lovingscience/chembl-v37-raw-activities huggingface dataset:
Compound (SMILES): N#Cc1ccc(N2CCN(C(=O)c3ccnc(OC[C@H]4c5ccccc5CN4c4cn[nH]c(=O)c4C(F)(F)F)c3)CC2)nc1
InChIKey: KLOCMIZXYDMYAT-DEOSSOPVSA-N
Target: Protein mono-ADP-ribosyltransferase TIPARP (SINGLE PROTEIN), Homo sapiens
UniProt: Q7Z3E1
Assay: Enzymatic Assay: Displacement of Probe A, a biotinylated probe binding to the TIPARP active site, was measured using a time-resolved fluorescence energy transfer (TR-FRET) assay. 20 nL of a dose response curve of each test compound was spotted in black 384-well polystyrene proxiplates (Perkin Elmer) using a Mosquito (TTP Labtech). Reactions were performed in a 8 μL volume by adding 6 JAL of TIPARP and Probe A in assay buffer (20 mM HEPES pH=8, 100 mM NaCl, 0.1% bovine serum albumin, 2 mM DTT and 0.002% Tween20), incubating with test compound at 25° C. for 30 min, then adding 2 μL of ULight-anti 6×His and LANCE Eu-W1024 labeled streptavidin (Perkin Elmer). The final concentrations of TIPARP and Probe A were 6 nM and 2 nM, respectively. The final concentration of ULight-anti 6×His and LANCE Eu-W1024 labeled streptavidin were 4 nM and 0.25 nM, respectively. Reactions were incubated at 25° C. for an additional 30 min, then read on an Envision plate reader equipped with a LANCE/DELFIA top mirror (Perkin Elmer) using excitation of 320 nm and emission of 615 nm and 665 nM with a 90 μs delay. The ratio of the 665/615 nm emission were calculated for each well to determine the amount of complex of TIPARP and Probe A in each well.
Measurement: IC50 > 1000 nM (pChEMBL 6)
Descriptors: MW 602.58, ALogP 3.55, HBA 9, HBD 1, PSA 131.34, QED 0.35
Compound (SMILES): CNC(=O)c1cc(Nc2nc(-c3cnc4c(c3)N([C@H]3C[C@@H](N5CCCCC5)C3)C(=O)C4(C)C)cc3ncn(C(C)C)c23)ccc1C
InChIKey: AYPXLEBZUMKKPX-WMPKNSHKSA-N
Target: Mitogen-activated protein kinase kinase kinase kinase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q92918
Assay: TBHPK1 IC50 Assay: Ten nanoliters of test compounds dissolved in DMSO at various concentrations were dispensed into a 384-well ProxiPlate (PerkinElmer #6008289). Five microliters of a solution of recombinant HPK1 diluted in HPK1 kinase assay buffer (50 mM BES [N,N-Bis(2-hydroxyethyl)-2-aminoethanesulfonic acid], pH 7.0; 10 mM magnesium chloride; 0.01% Triton X-100; 1 mM dithiothreitol; 0.01% bovine serum albumin; 0.1 mM sodium orthovanadate) was added to the compound-containing plate and was incubated for 15 minutes at 25° C. Five microliters of a mixture of ATP (Sigma-Aldrich #A6559) and peptide substrate STK S1 (Cisbio #61ST1BLC) diluted in HPK1 kinase assay buffer was then added to start the reaction. Final concentrations were 0.15 nM for HPK1, 10 μM for ATP, and 1 μM for the STK S1 peptide substrate. The reaction mixture was incubated at 25° C. for 3 hours and was stopped with the addition of 10 μl of an EDTA (Ethylenediaminetetraacetic acid)-containing detection buffer (Cisbio #62SDBRDF) supplemented with Europium cryptate-labeled anti-phospho-serine/threonine antibodies (Cisbio #62ST1PEJ) and XL665-labeled streptavidin (Cisbio #610SAXLG). The mixture was incubated for 16 hours at room temperature and peptide phosphorylation was measured by time-resolved fluorescence energy transfer (665 nm/620 nm) on an Envision plate reader (PerkinElmer).
Measurement: IC50 = 0.4 nM (pChEMBL 9.399999618530273)
Descriptors: MW 620.8, ALogP 6.13, HBA 8, HBD 2, PSA 108.28, QED 0.26
Compound (SMILES): CC(C)Oc1ccc(S(=O)(=O)N2C[C@H](n3nc(CC(=O)O)c4ccc(F)cc43)C[C@H]2C(=O)N(C)C)cc1
InChIKey: HPLRXYAIJGKPMX-HXOBKFHXSA-N
Target: Prostaglandin D2 receptor 2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9Y5Y4
Assay: Receptor Binding Inhibition Assay: Cell membrane was prepared from CRTH2-expressing K562 cell. The cell membrane (20 μg/well) was mixed with reaction buffer (50 mM Tris/HCl, pH 7.4, 10 mM MgCl2). Then, [3H]-PGD2 was added to the mixture and incubated for 60 min at room temperature. The concentration of [3H]-PGD2 was determined as equal to Kd calculated from Scatchard plot. Then, the mixture was filtrated with glass paper immediately. The filter paper was washed several times and radio activity on the filter was measured. Specific binding was defined as difference between total and non-specific binding (radio activity with 10 μM PGD2). Ki values for each compound were calculated from displacement curve.
Measurement: Ki = 2505 nM (pChEMBL 5.599999904632568)
Descriptors: MW 532.59, ALogP 2.68, HBA 7, HBD 1, PSA 122.04, QED 0.47
Compound (SMILES): CNC(=O)c1ccc(C[C@@H](CNC(=O)C[C@H](c2cccnc2)C2(C(F)(F)F)CC2)N(C)C)c(Cl)c1F
InChIKey: KQVXTDAEHDRBSS-PKOBYXMFSA-N
Target: Mu-type opioid receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P35372
Assay: MOR cAMP Agonist and Antagonist Assays: CHO Tag-lite human mOR stable cell line from Cisbio (NCBI accession number: NM_000914.3, Bedford, Mass.) were seeded and grown to approximately 80% confluence in Ham F-12 with 10% FBS, 50 U/ml penicillin, 50 μg/ml streptomycin, 2 mM Hepes, and 1 mg/ml geneticin (Invitrogen, Carlsbad, Calif.). Cells were then harvested using accutase (Corning. Coming, N.Y.), centrifuged at 1300 RPM for 5 min, and plated at 5000 cells per 5 μL per well in a 5× dilution of stimulation buffer consisting of the HTRF cAMP Gi kit, water and IBMX at 0.5 mM (Cisbio, Bedford, Mass.) in white HTRF low volume 384 well plates (Cisbio, Bedford, Mass.). Plates were then incubated at 37° C. in 5% CO2 for 10 minutes. For the agonist assay, forskolin was added to a final concentration of 4 μM. For the antagonist assay, forskolin (4 μM) and DAMGO at EC90 final concentration were added. Test compounds were dissolved in DMSO and water, and then serially diluted to working concentrations such that the concentration of DMSO was less than 0.1%. Diluted test compounds were added at 2.5 μL per well, and plates were incubated at 37° C., and 5% CO2 for 15 minutes, and then at room temperature for 15 minutes. Next 5 μl/well of cAMP Eu-cryptate and 5 μl/well of anti-cAMP-d2 (both diluted 1:20 in lysis buffer) were added, and plates were incubated at room temperature for 1 hour. Following incubation, plates were read in a Synergy Neo2 multi-mode reader (Biotek, Winooski, Vt.). Plate reader settings were set to time resolved fluorescence with excitation at 330 nm and emissions of 620 nm and 665 nm. Emission fluorescence was normalized (665/620 nm signal×1000). For the agonist assay, data was normalized using the maximal DAMGO response. Measurements were performed in triplicate and the dose response was fit using nonlinear regression.
Measurement: IC50 > 3000 nM (pChEMBL 5.5229997634887695)
Descriptors: MW 528.98, ALogP 4.34, HBA 4, HBD 2, PSA 74.33, QED 0.45
Compound (SMILES): Cc1c(-c2cc3cc(NC(=O)O[C@H]4C[C@@H](N5CCOCC5)C4)ncc3c(N)c2F)cnc2c1NCCO2
InChIKey: SWCWGRIVGOTOIV-CALCHBBNSA-N
Target: Mitogen-activated protein kinase kinase kinase kinase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q92918
Assay: TBD: TBD
Measurement: Ki = 0.054 nM (pChEMBL 10.270000457763672)
Descriptors: MW 508.55, ALogP 3.54, HBA 9, HBD 3, PSA 123.86, QED 0.45
Compound (SMILES): CN(CCCS(C)(=O)=O)c1nncc(-c2ccc3ccccc3c2)n1
InChIKey: BKCCDALXEBIECM-UHFFFAOYSA-N
Target: Cytochrome P450 11B2, mitochondrial (SINGLE PROTEIN), Homo sapiens
UniProt: P19099
Assay: Inhibitory Effect on hCYP11B2: The pcDNA3.1-human CYP11B2 plasmid was transfected into a Chinese hamster lung fibroblast V79 cell line to produce a cell line stably expressing human CYP11B2 gene.The cells were cultured and grown in the Dulbecco's modified Eagle's/Ham's medium supplemented with 10% fetal bovine serum and 1% G418 disulfate solution under the environment of 37° C., 95% air, and 5% CO2, and the grown cells were harvested.Then, the cells were fractionated to obtain mitochondria by reference to a method described in Chabre et al. JCE 86 M 85 (11) 4060-68, 2000. In particular, the cells suspended in a 5 mmol/L Tris-HCl buffer (pH 7.4) containing 250 mmol/L sucrose were homogenized in a Teflon (Registered Trademark) Potter Elvehjem homogenizer, and then the suspension was centrifuged (800×g, 15 min.). The supernatant was separated and again centrifuged (10000×g, 15 min.) to obtain a pellet (mitochondrial fraction).The mitochondrial fraction diluted with a buffer containing 10 mmol/L KH2PO4, 10 mmol/L Tris, 20 mmol/L KCl, 25 mmol/L sucrose, 5 mmol/L MgCl2, and 0.05% bovine serum albumin was dispensed to a 96-well plate. 0.5 μmol/L Deoxycorticosterone, 150 μmol/L NADPH and a compound of each concentration were added to each well, and incubated for 1.5-2 hours at room temperature to produce aldosterone. An amount of the produced aldosterone in the incubated solution was determined by using HTRF (Homogeneous Time Resolved Fluorescence) method.IC 50 (nmol/L) was calculated by analyzing the aldosterone production inhibition rate (%) of each concentration of compounds by non-linear regression to a logistic curve.
Measurement: IC50 = 38 nM (pChEMBL 7.420000076293945)
Descriptors: MW 356.45, ALogP 2.56, HBA 6, HBD 0, PSA 76.05, QED 0.68
Compound (SMILES): CCCO[C@H]([C@H]1O[C@@H]2SC(N(C)C)=N[C@@H]2[C@@H](O)[C@@H]1O)C(F)(F)F
InChIKey: FYROIRGCKYQNBP-VFZPANTDSA-N
Target: Protein O-GlcNAcase (SINGLE PROTEIN), Homo sapiens
UniProt: O60502
Assay: Enzymatic Assay: Enzymatic reactions were carried out in a reaction containing 50 mM NaH2PO4, 100 mM NaCl and 0.1% BSA (pH 7.0) using 2 mM 4-Methylumbelliferyl N-acetyl-β-D-glucosaminide dihydrate (Sigma M2133) dissolved in ddH2O, as a substrate. The amount of purified human O-GlcNAcase enzyme used in the reaction was 0.7 nM. Test compound of varying concentrations was added to the enzyme prior to initiation of the reaction. The reaction was performed at room temperature in a 96-well plate and was initiated with the addition of substrate. The production of fluorescent product was measured every 60 sec for 45 min with a Tecan Infinite M200 plate-reader with excitation at 355 nM and emission detected at 460 nM, with 4-Methylumbelliferone (Sigma M1381) used to produce a standard curve. The slope of product production was determined for each concentration of compound tested and plotted, using standard curve fitting algorithms for sigmoidal dose response curves. The values for a four parameter logistic curve fit of the data was determined.
Measurement: Ki = 4.5 nM (pChEMBL 8.350000381469727)
Descriptors: MW 358.38, ALogP 0.82, HBA 7, HBD 2, PSA 74.52, QED 0.78
Compound (SMILES): CC(C)c1c(-c2cnn(-c3cccc(Cl)c3)c2)[nH]c2ncnn2c1=O
InChIKey: HSNJAABZLMWACZ-UHFFFAOYSA-N
Target: Lysine-specific demethylase 5B (SINGLE PROTEIN), Homo sapiens
UniProt: Q9UGL1
Assay: TR-FRET (Time-Resolved Fluorescence Resonance Energy Transfer) Assay: In this assay, the potency (IC50) of each compound was determined from a ten point (1:3 serial dilution; final compound concentration range in assay from 100000 nM to 5.08 nM) titration curve using the following outlined procedure. To each well of a white Greiner 1536 Lumitrac 1536 well-plate, 50 nL of compound (100 fold dilution in final assay volume of 5 μL) was dispensed, followed by the addition of 4 μL of 1× assay buffer (50 mM Hepes 7.3, 0.5 mM TCEP, 0.005% Brij-35, 0.02% BSA, 50 μM Na-L-Ascorbate and 2 μM AmFe(II)Sulfate) containing 5 nM of Full-length KDM5B enzyme (recombinant protein from baculovirus-transfected Sf21 cells: full-length KDM5B; MW=176.825 kDa). Following a 30 minutes compound and enzyme incubation in a humidified chamber, each reaction was initiated by the addition of 1 μL 1× assay buffer containing 50 nM biotinylated H3K4Me3 peptide, and 500 nM α-ketoglutarate. The final reaction in each well of 5 μL consists of 5 nM KDM5B, 500 nM biotinylated-peptide, and 500 nM α-ketoglutarate. De-methylation reactions were allowed to proceed for 60 minutes. Reactions were immediately quenched by the addition of 5 uL of 2× Lance Detection Buffer (PerkinElmer) with 0.5 mM EDTA and 2 nM of Eu-anti-H3K4Me1-2 antibody, and 30 nM of Streptavidin-conjugated Dylight 650 detection reagents. After 60 minutes incubation with detection reagents, reaction plates were read on a PerkinElmer EnVision plate reader using standard TR-FRET protocol. Briefly, excitation of donor molecules (Eu-chelate-anti-H3K4Me1-2-antibody) with a laser light source at 337 nm produces energy that can be transferred to Dylight-650 acceptor molecules if this donor:acceptor pair is within close proximity. Fluorescence intensity at both 665 nm (acceptor) and 615 nm (donor) are measured and a TR-FRET ratio calculated for each well (acceptor intensity/donor intensity).
Measurement: IC50 = 9.66 nM (pChEMBL 8.020000457763672)
Descriptors: MW 354.8, ALogP 3.05, HBA 6, HBD 1, PSA 80.87, QED 0.61
Compound (SMILES): CNc1nc(Nc2cc([C@]34C[C@H]3COC4=O)nn2C2CC2)ncc1C(F)(F)F
InChIKey: BVYBQDCDFOVHIU-PWJLMRLQSA-N
Target: Leucine-rich repeat serine/threonine-protein kinase 2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q5S007
Assay: Biochemical Assay: Materials:    LRRK2 G2019S enzyme    Substrate (LRRKtide)    ATP    TR-FRET dilution buffer    pLRRKtide antibody    384-well assay plate    DMSOEnzyme Reaction Conditions    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    10 μL total reaction volumeDetection Reaction Conditions    1× TR-FRET dilution buffer    10 mM EDTA    2 nM antibody    23° C. reaction temperature    10 μL total reaction volumeCompounds 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.To perform the enzyme reaction, 5 μL of LRRKtide substrate and kinase mixture in assay buffer was added to all wells of the assay plate. The plate was centrifuged to concentrate the mixture at the bottom of the wells. The assay plate was incubated at 23° C. for 20 minutes. Following incubation, 5 μL of 2× ATP in assay buffer was added to each well, and plates were centrifuged to concentrate the mixture at the bottom of the wells. The plate was incubated at 23° C. for 60 minutes.To perform the detection of the reaction, EDTA completely mixed in TR-FRET dilution buffer was added to antibody reagent. 10 μL of detection reagent was added to all wells of each well of the assay plate and the plate was centrifuged to concentrate the mixture at the bottom of the wells. The plate was then incubated at 23° C. for 60 minutes. Plates were read on Perkin Elmer Envision 2104 instrument in TR-FRET mode using a 340 nm excitation filter, 520 nm fluorescence emission filter, and 490 or 495 nm terbium emission filter.
Measurement: IC50 < 30 nM (pChEMBL 7.5229997634887695)
Descriptors: MW 394.36, ALogP 2.63, HBA 8, HBD 2, PSA 93.96, QED 0.75
Compound (SMILES): CC(C)c1ncnc(C(C)C)c1-n1c(=O)nc(N2CCN(C(=O)/C=C/CN(C)C)C[C@@H]2C)c2cc(Cl)c(-c3ccccc3F)nc21
InChIKey: AIGRNVGSFBEYPE-FLSMSTFGSA-N
Target: GTPase KRas (SINGLE PROTEIN), Homo sapiens
UniProt: P01116
Assay: Coupled Nucleotide Exchange Assay: Purified GDP-bound KRAS protein (aa 1-169), containing both G12C and C118A amino acid substitutions and an N-terminal His-tag, was pre-incubated in assay buffer (25 mM HEPES pH 7.4, 10 mM MgCl2, and 0.01% Triton X-100) with a compound dose-response titration for either 5 min or 2 hours (see Table 15). Following compound pre-incubation, purified SOS protein (aa 564-1049) and GTP (Roche 10106399001) were added to the assay wells and incubated for an additional 30 min (for 5 min compound pre-incubation) or 1 hour (for 2 hour compound pre-incubation). To determine the extent of inhibition of SOS-mediated nucleotide exchange, purified GST-tagged cRAF (aa 1-149), nickel chelate AlphaLISA acceptor beads (PerkinElmer AL108R), and AlphaScreen glutathione donor beads (PerkinElmer 6765302) were added to the assay wells and incubated for 10 minutes. The assay plates were then read on a PerkinElmer EnVision Multilabel Reader, using AlphaScreen® technology, and data were analyzed using a 4-parameter logistic model to calculate IC50 values.
Measurement: IC50 = 1230 nM (pChEMBL 5.909999847412109)
Descriptors: MW 647.2, ALogP 5.43, HBA 9, HBD 0, PSA 100.35, QED 0.23
Compound (SMILES): CCn1nc(C)c(Br)c1C(=O)N[C@@H](CC(C)(C)C)C(=O)N[C@H](C#N)C[C@@H]1CCNC1=O
InChIKey: PLMXOHHVTRBRNY-KKUMJFAQSA-N
Target: Replicase polyprotein 1ab (SINGLE PROTEIN), Severe acute respiratory syndrome coronavirus 2
UniProt: P0DTD1
Assay: SARS-CoV-2 Coronavirus 3C Protease FRET Assay: The proteolytic activity of the main protease, 3CLpro, of SARS-CoV-2 was monitored using a continuous fluorescence resonance energy transfer (FRET) assay. The SARS-CoV-2 3CLpro assay measures the activity of full-length SARS-CoV-2 3CL protease to cleave a synthetic fluorogenic substrate peptide with the following sequence: Dabcyl-KTSAVLQ-SGFRKME-Edans modelled on a consensus peptide (V. Grum-Tokars et al. Evaluating the 3C-like protease activity of SARS-coronavirus: recommendations for standardized assays for drug discovery. Virus Research 133 (2008) 63-73). The fluorescence of the cleaved Edans peptide (excitation 340 nm/emission 490 nm) is measured using a fluorescence intensity protocol on a Flexstation reader (Molecular Devices). The fluorescent signal is reduced in the present of PF-835231, a potent inhibitor of SARS-CoV-2 3CLpro. The assay reaction buffer contained 20 mM Tris-HCl (pH 7.3), 100 nM NaCl, 1 mM EDTA and 25 μM peptide substrate. Enzyme reactions were initiated with the addition of 15 nM SARS-CoV-2 3CL protease and allowed to proceed for 60 minutes at 23° C. Percent inhibition or activity was calculated based on control wells containing no compound (0% inhibition/100% activity) and a control compound (100% inhibition/0% activity). IC50 values were generated using a four-parameter fit model using ABASE software (IDBS). Ki values were fit to the Morrison equation with the enzyme concentration parameter fixed to 15 nM, the Km parameter fixed to 14 μM and the substrate concentration parameter fixed to 25 μM using ABASE software (IDBS).
Measurement: Ki = 297 nM (pChEMBL 6.53000020980835)
Descriptors: MW 495.42, ALogP 2.04, HBA 6, HBD 3, PSA 128.91, QED 0.51
Compound (SMILES): CC(=O)N1CCN(c2cc(-c3cc(-c4ccnn4CC(F)(F)F)c4c(N)ncnn34)ccc2C)C(=O)C1(C)C
InChIKey: HSCFDSIPJBMTGR-UHFFFAOYSA-N
Target: Phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit delta isoform (SINGLE PROTEIN), Homo sapiens
UniProt: O00329
Assay: ADP-Glo Format PI3K Assay: The ADP-Glo format PI3K assays were performed in Proxiplate 384-well plates (Perkin Elmer #6008280). The final assay volume was 2 μl prepared from 1 μl additions of enzyme/PIP2:PS lipid (Invitrogen #PV5100) mixture and 1 μl ATP (provided in kit, Promega #V9101) and test compounds in assay buffer (50 mM HEPES pH 7.5, 3 mM MgCl2, 100 mM NaCl, 0.5 mM EGTA, 2 mM DTT, 0.03% CHAPS). The reaction was initiated by the combination of enzyme/lipid, ATP, and test compounds. The reaction mixture was incubated at room temperature for 30 minutes (PI3K Alpha, Beta, Gamma) or 3 hours for PI3K Delta. ADP-Glo (2 μl), followed by Kinase Detection reagent (4 μl), were added to reactions following the initial incubation and allowed to incubate 40 minutes at room temperature. The reaction mixture was analyzed on the TOPCOUNT (Perkin Elmer). Inhibition data were calculated by comparison to no enzyme control reactions for 100% inhibition and vehicle-only reactions for 0% inhibition. The final concentration of enzyme in the assays are PI3K Alpha [0.5 nM], PI3K Beta [2 nM], PI3K Gamma [20 nM], PI3K Delta [0.5 nM]. ATP final concentrations are as follows: for Alpha [10 μM], for Beta [12.5 μM], for Gamma [6.5 μM], for Delta [100 μM]. Lipid final concentration was the same for all enzymes, [25 μM]. Dose response curves were generated to determine the concentration required to inhibit 50% of activity. Compounds were dissolved at 0.12 mM in dimethylsulfoxide (DMSO) and evaluated at eleven concentrations. The IC50 values were derived by non-linear regression analysis.
Measurement: IC50 = 2 nM (pChEMBL 8.699999809265137)
Descriptors: MW 540.55, ALogP 3.69, HBA 8, HBD 1, PSA 114.65, QED 0.42
Compound (SMILES): Clc1ccc2[nH]c(C[C@H]3CC[C@H](c4ccc5ncccc5c4)CC3)nc2c1
InChIKey: PIENHQQPIDAMOV-WKILWMFISA-N
Target: Indoleamine 2,3-dioxygenase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P14902
Assay: IDO1 Enzymatic Assay: Recombinant IDO1 was overexpressed and purified from E. coli cells with an N-terminal His tag. IDO1 enzymatic assay was carried out using a methodology similar to described in the literature (J. Biol. Chem. (1980), 255, 1339-1345). The reaction mixture contains 50 nM IDO1, 1.3 mM D-tryptophan, 5 mM L-ascorbic acid, 6.25 M methylene Blue, 0.4 mg/mL catalase and compound (or DMSO) in a buffer containing 50 mM potassium phosphate pH 7.5 and 0.1% BSA. After incubation at 24° C. for 1.5 hours, absorbance of the reaction mixture was continuously read at 321 nm to monitor the formation of N′-formylkynurenine by a FULOstar OMEGA plate reader (BMG LABTECH) for 1 hour. The enzymatic activity was determined by measuring the slope of the linear absorbance increase as a function of time. The IC50s are calculated based on remaining enzyme activity in the presence of increasing concentrations of compounds.
Measurement: IC50 > 10000 nM (pChEMBL 5)
Descriptors: MW 375.9, ALogP 6.28, HBA 2, HBD 1, PSA 41.57, QED 0.46
Compound (SMILES): COc1cc2c(Oc3cccc(NC(=O)Nc4cc(C(C)(C)C)on4)c3)ncnc2cc1OCCCN1CCN(CCO)CC1
InChIKey: PEPIATATVDHQQV-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 619.72, ALogP 4.74, HBA 11, HBD 3, PSA 147.34, QED 0.19
Compound (SMILES): c1cc2cc(-c3cncc(N4CCOCC4)n3)cnc2[nH]1
InChIKey: CHKUYSNXBONHIO-UHFFFAOYSA-N
Target: Leucine-rich repeat serine/threonine-protein kinase 2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q5S007
Assay: LRRK2 Kinase Assay: Procedure: Enzyme was incubated with substrate peptide in reaction buffer in the presence and absence of test compounds or Staurosporine. All additions were done on ice, followed by the addition of ATP mix. Wells were uniformly mixed using an Eppendorff plate shaker and incubated at 30° C. for 20 min, and stopped by the addition of 5 μl of 3% phosphoric acid. Volume was increased to 100 μl by adding 0.8% phosphoric acid which was then transferred to PC filter mats (Millipore), pre-equilibrated with 70% ethanol and water. Plates were washed thrice with 100 μl 0.8% phosphoric acid and dried for an hour at 60° C. 100 μl scintillation fluid was added into each well and reading taken in Perkin Elmer TOPCOUNT beta counter. The data analysis was performed by averaging the duplicate top count readings for each standard, negative, positive control (enzyme control) and samples and subtracting the average negative control from each reading which results in corrected values. A validation EC50 curve was generated by plotting CPM for each Staurosporine concentration on y-axis against the Log concentration of Staurosporine (nM) on the x-axis followed by a best fit curve through the points.
Measurement: IC50 < 500 nM (pChEMBL 6.301000118255615)
Descriptors: MW 281.32, ALogP 1.86, HBA 5, HBD 1, PSA 66.93, QED 0.78
Compound (SMILES): COc1nc(N[C@@H](C)c2cc3cc(Cl)ccc3n(C)c2=O)ncc1C#N
InChIKey: RDWBYCLMYSKOTA-JTQLQIEISA-N
Target: Isocitrate dehydrogenase [NADP] cytoplasmic (SINGLE PROTEIN), Homo sapiens
UniProt: O75874
Assay: IDH1-R132H and IDH1-R132C Enzymatic Assay: Assays were performed in a 384-well black plate. An aliquot of 250 nL of compound was incubated with 10 μL of 30 nM IDH1-R132H or 10 nM IDH1-R132C recombinant protein in assay buffer (50 mM Tris pH=7.5, 150 mM NaCl, 5 mM MgCl2, 0.1% (w/v) Bovine Serum Albumin, and 0.01% Triton X-100) in each well at 25° C. for 15 minutes. After the plate was centrifuged briefly, an aliquot of 10 μL of 2 mM α-ketoglutarate and 20 μM NADPH solution prepared in assay buffer was then added to each well and the reaction was maintained at 25° C. for 45 minutes. An aliquot of 10 μL of diaphorase solution (0.15 U/mL diaphorase and 30 M Resazurin in assay buffer) was added to each well. The plate was maintained at 25° C. for 15 minutes and then read on a plate reader with excitation and emission wavelengths at 535 nm and 590 nm, respectively. The IC50 of a given compound was calculated by fitting the dose response curve of inhibition of NADPH consumption at a given concentration with the four parameter logistic equation.
Measurement: IC50 = 5500 nM (pChEMBL 5.260000228881836)
Descriptors: MW 369.81, ALogP 3.04, HBA 7, HBD 1, PSA 92.83, QED 0.76
Compound (SMILES): Cc1ncc(C)n2nc(CCc3nc4c5nccnc5ccc4n3C)nc12
InChIKey: QDNFQCZMNOFXDU-UHFFFAOYSA-N
Target: cAMP and cAMP-inhibited cGMP 3',5'-cyclic phosphodiesterase 10A (SINGLE PROTEIN), Homo sapiens
UniProt: Q9Y233
Assay: In Vitro Pharmacology Assay: In one embodiment, the compounds provided herein were assayed for their ability to inhibit human PDE-10A. In one embodiment, the activities of the compounds were determined using the Molecular Devices IMAP PDE Fluorescence Polarization assay using recombinant human PDE-10 enzyme expressed in a baculoviral system. Briefly, 10 μL of a compound (0.2 nM-20 μM) was added to either a 96-well half area black plate or a 384-well black plate along with 10 μL of Fluorescein-labeled cAMP/cGMP substrate as per manufacturer's instructions and 10 μL of PDE enzyme (activity 0.1 U). Following a 40-minute incubation at 37° C., 60 μL of IMAP binding reagent was added. The plate was then read on a Perkin Elmer Victor (480-535 nm). The data was analyzed using Prism Software (GraphPad Inc, San Diego, Calif.).
Measurement: IC50 < 10 nM (pChEMBL 8)
Descriptors: MW 358.41, ALogP 2.36, HBA 8, HBD 0, PSA 86.68, QED 0.49
Compound (SMILES): CC(C)N(CC(F)(F)F)C(=O)CN1CN(c2ccccc2)C2(CCN(C(=O)c3cnc4[nH]ncc4c3)CC2)C1=O
InChIKey: LABDHZIYLNNWCF-UHFFFAOYSA-N
Target: Epithelial discoidin domain-containing receptor 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q08345
Assay: In Vitro Binding Assay for DDR1: This assay is based on the intracellular domain of the DDR1 protein which contains the kinase active site. The recombinant protein additionally carries a GST-tag that can be recognized by an Eu-labeled anti-GST antibody. A tracer compound binding to the active site is labeled with a dye so that a FRET donor acceptor pair can be formed. Excitation energy absorbed by the Europium complex (350 nm flash light or pulsed laser) is transferred to a suitable fluorescent dye, if it is in close proximity. Compounds binding competitively with the tracer molecule will displace the bound tracer molecules and reduce the FRET signal in a dose dependent manner. Due to the long lifetime of the Eu excited state, the emission of the donor and the acceptor can be measured in time-gated mode such that most of the intrinsic fluorescence contributions have already decayed. This results in high sensitivity, excellent reproducibility and high data quality. This sensitive detection method enables protein concentrations below 20 nM.Protein, tracer and labeled antibody were obtained from commercial sources. The assay was performed in 384 low volume microtiter-plates with a final volume of 15 μl. Dose response curves were generated from 16 compound dilutions in DMSO as solvent compound dilutions, a solution containing protein and labeled antibody, and a solution containing the tracer which is added in the last step. The fluorescence of donor and acceptor were then measured after one hour incubation at room temperature. Every assay run was quality-controlled with dose response curves for two reference compounds.
Measurement: IC50 = 17.2 nM (pChEMBL 7.760000228881836)
Descriptors: MW 557.58, ALogP 3.04, HBA 6, HBD 1, PSA 105.74, QED 0.5
Compound (SMILES): Cc1cc(F)cc2c1NCC[C@H]2C(=O)n1nc2c(c1N)COCC2
InChIKey: IYOIFBAFGMVXQJ-LLVKDONJSA-N
Target: Coagulation factor XII (SINGLE PROTEIN), Homo sapiens
UniProt: P00748
Assay: Factor XIIa (FXIIa) Inhibitory Activity: In a 96-well clear bottom plate, 80 μl of assay buffer was added to each well. Assay buffer consists of 0.5× Hank's Balanced Salt Solution (Invitrogen), buffered with 25 mM HEPES pH 7.4 (Invitrogen) and 0.5× Tris-buffered saline with Tween-20 0.05% (Santa Cruz Biotechnology). Test compounds were first dissolved in DMSO (Sigma) and then 4 μl were added to test wells containing assay buffer. Serial dilutions using an automated multi-channel pipette were used to generate a concentration range of approximately 1-100 μM. Human FXIIa (Enzyme Research Labs) was diluted in assay buffer to a final concentration of 12.5 nM. 80 μl of this enzyme solution was added to the assay wells. The enzyme/compound mixtures were incubated for 10 minutes at room temperature. Chromogenic substrate (Pefachrome XIIa; Enzyme Research Labs) was added to assay wells at a final concentration of 400 μM. The assay plate was spun for 1 minute at 1500 g and then read at 37° C. in a SpectraMax M2 plate reader (λ=405 nm). Activity was quantified as the rate of change in absorbance, which corresponds to the rate of substrate cleavage. IC50 values were determined as the concentration of inhibitor that produced 50% of the rate of change of control wells without any inhibitor. For compounds with activity <1 μM, the assay was repeated with a lower concentration range, typically from 10-1000 nM.
Measurement: IC50 = 12500 nM (pChEMBL 4.900000095367432)
Descriptors: MW 330.36, ALogP 2.23, HBA 6, HBD 2, PSA 82.17, QED 0.84
Compound (SMILES): O=c1nc(NCc2cnoc2)c2ccc(C(F)(F)F)nc2n1-c1ccccc1Cl
InChIKey: GAXNYDGVNPQLLD-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 421.77, ALogP 4.05, HBA 7, HBD 1, PSA 85.84, QED 0.53
Compound (SMILES): Cn1c(=O)c(C(=O)NCC(=O)O)c(O)c2ccc(Oc3cc(Cl)cc(Cl)c3)cc21
InChIKey: SOSZQCCXGFFYTD-UHFFFAOYSA-N
Target: Hypoxia-inducible factor 1-alpha (SINGLE PROTEIN), Homo sapiens
UniProt: Q16665
Assay: Erythropoietin (EPO) Biological Assay: The activities of compounds of the invention in inducing erythropoietin (EPO) production were assessed by using human live cancer cell strain Hep3B (ATCC: American type culture collection, Manassas, Va.). Hep3B cells seeded in a 96-well plate with a density of 2.5×104 cells/well were cultivated overnight in DMEM culture medium (Dulbecco's Modified Eagle's Medium) at 37° C. in the presence of 10% fetal bovine serum (FBS). The next day, the supernatant liquid of culture medium was abandoned by suction, and to the residue was added fresh DMEM (containing 0.5% DMSO, 0.5% FBS) containing compound of the invention with a series of concentrations (0.31160.00 μM) or containing solvent used for comparison. The cells were cultivated at 37° C. for 24 h. The supernatant liquid was collected and the EPO concentration of the supernatant was quantified by using human EPO ELISA kit (Abcam). The activity of each compound in inducing erythropoietin (EPO) production was represented by half maximal effective concentration (EC50).
Measurement: EC50 = 20200 nM (pChEMBL 4.699999809265137)
Descriptors: MW 437.24, ALogP 3.16, HBA 6, HBD 3, PSA 117.86, QED 0.56
Compound (SMILES): Cc1c(-c2ccc(Nc3cc(NC(=O)C4CC4)nc4[nH]c(C(F)F)nc34)c(S(C)(=O)=O)c2)nn(C)c1C
InChIKey: OYJSIZQWTBNWBU-UHFFFAOYSA-N
Target: Non-receptor tyrosine-protein kinase TYK2 (SINGLE PROTEIN), Homo sapiens
UniProt: P29597
Assay: Binding Assay: Binding constants for compounds of the present invention against the JH2 domain were determined by the following protocol for a KINOMEscan® assay (DiscoveRx). A fusion protein of a partial length construct of human TYK2 (JH2domain-pseudokinase) (amino acids G556 to D888 based on reference sequence NP_003322.3) and the DNA binding domain of NFkB was expressed in transiently transfected HEK293 cells. From these HEK 293 cells, extracts were prepared in M-PER extraction buffer (Pierce) in the presence of Protease Inhibitor Cocktail Complete (Roche) and Phosphatase Inhibitor Cocktail Set II (Merck) per manufacturers' instructions. The TYK2 (JH2domain-pseudokinase) fusion protein was labeled with a chimeric double-stranded DNA tag containing the NFkB binding site (5′-GGGAATTCCC-3′) fused to an amplicon for qPCR readout, which was added directly to the expression extract (the final concentration of DNA-tag in the binding reaction is 0.1 nM).
Measurement: Kd = 0.3 nM (pChEMBL 9.520000457763672)
Descriptors: MW 529.57, ALogP 4.41, HBA 8, HBD 3, PSA 134.66, QED 0.32
Compound (SMILES): Cc1noc2c1C(=O)N(CCN1CCN(c3nsc4ccccc34)CC1)CC2
InChIKey: BZUPCLAOTHNFTJ-UHFFFAOYSA-N
Target: 5-hydroxytryptamine receptor 1A (SINGLE PROTEIN), Homo sapiens
UniProt: P08908
Assay: Binding Activity Assay: Binding affinity of the present compound for human 5-HT1A receptor, human 5-HT2A receptor, and human D2 receptor was measured by the following procedures.CHO cell membrane fraction in which human 5-HT1A receptor, human 5-HT2A receptor, and human D2 receptor were expressed was purchased from PerkinElmer, Inc. In a test for evaluating binding affinity, a test compound dissolved in dimethylsulfoxide (DMSO) and each receptor membrane sample diluted in buffer were mixed with [3H]8-OH-DPAT, [3H]Ketanserin, or [3H]Spiperone (all purchased from PerkinElmer, Inc.) for 5-HT1A receptor, 5-HT2A receptor, and D2 receptor, respectively. Each mixture was incubated at room temperature for 60 minutes. Then, the mixture was added quickly on a glassfiber filter plate (Multiscreen FB, Millipore, Inc.) coated with 0.3% aqueous polyethylenimine, and vacuum-filtered. Radioactivity remaining on the filter was measured with a liquid scintillation counter (PerkinElmer, Inc.). Binding inhibition rate was calculated from the following formula.
Measurement: Ki = 6.99 nM (pChEMBL 8.15999984741211)
Descriptors: MW 397.5, ALogP 2.41, HBA 7, HBD 0, PSA 65.71, QED 0.67
Compound (SMILES): CCOC(=O)CCn1c(=O)[nH]/c(=N\c2ccc(Oc3ccncc3)cc2)n(Cc2ccc(Cl)cc2)c1=O
InChIKey: DDQSCQBHXAHRRJ-UHFFFAOYSA-N
Target: P2X purinoceptor 3 (SINGLE PROTEIN), Homo sapiens
UniProt: P56373
Assay: Human P2X3 Receptor Inhibition Assay: Stably expressing cell line (C6BU-1 transfected with human P2X3 receptor gene (GenBank accession number Y07683) was used. The cells were seeded in a 96-well microtiter plate at a concentration of 8000 cells/well and cultured in the medium (8.3% fetal bovine serum, 8.3% horse serum, 1% antibiotic and antifungal in DMEM) for one day at 37° C. under 5% carbon dioxide atmosphere. The medium was replaced with 4 μM Fluo-3-AM solution (20 mM HEPES, 137 mM NaCl, 2.7 mM KCl, 0.9 mM MgCl2, 5.0 mM CaCl2, 5.6 mM D-glucose, 2.5 mM probenecid, 10% BSA, and 0.08% Pluronic F-127, pH 7.5) and incubated at 37° C. under 5% dioxide carbon atmosphere for one hour. The plate was washed with washing buffer (20 mM HEPES, 137 mM NaCl, 2.7 KCl, 0.9 mM MgCl2, 5.0 mM CaCl2, 5.6 mM D-glucose, 2.5 mM probenecid, pH 7.5), each well was added with 40 μL of this buffer. The plate was placed in High-Throughput Screening System FDSS 3000 (Hamamatsu Photonics K.K.). Measurement of fluorescence intensity by FDSS 3000 was started, and 40 μl, of DMSO solutions containing different concentrations of the test, compound as prepared by dilution with dilution buffer (20 mM HEPES, 137 mM NaCl, 2.7 mM KCl, 0.9 mM MgCl2, 5.0 mM CaCl2, 5.6 mM D-glucose, 2.5 mM probenecid, 0.1.% Pluronic F-127, pH 7.5) were dispensed to each well through the built-in automatic dispenser. Five minutes after, 50 nM ATP solution (50 μL) prepared by dilution with the dilution buffer was dispensed through the built-in automatic dispenser, and the measurement of fluorescence intensity was continued for 3 min. For each well, the specific maximum fluorescence intensity was calculated as the ratio of the maximum fluorescence intensity after addition of the ATP solution to the fluorescence intensity at the starting of the measurement. The 50% inhibitory concentration (IC50) was calculated under the assumption that the specific maximum fluorescence intensity without test compound is 0% inhibition and that the specific maximum fluorescence intensity when the dilution buffer was added in place of ATP solution is 100% inhibition, to evaluate the inhibitory activity of the test compound. FDSS software (Hamamatsu Photonics K.K.) was used for calculation of the specific maximum fluorescence intensity. IC50 was calculated using Microsoft Excel (Microsoft Corporation) and Ltd.).
Measurement: IC50 = 2 nM (pChEMBL 8.699999809265137)
Descriptors: MW 521.96, ALogP 3.41, HBA 9, HBD 1, PSA 120.57, QED 0.34
Compound (SMILES): CC(C)C[C@@](C)(N)COc1cnc(-c2cc(Cl)ncc2F)cc1C(F)F
InChIKey: SISQPPCZDPNKAD-GOSISDBHSA-N
Target: AP2-associated protein kinase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q2M2I8
Assay: AAK1 Kinase Assay: The assays were performed in U-bottom 384-well plates. The final assay volume was 30 μl prepared from 15 μl additions of enzyme and substrates (fluoresceinated peptide (5-FAM)-Aha-KEEQSQITSQVTGQIGWR-NH2 and ATP) and test compounds in assay buffer (10 mM Tris-HCL pH 7.4, 10 mM MgCl2, 0.01% Tween-20 and 1.0 mM DTT). The reactions were initiated by the combination of bacterially expressed, GST-Xa-hAAK1 with substrates and test compounds. The reactions were incubated at room temperature for 3 hours and terminated by adding 60 μl of 35 mM EDTA buffer to each sample. The reactions were analyzed on the Caliper LabChip 3000 (Caliper, Hopkinton, Mass.) by electrophoretic separation of the fluorescent substrate and phosphorylated product. Inhibition data were calculated by comparison to EDTA quenched control reactions for 100% inhibition and vehicle-only reactions for 0% inhibition. The final concentration of reagents in the assays are ATP, 22 μM; (5-FAM)-Aha-KEEQSQITSQVTGQIGWR-NH2, 1.5 μM; GST-Xa-hAAK1, 3.5 nM; and DMSO, 1.6%. Dose response curves were generated to determine the concentration required inhibiting 50% of kinase activity (IC50). Compounds were dissolved at 10 mM in dimethylsulfoxide (DMSO) and evaluated at eleven concentrations. IC50 values were derived by non-linear regression analysis.
Measurement: IC50 = 58 nM (pChEMBL 7.239999771118164)
Descriptors: MW 387.83, ALogP 5.02, HBA 4, HBD 1, PSA 61.03, QED 0.68
Compound (SMILES): C[C@@H](C[C@H]1CN(S(=O)(=O)c2cn(C(F)F)nc2Cl)c2cc(NC(=O)OC(C)(C)C(F)(F)F)ccc2O1)C(=O)O
InChIKey: VJCOTIJZFWLYQR-JQWIXIFHSA-N
Target: Nuclear receptor ROR-gamma (SINGLE PROTEIN), Homo sapiens
UniProt: P51449
Assay: Biochemical TR-FRET Assay: To identify novel antagonists of RORgammaT, an assay was developed which employs the interaction of RORgammaT with its co-activator peptide SRC1_2. This peptide mimics the recruitment of co-activators to RORgammaT through its interaction with the LXXLL (SEQ ID NO:1) (e.g., NR box) motifs (Xie et al., J. Immunol. 175: 3800-09, 2005; Kurebayashi et al., Biochem. Biophys. Res. Commun. 315: 919-27, 2004; Jin et al., Mol. Endocrinology 24:923-29, 2010). The RORγ-Ligand Binding Domain TR-FRET Assay was run according to the following protocol.HIS-tagged RORγ-LBD protein was recombinantly expressed in Escherichia coli. The RORγ-LBD protein was purified by Ni2+-affinity resin. Purified protein was then diluted in assay buffer (50 mM Tris pH 7.0, 50 mM KCl, 1 mM EDTA, 0.1 mM DTT, 100 μg/mL bovine serum albumin, delipidated) to obtain a RORγ-LBD final concentration of 3 nM. Europium tagged anti-HIS antibody was also added to this solution (1.25 nM). Separately, SF9 cells not expressing any recombinant protein were lysed (32,000 cells per μl in 25 mM Tris, 50 mM NaCl) and the previously frozen lysate was added to the diluted RORγ-LBD solution at a ratio of 0.75 μl SF9 lysate per 15 μl of diluted RORγ-LBD.
Measurement: IC50 = 40.3 nM (pChEMBL 7.389999866485596)
Descriptors: MW 604.94, ALogP 4.89, HBA 8, HBD 2, PSA 140.06, QED 0.41
Compound (SMILES): CCC[C@H](NC(=O)Nc1ccc(Br)cc1)C(=O)NCC(=O)OC
InChIKey: SKHUYSZEXQZFCZ-LBPRGKRZSA-N
Target: N-formyl peptide receptor 2 (SINGLE PROTEIN), Homo sapiens
UniProt: P25090
Assay: FLIPR: HEK-Gα16 cells stably expressing the human FPR2 receptor was utilized. Cells were plated into 384-well poly-D-lysine coated plates at a density of 18,000 cells per well one day prior to use. The growth media was DMEM medium supplemented with 10% fetal bovine serum (FBS), 1% antibiotic-antimycotic, 50 μg/ml hygromycin, and 400 μg/ml geneticin. On the day of the experiment, the cells were washed twice with Hank's Balanced Salt Solution supplemented with 20 mM HEPES (HBSS/hepes buffer). The cells were then dye loaded with 2 μM Fluo-4 diluted in the HBSS/Hepes buffer and incubated at 37° C. for 40 minutes. Extracellular dye was removed by washing the cell plates four times prior to placing the plates in the FLIPR (Fluorometric Imaging Plate Reader, Molecular Devices). Ligands were diluted in HBSS/Hepes buffer and prepared in 384-well microplates.
Measurement: EC50 = 16400 nM (pChEMBL 4.789999961853027)
Descriptors: MW 386.25, ALogP 2.03, HBA 4, HBD 3, PSA 96.53, QED 0.63
Compound (SMILES): COc1cccc(S(=O)(=O)Nc2cc(-c3ccnc(NC(C)=O)c3)cnc2C)c1
InChIKey: YJPZKPGERFQUJS-UHFFFAOYSA-N
Target: Phosphatidylinositol 3-kinase catalytic subunit type 3 (SINGLE PROTEIN), Homo sapiens
UniProt: Q8NEB9
Assay: VPS34 Enzyme Assays: 100 nL compounds in DMSO are added to wells of a 384 well microtitre plate (Greiner 780076). At room temperature: 5 ul VPS34 reaction buffer (Invitrogen Assay Buffer Q (diluted 1 in 5 with nanopure water) plus 2 mM DTT and 2 mM MnCl2) containing ATP (20 uM, Promega) and 200 uM PI-PS substrate (Invitrogen PV5122) is added followed immediately by 5 ul VPS34 reaction buffer (as above) containing VPS34 (5 nM, Millennium Protein Sciences Group) and the mixture is incubated with shaking at room temperature for 1 hour. Then 5 ul VPS34 stop-detect mix (as per Invitrogen Adapta Assay kit (PV5009) instructions (contains kinase quench buffer, TR-FRET buffer, Adapta Eu anti-ADP antibody and Alexa Fluor 647 ADP tracer)) is added to quench the reaction. The plates are then incubated for 30 minutes at room temperature with shaking and then read on a BMG PheraStar Plus reader.
Measurement: IC50 < 10 nM (pChEMBL 8)
Descriptors: MW 412.47, ALogP 3.22, HBA 6, HBD 2, PSA 110.28, QED 0.64
Compound (SMILES): NS(=O)(=O)c1cccc(-c2ccc(O)nc2)c1-c1nn[nH]n1
InChIKey: IKFVZDHIEIWFHK-UHFFFAOYSA-N
Target: Metallo-beta-lactamase type 2 (SINGLE PROTEIN), Bacteria
UniProt: C7C422
Assay: Class B Enzyme Assay: The Class B enzyme activities were measured in the presence of the test inhibitor in a fluorescence assay against a commercially available substrate consisting of a cephalosporin core linking 7-hydroxycoumarin to fluorescein (CCF2-FA). The enzyme (NDM-1, IMP-1 or VIM-1) and the substrate were diluted in 100 mM KH2PO4 buffer (pH 7) containing 0.005% Tween-20 and 10 μM ZnSO4. In the assay, the final concentration of enzyme was 1 pM, 2 pM and 30 pM for NDM-1, IMP-1 and VIM-1, respectively, and the final concentration of CCF2-FA was 1.25 μM. The test inhibitor was dissolved in dimethylsulfoxide and diluted 1:50 in the assay, resulting in a final concentration range of 20 uM to 0.00063 uM. In a 384-well microplate, the test inhibitor was incubated with the metallo-β-lactamase enzyme and the substrate for 2 hours at 25° C. Fluorescence at 460 nm following excitation at 405 nm was measured. The IC50 value was determined from semi-logarithmic plots of enzyme inhibition versus inhibitor concentration, with a curve generated using a 4-parameter fit.
Measurement: IC50 = 81.8 nM (pChEMBL 7.090000152587891)
Descriptors: MW 318.32, ALogP 0.28, HBA 7, HBD 3, PSA 147.74, QED 0.63