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

Background

[PubMed]

Ac-rkkrrorrrGK(QSY21)DEVDAPC(Alexa Fluor 647)-NH₂ (T_CAPQ₆₄₇) is a membrane-permeable, activatable caspase substrate developed for optical imaging of programmed cell death (apoptosis) (1, 2). T_CAPQ₆₄₇ 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 T_CAPQ₆₄₇ 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 T_CAPQ₆₄₇, 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 T_CAPQ₆₄₇ provides better tissue diffusion properties than the larger annexin V probes. Bullok et al. (1, 2) showed that APC-AF647 was enzymatically cleaved from T_CAPQ₆₄₇ 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.

Synthesis

[PubMed]

Bullok et al. (1) reported the synthesis of T_CAPQ₆₄₇. Briefly, the peptide backbone was synthesized via solid-phase peptide synthesis with the use of standard 9-fluorenylmethyloxycarbonyl chemistry. With the protected amino acid remaining on resin, the 1-(4,4-dimethyl-2,6-dioxocyclohexylidene)ethyl protecting group on the lysine adjacent to the DEVDAP sequence was selectively removed using 2% hydrazine in dimethylformamide. The resin containing the peptide was then dried and mixed with QSY21 succinimide in anhydrous dimethylformamide. Coupling of QSY21 to the primary amine of the lysine was allowed to proceed overnight. After extensive washing, the QSY21 couple was simultaneously deprotected and cleaved from the resin with the use of trifluoroacetic acid. AF647 was then thiol-conjugated to the C-terminal cysteine of the peptide to yield the final T_CAPQ₆₄₇ probe. T_CAPQ₆₄₇ was purified with reverse-phase high-performance liquid chromatography (HPLC) and characterized by electrospray mass spectrometry (m/z: 3927.0; calc: 3927.2), absorption spectrometry, and fluorometry. The yield of synthesis was 23.5% and the purity of the product was >95% (see Supporting Information for (1))

In Vitro Studies: Testing in Cells and Tissues

[PubMed]

Bullok et al. (1, 2) determined the in vitro fluorescence properties of T_CAPQ₆₄₇. The study showed a blue-shifted form of the absorption maximum for AF647 from 650 nm to 605 nm. This indicated a strong Coulombic interaction between the QSY21 and AF647 chromophores. The extinction coefficient of T_CAPQ₆₄₇ was determined to be 211,500 M^–1•cm^–1 at 605 nm. The fluorescence quenching efficiencies of T_CAPQ₆₄₇ were calculated to be 92–99% after incubation at 37ºC in milliQ water, in DMEM media with 10% heated inactivated fetal bovine serum (FBS), and in 100% heat-inactivated FBS.

Bullok et al. (1, 2) studied the ability of effector caspases to recognize and cleave T_CAPQ₆₄₇ by an extensive end point assay with recombinant caspase 3 at 37ºC. HPLC analysis indicated sequence-specific cleavage by caspase 3. Enzymatic kinetic studies using continuous fluorometric analysis were also conducted with recombinant caspases 1–10 (2). The study showed that T_CAPQ₆₄₇ was preferentially cleaved by the effector caspases 3, 6, and 7 but not by the caspases 1, 2, 4, 5, 8, 9, and 10. The “k_cat/K_m” values in (FU min^–1•μM^–1)/nM were 60, 180, and 36 for caspases 3, 7, and 6, respectively. Caspases 3, 7 and 6 cleaved T_CAPQ₆₄₇ at a 1,100-fold, 295-fold and 70-fold higher rate than caspase 9, respectively (2). Caspase inhibition assays conducted by Bullok et al. (1) showed that the reversible caspase inhibitor, DEVD-CHO, inhibited cleavage by caspase 7 (inhibition concentration (IC₅₀) = 11 nM) and caspase 3 (IC₅₀ = 1.2 nM) in a concentration-dependent manner.

Bullok et al. (1) induced apoptosis in KB 3-1 tumor cells by vinblastine treatment to study the intracellular delivery, localization, and activation of T_CAPQ₆₄₇. Live-cell confocal microscopy was used to monitor fluorescence produced by T_CAPQ₆₄₇ activation. The study showed that T_CAPQ₆₄₇ activation was observed only in vinblastine-treated apoptotic cells. The results confirmed that T_CAPQ₆₄₇ fluorescence was enzyme-dependent and provided direct evidence for intracellular delivery and caspase-specific activation of T_CAPQ₆₄₇. Similar results were observed in three cell lines (KB 3-1, HeLa, and MCF-7) treated with doxorubicin, which has been shown to induce apoptosis via caspase activation (2). Only low fluorescence background was observed in doxorubicin-treated cells incubated with a noncleavable, quenched peptide (all d-amino acid dT_CAPQ₆₄₇). When the treated cells were incubated with the general caspase substrate (l-asparatate)₂-rhodamine 110 (D₂R), which was an indicator for both effector and initiator caspases, a larger number of cells demonstrated D2R activation. T_CAPQ₆₄₇ activation was inhibited when apoptotic cells were treated with the effector caspase inhibitor D(OMe)QMD(OMe)-FMK. In another study, flow cytometry analysis was used to quantitatively determine in cellulo T_CAPQ₆₄₇ activation in Jurkat cells with apoptosis induced by C₆-ceramide (1). Activation of caspase 3 in the C₆-ceramide–treated Jurkat cells was confirmed with Western blot analysis. Detection of 10% early- and late-stage apoptotic cells was reported in ceramide-treated cells incubated with T_CAPQ₆₄₇. In comparison, only 3% of apoptotic cells was detected in untreated cells; 4% was detected in treated cells incubated with noncleavable dT_CAPQ₆₄₇; and 22% was detected in treated cells incubated with D₂R. No cell toxicity was observed in any of the cell assays (1). When Renilla luciferase–expressing KB cells were incubated with 1 nM – 100 μM T_CAPQ₆₄₇ for 24 h, the 50% lethal dose of T_CAPQ₆₄₇ was determined to be 10 ± 2 μM, 10-fold to 20-fold higher than the concentrations useful for imaging. These results correlated well with visual observations that cell death was notable at 10 μM and complete cell death was seen at 100 μM.

Animal Studies

Human Studies

[PubMed]

No publication is currently available.

Supplemental Information

[Disclaimers]

TcapQ647 Targeting

NIH Support

NIH P50 CA94056, CA83841, RO1 82841, RO1 A130084.

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This MICAD chapter is not included in the Open Access Subset, because it was authored / co-authored by one or more investigators who was not a member of the MICAD staff.