- Koehler, MF; Bergeron, P; Blackwood, EM; Bowman, K; Clark, KR; Firestein, R; Kiefer, JR; Maskos, K; McCleland, ML; Orren, L; Salphati, L; Schmidt, S; Schneider, EV; Wu, J; Beresini, MH Development of a Potent, Specific CDK8 Kinase Inhibitor Which Phenocopies CDK8/19 Knockout Cells. ACS Med Chem Lett 7: 223-8 (2016)
- Boateng, CA; Bakare, OM; Zhan, J; Banala, AK; Burzynski, C; Pommier, E; Keck, TM; Donthamsetti, P; Javitch, JA; Rais, R; Slusher, BS; Xi, ZX; Newman, AH High Affinity Dopamine D3 Receptor (D3R)-Selective Antagonists Attenuate Heroin Self-Administration in Wild-Type but not D3R Knockout Mice. J Med Chem 58: 6195-213 (2015)
- ChEMBL_2184503 (CHEMBL5096585) Inhibition of human IGF1R expressed in mouse IGF1R knockout MEF cells
- ChEMBL_2184504 (CHEMBL5096586) Inhibition of human InsR expressed in mouse IGF1R knockout MEF cells
- ChEMBL_2184505 (CHEMBL5096587) Binding affinity to human IGF1R expressed in mouse IGF1R knockout MEF cells assessed as dissociation constant
- ChEMBL_2184506 (CHEMBL5096588) Binding affinity to human InsR expressed in mouse IGF1R knockout MEF cells assessed as dissociation constant
- ChEMBL_1825358 (CHEMBL4325122) Induction of IRF3 pathway in STING knockout human THP1 cells measured after 20 hrs by luciferase reporter gene assay
- ChEMBL_2269367 Inhibition of RyR2 in Casq2 gene knockout mouse cardiomyocytes assessed as reduction in calcium sparks frequency incubated for 30 mins
- ChEMBL_2377727 Inhibition of IDO1 in MTAP knockout human HCT-116 cells assessed as reduction in kynurenine level incubated for 48 hrs
- ChEMBL_1451385 (CHEMBL3363669) Agonist activity at human LPA2 expressed in LPA1xLPA2 double knockout mouse MEF cells by Fura-2AM dye based Ca2+ mobilization assay
- ChEMBL_1451389 (CHEMBL3363673) Agonist activity at human LPA3 expressed in LPA1xLPA2 double knockout mouse MEF cells by Fura-2AM dye based Ca2+ mobilization assay
- ChEMBL_2310369 Agonist activity at human STING in STING knockout human THP1-Blue ISG cells incubated for 24 hrs in presence of PMA by luciferase reporter assay
- ChEMBL_2310370 Agonist activity at mouse STING in STING knockout mouse RAW-Lucia ISG cells incubated for 24 hrs in presence of PMA by luciferase reporter assay
- ChEMBL_2324967 Inhibition of mTOR in Tsc1 knockout mouse neurons assessed as reduction in phosphorylated S6 level incubated for 4 hrs by Hoechst staining based immunofluorescence analysis
- ChEMBL_1451387 (CHEMBL3363671) Agonist activity at human LPA1 expressed in LPA1xLPA2 double knockout mouse MEF cells up to 10 uM by Fura-2AM dye based Ca2+ mobilization assay
- ChEMBL_1572464 (CHEMBL3796255) Inhibition of human TDP2 expressed in DT40 cells (TDP2-knockout) using 18-mer single stranded oligonucleotide DNA as substrate incubated for 15 mins by PAGE assay
- ChEMBL_840552 (CHEMBL2090017) Inhibition of human IGF1R phosphorylation expressed in IGF-1-stimulated knockout mouse R+ cells treated 30 mins before IGF-1 stimulation measured after 20 mins by immunoblotting
- ChEMBL_864728 (CHEMBL2174995) Inhibition of human recombinant Tdp1 expressed in Tdp1 knockout chicken DT40 cells using 5'-32P-labeled 5'-GATCTAAAAGACTT-pY-3' as substrate after 15 mins by PAGE analysis
- ChEMBL_2163197 (CHEMBL5048058) Inhibition of PRMT5 methyltransferase activity in MTAP knockout human HCT-116 cells assessed as inhibition of PRMT5- mediated SDMA modification level incubated for 96 hrs by Western blot analysis
- ChEMBL_1451396 (CHEMBL3363956) Antagonist activity at human LPA3 expressed in LPA1xLPA2 double knockout mouse MEF cells assessed as reduction in LP18:1-induced calcium mobilization by Fura-2AM dye based Ca2+ mobilization assay
- ChEMBL_2197017 (CHEMBL5109533) Inhibition of PRMT5 methyltransferase activity in MTAP knockout human HCT-116 cells assessed as inhibition of PRMT5-mediated SDMA modification level incubated for 96 hrs by In-cell Western analysis
- ChEMBL_2380513 Inhibition of human adenylate cyclase 2 expressed in ACdelta 3/6 knockout HEK293 cells assessed as cyclic AMP accumulation incubated for 30 min followed by FSK stimulation by HTR-FRET assay
- ChEMBL_2324911 Inhibition of mTOR in TSC1 knockout mouse MEF cells assessed as reduction in phosphorylated S6 level at serine 240/244 residues incubated for 2 hrs by Hoechst/Alexa Fluor 647 staining based analysis
- ChEMBL_2261676 (CHEMBL5216687) Agonist activity at STING in cGAS knockout human THP-1 cells harboring IRF-inducible luciferase reporter construct assessed as increase in relative fluorescence unit incubated for 12 hrs by Quanti-luc reagent based assay
- ChEMBL_2261677 (CHEMBL5216688) Agonist activity at STING in STING knockout human THP-1 cells harboring IRF-inducible luciferase reporter construct assessed as increase in relative fluorescence unit incubated for 12 hrs by Quanti-luc reagent based assay
- ChEMBL_2307186 Inhibition of tetracycline inducible FLAG tagged human PARL expressed in human PARL knockout Flp-In-T-REx-293 cells cotransfected human PGAM5-Myc assessed as inhibition of PGAM5 cleavage incubated over night by immunoblot analysis
- ChEMBL_2314579 Displacement of [3H]-1,3-di-O-tolylguanidine from human sigma 2 receptor/TMEM97 transfected in sigma 1 receptor knockout HEK293 cell membranes assessed as inhibition constant measured after 120 mins by microbeta scintillation counting analysis
- ChEMBL_2185360 (CHEMBL5097442) Displacement of [3H]-1,3-di-O-tolylguanidine from human sigma 2 receptor/TMEM97 transfected in sigma 1 receptor knockout HEK293 cell membranes assessed as inhibition constant measured after 120 mins by liquid scintillation counting analysis
- ChEMBL_2443087 Inhibition of recombinant human PREP proteolytic activity transfected in PREP knockout HEK293 cells using Suc-Gly-Pro-AMC as substrate preincubated for 30 mins followed by substrate addition measured after 60 mins by fluorescence based microplate reader analysis
- ChEMBL_2340734 Agonist activity at human STING in STING knockout human THP1-Blue ISG cells expressing IRF inducible SEAP reporter construct assessed as increase in interferon production by measuring SEAP activity incubated for 24 hrs by Fingerprinting based QUANTI-Blue colorimetric assay
- ChEMBL_2247166 (CHEMBL5161376) Inhibition of Ca2+/ CAM stimulated human AC1 activity expressed in AC3 and AC6-knockout HEK293 cells assessed as A23187 stimulated cAMP accumulation incubated for 30 mins followed by A2318 stimulation in presence of IBMX for 1 hr by HTRF assay
- ChEMBL_2247167 (CHEMBL5161377) Inhibition of Ca2+/ CAM stimulated human AC8 activity expressed in AC3 and AC6-knockout HEK293 cells assessed as A23187 stimulated cAMP accumulation incubated for 30 mins followed by A2318 stimulation in presence of IBMX for 1 hr by HTRF assay
- ChEMBL_2340729 Agonist activity at human wild type STING overexpressed in STING knockout human THP1-Blue ISG cells expressing IRF inducible SEAP reporter construct assessed as increase in interferon production by measuring SEAP activity incubated for 24 hrs by Fingerprinting based QUANTI-Blue colorimetric assay
- In Vivo Inhibition of Beta-Secretase Several animal models, including mouse, rat, dog, and monkey, may be used to screen for inhibition of beta-secretase activity in vivo following administration of a test compound sample. Animals used in this invention can be wild type, transgenic, or gene knockout animals. For example, the Tg2576 mouse model, prepared and conducted as described in Hsiao et al., 1996, Science 274, 99-102, and other non-transgenic or gene knockout animals are useful to analyze in vivo inhibition of Amyloid beta peptide (Abeta) production in the presence of inhibitory test compounds. Generally, 2 to 18 month old Tg2576 mice, gene knockout mice or non-transgenic animals are administered test compounds formulated in vehicles, such as cyclodextran, phosphate buffers, hydroxypropyl methylcellulose or other suitable vehicles. One to twenty-four hours following the administration of compound, animals are sacrificed, and brains as well as cerebrospinal fluid (CSF) and plasma are removed for analysis of A-beta levels and drug or test compound concentrations (Dovey et al., 2001, Journal of Neurochemistry, 76, 173-181) Beginning at time 0, animals are administered by oral gavage, or other means of delivery such as intravenous injection, an inhibitory test compound of up to 100 mg/kg in a standard, conventional formulation, such as 2% hydroxypropyl methylcellulose, 1% Tween80. A separate group of animals receive 2% hydroxypropyl methylcellulose, 1% Tween80 alone, containing no test compound, and serve as a vehicle-control group. At the end of the test period, animals are sacrificed and brain tissues, plasma or cerebrospinal fluid are collected. Brains are either homogenized in 10 volumes (w/v) of 0.2% diethylamine (DEA) in 50 mM NaCl (Best et al., 2005, Journal of Pharmacology and Experimental Therapeutics, 313, 902-908), or in 10 volumes of 0.5% TritonX-100 in Tris-buffered saline (pH at about 7.6). Homogenates are centrifuged at 355,000 g, 4° C. for 30 minutes. CSF or brain supernatants are then analyzed for the presence of A-beta peptide by specific sandwich ELISA assays based on ECL (Electrochemiluminescence) technology.
- Dose Response confirmation of Inhibitors of Mdm2/MdmX interaction in luminescent format Data Source: Sanford-Burnham Center for Chemical Genomics (SBCCG) Source Affiliation: Sanford-Burnham Medical Research Institute (SBMRI, San Diego CA) Network: NIH Molecular Libraries Probe Production Centers Network (MLPCN) Grant Number: R03 MH089489-01 Assay Provider: Dr. Geoffrey M. Wahl, Salk Institute for Biological Studies, San Diego, CA A wild type but attenuated p53 is retained in approximately 50% of human tumors, and reactivation of p53 in such tumors is an attractive chemotherapeutic strategy. p53 activity is restricted in vivo by mdm2 and mdmx, and knockout of either of these proteins is embryonic lethal in a p53-dependent manner (1, 2). Both proteins bind to p53 via a hydrophobic N-terminal pocket and block p53-dependent transcription of genes required for tumor suppression. Efforts to reactivate p53 with small molecules have focused on inhibition of the mdm2/p53 interaction, which leads to increased p53 levels and activity. However, recent reports indicate that targetin
- Fluorescence-based biochemical high throughput dose response assay to identify inhibitors of Trypanosoma brucei RNA editing ligase 1 (TbREL1). Source (MLPCN Center Name): The Scripps Research Institute Molecular Screening Center (SRIMSC) Center Affiliation: The Scripps Research Institute Assay Provider: Achim Schnaufer, The University of Edinburgh Network: Molecular Libraries Probe Production Centers Network (MLPCN) Grant Proposal Number: R21 NS06173301 Grant Proposal PI: Achim Schnaufer, The University of Edinburgh External Assay ID: TBREL1_INH_FRET_1536_3XIC50 DRUN Name: Fluorescence-based biochemical high throughput dose response assay to identify inhibitors of Trypanosoma brucei RNA editing ligase 1 (TbREL1). Description: The trypanosomatid parasites T. brucei ssp., T. cruzi and Leishmania spp. are the causative agents of human African trypanosomiasis (HAT), Chagas' disease, and leishmaniasis, and economically important diseases in livestock. All trypanosomatids require a unique form of RNA editing for mitochondrial gene expression, and through conditional knockout studies in T. brucei in culture and in mice we ha
- Dose Response confirmation of Inhibitors of Mdm2/MdmX interaction using a Brca1/Bard1 BiLC Counterscreen assay Data Source: Sanford-Burnham Center for Chemical Genomics (SBCCG) Source Affiliation: Sanford-Burnham Medical Research Institute (SBMRI, San Diego CA) Network: NIH Molecular Libraries Probe Production Centers Network (MLPCN) Grant Number: R03 MH089489-01 Assay Provider: Dr. Geoffrey M. Wahl, Salk Institute for Biological Studies, San Diego, CA A wild type but attenuated p53 is retained in approximately 50% of human tumors, and reactivation of p53 in such tumors is an attractive chemotherapeutic strategy. p53 activity is restricted in vivo by mdm2 and mdmx, and knockout of either of these proteins is embryonic lethal in a p53-dependent manner (1, 2). Both proteins bind to p53 via a hydrophobic N-terminal pocket and block p53-dependent transcription of genes required for tumor suppression. Efforts to reactivate p53 with small molecules have focused on inhibition of the mdm2/p53 interaction, which leads to increased p53 levels and activity. However, recent reports indicate that targetin
- TDP2 Assays with Whole Cell Extracts Ten-million cells (1 x 107), either human, chicken DT40 wild type, or knockout for TDP2 and complemented with human TDP2, were collected, washed, and centrifuged. Cell pellets were then resuspended in 100 uL of CelLytic M cell lysis reagent (SIGMA-Aldrich C2978). After 15 min on ice, lysates were centrifuged at 12 000g for 10 min, and supernatants were transferred to a new tube. Protein concentrations were determined using a Nanodrop spectrophotometer (Invitrogen), and whole cell extracts were stored at -80 °C. The TY19 single-stranded DNA oligonucleotide containing a 5'-phosphotyrosine (see above) was incubated at 1 nM with 10-20 ug/mL of whole cell extract to obtain 30-40% cleavage in the absence or presence of inhibitor for 15 min at RT in the reaction buffer containing 50 mM Tris-HCl, pH 7.5, 80 mM KCl, 5 mM MgCl2, 0.1 mM EDTA, 1 mM DTT, 40 ug/mL BSA, and 0.01% Tween 20. Reactions were terminated and samples analyzed.
- Pharmaceutical Assay To determine the HECT E3 ligase selectivity of the compounds, a panel of biochemical HECT E3 ligase autoubiquitinylation assays was employed (Smurf-1, Smurf-2, WWP1, WWP2, ITCH, Nedd4, Nedd4L and E6AP). The conjugation of ubiquitin to a protein substrate is a multistep process. In an initial ATP-requiring step, a thioester bond is formed between the carboxyl terminus of ubiquitin and an internal cystein residue of the ubiquitin-activating enzyme (E1). Activated ubiquitin is then transferred to a specific cystein residue of an ubiquitin-conjugating enzyme (E2). E2s donate ubiquitin to a HECT E3 ligase (E3) from which it is transferred to the substrate protein. HECT E3 ligases can auto-ubiquitinylate. This event is detected in the TR-FRET (Time-Resolved Fluorescence Resonance Energy Transfer) assay used in this panel. The reaction mix contains E1, E2, tagged-E3, biotin-conjugated ubiquitin, the compound and ATP in a suitable buffer and is incubated for 45 minutes to allow auto-ubiquitinylation of the E3 ligase. To measure the extent of ubiquitinylated E3 ligase by TR-FRET, the donor fluorophore Europium cryptate (Eu3+ cryptate), conjugated to streptavidin which subsequently binds to biotinylated ubiquitin, and the modified allophycocyanin XL665 (HTRF primary acceptor fluorophore) coupled to a tag-specific antibody (HA, His or GST), which recognizes the respective E3 ligase fusion proteins, are added after the reaction is complete. When these two fluorophores are brought together by a biomolecular interaction (in this case ubiquitinylation of the E3 ligase), a portion of the energy captured by the Cryptate during excitation is released through fluorescence emission at 620 nm, while the remaining energy is transferred to XL665. This energy is then released by XL665 as specific fluorescence at 665 nm. Light at 665 nm is emitted only through FRET with Europium. Because Europium Cryptate is present in the assay, light at 620 nm is detected even when the biomolecular interaction does not bring XL665 within close proximity.