lovingscience/chembl-v37-raw-activities huggingface dataset:
Compound (SMILES): O=C(NC1CC2(CCC2)C1)c1cc2cc(-c3ccc(F)cc3)cnc2n(CCN2CCOCC2)c1=O
InChIKey: DAAWBTLJVPHAJO-UHFFFAOYSA-N
Target: Cannabinoid receptor 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P21554
Assay: CB1 and CB2 Receptor Binding Assay: CB1R binding protocol involves the use of the same solution buffer used for both incubation and washing reaction (Tris-HCl, 50 mM; EDTA, 2.5 mM; MgCl2, 2.5 mM; BSA, 0.5 mg/mL at pH 7.4), 0.4 nM for [3H]CP-55,940, test compounds (concentrations from 0.001 to 10 μM), and finally 8 μg/sample membrane in a total volume of 200 μL. CB2R binding assays were carried out with two different buffers: incubation buffer (Tris-HCl, 50 mM; MgCl2, 5 mM; CaCl2 1 mM; BSA, 0.2% at pH 7.4) and washing buffer (Tris-HCl, 50 mM; NaCl 500 mM; BSA, 0.1% at pH 7.4). The assay mixture contained incubation buffer, 0.4 nM [3H]CP-55,940, test substances (concentrations from 0.001 to 10 μM), and 4 μg/sample membrane in a total assay volume of 200 μL. Assays were performed in duplicate and incubated for 120 mM at 37° C. After the incubation, the assay mixture is filtered through 96 GF/C filter plates (Perkin Elmer #6005174) using Perkin Elmer Filtermate Harvester, and then washed four times with ice-cold washing buffer.
Measurement: IC50 = 39 nM (pChEMBL 7.409999847412109)
Descriptors: MW 490.58, ALogP 3.6, HBA 6, HBD 1, PSA 76.46, QED 0.57
Compound (SMILES): CC(=O)OC1(C)CCN(C(=O)c2cnc(C3CC3)c(OCC3CC3)n2)C1
InChIKey: URASPVATSWONFK-UHFFFAOYSA-N
Target: Cannabinoid receptor 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P21554
Assay: cAMP Assay: CHO cells expressing human CB1 or CB2 receptors are seeded 17-24 hours prior to the experiment 50.000 cells per well in a black 96 well plate with flat clear bottom (Corning Costar #3904) in DMEM (Invitrogen No. 31331), 1xHT supplement, with 10% fetal calf serum and incubated at 5% CO2 and 37° C. in a humidified incubator. The growth medium was exchanged with Krebs Ringer Bicarbonate buffer with 1 mM IBMX and incubated at 30° C. for 30 min. Compounds were added to a final assay volume of 100 ul and incubated for 30 min at 30° C. Using the cAMP-Nano-TRF detection kit the assay (Roche Diagnostics) was stopped by the addition of 50 ul lysis reagent (Tris, NaCl, 1.5% Triton X100, 2.5% NP40, 10% NaN3) and 50 ul detection solutions (20 uM mAb Alexa700-cAMP 1:1, and 48 uM Ruthenium-2-AHA-cAMP) and shaken for 2 h at room temperature. The time-resolved energy transfer is measured by a TRF reader (Evotec Technologies GmbH), equipped with a ND:YAG laser as excitation source. The plate is measured twice with the excitation at 355 nm and at the emission with a delay of 100 ns and a gate of 100 ns, total exposure time 10 s at 730 (bandwidth 30 nm) or 645 nm (bandwidth 75 nm), respectively. The FRET signal is calculated as follows: FRET=T730-Alexa730-P (T645-B645) with P=Ru730-B730/Ru645-B645, where T730 is the test well measured at 730 nM, T645 is the test well measured at 645 nm, B730 and B645 are the buffer controls at 730 nm and 645 nm, respectively. cAMP content is determined from the function of a standard curve spanning from 10 uM to 0.13 nM cAMP. EC50 values were determined using Activity Base analysis (ID Business Solution, Limited). The EC50 values for a wide range of cannabinoid agonists generated from this assay were in agreement with the values published in the scientific literature.
Measurement: EC50 > 10000 nM (pChEMBL 5)
Descriptors: MW 359.43, ALogP 2.31, HBA 6, HBD 0, PSA 81.62, QED 0.73
Compound (SMILES): Cc1nnc(-n2c(=O)n(CC3CC3)c3ccc(S(=O)(=O)NC4(C)CC4)cc32)s1
InChIKey: ASRAPKGFINDMMP-UHFFFAOYSA-N
Target: ADP-ribosylhydrolase ARH3 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9NX46
Assay: ARH3 Assay: ARH3 In vitro selectivity assays were conducted in a total volume of 15 ul in a standard 384 well format. 5 ul of Human Full Length ARH3 (Enzo Life Sciences: ALX-201-292), used at a final reaction concentration of 17.5 nM, was added to 5 ul of Ribosylated PARP substrate (also produced internally by Astra Zeneca) at final reaction concentration of 4.5 nM in assay buffer (50 mM Tris pH7.4, 0.1 mg/ml BSA, 3 mM EDTA, 0.4 mM EGTA, 1 mM DTT, 0.01% Tween 20, 50 mM KCl). The reaction was incubated at room temperature for 30 minutes and then 5 ul detection reagent was added. Detection Reagent consists of 42 nM MAb Anti-6HIS XL665 (CisBio: 61HISXLB) and 2.25 nM Streptavidin Europium Cryptate (CisBio: 610SAKLB), both at 3× working stock concentrations (final concentrations of 14 nM and 0.75 nM respectively), in a detection buffer of 50 mM Tris pH7.4, BSA at 0.1 mg/ml and KF at 100 mM. Following incubation at room temperature for 60 minutes in the dark, TR-FRET signal was measured at Ex 340 and Em 665 and Em 620. A ratio was calculated as Em665/EM620×104 for each well and used to calculate percent inhibition for test compounds.
Measurement: IC50 > 100000 nM (pChEMBL 4)
Descriptors: MW 419.53, ALogP 2.19, HBA 8, HBD 1, PSA 98.88, QED 0.66
Compound (SMILES): OC(c1ncc[nH]1)c1ccc(F)cc1-c1cscn1
InChIKey: GMGFDPMZWLQZJC-UHFFFAOYSA-N
Target: Proteinase-activated receptor 2 (SINGLE PROTEIN), Homo sapiens
UniProt: P55085
Assay: Trypin Activated FLPR Assay: Calcium mobilization was measured using 1321N1 cells stably expressing human PAR2. Cells were seeded in 384-well plates at 4,000 cells per well in 20 μl DMEM with Glutamax supplemented with 10% FBS and incubated for 18-24 h at 37° C., 5% CO2 and 95% humidity. Cells were loaded with 20 μl Fluo-8 NW calcium dye (Cat: 36316, AAT Bioquest) and kept at 37° C. for 30 min prior to addition of 10 μl compound prepared in 20 mM HEPES pH 7.4, HBSS, 2.5% DMSO, 0.1% BSA and 6 μM Vorapaxar (to block Trypsin induced activity of PAR1). The addition was done using a FLIPRTETRA (384-well head, Molecular Devices) and the response was read simultaneously to detect any agonist activity. The cells were incubated for 30 min at room temperature with the compounds and were then evaluated for antagonist activity by addition of 10 μl 234 nM Trypsin prepared in 20 mM HEPES pH 7.4, HBSS and 0.1% BSA and simultaneous detection of calcium mobilization using the FLIPRTETRA.
Measurement: IC50 = 16800 nM (pChEMBL 4.78000020980835)
Descriptors: MW 275.31, ALogP 2.75, HBA 4, HBD 2, PSA 61.8, QED 0.77
Compound (SMILES): CC(C)(OCc1cc(-c2cccc(OCC3CCC3)c2)n(-c2ccccc2Cl)n1)C(=O)O
InChIKey: VELYGEBUNLBQGU-UHFFFAOYSA-N
Target: Monocarboxylate transporter 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P53985
Assay: MCT1-Mediated Lactate Transport in BT20 Breast Cancer Cells: MCT1 activity may be measured using BT-20 breast cancer cells that express high native levels of MCT1, but do not express MCT4 and are known to those with skill in the art. Preparation of BCECF loaded cells are as described for Assay 1. Lactate transport assay is as described for Assay 1, except 10 mM L-lactate (rather than 50 mM) is added.
Measurement: IC50 = 27000 nM (pChEMBL 4.570000171661377)
Descriptors: MW 454.95, ALogP 5.75, HBA 5, HBD 1, PSA 73.58, QED 0.45
Compound (SMILES): O=C(NC1CCC(F)(F)CC1)c1nc(C2CC2NCC2CC2)cs1
InChIKey: LLGXFJJDHQGQFX-UHFFFAOYSA-N
Target: Amine oxidase [flavin-containing] B (SINGLE PROTEIN), Homo sapiens
UniProt: P27338
Assay: Inhibitory Activity Assay: The MAO-B inhibitory activity evaluation described below followed the protocol of MAO-Glo (registered trademark) Assay of Promega KK. A test compound dissolved in DMSO was added to a reaction solution (100 mM HEPES (pH 7.5), 5% glycerol, 10% DMSO) containing MAO-B enzyme (Sigma-Aldrich Co. LLC.), and the mixture was reacted at room temperature for 15 min. MAO substrate (Promega KK) was added to start the reaction. After reaction at room temperature for 60 min, Luciferine detection reagent (Promega KK) (50 uL) was added to terminate the reaction. After reaction at room temperature for 20 min with stirring, the luminescence was measured by Envision (PerkinElmer). The MAO-B inhibitory rate (%) of the test compound was calculated by the following formula.inhibitory rate (%)=(1-(test compound count-blank)/(control-blank))x100The count of the MAO-B enzyme reaction mixture under compound non-addition conditions is indicated as control, and the count under compound non-addition and MAO-B enzyme non-addition conditions is indicated as blank. A concentration necessary for achieving 50% inhibitory rate was taken as IC50 value.
Measurement: IC50 > 100000 nM (pChEMBL 4)
Descriptors: MW 355.45, ALogP 3.31, HBA 4, HBD 2, PSA 54.02, QED 0.82
Compound (SMILES): CC[C@H](Cn1ccc2cnc(Nc3cnn(C(CCO)C(=O)O)c3)nc21)C(C)C
InChIKey: KFCIUDXZUPTHAL-XPCCGILXSA-N
Target: Tyrosine-protein kinase JAK2 (SINGLE PROTEIN), Homo sapiens
UniProt: O60674
Assay: JAK/TYK2 Assay: 10 mM test compound stock or 1 mM control compound stock (tofocitinib, ruxolitinib or staurosporine) in DMSO was diluted to 0.4 mM in DMSO. A 3-fold series dilution was then performed in DMSO to generate 10 different compound concentrations. The assay was carried out in 384-well white plate. 0.5 uL of 40× compound DMSO solution at different concentrations was mixed with 10 uL 2× enzyme prepared in reaction buffer (20 mM HEPES, 10 mM MgCl2, 0.01% Tween, 1 mM DTT, pH 7.5). 10 uL 2× substrate mixture prepared in reaction buffer was then added to start the reaction. A short spin was done to settle down all solutions to the bottom of the plate. Final concentrations of test compound in the reaction mixture were 10000, 3333, 1111, 370, 123, 41.2, 13.7, 4.57, 1.52and 0.51 nM. Concentrations of control compound were ten times less. Enzymatic reaction was conducted at 25° C. for 1-2 hours. 10 uL of Kinase Glo Reagents was added to stop the reaction and generate the luminescent signal which was measured using Envision. Luminescence signal was inversely related to kinase activity. Reaction mixture which did not contain enzyme served as negative control. The mixture without any compound was the positive control.
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 400.48, ALogP 3.06, HBA 8, HBD 3, PSA 118.09, QED 0.48
Compound (SMILES): COCCn1cc2c(n1)CN(C(=O)Nc1nc(-c3ccc(F)cc3F)ccc1N)C2
InChIKey: LFROWVFEEUGWON-UHFFFAOYSA-N
Target: Histone deacetylase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q13547
Assay: Enzymatic Assay: The following describes an assay protocol for measuring the deacetylation of a peptide substrate by the enzymes HDAC2 or HDAC1. Enzyme, substrate, and cofactors are combined in a well of a microtiter plate and incubated for 3 hours at 25° C. At the end of the incubation, the reaction is quenched by the addition of an SDS-containing buffer. Substrate and product are separated and quantified electrophoretically using the microfluidic-based LabChip 3000 Drug Discovery System from Caliper Life Sciences. The peptide substrate used in this assay is FAM-TSRHK(AC)KL-CONH2 (FAM is carboxyfluorescein). Peptide should be >98% purity by Capillary Electrophoresis.    1. To a well of a 384-well plate, add 5 μL of 2× enzyme buffer. Using Labcyte Echo 550, add 100 nl compound. Enzyme and compound may be pre-incubated at this time if desired.    2. Add 5 μL of 2× substrate buffer.    3. Incubate plate at 25° C. for 17 hours.    4. Terminate reaction by adding 40 μL of 1.55× stop buffer.    5. Create job on a Caliper LabChip® 3000 Drug Discovery System.    6. Load the plate and start electrophoresis using blue laser (480 nm) for excitation and green CCD (520 nm) for detection (CCD2).    Reaction time=17 hours; Reaction temperature=25° C.Final Assay Reaction Mixture100 mM HEPES, pH 7.5 0.1% BSA 0.01% Triton X-100 25 mM KCl1% DMSO (from compound) 1 μM FAM-TSRHK(AC)KL-CONH2 5 nM HDAC Enzyme (specific activity may vary from lot-to-lot, and enzyme concentration may need to be adjusted to yield10-20% conversion of substrate to product).
Measurement: IC50 = 1250 nM (pChEMBL 5.900000095367432)
Descriptors: MW 414.42, ALogP 3, HBA 6, HBD 2, PSA 98.3, QED 0.67
Compound (SMILES): CC(C)NC(=O)C1CCC(CS(=O)(=O)c2ccc(OC/C(=C/F)CN)cc2)CC1
InChIKey: UXXWVMVAMGRIKP-GZTJUZNOSA-N
Target: Amine oxidase [flavin-containing] A (SINGLE PROTEIN), Homo sapiens
UniProt: P21397
Assay: SSAO Activity Assay: Amine oxidase activity of recombinant hSSAO, hMAO-A, and hMAO-B isoforms are measured using the MAO-Glo assay kit (Promega, V1402). Ten nL test compounds (with DMSO as vehicle, 0.1% v/v for hSSAO, hMAO-A and hMAO-B) were added into assay plates by Echo, and pre-incubated with 5 μL of enzyme for 30 mins at room temperature before the addition of 5 μL of luminogenic substrate. The substrate concentration is 40 μM for human recombinant SSAO, 40 μM for human recombinant MAO-A, and 4 μM for human recombinant MAO-B. The hSSAO and hMAO-A assays were conducted in the MAO-A reaction buffer in the kit, and the MAO-B assay was conducted in the MAO-B reaction buffer. Oxidation of the substrate was conducted for 1 hr, before the addition of detecting reagent according the manufacture's protocol. The IC50 value of the tested compounds was calculated by fitting the dose response curve using a 4-parameter non-linear regression routine.
Measurement: IC50 > 30000 nM (pChEMBL 4.5229997634887695)
Descriptors: MW 426.55, ALogP 2.98, HBA 5, HBD 2, PSA 98.49, QED 0.63
Compound (SMILES): Nc1ncc(-c2cccc(C(=O)N3CCN(CCO)CC3)c2)cc1C(=O)NC1CCCCC1
InChIKey: YPNVOEHUQFQAFV-UHFFFAOYSA-N
Target: TGF-beta receptor type-1 (SINGLE PROTEIN), Homo sapiens
UniProt: P36897
Assay: Caliper Assay: A kinase selectivity panel which measures substrate peptide phosphorylation was set-up for wild-type ALK2 (aa172-499), ALK2 FOP mutant (aa172-499 R206H), ALK1 (aa166-493), ALK5 (aa162-503) and ALK6 (aa168-495). The technology used for the described assay is based on the separation and quantification of substrate and product in an electrical field. In the course of the kinase reaction the peptide substrate is phosphorylated by a kinase. The transfer of a phosphate residue also causes the introduction of two additional negative charges and hence to a change in the net charge of the phospho-peptide compared to the unphosphorylated peptide. Due to this difference in charge the phosphorylated und unphosphorylated peptides migrate with different velocities in an electrical field.In the applied method, this separation takes place inside a chip that contains a complex capillary system for simultaneous analysis of 12 samples (12-sipper chip, Caliper Technologies Corp., Mountain View, USA). In order to allow the detection and quantification of the peptides in the capillary system, the peptides carry a fluorescent label (fluorescein). With this label the peptides can be quantified by fluorescence intensity through the instruments laser and detection system (LC3000, Caliper Life Sciences).The assays were performed in 384-well, low volume microtiter assay plates in a final reaction volume of 9 ul. Dose-response curves were generated by incubating 10 nM of each kinase together with 2 μm of the fluorescently labeled substrate peptide 5-Fluo-Ahx-KKYQAEEN-T-YDEYENKK-amid (10 mM stock solution in DMSO) in 50 mM Hepes pH 7.5, 0.02% Tween 20, 0.02% BSA, 1 mM DTT, 10 m Na3VO4, 10 mM If-Glycerolphosphate, 1 mM MgCl2, 12 mM MnCl2 (ALK1 and ALK6 7 mM) and 15 μm ATP for 60 min at 30° C. in the presence or absence of compound diluted in DMSO. Kinase reaction were terminated by adding 15 ul STOP buffer (100 mM HEPES pH 7.5, 5% DMSO, 0.1% Caliper coating reagent, 10 mM EDTA, and 0.015% Brij35.Plates with terminated kinase reactions were transferred to the Caliper LC3000 workstation (Caliper Technologies Corp., Mountain View, USA) for reading. The relative amount of phosphorylated peptide r, was calculated using the heights of the substrate peak, s, and the product peak, p: r=p/(p+s).
Measurement: IC50 = 20200 nM (pChEMBL 4.699999809265137)
Descriptors: MW 451.57, ALogP 2.14, HBA 6, HBD 3, PSA 111.79, QED 0.62
Compound (SMILES): COc1ccc(CN2C[C@]3(CC[C@](c4ccccc4)(N(C)C)CC3)N(CC3(C#N)CCC3)C2=O)cc1
InChIKey: MUCAUEQZECUJHN-RNPORBBMSA-N
Target: Nociceptin receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P41146
Assay: Receptor Binding Assay: The hNOP receptor binding assay was performed as homogeneous SPA-assay (scintillation proximity assay) using the assay buffer 50 mM TRIS-HCl. 10 mM MgCl2. 1 mM EDTA (pH 7.4). The final assay volume (250 ul/well) included 0.5 nM of [leucyl-3H]nociceptin as ligand (PerkinElmer Life Sciences. Inc. Boston. Mass. USA). and either test compound in dilution series or 1 uM unlabelled nociceptin for determination of unspecific binding. The test compound was diluted with 25% DMSO in H2O to yield a final 0.5% DMSO concentration. which also served as a respective vehicle control. The assay was started by adding wheat germ agglutinin coated SPA beads (GE Healthcare UK Ltd. Buckinghamshire. UK) which had been preloaded with hMOP receptor membranes (PerkinElmer Life Sciences. Inc. Boston. Mass. USA). After incubation for 60 minutes at RT and centrifugation for 20 minutes at 500 rpm the signal rate was measured by means of a 1450 Microbeta Trilux beta-counter.
Measurement: Ki = 310 nM (pChEMBL 6.510000228881836)
Descriptors: MW 486.66, ALogP 5.4, HBA 4, HBD 0, PSA 59.81, QED 0.53
Compound (SMILES): COc1ccc(CN2CCN(c3ccc(-c4cc(OC[C@@H](C)OC)cn5ncc(C#N)c45)cn3)CC2)cn1
InChIKey: FOPREYKQAULQJB-HXUWFJFHSA-N
Target: Proto-oncogene tyrosine-protein kinase receptor Ret (SINGLE PROTEIN), Homo sapiens
UniProt: P07949
Assay: RET Enzyme Assay: Compounds of Formula I were screened for their ability to inhibit wildtype and V804M mutant RET kinase using CisBio's HTRF® KinEASE¿-TK assay technology. Briefly, N-terminal GST tagged recombinant human RET cytoplasmic domain (aa 658-end) from Eurofins (0.25 nM RET; Catalog No. 14-570M) or N-terminal GST tagged recombinant human V804M mutant RET cytoplasmic domain (aa 658-end) from Millipore (0.25 nM enzyme; Catalog No. 14-760) was incubated with 250 nM TK-substrate biotin (CisBio, part of Catalog No. 62TK0PEC) and 1 mM ATP along with test compound in a buffer consisting of 25 mM HEPES pH 7.4, 10 mM MgCl2, 0.01% Triton X-100, and 2% DMSO in a volume of 8 μL. Compounds were typically prepared in a threefold serial dilution in DMSO and added to the assay to give the appropriate final concentration. After a 30-minute incubation at 22° C., the reaction was quenched by adding 8 μL of quench solution containing 31.25 nM Sa-XL665 and 1×TK-ab-Cryptate in HTRF detection buffer (all from CisBio, part of Cat. No. 62TK0PEC). After a 1 hour incubation at 22° C., the extent of reaction was determined using a PerkinElmer EnVision multimode plate reader via HTRF dual wavelength detection, and the percent of control (POC) was calculated using a ratiometric emission factor. 100 POC was determined using no test compounds and 0 POC was determined using pre-quenched control reactions. The POC values were fit to a 4 parameter logistic curve, and the IC50 is defined as the concentration of inhibitor at which the POC equals 50 for the fitted curve.
Measurement: IC50 = 348 nM (pChEMBL 6.460000038146973)
Descriptors: MW 513.6, ALogP 3.41, HBA 10, HBD 0, PSA 101.04, QED 0.33
Compound (SMILES): OCCn1cc(-c2cnc(Nc3cc(F)cc(F)c3)nc2Nc2ccc(C3CCNCC3)cc2)cn1
InChIKey: FKZLDSCDUWVAGZ-UHFFFAOYSA-N
Target: Tyrosine-protein kinase receptor UFO (SINGLE PROTEIN), Homo sapiens
UniProt: P30530
Assay: Verification on Inhibitory Activities of TAM Receptor Inhibiting Compounds on Tyro 3, Axl, and Mer: For evaluation of inhibitory activities of the compounds according to the present invention on Tyro 3, Axl, and Mer, the following test was carried out.The specific test method followed the method provided by Cisbio. The compounds of the examples were prepared at various concentrations, followed by addition of Tyro 3, Axl, or Mer and substrate peptides, and then ATP was added to initiate a reaction. After 1 hour, the reaction was stopped by addition of a solution containing EDTA. Thereafter, the amount of phosphorylated peptides was measured. Here, the phosphorylated peptides were treated with an europium (Eu)-labeled antibody recognizing phosphorylated peptides, and then after 1 hour, excited by irradiation of the light of a wavelength of 320 or 340 nm using an Envision Reader. The amount of light emitted at 665 nm was measured to investigate the degrees of inhibition of TAM receptors. The TAM receptor inhibitory activities (IC50 values) of the respective compounds were analyzed using the GraphPad Prism program, and tabulated in Tables 1 to 3 below. LDC1267 (CAS no. 1361030-48-9, Calbiochem), which is a TAM receptor inhibitor compound, was used as a positive control.
Measurement: IC50 = 870 nM (pChEMBL 6.059999942779541)
Descriptors: MW 491.55, ALogP 4.56, HBA 8, HBD 4, PSA 99.92, QED 0.29
Compound (SMILES): Cc1c(Cl)c2c(Cl)c(C)c1-c1c(-c3ccc(F)cc3)sc3ncnc(c13)O[C@@H](C(=O)O)Cc1cc(ccc1OCc1ccnc([C@H]3CC[C@H](OC[C@@H]4COCCO4)CC3)n1)OC[C@@H](CN1CCN(C)CC1)O2
InChIKey: BZMUNKANRLQJPB-STTXDENMSA-N
Target: Induced myeloid leukemia cell differentiation protein Mcl-1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q07820
Assay: Exemplary MCL-1 Inhibitors Bind MCL-1 in the presence of 10% HS: The ability of the exemplary MCL-1 inhibitors of Examples 1 through 151 to bind MCL-1 was demonstrated using the Time Resolved-Fluorescence Resonance Energy Transfer (TR-FRET) Assay. Tb-anti-GST antibody was purchased from Invitrogen (Catalog No. PV4216). The ability of exemplary MCL-1 inhibitors Example 1 to Example 3 to compete with probe F-Bak for binding MCL-1 was demonstrated using a Time Resolved Fluorescence Resonance Energy Transfer (TR-FRET) binding assay.MethodFor the assay, an acoustic dispenser was used to prepare dilution series from 10 mM test compounds in 100% DMSO and directly transfer 160 nL into low volume 384-well assay plates. 8 μL of a protein/probe/antibody mix was then added to each well resulting in final concentrations listed below: Test compound: 11 three-fold dilutions beginning at 25 μMProtein: GST-MCL-1  1 nM Antibody Tb-anti-GST  1 nM Probe: F-Bak 100 nMThe samples were then mixed on a shaker for 1 minute and incubated for an additional 2 hours at room temperature. For each assay plate, a probe/antibody and protein/antibody/probe mixture were included as a negative and a positive control, respectively. Fluorescence was measured on the Envision (Perkin Elmer) using a 340/35 nm excitation filter and 520/525 (F-Bak) and 495/510 nm (Tb-labeled anti-his antibody) emission filters. Dissociation constants (Ki) were determined using Wang's equation (Wang, 1995, FEBS Lett. 360:111-114). The TR-FRET assay can be performed in the presence of varying concentrations of human serum (HS) or fetal bovine serum (FBS). Compounds were tested in the presence of 10% HS.
Measurement: Ki = 0.16 nM (pChEMBL 9.800000190734863)
Descriptors: MW 1044.04
Compound (SMILES): CCC1(C)N=C(c2cc(OCC(F)(F)F)c(C3CC3)cn2)NC1=O
InChIKey: VUIOGDKXTPVFSW-UHFFFAOYSA-N
Target: Cannabinoid receptor 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P21554
Assay: Radioligand Binding Assay: The affinity of the compounds of the invention for cannabinoid CB1 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. The compounds of formula (I) show an excellent affinity for the CB2 receptor with affinities below 10 μM, more particularly of 1 nM to 3 μM and most particularly of 1 nM to 100 nM.
Measurement: EC50 > 10000 nM (pChEMBL 5)
Descriptors: MW 341.33, ALogP 2.95, HBA 4, HBD 1, PSA 63.58, QED 0.9
Compound (SMILES): Cn1cc(-c2ccc(Nc3nn([C@H]4CCCC[C@@H]4C#N)c4cc[nH]c(=O)c34)cc2Cl)cn1
InChIKey: UMDUAFFINPREKZ-KUHUBIRLSA-N
Target: Tyrosine-protein kinase JAK2 (SINGLE PROTEIN), Homo sapiens
UniProt: O60674
Assay: Biochemical HTRF Assay Protocol: The ability of compounds to inhibit the activity of JAK1, JAK2, JAK3, and Tyk2 was measured using a recombinant purified GST-tagged catalytic domain for each enzyme (Invitrogen JAK1 #M4290, JAK2 #M4290, JAK3 #M4290, Tyk2 #M4290) in an HTRF format biochemical assay. The reactions employed a common peptide substrate, LCB-EQEDEPEGDYFEWLW-NH2 (in-house). The basic assay protocol is as follows: First, 250 nL of diluted compounds in DMSO were dispensed into the wells of a dry 384-well Black plate (Greiner #781076) using a Labcyte Echo 555 acoustic dispenser. Subsequent reagent additions employed an Agilent Bravo. Next, 18 μL of 1.11× enzyme and 1.11× substrate in 1× assay buffer (Invitrogen kinase buffer #PV3189, 2 mM DTT, 0.05% BSA) were added to the wells and shaken and then preincubated for 30 minutes at room temperature to allow compound binding to equilibrate. After equilibration, 2 μL of 10×ATP in 1× assay buffer was added to initiate the kinase reaction and the plates were shaken and then incubated at room temperature for 120 minutes. At the end of the incubation, 20 μL of 2× stop buffer (streptavidin-Dylight 650 (Thermo #84547B/100 mL), Eu-tagged pY20 antibody (Perkin Elmer #AD0067), EDTA, HEPES, and Triton) was added to quench the reaction. Plates were shaken and centrifuged and then incubated 60 minutes at room temperature and then read on a Perkin Elmer Envision (λex=337 nm, λem=665 and 615 nm, TRF delay time=20 μs). HTRF signal=10,000*665 nm reading/615 nm reading. After normalization to untreated controls, the percent inhibition of the HTRF signal at each compound concentration was calculated.
Measurement: IC50 = 6400 nM (pChEMBL 5.190000057220459)
Descriptors: MW 447.93, ALogP 4.78, HBA 7, HBD 2, PSA 104.32, QED 0.47
Compound (SMILES): COc1cc2ncnc(N3CCN(c4ncc(O[C@@H](C)c5ccccc5)cn4)CC3)c2cc1OC
InChIKey: UNUFFZFMDJHRCB-SFHVURJKSA-N
Target: Mast/stem cell growth factor receptor Kit (SINGLE PROTEIN), Homo sapiens
UniProt: P10721
Assay: Biochemical Activity Assay: Kit D816V assay: In each well of a 384-well plate, 0.04 ng/ul (0.5 nM) of D816V Kit (Carna Bioscience 08-156) was incubated in a total of 12.5 ul of buffer (100 mM HEPES pH 7.5, 0.015% Brij 35, 10 mM MgCl2, 1 mM DTT) with 1 uM Srctide (5-FAM-GEEPLYWSFPAKKK-NH2) and 15 uM ATP at 25 C for 90 minutes in the presence or absence of a dosed concentration series of compound (1% DMSO final concentration). The reaction was stopped by the addition of 70 ul of Stop buffer (100 mM HEPES pH 7.5, 0.015% Brij 35, 35 mM EDTA and 0.2% of Coating Reagent 3 (Caliper Lifesciences)). The plate was then read on a Caliper EZReader 2 (protocol settings: −1.9 psi, upstream voltage −700, downstream voltage −3000, post sample sip 35 s). Data was normalized to 0% and 100% inhibition controls and the IC50 or EC50 calculated using a 4-parameter fit using GraphPad Prism.
Measurement: IC50 > 100 nM (pChEMBL 7)
Descriptors: MW 472.55, ALogP 3.9, HBA 9, HBD 0, PSA 85.73, QED 0.4
Compound (SMILES): CC1(NC(=O)c2ncccc2Cl)CCN(c2ccc(-c3cc(OCCN4CCCC4)cn4ncc(C#N)c34)cn2)CC1
InChIKey: ATMMVNQFNYZGOF-UHFFFAOYSA-N
Target: Proto-oncogene tyrosine-protein kinase receptor Ret (SINGLE PROTEIN), Homo sapiens
UniProt: P07949
Assay: RET Enzyme Assay: Compounds of Formula I were screened for their ability to inhibit wildtype and V804M mutant RET kinase using CisBio's HTRF KinEASE-TK assay technology. Briefly, N-terminal GST tagged recombinant human RET cytoplasmic domain (aa 658-end) from Eurofins (0.25 nM RET; Catalog No. 14-570M) or N-terminal GST tagged recombinant human V804M mutant RET cytoplasmic domain (aa 658-end) from Millipore (0.25 nM enzyme; Catalog No. 14-760) was incubated with 250 nM TK-substrate biotin (CisBio, part of Catalog No. 62TK0PEC) and 1 mM ATP along with test compound in a buffer consisting of 25 mM HEPES pH 7.4, 10 mM MgCl2, 0.01% Triton X-100, and 2% DMSO in a volume of 8 μL. Compounds were typically prepared in a threefold serial dilution in DMSO and added to the assay to give the appropriate final concentration. After a 30-minute incubation at 22° C., the reaction was quenched by adding 8 μL of quench solution containing 31.25 nM Sa-XL665 and 1×TK-ab-Cryptate in HTRF detection buffer (all from CisBio, part of Cat. No. 62TK0PEC). After a 1 hour incubation at 22° C., the extent of reaction was determined using a PerkinElmer EnVision multimode plate reader via HTRF dual wavelength detection, and the percent of control (POC) was calculated using a ratiometric emission factor. 100 POC was determined using no test compounds and 0 POC was determined using pre-quenched control reactions. The POC values were fit to a 4 parameter logistic curve, and the IC50 is defined as the concentration of inhibitor at which the POC equals 50 for the fitted curve.
Measurement: IC50 = 85.6 nM (pChEMBL 7.070000171661377)
Descriptors: MW 585.11, ALogP 4.58, HBA 9, HBD 1, PSA 111.68, QED 0.32
Compound (SMILES): CC(C)(Nc1ncc(C(=O)c2cccc(C(F)(F)F)c2)cn1)c1ccccc1
InChIKey: VPQSHZNXENRMQC-UHFFFAOYSA-N
Target: Pantetheinase (SINGLE PROTEIN), Homo sapiens
UniProt: O95497
Assay: Human Vanin-1 Enzyme Assay 2: The vanin-1 protein was prepared in-house from a construct expressing the extracellular domain of human vanin-1 (GenBank ID NM_004666) preceded N-terminally by the honey bee melittin signal peptide, a GSG linker sequence, a His6X tag and a FLAG tag. The secreted, soluble enzyme was purified from the conditioned medium from a CHO cell line stably expressing the resulting protein. Enzyme purification was performed through sequential Ni NTA and size-exclusion chromatography steps.On the day of the assay, dose response plates were prepared by diluting the inhibitors in DMSO at compound concentration 100-fold the final in-assay concentration. Concentration series were prepared by serially diluting in DMSO in a half-log series for a total of 11 data points. Intermediate compound plates containing compound in 10% DMSO were then created by diluting the compounds 10-fold in assay buffer consisting of 50 mM Tris-HCl pH=8.0, 50 mM KCl, 0.005% Brij-35 and 1.5 mM cysteamine. To begin the assay 3 μL were transferred from the intermediate compound plate to the assay plate.A working solution of human vanin-1 was prepared by diluting the enzyme stock to 1.25 nM in assay buffer. Next, 24 μL of the vanin-1 working solution were transferred to the assay plate. The enzyme reaction was then initiated by the addition of 3 μL of 100 μM pantetheine 7-amino-4-trifluoromethylcoumarin prepared in assay buffer. The final concentrations in the assay were 1 nM human vanin-1 and 10 uM substrate. The final concentration of DMSO was 1%. The assay plates were incubated for 45 minutes and before they were read on a Spectramax M5 using a 405 nm excitation wavelength and a 505 nm emission wavelength for detection.
Measurement: IC50 = 50.4 nM (pChEMBL 7.300000190734863)
Descriptors: MW 385.39, ALogP 5.07, HBA 4, HBD 1, PSA 54.88, QED 0.62
Compound (SMILES): CCN1CCN(Cc2ccc(NC(=O)c3ccc(C)c(Oc4cc(NC(=O)C5CC5)ncn4)c3)cc2C(F)(F)F)CC1
InChIKey: GJAQIFQPFSPOOM-UHFFFAOYSA-N
Target: Nuclear receptor subfamily 2 group C member 2 (SINGLE PROTEIN), Homo sapiens
UniProt: P49116
Assay: In Vitro Activity Assay: The in vitro activity of the compounds described herein in inhibiting TAK1, HCK and other kinases were obtained using an Invitrogen Select Screening assay as known in the art.
Measurement: IC50 = 92 nM (pChEMBL 7.039999961853027)
Descriptors: MW 582.63, ALogP 5.33, HBA 7, HBD 2, PSA 99.69, QED 0.35
Compound (SMILES): N[C@H]1CC[C@](F)(CNC2CC2c2ccccc2)CC1
InChIKey: YKXHGMBEVCIFNN-XNBUQLGVSA-N
Target: Amine oxidase [flavin-containing] A (SINGLE PROTEIN), Homo sapiens
UniProt: P21397
Assay: MAOA and MAOB In Vitro Activity Experiments: Screening method: Monoamine oxidase MAOA and MAOB activity screeningInstrument: microplate reader Envision (PerkinElmer, USA).MATERIALS: Human recombinant MAOA, purchased from Promega; human recombinant MAOB, purchased from Sigma;The MAOA and MAOB activity assay kit MAO-Glo was purchased from Promega. Principle: This method uses a specific luciferin derivative as a substrate, MAOA or MAOB can catalyze the conversion of substrate to luciferin methyl ester, and the product, luciferin methyl ester can produce fluorescence under the action of luciferase, thus reflecting the activity of MAOA or MAOB by the intensity of the fluorescent signal. Meanwhile, a blank control was set to determine the strength of the enzyme activity. Tranylcypromine (TCP) was employed as a positive inhibitor in the experiment.Sample processing: Samples were dissolved in DMSO, stored at low temperature, and the concentration of DMSO in the final system was controlled within a range that won't affect the activity of the assay.The activity of the sample was tested by primary screening at a single concentration, for example 100 μM. For samples exhibiting activity under certain conditions, for example, the inhibition rate (% Inhibition) being greater than 50, the active dose-dependent relationship, i.e., the IC50 value, was obtained by nonlinearly fitting the sample activity vs the sample concentration, the software used for the calculation was Graphpad Prism 5, the model used for the fit was sigmoidal dose-response (variable slope), and for most inhibitor screening models, the bottom and top of the fitted curve were set to 0 and 100.
Measurement: IC50 > 100000 nM (pChEMBL 4)
Descriptors: MW 262.37, ALogP 2.74, HBA 2, HBD 2, PSA 38.05, QED 0.88
Compound (SMILES): CC1(c2nc(-c3cc(OCC(F)(F)F)c(N4CC(F)(F)C4)cn3)no2)CC1
InChIKey: UCTPRPVWILHNJG-UHFFFAOYSA-N
Target: Cannabinoid receptor 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P21554
Assay: Radioligand Binding Assay: The affinity of the compounds of the invention for cannabinoid CB1 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. The compounds of formula (I) show an excellent affinity for the CB2 receptor with affinities below 10 μM, more particularly of 1 nM to 3 μM and most particularly of 1 nM to 100 nM.
Measurement: EC50 > 10000 nM (pChEMBL 5)
Descriptors: MW 390.31, ALogP 3.58, HBA 6, HBD 0, PSA 64.28, QED 0.73
Compound (SMILES): CCNC(=O)c1cc2c(-c3cc(C(C)(O)c4ccc(F)cc4)cnc3Oc3c(C)cccc3C)cn(C)c(=O)c2[nH]1
InChIKey: XQCKRPBZTFUCBF-UHFFFAOYSA-N
Target: Bromodomain-containing protein 4 (SINGLE PROTEIN), Homo sapiens
UniProt: O60885
Assay: TR-FRET Assay: Compound dilution series were prepared in DMSO via an approximately 3-fold serial dilution. Compound dilutions were added directly into white, low-volume assay plates (Perkin Elmer Proxiplate 384 Plus#6008280) using a Labcyte Echo in conjunction with Labcyte Access and Thermo Multidrop CombinL robotics. Compounds were then suspended in eight microliters (μL) of assay buffer (20 mM Sodium Phosphate, pH 6.0, 50 mM NaCl, 1 mM Ethylenediaminetetraacetic acid disodium salt dihydrate, 0.01% Triton X-100, 1 mM DL-Dithiothreitol) containing His-tagged bromodomain, Europium-conjugated anti-His antibody (Invitrogen PV5596) and Alexa-647-conjugated probe.The final concentration of 1× assay mixture contained 2% DMSO, 12 nM His tagged BRD4 (BDI_K57-E168) and 100 nM probe or 4 nM His tagged BRD4 (BDII_E352-M457) and 30 nM probe, and 1 nM Europium-conjugated anti-His-tag antibody, and compound concentrations in the range of: 49.02 μM-0.61 nM or 0.98 μM-0.15 nM.After a one-hour equilibration at room temperature, TR-FRET ratios were determined using an Envision multilabel plate reader (Ex 340, Em 495/520).
Measurement: Ki = 3 nM (pChEMBL 8.520000457763672)
Descriptors: MW 554.62, ALogP 5.48, HBA 6, HBD 3, PSA 109.24, QED 0.25
Compound (SMILES): CC(C)(C)c1onc(-c2c(Cl)cccc2Cl)c1C(=O)n1ccc2c(/C=C/C(=O)O)cccc21
InChIKey: PTWCGRGZFAKIRK-ZHACJKMWSA-N
Target: Isocitrate dehydrogenase [NADP] cytoplasmic (SINGLE PROTEIN), Homo sapiens
UniProt: O75874
Assay: Inhibitory Activity Against IDH1R132H and IDH1R132C Enzymes: The IDH1R132H protein and the IDH1R132C protein were prepared as follows: the IDH1R132H or IDH1R132C gene was integrated into a pET24b(+) vector (Novagen) to prepare a construct for C-terminal fusion of 6× histidine tag. After transformation of Rosetta 2 (DE3) E. coli, the expression of the protein was induced with IPTG. The E. coli was collected and homogenized, followed by the affinity purification of the 6× histidine fusion protein using HisTrap HP columns (GE Healthcare Japan Corp.) and gel filtration purification using Superdex 200 columns (GE Healthcare Japan Corp.) to obtain the IDH1R132H or IDH1R132C protein of interest.IDH1R132H and IDH1R132C each convert 2-oxoglutarate and NADPH to D-2-hydroxyglutarate (2-HG) and NADP+. Therefore, the activity of the IDH1R132H and IDH1R132C enzymes can be measured by detecting NADPH levels.The enzyme inhibitory activity evaluation was carried out as follows: 40 μL each of reaction solutions containing different concentrations of each compound (100 mM Tris-HCl (pH 7.5), 150 mM NaCl, 20 mM MgCl2, 0.5 mg/mL bovine serum albumin, 1 mM reduced glutathione, 40 μM NADPH, 0.5 mM 2-oxoglutarate, 0.5% dimethyl sulfoxide, 50000 to 0.128 nM compound, and 12 nM IDH1R132H or 10 nM IDH1R132C as the enzyme) was added to each well of 384-well plates (Greiner Bio One International GmbH, #781096) and incubated at room temperature. While NADPH-derived fluorescence was occasionally monitored, the reaction was terminated by the addition of 5 μL of 0.5 M EDTA before consumption of NADPH. 5 μL of WST-8 reagent (Dojindo Laboratories, #CK04) was further added and mixed therewith. 15 minutes later, the absorbance at 450 nm was measured using a plate reader (PerkinElmer, Inc., EnVision). The observed absorbance value reflects the amount of residual NADPH. From the absorbance data, the enzyme inhibitory activity of each compound at each concentration was calculated and analyzed using medical statistical analysis software GraphPad Prism to calculate an IC50 value.
Measurement: IC50 = 182 nM (pChEMBL 6.739999771118164)
Descriptors: MW 483.35, ALogP 6.69, HBA 5, HBD 1, PSA 85.33, QED 0.33
Compound (SMILES): CC(O)([C@H](F)CNC(=O)c1cnc(Nc2ccc3ncsc3c2)cc1N[C@@H]1CCC[C@H]1O)C(F)(F)F
InChIKey: LSIJAJUHLNGISU-WAZFGYGUSA-N
Target: Interleukin-1 receptor-associated kinase 4 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9NWZ3
Assay: Inhibition Assay: The assays were performed in U-bottom 384-well plates. The final assay volume was 30 μL prepared from 15 μL additions of enzyme and substrates (fluoresceinated peptide and ATP) and test compounds in assay buffer (20 mM HEPES pH 7.2, 10 mM MgCl2, 0.015% Brij35 and 4 mM DTT). The reaction was initiated by the combination of IRAK4 with substrates and test compounds. The reaction was incubated at room temperature for 60 min. and terminated by adding 45 μL of 35 mM EDTA to each sample. The reaction mixture was analyzed on the Caliper LABCHIP® 3000 (Caliper, Hopkinton, Mass.) by electrophoretic separation of the fluorescent substrate and phosphorylated product. Inhibition data were calculated by comparison to no enzyme control reactions for 100% inhibition and vehicle-only reactions for 0% inhibition. The final concentrations of reagents in the assays are ATP, 500 μM; FL-IPTSPITTTYFFFKKK peptide 1.5 μM; IRAK4, 0.6 nM; and DMSO, 1.6%.
Measurement: IC50 = 4.5 nM (pChEMBL 8.350000381469727)
Descriptors: MW 527.54, ALogP 4.14, HBA 8, HBD 5, PSA 119.4, QED 0.28
Compound (SMILES): C[C@H](NC(=O)c1nc(N[C@@H](C)c2ccncc2)nc2ccsc12)c1ccncc1
InChIKey: KWYWAWAMLVYZRH-KBPBESRZSA-N
Target: Adenosine receptor A2a (SINGLE PROTEIN), Homo sapiens
UniProt: P29274
Assay: Activity Assay: Compounds (Adenosine Receptor modulators) and activity assay.
Measurement: IC50 > 500 nM (pChEMBL 6.301000118255615)
Descriptors: MW 404.5, ALogP 4.15, HBA 7, HBD 2, PSA 92.69, QED 0.5
Compound (SMILES): Brc1ccc(CSc2nnc(Cc3c[nH]c4ccccc34)n2CCCc2c[nH]cn2)cc1
InChIKey: SRWHXHFNUKAGQG-UHFFFAOYSA-N
Target: Somatostatin receptor type 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P30872
Assay: Radioligand Binding Assay: Competitive radioligand binding experiments were performed using MEMBRANE TARGET Systems (Perkin-Elmer, Boston, Mass.) for human somatostatin receptors: SSTR1 (ES-520-M400UA), SSTR2A (ES-521-M400UA), SSTR3 (ES523-M400UA), SSTR4 (ES-524-M400UA) and SSTR5 (ES-522-M400UA) receptors. (See Table 1). Respective membrane receptor preparations were prepared in assay buffer (25 mM HEPES, 10 mM MgCl2, 1 mM CaCl2, 0.5% BSA, pH=7.4) at a 1:150 dilution. Binding assays were performed using 1251-Tyr-SRIF 14 (Perkin-Elmer, NEX 3890) dissolved in 1 mM HCl. Binding assays were performed in triplicate for each concentration of ligand in a total volume of 200 μL radioligand (25 μL ligand, 25 μL radioligand, 150 μL receptors) and incubated at room temperature for 90 minutes using a shaking table. Binding was terminated by filtration through GF/B glass fiber filters that were presoaked in 0.5% polyethyleneimine for a minimum of 4 hours. Filters were washed 9× with 1000 μL ice cold wash buffer (50 mM Tris-HCl, pH=7.4, 0.2% BSA). Filters were scored, transferred into plastic test tubes and counted in a gamma counter (Wizard2, Perkin-Elmer). Determination of the Ki for each compound was performed using non-linear regression with GraphPad PRISM-5 software (GraphPad Software, Inc., La Jolla, Calif.).
Measurement: Ki = 4645 nM (pChEMBL 5.329999923706055)
Descriptors: MW 507.46, ALogP 5.76, HBA 5, HBD 2, PSA 75.18, QED 0.25
Compound (SMILES): CC(C)c1ccc(N(C)c2ccc3c(c2)CN[C@H]3CNc2cnccc2C(=O)O)cc1
InChIKey: KCAXPJOFGCTLAL-DEOSSOPVSA-N
Target: Lysine-specific demethylase 6B (SINGLE PROTEIN), Homo sapiens
UniProt: O15054
Assay: JMJD3 Assay: The enzymatic assay of JMJD3 activity is based upon Time Resolved-Fluorescence Resonance Energy Transfer (TR-FRET) detection. The ability of test compounds to inhibit the activity of JMJD3 was determined in 384-well plate format under the following reaction conditions: 5 nM JMJD3, 250 nM H3K27me3-biotin labeled peptide (Anaspec cat #64367), 0.4 to 2 μM α-ketoglutaric acid in assay buffer of 50 mM HEPES, pH7.3, 0.005% Brij35, 0.5 mM TCEP, 0.2 mg/ml BSA, 50 μM sodium L-ascorbate, and 5 μM ammonium iron(II) sulfate. Reaction product was determined quantitatively by TR-FRET after the addition of detection reagent Phycolink Streptavidin-allophycocyanin (Prozyme) and Europium-anti-H3K27me2 antibody (PerkinElmer) in the presence of 5 mM EDTA in LANCE detection buffer (PerkinElmer) at a final concentration of 50 nM and 1 nM, respectively.The assay reaction was initiated by the following: 2 μM of the mixture of 750 nM H3K27me3-biotin labeled peptide and 1.2 to 6 μM alpha-ketoglutaric acid with 2 μL of 11-point serial diluted inhibitor in 3% DMSO were added to each well of plate, followed by the addition of 2 μl of 15 nM JMJD3 to initiate the reaction. The reaction mixture was incubated at RT for 30 min, and terminated by the addition of 6 μL of 5 mM EDTA in LANCE detection buffer containing 100 nM Phycolink Streptavidin-allophycocyanin and 2 nM Europium-anti-H3K27me2 antibody. Plates were read by EnVisionMultilabel Reader in TR-FRET mode (excitation at 320 nm, emission at 615 nm and 665 nm) after 1 hr incubation at RT. A ratio from the readout of 665/615 was calculated for each well and fitted to determine inhibition constant (IC50).
Measurement: IC50 = 5500 nM (pChEMBL 5.260000228881836)
Descriptors: MW 416.53, ALogP 4.93, HBA 5, HBD 3, PSA 77.49, QED 0.51
Compound (SMILES): O=C(O)[C@H](CCN(CCCCc1ccc2c(n1)NCCC2)CCOc1ccccn1)Nc1ncnc2c(Cl)cccc12
InChIKey: VRSJTCUXGCVOIQ-SANMLTNESA-N
Target: Integrin alpha-V/beta-6 (PROTEIN COMPLEX), Homo sapiens
UniProt: P06756
Assay: Solid Phase Integrin alphav/beta6 Binding Assay (B-1): Microplates were coated with recombinant human integrin αvβ6 (2 μg/mL) in PBS (100 μL/well 25° C., overnight). The coating solution was removed, washed with wash buffer (0.05% Tween 20; 0.5 mM MnCl2; in 1×TBS). The plate was blocked with 200 μL/well of Block Buffer (1% BSA; 5% sucrose; 0.5 mM MnCl2; in 1×TBS) at 37° C. for 2 h. Dilutions of testing compounds and recombinant TGFβ1 LAP (0.67 μg/mL) in binding buffer (0.05% BSA; 2.5% sucrose; 0.5 mM MnCl2; in 1×TBS) were added. The plate was incubated for 2 hours at 25° C., washed, and incubated for 1 hour with Biotin-Anti-hLAP. Bound antibody was detected by peroxidase-conjugated streptavidin. The IC50 values for testing compounds were calculated by four-parameter logistic regression.
Measurement: IC50 < 50 nM (pChEMBL 7.301000118255615)
Descriptors: MW 590.13, ALogP 5.09, HBA 9, HBD 3, PSA 125.39, QED 0.16
Compound (SMILES): Oc1cc(-c2ncc3c(N4CC5CCC(C4)N5)nc(OCCc4ccccc4)nc3c2F)c2ccccc2c1
InChIKey: YOJMTSIHDGMCSU-UHFFFAOYSA-N
Target: GTPase KRas (SINGLE PROTEIN), Homo sapiens
UniProt: P01116
Assay: KRas G12D Binding Assay: Table 2: The ability of a compound to bind to KRAS G12D was measured using a TR-FRET displacement assay. Biotinylated GDP-loaded recombinant human KRAS G12D (corresponding to amino acids 1:169, produced at Array BioPharma) was incubated with a custom-made Cy5 labelled tracer, europium labelled streptavidin and compound (2% DMSO final) in buffer (50 mM HEPES [pH 7.5], 5mM MgCl2, 0.005% Tween-20 & 1 mM DTT). After a 60 minute incubation at 22° C., the reaction was measured using a PerkinElmer EnVision multimode plate reader via TR-FRET dual wavelength detection, and the percent of control (POC) calculated using a ratiometric emission factor. 100 POC is determined using no test compound and 0 POC is determined using a concentration of control compound that completely inhibits binding of the tracer to KRAS. The POC values were fit to a 4-parameter logistic curve and the IC50 value was determined as the concentration where the curve crosses 50 POC.
Measurement: IC50 = 2221 nM (pChEMBL 5.650000095367432)
Descriptors: MW 521.6, ALogP 5.25, HBA 7, HBD 2, PSA 83.4, QED 0.32
Compound (SMILES): CNS(=O)(=O)c1cc(-c2sc(Nc3cccc(N4CCN(CCO)CC4=O)n3)nc2C)cc2c1C(=O)N([C@@H](C)C1CC1)C2
InChIKey: RNKHOYDAZIEJFF-SFHVURJKSA-N
Target: Phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit alpha isoform (SINGLE PROTEIN), Homo sapiens
UniProt: P42336
Assay: Enzymatic Activity Assay : The activity of recombinant human PI3Kγ ((aa144-1102)-6His) and PI3Kα, β, δ (6-His(p110-p85α)) was determined by measuring the ADP level after phosphorylation of DiC8-PIP2 using a commercially available ADP-Glo kit from Promega. The assay was carried out in white low volume 384 well plates in a final volume of 14 μl at R.T. The assay conditions contained the following: 50 mM Tris buffer pH 7.4, 2.1 mM DTT, 3 mM MgCl2, 0.05% CHAPS, 20 μM ATP, 80 μM DiC8-PIP2 and 1.2 nM PI3Kα, β, γ or 0.6 nM PI3Kδ. Potential inhibitors were made up in DMSO and then diluted in the assay to give a final concentration of not exceeding 1% (v/v) DMSO. A 10-point half-log dilution series of the inhibitors (highest concentration typically 0.1 μM for δ or γ and 33 μM for a or 1) was tested and the pIC50 determined using a 4-parameter logistic equation in a non-linear curve fitting routine. Routinely, inhibitors were pre-incubated with 3 μl of enzyme for 15 min prior to the addition of 2 μl substrate mixture for a further 60 min enzyme reaction. The phosphorylation was stopped with the addition of 3 μl ADP-Glo reagent (stop solution) followed by a 40 min incubation. Prior to detection 6 μl of ADP-Glo Kinase Detection Reagent was added and the plates were read in a micro plate reader using a Luminescence filter. All additions were followed by a short centrifugation step.
Measurement: IC50 = 27 nM (pChEMBL 7.570000171661377)
Descriptors: MW 625.78, ALogP 2.56, HBA 10, HBD 3, PSA 148.07, QED 0.31