Radiopharmacological evaluation of (18)F-labeled phosphatidylserine-binding peptides for molecular imaging of apoptosis.

INTRODUCTION: Radiolabeled phosphatidylserine (PS)-binding peptides represent an innovative strategy for molecular imaging of apoptosis with positron emission tomography (PET). The goal of this study was the radiopharmacological evaluation of radiolabeled peptides for their binding to PS on apoptotic cancer cells, involving metabolic stability, cellular uptake, biodistribution, and dynamic PET imaging experiments. METHODS: Binding of peptides LIKKPF, PGDLSR, FBz-LIKKPF, FBz-PGDLSR, FBAM-CLIKKPF and FBAM-CPGDLSR to PS was analyzed in a newly developed radiometric binding assay using (64)Cu-labeled wild-type annexin-V as radiotracer. Radiolabeling of most potent peptides with fluorine-18 was carried out with thiol-selective prosthetic group [(18)F]FBAM to give [(18)F]FBAM-CLIKKPF and [(18)F]FBAM-CPGDLSR. [(18)F]FBAM-labeled peptides were studied in camptothecin-induced apoptotic human T lymphocyte Jurkat cells, and in a murine EL4 tumor model of apoptosis using dynamic PET imaging and biodistribution. RESULTS: Peptides LIKKPF and PGDLSR inhibited binding of (64)Cu-labeled annexin-V to immobilized PS in the millimolar range (IC50 10-15 mM) compared to annexin-V (45 nM). Introduction of FBAM prosthetic group slightly increased inhibitory potencies (FBAM-CLIKKPF: IC50 = 1 mM; FBAM-CPGDLSR: IC50 = 6 mM). Radiolabeling succeeded in good radiochemical yields of 50-54% using a chemoselective alkylation reaction of peptides CLIKKPF and CPGDLSR with [(18)F]FBAM. In vivo metabolic stability studies in mice revealed 40-60% of intact peptides at 5 min p.i. decreasing to 25% for [(18)F]FBAM-CLIKKPF and less than 5% for [(18)F]FBAM-CPGDLSR at 15 min p.i.. Cell binding of [(18)F]FBAM-CLIKKPF in drug-treated Jurkat cells was significantly higher compared to untreated cells, but this was not observed for [(18)F]FBAM-CPGDLSR. Dynamic PET imaging experiments showed that baseline uptake of [(18)F]FBAM-CLIKKPF in EL4 tumors was higher (SUV(5min) 0.46, SUV(60min) 0.13) compared to [(18)F]FBAM-CPGDLSR (SUV(5min) 0.16, SUV(60min) 0.10). Drug-treated EL4 tumors did not show an increased uptake for both [(18)F]FBAM-labeled peptides. CONCLUSION: Although both (18)F-labeled peptides [(18)F]FBAM-CLIKKPF and [(18)F]FBAM-CPGDLSR showed higher binding to apoptotic Jurkat cells in vitro, their in vivo uptake profiles were not different in apoptotic EL4 tumors. This may explained by the relatively low potency of both compounds to compete with binding of (64)Cu-labeled annexin-V to PS. Overall the novel competitive radiometric PS-binding assay with (64)Cu-labeled annexin-V represents a versatile and very robust screening platform to analyze potential PS-binding compounds in vitro. Further studies will be necessary to evaluate alternative peptide structures toward their use as PET radiotracers imaging apoptosis in vivo. ADVANCES IN KNOWLEDGE AND IMPLICATIONS FOR PATIENT CARE: Development of peptide-based radiotracers for imaging apoptosis in vivo remains a significant challenge.

A computationally designed DNA aptamer template with specific binding to phosphatidylserine.

The phospholipid phosphatidylserine (PS) is an early marker exploited for detecting apoptosis (PS externalization in the cell membrane bilayer) and one factor that is associated with increased amyloid plaque deposition in transmissible spongiform encephalopathies (TSEs). PS can therefore be considered as a promising target for diagnosis or treatment of diseases. Aptamers (short nucleic acid sequences) are a particularly attractive class of materials among those currently considered for targeting PS. Here we applied an entropy based seed-and-grow strategy to design a DNA aptamer template to bind specifically to PS. The binding properties of designed aptamers were investigated computationally and experimentally. The studies identify the sequence, 5'-AAAGAC-3', as the preferred template for further modifications and studies toward its practical implementations.

Evaluation of phosphatidylserine-binding peptides targeting apoptotic cells.

The inhibition or dysregulation of apoptosis plays an intimate role in the initiation and progression of cancer by confounding normal tissue homeostasis. We currently do not have a clinical method to assess apoptosis induced by cancer therapies. Phosphatidylserine (PS) is an attractive target for imaging apoptosis because it is on the exterior of the apoptotic cells and PS externalization is an early marker of apoptosis. PS-binding peptides are an attractive option for developing an imaging probe to detect apoptosis using positron emission tomography. In this study, four peptides were evaluated for PS-binding characteristics using a plate-based assay system, a liposome mimic of cell membrane PS presentation, and a cell assay of apoptosis. This work also describes two screening techniques to enable researchers to identify and optimize compounds that bind to PS. The results of our study indicate that all four peptides bind to PS and are specific to apoptotic cells. Two of the peptides in particular that have an additional cysteine residue are good potential candidates for development into imaging probes because they bind to PS with high affinity and specificity and they can be easily radiolabelled with (18)F.

Radiolabeling of phosphatidylserine-binding peptides with prosthetic groups N-[6-(4-[18F]fluorobenzylidene)aminooxyhexyl]maleimide ([18F]FBAM) and N-succinimidyl-4-[18F]fluorobenzoate ([18F]SFB).

The widely used (18)F-prosthetic group N-succinimidyl-4-[(18)F]fluorobenzoate ([(18)F]SFB) and the recently developed N-[6-(4-[(18)F]fluorobenzylidene)aminooxyhexyl]maleimide ([(18)F]FBAM) were investigated for radiolabeling of two representative phosphatidylserine-binding peptides. The prosthetic groups were compared with respect to required reactions conditions for optimum labeling, radiolabeling yield and chemoselectivity. The N-terminus labeled product produced by reaction of [(18)F]SFB with binding peptide LIKKPF was produced in 18% radiochemical yield while no N-terminus labeled product could be isolated following [(18)F]SFB reaction with PDGLSR. When the peptides were modified by addition of a cysteine residue at the N-terminus they provided almost quantitative radiochemical yields with [(18)F]FBAM. Results indicate that for the peptides in this study, [(18)F]FBAM is a more useful prosthetic group compared to [(18)F]SFB due to its excellent chemoselectivity and high radiochemical yield.

Entropic fragment-based approach to aptamer design.

Aptamers are short RNA/DNA sequences that are identified through the process of systematic evolution of ligands by exponential enrichment and that bind to diverse biomolecular targets. Aptamers have strong and specific binding through molecular recognition and are promising tools in studying molecular biology. They are recognized as having potential therapeutic and diagnostic clinical applications. The success of the systematic evolution of ligands by exponential enrichment process requires that the RNA/DNA pools used in the process have a sufficient level of sequence diversity and structural complexity. While the systematic evolution of ligands by exponential enrichment technology is well developed, it remains a challenge in the efficient identification of correct aptamers. In this article, we propose a novel information-driven approach to a theoretical design of aptamer templates based solely on the knowledge regarding the biomolecular target structures. We have investigated both theoretically and experimentally the applicability of the proposed approach by considering two specific targets: the serum protein thrombin and the cell membrane phospholipid phosphatidylserine. Both of these case studies support our method and indicate a promising advancement in theoretical aptamer design. In unfavorable cases where the designed sequences show weak binding affinity, these template sequences can be still modified to enhance their affinities without going through the systematic evolution of ligands by exponential enrichment process.

Radiotracers for noninvasive molecular imaging of tumor cell death.

The need to monitor cancer therapy-induced cellular and tissue changes using noninvasive imaging techniques continues to stimulate both basic and clinical research. Monitoring changes in cellular proliferative capacity that occur after treatment with radiation and/or chemotherapy has the potential to provide longitudinal information on the cellular dynamics of tumors before, during, and after therapeutic intervention. Cells can lose their reproductive potential through one of several mechanisms, including apoptosis and autophagy (which are forms of programmed cell death), premature senescence, or necrosis. When a tumor responds to therapy, current imaging methods do not provide information about the exact mechanism of cell death executed. We are now beginning to develop the molecular imaging tools that will enable us to noninvasively image cell death mechanisms both in experimental models and in the clinical cancer environment. Studies with these imaging tools will contribute to a better understanding of therapeutic responses and assist in the design and evaluation of more effective treatments. This review examines the state-of-the-art in the use of (radio)tracers for the purpose of imaging mechanisms of tumor cell inactivation (cell death) in animal models and in clinical trials.