lovingscience/chembl-v37-raw-activities huggingface dataset:
Compound (SMILES): Cc1nc2sccn2c1-c1csc(NC(=O)[C@@H]2C[C@@H](F)CN2C(=O)CC2N=C(C(N)=O)c3cc(-c4ccnnc4)ccc32)n1
InChIKey: LLZOOTXWYIGCGQ-BVWPGQGPSA-N
Target: Complement factor D (SINGLE PROTEIN), Homo sapiens
UniProt: P00746
Assay: Factor D Assay: Human factor D (purified from human serum, Complement Technology, Inc.) at 80 nM final concentration is incubated with test compound at various concentrations for 5 minutes at room temperature in 50 mM Tris, 1M NaCl, pH 7.5. A synthetic substrate Z-L-Lys-SBzl and DTNB (Ellman's reagent) are added to final concentrations of 100 μM each. The increase in color is recorded at OD405 nm in a microplate in kinetic mode over 30 minutes with 30 second time points in a spectrofluorimeter. IC50 values are calculated by non-linear regression from the percentage of inhibition of complement factor D activity as a function of test compound concentration.
Measurement: IC50 > 10000 nM (pChEMBL 5)
Descriptors: MW 629.7, ALogP 3.58, HBA 11, HBD 2, PSA 160.83, QED 0.28
Compound (SMILES): COC(=O)C[C@@H]1COc2cc(O[C@@H]3CCc4c(Oc5ccc6c(c5)OCCO6)ccc(F)c43)ccc21
InChIKey: SZKUVGZYJGRZOJ-VOIUYBSRSA-N
Target: Free fatty acid receptor 1 (SINGLE PROTEIN), Homo sapiens
UniProt: O14842
Assay: Pharmacological Assay: IP1 accumulation measurements using the IPOne assay system 1321N1 cells stably expressing human GPR40 receptor (Euroscreen, Belgium) are seeded 24 h before the assay in white 384-well plates in culture medium containing 10% FCS, 1% Na-Pyruvate and 400 μg/mL G418. IP1 is assayed according to the manufacturer's description (Cisbio Bioassays, France). In brief, the assay is started by substitution of the culture medium by stimulation buffer (Hepes 10 mM, CaCl2 1 mM, MgCl2 0.5 mM, KCl 4.2 mM, NaCl 146 mM, glucose 5.5 mM and LiCl 50 mM, pH 7.4). Cells are stimulated for 1 h at 37° C., 5% CO2 by addition of the compounds that are diluted in stimulation buffer containing LiCl. Assays are stopped by adding HTRF-conjugates (IP1-d2 and Anti-IP1 cryptate Tb) and lysis buffer, provided by the manufacturer. After an incubation time of 1 h at room temperature plates are measured using an EnVision, Perkin Elmer. The obtained fluorescence ratios at 665/615 nM are then used to calculate the pEC50 values using Assay Explorer 3.3 Software (Accelrys, Inc.) by interpolation using an IP1 reference curve and subsequent sigmoidal curve fitting allowing for a variable hill slope.
Measurement: EC50 = 9 nM (pChEMBL 8.050000190734863)
Descriptors: MW 492.5, ALogP 5.49, HBA 7, HBD 0, PSA 72.45, QED 0.42
Compound (SMILES): Cc1nc(NS(=O)(=O)c2cc(Cl)c(N[C@H]3CCCC[C@@H]3N(C)C)cc2F)sc1Cl
InChIKey: QXLXDVRPFVZCAF-ZFWWWQNUSA-N
Target: Sodium channel protein type 9 subunit alpha (SINGLE PROTEIN), Homo sapiens
UniProt: Q15858
Assay: Electrophysiological Assay (EP) (In Vitro Assay): Patch voltage clamp electrophysiology allows for the direct measurement and quantification of block of voltage-gated sodium channels (NaV's), and allows the determination of the time- and voltage-dependence of block which has been interpreted as differential binding to the resting, open, and inactivated states of the sodium channel (Hille, B., Journal of General Physiology (1977), 69: 497-515).The following voltage clamp electrophysiology studies were performed on representative compounds using cells heterologously expressing Nav1.7 or Nav1.5 channels. cDNAs for Nav1.7 (NM_002977) and Nav1.5 (AC137587) were stably expressed in Chinese Hamstr Ovary (CHO) cells and CHL (Chinese Hamster Lung) cells respectively. Sodium currents were measured in the whole-cell configuration using Syncropatch 384PE (NanIon Technologies, Germany). 1NPC®-384 chips with custom medium resistance and single hole mode are used. Internal solution consists of (in mM): 110 CsCl, 10 CsCl, 20 EGTA, and 10 Hepes (pH adjusted to 7.2); and external solution contains (in mM): 60 NMDG, 80 NaCl, 4 KCl, 1 MgCl2, 2 CaCl2, 2 D-Glucose monohydrate, 10 Hepes (pH adjusted to 7.4 with NaOH).After system flushing, testing compounds are dissolved in external solution containing 0.1% Pluronic F-127. The chip is moved into the measuring head and the instrument primes the chip with external and internal solutions. 10l cells are added to the chip from a cell hotel, and a negative pressure of −50 mBar is applied to form a seal. Following treatment with seal enhancer solution and wash-off with external solution, negative pressure of −250 mbar is applied for 1 second to achieve the whole-cell configuration, followed by three washing steps in external solution. 20 μl of compounds is added to 40 μl in each well (1:3 dilution of compounds), and after mixing, 20 μl is removed so the volume is retained at 40 μl. After approximately 13 minutes recordings, 20 μl/well of 2 uM TTX, or 333 uM Tetracaine (for Nav1.5) is added to achieve full block.For voltage protocol, an holding potential of −50 mV is applied during the whole experiment. A depolarizing step is applied to −10 mV for 10 ms, followed by a hyperpolarization step to −150 mV for 20 ms to allow channel recovery from inactivation. A second depolarizing step is applied from −150 mV to −10 mV for 10 ms, where currents were measured to derive blocking effects of compounds. Inhibition is determined based on 7.5 min of compound incubation.
Measurement: IC50 = 12.3 nM (pChEMBL 7.909999847412109)
Descriptors: MW 481.45, ALogP 4.98, HBA 6, HBD 2, PSA 74.33, QED 0.61
Compound (SMILES): Cc1cc(Nc2cc3ncccc3c(N[C@@H]3C[C@H]4CC[C@@H](C3)N4CC(C)C)n2)n[nH]1
InChIKey: LDQPCFGSOSMMKQ-NNZMDNLPSA-N
Target: Tyrosine-protein kinase JAK1 (SINGLE PROTEIN), Homo sapiens
UniProt: P23458
Assay: Biochemical JAK Assay: 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 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 lh. 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 lh 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.Test compounds having a higher pKi value in each of the four JAK assays show greater inhibition of JAK activity. Compounds of the invention tested in this assay typically exhibited pKi values between about 7 and about 10.3
Measurement: Ki = 0.55 nM (pChEMBL 9.260000228881836)
Descriptors: MW 405.55, ALogP 4.47, HBA 6, HBD 3, PSA 81.76, QED 0.56
Compound (SMILES): CC(=O)N1CCN(c2ccc(NC(=O)Cc3cnc(-c4ccnc(C)c4)nc3)nc2)CC1
InChIKey: OUJYUYOINLETQL-UHFFFAOYSA-N
Target: Protein Wnt-3a (SINGLE PROTEIN), Homo sapiens
UniProt: P56704
Assay: Wnt-Luc Reporter Assay: Human embryonic kidney 293 cells (obtained from American Type Culture Collection, ATCC, Manassas, Va.) are cultured in DMEM medium (Gibco/Invitrogen, Carlsbad, Calif.) supplemented with 10% FBS (Gibco/Invitrogen, Carlsbad, Calif.), 50 unit/mL penicillin and 50 μg/mL of streptomycin (Gibco/Invitrogen, Carlsbad, Calif.) at 37° C. with 5% CO2 in air atmosphere. 293 cells in a 10 cm dish are co-transfected with 8 μg of STF-reporter plasmid containing a luciferase gene driven by Wnt-responsive elements and 2 μg of pcDNA3.1-Neo (Gibco/Invitrogen, Carlsbad, Calif.) with 30 μL of FuGENE6 (Roche Diagnostics, Indianapolis, Ind.) following the manufacturer's protocol. Stable cell lines (293 Wnt-Luc) were selected with 400 μg/mL of G418 (Gibco/Invitrogen, Carlsbad, Calif.). The 293 Wnt-Luc cells and L-cell Wnt3a cells (obtained from American Type Culture Collection, ATCC, Manassas, Va.) are trypsinized and co-cultured into a 384-well plate with DMEM medium supplemented with 2% FBS, and treated with different concentrations of a compound of the invention. After 24 hours, the firefly luciferase activities are assayed with the Bright-Glo Luciferase Assay System (Promega, Madison, Wis.). The IC50 is measured when the effect of the compound reduces the luminescence signal by 50%.
Measurement: IC50 = 0.3 nM (pChEMBL 9.520000457763672)
Descriptors: MW 431.5, ALogP 2.09, HBA 7, HBD 1, PSA 104.21, QED 0.66
Compound (SMILES): CC(C)(C)CN1CCC(c2ccc(NC(=O)Nc3ccccc3N3CC(C)(C)c4c(-c5ccc(Cl)cc5)c(F)cc(O)c43)cc2)CC1
InChIKey: PVZZYZHYQOTDBH-UHFFFAOYSA-N
Target: P2Y purinoceptor 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P47900
Assay: Scintillation Proximity Assay (SPA): A SPA membrane binding assay was used to identify inhibitors of [33P] 2MeS-ADP binding to cloned human P2Y1 receptors (The P2Y1 receptor membranes were provided by Biology and the cloning of the receptor and P2Y1 receptor membrane preparation is same as described by Biology). Binding reactions were performed in 384-well OptiPlates (PerkinElmer Life Sciences, Cat #6007299) in a volume of 50 μL containing 15 fmol of P2Y1 receptor (1.7 μg of total protein), 0.3 nM [33P] 2MeS-ADP (PerkinElmer; 2,000 Ci/mmol), various concentrations of the test compound (usually between 10 μM and 160 pM) in Buffer B containing 1% DMSO in assay buffer (15 mM, HEPES, 145 mM potassium chloride, 5 mM sodium chloride, 5 mM EDTA, 0.1 mM MgCl2, pH 7.4) and 100 μg of SPA bead (WGA polystyrene Image beads, #RPNQ 0260V, Amersham). Reactions were allowed to proceed to completion at room temperature for 1 hour followed by centrifugation of the plate for 5 min. About 40 μL of the aqueous solution was aspirated. Plates were sealed and the [33P] 2MeS-ADP bound to the P2Y1 receptor membranes that were bound to the SPA bead were determined in a Gen 4 LEADSEEKER (Amersham) Image Reader.
Measurement: Ki = 14 nM (pChEMBL 7.849999904632568)
Descriptors: MW 655.26, ALogP 10.15, HBA 4, HBD 3, PSA 67.84, QED 0.19
Compound (SMILES): CC(C)C[C@H](NC(=O)[C@H](Cc1ccccc1)NC(=O)c1cnccn1)B1OC(C)(C)C(C)(c2ccc(C(=O)N3CCC(COc4ccc(-n5c(O)nnc5-c5cc(C(C)C)c(O)cc5O)cc4)CC3)cc2)O1
InChIKey: MVOTXSVDLGESOV-QCAINQIKSA-N
Target: Receptor tyrosine-protein kinase erbB-2 (SINGLE PROTEIN), Homo sapiens
UniProt: P04626
Assay: HER2 Degradation Assay with BT-474 Cell Line: HER2 has emerged as a key target for anticancer drugs due to its intrinsic involvement in the phosphatidylinositol-3-kinase-Akt/protein kinase B (PI3 K-Akt) and the mitogen-activated protein kinase (MAPK) pathways, both of which suppress apoptosis and promote tumor cell survival, gene transcription, angiogenesis, cellular proliferation, migration, mitosis, and differentiation. The degradation of HER2 is a measure of efficacy of anticancer therapeutics that target Hsp90. Accordingly, the SDC-TRAP molecules of the invention that comprise a binding moiety that binds Hsp90 were tested in the following HER2 degradation assay.BT-474 cells (human breast cancer cell line ATCC HTB-20) were obtained from ATCC and seeded into 12-well tissue culture plates at 0.2x106/1.8 mL/well. The cells were incubated for more than 6 hours at 37° C. in DMEM+10% FBS, +1% P/S, +1.5 g/L sodium bicarbonate. Each test compound was titrated in 4-fold dilutions from 5 μM to 78 nM with DMSO and 200 μL of the titration was added to each well of the cell plate. The DMSO final concentration was 0.2%. Cells were incubated overnight at 37° C. in 5% CO2.Media was decanted from the plate, cells were washed 1x in PBS. 400 μL trypsin (EDTA) per well was added, and the cells were incubated for 2 to 3 minutes. Cells were collected into FACS tubes containing 1 ml culture medium to neutralize the trypsin and were centrifuged for 5 minutes at 1200 rpm. Supernatant was decanted and the cells were resuspended in 5 μL FITC (anti HER2/nu)/200 μL staining buffer (1×PBS+1% FBS+0.05% Sodium Azide)/tube. Controls were 5 μL IgG isotype control and staining buffer only. Tubes were incubated for 30 minutes in the dark at room temperature. 1 mL staining buffer was added to each tube and the tubes were centrifuged for 6 minutes at 1200 rpm. The supernatant was decanted and 300 μL staining buffer was added to each tube, which was stored at 4° C. for FACS (cytometer) analysis. The cytometer readout was normalized and the potency of each compound was evaluated with IC50 calculated with XLfit software.
Measurement: IC50 = 407 nM (pChEMBL 6.389999866485596)
Descriptors: MW 978.96
Compound (SMILES): COC(=O)N1CCc2ccc(Nc3nc(N[C@@H]4CCCC[C@@H]4N)ncc3C(N)=O)cc2C1
InChIKey: YJRFUVGAJGRHKT-ZWKOTPCHSA-N
Target: Tyrosine-protein kinase SYK (SINGLE PROTEIN), Homo sapiens
UniProt: P43405
Assay: ADP-Glo Kinase Assay: The Promega ADP-Glo Kinase Assay is a luminescent ADP detection assay that provides a universal, homogeneous, high-throughput screening method to measure kinase activity by quantifying the amount of ADP produced during a kinase reaction.
Measurement: IC50 = 12.4 nM (pChEMBL 7.909999847412109)
Descriptors: MW 439.52, ALogP 2.13, HBA 8, HBD 4, PSA 148.49, QED 0.55
Compound (SMILES): COCCN1C[C@@H](NC(=O)Nc2c(-c3ccccc3)nn(-c3cnccn3)c2C)[C@H](c2ccc(F)c(F)c2)C1
InChIKey: KUSVTESUNBYOGM-XUZZJYLKSA-N
Target: High affinity nerve growth factor receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P04629
Assay: TrkA Omnia Assay: Trk enzymatic selectivity was assessed using Omnia Kinase Assay reagents from Invitrogen Corp. Enzyme (TrkA from Invitrogen Corp.) and test compound (various concentrations) were incubated for 10 minutes at ambient temperature in a 384-well white polypropylene plate (Nunc catalog#267462). Omnia Tyr Peptide #4, as well as ATP, were then added to the plate. Final concentrations were as follows: 20 nM enzyme, 500 μM of ATP, 10 μM peptide substrate. The assay buffer consisted of 25 mM MOPS pH 7.5, 0.005% (v/v) Triton X-100 and 5 mM MgCl2. The production of phosphorylated peptide was monitored continuously for 70 minutes using a Molecular Devices FlexStation II384 microplate reader (excitation=360 nm; emission=485 nm). Initial rates were calculated from the progress curves. IC50 values were calculated from these rates using either a 4 or 5-parameter logistic curve fit.
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 533.58, ALogP 4.15, HBA 7, HBD 2, PSA 97.2, QED 0.35
Compound (SMILES): CC(C)Oc1cccc2c1CN(c1cn[nH]c(=O)c1C(F)(F)F)C2
InChIKey: DZKUFLDNSZGPAY-UHFFFAOYSA-N
Target: Protein mono-ADP-ribosyltransferase TIPARP (SINGLE PROTEIN), Homo sapiens
UniProt: Q7Z3E1
Assay: Enzymatic Assay: Displacement of Probe A, a biotinylated probe binding to the TIPARP active site, was measured using a time-resolved fluorescence energy transfer (TR-FRET) assay. 20 nL of a dose response curve of each test compound was spotted in black 384-well polystyrene proxiplates (Perkin Elmer) using a Mosquito (TTP Labtech). Reactions were performed in a 8 μL volume by adding 6 JAL of TIPARP and Probe A in assay buffer (20 mM HEPES pH=8, 100 mM NaCl, 0.1% bovine serum albumin, 2 mM DTT and 0.002% Tween20), incubating with test compound at 25° C. for 30 min, then adding 2 μL of ULight-anti 6×His and LANCE Eu-W1024 labeled streptavidin (Perkin Elmer). The final concentrations of TIPARP and Probe A were 6 nM and 2 nM, respectively. The final concentration of ULight-anti 6×His and LANCE Eu-W1024 labeled streptavidin were 4 nM and 0.25 nM, respectively. Reactions were incubated at 25° C. for an additional 30 min, then read on an Envision plate reader equipped with a LANCE/DELFIA top mirror (Perkin Elmer) using excitation of 320 nm and emission of 615 nm and 665 nM with a 90 μs delay. The ratio of the 665/615 nm emission were calculated for each well to determine the amount of complex of TIPARP and Probe A in each well.
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 339.32, ALogP 3.1, HBA 4, HBD 1, PSA 58.22, QED 0.93
Compound (SMILES): Cc1nc(-c2nc(NC3CCN(S(C)(=O)=O)CC3)ncc2C(F)(F)F)cn1-c1ccc(N2CC[C@H](N(C)C)C2)cc1F
InChIKey: PUTKJJSSZHRACA-FQEVSTJZSA-N
Target: Cyclin-dependent kinase 2/cyclin E1 (PROTEIN COMPLEX), Homo sapiens
UniProt: P24864
Assay: HTRF Enzyme Activity Assay: CDK2/Cyclin E1 enzyme activity assays utilize full-length human CDK2 co-expressed as N-terminal GST-tagged protein with FLAG-Cyclin E1 in a baculovirus expression system (Carna Product Number 04-165). Assays were conducted in white 384-well polystyrene plates in a final reaction volume of 8 μL. CDK2/Cyclin E1 (0.25 nM) was incubated with the compounds of the Examples (40 nL serially diluted in DMSO) in the presence of ATP (50 μM or 1 mM) and 50 nM ULight-labeled eIF4E-binding protein 1 (THR37/46) peptide (PerkinElmer) in assay buffer (containing 50 mM HEPES pH 7.5, 1 mM EGTA, 10 mM MgCl2, 2 mM DTT, 0.05 mg/mL BSA, and 0.01% Tween 20) for 60 minutes at room temperature. The reactions were stopped by the addition of EDTA and Europium-labeled anti-phospho-4E-BP1 antibody (PerkinElmer), for a final concentration of 15 mM and 1.5 nM, respectively. HTRF signals were read after 1 hour at room temperature on a PHERAstar FS plate reader (BMG Labtech). Data was analyzed with IDBS XLFit and GraphPad Prism 5.0 software using a three or four parameter dose response curve to determine IC50 for each compound.
Measurement: IC50 < 50 nM (pChEMBL 7.301000118255615)
Descriptors: MW 610.68, ALogP 3.77, HBA 9, HBD 1, PSA 99.49, QED 0.4
Compound (SMILES): COc1ccc(/C=C/n2c(C)cc(=O)cc2C)cc1OCCS(C)(=O)=O
InChIKey: JHBLMRZYURZCEB-BQYQJAHWSA-N
Target: 3',5'-cyclic-AMP phosphodiesterase 4B (SINGLE PROTEIN), Homo sapiens
UniProt: Q07343
Assay: Determination of the inhibition of the compounds on PDE4B1 activity by using cAMP HTRF assay: The inhibitory effect of the compounds on human PDE4B1 enzyme activity was determined by quantifying the 5′-adenosine monophosphate (5′-AMP) formed from 3′,5′-cyclic adenosine monophosphate (cAMP).The test compound or water (control) and human recombinant PDE4B1 enzyme (4.8 U) were mixed in a buffer solution (pH 7.4) consisting of 1× Hanks' balanced salt solution (HBSS), 5 mM HEPES, 3 mM MgCl2, and 0.1% BSA and incubated for 10 min. A cAMP enzyme substrate (final concentration 40 nM) was added, and the mixture was incubated at room temperature for 60 min. Then a fluorescent acceptor (Dye2 labeled with cAMP), a fluorescent donor (anti-cAMP antibody labeled with europium cryptate) and a non-specific phosphodiesterase inhibitor IBMX (3-isobutyl-1-methyl xanthine; final concentration 1 mM) were added. After 60 min, the fluorescence transfer related to the amount of remaining cAMP was measured on a microplate reader (Rubystar, BMG) at λex=337 nm, λem=620 nm and λem=665 nm. The enzyme activity is calculated from the ratio of the signals measured at 665 nm and 620 nm. The results are expressed as percent inhibition of the enzyme activity of the control (without PDE4 inhibitor). The enzyme was omitted for measurement of the basic control. The IC50 value (IC50=the concentration that caused the half-maximum inhibition of the specific activity of the control) is derived from dose-response measurements with eight different concentrations (n=2; repeated 2 times).
Measurement: IC50 = 160 nM (pChEMBL 6.800000190734863)
Descriptors: MW 377.46, ALogP 2.53, HBA 6, HBD 0, PSA 74.6, QED 0.74
Compound (SMILES): Cn1cnc(S(=O)(=O)N2C[C@@H]([C@@H]3CCCOC3)[C@H](Nc3cccc(-c4ccccc4)c3)C2)c1
InChIKey: LMOMWOCSJBTHLT-YFNKSVMNSA-N
Target: Alpha-1,3-mannosyl-glycoprotein 2-beta-N-acetylglucosaminyltransferase (SINGLE PROTEIN), Homo sapiens
UniProt: P26572
Assay: Radioligand Binding Assay: Radioligand binding assays using recombinant CHO cell membranes expressing human GlyT1: Radioligand binding to human GlyT1c transporter-expressing membranes was determined as described in Mezler et al., Molecular Pharmacology 74:1705-1715, 2008.
Measurement: Ki < 1000 nM (pChEMBL 6)
Descriptors: MW 466.61, ALogP 3.61, HBA 6, HBD 1, PSA 76.46, QED 0.6
Compound (SMILES): COc1ccc(CNC(=O)c2nc(N[C@@H](C)c3ccncc3)nc3ccsc23)cn1
InChIKey: DXTNGXASLMGAON-ZDUSSCGKSA-N
Target: Adenosine receptor A2b (SINGLE PROTEIN), Homo sapiens
UniProt: P29275
Assay: Activity Assay: Compounds (Adenosine Receptor modulators) and activity assay.
Measurement: IC50 = 75 nM (pChEMBL 7.119999885559082)
Descriptors: MW 420.5, ALogP 3.59, HBA 8, HBD 2, PSA 101.92, QED 0.47
Compound (SMILES): COCCN(C)c1cc(C(=O)Nc2ccc(C)c(-c3cc(N4CCOCC4)c(=O)n(C)c3)c2)ccn1
InChIKey: IYNXRZBFWPYIAK-UHFFFAOYSA-N
Target: Serine/threonine-protein kinase B-raf (SINGLE PROTEIN), Homo sapiens
UniProt: P15056
Assay: Raf Activity Assay: Raf and biotinylated Mek, kinase dead, were combined at 2x final concentrations in assay buffer (50 mM Tris, pH 7.5, 15 mM MgCl2, 0.01% BSA and 1 mM DTT) and dispensed 5 ml per well in assay plates (Greiner white 384 well assay plates #781207) containing 0.25 ml of 40x of a Raf kinase inhibitor test compound diluted in 100% DMSO. The plate was incubated for 60 minutes at room temperature. The Raf kinase activity reaction was started by the addition of 5 mL per well of 2xATP diluted in assay buffer. After 3 hours (b-Raf(V600E)) or 1 hour (c-Raf). The reactions were stopped and the phosphorylated product was measured using a rabbit anti-p-MEK (Cell Signaling, #9121) antibody and the Alpha Screen IgG (ProteinA) detection Kit (PerkinElmer #6760617R), by the addition of 10 mL to the well of a mixture of the antibody (1:2000 dilution) and detection beads (1:2000 dilution of both beads) in Stop/bead buffer (25 mM EDTA, 50 mM Tris, pH 7.5, 0.01% Tween20). The additions were carried out under dark conditions to protect the detection beads from light. A lid was placed on top of the plate and incubated for 1 hour at room temperature, then the luminescence was read on a PerkinElmer Envision instrument. The concentration of each compound for 50% inhibition (IC50) was calculated by non-linear regression using XL Fit data analysis software.
Measurement: IC50 = 2.8 nM (pChEMBL 8.550000190734863)
Descriptors: MW 491.59, ALogP 2.93, HBA 8, HBD 1, PSA 88.93, QED 0.52
Compound (SMILES): Cc1ncc(-c2ccc3c(c2)c(C(C)O)nn3CC(=O)N2C[C@H]3C[C@H]3[C@H]2C(=O)Nc2nc(Br)ccc2C)cn1
InChIKey: BMQKUJONDKALCF-OWJAULCESA-N
Target: Complement factor D (SINGLE PROTEIN), Homo sapiens
UniProt: P00746
Assay: Factor D Assay: Human Factor D (purified from human serum, Complement Technology, Inc.) at 80 nM final concentration is incubated with test compound at various concentrations for 5 min at room temperature in 50 mM Tris, 1M NaCl, pH 7.5. A synthetic substrate Z-L-Lys-SBz and DTNB (Ellman's reagent) are added to final concentrations of 100 μM each. Absorbance at 405 nm (A405) is recorded at 30 second intervals for 30 min using a microplate spectrophotometer. IC50 values are calculated by nonlinear regression of complement Factor D reaction rates as a function of test compound concentration.
Measurement: IC50 = 5500 nM (pChEMBL 5.260000228881836)
Descriptors: MW 590.48, ALogP 3.81, HBA 8, HBD 2, PSA 126.13, QED 0.33
Compound (SMILES): Cc1ncc(-c2cc(C(F)(F)F)c3c(N)ncnn23)cc1C(=O)Nc1cn(C(c2ccccc2)C(F)(F)F)cn1
InChIKey: ISHHNTYILWUHCR-UHFFFAOYSA-N
Target: Receptor-interacting serine/threonine-protein kinase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q13546
Assay: RIPK1 HTRF Binding Assay: A solution was prepared containing 0.2 nM Anti GST-Tb (Cisbio, 61GSTTLB), 90.6 nM probe and 1 nM His-GST-TVMV-hRIPK1(1-324) in FRET Buffer (20 mM HEPES, 10 mM MgCl2, 0.015% Brij-35, 4 mM DTT, 0.05 mg/mL BSA). Using Formulatrix Tempest, the detection antibody/enzyme/probe solution (2 mL) was dispensed into wells of a 1536 plate (Black Low Binding Polystyrene 1536 Plate (Corning, 3724)) containing 10 nL of compounds of interest at appropriate concentration in DMSO. The plate was incubated at rt for 1 h. FRET was measured using the EnVision plate reader (Excitation: 340 nM, Emission: 520 nM/495 nM). Total signal (0% inhibition) was calculated from wells containing 10 nL DMSO only. Blank signal (100% inhibition) calculated from wells containing 10 nL of 15 nM staurosporine and internal controls.
Measurement: IC50 = 9900 nM (pChEMBL 5)
Descriptors: MW 560.46, ALogP 5.3, HBA 8, HBD 2, PSA 116.02, QED 0.28
Compound (SMILES): C[C@H](NC(=O)c1nccnc1NCc1ccc(-c2cnc3[nH]c(=O)[nH]c3c2)cc1)c1ccc(F)cc1
InChIKey: ZIGHNBZMMNJUNQ-HNNXBMFYSA-N
Target: 3-phosphoinositide-dependent protein kinase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: O15530
Assay: PDK1 kinase assay: PDK1 (amino acids 51-359) and AKT2 (amino acids 140-467 fused to PIFtide, amino acids EEQEMFRDFDYIADW) were expressed as N-terminally tagged GST fusion proteins in insect cells and purified to homogeneity. Enzyme activity was determined in a coupled PDK1/AKT/FAM-crosstide assay and phosphorylation of FAM-crosstide was determined by standard IMAP protocol (Molecular Devices). For inhibition studies, compounds were titrated 4-fold in DMSO and diluted 40-fold into assay buffer (10 mM Tris HCl pH7.2; 10 mM MgCl2; 0.01% Triton X-100; 1 mM DTT) containing PDK1, AKT2, and FAM-crosstide (final concentrations: 0.75 nM PDK1, 10 nM unphosphorylated AKT2, and 100 nM crosstide substrate). The kinase reaction was initiated by adding ATP to a final concentration of 40 μM of PDK1 and incubated at 25° C. for 30 min. To detect assay product, the kinase reaction was combined with Progressive Binding Solution (1:600 Progressive Binding Reagent, 50% Buffer A, 50% Buffer B, Molecular Devices) in a 1:3 ratio. The mixture was incubated for 2 hours at 25° C. and the plate was scanned on an Analyst AD with excitation at 485 nm and emission at 530 nm. The fluorescence polarization value P is defined by the equation below. The value mP is generated by multiplying the P value for each reaction well by a factor of 1000. The value ΔmP for each well is the mP value for that well minus the mP value for the average negative control. P=(Fpar−Fperp)/(Fpar+Fperp)  Eq.:Where par is fluorescence intensity parallel to the excitation plane; and perp is fluorescence intensity perpendicular to the excitation plane. ΔmP values were plotted as a function of compound concentration and the data were analyzed with a 4-parameter fit using GraphPad Prism software.
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 483.51, ALogP 3.95, HBA 6, HBD 4, PSA 128.45, QED 0.28
Compound (SMILES): NC(=O)c1nn(CC(=O)N2C[C@H](F)C[C@H]2C(=O)Nc2nccc(Cl)n2)c2ccc(-c3ccnnc3)cc12
InChIKey: CYVGBPPYZDRYGW-PBHICJAKSA-N
Target: Complement factor D (SINGLE PROTEIN), Homo sapiens
UniProt: P00746
Assay: Human Factor D Assay: Human Factor D (purified from human serum, Complement Technology, Inc.) at 80 nM final concentration was incubated with test compound at various concentrations for 5 minutes at room temperature in 50 mM Tris, 1M NaCl, pH 7.5. A synthetic substrate Z-L-Lys-SBzl and DTNB (Ellman's reagent) are added to final concentrations of 100 μM each. Absorbance at 405 nm (A405) was recorded at 30 second intervals for 30 minutes using a microplate spectrophotometer. IC50 values are calculated by nonlinear regression of complement Factor D reaction rates as a function of test compound concentration.
Measurement: IC50 > 10000 nM (pChEMBL 5)
Descriptors: MW 523.92, ALogP 1.61, HBA 9, HBD 2, PSA 161.88, QED 0.36
Compound (SMILES): CN1CCN(Cc2ccc(C(=O)Nc3cnn4ccc(N5CCC[C@@H]5c5cc(F)ccc5F)nc34)cc2)CC1
InChIKey: RUNUMABDVCXWMO-AREMUKBSSA-N
Target: High affinity nerve growth factor receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P04629
Assay: In Vitro Assay: I. Prepare 1× Kinase Base Buffer and Stop Buffer for Testing Kinases1) 1× Kinase base buffer (for TRK-A)50 mM HEPES, pH 7.50.0015% Brij-352) Stop buffer100 mM HEPES, pH 7.50.015% Brij-350.2% Coating Reagent #350 mM EDTAII. Prepare Source Plate with Compound1) Dilute the compound to 50× of the final concentration in reaction by 100% DMSO. Transfer 100 μl of this compound dilution to a well in a 96-well plate. For example, if the desired highest inhibitor concentration is 10 μM, then prepare a 500 μM of compound DMSO solution in this step.2) Perform 3× series dilution of the compound dilution obtained in step 1) with 100% DMSO to provide 9 more diluted solutions of the compound.3) Add 100 μl of 100% DMSO to two empty wells for no compound control and no enzyme control in the same 96-well plate. Mark the plate as source plate.4) Prepare intermediate plateTransfer 10 μl of compound from the source plate to a new 96-well plate as the intermediate plate.Add 90 μl of 1× Kinase base buffer to each well of the intermediate plate.Mix the compounds in intermediate plate for 10 mM on a shaker.5) Prepare assay plateTransfer 5 μl of each well from the 96-well intermediate plate to a 384-well plate in duplicates. For example, A1 of the 96-well plate is transferred to A1 and A2 of the 384-well plate. A2 of the 96-well plate is transferred to A3 and A4 of the 384-well plate, and so on.III. Kinase Reaction1) Prepare 2.5× enzyme solution Add kinase in 1× kinase base buffer.2) Prepare 2.5× peptide solutionAdd FAM-labeled peptide (SEQ ID NO:1) and ATP in the 1× kinase base buffer.3) Transfer 2.5× enzyme solution to the assay plateAssay plate already contains 5 μl of the compound in 100% DMSO.Add 10 μl of 2.5× enzyme solution to each well of the 384-well assay plate.Incubate at room temperature for 10 min4) Transfer 2.5× peptide solution to the assay plateAdd 10 μl of 2.5× peptide solution to each well of the 384-well assay plate.5) Kinase reaction and stopIncubate at 28° C. for a specified period of time.Add 25 μl of Stop buffer to stop reaction.IV. Caliper ReadingCollect data on Caliper.V. Curve Fitting
Measurement: IC50 = 1.8 nM (pChEMBL 8.739999771118164)
Descriptors: MW 531.61, ALogP 4.35, HBA 7, HBD 1, PSA 69.01, QED 0.4
Compound (SMILES): C[C@@H]1CCOc2nc3cc(C(=O)Nc4ccc(OC(F)(F)Cl)cc4)cc(-c4cnsc4)c3n21
InChIKey: FDAODMPZZXVQBF-GFCCVEGCSA-N
Target: Tyrosine-protein kinase ABL1 (SINGLE PROTEIN), Homo sapiens
UniProt: P00519
Assay: Biochemical Kinase Assay: ABL1 WT protein (64-515aa) containing an N-terminal His tag was produced by co-expression with YopH in Sf9 insect cells. Cells were harvested by centrifugation and resuspended in 50 mM Tris, 500 mM NaCl, 5 mM β-ME, pH 8.2. Cells were lysed by sonication and clarified by centrifugation. ABL1 was purified by affinity chromatography using a HisTrap column with a wash step in 4% wash buffer (50 mM Tris, 500 mM NaCl, 500 mM imidazole, 5 mM β-ME, pH 8.2) and eluted in a linear gradient of the same buffer. Fractions containing ABL1 were pooled, concentrated and further purified using an ion exchange column washed with 50 mM Tris, pH 8.3 and eluted with a linear gradient of elution buffer (50 mM Tris, 1M NaCl, pH 8.3). Purified protein was stored at -80° C. in 50 mM Tris (pH 8.2), 300 mM NaCl, 1 mM DTT and 20% glycerol.
Measurement: IC50 = 0.43 nM (pChEMBL 9.369999885559082)
Descriptors: MW 490.92, ALogP 5.92, HBA 7, HBD 1, PSA 78.27, QED 0.35
Compound (SMILES): Cc1nnn(C)c1-c1cnc2c3ccc(C(C)(C)O)c(F)c3n(C(c3ncccc3F)C3CCOCC3)c2c1
InChIKey: GJGSUFHIMBFZFP-UHFFFAOYSA-N
Target: Bromodomain-containing protein 4 (SINGLE PROTEIN), Homo sapiens
UniProt: O60885
Assay: TR-FRET Assay: All assay components were dissolved in buffer composition 20 mM Hepes pH 7.5, 150 mM NaCl, 5 mM DTT, 0.005% Tween 20, and 100 ug/ml BSA for BRD4 (1-477 and 44-460). The final concentrations of the bromodomain proteins are 1.6 nM BRD4(44-168), 1 nM BRD4(333-460), and 1 nM BRD4(1-477 or 44-460), and the fluorescent probe molecule is 100 nM, 50 nM, and 7.5 nM respectively. All proteins were biotinylated. A streptavidin labeled with terbium cryptate (Cisbio SA-Tb) was used as detection, and pre-mixed with the bromodomain protein at a final concentration of 0.2 nM. In some instances for BRD4 (44-460), anti-His terbium cryptate was used as a detection. 7.5 nl of dose-responsed test compound or dmso vehicle (0.0375%) was pre-spotted in a black Corning 384 well plate and 10 ul each of bromodomain/detection reagent and fluorescent small molecule solution were added to the plate, and the reaction incubated for 60 min at room temperature. Plates were then read on EnVision plate reader, (λex=340 nm, acceptor λEm=520 nm, and donor λEm=615 nm, LANCE D400 mirror). Time resolved fluorescence intensity measurements were made at both emissions, and the ratio of acceptor/donor was calculated and used for data analysis.
Measurement: IC50 < 5 nM (pChEMBL 8.300999641418457)
Descriptors: MW 532.6, ALogP 5.21, HBA 8, HBD 1, PSA 90.88, QED 0.33
Compound (SMILES): CC(C)C[C@](C)(N)COc1cnc(-c2cc(Cl)ncc2F)cc1C(F)F
InChIKey: SISQPPCZDPNKAD-SFHVURJKSA-N
Target: AP2-associated protein kinase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q2M2I8
Assay: Kinase 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 (5-FAM)-Aha-KEEQSQITSQVTGQIGWR-NH2 and ATP) and test compounds in assay buffer (10 mM Tris-HCL pH 7.4, 10 mM MgCl2, 0.01% Tween-20 and 1.0 mM DTT). The reactions were initiated by the combination of bacterially expressed, GST-Xa-hAAK1 with substrates and test compounds. The reactions were incubated at room temperature for 3 hours and terminated by adding 60 μl of 35 mM EDTA buffer to each sample. The reactions were 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 EDTA quenched control reactions for 100% inhibition and vehicle-only reactions for 0% inhibition. The final concentration of reagents in the assays are ATP, 22 μM; (5-FAM)-Aha-KEEQSQITSQVTGQIGWR-NH2, 1.5 μM; GST-Xa-hAAK1, 3.5 nM; and DMSO, 1.6%. Dose response curves were generated to determine the concentration required inhibiting 50% of kinase activity (IC50). Compounds were dissolved at 10 mM in dimethylsulfoxide (DMSO) and evaluated at eleven concentrations.
Measurement: IC50 = 0.45 nM (pChEMBL 9.350000381469727)
Descriptors: MW 387.83, ALogP 5.02, HBA 4, HBD 1, PSA 61.03, QED 0.68
Compound (SMILES): CC(=O)c1nn(CC(=O)N2[C@H](C(=O)Nc3nc(Br)ccc3C)C[C@@]3(C)C[C@@H]23)c2ccc(-c3cnc(OC4CS(=O)(=O)C4)nc3)cc12
InChIKey: IYENTOKSPOEAKG-JLELKNTQSA-N
Target: Complement factor D (SINGLE PROTEIN), Homo sapiens
UniProt: P00746
Assay: Factor D Assay: Human Factor D (purified from human serum, Complement Technology, Inc.) at 80 nM final concentration is incubated with test compound at various concentrations for 5 min at room temperature in 50 mM Tris, 1M NaCl, pH 7.5. A synthetic substrate Z-L-Lys-SBz and DTNB (Ellman's reagent) are added to final concentrations of 100 μM each. Absorbance at 405 nm (A405) is recorded at 30 second intervals for 30 min using a microplate spectrophotometer. IC50 values are calculated by nonlinear regression of complement Factor D reaction rates as a function of test compound concentration.
Measurement: IC50 < 1000 nM (pChEMBL 6)
Descriptors: MW 708.59, ALogP 3.36, HBA 11, HBD 1, PSA 166.34, QED 0.21
Compound (SMILES): COC(=O)[C@H](C)[C@H](C)n1c(=O)[nH]/c(=N\c2ccc(CC(C)C)c(OC)c2)n(Cc2ccc(C)cc2)c1=O
InChIKey: TVEQWYQDBNIIQQ-UXHICEINSA-N
Target: P2X purinoceptor 3 (SINGLE PROTEIN), Homo sapiens
UniProt: P56373
Assay: Human P2X3 Receptor Inhibition Assay: Stably expressing cell line (C6BU-1 transfected with human P2X3 receptor gene (GenBank accession number Y07683) was used. The cells were seeded in a 96-well microtiter plate at a concentration of 8000 cells/well and cultured in the medium (8.3% fetal bovine serum, 8.3% horse serum, 1% antibiotic and antifungal in DMEM) for one day at 37° C. under 5% carbon dioxide atmosphere. The medium was replaced with 4 μM Fluo-3-AM solution (20 mM HEPES, 137 mM NaCl, 2.7 mM KCl, 0.9 mM MgCl2, 5.0 mM CaCl2, 5.6 mM D-glucose, 2.5 mM probenecid, 10% BSA, and 0.08% Pluronic F-127, pH 7.5) and incubated at 37° C. under 5% dioxide carbon atmosphere for one hour. The plate was washed with washing buffer (20 mM HEPES, 137 mM NaCl, 2.7 KCl, 0.9 mM MgCl2, 5.0 mM CaCl2, 5.6 mM D-glucose, 2.5 mM probenecid, pH 7.5), each well was added with 40 μL of this buffer. The plate was placed in High-Throughput Screening System FDSS 3000 (Hamamatsu Photonics K.K.). Measurement of fluorescence intensity by FDSS 3000 was started, and 40 μl, of DMSO solutions containing different concentrations of the test, compound as prepared by dilution with dilution buffer (20 mM HEPES, 137 mM NaCl, 2.7 mM KCl, 0.9 mM MgCl2, 5.0 mM CaCl2, 5.6 mM D-glucose, 2.5 mM probenecid, 0.1.% Pluronic F-127, pH 7.5) were dispensed to each well through the built-in automatic dispenser. Five minutes after, 50 nM ATP solution (50 μL) prepared by dilution with the dilution buffer was dispensed through the built-in automatic dispenser, and the measurement of fluorescence intensity was continued for 3 min. For each well, the specific maximum fluorescence intensity was calculated as the ratio of the maximum fluorescence intensity after addition of the ATP solution to the fluorescence intensity at the starting of the measurement. The 50% inhibitory concentration (IC50) was calculated under the assumption that the specific maximum fluorescence intensity without test compound is 0% inhibition and that the specific maximum fluorescence intensity when the dilution buffer was added in place of ATP solution is 100% inhibition, to evaluate the inhibitory activity of the test compound. FDSS software (Hamamatsu Photonics K.K.) was used for calculation of the specific maximum fluorescence intensity. IC50 was calculated using Microsoft Excel (Microsoft Corporation) and Ltd.).
Measurement: IC50 = 43 nM (pChEMBL 7.369999885559082)
Descriptors: MW 508.62, ALogP 3.5, HBA 8, HBD 1, PSA 107.68, QED 0.45
Compound (SMILES): NC(=O)[C@@H](Nc1cc2c3c(c1)nc(-c1cncnc1)n3CCCO2)C1CC1
InChIKey: VVNIKKCSIVONGE-INIZCTEOSA-N
Target: Phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit alpha isoform (SINGLE PROTEIN), Homo sapiens
UniProt: P42336
Assay: p110alpha (alpha) PI3K Binding Assay: PI3K Binding assays are intended for determining the biochemical potency of small molecule PI3K inhibitors. The PI3K lipid kinase reaction is performed in the presence of PIP2:3PS lipid substrate (Promega #V1792) and ATP. Following the termination if the kinase reaction, turnover of ATP to ADP by the phosphorylation of the lipid substrate is detected using the Promega ADP-Glo¿ (Promega #V1792) assay.
Measurement: Ki = 242 nM (pChEMBL 6.619999885559082)
Descriptors: MW 364.41, ALogP 1.95, HBA 7, HBD 2, PSA 107.95, QED 0.72
Compound (SMILES): CC(=O)N1CCc2c(c(Nc3ccc4c(c3)CCCC4)nn2CC2CC2)C1
InChIKey: XMJPUHSMQHGQKV-UHFFFAOYSA-N
Target: CREB-binding protein (SINGLE PROTEIN), Homo sapiens
UniProt: Q92793
Assay: TR-FRET Binding Assay: His/Flag epitope tagged CBP was cloned, expressed, and purified to homogeneity. CBP binding and inhibition was assessed by monitoring the engagement of a biotinylated small molecule compound with the target using the TR-FRET assay technology (Perkin-Elmer). Specifically, in a 384 well ProxiPlate CBP (4 nM final) was combined with biotin-ligand (60 nM final) in 50 mM HEPES (pH 7.5), 50 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 10 minutes incubation at room temperature, a mixture Eu-W1024 Anti-6×His antibody (6×His disclosed as SEO ID NO: 3) (Perkin Elmer AD0110) and SureLight Allophycocyanin-Streptavidin (APC-SA, Perkin Elmer CR130-100) were added to a final concentrations of 0.2 nMolar antibody and 50 nMolar APC-SA, respectively. After twenty minutes of equilibration, the plates were read on an Envision instrument and IC50s calculated using a four parameter non-linear curve fit.
Measurement: IC50 = 30 nM (pChEMBL 7.519999980926514)
Descriptors: MW 364.49, ALogP 3.82, HBA 4, HBD 1, PSA 50.16, QED 0.9
Compound (SMILES): Cc1cc(-c2n[nH]c3cc4c(cc23)CN([C@@H]2CN(CC(F)(F)F)C[C@H]2c2ccccc2F)C(=O)N4)ccn1
InChIKey: LPXHQAMCALNIMD-GBXCKJPGSA-N
Target: Mitogen-activated protein kinase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P28482
Assay: Z′-LYTE Kinase Assay: The inhibition of ERK activity by the compounds disclosed herein was determined using the Z′-LYTE kinase assay kit (Life Technologies) with a Ser/Thr 3 peptide substrate (Life Technologies) according to manufacturers' instructions. The assay was run with an ERK2 enzyme (Life Technologies) concentration of 0.47 ng/μL at 100 μM ATP (approximately the ATP Km for ERK2). The IC50 values for the compounds were determined with 3-fold serial dilutions in duplicate. The compounds were first diluted in 1:3 dilutions in 100% DMSO at 100× the desired concentration, and then further diluted (1:25) in 20 mM HEPES buffer (Invitrogen) to make 4× solutions prior to adding to the enzyme solution. The final DMSO concentration in the assay was 1%. Final reaction volume was 20 μL/well in 384-well plates. Kinase reactions were conducted or 1 hour followed by the assay development reaction (1 hour) in a 20 ul/well in a 384 well plate format.
Measurement: IC50 = 150 nM (pChEMBL 6.820000171661377)
Descriptors: MW 524.52, ALogP 5.45, HBA 4, HBD 2, PSA 77.15, QED 0.35
Compound (SMILES): C=C1C[C@@H](c2nc3cccc(C)c3[nH]2)N(C(=O)c2nc(C)sc2-c2ccc(F)cc2)C1
InChIKey: LKJVMTZGYDHCII-IBGZPJMESA-N
Target: Orexin/Hypocretin receptor type 1 (SINGLE PROTEIN), Homo sapiens
UniProt: O43613
Assay: 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 μg/ml G418, 100 U/ml penicillin, 100 μg/ml streptomycin and 10% heat inactivated fetal calf serum (FCS). The cells are seeded at 20,000 cells/well into 384-well black clear bottom sterile plates (Greiner). The seeded plates are incubated overnight at 37° C. in 5% CO2.Human orexin-A as an agonist is prepared as 1 mM stock solution in MeOH: water (1:1), diluted in HBSS containing 0.1% bovine serum albumin (BSA), NaHCO3: 0.375 g/l and 20 mM HEPES for use in the assay at a final concentration of 3 nM.Antagonists are prepared as 10 mM stock solution in DMSO, then diluted in 384-well plates using DMSO followed by a transfer of the dilutions into in HBSS containing 0.1% bovine serum albumin (BSA), NaHCO3: 0.375 g/l and 20 mM HEPES. On the day of the assay, 50 μl of staining buffer (HBSS containing 1% FCS, 20 mM HEPES, NaHCO3: 0.375 g/l, 5 mM probenecid (Sigma) and 3 M of the fluorescent calcium indicator fluo-4 AM (1 mM stock solution in DMSO, containing 10% pluronic) is added to each well. The 384-well cell-plates are incubated for 50 min at 37° C. in 5% CO2 followed by equilibration at RT for 30 min before measurement.
Measurement: IC50 = 99 nM (pChEMBL 7)
Descriptors: MW 432.52, ALogP 5.59, HBA 4, HBD 1, PSA 61.88, QED 0.43
Compound (SMILES): Cn1cc(-c2cnc(N)c(C(=O)NC3CCC[C@H]3O)c2)cn1
InChIKey: CARFAFSQEPQUGN-ZGTCLIOFSA-N
Target: Tyrosine-protein kinase Mer (SINGLE PROTEIN), Homo sapiens
UniProt: Q12866
Assay: TBD: TBD
Measurement: IC50 > 1000 nM (pChEMBL 6)
Descriptors: MW 301.35, ALogP 0.71, HBA 6, HBD 3, PSA 106.06, QED 0.77
Compound (SMILES): CN(c1ncnc2[nH]ccc12)[C@H]1C[C@@H](CS(=O)(=O)CCCCO)C1
InChIKey: GOVBNJVVLBICAP-BETUJISGSA-N
Target: Tyrosine-protein kinase JAK1 (SINGLE PROTEIN), Homo sapiens
UniProt: P23458
Assay: Caliper Enzyme Assay: Test article and assay controls were added to a 384-well plate. Reaction mixtures contained 20 mM HEPES, pH 7.4, 10 mM magnesium chloride, 0.01% bovine serum albumin (BSA), 0.0005% Tween 20, 1 mM ATP and 1 μM peptide substrate. The JAK1 and TYK2 assays contained 1 μM of the IRStide peptide (5FAM-KKSRGDYMTMQID) and the JAK2 and JAK3 assays contained 1 μM of the JAKtide peptide (FITC-KGGEEEEYFELVKK). The assays were initiated by the addition of 20 nM JAK1, 1 nM JAK2, 1 nM JAK3 or 1 nM TYK2 enzyme and were incubated at room temperature for three hours for JAK1, 60 minutes for JAK2, 75 minutes for JAK3 or 135 minutes for TYK2. Enzyme concentrations and incubation times were optimized for each new enzyme preps and were modified slightly over time to ensure 20%-30% phosphorylation. The assays were stopped with a final concentration of 10 mM EDTA, 0.1% Coating Reagent and 100 mM HEPES, pH=7.4. The assay plates were placed on a Caliper Life Science Lab Chip 3000 (LC3000) instrument, and each well was sampled using appropriate separation conditions to measure the unphosphorylated and phosphorylated peptide.
Measurement: IC50 = 0.039 nM (pChEMBL 10.40999984741211)
Descriptors: MW 352.46, ALogP 1.36, HBA 6, HBD 2, PSA 99.18, QED 0.7
Compound (SMILES): CC(C(=O)N1C[C@H](F)[C@H](NC(=O)c2cc(-c3cc(C(F)(F)F)c4c(N)ncnn34)cc(F)c2F)C1)C(F)(F)F
InChIKey: XDCDJKSTCMOYRZ-AZGGKPGBSA-N
Target: Receptor-interacting serine/threonine-protein kinase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q13546
Assay: RIPK1 HTRF Binding Assay: A solution was prepared containing 0.2 nM Anti GST-Tb (Cisbio, 61GSTTLB), 90.6 nM probe and 1 nM His-GST-TVMV-hRIPK1(1-324) in FRET Buffer (20 mM HEPES, 10 mM MgCl2, 0.015% Brij-35, 4 mM DTT, 0.05 mg/mL BSA). Using Formulatrix Tempest, the detection antibody/enzyme/probe solution (2 mL) was dispensed into wells of a 1536 plate (Black Low Binding Polystyrene 1536 Plate (Corning, 3724)) containing 10 nL of compounds of interest at appropriate concentration in DMSO. The plate was incubated at rt for 1 h. FRET was measured using the EnVision plate reader (Excitation: 340 nM, Emission: 520 nM/495 nM). Total signal (0% inhibition) was calculated from wells containing 10 nL DMSO only. Blank signal (100% inhibition) calculated from wells containing 10 nL of 15 nM staurosporine and internal controls.
Measurement: IC50 = 18000 nM (pChEMBL 4.75)
Descriptors: MW 568.4, ALogP 3.75, HBA 6, HBD 2, PSA 105.62, QED 0.47
Compound (SMILES): CCCC[C@@H](Cc1c[nH]c2ccccc12)NC(=O)c1cnc(N2CCN(C)CC2)s1
InChIKey: GDFWCSZNQVAQGR-SFHVURJKSA-N
Target: Alpha-synuclein (SINGLE PROTEIN), Homo sapiens
UniProt: P37840
Assay: In-Vitro Fluorescence Polarization Assay: The fluorescence polarization assay tests the ability of compounds to inhibit the self-aggregation of α-synuclein peptide fragments. Peptides were incubated for 60 min at room temperature in the presence or absence of test compounds (compound concentrations were 33.3 to 0.3 μM). Samples were read on a BMG Pherastar plate reader in fluorescence polarization mode using excitation at 485 nm and emission at 520 nm. Data was analyzed using a four-parameter logistic fit (XLFit, IDBS Software). Peptide 4F (CTGFVKKDQLGK (SEQ ID NO: 1)) was prepared by American Peptide. Fresh peptide samples were reconstituted in purified water at 5 mM and diluted into 50 mM HEPES pH 7.4 with 50 mM NaCl to 100 nM final concentration. Solid compounds were dissolved in DMSO (10 mM), and then diluted in buffer.
Measurement: IC50 = 400 nM (pChEMBL 6.400000095367432)
Descriptors: MW 425.6, ALogP 3.91, HBA 5, HBD 2, PSA 64.26, QED 0.57