Myriam Létourneau

Design and in vitro characterization of PAC1/VPAC1-selective agonists with potent neuroprotective effects

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a pleiotropic neuropeptide that exerts a large array of actions in the central nervous system and periphery. Through the activation of PAC1 and VPAC1, PACAP is able to exert neuroprotective, as well as anti-inflammatory effects, two phenomena involved in the pathogenesis and the progression of neurodegenerative diseases. The aim of the current study was to provide insights into the molecular arrangement of the amino terminus of PACAP and to develop new potent and selective PAC1/VPAC1 agonists promoting neuronal survival. We have synthesized a series of PACAP derivatives and measured their binding affinity and their ability to induce intracellular calcium mobilization for each receptor, i.e. PAC1, VPAC1, and VPAC2. Ultimately, analogs with an improved pharmacological profile were evaluated in an in vitro model of neuronal loss. Results showed that introduction of a hydroxyproline or an alanine moiety, respectively, at position 2 or 7 generated derivatives without significant VPAC2 agonistic activity. Moreover, the structure-activity relationship study suggests the presence of common (Asx-turn like) and distinct (different N-capping type) secondary structures that might be responsible for receptor recognition, selectivity and activation. Finally, evaluation of the neuroprotective activity of [Ala(7)]PACAP27 and [Hyp(2)]PACAP27 demonstrated their ability to protect potently human dopaminergic SH-SY5Y neuroblasts against the toxicity of MPP(+), in pre- and co-treatment experiments. These new pharmacological and structural data should prove useful for the rational design of PACAP-derived compounds that could be putative therapeutic agents for the treatment of neurodegenerative diseases.

Biochemical and pharmacological characterization of nuclear urotensin-II binding sites in rat heart

BACKGROUND AND PURPOSE During the past decade, a few GPCRs have been characterized at the nuclear membrane where they exert complementary physiological functions. In this study, we investigated (1) the presence of a functional urotensin‐II (U‐II) receptor (UT) in rat heart nuclear extracts and (2) the propensity of U‐II and U‐II‐related peptide (URP) to cross the plasma membrane in a receptor‐independent manner.

Discovery of new antagonists aimed at discriminating UII and URP‐mediated biological activities: insight into UII and URP receptor activation

Recent evidence suggested that urotensin II (UII) and its paralog peptide UII‐related peptide (URP) might exert common but also divergent physiological actions. Unfortunately, none of the existing antagonists were designed to discriminate specific UII‐ or URP‐associated actions, and our understanding, on how these two endogenous peptides can trigger different, but also common responses, is limited.

Design and characterization of novel cell-penetrating peptides from pituitary adenylate cyclase-activating polypeptide.

The discovery of cell-penetrating peptide opened up new promising avenues for the non-invasive delivery of non-permeable biomolecules within the intracellular compartment. However, some setbacks such as possible toxic effects or unexpected immunological responses have limited their use in clinic. To overcome these obstacles, we investigated the use of novel cell-penetrating peptides (CPPs) derived from the endogenous neuropeptide Pituitary adenylate cyclase-activating polypeptide (PACAP). First, we demonstrated the propensity of native PACAP isoforms (PACAP27 and PACAP38) to efficiently deliver a large and non-permeable molecule, i.e. streptavidin, into cells. An inactive modified fragment of PACAP38, i.e. [Arg(17)]PACAP(11-38), with preserved cell-penetrating physico-chemical properties, was also synthesized and successfully use for the intracellular delivery of various cargoes such as small molecules, peptides, proteins, and polynucleotides. Especially, its effectiveness as a transfection agent was comparable to Lipofectamine 2000 while being non-toxic for cells. Uptake mechanism studies demonstrated that direct translocation, caveolae-dependent endocytosis and macropinocytosis were involved in the internalization of [Arg(17)]PACAP(11-38). This study not only opened up a new aspect in the usefulness of PACAP and its derivatives for therapeutic application but also contributed to the identification of new members of the CPP family. As such, inactive PACAP-related analogs could represent excellent vectors for in vitro and in vivo applications.

Update on the urotensinergic system: new trends in receptor localization, activation, and drug design

The urotensinergic system plays central roles in the physiological regulation of major mammalian organ systems, including the cardiovascular system. As a matter of fact, this system has been linked to numerous pathophysiological states including atherosclerosis, heart failure, hypertension, diabetes as well as psychological, and neurological disorders. The delineation of the (patho)physiological roles of the urotensinergic system has been hampered by the absence of potent and selective antagonists for the urotensin II-receptor (UT). Thus, a more precise definition of the molecular functioning of the urotensinergic system, in normal conditions as well as in a pathological state is still critically needed. The recent discovery of nuclear UT within cardiomyocytes has highlighted the cellular complexity of this system and suggested that UT-associated biological responses are not only initiated at the cell surface but may result from the integration of extracellular and intracellular signaling pathways. Thus, such nuclear-localized receptors, regulating distinct signaling pathways, may represent new therapeutic targets. With the recent observation that urotensin II (UII) and urotensin II-related peptide (URP) exert different biological effects and the postulate that they could also have distinct pathophysiological roles in hypertension, it appears crucial to reassess the recognition process involving UII and URP with UT, and to push forward the development of new analogs of the UT system aimed at discriminating UII- and URP-mediated biological activities. The recent development of such compounds, i.e. urocontrin A and rUII(1–7), is certainly useful to decipher the specific roles of UII and URP in vitro and in vivo. Altogether, these studies, which provide important information regarding the pharmacology of the urotensinergic system and the conformational requirements for binding and activation, will ultimately lead to the development of potent and selective drugs.

Receptor-independent cellular uptake of pituitary adenylate cyclase-activating polypeptide

Pituitary adenylate cyclase-activating polypeptide (PACAP), a hypophysiotropic neurohormone, participates in the regulation of pleiotropic functions. The recent discovery of intracellular PACAP receptors in the brain and the testis as well as the physico-chemical characteristics of PACAP, i.e. extended α-helix containing basic residues, prompted us to evaluate the propensity of PACAP to cross the plasma membrane in a receptor-independent manner. Using confocal microscopy and flow cytometry, we demonstrated the ability of FITC-conjugated PACAP to efficiently penetrate into the internal cell compartment by direct translocation and endocytosis through clathrin-coated pits and macropinocytosis. Our study also revealed that, once inside the cells, PACAP38 is not entirely degraded by intracellular enzymes and that a significant amount of intact PACAP38 is also able to exit cells. Moreover, using binding assay on rat nuclear fractions from various tissues, PACAP nuclear receptors were identified. We also found that PACAP stimulates calcium release in rat testis nuclei. Interestingly, PACAP27 and PACAP38 but not VIP were able to upregulate de novo DNA synthesis in testis nuclei and that this effect was abolished by PACAP(6-38). These results support the presence of PAC1 receptors at the nuclear membrane and raise questions about their role in the biological activity of the peptide. These findings contribute to the characterization of PACAP as an intracrine factor and suggest that these intracellular PAC1 binding sites, probably associated with specific biological activities, should be taken into account during the development of PACAP-based drugs.

Development of photolabile caged analogs of endothelin-1

Photoactivable caged analogs of endothelin-1 (ET-1) were obtained after derivatization with the photolabile 4,5-dimethoxynitrobenzyl (DMNB) group. This was achieved by the incorporation of N-alpha-Fmoc caged building blocks of Lys, Asp, Glu and Tyr during the solid phase peptide synthesis step. The C-terminal carboxylic function was also derivatized. However, difficulties were encountered with the introduction of the Asp and Glu photoactivable building blocks. As a matter of fact, formation of an aminosuccinyl derivative, through cyclization of the Asp(ODMNB) residue, and the formation of a pyrrolidone ring from the Glu(ODMNB) residue were highly favored by the electronic properties of the photocleavable function. ET-1 analogs were also tested in the ET(A) and ET(B) paradigms and specific pharmacological profiles were obtained for each peptide.

The Expression of Multiple Connexins Throughout Spermatogenesis in the Rainbow Trout Testis Suggests a Role for Complex Intercellular Communication

Abstract Certain fish, such as rainbow trout (Oncorhynchus mykiss), are seasonal breeders. Spermatogenesis in rainbow trout is synchronous; therefore, at any time point during this process, germ cells are predominantly at the same stage of development. As such, rainbow trout represent an excellent model in which to study spermatogenesis. Gap junctions are composed of connexons, which are themselves formed by six transmembrane proteins termed connexins (Cxs). The objectives of this study were to assess which Cxs are expressed in the rainbow trout testis, and if their expression was stage specific during gonadal maturation. Rainbow trout were killed at various stages of maturation, and total cellular RNA was isolated from the testes. RT-PCR using degenerate primers recognizing all vertebrate Cxs indicates that there are several different Cxs in trout testes. Amplicons were cloned and sequenced. Homology comparisons indicate that these were cx43, cx43.4, cx31, and cx30. Immunolocalization of these Cxs indicate that Cx43 was localized primarily to Sertoli cells, while Cx43.4 was localized along the lateral plasma membranes between adjacent spermatocytes. Cx30 was localized to the interstitial Leydig cells, and Cx31 was localized primarily to the endothelium of interstitial blood vessels. The expression of each Cx varied as a function of the stage of spermatogenesis, suggesting that the expression of these proteins is highly regulated. Together, these results indicate that intercellular communication in the testis is complex, involves several different Cxs, and is a highly regulated process.

Identification by photoaffinity labeling of the extracellular N-terminal domain of PAC1 receptor as the major binding site for PACAP

Pituitary adenylate cyclase-activating polypeptide (PACAP) exerts many crucial biological functions through the interaction with its specific PAC1 receptor (PAC1-R), a class B G protein-coupled receptor (GPCR). To identify the binding sites of PACAP in the PAC1-R, three peptide derivatives containing a photoreactive p-benzoyl-phenylalanine (Bpa) residue were developed. These photosensitive PACAP analogs were fully biologically active and competent to displace radiolabeled Ac-PACAP27 from the PAC1-R. Subsequently, the (125)I-labeled photoprobes were used to anchor the PAC1-R expressed in Chinese hamster ovary cells. Photolabeling led to the formation of two protein complexes of 76 and 67 kDa, representing different glycosylated forms of the receptor. Proteinase and chemical cleavages of the peptide-receptor complexes revealed that (125)I[Bpa(0), Nle(17)]PACAP27, (125)I[Bpa(6), Nle(17)]PACAP27 and (125)I[Nle(17), Bpa(22)]PACAP27 covalently labeled the Ser(98) - Met(111) segment, the Ser(124) - Glu(125) dipeptide and the Ser(141) - Met(172) fragment, respectively. Taking into account the topology of the PAC1-R, these segments are mainly located within the extracellular N-terminal domain, indicating that this PAC1-R domain is the major binding site of PACAP27. The present study constitutes the first characterization of the binding domains of PACAP to its specific receptor and suggests heterogeneity within the binding mode of peptide ligands to class B GPCRs.

Urocontrin, a novel UT receptor ligand with a unique pharmacological profile

In recent years, several studies have demonstrated that urotensin II (UII) and urotensin II-related peptide (URP) can exhibit differential biological activity. So far, known antagonists of the urotensin II receptor (UT) are of limited usefulness for investigating the specific pathophysiological role of UII or URP. Therefore, identification of new compounds able to discriminate UII- and URP-associated biological activities is crucially needed. In the present study, we report preliminary data regarding the pharmacological properties of a novel UT ligand termed urocontrin, i.e. [Bip(4)]URP, that is able to reduce the ex vivo efficacy of hUII- but not URP-induced vasoconstriction in rat aortic rings. In vivo studies support the pharmacological profile described above. Although urocontrin exert some residual agonist activity, this compound should be useful for the rational design of potent molecules that would allow discriminating specific biological action mediated by UII or URP.