Subject Chemistry

Paper No and Title 16, Bio-organic and Biophysical Chemistry

Module No and Title 30, as targets or drug design

Module Tag CHE_P16_M30

Chemistry PAPER No. : 16, Bioorganic and biophysical chemistry MODULE No. : 30, Enzymes as targets of drug design

TABLE OF CONTENTS

1. Learning outcomes 2. Why Enzymes as Drug Targets? 3. structure and Catalysis 4. Concept of Enzyme Inhibition 5. Types of Enzyme Inhibition 5.1. Nonspecific Inhibitors 5.2. Specific Inhibitors 6. Summary

Chemistry PAPER No. : 16, Bioorganic and biophysical chemistry MODULE No. : 30, Enzymes as targets of drug design

1. Learning outcomes

After studying this module you shall be able to:

 Know about enzymes as drug targets  Learn about enzyme structure and catalysis  Know the concept of enzyme inhibition  Understand the different types of enzyme inhibition

2. Introduction

2.1 Why Enzymes as Drug Targets?

Medicine in twenty first century has become a science in which drug molecules are directed against the macromolecules. Enzymes hold a prominent position among the biological macromolecules that can be used as drug targets. Some features of enzyme structures and reaction pathways which make them attractive and primary target for drug action are as follows  They play an essential role in biological life processes and pathophysiology  The structures of active sites of enzymes and the ligand binding pockets are highly amenable for high affinity interactions with small drug like molecules  Presence of potential allosteric sites  Conformational variations in the binding sites  Can be directly used as a therapeutic agent

3. Enzyme structure and catalysis

Enzymes are biological catalysts as well as can act as receptors by acting with the substrates. These are the most efficient catalysts known in nature. They have the ability to enhance reaction rates by lowering the activation energy of reactions and by stabilizing the reacting molecules at their activated complex states. Stabilization theory of the activated complex by enzymes was first proposed by Pauling. He concluded that the active site of enzyme is complementary to the structure of the activated complex so that the binding of the enzyme to the activated complex is extremely tight, which reduces the activation energy and enhance the reaction rate.

Chemistry PAPER No. : 16, Bioorganic and biophysical chemistry MODULE No. : 30, Enzymes as targets of drug design

4. Concept of Enzyme Inhibition

Although activation of enzymes may be exploited therapeutically, most effects are produced by enzyme inhibition. A survey reported in 2002 found that close to 30% of all drugs in clinical use derive their therapeutic efficacy through enzyme inhibition and then it was updated to include newly launched drugs and found that ~47% of all marketed small molecule drugs inhibit enzymes as their molecular target. Any compound that slows down or blocks enzyme catalysis is termed as . This strategy can be exploited in correcting chemical deficiencies by inhibiting the enzyme which uses that chemical as its substrate. Like in Seizures which can be caused by insufficient gamma amino butyric acid (GABA) levels. Inhibition of the GABA amino enzyme results in the increased concentration of GABA, ultimately leading to producing effects. (Fig. 1)

Fig. 1 Chemical excess can also be corrected by inhibiting the enzyme that produces the molecule, like Gout results from excess uric acid concentration. Inhibition of enzyme xanthine oxidase, which converts xanthine to uric acid, would result in the antihyperuricemic effects. (Fig. 2)

Chemistry PAPER No. : 16, Bioorganic and biophysical chemistry MODULE No. : 30, Enzymes as targets of drug design

Fig. 2 Inhibition of biochemical pathways unique to a pathogen can reduce the growth or kill the pathogen (Bacteria, virus or parasite). Like Sulfa drugs act as bacteriostatic by inhibiting dihydropteroate synthetase enzyme which is required for folic acid synthesis in bacteria. Humans don’t require this enzyme as they can absorb folic acid directly from the diet. (Fig. 3)

Chemistry PAPER No. : 16, Bioorganic and biophysical chemistry MODULE No. : 30, Enzymes as targets of drug design

Fig. 3 Table 1. Selected Enzyme inhibitors in Clinical use Compound Target Enzyme Clinical Use Acetazolamide Carbonic anhydrase Glaucoma Acyclovir Viral DNA Polymerase Herpes Allopurinol Xanthine oxidase Gout Inflammation, Pain, fever Amoxicillin binding proteins Bacterial infections Enalapril Angiotensin converting Hypertension enzyme Carbidopa Dopa decarboxylase Parkinson’s disease Digoxin Sodium Potassium ATPase Heart disease Lovastatin HMG-CoA reductase Cholesterol lowering Cancer, Immunosupression Norfloxacin DNA gyrase Urinary tract infections Omeperazole H+, K+ ATPase Peptic ulcers

Chemistry PAPER No. : 16, Bioorganic and biophysical chemistry MODULE No. : 30, Enzymes as targets of drug design

Viagra Phosphodiesterase Erectile dysfunction

5. Types of Enzyme inhibition

Enzyme inhibitors are molecules that interact in some way with the enzyme to prevent it from working in the normal manner. There are a variety of types of inhibitors including: nonspecific, irreversible, reversible - competitive and noncompetitive. (Fig.4) Poisons and drugs are examples of enzyme inhibitors.

5.1 Nonspecific Inhibitors

A nonspecific inhibition affects all enzymes in the same way. Non-specific methods of inhibition include any physical or chemical changes which ultimately denature the protein portion of the enzyme and are therefore irreversible.

5.1.1. Temperature

Usually, the reaction rate increases with temperature, but with enzyme reactions, a point is reached when the reaction rate decreases with increasing temperature. At high temperatures the protein part of the enzyme begins to denature, thus inhibiting the reaction.

Chemistry PAPER No. : 16, Bioorganic and biophysical chemistry MODULE No. : 30, Enzymes as targets of drug design

Fig. 4 Flowchart showing types of Enzyme Inhibitors 5.1.2. Acids and Bases Enzyme activity is also controlled by pH. As the pH is decreased or increased, the nature of the various acid and amine groups on side chains is altered with resulting changes in the overall shape structure of the enzyme.

5.2 Specific Inhibitors

Specific Inhibitors exert their effects upon a single enzyme. Most of the poisons and drugs act by specifically acting on a single enzyme.

5.2.1. Reversible Inhibition

Reversible inhibitors can bind to enzymes through weak non-covalent interactions such as ionic bonds, hydrophobic interactions, and hydrogen bonds. Because reversible inhibitors do not form any chemical bonds or reactions with the enzyme, they are formed rapidly and can be easily removed; thus the enzyme and inhibitor complex is rapidly dissociated.  Competitive  Noncompetitive  Uncompetitive

5.2.1.1 A competitive inhibitor is any compound which closely resembles the chemical structure and molecular geometry of the substrate. The inhibitor competes for the same active site as the substrate molecule. The inhibitor may interact with the enzyme at the active site, but no reaction takes place. The inhibitor is "stuck" on the enzyme and prevents any substrate molecules from reacting with the enzyme. However, a competitive inhibition is usually reversible if sufficient substrate molecules are available to ultimately displace the inhibitor. Therefore, the amount of enzyme inhibition depends upon the inhibitor concentration, substrate concentration, and the relative affinities of the inhibitor and substrate for the active site.

Example 1: Ethanol is metabolized in the body by oxidation to acetaldehyde, which is in turn further oxidized to acetic acid by aldehyde oxidase enzymes. Normally, the second reaction is rapid so that acetaldehyde does not accumulate in the body. A drug, (Antabuse) inhibits the aldehyde oxidase which causes the accumulation of acetaldehyde with subsequent unpleasant side- effects of nausea and vomiting. This drug is sometimes used to help people overcome the drinking habit.

Chemistry PAPER No. : 16, Bioorganic and biophysical chemistry MODULE No. : 30, Enzymes as targets of drug design

Example 2: Folic acid, folacin or pteroil glutamic acid belongs to the group of water soluble vitamins. Fresh leafy green vegetables, cauliflower, kidney and liver are rich sources of folic acid. The physiological function of folic acid coenzymes is in the synthesis of purine nucleotides and thymine, precursors in the synthesis of RNA and DNA intracellulary, respectively. The folic acid coenzymes are specifically concerned with biochemical reactions involving the transfer and utilization of the single carbon (C1)) moiety. Before functioning as a C1 carrier, folic acid must be reduced, first to 7,8-dihydrofolic acid (H2 - folate) and then to the tetrahydro compound (H4 - 5,6,7,8- tetrahydrofolic acid) catalyzed by folic acid reductase which uses NADPH as hydrogen donor. The participation of folic acid coenzymes in reaction leading to synthesis of purines and to thymine, the methylated pyrimidine of DNA, emphasizes the fundamental role of folic acid in the growth and replication of cells. Cancer cells grow more rapidly than the cells of most normal tissues and thus they have greater requirements for nucleotides as precursors of DNA and RNA synthesis. Consequently, cancer cells are generally more sensitive to inhibitors of nucleotide biosynthesis than are normal cells.

Fig. 5 Methotrexate Antifolates, folate analogs, aminopterin (4-amino folic acid) and methotrexate (amethopterin, 4- amino-10-methylfolicacid) (Fig. 5) are extremely potent competitive inhibitors of the dihydrofolate reductase and thymidylate synthetase and because of that inhibits the synthesis of RNA and DNA. The application of methotrexate disturbs the metabolism of polyamines in rapidly growing tissues. Inhibition of polyamine oxidase, the key enzyme in biodegradation pathway of spemine and spermidine, induced by methotrexate in regenerating rat liver tissue is probably the consequence of the inhibition of nucleic acids and protein synthesis.

5.2.1.2 Non-Competitive inhibition

A noncompetitive inhibitor is a substance that interacts with the enzyme, but usually not at the active site. The noncompetitive inhibitor reacts either remote from or very close to the active site. The net effect of a noncompetitive inhibitor is to change the shape of the enzyme and thus the active site, so that the substrate can no longer interact with the enzyme to give a reaction. Noncompetitive inhibitors are usually reversible, but are not influenced by concentrations of the substrate as is the case for a reversible competitive inhibitor.

Chemistry PAPER No. : 16, Bioorganic and biophysical chemistry MODULE No. : 30, Enzymes as targets of drug design

Example 1: Chymotrypsin is an enzyme which hydrolyzes peptides at the carbonyl side of tyr or phe or trp (i.e. those that have an aromatic side chain. In the graphic on the left, the substrate and the irreversible inhibitor are shown in the active site pocket. In the case of the inhibitor the reaction starts in the same way as with the substrate, but the end result is that the inhibitor is covalently bonded to the histidine-57 in the active site and is not reversible. (Fig. 6)

Fig.6. Showing Noncompetitive inhibition mechanism of Chymotrypsin

5.2.1.3. Uncompetitive Inhibition

Those molecules that can only bind reversibly to an enzyme when the substrate is already bound to the active site are termed as uncompetitive inhibitors. In other words the inhibitor binds to the enzyme substrate complex. Increasing the concentration of substrate will not overcome the inhibition in this case. Indeed the level of inhibition would depend upon sufficient substrate being present at the active site to make enzyme substrate complex. This inhibition is not very common.

5.2.2. Irreversible Inhibition

Chemistry PAPER No. : 16, Bioorganic and biophysical chemistry MODULE No. : 30, Enzymes as targets of drug design

Irreversible inhibitors covalently bind to an enzyme, because chemical changes to the active sites of enzymes, and cannot be reversed. A main role of irreversible inhibitors includes modifying key amino acid residues needed for enzymatic activity. They often contain reactive functional groups such as aldehydes, alkenes, or phenyl sulphonates. These electrophilic groups are able to react with amino acid side chains to form covalent adduct. The amino acid components are residues containing nucleophilic side chains such as hydroxyl or sulfhydryl groups such as amino acids serine, cysteine, threonine, or tyrosine.

Irreversible Inhibitors form strong covalent bonds with an enzyme. These inhibitors may act at, near, or remote from the active site. Consequently, they may not be displaced by the addition of excess substrate. In any case, the basic structure of the enzyme is modified to the degree that it ceases to work. Since many enzymes contain sulfhydryl (-SH), alcohol, or acid groups as part of their active sites, any chemical which can react with them acts as an irreversible inhibitor. Heavy metals such as Ag+, Hg2+, Pb2+ have strong affinities for -SH groups.

Nerve gases such as diisopropylfluorophosphate (DFP) inhibit the active site of acetylcholine esterase by reacting with the hydroxyl group of serine to make an ester. Oxalic and citric acid inhibit blood clotting by forming complexes with calcium ions necessary for the enzyme metal ion activator.

5.2.2.1. Affinity labels or active site directed irreversible inhibition

Affinity labels are molecules that are structurally similar to the substrate for the enzyme that covalently modify active site residues. They contain a reactive functional group so not only they can react with the active site of target enzyme but also can react with many nucleophiles associated with many other enzymes and biomolecules. Consequently these inhibitors display potential toxicity. Many cancer chemotherapy drugs are affinity labeling agents so are not as common in drug design as other type of inhibitors.

Example 1: Aspirin (Fig. 7) irreversibly acetylates the COX binding site preventing formation of prostaglandins. Binding to COX1 inhibits its prostaglandin production by preventing catalysis of arachidonic acid, responsible for assisting platelet formation thus causing the blood to thin and less clotting.

Chemistry PAPER No. : 16, Bioorganic and biophysical chemistry MODULE No. : 30, Enzymes as targets of drug design

Fig. 7 Aspirin

Example 2: Tosyl-l-phenylalanine chloromethyl ketone (TPCK) is a reactive analog of the normal substrate for the enzyme chymotrypsin. TPCK binds at the active site of chymotrypsin and modifies an essential histidine residue.

5.2.2.2. Suicide Inhibition

These are modified substrates that provide the most specific means to modify an enzyme active site. The inhibitor binds to the enzyme as a substrate and is initially processed by the normal catalytic mechanism. The mechanism of catalysis then generates a chemically reactive intermediate that inactivates the enzyme through covalent modification. This is called as suicide inhibition because the enzyme is committing suicide by reacting with these drugs. The difference from affinity labels is the requirement for an enzyme which reacts on it and converts it into a product which is actually an inactivator species.

Example 1: Monoamine oxidase deaminates neurotransmitters such as dopamine and serotonin, lowering their levels in the brain. The drug , (Fig. 8) which is used to treat Parkinson disease and depression, is a suicide inhibitor of monoamine oxidase. It binds selectively to monoamine oxidase B.

Fig. 8 Selegiline

6. Summary

In this chapter we have attempted to describe some of the features of enzyme structure and reaction pathway that make enzymes particularly attractive targets for drug discovery and design efforts. The essentials of drug discovery through enzyme inhibition have been outlined with the help of few examples.

Chemistry PAPER No. : 16, Bioorganic and biophysical chemistry MODULE No. : 30, Enzymes as targets of drug design