lovingscience/chembl-v37-raw-activities huggingface dataset:
Compound (SMILES): N#CC1(CC(C(=O)Nc2ccc(C(=O)O)cc2)c2ccc(-c3c(C(F)(F)F)ccc(Cl)c3F)c[n+]2[O-])CC1
InChIKey: IABKOYWFXCSAPK-UHFFFAOYSA-N
Target: Plasma kallikrein (SINGLE PROTEIN), Homo sapiens
UniProt: P03952
Assay: Kallikrein Assay: The effectiveness of a compound of the present invention as an inhibitor of Kallikrein can be determined using a relevant purified serine protease, and an appropriate synthetic substrate. The rate of hydrolysis of the chromogenic or fluorogenic substrate by the relevant serine protease was measured both in the absence and presence of compounds of the present invention. Assays were conducted at room temperature or at 37° C. Hydrolysis of the substrate resulted in release of amino trifluoromethylcoumarin (AFC), which was monitored spectrofluorometrically by measuring the increase in emission at 510 nm with excitation at 405 nm. A decrease in the rate of fluorescence change in the presence of inhibitor is indicative of enzyme inhibition. Such methods are known to one skilled in the art. The results of this assay are expressed as the half-maximal inhibitory concentrations (IC50), or the inhibitory constant, Ki.Kallikrein determinations were made in 50 mM HEPES buffer at pH 7.4 containing 150 mM NaCl, 5 mM CaCl2, and 0.1% PEG 8000 (polyethylene glycol; Fisher Scientific). Determinations were made using purified Human plasma kallikrein at a final concentration of 0.5 nM (Enzyme Research Laboratories) and the synthetic substrate, Acetyl-K-P-R-AFC (Sigma # C6608) at a concentration of 100 mM.Activity assays were performed by diluting a stock solution of substrate at least tenfold to a final concentration ≤0.2 Km into a solution containing enzyme or enzyme equilibrated with inhibitor. Times required to achieve equilibration between enzyme and inhibitor were determined in control experiments. The reactions were performed under linear progress curve conditions and fluorescence increase measured at 405 Ex/510 Em nm. Values were converted to percent inhibition of the control reaction (after subtracting 100% Inhibition value). IC50 was determined by inflection point from a four parameter logistic curve fit. Ki was calculated using the Cheng Prusoff equation, Ki=IC50/(1+([S]/Km)).
Measurement: IC50 = 155 nM (pChEMBL 6.809999942779541)
Descriptors: MW 547.89, ALogP 5.91, HBA 4, HBD 2, PSA 117.13, QED 0.22
Compound (SMILES): CC1(C)CN(c2ccc(-c3ccccc3P(C)(C)=O)c(F)c2F)C(=O)[C@@H]1NC(=O)Nc1ccc(C(F)(F)F)cc1F
InChIKey: FWWVYQFMSHSYOO-DEOSSOPVSA-N
Target: fMet-Leu-Phe receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P21462
Assay: FPR2 and FPR1 Cyclic Adenosine Monophosphate (cAMP) Assays: A mixture of forskolin (5 μM final for FPR2 or 10 μM final for FPR1) and IBMX (200 μM final) were added to 384-well Proxiplates (Perkin-Elmer) pre-dotted with test compounds in DMSO (1% final) at final concentrations in the range of 0.020 nM to 100 μM. Chinese Hamster Ovary cells (CHO) overexpressing human FPR1 or human FPR2 receptors were cultured in F-12 (Ham's) medium supplemented with 10% qualified FBS, 250 μg/ml zeocin and 300 μg/ml hygromycin (Life Technologies). Reactions were initiated by adding 2,000 human FPR2 cells per well or 4,000 human FPR1 cells per well in Dulbecco's PBS (with calcium and magnesium) (Life Technologies) supplemented with 0.1% BSA (Perkin-Elmer). The reaction mixtures were incubated for 30 min at room temperature. The level of intracellular cAMP was determined using the HTRF HiRange cAMP assay reagent kit (Cisbio) according to manufacturer's instruction. Solutions of cryptate conjugated anti-cAMP and d2 fluorophore-labelled cAMP were made in a supplied lysis buffer separately. Upon completion of the reaction, the cells were lysed with equal volume of the d2-cAMP solution and anti-cAMP solution. After a 1-h room temperature incubation, time-resolved fluorescence intensity was measured using the Envision (Perkin-Elmer) at 400 nm excitation and dual emission at 590 nm and 665 nm. A calibration curve was constructed with an external cAMP standard at concentrations ranging from 1 μM to 0.1 pM by plotting the fluorescent intensity ratio from 665 nm emission to the intensity from the 590 nm emission against cAMP concentrations. The potency and activity of a compound to inhibit cAMP production was then determined by fitting to a 4-parametric logistic equation from a plot of cAMP level versus compound concentrations.
Measurement: EC50 = 570 nM (pChEMBL 6.239999771118164)
Descriptors: MW 597.5, ALogP 6.6, HBA 3, HBD 2, PSA 78.51, QED 0.26
Compound (SMILES): COC(=O)N1c2ccc3c(nc(COC4CCCC4)n3[C@@H]3CCC[C@@H](C(=O)O)C3)c2CC[C@@H]1C
InChIKey: AXDSDHVCYVJVHC-RCCFBDPRSA-N
Target: Bromodomain-containing protein 4 (SINGLE PROTEIN), Homo sapiens
UniProt: O60885
Assay: HTRF Biochemical Assay for CBP and BRD4 Activity: The assay was performed in a final volume of 6 μL in assay buffer containing 50 mM Hepes (pH 7.5, (0.5M Hepes, pH 7.5 solution; Teknova H1575)), 0.5 mM GSH, 0.01% BGG (0.22 μM filtered, Sigma, G7516-25G), 0.005% BSA (0.22 μM filtered, EMD Millipore Corporation, 126575) and 0.01% Triton X-100 (Sigma, T9284-10L). Nanoliter quantities of 10-point, 3-fold serial dilution in DMSO were pre-dispensed into 1536 assay plates (Corning, #3724BC) for a final test concentration of 33 μM to 1.7 nM, top to lowest dose, respectively. 3 of 2× Protein and 3 μL of 2× Peptide Ligand were added to assay plates (pre-stamped with compound). Plates were incubated for varying times at room temperature prior to measuring the signal. TR-FRET (Time-Resolved Fluorescence Resonance Energy Transfer) was measured on the PHERAstar (BMG, equipped with HTRF optic module [337/520/490]) or on the Envision (PerkinElmer, equipped with the TRF Laser unit, TRF dual mirror D400/D505 and emission filters M520 and M495). Data were reported as percent inhibition compared with control wells based on the following equation: % inh=1−((TR-FRET ratio−AveLow)/(AveHigh−AveLow)) where TR-FRET ratio=(Fluorescence at 520 nm/Fluorescence at 490 nm)*10000), AveLow=average TR-FRET ratio of no enzyme control (n=32), and AveHigh=average TR-FRET ratio of DMSO control (n=32). IC50 values were determined by curve fitting of the standard 4 parameter logistic fitting algorithm included in the Activity Base software package: IDBS XE Designer Model 205. Data is fitted using the Levenburg Marquardt algorithm. For all assay formats data were reported as percent inhibition compared with control wells based on the following equation: % inh=100*((FLU−AveLow)/(AveHigh−AveLow)) where FLU=measured Fluorescence, AveLow=average Fluorescence of no enzyme control (n=32), and AveHigh=average Fluorescence of DMSO control (n=32). IC50 values were determined by curve fitting of the standard 4 parameter logistic fitting algorithm included in the Activity Base software package: IDBS XE Designer Model 205. Data is fitted using the Levenburg Marquardt algorithm.
Measurement: IC50 = 50500 nM (pChEMBL 4.300000190734863)
Descriptors: MW 469.58, ALogP 5.22, HBA 6, HBD 1, PSA 93.89, QED 0.65
Compound (SMILES): COc1cc(N2CCN(C(=O)[C@@H](C)C[C@@]3(c4ccccn4)NC(=O)NC3=O)C[C@@H]2C)cc(F)c1F
InChIKey: NAZJMGDZKSUZPA-ISQRANHSSA-N
Target: A disintegrin and metalloproteinase with thrombospondin motifs 5 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9UNA0
Assay: In Vitro Assay: TABLE IX: The basis for the assay is the cleavage of the substrate TBIS-1 (5 FAM-TEGEARGSVILLK (5TAMRA)K-NH2) (SEQ ID NO: 2) by human ADAMTS-5.For the dose response (10 point), 4 μL of a dilution series of compound (2 mM highest concentration, ⅕ dilution in DMSO further diluted 1 in 10 in water, corresponding to a final highest concentration of 20 μM), is transferred to 384 well Fluotrac 200 plate (Greiner, cat #781076) and incubated at room temperature for 30 min with a 26 μL buffer solution (50 mM Hepes pH 7.5, 100 mM NaCl, 5 mM CaCl2, 0.1% CHAPS) containing hADAMTS-5 (0.63 ng/μL) (it will be appreciated by the skilled person that the potency read out is independent of the enzyme concentration).The reaction is initiated by adding to the assay plate TBIS-1 (10 μL, 4.5 μM, Anaspec) in the same buffer.Finally, the fluorescence is read on the Envision (Perkin Elmer) after an incubation of 90 min at 37° C. (Excitation 485 nm, emission 530).
Measurement: IC50 = 37.5 nM (pChEMBL 7.429999828338623)
Descriptors: MW 487.51, ALogP 2.17, HBA 6, HBD 2, PSA 103.87, QED 0.61
Compound (SMILES): O=C1CCC(c2ccccc2)N1c1ccc(-c2noc(C(F)(F)F)n2)cc1
InChIKey: FDNJKUCJVFAHFE-UHFFFAOYSA-N
Target: Histone deacetylase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q13547
Assay: Enzymatic Assay: HDAC1 and 6 reagents: FLAG-tagged HDACs 1 and 6 were prepared in-house by protein expression in HEK293F cells followed by anti-FLAG affinity purification. Assays were performed with buffer containing 20 mM HEPES, pH 8.0 [Boston BioProducts, catalog #BB-104, 1M stock], 137 mM NaCl [Sigma, catalog #S5150, 5M stock], 2.7 mM KCl [BioChemika, catalog #87526, 4M stock], 1 mM MgCl2 [Fluka, catalog #63020, 1M stock], and 0.05% BSA (Fraction V) [Invitrogen, catalog #15260, 7.5% stock]. In addition to the above buffer ingredients, TCEP [CalBiochem, catalog #580561, 500 mM stock] was added at a final concentration of 0.5 mM to the buffer for the HDAC6 assays. HDAC1, 2, 3, and 6 enzymes were run at the final concentrations of 0.3 nM, 1.5 nM, 0.3 nM, and 1.333 nM, respectively. Fluor-de-Lys substrate [BioMol Research Laboratories, catalog #KI-104], used to evaluate enzyme activity, was added at the final concentrations of 20 uM, 40 uM, 20 uM, and 2.5 uM for HDACs 1, 2, 3, and 6. To enable detection of the signal, Developer [BioMol Research Laboratories, catalog #KI-105] was added at a 1:250 dilution to the stop solution, which also included 10 uM SAHA [Sigma, catalog #SML0061] to ensure complete termination of the reaction.
Measurement: IC50 = 1000 nM (pChEMBL 6)
Descriptors: MW 373.33, ALogP 4.62, HBA 4, HBD 0, PSA 59.23, QED 0.67
Compound (SMILES): N#C[C@H]1CCOC[C@@H]1n1nc(Nc2ccc(C(N3CCCC3)C(F)(F)F)cc2)c2c(=O)[nH]ccc21
InChIKey: TVAFKVGYDWDABI-NTEFNCDNSA-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. After normalization to untreated controls, the percent inhibition of the HTRF signal at each compound concentration was calculated.
Measurement: IC50 = 130 nM (pChEMBL 6.889999866485596)
Descriptors: MW 486.5, ALogP 4.27, HBA 7, HBD 2, PSA 98.97, QED 0.56
Compound (SMILES): COCCOc1nc(Nc2ccc(CN3CCN(C)CC3)cc2)nc2[nH]cc(-c3ccc(F)cc3)c12
InChIKey: GBJMPZOLRHFDND-UHFFFAOYSA-N
Target: Tyrosine-protein kinase Mer (SINGLE PROTEIN), Homo sapiens
UniProt: Q12866
Assay: Mer Kinase Assay: Certain compounds of formula (I) were tested for their potency in inhibiting Mer kinase. Inhibition of Mer kinase activity by a test compound was calculated by AlphaScreen (PerkinElmer 6760620M). Standard assay conditions included 2 ng of recombinant Mer kinase (SignalChem) with 5 ng Poly-(GT)-Biotin (Cisbio) in the assay buffer, i.e., 10 μM ATP (Sigma), 8 mM MOPs, pH7.0 (Sigma M-1254), 0.2 mM EDTA (Sigma E-6758), 0.5% glycerol (J. T Baker 2136-1), 0.1 mg/ml BSA (Bio-Rad 500-0007), 0.01% 2-mercaptoethanol (BDH 441433A), 0.001% Brij35 (Sigma P1254), 10 mM MgCl2, and 10 mM MnCl2 in a final volume of 25 μL. Reactions were incubated at 30° C. for 30 minutes and stopped by adding 5 μL of 50 mM EDTA. Product analysis was performed using the AlphaScreen kit and counted with Enspire Alpha (PerkinElmer). Screen values of inhibitors were determined after carrying out assays with each compound at serially diluted concentrations in duplication. Results were analyzed using linear regression software (GraphPad Prism 4; GraphPad Software Inc.).
Measurement: IC50 = 250 nM (pChEMBL 6.599999904632568)
Descriptors: MW 490.58, ALogP 4.28, HBA 7, HBD 2, PSA 78.54, QED 0.34
Compound (SMILES): CN1CCN(c2ccc(Nc3nc(-n4ccc(C(=O)NC5CC5)c4)c4ccsc4n3)cc2F)CC1
InChIKey: YRDQOXAUSNHEKH-UHFFFAOYSA-N
Target: Tyrosine-protein kinase JAK2 (SINGLE PROTEIN), Homo sapiens
UniProt: O60674
Assay: AlphaLisa Assay: 1. Add 20 μl of the diluted compound to each well of the 384-well opti plate2. Tap the plate gently3. Add 10 μl of the enzyme to each well of the opti plate4. Cover with plate sealer5. Spin the plate briefly (640 rpm for 30 s) in a plate centrifuge.6. Incubate the plate at room temperature for 10 minutes7. Add 10 μl of the substrate8. Cover with plate sealer9. Spin the plate briefly (640 rpm for 30 s) in a plate centrifuge.10. Cover with plate sealer and incubate at room temperature for 1 hour11. After incubation, transfer 10 μl of reaction mix to fresh wells of a 384-well plate.12. Add 10 μl acceptor bead from working stock.13. Cover with plate sealer14. Spin the plate briefly (640 rpm for 30 s) in a plate centrifuge.15. Cover with plate sealer and incubate at room temperature for 30 minutes16. Add 10 μl donor bead from working stock (in dark).17. Cover with plate sealer18. Spin the plate briefly (640 rpm for 30 s) in a plate centrifuge.19. Cover with plate sealer and incubate at room temperature for 15-30 minutes20. Read the plate in pherastar (alpha screen protocol)
Measurement: IC50 = 10000 nM (pChEMBL 5)
Descriptors: MW 491.6, ALogP 4.01, HBA 8, HBD 2, PSA 78.32, QED 0.42
Compound (SMILES): CCOC(=O)Cn1cc(Nc2ncc3ccn(CC4CCCCC4C)c3n2)cn1
InChIKey: CQCDCUPMRWRWDC-UHFFFAOYSA-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 396.5, ALogP 3.76, HBA 8, HBD 1, PSA 86.86, QED 0.61
Compound (SMILES): COc1ccc(F)c(C(=O)N2CC3CN(Cc4c(-c5ccc(Cl)cc5)nc5ccccn45)CC3C2)n1
InChIKey: YXSUHEIMCHWLAT-UHFFFAOYSA-N
Target: Potassium channel subfamily K member 9 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9NPC2
Assay: In Vitro Electrophysiological Assay: In Vitro Electrophysiological Analysis of the Human TASK-1 and TASK-3 Channels Via Two-Electrode Voltage Clamp Technique in Xenopus laevis OocytesSubsequently, the oocytes were injected with 0.5-5 ng of a cRNA solution coding for TASK-1 or TASK-3. For the electrophysiological analysis of the channel proteins expressed in the oocytes, the two-electrode voltage clamp technique was used. The measurements were conducted at room temperature (21-22° C.) using a Turbo TEC 10CD amplifier (NPI), recorded at 2 kHz and filtered with 0.4 kHz. Substance administration was performed using a gravitation-driven perfusion system. Here, the oocyte is located in a measuring chamber and exposed to the solution stream of 10 ml/min. The level in the measuring chamber is monitored and regulated by sucking off the solution using a peristaltic pump.
Measurement: IC50 = 110 nM (pChEMBL 6.960000038146973)
Descriptors: MW 505.98, ALogP 4.4, HBA 6, HBD 0, PSA 62.97, QED 0.4
Compound (SMILES): COc1cc2nccc(Oc3ccc(NC(=O)C4(C(=O)Nc5ccc(F)cc5)CC4)cc3)c2cc1C(=O)O
InChIKey: KDSURWWNWSNQLU-UHFFFAOYSA-N
Target: Tyrosine-protein kinase Mer (SINGLE PROTEIN), Homo sapiens
UniProt: Q12866
Assay: Mer Kinase Assay: Human Mer (residues R557-E882 with H628Q and R794A, 0.7 nM) was incubated with 8 mM MOPS pH 7.0, 0.2 mM EDTA, 30 mM NaCl, 250 μM GGMEDIYFEFMGGKKK (SEO ID NO. 44). 10 mM magnesium acetate, and 10 μM [γ-33P-ATP]. The reaction was initiated by the addition of the Mg/ATP mix. After incubation for 40 minutes at room temperature, the reaction was stopped by the addition of phosphoric acid to a concentration of 0.5%. A reaction aliquot of 10 μL was then spotted onto a P30 filtermat and washed four times for 4 minutes in 0.425% phosphoric acid and once in methanol prior to drying and scintillation counting. Incorporated 33P was measured using the Wallac Microbeta scintillation counter (Perkin Elmer).
Measurement: IC50 = 55 nM (pChEMBL 7.260000228881836)
Descriptors: MW 515.5, ALogP 5.23, HBA 6, HBD 3, PSA 126.85, QED 0.27
Compound (SMILES): O=C(CCCc1nc2ccccc2c(=O)[nH]1)N1CCC(c2nnc(-c3cnccn3)o2)CC1
InChIKey: OUWQQYPQZGBQTQ-UHFFFAOYSA-N
Target: Poly [ADP-ribose] polymerase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P09874
Assay: Biochemical Assay: PARP1 biochemical assay was purchased as a kit from Trevigen (cat.#4676-096-K) and used per manufacturers recommendations.PARP2 biochemical assay was purchased as a kit from BPS (cat.#80552) and used per manufacturers recommendations.
Measurement: IC50 = 8540 nM (pChEMBL 5.070000171661377)
Descriptors: MW 445.48, ALogP 2.49, HBA 8, HBD 1, PSA 130.76, QED 0.48
Compound (SMILES): N#CC1(C(=O)N2CCC(n3cnc(-c4ccc(F)cc4)c3-c3ccc4nc(NC(=O)c5ccnc(F)c5)cn4n3)CC2)CC1
InChIKey: SLLHJEGXTPYIOI-UHFFFAOYSA-N
Target: Casein kinase I isoform delta (SINGLE PROTEIN), Homo sapiens
UniProt: P48730
Assay: Kinase Assay: The kinase assay was performed in V-bottom 384-well plates. The final assay volume was 30 μl prepared from 15 μl additions of enzyme, substrates (fluoresceinated peptide FL-AHA-KRRRAL-PSER-VASLPGL-OH and ATP) and test compounds in assay buffer (20 mM HEPES pH 7.4, 30 mM MgCl2, 0.015% Brij35 and 4 mM DTT). The reaction was incubated at room temperature for 22 hours and terminated by adding 45 μl of 35 mM EDTA to each sample. The reaction mixture was analyzed on the Caliper LABCHIP®3000 (Caliper, Hopkinton, Mass.) by electrophoretic separation of the unphosphorylated substrate and phosphorylated product. Inhibition data were calculated by comparison of the no enzyme control reactions for 100% inhibition and vehicle-only reactions for 0% inhibition. The final concentration of reagents in the assay were 200 pM CK1ε or CK1δ, 50 μM ATP, 1.5 μM FL-AHA-KRRRAL-PSER-VASLPGL-OH, and 1.6% DMSO. Dose response curves were generated to determine the concentration required to inhibit 50% of the kinase activity (IC50). Compounds were dissolved at 10 mM in dimethylsulfoxide (DMSO) and evaluated at eleven concentrations.
Measurement: IC50 = 0.172 nM (pChEMBL 9.760000228881836)
Descriptors: MW 593.6, ALogP 4.65, HBA 9, HBD 1, PSA 134.1, QED 0.28
Compound (SMILES): Cc1cccc(-n2nccn2)c1C(=O)N1CCC[C@H]1c1nc(-c2ccccc2OC(F)(F)F)no1
InChIKey: MPHXVTBJXAJZHY-KRWDZBQOSA-N
Target: Orexin receptor type 2 (SINGLE PROTEIN), Homo sapiens
UniProt: O43614
Assay: In Vitro Assay: Human orexin-A as an agonist is prepared as 1 mM stock solution in MeOH: water (1:1), diluted in HBSS containing 0.1% bovine serum albumin (BSA), NaHCO3: 0.375 g/L and 20 mM HEPES for use in the assay at a final concentration of 3 nM.Antagonists are prepared as 10 mM stock solution in DMSO, then diluted in 384-well plates using DMSO followed by a transfer of the dilutions into in HBSS containing 0.1% bovine serum albumin (BSA), NaHCO3: 0.375 g/L and 20 mM HEPES. On the day of the assay, 50 uL of staining buffer (HBSS containing 1% FCS, 20 mM HEPES, NaHCO3: 0.375 g/L, 5 mM probenecid (Sigma) and 3 uM of the fluorescent calcium indicator fluo-4 AM (1 mM stock solution in DMSO, containing 10% pluronic) is added to each well. The 384-well cell-plates are incubated for 50 min at 37° C. in 5% CO2 followed by equilibration at rt for 30 min before measurement.Within the Fluorescent Imaging Plate Reader (FLIPR Tetra, Molecular Devices), antagonists are added to the plate in a volume of 10 uL/well, incubated for 120 min and finally 10 uL/well of agonist is added. Fluorescence is measured for each well at 1 second intervals, and the height of each fluorescence peak is compared to the height of the fluorescence peak induced by 3 nM orexin-A with vehicle in place of antagonist. The IC50 value (the concentration of compound needed to inhibit 50% of the agonistic response) is determined and may be normalized using the obtained IC50 value of an on-plate reference compound. Optimized conditions were achieved by adjustment of pipetting speed and cell splitting regime. The calculated IC50 values may fluctuate depending on the daily cellular assay performance. Fluctuations of this kind are known to those skilled in the art. In the case where IC50 values have been determined several times for the same compound, the geometric mean has been given.
Measurement: IC50 = 85 nM (pChEMBL 7.070000171661377)
Descriptors: MW 484.44, ALogP 4.5, HBA 8, HBD 0, PSA 99.17, QED 0.41
Compound (SMILES): C[C@@H](Cn1c(=O)[nH]/c(=N\c2ccc(Oc3cnc(Cl)cn3)cc2)n(Cc2ccc(Cl)cc2)c1=O)C(=O)O
InChIKey: HAMFTESJQYPRTL-AWEZNQCLSA-N
Target: P2X purinoceptor 3 (SINGLE PROTEIN), Rattus norvegicus
UniProt: P49654
Assay: Rat P2X3 Binding Assay in Rat Serum Albumin (HSA): Rat P2X3 receptor gene (GenBank accession number NM_031075) was expressed in C6BU-1 cell. The C6BU-1 cells were seeded in a 96-well microtiter plate at a concentration of 2500 cells/well and cultured in the medium (7.0% fetal bovine serum, 7.0% horse serum, and 1% antibiotic and antifungal in DMEM) for one day at 37° C. under 5% carbon dioxide atmosphere. The plasmid was transfected into the cells using transfection reagent FuGENE6 (Promega). The transfected cells were cultured in the medium for one day at 37° C. under 5% carbon dioxide atmosphere. The medium was replaced with 4 μM Fluo-4-AM solution (pH7.5) containing 20 mM HEPES, 137 mM NaCl, 5.27 mM KCl, 0.9 mM MgCl2, 1.26 mM CaCl2, 5.6 mM D-glucose, 2.5 mM probenecid, 10% BSA, and 0.08% Pluronic F-127, and incubated at 37° C. under 5% dioxide carbon atmosphere for one hour. The plate was washed with washing buffer (20 mM HEPES, 137 mM NaCl, 5.27 mM KCl, 0.9 mM MgCl2, 1.26 mM CaCl2, 5.6 mM D-glucose, 2.5 mM probenecid, pH7.5), and each well was added with 40 μL of this buffer. The plate was placed in High-Throughput Screening System FDSS 3000 (Hamamatsu Photonics K.K.). Measurement of fluorescence intensity by FDSS 3000 was started, and 40 μL of DMSO solutions containing 1% RSA (final concentrations) and different concentrations of the test compound as prepared by dilution with dilution buffer (20 mM HEPES, 137 mM NaCl, 5.27 mM KCl, 0.9 mM MgCl2, 1.26 mM CaCl2, 5.6 mM D-glucose, 2.5 mM probenecid, 0.1% Pluronic F-127, pH7.5) were dispensed to each well through the built-in automatic dispenser. Five minutes after, 50 nM ATP solution (50 μL) prepared by dilution with the dilution buffer was dispensed through the built-in automatic dispenser, and the measurement of fluorescence intensity was continued for 4 min. For each well, the specific maximum fluorescence intensity was calculated as the ratio of the maximum fluorescence intensity after addition of the ATP solution to the fluorescence intensity at the starting of the measurement. The 50% inhibitory concentration (IC50) was calculated under the assumption that the specific maximum fluorescence intensity without test compound is 0% inhibition and that the specific maximum fluorescence intensity when the dilution buffer was added in place of ATP solution is 100% inhibition, to evaluate the inhibitory activity of the test compound. FDSS software (Hamamatsu Photonics K.K.) was used for calculation of the specific maximum fluorescence intensity. IC50 was calculated using Microsoft Excel (Microsoft Corporation) and XLfit (idbs Ltd.).
Measurement: IC50 = 6 nM (pChEMBL 8.220000267028809)
Descriptors: MW 543.37, ALogP 3.23, HBA 9, HBD 2, PSA 144.46, QED 0.35
Compound (SMILES): CC(C(=O)Nc1cc(C(=O)Nc2ccc(-c3ccc(C(F)F)cc3)nc2)ccc1OC(F)(F)F)N1CCOCC1
InChIKey: FJXNQTNRVQCBSY-UHFFFAOYSA-N
Target: Protein Wnt-3a (SINGLE PROTEIN), Homo sapiens
UniProt: P56704
Assay: HCT116 FOPFlash : In preparation of the assay, the two cell lines were plated 24 hours before at 10000 cells per well of a 384 micro titre plate (MTP) in 30 μL growth medium. Selective inhibitory activity for small molecules on the mutated Wnt pathway was determined after parallel incubation of both (TOP and FOP) HCT116 reporter cell lines with a compound dilution series from 50 μM to 15 nM in steps of 3.16-fold dilutions in CAFTY buffer (130 mM NaCl, 5 mM KCl, 20 mM HEPES, 1 mM MgCl2, 5 mM NaHCO3, pH 7.4) containing 2 mM Ca2+ and 0.01% BSA. The compounds were thereby serially prediluted in 100% DMSO and thereafter in addition 50 fold into the CAFTY compound dilution buffer (described above). From this dilution 10 μL were added to the cells in 30 μL growth medium and incubated for 36 hours at 37° C. and 5% CO2. Thereafter luciferase assay buffer (1:1 mixture of luciferase substrate buffer (20 mM Tricine, 2.67 mM MgSO4, 0.1 mM EDTA, 4 mM DTT, 270 μM Coenzyme A, 470 μM Luciferin, 530 μM ATP, ph adjusted to pH 7.8 with a sufficient volume of 5M NaOH) and Triton buffer (30 mL Triton X-100, 115 mL glycerol, 308 mg Dithiothreitol, 4.45 g Na2HPO4.2H2O, 3.03 g TRIS HCl, ad 1I H2O, pH 7.8) was added as equal volume to the compound solution on the cells to determine luciferase expression as a measure of Wnt signaling activity in a luminometer.n order to determine the inhibitory activity of compounds for the WT Wnt signaling pathway, the Super FopFlash vector were cotransfected with pcDNA3 into HEK293 cells.
Measurement: IC50 > 50000 nM (pChEMBL 4.301000118255615)
Descriptors: MW 564.51, ALogP 5.5, HBA 6, HBD 2, PSA 92.79, QED 0.35
Compound (SMILES): Cc1cc(C(F)(F)F)cc(O)c1-c1ccc(NC[C@H]2CC(F)(F)CN2)nn1
InChIKey: DYYNZRAPERRPBZ-LLVKDONJSA-N
Target: Tumor necrosis factor (SINGLE PROTEIN), Homo sapiens
UniProt: P01375
Assay: TNF-alpha Secretion Assay: Monocytic THP-1 cells were maintained according to providers' instructions in RPMI media as described above. Undifferentiated cells were plated at 50,000 cells per well in a 384-well flat-bottom cell culture plates (Greiner, #781986), and allowed to rest overnight. Experimental compounds were prepared and added as described above. TNF-α secretion was triggered by the addition of either 1 μg/mL LPS (Sigma, #L4391) or 100 ng/mL Pam3CSK4 (Invivogen, #tlrl-pms) depending on the experiment, and cells were incubated for 3 hours. 10 μL supernatant was removed, and TNF-α levels were monitored using an HTRF assay (CisBio, #62TNFPEC) according to manufacturers' instructions.
Measurement: IC50 > 100000 nM (pChEMBL 4)
Descriptors: MW 388.34, ALogP 3.59, HBA 5, HBD 3, PSA 70.07, QED 0.7
Compound (SMILES): CCNc1cc2c(cn1)c(-c1cnn(C)c1)nn2C1CCC(Oc2nccc(C)c2F)CC1
InChIKey: FPFXKHUOJATUAN-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 449.53, ALogP 4.67, HBA 8, HBD 1, PSA 82.68, QED 0.46
Compound (SMILES): O=C(N[C@@H](c1ccc(OC(F)(F)F)cc1)C1CC1)c1cc(=O)[nH]c(-c2ncccn2)n1
InChIKey: MAWBTKSIPKCZPV-MRXNPFEDSA-N
Target: cGMP-dependent 3',5'-cyclic phosphodiesterase (SINGLE PROTEIN), Homo sapiens
UniProt: O00408
Assay: Fluorescence Polarization (FP) Assay: In a typical experiment the PDE2 inhibitory activity of the compounds of the present invention was determined in accordance with the following experimental method. Rhesus PDE2A3 was amplified from rhesus macaque brain cDNA (Biochain Institute, Hayward, Calif.) using primers based on human PDE2A sequence (accession NM_002599.3) where the forward primer containing a Kozak consensus was 5′-gccaccatggggcaggcatgtggc-3′ and the reverse primer was 5′-tcactcagcatcaaggctgca-3′. Amplification with Easy-A High-Fidelity PCR cloning enzyme (Stratagene, La Jolla, Calif.) was 95° C. for 2 minutes followed by thirty three cycles of 95° C. for 40 seconds, 52° C. for 30 seconds, and 72° C. for 2 minutes 48 seconds. Final extension was 72° C. for 7 minutes. The PCR product was TA cloned into pcDNA3.3-TOPO (Invitrogen, Carlsbad, Calif.) according to standard protocol. A consensus sequence was developed from multiple clones and then deposited into GenBank (EU812167). AD293 cells (Stratagene, La Jolla, Calif.) with 70-80% confluency were transiently transfected with rhesus PDE2A3/pcDNA3.3-TOPO using Lipofectamine 2000 according to manufacturer specifications (Invitrogen, Carlsbad, Calif.).
Measurement: Ki = 21 nM (pChEMBL 7.679999828338623)
Descriptors: MW 431.37, ALogP 3.01, HBA 6, HBD 2, PSA 109.86, QED 0.62
Compound (SMILES): Cc1ccnn1-c1ccncc1NC(=O)c1c(N)nn2cc(F)cnc12
InChIKey: PTQCIVUYWYWRTH-UHFFFAOYSA-N
Target: Serine/threonine-protein kinase ATR (SINGLE PROTEIN), Homo sapiens
UniProt: Q13535
Assay: Inhibition Assay: Table 5: Compounds were screened for their ability to inhibit ATR kinase using a radioactive-phosphate incorporation assay. Assays were carried out in a mixture of 50 mM Tris/HCl (pH 7.5), 10 mM MgCl2 and 1 mM DTT. Final substrate concentrations were 10 μM [y-33P]ATP (3 mCi 33P ATP/mmol ATP, Perkin Elmer) and 800 μM target peptide(ASELPASQPQPFSAKKK) (SEQ ID NO:1).Assays were carried out at 25° C. in the presence of 5 nM full-length ATR. An assay stock buffer solution was prepared containing all of the reagents listed above, with the exception of ATP and the test compound of interest. 13.5 μL of the stock solution was placed in a 96 well plate followed by addition of 2 μL of DMSO stock containing serial dilutions of the test compound (typically starting from a final concentration of 15 μM with 3-fold serial dilutions) in duplicate (final DMSO concentration 7%). The plate was pre-incubated for 10 minutes at 25° C. and the reaction initiated by addition of 15 μL [γ-33P]ATP (final concentration 10 μM).
Measurement: Ki = 550 nM (pChEMBL 6.260000228881836)
Descriptors: MW 352.33, ALogP 1.59, HBA 8, HBD 2, PSA 116.02, QED 0.58
Compound (SMILES): NS(=O)(=O)N1CCC(c2n[nH]c3ccc(C(c4ccc(Cl)cc4)c4ccc(Cl)cc4)cc23)CC1
InChIKey: IWRKTSVEBQDGHV-UHFFFAOYSA-N
Target: Cannabinoid receptor 2 (SINGLE PROTEIN), Homo sapiens
UniProt: P34972
Assay: Biological Assay: CB1 and CB2 receptors are Gi-coupled GPCR. Activation of CB1 and CB2 receptors results in a decrease in cAMP production. An inverse agonist of the CB1 or CB2 receptor results in the opposite effect, an increase of cAMP production. The principle of this assay is based on HTRF® technology (Homogeneous Time-Resolved Fluorescence). The method is a competitive immunoassay between native cAMP produced by cells and the cAMP labeled with the fluorophore d2. The tracer binding is quantified by a Mab anti-cAMP labeled with Eu3+TBP-NHS Cryptate (supplied as part of the assay kit). The specific signal (i.e. energy transfer) is inversely proportional to the concentration of cAMP in the standard or sample.
Measurement: EC50 > 10000 nM (pChEMBL 5)
Descriptors: MW 515.47, ALogP 5.43, HBA 3, HBD 2, PSA 92.08, QED 0.35
Compound (SMILES): O=CO[C@H](CCCCNC(=O)c1ccc(F)cc1)OC(=O)N[C@@H](CCC(=O)O)C(=O)O
InChIKey: RYHAFNZTGXXJNZ-HOCLYGCPSA-N
Target: Glutamate carboxypeptidase 2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q04609
Assay: Fluorescence-Based Amplex Red Glutamic Acid Assay : The PSMA inhibitory activity was determined using a modification of the fluorescence-based Amplex Red Glutamic Acid Assay. Briefly, lysates of LNCaP cell extracts (25 μL) were incubated with the inhibitor (12.5 μL) in the presence of 4 μM N-acetylaspartylglutamate (NAAG) (12.5 μL) for 120 min. The amount of the glutamate released by NAAG hydrolysis was measured by incubating with a working solution (50 μL) of the Amplex Red Glutamic Acid Kit for 60 min. Fluorescence was measured with a VICTOR3V multilabel plate reader with excitation at 490 nm and emission at 642 nm. Inhibition curves were determined using semi-log plots and IC50 values were determined at the concentration at which enzyme activity was inhibited by 50%. Enzyme inhibitory constants (Ki values) were generated using the Cheng-Prusoff conversion. Assays were performed in triplicate.
Measurement: Ki = 42 nM (pChEMBL 7.380000114440918)
Descriptors: MW 442.4, ALogP 1.27, HBA 7, HBD 4, PSA 168.33, QED 0.19
Compound (SMILES): O=C(Nc1ccc(C(O)(C(F)(F)F)C(F)(F)F)cc1)C1NCc2cc(S(=O)(=O)CC3(O)CC3)ccc21
InChIKey: ZEJMRHIMGVKOSB-UHFFFAOYSA-N
Target: Nuclear receptor ROR-gamma (SINGLE PROTEIN), Homo sapiens
UniProt: P51449
Assay: FRET Assay: The assay was run in black 384 well plates (Greiner cat no: 784900). Various concentrations of test ligands in 0.1 microlitres DMSO were dispensed to assay plates using an Echo acoustic dispenser. Two pre-mixes were prepared and incubated for 1 h at room temp in the dark. Pre-mix 1 comprised 100 nM Protein (Biotinylated HN-Avi-MBP-TCS-hRORÎ³ (258-518)) and 60 nM Streptavidin APC in assay buffer, 50 mM MOPS pH7.4, 50 mM KF, 0.003% (w/v) CHAPS, 10 mM DTT and 0.01% (w/v) BSA and pre-mix 2 comprised 160 nM biotinylated SRC-1 peptide (NCOA1-677-700) and 20 nM Europium-W8044 labelled Streptavidin in assay buffer. Five microlitres of pre-mix 2 was dispensed to assay plates containing 0.1 microlitres of test compound and was incubated for 15 minutes prior to adding five microlitres of pre-mix 1. Plates were incubated at room temperature for 1 hour in the dark, prior to reading in a Pherastar multi-mode plate reader using HTRF filter set (ex 320, em 612 and 665). The FRET signal at 665 nm was divided by the signal at 612 nm and multiplied by 10,000 to generate a signal ratio value for each well. The raw data was transformed to % effect using the equation:Compound % effect=100*[(Xâmin)/(maxâmin)],where X represents the normalized value for the compound based on the Min (vehicle) and Max (reference compound) inhibition control.
Measurement: IC50 = 1000 nM (pChEMBL 6)
Descriptors: MW 538.47, ALogP 3.08, HBA 6, HBD 4, PSA 115.73, QED 0.42
Compound (SMILES): COCC(=O)Nc1ccc2c(c1)[C@]1(CCO2)CS(=O)(=O)N(C)C(=N)N1
InChIKey: VTEPBMKUHQXDEL-HNNXBMFYSA-N
Target: Beta-secretase 2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9Y5Z0
Assay: BACE-2 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 (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 (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. IC50s are determined by nonlinear regression analysis (sigmoidal dose response, variable slope) of percent inhibition data with minimum and maximum values set to 0 and 100 percent respectively.
Measurement: Ki = 341 nM (pChEMBL 6.46999979019165)
Descriptors: MW 368.42, ALogP 0.05, HBA 6, HBD 3, PSA 120.82, QED 0.69
Compound (SMILES): Cn1c(=O)n(CC(=O)N2CC(F)C2)c2cc(-c3ccc(CO)s3)cnc21
InChIKey: FDLIKLKMLSBNAT-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 = 915 nM (pChEMBL 6.039999961853027)
Descriptors: MW 376.41, ALogP 1.14, HBA 7, HBD 1, PSA 80.36, QED 0.74
Compound (SMILES): CN1CC2N(C(=O)C3CCS(=O)(=O)CC3)CCC2(S(=O)(=O)c2ccccc2)c2ccc(C(F)(C(F)(F)F)C(F)(F)F)cc21
InChIKey: SLRWDASWAUKALJ-UHFFFAOYSA-N
Target: Nuclear receptor ROR-gamma (SINGLE PROTEIN), Homo sapiens
UniProt: P51449
Assay: Binding Assay : The binding of potential ligands to RORγ is measured by competition with [3H]25-hydroxycholesterol (Perkin Elmer NET674250UC) using a scintillation proximity assay (SPA). The ligand binding domain of human RORγ (A262-S507) with an N-terminal His tag is expressed in E. coli and purified using nickel affinity chromatography. 15 μg/well RORγ (A262-S507) is incubated with test compound at varying concentrations in 3-fold serial dilution, with final concentrations ranging from 16.6 μM to 0.28 nM, for 10 min at rt in PBS buffer (Invitrogen #14190-144) containing 0.5% fatty acid free BSA (Gemini Bio-Products, Cat. #700-107P) and 0.1% glycerol (Sigma Cat # G5516). 10 nM of [3H] 25-hydroxycholesterol is then added, and the reaction is incubated for 10 min. 10 mg/mL of Copper-His Tag-PVT beads (Perkin Elmer cat # RPNQ0095) are added, and the mixture is incubated for 60 min. The reaction is read on a TopCount Microplate scintillation plate reader (Perkin Elmer). The competition data of the test compound over a range of concentrations was plotted as percentage inhibition of radioligand specifically bound in the absence of test compound (percent of total signal). After correcting for non-specific binding, IC50 values were determined.
Measurement: IC50 = 43 nM (pChEMBL 7.369999885559082)
Descriptors: MW 656.64, ALogP 4.52, HBA 6, HBD 0, PSA 91.83, QED 0.45
Compound (SMILES): CC(F)(F)c1cc(-n2cc(COc3ncc(C(F)F)cn3)nn2)ccc1Cl
InChIKey: SCHOYBYKAYKJLP-UHFFFAOYSA-N
Target: Retinoic acid receptor RXR-alpha (SINGLE PROTEIN), Homo sapiens
UniProt: P19793
Assay: NR2B standard assay: 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 149 mM NaCl, 4 mM KCl, 2 mM CaCl2, and 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 (149 mM NaCl (standard assay) or 150 mM (HTS assay), 4 mM KCl (standard assay) or 3 mM (HTS assay), 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 ¿EC80 (standard assay) or EC40 (HTS assay) is used to maximize the assay's signal window or the ability to detect NMDA receptor antagonists and negative allosteric modulators, respectively. 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 = 9 nM (pChEMBL 8.050000190734863)
Descriptors: MW 401.75, ALogP 4.34, HBA 6, HBD 0, PSA 65.72, QED 0.57
Compound (SMILES): Cc1cnc(CC(=O)Nc2cc([C@H]3CC[C@@H](OC(=O)NCCC(F)(F)F)C3)[nH]n2)o1
InChIKey: RYALJKKPLGHHOZ-NWDGAFQWSA-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 = 30.6 nM (pChEMBL 7.510000228881836)
Descriptors: MW 429.4, ALogP 3.2, HBA 6, HBD 3, PSA 122.14, QED 0.62
Compound (SMILES): O=C(NC[C@H](O)c1ccc(F)cn1)[C@H]1CCN(c2ncnc3[nH]ccc23)CC12CC2
InChIKey: HUGPLCWWVOYGRE-WBVHZDCISA-N
Target: Tyrosine-protein kinase JAK2 (SINGLE PROTEIN), Homo sapiens
UniProt: O60674
Assay: JAK Kinase Assays: Human baculovirus-expressed JAK1, 2, 3 and TYK2 were purchased from Carna Biosciences, Inc. All four purified enzymes contain only the catalytic domain. JAK1 (aa 850-1154) and TYK2 (aa 871-1187) are expressed with an N-terminally fused GST-tag, and JAK2 and JAK3 with an N-terminally fused His-tag.Inhibition of phosphorylation of a synthetic peptide was measured in an HTRF-based assay using the TK substrate-Biotin from the Cisbio HTRFKinEASE TK kit. First, 2 μl of TK solution (TK substrate-biotin in kinase buffer [1× enzymatic buffer from HTRFKinEASE TK kit, 1 mM DTT]) is added to a plate containing 1 μl prediluted compound (final assay concentration DMSO: 0.75%). Then, 5 μl kinase-ATP mix (prepared in kinase buffer) is added to the wells and the plates are incubated at RT for 20-30 min. For all four kinases a concentration of ATP that corresponded to the Km for ATP was used. The final concentrations of buffers, substrate, kinase and ATP were: JAK1: 50 mM Hepes buffer pH 7.0, 0.01% BSA, 10 mM MgCl2, 1 mM DTT, 7 μM ATP, 50 nM SEB, 1 μM TK Substrate-Biotin and 5 ng JAK1; JAK2: 50 mM Hepes buffer pH 7.0, 0.01% BSA, 5 mM MgCl2, 1 mM DTT, 4 μM ATP, 1 μM TK Substrate-Biotin and 0.1 ng JAK2; JAK3: 50 mM Hepes buffer pH 7.0, 0.01% BSA, 5 mM MgCl2, 1 mM DTT, 2 μM ATP, 1 μM TK Substrate-Biotin and 0.3 ng JAK3; TYK2: 50 mM Hepes buffer pH 7.0, 0.01% BSA, 5 mM MgCl2, 1 mM DTT, 13 μM ATP, 50 nM SEB, 1 μM TK Substrate-Biotin and 0.8 ng TYK2. Thereafter, the kinase reaction is stopped by adding 4 μl detection mix (final concentrations: 50 mM Hepes buffer pH 7.0, 0.01% BSA, 0.8 M KF, 20 mM EDTA, 42 nM Streptavidin-XL665 and 1:400 STK Ab Cryptate) and the plates are incubated overnight in the dark. The HTRF signal is read using an Envision plate reader.
Measurement: EC50 = 8.8 nM (pChEMBL 8.0600004196167)
Descriptors: MW 410.45, ALogP 1.95, HBA 6, HBD 3, PSA 107.03, QED 0.59