PACAP key interactions with PAC1, VPAC1, and VPAC2 identified by molecular dynamics simulations.

The neuropeptide pituitary adenylate cyclase-activating polypeptide (PACAP) belongs to the glucagon/secretin family. PACAP interacts with the pituitary adenylate cyclase-activating polypeptide receptor type 1 (PAC1) and vasoactive intestinal peptide receptors 1 and 2 (VPAC1 and VPAC2), exhibiting functions in the immune, endocrine, and nervous systems. This peptide is upregulated in numerous instances of brain injury, acting as a neuroprotective agent. It can also suppress HIV-1 and SARS-CoV-2 viral replication in vitro. This work aimed to identify, in each peptide-receptor system, the most relevant residues for complex stability and interaction energy communication via Molecular Dynamics (MD), Free Energy calculations, and Protein-energy networks, thus revealing in detail the underlying mechanisms of activation of these receptors. Hydrogen bond formation, interaction energies, and computational alanine scanning between PACAP and its receptors showed that His1, Asp3, Arg12, Arg14, and Lys15 are crucial to the peptide's stability. Furthermore, several PACAP interactions with structurally conserved positions deemed necessary in GPCR B1 activation, including Arg2.60, Lys2.67, and Glu7.42, were significant for the peptide's stability within the receptors. According to the protein-energy network, the connection between Asp3 of PACAP and the receptors' conserved Arg2.60 represents a critical energy communication hub in all complexes. Additionally, the ECDs of the receptors were also found to function as energy communication hubs for PACAP. Although the overall binding mode of PACAP in the three receptors was found to be highly conserved, Arg12 and Tyr13 of PACAP were more prominent in complex with PAC1, while Ser2 of PACAP was with VPAC2. The detailed analyses performed in this work pave the way for using PACAP and its receptors as therapeutic targets.Communicated by Ramaswamy H. Sarma.

Early Alterations of PACAP and VIP Expression in the Female Rat Brain Following Spinal Cord Injury.

Previous evidence shows that rapid changes occur in the brain following spinal cord injury (SCI). Here, we interrogated the expression of the neuropeptides pituitary adenylyl cyclase-activating peptide (PACAP), vasoactive intestinal peptides (VIP), and their binding receptors in the rat brain 24 h following SCI. Female Sprague-Dawley rats underwent thoracic laminectomy; half of the rats received a mild contusion injury at the level of the T10 vertebrate (SCI group); the other half underwent sham surgery (sham group). Twenty-four hours post-surgery, the hypothalamus, thalamus, amygdala, hippocampus (dorsal and ventral), prefrontal cortex, and periaqueductal gray were collected. PACAP, VIP, PAC1, VPAC1, and VPAC2 mRNA and protein levels were measured by real-time quantitative polymerase chain reaction and Western blot. In SCI rats, PACAP expression was increased in the hypothalamus (104-141% vs sham) and amygdala (138-350%), but downregulated in the thalamus (35-95%) and periaqueductal gray (58-68%). VIP expression was increased only in the thalamus (175-385%), with a reduction in the amygdala (51-68%), hippocampus (40-75%), and periaqueductal gray (74-76%). The expression of the PAC1 receptor was the least disturbed by SCI, with decrease expression in the ventral hippocampus (63-68%) only. The expression levels of VPAC1 and VPAC2 receptors were globally reduced, with more prominent reductions of VPAC1 vs VPAC2 in the amygdala (21-70%) and ventral hippocampus (72-75%). In addition, VPAC1 downregulation also extended to the dorsal hippocampus (69-70%). These findings demonstrate that as early as 24 h post-SCI, there are region-specific disruptions of PACAP, VIP, and related receptor transcript and protein levels in supraspinal regions controlling higher cognitive functions.

Effects of alpha-melanocyte-stimulating hormone on mitochondrial energy metabolism in rats of different age-groups.

Hypothalamic alpha-melanocyte-stimulating hormone (α-MSH) is a key catabolic mediator of energy homeostasis. Its anorexigenic and hypermetabolic effects show characteristic age-related alterations that may be part of the mechanism of middle-aged obesity and geriatric anorexia/cachexia seen in humans and other mammals. We aimed to investigate the role of α-MSH in mitochondrial energy metabolism during the course of aging in a rodent model. To determine the role of α-MSH in mitochondrial energy metabolism in muscle, we administered intracerebroventricular (ICV) infusions of α-MSH for 7-days to different age-groups of male Wistar rats. The activities of oxidative phosphorylation complexes I to V and citrate synthase were determined and compared to those of age-matched controls. We also quantified mitochondrial DNA (mtDNA) copy number and measured the expression of the master regulators of mitochondrial biogenesis, peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α) and peroxisome proliferator-activated receptor gamma (PPARγ). The peptide reduced weight gain in juvenile rats to one fifth of that of controls and increased the weight loss in older animals by about five fold. Mitochondrial DNA copy number inversely correlated with changes in body weight in controls, but not in α-MSH-treated animals. The strong increase in body weight in young rats was associated with a low mtDNA copy number and high PPARγ mRNA levels in controls. Expression of PGC-1α and PPARγ declined with age, whereas OXPHOS and citrate synthase enzyme activities were unchanged. In contrast, α-MSH treatment suppressed OXPHOS enzyme and citrate synthase activity. In conclusion, our results showed age-related differences in the metabolic effects of α-MSH. In addition, administration of α-MSH suppressed citrate synthase and OXPHOS activities independent of age. These findings suggest that α-MSH exposure may inhibit mitochondrial biogenesis.

Novel evidence that pituitary gonadotropins directly stimulate human leukemic cells-studies of myeloid cell lines and primary patient AML and CML cells.

We recently reported that normal hematopoietic stem cells express functional pituitary sex hormone (SexH) receptors. Here we report for the first time that pituitary-secreted gonadotrophins stimulate migration, adhesion, and proliferation of several human myeloid and lymphoid leukemia cell lines. Similar effects were observed after stimulation of human leukemic cell lines by gonadal SexHs. This effect seems to be direct, as the SexH receptors expressed by leukemic cells responded to stimulation by phosphorylation of MAPKp42/44 and AKTser473. Furthermore, in parallel studies we confirmed that human primary patient-derived AML and CML blasts also express several functional SexH receptors. These results shed more light on the potential role of SexHs in leukemogenesis and, in addition, provide further evidence suggesting a developmental link between hematopoiesis and the germline.

Expression of melanin-concentrating hormone receptor 2 protects against diet-induced obesity in male mice.

Melanin-concentrating hormone (MCH) is an orexigenic neuropeptide that is a ligand for two subtypes of MCH receptors, MCHR1 and MCHR2. MCHR1 is universally expressed in mammals ranging from rodents to humans, but the expression of MCHR2 is substantially restricted. In mammals, MCHR2 has been defined in primates as well as other species such as cats and dogs but is not seen in rodents. Although the role of MCHR1 in mediating the actions of MCH on energy balance is clearly defined using mouse models, the role of MCHR2 is harder to characterize because of its limited expression. To determine any potential role of MCHR2 in energy balance, we generated a transgenic MCHR1R2 mouse model, where human MCHR2 is coexpressed in MCHR1-expressing neurons. As shown previously, control wild-type mice expressing only native MCHR1 developed diet-induced obesity when fed a high-fat diet. In contrast, MCHR1R2 mice had lower food intake, leading to their resistance to diet-induced obesity. Furthermore, we showed that MCH action is altered in MCHR1R2 mice. MCH treatment in wild-type mice inhibited the activation of the immediate-early gene c-fos, and coexpression of MCHR2 reduced the inhibitory actions of MCHR1 on this pathway. In conclusion, we developed an experimental animal model that can provide insight into the action of MCHR2 in the central nervous system and suggest that some actions of MCHR2 oppose the endogenous actions of MCHR1.

Oestradiol decreases melanin-concentrating hormone (MCH) and MCH receptor expression in the hypothalamus of female rats.

Previous studies have shown that oestradiol (E2) decreases the orexigenic effect of melanin-concentrating hormone (MCH). In the present study, we examined whether this action of E2 is mediated by its ability to decrease the expression of MCH or its receptor (MCHR1). Using immunocytochemistry and western blotting, we examined whether E2 decreases MCH-immunoreactive neurones or MCHR1 protein content in the hypothalamus of female rats. We found that both MCH and MCHR1 protein expression was decreased by acute E2 treatment in ovariectomised rats, and by the peri-ovulatory increase in circulating E2 in pro-oestrous rats, relative to rats at other cycle stages. To determine whether these changes in MCH/MCHR1 protein expression may be mediated by E2's ability to directly regulate the transcription of MCH and MCHR1 genes, the effect of E2 treatment on MCH and MCHR1 mRNA expression in a neuronal hypothalamic cell line was examined using real-time reverse transcriptase-polymerase chain reaction. We also determined whether MCH and oestrogen receptor (ER)α are co-expressed in the hypothalamus of female rats. E2 treatment did not decrease MCH or MCHR1 mRNA expression in vitro, and no hypothalamic neurones were identified that co-expressed MCH and ERα. We conclude that E2-dependent decreases in hypothalamic MCH/MCHR1 protein expression mediate the ability of E2 to decrease MCH-induced feeding. The current findings suggest, however, that E2 exerts these actions indirectly, most likely though interactions with other neuronal systems that provide afferent input to MCH and MCHR1 neurones.

Chromosomal alterations, prognostic factors, and targeted molecular therapies for malignant meningiomas.

Meningiomas are the second most common intracranial neoplasm in adults and originate from arachnoidal cap cells. Malignant meningiomas are resistant to conventional treatments such as surgery, chemotherapy, and radiotherapy. Unlike benign meningiomas, atypical and anaplastic tumors generally display more complex karyotypes associated with aggressive behavior. While these chromosomal anomalies are associated with greater malignancy in meningiomas, the specific genes involved remain unknown. Malignant meningiomas are characterized by increased tumor aggressiveness, rapid recurrence, local invasion, atypical histological appearance, and a high mitotic index. Potential prognostic factors include extent of resection, treatment with radiotherapy or stereotactic radiosurgery, Ki-67/MIB-1 labeling index, p53 overexpression, percentage of tumor cells in the S-phase, telomerase activity, and numerous genetic expression profiles. A greater understanding of prognostic factors and molecular markers involved in critical signaling pathways may aid in the identification of novel therapeutic targets. As such, further studies are needed to establish reliable prognostic factors and develop more effective treatments for malignant meningiomas.

Expression profiles of key candidate genes involved in steroidogenesis during follicular atresia in the pig ovary.

More than 99 % of follicles in mammalian ovaries undergo a degenerative process known as atresia, and thus only a limited number of ovarian follicles actually ovulate after full growth and development. The endocrinological regulatory mechanisms involved in follicular development have been studied extensively, but the precise and systematic molecular mechanisms of steroidogenesis enzymes involved in atresia are unclear. In the present study, we examined whether and how the steroidogenesis enzymes are involved in porcine ovary follicular atresia. Expression of steroidogenic acute regulatory protein, CYP11, CYP17, 3ß-hydroxysteroid dehydrogenase (3ß-HSD), CYP19, as well as related pituitary and ovarian hormone receptors were quantified in ovaries. During porcine follicular atresia, expressions of P450 cholesterol side chain cleavage enzyme, progesterone and androgen receptors increased significantly during the late atretic stage, while the expression of aromatase and follicle-stimulating hormone receptors decreased significantly in the early stage. These data suggested that the regulation of aromatase by follicle-stimulating hormone might induce follicular atresia, and that progesterone and androgen production further promoted follicular atresia. Additionally, a correlation analysis indicated a large and complex interactive network among these genes and the endocrinological microenvironment of the follicles. Significant correlations were observed between expression of steroidogenic enzymes and their receptors, and also between progesterone and 17ß-estradiol (E2) levels in follicular fluid. Taken together, these results suggest that CYP19 plays a role during early atresia by regulating the production of E2, whereas CYP11 and 3ß-HSD increase atresia progression by increasing progesterone levels.

Discovery of a novel melanin concentrating hormone receptor 1 (MCHR1) antagonist with reduced hERG inhibition.

An initial SAR study resulted in the identification of the novel, potent MCHR1 antagonist 2. After further profiling, compound 2 was discovered to be a potent inhibitor of the hERG potassium channel, which prevented its further development. Additional optimization of this structure resulted in the discovery of the potent MCHR1 antagonist 11 with a dramatically reduced hERG liability. The decrease in hERG activity was confirmed by several in vivo preclinical cardiovascular studies examining QT prolongation. This compound demonstrated good selectivity for MCHR1 and possessed good pharmacokinetic properties across preclinical species. Compound 11 was also efficacious in reducing body weight in two in vivo mouse models. This compound was selected for clinical evaluation and was given the code AMG 076.

Synthesis, structure-activity relationship, and pharmacological studies of novel melanin-concentrating hormone receptor 1 antagonists 3-aminomethylquinolines: reducing human ether-a-go-go-related gene (hERG) associated liabilities.

Recently, we discovered 3-aminomethylquinoline derivative 1, a selective, highly potent, centrally acting, and orally bioavailable human MCH receptor 1 (hMCHR1) antagonist, that inhibited food intake in F344 rats with diet-induced obesity (DIO). Subsequent investigation of 1 was discontinued because 1 showed potent hERG K(+) channel inhibition in a patch-clamp study. To decrease hERG K(+) channel inhibition, experiments with ligand-based drug designs based on 1 and a docking study were conducted. Replacement of the terminal p-fluorophenyl group with a cyclopropylmethoxy group, methyl group introduction on the benzylic carbon at the 3-position of the quinoline core, and employment of a [2-(acetylamino)ethyl]amino group as the amine portion eliminated hERG K(+) channel inhibitory activity in a patch-clamp study, leading to the discovery of N-{3-[(1R)-1-{[2-(acetylamino)ethyl]amino}ethyl]-8-methylquinolin-7-yl}-4-(cyclopropylmethoxy)benzamide (R)-10h. The compound (R)-10h showed potent inhibitory activity against hMCHR1 and dose-dependently suppressed food intake in a 2-day study on DIO-F344 rats. Furthermore, practical chiral synthesis of (R)-10h was performed to determine the molecule's absolute configuration.

Effects of quinestrol on reproductive hormone expression, secretion, and receptor levels in female Mongolian gerbils (Meriones unguiculatus).

Quinestrol, a synthetic estrogen with marked estrogenic effects and prolonged activity, has potential as a contraceptive for Mongolian gerbils. The objective of this study was to describe the effects of quinestrol on reproductive hormone expression, secretion, and receptor levels in female Mongolian gerbils. Serum and pituitary concentrations of follicle stimulating hormone (FSH) and luteinizing hormone (LH) were decreased, whereas serum concentrations of estradiol (E2) and progesterone (P4) were increased after quinestrol treatment; the effects were both time- and dose-dependent. Furthermore, quinestrol downregulated expression of FSHß and LHß mRNA in the pituitary gland, as well as FSH receptor (FSHR) and estrogen receptor (ER) ß in the ovary. However, it up-regulated mRNA expression levels of ERα and progesterone receptor (PR) in the pituitary gland and uterus, as well as mRNA for LH receptor (LHR) and PR in the ovary (these effects were time- and dose-dependent). In contrast, quinestrol had no significant effects on the mRNA expression levels of ERα in the ovary, or the gonadotropin α (GtHα) subunit in the pituitary gland. We inferred that quinestrol impaired synthesis and secretion of FSH and LH and that the predominant ER subtype in the pituitary gland of Mongolian gerbils may be ERα. Overall, quinestrol disrupted reproductive hormone receptor expression at the mRNA level in the pituitary-gonadal axis of the Mongolian gerbil.

Signalling pathway of goldfish melanin-concentrating hormone receptors 1 and 2.

Melanin-concentrating hormone (MCH) is the natural ligand for the MCH-1 receptor (MCHR1) and MCH-2 receptor (MCHR2). The MCH-MCHR1 system plays a central role in energy metabolism in rodents. Recently, we identified MCHR1 and MCHR2 orthologues in goldfish, designated gfMCHR1 and gfMCHR2. In a mammalian cell-based assay, calcium mobilization was evoked by gfMCHR2 via both Gαi/o and Gαq, while the gfMCHR1-mediated response was exclusively dependent on Gαq. This coupling capacity to G proteins is in contrast to human MCHR1 and MCHR2. Here, we extended our previous characterization of the two gfMCHRs by examining their different signalling pathway. We found that MCH caused activation of extracellular signal-regulated kinases 1 and 2 (ERK1/2) via both gfMCHR1 and gfMCHR2 in dose-dependent manners. Unlike the case for gfMCHR2, gfMCHR1 signalling was not sensitive to pertussis toxin, suggesting Gαq coupling of gfMCHR1 in the ERK1/2 pathway as well as a calcium mobilization system. Cyclic AMP assays revealed that gfMCHR2 was efficiently coupled to Gαi/o, while gfMCHR1 was weakly coupled to Gαs. Finally, we investigated the transduction features stimulated by two mammalian MCH analogues. As expected, Compound 15, which is a full agonist of human MCHR1, was a potent gfMCHR1 agonist in multiple signalling pathways. On the other hand, Compound 30, which is a human MCHR1-selective antagonist with negligible agonist potency, unexpectedly acted as a selective agonist of gfMCHR1. These results are the first to demonstrate that gfMCHR1 and gfMCHR2 have quite different signalling properties from human MCHRs.

Cellular models for the study of the pharmacology and signaling of melanin-concentrating hormone receptors.

Cellular models for the study of the neuropeptide melanin-concentrating hormone (MCH) have become indispensable tools for pharmacological profiling and signaling analysis of MCH and its synthetic analogues. Although expression of MCH receptors is most abundant in the brain, MCH-R(1) is also found in different peripheral tissues. Therefore, not only cell lines derived from nervous tissue but also from peripheral tissues that naturally express MCH receptors have been used to study receptor signaling and regulation. For screening of novel compounds, however, heterologous expression of MCH-R(1) or MCH-R(2) genes in HEK293, Chinese hamster ovary, COS-7, or 3T3-L1 cells, or amplified MCH-R(1) expression/signaling in IRM23 cells transfected with the G(q) protein gene are the preferred tools because of more distinct pharmacological effects induced by MCH, which include inhibition of cAMP formation, stimulation of inositol triphosphate production, increase in intracellular free Ca(2+) and/or activation of mitogen-activated protein kinases. Most of the published data originate from this type of model system, whereas data based on studies with cell lines endogenously expressing MCH receptors are more limited. This review presents an update on the different cellular models currently used for the analysis of MCH receptor interaction and signaling.

Functional divergence of glycoprotein hormone receptors.

Two lamprey glycoprotein hormone receptors (lGpH-R I and II) highly similar with gnathostome GpH-Rs were cloned from sea lamprey testes and thyroid, respectively. Vertebrate glycoprotein protein receptors have a large extracellular domain (ED) containing a leu rich domain (LRD) linked to a rhodopsin-like transmembrane domain (TMD) through a highly divergent linker region (signal specificity domain, SSD or 'hinge' region) and a third major segment, the intracellular domain. To determine the potential roles of the different domains in the activation of the receptor following ligand-receptor binding, functional assays were performed on lGpH-R I/rat luteinizing hormone (LH)-R domain swapped chimeric receptors. These results show that the functional roles of the lamprey glycoprotein-receptor I (lGpH-R I) domains are conserved compared with its Gnathostome homologs. The ability of different glycoprotein hormones to activate chimeric lamprey/rat receptors suggests that the selectivity of the GpH-Rs in respect to their ligands is not controlled exclusively by a single domain but is the result of specific interactions between domains. We hypothesize that these interactions were refined during millions of years of co-evolution of the receptors with their cognate ligands under particular intramolecular, intermolecular and physiological constraints.

Melanin-concentrating hormone induces neurite outgrowth in human neuroblastoma SH-SY5Y cells through p53 and MAPKinase signaling pathways.

Melanin-concentrating hormone (MCH) peptide plays a major role in energy homeostasis regulation. Little is known about cellular functions engaged by endogenous MCH receptor (MCH-R1). Here, MCH-R1 mRNA and cognate protein were found expressed in human neuroblastoma SH-SY5Y cells. Electrophysiological experiments demonstrated that MCH modulated K(+) currents, an effect depending upon the time of cellular growth. MCH treatments induced a transient phosphorylation of MAPKinases, abolished by PD98059, and partially blocked by PTX, suggesting a Galphai/Galphao protein contribution. MCH stimulated expression and likely nuclear localization of phosphorylated p53 proteins, an effect fully dependent upon MAPKinase activities. MCH treatment also increased phosphorylation of Elk-1 and up-regulated Egr-1, two transcriptional factors targeted by the MAPKinase pathway. Finally, MCH provoked neurite outgrowth after 24h-treatment of neuroblastoma cells. This effect and transcriptional factors activation were partly prevented by PD98059. Collectively, our results provide the first evidence for a role of MCH in neuronal differentiation of endogenously MCH-R1-expressing cells via non-exclusive MAPKinase and p53 signaling pathways.

Melanin-concentrating hormone and melanin-concentrating hormone receptors in mammalian skin physiopathology.

To date, there is a dearth of evidence to support functions for melanin-concentrating hormone (MCH) and melanin-concentrating hormone receptors (MCH-R) in mammalian skin physiology including pigmentation, inflammation and immune responses and skin cell proliferation. Much research is therefore still needed to define the roles of the hormone and its receptors in mammalian skin. This will be a crucial step to identifying pathogenic mechanisms that may involve the MCH/MCH-R system in the context of inflammatory and autoimmune skin diseases as well as skin cancers. The following review summarizes the studies which have been carried out to examine the expression and function of MCH and MCH-R in mammalian skin. Recent findings with regard to humoral immune responses to the MCH-R1 in patients with the skin depigmenting disease vitiligo are also discussed.

Lead optimization of melanin concentrating hormone receptor 1 antagonists with low hERG channel activity.

The discovery of small molecule melanin concentrating hormone receptor (MHCr1) antagonists as novel therapeutic agents has been widely pursued across the pharmaceutical industry. While multiple chemotypes of small molecule MCHr1 antagonists have been identified and shown to induce weight loss in rodent models of obesity, many of these lead compounds have been found to cross react with the hERG channel. This review describes efforts that led to the identification of two sub-series of MCHr1 antagonists with low affinity for the hERG channel. Ultimately, however, the modifications introduced to thwart hERG channel activity resulted in lead compounds with sub-optimal CNS behavior.

Peptide ligands for the melanin-concentrating hormone (MCH) receptor 1.

The melanin-concentrating hormone receptor 1 (MCH-1R) has been recognized as a receptor which mediates effects of the endogenous melanin-concentrating hormone (MCH) on appetite and body weight gain in rodents. In the last several years, a number of hMCH analogs have been designed which were potent and selective ligands for hMCH-1R. These peptidic agonists and antagonists have served as research tools in animal studies that showed a key role of the MCH-1R in the development of obesity and proved that MCH-1R antagonism can produce anti-obesity effects in rodents.

SAR study of bicyclo[4.1.0]heptanes as melanin-concentrating hormone receptor R1 antagonists: taming hERG.

To improve the ex vivo potency of MCH inhibitor 1a and to address its hERG liability, a structure-activity study was carried out, focusing on three regions of the lead structure. Introduction of new side chains with basic nitrogen improved in vitro and ex vivo bindings. Many potent compounds with K(i)<10nM were discovered (compounds 6a-j) and several compounds (14-17) had excellent ex vivo binding at 6h and 24h. Attenuating the basicity of nitrogen on the side chain, and in particular, introduction of a polar group such as aminomethyl on the distal phenyl ring significantly lowered the hERG activity. Further replacement of the distal phenyl group with heteroaryl groups in the cyclohexene series provided compounds such as 28l with excellent ex vivo activity with much reduced hERG liability.

Hippocampal formation: shedding light on the influence of sex and stress on the brain.

The hippocampus is a malleable brain region that responds to external agents such as hormones and stressors. Investigations that began in our laboratories with the Golgi technique and an appreciation of hippocampal neuroanatomy at the light and electron microscopic levels have led us down a path that has uncovered unexpected structural plasticity in the adult brain along with unanticipated cellular and molecular mechanisms of this plasticity and of hormone mediation of these effects. This chapter reviews the history of discoveries in our two laboratories involving the actions of estradiol and stress hormones on neuronal structure and function and then discusses the insight to hormone-brain interactions that this has engendered. These discoveries have led us to a new view of brain structural plasticity and the role and mechanism of steroid hormone action involving both genomic and non-genomic pathways. This new view is consistent with the predictions of Cajal in his book "The Structure of Ammon's horn", 1892.