lovingscience/chembl-v37-raw-activities huggingface dataset:
Compound (SMILES): COc1cc2c(Nc3ccc(Br)c(Cl)c3)ncnc2cc1O[C@@H]1C[C@H]2OCC3(OCCO3)[C@H]2O1
InChIKey: XCAOLOJNZVNRSK-HKBOAZHASA-N
Target: Epidermal growth factor receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P00533
Assay: Inhibition of recombinant human N-terminal GST His6-tagged thrombin cleavage site-fused EGFR (H672 to A1210 residues) expressed in baculovirus using poly-EY as substrate after 210 mins by luciferase coupled chemiluminescence assay
Measurement: IC50 < 50 nM (pChEMBL 7.301000118255615)
Descriptors: MW 550.79, ALogP 4.43, HBA 9, HBD 1, PSA 93.19, QED 0.5
Compound (SMILES): N#Cc1ccc(Cl)cc1-c1ccn(C(Cc2ccccc2)C(=O)Nc2ccc(-c3nnn[nH]3)cc2)c(=O)c1
InChIKey: QLAFRGZBWLGFNO-UHFFFAOYSA-N
Target: Coagulation factor XI (SINGLE PROTEIN), Homo sapiens
UniProt: P03951
Assay: Inhibition of human FXIa using Boc-Glu(OBzl)-Ala-Arg-AMC as substrate measured for 30 mins by fluorometric assay
Measurement: IC50 = 8 nM (pChEMBL 8.100000381469727)
Descriptors: MW 521.97, ALogP 4.64, HBA 7, HBD 2, PSA 129.35, QED 0.32
Compound (SMILES): COc1ccc(C2(C(F)(F)F)CC(c3ccc(C)cc3)=C(C#N)C(=O)N2)cc1
InChIKey: JWJSLVALDGJEQA-UHFFFAOYSA-N
Target: Alpha-1,6-mannosyl-glycoprotein 2-beta-N-acetylglucosaminyltransferase (SINGLE PROTEIN), Homo sapiens
UniProt: Q10469
Assay: MGAT SPA Assay: MGAT2 enzyme was assayed using membranes isolated from Sf9 cells expressing the recombinant human MGAT2 cDNA with 2-monooleoylglycerol and [3H]-oleoyl-CoA as substrates as described by Seethala et al. [Anal. Biochem., 383(2):144-150 (Dec. 15, 2008)]. Briefly, the assays were conducted in 384-well plates in a total volume of 30 uL at 25° C. In each assay, 200 ng of recombinant human MGAT2 membrane was incubated with 10 uM of 2-monooleoylglycerol and 15 uM of [3H]-oleoyl-CoA in 100 mM potassium phosphate (pH 7.4) for 20 min with various concentrations of compounds delivered in DMSO. The assay was terminated by the addition of 20 ul of Stopping Solution (7.5 mg/ml Yttrium Oxide Polylysine beads, 3.3 mg/ml Fraction V BSA and 200 uM Mercuric chloride in 50 mM HEPES, pH 7.4). The signal was measured 1 h after quenching the reaction using LEADSEEKERSM for 5 minutes. To calculate the degree of inhibition, the zero level of enzyme activity (blank) was defined.
Measurement: IC50 = 774 nM (pChEMBL 6.110000133514404)
Descriptors: MW 386.37, ALogP 4.26, HBA 3, HBD 1, PSA 62.12, QED 0.86
Compound (SMILES): CCOC(C(=O)c1ccc(-c2cc(OC)c(Cl)c(OC)c2)o1)c1ccc(-c2nnc(C)s2)cc1
InChIKey: QQHJRLKSCSRMEM-UHFFFAOYSA-N
Target: cGMP-inhibited 3',5'-cyclic phosphodiesterase 3A (SINGLE PROTEIN), Homo sapiens
UniProt: Q14432
Assay: Inhibtion Assay: The PDE assay medium consisted of (final concentrations); 50 mM Tris-HCL, pH 7.5; 8.3 mM MgCl2; 1.7 mM EGTA; 0.5 mg/mL bovine serum albumin; and substrate (H-cAMP or H-cGMP). The BSA was lyophilized powder, essentially fatty acid free, >=96% pure from Sigma (A6003). 89 ul of assay medium and 1 ul of Compound 1 in 100% DMSO were added to a 96-well SPA plate and incubated for 1 minute at 30° C. The assay was initiated by addition of 10 ul of PDE10, and incubated for 21 minutes. Then 50 ul of SPA beads were added and the plate was sealed, shaken, and incubated at room temperature for 1 hour. The plate was then counted in a Wallac 1450 Microbeta Trilux plate scintillation counter. For the PDE10 IC50 experiment, the substrate was 125 nM H-cGMP. For the selectivity experiments, the substrates were 37 nM H-cAMP for PDEs 3, 4, 7, and 8; and 37 nM H-cGMP for PDEs 1, 2, 5, 9, 10, and 11. In all cases substrate concentrations were below the KM. The consumption of substrate was less than 6%, indicating that substrate concentrations did not change appreciably during the assay.
Measurement: IC50 > 10000 nM (pChEMBL 5)
Descriptors: MW 498.99, ALogP 6.4, HBA 8, HBD 0, PSA 83.68, QED 0.25
Compound (SMILES): CC(C)(CO)C(=O)N(CCN1[C@@H]2CC[C@H]1C[C@@H](c1cccc(C(N)=O)c1)C2)CC1CCCC1.O=C(O)C(F)(F)F
InChIKey: TUKISRANEWHWBZ-MGJLCPRGSA-N
Target: Mu-type opioid receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P35372
Assay: Displacement of [3H]-diprenorphine from human mu opioid receptor expressed in CHOK1 cell membranes
Measurement: Ki = 10 nM (pChEMBL 8)
Descriptors: MW 455.64, ALogP 3.53, HBA 4, HBD 2, PSA 86.87, QED 0.6
Compound (SMILES): CC[C@H](C)CN(Cc1nc2c(c(=O)[nH]1)COCC2)C(=O)CN1CCC(C(=O)c2ccc(F)cc2)CC1
InChIKey: DUYFEUQQISPFMM-SFHVURJKSA-N
Target: Poly [ADP-ribose] polymerase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P09874
Assay: Biochemical Assay: The autoparsylation activity of the TNKS 1/2 or PARP1/2 enzymes was measured by the liquid chromatography-mass spectrometry (LC/MS) detection of nicotinamide as readout. Compound activity in inhibiting the TNKS and PARP autoparsylation was evaluated by IC50 measurements. In the compound screening assays, the reaction is composed of 5 μL of compound in 8-point serial dilutions with concentrations ranging from 0.0086 to 18.75 μM, 20 nM of purified enzyme, and 250 μM of β-NAD+ in the 1× Assay Buffer. After 60 min incubation at room temperature, the reactions were quenched by the addition of 10 μL of 5× quenching solution (20% formic acid and 500 nM [d]-nicotinamide in water). For the background control wells, 10 μL of the 5× quenching solution per well was added prior to the addition of β-NAD+.
Measurement: IC50 > 19000 nM (pChEMBL 4.7210001945495605)
Descriptors: MW 498.6, ALogP 2.95, HBA 6, HBD 1, PSA 95.6, QED 0.53
Compound (SMILES): Cc1nc(C(=O)O)ccc1-c1nnc(SCCCN2CC[C@]3(C[C@@H]3c3ccc(C(F)(F)F)cc3)C2)n1C
InChIKey: FXIOLEMXUYMYEG-NLFFAJNJSA-N
Target: Voltage-gated inwardly rectifying potassium channel KCNH2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q12809
Assay: Inhibition of human ERG transfected in HEK293 cells assessed as reduction in tail current by patch clamp assay
Measurement: IC50 = 10000 nM (pChEMBL 5)
Descriptors: MW 531.6, ALogP 5.26, HBA 7, HBD 1, PSA 84.14, QED 0.31
Compound (SMILES): Nc1nnc(-c2ccc3[nH]cc(-c4cncc(N5CCOCC5)n4)c3c2)o1
InChIKey: JYUCZTGADIKZPX-UHFFFAOYSA-N
Target: Serine/threonine-protein kinase pim-3 (SINGLE PROTEIN), Homo sapiens
UniProt: Q86V86
Assay: Enzyme Assay: The assay for the determination of Pim activity is based on the formation of phosphorylated biotinylated-BAD peptide at the Serine 112 residue (S112) and employs HTRF® (homogeneous time resolved fluorescence) technology to detect the product in a 96-well plate format. The phosphorylation of biotinylated-BAD (S112) peptide by full length recombinant Pim-1, Pim-2, or Pim-3 protein was detected with streptavidin:Allophycocyanin (APC) conjugate and a europium (Eu) labeled antibody directed against phosphorylated-BAD (S112). Excitation of Eu by a high energy laser light (337 nm) leads to a transfer of energy to the APC molecule, and results in an emission at 665 nm. The fluorescence is directly proportional to the amount of phosphorylated BAD peptide present in the reaction.Compounds were prepared in DMSO by conducting 3-fold serial dilutions to give a 10-point dosing curve having a high dose of 1 uM. A reference compound was included on each assay plate in order to validate that plate; on
Measurement: IC50 = 6.455 nM (pChEMBL 8.1899995803833)
Descriptors: MW 363.38, ALogP 2.09, HBA 8, HBD 2, PSA 118.98, QED 0.57
Compound (SMILES): COc1cc(OC)cc(N(CC2CC2)c2ccc3ncc(-c4cnn(CC(O)CN5CCCCC5)c4)nc3c2)c1
InChIKey: BFQGJTMYIGHGLT-UHFFFAOYSA-N
Target: Vascular endothelial growth factor receptor 2 (SINGLE PROTEIN), Homo sapiens
UniProt: P35968
Assay: Inhibition of VEGFR2 (unknown origin) transfected in mouse BA/F3 cells assessed as decrease in cell proliferation in absence of mouse IL3 after 24 hrs by Alamar blue assay
Measurement: IC50 = 5754.4 nM (pChEMBL 5.239999771118164)
Descriptors: MW 542.68, ALogP 4.91, HBA 9, HBD 1, PSA 88.77, QED 0.29
Compound (SMILES): COc1nc2ccc(C(O)(c3ccccc3)C3CCN(C(C)=O)CC3)cc2c(Cl)c1Cc1ccc(-n2cccn2)cc1
InChIKey: QBIGUDRHHJTXKG-UHFFFAOYSA-N
Target: Nuclear receptor ROR-gamma (SINGLE PROTEIN), Homo sapiens
UniProt: P51449
Assay: ThermoFluor® Assay: Bar-coded assay plates are robotically loaded onto a thermostatically controlled PCR-type thermal block and then heated at a typical ramp-rate of 1 °C./min for all experiments. Fluorescence was measured by continuous illumination with UV light (Hamamatsu LC6) supplied via fiber optic and filtered through a band-pass filter (380-400 nm; >6 OD cutoff). Fluorescence emission of the entire 384-well plate was detected by measuring light intensity using a CCD camera (Sensys, Roper Scientific) filtered to detect 500±25 nm, resulting in simultaneous and independent readings of all 384 wells. Images were collected at each temperature, and the sum of the pixel intensity in a given area of the assay plate was recorded versus temperature. Reference wells contained RORγt without compounds, and the assay conditions were as follows: 0.065 mg/mL RORγt, 60 μM 1,8-ANS, 100 mM Hepes, pH 7.0, 10 mM NaCl, 2.5 mM GSH, 0.002% Tween-20. Project compounds were arranged in a pre-dosed mother plate (Greiner Bio-one) wherein compounds are serially diluted in 100% DMSO by 1:2 from a high concentration of 10 mM over 12 columns within a series (column 12 is a reference well containing DMSO, no compound). The compounds were robotically dispensed directly into assay plates (1×=46 nL) using a Hummingbird capillary liquid handling instrument (Digilab). Following compound dispense, protein and dye in buffer was added to achieve the final assay volume of 3 μL, followed by 1 μL of silicone oil.
Measurement: Kd = 77 nM (pChEMBL 7.110000133514404)
Descriptors: MW 581.12, ALogP 6.17, HBA 6, HBD 1, PSA 80.48, QED 0.25
Compound (SMILES): COc1cc(OC)cc(N(C[C@@H](O)C(F)(F)F)c2ccc3ncc(-c4cnn(CCCN)c4)nc3c2)c1
InChIKey: WRSYRDYMLSQTFT-XMMPIXPASA-N
Target: Vascular endothelial growth factor receptor 2 (SINGLE PROTEIN), Homo sapiens
UniProt: P35968
Assay: Inhibition of VEGFR2 (unknown origin) transfected in mouse BA/F3 cells assessed as decrease in cell proliferation in absence of mouse IL3 after 24 hrs by Alamar blue assay
Measurement: IC50 = 6606.93 nM (pChEMBL 5.179999828338623)
Descriptors: MW 516.52, ALogP 3.92, HBA 9, HBD 2, PSA 111.55, QED 0.33
Compound (SMILES): O=C(c1ccccc1-n1nccn1)N1CCOC[C@H]1Cc1cccc(-c2ncon2)c1
InChIKey: CSOMRAVZERRDSF-GOSISDBHSA-N
Target: Cytochrome P450 3A4 (SINGLE PROTEIN), Homo sapiens
UniProt: P08684
Assay: CYP3A4 Inhibition Assay: The CYP3A4 inhibition assay was performed using human liver microsomes and testosterone 6'-hydroxylation as a P450 isoform-specific marker. In a total volume of 150 uL, 14C-testosterone at a final concentration of 40 uM in a 100 mM phosphate buffer (pH 7.4) was incubated in a 96-well plate with 0.3 mg/mL of human liver microsomes in an Eppendorf thermomixer at 37° C. and 400 rpm. A 1.0 uL-aliquot of the 150-fold concentrated compound stock solution, prepared in DMSO, was added to yield final inhibitor concentrations of 0, 0.1, 0.5, 1.0, 5.0, 10, 25, and 50 uM. The reaction was initiated by addition of 15 uL of the NADPH-regenerating system containing the glucose-6-phosphate dehydrogenase and terminated after 7 min with a 75 uL-aliquot of methanol. After centrifugation at 465 g and 4° C. for 20 min, a 50 uL-aliquot of the supernatant was submitted to HPLC according to the method described below.
Measurement: IC50 > 50000 nM (pChEMBL 4.301000118255615)
Descriptors: MW 416.44, ALogP 2.4, HBA 8, HBD 0, PSA 99.17, QED 0.49
Compound (SMILES): Cn1cc(-c2ccc(F)c(C(F)(F)F)c2)nc1C1CCN(c2ncnc(N)c2C(N)=O)CC1
InChIKey: ODHFZINJDDSFIB-UHFFFAOYSA-N
Target: Voltage-gated inwardly rectifying potassium channel KCNH2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q12809
Assay: Inhibition of human ERG by patch clamp assay
Measurement: IC50 = 1900 nM (pChEMBL 5.71999979019165)
Descriptors: MW 463.44, ALogP 3.1, HBA 7, HBD 2, PSA 115.95, QED 0.57
Compound (SMILES): COc1cc(OC)cc(N(CC#Cc2cc(Cl)nc(N)n2)c2ccc3ncc(-c4cnn(C)c4)nc3c2)c1
InChIKey: XHSUXSHBEVITPC-UHFFFAOYSA-N
Target: Vascular endothelial growth factor receptor 2 (SINGLE PROTEIN), Homo sapiens
UniProt: P35968
Assay: Inhibition of VEGFR2 (unknown origin) transfected in mouse BA/F3 cells assessed as decrease in cell proliferation in absence of mouse IL3 after 24 hrs by Alamar blue assay
Measurement: IC50 > 10000 nM (pChEMBL 5)
Descriptors: MW 526.99, ALogP 4.26, HBA 10, HBD 1, PSA 117.1, QED 0.26
Compound (SMILES): O=c1c(O)cccc2ccc(O)c(O)c12
InChIKey: OLSYHLURTGEIBH-UHFFFAOYSA-N
Target: Cyclin-dependent kinase 2 (SINGLE PROTEIN), Homo sapiens
UniProt: P24941
Assay: Inhibition of 8-((4-chlorophenyl)amino)naphthalene-1-sulfonic acid binding to CDK2 (unknown origin) incubated for 2 hrs in presence of staurosporine by fluorescence based analysis
Measurement: IC50 = 9100 nM (pChEMBL 5.039999961853027)
Descriptors: MW 204.18, ALogP 1.32, HBA 4, HBD 3, PSA 77.76, QED 0.57
Compound (SMILES): COC(=O)Nc1ccc(-c2[nH]c([C@@H]3C[C@H](C4CCN(S(C)(=O)=O)CC4)CN3C(=O)C3CCN(C(=N)N)CC3)nc2Cl)cc1
InChIKey: HOTFNPNTKCEFJL-UNMCSNQZSA-N
Target: Coagulation factor X (SINGLE PROTEIN), Homo sapiens
UniProt: P00742
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 > 100000 nM (pChEMBL 4)
Descriptors: MW 635.19, ALogP 3.08, HBA 7, HBD 4, PSA 177.81, QED 0.28
Compound (SMILES): CC(=S)NCCCC[C@@H]1NC(=O)[C@H](Cc2csc3ccccc23)NC(=O)CCCCCCC(=O)NCCC[C@@H](C(N)=O)NC1=O
InChIKey: FMJMJQGMJXXPLJ-GSDHBNRESA-N
Target: NAD-dependent protein deacetylase sirtuin-3, mitochondrial (SINGLE PROTEIN), Homo sapiens
UniProt: Q9NTG7
Assay: Inhibition of recombinant human His6-tagged SIRT3 using H2N-HK-[Nepsilon-acetyl-lysine]-LM-COOH as substrate incubated for 10 mins in presence of beta-NAD+ by HPLC based assay
Measurement: IC50 = 3180 nM (pChEMBL 5.5)
Descriptors: MW 658.89, ALogP 2.74, HBA 7, HBD 6, PSA 171.52, QED 0.19
Compound (SMILES): CC(C)(C)C(=O)OCOC(=O)C1(C/C=C/c2ccccc2)/C(=C/C=O)O[C@@H]2CC(=O)N21
InChIKey: RTZMBJMWCNEYEE-HUCLUGSFSA-N
Target: Beta-lactamase TEM (SINGLE PROTEIN), Bacteria
UniProt: P62593
Assay: Cell Free Inhibition Assay: The inhibitory concentrations (IC50, [uM]) of the beta -lactamase inhibitors against purified TEM-1, SHV-1 and AmpC beta -lactamases are assessed by determining the concentration of inhibitor at which 50% of the nitrocefin hydrolysis is inhibited by the particular enzyme. Assays are performed with beta -lactamases expressed in the pET system (Novagen, San Diego, Calif.) without signal peptides. They contain an N-terminal hexa-Histidine tag that is used for purification on Ni-NTA (Qiagen, Hilden, Germany). The compounds are prepared as 50 mM stocks in DMSO and diluted into buffer P1 (50 mM phosphate, pH 7) to a final concentration of 10% DMSO. All further dilutions are done in P2 (P1 with 10% DMSO). Enzyme and compound dilutions are pre-incubated for 10 min at 37 C. and the reaction is started with the addition of pre-warmed (37 C.) nitrocefin at a final concentration of 50 mM. The change in absorption at 490 nm is followed at 37 C. for 10 min with 30 s intervals.
Measurement: IC50 = 870.6 nM (pChEMBL 6.059999942779541)
Descriptors: MW 427.45, ALogP 2.59, HBA 7, HBD 0, PSA 99.21, QED 0.22
Compound (SMILES): C[C@H](Nc1ncnc(N)c1C#Cc1ccc(F)cn1)c1nc2c(Cl)ccc(Cl)c2c(=O)n1-c1ccccc1
InChIKey: IDDSEYKZFGQZJQ-HNNXBMFYSA-N
Target: Phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit delta isoform (SINGLE PROTEIN), Homo sapiens
UniProt: O00329
Assay: Time-Resolved Fluorescence Resonance Energy Transfer (TR-FRET) Assay: PI3K isoforms were assayed under initial rate conditions in the presence of 25 mM Hepes (pH 7.4), and 2xKm ATP (100-300 uM), 10 uM PIP2, 5% glycerol, 5 mM MgCl2, 50 mM NaCl, 0.05% (v/v) Chaps, 1 mM dithiothreitol, 1% (v/v) DMSO at the following concentrations for each isoform: PI3K alpha, beta, delta, and gamma at 50 picomolar (pM) and PI3Kgamma at 2 nanomolar (nM). After an assay reaction time of 30 minutes at 25° C., reactions were terminated with a final concentration of 10 mM EDTA, 10 nM labeled-PIP3, and 35 nM Europium labeled GRP-1 detector protein before reading TR-FRET on an Envision plate reader (Ex: 340 nm; Em: 615/665 nm; 100 us delay and 500 us read window).
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 546.39, ALogP 5.17, HBA 8, HBD 2, PSA 111.61, QED 0.3
Compound (SMILES): COc1cccnc1C#Cc1c(N)ncnc1N[C@@H](C)c1nc2cccc(Cl)c2c(=O)n1-c1ccccc1
InChIKey: NNKJDDUDVQCZJC-KRWDZBQOSA-N
Target: Phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit delta isoform (SINGLE PROTEIN), Homo sapiens
UniProt: O00329
Assay: Time-Resolved Fluorescence Resonance Energy Transfer (TR-FRET) Assay: PI3K isoforms were assayed under initial rate conditions in the presence of 25 mM Hepes (pH 7.4), and 2xKm ATP (100-300 uM), 10 uM PIP2, 5% glycerol, 5 mM MgCl2, 50 mM NaCl, 0.05% (v/v) Chaps, 1 mM dithiothreitol, 1% (v/v) DMSO at the following concentrations for each isoform: PI3K alpha, beta, delta, and gamma at 50 picomolar (pM) and PI3Kgamma at 2 nanomolar (nM). After an assay reaction time of 30 minutes at 25° C., reactions were terminated with a final concentration of 10 mM EDTA, 10 nM labeled-PIP3, and 35 nM Europium labeled GRP-1 detector protein before reading TR-FRET on an Envision plate reader (Ex: 340 nm; Em: 615/665 nm; 100 us delay and 500 us read window).
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 523.98, ALogP 4.39, HBA 9, HBD 2, PSA 120.84, QED 0.33
Compound (SMILES): COc1ccc(CNc2nc(N3CC4CC4C3)ncc2C(=O)NCc2ccccc2)cc1Cl
InChIKey: QFAMNDUERIPOEM-UHFFFAOYSA-N
Target: Rod cGMP-specific 3',5'-cyclic phosphodiesterase subunit alpha (SINGLE PROTEIN), Bos taurus
UniProt: P11541
Assay: Enzyme Assay: Method: The test compound or solvent was incubated at 25° C. for 15 min with enzyme solution (0.2 μg/mL) in Tris-HCl buffer of pH 7.5. 100 μM cGMP and 0.03 μM [3H] cGMP were then added to activate the reaction. The mixture was incubated for 20 min. The reaction was stopped at 100° C. By addition of snake venom nucleotidase, the product [3H] GMP was converted into [3H] Guanosine, which was separated with AG1-X2 resin. The amount of [3H] Guanosine was measured.
Measurement: IC50 = 8.89 nM (pChEMBL 8.050000190734863)
Descriptors: MW 463.97, ALogP 4.14, HBA 6, HBD 2, PSA 79.38, QED 0.52
Compound (SMILES): CCc1nsc(-c2nnc3n2CCN(C(=O)c2ccc(F)cc2)C3C)n1
InChIKey: UOMUYZXEONWAOD-UHFFFAOYSA-N
Target: Substance-P receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P25103
Assay: Binding Assay: The affinity of compounds of the invention for the NK-1 receptor was evaluated in CHO recombinant cells which express the human NK-1 receptor. Membrane suspensions were prepared from these cells. The following radioligand: [3H] substance P (PerkinElmer Cat#NET111520) was used in this assay. Binding assays were performed in a 50 mM Tris/5 mM MnCl2/150 mM NaCl/0.1% BSA at pH 7.4. Binding assays consisted of 25 μl of membrane suspension (approximately 5 μg of protein/well in a 96 well plate), 50 μl of compound or reference ligand (Substance P) at increasing concentrations (diluted in assay buffer) and 2 nM [3H] substance P. The plate was incubated 60 min at 25° C. in a water bath and then filtered over GF/C filters (Perkin Elmer, 6005174, presoaked in 0.5% PEI for 2 h at room temperature) with a Filtration unit (Perkin Elmer). The radioactivity retained on the filters was measured by using the TopCount-NXT reader (Packard).
Measurement: Ki > 30000 nM (pChEMBL 4.5229997634887695)
Descriptors: MW 372.43, ALogP 2.72, HBA 7, HBD 0, PSA 76.8, QED 0.71
Compound (SMILES): Cc1ccnc(-n2nnc3c2CCN(C(=O)c2cccc(Cl)c2Cl)C3C)c1
InChIKey: YMZRPAMOYRNYTO-UHFFFAOYSA-N
Target: P2X purinoceptor 7 (SINGLE PROTEIN), Rattus norvegicus
UniProt: Q64663
Assay: FLIPR Assay: 1321 N1 cells expressing the recombinant human, rat or mouse P2X7 channel was cultured in HyQ DME/(HyClone/Dulbecco's Modified Eagle Medium) high glucose supplemented with 10% Fetal Bovine Serum (FBS) and appropriate selection marker. Cells were seeded at a density of 25000 cells/well (96-well clear bottom black walled plates) in 100 ul volume/well. On the day of the experiment, cell plates were washed with assay buffer, containing (in mM): 130 NaCl, 2 KCl, 1 CaCl2, 1 MgCl2, 10 HEPES, 5 glucose; pH 7.40 and 300 mOs. After the wash, cells were loaded with the Calcium-4 dye (Molecular Device) and incubated in the dark for 60 minutes. Test compounds were prepared at 250Ã¿ the test concentration in neat DMSO. Intermediate 96-well compound plates were prepared by transferring 1.2 uL of the compound into 300 uL of assay buffer. A further 3x dilution occurred when transferring 50 uL/well of the compound plate to 100 uL/well in the cell plate. Cells were incubateÂ  with test compounds and dye for 30 minutes. Calcium dye fluorescence was monitored in FLIPR as the cells were challenged by adding 50 uL/well of BzATP (final concentration is 250 uM BzATP (human and rat) or 600 (mouse)). The fluorescence change was measured 180 seconds after adding the agonist.
Measurement: IC50 = 127.6 nM (pChEMBL 6.889999866485596)
Descriptors: MW 402.29, ALogP 4.04, HBA 5, HBD 0, PSA 63.91, QED 0.65
Compound (SMILES): N#Cc1ccc(Oc2ccc3nc(C(=O)N4CCNCC4)ccc3c2)cc1
InChIKey: BZEQTXGNCCKNKH-UHFFFAOYSA-N
Target: Voltage-gated inwardly rectifying potassium channel KCNH2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q12809
Assay: Inhibition of hERG channel by electrophysiology
Measurement: IC50 = 5200 nM (pChEMBL 5.28000020980835)
Descriptors: MW 358.4, ALogP 2.94, HBA 5, HBD 1, PSA 78.25, QED 0.78
Compound (SMILES): CO[C@H]1CC[C@]2(CC1)Cc1ccc(NC(=O)c3ccc(F)cn3)cc1C21N=C(C)C(N)=N1
InChIKey: XGZLGNFGLZOBER-SCRAKJMHSA-N
Target: Amyloid-beta precursor protein (SINGLE PROTEIN), Homo sapiens
UniProt: P05067
Assay: sAPPbeta Release Assay: SH-SY5Y cells were cultured in DMEM/F-12 with Glutamax, 10% FCS and 1% non-essential amino acids and cryopreserved and stored at −140° C. at a concentration of 7.5-9.5×106 cells per vial. Thaw cells and seed at a conc. of around 10000 cells/well in DMEM/F-12 with Glutamax, 10% FCS and 1% non-essential amino acids to a 384-well tissue culture treated plate, 100 μL cell susp/well. The cell plates were then incubated for 7-24 h at 37° C., 5% CO2. The cell medium was removed, followed by addition of 30 μL compound diluted in DMEM/F-12 with Glutamax, 10% FCS, 1% non-essential amino acids and 1% PeSt to a final conc. of 1% DMSO. The compounds were incubated with the cells for 17 h (overnight) at 37° C., 5% CO2. Meso Scale Discovery (MSD) plates were used for the detection of sAPPβ release. MSD sAPPβ plates were blocked in 1% BSA in Tris wash buffer (40 μL/well) for 1 h on shake at r.t. and washed 1 time in Tris wash buffer (40 μL/well). 20 μL of medium was transferred to the pre-blocked and washed MSD sAPPβ microplates, and the cell plates were further used in an ATP assay to measure cytotoxicity. The MSD plates were incubated with shaking at r.t. for 2 h and the media discarded. 10 μL detection antibody was added (1 nM) per well followed by incubation with shaking at r.t. for 2 h and then discarded. 40 μL Read Buffer was added per well and the plates were read in a SECTOR Imager.
Measurement: IC50 = 29 nM (pChEMBL 7.539999961853027)
Descriptors: MW 435.5, ALogP 3.59, HBA 6, HBD 2, PSA 101.96, QED 0.77
Compound (SMILES): Cc1cccc2cc(Cn3nc(-c4cccc(Cl)c4)c4c(N)ncnc43)n(-c3ccccc3F)c(=O)c12
InChIKey: PYKOOVIJONGULF-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 = 10000 nM (pChEMBL 5)
Descriptors: MW 510.96, ALogP 5.53, HBA 7, HBD 1, PSA 91.62, QED 0.34
Compound (SMILES): O=S(=O)(NCC1CC1)c1ccc(NC2CC3CCC2C3)cc1C(F)(F)F
InChIKey: FJHVEQHZHSLVPQ-UHFFFAOYSA-N
Target: Cannabinoid receptor 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P21554
Assay: Binding Assay: The binding assays were performed by testing the ability of the new compounds to displace the radiolabeled synthetic non-selective cannabinoid agonist [3H]CP55940 (168 Ci/mmol; PerkinElmer) from the human CB1 (hCB1) or human CB2 (hCB2) receptors on membranes derived from stably transfected HEK-293 cells (PerkinElmer). Membranes were diluted in assay buffer (50 mM Tris-HCl, 1 mM EDTA, 1 mM MgCl2, 1 mg/ml BSA, pH=7.4). The amount of membrane was determined for each batch of membranes according to protein binding assay. The minimum amount of membrane that gave 50% specific binding was used for the binding assay. In most assays, binding was tested using 8 μg and 4 μg protein of hCB1 and hCB2 membranes, respectively. The tested compounds were dissolved in DMSO and diluted in assay buffer to a final concentration of 0.1% solvent. Total binding of [3H]CP55940 was evaluated with 1.5 nM to hCB1 and with 0.5 nM to hCB2, according to Kd affinity of [3H]CP55940 for the respective membranes. The ability of the tested compounds to displace [3H]CP55940 was evaluated first at a single concentration point of 100 nM for binding toward hCB2 or hCB1. In certain cases, the displacement was tested at compound concentrations ranging from 0.03 nM to 10 μM. Non-specific binding was measured by the addition of 1 μM of unlabelled CP55940 to the tubes. Binding assays were performed in triplicate in a total volume of 500 μl for 60 minutes at 30° C., in a shaking bath. Free and bound radioligands were separated by rapid filtration through GF/C filter plates (PerkinElmer) that had been presoaked with 0.1% Polyethylenimine (Sigma). Filters were shaken for 1 hour in 7 ml scintillation fluid (PerkinElmer) and radioactivity was determined by liquid scintillation counter (Wallac; PerkinElmer).
Measurement: Ki = 2720 nM (pChEMBL 5.570000171661377)
Descriptors: MW 388.46, ALogP 3.99, HBA 3, HBD 2, PSA 58.2, QED 0.77
Compound (SMILES): CC1c2ncn(-c3cnccn3)c2C=CN1C(=O)c1cccc(C(F)(F)F)c1Cl
InChIKey: PHBHCPMOVBUSRC-UHFFFAOYSA-N
Target: P2X purinoceptor 7 (SINGLE PROTEIN), Rattus norvegicus
UniProt: Q64663
Assay: FLIPR Assay: 1321 N1 cells expressing the recombinant human, rat or mouse P2X7 channel was cultured in HyQ DME/(HyClone/Dulbecco's Modified Eagle Medium) high glucose supplemented with 10% Fetal Bovine Serum (FBS) and appropriate selection marker. Cells were seeded at a density of 25000 cells/well (96-well clear bottom black walled plates) in 100 ul volume/well. On the day of the experiment, cell plates were washed with assay buffer, containing (in mM): 130 NaCl, 2 KCl, 1 CaCl2, 1 MgCl2, 10 HEPES, 5 glucose; pH 7.40 and 300 mOs. After the wash, cells were loaded with the Calcium-4 dye (Molecular Device) and incubated in the dark for 60 minutes. Test compounds were prepared at 250Ã¿ the test concentration in neat DMSO. Intermediate 96-well compound plates were prepared by transferring 1.2 uL of the compound into 300 uL of assay buffer. A further 3x dilution occurred when transferring 50 uL/well of the compound plate to 100 uL/well in the cell plate. Cells were incubateÂ  with test compounds and dye for 30 minutes. Calcium dye fluorescence was monitored in FLIPR as the cells were challenged by adding 50 uL/well of BzATP (final concentration is 250 uM BzATP (human and rat) or 600 (mouse)). The fluorescence change was measured 180 seconds after adding the agonist.
Measurement: IC50 = 398.1 nM (pChEMBL 6.400000095367432)
Descriptors: MW 419.79, ALogP 4.52, HBA 5, HBD 0, PSA 63.91, QED 0.61
Compound (SMILES): O=C(O)C1CN(Cc2ccc3c(c2)CCc2c-3n[nH]c2-c2onc(-c3ccccc3)c2C(F)(F)F)C1
InChIKey: NOCZLXSAZCFIBP-UHFFFAOYSA-N
Target: Sphingosine 1-phosphate receptor 3 (SINGLE PROTEIN), Homo sapiens
UniProt: Q99500
Assay: GTPgammaS Binding Assay: Compounds were loaded in a 384 Falcon v-bottom plate (0.5 ul/well in a 11 point, 3-fold dilution). Membranes prepared from S1P1/CHO cells or EDG3-Ga15-bla HEK293T cells were added to the compound plate (40 ul/well, final protein 3 ug/well) with multidrop. [35S]GTP (1250 Ci/mmol, Perkin Elmer) was diluted in assay buffer: 20 mM HEPES, pH7.5, 10 mM MgCl2, 150 mM NaCl, 1 mM EGTA, 1 mM DTT, 10 uM GDP, 0.1% fatty acid free BSA, and 10 ug/ml Saponin to 0.4 nM. 40 ul of the [35S] GTP solution was added to the compound plate with a final concentration of 0.2 nM. The reaction was kept at room temperature for 45 min. At the end of incubation, all the mixtures in the compound plate were transferred to Millipore 384-well FB filter plates via the Velocity11 Vprep liquid handler. The filter plate was washed with water 4 times by using the manifold Embla plate washer and dried at 60° C. for 45 min. MicroScint 20 scintillation fluid (30 u) was added to each well.
Measurement: EC50 > 63000 nM (pChEMBL 4.201000213623047)
Descriptors: MW 494.47, ALogP 5.03, HBA 5, HBD 2, PSA 95.25, QED 0.4
Compound (SMILES): COc1cc(OC)cc(N(CC#Cc2nccnc2Cl)c2ccc3ncc(-c4cnn(C)c4)nc3c2)c1
InChIKey: XWGYUZGILVRSRS-UHFFFAOYSA-N
Target: Vascular endothelial growth factor receptor 2 (SINGLE PROTEIN), Homo sapiens
UniProt: P35968
Assay: Inhibition of VEGFR2 (unknown origin) transfected in mouse BA/F3 cells assessed as decrease in cell proliferation in absence of mouse IL3 after 24 hrs by Alamar blue assay
Measurement: IC50 = 3311.31 nM (pChEMBL 5.480000019073486)
Descriptors: MW 511.97, ALogP 4.68, HBA 9, HBD 0, PSA 91.08, QED 0.3
Compound (SMILES): COc1cc(N2CCN(C(C)C)CC2)ccc1Nc1nccc(-c2c(-c3cccc(C(=O)Nc4c(F)cccc4F)c3)nc3ccccn23)n1
InChIKey: AAYJEKJTXVXYTO-UHFFFAOYSA-N
Target: Epidermal growth factor receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P00533
Assay: Inhibition of recombinant human full length EGFR expressed in baculovirus expression system assessed as decrease in transfer of gamma-phosphate from ATP onto tyrosine residue of biotinylated peptide substrate preincubated for 1 hr followed by substrate addition measured after 90 mins by HTRF method
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 674.76, ALogP 7.27, HBA 9, HBD 2, PSA 99.92, QED 0.17
Compound (SMILES): CN1CN([C@H]2CC[C@H](C(F)F)CC2)c2c(cnc3[nH]ccc23)C1=O
InChIKey: CSRWBCFTYXBWIL-XYPYZODXSA-N
Target: Tyrosine-protein kinase JAK3 (SINGLE PROTEIN), Homo sapiens
UniProt: P52333
Assay: Enzyme Assay: The inhibitory activity of the compounds of the present invention against JAK was measured.The respective enzymes (JAK1, JAK2, JAK3 and Tyk2) were purchased from Carna Biosciences, Inc.As a substrate of the enzymes (hereinafter referred to as substrate), LANCE Ultra ULight-JAK-1 (Tyr1023) Peptide (manufactured by PerkinElmer Inc.) was used.As an antibody for detecting phosphorylation of the substrate, LANCE Ultra Europium-anti-phospho tyrosine antibody (PT66) (manufactured by PerkinElmer Inc.) was used.The other reagents were purchased from the following.Adenosine triphosphate (ATP): Sigma-Aldrich4-(2-Hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES): DOJINDO LABORATORIESGlycol ether diamine tetraacetic acid (EGTA): DOJINDO LABORATORIES Magnesium chloride (MgCl2): Wako Pure Chemical Industries, Ltd.Dithiothreitol (DTT): Wako Pure Chemical Industries, Ltd.Tween 20: Sigma-AldrichEthylenediaminetetraacetic acid (EDTA): DOJINDO LABORATORIESThe compounds of the present invention, the enzymes (JAK1, JAK2, JAK3 and Tyk2), the substrate and ATP were used for assay as diluted with assay buffer.Â  As the assay buffer, one having the following composition was used.Â  HEPES (pH7.5): 50 mM EGTA: 1 mM MgCl2: 10 mM DTT: 2 mM Tween 20: 0.01% (wt/wt). The dilute concentration and the addition amount on a well plate as described hereinafter were adjusted so that the following final concentrations were achieved on the well plate.
Measurement: IC50 = 32 nM (pChEMBL 7.5)
Descriptors: MW 334.37, ALogP 3.24, HBA 3, HBD 1, PSA 52.23, QED 0.92
Compound (SMILES): Cn1cc(-c2ccc3c(c2)ncn3-c2cc(NS(=O)(=O)C3CC3)cc(-c3ccc(F)cc3F)c2)cn1
InChIKey: ZJFCBQXPTQSTCZ-UHFFFAOYSA-N
Target: Fibroblast growth factor receptor 2 (SINGLE PROTEIN), Homo sapiens
UniProt: P21802
Assay: Inhibition of recombinant FGFR2 (unknown origin) in the presence of [p33]-ATP
Measurement: IC50 = 16 nM (pChEMBL 7.800000190734863)
Descriptors: MW 505.55, ALogP 5.28, HBA 6, HBD 1, PSA 81.81, QED 0.34
Compound (SMILES): C=CC(=O)Nc1cccc(-c2cnc3[nH]cc(Cl)c3c2)n1
InChIKey: SCGPRWAOEPYMGB-UHFFFAOYSA-N
Target: Tyrosine-protein kinase ITK/TSK (SINGLE PROTEIN), Homo sapiens
UniProt: Q08881
Assay: Biochemical Inhibition Assay: 1H-pyrrolo[2,3-b]pyridine, 1H-pyrazolo[3,4-b]pyridine and 5H-pyrrolo[2,3-b]pyrazine series of compounds exhibited over >100 fold selective against Janus and TEC family of kinases except for the BMX and TXK it had activity of 30 and 45 nM respectively.
Measurement: IC50 = 30 nM (pChEMBL 7.519999980926514)
Descriptors: MW 298.73, ALogP 3.4, HBA 3, HBD 2, PSA 70.67, QED 0.73
Compound (SMILES): Cc1cccc2c1C(c1ccc3ccccc3c1)CN(C)C2
InChIKey: JSHASAWQPDVBRE-UHFFFAOYSA-N
Target: Sodium-dependent serotonin transporter (SINGLE PROTEIN), Homo sapiens
UniProt: P31645
Assay: Binding Assay: In order to evaluate the relative affinity of the various compounds at the NE, DA and 5HT transporters, HEK293E cell lines were developed to express each of the three human transporters. cDNAs containing the complete coding regions of each transporter were amplified by PCR from human brain libraries. The cDNAs contained in pCRII vectors were sequenced to verify their identity and then subcloned into an Epstein-Barr virus based expression plasmid (Shen et al., Gene 156:235-239 (1995), which is hereby incorporated by reference in its entirety). This plasmid containing the coding sequence for one of the human transporters was transfected into HEK93E cells. Successful transfection was verified by the ability of known reuptake blockers to inhibit the uptake of tritiated NE, DA or 5HT.For binding, cells were homogenized, centrifuged, and then resuspended in incubation buffer (5 mM Tris, 120 mM NaCl, 5 mM KCl, pH 7.4). Then, the appropriate radioligand was added.
Measurement: Ki = 3.4 nM (pChEMBL 8.470000267028809)
Descriptors: MW 287.41, ALogP 4.73, HBA 1, HBD 0, PSA 3.24, QED 0.63
Compound (SMILES): O=C(O)c1ccc(C2CCN(c3cc(OC(F)(F)F)ccc3F)CC2)cc1
InChIKey: FVEGBGXNQWDPFY-UHFFFAOYSA-N
Target: Free fatty acid receptor 4 (SINGLE PROTEIN), Homo sapiens
UniProt: Q5NUL3
Assay: Agonist activity at human GPR120 assessed as induction of IP1 accumulation
Measurement: EC50 > 10000 nM (pChEMBL 5)
Descriptors: MW 383.34, ALogP 4.81, HBA 3, HBD 1, PSA 49.77, QED 0.77
Compound (SMILES): COc1ccc(C(=O)C2CCN([C@@H]3CCN(CC(=O)Nc4nnc5n4CCS5)C3=O)CC2)cc1
InChIKey: PADREXIRIZBNMQ-GOSISDBHSA-N
Target: Poly [ADP-ribose] polymerase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P09874
Assay: Biochemical Assay: The autoparsylation activity of the TNKS 1/2 or PARP1/2 enzymes was measured by the liquid chromatography-mass spectrometry (LC/MS) detection of nicotinamide as readout. Compound activity in inhibiting the TNKS and PARP autoparsylation was evaluated by IC50 measurements. In the compound screening assays, the reaction is composed of 5 uL of compound in 8-point serial dilutions with concentrations ranging from 0.0086 to 18.75 uM, 20 nM of purified enzyme, and 250 uM of p-NAD+ in the 1x Assay Buffer. After 60 min incubation at room temperature, the reactions were quenched by the addition of 10 uL of 5x quenching solution (20% formic acid and 500 nM [d]-nicotinamide in water). For the background control wells, 10 uL of the 5x quenching solution per well was added prior to the addition of p-NAD+. The % Inhibition was calculated as: (Control-Sample)/(Control-Background)*100. "Control" is the average value of 8 wells without compound; and "Background" is the average of 8 wells mixed with 5x quenching solution measured prior to initiation of the reaction.
Measurement: IC50 > 19000 nM (pChEMBL 4.7210001945495605)
Descriptors: MW 484.58, ALogP 1.53, HBA 9, HBD 1, PSA 109.66, QED 0.59
Compound (SMILES): CC1CCN(c2cncc(-c3c[nH]c4ccc(-c5nnc(N)s5)cc34)n2)CC1
InChIKey: IIFMWBFXGMFSOH-UHFFFAOYSA-N
Target: Serine/threonine-protein kinase pim-3 (SINGLE PROTEIN), Homo sapiens
UniProt: Q86V86
Assay: Enzyme Assay: The assay for the determination of Pim activity is based on the formation of phosphorylated biotinylated-BAD peptide at the Serine 112 residue (S112) and employs HTRF® (homogeneous time resolved fluorescence) technology to detect the product in a 96-well plate format. The phosphorylation of biotinylated-BAD (S112) peptide by full length recombinant Pim-1, Pim-2, or Pim-3 protein was detected with streptavidin:Allophycocyanin (APC) conjugate and a europium (Eu) labeled antibody directed against phosphorylated-BAD (S112). Excitation of Eu by a high energy laser light (337 nm) leads to a transfer of energy to the APC molecule, and results in an emission at 665 nm. The fluorescence is directly proportional to the amount of phosphorylated BAD peptide present in the reaction.Compounds were prepared in DMSO by conducting 3-fold serial dilutions to give a 10-point dosing curve having a high dose of 1 uM. A reference compound was included on each assay plate in order to validate that plate; on
Measurement: IC50 = 8.537 nM (pChEMBL 8.069999694824219)
Descriptors: MW 391.5, ALogP 3.96, HBA 7, HBD 2, PSA 96.61, QED 0.55
Compound (SMILES): O=C(CN[C@H]1CC[C@H](c2nnn3cnc4[nH]ccc4c23)CC1)NCC(F)(F)F
InChIKey: XNSIBQZZWITRMV-XYPYZODXSA-N
Target: Tyrosine-protein kinase JAK3 (SINGLE PROTEIN), Homo sapiens
UniProt: P52333
Assay: Enzyme Assay: JAK1, JAK2, JAK3 and Tyk2 were purchased from Carna Biosciences, Inc. As the substrate, LANCE Ultra ULight-JAK1 Peptide (manufactured by PerkinElmer Co., Ltd. (PE)) was used. Dilute solutions of compounds and enzymes in assay buffer (50 mM HEPES pH7.5, 1 mM EGTA, 1 mM MgCl2, 2 mM DTT, 0.01% Tween20) were dispensed into wells of a 384-well black plate. After 5 minutes of preincubation, dilute solutions of the substrate and ATP (adenosine triphosphate) were added at a final concentration of 100 μM, and the plate was incubated at room temperature for 2 hours. After addition of a termination reagent containing EDTA (ethylenediamine tetraacetic acid) at a final concentration of 10 mM, LANCE Eu-W1024 Anti-phosphotyrosine (PT66) (manufactured by PE) was added, and after 1 hour of incubation, the fluorescences were measured with ARVO-HTS.
Measurement: IC50 > 1000 nM (pChEMBL 6)
Descriptors: MW 395.39, ALogP 1.9, HBA 6, HBD 3, PSA 100, QED 0.61
Compound (SMILES): Nc1nnc(-c2ccc3[nH]cc(-c4nccc(-c5ccccc5)n4)c3c2)o1
InChIKey: UYWOVNUCOZQXAD-UHFFFAOYSA-N
Target: Serine/threonine-protein kinase pim-3 (SINGLE PROTEIN), Homo sapiens
UniProt: Q86V86
Assay: Enzyme Assay: The assay for the determination of Pim activity is based on the formation of phosphorylated biotinylated-BAD peptide at the Serine 112 residue (S112) and employs HTRF® (homogeneous time resolved fluorescence) technology to detect the product in a 96-well plate format. The phosphorylation of biotinylated-BAD (S112) peptide by full length recombinant Pim-1, Pim-2, or Pim-3 protein was detected with streptavidin:Allophycocyanin (APC) conjugate and a europium (Eu) labeled antibody directed against phosphorylated-BAD (S112). Excitation of Eu by a high energy laser light (337 nm) leads to a transfer of energy to the APC molecule, and results in an emission at 665 nm. The fluorescence is directly proportional to the amount of phosphorylated BAD peptide present in the reaction.Compounds were prepared in DMSO by conducting 3-fold serial dilutions to give a 10-point dosing curve having a high dose of 1 uM. A reference compound was included on each assay plate in order to validate that plate; on
Measurement: IC50 = 2.397 nM (pChEMBL 8.619999885559082)
Descriptors: MW 354.37, ALogP 3.92, HBA 6, HBD 2, PSA 106.51, QED 0.51
Compound (SMILES): N#CCC1(n2cc(-c3ncnc4[nH]ccc34)cn2)CN(C2CCN(C(=O)c3sc4cc(F)ccc4c3Cl)CC2)C1
InChIKey: GPKMLSFODGOOPI-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 575.07, ALogP 5.06, HBA 8, HBD 1, PSA 106.73, QED 0.31
Compound (SMILES): C[C@@H](NC(=O)c1ccc2c(=O)n(-c3ccc(F)cc3)c(CCCCC(=O)O)cc2c1)c1ccc(F)cc1
InChIKey: NXJVSPCZMLVTEC-GOSISDBHSA-N
Target: Prostaglandin D2 receptor 2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9Y5Y4
Assay: Antagonist activity at CRTh2 (unknown origin) assessed as inhibition of CD11b activation
Measurement: Ki = 0.23 nM (pChEMBL 9.640000343322754)
Descriptors: MW 504.53, ALogP 5.56, HBA 4, HBD 2, PSA 88.4, QED 0.29
Compound (SMILES): CCN1CCN(C2CCN(C(=O)COc3ccc(-c4ccccc4)cc3)CC2)CC1
InChIKey: BODMOCLWYAYDCG-UHFFFAOYSA-N
Target: Acetylcholinesterase (SINGLE PROTEIN), Homo sapiens
UniProt: P22303
Assay: Inhibition of human recombinant AChE using acetylthiocholine iodide as substrate measured over 60 secs by Ellman assay
Measurement: IC50 = 31000 nM (pChEMBL 4.510000228881836)
Descriptors: MW 407.56, ALogP 3.36, HBA 4, HBD 0, PSA 36.02, QED 0.74
Compound (SMILES): N#CCC1(n2cc(-c3ncnc4[nH]ccc34)cn2)CN(C2CCN(C(=O)c3c(F)cc(F)cc3F)CC2)C1
InChIKey: WWYPEOYWKHWVHB-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 520.52, ALogP 3.47, HBA 7, HBD 1, PSA 106.73, QED 0.43
Compound (SMILES): CCCC[C@H](NC(C)=O)C(=O)N[C@H]1CCC(=O)NCC[C@@H](C(=O)N[C@@H](C)C(N)=O)NC(=O)[C@H](CCCNC(=N)N)NC(=O)[C@@H](Cc2ccccc2)NC(=O)[C@H](Cc2c[nH]cn2)NC1=O
InChIKey: OPKOWPIJJAPYJZ-ZYELEKFWSA-N
Target: Melanocyte-stimulating hormone receptor (SINGLE PROTEIN), Homo sapiens
UniProt: Q01726
Assay: cAMP Assay: Accumulation of intracellular cAMP was examined as a measure of the ability of the peptides of the present invention to elicit a functional response in a human melanoma cell line, HBL, that express hMCR-1 (see Kang, L., et al., A selective small molecule agonist of melanocortin-1 receptor inhibits lipopolysaccharide-induced cytokine accumulation and leukocyte infiltration in mice, J. Leuk. Biol. 80:897-904 (2006)) or HEK-293 cells that express hMCR-4. Confluent HBL cells that express hMCR-1 or HEK-293 cells that express recombinant hMCR-4 were detached from culture plates by incubation in enzyme-free cell dissociation buffer. Dispersed cells were suspended in Earle's Balanced Salt Solution containing 10 mM HEPES (pH 7.5), 1 mM MgCl2, 1 mM glutamine, 0.5% albumin and 0.3 mM 3-isobutyl-1-methyl-xanthine (IBMX), a phosphodiesterase inhibitor. The cells were plated in 96-well plates at a density of 0.4Ã¿105 cells per well for HBL cells and 0.5x105 cells per well for HEK-293 cells and pre-incubated for 10 minutes. Cells were exposed for 15 minutes at 37° C. to peptides of the present invention dissolved in DMSO (final DMSO concentration of 1%) at a concentration range of 0.05-5000 nM in a total assay volume of 200 uL. NDP-α-MSH was used as the reference agonist. cAMP levels were determined by an HTRF cAMP cell-based assay system from Cisbio Bioassays utilizing cryptate-labeled anti-cAMP and d2-labeled cAMP, with plates read on a Perkin-Elmer Victor plate reader at 665 and 620 nM. Data analysis was performed by nonlinear regression analysis with Graph-Pad Prism software. Maximum efficacy (Emax) values were determined for each test peptide of the present invention, compared to that achieved by the reference melanocortin agonist NDP-α-MSH.
Measurement: EC50 = 0.08 nM (pChEMBL 10.100000381469727)
Descriptors: MW 895.04, ALogP -3.13, HBA 11, HBD 13, PSA 366.47, QED 0.04
Compound (SMILES): CC[C@]1(C(=O)N2CC[C@](c3ccc(C(F)(C(F)(F)F)C(F)(F)F)cc3)(S(=O)(=O)c3ccc(F)cc3)C2)CC[C@H](C(=O)O)CC1
InChIKey: SVRMKEVXYJZNLQ-ATANMQQVSA-N
Target: Nuclear receptor subfamily 1 group I member 2 (SINGLE PROTEIN), Homo sapiens
UniProt: O75469
Assay: Transactivation of PXR (unknown origin) expressed in human HepG2 cells
Measurement: EC50 > 50000 nM (pChEMBL 4.301000118255615)
Descriptors: MW 655.6, ALogP 6.69, HBA 4, HBD 1, PSA 91.75, QED 0.27
Compound (SMILES): CCOCc1ccc2c(c1)nc(NC(=O)c1cccc(C(F)(F)F)c1)n2[C@H]1CC[C@H](O)CC1
InChIKey: NDHWKINWBPLWGB-WGSAOQKQSA-N
Target: Epidermal growth factor receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P00533
Assay: Inhibition of N-terminal 6x-HIS-tagged wild type human recombinant EGFR (696 to 1022 residues) expressed in Sf9 cells pre-incubated for 90 mins followed by 10 uM ATP addition by HTRF assay
Measurement: IC50 > 42000 nM (pChEMBL 4.376999855041504)
Descriptors: MW 461.48, ALogP 5.32, HBA 5, HBD 2, PSA 76.38, QED 0.52
Compound (SMILES): Cc1ccc(C2CC(C(C)(C)C)=NN2c2ccc(C(=O)O)cc2)cc1
InChIKey: MJSWJWIRIXUTEM-UHFFFAOYSA-N
Target: Prostaglandin G/H synthase 2 (SINGLE PROTEIN), Homo sapiens
UniProt: P35354
Assay: Colorimetric Assay: The activities of COX-1 and COX-2 were measured after the addition of arachidonic acid and incubation at 25° C. for 5 min by absorbance at 590 nm as specified by the manufacturer.
Measurement: IC50 > 100000 nM (pChEMBL 4)
Descriptors: MW 336.44, ALogP 5.05, HBA 3, HBD 1, PSA 52.9, QED 0.85
Compound (SMILES): C[C@](N)(CSCCNC(=N)N)C(=O)O.Cl.Cl
InChIKey: GGNXGWZVWYPKDE-KLXURFKVSA-N
Target: Nitric oxide synthase 3 (SINGLE PROTEIN), Homo sapiens
UniProt: P29474
Assay: Inhibition of human endothelial constitutive NOS expressed in Sf9 insect cells assessed as reduction in conversion of L-[2,3-3H]-arginine to L-[2,3-3H]-citrulline incubated for 15 mins by liquid scintillation counting method
Measurement: IC50 = 77000 nM (pChEMBL 4.110000133514404)
Descriptors: MW 220.3, ALogP -1, HBA 4, HBD 5, PSA 125.22, QED 0.22
Compound (SMILES): COc1cc2c(Nc3ccc(Cl)c(Cl)c3)ncnc2cc1OCC1CN(C2CCCC2)CCO1
InChIKey: JZVGJCJOXIONNM-UHFFFAOYSA-N
Target: Epidermal growth factor receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P00533
Assay: Inhibition of recombinant human N-terminal GST His6-tagged thrombin cleavage site-fused EGFR (H672 to A1210 residues) expressed in baculovirus using poly-EY as substrate after 210 mins by luciferase coupled chemiluminescence assay
Measurement: IC50 < 50 nM (pChEMBL 7.301000118255615)
Descriptors: MW 503.43, ALogP 5.71, HBA 7, HBD 1, PSA 68.74, QED 0.44