Structure activity studies of mast cell activation and hypotension induced by neuropeptide Y (NPY), centrally truncated and C‐terminal NPY analogues

Abstract

1 Neuropeptide-induced histamine release is thought to occur via receptor-independent mechanisms, with net charge and lipophilicity being important factors. 2 In this study, the histamine releasing ability of neuropeptide Y (NPY), two C-terminal segments of NPY and 13 centrally truncated NPY analogues was examined. These results were compared with the ability of the peptides to bind to the Y2 receptor in the rabbit kidney membrane model and with their hypotensive actions in the anaesthetized-rat model. 3 All analogues tested, with the exception of [Glu4,25'33,35]-NPY(1-4)-Ahx-(25-36) and [Asp4'25'33'35]NPY(1-4)-Ahx-(25-36) which were devoid of histamine releasing activity, evoked a dosedependent histamine release but there were marked differences between the peptides. The native peptide was the least active. 4 Histamine release was not linked to the ability of the peptides to displace NPY from Y2 receptors.There was a statistical correlation between the hypotensive effects expressed as ED10 values (pmol kg-', which induced a blood pressure decrease of 10 mmHg) and the EC25 for histamine release (r=0.62, P=0.04), although histamine release may not be the sole determinant of the alterations in blood pressure.5 There was a strong negative correlation between EC25 for histamine release and net positive charge (r = -0.93, P = 5.7 x 10-7), i.e. increasing the net positive charge caused greater histamine release.However, there was a 12 fold difference in activity amongst the most positively charged analogues (+ 5). Helicity did not correlate with histamine releasing ability. 6 In the development of NPY-related drugs the avoidance of compounds with net positive charge is recommended.

Neuropeptides

“…[86][87][88][89] Several neuropeptides (eg, SP, VIP, and neuropeptide Y) can bind to and activate mast cells, resulting in degranulation and histamine release. [90][91][92] Furthermore, Ansel and coworkers 93 demonstrated that mast cell TNF-␣ mRNA is selectively upregulated by SP in a dose-dependent manner. Substance P increased TNF-␣ secreted from cloned murine mast cells and freshly isolated peritoneal mast cells.…”

Design of Y2 Receptor Selective and Proteolytically Stable PYY3–36 Analogues

“…Although peptides 35−37 were improved with respect to Y 2 receptor selectivity against the Y 1 or the Y 5 receptor, the Y 2 receptor affinity was lower compared to peptide 28 and 29, with peptide 37 being the best with a Y 2 receptor binding of 2.0 nM and potency of 7.6 nM. This could be slightly improved to 1.3 nM (binding) and 2.0 nM (potency) if Ser 3 and Ile 28,31 in NPY were replaced with Ile 3 , Leu 28 , and Val 31 in PYY, as represented by peptide 38, but the achieved selectivity versus the other receptors was lower than for peptides 28 and 29. A more recent truncated analogue, Abz-PYY 25−36 14 (34), notably displayed very increased selectivity against the Y 1 and Y 5 receptor, with a ratio of Y 1 /Y 2 and Y 5 /Y 2 of approximately 2500 and 650.…”

“…The potency of [Trp 30 ]PYY 3−36 (27) follows that of the binding data with equal Y 2 receptor potency as PYY 3−36 but increased selectivity toward the Y 1 receptor of approximately 50-fold, however, with no improvement in Y 5 receptor selectivity. By combining Trp 30 with either MeArg 35 or β-homoArg 35 , we obtained two analogues [Trp 30 ,MeArg 35 ]-PYY 3−36 (28) and [Trp 30 ,β-hArg 35 ]PYY 3−36 (29), that showed even greater selectivity than the analogues with one substitution while good Y 2 receptor affinity and potency were maintained. For the analogue [Trp 30 (29), modest improvement in selectivity and Y 2 receptor affinity was observed for the combination of Trp 30 and β-hArg 35 (Table 4).…”

“…In Vitro Binding for the Mouse Receptors. The four analogues [MeArg 35 ]PYY 3−36 (4), [hArg 35 ]PYY 3−36 (10), [Trp 30 ,MeArg 35 ]PYY 3−36 (28), and [Trp 30 ,β-hArg 35 ]PYY 3−36 (29) were also tested against the mouse NPY receptors (Table 5). The two most potent and selective analogues, [Trp 30 ,MeArg 35 ]PYY 3−36 (28) and [Trp 30 ,β-hArg 35 ]PYY 3−36 (29), showed an affinity for mouse Y 2 receptor of 0.5 and 1 nM, respectively, which is similar to that of PYY 3−36 (0.4 nM).…”

“…While many truncated analogues may display improved selectivity compared to PYY 3−36 , the pI of these shortened Y 2 receptor agonists is very high (approximately pI 12), and this imposes a potential risk of injection site reactions, as reported earlier with truncated NPY analogues. 28 With the aim to identify proteolytically stable and Y 2 receptor selective analogues that can be the basis for the development of longacting Y 2 receptor selective analogues for the treatment of obesity, we generated a structure−activity relationship around the C-terminal part of PYY 3−36 . We decided to use the PYY 3−36 scaffold for the design of PYY 3−36 analogues with particular attention to the very C-terminal portion of PYY 3−36 , since this is the essential part docking into the deep receptor binding pocket of the Y 2 receptor.…”