ALL AMERICAN INSTITUTE OF MEDICAL SCIENCES

Enzymes
Inhibition of Enzymes: Use of Enzymes in the Medical Field
Melissa Peart 28-Oct-11

Modern medicine is defined as mainstream orthodox of western medicine of the twentieth century. It has its roots in the Hippocratic which emphasizes empirical evidence based medicine in the identification and treatment of a condition. This field utilizes drugs and varying therapies to treat varying conditions is essential in combating their effects. The study of proteins in particular, enzymes, has been an important part of this process as in many cases understanding their composition, three-dimensional shape and chemical activity may be key to meeting some of the most pressing scientific challenges. The authors Garrett and Grisham stated Enzymes are proteins that catalyze, (or increase the rate of) chemical reactions [1]. Horton et al, reemphasized that enzymes are extraordinarily efficient and selective biological catalysts, in that they are agents that speed up the approach of a reaction towards equilibrium without changing the position of that equilibrium [2]. Their action is related to their conformation and is maintained by interactions between the various amino acids that compose them. This conformation is sensitive to changes in the enzyme's environment which have important influences on an enzymes conformation. These changes include pH, temperature and the presence of inhibitors, which can significantly alter their conformation thus making the enzymes unable to catalyze reactions. Temperature, a measure of the intensity of heat, is an important factor in the activity of enzymes. This is because substrates collide with active sites frequently in the presence of rapidly moving molecules. In addition, although these molecules do move rapidly the speed of the reaction drops sharply. In short, thermal agitation causes protein molecules (enzymes) to denature (breakdown of protein structures). All enzymes have an optimal temperature at which reaction rates go fastest without denaturing the enzyme [3]. pH, a measure of hydrogen ion concentration, is a second important factor in the activity of enzymes. Changes in pH can change the shape of the active site in an enzyme. Extremely high or

low pH concentrations usually result in complete loss of enzyme activity due to denaturation [4]. Enzyme inhibitors are substances which alter the catalytic action of the enzyme and consequently slow down, or in some cases, stop catalysis. There are three common types of enzyme inhibition - competitive, non-competitive and substrate inhibition. In this paper the focus will mainly be placed on how the presence of inhibitors can affect the activity of an enzyme. Enzymes have well been exploited as medicinal targets, leading to discoveries that have greatly improved the quality of life of patients [5]. The study of the consequences of the action of enzyme inhibitors has had profound implications for medical practice. The inhibition of enzyme in particular, plays an integral role in the improvement of the quality of life for patients and the world at large. Their introduction has transformed attitudes to and expectations of the effects of many diseases and their outcomes. Studies of their action have led to a greater understanding of the causes of diseases. This paper will delve into the description of a few enzyme inhibitors which have been used in medicine and their mode of action. There are two broad classes of enzyme inhibitors: 1. Irreversible enzyme inhibitors are life threatening and do not pose much opportunity for beneficial drug research. 2. Reversible enzyme inhibitors -when the inhibitor concentration drops, enzyme activity is regenerated. These reversible enzyme inhibitors are divided into two groups: 1. Competitive these enzyme inhibitors are called competitive because they compete with the substrate for binding at the active site of the enzyme. Most enzyme inhibitor drugs are competitive enzyme inhibitors.

2. Noncompetitive - these inhibitors bind at places other than the active site of the enzyme so they dont compete with the substrate. Almost all therapeutic drugs are enzyme inhibitors, from aspirin and penicillin to the newest compounds used to treat HIV infection. Other examples include, enzyme inhibition seen in cases like the treatment of the rare condition Myasthenia gravis, and the enzymes Cyclooxygenase, and the Aurora Kinase family, which will all be examined in the latter part of this paper. The study of enzyme kinetics therefore, plays an outstanding role in the effort to produce effective therapeutics as it can quantify the degree that inhibitors inactivate or slow down the targeted enzymes catalytic rate and describe its potential efficacy as a drug. Likewise, there are many naturally produced toxic substances and chemical warfare agents which can act as enzyme inhibitors. An example is a peptide found in snake venom which was essential in the development of a class of hypertension drugs that inhibit angiotensinconverting enzyme (ACE inhibitors). The nerve gas sarin is also a potent inhibitor of an enzyme important for synaptic transmission in nerve tissue. HIV (human immunodeficiency virus), the virus that causes AIDS, is one of the hottest areas of medical research today. HIV is the cause of Acquired Immunodeficiency Syndrome (AIDS). HIV directs the synthesis of several polyproteins, which each consist of several tandemly linked proteins. The maturation of the virus to its infectious form requires that these polyproteins be cleaved to their component proteins. HIV-1 protease, a homodimeric enzyme, is responsible for doing so and is therefore crucial to the virus's infectious capacity [6]. After a person has been infected with HIV, the virus usually remains dormant for long periods of time.

Then the virus begins a cycle of attacking cells of the immune system by incorporating its genetic material into the cells, using the immune cells' machinery to make more viruses from the incorporated genetic material, and then breaking the cells apart (killing them) so that the new viruses can infect more cells. In this manner, the immune system is weakened, so that the body can no longer defend itself against the pathogens that it encounters every day. There currently is no cure or vaccine against HIV. Researchers, however, have discovered treatments that can halt and even reverse the progression of AIDS, due in large part to our understanding of the structure of HIV-1 protease. Saquinavir (Invirase) was the first protease inhibitor approved by the FDA for the treatment of HIV. It inhibits HIV protease by binding tightly in the active site tunnel, preventing the binding of polyproteins. Its chemical structure mimics the tetrahedral intermediate of the hydrolytic reaction, thereby interacting strongly with the catalytic Asp residues. Saquinavir is essentially an uncleavable ligand, as indicated by the similar conformational changes in the protease flaps on binding saquinavir or a polypeptide [7]. Other drugs used to treat HIV infection that inhibit HIV protease include Indinavir (Crixivan), Ritonavir (Norvir), and Nelfinavir (Viracept) Myasthenia gravis (MG) is an autoimmune syndrome caused by the failure of neuromuscular transmission, which results from the binding of auto antibodies to proteins involved in signaling at the neuromuscular junction (NMJ). These proteins include the nicotinic AChR or, less frequently, a muscle-specific tyrosine kinase (MuSK) involved in AChR clustering. Much is known about the mechanisms that maintain self tolerance and modulate antiAChR Ab synthesis, AChR clustering, and AChR function as well as those that cause neuromuscular transmission failure upon Ab binding. This insight has led to the development of

improved diagnostic methods and to the design of specific immunosuppressive or immunomodulatory treatments [8]. Pyridostigmine is one of the inhibitors of choice used to treat muscle weakness in people who are diagnosed with Myasthenia gravis. In a synapse,action potentials are conducted along motor nerves to their terminals where they initiate a Ca2+ influx and the release of acetylcholine (ACh). The ACh diffuses across the synaptic cleft and binds to receptors on the post synaptic membrane, causing an influx of Na+ and K+ ions, resulting in depolarization. If large enough, this depolarization results in an action potential. To prevent constant stimulation once the ACh is released, an enzyme calledacetylcholinesterase is present in the endplate membrane close to the receptors on the post synaptic membrane, and quickly hydrolizes ACh. Pyridostigmine inhibits acetylcholinesterase in the synaptic cleft, thus slowing down the hydrolysis of acetylcholine. It is a quaternary carbamate inhibitor of cholinesterase that does not cross the blood-brain barrier, and is taken daily in anticipation of an attack, which carbamylates about 30% of peripheral cholinesterase enzyme. The carbamylated enzyme eventually regenerates by natural hydrolysis and excess ACh levels revert to normal. The Aurora kinases are a family of oncogenic three (A, B, and C) enzymes that are important for the accurate segregation of chromosomes into daughter cells during the mitotic stage of the cell cycle (cytokinesis). Aurora A and B are of particular interest since they are strongly associated with cancer and are therefore attractive targets for small-molecule therapeutics. Aurora A and B have been shown to be over-expressed in a broad range of human tumors.1 Inhibition of Aurora B kinase and resulting inhibition of cytokinesis (or anticytokinesis), is an attractive anticancer strategy. Inhibition of Aurora B kinase does not arrest the cell cycle but leads to failure of cytokinesis resulting in polyploidy and, ultimately, cell death

[9]. Inhibition of the enzyme AK disrupts cell division and multiplication and causes cells to undergo apoptosis. Aurora kinase activity is over expressed or amplified in several types of tumor cells. Thus it offers a valid target for a broad range of cancer. Aurora kinase A is localized to the spindle poles of the cell and is essential for mitosis. Its activity is over expressed in bladder, breast, cervical, colon, gastric, ovarian and pancreatic cancer. Aurora kinase B is associated with chromosomes and is over expressed in different tumor types [10]. Cyclooxygenase, abbreviated as COX, is an enzyme that is responsible for the production of prostanoids, such as prostaglandins, prostacyclin and thromboxane. These eicosanoids, or more simply signaling molecules, are responsible for inflammatory and anaphylactic reactions, vasoconstriction, and the resolution of inflammation respectively. Inhibition of this enzyme can therefore lead to temporary relief of pain and inflammation. Naproxen sodium is a nonsteroidal anti-inflammatory drug (NSAID) commonly used for the reduction of pain, fever, inflammation and stiffness. Nonsteroidal anti-inflammatory drugs, NSAID, target and inhibit the COX enzyme to achieve these desired effects. Of the three variants, (COX-1, COX-2, COX-3), Naproxen targets COX-1 and COX-2. COX-1 is an essential enzyme in the biosynthesis of prostaglandins, responsible for the inflammatory and anaphylactic reations, and is found in the blood, kidneys, and stomach. But COX-1 is also involved in the synthesis of the natural mucus lining that protects the stomach, hence why an overdose, or frequent doses can cause stomach ulcers and bleeding. COX-2 is involved in prostagladin synthesis (all three types) but is only found at the site of inflammation, therefore is not responsible for the notable GI tract side effects that occur with the COX-1 inhibitors. As COX-2 enzymes are found strictly at the site of inflammation, they make an excellent drug target as side effects are limited.

Although Naproxen has been on the market for a number of years, little is known about its method of inhibition. While researchers have recently discovered how naproxen binds to the COX-2 enzyme, they have not yet been able to prove how this inhibits the enzymes function. Observing the COX-2 naproxen complex, one can find the naproxen molecules buried deep within the proteins structure. Looking at the amino acids that surround the active site, researchers have found that the NH2 group on Arg 120 and the alcohol on Tyr 355 stabilize naproxen through their hydrogen bonding with naproxens carboxylic acid. This can easily be seen through the ball and stick structure of the two amino acids and the naproxen molecule. The binding of naproxen is also stabilized by the hydrophobic interactions that occur between naproxens aromatic residues and the various hydrophobic amino acid residues of the active site. Ser 530, Val 349, Leu 531, Ala 527, Val 523, and Leu 352 all work to create a hydrophobic pocket in which the nonpolar region of naproxen can sit. In this active site model these interactions can easily be seen as hydrophobic amino acid residues are pictured in green, Arg in blue, and Try in red, one can grasp a greater understanding of how Naproxen binds to the active site[11]. With these examples, it is only logical to state the importance of enzyme inhibition in improving the life of patience. Enzyme inhibition is an important tool in the pharmaceutical sector. Without it, life would not be the same as we know it.

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[11] Trelle S, Reichenbach S, Wandel S, Hildebrand P, Tschannen B, Villiger PM, Egger M, Jni P. (2011). "Cardiovascular safety of non-steroidal anti-inflammatory drugs: network metaanalysis". BMJ 342: c7086. [12] Duggan, K.C., et al. Molecular basis for cyclooxygenase inhibition by the non-steroidal anti-inflammatory drug naproxen. (2010) J.Biol.Chem. 285: 34950-34959