Development of leptin derivatives with enhanced stability and bioefficacy

Abstract

Leptin, a protein hormone secreted by adipocytes, plays a key role in regulating body energy homeostasis and weight loss by inhibiting feeding behaviors. Leptin is currently being used in several different clinical trials to treat leptin-deficient diseases. It is necessary to prevent aggregation or denaturation of therapeutic proteins; however, many studies have demonstrated that leptin is easily aggregated during preparation and storage steps. We previously demonstrated that a stabilizing peptide (SP) fusion protein of leptin (SP-leptin) appeared to resist aggregation induced by agitation, freezing/thawing, or heat stress. In the first study, we fused mouse leptin with the stabilizing peptide and compared the biological activities of leptin and SP-leptin in vivo using a male C57Bl mouse model and ex vivo using MCF7 breast cancer cell lines. The SP-leptin treated mouse groups showed decreased body weight and food intake more significantly than treating wild type leptin, and the SP-leptin treated MCF7 cells displayed better cell proliferation than leptin. SP-leptin treated mouse groups also showed decreased abdominal fat contents and low triglyceride (TG) concentration, and contained fewer and smaller lipid droplets in the liver. These results demonstrated that SP-leptin is more effective than wild type leptin in normal mice in lowering their body weight and fat contents in the abdominal region, the serum, and the liver.In the second study, we demonstrated the development of leptin mutants with enhanced bioefficacy by site-directed mutagenesis. We systematically mutated 35 surface residues, and then compared the biological activities of wild type (WT) leptin and 35 leptin mutants ex vivo using MCF7 cell line and in vivo using C57Bl mice. Through the initial screening, we selected two leptin mutants (T12A and Q56S) and conducted additional in vitro and in vivo experiments. The T12A and Q56S leptin mutants appeared to induce more STAT3 and ERK phosphorylation in MCF7 cells. Furthermore, the T12A leptin mutant showed significantly increased thermal stability. For the in vivo studies, each group of mice was treated with saline, WT leptin, T12A, or Q56S leptin. As a result, both leptin mutants appeared to decrease body weight, food intake, abdominal fat and triglyceride (TG) concentration more significantly than WT leptin. Consistent with the thermal stability data, the in vivo effects of T12A appeared to be greater than Q56S. These results indicate that the T12A leptin mutant is more effective than both Q56S and WT leptin in terms of bioefficacy and protein stability. These findings showed that the SP-leptin and the mutant leptin (T12A) were more stable and effective compared to WT leptin, thus these leptin derivatives would be more useful in therapeutic applications of leptin.