Recent reports have revealed that leptins effects are not restricted to central control of body weight, demonstrating instead that leptin is a pleiotropic hormone with a wide variety of different biological actions. Leptin exhibits undesirable effects in autoimmune diseases, in atherosclerosis, and possibly in several types of cancer, and increases the risk of cardiovascular disease in obese people. Therefore, preparation of reagents capable of abolishing leptins action is both valid and timely.
As no structural information on the 3D structure of leptin receptor (LEPR) is available, the model of interleukin 6 (IL6) was applied. We identified leptins putative binding site III, which does not affect binding but is necessary for receptor activation, by modeling LEPR on the basis of its alignment with gp130, and fitting leptin on IL6 in the IL6/gp130 complex.
Six muteins of human, ovine, rat, and mouse leptins, mutated to Ala in amino acids 3941 or 3942, were prepared by site-directed mutagenesis of the putative site III, and purified to homogeneity. All muteins had typical cytokine secondary structure, acted as true antagonistsnamely, they interacted with LEPR with an affinity similar to that of the wild-type hormone (as evidenced by SPR and RRA), were devoid of biological activity in several leptin-response bioassays, and specifically inhibited leptin action in vitro and in vivo. These muteins can be prepared in gram amounts and thus serve as a novel tool for studying leptin function in vitro and in vivo. To prolong their lives in circulation, some muteins were pegylated using 40-, 30- and 20-kDa polyethylene glycol. Although pegylation decreased their in-vitro activity, increasing circulation half-life can compensate for this deficit in vivo.
Antagonizing leptin has been suggested as a possible therapy in autoimmune diseases and heart failure. Thus, leptin antagonists not only offer a novel tool to elucidate the role of leptin in mammalian physiology but have a potential role as a therapeutic drug.