Metal ions often play critical roles in protein structure and function. Engineered metal-binding sites in peptides and proteins have been widely used to enhance structural integrity, stabilize biologically active conformations, and confer novel enzymatic activities (1-3). Biochemical and structural analyses of transition-metal coordination by proteins and peptides have traditionally focused on zinc, copper, manganese, and iron because of their roles in important biological processes (4-7). Other transition metals not found in natural proteins have coordination, isotopic, and chemical properties that make them attractive for peptide and protein engineering. Rhenium (Re) and technetium (Tc) are group VIIB transition metals that share similar coordination geometries and form stable complexes with amine and amide nitrogens, carboxylate oxygens, and thiolate and thioether sulfurs, with a strong preference for thiolate sulfurs (8). Radioactive isotopes of Re and Tc have significant medical applications because of the nature of their associated radiation and physical half-life properties.The synthesis and characterization of radiolabeled antibodies, peptides, and steroid hormones as in vivo tumor-imaging and therapeutic agents is an active area of cancer research today. These molecules specifically target tumor cells by virtue of their high specificities for receptors and antigens present on the surfaces of these cells. In one commonly used approach, metallic radionuclides such as 186 Re, 188

No abstract

Receptor binding peptides labeled with medically important radionuclides such as technetium and rhenium are an important tool for the imaging and treatment of many forms of cancer. This paper describes a method of labeling peptides with rhenium using a natural amino acid chelating moiety. The structural characteristics of this chelate moiety, N-acetyl-cysteine-glycine-cysteine-glycine (NAc-CGCG) complexed with nonradioactive rhenium, have been investigated. The stability of this peptide-metal complex has been evaluated on the tracer level using radioactive rhenium-186. The rhenium-bound peptide has been appended to the N termini of receptor binding alpha-melanocyte stimulating hormone (alpha-MSH, NAc-Ser-Tyr-Ser-Met-Glu-His-Phe-Arg-Trp-Gly-Lys-Pro-Val-NH2) fragments via solid phase peptide synthesis. Bioassays and receptor binding studies of the resulting complexes demonstrate that the fragments retained biological activity and exhibited receptor binding constants ranging from 0.3 to 1.1 nM. This method could provide a general means of labeling bioactive peptide fragments that would simplify product purification and characterization.

This study describes the synthesis and preliminary biologic evaluation of an 111 Inlabeled peptide antagonist of the urokinase-type plasminogen activator receptor (uPAR) as a potential probe for assessing metastatic potential of human breast cancer in vivo. The peptide (NAc-dD-CHA-F-dS-dR-Y-L-W-S-βAla) 2 -K-K(DOTA)-NH 2 was synthesized and conjugated with the DOTA chelating moiety via conventional Solid-Phase Peptide Synthesis (SPPS), purified by reversed-phase HPLC, and characterized by MALDI-TOF MS and receptor binding assay. In vitro receptor binding studies demonstrated an IC 50 of 240 ± 125 nM for the peptide, compared with IC 50 's of 0.44 ± 0.02 and 0.75 ± 0.01 nM for the amino terminal fragment (ATF) of the urokinase-type plasminogen activator (uPA) and full-length uPA, respectively. In vivo biodistribution studies were carried out using SCID mice bearing MDA-MB-231 human breast cancer xenografts. Biodistribution data was collected at 1, 4, and 24 hr post-injection of 111 In-DOTA-peptide, and compared with data obtained using a scrambled control peptide, as well as with data obtained using wild-type ATF radiolabeled with I-125. Biodistribution studies showed rapid elimination of the 111 In-labeled peptide from the blood pool, with 0.12 ± 0.06% ID/g remaining in blood at 4 hr pi. Elimination was seen primarily via the renal/ urinary route, with 83.9 ± 2.2%ID in the urine at the same timepoint. Tumor uptake at this time was 0.53 ± 0.11%ID/g, resulting in tumor: blood and tumor: muscle ratios of 4.2 and 9.4, respectively. Uptake in tumor was significantly higher than that obtained using a scrambled control peptide that showed no specific binding to uPAR (p < 0.05). In vitro and ex vivo results both suggested that the magnitude of tumor-specific binding was reduced in this model by endogenous expression of uPA. The results indicate that radiolabeled peptide uPAR antagonists may find application in the imaging and therapy of uPAR-expressing breast cancers in vivo.