Subject Chemistry Paper No and Title 16, Bio-organic and Biophysical Chemistry Module No and Title 30, Enzymes 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. Enzyme 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 enzyme inhibitor. 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 transferase enzyme results in the increased concentration of GABA, ultimately leading to producing anticonvulsant 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 Aspirin Cyclooxygenases Inflammation, Pain, fever Amoxicillin Penicillin 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 Methotrexate Dihydrofolate reductase 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 Competitive Inhibition 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, disulfiram (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