Role of enzymes in drug discovery

Role of enzymes in drug discovery

1. Dr. Kumbhare Manoj R. Professor S.M.B.T . College of Pharmacy Dhamangaon Nashik (M.S .). Role of Enzyme Inhibition in Drug Discovery

2. What is a drug? Defined composition with a pharmacological effect Regulated by the Food and Drug Administration (FDA) What is the process of Drug Discovery and Development? 2

3. For the past 20 years target-based drug discovery has been the main research paradigm used by the pharmaceutical industry and billions of dollars have been invested into this approach. Drug discovery approaches Drug discovery can essentially follow two different approaches 1. The physiology-based (or phenotypic) approach . On the basis of information about diseases and existing treatments, one or more screening models. 2. The target-based approach. On the basis of information about diseases, biological mechanisms and existing treatments, the researcher decides to develop a drug that affects a specific target and with a specific mode-of-action. 3 Drug discovery

4. Drug discovery Multi-stage process Identification of target Lead generation, Lead optimization, Preclinical evaluation Clinical testing New medicine Industry/ Researchers : Significant challenge 4

5. Drug Discovery Process Small Molecules Natural products fermentation broths plant extracts animal fluids (e.g., snake venoms) Synthetic products Project medicinal chemistry derived Combinatorial chemistry derived Biologicals Natural products (isolation) Recombinant products 5

6. Drug Development Process: Discovery-Approval Time (yr) : 4 2 1.5 2 3.5 1 #’s : 30,000 2000 200 40 12 8 Drug Development Process- 10-15 years 800-1300 million dollars 0.003% chance of a return on investment (1/30,000) 6

7. Enzymes Enzymes   are macromolecular biological catalysts. Enzymes accelerate, or catalyze, chemical reactions. The molecules at the beginning of the process upon which enzymes may act are called substrates and the enzyme converts these into different molecules, called products.

8. Why Enzymes as Drug Targets? • Enzymes are excellent targets for pharmacological intervention, owing to their essential roles in life processes and pathophysiology. • The structures of enzyme active sites, and other ligand binding pockets on enzymes , are ideally suited for high-affinity interactions with drug-like inhibitors. 8 ENZYMES ARE ESSENTIAL FOR LIFE Deregulated enzyme activity can also lead to disease states.

9. Distribution of marketed drugs by biochemical target class. 9

10. HTS screening success rates by target class in AstraZeneca 2004–2008. Success rate is defined as the percentage of screening campaigns that led to a project transitioning into the lead identification phase – typically demonstrating on-target effects in a cellular assay, with evidence of exposure in a rodent species. Unsuccessful screens are broadly categorised according to reason for failure – chemistry, where no tractable hits were found, or target validation. Numbers indicate the total number of screens run in each class and/or category. 10 HTS screening

11. Many of the top 100 drugs sold worldwide are enzyme inhibitors. In recent years, enzyme inhibitors not only have provided an increasing number of potent therapeutic agents for the treatment of diseases, but also have significantly advanced the understanding of enzymatic transformations. Enzyme inhibitors in Medicine A human cell contains thousands of enzymes each of which can, theoretically, be selectively inhibited. These enzymes constitute the various metabolic pathways that, in concert, provide the requirements for the viability of the cell. A selective inhibitor may block either a single enzyme or a group of enzymes, leading to the disruption of a metabolic pathway(s). 11

12. Enzyme Inhibition: Mechanisms and Scope Enzyme inhibition is a science of enzyme-substrate reaction influenced by the presence of any organic chemical or inorganic metal or biosynthetic compound due to their covalent or non-covalent interactions with enzyme active site. The enzyme inhibitors are low molecular weight chemical compounds. They can reduce or completely inhibit the enzyme catalytic activity either reversibly or permanently (irreversibly). In drug discovery, several drug analogues are chosen and/or designed to inhibit specific enzymes. 12

13. Reversible inhibition: E ffect of an inhibitor can be reversed by decreasing the concentration of inhibitor Irreversible inhibition: N o reversal of inhibition on decreasing the inhibitor concentration: an example of enzyme inactivation Example: Cyanide : by covalently binding mitochondrial cytochrome oxidase, it inhibits all the reactions associated with electron transport Penicillin for bacterial peptidase 13

14. Reversible Modes of Inhibitor Interactions with Enzymes • Most drugs bind to their enzyme target through reversible interactions. • Inhibitors can bind directly to the free form of the enzyme, to an enzyme species that follows formation of the enzyme-substrate complex, or to both. • Drug affinity is best quantified in terms of the equilibrium dissociation constant for these varied forms of the target enzyme. • Comparisons of affinity among different inhibitors for a common enzyme, or among different enzymes for a common inhibitor, are best done in terms of the relative dissociation constants or the related Gibbs free energy of binding. 14

15. ENZYME INHIBITORS AS DRUGS Competitive Inhibitors compete with the substrate for the active site Non-Competitive inhibitors bind near but not at the active site Irreversible inhibitors bind at the active site of the enzyme and form a covalent bond with a group at this site 15

16. 16 Enzyme Inhibition

17. Enzyme Inhibition (Mechanism) Competitive Non-competitive Uncompetitive E E Different site Compete for active site Inhibitor Substrate Cartoon Guide Equation and Description [ I ] binds to free [E] only, and competes with [S]; increasing [S] overcomes Inhibition by [ I ]. [ I ] binds to free [E] or [ES] complex; Increasing [S] can not overcome [ I ] inhibition. [ I ] binds to [ES] complex only, increasing [S] favors the inhibition by [ I ]. E + S → ES → E + P + I ↓ E I ← ↑ E + S → ES → E + P + + I I ↓ ↓ E I + S →E I S ← ↑ ↑ E + S → ES → E + P + I ↓ E I S ← ↑ X Juang RH (2004) BCbasics 17

18. 18 C ompetitive inhibitors as drugs Doxycycline : Competitive inhibitor of collagenase - control of periodontal disease AZT : Competitive inhibitor of HIV RT Mozenavir and Tipranavir : Competitive inhibitors of HIV protease Allopurinol (used in gout)— Oxypurinol —inhibits xanthine oxidase 18

19. 19 COMPETITIVE INHIBITORS AS DRUGS Adenine Thioadenine

20. Angiotensin converting enzyme inhibitors Enalapril Benzapril Lisinopril Moexipril Perindopril Quinapril Captopril ( 1965 Ferreira ) Ramipril Spirapril Trandolapril 20

21. Statins are a group of cholesterol-lowering agents largest selling drugs in the world. lowers serum cholesterol levels by competitively inhibiting 3- hydroxy-3-methylglutaryl-coenzyme-A (HMG- CoA ) reductase , a key enzyme in cholesterol biosynthesis. Eg . Atorvastatin , simvastatin , fluvastatin 21

22. The cGMP substrate and sildenafil a competitive inhibitor of the cGMPphosphodiesterase-5. 22

23. Sulfanilamides – Simplest form of Sulfa drugs. Earliest antibacterial chemotherapeutic drugs classified as enzyme inhibitors. Competitive inhibitors of the bacterial folic acid synthesizing enzyme system from p- aminobenzoic acid. Bacterial cannot absorb pre-made folate that is necessary to be synthesized de novo. Structural similarity of sulfanilamide (and other sulfas derived from it) to p- aminobenzoic acid made them competitive inhibitors to the enzyme. 23

24. Organophosphorus compounds and the suicidal irreversible mechanism-based inhibition of the enzyme acetylcholine esterase by diisopropylfluorophosphate . Malathion and parathion are organophosphorus insecticides. The nerve gases Tabun and Sarin are other organophosphorus compounds. 24

25. Methotrexate – competitive inhibitor of dihydrofolate reductase (DHFR) Anticancer antimetabolite chemotherapy particularly for pediatric leukemia. It hinders the availability of tetrahydrofolate as a carrier for one-carbon moieties important for anabolic pathways -particularly synthesis of purine nucleotides for DNA replication 25

26. Substrate mimics competitive inhibitors are the peptide-based protease inhibitors, a very successful class of antiretroviral drugs used to treat HIV, ritonavir that contains three peptide bonds. 26

27. Guanosine analogue antiviral drug aciclovir - acycloguanosine (2-amino-9-((2- hydroxyethoxy )methyl)-1H-purin-6(9H)-one), as one of the most commonly-used antiviral drugs, it is primarily used for the treatment of herpes simplex and herpes zoster (shingles) viral infections. Acyclo -GTP is a very potent inhibitor of viral DNA polymerase Aciclovir : Prodrug for the suicidal irreversible inhibition of the viral DNA polymerase 27

28. Selected enzyme inhibitors in clinical use or trials 28

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30. Some examples of mechanism-based inactivators as drugs 30

31. Enzyme Inhibitors Used in Human Diseases 31 Clinical Use Enzyme Inhibited Inhibitor Epilepsy GABA transaminase Carbonic anhydrase Succinic semialdehyde dehydrogenase y-Vinyl GABA Sulthiame Sodium valproate Antidepressant Monoamine oxidase (MAO) Tranylcypromine, phenelzine Antihypertensive Angiotensin converting enzyme Captopril, enalaprilat Cardiac disorders Na',K'-ATPase Cardiac glycosides Gout Xanthine oxidase Allopurinol Hyperlipidemia HMG-CoA reductase Atorvastatin, simvastatin Anti-inflammatory Prostaglandin synthase, Aspirin, naproxen, Arthritis Cyclooxygenase (COX) I and II Cyclooxygenase (COX) II Celecoxib Neostigmine Glaucoma Acetylcholinesterase Acetazolamide ,

32. Enzyme Inhibitors Used in the Treatment of Bacterial , Fungal, Viral, and Parasitic Diseases 32

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34. 34 Development of ACE inhibitors for controlling hypertension

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36. Kinases Class of enzymes that play a key role in intracellular responses to stress and growth factors. Catalyse the transfer of phosphate from ATP to proteins Activation leads to a cascade of activating signals that regulate many physiological processes, such as cell division and differentiation. Aberrant activity is associated with many diseases like cancer, diabetes, and chronic inflammatory disorders. They have been identified by the pharmaceutical industry as important targets for drug development. 36

37. Enzyme Inhibitors Used in the Treatment of Cancer Type of Cancer Enzyme Inhibited Inhibitor 37

38. Imatinib mesylate (Gleevec1, Novartis) is a small-molecule compound that inhibits a specific tyrosine kinase enzyme, the Bcr–Abl fusion oncoprotein . It is used for gastrointestinal stromal tumor and chronic myeloid leukemia. Gefitinib (Iressa1, AstraZeneca & Teva ) is a small-molecule inhibitor of the epidermal growth factor receptor’s (EGFR, or erbB1) tyrosine kinase domain. It is used for non-small-cell lung cancer. Bortezomib (Velcade1, Millenium Pharmaceuticals) is a small-molecule proteasome inhibitor used for the treatment of multiple myeloma refractory to other treatments. 38 Rituximab (Rituxan1, Biogen Idec & Genentech) is a monoclonal antibody used in the treatment of B-cell non-Hodgkin’s lymphoma and B-cell leukemia.It binds the CD20 antigen on the CD20+ B-cells, causing their apoptosis.

39. CYP inhibition and its relation to chemoprevention Chemoprevention is the ability of compounds to protect healthy tissues via the prevention, inhibition or reversal of caricnogenesis . The inhibition of CYP1 enzymes is one such route among others that include the induction of cell cycle arrest, the induction of phase II enzymes and the inhibition of inflammatory 39 The CYP1 family has been linked with the activation of pro-carcinogens which is facilitated by the regulation of the aryl hydrocarbon receptor. As such research has shown that inhibiting CYP1 enzymes plays a key role in protecting healthy cells from the harmful effects of activated carcinogens.

40. A schematic representation of carcinogenesis via the activation of CYP1 enzymes. Upon the activation of the pro-carcinogens by the CYP1 enzymes, they have the ability to bind to DNA, which can lead to mutations and then the formation of cancer cells. 40

41. Interaction of quassinoids with CYP1A1 Review of compounds that have potent and selective inhibitory properties against the activities of CYP1 family in particular CYPs 1A1 and 1B1 aid in identification of usefuchemoprotectors . 41

42. New developments in art of enzyme inhibition Immobilized enzymes : Medicinal and industrial enzyme products . amylase , glucoamylase , trypsin , pepsin, rennet, glucose isomerase , penicillinase , glucose oxidase , lipase, invertase , pectinase , cellulase in medicinal use. 42

43. Softwares and computerization in enzyme inhibition kinetics The data and results analysis is transferred in user friendly lay-out, ANOVA window, % inhibition using Monte-Carlos fits, and receptor or ligand binding calculator. For interested readers, VISUALENZYMICS 2010® is available for statistical analysis for enzyme kinetics . [ http://www.softzymics.com/visualenzymics.htm]. 43

44. 44 CONCLUSIONS Enzyme inhibitors have long played an important role in medicine, pharmacology, and basic research. The advances in DNA technology have enabled cloning and overexpression of large numbers of enzymes. However, in the postgenomics era, large numbers of new targets have been identified. Although the drug discovery process moves toward structure-based drug design as its prime tool, even with high-throughput crystallography, not all target proteins will be readily accessible.