Drug development-targeted screening of leptin agonist glycopeptides

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.