1. Impact of novel lipidized analogs of neuropeptides on food intake decrease and metabolic changes in rodents Institute of Organic Chemistry and Biochemistry Academy of Sciences of the Czech Republic Prague, Czech Republic Lenka Maletínská ENDOCRINOLOGY-2014 Chicago, October 22 nd, 2014

2. Food intake regulation by peripheral hormones and central neuropeptides hypothalamus brainstem CCK Schwartz, Nature 404 (2000) Zanella, Arq Bras Endocrin Metab 53 (2009) PP Amylin anorexigenic = decreasing food intake orexigenic = increasing food intake PrRP

3. Lipidization of neuropeptides: possibility of transport through BBB Peripheral administration – how to cross blood brain barrier? Neuropeptides as potential anti-obesity agents Advantages: specific, centrally acting Disadvantages: not able to cross BBB from the periphery Central administration Peripheral administration

4. central anorexigenic neuropeptide (hypothalamus, brainstem) receptor GPR10 – PrRP discovered 1998 ( Hinuma et al., Nature 393:272 (1998)) affinity also to neuropeptide FF receptor (NPFF2) effect on prolactin release questioned PrRP KO mice are obese PrRP receptor KO mice are obese PrRP – prolactin-releasing peptide Takayanagi et al., J Clin Invest 118: 1 (2009) Bjursell et al., BBRC 363: 633 (2007) ↑ PrRP

5. PrRP – lipidization Phe 2 natural forms with equal biological activity: PrRP31, PrRP20 - identical C-terminus important for biological activity „RF-amide peptides“

6. PrRP – lipidization Phe Lipidization with fatty acid attached by amid bound at N-terminal amino acid of PrRP31 Fatty acid Solid phase Fmoc synthesis, lipidization on resin Fatty acid: Octanoyl Decanoyl Dodecanoyl Myristoyl Palmitoyl Stearoyl „RF-amide peptides“

7. PrRP – lipidization Phe Lipidization with fatty acid attached by amid bound at N-terminal amino acid of PrRP20 Solid phase Fmoc synthesis, lipidization on resin Fatty acid: Octanoyl Decanoyl Dodecanoyl Myristoyl „RF-amide peptides“ Fatty acid Thr

8. High affinity binding of lipidized PrRP analogs to human PrRP and NPFF2 receptors Analog Human PrRP rec 125 I-human PrRP31 binding K i (nM) Human NPFF2 rec 125 I-1DMe binding K i (nM) PrRP313.91 ± 0.2142.2 ± 6.76 Oct-PrRP311.49 ± 0.0724.8 ± 13.2 Dec-PrRP311.42 ± 0.5514.7 ± 3.10 Dodec-PrRP311.15 ± 0.3514.2 ± 6.40 Myr-PrRP310.69 ± 0.091.59 ± 0.32 Palm-PrRP312.94 ± 0.330.69 ± 0.36 Stear-PrRP315.24 ± 0.57 15.9 ± 14.4 PrRP204.40 ± 0.7721.8 ± 9.91 Oct-PrRP201.88 ± 0.31174.9 ±132.7 Dec-PrRP202.94 ± 0.473.60 ± 2.57 Dodec-PrRP202.34 ± 0.259.97 ± 3.48 Myr-PrRP204.21 ± 0.248.23 ± 1.97

9. Food intake in fasted mice (dose 5 mg/kg SC) Statistika:One-way ANOVA, Dunnett’s post hoc test, vs saline (n = 6-8 ) Lipidized PrRP31 analogs: Food intake after peripheral administration *

10. Statistika:One-way ANOVA, Dunnett’s post hoc test *** vs saline (n = 6-8 ) Food intake in fasted mice (dose 5 mg/kg SC) Lipidized PrRP20 analogs: Food intake after peripheral administration

11. saline 3V PVN PrRP31Oct-PrRP31 Myr-PrRP31 Palm-PrRP31 Imunohistochemistry: Fos 90 min after lipo-PrRP injection to fasted mice (s.c. administration 5 mg/kg) Lipidized PrRP analogs with anorexigenic activity increase neuronal activity in the brain areas regulating food intake PVN Paraventricular nucleus

12. Wistar rats, repeated IP administration – once daily (before lights out) (n=5, 10mg/kg in saline) Palm-PrRP31 lowers food intake in free fed rats t-test: *** vs saline

13. Lipidization increases stability of PrRP in rat plasma (Measured by PrRP EIA kit)

14. Long-term effect of lipidized PrRP analogs in mouse DIO (diet-induced obesity) model (repeated SC administration) C57BL mice: 3 months on high-fat diet (60% fat) 14 days s.c. administration 2-times per day: saline Myr-PrRP20, 5mg/kg Palm-PrRP31, 5mg/kg

15. Statistics:One-way ANOVA, Dunnett’s post hoc test (n = 9-10 ) Anorexigenic lipidized PrRP analogs lower body weight of DIO mice Food intake, body weight, fat, liver weight Glucose, insulin, leptin in blood plasma DIO mouse model: body weight decrease

16. Lipidized PrRP analogs: Summary  New PrRP analogs lipidized at N-terminus are stable agonists with high affinity to PrRP and NPFF2 receptors  Palmitoylated and myristoylated PrRP analogs after acute peripheral administration showed long-lasting decrease in food intake and increased neuronal activity in hypothalamus and brainsten of fasted mice  In lean free fed rats, repeated IP treatment with palm-PrRP31 very significantly decreased food intake  In mice with diet-induced obesity, repeated SC treatment with myr-PrRP20 and palm-PrRP31 caused significant decrease in food intake, body weight and improvement of metabolic parameters connected with obesity  No possible side effects found: no anxiety, sedation, analgesia no signs of toxicity no prolactin release after peripheral administration

17. Conclusions Palmitoylated PrRP31 and myristoylated PrRP20 are:  Stable, selective and long-lasting anorexigenic peptides  Centrally active after peripheral administration  Potential candidates for antiobesity treatment

18. Acknowledgements Food intake – rat models: Jaroslav Kuneš Institute of Physiology IOCB Group of Antiobesity peptides Blanka Železná, Martina Holubová, Andrea Špolcová, Veronika Nagelová Jana Zemenová, Barbora Mikulášková, Zdeno Pirník, Hedvika Vysušilová Peptide synthesis: Mirka Blechová Radioiodination: Tomáš Elbert Insulin resistance, clinical diabetology and obesitology: Martin Haluzík, Zdena Lacinová First Faculty of Medicine, Charles University, Prague Stability and pharmacokinetics by LC-MS: David Sýkora University of Chemical Technology, Prague Grant Czech Science Foundation P303/12/0576 TACR TE01020028 / Center for Development of Original Drugs and RVO:61388963