Peter W. Schiller

Characterization of specific receptors for atrial natriuretic factor in bovine adrenal zona glomerulosa

We have recently shown that synthetic rat atrial natriuretic factor (ANF) directly inhibits mineralocorticoid and glucocorticoid secretion in cultured bovine adrenal cells with a potency of 100 pM. [125I]iodo-ANF was used in the present study to characterize potential receptor sites in bovine zona glomerulosa membranes. ANF binds to a class of high affinity binding sites with a pK of 10.2 and a density of 1.3 pmol/mg protein. Detailed competition curves with ANF document a class of high affinity sites with a pK of 10.2 and also a second class of lower affinity sites with a pK of 8.5. Nonspecific binding amounts to less than 10% of [125I]iodo-ANF binding at concentrations less than 100 pM. High affinity binding of [125I]iodo-ANF is reversible with a half-time of association of 15 minutes at 25 pM and a half-time of dissociation of 140 minutes. Monovalent cations Na, Li and K equipotently enhance [125I]iodo-ANF specific binding. Divalent cations Mg, Ca and Mn also increase [125I]iodo-ANF specific binding, with Mn being the most active cation. No effect of guanine nucleotide could be detected on ANF binding. The binding of [125I]iodo-ANF is very specific and is not inhibited by 1 microM angiotensin II, ACTH, VIP, somatostatin, Leu-enkephalin, dynorphin or by the N-terminal of POMC. The N-terminal fragment ANF-(1-16) is also completely inactive. Reduction of the disulfide bridge of ANF inactivates the peptide. This enabled the development of a highly specific radio-receptor assay for ANF with a minimum detectable dose of 2 femtomoles. The results document the specific receptor involved in the potent inhibitory effect of ANF on adrenal steroidogenesis and indicate that bovine adrenal zonal glomerulosa provide a highly sensitive system for studying the recently discovered atrial natriuretic factor.

Attenuation of morphine tolerance and dependence with the highly selective δ-opioid receptor antagonist TIPP[ψ]

Abstract We examined the effects of i.c.v. treatment with naltrindole, and the two highly selective peptide δ-opioid receptor antagonists H-Tyr-Tic-Phe-Phe-OH (TIPP) and H-Tyr-Ticψ[CH2-NH]-Phe-Phe-OH (TIPP[ψ]), on the development of morphine tolerance and dependence. Each treatment significantly decreased naloxone-precipitated withdrawal, with TIPP[ψ] reducing the most symptoms. TIPP[ψ], but neither naltrindole nor TIPP, attenuated the development of analgesic tolerance in the tail-flick test. These results suggest that δ-opioid receptors are critically involved in the development of morphine tolerance and dependence.

Novel Therapies Targeting Inner Mitochondrial Membrane—From Discovery to Clinical Development

ABSTRACTMitochondrial oxidative stress and dysfunction have been implicated in the aging process and in numerous chronic diseases. The need for therapies that can protect and/or improve mitochondrial function is obvious. However, the development of mitoprotective drugs has been hampered by a number of challenges, and there are at present no approved therapies for mitochondrial dysfunction. This article describes the original discovery, preclinical development, and clinical development of a novel class of small peptide molecules that selectively target the inner mitochondrial membrane and protect mitochondrial function. These compounds have the potential to be a paradigm-shifting approach to the treatment of mitochondrial dysfunction, which underlies many common diseases, including cardiorenal, neurologic, and metabolic disorders.

Bioluminescence Resonance Energy Transfer Assays Reveal Ligand-specific Conformational Changes within Preformed Signaling Complexes Containing δ-Opioid Receptors and Heterotrimeric G Proteins

Heptahelical receptors communicate extracellular information to the cytosolic compartment by binding an extensive variety of ligands. They do so through conformational changes that propagate to intracellular signaling partners as the receptor switches from a resting to an active conformation. This active state has been classically considered unique and responsible for regulation of all signaling pathways controlled by a receptor. However, recent functional studies have challenged this notion and called for a paradigm where receptors would exist in more than one signaling conformation. This study used bioluminescence resonance energy transfer assays in combination with ligands of different functional profiles to provide in vivo physical evidence of conformational diversity of δ-opioid receptors (DORs). DORs and αi1β1γ2 G protein subunits were tagged with Luc or green fluorescent protein to produce bioluminescence resonance energy transfer pairs that allowed monitoring DOR-G protein interactions from different vantage points. Results showed that DORs and heterotrimeric G proteins formed a constitutive complex that underwent structural reorganization upon ligand binding. Conformational rearrangements could not be explained by a two-state model, supporting the idea that DORs adopt ligand-specific conformations. In addition, conformational diversity encoded by the receptor was conveyed to the interaction among heterotrimeric subunits. The existence of multiple active receptor states has implications for the way we conceive specificity of signal transduction.

Cyclic enkephalin analogs containing a cystine bridge

Abstract Two conformationally constrained enkephalin analogs were synthesized by substitution of cysteines in positions 2 and 5 and oxidative disulfide bond formation. In the guinea pig ileum assay the obtained cyclic analogs, [D-Cys 2 -L-Cys 5 ]enkephalinamide and [D-Cys 2 -D-Cys 5 ]enkephalinamide, showed potency ratios of 37.9 ± 0.8 and 73.3 ± 0.9, respectively, relative to [Met 5 ]enkephalin. The extremely high potency of the analogs was shown to be a consequence of the conformational restrictions introduced by cyclization. Rat brain membrane binding studies with [ 3 H]naloxone and [ 3 H](D-Ala 2 , D-Leu 5 )enkephalin as radiolabels revealed a moderate preference of both analogs for μ-receptors over δ-receptors. Furthermore, the cystine-containing analogs were shown to be highly resistant to enzymatic degradation.

Structural basis for bifunctional peptide recognition at human δ-opioid receptor

Bifunctional μ- and δ-opioid receptor (OR) ligands are potential therapeutic alternatives, with diminished side effects, to alkaloid opiate analgesics. We solved the structure of human δ-OR bound to the bifunctional δ-OR antagonist and μ-OR agonist tetrapeptide H-Dmt-Tic-Phe-Phe-NH2 (DIPP-NH2) by serial femtosecond crystallography, revealing a cis-peptide bond between H-Dmt and Tic. The observed receptor-peptide interactions are critical for understanding of the pharmacological profiles of opioid peptides and for development of improved analgesics.

Fluorescent dyes alter intracellular targeting and function of cell-penetrating tetrapeptides

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 (Lys4) in a tetrapeptide ([Dmt1]DALDA, Dmt‐D‐Arg‐Phe‐ Lys‐NH2 where Dmt = 2’,6’‐dimethyltyrosine) with two different fluorescent amino acids (β‐ dansyl‐L‐α,β‐diaminopropionic acid (dnsDap4) or β‐anthraniloyl‐L‐α,β‐diaminopropionic acid (atnDap4)). Initial studies with confocal laser scanning microscopy (CLSM) showed very different localization patterns for the two fluorescent analogs, with [Dmt1,atnDap4]DALDA showing mitochondrial localization and [Dmt1,dnsDap4]DALDA showing diffuse cytoplasmic localization. Studies with isolated mouse liver mitochondria suggested that [Dmt1,dnsDap4]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, [Dmt1,atnDap4]DALDA was retained in the inner mitochondrial membrane and did not induce mitochondrial swelling. Furthermore, [Dmt1,atnDap4]DALDA protected mitochondria against Ca2+‐induced swelling. Importantly, the unlabeled parent peptide [Dmt1]DALDA behaved like [Dmt1,atnDap4]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.

Histone Deacetylase-3 Activation Promotes Tumor Necrosis Factor-α (TNF-α) Expression in Cardiomyocytes during Lipopolysaccharide Stimulation

Lipopolysaccharides (LPS) induce tumor necrosis factor-α (TNF-α) production in cardiomyocytes, which contributes to myocardial depression during sepsis. However, the underlying mechanisms remain not fully understood. This study was undertaken to investigate the contribution of histone deacetylase (HDAC) to TNF-α expression in cardiomyocytes and the signaling mechanism of LPS-induced HDAC activation. Here, we show for the first time that LPS increases HDAC activity and that inhibition of HDAC decreases LPS-stimulated TNF-α expression via the accumulation of NF-κB/p65 at the TNF-α promoter in cardiomyocytes. Using a positive screen, we have further identified HDAC3 as a specific member of the HDAC family able to regulate TNF-α production. Furthermore, our data reveal that LPS-induced HDAC activity is mediated through reactive oxygen species from mitochondria and c-Src signaling. In summary, this study demonstrates a novel signaling mechanism by which LPS via mitochondrial reactive oxygen species/c-Src/HDAC3 pathways mediate TNF-α expression in cardiomyocytes.

A novel cyclic opioid peptide analog showing high preference for μ-receptors

The side-chain to side-chain cyclized opioid peptide analogs H-Tyr-D-Orn-Phe-Asp-NH2 (I) and H-Tyr-D-Lys-Phe-Glu-NH2 (II) were synthesized and tested in the guinea pig ileum and mouse vas deferens assays and in binding assays based on displacement of mu- and delta-opioid receptor-selective radioligands from rat brain membranes. The more rigid cyclic analog I containing a 13-membered ring structure showed very high preference for mu-receptors over delta-receptors, whereas the more flexible cyclic peptide II (15-membered ring) was non-selective. These results indicate that variation in the degree of conformational restriction of opioid peptides can produce drastic shifts in their receptor selectivity profile. Because of its high mu-receptor selectivity and rigidity cyclic analog I will be useful for determining the conformational requirements of mu-opioid receptors.

Pharmacological characterization of the dermorphin analog [Dmt1]DALDA, a highly potent and selective μ-opioid peptide

The dermorphin-derived peptide [Dmt(1)]DALDA (H-Dmt-D-Arg-Phe-Lys-NH(2)), labels mu-opioid receptors with high affinity and selectivity in receptor binding assays. In mouse, radiant heat tail-flick assay [Dmt(1)]DALDA produced profound spinal and supraspinal analgesia, being approximately 5000- and 100-fold more potent than morphine on a molar basis, respectively. When administered systemically, [Dmt(1)]DALDA was over 200-fold more potent than morphine. Pharmacologically, [Dmt(1)]DALDA was distinct from morphine. [Dmt(1)]DALDA displayed no cross-tolerance to morphine in the model used and it retained supraspinal analgesic activity in morphine-insensitive CXBK mice. Supraspinally, it also differed from morphine in its lack of sensitivity towards naloxonazine. Finally, in antisense mapping studies, [Dmt(1)]DALDA was insensitive to MOR-1 exon probes that reduced morphine analgesia, implying a distinct receptor mechanism of action. Thus, [Dmt(1)]DALDA is an interesting and extraordinarily potent, systemically active peptide analgesic, raising the possibility of novel approaches in the design of clinically useful drugs.