Charleen Miller

A New Family of Conus Peptides Targeted to the Nicotinic Acetylcholine Receptor

In this work, a new family of Conus peptides, the αA-conotoxins, which target the nicotinic acetylcholine receptor, is defined. The first members of this family have been characterized from the eastern Pacific species, Conus purpurascens (the purple cone); three peptides that cause paralysis in fish were purified and characterized from milked venom. The sequence and disulfide bonding pattern of one of these, αA-conotoxin PIVA, is as follows: where O represents trans-4-hydroxyproline. The two other peptides purified from C. purpurascens venom are the under-hydroxylated derivatives, αA-conotoxin PIVA and [Pro]αA-conotoxin PIVA. The peptides have been chemically synthesized in a biologically active form. Both electrophysiological experiments and competition binding with α-bungarotox- in demonstrate that αA-PIVA acts as an antagonist of the nicotinic acetylcholine receptor at the postsynaptic membrane.

A Uniquely Selective Inhibitor of the Mammalian Fetal Neuromuscular Nicotinic Acetylcholine Receptor

We have purified and characterized a novel conotoxin from the venom of Conus obscurus, which has the unique property of selectively and potently inhibiting the fetal form of the mammalian neuromuscular nicotinic acetylcholine receptor (nAChR) (α1β1γδ-subunits). Although this conotoxin, αA-conotoxin OIVB (αA-OIVB), is a high-affinity antagonist (IC50 of 56 nm) of the fetal muscle nAChR, it has >1800-fold lower affinity for the adult muscle nAChR (α1β1ϵδ-subunits) and virtually no inhibitory activity at a high concentration on various neuronal nAChRs (IC50 > 100 μm in all cases). The peptide (amino acid sequence, CCGVONAACPOCVCNKTCG), with three disulfide bonds, has been chemically synthesized in a biologically active form. Although the neuromuscular nAChRs are perhaps the most extensively characterized of the receptors/ion channels of the nervous system, the precise physiological roles of the fetal form of the muscle nAChR are essentially unknown.αA-OIVB is a potentially important tool for delineating the functional roles ofα1β1γδ receptors in normal development, as well as in various adult tissues and in pathological states. In addition to its potential as a research tool, αA-OIVB may have some direct biomedical applications.

Separation of neuropeptide Y diastereomers by high-performance liquid chromatography and capillary zone electrophoresis

Separation of analogues of neuropeptide Y (NPY) in which a single D-amino acid replaced the corresponding naturally occurring residue was performed by chromatographic techniques to ensure the quality of the synthetic peptides to be used for structural and biological studies. Of the 35 compounds, 28 were easily separated (alpha = 1.02-2.76) from native NPY by standard reversed-phase high-performance chromatography (RP-HPLC) methods using a Vydac C18 column and a gradient buffer system developed in our laboratory comprised of triethylammonium phosphate (TEAP) at pH 2.25 and acetonitrile at 40 degrees C. The identical diastereomers could be separated on the same solid support and by using 0.1% trifluoroacetic acid (TFA) as the mobile phase modifier, however separation factors were smaller and retention times were longer. Three of the remaining seven unresolved analogues were separated (alpha = 1.02-1.96) by changing the solid-phase support to Vydac diphenyl derivatized silica and a buffer system consisting of 0.1% TFA and acetonitrile. Of the four remaining unresolved analogues, only two could be separated by capillary zone electrophoresis (CZE) in 0.1 M sodium phosphate at pH 2.5, but all four were finally resolved by changing the electrophoretic buffer to 0.1 M TEAP buffer at pH 2.5. Migration times of the diastereomers differed by 0.2-2.0 min from that of the natural NPY. In addition to confirming the uniqueness of each isomer, this investigation demonstrated the expansive utility and high efficiency of the TEAP buffer system for both RP-HPLC and CZE as well as the difference in selectivity produced by the TEAP and TFA buffers in RP-HPLC.(ABSTRACT TRUNCATED AT 250 WORDS)

A minimized human insulin-receptor-binding motif revealed in a Conus geographus venom insulin

Insulins in the venom of certain fish-hunting cone snails facilitate prey capture by rapidly inducing hypoglycemic shock. One such insulin, Conus geographus G1 (Con-Ins G1), is the smallest known insulin found in nature and lacks the C-terminal segment of the B chain that, in human insulin, mediates engagement of the insulin receptor and assembly of the hormones hexameric storage form. Removal of this segment (residues B23–B30) in human insulin results in substantial loss of receptor affinity. Here, we found that Con-Ins G1 is monomeric, strongly binds the human insulin receptor and activates receptor signaling. Con-Ins G1 thus is a naturally occurring B-chain-minimized mimetic of human insulin. Our crystal structure of Con-Ins G1 reveals a tertiary structure highly similar to that of human insulin and indicates how Con-Ins G1s lack of an equivalent to the key receptor-engaging residue PheB24 is mitigated. These findings may facilitate efforts to design ultrarapid-acting therapeutic insulins.

Structural modifications of non-mammalian gonadotropin-releasing hormone (GnRH) isoforms: design of novel GnRH analogues.

Three natural forms of vertebrate gonadotropin-releasing hormone (GnRH) provided the structural basis upon which to design new GnRH agonists: [His5,Trp7,Leu8]-GnRH, dogfish (df) GnRH; [His5,Asn8]-GnRH, catfish (cf) GnRH; and [His5,Trp7,Tyr8]-GnRH, chicken (c) GnRH-II. The synthetic peptides incorporated the position 6 dextro (D)-isomers D-arginine (D-Arg) or D-naphthylalanine (D-Nal) in combination with an ethylamide substitution of position 10. The in vitro potencies for LH and FSH release of these analogues were assessed using static cultures of rat anterior pituitary cells. Efficacious peptides were examined for their gonadotropin-II and growth hormone releasing abilities from perifused goldfish pituitary fragments. Rat LH and FSH release was measured using homologous radioimmunoassays, whereas goldfish growth hormone and gonadotropin-II release were determined using heterologous carp hormone radioimmunoassays. The receptor binding of the most potent analogues was determined in bovine pituitary membrane preparations. Substitution of D-Nal6 into [His5,Asn8]-GnRH increased the potency over 2200-fold compared with the native ligand (cfGnRH) in cultured rat pituitary cells. This was equivalent to a 55-fold greater potency than that of the native mammal (m) GnRH peptide. Substitution of D-Nal6 or D-Arg6 into dfGnRH or cGnRH-II resulted in potencies that were related to the overall hydrophobicity of the analogues. The [D-Nal6,Pro9NEt]-cfGnRH bound to the bovine membrane preparation with an affinity statistically similar to that of [D-Nal6,Pro9NEt]-mGnRH (kd = 0.40 +/- 0.04 and 0.55 +/- 0.10 nM, respectively) in cultured rat pituitary cells. All analogues tested released the same ratio of FSH to LH. In goldfish, the analogues did not possess superagonistic activity but instead desensitized the pituitary fragments at lower analogue doses than that of the sGnRH standard suggesting differences in receptor affinity or signal transduction.

Synthesis, purification and characterization of rat histone H2A (1–53)-NH2

Abstract Synthetic peptides (up to 40 residues in length) are routinely purified and characterized by reversed-phase liquid chromatography (RP-LC). However, as the peptide size is further increased, this technique becomes less efficient in separating micro-heterogeneities despite the extensive use of columns or buffer systems known to exhibit different selectivities. As a result, alternative analytical and preparative methodologies were sought for the purification and characterization of synthetic histone H2A (1–53)-NH 2 . This 53-peptide was synthesized on a 2,4-dimethoxybenzhydrylamine resin using the fluorenylmethyloxycarbonyl strategy. Interestingly, the crude histone, after trifluoroacetic acid cleavage from the resin and deprotection, appeared to be remarkably pure. This result being unexpected, this sample was also analyzed by cation-exchange chromatography using a recently developed narrow-bore Pharmacia Mono S Precision Column 1.6/5 and an organic modifier (acetonitrile) in the buffer. Under the experimental conditions that were used, the presence of the desired peptide was determined by mass spectrometry and accounted for ca. 7% of the total absorbance at 214 nm. Scale-up studies allowed the isolation of significant amounts of highly purified histone H2A (1–53)-NH 2 . This preparation was characterized using RP-LC, capillary zone electrophoresis, amino acid analysis, mass spectrometry and sequence analysis using automated Edman degradation.

Total synthesis, purification, and characterization of human [Phe(p-CH2SO3Na)52, Nle32,53,56, Nal55]-CCK20–58, [Tyr52, Nle32,53,56,Nal55]-CCK-58, and [Phe(p-CH2SO3Na)52, Nle32,53,56, Nal55]-CCK-58

The synthesis of [Phe(p-CH2SO3Na)52, Nle32,53,56 Nal55]-CCK20–58, [Tyr52, Nle32,53,56, Nal55]-CCK-58 and of [Phe(p-CH2SO3Na)52, Nle32,53,56, Nal55]-CCK-58 using the (9-fluorenylmethyloxy)-carbonyl (Fmoc) strategy on a 2,4-DMBHA resin is described. The crude peptide preparations were extremely complex when analyzed by RP-HPLC, capillary zone electrophoresis (CZE), and ion-exchange chromatography (IE-FPLC). We found that the most effective strategy for purification included cation-exchange chromatography followed by a RP-HPLC desalting step. The highly purified peptides (purity greater than 90%) were characterized by RP-HPLC, size exclusion HPLC (SEC), IE-FPLC, CZE, mass spectrometry, amino acid analysis, and Edman sequence analysis {for [Tyr52, Nle32,53,56, Nal55]-CCK-58}. The results demonstrate the applicability of the 2,4-DMBHA resin for Fmoc solid-phase synthesis of long peptides amides (58 residues in length in this case) as well as the efficacy of an FPLC/RP-HPLC approach for the purification of very long, heterogeneous crude peptides, allowing a true assessment of the biological properties of these analogs to be carried out. [Phe(p-CH2SO3Na)52, Nle32,53,56, Nal55]-CCK20–58 was less than 1% as potent as CCK-8 while [Tyr52, Nle32,53,56, Nal55]-CCK-58 and [Phe(p-CH2SO3Na)52, Nle32,53,56, Nal55]-CCK-58 were inactive at the doses tested (<0.01%).

Characterization of a Pachymedusa dacnicolor–Sauvagine Analog as a New High-Affinity Radioligand for Corticotropin-Releasing Factor Receptor Studies

The corticotropin-releasing factor (CRF) peptide family comprises the mammalian peptides CRF and the urocortins as well as frog skin sauvagine and fish urophyseal urotensin. Advances in understanding the roles of the CRF ligand family and associated receptors have often relied on radioreceptor assays using labeled CRF ligands. These assays depend on stable, high-affinity CRF analogs that can be labeled, purified, and chemically characterized. Analogs of several of the native peptides have been used in this context, most prominently including sauvagine from the frog Phyllomedusa sauvageii (PS-Svg). Because each of these affords both advantages and disadvantages, new analogs with superior properties would be welcome. We find that a sauvagine-like peptide recently isolated from a different frog species, Pachymedusa dacnicolor (PD-Svg), is a high-affinity agonist whose radioiodinated analog, [125ITyr0-Glu1, Nle17]-PD-Svg, exhibits improved biochemical properties over those of earlier iodinated agonists. Specifically, the PD-Svg radioligand binds both CRF receptors with comparably high affinity as its PS-Svg counterpart, but detects a greater number of sites on both type 1 and type 2 receptors. PD-Svg is also ∼10 times more potent at stimulating cAMP accumulation in cells expressing the native receptors. Autoradiographic localization using the PD-Svg radioligand shows robust specific binding to rodent brain and peripheral tissues that identifies consensus CRF receptor–expressing sites in a greater number and/or with greater sensitivity than its PS-Svg counterpart. We suggest that labeled analogs of PD-Svg may be useful tools for biochemical, structural, pharmacological, and anatomic studies of CRF receptors.

Large-Scale Synthesis of Gonadotropin-Releasing Hormone Antagonists for Clinical Investigations

Publisher Summary This chapter discusses large-scale synthesis of gonadotropin-releasing hormone antagonists for clinical investigations. Once a new peptide has been discovered and its biological activities identified, a logical extension is to design analogs for the study and understanding of its structure–activity relationships and to identify molecules that are either more potent, longer acting, or competitive antagonists. Either an agonist or an antagonist of a given peptide may be examined as a potential candidate for the treatment of diseases arising from defects in the normal pathways that the native peptide governs. Such synthetic peptides and their analogs can be synthesized and purified in large-enough quantities for their successful and safe use in a clinical setting. The chapter discusses the development of two antagonists of gonadotropin-releasing hormone (GnRH): (1) Ac- d -2-Nal- d -Cpa- d -3-Pal-Ser-Arg- d -2-amino-5-oxo-5-(4-methoxyphenyl) pentanoic acid-Leu-Arg-Pro- d -Ala-NH 2 (Nal-Glu antagonist), which has already been tested extensively in humans; and (2) Ac- d -2-Nal- d -Cpa- d -3-Pal-Ser-Lys(atz)- d -Lys(atz)-Leu-ILys-Pro- d -Ala-NH 2 (Azaline), a member of the most recent generation of potent, water-soluble GnRH antagonists with little or no histamine-releasing activity.

Characterization of Multisubstituted Corticotropin Releasing Factor (CRF) Peptide Antagonists (Astressins)

CRF mediates numerous stress-related endocrine, autonomic, metabolic, and behavioral responses. We present the synthesis and chemical and biological properties of astressin B analogues {cyclo(30-33)[D-Phe(12),Nle(21,38),C(α)MeLeu(27,40),Glu(30),Lys(33)]-acetyl-h/r-CRF(9-41)}. Out of 37 novel peptides, 17 (2, 4, 6-8, 10, 11, 16, 17, 27, 29, 30, 32-36) and 16 (3, 5, 9, 12-15, 18, 19, 22-26, 28, 31) had k(i) to CRF receptors in the high picomolar and low nanomole ranges, respectively. Peptides 1, 2, and 11 inhibited h/rCRF and urocortin 1-induced cAMP release from AtT20 and A7r5 cells. When Astressin C 2 was administered to adrenalectomized rats at 1.0 mg subcutaneously, it inhibited ACTH release for >7 d. Additional rat data based on the inhibitory effect of (2) on h/rCRF-induced stimulation of colonic secretory motor activity and urocortin 2-induced delayed gastric emptying also indicate a safe and long-lasting antagonistic effect. The overall properties of selected analogues may fulfill the criteria expected from clinical candidates.