Identification of a novel snake peptide toxin displaying high affinity and antagonist behaviour for the α₂‐adrenoceptors

Abstract

BACKGROUND AND PURPOSEMuscarinic and adrenergic G protein-coupled receptors (GPCRs) are the targets of rare peptide toxins isolated from snake or cone snail venoms. We used a screen to identify novel toxins from Dendroaspis angusticeps targeting aminergic GPCRs. These toxins may offer new candidates for the development of new tools and drugs. EXPERIMENTAL APPROACHIn binding experiments with 3 H-rauwolscine, we studied the interactions of green mamba venom fractions with a2-adrenoceptors from rat brain synaptosomes. We isolated, sequenced and chemically synthesized a novel peptide, r-Da1b. This peptide was pharmacologically characterized using binding experiments and functional tests on human a2-adrenoceptors expressed in mammalian cells. KEY RESULTSr-Da1b, a 66-amino acid peptide stabilized by four disulphide bridges, belongs to the three-finger-fold peptide family. Its synthetic homologue inhibited 80% of 3 H-rauwolscine binding to the three a2-adrenoceptor subtypes, with an affinity between 14 and 73 nM and Hill slopes close to unity. Functional experiments on a2A-adrenoceptor demonstrated that r-Da1b is an antagonist, shifting adrenaline activation curves to the right. Schild regression revealed slopes of 0.97 and 0.67 and pA2 values of 5.93 and 5.32 for yohimbine and r-Da1b, respectively. CONCLUSIONS AND IMPLICATIONSr-Da1b is the first toxin identified to specifically interact with a2-adrenoceptors, extending the list of class A GPCRs sensitive to toxins. Additionally, its affinity and atypical mode of interaction open up the possibility of its use as a new pharmacological tool, in the study of the physiological roles of a2-adrenoceptor subtypes. AbbreviationsBRET, bioluminescence resonance energy transfer; EPA, 5-(N-ethyl-N-isopropyl)-amiloride; GPCR, G protein-coupled receptor; RBS, rat brain synaptosomes BJP British Journal of Pharmacology

Orthosteric Binding of ρ-Da1a, a Natural Peptide of Snake Venom Interacting Selectively with the α1A-Adrenoceptor

“…Several three-finger snake toxins display similar incomplete competition for radioligand binding to GPCRs, however in the case of MT7 binding to the M1 muscarinic receptor, this residual binding is readily explained by an allosteric mode of interaction [11], [14], [16]. ρ-Da1b and MTα are also unable to fully inhibit 3 H-rauwolscine binding to α 2 -ARs despite showing no effect on the 3 H-rauwolscine dissociation rate, but their modes of action have still not been fully established [10], [57]. A third interesting case is that of ρ-TIA, which has an allosteric mode of action at α 1B -ARs but has been described as a competitive antagonist of the α 1A -AR in functional assays [19].…”

“…The modes of action of these peptide ligands on ARs are not clear. In equilibrium binding experiments, neither ρ-Da1a nor ρ-Da1b fully inhibits radioligand binding [9],[10]. In addition, in isolated prostatic muscle, ρ-Da1a acts as an insurmountable antagonist [9], and cell-based assays indicate that ρ-Da1b is a non-competitive antagonist at the human α 2A -AR [10].…”

“…We suspected that animal venoms are a potential source of novel GPCR binding agents, and developed a screening strategy, initially focused on the binding of green mamba venom to ARs. This screening led to the isolation of two novel snake toxins from Dendroaspis angusticeps : ρ-Da1a, previously called AdTx1, which is highly selective for the α 1A -AR [9], and ρ-Da1b, selective for α 2 -ARs [10]. ρ-Da1a and ρ-Da1b are peptides of 65 and 66 residues, respectively, reticulated by four disulfide bridges, and are members of the three-finger-fold toxin family.…”

Engineering of Three-Finger Fold Toxins Creates Ligands with Original Pharmacological Profiles for Muscarinic and Adrenergic Receptors

“…Despite high primary structure identity, often greater than 55%, muscarinic and adrenergic toxins exhibit distinct pharmacological profiles and clear functional differences [17], [18], [20], [21], [22], [23]. For example, MT7 acts as a highly potent and selective antagonist of the M1 receptor subtype [24], [25], [26], [27], [28], through a very stable interaction with the allosteric binding site on the M1 receptor [29].…”

Ancestral protein resurrection and engineering opportunities of the mamba aminergic toxins

“…The black widow spider and the gila monster provide further examples of GPCRs interacting toxins, namely, α-latrotoxin interacting with the latrophilin receptor 11 and exenatide, exploited as an anti-diabetic drug 12 , that targets the GLP-1 receptor, respectively. In addition, mamba venoms contain aminergic toxins that recognize various bioaminergic receptors [13][14][15][16][17][18][19] . Aminergic toxins belong to the three-finger fold toxin (3FT) superfamily, a structural fold known to support a large diversity of biological functions 20 .…”