Background and purpose: Venoms are a rich source of ligands for ion channels, but very little is known about their capacity to modulate G-protein coupled receptor (GPCR) activity. We developed a strategy to identify novel toxins targeting GPCRs. Experimental approach: We studied the interactions of mamba venom fractions with a1-adrenoceptors in binding experiments with 3 H-prazosin. The active peptide (AdTx1) was sequenced by Edman degradation and mass spectrometry fragmentation. Its synthetic homologue was pharmacologically characterized by binding experiments using cloned receptors and by functional experiments on rabbit isolated prostatic smooth muscle. Key results: AdTx1, a 65 amino-acid peptide stabilized by four disulphide bridges, belongs to the three-finger-fold peptide family. It has subnanomolar affinity (Ki = 0.35 nM) and high specificity for the human a1A-adrenoceptor subtype. We showed high selectivity and affinity (Kd = 0.6 nM) of radio-labelled AdTx1 in direct binding experiments and revealed a slow association constant (kon = 6 ¥ 10) with an unusually stable a1A-adrenoceptor/AdTx1 complex (t1/2diss = 3.6 h). AdTx1 displayed potent insurmountable antagonism of phenylephrine's actions in vitro (rabbit isolated prostatic muscle) at concentrations of 10 to 100 nM. Conclusions and implications:AdTx1 is the most specific and selective peptide inhibitor for the a1A-adrenoceptor identified to date. It displays insurmountable antagonism, acting as a potent relaxant of smooth muscle. Its peptidic nature can be exploited to develop new tools, as a radio-labelled-AdTx1 or a fluoro-labelled-AdTx1. Identification of AdTx1 thus offers new perspectives for developing new drugs for treating benign prostatic hyperplasia.

Antibody detection in autoimmune disorders, such as multiple sclerosis (MS) and Rett syndrome (RTT) can be achieved more efficiently using synthetic peptides. The previously developed synthetic antigenic probe CSF114(Glc), a type I' β-turn N-glucosylated peptide structure, is able to recognize antibodies in MS and RTT patients' sera as a sign of immune system derangement. We report herein the design, synthesis, conformational analysis, and immunological evaluation of a collection of glycopeptide analogs of CSF114(Glc) to characterize the specific role of secondary structures in MS and RTT antibody recognition. Therefore, we synthesized a series of linear and cyclic short glucosylated sequences, mimicking different β-turn conformations, which were evaluated in inhibition enzyme-linked immunosorbent assays (ELISA). Calculated IC50 ranking analysis allowed the selection of the candidate octapeptide containing two (S)-2-amino-4-pentynoic acid (L-Pra) residues Ac-Pra-RRN(Glc)GHT-Pra-NH2 , with an IC50 in the nanomolar range. This peptide was adequately modified for solid-phase ELISA (SP-ELISA) and surface plasmon resonance (SPR) experiments. Pra-RRN(Glc)GHT-Pra-NH2 peptide was modified with an alkyl chain linked to the N-terminus, favoring immobilization on solid phase in SP-ELISA and differentiating IgG antibody recognition between patients and healthy blood donors with a high specificity. However, this peptide displayed a loss in IgM specificity and sensitivity. Moreover, an analog was obtained after modification of the octapeptide candidate Ac-Pra-RRN(Glc)GHT-Pra-NH2 to favor immobilization on SPR sensor chips. SPR technology allowed us to determine its affinity (KD  = 16.4 nM), 2.3 times lower than the affinity of the original glucopeptide CSF114(Glc) (KD  = 7.1 nM).

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We report on the characteristics of the radical-ion-driven dissociation of a diverse array of β-amino acids incorporated into α-peptides, as probed by tandem electron-capture and electron-transfer dissociation (ECD/ETD) mass spectrometry. The reported results demonstrate a stronger ECD/ETD dependence on the nature of the amino acid side chain for β-amino acids than for their α-form counterparts. In particular, only aromatic (e.g., β-Phe), and to a substantially lower extent, carbonyl-containing (e.g., β-Glu and β-Gln) amino acid side chains, lead to N-Cβ bond cleavage in the corresponding β-amino acids. We conclude that radical stabilization must be provided by the side chain to enable the radical-driven fragmentation from the nearby backbone carbonyl carbon to proceed. In contrast with the cleavage of backbones derived from α-amino acids, ECD of peptides composed mainly of β-amino acids reveals a shift in cleavage priority from the N-Cβ to the Cα-C bond. The incorporation of CH2 groups into the peptide backbone may thus drastically influence the backbone charge solvation preference. The characteristics of radical-driven β-amino acid dissociation described herein are of particular importance to methods development, applications in peptide sequencing, and peptide and protein modification (e.g., deamidation and isomerization) analysis in life science research.