Synthesis and Characterization of a Small, Membrane-Permeant, Caspase-Activatable Far-Red Fluorescent Peptide for Imaging Apoptosis

Bullok, Kristin; Piwnica‐Worms, David

doi:10.1021/jm050008p

Cited by 133 publications

(137 citation statements)

References 14 publications

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“…This probe is identical to that of TcapQ, except that the entire peptide, including the DEVD cleavage sequence, consists entirely of nonnative d, as opposed to l, amino acids. In vitro experiments have previously confirmed that the all-d probe is not activated by effector caspases (19). Eyes in which dTcapQ were injected following NMDA injection showed little to no fluorescent labeling in the retina (data not shown), consistent with stereospecific probe activation by effector caspases in vivo.…”

Section: Injection Of Noncleavable Dtcapq To Rule Out Nonspecific Probesupporting

confidence: 55%

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Single-cell imaging of retinal ganglion cell apoptosis with a cell-penetrating, activatable peptide probe in an in vivo glaucoma model

Barnett

Zhang

Maxwell

et al. 2009

Proc. Natl. Acad. Sci. U.S.A.

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126

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Molecular imaging probes have potential for in vivo identification of apoptosis and other intracellular processes. TcapQ, a cell-penetrating, near-infrared fluorescent peptide probe designed to be optically silent through intramolecular fluorescence quenching and activated by effector caspases, has been previously described and validated in vitro. Herein, using NMDA-induced apoptosis of retinal ganglion cells (RGCs), representing an in vivo rat model of glaucoma, we assessed the ability of TcapQ to image single-cell apoptosis through effector caspase activity. Following intravitreal injection, intracellular TcapQ activation occurred specifically in RGCs, identified individual apoptotic cells, showed a clear dose-response relationship with NMDA, and colocalized with TUNEL labeling in the retina. There was a significant diminution of probe activation following pretreatment with a specific inhibitor of caspase-3. Stereospecificity was also exhibited by the lack of intracellular fluorescence upon administration of the noncleavable isomer, d TcapQ. TcapQ has potential utility in detecting and monitoring single-cell apoptosis in glaucoma in vivo.

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Section: Injection Of Noncleavable Dtcapq To Rule Out Nonspecific Probesupporting

confidence: 55%

“…The in vitro and preliminary in vivo validation of a cell-penetrating effector caspase imaging probe, TcapQ, has been previously described (18,19). This probe utilizes a modified Tat peptide cell-penetrating moiety, which confers a surprisingly selective uptake by RGCs following intravitreal injection (20).…”

mentioning

confidence: 99%

Single-cell imaging of retinal ganglion cell apoptosis with a cell-penetrating, activatable peptide probe in an in vivo glaucoma model

Barnett

Zhang

Maxwell

et al. 2009

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

126

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“…Bullok & Piwnica-Worms [41] developed a near-IR probe for caspase-3 to image apoptosis and showed successful application in vitro and in vivo. The C-terminal side of caspase-3 substrate was tagged with Alexa Fluor 647 as the energy donor, and the quencher QSY21 as the energy acceptor was placed at the N-terminal side of the substrate.…”

Section: (D) Substrate Reporter Based On Fluorescence Resonance Energmentioning

confidence: 99%

Optical imaging for the new grammar of drug discovery

Krucker¹,

Sandanaraj²

2011

Phil. Trans. R. Soc. A.

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Optical technologies used in biomedical research have undergone tremendous development in the last decade and enabled important insight into biochemical, cellular and physiological phenomena at the microscopic and macroscopic level. Historically in drug discovery, to increase throughput in screening, or increase efficiency through automation of image acquisition and analysis in pathology, efforts in imaging were focused on the reengineering of established microscopy solutions. However, with the emergence of the new grammar for drug discovery, other requirements and expectations have created unique opportunities for optical imaging. The new grammar of drug discovery provides rules for translating the wealth of genomic and proteomic information into targeted medicines with a focus on complex interactions of proteins. This paradigm shift requires highly specific and quantitative imaging at the molecular level with tools that can be used in cellular assays, animals and finally translated into patients. The development of fluorescent targeted and activatable 'smart' probes, fluorescent proteins and new reporter gene systems as functional and dynamic markers of molecular events in vitro and in vivo is therefore playing a pivotal role. An enabling optical imaging platform will combine optical hardware refinement with a strong emphasis on creating and validating highly specific chemical and biological tools.

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“…In one study a construct containing the TAT-PTD sequence, a tetrapeptide from the amino side of a target recognition/cleavage site, and both a donor and a quencher was synthesized as a probe for proteases inside live cells [19]. The TAT-PTD is believed to transport macromolecules into cells by an initial interaction with cell membrane lipid rafts in a receptor-independent manner, followed by a rapid internalization through macropinocytosis, then a pH drop and destabilization of the integrity of the macropinosome vesicle lipid bilayer, and, ultimately, release of the TATcargo into the cytosol [20].…”

Section: Approaches To Protease Probe and Inhibitor Designmentioning

confidence: 99%

Intracellular protease activation in apoptosis and cell-mediated cytotoxicity characterized by cell-permeable fluorogenic protease substrates

Packard

Komoriya

2008

Cell Res

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Over the past decade the importance of signaling from reporter molecules inside live cells and tissues has been clearly established. Biochemical events related to inflammation, tumor metastasis and proliferation, and viral infectivity and replication are examples of processes being further defined as more molecular tools for live cell measurements become available. Moreover, in addition to quantitating parameters related to physiologic processes, real-time imaging of molecular interactions that compose basic cellular activities are providing insights into understanding disease mechanisms as well as extending clinical efficacy of therapeutic regimens. In this review the use of highly cell-permeable fluorogenic substrates that report protease activities inside live cells is described; applications to defining the molecular events of two cellular processes, i.e., apoptosis and cell-mediated cytotoxicity, are then illustrated.

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Synthesis and Characterization of a Small, Membrane-Permeant, Caspase-Activatable Far-Red Fluorescent Peptide for Imaging Apoptosis

Cited by 133 publications

References 14 publications

Single-cell imaging of retinal ganglion cell apoptosis with a cell-penetrating, activatable peptide probe in an in vivo glaucoma model

Single-cell imaging of retinal ganglion cell apoptosis with a cell-penetrating, activatable peptide probe in an in vivo glaucoma model

Optical imaging for the new grammar of drug discovery

Intracellular protease activation in apoptosis and cell-mediated cytotoxicity characterized by cell-permeable fluorogenic protease substrates

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