Peptide leucine arginine

We developed a Tc-99m and TAMRA-labeled peptide, Tc-99m arginine-arginine-leucine (RRL) peptide (TAMRA-GHEG-ECG-RRL), to target tumor cells and evaluated the diagnostic performance of Tc-99m TAMRA-GHEG-ECG-RRL as a dual-modality imaging agent for tumor in a murine model. TAMRA-GHEG-ECG-RRL was synthesized using Fmoc solid-phase peptide synthesis. Binding affinity and in vitro cellular uptake studies were performed. Gamma camera imaging, biodistribution, and ex vivo imaging studies were performed in murine models with PC-3 tumors. Tumor tissue slides were prepared and analyzed with immunohistochemistry using confocal microscopy. After radiolabeling procedures with Tc-99m, Tc-99m TAMRA-GHEG-ECG-RRL complexes were prepared in high yield (>96%). The Kd of Tc-99m TAMRA-GHEG-ECG-RRL determined by saturation binding was 41.7 ± 7.8 nM. Confocal microscopy images of PC-3 cells incubated with TAMRA-GHEG-ECG-RRL showed strong fluorescence in the cytoplasm. Gamma camera imaging revealed substantial uptake of Tc-99m TAMRA-GHEG-ECG-RRL in tumors. Tumor uptake was effectively blocked by the coinjection of an excess concentration of RRL. Specific uptake of Tc-99m TAMRA-GHEG-ECG-RRL was confirmed by biodistribution, ex vivo imaging, and immunohistochemistry stain studies. In conclusion, in vivo and in vitro studies revealed substantial uptake of Tc-99m TAMRA-GHEG-ECG-RRL in tumors. Tc-99m TAMRA-GHEG-ECG-RRL has potential as a dual-modality tumor imaging agent.

The peptide leucine arginine (pLR) belongs to a new class of cyclic peptides isolated from frog skin. Its primary sequence is similar to the reactive loop of plant Bowman-Birk inhibitors (BBI), and the recently discovered circular sunflower trypsin inhibitor-1 (SFTI-1). The conformational properties of pLR in solution were determined by NMR spectroscopy and revealed excellent structural similarity to BBI and SFTI-1. Moreover, pLR is a highly potent trypsin inhibitor, with Ki values in the nanomolar range, and, due to its small size, a potential inhibitor of the serine protease tryptase. Since tryptase plays a crucial role in the development of allergic airway inflammation, the therapeutic potential of pLR in a murine asthma model was investigated. Treatment of ovalbumin-sensitized mice with pLR during allergen challenge reduced the acute asthma phenotype. Most importantly, application even at the end of a long-lasting chronic asthma model decreased the development of chronic airway inflammation and tissue remodeling.

On the basis of histamine release from rat peritoneal mast cells, an octadecapeptide was isolated from the skin extract of the Northern Leopard frog (Rana pipiens). This peptide was purified to homogeneity using reversed-phase high performance liquid chromatography and found to have the following primary structure by Edman degradation and pyridylethylation: LVRGCWTKSYPPKPCFVR, in which Cys(5) and Cys(15) are disulfide bridged. The peptide was named peptide leucine-arginine (pLR), reflecting the N- and C-terminal residues. Molecular modeling predicted that pLR possessed a rigid tertiary loop structure with flexible end regions. pLR was synthesized and elicited rapid, noncytolytic histamine release that had a 2-fold greater potency when compared with one of the most active histamine-liberating peptides, namely melittin. pLR was able to permeabilize negatively charged unilamellar lipid vesicles but not neutral vesicles, a finding that was consistent with its nonhemolytic action. pLR inhibited the early development of granulocyte macrophage colonies from bone marrow stem cells but did not induce apoptosis of the end stage granulocytes, i.e. mature neutrophils. pLR therefore displays biological activity with both granulopoietic progenitor cells and mast cells and thus represents a novel bioactive peptide from frog skin.