Alpha-helical small molecular size analogues of neuropeptide Y: structure-activity relationships.

C-terminal analogues of neuropeptide Y (NPY) of small molecular size have been synthesized. The influence of chain length, single or multiple amino acid substitution, and segment substitutions on receptor binding, pre- and postsynaptic biological activity, and conformational properties have been investigated. Receptor binding and in vivo assays revealed biological activity for NPY Ac-25-36 that increased with increasing alpha-helicity. In attempts to stabilize the alpha-helical content, three independent types of modified NPY Ac-25-36 analogues were synthesized. Strong agonistic activities could be detected in a series of discontinuous analogues, which are constructs of N-terminal parts linked via different spacer molecules to C-terminal segments. One of the most active molecules was NPY 1-4-Aca-25-36 (Aca, epsilon-aminocaproic acid). For the first time conformational properties of a series of small NPY analogues have been investigated by CD, and correlated with biological activity and receptor binding. A C-terminal dodecapeptide segment of NPY with an amount of 50% substitution to the native C-terminal sequence of NPY was found to exhibit significant receptor binding.     

Conformational Analysis of Neuropeptide Y Segments by CD,NMR Spectroscopy and Restrained Molecular Dynamics

Neuropeptide Y (NPY), a peptide amide comprising 36 residue has been shown to act as a potent vasoconstrictor. In order to shed light on the structural requirements for the biological activities with respect to the different prerequisites for affinity to the NPY receptor subtypes Y₁ and Y₂, in the present study the syntheses and conformational analyses of two C-terminal segments, NPY(18–36) and NPY(13–36), are described. The results obtained by CD measurements, two-dimensional NMR spectros copy and a conformational refinement of the NMR-derived structure by molecular mechanics simulations support the findings of previously published structure –activity relationship studies for biologically active and selective compounds. In particular, the α-helical conformation as well as an appropriate exposure of the side chains of the critical C-terminal dipeptide within NPY(18–36) are in agreement with the prerequisites proposed for Y₂ receptor binding of that segment.     

Synthesis and biological evaluation of pNPY fragments

Peptide fragments of pNPY corresponding to the C-terminal segments (13-36) and (25-36), the N-terminal segments (1-12) and (1-24), the segments (6-14) and (7-20), which contain a putative beta-turn, and the internal segments (13-24) and (20-30) were synthesized using solid phase methodology. These fragments were assayed for NPY receptor binding activity in the rat hypothalamus membrane preparation, enhancement of food intake in the rat following ivt administration and inhibition of electrically stimulated muscle contraction in the rat vas deferens. Only the C-terminal fragment (13-36) retained some of the activities of pNPY, appearing to act as a weak agonist, having an additive effect with pNPY on the inhibition of muscle contraction and prolonging the duration of action of pNPY in the feeding assay. It also had considerable alpha-helical character, as did pNPY. None of the other peptide fragments had any agonist or antagonist activity. These results suggest that the expression of full biological NPY activity requires both the C- and the N-terminal segments as well as a putative amphiphilic alpha-helical segment (14-31).     

The contribution of helical potential to the in vitro receptor binding activity of a neuropeptide Y N-terminal deletion fragment

In its dimeric form neuropeptide Y (NPY) folds into a compact structure in which the antiparallel oriented proline and α-helices apparently associate to form a primitive hydrophobic core. To investigate the contribution of helical stability to the receptor binding activity of NPY and its N-terminal deletion fragments, we synthesized and studied the solution conformational properties and in vitro activities of NPY, N^α-acetyl-NPY_2–36, NPY_15–36, N^α-propinonly-NPY_15–36, and N^α-succinyl-NPY_15–36 is significantly less helical than both NPY and N^α-acetyl-NPY_2–36, and this decreased helical potential is attributed of the absence of the intramolecular stabilizing interaction afforded by the proline helix in the latter analogues. However, in accord with the helix dipole model, the helical potential of NPY_15–36 is significantly increased by N-terminal succinlyation, whereas propionylation has no effect. In addition to an increase in helical potential, N^α-succinyl-NPY_15–36 is 2.5 and 4.6 times more active than NPY_15–36 and N^α-propionly-NPY_15–36, respectively and is equipotent with N^α-acteyl-NPY_2–36 in displacing 1mM[³H]-NPY from specific binding sites in rat brain membranes. The demonstration of positive correlation between % α-helix content and in vitro binding activity suggests that the helical potential of N-terminal NPY deletion fragments contributes to their in vitro activity in the rat brain, and that a second role of the proline helix might be to stabilize the receptor-active conformation of the NPY α-helix. © 1993 John Wiley & Sons, Inc.     

Interaction of neuropeptide Y and Hsp90 through a novel peptide binding region

Hsp90 is a molecular chaperone that binds and assists refolding of non-native and/or labile polypeptides and also bind various peptides. However, the rules of how Hsp90 recognizes substrates have not been well characterized. By surface plasmon resonance measurements, a physiologically active peptide, neuropeptide Y (NPY), with a strong binding property to Hsp90 was identified from screening of 38 randomly selected peptide candidates. We showed that the carboxy-terminal fragment of NPY (NPY13-36), which forms an amphipathic alpha-helix structure, preserved the strong binding to Hsp90. Immunoprecipitation and immunoblotting using HeLa cell extracts revealed that newly synthesized NPY precursors bound to Hsp90, suggesting that the in vitro binding experiments identified an interactive peptide in vivo. Proteolytic cleavage of the NPY13-36/Hsp90 complex, as well as binding site analysis using deletion mutants of Hsp90, revealed the NPY binding locus on Hsp90alpha as the 192 amino acid region following the N-terminal domain. By electron microscopic analysis using an anti-Hsp90 antibody against the sequence proximal to the highly charged region, we showed that the Hsp90 dimer bound to NPY13-36 at both ends. Mutation of arginine residues in NPY13-36 to alanine abrogated binding to Hsp90. Our studies indicate that the hinge region after the N-terminal domain of Hsp90 and the positive charges on NPY are important for this interaction.     

Structure/activity relationships of C-terminal neuropeptide Y peptide segments and analogues composed of sequence 1-4 linked to 25-36

C-terminal analogues of neuropeptide Y have been synthesized. The influence of chain length, single-amino-acid substitutions and segment substitutions on receptor binding, biological activity and conformational properties has been investigated. Receptor binding and in vivo assays revealed biological activity already for amino acids 28-36 of neuropeptide Y [neuropeptide Y-(Ac-28-36)-peptide] which increased with increasing chain length. Replacement of Arg25 in neuropeptide Y-(Ac-25-36)-peptide had no influence on binding, whereas Arg33 and Arg35 cannot be replaced by lysine or ornithine without considerable decrease in receptor binding. The introduction of conformational constraints by the 2-aminoisobutyric acid residue (Aib) in position 30 and replacing the amino acids 28-32 by Ala-Aib-Ala-Aib-Ala decreased receptor binding. However, the corresponding Aib-Ala-Aib-Ala-Aib-substituted analogue and a more flexible analogue with Gly5 at position 28-32 exhibited considerable affinity for the receptor. All these substitutions led to a decrease in postsynaptic activity. Strong agonistic activities could be detected in a series of 10 discontinuous analogues, which are constructs of N-terminal parts linked via different spacer molecules to C-terminal segments. One of the most active molecules was neuropeptide Y amino acids 1-4 linked to amino acids 25-36 through aminohexanoic acid (Ahx) [neuropeptide Y-(1-4-Ahx-25-36)-peptide].     

Characterization of NPY receptor subtypes Y2 and Y7 in rainbow trout Oncorhynchus mykiss

We report the cloning and pharmacological characterization of two neuropeptide Y (NPY) receptor subtypes, Y2 and Y7, in rainbow trout (Oncorhynchus mykiss). These subtypes are approximately 50% identical to each other and belong to the Y2 subfamily of NPY receptors. The binding properties of the receptors were investigated after expression in human HEK-293 EBNA cells. Both receptors bound the three zebrafish peptides NPY, PYYa, and PYYb, as well as porcine NPY and PYY, with affinities in the nanomolar range that are similar to mammalian Y2. The affinity of the truncated porcine NPY fragments, NPY 13-36 and NPY 18-36 was markedly lower compared to mammalian and chicken Y2. This suggests that mammalian and chicken Y2 are unique among NPY receptors in their ability to bind truncated peptide fragments. The antagonist BIIE0246, developed for mammalian Y2, did not bind either of the two rainbow trout receptors. Our results support the proposed expansion of this gene family by duplications before the gnathostome radiation. They also reveal appreciable differences in the repertoire and characteristics of NPY receptors between fish and tetrapods stressing the importance of lineage-specific gene loss as well as sequence divergence after duplication.     

Y2 receptors for neuropeptide Y in the nucleus of the solitary tract mediate depressor responses.

In anesthetized, spontaneously breathing rats, microinjections of selective agonists of neuropeptide Y (NPY) receptor subtypes were made into the medial region of the caudal nucleus of the solitary tract (NTS) at the level of the area postrema. This region of the rat NTS exhibits very high densities of NPY binding sites. Microinjections of the long C-terminal NPY fragment, NPY(13-36), a selective agonist at Y2 receptors, into the caudal NTS elicited pronounced, dose-related reductions in blood pressure and respiratory minute volume. Moreover, the specific pattern of cardiorespiratory responses elicited by NPY(13-36) was remarkably similar, over approximately the same dosage range, with the cardiorespiratory response pattern elicited by intact NPY. In contrast to the potent NTS-mediated responses evoked by NPY(13-36), similar microinjections conducted with either NPY(26-36), an inactive C-terminal NPY fragment, or [Leu31,Pro34]NPY, a NPY analog with specific agonist properties at Y1 receptors, into the same caudal NTS sites did not appreciably affect cardiorespiratory parameters even at 10-20-fold higher dosages. The present results with selective agonists for NPY receptor subtypes suggest that the depressor responses and reductions in minute volume elicited by microinjections of intact NPY and NPY(13-36) were mediated by Y2 receptors in the caudal NTS, likely distributed at presynaptic sites in the medial region of the subpostremal NTS.     

Neuropeptide Y and neuropeptide Y18-36. Structural and biological characterization

Neuropeptide Y (NPY), a 36-residue peptide amide, has been shown by numerous studies to be a potent vasoconstrictor. In order to gain an appreciation of the structural requirements for this action, we have previously synthesized a number of fragments of NPY. It had been shown that sequential deletions from the N-terminus resulted in peptides with decreasing hypertensive activity. In the present study we present data supporting the unexpected finding of two fragments, NPY17-36 and NPT18-36 with substantial hypotensive action in vivo. This action was dose dependent (data not shown) and was also observed to a lesser extent with NPY19-36 but not NPY16-36 or NPY20-36. It was, however, slower in onset and of longer duration than the hypertensive action of NPY. These differing kinetics of action may suggest that NPY and NPY18-36 act through different mechanisms. Structural studies using circular dichroism were performed. While NPY was found to assume an ordered helical structure in both aqueous buffer and trifluoroethanol (TFE), 30% TFE in aqueous buffer was required to induce substantial helicity for NPY18-36. This structural investigation suggests that both NPY and NPY18-36 assume an ordered conformation upon reaching the lipid rich receptor environment.     

Effects of neuropeptide Y (NPY) at the sympathetic neuroeffector junction. Can pre- and postjunctional receptors be distinguished?

Neuropeptide Y (NPY) is widely distributed in central and peripheral neurons. In sympathetic postganglionic neurons, NPY coexists with noradrenaline. NPY and its structural relative peptide YY (PYY) appear to exert three principally different effects at the sympathetic neuroeffector junction. Firstly, NPY has a direct postjunctional effect; this effect is manifested as a vasoconstriction when studied on the guinea pig iliac vein. Secondly, NPY has an indirect postjunctional effect in that it potentiates the response to various vasoconstrictors; this was studied on the rabbit femoral artery and vein, using noradrenaline and histamine, respectively, as vasoconstrictors. Thirdly, NPY acts prejunctionally in that it suppresses the release of noradrenaline from sympathetic nerve terminals; this was studied in the rat vas deferens. The aim of the investigation was to examine whether the three effects of NPY were mediated by the same type of receptor. For this purpose, we examined the effects of a series of NPY-related peptides, namely NPY, PYY, desamido-NPY, and five C-terminal fragments (NPY 19-36, NPY 24-36, PYY 13-36, PYY 24-36 and PYY 27-36). NPY and PYY were active in all three assay systems. The C-terminal amide appears to be crucial for maintaining the biological activity, since desamido-NPY was inactive in the three test systems. Interestingly, PYY 13-36 was almost as active as NPY and PYY in suppressing the electrically evoked contractions of the vas deferens; PYY 13-36 was inactive in the two other test systems. None of the shorter fragments had any biological activity.(ABSTRACT TRUNCATED AT 250 WORDS)     

Receptor binding profiles of NPY analogues and fragments in different tissues and cell lines

To investigate receptor selectivity and possible species selectivity of a number of NPY analogues and fragments, receptor binding studies were performed using cell lines and membranes of several species. NPY displays 4–25-fold higher affinity for the Y₂ receptor than for the Y₁ receptor. The affinity of [Leu³¹,Pro³⁴]NPY is 7–60-fold higher for the Y₁ receptor when compared with the Y₂ subtype. Species selectivity within the Y₂ receptors is demonstrated by PYY(3–36), NPY(2–36), NPY(22–36), and NPY(26–36). It is shown that NPY(22–36) is species selective for the human Y₂ subtype (K_i of 0.3 nM) compared with the rabbit and rat Y₂ receptor (K_i of 2 and 10 nM, respectively). PYY(3–36) displays highest affinity for the human and rabbit Y₂ subtype (K_i of 0.03 and 0.17 nM). The screening of NPY analogues and fragments revealed that highest affinity for the human Y₂ receptor is shown by NPY(2–36) and PYY(3–36). In addition, PYY(3–36) and NPY(2–36) are not only subtype selective, but also species selective.     

Highly potent and small neuropeptide Y agonist obtained by linking NPY 1-4 via spacer to alpha-helical NPY 25-36

Analogues of neuropeptide Y (NPY) containing small N- and C-terminal segments linked via flexible spacer arms were found to exhibit receptor binding affinity constants almost as high as NPY as well as post- and presynaptic NPY-agonistic activities. One of the most active analogues contains N-terminal NPY segment 1-4 linked via epsilon-aminocaproic acid (Aca) to the C-terminal partially alpha-helical peptide amide segment 25-36. NPY 1-4-Aca-25-36 is the first highly potent NPY agonist, which is of considerably reduced size in comparison to the native hormone. The analogues are accessible by solid-phase synthesis using Fmoc strategy.     

Inhibitory and stimulatory effects of neuropeptide Y(17-36) on rat cardiac adenylate cyclase activity. Structure-function studies.

Neuropeptide Y (NPY) inhibits cardiac adenylate cyclase activity by interacting with specific receptors coupled to a pertussis toxin-sensitive G protein. Structure-activity studies revealed that only C-terminal fragments can exhibit an NPY-like inhibitory effect on 125I-NPY binding and adenylate cyclase activity of rat cardiac ventricular membranes. Although NPY(17-36) inhibited 125I-NPY binding with high potency, it produced a biphasic effect on basal (GTP, 10 and 100 microM or guanosine 5'-gamma-O-(thio)triphosphate (GTP gamma S, 10 microM) adenylate cyclase activity. Low concentrations (less than 1 nM) of NPY(17-36) inhibited the adenylate cyclase activity whereas high concentrations (greater than 1 nM) reversed this action. GTP gamma S (100 microM) reversed the biphasic effect of NPY(17-36). NPY(17-36) exhibited only a stimulatory effect in the membranes from pertussis toxin-treated rats and an inhibitory effect with membranes from cholera toxin-treated rats. Low concentrations (less than 1 nM) of NPY(17-36) inhibited isoproterenol-stimulated adenylate cyclase activity whereas high doses (greater than 1 nM) reversed this activity. The cardiac NPY receptor antagonist, NPY(18-36) (1 microM), completely blocked the biphasic effect of NPY(17-36) on isoproterenol-stimulated activity. The inhibitory dose-response curve of NPY on isoproterenol-stimulated adenylate cyclase activity was shifted parallel to the right by NPY(17-36) (1 microM), suggesting that it is an antagonist of NPY at high concentrations. N-alpha-acetylated and C-terminally deamidated analogs of NPY(17-36) had no effect on the adenylate cyclase activity. [im-DNP-His26] NPY exhibited a more pronounced biphasic effect whereas N-alpha-myristoyl-NPY(17-36) elicited only a stimulatory effect. These investigations suggest that: 1) the inhibitory and stimulatory effects of NPY(17-36) are mediated by high affinity NPY receptors coupled to a pertussis toxin-sensitive G protein and a distinct population of low affinity receptors coupled to a cholera toxin-sensitive G protein, respectively; and 2) the stimulatory effect of NPY(17-36) is dissociable.     

Structural and dynamic properties of thymopoietin mimetics

We propose a hypothesis that the T-cell receptor is a possible target of thymic hormones. We modelled the conformational dynamics of thymopentin and its structural variants in solution, as well as the interactions of these short peptides with the proposed molecular target. Thymopentin is a five-amino-acid fragment of the thymic hormone thymopoietin (residues 32 to 36) that reproduces the immunomodulatory activity of the complete hormone. Using molecular dynamics and flexible docking methods, we demonstrated high-affinity binding of thymopentin and its prospective mimetics with the T-cell receptor. The calculated biological activity spectra of thymopentin and its two promising modifications can be used in immunomodulatory activity screenings with live systems.     

Interaction of NPY compounds with the rat glucocorticoid-induced receptor (GIR) reveals similarity to the NPY-Y2 receptor

The rat glucocorticoid-induced receptor (rGIR) is an orphan G protein-coupled receptor awaiting pharmacological characterization. Among known receptors, rGIR exhibits highest sequence similarity to the neuropeptide Y (NPY)-Y(2) receptor (38-40%). The pharmacological profile of rGIR was investigated using (125)I-PYY(3-36), a Y(2)-preferring radioligand and several NPY analogs. rGIR displayed a similar displacement profile as reported for the Y(2) receptor, in that the Y(2)-selective C terminus fragments of NPY and PYY (NPY(3-36) and PYY(3-36)) showed high affinity binding and activation of rGIR (low nanomolar range). The rank order potency for displacement was NPY(3-36)>PYY(3-36)=NPY>NPY(13-36)>Ac, Leu NPY(24-36)>[D-Trp(32)]-NPY>Leu(31), Pro(34)-NPY=hPP. NPY and Y(2)-selective agonists NPY(3-36) and PYY(3-36) led to significant activation of (35)S-GTPgammaS binding to rGIR transfected cells. BIIE0246, a specific Y(2) antagonist, displaced (125)I-PYY(3-36) binding to rGIR with high affinity (95nM). Activation of (35)S-GTPgammaS binding by Y(2)-selective agonist in rGIR transfected cells was also completely abolished by BIIE0246. Our data report, for the first time, an interaction of NPY ligands with rGIR expressed in vitro, and indicate similarities between GIR and the NPY-Y(2) receptor.     

Y1 receptors for neuropeptide Y are coupled to mobilization of intracellular calcium and inhibition of adenylate cyclase

Two types of binding sites have previously been described for neuropeptide Y (NPY), called Y1 and Y2 receptors. The intracellular events following Y1 receptor activation was studied in the human neuroblastoma cell line SK-N-MC. Both NPY and the specific Y1 receptor ligand, [Leu31,Pro34]-NPY, caused a rapid and transient increase in the concentration of free calcium in the cytoplasm as measured by the fluorescent probe, Fura-2. The effect of both peptides was independent of extracellular calcium as addition of EGTA or manganese neither changed the size nor the shape of the calcium response. The calcium response to NPY was abolished by pretreatment with thapsigargin, which can selectively deplete a calcium store in the endoplasmic reticulum. Y1 receptor stimulation, by both NPY and [Leu31,Pro34]NPY, also inhibited the forskolin-stimulated cAMP production with an EC50 of 3.5 nM. There was a close relation between the receptor binding and the cellular effects as half-maximal displacement of [125I-Tyr36]monoiodoNPY from the receptor was obtained with 2.1 nM NPY. The Y2-specific ligand NPY(16-36)peptide had no effect on either intracellular calcium or cAMP levels in the SK-N-MC cells. It is concluded that Y1 receptor stimulation is associated with both mobilization of intracellular calcium and inhibition of adenylate cyclase activity.     

Neuropeptide Y and its receptor subtypes specifically modulate rat peritoneal macrophage functions in vitro: counter regulation through Y1 and Y2/5 receptors

It is well documented that neuropeptide Y (NPY) exerts a wide range of biological functions through at least five NPY Y receptor subtypes (Y1-Y5), but its immunological effects only recently came into focus. Using NPY family peptides and NPY-related receptor-specific peptides as well as Y1 and Y2 receptor antagonists, we have tested which NPY Y receptors are involved in NPY-induced modulation of rat peritoneal macrophage function in vitro. NPY and PYY increased oxidative burst in phorbol myristate acetate (PMA)-stimulated macrophages involving activation of protein kinase C (PKC), and decreased it in zymosan-stimulated cells resembling inhibition of signaling pathways subsequent to binding of zymosan particles for the iC3b fragment receptor on macrophages. The combined treatment with NPY and NPY Y receptor antagonists revealed that NPY-induced potentiation of oxidative burst in PMA-stimulated cells is mediated through Y1 and Y2 receptors, while NPY-induced suppression in zymosan-stimulated cells is mediated through Y2 receptors only. NPY-related peptides differently modulated macrophage function, confirming involvement of NPY Y2 receptor in both potentiation and suppression of oxidative burst in these cells. Additionally, it was shown that NPY Y5 receptor mediated suppression of oxidative burst in PMA- and zymosan-stimulated macrophages. Taken together, the present data reveal an NPY Y1 and Y2/Y5 receptor interaction in NPY-induced modulation of macrophage functions related to inflammation.     

Neuropeptide Y (NPY), an endogenous presynaptic modulator of adrenergic neurotransmission in the rat vas deferens: structural and functional studies

The role of neuropeptide tyrosine (NPY) on adrenergic neurotransmission was assessed in the rat vas deferens transmurally stimulated with square pulses of 0.15 or 15 Hz. Nanomoles of NPY inhibited the electrically-induced contractions on the prostatic half but not on the epididymal end of the ductus. NPY was at least 200-fold more potent than norepinephrine or adenosine to produce an equivalent inhibition. Complete amino acid sequence of NPY is required for full agonist activity; deletion of tyrosine at the amino terminus, i.e., NPY fragment 2-36 was 3-fold less potent than the native peptide. NPY fragment 5-36, 11-36 or 25-36 were proportionally less potent than NPY. Avian pancreatic polypeptide was inactive. The presynaptic nature of the NPY activity was established measuring the outflow of 3H-norepinephrine from the adrenergic varicosities of the vas deferens electrically stimulated. In this assay, NPY was more potent than NPY 2-36 or NPY fragment 5-36. No inhibitory action of NPY was detected in K+ depolarized tissues. The inhibitory effect of NPY on the rat vas deferens neurotransmission was not significantly modified by yohimbine, theophylline or naloxone, indicating that the effect of NPY is not due to the activation of alpha 2-adrenoceptors, adenosine receptors or opiate receptors respectively. Picrotoxin or apamin did not modify the inhibitory potency of NPY; verapamil or methoxyverapamil significantly reduced its potency. The inhibitory action of NPY is best explained through the activation of presynaptic NPY receptors that regulate norepinephrine release via a negative feedback mechanism. Structure activity studies give support to the notion of NPY receptors.     

Conformational and receptor binding properties of human EGF and TGF-α second loop fragments

The solution conformation of the second loop fragment of human EGF, [Ala²⁰]EGF(14–31), was determined using two-dimensional NMR homonuclear Hartmann–Hahn and rotating frame nuclear Overhauser enhancement spectroscopy. The results are compared with the conformation of the second loop fragment of human TGF-α, [Ala²¹] TGF-α(16–32), and with that of the second loop of intact EGF. Comparison of the two experimentally determined structures of the second loop fragments shows significant differences in the turn regions of each peptide. For the EGF fragment, hydrophobic side chain groups protrude away from the ring, whereas for the TGF-α fragment hydrophilic groups are directed away from the ring. Although these turn regions represent the putative receptor binding sites, neither second loop fragment binds to the EGF receptor. The biological activity is discussed in terms of the conformational differences found for the two second loop fragments.