A Model for Anesthetic Binding to GABA and Nicotinic Acetylcholine Receptors Richard J. Law McCammon Lab.

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Presentation transcript:

A Model for Anesthetic Binding to GABA and Nicotinic Acetylcholine Receptors Richard J. Law McCammon Lab

Anesthetics “Suffering so great as I underwent cannot be expressed in words... but the blank whirlwind of emotion, the horror of great darkness, and the sense of desertion by God and man, which swept through my mind, and overwhelmed my heart, I can never forget.” - Professor George Wilson to James Young Simpson, the Edinburgh doctor who discovered the use of chloroform as an anaesthetic in1846.

What Am I Going To Talk About? Introduction to Cys loop Ligand Gated Ion Channels. Introduction to Cys loop Ligand Gated Ion Channels. An introduction to Anesthetics. An introduction to Anesthetics. Searching for the Anesthetic Binding Site. Searching for the Anesthetic Binding Site. Docking Anesthetics to the Ligand Gated Ion Channels. Docking Anesthetics to the Ligand Gated Ion Channels. Docking Agonists to their binding sites – can we trust AutoDock? Docking Agonists to their binding sites – can we trust AutoDock? Some Conclusions Some Conclusions Some Future Ideas Some Future Ideas All along – is there a better way to do this? All along – is there a better way to do this?

Cys loop family of Ligand Gated Ion Channels. There are four members of the cys loop LIGC family: There are four members of the cys loop LIGC family: GABA-A GABA-A Glycine Glycine 5-HT 3 (Serotonin) 5-HT 3 (Serotonin) Nicotinic Nicotinic

Cys loop family of Ligand Gated Ion Channels. Binding Domain - Xray TM Domain - EM Complete Homology Model – nAChR and GABAR Topology of the Channel Structure

Cys loop family of Ligand Gated Ion Channels. Family of excitable cell, ligand gated, transmembrane ion channels. Family of excitable cell, ligand gated, transmembrane ion channels. Main Family members: Main Family members:  Depolarising - Nicotinic AcetylCholine Recptor (exciting)- 5HT 3 (Serotonin) Receptor (cation selective)  Hyperpolarising - GABA Receptor (repressing) - Glycine Receptor (anion selective) These members share high sequence similarity, and undoubtedly also share structural and functional details. These members share high sequence similarity, and undoubtedly also share structural and functional details.

Anesthetics; their use and effects. Hospitals (and dentists) use various anesthetics to produce different types of anesthesia for different types of operation. Hospitals (and dentists) use various anesthetics to produce different types of anesthesia for different types of operation. Many types – mostly small molecules – simple hydrocarbons and ethers. Many types – mostly small molecules – simple hydrocarbons and ethers. Most comman are propofol, ketamine, halothane and Isoflurane – often mixed to produce ideal effect. Eg. Propofol with ketamine (ketamine targets Glutamate receptors). Most comman are propofol, ketamine, halothane and Isoflurane – often mixed to produce ideal effect. Eg. Propofol with ketamine (ketamine targets Glutamate receptors). Propofol, halothane, and other anesthetics have been shown to inhibit nerve action potential production by altering the activity of the ligand gated ion channels. Propofol, halothane, and other anesthetics have been shown to inhibit nerve action potential production by altering the activity of the ligand gated ion channels. Ethanol has been shown to have similar effects and interactions. Ethanol has been shown to have similar effects and interactions.

Anesthetics; their use and effects. Decrease currents in excitory channels – eg. Nicotinic AcetylCholine Receptor. Increase currents in inhibitory channels such as the GABA Receptor.

Anesthetics; effects at a molecular level Spectroscopy (and one MD sim. with Gramicidin) seem to suggest that anesthetics reduce vibrational motion in membrane proteins with which they interact. Spectroscopy (and one MD sim. with Gramicidin) seem to suggest that anesthetics reduce vibrational motion in membrane proteins with which they interact. They stabilize the closed/desensitized state of the Nicotinic Receptor. They stabilize the closed/desensitized state of the Nicotinic Receptor. They stabilize the open state of the GABA Receptor. They stabilize the open state of the GABA Receptor. So how can such simple molecules produce such specific and opposite events in such similar channel structures?? So how can such simple molecules produce such specific and opposite events in such similar channel structures??

What is the mode of Anesthetic Action? Via Perturbation of membrane Via Perturbation of membrane  Molecular simplicity of molecules (often simple alcohols)  Correlation of potency with fat solubility  Such a wide range of molecules could affect such similar receptors, so differently.  Estimated K d s seem to indicate non-specific binding Via Binding to an allosteric site on the receptor Via Binding to an allosteric site on the receptor  Small potencies  Molecular size cut-off  Stereo-selective  Labelling  Mutagenesis

What are the aims of this study? What is the mode of binding of various anesthetic compounds? How does agree with experimental data? What is the mode of binding of various anesthetic compounds? How does agree with experimental data? How does this mode differ such that anesthetics affect different members of the cys loop family differently? How does this mode differ such that anesthetics affect different members of the cys loop family differently? What might be the mechanism by which these anesthetics work? What might be the mechanism by which these anesthetics work? (Far off goal) Perhaps to provide information that might be useful is designing new compounds that might be useful in the treatment of various neuronal diseases and addictions (nicotine/alcohol). (Far off goal) Perhaps to provide information that might be useful is designing new compounds that might be useful in the treatment of various neuronal diseases and addictions (nicotine/alcohol). Investigate how effective AutoDock is at answering these questions and how “good” my homology models are. Investigate how effective AutoDock is at answering these questions and how “good” my homology models are.

Residues Shown Experimentally To Be Involved Halothane Ethanol Azioctanol Propofol, Propanethiol, PMTS, isoflurane, sevoflurane, + others. Sevoflurane, loreclezole Etomidate Propofol, Propanethiol, PMTS, isoflurane, sevoflurane, + others. Etomidate Halothane (M2- M3 loop) M4 M1 M3 M2

Searching for the binding Site Protein structures often contain cavities (imperfections in ideal packing of the structure) that allow small ligands to bind. This can cause allosteric changes in the protein’s structure/activity. Protein structures often contain cavities (imperfections in ideal packing of the structure) that allow small ligands to bind. This can cause allosteric changes in the protein’s structure/activity. These cavities are generally quite hydrophobic in nature, although may contain some more polar moieties that might restrain the nature of the ligands that can bind there. These cavities are generally quite hydrophobic in nature, although may contain some more polar moieties that might restrain the nature of the ligands that can bind there. There are various programs that attempt to analyse a protein structure and search for candidate binding sites (cavities) – MOE SiteFinder, PASS, and Ligsite. There are various programs that attempt to analyse a protein structure and search for candidate binding sites (cavities) – MOE SiteFinder, PASS, and Ligsite.

Binding Site Finder (MOE SiteFinder) Results SiteFinder Variables: Probe radius 1.4 Min. site radius2.5 Min. site size150 GABA (or Ach) Binding Site Toxin/Blocker binding site in the pore. Toxin Binding Site (snake toxins) Putative Anesthetic Binding Site

Anesthetics Used in the Study (Or will be!) O CH3-CH2-CH2-SH CH3-CH2-CH2-S-S-CH3 O Propofol Propanethiol PMTS Propofol Propanethiol PMTS Other I need/want to do: halothane (CF3CHBRCL) Etomidate Isoflurane Ethanol Ketamine Cocaine All structures were made using Sybyl.

Docking Anesthetics to the Channel Structures (Monomers) AutoDock Settings Grid spacing0.375 Used the largest grid ADT would allow. Used AutoDock Tools for most of the set-up. Fully Flexible ligand. Docking to the TM domain and base of the binding domain of GABAR and nAChR monomers. (Did try docking to whole monomer with 0.8 grid but results weren’t good.)

Docking Anesthetics to the Channel Structures - Results Xcscore and AutoDock scoring are consistent in terms of ranking. Usually, the highest ranked clusters, in the case of both receptors, are the ones within the putative anesthetic binding site – both in terms of mode and estimated energy. Comparing the binding energies for each ligand; usually lower for the nAChR structure. Closed state???

Docking Anesthetics to the Channel Structures - Results

Docking the Agonists to the Dimer Interface Binding Site (How good is AutoDock?!) AutoDock Settings Grid spacing0.375 Used the largest grid ADT would allow. Used AutoDock Tools for most of the set-up. Fully Flexible ligand. Docking to the centre of the binding domain of GABAR and nAChR dimers. Acetyl Choline (ACh)  -amino butyric acid (GABA)

Docking the Agonist the Binding Site – Results. Xcscore and AutoDock scoring results are consistent Highest ranked clusters, in the case of both receptors, are their native ligands – both in terms of mode and estimated energy. Comparing the binding energies, always lower for the native complex.

Conclusions of docking studies Anesthetics bind most favorably to a site that is consistent with the experimental evidence. Anesthetics bind most favorably to a site that is consistent with the experimental evidence. Anesthetics dock slight more favorably to the Ach receptor? Closed state? Anesthetics dock slight more favorably to the Ach receptor? Closed state? Docking of agonists (GABA and ACh) to the receptor models follows expected profile, in terms of mode and relative binding energies. Docking of agonists (GABA and ACh) to the receptor models follows expected profile, in terms of mode and relative binding energies.

Unresolved Issues from Anesthetic binding study. How much can these results be believed? (Somewhat non-specific binding) How much can these results be believed? (Somewhat non-specific binding) We cannot completely discount the possibility of multiple binding sites – non-specific binding. We cannot completely discount the possibility of multiple binding sites – non-specific binding. Solvation term – docking to a TM region of a protein. Solvation term – docking to a TM region of a protein. How does the binding really differentially effect gating of the two channels? How does the binding really differentially effect gating of the two channels? Not investigated - Different effects of anesthetic binding on different multimeric forms of the receptors. Also; stereoselectivity. Not investigated - Different effects of anesthetic binding on different multimeric forms of the receptors. Also; stereoselectivity. Binding preferentially to open/closed states?? Binding preferentially to open/closed states??

Unresolved issue – Open State of the Channel. Nigel Unwin Lab – Pictoral Model of channel opening.

Unresolved issue – Open State of the Channel. EM data provided a closed/desensitized structure for the TM region of these channels. EM data provided a closed/desensitized structure for the TM region of these channels. Ion pulling simulations provided a model of the open state of the M2 pore lining helices in the channel. Ion pulling simulations provided a model of the open state of the M2 pore lining helices in the channel. Could use targeted MD to convert one M2 state into the other and form a model of the open state of the TM portion of the channel. Could use targeted MD to convert one M2 state into the other and form a model of the open state of the TM portion of the channel. EM of M2 Helices (closed) Model of M2 Helices (Open)

Acknowledgements Andy McCammon Andy McCammon Stewart Adcock Stewart Adcock Alex Perryman Alex Perryman Julie Schames Julie Schames Richard Henchman Richard Henchman Justin Gullingsrud Justin Gullingsrud HHMI. HHMI.