lovingscience/chembl-v37-raw-activities huggingface dataset:
Compound (SMILES): O=c1c2cccnc2nc2n1-c1ccc([N+](=O)[O-])cc1C2=NOCCO
InChIKey: IEPIPMXUKONYJP-UHFFFAOYSA-N
Target: Indoleamine 2,3-dioxygenase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P14902
Assay: Indoleamine-2,3-Dioxygenase 1 Inhibitory Activity: Experimental method: IDO-1 can oxidatively cleave the indole ring of tryptophan to form N-formylkynurenine. Referring to the method in the literature (Eddy W. Yue, et al. J. Med. Chem. 2009, 52, 7364-7367), 20 nM IDO-1, 2 mM D-tryptophan, 20 mM ascorbic acid, 3.5M methyl blue and 0.2 mg/mL antioxidant enzyme were sequentially added to 50 mM potassium phosphate buffer at room temperature. Due to the formation of N-formylkynurenine, the reaction rate was recorded by the increasing absorbance of the solution at 321 nm. IC50 values were calculated using Prism GraphPad software.
Measurement: IC50 = 550 nM (pChEMBL 6.260000228881836)
Descriptors: MW 353.29, ALogP 0.76, HBA 9, HBD 1, PSA 132.74, QED 0.33
Compound (SMILES): Cn1cc(-c2cc(-c3ccc(N4CC(CC(=O)O)C4)nc3)c3c(C#N)cnn3c2)cn1
InChIKey: HAMNMZPSZGMPMK-UHFFFAOYSA-N
Target: Proto-oncogene tyrosine-protein kinase receptor Ret (SINGLE PROTEIN), Homo sapiens
UniProt: P07949
Assay: RET Enzyme Assay: Compounds of General Formula I were screened for their ability to inhibit wild type and V804M mutant RET kinase using CisBio's HTRF® KinEASE -TK assay technology. Briefly, N-terminal GST tagged recombinant human RET cytoplasmic domain (aa 658-end) from Eurofins (0.25 nM RET; Cat. No. 14-570M) or N-terminal GST tagged recombinant human V804M mutant RET cytoplasmic domain (aa 658-end) from Millipore (0.25 nM enzyme; Cat. No. 14-760) was incubated with 250 nM TK-substrate biotin (CisBio, part of Cat. No. 62TK0PEC) and 1 mM ATP along with test compound in a buffer consisting of 25 mM HEPES pH 7.4, 10 mM MgCl2, 0.01% Triton X-100, and 2% DMSO in a volume of 8 μL. Compounds were typically prepared in a threefold serial dilution in DMSO and added to the assay to give the appropriate final concentration. After a 30-minute incubation at 22° C., the reaction was quenched by adding 8 μL of quench solution containing 31.25 nM Sa-XL665 and 1×TK-ab-Cryptate in HTRF detection buffer (all from CisBio, part of Cat. No. 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. 100 POC was determined using no test compounds and 0 POC was determined using pre-quenched control reactions. The POC values were fit to a 4 parameter logistic curve, and the IC50 is defined as the concentration of inhibitor at which the POC equals 50 for the fitted curve.
Measurement: IC50 = 498 nM (pChEMBL 6.300000190734863)
Descriptors: MW 413.44, ALogP 2.58, HBA 8, HBD 1, PSA 112.34, QED 0.53
Compound (SMILES): Cc1c(C[C@@H](CNC(=O)C[C@@H](c2cncc(F)c2)C(C)(C)C)N(C)C)ccc(C(N)=O)c1F
InChIKey: ABFZQEVJKKGYFO-FPOVZHCZSA-N
Target: Mu-type opioid receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P35372
Assay: MOR cAMP Agonist and Antagonist Assays: CHO Tag-lite human mOR stable cell line from Cisbio (NCBI accession number: NM_000914.3, Bedford, Mass.) were seeded and grown to approximately 80% confluence in Ham F-12 with 10% FBS, 50 U/ml penicillin, 50 μg/ml streptomycin, 2 mM Hepes, and 1 mg/ml geneticin (Invitrogen, Carlsbad, Calif.). Cells were then harvested using accutase (Corning. Coming, N.Y.), centrifuged at 1300 RPM for 5 min, and plated at 5000 cells per 5 μL per well in a 5× dilution of stimulation buffer consisting of the HTRF cAMP Gi kit, water and IBMX at 0.5 mM (Cisbio, Bedford, Mass.) in white HTRF low volume 384 well plates (Cisbio, Bedford, Mass.). Plates were then incubated at 37° C. in 5% CO2 for 10 minutes. For the agonist assay, forskolin was added to a final concentration of 4 μM. For the antagonist assay, forskolin (4 μM) and DAMGO at EC90 final concentration were added. Test compounds were dissolved in DMSO and water, and then serially diluted to working concentrations such that the concentration of DMSO was less than 0.1%. Diluted test compounds were added at 2.5 μL per well, and plates were incubated at 37° C., and 5% CO2 for 15 minutes, and then at room temperature for 15 minutes. Next 5 μl/well of cAMP Eu-cryptate and 5 μl/well of anti-cAMP-d2 (both diluted 1:20 in lysis buffer) were added, and plates were incubated at room temperature for 1 hour. Following incubation, plates were read in a Synergy Neo2 multi-mode reader (Biotek, Winooski, Vt.). Plate reader settings were set to time resolved fluorescence with excitation at 330 nm and emissions of 620 nm and 665 nm. Emission fluorescence was normalized (665/620 nm signal×1000). For the agonist assay, data was normalized using the maximal DAMGO response. Measurements were performed in triplicate and the dose response was fit using nonlinear regression.
Measurement: IC50 > 3000 nM (pChEMBL 5.5229997634887695)
Descriptors: MW 460.57, ALogP 3.58, HBA 4, HBD 2, PSA 88.32, QED 0.6
Compound (SMILES): C[C@@]1(c2cc(Nc3ccnc4cc(Cl)cnc34)ncc2F)CS(=O)(=O)[C@@](C)(C2CC2)C(N)=N1
InChIKey: FCXFMBYBPKCVQJ-VXKWHMMOSA-N
Target: Beta-secretase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P56817
Assay: HTRF FRET Assay: The compounds of the invention were determined to be potent inhibitors of BACE-1 using the following assay.The following reagents were used in this assay. Na+-Acetate pH 5.0; 1% Brij-35; Glycerol; Dimethyl Sulfoxide (DMSO); Recombinant human soluble BACE-1 catalytic domain (>95% pure); APP Swedish mutant peptide substrate (QSY7-APPswe-Eu): QSY7-EISEVNLDAEFC-Europium-amide.A homogeneous time-resolved FRET assay can be used to determine IC50 values for inhibitors of the soluble human BACE-1 catalytic domain. This assay monitors the increase of 620 nm fluorescence that resulted from BACE-1 cleavage of an APPswedish APPswe mutant peptide FRET substrate (QSY7-EISEVNLDAEFC-Europium-amide). This substrate contains an N-terminal QSY7 moiety that serves as a quencher of the C-terminal Europium fluorophore (620 nm Em). In the absence of enzyme activity, 620 nm fluorescence is low in the assay and increased linearly over 3 hours in the presence of uninhibited BACE-1 enzyme. Inhibition of BACE-1 cleavage of the QSY7-APPswe-Eu substrate by inhibitors is manifested as a suppression of 620 nm fluorescence.
Measurement: Ki = 6.2 nM (pChEMBL 8.210000038146973)
Descriptors: MW 488.98, ALogP 3.73, HBA 8, HBD 2, PSA 123.22, QED 0.57
Compound (SMILES): CO[C@@H]1C[C@@H](c2nc3ccc(Cl)c(C)c3[nH]2)N(C(=O)c2c(-n3nccn3)ccc(C)c2F)C1
InChIKey: OHDRBNXNNCSTBA-KDOFPFPSSA-N
Target: Orexin/Hypocretin receptor type 1 (SINGLE PROTEIN), Homo sapiens
UniProt: O43613
Assay: Inhibition Assay: Chinese hamster ovary (CHO) cells expressing the human orexin-1 receptor and the human orexin-2 receptor, respectively, are grown in culture medium (Ham F-12 with L-Glutamine) containing 300 μg/ml G418, 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 384-well black clear bottom sterile plates (Greiner). The seeded plates are incubated overnight at 37° C. in 5% CO2.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 μl of staining buffer (HBSS containing 1% FCS, 20 mM HEPES, NaHCO3: 0.375 g/l, 5 mM probenecid (Sigma) and 3 M 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.
Measurement: IC50 = 16 nM (pChEMBL 7.800000190734863)
Descriptors: MW 468.92, ALogP 4.16, HBA 6, HBD 1, PSA 88.93, QED 0.49
Compound (SMILES): COc1cc2c(cc1-c1c(C)noc1C)[nH]c1nc(C)nc(-c3ccc(C(=O)NC4CCN(C5COC5)CC4)c4ccccc34)c12
InChIKey: NLHZWVYLQRZHDF-UHFFFAOYSA-N
Target: Bromodomain-containing protein 2 (SINGLE PROTEIN), Homo sapiens
UniProt: P25440
Assay: FP Competitive Binding Assay (No.350): Fluorescence Polarization (FP) competitive binding studies (see above) were carried out using the FAM labeled fluorescent probe Cpd. No. 350 to determine binding affinities of representative compounds to both BD1 and BD2 of BRD2, BRD3, and BRD4 proteins. Equilibrium dissociation constants (Id) values of Cpd. No. 350 to these six proteins were determined from protein saturation experiments by monitoring the total fluorescence polarization of mixtures composed with the fluorescent probe at a fixed concentration and proteins with increasing concentrations up to full saturation. Serial dilutions of testing protein were mixed with Cpd. No. 350 to a final volume of 200 μl in the assay buffer. In order to achieve large dynamic rages, particularly for BD1 bromodomains, 100 mM phosphate buffer (pH=6.5, 0.01% Triton X-100 (Sigma, 282103) being added right before assays) was used as the assay buffer. Final Cpd. No. 350 concentration was 1.5 nM for all proteins. Plates were incubated at room temperature for 30 minutes with gentle shaking to assure equilibrium. FP values in millipolarization units (mP) were measured using the Infinite M-1000 plate reader (Tecan U.S., Research Triangle Park, N.C.) in Microfluor 1 96-well, black, round-bottom plates (Thermo Scientific, Waltham, Mass.) at an excitation wavelength of 485 nm and an emission wavelength of 530 nm. Kd values of Cpd. No. 350, which were calculated by fitting the sigmoidal dose-dependent FP increases as a function of protein concentrations using Graphpad Prism 6.0 software (Graphpad Software, San Diego, Calif.), are 2.0, 2.2, 6.5, 0.6, 5.5, and 3.0 nM to BRD2 BD1 and 2, BRD3 BD1 and 2, and BDR4 BD1 and 2, respectively.
Measurement: Ki = 6.2 nM (pChEMBL 8.210000038146973)
Descriptors: MW 616.72, ALogP 6.11, HBA 8, HBD 2, PSA 118.4, QED 0.23
Compound (SMILES): Cc1c(C(=O)CN2C3CCC2CC3)cc(C#CCCC#N)n1-c1ccc(C#N)cc1
InChIKey: ZWVCJEUZTWICPB-UHFFFAOYSA-N
Target: Ubiquitin carboxyl-terminal hydrolase 14 (SINGLE PROTEIN), Homo sapiens
UniProt: P54578
Assay: Usp14 Inhibition Assay: Usp14 Inhibition Assay was described in B. H. Lee et al. Nature 2010, 467 (9), 179.
Measurement: IC50 < 50 nM (pChEMBL 7.301000118255615)
Descriptors: MW 396.49, ALogP 4.12, HBA 5, HBD 0, PSA 72.82, QED 0.43
Compound (SMILES): CC(=O)Nc1ccc(S(=O)(=O)N2CCN=C2N2CCN(C3CCC3)CC2)cc1Cl
InChIKey: RWJVIFMHWXPOFD-UHFFFAOYSA-N
Target: Lysine-specific demethylase 2B (SINGLE PROTEIN), Homo sapiens
UniProt: Q8NHM5
Assay: KDM2B TR-FRET Assay: Full length KDM2B was cloned, expressed, and purified to homogeneity. Compound inhibition of KDM2B demethylase activity was assessed by monitoring the methylation status of a biotin-H3K36me2 peptide substrate (H2N-RKSAPATGGV(KMe2)KPHRYRPGTV-NTPEGBiot; New England Peptide) in the presence of α-keotglutarate (2-OG) and iron (Fe2+) using the TR-FRET assay technology (Cisbio). Specifically, in a 384 well ProxiPlate KDM2B (5 mM final), ascorbate (500 μM final) and DTT (2 mM final) were combined with the biotin-H3K36me2 peptide substrate (200 nM final), 2-OG (0.3 μM or 6 μM final; Sigma K2010) and Fe2+ (100 μM final; Sigma F1543) in 50 mM HEPES (pH 6.5) and 0.01% Triton-X 100 either in the presence of DMSO (final 0.25% DMSO) or compound dilution series in DMSO and mixed. After a two hour incubation at room temperature, a mixture of EU-anti-H3K36mel antibody (2 nM final; Cisbio #64CUSKAZ), and Streptavidin-d2 (50 nM final; Cisbio #64CUS000) in 200 mM KF, 200 mM EDTA, 0.1% BSA and 50 mM HEPES (pH 6.5) was added. After 1 hour incubation, the plates were read on an Envision instrument, the readouts were transformed into % inhibition, and IC50 values were generated using a four parameter logistic model (XLFIT5). The KDM2B TR-FRET Assay described above represents an additional embodiment of the invention.
Measurement: IC50 = 0.127 nM (pChEMBL 9.899999618530273)
Descriptors: MW 439.97, ALogP 1.83, HBA 6, HBD 1, PSA 85.32, QED 0.77
Compound (SMILES): C[Si](C)(O)CCCNc1nonc1/C(=N/O)N[C@H]1Cc2ccc(F)cc21
InChIKey: UJPMXSCKNNTKOT-ZDUSSCGKSA-N
Target: Indoleamine 2,3-dioxygenase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P14902
Assay: Enzyme Assay: HIS-tagged IDO1 protein was recombinantly expressed in Escherichia coli using ZYP5052 autoinduction media supplemented with 500 μM delta aminolevulinic acid for 48 hours at 16 degrees Celsius. IDO1 protein was purified using Ni2+-affinity resin and size exclusion chromatography. Purified protein was then diluted in assay buffer (50 mM Tris pH 7.0, 1% glycerol, 20 μM methylene blue, 0.05% Tween-20, 20 mM sodium ascorbate, 100 units/mL catalase to obtain a final IDO1 concentration of 40 nM. IDO1 solution (30 μM) or buffer alone (30 μM) were dispensed to wells of the assay plate using a BioRAPTR liquid dispenser (Beckman Coulter). Assay plates containing compound and IDO1 enzyme were incubated at room temperature for 30 minutes. Afterwards, 10 μL of 400 μM tryptophan in assay buffer were added to each well of the assay plate using a BioRAPTR liquid dispenser. Plates were incubated at room temperature for 60 minutes and reactions were quenched by addition of 10 μL of 0.5 M methyl isonipecotate in dimethyl sulfoxide. Plates were sealed and incubated at 37° C. for 4 hours or 50° C. for 2 h. The plates are allowed to cool and then centrifuged for 1 minute at 1000×g. The resulting fluoresence was measured in an Envision plate reader (Perkin Elmer) with a 400/25 nm excitation filter and an 510/20 nm emission filter.
Measurement: IC50 = 900 nM (pChEMBL 6.050000190734863)
Descriptors: MW 379.47
Compound (SMILES): O=C(NO)C(Cc1ccccc1)n1cc2cccc(CN3CCOCC3)c2n1
InChIKey: LXODKNHJOXPHBB-UHFFFAOYSA-N
Target: Histone deacetylase 8 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9BY41
Assay: Enzymatic Assay: HDAC-Glo I/II Assay Kit was purchased from Promega Corporation (Madison, Wis.), including the following components: HDAC-Glo I/II Buffer (25 mM Tris buffer, pH 8.0, supplemented with 137 mM: NaCl, 2.7 mM KCl, 1% v/v Triton X-100 and 1 mM: MgCl2), luminogenic substrates Boc-GAK(Ac)-aminoluciferin (HDAC-Glo I/II substrate), proprietary developer reagent (containing trypsin). Assay:The HDAC8 reaction was performed at room temperature in white 384-well polystyrene, flat-bottom microtiter plate (Greiner Bio-one, Monroe, N.C.) in a final volume of 20 μL. Test compounds were first diluted serially in DMSO and 100 nL were transferred to the plate wells by Echo 550 (Labcyte, Sunnyvale, Calif.) before the addition of other reaction components. The final concentration of DMSO in the assay was 0.5%. The HDAC-Glo I/II assay reagent was prepared by rehydration of lyophilized HDAC-Glo I/II substrate in 10 mL. HDAC-Glo I/II assay buffer followed with the addition of 10 μL of developer reagent. 10 uL of 3 nM HDAC8 in the HDAC-Glo I/II assay buffer were first dispensed into the microliter plate containing the compounds and incubated at room temperature for 30 min. The reactions were initiated with the addition of 10 uL HDAC-Glo I/II assay reagent with Multidrop.
Measurement: IC50 = 375 nM (pChEMBL 6.429999828338623)
Descriptors: MW 380.45, ALogP 2.16, HBA 6, HBD 2, PSA 79.62, QED 0.51
Compound (SMILES): Nc1cc(Sc2ncc(N3CCC4(CC3)C[C@H](O)C[C@H]4N)nc2N)c(Cl)c(Cl)n1
InChIKey: VPAOHNZMBRWXLI-MWLCHTKSSA-N
Target: Tyrosine-protein phosphatase non-receptor type 11 (SINGLE PROTEIN), Homo sapiens
UniProt: Q06124
Assay: SHP2 Allosteric Inhibition Assay: SHP2 is allosterically activated through binding of bis-tyrosyl-phorphorylated peptides to its Src Homology 2 (SH2) domains. The latter activation step leads to the release of the auto-inhibitory interface of SHP2, which in turn renders the SHP2 protein tyrosine phosphatase (PTP) active and available for substrate recognition and reaction catalysis. The catalytic activity of SHP2 was monitored using the surrogate substrate DiFMUP in a prompt fluorescence assay format.More specifically, the phosphatase reactions were performed at room temperature in 384-well black polystyrene plate, flat bottom, low flange, non-binding surface (Corning, Cat#3575) using a final reaction volume of 25 μL and the following assay buffer conditions: 60 mM HEPES, pH 7.2, 75 mM NaCl, 75 mM KCl, 1 mM EDTA, 0.05% P-20, 5 mM DTT.The inhibition of SHP2 by compounds of the invention (concentrations varying from 0.003-100 μM) was monitored using an assay in which 0.5 nM of SHP2 was incubated with of 0.5 μM of peptide IRS1_pY1172(dPEG8)pY1222 (sequence: H2N-LN(pY)IDLDLV(dPEG8)LST(pY)ASINFQK-amide) (SEQ ID NO:1). After 30-60 minutes incubation at 25° C., the surrogate substrate DiFMUP (Invitrogen, cat# D6567) was added to the reaction and incubated at 25° C. for 30 minutes. The reaction was then quenched by the addition of 5 μl of a 160 μM solution of bpV(Phen) (Enzo Life Sciences cat# ALX-270-204). The fluorescence signal was monitored using a microplate reader (Envision, Perki-Elmer) using excitation and emission wavelengths of 340 nm and 450 nm, respectively. The inhibitor dose response curves were analyzed using normalized IC50 regression curve fitting with control based normalization.
Measurement: IC50 = 4 nM (pChEMBL 8.399999618530273)
Descriptors: MW 456.4, ALogP 2.56, HBA 9, HBD 4, PSA 140.2, QED 0.51
Compound (SMILES): CCCCN1CCC(CCC(=O)c2cc(I)c(N)cc2OC)CC1
InChIKey: AEHUGLUJLXQXMZ-UHFFFAOYSA-N
Target: 5-hydroxytryptamine receptor 4 (SINGLE PROTEIN), Homo sapiens
UniProt: Q13639
Assay: Competition Assay: Prior to these competition studies, a series of saturation curves were performed in order to check whether the pharmacological parameters Kd, Bmax and the Hill Coefficient of [3H]-GR113808, obtained in our experimental conditions, are consistent with those published in the literature. For that purpose, 7.5 μg/μL membrane preparation samples are incubated in duplicate (50 mM HEPES buffer, pH 7.4, +37° C.) at +37° C. for 30 minutes, in the presence of 7 increasing concentrations (0.05-1.5 nM, 200 μl of final volume) of [3H]-GR113808 (Grossman et al., 1993). Using a Brandel Cell Harvester, the binding of the radioligand with the proteins is interrupted by rapid filtration of the incubation medium through a filter strip (WHATMAN GF/B FP-100) pre-incubated in polyethylenimine (PEI 0.5% in water); filtration is followed by 3 rinses with 4 ml of 50 mM HEPES buffer, pH 7.4, at +4° C. The non-specific binding of [3H]-GR113808 is quantified in the presence of 30 μM of serotonin, specific binding thus being estimated as the difference: (binding in the absence of serotonin)−(binding in the presence of serotonin).
Measurement: Ki = 44.7 nM (pChEMBL 7.349999904632568)
Descriptors: MW 444.36, ALogP 4.36, HBA 4, HBD 1, PSA 55.56, QED 0.37
Compound (SMILES): CC(C)C[C@@H](N)c1nc2cc(-c3cc(F)nc(F)c3)ccc2[nH]1
InChIKey: NYGIVTZBRSIGJP-GFCCVEGCSA-N
Target: AP2-associated protein kinase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q2M2I8
Assay: AAK1 Kinase Assay: The assays were performed in U-bottom 384-well plates. The final assay volume was 30 μl prepared from 15 μl additions of enzyme and substrates (fluoresceinated peptide (5-FAM)-Aha-KEEQSQITSQVTGQIGWR-NH2 and ATP) and test compounds in assay buffer (10 mM Tris-HCL pH 7.4, 10 mM MgCl2, 0.01% Tween-20 and 1.0 mM DTT). The reactions were initiated by the combination of bacterially expressed, GST-Xa-hAAK1 with substrates and test compounds. The reactions were incubated at room temperature for 3 hours and terminated by adding 60 μl of 35 mM EDTA buffer to each sample. The reactions were analyzed on the Caliper LabChip 3000 (Caliper, Hopkinton, Mass.) by electrophoretic separation of the fluorescent substrate and phosphorylated product. Inhibition data were calculated by comparison to EDTA quenched control reactions for 100% inhibition and vehicle-only reactions for 0% inhibition. The final concentration of reagents in the assays are ATP, 22 μM; (5-FAM)-Aha-KEEQSQITSQVTGQIGWR-NH2, 1.5 μM; GST-Xa-hAAK1, 3.5 nM; and DMSO, 1.6%. 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 = 3940 nM (pChEMBL 5.409999847412109)
Descriptors: MW 316.36, ALogP 3.95, HBA 3, HBD 2, PSA 67.59, QED 0.72
Compound (SMILES): CCCOc1cnc(/C(F)=C/c2ccc(F)c([C@@]3(C)N=C(N)[C@@]4(CF)CC[C@@H]3S4(=O)=O)c2)cn1
InChIKey: HFDHAAAQGHEMJE-NKUXOPIZSA-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 = 86 nM (pChEMBL 7.070000171661377)
Descriptors: MW 494.54, ALogP 3.74, HBA 7, HBD 1, PSA 107.53, QED 0.63
Compound (SMILES): CC(C)(O)COc1cc(-c2ccc(N3CCC(C)(NC(=O)c4cccc(Cl)c4)CC3)nc2)c2c(C#N)cnn2c1
InChIKey: KWSSCMRTZNTEIG-UHFFFAOYSA-N
Target: Proto-oncogene tyrosine-protein kinase receptor Ret (SINGLE PROTEIN), Homo sapiens
UniProt: P07949
Assay: RET Enzyme Assay: Compounds of Formula I were screened for their ability to inhibit wildtype and V804M mutant RET kinase using CisBio's HTRF KinEASE-TK assay technology. Briefly, N-terminal GST tagged recombinant human RET cytoplasmic domain (aa 658-end) from Eurofins (0.25 nM RET; Catalog No. 14-570M) or N-terminal GST tagged recombinant human V804M mutant RET cytoplasmic domain (aa 658-end) from Millipore (0.25 nM enzyme; Catalog No. 14-760) was incubated with 250 nM TK-substrate biotin (CisBio, part of Catalog No. 62TK0PEC) and 1 mM ATP along with test compound in a buffer consisting of 25 mM HEPES pH 7.4, 10 mM MgCl2, 0.01% Triton X-100, and 2% DMSO in a volume of 8 μL. Compounds were typically prepared in a threefold serial dilution in DMSO and added to the assay to give the appropriate final concentration. After a 30-minute incubation at 22° C., the reaction was quenched by adding 8 μL of quench solution containing 31.25 nM Sa-XL665 and 1×TK-ab-Cryptate in HTRF detection buffer (all from CisBio, part of Cat. No. 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. 100 POC was determined using no test compounds and 0 POC was determined using pre-quenched control reactions. The POC values were fit to a 4 parameter logistic curve, and the IC50 is defined as the concentration of inhibitor at which the POC equals 50 for the fitted curve.
Measurement: IC50 = 16.7 nM (pChEMBL 7.78000020980835)
Descriptors: MW 559.07, ALogP 4.86, HBA 8, HBD 2, PSA 115.78, QED 0.33
Compound (SMILES): O=C(N[C@@H]1CCCc2cc(C(F)(F)F)ccc21)c1cc(=O)[nH]c(-c2ncccn2)n1
InChIKey: NDVDOANIORNTMZ-CQSZACIVSA-N
Target: GMP-stimulated 3',5'-cyclic nucleotide phosphodiesterase (SINGLE PROTEIN), Macaca mulatta
UniProt: B3VTJ2
Assay: Fluorescence Polarization (FP) Assay: In a typical experiment the PDE2 inhibitory activity of the compounds of the present invention was determined in accordance with the following experimental method. Rhesus PDE2A3 was amplified from rhesus macaque brain cDNA (Biochain Institute, Hayward, Calif.) using primers based on human PDE2A sequence (accession NM_002599.3) where the forward primer containing a Kozak consensus was 5′-gccaccatggggcaggcatgtggc-3′ and the reverse primer was 5′-tcactcagcatcaaggctgca-3′. Amplification with Easy-A High-Fidelity PCR cloning enzyme (Stratagene, La Jolla, Calif.) was 95° C. for 2 minutes followed by thirty three cycles of 95° C. for 40 seconds, 52° C. for 30 seconds, and 72° C. for 2 minutes 48 seconds. Final extension was 72° C. for 7 minutes. The PCR product was TA cloned into pcDNA3.3-TOPO (Invitrogen, Carlsbad, Calif.) according to standard protocol. A consensus sequence was developed from multiple clones and then deposited into GenBank (EU812167). AD293 cells (Stratagene, La Jolla, Calif.) with 70-80% confluency were transiently transfected with rhesus PDE2A3/pcDNA3.3-TOPO using Lipofectamine 2000 according to manufacturer specifications (Invitrogen, Carlsbad, Calif.).
Measurement: Ki = 1431 nM (pChEMBL 5.840000152587891)
Descriptors: MW 415.38, ALogP 3.05, HBA 5, HBD 2, PSA 100.63, QED 0.68
Compound (SMILES): CCc1nc(C(=O)NC[C@H]2CC[C@H](S(C)(=O)=O)CC2)c(C)n1-c1ncc(C[C@@H](C)C(F)(F)F)cc1OC(F)F
InChIKey: RDQCEOCZQDVPFA-QGPMSJSTSA-N
Target: Nuclear receptor ROR-gamma (SINGLE PROTEIN), Homo sapiens
UniProt: P51449
Assay: Fluorescence Based Assay: ThermoFluor is a fluorescence based assay that estimates ligand binding affinities by measuring the effect of a ligand on protein thermal stability (Pantoliano, M. W., Petrella, E. C., Kwasnoski, J. D., Lobanov, V. S., Myslik, J., Graf, E., Carver, T., Asel, E., Springer, B. A., Lane, P., and Salemme, F. R. (2001) High-density miniaturized thermal shift assays as a general strategy for drug discovery. J Biomol Screen 6, 429-40, and Matulis, D., Kranz, J. K., Salemme, F. R., and Todd, M. J. (2005) Thermodynamic stability of carbonic anhydrase: measurements of binding affinity and stoichiometry using ThermoFluor. Biochemistry 44, 5258-66). This approach is applicable to a wide variety of systems, and rigorous in theoretical interpretation through quantitation of equilibrium binding constants (KD).
Measurement: Kd = 58 nM (pChEMBL 7.239999771118164)
Descriptors: MW 580.62, ALogP 4.81, HBA 7, HBD 1, PSA 103.18, QED 0.4
Compound (SMILES): COc1cccc(-c2nnc(NS(=O)(=O)[C@@H](C)[C@H](OC)c3ncc(Cl)cn3)n2C2CCC2)n1
InChIKey: SIGBMXYETLTOFM-SJCJKPOMSA-N
Target: Apelin receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P35414
Assay: 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 9 μL assay buffer [20 mM HEPES, pH 7.5, 5 mM MgCl2, 100 mM NaCl and 0.1% (w/v) BSA], 1 μL of diluted test compound (starting with 0.75 mM, 2-fold serial dilution with DMSO, total 22 points), 10 μL of 18 μM GDP (final concentration of 3 μM GDP), 20 μL of 0.25 μg/mL membrane protein expressing human APJ receptor captured with WGA PS beads (final concentration of 5 μg per well), and 20 μL of 0.3 nM [35S]GTPγS (final concentration is 0.1 nM [35S]GTPγS)(Perkin Elmer Life and Analytical Sciences, Waltham USA). One column of the plate was 1 μL of DMSO as background and another column of the plate was 1 μL of 180 μM Pyr-Apelin-13 which was used as control at a final concentration of 3 μM. Incubation was at RT for 90 min and the microplate was read using a ViewLux ultra HTS Microplate Imager.
Measurement: EC50 = 25 nM (pChEMBL 7.599999904632568)
Descriptors: MW 493.98, ALogP 3.04, HBA 10, HBD 1, PSA 134.01, QED 0.48
Compound (SMILES): C=CC(=O)N1CC2(CC(n3nc(-c4ccc5nn(CC(C)(C)O)cc5c4)c(-c4c(Cl)c(C)cc5[nH]ncc45)c3C)C2)C1
InChIKey: VORISWHHRHZGEE-UHFFFAOYSA-N
Target: GTPase KRas (SINGLE PROTEIN), Homo sapiens
UniProt: P01116
Assay: Scintillation Proximity Assay (SPA): Assays were run using 384-well plates (781207/Greiner) in which one column was designated as the high signal (no inhibition) control, and contained DMSO with no test compound, and another column was designated as the low signal control (maximal inhibition), and contained no protein. Serial dilutions of compounds to be tested were added to the assay plate (resulting in duplicate 11-point dose response with semi-log compound dilutions from 50 μM to 0.5 nM or from 5 μM to 0.05 nM for the most potent compounds). A 1/20 isotopic dilution of labeled (Compound B) and non-labeled covalent (Compound A) probe was prepared and added to all wells on the plate. The reaction was started by addition of KRasG12C (M1-K169, biotinylated on the N-terminus) to the compounds and incubated for 2 hours with continuous agitation allowing for full modification of KRasG12C with probe or test compounds. Final concentrations in an assay volume of 40 μL were 10 nM KRasG12C, 25 nM radio-ligand and 475 nM unlabelled ligand. The assay buffer contained 20 mM Tris-HCl pH 7.5 (Invitrogen), 150 mM NaCl (Sigma Aldrich), 0.1 mM MgCl2 (Sigma Aldrich), and 0.01% Tween-20 (Sigma Aldrich). Following addition of 50 μL of a 400 μg/ml. suspension of streptavidin-coated YSi beads (Perkin Elmer), plates were incubated for a further 30 min with continuous agitation before reading the plates on a scintillation counter (Topcount NXT 384 (Packard).
Measurement: IC50 = 12 nM (pChEMBL 7.920000076293945)
Descriptors: MW 584.12, ALogP 5.83, HBA 7, HBD 2, PSA 104.86, QED 0.24
Compound (SMILES): Cn1cnc(-c2cc(Cl)c(F)c(C(=O)NNS(=O)(=O)c3cccc(O)n3)c2)c1
InChIKey: VQIZJLDXUOEROM-UHFFFAOYSA-N
Target: Histone acetyltransferase KAT5 (SINGLE PROTEIN), Homo sapiens
UniProt: Q92993
Assay: KAT-5 Enzyme Assay: Enzyme assay buffer was 50 mM Tris pH 8.0, 0.002% Tween20, 0.005% bovine skin gelatin, and 1 mM dithiothreitol (DTT). For determination of IC50values, compounds were serially diluted with 2% (v/v) DMSO in the final reaction, pre-incubating each dilution of each compound with 40 μL of assay buffer containing KAT-5 enzyme (9 nM final concentration). 10 μL of assay buffer containing 1 μM peptide substrate and 0.5 μM acetyl coenzyme A (final concentrations) was added. Reactions (50 μL total) were then carried out at 25° C. for 90 minutes. Reactions were terminated by the addition of 0.5% formic acid (final concentration), and a sample of each reaction was analyzed by SAMDI Tech, Inc. (Chicago, Ill.) using self-assembled monolayer desorption/ionization time-of-flight mass spectrometry (Mrksich, M. (2008) Mass spectrometry of self-assembled monolayers: a new tool for molecular surface science. ACS Nano 2, 7-18).
Measurement: IC50 < 10000 nM (pChEMBL 5)
Descriptors: MW 425.83, ALogP 1.6, HBA 7, HBD 3, PSA 126.21, QED 0.53
Compound (SMILES): Cc1ccn2c(-c3ccc(Nc4ccc(N5CCN(C)CC5)cn4)c4c3CNC4=O)cnc2c1
InChIKey: DNVLFVYYLXBGLY-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 μM 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 453.55, ALogP 3.44, HBA 7, HBD 2, PSA 77.8, QED 0.49
Compound (SMILES): Cc1ccc(C#N)c(S(=O)(=O)Nc2ccc(-c3nc(OC4CCCNC4)c4c(C)n[nH]c4n3)cc2)c1
InChIKey: JSKBFEJDMAMLAA-UHFFFAOYSA-N
Target: Serine/threonine-protein kinase Sgk1 (SINGLE PROTEIN), Homo sapiens
UniProt: O00141
Assay: Enzymatic Activity Assay: The compounds were tested for serum and glucocorticoid-regulated kinase 1 (SGK-1) inhibitory activity in a substrate phosphorylation assay designed to measure the ability of the isolated enzyme to catalyze the transfer of phosphate from ATP to serine/threonine residues in a fluorescein-labeled substrate peptide, using recombinant human SGK-1 enzyme produced in a baculovirus system (Biomol, Hamburg, Germany, Cat. No. 4-331). The synthesized fluorescent labeled peptide substrate contained (5(6)-Carboxyfluorescein)-RPRAATF-NH2. The phosphorylated substrate peptide and non-phosphorylated substrate peptide were separated with caliper life science's lab-chip technology based on a micro fluidics method. All fluid flow was established on the chip by applying a vacuum of a few psi to the waste well transporting fluid from various sources through interconnecting channels. Because the phosphoryl group is doubly negatively charged, under the pressure-driven hydrodynamic flow and the voltage-driven flow within the electric field, the fluorescent labeled peptide substrate and its phosphorylation product appear at different times in the detection window to the detection point. Substrate turnover can thus be determined as the ratio of the product peak area and the sum of substrate peak area and product peak area.The enzyme reaction was carried out in a buffer containing 25 mM Tris-HCl (pH 7.4), 5 mM MgCl2, 2 mM MnCl2, 2 mM DTT, and 0.03% bovine serum albumine. The enzyme was pre-incubated with the test compound for 30 min at 24° C. The kinase reaction was initiated by addition of the substrate mixture containing the peptide substrate (final concentration 1 μM) and ATP (final concentration 10 μM). After 60 min incubation at 37° C., the enzyme reaction was terminated by adding a buffer containing 100 mM Hepes (pH 7.4) and 35 mM EDTA.
Measurement: IC50 = 3.7 nM (pChEMBL 8.430000305175781)
Descriptors: MW 503.59, ALogP 3.44, HBA 8, HBD 3, PSA 145.68, QED 0.36
Compound (SMILES): COC1(c2cccc(C(N)=O)c2)C2CCCC1CN(CC1CCS(=O)(=O)CC1)C2
InChIKey: QAWGWXQXDNAGGI-UHFFFAOYSA-N
Target: Mu-type opioid receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P35372
Assay: Competitive Displacement Assay: Membrane protein from CHO (Chinese Hamster Ovarian) cells that stably expressed one type of the cloned human opioid receptor were incubated with 12 different concentrations of the compound in the presence of 0.25 nM [3H]DAMGO, 0.2 nM [3H]naltrindole or 1 nM [3H]U69,593 in a final volume of 1 mL of 50 mM Tris-HCl, pH 7.5 at 25° C. Incubation times of 60 min were used for [3H]DAMGO and [3H]U69,593. Because of a slower association of [3H]naltrindole with the receptor, a 3 h incubation was used with this radioligand. Samples incubated with [3H]naltrindole also contained 10 mM MgCl2 and 0.5 mM phenylmethylsulfonyl fluoride. Nonspecific binding was measured by inclusion of 10 μM naloxone. The binding was terminated by filtering the samples through Schleicher & Schuell No. 32 glass fiber filters using a Brandel 48-well cell harvester. The filters were subsequently washed three times with 3 mL of cold 50 mM Tris-HCl, pH 7.5, and were counted in 2 mL Ecoscint A scintillation fluid. For [3H]naltrindole and [3H]U69,593 binding, the filters were soaked in 0.1% polyethylenimine for at least 60 min before use.
Measurement: Ki = 3 nM (pChEMBL 8.520000457763672)
Descriptors: MW 420.58, ALogP 2.18, HBA 5, HBD 1, PSA 89.7, QED 0.79
Compound (SMILES): Cc1ccc(NC(=O)N2CC[C@@H](CC(F)(F)F)C2)cc1-c1cc(N2CCOCC2)nc(NC2CCC(O)C2)n1
InChIKey: MPEIIRBJUDGAAW-JSOIOWGOSA-N
Target: RAF proto-oncogene serine/threonine-protein kinase (SINGLE PROTEIN), Homo sapiens
UniProt: P04049
Assay: Enzymatic RAF1 Kinase: Small molecule inhibition of RAF1 kinases was measured using ADP-Glo assay. In the assay, ADP is converted to ATP in the presence of test kinase and substrate, resulting in luciferase reaction and luminescent readout with light generated proportional to the relative kinase activity. Compounds diluted in DMSO were used in 10-point, 3-fold dose curve for both assays. Final concentrations of 3 nM RAF1 (CarnaBio, Cat. 09-125) and 30 nM MEK1 substrate (Millipore, Cat. 14-420) were incubated with 3 μM ATP, 10 mM MgCl2, 0.003% Brij-35, 2 mM DTT, 0.05% BSA, 1 mM EGTA, and 50 mM HEPES for 90 minutes at room temp prior to addition of ADP-Glo reagent (Promega, Cat. V9102) for 40 minutes, and detection reagent for 45 minutes. Luminescence was read on an Envision plate reader (PerkinElmer) and percent remaining activity was used to calculate IC50 using a four-parameter fit model using Dotmatics Knowledge Solutions Studies curve fitting (Dotmatics, Bishops Stortford, UK, CM23).
Measurement: IC50 = 55 nM (pChEMBL 7.260000228881836)
Descriptors: MW 548.61, ALogP 4.42, HBA 7, HBD 3, PSA 102.85, QED 0.49
Compound (SMILES): Cc1cnn(Cc2ccc(Cn3cc(C(=O)NCc4ccc(Cl)cc4F)c(C(F)(F)F)n3)cc2)c1
InChIKey: MPXDOEQMYFYSIT-UHFFFAOYSA-N
Target: Plasma kallikrein (SINGLE PROTEIN), Homo sapiens
UniProt: P03952
Assay: Inhibitory Activity In Vitro Assay : Plasma kallikrein inhibitory activity in vitro was determined using standard published methods. Human plasma kallikrein (Protogen) was incubated at 25° C. with the fluorogenic substrate H-DPro-Phe-Arg-AFC and various concentrations of the test compound. Residual enzyme activity (initial rate of reaction) was determined by measuring the change in optical absorbance at 410 nm and the IC50 value for the test compound was determined.
Measurement: IC50 = 865 nM (pChEMBL 6.059999942779541)
Descriptors: MW 505.9, ALogP 5.23, HBA 5, HBD 1, PSA 64.74, QED 0.35
Compound (SMILES): Cn1ccc2c(-c3ncc(Nc4cccc(N5CC6(CCN6)C5)n4)c4c3CNC4=O)ccnc21
InChIKey: VUGAQBVZEVJFKZ-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 μM), 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 μM 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 aCaliper EZ Reader (Perkin Elmer, UK).
Measurement: IC50 = 550 nM (pChEMBL 6.260000228881836)
Descriptors: MW 452.52, ALogP 2.57, HBA 8, HBD 3, PSA 100, QED 0.44
Compound (SMILES): CCCS(=O)(=O)Nc1ccc(F)c(C=O)c1F
InChIKey: RWAQKKCELAQLHO-UHFFFAOYSA-N
Target: Serine/threonine-protein kinase B-raf (SINGLE PROTEIN), Homo sapiens
UniProt: P15056
Assay: Biochemical Assay: Assays for biochemical and cell based activity are known in the art, for example, as described in PCT publication WO 2007/002433, the disclosure of which is hereby incorporated by reference as it relates to such assays. For example, the biochemical activity IC50 values are determined with respect to inhibition of B-Raf kinase activity, c-Raf-1 kinase activity, or B-Raf V600E kinase activity, where inhibition of phosphorylation of a peptide substrate is measured as a function of compound concentration. Compounds to be tested are diluted in dimethyl sulfoxide to a concentration of 0.1 mM. These are serially diluted 15 μL into 30 μL of dimethyl sulfoxide seven times in 96 well plates for a total of 8 dilution points, and for each dilution point 1 μL is added to a well of an assay plate. Plates are prepared such that each well in a 384 well plate contains 1 μL of compound in 10 μL volume with 0.1 ng Raf enzyme (i.e. any of B-Raf, c-Raf-1 or B-Raf V600E, Upstate Biotechnology or prepared by methods known to one of skill in the art), 50 mM HEPES, pH 7.0, 50 mM NaCl, 2 mM MgCl2, 1 mM MnCl2, 0.01% Tween-20, 1 mM DTT, and 100 nM biotin-MEK1 as substrate. The reaction is started with addition of 10 μL of 200 μM ATP (i.e. final 100 μM ATP). After incubation of the kinase reaction for 45 minutes at room temperature, 5 μL/well of Stop Solution is added (25 mM Hepes pH 7.5, 100 mM EDTA, 0.01% BSA with donor beads (Streptavidin coated beads, Perkin Elmer), acceptor beads (Protein A coated, Perkin Elmer), and anti phosphor MEK1/2 antibody (CellSignal), each at final concentration 10 μg/mL). The plates are incubated for 3 hours at room temperature and read on Envision reader (Perkin Elmer). Phosphorylation of Mek1 results in binding of the anti-phosphor-MEK1/2 antibody and association of the donor and acceptor beads such that signal correlates with kinase activity.
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 263.26, ALogP 1.93, HBA 3, HBD 1, PSA 63.24, QED 0.83
Compound (SMILES): N#CC[C@@H](C1CC1)n1nc(Nc2ccc(C(O)(c3ccncc3)C(F)(F)F)cc2)c2c(=O)[nH]ccc21
InChIKey: LJKNVXRQRRMRNP-XGLRFROISA-N
Target: Tyrosine-protein kinase JAK1 (SINGLE PROTEIN), Homo sapiens
UniProt: P23458
Assay: Jak Biochemical HTRF Assay Protocol: The ability of compounds to inhibit the activity of JAK1, JAK2, JAK3, and Tyk2 was measured using a recombinant purified GST-tagged catalytic domain for each enzyme (Invitrogen JAK1 #M4290, JAK2 #M4290, JAK3 #M4290, Tyk2 #M4290) in an HTRF format biochemical assay. The reactions employed a common peptide substrate, LCB-EQEDEPEGDYFEWLW-NH2 (in-house). The basic assay protocol is as follows: First, 250 nL of diluted compounds in DMSO were dispensed into the wells of a dry 384-well Black plate (Greiner #781076) using a Labcyte Echo 555 acoustic dispenser. Subsequent reagent additions employed an Agilent Bravo. Next, 18 μL of 1.11× enzyme and 1.11× substrate in 1× assay buffer (Invitrogen kinase buffer #PV3189, 2 mM DTT, 0.05% BSA) were added to the wells and shaken and then preincubated for 30 minutes at room temperature to allow compound binding to equilibrate. After equilibration, 2 μL of 10×ATP in 1× assay buffer was added to initiate the kinase reaction and the plates were shaken and then incubated at room temperature for 120 minutes. At the end of the incubation, 20 μL of 2× stop buffer (streptavidin-Dylight 650 (Thermo #84547B/100 mL), Eu-tagged pY20 antibody (Perkin Elmer #AD0067), EDTA, HEPES, and Triton) was added to quench the reaction. Plates were shaken and centrifuged and then incubated 60 minutes at room temperature and then read on a Perkin Elmer Envision (λex=337 nm, λem=665 and 615 nm, TRF delay time=20 μs). HTRF signal=10,000*665 nm reading/615 nm reading. After normalization to untreated controls, the percent inhibition of the HTRF signal at each compound concentration was calculated.
Measurement: IC50 = 200 nM (pChEMBL 6.699999809265137)
Descriptors: MW 494.48, ALogP 4.53, HBA 7, HBD 3, PSA 119.62, QED 0.35
Compound (SMILES): CS(C)(=O)=NC(=O)c1cnc(N)c(-c2cc3cc(NC(=O)Nc4cc(C(F)(F)F)ccc4F)ccc3s2)c1
InChIKey: XTCSSXNPALQZJQ-UHFFFAOYSA-N
Target: Platelet-derived growth factor receptor beta (SINGLE PROTEIN), Homo sapiens
UniProt: P09619
Assay: Biochemical Assay: Biochemical PDGFRβ kinase assays were performed in 96 well microtiter plates that were coated overnight with 75 μg of poly-Glu-Tyr (4:1) in 10 mM Phosphate Buffered Saline (PBS), pH 7.4. The coated plates were washed with 2 mls per well PBS+0.05% Tween-20 (PBS-T), blocked by incubation with PBS containing 1% BSA, then washed with 2 mls per well PBS-T prior to starting the reaction. Reactions were carried out in 100 μL reaction volumes containing 36 μM ATP in kinase buffer (50 mM Hepes buffer pH 7.4, 20 mM MgCl2, 0.1 mM MnCl2 and 0.2 mM Na3VO4). Test compounds were reconstituted in 100% DMSO and added to the reaction to give a final DMSO concentration of 5%. Reactions were initiated by the addition 20 μl per well of kinase buffer containing 200-300 ng purified cytoplasmic domain PDGFR-b protein (Millipore). Following a 60 minute incubation at 30° C., the reactions were washed 2 mls per well PBS-T. 100 μl of a monoclonal anti-phosphotyrosine antibody-peroxidase conjugate diluted 1:10,000 in PBS-T was added to the wells for 30 minutes. Following a 2 mls per well wash with PBS-Tween-20, 100 μl of O-Phenylenediamine Dihydrochloride in phosphate-citrate buffer, containing urea hydrogen peroxide, was added to the wells for 7-10 minutes as a colorimetric substrate for the peroxidase. The reaction was terminated by the addition of 100 μl of 2.5N H2SO4 to each well and read using a microplate ELISA reader set at 492 nm. IC50 values for compound inhibition were calculated directly from graphs of optical density (arbitrary units) versus compound concentration following subtraction of blank values.
Measurement: IC50 = 6 nM (pChEMBL 8.220000267028809)
Descriptors: MW 565.57, ALogP 6.22, HBA 6, HBD 3, PSA 126.54, QED 0.25
Compound (SMILES): COC(=O)Nc1c(C#N)cc(F)c(F)c1-c1ccc2ncc(-c3cc(C)cc(F)c3)c(N3CCC(N)CC3)c2c1
InChIKey: PLGWYRNGIYQMGN-UHFFFAOYSA-N
Target: Somatostatin receptor type 2 (SINGLE PROTEIN), Homo sapiens
UniProt: P30874
Assay: cAMP Assay : Four days prior to the assay, 5,000 Chinese hamster ovary cells (CHO-K1, ATCC #CCL-61) stably expressing the human SSTR2 are plated in each well of a 96-well tissue culture-treated plate in Ham's F12 growth media (ThermoFisher #10-080-CM) supplemented with 10% donor bovine serum (Gemini Bio-Products #100-506), 100 U/mL penicillin; 100 ug/mL streptomycin; 2 mM L-glutamine (Gemini Bio-Products #400-110) and 0.2 mg/mL hygromycin B (GoldBio #31282-04-9). The cells are cultured at 37° C., 5% CO2 and 95% humidity. On the day of the assay, the media is aspirated and the cells are treated with 50 μL of 1.6 μM NKH477 (Sigma #N3290) plus various dilutions of compounds of the invention in assay buffer [1× Hank's Balanced Salt Solution (ThermoFisher #SH3058802), 0.5 mM HEPES pH 7.4, 0.1% bovine serum albumin, 0.2 mM 3-Isobutyl-1-methylxanthine (IBMX, VWR #200002-790)]. The cells are incubated for 20 minutes at 37° C. (the final concentration of the compounds of the invention are typically 0-10,000 nM). The cells are treated with 50 μL of lysis buffer (HRTF cAMP kit, Cisbio). The lysate is transferred to 384-well plates and cAMP detection and visualization antibodies are added and incubated for 1-24 hours at room temperature. The time-resolved fluorescent signal is read with a Tecan M1000Pro multiplate reader. The intracellular cAMP concentrations are calculated by regression to a standard curve and are plotted vs. the concentration of the compounds of the invention and the EC50 of the compounds are calculated using standard methods. All data manipulations are in GraphPad Prism v6.
Measurement: EC50 < 10 nM (pChEMBL 8)
Descriptors: MW 545.57, ALogP 6.27, HBA 6, HBD 2, PSA 104.27, QED 0.31