The goal of the Chemistry Core is to provide chemistry support to the three projects involved in the Program Project Grant "General Anesthetic Sites on Ligand-Gated Ion Channels". The overall mission of this PPG is to identify binding sites for common general anesthetics - modulators of GABAA and glycine receptors, exemplified by anesthetic steroids, barbiturates, propofol and etomidate. The general strategy is to synthesize and apply analogs of the known anesthetics equipped with the photo-reactive chemical residues. Illumination of the receptor - modulator complexes with UV light forms reactive carbene or nitrene species that indiscriminately modify chemical residues at the modulator binding site. Determining the location and make up of these sites will advance understanding of molecular mechanisms underlying general anesthesia. The goal of Chemistry Core will be realized in four specific aims, each targeting a different class of allosteric modulators of GABAA and glycine receptors. (1) To synthesize diazirine and azide analogs of alphaxalone, a known steroid anesthetic. The goal is to reproduce the pharmacological properties of the parent steroid, but build-in the photo-reactive residues at four different locations of the steroid framework within the confines of the binding sites. Photolabeling patterns by these analogs should enable determination of the binding site location and the binding poses of the ligands within the site. (2) To synthesize subunit-interface selective analogs of mephobarbital. The structural design involves optimization of the substituents at the N-1 and C-5 positions of barbituric acid for highest subunit-interface-selectivity and highest affinity. Our design will involve substituents of varying length and size at N-1, and alkene, vinyl and cyclopropyl chains of varying rigidity and geometry at C-5. (3) To synthesize photo-reactive analogs of propofol for identification of the binding sites on GABAA and glycine receptors. Analogs in which phenol OH group in propofol is separated from the phenyl ring will be synthesized to avoid stabilization of the carbene at the benzylic positions in ortho- or para-positions. sec-Butyl analogs of diazirine-modified propofols with higher affinity will also be synthesized as separate enantiomers. Additionally, p-halogenated analogs will be synthesized as potential photoprobes for GABAA and glycine receptors. In view of the disparity of the published results and those recently obtained by the PPG on the potency of p-chloro-propofol as a modulator of the glycine receptor, we will identify products of the photolytic breakdown of this compound and synthesize them as possible novel potent modulators of this receptor. (4) To synthesize smaller-sized photoreactive analogs of etomidate. The assessment of the available space in the etomidate binding site will be performed first by analogs with varying size of substitution in the phenyl ring. Finally, analogs with smaller five-membered aromatic residues, such as furane, bearing a smaller fluorodiazirine and diazo photo-reactive residues will also be synthesized.

Agency
National Institute of Health (NIH)
Type
Research Program Projects (P01)
Project #
2P01GM058448-16
Application #
8742129
Study Section
Special Emphasis Panel (ZGM1)
Project Start
Project End
Budget Start
Budget End
Support Year
16
Fiscal Year
2014
Total Cost
Indirect Cost
Name
Massachusetts General Hospital
Department
Type
DUNS #
City
Boston
State
MA
Country
United States
Zip Code
02199
Forman, Stuart A; Chiara, David C; Miller, Keith W (2015) Anesthetics target interfacial transmembrane sites in nicotinic acetylcholine receptors. Neuropharmacology 96:169-77
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Jayakar, Selwyn S; Zhou, Xiaojuan; Chiara, David C et al. (2014) Multiple propofol-binding sites in a ?-aminobutyric acid type A receptor (GABAAR) identified using a photoreactive propofol analog. J Biol Chem 289:27456-68
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Chiara, David C; Gill, Jonathan F; Chen, Qiang et al. (2014) Photoaffinity labeling the propofol binding site in GLIC. Biochemistry 53:135-42
Jayakar, Selwyn S; Dailey, William P; Eckenhoff, Roderic G et al. (2013) Identification of propofol binding sites in a nicotinic acetylcholine receptor with a photoreactive propofol analog. J Biol Chem 288:6178-89
Stewart, Deirdre S; Hotta, Mayo; Desai, Rooma et al. (2013) State-dependent etomidate occupancy of its allosteric agonist sites measured in a cysteine-substituted GABAA receptor. Mol Pharmacol 83:1200-8
Forman, Stuart A (2013) A paradigm shift from biophysical to neurobiological: the fading influence of Claude Bernard's ideas about general anesthesia. Anesthesiology 118:984-5

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