# Diversity in the binding interactions of nicotinic ligands to the nAChRs and associated conformational fluctuations: Insights into the core motif dictating antagonism (JMC 2019, Poster)

Yves Bourne, Gerlind Sulzenbacher, Zoran Radić, Laurent Chabaud, Rómulo Aráoz, Evelyne Benoit, Armen Zakarian, Denis Servent, Catherine Guillou, Palmer Taylor, et al.

► To cite this version:

Yves Bourne, Gerlind Sulzenbacher, Zoran Radić, Laurent Chabaud, Rómulo Aráoz, et al. Diversity in the binding interactions of nicotinic ligands to the nAChRs and associated conformational fluctuations: Insights into the core motif dictating antagonism (JMC 2019, Poster). Canaux ioniques activés par les ligands: de la structure atomique à la transmission synaptique / Ligand-gated ion channels: from atomic structure to synaptic transmission, May 2019, Roscoff, France., 2024. hal-04771646

HAL Id: hal-04771646

https://hal.science/hal-04771646v1

Submitted on 8 Nov 2024

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# Diversity in the binding interactions of nicotinic ligands to the nAChRs and associated conformational fluctuations: Insights into the core motif dictating antagonism

Bourne Y¹, Sulzenbacher G¹, Radić Z², Chabaud L³, Aráoz R⁴, Benoit E⁴, Zakarian A⁵, Servent D⁴, Guillou C³, Taylor P², Molgo J⁴, & Marchot P¹

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¹Lab 'Architecture & Fonction des Macromolécules Biologiques', CNRS/AMU, Faculté des Sciences - Campus Luminy, Marseille, FR. ²Skaggs School of Pharmacy & Pharmaceutical Sciences, Dept of Pharmacology, UC San Diego, CA, USA. ³Institut de Chimie des Substances Naturelles, CNRS, Gif/Yvette, FR. ⁴Institut des Sciences du Vivant Frédéric Joliot, CEA Paris-Saclay, Gif/Yvette, FR. ⁵Dept of Chemistry & Biochemistry, UC Santa Barbara, CA, USA.

## 1- Binding of nicotinic antagonists and agonists to the soluble, homopentameric nAChR surrogate, AChBP

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Central to the subunit interface:
an aromatic nest for agonist binding

Conformational changes in AChBP loop C associated w/ ligand binding

ANTAGONISTS

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## 4- Spirolides and gymnodimines: high affinity, low nAChR subtype specificity

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Functional analysis

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Imine toxins acting as antagonists:

* nM affinities dictated by slow dissociation

* low specificity for the various nAChR subtypes

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Spirolide

Gymnodimine

"Mb" side

* Main anchoring point for the common cyclic imine: central to the subunit interface

* Additional anchoring points in the apical and "membrane" directions: promote high-affinity binding

* The sequence-variable loop F hardly contributes to toxin binding: low specificity

## 2- Marine phycotoxins from dinoflagellate organisms

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Apical
subsite
(Im1)

Agonist
core

'Mb side'
subsite
(MLA)

* The binding pocket extends beyond the agonist core to provide additional binding subsites for the antagonists

* The additional binding loci govern subtype specificity for the antagonists

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## 3- A combination of experimental approaches

* Synthesize toxins and analogs with controlled (+/-) and (R/S) properties

* Define mode of action, affinity, and subtype specificity for nAChR subtypes* in the membrane state (transfected oocytes), using voltage-clamp electrophysiology

* Record affinity and subtype specificity by competition binding at equilibrium to nAChR subtypes* in the membrane state (transfected HEK cells)

* Record kinetics and affinities for binding to soluble AChBP subtypes and mutants

* Crystallize (by hand!) the preformed AChBP complexes and solve their crystal structures

## 6- Is the imine core found in the macrocyclic toxins sufficient for dictating nicotinic antagonism?

<figure><img src="image_27.png" /><figcaption>(R)-(+)- and (S)-(-) spiroimine</figcaption></figure>

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Functional analysis

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α₁₂βγδ nAChR

α₁₂βγδ, α7, α4β2

ΔIC50 (+/-) ≈ 10 to 35-fold

Compared with the macrocyclic imines:

* much lower affinity for the nAChRs and AChBPs, associated with limited interaction networks

* moderate nAChR subtype specificity

* longer term antagonistic properties

Inhibition of ACh-evoked currents

Muscle-type
α₁₂βγδ nAChR

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Neuronal
α4β2 nAChR

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Full versus partial antagonism at 10 µM

Structural analysis

Apical side

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<figure><img src="image_33.png" /><figcaption>(S)-(-)-spiroimine</figcaption></figure>

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* Main anchoring point still in the agonist core

* (R)-(+)-spiroimine displays a mixed antagonist/agonist mode of binding (open/closed loop C) not observed for (S)-(-)-spiroimine

## 5- Pinnatoxins: high affinity, high nAChR subtype specificity

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Functional analysis

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Apical side

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PnTx-A

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"Mb" side

PnTx-A vs SPX

Compared with SPX / GYM:

* similar high (0.1-10 nM) affinities dictated by slow dissociation

* higher nAChR subtype specificity

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AGENCE NATIONALE DE LA RECHERCHE
ANR

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National Institute of
General Medical Sciences

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Aix*Marseille
université

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SPINE 2

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MPLEXES

<footnote>*muscle-type α₁₂βγδ and neuronal α7, α4β2, α3β4</footnote>