Dissection of Kisspeptin's Role in the Neuroendocrine Regulation of Gonadotropin Using Antagonists.Robert P. Millar, Ph.D.

Abstract

Kisspeptin and its cognate receptor (GPR54) are central regulators of reproduction and are key mediators of nutritional, photoperiod, stress and steroid hormone regulation of the GnRH neuron. Kisspeptin also regulates trophoblast invasion and stimulates vasoconstriction. Thus, specific antagonists at GPR54 would be useful as tools to interrogate physiological and patho-physiological roles of kisspeptin. Antagonists would also have potential as therapeutic agents for treating a wide range of clinical conditions. We have systematically substituted single and combinations of amino acids in kisspeptin-10 in a detailed structure-activity-relationship study using receptor binding and inhibition of kisspeptin-10 stimulation of inositol phosphate (IP) mobilisation as outputs. These studies revealed an essential binding pharmacophore and identified residues required for GPR54 activation allowing for the subsequent derivation of a series of antagonists and partial agonists. The most potent antagonists had IC</SUB50>s in the nM range as measured by the inhibition of 10nM kisspeptin stimulation of IP. A selected antagonist potently inhibited (1nM) kisspeptin-10 stimulation of GnRH neuronal firing in mouse brain slices and GnRH secretion in thesus monkeys. It also inhibited kisspeptin-induced LH secretion in male rats and mice. The antagonist inhibited the post-castration LH rise in mice and rats when administered intracerebroventricularly, confirming that inhibition of kisspeptin signalling mediates gonadal steroid negative feedback. The antagonist had no effect on basal LH secretion in male rats and mice possibly indicating a lower tone of kisspeptin input. LH pulse frequency and amplitude were inhibited by intracerebroventricular antagonist administration to ovariectomized ewes. Antagonist also delayed puberty in female rats and inhibited the ovulatory LH surge confirming a role in these processes. In summary, we have developed GPR54 antagonists that provide useful tools to investigate the neuroendocrine regulation of reproduction and for potential therapeutic applications.