Drug discovery thr enzyme inhibition

Drug discovery through enzyme inhibition

1. Dr. Kumbhare Manoj R. Professor, Head Dept. of Pharmaceutical Chemistry S.M.B.T. College of Pharmacy, Dhamangaon Tal- Igatpuri Dist. Nashik (M.S.). [email protected] =================== Drug Discovery through Enzyme Inhibition

2. Drug Discovery 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 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. 2

3. Drug discovery proceeds as a multi-stage process, from the identification of a potential therapeutic target through lead generation, lead optimization, preclinical evaluation to clinical testing, before a new medicine is marketed. Uniting synthetically accessible chemical space with disease relevant biological space represents a significant challenge. Industrial chemical biology approaches have been recognized by pharmaceutical companies as a novel strategy in drug discovery. 3

4. 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? 4

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

6. 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 lnhibitors 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). 6

7. Examples of Enzyme Inhibitors Used in the Treatment of Bacterial, Fungal, Viral, and Parasitic Diseases 7

8. Examples of Enzyme Inhibitors Used in Various Human Disease States Clinical Use Enzyme Inhibited Inhibitor Epilepsy Epilepsy Epilepsy Antidepressant Antihypertensive Cardiac disorders Gout Ulcer Hyperlipidemia Anti-inflammatory Arthritis Glaucoma Glaucoma GABA transaminase Carbonic anhydrase Succinic semialdehyde dehydrogenase Monoamine oxidase (MAO) Angiotensin converting enzyme Na',K'-ATPase Xanthine oxidase Hf ,K+- ATPase HMG- CoA reductase Prostaglandin synthase , Cyclooxygenase (COX) I and II Cyclooxygenase (COX) II Acetylcholinesterase Carbonic anhydrase II y-Vinyl GABA Sulthiame Sodium valproate Tranylcypromine , phenelzine Captopril , enalaprilat Cardiac glycosides Allopurinol Omeprazole Atorvastatin , simvastatin Aspirin, naproxen, Celecoxib Neostigmine Acetazolamide , dichlorphenamide 8

9. Drug Development Process: Discovery-Approval PI P2 P3 Discovery Preclinical Clinical FDA 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 500-800 million dollars 0.003% chance of a return on investment (1/30,000) 9

10. 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. 10 ENZYMES ARE ESSENTIAL FOR LIFE Deregulated enzyme activity can also lead to disease states

11. Enzyme Inhibition: Mechanisms and Scope Enzyme is a protein molecule acting as catalyst in enzyme reaction. 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. 11

12. 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. 12

13. 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. 13

14. Reversible inhibition : the effect of an inhibitor can be reversed by decreasing the concentration of inhibitor Irreversible inhibition : there is no reversal of inhibition on decreasing the inhibitor concentration: an example of enzyme inactivation e.g. Cyanide: by covalently binding mitochondrial cytochrome oxidase, it inhibits all the reactions associated with electron transport Penicillin for bacterial peptidase The distinction between reversible and irreversible inhibition is not absolute and may be difficult to make if the inhibitor binds very tightly to the enzyme and is released very slowly ( tight-binding inhibitors ) 14

15. Distribution of marketed drugs by biochemical target class. 15

16. 16 Enzyme Inhibition-Drug Discovery Aspirin (anti-inflammatory) Penicillin (effective against gram positive) Sulfonamides Anti HIV drugs

17. 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 17

18. 18 Enzyme inhibition

19. 19 COMPETITIVE INHIBITORS AS DRUGS Adenine Thioadenine

20. 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 20

21. Kinases are a class of enzymes that play a key role in intracellular responses to stress and growth factors. Kinases catalyse the transfer of phosphate from adenosine triphosphate (ATP) to proteins, which may be either other kinases , transcription factors or other regulatory proteins. As such, activation of kinases leads to a cascade of activating signals that regulate many physiological processes, such as cell division and differentiation. Aberrant kinase activity is associated with many diseases like cancer, diabetes, and chronic inflammatory disorders. Kinases have been identified by the pharmaceutical industry as important targets for drug development. 21

22. 22 competitive 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 22

23. Selected enzyme inhibitors in clinical use or trials 23

24. 24

25. Some examples of mechanism-based inactivators as drugs 25

26. 26 Development of ACE inhibitors for controlling hypertension

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29. Control of hypertension Alternatives for the control of hypertension were considered: –Finding an inhibitor of renin •efforts resulted in the finding of inhibitors with powerful in vitro potency. In addition much was learned on how to convert peptides into more drug-like molecules, however no orally active agent was found. –Potentiating the formation of bradykinin •the enhancement of enzyme action (i.e. stimulation of the production of more bradykinin ) was known to be far more difficult than inhibition of an enzyme and so this route was rejected. –Finding an inhibitor of ACE •Early work suggested that bradykinin was inactivated by an enzyme similar to ACE. When this enzyme was proved to be ACE the inhibition of ACE became the target for a new anti-hypertensive agent. 29

30. Examples of angiotensin converting enzyme inhibitors Enalapril Benzapril Lisinopril Moexipril Perindopril Quinapril Captopril (1965 Ferreira ) Ramipril Spirapril Trandolapril 30

31. The statins are a group of cholesterol-lowering agents that have become some of the largest selling drugs in the world. They lower serum cholesterol levels by competitively inhibiting 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase, a key enzyme in cholesterol biosynthesis. Ex mevastatin , simvastatin, fluvastatin 31

32. 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. 32

33. Methotrexate - competitive inhibitor of dihydrofolate reductase (DHFR) is another example. The drug is used as 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 33

34. Sulfanilamides - the simplest form of Sulfa drugs - were among earliest antibacterial chemotherapeutic drugs classified as enzyme inhibitors. They are 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. 34

35. The cGMP substrate and sildenafil a competitive inhibitor of the cGMPphosphodiesterase-5. 35

36. Another example of these substrate mimics competitive inhibitors are the peptide-based protease inhibitors, a very successful class of antiretroviral drugs used to treat HIV, e.g., ritonavir that contains three peptide bonds. 36

37. 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; the prodrug for the suicidal irreversible inhibition of the viral DNA polymerase 37

38. Anticancer Drug Development Around the world, tremendous resources are being invested in prevention, diagnosis, and treatment of cancer. Cancer is the second leading cause of death in Europe and North America. Discovery and development of anticancer agents are the key focus of several pharmaceutical companies as well as nonprofit government and non-government organizations, like the National Cancer Institute (NCI) in the United States, the European Organization for Research and Treatment of Cancer (EORTC) , and the British Cancer Research Campaign (CRC) . The discovery and development of anticancer drugs, especially cytotoxic agents, differ significantly from the drug development process for any other indication. The unique challenges and opportunities in working with these agents are reflected in each stage of the drug development process 38

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 useful chemoprotectors. 41

42. Examples of Enzyme Inhibitors Used in the Treatment of Cancer Type of Cancer Enzyme Inhibited Inhibitor 42

43. 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. 43

44. 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. Trastuzumab (Herceptin1, Genentech) is a monoclonal antibody that binds the cell surface HER2/neu (erbB2) receptor and is used in the therapy of erbB2+ breast cancer 44

45. New developments in art of enzyme inhibition Now a day, immobilized enzymes are used in industries and have value as medicinal and industrial enzyme products. Good examples of industrial enzymes are amylase, glucoamylase , trypsin , pepsin, rennet, glucose isomerase , penicillinase , glucose oxidase , lipase, invertase , pectinase , cellulase in medicinal use. With emergence of new inhibitors in the quest of drug discovery, several new inhibition mechanisms are expected in case of new substrate analogues. New substrate–enzyme active site interactions are envisaged due to different binding intricacies Slow-tight inhibition: Slow-tight inhibition occurs when the initial enzyme-inhibitor complex EI undergoes isomerizing conformational change to a more tightly binding complex. However, the overall inhibition process is reversible. 45

46. Substrate and product inhibition : Substrate and product inhibition is where either the substrate or product of an enzyme reaction inhibits the enzyme's activity. This inhibition may follow the competitive, uncompetitive or mixed patterns. Antimetabolites: They are chemicals that interfere with the normal metabolism of normal biochemical metabolite(s). This in most of case is due to their structural similarity to such physiological substrates and therefore works as competitive enzyme inhibitors. They include antifolates such as methotrexate, hydroxyurea and purine and pyrimidine analogues. They are mainly used as cytotoxic anticancer drugs through inhibiting DNA and RNA synthesis and cell division. 46

47. Antienzyme: Intestinal parasites, e.g., Ascaris, protect themselves from digestion by expressing on their surface substances that are protein in nature which inhibit the action of digestive enzymes, e.g., pepsin and trypsin. The blood plasma and extracellular fluids are containing several types of protease inhibitors particularly important in controlling the blood clot formation and dissolution and matrix and cytokine homeostasis. Antibodies against several nonfunctional plasma enzymes have clinical diagnostic importance since they are longer living than the enzyme itself and hence reflect the disease history better. In this respect, autoimmune antibodies are clinically important in diagnosis of autoimmune diseases, e.g., anti- glutamic acid decarboxylase antibodies in type 1 diabetes mellitus. 47

48. Biosensors : Light inhibits most enzyme activity although some enzymes, e.g., amylase are activated by red or green light and also specific DNA repairing enzymes (e.g., Uvspecific endonuclease ) are activated by the blue and UV light. Ultraviolet rays and ionizing radiations cause denaturation of most enzymes. Most enzymes contain sulfhydryl (-SH) groups at their active sites which upon oxidation by oxidants and free radicals by oxidants and free radicals inactivate the enzyme. Examples: Effect of radiations, light and oxidants on the rate of the enzyme catalyzed reaction. 48

49. Searching for new allosteric sites in enzymes The discovery of new allosteric sites generates opportunities for the identification of novel pharmaceuticals and increases our understanding of basic biological processes. Increasingly, allosteric sites are being discovered in various families of proteins by several methods, paving the way for the development of entirely new classes of drugs with a wide range of chemotypes . New allosteric sites in enzymes have been discovered both incidentally and by directed means, and the mechanisms by which allosteric activation and inhibition occur at these sites have been investigated. By exploring recent structurally well-characterized examples, trends begin to emerge for both the modes of binding and mechanisms of inhibition. 49

50. New allosteric sites occur distal from the active site in HIV-1 RT, GlyP and F16BPase. (a) The binding site in HIV-1 RT for the NNRTIs ( pink spheres ) is non-overlapping with the three active site aspartate residues ( green spheres ). (b) GlyP monomers are drawn in pink and blue. The new allosteric site at the dimer interface is shown with the allosteric indole -based compounds drawn as black spheres. The active site is in the region of the pyridoxyl phosphate cofactor (red) and substrate (green). Other regulatory sites are the AMP-binding site (yellow) and the Ser14 phosphorylation site (bright pink). (c) The four F16BPase monomers are drawn as pink or blue ribbons . The sites bound by allosteric inhibitors (black spheres) are distal from the AMP-binding site (yellow spheres), the active site manganese ( orange ) and the fructose-6-phosphate product ( green) . 50

51. Softwares and computerization in enzyme inhibition kinetics Recently softwares have popped up to visualize custom visual interface to see curve fits in real-time, graph transforms, equations using kinetic data entry in terms of substrate, inhibitor, activator, velocity, and standard deviation of the velocity. Data tables are directly generated linked to the Fitting Panel of software. 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]. 51

52. 52 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.

53. The evolution of algorithms that can predict enzyme function and mechanism will ensure that the rational design of enzyme inhibitors not only complements structure-based approaches but continues to play a stand-alone role in the discovery of novel therapeutics . 53