The interaction of neuropeptide Y with negatively charged and zwitterionic phospholipid membranes.

The interaction of the 36 amino acid neuropeptide Y (NPY) with liposomes was studied using the intrinsic tyrosine fluorescence of NPY and an NPY fragment comprising amino acids 18-36. The vesicular membranes were composed of phosphatidylcholine and phosphatidylserine at varying mixing ratios. From the experimentally measured binding curves, the standard Gibbs free energy for the peptide transfer from aqueous solution to the lipid membrane was calculated to be around -30 kJ/mol for membrane mixtures containing physiological amounts of acidic lipids at pH 5. The effective charge of the peptide depends on the pH of the buffer and is about half of its theoretical net charge. The results were confirmed using the fluorescence of the NPY analogue [Trp(32)]-NPY. Further, the position of NPY's alpha-helix in the membrane was estimated from the intrinsic tyrosine fluorescence of NPY, from quenching experiments with spin-labelled phospholipids using [Trp(32)]-NPY, and from (1)H magic-angle spinning NMR relaxation measurements using spin-labelled [Ala(31), TOAC(32)]-NPY. The results suggest that the immersion depth of NPY into the membrane is triggered by the membrane composition. The alpha-helix of NPY is located in the upper chain region of zwitterionic membranes but its position is shifted to the glycerol region in negatively charged membranes. For membranes composed of phosphatidylcholine and phosphatidylserine, an intermediate position of the alpha-helix is observed.

Radiation dosimetry and biodistribution of 11C-ABP688 measured in healthy volunteers.

INTRODUCTION: In this study, we assessed the whole-body biodistribution and radiation dosimetry of the new glutamatergic ligand (11)C-ABP688. This ligand binds specifically to the metabotropic glutamatergic receptor of subtype 5 (mGluR5). MATERIALS AND METHODS: The study included five healthy male volunteers aged 20-29 years. After intravenous injection of 240-260 MBq, a series of four to ten whole-body positron emission tomography/computed tomography scans were initiated, yielding 60-80 min of data. Residence times were then calculated in the relevant organs, and the software packages Mirdose and Olinda were used to calculate the absorbed radiation dose and the effective dose equivalent. RESULTS: Of the excreted (11)C activity at 1 hour, approximately 80% were eliminated via the hepato-biliary pathway and 20% through the urinary tract. The absorbed dose (mGy/MBq) was highest in the liver (1.64 E (-2) +/- 5.08 E (-3)), gallbladder (8.13 E (-3) +/- 5.6 E (-3)), and kidneys (7.27 E (-3) +/- 2.79 E (-3)). The effective dose equivalent was 3.68 +/- 0.84 microSv/MBq. Brain uptake in the areas with high mGluR5 density was 2-3 (SUV). The agreement between the values obtained from Mirdose and the Olinda was excellent. CONCLUSION: (11)C-ABP688 is a very promising ligand for the investigation of mGluR5 receptors in humans. Brain uptake is high and the effective dose equivalent so low that serial examinations in the same subject seem feasible.

Evaluation of the metabotropic glutamate receptor subtype 5 using PET and 11C-ABP688: assessment of methods.

UNLABELLED: (11)C-ABP688 is a new PET ligand to assess the subtype 5 metabotropic glutamate receptor (mGlu(5)). The purpose of this study was to evaluate different methods for the analysis of human (11)C-ABP688 data acquired from 6 healthy, young volunteers. METHODS: The methods were a 1-tissue-compartment model (K(1), k(2)''), a 2-tissue-compartment model (K1-k4), and the noncompartmental method developed by Logan. Parameters related to receptor density were the total distribution volume (DV), DV'' (= K(1)/k(2)'', 1 tissue compartment); specific DV, DV(C2) (= K(1)/k(2)' x k(3)'/k(4), 2 tissue compartments); and DV(tot) for the noncompartmental method. RESULTS: The 1-tissue-compartment model was too simple to adequately fit the data. DV(C2) calculated with the 2-tissue-compartment model ranged from 5.45 +/- 1.47 (anterior cingulate) to 1.91 +/- 0.32 (cerebellum). The corresponding values for DV(tot), calculated with the 2-tissue-compartment model and the Logan method (in parentheses), were 6.57 +/- 1.45 (6.35 +/- 1.32) and 2.93 +/- 0.53 (2.48 +/- 0.40). There was no clear evidence of a region devoid of mGlu(5) receptors. The first-pass extraction fraction exceeded 95%. The minimal scan duration to obtain stable results was estimated to be 45 min. CONCLUSION: (11)C-ABP688 displays favorable kinetics for assessing mGlu(5) receptors. For tracer kinetic modeling, 2-tissue-compartment models are clearly superior to models with only 1 tissue compartment. In comparison to the compartmental models, the Logan method is equally useful if only DV(tot) values are required and fast pixelwise parametric maps are desired. The lack of regions devoid of receptors limits the use of reference region methods that do not require arterial blood sampling. Another advantage of the tracer is the fast kinetics that allow for relatively short acquisitions.

Quantitative evaluation of 11C-ABP688 as PET ligand for the measurement of the metabotropic glutamate receptor subtype 5 using autoradiographic studies and a beta-scintillator.

In this study we assessed the new glutamatergic ligand (11)C-ABP688 with regard to the following characteristics: (A) brain distribution, (B) first pass extraction fraction, (C) suitable model to describe tracer kinetics and (D) specificity for the mGlu5 receptor. These parameters were assessed using autoradiography and a beta-scintillator positioned in the striatum. The study included 13 male rats. In 2 animals cerebral blood flow was measured using H(2)(15)O. The (11)C-ABP688 data were analyzed using compartmental modeling. A two-tissue compartment model turned out to fit the data more adequately (parameters: K(1), k(2)('), k(3)('), k(4), total distribution volume DV(tot)=K(1)/k(2)(') (1+k(3)(')/k(4)) than a one-tissue compartment model. The autoradiographic studies revealed high uptake in hippocampus, striatum and cortex and low accumulation in thalamus and cerebellum. The uptake was markedly reduced following blockade with the mGlu5 antagonist M-MPEP. The first pass extraction fraction exceeded 85%. Baseline DV(tot) was 15.16+/-2.67 ml plasma/ml tissue and decreased by 56, 67 and 72% following blockade with 1, 2 and 6 mg/kg M-MPEP, respectively. These results show that (11)C-ABP688 is a promising PET ligand for the quantification of mGlu5 receptors in humans and animals. It readily crosses the blood-brain barrier and binds with high specificity to the mGlu5 receptor. The study furthermore demonstrates the usefulness of a beta-scintillator, if necessary in connection with autoradiography, to evaluate new receptor tracers.

Synthesis and preclinical evaluation of a folic acid derivative labeled with 18F for PET imaging of folate receptor-positive tumors.

UNLABELLED: Folic acid was linked regioselectively through its alpha- and gamma-carboxyl groups to 4-fluorobenzylamine (FBA), and the alpha- and gamma-FBA-folate regioisomers were evaluated for their ability to bind to folate receptor-positive cells. The 18F-labeled alpha/gamma-FBA-folate counterpart was examined for in vivo tumor targeting efficiency in nude mice bearing folate receptor-positive tumor cells. METHODS: 18F-alpha/gamma-FBA-folate was prepared in a 4-step reaction sequence starting from folic acid. The relative binding affinities of the alpha- and gamma-FBA-folates to the folate receptor with respect to parent folic acid were determined in cultured KB-31 cells (nasopharyngeal epidermal carcinoma cell line) overexpressing the folate receptor using 3H-folic acid. Tumor accumulation of the 18F-labeled alpha/gamma-FBA-folate and 18F-FDG was analyzed in vivo by high-resolution PET. Biodistribution and PET studies were performed under baseline and blockage conditions. RESULTS: The radiochemical yield of the coupling step ranged from 15% to 44%, and the maximum specific radioactivity was 24 GBq/micromol. The in vitro binding affinities of the alpha- and gamma-isomers and folic acid were 71, 62, and 41 nmol/L, respectively. PET revealed heterogeneous uptake of the radioligand, with the highest activity concentrations found in the tumor rim. In contrast, 18F-FDG uptake in a nude mouse bearing KB-31 folate receptor-positive tumors was negligible. Radioligand uptake in tumors at 125 min after injection amounted to 6.56% of the injected dose per gram of tissue (%ID/g) in control animals, whereas radioactivity accumulation in the tumors of folic acid-treated animals was significantly reduced by more than 80%-to 1.07 %ID/g (P = 0.001). CONCLUSION: This new 18F-labeled folic acid derivative is a promising tool for PET imaging of folate receptor-positive tumors.

Membrane interaction of neuropeptide Y detected by EPR and NMR spectroscopy.

Neuropeptide Y (NPY) is one of the most abundant peptides in the central nervous system of mammals. It belongs to the best-conserved peptides in nature, i.e., the amino acid sequences of even evolutionary widely separated species are very similar to each other. Using porcine NPY, which differs from human NPY only at position 17 (a leucine residue exchanged for a methionine), labeled with a TOAC spin probe at the 2nd, 32nd, or 34th positions of the peptide backbone, the membrane binding and penetration of NPY was determined using EPR and NMR spectroscopy. The vesicular membranes were composed of phosphatidylcholine and phosphatidylserine at varying mixing ratios. From the analysis of the EPR line shapes, the spectral contributions of free, dimerized, and membrane bound NPY could be separated. This analysis was further supported by quenching experiments, which selected the contributions of the bound NPY fraction. The results of this study give rise to a model where the alpha-helical part of NPY (amino acids 13-36) penetrates the membrane interface. The unstructured N-terminal part (amino acids 1-12) extends into the aqueous phase with occasional contacts with the lipid headgroup region. Besides the mixing ratio of zwitterionic and negatively charged phospholipid species, the electrostatic peptide membrane interactions are influenced by the pH value, which determines the net charge of the peptide resulting in a modified membrane binding affinity. The results of these variations indicate that NPY binding to phospholipid membranes depends strongly on the electrostatic interactions. An estimation of the transfer energy of the peptide from aqueous solution to the membrane interface DeltaG supports the preferential interaction of NPY with negatively charged membranes.

Disruption of the beta-sheet structure of a protected pentapeptide, related to the beta-amyloid sequence 17-21, induced by a single, helicogenic C(alpha)-tetrasubstituted alpha-amino acid.

Fifteen years ago it was shown that an alpha-aminoisobutyric acid (Aib) residue is significantly more effective than an L-Pro or a D-amino acid residue in inducing beta-sheet disruption in short model peptides. As this secondary structure element is known to play a crucial role in the neuropathology of Alzheimer's disease, it was decided to check the effect of Aib (and other selected, helix inducer, C(alpha)-tetrasubstituted alpha-amino acids) on the beta-sheet conformation adopted by a protected pentapeptide related to the sequence 17-21 of the beta-amyloid peptide. By use of FT-IR absorption and 1H NMR techniques it was found that the strong self-association characterizing the pentapeptide molecules in weakly polar organic solvents is completely abolished by replacing a single residue with Aib or one of its congeners.

The neuropeptide Y monomer in solution is not folded in the pancreatic-polypeptide fold.

Fluorescence-labelled analogs of NPY, a 36-amino acid peptide amide, were synthesized by solid-phase peptide synthesis and used for fluorescence-resonance energy transfer studies to investigate the conformation. Energy-transfer efficiency measurements in different media at the concentration of 10 microM are in agreement with a model of the NPY structure proposed by NMR studies (performed at millimolar concentration) in which the C-terminal part of the molecule adopts an alpha-helical conformation while the N-terminal part is flexible. According to this model, the alpha-helix is stabilized by intermolecular hydrophobic interactions because of the formation of dimers. The decrease of the peptide concentration causes a shift of the dimerization equilibrium toward the monomeric form. Energy-transfer efficiency measurements performed at lower concentrations do not support the hypothesis of the folding back of the N-terminal tail onto the C-terminal alpha-helix to yield the so-called "PP-fold" conformation. This structure is observed in the crystal structure of avian pancreatic polypeptide, a member of the NPY peptide hormone family, and it has been considered to be the bioactive one. Our results complete the structural characterization of NPY in solution at concentration ranges in which NMR experiments are not feasible. Furthermore, these results open the way to study the conformation of the receptor-bound ligand.

Electron paramagnetic resonance backbone dynamics studies on spin-labelled neuropeptide Y analogues.

Neuropeptide Y (NPY) is one of the most abundant peptides in the central nervous system of mammalians. NPY acts by binding to at least five G-protein coupled receptors (GPCRs) which have been named Y1, Y2, Y4, Y5 and Y6. Three spin-labelled NPY analogues containing the nitroxide group of the amino acid TOAC (2.2.6.6-tetramethylpiperidine-1-oxyl-4-amino-4-carboxylic acid) as a paramagnetic probe were synthesized by solid-phase peptide synthesis. Synthetic problems owing to the sensitivity of nitroxide towards acidic and reducing conditions have been overcome by using a cleavage cocktail that contains anisole and cresol scavengers. Concerning the receptor binding preferences, the analogues [TOAC34]-pNPY and [Ala31, TOAC32]-pNPY showed a marked selectivity for the Y5 receptor, while [TOAC2]-pNPY maintained a significant binding also to the Y2 receptor subtype. The modifications of the native peptide structure caused by the introduction of TOAC were examined by circular dichroism. In order to determine the rotational correlation time of the spin probes, electron paramagnetic resonance measurements were performed in solution and in the presence of liposomes. This allowed us to evaluate the backbone dynamics of the different parts of the NPY molecule in the free and membrane bound states. The results of these studies showed that NPY Interacts with liposomes by using the C-terminal alpha-helix while the N-terminal tail retains a flexibility that is comparable to that of the peptide in solution as already shown by NMR studies on DPC micelles. Furthermore, we demonstrated that TOAC-labelllng is a valuable tool to investigate changes in the backbone conformation and dynamics. This may be of major importance for peptides and small proteins when they bind to cell membranes.