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

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

3. The  -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  -lactam antibiotics are believed to resemble the D-Ala-D-Ala terminus of the pentapeptide side chain (Strominger Hypothesis) 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.

5. Link

9. Why are  -lactam antibiotics such good drugs?  -Lactam antbiotics 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.  -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  -lactamase(s)

10. Resistance to  -Lactam Antibiotics 1)Production of one or more enzymes (  -lactamases) that hydrolytically destroy  -lactam antibiotics 2)Produce PBPs that do not recognize penicillin 3)In the case of Gram-negative strains delete outer membrane porins, which are responsible for the allowing the  -lactams to reach the periplasm and hence the cell wall 4)In the case of Gram-negative strains, upregulate efflux pumps, which are responsible for pumping out foreign substances (including  -lactams). Action of Serine  -Lactamases

13. The  -Lactamases More than 600 different  -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. One early strategy for countering  -lactamase mediated resistance was to design  - lactam antibiotics which would also be poor  -lactamase substrates. This was achieved by incorporating sterically large substituents at C6 (penicillin) or C7 (cephalosporin).Bulkygroup Enzyme

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

16. Recent Trends in  -Lactamase-mediated Resistance Broad spectrum  -lactamases, known as extended spectrum  -lactamases (ESBLs) capable of hydrolyzing third generation cephalosporins, are disseminated widely (e.g. class A, CTX-M) Class C  -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-  -lactamases are disseminating widely. These enzymes were originally seen in Asia and in Europe, but cases of resistance due to class B  -lactamases are now appearing in the US (e.g. IMP and VIM).

17. Current Commercial  -Lactamase Inhibitors A second approach was to develop inhibitors of  -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  -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  -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. enzymatic mechanism active site dimensions and binding characteristics synthetic feasibility generate a library of prospective inhibitors Assay against all relevant enzymes The Inhibitor Design Process

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

24. IC 50 Values against the class C  -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. Table 1. Inhibitory activity on Three Representative Serine  -Lactamases IC 50 (  M) R1R1 R2R2 P99TEM-1PC1 None (Tazo) CH 2 C 2 H 2 N 3 51.90.2972.57 CO 2 Na CH 3 4.501.8108 CO 2 Na CH 2 O 2 CCH 3 0.7080.18076.53 CO 2 Na CH 2 O 2 CCH 2 ClNT0.1967.2 CO 2 Na CH 2 O 2 CH0.5921.84173 CO 2 Na CH 2 O 2 CCH 2 Ph0.540.0154579.0 CO 2 Na CH 2 O 2 CCH 2 3’,4’-C 6 H 3 (OH) 2 0.370.105116 CO 2 Me CH 2 O 2 CCH 3 9.512.72NT CO 2 NH 2 CH 2 O 2 CH 3 8.480.312.21 CO 2 Na CH 2 Cl527.0120.52100 CO 2 Me CH 2 Cl13.9144.51432 CO 2 Na CH=CHCN6.7621.67504 CO 2 Na CH 2 O 2 CCH 2 -S-tet0.640.233NT CO 2 Me CH 2 O 2 CCH 2 -S-tet13.22.37939.7  ’-pyr CH 2 O 2 CCH 3 0.0620.0040.66  ’-pyr CH 2 O 2 CCH 2 Ph0.0010.040.39  ’-pyr CH 2 O 2 CCH 2 -3’,4’-C 6 H 3 (OH) 2 0.0260.06 0.7 Buynak, J. D. et. al. BMCL 1999, 9, 1997-2002.

33. PiperacillinPIP:TAZPIP:JDB/LN-1-255 P. aeruginosa Ps505A1 (AmpC derepressed)>6416 2 A. sobria ((Asb A, OXA-12, AsbM) 64 1 S. marcescens GC 4132 (Amp C, in vivo)64 32 4 E. coli C600N (no b-lactamase)221 E. coli C600N +(TEM-1)>6442 E. coli C600N + (IRT – 2)>6482 E. coli C600N + (SHV – 4)>6422 E. coli C600N + (PSE – 1)3212 E. coli C600N + (OXA-10) {PSE-2}>6422 E. coli C600N + (MIR-1)6488 E. coli C600N + (Imi-1)>64168 E. coli 300 + (TEM-1)>6441 E. coli 300 + (ampRampC)1642 K. Pneumoniae KC 2 (TEM-10)>6424 E. coli GC6265 (TEM-1, in vivo)>64 4 4

34. Inhibition of Representative  -lactamases (IC 50,  M) InhibitorTEM-1 E. Coli AmpC P. aeruginosa AmpC A. baumannii OXA-40 A. baumannii In serum AmpC P. aeruginosa JDB/SA-3-180.00040.0080.0170.00600.012 JDB/SA-4-110.000100.1850.191 0.028 JDB/SA-4-170.000030.0120.0200.0070.014 JDB/SA-4-1410.00020.0650.0710.5830.029 JDB/SA-4-1570.00060.2010.5150.8880.080 JDB/SA-4-1960.00010.0060.0150.0460.003 JDB/SA-4-1980.00010.0390.0520.0790.015 JDB/LN-1-2550.000030.0060.0040.0110.082

35. Synergy of Inhibitors with Imipenem Against Resistant P. aeruginosa Imipenem (mg/L) JDB/SA- 3-18 (mg/L) JDB/SA- 4-11 (mg/L) JDB/SA- 4-17 (mg/L) JDB/SA- 4-141 (mg/L) JDB/SA- 4-157 (mg/L) JDB/SA- 4-196 (mg/L) JDB/SA- 4-198 (mg/L) JDB/LN- 1-255 (mg/L) MIC2000000000 0.5 MIC1012.525 12.53.1256.2512.56.25 0.25 MIC510050 2512.5 25 0.125 MIC 2.5100 25 12.550 0.0625 MIC 1.25>100 50 25100 0.0313 MIC 0.625>100

36. Inhibition of  -Lactamase (IC 50  M) REscherichia coli W3310 (Class A) Enterobacter cloacae P99 (Class C) t-butylmethylidene (allene)>200011.8  -pyridyl 441.30 CO 2 Bu t 0.28429 tazobactam1.3751.9 clavulanic acid3.3>2000

37. Initial attempts to improve the cephalosporin series of  -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.

40. TypeR1R2TEM-1PC1P99GC1 Tazo0.322.849.83.4 I2’-pyE-CH=CH-CN0.0140.720.010.012 I2’-pyE-CH=CHCO 2 Me0.020.300.200.30 I2’-pyE-CH=CHCONH 2 0.090.100.0260.01 I2’-pyZ-CH=CClCO 2 Me0.071.40.900.18 I2’-pyE-CH=CH-CH=CH 2 687524NT I2’-pyE-CH=CHCO 2 ButNT2401.48NT I2’-pyE-CH=CHCO 2 Na2.5310.31NT I2’-pyE-CH=CHNO 2 0.070.200.020.10 I2’-pyE-CH=CH-2’-py0.204.30.18NT I2’-pyE-CH=CH-2”py-N-ox0.0068.60.600.10 I2’-pyCN2.342800.029NT I2’-thzlE-CH=CHCONH 2 0.901540.29NT II2’pyE-CH=CHCO 2 Me2.96.00.030.06 II2’-pyE-CH=CH-CO 2 ButNT 440150 II2’-pyE-CH=CHCO 2 Na2.5NT6.60NT

41. RTEM-1 Inhibition IC 50,  M P99 Inhibition IC 50,  M Tazobactam0.25101.6 CH=CH-CONH 2 0.26150.022 CH=CH-CONHCH 2 CF 3 0.0781.18 CH=CH-CONHCH 2 CH 2 OH0.07010.212 CH=CH-CONHCH(CH 2 ) 2 0.2400.824 CH=CH-CONH-CH 2 CH 2 (CN 3 H 4 )0.00830.0055 CH=CH-CONHOH7.690.128 CH=CH-CONHC 6 F 5 4.280.127 CH=CH-CON(CH 2 CH 2 ) 2 NMe 0.0536.34 CH=CH-CONHCH 2 Ph1.40.11 CH=CH-CONHNH 2 0.391.1 CH=CH-CO-NHC 6 H 4 OH0.110.035 CH=CH-CONHCH 2 CO 2 Na4.20.31 CH=CH-CONH(CH 2 ) 3 NH 2 1.594.2

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)  -lactamases, since inhibitory mechanism does not depend on enzyme active site groups

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

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

48. Inhibiting metallo-  -lactamases Like most metalloenzymes, metallo-  -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

55. Compound TEM-1 (class A) (Serine) P99 (class C) (Serine) L1 (class B) (Metallo) BCII (Class B) (Metallo) Tazobactam 0.122 53.2>200 752 409>200 275 96.2>200 0.65 3.972.3>200 14.6 10.0>200 Inhibition of Serine and Metallo-  -lactamases IC 50 (  M)

56. TEM-1 (class A) (Serine) P99 (class C) (Serine) L1 (class B) (Metallo) BC1 (Class B) (Metallo) Tazobactam 0.12253.2>200 6010.1032.12.9 6483.7510.91.7 6.810.50.101.4 51.77.50.302.0 Inhibition of Serine and Metallo-  -lactamases IC 50 (  M)

57. Van den Akker Strategy: Stabilize the E-  -aminoacrylate intermediate in the active site. Designed by analogy with acyl-enzyme of Tazobactam. This should result in an acyl-enzyme with increased affinity for the site. May retain occupancy of the site subsequent to hydrolysis of the covalent ester linkage of the acyl-enzyme. Focus Here

58. Design a 2’-substituent that stabilized the E-form of the  -aminoacrylate

62. Thanks to my collaborators: Robert Bonomo Paul Carey Marion Helfand Focco van den Akker And my funding sources: Robert A. Welch Foundation National Institutes of Health