ENZYME INHIBITION

RATIONAL DESIGN OF COVALENTLY BINDING ENZYME INHIBITORS

1. RATIONAL DESIGN OF COVALENTLY BINDING ENZYME INHIBITORS 1

2. Enzyme inhibitors are the agents which inhibit the enzyme. Binding of inhibitors with enzyme’s active site with forming a covalent bond. 2

3. Types of covalently binding enzyme inhibitors Chemical modifiers Affinity label Mechanism based inhibitors Pseudoirreversible inhibitors 3

4. TARGET: chemically reactive groups in the enzyme's active site. Nucleophiles –OH - serine, threonine , tyrosine -SH - cysteine –COOH - aspartic and glutamic acid -NH 2 of lysine Arginine - electrophilic side chain 4

5. CHEMICAL MODIFIERS Small organic molecules, generally electrophiles , that are used to modify the enzyme's side chains in such a way as to produce a stable covalent bond. These are used to study enzyme inactivation and to identify residues potentially involved in binding and catalysis. 5

6. RESIDUE TAEGETTED REAGENTS OTHER RESIDUE LABELED Lysine Acetic anhydride Isothiocyanates Trinitrobenzenesulfonate (TNBS) Cyanate These reagents can also react with the N-terminal amino group Histidine Diethylpyrocarbonate (DEPC) DEPC should be used at neutral pH to minimize reaction with lysines , cysteines , and tyrosines Arginine Phenylglyoxal   Butanedione Phenylglyoxal can react with lysine Butanedione should be used in the dark to prevent reaction with tryptophans , histidines , and tyrosines Tyrosine Tetranitromethane   Chloramine T Chloramine T also modifies histidines and methionines 6

7. compounds are chemically reactive and may lead to the modification of both catalytic and nonessential residues. Usually not specific for a given enzyme.   7

8. AFFINITY LABEL RSA are molecules that have a similar structure to the substrate allowing them to covalently bind to the active site of the enzyme. React with the substrate , inhibiting the enzyme. Mimics the functional properties of the substrate, but consequently irreversibly inhibits the enzyme through the strong covalent bonding. 8

9. First bind to the enzyme's active site in a noncovalent fashion . Formation of the enzyme-inhibitor complex. React by various mechanisms with one or more amino acid residues in the enzyme's active site. Covalent bond formation between the enzyme and the inhibitor. 9

10. Eg : TPCK ( tosyl phenyl alanine chloromethyl ketone ) Mimic substrates of chymotrypsin - ( tosyl phenyl alanine methyl ester). Presence of a halomethyl group- covalent attachment. 10

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12. Mechanism-Based lnhibitors Have great potential as drugs Designed to be specific toward their target enzyme. Compounds are unreactive until activated within their target enzyme Little or no cellular toxicity. 12

13. Design requires an understanding of the binding specificity requirements for the ligand -recognition site of the enzyme, to promote the formation of the initial noncovalent enzyme-inhibitor complex E . I 13

14. The choice of an appropriate latent functional group requires knowledge of the catalytic mechanism of the target enzyme with its normal substrate. Covalent bond formation by the activated inhibitor-chemical reactivity, and its proximity to a susceptible amino acid residue or cofactor. 14

15. Eg : Pyridoxal phosphate (PLP)-dependent enzymes. MECHANISM Step 1 Schiff base formation - by the amino group of the substrate reading with pyridoxal phosphate to form an aldimine . Step 2 Loss of a functional group usually by abstraction by an active-site base, to form a resonance-stabilized carbocation . 15

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17. Examples of PLP-dependent enzyme. GABA-T 17

18. Ornithine decarboxylase (ODC) Catalyzes the rate limiting step in the biosynthesis of polymines . Conversion of ornithine to putrescine African sleeping sickness Eflomithine (a- difluoromethylomithine , DFMO) 18

19. Inactivation of ODC by DFMO involves The decarboxylation of DFMO by the enzyme, with subsequent stoichiometric binding of a reactive species to the enzyme 19

20. Pseudoirreversible lnhibitors Least common of the covalently binding enzyme inhibitors. Covalent bond formed between the enzyme and the inhibitor is reversible. The utility of a pseudoirreversible inhibitor will be determined by a combination of the rate of formation of the covalent enzyme inhibitor adduct and the half-life for reactivation. 20

21. The inherent reversibility of pseudoirreversible inhibitors, it may be more difficult to obtain structural evidence for the covalent enzyme inhibitor adduct. Determination of the rate of reactivation and characterization of the products 21

22. Different intermediates are formed Acetyl- serine intermediate Serine- phosphate esters Serine- carbamate 22

23. Eg : Irreversible inhibition : Acetylcholinesterase ( AcChE ) Decrease the conc. in synaptic cleft 23

24. Agents such as parathion(insecticides) and sarin (nerve gases) With enzyme to form the active-site -serine-phosphate esters. Hydrolyzed extremely slowly by water, making the inhibition effectively irreversible. 24

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26. Carbamate inhibitors – physostigmine , rivastigmine React with AcChE to form a carbamylated serine. Rapidly hydrolyzed, thereby regenerating AcChE . Rivastigmine .a more useful therapeutic agent(half-life more than 10 h) 26

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28. REFERENCE Burger’s Medicinal Chemistry and Discovery sixth edition, volume 1,p.no:754-774 28

29. THANK YOU... 29