Molecular Design Of Conantokin-G Analogues And Against Morphine Dependence Effect

1 ObjectiveDrug dependence is not only a medical problem,biological problem,but also social issue,and it is a chronic relapsing encephalopathy.Causes of dependency include positive reinforcement factors(euphoria,reward effect),negative reinforcement factors(escape from reality,to reduce withdrawal symptoms) and conditions of the reinforcement factors.Opioid dependence is the essence of compensatory adaptation,involving multiple brain regions,a variety of changes in neurotransmitters and their receptors(including the opioid system itself) norepinephrine system,excitatory amino acid systems(such as N-methyl-D-aspartate (NMDA) receptors),etc.NMDAR is the excitatory glutamate receptor subtype,which consists of a different proportion of NR2,NR3 and NR1 subunits,and it has a high-pass permeability to Ca²⁺.Studies have shown that NMDAR in synaptic signal transduction,neural plasticity,learning and memory,etc.play an important physiological role,while many acute and chronic pathological changes of the nervous system are closely related to NMDA receptor dysfunction.In view of the central nervous system,NMDA receptors are involved in reward-dependent effects of addictive drugs,as the central nervous system,NMDA receptor-specific antagonist Conantokins family may be applied to treat drug addiction. Our experiments confirmed that Con-G can effectively interfere with the expression of morphine-induced CPP and reconstruction,which may have prevention and treatment of morphine dependence and relapse potential.Since there are some disadvantages on Con-G,such as the inconvenient administration,low absorption rate and the high cost disadvantages.Therefore,this study is designed using computer-aided to design Con-G analogues,so as to overcome some shortcomings of the original peptide and used using the computer based video tracking CPP system to analyze its anti-assess the effects of drug addiction,which is used to strive for effective screening of the Con-G analogs.2 Materials and methods2.1 Animals:Adult male Kun-Ming mice(18-22g) were supplied by Shanghai Centre of Experimental Animals,Chinese Academy of Sciences.2.2 Chemical reagents:Conantokin G(purity＞90%)(Sigma)[Glu^3,4,7,10,14]-Conantokin G(purity＞90%)(Sigma)[Glu^3,4,7,10,14]-Conantokin G[1-13](purity＞90%)(Sbsgene)Morphine hydrochloride(First Pharmaceutical Company of Shenyang,China);2.3 Apparatus:Computer simulation softwareZDOCK in Insightâ…¡softwareModeller 8v2NACCESSComputer based video tracking CPP systemSPSS software2.4 Methods2.4.1 Homology modelingThe protein sequence of human NR2B subunit(404-802) was obtained from the National Center for Biotechnology Information(NCBI) server.Homologous proteins with known crystal structures were found by performing a position- specific iterated BLAST(PSI-BLAST) search.Using Modeller 8v2,protein 3D structures were generated by satisfying spatial restraints imposed by the sequence alignment with the template structure.To optimize the local interactions,several models obtained were subjected to a short simulated annealing refinement protocol using Charmml9 force-field.Stereochemical quality of the polypeptide backbone and side chains was evaluated using Ramachandran plots obtained from the RAMPAGE server.Amino acid environment was evaluated using Verify 3D and Errat plots.2.4.2 Ligand dockingProtein Docking was performed with the ZDOCK software bundled with the Insightâ…¡package version 2005(Accelrys^TM Inc.).Con-G was first docked manually by ZDOCK,then computationally by RDOCK for the top 50 ranks.The resulting NR2B/ligand complex was energy-minimized in the absence of solvent using Charmm19 force-field.The accessible surface area(ASA) of NR2B subunit (uncomplexed) and its docked complex with Con-G were calculated using the program NACCESS.Composite coordinates of ligand and NR2B subunit were generated to form the docked complex.The change in accessible surface area for residue was calculated using:â–³ASAⁱ=ASAⁱ_NR2B-ASAⁱ_NR2B-Con-G.2.4.3 Molecular DesignBased on molecular docking complex combination of model,the geometric model and structural characteristics between NR2B subunit Con-G analogues were designed according to model and structure activity relationship2.4.4 Effect of Glu-Con-G[1-13],Con-G and Glu-Con-G on the expression of CPP induced by morphine:Male Kun-Ming mice were administered morphine(5mg/kg,i.p.) in white chamber for 50 mins on day1,3,5 and 7.On day2,4,6,8 mice were given saline in black chamber for 50 mins.The control group was treated with a daily saline injection for eight consecutive days both in the morphine and saline paired chambers.To test the effects of Con-G and its analogues Glu-Con-G,Glu-Con-G[1-13]on the expression of morphine-induced CPP,different dose of Con-G and its analogues Glu-Con-G, Glu-Con-G[1-13](0,30,60,120pmol) were given by intracranial administration 30 mins before the test on day 9.Total volume was 2μl. 2.5 Data analysis and statisticsPlace Preference:the time spent in white chamber(TW);Locomotor Activity:the total distance that mice moved in different chambers(TD) and shuttle time(ST);All the data were expressed as the mean±S.E.M,and analyzed with SPSS software.P＜0.05 was considered statistically significant.3 Results:3.1 Homology ModelingIn the Ramachandran plot,a total of 79.0%of the residues were in the most favoured region,a total of 95.7%of the residues were in the allowed region.Verify 3D score and Errat plots score were 136.58 and 79.3%,and its reference protein score are 114.98 and 94.7%.3.2 Ligand DockingWhen Con-G structure was docked into the glutamate binding site of NR2B subunit by energy minimization,Con-G structure was nicely fitted into the agonist binding cleft on the NR2B subunit.Several H-bonds were found between NR2B subunit and Con-G.Connolly surfaces of the NR2B subunit and of Con-G were calculated.The decreases of accessible surface area(â–³ASA) of the docking complex were observed.Considering NR2B subunit,significant change of accessible surface area of Glu420,Ser421,Asp423,Lys458 and Asp715 occurred,Lys458 and Asp715 lost more than 50A ASA when going from the uncomplexed to the complexed state.3.3 Test of preference in preconditioning phasePre-conditioning test showed that all groups mice spent more time in black chamber than in white chamber(P＜0.05),which suggested that mice prefer the black chamber;Meanwhile,mice of the different groups did not show any significant difference(P＞0.05) in the time spent in black chamber.3.4 Effect of Con-G and its analogues on the expression of morphine induced CPPAfter conditioning with morphine for 8 days,on the CPP expression day(d9), compared with the mice in morphine control group(M-G0),A pretreatment with Con-G and its analogue(30,60,120pmol) 30 min before the test,Con-G displayed a dose-effect dependent decrease the expression of morphine induced CPP(30pmol, P＜0.01;60pmol,P＜0.01;120pmol;P＜0.01);Glu-Con-G(30,60,120pmol) displayed a dose-effect dependent decrease the expression of morphine induced CPP(30pmol, P＜0.01;60pmol,P＜0.01;120pmol;P＜0.01);Glu-Con-G[1-13](30,60,120pmol) also displayed a dose-effect dependent decrease the expression of morphine induced CPP (30pmol,P＜0.01;60pmol,P＜0.01;120pmol;P＜0.01).Con-G and its analogues had the same lowest efficient dose.4 Conclusions4.1 The glutamate binding site is built according to the sequence of NR2B 404-802.4.2 Structurally and functionally important residues were identified,including E2, Gla4,L5,Q9,I12 and Q13 of Con-G,they interacted with E420,S421,D423,K458 and D715 of NR2B.Seven H-bonds were also found.4.3 Glu-Con-G[1-13]is in line with docking model.4.4 Glu-Con-G[1-13],Con-G and Glu-Con-G,displayed a dose-effect dependent decrease the expression of morphine induced CPP.4.5 Glu-Con-G[1-13]has an improvement in the molecular size and the difficulty and cost of synthesis which can be used as the next phase of the study key.