The hemagglutinin-tagged human trace amine-associated receptor1 (TAAR1) was stably coexpressed with rat G␣ s in the AV12-664 cell line, and receptor activation was measured as the stimulation of cAMP formation. After blockade of endogenously expressed ␣ 2 -and ␤-adrenoceptors with 2-[2-(2-methoxy-1,4-benzodioxanyl)]-imidazoline hydrochloride (2-methoxyidazoxan, RX821002) and alprenolol, respectively, the resulting pharmacology was consistent with that of a unique receptor subtype. ␤-Phenylethylamine (␤-PEA), the putative endogenous ligand, gave an EC 50 of 106 Ϯ 5 nM in the assay. For a series of ␤-PEA analogs used to explore the pharmacophore, small substituents at ring positions 3 and/or 4 generally resulted in compounds having lower potency than ␤-PEA, although several were as potent as ␤-PEA. However, small substituents at ring position 2 resulted in a number of compounds having potencies as good as or better than ␤-PEA. A number of nonselective antagonists known to share affinity for multiple monoaminergic receptors were evaluated for their ability to inhibit ␤-PEA stimulation of the human TAAR1. None had an IC 50 Ͻ10 M. For comparison, the rat TAAR1 receptor was expressed in the AV12-664 cell line. A number of agonist compounds had significantly different relative potencies between the rat and human TAAR1, demonstrating a significant species difference between the rat and human TAAR1. The TAAR1 receptor exhibits a pharmacologic profile uniquely different from those of classic monoaminergic receptors, consistent with the structural information that places them in a distinct family of receptors. This unique pharmacologic profile suggests the potential for development of TAAR-selective agonists and antagonists to study their physiologic roles.The trace amines are congeners of the so-called classic monoamine or biogenic amine neurotransmitters, e.g., dopamine, norepinephrine and serotonin, but are found in the brain in much lower concentrations (nanograms per gram or less) than the classic neurotransmitters (Baldessarini and Fischer, 1978;Philips et al., 1978). Compounds typically discussed under the category of trace amines include (but are not limited to) ␤-phenylethylamine, m-and p-tyramine, octopamine, and tryptamine. Hypotheses regarding the possible actions of the trace amines in normal physiology and disease states were published as early as the 1970s (Baldessarini and Fischer, 1978;Philips et al., 1978;Boulton, 1980). However, this field of study remained on the fringes of neurotransmitter research because of the lack of tools that would differentiate the actions of trace amines from those of other biogenic amine neurotransmitters. In 2001, cloning studies revealed the existence of a group of receptors described as the trace amine receptor family (Borowsky et al., 2001;Bunzow et al., 2001). These initial reports have been followed by several reviews and additional characterizations of these receptors (Branchek and Blackburn, 2003;Lewin, 2006;Navarro et al., 2006). However, given the time since ...

The endothelins (ETs) are a family of bicyclic 21-amino acid peptides that are potent and prolonged vasoconstrictors. It has been shown that highly potent combined ETA/ETB receptor antagonists can be developed from the C-terminal hexapeptide of ET (His16-Leu17-Asp18-Ile19-Ile20-Trp21), such as Ac-(D)Dip16-Leu-Asp-Ile-Ile-Trp21 (PD 142893) and Ac-DBhg16-Leu-Asp-Ile-Ile-Trp21 (PD 145065). However, these compounds are relatively unstable to enzymatic proteolysis as determined in an in vitro rat intestinal perfusate assay. This instability is thought to be due to carboxypeptidase activity. In fact, incubation of PD 145065 with carboxypeptidase inhibitors greatly increased its half-life in rat intestinal perfusate. By performing a reduced amide bond and N-methyl amino acid scan, it was discovered that N-methylation of Ile-20 resulted in a compound (Ac-DBhg16-Leu-Asp-Ile-[NMe]Ile-Trp21, PD 156252) that retained full receptor affinity at both endothelin receptor subtypes along with enhanced proteolytic stability and cellular permeability. Interestingly, N-methylation of this bond allows the cis configuration to be readily accessible which greatly alters the preferred structure of the entire molecule and may be responsible for the observed enhanced metabolic stability.

As new methods for volatilizing and ionizing biological molecules for mass spectrometry (MS) analysis are developed, the conformation of gas-phase biomolecule ions and how it relates to solution-phase reactivity and structure has been generating considerable interest. 1 The conformations of gas-phase ions of large polypeptides formed by electrospray ionization (ESI) have been probed by a variety of means, including ion-molecule reactions, 2 collision cross-section measurements, 3 and hydrogendeuterium-exchange (H/D). 4 The number of exchangeable protons reflects the openness of the protein conformation. Ion mobility measurements have resolved gas-phase structural isomers for a number of proteins. 5 The charge state distribution produced by ESI as a function of pH, 6 solvent content, 7 and metal-binding properties 8 has also been related to the protein conformation in solution. Another recent application of ESI-MS includes the study of noncovalently bound complexes, 9,10 for which MS has unique advantages in determining complex stoichiometry of the binding partners. Characteristics of the native solution state of proteins and oligonucleotides and their interactions with other biomolecules can be also captured by MS measurements.Tandem mass spectrometry (MS/MS) with collisionally activated dissociation (CAD) has been used also to explore the reactivities of large ions. Mass-analyzed kinetic energy (MIKE) spectrometry was used to study the stability of secondary structure in the absence of solvent for the peptide melittin. 11 The CAD mass spectra of peptide-metal ion complexes show that relatively selective bond cleavages can be induced, as specific complexation with metal ions can direct specific dissociation pathways. 12 Smith and co-workers' CAD study of 12 kDa cytochrome c proteins suggests that the fragmentation pattern could be influenced by the protein's higher order structure. 13 However, from these studies, it is not clear whether these three-dimensional structures in a solvent-less environment bear any resemblance to the conformation in the solution phase. We present an example in which the gas-phase MS/MS-based measurements of a polypeptide are consistent with its known solution-phase structure.RES-701-1 (I), isolated from Streptomyces sp. RE-701, is a potent and selective endothelin ET B receptor antagonist. 14 The hexadecapeptide possesses a unique linkage between the -carbonyl carbon of Asp-9 and the R-amino group of Gly-1, forming a 9-residue cyclic "head" structure and a 7-residue "tail" (see Scheme 1). However, a synthetic version of the peptide (II) is characterized by an affinity for the ET B receptor reduced by nearly 3 orders of magnitude (IC 50 of 10 nM for the natural peptide and 6.5 µM for the synthetic peptide). 15,16 High-resolution, exact mass measurements with ESI show the two peptides to be within (0.004 mass units ((2 ppm) of the expected value (measured monoisotopic mass of I was 2041.8520, and a mass of 2041.8492 for II; expected mass from sequence is 2041.8536). 17 Dramatic differ...

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Communications to the Editor Design of a Functional Hexapeptide Antagonist of Endothelin Endothelin-1 (ET-1, Figure 1), a bicyclic 21-amino acid peptide, is a potent constrictor of vascular smooth muscle.1 2"3 Since the isolation of ET-1 from the supernatant of cultured porcine endothelial aortic cells, human genomic analysis has identified two structurally and functionally related isopeptides (ET-2 and ET-3).4 56Previous structure-activity analyses have shown the importance of the C-terminal L-tryptophan indole ring, its carboxylate, and the two cystine bridges (1-15 and 3-11

Neurotensin (NT) is a linear tridecapeptide with a broad range of central and peripheral pharmacological effects. The C-terminal hexapeptide of NT (NT8-13) has been shown to possess similar properties to NT itself, and in fact, an analogue of NT8-13 (N alpha MeArg8-Lys-Pro-Trp-Tle-Leu13, Tle = tert-leucine) has been reported to possess central activity after peripheral administration. Cyclic derivatives of this hexapeptide were synthesized by a combination of solution and solid-phase peptide synthetic methodologies, and several analogues had low nanomolar binding affinity for the NT receptor. In particular, cyclo[Arg-Lys-Pro-Trp-Glu]-Leu (cyclized between the alpha amine of Arg and the gamma carboxylate of Glu) possessed 16 nM NT receptor affinity and was determined to be an agonist in vitro. 1H-NMR and 13C-edited 1H-NMR spectroscopy were performed on this and related cyclic analogues to help identify structural properties which may be important for receptor recognition. These cyclic peptides represent novel molecular probes to further investigate NT receptor pharmacology, as well as to advance our understanding of the structure-conformation relationships of NT and to help establish a working basis for additional pharmacophore mapping studies.

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