The N‐terminal tetrapeptide segments of dermorphin (Tyr–D‐Ala–Phe–Gly–Tyr–Pro–Ser–NH2) and deltor... more The N‐terminal tetrapeptide segments of dermorphin (Tyr–D‐Ala–Phe–Gly–Tyr–Pro–Ser–NH2) and deltorphin (Tyr–D‐Ala–Phe–Asp/Glu–Val–Val–Gly–NH2) are agonists at the opioid receptors µ and δ, respectively. [D‐Arg2, Lys4]‐dermorphin‐(1–4) amide (Tyr–D‐Arg–Phe–Lys–NH2, DALDA) and [Dmt1]DALDA (where Dmt is 2′,6′‐dimethyltyrosine) are among the most potent and selective µ‐agonists reported to date, both in vitro (having picomolar µ receptor affinity) and in vivo. In this communication, conformation‐activity studies of the following four cyclic analogs of DALDA are presented and discussed: the lead peptide S2,S4‐cyclo (Tyr–D‐Cys–Phe–Cys–NH2), constrained by means of an S4.2S4.4 disulfide between Cys2 and Cys4; its two cis and trans C4.2C4.4‐olefinic dicarba analogs, and the product of saturation of them both. They are potent nonselective or moderately µ‐selective opioid agonists in vitro.They have been synthesized and tested earlier [Berezowska I, Chung NN, Lemieux C, Wilkes BC, and Schill...
Reactive oxygen species (ROS) play an important role in the development of complex regional pain ... more Reactive oxygen species (ROS) play an important role in the development of complex regional pain syndrome-Type I (CRPS-I), as also demonstrated with the chronic post ischemia pain (CPIP) animal model of CRPS-I. We show that morphine and the antioxidant N-acetylcysteine (NAC) act synergistically to reduce mechanical allodynia in CPIP rats. The tetrapeptide amide [Dmt(1)]DALDA (H-Dmt-d-Arg-Phe-Lys-NH2) is a potent and selective μ opioid receptor (MOR) agonist with favorable pharmacokinetic properties and with antioxidant activity due to its N-terminal Dmt (2',6'-dimethyltyrosine) residue. In the CPIP model, [Dmt(1)]DALDA was 15-fold more potent than morphine in reversing mechanical allodynia and 4.5-fold more potent as analgesic in the heat algesia test. The results indicate that bifunctional compounds with MOR agonist/antioxidant activity have therapeutic potential for the treatment of CRPS-I.
The retina of the human eye is extremely vulnerable to oxidative damage. Previous studies have de... more The retina of the human eye is extremely vulnerable to oxidative damage. Previous studies have demonstrated that oxidative stress is the predominant mechanism associated with the pathogenesis of age‑related macular degeneration, diabetic retinopathy, glaucoma and retinitis pigmentosa. MTP‑131, a novel mitochondria‑targeted peptide, has been demonstrated to specifically concentrate in the inner mitochondria membrane and to exhibit remarkable antioxidant effects both in vitro and in animal models. In the present study, the protective effect of MTP‑131 was evaluated in response to hydrogen peroxide (H2O2)‑induced oxidative damage in a retinal ganglion cell line, RGC‑5. Cell viability was measured by lactate dehydrogenase (LDH) assay. Changes of mitochondrial membrane potential and generation of intracellular reactive oxygen species (ROS) were measured by flow cytometry and confocal microscopy, respectively. Annexin V‑fluorescein isothiocyanate/propidium iodide staining was used for ass...
Opioid receptors are the primary sites of actions of opiates and endogenous opioid peptides, whic... more Opioid receptors are the primary sites of actions of opiates and endogenous opioid peptides, which have a wide variety of pharmacological and physiological effects. The opioid receptors are classified into at least three subtypes, p, 6, and x, and their cDNAs have been cloned. In this review, we describe the molecular cloning of opioid receptor gene family and studies of the structure-function relationships, modes of coupling to second messenger systems, pharmacological effects of antisense oligonucleotides, and anatomical distribution of opioid receptor mRNAs.
Peptide therapeutics offer numerous advantages in the treatment of diseases and disorders of the ... more Peptide therapeutics offer numerous advantages in the treatment of diseases and disorders of the central nervous system (CNS). However, they are not without limitations, especially in terms of their pharmacokinetics where their metabolic lability and low blood–brain barrier penetration hinder their application. Targeted nanoparticle delivery systems are being tapped for their ability to improve the delivery of therapeutics into the brain non-invasively. We have developed a family of mannosylated glycoliposome delivery systems for targeted drug delivery applications. Herein, we demonstrate via in vivo distribution studies the potential of these glycoliposomes to improve the utility of CNS active therapeutics using dynantin, a potent and selective dynorphin peptide analogue antagonist of the kappa opioid receptor (KOR). Glycoliposomal entrapment protected dynantin against known rapid metabolic degradation and ultimately improved brain levels of the peptide by approximately 3–3.5-fold....
The therapeutic application of peptide-based drugs is significantly limited by the rapid proteoly... more The therapeutic application of peptide-based drugs is significantly limited by the rapid proteolytic degradation that occurs when in blood. Encapsulation of these peptide structures within a delivery system, such as liposomes, can greatly improve both stability and target delivery. As part of our work focused on novel ambiphilic mannosylated neoglycolipids as targeted drug delivery systems, we have developed a C14-alkyl-mannopyranoside that forms self-assembled monodisperse liposomes. Herein, these glycoliposomes are investigated as a potential method to improve the plasma stability of peptide-based drugs. Reversed phase high-performance liquid chromatography (RP-HPLC) and mass spectrometry (MS) methods were developed to assess the in vitro plasma stability of two structurally diverse peptides, including the kappa opioid receptor selective antagonist dynantin, and the NOD2 innate immune receptor ligand muramyl dipeptide (MDP). The RP-HPLC methods developed were able to resolve the p...
Opioid receptors (ORs) precisely modulate behavior when activated by native peptide ligands but d... more Opioid receptors (ORs) precisely modulate behavior when activated by native peptide ligands but distort behaviors to produce pathology when activated by non-peptide drugs. A fundamental question is how drugs differ from peptides in their actions on target neurons. Here, we show that drugs differ in the subcellular location at which they activate ORs. We develop a genetically encoded biosensor that directly detects ligand-induced activation of ORs and uncover a real-time map of the spatiotemporal organization of OR activation in living neurons. Peptide agonists produce a characteristic activation pattern initiated in the plasma membrane and propagating to endosomes after receptor internalization. Drugs produce a different activation pattern by additionally driving OR activation in the somatic Golgi apparatus and Golgi elements extending throughout the dendritic arbor. These results establish an approach to probe the cellular basis of neuromodulation and reveal that drugs distort the ...
Administration of the handle region peptide (HRP), a (pro)renin receptor blocker, decreases body ... more Administration of the handle region peptide (HRP), a (pro)renin receptor blocker, decreases body weight gain and visceral adipose tissue (VAT) in high-fat/high-carbohydrate (HF/HC) diet-fed mice. The objective of this study was to elucidate potential mechanisms implicated in these observations. Mice were given a normal or a HF/HC diet along with saline or HRP for 10 weeks. In HF/HC-fed mice, HRP increased the expression of several enzymes implicated in lipogenesis and lipolysis in subcutaneous fat (SCF) while the expression of the enzyme implicated in the last step of lipogenesis decreased in VAT. A reduction was also observed in circulating free fatty acids in these animals which was accompanied by normalized adipocyte size in VAT and increased adipocyte size in SCF. ''Beiging'' is the evolution of a white adipose tissue toward a brown-like phenotype characterized by an increased mitochondrial density and small lipid droplets. HRP increased the expression of'…
International Journal of Peptide and Protein Research, 2009
Conformational features of a series of cyclic, penicillamine-containing enkephalin analogs, all o... more Conformational features of a series of cyclic, penicillamine-containing enkephalin analogs, all of which display selectivity for the delta opioid receptor, were studied by 'H n.m.r. in aqueous solution. Comparison of chemical shifts, coupling constants, and temperature dependence of amide proton chemical shifts suggests different conformational features among the analogs, some of which can be related to the different primary sequences of these peptides. The observation that some of the analogs display disparate individual conformational features while exhibiting similar opioid potency and receptor selectivity suggests that such analogs may share a similar overall topography or at the least maintain the same relative orientations of key portions of the molecule.
Fluorescent labels are commonly used to investigate the mechanisms of cellular uptake and intrace... more Fluorescent labels are commonly used to investigate the mechanisms of cellular uptake and intracellular distribution of cell-penetrating peptides. However, labels such as fluorescein and rhodamine are relatively large and very lipophilic and may significantly alter physicochemical properties of small peptides. To minimize the impact of the fluorescent probe on a tetrapeptide, we substituted one of the amino acids (Lys 4) in a tetrapeptide ([Dmt 1 ]DALDA, Dmt-D-Arg-Phe-Lys-NH 2 where Dmt = 2',6'-dimethyltyrosine) with two different fluorescent amino acids (βdansyl-L-α,β-diaminopropionic acid (dnsDap 4) or β-anthraniloyl-L-α,β-diaminopropionic acid (atnDap 4)). Initial studies with confocal laser scanning microscopy (CLSM) showed very different localization patterns for the two fluorescent analogs, with [Dmt 1 ,atnDap 4 ]DALDA showing mitochondrial localization and [Dmt 1 ,dnsDap 4 ]DALDA showing diffuse cytoplasmic localization. Studies with isolated mouse liver mitochondria suggested that [Dmt 1 ,dnsDap 4 ]DALDA targeted the mitochondrial matrix resulting in mitochondrial depolarization, opening of the permeability transition pore, mitochondrial swelling, and rapid release of the peptide into the cytoplasm. In contrast, [Dmt 1 ,atnDap 4 ]DALDA was retained in the inner mitochondrial membrane and did not induce mitochondrial swelling. Furthermore, [Dmt 1 ,atnDap 4 ]DALDA protected mitochondria against Ca 2+-induced swelling. Importantly, the unlabeled parent peptide [Dmt 1 ]DALDA behaved like [Dmt 1 ,atnDap 4 ]DALDA and was mitoprotective. These findings suggest that experimental results obtained with fluorescent labels must be interpreted with caution, and the use of multiple fluorophores, together with confirmation using the original or radiolabeled molecule, is recommended. Key words: membrane transduction • mitochondria • permeability transition • cytoplasm • confocal microscopy ell-penetrating peptides have recently received much attention because of their ability to penetrate cell membranes and assist in the delivery of biomolecules such as DNA, RNA, and proteins into cells (1). Fluorescent labels have generally been used to demonstrate C
Proceedings of the National Academy of Sciences, 1980
A conformationally restricted analog of [Leu5]enkephalin was synthesized by cyclization of the CO... more A conformationally restricted analog of [Leu5]enkephalin was synthesized by cyclization of the COOH-terminal carboxyl group of leucine to the gamma-amino moiety of alpha, gamma-diaminobutyric acid (A2bu) substituted in position 2 of the peptide. Relative to [Leu5]enkephalin, the cyclic analog with D configuration in position 2, H-Tyr-cyclo(-N gamma-D-A2bu-Gly-Phe-Leu-), was 17.5 times more potent in the guinea pig ileum assay and twice as potent in the rat brain receptor binding assay, whereas its diastereomer H-Tyr-cyclo(-N gamma-L-A2bu-Gly-Phe-Leu-) showed low activity. The cyclic D isomer was also slightly more active than the open-chain reference compound [D-Nva2, Leu5]enkephalinamide in both assays, and it proved to be highly resistant to degradation by brain "enkephalinases." The steric constraints introduced in H-Tyr-cyclo(-N gamma-D-A2bu-Gly-Phe-Leu-) were shown to prevent the realization of most of the conformational features ascribed to linear enkephalin in solut...
This study assessed how conformational information encoded by ligand binding to d-opioid receptor... more This study assessed how conformational information encoded by ligand binding to d-opioid receptors (DORs) is transmitted to Kir3.1/Kir3.2 channels. Human embryonic kidney 293 cells were transfected with bioluminescence resonance energy transfer (BRET) donor/acceptor pairs that allowed us to evaluate independently reciprocal interactions among signaling partners. These and coimmunoprecipitation studies indicated that DORs, Gbg, and Kir3 subunits constitutively interacted with one another. GaoA associated with DORs and Gbg, but despite being part of the complex, no evidence of its direct association with the channel was obtained. DOR activation by different ligands left DOR-Kir3 interactions unmodified but modulated BRET between DOR-GaoA, DOR-Gbg, GaoA-Gbg, and Gbg-Kir3 interfaces. Ligand-induced BRET changes assessing Gbg-Kir3.1 subunit interaction 1) followed similar kinetics to those monitoring the GaoA-Gbg interface, 2) displayed the same order of efficacy as those observed at the DOR-Gbg interface, 3) were sensitive to pertussis toxin, and 4) were predictive of whether a ligand could evoke channel currents. Conformational changes at the Gbg/Kir3 interface were lost when Kir3.1 subunits were replaced by a mutant lacking essential sites for Gbg-mediated activation. Thus, conformational information encoded by agonist binding to the receptor is relayed to the channel via structural rearrangements that involve repositioning of Gbg with respect to DORs, GaoA, and channel subunits. Further, the fact that BRET changes at the Gbg-Kir3 interface are predictive of a ligand's ability to induce channel currents points to these conformational biosensors as screening tools for identifying GPCR ligands that induce Kir3 channel activation.
Strategies for the design of bi-or multifunctional drugs are reviewed. A distinction is made betw... more Strategies for the design of bi-or multifunctional drugs are reviewed. A distinction is made between bifunctional drugs interacting in a monovalent fashion with two targets and ligands containing two distinct pharmacophores binding in a bivalent mode to the two binding sites in a receptor heterodimer. Arguments are presented to indicate that some of the so-called "bivalent" ligands reported in the literature are unlikely to simultaneously interact with two binding sites. Aspects related to the development of bi-or multifunctional drugs are illustrated with examples from the field of opioid analgesics. The drug-like properties of the tetrapeptide Dmt 1 [DALDA] with triple action as a μ opioid agonist, norepinephrine uptake inhibitor and releaser of endogenous opioid peptides to produce potent spinal analgesia are reviewed. Rationales for the development of opioid peptides with mixed agonist/antagonist profiles as analgesics with reduced side effects are presented. Progress in the development of mixed μ opioid agonist/δ opioid antagonists with low propensity to produce tolerance and physical dependence is reviewed. Efforts to develop bifunctional peptides containing a μ opioid agonist and a cholecystokinin antagonist or an NK1 receptor antagonist as analgesics expected to produce less tolerance and dependence are also reviewed. A strategy to improve the drug-like properties of bifunctional opioid peptide analgesics is presented.
3-(2,6-Dimethyl-4-carbamoylphenyl)propanoic acid (Dcp), a 2&a... more 3-(2,6-Dimethyl-4-carbamoylphenyl)propanoic acid (Dcp), a 2',6'-dimethyltyrosine analogue containing a carbamoyl group in place of the hydroxyl function and lacking the amino group, was synthesized. The replacement of Tyr1 in an enkephalin analogue and in dynorphin A(1-11)-NH2 with Dcp resulted in the first opioid peptide-derived antagonists that do not contain a phenolic hydroxyl group at the 1-position residue. The cyclic peptide Dcp-c[D-Cys-Gly-Phe(pNO2)-D-Cys]NH2 represents a novel, potent mu opioid antagonist.
The opioid peptide H-Tyr-c[D-Cys-Phe-Phe-Cys]NH 2 cyclized via a methylene dithiother is a potent... more The opioid peptide H-Tyr-c[D-Cys-Phe-Phe-Cys]NH 2 cyclized via a methylene dithiother is a potent and selective μ opioid agonist (Przydial M.J. et al., J Peptide Res, 66, 2005, 255). Dicarba analogues of this peptide with Tyr, 2′,6′-dimethyltyrosine (Dmt), 3-[2,6-dimethyl-4-hydroxyphenyl)propanoic acid (Dhp) or (2S)-2-methyl-3-(2,6-dimethyl-4-hydroxyphenyl)propanoic acid [(2S)-Mdp] in the 1position were prepared. The peptides were synthesized on solid-phase by substituting D-allylglycine and (2S)-2-amino-5-hexenoic acid in position 2 and 5, respectively, followed by ring-closing metathesis. Mixtures of cis and trans isomers of the resulting olefinic peptides were obtained, and catalytic hydrogenation yielded the saturated-CH 2-CH 2-bridged peptides. All six Tyr 1-and Dmt 1-dicarba analogues retained high μ and δ opioid agonist potency and showed only slight or no preference for μ over δ receptors. As expected, the six Dhp 1-and (2S)-Mdp 1-dicarba analogues turned out to be μ opioid antagonists but, surprisingly, displayed a range of different efficacies (agonism, partial agonism or antagonism) at the δ receptor. The obtained results indicate that the μ versus δ receptor selectivity and the efficacy at the δ receptor of these cyclic peptides depend on distinct conformational characteristics of the 15-membered peptide ring structure, which may affect the spatial positioning of the exocyclic residue and of the Phe 3 and Phe 4 side chains.
Previously, we reported that H-2 0 ,6 0-dimethyltyrosine [Dmt 1 ]-D-Arg-Phe-Lys-NH 2 (DALDA), an ... more Previously, we reported that H-2 0 ,6 0-dimethyltyrosine [Dmt 1 ]-D-Arg-Phe-Lys-NH 2 (DALDA), an analogue of the naturally occurring opioid peptide dermorphin, is a highly potent and selective mu receptor agonist with low cross-tolerance to morphine. In the present study, we investigated the effect of treating mice chronically with [Dmt 1 ]DALDA. The AD 50 of [Dmt 1 ]DALDA (s.c.) increased eightfold in animals given this drug chronically; in contrast, the AD 50 increased twofold in mice chronically treated with morphine. The AD 50 of morphine (s.c.) in these [Dmt 1 ]DALDA-treated animals was increased more than 120 times, while that of the more selective m agonist [D-Ala 2-MePhe 4-Gly-ol 5 ]enkephalin (DAMGO) given intrathecally was increased more than 240 times. However, the AD 50 of DAMGO given intracerebroventricularly was essentially the same in animals treated chronically with [Dmt 1 ]DALDA as in naı¨ve animals. The dose of naloxone required to precipitate withdrawal in [Dmt 1 ]DALDA-treated animals was 20 times lower than that in morphine-tolerant animals. 2 Using real-time quantitative PCR, we found that expression of the m opioid receptor, d opioid receptor, preproenkephalin and preprodynorphin genes was upregulated in the brain by [Dmt 1 ]DALDA treatment. No significant changes in expression of opioid receptor or opioid peptide genes were detected in the spinal cord of [Dmt 1 ]DALDA-treated mice, nor in the brain or spinal cord of morphine-treated mice. We conclude that a high degree of tolerance to [Dmt 1 ]DALDA develops in the spinal cord but not brain, and cannot be accounted for by changes in expression of opioid receptors or opioid peptides in these tissues.
The N‐terminal tetrapeptide segments of dermorphin (Tyr–D‐Ala–Phe–Gly–Tyr–Pro–Ser–NH2) and deltor... more The N‐terminal tetrapeptide segments of dermorphin (Tyr–D‐Ala–Phe–Gly–Tyr–Pro–Ser–NH2) and deltorphin (Tyr–D‐Ala–Phe–Asp/Glu–Val–Val–Gly–NH2) are agonists at the opioid receptors µ and δ, respectively. [D‐Arg2, Lys4]‐dermorphin‐(1–4) amide (Tyr–D‐Arg–Phe–Lys–NH2, DALDA) and [Dmt1]DALDA (where Dmt is 2′,6′‐dimethyltyrosine) are among the most potent and selective µ‐agonists reported to date, both in vitro (having picomolar µ receptor affinity) and in vivo. In this communication, conformation‐activity studies of the following four cyclic analogs of DALDA are presented and discussed: the lead peptide S2,S4‐cyclo (Tyr–D‐Cys–Phe–Cys–NH2), constrained by means of an S4.2S4.4 disulfide between Cys2 and Cys4; its two cis and trans C4.2C4.4‐olefinic dicarba analogs, and the product of saturation of them both. They are potent nonselective or moderately µ‐selective opioid agonists in vitro.They have been synthesized and tested earlier [Berezowska I, Chung NN, Lemieux C, Wilkes BC, and Schill...
Reactive oxygen species (ROS) play an important role in the development of complex regional pain ... more Reactive oxygen species (ROS) play an important role in the development of complex regional pain syndrome-Type I (CRPS-I), as also demonstrated with the chronic post ischemia pain (CPIP) animal model of CRPS-I. We show that morphine and the antioxidant N-acetylcysteine (NAC) act synergistically to reduce mechanical allodynia in CPIP rats. The tetrapeptide amide [Dmt(1)]DALDA (H-Dmt-d-Arg-Phe-Lys-NH2) is a potent and selective μ opioid receptor (MOR) agonist with favorable pharmacokinetic properties and with antioxidant activity due to its N-terminal Dmt (2',6'-dimethyltyrosine) residue. In the CPIP model, [Dmt(1)]DALDA was 15-fold more potent than morphine in reversing mechanical allodynia and 4.5-fold more potent as analgesic in the heat algesia test. The results indicate that bifunctional compounds with MOR agonist/antioxidant activity have therapeutic potential for the treatment of CRPS-I.
The retina of the human eye is extremely vulnerable to oxidative damage. Previous studies have de... more The retina of the human eye is extremely vulnerable to oxidative damage. Previous studies have demonstrated that oxidative stress is the predominant mechanism associated with the pathogenesis of age‑related macular degeneration, diabetic retinopathy, glaucoma and retinitis pigmentosa. MTP‑131, a novel mitochondria‑targeted peptide, has been demonstrated to specifically concentrate in the inner mitochondria membrane and to exhibit remarkable antioxidant effects both in vitro and in animal models. In the present study, the protective effect of MTP‑131 was evaluated in response to hydrogen peroxide (H2O2)‑induced oxidative damage in a retinal ganglion cell line, RGC‑5. Cell viability was measured by lactate dehydrogenase (LDH) assay. Changes of mitochondrial membrane potential and generation of intracellular reactive oxygen species (ROS) were measured by flow cytometry and confocal microscopy, respectively. Annexin V‑fluorescein isothiocyanate/propidium iodide staining was used for ass...
Opioid receptors are the primary sites of actions of opiates and endogenous opioid peptides, whic... more Opioid receptors are the primary sites of actions of opiates and endogenous opioid peptides, which have a wide variety of pharmacological and physiological effects. The opioid receptors are classified into at least three subtypes, p, 6, and x, and their cDNAs have been cloned. In this review, we describe the molecular cloning of opioid receptor gene family and studies of the structure-function relationships, modes of coupling to second messenger systems, pharmacological effects of antisense oligonucleotides, and anatomical distribution of opioid receptor mRNAs.
Peptide therapeutics offer numerous advantages in the treatment of diseases and disorders of the ... more Peptide therapeutics offer numerous advantages in the treatment of diseases and disorders of the central nervous system (CNS). However, they are not without limitations, especially in terms of their pharmacokinetics where their metabolic lability and low blood–brain barrier penetration hinder their application. Targeted nanoparticle delivery systems are being tapped for their ability to improve the delivery of therapeutics into the brain non-invasively. We have developed a family of mannosylated glycoliposome delivery systems for targeted drug delivery applications. Herein, we demonstrate via in vivo distribution studies the potential of these glycoliposomes to improve the utility of CNS active therapeutics using dynantin, a potent and selective dynorphin peptide analogue antagonist of the kappa opioid receptor (KOR). Glycoliposomal entrapment protected dynantin against known rapid metabolic degradation and ultimately improved brain levels of the peptide by approximately 3–3.5-fold....
The therapeutic application of peptide-based drugs is significantly limited by the rapid proteoly... more The therapeutic application of peptide-based drugs is significantly limited by the rapid proteolytic degradation that occurs when in blood. Encapsulation of these peptide structures within a delivery system, such as liposomes, can greatly improve both stability and target delivery. As part of our work focused on novel ambiphilic mannosylated neoglycolipids as targeted drug delivery systems, we have developed a C14-alkyl-mannopyranoside that forms self-assembled monodisperse liposomes. Herein, these glycoliposomes are investigated as a potential method to improve the plasma stability of peptide-based drugs. Reversed phase high-performance liquid chromatography (RP-HPLC) and mass spectrometry (MS) methods were developed to assess the in vitro plasma stability of two structurally diverse peptides, including the kappa opioid receptor selective antagonist dynantin, and the NOD2 innate immune receptor ligand muramyl dipeptide (MDP). The RP-HPLC methods developed were able to resolve the p...
Opioid receptors (ORs) precisely modulate behavior when activated by native peptide ligands but d... more Opioid receptors (ORs) precisely modulate behavior when activated by native peptide ligands but distort behaviors to produce pathology when activated by non-peptide drugs. A fundamental question is how drugs differ from peptides in their actions on target neurons. Here, we show that drugs differ in the subcellular location at which they activate ORs. We develop a genetically encoded biosensor that directly detects ligand-induced activation of ORs and uncover a real-time map of the spatiotemporal organization of OR activation in living neurons. Peptide agonists produce a characteristic activation pattern initiated in the plasma membrane and propagating to endosomes after receptor internalization. Drugs produce a different activation pattern by additionally driving OR activation in the somatic Golgi apparatus and Golgi elements extending throughout the dendritic arbor. These results establish an approach to probe the cellular basis of neuromodulation and reveal that drugs distort the ...
Administration of the handle region peptide (HRP), a (pro)renin receptor blocker, decreases body ... more Administration of the handle region peptide (HRP), a (pro)renin receptor blocker, decreases body weight gain and visceral adipose tissue (VAT) in high-fat/high-carbohydrate (HF/HC) diet-fed mice. The objective of this study was to elucidate potential mechanisms implicated in these observations. Mice were given a normal or a HF/HC diet along with saline or HRP for 10 weeks. In HF/HC-fed mice, HRP increased the expression of several enzymes implicated in lipogenesis and lipolysis in subcutaneous fat (SCF) while the expression of the enzyme implicated in the last step of lipogenesis decreased in VAT. A reduction was also observed in circulating free fatty acids in these animals which was accompanied by normalized adipocyte size in VAT and increased adipocyte size in SCF. ''Beiging'' is the evolution of a white adipose tissue toward a brown-like phenotype characterized by an increased mitochondrial density and small lipid droplets. HRP increased the expression of'…
International Journal of Peptide and Protein Research, 2009
Conformational features of a series of cyclic, penicillamine-containing enkephalin analogs, all o... more Conformational features of a series of cyclic, penicillamine-containing enkephalin analogs, all of which display selectivity for the delta opioid receptor, were studied by 'H n.m.r. in aqueous solution. Comparison of chemical shifts, coupling constants, and temperature dependence of amide proton chemical shifts suggests different conformational features among the analogs, some of which can be related to the different primary sequences of these peptides. The observation that some of the analogs display disparate individual conformational features while exhibiting similar opioid potency and receptor selectivity suggests that such analogs may share a similar overall topography or at the least maintain the same relative orientations of key portions of the molecule.
Fluorescent labels are commonly used to investigate the mechanisms of cellular uptake and intrace... more Fluorescent labels are commonly used to investigate the mechanisms of cellular uptake and intracellular distribution of cell-penetrating peptides. However, labels such as fluorescein and rhodamine are relatively large and very lipophilic and may significantly alter physicochemical properties of small peptides. To minimize the impact of the fluorescent probe on a tetrapeptide, we substituted one of the amino acids (Lys 4) in a tetrapeptide ([Dmt 1 ]DALDA, Dmt-D-Arg-Phe-Lys-NH 2 where Dmt = 2',6'-dimethyltyrosine) with two different fluorescent amino acids (βdansyl-L-α,β-diaminopropionic acid (dnsDap 4) or β-anthraniloyl-L-α,β-diaminopropionic acid (atnDap 4)). Initial studies with confocal laser scanning microscopy (CLSM) showed very different localization patterns for the two fluorescent analogs, with [Dmt 1 ,atnDap 4 ]DALDA showing mitochondrial localization and [Dmt 1 ,dnsDap 4 ]DALDA showing diffuse cytoplasmic localization. Studies with isolated mouse liver mitochondria suggested that [Dmt 1 ,dnsDap 4 ]DALDA targeted the mitochondrial matrix resulting in mitochondrial depolarization, opening of the permeability transition pore, mitochondrial swelling, and rapid release of the peptide into the cytoplasm. In contrast, [Dmt 1 ,atnDap 4 ]DALDA was retained in the inner mitochondrial membrane and did not induce mitochondrial swelling. Furthermore, [Dmt 1 ,atnDap 4 ]DALDA protected mitochondria against Ca 2+-induced swelling. Importantly, the unlabeled parent peptide [Dmt 1 ]DALDA behaved like [Dmt 1 ,atnDap 4 ]DALDA and was mitoprotective. These findings suggest that experimental results obtained with fluorescent labels must be interpreted with caution, and the use of multiple fluorophores, together with confirmation using the original or radiolabeled molecule, is recommended. Key words: membrane transduction • mitochondria • permeability transition • cytoplasm • confocal microscopy ell-penetrating peptides have recently received much attention because of their ability to penetrate cell membranes and assist in the delivery of biomolecules such as DNA, RNA, and proteins into cells (1). Fluorescent labels have generally been used to demonstrate C
Proceedings of the National Academy of Sciences, 1980
A conformationally restricted analog of [Leu5]enkephalin was synthesized by cyclization of the CO... more A conformationally restricted analog of [Leu5]enkephalin was synthesized by cyclization of the COOH-terminal carboxyl group of leucine to the gamma-amino moiety of alpha, gamma-diaminobutyric acid (A2bu) substituted in position 2 of the peptide. Relative to [Leu5]enkephalin, the cyclic analog with D configuration in position 2, H-Tyr-cyclo(-N gamma-D-A2bu-Gly-Phe-Leu-), was 17.5 times more potent in the guinea pig ileum assay and twice as potent in the rat brain receptor binding assay, whereas its diastereomer H-Tyr-cyclo(-N gamma-L-A2bu-Gly-Phe-Leu-) showed low activity. The cyclic D isomer was also slightly more active than the open-chain reference compound [D-Nva2, Leu5]enkephalinamide in both assays, and it proved to be highly resistant to degradation by brain "enkephalinases." The steric constraints introduced in H-Tyr-cyclo(-N gamma-D-A2bu-Gly-Phe-Leu-) were shown to prevent the realization of most of the conformational features ascribed to linear enkephalin in solut...
This study assessed how conformational information encoded by ligand binding to d-opioid receptor... more This study assessed how conformational information encoded by ligand binding to d-opioid receptors (DORs) is transmitted to Kir3.1/Kir3.2 channels. Human embryonic kidney 293 cells were transfected with bioluminescence resonance energy transfer (BRET) donor/acceptor pairs that allowed us to evaluate independently reciprocal interactions among signaling partners. These and coimmunoprecipitation studies indicated that DORs, Gbg, and Kir3 subunits constitutively interacted with one another. GaoA associated with DORs and Gbg, but despite being part of the complex, no evidence of its direct association with the channel was obtained. DOR activation by different ligands left DOR-Kir3 interactions unmodified but modulated BRET between DOR-GaoA, DOR-Gbg, GaoA-Gbg, and Gbg-Kir3 interfaces. Ligand-induced BRET changes assessing Gbg-Kir3.1 subunit interaction 1) followed similar kinetics to those monitoring the GaoA-Gbg interface, 2) displayed the same order of efficacy as those observed at the DOR-Gbg interface, 3) were sensitive to pertussis toxin, and 4) were predictive of whether a ligand could evoke channel currents. Conformational changes at the Gbg/Kir3 interface were lost when Kir3.1 subunits were replaced by a mutant lacking essential sites for Gbg-mediated activation. Thus, conformational information encoded by agonist binding to the receptor is relayed to the channel via structural rearrangements that involve repositioning of Gbg with respect to DORs, GaoA, and channel subunits. Further, the fact that BRET changes at the Gbg-Kir3 interface are predictive of a ligand's ability to induce channel currents points to these conformational biosensors as screening tools for identifying GPCR ligands that induce Kir3 channel activation.
Strategies for the design of bi-or multifunctional drugs are reviewed. A distinction is made betw... more Strategies for the design of bi-or multifunctional drugs are reviewed. A distinction is made between bifunctional drugs interacting in a monovalent fashion with two targets and ligands containing two distinct pharmacophores binding in a bivalent mode to the two binding sites in a receptor heterodimer. Arguments are presented to indicate that some of the so-called "bivalent" ligands reported in the literature are unlikely to simultaneously interact with two binding sites. Aspects related to the development of bi-or multifunctional drugs are illustrated with examples from the field of opioid analgesics. The drug-like properties of the tetrapeptide Dmt 1 [DALDA] with triple action as a μ opioid agonist, norepinephrine uptake inhibitor and releaser of endogenous opioid peptides to produce potent spinal analgesia are reviewed. Rationales for the development of opioid peptides with mixed agonist/antagonist profiles as analgesics with reduced side effects are presented. Progress in the development of mixed μ opioid agonist/δ opioid antagonists with low propensity to produce tolerance and physical dependence is reviewed. Efforts to develop bifunctional peptides containing a μ opioid agonist and a cholecystokinin antagonist or an NK1 receptor antagonist as analgesics expected to produce less tolerance and dependence are also reviewed. A strategy to improve the drug-like properties of bifunctional opioid peptide analgesics is presented.
3-(2,6-Dimethyl-4-carbamoylphenyl)propanoic acid (Dcp), a 2&a... more 3-(2,6-Dimethyl-4-carbamoylphenyl)propanoic acid (Dcp), a 2',6'-dimethyltyrosine analogue containing a carbamoyl group in place of the hydroxyl function and lacking the amino group, was synthesized. The replacement of Tyr1 in an enkephalin analogue and in dynorphin A(1-11)-NH2 with Dcp resulted in the first opioid peptide-derived antagonists that do not contain a phenolic hydroxyl group at the 1-position residue. The cyclic peptide Dcp-c[D-Cys-Gly-Phe(pNO2)-D-Cys]NH2 represents a novel, potent mu opioid antagonist.
The opioid peptide H-Tyr-c[D-Cys-Phe-Phe-Cys]NH 2 cyclized via a methylene dithiother is a potent... more The opioid peptide H-Tyr-c[D-Cys-Phe-Phe-Cys]NH 2 cyclized via a methylene dithiother is a potent and selective μ opioid agonist (Przydial M.J. et al., J Peptide Res, 66, 2005, 255). Dicarba analogues of this peptide with Tyr, 2′,6′-dimethyltyrosine (Dmt), 3-[2,6-dimethyl-4-hydroxyphenyl)propanoic acid (Dhp) or (2S)-2-methyl-3-(2,6-dimethyl-4-hydroxyphenyl)propanoic acid [(2S)-Mdp] in the 1position were prepared. The peptides were synthesized on solid-phase by substituting D-allylglycine and (2S)-2-amino-5-hexenoic acid in position 2 and 5, respectively, followed by ring-closing metathesis. Mixtures of cis and trans isomers of the resulting olefinic peptides were obtained, and catalytic hydrogenation yielded the saturated-CH 2-CH 2-bridged peptides. All six Tyr 1-and Dmt 1-dicarba analogues retained high μ and δ opioid agonist potency and showed only slight or no preference for μ over δ receptors. As expected, the six Dhp 1-and (2S)-Mdp 1-dicarba analogues turned out to be μ opioid antagonists but, surprisingly, displayed a range of different efficacies (agonism, partial agonism or antagonism) at the δ receptor. The obtained results indicate that the μ versus δ receptor selectivity and the efficacy at the δ receptor of these cyclic peptides depend on distinct conformational characteristics of the 15-membered peptide ring structure, which may affect the spatial positioning of the exocyclic residue and of the Phe 3 and Phe 4 side chains.
Previously, we reported that H-2 0 ,6 0-dimethyltyrosine [Dmt 1 ]-D-Arg-Phe-Lys-NH 2 (DALDA), an ... more Previously, we reported that H-2 0 ,6 0-dimethyltyrosine [Dmt 1 ]-D-Arg-Phe-Lys-NH 2 (DALDA), an analogue of the naturally occurring opioid peptide dermorphin, is a highly potent and selective mu receptor agonist with low cross-tolerance to morphine. In the present study, we investigated the effect of treating mice chronically with [Dmt 1 ]DALDA. The AD 50 of [Dmt 1 ]DALDA (s.c.) increased eightfold in animals given this drug chronically; in contrast, the AD 50 increased twofold in mice chronically treated with morphine. The AD 50 of morphine (s.c.) in these [Dmt 1 ]DALDA-treated animals was increased more than 120 times, while that of the more selective m agonist [D-Ala 2-MePhe 4-Gly-ol 5 ]enkephalin (DAMGO) given intrathecally was increased more than 240 times. However, the AD 50 of DAMGO given intracerebroventricularly was essentially the same in animals treated chronically with [Dmt 1 ]DALDA as in naı¨ve animals. The dose of naloxone required to precipitate withdrawal in [Dmt 1 ]DALDA-treated animals was 20 times lower than that in morphine-tolerant animals. 2 Using real-time quantitative PCR, we found that expression of the m opioid receptor, d opioid receptor, preproenkephalin and preprodynorphin genes was upregulated in the brain by [Dmt 1 ]DALDA treatment. No significant changes in expression of opioid receptor or opioid peptide genes were detected in the spinal cord of [Dmt 1 ]DALDA-treated mice, nor in the brain or spinal cord of morphine-treated mice. We conclude that a high degree of tolerance to [Dmt 1 ]DALDA develops in the spinal cord but not brain, and cannot be accounted for by changes in expression of opioid receptors or opioid peptides in these tissues.
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Papers by Peter Schiller