The Prediction And Biological Activity Validation Of Selective Antagonists For Grass Carp Somatostatin Receptors

Somatostatin（SS）is a cyclic peptide with potent inhibitory effects on hormone secretion and nerve excitation,and its inhibitory effects are mediated by somatostatin receptor（SST）1-5.In mammals and fish,because expression of SSTs is tissue-specific,SS can inhibit the expression and secretion of hormones in the growth hormone（GH）-insulin-like growth factor（IGF）axis via specific SSTs,leading to the inhibition of the growth and development of the body.Consistently attenuation of the effects of SS can promote the growth of fish,but the non-specific attenuation of SS effects leads to some side effects.Therefore,screening the antagonists specific for the SST subtypes expressed in the GH-IGF axis and applying the antagonists to the fish not only can promote the body growth and development,but also reduce its side effects.In this thesis,we used grass carp as the animal model and identified the SST subtype（s）expressed with high levels in GH-IGF axis（pituitary and liver）.Then,a selective antagonist for the SST subtype（s）was predicted from the available antagonists in the literatures by virtual screening,and its biological activity was verified.At last,growth-promoting effect of the antagonist was explored.Firstly,based on the protein sequences of seven grass carp SSTs subtypes,their 3D models were constructed by homologous modeling.To evaluate their binding abilities with the ligand,online program,Cluspro,was used to predict the affinity of SS-14 for grass carp SSTs.In addition,the expression plasmids for seven grass carp SSTs were constructed and the abilities of SS-14 to activate these SSTs were evaluated by the luciferase reporter assay.Compared with the predicted results,except for SST2b2,the activation of SST receptors by SS-14 was nearly consistent with the predicted affinity between them.This result proved that the grass carp SST models could be used for subsequent virtual screening of their antagonists.Secondly,RT-PCR was carried out to detect the expression of seven grass carp SSTs in various tissues.The results showed that SST2 a was expressed with highest levels in both pituitary and liver,indicating that it might played an important role in mediating the inhibition of SS on GH-IGF axis.Then,the antagonist,Cyclosomatostatin（C-SOM）,with higher affinity to this receptor was virtually screened by molecular docking.The antagonistic ability of this antagonist was evaluated by the luciferase reporter assay.The results showed that it could strongly inhibit the activation of SST2 a by SS-14,while it had a weaker inhibition on the activation of SS-14 on SST3,suggesting that C-SOM might have a higher selectivity for SST2 a.Finally,a preliminary exploration of the effect of this antagonist on the growth of grass carp was conducted.The results showed that although four weeks of continuous intraperitoneal injection of C-SOM did not significantly increase the body weight,hepatosomatic index and fullness of the juvenile grass carp,the administration could significantly up-regulate the gene expression of IGF-2a in the liver of the fish within 12 hours.This proved that this drug had the potential to antagonize the effects of SS in grass carp.In summary,this study provides the first evaluation of the binding abilities between SS-14 and seven grass carp SSTs subtypes through computer-assisted drug screening and cell biology experiments.On this basis,C-SOM,a selective antagonist of SST2 a highly expressed in the grass carp growth axis,was obtained and its ability to block SST2 a activation by SS-14 was also demonstrated.Although this antagonist significantly enhanced IGF-2a expression in the liver of juvenile grass carp,further experiments are needed to determine its growth-promoting effect.The method used in this study greatly saves the time and the economic cost for drug screening,and provides a new strategy to screen new drugs to promote the fish growth.