lovingscience/chembl-v37-raw-activities huggingface dataset:
Compound (SMILES): CCOc1cc(-c2ccc(N3C[C@@H](N)[C@H](Oc4ccc(OC)nc4)C3)nc2)c2c(C#N)cnn2c1
InChIKey: CFAYPODXCWHPES-FGZHOGPDSA-N
Target: Proto-oncogene tyrosine-protein kinase receptor Ret (SINGLE PROTEIN), Homo sapiens
UniProt: P07949
Assay: HTRF Kinease-TK Assay: The potency of a compound inhibiting G810R mutant RET kinase was determined using CisBio's HTRF Kinease-TK assay technology. The assays contained G810R mutant RET produced at Array Biopharma, Inc. (1 nM enzyme p1982 Lot. No. 160713. The kinase was incubated with 250 nM TK-substrate biotin (CisBio, part of Catalog #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 as a three-fold serial dilution in DMSO and added to the assay to give the appropriate final concentration. After a 60-min 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 #62TK0PEC). After a 1-h 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. One hundred POC was determined using no test compounds, and 0 POC was determined using pre-quenched control reactions. A 4-parameter logistic curve was fit to the POC values as a function of the concentration of compound, and the IC50 value was the point where the best-fit curve crossed 50 POC.
Measurement: IC50 = 123 nM (pChEMBL 6.909999847412109)
Descriptors: MW 471.52, ALogP 2.67, HBA 10, HBD 1, PSA 123.82, QED 0.43
Compound (SMILES): Cc1cscc1-c1ccc([C@H]2C[C@@H]2N)cn1
InChIKey: TWVXGTLPXBMRAJ-PWSUYJOCSA-N
Target: Amine oxidase [flavin-containing] B (SINGLE PROTEIN), Homo sapiens
UniProt: P27338
Assay: Monoamine Oxidase Assays: Assays were conducted in 96-well black plates with clear bottom (Corning) in a final volume of 100 μL. The assay buffer was 100 mM HEPES, pH 7.5. Each experiment was performed in triplicate within the same experiment. Briefly, a fixed amount of MAO (0.25 μg for MAO-A and 0.5 μg for MA-B) was incubated on ice for 15 minutes in the reaction buffer, in the absence and/or in the presence of various concentrations of inhibitor (e.g., from 0 to 50 μM, depending on the inhibitor strength). Tranylcypromine (Biomol International) was used as a control for inhibition. After leaving the enzyme(s) interacting with the inhibitor, 60 to 90 μM of kynuramine was added to each reaction for MAO-B and MAO-A assay respectively, and the reaction was left for 1 hour at 37° C. in the dark. The oxidative deamination of the substrate was stopped by adding 50 μL (v/v) of NaOH 2N. The conversion of kynuramine to 4-hydroxyquinoline, was monitored by fluorescence (excitation at 320 nm, emission at 360 nm) using a microplate reader (Infinite 200, Tecan).
Measurement: Ki > 1000 nM (pChEMBL 6)
Descriptors: MW 230.34, ALogP 2.93, HBA 3, HBD 1, PSA 38.91, QED 0.86
Compound (SMILES): Cc1ccc(CNC(=O)C2CN(c3ncnn4cc(-c5cc(C)cs5)cc34)C2)cc1
InChIKey: OXTANBFFJYZLCQ-UHFFFAOYSA-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 417.54, ALogP 3.83, HBA 6, HBD 1, PSA 62.53, QED 0.54
Compound (SMILES): COc1ccc(-c2c(C)noc2-c2cc(-c3cccnc3)c(O)cc2O)cc1
InChIKey: LOBGOMVJTPDQSA-UHFFFAOYSA-N
Target: ATP-dependent molecular chaperone HSP82 (SINGLE PROTEIN), Saccharomyces cerevisiae S288c
UniProt: P02829
Assay: Malachite Green ATPase Assay: 1. Dilute test compounds to 500 uM in AR water (DMSO concentration will be 2.5%). Transfer 2.5 ul of these compounds directly from the daughter plate to the assay plate, giving a final assay concentration of 100 uM. To obtain 12 point IC50 values, perform serial dilutions 1:2 to produce a range of assay concentrations from 100 uM to 97.6 nM (2.5% DMSO), and transfer 2.5 uM of each concentration into the assay plate. Column 1 in the assay plate contains no compound, as a negative control. An additional row with no compound is also used as a background.2. Prepare ATP by diluting 100 mM stock to 925 uM with assay buffer, and aliquot 5 ul of diluted ATP to each well including controls (final assay concentration 370 uM).3. Add 5 ul of buffer to background row.4. Dilute enzyme preparation to 1.05 uM with assay buffer, and aliquot 5 uM into each compound well and to the negative control column.5. Collect the reagents to the bottom of the well, cover plate with plate seal and incubate overnight at 37deg C.6. First thing in the morning prepare the Malachite Green Reagent. Add 2 parts of Malachite Green Solution, 1 part of Polyvinyl Alcohol Solution, 1 part of Ammonium Molybdate Solution, and 2 parts of AR water. 7. Invert to mix, and leave for approximately 1 hour until the colour turns from brown to golden yellow.8. Add 40 uM of Malachite Green Reagent to each well, allow 5 mins for colour to develop.9. Add 5 ul of Sodium Citrate Reagent to each well (see comment 2)10. Re-cover with plate seal and shake on plate shaker for at least 15 mins.11. Measure Absorbance at 620 nM using a suitable plate reader (e.g. Victor, Perkin Elmer Life Sciences, Milton Keynes, UK). Under these conditions, the control absorbance is 0.9 to 1.4, and the background is 0.2-0.35 giving a signal to noise ratio of 12. The Z factor calculated from data obtained using these conditions is between 0.6
Measurement: IC50 > 50000 nM (pChEMBL 4.301000118255615)
Descriptors: MW 374.4, ALogP 4.8, HBA 6, HBD 2, PSA 88.61, QED 0.53
Compound (SMILES): Cc1sc2c(c1C)C(c1ccc(Cl)cc1)=N[C@@H](CC(=O)Nc1ccc3c(c1)COC3=O)c1nnc(C)n1-2
InChIKey: OLAQRMVYAWHVED-NRFANRHFSA-N
Target: Bromodomain-containing protein 4 (SINGLE PROTEIN), Homo sapiens
UniProt: O60885
Assay: BRD4 Biochemical Activity Test: The experiment plate was a plate that contained a gradient concentration of the compound and a corresponding DMSO solution and was prepared with ECHO before the experiment:a) The experiment plate was taken out, and 5 μL/well of solution A (protein solution) was added to columns 2-23 of the experiment plate, then 5 μL/well of 1× assay buffer was added to columns 1 and 24 of the experiment plate, and columns 1 and 24 were used as Min control in the experiment system;b) Centrifugation at 1000 rpm was carried out for 30 seconds;c) The plate was incubated at 23° C. for 20 minutes;d) After 20 minutes of incubation, 5 μL/well of solution B (polypeptide solution) was added to columns 1-24 of the experiment plate;e) Centrifugation at 1000 rpm was carried out for 30 seconds;f) The plate was incubated at 23° C. for 20 minutes;g) After 20 minutes of incubation, 5 μL/well of solution C (test reagent solution) was added to columns 1-24 of the experiment plate;h) Centrifugation at 1000 rpm was carried out for 30 seconds;i) The plate was incubated at 23° C. for 40 minutes;j) The experiment plate was placed on EnVision to read the plate.
Measurement: IC50 = 10 nM (pChEMBL 8)
Descriptors: MW 532.03, ALogP 5.5, HBA 8, HBD 1, PSA 98.47, QED 0.35
Compound (SMILES): CNc1cc2c(cn1)CN(c1c(F)c(OC)cc(OC)c1F)C(=O)C21CC1
InChIKey: NWBYJLBUUNLMHP-UHFFFAOYSA-N
Target: Fibroblast growth factor receptor 2 (SINGLE PROTEIN), Homo sapiens
UniProt: P21802
Assay: Enzymatic Assay: The inhibitor potency of the exemplified compounds was measured in an enzyme assay that measures peptide phosphorylation using FRET measurements to detect product formation. Inhibitors were serially diluted in DMSO and a volume of 0.5 μL was transferred to the wells of a 384-well plate. For FGFR3, a 10 μL volume of FGFR3 enzyme (Millipore) diluted in assay buffer (50 mM HEPES, 10 mM MgCl2, 1 mM EGTA, 0.01% Tween-20, 5 mM DTT, pH 7.5) was added to the plate and pre-incubated for 5-10 minutes. Appropriate controls (enzyme blank and enzyme with no inhibitor) were included on the plate. The assay was initiated by the addition of a 10 μL solution containing biotinylated EQEDEPEGDYFEWLE peptide substrate (SEQ ID NO: 1) and ATP (final concentrations of 500 nM and 140 μM respectively) in assay buffer to the wells. The plate was incubated at 25° C. for 1 hr. The reactions were ended with the addition of 10 μL/well of quench solution (50 mM Tris, 150 mM NaCl, 0.5 mg/mL BSA, pH 7.8; 30 mM EDTA with Perkin Elmer Lance Reagents at 3.75 nM Eu-antibody PY20 and 180 nM APC-Streptavidin). The plate was allowed to equilibrate for 1 hr before scanning the wells on a PheraStar plate reader (BMG Labtech).FGFR1 and FGFR2 were measured under equivalent conditions with the following changes in enzyme and ATP concentrations: FGFR1, 0.02 nM and 210 μM, respectively and FGFR2, 0.01 nM and 100 μM, respectively. The enzymes were purchased from Millipore or Invitrogen.
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 375.38, ALogP 3, HBA 5, HBD 1, PSA 63.69, QED 0.89
Compound (SMILES): OC(c1cc(-c2nc3ccc(F)cc3n2C2CC2)cnn1)C(F)(F)F
InChIKey: QIPCBUDMPMEIJS-UHFFFAOYSA-N
Target: Cytochrome P450 11B1, mitochondrial (SINGLE PROTEIN), Homo sapiens
UniProt: P15538
Assay: Metalloenzyme Activity: V79-4 cells expressing recombinant andrenodoxin and andrenodoxin reductase with either recombinant human CYP11B2 or CYP11B1 were prepared according to methods previously described (LaSala et al 2009 Anal Bioch 394:56-61). An enzyme enriched microsomal fraction was prepared from cellular lysates and subsequently used as the enzyme source for determining inhibitor IC50s. The substrate Km values were experimentally determined for 11-deoxycorticosterone (CYP11B2 substrate) and 11-deoxycortisol (CYP11B1 substrate). Enzyme assays for inhibitor screening employed CYP11B2 and CYP11B1 enzyme enriched microsomes and were run at the Km of the respective substrates. Products of the enzyme reactions, aldosterone for CYP11B2 or cortisol for CYP11B1, were measured by LC-MS. Assays were run under conditions of less than 20% substrate turnover. Inhibitor IC50s were generated by determining the product formation in the absence or presence of inhibitor at various concentrations. In the absence of the test compound, the product formed (Pt) in each data set was defined as 100% activity. In the absence of enzyme, the product formed (Pb) in each data set was defined as 0% activity. The percent activity in the presence of each inhibitor was calculated according to the following equation: % activity=(P−Pb)/(Pt−Pb), where P=the product formed in the presence of the inhibitor. The IC50 value was defined as the inhibitor concentration causing a 50% decrease in activity relative to the no inhibitor control reaction.
Measurement: IC50 = 750 nM (pChEMBL 6.119999885559082)
Descriptors: MW 352.29, ALogP 3.56, HBA 5, HBD 1, PSA 63.83, QED 0.73
Compound (SMILES): CN(Cc1cccc(F)c1)C(=O)c1cc(-c2ccccc2)on1
InChIKey: CKYXRBUUUYNHKH-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 = 15670 nM (pChEMBL 4.800000190734863)
Descriptors: MW 310.33, ALogP 3.75, HBA 3, HBD 0, PSA 46.34, QED 0.74
Compound (SMILES): CCOC(=O)CCn1cc(Nc2ncc3ccn(C[C@H]4CC[C@H](O)CC4)c3n2)cn1
InChIKey: VZPYTBIIQFPUIC-RZDIXWSQSA-N
Target: Tyrosine-protein kinase JAK2 (SINGLE PROTEIN), Homo sapiens
UniProt: O60674
Assay: JAK/TYK2 Assay: 10 mM test compound stock or 1 mM control compound stock (tofocitinib, ruxolitinib or staurosporine) in DMSO was diluted to 0.4 mM in DMSO. A 3-fold series dilution was then performed in DMSO to generate 10 different compound concentrations. The assay was carried out in 384-well white plate. 0.5 uL of 40× compound DMSO solution at different concentrations was mixed with 10 uL 2× enzyme prepared in reaction buffer (20 mM HEPES, 10 mM MgCl2, 0.01% Tween, 1 mM DTT, pH 7.5). 10 uL 2× substrate mixture prepared in reaction buffer was then added to start the reaction. A short spin was done to settle down all solutions to the bottom of the plate. Final concentrations of test compound in the reaction mixture were 10000, 3333, 1111, 370, 123, 41.2, 13.7, 4.57, 1.52and 0.51 nM. Concentrations of control compound were ten times less. Enzymatic reaction was conducted at 25° C. for 1-2 hours. 10 uL of Kinase Glo Reagents was added to stop the reaction and generate the luminescent signal which was measured using Envision. Luminescence signal was inversely related to kinase activity. Reaction mixture which did not contain enzyme served as negative control. The mixture without any compound was the positive control.
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 412.49, ALogP 2.88, HBA 9, HBD 2, PSA 107.09, QED 0.55
Compound (SMILES): CC1(C)COc2c(-c3cc4nccc(N[C@@H]5C[C@H](COS(N)(=O)=O)[C@@H](O)[C@H]5O)n4n3)cccc21
InChIKey: NUWGQHGWOIICDS-KRVNLHHGSA-N
Target: Ubiquitin-like modifier-activating enzyme 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P22314
Assay: Ubiquitin Activating Enzyme (UAE) HTRF Assay: The UAE enzymatic reaction totals 50 μL and contains 50 mmol HEPES (pH 7.5), 0.05% BSA, 2.5 mM MgCl2, 0.1 uM ATP, 8 nM GST-Ubc-2, 35 nM flag-ubiquitin, 1 nM recombinant human UAE or mouse UAE. Compounds for this hUAE IC50 assay are tested at 10 point 3-fold dilution. The top concentration for this assay is 1 μM. Each compound is ordered in duplicate on the same plate. The enzymatic reaction mixture is incubated for 90 minutes at room temperature (24 degrees C.) in a 384 well plate prior to termination with a stop solution (0.1 M HEPES/0.05% Tween20, 20 mmol EDTA, 410 mM KF, 0.5 nM Eu Cryptate anti-FLAG M2-K antibody (Cis-bio International), 8 ug/mL Anti-GST XL-APC (Prozyme)). After incubation for 120 minutes, quantification of the FRET is performed on the Pherostar (BMG).
Measurement: IC50 = 55 nM (pChEMBL 7.260000228881836)
Descriptors: MW 489.55, ALogP 0.81, HBA 10, HBD 4, PSA 161.3, QED 0.39
Compound (SMILES): Cn1cc(-c2cn3ncc(C#N)c3c(-c3ccc(N4CCC[C@H](CN)C4)nc3)n2)cn1
InChIKey: VODPNPMPPJNVGA-OAHLLOKOSA-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. 62TKOPEC) 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. 62TKOPEC). 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 = 229 nM (pChEMBL 6.639999866485596)
Descriptors: MW 413.49, ALogP 2.24, HBA 9, HBD 1, PSA 113.95, QED 0.55
Compound (SMILES): COCc1ccc(NC(=O)c2c(C#Cc3nccn3C)n(C3(C)CC3)c3ncnc(N)c23)cc1
InChIKey: JJQNHDJJWOQICB-UHFFFAOYSA-N
Target: Proto-oncogene tyrosine-protein kinase receptor Ret (SINGLE PROTEIN), Homo sapiens
UniProt: P07949
Assay: Inhibitory Activity Against Resistance Mutation of RET (In Vitro): Regarding the conditions for measurement of in vitro inhibitory activity of compounds against RET (V804L) (i.e., RET with V804L mutation) kinase activity, the website of AnaSpec states that Srctide (GEEPLYWSFPAKKK) corresponds to the substrate peptide for reaction to measure RET kinase activity. Thus, the amino acid sequence was partly modified and biotinylated to prepare biotinylated peptides (biotin-EEPLYWSFPAKKK). The purified recombinant human RET (V804L) protein used in the test was purchased from Eurofins.To measure the inhibitory activity, first, the test compounds were individually diluted with dimethyl sulfoxide (DMSO) stepwise. Subsequently, RET (V804L) protein, the substrate peptides (the final concentration: 250 nM), magnesium chloride (the final concentration: 10 mM), ATP (the final concentration: 10 μM), and a solution of a test compound in DMSO (the final concentration of DMSO: 5%) were added to a buffer for kinase reaction (13.5 mM Tris, pH of 7.5, 2 mM dithiothreitol, 0.009% Tween-20). Each of the mixtures was incubated at 25° C. for 120 minutes to perform a kinase reaction. EDTA was then added thereto to give a final concentration of 40 mM so that the reaction was terminated. A detection solution containing Eu-labeled antiphosphotyrosine antibody PT66 (PerkinElmer) and SureLight APC-SA (PerkinElmer) was added thereto, and each mixture was allowed to stand at room temperature for 2 hours or more. Finally, the intensity of fluorescence under excitation light with a wavelength of 337 nm was measured with a PHERAstar FS (BMG Labtech) at the two wavelengths of 620 nm and 665 nm. The phosphorylation level was calculated from the ratio of the fluorescence intensity at the two wavelengths, and the compound concentration at which phosphorylation was inhibited by 50% was defined as the IC50 value (nM).
Measurement: IC50 = 1.8 nM (pChEMBL 8.739999771118164)
Descriptors: MW 455.52, ALogP 3.05, HBA 8, HBD 2, PSA 112.88, QED 0.45
Compound (SMILES): CCOc1nn(C)cc1Nc1ncc(C)c(-c2c[nH]c3c(NC(=O)[C@H](CC)N4CCN(C)CC4)cccc23)n1
InChIKey: MFLOYXVMLPUWDM-QHCPKHFHSA-N
Target: Tyrosine-protein kinase JAK2 (SINGLE PROTEIN), Homo sapiens
UniProt: O60674
Assay: JAK Enzymatic Assay: Enzyme inhibition studies were performed using recombinant JAK1 (amino acids 866-1154, Life Technologies, #PV4774, Carlsbad, Calif.), JAK2 (amino acids 831-1132), or JAK3 (amino acids 781-1124) under buffer conditions of 50 mM HEPES pH 7.3, 1 mM DTT, 0.01% Tween® 20, 50 μg/mL BSA, and 10 mM MgCl2. JAK enzyme was expressed as an N-terminal GST fusion in insect cells and purified by glutathione-affinity and size-exclusion chromatographies. Enzymes were assayed both at their respective ATP Km (JAK1: 55 μM, JAK2: 15 μM, JAK3: 3 μM) and the approximated high end of physiological ATP concentration of 5 mM, in the presence of inhibitor dosed at 30, 3, 0.3, 0.03, 0.003 and 0 μM final test concentrations. For JAK1, 6 nM of enzyme (for Km ATP assay) or 4 nM enzyme (for high ATP assay) was incubated with 1.5 μM peptide substrate (FITC-C6-KKHTDDGYMPMSPGVA-NH2 (SEQ ID NO:1), Intonation, Boston, Mass.). For JAK2, 0.8 nM of enzyme (for Km ATP assay) or 0.3 nM enzyme (for high ATP assay) was incubated with 1.5 μM peptide substrate (5FAM-GEEPLYWSFPAKKK-NH2 (SEQ ID NO:2), Intonation, Boston, Mass.). For JAK3, 0.2 nM of enzyme (for Km ATP assay) or 0.1 nM enzyme (for high ATP assay) was incubated with 1.5 μM peptide substrate (5FAM-GEEPLYWSFPAKKK-NH2 (SEQ ID NO:2), Intonation, Boston, Mass.). Phosphorylated and unphosphorylated peptides were separated and quantified by a Caliper LC3000 system (Caliper Life Sciences, MA) for calculating percent inhibition. The results of this assay are shown in Table 16 and indicate that the compounds of Formula (I), (Ia), (Ib) and Table 1 exhibit preferential inhibition of JAK1 over JAK2 (in many cases demonstrating over 100 times selectivity for inhibition of JAK1 vs. JAK2).
Measurement: IC50 > 30000 nM (pChEMBL 4.5229997634887695)
Descriptors: MW 531.67, ALogP 3.77, HBA 9, HBD 3, PSA 116.23, QED 0.3
Compound (SMILES): CC(C)C(=O)N1CCC(c2cc(-c3ccc(NC(=O)c4cn(C(C)C)cc(-c5cncc(F)c5)c4=O)cc3)c3c(N)ncnn23)CC1
InChIKey: DXYZJNSIYAPTJV-UHFFFAOYSA-N
Target: Tyrosine-protein kinase Mer (SINGLE PROTEIN), Homo sapiens
UniProt: Q12866
Assay: Enzymatic Assay: Tyro3, and Mer:The kinase assay buffer contained 50 mM HEPES, pH7.5, 10 mM MgCl2, 1 mM EGTA, 0.01% NP-40 and 2 mM DTT. 0.1 ul test compounds dissolved in DMSO were transferred from compound plates to white 384-well assay plates (Greiner LUMITRAC plates). The final concentration of DMSO was 1.25%. Enzyme solutions of 5.1 nM phosphor-Axl, or 0.0625 nM c-Mer (Carna Biosciences, 08-108), or 0.366 nM Tyro3 (Life Technologies, PR7480A) were prepared in assay buffer. A 1 mM stock solution of peptide substrate Biotin-EQEDEPEGDYFEWLE-amide SEQ ID NO: 1 (Quality Controlled Biochemicals, MA) dissolved in DMSO was diluted to 1 uM in assay buffer containing 2000 uM ATP. 4 ul enzyme solution (or assay buffer for the enzyme blank) was added to the appropriate wells in each plate, and then 4 ul/well substrate solution was added to initiate the reaction. The plate was protected from light and incubated at room temperature for 60 min. The reaction was stopped by adding 4 ul detection solution containing 50 mM Tris-HCl, pH7.8, 150 mM NaCl, 0.05% BSA, 45 mM EDTA, 180 nM SA-APC (Perkin Elmer, CR130-100) and 3 nM Eu-W1024 anti-phosphotyrosine PY20 (Perkin Elmer, AD0067). The plate was incubated for 1 h at room temperature, and HTRF (homogenous time resolved fluorescence) signal was measured on a PHERAstar FS plate reader (BMG labtech).
Measurement: IC50 = 7.5 nM (pChEMBL 8.119999885559082)
Descriptors: MW 636.73, ALogP 5.54, HBA 9, HBD 2, PSA 140.51, QED 0.24
Compound (SMILES): Cc1ncc(NC(=O)c2ccnc(C(C)(C)F)c2)cc1-c1cnc(OC2CC3(COC3)C2)c(N2CCOCC2)c1
InChIKey: IRIGYQNVLRIJJA-UHFFFAOYSA-N
Target: RAF proto-oncogene serine/threonine-protein kinase (SINGLE PROTEIN), Homo sapiens
UniProt: P04049
Assay: In Vitro Raf Activity: Raf and biotinylated Mek, kinase dead, were combined at 2× final concentrations in assay buffer (50 mM Tris, pH 7.5, 15 mM MgCl2, 0.01% BSA and 1 mM DTT) and dispensed 5 ml per well in assay plates (Greiner white 384 well assay plates #781207) containing 0.25 ml of 40× of a Raf kinase inhibitor test compound diluted in 100% DMSO. The plate was incubated for 60 minutes at room temperature. The Raf kinase activity reaction was started by the addition of 5 mL per well of 2×ATP diluted in assay buffer. After 3 hours (b-Raf(V600E)) or 1 hour (c-Raf). The reactions were stopped and the phosphorylated product was measured using a rabbit anti-p-MEK (Cell Signaling, #9121) antibody and the Alpha Screen IgG (ProteinA) detection Kit (PerkinElmer #6760617R), by the addition of 10 mL to the well of a mixture of the antibody (1:2000 dilution) and detection beads (1:2000 dilution of both beads) in Stop/bead buffer (25 mM EDTA, 50 mM Tris, pH 7.5, 0.01% Tween20). The additions were carried out under dark conditions to protect the detection beads from light. A lid was placed on top of the plate and incubated for 1 hour at room temperature, then the luminescence was read on a PerkinElmer Envision instrument. The concentration of each compound for 50% inhibition (IC50) was calculated by non-linear regression using XL Fit data analysis software.
Measurement: IC50 = 1.04 nM (pChEMBL 8.979999542236328)
Descriptors: MW 547.63, ALogP 4.7, HBA 8, HBD 1, PSA 98.7, QED 0.45
Compound (SMILES): O[C@@H]1[C@H](O)CS[C@H]1c1nnc2c(NC3CCC3)nccn12
InChIKey: MQQOLPKMJHTAOJ-OPRDCNLKSA-N
Target: Adenosine receptor A3 (SINGLE PROTEIN), Homo sapiens
UniProt: P0DMS8
Assay: Assay for Binding Affinity for Adenosine Receptor A3: HEK-293 cell membrane homogenates (32 μg protein), in which A3 adenosine receptors were expressed, are incubated for 120 min at 22° C. with 0.15 nM [1251]AB-MECA in the absence or presence of the test compound in a buffer containing 50 mM Tris-HCl (pH 7.4), 5 mM MgCl2, 1 mM EDTA and 2 UI/ml ADA. Nonspecific binding is determined in the presence of 1 μM IB-MECA. Following incubation, the samples are filtered rapidly under vacuum through glass fiber filters (GF/B, Packard) presoaked with 0.3% PEI and rinsed several times with ice-cold 50 mM Tris-HCl using a 96-sample cell harvester (Unifilter, Packard). The filters are dried then counted for radioactivity in a scintillation counter (Topcount, Packard) using a scintillation cocktail (Microscint 0, Packard). The results are expressed as a percent inhibition of the control radioligand specific binding. The standard reference compound is IB-MECA, which is tested in each experiment at several concentrations to obtain a competition curve from which its IC50 is calculated.
Measurement: IC50 = 550 nM (pChEMBL 6.260000228881836)
Descriptors: MW 307.38, ALogP 0.6, HBA 8, HBD 3, PSA 95.57, QED 0.76
Compound (SMILES): CNc1cc2c(cn1)c(-c1cnn(C)c1)nn2C1CCC(Oc2ccnn2C)CC1
InChIKey: IPXSULNOCXFCFG-UHFFFAOYSA-N
Target: Tyrosine-protein kinase Mer (SINGLE PROTEIN), Homo sapiens
UniProt: Q12866
Assay: Enzyme Assay: MER: Compounds of Formula I were screened for their ability to inhibit MER kinase using Invitrogen's LanthaScreen Eu Kinase Binding technology. His-tagged recombinant human MER cytoplasmic domain (5 nM) was incubated with 20 nM Alexa-Fluor® Tracer 236 (PR9078A), 2 nM biotinylated anti-His (Cat. No. M4408), and 2 nM europium-labeled Streptavidin (Cat. No. PV5899) 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. Compounds were typically prepared in a threefold serial dilution in DMSO and added to the assay to give the appropriate final concentration. After a 60-minute incubation at 22° C., the reaction was measured using a PerkinElmer EnVision multimode plate reader via TR-FRET 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 a concentration of control compound that completely inhibits the enzyme. The POC values are fit to a 4 parameter logistic curve and the IC50 value is point where the curve crosses 50 POC.
Measurement: IC50 < 20 nM (pChEMBL 7.698999881744385)
Descriptors: MW 406.49, ALogP 3.17, HBA 9, HBD 1, PSA 87.61, QED 0.55
Compound (SMILES): Cc1cc(-c2cnc(C(=O)N[C@H](C(=O)N3C[C@H](Cl)[C@H]4OCC(=O)[C@H]43)C3CCCCC3)s2)cnn1
InChIKey: PHKGTCBBQTUGFZ-KCUHQSDYSA-N
Target: Cathepsin K (SINGLE PROTEIN), Homo sapiens
UniProt: P43235
Assay: Enzyme Assay: Recombinant human cathepsins (CatS, CatK, CatL) were purchased from a Enzo Life Sciences. All assays were carried out in 96-well format using a buffer of 50 mM KH2PO4, 50 mM NaCl, 2 mM EDTA, 0.5 mM DTT and 1% Triton-X-100, pH 6.5 for Cathepsin S and a buffer of 50 mM NaOAc, 10 mM EDTA, 1 mM DTT and 0.01% Triton-X-100, pH 5.5 for CatK/L/B. For CatS, the enzyme (0.0007 mU/well) was incubated with fluorogeninc substrate (Z-VVR-AMC, 5 μM) at RT for 10 min. For CatK the enzyme (0.00175 mU/well) was incubated with fluorogeninc substrate (Z-FR-AMC, 40 μM) at RT for 10 min. For CatL, the enzyme (0.000874 mU/well) was incubated with fluorogeninc substrate (Z-VVR-AMC, 40 μM) at RT for 10 min. Flourogenic substrate turnover was detected using a microplate reader (Synergy H4, BioTek). Ki values were calculated using the Cheng Prusoff equation (Cheng & Prosoff 1973).
Measurement: Ki = 18 nM (pChEMBL 7.75)
Descriptors: MW 504.01, ALogP 2.37, HBA 8, HBD 1, PSA 114.38, QED 0.62
Compound (SMILES): Cc1cc(COc2ccc(-c3cccc4nc(NC(=O)C5CC5)nn34)cc2)n(C)n1
InChIKey: ZAKKBFGSGZDFOC-UHFFFAOYSA-N
Target: Tyrosine-protein kinase JAK2 (SINGLE PROTEIN), Homo sapiens
UniProt: O60674
Assay: JAK2 Inhibition Assay: Recombinant human JAK2 catalytic domain (amino acids 808-1132; catalog number PV4210) was purchased from Invitrogen. 0.025 mU of JAK2 was incubated with 2.5 μg polyGT substrate (Sigma catalog number P0275) in kinase reaction buffer (5 mM MOPS pH 7.5, 9 mM MgAc, 0.3 mM EDTA, 0.06% Brij and 0.6 mM DTT, 1 μM non-radioactive ATP, 0.25 μCi 33P-gamma-ATP (GE Healthcare, catalog number AH9968) final concentrations) with or without 5 μL containing test compound or vehicle (DMSO, 1% final concentration), in a total volume of 25 μL, in a polypropylene 96-well plate (Greiner, V-bottom). After 90 min at 30° C., reactions were stopped by adding of 25 μL/well of 150 mM phosphoric acid. All of the terminated kinase reaction was transferred to prewashed (75 mM phosphoric acid) 96 well filter plates (Perkin Elmer catalog number 6005177) using a cell harvester (Perkin Elmer). Plates were washed 6 times with 300 μL per well of a 75 mM phosphoric acid solution and the bottom of the plates was sealed. 40 μL/well of Microscint-20 was added, the top of the plates was sealed and readout was performed using the Topcount (Perkin Elmer). Kinase activity was calculated by subtracting counts per minute (cpm) obtained in the presence of a positive control inhibitor (10 μM staurosporine) from cpm obtained in the presence of vehicle. The ability of a test compound to inhibit this activity was determined as:Percentage inhibition=((cpm determined for sample with test compound present−cpm determined for sample with positive control inhibitor) divided by (cpm determined in the presence of vehicle−cpm determined for sample with positive control inhibitor))*100%.
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 402.46, ALogP 3.37, HBA 7, HBD 1, PSA 86.34, QED 0.53
Compound (SMILES): CN(C)[C@H](CNC(=O)NCc1cccc([N+](=O)[O-])c1)Cc1ccccc1
InChIKey: BCGAAVLXDMFNFW-SFHVURJKSA-N
Target: Kappa-type opioid receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P41145
Assay: Radioligand Binding Assay: For a primary screen of selected molecules, binding to Î¼OR was assessed by measuring competition against the radioligand 3H-diprenorphine (3H-DPN). Each compound was initially tested at 20 Î¼M and was incubated with 3H-DPN at a concentration equal to the Kd (0.4 nM) of the radioligand in Î¼OR containing Sf9 insect cell membranes. The reaction contained 40 fmol of Î¼OR and was incubated in a buffer of 20 mM HEPES pH 7.5, 100 mM sodium chloride, and 0.1% bovine serum albumin for 1 hour at 25Â° C. To separate free from bound radioligand, reactions were rapidly filtered over Whatman GF/B filters with the aid of a Brandel harvester and 3H-DPN counts were measured by liquid scintillation. Compounds with more than 25% of 3H-DPN radioactivity were further tested in full dose-response to determine the affinity (Ki) in HEK293 membranes. Subsequently, the 15 analogs were tested in full dose-response for affinity at the Î¼OR and the ÎºOR by the National Institutes of Mental Health Psychoactive Drug Screen Program (PDSP), as were the affinities of compounds 12, PZM21, and their stereoisomers at the Î¼OR, Î´OR, ÎºOR and nociception receptor.
Measurement: Ki = 510 nM (pChEMBL 6.289999961853027)
Descriptors: MW 356.43, ALogP 2.57, HBA 4, HBD 2, PSA 87.51, QED 0.56
Compound (SMILES): CS(=O)(=O)c1ccc(Nc2oc(-c3c(Cl)cccc3Cl)nc2C(N)=O)cc1
InChIKey: VSESJZFXWMRDNW-UHFFFAOYSA-N
Target: Tyrosine-protein kinase JAK1 (SINGLE PROTEIN), Homo sapiens
UniProt: P23458
Assay: Enzyme Inhibition Assay: Kinase assays were performed at Reaction Biology Corp., Malvern, Pa., USA, using the following general procedure:  1) Prepare indicated substrate in freshly prepared Base Reaction Buffer (20 mM Hepes pH 7.5, 10 mM MgCl2, 1 mM EGTA, 0.02% Brij35, 0.02 mg/ml BSA, 0.1 mM Na3VO4, 2 mM DTT, 1% DMSO).  2) Deliver cofactors (1.5 mM CaCl2, 16 ug/mL Calmodulin, 2 mM MnCl2) to the substrate solution above  3) Deliver indicated kinase into the substrate solution and gently mix  4) Deliver varying concentrations of test compound in DMSO into the kinase reaction mixture  5) Deliver 33P-ATP (specific activity 0.01 μCi/μL final) into the reaction mixture to initiate the reaction  6) Incubate kinase reaction for 120 min at room temperature  7) Reactions are spotted onto P81 ion exchange filter paper (Whatman #3698-915)  8) Unbound phosphate is removed by washing filters extensively in 0.75% Phosphoric acid.  9) 33P signal was determined using Typhoon phosphorimagers (GE Healthcare). After subtraction of background derived from control reactions containing inactive enzyme, IC50 values were determined using the nonlinear regression function in Prism (Graphpad software).
Measurement: IC50 = 21.9 nM (pChEMBL 7.659999847412109)
Descriptors: MW 426.28, ALogP 3.89, HBA 6, HBD 2, PSA 115.29, QED 0.64
Compound (SMILES): NC(=O)c1c(-c2ccc(Oc3ncc(Cl)cc3F)cc2)nn([C@@H]2CCCN(C(=O)/C=C/CF)C2)c1N
InChIKey: ANQSRGWBVINOHT-JHBSALDVSA-N
Target: Epidermal growth factor receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P00533
Assay: TR-FRET LanthaScreen Assay: EGFR: TR-FRET LanthaScreen assays were performed by incubating a dilution series of inhibitor concentrations with 20 μM ATP, 100 nM peptide substrate (FITC-C6-KKAEEEEYFELVAKK-NH2 (SEQ ID NO.: 2, American Peptide, #333778) and 600 pM of human EGFR kinase domain (Invitrogen). The assays were performed with and without pre-incubating the inhibitors with the enzyme for 60 minutes before starting the kinase reaction by adding ATP and the peptide substrate. Samples containing enzyme but no inhibitor were included to determine the maximal extent of reaction. Samples containing no enzyme served as the negative control. The kinase reaction mixtures were incubated at room temperature for 60 minutes before stopping the kinase activity by the addition of 15 mM EDTA. The extent of peptide phosphorylation by EGFR was detected using a Terbium-conjugated anti-phospho-Tyrosine antibody (Tb-PT66 antibody, Invitrogen # PV3557). Phosphorylation of peptide substrate was measured by determining the ratio of 520/495 nm on an Envision Multi-label Reader (Perkin Elmer) and IC50 values were calculated by fitting the data to a four-parameter equation using XLFit4 (IDBS).
Measurement: IC50 = 381 nM (pChEMBL 6.420000076293945)
Descriptors: MW 516.94, ALogP 3.9, HBA 7, HBD 2, PSA 129.36, QED 0.46
Compound (SMILES): COc1cc(N2CCN(C)CC2)ccc1Nc1ncc(Cl)c(NCCc2c(F)cccc2Cl)n1
InChIKey: DKNACWKOKCXDEJ-UHFFFAOYSA-N
Target: ALK tyrosine kinase receptor (SINGLE PROTEIN), Homo sapiens
UniProt: Q9UM73
Assay: Enzyme-Linked Immunosorbent Assay: Enzyme-Linked Immunosorbent Assay.
Measurement: IC50 = 7 nM (pChEMBL 8.149999618530273)
Descriptors: MW 505.43, ALogP 5.08, HBA 7, HBD 2, PSA 65.55, QED 0.44
Compound (SMILES): Cn1cc(CC(=O)N[C@H]2CC[C@H](CCN3C[C@@H]4C[C@]4(c4cc(C5CC5)on4)C3)CC2)cn1
InChIKey: TWAGQMOSGNQWTA-WDRWKGISSA-N
Target: D(2) dopamine receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P14416
Assay: Binding Inhibition for Dopamine D2 Receptor: Buffer solution: 50 mM Tris-HCl (35409-45, Nacalai Tesque) (pH 7.4) containing 120 mM NaCl (31320-05, Nacalai Tesque), 1 mM MgCl2.6H2O (20909-55, Nacalai Tesque), 5 mM KCl (28514-75, Nacalai Tesque) and 2 mM CaCl2 (067-31, NAKARAI CHEMICALS, LTD.)Radioligand: (final concentration) 1.2 nM [3H]-Methylspiperone ([3HN-methyl-]-Methylspiperone, NET-856, 83.8 Ci/mmol, PerkinElmer)Non-specific ligand: (final concentration) 10 μM Butaclamol [(+)-Butaclamol Hydrochloride, D033; Sigma]SPA beads solution: SPA beads [WGA PVT SPA Scintillation Beads, RPNQ0001 (500 mg), RPNQ0060 (2 g), PerkinElmer] (0.2 mg/well)Incubation time and temperature: 120 min at 25° C.
Measurement: Ki = 730 nM (pChEMBL 6.139999866485596)
Descriptors: MW 437.59, ALogP 3.17, HBA 6, HBD 1, PSA 76.19, QED 0.69
Compound (SMILES): O=c1c2cnc(C3CCOCC3)n2ccn1CC1CCCCC1
InChIKey: XXIPRLNKXMMFIK-UHFFFAOYSA-N
Target: Dual specificity calcium/calmodulin-dependent 3',5'-cyclic nucleotide phosphodiesterase 1B (SINGLE PROTEIN), Homo sapiens
UniProt: Q01064
Assay: PDE1 Inhibition Assay: PDE1A, PDE1B and PDE1C assays were performed as follows: the assays was performed in 60 μL samples containing a fixed amount of the PDE1 enzyme (sufficient to convert 20-25% of the cyclic nucleotide substrate), a buffer (50 mM HEPES pH 7.6; 10 mM MgCl2; 0.02% Tween20), 0.1 mg/ml BSA, 15 nM tritium labelled cAMP and varying amounts of inhibitors. Reactions were initiated by addition of the cyclic nucleotide substrate, and reactions were allowed to proceed for 1 h at room temperature before being terminated through mixing with 20 μL (0.2 mg) yttrium silicate SPA beads (PerkinElmer). The beads were allowed to settle for 1 h in the dark before the plates were counted in a Wallac 1450 Microbeta counter. The measured signals were converted to activity relative to an uninhibited control (100%) and IC50 values were calculated using XIFit (model 205, IDBS).
Measurement: IC50 = 72 nM (pChEMBL 7.139999866485596)
Descriptors: MW 315.42, ALogP 2.97, HBA 5, HBD 0, PSA 48.53, QED 0.87
Compound (SMILES): OC(c1cccnn1)c1cc(-c2ncnc3cc(N4CCOCC4)ccc23)ccc1Cl
InChIKey: NKTVIVGVBABPLJ-UHFFFAOYSA-N
Target: Voltage-gated inwardly rectifying potassium channel KCNH2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q12809
Assay: Patch Clamp Assay: The patch-clamp measurement was carried out at room temperature in whole-cell configuration on human embryonic kidney cells (HEK293) which have been transfected in a stable manner with the hERG gene.The whole-cell configurations were carried out using an automated patch clamp device (Patchliner, Nanion Technologies, Munich). This is a glass chip-based system with which automated whole-cell measurements on up to 8 cells simultaneously are possible. The glass chip has a hole of defined size to which the cell is transferred into the Gigaseal by application of a reduced pressure and brought into the whole-cell configuration. Buffer, cell suspension and test substances were added to microchannels of the chip using a Teflon-coated pipette. The cells were clamped to a holding potential of -80 mV. For measurement of substance-promoted inhibition of the Kv11.1 channel, the following voltage protocol was applied at 10-second intervals: 51 ms/-80 mV, 500 ms/+40 mV, 500 ms/-40 mV, 200 ms/-80 mV. The leakage current is subtracted by means of the P4 method. The cells were resuspended in extracellular buffer (EC) and applied to the chip. After the cell had been collected, the seal was improved by addition of a seal enhancer buffer. As soon as the whole-cell configuration had been reached, the seal enhancer buffer was washed out and replaced by extracellular buffer. The measurement started in EC for 1.5 min. DMSO (vehicle control, 0.1% of DMSO) was then applied, and the control current was recorded for 3 min. The test substance was subsequently added twice in the same concentration, and the potassium current was measured for 3.5 min in each case.
Measurement: Ki = 12500 nM (pChEMBL 4.900000095367432)
Descriptors: MW 433.9, ALogP 3.66, HBA 7, HBD 1, PSA 84.26, QED 0.53
Compound (SMILES): Cc1ccc2c3c(nn2c1)CCC(NC(=O)c1c(Cl)cc(-n2cnc(C)n2)cc1Cl)(C(C)O)C3
InChIKey: PJDJHOJIOCPXKM-UHFFFAOYSA-N
Target: Coagulation factor IX (SINGLE PROTEIN), Homo sapiens
UniProt: P00740
Assay: TBD: TBD
Measurement: IC50 = 12.6 nM (pChEMBL 7.900000095367432)
Descriptors: MW 499.4, ALogP 3.88, HBA 7, HBD 2, PSA 97.34, QED 0.44
Compound (SMILES): CC(C)CN1[C@H](c2ccc(NC3CN(CCCF)C3)cn2)c2ccc3[nH]ncc3c2C[C@H]1C
InChIKey: VQGLUTISKQTZKB-DWXRJYCRSA-N
Target: Estrogen receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P03372
Assay: MCF-7 ER Down-Regulation Assay: The ability of compounds to down-regulate Estrogen Receptor (ER) numbers was assessed in a cell based immuno-fluorescence assay using the MCF-7 human ductal carcinoma breast cell line. MCF-7 cells were revived directly from a cryovial (approx 5×106 cells) in Assay Medium (phenol red free Dulbecco's Modified Eagle's medium (DMEM); Sigma D5921) containing 2 mM L-Glutamine and 5% (v/v) Charcoal/Dextran treated foetal calf serum. Cells were syringed once using a sterile 18G×1.5 inch (1.2×40 mm) broad gauge needle and cell density was measured using a Coulter Counter (Beckman). Cells were further diluted in Assay Medium to a density of 3.75×104 cells per mL and 40 μL per well added to transparent bottomed, black, tissue culture-treated 384 well plates (Costar, No. 3712) using a Thermo Scientific Matrix WellMate or Thermo Multidrop. Following cell seeding, plates were incubated overnight at 37° C., 5% CO2 (Liconic carousel incubator). Test data was generated using the LabCyte Echo model 555 compound reformatter which is part of an automated workcell (Integrated Echo 2 workcell). Compound stock solutions (10 mM) of the test compounds were used to generate a 384 well compound dosing plate (Labcyte P-05525-CV1). 40 μL of each of the 10 mM compound stock solutions was dispensed into the first quadrant well and then 1:100 step-wise serial dilutions in DMSO were performed using a Hydra II (MATRIX UK) liquid handling unit to give 40 μL of diluted compound into quadrant wells 2 (0.1 mM), 3 (1 μM) and 4 (0.01 μM), respectively. 40 μL of DMSO added to wells in row P on the source plate allowed for DMSO normalisation across the dose range. To dose the control wells 40 μL of DMSO was added to row O1 and 40 μL of 100 μM fulvestrant in DMSO was added to row O3 on the compound source plate.
Measurement: IC50 = 0.55 nM (pChEMBL 9.260000228881836)
Descriptors: MW 450.61, ALogP 4.41, HBA 5, HBD 2, PSA 60.08, QED 0.53
Compound (SMILES): CN1CCC(N(C)C(=O)c2ccc(Nc3nc4c(N5CCC(CCC#N)(c6ccc(Cl)cc6)CC5)cccn4n3)cc2)CC1
InChIKey: AIYARRXLUTYUKK-UHFFFAOYSA-N
Target: Tyrosine-protein kinase JAK2 (SINGLE PROTEIN), Homo sapiens
UniProt: O60674
Assay: Enzyme Assay: The activity of the isolated recombinant JAK1 and JAK2 kinase domain was measured by monitoring phosphorylation of a peptide derived from JAK3 (Val-Ala-Leu-Val-Asp-Gly-Tyr-Phe-Arg-Leu-Thr-Thr, fluorescently labeled on the N-terminus with 5-carboxyfluorescein) using the Caliper LabChip® technology (Caliper Life Sciences, Hopkinton, Mass.). To determine inhibition constants (Ki), compounds were diluted serially in DMSO and added to 50 μL kinase reactions containing purified enzyme (1.5 nM JAK1, or 0.2 nM JAK2), 100 mM HEPES buffer (pH 7.2), 0.015% Brij-35, 1.5 μM peptide substrate, ATP (25 μM), 10 mM MgCl2, 4 mM DTT at a final DMSO concentration of 2%. Reactions were incubated at 22° C. in 384-well polypropylene microtiter plates for 30 minutes and then stopped by addition of 25 μL of an EDTA containing solution (100 mM HEPES buffer (pH 7.2), 0.015% Brij-35, 150 mM EDTA), resulting in a final EDTA concentration of 50 mM. After termination of the kinase reaction, the proportion of phosphorylated product was determined as a fraction of total peptide substrate using the Caliper LabChip® 3000 according to the manufacturer's specifications.
Measurement: Ki = 0.33 nM (pChEMBL 9.479999542236328)
Descriptors: MW 611.19, ALogP 6.13, HBA 8, HBD 1, PSA 92.8, QED 0.26
Compound (SMILES): C[C@H](N)C(=O)N1CCN(c2cnc3ccc(-c4ccc5oc(N)nc5c4)cc3n2)CC1
InChIKey: SLNSZBRVIDWVBZ-ZDUSSCGKSA-N
Target: Phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit delta isoform (SINGLE PROTEIN), Homo sapiens
UniProt: O00329
Assay: PI 3-Kinase HTRF Assay: Briefly, the assay is a time resolved FRET assay that indirectly measures PIP3 product formed by the activity of a PI3-K. The kinase reaction is performed in a microtitre plate (e.g., a 384 well microtitre plate). The total reaction volume is approximately 20 ul per well. In the first step, each well receives 2 ul of test compound in 20% dimethylsulphoxide resulting in a 2% DMSO final concentration. Next, approximately 14.5 ul of a kinase/PIP2 mixture (diluted in 1x reaction buffer) is added per well for a final concentration of 0.25-0.3 ug/ml kinase and 10 uM PIP2. The plate is sealed and incubated for 15 minutes at room temperature. To start the reaction, 3.5 ul of ATP (diluted in 1x reaction buffer) is added per well for a final concentration of 10 uM ATP. The plate is sealed and incubated for 1 hour at room temperature. The reaction is stopped by adding 5 ul of Stop Solution per well and then 5 ul of Detection Mix is added per well. The plate is sealed, incubated for 1 hour at room temperature, and then read on an appropriate plate reader.
Measurement: IC50 < 1000 nM (pChEMBL 6)
Descriptors: MW 417.47, ALogP 2.02, HBA 8, HBD 2, PSA 127.4, QED 0.52