A Venomics Approach Coupled to High-Throughput Toxin Production Strategies Identifies the First Venom-Derived Melanocortin Receptor Agonists

Abstract

Animal venoms are rich in hundreds of toxins with extraordinary biological activities. Their exploitation is difficult due to their complexity and the small quantities of venom available from most venomous species. We developed a Venomics approach combining transcriptomic and proteomic characterization of 191 species and identified 20,206 venom toxin sequences. Two complementary production strategies based on solid-phase synthesis and recombinant expression in Escherichia coli generated a physical bank of 3597 toxins. Screened on hMC4R, this bank gave an incredible hit rate of 8%. Here, we focus on two novel toxins: N-TRTX-Preg1a, exhibiting an inhibitory cystine knot (ICK) motif, and N-BUTX-Ptr1a, a short scorpion-CSαβ structure. Neither N-TRTX-Preg1a nor N-BUTX-Ptr1a affects ion channels, the known targets of their toxin scaffolds, but binds to four melanocortin receptors with low micromolar affinities and activates the hMC1R/Gs pathway. Phylogenetically, these two toxins form new groups within their respective families and represent novel hMC1R agonists, structurally unrelated to the natural agonists.

Strategies for Heterologous Expression, Synthesis, and Purification of Animal Venom Toxins

“…As an example, heterologous expression allows for the production of thousands of toxins, which can be screened for interesting bioactivity in a high-throughput setup. Having such expression and purification workflows in place, ideally in an automated fashion, can also allow for the identification of new targets for previously uncharacterized toxins ( Sequeira et al, 2017 ; Duhoo et al, 2019 ; Reynaud et al, 2020 ).…”

Structural and Functional Diversity of Animal Toxins Interacting With GPCRs

“…Toxins with two disulfide bridges are much less present in venoms as compared to toxins with three or four disulfide bridges (Reynaud et al, 2020), and most of them are found in conus venoms. Exceptionally, they were identified in arthropods (Smith et al, 2011;Daly and Wilson, 2018) as disulfide directed β-hairpin (DDH: C-C-C-C framework with the I-III, II-IV connectivity), a fold which seems to be evolutionarily linked to three-disulfide-bridge ICK toxins (Undheim et al, 2016).…”

“…Screened on membrane preparations of COS cells stably expressing the human melanocortin receptor 4 (hMC4R) by competition with 125 I-NDP-α-MSH, this bank gave an incredible hit rate of 8%. Two toxins isolated from this screening have been studied in depth, the spider N-TRTX-Preg1a toxin, exhibiting an inhibitory cystine knot (ICK) motif, and N-BUTX-Ptr1a, a short scorpion toxin with a CSαβ structure (Reynaud et al, 2020).…”

Towards a generic prototyping approach for therapeutically-relevant peptides and proteins in a cell-free translation system

“…Solid-phase synthesis of disulfide-rich peptides either relies on thiol protecting groups to avoid side reactions or demands high initial yields to allow downstream refolding and purification 26 . Also, it can be reliably applied only to peptides shorter than 35 27 or 50 28 residues dependent on beta-sheet proportion. Successful heterologous peptide production generally relies on fusion with a carrier protein to confer solubility 29 and protease protection 30 .…”

“…For both chemical and biological synthetic approaches, it is challenging to produce large amounts of soluble disulfide-rich peptides omitting the refolding step. Robust statistics provided by Venomics platform 27 , 33 , the success rate of peptide refolding following heterologous expression 31 or chemical synthesis 26 suggest that under the universal folding conditions only ~half of the peptides could be produced in a soluble form. This is consistent with the existence of two principal folding modes—“framework” and “collapsed”, representing either the hierarchic condensation of native-like elements or the slow flux through the off-path folding states 34 , respectively.…”