Drug Development-targeted Screening of Leptin Agonist Glycopeptides |
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Authors: | Daniel Knappe Laszlo Otvos Jr. Francesco De Pascali Marco Cassone Laura Scolaro Giovanni Abbadessa Cynthia Carrillo-Infante Mohammad F. Kiani Fred Donelson Ralf Hoffmann Eva Surmacz |
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Affiliation: | (1) Institute of Bioanalytical Chemistry, Center for Biotechnology and Biomedicine, Leipzig University, Leipzig, 04103, Germany;(2) Sbarro Institute for Cancer Research and Molecular Medicine, Temple University, Philadelphia, PA 19122, USA;(3) Department of Biology, Temple University, 1900 North 12th Street, Philadelphia, PA 19122, USA;(4) Department of Mechanical Engineering, Temple University, Philadelphia, PA 19122, USA |
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Abstract: | A glycosylated dodecapeptide fragment corresponding to the hypothalamus-active cytokine leptin exhibits agonistic properties to the leptin receptor (ObR) in vitro and penetrates into the brain in vivo. In order to characterize the drug development potential of the lead peptide and to optimize it for pharmacological applicability, a series of biochemical screening assays were custom-tailored to the leptin/ObR system. To identify peptides that bind the extracellular domain of ObR, we characterized the optimal conditions for an ELISA-type assay where the leptin fragments were immobilized to the plates. With this technology we could identify low-dose binder peptidomimetics which, according to a comparison of the conventional cell proliferation assay and a measure of metabolically active cells, revealed that agonists identified by these cellular assays may not necessarily induce the expected growth characteristics in ObR expressing cells. The original glycopeptide lead displayed a 2 h half life in 25% diluted mouse serum but poor stability in mouse brain extract. Fifteen percent of the glycopeptide crossed a dual endothelial/astrocyte cell layer (representing an in vitro model of blood-brain-barrier) in 30 min, and the coexistence of the two cell types appeared necessary to quantify the level of brain accessibility. Finally, in an in vivo mouse model, a Cy5.5 labeled glycopeptide was more evenly distributed all over the body, including the brain, than a similarly labeled full-sized leptin protein. |
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Keywords: | KeywordHeading" >Kewords Binding Biodistribution Blood-brain-barrier penetration Glycoamino acid Leptin deficiency Leptin receptor Obesity Signal transduction |
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