Johnny Cnudde

Development of High Affinity Selective VIP1 Receptor Agonists

The biological effects of VIP are mediated by at least two VIP receptors: the VIP1 and the VIP2 receptors that were cloned in rat, human and mice. As the mRNA coding for each receptor are located in different tissues, it is likely that each receptor modulates different functions. It is therefore of interest to obtain selective agonists for each receptor subtype. In the present work, we achieved the synthesis of two VIP1 receptor selective agonsits derived from secretin and GRF. [R16]chicken secretin had IC50 values of binding of 1,10,000, 20, and 3000 nM for the rat VIP1-, VIP2-, secretion- and PACAP receptors, respectively. This peptide, however, had a weaker affinity for the human VIP1 receptor (IC50 of 60 nM). The chimeric, substituted peptide [K15, R16, L27]VIP(1-7)/GRF(8-27) had IC50 values of binding of 1,10,000, 10,000 and 30,000 nM for the rat VIP1-, VIP2-, secretin- and PACAP receptors, respectively. Furthermore, its also showed an IC50 of 0.8 nM for the human VIP1 receptor and a low affinity for the human VIP2 receptor. It is unlikely that this GRF analogue interacted with a high affinity to the pituitary GRF receptors as it did not stimulate rat pituitary adenylate cyclase activity. The two described analogues stimulated maximally the adenylate cyclase activity on membranes expressing each receptor subtype.

In Vitro Properties of a High Affinity Selective Antagonist of the VIP1 Receptor

A selective high affinity VIP1 receptor antagonist [Acetyl-His1, D-Phe2, Lys15, Arg16, Leu17] VIP(3-7)/GRF(8-27) or PG 97-269 was synthesized, by analogy with recently obtained selective VIP1 receptor agonists. The properties of the new peptide were evaluated on Chinese hamster ovary (CHO) cell membranes expressing either the rat VIP1-, rat VIP2- or the human VIP2-recombinant receptors and on LoVo cell membranes expressing exclusively the human VIP1 receptor. The IC50 values of 125I-VIP binding inhibition by PG 97-269 were 10, 2000, 2 and 3000 nM on the rat VIP1-, rat VIP2-, human VIP1- and human VIP2 receptors, respectively. PG 97-269 had a negligible affinity for the PACAP I receptor type. It did not stimulate adenylate cyclase activity, but inhibited competitively effect of VIP on the VIP1 receptor mediated stimulation of adenylate cyclase activity. The Ki values were respectively of 15 +/- 5 nM and 2 +/- 1 nM for the rat and human VIP1 receptors. Thus the described molecule in the first reported VIP antagonist with an affinity in the nM range and with a high selectivity for the VIP1 receptor subclass. It may be useful for evaluation of the physiological role of VIP in rat and human tissues.

Development of selective agonists and antagonists for the human vasoactive intestinal polypeptide VPAC2 receptor

Ro 25-1553 is a cyclic VIP derivative with a high affinity for the VPAC(2) receptor subtype. Our goal was to identify the modifications that support its selectivity for VPAC(2) receptors, and to develop a VIP or Ro 25-1553 analog behaving as a high affinity, VPAC(2) selective antagonist. The selectivity of Ro 25-1553 for the human receptor was supported mainly by the acetylation of the amino-terminus, by the introduction of a lysine residue in position 12, and by the carboxyl-terminal extension. The lactam bridge created between positions 21 and 25 contributed to the affinity of the compound for the VIP receptors but participated only marginally to its selectivity. Deletion of the first five aminoacid residues led to a low affinity antagonist with a low selectivity. Introduction of a D-Phe residue in position 2 reduced the affinity, the selectivity and the intrinsic activity, the compound being a partial agonist. Myristoylation of the amino-terminus of [K(12)]VIP(1-26) extended carboxyl-terminally with the -K-K-G-G-T sequence of Ro 25-1553 led to a high affinity, selective VPAC(2) receptor antagonist. This molecule represents the first selective human VPAC(2) receptor antagonist described to date.

Interaction of lipophilic VIP derivatives with recombinant VIP1/PACAP and VIP2/PACAP receptors

Stearyl vasoactive intestinal polypeptide has been reported to be a VIP (vasoactive intestinal polypeptide) receptor agonist of high potency with an original bioavailability and action. We synthesized three fatty acyl derivatives, myristyl-, palmityl- and stearyl-[Nle17]VIP, and tested their capacity to recognize recombinant rat- and human VIP1- and VIP2/PACAP (pituitary adenylate cyclase-activating polypeptide) receptors and to stimulate adenylate cyclase activity. The three lipophilic analogues bound with high affinity (from 0.5 to 20 nM) to both receptor subtypes but did not distinguish between them. In preparations expressing a high density of human VIP1/PACAP receptors, the three lipophilic analogues had the same efficacy as VIP and [Nle17]VIP. In preparations expressing the rat receptors, stearyl-[Nle17]VIP had a lower efficacy than the other peptides tested. In preparations expressing a low level of VIP1/PACAP receptors and in those expressing VIP2/PACAP receptors, all analogues behaved like partial agonists. The lowest efficacy was observed for stearyl-[Nle17]VIP on the VIP2/PACAP receptor subclass. Based on our results, a complex pattern of in vivo biological effects of the lipophilic VIP derivatives should be expected: these compounds might behave as full agonists, partial agonists, or antagonists of the VIP response, depending on the number and the subtype of receptor expressed.

Analogues of VIP, Helodermin, and PACAP Discriminate between Rat and Human VIP1 and VIP2 Receptorsa

Abstract: Vasoactive intestinal polypeptide (VIP) acts through interaction with two subclasses of seven transmembrane G protein‐coupled receptors named VIP1 and VIP2 receptors. These receptors have been cloned in different species, such as rat and human. Considering the different distribution of both receptor subclasses, there is considerable interest in the development of selective agonists and antagonists. The present study compares the binding properties of VIP, PACAP, GRF, secretin, and helodermin analogues on recombinant rat and human VIP1 and VIP2 receptors. On both rat and human receptors, secretin and GRF had a higher affinity for the VIP1 receptor subtypes. The amino‐shortened VIP, and the carboxy terminal‐shortened VIP and PACAP analogues also presented a higher affinity for the VIP1 receptor. PHI, PHV, helodermin, and helospectin were selective for the human VIP2 receptor subtypes. These results suggest that the helical structure of the carboxy terminal end is necessary for VIP2 recognition. The differences between species were the following: PHI, PHV, helodermin, and helospectin had a higher affinity for the rat VIP1 receptor than for the human VIP1 receptor. On both rat and human receptors, D‐Ala4 VIP and D‐Phe4 VIP had a high affinity for the VIP1 receptor and a low affinity for the VIP2 receptor. Thus, three domains of the ligand involved in VIP1/VIP2 receptor discrimination were identified: the amino acid residue in position 4 ([D‐Ala4], [D‐Phe4]VIP), in positions 8 and 9 (the effects of helodermin and helospectin), and the carboxy terminal end (the effects of the shortened VIP and pituitary adenylate cyclase activating polypeptide analogues).

Vasoactive intestinal peptide modification at position 22 allows discrimination between receptor subtypes

Secretin and growth hormone releasing factor (GRF) have a weak affinity for VIP (vasoactive intestinal peptide)/PACAP (pituitary adenylate cyclase activating polypeptide) receptors, but discriminate between VIP1/PACAP and VIP2/PACAP receptors. This previously allowed us to develop modified secretin and GRF derivatives as high affinity and highly selective VIP1/PACAP receptor ligands. We tested the hypothesis that the presence of a Gln residue at position 24 and a Leu residue at position 22 was responsible for their VIP1/PACAP receptor selectivity. [Gln24]VIP was not different from VIP but [Leu22]VIP had a 100-fold lower affinity for VIP2/PACAP receptors as compared to VIP1/PACAP receptors. The substitution of Tyr22 by Phe22 in VIP had no significant effect on the recognition of both receptors but [Ala22]VIP had a reduced affinity for the VIP2/PACAP receptor. This indicated that an aromatic residue at position 22 of VIP was required for a high affinity for the VIP2/PACAP receptor but not for the VIP1/PACAP receptor.

Mutational and Cysteine Scanning Analysis of the Glucagon Receptor N-terminal Domain

The glucagon receptor belongs to the B family of G-protein coupled receptors. Little structural information is available about this receptor and its association with glucagon. We used the substituted cysteine accessibility method and three-dimensional molecular modeling based on the gastrointestinal insulinotropic peptide and glucagon-like peptide 1 receptor structures to study the N-terminal domain of this receptor, a central element for ligand binding and specificity. Our results showed that Asp63, Arg116, and Lys98 are essential for the receptor structure and/or ligand binding because mutations of these three residues completely disrupted or markedly impaired the receptor function. In agreement with these data, our models revealed that Asp63 and Arg116 form a salt bridge, whereas Lys98 is engaged in cation-π interactions with the conserved tryptophans 68 and 106. The native receptor could not be labeled by hydrophilic cysteine biotinylation reagents, but treatment of intact cells with [2-(trimethylammonium)ethyl]methanethiosulfonate increased the glucagon binding site density. This result suggested that an unidentified protein with at least one free cysteine associated with the receptor prevented glucagon recognition and that [2-(trimethylammonium)ethyl]methanethiosulfonate treatment relieved this inhibition. The substituted cysteine accessibility method was also performed on 15 residues selected using the three-dimensional models. Several receptor mutants, despite a relatively high predicted cysteine accessibility, could not be labeled by specific reagents. The three-dimensional models show that these mutated residues are located on one face of the protein. This could be part of the interface between the receptor and the unidentified inhibitory protein, making these residues inaccessible to biotinylation compounds.

Two tyrosine residues in the first transmembrane helix of the human vasoactive intestinal peptide receptors play a role in supporting the active conformation

We investigated the human vasoactive intestinal polypeptide (VIP) receptors VPAC1 and VPAC2 mutated at conserved tyrosine residues in the first transmembrane helix (VPAC1 receptor Y146A and Y150A and VPAC2 receptor Y130A and Y134A). [125I]‐Acetyl‐His1 [D‐Phe2, K15, R16, L27]‐VIP (1–7)/GRF (8–27) (referred to as [125I]‐VPAC1 antagonist) labelled VPAC1 binding sites, that displayed high and low affinities for VIP (IC50 values and per cent of high affinity binding sites: wild‐type, 1 nM (57±9%) and 160 nM; Y146A, 30 nM (40±8%) and 800 nM; Y150A, 4 nM (27±8%) and 300 nM). [R16]‐VIP behaved as a ‘super agonist’ at both mutated VPAC1 receptors and the efficacies of VIP analogues modified in positions 1, 3 and 6 were significantly decreased. VIP was less potent at the Y130A and Y134A mutated VPAC2 receptors (EC50 200 and 400 nM, respectively) than at the wild‐type VPAC2 receptor (EC50 7 nM). Furthermore, [hexanoyl‐His1]‐VIP behaved as a ‘super agonist’ at the two mutated VPAC2 receptors, and VIP analogues modified in positions 1, 3 and 6 were less potent and efficient at the mutated than at wild‐type VPAC2 receptors. However, the Y130A and Y134A mutants could not be studied in binding assays Our results suggest that the conserved tyrosine residues do not interact directly with the VIP His1, Asp3 or Phe6 residues (that are necessary for receptor activation), but stabilize the correct active receptor conformation.

VPAC1 receptors have different agonist efficacy profiles on membrane and intact cells

The vasoactive intestinal peptide receptor VPAC(1) is preferentially coupled to G(alpha s) protein but also increases [Ca(2+)](i) through interaction with G(alpha i)/G(alpha q) protein. We evaluated a panel of full, partial and null agonists for their capability to stimulate adenylate cyclase activity in both intact cells and membrane and [Ca(2+)](i) in intact cells transfected with the reporter gene aequorin. In intact cells, the agonists efficacy for cAMP and calcium increase were well, but not linearly correlated: VPAC(1) receptors activated G(alpha s) protein more efficiently but with the same pharmacological profile as the other G proteins. In contrast, there was a difference between cAMP increase in intact and broken cell membranes: EC(50) values were generally lower in intact cells whereas the efficacy was higher. There was, however, no correlation between the shift in the EC(50) value and the intrinsic activity. Of interest, the (4-28) fragment, a reported antagonist on cell membrane, was a full agonist in intact cells. We concluded that the active states of the VPAC(1) receptor resulting from the coupling to different effector are undistinguishable by the VIP analogs tested but that receptor properties are different when evaluated in intact cells or cell membranes.

Properties of a recombinant human secretin receptor: A comparison with the rat and rabbit receptors

A secretin receptor was cloned from a commercial human pancreatic complementary DNA (cDNA) bank. The amino acid sequence deduced from the nucleotide sequence differed slightly from the three different sequences previously published, suggesting a genetic polymorphism of the human receptor. The binding properties of the receptor were evaluated by testing natural secretin, related peptides, and synthetic analogs or fragments on membranes of Chinese hamster ovary (CHO) cells expressing the receptor after transfection. The second-messenger coupling was evaluated by adenylate cyclase measurement. The human secretin receptor was compared with the rat and the rabbit receptors. In the three animals species, rat and human secretin were equipotent; rabbit secretin was equipotent on human and rabbit secretin receptors and less potent on the rat receptor. Similar data were obtained for the [Arg16]-secretin analog. Deletion of histidine 1 and replacement of aspartate 3 reduced the affinity of the peptides for the three receptors; however, the reduction was more pronounced on rat than on human and rabbit secretin receptors. Finally, the low affinity of the rat and human receptors for vasoactive intestinal peptide (VIP) was identical; the rabbit receptor, however, had a 20-fold higher affinity. Thus the human secretin receptor shows properties of both rat and rabbit receptors. Evaluation of the properties of chimeric receptors will be useful to fit the ligand on the receptors.