A Model for Anesthetic A Model for Anesthetic Binding to GABA and Binding to GABA and

Nicotinic Acetylcholine Nicotinic Acetylcholine ReceptorsReceptors

Richard J. LawRichard J. Law

McCammon LabMcCammon Lab

AnestheticsAnesthetics

““Suffering so great as I Suffering so great as I underwent cannot be expressed underwent cannot be expressed in words . . . but the blank in words . . . but the blank whirlwind of emotion, the horror whirlwind of emotion, the horror of great darkness, and the sense of great darkness, and the sense of desertion by God and man, of desertion by God and man, which swept through my mind, which swept through my mind, and overwhelmed my heart, I can and overwhelmed my heart, I can never forget.” never forget.”

- - Professor George Wilson to Professor George Wilson to James Young Simpson, the Edinburgh doctor who James Young Simpson, the Edinburgh doctor who discovered the use of chloroform as an discovered the use of chloroform as an anaesthetic in1846.anaesthetic in1846.

What Am I Going To Talk About?What Am I Going To Talk About?

• Introduction to Cys loop Ligand Gated Ion Introduction to Cys loop Ligand Gated Ion Channels.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 Docking Anesthetics to the Ligand Gated Ion Channels.Channels.

• Docking Agonists to their binding sites – can we Docking Agonists to their binding sites – can we trust AutoDock?trust AutoDock?

• Some ConclusionsSome Conclusions

• Some Future IdeasSome 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 Cys loop family of Ligand Gated Ion Channels.Channels.

• There are four members There are four members of the cys loop LIGC of the cys loop LIGC family:family:

• GABA-AGABA-A

• GlycineGlycine

• 5-HT5-HT33 (Serotonin) (Serotonin)

• NicotinicNicotinic

Cys loop family of Ligand Gated Ion Cys loop family of Ligand Gated Ion Channels.Channels.

Binding Domain - Binding Domain - XrayXray

TM Domain - EMTM Domain - EMComplete Homology Complete Homology Model – nAChR and Model – nAChR and GABARGABAR

Topology of the Topology of the Channel Channel StructureStructure

Cys loop family of Ligand Gated Ion Cys loop family of Ligand Gated Ion Channels.Channels.

• Family of excitable cell, ligand gated, transmembrane ion Family of excitable cell, ligand gated, transmembrane ion channels.channels.

• Main Family members:Main Family members: Depolarising Depolarising - Nicotinic AcetylCholine Recptor- Nicotinic AcetylCholine Recptor

(exciting)(exciting) - 5HT- 5HT33 (Serotonin) Receptor (Serotonin) Receptor

(cation selective)(cation selective)

Hyperpolarising - GABA ReceptorHyperpolarising - GABA Receptor

(repressing)(repressing) - Glycine Receptor - Glycine Receptor

(anion selective)(anion selective)

• These members share high sequence similarity, and These members share high sequence similarity, and

undoubtedly also share structural and functional details.undoubtedly also share structural and functional details.

Anesthetics; their use and Anesthetics; their use and effects.effects.

• Hospitals (and dentists) use various anesthetics to produce different Hospitals (and dentists) use various anesthetics to produce different types of anesthesia for different types of operation.types of anesthesia for different types of operation.

• Many types – mostly small molecules – simple hydrocarbons and Many types – mostly small molecules – simple hydrocarbons and ethers.ethers.

• Most comman are propofol, ketamine, halothane and Isoflurane – Most comman are propofol, ketamine, halothane and Isoflurane – often mixed to produce ideal effect. Eg. Propofol with ketamine often mixed to produce ideal effect. Eg. Propofol with ketamine (ketamine targets Glutamate receptors).(ketamine targets Glutamate receptors).

• Propofol, halothane, and other anesthetics have been shown to inhibit Propofol, halothane, and other anesthetics have been shown to inhibit nerve action potential production by altering the activity of the ligand nerve action potential production by altering the activity of the ligand gated ion channels.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 Anesthetics; their use and effects.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 Anesthetics; effects at a molecular levellevel

• Spectroscopy (and one MD sim. with Gramicidin) seem to Spectroscopy (and one MD sim. with Gramicidin) seem to suggest that anesthetics reduce vibrational motion in suggest that anesthetics reduce vibrational motion in membrane proteins with which they interact.membrane proteins with which they interact.

• They stabilize the closed/desensitized state of the They stabilize the closed/desensitized state of the Nicotinic Receptor.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 So how can such simple molecules produce such specific and opposite events in such similar channel structures??and opposite events in such similar channel structures??

What is the mode of Anesthetic What is the mode of Anesthetic Action?Action?

• Via Perturbation of membraneVia Perturbation of membrane Molecular simplicity of molecules (often simple alcohols)Molecular simplicity of molecules (often simple alcohols) Correlation of potency with fat solubilityCorrelation of potency with fat solubility Such a wide range of molecules could affect such similar Such a wide range of molecules could affect such similar

receptors, so differently.receptors, so differently. Estimated KEstimated Kdds seem to indicate non-specific bindings seem to indicate non-specific binding

• Via Binding to an allosteric site on the receptorVia Binding to an allosteric site on the receptor Small potenciesSmall potencies Molecular size cut-offMolecular size cut-off Stereo-selectiveStereo-selective LabellingLabelling MutagenesisMutagenesis

What are the aims of this study?What are the aims of this study?

• What is the mode of binding of various anesthetic compounds? How does What is the mode of binding of various anesthetic compounds? How does agree with experimental data?agree with experimental data?

• How does this mode differ such that anesthetics affect different How does this mode differ such that anesthetics affect different members of the cys loop family differently?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 (Far off goal) Perhaps to provide information that might be useful is designing new compounds that might be useful in the treatment of designing new compounds that might be useful in the treatment of various neuronal diseases and addictions (nicotine/alcohol).various neuronal diseases and addictions (nicotine/alcohol).

• Investigate how effective AutoDock is at answering these questions and Investigate how effective AutoDock is at answering these questions and how “good” my homology models are.how “good” my homology models are.

Residues Shown Experimentally To Be Residues Shown Experimentally To Be InvolvedInvolved

HalothanHalothanee

EthanolEthanol

AzioctanAzioctanolol

Propofol, Propofol, PropanethiolPropanethiol, PMTS, , PMTS, isoflurane, isoflurane, sevoflurane, sevoflurane, + others.+ others.

SevofluranSevoflurane, e, loreclezoleloreclezole

EtomidateEtomidate

Propofol, Propofol, PropanethiolPropanethiol, PMTS, , PMTS, isoflurane, isoflurane, sevoflurane, sevoflurane, + others.+ others.

EtomidatEtomidatee

Halothane Halothane (M2-M3 loop)(M2-M3 loop)

M4M4

M1M1

M3M3 M2M2

Searching for the binding SiteSearching for the binding Site

• Protein structures often contain cavities Protein structures often contain cavities (imperfections in ideal packing of the structure) that (imperfections in ideal packing of the structure) that allow small ligands to bind. This can cause allosteric allow small ligands to bind. This can cause allosteric changes in the protein’s structure/activity.changes in the protein’s structure/activity.

• These cavities are generally quite hydrophobic in These cavities are generally quite hydrophobic in nature, although may contain some more polar nature, although may contain some more polar moieties that might restrain the nature of the ligands moieties that might restrain the nature of the ligands that can bind there.that can bind there.

• There are various programs that attempt to analyse a There are various programs that attempt to analyse a protein structure and search for candidate binding protein structure and search for candidate binding sites (cavities) – MOE SiteFinder, PASS, and Ligsite.sites (cavities) – MOE SiteFinder, PASS, and Ligsite.

Binding Site Finder (MOE SiteFinder) Binding Site Finder (MOE SiteFinder) ResultsResults

SiteFinder Variables:

Probe radius 1.4

Min. site radius 2.5

Min. site size 150

GABA (or Ach) Binding GABA (or Ach) Binding SiteSite

Toxin/Blocker binding site in Toxin/Blocker binding site in the pore.the pore.

Toxin Binding Site (snake Toxin Binding Site (snake toxins)toxins)

Putative Anesthetic Binding SitePutative Anesthetic Binding Site

Anesthetics Used in the Study (Or will be!)Anesthetics Used in the Study (Or will be!)

O

CH3-CH2- CH2-SH CH3-CH2-CH2-S-S-CH3

O

PropofolPropofol Propanethiol Propanethiol PMTS PMTS

Other I need/want to Other I need/want to do:do:

halothane (CF3CHBRCL)

Etomidate

Isoflurane

Ethanol

Ketamine

CocaineCocaineAll structures were made using Sybyl.

Docking Anesthetics to the Channel Docking Anesthetics to the Channel Structures (Monomers)Structures (Monomers)

AutoDock Settings

Grid spacing 0.375

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 Docking Anesthetics to the Channel Structures - ResultsStructures - 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???

Receptor Ligand Rank Energy (kcal/mol.) Rank Energy (kcal/mol.)nAChR Propofol 1st/2 -5.98 2nd/2 -7.40GABAR Propofol 1st/4 -5.67 1st/4 -6.57nAChR PMTS 1st/7 -4.91 1st/7 -6.53GABAR PMTS 2nd/2 -4.47 2nd/2 -3.31nAChR Propanethiol 1st/6 -3.41 1st/6 -6.19GABAR Propanethiol 1st/4 -3.92 1st/4 -5.51nAChR Cocaine 1 -24.56 1 -5.44GABAR Cocaine 1st/6 -20.22 1st/6 -6.71

Docking ComplexAutoDock Scoring of binding site

clusterXcscore Scoring of binding site

cluster

Docking Anesthetics to the Channel Docking Anesthetics to the Channel Structures - ResultsStructures - Results

Docking the Agonists to the Dimer Interface Binding Docking the Agonists to the Dimer Interface Binding

SiteSite (How good is AutoDock?!)(How good is AutoDock?!)

AutoDock Settings

Grid spacing 0.375

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)Acetyl Choline (ACh)

-amino butyric acid -amino butyric acid (GABA)(GABA)

Docking the Agonist the Binding Site – Docking the Agonist the Binding Site – Results.Results.

Receptor Ligand Rank Energy (kcal/mol.) Rank Energy (kcal/mol.)nAChR GABA 7th/7 -6.91 7th/7 -3.30nAChR ACh 1st/8 -6.70 1st/8 -4.59GABAR GABA 1st/8 -8.26 1st/8 -5.53GABAR ACh 3rd/5 -5.48 2nd/5 -3.32

Docking ComplexAutoDock Scoring of binding site

clusterXcscore Scoring of binding site

cluster

• 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 studiesConclusions of docking studies

• Anesthetics bind most favorably to a site that Anesthetics bind most favorably to a site that is consistent with the experimental evidence.is consistent with the experimental evidence.

• Anesthetics dock slight more favorably to the Anesthetics dock slight more favorably to the Ach receptor? Closed state?Ach receptor? Closed state?

• Docking of agonists (GABA and ACh) to the Docking of agonists (GABA and ACh) to the receptor models follows expected profile, in receptor models follows expected profile, in terms of mode and relative binding energies.terms of mode and relative binding energies.

Unresolved Issues from Anesthetic Unresolved Issues from Anesthetic binding study.binding study.

• How much can these results be believed? (Somewhat non-specific How much can these results be believed? (Somewhat non-specific binding)binding)

• We cannot completely discount the possibility of multiple binding sites We cannot completely discount the possibility of multiple binding sites – non-specific binding.– 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 How does the binding really differentially effect gating of the two channels?channels?

• Not investigated - Different effects of anesthetic binding on different Not investigated - Different effects of anesthetic binding on different multimeric forms of the receptors. Also; stereoselectivity.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 Unresolved issue – Open State of the Channel.Channel.

Nigel Unwin Lab – Pictoral Model of channel Nigel Unwin Lab – Pictoral Model of channel opening.opening.

Unresolved issue – Open State of the Unresolved issue – Open State of the Channel.Channel.

• EM data provided a closed/desensitized structure for the TM EM data provided a closed/desensitized structure for the TM region of these channels.region of these channels.

• Ion pulling simulations provided a model of the open state Ion pulling simulations provided a model of the open state of the M2 pore lining helices in the channel.of the M2 pore lining helices in the channel.

• Could use targeted MD to convert one M2 state into the Could use targeted MD to convert one M2 state into the other and form a model of the open state of the TM portion other and form a model of the open state of the TM portion of the channel.of the channel.

EM of M2 EM of M2 Helices (closed)Helices (closed)

Model of M2 Model of M2 Helices (Open)Helices (Open)

AcknowledgementsAcknowledgements

• Andy McCammonAndy McCammon

• Stewart AdcockStewart Adcock

• Alex PerrymanAlex Perryman

• Julie SchamesJulie Schames

• Richard HenchmanRichard Henchman

• Justin GullingsrudJustin Gullingsrud

• HHMI.HHMI.