lovingscience/chembl-v37-raw-activities huggingface dataset:
Compound (SMILES): C[C@H](c1ccc(F)cc1)N(Cc1nc2c(c(=O)[nH]1)COCC2)C(=O)CN1CCC(C(=O)c2ccc(F)cc2)CC1
InChIKey: QDMLZFDXQMJAJN-LJQANCHMSA-N
Target: Poly [ADP-ribose] polymerase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P09874
Assay: Biochemical Assay: The autoparsylation activity of the TNKS 1/2 or PARP1/2 enzymes was measured by the liquid chromatography-mass spectrometry (LC/MS) detection of nicotinamide as readout. Compound activity in inhibiting the TNKS and PARP autoparsylation was evaluated by IC50 measurements. In the compound screening assays, the reaction is composed of 5 μL of compound in 8-point serial dilutions with concentrations ranging from 0.0086 to 18.75 μM, 20 nM of purified enzyme, and 250 μM of β-NAD+ in the 1× Assay Buffer. After 60 min incubation at room temperature, the reactions were quenched by the addition of 10 μL of 5× quenching solution (20% formic acid and 500 nM [d]-nicotinamide in water). For the background control wells, 10 μL of the 5× quenching solution per well was added prior to the addition of β-NAD+.
Measurement: IC50 > 19000 nM (pChEMBL 4.7210001945495605)
Descriptors: MW 550.61, ALogP 3.81, HBA 6, HBD 1, PSA 95.6, QED 0.43
Compound (SMILES): CC1=C[C@@H]2Cc3nc4cc(Cl)ccc4c(NCCCCCCCCCNC(=O)c4cc(O)c5c(c4)C(=O)c4cccc(O)c4C5=O)c3[C@H](C1)C2
InChIKey: VZYCFKMNVCPCHV-SKCDSABHSA-N
Target: Acetylcholinesterase (SINGLE PROTEIN), Homo sapiens
UniProt: P22303
Assay: Inhibition Assay: AChE inhibitory activity of compounds (Ia)-(Ie) and (-)-(Ib) and (+)-(Ib) was evaluated spectrophotometrically at 25° C. by the method of Ellman et al., using recombinant human AChE (hAChE) and Electrophorus electricus (ee) AChE and acetylthiocholine iodide as substrate (cf. G. L. Ellman et al., New and Rapid Colorimetric Determination of Acetylcholinesterase Activity Biochem. Pharmacol. 1961, vol. 7, pp. 88-95). For eeAChE inhibitory activity determination, the reaction took place in a final volume of 300 uL of 0.1 M phosphate-buffered solution pH 8.0, containing 0.03 unit/mL of ee AChE and 333 uM 5,5'-dithiobis(2-nitrobenzoic) acid (DTNB) solution used to produce the yellow anion of 5-thio-2-nitrobenzoic acid. Inhibition curves were performed in duplicates using at least 10 increasing concentrations of inhibitor and preincubating at 37° C. for 20 min. One duplicate sample without inhibitor was always present to yield 100% of AChE activity. Then, substrate acetylthiocholine (450 uM) was added and the reaction was developed at 37° C. for 5 min. The color production was measured at 414 nm.
Measurement: IC50 = 2.4 nM (pChEMBL 8.619999885559082)
Descriptors: MW 692.26, ALogP 8.64, HBA 7, HBD 4, PSA 128.62, QED 0.08
Compound (SMILES): C[C@@H]1c2nnc(-c3nc(CC(F)(F)F)no3)n2CCN1C(=O)c1ccc(F)cc1
InChIKey: BGZYIPYUGYCEIV-SECBINFHSA-N
Target: Substance-P receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P25103
Assay: Binding Assay: The affinity of compounds of the invention for the NK-1 receptor was evaluated in CHO recombinant cells which express the human NK-1 receptor. Membrane suspensions were prepared from these cells. The following radioligand: [3H] substance P (PerkinElmer Cat#NET111520) was used in this assay. Binding assays were performed in a 50 mM Tris/5 mM MnCl2/150 mM NaCl/0.1% BSA at pH 7.4. Binding assays consisted of 25 μl of membrane suspension (approximately 5 μg of protein/well in a 96 well plate), 50 μl of compound or reference ligand (Substance P) at increasing concentrations (diluted in assay buffer) and 2 nM [3H] substance P. The plate was incubated 60 min at 25° C. in a water bath and then filtered over GF/C filters (Perkin Elmer, 6005174, presoaked in 0.5% PEI for 2 h at room temperature) with a Filtration unit (Perkin Elmer). The radioactivity retained on the filters was measured by using the TopCount-NXT reader (Packard).
Measurement: Ki > 30000 nM (pChEMBL 4.5229997634887695)
Descriptors: MW 410.33, ALogP 2.79, HBA 7, HBD 0, PSA 89.94, QED 0.62
Compound (SMILES): COc1ccc2c3c1O[C@H]1[C@@]4(OC)CC[C@@]5(C[C@@H]4COC(=O)[C@@H](N)CCC(N)=O)[C@@H](C2)N(CC2CC2)CC[C@]315
InChIKey: YSAXIOGYJGEWRO-RQDKNVRVSA-N
Target: Kappa-type opioid receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P41145
Assay: Functional Assay: Functional [35S]GTPgammaS binding assays were conducted as follows. kippa opioid receptor membrane solution was prepared by sequentially adding final concentrations of 0.026 ug/ul kippa membrane protein (in-house), 10 ug/mL saponin, 3 uM GDP and 0.20 nM [35S]GTPgammaS to binding buffer (100 mM NaCl, 10 mM MgCl2, 20 mM HEPES, pH 7.4) on ice. The prepared membrane solution (190 ul/well) was transferred to 96-shallow well polypropylene plates containing 10 ul of 20x concentrated stock solutions of agonist prepared in DMSO. Plates were incubated for 30 min at a temperature of about 25° C. with shaking. Reactions were terminated by rapid filtration onto 96-well Unifilter GF/B filter plates (Perkin Elmer, Shelton, Conn.) using a 96-well tissue harvester (Packard) and followed by three filtration washes with 2001 ice-cold binding buffer (10 mM NaH2PO4, 10 mM Na2HPO4, pH 7.4). Filter plates were subsequently dried at 50° C. for 2-3 hours.
Measurement: EC50 = 316.93 nM (pChEMBL 6.5)
Descriptors: MW 539.67, ALogP 2.06, HBA 8, HBD 2, PSA 126.34, QED 0.43
Compound (SMILES): O[C@@H]1CCN(C[C@H]2CC[C@H](c3nnn4cnc5[nH]ccc5c34)CC2)C1
InChIKey: FKHKOSJBNTWNIX-MGPQQGTHSA-N
Target: Tyrosine-protein kinase JAK3 (SINGLE PROTEIN), Homo sapiens
UniProt: P52333
Assay: Enzyme Assay: JAK1, JAK2, JAK3 and Tyk2 were purchased from Carna Biosciences, Inc. As the substrate, LANCE Ultra ULight-JAK1 Peptide (manufactured by PerkinElmer Co., Ltd. (PE)) was used. Dilute solutions of compounds and enzymes in assay buffer (50 mM HEPES pH7.5, 1 mM EGTA, 1 mM MgCl2, 2 mM DTT, 0.01% Tween20) were dispensed into wells of a 384-well black plate. After 5 minutes of preincubation, dilute solutions of the substrate and ATP (adenosine triphosphate) were added at a final concentration of 100 μM, and the plate was incubated at room temperature for 2 hours. After addition of a termination reagent containing EDTA (ethylenediamine tetraacetic acid) at a final concentration of 10 mM, LANCE Eu-W1024 Anti-phosphotyrosine (PT66) (manufactured by PE) was added, and after 1 hour of incubation, the fluorescences were measured with ARVO-HTS.
Measurement: IC50 = 790 nM (pChEMBL 6.099999904632568)
Descriptors: MW 340.43, ALogP 1.95, HBA 6, HBD 2, PSA 82.34, QED 0.76
Compound (SMILES): Cc1cccnc1N(C(=O)c1ccc(-c2cnn(C)c2-c2nnn(C(OC(=O)OC(C)C)C(C)C)n2)cc1)[C@@H]1CCCNC1
InChIKey: FHLUJSHYVXOHKA-DDAUYOFQSA-N
Target: Proprotein convertase subtilisin/kexin type 9 (SINGLE PROTEIN), Homo sapiens
UniProt: Q8NBP7
Assay: Cell Free Assay: In order to eliminate the permeability barrier inherent to the WT7 and SCHH cell-based assays a cell-free system was also established to access compound activity. A sequence containing the full length human PCSK9 (NCBI reference identifier, NM-174936.3, where coding sequence start annotated at position 363) along with 84 additional 3â¿² nucleotides, comprising a V5 tag and polylinkinker followed by an in frame modified firefly luciferase reporter (corresponding to nucleotide positions 283-1929 of pGL3, Gen Bank reference identifier JN542721.1) was cloned into the pT7CFE1 expression vector (ThermoScientific). The construct was then in-vitro transcribed using the MEGAscript T7 Kit (Life Technologies) and RNA subsequently purified incorporating the MEGAclear Kit (Life Technologies) according to manufacturer's protocols. HeLa cell lysates were prepared following the protocols described by Mikami (reference is Cell-Free Protein Synthesis Systems with Extracts from Cultured Human Cells, S. Mikami, T. Kobayashi and H. Imataka; from Methods in Molecular Biology, vol. 607, pages 43-52, Y. Endo et al. (eds.), Humana Press, 2010) with the following modifications.
Measurement: IC50 = 7380 nM (pChEMBL 5.130000114440918)
Descriptors: MW 601.71, ALogP 4.56, HBA 12, HBD 1, PSA 142.18, QED 0.27
Compound (SMILES): CN[C@H](C)C(=O)Nc1cc(-c2ccc(OC)nc2)cc(C#Cc2ccccc2)n1
InChIKey: SQIFKCAJUJSUEH-MRXNPFEDSA-N
Target: Baculoviral IAP repeat-containing protein 2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q13490
Assay: XIAP BIR3 & cIAP1 BIR3 Binding Assays (DELFIA): BIR3 domains (10 nM) were incubated with SMAC peptide (10 nM) in assay buffer (50 mM Tris, 120 mM NaCl, 0.1% BSA, 1 mM DTT, 0.05% TritonX100) for one hour at room temperature in the presence of inhibitory compounds. The assay mixture was transferred to a strepatvidin coated plate and incubated for one hour at room temperature to allow binding of the biotinylated peptide and associated BIR3 domains to the plate. After several washing steps Eu labeled anti-GST antibody (e.g. Perkin Elmer DELFIA Eu-N1-antiGST AD0250) was added to detect BIR3 domain-SMAC peptide interactions according to Perkin Elmer's instructions. Briefly, the antibody was added (dilution 1:5000 in Perkin Elmer DELFIA Assay Buffer 2013-01) and incubated for one hour. After 3 washing steps using Delfia Washing Buffer (Perkin Elmer DELFIA Wash 2013-05), Enhancement Solution (Perkin Elmer Enhancement Asolution 2013-02) was added and incubation continued for 10 minutes. Time resolved Europium fluorescence was measured in a Wallac Victor using Standard assay settings.
Measurement: IC50 = 325 nM (pChEMBL 6.489999771118164)
Descriptors: MW 386.46, ALogP 3.1, HBA 5, HBD 2, PSA 76.14, QED 0.66
Compound (SMILES): CCO[C@@H]1CC(CCO)N(C(=O)c2cc(OC)c3oc(NC(CF)c4cccc(Cl)c4)nc3c2)C1
InChIKey: SQGQDMQHBJIGDA-OBXNSHGQSA-N
Target: Prothrombin (SINGLE PROTEIN), Homo sapiens
UniProt: P00734
Assay: Inhibition Assay: To determine the thrombin inhibition of the substances listed above, a biochemical test system is constructed in which the conversion of a thrombin substrate is used for determining the enzymatic activity of human thrombin. Here, thrombin cleaves aminomethylcoumarin, which is measured fluorescently, from the peptic substrate. The determinations are carried out in microtitre plates.Substances to be tested are dissolved in various concentrations in dimethyl sulphoxide and incubated for 15 min with human thrombin (0.06 nmol/l dissolved in 50 mmol/l of Tris buffer [C,C,C-tris(hydroxymethyl)aminomethane], 100 mmol/l of sodium chloride, 0.1% BSA [bovine serum albumin], pH 7.4) at 22° C. The substrate (5 umol/l Boc-Asp(OBzl)-Pro-Arg-AMC from Bachem) is then added. After 30 min of incubation, the sample is excited at a wavelength of 360 nm and the emission is measured at 460 nm. The measured emissions of the test batches with test substance are compared to the control batches without test substance (only dimethyl sulphoxide instead of test substance in dimethyl sulphoxide) and the IC50 values are calculated from the concentration/activity relationships.
Measurement: IC50 = 0.54 nM (pChEMBL 9.270000457763672)
Descriptors: MW 505.97, ALogP 4.61, HBA 7, HBD 2, PSA 97.06, QED 0.41
Compound (SMILES): CC[C@@H](NC(=O)c1cc(C(=O)NC(C)c2nnn(C)n2)n2c1COCC2)c1ccccc1
InChIKey: QUIIABVJOCTSBL-FBMWCMRBSA-N
Target: Potassium channel subfamily K member 3 (SINGLE PROTEIN), Homo sapiens
UniProt: O14649
Assay: Activity Assay: Human TASK-1 channels were expressed in Xenopus oocytes. For this purpose, oocytes were isolated from Xenopus laevis and defoliculated. Subsequently, TASK-1-encoding RNA synthesized in vitro was injected into the oocytes. After two days of TASK-1 protein expression, TASK-1 currents were measured by two-microelectrode voltage clamp. Data were acquired and analyzed using a TEC-10cx amplifier (NPI Electronic, Tamm, Germany) connected to an ITC-16 interface (Instrutech Corp., Long island, USA) and Pulse software (HEKA Elektronik, Lambrecht, Germany). Oocytes were clamped to −90 mV and TASK-1 mediated currents were measured during 500 ms voltage pulses to 40 mV. Oocytes were continuously superfused with ND96 buffer containing 98 mM sodium chloride, 2 mM potassium chloride, 1.8 mM calcium chloride, 1 mM magnesium chloride, 5 mM 4-(2-hydroxyethyl)-piperazine-1-ethanesulfonic acid (HEPES: pH adjusted to 7.4 with sodium hydroxide). All experiments were performed at room temperature. Test compounds were consecutively added to the bath solution at rising concentrations.
Measurement: IC50 = 1600 nM (pChEMBL 5.800000190734863)
Descriptors: MW 437.5, ALogP 1.91, HBA 8, HBD 2, PSA 115.96, QED 0.58
Compound (SMILES): NC1CCC(N[C@H]2C[C@@H]2c2cccnc2)CC1
InChIKey: QAZSEXHFQQZAIT-PQAZSJQKSA-N
Target: Lysine-specific histone demethylase 1A (SINGLE PROTEIN), Homo sapiens
UniProt: O60341
Assay: Biological Assay: The compounds of the invention can be tested for their ability to inhibit LSD1. The ability of the compounds of the invention to inhibit LSD1 can be tested as follows. Human recombinant LSD1 protein was purchased from BPS Bioscience Inc (catalog reference number 50100: human recombinant LSD1, GenBank accession no. NM_015013, amino acids 158-end with N-terminal GST tag, MW: 103 kDa). In order to monitor LSD1 enzymatic activity and/or its inhibition rate by our inhibitor(s) of interest, di-methylated H3-K4 peptide (Anaspec) was chosen as a substrate. The demethylase activity was estimated, under aerobic conditions, by measuring the release of H2O2 produced during the catalytic process, using the Amplex Red hydrogen peroxide/peroxidase assay kit (Invitrogen).Briefly, a fixed amount of LSD1 was incubated on ice for 15 minutes, in the absence and/or in the presence of at least eight 3-fold serial dilutions of the respective inhibitor (e.g., from 0 to 75 uM, depending on the inhibitor strength). Tranylcypromine (Biomol International) was used as a control for inhibition. Within the experiment, each concentration of inhibitor was tested in duplicate. After leaving the enzyme interacting with the inhibitor, KM of di-methylated H3-K4 peptide was added to each reaction and the experiment was left for 30 minutes at 37° C. in the dark. The enzymatic reactions were set up in a 50 mM sodium phosphate, pH 7.4 buffer. At the end of the incubation, Amplex Red reagent and horseradish peroxidase (HPR) solution were added to the reaction according to the recommendations provided by the supplier (Invitrogen), and left to incubate for 5 extra minutes at room temperature in the dark. A 1 uM H2O2 solution was used as a control of the kit efficiency. The conversion of the Amplex Red reagent to resorufin due to the presence of H2O2 in the assay, was monitored by fluorescence (excitation at 540 nm, emission at 590 nm) using a microplate reader (Infinite 200, Tecan). Arbitrary units were used to measure level of H2O2 produced in the absence and/or in the presence of inhibitor. The maximum demethylase activity of LSD1 was obtained in the absence of inhibitor and corrected for background fluorescence in the absence of LSD1. The IC50 value of each inhibitor was calculated with GraphPad Prism Software.
Measurement: IC50 = 69 nM (pChEMBL 7.159999847412109)
Descriptors: MW 231.34, ALogP 1.8, HBA 3, HBD 2, PSA 50.94, QED 0.83
Compound (SMILES): C[C@@H](Oc1cc(-c2cnn(C3CC4(CCNCC4)C3)c2)cnc1N)c1c(Cl)ccc(F)c1Cl
InChIKey: MXQDRRGPOHEUMT-CQSZACIVSA-N
Target: ALK tyrosine kinase receptor (SINGLE PROTEIN), Homo sapiens
UniProt: Q9UM73
Assay: Inhibition Assay: The inhibitory activities of compounds of Formula I were assay at Reaction Biology Corporation, One Great Valley Parkway, Malvern, Pa., USA. Human ALK and cMet enzymes were used and the substrate was a peptide substrate, poly[Glu:Tyr] (4:1) at a concentration of 0.2 mg/ml. The ATP concentration for the assay was 10 uM and Staurosporine was used as a standardwith an IC50 of 2.3 nM, and 75 nM, respectively for ALK and cMet.
Measurement: IC50 < 1.2 nM (pChEMBL 8.920999526977539)
Descriptors: MW 490.41, ALogP 5.82, HBA 6, HBD 2, PSA 77.99, QED 0.44
Compound (SMILES): CCCc1nn(C)c(C)c1NS(=O)(=O)c1ccc(Br)cc1
InChIKey: LZJUEYNZAAZMEH-UHFFFAOYSA-N
Target: Glycylpeptide N-tetradecanoyltransferase (SINGLE PROTEIN), Leishmania major
UniProt: Q4Q5S8
Assay: Enzyme Inhibition Assay: Measurements were performed using a modification of the scintillation proximity assay platform described previously by Georgopapadakou, N. H. et al. (22nd International Congress on Chemotherapy, 2001, Abstract P16.001). Compounds were solubilised in DMSO at a top concentration of 10 mM and serially diluted in half log steps to achieve a range of final assay concentrations of 100 uM to 1 nM. Compound at each concentration (100-fold final) was added to white 384 well plates in a volume of 0.5 ml. Human, A. fumigatus, T. brucei or L. major N-myristoyl transferase enzyme, dissolved to a working concentration of 10 nM in assay buffer (30 mM Tris/HCl pH 7.4, 0.5 mM EGTA, 0.5 mM EDTA, 1.25 mM DTT, 0.1% Triton X-100), was then added to columns 1 to 11 and 13 to 23 of the plates in a volume of 20 ml. To columns 12 and 24, 20 ml assay buffer was added to provide a no enzyme control. Following a 5 minute incubation at room temperature the substrates (GCGGSKVKPQPPQAK(Biotin)-Amide and myristoyl coenzyme A), dissolved in assay buffer, were added to all wells in a volume of 20 ml to start the reaction. The final concentrations of peptide and 3H-myristoyl coenzyme A were 0.5 mM and 125 nM respectively and the specific activity of the radiolabel was 8 Ci/mmol. Plates were then incubated at room temperature for up to 50 minutes (dependant upon the period of linearity for the different enzyme species) before SPA beads, suspended to 1 mg/ml in a stop solution (200 mM Phosphoric Acid/NaOH pH 4, 750 mM MgCl2), were added in a volume of 40 ml.
Measurement: IC50 > 1000 nM (pChEMBL 6)
Descriptors: MW 372.29, ALogP 3.24, HBA 4, HBD 1, PSA 63.99, QED 0.88
Compound (SMILES): CN(c1ccc(S(N)(=O)=O)cn1)[C@@H]1CCN(c2ncnc3[nH]ccc23)C1
InChIKey: GPWLOCKZXAMXFZ-LLVKDONJSA-N
Target: Tyrosine-protein kinase JAK3 (SINGLE PROTEIN), Homo sapiens
UniProt: P52333
Assay: Enzymatic Assay: JAK1/2/3 kinase assay are performed in vitro using Kit-Tyr 6 Peptide (Invitrogen, Cat. No. PV4122). TYK2 kinase assay are performed in vitro using Z'-LYTE™ Kinase Assay Kit-Tyr 3 Peptide (Invitrogen, Cat. No. PV3192). Recombinant human JAK1/2/3 or TYK2 catalytic domains are from Invitrogen (Cat No. PV4774/PV4210/PV3855/PV4790); All reactions (20 μL) are started by adding 2.5 μL of the testing compound in 4% DMSO solution, 5 μL of Kinase/Peptide substrate Mixture (3.2, 0.04, 0.2 or 8 μg/mL for Recombinant human JAK1/2/3 catalytic domains, 4 μM for Z-LYTE™ Tyr 6 peptide or Z-LYTE™ Tyr 3 peptide) or Phospho-Peptide solution (Invitrogen, Cat. No. PV3192, diluted with 1.33× Kinase Buffer), 2.5 μL ATP Solution (300/100/40/100M, JAK1/JAK2/JAK3/TYK2) or 1.33× Kinase Buffer (Invitrogen, Cat. No. PV3189, 5× diluted with distilled water). The 384-well assay plate (Corning, Cat. No. 3575) is mixed and incubated at room temperature for 1 hour. 5 μL of the Development Solution (Dilute Development Reagent A (Cat. No. PV3297) is diluted to 1/64 with Development Buffer (Cat. No. PV3127) for JAK1, JAK2 and JAK3 assay; Development Reagent A (Cat. No. PV3297) is diluted to 1/2048 with Development Buffer (Cat. No. PV3127) for TYK2 assay. The diluted Development Solution is then added to each well, mixed and incubated at room temperature for another 1 hour. The kinase reaction is then stopped by adding 5 μL of the Stop Reagent (Invitrogen, Cat. No. PV3094), and the plate is read with Wallac 1420 VICTOR3 Multilabel Counter (PerkinElmer™) at 445 nm and 520 nm fluorescence. All compounds are initially tested at 8 different concentrations (1 μM down to 0.0003 μM) using a 1:3 serial dilution scheme.
Measurement: IC50 = 86.3 nM (pChEMBL 7.059999942779541)
Descriptors: MW 373.44, ALogP 0.72, HBA 7, HBD 2, PSA 121.1, QED 0.69
Compound (SMILES): C[C@H](Cc1cccc(N)c1)NC[C@H](O)c1cc(O)cc(O)c1
InChIKey: YSDUJIGQJOLYND-DIFFPNOSSA-N
Target: Beta-2 adrenergic receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P07550
Assay: Beta2-AR Binding Assay: HEK 293 cells stability transfected with cDNA encoding human beta2-AR (provided by Dr. Brian Kobilka, Stanford Medical Center, Palo Alto, Calif.) were grown in Dulbecco's Modified Eagle Medium (DMEM) containing 10% fetal bovine serum (FBS), 0.05% penicillin-streptomycin, and 400 Î¼g/ml G418 as previously described (Pauwels et al., Biochem. Pharmacol. 42: 1683-1689, 1991). The cells were scraped from the 150x25 mm plates and centrifuged at 500xg for 5 minutes. The pellet was homogenized in 50 mM Tris-HCl, pH 7.7, with a Polytron, centrifuged at 27,000xg, and resuspended in the same buffer. The latter process was repeated, and the pellet was resuspended in 25 mM Tris-HCl containing 120 mM NaCl, 5.4 mM KCl, 1.8 mM CaCl2, 0.8 mM MgCl2, and 5 mM glucose, pH 7.4. The binding assays contained 0.3 nM [3H]CGP-12177 in a volume of 1.0 ml. Nonspecific binding was determined by 1 uM propranolol.
Measurement: Kd = 1862 nM (pChEMBL 5.730000019073486)
Descriptors: MW 302.37, ALogP 1.93, HBA 5, HBD 5, PSA 98.74, QED 0.53
Compound (SMILES): CC#CCn1c(N2CCC[C@@H](N)C2)nc2c1c(=O)n(Cc1nc3cc(F)ccc3s1)c(=O)n2C
InChIKey: KNVUDUILISDHBC-OAHLLOKOSA-N
Target: Dipeptidyl peptidase 4 (SINGLE PROTEIN), Homo sapiens
UniProt: P27487
Assay: DPP-IV Activity InhibitoryAssay: Gly-Pro-7-amido-4-methylcoumarin can be hydrolyzed by dipeptidyl peptidase IV (DPP-IV) at room temperature, to generate 7-amido-4-methyl coumarin, which can emit fluorescence with wavelength of 460 nm at excitation wavelength of 355 nm. The variation of the product amount can be determined by the variation of fluorescence intensity, so as to reflect the activity level of the enzyme. The dipeptidyl peptidase IV (DPP-IV), DPP-IV buffer and test samples were used to construct the reaction system of 200 uL, while the blank control (without enzyme and samples) and negative control (without samples) having the same volume were set up. The reaction system and the controls were reacted at room temperature for 10 min, and then dipeptidyl peptidase IV substrate was added thereto respectively, then reacted at room temperature for 30 min. The fluorescence intensity F (excitation wavelength of 355 nm, emission wavelength of 460 nm) was determined.
Measurement: IC50 = 0.08 nM (pChEMBL 10.100000381469727)
Descriptors: MW 481.56, ALogP 1.64, HBA 10, HBD 1, PSA 103.97, QED 0.44
Compound (SMILES): Cc1c(C(=O)N2CCc3c(nnn3-c3ncccn3)[C@H]2C)cccc1C(F)(F)F
InChIKey: URMMXPJYAWCRNC-GFCCVEGCSA-N
Target: P2X purinoceptor 7 (SINGLE PROTEIN), Rattus norvegicus
UniProt: Q64663
Assay: FLIPR Assay: 1321 N1 cells expressing the recombinant human, rat or mouse P2X7 channel was cultured in HyQ DME/(HyClone/Dulbecco's Modified Eagle Medium) high glucose supplemented with 10% Fetal Bovine Serum (FBS) and appropriate selection marker. Cells were seeded at a density of 25000 cells/well (96-well clear bottom black walled plates) in 100 ul volume/well. On the day of the experiment, cell plates were washed with assay buffer, containing (in mM): 130 NaCl, 2 KCl, 1 CaCl2, 1 MgCl2, 10 HEPES, 5 glucose; pH 7.40 and 300 mOs. After the wash, cells were loaded with the Calcium-4 dye (Molecular Device) and incubated in the dark for 60 minutes. Test compounds were prepared at 250Ã¿ the test concentration in neat DMSO. Intermediate 96-well compound plates were prepared by transferring 1.2 uL of the compound into 300 uL of assay buffer. A further 3x dilution occurred when transferring 50 uL/well of the compound plate to 100 uL/well in the cell plate. Cells were incubateÂ  with test compounds and dye for 30 minutes. Calcium dye fluorescence was monitored in FLIPR as the cells were challenged by adding 50 uL/well of BzATP (final concentration is 250 uM BzATP (human and rat) or 600 (mouse)). The fluorescence change was measured 180 seconds after adding the agonist.
Measurement: IC50 = 8.5 nM (pChEMBL 8.069999694824219)
Descriptors: MW 402.38, ALogP 3.14, HBA 6, HBD 0, PSA 76.8, QED 0.66
Compound (SMILES): CCc1nsc(-c2nnc3n2CCN(C(=O)c2ccc(F)cc2)[C@@H]3C)n1
InChIKey: UOMUYZXEONWAOD-SNVBAGLBSA-N
Target: Substance-P receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P25103
Assay: Binding Assay: The affinity of compounds of the invention for the NK-1 receptor was evaluated in CHO recombinant cells which express the human NK-1 receptor. Membrane suspensions were prepared from these cells. The following radioligand: [3H] substance P (PerkinElmer Cat#NET111520) was used in this assay. Binding assays were performed in a 50 mM Tris/5 mM MnCl2/150 mM NaCl/0.1% BSA at pH 7.4. Binding assays consisted of 25 μl of membrane suspension (approximately 5 μg of protein/well in a 96 well plate), 50 μl of compound or reference ligand (Substance P) at increasing concentrations (diluted in assay buffer) and 2 nM [3H] substance P. The plate was incubated 60 min at 25° C. in a water bath and then filtered over GF/C filters (Perkin Elmer, 6005174, presoaked in 0.5% PEI for 2 h at room temperature) with a Filtration unit (Perkin Elmer). The radioactivity retained on the filters was measured by using the TopCount-NXT reader (Packard).
Measurement: Ki = 23800 nM (pChEMBL 4.619999885559082)
Descriptors: MW 372.43, ALogP 2.72, HBA 7, HBD 0, PSA 76.8, QED 0.71
Compound (SMILES): CCOC(=O)C[C@H]1C[C@@]2(O)[C@H]3Cc4ccc(OC)cc4[C@@]2(CCN3CC2CC2)CC1=O
InChIKey: BLZWGGQJDWDCCU-VCRBZLFYSA-N
Target: Kappa-type opioid receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P41145
Assay: [35S]GTPγS Functional Assay: Functional [35S]GTPγS binding assays were conducted as follows. κ opioid receptor membrane solution was prepared by sequentially adding final concentrations of 0.026 μg/μl κ membrane protein (in-house), 10 μg/mL saponin, 3 μM GDP and 0.20 nM [35S]GTPγS to binding buffer (100 mM NaCl, 10 mM MgCl2, 20 mM HEPES, pH 7.4) on ice. The prepared membrane solution (190 μl/well) was transferred to 96-shallow well polypropylene plates containing 10 μl of 20× concentrated stock solutions of agonist prepared in DMSO. Plates were incubated for 30 min at a temperature of about 25° C. with shaking. Reactions were terminated by rapid filtration onto 96-well Unifilter GF/B filter plates (Perkin Elmer, Shelton, Conn.) using a 96-well tissue harvester (Packard) and followed by three filtration washes with 200 μl ice-cold binding buffer (10 mM NaH2PO4, 10 mM Na2HPO4, pH 7.4). Filter plates were subsequently dried at 50° C. for 2-3 hours. Fifty μl/well scintillation cocktail (Perkin Elmer, Shelton, Conn.) was added and plates were counted in a Packard Top-Count for 1 min/well.
Measurement: EC50 = 5114.25 nM (pChEMBL 5.289999961853027)
Descriptors: MW 427.54, ALogP 2.64, HBA 6, HBD 1, PSA 76.07, QED 0.7
Compound (SMILES): NC(=O)C1CCC(N[C@H]2C[C@@H]2c2ccc(-c3cccc(C(F)(F)F)c3)nc2)CC1
InChIKey: WVVHNLMQBRBRGZ-DTYDVHMXSA-N
Target: Lysine-specific histone demethylase 1A (SINGLE PROTEIN), Homo sapiens
UniProt: O60341
Assay: Biological Assay: The compounds of the invention can be tested for their ability to inhibit LSD1. The ability of the compounds of the invention to inhibit LSD1 can be tested as follows. Human recombinant LSD1 protein was purchased from BPS Bioscience Inc (catalog reference number 50100: human recombinant LSD1, GenBank accession no. NM_015013, amino acids 158-end with N-terminal GST tag, MW: 103 kDa). In order to monitor LSD1 enzymatic activity and/or its inhibition rate by our inhibitor(s) of interest, di-methylated H3-K4 peptide (Anaspec) was chosen as a substrate. The demethylase activity was estimated, under aerobic conditions, by measuring the release of H2O2 produced during the catalytic process, using the Amplex Red hydrogen peroxide/peroxidase assay kit (Invitrogen).Briefly, a fixed amount of LSD1 was incubated on ice for 15 minutes, in the absence and/or in the presence of at least eight 3-fold serial dilutions of the respective inhibitor (e.g., from 0 to 75 uM, depending on the inhibitor strength). Tranylcypromine (Biomol International) was used as a control for inhibition. Within the experiment, each concentration of inhibitor was tested in duplicate. After leaving the enzyme interacting with the inhibitor, KM of di-methylated H3-K4 peptide was added to each reaction and the experiment was left for 30 minutes at 37° C. in the dark. The enzymatic reactions were set up in a 50 mM sodium phosphate, pH 7.4 buffer. At the end of the incubation, Amplex Red reagent and horseradish peroxidase (HPR) solution were added to the reaction according to the recommendations provided by the supplier (Invitrogen), and left to incubate for 5 extra minutes at room temperature in the dark. A 1 uM H2O2 solution was used as a control of the kit efficiency. The conversion of the Amplex Red reagent to resorufin due to the presence of H2O2 in the assay, was monitored by fluorescence (excitation at 540 nm, emission at 590 nm) using a microplate reader (Infinite 200, Tecan). Arbitrary units were used to measure level of H2O2 produced in the absence and/or in the presence of inhibitor. The maximum demethylase activity of LSD1 was obtained in the absence of inhibitor and corrected for background fluorescence in the absence of LSD1. The IC50 value of each inhibitor was calculated with GraphPad Prism Software.
Measurement: IC50 = 91 nM (pChEMBL 7.039999961853027)
Descriptors: MW 403.45, ALogP 4.26, HBA 3, HBD 2, PSA 68.01, QED 0.78
Compound (SMILES): C[C@]1(c2nc(NC(=O)c3ncc(C#CCO)cc3Cl)ccc2F)N=C(N)OCC1(F)F
InChIKey: GNNDIRZJLBDRCO-GOSISDBHSA-N
Target: Amyloid-beta precursor protein (SINGLE PROTEIN), Homo sapiens
UniProt: P05067
Assay: Inhibition Assay: Chinese hamster ovary cells are transfected with the human gene for amyloid precursor protein. The cells are plated at a density of 8000 cells/well into 96-well microtiter plates and cultivated for 24 hours in DMEM cell culture medium containing 10% FCS. The test compound is added to the cells at various concentrations, and the cells are cultivated for 24 hours in the presence of the test compound. The supernatants are collected, and the concentration of amyloid peptide 1-40 is determined using state of the art immunoassay techniques, for example sandwich ELISA, homogenous time-resolved fluorescence (HTRF) immunoassay, or electro-chemiluminescence immunoassay. The potency of the compound is calculated from the percentage of inhibition of amyloid peptide release as a function of the test compound concentration.
Measurement: IC50 = 19 nM (pChEMBL 7.71999979019165)
Descriptors: MW 453.81, ALogP 2.06, HBA 7, HBD 3, PSA 122.72, QED 0.61
Compound (SMILES): NC1CCC(N[C@H]2C[C@@H]2c2ccc(OCc3ccccc3)cc2)CC1
InChIKey: HPMSXSJJPUNVQO-FGGKUVOCSA-N
Target: Lysine-specific histone demethylase 1A (SINGLE PROTEIN), Homo sapiens
UniProt: O60341
Assay: Biological Assay: The compounds of the invention can be tested for their ability to inhibit LSD1. The ability of the compounds of the invention to inhibit LSD1 can be tested as follows. Human recombinant LSD1 protein was purchased from BPS Bioscience Inc (catalog reference number 50100: human recombinant LSD1, GenBank accession no. NM_015013, amino acids 158-end with N-terminal GST tag, MW: 103 kDa). In order to monitor LSD1 enzymatic activity and/or its inhibition rate by our inhibitor(s) of interest, di-methylated H3-K4 peptide (Anaspec) was chosen as a substrate. The demethylase activity was estimated, under aerobic conditions, by measuring the release of H2O2 produced during the catalytic process, using the Amplex Red hydrogen peroxide/peroxidase assay kit (Invitrogen).Briefly, a fixed amount of LSD1 was incubated on ice for 15 minutes, in the absence and/or in the presence of at least eight 3-fold serial dilutions of the respective inhibitor (e.g., from 0 to 75 uM, depending on the inhibitor strength). Tranylcypromine (Biomol International) was used as a control for inhibition. Within the experiment, each concentration of inhibitor was tested in duplicate. After leaving the enzyme interacting with the inhibitor, KM of di-methylated H3-K4 peptide was added to each reaction and the experiment was left for 30 minutes at 37° C. in the dark. The enzymatic reactions were set up in a 50 mM sodium phosphate, pH 7.4 buffer. At the end of the incubation, Amplex Red reagent and horseradish peroxidase (HPR) solution were added to the reaction according to the recommendations provided by the supplier (Invitrogen), and left to incubate for 5 extra minutes at room temperature in the dark. A 1 uM H2O2 solution was used as a control of the kit efficiency. The conversion of the Amplex Red reagent to resorufin due to the presence of H2O2 in the assay, was monitored by fluorescence (excitation at 540 nm, emission at 590 nm) using a microplate reader (Infinite 200, Tecan). Arbitrary units were used to measure level of H2O2 produced in the absence and/or in the presence of inhibitor. The maximum demethylase activity of LSD1 was obtained in the absence of inhibitor and corrected for background fluorescence in the absence of LSD1. The IC50 value of each inhibitor was calculated with GraphPad Prism Software.
Measurement: IC50 = 40 nM (pChEMBL 7.400000095367432)
Descriptors: MW 336.48, ALogP 3.98, HBA 3, HBD 2, PSA 47.28, QED 0.84
Compound (SMILES): CC1(N)CCC(N[C@H]2C[C@@H]2c2ccccc2)CC1
InChIKey: XWEAHNZIYFXBRU-FYRZTWFESA-N
Target: Lysine-specific histone demethylase 1A (SINGLE PROTEIN), Homo sapiens
UniProt: O60341
Assay: Biological Assay: The compounds of the invention can be tested for their ability to inhibit LSD1. The ability of the compounds of the invention to inhibit LSD1 can be tested as follows. Human recombinant LSD1 protein was purchased from BPS Bioscience Inc (catalog reference number 50100: human recombinant LSD1, GenBank accession no. NM_015013, amino acids 158-end with N-terminal GST tag, MW: 103 kDa). In order to monitor LSD1 enzymatic activity and/or its inhibition rate by our inhibitor(s) of interest, di-methylated H3-K4 peptide (Anaspec) was chosen as a substrate. The demethylase activity was estimated, under aerobic conditions, by measuring the release of H2O2 produced during the catalytic process, using the Amplex Red hydrogen peroxide/peroxidase assay kit (Invitrogen).Briefly, a fixed amount of LSD1 was incubated on ice for 15 minutes, in the absence and/or in the presence of at least eight 3-fold serial dilutions of the respective inhibitor (e.g., from 0 to 75 uM, depending on the inhibitor strength). Tranylcypromine (Biomol International) was used as a control for inhibition. Within the experiment, each concentration of inhibitor was tested in duplicate. After leaving the enzyme interacting with the inhibitor, KM of di-methylated H3-K4 peptide was added to each reaction and the experiment was left for 30 minutes at 37° C. in the dark. The enzymatic reactions were set up in a 50 mM sodium phosphate, pH 7.4 buffer. At the end of the incubation, Amplex Red reagent and horseradish peroxidase (HPR) solution were added to the reaction according to the recommendations provided by the supplier (Invitrogen), and left to incubate for 5 extra minutes at room temperature in the dark. A 1 uM H2O2 solution was used as a control of the kit efficiency. The conversion of the Amplex Red reagent to resorufin due to the presence of H2O2 in the assay, was monitored by fluorescence (excitation at 540 nm, emission at 590 nm) using a microplate reader (Infinite 200, Tecan). Arbitrary units were used to measure level of H2O2 produced in the absence and/or in the presence of inhibitor. The maximum demethylase activity of LSD1 was obtained in the absence of inhibitor and corrected for background fluorescence in the absence of LSD1. The IC50 value of each inhibitor was calculated with GraphPad Prism Software.
Measurement: IC50 = 148 nM (pChEMBL 6.829999923706055)
Descriptors: MW 244.38, ALogP 2.79, HBA 2, HBD 2, PSA 38.05, QED 0.86
Compound (SMILES): COc1ccc2c3c1O[C@H]1[C@@]4(OC)CC[C@@]5(C[C@@H]4COc4ccc(-n6ccnc6)cc4)[C@@H](C2)N(CC2CC2)CC[C@]315
InChIKey: RLFBLDXDGTVVAF-KKASODRESA-N
Target: Delta-type opioid receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P41143
Assay: Radioligand Dose-Displacement Binding Assay: Delta-opioid Receptor Binding Assay Procedures were conducted as follows. Radioligand dose-displacement assays used 0.3 nM [3H]-Naltrindole (Perkin Elmer, Shelton, Conn.; 33.0 Ci/mmole) with 5 ug membrane protein (Perkin Elmer, Shelton, Conn.) in a final volume of 500 ul binding buffer (5 mM MgCl2, 5% DMSO, 50 mM Trizma base, pH 7.4). Non-specific binding was determined in the presence of 25 uM unlabeled naloxone. All reactions were performed in 96-deep well polypropylene plates for 1 hr at a temperature of about 25° C. Binding reactions were terminated by rapid filtration onto 96-well Unifilter GF/C filter plates (Perkin Elmer, Shelton, Conn.) presoaked in 0.5% polyethylenimine (Sigma). Harvesting was performed using a 96-well tissue harvester (Perkin Elmer, Shelton, Conn.) followed by five filtration washes with 500 ul ice-cold binding buffer. Filter plates were subsequently dried at 50° C. for 1-2 hours.
Measurement: Ki = 211.03 nM (pChEMBL 6.679999828338623)
Descriptors: MW 553.7, ALogP 5.18, HBA 7, HBD 0, PSA 57.98, QED 0.38
Compound (SMILES): COCCOCCOCCOC[C@H]1C[C@@]23CC[C@]1(OC)[C@@H]1Oc4c(OC)ccc5c4[C@@]12CCN(CC1CC1)[C@@H]3C5
InChIKey: AJOMQFBUCVXNGB-BUVWRSTLSA-N
Target: Delta-type opioid receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P41143
Assay: Radioligand Dose-Displacement Binding Assay: Delta-opioid Receptor Binding Assay Procedures were conducted as follows. Radioligand dose-displacement assays used 0.3 nM [3H]-Naltrindole (Perkin Elmer, Shelton, Conn.; 33.0 Ci/mmole) with 5 ug membrane protein (Perkin Elmer, Shelton, Conn.) in a final volume of 500 ul binding buffer (5 mM MgCl2, 5% DMSO, 50 mM Trizma base, pH 7.4). Non-specific binding was determined in the presence of 25 uM unlabeled naloxone. All reactions were performed in 96-deep well polypropylene plates for 1 hr at a temperature of about 25° C. Binding reactions were terminated by rapid filtration onto 96-well Unifilter GF/C filter plates (Perkin Elmer, Shelton, Conn.) presoaked in 0.5% polyethylenimine (Sigma). Harvesting was performed using a 96-well tissue harvester (Perkin Elmer, Shelton, Conn.) followed by five filtration washes with 500 ul ice-cold binding buffer. Filter plates were subsequently dried at 50° C. for 1-2 hours.
Measurement: Ki = 3024.75 nM (pChEMBL 5.519999980926514)
Descriptors: MW 557.73, ALogP 3.62, HBA 8, HBD 0, PSA 67.85, QED 0.3
Compound (SMILES): Cc1cc(N=S(C)(C)=O)cc2ncnc(Nc3ccc(F)cc3O[C@H](C)C#N)c12
InChIKey: ACCGDLRUTZSFSS-CYBMUJFWSA-N
Target: MAP kinase-interacting serine/threonine-protein kinase 2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9HBH9
Assay: In vitro Kinase Assay: The inhibition of kinase activity of MNK2a was assessed using pre-activated GST-MNK2a. The white, 384-well OptiPlate F plates were purchased from PerkinElmer. The ADP-Glo Kinase Assay (including ultra pure ATP) was purchased from Promega (V9103). Activated MNK2a was obtained as described in WO2011/104340. The unlabeled eIF4E peptide (NH2-TATKSGSTTKNR-CONH2), differing from Seq. ID No. 5 of WO 2011/104340 by the C-terminal CONH2 group, was purchased from Thermo Fisher Scientific. All other materials were of highest grade commercially available. Compounds are tested in either serial dilutions or single dose concentrations. The compound stock solutions are 10 mM in 100% DMSO The serial compound dilutions are prepared in 100% DMSO followed by 1:27.3 intermediate dilution in assay buffer. The final DMSO concentration in assay will be <3%.
Measurement: IC50 = 3 nM (pChEMBL 8.520000457763672)
Descriptors: MW 413.48, ALogP 4.47, HBA 7, HBD 1, PSA 100.26, QED 0.66
Compound (SMILES): CC(C)C[C@@H](C(=O)N1CCC(Nc2ncc(C(=O)NO)cn2)(c2ccccc2)CC1)c1ccccc1
InChIKey: LBBABBCYTOTUAF-XMMPIXPASA-N
Target: Histone deacetylase 3 (SINGLE PROTEIN), Homo sapiens
UniProt: O15379
Assay: HDAC Enzyme Assay: The compounds were diluted in assay buffer (50 mM HEPES, pH 7.4, 100 mM KCl, 0.001% Tween-20, 0.05% BSA, 20 uM TCEP) to 6 fold their final concentration. The HDAC enzymes (purchased from BPS Biosciences) were diluted to 1.5 fold their final concentration in assay buffer. The tripeptide substrate and trypsin at 0.05 uM final concentration were diluted in assay buffer at 6 fold their final concentration. The final enzyme concentrations used in these assays were 3.3 ng/ml (HDAC1), 0.2 ng/ml (HDAC2), 0.08 ng/ml (HDAC3) and 2 ng/ml (HDAC6). The final substrate concentrations used were 16 uM (HDAC1), 10 uM (HDAC2), 17 uM (HDAC3) and 14 uM (HDAC6). Five ul of compounds and 20 ul of enzyme were added to wells of a black, opaque 384 well plate in duplicate. Enzyme and compound were incubated together at room temperature for 10 minutes. Five u1 of substrate was added to each well, the plate was shaken for 60 seconds and placed into a Victor 2 microtiter plate reader. The development of fluorescence was monitored for 60 min and the linear rate of the reaction was calculated.
Measurement: IC50 = 114 nM (pChEMBL 6.940000057220459)
Descriptors: MW 487.6, ALogP 4.36, HBA 6, HBD 3, PSA 107.45, QED 0.32
Compound (SMILES): N[C@H](CCP(=O)(O)C(O)c1ccc(OCC(=O)O)c(C(F)(F)F)c1)C(=O)O
InChIKey: UBMMJJHZNWYYJF-CGCSKFHYSA-N
Target: Metabotropic glutamate receptor 7 (SINGLE PROTEIN), Rattus norvegicus
UniProt: P35400
Assay: Pharmacological Assay: Metabotropic glutamate receptors were transiently transfected in HEK293 cells by electroporation as described elsewhere (Brabet I. et al., 1998) and plated in 96-well microplates. The high affinity glutamate transporter EAAC1 was co-transfected with the receptor in order to avoid any influence of glutamate released by the cells in the assay medium. In the experiments carried out by the inventors, Group-III mGluRs were co-transfected with a chimeric G-protein which couples the activation of the receptor to the phospholipase-C (PLC) pathway. Thus receptor activation induces production of inositol phosphate (IP) which in turn induces intracellular Ca2+ release. Receptor activity was then determined by measurement of the IP production or Ca release as already described (Goudet C. et al., PNAS 2004). For intracellular calcium measurements, cells expressing mGluRs were loaded with Ca2+-sensitive fluorescent dye Fluo-4 AM (Invitrogen, Cergy-Pontoise, France) dissolved in Hanks' balanced Salt Solution.
Measurement: EC50 = 35500 nM (pChEMBL 4.449999809265137)
Descriptors: MW 415.26, ALogP 1.23, HBA 6, HBD 5, PSA 167.38, QED 0.37
Compound (SMILES): NC1CC(N[C@H]2C[C@@H]2c2ccccc2)c2ccccc21
InChIKey: ZFWZJXPRYBXQIV-ZLZJIPBDSA-N
Target: Lysine-specific histone demethylase 1A (SINGLE PROTEIN), Homo sapiens
UniProt: O60341
Assay: Biological Assay: The compounds of the invention can be tested for their ability to inhibit LSD1. The ability of the compounds of the invention to inhibit LSD1 can be tested as follows. Human recombinant LSD1 protein was purchased from BPS Bioscience Inc (catalog reference number 50100: human recombinant LSD1, GenBank accession no. NM_015013, amino acids 158-end with N-terminal GST tag, MW: 103 kDa). In order to monitor LSD1 enzymatic activity and/or its inhibition rate by our inhibitor(s) of interest, di-methylated H3-K4 peptide (Anaspec) was chosen as a substrate. The demethylase activity was estimated, under aerobic conditions, by measuring the release of H2O2 produced during the catalytic process, using the Amplex Red hydrogen peroxide/peroxidase assay kit (Invitrogen).Briefly, a fixed amount of LSD1 was incubated on ice for 15 minutes, in the absence and/or in the presence of at least eight 3-fold serial dilutions of the respective inhibitor (e.g., from 0 to 75 uM, depending on the inhibitor strength). Tranylcypromine (Biomol International) was used as a control for inhibition. Within the experiment, each concentration of inhibitor was tested in duplicate. After leaving the enzyme interacting with the inhibitor, KM of di-methylated H3-K4 peptide was added to each reaction and the experiment was left for 30 minutes at 37° C. in the dark. The enzymatic reactions were set up in a 50 mM sodium phosphate, pH 7.4 buffer. At the end of the incubation, Amplex Red reagent and horseradish peroxidase (HPR) solution were added to the reaction according to the recommendations provided by the supplier (Invitrogen), and left to incubate for 5 extra minutes at room temperature in the dark. A 1 uM H2O2 solution was used as a control of the kit efficiency. The conversion of the Amplex Red reagent to resorufin due to the presence of H2O2 in the assay, was monitored by fluorescence (excitation at 540 nm, emission at 590 nm) using a microplate reader (Infinite 200, Tecan). Arbitrary units were used to measure level of H2O2 produced in the absence and/or in the presence of inhibitor. The maximum demethylase activity of LSD1 was obtained in the absence of inhibitor and corrected for background fluorescence in the absence of LSD1. The IC50 value of each inhibitor was calculated with GraphPad Prism Software.
Measurement: IC50 = 34 nM (pChEMBL 7.46999979019165)
Descriptors: MW 264.37, ALogP 3.28, HBA 2, HBD 2, PSA 38.05, QED 0.89
Compound (SMILES): COc1ccc2c3c1O[C@H]1[C@@]4(OC)CC[C@@]5(C[C@@H]4COCc4ccccc4)[C@@H](C2)N(Cc2nn[nH]n2)CC[C@]315
InChIKey: LJKWSKUTFPQAJW-LDGIGSNBSA-N
Target: Mu-type opioid receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P35372
Assay: Functional Assay: [35S]GTPgammaS functional assays were conducted using freshly thawed u-receptor membranes (Perkin Elmer, Shelton, Conn.). Assay reactions were prepared by sequentially adding the following reagents to binding buffer (100 mM NaCl, 10 mM MgCl2, 20 mM HEPES, pH 7.4) on ice (final concentrations indicated): membrane protein (0.026 mg/mL), saponin (10 mg/mL), GDP (3 mM) and [35S]GTPgammaS (0.20 nM; Perkin Elmer, Shelton, Conn.). The prepared membrane solution (190 ul/well) was transferred to 96-shallow well polypropylene plates containing 10 ul of 20x concentrated stock solutions of the agonist [D-Ala2, N-methyl-Phe4 Gly-ol5]-enkephalin (DAMGO) prepared in dimethyl sulfoxide (DMSO). Plates were incubated for 30 min at about 25° C. with shaking. Reactions were terminated by rapid filtration onto 96-well Unifilter GF/B filter plates (Perkin Elmer, Shelton, Conn.) using a 96-well tissue harvester (Perkin Elmer, Shelton, Conn.) followed by three filtration washes with 2001 of ice-cold binding buffer.
Measurement: EC50 = 1364.35 nM (pChEMBL 5.869999885559082)
Descriptors: MW 529.64, ALogP 3.44, HBA 8, HBD 1, PSA 94.62, QED 0.47
Compound (SMILES): CC(C)(C)[C@@]1(C)OC(c2ccccc2)O[C@]23CC[C@@]4(C[C@@H]21)[C@H]1Cc2ccc(O)c5c2[C@@]4(CCN1CC1CC1)[C@H]3O5
InChIKey: HGBJMGMRNYTIHF-CODBXGTISA-N
Target: Kappa-type opioid receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P41145
Assay: κ-Opioid Receptor Binding Assay: Radioligand dose displacement assays used 0.4 nM [3H]-U69,593 (GE Healthcare, Piscataway, N.J.; 40 Ci/mmole) with 15 μg membrane protein (recombinant κ opioid receptor expressed in HEK 293 cells; in-house prep) in a final volume of 200 μl binding buffer (5% DMSO, 50 mM Trizma base, pH 7.4). Non-specific binding was determined in the presence of 10 μM unlabeled naloxone or U69,593. All reactions were performed in 96-well polypropylene plates for 1 hour at a temperature of about 25 °C. Binding reactions were terminated by rapid filtration onto 96-well Unifilter GF/C filter plates (Perkin Elmer, Shelton, Conn.) presoaked in 0.5% polyethylenimine (Sigma). Harvesting was performed using a 96-well tissue harvester (Perkin Elmer, Shelton, Conn.) followed by five filtration washes with 200 μl ice-cold binding buffer. Filter plates were subsequently dried at 50 °C. for 1-2 hours. Fifty μl/well scintillation cocktail (Perkin Elmer, Shelton, Conn.) was added and plates were counted in a Packard Top-Count for 1 min/well.
Measurement: Ki = 4.61 nM (pChEMBL 8.34000015258789)
Descriptors: MW 541.73, ALogP 6.52, HBA 5, HBD 1, PSA 51.16, QED 0.48