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

Fruchart-Gaillard, Carole; Mourier, Gilles; Blanchet, Guillaume; Vera, Laura; Gilles, Nicolas; Ménez, R.; Marcon, Elodie; Stura, E.A.; Servent, Denis

doi:10.1371/journal.pone.0039166

Cited by 30 publications

(21 citation statements)

References 45 publications

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“…Interestingly, such a chain termination, associated with low coupling efficiency, was already observed in our laboratory during the synthesis of the three-finger fold polypeptides, such as muscarinic and adrenergic toxins (28,29), and was located (i) in a ␤-sheet rich area in the central second loop of the molecules, and (ii) in the N-terminal part of the sequences. We addressed this difficulty by replacing Ser or Thr residues located during the synthetic process just before the critical regions by their corresponding dimethyloxazolidine.…”

Section: Discussionmentioning

confidence: 87%

Mambalgin-1 Pain-relieving Peptide, Stepwise Solid-phase Synthesis, Crystal Structure, and Functional Domain for Acid-sensing Ion Channel 1a Inhibition

Mourier

Salinas

Kessler

et al. 2016

Journal of Biological Chemistry

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Mambalgins are peptides isolated from mamba venom that specifically inhibit a set of acid-sensing ion channels (ASICs) to relieve pain. We show here the first full stepwise solid phase peptide synthesis of mambalgin-1 and confirm the biological activity of the synthetic toxin both in vitro and in vivo. We also report the determination of its three-dimensional crystal structure showing differences with previously described NMR structures. Finally, the functional domain by which the toxin inhibits ASIC1a channels was identified in its loop II and more precisely in the face containing Phe-27, Leu-32, and Leu-34 residues. Moreover, proximity between Leu-32 in mambalgin-1 and Phe-350 in rASIC1a was proposed from double mutant cycle analysis. These data provide information on the structure and on the pharmacophore for ASIC channel inhibition by mambalgins that could have therapeutic value against pain and probably other neurological disorders.

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Section: Discussionmentioning

confidence: 87%

Mambalgin-1 Pain-relieving Peptide, Stepwise Solid-phase Synthesis, Crystal Structure, and Functional Domain for Acid-sensing Ion Channel 1a Inhibition

Mourier

Salinas

Kessler

et al. 2016

Journal of Biological Chemistry

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“…Muscarinic toxins from mamba venoms, such as MT1 and MT7 (Figures 4G,H), act as highly potent and selective antagonists of M1 receptor subtype through allosteric interactions with the M1 receptor. Fruchart-Gaillard et al (2012) synthesized seven chimeric 3FTXs based on MT1 and MT7 proteins that have remarkable affinity for α1 A -adrenoceptor receptor subtypes but low affinity for M1 receptor. In this study, substitution within loop 1 and loop 3 weaken the toxin interactions with the M1 receptor, resulting in a 2-fold decrease in affinity (Figures 4I,J).…”

Section: Three-finger Toxins (3ftxs)mentioning

confidence: 99%

“…Furthermore, modifications in loop 2 of the MT1 and MT7 significantly reduce the affinity for the M1 receptor. Interestingly, a significant increase in affinity was achieved on the α1 A -adrenoceptor by combined modifications in loops 1 and 3, where loop 1 forms a critical interaction with the receptor (Fruchart-Gaillard et al, 2012). Another muscarinic toxin named MTβ was designed based on ρ-Da1a protein from Black Mamba which is known to have affinity for the muscarinic receptor.…”

Section: Three-finger Toxins (3ftxs)mentioning

confidence: 99%

Multifunctional Toxins in Snake Venoms and Therapeutic Implications: From Pain to Hemorrhage and Necrosis

et al. 2019

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Animal venoms have evolved over millions of years for prey capture and defense from predators and rivals. Snake venoms, in particular, have evolved a wide diversity of peptides and proteins that induce harmful inflammatory and neurotoxic effects including severe pain and paralysis, hemotoxic effects, such as hemorrhage and coagulopathy, and cytotoxic/myotoxic effects, such as inflammation and necrosis. If untreated, many envenomings result in death or severe morbidity in humans and, despite advances in management, snakebite remains a major public health problem, particularly in developing countries. Consequently, the World Health Organization recently recognized snakebite as a neglected tropical disease that affects ∼2.7 million p.a. The major protein classes found in snake venoms are phospholipases, metalloproteases, serine proteases, and three-finger peptides. The mechanisms of action and pharmacological properties of many snake venom toxins have been elucidated, revealing a complex multifunctional cocktail that can act synergistically to rapidly immobilize prey and deter predators. However, despite these advances many snake toxins remain to be structurally and pharmacologically characterized. In this review, the multifunctional features of the peptides and proteins found in snake venoms, as well as their evolutionary histories, are discussed with the view to identifying novel modes of action and improving snakebite treatments.

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“…We applied this strategy on aminergic toxins using a loop permutation approach and create chimeric toxins with new functional profiles on muscarinic and adrenergic receptors 23 . Nevertheless, within a protein family with many functionally diverse members, a vertical strategy which reconstruct ancestral genes and study the effect of mutations on functional diversification should allow the identification of the structural determinants of protein function and the exploitation of this knowledge for protein engineering 24 .…”

Section: Discussionmentioning

confidence: 99%

Ancestral protein resurrection and engineering opportunities of the mamba aminergic toxins

Blanchet

Stura

Mourier

et al. 2018

Toxicon

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

Cited by 30 publications

References 45 publications

Mambalgin-1 Pain-relieving Peptide, Stepwise Solid-phase Synthesis, Crystal Structure, and Functional Domain for Acid-sensing Ion Channel 1a Inhibition

Mambalgin-1 Pain-relieving Peptide, Stepwise Solid-phase Synthesis, Crystal Structure, and Functional Domain for Acid-sensing Ion Channel 1a Inhibition

Multifunctional Toxins in Snake Venoms and Therapeutic Implications: From Pain to Hemorrhage and Necrosis

Ancestral protein resurrection and engineering opportunities of the mamba aminergic toxins

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