lovingscience/chembl-v37-raw-activities huggingface dataset:
Compound (SMILES): CC1CCC(NC(=O)c2cc3cc(-c4ccc(OC(F)F)cc4)cnc3n(CCN3CCOCC3)c2=O)CC1
InChIKey: XFGCLOMELVRHGB-UHFFFAOYSA-N
Target: Cannabinoid receptor 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P21554
Assay: CB1 and CB2 Receptor Binding Assay: CB1R binding protocol involves the use of the same solution buffer used for both incubation and washing reaction (Tris-HCl, 50 mM; EDTA, 2.5 mM; MgCl2, 2.5 mM; BSA, 0.5 mg/mL at pH 7.4), 0.4 nM for [3H]CP-55,940, test compounds (concentrations from 0.001 to 10 μM), and finally 8 μg/sample membrane in a total volume of 200 μL. CB2R binding assays were carried out with two different buffers: incubation buffer (Tris-HCl, 50 mM; MgCl2, 5 mM; CaCl2 1 mM; BSA, 0.2% at pH 7.4) and washing buffer (Tris-HCl, 50 mM; NaCl 500 mM; BSA, 0.1% at pH 7.4). The assay mixture contained incubation buffer, 0.4 nM [3H]CP-55,940, test substances (concentrations from 0.001 to 10 μM), and 4 μg/sample membrane in a total assay volume of 200 μL. Assays were performed in duplicate and incubated for 120 mM at 37° C. After the incubation, the assay mixture is filtered through 96 GF/C filter plates (Perkin Elmer #6005174) using Perkin Elmer Filtermate Harvester, and then washed four times with ice-cold washing buffer.
Measurement: IC50 > 10000 nM (pChEMBL 5)
Descriptors: MW 540.61, ALogP 4.31, HBA 7, HBD 1, PSA 85.69, QED 0.46
Compound (SMILES): CCC(CO)CNC(=O)O[C@@H]1CC[C@H](c2cc(NC(=O)Cc3ccnc(OC)c3)n[nH]2)C1
InChIKey: OKGXRGDRQODOSG-MWSTZMHHSA-N
Target: Glycogen synthase kinase-3 beta (SINGLE PROTEIN), Homo sapiens
UniProt: P49841
Assay: Biochemical Assay: The purpose of GSK3β assay is to evaluate the inhibition (% inhibition, Kiapp and Ki values) of small molecule inhibitors by using a fluorescence-based microfluidic mobility shift assay. GSK3β catalyzes the production of ADP from ATP that accompanies the phosphoryl transfer to the substrate peptide FL-Peptide-15 (5-FAM-KRREILSRRPpSYR-COOH, CPC Scientific, Sunnyvale, Calif.) (SEQ ID NO:2). The mobility shift assay electrophoretically separates the fluorescently labeled peptides (substrate and phosphorylated product) following the kinase reaction. Both substrate and product are measured and the ratio of these values is used to generate % conversion of substrate to product by the LabChip EZ Reader. Active GSK3β (H350L) was purchased from Upstate/Millipore. Typical reaction solutions (50 μL final reaction volume) contained 2% DMSO (±inhibitor), 4 mM MgCl2, 1 mM DTT, 40 μM ATP (ATP Km=9.43 μM), 0.005% Tween-20, 2 μM FL-Peptide-15, and 0.6 nM GSK3β in 25 mM HEPES buffer at pH 7.5. The assay was initiated with the addition of ATP, following 15 minutes pre-incubation of enzyme and inhibitor at room temperature in the reaction mixture. The reaction was stopped after 30 minutes at room temperature by the addition of 50 μL of 80 mM EDTA. The Ki value was determined from the fit of the data to the Morrison tight-binding competitive inhibition equation with the enzyme concentration as a variable.
Measurement: Ki = 4.63 nM (pChEMBL 8.329999923706055)
Descriptors: MW 445.52, ALogP 2.38, HBA 7, HBD 4, PSA 138.46, QED 0.44
Compound (SMILES): C[C@H](CO)N(CC(=O)N[C@H](CN(C)C)c1cccc(Cl)c1F)C(=O)Cn1nc(C(N)=O)c2cc(N)ccc21
InChIKey: LAYBRZDNGAFBFW-AUUYWEPGSA-N
Target: Complement factor D (SINGLE PROTEIN), Homo sapiens
UniProt: P00746
Assay: Factor D Esterolytic Assay: An established esterolytic assay for the measurement of Factor D activity and inhibition of Factor D activity was used (Kam, C. M.; McRae, B. J.; Harper, J. W.; Niemann, M. A.; Volanakis, J. E.; Powers, J. C. Human complement proteins D, C2, and B Active site mapping with peptide thioester substrates. J Biol. Chem. 1987, 262, 3444-3451). For this assay Z-Lys-SBzl, 1.29 mM (Kim, S.; Narayana, S. V. L; Volanakis, J. E. Mutational analysis of the substrate binding site of human complement Factor D. Biochemistry. 1994, 33, 14393-14399) was used as the substrate for Factor D (104 mM). Hydrolysis of this compound by Factor D liberated a free sulfhydryl group which is then reacted with 5,5′-dithiobis(2nitrobenzoic acid) producing an intense yellow color (Habeeb, A. F. S. A. Reaction of protein sulfhydryl groups with Ellman's Reagent. Methods in Enzymol. 1976, 25, 457-464). The assays were performed in 96 well microtiter plates and rates of hydrolysis were monitored at 405 nm on a Biotek Synergy H1 plate reader. Hydrolysis rates were reported as change in mOD/min. The assay was conducted in 100 mM HEPES, 500 mM NaCl, pH 7.5 containing 10% DMSO in a final volume of 50 μL per well.
Measurement: IC50 > 10000 nM (pChEMBL 5)
Descriptors: MW 548.02, ALogP 1.14, HBA 8, HBD 4, PSA 159.81, QED 0.26
Compound (SMILES): CC(C)(C)S(=O)(=O)c1ccc(Nc2nn(C3(CC#N)CCOCC3)c3cc[nH]c(=O)c23)cc1
InChIKey: CGCSXHOCRVBMML-UHFFFAOYSA-N
Target: Tyrosine-protein kinase JAK2 (SINGLE PROTEIN), Homo sapiens
UniProt: O60674
Assay: Biochemical HTRF Assay : The ability of compounds to inhibit the activity of JAK1, JAK2, JAK3, and Tyk2 was measured using a recombinant purified GST-tagged catalytic domain for each enzyme (Invitrogen JAK1 #M4290, JAK2 #M4290, JAK3 #M4290, Tyk2 #M4290) in an HTRF format biochemical assay. The reactions employed a common peptide substrate, LCB-EQEDEPEGDYFEWLW-NH2 (in-house). The basic assay protocol is as follows: First, 250 nL of diluted compounds in DMSO were dispensed into the wells of a dry 384-well Black plate (Greiner #781076) using a Labcyte Echo 555 acoustic dispenser. Subsequent reagent additions employed an Agilent Bravo. Next, 18 μL of 1.11× enzyme and 1.11× substrate in 1× assay buffer (Invitrogen kinase buffer #PV3189, 2 mM DTT, 0.05% BSA) were added to the wells and shaken and then preincubated for 30 minutes at room temperature to allow compound binding to equilibrate. After equilibration, 2 μL of 10×ATP in 1× assay buffer was added to initiate the kinase reaction and the plates were shaken and then incubated at room temperature for 120 minutes. At the end of the incubation, 20 μL of 2× stop buffer (streptavidin-Dylight 650 (Thermo #84547B/100 mL), Eu-tagged pY20 antibody (Perkin Elmer #AD0067), EDTA, HEPES, and Triton) was added to quench the reaction. Plates were shaken and centrifuged and then incubated 60 minutes at room temperature and then read on a Perkin Elmer Envision (λex=337 nm, λem=665 and 615 nm, TRF delay time=20 μs). HTRF signal=10,000*665 nm reading/615 nm reading.
Measurement: IC50 = 0.84 nM (pChEMBL 9.079999923706055)
Descriptors: MW 469.57, ALogP 3.46, HBA 8, HBD 2, PSA 129.87, QED 0.59
Compound (SMILES): O=C(c1cccnc1-n1nccn1)N1CC2CC(Nc3cnc(C(F)(F)F)cn3)C1C2
InChIKey: UJMHBRGQUWGCJU-UHFFFAOYSA-N
Target: Orexin/Hypocretin receptor type 1 (SINGLE PROTEIN), Rattus norvegicus
UniProt: P56718
Assay: Radioligand Binding Assay: Human Embryonic Kidney 293 cells (HEK293) stably expressing rat orexin 1 receptor (Genebank accession number NM_001525) or Chinese ovary cells (CHO) stably expressing human orexin 1 receptor (Genebank accession number NM_001526) were grown to confluency in DMEM (Hyclone, cat # SH30022), 10% FBS, 1× Pen/Strep, 1× sodium pyruvate, 10 mM HEPES, 600 μg/mL G418 and DMEM/F12 (Gibco, Cat #11039), 10% FBS, 1× Pen/Strep, 600 μg/mL G418 media, respectively on 150 cm2 tissue culture plates, washed with 5 mM EDTA in PBS (HyClone Dulbecco's Phosphate Buffered Saline 1× with Calcium and Magnesium, Cat # SH30264.01, hereafter referred to simply as PBS) and scraped into 50 ml tubes. After centrifugation (2K×G, 5 min at 4° C.), the supernatant was aspirated and the pellets frozen and stored at −80° C. Cells were resuspended in PBS in the presence of 1 tablet of protease inhibitor cocktail (Roche, Cat. #11836145001) per 50 mL. Each cell pellet from a 15 cm plate was resuspended in 10 mL, stored on ice, and homogenized for 45 sec prior to addition to the reactions. Competition binding experiments in 96 well polypropylene plates were performed using [3H]-(1-(5-(2-fluoro-phenyl)-2-methyl-thiazol-4-yl)-1-((S)-2-(5-phenyl-(1,3,4)oxadiazol-2-ylmethyl)-pyrrolidin-1-yl)-methanone) (Moraveck Corporation, specific activity=35.3 Ci/mmol), diluted to a 10 nM concentration in PBS (4 nM final). Compounds were solubilized in 100% DMSO (Acros Organics, Cat. #61042-1000) and tested over a range of 7 concentrations (from 0.1 nM to 10 μM). The final concentration of DMSO in the reactions is equal to or less than 0.1%. Total and nonspecific binding was determined in the absence and presence of 10 μM almorexant. The total volume of each reaction is 200 μL (20 μL of diluted compounds, 80 μL of [3H]-(1-(5-(2-fluoro-phenyl)-2-methyl-thiazol-4-yl)-1-((S)-2-(5-phenyl-(1,3,4)oxadiazol-2-ylmethyl)-pyrrolidin-1-yl)-methanone) diluted in PBS and 100 μL of the cell suspension). Reactions were run for 60 min at room temperature and terminated by filtration through GF/C filter plates (PerkinElmer, Cat. #6005174) presoaked in 0.3% polyethylenimine using the cell harvester (PerkinElmer Filtermate). The plates were washed 3 times by aspirating 30 ml PBS through the plates. Plates were dried in 55° C. oven for 60 min, scintillation fluid was added, and the radioactivity was counted on a Topcount (Packard).
Measurement: Ki = 191 nM (pChEMBL 6.71999979019165)
Descriptors: MW 430.39, ALogP 2.19, HBA 8, HBD 1, PSA 101.72, QED 0.68
Compound (SMILES): O=C(c1cc(-c2ccc3c(c2)OCO3)on1)N1Cc2ccccc2C1
InChIKey: OWWAFNYKNXRLBS-UHFFFAOYSA-N
Target: Stearoyl-CoA desaturase 5 (SINGLE PROTEIN), Homo sapiens
UniProt: Q86SK9
Assay: Compound Profiling Methods: Strains expressing SCD1 or SCD5 as the sole desaturase, the human SCD1 and SCD5 genes were used to evaluate inhibition of SCD1/SCD5 using reduced growth as a surrogate for SCD inhibition. These yeast strains express human SCD1 or SCD5 from a plasmid harbored in a strain in which the yeast OLE1 gene is deleted.All compound profiling experiments were performed using the same basic protocol. Yeast were cultured using standard techniques in complete synthetic media lacking uracil and containing yeast nitrogen base supplemented with 2% (w/v) glucose (SD-Ura) Starter cultures were inoculated in 3 mL SD-Ura media containing 0.01% tween and 0.2 mM palmitoleic and oleic acid. Cultures were incubated overnight in a 30° C. shaker incubator (225 rpm). Saturated morning cultures were centrifuged, washed in SD-Ura media lacking TWEEN-20 and fatty acids, and then diluted 1:20 in fresh SD-Ura media also lacking TWEEN-20 and fatty acids. Cells were grown for 6 h to an  (optical density) of 0.4-0.8 at 30° C. with shaking.Compound stocks (10 mM in 100% DMSO) were arrayed into 384-round well, v-bottom polypropylene plates and diluted according to indicated dilution factors. Compound administration was performed in two separate steps. First, 15 μL of SD-Ura was dispensed into clear 384-well assay plates using a MULTIDROP Combi reagent dispenser. The diluted compound stock plates were then applied to the assay plates using an automated workstation (Perkin Elmer JANUS) outfitted with a 384-pin tool containing slotted pins that deliver 100 nL of compound. The cultures described above were centrifuged and washed with media lacking TWEEN-20 or oleic and palmitoleic acids. Cultures were then resuspended at a 2-fold concentrated  of 0.02 (final  of 0.0.01) in SD-Ura. 15 μL of diluted culture was then dispensed into the pinned assay plate to achieve 30 μL of the 1× culture (0.01) and a top drug concentration of 33.3 μM.After yeast delivery, assay plates were incubated under humidified conditions at 30° C. for 40 h. Yeast growth was monitored by reading the  of each well using a microplate reader (Perkin Elmer EnVision). Data were analyzed as follows. Raw data were processed by background subtracting and converting values to a percent of the nontreated condition for that strain [(EXP-0.035)/(DMSO-0.035)×100%].
Measurement: IC50 > 45000 nM (pChEMBL 4.3470001220703125)
Descriptors: MW 334.33, ALogP 3.23, HBA 5, HBD 0, PSA 64.8, QED 0.72
Compound (SMILES): CN1C[C@@](C)(c2cc(NC(=O)CC3CC3)ccc2F)NC(=N)C(C)(C)S1(=O)=O
InChIKey: WBHPJNUIYLHJGA-IBGZPJMESA-N
Target: Beta-secretase 2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9Y5Z0
Assay: Time-Resolved Endpoint Proteolysis Assay: Inhibitor IC50s at purified human autoBACE-2 are determined in a time-resolved endpoint proteolysis assay that measures hydrolysis of the QSY7-EISEVNLDAEFC-Eu-amide FRET peptide substrate (SEQ ID 1) (BACE-HTRF assay). BACE-mediated hydrolysis of this peptide results in an increase in relative fluorescence (RFU) at 620 nm after excitation with 320 nm light. Inhibitor compounds, prepared at 3× the desired final concentration in 1×BACE assay buffer (20 mM sodium acetate pH 5.0, 10% glycerol, 0.1% Brij-35) supplemented with 7.5% DMSO are pre-incubated with an equal volume of autoBACE-2 enzyme diluted in 1× BACE assay buffer (final enzyme concentration 1 nM) in black 384-well NUNC plates for 30 minutes at 30° C. The assay is initiated by addition of an equal volume of the QSY7-EISEVNLDAEFC-Eu-amide substrate (SEQ ID 1) (200 nM final concentration, Km=8 μM for 4 μM for autoBACE-2) prepared in 1×BACE assay buffer supplemented with 7.5% DMSO and incubated for 90 minutes at 30° C. DMSO is present at 5% final concentration in the assay. Following laser excitation of sample wells at 320 nm, the fluorescence signal at 620 nm is collected for 400 ms following a 50 μs delay on a RUBYstar HTRF plate reader (BMG Labtechnologies). Raw RFU data is normalized to maximum (1.0 nM BACE/DMSO) and minimum (no enzyme/DMSO) RFU values.
Measurement: Ki = 2190 nM (pChEMBL 5.659999847412109)
Descriptors: MW 410.52, ALogP 2.4, HBA 4, HBD 3, PSA 102.36, QED 0.71
Compound (SMILES): Cc1cccc(-c2cc(Cl)c(F)c(C(=O)NNS(=O)(=O)CC3CNCCN3C)c2)c1
InChIKey: XYLFDTVOOKEYBC-UHFFFAOYSA-N
Target: Histone acetyltransferase KAT6A (SINGLE PROTEIN), Homo sapiens
UniProt: Q92794
Assay: KAT-6A Enzyme Assay: KAT-6A. Enzyme assay buffer was 50 mM Tris pH 8.0, 0.002% Tween20, 0.005% bovine skin gelatin, and 1 mM dithiothreitol (DTT). For determination of IC50values, compounds were serially diluted with 2% (v/v) DMSO in the final reaction, pre-incubating each dilution of each compound with 40 μL of assay buffer containing KAT-6A enzyme (12.5 nM final concentration). 10 μL of assay buffer containing 1 μM peptide substrate and 1 μM acetyl coenzyme A (final concentrations) was added. Reactions (50 μL total) were then carried out at 25° C. for 90 minutes. Reactions were terminated by the addition of 0.5% formic acid (final concentration), and a sample of each reaction was analyzed by SAMDI Tech, Inc. (Chicago, Ill.) using self-assembled monolayer desorption/ionization time-of-flight mass spectrometry (Mrksich, M. (2008) Mass spectrometry of self-assembled monolayers: a new tool for molecular surface science. ACS Nano 2, 7-18).
Measurement: IC50 > 100000 nM (pChEMBL 4)
Descriptors: MW 454.96, ALogP 1.92, HBA 5, HBD 3, PSA 90.54, QED 0.58
Compound (SMILES): CC(=O)N1CC[C@H](Oc2ccc(-c3ncnc(Nc4ccc(S(C)(=N)=O)cc4)n3)cc2C#N)C(F)(F)C1
InChIKey: SUGGCBUBUXYRSO-CTRYDFCHSA-N
Target: Tyrosine-protein kinase JAK2 (SINGLE PROTEIN), Homo sapiens
UniProt: O60674
Assay: Biological for hJAK2: Enzymatic activity of hJAK2 was measured using a LANCE homogeneous time resolved fluorescence resonance energy transfer (TR-FRET) assay that monitors enzyme dependent phosphorylation of a biotinylated serine/threonine peptide substrate (PTK). An increase in the amount of phopshorylated peptide results in an increase in TR-FRET signal. hJAK2 was expressed and purified as full length recombinant proteins. Detection reagents for the assay were purchased from PerkinElmer. hJAK2 enzyme was assayed under initial rate conditions in the presence of 2×Km ATP (30 μM) and 1 μM peptide, 50 mM Tris-Cl (pH 7.5), 10 mM MgCl2, 1mM dithiothreitol, 0.01% BSA, 0.05% (v/v) DMSO at the following concentration of enzyme: hJAK2 at 0.3 nM. After an assay reaction time of 40 minutes at 25° C., reactions were terminated with EDTA and LANCE Detection Buffer.Amount of phosphorylated peptide was determined by the addition of 20 nM SA-APC and 1 nM europium cryptate labeled anti-phospho PTK monoclonal antibody PY66 and the resulting TR-FRET signal was recorded on an Envision plate reader (Ex: 340 nm; Em: 615/665 nm; 100 μs delay and 200 μs read window). Data was normalized based on a positive (1 μM Staurosporine) and negative (DMSO) controls and IC50 values calculated from the fit of the doseresponse curves to a four-parameter equation. All IC50 values represent geometric mean values of a minimum of four determinations. These assays generally produced results within 3-fold of the reported mean.
Measurement: IC50 = 320 nM (pChEMBL 6.5)
Descriptors: MW 527.56, ALogP 3.82, HBA 9, HBD 2, PSA 144.95, QED 0.49
Compound (SMILES): CN(c1nc(N2CC(OC(=O)C3CCS(=O)(=O)CC3)C2)nc2c1[S+]([O-])CC2)C1CCOCC1
InChIKey: GWGMGGRSNWDNNB-UHFFFAOYSA-N
Target: Tumor necrosis factor (SINGLE PROTEIN), Homo sapiens
UniProt: P01375
Assay: TNF-alpha RELEASE: Human peripheral blood mononuclear cells (PBMC) were isolated from fresh buffy coats by density centrifugation using lymphoprep tubes (Medinor). Frozen PBMC's were washed in serum free assay buffer (RPMI1640 with 25 mM HEPES, 1% pen/strep, 200 mM L glutamine, 0.5% human serum albumin) and living cells counted. Lipopolysaccharide (1 microg/ml; SIGMA) was added to the cells which were then transferred to 384 well tissue culture plates (5×105 c/ml) containing titrated test compounds. The cells were incubated for 18 hours at 37° C. in serum free assay buffer and the level of TNFalpha in the supernatant was quantitated by AlphaLISA (PerkinElmer) by measuring fluorescence intensity at 615 nm.Results were expressed as IC50 values calculated from inhibition curves using as controls the secretion in LPS stimulated wells and the secretion in cells incubated with 10 microM of a PDE4 inhibitor reference compound.
Measurement: IC50 = 300 nM (pChEMBL 6.519999980926514)
Descriptors: MW 498.63, ALogP 0.31, HBA 10, HBD 0, PSA 124.99, QED 0.41
Compound (SMILES): CC(C)NC(=O)Cn1cc(-c2ccc(CCN3CCC(O)CC3)cc2)nc1-c1cccc(Cl)c1
InChIKey: RWONUQUBUQNOIA-UHFFFAOYSA-N
Target: Vasopressin V1b receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P47901
Assay: Binding Assay: Human V1b receptor was transiently expressed in 293FT cells (Invitrogen). The cells were collected and then homogenated in a 15 mmol/L tris-hydrochloric acid buffer (pH 7.4 and containing 2 mmol/L magnesium chloride, 0.3 mmol/L ethylenediaminetetracetic acid, and 1 mmol/L glycol ether diaminetetraacetic acid). The resulting homogenate was centrifuged at 50,000xg at 4° C. for 20 minutes. The precipitate was resuspended in a 75 mmol/L tris-hydrochloric acid buffer (pH 7.4 and containing 12.5 mmol/L magnesium chloride, 0.3 mmol/L ethylenediaminetetracetic acid, 1 mmol/L glycol ether diaminetetraacetic acid, and 250 mmol/L sucrose) to give a crude membrane preparation, which was stored at â¿¿80° C. until the binding test was initiated. In the binding test, the crude membrane preparation was diluted with a 50 mmol/L tris-hydrochloric acid buffer (pH 7.4 and containing 10 mmol/L magnesium chloride and 0.1% bovine serum albumin) and mixed with each test compound and [3H]AVP (final concentration: 0.4 to 1 nmol/L), followed by incubation at room temperature for 60 minutes. The test compound was serially diluted with DMSO so that it would have final concentrations of 0.01 nmol/L to 1 umol/L at the time of mixing. After the incubation, the mixture was suction filtered through a GF/C filter that was preliminarily impregnated with 0.3% polyethyleneimine. The GF/C filter was dried and after adding a scintillator, the residual radioactivity on the filter was measured using TopCount (PerkinElmer Inc.). The radioactivity in the presence of unlabeled AVP at 10 mmol/L was defined as 0%, and the radioactivity in the absence of unlabeled AVP was defined as 100%. A dose-response curve was plotted from radio activities in the presence of a test compound at various concentrations, and the 50% inhibitory concentration (IC50 value) of the test compound was calculated. The IC50 values of the compounds of the present invention were in the range of 0.1 to 1000 nM.
Measurement: IC50 = 55 nM (pChEMBL 7.260000228881836)
Descriptors: MW 481.04, ALogP 4.39, HBA 5, HBD 2, PSA 70.39, QED 0.5
Compound (SMILES): COc1ccc(CN2C3CC2CN(c2ccc(-c4cc(OC5CCN(C)CC5)cn5ncc(C#N)c45)cn2)C3)cn1
InChIKey: XZHHPRQRTYTTHU-UHFFFAOYSA-N
Target: Proto-oncogene tyrosine-protein kinase receptor Ret (SINGLE PROTEIN), Homo sapiens
UniProt: P07949
Assay: RET Enzyme Assay: Compounds of Formula I were screened for their ability to inhibit wildtype and V804M mutant RET kinase using CisBio's HTRF® KinEASE¿-TK assay technology. Briefly, N-terminal GST tagged recombinant human RET cytoplasmic domain (aa 658-end) from Eurofins (0.25 nM RET; Catalog No. 14-570M) or N-terminal GST tagged recombinant human V804M mutant RET cytoplasmic domain (aa 658-end) from Millipore (0.25 nM enzyme; Catalog No. 14-760) was incubated with 250 nM TK-substrate biotin (CisBio, part of Catalog No. 62TK0PEC) and 1 mM ATP along with test compound in a buffer consisting of 25 mM HEPES pH 7.4, 10 mM MgCl2, 0.01% Triton X-100, and 2% DMSO in a volume of 8 μL. Compounds were typically prepared in a threefold serial dilution in DMSO and added to the assay to give the appropriate final concentration. After a 30-minute incubation at 22° C., the reaction was quenched by adding 8 μL of quench solution containing 31.25 nM Sa-XL665 and 1×TK-ab-Cryptate in HTRF detection buffer (all from CisBio, part of Cat. No. 62TK0PEC). After a 1 hour incubation at 22° C., the extent of reaction was determined using a PerkinElmer EnVision multimode plate reader via HTRF dual wavelength detection, and the percent of control (POC) was calculated using a ratiometric emission factor. 100 POC was determined using no test compounds and 0 POC was determined using pre-quenched control reactions. The POC values were fit to a 4 parameter logistic curve, and the IC50 is defined as the concentration of inhibitor at which the POC equals 50 for the fitted curve.
Measurement: IC50 = 434 nM (pChEMBL 6.360000133514404)
Descriptors: MW 550.67, ALogP 3.61, HBA 10, HBD 0, PSA 95.05, QED 0.34
Compound (SMILES): Nc1nc2c(ncn2[C@@H]2O[C@@H]3COP(=O)(S)OCC(F)(F)Cn4c(nc5cncnc54)COP(=O)(S)O[C@@H]2[C@@H]3F)c(=O)[nH]1
InChIKey: FZXXRNQUODHWKP-DONFFWFGSA-N
Target: Stimulator of interferon genes protein (SINGLE PROTEIN), Homo sapiens
UniProt: Q86WV6
Assay: Binding Assay: A human STING [3H] 2′,3′-cGAMP scintillation proximity competition binding assay was utilized to detect binding of compounds to STING protein. The competition binding assay was conducted with either a wild-type (WT, SEQ ID NO: 1) or an AQ variant (SEQ ID NO: 2) of truncated human STING protein that lacks the N-terminal four transmembrane domains and contains amino acids 155-379. The protein encoding plasmid contained a HIS tag, followed by an Avi tag and then a SUMO tag, which were followed by the STING coding sequence within the pET-28a (+) vector. The plasmid was transfected into a BL21 (DE3)/BirA E. coli co-expression strain. Protein was purified from the bacterial lysate using a cobalt-affinity column. The purified protein was dialyzed against PBS and then 20% glycerol was added prior to storage at −80° C.The assay was conducted in a 384-well plate containing final concentration of 25 nM biotin-STING protein and 17.5 nM [3H] 2′,3′-cGAMP in assay buffer (50 mM Tris pH 7.5, 100 mM NaCl, 0.1% Fatty Acid Free BSA). Test compounds (2.5 μL) were added to the plate first followed by a 25 μL mixture of STING protein, streptavidin PVT SPA beads (Perkin Elmer) and 10 μL [3H] 2′,3′-cGAMP. Plates were incubated at room temperature for 18 hrs and read on a Wallac MicroBeta TriLux (Perkin Elmer). 2′,2′-cGAMP was used as a reference compound and had an IC50 of 0.562 μM using the WT STING protein and 0.098 M using the AQ STING protein.
Measurement: IC50 = 5500 nM (pChEMBL 5.260000228881836)
Descriptors: MW 685.5, ALogP 2.44, HBA 16, HBD 4, PSA 213.48, QED 0.17
Compound (SMILES): Nc1ncnc2ccc(-c3cccnc3-c3cccc(C(F)F)n3)nc12
InChIKey: DBWHTIBDVVUYEM-UHFFFAOYSA-N
Target: TGF-beta receptor type-2 (SINGLE PROTEIN), Homo sapiens
UniProt: P37173
Assay: Biological Assay: Assays for the compounds reported below were conducted in 1536-well plates and 2 mL reactions are prepared from addition of HIS-TGFβR1 T204D or HIS-TGFβR2 WT, anti-HIS detection antibody, a labeled small molecule probe (Kd=<100 nM; koff=<0.001 s−1.) and test compounds in assay buffer (20 mM HEPES pH 7.4, 10 mM MgCl2, 0.015% Brij35, 4 mM DTT, and 0.05 mg/ml BSA). The reaction is incubated for 1 hour at room temperature and the HTRF signal was measured on an Envision plate reader (Ex: 340 nm; Em: 520 nm/495 nm). 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 reagents in the assay are 1 nM HIS-TGFβR1 T204D or HIS-TGFβR2 WT, 0.2 nM anti-HIS detection antibody, labeled small molecule probe (at Kd) and 0.5% DMSO. 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.
Measurement: IC50 > 15000 nM (pChEMBL 4.823999881744385)
Descriptors: MW 350.33, ALogP 3.67, HBA 6, HBD 1, PSA 90.47, QED 0.61
Compound (SMILES): CC(C)(CO)CN1Cc2cc(Nc3nn([C@H]4CCCC[C@@H]4C#N)c4cc[nH]c(=O)c34)ccc2S1(=O)=O
InChIKey: HQONIIZFENJQET-APWZRJJASA-N
Target: Tyrosine-protein kinase JAK2 (SINGLE PROTEIN), Homo sapiens
UniProt: O60674
Assay: Biochemical HTRF Assay Protocol: The ability of compounds to inhibit the activity of JAK1, JAK2, JAK3, and Tyk2 was measured using a recombinant purified GST-tagged catalytic domain for each enzyme (Invitrogen JAK1 #M4290, JAK2 #M4290, JAK3 #M4290, Tyk2 #M4290) in an HTRF format biochemical assay. The reactions employed a common peptide substrate, LCB-EQEDEPEGDYFEWLW-NH2 (in-house). The basic assay protocol is as follows: First, 250 nL of diluted compounds in DMSO were dispensed into the wells of a dry 384-well Black plate (Greiner #781076) using a Labcyte Echo 555 acoustic dispenser. Subsequent reagent additions employed an Agilent Bravo. Next, 18 μL of 1.11× enzyme and 1.11× substrate in 1× assay buffer (Invitrogen kinase buffer #PV3189, 2 mM DTT, 0.05% BSA) were added to the wells and shaken and then preincubated for 30 minutes at room temperature to allow compound binding to equilibrate. After equilibration, 2 μL of 10×ATP in 1× assay buffer was added to initiate the kinase reaction and the plates were shaken and then incubated at room temperature for 120 minutes. At the end of the incubation, 20 μL of 2× stop buffer (streptavidin-Dylight 650 (Thermo #84547B/100 mL), Eu-tagged pY20 antibody (Perkin Elmer #AD0067), EDTA, HEPES, and Triton) was added to quench the reaction. Plates were shaken and centrifuged and then incubated 60 minutes at room temperature and then read on a Perkin Elmer Envision (λex=337 nm, λem=665 and 615 nm, TRF delay time=20 μs). HTRF signal=10,000*665 nm reading/615 nm reading. After normalization to untreated controls, the percent inhibition of the HTRF signal at each compound concentration was calculated.
Measurement: IC50 = 850 nM (pChEMBL 6.070000171661377)
Descriptors: MW 510.62, ALogP 3.25, HBA 8, HBD 3, PSA 144.11, QED 0.46
Compound (SMILES): CC1C(C(C)(C)O)CCN1c1cc(-n2ncc3ccc([C@@]4(C#N)CC45CC5)cc32)ncn1
InChIKey: QEEQAZIZVOESQO-QKJKHRQNSA-N
Target: Leucine-rich repeat serine/threonine-protein kinase 2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q5S007
Assay: LanthaScreen Assay: The LRRK2 kinase activity reported herein as IC50 values was determined with LanthaScreen technology from Life Technologies Corporation (Carlsbad, Calif.) using GST-tagged truncated human mutant G2019S LRRK2 in the presence of the fluorescein-labeled peptide substrate LRRKtide, also from Life Technologies. The data presented for the Km ATP LanthaScreen Assay represents mean IC50 values based on several test results and may have reasonable deviations depending on the specific conditions and reagents used. Assays were performed in the presence of 134 μM ATP (Km ATP). Upon completion, the assay was stopped and phosphorylated substrate detected with a terbium (Tb)-labeled anti-pERM antibody (cat. no. PV4898). The compound dose response was prepared by diluting a 10 mM stock of compound to a maximum concentration of 9.99 M in 100% dimethylsulfoxide followed by custom fold serial dilution in dimethylsulfoxide nine times. Twenty nanoliters of each dilution was spotted via a Labcyte Echo onto a 384-well black-sided plate (Corning 3575) followed by 15 μl of a 1.25 nM enzyme solution in 1× assay buffer (50 mM Tris pH 8.5, 10 mM MgCl2, 0.01% Brij-35, 1 mM EGTA, 2 mM dithiothreitol, 0.05 mM sodium orthovanadate). Following a 15-minute incubation at room temperature, the kinase reaction was started with the addition of 5 μl of 400 nM fluorescein-labeled LRRKtide peptide substrate and 134 μM ATP solution in 1× assay buffer. The reaction was allowed to progress at ambient temperature for 90 minutes. The reaction was then stopped by the addition of 20 μl of TR-FRET Dilution Buffer (Life Technologies, Carlsbad, Calif.) containing 2 nM Tb-labeled anti-phospho LRRKtide antibody and 10 mM EDTA (Life Technologies, Carlsbad, Calif.). After an incubation of 1 hour at room temperature, the plate was read on an EnVision multimode plate reader (Perkin Elmer, Waltham, Mass.) with an excitation wavelength of 337 nm (Laser) and a reading emission at both 520 and 495 nm. Compound IC50s were interpolated from nonlinear regression best fits of the log of the final compound concentration, plotted as a function of the 520/495-nm emission ratio using Activity base. Abase uses a 4 parameter (4P) logistic fit based on the Levenberg-Marquardt algorithm.
Measurement: IC50 = 1.9 nM (pChEMBL 8.720000267028809)
Descriptors: MW 428.54, ALogP 3.75, HBA 7, HBD 1, PSA 90.86, QED 0.68
Compound (SMILES): O=C(Cn1c(=O)[nH]c2ncc(-c3cc(OCCF)cc(C(F)(F)F)c3)cc21)N1CCC1
InChIKey: WMIBMWDMMZPQRG-UHFFFAOYSA-N
Target: Glutamate NMDA receptor; GRIN1/GRIN2B (PROTEIN COMPLEX), Homo sapiens
UniProt: Q05586
Assay: Biological Assay (standard): NMDA receptors are ion channels that are highly permeable to Ca2+ ions, rendering it possible to monitor NMDA receptor function using cell-based calcium flux assay. In this assay, co-agonists glutamate and glycine are added to cells heterologously expressing human GluN1/GluN2B NMDA receptors to initiate cellular Ca2+ influx. The time course of the changes in intracellular calcium is measured using a fluorescent dye and a FLIPR (Fluorometric Imaging Plate Reader) device.Twenty four hours before measurements, the expression of the NMDA receptors in the stable cell line is induced with Tet-On inducible system in the presence of a non-selective NMDA receptor blocker. On the day of the experiment, cell culture media is carefully washed and the cells are loaded with Calcium 5 Dye Kit (Molecular Devices) in dye loading buffer containing 137 mM NaCl, 4 mM KCl, 2 mM CaCl2), 0.5 mM MgCl2, 10 mM HEPES and 5 mM D-glucose; pH 7.4. After 1 h incubation at the room temperature, the dye is washed away with the assay buffer (137 mM NaCl, 4 mM KCl, 2 mM CaCl2), 0.01 mM EDTA, 10 mM HEPES and 5 mM D-glucose; pH 7.4) In the FLIPR TETRA reader, various concentrations of the test compounds are added to the cells for 5 min while fluorescence is monitored to detect potential agonist activity. Next, co-agonists, glutamate and glycine are added for another 5 minutes. The concentration of glutamate corresponding to ~EC40 used to maximize the assay's signal window and ability to detect NMDA receptor antagonists and negative allosteric modulators. A saturating concentration (10 μM) of glycine is also present in the assay. A non-selective NMDA receptor antagonist, (+)MK-801 is used as a positive control for antagonist activity. The fluorescent signal in the presence of test compounds is quantified and normalized to the signal defined by the appropriate control wells.
Measurement: IC50 = 333 nM (pChEMBL 6.480000019073486)
Descriptors: MW 438.38, ALogP 2.99, HBA 5, HBD 1, PSA 80.22, QED 0.6
Compound (SMILES): COC1(C(=O)N[C@@H](C)c2ccc(Cl)cc2)CN(c2ncnn3cc(-c4cnn(C)c4)cc23)C1
InChIKey: RUDMTIRUTGDKSK-HNNXBMFYSA-N
Target: Mast/stem cell growth factor receptor Kit (SINGLE PROTEIN), Homo sapiens
UniProt: P10721
Assay: Kit D816V Assay: In each well of a 384-well plate, 0.04 ng/ul (0.5 nM) of D816V Kit (Carna Bioscience 08-156) was incubated in a total of 12.5 ul of buffer (100 mM HEPES pH 7.5, 0.015% Brij 35, 10 mM MgCl2, 1 mM DTT) with 1 uM Srctide (5-FAM-GEEPLYWSFPAKKK-NH2) and 15 uM ATP at 25 C for 90 minutes in the presence or absence of a dosed concentration series of compound (1% DMSO final concentration). The reaction was stopped by the addition of 70 ul of Stop buffer (100 mM HEPES pH 7.5, 0.015% Brij 35, 35 mM EDTA and 0.2% of Coating Reagent 3 (Caliper Lifesciences)). The plate was then read on a Caliper EZReader 2 (protocol settings: −1.9 psi, upstream voltage −700, downstream voltage −3000, post sample sip 35s).
Measurement: IC50 = 550 nM (pChEMBL 6.260000228881836)
Descriptors: MW 465.95, ALogP 2.87, HBA 8, HBD 1, PSA 89.58, QED 0.47
Compound (SMILES): CCOC(=O)N1Cc2cc(Cl)ccc2-n2c(C3CC4(C3)CN(c3ncc(F)cn3)C4)nnc2C1C
InChIKey: INDATFYLTWKBKV-UHFFFAOYSA-N
Target: Vasopressin V1b receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P47901
Assay: Vasopressin V1B Receptor Antagonist Assay: The purpose of the assay is to determine the inhibitory effect of synthesized compounds on the Vasopressin V1b receptor. The assay was performed in Chinese Hamster Ovary (CHO) cells expressing the human Arginine Vasopressin Receptor 1b (AVPR1b). Arginine Vasopressin (AVP) evokes an increase in intracellular calcium in CHO-AVPR1b cells which is measured in a fluorescence assay on the FLIPRTETRA using calcium sensitive dyes. Test compounds were assessed for their ability to affect the magnitude of the response to AVP, with antagonists showing a concentration-dependent reduction in the AVP-mediated fluorescence.CHO-AVPR1b cells were maintained in routine culture in T175 Flasks at 37° C., 5% CO2. The growth medium consisted of Ham's F12 media supplemented with 10% v/v fetal bovine serum, 1× non-essential amino acids, and 0.4 mg/ml Geneticin G418.
Measurement: IC50 > 1000 nM (pChEMBL 6)
Descriptors: MW 497.96, ALogP 4.27, HBA 8, HBD 0, PSA 89.27, QED 0.53
Compound (SMILES): N[C@@H](c1ccc(-c2cccnc2)cc1)[C@](O)(Cc1cc(OC2CCCCC2)ccn1)C(=O)O
InChIKey: AWWHKHPXABJLRH-AZGAKELHSA-N
Target: Leucyl-cystinyl aminopeptidase (SINGLE PROTEIN), Homo sapiens
UniProt: Q9UIQ6
Assay: Inhibition of Human P-LAP (hP-LAP) Assay: HEK293 cells forced to transiently express hP-LAP (J Biol Chem 1996; 271: 56-61) were prepared by lipofection, homogenized, and then subjected to ultracentrifugation at 100,000×g for 30 minutes. Microsomes containing hP-LAP were thereby prepared. The microsomes (with a total protein content of 0.5 to 1.5 μg/well) were mixed with a solvent (DMSO; final concentration: 0.1%) or with each test compound (common ratio: 3; maximum concentration: 10 μM). AVP was then added to the solution into a final concentration of 25 μM, and the resulting solution was allowed to react for one hour at 37° C. An aqueous TFA solution was then added to the solution (final concentration: 1%) to stop the enzymatic reaction. Residual AVP was then determined by mass spectrometry (MALDI-MS). Based on the results, IC50 values (nM), i.e. concentrations required for 50% inhibition of decrease in AVP level in the solvent control group, of the individual test compounds were calculated by logistic regression to evaluate inhibition of human P-LAP (hP-LAP) activity. The results are shown in Tables 1 and 2 and indicate that the example compounds effectively inhibit AVP degradation by hP-LAP.
Measurement: IC50 = 1.8 nM (pChEMBL 8.739999771118164)
Descriptors: MW 447.54, ALogP 3.91, HBA 6, HBD 3, PSA 118.56, QED 0.48
Compound (SMILES): COc1cc(F)ccc1-c1cnc2[nH]c(=O)n(Cc3ccc(F)cc3)c2c1
InChIKey: HFXRBYHYHTVUSY-UHFFFAOYSA-N
Target: Glutamate NMDA receptor; GRIN1/GRIN2B (PROTEIN COMPLEX), Homo sapiens
UniProt: Q05586
Assay: Biological Assay (HTS): NMDA receptors are ion channels that are highly permeable to Ca2+ ions, rendering it possible to monitor NMDA receptor function using cell-based calcium flux assay. In this assay, co-agonists glutamate and glycine are added to cells heterologously expressing human GluN1/GluN2B NMDA receptors to initiate cellular Ca2+ influx. The time course of the changes in intracellular calcium is measured using a fluorescent dye and a FLIPR (Fluorometric Imaging Plate Reader) device. Twenty four hours before measurements, the expression of the NMDA receptors in the stable cell line is induced with Tet-On inducible system in the presence of a non-selective NMDA receptor blocker. On the day of the experiment, cell culture media is carefully washed and the cells are loaded with Calcium 5 Dye Kit (Molecular Devices) in dye loading buffer containing 137 mM NaCl, 4 mM KCl, 2 mM CaCl2), 1.5 mM MgCl2, 10 mM HEPES and 5 mM D-glucose; pH 7.4. After 1 h incubation at the room temperature, the dye is washed away with the assay buffer (150 mM, 3 mM, 2 mM CaCl2), 0.01 mM EDTA, 10 mM HEPES and 5 mM D-glucose; pH 7.4) In the FLIPR TETRA reader, various concentrations of the test compounds are added to the cells for 5 min while fluorescence is monitored to detect potential agonist activity. Next, co-agonists, glutamate and glycine are added for another 5 minutes. The concentration of glutamate corresponding to EC40 is used to maximize the assay's signal window and ability to detect NMDA receptor antagonists and negative allosteric modulators. A saturating concentration (10 μM) of glycine is also present in the assay. A non-selective NMDA receptor antagonist, (+)MK-801 is used as a positive control for antagonist activity. The fluorescent signal in the presence of test compounds is quantified and normalized to the signal defined by the appropriate control wells.
Measurement: IC50 = 790 nM (pChEMBL 6.099999904632568)
Descriptors: MW 367.36, ALogP 3.73, HBA 4, HBD 1, PSA 59.91, QED 0.6
Compound (SMILES): Fc1cc(F)cc(Nc2ncc(-c3cnn(C4CCNCC4)c3)c(Nc3ccc4c(c3)CNCC4)n2)c1
InChIKey: YULMPWFRJYANBE-UHFFFAOYSA-N
Target: Tyrosine-protein kinase receptor UFO (SINGLE PROTEIN), Homo sapiens
UniProt: P30530
Assay: Verification on Inhibitory Activities of TAM Receptor Inhibiting Compounds on Tyro 3, Axl, and Mer: For evaluation of inhibitory activities of the compounds according to the present invention on Tyro 3, Axl, and Mer, the following test was carried out.The specific test method followed the method provided by Cisbio. The compounds of the examples were prepared at various concentrations, followed by addition of Tyro 3, Axl, or Mer and substrate peptides, and then ATP was added to initiate a reaction. After 1 hour, the reaction was stopped by addition of a solution containing EDTA. Thereafter, the amount of phosphorylated peptides was measured. Here, the phosphorylated peptides were treated with an europium (Eu)-labeled antibody recognizing phosphorylated peptides, and then after 1 hour, excited by irradiation of the light of a wavelength of 320 or 340 nm using an Envision Reader. The amount of light emitted at 665 nm was measured to investigate the degrees of inhibition of TAM receptors. The TAM receptor inhibitory activities (IC50 values) of the respective compounds were analyzed using the GraphPad Prism program, and tabulated in Tables 1 to 3 below. LDC1267 (CAS no. 1361030-48-9, Calbiochem), which is a TAM receptor inhibitor compound, was used as a positive control.
Measurement: IC50 = 41 nM (pChEMBL 7.389999866485596)
Descriptors: MW 502.57, ALogP 4.68, HBA 8, HBD 4, PSA 91.72, QED 0.3
Compound (SMILES): Brc1ccc(-c2cn3cccc(-c4nnc(Nc5ccc6[nH]ncc6c5)s4)c3n2)cc1
InChIKey: WZZGNGXQIDMNMZ-UHFFFAOYSA-N
Target: Rho-associated protein kinase 2 (SINGLE PROTEIN), Homo sapiens
UniProt: O75116
Assay: Biological Assays for Inhibition of Rho-Associated Protein Kinase: Assays for ROCK inhibition were performed using the following protein constructs: glutathione S-transferase (GST)-tagged human ROCK1 catalytic domain 1-477 from Carna Biosciences (cat #01-109; apparent Km value for ATP is 10 μM) and GST-tagged human ROCK2 catalytic domain 1-553 from Carna Biosciences (Cat#01-110; apparent Km value for ATP is 15 μM). Protein constructs were purified from a baculovirus expression system. The peptide substrate was fluorescent LANCE® Ultra ULight-CREBtide: CKRREILSRRPSYRK (PerkinElmer, # TRF0107-D). Kinase reactions were carried out in in a 10 μL volume in 384-well plates: 50 nM ULight-CREBtide substrate, 2 nM constitutively active ROCK1 or ROCK2 kinase, and test compound in DMSO (or DMSO only for controls) were diluted into assay buffer containing 50 mM Tris-HCl (pH=7.5), 10 mM MgCl2, 1 mM EGTA, 0.01% Tween-20, and 2 mM DTT such that the final concentration of DMSO was 0.5%. After a 90 minute incubation at room temperature on a shaker table, the kinase reaction was stopped by addition of 10 mM EDTA, and phosphorylation of the substrate was detected by adding 1 nM LANCE® Ultra Europium-anti-phospho-CREB (ser133) antibody (PerkinElmer, # TRF0200-D) and incubating for 60 minutes on a shaker at room temperature. The flurorescence resonance energy transfer (FRET) signals were read and analyzed on an Envision¿ 2103 Multilabel Reader (Perkin Elmer). The concentration of test compound required to inhibit substrate phosphorylation by 50% (the IC50) was calculated by non-linear regression using GraphPad PRIZM.
Measurement: IC50 < 1000 nM (pChEMBL 6)
Descriptors: MW 488.37, ALogP 5.9, HBA 7, HBD 2, PSA 83.79, QED 0.32
Compound (SMILES): CN(CC(F)(F)F)S(=O)(=O)C[C@H]1C[C@@H](N(C)c2ncnc3[nH]ccc23)C1
InChIKey: MRBWMMOSKVWAGW-PHIMTYICSA-N
Target: Tyrosine-protein kinase JAK2 (SINGLE PROTEIN), Homo sapiens
UniProt: O60674
Assay: Caliper Enzyme Assay: 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, 1 mM ATP and 1 μM peptide substrate. The JAK1 and TYK2 assays contained 1 μM of the IRStide peptide (5FAM-KKSRGDYMTMQID) and the JAK2 and JAK3 assays contained 1 μM of the JAKtide peptide (FITC-KGGEEEEYFELVKK). The assays were initiated by the addition of 20 nM JAK1, 1 nM JAK2, 1 nM JAK3 or 1 nM TYK2 enzyme and were incubated at room temperature for three hours for JAK1, 60 minutes for JAK2, 75 minutes for JAK3 or 135 minutes for TYK2. 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 = 162 nM (pChEMBL 6.789999961853027)
Descriptors: MW 391.42, ALogP 2, HBA 5, HBD 1, PSA 82.19, QED 0.81
Compound (SMILES): FC1(CNC2CC2c2ccccc2)CCN(CCCC2CCCCC2)CC1
InChIKey: NGCAFPIUAYVGON-UHFFFAOYSA-N
Target: Amine oxidase [flavin-containing] A (SINGLE PROTEIN), Homo sapiens
UniProt: P21397
Assay: MAOA and MAOB In Vitro Activity Experiments: Screening method: Monoamine oxidase MAOA and MAOB activity screeningInstrument: microplate reader Envision (PerkinElmer, USA).MATERIALS: Human recombinant MAOA, purchased from Promega; human recombinant MAOB, purchased from Sigma;The MAOA and MAOB activity assay kit MAO-Glo was purchased from Promega. Principle: This method uses a specific luciferin derivative as a substrate, MAOA or MAOB can catalyze the conversion of substrate to luciferin methyl ester, and the product, luciferin methyl ester can produce fluorescence under the action of luciferase, thus reflecting the activity of MAOA or MAOB by the intensity of the fluorescent signal. Meanwhile, a blank control was set to determine the strength of the enzyme activity. Tranylcypromine (TCP) was employed as a positive inhibitor in the experiment.Sample processing: Samples were dissolved in DMSO, stored at low temperature, and the concentration of DMSO in the final system was controlled within a range that won't affect the activity of the assay.The activity of the sample was tested by primary screening at a single concentration, for example 100 μM. For samples exhibiting activity under certain conditions, for example, the inhibition rate (% Inhibition) being greater than 50, the active dose-dependent relationship, i.e., the IC50 value, was obtained by nonlinearly fitting the sample activity vs the sample concentration, the software used for the calculation was Graphpad Prism 5, the model used for the fit was sigmoidal dose-response (variable slope), and for most inhibitor screening models, the bottom and top of the fitted curve were set to 0 and 100.
Measurement: IC50 > 100000 nM (pChEMBL 4)
Descriptors: MW 372.57, ALogP 5.3, HBA 2, HBD 1, PSA 15.27, QED 0.67
Compound (SMILES): CN(c1cc(F)c(S(=O)(=O)Nc2cscn2)cc1Cl)[C@H]1CCN(Cc2ccc[nH]2)C1
InChIKey: KNGYMSOCAAKEER-AWEZNQCLSA-N
Target: Sodium channel protein type 1 subunit alpha (SINGLE PROTEIN), Homo sapiens
UniProt: P35498
Assay: Sodium Influx Assay (In Vitro Assay): This sodium influx assay employs the use of the cell permeable, sodium sensitive dye ANG2 to quantify sodium ion influx through sodium channels which are maintained in an open state by use of sodium channel modulators. This high throughput sodium influx assay allows for rapid profiling and characterization of sodium channel blockers.In general, Trex HEK293 cells were stably transfected with an inducible expression vector containing the full-length cDNA coding for the desired human sodium channel α-subunit and with an expression vector containing full length cDNA coding for the β1-subunit. Sodium channel expressing cell lines were induced with tetracycline (1 μg/mL) and plated on 384-well PDL-coated plates at a density of 25K-30K cells/well in culture media (DMEM, containing 10% FBS and 1% L-glutamine). After overnight incubation (37° C., 5% CO2), culture media was removed and cells were loaded with 5 uM ANG2 dye for 1-1.5 h in Buffer 1 (155 mM NMDG, 5 mM KCl, 2 mM CaCl2, 1 mM MgCl2, 10 mM HEPES, 10 mM glucose, adjusted with Tris to pH 7.4). Access dye was removed and cells were incubated with test compounds for 1 hr in buffer 1 containing sodium channel modulator(s) at room temperature. Hamamatsu FDSS ρCell was used to perform a 1:1 addition of Na/K challenge buffer (140 mM NaCl, 20 mM HEPES, 1 mM CaCl2, 15 mM KCl, 1 mM MgCl2, 10 mM glucose, adjusted with Tris to pH 7.4) and simultaneously read plates at excitation wavelength of 530 nm and emission wavelength set at 558 nm. Percent inhibition of sodium ion influx was calculated for each test compound at each test concentration to determine the IC50 values.
Measurement: IC50 = 25069 nM (pChEMBL 4.599999904632568)
Descriptors: MW 470, ALogP 3.78, HBA 6, HBD 2, PSA 81.33, QED 0.55
Compound (SMILES): CNC(=O)c1cncc(-c2ncc3[nH]nc(-c4ccc(N5CCN(C)CC5)cc4)c3n2)c1
InChIKey: KETUQSHPCQUVFR-UHFFFAOYSA-N
Target: Mitogen-activated protein kinase kinase kinase kinase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q92918
Assay: Kinase Binding Assay: A stock solution of 1 mM test compound was prepared in DMSO. The compound plate was prepared by 3-fold and 11-point serial dilutions. 0.1 μL of the compound in DMSO was transferred from the compound plate to the white 384 well polystyrene plates. The assay buffer contained 50 mM HEPES, pH 7.5, 0.01% Tween-20, 5 mM MgCl2, 0.01% BSA, and 5 mM DTT. 5 μl of 4 nM active HPK1 (SignalChem M23-11G) prepared in the buffer was added to the plate. The enzyme concentration given was based on the given stock concentration reported by the vender. 5 μl of 18 nM tracer 222 (ThermoFisher PV6121) and 4 nM LanthaScreen Eu-Anti GST antibody (ThermoFisher PV5595) were added. After one hour incubation at 25° C., the plates were read on a PHERAstar FS plate reader (BMG Labtech). Ki values were determined.
Measurement: Ki < 100 nM (pChEMBL 7)
Descriptors: MW 428.5, ALogP 2.19, HBA 7, HBD 2, PSA 102.93, QED 0.51