UMass Chem 423 Student Projects 2011-1

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Spring 2011 Chem423 Team Projects: Understanding Drug Mechanisms Instructions posted here: Student Projects for UMass Chemistry 423 Spring 2011

Student projects continued below.

Contents 1 Acetylcholinesterase bound by Tacrine 1.1 Introduction 1.2 Overall structure 1.3 Binding 1.4 Additional Features 1.5 References 1.6 Credits 2 Cyclooxygenase 2.1 Introduction 2.2 Overall Structure 2.3 Drug Binding Site 2.4 Additional Features 2.5 References 2.6 Credits

Acetylcholinesterase bound by Tacrine

Introduction

Acetylcholinesterase breaks down acetylcholine into acetic acid and choline via a hydrolysis reaction.^[1] Acetylcholine is a neurotransmitter that signals muscle contraction. If acetylcholine is not broken down, then the chemical builds up in the synapses between nerve cells and muscle cells resulting in loss of muscle function and ultimately paralysis. The enzyme’s extremely fast reaction rate (approaching the diffusion limit) for breaking one acetylcholine into its two components indicates acetylcholinesterase’s biological importance. Many natural poisons and toxins work by inhibiting this enzyme, thus, paralyzing the victim.^[2]

Intentionally inhibiting acetylcholinesterase is a treatment for Alzheimer’s disease. Alzheimer’s is basically the progressive breakdown of the nervous system. Worldwide, there are an estimated 20 million individuals diagnosed with Alzheimer’s, most of whom are over the age of 65.^[3] Symptoms of the disease include confusion, irritability and aggressioin, mood swings, language breakdown, long-term memory loss, and eventually loss of bodily functions.^[4] To help combat nerve cell degeneration, these acetylcholinesterase inhibitors partially block the enzyme so that excess neurotransmitters remain in the synapse and strengthen the signal.^[5]

The drug Tacrine, also known as Cognex, was the first acetylcholinesterase inhibitor approved to treat Alzheimer’s disease. Studies show that the drug only led to slight improvements in people who took it during early stages of the disease, but the drug did nothing to delay the onset of the disease.^[6] Tacrine is not often used anymore because it has to be taken four times a day and has adverse side effects, including nausea, diarrhea, heartburn, muscle aches and headaches.^[7]

Overall structure

Acetylcholinesterase (AChE) is an monomeric enzyme. Most often, AChE forms a tetramer and binds with a molecule, collagen Q, to connect to the membrane of the neuromuscular junction. ^[8]. From the , it can be seen that there are 17 and 14 . There are 2 beta sheets formed from 3 anti-parallel and 11 anti-parallel beta sheets, respectively. As the shows, turns, alpha helices, and beta sheets all occupy a portion of the exterior of the protein. The means that the turns must be composed primarily of polar side chains. On the other hand, the alpha helices will be amphipathic with side chain order designated by the helical wheel; the exterior will be filled with polar side chains that can hydrogen bond with water while the inside of the alpha helix will have nonpolar, hydrophobic groups. The beta sheets must also be amphipathic, but the pattern of side chains is alternating polar and nonpolar. In addition, in order to maintain its tertiary structure, the protein has three sulfide bonds, which are covalent bonds that form between cysteine residues. The between cysteine 67 and cysteine 94 is 5.03 angstroms.

Binding

The active site of Torpedo californica acetylcholinesterase (TcAChE) is buried at the bottom of a narrow, deep gorge in the enzyme, and contains a consisting of Ser200, Glu327, and His440. When complexed with tacrine (THA), the aromatic rings of sandwich the THA’s acridine ring . The phenyl ring of Phe330 lies parallel to and in contact with THA. THA is stacked against Trp-84. Its ring nitrogen is H-bonded to the main chain carbonyl oxygen of Hist-440 and it’s amino nitrogen is H-bonded to a water molecule.

Additional Features

Huperzine A reversible binds with AChE at Ser200 but forms hydrogen bonds with Tyr130, Gly117, and Glu199.^[9]

Decamethonium is similar to acetylcholine in that it contains trimethylammonium cations allowing it to bind to the nicotinic acetylcholine receptor.^[10]

Soman binds to Ser200, in the active gorge. After binding the acetylcholinesterase(AChE) catalyzes the cleavage of the ether bond on the carbon side causing irreversible inhibition.^[11]

Rivastigmine reversible inhibits AChE, binding at the active gorge. The AChE cleaves the rivastigmine into carbamyl moiety and NAP.^[12]

References

Credits

Introduction - Tyler Vlass

Overall Structure - Zach Brentzel

Drug Binding Site - Andy Kim

Additional Features - Zach Hitzig

Cyclooxygenase

spinqualitylabelsall models PDB ID 3hs5 Show:Asymmetric Unit Biological Assembly Drag the structure with the mouse to rotate   Export Animated Image
3hs5, resolution 2.10Å ()
Ligands:	, , , , , ,
Gene:	Ptgs2, Cox-2, Cox2, Pghs-b, Tis10 (Mus musculus)
Activity:	Prostaglandin-endoperoxide synthase, with EC number 1.14.99.1
Related:	1cvu, 1ddx, 1diy, 5cox, 3hs6, 3hs7
Structural annotation: Resources: InterPro : Ipr000742, Ipr006210, Ipr019791, Ipr010255, Ipr002007 Pfam : PF03098
Resources:	FirstGlance, OCA, RCSB, PDBsum
Coordinates:	save as pdb, mmCIF, xml

Introduction

Cyclooxygenase, abbreviated COX, is an enzyme involved in the formation of biological mediators and takes part in the pain and inflammatory response. It serves as an effective pain and inflammation signal in the body to indicate a fault in the body’s homeostatic balance. Drugs target the binding sites of COX to prevent substrate binding and therefore reduce pain and inflammation in the body.

There are two commonly used forms of cyclooxygenase in animals which are denoted as COX-1 and COX-2. COX-1 carries out normal, physiological production of prostaglandins and serves as a basic housekeeping messages throughout the body. Alternatively, COX-2 is constructed in specialty cells and is used in pain and inflammation signaling. COX-2 is “induced by cytokines, mitogens and endotoxins in inflammatory cells, and which is responsible for the production of prostaglandins in inflammation.”

COX has been of research interest because of the value it provides in particular signaling pathways. Currently, cyclooxygenase is widely targeted in the production of a group of drugs called non-steroidal anti-inflammatory drugs (NSAIDS). This group of drugs inclue asprin, ibuprofecn, flurbiprofen and acetaminophen. These drugs attack the binding site of cyclooxygenase and prevent the substrate binding to reduce pain and fever. NSAIDS are broken down into four different classes. Asprin is categorized in class one, ibuprofem is in class two, flurbiprofen and indomethacin are examples of class three, and Vioxx and Celebrex are components of class four. Latest research shows that cyclooxygenase can possibly serve as an effective target in battling cancer including lung and bladder cancer. Additionally, research is being conducted to evaluate the effectiveness of targeting COX in studying Alzhemier’s disease and cardiovascular disease.

Structurally, cyclooxygenase is composed primarily of alpha helices with few beta sheets. The protein contains two binding sites, the cyclooxygenase active site and the peroxidase site.

Overall Structure

COX-2 is a homodimer membrane protein with two identical subunits. The of each subunit contains primarily alpha helices, shown in light blue, with a few beta sheets, shown in yellow. Each subunit contains 587 amino acids.

Each subunit contains three , the epidermal growth factor (red) beginning at the N-terminus, followed by a membrane binding domain (green), and a large catalytic domain at the C-terminus which contains 480 amino acids (blue). The catalytic domain contains two active sites, the cyclooxygenase and peroxidase. The membrane binding domain is made up of four alpha helices. The alpha helices are amphipathic, creating a , shown in gray, which integrates into the membrane bilayer.

COX-1 and COX-2 are very conserved, being 67% identical in their amino acid sequences. The greatest difference occurs in the membrane binding domain which is only 33% identical.

Drug Binding Site

Many drugs such as aspirin, tylenol, and ibuprofen help regulate pain and and the inflammatory response in the body by blocking the active site of COX. These drugs along with others not only inhibit COX-2 but also inhibit COX-1, causing severe side effects in a small percentage of patients. Recently there has been advances in selectively regulating COX-2 without affecting COX-1 with a drug such as Vioxx. The enzyme COX-2 breaks down arachidonic acid to initiate the production of prostaglandins. Arachidonic acid enters the enzyme through a hydrophobic tunnel formed by the four alpha-helices in the second domain. Once the arachidonic acid reaches the , a hydrogen is believed to be ripped off by the . The heme group located near the peroxidase active site which is believed to help stabilize the radical formed. This radical goes on to further processes that create prostaglandins which intensify pain signals and induce inflammation in damaged parts of the body.

The COX enzyme can be regulated by four different classes of non-steroidal anti-inflammatory drugs (NSAIDS). The first class of inhibitors such as Aspirin regulate the enzyme by irreversibly inactivating the enzyme through covalent modification. A second class of NSAIDS like Motrin or Advil competitively regulates the enzyme. The third class of NSAIDS, for example Flurbiprofen and Indomethacin, forms salt bridges with the enzyme resulting in a slow, time dependent regulation. Finally the fourth class of drugs namely Vioxx and Celebrex selectively regulates COX-2.

Aspirin inhibits the COX enzyme by acetylating the serine residue in the catalytic site preventing the substrate from being catalyzed. Flurbiprofen, a class three inhibitor, binds to the hydrophobic tunnel preventing the substrate from reaching the active site. Flurbiprofen does this with a number of interactions. First it binds to the 120 arginine residue with the formation of a salt bridge. It then hydrogen bonds with the 355 tyrosine residue. An example of a class four inhibitor is SC-558. This drug works in a very similar way as the class three drugs in that it blocks the hydrophobic tunnel but it selectively affects the COX-2. Due to an exposed pocket in COX-2 that is not accessible in COX-1, this drug selectively regulates COX-2 19,000 times more effectively then COX-1. The phenylsulphonamide group of .

Additional Features

Role in other conditions and diseases

COX-2 does not only aid in pain response but plays a role in numerous conditions and diseases including Alzheimer's disease, cardiovascular disease, and cancers such as of the lung and bladder.

Recent discoveries have shown that COX-2 has indirectly played a role in smoker related cancers. Cigarette smoke has been proven to decrease COX-2 expression and cause an increase of PGE2 and TxA2 release. This imbalance causes the progression of tumors and carcinogenesis. This imbalance also contributed to progression of cardiovascular disease. Particularly, more COX-2 positive tumors were found in lung cancer patients than COX-1 positive tumors. Furthermore, smokers of non-cancerous and cancerous patients both had higher expression and imbalance of COX-2, PGE2, and TxA2. Thus, COXIBs are a possible candidate for research in some possible anti-tumor reagents.

Overexpression of COX-2 is observed in later and severe stages of Alzheimer's disease. COX-2 is found to be expressed normally in brain neurons; however, it is questioned as to whether COX-2 prevents or causes neuronal cell death. Some research indicates that an increase in COX-2 and PG synthesis may cause neuronal cell death. Other research has found that NSAID treatment in rats with Alzheimer's disease decreased activated microglial cells and could be used to treat Alzheimer's disease in small doses. Further research must be clarified about the role of COX-2 in the hippocampus, as its role is not clearly defined.

NSAIDS have been shown to have adverse side effects on the kidneys and cause fluid retention, hypertension, edema, and hyperkalemia through the alterations of renal blood flow, and sodium and potassium excretion. COX-2 has selective inhibition and is expressed in the kidneys, thus, with NSAID use severe side effects can occur.

References

Wang, Jane; Limburg, David; Graneto, Matthew; Springer, John; Hamper, Joseph; Liao, Subo; Pawlitz, Jennifer; Kurubail, Ravi; Maziasz, Timothy; Talley, John; Kiefer, James; Carter, Jeffrey. The novel benzopyran class of selective cyclooxygenase-2 inhibitors. Part 2: The second clinical candidate having a shorter and favorable human half-life. Bioorganic and Medicinal Chemistry Letters. 2010, 20, 7159-7163.

Cyclooxygenase Structure and Mechanism. University of Virginia. http://cti.itc.virginia.edu/~cmg/Demo/pdb/cycox/cycox.html (Accessed April 19, 2011).

Cyclooxygenase. Worldwide Protein Data Bank. http://www.rcsb.org/pdb/static.do?p=education_discussion/molecule_of_the_month/pdb17_1.html (Accessed April 19, 2011).

Kurumbail, R. G.; Stevens, A. M.; Gierse, J. K.; McDonald, J. J.; Stegeman, R. A.; Pak, J. Y.; Gildehaus, D.; Miyashiro, J. M.; Penning, T. D.; Seibert, K.; Isakson, P. C.; Stallings, W. C. Structural basis for selective inhibition of cyclooxygenase-2 by anti-inflammatory agents. Nature. 1996, 384, 644-648.

Luong, C.; Miller, A.; Barnett, J.; Chow, J.; Ramesha, C.; Browner, M. F. Flexibility of the NSAID binding site in the structure of human cyclooxygenase-2. Nat. Struct. Biol. 1996, 3, 927-933.

Messiah College. http://www.messiah.edu/departments/chemistry/molscilab/my_molecules/molslides/my_slides/COX/COX.htm

Chen, George; Huang, Run-Yue. Cigarette smoking, cyclooxygenase-2 pathway and cancer. Biochimica et Biophysica Acta, 2010, 1815, 158-169.

Credits

Introduction: Varun Chalupadi

Overall Structure: Alan Stebbins

Drug Binding Site: Anthony Laviola

Additional Features: Tiffany Brucker

References (edited by): Tiffany Brucker