680622-70-2Relevant articles and documents
PHOTOPROXIMITY PROFILING OF PROTEIN-PROTEIN INTERACTIONS IN CELLS
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Page/Page column 73-75, (2021/04/01)
Photoactive probes and probe systems for detecting biological interactions are described. The photoactive probes include probes that combine both photocleavable and photoreactive moieties. The photoactive probe systems can include a first probe comprising a photocatalytic group and a second probe comprising a group that can act as a substrate for the reaction catalyzed by the photocatalytic group. The probes and probe systems can also include groups that can specifically bind to a binding partner on a biological entity of interest and a detectable group or a precursor thereof. The probes and probe systems can detect spatiotemporal interactions of proteins or cells. In some embodiments, the interactions can be detected in live cells. Also described are methods of detecting the biological interactions.
Photoproximity Profiling of Protein-Protein Interactions in Cells
Carlos, Anthony,Lee, Gihoon,McCutcheon, David C.,Moellering, Raymond E.,Montgomery, Jeffrey E.
, p. 146 - 153 (2020/01/31)
We report a novel photoproximity protein interaction (PhotoPPI) profiling method to map protein-protein interactions in vitro and in live cells. This approach utilizes a bioorthogonal, multifunctional chemical probe that can be targeted to a genetically encoded protein of interest (POI) through a modular SNAP-Tag/benzylguanine covalent interaction. A first generation photoproximity probe, PP1, responds to 365 nm light to simultaneously cleave a central nitroveratryl linker and a peripheral diazirine group, resulting in diffusion of a highly reactive carbene nucleophile away from the POI. We demonstrate facile probe loading, and subsequent interaction- A nd light-dependent proximal labeling of a model protein-protein interaction (PPI) in vitro. Integration of the PhotoPPI workflow with quantitative LC-MS/MS enabled unbiased interaction mapping for the redox regulated sensor protein, KEAP1, for the first time in live cells. We validated known and novel interactions between KEAP1 and the proteins PGAM5 and HK2, among others, under basal cellular conditions. By contrast, comparison of PhotoPPI profiles in cells experiencing metabolic or redox stress confirmed that KEAP1 sheds many basal interactions and becomes associated with known lysosomal trafficking and proteolytic proteins like SQSTM1, CTSD, and LGMN. Together, these data establish PhotoPPI as a method capable of tracking the dynamic subcellular and protein interaction "social network" of a redox-sensitive protein in cells with high temporal resolution.
Synergic "click" Boronate/Thiosemicarbazone System for Fast and Irreversible Bioorthogonal Conjugation in Live Cells
Akgun, Burcin,Li, Caishun,Hao, Yubin,Lambkin, Gareth,Derda, Ratmir,Hall, Dennis G.
supporting information, p. 14285 - 14291 (2017/10/17)
Fast, high-yielding, and selective bioorthogonal "click" reactions employing nontoxic reagents are in high demand for their great utility in the conjugation of biomolecules in live cells. Although a number of click reactions were developed for this purpose, many are associated with drawbacks and limitations that justify the development of alternative systems for both single- or dual-labeling applications. Recent reports have highlighted the potential of boronic ester formation as a bioorthogonal click reaction between abiotic boronic acids and diols. Boronic ester formation is a fast dehydrative process; however it is intrinsically reversible in aqueous medium. We designed and optimized a synergic system based on two bifunctional reagents, a thiosemicarbazide-functionalized nopoldiol and an ortho-acetyl arylboronic acid. Both reagents were shown to be chemically stable and nontoxic to HEK293T cells at concentrations as high as 50 μM. The resulting boronate/thiosemicarbazone adduct is a medium-sized ring that forms rapidly and irreversibly without any catalyst at low μM concentrations, in neutral buffer, with a rate constant of 9 M-1 s-1 as measured by NMR spectroscopy. Control experiments in the presence of competing boronic acids showed no crossover side-products and confirmed the stability and lack of reversibility of the boronate/thiosemicarbazone conjugates. Formation of the conjugates is not affected by the presence of biological diols such as fructose, glucose, and catechol, and the thiosemicarbazide-functionalized nopoldiol is inert to aldehyde electrophiles of the sort found on protein-bound glyoxylyl units. The suitability of this system in the cell-surface labeling of live cells was demonstrated using a SNAP-tag approach to install the boronic acid reagent onto the extracellular domain of the Beta-2 adrenergic receptor in HEK293T cells, followed by incubation with the optimal thiosemicarbazide-functionalized nopoldiol reagent labeled with fluorescein dye. Successful visualization by fluorescence microscopy was possible with a reagent concentration as low as 10 μM, thus confirming the potential of this system in biological applications.
Benzyl guanine derivative, an organic salt compound thereof, a medicine composition, and application thereof
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Paragraph 0052; 0056; 0057; 0061-0064; 0073-0076, (2017/09/26)
The invention relates to the field of antitumor medicines and especially relates to a benzyl guanine derivative, an organic salt compound thereof, a medicine composition, and an application thereof. The benzyl guanine derivative is easy to prepare, has good druggability, can inhibit enzyme-catalysis activity of Pin 1 protein, can improve biosynthesis of mature microRNA at cellular and animal levels, and increases the types of antitumor medicines.
A β-chloro ethylnitrosourea compound and its synthetic method and use
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Paragraph 0170; 0171, (2017/01/17)
The invention relates to novel beta-chloroethylnitrosourea compounds, and a synthesis method and application thereof. The structure of the beta-chloroethylnitrosourea compounds is disclosed as general formula (II). The in-vitro antitumor screening test on the compounds disclosed as general formula II proves that the compounds disclosed as general formula I have obvious inhibiting action on human cerebral nerve glioma cells SF763, SF767, SF126 and SF188, human colon cancer cell HT29, mouse leukaemia cell L1210 and many other tumor cell lines and have higher tumor inhibiting activity than the existing CENU and CENU/O6-benzylguanine combined medicine.
LASERTAG: A TOOLKIT ALLOWING THE SPACE-SPECIFIC RECOVERY, CONTROL AND MODIFICATION OF SINGLE CELLS AND BIOLOGICAL MOLECULES IN VIVO
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Page/Page column 28, (2016/06/15)
In some aspects, the disclosure relates to self-ligating protein tags conjugated to a photocaging molecule and methods of their use.
A rapidly reversible chemical dimerizer system to study lipid signaling in living cells
Feng, Suihan,Laketa, Vibor,Stein, Frank,Rutkowska, Anna,MacNamara, Aidan,Depner, Sofia,Klingmueller, Ursula,Saez-Rodriguez, Julio,Schultz, Carsten
, p. 6720 - 6723 (2014/07/08)
Chemical dimerizers are powerful tools for non-invasive manipulation of enzyme activities in intact cells. Here we introduce the first rapidly reversible small-molecule-based dimerization system and demonstrate a sufficiently fast switch-off to determine kinetics of lipid metabolizing enzymes in living cells. We applied this new method to induce and stop phosphatidylinositol 3-kinase (PI3K) activity, allowing us to quantitatively measure the turnover of phosphatidylinositol 3,4,5-trisphosphate (PIP 3) and its downstream effectors by confocal fluorescence microscopy as well as standard biochemical methods. Little helper: Chemical dimerizers are powerful tools for manipulation of enzyme activities in intact cells. The first rapidly reversible chemical dimerization system is introduced, which permits to determine kinetics of lipid metabolizing enzymes in living cells. This new method was applied to induce and stop phosphatidylinositol 3-kinase activity, allowing to quantitatively measure 3,4,5-trisphosphate turnover.
DYE COMPOSITIONS, METHODS OF PREPARATION, CONJUGATES THEREOF, AND METHODS OF USE
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, (2013/07/31)
Dye compounds of the formula (1) wherein A is a protective agent group that has a characteristic of modifying the singlet-triplet occupancy of the shown cyanine moiety, and M is a reactive crosslinking group or a group that can be converted to a reactive crosslinking group. Methods for synthesizing the dye compounds and applications for their use are also described.
Highly activatable and environment-insensitive optical highlighters for selective spatiotemporal imaging of target proteins
Kobayashi, Tomonori,Komatsu, Toru,Kamiya, Mako,Campos, Claudia,Gonzalez-Gaitan, Marcos,Terai, Takuya,Hanaoka, Kenjiro,Nagano, Tetsuo,Urano, Yasuteru
, p. 11153 - 11160 (2012/09/22)
Optical highlighters are photoactivatable fluorescent molecules that exhibit pronounced changes in their spectral properties in response to irradiation with light of a specific wavelength and intensity. Here, we present a novel design strategy for a new class of caged BODIPY (4,4-difluoro-4-bora-3a, 4a-diaza-s-indacene) fluorophores, based on the use of photoremovable protecting groups (PRPGs) with high reduction potentials that serve as both a photosensitive unit and a fluorescence quencher via photoinduced electron transfer (PeT). 2,6-Dinitrobenzyl (DNB)-caged BODIPY was efficiently photoactivated, with activation ratios exceeding 600-fold in aqueous solutions. We then combined this photoactivatable fluorophore with a SNAP (mutant of O 6-alkylguanine DNA alkyltransferase) ligand to obtain a small-molecule-based optical highlighter for visualization of protein dynamics, using the well-established SNAP tag technology. As proof of concept, we demonstrate spatiotemporal imaging of the fusion protein of epidermal growth factor receptor (EGFR) with SNAP tag in living cells. We also demonstrate highlighting of cells of interest in live zebrafish embryos, using the fusion protein of histone 2A with SNAP tag.
Syntheses and characterizations of novel pyrrolocoumarin probes for SNAP-tag labeling technology
Mei, De-Sheng,Qu, Yi,He, Jin-Xiang,Chen, Lei,Yao, Zhu-Jun
, p. 2251 - 2259 (2011/04/22)
SNAP-tag technology is a revolutionary protein labeling technology employing in various biological studies. Since low signal/noise ratio and severe overlap between the FRET donors/acceptors often occurred in applying present fluorescent probes and thus limited the further applications, development of new fluorescent probes with excellent fluorescent properties is still of request by today's SNAP-tag technology. In this paper, a number of SNAP-tag protein probes have been developed by incorporating a novel pyrrolocoumarin fluorophore recently developed by our group. Examination of these novel synthetic compounds shows all these materials possess satisfactory fluorescent properties. Among these, probe 7 exhibits the most excellent characters, and its quantum yield, maximum emission wavelength and Stocks shift reach to 0.44, 534 nm and 112 nm, respectively. Further analysis of structure-property relationship indicates that the probes with a longer C3-substituted alkyl (such as pentyl) give stronger fluorescence.
