lovingscience/chembl-v37-raw-activities huggingface dataset:
Compound (SMILES): Cc1cnc([C@H](C)[C@@H](C)S(=O)(=O)Nc2nnc(-c3cncc(C)c3)n2-c2c(F)cccc2F)nc1
InChIKey: RELSLJYCYRJWFM-HZPDHXFCSA-N
Target: Apelin receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P35414
Assay: [35S]GTPgammaS Binding Assay: The human APJ receptor was cloned by polymerase chain reaction and the gene encoding the receptor was subcloned in pFLAG-CMV¿-3 expression vector (Sigma, Saint Louis, Mo. USA) in-house at Amgen. A GTPγS binding assay was performed on membranes prepared from CHO cells stably expressing human APJ receptor. The optimum experimental conditions for the concentrations of GDP, MgCl2, and NaCl in the assay buffer were initially determined. The assay was performed in assay buffer [20 mM HEPES, pH 7.5, 5 mM MgCl2, and 0.1% (w/v) BSA with 200 mM NaCl, 3 μM GDP] and membranes expressing human APJ receptor/well along with WGA PS beads. The reaction was initiated by addition of 0.2 nM [35S]GTPγS (Perkin Elmer Life and Analytical Sciences, Waltham USA) in the absence or presence of various ligands and incubated at RT for 90 min. Nonspecific binding was determined in the presence of 100 μM GTPγS and was always less than 0.2% of total binding. All the results presented are means of several independent experiments and analyzed by non-linear regression methods using commercially available program Prism (GraphPad, San Diego, Calif.) to obtain EC50.
Measurement: EC50 = 1.7 nM (pChEMBL 8.770000457763672)
Descriptors: MW 499.55, ALogP 3.95, HBA 8, HBD 1, PSA 115.55, QED 0.41
Compound (SMILES): CN(C)C(=O)c1ccc(N2CCN(c3nccc(COC(=O)NC(=N)N)c3F)CC2)cc1
InChIKey: GARLZCZMRRKADA-UHFFFAOYSA-N
Target: Amine oxidase [copper-containing] 3 (SINGLE PROTEIN), Homo sapiens
UniProt: Q16853
Assay: TBD: TBD
Measurement: IC50 = 5000 nM (pChEMBL 5.300000190734863)
Descriptors: MW 443.48, ALogP 1.37, HBA 7, HBD 3, PSA 127.88, QED 0.47
Compound (SMILES): Cc1noc(C)c1-c1cc(-c2ccc(O)nc2)c2[nH]c(=O)n3c2c1OC[C@@H]3c1ccccn1
InChIKey: DKSCMWYAKBTGLO-GOSISDBHSA-N
Target: Bromodomain-containing protein 4 (SINGLE PROTEIN), Homo sapiens
UniProt: O60885
Assay: AlphaScreen Assay: BRD4-BD1 and BRD4-BD2 assays were conducted in white 384-well polystyrene plate in a final volume of 20 μL for BD1 and 40 μL for BD2. Inhibitors were first serially diluted in DMSO and added to the plate wells before the addition of other reaction components. The final concentration of DMSO in the assay was 1.25% (BD1) and 0.83% (BD2). The assays were carried out at room temperature for 75 min. in the assay buffer (50 mM HEPES, pH 7.4, 100 mM NaCl, 0.05% CHAPS, 0.01% BSA), containing 50 nM Biotin-labeled tetra-acetylated histone H4 peptide (H4Ac4), 3.8 nM (BRD4-BD1, BPS Bioscience #31040) or 20 nM (BRD4-BD2, BPS Bioscience #31041). The reaction followed by the addition of 20 μL of assay buffer supplemented with Streptavidin donor beads (PerkinElmer 6760002) and GSH Acceptor beads (PerkinElmer-AL109C) at 4 μg/mL under reduced light. After plate sealing, the plate was incubated in the dark at room temperature for 75 min. before reading on a PHERAstar FS plate reader (BMG Labtech).
Measurement: IC50 = 550 nM (pChEMBL 6.260000228881836)
Descriptors: MW 441.45, ALogP 3.75, HBA 8, HBD 2, PSA 119.06, QED 0.44
Compound (SMILES): C[C@H](C(=O)N[C@H](C)c1ccnc(N2CCN(C)CC2)c1)N1Cc2ccc(-c3nc(NC4CCOCC4)ncc3Cl)cc2C1=O
InChIKey: HOCNVFJMPNVJIT-NHCUHLMSSA-N
Target: Mitogen-activated protein kinase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P28482
Assay: ERK2 In vitro Inhibition Assay: Activity of ERK2 enzyme (Life Technologies) was determined using a time-resolved fluorescence format measuring the phosphorylation of a truncated version of Activating transcription factor 2 labelled with green fluorescent protein (ATF2-GFP) (Life Technologies). Assay reactions containing 50 mM Tris pH 7.5, 10 mM MgC2, 1 mM EGTA, 0.01% Triton X-100, 1 mM DTT, 2.5% DMSO, 0.4 μM ATF2-GFP, 20 μM ATP and 0.25 nM ERK2 were set up in the presence of compound and allowed to proceed for 30 min at room temperature. Reactions were then stopped using TR-FRET dilution buffer (Life Technologies), 25 mM EDTA and 2 nM Tb-Anti-pATF2 (Thr71) (Life Technologies). After a further incubation period of at least 30 minutes, fluorescence was read on a Pherastar reader (Lanthascreen optic module; excitation 340 nm, emission 520 nm (channel A), 495 nm (channel B)). The ratio between A and B counts was used to calculate signal. IC50 values were calculated using a sigmoidal dose response equation (Prism GraphPad software, La Jolla, Calif., USA).In the assays using ERK2, the compounds of Examples 125, 127, 162, 181, 183, 289, 299, 310 and 939 have IC50 values in the range from 1 μM to 10 μM, provide at least 50% inhibition of the activity at a concentration of 10 μM in the assay against ERK2 or would be expected to provide at least 50% inhibition of the activity at a concentration of 10 μM (based on the level of inhibition of the activity at concentrations of 3 μM) in the assay against ERK2.
Measurement: IC50 = 5.5 nM (pChEMBL 8.260000228881836)
Descriptors: MW 619.17, ALogP 3.76, HBA 9, HBD 2, PSA 115.82, QED 0.39
Compound (SMILES): CC(N)c1nc2cnc3ccsc3c2n1[C@H]1CC[C@H](CC#N)OC1
InChIKey: MEYXZFYNDTXKRD-GLXQMMQGSA-N
Target: Tyrosine-protein kinase JAK1 (SINGLE PROTEIN), Homo sapiens
UniProt: P23458
Assay: In Vitro JAK Kinase Assay: Compounds herein were tested for inhibitory activity of JAK targets according to the following in vitro assay described in Park et al., Analytical Biochemistry 1999, 269, 94-104. The catalytic domains of human JAK1 (a.a. 837-1142), JAK2 (a.a. 828-1132) and JAK3 (a.a. 781-1124) were expressed using baculovirus in insect cells and purified. The catalytic activity of JAK1, JAK2 or JAK3 was assayed by measuring the phosphorylation of a biotinylated peptide. The phosphorylated peptide was detected by homogenous time resolved fluorescence (HTRF). IC50s of compounds were measured for each kinase in the 40 μL reactions that contain the enzyme, ATP and 500 nM peptide in 50 mM Tris (pH 7.8) buffer with 100 mM NaCl, 5 mM DTT, and 0.1 mg/mL (0.01%) BSA. For the 1 mM IC50 measurements, ATP concentration in the reactions was 1 mM. Reactions were carried out at room temperature for 1 hour and then stopped with 20 μL 45 mM EDTA, 300 nM SA-APC, 6 nM Eu-Py20 in assay buffer (Perkin Elmer, Boston, Mass.). Binding to the Europium labeled antibody took place for 40 minutes and HTRF signal was measured on a PHERA star plate reader (BMG, Cary, N.C.).
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 341.44, ALogP 3.3, HBA 7, HBD 1, PSA 89.75, QED 0.79
Compound (SMILES): Cc1ccc(S[C@H]2OC(CO)[C@H](O)C(n3cc(-c4cc(F)c(F)c(F)c4)nn3)[C@@H]2O)cc1
InChIKey: QNVXTRRKWGDVAN-ZRTAOYRGSA-N
Target: Galectin-3 (SINGLE PROTEIN), Homo sapiens
UniProt: P17931
Assay: Fluorescence Anisotropy Assay: The assay was adapted to be able to measure the high affinity of compounds for galectin-3 by using the below probe constructed to have high affinity for galectin-3 which made it possible to use a low concentration of galectin-3 (50 nM). 100 nM albumin was included as a carrier to prevent protein loss at such low concentration of galectin.
Measurement: Kd = 460 nM (pChEMBL 6.340000152587891)
Descriptors: MW 467.47, ALogP 2.44, HBA 8, HBD 3, PSA 100.63, QED 0.5
Compound (SMILES): CC(C)[C@H](NC(=O)C(F)(F)F)[C@H](Oc1ccc2c(cnn2-c2ccc(=O)n(C)c2)c1)c1cc(Cl)cc(Cl)c1
InChIKey: NQHPYKFDRDDNNA-BJKOFHAPSA-N
Target: Glucocorticoid receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P04150
Assay: GRE Agonist Assay: A reporter cell line (ChagoK1 18:7:2 s4/GRE) was established by stable transfection of the human bronchogenic carcinoma cell line, ChaGo K1 (ATCC: HTB 168) with a MMTV-GRE-LacZ reporter construct. The generated cell line allows for identification of compounds showing agonist activity at the human glucocorticoid receptor (GR) via induction of LacZ gene expression. Ligand-activated GR binds to the Glucocorticoid Response Element (GRE) in the promoter of the LacZ gene and transcription is initiated. The resulting beta-galactosidase activity is measured through a colour reaction (change in absorbance).Cryo-preserved ChagoK1 18:7:2 s4/GRE cells were suspended in RPMI medium with 10% FBS, 1% NEAA and 1% sodium pyruvate, and seeded as 50000 cells/200 ul/well in 96-well plates and cultured at 37° C. with 5% CO2 and 95% humidity for 24 hours. 1 μl compound was added at different concentrations to the cells and incubated for another 24 hours. Cells were washed once in PBS and lysed with 50 μl of 0.1% Triton-X for 10 min at room temperature. 40 μl of reaction mixture (2.5 mM MgCl2, 0.1 M β-mercapto ethanol, 1.7 mg/ml ONPG and 42.5 mM sodium phosphate, pH 7.5), was added to each well and kept at 37° C. for 60 min. The reaction was then terminated by addition of 100 μl stop solution (300 mM glycine, 15 mM EDTA, pH 11.3, adjusted with NaOH). The plates were measured at 420 nm for absorbance in a SpectraMax reader (Molecular Device).The relative efficacy (% effect) of a compound is calculated based on the full agonist effect of dexamethasone: % Effect=((Sample abs−min abs)/(max abs−min abs))×100To calculate EC50, max, min and slope factor for each compound, a concentration response curve is fitted by plotting % Effect versus compound concentration using the 4 parameter logistic equation: y=A+(B−A)/(1+((10C)/x)D)Where A=min Y, B=max Y, C=log EC50 and D=Slope factor.
Measurement: EC50 = 20 nM (pChEMBL 7.699999809265137)
Descriptors: MW 567.4, ALogP 5.85, HBA 6, HBD 1, PSA 78.15, QED 0.3
Compound (SMILES): O=C(c1cccc(N2CC3(COC3)C2)c1Cl)N1CCN2C[C@@](O)(c3ccc(Cl)cc3)CC[C@@H]2C1
InChIKey: NISJSXPVUNPGRC-FQRUVTKNSA-N
Target: Monoglyceride lipase (SINGLE PROTEIN), Homo sapiens
UniProt: Q99685
Assay: Inhibition Assay: In one embodiment, the present invention provides compounds of formula (Ie) and their pharmaceutically acceptable salts as described herein, wherein said compounds of formula (Ie) and their pharmaceutically acceptable salts have an IC50 for MAGL below 25 μM, preferably below 10 μM, more preferably below 5 μM as measured in an assay comprising the steps of: list-style a) providing a solution of a compound formula (e), or a pharmaceutically acceptable salt thereof, in DMSO; b) providing a solution of MAGL (recombinant wild-type) in assay buffer (50 mM tris(hydroxymethyl)aminomethane; 1 mM ethylenediaminetetraacetic acid); c) adding 1 μL of compound solution from step a) to 19 μL of MAGL solution from step b); d) shaking the mixture for 1 min at 2000 rpm; e) incubating for 15 min at RT; f) adding 20 μL of a solution of 4-nitrophenlyacetate in assay buffer (50 mM tris(hydroxymethyl)aminomethane; 1 mM ethylenediaminetetraacetic acid, 6% EtOH); g) shaking the mixture for 1 min at 2000 rpm;h) incubating for 5 min at RT; i) measuring the absorbance of the mixture at 405 nm a first time; j) incubating a further 80 min at RT;k) measuring the absorbance of the mixture at 405 nm a second time; l) substracting the absorbance measured under i) from the absorbance measured under k) and calculating the slope of absorbance; wherein: i) the concentration of the compound of formula (Ie), or the pharmaceutically acceptable salt thereof in the assay after step f) is in the range of 25 μM to 1.7 nM; ii) the concentration of MAGL in the assay after step f) is 1 nM; >iii) the concentration of 4-nitrophenylacetate in the assay after step f) is 300 μM; and iv) steps a) to 1) are repeated for at least 3 times, each time with a different concentration of the compound of formula (Ie), or the pharmaceutically acceptable salt thereof.
Measurement: IC50 = 39.7 nM (pChEMBL 7.400000095367432)
Descriptors: MW 502.44, ALogP 3.64, HBA 5, HBD 1, PSA 56.25, QED 0.7
Compound (SMILES): Cc1ccc(NC(=O)N2CC[C@@H](CC(F)(F)F)C2)cc1-c1cc(N2CCOCC2)nc(NCCO)n1
InChIKey: IQVKBQFYWLAYAD-KRWDZBQOSA-N
Target: RAF proto-oncogene serine/threonine-protein kinase (SINGLE PROTEIN), Homo sapiens
UniProt: P04049
Assay: Enzymatic RAF1 Kinase: Small molecule inhibition of RAF1 kinases was measured using ADP-Glo assay. In the assay, ADP is converted to ATP in the presence of test kinase and substrate, resulting in luciferase reaction and luminescent readout with light generated proportional to the relative kinase activity. Compounds diluted in DMSO were used in 10-point, 3-fold dose curve for both assays. Final concentrations of 3 nM RAF1 (CarnaBio, Cat. 09-125) and 30 nM MEK1 substrate (Millipore, Cat. 14-420) were incubated with 3 μM ATP, 10 mM MgCl2, 0.003% Brij-35, 2 mM DTT, 0.05% BSA, 1 mM EGTA, and 50 mM HEPES for 90 minutes at room temp prior to addition of ADP-Glo reagent (Promega, Cat. V9102) for 40 minutes, and detection reagent for 45 minutes. Luminescence was read on an Envision plate reader (PerkinElmer) and percent remaining activity was used to calculate IC50 using a four-parameter fit model using Dotmatics Knowledge Solutions Studies curve fitting (Dotmatics, Bishops Stortford, UK, CM23).
Measurement: IC50 < 10 nM (pChEMBL 8)
Descriptors: MW 508.55, ALogP 3.5, HBA 7, HBD 3, PSA 102.85, QED 0.53
Compound (SMILES): CC(C)(C#Cc1cccc(-n2nc(C(N)=O)c3ccccc32)c1)c1nccs1
InChIKey: RDZTYHXUOBRBHS-UHFFFAOYSA-N
Target: Mitogen-activated protein kinase kinase kinase 14 (SINGLE PROTEIN), Homo sapiens
UniProt: Q99558
Assay: NIK Enzyme Inhibition Assay: The ability of the nuclear factor-kappa B (NF-kB)-inducing kinase (NIK) to catalyze the hydrolysis of adenosine-5′-triphosphate (ATP) was monitored using the Transcreener ADP (adenosine-5′-diphosphate) assay (BellBrook Labs). Purified NIK (0.5 nM) derived from a baculovirus-infected insect cell expression system was incubated with test compounds for 1-3.5 hours in 50 mM 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid buffer (pH 7.2) containing 10 mM MgCl2, 2 mM dithiothreitol, 10 μM ATP, 0.01% Triton X-100, 0.1% gamma-globulins from bovine blood, 1% dimethylsulfoxide (DMSO), 7 μg/mLADP antibody and 5 nM ADP-MR121 633 tracer. Reactions were quenched by the addition of 20 mM 2,2′,2″,2′″-(ethane-1,2-diyldinitrilo)tetraacetic acid and 0.01% Brij 35. The tracer bound to the antibody was displaced by the ADP generated during the NIK reaction, which causes a decrease in fluorescence polarization that was measured by laser excitation at 633 nm with a Fluorescence Correlation Spectroscopy Plus reader (Evotec AG). Equilibrium dissociation constant (Ki) values for NIK inhibitors are calculated from plots of activity vs. inhibitor concentration using Morrison's quadratic equation that accounts for the potential of tight binding, and by also applying the conversion factor that accounted for competitive inhibition and the concentration of substrate used in the assay relative to its Michaelis constant (Km).
Measurement: Ki = 0.75 nM (pChEMBL 9.119999885559082)
Descriptors: MW 386.48, ALogP 3.91, HBA 5, HBD 1, PSA 73.8, QED 0.54
Compound (SMILES): Oc1ccc(-c2cc(N3CCOCC3)nc3c(-c4ccn[nH]4)nccc23)cc1
InChIKey: WPYZORODUUZGDB-UHFFFAOYSA-N
Target: Serine/threonine-protein kinase ATR (SINGLE PROTEIN), Homo sapiens
UniProt: Q13535
Assay: Time-Resolved Fluorescence Energy Transfer (TR-FRET Tracer A): To determine of binding activity of the test compounds, full-length human ATR protein was expressed and purified together with ATRIP as described above. Furthermore, a fluorescently labelled compound (tracer A) was used as a tracer molecule. Detection of the binding event of the tracer was achieved by time-resolved fluorescence energy transfer (TR-FRET). We used an anti-GST-Terbium antibody (CisBio) that binds to the GST-tag at the N-terminus of ATR-kinase. Excitation of Terbium with 337 nm light results in emission of fluorescent light with 545 nm. In case a tetrameric complex has formed (antiGST-Tb+GST-ATR+Strp2-ATRIP+tracer), part of the energy will be transferred from the Terbium to the fluorophore that itself emits light of 570 nm. Displacement of the fluorescent tracer by a test compound leads to a reduction of the TR-FRET-signal.For the assay 50 nl of a 100-fold concentrated solution of the test compound in DMSO was pipetted into a black low volume 384well microtiter plate (MTP, Greiner Bio-One, Frickenhausen, Germany). To prepare the ATR-working solution, ATR/ATRIP stock solution was diluted in assay buffer [50 mM HEPES (pH 7.0), 10 mM MgCl2, 1 mM DTT, 0.01% (w/v) Igepal, 0.01% (w/v) BSA] to 4.2 nM protein concentration (concentration may vary from lot to lot of protein preparation). AntiGST-Tb antibody was diluted to 4.2 nM. The ATR-working solution was incubated for 30 min at 22° C. prior to dispensing to pre-form the complex of antiGST-Tb+GST-ATR+ATRIP. Then, 3 μl of the ATR-working solution were added to the test compound and the mixture was incubated for 10 min at 22° C. to allow pre-binding of the test compounds to ATR/ATRIP. Then, 2 μl of a 100 nM solution of either tracer A or B in assay buffer were added to the ATR-working solution. The resulting mixture was incubated for 30 min at 22° C. The measurement of the TR-FRET signal was performed in a standard HTRF-compatible MTP reader instrument (e.g. BMG Pherastar) by recording the fluorescence emissions at 545 nm and 570 nm after excitation at 337-350 nm. The ratio between emission at 570 nm divided by emission at 545 nm was calculated to give the well ratio. The experimental data (well ratios) were normalised by the following way: positive control contained ATR-working solution plus either tracer A or B solution (=0% inhibition), the negative control contained all components except GST-ATR/ATRIP (=100% inhibition).
Measurement: IC50 = 73.2 nM (pChEMBL 7.130000114440918)
Descriptors: MW 373.42, ALogP 3.23, HBA 6, HBD 2, PSA 87.16, QED 0.57
Compound (SMILES): CCOC(=O)Cn1c(=O)[nH]/c(=N\c2ccc(Oc3ccc(F)cn3)cc2)n(Cc2ccc(Cl)cc2)c1=O
InChIKey: LZXTWYDQLCXYBJ-UHFFFAOYSA-N
Target: P2X purinoceptor 3 (SINGLE PROTEIN), Homo sapiens
UniProt: P56373
Assay: Receptor Inhibition Assay: Stably expressing cell line (C6BU-1 cell transfected with human P2X3 receptor gene (GenBank accession number Y07683) was used. The cells were seeded in a 384-well PDL-coated microtiter plate at a concentration of 3000 cells/well and cultured in the medium (8.3% fetal bovine serum, 8.3% horse serum, and 1% antibiotic and antifungal in DMEM) for one day at 37° C. under 5% carbon dioxide atmosphere. The medium was replaced with 4 μM Fluo-3-AM solution (20 mM HEPES, 137 mM NaCl, 2.7 mM KCl, 0.9 mM MgCl2, 5.0 mM CaCl2, 5.6 mM D-glucose, 2.5 mM probenecid, 0.5% BSA, and 0.04% Pluronic P-127, pH 7.5) and incubated at 37° C. under 5% dioxide carbon atmosphere for one hour. The plate was washed with washing buffer (20 mM HEPES, 137 mM NaCl, 2.7 mM KCl, 0.9 mM MgCl2, 5.0 mM CaCl2, 5.6 mM D-glucose, 2.5 mM probenecid, pH 7.5), and each well was added with 20 μL of the washing buffer. The plate was placed in High-Throughput Screening System FLIPR 384 (Molecular Device Co.). Measurement of fluorescence intensity by FLIPR 884 was started, and 20 μL of DMSO solutions containing different concentrations of the test compound as prepared by dilution with dilution buffer (20 mM HEPES, 137 mM NaCl, 2.7 mM KCl, 0.9 mM MgCl2, 5.0 mM CaCl2, 5.6 mM D-glucose, 2.5 mM probenecid, 0.1% Pluronic F-127, pH 7.5) were dispensed to each well through the built-in automatic dispenser. Five minutes after, 150 nM ATP solution (25 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.
Measurement: IC50 = 4 nM (pChEMBL 8.399999618530273)
Descriptors: MW 525.92, ALogP 3.16, HBA 9, HBD 1, PSA 120.57, QED 0.35
Compound (SMILES): O=C1CC(c2ccc(F)cc2)Oc2cc(OCCCCN3CCC(c4noc5cc(F)ccc45)CC3)cc(O)c21
InChIKey: KUOYFTRGKVRORO-UHFFFAOYSA-N
Target: D(2) dopamine receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P14416
Assay: Effects of the Compounds of the Invention Against Activity of Dopamine D2 Receptor: Objective: positive symptoms (hallucination, delusion, etc.) in patients with schizophrenia may be associated with dopamine (DA) hyperfunction in subcortical limbic system, and antipsychotic drugs, which block Dopamine D2 Receptor (DRD2), can effectively control positive symptoms of schizophrenia. By establishing a cell line co-transfected with DRD2 and Gα16, the activated DRD2 can activate Gα16 protein, thereby activating phospholipase C (PLC) to produce inositol 1,4,5-triphosphate (IP3) and diacylglycerol (DAG), wherein IP3 can bind to the IP3 receptor on endoplasmic reticulum and mitochondria, resulting in intracellular calcium release. Therefore, the determination of a change in intracellular calcium can be used as a method for detecting the activated state of DRD2.Fluo-4/AM was a calcium fluorescent probe indicator for determining calcium ions. In this experiment, a Fluo-4 fluorescence method was used to determine the level of activated Gα protein by measuring the fluorescence intensity excited by intracellular calcium ions. If a compound could activate DRD2, the calcium influx was enhanced; on the contrary, if a compound could antagonize DRD2, the calcium influx was reduced.Methods: HEK293 cells stably expressing DRD2/Gα16 (a human embryonic kidney cell line, derived from Shanghai Institute of Materia Medica, Chinese Academy of Sciences) were seeded in a 96-well plate (the culture solution was 90% DMEM+10% fetal bovine serum), and incubated overnight. The culture solution was pipetted off, and a freshly prepared dye Fluo-4/AM was added. The cells were incubated in a 37° C. incubator for 40 min. The dye was completely pipetted off. After the cells were washed with a freshly prepared calcium buffer, a calcium buffer (50 μl) dissolved with a test drug was added. FlexStation II instrument was used in the determination. A calcium buffer (25 μl) dissolved with a known agonist was added automatically by the instrument at the fifteenth second, and the fluorescence value at 525 nm (an excitation wavelength of 485 nm) was finally read. Dopamine was used as agonist, Eticlopride (D2 receptor antagonist) was used as antagonist, and the cell response (% Response) of each sample at each concentration was calculated by the following formula: % Response=(LSample−LBlank)/(LDopamine−LBlank), wherein LSample represents the detected signal value of a test sample, LBlank represents the detected signal value as completely inhibited by Eticlopride, and LDopamine represents the detected signal value after the stimulation of the DMSO group with 50 nM Dopamine (agonist). IC50 value was calculated by GraphPad Prism.
Measurement: IC50 = 87.7 nM (pChEMBL 7.059999942779541)
Descriptors: MW 548.59, ALogP 6.56, HBA 7, HBD 1, PSA 85.03, QED 0.25
Compound (SMILES): CC(C)(C(=O)O)C1CN(S(=O)(=O)c2ccc(F)cc2)c2cc(NC(=O)OC(C)(C)C(F)(F)F)ccc2O1
InChIKey: WMGAORNONADGSR-UHFFFAOYSA-N
Target: Nuclear receptor ROR-gamma (SINGLE PROTEIN), Homo sapiens
UniProt: P51449
Assay: biochemical TR-FRET assay: HIS-tagged RORγ-LBD protein was recombinantly expressed in Escherichia coli. The RORγ-LBD protein was purified by Ni2+-affinity resin. Purified protein was then diluted in assay buffer (50 mM Tris pH 7.0, 50 mM KCl, mM EDTA, 0.1 mM DTT, 100 μg/mL bovine serum albumin, delipidated) to obtain a RORγ-LBD final concentration of 3 nM. Europium tagged anti-HIS antibody was also added to this solution (1.25 nM). Separately, SF9 cells not expressing any recombinant protein were lysed (32,000 cells per μl in 25 mM Tris, 50 mM NaCl) and the previously frozen lysate was added to the diluted RORγ-LBD solution at a ratio of 0.75 μl SF9 lysate per 15 μl of diluted RORγ-LBD.Compounds to be tested were injected to the 384-well assay plate using Acoustic Droplet Ejection technology by Echo 550 liquid handler (Labcyte, Calif.).A stock of biotinylated-LXXLL peptide from coactivator SRC1 (Biotin-SPSSHSSLTERHKILHRLLQEGSP) (SEQ ID NO:2) and APC-conjugated streptavidin (final concentrations 100 nM and 8 nM respectively) were also added to each well.The final assay mixture was incubated overnight at 4° C., warmed to room temperature and the fluorescence signal was measured on an Envision plate reader: (Excitation filter=340 nm; APC emission=665 nm; Europium emission=615 nm; dichroic mirror=D400/D630; delay time=100 μs, integration time=200 μs). IC50 values for test compounds were calculated from the quotient of the fluorescence signal at 665 nm divided by the fluorescence signal at 615 nm.
Measurement: IC50 = 1136 nM (pChEMBL 5.949999809265137)
Descriptors: MW 548.51, ALogP 4.78, HBA 6, HBD 2, PSA 122.24, QED 0.5
Compound (SMILES): CCN(CC)C(=O)c1ccc2c(c1)N=C(N)N(C1Cc3ccccc3C1)C2
InChIKey: IPZWZCNMWSVBLQ-UHFFFAOYSA-N
Target: Cathepsin D (SINGLE PROTEIN), Homo sapiens
UniProt: P07339
Assay: IN-VITRO FLUORESCENCE ASSAY: In order to identify modulators of cathepsin D activity, a continuous enzymatic test was carried out with a synthetic peptide which carries a fluorescent group (MCA=(7-methoxycoumarin-4-yl)acetyl) which is quenched by energy transfer from a Dpn (2,4 dinitrophenyl) group on the same molecule, in Greiner 384-well nb microtitre plates. Cleavage of the peptidic substrate by cathepsin D causes an increase in the fluorescence intensity. In order to determine the efficacy of substances, the time-dependent increase in the fluorescence intensity in the presence of the substance was compared with the time-dependent increase in fluorescence in the absence of substances. The reference substance used was pepstatin A (Sigma-Aldrich). The substrate used was MCA-GKPILFFRLK(Dnp)d-R—NH2 (Enzo Life Sciences, Lörach). The enzyme employed was cathepsin D isolated from the human liver (Sigma-Aldrich) in a final concentration of 1.4 nM. The test was carried out in 100 mM sodium acetate buffer, 1.25% (v/v) of DMSO, 0.25% (w/v) of Chaps, pH 5.5. 2 μl of each substance solution with serially diluted substance concentration were added to in each case 4 μl of cathepsin D solution and incubated at room temperature for 10 min. The reaction was started by addition of 2 μl of substrate solution (final concentration 5 μM). After carrying out a starting-point fluorescence measurement (excitation wavelength 340 nm/emission wavelength 450 nm) using an Envision multilabel reader (Perkin Elmer), the reaction was incubated at room temperature for 60 min. The amount of peptide fragment cleaved off during the reaction time was subsequently measured by determination of the increase in the fluorescence intensity at 450 nm (excitation wavelength 340 nm).
Measurement: IC50 = 2900 nM (pChEMBL 5.539999961853027)
Descriptors: MW 362.48, ALogP 3.1, HBA 4, HBD 1, PSA 61.93, QED 0.91
Compound (SMILES): C[C@@H](Nc1nc(-c2noc(=O)[nH]2)nc2nc(N3CC(C)(O)C3)n(C[C@H]3CC[C@H](C)CC3)c12)C1CCC1
InChIKey: FHJYNRCXPRNWEQ-BZUAXINKSA-N
Target: E3 ubiquitin-protein ligase Mdm2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q00987
Assay: FRET Assay: An HDM2 FRET assay was developed to assess the compounds' inhibitory activity towards binding of p53 protein. A truncated version of HDM2 with residues 17 to 125 (containing p53 binding surface, Science (1994) 265, 346-355), with N-terminal His and Thioredoxin tag was generated in pET32a expression vector and expressed in E. coli strain BL21(DE3)Rosetta. Protein was purified using Ni-affinity chromatography, followed by size exclusion chromatography using Superdex 75 26/60 column. To assess inhibition of p53 binding to HDM2, a FITC labeled 8-mer peptide (sequence: Ac-Phe-Arg-Dpr-Ac6c-(6-Br)Trp-Glu-Glu-Leu-NH2; Anal Biochem. 2004 Aug. 1; 331(1):138-46) with strong affinity towards p53 binding pocket of HDM2 was used. The HDM2 assay buffer contained 1× Phosphate Buffered Saline (Invitrogen, Cat#14190), 0.01% BSA (Jackson ImmunoResearch, Cat#001-000-162), 0.01% Tween-20. In the 1× assay buffer recombinant HDM2 protein, peptide and Lumi4-Tb Cryptate-conjugate mouse anti-6×His antibody (cisbio, Cat# Tb61HISTLB) were added and transferred to ProxiPlate PLUS (PerkinElmer, Cat#6008269), containing compounds so that final DMSO concentration is 0.1%. Final concentrations of reagents in the assay wells are 0.5 nM HDM2, 0.25 nM anti HIS (Tb label) antibody and 3 nM peptide. After two hour incubation at room temperature in a humidified chamber plates were read on EnVision plate reader with the following settings: excitation: UV, 340 nM, two emission filters: 520 nm and 495 nm respectively. Ratio of em520/em495 was used to calculate % inhibition and to obtain IC50 with 4-parameter logistic equation.
Measurement: IC50 = 36.21 nM (pChEMBL 7.440000057220459)
Descriptors: MW 496.62, ALogP 3.17, HBA 10, HBD 3, PSA 137.99, QED 0.45
Compound (SMILES): C/C=C/C[C@@H](C)[C@@H](O)C1C(=O)N[C@@H](CC)C(=O)N(C)[C@H](CCCO)C(=O)N(C)[C@@H]([C@H](C)CS(C)(=O)=O)C(=O)N[C@@H](C(C)C)C(=O)N(C)[C@@H](CC(C)C)C(=O)N[C@@H](C)C(=O)N[C@H](C)C(=O)N(C)[C@@H](CC(C)C)C(=O)N(C)[C@@H](CC(C)C)C(=O)N(C)[C@@H](C(C)C)C(=O)N1C
InChIKey: VIDPGLCDGOXIAU-LZOUNNMPSA-N
Target: Peptidyl-prolyl cis-trans isomerase A (SINGLE PROTEIN), Homo sapiens
UniProt: P62937
Assay: Surface Plasmon Resonance (SPR) Assay: Binding of inhibitors to expressed cyclophilins was determined using surface plasmon resonance (SPR) experiments. Briefly, avi-tagged cyclophilin proteins with mono-biotinylation were immobilized onto a Biotin CAPture chip (GE Healthcare, cat. #28920234). SPR experiments were carried out on an upgraded Biacore T200 system using a running buffer containing the inhibitor, 10 mM HEPES, pH 7.4, 150 mM NaCl, 0.05% P20, and 3% DMSO. Single-cycle kinetics measurements were used to study the interactions between cyclophilin inhibitors and different cyclophlins. Accordingly, compound at various concentrations was injected to the flow cells with a contact time of 1 min each at a flow rate of 50 μL/min. The final dissociation phase lasts for 10 min following the sample injection. Data was analyzed using Biacore T200 Evaluation Software Version 1.0 and a 1:1 binding model was applied to fit the data to obtain kon, koff, and KD.
Measurement: Kd = 7 nM (pChEMBL 8.149999618530273)
Descriptors: MW 1324.78
Compound (SMILES): N=S1(=O)CCC1CNc1nonc1/C(=N/O)N[C@H]1Cc2ccc(F)cc21
InChIKey: NRDODOMPUMOKAD-MUDXCBLSSA-N
Target: Indoleamine 2,3-dioxygenase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P14902
Assay: Enzyme Assay: HIS-tagged IDO1 protein was recombinantly expressed in Escherichia coli using ZYP5052 autoinduction media supplemented with 500 μM delta aminolevulinic acid for 48 h at 16° C. IDO1 protein was purified using Ni2+-affinity resin and size exclusion chromatography. Purified protein was then diluted in assay buffer (50 mM Tris pH 7.0, 1% glycerol, 20 μM methylene blue, 0.05% Tween-20, 20 mM sodium ascorbate, 100 units/mL catalase to obtain a final IDO1 concentration of 40 nM. IDO1 solution (30 μM) or buffer alone (30 μM) were dispensed to wells of the assay plate using a BioRAPTR liquid dispenser (Beckman Coulter). Assay plates containing compound and IDO1 enzyme were incubated at RT for 30 min. Afterwards, 10 μL of 400 μM tryptophan in assay buffer were added to each well of the assay plate using a BioRAPTR liquid dispenser. Plates were incubated at RT for 60 min and reactions were quenched by addition of 10 μL of 0.5 M methyl isonipecotate in dimethyl sulfoxide. Plates were sealed and incubated at 37° C. for 4 h or 50° C. for 2 h. The plates are allowed to cool and then centrifuged for 1 min at 1000×g. The resulting fluoresence was measured in an Envision plate reader (Perkin Elmer) with a 400/25 nm excitation filter and an 510/20 nm emission filter.
Measurement: IC50 = 137 nM (pChEMBL 6.860000133514404)
Descriptors: MW 380.41, ALogP 1.46, HBA 8, HBD 4, PSA 136.49, QED 0.27
Compound (SMILES): N#Cc1ccnc(-c2cc(-c3nc(C4CCCN4C#N)no3)ccn2)c1
InChIKey: WYJJRHZAARUPOH-UHFFFAOYSA-N
Target: Ubiquitin carboxyl-terminal hydrolase 30 (SINGLE PROTEIN), Homo sapiens
UniProt: Q70CQ3
Assay: Biochemical IC50 Assay: Dilution plates were prepared at 21 times the final concentration (2100 μM for a final concentration of 100 μM) in 50% DMSO in a 96-well polypropylene V-bottom plate (Greiner #651201). A typical 8-point dilution series would be 100, 30, 10, 3, 1, 0.3, 0.1, 0.03 μM final. Reactions were performed in duplicate in black 384 well plates (small volume, Greiner 784076) in a final reaction volume of 21 μl. Either 1 μl of 50% DMSO or diluted compound was added to the plate. USP30 was diluted in reaction buffer (40 mM Tris, pH 7.5, 0.005% Tween 20, 0.5 mg/ml BSA, 5 mM beta-mercaptoethanol) to the equivalent of 0.05 μl/well and 10 μl of diluted USP30 was added to the compound. Enzyme and compound were incubated for 30 min at room temp. Reactions were initiated by the addition of 50 nM of TAMRA labelled peptide linked to ubiquitin via an iso-peptide bond as fluorescence polarisation substrate. Reactions were read immediately after addition of substrate and following a 2 h incubation at room temperature. Readings were performed on a Pherastar Plus (BMG Labtech). λ Excitation 540 nm; λ Emission 590 nm.
Measurement: IC50 = 5500 nM (pChEMBL 5.260000228881836)
Descriptors: MW 343.35, ALogP 2.68, HBA 8, HBD 0, PSA 115.52, QED 0.67
Compound (SMILES): Cc1cc(S(=O)(=O)NCCOCCOCCNC(=O)NCCCCNC(=O)NCCOCCOCCNS(=O)(=O)c2ccc(O[C@H]3c4cc(Cl)cc(F)c4C[C@@H]3N3CCC[C@@H](N)C3)c(C)c2)ccc1O[C@H]1c2cc(Cl)cc(F)c2C[C@@H]1N1CCC[C@@H](N)C1
InChIKey: AFOUNHXQRIJJMQ-JPUKWVNYSA-N
Target: Sodium/hydrogen exchanger 3 (SINGLE PROTEIN), Homo sapiens
UniProt: P48764
Assay: Cell-Based Assay of NHE-3 Activity (Pre-Incubation Inhibition): Rat and human NHE-3-mediated Na+-dependent H+ antiport was measured using a modification of the pH sensitive dye method originally reported by Paradiso (Proc. Natl. Acad. Sci. USA. (1984) 81(23): 7436-7440). PS120 fibroblasts stably expressing human NHE3 and NHERF2 were obtained from Mark Donowitz (Baltimore, Md.). Opossum kidney (OK) cells were obtained from the ATCC and propagated per their instructions. The rat NHE-3 gene (GenBank M85300) was introduced into OK cells via electroporation, and cells were seeded into 96 well plates and grown overnight. Medium was aspirated from the wells then incubated for 30 min at 37° C. with NH4Cl-HEPES buffer (20 mM NH4Cl, 80 mM NaCl, 50 mM HEPES, 5 mM KCl, 2 mM CaCl2, 1 mM MgCl2, pH 7.4) containing 5 μM BCECF-AM. Cells were washed once with Ammonium free, Na+-free HEPES (100 mM choline, 50 mM HEPES, 10 mM glucose, 5 mM KCl, 2 mM CaCl2, 1 mM MgCl2, pH 7.4) and incubated in the same buffer for 10 minutes at room temperature to lower intracellular pH with 0-30 μM test compound. After incubation, NHE-3-mediated recovery of neutral intracellular pH was initiated by addition of Na-HEPES buffer containing 0.4 μM ethyl isopropyl amiloride (EIPA, a selective antagonist of NHE-1 activity that does not inhibit NHE-3). Changes in intracellular pH were monitored using a FLIPR Tetra® (Molecular Devices, Sunnyvale, Calif.) by excitation at λex 439 to 505 nm, and measuring BCECF fluorescence at λem 538 nm. The initial rate of the fluorescence ratio change was used as a measure of NHE-mediated Na+/H+ activity, and reported as the change in fluorescence ratio per minute. Initial rates were plotted as the average of 2 or more replicates, and pIC50 values were estimated using GraphPad Prism.
Measurement: IC50 > 1000 nM (pChEMBL 6)
Descriptors: MW 1310.43
Compound (SMILES): NC(=O)c1ccc(-c2ccc3ncc(-c4cccs4)n3n2)cc1
InChIKey: SHUKLFNPWOBASC-UHFFFAOYSA-N
Target: Tyrosine-protein kinase Fyn (SINGLE PROTEIN), Homo sapiens
UniProt: P06241
Assay: ADP-Glo Kinase Assay: The assay procedure determines the IC50 of each potential FYN kinase inhibitor by measuring the enzyme catalyzed ATP-dependent phosphorylation of the FYN substrate peptide. The ADP-Glo Kinase Assay is specifically designed to quantify kinase activity by measuring the ADP produced in the reaction The reaction buffer comprises 40 mM Tris.HCl (pH 7.5), 20 mM MgCl2, 0.1 mg/ml BSA and 5 microM DTT. Individual compounds within the chemical library were dissolved in DMSO at known concentrations. Briefly, 2.5 microliters of reaction buffer containing 3 picograms of FYN kinase and serial dilutions of each potential FYN kinase inhibitor (9 3-fold dilutions of a 10 micromolar top sample) are placed into each well of a 384 well microtiter plate and incubated at room temperature for 30 minutes. An additional 2.5 microliters of reaction buffer containing 100 picograms of the FYN substrate and 12.5 picomoles of ATP are then added to each well and the reaction carried out for 90 minutes at room temperature.
Measurement: IC50 = 951 nM (pChEMBL 6.019999980926514)
Descriptors: MW 320.38, ALogP 3.22, HBA 5, HBD 1, PSA 73.28, QED 0.63
Compound (SMILES): CCO[C@H]1CC[C@H](CN2Cc3cc(C(=O)NCc4ccc(S(=O)(=O)CC)cn4)ccc3[C@H]2C(C)C)CC1
InChIKey: HUTNJMJQIWXNOB-LFGPKHSOSA-N
Target: Nuclear receptor ROR-gamma (SINGLE PROTEIN), Homo sapiens
UniProt: P51449
Assay: Binding Assay: Compounds of the present invention were tested for ability to bind to RORγ in a cell-free competition assay with commercially available radio-ligand (RL), 25-hydroxy[26,27-3H]-cholesterol (PerkinElmer, Cat. #NET674250UC), for a ligand binding site on a recombinant RORγ Ligand Binding Domain (LBD) protein expressed as a 6×His-Glutathione-S-Transferase (GST) fusion. The assay was performed in 96-well SPA plates (PerkinElmer, Cat. #1450-401) in 50 mM HEPES buffer, pH 7.4, containing 150 mM NaCl, 5 mM MgCl2, 10% (v/v) glycerol, 2 mM CHAPS, 0.5 mM β-octylglucopyranoside and 5 mM DTT. Tested compounds were dissolved in DMSO, and semi-log (3.162×) serial dilutions of the compounds were prepared in the same solvent. Two μL of the DMSO solutions were mixed with 28 μL of 8.6 nM 25-hydroxy[26,27-3H]-cholesterol and 50 μL of 24 nM RORγ LBD. The plate was shaken at 700 rpm for 20 min and incubated for 10 min at rt, after which 40 μL of poly-Lys YSi SPA beads (PerkinElmer, Cat. #RPNQ0010) were added to achieve 50 μg of the beads per well. The plate was incubated on an orbital shaker for 20 min and then for 10 min without agitation at rt. SPA signal for tritium beta radiation was registered on PerkinElmer Microbeta plate reader. Percent inhibition values were calculated based on the high signal obtained with DMSO control and the low signal observed with 10 μM standard RORγ inverse agonist T0901317 (SigmaAldrich, Cat. #T2320). The percent inhibition vs. concentration data were fit into a four-parameter model, and IC50 values were calculated from the fit as the concentrations corresponding to the inflection points on the dose-response curves.
Measurement: Ki > 1000 nM (pChEMBL 6)
Descriptors: MW 527.73, ALogP 4.91, HBA 6, HBD 1, PSA 88.6, QED 0.47
Compound (SMILES): COC(=O)Nc1cc(-c2cncc(S(=O)(=O)N3CCOCC3)c2)ccn1
InChIKey: WUJWIJFEOFDXJU-UHFFFAOYSA-N
Target: Phosphatidylinositol 3-kinase catalytic subunit type 3 (SINGLE PROTEIN), Homo sapiens
UniProt: Q8NEB9
Assay: Enzyme Assay : 100 nL compounds in DMSO are added to wells of a 384 well microtitre plate (Greiner 780076). At room temperature: 5 ul VPS34 reaction buffer (Invitrogen Assay Buffer Q (diluted 1 in 5 with nanopure water) plus 2 mM DTT and 2 mM MnCl2) containing ATP (20 uM, Promega) and 200 uM PI-PS substrate (Invitrogen PV5122) is added followed immediately by 5 ul VPS34 reaction buffer (as above) containing VPS34 (5 nM, Millennium Protein Sciences Group) and the mixture is incubated with shaking at room temperature for 1 hour. Then 5 ul VPS34 stop-detect mix (as per Invitrogen Adapta Assay kit (PV5009) instructions (contains kinase quench buffer, TR-FRET buffer, Adapta Eu anti-ADP antibody and Alexa Fluor 647 ADP tracer)) is added to quench the reaction. The plates are then incubated for 30 minutes at room temperature with shaking and then read on a BMG PheraStar Plus reader.For the assay methods described above, test compound percent inhibition, at various concentrations, is calculated relative to control (DMSO and EDTA) treated samples. Compound concentration versus percent inhibition curves are fitted to generate IC50 values. One skilled in the art will appreciate that the values generated either as percentage inhibition at a single concentration or IC50 values are subject to experimental variation.
Measurement: IC50 = 30 nM (pChEMBL 7.519999980926514)
Descriptors: MW 378.41, ALogP 1.34, HBA 7, HBD 1, PSA 110.72, QED 0.85
Compound (SMILES): Cn1ncc(NC(=O)c2csc(-c3c(O)cccc3F)n2)c1[C@@H]1CC[C@@H](N)[C@H](F)CO1
InChIKey: IAJKHASECAMBKL-HSMVNMDESA-N
Target: Serine/threonine-protein kinase pim-1 (SINGLE PROTEIN), Homo sapiens
UniProt: P11309
Assay: Biological Assay: Determination of the Pim kinase activity of a Formula I compound is possible by a number of direct and indirect detection methods. Certain exemplary compounds described herein were assayed for their Pim kinase binding activity, including isoforms Pim-1, Pim-2, andPim-3, (Example 901) and in vitro activity against tumor cells (Example 902). Certain exemplary compounds of the invention had Pim binding activity IC50 values less than about 1 micromolar (μM). Certain compounds of the invention had tumor cell-based activity EC50 values less than about 1 micromolar (μM).
Measurement: Ki = 0.121 nM (pChEMBL 9.920000076293945)
Descriptors: MW 449.48, ALogP 3.16, HBA 8, HBD 3, PSA 115.29, QED 0.56
Compound (SMILES): Cc1cc(Cl)cc(C(=O)N(C)c2cnccc2-c2ccccc2C)n1
InChIKey: LXIFEGLLBHDFQQ-UHFFFAOYSA-N
Target: G-protein coupled bile acid receptor 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q8TDU6
Assay: cAMP Assay: CHO-dhfr(minus) cells expressing human GPBAR1 receptors are seeded 17-24 hours prior to the experiment 50.000 cells per well in a black 96 well plate with flat clear bottom (Corning Costar #3904) in DMEM (Invitrogen No. 31331), lx HT supplement, with 10% fetal calf serum and incubated at 5% CO2 and 37° C. in a humidified incubator. The growth medium was exchanged with Krebs Ringer Bicarbonate buffer with 1 mM IBMX and incubated at 30° C. for 30 min Compounds were added to a final assay volume of 100 μl and incubated for 30 min at 30° C. The assay was stopped by the addition of 50 μl lysis reagent (Tris, NaCl, 1.5% Triton X100, 2.5% NP40, 10% NaN3) and 50 detection solutions (20 μM mAb Alexa700-cAMP 1:1, and 48 μM Ruthenium-2-AHA-cAMP) and shaked for 2 h at room temperature. The time-resolved energy transfer is measured by a TRF reader (Evotec Technologies GmbH, Hamburg Germany), equipped with a ND:YAG laser as excitation source. The plate is measured twice with the excitation at 355 nm and at the emission with a delay of 100 ns and a gate of 100 ns, total exposure time 10 s at 730 (bandwidth 30 nm) or 645 nm (bandwidth 75 nm), respectively. The measured signal at 730 nm has to be corrected for the ruthenium background, the direct excitation of Alexa and the buffer control. The FRET signal is calculated as follows: FRET=T730−Alexa730−P(T645−B645) with P=Ru730−B730/Ru645−B645, where T730 is the test well measured at 730 nM, T645 is the test well measured at 645 nm, B730 and B645 are the buffer controls at 730 nm and 645 nm, respectively. cAMP content is determined from the function of a standard curve spanning from 10 μM to 0.13 nM cAMP.
Measurement: EC50 = 2433 nM (pChEMBL 5.610000133514404)
Descriptors: MW 351.84, ALogP 4.69, HBA 3, HBD 0, PSA 46.09, QED 0.69
Compound (SMILES): Cc1cc(N)nc(C)c1CNC(=O)c1cn(Cc2ccc3ncc(Cl)cc3c2)c2ncccc12
InChIKey: KAHFCXDBNMIWGS-UHFFFAOYSA-N
Target: Plasma kallikrein (SINGLE PROTEIN), Homo sapiens
UniProt: P03952
Assay: In Vitro Enzyme Inhibition: A 10 mM solution of the test compound was made in DMSO. This solution was serially diluted 1:5 in DMSO to yield 2000, 400, 80, 16, 3.2, 0.64, 0.128, 0.0256 and 0.00512 μM compound test solutions. A control tube containing only DMSO is included. 16 μL of each compound test solution was combined with 384 μL of assay buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.01% Triton X-100) to yield a 4× test compound buffer stock.Separately, a 40 nM solution of human Plasma Kallikrein (Abcam) and a 93.6 μM solution Pro-Phe-Arg-AMC (Bachem) were made using assay buffer. These solutions are hereby referred to as 4×hPK and 2×PFR-AMC, respectively.60 μL of each 4× test compound buffer stock was combined with 60 μL of 4×hPK to yield 120 μL of 2× test compound buffer stock/2×hPK. 50 μL was removed from this mixture and placed into duplicate wells on a Microfluor 1Black U-bottom microtiter plate (Thermo Scientific). This plate was incubated for 5 minutes at 37° C. To each well, 50 μL of pre-warmed 2×PFR-AMC was added to start the enzymatic reaction. Cleavage of PFR-AMC was monitored in a Biotek Synergy H4 reader set at 37° C. Readings are taken every 43 seconds for 1 hour. The highest mean velocity over 20 reads (15 minutes) is used to calculate the IC50. The IC50 is calculated using the Gen5 (Biotek Instruments).
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 470.96, ALogP 4.81, HBA 6, HBD 2, PSA 98.72, QED 0.38
Compound (SMILES): CC(C)c1cnn2c(Nc3cccc(NC(=O)C4CCC(NC(=O)/C=C/CN(C)C)CC4)c3)nc(OC3CCN(C)CC3)nc12
InChIKey: LFQSGWPPJFRUFY-JXMROGBWSA-N
Target: CDK7/Cyclin H/MNAT1 (PROTEIN COMPLEX), Homo sapiens
UniProt: P51948
Assay: Biochemical Assay for CDK7: The ability of compounds to inhibit CDK7 kinase activity was tested in a TR-FRET assay using 5 nM of CDK7/CycH/MNAT1 obtained from Invitrogen, USA. Test compounds were pre-incubated with the kinase at room temperature for 60 min. After incubation, substrate mix [100 nM Ultra-light MBP (Perkin Elmer, USA) and 1 mM ATP (Sigma)] was added. The above reaction was stopped by the addition of 40 mM EDTA after 60 minutes of kinase reaction. 1 nM Eu-labelled antiphospho-MBP antibody [Perkin Elmer, USA] was added, mixed well and the fluorescence emission at 615 nm and 665 nm [excitation at 340 nm] was measured. The final DMSO concentration in the assay was 1%. For IC50 determination, appropriate concentrations were made by ⅓rd serial dilutions of 10 mM DMSO stock solution of test compound. All the fluorescence measurements were made in a Victor 3 Multilabel Counter [Perkin Elmer, USA]. The IC50 was determined by fitting the dose response data to sigmoidal curve fitting equation using GraphPad Prism software V5. To identify compounds that inhibit CDK7 irreversibly, time depended inhibition studies were carried by pre-incubating compound with the enzyme at three time points (20, 60 and 180 min) and carrying out assay as described above.
Measurement: IC50 = 650 nM (pChEMBL 6.190000057220459)
Descriptors: MW 617.8, ALogP 4.2, HBA 10, HBD 3, PSA 129.02, QED 0.27