Allosteric α1-Adrenoreceptor Antagonism by the Conopeptide ρ-TIA

Abstract

A peptide contained in the venom of the predatory marine snail Conus tulipa, -TIA, has previously been shown to possess ␣ 1 -adrenoreceptor antagonist activity. Here, we further characterize its pharmacological activity as well as its structure-activity relationships. In the isolated rat vas deferens, -TIA inhibited ␣ 1 -adrenoreceptor-mediated increases in cytosolic Ca 2؉ concentration that were triggered by norepinephrine, but did not affect presynaptic ␣ 2 -adrenoreceptor-mediated responses. In radioligand binding assays using [125 I]HEAT, -TIA displayed slightly greater potency at the ␣ 1B than at the ␣ 1A or ␣ 1D subtypes. Moreover, although it did not affect the rate of association for [ 3 H]prazosin binding to the ␣ 1B -adrenoreceptor, the dissociation rate was increased, indicating non-competitive antagonism by -TIA. N-terminally truncated analogs of -TIA were less active than the full-length peptide, with a large decline in activity observed upon removal of the fourth residue of -TIA (Arg 4 ). An alanine walk of -TIA confirmed the importance of Arg 4 for activity and revealed a number of other residues clustered around Arg 4 that contribute to the potency of -TIA. The unique allosteric antagonism of -TIA resulting from its interaction with receptor residues that constitute a binding site that is distinct from that of the classical competitive ␣ 1 -adrenoreceptor antagonists may allow the development of inhibitors that are highly subtype selective.␣ 1 -Adrenoceptors, members of the G protein-coupled receptor superfamily, are the predominant mediators of the response to norepinephrine released from the sympathetic nerves that innervate resistance vessels (1). Norepinephrine release modulates vascular tone and, as such, ␣ 1 -adrenoreceptors are critically involved in circulatory homeostasis. Several ␣ 1 -adrenoreceptor antagonists, such as the quinazoline derivative, prazosin, are widely used for the treatment of hypertension. ␣ 1 -Adrenoreceptor antagonists are also used to treat bladder outlet obstruction in benign prostatic hyperplasia (for review, see Ref.2) because of their ability to relax smooth muscle.Nevertheless, the ␣ 1 -adrenoreceptor ligands developed to date interact largely with residues of the transmembrane segments that are homologous between the various receptor subtypes, rather than with residues forming the framework regions (the intra-and extracellular loops). It is not surprising, therefore, that available agonists, and also antagonists, show limited subtype selectivity (affinities differing by 50-fold or less between the various subtypes). For this reason, we sought to identify novel ligands that are likely to interact allosterically and, thus, more likely with the framework residues that are distinct between the three ␣ 1 -adrenoreceptor subtypes (␣ 1A , ␣ 1B , and ␣ 1D ).The venoms of cone snails (marine gastropods of the genus Conus) contain bioactive peptides that disrupt neurotransmission. These compounds are referred to generically as "conopeptides" or "conotoxins." Individual ...

Subtype-selective Noncompetitive or Competitive Inhibition of Human α1-Adrenergic Receptors by ρ-TIA

“…1). However, unlike the limited selectivity observed previously with the rodent clones (27), -TIA was more potent and showed a 10-fold ␣ 1B selectivity. IC 50 values for -TIA were 18 nM (logIC 50 Ϫ7.4 Ϯ 0.08) at ␣ 1A -ARs; 2 nM (logIC 50 Ϫ8.4 Ϯ 0.10) at ␣ 1B -ARs; and 25 nM (logIC 50 Ϫ7.3 Ϯ 0.13) at ␣ 1D -ARs.…”

“…Studies on recombinant hamster ␣ 1B -ARs found that increasing concentrations of -TIA progressively reduced the density of radioligand binding sites without changing radioligand affinity, suggesting that -TIA is a noncompetitive, possibly allosteric, inhibitor of ␣ 1B -ARs. In addition, radioligand binding assays performed using recombinant rodent ␣ 1 -AR subtypes revealed a 2-5-fold higher affinity for -TIA at ␣ 1B -ARs (27).…”

“…This highly charged peptide (amino acid sequence FN-WRCCLIPACRRNHKKFC) forms a distinct structure with two disulfide bonds between cysteines 5 and 11 and cysteines 6 and 19 (26). -TIA was found to be a selective ␣ 1 -AR antagonist, as it inhibited ␣ 1 -AR-mediated contraction of rat vas deferens without affecting ATP or ␣ 2 -AR-mediated responses (26,27). Studies on recombinant hamster ␣ 1B -ARs found that increasing concentrations of -TIA progressively reduced the density of radioligand binding sites without changing radioligand affinity, suggesting that -TIA is a noncompetitive, possibly allosteric, inhibitor of ␣ 1B -ARs.…”

“…Recently, a 19-amino acid conopeptide, -TIA, was isolated from the venom of the fish-hunting cone snail Conus tulipa (26,27). This highly charged peptide (amino acid sequence FN-WRCCLIPACRRNHKKFC) forms a distinct structure with two disulfide bonds between cysteines 5 and 11 and cysteines 6 and 19 (26).…”

Developmental Biology of Neoplastic Growth

“…An analogue of χ-MrIA, with the unstable asparagine replaced with a pyroglutamate (named Xen2174), is currently in the clinical in a Phase I/IIa trial in severe cancer pain patients Paczkowski et al (2007) with permission α 1D -adrenoceptors (Sharpe et al 2001). Interestingly, TIA noncompetitively inhibits α 1B -adrenoceptors but competitively inhibits α 1A -and α 1D -adrenoceptors (Sharpe et al 2003;Chen et al 2004). Given its unusual mode of action and specificity for the α 1B subtype, TIA has been used to determine the extent of α 1B -adrenoceptor involvement in the contractions of different tissues Lima et al 2005).…”