New α-adrenergic property for synthetic MTβ and CM-3 three-finger fold toxins from black mamba

Blanchet, Guillaume; Upert, Grégory; Mourier, Gilles; Gilquin, Bernard; Gilles, Nicolas; Servent, Denis

doi:10.1016/j.toxicon.2013.04.017

“…AncTx5 preserved only the interactions with the α 2A and α 2C adrenoceptors, while those with M 1 , α 1A , α 1B and α 1D receptor subtypes were lost and the affinity for M 4 suffered a massive decrease (pK i = 6.81 vs. 9.08 for MT3). However, AncTx5 recognizes the dopamine D 3 receptor, a subfamily of receptor targeted until now by only one aminergic toxin 36 . The pK i value of AncTx5 for this subtype was slightly higher (6.81) than for ρ-Da1b (6.12) (Table 2, Supplementary Fig.…”

Section: Resultsmentioning

confidence: 99%

“…These higher pK i values arise from the only five substitutions that distinguish these two toxins. These include charge modifications in finger I (N7D, E15Q) and modifications at positions 26 (K to I) and 28 (W to R), already shown as key determinants for target modulation 36 .…”

Section: Resultsmentioning

confidence: 99%

Ancestral protein resurrection and engineering opportunities of the mamba aminergic toxins

Blanchet

¹

,

Alili

²

,

Protte

³

et al. 2017

Sci Rep

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Mamba venoms contain a multiplicity of three-finger fold aminergic toxins known to interact with various α-adrenergic, muscarinic and dopaminergic receptors with different pharmacological profiles. In order to generate novel functions on this structural scaffold and to avoid the daunting task of producing and screening an overwhelming number of variants generated by a classical protein engineering strategy, we accepted the challenge of resurrecting ancestral proteins, likely to have possessed functional properties. This innovative approach that exploits molecular evolution models to efficiently guide protein engineering, has allowed us to generate a small library of six ancestral toxin (AncTx) variants and associate their pharmacological profiles to key functional substitutions. Among these variants, we identified AncTx1 as the most α1A-adrenoceptor selective peptide known to date and AncTx5 as the most potent inhibitor of the three α2 adrenoceptor subtypes. Three positions in the ρ-Da1a evolutionary pathway, positions 28, 38 and 43 have been identified as key modulators of the affinities for the α1 and α2C adrenoceptor subtypes. Here, we present a first attempt at rational engineering of the aminergic toxins, revealing an epistasis phenomenon.

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“…As previously described in Blanchet et al 36 , the six AncTxs and the two engineered AncTx1 variants were synthesized on a 50 µmol scale using Fmoc chemistry on a Prelude synthesizer (Protein Technologies ® ) using HMPB ChemMatrix ® resin functionalized with the appropriate protected amino acid (AA). Peptides were purified by reverse phase semi-preparative HPLC using a Vydac C18 column (250 × 10 mm) with an optimized gradient (usually 40 to 60% of solvent B in 40 min (A: 0.1% TFA in H 2 O, B: 60% acetonitrile and 0.1% TFA in H 2 O).…”

Section: Methodsmentioning

confidence: 99%

“…CD spectra were recorded on a Jasco J-815 CD spectropolarimeter. As previously described in Blanchet et al 36 , measurements were routinely performed at 20 °C in 0.1 cm path length quartz cells (Hellma) with a peptide concentration of 20.10 −6 M in 5 mM sodium phosphate buffer pH 7.4. Spectra were recorded in the 190 to 260 nm wavelength range.…”

Section: Methodsmentioning

confidence: 99%

“…These higher pK i values arise from the only five substitutions that distinguish these two toxins. These include charge modifications in finger I (N7D, E15Q) and modifications at positions 26 (K to I) and 28 (W to R), already shown as key determinants for target modulation 36 . Due to the high number of substitutions at the other nodes (10 with an insertion at the AncTx5 node, see Figs 1A and 3), understanding the SAR was more difficult.…”

Section: Ancestral Resurrection and Engineering Opportunitiesmentioning

confidence: 99%

See 1 more Smart Citation

Ancestral protein resurrection and engineering opportunities of the mamba aminergic toxins

Blanchet

¹

,

Stura

²

,

Mourier

³

et al. 2018

Toxicon

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0

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Mamba venoms contain a multiplicity of three-finger fold aminergic toxins known to interact with various α-adrenergic, muscarinic and dopaminergic receptors with different pharmacological profiles. In order to generate novel functions on this structural scaffold and to avoid the daunting task of producing and screening an overwhelming number of variants generated by a classical protein engineering strategy, we accepted the challenge of resurrecting ancestral proteins, likely to have possessed functional properties. This innovative approach that exploits molecular evolution models to efficiently guide protein engineering, has allowed us to generate a small library of six ancestral toxin (AncTx) variants and associate their pharmacological profiles to key functional substitutions. Among these variants, we identified AncTx1 as the most α 1A -adrenoceptor selective peptide known to date and AncTx5 as the most potent inhibitor of the three α2 adrenoceptor subtypes. Three positions in the ρ-Da1a evolutionary pathway, positions 28, 38 and 43 have been identified as key modulators of the affinities for the α 1 and α 2C adrenoceptor subtypes. Here, we present a first attempt at rational engineering of the aminergic toxins, revealing an epistasis phenomenon.Evolutionary processes of venomous animals have selected enzymes and disulfide-rich peptides in their venoms to improve their ability to subdue their prey and defend against predators. These compounds act predominantly on few, well characterized and physiologically-relevant molecular targets of the envenomed animals 1 . Recruited by convergent evolution, toxins impact mainly the haemostatic, nervous and cardiovascular systems 2 . Some toxins present in venoms, in spite of their debilitating effects, have become live-saving drugs [3][4][5] . To exert their biological activities, toxins interact mainly with ion channels, coagulation factors, nicotinic receptors, cell membrane or enzymes but only rarely with the most important class of physiological targets, i.e, the G protein-coupled receptors (GPCRs) 6 . Compared to numerous toxins that act on voltage-gated or ligand-gated ion channels, only a few toxins that interact with GPCRs have been identified. These toxins have been isolated mainly from cone snails venoms such as conopressin interacting with vasopressin receptor 7 , contulakin-G which binds to neurotensin receptor 8 , ρ-conotoxin TIA specific of the α 1A -adrenoceptor 9 and the τ-conotoxin CnVA that interacts with the somatostatin sst3 receptor 10 . 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...

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Snake Venom Toxins Targeted at the Nervous System

Osipov¹,

Utkin²

2017

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New α-adrenergic property for synthetic MTβ and CM-3 three-finger fold toxins from black mamba

Cited by 19 publications

References 37 publications

Ancestral protein resurrection and engineering opportunities of the mamba aminergic toxins

Ancestral protein resurrection and engineering opportunities of the mamba aminergic toxins

Ancestral protein resurrection and engineering opportunities of the mamba aminergic toxins

Snake Venom Toxins Targeted at the Nervous System

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