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Class B G-protein coupled receptors (GPCR) PAC1-R is a neuropeptide pituitary adenylate cyclase activating polypeptide (PACAP)-preferring receptor that mediates the effective neuroprotective activity.Based on our previous data showing that doxycycline and minocycline work as the positive allosteric modulator (PAM) of PAC1-R,we used computer molecular docking and isothermal titration calorimetry assay to further determine the bindings of doxycycline/minocycline’s derivatives including tetracycline/tigecycline with the N-terminal extracellular domain of PAC1-R (PAC1-EC1).Then the cAMP assay combined with the PAC1-R natural agonist PACAP27 was used to confirm the possible PAM roles of the small-molecule antibiotics.The results showed that tetracycline/tigecycline had significant lower affinity to PAC1-EC1 than doxycycline/minocycline,which was consistent with their non-positive allosteric modulation activity on PAC1-R.Furthermore,by comparing the key residues contributing to the PAM binding with the predicted allosteric site in PAC1-EC1,we characterized four motifs contributing to PAM binding in PAC1-EC1.The site-directed mutation results showed that ASN60 played the most important role in the PAM binding of the small-molecule antibiotics,while ASP116 played a sensitive marginal role in the PAM binding.These results not only help to explain the clinical and experimental neuroprotective effects of doxycycline/minocycline,but also help to characterize the PAM binding site in PAC1-EC1,which will promote the screening and characterization of novel small-molecule PAMs targeting PAC1-EC1 with drug development potency in nerve system disease.