Pituitary adenylate cyclase activating polypeptide (PACAP) is a neurotrophin. However, its role in human Alzheimer's disease (AD) is largely unknown. We examined PACAP expression in postmortem human AD and triple transgenic mouse (3xTG, Psen1/APPSwe/TauP301L) brains. We established an in vitro model of primary neuronal cell culture to study the protective effects of PACAP against β-amyloid (Aβ) toxicity. We further studied the PACAP-Sirtuin 3 (Sirt3) pathway on mitochondrial function. PACAP expression was reduced in AD and 3xTG mouse brains. This reduction was inversely correlated with Aβ and tau protein levels. Treatment with PACAP effectively protected neurons against Aβ toxicity. PACAP stimulated mitochondrial Sirt3 production. Similar to PACAP, Sirt3 was reduced in AD and 3xTG brains. Knocking down Sirt3 compromised the neuroprotective effects of PACAP, and this was reversed by over-expressing Sirt3. PACAP is reduced in AD and may represent a novel therapeutic strategy.

Diabetic nephropathy (DN) is a leading cause of end-stage kidney disease; however, there are few treatment options. Inflammation plays a crucial role in the initiation and/or progression of DN. Pituitary adenylate cyclase-activating polypeptide (PACAP) is a neuropeptide, which was originally isolated from the ovine hypothalamus and reportedly has diverse biological functions. It has been reported that PACAP has renoprotective effects in different models of kidney pathology. However, the specific cell types within the kidney that are protected by PACAP have not yet been reported. In this study, we localized VPAC1, one of the PACAP receptors, to glomerular podocytes, which also reportedly has crucial roles not only in glomerular physiology but also in pathology. PACAP was effective in the downregulation of proinflammatory cytokines, such as monocyte chemoattractant protein-1 (MCP-1) and interleukin-6, which had been induced by the activation of toll-like receptor (TLR) with lipopolysaccharide. PACAP also had downregulated the expression of MCP-1 through the protein kinase A signaling pathway; this led to the attenuation of the activation of extracellular signal-regulated kinase and nuclear factor-kappa B signaling. Our results suggested that PACAP could be a possible treatment option for DN through the use of anti-inflammation effects on glomerular podocytes.

Pituitary adenylate cyclase-activating polypeptide (PACAP) is an inhibitor of megakaryopoiesis and platelet function. Recently, PACAP deficiency was observed in children with nephrotic syndrome (NS), associated with increased platelet count and aggregability and increased risk of thrombosis. To further study PACAP deficiency in NS, we used transgenic Tg(cd41:EGFP) zebrafish with GFP-labeled thrombocytes. We generated two models for congenital NS, a morpholino injected model targeting nphs1 (nephrin), which is mutated in the Finnish-type congenital NS. The second model was induced by exposure to the nephrotoxic compound adriamycin. Nephrin RNA expression was quantified and zebrafish embryos were live-screened for proteinuria and pericardial edema as evidence of renal impairment. Protein levels of PACAP and its binding-protein ceruloplasmin were measured and GFP-labeled thrombocytes were quantified. We also evaluated the effects of PACAP morpholino injection and the rescue effects of PACAP-38 peptide in both congenital NS models. Nephrin downregulation and pericardial edema were observed in both nephrin morpholino injected and adriamycin exposed congenital NS models. However, PACAP deficiency was demonstrated only in the adriamycin exposed condition. Ceruloplasmin levels and the number of GFP-labeled thrombocytes remained unchanged in both models. PACAP morpholino injections worsened survival rates and the edema phenotype in both congenital NS models while injection with human PACAP-38 could only rescue the adriamycin exposed model. We hereby report, for the first time, PACAP deficiency in a NS zebrafish model as a consequence of adriamycin exposure. However, distinct from the human congenital NS, both zebrafish models retained normal levels of ceruloplasmin and thrombocytes. We further extend the renoprotective effects of the PACAP-38 peptide against adriamycin toxicity in zebrafish.

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a neuropeptide acting as a neurotransmitter, neuromodulator, or neurotrophic factor. PACAP is widely expressed throughout the brain and exerts its functions through the PACAP-specific receptor (PAC(1)). Recent studies reveal that genetic variants of the PACAP and PAC(1) genes are associated with mental disorders, and several behavioral abnormalities of PACAP knockout (KO) mice are reported. However, an insufficient number of backcrosses was made using PACAP KO mice on the C57BL/6J background due to their postnatal mortality. To elucidate the effects of PACAP on neuropsychiatric function, the PACAP gene was knocked out in F1 hybrid mice (C57BL/6J × 129SvEv) for appropriate control of the genetic background. The PACAP KO mice were then subjected to a behavioral test battery. PACAP deficiency had no significant effects on neurological screen. As shown previously, the mice exhibited significantly increased locomotor activity in a novel environment and abnormal anxiety-like behavior, while no obvious differences between genotypes were shown in home cage (HC) activity. In contrast to previous reports, the PACAP KO mice showed normal prepulse inhibition (PPI) and slightly decreased depression-like behavior. Previous study demonstrates that the social interaction (SI) in a resident-intruder test was decreased in PACAP KO mice. On the other hand, we showed that PACAP KO mice exhibited increased SI in Crawley's three-chamber social approach test, although PACAP KO had no significant impact on SI in a HC. PACAP KO mice also exhibited mild performance deficit in working memory in an eight-arm radial maze (RM) and the T-maze (TM), while they did not show any significant abnormalities in the left-right discrimination task in the TM. These results suggest that PACAP has an important role in the regulation of locomotor activity, social behavior, anxiety-like behavior and, potentially, working memory.

While pituitary adenylate cyclase activating polypeptide (PACAP) signaling in the hypothalamic ventromedial nuclei (VMN) has been shown to regulate feeding, a challenge in unmasking a role for this peptide in obesity is that excess feeding can involve numerous mechanisms including homeostatic (hunger) and hedonic-related (palatability) drives. In these studies, we first isolated distinct feeding drives by developing a novel model of binge behavior in which homeostatic-driven feeding was temporally separated from feeding driven by food palatability. We found that stimulation of the VMN, achieved by local microinjections of AMPA, decreased standard chow consumption in food-restricted rats (e.g., homeostatic feeding); surprisingly, this manipulation failed to alter palatable food consumption in satiated rats (e.g., hedonic feeding). In contrast, inhibition of the nucleus accumbens (NAc), through local microinjections of GABA receptor agonists baclofen and muscimol, decreased hedonic feeding without altering homeostatic feeding. PACAP microinjections produced the site-specific changes in synaptic transmission needed to decrease feeding via VMN or NAc circuitry. PACAP into the NAc mimicked the actions of GABA agonists by reducing hedonic feeding without altering homeostatic feeding. In contrast, PACAP into the VMN mimicked the actions of AMPA by decreasing homeostatic feeding without affecting hedonic feeding. Slice electrophysiology recordings verified PACAP excitation of VMN neurons and inhibition of NAc neurons. These data suggest that the VMN and NAc regulate distinct circuits giving rise to unique feeding drives, but that both can be regulated by the neuropeptide PACAP to potentially curb excessive eating stemming from either drive.

The blood-brain barrier (BBB) regulates the traffic of molecules into the central nervous system (CNS) and also limits the drug delivery. Due to their flexible properties, liposomes are an attractive tool to deliver drugs across the BBB. We previously characterized gH625, a peptide derived from Herpes simplex virus 1. The present study investigates the efficiency of liposomes functionalized on their surface with gH625 to promote the brain uptake of neuroprotective peptide PACAP (pituitary adenylate cyclase-activating polypeptide). Using a rat in vitro BBB model, we showed that the liposomes preparations were non-toxic for the endothelial cells, as assessed by analysis of tight junction protein ZO1 organization and barrier integrity. Next, we found that gH625 improves the transfer of liposomes across endothelial cell monolayers, resulting in both low cellular uptake and increased transport of PACAP. Finally, in vivo results demonstrated that gH625 ameliorates the efficiency of liposomes to deliver PACAP to the mouse brain after intravenous administration. gH625-liposomes improve both PACAP reaching and crossing the BBB, as showed by the higher number of brain cells labelled with PACAP. gH625-liposomes represent a promising strategy to deliver therapeutic agents to CNS and to provide an effective imaging and diagnostic tool for the brain.

The highly conserved neuropeptide pituitary adenylate cyclase activating polypeptide (PACAP) has been implicated in a broad variety of physiological processes. The PACAP precursor protein gives rise to three different peptides, the cryptic peptide, GHRH, and PACAP, respectively, and here we dissect their functional properties using Xenopus as model system. PACAP and GHRH but not the cryptic peptide directly neuralize animal caps. In contrast to GHRH, the neuralizing effect mediated by PACAP is independent of the PKA pathway. Moreover, PACAP but not GHRH behaves like a BMP-4 antagonist. Blastocoel injection of PACAP-38 but not of the closely related peptides PACAP-27 and VIP leads to strong anteriorization of the injected embryos suggesting the possible involvement of a novel PACAP-preferring receptor.

Pituitary adenylate cyclase activating polypeptide (PACAP) isolated from ovine hypothalamus is considered to be a member of the vasoactive intestinal peptide/glucagon/secretin/growth hormone-releasing hormone family of peptides. Two forms of PACAP, PACAP38 and PACAP27, have been demonstrated in the rat hypothalamus. The PACAP precursor contains another peptide called PACAP-related peptide (PRP), but so far no information on this peptide in tissue exists. We have developed three radioimmunoassays specific for PACAP38, PACAP27 and PRP and demonstrate that all three preproPACAP peptides are expressed in the rat hypothalamus, the PACAP38/PACAP27 ratio being around 60 and the PACAP38/PRP ratio being around 10. HPLC analysis of hypothalamic extract showed that PACAP38 and PACAP27 are found in only one form corresponding to the respective synthetic peptides, whereas PRP eluted in two peaks, the predominant form corresponding to synthetic PRP1-29. The cellular distribution of PACAP38, PACAP27, and PRP and corresponding mRNA in the hypothalamus was determined with immunohistochemistry and in situ hybridization histochemistry. PACAP- and PRP-immunoreactive neuronal perikarya were observed in the medial parvocellular part of the paraventricular nucleus (PVN) in colchicine pretreated rats. Some cell bodies of magnocellular variety were found in the PVN. PACAP mRNA containing cells were observed in moderate numbers in the same parts of the paraventricular nucleus. PACAP- and PRP immunoreactive fibres and varicosities were distributed in the PVN and in the periventricular nucleus. These data show that PACAP38, PACAP27 and PRP are expressed in the parvocellular part of the PVN, implying roles as hypothalamic regulatory peptides.

Depression is one of the foremost psychological illness, and the exact mechanism is unclear. Recent studies have reported that the pituitary adenylate cyclase-activating polypeptide (PACAP) signaling pathway is involved in the progression of depression. In the present study, we extracted crocin from the traditional Chinese medicine (TCM), Gardenia jasminoides Ellis, to evaluate its antidepressant effect and clarify the underlying mechanism. Here, we established a chronic unpredictable mild stress (CUMS) mouse model to assess whether crocin can improve depression-like behavior in an open field test (OFT), tail suspension test (TST), forced swimming test (FST), and sucrose preference test (SPT). A corticosterone (CORT) model of PC12 was set up to explore the antidepressant mechanism of crocin. We pretreated PC12 cells with crocin for 1 hour and then stimulated the cells with CORT for 24 hours. Cell survival was detected by Hoechst staining and MTT assay. The expression of PACAP, cyclic adenosine monophosphate (cAMP) response element binding protein (CREB), and extracellular regulated protein kinases (ERK) were analyzed by western blotting. PACAP RNAi was used to interfere with PC12 cells to downregulate the content of PACAP. The results showed that crocin (30 mg/kg) significantly reversed the decrease of body weight and elevation of serum CORT, mitigated CUMS induced depression-like behaviors of mice, and crocin (12.5 μmol/L) protected PC12 cells against CORT (200 μmol/L)-induced injury. Furthermore, crocin greatly increased the protein expression of PACAP and phosphorylation of ERK and CREB in the CORT model. PACAP RNAi cancelled the neuroprotective effect of crocin. In conclusion, these results indicated that crocin exerted an antidepressant effect via upregulating PACAP and its downstream ERK and CREB signaling pathways.

Sensory nerves regulate cutaneous local inflammation indirectly through induction of pruritus and directly by acting on local immune cells. The underlying mechanisms for how sensory nerves influence cutaneous acquired immune responses remain to be clarified.

The neuropeptide Pituitary adenylate cyclase-activating polypeptide (PACAP) plays pivotal roles in immunity and inflammation. So far, potential immune-modulatory properties of PACAP have not been investigated in experimental ileitis.

Pituitary Adenylate Cyclase-Activating Polypeptide (PACAP) is an endogenous neuropeptide with distinct functions including the regulation of inflammatory processes. PACAP is able to modify the immune response by directly regulating macrophages and monocytes inhibiting the production of inflammatory cytokines, chemokines and free radicals. Here, we analyzed the effect of exogenous PACAP on peripheral immune cell subsets upon acute infection with the parasite Toxoplasma gondii (T. gondii). PACAP administration was followed by diminished innate immune cell recruitment to the peritoneal cavity of T. gondii-infected mice. PACAP did not directly interfere with parasite replication, instead, indirectly reduced parasite burden in mononuclear cell populations by enhancing their phagocytic capacity. Although proinflammatory cytokine levels were attenuated in the periphery upon PACAP treatment, interleukin (IL)-10 and Transforming growth factor beta (TGF-β) remained stable. While PACAP modulated VPAC1 and VPAC2 receptors in immune cells upon binding, it also increased their expression of brain-derived neurotrophic factor (BDNF). In addition, the expression of p75 neurotrophin receptor (p75NTR) on Ly6Chi inflammatory monocytes was diminished upon PACAP administration. Our findings highlight the immunomodulatory effect of PACAP on peripheral immune cell subsets during acute Toxoplasmosis, providing new insights about host-pathogen interaction and the effects of neuropeptides during inflammation.

The rate of atmospheric vanadium is constantly increasing due to fossil fuel combustion. This environmental pollution favours vanadium exposure in particular to its vanadate form, causing occupational bronchial asthma and bronchitis. Based on the well admitted bronchodilator properties of the pituitary adenylate cyclase-activating polypeptide (PACAP), we investigated the ability of this neuropeptide to reverse the vanadate-induced airway hyperresponsiveness in rats. Exposure to ammonium metavanadate aerosols (5 mg/m(3)/h) for 15 minutes induced 4 hours later an array of pathophysiological events, including increase of bronchial resistance and histological alterations, activation of proinflammatory alveolar macrophages, and increased oxidative stress status. Powerfully, PACAP inhalation (0.1 mM) for 10 minutes alleviated many of these deleterious effects as demonstrated by a decrease of bronchial resistance and histological restoration. PACAP reduced the level of expression of mRNA encoding inflammatory chemokines (MIP-1α, MIP-2, and KC) and cytokines (IL-1α and TNF-α) in alveolar macrophages and improved the antioxidant status. PACAP reverses the vanadate-induced airway hyperresponsiveness not only through its bronchodilator activity but also by counteracting the proinflammatory and prooxidative effects of the metal. Then, the development of stable analogs of PACAP could represent a promising therapeutic alternative for the treatment of inflammatory respiratory disorders.

A number of efforts have been made to understand how pituitary adenylate cyclase activating polypeptide (PACAP) functions as a neurotrophic and neuroprotective factor in Parkinson's disease (PD). Recently its effects on neurotransmission and underlying mechanisms have generated interest. In the present study, we investigate the effects of PACAP on catecholamine storage and secretion in PC12 cells with amperometry and transmission electron microscopy (TEM). PACAP increases quantal release induced by high K+ without significantly regulating the frequency of vesicle fusion events. TEM data indicate that the increased volume of the vesicle is mainly the result of enlargement of the fluidic space around the dense core. Moreover, the number of docked vesicles isn't modulated by PACAP. When cells are acutely treated with L-DOPA, the vesicular volume and quantal release both increase dramatically. It is likely that the characteristics of amperometric spikes from L-DOPA treated cells are associated with increased volume of individual vesicles rather than a direct effect on the mechanics of exocytosis. Treatment with PACAP versus L-DOPA results in different profiles of the dynamics of exocytosis. Release via the fusion pore prior to full exocytosis was observed with the same frequency following treatment with PACAP and L-DOPA. However, release events have a shorter duration and higher average current after PACAP treatment compared to L-DOPA. Furthermore, PACAP reduced the proportion of spikes having rapid decay time and shortened the decay time of both fast and slow spikes. In contrast, the distributions of the amperometric spike decay for both fast and slow spikes were shifted to longer time following L-DOPA treatment. Compared to L-DOPA, PACAP may produce multiple favorable effects on dopaminergic neurons, including protecting dopaminergic neurons against neurodegeneration and potentially regulating dopamine storage and release, making it a promising therapeutic agent for the treatment of PD.

NLR Family Pyrin Domain Containing 3 (NLRP3) inflammasome-induced neuroinflammation is the main pathogenic mechanism of dopaminergic (DA) neuron degeneration in Parkinson's disease (PD). Hyperoside (quercetin-3-O-β-D-galactoside), an active compound obtained from the traditional Chinese medicinal herb Abelmoschus manihot, is a potential inflammasome inhibitor. Besides, pituitary adenylate cyclase-activated peptide (PACAP) is an endogenous neuropeptide with neuroprotective effects in various neurodegenerative diseases, such as PD. This study aimed to explore the effects of hyperoside on inflammasome-induced neuroinflammation, and its relationship with PACAP in PD. N-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) was used to induce PD-like lesions in mice. Behavioral methods, including the pole test and rotarod test, were used to evaluate the hyperoside effects on MPTP-induced motor dysfunction. Immunohistochemistry was done to detect the loss of DA neurons and activation of glia in the substantia nigra compacta (SNpc). Besides, an enzyme-linked immunosorbent assay (ELISA) was used to detect pro-inflammatory cytokines and Western blotting to detect the inflammasome components. PACAP 6-38, a non-irritating competitive antagonist of PACAP, was used to explore the anti-inflammation mechanism of hyperoside. The results showed that hyperoside inhibited the activation of glia and reduced the secretion of inflammatory factors, protecting DA neurons and reversing the motor dysfunction caused by MPTP. Hyperoside also inhibited the inflammasome activation by reducing the expression of NLRP3, apoptosis-associated speck-like protein containing caspases recruitment domain (ASC), and caspase-1 and increased PACAP content and CREB phosphorylation in the SNpc of the mice. PACAP 6-38 reversed the inhibitory effect of hyperoside on the microglia proliferation and activation of the NLRP3 inflammasome. These results indicate that hyperoside can inhibit the activation of the NLRP3 inflammasome by up-regulating PACAP, thus effectively inhibiting MPTP-induced neuroinflammation and protecting DA neurons. Therefore, hyperoside can be used to treat PD.

The Pituitary Adenylate Cyclase-Activating Polypeptide (PACAP), a polycationic, amphiphilic and helical neuropeptide, is well known for its neuroprotective actions and cell penetrating properties. In the present study, we evaluated the potent antibacterial property of PACAP38 and related analogs against various bacterial strains. Interestingly, PACAP38 and related analogs can inhibit the growth of various bacteria including Escherichia coli (JM109), Bacillus subtilis (PY79), and the pathogenic Burkholderia cenocepacia (J2315). Investigation of the mechanism of action suggested that a PACAP metabolite, identified as PACAP(9-38), might indeed be responsible for the observed PACAP38 antibacterial action. Surprisingly, PACAP(9-38), which does not induce haemolysis, exhibits an increased specificity toward Burkholderia cenocepacia J2315 compared to other tested bacteria. Finally, the predisposition of PACAP(9-38) to adopt a π-helix conformation rather than an α-helical conformation like PACAP38 could explain this gain in specificity. Overall, this study has revealed a new function for PACAP38 and related derivatives that can be added to its pleiotropic biological activities. This innovative study could therefore pave the way toward the development of new therapeutic agents against multiresistant bacteria, and more specifically the Burkholderia cenocepacia complex.

This study aimed to investigate the effect of pituitary adenylate cyclase-activating peptide (PACAP) on the pacemaker activity of interstitial cells of Cajal (ICC) in mouse colon and to identify the underlying mechanisms of PACAP action. Spontaneous pacemaker activity of colonic ICC and the effects of PACAP were studied using electrophysiological recordings. Exogenously applied PACAP induced hyperpolarization of the cell membrane and inhibited pacemaker frequency in a dose-dependent manner (from 0.1 nM to 100 nM). To investigate cyclic AMP (cAMP) involvement in the effects of PACAP on ICC, SQ-22536 (an inhibitor of adenylate cyclase) and cell-permeable 8-bromo-cAMP were used. SQ-22536 decreased the frequency of pacemaker potentials, and cell-permeable 8-bromo-cAMP increased the frequency of pacemaker potentials. The effects of SQ-22536 on pacemaker potential frequency and membrane hyperpolarization were rescued by co-treatment with glibenclamide (an ATP-sensitive K(+) channel blocker). However, neither N (G)-nitro-L-arginine methyl ester (L-NAME, a competitive inhibitor of NO synthase) nor 1H-[1,2,4]oxadiazolo[4,3-α]quinoxalin-1-one (ODQ, an inhibitor of guanylate cyclase) had any effect on PACAP-induced activity. In conclusion, this study describes the effects of PACAP on ICC in the mouse colon. PACAP inhibited the pacemaker activity of ICC by acting through ATP-sensitive K(+) channels. These results provide evidence of a physiological role for PACAP in regulating gastrointestinal (GI) motility through the modulation of ICC activity.

: Pituitary adenylate cyclase activating polypeptide (PACAP) is a neuropeptide with diverse developmental roles, including differentiation of skeletal elements. It is a positive regulatory factor of chondrogenesis and osteogenic differentiation in vitro, but little is known about its in vivo role in bone formation. In our experiments, diaphyses of long bones from hind limbs of PACAP gene-deficient mice showed changes in thickness and increased staining intensity. Our main goal was to perform a detailed morphological and molecular biological analysis of femurs from PACAP knockout (KO) and wild type (WT) mice. Transverse diameter and anterior cortical bone thickness of KO femurs showed significant alterations with disturbed Ca2+ accumulation and collagen type I expression. Higher expression and activity of alkaline phosphatase were also observed, accompanied by increased fragility PACAP KO femurs. Increased expression of the elements of bone morphogenic protein (BMP) and hedgehog signalling was also observed, and are possibly responsible for the compensation mechanism accounting for the slight morphological changes. In summary, our results show that lack of PACAP influences molecular and biomechanical properties of bone matrix, activating various signalling cascade changes in a compensatory fashion. The increased fragility of PACAP KO femur further supports the role of endogenous PACAP in in vivo bone formation.

Pituitary adenylate cyclase-activating peptide (PACAP) is an important neuropeptide and immunomodulator in various tissues. Although this peptide and its receptors (ie, VPAC1R, VPAC2R, and PAC1R) are expressed in human skin, their biological roles are unknown. Therefore, we tested whether PACAP regulates vascular responses in human skin in vivo. When injected intravenously, PACAP induced a significant, concentration-dependent vascular response (ie, flush, erythema, edema) and mediated a significant and concentration-dependent increase in intrarectal body temperature that peaked at 2.7°C. Topical application of PACAP induced marked concentration-dependent edema. Immunohistochemistry revealed a close association of PACAP-immunoreactive nerve fibers with mast cells and dermal blood vessels. VPAC1R was expressed by dermal endothelial cells, CD4+ and CD8+ T cells, mast cells, and keratinocytes, whereas VPAC2R was expressed only in keratinocytes. VPAC1R protein and mRNA were also detected in human dermal microvascular endothelial cells. The PACAP-induced change in cAMP production in these cells demonstrated VPAC1R to be functional. PACAP treatment of organ-cultured human skin strongly increased the number of CD31+ vessel cross-sections. Taken together, these results suggest that PACAP directly induces vascular responses that may be associated with neurogenic inflammation, indicating for the first time that PACAP may be a crucial vascular regulator in human skin in vivo. Antagonists to PACAP function may be beneficial for the treatment of inflammatory skin diseases with a neurogenic component.

Pituitary adenylate cyclase-activating peptide I receptor (PAC1R) is member of the B class of G protein-coupled seven-transmembrane receptors, with molecular functions associated with neural cell differentiation, regeneration and the inhibition of apoptosis. However, the integrity of the protein structure is difficult to be determined in vitro. In the present study, the physicochemical properties of PAC1R were analyzed, the extracellular, transmembrane and intracellular regions were constructed and a three-dimensional structure model of PAC1R was produced using extracellular loop region optimization and the energy minimization homology modeling method. Preliminary studies on the PAC1R protein and ligand interactions used a molecular docking method. The results indicated that the interaction sites of PAC1R were at Ile63, Ser100 and Gln105. These were the sites where the PAC1R combined with a hydrazide small molecule inhibitor. This study provides a theoretical basis for further studies on the model for the development of PAC1R target drugs.