Leptin is an adipocyte-derived protein hormone (adipokine) with important role in energy balance regulation. Its plasma concentration depends on the amount of white adipose tissue and is also influenced by changes in the energy status of the organism. During the periods of excessive food intake plasma leptin levels increase while they decrease with fasting. Such changes are sensed by specific leptin-receptor expressing hypothalamic neurons, which project to many other centers in the brain to activate several compensatory mechanisms that rebalance organism’s energy status. Therefore, leptin represents the most important factor of the feedback loop which regulates the amount of energy reserves in the form of fat. Despite its elevated plasma concentrations in most obese people leptin fails to activate central compensatory mechanisms to reduce food intake and increase energy expenditure due to central leptin resistance. Increased plasma leptin concentration (hyperleptinemia) leads to excessive peripheral leptin signaling as leptin receptors are also present on diverse cells outside the central nervous system. There is accumulating evidence that excessive peripheral leptin activity contributes to the onset and progression of serious obesity-related conditions, such as certain types of cancer, autoimmune and cardiovascular diseases. In light of these findings, there is a growing interest in the development of leptin antagonists as potential novel class of drugs for use in therapy of aforementioned diseases. On the other hand, leptin agonists also hold potential as therapeutics for lipodystrophy, hypothalamic amenorrhea and general immune deficiency due to starvation. Moreover, recent studies also support the use of leptin agonists in therapy of type 2 diabetes. Because both, hyperleptinemia and leptin deficiency can have harmful consequences, we decided to screen phage display libraries with the main goal to identify biologically active peptide ligands of leptin or leptin receptor. Such peptides would be considered leads for development of therapeutically useful leptin agonists or antagonists. Even biologically inactive leptin or leptin receptor ligands might still find use as detection molecules in analytical or diagnostic techniques. We were unable to isolate any leptin or leptin receptor ligands from phage display libraries of random short peptides. While affinity selections towards anti-leptin neutralizing antibodies did yield binders, none of the mimotopes showed cross-reactivity with leptin receptor. Based on extended analysis of results from previous affinity selections performed in our laboratory, we conclude that the nature of target molecule critically affects the outcome of biopanning in terms of successful isolation of specific short peptide ligands. According to our findings, the most suitable targets for selection of short peptide ligands are those whose natural binding partners are peptides or proteins, and that contain specific topological features (e.g., clefts or pockets) allowing anchoring of short peptides. In contrast, proteins engaged in protein-protein interactions, which are generally characterized by relatively large and flat contact surfaces, proved to be less suitable targets for selection of short peptide ligands from phage display libraries. Therefore, we focused on screening phage display libraries of synthetic single-chain variable fragments (scFvs) instead. Through biopanning we isolated four different scFvs which specifically recognize the native form of extracellular region of the human leptin receptor, and compete with leptin for binding to the receptor. One of them (3L5) showed moderate selectivity for human leptin receptor as compared to its murine orthologue. In addition, 3L5 scFv inhibited leptin-stimulated proliferation of THP-1 cells in preliminary assay indicating it acts as leptin antagonist. Estimated values of the constants IC50 (for 3L5 scFv-mediated inhibition of leptin binding to the leptin receptor) and Kd (for 3L5 scFv-leptin receptor interaction in solution) are in the nanomolar range. Therefore, we believe that 3L5 scFv represents a promising lead for further development of therapeutically useful antibody-based leptin antagonists and molecular probes for leptin receptor detection. Last but not least, we developed a model system for simultaneous presentation of two different proteins on the surface of filamentous phage virions. We designed and constructed three different type 88 helper phages and evaluated their ability to introduce green fluorescent protein into the capsid of phagemid-based viral particles simultaneously expressing scFv fragments on their surface. scFvs were chosen to allow the recognition of specific target molecules, while green fluorescent protein would allow detection of target bound virions. Successful presentation of both functional proteins on recombinant viral particles was confirmed by fluorescence microscopy, where sepharose particles with covalently bound target protein were observed under the microscope after incubation with recombinant bifunctional virions. However, helper phages at this stage do not allow for the introduction of fluorescent protein onto the phage particles in sufficient copy number that would enable their use as a detection component in fluorescence-linked immunosorbent assay (FLISA). Nevertheless, the described prototype represent the basic platform for further development of more sensitive phage particle-based detection probes for analytical and diagnostic purposes, such as determining the abundance of leptin receptor on cells by immunofluorescence and immunochemical techniques. Taken together, we have identified four competitive scFv fragments targeting leptin receptor, thereby contributing new leads to the collection of candidates for the development of therapeutically useful leptin antagonists. In addition, the described scFvs can be used as components of molecular probes for detection of leptin receptors on cell surface, thus forming the basis for development of novel diagnostic tests based on determining expression level of leptin receptor. The latter is overexpressed on cells of certain types of tumors, atherosclerotic plaques and the affected tissues in autoimmune disorders.
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