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Revision as of 02:32, 8 May 2011

This sandbox is in use until August 1, 2011 for UMass Chemistry 423. Others please do not edit this page. Thanks! Chem423 Team Projects: Understanding Drug Mechanisms

Group Members: Chris Brueckner, Daniel Roy, John Clarkson, Justin Srodulski

Template:STRUCTURE 1pbq Template:STRUCTURE 2a5t Template:STRUCTURE 3jpy Template:STRUCTURE 3jpw

N-methyl-D-aspartate (NMDA) receptor in binding complex with Ketamine

JMol Applet legend:

1. NR1 Ligand Binding Domain (Closed Ion-Channel)
2. NR1/NR2a Ligand Binding Domains bound with Glutamate and Glycine (Open Ion-Channel)
3. Zinc-Bound Amino Terminal Domain of NR2B subunit
4. Unbound Amino Terminal Domain of NR2B subunit

Introduction

The drug ketamine is used for medicinal purposes and also, because of its hallucinatory effects, used recreationally. Ketamine is classified as an NMDA receptor antagonist. Glutamate is released and then binds to the NMDA receptor. This triggers the opening of the ion channel. However in the ionotropic pore there are magnesium ions, which greatly limits the ion flow.(1) To counter this the AMPA (α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid) receptor also binds glutamate and flows freely. A voltage is built up by the flowing of sodium and potassium ions that eventually the post-synaptic cell gets depolarized and expels the magnesium, allowing for sodium, potassium, and calium ions to pass through.(2)

When ketamine binds to the NMDA receptor, the ion channel becomes plugged whether or not the magnesium is expelled, in particular blocking the flow of calcium. This means more AMPA receptors would be created, as well as Kainate receptors, which are also glutamate receptors that are not open as long.(6) Ions that pass through the NMDA receptor also modulate the activity of the receptor. In addition, calcium ions fucntion in the signaling pathway as a second messenger. In that respect, the blocking of the NMDA receptor has been known to cause problems with memory.(3) In fact katamine and PCP, another NMDA receptor antagonist drug, were used to model the hypoglutamate state of schizophrenia.(4)

Today, ketamine is primarily used as a general anesthetic, but is also used as an analgesic and a bronchodilator to help breathing. It has even been proven effective in decreasing depression symptoms that accompany bipolar disorder.(5) In knowing in greater detail the structure and function of the NMDA receptor, as well as other receptors, we can gather a better understanding of how drugs like ketamine and PCP effect the body by binding to this site.

Overall Structure

To date, the entire X-ray or NMR crystal structure of the has not been produced. However, there are many structural subunits of NMDA that have successfully been crystallized and analyzed which provide some structural information about the NMDA receptor. The architecture of NMDA receptors is modular and is composed of multiple domains with distinct functional roles. The large extracellular region of the receptor is partitioned into two domains: an (ATD) and a (LBD) (7). Each domain consists of 8 and antiparallel . The alpha helices are located on the outside while the beta sheets are found more toward the center. The NMDA receptor has polar amino acid side-chains located extracellularly and at the ion-pore, but also many non-polar side chains at points where the protein passes through the phospholipid bilayer.

The ligand-binding domain of NMDA receptors are heterotetrameric ion channels composed of two copies of the glycine-binding NR1 subunit and two copies of the L-glutamate-binding NR2 subunit. The NR1 subunit is further divided up into splice units while the NR2 subunit has four sub-variants (NR2A-NR2D). The receptor as a whole has four general ligand binding sites (8).

The amino-terminal domain has an overall clamshell shaped structure and is notably distinct from non-NMDA receptor ATD's. The most important ATD is the NR2B ATD and it is particularly important in current research. It has been shown that the binding of Zn2+ provides neuroprotective agents without the adverse side effects that are more commonly observed with LBD agonists. NR2B ATD has the typical clamshell-like architecture composed of two domains, R1 and R2, which are tied together by three well-structured loops. There is a distinct R1–R2 domain orientation, which in NR2B ATD, is ‘twisted’ by a striking rotation of B45 and 541 compared with the R1–R2 orientation in GluR2 ATD or GluR6 ATD (7).

There are three types of sub units of an NMDA receptor, but not all receptors have the same composition of subtypes. Each subunit consists of three transmembrane segments, a P loop, and an intracellular C-terminus domain (CTD). The segments S1 and S2 in the LBD form a venus-flytrap structure and define the region for agonist recognition (8).

The first molecule is the closed NMDA receptor in its natural state. The second is the open NMDA receptor when it is bound by glutamate and co-agonist glycine. The third is the closed form of the NR2B subunit when it is bound by Zn2+. The fourth is the open state of the NR2B subunit.

Drug Binding Site

The NMDA Receptor primarily functions as a specific type of . In addition to binding NMDA, its namesake, the receptor also contains binding sites for a wide variety of other molecules, most notably Benzodiazepines, D-serine, and Glycine. These binding sites occur extracellularly along the ligand binding domain (LBD). Glutamate binding alone is not enough to trigger a conformational change. In order to open the ion-channel, the NMDA receptor makes use of either Glycine or D-Serine, which are co-agonists. These two molecules compete for the same binding site, and only their binding in conjunction with Glutamate can alter the protein's shape to expose the ion-channel. Furthermore, this allows the Mg2+ plug to be ejected from the pore during cellular depolarization-- the final stage before ion transport can begin(8). It is also known that the NR2B subunit along the Amino Terminal Domain contains several binding sites for small molecules such as . When bound, these zinc ions can also cause a conformational change, resulting in a tightly wound, closed-channel state.

Molecules such as Phencyclidine (PCP), Ketamine, and dizocilpine (MK-801) are known to block the flow of ions through the NMDA receptor. These molecules are non-competitive antagonists because they do not interfere with either the Glutamate or Glycine/D-Serine active sites (8)(9). Instead, they bind to the ion-channel in order to block the flow of ions, much in the same way that magnesium blocks transport. Given that the crystal structure of the ion-channel binding site has not yet been proven, it is difficult to pin down the roles of each residue in channel-blockage binding. However, there is evidence that the ideal binding site for these molecules occurs along side-chain oxygens of Asn 0 Residues, Phe and Leu at -1, Met +25, Val +28, and Ala +29. These are labeled relative to the N/Q/R site (position 0) as their proper orientation within the protein as a whole is still undetermined. The biding reaction of PCP and Ketamine to the NMDA receptor is thought to be stabilized via binding of the antagonist's amino group to these Asn oxygens, while Phe and Leu multi-carbon side-chains form bonds with the hydrophobic edges of the molecules. The pore itself is about 5.5 Angstroms in diameter which suggests the vast majority of blockers bind at the entrance to this very narrow section (10).

As shown in figures 1-3, Ketamine, PCP, and MK-801 all share similar cyclic carbon structures which can easily bind and block such a narrow opening (10). Ketamine itself has racemic properties, and it has been shown that (s)-Ketamine has a much higher binding affinity for the NMDA receptor than (r)-Ketamine, yet PCP has shown to have an even greater binding affinity than either. This can cause problems as PCP will bind so strongly that it eventually causes severe damage to the protein structure through its inhibitory nature (8)(11). Given that PCP is often smoked in recreational abuse, the applied heat causes a reaction that may break the molecule down into 1-phenyl-1-cyclohexene (PC) and piperidine. Despite this, the remaining PCP is most likely the molecule that ultimate binds to the ion-channel, given it's close structural similarities to Ketamine. This binding reaction, and subsequent destruction of the NMDA receptor is thought to be the cause of many neurological disorders involving psychosis such as Schizophrenia. A natural degradation of NMDA receptors may be involved in Alzheimer's Disease (AD) given the receptor's importance in learning and memory (8)(11)(12).

Image:(S)Ketamine3.png

Figure 1: S-Ketamine Structure

Image:PCPMetabolism.png

Figure 2: PCP in reaction with heat. (By Magnus Manske

11:40, 27 August 2002 Magnus Manske 522x221 (2,175 bytes) (from meta))

Image:MK801.png

Figure 3: Structure of MK-801

Additional Features

NMDA receptors are crucial for certain types of long term potentiation (LTP) to occur. LTP is an extended period of enhanced synaptic transmission between two neurons. Ca2+ is required to initiate the activation of LTP. With the opening of calcium channels due to NMDA receptor activation Ca2+ is allowed to flow through which activates calcium-dependant protein kinase II (CaMKII.) CaMKII have two main functions in initiating LTP, the first is to phosphorylate AMPA receptors, and the second is to mediate the addition of AMPA receptors. Protein synthesis is activated by various protein kinases. Scientists are unsure of the proteins produced but are sure that they contribute to the cell entering LTP. (14)

There is a known correlation between LTP and memory storage. Memory storage is found to increase when LTP is activated. Since the opening of Calcium channels is required for the activation of LTP, NMDA receptor binding is thus linked to memory storage. Studies in rats have shown that instances where NMDA receptors have been blocked the rats had issues recalling tasks they have previously performed. (15)

Credits

John Clarkson: Introduction

Daniel Roy: Overall Structure

Chris Brueckner: Drug Binding Site

Justin Srodulksi: Additional Features

References

1. Paoletti P, Neyton J. "NMDA receptor subunits: function and pharmacology" Curr Opin Pharmacol vol 7 (1), 39–47, February 2007.
2. Mark L. Mayer, Gary L. Westbrook, Peter B. Guthrie. "Voltage-dependent block by Mg2+ of NMDA responses in spinal cord neurones" Nature vol 309, 261 - 263, 17 May 1984.
3. Fei Li and Joe Z. Tsien. "Clinical Implications of Basic Research: Memory and the NMDA receptors" New England Journal of Medicine, 361:302, July 16, 2009
4. S Kapur1, P Seeman. "NMDA receptor antagonists ketamine and PCP have direct effects on the dopamine D2 and serotonin 5-HT2 receptors implications for models of schizophrenia" Molecular Psychiatry vol 7, 8, 837-844, 2002.
5. Nancy Diazgranados et al. "A Randomized Add-on Trial of an N-methyl-D-aspartate Antagonist in Treatment-Resistant Bipolar Depression" Archives of General Psychiatry vol 67 (8), 793–802, August 2010.
6. Huettner JE. "Kainate receptors and synaptic transmission" Prog. Neurobiol. vol 70 (5), 387–407, 2003.
7. "Structure of the zinc-bound amino-terminal domain of the NMDA receptor NR2B subunit"
8. http://chemwiki.ucdavis.edu/Wikitexts/Truman_Chem_421%3A_Nagan/N-Methyl-D-Aspartate_Receptor#Subunits
9. Meyer J. & Quenzer L., Pharmacology: Drugs, the Brain, and Behavior. Sinauer Associates, Sunderland, MA. 2005. p. 166-169.
10. Chen, HS V., Lipton, S.A., "The Chemical Biology of Clinically Tolerated NMDA Receptor Antagonists" Journal of Neurochemitry Vol 97, 6, 1611-1626. June 2006.
11. Tikhonov, Denis B. "Ion Channels of Glutamate Receptors: Structural Modeling" Molecular Membrane Biology, March-April 2007; 24(2): 135-147.
12. Newcomer, John W., Farber, Nuri B., Jevtovic-Todorovic, Vesna., Selke, Greg., Melson, Angela Kelly., Hershey, Tamara., Craft, Suzanne., Olney, John W., "Ketamine-Induced NMDA Receptor Hypofunction as a Model of Memory Impairment and Psychosis" Copyright 1999 American College of Neuropsychopharmacology. Elsevier Science Inc. Neuropsychopharmacology. 1999 Vol. 20, (3)
13. Jentsch, J. David, Roth, Robert H., "The Neuropsychopharmacology of Phencyclidine: From NMDA Receptor Hypofunction to the Dopamine Hypothesis of Schizophrenia" Copyright 1999 American College of Neuropsychopharmacology. Elsevier Science Inc. Neuropsychopharmacology. 1999 Vol. 20, (106-118.10.1038).
14. Sweatt J (1999). "Toward a molecular explanation for long-term potentiation". Learn Mem 6 (5): 399–416.doi:10.1101/lm.6.5.399. PMID 10541462.
15. McHugh T, Blum K, Tsien J, Tonegawa S, Wilson M (1996). "Impaired hippocampal representation of space in CA1-specific NMDAR1 knockout mice". Cell 87 (7): 1339–49.doi:10.1016/S0092-8674(00)81828-0. PMID 8980239