Fluorescent-labeled bioconjugates of the opioid peptides biphalin and DPDPE incorporating fluorescein-maleimide linkers.

AIM: The conjugation of fluorescent labels to opioid peptides is an extremely challenging task, which needs to be overcome to create new classes of probes for biological assays. MATERIALS & METHODS: Three opioid peptide analogs of biphalin and [D-Pen2,5]-Enkephalin (DPDPE) containing a fluorescein-maleimide motif were synthesized. RESULTS & DISCUSSION: The biphalin analog 17 binds to opioid receptors with Kiµ = 530 ± 90 nM and Kiδ = 69.8 ± 16.4 nM. We then tested the ability of the compounds to stimulate G-protein-coupling, 17 activated µ-receptor expressing cells (EC50 = 16.7 ± 6.7 nM, EMax = 76 ± 4%) as well as δ-receptor expressing cells (EC50 = 42 ± 10 nM, EMax = 34 ± 8%). However, 17 was not able to fluorescently label receptor in live or fixed cells. CONCLUSION: Our data suggest that the biphalin scaffold could be employed to develop fluorescent ligands with the appropriate fluorescent motif, and suggest a means for further probe development.

In vitro and in vivo efficacy of a potent opioid receptor agonist, biphalin, compared to subtype-selective opioid receptor agonists for stroke treatment.

To meet the challenge of identification of new treatments for stroke, this study was designed to evaluate a potent, nonselective opioid receptor (OR) agonist, biphalin, in comparison to subtype selective OR agonists, as a potential neuroprotective drug candidate using in vitro and in vivo models of ischemic stroke. Our in vitro approach included mouse primary neuronal cells that were challenged with glutamate and hypoxic/aglycemic (H/A) conditions. We observed that 10nM biphalin, exerted a statistically significant neuroprotective effect after glutamate challenge, compared to all selective opioid agonists, according to lactate dehydrogenase (LDH) and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays. Moreover, 10nM biphalin provided superior neuroprotection after H/A-reoxygenation compared to selective opioid agonists in all cases. Our in vitro investigations were supported by in vivo studies which indicate that the nonselective opioid agonist, biphalin, achieves enhanced neuroprotective potency compared to any of the selective opioid agonists, evidenced by reduced edema and infarct ratios. Reduction of edema and infarction was accompanied by neurological improvement of the animals in two independent behavioral tests. Collectively these data strongly suggest that concurrent agonist stimulation of mu, kappa and delta ORs with biphalin is neuroprotective and superior to neuroprotection by activation of any single OR subtype.

Characterization of cyclic peptides containing disulfide bonds.

Unlike linear peptides, analysis of cyclic peptides containing disulfide bonds is not straightforward and demands indirect methods to achieve a rigorous proof of structure. Three peptides that belong to this category, p-Cl-Phe-DPDPE, DPDPE, and CTOP, were analyzed and the results are presented in this paper. The great potential of two dimensional NMR and ESI tandem mass spectrometry was harnessed during the course of peptide characterizations. A new RP-HPLC method for the analysis of trifluoroacetic acid is also presented. It is robust, simple, and efficient compared to the currently available methods.

Lipid bilayers on polyacrylamide brushes for inclusion of membrane proteins.

The ability of neutral polymer cushions to support neutral lipid bilayers for the incorporation of mobile transmembrane proteins was investigated. Polyacrylamide brush layers were grown on fused silica using atom-transfer radical polymerization to provide polymer layers of 2.5-, 5- and 10-nm thickness. Lipid bilayers composed of POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine) were formed by vesicle fusion onto bare fused silica and onto each of the polyacrylamide layers. Bilayer fluidity was assessed by the diffusion of a probe, NBD-labeled phosphatidylcholine, using fluorescence recovery after photobleaching. A transmembrane protein, the human delta-opioid receptor, was inserted into each lipid bilayer, and its ability to bind a synthetic ligand, DPDPE, cyclic[2-d-penicillamine, 5-d-penicillamine]enkephalin, was detected using single-molecule fluorescence spectroscopy by labeling this ligand with a rhodamine dye. The transmembrane protein was observed to bind the ligand for all bilayers tested. The protein's electrophoretic mobility was probed by monitoring the fluorescence from the bound ligand. The 5-nm polyacrylamide thickness gave the fastest diffusion for the fluorescent lipid probe (D(1) = 2.0(+/-1.2) x 10(-7) and D(2) = 1.2(+/-0.5) x 10(-6) cm(2)/s) and also the largest electrophoretic mobility for the transmembrane protein (3 x 10(-8) cm(2)/V.s). The optimum in polymer thickness is suggested to be a tradeoff between decoupling from the substrate and increasing roughness of the polymer surface.

Effect of peptide conformation on membrane permeability.

The effect of peptide conformational constraint on the peptide permeation across the model membranes was examined by determining the permeability of pairs of cyclic and acyclic peptides related to c[d-Pen2, d-Pen5] enkephalin (DPDPE). The peptides were cyclized by formation of an intramolecular disulfide bridge between the second and fifth residues composed of either d-penicillamine or cysteine. In each case the acyclic peptide was three to seven times more permeable than corresponding cyclic peptide. The possibility that the differences in permeability of cyclic and acyclic peptides is based on the greater conformational freedom of the acyclic peptides in the presence of membrane was examined in more detail by isothermal titration calorimetric studies of Trp6-DPDPE and its acyclic analog. The membrane binding of the acyclic peptide is a more exothermic process than binding of its cyclic Trp6-DPDPE. The transfer of acyclic peptide from water to membrane is an enthalpy driven process, whereas the transfer of the cyclic peptide is driven by entropy.

Simulation of the bis-(penicillamine) enkephalin in ammonium chloride solution: a comparison with sodium chloride.

In order to quantify specific ion effects, a simulation study of bis(penicllamine) enkephalin, also known as DPDPE, has been performed in aqueous ammonium chloride solution and has been compared to a previous simulation of DPDPE in aqueous sodium chloride solution. Global thermodynamics have been calculated for a model system and the solution environment around DPDPE has been characterized. Associations of ions with DPDPE have been investigated. The observed differences between sodium chloride solution and ammonium chloride solution suggest that individual cations affect the solvation and peptide binding properties of a given anion.

Interaction of enkephalin peptides with anionic model membranes.

According to the model for passive transport across the membranes, the total flow of permeant molecules is related to the product of the water-membrane partition coefficient and the diffusion coefficient, and to the water-membrane interfacial barrier. The effect of membrane surface charge on the permeability and interaction of analgesic peptide ligands with model membranes was investigated. A mixture of zwitterionic phospholipids with cholesterol was used as a model membrane. The lipid membrane charge density was controlled by the addition of anionic 1-palmitoyl-2-oleoylphosphatidylserine. Two classes of highly potent analgesic peptides were studied, c[D-Pen(2),D-Pen(5)]enkephalin (DPDPE) and biphalin, a dimeric analog of enkephalin. The effect of increased surface charge on the permeability of the zwitterionic DPDPE is a relatively modest decrease, that appears to be due to a diminished partition coefficient. On the other hand the binding of the dicationic biphalin ligands to membranes increases proportionally with increased negative surface charge. This effect translates into a significant reduction of biphalin permeability by reducing the diffusion of the peptide across the bilayer. These experiments show the importance of electrostatic effects on the peptide-membrane interactions and suggest that the negative charge naturally present in cell membranes may hamper the membrane transport of some peptide drugs, especially cationic ones, unless there are cationic transporters present.

Pharmacodynamic and pharmacokinetic characterization of poly(ethylene glycol) conjugation to met-enkephalin analog [D-Pen2, D-Pen5]-enkephalin (DPDPE).

Poly(ethylene glycol), or PEG, conjugation to proteins and peptides is a growing technology used to enhance efficacy of therapeutics. This investigation assesses pharmacodynamic and pharmacokinetic characteristics of PEG-conjugated [D-Pen2,D-Pen5]-enkephalin (DPDPE), a met-enkephalin analog, in rodent (in vivo, in situ) and bovine (in vitro) systems. PEG-DPDPE showed increased analgesia (i.v.) compared with nonconjugated form (p < 0.01), despite a 172-fold lower binding affinity for the delta-opioid receptor. [125I]PEG-DPDPE had a 36-fold greater hydrophilicity (p < 0.01) and 12% increase in the unbound plasma protein fraction (p < 0.01), compared with [(125)I]DPDPE. [125I]PEG-DPDPE had a 2.5-fold increase in elimination half-life (p < 0.01), 2.7-fold decrease in volume of distribution (p < 0.01), and a 7-fold decrease in plasma clearance rate (p < 0.01) to [125I]DPDPE. Time course distribution showed significant concentration differences (p < 0.01) in plasma, whole blood, liver, gallbladder, gastrointestinal (GI) content, GI tract, kidneys, spleen, urine, and brain (brain, p < 0.05), between the conjugated and nonconjugated forms. Increased brain uptake of [(125)I]PEG-DPDPE corresponded to analgesia data. [125I]PEG-DPDPE in brain was shown to be 58.9% intact, with 41.1% existing as [125I]DPDPE (metabolite), whereas [125I]DPDPE was 25.7% intact in the brain (at 30 min). In vitro P-glycoprotein affinity was shown for [125I]DPDPE (p < 0.01) but not shown for [125I]PEG-DPDPE. In vitro saturable uptake, with 100 microM DPDPE, was shown for [125I]PEG-DPDPE (p < 0.05). In this study, PEG-conjugated DPDPE seems to act as a prodrug, enhancing peripheral pharmacokinetics, while undergoing hydrolysis in the brain and allowing nonconjugated DPDPE to act at the receptor.

Simulations of the bis-penicillamine enkephalin in sodium chloride solution: a parameter study.

A simulation study of DPDPE in sodium chloride solution has been performed and compared with previous simulations using a different interaction potential for the ions. Both global thermodynamics as well as a characterization of association to DPDPE have been calculated. We show that the parameters used for the ions have a profound effect on the association to the peptide in 1M NaCl. The observed differences suggest that individual associations in these and previous simulations are sensitive to parameters.

Assessment of stereoselectivity of trimethylphenylalanine analogues of delta-opioid [D-Pen(2),D-Pen(5)]-enkephalin.

[D-Pen(2),D-Pen(5)]-Enkephalin (DPDPE) is an enzymatically stable delta-opioid receptor-selective peptide, which was modified by the trimethylation of the Phe(4) residue to give beta-methyl-2', 6'-dimethylphenylalanine (TMP), resulting in four conformations : (2R,3S)-beta-Phe-DPDPE, (2R,3R)-beta-Phe-DPDPE, (2R, 3S)-beta-Phe-DPDPE, and (2S,3R)-beta-Phe-DPDPE. Synthesis was by solid-phase techniques using enantiomerically pure amino acids to give the four optically pure diastereoisomer peptides. The potency and selectivity (delta- versus mu-opioid receptor) were evaluated by radioreceptor binding in rat brain, with a mu/delta ratio decrease for all TMP conformations, compared with the parent compound (DPDPE). Octanol/buffer distribution analysis showed enhanced lipophilicity of all TMP forms, with a sixfold enhancement associated with (2S,3S)-TMP. In situ vascular perfusion in anesthetized rats showed a 1.6-fold (p < 0.01) increase in the ratio of brain uptake for (2S,3S)-TMP and a 1.5-fold (p < 0.01) decrease in uptake for (2R,3R)-TMP. Saturability of (2S,3S)-TMP was shown (p < 0.01) against 100 microM unlabeled DPDPE, showing a shared nondiffusionary transport system. P-glycoprotein affinity was shown in situ for the parent and (2S,3S)-TMP (p < 0.01). Protein binding capacity of the TMP compounds in rat plasma and in situ mammalian bovine serum albumin-Ringer showed (2R,3S)-TMP and (2S,3R)-TMP with the lowest degree of protein binding (p < 0.01), and (2S,3S)-TMP and (2R,3R)-TMP with comparable affinities to DPDPE. Analgesia, via intravenous administration, showed significantly reduced (p < 0.01) end effect and time course for (2R,3R)-TMP, (2R,3S)-TMP, and (2S, 3R)-TMP as compared with DPDPE. These results demonstrate that topographical modification in a conformationally restricted peptide can significantly modulate potency and receptor selectivity, binding capacity, enzymatic stability, lipophilicity, P-glycoprotein affinity, and blood-brain barrier permeability, resulting in a change of bioavailability, and thereby provides insight for future peptide drug design.

A three-dimensional model of the delta-opioid pharmacophore: comparative molecular modeling of peptide and nonpeptide ligands.

A comparative molecular modeling study of delta-opioid ligands was performed under the assumption that potent peptide and nonpeptide agonists may have common three-dimensional (3D) arrangement of pharmacophore groups upon binding to the delta-receptor. Low-energy conformations of the agonists 7-spiroindanyloxymorphone (SIOM) and 2-methyl-4a-alpha-(3-hydroxyphenyl)-1,2,3,4,4a,5,12, 12a-alpha-octahydro-quinolino[2,3,3-g]isoquinoline (TAN-67), and a partial agonist oxomorphindole (OMI) were determined by high-temperature molecular dynamics (MD). A good spatial overlap was found for the pharmacophore groups of SIOM, TAN-67, and OMI, including the basic nitrogen, phenol hydroxyl, and two aromatic ring. Based on this overlap we proposed a 3D pharmacophore model for nonpeptide delta-opioid agonists with a distance of 7.0 +/- 1.3 A between the two aromatic rings and of 8.2 +/- 1.0 A between the nitrogen and phenyl ring. The potent and highly delta-opioid receptor selective agonist [(2S,3R)-TMT(1)]DPDPE, which shares global backbone constraints of the 14-membered disulfide cycle and a strong preference for the trans rotamer of the TMT(1) side chain, was chosen as a peptide template of the delta-opioid pharmacophore. Extensive MD simulations at 300 K with the AMBER force field were performed for [(2S,3R)-TMT(1)]DPDPE and the less potent [(2S, 3S)-TMT(1)]DPDPE analogue. Multiple MD trajectories were collected for each peptide starting from the x-ray structures of DPDPE and [L-Ala(3)]DPDPE and from models proposed in the literature. Low-energy MD conformations were filtered by the nonpeptide pharmacophore query and then directly superimposed with SIOM, OMI, and TAN-67. Two conformers of [(2S,3R)-TMT(1)]DPDPE that showed the best overlap with the nonpeptide pharmacophore (rms deviation

The effect of halogenation on blood-brain barrier permeability of a novel peptide drug.

The utility of a drug depends on its ability to reach appropriate receptors at the target tissue and remain metabolically stable to produce the desired effect. To improve central nervous system entry of the opioid analgesic [D-Pen2, L-Pen5, Phe6] Enkephalin (DPLPE-Phe), our research group synthesized analogs that had chloro, bromo, fluoro, and iodo halogens on the para positions of the phenylalanine-4 residue. This study reports on investigation of the effect of halogenation on stability, lipophilicity, and in vitro blood-brain barrier permeability of a novel enkephalin analog DPLPE-Phe. The stability of each halogenated DPLPE-Phe analog as well as the amidated and nonamidated parent peptide was tested in plasma and brain. All peptides tested had a half-time disappearance >300 min except for DPLPE-Phe-NH2, which was found to have a half-life of 30 min in plasma. Octanol/saline distribution studies indicated addition of halogens to DPLPE-Phe-OH significantly increased lipophilicity except for p-[F-Phe4]DPLPE-Phe-OH. p-[Cl-Phe4]DPLPE-Phe-OH exhibited the most pronounced increase in lipophilicity. Para-bromo and para-chloro halogen additions significantly enhanced in vitro blood-brain barrier permeability, providing evidence for improved delivery to the central nervous system.