Virtual Screening And Activity Identification Of Positive Small Molecule Allosteric Regulators Targeting The N-terminal Extracellular Domain Of PAC1-R

Objective:As G-protein-coupled receptor(GPCR),PAC1-R mediates the neuroprotective effect of pituitary adenylate cyclase-activating polypeptide(PACAP),which is an important target for drug development in nervous system diseases.At present,most of the PAC1-R receptors are polypeptide drugs,which have short response time.Therefore,the regulation of PAC1-R activity by chemical small molecule allosteric regulators is considered as a new effective treatment for related diseases.Based on the study of doxycycline(DOX)targeting N-terminal extracellular allosteric regulatory sites of PAC1-R and acting as PAC1-R positive allosteric modulator(PAM),a series of compounds that can bind to PAC1-R positive allosteric regulatory sites were screened by computer virtual screening,and the compounds were detected,analyzed and identified at the molecular and cellular levels.Therefore,a set of techniques and schemes for predicting,screening and identifying PAC1-R isomeric regulators are established.Methods:First,we used 1-methyl-4-phenylpyridinium(MPP~+)to induce apoptosis of human neuroblastoma SHSY-5Y cells,and then established a Parkinson's disease cell model,and tested whether the model could be used as a screening tool for positive small molecule allosteric regulators.Secondly,we simulated the N-terminal extracellular allosteric regulatory sites of PAC1-R combined with DOX,a series of candidate positive small molecule allosteric regulators were obtained by virtual screening of compounds targeting the N-terminal extracellular regulatory sites of PAC1-R using virtual docking program.Then the affinity of candidate positive small molecule allosteric regulators to the N-terminal extracellular domain of PAC1-R was detected by Microscale Thermophoresis(MST),and the anti-apoptotic activity of candidate small molecule allosteric regulators mediated by PAC1-R was detected by Parkinson's disease cell model,We screened out the most active small molecule allosteric regulators.Finally,aiming at the optimal small molecule allosteric regulators,PAC1-CHO cells with high expression of PAC1-R and cAMP assay were used to determine their synergistic activity in promoting the activation efficiency of PAC1-R natural ligand PACAP27 as PAM.and the neuroprotective effect of the optimal small molecule allosteric regulator on PAC1-R was determined at the cellular level by using the cell model of Parkinson's disease.Results:1)MPP~+(8mM)can effectively induce SHSY-5Y cell apoptosis and decrease dopamine and dopamine transporters and a cell model of SHSY-5Y Parkinson's disease induced by MPP~+(8mM)was established.2)DOX and minocycline can effectively resist MPP~+induced SHSY-5Y cell apoptosis through PAC1-R,indicating that MPP~+induced SHSY-5Y Parkinson's disease cell model can be used for the screening of PAC1-R positive small molecule allosteric modulators.3)MST assay and MPP~+induced SHSY-5Y Parkinson's disease cell model screening were performed on the small molecule allosteric regulators targeting the N-terminal extracellular domain of PAC1-R obtained by a series of virtual screening,and a positive small molecule allosteric regulator named SPAM1(Small Molecular Positive Allosteric Modulor 1)was obtained,which has the strongest N-terminal extracellular affinity with PAC1-R and can effectively protect SHSY-5Y cells.4)For 0.1nM PACAP27,the EC50 of DOX as PAM was0.81+0.18?M,and that of SPAM1 as PAM was 0.59+0.14?M.In the presence of 1?M SPAM1/DOX,the dose-response curve of PACAP27 shifted significantly to the left,and the effect of SPAM1 was slightly better than that of DOX,indicating that SPAM1 was a new type of PAC 1-R with high efficiency.5)The SHSY-5Y Parkinson's disease cell model induced by MPP~+was used to confirm the allosteric regulation of SPAM1 on PAC1-R and its neuroprotective effect at cellular level.Conclusion:This study is the first attempt to carry out virtual screening of novel positive small molecule allosteric regulators targeting PAC1-R in N-terminal extracellular domain.Secondly,the positive small molecule allosteric regulators obtained by virtual screening are validated in the laboratory at the molecular and cellular levels,so as to obtain the best positive small molecule allosteric regulators(SPAM1)targeting PAC1-R.Finally,the positive allosteric regulation effect and neuroprotective effect of the positive small molecule allosteric regulator were determined,and a set of effective screening and testing methods and experimental schemes for PAC1-R allosteric regulators were preliminarily established,which laid a foundation for the discovery and creation of more targeted PAC1-R allosteric regulators.