lovingscience/chembl-v37-raw-activities huggingface dataset:
Compound (SMILES): CCn1c(-c2cccnc2[C@H](C)OC)c2c3cc(ccc31)-c1cccc(c1)C[C@H](NC(=O)[C@H](C(C)C)N1CC[C@@]3(CCN(C(=O)C#C[C@H]4COCCN4C)C3)C1=O)C(=O)N1CCC[C@H](N1)C(=O)OCC(C)(C)C2
InChIKey: MWHJAHJNBCRHKM-GSOCGLHPSA-N
Target: GTPase KRas (SINGLE PROTEIN), Homo sapiens
UniProt: P01116
Assay: Biochemical Assay: A variety of Ras proteins may be inhibited by compounds of the present invention (e.g., K-Ras, N-Ras, H-Ras, and mutants thereof at positions 12, 13 and 61, such as G12C, G12D, G12V, G12S, G13C, G13D, and Q61L, and others described herein). The purpose of this biochemical assay is to measure the ability of test compounds to covalently label nucleotide-loaded K-Ras isoforms. In assay buffer containing 12.5 mM HEPES pH 7.4, 75 mM NaCl, 1 mM MgCl2, 1 mM BME, 5 uM Cyclophilin A and 2 uM test compound, a 5 uM stock of GMP-PNP-loaded K-Ras (1-169) G12C is diluted 10-fold to yield a final concentration of 0.5 uM; with final sample volume being 100 uL. The sample is incubated at 25�� C. for a time period of up to 24 hours prior to quenching by the addition of 10 uL of 5% Formic Acid. Quenched samples are centrifuged at 15000 rpm for 15 minutes in a benchtop centrifuge before injecting a 10 uL aliquot onto a reverse phase C4 column and eluting into the mass spectrometer with an increasing acetonitrile gradient in the mobile phase. Analysis of raw data may be carried out using Waters MassLynx MS software, with % bound calculated from the deconvoluted protein peaks for labeled and unlabeled K-Ras.
Measurement: IC50 < 10 nM (pChEMBL 8)
Descriptors: MW 997.25, ALogP 5.56, HBA 12, HBD 2, PSA 167.88, QED 0.17
Compound (SMILES): N[C@H]1CC[C@@H](Nc2nc(Nc3cc(Cl)cc(Cl)c3)ncc2-c2cnn(C3CCNCC3)c2)CC1
InChIKey: WLCODBRLUVZHCP-KDURUIRLSA-N
Target: Tyrosine-protein kinase Mer (SINGLE PROTEIN), Homo sapiens
UniProt: Q12866
Assay: Verification on Inhibitory Activities of TAM Receptor Inhibiting Compounds on Tyro 3, Axl, and Mer: For evaluation of inhibitory activities of the compounds according to the present invention on Tyro 3, Axl, and Mer, the following test was carried out.The specific test method followed the method provided by Cisbio. The compounds of the examples were prepared at various concentrations, followed by addition of Tyro 3, Axl, or Mer and substrate peptides, and then ATP was added to initiate a reaction. After 1 hour, the reaction was stopped by addition of a solution containing EDTA. Thereafter, the amount of phosphorylated peptides was measured. Here, the phosphorylated peptides were treated with an europium (Eu)-labeled antibody recognizing phosphorylated peptides, and then after 1 hour, excited by irradiation of the light of a wavelength of 320 or 340 nm using an Envision Reader. The amount of light emitted at 665 nm was measured to investigate the degrees of inhibition of TAM receptors. The TAM receptor inhibitory activities (IC50 values) of the respective compounds were analyzed using the GraphPad Prism program, and tabulated in Tables 1 to 3 below. LDC1267 (CAS no. 1361030-48-9, Calbiochem), which is a TAM receptor inhibitor compound, was used as a positive control.
Measurement: IC50 = 45 nM (pChEMBL 7.349999904632568)
Descriptors: MW 501.47, ALogP 5, HBA 8, HBD 4, PSA 105.71, QED 0.38
Compound (SMILES): C#Cc1ccc(CN2C(=O)c3cc(C(C)(C)O)cc(F)c3[C@]2(OCC2(CO)CC2)c2ccc(Cl)cc2)cc1
InChIKey: NBPAQCMFLGCFBK-WJOKGBTCSA-N
Target: E3 ubiquitin-protein ligase Mdm2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q00987
Assay: Binding Assay (ELISA): The ELISA assay was performed in streptavidin coated plates which were preincubated with 200 ul per well of 1 μg ml−1 biotinylated IP3 peptide. The plates were ready to use for MDM2 binding after washing the plate with PBS.Compounds and control solutions in DMSO aliquoted in 96-well plates were pre-incubated in a final 2.5-5% (v/v) DMSO concentration at room temperature (for example 20C.) for 20 min with 190 ul aliquots of optimized concentrations of in vitro translated MDM2, before transfer of the MDM2-compound mixture to the b-IP3 streptavidin plates, and incubation at 4C. for 90 min. After washing three times with PBS to remove unbound MDM2, each well was incubated at 20C. for 1 hour with a TBS-Tween (50 mM Tris pH7.5; 150 mM NaCl; 0.05% Tween 20 nonionic detergent) buffered solution of primary mouse monoclonal anti-MDM2 antibody (Ab-5, Calbiochem, used at a 1/10000 or 1/200 dilution depending on the antibody stock solution used), then washed three times with TBS-Tween before incubation for 45 mins at 20C. with a TBS-Tween buffered solution of a goat-anti-mouse horseradish peroxidase (HRP) conjugated secondary antibody (used at 1/20000 or 1/2000 depending on the antibody stock solution). The unbound secondary antibody was removed by washing three times with TBS-Tween. The bound HRP activity was measured by enhanced chemiluminesence (ECL, Amersham Biosciences) using the oxidation of the diacylhydrazide substrate, luminol, to generate a quantifiable light signal. The percentage of MDM2 inhibition at a given concentration is calculated as the [1−(RLU detected in the compound treated sample−RLU negative DMSO control)÷(RLU of DMSO positive and negative controls)]x100 or as the (RLU detected in the compound treated sample ÷ RLU of DMSO controls)x100.
Measurement: IC50 = 5.2 nM (pChEMBL 8.279999732971191)
Descriptors: MW 534.03, ALogP 5.33, HBA 4, HBD 2, PSA 70, QED 0.38
Compound (SMILES): CCCn1cc(-c2cnc(Nc3cc(Cl)cc(Cl)c3)nc2Nc2ccc3c(c2)CNCC3)cn1
InChIKey: DYTOVPQOWGLXKV-UHFFFAOYSA-N
Target: Tyrosine-protein kinase Mer (SINGLE PROTEIN), Homo sapiens
UniProt: Q12866
Assay: Verification on Inhibitory Activities of TAM Receptor Inhibiting Compounds on Tyro 3, Axl, and Mer: For evaluation of inhibitory activities of the compounds according to the present invention on Tyro 3, Axl, and Mer, the following test was carried out.The specific test method followed the method provided by Cisbio. The compounds of the examples were prepared at various concentrations, followed by addition of Tyro 3, Axl, or Mer and substrate peptides, and then ATP was added to initiate a reaction. After 1 hour, the reaction was stopped by addition of a solution containing EDTA. Thereafter, the amount of phosphorylated peptides was measured. Here, the phosphorylated peptides were treated with an europium (Eu)-labeled antibody recognizing phosphorylated peptides, and then after 1 hour, excited by irradiation of the light of a wavelength of 320 or 340 nm using an Envision Reader. The amount of light emitted at 665 nm was measured to investigate the degrees of inhibition of TAM receptors. The TAM receptor inhibitory activities (IC50 values) of the respective compounds were analyzed using the GraphPad Prism program, and tabulated in Tables 1 to 3 below. LDC1267 (CAS no. 1361030-48-9, Calbiochem), which is a TAM receptor inhibitor compound, was used as a positive control.
Measurement: IC50 = 150 nM (pChEMBL 6.820000171661377)
Descriptors: MW 494.43, ALogP 6.19, HBA 7, HBD 3, PSA 79.69, QED 0.28
Compound (SMILES): CCCCNc1ncc(-c2ccc(C(=O)N(C)C3CCN(C)CC3)cn2)c(NC2CCC(O)CC2)n1
InChIKey: PXHDQHNAGHIZGO-UHFFFAOYSA-N
Target: Receptor-type tyrosine-protein kinase FLT3 (SINGLE PROTEIN), Homo sapiens
UniProt: P36888
Assay: Activity Assay: Briefly, activity assays were performed in a 384 well, polypropylene microplate in a final volume of 50 μL of 50 mM Hepes, Ph 7.4 containing 10 mM MgCl2, 1.0 mM DTT, 0.01% Triton X-100, 0.1% Bovine Serum Albumin (BSA), containing 1.0 μM fluorescent substrate and ATP at the Km for each enzyme. All reactions were terminated by addition of 20 μL of 70 mM EDTA. After a 180 min incubation, phosphorylated and unphosphorylated substrate peptides were separated in buffer supplemented with 1×CR-8 on a LabChip EZ Reader equipped with a 12-sipper chip. Data were analyzed using EZ Reader software. Assay conditions for MCE assays
Measurement: IC50 = 5.1 nM (pChEMBL 8.289999961853027)
Descriptors: MW 495.67, ALogP 3.63, HBA 8, HBD 3, PSA 106.51, QED 0.45
Compound (SMILES): CC1=NN(C(C)C)/C(=C(/C=C/c2cnn(C)c2C)NCc2cnn(C)c2)C1
InChIKey: DURMBXZBTJGGSF-DFCCZADTSA-N
Target: Dual specificity calcium/calmodulin-dependent 3',5'-cyclic nucleotide phosphodiesterase 1C (SINGLE PROTEIN), Homo sapiens
UniProt: Q14123
Assay: Inhibition Assay: PDE1A, PDE1B and PDE1C assays were performed as follows: the assays was performed in 60 uL samples containing a fixed amount of the PDE1 enzyme (sufficient to convert 20-25% of the cyclic nucleotide substrate), a buffer (50 mM HEPES pH 7.6; 10 mM MgCl2; 0.02% Tween20), 0.1 mg/ml BSA, 15 nM tritium labeled cAMP and varying amounts of inhibitors. Reactions were initiated by addition of the cyclic nucleotide substrate, and reactions were allowed to proceed for 1 hr at room temperature before being terminated through mixing with 20 uL (0.2 mg) yttrium silicate SPA beads (PerkinElmer). The beads were allowed to settle for 1 hr in the dark before the plates were counted in a Wallac 1450 Microbeta counter. The measured signals were converted to activity relative to an uninhibited control (100%) and IC50 values were calculated using XIFit (model 205, IDBS).
Measurement: IC50 = 220 nM (pChEMBL 6.659999847412109)
Descriptors: MW 367.5, ALogP 2.97, HBA 7, HBD 1, PSA 63.27, QED 0.85
Compound (SMILES): COc1cc(OC)cc(N(CCNC(C)C)c2cc3cc(-c4cnn(C)c4)cnc3cn2)c1
InChIKey: FRKAOJSMULEWJT-UHFFFAOYSA-N
Target: Fibroblast growth factor receptor 3 (SINGLE PROTEIN), Homo sapiens
UniProt: P22607
Assay: FGFR3 (Enzymatic Assay): In a final reaction volume of 30 μL, FGFR3 (h) (40 ng/ml) was incubated with 50 mM HEPES pH 7.5, 6 mM MnCl2, 1 mM DTT, 0.1 mM Na3VO4, 0.01% Triton-X-100, 500 nM Btn-Flt3 and 25 μM ATP in the presence of compound (1% DMSO final). After incubation for 60 minutes at room temperature the reaction was stopped with 2.27 nM EU-anti P-Tyr, 7 mM EDTA, 31.25 nM SA-XL-665 and 0.02% BSA which was present for 60 minutes at room temperature. Time-Resolved Fluorescence Resonance Energy Transfer (TR-FRET) signal (ex340 nm. Em 620 nm, em 655 nm) was measured afterwards and results are expressed in RFU (Relative Fluorescence Units). In this assay, the inhibitory effect of different compound concentrations (range 10 μM to 0.1 nM) was determined and used to calculate an IC50 (M) and pIC50 (−log IC50) value.
Measurement: IC50 = 174 nM (pChEMBL 6.760000228881836)
Descriptors: MW 446.56, ALogP 4.18, HBA 8, HBD 1, PSA 77.33, QED 0.41
Compound (SMILES): N#CCC1(n2ccc(-c3nc(-c4cnc5c(c4)CCC5)cc4ncccc34)n2)CN(CC(F)(F)F)C1
InChIKey: KLQJEKRSINSXAG-UHFFFAOYSA-N
Target: Tyrosine-protein kinase JAK2 (SINGLE PROTEIN), Homo sapiens
UniProt: O60674
Assay: JAK Caliper Enzyme Assay at 1mM ATP: Compounds were added to a 384-well plate. Reaction mixtures contained 10 mM HEPES, pH 7.4, 10 mM MgCl2, 0.01% BSA, 0.0005% Tween 20, 1 mM ATP and 1 μM peptide substrate. The JAK1 and TYK2 assays contained 1 μM of the IRStide peptide (5FAM-KKSRGDYMTMQID) and the JAK2 and JAK3 assays contained 1 μM of the JAKtide peptide (FITC-KGGEEEEYFELVKK). The assays were initiated by the addition of 20 nM JAK1, 1 nM JAK2, 1 nM JAK or 1 nM TYK2 enzyme and were incubated at room temperature for three hours for JAK1, 60 minutes for JAK2, 75 minutes for JAK3 or 135 minutes for TYK2. Enzyme concentrations and incubation times were optimized for each new enzyme preps and were modified slightly over time to ensure 20% to 30% phosphorylation. The assays were stopped with 15 μL of 180 mM HEPES, pH 7.4, 20 mM EDTA, and 0.2% Coating Reagent 3. The assay plates were placed on a Caliper Life Science LC3000 instrument, and each well was sampled using appropriate separation conditions to measure the unphosphorylated and phosphorylated peptide.
Measurement: IC50 = 5816 nM (pChEMBL 5.239999771118164)
Descriptors: MW 489.51, ALogP 4.53, HBA 7, HBD 0, PSA 83.52, QED 0.41
Compound (SMILES): CS(=O)(=O)C1CN(Cc2ccc(-c3cccn4nc(NC(=O)C5CC5)nc34)cc2F)C1
InChIKey: QAIJWLIWVCJZOT-UHFFFAOYSA-N
Target: Tyrosine-protein kinase JAK3 (SINGLE PROTEIN), Homo sapiens
UniProt: P52333
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 > 50 nM (pChEMBL 7.301000118255615)
Descriptors: MW 443.5, ALogP 2.11, HBA 7, HBD 1, PSA 96.67, QED 0.63
Compound (SMILES): COc1cc2nccc(Oc3ccc(NC(=O)C4(C(=O)Nc5ccc(F)cc5)CC4)cc3)c2cc1C1CC1
InChIKey: MOIHTTVIDKDORZ-UHFFFAOYSA-N
Target: Hepatocyte growth factor receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P08581
Assay: Met Kinase Assay: Human Met (residues R974-S1390 with A1209G and V1290L, 3.4 nM) was incubated with 8 mM MOPS pH 7.0, 0.2 mM EDTA, 250 μM KKKGQEEEYVFIE (SEO ID NO. 45), 1 mM sodium orthovanadate, 5 mM sodium-6-glycerophosphate, 10 mM magnesium acetate, and 10 μM [γ-33P-ATP]. The reaction was initiated by the addition of the Mg/ATP mix. After incubation for 40 minutes at room temperature, the reaction was stopped by the addition of phosphoric acid to a concentration of 0.5%. A reaction aliquot of 10 sL was then spotted onto a P30 filtermat and washed four times for 4 minutes in 0.425% phosphoric acid and once in methanol prior to drying and scintillation counting. Incorporated 33P was measured using the Wallac Microbeta scintillation counter (Perkin Elmer).
Measurement: IC50 = 55 nM (pChEMBL 7.260000228881836)
Descriptors: MW 511.55, ALogP 6.41, HBA 5, HBD 2, PSA 89.55, QED 0.27
Compound (SMILES): Nc1ncnc2c1c(/C=C1/CCNC1=O)nn2Cc1cc2ccccc2nc1-c1ccccc1F
InChIKey: KMERYFXNUAIPJF-VBKFSLOCSA-N
Target: Phosphatidylinositol 4-phosphate 3-kinase C2 domain-containing subunit beta (SINGLE PROTEIN), Homo sapiens
UniProt: O00750
Assay: Inhibition Assay: Class I PI3-Ks can be either purchased (p110α/p85α, p110β/p85α, p110δ/p85α from Upstate, and p110γ from Sigma) or expressed as previously described (Knight et al., 2004). IC50 values are measured using either a standard TLC assay for lipid kinase activity (described below) or a high-throughput membrane capture assay. Kinase reactions are performed by preparing a reaction mixture containing kinase, inhibitor (2% DMSO final concentration), buffer (25 mM HEPES, pH 7.4, 10 mM MgCl2), and freshly sonicated phosphatidylinositol (100 μg/ml). Reactions are initiated by the addition of ATP containing 10 μCi of γ-32P-ATP to a final concentration 10 or 100 μM and allowed to proceed for 5 minutes at room temperature. For TLC analysis, reactions are then terminated by the addition of 105 l 1N HCl followed by 160 μl CHCl3:MeOH (1:1). The biphasic mixture is vortexed, briefly centrifuged, and the organic phase is transferred to a new tube using a gel loading pipette tip precoated with CHCl3. This extract is spotted on TLC plates and developed for 3-4 hours in a 65:35 solution of n-propanol:1M acetic acid. The TLC plates are then dried, exposed to a phosphorimager screen (Storm, Amersham), and quantitated. For each compound, kinase activity is measured at 10-12 inhibitor concentrations representing two-fold dilutions from the highest concentration tested (typically, 200 μM). For compounds showing significant activity, IC50 determinations are repeated two to four times, and the reported value is the average of these independent measurements.
Measurement: IC50 > 1000 nM (pChEMBL 6)
Descriptors: MW 465.49, ALogP 3.71, HBA 7, HBD 2, PSA 111.61, QED 0.39
Compound (SMILES): CC1C2CC(Oc3ccc(C(F)(F)F)cn3)C(C2)N1C(=O)c1cc(F)ccc1-c1ncccn1
InChIKey: VWQZZPXHGNEBLW-UHFFFAOYSA-N
Target: Orexin/Hypocretin receptor type 1 (SINGLE PROTEIN), Rattus norvegicus
UniProt: P56718
Assay: Receptor Radioligand Binding Assay: Human Embryonic Kidney 293 cells (HEK293) stably expressing rat orexin 1 receptor (Genebank accession number NM_001525) or Chinese ovary cells (CHO) stably expressing human orexin 1 receptor (Genebank accession number NM_001526) were grown to confluency in DMEM (Hyclone, cat # SH30022), 10% FBS, 1× Pen/Strep, 1× sodium pyruvate, 10 mM HEPES, 600 μg/mL G418 and DMEM/F12 (Gibco, Cat #11039), 10% FBS, 1× Pen/Strep, 600 μg/mL G418 media, respectively on 150 cm2 tissue culture plates, washed with 5 mM EDTA in PBS (HyClone Dulbecco's Phosphate Buffered Saline 1× with Calcium and Magnesium, Cat # SH30264.01, hereafter referred to simply as PBS) and scraped into 50 ml tubes. After centrifugation (2K×G, 5 min at 4° C.), the supernatant was aspirated and the pellets frozen and stored at −80° C. Cells were resuspended in PBS in the presence of 1 tablet of protease inhibitor cocktail (Roche, Cat. #11836145001) per 50 mL. Each cell pellet from a 15 cm plate was resuspended in 10 mL, stored on ice, and homogenized for 45 sec prior to addition to the reactions. Competition binding experiments in 96 well polypropylene plates were performed using [3H] (1-(5-(2-fluoro-phenyl)-2-methyl-thiazol-4-yl)-1-((S)-2-(5-phenyl-(1,3,4)oxadiazol-2-ylmethyl)-pyrrolidin-1-yl)-methanone) (Moraveck Corporation, specific activity=35.3 Ci/mmol), diluted to a 10 nM concentration in PBS (4 nM final). Compounds were solubilized in 100% DMSO (Acros Organics, Cat. #61042-1000) and tested over a range of 7 concentrations (from 0.1 nM to 10 μM). The final concentration of DMSO in the reactions is equal to or less than 0.1%. Total and nonspecific binding was determined in the absence and presence of 10 μM almorexant. The total volume of each reaction is 200 μL (20 μL of diluted compounds, 80 μL of [3H]¿ (1-(5-(2-fluoro-phenyl)-2-methyl-thiazol-4-yl)-1-((S)-2-(5-phenyl-(1,3,4)oxadiazol-2-ylmethyl)-pyrrolidin-1-yl)-methanone) diluted in PBS and 100 μL of the cell suspension). Reactions were run for 60 min at room temperature and terminated by filtration through GF/C filter plates (PerkinElmer, Cat. #6005174) presoaked in 0.3% polyethylenimine using the cell harvester (PerkinElmer Filtermate).
Measurement: Ki = 3 nM (pChEMBL 8.520000457763672)
Descriptors: MW 472.44, ALogP 4.77, HBA 5, HBD 0, PSA 68.21, QED 0.51
Compound (SMILES): C[C@]1(c2cc(/C=C(\F)c3cnc(OCC(F)(F)F)cn3)ccc2F)N=C(N)[C@@]2(CF)CC[C@@H]1S2(=O)=O
InChIKey: LJHVTKLLYRJZCG-KUDAXZBBSA-N
Target: Beta-secretase 2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9Y5Z0
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 = 167 nM (pChEMBL 6.78000020980835)
Descriptors: MW 534.48, ALogP 3.9, HBA 7, HBD 1, PSA 107.53, QED 0.56
Compound (SMILES): C[C@H](NC(=O)c1c(N)nn2cccnc12)c1cc2cccc(Cl)c2c(=O)n1CCNS(=O)(=O)C(F)(F)F
InChIKey: IHKFWOVCWDDPID-NSHDSACASA-N
Target: Phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit gamma isoform (SINGLE PROTEIN), Homo sapiens
UniProt: P48736
Assay: Inhibition Assay: Class I PI3-Ks can be either purchased (p110α/p85α, p110β/p85α, p110δ/p85α from Upstate, and p110γ from Sigma) or expressed as previously described (Knight et al., 2004). IC50 values are measured using either a standard TLC assay for lipid kinase activity (described below) or a high-throughput membrane capture assay. Kinase reactions are performed by preparing a reaction mixture containing kinase, inhibitor (2% DMSO final concentration), buffer (25 mM HEPES, pH 7.4, 10 mM MgCl2), and freshly sonicated phosphatidylinositol (100 μg/ml). Reactions are initiated by the addition of ATP containing 10 μCi of γ-32P-ATP to a final concentration of 10 or 100 μM and allowed to proceed for 5 minutes at room temperature. For TLC analysis, reactions are then terminated by the addition of 105 μL 1N HCl followed by 160 μL CHCl3:MeOH (1:1). The biphasic mixture is vortexed, briefly centrifuged, and the organic phase is transferred to a new tube using a gel loading pipette tip precoated with CHCl3. This extract is spotted on TLC plates and developed for 3-4 hours in a 65:35 solution of n-propanol:1M acetic acid. The TLC plates are then dried, exposed to a phosphorimager screen (Storm, Amersham), and quantitated. For each compound, kinase activity is measured at 10-12 inhibitor concentrations representing two-fold dilutions from the highest concentration tested (typically, 200 μM). For compounds showing significant activity, IC50 determinations are repeated two to four times, and the reported value is the average of these independent measurements.
Measurement: IC50 = 750 nM (pChEMBL 6.119999885559082)
Descriptors: MW 557.94, ALogP 2.21, HBA 9, HBD 3, PSA 153.48, QED 0.31
Compound (SMILES): COc1ccc2c(c1)[C@]1(C[C@H]1c1ccc3c(C#Cc4ccc(CN5CCN(C)CC5)cc4)n[nH]c3c1)C(=O)N2
InChIKey: JDGANQKXCPYAQB-UCGGBYDDSA-N
Target: Aurora kinase B (SINGLE PROTEIN), Homo sapiens
UniProt: Q96GD4
Assay: Inhibition Assay: Aurora B inhibition was determined using the Z-Lyte assay kit from Invitrogen. The assay was performed using the recommended manufacturer's instructions with 128 μM ATP and 28 nM Aurora B (Invitrogen cat #PV3970). The % inhibition values were determined according to the manufacturer's directions and IC50 values were obtained using a non-linear 4 point logistic curve fit (XLfit4, IDBS)
Measurement: IC50 = 550 nM (pChEMBL 6.260000228881836)
Descriptors: MW 517.63, ALogP 4.1, HBA 5, HBD 2, PSA 73.49, QED 0.4
Compound (SMILES): [C-]#[N+]CN1CCC(c2ccc3[nH]c(-c4cc(F)c5ncnn5c4)c(C(C)C)c3c2)CC1
InChIKey: PJUKAVNPKCVRQK-UHFFFAOYSA-N
Target: Toll-like receptor 9 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9NR96
Assay: TLR7/8/9 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. The percent inhibition is determined as the % reduction in the HEK-Blue signal present in wells treated with agonist plus DMSO alone compared to wells treated with a known inhibitor.
Measurement: IC50 = 3957 nM (pChEMBL 5.400000095367432)
Descriptors: MW 416.5, ALogP 5.2, HBA 4, HBD 1, PSA 53.58, QED 0.47
Compound (SMILES): O=C1COc2c(F)cc(CCN3CCN(c4cccc5sccc45)CC3)cc2N1
InChIKey: GCEJUCPMFGNYTI-UHFFFAOYSA-N
Target: 5-hydroxytryptamine receptor 2A (SINGLE PROTEIN), Homo sapiens
UniProt: P28223
Assay: 5-HT2A Receptor Antagonism Activity Test: The 5-HT2A receptor antagonism activity test (The antagonism activity of test compounds on 5-HT2A receptor expressing human recombinant 5-HT2A receptor in CHO-K1 cells) was performed using FLIPR Calcium 5 Assay Kit (Product of Molecular Devices USA Inc.) according to the method documented in the kit instructions. The concentration of the test compounds was 0.1 nM-10000 nM and risperidone was used as a positive control. Test method is as follows: On the first day the seed cells are placed in T-175 flask containing 25 ml growth medium (F-12 nutrient mixture+10% FBS+1% penicillin/streptomycin+1.2% 50 mg/ml Geneticin) at a density of 14 million per bottle, cells were cultured under 37° C./5% CO2/humidified conditions for 24 hours; On the next day the seed cells were inoculated to the 384-well cell culture plate (each well containing 20,000 cells), the growth medium was replaced by 50 μL detecting medium (F-12 nutrient mixture+1.5% activated carbon-treated FBS), the cells were cultured under 37° C./5% CO2/humidified conditions for 16 hours; On the third day the medium was removed, each well of the cells plates were added with 24 μL freshly prepared loading dye solution (formulated according to the instructions), the plates were placed in an incubator and incubated under 37° C./5% CO2/humidified conditions for 120 minutes; transfer 6 μL test compound solution to the assay plate and gently shaking for one minute, incubated under 37° C./5% CO2/humidified conditions for 30 minutes; each well of the assay plate was added with 10 μL freshly prepared α-methyl-5-hydroxytryptamine solution (1.2 M, the final concentration of α-methyl-5-hydroxytryptamine is 300 nM), data were detected and analyzed by FLIPR (Product of US Molecular Devices Corporation). Inhibition rate of the compounds at different concentrations was calculated, IC50 value was calculated by the Excelfit software
Measurement: IC50 = 104 nM (pChEMBL 6.980000019073486)
Descriptors: MW 411.5, ALogP 3.74, HBA 5, HBD 1, PSA 44.81, QED 0.71
Compound (SMILES): CN1CCO[C@H](COc2cc(-c3ccc(N4CCC(C)(NC(=O)c5ncccc5Cl)CC4)nc3)c3c(C#N)cnn3c2)C1
InChIKey: KXPJVOALQYUCLC-DEOSSOPVSA-N
Target: Proto-oncogene tyrosine-protein kinase receptor Ret (SINGLE PROTEIN), Homo sapiens
UniProt: P07949
Assay: RET G810R Mutant Assay: The potency of a compound inhibiting G81 OR mutant RET kinase was determined using CisBio's HTRF Kinease-TK assay technology. The assays contained G81OR mutant RET produced at Array Biopharma, Inc. (1 nM enzyme p1982 Lot. No. 160713. The kinase was incubated with 250 nM TK-substrate biotin (CisBio, part of Catalog #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 as a three-fold serial dilution in DMSO and added to the assay to give the appropriate final concentration. After a 60-min incubation at 22° C., the reaction was quenched by adding 8 μL of quench solution containing 31.25 nM Sa-XL665 and 1×TK-Ab-Cryptate in HTRF detection buffer (all from CisBio, part of cat #62TK0PEC). After a 1-h incubation at 22° C., the extent of reaction was determined using a PerkinElmer EnVision multimode plate reader via HTRF dual wavelength detection, and the percent of control (POC) was calculated using a ratiometric emission factor. One hundred POC was determined using no test compounds, and 0 POC was determined using pre-quenched control reactions. A 4-parameter logistic curve was fit to the POC values as a function of the concentration of compound, and the IC50 value was the point where the best-fit curve crossed 50 POC.
Measurement: IC50 = 55 nM (pChEMBL 7.260000228881836)
Descriptors: MW 601.11, ALogP 3.81, HBA 10, HBD 1, PSA 120.91, QED 0.34
Compound (SMILES): Cc1ccc(-n2nc(C(C)C)cc2NC(=O)Nc2ccc(Oc3ccnc(Nc4cc(C)cc(C(=O)NCCN5CCOCC5)c4)n3)c3ccccc23)cc1
InChIKey: IHERTXREJRKYHR-UHFFFAOYSA-N
Target: Tyrosine-protein kinase SYK (SINGLE PROTEIN), Homo sapiens
UniProt: P43405
Assay: Syk Enzyme Inhibition 1: The inhibitory activities of compounds of the invention against Syk enzymes (Invitrogen), are evaluated in a similar fashion to that described hereinabove. The relevant enzyme (2000 ng/mL, 2.5 μL) is incubated with the test compound (either 4 μg/mL, 0.4 μg/mL, 0.04 μg/mL, or 0.004 μg/mL, 2.5 μL each) for 2 hr at RT. The FRET peptides (8 μM, 2.5 μL), and appropriate ATP solutions (2.5 μL, 60 μM ATP) are then added to the enzymes/compound mixtures and incubated for 1 hr. Development reagent (protease, 5 μL) is added for 1 hr prior to detection in a fluorescence microplate reader (Varioskan Flash, ThermoFisher Scientific).
Measurement: IC50 = 17 nM (pChEMBL 7.769999980926514)
Descriptors: MW 739.88, ALogP 7.8, HBA 10, HBD 4, PSA 147.56, QED 0.1
Compound (SMILES): CCOc1nn(C)cc1Nc1ncc(F)c(-c2c[nH]c3c(NC(=O)[C@@H](C)N4CCN(CC)CC4)cccc23)n1
InChIKey: ILENBJYJIMEMLK-QGZVFWFLSA-N
Target: Tyrosine-protein kinase JAK3 (SINGLE PROTEIN), Homo sapiens
UniProt: P52333
Assay: JAK Enzymatic Assay: Enzyme inhibition studies were performed using recombinant JAK1 (amino acids 866-1154, Life Technologies, #PV4774, Carlsbad, Calif.), JAK2 (amino acids 831-1132), or JAK3 (amino acids 781-1124) under buffer conditions of 50 mM HEPES pH 7.3, 1 mM DTT, 0.01% Tween® 20, 50 μg/mL BSA, and 10 mM MgCl2. JAK enzyme was expressed as an N-terminal GST fusion in insect cells and purified by glutathione-affinity and size-exclusion chromatographies. Enzymes were assayed both at their respective ATP Km (JAK1: 55 μM, JAK2: 15 μM, JAK3: 3 μM) and the approximated high end of physiological ATP concentration of 5 mM, in the presence of inhibitor dosed at 30, 3, 0.3, 0.03, 0.003 and 0 μM final test concentrations. For JAK1, 6 nM of enzyme (for Km ATP assay) or 4 nM enzyme (for high ATP assay) was incubated with 1.5 μM peptide substrate (FITC-C6-KKHTDDGYMPMSPGVA-NH2 (SEQ ID NO:1), Intonation, Boston, Mass.). For JAK2, 0.8 nM of enzyme (for Km ATP assay) or 0.3 nM enzyme (for high ATP assay) was incubated with 1.5 μM peptide substrate (5FAM-GEEPLYWSFPAKKK-NH2 (SEQ ID NO:2), Intonation, Boston, Mass.). For JAK3, 0.2 nM of enzyme (for Km ATP assay) or 0.1 nM enzyme (for high ATP assay) was incubated with 1.5 μM peptide substrate (5FAM-GEEPLYWSFPAKKK-NH2 (SEQ ID NO:2), Intonation, Boston, Mass.). Phosphorylated and unphosphorylated peptides were separated and quantified by a Caliper LC3000 system (Caliper Life Sciences, MA) for calculating percent inhibition. The results of this assay are shown in Table 16 and indicate that the compounds of Formula (I), (Ia), (Ib) and Table 1 exhibit preferential inhibition of JAK1 over JAK2 (in many cases demonstrating over 100 times selectivity for inhibition of JAK1 vs. JAK2).
Measurement: IC50 > 30000 nM (pChEMBL 4.5229997634887695)
Descriptors: MW 535.63, ALogP 3.6, HBA 9, HBD 3, PSA 116.23, QED 0.3
Compound (SMILES): Cn1cc(-c2cc3c(N4CC(C(=O)N[C@@H](CO)c5ccc(Cl)cc5)C4)ncnn3c2)cn1
InChIKey: IEPIIOREPZMDKX-IBGZPJMESA-N
Target: Mast/stem cell growth factor receptor Kit (SINGLE PROTEIN), Homo sapiens
UniProt: P10721
Assay: Autophosphorylation Assay: 10,000 HMC1.2 cells were incubated in 22 ul culture media (phenol-red free IMDM, no serum) in each well of a 384-well plate and serum starved overnight in a tissue culture incubator (5% CO2, 37° C.). A 10-point dose concentration series of compound (25 uM-95.4 pM) were then added to the cells in a volume of 3.1 ul to each well (0.25% DMSO final concentration). After 90 minutes, 6 ul of 5× AlphaLISA Lysis Buffer (Perkin Elmer) supplemented with a protease and phosphatase inhibitor cocktail (Cell Signaling Technologies) was added to each well and shaken at 450 rpm for 15 minutes at 4° C. 10 ul of phospho-Y719 c-Kit and total c-Kit antibodies (15 nM final concentration, Cell Signaling Technologies) and 50 ug/ml AlphaLISA rabbit acceptor beads (Perkin Elmer) were added to each well and shaken at 300 rpm at room temperature for 2 hours. 10 ul of 100 ug/ml streptavidin donor beads (Perkin Elmer) were added to each well, blocked from light with aluminum adhesive and shaken at 300 rpm at room temperature for 2 hours. Fluorescence signal was obtained on Envision (Perkin Elmer) by AlphaScreen 384 well HTS protocol.
Measurement: IC50 = 550 nM (pChEMBL 6.260000228881836)
Descriptors: MW 451.92, ALogP 2.07, HBA 8, HBD 2, PSA 100.58, QED 0.47
Compound (SMILES): CC(C)N1CCOc2c(F)cc(-c3nc(Nc4ccc5c(n4)CCNC5)ncc3F)cc21
InChIKey: PSWOMNFIVDHMDM-UHFFFAOYSA-N
Target: CDK2/Cyclin A2 (PROTEIN COMPLEX), Homo sapiens
UniProt: P20248
Assay: Z′-LYTE Assay: CDK5 (p25): IC50 values of compounds against CDK5 (p25) were determined by Z′-LYTE™. These screening assays were performed at Invitrogen Life Technologies (Grand Island, N.Y.) on a low volume NBS, black 384-well plate (#4514, Corning). 0.1 μL of 100× test compound in 100% DMSO (at specific solutions) were mixed with 2.4 μL of Kinase Buffer (50 mM HEPES pH 7.5, 0.01% BRIJ-35, 10 mM MgCl2, 1 mM EGTA), 5 μL of 2× Kinase (0.18-2 ng CDK5/p25)/Peptide (2 μM Ser/Thr 12), and 2.5 μL of 4×ATP solution (17 μM). The plates were shaken for 30 seconds, and incubated for 60 minutes at room temperature. Development Reagent Solution (5 μL of 1:4096 dilution) was added to the plates followed with another 30-second plate shake and the plates were further incubated at room temperature for one hour. The plates were read on fluorescence plate reader with Dual emission at 445 nm and 520 nm.
Measurement: IC50 = 24 nM (pChEMBL 7.619999885559082)
Descriptors: MW 438.48, ALogP 3.81, HBA 7, HBD 2, PSA 75.2, QED 0.64
Compound (SMILES): CCn1c(=O)c(-c2cccs2)cc2cnc(Nc3ccc(C)cn3)cc21
InChIKey: RGUUUSHXRMXJNC-UHFFFAOYSA-N
Target: Tyrosine-protein kinase Lyn (SINGLE PROTEIN), Homo sapiens
UniProt: P07948
Assay: Kinase Assays: A concentration of a drug that reduces the observed activity of an enzyme by 50%. An IC50 is not a true affinity constant and the specific value determined can vary depending on the particular assay used. Nonetheless the rank order of IC50 for a set of compounds will typically be similar from one assay to another. (The true affinity constant for an inhibitor binding to an enzyme, Ki, can of course also be used for the same purposes as described herein for IC50). With respect to the kinases that are targeted by the compounds disclosed herein, kinase activity can be assayed, without limitation, as consumption of ATP or as incorporation of phosphate into a substrate. For example, the assay used in Example 76 assesses consumption of ATP based on luminescent detection of ADP. As an alternate example, the assay used in Example 78 assesses incorporation of phosphate based on the radiologic detection of 33P-labeled artificial substrates. Myriad other kinase assays are known in the art many of which are commercially available either as a kit or a service and one of skill in the art will recognize their particular usefulness for generating IC50 data.
Measurement: IC50 > 5000 nM (pChEMBL 5.301000118255615)
Descriptors: MW 362.46, ALogP 4.59, HBA 6, HBD 1, PSA 59.81, QED 0.58
Compound (SMILES): COC(=O)c1ccc2c(c1)nc(Nc1ccc(OC(F)(F)F)cc1)n2[C@H]1C[C@@H](C)CC(C)(C)C1
InChIKey: BUONBQRWWDSYIP-QAPCUYQASA-N
Target: Isocitrate dehydrogenase [NADP] cytoplasmic (SINGLE PROTEIN), Homo sapiens
UniProt: O75874
Assay: Biochemical Assay: mIDH1 catalyzes the NADPH-dependent reduction of alpha-ketoglutarate (α-KG) to (2R)-2-hydroxyglutarate (2-HG). NADPH consumption was measured by luminescent readout.The biochemical reactions were performed at 32° C. in 384-well plates using a reaction volume of 41 μl and the following assay buffer conditions: 50 mM Tris pH 7.5, 100 mM NaCl, 20 mM MgCl2, 0.05% BSA, 0.01% Brij, 1 μM NADPH, and 250 μM α-KG. The IDH1 R132H enzyme was used in a final concentration of 1.5 nM. Test compounds were used in a concentration range between 0.002 and 10 μM. The final DMSO concentration was 2.4%.The reaction was incubated for 30 minutes, then 40 μl of detection mix (0.75 μg/ml Luciferase, 0.02 U/ml Oxidoreductase, 4 μg/ml FMN, 2 μl/ml Decanal/Ethanol, 50 mM Tris pH 7.5, 0.5% Glycerin, 0.01% Tween-20, 0.05% BSA) was added. Luminescence was measured on a luminescent reader (10 seconds measuring time, 1 second integration period, 30% sensitivity). The decrease in luminescence is proportional to mIDH1 activity. IC50 values are determined by interpolation from plots of relative luminescence versus inhibitor concentration.
Measurement: IC50 = 460 nM (pChEMBL 6.340000152587891)
Descriptors: MW 475.51, ALogP 6.85, HBA 6, HBD 1, PSA 65.38, QED 0.41
Compound (SMILES): O=C(NC1CCOCC1)c1cccc(C2CC2NCC2CC2)c1
InChIKey: IPBBEAIDXLVCLZ-UHFFFAOYSA-N
Target: Amine oxidase [flavin-containing] B (SINGLE PROTEIN), Homo sapiens
UniProt: P27338
Assay: MAO-B Inhibition Assay: The MAO-B inhibitory activity evaluation described below followed the protocol of MAO-Glo (registered trademark) Assay of Promega KK.A test compound dissolved in 4% DMSO was added by 12.5 μL to 25 μL reaction solution (100 mM HEPES (pH 7.5), 5% glycerol, 10% DMSO) containing 400 ng of MAO-B enzyme (Sigma-Aldrich Co. LLC.), and the mixture was reacted at room temperature for 10 min. MAO substrate (Promega KK) (16 μM) was added by 12.5 μL to start the reaction. After reaction at room temperature for 60 min, Luciferine detection reagent (Promega KK) (50 μL) was added to terminate the reaction. After reaction at room temperature for 20 min with stirring, the luminescence was measured by Envision (PerkinElmer). The MAO-B inhibitory rate (%) of the test compound was calculated by the following formula.inhibitory rate(%)=(1−(test compound count−blank)÷(control−blank))×100
Measurement: IC50 > 10000 nM (pChEMBL 5)
Descriptors: MW 314.43, ALogP 2.45, HBA 3, HBD 2, PSA 50.36, QED 0.85
Compound (SMILES): COCc1ccc(NC(=O)c2c(C#Cc3cnc4ccccn34)n(C3(C)CC3)c3ncnc(N)c23)cc1
InChIKey: ZCOHTTBWJKQIKO-UHFFFAOYSA-N
Target: Proto-oncogene tyrosine-protein kinase receptor Ret (SINGLE PROTEIN), Homo sapiens
UniProt: P07949
Assay: Measurement of Inhibitory Activity Against RET (V804M): RET (V804M) (i.e., RET with V804M mutation) kinase activity was measured using purified recombinant human RET (V804M) protein purchased from Eurofins with the final concentration of ATP being 13 μM in the kinase reaction system, following procedure 1) for the other part.
Measurement: IC50 = 5.8 nM (pChEMBL 8.239999771118164)
Descriptors: MW 491.56, ALogP 3.97, HBA 8, HBD 2, PSA 112.36, QED 0.36
Compound (SMILES): CCc1ccc(Oc2ccccc2)c(O)c1
InChIKey: XZOPHRPAUNJURP-UHFFFAOYSA-N
Target: Enoyl-[acyl-carrier-protein] reductase [NADPH] FabI (SINGLE PROTEIN), Staphylococcus aureus
UniProt: Q6GI75
Assay: Tight-binding assay: Kinetics were performed on a Cary 100 spectrophotometer (Varian) at 20° C. Reaction velocities were measured by monitoring the oxidation of NAD(P)H to NAD(P)+ at 340 nm (Figure US10071965-20180911-P00001=6220 M−1 cm−1). For saFabI, the reaction mixture was identical to that described previously for progress curve experiments (Schiebel et al. (2012). Structure 20:802-13). For ecFabI, the final reaction mixture contained ecFabI (75 nM), trans-2-butenoyl-CoA (800 μM; Sigma and Advent Bio), NADH (300 μM; Sigma), NAD+ (400 μM; Sigma), and inhibitor (2 v/v % DMSO) in 50 mM potassium phosphate, pH 7.5, 150 mM NaCl, 8 v/v % glycerol. For InhA, the final reaction mixture contained InhA (100 nM), trans-2-octenoyl-CoA (200 μM), NADH (250 μM), NAD+ (200 μM), and inhibitor (2 v/v % DMSO) in 30 mM PIPES, pH 6.8, 150 mM NaCl, 1 mM EDTA, 8 v/v % glycerol. The resulting curves were fit to the Morrison and Walsh integrated rate equation (Equation 1) (Morrison et al.
Measurement: Ki = 33.6 nM (pChEMBL 7.46999979019165)
Descriptors: MW 214.26, ALogP 3.75, HBA 2, HBD 1, PSA 29.46, QED 0.84
Compound (SMILES): C[C@H]1C[C@@H](F)CCN1C(=O)c1nc(C(=O)NCC(C)(C)O)sc1-c1ccc(C(O)(C(F)(F)F)C(F)(F)F)c(Cl)c1Cl
InChIKey: FSXDWHIVGZZCPI-QWRGUYRKSA-N
Target: Nuclear receptor ROR-beta (SINGLE PROTEIN), Homo sapiens
UniProt: Q92753
Assay: Inverse agonist activity at human RORbeta assessed as dissociation constant by thermofluor assay
Measurement: Kd = 1000 nM (pChEMBL 6)
Descriptors: MW 654.43, ALogP 5.89, HBA 6, HBD 3, PSA 102.76, QED 0.34
Compound (SMILES): COC1CN(C(=O)c2nc(N[C@@H](C)c3ccccn3)nc3cc(C)sc23)C1
InChIKey: PBAIIPVGKBUNMB-LBPRGKRZSA-N
Target: Adenosine receptor A1 (SINGLE PROTEIN), Homo sapiens
UniProt: P30542
Assay: Activity Assay: Compounds (Adenosine Receptor modulators) and activity assay.
Measurement: IC50 = 75 nM (pChEMBL 7.119999885559082)
Descriptors: MW 383.48, ALogP 3.04, HBA 7, HBD 1, PSA 80.24, QED 0.73
Compound (SMILES): C=CC(=O)Nc1cn(CC2CCOCC2)nc1-c1cc2c(cn1)CN(c1c(F)c(OC)cc(OC)c1F)C(=O)C21CC1
InChIKey: JJNWMRISEGJDNT-UHFFFAOYSA-N
Target: Fibroblast growth factor receptor 2 (SINGLE PROTEIN), Homo sapiens
UniProt: P21802
Assay: Enzymatic Assay: The inhibitor potency of the exemplified compounds was measured in an enzyme assay that measures peptide phosphorylation using FRET measurements to detect product formation. Inhibitors were serially diluted in DMSO and a volume of 0.5 μL was transferred to the wells of a 384-well plate. For FGFR3, a 10 μL volume of FGFR3 enzyme (Millipore) diluted in assay buffer (50 mM HEPES, 10 mM MgCl2, 1 mM EGTA, 0.01% Tween-20, 5 mM DTT, pH 7.5) was added to the plate and pre-incubated for a time between 5-10 minutes and up to 4 hours. Appropriate controls (enzyme blank and enzyme with no inhibitor) were included on the plate. The assay was initiated by the addition of a 10 μL solution containing biotinylated EQEDEPEGDYFEWLE peptide substrate (SEQ ID NO: 1) and ATP (final concentrations of 500 nM and 140 μM respectively) in assay buffer to the wells. The plate was incubated at 25° C. for 1 hr. The reactions were ended with the addition of 10 μL/well of quench solution (50 mM Tris, 150 mM NaCl, 0.5 mg/mL BSA, pH 7.8; 30 mM EDTA with Perkin Elmer Lance Reagents at 3.75 nM Eu-antibody PY20 and 180 nM APC-Streptavidin). The plate was allowed to equilibrate for 1 hr before scanning the wells on a PheraStar plate reader (BMG Labtech).FGFR1, FGFR2, and FGFR4 are measured under equivalent conditions with the following changes in enzyme and ATP concentrations: FGFR1, 0.02 nM and 210 uM respectively, FGFR2, 0.01 nM and 100 uM, respectively, and FGFR4, 0.04 nM and 600 uM respectively. The enzymes were purchased from Millipore or Invitrogen.
Measurement: IC50 > 200 nM (pChEMBL 6.698999881744385)
Descriptors: MW 579.6, ALogP 4.37, HBA 8, HBD 1, PSA 107.81, QED 0.4
Compound (SMILES): Cc1c(-c2cc(O)cc3ccccc23)ncc2c(N3CC4CCC(C3)N4)nc(OC[C@@H]3CCCN3C)nc12
InChIKey: XGUHBRVEFLTIQZ-HRTMPFAESA-N
Target: GTPase KRas (SINGLE PROTEIN), Homo sapiens
UniProt: P01116
Assay: KRas G12D Binding Assay: Table 2: The ability of a compound to bind to KRAS G12D was measured using a TR-FRET displacement assay. Biotinylated GDP-loaded recombinant human KRAS G12D (corresponding to amino acids 1:169, produced at Array BioPharma) was incubated with a custom-made Cy5 labelled tracer, europium labelled streptavidin and compound (2% DMSO final) in buffer (50 mM HEPES [pH 7.5], 5mM MgCl2, 0.005% Tween-20 & 1 mM DTT). After a 60 minute incubation at 22° C., the reaction was measured using a PerkinElmer EnVision multimode plate reader via TR-FRET dual wavelength detection, and the percent of control (POC) calculated using a ratiometric emission factor. 100 POC is determined using no test compound and 0 POC is determined using a concentration of control compound that completely inhibits binding of the tracer to KRAS. The POC values were fit to a 4-parameter logistic curve and the IC50 value was determined as the concentration where the curve crosses 50 POC.
Measurement: IC50 = 16314 nM (pChEMBL 4.789999961853027)
Descriptors: MW 510.64, ALogP 4.27, HBA 8, HBD 2, PSA 86.64, QED 0.41
Compound (SMILES): Cc1cc(S(=O)(=O)Nc2cscn2)cnc1NCc1c(F)cccc1CN(C)C(C)(C)C
InChIKey: WWLCGLSOEPKESV-UHFFFAOYSA-N
Target: Sodium channel protein type 1 subunit alpha (SINGLE PROTEIN), Homo sapiens
UniProt: P35498
Assay: Sodium Influx Assay (In Vitro Assay): This sodium influx assay employs the use of the cell permeable, sodium sensitive dye ANG2 to quantify sodium ion influx through sodium channels which are maintained in an open state by use of sodium channel modulators. This high throughput sodium influx assay allows for rapid profiling and characterization of sodium channel blockers.In general, Trex HEK293 cells were stably transfected with an inducible expression vector containing the full-length cDNA coding for the desired human sodium channel α-subunit and with an expression vector containing full length cDNA coding for the β1-subunit. Sodium channel expressing cell lines were induced with tetracycline (1 μg/mL) and plated on 384-well PDL-coated plates at a density of 25K-30K cells/well in culture media (DMEM, containing 10% FBS and 1% L-glutamine). After overnight incubation (37° C., 5% CO2), culture media was removed and cells were loaded with 5 uM ANG2 dye for 1-1.5h in Buffer 1 (155 mM NMDG, 5 mM KCl, 2 mM CaCl2, 1 mM MgCl2, 10 mM HEPES, 10 mM glucose, adjusted with Tris to pH 7.4). Access dye was removed and cells were incubated with test compounds for 1 hr in buffer 1 containing sodium channel modulator(s) at room temperature. Hamamatsu FDSS μCell was used to perform a 1:1 addition of Na/K challenge buffer (140 mM NaCl, 20 mM HEPES, 1 mM CaC2, 15 mM KCl, 1 mM MgCl2, 10 mM glucose, adjusted with Tris to pH 7.4) and simultaneously read plates at excitation wavelength of 530 nm and emission wavelength set at 558 nm. Percent inhibition of sodium ion influx was calculated for each test compound at each test concentration to determine the IC50 values.
Measurement: IC50 = 14879 nM (pChEMBL 4.829999923706055)
Descriptors: MW 477.63, ALogP 4.63, HBA 7, HBD 2, PSA 87.22, QED 0.49
Compound (SMILES): Clc1ccc(OCC2CCCCC2)c(OC2CNC2)c1
InChIKey: MHUOQHAUHJOSBS-UHFFFAOYSA-N
Target: 5-hydroxytryptamine receptor 6 (SINGLE PROTEIN), Homo sapiens
UniProt: P50406
Assay: Binding Assay: Receptor binding was performed using membrane fractions prepared from the HEK-293 cell line recombinantly expressing rat 5-HT7 receptors (NCBI accession NM_022938). Compound affinity for the rat 5-HT7 receptor subtype was evaluated by competitive radioligand binding assays using 5-carboxamido[3H]tryptamine ([3H]5-CT) (Amersham Biosciences, cat. 90000403) detection. HitHunter cAMP assays are in-vitro based competitive immunoassays. The assay was performed on the HEK-293 cell line stably transfected with r5-HT7 receptor. Cells were pre-incubated with test compounds for 10 minutes. For antagonist testing, the cells were then challenged with 100 nM 5-CT for 20 minutes. Cells were then lysed and cAMP measured according to manufacturers protocol (Amersham, cat. NET791250UC) or [3H]mesulergine (Amersham, cat. TRK1041). The assay was performed on membranes prepared from HEK-293 cells stably transfected with h5-HT6. Following centrifugation, membranes were resuspended and incubated for 60 min at room temperature with 1.7 nM [3H]LSD in the presence of increasing concentration of test compounds. Nonspecific binding was defined in the presence of 10 μM clozapine (Tocris, cat. TRK1068). Homogenized HEK-293 membranes expressing the human SERT were incubated in 50 mM Tris-HCl (pH 7.5), 120 mM NaCl, 5 mM KCl with [3H]-citalopram (3 nM) with or without test compounds. Nonspecific binding was determined in the presence of 10 μM fluoxetine. Radioactivity readouts and Ki values were performed as previously described for r5-HT7.
Measurement: Ki = 12 nM (pChEMBL 7.920000076293945)
Descriptors: MW 295.81, ALogP 3.65, HBA 3, HBD 1, PSA 30.49, QED 0.9