Neurosteroids are steroidal compounds produced by the nervous tissue and thought to regulate neural activity locally through direct modulation of membrane receptors. NMDA receptors, in contrast to AMPA/kainate receptors, are significantly influenced by neurosteroids. Pregnanolone sulfate (PA-S) inhibits NMDA receptors, while structurally similar pregnenolone sulfate (PE-S) has a potentiating effect. We are focusing on the following areas.

Molecular mechanism of steroid action at NMDA receptors

Our goal is to identify the site(s) and the molecular mechanism(s) of steroid action at the NMDA receptor. Our results suggest that PA-S binds in the extracellular vestibule of the ion channel pore. Kinetic analysis of NMDA receptor inhibition by PA-S and its synthetic analogues suggested that these compounds may inhibit tonically activated NMDA receptors more potently than synaptically activated receptors, a pharmacological profile that would be very useful clinically. Our preliminary results confirm these predictions and suggest that PA-S is neuroprotective without psychomimetic side effects characteristic of other NMDA receptor inhibitors. We are also trying to identify the binding site for neurosteroids with potentiating action at the NMDA receptor.

Role of cytoplasmic membrane in the access and action of steroids at the NMDA receptor

NMDA receptors are transmembrane protein structures and their membrane domains are in direct in contact with and influenced by membrane lipids. Neurosteroids at concentrations active at NMDA receptors form micelles in aqueous solutions. We have developed a model describing the conversion of micellar steroid to its molecular form. Using molecular dynamics simulations we continue to study the interactions of steroids with the membrane.

Relationship between the structure and the function of NMDA receptor-modulating steroids

In collaboration with Eva Kudová, Ph.D. and Hana Chodounská, Ph.D. (IOCB, CAS) we are testing the properties of synthetic analogues of PA-S and PE-S. One of our goals is to identify the structural basis for steroid inhibitory vs. potentiating action at NMDA receptors. In addition, we are searching for highly potent steroids; out of ~200 newly synthesized compounds, several are active at nM concentrations. The most promising compounds are subsequently tested in behavioral assays.

Modeling of NMDA receptors

Trying to complement the above mentioned experimental approaches we study the structure function relationships of NMDA receptors also theoretically. A combination of tools of molecular modeling is employed for understanding the molecular mechanism of opening and modulation of function by interaction with neurosteroids and membrane components. The tools we are using cover the homology modeling, docking of proteins and neurosteroid ligands, molecular dynamics simulation as well as accurate quantum chemical calculations. The computational approach is realized by Jiří Černý, Ph.D. and Michaela Nekardová (Faculty of Mathematics and Physics, Charles University in Prague).