lovingscience/chembl-v37-raw-activities huggingface dataset:
Compound (SMILES): CC(=O)N1CCN(c2ccc3ncn(CC(=O)NCc4ccc(C(F)(F)F)nc4)c(=O)c3c2)CC1
InChIKey: CSWBLMUCVMMHEY-UHFFFAOYSA-N
Target: Autotaxin (SINGLE PROTEIN), Homo sapiens
UniProt: Q13822
Assay: Human ATX Enzyme Inhibition Assay: ATX inhibition was measured by a fluorescence quenching assay using a specifically labeled substrate analogue (MR121 substrate). To obtain this MR121 substrate, BOC and TBS protected 6-amino-hexanoic acid (R)-3-({2-[3-(2-{2-[2-(2-amino-ethoxy)-ethoxy]-ethoxy}-ethoxy)-propionylamino]-ethoxy}-hydroxy-phosphoryloxy)-2-hydroxy-propyl ester (Ferguson et al., Org. Lett. 2006, 8, 2023) was labeled with MR121 fluorophore [CAS RN 185308-24-1], 1-(3-carboxypropyl)-11-ethyl-1,2,3,4,8,9,10,11-octahydro-dipyrido[3,2-b:2′,3′-i]phenoxazin-13-ium) on the free amine of the ethanolamine side and then, after deprotection, subsequently with tryptophan on the side of the aminohexanoic acid.Assay working solutions were made as follows:Assay buffer (50 mM Tris-HCl, 140 mM NaCl, 5 mM KCl, 1 mM CaCl2, 1 mM MgCl2, 0.01% Triton-X-100, pH 8.0;ATX solution: ATX (human His-tagged) stock solution (1.08 mg/mL in 20 mM bicine, pH 8.5, 0.15 M NaCl, 10% glycerol, 0.3% CHAPS, 0.02% NaN3), diluted to 1.4-2.5× final concentration in assay buffer;MR121 substrate solution: MR121 substrate stock solution (800 μM MR121 substrate in DMSO), diluted to 2-5× final concentration in assay buffer.Test compounds (10 mM stock in DMSO, 8 μL) were obtained in 384 well sample plates (Corning Costar #3655) and diluted with 8 μL DMSO. Row-wise serial dilutions were made by transferring 8 μL cpd solution to the next row up to row O. The compound and control solutions were mixed five times and 2 μL were transferred to 384 well assay plates (Corning Costar #3702). Then, 15 μL of 41.7 nM ATX solution was added (30 nM final concentration), mixed five times and then incubated for 15 minutes at 30° C. 10 μL of MR121 substrate solution was added (1 μM final concentration), mixed 30 times and then incubated for 15 minutes at 30° C. Fluorescence was then measured every 2 min for 1 h (Perkin Elmer plate: vision multimode reader); light intensity: 2.5%; exp. time: 1.4 s, Filter: Fluo_630/690 nm) and IC50 values were calculated from these readouts.
Measurement: IC50 = 207 nM (pChEMBL 6.679999828338623)
Descriptors: MW 488.47, ALogP 1.8, HBA 7, HBD 1, PSA 100.43, QED 0.59
Compound (SMILES): CN1CCC(c2ccc3c(c2)[nH]/c(=N\C(=O)c2ccnc(-c4cnn(C)c4)c2)n3CC(C)(C)O)CC1=O
InChIKey: RCOIDSYWOGWACZ-UHFFFAOYSA-N
Target: Epidermal growth factor receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P00533
Assay: Biochemical EGFR Inhibition Assays (100 mM ATP): 5 μL of EGFR enzyme form in assay buffer (50 mM HEPES pH 7.3, 10 mM MgCl2, 1 mM EGTA, 0.01% Tween 20, 2 mM DTT) are dispensed into columns 1-23, than 5 μL of ATP and ULight-poly-GT substrate (PerkinElmer; TRF0100-M) mix in assay buffer is added to all wells (final assay concentration of the ULight-poly-GT substrate 200 nM). Each of the different EGFR enzyme form assays is available at high ATP levels (final assay concentration 100 μM). After 90 minutes incubation at room temperature 5 μL EDTA (final assay concentration 50 mM) and LANCE Eu-anti-P-Tyr (PT66) antibody (PerkinElmer, AD0069) (final assay concentration 6 nM) mix are added to stop the reaction and start the binding of the antibody. After additional 60 minutes incubation at room temperature the signal is measured in a PerkinElmer Envision HTS Multilabel Reader using the TR-FRET LANCE Ultra specs of PerkinElmer (used wavelengths: excitation 320 nm, emission 1 665 nm, emission 2 615 nm). Each plate contains 16 wells of a negative control (diluted DMSO instead of test compound; w EGFR enzyme form; column 23) and 16 wells of a positive control (diluted DMSO instead of test compound; w/o EGFR enzyme form; column 24). Negative and positive control values are used for normalization and IC50 values are calculated and analysed using a 4 parametric logistic model.
Measurement: IC50 = 5.6 nM (pChEMBL 8.25)
Descriptors: MW 501.59, ALogP 2.61, HBA 7, HBD 2, PSA 121.4, QED 0.44
Compound (SMILES): COc1ccc(Cn2ncn(-c3ccc(C4CCN(C(=O)Nc5c(Cl)cccc5Cl)CC4)cc3)c2=O)cc1
InChIKey: URMGMUGAVHSFAQ-UHFFFAOYSA-N
Target: Diacylglycerol O-acyltransferase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: O75907
Assay: FlashPlate™ Assay: DGAT activity in membrane preparations was assayed in 50 mM Tris-HCl (pH 7.4), 150 mM MgCl2, 1 mM EDTA and 0.2% BSA, containing 50 μM DAG, 32 μg/ml PC/PS and 8.4 μM [3H]-oleoylCoA (at a specific activity of 30 nCi/well) in a final volume of 50 μl in 384-well format using the red shifted Basic Image FlashPlate™ (Perkin Elmer Cat.No. SMP400). In detail, 10 μl enzyme mix and 10 μl substrate mix were added to 30 μl of assay buffer, optionally in the presence of 1 μl DMSO (blank and controls) or 1 μl of the compound to be tested. This reaction mixture was incubated for 120 minutes at 37° C. and the enzymatic reaction stopped by adding 20 μl of the stop mix. The plates were sealed and the vesicles allowed to settle overnight at room temperature. Plates were centrifuged for 5 minutes at 1500 rpm and measured in Leadseeker.
Measurement: IC50 = 1170 nM (pChEMBL 5.929999828338623)
Descriptors: MW 552.46, ALogP 5.81, HBA 6, HBD 1, PSA 81.39, QED 0.33
Compound (SMILES): O=C(NCc1ccc(F)c(F)c1Cl)[C@H]1CC[C@H](O)c2ncccc21
InChIKey: FKYKRHISLNTHPE-AAEUAGOBSA-N
Target: P2X purinoceptor 7 (SINGLE PROTEIN), Homo sapiens
UniProt: Q99572
Assay: Pharmacological Assay: 1. Wash each well with 80 microL of assay buffer (20 mM HEPES, 1×HBSS, pH 7.4 adjusted with NaOH) three times and leave 20 microL using plate washer, ELx-405 Select CW (BIO-TEK).2. Add 20 microL of assay buffer containing 2.5 mM probenecid, 0.5 microM Fluo-4-AM (Molecular Probes) and 0.1% Pluronic F-127 to each well.3. Incubate the plate at 37° C. in 5% CO2 for 1 h.4. Wash each well with 80 microL of assay buffer (see below) three times and leave 20 microL using plate washer, ELx-405 Select CW (BIO-TEK).5. Test compounds were prepared at 100× the test concentration in DMSO by serial dilution with Biomek-FX liquid handling instrument. 33× diluted compound solutions in assay buffer were prepared in intermediate compound plate with Biomek-NX liquid handling instrument. A further 3× dilution occurred in below steps 6 and 7.6. Add 20 microL of 33× diluted compound solutions into each well and leave the plate for 10 min under the dark at room temperature.7. Measure activity by FDSS as follows:Set the assay plate on the stacker of FDSS.Start the detection of fluorescence intensity at 540 nm by 480 nm exicitation.After 30 seconds, add 20 microL of assay buffer containing 240 microM BzATP (final concentration 80 microM).
Measurement: IC50 < 50 nM (pChEMBL 7.301000118255615)
Descriptors: MW 352.77, ALogP 3.24, HBA 3, HBD 2, PSA 62.22, QED 0.83
Compound (SMILES): O=C(CC1(n2cc(-c3c[nH]c4ncccc34)cn2)CCCC1)NCC(F)(F)F
InChIKey: LUGCYYGYISQAJZ-UHFFFAOYSA-N
Target: Tyrosine-protein kinase JAK1 (SINGLE PROTEIN), Homo sapiens
UniProt: P23458
Assay: Inhibition of JAK: The study of the effect of compounds on the activity of purified recombinant JAK was performed by studying the inhibitory activity of the compounds on JAK from the enzymatic level. The experimental principle is to use a luminescence kinase assay to detect the ADP content produced by the reaction of JAK with the substrate Poly (4:1 Glu, Tyr) peptide: after ADP is converted to ATP, ATP can act as a substrate for the Ultra-Glo luciferase catalytic reaction, producing an optical signal. The luminescence signal is positively correlated with the amount of ADP and kinase activity. Therefore, the inhibitory effect of the compounds on the recombinant JAK was determined by observing the luminescence signal produced by the reaction of JAK and the substrate, and was expressed by IC50.
Measurement: IC50 = 35 nM (pChEMBL 7.460000038146973)
Descriptors: MW 391.4, ALogP 3.76, HBA 4, HBD 2, PSA 75.6, QED 0.7
Compound (SMILES): O=C1CCc2c(Oc3ccc4c(c3)[C@H]3[C@H](NC(=O)Nc5cccc([N+](=O)[O-])c5)[C@H]3O4)ccnc2N1
InChIKey: DGSAFNDAWPHKBN-FKBYEOEOSA-N
Target: Serine/threonine-protein kinase B-raf (SINGLE PROTEIN), Homo sapiens
UniProt: P15056
Assay: Raf IC50 Assay: Compounds disclosed herein were tested against B-Raf (V600E) (PV3849, from Invitrogen) or C-Raf (Y340D/Y341D) (PV3805, from Invitrogen) in a time-resolved fluorescence energy transfer assay. The assay was carried out in reactions (10 μL) containing 0.0625 nM B-Raf or 0.5 nM C-Raf, 25 mM Tris pH7.4, 10 mM MgCl2, 0.5 mM EGTA, 0.5 mM Na3VO4, 5 mM beta-glycerophosphate, 0.01% Triton X-100, 2.5 mM DTT, 0.1% BSA, 0.1 mM ATP, 13.7 nM GST-tagged MEK1 (Full-length protein with K97R mutation, recombinant protein purified from bacterial expression system) and 0-5 μM compounds disclosed herein (final concentration of 1% DMSO). The enzyme was incubated with the compounds at room temperature for 60 minutes and the reactions were initiated by the addition of ATP and GST-MEK1. After reaction at room temperature for 60 minutes, an equal volume of stop/detection solution was added according to the manufacture's instruction (CisBio Bioassays). The stop/detection solution contained Eu3+ cryptate-conjugated anti-phospho MEK1/2 (Ser217/221) rabbit polyclonal antibody and d2-conjugated anti-GST mouse monoclonal antibody in buffer containing 25 mM Tris pH7.4, 400 mM KF, 50 mM EDTA, 0.01% BSA and 0.01% Triton X-100. Plates were sealed and incubated at room temperature for 2 hours, and the TR-FRET signals (ratio of fluorescence emission at 665 nm over emission at 620 nm with excitation at 337 nm wavelength) were recorded on a PHERAstar FS plate reader (BMG Labtech). Phosphorylation of MEK1 led to the binding of anti-phospho-MEK1/2 antibody to GST-MEK1 protein that place fluorescent donor (Eu3+ crypate) in close proximity to the accepter d2 on the anti-GST antibody, thus resulting in a high degree of fluorescence resonance energy transfer from the donor fluorophore (at 620 nm) to the acceptor fluorophore (at 665 nm). Inhibition of RAF kinase activity resulted in decrease of the TR-FRET signal. The IC50 for each compound was derived from fitting the dose-response % inhibition data to the four-parameter logistic model by Graphpad Prism software.
Measurement: IC50 = 1800 nM (pChEMBL 5.75)
Descriptors: MW 473.45, ALogP 3.72, HBA 7, HBD 3, PSA 144.72, QED 0.38
Compound (SMILES): C[C@@H]1CN(c2ccc(Cl)c(F)c2)CCN1C(=O)NCCC1CCN(Cc2ccccc2)CC1
InChIKey: YWBLUYIJHOWRBA-HXUWFJFHSA-N
Target: Muscarinic acetylcholine receptor M4 (SINGLE PROTEIN), Homo sapiens
UniProt: P08173
Assay: Binding Assay: Binding affinity (Ki) of compounds was measured by inhibition of radioligand binding to membranes from CHO cells expressing human M1, M2, M3, M4 and M5 receptors. Membranes were prepared by nitrogen cavitation and differential centrifugation as previously described (Hoare et al., Mol. Pharmacol. 2003 March; 63(3): 751-65). The radioligand employed was tritiated N-methylscopolamine, used at a concentration of 1.5 nM. A dose-response of twelve concentrations of compound was used, ranging from 10 Î¼M to 32 Î¼M. The assay buffer was 50 mM HEPES, 100 mM NaCl, 5 mM MgCl2, 1 mM ethylenediaminetetraacetic acid, pH-adjusted to pH 7.4.
Measurement: Ki < 10 nM (pChEMBL 8)
Descriptors: MW 473.04, ALogP 5, HBA 3, HBD 1, PSA 38.82, QED 0.64
Compound (SMILES): CCOc1ccc(Cn2cc(C(=O)NCc3ccc4c(N)nccc4c3)c(NC(=O)COC)n2)cn1
InChIKey: JSUZMXJTHHQZII-UHFFFAOYSA-N
Target: Plasma kallikrein (SINGLE PROTEIN), Homo sapiens
UniProt: P03952
Assay: Inhibitory Activity Assay: Plasma kallikrein inhibitory activity in vitro was determined using standard published methods (see e.g. Johansen et al., Int. J. Tiss. Reac. 1986, 8, 185; Shori et al., Biochem. Pharmacol., 1992, 43, 1209; Sturzebecher et al., Biol. Chem. Hoppe-Seyler, 1992, 373, 1025). Human plasma kallikrein (Protogen) was incubated at 37° 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 = 128 nM (pChEMBL 6.889999866485596)
Descriptors: MW 489.54, ALogP 2.37, HBA 9, HBD 3, PSA 146.28, QED 0.31
Compound (SMILES): N#Cc1ccc([C@H](O)CNC(=O)[C@H]2CCN(c3ncnc4[nH]ccc34)CC23CC3)cc1
InChIKey: HZWQWCYZVUNWOR-RTBURBONSA-N
Target: Tyrosine-protein kinase JAK1 (SINGLE PROTEIN), Homo sapiens
UniProt: P23458
Assay: JAK Kinase Assays: Human baculovirus-expressed JAK1, 2, 3 and TYK2 were purchased from Carna Biosciences, Inc. All four purified enzymes contain only the catalytic domain. JAK1 (aa 850-1154) and TYK2 (aa 871-1187) are expressed with an N-terminally fused GST-tag, and JAK2 and JAK3 with an N-terminally fused His-tag.Inhibition of phosphorylation of a synthetic peptide was measured in an HTRF-based assay using the TK substrate-Biotin from the Cisbio HTRFKinEASE TK kit. First, 2 μl of TK solution (TK substrate-biotin in kinase buffer [1× enzymatic buffer from HTRFKinEASE TK kit, 1 mM DTT]) is added to a plate containing 1 μl prediluted compound (final assay concentration DMSO: 0.75%). Then, 5 μl kinase-ATP mix (prepared in kinase buffer) is added to the wells and the plates are incubated at RT for 20-30 min. For all four kinases a concentration of ATP that corresponded to the Km for ATP was used. The final concentrations of buffers, substrate, kinase and ATP were: JAK1: 50 mM Hepes buffer pH 7.0, 0.01% BSA, 10 mM MgCl2, 1 mM DTT, 7 μM ATP, 50 nM SEB, 1 μM TK Substrate-Biotin and 5 ng JAK1; JAK2: 50 mM Hepes buffer pH 7.0, 0.01% BSA, 5 mM MgCl2, 1 mM DTT, 4 μM ATP, 1 μM TK Substrate-Biotin and 0.1 ng JAK2; JAK3: 50 mM Hepes buffer pH 7.0, 0.01% BSA, 5 mM MgCl2, 1 mM DTT, 2 μM ATP, 1 μM TK Substrate-Biotin and 0.3 ng JAK3; TYK2: 50 mM Hepes buffer pH 7.0, 0.01% BSA, 5 mM MgCl2, 1 mM DTT, 13 μM ATP, 50 nM SEB, 1 μM TK Substrate-Biotin and 0.8 ng TYK2. Thereafter, the kinase reaction is stopped by adding 4 μl detection mix (final concentrations: 50 mM Hepes buffer pH 7.0, 0.01% BSA, 0.8 M KF, 20 mM EDTA, 42 nM Streptavidin-XL665 and 1:400 STK Ab Cryptate) and the plates are incubated overnight in the dark. The HTRF signal is read using an Envision plate reader.
Measurement: EC50 = 0.6 nM (pChEMBL 9.220000267028809)
Descriptors: MW 416.49, ALogP 2.29, HBA 6, HBD 3, PSA 117.93, QED 0.59
Compound (SMILES): COCCC1CCN(C(=O)c2ccc(Cl)c(C(=O)c3cc4c(C)cc(C(F)(F)F)nc4n3C)c2Cl)CC1
InChIKey: PEOOGPKLIIUWDL-UHFFFAOYSA-N
Target: Nuclear receptor ROR-gamma (SINGLE PROTEIN), Homo sapiens
UniProt: P51449
Assay: transient transactivation assay: The primary screen was performed by transient transactivation assays. These cell-based assays were carried out using Cos-7 cells transfected with a chimeric human RORγ-Gal4 receptor expression plasmid and a 5Gal4 pGL3 TK Luc reporter plasmid. Transfections were performed by a chemical agent (Jet PEI). Transfected cells were distributed in 384-wells plates and were allowed to recover for 24 h. The culture medium was then removed and fresh medium containing the compounds to be tested was added (final concentration ranging from 10−4 M to 3 10−10 M). After an overnight incubation, luciferase expression was measured by adding SteadyGlo according to the manufacturer's instructions (Promega). T0901317 at 10−5 M was used as reference. Results were expressed as fold induction compared to basal level or as percentage activity compared to references taken as 100% (basal) or 0% (T0901317). Dose-effect curves and IC50 were calculated using the software Assay Explorer (MDL).
Measurement: IC50 = 300 nM (pChEMBL 6.519999980926514)
Descriptors: MW 556.41, ALogP 6.33, HBA 5, HBD 0, PSA 64.43, QED 0.33
Compound (SMILES): CCNC(=O)Nc1cccc(-n2c([C@H](C)Nc3ncnc(N)c3C#Cc3ccccn3)nc3cccc(Cl)c3c2=O)c1
InChIKey: OXBQAPCAOHOXBA-SFHVURJKSA-N
Target: Phosphatidylinositol 3-kinase regulatory subunit beta (SINGLE PROTEIN), Homo sapiens
UniProt: O00459
Assay: TR-FRET assays for PI3K isoforms: TR-FRET monitored the formation of 3,4,5-inositol triphosphate molecule that competed with fluorescently labeled PIP3 for binding to the GRP-1 pleckstrin homology domain protein. An increase in phosphatidylinositide 3-phosphate product resulted in a decrease in TR-FRET signal as the labeled fluorophore was displaced from the GRP-1 protein binding site.Class I PI3K isoforms were expressed and purified as heterodimeric recombinant proteins. All assay reagents and buffers for the TR-FRET assay were purchased from Millipore. PI3K isoforms were assayed under initial rate conditions in the presence of 25 mM Hepes (pH 7.4), and 2×Km ATP (75-500 μM), 2 μM PIP2, 5% glycerol, 5 mM MgCl2, 50 mM NaCl, 0.05% (v/v) Chaps, 1 mM dithiothreitol, and 1% (v/v) DMSO at the following concentrations for each isoform: PI3Kα, PI3Kβ, and PI3Kδ between 25 and 50 pM, and PI3Kγ at 2 nM. The compounds of Table 1 and Compound X ((S)-2,4-diamino-6-((1-(5-chloro-4-oxo-3-phenyl-3,4-dihydroquinazolin-2-yl)ethyl)amino)pyrimidine-5-carbonitrile) were added to the assay solution and incubated for 30 minutes at 25° C. Additionally, Compounds 24-164 were added to the assay solution and incubated for 30 minutes at 250 in a similar manner. The reactions were terminated with a final concentration of 10 mM EDTA, 10 nM labeled-PIP3, and 35 nM Europium labeled GRP-1 detector protein before reading TR-FRET on an Envision plate reader (Ex: 340 nm; Em: 615/665 nm; 100 μs delay and 500 μs read window).The results were normalized based on positive (1 μM wortmanin) and negative (DMSO) controls, and the IC50 values for PI3Kα, β, δ, and γ were calculated from the fit of the dose-response curves to a four-parameter equation. These assays generally produced results within 3-fold of the reported mean.
Measurement: IC50 = 32000 nM (pChEMBL 4.5)
Descriptors: MW 580.05, ALogP 4.52, HBA 9, HBD 4, PSA 152.74, QED 0.21
Compound (SMILES): COc1cc(COC2CCN(C)CC2)ccc1-c1cc(Nc2cnc(C#N)cn2)n[nH]1
InChIKey: BHHJNFKYSJQQIG-UHFFFAOYSA-N
Target: Serine/threonine-protein kinase Chk1 (SINGLE PROTEIN), Homo sapiens
UniProt: O14757
Assay: Kinase Inhibiting Activity Assay: Chk-1 Kinase Inhibiting ActivityThe compounds of the invention were tested for activity against Chk-1 kinase using the materials and protocols set out below.Reaction Buffer:Base Reaction buffer: 20 mM Hepes (pH 7.5), 10 mM MgCl2, 1 mM EGTA, 0.02% Brij35, 0.02 mg/ml BSA, 0.1 mM Na3VO4, 2 mM DTT, 1% DMSO*Required cofactors are added individually to each kinase reactionReaction Procedure:(i) Prepare indicated substrate in freshly prepared Base Reaction Buffer(ii) Deliver any required cofactors to the substrate solution above(iii) Deliver indicated kinase into the substrate solution and gently mix(iv) Deliver compounds in DMSO into the kinase reaction mixture(v) Deliver 33P-ATP (specific activity 0.01 μCi/μl final) into the reaction mixture to initiate the reaction.(vi) Incubate kinase reaction for 120 minutes at room temperature(vii) Reactions are spotted onto P81 ion exchange paper (Whatman #3698-915)(viii) Wash filters extensively in 0.1% phosphoric acid.(ix) Dry filters and measure counts in scintillation counterKinase Information:CHK-1 Genbank Accession # AF016582Recombinant full length construct, N-terminal GST tagged, purified from insect cells.No special measures were taken to activate this kinase.Final concentration in assay=0.5 nMSubstrate: CHKtidePeptide sequence: [KKKVSRSGLYRSPSMPENLNRPR]Final concentration in assay=20 μM
Measurement: IC50 = 0.27 nM (pChEMBL 9.569999694824219)
Descriptors: MW 419.49, ALogP 3.1, HBA 8, HBD 2, PSA 111.98, QED 0.6
Compound (SMILES): CCN(C[C@@]12CC[C@@H](c3cc(-c4ccccc4F)nnc31)C2(C)C)C(=O)c1c[nH]c(=O)cn1
InChIKey: BFPSBGHYVIZQBY-GKVSMKOHSA-N
Target: Nuclear receptor ROR-gamma (SINGLE PROTEIN), Homo sapiens
UniProt: P51449
Assay: In Vitro RORc Ligand Binding Assay: Consumable Supplier and product code GFB Unifilter plates Perkin Elmer 6005177 3-[(3- Sigma C5070 Cholamidopropyl)dimethylammonio]- 1-propanesulfonate (CHAPS) 96-well polypropylene U-bottom Nunc 267245 assay plate HEPES buffer, 1M Sigma H3375 Magnesium chloride (MgCl2) Sigma M8266 D,L-Dithiothreitol (DTT) Sigma D0632 Sodium chloride (NaCl) Sigma 71382 Bovine serum albumin (BSA) Sigma A7030 [lyophilized powder, ≥98% (agarose gel electrophoresis), Essentially fatty acid free, essentially globulin free] 25-hydroxycholesterol Sigma H1015 25-[26,27-3H]hydroxycholesterol Perkin Elmer NET674250UC American Radiolabeled Chemicals ART0766 RORc ligand binding domain Genentech (e.g., PUR 28048), expressed in E. coli Plate seals Perkin Elmer 6005185 Microscint 0 Perkin Elmer 6013611. Final concentrations were as follows: 50 mM HEPES buffer (pH 7.4); 150 mM NaCl; 1 mM DTT; 5 mM MgCl2, 0.01% BSA; 5% DMSO; 0.6 ug/mL RORc receptor; 6 nM 25-[3H]hydroxycholesterol. For NSB wells, 1 uM 25-hydroxycholesterol was also present.
Measurement: IC50 = 30 nM (pChEMBL 7.519999980926514)
Descriptors: MW 447.51, ALogP 3.68, HBA 5, HBD 1, PSA 91.84, QED 0.64
Compound (SMILES): NC/C(=C\F)COc1ccc(S(=O)(=O)Cn2ccccc2=O)cc1
InChIKey: AOBUSEKEDCFFNH-UKTHLTGXSA-N
Target: Amine oxidase [copper-containing] 2 (SINGLE PROTEIN), Homo sapiens
UniProt: O75106
Assay: SSAO Activity Assay: Amine oxidase activity of recombinant hSSAO, hMAO-A, and hMAO-B isoforms are measured using the MAO-Glo assay kit (Promega, V1402). Ten nL test compounds (with DMSO as vehicle, 0.1% v/v for hSSAO, hMAO-A and hMAO-B) were added into assay plates by Echo, and pre-incubated with 5 μL of enzyme for 30 mins at room temperature before the addition of 5 μL of luminogenic substrate. The substrate concentration is 40 μM for human recombinant SSAO, 40 μM for human recombinant MAO-A, and 4 μM for human recombinant MAO-B. The hSSAO and hMAO-A assays were conducted in the MAO-A reaction buffer in the kit, and the MAO-B assay was conducted in the MAO-B reaction buffer. Oxidation of the substrate was conducted for 1 hr, before the addition of detecting reagent according the manufacture's protocol. The IC50 value of the tested compounds was calculated by fitting the dose response curve using a 4-parameter non-linear regression routine.
Measurement: IC50 = 60 nM (pChEMBL 7.21999979019165)
Descriptors: MW 352.39, ALogP 1.47, HBA 6, HBD 1, PSA 91.39, QED 0.81
Compound (SMILES): COc1cc(N2CCN(C(=O)CCC3(C)NC(=O)NC3=O)CC2)ccc1Cl
InChIKey: MDIPYMUNUNAQJB-UHFFFAOYSA-N
Target: A disintegrin and metalloproteinase with thrombospondin motifs 4 (SINGLE PROTEIN), Homo sapiens
UniProt: O75173
Assay: In Vitro Assay: TABLE VI: The basis for the assay is the cleavage of the substrate TBIS-1 (5 FAM-TEGEARGSVILLK (5TAMRA)K-NH2) (SEQ ID NO: 2) by human ADAMTS4.For the dose response (10 point), 4 μL of a dilution series of compound (2 mM highest concentration, ⅕ dilution in DMSO further diluted 1 in 10 in water corresponding to a final highest concentration of 20 μM), is transferred to 384 well Fluotrac 200 plate (Greiner, cat #781076) and incubated at room temperature for 30 min with a 26 μL buffer solution (50 mM Hepes pH 7.5, 100 mM NaCl, 5 mM CaCl2, 0.1% CHAPS) containing hADAMTS4 (0.38 ng/μL) (it will be appreciated by the skilled person that the potency read out is independent of the enzyme concentration).The reaction is initiated by adding to the assay plate TBIS-1 (10 μL, 4.5 μM, Anaspec) in the same buffer.Finally, the fluorescence is read on the Envision (Perkin Elmer) after an incubation of 180 min at 37° C. (Excitation 485 nm, emission 535).
Measurement: IC50 > 10000 nM (pChEMBL 5)
Descriptors: MW 394.86, ALogP 1.38, HBA 5, HBD 2, PSA 90.98, QED 0.74
Compound (SMILES): CC(C)Nc1cc(Nc2ccc3ncsc3c2)ncc1C(=O)N[C@H]1CC[C@H](C(=O)NC(C)C)CC1
InChIKey: NIBUWLIKDGKNQM-IYARVYRRSA-N
Target: Interleukin-1 receptor-associated kinase 4 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9NWZ3
Assay: Inhibition 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 and ATP) and test compounds in assay buffer (20 mM HEPES pH 7.2, 10 mM MgCl2, 0.015% Brij35 and 4 mM DTT). The reaction was initiated by the combination of IRAK4 with substrates and test compounds. The reaction was incubated at room temperature for 60 min. and terminated by adding 45 μL of 35 mM EDTA to each sample. The reaction mixture was analyzed on the Caliper LABCHIP® 3000 (Caliper, Hopkinton, Mass.) by electrophoretic separation of the fluorescent substrate and phosphorylated product. Inhibition data were calculated by comparison to no enzyme control reactions for 100% inhibition and vehicle-only reactions for 0% inhibition. The final concentrations of reagents in the assays are ATP, 500 μM; FL-IPTSPITTTYFFFKKK peptide 1.5 μM; IRAK4, 0.6 nM; and DMSO, 1.6%.
Measurement: IC50 = 2 nM (pChEMBL 8.699999809265137)
Descriptors: MW 494.67, ALogP 5.07, HBA 7, HBD 4, PSA 108.04, QED 0.35
Compound (SMILES): Cc1cc(C#N)ccc1NC(=O)C1CN(c2ncnn3cc(-c4cnn(C)c4)cc23)C1
InChIKey: NSFXTYHVBZRZFH-UHFFFAOYSA-N
Target: Mast/stem cell growth factor receptor Kit (SINGLE PROTEIN), Homo sapiens
UniProt: P10721
Assay: Kit Wild Type Assay: In each well of a 384-well plate, 0.2 ng/ul final (2 nM) of wild type Kit (Carna Bioscience 08-156) was incubated in a total of 12.5 ul of buffer (100 mM HEPES pH 7.5, 0.015% Brij 35, 10 mM MgCl2, 1 mM DTT) with 1 uM Srctide (5-FAM-GEEPLYWSFPAKKK-NH2) and 400 uM ATP at 25 C for 90 minutes in the presence or absence of a dosed concentration series of compound (1% DMSO final concentration). The reaction was stopped by the addition of 70 ul of Stop buffer (100 mM HEPES pH 7.5, 0.015% Brij 35, 35 mM EDTA and 0.2% of Coating Reagent 3 (Caliper Lifesciences)). The plate was then read on a Caliper EZReader 2 (protocol settings: −1.9 psi, upstream voltage −700, downstream voltage −3000, post sample sip 35s).
Measurement: IC50 = 5.5 nM (pChEMBL 8.260000228881836)
Descriptors: MW 412.46, ALogP 2.38, HBA 8, HBD 1, PSA 104.14, QED 0.55
Compound (SMILES): Cc1nc(-c2nc(C)n(C)c2NC(=O)c2ccc(C(F)(F)F)c(-c3ccn(C)n3)c2)cs1
InChIKey: SHUHFRBHANTVNS-UHFFFAOYSA-N
Target: High affinity nerve growth factor receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P04629
Assay: DiscoverX PathHunter Assay: In this assay, U2OS cells express the human TrkA receptor as a fusion with the weakly complementing fragment of B-galactosidase, which DiscoverX calls Prolink (PK); additionally, Shc1 is fused with a larger fragment, which is called Enzyme Acceptor (EA). Activation of the TrkA receptor, upon NGF addition, results in the kinase domain being phosphorylated, resulting in subsequent recruitment of Shc1-EA protein. That recruitment results in an active B-galactosidase enzyme that is detected by addition of a chemiluminescent substrate. The human p75NTR protein was also expressed as a co-receptor for NGF.All reagents were purchased from DiscoverX, except for the receptor agonists (NGF, BDNF, NT3) which were purchased from Peprotech. Cells were expanded and frozen into cryovials, and stored in the vapor phase of liquid nitrogen, and thawed immediately before use. Thawed cells were added to a 384-well plate at 7500 cells/well, and allowed to incubate overnight. Compound at various concentrations was added the following morning and allowed to incubate on cells for 1 h. Then, NGF was added at a concentration sufficient to elicit 80% of a maximal response and allowed to incubate for 3 h at ambient temperature. DiscoverX PathHunter detection reagent was then added and the plate was further incubated for 1 h in the dark. The plate was then read via luminescence on the Perkin Elmer Envision.
Measurement: IC50 = 590 nM (pChEMBL 6.230000019073486)
Descriptors: MW 460.49, ALogP 4.83, HBA 7, HBD 1, PSA 77.63, QED 0.47
Compound (SMILES): O=C(O)c1cc2cc(C(F)(F)P(=O)(O)O)c(Br)cc2s1
InChIKey: TYTJRJKCKZRLFW-UHFFFAOYSA-N
Target: Tyrosine-protein phosphatase non-receptor type 2 (SINGLE PROTEIN), Homo sapiens
UniProt: P17706
Assay: Enzyme Assay Data on TC-PTP: The assay was carried out at room temperature in 96 well plates. The reaction mixture in 170 μl contained 50 mM Bis-Tris (pH=6.3), 2 mM EDTA, 5 mM N,N′-dimethyl-N,N′bis(mercaptoacetyl)hydrazine (DMH) and 10 μM fluorescein diphosphare (FDP) or 6,8-difluoro-4-methylumbelliferyl phosphate (DiFMUP). 10 μl of 10 concentrations (serial dilution) of the test compound (inhibitor) dissolved in DMSO or DMSO alone for control was added to each well and the plate was mixed for 2 min. The reaction was initiated by adding 20 μl of diluted TC-PTP (50 nM for FDP, 0.5 nM for DiFMUP in 50 mM Bis/Tris (pH=6.3), 2 mM EDTA, 5 mM DMH, 20% glycerol and 0.01% Triton X-100. The phosphatase activity was followed by monitoring the appearance of the fluorescent product fluorescein monophosphate (FMP) or 6,8-difluoro-7-hydroxyl-4-coumarin (DiFMU) continuously for 15-30 min, using the Spectromax Gemini fluorescent plate reader (Molecular probes) with excitation of 440 nm and emission at 530 nm (cutoff filter at 525 nm) for FDP and excitation at 360 nm and emission at 450 nm (cutoff filter at 435 nm) for DiFMUP. All the assays were done at least in duplicate. The initial rate of FMP or DiFMU formation is plotted against the concentration of inhibitor and the data was fitted to 4-parameter equation and the inflection point of the fit is the IC50.
Measurement: IC50 = 260 nM (pChEMBL 6.579999923706055)
Descriptors: MW 387.09, ALogP 3.59, HBA 3, HBD 3, PSA 94.83, QED 0.7
Compound (SMILES): COc1cccc(OC)c1-n1c(NS(=O)(=O)[C@@H](C)[C@H](C)c2ncc(F)c([C@@H]3COCCO3)n2)nnc1-c1cccc(OCC(O)(C(F)(F)F)C(F)(F)F)n1
InChIKey: ABUJXGZXWOPYQX-HOIFWPIMSA-N
Target: Apelin receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P35414
Assay: [35S]GTPγS Binding: The human APJ receptor was cloned by polymerase chain reaction and the gene encoding the receptor was subcloned in pFLAG-CMV-3 expression vector (Sigma, Saint Louis, Mo. USA) in-house at Amgen. A GTPγS binding assay was performed on membranes prepared from CHO cells stably expressing human APJ receptor. The optimum experimental conditions for the concentrations of GDP, MgCl2, and NaCl in the assay buffer were initially determined. The assay was performed in assay buffer [20 mM HEPES, pH 7.5, 5 mM MgCl2, and 0.1% (w/v) BSA with 200 mM NaCl, 3 M GDP] and membranes expressing human APJ receptor/well along with WGA PS beads. The reaction was initiated by addition of 0.2 nM [35S]GTPγS (Perkin Elmer Life and Analytical Sciences, Waltham USA) in the absence or presence of various ligands and incubated at RT for 90 min. Nonspecific binding was determined in the presence of 100 M GTPγS and was always less than 0.2% of total binding. All the results presented are means of several independent experiments and analyzed by non-linear regression methods using commercially available program Prism (GraphPad, San Diego, Calif.) to obtain EC50.
Measurement: EC50 = 160 nM (pChEMBL 6.800000190734863)
Descriptors: MW 795.69, ALogP 4.53, HBA 14, HBD 2, PSA 181.93, QED 0.18
Compound (SMILES): CCCCC(C(=O)Nc1ccc(C(=O)NC)nc1)n1cc(OC)c(-c2cc(Cl)ccc2-n2cnnn2)cc1=O
InChIKey: ORRIMSVBDDRCMC-UHFFFAOYSA-N
Target: Coagulation factor XI (SINGLE PROTEIN), Homo sapiens
UniProt: P03951
Assay: Inhibition Assay: The factor XIa inhibition of the substances according to the invention is determined using a biochemical test system which utilizes the reaction of a peptidic factor XIa substrate to determine the enzymatic activity of human factor XIa. Here, factor XIa cleaves from the peptic factor XIa substrate the C-terminal aminomethylcoumarin (AMC), the fluorescence of which is measured. The determinations are carried out in microtitre plates.Test substances are dissolved in dimethyl sulphoxide and serially diluted in dimethyl sulphoxide (3000 μM to 0.0078 μM; resulting final concentrations in the test: 50 μM to 0.00013 μM). In each case 1 μl of the diluted substance solutions is placed into the wells of white microtitre plates from Greiner (384 wells). 20 μl of assay buffer (50 mM of Tris/HCl pH 7.4; 100 mM of sodium chloride; 5 mM of calcium chloride; 0.1% of bovine serum albumin) and 20 μl of factor XIa from Kordia (0.45 nM in assay buffer) are then added successively. After 15 min of incubation, the enzyme reaction is started by addition of 20 μl of the factor XIa substrate Boc-Glu(OBzl)-Ala-Arg-AMC dissolved in assay buffer (10 μM in assay buffer) from Bachem, the mixture is incubated at room temperature (22° C.) for 30 min and fluorescence is then measured (excitation: 360 nm, emission: 460 nm).
Measurement: IC50 = 0.71 nM (pChEMBL 9.149999618530273)
Descriptors: MW 551.01, ALogP 3.28, HBA 10, HBD 2, PSA 145.92, QED 0.31
Compound (SMILES): Nc1ccc(-c2ccc(F)cc2)cc1NC(=O)c1nc2c(s1)CNCC2
InChIKey: ZNLJIPYPDKZHSX-UHFFFAOYSA-N
Target: Histone deacetylase 2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q92769
Assay: Reporter Displacement Assay: HDAC2 containing a C-terminal HIS-Tag (Proteros) and fluorescently-labeled anti-HIS-antibody are diluted in one vial in assay buffer 50 mM Tris, pH 8.0, 1 mM DTT, 150 mM NaCl, and 0.01% Tween20. Components are pre-incubated for 30 min, then a small volume of reporter probe (Proteros) is added from a highly concentrated stock and incubation is continued for another 30 min. Final concentrations after adding the reporter probe amount to 20 nM HDAC2, 4 nM antibody, and 180 nM probe.
Measurement: Kd = 300 nM (pChEMBL 6.519999980926514)
Descriptors: MW 368.44, ALogP 3.43, HBA 5, HBD 3, PSA 80.04, QED 0.62
Compound (SMILES): COc1cc2[nH]c([C@H](NC(=O)OC(C)(C)C)C3CCCC3)nc2cc1-c1ccncc1
InChIKey: APUCMDSYCNHJOL-OAQYLSRUSA-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 = 1803 nM (pChEMBL 5.739999771118164)
Descriptors: MW 422.53, ALogP 5.39, HBA 5, HBD 2, PSA 89.13, QED 0.58
Compound (SMILES): Cc1ccccc1-c1cc(C2CCC(CC(=O)O)CC2)nc(C#N)c1N(C)C(=O)C(C)(C)c1cc(C(F)(F)F)cc(C(F)(F)F)c1
InChIKey: RXOBEDAYMSZORF-UHFFFAOYSA-N
Target: Substance-P receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P25103
Assay: Receptor Binding Assay: The buffer solution for the receptor binding test was dispensed to the wells of a 96-well assay plate (Greiner) at 22.5 μL/well. DMSO solutions of a test compound, which were prepared at an 80-time higher concentration using 100% dimethyl sulfoxide (DMSO), were added to the wells at 2.5 μL/well (final concentrations of 1 nM to 100 nM), and the solutions were mixed. As a radiolabeled ligand, 125I-substance P (Substance P, [125I]Tyr8-, PerkinElmer) was used. 125I-substance P was diluted with the buffer solution for the receptor binding test to a concentration resulting in 125 pmol/25 μL/well and added to the 96-well assay plate, and the solutions were mixed. The membrane fraction prepared from the human NK1 receptor-expressing cells was diluted with the buffer solution for the receptor binding test to a concentration resulting in 8 to 10 μg/well, suspended until the suspension became in such a homogenous state that the suspension could flow through a 27 G injection needle smoothly and then added to the 96-well assay plate at 150 μL/well. Then, the plate was incubated at room temperature for 60 minutes while shaking the plate. The reaction solutions were suction-filtered through a multiscreen 96-well filter plate (Millipore) which had been pre-treated with 0.3% polyethyleneimine, and the reaction was terminated by washing with a washing solution (50 mM Tris and 0.02% bovine serum albumin, pH 7.4) four times. The bottom of the microplate was dried at 60° C., and then 100 μL/well of MicroScint 20 (PerkinElmer) was dispensed to the wells. The top of the plate was sealed with TopSeal A (PerkinElmer), and the plate was shaken for 5 to 10 minutes. Then, the radioactivities were measured with TopCount NXT (registered trademark) (PerkinElmer). The radioactivity of each well was calculated by subtracting the radioactivity of the well to which 10 μM aprepitant was added (non-specific binding). The binding rate (%) of 125I-substance P=(the radioactivity of the group to which the test compound was added)/(the radioactivity of the group to which the vehicle was added)×100 was calculated. Using analysis software, GraphPad Prism (GraphPad Software), the binding rate (%) was plotted against the concentration of the test compound and linearly approximated, and the concentration required for 50% inhibition, IC50, was calculated.
Measurement: IC50 = 2.78 nM (pChEMBL 8.5600004196167)
Descriptors: MW 645.64, ALogP 8.66, HBA 4, HBD 1, PSA 94.29, QED 0.26
Compound (SMILES): CC(C)O[C@H]1C[C@]2(CN(C(C)C)C(=O)c3c(O)c(=O)c(C(=O)NCc4ccc(F)cc4F)cn32)C1
InChIKey: FGCRCCUZOWTBAK-CKQQTVSASA-N
Target: Protein Nef (SINGLE PROTEIN), Human immunodeficiency virus 1
UniProt: P03406
Assay: Measurement of Antiviral (HIV-1) Activity: The medium (40 μL), a test substance (10 μL) diluted with the medium, and a 1×105 cells/mL MT-4 cell suspension (50 μL) wherein HIV-1 NL4-3 strain was infected with MOI (infection multiplicity) 0.05 were added to each well of a 96-well black plate (manufactured by Corning Incorporated), and the mixture was cultured at 37° C. for 5 days.medium composition: RPMI1640, 10% FBS, 100 U/mL penicillin, 100 μg/mL streptomycin.Then, Cell Titer-Glo (manufactured by Promega Corporation, 100 μL) was added to each well, and the mixture was stood at room temperature for 10 min, and the luminescence intensity was measured.
Measurement: EC50 = 6.4 nM (pChEMBL 8.1899995803833)
Descriptors: MW 489.52, ALogP 2.91, HBA 6, HBD 2, PSA 100.87, QED 0.65
Compound (SMILES): Nc1nnc(N2CCC(N3C[C@H](CN4CCC(F)(F)C4)OC[C@@H]3Cc3ccc(Cl)cc3)CC2)[nH]1
InChIKey: ICJVQDGNTVIVJX-PMACEKPBSA-N
Target: Chitotriosidase-1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q13231
Assay: AMCase Activity Assay: An enzymatic assay with recombinant human AMCase was used in order to establish inhibitory activity of the compounds (Boot et al., 2001, JBC:276). The assay was run in the 96-well plate format, each reaction in the total volume of 100 μL. 4-Methylumbelliferyl B-D-N,N′-diacetylchitobioside hydrate was used as a substrate for the enzyme. Upon hydrolysis by AMCase, the substrate releases 4-methylumbelliferyl (4MU) that, when ionized in basic pH, emits fluorescence at 460 nm. Briefly, 40 μL of a substrate was added to each well, followed by 10 μL of compound dilution and 50 μL of hAMCase recombinant enzyme solution. The reaction was carried out in citrate buffer, pH 5.2, in the dark, at 37° C. for 60 minutes with shaking. After that time the reaction was stopped by adding 195 μL of Stop Buffer (pH 10.5) to each well. The fluorescence of the reaction product was measured in Tecan Spark multimode plate reader at an excitation wavelength of 355 nm. The IC50 values were calculated using GraphPad Prism.
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 496.01, ALogP 2.66, HBA 7, HBD 2, PSA 86.54, QED 0.64
Compound (SMILES): O=C1O[C@@]23CCC1(O)C[C@@]21CCN(CC2CC2)[C@@H]3Cc2ccc(O)cc21
InChIKey: VHGDSYISPRNXMO-QKNTZHCYSA-N
Target: Mu-type opioid receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P35372
Assay: Mu-Opioid Receptor Binding Assay: Radioligand dose-displacement binding assays for μ-opioid receptors used 0.3 nM [3H]-diprenorphine (Perkin Elmer, Shelton, Conn.), with 5 mg membrane protein/well in a final volume of 500 μl binding buffer (10 mM MgCl2, 1 mM EDTA, 5% DMSO, 50 mM HEPES, pH 7.4). Reactions were carried out in the absence or presence of increasing concentrations of unlabeled naloxone. All reactions were conducted in 96-deep well polypropylene plates for 2 hours at room temperature. Binding reactions were terminated by rapid filtration onto 96-well Unifilter GF/C filter plates (Perkin Elmer, Shelton, Conn.), presoaked in 0.5% polyethylenimine using a 96-well tissue harvester (Perkin Elmer, Shelton, Conn.) followed by performing three filtration washes with 500 μl of ice-cold binding buffer. Filter plates were subsequently dried at 50° C. for 2-3 hours. BetaScint scintillation cocktail (Perkin Elmer, Shelton, Conn.) was added (50 μl/well), and plates were counted using a Packard Top-Count for 1 min/well. The data were analyzed using the one-site competition curve fitting functions in GraphPad PRISM v. 3.0 or higher (San Diego, Calif.), or an in-house function for one-site competition curve-fitting.
Measurement: Ki = 10.1 nM (pChEMBL 8)
Descriptors: MW 355.43, ALogP 1.88, HBA 5, HBD 2, PSA 70, QED 0.79
Compound (SMILES): COOCOC[C@H]1C[C@H]2COc3c(Sc4cnc(N5CCC6(CC5)CO[C@@H](C)[C@H]6N)c(CO)n4)ccnc3N2C1
InChIKey: GLWVMMIZBMQCRV-GSRZOBFVSA-N
Target: Tyrosine-protein phosphatase non-receptor type 11 (SINGLE PROTEIN), Homo sapiens
UniProt: Q06124
Assay: SHP2 Allosteric Inhibition Assay: SHP2 possesses two N-terminal Src homology 2 (SH2) domains, a central protein-tyrosine phosphatase (PTP) domain, and C-terminal tail. At the basal state, SHP2 is auto-inhibited and access of substrates to the catalytic site is blocked by the intermolecular interactions between the SH2 domains and the PTP domain. When bis-tyrosyl-phosphorylated peptides bind to SH2 domain of SHP2, the PTP domain becomes available for substrate recognition and reaction catalysis and SHP2 is allosterically activated. SHP2 catalytic activity can be measured using a fluorogenic artificial substrate DiFMUP.The phosphatase reactions were carried out at room temperature in 384-well black polystyrene plates (Greiner Bio-One, Cat #784076) using assay buffers containing 60 mM HEPES, pH 7.2, 75 mM NaCl, 75 mM KCl, 1 mM EDTA, 0.05% P-20, and 5 mM DTT.0.33 nM of SHP2 was co-incubated with of 0.5 μM of bisphos-IRS 1 peptide (sequence: H2N-LN(pY)IDLDLV(dPEG8)LST(pY)ASINFQK-amide) and various concentrations of compounds for 30-60 min at room temperature. Then the reaction was initiated by addition of the surrogate substrate DiFMUP (Invitrogen, Cat #D6567, 100 uM final).The real-time conversion of DiFMUP to DiFMU (6,8-difluoro-7-hydroxyl-4-methyl-coumarin) was measured every 5 min for 30 min using a microplate reader (CLARIOstar, BMG Labtech) with excitation and emission wavelengths of 340 nm and 450 nm, respectively. Initial reaction rates were determined by linear fitting of the data and 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 574.7, ALogP 1.99, HBA 13, HBD 2, PSA 137.55, QED 0.2
Compound (SMILES): CNCCN1CCC(c2ccc3[nH]c(-c4ccnc(OC)c4)c(C(C)C)c3c2)CC1
InChIKey: MYXBEUCLMYJADF-UHFFFAOYSA-N
Target: Toll-like receptor 7 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9NYK1
Assay: Inhibition Reporter Assay: HEK-Blue-cells (Invivogen) overexpressing human TLR7, TLR8 or TLR9 receptors were used for screening inhibitors of these receptors using an inducible SEAP (secreted embryonic alkaline phosphatase) reporter gene under the control of the IFN-β minimal promoter fused to five NF-κB and AP-1-binding sites. Briefly, cells are seeded into Greiner 384 well plates (15000 cells per well for TLR7, 20,000 for TLR8 and 25,000 for TLR9) and then treated with test compounds in DMSO to yield a final dose response concentration range of 0.05 nM-50 μM. After a 30 minute compound pre-treatment at room temperature, the cells are then stimulated with a TLR7 ligand (gardiquimod at a final concentration of 7.5 μM), TLR8 ligand (R848 at a final concentration of 15.9 μM) or TLR9 ligand (ODN2006 at a final concentration of 5 nM) to activate NF-κB and AP-1 which induce the production of SEAP. After a 22 hour incubation at 37° C., 5% CO2, SEAP levels are determined with the addition of HEK-Blue Detection reagent (Invivogen), a cell culture medium that allows for detection of SEAP, according to manufacturer's specifications.
Measurement: IC50 = 55.1 nM (pChEMBL 7.260000228881836)
Descriptors: MW 406.57, ALogP 4.76, HBA 4, HBD 2, PSA 53.18, QED 0.59
Compound (SMILES): Cc1nc2cnc(N(C)C3CCN(CC4CC4)CC3)c(C#N)c2c(=O)n1CCOc1ccc(Cl)cc1-c1ccnc2c(C(=O)O)csc12
InChIKey: MCDLKDZRWLJGRQ-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 = 19.9 nM (pChEMBL 7.699999809265137)
Descriptors: MW 684.22, ALogP 5.99, HBA 11, HBD 1, PSA 137.47, QED 0.19
Compound (SMILES): CO[C@H]1[C@@H](C(=O)N[C@H]2CCCC(C)(C)C2)N(C(=O)Cn2nc(C(N)=O)c3ccccc32)C[C@@H]1F
InChIKey: VNIUXPGGFOMUNC-PQOQACGUSA-N
Target: Complement factor D (SINGLE PROTEIN), Homo sapiens
UniProt: P00746
Assay: Human Complement Factor D Assay: Method 1: Recombinant human factor D (expressed in E. coli and purified using standard methods) at 10 nM concentration is incubated with test compound at various concentrations for 1 hour at room temperature in 0.1 M Hepes buffer, pH 7.5, containing 1 mM MgCl2, 1 M NaCl and 0.05% CHAPS. A synthetic substrate Z-Lys-thiobenzyl and 2,4-dinitrobenzenesulfonyl-fluoresceine are added to final concentrations of 200 μM and 25 μM, respectively. The increase in fluorescence is recorded at excitation of 485 nm and emission at 535 nm in a microplate spectrofluorimeter. IC50 values are calculated from percentage of inhibition of complement factor D-activity as a function of test compound concentration.
Measurement: IC50 = 585 nM (pChEMBL 6.230000019073486)
Descriptors: MW 473.55, ALogP 1.78, HBA 6, HBD 2, PSA 119.55, QED 0.66