Leptin is a pleiotropic hormone acting both centrally and peripherally. It participates in variety of biological processes including energy metabolism, reproduction and modulation of the immune response. So far structural elements affecting leptin binding to its receptor remain unknown. We employed random mutagenesis of leptin, followed by selection of high-affinity mutants by yeast-surface display and discovered that replacing residue D23 with a non-negatively charged amino acid leads to dramatically enhanced affinity of leptin for its soluble receptor. Rational mutagenesis of D23 revealed the D23L substitution to be most effective. Coupling the D23 mutation with alanine mutagenesis of three amino acids (L39A/D40A/F41A) previously reported to convert leptin into antagonist resulted in potent antagonistic activity. These novel superactive mouse and human leptin antagonists (D23L/L39A/D40A/F41A), termed respectively SMLA and SHLA, exhibited over 60-fold increased binding to leptin receptor and 14-fold higher antagonistic activity in vitro relative to the L39A/D40A/F41A mutants. To prolong and enhance in vivo activity, SMLA and SHLA were monopegylated mainly at the N-terminus. Administration of the pegylated SMLA to mice resulted in a remarkably rapid, significant and reversible 27-fold more potent increase in body weight (as compared to pegylated MLA), due to increased food consumption. Thus, recognition and mutagenesis of D23 enabled construction of novel compounds that induce potent and reversible central and peripheral leptin deficiency. In addition to enhancing our understanding of leptin interactions with its receptor, these antagonists enable in vivo study of leptins role in metabolic and immune processes, and hold potential for future therapeutic use in disease pathologies involving leptin.
30 Apr - 04 May 2011
European Society of Endocrinology