Kisspeptin-10 Analogue Receptor Pharmacology: A Research Literature Review

Kisspeptin-10 (KP-10) is the minimal C-terminally amidated fragment of the KISS1 gene product that retains full functional activity at the G protein-coupled receptor KISS1R, and it has been reported to exhibit higher receptor affinity and biopotency than longer kisspeptin forms [1]. Systematic alanine substitution of rat kp-10, assessed by intracellular Ca2+ responses in rat kiss1r-transfected Chinese hamster ovary cells, showed that substitutions at positions 6 and 10 increased EC50 values by more than two orders of magnitude and reduced the proportion of responsive cells, while in rats the Ala6 substitution diminished and the Ala10 substitution eliminated luteinising hormone (LH) secretory responses [1]. NMR-restrained molecular modelling in the same work indicated that kp-10 adopts a helicoidal structure between Asn4 and Tyr10, with Ala10 substitution disrupting the C-terminal helix entirely [1]. Several groups have modified the KP-10 scaffold to alter metabolic handling while retaining receptor engagement. In one comparative study, the analogue [dY]1KP-10 bound KISS1R with lower affinity than KP-10 and produced similar ERK1/2 phosphorylation in vitro, yet peripheral administration in mice raised plasma LH and testosterone more potently than KP-10 itself [2]. In a dual luciferase reporter assay in KISS1R-transfected HEK293T cells, beta-amino acid-modified and fluorescently labelled kisspeptin analogues were identified as potent agonists, and the authors reported that proteolysis of kisspeptin within the assay itself can diminish apparent agonist output unless protease inhibitors are present [3]. Conjugation of the chelator DOTA to the NH2 terminus of KP10 preserved biological activity as monitored by inositol phosphate accumulation in HEK293 cells transfected with the KISS1R gene, and radiolabelled DOTA-KP10 showed rapid background clearance with a blood half-life of 18 ± 3 minutes in micro-PET/CT studies in animals [4]. Direct side-by-side comparison of agonists in HEK293 cells stably expressing the human kisspeptin receptor found that the synthetic analogue C6 produced the most sustained intracellular Ca2+ mobilisation and the most pronounced tachyphylaxis, which the authors attributed to greater depletion of intracellular Ca2+ stores [5]. In that same in vitro system, a second stimulation with low concentrations of hKp54, but not hKp10, elicited an increased response, and C6 induced the strongest and most sustained transcriptional response across immediate-early genes (IER2, EGR1, KLF10), transcription factors (NR4A1, NR4A2, NFKB1A), and inflammatory mediators (CXCL8, CXCL1, CCL2) [5]. Desensitisation has also been examined in vivo: in agonadal juvenile male rhesus monkeys, continuous infusion of kisspeptin-10 desensitised KISS1R such that a subsequent kisspeptin-10 bolus failed to elicit gonadotropin-releasing hormone (GnRH) release, with responsiveness progressively restored over the following 72 hours [6]. Analogue work has also produced antagonists and revealed selectivity limits. Kisspeptin-10 analogues bearing amino acid substitutions yielded potent and specific antagonists that inhibited GnRH neuron firing in the mouse brain, reduced pulsatile GnRH secretion in female pubertal monkeys, blocked kisspeptin-induced LH release in rats and mice, and blocked the post-castration LH rise in sheep, rats, and mice [7]. Separately, RF9 was shown to bind KISS1R specifically in stably transfected Chinese hamster ovary cells and to stimulate intracellular calcium, inositol phosphate accumulation, and ERK phosphorylation in a KISS1R-dependent manner, while in mice its LH-stimulating effect was preserved in Npffr1-null animals but markedly reduced in Kiss1r-null animals [8]. Cross-reactivity in the opposite direction has also been reported: human kisspeptin-13 and kisspeptin-8 activated the human neuropeptide FF2 receptor in Xenopus oocytes with roughly 25% of the relative efficacy of NPFF, and induced GTP-gamma-[35S] binding in Chinese hamster ovary cells expressing hNPFF2R, whereas the corresponding mouse peptides produced only modest activation [9]. Functional readouts of KP-10 in intact animals have been characterised across species and physiological states. Intravenous kisspeptin-10 in conscious male rats evoked dose-dependent LH bursts, repeated injections evoked LH pulses of constant magnitude across the sampling period, and kisspeptin-52 produced responses of slightly greater magnitude and duration [10]. In adult female rats, central kisspeptin-10 elicited LH bursts at all phases of the oestrous cycle with maximal responses at oestrus and greatest sensitivity at dioestrus, responses persisted after removal of ovarian steroids and during pregnancy and lactation, and hypothalamic KiSS-1 expression increased during pregnancy while GPR54 mRNA was unaltered [11]. In male rats, intraperitoneal kisspeptin-10 altered both testosterone concentration and sexual motivation, whereas intranasal administration increased sexual motivation without changing testosterone [12]. In fetal sheep, intravenous KP-10 elicited LH release followed by a delayed rise in serum testosterone in males, and pretreatment with the GnRH receptor antagonist acyline abolished the LH response, indicating a hypothalamic site of action [13]. In female rhesus monkeys, the KISS1R agonist human kisspeptin-10 stimulated GnRH release dose-responsively, and reciprocal blockade experiments with the KISS1R antagonist peptide 234 and the NK3R antagonist SB222200 indicated that kisspeptin and neurokinin B signalling become interdependent at puberty but are independent prepubertally [14]. Reviews of this circuitry describe kisspeptin, neurokinin B, and dynorphin A co-expression in arcuate KNDy neurons in humans and rodents [15], and the kisspeptin/GPR54 pathway has been discussed in the context of GnRH neuronal development and pubertal disorders [16]. Beyond the reproductive axis, KISS1R-directed analogues have been profiled in other models. Ten kisspeptin-10 analogues were screened in cervical, prostate, breast, and gastric cancer cell lines, with the most pronounced cytotoxic effects in cervical cancer cells; kinase array and migration assays showed altered kinase signalling and reduced cell motility, and bioluminescence resonance energy transfer assays confirmed analogue interaction with the kisspeptin receptor [17]. In a rat model of experimental autoimmune myasthenia gravis, intraperitoneal KP10 modulated Th1/Th17/Treg balance through inhibition of NF-kappaB signalling in CD4+ T cells, with serum kisspeptin reported to be lower in myasthenia gravis patients and in the rat model [18]. Immunohistochemical work in prepubertal bitches localised GPR54 mainly to luminal epithelial cells and superficial and deep uterine glands, with kisspeptin-10 immunoreactivity low in all uterine cell types [19]. In humans, an intravenous bolus of the kisspeptin analogue kisspeptin-112-121 given to 12 healthy adults was followed by an 18.8% rise in serum oxytocin at 10 minutes, with a larger increase in males than in females [20].