lovingscience/chembl-v37-raw-activities huggingface dataset:
Compound (SMILES): CC(=O)N1CCC(c2ccccc2[C@H]2CCCN2c2ccc(-c3ccc(C(=O)NS(=O)(=O)c4ccc(NCC5CCOCC5)c([N+](=O)[O-])c4)c(Oc4cnc5[nH]ccc5c4)c3)cc2)CC1
InChIKey: SARKDBRGPPXLPC-WBVITSLISA-N
Target: Apoptosis regulator Bcl-2 (SINGLE PROTEIN), Homo sapiens
UniProt: P10415
Assay: Bcl-2/Bcl-X TR-FRET Assay: Compounds disclosed herein were tested for blocking of Bcl-2/Bcl-X protein with its ligand in an assay based on Time-Resolved Fluorescence Resonance Energy Transfer (TR-FRET) methodology. Recombinant human 0.05 nM Bcl-2/0.03 nM Bcl-X protein was pre-incubated with a serial dilution of compounds disclosed herein (maximum concentration is 0.1 μM for Bcl-2 assay, and 10 μM for Bcl-xl assay, 3-fold serially diluted, 10 points; or maximum concentration is 0.02 μM for Bcl-2 assay, and 2 μM for Bcl-xl assay, 3-fold serially diluted, 10 points) at room temperature for 0.5 hour in an assay buffer containing 20 mM potassium phosphate buffer, pH 7.5, 50 mM NaCl, 1 mM EDTA, 0.05% Tween-20, 0.01% BSA. Then the FITC labeled Bak peptide Ac-GQVGRQLAIIGDK(FITC)INR-amide (0.5 nM for Bcl-2, 0.3 nM for Bcl-xl) and MAb Anti 6His Tb cryptate Gold were added to plate and further incubated at room temperature for 1 hour. The TR-FRET signals (337 nm-520 nm-490 nm) were read on BMG PHERAstar FSX instrument. The inhibition percentage of Bcl-2/Bcl-X interaction with its ligand in presence of increasing concentrations of compounds was calculated based on the TR-FRET signals. The IC50 for each compound was derived from fitting the data to the four-parameter logistic equation by Graphpad Prism software. To improve the assay sensitivity and test more potent compounds in the present application, the bcl-2 concentration was reduced in method B.
Measurement: IC50 = 3 nM (pChEMBL 8.520000457763672)
Descriptors: MW 898.05, ALogP 8.96, HBA 11, HBD 3, PSA 189.1, QED 0.07
Compound (SMILES): CC(C)(O)CCc1cn(-c2cnc(-n3ncc4cc(C#N)cnc43)cc2NC2COCC2F)nn1
InChIKey: LNKUFPHUASVECL-UHFFFAOYSA-N
Target: Interleukin-1 receptor-associated kinase 4 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9NWZ3
Assay: IRAK4 Inhibition Assay: The assays were performed in U-bottom 384-well plates. The final assay volume was 30 μL prepared from 15 μL additions of enzyme and substrates (fluoresceinated peptide and ATP) and test compounds in assay buffer (20 mM HEPES pH 7.2, 10 mM MgCl2, 0.015% Brij 35 and 4 mM DTT). The reaction was initiated by the combination of IRAK4 with substrates and test compounds. The reaction mixture was incubated at room temperature for 60 min. and terminated by adding 45 μL of 35 mM EDTA to each sample. The reaction mixture was analyzed on the Caliper LABCHIP® 3000 (Caliper, Hopkinton, Mass.) by electrophoretic separation of the fluorescent substrate and phosphorylated product. Inhibition data were calculated by comparison to no enzyme control reactions for 100% inhibition and vehicle-only reactions for 0% inhibition. The final concentrations of reagents in the assays are ATP, 500 μM; FL-IPTSPITTTYFFFKKK peptide 1.5 μM; IRAK4, 0.6 nM; and DMSO, 1.6%.
Measurement: IC50 = 5 nM (pChEMBL 8.300000190734863)
Descriptors: MW 477.5, ALogP 2.12, HBA 11, HBD 2, PSA 139.59, QED 0.41
Compound (SMILES): C=CC(=O)N[C@H]1CCC[C@H]1NC(=O)c1sc2nccc3c2c1NC(=O)N3c1ccc(Oc2cccnc2)cn1
InChIKey: ZFZDSKFHERYTNT-ZWKOTPCHSA-N
Target: Tyrosine-protein kinase BTK (SINGLE PROTEIN), Homo sapiens
UniProt: Q06187
Assay: Kinase Lanthascreen Binding Assay: A BTK kinase lanthascreen binding assay monitors compound binding to unphosphorylated-BTK kinase domain (UP-BTK), by competing with a fluorescent labeled tracer. UP-BTK, consisting of the kinase domain of non-phosphorylated BTK protein (389-659aa), was produced in a Baculovirus/insect cell expression system. Into a 384-well plate, 2 ng of GST-tagged human BTK (389-659aa) was incubated with compound, 50 nM of Tracer 236 and 2 nM anti-GST antibody for 60 minutes using an optimized Lanthascreen assay. After 60 minutes, plates were read at 340 nM and 615/665 nM in an Infinite F500 (Tecan).
Measurement: IC50 = 37.2 nM (pChEMBL 7.429999828338623)
Descriptors: MW 541.59, ALogP 4.52, HBA 8, HBD 3, PSA 138.44, QED 0.29
Compound (SMILES): COc1ccc(Cn2c(=O)n3ncnc3c3cc(N4CCC(N(C)C)CC4)cnc32)cc1
InChIKey: NPXNBDQDCHRUQN-UHFFFAOYSA-N
Target: Dual specificity calcium/calmodulin-dependent 3',5'-cyclic nucleotide phosphodiesterase 1B (SINGLE PROTEIN), Homo sapiens
UniProt: Q01064
Assay: PDE1B Inhibitory Assay: PDE1B inhibition was determined by an IMAP TR-FRET assay. The IMAP TR-FRET PDE assay was optimized for concentration of enzyme, Calmodulin, cAMP or cGMP substrate, DMSO tolerance, and incubation time. Into each well of a solid white 1536 well plate (Corning) was dispensed 250 pg full-length recombinant NH-terminal GST tagged human PDE1B enzyme (BPS Bioscience Cat #60011, San Diego, Calif.) in 2.5 μL IMAP BSA reaction buffer (Molecular Devices, Sunnyvale, Calif.) containing 10 U/ml Calmodulin and 2.5 mM CaCl2 (Sigma Aldrich.) After a brief centrifugation, 30 nl compound was added by transfer from 1 mM stock in DMSO using a Kalypsys 1536 Pintool. Plates were incubated for 5 minutes at room temperature before dispensing 1.5 μL of 533 nM 5-carboxy fluorescein (FAM)-labeled cAMP (Molecular Devices, Sunnyvale, Calif.) for a final concentration of 200 nM. After a brief centrifugation, the plates were incubated for 30 minutes at room temperature. The assay was terminated by adding 5 μL IMAP binding reagent/Tb complex (Molecular Devices, Sunnyvale, Calif.) to each well. Plates were incubated 1 hour at room temperature and read on a Viewlux multimode plate reader (Perkin Elmer). The instrument was set to excite using the DUG11 filter and measure using 490/10 nm and 520/10 nm filters. Ratios of acceptor and donor were then calculated.
Measurement: IC50 = 550 nM (pChEMBL 6.260000228881836)
Descriptors: MW 433.52, ALogP 2.03, HBA 9, HBD 0, PSA 80.79, QED 0.48
Compound (SMILES): N#Cc1cc(-c2ccnc3[nH]c(C4=CCNC4)cc23)ccc1OC1CCOCC1
InChIKey: JSJHGXYXYWXMNC-UHFFFAOYSA-N
Target: Serine/threonine-protein kinase TBK1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9UHD2
Assay: Biological Assay for TBK1 and IKKs: Enzymatic activity of IKKε and TBK1 was measured using a homogeneous time resolved fluorescence resonance energy transfer (TR-FRET) assay that monitors enzyme dependent phosphorylation of a biotinylated serine/threonine peptide substrate. An increase in the amount of phopshorylated peptide results in an increase in TR-FRET signal. TBK1 and IKKs were expressed and purified as full length recombinant proteins. Detection reagents for the assay were purchased from Cisbio. TBK1 and IKKε enzymes were assayed under initial rate conditions in the presence of 2× Km ATP (40-80 μM) and 1 μM peptide, hepes (pH 7), 0.1 mM orthovanadate, 0.02% NaN3, 0.01% BSA, 10 mM MgCl2, 0.01% (v/v) tritonX, 1 mM dithiothreitol, 0.5% (v/v) DMSO at the following concentrations for each enzyme: TBK1 at 2.5 nM and IKKε at 0.3 nM. After an assay reaction time of 240 minutes at 25° C., reactions were terminated with EDTA.Amount of phosphorylated peptide was determined by the addition of 125 nM streptavidin XL665 and europium cryptate labeled anti-phospho monoclonal antibody and the resulting TR-FRET signal was recorded on an Envision plate reader (Ex: 340 nm; Em: 615/665 nm; 100 μs delay and 200 μs read window). Data was normalized based on a positive (1 μM Staurosporine) and negative (DMSO) controls and IC50 values calculated from the fit of the dose-response curves to a four-parameter equation. All IC50 values represent geometric mean values of a minimum of four determinations.
Measurement: IC50 = 13.4 nM (pChEMBL 7.869999885559082)
Descriptors: MW 386.46, ALogP 3.65, HBA 5, HBD 2, PSA 82.96, QED 0.72
Compound (SMILES): O=C(c1ccc(-c2ccccc2)cc1)N1CCn2c(nnc2-c2nc(-c3ccc(F)cc3)no2)C1
InChIKey: JLEYSSCROZCMCB-UHFFFAOYSA-N
Target: Neuromedin-K receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P29371
Assay: 3H-SB222200 Binding Competition Assay with Human NK-3 Receptor: The ability of the compounds of the invention to inhibit the binding of the NK-3 receptor selective antagonist SB222200 was assessed by an in vitro radioligand binding assay. Membranes were prepared from Chinese hamster ovary recombinant cells which express the human NK3 receptor. The membranes were incubated with 5 nM 3H-SB222200_(ARC) in a HEPES 25 mM/NaCl 0.1M/CaCl2 1 mM/MgCl2 5 Mm/BSA 0.5%/Saponin 10 μg/ml buffer at pH 7.4 and various concentrations of the compounds of the invention. The amount of tritiated SB222200 bound to the receptor was determined after filtration by the quantification of membrane associated radioactivity using the TopCount-NXT reader (Packard). Competition curves were obtained for compounds of the invention and the concentrations of compounds which displaced 50% of bound radioligand (IC50) were determined and then apparent inhibition constant Ki values were calculated by the following equation: Ki=IC50/(1+[L]/KD) where [L] is the concentration of free radioligand and KD is its dissociation constant at the receptor, derived from saturation binding experiments (Cheng and Prusoff, 1973).
Measurement: Ki < 500 nM (pChEMBL 6.301000118255615)
Descriptors: MW 466.48, ALogP 4.46, HBA 7, HBD 0, PSA 89.94, QED 0.39
Compound (SMILES): COc1cc2nc(N)n3nc([C@@H]4CCCN(c5cnn(CCCC(C)=O)c5)C4)nc3c2cc1F
InChIKey: BIIUSNNKNOZUOG-OAHLLOKOSA-N
Target: Adenosine receptor A2a (SINGLE PROTEIN), Homo sapiens
UniProt: P29274
Assay: Measurement of A2a Binding Affinity Using Radioligand Binding: 148 μL (5 μg/mL) membranes (Perkin Elmer, Cat. No. RBHA2aM400UA) and 2 μL compounds of the invention to be tested (test compound) were transferred to individual wells of a 96-well polypropylene assay plate and incubated for 15 to 30 minutes at room temperature. [3H] SCH58261 ((7-(2-phenylethyl)-5-amino-2-(2-furyl)-pyrazolo-[4,3-e]-1,2,4-triazolo[1,5-c]pyrimidine)) was diluted in assay buffer (50 mM Tris pH 7.4, 10 mM MgCl2, 0.005% Tween20) to a concentration of 4 nM and 50 μL transferred to each well of the assay plate. To define total and non-specific binding, wells containing 1% DMSO and 1 μM ZM241385 (Tocris Bioscience, Cat. No. 1036) respectively, were also included. The assay plate was incubated at room temperature for 60 minutes with agitation. Using a FilterMate Harvester® (Perkin Elmer), the contents of the assay plate were filtered through a UniFilter-96® PEI coated plate (Perkin Elmer Cat. No. 6005274 or 6005277). Filtering was achieved by aspirating the contents of the assay plate for 5 seconds, then washing and aspirating the contents three times with ice-cooled wash buffer (50 mM Tris-HCl pH 7.4, 150 mM NaCl) and allowing the vacuum manifold to dry the plate for 30 seconds. The filter plate was incubated for at least 1 hour at 55° C., and allowed to dry. The bottom of the filter plate was sealed with backing tape. 40 μL Ultima Gold (Perkin Elmer, Cat. No. 6013329) was added to each well of the filter plate and the top of the plate was sealed with TopSeal-A PLUS® clear plate seal (Perkin Elmer, Cat. No. 6050185). The plate was incubated for at least 20 minutes, and then the amount of radioactivity remaining in each well was determined using a TopCount® (Perkin Elmer) scintillation counter. After normalization to total and non-specific binding, the percent effect at each compound concentration was calculated. The plot of percent effect versus the log of compound concentration was analyzed electronically using a 4-parameter logistic fit based on the Levenberg-Marquardt algorithm to generate IC50 values.
Measurement: IC50 = 5.8 nM (pChEMBL 8.239999771118164)
Descriptors: MW 466.52, ALogP 2.96, HBA 10, HBD 1, PSA 116.46, QED 0.44
Compound (SMILES): Cc1cc(Nc2nccc(-c3cc(C#N)c4c(c3)[C@@](C)(CO)CN4)n2)n(CC2CCCOC2)n1
InChIKey: OGHTWIMFXMQQDO-DNIBEGIYSA-N
Target: Mitogen-activated protein kinase kinase kinase 14 (SINGLE PROTEIN), Homo sapiens
UniProt: Q99558
Assay: Inhibition of auto-phosphorylation of recombinant human NF-kappaB-inducing kinase (NIK/MAP3K14) activity: NIK/MAP3K14 auto-phosphorylation activity was measured using the AlphaScreen® (αscreen) format (Perkin Elmer). All compounds tested were dissolved in dimethyl sulfoxide (DMSO) and further dilutions were made in assay buffer. Final DMSO concentration was 1% (v/v) in assays. Assay buffer was 50 mM Tris pH 7.5 containing 1 mM EGTA (ethylene glycol tetraacetic acid), 1 mM DTT (dithiothreitol), 0.1 mM Na3VO4, 5 mM MgCl2, 0.01% Tween® 20. Assays were carried out in 384 well Alphaplates (Perkin Elmer). Incubations consisted of compound, 25 microM Adenosine-5′-triphosphate (ATP), and 0.2 nM NIK/MAP3K14. Incubations were initiated by addition of GST-tagged NIK/MAP3K14 enzyme, carried out for 1 h at 25° C. and terminated by addition of stop buffer containing anti-phospho-IKK Ser176/180 antibody. Protein A Acceptor and Glutathione-Donor beads were added before reading using an EnVision® Multilabel Plate Reader (Perkin Elmer). Signal obtained in the wells containing blank samples was subtracted from all other wells and IC50's were determined by fitting a sigmoidal curve to % inhibition of control versus Log10 compound concentration.
Measurement: IC50 = 5.6 nM (pChEMBL 8.25)
Descriptors: MW 459.55, ALogP 3.37, HBA 9, HBD 3, PSA 120.91, QED 0.51
Compound (SMILES): CO[C@H]1C[C@@H](c2ncc3nc(C)n(CCOc4ccc(Cl)cc4-c4cc(C)nc5c(C(=O)O)csc45)c(=O)c3c2C#N)C1
InChIKey: UNRHAGMTYYITKV-IZAXUBKRSA-N
Target: Eukaryotic translation initiation factor 4E-binding protein 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q13541
Assay: Fluorescence Polarization Assay: eIF4E protein was pre-incubated with EDA-m7GDP-ATTO-550 in FPB at 2xconcentrations for 5 minutes prior to the addition of compounds. Compounds (100x) were prepared using 3-fold serial dilutions in 100% DMSO, and subsequently diluted 1:50 in FPB to produce 2x stocks. 50 ul of 2x eIF4E/EDA-m7GDP-ATTO-550 were transferred to the wells of a 96-well half-area black flat bottom polystyrene plate. 50 ul of 2x test compound were added, and binding reactions were allowed to equilibrate for 30 minutes at room temperature while protected from light. The fluorescent polarization signal was detected using a Victor 2 multi-label counter (Perkin Elmer) and the concentration necessary to achieve inhibition of eIF4E/EDA-m7GDP-ATTO-550 binding by 50% (IC50) was calculated using data from an 8-point compound dilution series.
Measurement: IC50 = 3.8 nM (pChEMBL 8.420000076293945)
Descriptors: MW 616.1, ALogP 5.88, HBA 10, HBD 1, PSA 140.22, QED 0.23
Compound (SMILES): Cc1c(Cl)c2c(Cl)c(C)c1-c1c(-c3ccc(F)cc3)sc3ncnc(c13)O[C@@H](C(=O)O)Cc1cc(ccc1OCc1ccnc(N3CCO[C@@H](COCC4COCCO4)C3)n1)OC[C@@H](CN1CCN(C)CC1)O2
InChIKey: VPNQTVBAKONRDL-VSMJZUQOSA-N
Target: Induced myeloid leukemia cell differentiation protein Mcl-1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q07820
Assay: Exemplary MCL-1 Inhibitors Bind MCL-1: The ability of the exemplary MCL-1 inhibitors of Examples 1 through 151 to bind MCL-1 was demonstrated using the Time Resolved-Fluorescence Resonance Energy Transfer (TR-FRET) Assay. Tb-anti-GST antibody was purchased from Invitrogen (Catalog No. PV4216). The ability of exemplary MCL-1 inhibitors Example 1 to Example 3 to compete with probe F-Bak for binding MCL-1 was demonstrated using a Time Resolved Fluorescence Resonance Energy Transfer (TR-FRET) binding assay.MethodFor the assay, an acoustic dispenser was used to prepare dilution series from 10 mM test compounds in 100% DMSO and directly transfer 160 nL into low volume 384-well assay plates. 8 μL of a protein/probe/antibody mix was then added to each well resulting in final concentrations listed below: Test compound: 11 three-fold dilutions beginning at 25 μMProtein: GST-MCL-1  1 nM Antibody Tb-anti-GST  1 nM Probe: F-Bak 100 nMThe samples were then mixed on a shaker for 1 minute and 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 (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, 1995, FEBS Lett. 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 = 0.058 nM (pChEMBL 10.239999771118164)
Descriptors: MW 1061.03
Compound (SMILES): O=C(NCc1cc(N2CCC(C(F)(F)F)CC2)c(C(F)(F)F)cn1)[C@@H]1C[C@@H](F)CN1S(=O)(=O)c1ccc(F)cc1
InChIKey: LOEVTFQCTSNTAC-IERDGZPVSA-N
Target: Transient receptor potential cation channel subfamily A member 1 (SINGLE PROTEIN), Homo sapiens
UniProt: O75762
Assay: FLIPR Assay: IC50s (effective concentration) of compounds on the human and rat TRPA1 channels were determined using a FLIPR Tetra instrument. CHO cells expressing TRPA1 were plated into 384-well plates, incubated overnight at 37C, and loaded with BD calcium indicator dye for 1 hr at 37° C. followed by 15 minutes. at room temperature. The assay buffer was Hank's Balanced Salt Solution (HBSS) containing 20 mM HEPES (pH readjusted to 7.4) along with 0.02% BSA.Following dye load and plate cool down, compounds were added to the cells using FLIPR Tetra. Plates were then incubated with compounds for 20 minutes at room temperature prior to adding agonist. Following this incubation, EC80 concentration of cinnamaldehyde (75 uM for human TRPA1 and 45 uM for rat TRPA1) was added to active the channels and block of cinnamaldehyde induced calcium influx was measured.
Measurement: IC50 = 52 nM (pChEMBL 7.28000020980835)
Descriptors: MW 600.53, ALogP 4.44, HBA 5, HBD 1, PSA 82.61, QED 0.5
Compound (SMILES): COc1ccc(Br)cc1C(=O)Nc1cc(-c2nnc(-c3ccco3)o2)ccc1F
InChIKey: ZHRXLPORGAYZQI-UHFFFAOYSA-N
Target: Lysocardiolipin acyltransferase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q6UWP7
Assay: In Vitro Acyltransferase Assays: ALCAT1 enzymatic activity was determined by measuring the conversion of monolysocardiolipin (MLCL) to cardiolipin or lysophosphatidylglycerol (LPG) to phosphatidylglycerol (PG) in an enzymatic reaction mixture that contained 50 mm Tris/HCl, pH 7.0, 100 μm lysophospholipids, 25 μm [14C]acyl-CoA (50 mCi/mmol, American Radiolabeled Chemicals, Inc), and membrane fraction (0.5-2.5 μg) in a total volume of 200 μl. The reaction was incubated at room temperature for 30 min. The lipids were extracted will chloroform, dried, and separated by thin layer chromatography (TLC) with chloroform:hexane:methanol:acetic acid (50:30:10:5, v/v) or chloroform:methanol:water (65:25:4, v/v). After separation, TLC plates were exposed to a Phosphorlmager screen to visualize the radiolabeled products with a Molecular Dynamics Typhoon Scanner (Sunnyvale, Calif.). In some experiments, NBD-CoA was used at 100 μM to replace the [14C]acyl-CoA in the enzymatic reaction. All quantitative data were expressed as mean±S.E. Statistical analyses for differences between two groups were carried out using a Student's t test.
Measurement: IC50 < 35 nM (pChEMBL 7.455999851226807)
Descriptors: MW 458.24, ALogP 5.16, HBA 6, HBD 1, PSA 90.39, QED 0.44
Compound (SMILES): O=C(O)c1nn(CC(=O)C2CCc3[nH]nnc3C2)cc1-c1cnc(NC2Cc3ccccc3C2)nc1
InChIKey: BOMBLUPDZJCKAC-UHFFFAOYSA-N
Target: Autotaxin (SINGLE PROTEIN), Homo sapiens
UniProt: Q13822
Assay: Inhibitory Activity on Human ENPP2: Two-fold dilution of each test compound solution (10 μM, 100% dimethyl sulfoxide) is carried out on 96-well V bottom plate (Costar 3363). After ten-fold dilution of each the test compound solution (100% dimethyl sulfoxide) with deionized distilled water, 10 μL of the diluted each compound solution (10% dimethyl sulfoxide) was aliquoted to a black flat bottom 96-well plate (Costar 3915). 50 μL of 1.6× Assay solution (224 mM NaCl, 80 mM Tris-HCl (pH 8.0), 8 mM KCl, 1.6 mM CaCl2, 1.6 mM MgCl2, and 1.6 mg/mL fatty acid free BSA) was added thereto, and subsequently 20 μL of 20 nM human ENPP2 solution (buffer solution: 140 mM NaCl, 50 mM Tris-HCl (pH 8.0), 5 mM KCl, 1 mM CaCl2, 1 mM MgCl2, and 1 mg/mL fatty acid free BSA) and 20 μL of 5 μM FS-3 solution (buffer solution: deionized distilled water) were added thereto, respectively, followed by mixing. Under reaction for 30 minutes at 37° C., fluorescence intensity measurement (Ex: 485 mm, Em: 528 mm) was carried out at every 5 minutes by Envision Xcite Multilabel Reader. ΔCFU30 min value (CFU value measured at 30 minutes−CFU value measured at 0 minute) is obtained for each test solution and based on the equation of 100−(ΔCFU30 min of test solution/Mean value of ΔCFU30 min of control group)×100, the inhibitory activity percentage ratio (i.e., % inhibition) is obtained.
Measurement: IC50 = 1.5 nM (pChEMBL 8.819999694824219)
Descriptors: MW 484.52, ALogP 2.11, HBA 9, HBD 3, PSA 151.57, QED 0.36
Compound (SMILES): O=C(O)C1CN(c2ccnc(-c3cnc4ccc(C(F)(F)F)cn34)n2)CC(F)(F)C1
InChIKey: BMCQRCVHHKZWJG-UHFFFAOYSA-N
Target: eIF-2-alpha kinase GCN2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9P2K8
Assay: Enzyme Inhibition Assay: An assay buffer containing GCN2 kinase and (RS)7 was prepared. 4.7 μL of this stock solution was placed per well of a black, low volume, 384-well microtitre plate (e.g. catalogue number 3676, Corning Inc., NY). To this was added 0.65 μM of DMSO containing serial dilutions of the test compound (typical final concentrations of test compound were 0 to 8 μM). The plate was incubated for 10 minutes at 25° C. prior to the addition of 4.7 μL of ATP stock buffer to initiate the enzyme reaction. The reaction was allowed to proceed for 1 hour at 25° C., prior to the addition of 10 μL detection buffer (consisting of appropriate concentrations of ADP2 antibody and ADP Alexa633 tracer in 1× stop and detect buffer as supplied by BellBrook Labs). The reaction was left to incubate for 1 hour at 25° C., prior to measuring the fluorescence polarisation signal (mP) in each well using a PHERAstar FS reader (BMG Labtech, Germany).
Measurement: IC50 = 55 nM (pChEMBL 7.260000228881836)
Descriptors: MW 427.33, ALogP 3.36, HBA 6, HBD 1, PSA 83.62, QED 0.65
Compound (SMILES): CCn1c(C)nnc1-c1cccc(N2Cc3c(cc(N4CCC[C@H]4C)nc3[C@@H](C)N)C2=O)n1
InChIKey: YSLGZDDMLIYNIJ-HUUCEWRRSA-N
Target: Mitogen-activated protein kinase kinase kinase kinase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q92918
Assay: Biochemical Selectivity Kinase Assay: PRKD2 (PKD2) and RPS6KA2 (RSK3) kinase assays were performed with Km-levels of ATP at the Thermo Fisher Scientific, Inc. (Madison, Wis.), using their Invitrogen SelectScreen fluorescence resonance energy transfer (FRET) Z′-LYTE technology, based on the differential sensitivity of phosphorylated and nonphosphorylated peptides to proteolytic cleavage. Human recombinant full-length GST-tagged PRKD2 (PKD2) and RPS6KA2 (RSK3) were produced by Thermo Fisher Scientific, Inc. (Madison, Wis.). The kinase assays were conducted in 10-μL reactions. PRKD2 (PKD2) reactions contained 0.64-5.84 ng enzyme, 25 μM ATP (Km), 2 μM of Ser/Thr 17 (Z′-LYTE peptide substrate), 10 mM MgCl2, 0.01% BRIJ-35, 1 mM EGTA in 50 mM HEPES, pH 7.5. RPS6KA2 (RSK3) reactions were conducted similarly, and contained 0.5-9 ng enzyme, or 10 μM ATP (Km) and 2 μM Ser/Thr 06 peptide. After the 1-hour kinase reaction incubation, 5 μL of a 1:256 and 1:4096 dilution of Z′-LYTE Development Reagent A was added to the PRKD2 (PKD2) and RPS6KA2 (RSK3) reactions, respectively. The extent of kinase reactions, resulting in a change of FRET signal of the peptide substrate, was measured, and inhibition for each kinase was measured with respect to DMSO control and reported as an average of duplicate measurements.
Measurement: Ki = 0.25 nM (pChEMBL 9.600000381469727)
Descriptors: MW 446.56, ALogP 3.23, HBA 8, HBD 1, PSA 106.06, QED 0.64
Compound (SMILES): C=CC(=O)N1CCN(CCn2c(=O)ccc3cnc(Nc4cnn(C5CCN(C)CC5)c4)nc32)[C@@H](CC)C1
InChIKey: ABMHMKULTJNRRU-QFIPXVFZSA-N
Target: Tyrosine-protein kinase JAK1 (SINGLE PROTEIN), Homo sapiens
UniProt: P23458
Assay: Biochemical JAK and Tyk2 Kinase Assays: A panel of four LanthaScreen JAK biochemical assays (JAK1, 2, 3 and Tyk2) were carried in a common kinase reaction buffer (50 mM HEPES, pH 7.5, 0.01% Brij-35, 10 mM MgCl2, and 1 mM EGTA). Recombinant GST-tagged JAK enzymes and a GFP-tagged STAT1 peptide substrate were obtained from Life Technologies. Serially or discretely diluted compounds were pre-incubated with each of the four JAK enzymes and the substrate in white 384-well microplates (Corning) at ambient temperature for 1 h. ATP was subsequently added to initiate the kinase reactions in 10 μL total volume, with 1% DMSO. The final enzyme concentrations for JAK1, 2, 3 and Tyk2 are 4.2 nM, 0.1 nM, 1 nM, and 0.25 nM respectively; the corresponding Km ATP concentrations used are 25 μM, 3 μM, 1.6 μM, and 10 μM; while the substrate concentration is 200 nM for all four assays. Kinase reactions were allowed to proceed for 1 hour at ambient temperature before a 10 μL preparation of EDTA (10 mM final concentration) and Tb-anti-pSTAT1 (pTyr701) antibody (Life Technologies, 2 nM final concentration) in TR-FRET dilution buffer (Life Technologies) was added. The plates were allowed to incubate at ambient temperature for 1 h before being read on the EnVision reader (Perkin Elmer). Emission ratio signals (520 nm/495 nm) were recorded and utilized to calculate the percent inhibition values based on DMSO and background controls.For dose-response analysis, percent inhibition data were plotted vs. compound concentrations, and IC50 values were determined from a 4-parameter robust fit model with the Prism software (GraphPad Software). Results were expressed as pIC50 (negative logarithm of IC50) and subsequently converted to pKi (negative logarithm of dissociation constant, Ki) using the Cheng-Prusoff equation.
Measurement: Ki > 100 nM (pChEMBL 7)
Descriptors: MW 519.65, ALogP 2.11, HBA 10, HBD 1, PSA 104.42, QED 0.45
Compound (SMILES): Cc1cn(-c2cc3cccc(-c4nc(C5CC5)n5c4CN(C)C(=O)C5C)c3cn2)cn1
InChIKey: FOLIJXOPRUSFGM-UHFFFAOYSA-N
Target: Bromodomain-containing protein 4 (SINGLE PROTEIN), Homo sapiens
UniProt: O60885
Assay: AlphaScreen Assay: BRD4: 2.5 nM of BRD4(49-170) and 10 nM biotin-H4(1-21) Ac-K5/8/12/16 (AnaSpec. 64989) were incubated with varying concentrations of CBP inhibitors in 15 μL of buffer containing 50 mM HEPES 7.5, 100 nM NaCl, 1 mM TCEP, and 0.003% Tween-20. After 30 minutes incubation at room temperature, 15 μL of detection buffer (BPS Bio. 33006) containing 7 μg/mL of Glutathione AlphaLisa acceptor beads (Perkin Elmer AL109) and 14 μg/mL of Streptavidin donor beads (Perkin Elmer 676002) was then added to the previous mixture. The reaction was incubated for an additional 2 hours at at room temperature, and the AlphaScreen signal was quantified using the Envision Multilabel plate reader. As negative control, GST-CBP(1081-1197) was incubated with the non-acetylated biotin-H4(1-21) peptide(AnaSpec. 62555) and in presence of 0.25% of final DMSO concentration.
Measurement: IC50 = 2 nM (pChEMBL 8.699999809265137)
Descriptors: MW 412.5, ALogP 4, HBA 6, HBD 0, PSA 68.84, QED 0.51
Compound (SMILES): CCC(C)S(=O)(=O)Nc1ccc(F)c(C(=O)c2c[nH]c3ncc(-c4ccc(Cl)cc4)cc23)c1F
InChIKey: LFTFZFQSIWBYQC-UHFFFAOYSA-N
Target: Serine/threonine-protein kinase B-raf (SINGLE PROTEIN), Homo sapiens
UniProt: P15056
Assay: Biochemical activity: Assays for biochemical and cell based activity are known in the art, for example, as described in PCT publication WO 2007/002433, the disclosure of which is hereby incorporated by reference as it relates to such assays. For example, the biochemical activity IC50 values are determined with respect to inhibition of B-Raf V600E kinase activity or p-Erk kinase activity, where inhibition of phosphorylation of a peptide substrate is measured as a function of compound concentration. Compounds to be tested are diluted in dimethyl sulfoxide to a concentration of 0.1 mM. These are serially diluted 15 uL into 30 uL of dimethyl sulfoxide seven times in 96 well plates for a total of 8 dilution points, and for each dilution point 1 uL is added to a well of an assay plate. Plates are prepared such that each well in a 384 well plate contains 1 uL of compound in 10 uL volume with 0.1 ng Raf enzyme (i.e. any of B-Raf, c-Raf-1 or B-Raf V600E, Upstate Biotechnology or prepared by methods known to one of skill in the art), 50 mM HEPES, pH 7.0, 50 mM NaCl, 2 mM MgCl2, 1 mM MnCl2, 0.01% Tween-20, 1 mM DTT, and 100 nM biotin-MEK1 as substrate. The reaction is started with addition of 10 uL of 200 uM ATP (i.e. final 100 uM ATP). After incubation of the kinase reaction for 45 minutes at room temperature, 5 uL/well of Stop Solution is added (25 mM Hepes pH 7.5, 100 mM EDTA, 0.01% BSA with donor beads (Streptavidin coated beads, Perkin Elmer), acceptor beads (Protein A coated, Perkin Elmer), and anti phosphor MEK1/2 antibody (CellSignal), each at final concentration 10 ug/mL).
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 503.96, ALogP 5.93, HBA 4, HBD 2, PSA 91.92, QED 0.31
Compound (SMILES): O=C(NCC(F)(F)F)Nc1cccc(-c2cnc3cc(-c4cnn(C5CCN(C(=O)CC6CCCC6)CC5)c4)cnn23)c1
InChIKey: UAKWIHGQWDDSRN-UHFFFAOYSA-N
Target: Fibroblast growth factor receptor 3 (SINGLE PROTEIN), Homo sapiens
UniProt: P22607
Assay: FGFR3 Enzymatic Assay: The inhibitor potency of compounds of the invention was measured in an enzyme assay that measures peptide phosphorylation using FRET measurements to detect product formation. Inhibitors were serially diluted in DMSO and a volume of 0.5 μL was transferred to the wells of a 384-well plate. A 10 μL volume of FGFR3 enzyme (Millipore) diluted in assay buffer (50 mM HEPES, 10 mM MgCl2, 1 mM EGTA, 0.01% Tween-20, 5 mM DTT, pH 7.5) was added to the plate and pre-incubated for 5-10 minutes. Appropriate controls (enzyme blank and enzyme with no inhibitor) were included on the plate. The assay was initiated by the addition of a 10 μL solution containing biotinylated peptide and ATP (final concentrations of 500 nM and 140 μM respectively) in assay buffer to the wells. The plate was incubated at 25° C. for 1 hr. The reactions were ended with the addition of 10 μL/well of quench solution (50 mM Tris, 150 mM NaCl, 0.5 mg/ml BSA, pH 7.8; added fresh 30 mM EDTA and Perkin Elmer Lance Reagents for HTRF at 3.75 nM Eu-antibody PY20 and 180 nM APC-Streptavidin). The plate was allowed to equilibrate for 1 hr before scanning the wells on a PheraStar plate reader.GraphPad prism3 was used to analyze the data. The IC50 values were derived by fitting the data to the equation for a sigmoidal dose-response with a variable slope. Y=Bottom+(Top−Bottom)/(1+10^((Log IC50−X)*HillSlope)) where X is the logarithm of concentration and Y is the response.
Measurement: IC50 < 10 nM (pChEMBL 8)
Descriptors: MW 594.64, ALogP 5.69, HBA 7, HBD 2, PSA 109.45, QED 0.29
Compound (SMILES): C[C@@H]1OCC2(CCN(c3cnc4c(N5CCCc6nc(C7CCOC7)ccc65)n[nH]c4n3)CC2)[C@@H]1N
InChIKey: RMAXQIPQKVVTID-BJZPFVDKSA-N
Target: Tyrosine-protein phosphatase non-receptor type 11 (SINGLE PROTEIN), Homo sapiens
UniProt: Q06124
Assay: Inhibition Assay: SHP2 is allosterically activated through binding of bis-tyrosyl-phorphorylated peptides to its Src Homology 2 (SH2) domains. The latter activation step leads to the release of the auto-inhibitory interface of SHP2, which in turn renders the SHP2 protein tyrosine phosphatase (PTP) active and available for substrate recognition and reaction catalysis. The catalytic activity of SHP2 was monitored using the surrogate substrate DiFMUP in a prompt fluorescence assay format.The inhibition of SHP2 by compounds of the disclosure (concentrations varying from 0.003-100 μM) was monitored using an assay in which 0.25 nM of SHP2 was incubated with of 0.5 μM of peptide IRS1_pY1172(dPEG8)pY1222 (sequence: H2N-LN(pY)IDLDLV(dPEG8)LST(pY)ASINFQK-amide) After 30-60 minutes incubation at 25° C., the surrogate substrate DiFMUP (Invitrogen, cat #D6567, 100 μM final) was added to the reaction and the conversion of DiFMUP to 6,8-difluoro-7-hydroxyl-4-methyl coumarin (DiFMU) was monitored continuously for 10 minutes with excitation at 355 nm and emission at 460 nm using a microplate reader (PolarStar, BMG).
Measurement: IC50 < 1000 nM (pChEMBL 6)
Descriptors: MW 490.61, ALogP 2.67, HBA 9, HBD 2, PSA 118.31, QED 0.57
Compound (SMILES): Cn1cncc1-c1ccc2cnc(NC(=O)C3C(C)(C)C3(C)C)cc2c1
InChIKey: LTAUSRXHMFPKTC-UHFFFAOYSA-N
Target: Glycogen synthase kinase-3 beta (SINGLE PROTEIN), Homo sapiens
UniProt: P49841
Assay: The screening for GSK3beta kinase activity: Each compound is dissolved in DMSO as a 10 mM stock and used to prepare compound source plates. Serial dilution (1:3, 11-point dose-response curves from 10 μM to 0.0003 μM) and compound transfer was performed using the ECHO 550 (Labcyte, Sunnyvale, Calif.) into 1536-well black-walled round bottom plates (Corning). The GSK3β kinase assay is run using the Ser/Thr 09 peptide Z-lyte assay kit according to manufacturer's instructions (Life Technologiesa Division of Thermo-Fisher). This is a non-radioactive assay using fluorescence resonance energy transfer (FRET) between coumarin and fluorescein to detect kinase activity which is represented as ratio of coumarin emission/fluorescein emission. Briefly, recombinant GSK3β kinase, ATP and Ser/Thr peptide 09 are prepared in 1× Kinase buffer to final concentrations of 0.04 μg/mL, 46 μM, and 4 μM respectively. The mixture is allowed to incubate with the representative compounds for one hour at room temperature. All reactions were performed in duplicate. Unphosphorylated (0% Control) and phosphorylated (100% control) forms of Ser/Thr 18 serve as control reactions. After incubation, diluted Development Buffer is added to the reaction and allowed to further incubate for one hour at room temperature. The plate is read at Ex 400 Em 455 to detect the coumarin signal and Ex 400 Em 520 to measure the signal (EnVision Multilabel Plate Reader, PerkinElmer). The Emission ratio (Em) is calculated as a ratio of the coumarin (C) emission signal (at 445 nm)/Fluorescein (F) emission signal (at 520 nm). The percent phosphorylation is then calculated using the following formula: [1−((Em ratio×F100%)−C100%)/((C0%−C100%)+(Em ratio×(F100%−F0%)))]. Dose-response curves are generated and inhibitory concentration (IC50) values are calculated using non-linear regression curve fit in the Dotmatics' Studies Software (Bishops Stortford, UK).
Measurement: EC50 = 1022 nM (pChEMBL 5.989999771118164)
Descriptors: MW 348.45, ALogP 4.26, HBA 4, HBD 1, PSA 59.81, QED 0.77
Compound (SMILES): Cn1ncc(NC2CCCN(Cc3ncc[nH]3)C2)c(Cl)c1=O
InChIKey: YDGCKWBQJIHFDW-UHFFFAOYSA-N
Target: Histone acetyltransferase KAT2B (SINGLE PROTEIN), Homo sapiens
UniProt: Q92831
Assay: PCAF AlphaLisa Binding Assay: His/Flag epitope tagged PCAF719-832 bromodomain was cloned, expressed and purified to homogeneity in-house. PCAF bromodomain binding and inhibition of the compounds disclosed herein was assessed by monitoring the engagement of biotinylated small molecule ligand (known to bind to the PCAF bromodomain) with the target using the AlphaLisa technology (Perkin-Elmer). Specifically, in a 384 well ProxiPlate PCAF bromodomain (225 nM final) was combined with the biotinylated small molecule ligand (6 nM final) in 50 mM HEPES (pH 7.5), 75 mM NaCl, 1 mM TCEP, 0.01% (w/v) BSA, and 0.008% (w/v) Brij-35 either in the presence of DMSO (final 0.2% DMSO) or compound dilution series in DMSO. After 15 minute incubation at room temperature AlphaLisa streptavidin acceptor beads and AlphaLisa nickel chelate donor beads were added to a final concentration of 12.5 μg/mL each. After 90 minutes of equilibration plates were read on an Envision instrument and IC50s calculated using a four parameter non-linear curve fit.
Measurement: IC50 = 379 nM (pChEMBL 6.420000076293945)
Descriptors: MW 322.8, ALogP 1.23, HBA 6, HBD 2, PSA 78.84, QED 0.88
Compound (SMILES): O=C(c1ccc2c(n1)N(c1ncc(-c3ccccc3F)cn1)CC21CC1)N1CCOCC1
InChIKey: CHUJJAIJKSEKDR-UHFFFAOYSA-N
Target: 3',5'-cyclic-AMP phosphodiesterase 4B (SINGLE PROTEIN), Homo sapiens
UniProt: Q07343
Assay: cAMP HTRF Assay to Determine the Activity of hPDE4B1: The inhibiting effect of the compounds on the enzyme activity of human PDE4B1 was measured by the quantification of 5′-adenosine monophosphate (5′-AMP), which is formed from 3′,5′-cyclic adenosine monophosphate (cAMP). Human recombinant enzyme, expressed in Sf9 cells, and the HTRF (homogeneous time-resolved fluorescence) detection method were used in the assay.The test compound or water (control) was mixed with the human recombinant PDE4B1 enzyme (4.8 U) in a buffer consisting of 44.4 mM tris-HCl, 5.28 mM MgCl2, 2.64 mM DTT and 0.044% Tween 20 (pH 7.8). After adding the cAMP enzyme substrate (final concentration 40 nM), the mixture was incubated for 30 minutes at room temperature. Then a fluorescence acceptor (Dye2 marked with cAMP), a fluorescence donor (anti-cAMP antibody marked with a europium cryptate) and the non-specific phosphodiesterase inhibitor IBMX (3-isobutyl-1-methylxanthine; final concentration 1 mM) were added. After 60 min the fluorescence transfer, which correlates with the amount of remaining cAMP, was measured with a microplate reader (Rubystar, BMG) at λex=337 nm, λem=620 nm and λem=665 nm. The enzyme activity was calculated from the quotient formed from the measured signal at 665 nm and that at 620 nm. The result was expressed as the percentage inhibition of enzyme activity of the control (without PDE4 inhibitor). The enzyme was omitted for measurement of the basal control.[N. Saldou et al., Comparison of recombinant human PDE4 isoforms: interaction with substrate and inhibitors, Cell. Signal. Vol. 10, No. 6, 427-440, 1998].
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 431.47, ALogP 3.33, HBA 6, HBD 0, PSA 71.45, QED 0.63
Compound (SMILES): Cc1cnc(-c2cc(OC[C@@H]3CCOC3)cc(C(=O)N[C@H](C)c3ccc(C(F)(F)F)nn3)c2)s1
InChIKey: BICIIZABCQYPPY-HUUCEWRRSA-N
Target: P2X purinoceptor 3 (SINGLE PROTEIN), Homo sapiens
UniProt: P56373
Assay: Intracellular Calcium Measurement to Assess Antagonist Activity at Human P2X3 and Human P2X2/3 Receptors: A fluorescent imaging plate reader (FLEX/FLIPR station; Molecular Devices) was used to monitor intracellular calcium levels using the calcium-chelating dye Fluo-4 (Molecular Probes). The excitation and emission wavelengths used to monitor fluorescence were 470-495 nm and 515-575 nm, respectively. Cells expressing purinergic receptors P2X3 (human) or P2X2/3 (human) were plated at a density of 15,000 cells/well in collagen-coated 384-well plates approximately 20 hours before beginning the assay. On the day of the assay, 20 μl of Loading buffer (Hank's balanced salt solution, 20 mM HEPES, 0.5 mM CaCl2, 0.5 mM MgCl2, 0.1% BSA, 5 mM probenecid, 10 mM D-glucose monohydrate, 2 μM Fluo-4, and 5 units/mL, hexokinase, pH=7.4) was added and cells dye-loaded for 90 min at 37° C. The dye supernatant was removed and replaced with 45 μl probenecid buffer (Hank's balanced salt solution, 20 mM HEPES, 0.5 mM CaCl2, 0.5 mM MgCl2, 0.1% BSA, 5 mM probenecid, 10 mM D-glucose monohydrate, pH=7.4). The test compound was added in a volume of 5 μl and allowed to incubate for 30 min at 37° C. The final assay DMSO concentration is 1%. The agonist, α,β-Me-ATP, was added in a volume of 20 μl at a concentration representing the EC80 value. The fluorescence was measured for an interval of 90 sec at 2 sec intervals and analyzed based on the increase in peak relative fluorescence units (RFU) compared to the basal fluorescence. Peak fluorescence was used to determine the response to agonist obtained at each concentration of test compound by the following equation: % Response=100*(RFU(test compound)−RFU(control)/RFU(DMSO)−RFU(control)).
Measurement: IC50 = 9 nM (pChEMBL 8.050000190734863)
Descriptors: MW 492.52, ALogP 4.83, HBA 7, HBD 1, PSA 86.23, QED 0.51
Compound (SMILES): COCCNC(=O)c1ccc(Nc2cc(-c3c(F)cccc3F)nc3c2C(=O)NC3)nc1
InChIKey: VFRZAKFHTBYKAX-UHFFFAOYSA-N
Target: Non-receptor tyrosine-protein kinase TYK2 (SINGLE PROTEIN), Homo sapiens
UniProt: P29597
Assay: Tyk2 & JAK2 Caliper Assay: The caliper machine employs an off chip mobility shift assay to detect phosphorylated peptide substrates from kinase assays, using microfluidics technology. The assays are carried out at ATP concentration equivalent to the ATP Km, and at 1 mM ATP. Compounds are serially diluted in DMSO then further diluted in assay buffer (25 mM HEPES, pH 7.5, 0.01% Brij-35, 0.01% Triton, 0.5 mM EGTA). 5 ul of diluted compound was added into wells first, then 10 ul of enzyme mix was added into wells, followed by 10 uL of substrate mix (peptide and ATP in 10 mM MgCl2) to start reaction. Reaction was incubated at 28° C. for 25 min and then added 25 ul stop buffer (100 mM HEPES, 0.015% Brij-35, 50 mM EDTA), followed by reading with Caliper. JAK2 at 1 nM final concentration and TYK2 at 9.75 nM are from Carna, and substrates used are ATP at 20 and 16 uM, respectively. JAK2 assay uses peptide 22 and TYK2 uses peptide 30 (Caliper), each at 3 uM.
Measurement: Ki < 10 nM (pChEMBL 8)
Descriptors: MW 439.42, ALogP 2.79, HBA 6, HBD 3, PSA 105.24, QED 0.49
Compound (SMILES): Cc1ccc(S(=O)(=O)c2ccc(NC(=O)NCc3cn[nH]c3)cc2)cn1
InChIKey: YAACISUIVOHBHM-UHFFFAOYSA-N
Target: Nicotinamide phosphoribosyltransferase (SINGLE PROTEIN), Homo sapiens
UniProt: P43490
Assay: Enzyme Assay and NMN Detection: NAMPT reaction was performed in 50 mM HEPES, pH 7.5, containing 5 mM MgCl2, 0.1% Prionex, 0.005% Tween 20, and 1 mM TCEP at room temperature in Greiner 384-well black polypropylene plates. The concentration of substrates and enzymes in the final assay were as follows: ATP (120 uM), nicotinamide (5 uM), phosphoribosylpyrophosphate (6.25 uM), NAMPT enzyme (16 nM), pyrophosphatase (0.04 U/ml) in the final reaction volume 6 uL. Following 2 h incubation the reaction was stopped by addition 2.5 ul 20% acetophenone in dimethyl sulfoxide (DMSO) and 2.5 ul 2 MKOH. Plates were centrifuges and added with 10.5 ul 88% formic acid. The fluorescence (ex380/em460) was measured following another short incubation and 30 min incubation. The increased concentration of NMN was determined from a calibration curve obtained at the time of experiment.
Measurement: EC50 = 1050 nM (pChEMBL 5.980000019073486)
Descriptors: MW 371.42, ALogP 2.27, HBA 5, HBD 3, PSA 116.84, QED 0.64
Compound (SMILES): O=C(C1CCOC1)N1CC[C@@]2(S(=O)(=O)c3ccc(F)cc3)c3ccc(C(F)(C(F)(F)F)C(F)(F)F)cc3CC[C@@H]12
InChIKey: BKWHSBXYDDIEGE-KDTADFBSSA-N
Target: Nuclear receptor ROR-gamma (SINGLE PROTEIN), Mus musculus
UniProt: P51450
Assay: General RORgamma SPA binding assay : The binding of potential ligands to RORγ is measured by competition with [3H]25-hydroxycholesterol (Perkin Elmer NET674250UC) using a scintillation proximity assay (SPA). The ligand binding domain of human RORγ (A262-S507) with an N-terminal His tag is expressed in E. coli and purified using nickel affinity chromatography. 15 μg/well RORγ (A262-S507) is incubated with test compound at varying concentrations in 3-fold serial dilution, with final concentrations ranging from 16.6 μM to 0.28 nM, for 10 min at rt in PBS buffer (Invitrogen #14190-144) containing 0.5% fatty acid free BSA (Gemini Bio-Products, Cat. #700-107P) and 0.1% glycerol (Sigma Cat#G5516). 10 nM of [3H] 25-hydroxycholesterol is then added, and the reaction is incubated for 10 min. 10 mg/mL of Copper-His Tag-PVT beads (Perkin Elmer cat # RPNQ0095) are added, and the mixture is incubated for 60 min. The reaction is read on a TopCount Microplate scintillation plate reader (Perkin Elmer). The competition data of the test compound over a range of concentrations was plotted as percentage inhibition of radioligand specifically bound in the absence of test compound (percent of total signal). After correcting for non-specific binding, IC50 values were determined. The IC50 value is defined as the concentration of test compound needed to reduce [3H] 25-hydroxycholesterol specific binding by 50% and is calculated using the four parameter logistic equation to fit the normalized data.
Measurement: IC50 = 52 nM (pChEMBL 7.28000020980835)
Descriptors: MW 597.52, ALogP 5.37, HBA 4, HBD 0, PSA 63.68, QED 0.35
Compound (SMILES): Cc1noc(C)c1-c1cnc2c(-c3cnn(C)c3)cn(S(=O)(=O)c3ccccc3)c2c1
InChIKey: YYYGWRYVZPKJJQ-UHFFFAOYSA-N
Target: Bromodomain-containing protein 4 (SINGLE PROTEIN), Homo sapiens
UniProt: O60885
Assay: Alphascreen Binding Assay: Binding of compounds of formula (I) with bromodomain 2, 3 4 was assessed using Alphascreen binding assay. The inhibition of the interaction between bromodomain and its acetylated target protein (Filippakopoulos P et al. 2012) was measured quantitatively using recombinant BRD proteins, an acetylated Histone 4 peptide and AlphaScreen technology. In absence of inhibition the BRD protein bound to AlphaScreen nickel chelate acceptor beads can interact with the acetylated Histone 4 peptide which is immobilized by the AlphaScreen Streptavidin coated beads. This interaction brings donor and acceptor beads in proximity. The close proximity allows the singlet oxygen produced by laser excitation of the donor beads to reach the acceptor beads and generate a luminescence signal. BRD inhibitors result in a decrease in the proximity signal through an inhibition of the BRD-acetylated peptide interaction.Recombinant human bromodomains containing the N-terminal bromodomain (BRD2-BD1 (71-194), BRD3-BD1 (24-144) and BRD4-BD1 (44-164)) or dual bromodomains (BRD4-BD12 (1-477), BRD4-BD12 (1-472)) were prepared and purified as described in protein expression and purification session.
Measurement: IC50 < 1000 nM (pChEMBL 6)
Descriptors: MW 433.49, ALogP 3.95, HBA 8, HBD 0, PSA 95.81, QED 0.43
Compound (SMILES): CCNCC1(c2ccc(Cl)c(Cl)c2)CCCCC1CO
InChIKey: MILNRKCCACSUTQ-UHFFFAOYSA-N
Target: Sodium-dependent serotonin transporter (SINGLE PROTEIN), Homo sapiens
UniProt: P31645
Assay: Functional Uptake Assay (hSERT): Inhibition of human serotonin reuptake transporter was assayed using the recombinant human serotonin transporter expressed in HEK-293 cells. HEK-293 cells expressing human serotonin transporter were plated before the assay. Test compound and/or vehicle was preincubated with cells in modified HEPES buffer pH 7.1 or pH 7.4 for 20 minutes at 18 to 25° C. and 65 nM [3H]serotonin was then added for an additional timed incubation period (ten to thirty minutes). Cells with internalized [3H]serotonin were washed and the amount of tritium taken into cells is counted using a liquid scintillation counter to determine [3H]serotonin uptake. Non-specific binding of tritium was measured in a control reaction containing 10 uM fluoxetine, and was subtracted from the counts for assays to correct for non-specific binding of tritium. Reduction of [3H]serotonin uptake by 50 percent or more (50%) relative to an uninhibited control reaction indicates significant inhibitory activity. Compounds were screened at 10, 1, 0.1, 0.01 and 0.001 uM. The reference compound for the assay was fluoxetine, for which the IC50 value of 7.1 nM was obtained in a typical experiment.
Measurement: IC50 = 332 nM (pChEMBL 6.480000019073486)
Descriptors: MW 316.27, ALogP 4.02, HBA 2, HBD 2, PSA 32.26, QED 0.86