# Is the cyclic imine core common to the marine macrocyclic toxins sufficient to dictate nicotinic ACh receptor antagonism? (BSI 2023, Session "Neurons", Poster P80)

Yves Bourne, Gerlind Sulzenbacher, Laurent Chabaud, Rómulo Aráoz, Zoran Radić, Sandrine Conrod, Palmer Taylor, Catherine Guillou, Jordi Molgó, Pascale Marchot

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Yves Bourne, Gerlind Sulzenbacher, Laurent Chabaud, Rómulo Aráoz, Zoran Radić, et al. Is the cyclic imine core common to the marine macrocyclic toxins sufficient to dictate nicotinic ACh receptor antagonism? (BSI 2023, Session "Neurons", Poster P80). BSI 2023 - 3rd edition of the Integrative Structural Biology Meeting, Nov 2023, Marseille, France. . [hal-04752741](https://hal.science/hal-04752741v1)

HAL Id: hal-04752741

https://hal.science/hal-04752741v1

Submitted on 24 Oct 2024

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Distributed under a Creative Commons Attribution 4.0 International License
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AFMB

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ICSN

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Institut
des sciences du vivant
Frédéric Joliot

UC San Diego

SKAGGS SCHOOL OF PHARMACY
AND PHARMACEUTICAL SCIENCES

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Centre de Recherche en Neurobiologie et Neurophysiologie de Marseille

# Is the cyclic imine core common to the marine macrocyclic toxins sufficient to dictate nicotinic acetylcholine receptor antagonism?

Y. Bourne¹, G. Sulzenbacher¹, L. Chabaud²,§, R. Aráoz³, Z. Radić⁴, S. Conrod⁵,&, P. Taylor⁴, C. Guillou², J. Molgó³, P. Marchot¹,⁵,*

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¹Lab "Architecture et Fonction des Macromolécules Biologiques" (AFMB), CNRS, Aix-Marseille Univ, Faculté des Sciences Campus Luminy, Marseille, France. ²Institut de Chimie des Substances Naturelles (ICSN), CNRS, Univ Paris-Sud, Univ Paris-Saclay, Gif-sur-Yvette, France. ³CEA, INRAE, Univ Paris-Saclay, DMTS, SIMoS, ERL CNRS, Gif-sur-Yvette, France. ⁴Skaggs School of Pharmacy and Pharmaceutical Sciences (SSPPS), Univ of California San Diego, La Jolla, CA, USA. ⁵Centre de Recherche en Neurobiologie et Neurophysiologie de Marseille (CRN2M), CNRS, Aix Marseille Univ, Marseille, France.

[Now at: §Univ Bordeaux, CNRS, Bordeaux INP, ISM, Talence, France. &Aix-Marseille Univ, CNRS, IRD, Collège de France, INRAE, CEREGE, Aix-en-Provence, France.]

## 1- Marine phycotoxins from dinoflagellate organisms

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1970

2006

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## 4- Macrocyclic imine toxins from shellfish

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Binding (not shown) and functional analyses on nAChRs

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Display high affinities (nM range) for the nAChRs and AChBPs and either low (gymnodimines & spirolides) or high (pinnatoxins) nAChR subtype specificities

Structural analysis on Aplysia AChBPs

<figure><img src="image_15.png" /><figcaption>Spirolide</figcaption></figure>

<figure><img src="image_16.png" /><figcaption>Gymnodimine</figcaption></figure>

<figure><img src="image_17.png" /><figcaption>Pinnatoxin-A</figcaption></figure>

<figure><img src="image_18.png" /><figcaption>Pinnatoxin-A vs Spirolide</figcaption></figure>

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

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

* The sequence-variable loop F hardly contributes to toxin binding --> low nAChR subtype specificity

* The bridged ketal group specific to the pinnatoxins extends radially toward the edge of the subunit interface and loop F

* The sequence-variable loop F largely contributes to toxin binding and governs high nAChR subtype specificity

## 2- A combination of experimental approaches

* Identify the toxins and their chemical structure

* Identify the toxins' pharmacological target(s)

* Synthesize toxins and analogs with controlled iso/enantiomeric 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 (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

* Correlate functional and structural data to explain the toxins' mode of action

## 3- nAChRs versus AChBPs

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Apical
subsite
(antag.)

Central
core
(agonists)

'Mb side'
subsite
(antag.)

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## 4- YES, the imine core is sufficient for dictating nicotinic antagonism!

(+)-4 R and (-)-4 S spiroimines

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<figure><img src="image_23.png" /><figcaption>Spiroimines</figcaption></figure>

Functional analysis on nAChRs

<table><thead><tr><th rowspan="2">nAChR<br>subtype</th><th colspan="3">IC50 (µM)</th></tr><tr><th>(±)-4</th><th>(+)-4 R</th><th>(–)-4 S</th></tr></thead><tbody><tr><td>Torpedo α₁βγδ</td><td>0.22</td><td>0.37</td><td>0.33</td></tr><tr><td>Human α7</td><td>2.04</td><td>5.15</td><td>1.31</td></tr><tr><td>Human α4β2</td><td>6.93</td><td>10.09</td><td>1.50</td></tr></tbody></table>

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Compared with the parental macrocyclic imine toxins: the spiroimines display preserved antagonism for both enantiomers, despite lower nAChR subtype specificity and binding affinities associated with faster reversibility.

Binding & structural analyses on Aplysia AChBP

<table><thead><tr><th></th><th>Kinetic/equilibrium<br>parameters</th><th>(±)-4</th><th>(+)-4 R</th><th>(–)-4 S</th></tr></thead><tbody><tr><td rowspan="5">A-AChBP</td><td>k<sub>on</sub> (10<sup>9</sup> M<sup>-1</sup>min<sup>-1</sup>)</td><td>6.3</td><td>7.4</td><td>6.1</td></tr><tr><td>k<sub>off</sub> (10<sup>3</sup> min<sup>-1</sup>)</td><td>18</td><td>16</td><td>26</td></tr><tr><td>k<sub>off</sub>/GAL (10<sup>3</sup> min<sup>-1</sup>)</td><td>15</td><td>14</td><td>24</td></tr><tr><td>K<sub>d</sub>/(k<sub>off</sub>/kon) (µM)</td><td>2.9</td><td>2.2</td><td>4.3</td></tr><tr><td>K<sub>d</sub>/SF<sub>eq</sub> (µM)</td><td>4.2</td><td>3.1</td><td>5.4</td></tr><tr><td></td><td>K<sub>d</sub>/SPA<sub>eq</sub> (µM)</td><td>2.4</td><td>2.3</td><td>2.4</td></tr></tbody></table>

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Modest affinities for AChBP, associated with positional variability along with a range of AChBP loop-C conformations, denoting prevalence of antagonistic properties

This study identifies a minimal set of functional pharmacophores and binding determinants as templates for designing new antagonists targeting disease-associated nAChR subtypes.

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Aix*Marseille
université

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SPINE
2
MPLEXES

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anr

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<footnote>*muscle-type α₁βγδ from *Torpedo* electrocyte neuronal α7, α4β2, α3β4 from human cDNAs</footnote>