Studies On Synthesis And Biological Activities Of Analogues Of Neuropeptide S(NPS)with Fatty Acid Conjugation In Vitro And In Vivo

Neuropeptide S(NPS)has recently been extensively studied as a 20 amino acid neuropeptide,which can regulate many biological functions such as anxiety,arousal,locomotion,learning and memory,and drug abuse through activating its receptor NPSR.Previous articles displayed that the NPS was a short-effect drug,thus we intended to pay more attention to study long-efficient analogues of NPS.Therefore we researched the half-life of NPS in brain homogenates,the study demonstrated the half-life of NPS has only 15min in the pre-experiment.And previously reported fatty acid modified peptides firstly combined with albumin to prolong its stability.In this paper we modified the side chain of 11,12,19 of Lys,and with Aib subsitituted Phe15,designed 13 analogues,detected the calcium flow and enzymatic stability of analogues in vitro,and investigated the activity of M-3 in mice.The result of cell calcium ions showed that the activity of NPS modified by palmitic acid at 11,12,19 was better than the analogues of stearyl acid modified,and the analogues of fatty acid conjugation 12 of NPS have higher effect than compound modified at Lys11,Lys19.Interestingly,Aib-substitute analogues at position 15 and fatty acid modified side chain of Lys11,Lys19 in combination produced a small increased than alone modified,but analogues modified at 12 generated a weak reduction;M-13 showed a statistically significant loss of potency.The results of enzymatic stability illustrated that the half-life of all analogues of fatty acid modified were longer than NPS,and the half-life of M-3 and M-4 are 525min and 1939min,which respectively extended 44 and 101 folded compared with NPS.However,the peptides M-3 and M-4,which are the Aib-substitute analogues at position 15 and fatty acid modified side chain of Lys12 in combination,prolonged the half-life only 4.6 and 6.7 folded than NPS.This results indicated Aib-substitute analogues at position 15 affected the enzymatic stability.We screened M-3 had the higher efficacy activity and the longer half-life compared with NPS.And we studied the anxiety and locomotion activity of M-3 through intracerebroventricular(i.c.v)administration in the elevated plus maze and open field.It was found that M-3 showed a same anxiolytic-like effect compared with NPS 15min after i.c.v injection,and it still has anxiolytic effect 1h,4h after administration,but NPS has no efficacy at 4h.In the open field,M-3(0.01-lnmol)displayed a dose-dependent,and the effect of M-3 improved 10 fold than NPS in vivo.M-3 still promoted locomotion 1h after injection,but NPS didn't.Both NPS and M-3 have no effect after 4h.Furthermore,M-3 has anxiolytic-like effect 2h after nasal,NPS has no efficacy after nasal.In conclusion,we find conjugation with fatty acid that can prolong the duration of NPS action and transmembrane capacity,it is beneficial for targeting study of drug and provide date for Clinical Research.