Description of Research Expertise:
MOLECULAR PHARMACOLOGY OF INHALED ANESTHETICS
The inhaled anesthetics are considered one of the most important medical advances of all time, are used in over 100 million patients every year, and yet remain the most toxic and poorly understood of all drugs. The goal of my laboratory is a translational understanding of inhaled anesthetic pharmacology. Most of the current work is focused on the biophysics of anesthetic/macromolecular interactions because of the importance of establishing a foundation of knowledge at this most basic level, on which the subsequent superstructure of molecular, cellular and organism understanding will be built. We have developed a wide variety of experimental approaches to study inhaled anesthetics binding to proteins, and the structural and dynamic consequences. Thus, photoaffinity labeling, fluorescence spectroscopy, amide hydrogen exchange, low-affinity elution chromatography and differential/isothermal calorimetry have all been introduced and validated for this purpose. Many protein and peptide models are used, including serum albumin and its domains, odorant binding protein, rhodopsin and other G-protein-coupled receptors, ferritin, and de novo designed helical bundles. Our group also uses NMR spectroscopy, x-ray crystallography and molecular dynamic simulations via close collaborations to gain a detailed atomic-level appreciation for the interactions and consequences in both time and space. In collaboration with Pat Loll of Drexel University, we have completed high resolution characterization of halothane, isoflurane and propofol binding sites in apoferritin - the highest affinity inhaled anesthetic binding protein yet described (see Figures 1 & 2). This has yielded considerable insight into the features underlying anesthetic binding, and provided a template for anesthetic discovery. In turn, discovered reagents are providing for novel anesthetic targets discovery. Proteomic and genomic approaches have permitted initial forays into cell and organism implications of our binding results. Wide collaborations with many other departments and institutions have facilitated a rapid, multidisciplinary attack on some of the most fundamental questions in anesthetic pharmacology.
The apoferritin anesthetic site is a buried cavity at the dimer interface: panel A. Note the secondary, tertiary and quarternary structural similarity of the motif in panel A with the presumed anesthetic site in GABAA receptor TM regions, panel B. (FASEB Journal 2005; 19:567-576). This, combined with its ability to bind a range of anesthetics according to their potency (a propofol analogue shown in Figure 2), makes the apoferritin site a suitable template for anesthetic discovery using high throughput approaches (Figure 3).
Our photoaffinity work has allowed us to define binding sites in complex proteins, permitting mechanistic hypotheses and drug optimization. Shown in panel A is the halothane-binding residue from photolabeling, and in panel B, the same residue is implicated from molecular dynamic simulations. This integration of experimental and theoretical approaches is a hallmark of work done in this lab.
Our studies of inhaled anesthetics led to the observation that they can potently promote aggregation of selected peptides and proteins. Since a common feature of most neurodegenerative disorders is aggregation of endogenous peptide, inhaled anesthetics may enhance this process, and accelerate the onset of the disorder. Examination of this possibility in a fully translational manner is a growing focus of my laboratory. Current efforts include cell culture, transgenic animals, clinical biomarker studies and associative database studies. The figure below is a section of transgenic mouse brain showing plaque, and the graph shows that a halothane anesthetic enhances plaque number only 2 weeks after exposure.
(left-right): Maryellen Eckenhoff, Lindsay Roth, Wesley Peng, Emmanuel Suarez, Tim DeYoung, Daniel Xu, Kellie Woll, Jack Jacobs, Rod Eckenhoff, Weiming Bu, and John Li
Woll Kellie A, Murlidaran Sruthi, Pinch Benika J, Hénin Jérôme, Wang Xiaoshi, Salari Reza, Covarrubias Manuel, Dailey William P, Brannigan Grace, Garcia Benjamin A: A Novel Bifunctional Alkylphenol Anesthetic Allows Characterization of γ-Aminobutyric Acid, Type A (GABAA), Receptor Subunit Binding Selectivity in Synaptosomes. The Journal of biological chemistry 291 (39): 20473-86,2016.
Ho J, Perez-Aguilar JM, Gao L, Saven JG, Matsunami H: Molecular recognition of ketamine by a subset of olfactory G protein–coupled receptors. Sci. Signal. 8 (370): 2015.
Weiser Brian P: Propofol Inhibits SIRT2 Deacetylase through a Conformation-specific, Allosteric Site. The Journal of biological chemistry 290 (13): 8559-68,2015.
McKinstry-Wu Andrew R, Bu Weiming, Rai Ganesha, Lea Wendy A, Weiser Brian P, Liang David F, Simeonov Anton, Jadhav Ajit, Maloney David J: Discovery of a novel general anesthetic chemotype using high-throughput screening. Anesthesiology 122 (2): 325-33,2015.
Tang Junxia X, Mardini Feras, Janik Luke S, Garrity Sean T, Li Rosie Q, Bachlani Gulnaz, Eckenhoff Roderic G: Modulation of murine Alzheimer pathogenesis and behavior by surgery. Annals of surgery 257 (3): 439-48,2013.
Emerson DJ, Weiser BP, Psonis J, Liao Z, Taratula O, Fiamengo A, Wang X, Sugasawa K, Smith A, Eckenhoff RG: Direct modulation of microtubule stability contributes to anthracene general anesthesia.
J. Am. Chem. Soc. : 2013.
Hall Michael A, Xi Jin, Lor Chong, Dai Shuiping, Pearce Robert, Dailey William P: m-Azipropofol (AziPm) a photoactive analogue of the intravenous general anesthetic propofol. Journal of medicinal chemistry 53 (15): 5667-75,2010.
Liu R, Loll PJ: Structural basis for high-affinity volatile anesthetic binding in a natural 4-helix bundle protein. FASEB J. 19 (6): 567-76,2005.
Eckenhoff RG, Johansson JS, Wei H, Carnini A, Kang B, Wei W, Pidikiti R, Keller JM: Inhaled anesthetic enhancement of amyloid-beta oligomerization and cytotoxicity. Anesthesiology 101 (3): 703-9,2004.
Promiscuous ligands and attractive cavities; How do the inhaled anesthetics work? Mol. Interventions 1 : 258-268,2001.
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