lovingscience/chembl-v37-raw-activities huggingface dataset:
Compound (SMILES): COc1cc(-c2cc3c(-c4ccc(OC5CCS(=O)(=O)CC5)c(C#N)c4)ncnc3[nH]2)ccc1N1CCN(C2COC2)CC1
InChIKey: OWTXNTZZHGTTPX-UHFFFAOYSA-N
Target: Serine/threonine-protein kinase TBK1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9UHD2
Assay: Biological Assay for TBK1 and IKKs: Enzymatic activity of IKKε and TBK1 was measured using a homogeneous time resolved fluorescence resonance energy transfer (TR-FRET) assay that monitors enzyme dependent phosphorylation of a biotinylated serine/threonine peptide substrate. An increase in the amount of phopshorylated peptide results in an increase in TR-FRET signal. TBK1 and IKKs were expressed and purified as full length recombinant proteins. Detection reagents for the assay were purchased from Cisbio. TBK1 and IKKε enzymes were assayed under initial rate conditions in the presence of 2× Km ATP (40-80 μM) and 1 μM peptide, hepes (pH 7), 0.1 mM orthovanadate, 0.02% NaN3, 0.01% BSA, 10 mM MgCl2, 0.01% (v/v) tritonX, 1 mM dithiothreitol, 0.5% (v/v) DMSO at the following concentrations for each enzyme: TBK1 at 2.5 nM and IKKε at 0.3 nM. After an assay reaction time of 240 minutes at 25° C., reactions were terminated with EDTA.Amount of phosphorylated peptide was determined by the addition of 125 nM streptavidin XL665 and europium cryptate labeled anti-phospho monoclonal antibody and the resulting TR-FRET signal was recorded on an Envision plate reader (Ex: 340 nm; Em: 615/665 nm; 100 μs delay and 200 μs read window). Data was normalized based on a positive (1 μM Staurosporine) and negative (DMSO) controls and IC50 values calculated from the fit of the dose-response curves to a four-parameter equation. All IC50 values represent geometric mean values of a minimum of four determinations.
Measurement: IC50 = 3.68 nM (pChEMBL 8.430000305175781)
Descriptors: MW 614.73, ALogP 3.65, HBA 10, HBD 1, PSA 133.67, QED 0.33
Compound (SMILES): C#CCCOCCO[C@H]1CCc2cc([C@@H]3CC[C@@](N)(COP(=O)(O)O)C3)ccc2C1
InChIKey: BENUMICRHCXHIG-FSSWDIPSSA-N
Target: Sphingosine 1-phosphate receptor 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P21453
Assay: Binding Assay: Membranes were prepared from CHO cells expressing human S1P1. Cells pellets (1x10^9 cells/pellet) were suspended in buffer containing 20 mM HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid), pH 7.5, 50 mM NaCl, 2 mM EDTA (Ethylenediaminetetraacetic acid) and Protease Inhibitor cocktail (Roche), and disrupted on ice using the Polytron homogenizer. The homogenate was centrifuged at 20,000 rpm (48,000 g) and the supernatant was discarded. The membrane pellets were resuspended in buffer containing 50 mM HEPES, pH 7.5, 100 mM NaCl, 1 mM MgCl2, 2 mM EDTA and stored in aliquots at -80Â° C. after protein concentration determination.Membranes (2 ug/well) and 0.03 nM final concentration of 33P-S1P ligand (1 mCi/ml, Perkin elmer or American Radiolabeled Chemicals) diluted in assay buffer (50 mM HEPES, pH7.4, 5 mM MgCl2, 1 mM CaCl2, 0.5% fatty acid free BSA (bovine serum albumin), 1 mM NaF) were added to the compound plates (384 Falcon v-bottom plate (0.5 ul/well in a 11 point, 3-fold dilution). Binding was performed for 45 minutes at room temperature, terminated by collecting the membranes onto 384-well Millipore FB filter plates, and radioactivity was measured by TOPCOUNT. The competition data of the test compounds over a range of concentrations was plotted as percentage inhibition of radioligand specific binding. The IC50 is defined as the concentration of competing ligand needed to reduce specific binding by 50%.
Measurement: IC50 < 10 nM (pChEMBL 8)
Descriptors: MW 437.47, ALogP 2.67, HBA 5, HBD 3, PSA 111.24, QED 0.29
Compound (SMILES): COc1cccc(OC)c1-n1c(NS(=O)(=O)[C@H](C)[C@@H](O)c2ncc(C)cn2)nnc1-c1cncc(C)c1
InChIKey: CMOYZDQFCLGCRI-IIBYNOLFSA-N
Target: Apelin receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P35414
Assay: [35S]GTPγS Binding: 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 M 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 M 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 = 12 nM (pChEMBL 7.920000076293945)
Descriptors: MW 525.59, ALogP 2.62, HBA 11, HBD 2, PSA 154.24, QED 0.33
Compound (SMILES): CCc1nc(O)ncc1-c1nc2cc(C(=O)N3C[C@H]4CC[C@@H]3[C@@H]4N)cc(OC)c2n1C
InChIKey: JOGADVNEUUWXSC-XNAIMREJSA-N
Target: Protein-arginine deiminase type-4 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9UM07
Assay: Biological Assay (AR): Compounds were solubilised in 100% DMSO to achieve 100 mM final compound concentration. Compound stock solutions were stored at RT. A series of dilutions were prepared in DMSO and mixed 8 times with 20 μL mixing volume. Final assay conditions were as follows:    Reaction volume: 20 μl    Assay buffer (as aforementioned): 100 mM Tris-HCl (pH 7.6), 2 mM DTT, 1 mM CaCl2)    Final concentrations:      −100 nM hPAD4 enzyme      −50 μM (8-fold sub-Km) substrate peptide      −0.5% DMSO    Total incubation time: 65 mins at 37° C.    Stop solution: 40 μl 5% TCA in ACN0.25 μL of compound solution was added to 10 μL of 200 nM PAD4 in assay buffer (100 mM Tris-HCl pH 7.6, 2 mM DTT). After 5 mins, 10 μL of 100 μM of substrate in buffer (100 mM Tris-HCl pH 7.6, 2 mM DTT, 2 mM CaCl2)) was added and the reaction incubated for 60 mins at 37° C. The enzymatic reaction was quenched by addition of 40 μl of 5% TCA in ACN (1.7% TCA final concentration) stop solution. Arginine containing substrate were subjected to solid phase extraction on Agilent RapidFire (RF) 300 system and detected on a coupled, triple quadrupole Agilent 6460 QQQ mass spectrometry (MS) device under application of multiple reaction monitoring (MRM) for quantitation.
Measurement: IC50 = 10000 nM (pChEMBL 5)
Descriptors: MW 422.49, ALogP 1.87, HBA 8, HBD 2, PSA 119.39, QED 0.66
Compound (SMILES): CCOc1ccc(C[C@@H]2CCc3cc([C@H]4CC[C@](N)(COP(=O)(O)O)C4)ccc3C2)cc1
InChIKey: FWEPWVHUTLNWNX-BFMBIBTESA-N
Target: Sphingosine 1-phosphate receptor 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P21453
Assay: Binding Assay: Membranes were prepared from CHO cells expressing human S1P1. Cells pellets (1x10^9 cells/pellet) were suspended in buffer containing 20 mM HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid), pH 7.5, 50 mM NaCl, 2 mM EDTA (Ethylenediaminetetraacetic acid) and Protease Inhibitor cocktail (Roche), and disrupted on ice using the Polytron homogenizer. The homogenate was centrifuged at 20,000 rpm (48,000 g) and the supernatant was discarded. The membrane pellets were resuspended in buffer containing 50 mM HEPES, pH 7.5, 100 mM NaCl, 1 mM MgCl2, 2 mM EDTA and stored in aliquots at -80Â° C. after protein concentration determination.Membranes (2 ug/well) and 0.03 nM final concentration of 33P-S1P ligand (1 mCi/ml, Perkin elmer or American Radiolabeled Chemicals) diluted in assay buffer (50 mM HEPES, pH7.4, 5 mM MgCl2, 1 mM CaCl2, 0.5% fatty acid free BSA (bovine serum albumin), 1 mM NaF) were added to the compound plates (384 Falcon v-bottom plate (0.5 ul/well in a 11 point, 3-fold dilution). Binding was performed for 45 minutes at room temperature, terminated by collecting the membranes onto 384-well Millipore FB filter plates, and radioactivity was measured by TOPCOUNT. The competition data of the test compounds over a range of concentrations was plotted as percentage inhibition of radioligand specific binding. The IC50 is defined as the concentration of competing ligand needed to reduce specific binding by 50%.
Measurement: IC50 = 550 nM (pChEMBL 6.260000228881836)
Descriptors: MW 459.52, ALogP 4.51, HBA 4, HBD 3, PSA 102.01, QED 0.5
Compound (SMILES): COc1ccc(F)cc1-c1ccnc2[nH]c(C3=CCN(Cc4ccccn4)CC3)cc12
InChIKey: LSFFUPCKIREDGJ-UHFFFAOYSA-N
Target: CDK9/cyclin T1 (PROTEIN COMPLEX), Homo sapiens
UniProt: P50750
Assay: LANCE ULight TR-FRET Kinase Assay: CDK9 enzyme activities were measured using LANCE ULight TR-FRET kinase assay reagents (PerkinElmer, Waltham, Mass.). Compounds were directly added in 100% DMSO to white low volume assay plates (Perkin Elmer Proxiplate 6008289) using a Labcyte Echo acoustic dispenser. Assay reagents in serine/threonine kinase assay buffer containing 20 mM HEPES, 10 mM MgCl2, 100 mM Na3VO4, and 0.0075% Triton X-100. were added for final reaction mixture concentrations of 1000 μM ATP, 100 nM U-light MBP peptide (Perkin Elmer TRF0109M) and reaction initiated with 4 nM CDK9/Cyclin T1 (Carna Biosciences 04-110). The kinase reaction was carried out for 30 minutes before addition of stopping buffer to a final of 20 mM EDTA and 0.5 nM of LANCE Ultra Europium anti-phospho-MBP antibody (PerkinElmer TRF0201M) in LANCE detection buffer (PerkinElmer CR97-100). The reaction was equilibrated for 1 hour and the signal read in the Perkin Elmer Envision in TR-FRET mode (excitation at 320 nm and emission at 615/665 nm).
Measurement: IC50 = 55 nM (pChEMBL 7.260000228881836)
Descriptors: MW 414.48, ALogP 5.06, HBA 4, HBD 1, PSA 54.04, QED 0.5
Compound (SMILES): Cc1cc(N)nc(C)c1CNC(=O)c1ccc2c(c1)C1OC2c2ccc(-c3ccc(F)cc3F)cc21
InChIKey: ULFZKTTWSDEBHE-UHFFFAOYSA-N
Target: Plasma kallikrein (SINGLE PROTEIN), Homo sapiens
UniProt: P03952
Assay: In Vitro Plasma Kallikrein Inhibition Assay: The chromogenic substrate D-Pro-Phe-Arg-pNa, 2HCl (BIOPHEN CS-31(02) from Hyphen BioMed, Neuville-Sur-oise, France) was dissolved in 5 mL deionized water and stored at 4° C. Concentration was determined in the spectrophotometer at 342 nm using an extinction coefficient of 8270. All other chemicals were of analytical grade.Human plasma kallikrein was purchased from Enzyme Research Labs (South Bend, Ind., USA, batch HPKa 2830). A stock solution of 7 μM in 50% glycerol was stored at −20° C.Enzyme reactions were conducted in assay buffer comprised of 20 mM HEPES at pH 7.4, 150 mM NaCl, 0.1% PEG-8000 and 0.01% Triton X-100.Both enzyme and substrate were diluted in assay buffer.The compound solutions as well as the enzyme and the substrate solutions were transferred to 96-well plates (Clear, UV-Star, Flat-bottom, Half-Area plates; cat. No. 675801 Greiner Bio-one, purchased from VWR International, Arlington Heights, Ill., USA) using a Rainin LTS 96-channel pipettor (Rainin, Columbus, Ohio, USA). Plate measurements were conducted using a SPECTROStar Nano reader (BMG Labtech, San Francisco, Calif., USA). The SPECTROStar Nano is a spectrophotometer and absorbance was measured at 405 nm. We used discrete wavelength, precise, kinetic reads of 15 cycles with a 60 sec cycle time.Determination of IC50 valuesFor the determination of IC50 values, the assays were performed at room temperature in 96-well plates with a total assay volume of 85 μL per well. The test compound was dissolved in 100% DMSO. The compounds were serially diluted in DMSO in a 7 point dose response. For the assays, 66.5 μL of protease solution (protease in assay buffer) was added per well followed by the addition of 8.5 μL of compound in 100% DMSO. The final assay concentration of the human plasma kallikrein was 250 μM. After 30 min. incubation at room temperature on an orbital shaker, the reactions were started by the addition of 10 μL substrate solution (in assay buffer, final assay concentration was 600 uM). After the addition of the substrate solution the final DMSO concentration was 10%. The plate was placed again on the shaker for 5 sec, spun at 2000 rpm for 5 sec and read on the spectrophotometer. The effect of the compound on the enzymatic activity was obtained from the linear part of the progress curves and determined after 15 minutes.
Measurement: IC50 = 117 nM (pChEMBL 6.929999828338623)
Descriptors: MW 483.52, ALogP 5.68, HBA 4, HBD 2, PSA 77.24, QED 0.39
Compound (SMILES): CC[C@H](C)[C@H](NC(C)c1ccccc1)C(=O)N1C[C@H](O)C[C@H]1C(=O)NCc1ccc(-c2scnc2C)cc1
InChIKey: AXZJILSTQJORSE-YLQURUFKSA-N
Target: von Hippel-Lindau disease tumor suppressor (SINGLE PROTEIN), Homo sapiens
UniProt: P40337
Assay: Fluorescence Polarization Assay (1% DMSO): Ability of VHL ligands to compete for the HIF 1 a binding site on VCB was determined through a fluorescence polarization competition assay as described in Buckley et al. JACS, 2012, 134, 4465-4468, WO 2013/106643, and US 2014-0356322, which are incorporated herein by reference in their entirety for all purposes (1% DMSO).
Measurement: IC50 = 37700 nM (pChEMBL 4.420000076293945)
Descriptors: MW 534.73, ALogP 4.46, HBA 6, HBD 3, PSA 94.56, QED 0.36
Compound (SMILES): CCCCS[C@H]1OC(CO)[C@H](O)C(n2cc(-c3cc(F)c(F)c(F)c3)nn2)[C@@H]1O
InChIKey: BADMWMVWCSARMQ-QSRQOKPUSA-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 = 450 nM (pChEMBL 6.349999904632568)
Descriptors: MW 433.45, ALogP 1.88, HBA 8, HBD 3, PSA 100.63, QED 0.45
Compound (SMILES): CNCc1cccnc1-c1cnc2c3[nH]ncc3c(=O)n(CC(F)(F)F)c2c1
InChIKey: GMCPSQPZQNXCAP-UHFFFAOYSA-N
Target: Methionine aminopeptidase 2 (SINGLE PROTEIN), Homo sapiens
UniProt: P50579
Assay: Enzyme Assay: For the enzymatic test, human MetAP2 protein was obtained from a culture supernatant of insect cells (sf9) infected with MetAP2 recombinant baculovirus.Before performing the experiment, dialysis of the MetAP2 supernatant was performed over 24 hours at 4° C. in a buffer (10 mM Hepes, 100 mM KCl, 10% glycerol, pH 7.4) in the presence of EDTA (1 mM) over the first 12 hours.The dialysis supernatant was recovered and manganese, used as cofactor, was added to a final concentration of 300 μM.The enzymatic test is a two steps procedure.In a first step, it consists in placing in contact the compound according to the invention, the dialysed MetAP2 protein and the substrate (Met-Pro-Arg-pNa peptide synthesized by Neosystem), the N-terminal methionine of which can be cleaved with MetAP2, and which bears at the C-terminal end a para-nitroaniline (pNa) chromophore, which can itself be released by another peptidase only when the N-terminal methionine has been cleaved beforehand.Consequently, the second step consists in reacting the peptides cleaved in the preceding step with a second peptidase in order to release the chromophore. The peptidase used in this second step is cathepsin, which comes from the TagZyme DAPase kit (Quiagen, 34366).The MetAP2 activity is proportional to the amount of para-nitroaniline released, which is measured by absorbance at 405 nm.
Measurement: IC50 = 459 nM (pChEMBL 6.340000152587891)
Descriptors: MW 388.35, ALogP 2.62, HBA 6, HBD 2, PSA 88.49, QED 0.56
Compound (SMILES): CN(C)C(=O)N[C@@H]1CCCN(c2cnc(C(N)=O)c(Nc3ccc(OC4CCCC4)cc3)n2)C1
InChIKey: PBNBZMPHPCWEFT-QGZVFWFLSA-N
Target: Tyrosine-protein kinase BTK (SINGLE PROTEIN), Homo sapiens
UniProt: Q06187
Assay: Btk In Vitro Inhibitory Assay: Human Btk kinase (Genbank accession # NP_000052) was purified from insect cells as a full-length construct containing a N-terminal 6×-His tag. Btk kinase activity was determined using a radiometric filter binding assay. Measurements are performed in a low μL reaction volume 384-well assay plates. BTK enzyme (8 nM final in reaction), inhibitor (at requested doses), and 0.2 mg/mL peptide substrate (Poly-Glu-Tyr, 4:1 ratio) are incubated in a reaction buffer composed of 20 mM Hepes (pH 7.5), 10 mM MgCl2, 1 mM EGTA, 0.02% Brij35, 0.02 mg/ml BSA, 0.1 mM Na3VO4, 2 mM DTT, 1% DMSO for 15 min. followed by addition of 1 μM ATP to start the assay. Kinase reactions are carried out for 120 min. at room temperature. The reaction was stopped by spotting of reaction sample onto P81 cationic exchange paper (Whatman). Unbound phosphate was removed by extensive washing of filters in 0.75% Phosphoric acid. After subtraction of background derived from control reactions containing inactive enzyme (via addition of saturating EDTA), kinase activity data for each dose of compound tested was expressed as the percent of remaining kinase activity in test samples compared to vehicle (dimethyl sulfoxide) reactions.
Measurement: IC50 = 55 nM (pChEMBL 7.260000228881836)
Descriptors: MW 467.57, ALogP 2.88, HBA 7, HBD 3, PSA 125.71, QED 0.57
Compound (SMILES): CC(C)(OC(=O)Nc1ccc2c(c1)N(S(=O)(=O)c1ccc(F)cc1)C[C@@H](CNC(=O)C1CC(F)(F)C1)O2)C(F)(F)F
InChIKey: ROFFWMQZBSXXOL-QGZVFWFLSA-N
Target: Nuclear receptor ROR-gamma (SINGLE PROTEIN), Homo sapiens
UniProt: P51449
Assay: Biochemical TR-FRET assay: The compounds of the invention inhibit RORgammaT activity. Activation of RORgammaT activity can be measured using, e.g., a biochemical TR-FRET assay. In such an assay, interaction of cofactor-derived peptides with human RORgammaT-Ligand Binding Domain (LBD) can be measured. The TR-FRET technique is a sensitive biochemical proximity assay that will give information concerning the interaction of a ligand with the LBD, in the presence of cofactor-derived peptides (Zhou et al., Methods 25:54-61, 2001).To identify novel antagonists of RORgammaT, an assay was developed which employs the interaction of RORgammaT with its co-activator peptide SRC1_2. This peptide mimics the recruitment of co-activators to RORgammaT through its interaction with the LXXLL (SEQ ID NO:1) (e.g., NR box) motifs (Xie et al., J. Immunol. 175: 3800-09, 2005; Kurebayashi et al., Biochem. Biophys. Res. Commun. 315: 919-27, 2004; Jin et al., Mol. Endocrinology 24:923-29, 2010). The RORγ-Ligand Binding Domain TR-FRET Assay was run according to the following protocol.HIS-tagged RORγ-LBD protein was recombinantly expressed in Escherichia coli. The RORγ-LBD protein was purified by Ni2+-affinity resin. Purified protein was then diluted in assay buffer (50 mM Tris pH 7.0, 50 mM KCl, 1 mM EDTA, 0.1 mM DTT, 100 μg/mL bovine serum albumin, delipidated) to obtain a RORγ-LBD final concentration of 3 nM. Europium tagged anti-HIS antibody was also added to this solution (1.25 nM). Separately, SF9 cells not expressing any recombinant protein were lysed (32,000 cells per μl in 25 mM Tris, 50 mM NaCl) and the previously frozen lysate was added to the diluted RORγ-LBD solution at a ratio of 0.75 μl SF9 lysate per 15 μl of diluted RORγ-LBD.
Measurement: IC50 = 9.34 nM (pChEMBL 8.029999732971191)
Descriptors: MW 609.55, ALogP 4.83, HBA 6, HBD 2, PSA 114.04, QED 0.44
Compound (SMILES): COc1ccc(C(=O)Nc2cc3cc(-c4cncn4C)ccc3cn2)cc1
InChIKey: RSXAFQXWBZVZMK-UHFFFAOYSA-N
Target: Dual specificity tyrosine-phosphorylation-regulated kinase 1A (SINGLE PROTEIN), Homo sapiens
UniProt: Q13627
Assay: Kinase Assay: Briefly, recombinant DYRK1A kinase, ATP and Ser/Thr peptide 18 were prepared in 1Ã Kinase buffer to final concentrations of 0.19 μg/mL, 30 μM, and 4 μM respectively. The mixture was allowed to incubate with the representative compounds for one hour at room temperature. All reactions were performed in duplicate. Unphosphorylated and phosphorylated forms of Ser/Thr 18 served as control reactions. Additionally, an 11-point dose-response curve of Staurosporine (1 uM top) was run to serve as a positive compound control.After incubation, Development Reagent A was diluted in Development Buffer then added to the reaction and allowed to further incubate for one hour at room temperature. The plate was read at Ex 400 Em 455 to detect the coumarin signal and Ex 400 Em 520 to measure the signal (EnVision Multilabel Plate Reader, PerkinElmer).
Measurement: EC50 = 3 nM (pChEMBL 8.520000457763672)
Descriptors: MW 358.4, ALogP 3.9, HBA 5, HBD 1, PSA 69.04, QED 0.6
Compound (SMILES): C[C@@H]1CC[C@@H](c2ncc(C3CCC3)o2)CN1C(=O)c1ccccc1-n1nccn1
InChIKey: RVFLBWKCKGGYJM-NVXWUHKLSA-N
Target: Orexin receptor type 2 (SINGLE PROTEIN), Homo sapiens
UniProt: O43614
Assay: FLIPR Ca2+ Flux Assay: For intracellular calcium measurements, Chinese hamster ovary (CHO) cells expressing the rat orexin-1 receptor or the human orexin-2 receptor, are grown in Iscove's modified DMEM containing 2 mM L-glutamine, 0.5 g/ml G418, 1% hypoxanthine-thymidine supplement, 100 U/ml penicillin, 100 μg/ml streptomycin and 10% heat-inactivated fetal calf serum (FCS). The cells are seeded at 20,000 cells/well into Becton-Dickinson black 384-well clear bottom sterile plates coated with poly-D-lysine. All reagents were from GIBCO-Invitrogen Corp. The seeded plates are incubated overnight at 37° C. and 5% CO2. Ala-6,12 human orexin-A as the agonist is prepared as a 1 mM stock solution in 1% bovine serum albumin (BSA) and diluted in assay buffer (HBSS containing 20 mM HEPES, 0.1% BSA and 2.5 mM probenecid, pH7.4) for use in the assay at a final concentration of 70 pM. Test compounds are prepared as 10 mM stock solution in DMSO, then diluted in 384-well plates, first in DMSO, then assay buffer. On the day of the assay, cells are washed 3 times with 100 μl assay buffer and then incubated for 60 min (37° C., 5% CO2) in 60 μl assay buffer containing 1 μM Fluo-4AM ester, 0.02% pluronic acid, and 1% BSA. The dye loading solution is then aspirated and cells are washed 3 times with 100 μl assay buffer. 30 μl of that same buffer is left in each well. Within the Fluorescent Imaging Plate Reader (FLIPR, Molecular Devices), test compounds are added to the plate in a volume of 25 μl, incubated for 5 min and finally 25 μl of agonist is added.
Measurement: IC50 = 16 nM (pChEMBL 7.800000190734863)
Descriptors: MW 391.48, ALogP 3.93, HBA 6, HBD 0, PSA 77.05, QED 0.67
Compound (SMILES): COCc1noc(CNC(=O)c2ccc3c(c2)OCc2cnccc2-3)n1
InChIKey: ASUOYIRQTMIDQA-UHFFFAOYSA-N
Target: Rho-associated protein kinase 2 (SINGLE PROTEIN), Homo sapiens
UniProt: O75116
Assay: In Vitro Assay: The effectiveness of compounds of the present invention as ROCK inhibitors can be determined in a 30 μL assay containing 20 mM HEPES, pH 7.5, 20 mM MgCl2, 0.015% Brij-35, 4 mM DTT, 5 μM ATP and 1.5 μM peptide substrate (FITC-AHA-AKRRRLSSLRA-OH). Compounds were dissolved in DMSO so that the final concentration of DMSO was <2%, and the reaction was initiated with Rho kinase variants. After incubation, the reaction was terminated by the addition of EDTA and the phosphorylated and non-phosphorylated peptides separated using a LABCHIP® 3000 Reader (Caliper Life Sciences). Controls consisted of assays that did not contain compound, and backgrounds consisted of assays that contained enzyme and substrate but had EDTA from the beginning of the reaction to inhibit kinase activity. Compounds were tested in dose-response format, and the inhibition of kinase activity was calculated at each concentration of compound. The inhibition data were fit using a curve-fitting program to determine the IC50; i.e., the concentration of compound required to inhibit 50% of kinase activity.
Measurement: IC50 = 550 nM (pChEMBL 6.260000228881836)
Descriptors: MW 352.35, ALogP 2.1, HBA 7, HBD 1, PSA 99.37, QED 0.75
Compound (SMILES): C#C[C@@H](CC)Oc1cnc(/C(F)=C/c2ccc(F)c([C@]3(C)CS(=O)(=O)C(C)(C)C(N)=N3)c2)cn1
InChIKey: NLPJNTRVQWYDNT-QQMPJMTPSA-N
Target: Beta-secretase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P56817
Assay: In Vitro Enzymatic BACE1 and BACE2 FRET (Fluorescence Resonance Energy Transfer) Assays: The cDNAs for both human recombinant BACE1 and 2 with C-terminal 6-His Tags were cloned into transient protein expression vectors, which were subsequently transfected into mammalian cell lines. These recombinant proteins were further purified using Ni-NTA affinity chromatography (Qiagen). The assay buffer used in these screens was 0.05 M acetate, pH 4.5, 8% DMSO final, 100 μM genapol (which is a nonionic detergent, below its Critical Micelle Concentration). The β-secretase enzyme (0.02 nM for BACE1 and 0.64 nM for BACE2), which was pre-incubated for one hour with the test compound, typically in about 1 uL of DMSO according to a serial dilution, was added thereto. The assay was effectively started by the addition of FRET substrate (50 nM) and the combination was incubated for one hour. The FRET assay was terminated by the addition of tris buffer, which raised the pH to neutrality, and the fluorescence was determined. The FRET substrate was a peptide with commercially available fluorophore and quencher, on opposite sides of the BACE cleavage site. The specific FRET substrate used in this assay was made by Amgen in-house. Commercially available FRET substrates, for example, the FRET substrate offered with the BACE1 FRET Assay Kit sold by ThermoFisher Scientific (Catalog Number P2985), may be used in this assay with the appropriate modifications, which are within the purview of the ability of a person with ordinary skill in the art. Proteolytic cleavage of the FRET substrate released quenching of fluorescence (excitation 488 nm and emission 590 nm).
Measurement: IC50 = 208 nM (pChEMBL 6.679999828338623)
Descriptors: MW 488.56, ALogP 3.65, HBA 7, HBD 1, PSA 107.53, QED 0.62
Compound (SMILES): COc1cncc(OC)c1-c1ccc(-c2cnn(C(=O)N3CCCCC3Cc3ccccc3)n2)cc1
InChIKey: IWHRXPZMJPSJPM-UHFFFAOYSA-N
Target: Diacylglycerol lipase-alpha (SINGLE PROTEIN), Homo sapiens
UniProt: Q9Y4D2
Assay: In Vitro Inhibition of DAGL: Briefly, membrane proteome (1 mg/ml, 20 μL) was prepared from HEK293T cells (transiently transfected with hDAGLα-FLAG or hDAGLα-S472A-FLAG, hDAGLβ-FLAG or hDAGLβ-S443A-FLAG) as described in Example 16. The proteome was incubated at room temperature with vehicle (DMSO) or compound in 0.5 μL DMSO for 30 min. The membrane proteome sample was subsequently treated for 30 min with HT-01 probe (1 μM) or FP-Rh probe (1 μM). The reactions were quenched with 10 μL 3× Laemmli sample buffer (final concentrations: 60 mM Tris-Cl pH 6.8, 2% (w/v) SDS, 10% (v/v) glycerol, 5% (v/v) (3-mercaptoethanol, 0.01% (v/v) bromophenol blue). The samples were directly loaded and resolved on SDS page gel (10% acrylamide). The gels were scanned using a ChemiDoc MP system (Cy3 settings, 605/50 filter).The resolved proteins were transferred from the gels to a polyvinyldifluoride membrane for Western Blotting using a Trans-Blot® Turbo (BioRad). FLAG-tagged enzymes were stained using rabbit anti-FLAG as primary antibody, and goat-anti-rabbit HRP as secondary antibody. The blot was developed in the dark using a 10 mL luminal solution, 100 μL ECL enhancer and 3 μL H2O2. Chemiluminescence was visualized using a ChemiDoc XRS (BioRad).The percentage of DAGL activity remaining in the assayed samples was determined by measuring the integrated optical intensity of the fluorescent protein bands of the Western Blot using image lab 4.1. The relative intensity was compared to the vehicle (DMSO) treated proteins, which were set to 100%. IC50 values were determined by plotting a log(inhibitor) vs. normalized response (Variable slope) dose-response curve generated using Prism software (GraphPad).
Measurement: IC50 = 6000 nM (pChEMBL 5.21999979019165)
Descriptors: MW 483.57, ALogP 5.09, HBA 7, HBD 0, PSA 82.37, QED 0.38
Compound (SMILES): Cc1cnc(CNCc2ccn3nc(-c4ccccn4)nc(Nc4ccncc4F)c23)cn1
InChIKey: JFQUUTOBSZUMCQ-UHFFFAOYSA-N
Target: TGF-beta receptor type-1 (SINGLE PROTEIN), Homo sapiens
UniProt: P36897
Assay: Biological Assays: Assays are conducted in 1536-well plates and 2 mL reactions are prepared from addition of HIS-TGFβR1 T204D or HIS-TGFβR2 WT, anti-HIS detection antibody, a labeled small molecule probe (Kd=<100 nM; koff=<0.001 s−1.) and test compounds in assay buffer (20 mM HEPES pH 7.4, 10 mM MgCl2, 0.015% Brij35, 4 mM DTT, and 0.05 mg/ml BSA). The reaction is incubated for 1 hour at room temperature and the HTRF signal was measured on an Envision plate reader (Ex: 340 nm; Em: 520 nm/495 nm). Inhibition data were calculated by comparison to no enzyme control reactions for 100% inhibition and vehicle-only reactions for 0% inhibition. The final concentration of reagents in the assay are 1 nM HIS-TGFβR1 T204D or HIS-TGFβR2 WT, 0.2 nM anti-HIS detection antibody, labeled small molecule probe (at Kd) and 0.5% DMSO. Dose response curves were generated to determine the concentration required inhibiting 50% of kinase activity (IC50). Compounds were dissolved at 10 mM in dimethylsulfoxide (DMSO) and evaluated at eleven concentrations. IC50 values were derived by non-linear regression analysis.
Measurement: IC50 = 2.6 nM (pChEMBL 8.59000015258789)
Descriptors: MW 441.47, ALogP 3.46, HBA 9, HBD 2, PSA 105.81, QED 0.4
Compound (SMILES): CN(C)CCN1CCC(c2cbc(Nc3ccc(-c4cnc5cc(F)ccn45)c4c3C(=O)NC4)cc2)CC1=O
InChIKey: WGIJKDJOSKUTCJ-UHFFFAOYSA-N
Target: Mitogen-activated protein kinase kinase kinase kinase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q92918
Assay: Biochemical Enzyme Assay: HPK1 biochemical enzyme assay: HPK1 enzyme inhibition was measured using a microfluidic mobility shift assay. Reactions were performed in a 384-well plate, containing 1.5 nM HPK1 (Invitrogen), in assay buffer (Carna Biosciences; pH 7.4). Test compounds were titrated in ten point curves (top final assay concentration 3 M), and preincubated with enzyme/substrate mix for 30 min prior to initiation of the reaction by addition of ATP (1 mM final concentration) and substrate (1 Î¼M final concentration; Carna Biosciences) diluted in assay buffer supplemented by MgCl2 (final assay concentration of 5 mM). Following 60 min incubation at RT, the reaction was terminated by addition of 60 l/well termination buffer (Carna Biosciences) and signal determination using a Caliper EZ Reader (Perkin Elmer, UK).
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 524.41
Compound (SMILES): Cc1noc(C)c1-c1cnc2c(-c3cnn(C(F)F)c3)cn(CC3CCC(F)(F)CC3)c2c1
InChIKey: DHEVUTUXGQZFDC-UHFFFAOYSA-N
Target: Bromodomain-containing protein 4 (SINGLE PROTEIN), Homo sapiens
UniProt: O60885
Assay: Alphascreen Binding Assay: Binding of compounds of formula (I) with bromodomain 2, 3 4 was assessed using Alphascreen binding assay. The inhibition of the interaction between bromodomain and its acetylated target protein (Filippakopoulos P et al. 2012) was measured quantitatively using recombinant BRD proteins, an acetylated Histone 4 peptide and AlphaScreen technology. In absence of inhibition the BRD protein bound to AlphaScreen nickel chelate acceptor beads can interact with the acetylated Histone 4 peptide which is immobilized by the AlphaScreen Streptavidin coated beads. This interaction brings donor and acceptor beads in proximity. The close proximity allows the singlet oxygen produced by laser excitation of the donor beads to reach the acceptor beads and generate a luminescence signal. BRD inhibitors result in a decrease in the proximity signal through an inhibition of the BRD-acetylated peptide interaction.Recombinant human bromodomains containing the N-terminal bromodomain (BRD2-BD1 (71-194), BRD3-BD1 (24-144) and BRD4-BD1 (44-164)) or dual bromodomains (BRD4-BD12 (1-477), BRD4-BD12 (1-472)) were prepared and purified as described in protein expression and purification session.
Measurement: IC50 < 1000 nM (pChEMBL 6)
Descriptors: MW 461.46, ALogP 6.39, HBA 6, HBD 0, PSA 61.67, QED 0.32
Compound (SMILES): COc1cc(C(=O)N2CC3CCC2[C@@H]3N)cc2nc(-c3cc4ccc(-c5ccc6ccc(=O)[nH]c6c5)cc4n3CC3CC3)n(C)c12
InChIKey: ZREUJCGWPDHLSI-GFXQXFAQSA-N
Target: Protein-arginine deiminase type-4 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9UM07
Assay: Rapid-Fire Mass Spectrum (RFMS) Assay: Compounds were solubilized in 100% DMSO to achieve a 10 mM compound concentration. Compound stock solutions were stored at RT. A series of dilutions were prepared in DMSO and mixed 8 times with 20 μL mixing volume. Final top concentration of compound in the assay is 50 μM. Final assay conditions were as follows:Reaction volume: 26 μlAssay buffer: 25 mM hepes, pH 7.5, 5 mM NaCl, 1 mM DTT, 0.2 mg/ml BSA, 0.01% CHAPS, 50 μM Calcium, and 5 μM TPENFinal concentrations: 5 nM hPAD4 enzyme, 250 μM BAEE, and 0.5% DMSOTotal incubation time: 30 mins compound and enzyme preincubation at 37° C., 90 min enzyme/substrate reaction, 30 min reaction with phenyl glyoxal at 37° C.Stop solution: 40 μl 5% TCA in ACN
Measurement: IC50 = 36 nM (pChEMBL 7.440000057220459)
Descriptors: MW 612.73, ALogP 5.68, HBA 7, HBD 2, PSA 111.17, QED 0.25
Compound (SMILES): COCCNC(=O)c1ccc(-c2ccc3sc(C(C(=O)NCC(=O)NC4CC4)S(=O)(=O)CCOC)nc3c2)cc1
InChIKey: SQRCLYSQIFWGQR-UHFFFAOYSA-N
Target: Endothelial lipase (SINGLE PROTEIN), Homo sapiens
UniProt: Q9Y5X9
Assay: Biological Activity: Endothelial lipase (EL) and hepatic lipase (HL) activities were measured using a fluorescent substrate, A10070, (Invitrogen, CA) doped into an artificial vesicle containing DMPG (Avanti Polar Lipids) as the excipient. Vesicles were prepared by combining 571 μL of 29 mM DMPG in a 1:1 mixture of MeOH and CHCl3 with 2000 μL of 1 mM A10070 in a 1:1 mixture of MeOH and CHCl3. The mixture was dried under nitrogen in multiple vials then resuspended in 20 mL total volume of 50 mM HEPES pH 8.0 buffer containing 50 mM NaCl and 0.2 mM EDTA. The sample was allowed to sit at room temperature for 15 min and then was sonicated 3×4 mins on ice with a Branson Sonicator using duty cycle 1. This preparation provides vesicles with a mole fraction of 0.11 for the FRET substrate.The enzymatic assay was measured using 384-well white Optiplates. Each well contained 20 μL of assay buffer (50 mM HEPES pH 8.0, 50 mM NaCl and 1 mM CaCl2) and 0.25 μL of a DMSO solution containing a compound of interest. EL or HL (10 μL) was added and allowed to incubate with the compound for 30 min at 37° C. The source of EL was conditioned media obtained from HT-1080 cells that were transformed using RAGE technology (Athersys) to overexpress endogenous EL, and HL was partially purified from conditioned media obtained from COS cells overexpressing HL. The reaction was started by the addition of 10 μL of a 1:10 dilution of vesicles. The final total reaction volume was 20.25 μL. The reaction rates were measured on a Gemini plate reader with an excitation wavelength of 490 nm and an emission wavelength of 530 nm. Readings were taken over a period of 60 minutes, and the slope between 300 and 900 secs of the readout was used to calculate the rate of the reaction.
Measurement: IC50 = 15 nM (pChEMBL 7.820000171661377)
Descriptors: MW 588.71, ALogP 1.84, HBA 9, HBD 3, PSA 152.79, QED 0.24
Compound (SMILES): CCCS(=O)(=O)Nc1ccc(F)c(Nc2ccc3ncn(-c4cccnc4)c(=O)c3c2C)c1Cl
InChIKey: AESNIHRYNSMXAB-UHFFFAOYSA-N
Target: Serine/threonine-protein kinase B-raf (SINGLE PROTEIN), Homo sapiens
UniProt: P15056
Assay: BRAF V600E and V600K Enzyme Assay: A competitive displacement assay was configured for B-Raf that monitors the amount of a fluorescently-tagged tracer bound to B-Raf via TR-FRET from an anti-tag Eu-labeled antibody also bound to B-Raf. For full-length FLAG-tagged B-Raf(V600E), the assay mixtures consisted of 25 mM K+HEPES, pH 7.4, 10 mM MgCl2, 0.01% Triton X-100, 1 mM DTT, 2% DMSO (from compound), 50 nM Tracer 1710 (ThermoFisher, PR9176A), 0.5 nM Eu anti-FLAG (M2)-cryptate Ab (Cisbio, 61FG2KLB) and 5 nM full-length, N-terminally FLAG-tagged B-Raf(V600E) (Origene Technologies, TP700031. When B-Raf(V600K) was assayed, the following substitutions were made: 15 nM Tracer 178 (ThermoFisher, PV5593), 2 nM Eu-anti-GST Ab (ThermoFisher, PV5595) and 5 nM N-terminally GST-tagged B-Raf(V600K) (331-end, SignalChem, B08-12DG). Compounds were typically diluted in DMSO across an 11-point dosing range created using a 3-fold serial dilution protocol at a top dose of 10 μM. The assay was run in 384-well, polystyrene, low-volume, non-treated, white microtiter plates (Costar 4512) in a final volume of 12 μL. Low control wells included 1 μM of a potent B-Raf inhibitor as a control.
Measurement: IC50 = 35.5 nM (pChEMBL 7.449999809265137)
Descriptors: MW 501.97, ALogP 4.78, HBA 7, HBD 2, PSA 105.98, QED 0.38
Compound (SMILES): Cc1nc2ccc(-c3c[nH]c4nc(Nc5ccc(CN6CCN(C)CC6)cc5)nc(OCC5CCCO5)c34)cc2o1
InChIKey: UNJQFDLOVSTMRV-UHFFFAOYSA-N
Target: Tyrosine-protein kinase receptor UFO (SINGLE PROTEIN), Homo sapiens
UniProt: P30530
Assay: Axl Kinase Assay: Certain compounds of formula (I) were tested for their potency in inhibiting Axl kinase. Inhibition of Axl kinase activity by a test compound was calculated by AlphaScreen (PerkinElmer 6760620M). Standard assay conditions included 1 ng of recombinant Axl kinase (SignalChem) with 3-10 ng Poly-(GT)-Biotin (Cisbio) in the assay buffer, i.e., 40 μM ATP (Sigma), 10 mM MOPs, pH7.0 (Sigma M-1254), 0.21 mM EDTA (Sigma E-6758), 0.5% glycerol (J. T Baker 2136-1), 1 mg/ml BSA (Bio-Rad 500-0007), 0.01% 2-mercaptoethanol (BDH 441433A), and 0.001% Brij35 (Sigma P1254) 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 eight serially diluted concentrations in duplication. Results were analyzed using linear regression software (GraphPad Prism 4; GraphPad Software Inc.).
Measurement: IC50 < 30 nM (pChEMBL 7.5229997634887695)
Descriptors: MW 553.67, ALogP 5.12, HBA 9, HBD 2, PSA 104.57, QED 0.27
Compound (SMILES): O=C(NCc1ccc(Cl)c(Cl)c1)c1cccc(CNC2=NCCN2)c1
InChIKey: DOIDLJPFNQHXFM-UHFFFAOYSA-N
Target: WD repeat-containing protein 5 (SINGLE PROTEIN), Homo sapiens
UniProt: P61964
Assay: FPA Binding Assay: FPA Assay protocol adopted from Karatas et al. (J. Med. Chem. 2010, 5179.; J. Amer. Chem. Soc. 2013, 669.): WDR5 (Δ23, residues 24-334), is expressed and purified in sufficient quantities for screening. FITC-MLL peptide (FITC-GSARAEVHLRKS) and 10mer-Thr-FAM (ARTEVHLRKS-(Ahx-Ahx)(Lys-(5-FAM))) were purchased from GeneScript and used without additional purification. FITC-MLL peptide is used at 50 nM, while WDR5 is added at the Ki value of the protein:peptide interaction (WDR5-WIN Ki=2.5 μM). 10mer-Thr-FAM peptide is used at 4 nM, while WDR5 is added at the Ki value of the protein:peptide interaction (WDR5-10mer-Thr Ki=4 nM).Stock compounds are dispensed in barcoded 384-well plates as 30 mM solutions in DMSO. This plate is used as the source plate for the Echo Liquid Handler, which distributes the compounds to the assay plate (black, flat-bottom; Greiner) in a 10-point, 3-fold dilution scheme with a top concentration of 100 μM (5 nM low concentration) in a final volume of 50 μL. Both the top concentration and the dilution scheme can be adjusted to fit the anticipated potency of the compounds.For the FITC-MLL assay, 2.5 μM WDR5 and 50 nM FITC-MLL peptide in assay buffer (1× Phosphate Buffered Saline, pH 6.0, 300 mM NaCl, 0.5 mM TCEP, 0.1% CHAPS) is added to all compound-containing wells and to columns 2, 24 (negative control, 0% inhibition). 2 μL of 50 nM FITC-MLL peptide alone in assay buffer is added to columns 1, 23 (positive control, 100% inhibition). For the 10mer-Thr-FAM assay, a similar addition protocol is performed, using 4 nM WDR5 and 4 nM 10mer-Thr-FAM peptide in assay buffer (1× Phosphate Buffered Saline pH 6.0, 300 mM NaCl, 0.5 mM TCEP, 0.1% CHAPS).The plate is covered, shielded from light, and incubated for 60 min at room temperature, with rocking. Anisotropy is measured at excitation wavelength 480 nm and emission wavelength 535 nm using an EnVision Multi-label plate reader (PerkinElmer, Wellesley, Mass., USA) or a BioTek Cytation 3 (BioTek, Winooski, Vt., USA). Fluorescence anisotropy is plotted against compound concentration to generate an IC50 (inhibitor concentration at which 50% of bound peptide is displaced) by fitting the data to a 4-parameter logistic model using XLFit software (Guildford, Surrey, UK). IC50 is converted to a binding dissociation constant (Ki value) according to the formula of Wang Z. FEBS Lett (1996) 3, 245.K i=[I]50/([L]50 /K d+[P]0 /K d+1)where [I]50 is the concentration of the free inhibitor at 50% inhibition, [L]50 is the concentration of the free labeled ligand at 50% inhibition, [P]0 is the concentration of the free protein at 0% inhibition, Kd represents the dissociation constant of the FITC-MLL or 10mer-Thr-FAM probe for WDR5. Total fluorescence is also measured, to rule out compounds that are inherently fluorescent or able to act as quenchers in the assay.
Measurement: Ki = 209 nM (pChEMBL 6.679999828338623)
Descriptors: MW 377.28, ALogP 2.97, HBA 4, HBD 3, PSA 65.52, QED 0.75
Compound (SMILES): C=CC(=O)Nc1cccc(-c2ccc(C(N)=O)c3[nH]c(-c4c(C)noc4C)cc23)c1C
InChIKey: AMHAIQCXLPPLGN-UHFFFAOYSA-N
Target: Tyrosine-protein kinase BTK (SINGLE PROTEIN), Homo sapiens
UniProt: Q06187
Assay: Time-Resolved Fluorescence Resonance Energy Transfer (TR-FRET) : The in-house BTK corresponds to recombinant human catalytic domain (aa 393-659), which was expressed in SF9 cells with an N-terminal his tag and purified by immobilized metal affinity chromatography. BTK was mixed with peptide substrate (biotin-TYR1, Sequence: Biotin-(Ahx)-GAEEEIYAAFFA-COOH, 0.2 μM final) at varying inhibitor concentrations in reaction buffer: 50 mM MOPSO pH 6.5, 10 mM MgCl2, 2 mM MnCl2, 2.5 mM DTT, 0.01% BSA, 0.1 mM Na3VO4 and 0.001 mM ATP. After about 60 mM incubation at room temperature, the reaction was quenched by addition of EDTA (final concentration: 100 mM) and developed by addition of detection reagents (final approximate concentrations: 30 mM HEPES pH 7.0, 0.06% BSA, 0.006% Tween-20, 0.24 M KF, 80 ng/mL PT66K (europium labeled anti-phosphotyrosine antibody cat #61T66KLB Cisbio, Bedford, Mass.) and 0.6 μg/mL SAXL (Phycolink streptavidin-allophycocyanin acceptor, cat #PJ25S, Prozyme, San Leandro, Calif.). The developed reaction was incubated in the dark for about 60 mM at room temperature, then read via a time-resolved fluorescence detector (Rubystar, BMG) using a 337 nm laser for excitation and monitoring emission wavelength at 665 nm. Within the linear range of the assay, the observed signal at 665 nm was directly related to phosphorylated product and can be used to calculate the IC50 values.
Measurement: IC50 = 550 nM (pChEMBL 6.260000228881836)
Descriptors: MW 414.47, ALogP 4.64, HBA 4, HBD 3, PSA 114.01, QED 0.41
Compound (SMILES): COc1cc([C@@H](CO)NC(=O)[C@@H](C)N2Cc3ccc(-c4nc(NC5CCOCC5)ncc4Cl)cc3C2=O)c(Cl)cn1
InChIKey: NZLHLWHXKQMSCP-IQMFZBJNSA-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 MgCl2, 1 mM EGTA, 0.01% Triton X-100, 1 mM DTT, 2.5% DMSO, 0.4 mM 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).
Measurement: IC50 = 59 nM (pChEMBL 7.230000019073486)
Descriptors: MW 601.49, ALogP 3.64, HBA 9, HBD 3, PSA 138.8, QED 0.34
Compound (SMILES): CCOc1c(F)cccc1-c1nc([C@@H]2CCCN2C(=O)c2cc(C)ccc2-n2nccn2)no1
InChIKey: SDAUGRXYUNFKEF-FQEVSTJZSA-N
Target: Orexin/Hypocretin receptor type 1 (SINGLE PROTEIN), Homo sapiens
UniProt: O43613
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 = 11 nM (pChEMBL 7.960000038146973)
Descriptors: MW 462.49, ALogP 4.14, HBA 8, HBD 0, PSA 99.17, QED 0.42
Compound (SMILES): CC1CCCCN(Cc2cc3c(c(C(F)(F)F)c2)CN(c2cccc([C@]4(Cc5nncn5C)C[C@@H](C#N)C4)c2)C3=O)C1
InChIKey: XRDGGFPSCNEHQL-AVCJDWODSA-N
Target: E3 ubiquitin-protein ligase CBL-B (SINGLE PROTEIN), Homo sapiens
UniProt: Q13191
Assay: Cbl-b Displacement Assay (Cbl-b Inhibition Assay low): The ability of candidate compounds to displace a known inhibitor and thereby inhibit Cbl-b activity was measured by monitoring the interaction of Cbl-b with a fluorophore-labeled probe in the presence of the candidate compound. A truncated variant of Cbl-b (UniProt number Q13191; SEQ ID NO:1) containing an Avitag at its N-terminus was co-expressed with BirA biotin ligase and purified using a standard protocol (see Dou et al., Nature Structural and Molecular Biology, 8: 982-987, 2013; Avidity LLC). Fluorescently-labeled inhibitor probe was synthesized and tagged with BODIPY FL (Example 59). Cbl-b displacement assays were performed in a 384-well plate at room temperature in a 10 μL reaction volume by pre-incubating 0.125 nM Cbl-b in an assay buffer containing 20 mM HEPES pH 7.5, 150 mM NaCl, 0.01% Triton X-100, 0.01% BSA and 0.5 mM TCEP in the presence of a candidate compound in 1% DMSO (final concentration) for 1 hour. After incubation in the presence of the candidate compound, the plate was incubated for an additional 1 hour in the presence of an approximate EC40 binding saturation consisting of 150 nM fluorescently-labeled inhibitor probe and 2 nM Streptavidin-Terbium (Cisbio) (final concentrations). Following the 1 hour incubation, the plates were read for TR-FRET signal at 520/620 nM using an Envision plate reader (Perkin Elmer). The presence of a TR-FRET signal indicated that the probe was not displaced from Cbl-b by the compound candidate. The absence of a FRET signal indicated that the probe was displaced from Cbl-b by the compound candidate.
Measurement: IC50 < 1 nM (pChEMBL 9)
Descriptors: MW 576.67, ALogP 6.03, HBA 6, HBD 0, PSA 78.05, QED 0.36