lovingscience/chembl-v37-raw-activities huggingface dataset:
Compound (SMILES): CN1CCC(n2cc(Nc3ncc(C(F)(F)F)c(CCc4ccccc4C4(C(N)=O)CC4)n3)cn2)CC1
InChIKey: FBOOAKYRDMGTPS-UHFFFAOYSA-N
Target: Vascular endothelial growth factor receptor 3 (SINGLE PROTEIN), Homo sapiens
UniProt: P35916
Assay: VEGFR3 Biochemical Assay: A biotin labelled peptide is used as substrate (amino acid sequence: Biotin-Glu-Gly-Pro-Trp-Leu-Glu-Glu-Glu-Glu-Glu-Ala-Tyr-Gly-Trp-Met-Asp-Phe-NH2). VEGFR3 cytoplasmic domain (amino acids 798-1298) was purchased as N-terminal GST-fusion protein (the enzyme). The 15 uL assay reactions are run in Greiner brand white 384-well low volume plates. All reactions contained 10 mM HEPES pH 7.4, mM MgCl2, 0.01% (v/v) Tween-20, 50 uM Na3VO4, 0.01% (w/v) albumin from chicken egg white, 1 mM Dithiothreitol, 111 nM peptide substrate, 500 uM ATP, and 3.8 ng/reaction enzyme, with the enzyme being omitted from negative control reactions. Compounds were added in a volume of 100 nL from dilution series prepared in DMSO, positive and negative control reactions receiving the same volume DMSO without compound. The plates were sealed with adhesive seals and incubated for 90 minutes at 30 degree Celsius.
Measurement: IC50 = 7 nM (pChEMBL 8.149999618530273)
Descriptors: MW 513.57, ALogP 4, HBA 7, HBD 2, PSA 101.96, QED 0.47
Compound (SMILES): Cc1ccc(NC(=O)c2cccc(C(F)(F)F)c2)cc1-c1cc(N2CCOCC2)nc(-n2ccnc2)n1
InChIKey: ZAYWARVMFZWGAP-UHFFFAOYSA-N
Target: Serine/threonine-protein kinase B-raf (SINGLE PROTEIN), Homo sapiens
UniProt: P15056
Assay: Raf Activity Assay: Raf and biotinylated Mek, kinase dead, were combined at 2x final concentrations in assay buffer (50 mM Tris, pH 7.5, 15 mM MgCl2, 0.01% BSA and 1 mM DTT) and dispensed 5 ml per well in assay plates (Greiner white 384 well assay plates #781207) containing 0.25 ml of 40x of a Raf kinase inhibitor test compound diluted in 100% DMSO. The plate was incubated for 60 minutes at room temperature. The Raf kinase activity reaction was started by the addition of 5 mL per well of 2xATP diluted in assay buffer. After 3 hours (b-Raf(V600E)) or 1 hour (c-Raf). The reactions were stopped and the phosphorylated product was measured using a rabbit anti-p-MEK (Cell Signaling, #9121) antibody and the Alpha Screen IgG (ProteinA) detection Kit (PerkinElmer #6760617R), by the addition of 10 mL to the well of a mixture of the antibody (1:2000 dilution) and detection beads (1:2000 dilution of both beads) in Stop/bead buffer (25 mM EDTA, 50 mM Tris, pH 7.5, 0.01% Tween20). The additions were carried out under dark conditions to protect the detection beads from light. A lid was placed on top of the plate and incubated for 1 hour at room temperature, then the luminescence was read on a PerkinElmer Envision instrument. The concentration of each compound for 50% inhibition (IC50) was calculated by non-linear regression using XL Fit data analysis software.
Measurement: IC50 = 0.5 nM (pChEMBL 9.300000190734863)
Descriptors: MW 508.5, ALogP 4.75, HBA 7, HBD 1, PSA 85.17, QED 0.42
Compound (SMILES): Clc1ccc2c(NC3=NCCC4(CN5CCC4CC5)O3)ncnn12
InChIKey: YKYKHUBLLYNPDU-UHFFFAOYSA-N
Target: Neuronal acetylcholine receptor subunit alpha-7 (SINGLE PROTEIN), Homo sapiens
UniProt: P36544
Assay: Agonist activity at alpha7 nACh receptor (unknown origin) expressed in HEK293 cells assessed as increase in Ca2+ flux by Fluo-4-AM dye based FLIPR assay
Measurement: EC50 = 1100 nM (pChEMBL 5.960000038146973)
Descriptors: MW 346.82, ALogP 2.04, HBA 7, HBD 1, PSA 67.05, QED 0.86
Compound (SMILES): CC1=C(C#N)C(c2ccc3[nH]ncc3c2)C(C#N)=C(C(F)(F)F)N1
InChIKey: PDMGTSVIIWGIAY-UHFFFAOYSA-N
Target: Hepatocyte growth factor receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P08581
Assay: Kinase Activity Assay (NADH Read-Out): Recombinant human c-Met protein (Invitrogen, Carlsbad, Calif., USA) is used. As substrate for the kinase reaction the peptide KKKSPGEYVNIEFG (JPT, Germany) is used. For the assay, 1 μL of a 51-fold concentrated solution of the test compound in DMSO is pipetted into a white 384-well microtiter plate (Greiner Bio-One, Frickenhausen, Germany). 25 μL of a solution of c-Met (final concentration 30 nM) and pyruvate kinase/lactate dehydrogenase (Roche Diagnostics, Mannheim, Germany; final concentration 8 mg/L) in assay buffer [3-(N-morpholino)propanesulfonic acid (MOPS), 50 mM, pH 7; MgCl2, 10 mM; bovine serum albumin (BSA), 0.01%; Triton X 100, 0.01%; DTT, 2 mM] are added, and the mixture is incubated for 5 min at room temperature. Then, the kinase reaction is started by the addition of 25 μL of a solution of adenosine triphosphate (ATP, final concentration 30 μM), substrate (final concentration 100 μM), nicotinamide adenine dinucleo-tide (NADH, final concentration 50 μM) and dithiothreitol (DTT, final concentration 2 mM) in assay buffer, and the resulting mixture is incubated for a reaction time of 100 min at 32° C. Subsequently, the amount of phosphorylated substrate is evaluated by measurement of the decrease of NADH fluorescence. Therefore, the fluorescence emissions at 465 nm after excitation at 340 nm is measured in a fluorescence reader, e.g. Tecan Ultra (Tecan, Männedorf, Switzerland).
Measurement: IC50 = 27 nM (pChEMBL 7.570000171661377)
Descriptors: MW 329.29, ALogP 3.39, HBA 4, HBD 2, PSA 88.29, QED 0.84
Compound (SMILES): COc1cc2c(N)nc(N3CCN(C(=O)C[C@@H](N)CC(C)C)CC3)nc2c(F)c1OC
InChIKey: ANCCVZUQUQIEJS-ZDUSSCGKSA-N
Target: Complement factor D (SINGLE PROTEIN), Homo sapiens
UniProt: P00746
Assay: ELISA Assay: CVF-Bb complex prepared from purified cobra venom factor (1 μM), recombinant human complement factor B (expressed in drosophila cells and purified using standard methods) and human complement factor D (expressed in E. Coli, refolded and purified using standard methods). CVF-Bb complex at 3 nM concentration was incubated with test compound at various concentrations for 1 hour at room temperature in PBS pH 7.4 containing 10 mM MgCl2 and 0.05% (w/v) CHAPS. Human complement C3 substrate purified from plasma was added to a final concentration of 1 μM. After 1 hour incubation at room temperature, the enzyme reaction was stopped by addition of a cocktail of concentrated pan-protease inhibitors. The product of the reaction, C3a, was quantified by means of an enzyme-linked-immunosorbent assay.
Measurement: IC50 = 680 nM (pChEMBL 6.170000076293945)
Descriptors: MW 434.52, ALogP 1.78, HBA 8, HBD 2, PSA 119.83, QED 0.68
Compound (SMILES): N#Cc1ccc2c3c([nH]c2c1)C1(CCOCC1)c1cc(OCCN2CCS(=O)(=O)CC2)ccc1C3=O
InChIKey: NXYOPKFWKLIRDM-UHFFFAOYSA-N
Target: ALK tyrosine kinase receptor (SINGLE PROTEIN), Homo sapiens
UniProt: Q9UM73
Assay: Inhibition Assay: ALK-inhibiting activity was measured by following an activity of inhibiting phosphorylation by biotinylated peptide (EGPWLEEEEEAYGWMDF). For the detection of phosphorylation of the biotinylated peptide, time-resolved fluorescence measurement was performed using an anti-phosphorylated tyrosine antibody labeled with europium cryptate and streptavidin conjugated to XL665, i.e., an allophycocyanin derivative.
Measurement: IC50 = 26.85 nM (pChEMBL 7.570000171661377)
Descriptors: MW 505.6, ALogP 2.79, HBA 7, HBD 1, PSA 112.49, QED 0.58
Compound (SMILES): O=c1[nH]nc(CCc2cccc(OC(F)(F)F)c2)cc1O
InChIKey: FIDAQQRAMNFEGF-UHFFFAOYSA-N
Target: D-amino-acid oxidase (SINGLE PROTEIN), Homo sapiens
UniProt: P14920
Assay: In Vitro DAAO Enzyme Assay: Human DAAO enzyme was supplied by the Takeda Pharmaceutical Company (Osaka) and each batch was tested and used at concentrations giving comparable levels of activity. The Km of D-Serine was measured for each enzyme batch to maintain consistency; this Km was used in subsequent assays.On the day of the assay compounds were serially diluted in DMSO before being diluted 1:20 with assay buffer (20 mM Tris ph 7.4). A 5 ul portion of assay buffer was added to the wells of a 384 clear base black-walled plate (Corning), 5 ul of diluted compound was then added via automated plate to plate transfer using the Bravo liquid handler (Agilent technologies) followed by 5 ul of human DAAO enzyme and then 5 ul D-Serine 50 mM was added to all but the negative control wells (final concentration of 10 mM). Finally 5 ul Amplex red reagent (Invitrogen) was added to all wells as per manufacturer's protocol. The plate was incubated for 60 minutes in the dark at 25° C., and the fluorescence in each well was measured in the Envision plate reader.
Measurement: IC50 = 22 nM (pChEMBL 7.659999847412109)
Descriptors: MW 300.24, ALogP 2.16, HBA 4, HBD 2, PSA 75.21, QED 0.91
Compound (SMILES): C[C@@H](NC(=O)c1nn(-c2ccc(Cl)c(Cl)c2)c2c1CCCC/C2=C\c1ccc(F)cc1)c1ccccn1
InChIKey: FNJXDPLNWFEMPF-RILMWAFASA-N
Target: Cannabinoid receptor 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P21554
Assay: Inverse agonist activity at human cannabinoid receptor 1 expressed in HEK293 cells assessed as increase in forskolin-induced cAMP production preincubated with cells followed by forskolin stimulation measured after 30 mins in presence of D2-labeled cAMP and Eu3+-TBP-NHS cryptate by HTRF assay
Measurement: EC50 = 35 nM (pChEMBL 7.460000038146973)
Descriptors: MW 535.45, ALogP 7.47, HBA 4, HBD 1, PSA 59.81, QED 0.27
Compound (SMILES): CN1C(=N)N[C@](C)(c2sc(-c3ccc(F)c(-c4nnco4)c3)cc2Cl)CS1(=O)=O
InChIKey: UKDMICDMAJRMJA-KRWDZBQOSA-N
Target: Beta-secretase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P56817
Assay: HTRF FRET Assay: Varying concentrations of inhibitors at 3× the final desired concentration in a volume of 10 μl are preincubated with purified human BACE1 catalytic domain (3 nM in 10 μl) for 30 minutes at 30° C. in reaction buffer containing 20 mM Na-Acetate pH 5.0, 10% glycerol, 0.1% Brij-35 and 7.5% DSMO. Reactions are initiated by addition of 10 μl of 600 nM QSY7-APPswe-Eu substrate (200 nM final) to give a final reaction volume of 30 in a 384 well Nunc HTRF plate. The reactions are incubated at 30° C. for 1.5 hours. The 620 nm fluorescence is then read on a Rubystar HTRF plate reader (BMG Labtechnologies) using a 50 millisecond delay followed by a 400 millisecond acquisition time window.
Measurement: Ki = 5.5 nM (pChEMBL 8.260000228881836)
Descriptors: MW 455.92, ALogP 3.27, HBA 7, HBD 2, PSA 112.18, QED 0.63
Compound (SMILES): COCCc1cccc(Cn2cc(C(=O)NCc3c(C)cc(N)nc3C)cn2)c1
InChIKey: VIGDTVWBASSIEN-UHFFFAOYSA-N
Target: Plasma kallikrein (SINGLE PROTEIN), Homo sapiens
UniProt: P03952
Assay: In-vitro Plasma Kallikrein Inhibition: Enzymatic reactions were conducted in â¿¿assay bufferâ¿¿, comprising 50 mM Hepes/NaOH at pH 7.8, 150 mM NaCl, 1 mM EDTA and 0.05% (w/v) CHAPS. For the determination of IC50 values, the assays were performed at room temperature in 384-well plates with a total assay volume of 25.25 ul per well. The test compound was dissolved in 90% (v/v) DMSO/water. For the assays, 250 nL of the 90% (v/v) DMSO/water solution or compound solution were added per well, followed by the addition of 12.5 ul protease solution (protease in assay buffer). The final assay concentration of the human plasma kallikrein was nominally 25 pM, the 11 compound concentrations in the dilution series were in the range form 1 nM to 100 uM. After 1 hour of pre-incubation at room temperature, the reactions were started by the addition of 12.5 ul substrate solution (in assay buffer, final assay concentration was 0.5 uM). After the addition of the substrate solution, the final DMSO concentration in the assay was 0.9% (v/v).
Measurement: IC50 = 190 nM (pChEMBL 6.71999979019165)
Descriptors: MW 393.49, ALogP 2.64, HBA 6, HBD 2, PSA 95.06, QED 0.61
Compound (SMILES): Cc1c(-c2ncnc3[nH]c(C4=CCCNC4)cc23)cccc1N1CCOc2cc(C3CC3)ccc2C1=O
InChIKey: YAHLLQYTUJBVIJ-UHFFFAOYSA-N
Target: Tyrosine-protein kinase BTK (SINGLE PROTEIN), Homo sapiens
UniProt: Q06187
Assay: Recombinant Human BTK Inhibition Assay: The inhibition assays were performed using the generic tyrosine kinase assay kit from C is Bio (HTRF KinEASE-TK #62TK0PEB). The assay is based on the principle of fluorescence resonance energy transfer (FRET) between the Europium Cryptate labeled anti-phosphotyrosine antibody and streptavidin labeled fluorophore, XL-665. In an uninhibited kinase reaction the biotinylated peptide substrate (Tk-Biotin) gets phosphorylated at an intermediate tyrosine site which is captured by anti-phosphotyrosine antibody which eventually acts as a FRET donor. The streptavidin labeled XL-665 binds strongly to the Tk-Biotin substrate and act as a FRET acceptor. The resultant FRET signal is measured at 665 nm. A ratio of this specific fluorescence (665 nm) to the background fluorescence (620 nm), conferred by the free antibody, is directly proportional to the kinase activity. Inhibition of the kinase activity by a pharmacological intervention such as a Btk inhibitor of the present disclosure will result into decreased FRET signal.
Measurement: IC50 > 1000 nM (pChEMBL 6)
Descriptors: MW 491.6, ALogP 5.23, HBA 5, HBD 2, PSA 83.14, QED 0.41
Compound (SMILES): O=C(OC(C(F)(F)F)C(F)(F)F)N1CCN(Cc2ccc(N3CCCC3)c(F)c2)CC1
InChIKey: KTFRRGGXJBLWHK-UHFFFAOYSA-N
Target: Fatty-acid amide hydrolase 1 (SINGLE PROTEIN), Mus musculus
UniProt: O08914
Assay: In Vitro Competitive Activity-Based Protein Assay: Proteomes (mouse brain membrane fraction or cell lysates) (50 uL, 1.0 mg/mL total protein concentration) were preincubated with varying concentrations of inhibitors at 37 C. After 30 min, FP-Rh (1.0 uL, 50 uM in DMSO) was added and the mixture was incubated for another 30 min at 37 C. Reactions were quenched with SDS loading buffer (50 uL-4x) and run on SDS-PAGE. Following gel imaging, serine hydrolase activity was determined by measuring fluorescent intensity of gel bands corresponding to MAGL, ABHD6 and FAAH using ImageJ 1.43u software.Preparation of Mouse Brain Proteomes from Inhibitor Treated Mice. Inhibitors were administered to wild-type C57Bl/6J by oral gavage in a vehicle of polyethylene glycol. Each animal was sacrificed 4 h following administration and brain proteomes were prepared and analyzed according to previously established methods (See Niphakis, M. J., et al. (2011) ACS Chem. Neurosci. and Long, J. Z., et al. Nat. Chem. Biol. 5:37-44).
Measurement: IC50 > 10000 nM (pChEMBL 5)
Descriptors: MW 457.39, ALogP 4.17, HBA 4, HBD 0, PSA 36.02, QED 0.64
Compound (SMILES): O=C(NS(=O)(=O)c1ccc(NCCCN2CCOCC2)cc1)c1ccc(N2CCN(Cc3ccccc3-c3ccc(Cl)cc3)CC2)cc1Oc1ccccc1
InChIKey: WVJAXJSOJWWUPR-UHFFFAOYSA-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 = 112.51 nM (pChEMBL 6.949999809265137)
Descriptors: MW 780.39, ALogP 7.37, HBA 9, HBD 2, PSA 103.45, QED 0.11
Compound (SMILES): CNc1nc(-c2nc3cnc(N4CCN(C)CC4)cc3n2Cc2ccccn2)cn2c(C)nnc12
InChIKey: UOBPZZRNSLHSFO-UHFFFAOYSA-N
Target: Histone H4 (SINGLE PROTEIN), Homo sapiens
UniProt: P62805
Assay: AlphaScreen Assay: Compounds are diluted in serial dilution 1:5 in assay buffer from 10 mM stock in DMSO (100 uM start concentration) in white OptiPlate-384 (PerkinElmer). A mix consisting of 15 nM GST-BRD4-BD1 protein (aa 44-168) or 150 nM GST-BRD4-BD2 (aa 333-460) and 15 nM biotinylated Acetyl-Histone H4 (Lys5, 8, 12, 16) peptide is prepared in assay buffer (50 mM HEPES pH=7.4; 25 mM NaCl; 0.05% Tween 20; 0.1% bovine serum albumin (BSA); 10 mM dithiothreitol (DTT)). 6 ul of the mix is added to the compound dilutions. Subsequently, 6 ul of premixed AlphaLISA Glutathione Acceptor Beads and AlphaScreen Streptavidin Donor Beads from PerkinElmer (in assay buffer at a concentration of 10 ug/ml each) are added and the samples are incubated for 30 min at RT in the dark (shaking 300 rpm). Afterwards, the signal is measured in a PerkinElmer Envision HTS Multilabel Reader using the AlphaScreen protocol from PerkinElmer. Each plate contains negative controls where biotinylated Acetyl-Histone H4 peptide and GST-BRD4-BD1 or GST-BRD4-BD2 are left out and replaced by assay buffer.
Measurement: IC50 = 13 nM (pChEMBL 7.889999866485596)
Descriptors: MW 469.56, ALogP 2.08, HBA 11, HBD 1, PSA 105.19, QED 0.41
Compound (SMILES): Cc1cccc2cc([C@H](C)Nc3ncnc4[nH]cnc34)n(C3CC3)c(=O)c12
InChIKey: XKHASMAALGBCSO-LBPRGKRZSA-N
Target: PI3-kinase p110-delta/p85-alpha (PROTEIN COMPLEX), Homo sapiens
UniProt: O00329
Assay: Inhibition of N-terminal His6-tagged recombinant full-length human p110delta/untagged recombinant full length p85alpha expressed in baculovirus infected Sf21 cells using phosphatidylinositol as substrate in presence of gamma-32P-ATP after 5 mins by thin layer chromatographic method
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 360.42, ALogP 3.48, HBA 6, HBD 2, PSA 88.49, QED 0.58
Compound (SMILES): CC[C@H](C)[C@@H]1NCCc2ccccc2/C=C\CCNC(=O)[C@@H](Cc2ccccc2)NC(=O)[C@@H](C)N(C)C1=O
InChIKey: DIZKGLGOKSGKDU-QSIVYGLDSA-N
Target: Growth hormone secretagogue receptor type 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q92847
Assay: Displacement of [125I]-Ghrelin from human GSHR1a transfected in HEK293 cell membranes after 30 mins by microplate scintillation and luminescence counting method
Measurement: Ki > 1000 nM (pChEMBL 6)
Descriptors: MW 518.7, ALogP 3.34, HBA 4, HBD 3, PSA 90.54, QED 0.58
Compound (SMILES): COC(=O)Nc1ccc(S(=O)(=O)C(C)C)c([C@H]2CCCN2C(=O)[C@H](Nc2ccc3c(c2F)C(=O)NC3)c2ccc(OC)c(OC)c2)c1
InChIKey: LDFNHHRVRPDDMT-AYWVHJORSA-N
Target: Coagulation factor VII/tissue factor (PROTEIN COMPLEX), Homo sapiens
UniProt: P13726
Assay: Inhibition of recombinant human factor-7a/TF using S2288 as substrate measured after 60 mins
Measurement: Ki = 6600 nM (pChEMBL 5.179999828338623)
Descriptors: MW 668.74, ALogP 4.96, HBA 9, HBD 3, PSA 152.37, QED 0.27
Compound (SMILES): CN(C)CCCOc1cccc(-c2cc(NC3CC3)n3ncc(/C=C4\NC(=O)NC4=O)c3n2)c1
InChIKey: XYHIAKXKQJOMIP-NDENLUEZSA-N
Target: Casein kinase II subunit beta (SINGLE PROTEIN), Homo sapiens
UniProt: P67870
Assay: Inhibition Assay: 5 uM ATP: Test compounds dissolved and diluted in DMSO (2 μl) were added to a reaction mixture comprising 10 μl of 5× Reaction Buffer (40 mM MOPS pH 7.0, 5 mM EDTA), 10 μl of recombinant human PIM2 solution (4 ng PIM-2 dissolved in dilution buffer (20 mM MOPS pH 7.0; EDTA 1 mM; 5% Glycerol; 0.01% Brij 35; 0.1%; 0.1% 2-mercaptoethanol; 1 mg/ml BSA)) and 8 ul of water. Reactions were initiated by the addition of 10 ul of ATP Solution (49% (15 mM MgCl2; 5 uM ATP) 1% ([γ-33P]ATP: Stock 1 mCi/100 μl; 3000 Ci/mmol (Perkin Elmer)) and 10 ul of substrate peptide solution (RSRSSYPAGT (SEQ ID NO.: 6), dissolved in water at a concentration of 1 mM), Reactions were maintained for 10 min at 30° C. The reactions were quenched with 100 ul of 0.75% Phosphoric acid, then transferred to and filtered through a Phosphocellulose filter plate (Millipore, MSPH-N6B-50). After washing each well 4 times with 0.75% Phosphoric acid, scintillation fluid (20 uL) was added to each well and
Measurement: IC50 = 2214.4 nM (pChEMBL 5.659999847412109)
Descriptors: MW 461.53, ALogP 2.48, HBA 8, HBD 3, PSA 112.89, QED 0.25
Compound (SMILES): O=C1CCCC(c2ccccc2-c2ccccc2)N1Cc1cccc(-c2ccccc2)c1
InChIKey: FQRZZXXWKKMRLD-UHFFFAOYSA-N
Target: Orexin/Hypocretin receptor type 1 (SINGLE PROTEIN), Homo sapiens
UniProt: O43613
Assay: In Vitro Assay: Intracellular Calcium Measurements : In Vitro Assay: Intracellular Calcium Measurements:Chinese hamster ovary (CHO) cells expressing the human orexin-1 receptor and the human orexin-2 receptor, respectively, are grown in culture medium (Ham F-12 with L-Glutamine) containing 300 g/ml G418, 100 U/ml penicillin, 100 g/ml streptomycin and 10% heat inactivated fetal calf serum (FCS). The cells are seeded at 20,000 cells/well into 384-well black clear bottom sterile plates (Greiner). The seeded plates are incubated overnight at 37° C. in 5% CO2. Human orexin-A as an agonist is prepared as 1 mM stock solution in MeOH:water (1:1), diluted in HBSS containing 0.1% bovine serum albumin (BSA), NaHCO3: 0.375 g/l and 20 mM HEPES for use in the assay at a final concentration of 3 nM.Antagonists are prepared as 10 mM stock solution in DMSO, then diluted in 384-well plates using DMSO followed by a transfer of the dilutions into in HBSS containing 0.1% bovine serum albumin (BSA), NaHCO3: 0.375 g/l and 20 mM HEPES. On the day of the assay, 50 μl of staining buffer (HBSS containing 1% FCS, 20 mM HEPES, NaHCO3: 0.375 g/l, 5 mM probenecid (Sigma) and 3 M of the fluorescent calcium indicator fluo-4 AM (1 mM stock solution in DMSO, containing 10% pluronic) is added to each well. The 384-well cell-plates are incubated for 50 min at 37° C. in 5% CO2 followed by equilibration at RT for 30 min before measurement.
Measurement: IC50 = 1450 nM (pChEMBL 5.840000152587891)
Descriptors: MW 417.55, ALogP 7.27, HBA 1, HBD 0, PSA 20.31, QED 0.33
Compound (SMILES): O=C(c1c(F)cccc1F)N1CCn2nc(COc3ccccc3)cc2C1
InChIKey: AWQLLPVALMFTAN-UHFFFAOYSA-N
Target: Metabotropic glutamate receptor 5 (SINGLE PROTEIN), Homo sapiens
UniProt: P41594
Assay: Cell-Based Functional Assay: HEK cells transfected with the human mGluR5a receptor (H10H cell line) were plated at 15,000 cells/well in clear-bottomed poly-D-lysine-coated assay plates (BD Falcon) in glutamate-glutamine-free growth medium and incubated overnight at 37° C. and 5% CO2. The following day, the growth medium was removed and the cells were washed with assay buffer containing 1× Hank's balanced salt solution (Invitrogen, Carlsbad, Calif.), 20 mM HEPES, 2.5 mM probenecid, pH 7.4 and left with 20 μL of this reagent. Following this step, the cells were loaded with calcium indicator dye, fluo-4 AM, to a final concentration of 2 μM and incubated for 40-45 min at 37° C. The dye solution was removed and replaced with assay buffer.
Measurement: EC50 = 292 nM (pChEMBL 6.539999961853027)
Descriptors: MW 369.37, ALogP 3.4, HBA 4, HBD 0, PSA 47.36, QED 0.71
Compound (SMILES): c1cc(-c2n[nH]c3cnc(-c4cnn(CC5COC5)c4)cc23)nc(N2CCNCC2)c1
InChIKey: GBKPXQXNHPVXSO-UHFFFAOYSA-N
Target: Serine/threonine-protein kinase pim-1 (SINGLE PROTEIN), Homo sapiens
UniProt: P11309
Assay: PIM Kinase Binding Activity Assay: PIM-1, PIM-2, and PIM-3 enzymes were generated as fusion proteins expressed in bacteria and purified by IMAC column chromatography (Sun, X., Chiu, J. F., and He, Q. Y. (2005) Expert Rev. Proteomics, 2:649-657). A fluorescent-labeled Pim-specific peptide substrate, was custom synthesized by American Peptide Company (Sunnyvale, Calif.). Reaction Buffer contained 10 mM HEPES, pH 7.2, 10 mM MgCl2, 0.01% Tween 20, 2 mM DTT. Termination Buffer contained 190 mM HEPES, pH 7.2, 0.015% Brij-35, 0.2% Coating Reagent 3 (Caliper Life Sciences, Hopkinton, Mass.), 20 mM EDTA. Separation Buffer contained 100 mM HEPES, pH 7.2, 0.015% Brij-35, 0.1% Coating Reagent 3, 1:200 Coating Reagent 8 (Caliper Life Sciences, Hopkinton, Mass.), 10 mM EDTA and 5% DMSO.
Measurement: Ki = 11.3 nM (pChEMBL 7.949999809265137)
Descriptors: MW 416.49, ALogP 1.94, HBA 8, HBD 2, PSA 96.78, QED 0.51
Compound (SMILES): O=C(Nc1ccc(C2=CCN(C(=O)[C@@H]3CCOC3)CC2)cc1)N1Cc2ccccc2C1
InChIKey: VKABRFSTVYJHCG-JOCHJYFZSA-N
Target: Nicotinamide phosphoribosyltransferase (SINGLE PROTEIN), Homo sapiens
UniProt: P43490
Assay: Time-Resolved Fluorescence Resonance Energy Transfer (TR-FRET) Binding Assay : Probe 2: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-1287128.0. 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 = 12.5 nM (pChEMBL 7.900000095367432)
Descriptors: MW 417.51, ALogP 3.89, HBA 3, HBD 1, PSA 61.88, QED 0.82
Compound (SMILES): CN1C(=O)CCc2cc(-c3cncc(COc4cccc(Cl)n4)c3)c(F)cc21
InChIKey: LLACDVWVHFYQHP-UHFFFAOYSA-N
Target: Cytochrome P450 11B2, mitochondrial (SINGLE PROTEIN), Homo sapiens
UniProt: P19099
Assay: G402-Based Assay: Herein we identified the use of the G-402 cell line as a host cell to ectopically express (transiently or stably) enzymes of the CYP11 family. Specifically we developed stable G-402 cells expressing ectopically human CYP11B1, human CYP11B2, human CYP11A1, cynmolgus CYP11B1 or cynomolgus CYP11B2 enzyme activity. Importantly the identified cell line G-402 expresses co-factors (adrenodoxin and adrenodoxin reductase) important for the activity of the CYP11 family and no relevant enzyme activity of the CYP11 family (in comparison to H295R cells) was detected in these cells. Therefore the G-402 cell line is uniquely suited as a host cell for the ectopic expression of enzymes from the CYP11 family.Cellular enzyme assays were performed in DMEM/F12 medium containing 2.5% charcoal treated FCS and appropriate concentration of substrate (0.3-10 uM 11-Deoxycorticosterone, 11-Deoxycortisol or Corticosterone). For assaying enzymatic activity, cells were plated onto 96 well plates and incubated for 16 h. An aliquot of the supernatant is then transferred and analyzed for the concentration of the expected product (Aldosterone for CYP11B2; Cortisol for CYP11B1). The concentrations of these steroids can be determined using HTRF assays from CisBio analyzing either Aldosterone or Cortisol.
Measurement: EC50 = 0.3 nM (pChEMBL 9.520000457763672)
Descriptors: MW 397.84, ALogP 4.42, HBA 4, HBD 0, PSA 55.32, QED 0.61
Compound (SMILES): CS(=O)(=O)n1cc(C(=O)NC2CC3COCC(C2)N3)c2cc(F)ccc21
InChIKey: LRLCEYPIWFDLJJ-UHFFFAOYSA-N
Target: 5-hydroxytryptamine receptor 3A (SINGLE PROTEIN), Homo sapiens
UniProt: P46098
Assay: In Vitro Inhibition of Ca Flux Assay: The 5HT3 antagonist activity of the compounds of the invention was determined by measuring the ability of the compounds to inhibit the calcium flux activity of 3HT3a receptor expressed in HEK-293T cells. HEK-293T cells were transfected with the 5HT3a expression construct using Xtreme Gene 9 (Roche) in 150 mm tissue culture treated plates and incubated for 24 hours at 37 °C. Cells were then split and plated at a density of 60K cells/well in poly-lysine coated, black 96-well plates with clear bottoms (BD BioSciences) and incubated overnight at 37 °C. Growth media was removed and cells loaded with 200 uL calcium indicator dye in HBSS containing 20 mM HEPES (Calcium 5 Assay kit, Molecular Devices) and incubated at 37 °C. for 1 hour. While cells were incubating, the 10× antagonist and agonist/antagonist addition plates were made. For 10× antagonist plate: half log serial dilutions (final concentrations range from 10−7 through 10−10 with the bottom well a negative, no ligand control) were made from test compounds in DMSO at a 1000× concentration and then diluted to 10× in HBSS/20 mM HEPES. For addition plate: 5HT was diluted to 100× in HBSS/20 mM HEPES (final concentration in the assay−216 nM) and 15 uL was added to each well of the addition plate, 15 uL of 10× compound was also added to the addition plate, and finally 120 uL of HBSS/20 mM HEPES (for a total of 150 uL). Cells were then removed from the incubator and equilibrated to room temperature for 10 minutes, then 22.5 uL of 10× test compounds were added in triplicate to the plates and incubated at room temperature for 10 minutes (Tropisetron was used as a positive control in every assay). Test plate and addition plate were loaded into the FlexStation III (Molecular Devices), and using the fluidics, 22.5 uL compound additions were made (at t=~17 seconds), and fluorescence was measured for 90 seconds, reading every 2.2 seconds.
Measurement: IC50 = 4.81 nM (pChEMBL 8.319999694824219)
Descriptors: MW 381.43, ALogP 0.84, HBA 6, HBD 2, PSA 89.43, QED 0.82
Compound (SMILES): CC(=O)N1C2CCC1c1c(-c3ccc(F)cc3)nn(Cc3ccc(F)cc3F)c1C2
InChIKey: HRNJZNLDXWNEHJ-UHFFFAOYSA-N
Target: Potassium channel subfamily K member 3 (SINGLE PROTEIN), Homo sapiens
UniProt: O14649
Assay: Biological Assay: Human TASK-1 channels were expressed in Xenopus oocytes. For this purpose, oocytes were isolated from Xenopus laevis and defoliculated. Subsequently, TASK-1-encoding RNA synthesized in vitro was injected into oocytes. After two days of TASK-1 protein expression, TASK-1 currents were measured by two-microelectrode voltage clamp. Data were acquired and analyzed using a TEC-10cx amplifier (NPI Electronic, Tamm, Germany) connected to an ITC-16 interface (Instrutech Corp., Long Island, USA) and Pulse software (HEKA Elektronik, Lambrecht, Germany). Oocytes were clamped to −90 mV and TASK-1 mediated currents were measured during 500 ms voltage pulses to 40 mV. Oocytes were continuously superfused with ND96 buffer containing: NaCl 96 mM, KCl 2 mM, CaCl2 1.8 mM, MgCl2 1 mM, HEPES 5 mM (pH adjusted to 7.4 with NaOH). All experiments were performed at room temperature.
Measurement: IC50 < 10000 nM (pChEMBL 5)
Descriptors: MW 411.43, ALogP 4.62, HBA 3, HBD 0, PSA 38.13, QED 0.63
Compound (SMILES): C[C@]12CC[C@H]3[C@@H](CC[C@H]4CC(=O)CC[C@@]43C)C1=CC=C2n1ccnc1
InChIKey: YKTFNUJFKZCZQJ-CFNFZEJTSA-N
Target: Steroid 17-alpha-hydroxylase/17,20 lyase (SINGLE PROTEIN), Homo sapiens
UniProt: P05093
Assay: Inhibition of CYP17 in human testicular microsomes using 17alpha-hydroxypregnenolone as substrate assessed as decrease in DHEA levels after 90 mins by ELISA
Measurement: IC50 <= 11 nM (pChEMBL 7.959000110626221)
Descriptors: MW 336.48, ALogP 4.87, HBA 3, HBD 0, PSA 34.89, QED 0.73
Compound (SMILES): COc1ccc([C@]2(CCO)CC(c3cccnc3)=NN2C(=O)Nc2ccc(Br)cc2)cc1
InChIKey: FKUVJZGPXYCCEB-DEOSSOPVSA-N
Target: Voltage-dependent T-type calcium channel subunit alpha-1H (SINGLE PROTEIN), Homo sapiens
UniProt: O95180
Assay: Inhibition of recombinant T-type calcium channel Cav3.2 (unknown origin) expressed in HEK293 cells assessed as inhibition of CaCl2-induced calcium influx preincubated for 3 mins prior CaCl2 addition by Fluo-4-AM dye-based FLIPR assay
Measurement: IC50 = 33 nM (pChEMBL 7.480000019073486)
Descriptors: MW 495.38, ALogP 4.77, HBA 5, HBD 2, PSA 87.05, QED 0.52
Compound (SMILES): CNC(=O)c1cccc(Nc2ncnc(N3CCC(n4cc(-c5ccc(Cl)cc5)cn4)CC3)n2)c1
InChIKey: MXJDPGGLPXGEIM-UHFFFAOYSA-N
Target: Sodium channel protein type 9 subunit alpha (SINGLE PROTEIN), Homo sapiens
UniProt: Q15858
Assay: Cell-Based Assay: Compounds were tested on human Nav1.7 (hNav1.7) expressed stably in 293 cells, which have no endogenous sodium channels. Two forms of whole-cell patch-clamp assays were used. In both, peak current through Nav1.7 was evoked by a depolarizing voltage pulse, and currents were monitored as a function of time in response to exposure to several different concentrations of drug. Automated electrophysiology uses the PatchXpress 7000A system. Up to sixteen cells at a time were voltage-clamped in parallel, and the steady-state voltage dependence of inactivation was determined experimentally for each cell. For each cell, fractional inactivation was set at 20%, and peak current through Nav1.7 as a function of time was measured in response to increasing concentrations of test compound. Manual electrophysiology refers to conventional whole-cell patch-clamping (Hamill et al., 1981). After formation of the whole-cell clamp, cells were lifted off the bottom of the dish with the patch pipette and positioned directly in front of an array of glass microperfusion tubes each with internal diameter about 250 microns, with individual compound concentrations flowing from each tube. Current as a function of time in response to increasing drug concentrations was measured with Nav1.7 fully in the resting state (holding voltage set to −140 mV) and on the same cells with the holding voltage set to produce about 20% inactivation. Compounds were not applied for a prespecified time, but for as long as required to achieve a stable level of block.
Measurement: IC50 = 4160 nM (pChEMBL 5.380000114440918)
Descriptors: MW 488.98, ALogP 4.33, HBA 8, HBD 2, PSA 100.86, QED 0.42
Compound (SMILES): COc1ccc2ccc3c(c2c1)C[C@H](N)[C@@H](c1cc(F)c(F)cc1F)O3
InChIKey: MDQFWLGJEDPEFU-AZUAARDMSA-N
Target: Dipeptidyl peptidase 4 (SINGLE PROTEIN), Homo sapiens
UniProt: P27487
Assay: Inhibition of DPP4 (unknown origin) expressed in Sf9 cells using Gly-Pro-AMC substrate
Measurement: IC50 = 60.61 nM (pChEMBL 7.21999979019165)
Descriptors: MW 359.35, ALogP 4.27, HBA 3, HBD 1, PSA 44.48, QED 0.7
Compound (SMILES): C=Cc1c(N)ncnc1N1CCC(c2nc(-c3cccc(F)c3)cn2CCN2CCCC2)CC1
InChIKey: USJIOUUNHSENIY-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 = 0.9 nM (pChEMBL 9.050000190734863)
Descriptors: MW 461.59, ALogP 4.18, HBA 7, HBD 1, PSA 76.1, QED 0.57
Compound (SMILES): CCCS(=O)(=O)Nc1ccc(F)c(C(=O)c2c[nH]c3ncc(-c4cnc(N5CCCC5)nc4)cc23)c1F
InChIKey: KIPKJEJCEBARJV-UHFFFAOYSA-N
Target: Serine/threonine-protein kinase B-raf (SINGLE PROTEIN), Homo sapiens
UniProt: P15056
Assay: Inhibition of B-Raf (unknown origin) assessed as decrease in biotin-tagged MEK1 phosphorylation after 45 mins
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 526.57, ALogP 4.28, HBA 7, HBD 2, PSA 120.94, QED 0.33
Compound (SMILES): COc1ccc(C2(OC/C=C/Cn3ccnc3CCC(=O)O)c3ccccc3-c3ccccc32)cc1
InChIKey: PIEZLFLTLKUGEC-VOTSOKGWSA-N
Target: GABA transporter 2 (SINGLE PROTEIN), Mus musculus
UniProt: P31651
Assay: Inhibition of mouse GAT2 expressed in HEK293 cells assessed as decrease in [3H]GABA uptake preincubated for 25 mins followed by [3H]GABA addition measured after 10 mins by liquid scintillation counter method
Measurement: IC50 = 32359.37 nM (pChEMBL 4.489999771118164)
Descriptors: MW 480.56, ALogP 5.45, HBA 5, HBD 1, PSA 73.58, QED 0.31
Compound (SMILES): O=C(Nc1nc2ccccc2s1)c1cccc2c1CN(c1ccc(-c3cnn(Cc4cccc(Cl)c4)c3)c(C(=O)O)n1)CC2
InChIKey: QHUOKCSYRBRCEQ-UHFFFAOYSA-N
Target: Bcl-2-like protein 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q07817
Assay: Time Resolved-Fluorescence Resonance Energy Transfer (TR-FRET) Assay: The measurement of competition of compounds of Formula (I) with F-Bak for a Bcl-2 family protein (Bcl-xL) binding site using a Time Resolved Fluorescence Resonance Energy Transfer (TR-FRET) binding assay: Test compounds were serially diluted in DMSO starting at 50 M (2x starting concentration; 10% DMSO) and 10 Î¼L transferred into a 384-well plate. Then 10 Î¼L of a protein/probe/antibody mix is added to each well at final concentrations listed in Table 1. Protein: GST-Bcl-xL; 1(nM) Probe: F-Bak (GQVGRQLAIIGDK (6-FAM)INR-amide) SEQ ID NO: 1; 100(nM) Antibody: Tb-anti-GST; 1(nM). The samples are then mixed on a shaker for 1 minute then incubated for an additional 2 hours at room temperature. For each assay plate, a probe/antibody and protein/antibody/probe mixture were included as a negative and a positive control, respectively. Fluorescence was measured on the ENVISION plate reader (Perkin Elmer) using a 340/35 nm excitation filter and 520/525 (F-Bak) and 495/510 nm (Tb-labeled anti-his antibody) emission filters. Dissociation constants (Ki) were determined using Wang's equation (Wang, Z.X. An 20 exact mathematical expression for describing competitive binding of two different ligands to protein molecule. FEBS Lett. 1995 360:111-114). The TR-FRET assay can be performed in the presence of varying concentrations of human serum (HS) or fetal bovine serum (FBS).
Measurement: Ki = 6 nM (pChEMBL 8.220000267028809)
Descriptors: MW 621.12, ALogP 6.77, HBA 8, HBD 2, PSA 113.24, QED 0.2
Compound (SMILES): CC(C)Oc1cccc(F)c1[C@@H]1C[C@@H](O)C(=O)N1Cc1ccc(OC(F)(F)F)cc1
InChIKey: MFPPCBZFUKRQFC-DLBZAZTESA-N
Target: Orexin/Hypocretin receptor type 1 (SINGLE PROTEIN), Homo sapiens
UniProt: O43613
Assay: In Vitro Assay: Intracellular Calcium Measurements : In Vitro Assay: Intracellular Calcium Measurements:Chinese hamster ovary (CHO) cells expressing the human orexin-1 receptor and the human orexin-2 receptor, respectively, are grown in culture medium (Ham F-12 with L-Glutamine) containing 300 g/ml G418, 100 U/ml penicillin, 100 g/ml streptomycin and 10% heat inactivated fetal calf serum (FCS). The cells are seeded at 20,000 cells/well into 384-well black clear bottom sterile plates (Greiner). The seeded plates are incubated overnight at 37° C. in 5% CO2. Human orexin-A as an agonist is prepared as 1 mM stock solution in MeOH:water (1:1), diluted in HBSS containing 0.1% bovine serum albumin (BSA), NaHCO3: 0.375 g/l and 20 mM HEPES for use in the assay at a final concentration of 3 nM.Antagonists are prepared as 10 mM stock solution in DMSO, then diluted in 384-well plates using DMSO followed by a transfer of the dilutions into in HBSS containing 0.1% bovine serum albumin (BSA), NaHCO3: 0.375 g/l and 20 mM HEPES. On the day of the assay, 50 μl of staining buffer (HBSS containing 1% FCS, 20 mM HEPES, NaHCO3: 0.375 g/l, 5 mM probenecid (Sigma) and 3 M of the fluorescent calcium indicator fluo-4 AM (1 mM stock solution in DMSO, containing 10% pluronic) is added to each well. The 384-well cell-plates are incubated for 50 min at 37° C. in 5% CO2 followed by equilibration at RT for 30 min before measurement.
Measurement: IC50 = 2547 nM (pChEMBL 5.590000152587891)
Descriptors: MW 427.39, ALogP 4.35, HBA 4, HBD 1, PSA 59, QED 0.7
Compound (SMILES): NC[C@@H](NC(=O)c1ccc(-c2nc(C3CCOCC3)cnc2N)cc1F)c1cccc(Cl)c1
InChIKey: CLDYKQRYJLMGMR-HXUWFJFHSA-N
Target: Mitogen-activated protein kinase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P28482
Assay: ERK2 (20 pM) Kinase Assay: Compound potency against activated ERK2 was determined using a kinase assay that measures ERK2-catalyzed phosphorylation of biotinylated ERKtide peptide substrate ([Biotin]-AHA-K-R-E-L-V-E-P-L-T-P-S-G-E-A-P-N-Q-A-L-L-R-[NH2], the peptide sequence derived from EGF receptor: SEQ ID NO:1). The assay was carried out in 20 mM HEPES [pH 7.5], 5 mM MgCl2, 1 mM DTT, 0.01% Tween-20, 0.05% BSA using 0.02 nM ERK2, 400 nM ERKtide peptide and 35 uM ATP (all concentrations are final in the reaction) in a total volume of 10.25 uL. A 16-point, half-log dilution series of compounds at 41x final concentration was used for generating IC50 curves. Compound dilution series were prepared in 100% DMSO. ERK2 was preincubated with compounds for 30 minutes at ambient temperature. Reaction was initiated by addition of a substrate cocktail of ERKtide peptide and ATP and was allowed to proceed for 4 hours at ambient temperature. Reaction was terminated by addition of 10 uL of a 2x stop buffer consisting of 100 mM Tris-Cl [pH 7.5], 25 mM EDTA, 0.01% Tween 20, 20 ug/mL of AlphaScreen Protein A Acceptor Beads, 20 ug/mL of Streptavidin Donor Beads (PerkinElmer, Waltham, Mass.), and 1:1000 dilution phospho-EGF Receptor (Thr669) antibody (Cat #8808, Cell Signaling Technology, Danvers, Mass.). Terminated reactions were read, after overnight incubation in the dark, on an EnVision Multilabel Plate Reader (PerkinElmer, Waltham, Mass.), with excitation and emission wavelengths set to 680 nm and 570 nm, respectively. IC50 values were determined using a four-parameter fit.
Measurement: IC50 = 0.021 nM (pChEMBL 10.680000305175781)
Descriptors: MW 469.95, ALogP 3.84, HBA 6, HBD 3, PSA 116.15, QED 0.5
Compound (SMILES): Cc1cc(C(=O)Nc2cccc(C(C)N3CCC(C(=O)NC4CCCCC4)CC3)c2)c2cnn(C(C)C)c2n1
InChIKey: XLNMNYIJNPXLPI-UHFFFAOYSA-N
Target: Atypical chemokine receptor 3 (SINGLE PROTEIN), Homo sapiens
UniProt: P25106
Assay: In Vitro Assay: CHO-K1 CXCR7 b-arrestin cells are detached from culture dishes with a cell dissociation buffer (Invitrogen, #13151-014) and collected in growing medium (F12 HAMS 90% (v/v)/FCS 10% (v/v), Penicilin/streptomycin 1% (v/v)). 5000 cells per well (in 20 μl) are seeded in a 384 well plate (white-walled, clear bottom; BD Falcon #353274). The plate is incubated at 37° C./5% CO2 for 24 hours. Medium is then replaced by 20 μl OPTIMEM (Invitrogen #31985) for 3 to 4 hours. Test compounds are dissolved at 10 mM in DMSO and serially diluted in DMSO to 200× of the final concentration for dose response curves. Compounds are then diluted 1:33.3 in HBSS1×. 5 μl/well of HBSS1×/20 mM HEPES/0.2% BSA are added to the assay plate followed by addition of 5 μl/well of diluted compounds. CXCL12 (Peprotech #300-28A) may be used as a reference agonist. The plate is incubated for 90 minutes at 37° C. 12 μl of detection reagent (Path Hunter Detection Kit, DiscoveRx, #93-0001) is transferred to the assay plate and to the plate is incubated for 1 hour at room temperature. Luminescent signal is read in a microplate reader (FLUOstar Optima, bmg).
Measurement: EC50 = 86 nM (pChEMBL 7.070000171661377)
Descriptors: MW 530.72, ALogP 5.79, HBA 6, HBD 2, PSA 92.15, QED 0.4
Compound (SMILES): O=C(NC1CCNCC1)c1cccc(-c2n[nH]c3cnc(-c4cccnc4)cc23)n1
InChIKey: BBVLEPANAGCAJE-UHFFFAOYSA-N
Target: Serine/threonine-protein kinase pim-1 (SINGLE PROTEIN), Homo sapiens
UniProt: P11309
Assay: PIM Kinase Binding Activity Assay: PIM-1, PIM-2, and PIM-3 enzymes were generated as fusion proteins expressed in bacteria and purified by IMAC column chromatography (Sun, X., Chiu, J. F., and He, Q. Y. (2005) Expert Rev. Proteomics, 2:649-657). A fluorescent-labeled Pim-specific peptide substrate, was custom synthesized by American Peptide Company (Sunnyvale, Calif.). Reaction Buffer contained 10 mM HEPES, pH 7.2, 10 mM MgCl2, 0.01% Tween 20, 2 mM DTT. Termination Buffer contained 190 mM HEPES, pH 7.2, 0.015% Brij-35, 0.2% Coating Reagent 3 (Caliper Life Sciences, Hopkinton, Mass.), 20 mM EDTA. Separation Buffer contained 100 mM HEPES, pH 7.2, 0.015% Brij-35, 0.1% Coating Reagent 3, 1:200 Coating Reagent 8 (Caliper Life Sciences, Hopkinton, Mass.), 10 mM EDTA and 5% DMSO.
Measurement: Ki = 30.5 nM (pChEMBL 7.519999980926514)
Descriptors: MW 399.46, ALogP 2.56, HBA 6, HBD 3, PSA 108.48, QED 0.49
Compound (SMILES): Cc1ccc(C2=C(C(=O)NS(=O)(=O)C(C)(C)C)C(=O)N[C@@](c3ccc(OCCCC(F)(F)F)cc3)(C(F)(F)F)C2)cc1
InChIKey: UFOLGFLZLZKWBB-SANMLTNESA-N
Target: Alpha-1,6-mannosyl-glycoprotein 2-beta-N-acetylglucosaminyltransferase (SINGLE PROTEIN), Homo sapiens
UniProt: Q10469
Assay: LCMS Assay: The MGAT enzyme reactions were performed in Corning FALCON® 96-well Polypropylene plates, in a total volume of 60 μL of 50 mM Potassium Phosphate buffer pH 7.4, containing a final concentration of 100 μM 2-oleoylglycerol, 15 μM oleoyl-Coenzyme A and 0.0013 μg/μL Human or Mouse MGAT-2 or 0.0026 μg/μL Rat recombinant MGAT-2 membranes expressed in Sf9 cells. Assay plates were run through a fully automated robotics system and shaken for 5 seconds every minute for a total 10 minutes. The reactions were then quenched with 120 μL of ice cold methanol containing 1 μg/mL 1,2-distearoyl-rac-glycerol as the internal standard. Plates were shaken for 2 minutes and spun down to remove protein precipitation. After the spin, samples were transferred to LC/MS compatible PCR plates. For LC/MS analysis, a ThermoFisher Surveyor pump, utilizing a Waters Symmetry C8, 50×2.1 mm column, was used for the chromatography of enzyme products. The buffer system consists of 0.1% formic acid in water with a mobile phase consisting 0.1% formic acid in methanol. The shallow gradient is 90-100% mobile phase in 0.2 min with a total run time of 2.3 min. The first 0.5 minutes of each injection was diverted to waste to eliminate the presence of Phosphate buffer in the enzymatic reaction. The column was run at 0.6 mL/min and a temperature of 65° C.
Measurement: IC50 = 348 nM (pChEMBL 6.460000038146973)
Descriptors: MW 620.61, ALogP 5.69, HBA 5, HBD 2, PSA 101.57, QED 0.22
Compound (SMILES): CC1(C)CCC(CN2CCN(c3ccc(C(=O)NS(=O)(=O)c4cnc(N[C@@H]5CCN(C(CF)CF)C5)c(Cl)c4)c(Oc4cnc5[nH]ccc5c4)c3)CC2)=C(c2ccc(Cl)cc2)C1
InChIKey: SUEBELNKMFIMDH-MGBGTMOVSA-N
Target: Apoptosis regulator Bcl-2 (SINGLE PROTEIN), Homo sapiens
UniProt: P10415
Assay: Time Resolved-Fluorescence Resonance Energy Transfer (TR-FRET) Assay: Representative compounds were serially diluted in dimethyl sulfoxide (DMSO) starting at 50 uM (2x starting concentration; 10% DMSO) and 10 uL were transferred into a 384-well plate. Then 10 uL of a protein/probe/antibody mix was added to each well at final concentrations listed in TABLE 1. Protein:GST-Bcl-2, Probe: F-Bak Peptide Probe Acetyl-GQVGRQLAIIGDK(6-FAM)INR-amide(SEQ ID NO: 1), Protein(nM): 1, Probe (nM): 100, Antibody: Tb-anit-GST, Antibody (nm): 1.  The samples are then mixed on a shaker for 1 minute and incubated for an additional 3 hours at room temperature. For each assay, the probe/antibody and protein/probe/antibody were included on each assay plate as negative and positive controls, respectively. Fluorescence was measured on the ENVISION plate reader (Perkin Elmer) using a 340/35 nm excitation filter and 520/525 (F-Bak peptide) and 495/510 nm (Tb-labeled anti-Histidine antibody) emission filters.
Measurement: Ki = 0.77 nM (pChEMBL 9.109999656677246)
Descriptors: MW 907.92, ALogP 8.76, HBA 10, HBD 3, PSA 135.79, QED 0.1
Compound (SMILES): CN[C@@H](C)C(=O)N[C@H]1CSc2c(NC(=O)C3CCNCC3)cccc2N(Cc2cccc3ccccc23)C1=O
InChIKey: GWNWLIHKMHHSTR-DFBJGRDBSA-N
Target: E3 ubiquitin-protein ligase XIAP (SINGLE PROTEIN), Homo sapiens
UniProt: P98170
Assay: TR-FRET Assay: Ten nanomolar of 6x Histidine-tagged BIR2 domain, corresponding to amino acids 124-240 of XIAP, or BIR3 domain, corresponding to amino acids 241-356 of XIAP, was mixed with 20 nM of the peptide AVPIAQKSEK-(8-biotin)-OH 1:2 TFA, in the presence of 50 mM Tris-C1, pH 7.5, 100 mM NaCl, 1 mM dithiothreitol (DTT) and 0.1 mg/mL bovine serum albumin (BSA). Following a 45 min. incubation at 37° C., Europium-Streptavidin and Allophycocyanin conjugated anti-Histidine antibody were added to a final concentration of 1.5 nM and 15 nM, respectively. Time-resolved fluorescence resonance energy transfer (TR-FRET) signals were measured 1 hour later at room temperature. Test compound potency was assessed at 10 serially diluted concentrations.
Measurement: IC50 = 14190 nM (pChEMBL 4.849999904632568)
Descriptors: MW 545.71, ALogP 3.51, HBA 6, HBD 4, PSA 102.57, QED 0.36