Cy5-Tat-Glu-Pro-Asp-acyloxymethyl ketone

Excerpt

Caspases are a group of cysteine proteases, which exist within cells as inactive zymogens, and are cleaved to form active enzymes after induction of apoptosis (1-3). There are two major pathways through which caspases are activated. One is the death signal–induced, death receptor–mediated extrinsic pathway, and the other is the stress-induced, mitochondria-directed intrinsic pathway (3, 4). Caspases convey the apoptotic signal in a proteolytic cascade, with caspases cleaving and activating other caspases. Caspase-8 and -9 act as initiators at the upper stream of this cascade, while caspase-3, -6, and -7 work as downstream executioners (1, 3, 4). Although physiological apoptosis takes place during embryonic development and in adult homeostasis, excessive apoptosis has been observed in some human diseases such as myocardial infarction, rheumatoid arthritis, ischemia, transplant rejection, and neurodegenerative disorders (Parkinson's and Alzheimer's) (5, 6). On the contrary, evading apoptosis is a hallmark of human cancer (7, 8).

Many different molecular probes have been developed for imaging apoptosis both at the cellular level and in vivo (2, 5, 7, 9, 10). Of them, a large number of the probes are annexin V–based. Annexin V is a 36-kDa protein that binds to externalized phosphatidylserine residues in the presence of calcium ions on the surface of apoptotic cells (5). Radiolabeled annexin V derivatives have already been used for clinical imaging in many diseases, most notably for imaging myocardial dysfunction. A drawback of annexin V–based probes is their low specificity to apoptotic cells. Annexin V cannot distinguish apoptosis from necrosis (5). Phosphatidylserine externalization is also associated with inflammation and platelet activation, causing further challenges with annexin V specificity. During recent years, much work has focused on the development of probes for caspase-3 and -7. Thornberry et al. have observed that most caspases prefer a tetrapeptide motif with an aspartyl residue at P4 (11). Asp-Glu-Val-Asp (DEVD) is the peptide sequence optimal for caspase-3 and -7, while the Val-Glu-His-Asp (VEHD) sequence is preferred by caspase-6 (5). These sequence-based probes demonstrate high caspase specificity; however, most probes suffer from poor cellular uptake in the intact cell, a requirement for early detection.

Edgington et al. developed a class of probes based on the acyloxymethyl ketone (AOMK) and the optimal sequence of caspase-3 and -7 (2). AB50-Cy5 is one of the probes, which contains a Glu-Pro-Asp-AOMK sequence labeled with the Cy5 fluorophore. This probe showed labeling of caspase-3 and legumain with no detectable cathepsin B labeling. To enhance the cell permeability of AB50-Cy5, the investigators synthesized the probe tAB50-Cy5, a version of the probe containing a tat peptide. For this probe, the tat peptide was used to increase the cell uptake of the probe through multiple positively charged amino acids, while AOMK was used to label caspases. This chapter summarized the synthesis and comparative analysis of tAB50-Cy5 and AB50-Cy5.