The Allosteric Modulation Effects Of Doxycycline/minocycline And Their Derivatives On The Neuropeptide Receptor PAC1-R

Objective:Class B G protein coupled receptor（GPCR）PAC1-R is a preferred receptor for the neuropeptide pituitary adenylate cyclase-activating polypeptide（PACAP）,which mediates effective neuroprotective activity.PAC1-R has a relatively large and complex N-terminal extracellular domain（PAC1-EC1）responsible for recognition and binding of ligand.Our existing studies have shown that doxycycline（DOX）and minocycline（MINO）can act as PAC1-R positive allosteric modulators（PAMs）by binding PAC1-EC1 and enhancing the orthosteric activation of PAC1-R（Yu R*,et al.Neuropharmacology.2015;103:1-15.）.Based on this research,our study used computer molecular docking,microscale thermophoresis（MST）,site-directed mutagenesis,and cAMP assays and so on to analyze and compare the allosteric effects of DOX/MINO and its derivatives,including various modifications of DOX,tetracycline（TET）and tigecycline（TIGE）on PAC1-R,tried to clarify the details of the positive allosteric modulator binding site in PAC1-EC1,which would lay the foundation for the subsequent screening of novel allosteric modulators targeting PAC1-R.Methods:Firstly Discovery Studio 2.5 Libdock was used to obtain the DOX/MINO/TET/TIGE3D molecular docking model with PAC1-EC1 for the analyzing and comparing the information of their binding sites.Subsequently,on the moleluclar level MST analysis technique was used to abtain the affinity information（dissociation constant KD,dissociation percentage DP）of DOX/MINO and its derivatives with PAC1-EC1.Then on the cellular level the established PAC1-CHO cell line with high expression of PAC1-R coupled enhanced green fluorescence protein（eGFP）in combination with cAMP assay and MTT assay was used to detect the allosteric regulatory effects of DOX/MINO and its derivatives on activation of PAC1-R by PAC1-R orthotopic agonist PACAP（1-13）.Afterwards AlloSite,a protein allosteric prediction software,was used to predict allosteric regulatory sites in PAC1-EC1.By analyzing the frequency of residues occurred in both prediction results and molecular docking results,four key motifs that mainly contribute to PAM were screened out.Through site-directed mutagenesis,the recombinant proteins of four mutants of PAC1-EC1 corresponding to the key motifs were obtained,and the key motifs were further identified by the MST assay.Results:MST results found the KD values for three types of chemically modified DOX are as followed:C₂₂H₂₄N₂O₈.1/2C₂H₆O.HCL.1/2H₂O（6940+/-274μM）>C₂₂H₂₄N₂O₈（203.0+/-25.2μM）>C₂₂H₂₄N₂O₈.H₂O（148.0+/-4.28μM）,indicating that the more complex chemical modifications the more seriously interfered with the binding of DOX to PAC1-EC1,without any modified DOX(C₂₂H₂₄N₂O₈)（203.0+/-25.2μM）and MINO(C₂₃H₂₇N₃O₇.HCL)（289.0+/-15.4μM）have similar binding affinity with PAC1-EC1,while TIGE(C₂₉H₃₉N₅O₈)（610.0+/-23.8μM）had the worst binding capacity.And the c AMP and MTT assays showed that DOX/MINO had EC₅₀ of 11.2±1.7nM and 7.1±1.1nM for its positive activity on the orthosteic activation of PACAP（1-13）as a high-efficiency PAMs of PAC1-R;TET had EC₅₀ of 44.3±2.4nM as a low-efficiency PAMs of PAC1-R;and TIGE had EC₅₀ of more than 1000nM as a silent allosteric modulator of PAC1-R.By comparing protein allosteric site prediction results used AlloSite with molecular docking results four key motifs that make up the small molecule PAMs binding sites in PAC1-EC1 were screened out.Four recombinant PAC1-EC1 derived mutants corresponding to four key motifs were obtained:rPEM1:I26F27--A26A27,rPEM2:N60I61--A60A61,rPEM3:F110D111--A110A111,rPEM4:F115D116--A115A116.The following MST analysis found that the mutation of motif N60I61 completely interferes with the binding of DOX/MINO to PAC1-EC1 considered as a key component of PAMs;while the mutation of motif F115D116on the one hand inhibits DOX binding and promotes MINO binding on the other hand,indicating that motif F115D116 plays a complex marginal role in the binding of small molecules PAMs to PAC1-EC1.Conclusion:In this study,it is the first time to identify at the experimental level the binding affinity of DOX/MINO and its derivatives with PAC1-EC1,to test the allosteric modulation effects of DOX/MINO and its derivatives on the orthosteric activation of PAC1-R,to validated the presence of allosteric modulation sites in the N-terminal ectodomain of PAC1-R predicted by bioinformatics;and try to detail the key amino acids contributing to the PAMs binding sites in the N-terminal extracellular domain of PAC1-R.All these results may lay the foundation for the subsequent screening of novel allosteric modulators targeting PAC1-R.