lovingscience/chembl-v37-raw-activities huggingface dataset:
Compound (SMILES): N=C(N)NCCCC(=O)Nc1ccc(C[C@H](NC(=O)[C@@H]2CCCN2S(=O)(=O)c2ccccc2)C(=O)O)cc1
InChIKey: CUGPAWBXPURXLJ-SFTDATJTSA-N
Target: Integrin alpha-5/beta-1 (PROTEIN COMPLEX), Homo sapiens
UniProt: P05556
Assay: Cell Adhesion Assay : A panel of cell lines and integrin ligands were developed to allow rapid examination of potency and specificity of inhibitors against all 8 integrins that recognize RGD sequences in ligands. A subset of these was used to generate the very encouraging data about 8 shown above. These cell lines were used to calculate IC50 concentrations for each inhibitor synthesized to rapidly generate structure activity information to drive subsequent modification and synthesis. Inhibitors were evaluated for showing better potency/selectivity in CCl4-induced liver fibrosis.
Measurement: IC50 < 10 nM (pChEMBL 8)
Descriptors: MW 544.63, ALogP 0.85, HBA 6, HBD 6, PSA 194.78, QED 0.13
Compound (SMILES): FC(F)(F)Oc1ccc(NC[C@H]2CC[C@H](c3nnn4cnc5[nH]ccc5c34)CC2)c(Cl)c1
InChIKey: OPECFSSECZXMDK-JOCQHMNTSA-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 464.88, ALogP 5.54, HBA 6, HBD 2, PSA 80.13, QED 0.4
Compound (SMILES): CCC#CC(=O)Nc1cccc(-c2cnc3[nH]cc(C)c3c2)c1
InChIKey: GNIWWRYROYEXHL-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 = 35 nM (pChEMBL 7.460000038146973)
Descriptors: MW 303.37, ALogP 3.89, HBA 2, HBD 2, PSA 57.78, QED 0.71
Compound (SMILES): COC(=O)NC[C@H](C)Nc1nccc(-c2cn(C(C)C)nc2-c2cc(Cl)cc(NS(C)(=O)=O)c2F)n1
InChIKey: HKXKEELLDFNXRA-ZDUSSCGKSA-N
Target: Mitogen-activated protein kinase 14 (SINGLE PROTEIN), Homo sapiens
UniProt: Q16539
Assay: Inhibition of IL1-beta-induced p38alpha phosphorylation in human A549 cells pre-incubated for 30 mins before IL1-beta stimulation and measured after 7 to 8 hrs post IL1-beta stimulation by Bright-Glo luciferase reporter gene assay
Measurement: IC50 = 690 nM (pChEMBL 6.159999847412109)
Descriptors: MW 540.02, ALogP 3.91, HBA 9, HBD 3, PSA 140.13, QED 0.37
Compound (SMILES): Cc1ccc(C(=O)N2CCOC[C@H]2Cc2cccc(-n3cccn3)c2)c(-n2nccn2)c1
InChIKey: WYFQHYNFGVNXFG-OAQYLSRUSA-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 = 41000 nM (pChEMBL 4.389999866485596)
Descriptors: MW 428.5, ALogP 2.85, HBA 7, HBD 0, PSA 78.07, QED 0.49
Compound (SMILES): O=S(=O)(Nc1ncns1)c1ccc2c(c1)OCCN2c1ccc(C(F)(F)F)cc1Br
InChIKey: OZHJKBUBNACKJW-UHFFFAOYSA-N
Target: Sodium channel protein type 5 subunit alpha (SINGLE PROTEIN), Homo sapiens
UniProt: Q14524
Assay: In Vitro Assay: HEK 293 cells stably transfected with either Nav 1.7 or with Nav 1.5 were recorded in population patch-clamp mode with the IonWorks® Quattro automated electrophysiology system in accordance with the manufacturer's specifications (Molecular Devices, LLC, Sunnyvale, Calif.). Sodium channel currents were measured in response to a train of depolarizations that induced successively greater inactivation. Cells were held at −110 mV for three seconds (Nav 1.7) or half a second (Nav 1.5) from a holding voltage of −15 mV, then put through a series of 26 pulses of 150 msec duration to −20 mV at a frequency of 5 Hz. Cells were then left unclamped for a period of 3 to 8 minutes while a single concentration of test compound was added. Cells were then reclamped and put through the same voltage protocol. Current at the end of the 26th pulse to −20 mV was subtracted from the peak current evoked by the 26th pulse to −20 mV to correct for leak current.
Measurement: IC50 > 10000 nM (pChEMBL 5)
Descriptors: MW 521.34, ALogP 4.65, HBA 7, HBD 1, PSA 84.42, QED 0.54
Compound (SMILES): COc1cc(OC)cc(N(CCCN2CCC(O)C2)c2ccc3ncc(-c4cnn(C)c4)nc3c2)c1
InChIKey: WXAXVITWIABKBN-UHFFFAOYSA-N
Target: Fibroblast growth factor receptor 3 (SINGLE PROTEIN), Homo sapiens
UniProt: P22607
Assay: Inhibition of TEL-fused FGFR3 (unknown origin) transfected in mouse BA/F3 cells assessed as decrease in cell proliferation in presence of mouse IL3 after 72 hrs by Alamar blue assay
Measurement: IC50 = 0.5754 nM (pChEMBL 9.239999771118164)
Descriptors: MW 488.59, ALogP 3.64, HBA 9, HBD 1, PSA 88.77, QED 0.38
Compound (SMILES): CC(=O)N1CCc2c(c(Nc3cccc(C)c3)nn2CC2CC2)C1
InChIKey: XVPFIRFAOOSQTK-UHFFFAOYSA-N
Target: Bromodomain-containing protein 4 (SINGLE PROTEIN), Homo sapiens
UniProt: O60885
Assay: AlphaLisa Binding Assay: His/Flag epitope tagged BRD4 BD142-168 was cloned, expressed, and purified to. BRD4 binding and inhibition was assessed by monitoring the engagement of biotinylated H4-tetraacetyl peptide (New England Peptide, NEP2069-1/13) with the target using the AlphaLisa technology (Perkin-Elmer). Specifically, in a 384 well ProxiPlate BRD4(BD1) (30 nM final) was combined with peptide (200 nM final) in 40 mM HEPES (pH 7.0), 40 mM NaCl, 1 mM DTT, 0.01% (w/v) BSA, and 0.008% (w/v) Brij-35 either in the presence of DMSO (final 1.2% DMSO) or compound dilution series in DMSO. After 20 minutes incubation at room temperature Alpha streptavidin donor beads and AlphaLisa anti-Flag acceptor beads were added to a final concentration of 10 ug/mL each. After three hours equilibration plates were read on an Envision instrument and IC50s calculated using a four parameter non-linear curve fit.
Measurement: IC50 = 9080 nM (pChEMBL 5.039999961853027)
Descriptors: MW 324.43, ALogP 3.25, HBA 4, HBD 1, PSA 50.16, QED 0.94
Compound (SMILES): CC(=O)c1ccc(Oc2ccc3nc(C(=O)N4CCNCC4)ccc3c2)cc1
InChIKey: BVCBGQASRGPDLU-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 = 23600 nM (pChEMBL 4.630000114440918)
Descriptors: MW 375.43, ALogP 3.28, HBA 5, HBD 1, PSA 71.53, QED 0.71
Compound (SMILES): Cc1cccc2cc(Cn3nc(-c4cn[nH]c4)c4c(N)ncnc43)n(-c3ccccc3C(F)(F)F)c(=O)c12
InChIKey: PFJNZGKJQBZJQB-UHFFFAOYSA-N
Target: PI3-kinase p110-alpha/p85-alpha (PROTEIN COMPLEX), Homo sapiens
UniProt: P42336
Assay: Inhibition of N-terminal His6-tagged recombinant full-length human p110alpha/untagged recombinant full length human 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 > 10000 nM (pChEMBL 5)
Descriptors: MW 516.49, ALogP 4.48, HBA 8, HBD 2, PSA 120.3, QED 0.36
Compound (SMILES): COCCNC(=O)C(C)(C)n1cc(-c2cnc3ccc(N(CC4CC4)c4cc(OC)cc(OC)c4)cc3n2)cn1
InChIKey: NGLRSTVZLVHSJO-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 544.66, ALogP 4.56, HBA 9, HBD 1, PSA 103.63, QED 0.26
Compound (SMILES): N#Cc1c(N)ncnc1N1CCC(c2nc(-c3ccc(F)c(C(F)(F)F)c3)cn2CCN2CCC2)CC1
InChIKey: BVYUJCVPGYLIMY-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 = 700 nM (pChEMBL 6.159999847412109)
Descriptors: MW 514.53, ALogP 4.04, HBA 8, HBD 1, PSA 99.89, QED 0.5
Compound (SMILES): CN1CCN(c2cccc(-c3c[nH]c4ccc(-c5nnc(N)s5)cc34)n2)CC1
InChIKey: CSYNBNGKBZGJAV-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 = 0.625 nM (pChEMBL 9.199999809265137)
Descriptors: MW 391.5, ALogP 3.08, HBA 7, HBD 2, PSA 86.96, QED 0.56
Compound (SMILES): Cc1ncc(NC(=O)c2cccc(C(F)(F)F)c2)cc1-c1cnc(OC2CCOCC2)c(N2CCOCC2)c1
InChIKey: FYNMINFUAIDIFL-UHFFFAOYSA-N
Target: RAF proto-oncogene serine/threonine-protein kinase (SINGLE PROTEIN), Homo sapiens
UniProt: P04049
Assay: In Vitro Enzyme Assay: Final assay conditions were 4 pM cRaf, 3 uM ATP, 10 nM biotin tagged MEK1 kinase dead protein substrate. Reactions were performed in Greiner384 well plates, catalog #784075. Reaction buffer was 50 mM Tris, pH 7.5, 50 mM NaCl, 10 mM MgCl2, 0.01% Tween-20, 0.05% BSA and 1 mM DTT. 5 ul of 2×cRaf plus MEK1 were dispensed into plates containing 0.25 ul of compound in 100% DMSO and incubated at room temperature for 30 minutes. Reactions were started by addition of 5 ul of 2×ATP. Reactions were run for two hours and terminated by addition of 10 ul of 2× antibody and Alphascreen bead mixture in Stop Buffer. Stop Buffer was 50 mM Tris, pH 7.5, 0.005% Tween-20, 25 mM EDTA.
Measurement: IC50 = 0.119 nM (pChEMBL 9.920000076293945)
Descriptors: MW 542.56, ALogP 5.12, HBA 7, HBD 1, PSA 85.81, QED 0.46
Compound (SMILES): COc1cc(OC)cc(N(CC2CC2)c2ccc3ncc(-c4cnn(CCN5CCCC5)c4)nc3c2)c1.Cl
InChIKey: PAWXGXHMRSTMFA-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 = 8912.51 nM (pChEMBL 5.050000190734863)
Descriptors: MW 498.63, ALogP 5.15, HBA 8, HBD 0, PSA 68.54, QED 0.3
Compound (SMILES): CC(=O)N[C@@H](Cc1ccccc1)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CO)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@H](C(=O)N[C@@H](Cc1ccccc1)C(=O)N[C@H](C(=O)N[C@@H](CCCNC(=N)N)C(=O)NCC(=O)N[C@@H](Cc1ccc(O)cc1)C(=O)N[C@@H](Cc1ccccc1)C(=O)N[C@@H](CCC(=O)O)C(=O)N[C@@H](CCCNC(=N)N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCNC(=N)N)C(=O)N[C@@H](C)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC(C)C)C(N)=O)C(C)C)C(C)C
InChIKey: YLPSCMDCADAOHB-IBTGVXJRSA-N
Target: Tissue factor pathway inhibitor (SINGLE PROTEIN), Homo sapiens
UniProt: P10646
Assay: Inhibition of C-terminus 10x-His-taged human TFPI-1 (29 to 282 residues) assessed as inhibition of factor 10a using S-2222 substrate incubated for 115 mins by micro-plate reader based assay
Measurement: EC50 = 5090 nM (pChEMBL 5.289999961853027)
Descriptors: MW 2528.01
Compound (SMILES): COc1ccc(-n2nccn2)c(C(=O)N2C3CCC2C(COc2ccc(F)cn2)C3)c1
InChIKey: NORCPTXRDGPWOZ-UHFFFAOYSA-N
Target: Orexin receptor type 2 (SINGLE PROTEIN), Homo sapiens
UniProt: O43614
Assay: Radioligand Binding Assay: HEK293 stably expressing human orexin 2 receptor (Genebank accession number NM_001526) were grown to confluency in DMEM (Hyclone, cat #SH30022), 10% FBS, 1x Pen/Strep, 1x NaPyruvate, 10 mM HEPES, 600 ug/ml G418 media on 150 cm2 tissue culture plates, washed with 5 mM EDTA in PBS (HyClone Dulbecco's Phosphate Buffered Saline 1x with Calcium and Magnesium, Cat #SH30264.01, hereafter referred to simply as PBS) and scraped into 50 ml tubes. After centrifugation (2KxG, 5 min at 40° C.), the supernatant was aspirated and the pellets frozen and stored at -800° C. Cells were resuspended in PBS in the presence of 1 tablet of protease inhibitor cocktail (Roche, Cat. #11836145001) per 50 mL. Each cell pellet from a 15 cm plate was resuspended in 10 mL, stored on ice, and homogenized for 45 sec just prior to addition to the reactions. Competition binding experiments in 96 well polypropylene plates were performed using [3H]-EMPA (Moraveck Corporation, specific activity=29.6 Ci/mmol), diluted to a 5 nM concentration in PBS (2 nM final concentration). Compounds were solubilized in 100% DMSO (Acros Organics, Cat. #61042-1000) and tested over a range of 7 concentration (from 0.1 nM to 10 uM). The final concentration of DMSO in the reactions is equal to or less than 0.1%. Total and nonspecific binding was determined in the absence and presence of 10 uM almorexant. The total volume of each reaction is 200 uL (20 uL of diluted compounds, 80 uL of [3H]-EMPA diluted in PBS and 100 uL of the cell suspension). Reactions were run for 60 min at room temperature and terminated by filtration through GF/C filter plates (PerkinElmer, Cat. #6005174) presoaked in 0.3% polyethylenimine using the cell harvester (PerkinElmer Filtermate). The plates were washed 3 times by aspirating 30 ml PBS through the plates. Plates were dried in 55° C. oven for 60 min, scintillation fluid was added, and the radioactivity was counted on a Topcount (Packard).
Measurement: Ki = 73 nM (pChEMBL 7.139999866485596)
Descriptors: MW 423.45, ALogP 2.88, HBA 7, HBD 0, PSA 82.37, QED 0.61
Compound (SMILES): CC1c2ncn(-c3ccc(F)cn3)c2CCN1C(=O)c1cccc(C(F)(F)F)c1Cl
InChIKey: PLIXFSPGDVKMDM-UHFFFAOYSA-N
Target: P2X purinoceptor 7 (SINGLE PROTEIN), Homo sapiens
UniProt: Q99572
Assay: Radioligand Binding Assay: human or rat P2X7-1321 N1 cells were collected and frozen @−80° C. On the day of the experiment, cell membrane preparations were made according to standard published methods. The total assay volume was 100 μl: 10 μl compound (10×)+(b) 40 μl tracer (2.5×)+50 μl membrane (2×). The tracer used for the assay was tritiated A-804598. The compound can be prepared as described in the literature. (Donnelly-Roberts, D. Neuropharmacology 2009, 56 (1), 223-229.) Compounds, tracer and membranes were incubated for 1 hour @ 4° C. The assay was terminated by filtration (GF/B filters pre-soaked with 0.3% PEI) and washed with washing buffer (Tris-HCl 50 mM).
Measurement: Ki = 125.9 nM (pChEMBL 6.900000095367432)
Descriptors: MW 438.81, ALogP 4.84, HBA 4, HBD 0, PSA 51.02, QED 0.54
Compound (SMILES): O=C(COc1cccc2cccnc12)NC[C@H](O)CN1CCc2ccccc2C1
InChIKey: ZVCIZCPAJITPQB-FQEVSTJZSA-N
Target: PRMT5/MEP50 complex (PROTEIN COMPLEX), Homo sapiens
UniProt: Q9BQA1
Assay: Inhibition of human full length FLAG-tagged PRMT5/human His6-tagged MEP50 expressed in baculovirus-infected Sf9 cells assessed as reduction in tritium incorporation into peptide substrate using human Histone H4 (1 to 15 residues) and [3H]SAM as substrate preincubated with enzyme and H4 for 30 mins followed by [3H]SAM addition and measured after 120 mins by radioactive flashplate assay
Measurement: IC50 = 144 nM (pChEMBL 6.840000152587891)
Descriptors: MW 391.47, ALogP 2.15, HBA 5, HBD 2, PSA 74.69, QED 0.65
Compound (SMILES): O=c1cc(/C=C/c2ccc(C(F)(F)F)cc2)c2cc(C(c3ccc(Cl)cc3)c3nccs3)ccc2[nH]1
InChIKey: LWHNNHZAUBLVTM-DAFODLJHSA-N
Target: Cytochrome P450 2C19 (SINGLE PROTEIN), Homo sapiens
UniProt: P33261
Assay: Inhibition of CYP2C19 in human liver microsomes using (S)-mephenytoin as substrate after 10 to 30 mins by LC/MS analysis
Measurement: IC50 > 10000 nM (pChEMBL 5)
Descriptors: MW 522.98, ALogP 8.01, HBA 3, HBD 1, PSA 45.75, QED 0.25
Compound (SMILES): NC(=O)c1cc(-c2cn3cc(O)ccc3n2)ccc1O
InChIKey: JPSJWJIHWSYPQG-UHFFFAOYSA-N
Target: Casein kinase I delta/epsilon (PROTEIN COMPLEX), Homo sapiens
UniProt: P49674
Assay: Inhibition of CK1 delta/epsilon (unknown origin)
Measurement: IC50 > 10000 nM (pChEMBL 5)
Descriptors: MW 269.26, ALogP 1.51, HBA 5, HBD 3, PSA 100.85, QED 0.66
Compound (SMILES): CN1C(=O)C2(NC1=S)c1cc(Br)ccc1C[C@]21CC[C@@H](OC(F)F)CC1
InChIKey: OKVJTSBYGLSMLN-KWDRTTAMSA-N
Target: Beta-secretase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P56817
Assay: BACE Assay: For each compound being tested, the BACE activity was monitored in a fluorescence quenching assay (FRET) using the ectodomain of BACE (aa 1-454) fused to a myc-his tag and secreted from HEK293/BACEect. cells into OptiMEM (Invitrogen) as enzyme source and a substrate peptide derived from the APP-Swedish mutation which possesses a Cy3-fluorophore at the N-terminus and a Cy5Q-quencher at the C-terminus (Cy3-SEVNLDAEFK-Cy5Q-NH2; Amersham). The substrate was dissolved at 1 mg/mL in DMSO.The assay was performed in the presence of 5 μl OptiMEM (supernatant collected over 24 hours and cleared from cellular debris by centrifugation) containing the ectodomain of BACE, 25 μl water containing the desired concentration of test compound and 1% DMSO, 1 μM substrate peptide, 20 mM NaOAc, pH 4.4 and 0.04% Triton-X100 in a total assay volume of 50 μl in a 384 well plate. In general, 25 μl of compound dilution were given to the plate followed by the addition of 10 μl of BACE containing OptiMEM diluted 1:2 in water with 0.2% Triton X-100. The reaction was started with the addition of 15 μl substrate in NaOAc buffer. The reaction was incubated at 30° C. in a fluorimeter and the cleavage of the substrate was recorded as kinetic for 60 min. at ex: 530 nm, em: 590 nm. Blank wells containing either no inhibitor or no enzyme were included on each plate.The intensity of fluorescence was regressed against time in order to derive velocities of reaction in all 384 wells. These velocities were used for calculating percent inhibition using an uninhibited control containing 1% DMSO and a fully inhibited control incubations performed in the absence of enzyme. IC50 values were calculated by fitting percent inhibition vs. inhibitor concentration using standard software like GraphPadPrism.
Measurement: IC50 = 8.4 nM (pChEMBL 8.079999923706055)
Descriptors: MW 445.33, ALogP 3.72, HBA 3, HBD 1, PSA 41.57, QED 0.71
Compound (SMILES): Cc1ccc(C(=O)N2C[C@@H](C)OC[C@H]2Cc2cc(-c3ncccn3)ccc2F)c(-n2nccn2)c1
InChIKey: HYSRABRBUHZYQJ-WIYYLYMNSA-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 = 31000 nM (pChEMBL 4.510000228881836)
Descriptors: MW 472.52, ALogP 3.64, HBA 7, HBD 0, PSA 86.03, QED 0.44
Compound (SMILES): NC(=O)c1nnc(N[C@@H]2CCCC[C@@H]2N)nc1Nc1cccc2[nH]ncc12
InChIKey: XDTVNHHINGTFKX-GXFFZTMASA-N
Target: Tyrosine-protein kinase SYK (SINGLE PROTEIN), Homo sapiens
UniProt: P43405
Assay: Inhibition of SYK in human Ramos cells assessed as reduction in anti IgM-induced phosphorylation of BLNK by flow cytometry assay
Measurement: IC50 = 14 nM (pChEMBL 7.849999904632568)
Descriptors: MW 367.42, ALogP 1.27, HBA 8, HBD 5, PSA 160.52, QED 0.45
Compound (SMILES): CC#CC(=O)Nc1cccc(-c2cnc3[nH]cc(Cl)c3c2)n1
InChIKey: SHWCVOJZLIWKHV-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 310.74, ALogP 3.24, HBA 3, HBD 2, PSA 70.67, QED 0.71
Compound (SMILES): CN(C)C1(c2ccccc2)CCC2(CC1)OCCc1c2[nH]c2ccc(F)cc12
InChIKey: CSMVOZKEWSOFER-UHFFFAOYSA-N
Target: Mu-type opioid receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P35372
Assay: Binding Assay: The receptor affinity for the human mu-opiate receptor was determined in a homogeneous batch in microtitre plates. For this, dilution series of the particular spirocyclic cyclohexane derivative to be tested were incubated in a total volume of 250 ul for 90 minutes at room temperature with a receptor membrane preparation (15-40 ug protein per 250 ul incubation batch) of CHO-K1 cells, which express the human mu-opiate receptor (RB-HOM receptor membrane preparation of NEN, Zaventem, Belgium), in the presence of 1 nmol/l of the radioactive ligand [3H]-naloxone (NET719, NEN, Zaventem, Belgium) and of 1 mg WGA-SPA beads (wheat germ agglutinin SPA beads from Amersham/Pharmacia, Freiburg, Germany). 50 mmoles/l Tris-HCl supplemented with 0.05 wt. % sodium azide and with 0.06 wt. % bovine serum albumin was used as the incubation buffer. 25 umoles/l naloxone was additionally added for determination of the non-specific binding.
Measurement: Ki = 1.5 nM (pChEMBL 8.819999694824219)
Descriptors: MW 378.49, ALogP 5.11, HBA 2, HBD 1, PSA 28.26, QED 0.67
Compound (SMILES): CC(=O)N1CCc2nc(N3CCC(Oc4ccc(F)cc4F)CC3)c(N[C@H]3CCOC3)nc2C1
InChIKey: HSWVJQBEXRKOBZ-KRWDZBQOSA-N
Target: G-protein coupled receptor 6 (SINGLE PROTEIN), Homo sapiens
UniProt: P46095
Assay: Binding affinity to GPR6 (unknown origin) expressed in CHO-K1 cells by liquid scintillation counting based competition binding assay
Measurement: Ki = 6 nM (pChEMBL 8.220000267028809)
Descriptors: MW 473.52, ALogP 2.91, HBA 7, HBD 1, PSA 79.82, QED 0.72
Compound (SMILES): COc1cn(C(C)C(=O)Nc2ccc(C(=O)O)cc2)c(=O)cc1-c1cc(Cl)ccc1C
InChIKey: CVTZPKKDPIRSKS-UHFFFAOYSA-N
Target: Coagulation factor XI (SINGLE PROTEIN), Homo sapiens
UniProt: P03951
Assay: Measurement of FXIa Inhibition: To determine the factor XIa inhibition of the substances according to the invention, a biochemical test system is used which utilizes the reaction of a peptidic factor XIa substrate to determine the enzymatic activity of human factor XIa. Here, factor XIa cleaves from the peptic factor XIa substrate the C-terminal aminomethylcoumarin (AMC), the fluorescence of which is measured. The determinations are carried out in microtitre plates.Test substances are dissolved in dimethyl sulphoxide and serially diluted in dimethyl sulphoxide (3000 μM to 0.0078 μM; resulting final concentrations in the test: 50 μM to 0.00013 μM). In each case 1 μl of the diluted substance solutions are placed into the wells of white microtitre plates from Greiner (384 wells). 20 μl of assay buffer (50 mM of Tris/HCl pH 7.4; 100 mM of sodium chloride solution; 5 mM of calcium chloride solution; 0.1% of bovine serum albumin) and 20 μl of factor XIa from Kordia (0.45 nM in assay buffer) are then added successively. After 15 min of incubation, the enzyme reaction is started by addition of 20 μl of the factor XIa substrate Boc-Glu(OBzl)-Ala-Arg-AMC dissolved in assay buffer (10 μM in assay buffer) from Bachem, the mixture is incubated at room temperature (22° C.) for 30 min and fluorescence is then measured (excitation: 360 nm, emission: 460 nm). The measured emissions of the test batches with test substance are compared to those of control batches without test substance (only dimethyl sulphoxide instead of test substance in dimethyl sulphoxide), and the IC50 values are calculated from the concentration/activity relationships.
Measurement: IC50 = 700 nM (pChEMBL 6.159999847412109)
Descriptors: MW 440.88, ALogP 4.38, HBA 5, HBD 2, PSA 97.63, QED 0.59
Compound (SMILES): C[C@H](O)C(=O)N(CCN1[C@@H]2CC[C@H]1C[C@@H](c1cccc(C(N)=O)c1)C2)CC1CCC(F)(F)CC1.O=C(O)C(F)(F)F
InChIKey: BGENIZOIPPPDKJ-GWTRQPKJSA-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 477.6, ALogP 3.53, HBA 4, HBD 2, PSA 86.87, QED 0.6
Compound (SMILES): N#CCC1(n2cc(-c3ncnc4[nH]ccc34)cn2)CN(C2CCN(C(=O)Nc3ccc(F)c(F)c3)CC2)C1
InChIKey: YPAZKLQPBCGWIR-UHFFFAOYSA-N
Target: Tyrosine-protein kinase JAK1 (SINGLE PROTEIN), Homo sapiens
UniProt: P23458
Assay: In Vitro JAK Kinase Assay: Compounds herein were tested for inhibitory activity of JAK targets according to the following in vitro assay described in Park et al., Analytical Biochemistry 1999, 269, 94-104. The catalytic domains of human JAK1 (a.a. 837-1142), Jak2 (a.a. 828-1132) and Jak3 (a.a. 781-1124) with an N-terminal His tag were expressed using baculovirus in insect cells and purified. The catalytic activity of JAK1, JAK2 or JAK3 was assayed by measuring the phosphorylation of a biotinylated peptide. The phosphorylated peptide was detected by homogenous time resolved fluorescence (HTRF). IC50s of compounds were measured for each kinase in the 40 microL reactions that contain the enzyme, ATP and 500 nM peptide in 50 mM Tris (pH 7.8) buffer with 100 mM NaCl, 5 mM DTT, and 0.1 mg/mL (0.01%) BSA. For the 1 mM IC50 measurements, ATP concentration in the reactions was 1 mM. Reactions were carried out at room temperature for 1 hr and then stopped with 20 μL 45 mM EDTA, 300 nM SA-APC, 6 nM Eu-Py20 in assay buffer (Perkin Elmer, Boston, Mass.). Binding to the Europium labeled antibody took place for 40 minutes and HTRF signal was measured on a Fusion plate reader (Perkin Elmer, Boston, Mass.).
Measurement: IC50 < 10 nM (pChEMBL 8)
Descriptors: MW 517.54, ALogP 3.72, HBA 7, HBD 2, PSA 118.76, QED 0.42
Compound (SMILES): CCCc1nn(C)c2c(=O)[nH]c(-c3cc(N4CCC(CCC(=O)NO)CC4)ccc3OCC)nc12
InChIKey: MLAVJWXMLJDXJL-UHFFFAOYSA-N
Target: Histone deacetylase 2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q92769
Assay: Inhibition of full length recombinant human C-terminal His-tagged HDAC2 expressed in baculovirus infected Sf9 cells using fluorogenic HDAC substrate after 30 mins by fluorescence analysis
Measurement: IC50 = 2030 nM (pChEMBL 5.690000057220459)
Descriptors: MW 482.59, ALogP 3.18, HBA 8, HBD 3, PSA 125.37, QED 0.32
Compound (SMILES): Cc1ncc(C(CNC(=O)c2ccccc2Cl)N2CCC(F)CC2)cn1
InChIKey: NKXKARQRCRKMSM-UHFFFAOYSA-N
Target: P2X purinoceptor 7 (SINGLE PROTEIN), Homo sapiens
UniProt: Q99572
Assay: Binding Assay: Cell culture: 293 HEK cells, stably transfected with plasmids capable of expressing human P2X7 receptor, were cultured by standard methods. Cells were plated to cell density of approximately 15,000 cells/well in 384-well assay plates (50 μl/well) with 1.5% low serum media (DMEM, 1.5% BCS, 1% L-glut (2 mM), 1% P/S).293 HEK cells, stably transfected with plasmids capable of expressing rat or mouse P2X7 receptor, were cultured by standard methods. Cells were plated to cell density of approximately 15,000 cells/well in 384-well assay plates (50 μl/well) with 1.5% low serum media (DMEM, 1.5% FBS, 1% L-glut (2 mM), 10 mM HEPES, 1% P/S). Cells were plated 24 hours prior to assay. Cells expressing human, rat or mouse P2X7 receptor were assayed in the following manner.Fluorescent Imaging Plate Reader (FLIPR) assay: Briefly, 293-human or mouse P2X7 stable cells were incubated in sucrose buffer, pH 7.4 [KCl (5 mM), NaH2PO4.2H2O (9.6 mM), HEPES (25 mM), sucrose (280 mM), glucose (5 mM), CaCl2 (0.5 mM), and probenecid (0.1425 g in 3 mL 1N NaOH was added for 500 mL solution)] in 384-well plates.293-rat P2X7 stable cells were incubated in HHPB (pH 7.4) [consisting of Hank's BSS (1×); HEPES (pH 7.4) (20 mM) (Sigma); probenecid (0.710 g/5 mL 1N NaOH) (Sigma); and BSA (0.05%) (Roche) which was added after the pH had been adjusted] in 384-well plates. Fluo-4 NW dye mix (Molecular Probes, Inc., Eugene, Oreg., USA) was prepared in buffer (see manufacturer's instructions). Cell plates were removed from the 37° C. incubator, the media discarded and then 30 μL of dye was added to each well. Plates were placed in the 37° C., non-CO2 incubator for 30 minutes and then room temperature for 30 minutes.
Measurement: IC50 = 53 nM (pChEMBL 7.28000020980835)
Descriptors: MW 376.86, ALogP 3.34, HBA 4, HBD 1, PSA 58.12, QED 0.87
Compound (SMILES): COc1ncc(-c2ccc3nccc(-c4ccc(CN(C)C)cc4)c3c2)cc1NS(=O)(=O)c1ccc(F)cc1F
InChIKey: KPHKHHHLIKEDLQ-UHFFFAOYSA-N
Target: Phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit beta isoform (SINGLE PROTEIN), Homo sapiens
UniProt: P42338
Assay: Inhibition of PI3Kbeta (unknown origin) using PIP2 as substrate after 1 hr in presence of ATP by ADP-glo based luminescence assay
Measurement: IC50 = 1.8 nM (pChEMBL 8.739999771118164)
Descriptors: MW 560.63, ALogP 6.11, HBA 6, HBD 1, PSA 84.42, QED 0.25
Compound (SMILES): Cc1ccc(C(=O)N2CCOC[C@H]2Cc2cc(-n3nccn3)ccc2C)c(-n2nccn2)c1
InChIKey: QRQCKASOXSZLKU-OAQYLSRUSA-N
Target: Orexin/Hypocretin receptor type 1 (SINGLE PROTEIN), Homo sapiens
UniProt: O43613
Assay: OX Inhibition Assay: 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 ug/mL G418, 100 U/mL penicillin, 100 ug/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.
Measurement: IC50 = 5 nM (pChEMBL 8.300000190734863)
Descriptors: MW 443.51, ALogP 2.55, HBA 8, HBD 0, PSA 90.96, QED 0.47
Compound (SMILES): CCC(C(=O)Nc1cc(F)c(C(=O)O)cc1F)n1cc(OC)c(-c2cc(Cl)ccc2C#N)cc1=O
InChIKey: ZVXCULUAQHRZBD-UHFFFAOYSA-N
Target: Plasma kallikrein (SINGLE PROTEIN), Homo sapiens
UniProt: P03952
Assay: Inhibition Assay: Test substances are dissolved in dimethyl sulphoxide and serially diluted in dimethyl sulphoxide (3000 μM to 0.0078 μM; resulting final concentrations in the test: 50 μM to 0.00013 μM). In each case 1 μl of the diluted substance solutions are placed into the wells of white microtitre plates from Greiner (384 wells). 20 μl of assay buffer (50 mM Tris/HCl pH 7.4; 100 mM sodium chloride solution; 5 mM of calcium chloride solution; 0.1% of bovine serum albumin) and 20 μl of plasma kallikrein from Kordia (0.6 nM in assay buffer) are then added successively. After 15 min of incubation, the enzyme reaction is started by addition of 20 μl of the substrate H-Pro-Phe-Arg-AMC dissolved in assay buffer (10 μl in assay buffer) from Bachem, the mixture is incubated at room temperature (22° C.) for 30 min and fluorescence is then measured (excitation: 360 nm, emission: 460 nm).
Measurement: IC50 = 130 nM (pChEMBL 6.889999866485596)
Descriptors: MW 501.87, ALogP 4.62, HBA 6, HBD 2, PSA 121.42, QED 0.49
Compound (SMILES): CC(C(=O)NC1COc2ncccc21)c1cccc(C(F)(F)F)c1Cl
InChIKey: CGZWZKJDTFHJQE-UHFFFAOYSA-N
Target: P2X purinoceptor 7 (SINGLE PROTEIN), Homo sapiens
UniProt: Q99572
Assay: In Vitro Assay: Cell line generation was performed in general according to established molecular cloning protocols. Specifically, RNA was extracted from human whole blood using the Qiagen RNeasy kit (Qiagen, CH) according to the manufacturer's instructions. Subsequently cDNA was made (Superscript II, Invitrogen AG, CH) and the human P2X7 gene (genbank ref. BC011913) was amplified with the following primers: ATCGCGGCCGCTCAGTAAGGACTCTTGAAGCCACT and CGCCGCTAGCACCACCATGCCGGCCTGCTGCAGCTGCA. The amplified sequence was subsequently ligated into a pcDNA3.1 (+) NotI, NheI digested plasmid. Human embryonic kidney (HEK) cells (ATCC CRL-1573, Manassas, Va., USA) were transfected with the pcDNA3.1 (+). hP2X7 plasmid using lipofectamine 2000 (Invitrogen AG, CH) according to the manufacturer's instructions. Following a 24 h exposure to DNA, cells were trypsinized and re-seeded at low density in the presence of 250 ug Geneticin. Geneticin resistant cells were then selected during two consecutive rounds of cloning by serial limiting dilution with visual inspection.
Measurement: IC50 = 156 nM (pChEMBL 6.809999942779541)
Descriptors: MW 370.76, ALogP 4.11, HBA 3, HBD 1, PSA 51.22, QED 0.88
Compound (SMILES): Cc1ccc(NC(=O)c2cccc(C(F)(F)F)c2)cc1-c1cc(N2CCOCC2)nc(NC(CO)CO)n1
InChIKey: JCQCZVGYVYKECZ-UHFFFAOYSA-N
Target: RAF proto-oncogene serine/threonine-protein kinase (SINGLE PROTEIN), Homo sapiens
UniProt: P04049
Assay: In Vitro Enzyme Assay: Final assay conditions were 4 pM cRaf, 3 uM ATP, 10 nM biotin tagged MEK1 kinase dead protein substrate. Reactions were performed in Greiner384 well plates, catalog #784075. Reaction buffer was 50 mM Tris, pH 7.5, 50 mM NaCl, 10 mM MgCl2, 0.01% Tween-20, 0.05% BSA and 1 mM DTT. 5 ul of 2×cRaf plus MEK1 were dispensed into plates containing 0.25 ul of compound in 100% DMSO and incubated at room temperature for 30 minutes. Reactions were started by addition of 5 ul of 2×ATP. Reactions were run for two hours and terminated by addition of 10 ul of 2× antibody and Alphascreen bead mixture in Stop Buffer. Stop Buffer was 50 mM Tris, pH 7.5, 0.005% Tween-20, 25 mM EDTA.
Measurement: IC50 = 0.322 nM (pChEMBL 9.489999771118164)
Descriptors: MW 531.54, ALogP 3.32, HBA 8, HBD 4, PSA 119.84, QED 0.35
Compound (SMILES): O=C(NO)c1cnc(NC2(c3ccccc3)CCN(C(=O)Nc3ccccc3)CC2)nc1
InChIKey: WFKHLNIJJUZWBT-UHFFFAOYSA-N
Target: Histone deacetylase 3 (SINGLE PROTEIN), Homo sapiens
UniProt: O15379
Assay: HDAC Enzyme Assay: The compounds were diluted in assay buffer (50 mM HEPES, pH 7.4, 100 mM KCl, 0.001% Tween-20, 0.05% BSA, 20 uM TCEP) to 6 fold their final concentration. The HDAC enzymes (purchased from BPS Biosciences) were diluted to 1.5 fold their final concentration in assay buffer. The tripeptide substrate and trypsin at 0.05 uM final concentration were diluted in assay buffer at 6 fold their final concentration. The final enzyme concentrations used in these assays were 3.3 ng/ml (HDAC1), 0.2 ng/ml (HDAC2), 0.08 ng/ml (HDAC3) and 2 ng/ml (HDAC6). The final substrate concentrations used were 16 uM (HDAC1), 10 uM (HDAC2), 17 uM (HDAC3) and 14 uM (HDAC6). Five ul of compounds and 20 ul of enzyme were added to wells of a black, opaque 384 well plate in duplicate. Enzyme and compound were incubated together at room temperature for 10 minutes. Five u1 of substrate was added to each well, the plate was shaken for 60 seconds and placed into a Victor 2 microtiter plate reader. The development of fluorescence was monitored for 60 min and the linear rate of the reaction was calculated.
Measurement: IC50 = 1010 nM (pChEMBL 6)
Descriptors: MW 432.48, ALogP 3.23, HBA 6, HBD 4, PSA 119.48, QED 0.36
Compound (SMILES): COC(=O)[C@H](CO)NC(=O)[C@H](CCCC[N+](C)(C)C)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](C)NC(=O)[C@H](Cc1ccccc1)N(C)C(=O)CNC(=O)c1ccccc1
InChIKey: INFJDHQAVTWBET-KMACTHOASA-O
Target: Chromobox protein homolog 8 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9HC52
Assay: Antagonist activity at recombinant human N-terminal His-tagged CBX8 chromodomain (8 to 61 residues) expressed in Escherichia coli Rosetta BL21(DE3)pLysS using biotin-labeled H3K9me3 measured after 30 mins by AlphaScreen assay
Measurement: IC50 > 60000 nM (pChEMBL 4.2220001220703125)
Descriptors: MW 796.99, ALogP 0.53, HBA 9, HBD 6, PSA 212.34, QED 0.05
Compound (SMILES): Clc1cncc(OC[C@@H]2CN(CCN3CCc4ccccc43)CCO2)c1
InChIKey: QURJBQQAYVMKHZ-IBGZPJMESA-N
Target: D(4) dopamine receptor (SINGLE PROTEIN), Mus musculus
UniProt: P51436
Assay: Radioligand Binding Assay: The detailed experimental protocols for the radioligand and functional receptor assays are available on the NIMH PDSP website at http://pdsp.med.unc.edu/UNC-CH %20Protocol %20Book.pdf. A. Serotonin receptors: 5-HT1A, 5-HT1B, 5-HT1D, 5-HT1E, 5-HT2A, 5-HT2B, 5-HT2C, 5-HT3, 5-HT5A, 5-HT6 and 5-HT7. Assay Buffer: Standard Binding Buffer (50 mM Tris-HCl, 10 mM MgCl2, 1 mM EDTA, pH 7.4) Membrane Fraction Source: Transiently or stably transfected cell lines (e.g., HEK293, COS, CHO, NIH3T3). Protocol adapted from Roth et al. (1986), J. Pharmacol. Exp. Ther., 238(2): 480-485; and Roth et al. (1994), J. Pharmacol. Exp. Ther., 268(3): 1403-1410. Adrenergic Receptors: alpha1A, alpha1B, alpha2A, alpha2B, alpha2C, beta1, beta2, beta3 Assay Buffers: For alpha1 receptors, alpha1 Binding Buffer (20 mM Tris-HCl, 145 mM NaCl, pH 7.4); for alpha2 receptors, alpha2 Binding Buffer (50 mM Tris-HCl, 5 mM MgCl2, pH 7.7); for beta receptors.
Measurement: Ki = 90 nM (pChEMBL 7.050000190734863)
Descriptors: MW 373.88, ALogP 2.88, HBA 5, HBD 0, PSA 37.83, QED 0.78
Compound (SMILES): CCCCCCC(C)(C)c1cc(OC)c(C2C(O)[C@]3(C)CC[C@H]2C3(C)C)c(OC)c1
InChIKey: VTTPPQRKLCGACC-CUISRYIQSA-N
Target: Cannabinoid receptor 2 (SINGLE PROTEIN), Homo sapiens
UniProt: P34972
Assay: [35S]GTPγS Binding Assay: Human CB2 expressing CHO-K1 membranes (5 μg per incubation mixture) were diluted in 50 mM Tris-HCl (pH 7.5) and 0.5 mM EDTA and added to HU compounds in a pre-mixed incubation cocktail. Final incubation concentrations were 55 mM Tris-HCl (pH 7.4), 1 mM EDTA, 100 mM NaCl, 5 mM MgCl2, 0.5% BSA, 50 μM GDP, 0.2 nM [35S]GTPγS (PerkinElmer) with varied HU compound concentration and 5 μg membrane. Incubations were continued for 60 minutes at 30° C. in a shaking water bath. Assays were terminated by addition of 2 ml ice cold wash buffer (50 mM Tris-HCl, pH 7.5 and 5 mM MgCl2) and filtration through pre-soaked GF/C filters (Whatman), followed by two further washes.
Measurement: EC50 = 576.8 nM (pChEMBL 6.239999771118164)
Descriptors: MW 416.65, ALogP 6.85, HBA 3, HBD 1, PSA 38.69, QED 0.45
Compound (SMILES): Cn1ccnc1-c1ccccc1OCC(=O)NCC(O)CN1CCc2ccccc2C1
InChIKey: LRUSRUIYLHEVPD-UHFFFAOYSA-N
Target: Protein arginine N-methyltransferase 5 (SINGLE PROTEIN), Homo sapiens
UniProt: O14744
Assay: Biochemical Assay: Molecular Biology: Full-length human PRMT5 (NM_006109.3) transcript variant 1 clone was amplified from a fetal brain cDNA library, incorporating flanking 5′ sequence encoding a FLAG tag (MDYKDDDDK) (SEQ ID NO.:4) fused directly to Ala 2 of PRMT5. Full-length human MEP50 (NM_024102) clone was amplified from a human testis cDNA library incorporating a 5′ sequence encoding a 6-histidine tag (MHHHHHH) (SEQ ID NO.:5) fused directly to Arg 2 of MEP50. The amplified genes were sublconed into pENTR/D/TEV (Life Technologies) and subsequently transferred by Gateway attL×attR recombination to pDEST8 baculvirus expression vector (Life Technologies).Protein Expression. Recombinant baculovirus and Baculovirus-Infected Insect Cells (BIIC) were generated according to Bac-to-Bac kit instructions (Life Technologies) and Wasilko, 2006, respectively. Protein over-expression was accomplished by infecting exponentially growing Spodoptera frugiperda (SF9) cell culture at 1.2×106 cell/ml with a 5000 fold dilution of BIIC stock. Infections were carried out at 27° C. for 72 hours, harvested by centrifugation, and stored at −80° C. for purification.
Measurement: IC50 = 550 nM (pChEMBL 6.260000228881836)
Descriptors: MW 420.51, ALogP 2, HBA 6, HBD 2, PSA 79.62, QED 0.58
Compound (SMILES): CNc1cc2nc(C)c(-c3cc(NC(=O)NCCC(C)(C)F)c(F)cc3C)cc2cn1
InChIKey: WMVXSEQUHAGMDB-UHFFFAOYSA-N
Target: Serine/threonine-protein kinase A-Raf (SINGLE PROTEIN), Homo sapiens
UniProt: P10398
Assay: KiNativ assay: Sample preparation: A375 cells from ATCC are lysed by sonication in commercially available lysis buffer, cleared by centrifugation, and the resulting supernatant gel filtered into a commercially available kinase reaction buffer containing 20 mM MnCl2. Final protein concentration of lysates are 10 mg/mL. 5 ul of each test compound is added from 100 uM, 10 uM, 1 uM, or 0.1 uM stock solutions in DMSO to 500 uL of lysate in duplicate for final concentrations of 1 uM, 0.1 uM, 0.01 uM, and 0.001 uM. 5 ul, of DMSO is added to 500 ul of lysate in quadruplicate for controls. After 15 minute incubation, desthiobiotin-ATP acylphosphate probe is added to each sample to a final concentration of 5 uM and incubated with the samples for 10 minutes. Following the probe reaction, samples are prepared for targeted mass spectrum analysis using ActivX standard protocol.
Measurement: IC50 = 120 nM (pChEMBL 6.920000076293945)
Descriptors: MW 427.5, ALogP 5.35, HBA 4, HBD 3, PSA 78.94, QED 0.5
Compound (SMILES): CC(C)(C)CC(=O)N1CCC2(CC1)CC2CNC(=O)N1Cc2ccncc2C1
InChIKey: UIWIJQHEDGPZIR-UHFFFAOYSA-N
Target: Cytochrome P450 2C9 (SINGLE PROTEIN), Homo sapiens
UniProt: P11712
Assay: Reversible inhibition of CYP2C9 in human liver microsomes using (S)-warfarin as substrate in presence of NADPH by LC-MS/MS analysis
Measurement: IC50 > 10000 nM (pChEMBL 5)
Descriptors: MW 384.52, ALogP 3.17, HBA 3, HBD 1, PSA 65.54, QED 0.87
Compound (SMILES): C[C@@H]1CN(Cc2ccccc2)c2cccnc2[C@H](C)N1C(=O)NCc1ccccc1
InChIKey: DVUQADUMMXGGPJ-UXHICEINSA-N
Target: Voltage-dependent T-type calcium channel subunit alpha-1I (SINGLE PROTEIN), Homo sapiens
UniProt: Q9P0X4
Assay: Channel blocking activity at recombinant human CaV 3.3 channel expressed in HEK293 cells assessed as inhibition of Ca2+ flux preincubated for 3 mins followed by CaCl2 addition by fluo-4 dye-based FLIPR assay
Measurement: IC50 = 91 nM (pChEMBL 7.039999961853027)
Descriptors: MW 400.53, ALogP 4.76, HBA 3, HBD 1, PSA 48.47, QED 0.69
Compound (SMILES): N[C@H]1CCC[C@@H]1Oc1cncc(-c2cc3c(cn2)cnn3-c2ccccn2)n1
InChIKey: FHESSCZVRQCSRX-KSSFIOAISA-N
Target: Serine/threonine-protein kinase pim-3 (SINGLE PROTEIN), Homo sapiens
UniProt: Q86V86
Assay: Kinase Binding Assay: PIM-1, -2, and -3 enzymes were generated as fusion proteins expressed in bacteria and purified by IMAC column chromatography (Sun, X., Chiu, J. F., and He, Q. Y. (2005) Expert Rev. Proteomics, 2:649-657). A fluorescent-labeled Pim-specific peptide substrate, was custom synthesized by American Peptide Company (Sunnyvale, Calif.). Reaction Buffer contained 10 mM HEPES, pH 7.2, 10 mM MgCl2, 0.01% Tween 20, 2 mM DTT. Termination Buffer contained 190 mM HEPES, pH 7.2, 0.015% Brij-35, 0.2% Coating Reagent 3 (Caliper Life Sciences, Hopkinton, Mass.), 20 mM EDTA. Separation Buffer contained 100 mM HEPES, pH 7.2, 0.015% Brij-35, 0.1% Coating Reagent 3, 1:200 Coating Reagent 8 (Caliper Life Sciences, Hopkinton, Mass.), 10 mM EDTA and 5% DMSO.PIM reactions were carried out in a final volume of 10 uL per well in a 384-well plate. A standard enzymatic reaction, initiated by the addition of 5 uL 2Ã¿Â¿ATP and test compound to 5 uL of 2x enzyme and FAM-peptide, contained 20 uM PIM1, 50 uM PIM2, or 55 uM PIM3, 1 uM FAM-peptide, and 10 uM ATP, in Reaction Buffer. After 90 minutes of incubation at room temperature, the phosphorylation reaction was stopped by the addition of 10 uL Termination Buffer. The product and substrate in each independent reaction were separated on a 12-sipper microfluidic chip (Caliper Life Sciences, Hopkinton, Mass.) run on a Caliper LC3000 (Caliper Life Sciences, Hopkinton, Mass.). The separation of product and substrate was optimized by choosing voltages and pressure using Caliper's Optimizer software (Hopkinton, Mass.). The separation conditions used a downstream voltage of -500V, an upstream voltage of -2150V, and a screening pressure of -1.2 psi. The product and substrate fluorophore were excited at 488 nm and detected at 530 nm. Substrate conversion was calculated from the electropherogram using HTS Well Analyzer software (Caliper Life Sciences, Hopkinton, Mass.). Ki values for the test compound were calculated.
Measurement: Ki = 0.07 nM (pChEMBL 10.149999618530273)
Descriptors: MW 373.42, ALogP 2.53, HBA 8, HBD 1, PSA 104.63, QED 0.59
Compound (SMILES): O=S(=O)(NCC1CC1)c1ccc(Nc2ccc(F)cc2F)cc1
InChIKey: UIXDCOGMNBQWMX-UHFFFAOYSA-N
Target: Cannabinoid receptor 2 (SINGLE PROTEIN), Homo sapiens
UniProt: P34972
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: IC50 = 1900 nM (pChEMBL 5.71999979019165)
Descriptors: MW 338.38, ALogP 3.4, HBA 3, HBD 2, PSA 58.2, QED 0.85