1. Enzymes as Drug Targets - a Closer Look Transition state analogs and “suicide substrates”

2. Enzymes as Drug Targets - a Closer Look Transition state analogs and “suicide substrates”

3. Enzymes - Background What do they do? Enzymes increase the rate of, but do not change equilibrium position of most reactions that occur in the cell. Catalysts! Relative transition-state stabilization lowers kinetic barrier, increases rate (k 2 )! S P (uncatalyzed) (enzyme catalyzed) S P

4. Enzymes - Background Enzymes can become saturated with substrate ([ES] = [E] tot ). (enzyme catalyzed) How is enzyme activity measured? Using kinetics (measuring rates: the rate of appearance of product or the rate of disappearance of substrate)

5. Enzymes as Drug Targets - a Closer Look Understanding the nature of enzyme catalysis AND the mechanism of a biochemical reaction can lead to the design of effective drugs Substrate and Transition-state structure Enzyme mechanism The majority of enzyme-targeted marketed drugs are related to the enzyme substrate structure.

6. Enzyme Inhibition by Small Molecules What small molecules inhibit enzymes?? Cellular regulators, drugs, toxic agents Inhibitors decrease the effectiveness of the enzyme as a catalyst (so inhibitors can be drugs; and some enzymes are good targets) Inhibitors can be Reversible or Irreversible. Reversible (competetive, noncompetitive, uncompetetive; kinetics experiments can distinguish between these modes of inhibition - see appendix):

7. Reversible enzyme inhibitors decrease enzyme activity reversibly

8. Reversible Inhibitors (continued) Lecture on Protein targets listed some examples of reversible inhibitors as drugs 1. Transition state mimic for adenosine deaminase (enzyme which degrades anticancer drugs) 2. Substrate mimic for dihydropteroate synthase (dihydrofolate synthesis) 3. Transition state mimic for HMG-CoA reductase (cholesterol synthesis) Substrate versus Transition-state analogs: Which approach should result in the highest affinity drug? Why? To design a reversible competetive inhibitor as a drug, design a mimic of the substrate or the transition state.

9. Reversible Inhibitors (continued). The transition state is stabilized more than the substrate Example 1: Isopentyl Diphosphate isomerase

10. Reversible Inhibitors (continued). Example 2: Purine nucleoside phosphorylase. Lower activity causes T-cell immunodeficiency. Potential therapy for T-cell cancer and T- cell autoimmune disorders. Transition state structure was determined with analogs of substrates: Inhibitors were designed with K D in the nanomolar range

11. Reversible Inhibitors (Ex. 2 continued). K i = 23pM

12. Reversible Inhibitors (Ex. 2 continued). Structures of bovine enzyme target + Substrate analogs Transition-state mimic Products These show how the enzyme binds the transition state more strongly than the substrate.

13. Reversible Inhibitors (Ex. 2 continued). What about the human enzyme??? 87% homologous to bovine enzyme Ki ~60pM (weaker binding than bovine enzyme) Active site structure is completely conserved, so it must have a different transition state structure (and therefore a different transition state analog) Ultimate inhibitor: Inhibits for lifetime of cell!!

14. Reversible Inhibitors (Ex. 2 continued). Ultimate inhibitor: Inhibits for lifetime of cell!! If the structure of target enzyme complex revealed additional potential binding interactions (empty hydrophobic pocket, etc), an even stronger drug could be designed. Recap: Reversible enzyme inhibitors bind reversibly! Competetive inhibitors’ structure should be more similar to that of the transition state for stronger binding Noncompetetive and uncompetetive inhibitors can’t be “designed”, because they don’t resemble the substrate or transition state.

15. Irreversible Inhibitors: Affinity labels, suicide substrates - form covalent bonds with the enzyme Affinity labels = molecules that Resemble the substrate, so targeted to binding site; Contain an electrophilic group (below, or alpha-halo ketones, or diazoketones) that reacts with a nucleophilic group of the enzyme in or near the active site to form a covalent bond.

16. Irreversible Inhibitors - affinity labels (continued) Ex. 1 Penicillin - resembles acyl D-ala-D-ala and it acylates the active site serine of transpeptidase. Steric bulk or conformational changes prevents hydrolysis or transamidation. Somewhat (or very) toxic because they are so reactive - they react at other sites than the enzyme binding site.

17. Irreversible Inhibitors - affinity label examples (continued) Ex. 2. TPCK (Tosyl-phenylalanyl-chloromethyl-ketone). Binds to active site of chymotrypsin (binds Phe, trp). Contains an electrophilic carbon that forms covalent bond with chymotrypsin active site histidine. Big problem - how to avoid reactions with other nucleophiles on other proteins? Mask the reactive electrophile until it is in the active site: Suicide Substrate/Trojan Horse Inhibitor/Mechanism-based Inhibitor!

18. Irreversible Inhibitors - Suicide substrates Ex. 1. Halo enol lactones and serine proteases

19. Irreversible Inhibitors - Suicide substrates Ex. 2. Vigabatrin, an anticonvulsant that inhibits a pyridoxyl phosphate-dependent enzyme that degrades GABA (neurotransmitter). Part of mechanism for amine substrates in pyridoxal-dependent enzymes: (Intermediate 4.19 can lose H+, CO 2, and may undergo further reactions).

20. Irreversible Inhibitors - Suicide substrates Ex. 2 Vigabatrin, (cont) Normal substrate for aminotransferase: Suicide substrate for aminotransferase: One new electrophilic center TWO new electrophilic centers!

21. Irreversible Inhibitors - Suicide substrates Ex. 2 Vigabatrin, (cont) Reactivity of cationic intermediates: N+ is a good electron “sink,” making the molecule susceptible to nucleophilic attack. The nucleophile may be a group on the enzyme, or another molecule Michael addition

22. Irreversible Inhibitors - Suicide substrates Ex. 2 Vigabatrin, (cont) Normal substrate: final products. Enzyme unchanged and active Suicide substrate: two pathways for products, one which inactivates the enzyme!

23. Recap: Irreversible Inhibitors: affinity labels, suicide substrates Affinity labels: Contain a reactive electrophile that reacts with an enzyme’s nucleophile to form a covalent (irreversible) bond Toxic because the electrophile is too reactive to be specific. Suicide substrates/mechanism-based inactivators: Designed to produce a reactive electrophile only upon binding to the correct enzyme and undergoing normal catalytic steps

24. Additional Example 1: JACS 2003, 125 p. 685 Inhibitors of AmpC beta lactamase were developed: Due to widespread resistance, inhibitors of beta-lactamases are sought. Clavulanic acid (d) is one inhibitor; ceftazidime (b) is a beta- lactam that is resistant to beta-lactamases. New substrate analogs “c” are found to inhibit new broad spectrum beta-lactamases. All have similar structure: resistance to these are also anticipated.

25. Alternate strategy: de novo structure-based design. Have found novel structures unlike natural substrate that circumvent traditional resistance mechanisms, but they are weak, with Ki = 25 micromolar. A third strategy: Transition state analogs. Beta-lactamase intermediate: Ki = 20nM The beta-lactam ring is replaced With the boronic acid; R1 can be changed to improve affinity. Investigators focused on “c”: carboxylate mimics cephalosporin Carboxylate in transition state. Additional Example 1 (cont)

26. 1nM inhibitor If the Carboxylate is removed, binding decreases by 30-fold Stereo view of the molecule above bound to the enzyme AmpC But now, except for boronic acid, the molecule looks a bit like a beta lactam…Will resistance be a problem? Best inhibitor: Additional Example 1 (cont)

27. Resistance is hardest to develop against analogs that resemble substrates….A resistant organism must distinguish between inhibitor and substrate (since it must act on the substrate!). Transition state analogs do resemble the substrate to some degree… Additional Example 1 (cont)

28. Additional Example 2 - Hepatitis C virus therapy. Target: HCV NS3 protease, a serine protease that is essential to viral replication. Serine proteases have a “catalytic triad” of residues in the active site. Mechanism: a.Substrate binds to active site b.Asp-his help make ser a better Nu. c. Ser attacks carbonyl of amide, forming a tetrahedral intermediate d.Asp-his-H+ helps makes amine a better leaving group (Peptide strand is broken; one part is released from enzyme) e. Asp-his make water a better Nu that attacks carbonyl of ester

29. Additional Example 2 (cont) f. New tetrahedral intermediate is formed g. Asp-his-H+ help make ser a better leaving group. h. Enzyme is back to original state. Other part of peptide is released. Suicide substrate for a serine protease: Alpha keto-amide may be attacked by serine, “trapping” the enzyme

30. Additional Example 2 (cont) Note: No leaving group attached to the carbonyl, so serine -OH will not cleave the drug.

31. References Robertson, J. G. “Mechanistic Basis of Enzyme-Targeted Drugs” Biochemistry, 2005, 44, 5561-5571. Silverman, R. B. The Organic Chemistry of Drug Design and Drug Action ; Academic Press: San Diego, CA, 1992 Schramm, V. L. “Enzymatic transition states: thermodynamics, dynamics and analogue design” Arch. Biochem. Biophys. 2005, 433, 13-26. Venkatraman, S.; Njoroge, F. G.; Girijavallabhan, V. M.; Madison, V. S.; Yao, N. H.; Prongay, A. J.; Butkeiwicz, N.; Pichardo, J.“Design and Synthesis of depeptidized macrocyclic inhibitors of Hepatitis C NS3-4A Protease using structure-based drug design” J. Med. Chem., 2005, 48, 5088-5091.

32. Appendix: Enzyme kinetics No inhibitor: Simplification of kinetic scheme (by rapid equilibrium or steady state approaches) leads to the Michealis- Menten equation.

33. Competetive Inhibition V

34. Noncompetetive (mixed)

35. Uncompetetive Inhibition

36. Molecular Diversity- Synthetic approaches: (Note: in synthesis, “target” is the molecule you want to synthesize; in drug discovery, “target” is the biological macromolecule you want to develop a drug to bind to) 1.Traditional (synthetic target-oriented; know structure of product; one product in one reaction vessel) A.Solution phase or Solid phase (beads) B.Protecting groups used, high yields desirable C.Parallel (can be solid or solution phase; simultaneous synthesis of many compounds) D.Location of active compound in a grid allows determination of structure of active compound 2.Combinatorial (many different products in one vessel) A.Use of solid phase, protecting groups, and “mix and split” is most common synthetic approach B.Deconvolution or encoding is required to determine structure of active compound 3.“Chemical structure space” versus “biological structure space”; how to improve your chances of getting a “hit”? A.Natural product-guided combichem B.Diversity-oriented synthesis (smaller libraries of more complex structures that look more like natural products than the simpler compounds made in standard combinatorial libraries. C.Click chemistry D.Dynamic combinatorial chemistry