The Design, Synthesis, and Evaluation of Mechanism-Based b - Lactamase Inhibitors

1. The Design, Synthesis, and Evaluation of Mechanism-Based b-Lactamase Inhibitors CWRU 2009

2. Major Classes of b-Lactam Antibiotics • Potent, broad-spectrum antibiotics • Usually well tolerated • Structural similarities include a negatively charged carboxylate, (usually fused bicylic) b-lactam, and C6 appendage

3. The b-lactam antibiotics interfere with one or more members of a crucial set of bacterial enzymes, known as the penicillin-binding-proteins (PBPs), that are responsible for cross-linking glycan strands through a protruding peptide side chain.

4. The b-lactam antibiotics are believed to resemble the D-Ala-D-Ala terminus of the pentapeptide side chain (StromingerHypothesis) • Bacterial transpeptidases cleave between the two D-Ala residues, to form an intermediate acyl-enzyme, which is then reacted with a free amino moiety (e.g. the w amino group of diaminopimelic acid) to form the cross link.

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9. Why are b-lactam antibiotics such good drugs? • b-Lactamantbiotics still comprise approximately half the commercial antibiotic market. • Formation of a covalent bond to the target(s) may be an effective strategy for avoiding resistance due to point mutations which lower affinity • Targeting the bacterial cell wall avoids the necessity to accumulate in cytoplasm, thus avoiding efflux pumps. • b-lactams do not penetrate most mammalian cell types, resulting in low toxicity (disadvantage when treating atypicals) • Most commonly observed resistance is due to production of b-lactamase(s)

10. Resistance to b-Lactam Antibiotics Production of one or more enzymes (b-lactamases) that hydrolytically destroy b-lactam antibiotics Produce PBPs that do not recognize penicillin In the case of Gram-negative strains delete outer membrane porins, which are responsible for the allowing the b-lactams to reach the periplasm and hence the cell wall In the case of Gram-negative strains, upregulate efflux pumps, which are responsible for pumping out foreign substances (including b-lactams). Action of Serine b-Lactamases

13. The b-Lactamases • More than 600 different b-lactamases, grouped into four classes A-D • Classes A, C, and D are serine enzymes • Class B are zinc metalloenzymes • Historically, the class A (serine) enzymes were the most prominent • Can be produced in large quantity (hyperexpressed) • Produced in the periplasm of Gram-negative organisms, or extracellularly in Gram-positive strains.

14. Bulky group Enzyme • One early strategy for countering b-lactamase mediated resistance was to design b-lactam antibiotics which would also be poor b-lactamase substrates. • This was achieved by incorporating sterically large substituents at C6 (penicillin) or C7 (cephalosporin).

15. Methicillin-resistant Staphylococcus aureus MRSA • Unfortunately, this gave rise to new forms of resistance, such as the appearance of a penicillin binding protein with reduced affinity for all b-lactam antibiotics (PBP2a in MRSA) and also the appearance of b-lactamases with enlarged active sites (extended spectrum b-lactamases or ESBLs) that could accommodate the larger antibiotics.

16. Recent Trends in b-Lactamase-mediated Resistance • Broad spectrum b-lactamases, known as extended spectrum b-lactamases (ESBLs) capable of hydrolyzing third generation cephalosporins, are disseminated widely (e.g. class A, CTX-M) • Class C b-lactamases (AmpC) are more widely disseminated, now including many plasmid-mediated AmpCs (e.g. FOX and CMY) • Classes A and D enzymes have evolved the ability to hydrolyze the carbapenem class of antibiotics. These serine carbapenemases are increasingly widespread (e.g. KPC). • Class B metallo-b-lactamases are disseminating widely. These enzymes were originally seen in Asia and in Europe, but cases of resistance due to class B b-lactamases are now appearing in the US (e.g. IMP and VIM).

17. Current Commercial b-Lactamase Inhibitors • A second approach was to develop inhibitors of b-lactamase • Unfortunately, current commercial inhibitors target only class A enzymes

18. Since the inhibitors have no independent antibacterial activity (i.e. ability to bind PBPs), they must be coadministered with b-lactam antibiotics

19. How do these commercial inhibitors work? • Placing sulfur at the sulfone oxidation state predisposes the thiazolidine ring to fragment, producing the iminium ion shown above. • The iminium ion can then tautomerize to the b-aminoacrylate, or be captured by a second active site serine, producing in both cases, a stabilized acyl-enzyme.

20. How can we build a better mousetrap? Irreversible inhibitors offer numerous opportunities for improving the inhibitory efficiency.

21. The Inhibitor Design Process enzymatic mechanism active site dimensions and binding characteristics synthetic feasibility generate a library of prospective inhibitors Assay against all relevant enzymes

22. Initially we focused on designing inhibitors which held the potential to quickly form very stable acyl-enzymes. Focus Here

24. IC50 Values against the class C b-lactamase derived from Enterobacter cloacae, strain P99

25. Further mechanistic investigations uncovered an isotope effect on the rate of inactivation. A mechanism consistent with this observation is shown below. Stabilized Acyl-Enzyme

26. New chemical methodology facilitated the preparation of new inhibitors.

27. The availability of 6-oxopenicillanate simplifies the synthesis of 6-alkylidene penams, as shown.

32. Buynak, J. D. et. al. BMCL 1999, 9, 1997-2002.

37. Initial attempts to improve the cephalosporin series of b-lactamase inhibitors relied on analogy with the cephalosporin antibiotics themselves.

38. But these efforts resulted in an abysmal failure!

39. Since the charge neutral pyridine moiety is a better leaving group than the negatively charged acetate, it is more likely to follow pathway 1 above. • Yet all the inhibitory mechanisms we have proposed follow pathway 2.

42. How do my inhibitors work? • Intramolecular capture of intermediate imine is more efficient than intermolecular capture (and/or tautomerization) • Inhibitors tend to be more general to all (serine) b-lactamases, since inhibitory mechanism does not depend on enzyme active site groups

46. Next goal: Prepare penicillin-derived inhibitors of metallo-b-lactamases Problem: Metallo-b-lactamases are still a small portion of total number of b-lactamase producing strains Solution: Prepare a single molecule that can function as dual inhibitor of both metallo- and serine-b-lactamases. Problem: Metallo and serine b-lactamases have profoundly different mechanisms of action.

47. Proposed series of events involved in the hydrolysis of a cephalosporin substrate by the L1 metallo-b-lactamase.

48. Inhibiting metallo-b-lactamases Like most metalloenzymes, metallo-b-lactamases are inactivated by good zinc chelators. Potential problem is that zinc chelating agents would likely be nonspecific, thus resulting in toxicity. Solution: Generate a zinc chelating moiety that relies on the action of the enzyme itself to achieve optimal inhibitory activity (i.e. generate a mechanism-based metalloenzyme inhibitor).

49. Proposed Mechanism-based Inhibitors of the Zinc Metallooenzymes