Annexin B12 Cys101,Cys260-N,N'-dimethyl-N-(iodoacetyl)-N'-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)ethylenediamine

Excerpt

Apoptosis, or programmed cell death, presents specific biochemical changes at its different stages (1-4). By targeting changes such as exposed phosphatidylserine (PS), dysfunctional mitochondria, activated caspases, fragmented DNA, and disrupted membrane integrity, various apoptosis-detecting methods have been developed and provide diagnostic and prognostic value for human diseases, most notably for cancer, myocardial dysfunction, and neurodegenerative disorders (2-5). For several reasons, exposed PS has been used as a single and direct target for apoptosis detection (2, 6-10) although it is not specific for apoptosis. First, PS is normally limited to the inner leaflet of the plasma membrane bilayer, whereas it is externalized to the outer leaflet of the membrane in the presence of calcium ions during apoptosis. Second, cell surface exposure of PS has been considered to be an indicator of early apoptotic processes in the absence of necrosis. Therefore, monitoring PS exposure provides a way to observe the initiation of the apoptotic pathway before other changes are present. Third, PS exposure provides extracellular binding targets that can be detected without the need to penetrate the cells.

Annexins are cellular proteins that are widely used to design PS-targeted, apoptosis-detecting probes (1, 7, 9). Annexins are characterized by their calcium-dependent ability to bind to negatively charged PS. All annexins are composed of two distinct regions: a C-terminal core and an amino N-terminal “head” region. The annexin core is highly conserved across the annexin family, and the N-terminus varies greatly. Radiolabeled annexin derivatives have already been used for clinical imaging in many diseases (2, 8, 10). However, annexin-based probes suffer from several drawbacks, such as slow delivery to the site of interest, slow clearance from tissue leading to high effective doses, and low specificity. Annexin-based probes do not appear to distinguish apoptosis from necrosis. Furthermore, PS externalization is associated with inflammation and platelet activation, causing further challenges with annexin-based probe specificity.

To address these problems, Kim et al. developed a class of polarity-sensitive annexin-based probes (pSIVAs) with built-in "on" (membrane-bound) and "off" (in solution) fluorescent states (1). The probes were generated by placing polarity-sensitive thiol-reactive fluorophores in the loop regions of annexin B12 (anxB12, also known as annexin XII) or annexin A5 (anx5, also known as annexin V), taking advantage of loop region mediation of Ca²⁺-dependent membrane interactions, transitioning from a polar (aqueous solution) to a nonpolar (lipid membrane) environment upon membrane binding. Two thiol-reactive fluorophores, N,N'-dimethyl-N-(iodoacetyl)-N'-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)ethylenediamine (IANBD) and 6-bromoacetyl-2-dimethylaminonaphthalene (BADAN), were tested, and the resulting probes were evaluated for apoptosis detection. The results showed that probe binding with PS on the apoptotic cells resulted in a measurable increase in fluorescence emission intensity. pSIVA_m (also known as anxB12 Cys101,Cys260-IANBD) is one of the promising probes for apoptosis detection (1).