min) and was counted and stored in the freezer. It was used within 48 hrs, as radioactive decay and ligand decomposition resulted in greatly reduced specific binding observed after 72 hrs. Incubation buffer: 25 mM HEPES-KOH (pH 7.0), 1.5 mM CaCl2, 1 mM MgSO4, 0.1 M NaCl, 1 mM 1,10-phenanthroline, and 1 Complete™ protease inhibitor tablet/100 mL (Roche, catalog number 1873580). Perkin Elmer frozen hMC5 membranes: catalog number RBXMC5M400UA, 0.4 mL/vial; 400 microassays/vial, 0.78 mg/mL protein concentration. Vials of frozen membranes were thawed rapidly immediately before use, diluted with binding buffer and vortexed. Resuspended membranes were kept on ice until they were added to the wells of the plate. Assays were performed in 96 well polypropylene plates. Membranes (0.78 μg 40 μL of a 1:40 dilution in incubation buffer) were added to [125I] NDP-MSH (0.84 nM; 2200 Ci/mmol) and test compounds in a total volume of 140 μL. This was incubated for 1 hr at 37° C. Non-specific binding was determined with 3 mM NDP-MSH. Plates were filtered using a Tomtec cell harvester with GF/A filters (Wallac) (presoaked in 0.6% polyethylenimine) and washed three times with 1.0 mL ice-cold wash buffer (the above incubation buffer without 1,10-phenanthroline and Complete™ protease inhibitor tablet). The filters were dried in a 37° C. oven, placed in a sample bag and 5 mL Betaplatescint (Wallac) was added. Prepared filters were counted in cassettes in a Microbeta Trilux (Wallac) for 1 min.
Measurement: IC50 < 1000 nM (pChEMBL 6)
Descriptors: MW 530.67, ALogP 2.78, HBA 5, HBD 5, PSA 132.57, QED 0.15
Compound (SMILES): CCN(C(=O)Oc1ccc(F)cc1)[C@@H]1CN(C(=O)C2CCN(C(=O)C3CCOC(C)(C)C3)CC2)C[C@H]1c1ccc(Cl)cc1
InChIKey: BPFKGAJXLJBBHS-RENJQWLISA-N
Target: Neuromedin-K receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P29371
Assay: [3H]SR142801 Competition Binding Assay: hNK3 receptor binding experiment were performed using [3H]SR142801 (Catalog No. TRK1035, specific activity: 74.0 Ci/mmol, Amersham, GE Healthcare UK limited, Buckinghamshire, UK) and membrane isolated from HEK293 cells transiently expressing recombinant human NK3 receptor. After thawing, the membrane homogenates were centrifuged at 48,000×g for 10 mM at 4° C., the pellets were resuspended in the 50 mM Tris-HCl, 4 mM MnCl2, 1 μM phosphoramidon, 0.1% BSA binding buffer at pH 7.4 to a final assay concentration of 5 μg protein/well. For inhibition experiments, membranes were incubated with [3H]SR142801 at a concentration equal to KD value of radioligand and 10 concentrations of the inhibitory compound (0.0003-10 μM) (in a total reaction volume of 500 μl) for 75 min at room temperature (RT). At the end of the incubation, membranes were filtered onto unitfilter (96-well white microplate with bonded GF/C filter preincubated 1 h in 0.3% PEI+0.3% BSA, Packard BioScience, Meriden, Conn.) with a Filtermate 196 harvester (Packard BioScience) and washed 4 times with ice-cold 50 mM Tris-HCl, pH 7.4 buffer. Nonspecific binding was measured in the presence of 10 μM SB222200 for both radioligands. The radioactivity on the filter was counted (5 min) on a Packard Top-count microplate scintillation counter with quenching correction after addition of 45 μl of microscint 40 (Canberra Packard S.A., Zürich, Switzerland) and shaking for 1 h.
Measurement: Ki = 4.7 nM (pChEMBL 8.329999923706055)
Descriptors: MW 614.16, ALogP 5.74, HBA 5, HBD 0, PSA 79.39, QED 0.42
Compound (SMILES): CC[C@H](C)[C@H](NC(=O)C[C@H](N)[C@H](Cc1ccccc1)NC(=O)[C@@H](NC(=O)Cc1cccc(NS(C)(=O)=O)c1)C(C)C)C(=O)N[C@H](C(=O)OC)C(C)C
InChIKey: FTMWUGWXNGDDBR-XAAQGLMKSA-N
Target: Cathepsin D (SINGLE PROTEIN), Homo sapiens
UniProt: P07339
Assay: In-Vitro Fluorescence Assay: In order to identify modulators of cathepsin D activity, a continuous enzymatic test was carried out with a synthetic peptide which carries a fluorescent group (MCA=(7-methoxycoumarin-4-yl)acetyl) which is quenched by energy transfer from a Dpn (2,4 dinitrophenyl) group on the same molecule, in Greiner 384-well nb microtitre plates. Cleavage of the peptidic substrate by cathepsin D causes an increase in the fluorescence intensity. In order to determine the efficacy of substances, the time-dependent increase in the fluorescence intensity in the presence of the substance was compared with the time-dependent increase in fluorescence in the absence of substances. The reference substance used was pepstatin A (Sigma-Aldrich). The substrate used was MCAGKPILFFRLK(Dnp)d-R-NH2 (Enzo Life Sciences, Lorrach). The enzyme employed was cathepsin D isolated from the human liver (Sigma-Aldrich) in a final concentration of 1.4 nM. The test was carried out in 100 mM sodium acetate buffer, 1.25% (v/v) of DMSO, 0.25% (w/v) of Chaps, pH 5.5. 2 ul of each substance solution with serially diluted substance concentration were added to in each case 4 ul of cathepsin D solution and incubated at room temperature for 10 min. The reaction was started by addition of 2 ul of substrate solution (final concentration 5 uM). After carrying out a starting-point fluorescence measurement (excitation wavelength 340 nm/emission wavelength 450 nm) using an Envision multilabel reader (Perkin Elmer), the reaction was incubated at room temperature for 60 min. The amount of peptide fragment cleaved off during the reaction time was subsequently measured by determination of the increase in the fluorescence intensity at 450 nm (excitation wavelength 340 nm).
Measurement: IC50 = 81 nM (pChEMBL 7.090000152587891)
Descriptors: MW 744.96, ALogP 2.03, HBA 9, HBD 6, PSA 214.89, QED 0.11
Compound (SMILES): CC(C)(C)c1cc(NC(=O)Nc2ccc(-c3cn4c(n3)sc3nc(OCC5(N)CC5)ccc34)cc2)no1
InChIKey: AWAWDBSSCRYBAO-UHFFFAOYSA-N
Target: Receptor-type tyrosine-protein kinase FLT3 (SINGLE PROTEIN), Homo sapiens
UniProt: P36888
Assay: Caliper's mobility shift assay (MSA): FLT3 is a receptor tyrosine kinase involved in survival and proliferation of leukemic cells. Constitutively activating FLT3 mutations has been found in about 30% of all patients with acute myeloid leukemia (AML). The tested compounds were screened for their ability to inhibit FLT3 kinase activity using Caliper's mobility shift assay (MSA). The assay uses a microfluidic chip to measure the conversion of a fluorescent peptide substrate to a phosphorylated product following separation by electrophoresis. The signature of the fluorescence signal over time reveals the extent of the reaction. Protein tyrosine kinase assays were carried out in a final volume of 25 ul containing 0.9 nM purified FLT3 (Carna, Cat 08-154) enzyme protein, 50 mM HEPES [pH=7.5], 0.0015% Brij-35, 10 mM MgCl2, 2 mM DTT, 2% DMSO, 97 uM ATP, 1.5 uM peptide 2. Each Compound was added into the reaction to final concentrations from 300 nM to 0.015 nM (10 concentration points in 3 fold serial dilution).
Measurement: IC50 = 38 nM (pChEMBL 7.420000076293945)
Descriptors: MW 517.62, ALogP 5.41, HBA 9, HBD 3, PSA 132.6, QED 0.27
Compound (SMILES): N#Cc1cc(-c2ccnc(Nc3cccc(=O)[nH]3)n2)cnc1N1CC[C@H](F)C1
InChIKey: VETBWYSGFIOLHB-AWEZNQCLSA-N
Target: Inhibitor of nuclear factor kappa-B kinase subunit epsilon (SINGLE PROTEIN), Homo sapiens
UniProt: Q14164
Assay: Time-Resolved Fluorescence-based Lanthascreen Assay: Kinase inhibition assays (10 μL) were performed at 20° C. in 384-well plate format. Compound IC50 values were determined at the apparent Km for ATP (20 μM) based on a radiometric assay (Invitrogen) using 8 or 10 point curves in duplicate. The final reaction conditions contained 400 nM fluorescein-IkBα substrate (DRHDSGLDSMKDE), 20 μM ATP, 2 nM or 8 nM IKKε or TBK1 kinase respectively, and 3% DMSO in kinase assay buffer consisting of 50 mM HEPES (pH 7.5), 10 mM MgCl, 1 mM EGTA, 0.01% Brij-35.Compound dilutions were prepared from 10 mM DMSO stocks by dilution into DMSO. Compound dilution series were further diluted in kinase assay buffer to give a 12% DMSO stock, the final concentration in the assay being 3% DMSO.The kinase phosphorylation assay was initiated by the addition of the kinase and the reaction was allowed to proceed for 1 h or 2.5 h for IKKε and TBK-1 kinase respectively.
Measurement: IC50 < 1000 nM (pChEMBL 6)
Descriptors: MW 377.38, ALogP 2.39, HBA 7, HBD 2, PSA 110.59, QED 0.72