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Ac-rkkrrorrrGK(QSY21)DEVDAPC(Alexa Fluor 647)-NH2.

Source

Molecular Imaging and Contrast Agent Database (MICAD) [Internet]. Bethesda (MD): National Center for Biotechnology Information (US); 2004-2013.
2008 Jan 23 [updated 2008 Apr 22].

Author information

1
National Center for Biotechnology, NLM, NIH, Bethesda, MD, Email: micad@ncbi.nlm.nih.gov
2
Molecular Imaging Center, Mallinckrodt Institute of Radiology, Department of Molecular Biology and Pharmacology, Washington University, St. Louis, MO, Corresponding Author, Email: piwnica-wormsd@mir.wustl.edu

Excerpt

Ac-rkkrrorrrGK(QSY21)DEVDAPC(Alexa Fluor 647)-NH2 (TCAPQ647) is a membrane-permeable, activatable caspase substrate developed for optical imaging of programmed cell death (apoptosis) (1, 2). TCAPQ647 is a small permeation peptide that comprises QSY21, a broad-spectrum quencher, and Alexa Fluor 647, a near-infrared fluorophore. Alexa Fluor 647 (AF647) is a fluorescence dye with a peak excitation of 650 nm, a peak emission maximum of 665 nm, and an extinction coefficient of 203,000 cm–1 M–1 (3). Apoptosis is an essential biological process that maintains homeostasis of tissues and organs in concert with proliferation, growth, and differentiation (4-6). Cell death can occur by the process of necrosis or by the process of apoptosis. Apoptosis is a highly regulated, genetically controlled, noninflammatory process that requires ATP (7). The apoptotic process can be triggered either by a decrease in factors required to maintain the cell in good health or by an increase in factors that cause cells to die (8). The two known mechanisms of apoptosis are the death receptor (extrinsic) and the mitochondrial (intrinsic) pathways (9, 10). Both pathways culminate in a mutual proteolytic cascade consisting of cysteine aspartic acid-specific proteases (caspases) (1, 2, 11, 12). These enzymes act as executors of the cell death process (13). Caspases are heterodimeric zymogens in all animal cells and are activated by proteolytic maturation or allosteric interactions (12). Fourteen mammalian caspases have been identified, and they function as either initiator or effector caspases. Initiator caspases receive and transmit signals to effector caspases, leading to eventual cell death. Annexin 5, labeled paramagnetically, optically, or with a radionuclide, can be used to image cell death on the basis of binding to phosphatidylserine, and when combined properly with a second probe documenting the integrity of an intact plasma membrane, the signal can signify externalized phosphatidylserine and apoptosis (9, 14, 15). However, probes such as Annexin 5 generally bind in a one-to-one ratio with limited signal generation. An alternative target for detection of apoptosis is the apoptosis-specific caspases. Because activation of effector caspases generally indicates cellular commitment to apoptosis, in vivo imaging of these caspases could offer a valuable and highly specific tool for early detection of apoptosis (2, 9). Bullock et al. (1) first described the development of TCAPQ647 as a small, membrane-permeant, caspase-activatable, near-infrared/far-red, fluorescent peptide for imaging apoptosis. To penetrate the plasma membrane of cells, the backbone of the probe is composed of a permeation peptide, an all d-amino acid human immunodeficiency virus-1Tat (HIV-1Tat) peptide-based sequence (16-18). Tat peptides likely enter cells through a non-receptor-mediated endocytic pathway or by macropinocytosis (18-21). Indeed, radiolabeled Tat peptides have been shown to rapidly accumulate and concentrate within cells in culture (22, 23). In addition, conjugation of the peptide with fluorescein could directly reveal the intracellular localization of the peptide within cells. In the design of TCAPQ647, the all d-amino acid permeation peptide sequence rkkrrqrrrg was placed at the N-terminus of an l-amino acid effector caspase recognition sequence, DEVD (1, 2). Bullok et al. (1) reported that the N-terminal placement showed enhanced kinetics over the C-terminal placement. The DEVDAPC-NH2 peptide fragment was flanked by AF647 and a spectrally complimented quencher QSY21. Conjugation of QSY21 near AF647 effectively absorbed and quenched fluorescence emission from AF647. Bullok et al. (1) suggested that the modest molecular weight of TCAPQ647 provides better tissue diffusion properties than the larger annexin V probes. Bullok et al. (1, 2) showed that APC-AF647 was enzymatically cleaved from TCAPQ647 in the presence of effector caspases 3, 6, and 7, and fluorescence emission of AF647 was effectively dequenched and the probe activated. As a result, this activatable fluorescent molecular probe provided an enhanced signal-to-background ratio and higher sensitivity for imaging caspase activity.

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