lovingscience/chembl-v37-raw-activities huggingface dataset:
Compound (SMILES): Cc1ncsc1-c1ccc(CNC(=O)[C@@H]2C[C@@H](O)CN2C(=O)[C@H](C(C)C)N2Cc3ccccc3C2=O)c(OCCOCCOCCOCCN2CCN(Cc3ccc(-c4cc5c(NCc6ccccc6Cl)ncnc5[nH]4)cc3)CC2)c1
InChIKey: RMVXIQSUEWGQPC-LXIAQAAISA-N
Target: Epidermal growth factor receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P00533
Assay: TR-FRET Assay for ERBB Kinases: All compounds and PROTACs were serially diluted in three-fold increments using 100% DMSO, followed by an intermediate 10-fold dilution using Buffer A (50 mM HEPES, pH 7.5, 50 mM NaCl, 10 mM MgCl2, 1 mM DTT, and 0.1% Pluronic F-68). Two microliters of serially diluted compound or PROTAC were then transferred to black 384-well Proxiplates (PerkinElmer, #6008260) using an Integra Viaflo96. Next, 10 uL of protein kinase in Buffer A was added to each well of the assay plate and pre-incubated with compound for 10 minutes. Kinase reactions were then initiated by addition of 5 uL substrate mix containing 3 mM ATP and 30 uM fluorescein-labeled Poly-GluTyr (Thermo Fisher, #PV3610) in Buffer A and allowed to proceed for 10 minutes at room temperature. Reactions were quenched by addition of a 5 uL mixture containing 5 nM LanthaScreen® Tb-pY20 Antibody (Thermo Fisher, #PV3552) and 40 mM EDTA in Buffer A. Assay plates were then read using a Synergy2 (Biotek Instruments, Winooski, Vt.) via excitation thru a 340/20 nm bandpass filter and emission collected thru 490/10 nm (donor) and 520/25 nm (acceptor) bandpass filters. The final kinase concentrations used for each 15 uL reaction were as follows: 0.2 nM EGFR Exon20NPG (SignalChem, #E10-132GG), 0.1 nM wild type EGFR (BPS Bioscience, #40187), 0.3 nM EGFR L858R/T790M/C797S (BPS Bioscience, #40351), 0.1 nM EGFR L858R (BPS Bioscience, #40189), 0.4 nM L858R/T790M (BPS Bioscience, #40350), InM EGFR Dell9 (SignalChem, #E10-122JG), 10 nM EGFR Dell9 T790M (SignalChem, #E10-122KG), 0.3 nM Her2 (BPS Bioscience, #40230), 15 nM Her2 InsYVMA (SignalChem, #E27-13BG).
Measurement: IC50 = 180 nM (pChEMBL 6.75)
Descriptors: MW 1139.82
Compound (SMILES): CC(C)(C)OC(=O)N1CCC(CC#N)(n2nc(Nc3cc(Cn4cccn4)cc(Cn4cccn4)c3)c3c(=O)[nH]ccc32)CC1
InChIKey: KIQCDFAOGRDXKR-UHFFFAOYSA-N
Target: Tyrosine-protein kinase JAK1 (SINGLE PROTEIN), Homo sapiens
UniProt: P23458
Assay: Biochemical HTRF Assay : 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.
Measurement: IC50 = 1.07 nM (pChEMBL 8.970000267028809)
Descriptors: MW 608.71, ALogP 4.6, HBA 11, HBD 2, PSA 151.68, QED 0.26
Compound (SMILES): COc1cc(F)cc2c1nc(N)n1nc(C3CCC(C)N(c4cn(CC(C)(C)O)nc4C)C3)nc21
InChIKey: NQRCZGYBNMUALG-UHFFFAOYSA-N
Target: Adenosine receptor A2a (SINGLE PROTEIN), Homo sapiens
UniProt: P29274
Assay: Measurement of A2a Binding Affinity Using Radioligand Binding: 148 μL (5 μg/mL) membranes (Perkin Elmer, Cat. No. RBHA2aM400UA) and 2 μL compounds of the invention to be tested (test compound) were transferred to individual wells of a 96-well polypropylene assay plate and incubated for 15 to 30 minutes at room temperature. [3H] SCH58261 ((7-(2-phenylethyl)-5-amino-2-(2-furyl)-pyrazolo-[4,3-e]-1,2,4-triazolo[1,5-c]pyrimidine)) was diluted in assay buffer (50 mM Tris pH 7.4, 10 mM MgCl2, 0.005% Tween20) to a concentration of 4 nM and 50 μL transferred to each well of the assay plate. To define total and non-specific binding, wells containing 1% DMSO and 1 μM ZM241385 (Tocris Bioscience, Cat. No. 1036) respectively, were also included. The assay plate was incubated at room temperature for 60 minutes with agitation. Using a FilterMate Harvester® (Perkin Elmer), the contents of the assay plate were filtered through a UniFilter-96® PEI coated plate (Perkin Elmer Cat. No. 6005274 or 6005277). Filtering was achieved by aspirating the contents of the assay plate for 5 seconds, then washing and aspirating the contents three times with ice-cooled wash buffer (50 mM Tris-HCl pH 7.4, 150 mM NaCl) and allowing the vacuum manifold to dry the plate for 30 seconds. The filter plate was incubated for at least 1 hour at 55° C., and allowed to dry. The bottom of the filter plate was sealed with backing tape. 40 μL Ultima Gold (Perkin Elmer, Cat. No. 6013329) was added to each well of the filter plate and the top of the plate was sealed with TopSeal-A PLUS® clear plate seal (Perkin Elmer, Cat. No. 6050185). The plate was incubated for at least 20 minutes, and then the amount of radioactivity remaining in each well was determined using a TopCount® (Perkin Elmer) scintillation counter. After normalization to total and non-specific binding, the percent effect at each compound concentration was calculated. The plot of percent effect versus the log of compound concentration was analyzed electronically using a 4-parameter logistic fit based on the Levenberg-Marquardt algorithm to generate IC50 values.
Measurement: IC50 = 50.6 nM (pChEMBL 7.300000190734863)
Descriptors: MW 482.56, ALogP 3.06, HBA 10, HBD 2, PSA 119.62, QED 0.45
Compound (SMILES): CC(C)(C)NC(=O)c1ccc(-n2ncc(-c3ccc(C#N)cc3)c2O)nc1
InChIKey: LZLHONJVBPVSSE-UHFFFAOYSA-N
Target: Egl nine homolog 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9GZT9
Assay: Inhibition Assay: The IC50 values for the PHD2 enzyme (residues 181-417) were determined by mixing increasing amounts of inhibitor with a fixed amount of enzyme (5 nM, final concentration) and Biotin labeled peptide (Biotin-Asp-Leu-Glu-Met-Leu-Ala-Pro-Tyr-Ile-Pro-Met-Asp-Asp-Asp-Phe-Gln-Leu, 1 uM final concentration) and 2-Oxyglutarate (2 uM final concentration) in 50 mM HEPES, 50 mM KCl, 0.5 mM TCEP, 2 uM FeCl2, 0.1 mg/ml BSA, at pH 7.3. The reaction was conducted by pre-incubating the enzyme in the presence of inhibitor for 60 min at room temperature. The activity of the free enzyme was measured by adding the peptide, the 2-Oxoglutarate (see above for final concentrations), and Ascorbic Acid (1 mM final concentration). The enzymatic activity was quenched after 60 min by adding an excess of a tight binding inhibitor to the assay mixture. The amount of product released was measured by using a LC/MS system (Agilent HPLC with Applied Biosystems API3000 Mass Spectrometer).
Measurement: IC50 = 58.9 nM (pChEMBL 7.230000019073486)
Descriptors: MW 361.41, ALogP 3.04, HBA 6, HBD 2, PSA 103.83, QED 0.75
Compound (SMILES): COc1ccccc1C(=O)NCc1ccc(-c2nn(C(C)(C)C)c3ncnc(N)c23)cc1
InChIKey: ZTQVREOZSGAIKH-UHFFFAOYSA-N
Target: Tyrosine-protein kinase BTK (SINGLE PROTEIN), Homo sapiens
UniProt: Q06187
Assay: BTKWT Binding Affinity: BTKWT binding affinity of each compound tested was determined using a time-resolved fluorescence resonance energy transfer (TR-FRET) methodology. 2.5 nM Recombinant BTKWT kinase, varying concentrations of inhibitor, 2 nM LanthaScreen Eu anti-His Antibody and 15 nM Kinase Tracer 236 was incubated in 1× LanthaScreen Kinase Buffer A for 5 h. Recombinant BTK kinase and all LanthaScreen components were purchased from Invitrogen. Measurements were performed in a reaction volume of 30 μL using half-area 96-well assay plates. The TR-FRET signal was read on a plate reader with an excitation wavelength of 340 nm and detection wavelengths of 615 and 665 nm. Binding affinity was determined for each compound by measuring TR-FRET signal at various concentrations of compound and plotting the relative fluorescence units against the inhibitor concentration to estimate the IC50 from log [Inhibitor] vs response using the Variable Slope model in Graphpad prism from Graphpad software (SanDiego, Calif.).
Measurement: IC50 < 10 nM (pChEMBL 8)
Descriptors: MW 430.51, ALogP 3.77, HBA 7, HBD 2, PSA 107.95, QED 0.5
Compound (SMILES): COc1ccc(-c2nc(C(=O)Nc3cc4cn(CC(=O)N5CCC(N6CCCC6)CC5)nc4cc3C)cs2)cc1
InChIKey: MBMNYTLZOXSVOC-UHFFFAOYSA-N
Target: Interleukin-1 receptor-associated kinase 4 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9NWZ3
Assay: Kinase Assay: For the assay, 11 different concentrations in the range from 20 μM to 0.073 nM were prepared from a 2 mM solution of the test substance in DMSO. For the assay, 50 nl of the respective solution were pipetted into a black low-volume 384-well microtitre plate (Greiner Bio-One, Frickenhausen, Germany), 2 μl of a solution of Irak4 in assay buffer [50 mM HEPES pH 7.5, 5 mM MgCl2, 1.0 mM dithiothreitol, 30 μM activated sodium orthovanadate, 0.1% (w/v) of bovine gamma-globulin (BGG) 0.04% (v/v) nonidet-P40 (Sigma)] were added and the mixture was incubated for 15 min to allow prebinding of the substances to the enzyme prior to the kinase reaction. The kinase reaction was then started by addition of 3 μl of a solution of adenosine triphosphate (ATP, 1.67 mM=final concentration in 5 μl of assay volume: 1 μM) and peptide substrate (0.83 μM=final concentration in 5 μl assay volume: 0.5 μM) in assay buffer, and the resulting mixture was incubated at 22° C. for the reaction time of 45 min. The concentration of the Irak4 was adjusted to the respective activity of the enzyme and set such that the assay was carried out in the linear range. Typical concentrations were in the order of about 0.2 nM. The reaction was stopped by addition of 5 μl of a solution of TR-FRET detection reagents [0.1 μM streptavidin-XL665 (Cisbio Bioassays; France, catalogue No. 610SAXLG) and 1.5 nM anti-phosphoserin antibody [Merck Millipore, STK Antibody, catalogue No. 35-002] and 0.6 nM LANCE EU-W1024-labelled anti-mouse-IgG antibody (Perkin-Elmer, product No. AD0077, alternatively it is possible to use a terbium cryptate-labelled anti-mouse-IgG antibody from Cisbio Bioassays) in aqueous EDTA solution (100 mM EDTA, 0.4% [w/v] bovine serum albumin [BSA] in 25 mM HEPES pH 7.5).
Measurement: IC50 = 28 nM (pChEMBL 7.550000190734863)
Descriptors: MW 558.71, ALogP 4.82, HBA 8, HBD 1, PSA 92.59, QED 0.35
Compound (SMILES): COc1ccc(S(=O)(=O)NC2CCN(c3ncnc4[nH]c(-c5cnn(C)c5)cc34)CC2)cc1
InChIKey: BKTJFNVDVXGUDQ-UHFFFAOYSA-N
Target: Mast/stem cell growth factor receptor Kit (SINGLE PROTEIN), Homo sapiens
UniProt: P10721
Assay: Cell-Based Assays of c-Kit Mutant Kinase Activity: The c-Kit mutant D816V inhibitors were assessed using an engineered BaF3-FL KIT D816V or BaF3-FL KIT V560G/D816V cell line. The BaF3-FL KIT D816V cell lines were created by introduction of KIT mutant (D816V) full length constructs that render the cells dependent on the introduced kinase for growth. Inhibitors of c-Kit mutant D816V kinase reduce or eliminate the mediated c-kit mutant D816V kinase activation, resulting in reduced cell proliferation of the BaF3-FL Kit mutant D816V cells. This inhibition is measured by the effect of compound concentration on cell growth to assess IC50 values. BaF3-FL KIT D816V cells were seeded at 1×104 cells per well of a 96 well cell culture plate in 50 μl of cell culture medium of RPMI Medium 1× (Invitrogen #11875-093) supplemented with 10% FBS (Invitrogen #10438), 1% Non Essential Amino Acids (Invitrogen #11140), 1% Penicillin Streptomycin (Invitrogen #15140), 1% L-Glutamine (Invitrogen #25030-081). Compounds were dissolved in DMSO at a concentration of 5 mM and were serially diluted 1:3 for a total of eight points and added to the cells to a final maximum concentration of 10 μM in 100 μl cell culture medium (final concentration 0.2% DMSO). Cells were also treated with Dasatinib as a positive control. The cells were incubated at 37° C., 5% CO2 for three days. ATPlite Buffer (Perkin Elmer #6016739) and substrate were equilibrated to room temperature, and enzyme/substrate Recombinant Firefly Luciferase/D-Luciferin was reconstituted. The cell plates were equilibrated to room temperature for 30 minutes, then lysed by addition of 25 uL per well of the ATPlite Reagent. The plate was mixed for 5 minutes on a plate shaker to lyse the cells. The plates were read on a Tecan Safire using Luminescence protocol modified to read 0.1s per well. The luminescence reading assesses the ATP content, which correlates directly with cell number such that the reading as a function of compound concentration is used to determine the IC50 value.
Measurement: Ki = 500 nM (pChEMBL 6.300000190734863)
Descriptors: MW 467.56, ALogP 2.31, HBA 8, HBD 2, PSA 118.03, QED 0.45
Compound (SMILES): CCC(CC)(NC(=O)c1ccc(N2CC(OC)C2)c(OC[C@H]2C[C@@H]2CO)n1)C(=O)OCCCOS(=O)(=O)c1ccc(C)cc1
InChIKey: CXMXMMNCGNLOML-DHIUTWEWSA-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 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 30Â° C. shaking. The reaction was terminated by rapid filtration through microfiltration plates coated with 0.5% polyethylenimine (UniFilter GFB 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.
Measurement: Ki = 21.6 nM (pChEMBL 7.670000076293945)
Descriptors: MW 633.76, ALogP 2.86, HBA 11, HBD 2, PSA 153.59, QED 0.15
Compound (SMILES): COc1ncc(CN2C3CC2CN(c2ccc(-c4cc(OCCN5CCOCC5)cn5ncc(C#N)c45)cn2)C3)cc1Cl
InChIKey: GYYIAZADTYTFGX-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 = 10.4 nM (pChEMBL 7.980000019073486)
Descriptors: MW 601.11, ALogP 3.5, HBA 11, HBD 0, PSA 104.28, QED 0.28
Compound (SMILES): CC(C)c1c(-c2ccc(N3CCCC3)cc2)[nH]c2ncnn2c1=O
InChIKey: RRBFMQYIXKWRPH-UHFFFAOYSA-N
Target: Lysine-specific demethylase 5B (SINGLE PROTEIN), Homo sapiens
UniProt: Q9UGL1
Assay: TR-FRET (Time-Resolved Fluorescence Resonance Energy Transfer) Assay: In this assay, the potency (IC50) of each compound was determined from a ten point (1:3 serial dilution; final compound concentration range in assay from 100000 nM to 5.08 nM) titration curve using the following outlined procedure. To each well of a white Greiner 1536 Lumitrac 1536 well-plate, 50 nL of compound (100 fold dilution in final assay volume of 5 μL) was dispensed, followed by the addition of 4 μL of 1× assay buffer (50 mM Hepes 7.3, 0.5 mM TCEP, 0.005% Brij-35, 0.02% BSA, 50 μM Na-L-Ascorbate and 2 μM AmFe(II)Sulfate) containing 5 nM of Full-length KDM5B enzyme (recombinant protein from baculovirus-transfected Sf21 cells: full-length KDM5B; MW=176.825 kDa). Following a 30 minutes compound and enzyme incubation in a humidified chamber, each reaction was initiated by the addition of 1 μL 1× assay buffer containing 50 nM biotinylated H3K4Me3 peptide, and 500 nM α-ketoglutarate. The final reaction in each well of 5 μL consists of 5 nM KDM5B, 500 nM biotinylated-peptide, and 500 nM α-ketoglutarate. De-methylation reactions were allowed to proceed for 60 minutes. Reactions were immediately quenched by the addition of 5 uL of 2× Lance Detection Buffer (PerkinElmer) with 0.5 mM EDTA and 2 nM of Eu-anti-H3K4Me1-2 antibody, and 30 nM of Streptavidin-conjugated Dylight 650 detection reagents. After 60 minutes incubation with detection reagents, reaction plates were read on a PerkinElmer EnVision plate reader using standard TR-FRET protocol. Briefly, excitation of donor molecules (Eu-chelate-anti-H3K4Me1-2-antibody) with a laser light source at 337 nm produces energy that can be transferred to Dylight-650 acceptor molecules if this donor:acceptor pair is within close proximity. Fluorescence intensity at both 665 nm (acceptor) and 615 nm (donor) are measured and a TR-FRET ratio calculated for each well (acceptor intensity/donor intensity).
Measurement: IC50 = 20.1 nM (pChEMBL 7.699999809265137)
Descriptors: MW 323.4, ALogP 2.81, HBA 5, HBD 1, PSA 66.29, QED 0.8
Compound (SMILES): Cn1c(=O)[nH]c2ncn(Cc3nc([C@@H]4CO[C@@H](c5ccc(Cl)cc5)C4)no3)c(=O)c21
InChIKey: WXUGHBCPMLOGEV-WCQYABFASA-N
Target: Transient receptor potential cation channel subfamily A member 1 (SINGLE PROTEIN), Homo sapiens
UniProt: O75762
Assay: FLIPR Assay (15 minutes): The IC50 (effective concentration) of compounds on the human TRPA1 channel was determined using a FLIPR Tetra instrument. CHO cells expressing TRPA1 were plated into 384-well plates, incubated overnight at 37° C., and loaded with BD calcium indicator dye for 1 hr at 37° C. followed by 15 minutes at room temperature. The assay buffer was Hank's Balanced Salt Solution (HBSS) containing 20 mM HEPES (pH readjusted to 7.4) along with 0.02% BSA.Following dye load and plate cool down, compounds were added to the cells using FLIPR Tetra. Plates were then incubated with compounds for 10 minutes or 90 minutes at room temperature prior to adding agonist. Following this incubation, about an EC80 concentration of cinnamaldehyde (75) was added to active the channels and block of cinnamaldehyde induced calcium influx was measured.The IC50 results were fit with a standard Hill function, keeping the Hill coefficient (n) fixed to 1.5. Fixing the Hill coefficient will generally reduce variability of the IC50 determination. The IC50 results were individually examined to make sure the MIN and MAX points were set correctly prior to validation of the results.
Measurement: IC50 = 40.4 nM (pChEMBL 7.389999866485596)
Descriptors: MW 428.84, ALogP 1.75, HBA 9, HBD 1, PSA 120.83, QED 0.53
Compound (SMILES): CC(C)(C)n1nc(-c2[nH]c3cccc(Cl)c3c2Cl)c2c(N)ncnc21
InChIKey: MHNZWTUHRIFXQQ-UHFFFAOYSA-N
Target: Proto-oncogene tyrosine-protein kinase receptor Ret (SINGLE PROTEIN), Homo sapiens
UniProt: P07949
Assay: Time-Resolved Fluorescence Transfer (FRET) Assay: RET and KDR: Kinase activity was detected using CisBio HTRF kinEASE kit based on time-resolved fluorescence transfer (FRET). The assay was performed in 384-well white plates (Corning #3574) in a reaction volume of 10 μL containing 1× CisBio enzymatic buffer supplemented with a final concentration of 5 mM MgCl2, 1 mM DTT, 10 nM SEB and 0.01% Triton X100 for RET. The same buffer was used for the KDR biochemical assay with the addition of 2 mM MnCl2.Inhibitors were pre-incubated in the plate for 15 mins with 5 μL kinase and assay buffer at the following concentrations; 13 pM RET (Carna Biosciences; 08-159) and 150 pM KDR (Millipore; 14-630). The reaction was initiated by the addition of 5 μL ATP and substrate at 2× final reaction concentrations. For RET, this was 18 pM and 2 μM; for KDR, this was 16 μM and 1 μM, respectively. Reactions were performed at ATP Km for each target. The assay was allowed to proceed at room temperature for 20 mins before terminating with the addition of 10 μL HTRF detection buffer containing EDTA supplemented with TK-antibody labelled with Eu3+-Cryptate (1:100 dilution) and streptavidin-XL665 (128 nM). Following incubation at room temperature for 1 hour, FRET signal was measured using the Pherastar FS Microplate Reader.
Measurement: IC50 = 424 nM (pChEMBL 6.369999885559082)
Descriptors: MW 375.26, ALogP 4.62, HBA 5, HBD 2, PSA 85.41, QED 0.51
Compound (SMILES): Cc1noc(CN2CCN(c3cc4c(cc3NC(=O)c3cnn5cccnc35)CC(C)(C)O4)CC2)n1
InChIKey: AYVJLWZYKNBTLV-UHFFFAOYSA-N
Target: Interleukin-1 receptor-associated kinase 4 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9NWZ3
Assay: Biological Assay: Kinase activities were assayed using the Transcreener-Fluorecescence polarization platform (BelBrook Labs, Madison, Wis., USA) that measures amounts of the reaction product, ADP. The IRAK4 reaction conditions were optimized using an IRAK1-derived peptide (sequence H-KKARFSRFAGSSPSQSSMVAR) to provide a linear reaction rate over the course of a 90 min incubation, which resulted in 10-12% conversion of the starting ATP to ADP. Final IRAK4 assay conditions were 1.25 nM IRAK4; 125 uM ATP; 10 uM MgCl2; 125 uM peptide in reaction buffer (25 mM HEPES (pH7.4); 2 mM Dithiothreitol; 0.015% Brij-35; and 0.5% dimethyl sulfoxide. The IRAK1 activity was optimized similarly, yielding final assay conditions of 3 mM IRAK1; 62.5 uM ATP; 5 uM MgCl2, and 62.5 uM IRAK1 peptide in reaction buffer for 60 min.Assays of compounds for kinase inhibition were performed using inhibitors serially-diluted in dimethyl sulfoxide, which was accomplished with a LabCyte Echo 555 liquid dispenser. 384 well assay plates spotted with compound received 4 ul of a 2× substrate (ATP+peptide) mix in reaction buffer, followed by 4 ul of 2× enzyme diluted in reaction buffer. Reactions were halted at 60 (IRAK1) or 90 (IRAK4) min by addition of 6 ul of detection buffer, containing EDTA (40 nM final concentration), 0.95 ug of the ADP-binding antibody ADP2, ADP tracer (3 nM final concentration), and 25 uM HEPES. Following a 1 hr incubation, fluorescence polarization of the ADP2-antibody TRACER complex was read on a Tecan M1000 plate reader using a 635/20 excitation filter in combination with a 670/20 emission filter.
Measurement: Ki = 16 nM (pChEMBL 7.800000190734863)
Descriptors: MW 488.55, ALogP 2.71, HBA 10, HBD 1, PSA 113.92, QED 0.45
Compound (SMILES): CCNC(=O)c1cc2c(-c3ccc(C(=O)N(CCN(CC)CC)c4ccccc4OC)cc3Oc3c(C)cccc3C)cn(C)c(=O)c2[nH]1
InChIKey: YXYZVRSYCZFMJV-UHFFFAOYSA-N
Target: Bromodomain-containing protein 4 (SINGLE PROTEIN), Homo sapiens
UniProt: O60885
Assay: TR-FRET Assay (BD1: amino acids K57-E168): Compound dilution series were prepared in DMSO via a 3-fold serial dilution from 2.5 mM to 42 nM. Compounds were then diluted 6:100 in 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) to yield 3× working solutions. Six microliters (μL) of the working solution was then transferred to white, low-volume assay plates (Costar #3673). A 1.5× assay mixture containing His-tagged bromodomain, Europium-conjugated anti-His antibody (Invitrogen PV5596) and the Alexa-647-conjugated probe molecule was also prepared. Twelve μL of this solution were added to the assay plate to reach a final volume of 18 μL. The final concentration of 1× assay buffer contains 2% DMSO, 50 μM-0.85 nM compound, 8 nM His-tagged bromodomain, 1 nM Europium-conjugated anti-His-tag antibody and 100 nM or 30 nM probe (for BDI or BDII, respectively). After a one-hour incubation at room temperature, TR-FRET ratios were determined using an Envision multilabel plate reader (Ex 340, Em 495/520).
Measurement: Ki = 592 nM (pChEMBL 6.230000019073486)
Descriptors: MW 663.82, ALogP 6.69, HBA 7, HBD 2, PSA 108.9, QED 0.15
Compound (SMILES): Cc1ccc(-c2cc3nn(C)cc3c(O[C@H](C)[C@H]3CNC(=O)C3)n2)s1
InChIKey: QBFSYMFIIHWPEL-VXGBXAGGSA-N
Target: Tyrosine-protein kinase SYK (SINGLE PROTEIN), Homo sapiens
UniProt: P43405
Assay: Syk Kinase Assay: Recombinant human Syk (amino acids 342-635) was expressed as a fusion protein with an N-terminal GST tag, affinity-purified and deep-frozen at a concentration of approx. 50-100 μM in storage buffer (25 mM HEPES pH7.5; 25 mM MgCl2; 5 mM MnCl2; 50 mM KCl; 0.2% BSA; 0.01% CHAPS; 100 μM Na3VO4; 0.5 mM DTT, 10% glycerol) at −80° C. until use.The catalytic activity of the GST-Syk kinase fusion protein was determined using the Kinase Glo® Luminescence Kinase test (Promega; V6712). In this homogeneous test the amount of ATP remaining after the kinase reaction is quantified by a luciferin-luciferase reaction using luminescence. The luminescence signal obtained correlates with the amount of ATP still present and thus correlates inversely with the activity of the kinase.
Measurement: IC50 = 89.2 nM (pChEMBL 7.050000190734863)
Descriptors: MW 356.45, ALogP 2.91, HBA 6, HBD 1, PSA 69.04, QED 0.78
Compound (SMILES): COc1ncc(-c2nc(N3CCOCC3)nc3c(CN4CCN(C(=O)C(F)(F)F)CC4)c(C)sc23)cc1NS(=O)(=O)C1CC1
InChIKey: CTZWNFBBCSXMAV-UHFFFAOYSA-N
Target: Phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit delta isoform (SINGLE PROTEIN), Homo sapiens
UniProt: O00329
Assay: Enzyme Activity Inhibition Assay: 1. Buffer preparation: 50 mM HEPES, pH 7.5, 3 mM MgCl2, 1 mM EGTA, 100 mM NaCl, 0.03% CHAPS.2. Compound was formulated in 100% DMSO in a concentration gradient, and added to a 384-well plate to a final DMSO concentration of 1%.3. PI3Kα, PI3Kδ, PI3Kβ and PI3Kγ enzymes were diluted to an optimum concentration with the following buffer: 50 mM HEPES, pH 7.5, 3 mM MgCl2, 1 mM EGTA, 100 mM NaCl, 0.03% CHAPS, 2 mM DTT, and transferred to a 384-well plate and incubated with the compound for a certain time.4. Substrate was diluted to an optimum concentration with the following buffer: 50 mM HEPES, pH 7.5, 3 mM MgCl2, 1 mM EGTA, 100 mM NaCl, 0.03% CHAPS, 2 mM DTT, 50 μM PIP2, 25 μM ATP; then added to a 384-well plate to initiate the reaction, and for PI3Kα, PI3Kβ and PI3Kγ at room temperature reacted for 1 hr, for PI3Kδ at room temperature reacted for 2 hrs. For PI3Kβ and PI3Kγ, it was still necessary to further add 10 μL ADP-Glo Detection Reagent, and then equilibrated at room temperature for 30 minutes.
Measurement: IC50 = 15 nM (pChEMBL 7.820000171661377)
Descriptors: MW 655.73, ALogP 3.02, HBA 11, HBD 1, PSA 130.09, QED 0.39
Compound (SMILES): CNC(=O)[C@H](c1ccccc1)n1ncc2c1nc(N)n1nc(-c3ccco3)nc21
InChIKey: YPLCTRRGZGFGAI-AWEZNQCLSA-N
Target: Adenosine deaminase (SINGLE PROTEIN), Homo sapiens
UniProt: P00813
Assay: Luciferase Reporter Assay: HEK293-A2AR-luc2p/CRE/Hygro cells were seeded at a density of 5,000 cells/well in DMEM with 1% FBS and 1 U/mL adenosine deaminase (ADA) (Sigma). After 18 h, the cells were treated with 3 nM CGS21680 plus a series dilution of A2AR antagonist, the compounds disclosed herein at the concentration of 0.110000 nM, prepared in DMEM with 1% FBS. After 5 h incubation, the luciferase activity in cells were measured using the Bright-Glo Luciferase Assay System (Promega) according to manufacturer's instructions. The luminescence signal was measured using a PHERAstar FS plate reader (BMG Labtech). Luminescence intensity from 10 μM preladenant treatment was set as 0%. Maximal luminescence intensity was determined in the presence of 3 nM CGS21680 and was set as 100%. The IC50 value was calculated from a dose dependent inhibition curve across the range of compound concentrations.
Measurement: IC50 = 2.2 nM (pChEMBL 8.65999984741211)
Descriptors: MW 388.39, ALogP 1.65, HBA 9, HBD 2, PSA 129.16, QED 0.48
Compound (SMILES): [O-][S@@+](CF)c1nc2n(n1)[C@H](c1cccc(F)c1F)C[C@@H]2F
InChIKey: KOYPBTOQXRFXMW-SYLNSOGPSA-N
Target: Receptor-interacting serine/threonine-protein kinase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q13546
Assay: Biochemical Assay: The ability of the receptor interacting protein kinase (RIPK1) to catalyze the hydrolysis of adenosine-5′-triphosphate (ATP) is monitored using the Transcreener ADP (adenosine-5′-diphosphate) assay (BellBrook Labs). Purified human RIP1 kinase domain (2-375) (50 nM) derived from a baculovirus-infected insect cell expression system is incubated with test compounds for 2 hours in 50 mM Hepes buffer (pH 7.5) containing 30 mM MgCl2, 1 mM dithiothreitol, 50 uM ATP, 0.002% Brij-35, and 0.5% dimethyl sulfoxide (DMSO). Reactions are quenched by the addition of 1× Bell Brooks Stop buffer B (20 mM Hepes (ph7.5), 40 mM ethylenediaminetetraacetic acid and 0.02% Brij-35) containing an additional 12 mM EDTA and 55 ug/mL ADP2 antibody and 4 nM ADP-AlexaFluor® 633 tracer. The tracer bound to the antibody is displaced by the ADP generated during the RIP1K reaction, which causes a decrease in fluorescence polarization that is measured by laser excitation at 633 nm with a FP microplate reader M1000. Fractional activity was plotted against test article concentration. Using Genedata Screener software (Genedata; Basel, Switzerland), the data were fit to the tight-binding apparent inhibition constant (Ki app) Morrison equation [Williams, J. W. and Morrison, J. F. (1979) The kinetics of reversible tight-binding inhibition. Methods Enzymol 63: 437-67]. The following equation was used to calculate fractional activity and Ki app:Fractional ⁢ ⁢ activity = V i V o = 1 - ( [ E ] T + [ I ] T + K i app ) - ( [ E ] T + [ I ] T + K i app ) 2 - 4 ⁡ [ E ] T ⁡ [ I ] T 2 ⁡ [ E ] Twhere [E]T and [I]T are the total concentrations of active enzyme and test article, respectively.
Measurement: Ki < 5 nM (pChEMBL 8.300999641418457)
Descriptors: MW 319.28, ALogP 2.59, HBA 4, HBD 0, PSA 53.77, QED 0.65
Compound (SMILES): CCCCNc1ncc2c(-c3ccc(CNC4CCCCC4)cc3)nc([C@H]3CC[C@@H](O)CC3)n2n1
InChIKey: LBFOYYYOENDKSM-QUPDYRNUSA-N
Target: Tyrosine-protein kinase Mer (SINGLE PROTEIN), Homo sapiens
UniProt: Q12866
Assay: TAM Enzymatic Assay: The kinase assay buffer contained 50 mM HEPES, pH7.5, 10 mM MgCl2, 1 mM EGTA, 0.01% NP-40 and 2 mM DTT. 0.1 ul test compounds dissolved in DMSO were transferred from compound plates to white 384-well assay plates (Greiner LUMITRAC plates). The final concentration of DMSO was 1.25%. Enzyme solutions of 5.1 nM phosphor-Axl, or 0.0625 nM c-Mer (Carna Biosciences, 08-108), or 0.366 nM Tyro3 (Life Technologies, PR7480A) were prepared in assay buffer. A 1 mM stock solution of peptide substrate Biotin-EQEDEPEGDYFEWLE-amide SEQ ID NO:1 (Quality Controlled Biochemicals, MA) dissolved in DMSO was diluted to 1 uM in assay buffer containing 2000 uM ATP. 4 ul enzyme solution (or assay buffer for the enzyme blank) was added to the appropriate wells in each plate, and then 4 ul/well substrate solution was added to initiate the reaction. The plate was protected from light and incubated at room temperature for 60 min. The reaction was stopped by adding 4 ul detection solution containing 50 mM Tris-HCl, pH7.8, 150 mM NaCl, 0.05% BSA, 45 mM EDTA, 180 nM SA-APC (Perkin Elmer, CR130-100) and 3 nM Eu-W1024 anti-phosphotyrosine PY20 (Perkin Elmer, AD0067). The plate was incubated for 1 h at room temperature, and HTRF (homogenous time resolved fluorescence) signal was measured on a PHERAstar FS plate reader (BMG labtech). Percentage of inhibition was calculated for each concentration and IC50 value was generated from curve fitting with GraphPad Prism software.
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 476.67, ALogP 5.44, HBA 7, HBD 3, PSA 87.37, QED 0.36
Compound (SMILES): C=CC(=O)N1CC[C@]2(C1)C[C@H](n1c(NC(=O)c3ccc(-c4cc[nH]n4)s3)nc3ccccc31)C2
InChIKey: FTMHKFYFKGDOSY-ODAJSGAPSA-N
Target: Tyrosine-protein kinase ITK/TSK (SINGLE PROTEIN), Homo sapiens
UniProt: Q08881
Assay: Kinase Inhibition Assay: The in vitro kinase assays were performed at Nanosyn (Santa Clara, Calif.) utilizing microfluidic detection technology. The test compounds were serially pre-diluted in DMSO and added, by the acoustic dispensing (Labcyte 550), directly to 384 well assay plates into 10 uL of a buffer with enzyme (ITK, TXK, BTK or TEC) comprising: 100 mM HEPES, pH7.5, 5 mM MgCl2, 0.1% bovine serum albumin, 1 mM DTT, 0.01% Triton X-100 and the enzyme. Final DMSO concentration was maintained at 1% in all samples, including the controls. The reactions were initiated by addition of ATP (1 mM final concentration) and the fluorescently labeled peptide substrate to a final concentration of 1 uM, and incubated for 3 hours at 25° C. Following incubation, the reactions were quenched by addition of 40 μL of termination buffer (100 mM HEPES, pH7.5, 0.01% Triton X-100, 50 mM EDTA). Terminated plates were analyzed using Caliper LabChip 3000 microfluidic electrophoresis instrument (Caliper Life Sciences/Perkin Elmer). The enzymatic modification of the peptide substrate (phosphorylation) results in a change of net charge enabling electrophoretic separation of product from substrate. As substrate and product are separated by electrophoresis, two peaks of fluorescence are observed. Change in the relative fluorescence intensity of the substrate and product peaks was the parameter measured, reflecting enzyme activity. In the presence of inhibitor, the ratio between product and substrate is altered: signal of the product decreases, while the signal of the substrate increases. Activity in each test sample was determined as the product to sum ratio (PSR): P/(S+P), where P is the peak height of the product and S is the peak height of the FAM-cAMP substrate. For each compound, enzyme activity was measured at 12 concentrations spaced by 3× dilution intervals. Negative control samples (0%-inhibition in the absence of inhibitor, DMSO only) and positive control samples (100%-inhibition, in the absence of enzyme or in the presence of control inhibitor) were assembled in replicates of four and were used to calculate %-inhibition values in the presence of compounds. Percent inhibition (Pinh) was determined using the following equation: Pinh=(PSR0%−PSRinh)/(PSR0%−PSR100%)*100, where PSRinh is the product sum ratio in the presence of inhibitor, PSR0% is the product sum ratio in the absence of inhibitor and PSR100% is the product sum ratio in 100%-inhibition control samples. To determine IC50 values, the inhibition curves (Pinh versus inhibitor concentration) were fitted by 4 parameter sigmoid dose-response model using XLfit software (IDBS).
Measurement: IC50 = 25.7 nM (pChEMBL 7.590000152587891)
Descriptors: MW 472.57, ALogP 4.48, HBA 6, HBD 2, PSA 95.91, QED 0.42
Compound (SMILES): CNC(=O)c1cc(-c2cc(N)c3cnc(NC(=O)[C@@H]4C[C@H]4C#N)cc3c2)n(C)n1
InChIKey: LFHVTFMUMYKOIV-QWHCGFSZSA-N
Target: Mitogen-activated protein kinase kinase kinase kinase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q92918
Assay: HPK Lantha Binding Assay: For the binding assay, 4 ul 2×HPK1 and Eu-anti-GST antibody were added to each well of the assay plate using a Multidrop reagent dispenser. The solutions were incubated in a 23 C incubator for 1 h. To each well of the assay plate was added 4 ul 2× Tracer-222 using a Multidrop reagent dispenser. The solutions were again incubated in a 23 C incubator for 1 h. The results of the assay were read using an Envision plate reader with the following parameters:TR_FRET, 340ex/615 and 665em; 100 usec Delay; and 200 usec integration.
Measurement: Ki = 24 nM (pChEMBL 7.619999885559082)
Descriptors: MW 389.42, ALogP 1.68, HBA 7, HBD 3, PSA 138.72, QED 0.58
Compound (SMILES): CC(=O)N1CCc2c(c(-c3cccc(C(=O)Nc4cccc(C#N)c4)c3)nn2C2CCOC2)C1
InChIKey: RPXCZDRMNPMDKT-UHFFFAOYSA-N
Target: CREB-binding protein (SINGLE PROTEIN), Homo sapiens
UniProt: Q92793
Assay: TR-FRET Binding Assay: His/Flag epitope tagged CBP was cloned, expressed, and purified to homogeneity. CBP binding and inhibition was assessed by monitoring the engagement of a biotinylated small molecule compound with the target using the TR-FRET assay technology (Perkin-Elmer). Specifically, in a 384 well ProxiPlate CBP (4 nM final) was combined with biotin-ligand (60 nM final) in 50 mM HEPES (pH 7.5), 50 mM NaCl, 1 mM TCEP, 0.01% (w/v) BSA, and 0.008% (w/v) Brij-35 either in the presence of DMSO (final 0.2% DMSO) or compound dilution series in DMSO. After 10 minutes incubation at room temperature, a mixture Eu-W1024 Anti-6xHis antibody (6xHis is disclosed as SEQ ID NO: 3) (Perkin Elmer ADO 110) and SureLight Allophycocyanin-Streptavidin (APC-SA, Perkin Elmer CR130-100) were added to a final concentrations of 0.2 nMolar antibody and 50 nMolar APC-SA, respectively. After twenty minutes of equilibration, the plates were read on an Envision instrument and IC50s calculated using a four parameter non-linear curve fit.
Measurement: IC50 = 24.8 nM (pChEMBL 7.610000133514404)
Descriptors: MW 455.52, ALogP 3.54, HBA 6, HBD 1, PSA 100.25, QED 0.65
Compound (SMILES): CCCCCC[C@H]1Cc2ccc([C@@H]3CC[C@](N)(COP(=O)(O)O)C3)cc2CO1
InChIKey: MJKUOBXNYRXRCS-HLAWJBBLSA-N
Target: Sphingosine 1-phosphate receptor 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P21453
Assay: Binding Assay: Membranes were prepared from CHO cells expressing human S1P1. Cells pellets (1x10^9 cells/pellet) were suspended in buffer containing 20 mM HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid), pH 7.5, 50 mM NaCl, 2 mM EDTA (Ethylenediaminetetraacetic acid) and Protease Inhibitor cocktail (Roche), and disrupted on ice using the Polytron homogenizer. The homogenate was centrifuged at 20,000 rpm (48,000 g) and the supernatant was discarded. The membrane pellets were resuspended in buffer containing 50 mM HEPES, pH 7.5, 100 mM NaCl, 1 mM MgCl2, 2 mM EDTA and stored in aliquots at -80Â° C. after protein concentration determination.Membranes (2 ug/well) and 0.03 nM final concentration of 33P-S1P ligand (1 mCi/ml, Perkin elmer or American Radiolabeled Chemicals) diluted in assay buffer (50 mM HEPES, pH7.4, 5 mM MgCl2, 1 mM CaCl2, 0.5% fatty acid free BSA (bovine serum albumin), 1 mM NaF) were added to the compound plates (384 Falcon v-bottom plate (0.5 ul/well in a 11 point, 3-fold dilution). Binding was performed for 45 minutes at room temperature, terminated by collecting the membranes onto 384-well Millipore FB filter plates, and radioactivity was measured by TOPCOUNT. The competition data of the test compounds over a range of concentrations was plotted as percentage inhibition of radioligand specific binding. The IC50 is defined as the concentration of competing ligand needed to reduce specific binding by 50%.
Measurement: IC50 < 10 nM (pChEMBL 8)
Descriptors: MW 411.48, ALogP 4.17, HBA 4, HBD 3, PSA 102.01, QED 0.42
Compound (SMILES): CC(C)(c1ccc(Nc2nn([C@H]3CCCC[C@@H]3C#N)c3cc[nH]c(=O)c23)cc1)n1nccn1
InChIKey: ONNLUDOADUVDMT-APWZRJJASA-N
Target: Tyrosine-protein kinase JAK1 (SINGLE PROTEIN), Homo sapiens
UniProt: P23458
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 = 140 nM (pChEMBL 6.849999904632568)
Descriptors: MW 442.53, ALogP 4.1, HBA 8, HBD 2, PSA 117.21, QED 0.48
Compound (SMILES): COc1ccc(-c2n[nH]c3cc4c(cc23)CN(CCc2ccc(F)cc2)C(=O)N4)cn1
InChIKey: DGBYGAQBSCYGRK-UHFFFAOYSA-N
Target: Mitogen-activated protein kinase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P28482
Assay: Z′-LYTE Kinase Assay: The inhibition of ERK activity by the compounds disclosed herein was determined using the Z′-LYTE kinase assay kit (Life Technologies) with a Ser/Thr 3 peptide substrate (Life Technologies) according to manufacturers' instructions. The assay was run with an ERK2 enzyme (Life Technologies) concentration of 0.47 ng/μL at 100 μM ATP (approximately the ATP Km for ERK2). The IC50 values for the compounds were determined with 3-fold serial dilutions in duplicate. The compounds were first diluted in 1:3 dilutions in 100% DMSO at 100× the desired concentration, and then further diluted (1:25) in 20 mM HEPES buffer (Invitrogen) to make 4× solutions prior to adding to the enzyme solution. The final DMSO concentration in the assay was 1%. Final reaction volume was 20 μL/well in 384-well plates. Kinase reactions were conducted or 1 hour followed by the assay development reaction (1 hour) in a 20 ul/well in a 384 well plate format.
Measurement: IC50 < 50 nM (pChEMBL 7.301000118255615)
Descriptors: MW 417.44, ALogP 4.36, HBA 4, HBD 2, PSA 83.14, QED 0.51
Compound (SMILES): CC(C)c1ncncc1-c1cnc2c3[nH]ncc3c(=O)n(CC(F)(F)F)c2c1
InChIKey: OTOSIRQCIQFQQV-UHFFFAOYSA-N
Target: Methionine aminopeptidase 2 (SINGLE PROTEIN), Homo sapiens
UniProt: P50579
Assay: Enzyme Assay: For the enzymatic test, human MetAP2 protein was obtained from a culture supernatant of insect cells (sf9) infected with MetAP2 recombinant baculovirus.Before performing the experiment, dialysis of the MetAP2 supernatant was performed over 24 hours at 4° C. in a buffer (10 mM Hepes, 100 mM KCl, 10% glycerol, pH 7.4) in the presence of EDTA (1 mM) over the first 12 hours.The dialysis supernatant was recovered and manganese, used as cofactor, was added to a final concentration of 300 μM.The enzymatic test is a two steps procedure.In a first step, it consists in placing in contact the compound according to the invention, the dialysed MetAP2 protein and the substrate (Met-Pro-Arg-pNa peptide synthesized by Neosystem), the N-terminal methionine of which can be cleaved with MetAP2, and which bears at the C-terminal end a para-nitroaniline (pNa) chromophore, which can itself be released by another peptidase only when the N-terminal methionine has been cleaved beforehand.Consequently, the second step consists in reacting the peptides cleaved in the preceding step with a second peptidase in order to release the chromophore. The peptidase used in this second step is cathepsin, which comes from the TagZyme DAPase kit (Quiagen, 34366).The MetAP2 activity is proportional to the amount of para-nitroaniline released, which is measured by absorbance at 405 nm.
Measurement: IC50 = 379 nM (pChEMBL 6.420000076293945)
Descriptors: MW 388.35, ALogP 3.42, HBA 6, HBD 1, PSA 89.35, QED 0.58
Compound (SMILES): CNc1nc(=O)n(-c2cccnc2)c2cc(C3CC3)cc(OC)c12
InChIKey: HIEQBDZMYQJPGZ-UHFFFAOYSA-N
Target: S-adenosylmethionine synthase isoform type-2 (SINGLE PROTEIN), Homo sapiens
UniProt: P31153
Assay: Phosphate Sensor Fluorescence Assay : MAT2A enzyme is incubated with a test compound in DMSO or DMSO and its substrates (L-methionine and ATP) in a microtiter plate. The enzymatic reaction is stopped by the addition of Working Phosphate Sensor Mixture. The plate is analyzed for fluorescence at 450 nm. The high control (DMSO with enzyme and its substrates) gives high fluorescence which represents no inhibition of enzymatic activity while the low control (DMSO with MAT2A substrates and no enzyme) gives low fluorescence which represents full inhibition of enzymatic activity.Materials:Human MAT2A: Cepter, amino acids 1-395Tris, pH 7.5: Invitrogen cat #15567-027KCl: Ambion cat #AM9640GMgCl2: Ambion cat #AM9530GBrij-35: Sigma cat B4184-10MLDTT: Goldbio cat #DTT100BGG: Sigma cat #G5009-25GPNP: Novus Biologicals cat #NBP1-508727-MEG: Cayman Chemical cat #15988L-Methionine: Alfa Aesar cat #J61904ATP: Alfa Aesar cat #J60336Phosphate Sensor: Thermo Fisher cat #PV4407EDTA: Life Tech cat #15575-038Assay plate: 384-well black polypropylene plate: Thomas Scientific cat #1149Q35Final Assay Conditions:Assay Buffer: 50 mM Tris, pH 7.5/50 mM KCl/10 mM MgCl2/0.01% Brij-35/1 mM DTT/0.1% BGG/40 nM PNP/6 uM 7-MEGMAT2A: 10 nM for Cepter clone ID 329, lot 00023-123 before the addition of Working Phosphate Sensor Mixture    5 nM for Cepter clone ID 334, lot 00023-148 before the addition of Working Phosphate Sensor MixtureL-methionine: 500 uM before the addition of Working Phosphate Sensor MixtureATP: 500 uM before the addition of Working Phosphate Sensor MixtureProcedure:For the assay, a mixture of 1 mM L-methionine/1 mM ATP (2× final pre-stopped concentration) in assay buffer; MAT2A (2× final pre-stopped concentration) in Assay Buffer and Working Phosphate Sensor Mixture (1.5 uM Phosphate Sensor/30 mM EDTA in Assay Buffer, which is 3× final concentrations) were prepared. Test compounds or DMSO were added to the appropriate well suing D300e digital dispenser. 5 μl/well of Assay Buffer was added to the wells corresponding to the negative control and 5 μl/well of MAT2A was added to all the wells except for those corresponding to the negative control. After incubating the plate at room temperature for 15 minutes, 5 μl/well of the 1 mM L-methionine/1 mM ATP mixture was added to all wells. The plate was centrifuged at 1000 rpm for 1 minute and then incubated at room temperature for 1 hour. 5 μl of the Working Phosphate Sensor Mixture was added to all wells and the plate was centrifuged at 1000 rpm for 1 minute. The plate was read for fluorescence at 450 nm after exciting at 430 nm.
Measurement: IC50 < 200 nM (pChEMBL 6.698999881744385)
Descriptors: MW 322.37, ALogP 2.71, HBA 6, HBD 1, PSA 69.04, QED 0.8