Natural aminosterols inhibit NMDA receptors with low nanomolar potency
Giulia Fania b, Elisabetta Coppic d, Silvia Erricoa b e f, Federica Cherchic d, Martina Gennaria b, Denise Barbutg h, Michele Vendruscoloe f, MICHAEL A. ZASLOFFg h i j, Anna Maria Pugliesec d, Fabrizio Chitia b✉
- aDepartment of Experimental and Clinical Biomedical Sciences, Section of Biochemistry University of Florence Italy
- bDepartment of Experimental and Clinical Biomedical Sciences, Section of Biochemistry, University of Florence, Italy
- cDepartment of Neuroscience, Psychology, Drug Research and Child Health (NEUROFARBA), Division of Pharmacology and Toxicology University of Florence Italy
- dDepartment of Neuroscience, Psychology, Drug Research and Child Health (NEUROFARBA), Division of Pharmacology and Toxicology, University of Florence, Italy
- eCentre for Misfolding Diseases, Yusuf Hamied Department of Chemistry University of Cambridge UK
- fCentre for Misfolding Diseases, Yusuf Hamied Department of Chemistry, University of Cambridge, UK
- gEnterin Research Institute Inc. Philadelphia PA USA
- hEnterin Research Institute Inc., Philadelphia, PA, USA
- iMedStar‐Georgetown Transplant Institute Georgetown University School of Medicine Washington DC USA
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Abstract
Abnormal functions of N‐methyl‐D‐aspartate receptors (NMDARs) are associated with many brain disorders, making them primary targets for drug discovery. We show that natural aminosterols inhibit the NMDAR‐mediated increase of intracellular calcium ions in cultured primary neurons and neuroblastoma cells. Structural comparison with known NMDAR‐negative allosteric modulators, such as pregnanolone‐sulfate‐2 (PAS), raises the hypothesis that aminosterols have the same mechanism of action. Fluorescence resonance energy transfer (FRET) measurements using labeled NMDAR and the labeled aminosterol trodusquemine (TRO) indicate close spatial proximity, likely arising from binding. Other indirect yet plausible mechanisms for NMDAR inhibition by TRO were excluded. Electrophysiological patch clamp measurements on primary neurons indicate that pre‐incubated TRO inhibits NMDA‐induced ion currents with a IC 50 of 5 n m . Inhibition is observed only after cell membrane pre‐adsorption, indicating accessibility to NMDAR from the cell membrane and binding to the transmembrane domains (TMDs) and TMD‐ligand‐binding domain (LBD) linkers, similarly to PAS. The TRO IC 50 is 5000‐fold higher than that of PAS and 20–16 000 times higher than those of other inhibitors binding to TMD/TMD‐LBD regions, identifying aminosterols as promising and potent NMDAR modulators.