A novel small positive allosteric modulator of neuropeptide receptor PAC1-R exerts neuroprotective effects in MPTP mouse Parkinson's disease model.

As a neuropeptide pituitary adenylate cyclase-activating polypeptide (PACAP)-preferring receptor, PAC1-R mediates effective neuroprotective activity. Based on the finding that the antibiotic doxycycline (DOX) with clinical neuroprotective activity functions as a positive allosteric modulator (PAM) of neuropeptide PACAP receptor 1 (PAC1-R), we use virtual and laboratory screening to search for novel small molecule PAMs of PAC1-R. Virtual screening is carried out using a small-molecule library TargetMol. After two-level precision screening with Glide, the top five compounds with the best predicted affinities for PAC1-R are selected and named small positive allosteric modulator 1‒5 (SPAM1‒5). Our results show that only 4-{[4-(4-Oxo-3,4-2-yl)butanamido]methyl}benzoic acid (SPAM1) has stronger neuroprotective activity than DOX in the MPP+ PD cell model and MPTP PD mouse model. SPAM1 has a higher affinity for PAC1-R than DOX, but has no antibiotic activity. Moreover, both SPAM1 and DOX block the decrease of PAC1-R level in mouse brain tissues induced by MPTP. The successful screening of SPAM1 offers a novel drug for the treatment of neurodegenerative disease targeting the PAC1-R.

Positive allosteric regulation of PAC1-R up-regulates PAC1-R and its specific ligand PACAP.

PAC1-R is a recognized preferential receptor for the neuropeptide of pituitary adenylate cyclase-activating polypeptide (PACAP), which mediates neuroprotective and nerve regenerative activities of PACAP. In this study, we found that in both PAC1R-CHO cells with high expression of PAC1R-eGFP and retinal ganglion cells (RGC-5) with the natural expression of PAC1-R, oligo-peptide PACAP(28-38) and the positively charged arginine-rich penetrating peptide TAT, as positive allosteric modulators of PAC1-R, significantly trigger the nuclear translocation of PAC1-R. The chromatin immunoprecipitation (ChIP)-PCR results show that the nuclear translocated PAC1-R binds with the promoter regions of PAC1-R and its specific ligand PACAP. The up-regulated promoter activities of PAC1-R and PACAP induced by PACAP(28-38) or TAT are positively correlative with the increase of the expression levels of PAC1-R and PACAP. Moreover, the nuclear translocation of PAC1-R induced by PACAP(28-38) or TAT is significantly inhibited by the mutation of PAC1-R on Cys25 and the palmitoylation inhibitor 2-bromopalmitate. Meanwhile, the increase in both PAC1-R and PACAP levels and the neuroprotective activities of PACAP(28-38) and TAT in MPP-induced cell model of Parkinson ' s disease are synchronously inhibited by 2-bromopalmitate, which are positively correlated with the nuclear translocation of PAC1-R induced by PACAP(28-38) or TAT. Bioinformatics analysis and motif enrichment analysis following ChIP-sequencing show that the transcription factors including SP1, Zic2, GATA1, REST and YY1 may be recruited by nuclear PAC1-R and involved in regulating the promoter activities of PAC1-R and PACAP. ChIP-sequencing and related bioinformatics analysis show that the downstream target genes regulated by the nuclear PAC1-R are mostly involved in the process of cellular stress and related to neuroprotection, neuronal genesis and development.

Blue light induces the nuclear translocation of neuropeptide receptor PAC1-R associated with the up-regulation of PAC1-R its own in reactive oxygen species associated way.

PAC1-R is neuropeptide PACAP (pituitary adenylate cyclase activating polypeptide) preferring receptor mediates the antioxidant and cytoprotective effects of PACAP. It was found in this research that in both PAC1R-CHO cells with high expression of PAC1R-eGFP and retinal ganglion cells (RGC-5) with natural expression of PAC1-R, blue light and hydrogen peroxide (H2O2) trigger the significant nuclear translocation of PAC1-R, and the nuclear translocation of PAC1-R was positive correlation with the up-regulation of expression level and promoter activity of PAC1-R its own, while red light worked much less efficiently than blue light. Reactive oxygen species (ROS) scavenger NAC (N-acetyl-L-cysteine) and palmitoylation inhibitor 2-bromopalmitate (2-BP) disturbed the nuclear shifting associated with the correlative up-regulation of PAC1 significantly, and PAC1-R mutant (M-PAC1-R) on Cys25/Ala25 displayed the significant decreased nuclear trafficking efficiency. Furthermore, the Western Blot results with the antibody raised against the C-terminal of PAC1-R showing PAC1-R in the nucleus was fragmentation hinting that C-terminal of PAC1-R with theoretical nuclear location signal (NLS) may be involved in activation of PAC1-R promoter in the nucleus. All above results indicated that PAC1-R makes the nuclear translocation to trigger the activation of PAC1-R itself promoter resulting into the up-regulation of of PAC1-R in response to the oxidative stress induced by blue light and ROS such as H2O2 .