Development of kisspeptin analogs for reproduction control – Kiss

To guide new analog synthesis it is important to predict the mode of interaction of Kp10 and the analogue with the kisspeptin receptor. Available docking software are not capable to predict correctly the interaction mode between the peptide and the receptor. To overcome this difficulty we developed a new tool using the fragment-based ligand design (FBLD). Fragment docking avoid taking into account the flexibility of the entire peptide when docking, this simplify the task. Fragments are than connected by peptide bonds at the time of the application of the covalent docking method. This is followed by an optimization of the new formed peptide. The covalent approach method has been compared to classic docking SP and SP-peptide methods (developed for the study of peptide-protein complexes) and resulted more accurate in the majority of the cases.
The structure of the human kisspeptin receptor obtained by homology was validated using bio-informatics methods and its stability tested by molecular dynamic simulation. The first docking results show an interaction of Kp10 Tyr10 with Trp276 of the receptor (toggle switch), an amino acid important for GPCR activation.
We have also created new analogues by using a classical medicinal chemistry approach. To check for efficacy and potency the pharmacological profile of the analogs is first studied in vitro using a calcium mobilization assay on cell line transfected with the kisspeptin receptor. The most interesting molecules are then injected intramuscularly in ewe and their action followed by dosing luteinizing hormone and progesterone.

The main aims of the present project is to proof that kisspeptin (Kp) analogs could induce ovulation in seasonal livestock during the non-breeding season. At first we replaced eCG (equine choriogonadotrophin) on a combined treatment with a progestogen, by our best analog (C6). Using this protocol we obtained fertile ovulation and a prolificity rate, 2 lambs per ewe, similar to that normally obtained in our farming facility. These results proof the concept that an intramuscular injection of a kisspeptin analog is capable, in the presence of a progestogen pretreatment, of inducing a fertile ovulation in the non-breeding season. This result strongly suggest that kisspeptin analogs could substitute for eCG in the management of reproduction.

We have developed a 3D homology model of the kisspeptin receptor and implemented a fragment-based docking system for kisspeptin. This allowed us to perform a first study of the interaction mode between kissepeptin and its receptor, which would permit to inform the optimization of new analogs.

To confirm the docking position of Kp10 and identify the important residue involved in the interaction between kisspeptin and its receptor simulation of molecular dynamic for longer time are ongoing. Once the interaction mode will be identified, this information will be used to help design the new analogs.
In addition, using the 3D model of the receptor we will perform an in silico screening of available chemical libraries in order to identify new molecular structures of interest.
On the other hand, we plan to perform a direct comparison in vivo of our molecule with eCG to establish the potential advantages of the analogs compared to the available treatment for managing reproduction in livestock.

Decourt et al. A synthetic kisspeptin analog that triggers ovulation and advances puberty. Scientific Reports 2016

KISS project associates increasing livestock productivity and sustainability with decreased health risk. Our ambition is to develop a new treatment for reproduction control improving reproductive performance and reducing hormone use. This new treatment will be based on the development of analogs of the endogenous neuropeptide kisspeptin (Kp).
A key objective of farming sustainable intensification is improvement of reproduction control in livestock. Our goal is to develop a new treatment to increase reproductive success and to obtain a superior control of birth timing while reducing hormone use and working cost.
Current treatments face 4 major problems: i) use of hormone which could persist in the food and in the environment (e.g. progesterone derivatives). If more restrictive laws banning hormonal treatment will be approved, farmers will be obliged to apply less efficient and more time consuming methods leading to income reduction. This could render economically untenable the management of small farm and results in a dramatic drop of their number causing a deconstruction of local social tissue. ii) sanitary risk due to potential diseases transmission (i.e. use of equine Chorionic Gonadotropin (eCG), also named PMSG for pregnant mare’s serum gonadotrophin, extracted from blood serum), iii) immunological response against eCG that affects efficacy of ensuing treatments, and iv) suboptimal efficiency.
To address these problems we designed a 1st generation of Kp analogs. Tests in sheep showed that these analogs are active at extremely low doses (tens of µg). Consequently any potential food contaminant issued from Kp analogs would be present in very low amount. In addition, thanks to their peptide nature, Kp analogs will be timely degraded and their inactive building block (simple amino acids) would be rapidly recycled or excreted. Furthermore, preliminary experiments in ewe showed that our 1st generation Kp analogs are superior to eCG in synchronizing ovulation and suggest that they may also improve efficiency. These results imply that replacement of eCG by Kp analogs is foreseeable. This would be by itself a major improvement eliminating sanitary risks and immunogenic liability. To complete the proof of concept tests to induce ovulation in sheep during the non breeding season are planned. Nonetheless, to assure an optimal response we anticipate the need to improve pharmacokinetics and pharmacodynamics of our 1st generation analogs. Therefore, we will develop a 2nd generation of molecules with refined pharmacokinetics and pharmacodynamics optimally suited for management of livestock reproduction.
On the other hand, relevant features of Kp system, that may have an impact on Kp analogs efficacy, have been poorly investigated (e.g. receptor desensitization mechanisms, existence of functionally selective agonists, precise distribution of Kp receptor, etc). The lack of a sufficient portfolio of Kp receptor ligands with varied chemical structures and pharmacological profiles (partial agonists, antagonists, and biased agonists) largely account for the paucity of information on these topics. Our chemistry effort is generating a series of analogs with different chemical structures, highly selective and with distinct pharmacological profiles. We have the unique opportunity to exploit these new pharmacological tools, and eventually modify them to create antagonists, to better understand Kp system properties. This information will represent an important advance in basic science and will help the design of optimized analogs.
Finally an added value of Kp analogs could be their use as a first step to develop new therapeutic agents to treat human diseases.

To summarize, the objectives of the present project are 1) to develop a 2nd generation of Kp analogs 2) to ameliorate livestock reproduction management and 3) to advance our understanding of Kp system physiological functions.