lovingscience/chembl-v37-raw-activities huggingface dataset:
Compound (SMILES): COc1cc(-c2cccc(-c3cccc(Nc4nccc5nccnc45)c3C)c2C)cc(F)c1CNCC(N)=O
InChIKey: LQHRTBDUAXRALS-UHFFFAOYSA-N
Target: Programmed cell death 1 ligand 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9NZQ7
Assay: Enzyme-Linked Immunosorbent AssayELISA: 96 Well plates were coated with 111 g/mL of human PD-L1 (obtained from R&D) in PBS overnight at 4° C. The wells were then blocked with 2% BSA in PBS (W/V) with 0.05% TWEEN-20 for 1 hour at 37° C. The plates were washed 3 times with PBS/0.05% TWEEN-20 and the compounds were serial diluted (1:5) in dilution medium and added to the ELISA plates. Human PD-1 and biotin 0.31 μg/mL (ACRO Biosystems) were added and incubated for 1 hour at 37° C. then washed 3 times with PBS/0.05% TWEEN-20. A second block was performed with 2% BSA in PBS (W/V)/0.05% TWEEN-20 for 10 min at 37° C. and the plates were washed 3 times with PBS/0.05% TWEEN-20. Streptavidin-HRP was added for 1 hour at 37° C. then the plates were washed 3 times with PBS/0.05% TWEEN-20. TMB substrate was added and reacted for 20 min at 37° C. A stop solution (2 N aqueous H2SO4) was added. The absorbance was read at 450 nm using a micro-plate spectrophotometer.
Measurement: IC50 = 505 nM (pChEMBL 6.300000190734863)
Descriptors: MW 536.61, ALogP 5.44, HBA 7, HBD 3, PSA 115.05, QED 0.23
Compound (SMILES): N#Cc1ccnc(-c2ccc3ncc(-c4cc(F)cc(F)c4)c(N4CC(C5COCCN5)C4)c3c2)c1N
InChIKey: ZXRWXLRFGIQEMI-UHFFFAOYSA-N
Target: Somatostatin receptor type 2 (SINGLE PROTEIN), Homo sapiens
UniProt: P30874
Assay: cAMP Assay: Four days prior to the assay, 5,000 Chinese hamster ovary cells (CHO-K1, ATCC # CCL-61) stably expressing the human SSTR2 are plated in each well of a 96-well tissue culture-treated plate in Ham's F12 growth media (ThermoFisher #10-080-CM) supplemented with 10% donor bovine serum (Gemini Bio-Products #100-506), 100 U/mL penicillin; 100 ug/mL streptomycin; 2 mM L-glutamine (Gemini Bio-Products #400-110) and 0.2 mg/mL hygromycin B (GoldBio #31282-04-9). The cells are cultured at 37° C., 5% CO2 and 95% humidity. On the day of the assay, the media is aspirated and the cells are treated with 50 μL of 1.6 μM NKH477 (Sigma # N3290) plus various dilutions of compounds of the invention in assay buffer [1× Hank's Balanced Salt Solution (ThermoFisher # SH3058802), 0.5 mM HEPES pH 7.4, 0.1% bovine serum albumin, 0.2 mM 3-Isobutyl-1-methylxanthine (IBMX, VWR #200002-790)]. The cells are incubated for 20 minutes at 37° C. (the final concentration of the compounds of the invention are typically 0-10,000 nM). The cells are treated with 50 μL of lysis buffer (HRTF cAMP kit, Cisbio). The lysate is transferred to 384-well plates and cAMP detection and visualization antibodies are added and incubated for 1-24 hours at room temperature. The time-resolved fluorescent signal is read with a Tecan M1000Pro multiplate reader.
Measurement: EC50 < 10 nM (pChEMBL 8)
Descriptors: MW 498.54, ALogP 4.12, HBA 7, HBD 2, PSA 100.09, QED 0.44
Compound (SMILES): O=C(N[C@@H]1CNCC[C@H]1O)c1sc2nccc3c2c1NC(=O)N3c1ccc(Oc2ccccc2)cc1
InChIKey: WVMIHTVLUJYHRV-UYAOXDASSA-N
Target: Tyrosine-protein kinase BTK (SINGLE PROTEIN), Homo sapiens
UniProt: Q06187
Assay: TBD: TBD
Measurement: IC50 = 251 nM (pChEMBL 6.599999904632568)
Descriptors: MW 501.57, ALogP 4.22, HBA 7, HBD 4, PSA 115.82, QED 0.33
Compound (SMILES): COc1cccc(-c2nnc(NS(=O)(=O)[C@@H](C)[C@H](OC)c3ncc(Cl)cn3)n2CC2CCOCC2)n1
InChIKey: BJOPCAOFTLNFLO-LIRRHRJNSA-N
Target: Apelin receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P35414
Assay: GTPγS Binding: The human APJ receptor was cloned by polymerase chain reaction and the gene encoding the receptor was subcloned in pFLAG-CMV-3 expression vector (Sigma, Saint Louis, Mo. USA) in-house at Amgen. A GTPγS binding assay was performed on membranes prepared from CHO cells stably expressing human APJ receptor. The optimum experimental conditions for the concentrations of GDP, MgCl2, and NaCl in the assay buffer were initially determined. The assay was performed in 9 μL assay buffer [20 mM HEPES, pH 7.5, 5 mM MgCl2, 100 mM NaCl and 0.1% (w/v) BSA], 1 μL of diluted test compound (starting with 0.75 mM, 2-fold serial dilution with DMSO, total 22 points), 10 μL of 18 μM GDP (final concentration of 3 μM GDP), 20 μL of 0.25 μg/mL membrane protein expressing human APJ receptor captured with WGA PS beads (final concentration of 5 μg per well), and 20 μL of 0.3 nM [35S]GTPγS (final concentration is 0.1 nM [35S]GTPγS)(Perkin Elmer Life and Analytical Sciences, Waltham USA). One column of the plate was 1 μL of DMSO as background and another column of the plate was 1 μL of 180 μM Pyr-Apelin-13 which was used as control at a final concentration of 3 μM. Incubation was at RT for 90 min and the microplate was read using a ViewLux ultra HTS Microplate Imager.
Measurement: EC50 = 53 nM (pChEMBL 7.28000020980835)
Descriptors: MW 538.03, ALogP 2.74, HBA 11, HBD 1, PSA 143.24, QED 0.41
Compound (SMILES): O=Cc1c(O)ccc2oc(C(=O)N3CCOCC3)cc12
InChIKey: PBBJJDBXYPPCSK-UHFFFAOYSA-N
Target: Serine/threonine-protein kinase/endoribonuclease IRE1 (SINGLE PROTEIN), Homo sapiens
UniProt: O75460
Assay: IRE-1alpha Assay: A fusion protein comprising glutathione S transferase (GST) and human IRE-1α (GST-IRE-1α) obtained from a 500 ml baculovirus-infected insect cell culture can be used to measure IRE-1α activity in vitro. Five μl of a reaction mixture comprising 1× reaction buffer (5× reaction buffer is 100 mM Hepes pH 7.5, 250 mM KOAc, 2.5 mM MgCl2), 3 mM DTT, and 0.4% polyethylene glycol water is added to each well of 384 well plates. Twenty-five nanoliters of a 1 mM test compound solution are added to test wells. Three μl of a 128 ng/ml IRE-1α preparation are added to each test well and to positive control wells (final concentration 5.82 ng/well). Negative control wells contain only reaction mixture and test compound. After spinning the plates at 1200 rpm for 30 seconds, 3 μl of an IRE-1α human mini-XBP-1 mRNA stem-loop substrate 5'-CAGUCCGCAGCACUG-3' (SEQ ID NO:1), labeled with the fluorescent dye Cy5 at the 5' end and Black Hole Quencher 2 (BH2) at the 3' end, are added to each well of a control plate. The plates are again spun at 1200 rpm for 30 seconds. Final concentrations for the assay are: 63 nM IRE-1α substrate, 5.82 ng IRE-1α protein, and 2.5 μM test compound. The plates are covered with lids and incubated for one hour at 30° C. The plates are then transferred to an ACQUEST™ microplate reader. Data is analyzed using data analysis software, and the percent activity of IRE-1α is calculated.
Measurement: EC50 > 10000 nM (pChEMBL 5)
Descriptors: MW 275.26, ALogP 1.42, HBA 5, HBD 1, PSA 79.98, QED 0.84
Compound (SMILES): CC(C)c1cc(C(=O)N2CC[C@@H](NC(=O)[C@H]3C[C@@H]3F)C2)[nH]n1
InChIKey: LXGAOGLHQMPZIY-VWYCJHECSA-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 = 0.33 nM (pChEMBL 9.479999542236328)
Descriptors: MW 308.36, ALogP 1.22, HBA 3, HBD 2, PSA 78.09, QED 0.88
Compound (SMILES): CCc1cnc(N2C(=O)N(c3c(F)c(OC)cc(OC)c3F)Cc3cnc(-c4cn(C)nc4C)cc32)cn1
InChIKey: LYBWHFGDCBIJOZ-UHFFFAOYSA-N
Target: Fibroblast growth factor receptor 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P11362
Assay: FGFR Enzymatic Assay 1: FGFR1, FGFR2, and FGFR4 are measured under equivalent conditions with the following changes in enzyme and ATP concentrations: FGFR1, 0.02 nM and 210 uM respectively, FGFR2, 0.01 nM and 100 uM, respectively, and FGFR4, 0.04 nM and 600 uM respectively. The enzymes were purchased from Millipore or Invitrogen. GraphPad prism3 was used to analyze the data. The IC50 values were derived by fitting the data to the equation for a sigmoidal dose-response with a variable slope. Y=Bottom+(Top−Bottom)/(1+10^((Log IC50−X)*HillSlope)) where X is the logarithm of concentration and Y is the response. Compounds having an IC50 of 1 μM or less are considered active. The compounds of the invention were found to be selective inhibitors of FGFR3 and/or FGFR4 according to the FGFR Enzymatic Assays. Compounds of Formula (I′) and (I) and all the compounds as described herein have been tested and exhibit an IC50 of less than 1 μM. Table 1 provides IC50 data for compounds of the invention assayed in the FGFR Enzymatic Assay after dilution in assay buffer, added to the plate and pre-incubated for 4 hours.
Measurement: IC50 = 115 nM (pChEMBL 6.940000057220459)
Descriptors: MW 521.53, ALogP 4.72, HBA 8, HBD 0, PSA 98.5, QED 0.36
Compound (SMILES): COc1cc2[nH]c(=O)sc2cc1Nc1ncnc2sc(C(=O)N(C)C)nc12
InChIKey: WHXOJFLHDLEWEL-UHFFFAOYSA-N
Target: MAP kinase-interacting serine/threonine-protein kinase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9BUB5
Assay: MKNK1 Kinase High ATP 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 MgCl2, 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, 3.3 mM=>final conc. in the 5 μL assay volume is 2 mM) 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 30 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 0.003 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. Therefore, the fluorescence emissions at 620 nm and 665 nm after excitation at 350 nm were measured in a TR-FRET reader, e.g. a Rubystar (BMG Labtechnologies, Offenburg, Germany) or a Viewlux (Perkin-Elmer). The ratio of the emissions at 665 nm and at 622 nm was taken as the measure for the amount of phosphorylated substrate.
Measurement: IC50 = 1080 nM (pChEMBL 5.96999979019165)
Descriptors: MW 402.46, ALogP 2.44, HBA 9, HBD 2, PSA 113.1, QED 0.54
Compound (SMILES): COc1ccc(F)cc1-c1ccnc2[nH]c(C3=CCC(NS(C)(=O)=O)CC3)cc12
InChIKey: BHVPGKAVDVPYPP-UHFFFAOYSA-N
Target: CDK9/cyclin T1 (PROTEIN COMPLEX), Homo sapiens
UniProt: P50750
Assay: LANCE ULight TR-FRET Kinase Assay: CDK9 enzyme activities were measured using LANCE ULight TR-FRET kinase assay reagents (PerkinElmer, Waltham, Mass.). Compounds were directly added in 100% DMSO to white low volume assay plates (Perkin Elmer Proxiplate 6008289) using a Labcyte Echo acoustic dispenser. Assay reagents in serine/threonine kinase assay buffer containing 20 mM HEPES, 10 mM MgCl2, 100 mM Na3VO4, and 0.0075% Triton X-100. were added for final reaction mixture concentrations of 1000 μM ATP, 100 nM U-light MBP peptide (Perkin Elmer TRF0109M) and reaction initiated with 4 nM CDK9/Cyclin T1 (Carna Biosciences 04-110). The kinase reaction was carried out for 30 minutes before addition of stopping buffer to a final of 20 mM EDTA and 0.5 nM of LANCE Ultra Europium anti-phospho-MBP antibody (PerkinElmer TRF0201M) in LANCE detection buffer (PerkinElmer CR97-100). The reaction was equilibrated for 1 hour and the signal read in the Perkin Elmer Envision in TR-FRET mode (excitation at 320 nm and emission at 615/665 nm).
Measurement: IC50 = 54 nM (pChEMBL 7.269999980926514)
Descriptors: MW 415.49, ALogP 3.86, HBA 4, HBD 2, PSA 84.08, QED 0.66
Compound (SMILES): Cc1ccc(C(=O)N(C)C)cc1-c1cc2cc(NC(=O)[C@H]3C[C@@H]3c3cnn(C)c3)ncc2c(N)n1
InChIKey: YLKSSXZZMDIBLX-UXHICEINSA-N
Target: Mitogen-activated protein kinase kinase kinase kinase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q92918
Assay: Binding Assay: Lanth: 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 = 3.36 nM (pChEMBL 8.470000267028809)
Descriptors: MW 469.55, ALogP 3.36, HBA 7, HBD 2, PSA 119.03, QED 0.46
Compound (SMILES): COc1cc(O[C@@H]2CCN(C(C)C)C2)ccc1-c1cc(Nc2cnc(C#N)cn2)n[nH]1
InChIKey: QHUFSBBIIKKNHZ-QGZVFWFLSA-N
Target: Serine/threonine-protein kinase Chk1 (SINGLE PROTEIN), Homo sapiens
UniProt: O14757
Assay: Kinase Inhibiting Activity Assay: Chk-1 Kinase Inhibiting ActivityThe compounds of the invention were tested for activity against Chk-1 kinase using the materials and protocols set out below.Reaction Buffer:Base Reaction buffer: 20 mM Hepes (pH 7.5), 10 mM MgCl2, 1 mM EGTA, 0.02% Brij35, 0.02 mg/ml BSA, 0.1 mM Na3VO4, 2 mM DTT, 1% DMSO*Required cofactors are added individually to each kinase reactionReaction Procedure:(i) Prepare indicated substrate in freshly prepared Base Reaction Buffer(ii) Deliver any required cofactors to the substrate solution above(iii) Deliver indicated kinase into the substrate solution and gently mix(iv) Deliver compounds in DMSO into the kinase reaction mixture(v) Deliver 33P-ATP (specific activity 0.01 μCi/μl final) into the reaction mixture to initiate the reaction.(vi) Incubate kinase reaction for 120 minutes at room temperature(vii) Reactions are spotted onto P81 ion exchange paper (Whatman #3698-915)(viii) Wash filters extensively in 0.1% phosphoric acid.(ix) Dry filters and measure counts in scintillation counterKinase Information:CHK-1 Genbank Accession # AF016582Recombinant full length construct, N-terminal GST tagged, purified from insect cells.No special measures were taken to activate this kinase.Final concentration in assay=0.5 nMSubstrate: CHKtidePeptide sequence: [KKKVSRSGLYRSPSMPENLNRPR]Final concentration in assay=20 μM
Measurement: IC50 = 0.15 nM (pChEMBL 9.819999694824219)
Descriptors: MW 419.49, ALogP 3.35, HBA 8, HBD 2, PSA 111.98, QED 0.6
Compound (SMILES): OCC1O[C@H](Sc2cccc(OC(F)(F)F)c2)C(O)[C@@H](n2cc(-c3cc(F)c(F)c(F)c3)nn2)[C@H]1O
InChIKey: KAXIWYDLBASFRY-LOVNCYQGSA-N
Target: Galectin-3 (SINGLE PROTEIN), Homo sapiens
UniProt: P17931
Assay: fluorescence anisotropy assay : The affinity of Example 1-106 for galectins were determined by a fluorescence anisotropy assay where the compound was used as an inhibitor of the interaction between galectin and a fluorescein tagged saccharide probe as described Sörme, P., Kahl-Knutsson, B., Huflejt, M., Nilsson, U. J., and Leffler H. (2004) Fluorescence polarization as an analytical tool to evaluate galectin-ligand interactions. Anal. Biochem. 334: 36-47, (Sörme et al., 2004) and Monovalent interactions of Galectin-1 By Salomonsson, Emma; Larumbe, Amaia; Tejler, Johan; Tullberg, Erik; Rydberg, Hanna; Sundin, Anders; Khabut, Areej; Frejd, Torbjorn; Lobsanov, Yuri D.; Rini, James M.; et al, From Biochemistry (2010), 49(44), 9518-9532, (Salomonsson et al., 2010). The assay was also adapted to be able to measure the high affinity of compounds for galectin-3 by using the below probe constructed to have high affinity for galectin-3 which made it possible to use a low concentration of galectin-3 (50 nM). 100 nM albumin was included as a carrier to prevent protein loss at such low concentration of galectin.
Measurement: Kd = 500 nM (pChEMBL 6.300000190734863)
Descriptors: MW 537.44, ALogP 3.03, HBA 9, HBD 3, PSA 109.86, QED 0.33
Compound (SMILES): CNc1ccc(-c2cc(NC)n3ncc(C(=O)NC4CCC4(F)F)c3n2)n2ccnc12
InChIKey: CJGPSWAGRQVISJ-UHFFFAOYSA-N
Target: Non-receptor tyrosine-protein kinase TYK2 (SINGLE PROTEIN), Homo sapiens
UniProt: P29597
Assay: TYK2 JH2 Domain Binding Assay: The binding reaction was assembled by combining 16 μl of DNA-tagged kinase extract, 3.8 μl liganded affinity beads, and 0.18 μl test compound (PBS/0.05% Tween 20/10 mM DTT/0.1% BSA/2 μg/ml sonicated salmon sperm DNA)]. Extracts were used directly in binding assays without any enzyme purification steps at a ≥10,000-fold overall stock dilution (final DNA-tagged enzyme concentration <0.1 nM). Extracts were loaded with DNA-tag and diluted into the binding reaction in a two step process. First extracts were diluted 1:100 in 1× binding buffer (PBS/0.05% Tween 20/10 mM DTT/0.1% BSA/2 μg/ml sonicated salmon sperm DNA) containing 10 nM DNA-tag. This dilution was allowed to equilibrate at room temperature for 15 minutes and then subsequently diluted 1:100 in 1× binding buffer. Test compounds were prepared as 111× stocks in 100% DMSO. Kds were determined using an 11-point 3-fold compound dilution series with three DMSO control points. All compounds for Kd measurements are distributed by acoustic transfer (non-contact dispensing) in 100% DMSO
Measurement: Kd < 0.2 nM (pChEMBL 9.699000358581543)
Descriptors: MW 426.43, ALogP 2.65, HBA 8, HBD 3, PSA 100.65, QED 0.45
Compound (SMILES): COc1cc(-c2[nH]c3ccc(C4CCC(N5CCC(F)C5)CC4)nc3c2C(C)C)cn2ncnc12
InChIKey: AHRRVLMWZSFVJS-UHFFFAOYSA-N
Target: Toll-like receptor 7 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9NYK1
Assay: TLR7/8/9 Inhibition Reporter Assays: 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 nM (pChEMBL 9)
Descriptors: MW 476.6, ALogP 5.47, HBA 6, HBD 1, PSA 71.34, QED 0.41
Compound (SMILES): CC#C[C@@H](CC(=O)O)c1ccc(OCc2ccc(CCN3Cc4ccccc4C3)cc2)cc1
InChIKey: XWZWUNOMCGYOQX-VWLOTQADSA-N
Target: Free fatty acid receptor 1 (SINGLE PROTEIN), Homo sapiens
UniProt: O14842
Assay: Activity Assay: The GPR40 protein activity according to the novel 3-(4-(benzyloxy)phenyl)hex-4-inoic acid derivative of the present invention was measured by investigating the changes in intracellular calcium concentration affected by the GRP40 activation. First, HEK-293 cells were transfected with human GPR40 DNA (Origene, RC218370) by using Fugene HD (Promega, E2311). The transfected HEK-293 cells were distributed in a 96-well black clear bottom floor plate (Costar, 3603), followed by culture. 24 hours later, the cell culture medium was replaced with Dulbecco's Modified Eagle Medium (DMEM, 50 μl/well) supplemented with 1% fetal bovine serum (FBS). To measure the calcium concentration, 50 μl of Fluo-4 reagent (Invitrogen, F10471) was added to each well, followed by culture in a 37° C. incubator for 2 hours. During the culture, the compounds of Examples and the compounds of Comparative Examples 1 and 2 were diluted with 1×HBSS (Hank's Buffered Salt Solution) containing 20 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer, resulting in the preparation of the samples for treating the cells. 2 hours after the culture began, those prepared samples were automatically injected to the cells by using Flexstation 3 (Molecular Devices). Then, intracellular calcium concentration was measured for 120 seconds by using SoftMax® Pro software. At this time, dimethylsulfoxide (DMSO) was injected to the cells for the non-treated group, followed by measuring the calcium concentration therein.
Measurement: EC50 > 300 nM (pChEMBL 6.5229997634887695)
Descriptors: MW 439.56, ALogP 5.41, HBA 3, HBD 1, PSA 49.77, QED 0.45
Compound (SMILES): COc1cccc2c(C(=O)NCc3cccc(COc4ccc(F)cc4)n3)nc(N)nc12
InChIKey: IDDCTUDFRLJCRE-UHFFFAOYSA-N
Target: Adenosine receptor A2a (SINGLE PROTEIN), Homo sapiens
UniProt: P29274
Assay: A2a Ki assay: Binding affinities of compounds of the invention for the human A2a receptor were determined in a competition binding assay using Scintillation Proximity technology. Thus, 1* μg, or preferably 0.25 μg of membranes from HEK293 cells expressing the human A2a receptor were incubated with a compound of the invention at concentrations ranging from 3000 nM to 0.15 nM in a reaction mixture containing also 0.5 nM of a tritiated form of 5-amino-7-[2-phenethyl]-2-(furan-2-yl)-7H-pyrazolo[4,3-e][1,2,4]triazolo[1,5-c]pyrimidine (the tritiated compound) and 25 μg of wheat germ agglutin-coated yttrium silicate SPA beads for one hour at room temperature with agitation. The beads were then allowed to settle to the bottom of the wells for 1 hr, after which the membrane-associated radioactivity was determined by scintillation counting in a TopCount microplate reader. Ki values were determined using the Cheng-Prusoff equation.
Measurement: Ki = 0.3 nM (pChEMBL 9.520000457763672)
Descriptors: MW 433.44, ALogP 3.26, HBA 7, HBD 2, PSA 112.25, QED 0.46
Compound (SMILES): CCc1cccnc1OCC(C)(C)C(=O)N[C@@H]1CCN(C)C1
InChIKey: IHQQZIZPRORHAM-CQSZACIVSA-N
Target: Somatostatin receptor type 4 (SINGLE PROTEIN), Homo sapiens
UniProt: P31391
Assay: Inhibition of Forskolin Stimulated cAMP in Cells Overexpressing SSTR4: This cell-based assay measures the ability of compounds to inhibit forskolin stimulated cAMP in CHO-K1 cells overexpressing SSTR4. CHO-K1 cells overexpressing SSTR4 (CHO-SSTR4) are purchased from DiscoveRx (product code 95-0059C2). The CHO-SSTR4 cells are maintained in F12K media with 10% Fetal Bovine Serum (Hyclone), 1% Pen/Strep (Life Technologies), and 800 μg/mL G418 (Life Technologies). To perform the assay, 3000 cells are plated per well in white 384-well plate (Corning 3570) in 50 μL complete media and the cells are allowed to attach for 16 hours in a 37° C., 5% CO2 incubator. The next day, the culture media is removed from the cells and the cells are washed (added then removed) with Kreb's Ringer Buffer (ZenBio, KRB-1000 mL). Test compounds are suspended in DMSO and diluted in stimulation buffer: Kreb's Ringer Buffer plus 0.5% BSA (Roche), 300 μM IBMX (Sigma), and 350 nM forskolin (Sigma). The cells are incubated in L compound/stimulation buffer for 30 minutes at room temperature. Cellular cAMP levels are detected with a HTRF LANCE Ultra cAMP kit (Perkin Elmer, catalog number TRF0264).The assay is performed in accordance with the manufacturer's instructions. Five μL of diluted Eu-W8044 labeled streptavidin (dilution: 1:50 in cAMP Detection Buffer) is added to each well. Then 5 μL of diluted biotin cAMP (dilution: 1:150 in cAMP Detection Buffer) is added to each well. The plates are covered and allowed to incubate for 60 minutes at room temperature on a shaker. HTRF (665 nm/615 nm) is read on a Perkin Elmer ENVISION plate reader. The pEC50 values are generated using Activity Base for Screening Data Management.
Measurement: EC50 = 50.1 nM (pChEMBL 7.300000190734863)
Descriptors: MW 305.42, ALogP 1.87, HBA 4, HBD 1, PSA 54.46, QED 0.87
Compound (SMILES): COc1ccc(Cn2c(=O)n3ncnc3c3c4c(sc32)N(Cc2ccc(Cl)cc2)CCC4)cc1OC
InChIKey: YSIHBQFXLASEFI-UHFFFAOYSA-N
Target: Dual specificity calcium/calmodulin-dependent 3',5'-cyclic nucleotide phosphodiesterase 1B (SINGLE PROTEIN), Homo sapiens
UniProt: Q01064
Assay: IMAP TR-FRET assay: The IMAP TR-FRET PDE assay was optimized for concentration of enzyme, Calmodulin, cAMP or cGMP substrate, DMSO tolerance, and incubation time. Into each well of a solid white 1536 well plate (Corning) was dispensed 250 pg full-length recombinant NH-terminal GST tagged human PDE1b enzyme (BPS Bioscience Cat #60011, San Diego, Calif.) in 2.5 μL IMAP BSA reaction buffer (Molecular Devices, Sunnyvale, Calif.) containing 10 U/mL Calmodulin and 2.5 mM CaCl2 (Sigma Aldrich.) After a brief centrifugation, 30 nL compound was added by transfer from 1 mM stock in DMSO using a Kalypsys 1536 Pintool. Plates were incubated for 5 minutes at room temperature before dispensing 1.5 μL of 533 nM 5-carboxy fluorescein (FAM)-labeled cAMP (Molecular Devices, Sunnyvale, Calif.) for a final concentration of 200 nM. After a brief centrifugation, the plates were incubated for 30 minutes at room temperature. The assay was terminated by adding 5 μL IMAP binding reagent/Tb complex (Molecular Devices, Sunnyvale, Calif.) to each well. Plates were incubated 1 hour at room temperature and read on a Viewlux multimode plate reader (Perkin Elmer). The instrument was set to excite using the DUG11 filter and measure using 490/10 nm and 520/10 nm filters.
Measurement: EC50 = 5500 nM (pChEMBL 5.260000228881836)
Descriptors: MW 522.03, ALogP 4.78, HBA 9, HBD 0, PSA 73.89, QED 0.32
Compound (SMILES): C[C@@H](Nc1nc(-c2noc(=O)[nH]2)nc2nc(N(C)C3CCCCC3)n(Cc3ccc(C(F)(F)F)cc3)c12)C1CCC1
InChIKey: BLPNHJFENAMQSE-MRXNPFEDSA-N
Target: E3 ubiquitin-protein ligase Mdm2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q00987
Assay: FRET Assay: An HDM2 FRET assay was developed to assess the compounds' inhibitory activity towards binding of p53 protein. A truncated version of HDM2 with residues 17 to 125 (containing p53 binding surface, Science (1994) 265, 346-355), with N-terminal His and Thioredoxin tag was generated in pET32a expression vector and expressed in E. coli strain BL21(DE3)Rosetta. Protein was purified using Ni-affinity chromatography, followed by size exclusion chromatography using Superdex 75 26/60 column. To assess inhibition of p53 binding to HDM2, a FITC labeled 8-mer peptide (sequence: Ac-Phe-Arg-Dpr-Ac6c-(6-Br)Trp-Glu-Glu-Leu-NH2; Anal Biochem. 2004 Aug. 1; 331(1):138-46) with strong affinity towards p53 binding pocket of HDM2 was used. The HDM2 assay buffer contained 1× Phosphate Buffered Saline (Invitrogen, Cat#14190), 0.01% BSA (Jackson ImmunoResearch, Cat#001-000-162), 0.01% Tween-20. In the 1× assay buffer recombinant HDM2 protein, peptide and Lumi4-Tb Cryptate-conjugate mouse anti-6×His antibody (cisbio, Cat# Tb61HISTLB) were added and transferred to ProxiPlate PLUS (PerkinElmer, Cat#6008269), containing compounds so that final DMSO concentration is 0.1%. Final concentrations of reagents in the assay wells are 0.5 nM HDM2, 0.25 nM anti HIS (Tb label) antibody and 3 nM peptide. After two hour incubation at room temperature in a humidified chamber plates were read on EnVision plate reader with the following settings: excitation: UV, 340 nM, two emission filters: 520 nm and 495 nm respectively. Ratio of em520/em495 was used to calculate % inhibition and to obtain IC50 with 4-parameter logistic equation.
Measurement: IC50 = 23.37 nM (pChEMBL 7.630000114440918)
Descriptors: MW 570.62, ALogP 5.61, HBA 9, HBD 2, PSA 117.76, QED 0.28
Compound (SMILES): Cc1n[nH]c(C)c1-c1cc2cc(Nc3cc4n(n3)CC(=O)N(C)CC4)ncc2c(N)c1F
InChIKey: ZNIXPMFKQLQSPL-UHFFFAOYSA-N
Target: Mitogen-activated protein kinase kinase kinase kinase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q92918
Assay: HPK1-FL HTRF Enzymatic Assay: To a 384 well Proxiplate with 80 nL compound or DMSO spotted on was added 4 μl/well kinase mix. The mixture was preincubated for 30 minutes and then 4 μl/well substrate mix was added. The solution was incubated for 60 min and then 4 μl/well detection mix was added. The solution was incubated for another 60 min. The plates were then loaded onto a Perkin Elmer Envision and the TR-FRET signal was measured at 615 and 665 nm. A ratio of 665/620 was used to calculate the % activity at each concentration of compound.
Measurement: Ki = 2.1 nM (pChEMBL 8.680000305175781)
Descriptors: MW 434.48, ALogP 2.92, HBA 7, HBD 3, PSA 117.75, QED 0.43
Compound (SMILES): COc1cc2ncnc(Oc3cccc(NC(=O)Nc4cc(C(C)(C)C)on4)c3)c2cc1OCCN1CCS(=O)(=O)CC1
InChIKey: OMNXUANWTOWGKV-UHFFFAOYSA-N
Target: Serine/threonine-protein kinase B-raf (SINGLE PROTEIN), Homo sapiens
UniProt: P15056
Assay: Competition Binding Assay: Competition binding assays used herein were developed, validated and performed as described in Fabian et al., Nature Biotechnology 2005, 23, 329-336. Kinases were produced as fusions to T7 phage (See, Fabian et al. or WO04/015142) or alternatively, the kinases were expressed in HEK-293 cells and subsequently tagged with DNA for PCR detection (See, WO08/005310). For the binding assays, streptavidin-coated magnetic beads were treated with biotinylated affinity ligands for 30 min at room temperature to generate affinity resins. The liganded beads were blocked with excess biotin and washed with blocking buffer (SeaBlock (Pierce), 1% BSA, 0.05% Tween 20, 1 mM DTT) to remove unbound ligand and to reduce non-specific binding. Binding reactions were assembled by combining kinase, liganded affinity beads, and test compounds in 1× binding buffer (20% SeaBlock, 0.17×PBS, 0.05% Tween 20, 6 mM DTT). Test compounds were prepared as 100× stocks in DMSO and rapidly diluted into the aqueous environment. DMSO was added to control assays lacking a test compound. Primary screen interactions were performed in polypropylene 384-well plates in a final volume of 34 μL, while Kd determinations were performed in polystyrene 96-well plates in a final volume of 135 μL. The assay plates were incubated at room temperature with shaking for 1 hour, long enough for binding reactions to reach equilibrium, and the affinity beads were washed extensively with wash buffer (1×PBS, 0.05% Tween 20) to remove unbound protein. The beads were then resuspended in elution buffer (1×PBS, 0.05% Tween 20, 2 μM non-biotinylated affinity ligand) and incubated at room temperature with shaking for 30 min. The kinase concentration in the eluates was measured by quantitative PCR. Each kinase was tested individually against each compound. KdS were determined using eleven serial threefold dilutions.
Measurement: Kd < 250 nM (pChEMBL 6.6020002365112305)
Descriptors: MW 610.69, ALogP 4.47, HBA 11, HBD 2, PSA 158.01, QED 0.28
Compound (SMILES): O=C(N[C@H]1CCN(S(=O)(=O)CCCN2CCOCC2)[C@@H](Cc2ccccc2)C1)c1cc(C2CC2)on1
InChIKey: STZZLAGCQXFFMJ-GOTSBHOMSA-N
Target: Histone-lysine N-methyltransferase SMYD3 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9H7B4
Assay: SMYD3 Biochemical Assay: The assays were all performed in a buffer consisting of 25 mM Tris-Cl pH 8.0, 1 mM TCEP, 0.005% BSG, and 0.005% Tween 20, prepared on the day of use. Compounds in 100% DMSO (1 ul) were spotted into a 384-well white opaque OptiPlate using a Bravo automated liquid handling platform outfitted with a 384-channel head (Agilent Technologies). DMSO (1 ul) was added to Columns 11, 12, 23, 24, rows A-H for the maximum signal control and 1 ul of SAH, a known product and inhibitor of SMYD3, was added to columns 11, 12, 23, 24, rows I-P for the minimum signal control. A cocktail (40 ul) containing the SMYD3 enzyme was added by Multidrop Combi (Thermo-Fisher). The compounds were allowed to incubate with SMYD3 for 30 min at room temperature, then a cocktail (10 ul) containing SAM and MEKK2 was added to initiate the reaction (final volume=51 ul). The final concentrations of the components were as follows: SMYD3 was 0.4 nM, 3H-SAM was 8 nM, MEKK2 was 12 nM, SAH in the minimum signal control wells was 1 mM, and the DMSO concentration was 2%. The assays were stopped by the addition of non-radiolabeled SAM (10 ul) to a final concentration of 100 uM, which dilutes the 3H-SAM to a level where its incorporation into MEKK2 is no longer detectable. Radiolabeled MEKK2 was detected using a scintillation proximity assay (SPA). 10 uL of a 10 mg/mL solution of SPA beads in 0.5 M citric acid was added and the plates centrifuged at 600 rpm for 1 min to precipitate the radiolabeled MEKK2 onto the SPA beads. The plates were then read in a PerkinElmer TopCount plate reader to measure the quantity of 3H-labeled MEKK2 as disintegrations per minute (dpm) or alternatively, referred to as counts per minute (cpm).% Inhibition Calculation% ⁢ ⁢ inh = 100 - ( dpm cmpd - dpm min dpm max - dpm min ) × 100Where dpm=disintegrations per minute, cmpd=signal in assay well, and min and max are the respective minimum and maximum signal controls.Four-Parameter IC50 FitY = Bottom + ( Top - Bottom ) ( 1 + ( X IC 50 ) Hill ⁢ ⁢ CoefficientWhere top and bottom are the normally allowed to float, but may be fixed at 100 or 0 respectively in a 3-parameter fit. The Hill Coefficient normally allowed to float but may also be fixed at 1 in a 3-parameter fit. Y is the % inhibition and X is the compound concentration.
Measurement: IC50 > 10000 nM (pChEMBL 5)
Descriptors: MW 516.66, ALogP 2.41, HBA 7, HBD 1, PSA 104.98, QED 0.52
Compound (SMILES): C[C@H]1CC[C@H](Cn2c(N3CCOCC3)nc3nc(-c4noc(=O)[nH]4)nc(-c4cccc(Cl)c4)c32)CC1
InChIKey: IUTCLYNCWIZGES-WKILWMFISA-N
Target: E3 ubiquitin-protein ligase Mdm2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q00987
Assay: FRET Assay: An HDM2 FRET assay was developed to assess the compounds' inhibitory activity towards binding of p53 protein. A truncated version of HDM2 with residues 17 to 125 (containing p53 binding surface, Science (1994) 265, 346-355), with N-terminal His and Thioredoxin tag was generated in pET32a expression vector and expressed in E. coli strain BL21(DE3)Rosetta. Protein was purified using Ni-affinity chromatography, followed by size exclusion chromatography using Superdex 75 26/60 column. To assess inhibition of p53 binding to HDM2, a FITC labeled 8-mer peptide (sequence: Ac-Phe-Arg-Dpr-Ac6c-(6-Br)Trp-Glu-Glu-Leu-NH2; Anal Biochem. 2004 Aug. 1; 331(1):138-46) with strong affinity towards p53 binding pocket of HDM2 was used. The HDM2 assay buffer contained 1× Phosphate Buffered Saline (Invitrogen, Cat#14190), 0.01% BSA (Jackson ImmunoResearch, Cat#001-000-162), 0.01% Tween-20. In the 1× assay buffer recombinant HDM2 protein, peptide and Lumi4-Tb Cryptate-conjugate mouse anti-6×His antibody (cisbio, Cat# Tb61HISTLB) were added and transferred to ProxiPlate PLUS (PerkinElmer, Cat#6008269), containing compounds so that final DMSO concentration is 0.1%. Final concentrations of reagents in the assay wells are 0.5 nM HDM2, 0.25 nM anti HIS (Tb label) antibody and 3 nM peptide. After two hour incubation at room temperature in a humidified chamber plates were read on EnVision plate reader with the following settings: excitation: UV, 340 nM, two emission filters: 520 nm and 495 nm respectively. Ratio of em520/em495 was used to calculate % inhibition and to obtain IC50 with 4-parameter logistic equation.
Measurement: IC50 = 71.75 nM (pChEMBL 7.139999866485596)
Descriptors: MW 510, ALogP 4.15, HBA 9, HBD 1, PSA 114.96, QED 0.43
Compound (SMILES): NC(=O)c1cccnc1Oc1ccc(CC(=O)N2CCc3cc(OCc4ccccc4)ccc32)cc1
InChIKey: JUFYIYMQFZNGBL-UHFFFAOYSA-N
Target: Rho-associated protein kinase 2 (SINGLE PROTEIN), Homo sapiens
UniProt: O75116
Assay: In Vitro Assay: The effectiveness of compounds of the present invention as ROCK inhibitors can be determined in a 30 μL assay containing 20 mM HEPES, pH 7.5, 20 mM MgCl2, 0.015% Brij-35, 4 mM DTT, 5 μM ATP and 1.5 μM peptide substrate (FITC-AHA-AKRRRLSSLRA-OH) (SEQ ID No. 1). Compounds were dissolved in DMSO so that the final concentration of DMSO was <2%, and the reaction was initiated with Rho kinase variants. After incubation, the reaction was terminated by the addition of EDTA and the phosphorylated and non-phosphorylated peptides separated using a LABCHIP® 3000 Reader (Caliper Life Sciences). Controls consisted of assays that did not contain compound, and backgrounds consisted of assays that contained enzyme and substrate but had EDTA from the beginning of the reaction to inhibit kinase activity. Compounds were tested in dose-response format, and the inhibition of kinase activity was calculated at each concentration of compound. The inhibition data were fit using a curve-fitting program to determine the IC50; i.e., the concentration of compound required to inhibit 50% of kinase activity.
Measurement: IC50 = 98 nM (pChEMBL 7.010000228881836)
Descriptors: MW 479.54, ALogP 4.68, HBA 5, HBD 1, PSA 94.75, QED 0.4
Compound (SMILES): C[C@@H]1CN(c2nc(-n3ccc(OCC4C5(CC5)C45CC5)n3)ccc2C(=O)NS(=O)(=O)c2ccccc2)C(C)(C)C1
InChIKey: IDAVUDFCPPOLFJ-FQEVSTJZSA-N
Target: Cystic fibrosis transmembrane conductance regulator (SINGLE PROTEIN), Homo sapiens
UniProt: P13569
Assay: Assays for Detecting and Measuring F508del-CFTR Modulator Properties of Compounds: The assay utilizes fluorescent voltage sensing dyes to measure changes in membrane potential using a fluorescent plate reader (e.g., FLIPR III, Molecular Devices, Inc.) as a readout for increase in functional F508del in NIH 3T3 cells. The driving force for the response is the creation of a chloride ion gradient in conjunction with channel activation and concurrent with compound treatment by a single liquid addition step after the cells have previously been loaded with a voltage sensing dye.
Measurement: EC50 = 550 nM (pChEMBL 6.260000228881836)
Descriptors: MW 561.71, ALogP 4.58, HBA 8, HBD 1, PSA 106.42, QED 0.43
Compound (SMILES): COCCOCCCNC(=O)CCCCCOc1cc(NC2CCN(S(=O)(=O)C(F)(F)F)CC2)c2cc(C(c3ccc(Cl)cc3)c3ccc(Cl)cc3)ccc2n1
InChIKey: IRABAKHPJIVQPF-UHFFFAOYSA-N
Target: Cannabinoid receptor 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P21554
Assay: cAMP Assay : Cells plated the day prior to the assay in clear bottom plates were viewed on an inverse microscope to ensure confluency in the range of 60-75%. The following mixtures and buffer solutions were prepared: (a) Buffer 1: HBSS (Mediatech Cat#21-023-CV) with 5 mM HEPES (1 mM stock, Gibco BRL Cat#15630-056) and 0.1% BSA (7.5% stock, Invitrogen Cat#15260-037); (b) Buffer 2: 0.5 mM IBMX (200 mM stock in DMSO, Sigma 15879) in Buffer 1; (c) 1 μM cAMP Standard (50 μM stock, Perkin Elmer Cat# AD0262) diluted in Buffer 2 and serially diluted in Buffer 2, 12 doses @ ½ dilutions resulting in a dose range of 1 μM to 0.5 nM; (d) d2 labelled cAMP (CisBio HTRF Detection Kit Cat #62AM4PEB reconstituted with 6 mL d-H2O) diluted 1/20 with lysis buffer (CisBio HTRF Detection Kit Cat #62AM4PEB); (e) anti-cAMP (CisBio HTRF Detection Kit Cat # 62AM4PEB reconstituted with 5 mL d-H2O) diluted 1/20 with lysis buffer (CisBio HTRF Detection Kit Cat #62AM4PEB); and (f) Forskolin (Sigma Cat# F6886, 10 mM in DMSO) diluted first in DMSO to 1 mM and then to 1.5 μM in Buffer 2.
Measurement: EC50 = 75 nM (pChEMBL 7.119999885559082)
Descriptors: MW 839.8, ALogP 8.56, HBA 8, HBD 2, PSA 119.09, QED 0.07
Compound (SMILES): CC1(C)N=C(c2ccc(-c3nc4ccccc4s3)cc2F)N(CC2CN(C(=O)C3CC3)C2)C1=O
InChIKey: BBQFPFZFKJDJQU-UHFFFAOYSA-N
Target: Fatty acid synthase (SINGLE PROTEIN), Homo sapiens
UniProt: P49327
Assay: Fatty Acid Synthase (FASN) Inhibition Scintillation Proximity Assay: In this assay, inhibition of FASN activity is measured using 3H-acetyl-CoA and malonyl-CoA as substrates. 3H-Acetyl CoA is converted to 3H-palmitate through a series of reactions by the FASN protein, which contains 7 functional domains and carries out 7 enzymatic reactions to ultimately produce 3H-palmitate. The assay principle is based upon the fact that 3H-acetyl-CoA is hydrophilic and the end product, 3H-palmitate is hydrophobic. The hydrophobic 3H-palmitate binds to scintillation proximity assay (SPA) imaging beads (resulting in light emission from the imaging beads) whereas the hydrophilic 3H-acetyl-CoA does not bind to the imaging beads (and therefore does not result in light emission from the imaging beads).10 μL assay buffer (100 mM KH2PO4 pH 7.5, 1 mM DTT) (20 μL in blanks) was added to a 384-well white Opti Plate plate (Perkin Elmer). 0.9 μL test compound (at concentrations of 30 μM, 10 μM, 3 μM, 1 μM, 0.30 μM, 0.10 μM, 0.03 μM and 0.01 μM)/DMSO and 10 μL hFASN enzyme (full length, 300 ng, purified in house) or 10 μL assay buffer was added to the wells. Then 10 μL 450 μM NADPH (Sigma N7505), 18.75 μM [3H]-acetyl-CoA (Perkin Elmer NET-290L), 150 μM malonyl-CoA (Sigma M4263) were added, mixed and incubated at room temperature for 60 minutes. The reaction was stopped by adding 20 μL Streptavidin coupled imaging beads (25 mg/ml). After incubation for 30 minutes at room temperature in the dark, the 384 well plate was centrifuged at 1500 rpm for 3 minutes and was measured after at least 24 hrs by the LEADseeker, measuring emission using a 610±20 nm pass filter. (Bays, N. W., et al., A simplified scintillation proximity assay for fatty acid synthase activity: development and comparison with other FAS activity assays, J. Biomol. Screen., 2009, pp 636-642, Vol. 14(6).)Raw data generated by the instrument were normalized to % Controlmin values, which were calculated as: % Controlmin=100*(x−mLC)/(mHC−mLC)where mLC and mHC were the means of the low control wells and high control wells on the plate, after manual exclusion of outliers. A plot of Controlmin versus test compound concentration was fitted to a 4-parameter logistic curve using a non-linear least squares regression method. From the plot, an IC50 (concentration at which 50% inhibition is achieved) was calculated. pIC50 values were calculated as −log(IC50), when IC50 is expressed in molar units.
Measurement: IC50 = 417 nM (pChEMBL 6.380000114440918)
Descriptors: MW 476.58, ALogP 4.34, HBA 5, HBD 0, PSA 65.87, QED 0.55
Compound (SMILES): O=C(N[C@@H]1CCCNC1)c1sc2nccc3c2c1NC(=O)N3c1cncc(-c2ccccc2)c1
InChIKey: AMCVLSIIVAQONW-QGZVFWFLSA-N
Target: Tyrosine-protein kinase BTK (SINGLE PROTEIN), Homo sapiens
UniProt: Q06187
Assay: TBD: TBD
Measurement: IC50 = 1000 nM (pChEMBL 6)
Descriptors: MW 470.56, ALogP 4.52, HBA 6, HBD 3, PSA 99.25, QED 0.41
Compound (SMILES): C/C=C/C(=O)N1CCC[C@@H](N2CC(C)c3c[nH]c4ncnc2c34)C1
InChIKey: SYCCBABYOOYLPC-OWTIGPFESA-N
Target: Tyrosine-protein kinase JAK3 (SINGLE PROTEIN), Homo sapiens
UniProt: P52333
Assay: JAK Caliper Enzyme Assay: Test article was solubilized in dimethyl sulfoxide (DMSO) to a stock concentration of 30 mM. An 11-point half log dilution series was created in DMSO with a top concentration of 600 μM. The test compound plate also contained positive control wells containing a known inhibitor to define 100% inhibition and negative control wells containing DMSO to define no inhibition. The compound plates were diluted 1 to 60 resulting in a top final assay compound concentration of 10 μM and a 2% DMSO concentration.Test article and assay controls were added to a 384-well plate. Reaction mixtures contained 20 mM HEPES, pH 7.4, 10 mM magnesium chloride, 0.01% bovine serum albumin (BSA), 0.0005% Tween 20, 4 μM ATP and 1 μM peptide substrate. The JAK3 assays contained 1 μM of the JAKtide peptide (FITC-KGGEEEEYFELVKK). The assays were initiated by the addition 1 nM JAK3 enzyme and were incubated at room temperature 75 minutes for JAK3. Enzyme concentrations and incubation times were optimized for each new enzyme preps and were modified slightly over time to ensure 20%-30% phosphorylation. The assays were stopped with a final concentration of 10 mM EDTA, 0.1% Coating Reagent and 100 mM HEPES, pH=7.4. The assay plates were placed on a Caliper Life Science Lab Chip 3000 (LC3000) instrument, and each well was sampled using appropriate separation conditions to measure the unphosphorylated and phosphorylated peptide.
Measurement: IC50 = 4937 nM (pChEMBL 5.309999942779541)
Descriptors: MW 325.42, ALogP 2.45, HBA 4, HBD 1, PSA 65.12, QED 0.86
Compound (SMILES): CN1CCC2(C1)OC(=O)c1cnc(-c3ccc(C[C@@H](C#N)NC(=O)[C@H]4N[C@@H]5CC[C@H]4C5)c(F)c3)cc12
InChIKey: YZPZXQUEXBVKKM-IKHSKEIOSA-N
Target: Dipeptidyl peptidase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P53634
Assay: Inhibition of Human DPPI (Cathepsin C): The recombinant human DPPI was diluted in TAGZyme buffer to 1 U/ml (38.1 μg/ml, respectively), and then activated by mixing in a 1:2 ratio with a Cysteamine aqueous solution (2 mM) and incubating for 5 min at room temperature. Five uL test compound (final concentration 0.1 nM to 100 μM) in aqua bidest 5 (containing 4% DMSO, final DMSO concentration 1%) were mixed with 10 μL of DPPI in MES buffer (final concentration 0.0125 ng/μL) and incubated for 10 min. Then, 5 μL of substrate in MES buffer (final concentration 50 μM) were added. The microtiter plates were then incubated at room temperature for 30 min. Then, the reaction was stopped by adding 10 μL of Gly-Phe-DMK in 10 MES-buffer (final concentration 1 μM). The fluorescence in the wells was determined using a Molecular Devices SpectraMax M5 Fluorescence Reader (Ex 360 nm, Em 460 nm) or an Envision Fluorescence Reader (Ex 355 nm, Em 460 nm).Each assay microtiter plate contained wells with vehicle controls (1% DMSO in bidest+0.075% 15 BSA) as reference for non-inhibited enzyme activity (100% Ctl; high values) and wells with inhibitor (Gly-Phe-DMK, in bidest+1% DMSO+0.075% BSA, final concentration 1 μM) as controls for background fluorescence (0% Ctl; low values). The analysis of the data was performed by calculating the percentage of fluorescence in the presence of test compound in comparison to the fluorescence of the vehicle control after 20 subtracting the background fluorescence using the following formula:(RFU(sample)−RFU(background))*100/(RFU(control)−RFU(background))
Measurement: IC50 = 1.6 nM (pChEMBL 8.800000190734863)
Descriptors: MW 489.55, ALogP 2.28, HBA 7, HBD 2, PSA 107.35, QED 0.62