Neurosteroid analogues produce general anesthesia by binding to GABA-A receptors and enhancing neuronal inhibition in the brain. The number of binding sites for neurosteroids on GABA-A receptors and their molecular localization has been incompletely defined and is based on indirect methods. Neurosteroids are also endogenous mediators of brain development and function whose sites of action remain undefined. This project will use photolabeling techniques to define the precise sites of neurosteroid binding on GABA-A receptors. Novel "click chemistry"-tagged, neurosteroid analogue photolabeling reagents will be developed and used in conjunction with isotopically tagged, "click chemistry" bifunctional linkers, to purify photolabeled proteins and peptides, providing the sensitivity and specificity to use high resolution mass spectrometry to detect and sequence low-abundance photolabeled peptides in biological samples. These techniques will also be applied as a discovery tool to identify additional proteins in brain that have neurosteroid binding sites. The Project has three specific aims:
In Aim 1, novel neurosteroid analogue photolabeling reagents containing an alkyne group and isotopically-tagged, bifunctional linker molecules containing both an azide group and an affinity tag will be synthesized for use in "click chemistry"-based visualization and purification of photolabeled proteins and peptides (Aims 2 and 3). The isotopic tag will provide a "signature" for specific mass spectrometric detection of photolabeled peptides. Neurosteroid analogues will be synthesized that differ in stereochemistry, mechanism of photolabeling and the location of the diazirine photolabeling moiety on the neurosteroid backbone.
In Aim 2 the number and structure of the neurosteroid binding sites on a defined GABA-A receptor (?1?3) will be assessed by photolabeling with the neurosteroid analogues prepared in Aim 1. Incorporating this photolabeling data in a molecular model of the receptor will define the geometry of the binding site(s).
Aim 2 will also quantitatively analyze which GABA-A receptor subunits are photolabeled by neurosteroids in native brain tissue.
In Aim 3, photolabeling will be used to identify additional proteins that have neurosteroid binding sites. Whole brain homogenates will be photolabeled with neurosteroid analogue photolabeling reagents and the labeled proteins will be identified either: 1) by using "click chemistry"-based enrichment of photolabeled proteins in conjunction with global proteomic methods or;2) by using bifunctional fluorescent linkers to track the photolabeled proteins through electrophoretic separation, and then identifying the proteins using gel-based mass spectrometry. The proposed work is expected to identify the sites of neurosteroid binding on GABA-A receptors and to discover new neurosteroid binding proteins. These data will provide a structural template for future synthetic chemistry and suggest novel mechanisms of neurosteroid action and potential pharmacological targets.

Public Health Relevance

Neurosteroids are important both as drugs and as naturally occurring substances that influence brain development and behavior. The proposed research will define the molecular sites of action for the anesthetic effects of neurosteroids and will identify novel neurosteroid binding proteins. The work will provide the basis for synthesizing more specific anesthetic and sedative drugs and will provide direction for new investigation in the biology of naturally occurring neurosteroids.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
1R01GM108799-01
Application #
8610535
Study Section
Molecular Neuropharmacology and Signaling Study Section (MNPS)
Program Officer
Cole, Alison E
Project Start
2014-05-01
Project End
2018-01-31
Budget Start
2014-05-01
Budget End
2015-01-31
Support Year
1
Fiscal Year
2014
Total Cost
$409,659
Indirect Cost
$140,834
Name
Washington University
Department
Anesthesiology
Type
Schools of Medicine
DUNS #
068552207
City
Saint Louis
State
MO
Country
United States
Zip Code
63130
Chen, Zi-Wei; Wang, Cunde; Krishnan, Kathiresan et al. (2014) 11-trifluoromethyl-phenyldiazirinyl neurosteroid analogues: potent general anesthetics and photolabeling reagents for GABAA receptors. Psychopharmacology (Berl) 231:3479-91