lovingscience/chembl-v37-raw-activities huggingface dataset:
Compound (SMILES): CCn1cc(-c2cc(Cn3ccsc3=N)cc(C(=O)N[C@H](c3ccc(F)c(OC)c3)C3CC3)c2)c(C(F)(F)F)n1
InChIKey: ATIXNZXSOBXEAZ-DEOSSOPVSA-N
Target: WD repeat-containing protein 5 (SINGLE PROTEIN), Homo sapiens
UniProt: P61964
Assay: TR-FRET Binding Assay : A Time-Resolved Fluorescence Resonance Energy Transfer (TR-FRET) assay that measures the displacement of the 10mer-Thr-FAM probe in response to compound treatment was performed for compounds wherein the IC50 from FPA assay using 10mer-Thr-FAM was below the lower assay IC50 limit 1 nM. Excess 10mer-Thr-FAM probe was utilized with His-tagged WDR5 in conjunction with a commercial anti-His antibody containing a Terbium label. The LanthaScreen Elite Tb-anti-HIS Antibody from ThermoFisher Scientific was used for this purpose. This Th-anti-HIS has an excitation/emission of 340 nm and 490 nm, respectively. The 10mer-Thr-FAM probe when bound to WDR5 will undergo a FRET interaction with the Tb-anti-HIS and emit at 520 nm. The ratio of the 520 and 495 signals are then utilized to generate a dose-response curve to calculate an IC50 value. By virtue of FRET there is little to no background fluorescence interference from 10mer-Thr-FAM probe allowing an excess of the probe to be used permitting an increase in the lower limit of the calculated Ki when testing against highly potent inhibitors with Ki<<1 nM. WDR5-His Tag (A23, residues 24-334) is expressed and purified in our lab in sufficient quantities for screening. 10mer-Thr-FAM peptide is used anywhere from 15 to 150 nM depending on the window of sensitivity required. WDR5-His tag protein is used at 2 nM. A source plate is prepared using an Echo Liquid Handler, which distributes the compounds to the assay plate (white, flat-bottom; OptiPlate) in a 10-point, 3-fold dilution schemes with a top concentration of either 5 or 20 μM depending on the anticipated potency of the compounds, in a final volume of 20 μL. A final target (WDR5)/Tb-Ab concentration of 2 nM/1 nM is dispensed from appropriate stock solutions, respectively. The final DMSO concentration in each well of the assay plate is 1% or lower. As before the plate is covered, shielded from light, and incubated for 60 minutes at room temperature with rocking. Anisotropy is then measured on a Biotek Cytation 3 at excitation wavelength of 340 nm, and emission wavelengths of 495 nm and 520 nm. Working buffer conditions (pH 7.0) are similar to that in FPA above. TR-FRET signal is plotted and IC50 and Ki values are calculated in the same manner as the fluorescence polarization anisotropy based competition assays.
Measurement: Ki < 0.1 nM (pChEMBL 10)
Descriptors: MW 573.62, ALogP 6.01, HBA 7, HBD 2, PSA 84.93, QED 0.24
Compound (SMILES): O=C(CCSCc1nc2ccccc2c(=O)[nH]1)N[C@H]1CC[C@H](c2nnc(-c3ccccc3)o2)CC1
InChIKey: HXWKRLPCYVNDNE-WGSAOQKQSA-N
Target: Poly [ADP-ribose] polymerase tankyrase-1 (SINGLE PROTEIN), Homo sapiens
UniProt: O95271
Assay: Biochemical Assay: The tankyrase 1 biochemical activity of the compounds was assayed in the following assay buffer (50 mM MOPS pH7.5, 100 mM NaCl, 2.5 mM MgCl2, 0.01% Tween-20, 0.05% BSA, and 1 mM DTT) as follows: 0.25 nM of 6×HIS-tankyrase1 (1091-1325) was incubated in the presence of compound (DMSO 1.85% final) in a Perkin Elmer 384 well Proxiplate Plus (cat.no. 6008289) with 400 nM of NAD for 60 minutes at RT. The assay was then stopped with the above assay buffer containing a 0.6 μM inhibitor and the following detection components: 0.05 μg/mL monoclonal anti-PAR antibody (Trevigen cat.no. 4335-MC-01K-AC) prebound for 60 minutes with 0.63 μg/mL protein G AlphaLisa® acceptor bead (Perkin Elmer cat.no. AL102M) and 5 μg/mL AlphaLisa® nickel chelate donor bead (Perkin Elmer cat.no. AS 101M). The assay was incubated for 16 hours at RT in the dark and read on a Perkin Elmer Envision® multi label reader using the default program set with laser excitation at 680 nM and emission at 615 nM.
Measurement: IC50 = 15.8 nM (pChEMBL 7.800000190734863)
Descriptors: MW 489.6, ALogP 4.44, HBA 7, HBD 2, PSA 113.77, QED 0.35
Compound (SMILES): N#Cc1ccc(NC2CC3CCC2N3C(=O)c2cccc(F)c2-c2ncccn2)nc1
InChIKey: RFROCCVRHRNSNQ-UHFFFAOYSA-N
Target: Orexin/Hypocretin receptor type 1 (SINGLE PROTEIN), Rattus norvegicus
UniProt: P56718
Assay: Rat and Human Orexin 1 Receptor Radioligand Binding Assay: Human Embryonic Kidney 293 cells (HEK293) stably expressing rat orexin 1 receptor (Genebank accession number NM_001525) or Chinese ovary cells (CHO) stably expressing human orexin 1 receptor (Genebank accession number NM_001526) were grown to confluency in DMEM (Hyclone, cat #SH30022), 10% FBS, 1× Pen/Strep, 1× sodium pyruvate, 10 mM HEPES, 600 μg/mL G418 and DMEM/F12 (Gibco, Cat #11039), 10% FBS, 1× Pen/Strep, 600 μg/mL G418 media, respectively on 150 cm2 tissue culture plates, washed with 5 mM EDTA in PBS (HyClone Dulbecco's Phosphate Buffered Saline 1× with Calcium and Magnesium, Cat #SH30264.01, hereafter referred to simply as PBS) and scraped into 50 ml tubes. After centrifugation (2K×G, 5 min at 4° C.), the supernatant was aspirated and the pellets frozen and stored at −800 C. Cells were resuspended in PBS in the presence of 1 tablet of protease inhibitor cocktail (Roche, Cat. #11836145001) per 50 mL. Each cell pellet from a 15 cm plate was resuspended in 10 mL, stored on ice, and homogenized for 45 sec prior to addition to the reactions. Competition binding experiments in 96 well polypropylene plates were performed using [3H]-(1-(5-(2-fluoro-phenyl)-2-methyl-thiazol-4-yl)-1-((S)-2-(5-phenyl-(1,3,4)oxadiazol-2-ylmethyl)-pyrrolidin-1-yl)-methanone) (Moraveck Corporation, specific activity=35.3 Ci/mmol), diluted to a 10 nM concentration in PBS (4 nM final). Compounds were solubilized in 100% DMSO (Acros Organics, Cat. #61042-1000) and tested over a range of 7 concentrations (from 0.1 nM to 10 μM). The final concentration of DMSO in the reactions is equal to or less than 0.1%. Total and nonspecific binding was determined in the absence and presence of 10 μM almorexant. The total volume of each reaction is 200 μL (20 μL of diluted compounds, 80 μL of [3H]-(1-(5-(2-fluoro-phenyl)-2-methyl-thiazol-4-yl)-1-((S)-2-(5-phenyl-(1,3,4)oxadiazol-2-ylmethyl)-pyrrolidin-1-yl)-methanone) diluted in PBS and 100 μL of the cell suspension). Reactions were run for 60 min at room temperature and terminated by filtration through GF/C filter plates (PerkinElmer, Cat. #6005174) presoaked in 0.3% polyethylenimine using the cell harvester (PerkinElmer Filtermate). The plates were washed 3 times by aspirating 30 ml PBS through the plates. Plates were dried in 55° C. oven for 60 min, scintillation fluid was added, and the radioactivity was counted on a Topcount (Packard).
Measurement: Ki = 1000 nM (pChEMBL 6)
Descriptors: MW 414.44, ALogP 3.41, HBA 6, HBD 1, PSA 94.8, QED 0.7
Compound (SMILES): CC(C)n1ccnc1CCOc1nc(N2CC3CCC(C2)N3)c2cnc(-c3cc(O)cc4ccccc34)c(F)c2n1
InChIKey: ASABWBJMDDAQPE-UHFFFAOYSA-N
Target: GTPase KRas (SINGLE PROTEIN), Homo sapiens
UniProt: P01116
Assay: KRas G12D Surface Plasmon Resonance (SPR) Binding Assay: Table 1: Briefly, 1 L of 1.05× HBS-Mg buffer (262.5 mM Bioultra Hepes, pH 7.5, 157.5 mM NaCl, 105 mM MgCl2, 0.525 mM TCEP, 0.0305% Brij-35) was prepared and filter sterilized using a 0.22 μm bottle top filter. Approximately 50 mL of 1.05× HBS-Mg buffer was removed and saved for future dilutions. A 50 mL aliquot of DMSO (Sigma Aldrich DMSO Lot. #SHBK2079) was added and continued to stir for 10 minutes, creating the final 1.0× HBS-Mg buffer (250 mM Bioultra Hepes pH 7.5, 150 mM NaCl, 100 mM MgCl2, 0.5mM TCEP, 0.03% Brij-35).
Measurement: Kd = 25.9 nM (pChEMBL 7.590000152587891)
Descriptors: MW 553.64, ALogP 5.03, HBA 9, HBD 2, PSA 101.22, QED 0.29
Compound (SMILES): C#Cc1cccc(C2(C(F)(F)F)N=N2)c1Cn1nc2nc(C(F)(F)c3ccccc3)nc(N3CCC(F)(F)C3)c2n1
InChIKey: NFXAIKGZPMXHTQ-UHFFFAOYSA-N
Target: Cannabinoid receptor 2 (SINGLE PROTEIN), Homo sapiens
UniProt: P34972
Assay: Radioligand Binding Assay: The affinity of the compounds of the invention for cannabinoid receptors was determined using recommended amounts of membrane preparations (PerkinElmer) of human embryonic kidney (HEK) cells expressing the human CNR1 or CNR2 receptors in conjunction with 1.5 or 2.6 nM [3H]-CP-55,940 (Perkin Elmer) as radioligand, respectively. Binding was performed in binding buffer (50 mM Tris, 5 mM MgCl2, 2.5 mM EDTA, and 0.5% (wt/vol) fatty acid free BSA, pH 7.4 for CB1 receptor and 50 mM Tris, 5 mM MgCl2, 2.5 mM EGTA, and 0.1% (wt/vol) fatty acid free BSA, pH 7.4 for CB2 receptor) in a total volume of 0.2 mL for 1 h at 30° C. shaking. The reaction was terminated by rapid filtration through microfiltration plates coated with 0.5% polyethylenimine (UniFilter GF/B filter plate; Packard). Bound radioactivity was analyzed for Ki using nonlinear regression analysis (Activity Base, ID Business Solution, Limited), with the Kd values for [3H]CP55,940 determined from saturation experiments.
Measurement: Ki = 1074 nM (pChEMBL 5.96999979019165)
Descriptors: MW 574.46, ALogP 5.42, HBA 8, HBD 0, PSA 84.45, QED 0.23
Compound (SMILES): C[C@H](NC(=O)C(C)(F)F)[C@H](Oc1ccc2c(cnn2-c2cccc(C(=O)N[C@H]3CCS(=O)(=O)C3)c2)c1)c1ccc2c(c1)OCOC2
InChIKey: UJHWQXHVXNNPAL-KADOZOMVSA-N
Target: Glucocorticoid receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P04150
Assay: Radioligand Binding Assay: In the GR radioligand binding assay, test compounds were serially diluted in semi-log steps (10 concentrations) with a final concentration of 10 μM. Test compounds (1 μL) and controls (1 μL) in 100% DMSO were added to 96 Greiner V-bottom polypropylene plates. 0% control was 6.7% DMSO (final concentration in assay) and 100% control was 6.7 μM Dexamethasone.The full length GR was diluted to a final concentration of 3.3% (0.495 mg/ml) in assay buffer (20 mM Tris-HCl, 1 mM EDTA, 10% (w/v) Glycerol, 20 mM Sodium molybdate, pH 7.4). 45 μL of GR was added to each well and the plates were incubated for 15 min at room temperature. 3H-dexamethasone solution was diluted to a concentration of 70 nM in assay buffer (7 nM final assay concentration) and 5 μL was added to each well. The samples were mixed for 5 min using a plate shaker at 700 rpm, before incubation for 2 h at room temperature.50 μL ice-cold charcoal solution (pH 7.4: 2% Charcoal, 0.2% Dextran T70 in 20 mM Tris-HCl, 1 mM EDTA and 20 mM Sodium molybdate) was added to each well and the samples were mixed on plate shaker for 5 minutes.
Measurement: IC50 = 1.4 nM (pChEMBL 8.850000381469727)
Descriptors: MW 654.69, ALogP 4.09, HBA 9, HBD 2, PSA 137.85, QED 0.28
Compound (SMILES): CC(=O)N1CCC(C(C)(O)c2cc(F)c3c(c2)C(=O)N(Cc2ccc(Cl)cn2)[C@@]3(OCC2(C(N)=O)CC2)c2ccc(Cl)cc2)CC1
InChIKey: JNWIBTVWXFDIHN-SZFCLFMZSA-N
Target: E3 ubiquitin-protein ligase Mdm2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q00987
Assay: Binding Assay (ELISA): The ELISA assay was performed in streptavidin coated plates which were preincubated with 200 ul per well of 1 μg ml−1 biotinylated IP3 peptide. The plates were ready to use for MDM2 binding after washing the plate with PBS.Compounds and control solutions in DMSO aliquoted in 96-well plates were pre-incubated in a final 2.5-5% (v/v) DMSO concentration at room temperature (for example 20C.) for 20 min with 190 ul aliquots of optimized concentrations of in vitro translated MDM2, before transfer of the MDM2-compound mixture to the b-IP3 streptavidin plates, and incubation at 4C. for 90 min. After washing three times with PBS to remove unbound MDM2, each well was incubated at 20C. for 1 hour with a TBS-Tween (50 mM Tris pH7.5; 150 mM NaCl; 0.05% Tween 20 nonionic detergent) buffered solution of primary mouse monoclonal anti-MDM2 antibody (Ab-5, Calbiochem, used at a 1/10000 or 1/200 dilution depending on the antibody stock solution used), then washed three times with TBS-Tween before incubation for 45 mins at 20C. with a TBS-Tween buffered solution of a goat-anti-mouse horseradish peroxidase (HRP) conjugated secondary antibody (used at 1/20000 or 1/2000 depending on the antibody stock solution). The unbound secondary antibody was removed by washing three times with TBS-Tween. The bound HRP activity was measured by enhanced chemiluminesence (ECL, Amersham Biosciences) using the oxidation of the diacylhydrazide substrate, luminol, to generate a quantifiable light signal. The percentage of MDM2 inhibition at a given concentration is calculated as the [1−(RLU detected in the compound treated sample−RLU negative DMSO control)÷(RLU of DMSO positive and negative controls)]x100 or as the (RLU detected in the compound treated sample ÷ RLU of DMSO controls)x100.
Measurement: IC50 = 1.6 nM (pChEMBL 8.800000190734863)
Descriptors: MW 669.58, ALogP 5.13, HBA 6, HBD 2, PSA 126.06, QED 0.33
Compound (SMILES): CN1C(=O)[C@@H](NC(=O)c2nn(Cc3ccccc3F)cc2F)COc2cccnc21
InChIKey: IQUMAYJHJVUKCF-HNNXBMFYSA-N
Target: Receptor-interacting serine/threonine-protein kinase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q13546
Assay: Fluorescent Polarization Binding Assay: Fluorescent polarization binding assay was performed in polystyrene low volume 384-well black plate, at Room Temperature (RT) in a final volume of 10.1 μl/well using 10 nM of GST-hRIPK1 (8-327) enzyme and 5 nM of fluorescent-labeled ligand (14-(2-{[3-({2-{[4-(cyanomethyl)phenyl]amino}-6-[(5-cyclopropyl-1H-pyrazol-3-yl)amino]-4-pyrimidinyl}amino) propyl]amino}-2-oxoethyl)-16,16,18,18-tetramethyl-6,7,7a,8a,9,10,16,18-octahydrobenzo[2″,3″ ]indolizino[8″,7″: 5′,6′]pyrano[3′,2′:3,4]pyrido[1,2-a]indol-5-ium-2-sulfonate.Test Compounds were serially diluted in DMSO at 100 fold final concentrations in the assay (1% DMSO final). In each well of a 384-well Plate were dispensed 0.1 μL of compound solution (or DMSO for controls) followed by 5 μL of GST-hRIPK1 (8-327) at twice the final concentrations in assay buffer (50 mM HEPES pH 7.5, 10 mM NaCl, 50 mM MgCl2, 0.02% CHAPS, 0.5 mM DTT and 0.01% Pluronic F127). For negative control the enzyme addition was replaced by assay buffer only.After addition of 5 μL of fluorescent-labeled ligand at twice the final concentrations in assay buffer, the plate was incubated at RT for 30 min. At the end, the binding was measured as FP value with the Envision (PerkinElmer) plate reader using filter for an excitation λ=531 nm FP and an emission λ=595 nm FP (S & P-pol).
Measurement: IC50 < 1000 nM (pChEMBL 6)
Descriptors: MW 413.38, ALogP 1.76, HBA 6, HBD 1, PSA 89.35, QED 0.7
Compound (SMILES): COc1ccc(-c2cc(=O)n(C)cc2-c2cnn(-c3ccccc3-c3nnn[nH]3)c2)cc1
InChIKey: KEGQJUMXRVTPLW-UHFFFAOYSA-N
Target: CREB-binding protein (SINGLE PROTEIN), Homo sapiens
UniProt: Q92793
Assay: In Vitro CBP Inhibition: The CBP-inhibitory activity of the compounds described herein was determined by calculating the IC50. More specifically, CBP inhibitor activity was assayed as follows: CBP was cloned and expressed in E. coli as His-tag protein and purified by Nickel affinity and gel-filtration chromatography. The protein was further characterized as a single band with the correct molecular weight by SDS-PAGE. CBP binding and inhibition is assessed by monitoring the interaction of biotinylated H4-tetraacetyl peptide (AnaSpec, H4K5/8/12/16(Ac), biotin-labeled) with the target using the AlphaScreen technology (Perkin Elmer). In a 384-well ProxiPlate CBP (50 nM final) was combined with peptide (20 nM final) in 50 mM HEPES (pH 7.3), 10 mM NaCl, 0.25 mM TCEP, 0.1% (w/v) BSA, and 0.005% (w/v) Brij-35 either in the presence of DMSO (final 0.4% DMSO) or compound dilution series in DMSO. After 20 min incubation at room temp, Alpha-streptavidin donor beads and Nickel Chelate acceptor beads were added to a final concentration of 5 μg/mL. After 2 hr of equilibration, plates were read on an Envision instrument and the IC50 calculated using a four parameter non-linear curve fit.
Measurement: IC50 < 500 nM (pChEMBL 6.301000118255615)
Descriptors: MW 425.45, ALogP 3.09, HBA 8, HBD 1, PSA 103.51, QED 0.46
Compound (SMILES): C=CC(=O)N1CC(N(C)C(=O)Cn2c(C)c(C(=O)N3CCN(c4ccc(C(F)(F)F)cn4)CC3)c3cc(Br)ccc32)C1
InChIKey: WPCMMQRUKKBPNP-UHFFFAOYSA-N
Target: GTPase KRas (SINGLE PROTEIN), Homo sapiens
UniProt: P01116
Assay: Coupled Nucleotide Exchange Assay (2 hours): Purified GDP-bound KRAS protein (aa 1-169), containing both G12C and C118A amino acid substitutions and an N-terminal His-tag, was pre-incubated in assay buffer (25 mM HEPES pH 7.4, 10 mM MgCl2, and 0.01% Triton X-100) with a compound dose-response titration for either 2 hours. Following compound pre-incubation, purified SOS protein (aa 564-1049) and GTP (Roche 10106399001) were added to the assay wells and incubated for an additional 30 min (for 5 min compound pre-incubation) or 1 hour (for 2 hour compound pre-incubation). To determine the extent of inhibition of SOS-mediated nucleotide exchange, purified GST-tagged cRAF (aa 1-149), nickel chelate AlphaLISA acceptor beads (PerkinElmer AL108R), and AlphaScreen glutathione donor beads (PerkinElmer 6765302) were added to the assay wells and incubated for 10 minutes. The assay plates were then read on a PerkinElmer EnVision Multilabel Reader, using AlphaScreen® technology, and data were analyzed using a 4-parameter logistic model to calculate IC50 values.
Measurement: IC50 = 820 nM (pChEMBL 6.090000152587891)
Descriptors: MW 647.5, ALogP 3.94, HBA 6, HBD 0, PSA 81.99, QED 0.38
Compound (SMILES): CN(C)[C@H]1C[C@@H](c2cccc(C(F)(F)F)c2)CC[C@@H]1Nc1ccc(S(=O)(=O)Nc2ccncn2)c(F)c1
InChIKey: OASKTWJFUMWDFE-HSQYWUDLSA-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 = 1.07 nM (pChEMBL 8.970000267028809)
Descriptors: MW 537.58, ALogP 5.11, HBA 6, HBD 2, PSA 87.22, QED 0.41
Compound (SMILES): O=C(c1ccccc1-c1ncccn1)N1CC2CC(Nc3ccc(C(F)(F)F)cn3)C1C2
InChIKey: ZVHPDMQTWUUYIB-UHFFFAOYSA-N
Target: Orexin/Hypocretin receptor type 1 (SINGLE PROTEIN), Homo sapiens
UniProt: O43613
Assay: Human Orexin 1 Receptor Ca2+ Mobilization Assay: CHO cells stably transfected with the human orexin 1 receptor (Genebank accession number NM_001526) were grown to confluency in DMEM/F12, 10% FBS, 1×pen-strep, 400 μg/ml G418. Cells were seeded on to 384-well Packard viewplates at a density of 10,000 cells/well and incubated overnight at 37° C., 5% CO2. The cells were dye-loaded with BD Calcium Assay kit (BD, cat #640178) in HBSS (Gibco, cat #14025-092) with 2.5 mM probenecid and incubated at 37° C., 5% CO2 for 45 min. Cells were pre-incubated with compounds (diluted in DMEM/F-12) for 15-30 minutes before agonist (orexin A, 10 nM) stimulation. Ligand-induced Ca2+ release was measured using a Fluorometric Imaging Plate Reader (FLIPR, Molecular Devices, Sunnyvale, Calif.). Functional responses were measured as peak fluorescence intensity minus basal. The concentration of agonist that produced a half-maximal response is represented by the EC50 value. Antagonistic potency values were converted to apparent pKB values using a modified Cheng-Prusoff correction. Apparent pKB=−log IC50/1+[conc agonist/EC50].
Measurement: Ki = 5 nM (pChEMBL 8.300000190734863)
Descriptors: MW 439.44, ALogP 4.27, HBA 5, HBD 1, PSA 71.01, QED 0.66
Compound (SMILES): COc1cccc2[nH]c(C(=O)N[C@@H](CC(C)C)C(=O)N3C[C@]4(C[C@H]3C=O)C(=O)Nc3ccccc34)cc12
InChIKey: QSKOPRQXDBWMCJ-IYRXFJASSA-N
Target: Replicase polyprotein 1ab (SINGLE PROTEIN), Severe acute respiratory syndrome coronavirus 2
UniProt: P0DTD1
Assay: Biological Activity: SARS-CoV-2 3C-like (3CL) protease fluorescence assay (FRET): Recombinant SARS-CoV-2 3CL-protease was expressed and purified. TAMRA-SITSAVLQSGFRKMK-Dabcyl-OH peptide 3CLpro substrate was synthesized. Black, low volume, round-bottom, 384 well microplates were used. In a typical assay, 0.85 μL of test compound was dissolved in DMSO then incubated with SARS-CoV-2 3CL-protease (10 nM) in 10 μL assay buffer (50 mM HEPES [pH 7.5], 1 mM DTT, 0.01% BSA, 0.01% Triton-X 100) for 30 min at RT. Next, 10 μL of 3CL-protease substrate (40 μM) in assay buffer was added and the assays were monitored continuously for 1 h in an Envision multimode plate reader operating in fluorescence kinetics mode with excitation at 540 nm and emission at 580 nm at RT. No compound (DMSO only) and no enzyme controls were routinely included in each plate. All experiments were run in duplicate. Data Analysis: SARS-CoV-2 3CL-protease enzyme activity was measured as initial velocity of the linear phase (RFU/s) and normalized to controlled samples DMSO (100% activity) and no enzyme (0% activity) to determine percent residual activity at various concentrations of test compounds (0-10 μM). Data were fitted to normalized activity (variable slope) versus concentration fit in GraphPad Prism 7 to determine IC50.
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 502.57, ALogP 3.01, HBA 5, HBD 3, PSA 120.6, QED 0.43
Compound (SMILES): Cc1n[nH]c2ncc(-c3cnn(C)c3)cc12
InChIKey: KRZISEJJNXEPAC-UHFFFAOYSA-N
Target: Leucine-rich repeat serine/threonine-protein kinase 2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q5S007
Assay: LRRK2 Kinase Assay: Procedure: Enzyme was incubated with substrate peptide in reaction buffer in the presence and absence of test compounds or Staurosporine. All additions were done on ice, followed by the addition of ATP mix. Wells were uniformly mixed using an Eppendorff plate shaker and incubated at 30° C. for 20 min, and stopped by the addition of 5 μl of 3% phosphoric acid. Volume was increased to 100 μl by adding 0.8% phosphoric acid which was then transferred to PC filter mats (Millipore), pre-equilibrated with 70% ethanol and water. Plates were washed thrice with 100 μl 0.8% phosphoric acid and dried for an hour at 60° C. 100 μl scintillation fluid was added into each well and reading taken in Perkin Elmer TOPCOUNT beta counter. The data analysis was performed by averaging the duplicate top count readings for each standard, negative, positive control (enzyme control) and samples and subtracting the average negative control from each reading which results in corrected values. A validation EC50 curve was generated by plotting CPM for each Staurosporine concentration on y-axis against the Log concentration of Staurosporine (nM) on the x-axis followed by a best fit curve through the points.
Measurement: IC50 > 500 nM (pChEMBL 6.301000118255615)
Descriptors: MW 213.24, ALogP 1.67, HBA 4, HBD 1, PSA 59.39, QED 0.67
Compound (SMILES): C[C@@H](Cn1c(=O)[nH]/c(=N\c2ccc(Oc3ccc(F)cn3)cc2)n(Cc2ccc(Cl)s2)c1=O)C(=O)O
InChIKey: XAICCSGFWFGSIM-ZDUSSCGKSA-N
Target: P2X purinoceptor 3 (SINGLE PROTEIN), Homo sapiens
UniProt: P56373
Assay: Receptor Inhibition Assay: Stably expressing cell line (C6BU-1 cell transfected with human P2X3 receptor gene (GenBank accession number Y07683) was used. The cells were seeded in a 384-well PDL-coated microtiter plate at a concentration of 3000 cells/well and cultured in the medium (8.3% fetal bovine serum, 8.3% horse serum, and 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, 0.5% BSA, and 0.04% Pluronic P-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 mM KCl, 0.9 mM MgCl2, 5.0 mM CaCl2, 5.6 mM D-glucose, 2.5 mM probenecid, pH 7.5), and each well was added with 20 μL of the washing buffer. The plate was placed in High-Throughput Screening System FLIPR 384 (Molecular Device Co.). Measurement of fluorescence intensity by FLIPR 884 was started, and 20 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, 150 nM ATP solution (25 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 4 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.
Measurement: IC50 = 5 nM (pChEMBL 8.300000190734863)
Descriptors: MW 531.95, ALogP 3.38, HBA 9, HBD 2, PSA 131.57, QED 0.36
Compound (SMILES): CCOC(=O)CCC1CCCN(C2CCN(c3cnc4c(C)nn([C@H](C)c5ccc(Cl)cc5Cl)c4n3)CC2C)C1
InChIKey: XJKVQFDJKPJAPQ-YHCRIBGTSA-N
Target: C-C chemokine receptor type 4 (SINGLE PROTEIN), Homo sapiens
UniProt: P51679
Assay: Ca Flux assay: To measure binding of 125I-TARC or 125I-MDC to cells expressing CCR4 (e.g., CEM cells (e.g., ATCC HB-12624)), the 125I-TARC or 125I-MDC is diluted to a concentration of approximately 200 pM in a buffered saline solution (e.g., RPMI supplemented with 0.5% BSA), and added to an equal volume of a suspension of cells (e.g., CEM cells at 5×106 cells/mL). The resulting mixture is incubated for a period of time (e.g., 2 hrs), and the unbound 125I-TARC or 125I-MDC is separated from the cells by filtration, e.g., by passage through GF/B filter plate (Packard Biosciences) pre-treated with 0.3% polyethyleneimine (Sigma), using a Packard Filtermate 96 (Packard Biosciences). The amount of 125I-TARC or 125I-MDC retained with the cells on the filterplate is measured by adding a small amount of scintillation fluid (e.g., 50 μL of Microscint-20 Packard Biosciences)), and reading scintillation on appropriate detection equipment, e.g., a Packard TopCount 383 (Packard Biosciences).
Measurement: IC50 > 500 nM (pChEMBL 6.301000118255615)
Descriptors: MW 587.6, ALogP 6.32, HBA 8, HBD 0, PSA 76.38, QED 0.29
Compound (SMILES): COc1cc(C(=O)N2CC3CCC2[C@@H]3N)cc2nc(-c3cc4ccc(-c5ccc(OCC(C)(C)O)c(F)c5)nc4n3CC3CC3)n(C)c12
InChIKey: BHAINUQDNIQWLZ-BYICHJTDSA-N
Target: Protein-arginine deiminase type-4 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9UM07
Assay: Rapid-Fire Mass Spectrum (RFMS) Assay: Compounds were solubilized in 100% DMSO to achieve a 10 mM compound concentration. Compound stock solutions were stored at RT. A series of dilutions were prepared in DMSO and mixed 8 times with 20 μL mixing volume. Final top concentration of compound in the assay is 50 μM. Final assay conditions were as follows:Reaction volume: 26 μlAssay buffer: 25 mM hepes, pH 7.5, 5 mM NaCl, 1 mM DTT, 0.2 mg/ml BSA, 0.01% CHAPS, 50 μM Calcium, and 5 μM TPENFinal concentrations: 5 nM hPAD4 enzyme, 250 μM BAEE, and 0.5% DMSOTotal incubation time: 30 mins compound and enzyme preincubation at 37° C., 90 min enzyme/substrate reaction, 30 min reaction with phenyl glyoxal at 37° C.Stop solution: 40 μl 5% TCA in ACN
Measurement: IC50 = 140 nM (pChEMBL 6.849999904632568)
Descriptors: MW 652.77, ALogP 5.53, HBA 9, HBD 2, PSA 120.66, QED 0.22
Compound (SMILES): O=C(Nc1cn2nc(-c3c(-c4ccc(F)cc4)ncn3C3CCN(C(=O)c4ccon4)CC3)ccc2n1)c1cncs1
InChIKey: KDNAEADOMKMVFP-UHFFFAOYSA-N
Target: Casein kinase I isoform epsilon (SINGLE PROTEIN), Homo sapiens
UniProt: P49674
Assay: Kinase Assay: The kinase assay was performed in V-bottom 384-well plates. The final assay volume was 30 μl prepared from 15 μl additions of enzyme, substrates (fluoresceinated peptide FL-AHA-KRRRAL-PSER-VASLPGL-OH and ATP) and test compounds in assay buffer (20 mM HEPES pH 7.4, 30 mM MgCl2, 0.015% Brij35 and 4 mM DTT). The reaction was incubated at room temperature for 22 hours and terminated by adding 45 μl of 35 mM EDTA to each sample. The reaction mixture was analyzed on the Caliper LABCHIP®3000 (Caliper, Hopkinton, Mass.) by electrophoretic separation of the unphosphorylated substrate and phosphorylated product. Inhibition data were calculated by comparison of the no enzyme control reactions for 100% inhibition and vehicle-only reactions for 0% inhibition. The final concentration of reagents in the assay were 200 pM CK1ε or CK1δ, 50 μM ATP, 1.5 μM FL-AHA-KRRRAL-PSER-VASLPGL-OH, and 1.6% DMSO. Dose response curves were generated to determine the concentration required to inhibit 50% of the kinase activity (IC50). Compounds were dissolved at 10 mM in dimethylsulfoxide (DMSO) and evaluated at eleven concentrations.
Measurement: IC50 = 0.372 nM (pChEMBL 9.430000305175781)
Descriptors: MW 583.61, ALogP 4.57, HBA 11, HBD 1, PSA 136.34, QED 0.3
Compound (SMILES): COC(=O)C/C=C/C(=O)N1CCC[C@@H](Nc2ncnc3[nH]ccc23)C1
InChIKey: PQTXNLFPIHTMFG-ROLFPQFPSA-N
Target: Tyrosine-protein kinase JAK3 (SINGLE PROTEIN), Homo sapiens
UniProt: P52333
Assay: JAK Caliper Enzyme Assay: Test article was solubilized in dimethyl sulfoxide (DMSO) to a stock concentration of 30 mM. An 11-point half log dilution series was created in DMSO with a top concentration of 600 μM. The test compound plate also contained positive control wells containing a known inhibitor to define 100% inhibition and negative control wells containing DMSO to define no inhibition. The compound plates were diluted 1 to 60 resulting in a top final assay compound concentration of 10 μM and a 2% DMSO concentration.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, 4 μM ATP and 1 μM peptide substrate. The JAK3 assays contained 1 μM of the JAKtide peptide (FITC-KGGEEEEYFELVKK). The assays were initiated by the addition 1 nM JAK3 enzyme and were incubated at room temperature 75 minutes for JAK3. 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 = 904 nM (pChEMBL 6.039999961853027)
Descriptors: MW 343.39, ALogP 1.48, HBA 6, HBD 2, PSA 100.21, QED 0.63
Compound (SMILES): CC1(c2ccccc2)CC1NC1CCNCC1
InChIKey: ZFEJPJCYTLSKFD-UHFFFAOYSA-N
Target: Amine oxidase [flavin-containing] A (SINGLE PROTEIN), Homo sapiens
UniProt: P21397
Assay: Biological Assay: Human recombinant monoamine oxidase proteins MAO-A and MAO-B were purchased from Sigma Aldrich. MAOs catalyze the oxidative deamination of primary, secondary and tertiary amines. In order to monitor MAO enzymatic activities and/or their inhibition rate by inhibitor(s) of interest, a fluorescence-based (inhibitor)-screening assay was set up. 3-(2-Aminophenyl)-3-oxopropanamine (kynuramine dihydrobromide, Sigma Aldrich), a non fluorescent compound was chosen as a substrate. Kynuramine is a non-specific substrate for both MAO-A and MAO-B activities. While undergoing oxidative deamination by MAO activities, kynuramine is converted into 4-hydroxyquinoline (4-HQ), a resulting fluorescent product.The monoamine oxidase activity was estimated by measuring the conversion of kynuramine into 4-hydroxyquinoline. Assays were conducted in 96-well black plates with clear bottom (Corning) in a final volume of 100 uL. The assay buffer was 100 mM HEPES, pH 7.5. Each experiment was performed in duplicate within the same experiment. Briefly, a fixed amount of MAO (0.25 ug for MAO-A and 0.5 ug for MAO-B) was incubated on ice for 15 minutes in the reaction buffer, in the absence and/or in the presence of at least eight 3-fold serial dilutions each. Clorgyline and Deprenyl (Sigma Aldrich) was used as a control for specific inhibition of MAO-A and MAO-B respectively. After leaving the enzyme(s) interacting with the inhibitor, KM of kynuramine was added to each reaction for MAO-B and MAO-A assay respectively, and the reaction was left for 1 hour at 37° C. In the dark. The oxidative deamination of the substrate was stopped by adding 50 uL of NaOH 2N. The conversion of kynuramine to 4-hydroxyquinoline, was monitored by fluorescence (excitation at 320 nm, emission at 360 nm) using a microplate reader (Infinite 200, Tecan). Arbitrary units were used to measure levels of fluorescence produced in the absence and/or in the presence of inhibitor. The maximum of oxidative deamination activity was obtained by measuring the amount of 4-hydroxyquinoline formed from kynuramine deamination in the absence of inhibitor and corrected for background fluorescence in the absence of MAO enzymes. The IC50 values of each inhibitor were calculated with GraphPad Prism Software.
Measurement: IC50 = 100000 nM (pChEMBL 4)
Descriptors: MW 230.35, ALogP 2.06, HBA 2, HBD 2, PSA 24.06, QED 0.83
Compound (SMILES): COc1ccc(/C(F)=C/c2ccc(F)c([C@@]3(C)N=C(N)S[C@@]4(C(=O)N5CCCC5)C[C@H]43)c2)nc1
InChIKey: YOAUIBLLTVYUGO-BJOHRBRLSA-N
Target: Beta-secretase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P56817
Assay: In Vitro Enzymatic BACE1 and BACE2 FRET (Fluorescence Resonance Energy Transfer) Assays: The cDNAs for both human recombinant BACE1 and 2 with C-terminal 6-His Tags were cloned into transient protein expression vectors, which were subsequently transfected into mammalian cell lines. These recombinant proteins were further purified using Ni-NTA affinity chromatography (Qiagen). The assay buffer used in these screens was 0.05M acetate, pH 4.5, 8% DMSO final, 100 uM genapol (which is a nonionic detergent, below its Critical Micelle Concentration). The Beta Secretase enzyme (0.02 nM for BACE1 and 0.64 nM for BACE2) is pre-incubated for one hour with inhibitors, typically in about 1 uL of DMSO according to a serial dilution, are added thereto. This assay is effectively started by the addition of FRET substrate (50 nM) and the combination is incubated for one hour. The FRET assay is terminated by addition of Tris buffer, which raises the pH to neutrality, and the fluorescence is determined. The FRET substrate is a peptide with commercially available fluorophore and quencher, on opposite sides of the BACE cleavage site. Proteolytic cleavage of the FRET substrate releases quenching of fluorescence (excitation 488 nm and emission 590 nm).
Measurement: IC50 = 22.6 nM (pChEMBL 7.650000095367432)
Descriptors: MW 484.57, ALogP 4.35, HBA 6, HBD 1, PSA 80.81, QED 0.68
Compound (SMILES): CCc1nc(S(=O)(=O)N2C[C@H](CC(C)(C)C(=O)O)Oc3ccc(NC(=O)OC(C)(C)C(F)(F)F)cc32)c(OCC(F)(F)F)s1
InChIKey: SCFYJXNSKSNTNW-AWEZNQCLSA-N
Target: Nuclear receptor ROR-gamma (SINGLE PROTEIN), Homo sapiens
UniProt: P51449
Assay: Biochemical TR-FRET Assay: To identify novel antagonists of RORgammaT, an assay was developed which employs the interaction of RORgammaT with its co-activator peptide SRC1_2. This peptide mimics the recruitment of co-activators to RORgammaT through its interaction with the LXXLL (SEQ ID NO:1) (e.g., NR box) motifs (Xie et al., J. Immunol. 175: 3800-09, 2005; Kurebayashi et al., Biochem. Biophys. Res. Commun. 315: 919-27, 2004; Jin et al., Mol. Endocrinology 24:923-29, 2010). The RORγ-Ligand Binding Domain TR-FRET Assay was run according to the following protocol.HIS-tagged RORγ-LBD protein was recombinantly expressed in Escherichia coli. The RORγ-LBD protein was purified by Ni2+-affinity resin. Purified protein was then diluted in assay buffer (50 mM Tris pH 7.0, 50 mM KCl, 1 mM EDTA, 0.1 mM DTT, 100 μg/mL bovine serum albumin, delipidated) to obtain a RORγ-LBD final concentration of 3 nM. Europium tagged anti-HIS antibody was also added to this solution (1.25 nM). Separately, SF9 cells not expressing any recombinant protein were lysed (32,000 cells per μl in 25 mM Tris, 50 mM NaCl) and the previously frozen lysate was added to the diluted RORγ-LBD solution at a ratio of 0.75 μl SF9 lysate per 15 μl of diluted RORγ-LBD.
Measurement: IC50 = 12.6 nM (pChEMBL 7.900000095367432)
Descriptors: MW 677.64, ALogP 5.99, HBA 9, HBD 2, PSA 144.36, QED 0.29
Compound (SMILES): Cc1ccc(S(=O)(=O)N[C@H]2CC[C@H](O)CC2)cc1-c1cnc2c(N)nc(-c3cccc(F)c3)cn12
InChIKey: YGNYRDHATHZTTO-WGSAOQKQSA-N
Target: Phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit gamma isoform (SINGLE PROTEIN), Homo sapiens
UniProt: P48736
Assay: Scintillation Proximity Assay: PI3K-γ:[γ-33P]ATP (10 mCi/mL) and Wheat Germ Agglutinin (WGA) YSi SPA Scintillation Beads was purchased from Perkin-Elmer (Waltham, Mass.). Lipid kinase substrate, D-myo-Phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2)D (+)-sn-1,2-di-O-octanoylglyceryl, 3-O-phospho linked (PIP2), CAS 204858-53-7, was purchased from Echelon Biosciences (Salt Lake City, Utah). PI3Kγ (p110γ) Recombinant Human Protein was purchased from Life technology (Grand Island, N.Y.). ATP, MgCl2, DTT, EDTA, MOPS and CHAPS were purchased from Sigma-Aldrich (St. Louis, Mo.).The kinase reaction was conducted in polystyrene 384-well Greiner Bio-one white plate from Thermo Fisher Scientific in a final volume of 25 μL. Inhibitors were first diluted serially in DMSO and added to the plate wells before the addition of other reaction components. The final concentration of DMSO in the assay was 2%. The PI3Kγ assay was carried out at room temperature in 20 mM MOPS, pH 6.7, 10 mM MgCl2, 5 mM DTT and CHAPS 0.03%. Reactions were initiated by the addition of ATP, the final reaction mixture consisted of 20 μM PIP2, 2 μM ATP, 0.5 μCi [γ-33P] ATP, 13 nM PI3Kγ. Reactions were incubated for 120 min and terminated by the addition of 40 μL SPA beads suspended in quench buffer: 163 mM potassium phosphate pH 7.8, 20% glycerol, 25 mM EDTA. The final concentration of SPA beads is 1.0 mg/mL. After the plate sealing, plates were shaken overnight at room temperature and centrifuged at 1500 rpm for 10 min, the radioactivity of the product was determined by scintillation counting on Topcount (Perkin-Elmer).
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 495.58, ALogP 3.67, HBA 7, HBD 3, PSA 122.61, QED 0.39
Compound (SMILES): Cc1nc(C(=O)N2CCC[C@@H](C)[C@H]2COc2ccc(C(F)(F)F)cn2)c(-c2ccc(F)cc2)s1
InChIKey: JMPIATXFWNAJIK-AUUYWEPGSA-N
Target: Orexin/Hypocretin receptor type 1 (SINGLE PROTEIN), Homo sapiens
UniProt: O43613
Assay: Cell-based Functional Assay: Measurement of [Ca2+]i using a FLIPR: CHO-OX1 or CHO-OX2 cells are seeded into black-walled clear-base 384-well plates (Costar) at a density of 20,000 cells per well in F12-K medium supplemented with 10% FBS and selection antibiotic (500 ug/ml G418 or 15 ug/ml Blasticidin) and cultured overnight. The cells are incubated with equal volume of calcium4 loading buffer (Molecular Devices, Inc.) containing 2.5 mM probenecid at 37° C. for 30 min, followed by putative OX1 or OX2 receptor antagonists (dose-range 0.1 nM-10 μM) for another 30 min.
Measurement: IC50 = 1 nM (pChEMBL 9)
Descriptors: MW 493.53, ALogP 5.99, HBA 5, HBD 0, PSA 55.32, QED 0.41
Compound (SMILES): Clc1ccc2c(c1)CN(C1CC1)Cc1nnc(C3CC4(C3)CN(c3ccccn3)C4)n1-2
InChIKey: JLVVDOLYKNJJKG-UHFFFAOYSA-N
Target: Vasopressin V1a receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P37288
Assay: Vasopressin V1A Receptor Antagonist Assay: The purpose of this assay was to determine the inhibitory effect of synthesized compounds on the Vasopressin V1a receptor. The assay as performed in Chinese Hamster Ovary (CHO) cells expressing the human Arginine Vasopressin Receptor 1a (AVPR1a). Arginine Vasopressin (AVP) evokes an increase in intracellular calcium in CHO-AVPR1a cells which is measured in a fluorescence assay on the FLIPRTETRA using calcium sensitive dyes. Test compounds were assessed for their ability to affect the magnitude of the response to AVP, with antagonists showing a concentration-dependent reduction in the AVP-mediated fluorescence. Compounds were tested in duplicate in a 10-point, 1:3 dilution series starting at a nominal concentration of 3 μM in the assay.CHO-AVPR1a cells were maintained in routine culture in T175 Flasks at 37° C., 5% CO2. The growth medium consists of Ham's F12 media supplemented with 10% v/v fetal bovine serum, 1× non-essential amino acids, and 0.4 mg/ml Geneticin G418.
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 432.96, ALogP 4.18, HBA 6, HBD 0, PSA 50.08, QED 0.62
Compound (SMILES): CNc1cc(-c2cnc3n(CC(=O)N4CC5(COC5)C4)cccc2-3)nc2c(C(=O)N[C@H]3CC[C@@H]3OC)cnn12
InChIKey: QGDDVCXNDIJVEK-FPOVZHCZSA-N
Target: Non-receptor tyrosine-protein kinase TYK2 (SINGLE PROTEIN), Homo sapiens
UniProt: P29597
Assay: TYK2 JH2 Domain Binding Assay: The binding reaction was assembled by combining 16 μl of DNA-tagged kinase extract, 3.8 μl liganded affinity beads, and 0.18 μl test compound (PBS/0.05% Tween 20/10 mM DTT/0.1% BSA/2 μg/ml sonicated salmon sperm DNA)]. Extracts were used directly in binding assays without any enzyme purification steps at a ≥10,000-fold overall stock dilution (final DNA-tagged enzyme concentration <0.1 nM). Extracts were loaded with DNA-tag and diluted into the binding reaction in a two step process. First extracts were diluted 1:100 in 1× binding buffer (PBS/0.05% Tween 20/10 mM DTT/0.1% BSA/2 μg/ml sonicated salmon sperm DNA) containing 10 nM DNA-tag. This dilution was allowed to equilibrate at room temperature for 15 minutes and then subsequently diluted 1:100 in 1× binding buffer. Test compounds were prepared as 111× stocks in 100% DMSO. Kds were determined using an 11-point 3-fold compound dilution series with three DMSO control points. All compounds for Kd measurements are distributed by acoustic transfer (non-contact dispensing) in 100% DMSO
Measurement: Kd < 0.2 nM (pChEMBL 9.699000358581543)
Descriptors: MW 530.59, ALogP 1.51, HBA 10, HBD 2, PSA 127.91, QED 0.37
Compound (SMILES): CNc1cc(-c2cn(CC(=O)N(C)C)c3ncccc23)nc2c(C(=O)N[C@@H]3CC[C@H]3OC)cnn12
InChIKey: HMCXZXWMBCONAO-IEBWSBKVSA-N
Target: Non-receptor tyrosine-protein kinase TYK2 (SINGLE PROTEIN), Homo sapiens
UniProt: P29597
Assay: TYK2 JH2 Domain Binding Assay: The binding reaction was assembled by combining 16 μl of DNA-tagged kinase extract, 3.8 μl liganded affinity beads, and 0.18 μl test compound (PBS/0.05% Tween 20/10 mM DTT/0.1% BSA/2 μg/ml sonicated salmon sperm DNA)]. Extracts were used directly in binding assays without any enzyme purification steps at a ≥10,000-fold overall stock dilution (final DNA-tagged enzyme concentration <0.1 nM). Extracts were loaded with DNA-tag and diluted into the binding reaction in a two step process. First extracts were diluted 1:100 in 1× binding buffer (PBS/0.05% Tween 20/10 mM DTT/0.1% BSA/2 μg/ml sonicated salmon sperm DNA) containing 10 nM DNA-tag. This dilution was allowed to equilibrate at room temperature for 15 minutes and then subsequently diluted 1:100 in 1× binding buffer. Test compounds were prepared as 111× stocks in 100% DMSO. Kds were determined using an 11-point 3-fold compound dilution series with three DMSO control points. All compounds for Kd measurements are distributed by acoustic transfer (non-contact dispensing) in 100% DMSO
Measurement: Kd = 5.5 nM (pChEMBL 8.260000228881836)
Descriptors: MW 476.54, ALogP 1.78, HBA 9, HBD 2, PSA 118.68, QED 0.42
Compound (SMILES): COc1cccc(OC)c1-n1c(NS(=O)(=O)C[C@H](O)c2ccc(F)cn2)nnc1-c1cncc(C)c1
InChIKey: PXNPYMLOXDTXEO-SFHVURJKSA-N
Target: Apelin receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P35414
Assay: [35S]GTPγS Binding: The human APJ receptor was cloned by polymerase chain reaction and the gene encoding the receptor was subcloned in pFLAG-CMV-3 expression vector (Sigma, Saint Louis, Mo. USA) in-house at Amgen. A GTPγS binding assay was performed on membranes prepared from CHO cells stably expressing human APJ receptor. The optimum experimental conditions for the concentrations of GDP, MgCl2, and NaCl in the assay buffer were initially determined. The assay was performed in assay buffer [20 mM HEPES, pH 7.5, 5 mM MgCl2, and 0.1% (w/v) BSA with 200 mM NaCl, 3 M GDP] and membranes expressing human APJ receptor/well along with WGA PS beads. The reaction was initiated by addition of 0.2 nM [35S]GTPγS (Perkin Elmer Life and Analytical Sciences, Waltham USA) in the absence or presence of various ligands and incubated at RT for 90 min. Nonspecific binding was determined in the presence of 100 M GTPγS and was always less than 0.2% of total binding. All the results presented are means of several independent experiments and analyzed by non-linear regression methods using commercially available program Prism (GraphPad, San Diego, Calif.) to obtain EC50.
Measurement: EC50 = 1 nM (pChEMBL 9)
Descriptors: MW 514.54, ALogP 2.66, HBA 10, HBD 2, PSA 141.35, QED 0.34
Compound (SMILES): C[C@@H]1C[C@@H](n2c(Nc3ccc(NC(=O)C4(O)CC4)cc3)nc3ccccc32)CC(C)(C)C1
InChIKey: LZSDHZZZWGZGPW-YLJYHZDGSA-N
Target: Isocitrate dehydrogenase [NADP] cytoplasmic (SINGLE PROTEIN), Homo sapiens
UniProt: O75874
Assay: Biochemical Assay: mIDH1 catalyzes the NADPH-dependent reduction of alpha-ketoglutarate (α-KG) to (2R)-2-hydroxyglutarate (2-HG). NADPH consumption was measured by luminescent readout.The biochemical reactions were performed at 32° C. in 384-well plates using a reaction volume of 41 μl and the following assay buffer conditions: 50 mM Tris pH 7.5, 100 mM NaCl, 20 mM MgCl2, 0.05% BSA, 0.01% Brij, 1 μM NADPH, and 250 μM α-KG. The IDH1 R132H enzyme was used in a final concentration of 1.5 nM. Test compounds were used in a concentration range between 0.002 and 10 μM. The final DMSO concentration was 2.4%.The reaction was incubated for 30 minutes, then 40 μl of detection mix (0.75 μg/ml Luciferase, 0.02 U/ml Oxidoreductase, 4 μg/ml FMN, 2 μl/ml Decanal/Ethanol, 50 mM Tris pH 7.5, 0.5% Glycerin, 0.01% Tween-20, 0.05% BSA) was added. Luminescence was measured on a luminescent reader (10 seconds measuring time, 1 second integration period, 30% sensitivity). The decrease in luminescence is proportional to mIDH1 activity. IC50 values are determined by interpolation from plots of relative luminescence versus inhibitor concentration.
Measurement: IC50 > 10000 nM (pChEMBL 5)
Descriptors: MW 432.57, ALogP 5.63, HBA 5, HBD 3, PSA 79.18, QED 0.49