ToxiCeptor

The pentameric type 3 serotonin receptors (5-HT3Rs) are involved in irritable-bowel syndrome and chemo- or radiotherapy-induced vomiting. Since most 5-HT3R agonists and competitive antagonists, aka orthosteric effectors, have low affinity/selectivity and generate critical side effects, the development of allosteric modulators with 5-HT3R-subtype selectivity is of great interest for the pharmaceutical industry. Snake venoms feature vast natural libraries of peptides/proteins displaying many molecular structures and even more pharmacological activities. Many of these “toxins”, which are not always lethal, have been used as tools for studying their receptor at the basic research level and/or as molecular templates for the design of new effectors/drugs with selected pharmacological properties. A proof-of-concept study involving ligand binding on solubilized 5-HT3R and electrophysiology measurements on oocyte-expressed 5-HT3R led us to identify, from a snake venom, a high-affinity binder that seems to act as an allosteric modulator of channel activity. Low resolution negative stain electron microscopy (EM) imaging of the toxin-receptor complex revealed toxin molecules bound far from the orthosteric binding site for agonists and competitive antagonists, consistent with our binding and functional data. To ascertain the binding site and mode of action of this new, peptidic 5-HT3R binder, we now propose to (i) complete/refine our electrophysiology study and complement our data set with binding data on 5-HT3R subtypes embedded in mammalian cell membranes; (ii) identify the precise toxin binding site and orientation at the receptor surface and characterize the interacting molecular determinants in both the toxin and receptor, by solving a high resolution cryo-EM structure of the pentameric complex; (iii) explore possible conformational changes in the extracellular ligand-binding domain and the ion-channel associated with toxin binding, by comparing the receptor conformations in presence versus absence of the bound toxin; (iv) use the same strategy to document the identity and mode of action of a second 5-HT3R binder present in a semi-purified fraction issued from the venom of another snake species. Availability of complementary binding, functional and structural data on these peptidic ligands of the 5-HT3R with atypical binding sites and original modes of action will provide new avenues through their use as pharmacological tools to further explore new pathways for modulation of 5-HT3R activity. Such data could also lead to the structure-based design of new peptidic or organic molecules with tailored pharmacological activities aimed at modulating this receptor allosterically, or to be associated with chemo- or radiotherapy treatments to attenuate/alleviate their undesirable side effects. This proposal involves three French teams with complementary expertise in cell biology, biochemistry, biophysics, pharmacology, toxinology and structural biology, and with established publications and collaboration records in the field.