# Natural aminosterols inhibit NMDA receptors with low nanomolar potency

## TL;DR

Natural aminosterols like trodusquemine inhibit NMDA receptors in brain cells with low nanomolar potency, offering a new approach for treating brain disorders.

## Contribution

Aminosterols are identified as potent NMDA receptor inhibitors with a novel mechanism involving cell membrane binding.

## Key findings

- Trodusquemine inhibits NMDA-induced ion currents with an IC50 of 5 nm.
- Aminosterols bind to transmembrane domains and TMD-ligand-binding domain linkers of NMDARs.
- TRO's IC50 is significantly higher than other NMDAR inhibitors, indicating its unique potency.

## 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 nm. 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 IC50 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.

Misfunction of N‐methyl‐D‐aspartate (NMDA) receptors is associated with many brain disorders, making them primary targets for drug discovery. Trodusquemine and other natural aminosterols inhibit NMDA receptors with low nanomolar potency through binding to the cell membranes followed by interaction with the channels. The IC50 value is 5000‐fold higher than that of the structurally related pregnanolone‐sulfate‐2 (PAS) and 20–16 000 times higher than inhibitors binding to the TMD/TMD‐LBD regions of the receptors.