lovingscience/chembl-v37-raw-activities huggingface dataset:
Compound (SMILES): COC[C@H](N)C(=O)N[C@H]1CN[C@@](CCCCB(O)O)(C(=O)O)C1
InChIKey: SLNMLNJTJAYSQQ-GBIKHYSHSA-N
Target: Arginase-1 (SINGLE PROTEIN), Homo sapiens
UniProt: P05089
Assay: Biological Activity of Examples 1 to 30: The inhibitory effects of Examples 1 to 30 on the activity of Human Arginase 1 and Arginase 2 activity were quantified by measuring the formation of the thiol group from thioarginine using recombinant Arginase 1 or Arginase 2 produced from E. coli. The thiol group was detected with Ellman's reagent, 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB). DTNB reacts with the thiol to give the mixed disulfide and 2-nitro-5-thiobenzoic acid (TNB) which is quantified by the absorbance of the anion (TNB2−) at 412 nm.The assays were run in clear 384 well plates (Greiner cat no: 781101). Various concentrations of Examples 1 to 30 in 300 nL DMSO were dispensed to assay plates using an Echo acoustic dispenser immediately followed by plate sealing and centrifugation. Two pre-mixes were prepared from reagents thawed immediately before addition to assay plates. Pre-mix one comprised human Arginase 1 or human Arginase 2, at a final concentration of 5 nM and 0.5 mM DTNB in assay buffer, 45 mM HEPES pH7.5, brij 35, 0.045% (w/v) and 100 μM MnCl2. Pre-mix two comprised freshly thawed 0.5 mM thioarginine in assay buffer. Fifteen microlitres of pre-mix one was dispensed to assay plates containing Examples 1 to 30, centrifuged and incubated for 30 minutes at room temperature prior to adding fifteen microlitres of pre-mix two.Assay plates were centrifuged prior to reading absorbance at 412 nm in a Pherastar multi-mode plate reader to collect data at time point 0 (T0). The plates were incubated at room temperature for 60 min prior to reading again to collect data at time point 1 (T1). Data is derived by subtracting the A412 signal measured at T0 (time point 0) from that measured at T1 (time point 1). The data was transformed to % effect using the equation:Compound % effect=100*[(X−min)/(max−min)], where X represents the normalized value for the compound based on the Min (vehicle) and Max (reference compound) inhibition control.
Measurement: IC50 = 320 nM (pChEMBL 6.5)
Descriptors: MW 331.18
Compound (SMILES): C[C@@](O)(C(=O)N1C[C@H](F)[C@H](NC(=O)c2cc(-c3cc(C(F)(F)F)c4c(N)ncnn34)c(F)cc2Cl)C1)C(F)(F)F
InChIKey: FGKUAOYCEOJFQE-LRDNONRASA-N
Target: Receptor-interacting serine/threonine-protein kinase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q13546
Assay: RIPK1 HTRF Binding Assay: A solution was prepared containing 0.2 nM Anti GST-Tb (Cisbio, 61GSTTLB), 90.6 nM probe and 1 nM His-GST-TVMV-hRIPK1(1-324) in FRET Buffer (20 mM HEPES, 10 mM MgCl2, 0.015% Brij-35, 4 mM DTT, 0.05 mg/mL BSA). Using Formulatrix Tempest, the detection antibody/enzyme/probe solution (2 mL) was dispensed into wells of a 1536 plate (Black Low Binding Polystyrene 1536 Plate (Corning, 3724)) containing 10 nL of compounds of interest at appropriate concentration in DMSO. The plate was incubated at rt for 1 h. FRET was measured using the EnVision plate reader (Excitation: 340 nM, Emission: 520 nM/495 nM). Total signal (0% inhibition) was calculated from wells containing 10 nL DMSO only. Blank signal (100% inhibition) calculated from wells containing 10 nL of 15 nM staurosporine and internal controls.
Measurement: IC50 = 8500 nM (pChEMBL 5.070000171661377)
Descriptors: MW 600.85, ALogP 3.38, HBA 7, HBD 3, PSA 125.85, QED 0.39
Compound (SMILES): N#Cc1ccc(-c2cc(C(=O)N3CC[C@H](N)C3)ccc2-c2ccc(CCO)cc2F)cc1
InChIKey: YEAXWSQNQRISIQ-NRFANRHFSA-N
Target: Lysine-specific histone demethylase 1A (SINGLE PROTEIN), Homo sapiens
UniProt: O60341
Assay: Inhibitory Activity Assay: The conditions for measuring inhibitory activity of compounds against LSD1 activity were determined with reference to a document available from the website of PerkinElmer (U-TRF #38) and a patent of GlaxoSmithKline (WO2012135113).To measure the inhibitory activity, first, the compound of the present invention was serially diluted in dimethylsulfoxide (DMSO). Sequentially, the solution of the compound of the present invention in DMSO (final concentration of DMSO: 5%) and human LSD1 protein (Abcam, ab80379) were added to a reaction buffer (25 mM Tris-HCl (pH 7.5), 50 mM KCl, 2 mM CHAPS, 1 mM DTT, 0.02% BSA). The mixture was preincubated at 25° C. for 30 minutes. Thereafter, a H3K4 (Me1)-biotin-labeled peptide (Anaspec #64355) (final concentration: 200 nM) was added thereto and reacted for 60 minutes. Tranylcypromine (final concentration: 3 mM) was then added thereto to terminate the reaction. Thereafter, a detection solution containing an Eu-labeled anti-H3K4 antibody (PerkinElmer, TRF0404) and Streptavidin Alexa Fluor 647 (Thermo Fisher Scientific, 521374) was added thereto, and the mixture was allowed to stand at room temperature for 1 hour. Finally, the intensity of fluorescence under the excitation light with a wavelength of 337 nm was measured with a PHERAstar FS (BMG Labtech) at two wavelengths: 620 nm and 665 nm. The demethylation level was calculated from the ratio of the fluorescence intensity at the two wavelengths, and the compound concentration at which demethylation was inhibited by 50% was defined as IC50 (nM).
Measurement: IC50 = 3.68 nM (pChEMBL 8.430000305175781)
Descriptors: MW 429.5, ALogP 3.74, HBA 4, HBD 2, PSA 90.35, QED 0.65
Compound (SMILES): Cc1cc(C)cc(N2CCN(c3nccc(COC(=O)NC(=N)N)c3F)CC2)c1
InChIKey: MKRURFLDNNAJBW-UHFFFAOYSA-N
Target: Amine oxidase [copper-containing] 3 (SINGLE PROTEIN), Homo sapiens
UniProt: Q16853
Assay: TBD: TBD
Measurement: IC50 = 8000 nM (pChEMBL 5.099999904632568)
Descriptors: MW 400.46, ALogP 2.28, HBA 6, HBD 3, PSA 107.57, QED 0.54
Compound (SMILES): C=CC(=O)N1CCC[C@@H](Nc2nc(Nc3ccc(N4CCOCC4)cc3F)nc3[nH]cc(Cl)c23)C1
InChIKey: WMZLDFWSWOAUNR-OAHLLOKOSA-N
Target: Tyrosine-protein kinase JAK3 (SINGLE PROTEIN), Homo sapiens
UniProt: P52333
Assay: Enzyme Inhibitory Activity: JAK3 and BTK kinase inhibitory activities were measured for the compounds prepared in the above Examples through in vitro analysis on the ADP Glow (Glo) platform.Specifically, the inhibitory activities of JAK3 and BTK kinase were measured using a JAK3 kinase assay kit (Promega, V9441) and a BTK kinase assay kit (Promega, V9071) which were purchased from Promega. Recombinant purified human JAK3 and BTK were diluted with 1×kinase reaction buffer (JAK3: 40 mM Tris-Cl, pH 7.5, 20 mM MgCl2, 0.1 mg/mL BSA and 50 uM DTT/BTK: 40 mM Tris-Cl, pH 7.5, 20 mM MgCl2, 0.1 mg/mL BSA, 2 mM MnCl2 and 50 uM DTT) and added to a 96 well plate (JAK3: final concentration of 4 ng per reaction/BTK: final concentration of 8 ng per reaction). The compounds were treated so as to be finally a 1% DMSO aqueous solution, and a substrate cocktail containing ATP (JAK3: final concentration of 5 uM/BTK: final concentration of 10 uM) and 0.2 μg/μL of Poly(Glu4, Tyr1) peptide (JAK3 and BTK final concentration) in the total 254 reactants was added to a 96-well plate to initiate enzymatic reaction. After incubation (30° C.) for 1 hour, equivalent volume (254 per reaction) of ADP Glo was added and incubated (30° C.) for 40 minutes at room temperature. Then, a kinase detection reagent (504 per reaction) was added and incubated (30° C.) for 30 minutes at room temperature. The kinase activity was measured by chemiluminescence according to the instructions of ADP Glo kinase assay kit, and the inhibitory activity of the compounds according to the present invention was calculated.
Measurement: IC50 > 80 nM (pChEMBL 7.0970001220703125)
Descriptors: MW 499.98, ALogP 3.92, HBA 7, HBD 3, PSA 98.41, QED 0.44
Compound (SMILES): CC(C)C[C@@H](C(=O)N1C[C@]2(CC1C#N)C(=O)Nc1ccccc12)N(C)C(=O)c1ccc(F)c(Cl)c1
InChIKey: QRHVMNPPUDWJBK-UTMPKSGFSA-N
Target: Replicase polyprotein 1ab (SINGLE PROTEIN), Severe acute respiratory syndrome coronavirus 2
UniProt: P0DTD1
Assay: Fluorescence Assay (FRET): SARS-CoV-2 3C-like (3CL) protease fluorescence assay (FRET): Recombinant SARS-CoV-2 3CL-protease was expressed and purified. TAMRA-SITSAVLQSGFRKMK-Dabcyl-OH peptide 3CLpro substrate was synthesized. Black, low volume, round-bottom, 384 well microplates were used. In a typical assay, 0.85 μL of test compound was dissolved in DMSO then incubated with SARS-CoV-2 3CL-protease (10 nM) in 10 μL assay buffer (50 mM HEPES [pH 7.5], 1 mM DTT, 0.01% BSA, 0.01% Triton-X 100) for 30 min at RT. Next, 10 μL of 3CL-protease substrate (40 μM) in assay buffer was added and the assays were monitored continuously for 1 h in an Envision multimode plate reader operating in fluorescence kinetics mode with excitation at 540 nm and emission at 580 nm at RT. No compound (DMSO only) and no enzyme controls were routinely included in each plate. All experiments were run in duplicate. Data Analysis: SARS-CoV-2 3CL-protease enzyme activity was measured as initial velocity of the linear phase (RFU/s) and normalized to controlled samples DMSO (100% activity) and no enzyme (0% activity) to determine percent residual activity at various concentrations of test compounds (0-10 μM). Data were fitted to normalized activity (variable slope) versus concentration fit in GraphPad Prism 7 to determine IC50.
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 496.97, ALogP 3.98, HBA 4, HBD 1, PSA 93.51, QED 0.68
Compound (SMILES): CC(C)c1ccc(CC(=O)Nc2cnn(Cc3ccc(F)c(F)c3)n2)cc1
InChIKey: MQEYCCRZJALFGO-UHFFFAOYSA-N
Target: Voltage-dependent T-type calcium channel subunit alpha-1H (SINGLE PROTEIN), Homo sapiens
UniProt: O95180
Assay: Measurement of Calcium Channel Flux by Means of FLIPR Assay: Stock solutions of test compounds are prepared to a concentration of 10 mM in DMSO. For the Cav3.2 assay, serial dilutions of the compounds are prepared in TEAC buffer (100 mM tetraethylammonium chloride; 20 mM Hepes; 2.5 mM CaCl2; 5 mM KCl; 1 mM MgCl2; 1% FBS; pH 7.2), for the Cav1.2 assay serial dilutions are prepared in assay buffer. Test compounds are added to the cells to give a 3-fold dilution range from 10 μM to 0.05 nM. The compounds are incubated with the cells for 3 minutes and Ca2+ entry is stimulated by adding either CaCl2 to a final concentration of 10 mM (Cav3.2 assay) or by adding KCl to a final concentration of 20 mM (Cav1.2 assay). The kinetics of fluorescence increase are recorded for every well and the area under the fluorescence trace for every compound concentration is used to generate inhibition curves using non-linear regression sigmoidal concentration-response curve analysis with in-house software. IC50 values are calculated and represent the compound concentration required to inhibit 50% of the signal that is obtained in the presence of vehicle instead of test compound. In analogy, antagonistic activities (IC50 values) of all exemplified compounds have been measured for the for the Cav3.1- and the Cav3.3-channel. Antagonistic activities (IC50 values) of all exemplified compounds are in the range of 1.7 to 970 nM with respect to Cav3.1; and in the range of 1.1 to 620 nM with respect to Cav3.3.
Measurement: IC50 = 30 nM (pChEMBL 7.519999980926514)
Descriptors: MW 370.4, ALogP 3.91, HBA 4, HBD 1, PSA 59.81, QED 0.72
Compound (SMILES): Cc1s/c(=N\C2CCCCC2)n(/N=C/c2cc(O)c(O)cc2O)c1C1CC1
InChIKey: NSNKCRLHDVZRNS-GRWXYWTESA-N
Target: Induced myeloid leukemia cell differentiation protein Mcl-1 homolog (SINGLE PROTEIN), Mus musculus
UniProt: P97287
Assay: High-Throughput Competitive Fluorescence Polarization (FP) Screening Assay: A high-throughput competitive fluorescence polarization (FP) screening assay (Z-factor, 0.62) was developed based on the direct binding interaction between FITC-MCL-1 SAHBA and MCL-1ΔNΔC (EC50, 14 nM) (FIGS. 1A, 2A, and 3A). A compilation of 71,296 small molecules was screened for the capacity to displace FITC-MCL-1 SAHBA from recombinant MCL-1ΔNΔC (aa 172-327) (FIG. 4). To enrich for MCL-1-selective molecules by detecting binding activity for the BCL-XL subclass of anti-apoptotic proteins, the library was also counterscreened using a competitive FP assay (Z-factor, 0.71) developed based on the direct and selective interaction between FITC-BAD BH3 and BCL-XLΔC (EC50, 26 nM) (FIGS. 1B, 2B, and 3B). Small molecules with an apparent preference for MCL-1ΔNΔC (208 compounds, 0.3% hit rate), as defined both by >50% displacement of the FITC-MCL-1 SAHBA/MCL-1ΔNΔC interaction and a >45% difference in peptide displacement from MCL-1ΔNΔC vs. BCL-XLΔC, were advanced to increasingly stringent confirmatory in vitro binding assays including: (1) repeat single-dose testing of 208 molecules in the differential competitive FP screen; (2) alternative single-dose selectivity screen for 130 confirmed MCL-1-directed antagonists comparing relative displacement of FITC-BID BH3, a dual binder,35 from MCL-1 ΔNΔC vs. BCL-XLΔC; and then (3) dose-responsive competitive binding of the 64 most selective molecules against the FITC-MCL-1 SAHBA/MCL-1 ΔNΔC complex (FIG. 4).
Measurement: IC50 = 587 nM (pChEMBL 6.230000019073486)
Descriptors: MW 387.51, ALogP 3.97, HBA 7, HBD 3, PSA 90.34, QED 0.42
Compound (SMILES): CC(C)n1cc(-c2noc(C3CC3)c2-c2ccccn2)c2c(N)ncnc21
InChIKey: BSMWNBXVSLLPQA-UHFFFAOYSA-N
Target: Proto-oncogene tyrosine-protein kinase receptor Ret (SINGLE PROTEIN), Homo sapiens
UniProt: P07949
Assay: RET Enzyme Assay: The potency of compounds inhibiting several different RET kinase forms (Wild Type, V804M, M918T, G810R, & G810S) were determined using CisBio's HTRF Kinease-TK assay technology. The kinases were incubated with 250 nM TK-substrate biotin (CisBio, part of cat #62TK0PEC) at Km ATP along with test compounds in a buffer consisting of 25 mM HEPES, pH 7.4, 10 mM MgCl2, 0.01% Triton X-100, and 2% DMSO in a volume of 8 μL. Compounds were typically prepared as a three-fold serial dilution in DMSO and added to the assay to give the appropriate final concentration. After a 30-min incubation at 22° C., the reaction was quenched by adding 8 μL of quench solution containing 31.25 nM Sa-XL665 and 1×TK-Ab-Cryptate in HTRF detection buffer (all from CisBio, part of cat #62TK0PEC). After a 1 hour incubation at 22° C., the extent of reaction was determined using a PerkinElmer EnVision multimode plate reader via HTRF dual wavelength detection, and the percent of control (POC) was calculated using a ratiometric emission factor. One hundred POC was determined using DMSO only samples (no compound present), and 0 POC was determined using pre-quenched control reactions. A 4-parameter logistic curve was fit to the POC values as a function of the concentration of compound, and the IC50 value was the point where the best-fit curve crossed 50 POC.
Measurement: IC50 = 0.4 nM (pChEMBL 9.399999618530273)
Descriptors: MW 360.42, ALogP 4.19, HBA 7, HBD 1, PSA 95.65, QED 0.59
Compound (SMILES): Cc1cc(-n2ncc3cc(NC(=O)N[C@@H]4CN(C)C[C@H]4c4nc(C)cs4)ncc32)ccn1
InChIKey: DOPXPPYQDBSDDP-QZTJIDSGSA-N
Target: Mitogen-activated protein kinase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P28482
Assay: IMAP-FP Assay: Activated ERK2 activity was determined in an IMAP-FP assay (Molecular Devices). Using this assay format, the potency (IC50) of each compound was determined from a 10 point (1:3 serial dilution, 3 μM starting compound concentration) titration curve using the following outlined procedure. To each well of a black Corning 384-well plate (Corning Catalog #3575), 7.5 nL of compound (3333 fold dilution in final assay volume of 25 μL) was dispensed, followed by the addition of 15 μL of kinase buffer (tween containing kinase buffer, Molecular Devices) containing 0.0364 ng/mL (0.833 nM) of phosphorylated active hERK2 enzyme. Following a 15 minute compound & enzyme incubation, each reaction was initiated by the addition of 10 μL kinase buffer containing 2.45 μM ERK2 IMAP substrate peptides and 75 μM ATP. The final reaction in each well of 25 μL consists of 0.5 nM hERK2, 900 nM unlabeled peptide, 80 nM labeled-peptide, and 30 μM ATP. Phosphorylation reactions were allowed to proceed for 60 minutes and were immediately quenched by the addition of 60 μL IMAP detection beads (1:1000 dilutions) in IMAP binding buffer (Molecular Devices) with 24 mM NaCl. Plates were read on EnVision reader after 60 minutes binding equilibration using Fluorescence Polarization protocol (Perkin Elmer).
Measurement: IC50 = 16 nM (pChEMBL 7.800000190734863)
Descriptors: MW 448.56, ALogP 3.11, HBA 8, HBD 2, PSA 100.86, QED 0.5
Compound (SMILES): COC(c1c[nH]c2cc(-c3c(C)noc3C)cnc12)C1CC1
InChIKey: ZTMYMPFUKNVXAT-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 = 5500 nM (pChEMBL 5.260000228881836)
Descriptors: MW 297.36, ALogP 3.93, HBA 4, HBD 1, PSA 63.94, QED 0.79
Compound (SMILES): Cc1cccc(-c2csc(NC(=O)C(C#N)Cc3ccc(O)c(O)c3)n2)n1
InChIKey: CIOBIGJDTXEEKZ-UHFFFAOYSA-N
Target: Double-strand break repair protein MRE11 (SINGLE PROTEIN), Homo sapiens
UniProt: P49959
Assay: HTS Assay: The conditions of the assay are as follows:Microplate type: 1536 Well Black Round Bottom Polystyrene Not Treated (Corning cat no. 3936)Total reaction volume: 5 μLMRE11 concentration in the reaction: 18 nMDNA substrate concentration in the reaction: 40 nMNumber of compounds to test: 257Inhibitor concentration range tested: 7 nM-50 μMMultiplicates: 3Concentration points: 13Dilution step: 2.1Each plate contains a series of high and low signal control wells, where no compound is added:High signal: MRE11+DNA substrateLow signal: DNA substrate onlyThese are used during data evaluation.Two Extra Assay Controls:1) To check whether unwinding of DNA by the compounds alone occurs.The DNA substrate [CY5+BBQ(650)] is mixed with the compounds with no protein present.This is done as a single measurement at 25 μM inhibitor concentration.2) To check whether the compounds are able to quench the CY5.The CY5 single stranded oligo is mixed with the compounds with no protein present. This is done as a single measurement at 25 μM inhibitor concentration.The layout of the plates is created by the in-house software (CZ-Openscreen Prague) and this information is transferred to the robotic HTS station.Assay Steps1) Prepare 50 mL of master mix:16.7 mL 5× reaction buffer (150 mM Bis Tris pH 7; 5 mM DTT)1042 μL 400 mM MnCl232.3 mL H2O2) Fill the plates with 3 μL of master mix per well using MultiDrop (Thermo Scientific)3) Transfer of compounds to the plates at the robotic station with the contactless Echo dispenser (Labcyte)4) Measurement of autofluorescence with the EnVision reader (PerkinElmer)5) Prepare 20 mL of 90 nM MRE11 in T+50 buffer (25 mM Tris-HCl pH7.5, 50 mM KCl 8.7% glycerol, 0.5 mM EDTA)6) Add 1 μL of 90 nM MRE11 to the corresponding wells using MultiDrop7) Preincubation at RT for 30 min8) Prepare 20 mL of a 200 nM solution of 5′ overhang DNA substrate: 480 μL 6 μM DNA+19.52 mL H2O9) Prepare 4 mL of a 200 nM solution of the single stranded DNA (oligo 1): 8 μL 100 μM DNA+4 mL H2O10) Add 1 μL of each 200 nM DNA solution to the corresponding wells with the MultiDrop11) Fluorescence measurement with the EnVision reader every 45 minutesFluorescence readout: CY5λex/em=620/665 nmAnalysis
Measurement: IC50 = 6000 nM (pChEMBL 5.21999979019165)
Descriptors: MW 380.43, ALogP 3.25, HBA 7, HBD 3, PSA 119.13, QED 0.58
Compound (SMILES): O=C(O)c1ccc(-n2c(Cn3cc(C(=O)NCc4ncn5ccc(Cl)c(F)c45)nn3)cc3cc(C4CC4)ccc32)cc1
InChIKey: HMQMBBYTPJFINU-UHFFFAOYSA-N
Target: Plasma kallikrein (SINGLE PROTEIN), Homo sapiens
UniProt: P03952
Assay: Inhibitory Activity Assay: Example compounds were evaluated for inhibition of the human activated kallikrein enzyme in two formats of an assay employing a fluorogenic peptide substrate. In one assay format, the concentrations of reagents were as follows: 20 mM Tris pH 7.5, 1 mM EDTA, 150 mM sodium chloride, 0.1% PEG-400, 0.1% Triton X-100, 500 pM activated kallikrein enzyme, 300 uM Pro-Phe-Arg-7-amido-4-methylcoumarin substrate. Prior to reaction initiation with substrate, enzyme and inhibitors were preincubated for 30 min at RT. After initiation with substrate, reactions were incubated for 10 min at RT and fluorescence emission at 460 nm from 380 nm excitation measured with a microplate reader. In another assay format, the concentrations of reagents were as follows: 20 mM Tris pH 7.5, 1 mM EDTA, 150 mM sodium chloride, 0.1% PEG-400, 0.1% Triton X-100, 5 pM activated kallikrein enzyme, 300 uM Pro-Phe-Arg-7-amido-4-methylcoumarin substrate. Prior to reaction initiation with substrate, enzyme and inhibitors were preincubated for 30 min at RT. After initiation with substrate, reactions were incubated for 18 h at RT and fluorescence emission at 460 nm from 380 nm excitation measured with a microplate reader.
Measurement: EC50 = 5.5 nM (pChEMBL 8.260000228881836)
Descriptors: MW 584.01, ALogP 5.22, HBA 8, HBD 2, PSA 119.34, QED 0.25
Compound (SMILES): C=CC(=C)N1C[C@H](C)N(C2=NC(=C)N(c3ccc(S(=C)C)cc3C(C)C)c3nc(-c4ccccc4F)c(Cl)cc32)C[C@H]1C
InChIKey: QPKBSVKOSXFWAH-PIAHPUASSA-N
Target: GTPase KRas (SINGLE PROTEIN), Homo sapiens
UniProt: P01116
Assay: Coupled Nucleotide Exchange Assay: Purified GDP-bound KRAS protein (aa 1-169), containing both G12C and C118A amino acid substitutions and an N-terminal His-tag, was pre-incubated in assay buffer (25 mM HEPES pH 7.4, 10 mM MgCl2, and 0.01% Triton X-100) with a compound dose-response titration for either 5 min or 2 hours (see Table 15). Following compound pre-incubation, purified SOS protein (aa 564-1049) and GTP (Roche 10106399001) were added to the assay wells and incubated for an additional 30 min (for 5 min compound pre-incubation) or 1 hour (for 2 hour compound pre-incubation). To determine the extent of inhibition of SOS-mediated nucleotide exchange, purified GST-tagged cRAF (aa 1-149), nickel chelate AlphaLISA acceptor beads (PerkinElmer AL108R), and AlphaScreen glutathione donor beads (PerkinElmer 6765302) were added to the assay wells and incubated for 10 minutes. The assay plates were then read on a PerkinElmer EnVision Multilabel Reader, using AlphaScreen® technology, and data were analyzed using a 4-parameter logistic model to calculate IC50 values.
Measurement: IC50 = 677 nM (pChEMBL 6.170000076293945)
Descriptors: MW 616.25, ALogP 8.82, HBA 5, HBD 0, PSA 34.97, QED 0.2
Compound (SMILES): COc1ccc([C@@H]2CCCC[C@@H]2NC(=O)C[C@@H]2CCc3cc(C(O)(C(F)(F)F)C(F)(F)F)ccc3N2S(=O)(=O)c2ccc(F)cc2)cn1
InChIKey: DFMRFDFSZDXRMP-RNXOBYDBSA-N
Target: Nuclear receptor ROR-gamma (SINGLE PROTEIN), Homo sapiens
UniProt: P51449
Assay: Spa Binding Assay: The binding of potential ligands to RORγ is measured by competition with [3H] 25-hydroxycholesterol using a scintillation proximity assay (SPA) binding assay. The ligand binding domain of human RORγ (A262-S507) with an N-terminal His tag is expressed in E. coli and purified using nickel affinity chromotography. 50 nM RORγ (A262-S507) is incubated with test compound at varying concentrations for 15 min at room temperature in PBS buffer containing 0.5% fatty acid free BSA. 10 nM of [3H] 25-hydroxycholesterol is then added, and the reaction is incubated for 15 min. 4 mg/mL of Ysi Copper HIS-TAG SPA Beads (Perkin Elmer) are added, and the mixture is incubated for 30 min. The reaction is read on a MicroBeta Trilux scintillation plate reader (Perkin Elmer).
Measurement: IC50 = 41.2 nM (pChEMBL 7.380000114440918)
Descriptors: MW 703.68, ALogP 6.28, HBA 6, HBD 2, PSA 108.83, QED 0.27
Compound (SMILES): O=C1c2cc(-c3ccc(-c4cn[nH]c4)cc3)ccc2Nc2ccc(F)cc2N1Cc1ccccc1
InChIKey: MZLBZYCTLBYVIC-UHFFFAOYSA-N
Target: Epidermal growth factor receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P00533
Assay: HTRF-Based EGFR Biochemical Assay: IC50 values were determined at both 1 μM and 1 mM ATP to identify both ATP competitive and non-competitive compounds. Hits were also counter-screened against wild type EGFR to evaluate the mutant selectivity. The HTRF-based screen was carried out using 1 μM ATP, and active compounds were counter-screened at 1 mM ATP and against wild type EGFR to identify those that were potentially non-ATP-competitive and mutant selective. This strategy identified several compounds of distinct chemical classes that were both selective for the L858R/T790M mutant over WT EGFR and relatively insensitive to ATP concentrations, suggesting an allosteric mechanism of action. HTRF-Based EGFR Biochemical Assays. EGFR biochemical assays were carried out using a homogeneous time-resolved fluorescence (HTRF) assay as described previously. The reaction mixtures contained 1 μM biotin-Lck-peptide substrate, wild type or mutant EGFR enzyme in reaction buffer (50 mM HEPES pH 7.1, 10 mM MgCl2, 0.01% BSA. 1 mM TCEP and 0.1 mM Na3VO4 at a final volume of 10 μL. Enzyme concentrations were adjusted to accommodate varying kinase activity and ATP concentrations (0.2-0.4 nM L858R/T790M; or 2-4 nM L858R, or 2-4 nM T790M, or 40 nM WT). All reactions were carried out at room temperature in white ProxiPlate 384-well Plus plates (PerkinElmer) and were quenched with 5 μL of 0.2 M EDTA at 60 min. Five μL per well of the detection reagent containing 2.5 ng PT66K (Cis-bio) and 0.05 μg SAXL (Prozyme) were added, and the plates were then incubated at room temperature for 1 hour and read with an EnVision plate reader.
Measurement: IC50 < 250 nM (pChEMBL 6.6020002365112305)
Descriptors: MW 460.51, ALogP 6.79, HBA 3, HBD 2, PSA 61.02, QED 0.31
Compound (SMILES): O=C(NN1CCN(C[C@@H](O)COc2ccc(Cl)c(F)c2)CC1)c1ccc2cc(Cl)ccc2n1
InChIKey: OOHNNNJXHAZGCG-QGZVFWFLSA-N
Target: Cyclic AMP-dependent transcription factor ATF-4 (SINGLE PROTEIN), Homo sapiens
UniProt: P18848
Assay: Expression Inhibition Assay: The ATF4 reporter was prepared by fusing the human full length 5′UTR of ATF4 (NCBI Accession No. BC022088.2) upstream of the firefly luciferase coding sequence lacking the initiator methionine. The fused sequence was cloned into pLenti-EF1a-C-Myc-DDK-IRES-Puro cloning vector (Origen #PS100085) using standard methods. Virus production was carried out by using Lenti-X Packaging Single Shots Protocol (Clonetech #631276). Viral particles were used to transduce HEK293T cells (ATCC #CRL-3216, ATCC Manassas, Va.), which were subsequently selected with puromycin to generate stable cell line. Cells were maintained at 37° C. and 5% CO2 in DMEM-F12 (Hyclone #SH30023.02) supplemented with 10% heat-inactivated fetal bovine serum (Gibco #16000-044), 2 mM L-glutamine (Gibco #25030-081), 100 U/ml penicillin, and 100 μg/ml streptomycin (Gibco #15140-122).
Measurement: IC50 = 5.7 nM (pChEMBL 8.239999771118164)
Descriptors: MW 493.37, ALogP 3.38, HBA 6, HBD 2, PSA 77.93, QED 0.53
Compound (SMILES): O=C(O)CC1CCC2(CC1)CCN(c1cc(Cl)cc(F)c1F)CC2
InChIKey: KHTOWAICSRWHBK-UHFFFAOYSA-N
Target: Free fatty acid receptor 4 (SINGLE PROTEIN), Homo sapiens
UniProt: Q5NUL3
Assay: FLIPR Assay: On the day before the measurement day, CHO cells expressing human GPR120 were plated at 10000 cells per well in a 384-well black plate (#3702, Corning) and incubated overnight in a CO2 incubator. On the day of the measurement, 4 μM Fluo-4 AM (fluorescence calcium indicator reagent) was incubated to be introduced into the human GPR120 expression CHO cells in the presence of 0.08% Pluronic F-127 in a CO2 incubator for 90 minutes. To the cells was added the test compound diluted with HBSS solution containing 20 mM HEPES and 2.5 mM probenecid. Variations in the intracellular calcium concentration were measured by Fluorescence Imaging Plate Reader (FLIPR; Molecular Devices) to examine the agonist action, and EC50 values were calculated.
Measurement: EC50 = 1799 nM (pChEMBL 5.75)
Descriptors: MW 357.83, ALogP 4.87, HBA 2, HBD 1, PSA 40.54, QED 0.78
Compound (SMILES): O=C(NCc1sc(C(F)(F)F)nc1-c1cccc(Cl)c1)Nc1ccc(OCCO)nc1
InChIKey: GMNHTDIOOCUTKE-UHFFFAOYSA-N
Target: Transient receptor potential cation channel subfamily V member 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q8NER1
Assay: FLIPR Assay: The FLIPR protocol consists of 2 substance additions during a kinetic measurement. First the compounds to be tested (5 μM) are pipetted onto the cells and the Ca2+ influx is determined in comparison to the control (capsaicin 10 μM), providing the result as % activation and representing the compound-alone effect (calculation at peak signal related to baseline). A Ca2+ influx of 10%-60% reveals a partial agonist (pAG), a Ca2+ influx of >60% relates to a pure agonist (AG). After 5 min incubation, the Ca2+ influx is related to an injection of 100 nM of capsaicin and thereby the antagonistic effect of the test compounds detected.
Measurement: Ki = 29.8 nM (pChEMBL 7.53000020980835)
Descriptors: MW 472.88, ALogP 4.57, HBA 6, HBD 3, PSA 96.37, QED 0.47
Compound (SMILES): CC(C)(COc1ncc(Nc2ncnc3c2CCN(c2ccc(C#N)c(C(F)(F)F)c2)C3)cc1Cl)n1cnnn1
InChIKey: RJEBOVKLRFOZCO-UHFFFAOYSA-N
Target: Androgen receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P10275
Assay: Antagonist Activity for AR: Antagonist activity for AR was evaluated according to the following method. COS-7 cells (ATCC) were transfected with pMMTV-luc vector (reporter plasmid having, as an androgen response element, murine mouse mammary virus long terminal repeat) and pEX-hAR vector (human androgen receptor expression vector: which expresses human AR gene under control of CMV promoter) by using Nucleofector (registered trademark) Kit R (Lonza) as a transfection reagent and Amaxa (Lonza). The COS-7 cells obtained after transfection were seeded in a clear bottom 96 well microplate (BD) at 1.5×104/well with phenol red free RPMI1640 containing 10% charcoal-treated fetal bovine serum (hereinbelow, DCC-FBS) (hereinbelow, the medium is referred to as an evaluation medium), and then cultured overnight. The culture was added with the evaluation medium containing dihydrotestosterone (DHT) (final concentration of DHT: 1 nmol/L) or the evaluation medium containing the compound of Examples or the compound of Comparative Examples (final concentration of the compound of Examples or the compound of Comparative Examples: 5, 14, 41, 123, 370, 1111, 3333, or 10000 nmol/L), followed by culture for 24 hours. Then, the transcription activity value was measured. The transcription activity was measured by using Bright-Glo Luciferase Assay System (Promega). From the measured transcription activity, 50% transcription activity inhibition concentration (IC50 value) was calculated by logistic regression when the transcription activity value obtained by using 1 nmol/L DHT was 100% and the transcription activity value obtained by using the evaluation medium only was 0%.
Measurement: IC50 = 150 nM (pChEMBL 6.820000171661377)
Descriptors: MW 570.97, ALogP 4.52, HBA 11, HBD 1, PSA 130.56, QED 0.34
Compound (SMILES): Cn1ccc2c(=O)n(CCOc3ccc(Cl)cc3-c3cccc(C(=O)O)c3)cnc21
InChIKey: YNXQQPVFLPCNAO-UHFFFAOYSA-N
Target: Eukaryotic translation initiation factor 4E-binding protein 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q13541
Assay: Fluorescence Polarization Assay: eIF4E protein was pre-incubated with EDA-m7GDP-ATTO-550 in FPB at 2xconcentrations for 5 minutes prior to the addition of compounds. Compounds (100x) were prepared using 3-fold serial dilutions in 100% DMSO, and subsequently diluted 1:50 in FPB to produce 2x stocks. 50 ul of 2x eIF4E/EDA-m7GDP-ATTO-550 were transferred to the wells of a 96-well half-area black flat bottom polystyrene plate. 50 ul of 2x test compound were added, and binding reactions were allowed to equilibrate for 30 minutes at room temperature while protected from light. The fluorescent polarization signal was detected using a Victor 2 multi-label counter (Perkin Elmer) and the concentration necessary to achieve inhibition of eIF4E/EDA-m7GDP-ATTO-550 binding by 50% (IC50) was calculated using data from an 8-point compound dilution series.
Measurement: IC50 = 64300 nM (pChEMBL 4.190000057220459)
Descriptors: MW 423.86, ALogP 3.83, HBA 6, HBD 1, PSA 86.35, QED 0.51
Compound (SMILES): CN1CCN(c2ccc(Nc3cc(-c4ccc(OC5CCN(C)C5)c(C#N)c4)ncn3)nc2)CC1
InChIKey: NGRIQWBQAPFNJW-UHFFFAOYSA-N
Target: Serine/threonine-protein kinase TBK1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9UHD2
Assay: TBK Biochemical Assay: Test compounds were transferred into Labcyte polypropylene 384 well plates (P055-25) and diluted to 3 mM using DMSO. 3 mM test compounds were dispensed using Labcyte ECHO dose response module into Greiner 784075 plates (columns 3-12 and 13-22, 10 point 1:4) so that high concentration was 30 uM final. 100 uM of a reference compound (1 uM final high concentration). Backfilling was performed if necessary so that all wells contain 1% DMSO final:add 75 nl DMSO/well into columns 1, 2 and 24 using Labcyte Echo.add 75 nl 1.0 mM staurosporine/well into column 23 using Labcyte Echo (10 uM final)add 4.5 ul enzyme/well using multidrop dispenseradd 3 ul substrate/well using multidrop dispenserincubate at 25° C. in Heidolph incubator for 90 min.add 7.5 ul 2× stop buffer using multidrop dispenserread on labchip ez reader II using TBK1.jobRaw data files were opened in the Caliper LabChip Reviewer program (Version 3.0.265.0 SP2) and peak assignments were adjusted to reflect substrate first with the software's post-run analysis options. A spline-fit baseline was applied using the software's analysis algorithm.
Measurement: IC50 = 550 nM (pChEMBL 6.260000228881836)
Descriptors: MW 470.58, ALogP 2.99, HBA 9, HBD 1, PSA 93.44, QED 0.58
Compound (SMILES): COc1ccc(-c2ccccc2)cc1NS(=O)(=O)c1cc(Cl)c[nH]c1=O
InChIKey: UIWPCQJEQYYXPG-UHFFFAOYSA-N
Target: ATP-citrate synthase (SINGLE PROTEIN), Homo sapiens
UniProt: P53396
Assay: ADP-Glo Assay: Materials and Instruments Used in the ADP-Glo AssayVendor Cat No.Materials ACL Sino Biological 11769-H07BCoANa2 Sigma C3144Potassium Citrate Sigma 89306ATP Promega V915BADP-GloTM Kinase Assay Promega V9102kitHEPES Life Technologies 15630080MgCl2 Sigma M1028Brij 35 detergent Merck 203728DTT Sigma 646563DMSO MP 196055Optiplate-384, White 384- Perkin Elmer 6007290well384 dilution plate Corning 3657Topseal A Perkin Elmer E534196-well plate Nunc 249944InstrumentPlate reader Perkin Elmer Envision 2104Centrifuge Eppendorf 5810RCompounds of the present invention were evaluated in an ADP-GLO assay as follows:a) Dilute cpd 1:3 in succession in DMSO by hand for each cpds for 12 ptsb) Add 0.1 μL diluted cpd solution to assay plate, each dose with 2 replicatesc) Centrifuge 1000 RPM for 1 mind) Add 5 μL ACL working solution to 384-well assay plate, centrifuge 1000 RPM for 1 mine) Incubate at 25° C. for 15 minf) Add 5 μL substrate working solution to initiate reactiong) Final ACL reaction concentrations: 3 nM ACL, 15 μM ATP, 3 μM CoA, 300 μM Citrate, 0.01% Brij35, 4 mM DTT, 1% DMSO;h) Reference final conc: 30 uM starting Conc, 3× dilution, 11+0 points. Test cpd conc: 30/100 uM starting Conc, 3× dilution, 11+0 points.i) Incubate at 25° C. for 60 minj) Add 10 μL ADP Glo reagent, centrifuge 1000 RPM for 1 mink) Incubate at 25° C. for 40 minl) Add 20 μL kinase detection reagent, centrifuge 1000 RPM for 1 minm) Incubate at 25° C. for 40 minn) Read on Envision for US LUM as RLU
Measurement: IC50 = 10250 nM (pChEMBL 4.989999771118164)
Descriptors: MW 390.85, ALogP 3.5, HBA 4, HBD 2, PSA 88.26, QED 0.7
Compound (SMILES): CC1(C)CC(n2c(Nc3ccc(OC(F)(F)F)cc3)nc3cc(NS(=O)(=O)C4CC4)ccc32)CC(C)(C)C1
InChIKey: CSPSICBBJQBXNJ-UHFFFAOYSA-N
Target: Isocitrate dehydrogenase [NADP] cytoplasmic (SINGLE PROTEIN), Homo sapiens
UniProt: O75874
Assay: Biochemical Assay: mIDH1 catalyzes the NADPH-dependent reduction of alpha-ketoglutarate (α-KG) to (2R)-2-hydroxyglutarate (2-HG). NADPH consumption was measured by luminescent readout.The biochemical reactions were performed at 32° C. in 384-well plates using a reaction volume of 41 μl and the following assay buffer conditions: 50 mM Tris pH 7.5, 100 mM NaCl, 20 mM MgCl2, 0.05% BSA, 0.01% Brij, 1 μM NADPH, and 250 μM α-KG. The IDH1 R132H enzyme was used in a final concentration of 1.5 nM. Test compounds were used in a concentration range between 0.002 and 10 μM. The final DMSO concentration was 2.4%.The reaction was incubated for 30 minutes, then 40 μl of detection mix (0.75 μg/ml Luciferase, 0.02 U/ml Oxidoreductase, 4 μg/ml FMN, 2 μl/ml Decanal/Ethanol, 50 mM Tris pH 7.5, 0.5% Glycerin, 0.01% Tween-20, 0.05% BSA) was added. Luminescence was measured on a luminescent reader (10 seconds measuring time, 1 second integration period, 30% sensitivity). The decrease in luminescence is proportional to mIDH1 activity. IC50 values are determined by interpolation from plots of relative luminescence versus inhibitor concentration.
Measurement: IC50 = 900 nM (pChEMBL 6.050000190734863)
Descriptors: MW 550.65, ALogP 7.36, HBA 6, HBD 2, PSA 85.25, QED 0.32
Compound (SMILES): O=P(O)(O)CS(=O)(=O)C[C@H]1O[C@@H](n2ncc3c(NCc4ccc(F)cc4)nc(Cl)nc32)[C@H](O)[C@@H]1O
InChIKey: BSNOKTQNGFBRDU-VMUDFCTBSA-N
Target: 5'-nucleotidase (SINGLE PROTEIN), Homo sapiens
UniProt: P21589
Assay: Biochemical Assay: Assay Reaction ConditionsAssay Volume: 70 μlReaction Volume: 50 μlCD73: 0.3208 nMAMP: 15 μMAssay Buffer: 2 5 mM Tris-HCL, pH 7.4, 0.01% Brij-35, 0.01% BSA, 5 mM MgCl2Assay Procedure:Used 384 clear plate.Made dose titration of testing compounds in assay buffer, 10 points ½ log titrations in duplicates starting at 100 μM.Added 25 μl of CD73 to each well for a final concentration of 320 pM.Incubated at RT for 15 min.Added 25 μl of AMP to each well for a final concentration of 15 μM.Incubated at RT for 10 min.Added 10 μl of Malachite Green Reagent A, incubate at RT for 10 min.Added 10 μl of Malachite Green Reagent B, incubate at RT for 45 min.Read the Absorbance on Envision plate reader using excitation filter: Cy5 620 nM.
Measurement: IC50 < 500 nM (pChEMBL 6.301000118255615)
Descriptors: MW 551.88, ALogP 0.4, HBA 11, HBD 5, PSA 196.99, QED 0.19
Compound (SMILES): Cn1cccc(-c2ccc(S(=O)(=O)c3c(O)n([C@H](c4cccc(C#N)c4)C4CC4)c(CC4CC4)nc3=O)cc2)c1=O
InChIKey: KJFUBASYSUYJBB-MHZLTWQESA-N
Target: Apelin receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P35414
Assay: Intracellular cAMP Accumulation Assay: HEK293 cells stably expressing human APJ receptor were used to assess the activity of compounds. Cultured cells were detached and resuspended in the cAMP Homogeneous Time-Resolved Fluorescence (HTRF) assay buffer (Cisbio cat; #62AM4PEJ). The assay was performed in 384-well assay plates (Perkin-Elmer; cat #6008289) according to assay protocol provided by the manufacturer. Serial dilutions of a compound together with assay buffer containing 0.2 nM IBMX and 2 μM forskolin were added to each well containing 5,000 cells and incubated for 30 minutes at room temperature. Subsequently, cAMP D2 reagent was added in the lysis buffer followed by the EuK antibody (Cisbio; cat #62AM4PEJ) and incubated for 60 min. The fluorescence emission ratio was measured using fluorometer. The intracellular cAMP concentrations (compound-stimulated inhibition of forskolin-mediated cAMP production) were calculated by extrapolation from a standard curve using known cAMP concentrations. The EC50 values were obtained by fitting the data to a sigmoidal concentration-response curve with variable slope. The maximal achievable inhibition of forskolin-induced cAMP levels (Ymax) for each compound was expressed as relative percentage of inhibition attained using pyroglutamated apelin-13 ((Pyrl)apelin-13) peptide, which was set to 100%.
Measurement: EC50 = 5 nM (pChEMBL 8.300000190734863)
Descriptors: MW 568.66, ALogP 3.97, HBA 9, HBD 1, PSA 135.05, QED 0.34
Compound (SMILES): Cc1nnc(N[C@H](C)c2cccc(C(F)(F)F)c2F)c2cc(N3CC(C)(O)C3)ncc12
InChIKey: OSYVPYYNQLACCE-LLVKDONJSA-N
Target: Son of sevenless homolog 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q07889
Assay: Inhibition of Labeled Tracer Ligand: Table 22: This Example illustrates that exemplary compounds of the present invention bind to SOS1 and prevent a labeled tracer ligand from occupying the S0S1 binding site. The ability of a compound of Formula (I) to bind to SOS1 was measured using a HTRF displacement assay. A recombinant human SOS1 polypeptide (corresponding to amino acids 560-1049, expressed in E. Coli with N-terminal His-TEV-AviTag-SOS1 (MW=59.4 kDa) and lanthanide labeled streptavidin (CisBio) was incubated with an exemplary compound of Formula (I) (in a DMSO stock solution) in buffer (25 mM HEPES pH 7.5, 25 mM NaCl, 1 mM DTT, 0.01% Brij 35, 0.02% BSA, 0.1% DMSO). After a 10-15 minute incubation at room temperature, a solution comprised of a custom-made Cy5 labelled tracer and MAb Anti-6HIS Tb cryptate Gold (Cisbio 61HI2TLA) in buffer was added to the solution containing the SOS1 polypeptide and exemplary compound of Formula (I). After a 1-hour incubation at room temperature, the HTRF signal was measured using Clairostar plate reader (BMG Labtech) according to the manufacturer's instructions. Excitation filter EX-TR was used, and emission 1 was detected at 650-610 nm and emission 2 detected at 620-610 nm. The HTRF ratio was calculated using the formula: [emission 1/emission 2]*10000.
Measurement: Ki = 1.68 nM (pChEMBL 8.779999732971191)
Descriptors: MW 435.43, ALogP 4.24, HBA 6, HBD 2, PSA 74.17, QED 0.6
Compound (SMILES): CN1CCc2cc(/C=C/c3n[nH]c4cc([C@@H]5C[C@@]56C(=O)Nc5ccccc56)ccc34)ccc2C1
InChIKey: ZLSDSWNCEJQZGL-VRETXUBVSA-N
Target: Serine/threonine-protein kinase PLK4 (SINGLE PROTEIN), Homo sapiens
UniProt: O00444
Assay: Inhibition Assay: PLK4 activity was measured using an indirect ELISA detection system. Dephosphorylated GST-PLK4 (4 nM) was incubated in the presence of 15 μM ATP (Sigma cat#A7699), 50 mM HEPES-Na2+ pH 7.4, 10 mM MgCl2, 0.01% Brij 35 (Sigma cat#03-3170), in a 96 well microtitre plate pre-coated with MBP (Millipore cat#30-011). The reaction was allowed to proceed for 30 minutes, followed by 5 washes of the plate with Wash Buffer (50 mM TRIS-C1 pH 7.4 and 0.2% Tween 20), and incubation for 30 minutes with a 1:3000 dilution of primary antibody (Cell Signaling cat#9381). The plate was washed 5 times with Wash Buffer, incubated for 30 minutes in the presence of secondary antibody coupled to horse radish peroxidase (BioRad cat#1721019, 1:3000 concentration), washed an additional 5 times with Wash Buffer, and incubated in the presence of TMB substrate (Sigma cat#T0440). The colourimetric reaction was allowed to continue for 5 minutes, followed by addition of stop solution (0.5 N sulphuric acid), and quantified by detection at 450 nm with either a monochromatic or filter based plate reader (Molecular Devices M5 or Beckman DTX880, respectively).
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 446.55, ALogP 5.1, HBA 3, HBD 2, PSA 61.02, QED 0.46
Compound (SMILES): CN(C)[C@H]1C[C@@H]2[C@H](C[C@@H]1Nc1cc(F)c(S(=O)(=O)Nc3nccs3)cc1Cl)C2(F)F
InChIKey: RTIMNYPBTWEYOC-OWLDWWDNSA-N
Target: Sodium channel protein type 9 subunit alpha (SINGLE PROTEIN), Homo sapiens
UniProt: Q15858
Assay: Electrophysiological Assay (EP) (In Vitro Assay): Patch voltage clamp electrophysiology allows for the direct measurement and quantification of block of voltage-gated sodium channels (NaV's), and allows the determination of the time- and voltage-dependence of block which has been interpreted as differential binding to the resting, open, and inactivated states of the sodium channel (Hille, B., Journal of General Physiology (1977), 69: 497-515).The following voltage clamp electrophysiology studies were performed on representative compounds using cells heterologously expressing Nav1.7 or Nav1.5 channels. cDNAs for Nav1.7 (NM_002977) and Nav1.5 (AC137587) were stably expressed in Chinese Hamstr Ovary (CHO) cells and CHL (Chinese Hamster Lung) cells respectively. Sodium currents were measured in the whole-cell configuration using Syncropatch 384PE (NanIon Technologies, Germany). 1NPC®-384 chips with custom medium resistance and single hole mode are used. Internal solution consists of (in mM): 110 CsCl, 10 CsCl, 20 EGTA, and 10 Hepes (pH adjusted to 7.2); and external solution contains (in mM): 60 NMDG, 80 NaCl, 4 KCl, 1 MgCl2, 2 CaCl2, 2 D-Glucose monohydrate, 10 Hepes (pH adjusted to 7.4 with NaOH).After system flushing, testing compounds are dissolved in external solution containing 0.1% Pluronic F-127. The chip is moved into the measuring head and the instrument primes the chip with external and internal solutions. 10l cells are added to the chip from a cell hotel, and a negative pressure of −50 mBar is applied to form a seal. Following treatment with seal enhancer solution and wash-off with external solution, negative pressure of −250 mbar is applied for 1 second to achieve the whole-cell configuration, followed by three washing steps in external solution. 20 μl of compounds is added to 40 μl in each well (1:3 dilution of compounds), and after mixing, 20 μl is removed so the volume is retained at 40 μl. After approximately 13 minutes recordings, 20 μl/well of 2 uM TTX, or 333 uM Tetracaine (for Nav1.5) is added to achieve full block.For voltage protocol, an holding potential of −50 mV is applied during the whole experiment. A depolarizing step is applied to −10 mV for 10 ms, followed by a hyperpolarization step to −150 mV for 20 ms to allow channel recovery from inactivation. A second depolarizing step is applied from −150 mV to −10 mV for 10 ms, where currents were measured to derive blocking effects of compounds. Inhibition is determined based on 7.5 min of compound incubation.
Measurement: IC50 = 9.25 nM (pChEMBL 8.029999732971191)
Descriptors: MW 480.97, ALogP 4.12, HBA 6, HBD 2, PSA 74.33, QED 0.65
Compound (SMILES): COc1ncccc1-c1nnc(NS(=O)(=O)[C@@H](C)[C@H](C)c2ncc(F)cn2)n1-c1cn(C)c2ccccc12
InChIKey: OOXBJRSZWJAZON-HOTGVXAUSA-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 = 45 nM (pChEMBL 7.349999904632568)
Descriptors: MW 536.59, ALogP 3.69, HBA 10, HBD 1, PSA 129.71, QED 0.32