76. Hayashi, T.; Kanehira, K.; Hagihara, T.; Kumada, M.J. Asymmetric synthesis catalyzed by chiral ferrocenylphosphine-transition metal complexes. 5. Palladium-catalyzed asymmetric allylation of active methine compounds. *J. Org. Chem.* **1998**, *53*, 113–120.
[CrossRef]

77. Hauser, T.A.; Hepler, C.D.; Kombo, D.C.; Grinevich, V.P.; Kiser, M.N.; Hooker, D.N.; Zhang, J.; Mountfort, D.; Selwood, A.; Akireddy, S.R.; et al. Comparison of acetylcholine receptor interactions of the marine toxins, 13-desmethylspirolidone C and gymnodimine. *Neuropharmacology* **2012**, *62*, 2239–2250. [CrossRef]

78. Tan, K.P.; Singh, K.; Hazra, A.; Madhusudhan, M.S. Peptide bond planarity constrains hydrogen bond geometry and influences secondary structure conformations. *Curr. Res. Struct. Biol.* **2020**, *3*, 1–8. [CrossRef] [PubMed]

79. Bourne, Y.; Radić, Z.; Taylor, P.; Marchot, P. Conformational remodeling of femtomolar inhibitor-acetylcholinesterase complexes in the crystalline state. *J. Am. Chem. Soc.* **2010**, *132*, 18292–18300. [CrossRef] [PubMed]

80. Nemecz, Á.; Taylor, P. Creating an α7 nicotinic acetylcholine recognition domain from the acetylcholine-binding protein: Crystallographic and ligand selectivity analyses. *J. Biol. Chem.* **2011**, *286*, 42555–42565. [CrossRef] [PubMed]

81. Kaczanowska, K.; Camacho Hernandez, G.A.; Bendiks, L.; Kohs, L.; Cornejo-Bravo, J.M.; Harel, M.; Finn, M.G.; Taylor, P. Substituted 2-aminopyrimidines selective for α7-nicotinic acetylcholine receptor activation and association with acetylcholine binding proteins. *J. Am. Chem. Soc.* **2017**, *139*, 3676–3684. [CrossRef] [PubMed]

82. Neudert, G.; Klebe, G. DSX: A knowledge-based scoring function for the assessment of protein-ligand complexes. *J. Chem. Inf. Model.* **2011**, *51*, 2731–2745. [CrossRef]

83. Rahman, M.M.; Basta, T.; Teng, J.; Lee, M.; Worrell, B.T.; Stowell, M.H.B.; Hibbs, R.E. Structural mechanism of muscle nicotinic receptor desensitization and block by curare. *Nat. Struct. Mol. Biol.* **2022**, *29*, 386–394. [CrossRef]

84. McCoy, A.J. Solving structures of protein complexes by molecular replacement with Phaser. *Acta Crystallogr. D Biol. Crystallogr.* **2007**, *63*, 32–41. [CrossRef] [PubMed]

85. Murshudov, G.N.; Vagin, A.A.; Dodson, E.J. Refinement of macromolecular structures by the maximum-likelihood method. *Acta Crystallogr. D Biol. Crystallogr.* **1997**, *53*, 240–255. [CrossRef] [PubMed]

86. Emsley, P.; Cowtan, K. Coot: Model-building tools for molecular graphics. *Acta Crystallogr. D Biol. Crystallogr.* **2004**, *60*, 2126–2132.
[CrossRef] [PubMed]

87. CCP4. The CCP4 suite: Programs for protein crystallography. *Acta Crystallogr. D. Biol. Crystallogr.* **1994**, *50*, 760–763. [CrossRef] [PubMed]

88. Krissinel, E.; Henrick, K. Inference of macromolecular assemblies from crystalline state. *J. Mol. Biol.* **2007**, *372*, 774–797. [CrossRef] [PubMed]

89. Davis, I.W.; Leaver-Fay, A.; Chen, V.B.; Block, J.N.; Kapral, G.J.; Wang, X.; Murray, L.W.; Arendall, W.B.; Snoeyink, J.; Richardson, J.S.; et al. MolProbity: All-atom contacts and structure validation for proteins and nucleic acids. *Nucleic Acids Res.* **2007**, *35*, W375–W383. [CrossRef] [PubMed]

90. Schrödinger, LLC. *The PyMOL Molecular Graphics System, Version 1.5.0.4*; Schrödinger, LLC: New York, NY, USA, 2010.

91. Robert, X.; Gouet, P. Deciphering key features in protein structures with the new ENDscript server. *Nucleic Acids Res.* **2014**, *42*, W320–W324. [CrossRef] [PubMed]

92. Taylor, P.; Shyong, Y.J.; Samskey, N.; Ho, K.Y.; Radić, Z.; Fenical, W.; Sharpless, K.B.; Kovarik, Z.; Camacho-Hernandez, G.A. Ligand design for human acetylcholinesterase and nicotinic acetylcholine receptors, extending beyond the conventional and canonical. *J. Neurochem.* **2021**, *158*, 1217–1222. [CrossRef] [PubMed]

93. Kem, W.R.; Andrud, K.; Bruno, G.; Xing, H.; Soti, F.; Talley, T.T.; Taylor, P. Interactions of nereistoxin and its analogs with vertebrate nicotinic acetylcholine receptors and molluscan ACh binding proteins. *Mar. Drugs* **2022**, *20*, 49. [CrossRef]

94. Sine, S.M.; Taylor, P. Relationship between reversible antagonist occupancy and the functional capacity of the acetylcholine receptor. *J. Biol. Chem.* **1981**, *56*, 6692–6699. [CrossRef]

95. Bouzat, C. New insights into the structural bases of activation of Cys-loop receptors. *J. Physiol. Paris* **2012**, *106*, 23–33. [CrossRef] [PubMed]

96. Dumont, J.N. Oogenesis in *Xenopus laevis* (Daudin). I. Stages of oocyte development in laboratory maintained animals. *J. Morphol.* **1972**, *136*, 153–179. [CrossRef] [PubMed]

97. Villeneuve, P.; Esnault, G.; Benoit, E.; Molgó, J.; Aráoz, R. A monitoring study of repetitive surgical oocyte harvest in *Xenopus laevis*. In *Toxins and Ion Transfers*; Collection Meetings in Toxinology; Barbier, J., Benoit, E., Gilles, N., Ladant, D., Martin-Eauclaire, M.F., Mattei, C., Molgó, J., Popoff, M.R., Servent, D., Eds.; SFET Publication: Gif-sur-Yvette, France, 2011; pp. 173–178. ISSN 1760-6004. Available online: http://sfet.asso.fr/international/e-book-rt/e-book-rt.html (accessed on 27 November 2011).

98. Kharrat, R.; Servent, D.; Girard, E.; Ouanounou, G.; Amar, M.; Marrouchi, R.; Benoit, E.; Molgó, J. The marine phycotoxin gymnodimine targets muscular and neuronal nicotinic acetylcholine receptor subtypes with high affinity. *J. Neurochem.* **2008**, *107*, 952–963. [CrossRef] [PubMed]

99. Krieger, F.; Mourot, A.; Aráoz, R.; Kotzyba-Hibert, F.; Molgó, J.; Bamberg, E.; Goeldner, M. Fluorescent agonists for the *Torpedo* nicotinic acetylcholine receptor. *ChemBioChem* **2008**, *9*, 1146–1153. [CrossRef] [PubMed]

100. Aráoz, R.; Ouanounou, G.; Iorga, B.I.; Goudet, A.; Alili, D.; Amar, M.; Benoit, E.; Molgó, J.; Servent, D. The neurotoxic effect of 13,19-didesmethyl and 13-desmethyl spirolidone C phycotoxins is mainly mediated by nicotinic rather than muscarinic acetylcholine receptors. *Toxicol. Sci.* **2015**, *147*, 156–167. [CrossRef]
76. Hayashi, T.; Kanehira, K.; Hagihara, T.; Kumada, M.J. Asymmetric synthesis catalyzed by chiral ferrocenylphosphine-transition metal complexes. 5. Palladium-catalyzed asymmetric allylation of active methine compounds. *J. Org. Chem.* **1998**, *53*, 113–120.
[CrossRef]

77. Hauser, T.A.; Hepler, C.D.; Kombo, D.C.; Grinevich, V.P.; Kiser, M.N.; Hooker, D.N.; Zhang, J.; Mountfort, D.; Selwood, A.; Akireddy, S.R.; et al. Comparison of acetylcholine receptor interactions of the marine toxins, 13-desmethylspirolidone C and gymnodimine. *Neuropharmacology* **2012**, *62*, 2239–2250. [CrossRef]

78. Tan, K.P.; Singh, K.; Hazra, A.; Madhusudhan, M.S. Peptide bond planarity constrains hydrogen bond geometry and influences secondary structure conformations. *Curr. Res. Struct. Biol.* **2020**, *3*, 1–8. [CrossRef] [PubMed]

79. Bourne, Y.; Radić, Z.; Taylor, P.; Marchot, P. Conformational remodeling of femtomolar inhibitor-acetylcholinesterase complexes in the crystalline state. *J. Am. Chem. Soc.* **2010**, *132*, 18292–18300. [CrossRef] [PubMed]

80. Nemecz, Á.; Taylor, P. Creating an α7 nicotinic acetylcholine recognition domain from the acetylcholine-binding protein: Crystallographic and ligand selectivity analyses. *J. Biol. Chem.* **2011**, *286*, 42555–42565. [CrossRef] [PubMed]

81. Kaczanowska, K.; Camacho Hernandez, G.A.; Bendiks, L.; Kohs, L.; Cornejo-Bravo, J.M.; Harel, M.; Finn, M.G.; Taylor, P. Substituted 2-aminopyrimidines selective for α7-nicotinic acetylcholine receptor activation and association with acetylcholine binding proteins. *J. Am. Chem. Soc.* **2017**, *139*, 3676–3684. [CrossRef] [PubMed]

82. Neudert, G.; Klebe, G. DSX: A knowledge-based scoring function for the assessment of protein-ligand complexes. *J. Chem. Inf. Model.* **2011**, *51*, 2731–2745. [CrossRef]

83. Rahman, M.M.; Basta, T.; Teng, J.; Lee, M.; Worrell, B.T.; Stowell, M.H.B.; Hibbs, R.E. Structural mechanism of muscle nicotinic receptor desensitization and block by curare. *Nat. Struct. Mol. Biol.* **2022**, *29*, 386–394. [CrossRef]

84. McCoy, A.J. Solving structures of protein complexes by molecular replacement with Phaser. *Acta Crystallogr. D Biol. Crystallogr.* **2007**, *63*, 32–41. [CrossRef] [PubMed]

85. Murshudov, G.N.; Vagin, A.A.; Dodson, E.J. Refinement of macromolecular structures by the maximum-likelihood method. *Acta Crystallogr. D Biol. Crystallogr.* **1997**, *53*, 240–255. [CrossRef] [PubMed]

86. Emsley, P.; Cowtan, K. Coot: Model-building tools for molecular graphics. *Acta Crystallogr. D Biol. Crystallogr.* **2004**, *60*, 2126–2132. [CrossRef] [PubMed]

87. CCP4. The CCP4 suite: Programs for protein crystallography. *Acta Crystallogr. D. Biol. Crystallogr.* **1994**, *50*, 760–763. [CrossRef] [PubMed]

88. Krissinel, E.; Henrick, K. Inference of macromolecular assemblies from crystalline state. *J. Mol. Biol.* **2007**, *372*, 774–797. [CrossRef] [PubMed]

89. Davis, I.W.; Leaver-Fay, A.; Chen, V.B.; Block, J.N.; Kapral, G.J.; Wang, X.; Murray, L.W.; Arendall, W.B.; Snoeyink, J.; Richardson, J.S.; et al. MolProbity: All-atom contacts and structure validation for proteins and nucleic acids. *Nucleic Acids Res.* **2007**, *35*, W375–W383. [CrossRef] [PubMed]

90. Schrödinger, LLC. *The PyMOL Molecular Graphics System, Version 1.5.0.4*; Schrödinger, LLC: New York, NY, USA, 2010.

91. Robert, X.; Gouet, P. Deciphering key features in protein structures with the new ENDscript server. *Nucleic Acids Res.* **2014**, *42*, W320–W324. [CrossRef] [PubMed]

92. Taylor, P.; Shyong, Y.J.; Samskey, N.; Ho, K.Y.; Radić, Z.; Fenical, W.; Sharpless, K.B.; Kovarik, Z.; Camacho-Hernandez, G.A. Ligand design for human acetylcholinesterase and nicotinic acetylcholine receptors, extending beyond the conventional and canonical. *J. Neurochem.* **2021**, *158*, 1217–1222. [CrossRef] [PubMed]

93. Kem, W.R.; Andrud, K.; Bruno, G.; Xing, H.; Soti, F.; Talley, T.T.; Taylor, P. Interactions of nereistoxin and its analogs with vertebrate nicotinic acetylcholine receptors and molluscan ACh binding proteins. *Mar. Drugs* **2022**, *20*, 49. [CrossRef]

94. Sine, S.M.; Taylor, P. Relationship between reversible antagonist occupancy and the functional capacity of the acetylcholine receptor. *J. Biol. Chem.* **1981**, *56*, 6692–6699. [CrossRef]

95. Bouzat, C. New insights into the structural bases of activation of Cys-loop receptors. *J. Physiol. Paris* **2012**, *106*, 23–33. [CrossRef] [PubMed]

96. Dumont, J.N. Oogenesis in *Xenopus laevis* (Daudin). I. Stages of oocyte development in laboratory maintained animals. *J. Morphol.* **1972**, *136*, 153–179. [CrossRef] [PubMed]

97. Villeneuve, P.; Esnault, G.; Benoit, E.; Molgó, J.; Aráoz, R. A monitoring study of repetitive surgical oocyte harvest in *Xenopus laevis*. In *Toxins and Ion Transfers*; Collection Meetings in Toxinology; Barbier, J., Benoit, E., Gilles, N., Ladant, D., Martin-Eauclaire, M.F., Mattei, C., Molgó, J., Popoff, M.R., Servent, D., Eds.; SFET Publication: Gif-sur-Yvette, France, 2011; pp. 173–178. ISSN 1760-6004. Available online: http://sfet.asso.fr/international/e-book-rt/e-book-rt.html (accessed on 27 November 2011).

98. Kharrat, R.; Servent, D.; Girard, E.; Ouanounou, G.; Amar, M.; Marrouchi, R.; Benoit, E.; Molgó, J. The marine phycotoxin gymnodimine targets muscular and neuronal nicotinic acetylcholine receptor subtypes with high affinity. *J. Neurochem.* **2008**, *107*, 952–963. [CrossRef] [PubMed]

99. Krieger, F.; Mourot, A.; Aráoz, R.; Kotzyba-Hibert, F.; Molgó, J.; Bamberg, E.; Goeldner, M. Fluorescent agonists for the *Torpedo* nicotinic acetylcholine receptor. *ChemBioChem* **2008**, *9*, 1146–1153. [CrossRef] [PubMed]

100. Aráoz, R.; Ouanounou, G.; Iorga, B.I.; Goudet, A.; Alili, D.; Amar, M.; Benoit, E.; Molgó, J.; Servent, D. The neurotoxic effect of 13,19-didesmethyl and 13-desmethyl spirolidone C phycotoxins is mainly mediated by nicotinic rather than muscarinic acetylcholine receptors. *Toxicol. Sci.* **2015**, *147*, 156–167. [CrossRef]
101. Miledi, R.; Palma, E.; Eusebi, F. Microtransplantation of neurotransmitter receptors from cells to *Xenopus* oocyte membranes: New procedure for ion channel studies. *Methods Mol. Biol.* **2006**, *322*, 347–355. [CrossRef]

102. Aráoz, R.; Servent, D.; Molgó, J.; Iorga, B.I.; Fruchart-Gaillard, C.; Benoit, E.; Gu, Z.; Stivala, C.; Zakarian, A. Total synthesis of pinnatoxins A and G, and revision of the mode of action of pinnatoxin A. *J. Am. Chem. Soc.* **2011**, *133*, 10499–10511. [CrossRef]

103. Sands, S.B.; Costa, A.C.; Patrick, J.W. Barium permeability of neuronal nicotinic receptor alpha 7 expressed in *Xenopus* oocytes. *Biophys. J.* **1993**, *65*, 2614–2621. [CrossRef] [PubMed]

104. Cieslikiewicz-Bouet, M.; Naldi, M.; Bartolini, M.; Pérez, B.; Servent, D.; Jean, L.; Aráoz, R.; Renard, P.-Y. Functional characterization of multifunctional ligands targeting acetylcholinesterase and alpha 7 nicotinic acetylcholine receptor. *Biochem. Pharmacol.* **2020**, *177*, 114010. [CrossRef]

105. Kabsch, W. XDS. *Acta Crystallogr. D Biol. Crystallogr.* **2010**, *66*, 125–132. [CrossRef]

106. Winn, M.D.; Ballard, C.C.; Cowtan, K.D.; Dodson, E.J.; Emsley, P.; Evans, P.R.; Keegan, R.M.; Krissinel, E.B.; Leslie, A.G.; McCoy, A.; et al. Overview of the CCP4 suite and current developments. *Acta Crystallogr. D Biol. Crystallogr.* **2011**, *67*, 235–242. [CrossRef] [PubMed]

107. Evans, P. Scaling and assessment of data quality. *Acta Crystallogr. D Biol. Crystallogr.* **2006**, *62*, 72–82. [CrossRef] [PubMed]

108. Evans, P.R.; Murshudov, G.N. How good are my data and what is the resolution? *Acta Crystallogr. D Biol. Crystallogr.* **2013**, *69*, 1204–1214. [CrossRef] [PubMed]

109. French, S.; Wilson, K. On the treatment of negative intensity observations. *Acta Crystallogr.* **1978**, *34*, 517–525. [CrossRef]

**Disclaimer/Publisher's Note:** The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.
101. Miledi, R.; Palma, E.; Eusebi, F. Microtransplantation of neurotransmitter receptors from cells to *Xenopus* oocyte membranes: New procedure for ion channel studies. *Methods Mol. Biol.* **2006**, *322*, 347–355. [CrossRef]

102. Aráoz, R.; Servent, D.; Molgó, J.; Iorga, B.I.; Fruchart-Gaillard, C.; Benoit, E.; Gu, Z.; Stivala, C.; Zakarian, A. Total synthesis of pinnatoxins A and G, and revision of the mode of action of pinnatoxin A. *J. Am. Chem. Soc.* **2011**, *133*, 10499–10511. [CrossRef]

103. Sands, S.B.; Costa, A.C.; Patrick, J.W. Barium permeability of neuronal nicotinic receptor alpha 7 expressed in *Xenopus* oocytes. *Biophys. J.* **1993**, *65*, 2614–2621. [CrossRef] [PubMed]

104. Cieslikiewicz-Bouet, M.; Naldi, M.; Bartolini, M.; Pérez, B.; Servent, D.; Jean, L.; Aráoz, R.; Renard, P.-Y. Functional characterization of multifunctional ligands targeting acetylcholinesterase and alpha 7 nicotinic acetylcholine receptor. *Biochem. Pharmacol.* **2020**, *177*, 114010. [CrossRef]

105. Kabsch, W. XDS. *Acta Crystallogr. D Biol. Crystallogr.* **2010**, *66*, 125–132. [CrossRef]

106. Winn, M.D.; Ballard, C.C.; Cowtan, K.D.; Dodson, E.J.; Emsley, P.; Evans, P.R.; Keegan, R.M.; Krissinel, E.B.; Leslie, A.G.; McCoy, A.; et al. Overview of the CCP4 suite and current developments. *Acta Crystallogr. D Biol. Crystallogr.* **2011**, *67*, 235–242. [CrossRef] [PubMed]

107. Evans, P. Scaling and assessment of data quality. *Acta Crystallogr. D Biol. Crystallogr.* **2006**, *62*, 72–82. [CrossRef] [PubMed]

108. Evans, P.R.; Murshudov, G.N. How good are my data and what is the resolution? *Acta Crystallogr. D Biol. Crystallogr.* **2013**, *69*, 1204–1214. [CrossRef] [PubMed]

109. French, S.; Wilson, K. On the treatment of negative intensity observations. *Acta Crystallogr.* **1978**, *34*, 517–525. [CrossRef]

**Disclaimer/Publisher's Note:** The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.
Supplementary Material

# The Cyclic Imine Core Common to the Marine Macrocyclic Toxins Is Sufficient to Dictate Nicotinic Acetylcholine Receptor Antagonism

Yves Bourne <fnref n="1" />, Gerlind Sulzenbacher <fnref n="1" />, Laurent Chabaud <fnref n="2" />,†, Rómulo Aráoz <fnref n="3" />, Zoran Radić <fnref n="4" />, Sandrine Conrod <fnref n="5" />,‡,
Palmer Taylor <fnref n="4" />, Catherine Guillou <fnref n="2" />, Jordi Molgó <fnref n="3" />, Pascale Marchot <fnref n="1" />,<fnref n="5" />,*

<footnote n="1">¹ Lab "Architecture et Fonction des Macromolécules Biologiques" (AFMB), Aix-Marseille Univ, CNRS, Faculté des Sciences Campus Luminy, 13288 Marseille cedex 09, France; yves.bourne@univ-amu.fr (Y.B.); gerlind.sulzenbacher@univ-amu.fr (G.S.)</footnote>

<footnote n="2">² Institut de Chimie des Substances Naturelles (ICSN), Univ Paris-Saclay, CNRS, 91198 Gif-sur-Yvette, France; catherine.guillou@cnrs.fr</footnote>

<footnote n="3">³ Service d'Ingénierie Moléculaire pour la Santé (SIMoS) EMR CNRS 9004, Département Médicaments et Technologies pour la Santé, Institut des Sciences du Vivant Frédéric Joliot, CEA, INRAE, Université Paris-Saclay, 91191 Gif-sur-Yvette, France; romulo.araoz@cea.fr (R.A.); jordi.molgo@cea.fr (J.M.)</footnote>

<footnote n="4">⁴ Skaggs School of Pharmacy and Pharmaceutical Sciences (SSPPS), University of California San Diego, La Jolla, CA, 92093-0751, USA; zradic@health.ucsd.edu (Z.R.); pwtaylor@health.ucsd.edu (P.T.)</footnote>

<footnote n="5">5 Centre de Recherche en Neurobiologie et Neurophysiologie de Marseille (CRN2M), Aix Marseille Univ, CNRS, 13344 Marseille, France</footnote>

<footnote>† Current address: Institut des Sciences Moléculaires (ISM), Univ Bordeaux, CNRS, Bordeaux INP, 33400 Talence, France; laurent.chabaud@u-bordeaux.fr</footnote>

<footnote>‡ Current address: CEREGE, Aix-Marseille Univ, CNRS, IRD, Collège de France, INRAE, 13545 Aix-en-Provence, France; conrod@cerege.fr</footnote>

<footnote>* Correspondence: pascale.marchot@univ-amu.fr</footnote>
Supplementary Material

# The Cyclic Imine Core Common to the Marine Macrocyclic Toxins Is Sufficient to Dictate Nicotinic Acetylcholine Receptor Antagonism

Yves Bourne <fnref n="1" />, Gerlind Sulzenbacher <fnref n="1" />, Laurent Chabaud <fnref n="2" />,†, Rómulo Aráoz <fnref n="3" />, Zoran Radić <fnref n="4" />, Sandrine Conrod <fnref n="5" />,‡,
Palmer Taylor <fnref n="4" />, Catherine Guillou <fnref n="2" />, Jordi Molgó <fnref n="3" />, Pascale Marchot <fnref n="1" />,<fnref n="5" />,*

<footnote n="1">¹ Lab "Architecture et Fonction des Macromolécules Biologiques" (AFMB), Aix-Marseille Univ, CNRS, Faculté des Sciences Campus Luminy, 13288 Marseille cedex 09, France; yves.bourne@univ-amu.fr (Y.B.); gerlind.sulzenbacher@univ-amu.fr (G.S.)</footnote>

<footnote n="2">² Institut de Chimie des Substances Naturelles (ICSN), Univ Paris-Saclay, CNRS, 91198 Gif-sur-Yvette, France; catherine.guillou@cnrs.fr</footnote>

<footnote n="3">³ Service d'Ingénierie Moléculaire pour la Santé (SIMoS) EMR CNRS 9004, Département Médicaments et Technologies pour la Santé, Institut des Sciences du Vivant Frédéric Joliot, CEA, INRAE, Université Paris-Saclay, 91191 Gif-sur-Yvette, France; romulo.araoz@cea.fr (R.A.); jordi.molgo@cea.fr (J.M.)</footnote>

<footnote n="4">⁴ Skaggs School of Pharmacy and Pharmaceutical Sciences (SSPPS), University of California San Diego, La Jolla, CA, 92093-0751, USA; zradic@health.ucsd.edu (Z.R.); pwtaylor@health.ucsd.edu (P.T.)</footnote>

<footnote n="5">5 Centre de Recherche en Neurobiologie et Neurophysiologie de Marseille (CRN2M), Aix Marseille Univ, CNRS, 13344 Marseille, France</footnote>

<footnote>† Current address: Institut des Sciences Moléculaires (ISM), Univ Bordeaux, CNRS, Bordeaux INP, 33400 Talence, France; laurent.chabaud@u-bordeaux.fr</footnote>

<footnote>‡ Current address: CEREGE, Aix-Marseille Univ, CNRS, IRD, Collège de France, INRAE, 13545 Aix-en-Provence, France; conrod@cerege.fr</footnote>

<footnote>* Correspondence: pascale.marchot@univ-amu.fr</footnote>
<figure><img src="image_18.png" /><figcaption>**Figure S1.** ¹H NMR spectrum (CDCl₃) of 2-acetyl-2-(3-azidopropyl)cyclohexanone (±)-2.</figcaption></figure>

<figure><img src="image_19.png" /><figcaption>**Figure S2.** ¹³C NMR spectrum (CDCl₃) of 2-acetyl-2-(3-azidopropyl)cyclohexanone (±)-2.</figcaption></figure>
<figure><img src="image_19.png" /><figcaption>**Figure S1.** ¹H NMR spectrum (CDCl₃) of 2-acetyl-2-(3-azidopropyl)cyclohexanone (±)-2.</figcaption></figure>

<figure><img src="image_20.png" /><figcaption>**Figure S2.** ¹³C NMR spectrum (CDCl₃) of 2-acetyl-2-(3-azidopropyl)cyclohexanone (±)-2.</figcaption></figure>
<figure><img src="image_20.png" /><figcaption>**Figure S3.** ¹H NMR spectrum (CDCl₃) of 1-[6-(3-azidopropyl)-1,4-dioxaspiro[4.5]dec-6-yl]ethanone (±)-3.</figcaption></figure>

<figure><img src="image_21.png" /><figcaption>**Figure S4.** ¹³C NMR spectrum (CDCl₃) of 1-[6-(3-azidopropyl)-1,4-dioxaspiro[4.5]dec-6-yl]ethanone (±)-3.</figcaption></figure>
<figure><img src="image_21.png" /><figcaption>**Figure S3.** ¹H NMR spectrum (CDCl₃) of 1-[6-(3-azidopropyl)-1,4-dioxaspiro[4.5]dec-6-yl]ethanone (±)-3.</figcaption></figure>

<figure><img src="image_22.png" /><figcaption>**Figure S4.** ¹³C NMR spectrum (CDCl₃) of 1-[6-(3-azidopropyl)-1,4-dioxaspiro[4.5]dec-6-yl]ethanone (±)-3.</figcaption></figure>
<figure><img src="image_22.png" /><figcaption>**Figure S5.** ¹H NMR spectrum (CDCl₃) of 7-methyl-1,4-dioxa-8-azadispiro[4.0.5.4]pentadec-7-ene (±)-4.</figcaption></figure>

<figure><img src="image_23.png" /><figcaption>**Figure S6.** ¹³C NMR spectrum (CDCl₃) of 7-methyl-1,4-dioxa-8-azadispiro[4.0.5.4]pentadec-7-ene (±)-4.</figcaption></figure>
<figure><img src="image_23.png" /><figcaption>**Figure S5.** ¹H NMR spectrum (CDCl₃) of 7-methyl-1,4-dioxa-8-azadispiro[4.0.5.4]pentadec-7-ene (±)-4.</figcaption></figure>

<figure><img src="image_24.png" /><figcaption>**Figure S6.** ¹³C NMR spectrum (CDCl₃) of 7-methyl-1,4-dioxa-8-azadispiro[4.0.5.4]pentadec-7-ene (±)-4.</figcaption></figure>
<figure><img src="image_24.png" /><figcaption>**Figure S7.** Separation of enantiomers (+)-4 and (−)-4 from the (±)-4 racemate using chiral HPLC (Chiralcel® OD 10 µm 4.6x250 mm column, Hept/EtOH 80:20 (v/v), flow 1 mL/min).</figcaption></figure>
<figure><img src="image_25.png" /><figcaption>**Figure S7.** Separation of enantiomers (+)-4 and (−)-4 from the (±)-4 racemate using chiral HPLC (Chiralcel® OD 10 µm 4.6x250 mm column, Hept/EtOH 80:20 (v/v), flow 1 mL/min).</figcaption></figure>
<table><caption>**Table S1.** X-ray crystallography - Data collection and refinement statistics.</caption><thead><tr><th></th><th>Spiroimine (+)-4 R</th><th>Spiroimine (-)-4 S</th><th>Racemic (±)-4</th></tr></thead><tbody><tr><td>Beamline</td><td>ESRF ID14-EH1</td><td>SOLEIL Proxima-1</td><td>ESRF ID23-1</td></tr><tr><td><strong>Data collection</strong></td><td></td><td></td><td></td></tr><tr><td>Space group</td><td>P2<sub>1</sub>2<sub>1</sub>2<sub>1</sub></td><td>P2<sub>1</sub>2<sub>1</sub>2<sub>1</sub></td><td>P2<sub>1</sub>2<sub>1</sub>2<sub>1</sub></td></tr><tr><td>Cell dimensions a, b, c (Å)</td><td>87.98, 115, 129.96</td><td>88.34, 116.03, 131.04</td><td>75.79, 123.63, 131.1</td></tr><tr><td>Resolution <sup>a</sup> (Å)</td><td>48.13-2.00 (2.04-2.00)</td><td>48.49-1.85 (1.88-1.85)</td><td>65-2.10 (2.15-2.10)</td></tr><tr><td>No. reflections</td><td>89606 (4509)</td><td>109510 (5565)</td><td>72272 (4374)</td></tr><tr><td>CC (1/2)</td><td>0.999 (0.699)</td><td>1.0 (0.618)</td><td>0.993 (0.687)</td></tr><tr><td>R<sub>meas</sub><sup>b</sup></td><td>0.087 (0.959)</td><td>0.050 (0.954)</td><td>0.128 (0.720)</td></tr><tr><td>I / σI</td><td>15.5 (1.8)</td><td>22.2 (1.7)</td><td>8.7 (2.1)</td></tr><tr><td>Completeness (%)</td><td>100 (99.9)</td><td>94.9 (98.3)</td><td>99.7 (100)</td></tr><tr><td>Redundancy</td><td>6.2 (6.2)</td><td>7.0 (4.4)</td><td>3.5 (2.6)</td></tr><tr><td>Wilson B (Å<sup>2</sup>)</td><td>23.85</td><td>32.22</td><td>21.85</td></tr><tr><td><strong>Refinement</strong></td><td></td><td></td><td></td></tr><tr><td>Resolution (Å)</td><td>48.18-2.00 (2.052-2.00)</td><td>47.97-1.85 (1.88-1.85)</td><td>65-2.1 (2.15-2.1)</td></tr><tr><td>No. reflections working set</td><td>84991 (6197)</td><td>103900 (7900)</td><td>70046 (5108)</td></tr><tr><td>No. reflections test set</td><td>4537 (317)</td><td>5509 (434)</td><td>2180 (148)</td></tr><tr><td>R<sub>work</sub> / R<sub>free</sub><sup>c</sup></td><td>0.1712/0.2060</td><td>0.1820/0.2114</td><td>0.18/0.21</td></tr><tr><td>No. atoms</td><td></td><td></td><td></td></tr><tr><td>Protein + N-glycans</td><td>8457</td><td>8440</td><td>8560</td></tr><tr><td>Ligands/Ions</td><td>85</td><td>90</td><td>80</td></tr><tr><td>Water</td><td>784</td><td>706</td><td>520</td></tr><tr><td>B-factors</td><td></td><td></td><td></td></tr><tr><td>Protein + N-glycans, main/side</td><td>32.92/38.29</td><td>38.67/43.39</td><td>33.7/38.5</td></tr><tr><td>Ligands/Ions</td><td>40</td><td>66.83</td><td>-</td></tr><tr><td>Water</td><td>37.38</td><td>42.06</td><td>36.4</td></tr><tr><td>R.m.s. deviations <sup>d</sup></td><td></td><td></td><td></td></tr><tr><td>Bond lengths (Å)</td><td>0.008</td><td>0.008</td><td>0.007</td></tr><tr><td>Bond angles (°)</td><td>1.472</td><td>1.447</td><td>1.271</td></tr><tr><td>Ramachandran</td><td></td><td></td><td></td></tr><tr><td>Favoured (%)</td><td>98.94</td><td>99.04</td><td>98.5</td></tr><tr><td>Allowed (%)</td><td>1.06</td><td>0.96</td><td>1.5</td></tr><tr><td>Disallowed (%)</td><td>0</td><td>0</td><td>0</td></tr><tr><td>PDB accession code</td><td>8Q1M</td><td>8QTL</td><td>8QX2</td></tr></tbody></table>

<footnote><sup>a</sup> Values in parentheses are those for the highest-resolution shell. <sup>b</sup> R<sub>meas</sub> = ΣhΣi(n<sub>h</sub>/(n<sub>h</sub> - 1))<sup>1/2</sup>|I<sub>hi</sub> - <I>h</sub>|/ΣhΣi<sub>h</sub>, where I is an individual reflection measurement and <I>h</i> is the mean intensity for symmetry-related reflections. <sup>c</sup> R<sub>work</sub> = Σhkl|Fo(hkl)| - |Fc|/Σhkl|Fo(hkl)|, where Fo and Fc are observed and calculated structure factors, respectively. R<sub>free</sub> is calculated for 5% of randomly selected reflections excluded from refinement. <sup>d</sup> Root-mean-square deviation from ideal values.</footnote>
<table><caption>**Table S1.** X-ray crystallography - Data collection and refinement statistics.</caption><thead><tr><th></th><th>Spiroimine (+)-4 R</th><th>Spiroimine (-)-4 S</th><th>Racemic (±)-4</th></tr></thead><tbody><tr><td>Beamline</td><td>ESRF ID14-EH1</td><td>SOLEIL Proxima-1</td><td>ESRF ID23-1</td></tr><tr><td><strong>Data collection</strong></td><td></td><td></td><td></td></tr><tr><td>Space group</td><td>P2<sub>1</sub>2<sub>1</sub>2<sub>1</sub></td><td>P2<sub>1</sub>2<sub>1</sub>2<sub>1</sub></td><td>P2<sub>1</sub>2<sub>1</sub>2<sub>1</sub></td></tr><tr><td>Cell dimensions a, b, c (Å)</td><td>87.98, 115, 129.96</td><td>88.34, 116.03, 131.04</td><td>75.79, 123.63, 131.1</td></tr><tr><td>Resolution <sup>a</sup> (Å)</td><td>48.13-2.00 (2.04-2.00)</td><td>48.49-1.85 (1.88-1.85)</td><td>65-2.10 (2.15-2.10)</td></tr><tr><td>No. reflections</td><td>89606 (4509)</td><td>109510 (5565)</td><td>72272 (4374)</td></tr><tr><td>CC (1/2)</td><td>0.999 (0.699)</td><td>1.0 (0.618)</td><td>0.993 (0.687)</td></tr><tr><td>R<sub>meas</sub><sup>b</sup></td><td>0.087 (0.959)</td><td>0.050 (0.954)</td><td>0.128 (0.720)</td></tr><tr><td>I / σI</td><td>15.5 (1.8)</td><td>22.2 (1.7)</td><td>8.7 (2.1)</td></tr><tr><td>Completeness (%)</td><td>100 (99.9)</td><td>94.9 (98.3)</td><td>99.7 (100)</td></tr><tr><td>Redundancy</td><td>6.2 (6.2)</td><td>7.0 (4.4)</td><td>3.5 (2.6)</td></tr><tr><td>Wilson B (Å<sup>2</sup>)</td><td>23.85</td><td>32.22</td><td>21.85</td></tr><tr><td><strong>Refinement</strong></td><td></td><td></td><td></td></tr><tr><td>Resolution (Å)</td><td>48.18-2.00 (2.052-2.00)</td><td>47.97-1.85 (1.88-1.85)</td><td>65-2.1 (2.15-2.1)</td></tr><tr><td>No. reflections working set</td><td>84991 (6197)</td><td>103900 (7900)</td><td>70046 (5108)</td></tr><tr><td>No. reflections test set</td><td>4537 (317)</td><td>5509 (434)</td><td>2180 (148)</td></tr><tr><td>R<sub>work</sub> / R<sub>free</sub><sup>c</sup></td><td>0.1712/0.2060</td><td>0.1820/0.2114</td><td>0.18/0.21</td></tr><tr><td>No. atoms</td><td></td><td></td><td></td></tr><tr><td>Protein + N-glycans</td><td>8457</td><td>8440</td><td>8560</td></tr><tr><td>Ligands/Ions</td><td>85</td><td>90</td><td>80</td></tr><tr><td>Water</td><td>784</td><td>706</td><td>520</td></tr><tr><td>B-factors</td><td></td><td></td><td></td></tr><tr><td>Protein + N-glycans, main/side</td><td>32.92/38.29</td><td>38.67/43.39</td><td>33.7/38.5</td></tr><tr><td>Ligands/Ions</td><td>40</td><td>66.83</td><td>-</td></tr><tr><td>Water</td><td>37.38</td><td>42.06</td><td>36.4</td></tr><tr><td>R.m.s. deviations <sup>d</sup></td><td></td><td></td><td></td></tr><tr><td>Bond lengths (Å)</td><td>0.008</td><td>0.008</td><td>0.007</td></tr><tr><td>Bond angles (°)</td><td>1.472</td><td>1.447</td><td>1.271</td></tr><tr><td>Ramachandran</td><td></td><td></td><td></td></tr><tr><td>Favoured (%)</td><td>98.94</td><td>99.04</td><td>98.5</td></tr><tr><td>Allowed (%)</td><td>1.06</td><td>0.96</td><td>1.5</td></tr><tr><td>Disallowed (%)</td><td>0</td><td>0</td><td>0</td></tr><tr><td>PDB accession code</td><td>8Q1M</td><td>8QTL</td><td>8QX2</td></tr></tbody></table>

<footnote><sup>a</sup> Values in parentheses are those for the highest-resolution shell. <sup>b</sup> R<sub>meas</sub> = ΣhΣi(n<sub>h</sub>/(n<sub>h</sub> - 1))<sup>1/2</sup> |I<sub>hi</sub> - <I>h</sub>| / ΣhΣi<sub>h</sub>, where I is an individual reflection measurement and <I>h</i> is the mean intensity for symmetry-related reflections. <sup>c</sup> R<sub>work</sub> = Σhkl|Fo(hkl)| - |Fc| / Σhkl|Fo(hkl)|, where Fo and Fc are observed and calculated structure factors, respectively. R<sub>free</sub> is calculated for 5% of randomly selected reflections excluded from refinement. <sup>d</sup> Root-mean-square deviation from ideal values.</footnote>
<table><caption>**Table S2.** Geometric parameters of key interactions between the (+)-4 R and (-)-4 S enantiomers and side chains in the A-AChBP binding pocket.</caption><thead><tr><th rowspan="3">Subunit interface</th><th colspan="3">Spiroimine (+)-4 R</th><th colspan="3">Spiroimine (-)-4 S</th></tr><tr><th colspan="3">Type of interaction</th><th colspan="3">Type of interaction</th></tr><tr><th>H-bonds*</th><th>π-π stacking</th><th>van der Waals**</th><th>H-bonds*</th><th>π-π stacking</th><th>van der Waals**</th></tr></thead><tbody><tr><td>A-B</td><td>N3-W147<br>carbonyl oxygen<br>2.6 Å / 140.7°</td><td>Cyclic imine-<br>W147<br>face-to-face</td><td>Y93, Y188,<br>C190, Y195<br>Y55, I118,<br>S167</td><td>N3-W147<br>carbonyl oxygen<br>2.9 Å / 123.8°</td><td>Cyclic imine-<br>W147<br>T-shaped</td><td>Y93, Y188,<br>C190, C191,<br>Y195<br>Y55, I118</td></tr><tr><td>B-C</td><td>N3-W147<br>carbonyl oxygen<br>2.7 Å / 144.2°</td><td>Cyclic imine-<br>W147<br>face-to-face</td><td>Y93, Y188,<br>C190, Y195<br>Y55, I118</td><td>N3-W147<br>carbonyl oxygen<br>3.5 Å / 121.2°</td><td>Cyclic imine-<br>W147<br>T-shaped</td><td>Y93, Y188,<br>C190, C191,<br>Y195<br>Y55, I118</td></tr><tr><td>C-D</td><td>N3-W147<br>carbonyl oxygen<br>2.7 Å / 142°</td><td>Cyclic imine-<br>W147<br>face-to-face</td><td>Y93, Y188,<br>C190, Y195<br>Y55, I118</td><td>N3-W147<br>carbonyl oxygen<br>2.8 Å / 125.3°</td><td>Cyclic imine-<br>W147<br>T-shaped</td><td>Y93, Y188,<br>C190, C191,<br>Y195<br>Y55, I118</td></tr><tr><td>D-E</td><td>N3-W147<br>carbonyl oxygen<br>2.7 Å / 144.4°</td><td>Cyclic imine-<br>W147<br>face-to-face</td><td>Y93, Y188,<br>C190, Y195<br>Y55, I118</td><td>N3-W147<br>carbonyl oxygen<br>3.0 Å / 126.9°</td><td>Cyclic imine-<br>W147<br>T-shaped</td><td>Y93, Y188,<br>C190, C191,<br>Y195<br>Y55, I118</td></tr><tr><td>E-A</td><td>N3-W147<br>carbonyl oxygen<br>2.7 Å / 142.1°</td><td>Cyclic imine-<br>W147<br>face-to-face</td><td>Y93, Y188,<br>C190, Y195<br>Y55, I118</td><td>N3-W147<br>carbonyl oxygen<br>2.9 Å / 125.6°</td><td>Cyclic imine-<br>W147<br>T-shaped</td><td>Y93, Y188,<br>C190, C191,<br>Y195<br>Y55, I118</td></tr></tbody></table>

<footnote>*The angle value refers to the *H* angle between the hydrogen bond donor, acceptor and acceptor antecedent.</footnote>

<footnote>**Non-italicized A-AChBP residues Y93, Y188, C190, C191, Y195 belong to the principal, (+) face of the subunit interface, while italicized residues Y55, I118, S167 belong to the complementary, (-) face of the interface (Figure A1).</footnote>
<table><caption>**Table S2.** Geometric parameters of key interactions between the (+)-4 R and (-)-4 S enantiomers and side chains in the A-AChBP binding pocket.</caption><thead><tr><th rowspan="3">Subunit interface</th><th colspan="3">Spiroimine (+)-4 R</th><th colspan="3">Spiroimine (-)-4 S</th></tr><tr><th colspan="3">Type of interaction</th><th colspan="3">Type of interaction</th></tr><tr><th>H-bonds*</th><th>π-π stacking</th><th>van der Waals**</th><th>H-bonds*</th><th>π-π stacking</th><th>van der Waals**</th></tr></thead><tbody><tr><td>A-B</td><td>N3-W147<br>carbonyl oxygen<br>2.6 Å / 140.7°</td><td>Cyclic imine-<br>W147<br>face-to-face</td><td>Y93, Y188,<br>C190, Y195<br>Y55, I118,<br>S167</td><td>N3-W147<br>carbonyl oxygen<br>2.9 Å / 123.8°</td><td>Cyclic imine-<br>W147<br>T-shaped</td><td>Y93, Y188,<br>C190, C191,<br>Y195<br>Y55, I118</td></tr><tr><td>B-C</td><td>N3-W147<br>carbonyl oxygen<br>2.7 Å / 144.2°</td><td>Cyclic imine-<br>W147<br>face-to-face</td><td>Y93, Y188,<br>C190, Y195<br>Y55, I118</td><td>N3-W147<br>carbonyl oxygen<br>3.5 Å / 121.2°</td><td>Cyclic imine-<br>W147<br>T-shaped</td><td>Y93, Y188,<br>C190, C191,<br>Y195<br>Y55, I118</td></tr><tr><td>C-D</td><td>N3-W147<br>carbonyl oxygen<br>2.7 Å / 142°</td><td>Cyclic imine-<br>W147<br>face-to-face</td><td>Y93, Y188,<br>C190, Y195<br>Y55, I118</td><td>N3-W147<br>carbonyl oxygen<br>2.8 Å / 125.3°</td><td>Cyclic imine-<br>W147<br>T-shaped</td><td>Y93, Y188,<br>C190, C191,<br>Y195<br>Y55, I118</td></tr><tr><td>D-E</td><td>N3-W147<br>carbonyl oxygen<br>2.7 Å / 144.4°</td><td>Cyclic imine-<br>W147<br>face-to-face</td><td>Y93, Y188,<br>C190, Y195<br>Y55, I118</td><td>N3-W147<br>carbonyl oxygen<br>3.0 Å / 126.9°</td><td>Cyclic imine-<br>W147<br>T-shaped</td><td>Y93, Y188,<br>C190, C191,<br>Y195<br>Y55, I118</td></tr><tr><td>E-A</td><td>N3-W147<br>carbonyl oxygen<br>2.7 Å / 142.1°</td><td>Cyclic imine-<br>W147<br>face-to-face</td><td>Y93, Y188,<br>C190, Y195<br>Y55, I118</td><td>N3-W147<br>carbonyl oxygen<br>2.9 Å / 125.6°</td><td>Cyclic imine-<br>W147<br>T-shaped</td><td>Y93, Y188,<br>C190, C191,<br>Y195<br>Y55, I118</td></tr></tbody></table>

<footnote>*The angle value refers to the $\hat{H}$ angle between the hydrogen bond donor, acceptor and acceptor antecedent.</footnote>

<footnote>**Non-italicized A-AChBP residues Y93, Y188, C190, C191, Y195 belong to the principal, (+) face of the subunit interface, while italicized residues Y55, I118, S167 belong to the complementary, (-) face of the interface (Figure A1).</footnote>