The Development of Kisspeptin Antagonists for Therapeutic Interventions

Antonia Roseweir1, Alexander Kauffman2, Jeremy Smith3, Kathryn Guerriero4, Kevin Morgan1, Justyna Pielecka-Fortuna5, Rafael Pineda6, Michelle Gottsch2, Manuel Tena-Sempere6, Suzanne Moenter5, Ei Terasawa4, Iain Clarke3, Robert Steiner2, Robert Millar1. 1MRC Human Reproductive Sciences Unit, Edinburgh, Scotland, United Kingdom, 2University of Washington, Seattle, Washington, United States, 3Monash University, Melbourne, Victoria, Australia, 4University of Wisconsin-Madison, Madison, Wisconsin, United States, 5Univesity of Virginia, Charlottesville, Virginia, United States, 6Cordoba Univesity, Cordoba, Andalusia, Spain.

Kisspeptin and its receptor (GPR54) are central regulators of reproduction and are key mediators of nutritional, photoperiod and steroid hormone regulation of the GnRH neuron. Specific antagonists at GPR54 would be useful tools to interrogate physiological and patho-physiological roles of kisspeptin, and 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) as outputs. These studies revealed an essential binding pharmacophore and identified residues required for GPR54 activation allowing for the subsequent derivation of antagonists. The most potent antagonists had IC50s in the nM range 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 rhesus monkeys. It also inhibited kisspeptin-induced LH secretion as well as 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. The inhibition of the rise in LH by antagonist after gonadectomy indicates that it is due to increased kisspeptin secretion. In summary, we have developed GPR54 antagonists that provide useful tools to investigate the neuroendocrine regulation of reproduction and for potential therapeutic applications.