lovingscience/chembl-v37-raw-activities huggingface dataset:
Compound (SMILES): CNc1nc(Nc2cn(-c3cn([C@H]4CCn5cncc54)nc3C)nc2C)ncc1C(F)(F)F
InChIKey: JRNOWWCJYALINT-HNNXBMFYSA-N
Target: Leucine-rich repeat serine/threonine-protein kinase 2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q5S007
Assay: Biochemical Assay : 50 mM Tris pH 7.5, 10 mM MgCl2, 1 mM EGTA, 0.01% Brij-35, 2 mM DTT, 5 nM LRRK2, 134 μM ATP, 60 minute reaction time, 23° C. reaction temperature, and 10 μL total reaction volume.Detection Reaction Conditions:1×TR-FRET dilution buffer, 10 mM EDTA, 2 nM antibody, 23° C. reaction temperature, and 10 μL total reaction volume.Compound solutions were prepared by initially diluting to 1 mM with DMSO. 35 μL of reference compound solution, 35 μL of test compound solution, and 35 μL HPE were successively added to the source plate (384-well assay plate, Labcyte). The plates were centrifuged at 2500 rpm for 1 minute and sealed in foil. POD was used to perform a 3.162 fold serial dilution and 100 nL of reference compound solution, test compound solution, HPE and ZPE were transferred to assay plates. The assay plate was centrifuged at 2500 rpm for 1 minute, and sealed with foil.
Measurement: IC50 = 45 nM (pChEMBL 7.349999904632568)
Descriptors: MW 458.45, ALogP 3.47, HBA 10, HBD 2, PSA 103.3, QED 0.47
Compound (SMILES): CN1CCN(c2cc(NC(=O)Cn3cc(-c4cc(Cl)c(O)c(C(N)=O)c4)c4c(=O)n(C)cnc43)c(Cl)cn2)C(C)(C)C1
InChIKey: NQKUWAIKXADZDV-UHFFFAOYSA-N
Target: N-formyl peptide receptor 2 (SINGLE PROTEIN), Homo sapiens
UniProt: P25090
Assay: Surface Plasmon Resonance (SPR) Assay: SPR studies were performed using a Biacore™ T200 instrument (GE Health Sciences Inc.). The BCL6 BTB protein used in the FP assay was biotinylated using the site specific biotinylating enzyme BirA, and then cleaved with TEV protease to produce the BCL6 BTB domain (biotin-thrombin-BCL6 amino acids 1-129; 17 kd) that were use in SPR. This protein was stably captured (1000RU) to streptavidin coupled SA chips (BR-1005-31, GE Health Sciences Inc.) according to the manufacture's protocol. Compounds were dissolved in 100% DMSO at 10 mM and 2-fold serial dilutions were done in 100% DMSO. For SPR analysis the serially titrated compounds were diluted 1/20 into buffer (10 mM HEPES pH7.4, 150 mM NaCl, 0.05% Tween-20, 3 mM EDTA) giving a final concentration of 5% DMSO. The Biacore flow rate was set at 100 ul/min. For KD determinations, single cycle kinetic analysis was performed with an on time of 60 seconds, and an off time of 300 seconds. Curve fitting and KD calculations were done with the Biacore T200 Evaluation software (GE Health Sciences Inc).
Measurement: Kd = 59.7 nM (pChEMBL 7.21999979019165)
Descriptors: MW 613.51, ALogP 3.08, HBA 10, HBD 3, PSA 151.61, QED 0.3
Compound (SMILES): CCS(=O)(=O)c1cc(C)ccc1Cn1cnc2ccc(C(F)(F)F)cc2c1=O
InChIKey: LWINECRLXNKIGF-UHFFFAOYSA-N
Target: Epithelial discoidin domain-containing receptor 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q08345
Assay: LanthaScreen Assay: DDR1 binding activity was measured using the LanthaScreen (Registered trademark) Eu Kinase Binding Assay (manufactured by Life Technologies Corporation). The test compound and the Alexa Fluor® 647-labeled Kinase Tracer 178 (manufactured by Life Technologies Corporation) were added to a mixture of DDR1 and the LanthaScreen (Registered trademark) Eu-anti-GST antibody. After one-hour reaction at room temperature, the fluorescence resonance energy transfer was measured. The 50% inhibitory concentration (IC50) was calculated from the inhibition rate relative to the test compound-free control.
Measurement: IC50 = 2640 nM (pChEMBL 5.579999923706055)
Descriptors: MW 410.42, ALogP 3.57, HBA 5, HBD 0, PSA 69.03, QED 0.66
Compound (SMILES): CC[C@@H](Nc1cc(-c2cnc(NC(=O)C3CC3)cn2)cnc1C#N)C(=O)N(C)C
InChIKey: RWKPXTMKESNKIJ-CQSZACIVSA-N
Target: Serine/threonine-protein kinase TBK1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9UHD2
Assay: Inhibition Assay: TBK1 inhibition is determined using a 384 well μlate format in buffer containing 20 mM Hepes, pH 7.4, 10 mM MgCl2, 1 mM EDTA, 0.01% Brij-L23, 1 mM DTT. Each test compound is prepared in DMSO using 2.5-fold serial dilutions for 11 data points, which are added to the buffer so that each dilution contains 1% DMSO. To each well is added 2 μL of 1 μM 5FAM-DRHDSGLDSMKDE-NH2 (in buffer), 2 μL of diluted test compound (1% DMSO in buffer), and 5 μL of 3 nM TBK1 and 25 μM ATP (in buffer). The reaction mixture is incubated at RT for 60 min, and quenched by adding 20 mM Hepes, pH 7.4, 10 mM MgCl2, 1 mM EDTA, 0.01% Brij-L23, 1 mM DTT+25 mM EDTA. To quantify the fluorescent-labeled substrate and product following reaction, the test plate is loaded on a Caliper LC-3000, which measures percent of conversion by microfluidic-based separation.
Measurement: IC50 = 501 nM (pChEMBL 6.300000190734863)
Descriptors: MW 393.45, ALogP 2.04, HBA 7, HBD 2, PSA 123.9, QED 0.74
Compound (SMILES): CN(C)C/C=C/C(=O)Nc1cccc(-c2c(N)ncnc2Oc2ccc(Oc3ccccc3)cc2)c1
InChIKey: UQJKGYDUTSFPQB-KPKJPENVSA-N
Target: Tyrosine-protein kinase BTK (SINGLE PROTEIN), Homo sapiens
UniProt: Q06187
Assay: Enzyme Assay: The following describes a microfluidic, off-chip mobility shift kinase assay used to measure inherent potency of compounds against BTK enzyme. Compounds described by embodiments of the present invention were assayed using this protocol and the data from the same is recorded in Table 2 within the column labeled: Time Dependent BTK Enzyme Assay IC50. These IC50 values are reported in ranges wherein: A<100 nM, B<1 uM, and C>1 uM.2.5× stocks of full-length human BTK (08-080) from CarnaBio USA, Inc., Natick, Mass., 1.6×ATP and appropriate kinKDR peptide substrate (FITC-AHA-EEPLYWSFPAKKK-NH2) were prepared in kinase reaction buffer consisting of 25 mM MgCl2, 0.015% Brij-35 (30%), 100 mM Hepes, pH 7.5, and 10 mM DTT.5 uL of enzyme buffer and 7.5 uL of ATP/kinKDR peptide substrate mix were added to Matrix (#115304) 384-well, sterile, polypropylene plates (Thermo Fisher Scientific, Hudson, N.H.) with 125 nL of serially diluted compounds prepared in 100% DMSO, and incubated for 90 min. at 27 C. Following the incubation period, reactions were stopped by adding 60 uL stop buffer consisting of 100 mM Hepes, pH 7.5, 0.015% Brij-35 (30%), 0.277% Coating Reagent #3 (Caliper Life Sciences, Mountain View, Calif.), 5% DMSO. Stopped reactions were monitored at −2 PSI, −3000 V/−700 V in a LabChip 3000 plate reader from Caliper Life Sciences, a PerkinElmer Company (Hopkinton, Mass.), and the activity was measured by off-chip mobility shift assay measuring the charge/mass difference between substrate and product resulting from peptide phosphorylation. IC50 and efficacy were determined by plotting log [Inhibitor] vs. % Activity in GeneData Screener (Basel, Switzerland). Compounds described by embodiments of the present invention were assayed using this protocol and the data from the same is recorded in Table 2 within the column labeled: Time Dependent PBMC BTK Enzyme Assay IC50.
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 481.56, ALogP 5.37, HBA 7, HBD 2, PSA 102.6, QED 0.31
Compound (SMILES): O=C1NC[C@H](c2ccc(-c3cnc(C(F)(F)F)s3)cc2)N1c1ccc2[nH]cnc2c1
InChIKey: XHMRZHYTEPRFPJ-MRXNPFEDSA-N
Target: Glutaminyl-peptide cyclotransferase (SINGLE PROTEIN), Homo sapiens
UniProt: Q16769
Assay: QC Inhibition Assay: An inhibition activity assay of QC inhibitors was conducted. See Huang et al., J. Biol. Chem. 2011, 286, 12439-12449. A reaction mixture containing 300 μM of Gln-βNA and 0.2 units of human PAP I was prepared. QC was first incubated with an inhibitor at 25° C. for 30 minutes and the enzyme-inhibitor mixture was then added to the reaction mixture to initiate the cyclization reaction. An IC50 value was obtained by fitting an initial reaction rate versus an inhibitor concentration using KaleidaGraph. A Ki value of the inhibitor was calculated according to an equation IC50=Ki(1+[S]/Km). See Segel, Enzyme Kinetics: Behavior and Analysis of Rapid Equilibrium and Steady-State Enzyme Systems, pp. 100-118. New York: John Wiley & Sons, 1993. In this equation, [S] is a substrate (i.e., Gln-βNA) concentration and Km is a Michaelis-Menten constant. The lower the Ki value, the higher the inhibitor's QC inhibition rate.
Measurement: Ki = 16 nM (pChEMBL 7.800000190734863)
Descriptors: MW 429.43, ALogP 4.98, HBA 4, HBD 2, PSA 73.91, QED 0.48
Compound (SMILES): CC1(Nc2nc(=O)n(-c3ccccc3Cl)c3nc(C(F)(F)F)ccc23)CCC1
InChIKey: XGLVANYOJZIOKG-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 408.81, ALogP 4.81, HBA 5, HBD 1, PSA 59.81, QED 0.68
Compound (SMILES): O=C(N[C@H]1CCN(C2CCCCC2)C[C@@H]1C(=O)NC(CO)c1ccccn1)c1cc(-c2ccc(F)cc2F)on1
InChIKey: VDAUERLLSCIHKP-OYICLKKBSA-N
Target: Atypical chemokine receptor 3 (SINGLE PROTEIN), Homo sapiens
UniProt: P25106
Assay: In Vitro Assay: Tango CXCR7-bla U2OS cells are detached from culture dishes with 0.05% trypsin-EDTA and collected in growing medium (McCoy's 5A 90% (v/v), dialyzed FCS 10% (v/v), 0.1 mM NEAA, 25 mM HEPES (pH7.3), 1 mM sodium pyruvate, P/S 1% (v/v) 50 μg/ml Hygromycin, 100 μg/ml Geneticin, 200 μg/ml Zeocin), spinned down and resuspended in assay medium (McCoy's 5A 90% (v/v), dialyzed FCS 1% (v/v), 0.1 mM NEAA, 25 mM HEPES (pH7.3), P/S 1% (v/v)). 10′000 cells per well (in 30 μl) are seeded in a 384 well plate (black-walled, clear bottom). The plate is incubated at 37° C./5% CO2 for 24 hours. Test compounds are dissolved to 10 mM in DMSO and serially diluted in DMSO to 500× of the final concentration for dose response curves. Compounds are then diluted 1:100 in assay medium to 5× of the final concentration. 10 μl/well of diluted compounds are added to the assay plate and incubated for 15 minutes at 37° C. Thereafter CXCL12/SDF1-α is diluted in assay medium to 5× of the final concentration (its EC80 value for receptor activation) and 10 μl/well are added to the assay plate. The agonist leads to activation of the receptor and therefore to b-arrestin recruitment. Compounds acting as antagonists reduce this activation. The plate is incubated for 22 hrs at 37° C. 10 μl/well of detection reagent (LiveBLAzer-FRET BIG (CCF4-AM) substrate) is transferred to the assay plate and the plate is incubated for 2 hours at room temperature protected from light. Fluorescent counts are determined (Scan1: Ex 409/20 nm, Em 460/30 nm, Scan 2: Ex 409/20 nm, Em 530/30 nm).
Measurement: IC50 = 145 nM (pChEMBL 6.840000152587891)
Descriptors: MW 553.61, ALogP 3.62, HBA 7, HBD 3, PSA 120.59, QED 0.39
Compound (SMILES): CC(=O)N1CCCC(Cn2c(=O)c(O)c(C(=O)O)c3sccc32)C1
InChIKey: YXBYSAYQSBHNKQ-UHFFFAOYSA-N
Target: Flap endonuclease 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P39748
Assay: Enzymatic IC50 Assays: FEN1, EXO1 or XPG enzyme was incubated with compound or vehicle (DMSO) and the FAM-labeled DNA oligomer substrate in a microtiter plate. The stop buffer contains EDTA to stop the enzymatic reaction. The plate is read for fluorescence intensity. The high control (DMSO) with high fluorescence intensity represents no inhibition of enzymatic reaction while the low control (10 μM) with low fluorescence intensity represents full inhibition of enzymatic reaction.
Measurement: IC50 = 6125 nM (pChEMBL 5.210000038146973)
Descriptors: MW 350.4, ALogP 1.73, HBA 6, HBD 2, PSA 99.84, QED 0.88
Compound (SMILES): CC(C)(C)OC(=O)N1CCC(CC#N)(n2nc(Nc3ccc([C@@]4(C(F)(F)F)CCCN4)cc3)c3c(=O)[nH]ccc32)CC1
InChIKey: DADRYUBONNHRSW-MUUNZHRXSA-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 = 0.55 nM (pChEMBL 9.260000228881836)
Descriptors: MW 585.63, ALogP 5.25, HBA 8, HBD 3, PSA 128.07, QED 0.37
Compound (SMILES): Nc1nc(-c2ccccc2)c(-c2ccnc(Cl)c2)c2nncn12
InChIKey: OQRHRHLGSRKOOX-UHFFFAOYSA-N
Target: Adenosine receptor A2a (SINGLE PROTEIN), Homo sapiens
UniProt: P29274
Assay: Adenosine A2a Receptor: CHO-K1/A2aR cells were cultured in DMEM/F12 medium containing 10% fetal bovine serum and 800 μg/ml bleomycin. The cells were digested with the cell separation buffer during the experiment. The cells were resuspended in the balanced salt buffer containing 20 mM HEPES and 0.1% bovine serum albumin and counted, and the cell density was adjusted to 106 cells/ml. In the 384-well plate, each well was added with 5 μl of cell suspension, and 2.5 μl of test compound (4× concentration) formulated with the balanced salt buffer containing 20 mM HEPES, 0.1% bovine serum albumin, 54 μM rolipram and 2.7 U/ml adenosine deaminase, and the plate was incubated at room temperature for 30 minutes. Each well was then added with 2.5 μl of ethyl carbazole (4× concentration) formulated with the balanced salt buffer containing 20 mM HEPES, 0.1% bovine serum albumin, 54 μM rolipram and 2.7 U/ml adenosine deaminase, and the plate was incubated at room temperature for 30 minutes. The final concentrations of the compounds were: 10000, 2000, 400, 80, 16, 3.2, 0.64, 0.128, 0.0256, 0.00512, and 0.001024 nM. The final concentration of ethyl carbazole was 20 nM. Intracellular cAMP concentration was detected with the cAMP dynamic 2 kit. cAMP-d2 and Anti-cAMP-Eu-Cryptate were diluted respectively with the cAMP lysis buffer at a ratio of 1:4. Each well was added with 5 μl of diluted cAMP-d2, followed by addition of 5 μl of diluted Anti-cAMP-Eu-Cryptate, and the plate was incubated at room temperature in the dark for 1 hour. The HTRF signal values were read by the PHERAstar multi-function microplate reader. IC50 values of inhibition activity of the compounds were calculated by Graphpad Prism software.
Measurement: IC50 = 2.18 nM (pChEMBL 8.65999984741211)
Descriptors: MW 322.76, ALogP 3.09, HBA 6, HBD 1, PSA 81.99, QED 0.57
Compound (SMILES): CN1C(=O)[C@@H](NC(=O)c2n[nH]c3c2CC2CC2(C)C3)COc2ccccc21
InChIKey: GRWRHAMUFRDCSK-QINJFGQTSA-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.
Measurement: Ki = 4700 nM (pChEMBL 5.329999923706055)
Descriptors: MW 366.42, ALogP 1.69, HBA 4, HBD 2, PSA 87.32, QED 0.85
Compound (SMILES): CC(C)c1cc(C(=O)N2CC[C@@H](n3cccc(C4CC4)c3=O)C2)[nH]n1
InChIKey: KCKPUNHLKNGJAR-CQSZACIVSA-N
Target: Lysine-specific demethylase 5A (SINGLE PROTEIN), Homo sapiens
UniProt: P29375
Assay: KDM5 Enzyme Assay: Full length KDM5A enzyme was expressed and purified inhouse. Biotin-H3K4me3 peptide was purchased from New England Biolabs. HTRF reagents (containing Eu-labeled H3K4me1-2 antibody, and streptavidin-XL665) were purchased from Cis-Bio International. Plates were read on an Envision multi-label plate reader (Perkin Elmer).The HTRF assay mixture contained 2 nM full length KDM5A enzyme, 100 nM H3K4Me3 peptide substrate, 1 uM 2-OG, 100 uM Fe2+, 500 uM ascorbate. 50 mM HEPES pH 7.0 buffer, 0.01% Triton-X 100, 2 mM DTT, 0.25% DMSO at a final volume of 10 uL.The enzyme reaction was carried out at room temperature in black Proxiplate 384-Plus plate (Corning. Costar) within 30 minutes, in the presence of varying concentration of a test compound. At the end of enzyme reaction, 5 uL of 1 mM EDTA were added to quench the reaction and then the detection reagents (5 uL) were added to give final concentrations of 0.5 nM Eu-labeled H3K4me1-2 antibody, and 50 nM streptavidin-XL665. The plates were incubated at room temperature for 60 minutes and then read in the Envision plate reader. The readouts were transformed into % inhibition, and IC50 value of a test compounds was generated by using four parameters curve fitting (Model 205 in XLFIT5, iDBS).
Measurement: IC50 = 0.051 nM (pChEMBL 10.289999961853027)
Descriptors: MW 340.43, ALogP 2.66, HBA 4, HBD 1, PSA 70.99, QED 0.93
Compound (SMILES): N#Cc1ccncc1-c1ccc(NC(=O)c2ccnc(-c3c(F)cccc3Cl)n2)c(N2C[C@@H](N)C[C@H]2CO)c1
InChIKey: PJPVNRZDRDNSLL-OALUTQOASA-N
Target: Mitogen-activated protein kinase kinase kinase kinase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q92918
Assay: HPK1 Kinase Binding Assay: A stock solution of 1 mM test compound was prepared in DMSO. The compound plate was prepared by 3-fold and 11-point serial dilutions. 0.1 μL of the compound in DMSO was transferred from the compound plate to the white 384 well polystyrene plates. The assay buffer contained 50 mM HEPES, pH 7.5, 0.01% Tween-20, 5 mM MgCl2, 0.01% BSA, and 5 mM DTT. 5 μL of 4 nM active HPK1 (SignalChem M23-11G) prepared in the buffer was added to the plate. The enzyme concentration given was based on the given stock concentration reported by the vender. 5 μl of 18 nM tracer 222 (ThermoFisher PV6121) and 4 nM LanthaScreen Eu-Anti GST antibody (ThermoFisher PV5595) were added. After one hour incubation at 25° C., the plates were read on a PHERAstar FS plate reader (BMG Labtech). Ki values were determined.
Measurement: Ki < 100 nM (pChEMBL 7)
Descriptors: MW 543.99, ALogP 4.02, HBA 8, HBD 3, PSA 141.05, QED 0.33
Compound (SMILES): O=C(Nc1ccc2ncc(-c3cc4ccccc4o3)n2n1)C(O)c1cccnc1
InChIKey: FQFWDRRIGZELJC-UHFFFAOYSA-N
Target: MAP kinase-interacting serine/threonine-protein kinase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9BUB5
Assay: Kinase Assay: For the assay 50 nL of a 100 fold concentrated solution of the test compound in DMSO was pipetted into a black low volume 384 well microtiter plate (Greiner Bio-One, Frickenhausen, Germany), 2 μL of a solution of MKNK1 in aqueous assay buffer [50 mM HEPES pH 7.5, 5 mM magnesium chloride, 1.0 mM dithiothreitol, 0.005% (v/v) Nonidet-P40 (Sigma)] was added and the mixture was incubated for 15 min at 22° C. to allow pre-binding of the test compounds to the enzyme before the start of the kinase reaction. Then the kinase reaction was started by the addition of 3 μL of a solution of adenosine-tri-phosphate (ATP, 16.7 μM=>final conc. in the 5 μL assay volume is 10 μM) and substrate (0.1 μM=>final conc. in the 5 μL assay volume is 0.06 μM) in assay buffer and the resulting mixture was incubated for a reaction time of 45 min at 22° C. The concentration of MKNK1 was adjusted depending of the activity of the enzyme lot and was chosen appropriate to have the assay in the linear range, typical concentrations were in the range of 0.05 μg/ml. The reaction was stopped by the addition of 5 μL of a solution of TR-FRET detection reagents (5 nM streptavidine-XL665 [Cisbio Bioassays, Codolet, France] and 1 nM anti-ribosomal protein S6 (pSer236)-antibody from Invitrogen [#44921G] and 1 nM LANCE EU-W1024 labeled ProteinG [Perkin-Elmer, product no. AD0071]) in an aqueous EDTA-solution (100 mM EDTA, 0.1% (w/v) bovine serum albumin in 50 mM HEPES pH 7.5).The resulting mixture was incubated for 1 h at 22° C. to allow the formation of complex between the phosphorylated biotinylated peptide and the detection reagents. Subsequently the amount of phosphorylated substrate was evaluated by measurement of the resonance energy transfer from the Eu-chelate to the streptavidine-XL.
Measurement: IC50 = 3 nM (pChEMBL 8.520000457763672)
Descriptors: MW 385.38, ALogP 3.21, HBA 7, HBD 2, PSA 105.55, QED 0.49
Compound (SMILES): CC1(C(=O)N2CCC(Oc3ccc(OCCCN4CCCCC4)cc3)CC2)CC1
InChIKey: OVOCBFDIARGWAI-UHFFFAOYSA-N
Target: Histamine H3 receptor (SINGLE PROTEIN), Rattus norvegicus
UniProt: Q9QYN8
Assay: In Vitro Binding Assay: Receptor binding assays were performed in at least 5 concentrations, with two parallel samples in each concentrations, in at least two independent experiments using the binding buffer (50 mM Tris-HCl, pH 7.4, 5 mM MgCl2), rat H3 membrane (140 μg protein/tube), and N-α-[Methyl-3H]methylhistamine dihydrochloride (1 nM) as radioligand. Non-specific binding was determined in the presence of 10 μM thioperamide. The samples were incubated in a final volume of 0.50 ml for 30 min at 25° C. Binding reactions were terminated by rapid filtration through UniFilter® GF/B fiber glass filters presoaked for at least 2 h in 0.5% polyethylene imine (PEI). The filterplates were washed nine times with 0.5 ml of ice-cold washing buffer (50 mM Tris-HCl, pH 7.4, 5 mM MgCl2, 10 μg/ml saponine). The filterplates were dried at 50° C. for 45 min and 40 μl of Microscint20 (Packard) scintillation cocktail was added to each well. Filters radioactivity was determined by
Measurement: Ki < 10 nM (pChEMBL 8)
Descriptors: MW 400.56, ALogP 4.11, HBA 4, HBD 0, PSA 42.01, QED 0.62
Compound (SMILES): CC(C)N1CCC(n2nc(-c3cc4cc(O)ccc4[nH]3)c3c(N)ncnc32)CC1
InChIKey: GEFONYPTJXAGRH-UHFFFAOYSA-N
Target: Phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit alpha isoform (SINGLE PROTEIN), Homo sapiens
UniProt: P42336
Assay: Inhibition Assay: Class I PI3-Ks can be either purchased (p110α/p85α, p110β/p85α, p110Î´/p85α from Upstate, and p110γ from Sigma) or expressed as previously described (Knight et al., 2004). IC50 values are measured using either a standard TLC assay for lipid kinase activity (described below) or a high-throughput membrane capture assay. Kinase reactions are performed by preparing a reaction mixture containing kinase, inhibitor (2% DMSO final concentration), buffer (25 mM HEPES, pH 7.4, 10 mM MgCl2), and freshly sonicated phosphatidylinositol (100 ug/ml). Reactions are initiated by the addition of ATP containing 10 uCi of γ-32P-ATP to a final concentration 10 or 100 uM and allowed to proceed for 5 minutes at room temperature. For TLC analysis, reactions are then terminated by the addition of 105 ul 1N HCl followed by 160 ul CHCl3:MeOH (1:1). The biphasic mixture is vortexed, briefly centrifuged, and the organic phase is transferred to a new tube using a gel loading pipette tip precoated with CHCl3. This extract is spotted on TLC plates and developed for 3-4 hours in a 65:35 solution of n-propanol:1M acetic acid. The TLC plates are then dried, exposed to a phosphorimager screen (Storm, Amersham), and quantitated. For each compound, kinase activity is measured at 10-12 inhibitor concentrations representing two-fold dilutions from the highest concentration tested (typically, 200 uM).
Measurement: IC50 > 500 nM (pChEMBL 6.301000118255615)
Descriptors: MW 391.48, ALogP 3.31, HBA 7, HBD 3, PSA 108.88, QED 0.49
Compound (SMILES): O=C(NC1CC2(CCC2)C1)c1cc2cc(Br)cnc2n(Cc2cccnc2)c1=O
InChIKey: BWMYVWJUVYBPMS-UHFFFAOYSA-N
Target: Cannabinoid receptor 2 (SINGLE PROTEIN), Homo sapiens
UniProt: P34972
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 < 100 nM (pChEMBL 7)
Descriptors: MW 453.34, ALogP 3.66, HBA 5, HBD 1, PSA 76.88, QED 0.65
Compound (SMILES): COCCS(=O)(=O)C(C(=O)NCC(=O)NCC(F)(F)F)c1nc2ccc(-c3ccc(F)nc3)cc2s1
InChIKey: MXRHQXCCLRWWRT-UHFFFAOYSA-N
Target: Endothelial lipase (SINGLE PROTEIN), Homo sapiens
UniProt: Q9Y5X9
Assay: Biological Activity: Endothelial lipase (EL) and hepatic lipase (HL) activities were measured using a fluorescent substrate, A10070, (Invitrogen, CA) doped into an artificial vesicle containing DMPG (Avanti Polar Lipids) as the excipient. Vesicles were prepared by combining 571 μL of 29 mM DMPG in a 1:1 mixture of MeOH and CHCl3 with 2000 μL of 1 mM A10070 in a 1:1 mixture of MeOH and CHCl3. The mixture was dried under nitrogen in multiple vials then resuspended in 20 mL total volume of 50 mM HEPES pH 8.0 buffer containing 50 mM NaCl and 0.2 mM EDTA. The sample was allowed to sit at room temperature for 15 min and then was sonicated 3×4 mins on ice with a Branson Sonicator using duty cycle 1. This preparation provides vesicles with a mole fraction of 0.11 for the FRET substrate.The enzymatic assay was measured using 384-well white Optiplates. Each well contained 20 μL of assay buffer (50 mM HEPES pH 8.0, 50 mM NaCl and 1 mM CaCl2) and 0.25 μL of a DMSO solution containing a compound of interest. EL or HL (10 μL) was added and allowed to incubate with the compound for 30 min at 37° C. The source of EL was conditioned media obtained from HT-1080 cells that were transformed using RAGE technology (Athersys) to overexpress endogenous EL, and HL was partially purified from conditioned media obtained from COS cells overexpressing HL. The reaction was started by the addition of 10 μL of a 1:10 dilution of vesicles. The final total reaction volume was 20.25 μL. The reaction rates were measured on a Gemini plate reader with an excitation wavelength of 490 nm and an emission wavelength of 530 nm. Readings were taken over a period of 60 minutes, and the slope between 300 and 900 secs of the readout was used to calculate the rate of the reaction.
Measurement: IC50 = 198 nM (pChEMBL 6.699999809265137)
Descriptors: MW 548.54, ALogP 2.39, HBA 8, HBD 2, PSA 127.35, QED 0.29
Compound (SMILES): C[C@@H](CC(F)(F)F)n1cc(-c2nc(-c3cnn(C[C@@H](O)CO)c3)cn3nccc23)cn1
InChIKey: YPBHFJWUPLVJAN-SWLSCSKDSA-N
Target: Non-receptor tyrosine-protein kinase TYK2 (SINGLE PROTEIN), Homo sapiens
UniProt: P29597
Assay: Tyk2 Inhibition Assay: Compounds of Formula I were screened for their ability to inhibit Tyk2 using the general enzyme inhibition assay method, in which the assay mixture contained 1 mM ATP, 8 μM Omnia® Y12 peptide (Catalog # IVGN KPZ3121C; Invitrogen Corporation, Carlsbad, Calif.) and 1 nM Tyk2 in a total volume of 25 μL. Human Tyk2 kinase domain, comprising amino acids 886 to 1187 with 10 additional histidine residues (histidine tag) on the carboxy terminus, was expressed and purified from bacculovirus in-house at Array BioPharma Inc. (Boulder, Colo.). The histidine tag was cleaved after purification using standard conditions.
Measurement: IC50 = 550 nM (pChEMBL 6.260000228881836)
Descriptors: MW 435.41, ALogP 2.32, HBA 9, HBD 2, PSA 106.29, QED 0.46
Compound (SMILES): C[C@@H]1CC(F)(F)CN(C(=O)c2nc(C(F)(F)F)ccc2-n2nccn2)C1CNc1ccc(C(F)(F)F)cn1
InChIKey: NPVCPCWYHFRHJG-KEKZHRQWSA-N
Target: Orexin receptor type 2 (SINGLE PROTEIN), Homo sapiens
UniProt: O43614
Assay: Orexin Receptor Cell-Based Functional Assay: Measurement of [Ca2+]i using a FLIPR: CHOOX1 or CHOOX2 cells were seeded into black-walled clear-base 384-well plates (Corning, catalog #3712) at a density of 20,000 cells per well in F12-K medium supplemented with 10% FBS and then incubated in a 5% CO2, 37° C. incubator overnight to reach 90% confluency. The cells were incubated with equal volume of calcium 6 loading buffer (Molecular Devices, Inc.) containing 2.5 mM probenecid at 37° C. for 2 h, followed by test compounds (dose-range 0.1 nM-10 μM) for another 30 min. The plates were then placed into a FLIPR (Molecular Devices, Inc.) to monitor fluorescence (λ excitation 488 nm, λ emission 540 nm) before and after the addition of EC90 of [OX].
Measurement: IC50 = 3700 nM (pChEMBL 5.429999828338623)
Descriptors: MW 549.43, ALogP 4.69, HBA 7, HBD 1, PSA 88.83, QED 0.46
Compound (SMILES): Cc1nc2c(C)cc(-c3[nH]c4ccc(C5CCN(C(=O)CN(C)C)CC5)cc4c3C(C)C)cn2n1
InChIKey: HLIRIKSXLBXUDR-UHFFFAOYSA-N
Target: Toll-like receptor 7 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9NYK1
Assay: TLR7/8/9 Inhibition Reporter Assay: HEK-Blue-cells (Invivogen) overexpressing human TLR7, TLR8 or TLR9 receptors were used for screening inhibitors of these receptors using an inducible SEAP (secreted embryonic alkaline phosphatase) reporter gene under the control of the IFN-β minimal promoter fused to five NF-κB and AP-1-binding sites. Briefly, cells are seeded into Greiner 384 well plates (15000 cells per well for TLR7, 20,000 for TLR8 and 25,000 for TLR9) and then treated with test compounds in DMSO to yield a final dose response concentration range of 0.05 nM-50 μM. After a 30 minute compound pre-treatment at room temperature, the cells are then stimulated with a TLR7 ligand (gardiquimod at a final concentration of 7.5 μM), TLR8 ligand (R848 at a final concentration of 15.9 μM) or TLR9 ligand (ODN2006 at a final concentration of 5 nM) to activate NF-κB and AP-1 which induce the production of SEAP. After a 22 hour incubation at 37° C., 5% CO2, SEAP levels are determined with the addition of HEK-Blue Detection reagent (Invivogen), a cell culture medium that allows for detection of SEAP, according to manufacturer's specifications. The percent inhibition is determined as the % reduction in the HEK-Blue signal present in wells treated with agonist plus DMSO alone compared to wells treated with a known inhibitor.
Measurement: IC50 = 1.1 nM (pChEMBL 8.960000038146973)
Descriptors: MW 472.64, ALogP 4.89, HBA 5, HBD 1, PSA 69.53, QED 0.45
Compound (SMILES): CCOc1cc(Nc2nc(=O)c(OC)cn2Cc2ccccc2)c(C)cc1C
InChIKey: XPXRNSYVBKAMRM-UHFFFAOYSA-N
Target: P2X purinoceptor 7 (SINGLE PROTEIN), Homo sapiens
UniProt: Q99572
Assay: Evaluation of a Human P2X7 Receptor Inhibitory Activity: Stably expressing cell line (1321N1 cell transfected with the human P2X7 receptor gene (GenBank accession number NM_002562.5 including T606C and G952A SNP)) was used. The cells were seeded in a 384-well microtiter plate at a concentration of 8000 cells/well and cultured in the medium (10% fetal bovine serum, 25 mM HEPES, 1% penicillin and streptomycin in DMEM) for one day at 37° C. under 5% carbon dioxide atmosphere. After replacing with 20 μL of the HBSS buffer (20 mM HEPES, 55.6 mM D-glucose, 1×HBSS(−), pH7.4-7.5), 15 μL of 17.3 μM Yo-Pro solution in the HBSS buffer was added. The plate was placed in high-throughput cellular screening system FLIPR TETRA (Molecullar Devices, LLC.) and 15 μL of 130 μM BzATP solution in the HBSS buffer was added. Measurement of fluorescence intensity by FLIPR TETRA was started. After eight minutes, 15 μL of DMSO solutions containing different concentrations of the compound of the present invention as prepared by dilution with the HBSS buffer were dispensed to each well through the built-in automatic dispenser, and the measurement of fluorescence intensity was continued for 20 minutes. The maximum fluorescence intensity without the compound of the present invention is calculated as 0% inhibition and the maximum fluorescence intensity when the reference compound was added is calculated as 100% inhibition. Changing values of fluorescence intensity by the compound of the present invention were calculated by difference between maximum and minimum fluorescence intensity for 20 minutes. IC50 was calculated using logistic approximation.
Measurement: IC50 < 10 nM (pChEMBL 8)
Descriptors: MW 379.46, ALogP 4.06, HBA 6, HBD 1, PSA 65.38, QED 0.67
Compound (SMILES): CN(C)/C=C(/Oc1ccc(Cl)nc1)C(=O)c1ccccc1Cl
InChIKey: DKSBPSFRMXMCGH-GXDHUFHOSA-N
Target: Cytochrome P450 11B2, mitochondrial (SINGLE PROTEIN), Homo sapiens
UniProt: P19099
Assay: Inhibition Assay: G-402 cells can be obtained from ATCC (CRL-1440) and were originally derived from a renal leiomyoblastoma.The expression plasmids contains the ORF for either human/cyno CYP11B1 or CYP11B2 under the control of a suitable promoter (CMV-promoter) and a suitable resistance marker (neomycin). Using standard techniques the expression plasmid is transfected into G-402 cells and these cells are then selected for expressing the given resistance markers. Individual cell-clones are then selected and assessed for displaying the desired enzymatic activity using 11-Deoxycorticosterone (Cyp11B2) or 11-Deoxycortisol (Cyp11B1) as a substrate.G-402 cells expressing CYP11 constructs were established as described above and maintained in McCoy's 5a Medium Modified, ATCC Catalog No. 30-2007 containing 10% FCS and 400 μg/ml G418 (Geneticin) at 37° C. under an atmosphere of 5% CO2/95% air. Cellular enzyme assays were performed in DMEM/F12 medium containing 2.5% charcoal treated FCS and appropriate concentration of substrate (0.3-10 uM 11-Deoxycorticosterone, 11-Deoxycortisol or Corticosterone). For assaying enzymatic activity, cells were plated onto 96 well plates and incubated for 16 h. An aliquot of the supernatant is then transferred and analyzed for the concentration of the expected product (Aldosterone for CYP11B2; Cortisol for CYP11B1). The concentrations of these steroids can be determined using HTRF assays from CisBio analyzing either Aldosterone or Cortisol.
Measurement: EC50 = 0.113 nM (pChEMBL 9.949999809265137)
Descriptors: MW 337.21, ALogP 4.05, HBA 4, HBD 0, PSA 42.43, QED 0.36
Compound (SMILES): Cc1ccc(C#Cc2ccccn2)cc1-c1ccc2nc(NC(=O)[C@@H]3C[C@@H]3F)sc2c1
InChIKey: ILSITRXXAPFTEA-RTWAWAEBSA-N
Target: Tyrosine-protein kinase ABL1 (SINGLE PROTEIN), Homo sapiens
UniProt: P00519
Assay: c-abl Kinase Assay: ADP-Glo assay kit was purchased from Promega. Magnesium chloride (MgCl2), bovine serum albumin (BSA), ethylene glycol-bis(β-aminoethyl ether)-N,N′,N′-tetraacetic acid (EGTA), tween-20, 1,4-dithiothreitol (DTT) and dimethyl sulfoxide (DMSO) were purchased from Sigma-Aldrich. HEPES buffer was purchased from Gibco. ABL1 kinase and Abltide were purchased from Signalchem.c-abl kinase activity was measured by Promega's ADP-Glo Assay. In this assay, His-tagged recombinant human ABL1 (0.25 ng/μl) is incubated with 5 μL of compounds (0.5% DMSO), 5 μL of Abltide (0.01 μg/μl) and 5 μL of ATP (25 μM) in buffer (50 mM HEPES, 7.5; 10 mM MgCl2; 1 mM EGTA; 0.05% BSA; 0.01% Tween-20; 2 mM DTT). The assay was started by incubating the reaction mixture in a 96-well plate at 30° C. for 30-min. After the incubation, 25 μL ADP-Glo reagent was added and the reaction was incubated at room temperature for 40-min to stop the reaction and degrade residual ATP. The ADP product was then converted to ATP by adding 50 μL per well of detection reagent. Luminescence was detected after 30-min room temperature incubation with the Molecular device I3X plate reader. The IC50 values were calculated from a series of percent inhibition values determined at a range of inhibitor concentration using software routines as implemented in the GraphPad Prism 7 software or SigmaPlot 13.0.
Measurement: IC50 = 172 nM (pChEMBL 6.760000228881836)
Descriptors: MW 427.5, ALogP 5.36, HBA 4, HBD 1, PSA 54.88, QED 0.45
Compound (SMILES): CC(=O)c1nn(CC(=O)N2[C@H]3C[C@]4(C[C@@H]24)CN(C)CCCC/C=C\Cc2ccc(Br)nc2NC3=O)c2c(CF)cc(-c3cnc(C)nc3)cc12
InChIKey: BUFDIZPLGBOIQS-RAVXFVBSSA-N
Target: Complement factor D (SINGLE PROTEIN), Homo sapiens
UniProt: P00746
Assay: Human Factor D Assay: Human Factor D (purified from human serum, Complement Technology, Inc.) at 80 nM final concentration is incubated with test compound at various concentrations for 5 min at room temperature in 50 mM Tris, 1M NaCl, pH 7.5. A synthetic substrate Z-L-Lys-SBzl and DTNB (Ellman's reagent) are added to final concentrations of 100 M each. Absorbance at 405 nm (A405) is recorded at 30 second intervals for 30 minutes using a microplate spectrophotometer. IC50 values are calculated by nonlinear regression of Complement Factor D reaction rates as a function of test compound concentration.
Measurement: IC50 < 1000 nM (pChEMBL 6)
Descriptors: MW 743.68, ALogP 5.84, HBA 9, HBD 1, PSA 126.21, QED 0.15
Compound (SMILES): O=C(CC1CCN(c2ccc(Cl)cn2)CC1)Nc1ccc(O)cn1
InChIKey: PBSOFGLNTMFLKC-UHFFFAOYSA-N
Target: Sphingolipid delta(4)-desaturase DES1 (SINGLE PROTEIN), Homo sapiens
UniProt: O15121
Assay: Des1 Activity Assays: Jurkat clone E6-1 cells were grown and then seeded at 106 cells/mL in a 96-well plate (400 μL in each well. The cells were administered 100 μL of cell culture media containing 50-μM NBD-C6-dihydroceramide (Des1 substrate), affording a final concentration of substrate of 10 μM. The cells were incubated with substrate at 4° C. for 30 minutes. Following the incubation at 4° C., the cell suspension was centrifuged at 1200 rpm for 3 minutes, and the cell pellet is resuspended in 400 μL of fresh media containing various concentrations of either fenretinide (known Des1 inhibitor control compound) or test article. The final concentrations of control compound and test compounds were tested in a range from 0-10 μM. The cells and compounds were incubated at 37° C. for 3 hours. Following the 3-hour incubation, the plate was centrifuged at 2500 g for 3 minutes at 4° C., followed by collection and transfer of 200 μL of the supernatant to a new 96-well plate with 300 μL of methanol an containing appropriate internal standards for liquid chromatography/tandem mass spectroscopy (LC/MS/MS) analysis (internal standard: 500 nM labetalol and 100 nM alprazolam). The samples were vortexed for 2 minutes followed by centrifugation at 3,220 g for 20 minutes. Following centrifugation, 200 μL of the supernatant was transferred to a new 96-well plate for LC-MS/MS analysis to determine the amount of NBD-C6-ceramide (Des1 product) produced. The assay was typically performed in duplicate. A reduction of at least 30% compared to vehicle control (0 μM test article) is indicative of an active compound, and a reduction of 75% compared to vehicle control is preferred.
Measurement: IC50 = 3.9 nM (pChEMBL 8.40999984741211)
Descriptors: MW 346.82, ALogP 3.08, HBA 5, HBD 2, PSA 78.35, QED 0.89
Compound (SMILES): Cc1ccc(CNC(=O)C2CN(c3ncnn4cc(-c5ccc(C(O)CO)cc5)cc34)C2)cc1
InChIKey: VSVMYESAENJCRO-UHFFFAOYSA-N
Target: Mast/stem cell growth factor receptor Kit (SINGLE PROTEIN), Homo sapiens
UniProt: P10721
Assay: Kit Wild Type Assay: In each well of a 384-well plate, 0.2 ng/ul final (2 nM) of wild type 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 400 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 35s).
Measurement: IC50 < 1 nM (pChEMBL 9)
Descriptors: MW 457.53, ALogP 2.48, HBA 7, HBD 3, PSA 102.99, QED 0.39
Compound (SMILES): C=CC(=O)Nc1cnc(C(=O)N(C)[C@H](C(=O)N[C@H]2Cc3cccc(c3)-c3ccc4c(c3)c(c(-c3cccnc3C(C)OC)n4CC)CC(C)(C)COC(=O)[C@@H]3CCCN(N3)C2=O)C(C)C)n1C
InChIKey: CSQMNMAPLGEGOZ-DVBKWRDNSA-N
Target: GTPase KRas (SINGLE PROTEIN), Homo sapiens
UniProt: P01116
Assay: Biochemical Assay: A variety of Ras proteins may be inhibited by compounds of the present invention (e.g., K-Ras, N-Ras, H-Ras, and mutants thereof at positions 12, 13 and 61, such as G12C, G12D, G12V, G12S, G13C, G13D, and Q61L, and others described herein). The purpose of this biochemical assay is to measure the ability of test compounds to covalently label nucleotide-loaded K-Ras isoforms. In assay buffer containing 12.5 mM HEPES pH 7.4, 75 mM NaCl, 1 mM MgCl2, 1 mM BME, 5 uM Cyclophilin A and 2 uM test compound, a 5 uM stock of GMP-PNP-loaded K-Ras (1-169) G12C is diluted 10-fold to yield a final concentration of 0.5 uM; with final sample volume being 100 uL. The sample is incubated at 25�� C. for a time period of up to 24 hours prior to quenching by the addition of 10 uL of 5% Formic Acid. Quenched samples are centrifuged at 15000 rpm for 15 minutes in a benchtop centrifuge before injecting a 10 uL aliquot onto a reverse phase C4 column and eluting into the mass spectrometer with an increasing acetonitrile gradient in the mobile phase. Analysis of raw data may be carried out using Waters MassLynx MS software, with % bound calculated from the deconvoluted protein peaks for labeled and unlabeled K-Ras.
Measurement: IC50 = 5500 nM (pChEMBL 5.260000228881836)
Descriptors: MW 914.12, ALogP 6.4, HBA 12, HBD 3, PSA 182.02, QED 0.1
Compound (SMILES): Cc1ccc(C(CC(=O)O)c2ccn(C)c(=O)c2)cc1CN1C[C@@H](C)Oc2ncccc2S1(=O)=O
InChIKey: XXWNAIROFABZMP-OQHSHRKDSA-N
Target: Nuclear factor erythroid 2-related factor 2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q16236
Assay: BEAS-2B NQO1 MTT Assay: NAD(P)H:quinone oxidoreductase 1 (NQO1), also called DT diaphorase, is a homodimeric FAD-containing enzyme that catalyzes obligatory NAD(P)H-dependent two-electron reductions of quinones and protects cells against the toxic and neoplastic effects of free radicals and reactive oxygen species arising from one-electron reductions. The transcription of NQO1 is finely regulated by Nrf2, and thus NQO1 activity is a good marker for Nrf2 activation. On day one, frozen BEAS-2B cells (ATCC) are thawed in a water bath, counted, and re-suspended at a concentration of 250,000 cells/mL. Fifty microliters of cells are plated in 384 well black clear-bottomed plates. Plates are incubated at 37° C., 5% CO2 overnight. On day two, plates are centrifuged and 50 nL of compound or controls are added to the cells. Plates are then incubated at 37° C., 5% CO2 for 48 h. On day four, medium is aspirated from the plate and crude cell lysates are made by adding 13 μL of 1× Cell Signaling Technologies lysis buffer with 1 Complete, Mini, EDTA-free Protease Inhibitor Tablet (Roche) for each 10 mL of lysis buffer. After lysis plates are incubated for 20 min at room temperature. Two microliters of lysate are removed for use in Cell Titer Glo assay (Promega) and MTT cocktail is prepared (Prochaska et. al. 1998) for measurement of NQO1 activity. Fifty microliters of MTT cocktail is added to each well, plate is centrifuged, and analyzed on an Envision plate reader (Perkin Elmer) using Absorbance 570 nm label for 30 min. Product formation is measured kinetically and the pEC50 of NQO1 specific activity induction is calculated by plotting the change in absorbance (Delta OD/min) versus the log of compound concentration followed by 3-parameter fitting.
Measurement: EC50 = 55000 nM (pChEMBL 4.260000228881836)
Descriptors: MW 497.57, ALogP 2.67, HBA 7, HBD 1, PSA 118.8, QED 0.56
Compound (SMILES): CN(C(=O)c1cc(-c2cc3c(cc2C(=O)N2Cc4ccccc4C[C@H]2CN2CCOCC2)CNCC3)n2c1CCCC2)c1ccc(O)cc1
InChIKey: GEPQHKOIEXNXGJ-YTTGMZPUSA-N
Target: Apoptosis regulator Bcl-2 (SINGLE PROTEIN), Homo sapiens
UniProt: P10415
Assay: Fluorescence Polarisation Assay: The fluorescence polarisation tests were carried out on microplates (384 wells). The Bcl-2 protein, labelled (histag-Bcl-2 such that Bcl-2 corresponds to the UniProtKB® primary accession number: P10415), at a final concentration of 2.50×10−8 M, is mixed with a fluorescent peptide (Fluorescein-REIGAQLRRMADDLNAQY), at a final concentration of 1.00×10−8 M in a buffer solution (Hepes 10 mM, NaCl 150 mM, Tween20 0.05%, pH 7.4), in the presence or in the absence of increasing concentrations of test compounds. After incubation for 2 hours, the fluorescence polarisation is measured.
Measurement: IC50 = 397 nM (pChEMBL 6.400000095367432)
Descriptors: MW 659.83, ALogP 5.02, HBA 7, HBD 2, PSA 90.28, QED 0.3