Three Decades of β-Lactamase Inhibitors · Non--Lactam Inhibitors ... hibiting enzymes involved in cell wall synthesis. The integrity of the bacterial cell wall is essential to maintaining

CLINICAL MICROBIOLOGY REVIEWS, Jan. 2010, p. 160–201 Vol. 23, No. 10893-8512/10/$12.00 doi:10.1128/CMR.00037-09Copyright © 2010, American Society for Microbiology. All Rights Reserved.

Three Decades of �-Lactamase InhibitorsSarah M. Drawz1 and Robert A. Bonomo2,3,4,5*

Departments of Pathology,1 Medicine,2 Pharmacology,3 and Molecular Biology and Microbiology,4

Case Western Reserve University School of Medicine, Cleveland, Ohio, and Research Service,Louis Stokes Cleveland Department of Veterans Affairs Medical Center, Cleveland, Ohio5

INTRODUCTION .......................................................................................................................................................161MECHANISM OF ACTION OF �-LACTAM ANTIBIOTICS .............................................................................161RESISTANCE TO �-LACTAM ANTIBIOTICS .....................................................................................................161�-LACTAMASES ........................................................................................................................................................164

Classification ...........................................................................................................................................................164Class A serine �-lactamases .............................................................................................................................164Class A ESBLs ....................................................................................................................................................164Class A serine carbapenemases ........................................................................................................................165Class B metallo-�-lactamases ...........................................................................................................................165Class C serine cephalosporinases.....................................................................................................................166Class D serine oxacillinases ..............................................................................................................................166

�-Lactamase Hydrolytic Mechanisms..................................................................................................................166Class A .................................................................................................................................................................166Class C .................................................................................................................................................................168Class D .................................................................................................................................................................168Class B .................................................................................................................................................................168

CIRCUMVENTING �-LACTAMASES ....................................................................................................................169�-Lactamase Inhibitors: Mechanistic Considerations.......................................................................................169�-Lactamase Inhibitors in Clinical Practice ......................................................................................................169

Clavulanic acid, sulbactam, and tazobactam .......................................................................................169Mechanism of inhibition....................................................................................................................................170�-Lactam–�-lactamase inhibitor combinations: clinical use .......................................................................171

(i) Amoxicillin-clavulanate ............................................................................................................................171(ii) Ticarcillin-clavulanate .............................................................................................................................172(iii) Ampicillin-sulbactam..............................................................................................................................173(iv) Piperacillin-tazobactam ..........................................................................................................................173

Inhibitory Activity of Carbapenems .....................................................................................................................174INHIBITOR-RESISTANT CLASS A �-LACTAMASES........................................................................................176

Epidemiology and Detection of �-Lactamase Inhibitor Resistance.................................................................176Substitutions in Class A Enzymes Conferring Inhibitor Resistance...............................................................178

Arginine 244.........................................................................................................................................................179Methionine 69......................................................................................................................................................179Asparagine 276 ....................................................................................................................................................179Serine 130 ............................................................................................................................................................180Arginine 275.........................................................................................................................................................180Lysine 234 ............................................................................................................................................................180

THE PROMISE OF NOVEL �-LACTAMASE INHIBITORS .............................................................................180Monobactam Derivatives .......................................................................................................................................180ATMO Derivatives ..................................................................................................................................................182Penems .....................................................................................................................................................................182

BRL 42715 and Syn 1012...................................................................................................................................182BRL 42715 derivatives........................................................................................................................................183Oxapenems...........................................................................................................................................................184Tricyclic carbapenems (trinems) ......................................................................................................................1841-�-Methylcarbapenems.....................................................................................................................................184

Penicillin and Cephalosporin Sulfone Derivatives.............................................................................................184C-2/C-3-substituted penicillin and cephalosporin sulfones ..........................................................................185C-6-substituted penicillin sulfones ...................................................................................................................185

* Corresponding author. Mailing address: Infectious Diseases Sec-tion, Louis Stokes Cleveland Department of Veterans Affairs MedicalCenter, 10701 East Blvd., Cleveland, OH 44106. Phone: (216) 791-3800, ext. 4399. Fax: (216) 231-3482. E-mail: [email protected].

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Non-�-Lactam Inhibitors.......................................................................................................................................186Boronic acid transition state analogs (TSAs).................................................................................................186Phosphonates.......................................................................................................................................................187NXL104.................................................................................................................................................................187Hydroxamates......................................................................................................................................................187Other non-�-lactam inhibitors .........................................................................................................................188

INHIBITION OF METALLO-�-LACTAMASES ...................................................................................................188Thiol Derivatives .....................................................................................................................................................189Pyridine Dicarboxylates .........................................................................................................................................189Trifluoromethyl Ketones and Alcohols ................................................................................................................189Carbapenem Analogs..............................................................................................................................................189Tricyclic Natural Products ....................................................................................................................................189Succinate Derivatives .............................................................................................................................................189C-6-Mercaptomethyl Penicillinates ......................................................................................................................190

LESSONS LEARNED ................................................................................................................................................190A PERSPECTIVE .......................................................................................................................................................191ACKNOWLEDGMENTS ...........................................................................................................................................191REFERENCES ............................................................................................................................................................191

INTRODUCTION

The development of antibiotics remains one of the most sig-nificant advances in modern medicine (364). Antibiotics havesaved countless lives and continue to be a mainstay of therapy forbacterial infections. The clinical success of the first �-lactam,penicillin G (benzylpenicillin [Fig. 1, compound 1), prompted thesearch for and development of additional derivatives. This questgave rise to the �-lactam antibiotics in clinical use today (penicil-lins, narrow- and extended-spectrum cephalosporins, monobac-tams, and carbapenems [Fig. 1, compounds 1 to 7) (14). Thecommon structural feature of these classes of antibiotics is thehighly reactive four-membered �-lactam ring.

Unfortunately, �-lactamase-mediated resistance to �-lactamantibiotics emerged as a significant clinical threat to these life-saving drugs. In response to this challenge, two strategies wereadvanced to preserve the utility of �-lactam antibiotics: (i) dis-cover or design �-lactam antibiotics that are able to evade bacte-rial enzymatic inactivation conferred by �-lactamases, or (ii) in-hibit �-lactamases so the partner �-lactam can reach the penicillinbinding proteins (PBPs), the target of �-lactam antibiotics.

In this review, we summarize 3 decades of investigation of�-lactamase inhibition. This perspective is framed by our back-ground in clinical infectious diseases. First, we highlight the fun-damental principles of �-lactamase enzymology. We then sum-marize the salient features of �-lactam–�-lactamase inhibitorcombinations that are used in clinical practice. Next, we define theproblem of resistance to �-lactamase inhibitors by explaining theimportant changes in class A �-lactamases that define this phe-notype. With this background, we review the �-lactamase inhib-itors that have been developed to this point and discuss the novel�-lactamase inhibitors that are hoped to extend the life span ofour current �-lactams. We view these agents as extremely impor-tant to the future of �-lactam therapy: inhibitors not only canpreserve our current armamentarium but may also be used asnovel �-lactams are introduced into the clinic. Finally, we con-clude with some “lessons learned.”

MECHANISM OF ACTION OF �-LACTAM ANTIBIOTICS

�-Lactam antibiotics exhibit their bactericidal effects by in-hibiting enzymes involved in cell wall synthesis. The integrity of

the bacterial cell wall is essential to maintaining cell shape in ahypertonic and hostile environment (249). Osmotic stability ispreserved by a rigid cell wall comprised of alternating N-acetyl-muramic acid (NAM) and N-acetylglucosamine (NAG) units.These glycosidic units are linked by transglycosidases. A pen-tapeptide is attached to each NAM unit, and the cross-linking oftwo D-alanine–D-alanine NAM pentapeptides is catalyzed byPBPs, which act as transpeptidases (142, 377). This cross-linkingof adjacent glycan strands confers the rigidity of the cell wall.

The �-lactam ring is sterically similar to the D-alanine–D-alanine of the NAM pentapeptide, and PBPs “mistakenly” usethe �-lactam as a “building block” during cell wall synthesis(459). This results in acylation of the PBP, which renders theenzyme unable to catalyze further transpeptidation reactions(125). As cell wall synthesis slows to a halt, constitutive pepti-doglycan autolysis continues. The breakdown of the mureinsacculus leads to cell wall compromise and increased perme-ability. Thus, the �-lactam-mediated inhibition of transpepti-dation causes cell lysis, although the specific details of penicil-lin’s bactericidal effects are still being unraveled (20).

RESISTANCE TO �-LACTAM ANTIBIOTICS

There are four primary mechanisms by which bacteria canovercome �-lactam antibiotics (14).

(i) Production of �-lactamase enzymes is the most commonand important mechanism of resistance in Gram-negative bac-teria and will be the focus of this review.

(ii) Changes in the active site of PBPs can lower the affinityfor �-lactam antibiotics and subsequently increase resistanceto these agents, such as those seen in PBP2x of Streptococcuspneumoniae (212). Through natural transformation and re-combination with DNA from other organisms, Neisseria spp.and Streptococcus spp. have acquired highly resistant, low-affinity PBPs (39, 313, 459). In a related manner, penicillinresistance in Streptococcus sanguis, Streptococcus oralis, andStreptococcus mitis developed from horizontal transfer of aPBP2b gene from Streptococcus pneumoniae (107, 348). Methicil-lin resistance in Staphylococcus spp. is also a significant clinicalchallenge. While there are many reasons for this resistance, the�-lactam resistance phenotype is also conferred by acquisition of

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the mecA gene which produces PBP2a (also denoted PBP2�) (76).PBP2a can assemble new cell wall in the presence of high con-centration of penicillins and cephalosporins.

(iii) Decreased expression of outer membrane proteins(OMPs) is another mechanism of resistance. In order to accessPBPs on the inner plasma membrane, �-lactams must either

FIG. 1. Chemical structures of compounds discussed in the text. Compounds 1 to 7, a representative penicillin (compound 1), an extended-spectrum cephalosporin (compound 2), a monobactam (compound 3), and carbapenems (compounds 4 to 7). The numbering scheme forpenicillins, cephalosporins, and monobactams is shown. Compounds 8 to 10, �-lactamase inhibitors in clinical practice. Compounds 11 to 38,investigational �-lactamase inhibitors: monobactam derivatives (compounds 11 to 14), a penicillin derivative (compound 15), penems (compounds16 to 20), penam sulfones (compounds 21 to 24), a boronic acid transition state analog (compound 25), non-�-lactams (compounds 26 to 28), andmetallo-�-lactamase inhibitors (compounds 29 to 38).

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diffuse through or directly traverse porin channels in the outermembrane of Gram-negative bacterial cell walls. Some Entero-bacteriaceae (e.g., Enterobacter spp., Klebsiella pneumoniae, andEscherichia coli) exhibit resistance to carbapenems based onloss of these OMPs; the loss of OprD is associated with imi-penem resistance and reduced susceptibility to meropenem inthe nonfermenter Pseudomonas aeruginosa (169, 187, 236, 286,306, 395). Resistance to imipenem and meropenem has alsobeen associated with the loss of the CarO OMP in clinicalisolates of multidrug-resistant Acinetobacter baumannii (278,346). Point mutations or insertion sequences in porin-encodinggenes can produce proteins with decreased function and thuslower permeability to �-lactams (106). Of note, the disruptionof porin proteins alone is not always sufficient for producingthe resistance phenotype, and typically this mechanism isfound in combination with �-lactamase expression (106, 235).

(iv) Efflux pumps, as part of either an acquired or intrinsicresistance phenotype, are capable of exporting a wide range ofsubstrates from the periplasm to the surrounding environment(347). These pumps are an important determinant of multidrugresistance in many Gram-negative pathogens, particularly P.aeruginosa and Acinetobacter spp. In P. aeruginosa, upregulationof the MexA-MexB-OprD system, in combination with the or-ganism’s low outer membrane permeability, can contribute todecreased susceptibility to penicillins and cephalosporins, as wellas quinolones, tetracycline, and chloramphenicol (224–226, 373,389). To illustrate, an increase in the carbenicillin MIC from 32�g/ml to 1,028 �g/ml is associated with overproduction of thisefflux pump (12, 225). Additionally, an upregulated efflux pump(e.g., AdeABC, an RND-type efflux pump, in A. baumannii) canaugment the carbapenem resistance conferred by a catalyticallypoor �-lactamase (e.g., OXA-23) (162, 333).

FIG. 1—Continued.



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�-LACTAMASES

The first �-lactamase enzyme was identified in Bacillus (Esch-erichia) coli before the clinical use of penicillin. In a sentinel paperpublished nearly 70 years ago, E. P. Abraham and E. Chaindescribed the B. coli “penicillinase” (1). The enzyme was notthought to be clinically relevant, since penicillin was targeted totreat staphylococcal and streptococcal infections, and Abraham,Chain, and their colleagues were unable to isolate the enzymefrom these Gram-positive organisms (2, 55). It is sobering now toconsider the ramifications of this observation.

Four years later, Kirby successfully extracted these cell-free“penicillin inactivators” from Staphylococcus aureus, which fore-shadowed the emergence of a significant clinical problem (202).The growing number of �-lactam antibiotics has since increasedthe selective pressure on bacteria, promoting the survival of or-ganisms with multiple �-lactamases (249, 256). Currently, morethan 850 �-lactamases are identified (K. Bush, personal commu-nication). As authors, we speculate that the rapid replication rate,recombination rates, and high mutation frequency permit bacte-ria to adapt to novel �-lactams by evolution of these �-lactamases(332).

Classification

Two major classification schemes exist for categorizing�-lactamase enzymes: Ambler classes A through D, based onamino acid sequence homology, and Bush-Jacoby-Medeirosgroups 1 through 4, based on substrate and inhibitor profile(Table 1) (10, 57). A “family portrait” reveals the structuralsimilarity of class A, C, and D serine �-lactamases (Fig. 2).Class B �-lactamases (“a class apart”) are metallo-�-lactama-ses (MBLs) (52). MBLs possess either a single Zn2� ion or apair of Zn2� ions coordinated to His/Cys/Asp residues in theactive site. In this review, the Ambler classification scheme will beused.

Class A serine �-lactamases. In general, class A enzymes aresusceptible to the commercially available �-lactamase inhibi-tors (clavulanate, tazobactam, and [less so] sulbactam), al-though the K. pneumoniae carbapenemase KPC may be animportant exception to this generalization (319a). The firstplasmid-mediated �-lactamase was identified in E. coli in 1963(and reported in 1965), and was named “TEM” after the pa-tient from whom it was isolated (100). SHV, another common�-lactamase found primarily in K. pneumoniae, was namedfrom the term “sulfhydryl reagent variable.” Early studies ofSHV-1 showed that p-chloromercuribenzoate inhibited the hy-drolysis of cephaloridine but not that of benzylpenicillin (251).TEM and SHV are common �-lactamases detected in clinicalisolates of E. coli and K. pneumoniae, pathogens responsiblefor urinary tract, hospital-acquired respiratory tract, andbloodstream infections (60, 145, 372). While SHV-1 andTEM-1 share 68% sequence homology, the active site ofSHV-1 is approximately 0.7 to 1.2 Å wider than that of TEM-1,which may have important structural implications, especiallyrelated to the substrate profiles of SHV variants (421).

Although blaTEM and blaSHV may be found on plasmids,other class A enzymes are encoded on the chromosome (e.g.,blaPenA from Burkholderia pseudomallei) or on integrons (e.g.,blaGES-1 from K. pneumoniae and blaVEB-1 in P. aeruginosa andA. baumannii) (57, 280).

Class A ESBLs. The growing number of �-lactamases in E.coli and K. pneumoniae, as well as the emergence of theseenzymes in other pathogens (e.g., Haemophilus influenzae andNeisseria gonorrhoeae), led to the development of extended-spectrum cephalosporins with an oxyimino side chain, carbap-enems, cephamycins, and monobactams (71, 115, 188, 321).Upon the introduction of the penems and cephems in the early1980s, these new agents were effective against many �-lactamresistant bacteria. However, selective pressure quickly fosteredthe emergence of extended-spectrum �-lactamases (ESBLs),which could hydrolyze many of the oxyimino-cephalosporins

TABLE 1. �-Lactamase classification schemesa

Ambler class Bush-Jacoby-Medeiros class Preferred substrates Inhibited by

clavulanate Representative enzyme(s)

A (serine penicillinases) 2a Penicillins � PC1 from S. aureus2b Penicillins, narrow-spectrum

cephalosporins� TEM-1, TEM-2, SHV-1

2be Penicillins, narrow-spectrum and extended-spectrum cephalosporins

� SHV-2 to SHV-6, TEM-3 toTEM-26, CTX-Ms

2br Penicillins � TEM-30, SHV-722c Penicillins, carbenicillin � PSE-12e Extended-spectrum cephalosporins � FEC-1, CepA2f Penicillins, cephalosporins, carbapenems � KPC-2, SME-1, NMC-A

B (metallo-�-lactamases) 3 Most �-lactams, including carbapenems � IMP-1, VIM-1, CcrA, and BcII(B1); CphA (B2); L1(B3)

C (cephalosporinases) 1 Cephalosporins � AmpC, CMY-2, ACT-1

D (oxacillinases) 2d Penicillins, cloxacillin � OXA-1, OXA-10

Not classified 4

a Based on data from reference 57. An updated classification system by Bush and Jacoby is in press (56a) at the time of this writing.



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(186, 188).In a dramatic parallel to the observations of Abraham and

Chain, within 2 years of the introduction of the extended-spectrum cephalosporins cefotaxime and ceftazidime, novelESBLs in E. coli and K. pneumoniae were reported (204).Interestingly, these “new �-lactamases” harbored point muta-tions in the parent blaTEM-1 and blaSHV-1 genes that led tosingle amino acid changes in the �-lactamases. Other ESBLs,such as CTX-M, arose by plasmid transfer from preexistingchromosomal ESBL genes from Kluyvera spp., which typicallyare nonpathogenic commensal organisms (14, 32). CTX-MESBLs now represent important enzymes found in isolatesfrom the community and are the most commonly isolatedESBLs in many parts of the world, particularly Europe (343).

ESBLs hydrolyze penicillins, narrow- and extended-spec-trum cephalosporins (including the anti-methicillin-resistant S.aureus [MRSA] cephalosporin ceftobiprole), and the mono-bactam aztreonam (11, 321, 357). In contrast, ESBLs cannotefficiently degrade cephamycins, carbapenems, and �-lacta-mase inhibitors. Since their initial description, more than 200different ESBLs have been identified, posing a significant riskto public health and to hospitalized patients in intensive careunits, where infection with an ESBL may lead to significantmorbidity and mortality (up-to-date listings of verified ESBLsequences are available on the website www.lahey.org/studies/webt.asp maintained by G. A. Jacoby and K. Bush) (321). Themajority of ESBLs are from the SHV, TEM, and CTX-Mfamilies; less frequently they are derived from BES, GES-1,

VEB, and PER enzymes, and sometimes these enzymes do notbelong to any defined family (280).

Class A serine carbapenemases. Class A serine carbapen-emases include the nonmetallocarbapenamase of class A(NMC-A), IMI, SME, and KPC. Members of this group of�-lactamases can hydrolyze carbapenems as well as cephalo-sporins, penicillins, and aztreonam (356). These carbapenem-hydrolyzing enzymes have been identified primarily in Entero-bacter cloacae, Serratia marcescens, and K. pneumoniae,bacteria which often harbor multiple resistance determinants,narrowing the range of treatment options (290, 291, 359, 454,455). The bla gene for the former two organisms is typically foundon the chromosome, while the K. pneumoniae carbapenemaseblaKPC gene is carried on plasmids containing Tn4401 (292).MICs of carbapenems in carbapenemase-expressing strains canvary from moderately increased (2 to 4 �g/ml) to resistant (� 32�g/ml) (116b, 356).

Class B metallo-�-lactamases. Class B enzymes are Zn2�-dependent �-lactamases that demonstrate a hydrolytic mech-anism different from that of the serine �-lactamases of classesA, C, and D (57). Organisms producing these enzymes usuallyexhibit resistance to penicillins, cephalosporins, carbapenems,and the clinically available �-lactamase inhibitors (432). Inter-estingly, the hydrolytic profile of MBLs does not typically in-clude aztreonam. MBLs likely evolved separately from theother Ambler classes, which have serine at their active site(249). The blaMBL genes are located on the chromosome, plas-mid, and integrons (133, 432). P. aeruginosa, K. pneumoniae,

FIG. 2. “Family portrait” of �-lactamase enzymes. (A) Class A SHV-1; (B) class B IMP-1; (C) class C E. coli AmpC; (D) class D OXA-1. Forclasses A, C, and D, the active-site serine is shown in yellow; for class B, the two Zn2� ions are shown. (Based on PDB entries 1SHV, 1DDK, 2BLS,and 1M6K, respectively.)



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and A. baumannii produce class B enzymes encoded by mobilegenetic elements (93, 175, 177). In contrast, Bacillus spp.,Chryseobacterium spp., and Stenotrophomonas maltophilia pos-sess chromosomally encoded MBLs, but the majority of thesehost pathogens are not frequently responsible for serious in-fections (432). The role that these �-lactamases in Bacillus spp.and Chryseobacterium spp. will play in the clinical arena is stillunknown.

Class C serine cephalosporinases. Class C AmpC �-lacta-mases include CMY-2, P99, ACT-1, and DHA-1, which areusually encoded by bla genes located on the bacterial chromo-some, although plasmid-borne AmpC enzymes are becomingmore prevalent (340). Organisms expressing the AmpC �-lac-tamase are typically resistant to penicillins, �-lactam–�-lacta-mase inhibitor combinations, and cephalosporins, includingcefoxitin, cefotetan, ceftriaxone, and cefotaxime. AmpC en-zymes poorly hydrolyze cefepime and are inhibited by cloxacil-lin, oxacillin, and aztreonam (57). Members of the Enterobac-teriaceae family, such as Enterobacter spp. and Citrobacter spp.,are AmpC �-lactamase producers that resist inhibition by cla-vulanate and sulbactam, although Klebsiella spp., Salmonellaspp., and Proteus spp. normally do not harbor chromosomalblaAmpC genes (19, 184).

Production of chromosomal AmpCs in Gram-negative bac-teria is at a low level (“repressed”) but can be “derepressed” byinduction with certain �-lactams, particularly cefoxitin (14, 24,148). The mechanism of this regulation has been the subject ofintense investigation (183). Of significant concern is the selec-tion of mutant bacterial populations that are genetically dere-pressed for AmpC production, which can cause a dramaticincrease in MICs during the course of �-lactam therapy (e.g.,after 14 days of ceftazidime therapy, a strain of P. aeruginosawith MICs increased from 1 to 32 �g/ml was selected) (184,193, 233).

Class D serine oxacillinases. Class D �-lactamases wereinitially categorized as “oxacillinases” because of their abilityto hydrolyze oxacillin at a rate of at least 50% of that ofbenzylpenicillin, in contrast to the relatively slow hydrolysis ofoxacillin by classes A and C (98). In bacteria, OXA �-lacta-mases can also confer resistance to penicillins, cephalosporins,extended-spectrum cephalosporins (OXA-type ESBLs), andcarbapenems (OXA-type carbapenemases). Generally speak-ing, OXA enzymes are resistant to inhibition by clavulanate,sulbactam, and tazobactam, (with some exceptions; e.g.,OXA-2 and OXA-32 are inhibited by tazobactam but not sul-bactam and clavulanate, and OXA-53 is inhibited by clavu-lanate) (98, 276, 279, 345). Interestingly, sodium chloride atconcentrations of �50 to 75 mM inhibits some carbapenem-hydrolyzing oxacillinases (e.g., OXA-25 and OXA-26) (6).Site-directed mutagenesis studies suggest that susceptibility toinhibition by sodium chloride is related to the presence of aTyr residue at position 144 (161, 279). Presumably, Tyr144 mayfacilitate sodium chloride binding better than the Phe residuefound in resistant oxacillinases, although the molecular mech-anism remains unexplained. Examples of OXA enzymes in-clude those rapidly emerging in A. baumannii (e.g., OXA-23and OXA-24/40) and constitutively expressed in P. aeruginosa(e.g., OXA-50) (141a, 346, 435).

�-Lactamase Hydrolytic Mechanisms

Serine �-lactamases acylate �-lactam antibiotics, much likePBPs, and then use strategically positioned water molecules tohydrolyze the acylated �-lactam (265). In this manner, the�-lactamase is regenerated and can inactivate additional �-lac-tam molecules. This enzymatic reaction may be represented bythe following equation:

E � SL|;k1

k�1

E:SO¡k2

E � SO¡k3, H2O

E � P (1)

In this scheme, E is a �-lactamase, S is a �-lactam substrate,E:S is the Michaelis complex, E � S is the acyl-enzyme, and Pis the product devoid of antibacterial activity. The rate con-stants for each step are represented by k1, k�1, k2, and k3. k1

and k�1 are the association and dissociation rate constants forthe preacylation complex, respectively; k2 is the acylation rateconstant, and k3 is the deacylation rate constant. More com-plicated branched reaction mechanisms are seen with some�-lactamases (129). In order to be uniform, we now definebasic terms that are often used to describe the kinetic behaviorof �-lactamases.

The Michaelis constant, Km, is defined as (129)

Km � k3Ks/�k2 � k3 (2)

where the kinetic constant, Ks, is (k�1 � k2)/k1. The turnovernumber, kcat, is a composite rate constant that represents mul-tiple chemical steps and is defined as (92)

kcat � k2k3/�k2 � k3 (3)

or

Vmax � kcatE� (4)

Km, expressed in terms of concentration, represents the rela-tive affinity of the ES encounter and the rate at which the ESis converted to P; a large Km value represents poor affinity(large Ks).

Class A. In Fig. 3, we represent the reaction scheme of atypical class A �-lactamase. In brief, (i) after formation of theHenri-Michaelis complex, the active-site serine performs a nu-cleophilic attack on the carbonyl of the �-lactam antibiotic thatresults in a high-energy tetrahedral acylation intermediate(‡1); (ii) this intermediate transitions into a lower-energy co-valent acyl-enzyme following protonation of the �-lactam ni-trogen and cleavage of the C-N bond; (iii) next, an activatedwater molecule attacks the covalent complex and leads to ahigh-energy tetrahedral deacylation intermediate (‡2); and (iv)hydrolysis of the bond between the �-lactam carbonyl and theoxygen of the nucleophilic serine regenerates the active en-zyme and releases the inactive �-lactam. Acylation and deacy-lation require the activation of the nucleophilic serine andhydrolytic water, respectively. An entire body of experimenta-tion is devoted to examining the mechanistic roles of the indi-vidual residues in the active sites of �-lactamases; here wepresent only several leading insights to provide a foundationfor the discussion of inhibitory pathways.

For class A enzymes, a leading theory holds that Glu166 actsas the activating base of the hydrolytic water in deacylation



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(166, 392). In contrast, the acylation mechanism is less clear,and several hypotheses and lines of evidence exist. The firstnotion proposes that Glu166, via a water molecule, deproto-nates the Ser70 hydroxyl before addition to the �-lactam bond.This Glu166 general base theory is supported by quantummechanical/molecular mechanical (QM/MM) calculations andthe protonated status of Glu166 in the ultra-high-resolution(0.85-Å) structure of TEM-1 in complex with an acylationtransition state analog (TSA) (Research Collaboratory forStructural Bioinformatics Protein Data Bank [PDB] entry1M40) (163, 265). Surprisingly, substitutions at residue 166 donot prevent acylation (139, 146, 207, 392).

A second view of acylation maintains that Lys73 can serve asthe general base deprotonating Ser70 (166, 261, 401). RecentQM/MM studies by Meroueh et al. demonstrate that the path-way involving Lys73 as the activating base is energetically fa-vorable and may exist in competition with the pathway whereGlu166 serves as the activating residue (261). Additional ideason the mechanism of acylation have been proposed, includingboth (i) a role for the C-3 or C-4 substrate carboxylate andSer130 in activating Ser70 and (ii) a general acid pathwaywhere protonation of the �-lactam nitrogen is the primaryevent (13, 103). Further clarification of the acylation mecha-nism may prove useful for the design of inhibitors that capi-talize on the enzymes’ native machinery.

Highly conserved structural motifs in class A enzymes in-

clude four polar regions: (i) the active-site pocket Ser70-Xaa-Xaa-Lys73, (ii) the “�-loop” residues 163 to 178, (iii) Ser130-Asp131-Asn132, and (iv) Lys234-Thr(Ser)235-Gly236. Theroles of residues in the “SDN” loop comprised of Ser130-Asp131-Asn132 are related to maintaining the structure of theactive site cavity, enzyme stability, and stabilization of theenzyme transition state, respectively (182, 305). The functionalexplanation for the conservation of the Lys234-Thr(Ser)235-Gly236 triad is less well understood. Removal of the hydroxylgroup from the middle residue has little impact on penicillinhydrolysis in TEM (but more on the catalytic efficiency ofcephalosporins), and despite what may be expected from crys-tal structure alignments, the study of modified �-lactams ar-gues against an interaction between the �-lactam carboxylateand the Thr(Ser) hydroxyl group (112, 181). In SHV, theThr235Ala substitution lowers MICs of both penicillins andcephalosporins (174). Interestingly, the expression of the struc-turally conserved SHV Thr235Ser variant has no effect on�-lactam MICs but does lead to an increased susceptibility tothe piperacillin-tazobactam combination (174).

The most common mechanism for transformation of abroad-spectrum �-lactamase to an ESBL is point mutationsthat result in amino acid sequence changes near the enzymeactive site, facilitating hydrolysis of oxyimino-cephalosporins(186, 341). However, the specific amino acid replacements andresistance mechanisms vary between enzymes (124). In TEM,

FIG. 3. Proposed reaction mechanism for a penicillin �-lactam substrate and a class A serine �-lactamase enzyme in which Glu166 participatesin activating a water molecule for both acylation and deacylation (additional evidence exists to suggest that this acylation scheme may exist incompetition with a mechanism where Lys73 acts as the general base to activate Ser70) (261). Dashed lines represent hydrogen bonds, and thenumber labels are included to help the reader appreciate the general order of events, not as absolute and discrete steps. Following activation ofthe hydroxyl group, Ser70 performs a nucleophilic attack on the carbonyl group of the �-lactam antibiotic, resulting in a high-energy acylationintermediate. Protonation of the �-lactam nitrogen leads to cleavage of the C-N bond and formation of the covalent acyl-enzyme, which adoptsa lower energy state. Attack by a catalytic water leads to a high-energy deacylation intermediate, with subsequent hydrolysis of the bond betweenthe �-lactam carbonyl and the oxygen of Ser70. Deacylation regenerates the active enzyme and releases the inactive �-lactam.



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changes at Ambler positions Arg164 (-His, -Ser), Gly238 (-Ser,-Ala), and Glu240 (-Lys) result in variants that confer theESBL phenotype (40; www.lahey.org/studies/webt.asp). Sev-eral crystallographic studies demonstrated that the “new”TEM active site is expanded or remodeled, compared toTEM-1, to accommodate the larger side chain of expanded-spectrum cephalosporins (83, 206, 298, 304). The crystal struc-ture of SHV-2 (Gly238Ser) at 0.91 Å resolution (PDB 1N9B),compared to SHV-1, showed a displacement in the b3 �-strandcontaining residues 238 to 242 which created an expanded�-lactam binding site but preserved the positioning of the es-sential catalytic residues (124, 298). Similarly, the PER-1ESBL active site is expanded by a novel fold of the �-loop andthe insertion of four residues after Lys240 (417).

In contrast, the crystal structure of the acyl-enzyme inter-mediate of the ESBL Toho-1 in complex with cefotaxime didnot resemble the enlarged active sites seen with the ESBLTEM and SHV �-lactamases. Rather, the Toho-1 enzyme fa-cilitates cephalosporin binding through movement of the�-loop toward the active site, interactions between conservedresidues Asn104 and Asp240 and the bulky side chain of cefo-taxime, and contacts with Ser237 that help position the �-lac-tam carbonyl in the oxyanion hole (176, 382). The high-reso-lution crystal structures of four additional CTX-M�-lactamases confirmed that the active sites of these enzymeswere not enlarged compared to those of the narrow-spectrumTEM-1 and SHV-1 (83, 85). Instead, the structural basis forthe extended substrate profiles in the CTX-M enzyme familyappears to be specific amino acid (i.e., Ser237 and Asp104)interactions with the oxyimino-cephalosporins and increasedmobility of the b3 �-strand (83). Overall, this increased mobil-ity and activity “costs the enzyme” thermal stability, a themerecapitulated by the evolution of many resistance phenotypes(383).

Point mutations are not the only mechanism for the ESBLphenotype; other alterations in enzyme regulation produceresistance, such as promoter sequence changes leading to in-creased enzyme expression or alterations in outer membraneporins (293, 302).

At this time, the biochemical basis for class A serine carbapen-emase activity is an area of active investigation. Current evidencesuggests that this property rests upon a remodeling of the active site.Unlike class A ESBLs (with expanded or enhanced mobility of theb3 �-strand), the active sites of class A carbapenemases such asKPC-2, NMC-A, and SME-1 reveal that there are multiple alter-ations in the spatial positions of catalytic residues (200, 338, 388, 402).We are just beginning to understand how this remodeling allowsenhanced carbapenemase activity compared to that of other class Aenzymes.

Class C. The prevailing evidence regarding the class C hy-drolytic mechanism suggests that Tyr150 behaves as a generalbase by increasing the nucleophilicity of Ser64 for acylation(299). Early studies of the deacylation mechanism indicatedthat Tyr150 also acts as the catalytic base, accepting a protonfrom the deacylating water (111, 299). For example, QM/MMcalculations suggested that Tyr150 interacts with Lys67 in aconjugate base manner (136). However, the crystal structure ofthe E. coli AmpC in complex with a deacylation transition stateanalog revealed that Tyr150 remains protonated throughoutthe reaction and therefore is unlikely to be the anionic base

that deprotonates the catalytic water for hydrolysis (84). In-stead, deacylation may proceed by “substrate-activated cataly-sis” whereby the nitrogen on the �-lactam acts as a base andtransiently accepts the proton from the hydrolytic water, whilethe Tyr150 proton helps stabilize the water’s developing neg-ative charge (51, 84). The crystallographic evidence and mu-tagenesis studies do not rule out a role of Lys67 in the coor-dinate base mechanism, and further studies exploring the roleof Lys67 are anticipated (84, 271).

Class D. Our understanding of the hydrolytic mechanism ofclass D �-lactamases is based on the careful study of OXA-10,-13, and -1 (253, 310, 334, 379, 398, 399). This class of enzymesis unique because of the direct role of carboxylation of theactive-site Lys70. The carbamic acid on Lys70 can ionize toyield a carbamate that hydrogen bonds with the nucleophilicSer67 residue. In this manner, the carboxylated Lys70 mayserve as the general base by activating both Ser67 for acylationand the hydrolytic water for deacylation (144, 252, 253).

Class B. In contrast to the serine �-lactamases describedabove, class B includes Zn2�-dependent enzymes that follow adifferent hydrolytic mechanism. In general, MBLs use the -OHgroup from a water molecule that is coordinated by Zn2� tohydrolyze the amide bond of a �-lactam. MBLs are dividedinto three classes based on their Zn2� dependency, i.e.,whether they (i) are fully active with either one or two ions(subclass B1, e.g., IMP-1, VIM-2, BcII, and CcrA), (ii) requiretwo ions (subclass B3, e.g., L1), or (iii) employ one ion and areinhibited by binding of an additional ion (subclass B2, e.g.,CphA) (130, 164, 165, 325, 326). Crowder and colleagues com-piled the recent structural and mechanistic data and summa-rized the common features for the three subclasses of MBLs(96). MBLs utilizing two Zn2� ions for hydrolysis, such as thesubclass B3 S. maltophilia L1, coordinate the �-lactam sub-strate by the carboxylate and carbonyl groups, bridged by ahydroxide ion. After substrate binding, one of the Zn2� ions, inconjunction with enzyme residues, polarizes the �-lactam car-bonyl for attack by the -OH group, which is hydrogen bondedto deprotonated Asp120. Nucleophilic attack by the -OHgroup creates a tetrahedral species which rapidly collapses intoan intermediate in which the �-lactam nitrogen is anionic.Protonation of the nitrogen leads to product formation. Thesource of the proton is not certain, and it may come fromAsp120 or a water molecule.

The B1 enzyme Bacillus cereus BcII is active in both itsmononuclear and dinuclear forms, and in the resting state, theZn2�-bound -OH group is hydrogen bonded to thedeprotonated Asp120 as well as several other active-site resi-dues (Fig. 4) (96, 440). After attack by this -OH group, the

FIG. 4. Schematic representation of the Zn2�-binding site ofdinuclear subclass B1 metallo-�-lactamases such as B. cereus BcII.



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breakdown of the tetrahedral intermediate requires protona-tion of the �-lactam nitrogen; the source of this proton is underinvestigation. In the case of the CphA B2 MBL from Aeromo-nas hydrophila, a second bound Zn2� ion is inhibitory. Theproposed mechanism includes a water molecule activated byeither His118 or Asp120, rather than a Zn2�-bound -OHgroup; the singular Zn2� appears to help coordinate the �-lac-tam nitrogen (132, 451).

CIRCUMVENTING �-LACTAMASES

�-Lactamase Inhibitors: Mechanistic Considerations

A successful strategy for combating �-lactamase-medi-ated resistance is the use of agents designed to bind at theactive site, which are frequently �-lactams. This strategy cantake two forms: (i) create substrates that reversibly and/orirreversibly bind the enzyme with high affinity but formunfavorable steric interactions as the acyl-enzyme or (ii)develop mechanism-based or irreversible “suicide inhibi-tors” (53). Examples of the former are extended-spectrumcephalosporins, monobactams, or carbapenems which formacyl-enzymes and adopt catalytically incompetent conforma-tions that are poorly hydrolyzed (Fig. 1, compounds 2, 3, and4 to 7, respectively). For reversible inhibition, the reactioncan be described as was shown above in equation 1, where Srepresents the (very) slowly hydrolyzed substrate. An equi-librium constant, Ki, can be calculated from the pre-steady-state rate constants, k�1/k1, and yields an estimate of affinity.

Irreversible “suicide inhibitors” can permanently inactivatethe �-lactamase through secondary chemical reactions in theenzyme active site. Equation 5 represents a general mechanismof irreversible inhibitors (I) leading to permanent enzyme in-activation (E � I*):

E � I L|;k1

k�1

E:I O¡k2

E � I O¡k3

E � I* (5)

Examples of these inactivators are the commercially avail-able class A inhibitors clavulanic acid, sulbactam, andtazobactam (Fig. 1, compounds 8 to 10). As will be describedbelow, these types of inactivators often display additionalpathways to inhibition. Irreversible inhibitors can be char-acterized by first-order rate constants for inhibition (kinact,the rate of inactivation achieved with an “infinite” concen-tration of inactivator) and KI values (the concentration ofinactivator which yields an inactivation rate that is half thevalue of kinact) (54, 92). While KI closely approximates themeaning of Km for enzyme substrates, depending on theindividual rate constants comprising the reaction, the KI

may or may not equal the equilibrium constant Ki ( k�1/k1)determined under pre-steady-state conditions.

The 50% inhibitory concentration (IC50) measures theamount of inhibitor required to decrease enzyme activity to50% of its uninhibited velocity. While an IC50 can reflect aninhibitor’s affinity or kcat/kinact ratio, these parameters are notalways congruent; e.g., an inhibitor can have a very poor “af-finity” and acylate the enzyme slowly but still yield a low IC50

because of very low deacylation rates.

�-Lactamase Inhibitors in Clinical Practice

Clavulanic acid, sulbactam, and tazobactam. Clavulanicacid, the first �-lactamase inhibitor introduced into clinicalmedicine, was isolated from Streptomyces clavuligerus in the1970s, more than 3 decades ago (360). Clavulanate (the saltform of the acid in solution) showed little antimicrobialactivity alone, but when combined with amoxicillin, clavu-lanate significantly lowered the amoxicillin MICs against S.aureus, K. pneumoniae, Proteus mirabilis, and E. coli (47).Sulbactam and tazobactam are penicillinate sulfones thatwere later developed by the pharmaceutical industry as syn-thetic compounds in 1978 and 1980, respectively (117, 121).

All three �-lactamase inhibitor compounds share struc-tural similarity with penicillin; are effective against manysusceptible organisms expressing class A �-lactamases (in-cluding CTX-M and the ESBL derivatives of TEM-1,TEM-2, and SHV-1); and are generally less effective againstclass B, C, and D �-lactamases (53, 60, 67, 341). The activityof an inhibitor can be evaluated by the turnover number (tn)(also equivalent to the partition ratio [kcat/kinact]), defined asthe number of inhibitor molecules that are hydrolyzed perunit time before one enzyme molecule is irreversibly inac-tivated (58). For example, S. aureus PC1 requires one clav-ulanate molecule to inactivate one �-lactamase enzyme,while TEM-1 needs 160 clavulanate molecules, SHV-1 re-quires 60, and B. cereus I requires more than 16,000 (53, 69,113, 123, 411). For comparison, sulbactam tns are 10,000 and13,000 for TEM-1 and SHV-1, respectively (180, 410).

The low KIs of the inhibitors for class A �-lactamases (nM to�M), the ability to occupy the active site “longer” than �-lactams(high acylation and low deacylation rates), and the failure to behydrolyzed efficiently are integral to their efficacy (158). Clavu-lanate, sulbactam, and tazobactam differ from �-lactam antibiot-ics as they possess a leaving group at position C-1 of the five-membered ring (sulbactam and tazobactam are sulfones, whileclavulanate has an enol ether oxygen at this position). The betterleaving group allows for secondary ring opening and �-lactamaseenzyme modification. Compared to clavulanate, the unmodifiedsulfone in sulbactam is a relatively poor leaving group, a propertyreflected in the high partition ratios for this inhibitor (e.g., forTEM-1, sulbactam tn 10,000 and clavulanate tn 160) (179,180). Tazobactam possesses a triazole group at the C-2 �-methylposition. This modification leads to tazobactam’s improved IC50s,partition ratios, and lowered MICs for representative class A andC �-lactamases (36, 58, 60).

The efficacy of the mechanism-based inhibitors can varywithin and between the classes of �-lactamases (Table 2).For class A, SHV-1 is more resistant to inactivation bysulbactam than TEM-1 but more susceptible to inactivationby clavulanate (328). Comparative studies of TEM- andSHV-derived enzymes, including ESBLs, found that theIC50s for clavulanate were 60- and 580-fold lower than thosefor sulbactam against TEM-1 and SHV-1, respectively (328).In our opinion, the explanations for these differences ininactivation chemistry are likely subtle, yet highly important,differences in the enzyme active sites. For example, Thom-son et al. compared the atomic structure models of TEM-1and SHV-1 and found that the distance between Val216 andArg244, residues responsible for positioning of the water



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molecule important in the inactivation mechanism of clavu-lanate, was more than 2 Å greater in SHV-1 than in TEM-1(411). This increased distance may be too great for coordi-nation of a water molecule, suggesting that the strategicwater is positioned elsewhere in SHV-1 and may be re-cruited into the active site with acylation of the substrate orinhibitor. This variation underscores the notion that mech-anism-based inhibitors may undergo different inactivationchemistry even in highly similar enzymes (410, 411).

Mechanism of inhibition. Evidence from X-ray crystallog-raphy, UV difference spectra, isoelectric focusing, massspectrometry (MS), and Raman microscopy suggests thatthe inactivators of class A �-lactamases undergo complexreaction schemes with multiple branch points after forma-tion of the acyl-enzyme (45, 78, 79, 157, 309, 396). As rep-resented in equations 5 and 6, the acyl-enzyme intermediatecan (i) undergo a reversible change that generates a tran-siently inhibited enzyme, a tautomer (E � T); (ii) lead topermanent inactivation as a covalent acyl-enzyme species(E � I*); or (iii) regenerate the active enzyme via hydrolysis(E � P):

E � T

k4/ k�4

E�I

k5

3 E � P

k32E � I*

(6)

The functional inhibition of the enzyme is determined by therelative rates (k3, k4, k�4, and k5) of each of these pathwaysand in particular by the formation of the E � I* species (53).

Figure 5 shows a more detailed mechanism describing clav-ulanate inhibition of PC1, TEM-1, or SHV-1 (78, 79, 81, 122,179, 180). Kinetic and mass spectrometry analysis of inactiva-tion mechanisms, combined with crystallographic studies,

suggests that clavulanate, sulbactam, and tazobactam followsimilar reaction pathways, beginning with formation of an acyl-enzyme species (48, 81, 307, 308). After acylation, opening ofthe five-membered ring leads to formation of a transient imineintermediate. This imine species is likely the common inter-mediate preceding the chemical conversions that lead to tran-sient enzyme inhibition (44, 179, 197). Raman spectroscopy ofSHV/inhibitor crystals show that the imine species then rear-ranges to form enamine intermediates (197, 307). This en-amine intermediate, in either the trans or cis conformation,represents a second important intermediate in the inactivationmechanism (69, 81, 368). Depending on the properties of theenzyme and inhibitor, the reaction will ultimately proceed todeacylation or irreversible (prolonged) inactivation. In the caseof deacylation of the enamine intermediate, the acyl-enzymeundergoes decarboxylation and ester bond hydrolysis, regen-erating the active �-lactamase, albeit very slowly (260).

The duration of transient inhibition is determined, in part,by the stability of the intermediate species. Preceding acylationof the inhibitor, a persistent noncovalent Michaelis complexmay account for inhibition of enzyme activity (196). Followingformation of the acyl-enzyme, stabilization of the enamineintermediate is a significant factor in prolonged enzyme inhi-bition. Crystals of tazobactam in complex with the SHVGlu166Ala variant (PDB 1RCJ) showed that tazobactamformed stoichiometric amounts of the trans-enamine (308).The trans-enamine intermediate of tazobactam, as opposed tothe trans-enamine intermediates of clavulanate and sulbactam,may be stabilized by intra- and intermolecular interactions.These interactions between the sulfone and triazolyl moietiesof the tazobactam intermediate and the enzyme active site mayexplain, in part, tazobactam’s potent in vitro and in vivo inhi-bition of many serine �-lactamases (36, 60, 308, 312).

Data from Raman crystallography also support the hypoth-esis that compared to clavulanate and sulbactam, tazobactammore readily forms the trans-enamine intermediate (197). Inthis case, Raman spectroscopy allows for the identification ofreaction intermediates and calculation of their rates of decayand accumulation by examining single crystals in solution.Studies of SHV-1 in complex with each of the mechanism-based inhibitors revealed that tazobactam forms a predomi-nant population of trans-enamine, as opposed to clavulanateand sulbactam, which form a mixture of trans-enamine and the

TABLE 2. Kinetic properties of representative �-lactamasesa

�-Lactamase Amblerclass

Clavulanate Sulbactam Tazobactam

Reference(s)KI (�M) IC50

(nM) tnKI

(�M)IC50(nM) tn

KI(�M)

IC50(nM) tn

TEM-1 A 0.1 60 160 1.6 900 10,000 0.01 97 140 73, 180, 283SHV-1 A 1 12 60 8.6 12,000 13,000 0.07 150 5 58, 153, 410, 411SHV-5 A 20 1,800 80 137PC1 A 30 1 80 27 1 47, 58, 69CTX-M-2 A 200 2,100 20 18CcrA B �500,000 �500,000 �500,000 400,000 4,000 58P99 C �100,000 �500,000 5,600 8.5 50 58CMY-2 C 4,365 101 50 116aOXA-1 D 1,800 4,700 380 1400 196, 328OXA-2 D 1,400 0.1 140 10 211, 328

a The definition of KI may not be uniform from laboratory to laboratory. See the cited references for the methods used in each determination.



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more chemically labile cis-enamine and imine species (197).Analysis of the inactivation reactions of PC1, TEM, and

SHV �-lactamases using mass spectrometry has identified aseries of intermediates beyond the acyl-enzyme, imine, andenamine (48, 81, 311, 396, 411). The terminal end products ofinhibition with these mechanism-based inhibitors suggest thata covalent modification of the active-site Ser70 persiststhrough the inhibition process. Mass adducts (in daltons) cor-respond to different inactivated enzyme species; e.g., (i) Ser70-Ser130 cross-linked enzyme or propynyl enzyme (� � 52 � 3),(ii) aldehyde (� � 70 � 3), (iii) hydrated aldehyde (� � 88 �3), and (iv) decarboxylated trans-enamine (� � 156 � 3) aresome of the products (Fig. 5).

�-Lactam–�-lactamase inhibitor combinations: clinical use.Generally, the inhibitors do not inactivate PBPs, but notableexceptions include (i) the intrinsic activities of sulbactamagainst Bacteroides spp., Acinetobacter spp., and N. gonor-rhoeae; (ii) clavulanate action against Haemophilus influenzaeand N. gonorrhoeae; and (iii) tazobactam inhibition of PBPs inBorrelia burgdorferi (53, 167, 222, 264, 285, 423, 424, 447). Asthese “antibacterial effects” are relatively weak, the inhibitorsare always combined with �-lactam antibiotics for clinical use.Currently, there are five �-lactam–�-lactamase inhibitor for-mulations available. Amoxicillin-clavulanate, ticarcillin-clavu-lanate, ampicillin-sulbactam, and piperacillin-tazobactam areavailable in the United States. Cefoperazone-sulbactam is usedin several European countries, Japan, and India but is notavailable in the United States, and therefore we will not in-clude this combination in this review. Table 3 summarizesselected clinical features of these available combinations.

Importantly, as in clinical usage, the �-lactamase inhibitorsare combined with �-lactams for in vitro susceptibility testing.The composition of these formulations can have a major im-

pact on susceptibility results, which may or may not reflectclinical efficacy. We present to the reader the following exam-ple. The ticarcillin-clavulanate preparation for parental admin-istration is 3.0 g-0.1 g, while ampicillin-sulbactam is at a 2.0g-1.0 g ratio. However, both combinations are tested at a 2:1ratio in vitro, and since clavulanate has higher “gram-for-gram”potency than sulbactam, clavulanate may “test better” in MICprofiling. However, clinically ampicillin-sulbactam may retainefficacy because of the relatively large amount of inhibitordelivered relative to the �-lactam and relative to the amount of�-lactamase in the cell. This scenario illustrates an importantissue in susceptibility testing and its correlate to clinical effi-cacy.

(i) Amoxicillin-clavulanate. Amoxicillin-clavulanate was thefirst �-lactam–�-lactamase inhibitor combination introducedinto clinical practice, in 1981 in the United Kingdom and in1984 in the United States, and remains the only combinationavailable for oral use. The activity of amoxicillin against sus-ceptible bacteria that do not possess a �-lactamase (e. g.,streptococci, enterococci, E. coli, and Listeria spp.) is not im-proved by the use of clavulanate. However, the addition ofclavulanate significantly expands amoxicillin’s spectrum to in-clude penicillinase-producing S. aureus, H. influenzae, Morax-ella catarrhalis, Bacteroides spp., N. gonorrhoeae, E. coli, Kleb-siella spp., and P. mirabilis (59, 387). The oral availability ofamoxicillin-clavulanate makes it well suited for the outpatientclinic, and this �-lactam–�-lactamase inhibitor combinationexerted its greatest impact on the treatment of community-acquired respiratory infections (59). Additionally, amoxicillin-clavulanate is sometimes used as an oral equivalent (“step-down therapy”) of ampicillin-sulbactam or ticarcillin-clavulanate in the treatment of skin, soft tissue, abdominal, andgynecological infections. Intravenous formulations of amoxicil-

FIG. 5. Proposed mechanism of inhibition of class A �-lactamases by clavulanate, showing the different acyl-enzyme fragmentation products(expressed in daltons), that have been experimentally observed. (Based on data from references 48, 108, 197, 411, and 453.)



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lin-clavulanate are available in Europe and have been studiedprimarily for their efficacy against ESBL-producing E. coli bac-teremia (239, 370).

(ii) Ticarcillin-clavulanate. When introduced in 1985, ticar-cillin-clavulanate was the first �-lactam–�-lactamase inhibitorcombination available for parenteral administration. Similar tothe case for amoxicillin, the addition of clavulanate does not

increase activity against pathogens for which ticarcillin alone iseffective, such as non-�-lactamase-producing Haemophilusspp., E. coli, Proteus spp., Enterobacter spp., Morganella spp.,Providencia spp., and P. aeruginosa. However, the combinationof ticarcillin and clavulanate does increase activity against�-lactamase-producing staphylococci, E. coli, H. influenzae,Klebsiella spp., Proteus spp., Pseudomonas spp., Providencia

TABLE 3. Selected clinical features of available �-lactam–�-lactamase inhibitor combinations

�-Lactam–�-lactamaseinhibitor

composition

Trade name;manufacturer

Route ofadministration,

formulations, anddosing intervalsa

t1/2, h (%protein bound)

Mean peak serumlevels (�g/ml)

Approved clinicalindications

Proven clinical efficacy forsusceptible causative

organism(s):

Piperacillin- Zosyn; Wyeth i.v., 2.0 g/0.25 g q6h, Piperacillin and 1 h after 3.375-g dose: Appendicitis E. coli, Bacteroides spp.tazobactam 3.0 g/0.375 g q6h, 4.0 tazobactam, piperacillin, 106; Skin and soft tissue S. aureus

g/0.5 g q6h 0.7–1.2 (30) tazobactam 10.7 infections, includingdiabetic foot infections

Postpartum endometritisor pelvic inflammatorydisease

E. coli

Community-acquiredpneumonia

H. influenzae

Hospital-acquired,ventilator-acquiredpneumonia

S. aureus, A. baumannii,H. influenzae, K.pneumoniae, P.aeruginosab

Ampicillin-sulbactam

Unasyn; Pfizer i.v./i.m., 1.0 g/0.5 g q6h,2.0 g/1.0 g q6h

Ampicillin, 1.0(28);sulbactam,1.0 (38)

After 15-min infusionof 1-g/0.5-g dose:ampicillin, 40-71;sulbactam, 21-40

Skin and skin structureinfections

S. aureus, E. coli,Klebsiella spp., P.mirabilis, B. fragilis,Enterobacter spp., A.calcoaceticus

Intra-abdominalinfections

E. coli, Klebsiella spp.,Bacteroides spp.,Enterobacter spp.

Gynecological infections E. coli, Bacteroides spp.

Amoxicillin-clavulanate

Augmentin;GlaxoSmithKline

Pediatric oralsuspension, 600 mg/42.9 mg/5 ml q12hc

Amoxicillin, 1.3(18);clavulanate,

1 h after 250-mg/62.5-mg dose:amoxicillin, 6.9;

Otitis media S. pneumoniae, H.influenzae, Moraxellacatarrhalis

Chewable tablet andoral suspension, 125mg/31.25 mg q8h,200 mg/28.5 mgq12h, 250 mg/62.5mg q8h, 400 mg/57.0mg q12h

1.0 (25) clavulanate, 1.6 Community-acquiredpneumonia or acutebacterial sinusitis

H. influenzae, M.catarrhalis, H.parainfluenzae, K.pneumoniae, methicillin-susceptible S. aureus, S.pneumoniae

Tablet, 250 mg/125 mgq8h, 500 mg/125 mgq8-12h, 875 mg/125mg q12h

Sinusitis H. influenzae, M.catarrhalis

Extended-releasetablet, 1,000 mg/62.5

Skin and skin structureinfections

S. aureus, E. coli,Klebsiella spp.

mg q12h Urinary tract infections E. coli, Klebsiella spp.,Enterobacter spp.

Ticarcillin-clavulanate

Timentin;GlaxoSmithKline

i.v., 3.0 g/0.1 g q4-6h Ticaracillin, 1.1(45);

1 h after 3.1-g dose:ticarcillin, 131;

Septicemia Klebsiella spp., E. coli, S.aureus, P. aeruginosa

clavulanate,1.1 (25)

clavulanate, 1.8 Lower respiratory tractinfections

S. aureus, H. influenzae,Klebsiella spp.

Bone and joint infections S. aureusSkin and skin structure

infectionsS. aureus, Klebsiella spp.,

E. coliUrinary tract infections E. coli, Klebsiella spp., P.

aeruginosa, Citrobacterspp., Enterobactercloacae, Serratiamarcescens, S. aureus

Gynecologic infections Prevotella melaninogenicus,Enterobacter spp., E.coli, K. pneumoniae, S.aureus, S. epidermidis

Intra-abdominalinfections

E. coli, K. pneumoniae, B.fragilis

a i.v., intravenous; i.m., intramuscular; q6h, every 6 h.b When treated with piperacillin-tazobactam, hospital-acquired and ventilator-acquired pneumonia caused by P. aeruginosa should be administered with an

aminoglycoside.c The dose of amoxicillin-clavulanate in the pedatric oral suspension is 45 mg/kg q12h based on information available in the material supplied by the manufacturer.



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spp., N. gonorrhoeae, Moraxella catarrhalis, and Bacteroides spp.(59, 127, 319). Surprisingly, ticarcillin-clavulanate exhibits ac-tivity against the multidrug-resistant, non-lactose-fermentingS. maltophilia, and the use of this combination in addition toother antimicrobials (e.g., aztreonam or trimethoprim-sulfa-methoxazole) leads to synergistic killing (118, 275). This phe-nomenon may represent a very interesting opportunity to studythe activity of ticarcillin-clavulanate against S. maltophilia.

Clavulanate induces the expression of chromosomally medi-ated AmpC �-lactamases in many Enterobacteriaceae and canantagonize the antibacterial effects of ticarcillin as a partner�-lactam (8, 203, 231, 237). This ticarcillin antagonism wasobserved in laboratory checkerboard studies with E. cloacaeand Morganella morganii strains (237). However, the impact of�-lactamase induction in the clinic is very hard to measure.When testing or evaluating antibiotics in preclinical or clinicaltrials, �-lactamase induction may bear importance as an even-tual predictor of efficacy. In our opinion, the ability of �-lac-tamase inhibitors to induce cephalosporinase productionshould be carefully examined and defined before clinical trialsare performed.

In response to the potential for increased AmpC productiondue to �-lactam inducers (such as clavulanate, but includingalso cefoxitin, imipenem, and ceftazidime) as well as the rela-tively poor performance of amoxicillin-clavulanate and ticar-cillin-clavulanate against ESBL expressors, Livermore et al.and Nikaido et al. have proposed the combination of clavu-lanate with a cephalosporin, such as cefepime or cefpirome,that is more stable against AmpC enzymes (239, 288).Cefepime-clavulanate and cefpirome-clavulanate combina-tions, with a constant concentration of 4 �g/ml clavulanate,were effective at �1 �g/ml against ESBL-producing Enterobac-teriaceae (239). The combination of cefepime and clavulanateenjoys a particular advantage due to the inability of class Cenzymes to hydrolyze cefepime and the ability of clavulanate toinhibit ESBLs. Currently, there are important obstacles tocombining these agents (i.e., all three agents are at the end ofpatent protection, and the required trials of the formulationswould be very expensive), but provided that the clinical re-search can be performed, these combinations may be an at-tractive alternative to carbapenem treatment of ESBL-produc-ing organisms. We hasten to add that attention should be paidto the pharmacokinetics and pharmacodynamics of these novelcombinations before randomized controlled trials are per-formed, as it is not possible to “fix” a concentration of clavu-lanate in vivo.

(iii) Ampicillin-sulbactam. Ampicillin shows activity againstmost streptococci, enterococci, Listeria spp., and strains of S.aureus, H. influenzae, E. coli, P. mirabilis, Salmonella spp., andShigella spp. that are devoid of �-lactamases (an importantexception are ampicillin-resistant strains of H. influenzae thatdo not contain a �-lactamase and often have substitutions inPBP sequences) (194, 257). In combination with sulbactam, theactivity extends to �-lactamase-containing S. aureus, H. influ-enzae, M. catarrhalis, E. coli, Proteus spp., Klebsiella spp., andanaerobes (59, 362, 444). When the combination of ampicillinat 2.0 g and sulbactam at 1.0 g was marketed in 1987, thisbroad-spectrum activity made ampicillin-sulbactam ideal ther-apy for polymicrobial infections such as abdominal and gyne-cological surgical infections, aspiration pneumonia, odonto-

genic abscesses, and diabetic foot infections. The early clinicalsuccess of this combination (and susceptibility of many patho-gens to ampicillin-sulbactam and ticarcillin-clavulanate) estab-lished confidence in the role of �-lactam–�-lactamase inhibitortherapy. Unfortunately, the resistance to ampicillin-sulbactamamong clinical isolates of E. coli is increasing (199).

Sulbactam is not well absorbed orally and must be admin-istered parenterally (126). However, the intravenous ampi-cillin-sulbactam combination is well tolerated, with very fewreported side effects (7, 66).

(iv) Piperacillin-tazobactam. Piperacillin in combinationwith tazobactam became available in the United States in 1993.Piperacillin is a broad-spectrum penicillin that is bactericidalagainst many Gram-positive and Gram-negative aerobes andanaerobes (140). As a single agent, piperacillin demonstratesactivity against P. aeruginosa, pneumococci, streptococci,anaerobes, and Enterococcus faecalis, and this activity is re-tained in combination with tazobactam (232). Clinicians mustremember that the addition of tazobactam does not alwaysincrease susceptibility of P. aeruginosa and other Gram-nega-tive bacilli expressing AmpC �-lactamases (140). However,tazobactam does extend piperacillin’s activity against most�-lactamase producing strains of Enterobacteriaceae, H. influ-enzae, N. gonorrhoeae, and M. catarrhalis and has the potentialto lower MICs against these strains expressing ESBLs (59,192). The in vitro inhibition of CMY-type �-lactamases bytazobactam is reported, but the clinical relevance of this phe-notype is still uncertain (34, 116a).

Several recent retrospective studies of clinical outcomesfrom E. coli and K. pneumoniae ESBL-producing isolatesargue for the efficacy of piperacillin-tazobactam in the treat-ment of these infections (135, 336, 370, 420). Gavin et al.showed that when in vitro testing revealed susceptibility topiperacillin-tazobactam (�16/4 �g/ml), successful outcomeswere seen in 10 out of 11 non-urinary tract infections (135).An examination of 43 bloodstream infections caused pri-marily by CTX-M ESBL-producing E. coli isolates revealedthat piperacillin-tazobactam, amoxicillin-clavulanate (i.v.),or carbapenem treatment led to lower mortality than withcephalosporin or fluoroquinolone treatment (9% versus35%) (370). Another study, including infections caused bySHV- and TEM-type ESBL-producing K. pneumoniae, es-tablished that empirical therapy with �-lactam–�-lactamaseinhibitor combinations was validated by in vitro susceptibil-ity testing in 75% of cases, compared to 0% for oxymino-cephalosporin treatment (420). Furthermore, initial treat-ment with an adequate antibiotic was associated with lowermortality rates (37%) than inadequate empirical therapy(67%).While this analysis did not stratify the outcome databased on ESBL type, other authors have reported thatTEM-type ESBLs are more susceptible to piperacillin-ta-zobactam than are SHV-type ESBLs (185, 361).

Several observational studies have strongly suggested thatthe replacement of extended-spectrum cephalosporins withpiperacillin-tazobactam can help lower the rates of infectionfrom Gram-negative ESBL producers and vancomycin-resis-tant Enterococcus spp. (16, 43, 215, 216, 323, 355, 365). Whilethe mechanism for decreasing ESBL-producing isolates is notclear, it must be noted that the efficacy of �-lactam–�-lacta-mase inhibitor combinations may be reduced for organisms



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producing multiple ESBLs, particularly if they also have anAmpC �-lactamase (42, 321, 381). For this reason, we cautionagainst the use of piperacillin-tazobactam for the treatment ofESBL-producing pathogens.

So, why do the in vitro susceptibility data and the clinicallycited data suggest that piperacillin-tazobactam can be usedin the treatment of ESBL producers? The reasons for thiscontradiction include that piperacillin, as a partner �-lactamfor �-lactamase inhibitors, is relatively resistant to hydroly-sis by certain plasmid-mediated �-lactamases (e.g., TEM)compared to amoxicillin, ampicillin, or ticarcillin. This sub-strate stability may be due, in part, to piperacillin’s loweraffinity for and lower turnover (kcats) by these enzymes andalso piperacillin’s greater affinity for bacterial PBPs (234).We also propose that the pharmacokinetics and pharmaco-dynamics of the 3.375-g and 4.5-g formulations lead to ex-tended periods of time when free drug concentrations(�100 �g/ml piperacillin in serum) are greater than theMIC for �50% of the dosing interval for many nosocomialpathogens (242). It is likely that the high serum concentra-tions combined with the superior activity of piperacillin andthe relatively “better” inhibitory activity of tazobactam ver-sus clavulanate or sulbactam is enough to inhibit a majorityof �-lactamases in a complex �-lactamase background (classA and C �-lactamases) (328). However, this efficacy is notalways clinically certain, and the physician must be awarethat the susceptibility pattern of an infecting Gram-negativepathogen does not always reveal the number and complexityof the �-lactamases present. Moreover, the formulation thatis tested in the lab may not mirror what is administered topatients.

Inhibitory Activity of Carbapenems

It is our opinion that consideration of carbapenems, al-though they are not explicitly �-lactamase inhibitors, is es-sential to a review of �-lactamase inhibition. These thien-amycin derivatives are among the “last line of defense”against many multidrug-resistant pathogens. An increasingbody of evidence suggests that carbapenems are effectivenot just for their broad-spectrum antibacterial activity butalso for their multiple �-lactamase-inhibitory mechanisms.The insight gleaned from the ability of carbapenems toinhibit serine �-lactamases may serve as a useful startingpoint for intelligent inhibitor design. Also, while we awaitthe development and release of novel �-lactamase inhibi-tors, it behooves us to wisely implement the �-lactams cur-rently in our armamentarium. With this in mind, a discus-sion of the inhibitory activity of carbapenems follows.

Meropenem, imipenem, ertapenem, and doripenem (dorip-enem was released in the U.S. and European markets in 2007)are often reserved for the most difficult-to-treat Gram-negativeinfections (Fig. 1, compounds 4 to 7) (320). Although resis-tance to carbapenems is rare in the clinic, several carbapenem-hydrolyzing serine �-lactamases are identified, such as KPC-2,NMC-A, SME-1, and IMI-1 (358, 455). These enzymes may befound in organisms that also produce additional �-lactamases,conferring broad substrate profiles. For example, E. cloacaeisolates can produce IMI-1 or NMC-A, which hydrolyze car-bapenems, and also TEM and AmpC-type enzymes (359).

In addition to their broad antibacterial spectrum, carbapenemsare also effective inhibitors, or “slow substrates,” of most serine�-lactamases.

The stability of thienamycins to �-lactamase hydrolysis wasnoted in early studies of imipenem, including low kcat valuesfor the class A �-lactamase of B. cereus and a class C P.aeruginosa �-lactamase (150, 208, 270, 366). Charnas andKnowles observed biphasic kinetics when RTEM was acylatedby carbapenem compounds; an initial burst was followed by aslower steady-state reaction, strongly suggestive of a branchedreaction pathway (80, 114). Further work by Mobashery’sgroup demonstrated that acylation of TEM-1 by imipenemoccurs readily and is followed by �2- to �1-tautomerization ofthe pyrroline double bond in the acyl-enzyme species (Fig. 6)(407). The rate of deacylation differs for the tautomers (the �1

tautomer is approximately 10-fold slower than the �2 tau-tomer), and it was initially accepted that molecular rearrange-ments to the more stable (i.e., more slowly deacylated) speciesaccounted for enzyme inhibition. Site-directed mutagenesisstudies of TEM-1 Arg244Ser showed that the guanidiniumgroup on Arg244 provides directly, or indirectly via a watermolecule, the necessary proton for tautomerization to the �1-pyrroline acyl-enzyme (407, 458). Incidentally, this water ap-pears to be the same molecule that serves as the proton donornecessary for clavulanate inhibition in class A enzymes (179).

Elucidation of the crystal structures of several �-lactamasesin complex with carbapenems has offered additional insightinto the versatile inhibition mechanism of these compounds.Maveyraud et al. found that in the structure of TEM-1 com-plexed with imipenem (PDB 1BT5), the ester carbonyl oxygenof the acyl-enzyme was not located in the oxyanion hole (255).This approximately 180° rotation of the carbonyl was also seenin the crystal structure of acyl-enzyme species of imipenemcomplexed via Ser64 with the AmpC �-lactamase (PDB 1LL5)(21). In contrast, the SHV-1/meropenem crystal demonstratedtwo conformations of the intermediate acyl species, one wherethe meropenem carbonyl oxygen of the acyl-enzyme is in theoxyanion hole and one where it is not (Fig. 7) (295). Properposition in this oxyanion hole, the binding pocket for the �-lac-tam carbonyl, is important for both initial enzyme acylationand hydrolysis of the ester bond (277, 425). A conformationwhich places the �-lactam carbonyl outside of this electrophiliccenter may be delayed in hydrolysis.

Computational models of TEM-1 with imipenem demon-strated that the covalent adduct formed first after acylation didhave its carbonyl in the oxyanion hole (255). However, thisconformation created a steric clash between the hydroxyethylgroup and residues 129 to 131, which forces the rearrangement

FIG. 6. Imipenem tautomers hypothesized to form after acylationof carbapenems by serine �-lactamases. The deacylation rate of the�1-pyrroline is significantly lower than that of the �2-pyrroline, whichis thought to play a large role in the inhibitory activity of these com-pounds (407, 458).



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of the acyl-enzyme rotating the carbonyl outside the oxyanionhole. Thus, while producing a conformational change in theenzyme, the C-6 hydroxyethyl substituent also facilitates rear-rangement of the carbapenem to a more slowly deacylatedform. Subsequent examination of the TEM Asn132Ala variantdemonstrated that replacement of the residue relieved the stericencumbrance of the TEM-1/imipenem acyl-enzyme and allowedthe carbonyl to rotate back into the oxyanion hole (437). Further-more, the TEM-1/imipenem structure indicated that hydrogenbonds were formed between the hydroxyl group of the C-6 �-1R-hydroxyethyl substituent on imipenem and both the Glu166-as-sociated hydrolytic water and the side chain oxygen of Asn132(255). Similarly, the SHV-1/meropenem and class D OXA-13/meropenem structures (PDB 2ZD8 and 1H8Y, respectively) re-vealed that the putative deacylation water molecule has an addi-tional hydrogen-bonding interaction with the -OH group ofmeropenem’s C-6 �-1R-hydroxyethyl substituent (295, 334). Thisinteraction weakens the nucleophilicity and/or changes the direc-tion of the lone pair of electrons of the water molecule and resultsin poor turnover of the carbapenems.

As individual hydrogen atoms are difficult to resolve byX-ray crystallography, the structural data for �-lactamasesand carbapenems have not been able to discriminate be-tween the �1- and �2-pyrroline tautomers. In contrast, Ra-man spectroscopy allows the monitoring of specific bondcharacter and compound reactivity. Recent studies of Ra-man difference spectra in SHV-1 reacted with meropenem,ertapenem, and imipenem propose the correlation that the�1-pyrroline tautomer corresponds to the acyl-enzyme spe-

cies with the carbonyl outside the oxyanion hole and the�2-pyrroline tautomer to the acyl-enzyme with the carbonylinside the electrophilic center (195). Further, this work sug-gests that in crystal form the �1- and �2-tautomers arepresent in equal amounts but that �1-pyrroline predomi-nates in solution. This ratio is consistent with the kineticdata implicating the �1-tautomer as the primary inhibitoryspecies.

The recent structure of OXA-1, a class D monomeric �-lac-tamase, inactivated by doripenem provided further support forthe role of carbapenems as inactivators of serine �-lactamases(PDB 3ISG) (379a). In this structure by Schneider and col-leagues, although the carbonyl oxygen is positioned in theoxyanion hole, water molecules are absent. The carbamate ofLys70 forms a hydrogen bond to the C-6 hydroxyethyl group,and Lys212 and Thr213 form a salt bridge and hydrogen bondto doripenem. Furthermore, the bond angles of the acyl-en-zyme suggest the �1-tautomer is formed.

In addition to the imipenem-induced conformation changeof class A and C �-lactamases, the (albeit slow) turnover ofcarbapenems leads to a stable enzyme-product species thatoccupies the �-lactamase active site for relatively longer thanthe enzyme-products of other �-lactams, such as penicillin(whose dissociation is limited by diffusion for class A enzymes)(149, 254). However, the deacylation rate was still lower thanthe dissociation rate for imipenem, suggesting that acyl-en-zyme rearrangement accounts most significantly for inhibition.But notably, the presence of hydrolyzed inhibitor in the activesite can contribute to enzyme inhibition.

FIG. 7. Molecular representation of SHV-1/meropenem acyl-enzyme, based on PDB coordinates 2ZD8. The conformation with the carbonylof the acyl-serine bond in the oxyanion hole (formed by NH amides of Ser70 and Ala237) is colored teal, while the conformation with the carbonylflipped out is colored purple. Residues 244, 130, and 105, which demonstrate significant orientation changes compared to the apo SHV-1�-lactamase, are labeled. Glu166 and Asn132, important for making hydrogen bonds with the deacylation water (the approximate location isrepresented as a red sphere) and C-6 hydroxyethyl substituent, respectively, are shown. The large R� carbamoyl-pyrrolidinyl group of meropenemwas disordered and thus is only partially illustrated.



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INHIBITOR-RESISTANT CLASS A �-LACTAMASES

Within several years of the introduction of clavulanate com-binations for clinical use, resistance to amoxicillin-clavulanateand ticarcillin-clavulanate was observed in isolates of E. coliand K. pneumoniae (247, 248, 376, 446). In 1987, Martinez etal. reported resistance rates in E. coli in Madrid hospitals andestimated that of amoxicillin-resistant strains, 20 to 30% wereco-amoxicillin-clavulanate resistant (248). Investigations in thelate 1980s demonstrated that this phenotype may result fromproduction of �-lactamases not susceptible to the inhibitors(e.g., AmpCs from Enterobacter spp. or P. aeruginosa or me-tallo-�-lactamases) or enzyme hyperproduction (68). Hyper-production of a �-lactamase can be mediated by mutations inthe promoter region of the gene and/or high copy numbers ofplasmids carrying the bla gene; both scenarios were identifiedfor the TEM-1 enzyme (247, 248, 446, 448, 449).

Additional resistance mechanisms were concurrently be-ing examined in research laboratories. In 1989, Oliphantand Struhl studied clavulanate and sulbactam resistance inTEM-1 by introducing a series of random substitutions intothe DNA segment which coded for the enzyme’s active site(300). Through mutagenesis of a 17-amino-acid segment ofthe TEM-1 active site (Arg61 to Cys77) and functional se-lection assays, they found that enzymes with an Ile, Leu, orVal substitution at Met69 had increased resistance to ampi-cillin-clavulanate and ampicillin-sulbactam. This foreshad-owed an important amino acid substitution in inhibitor-resistant (IR) TEM.

Three years later, Zafaralla et al. engineered by site-directedmutagenesis the Arg244Ser variant of TEM-1 to explore therole of this key amino acid and its interaction with the peni-cillin C-3 carboxylate (457). This work advanced the earlierhypothesis by Moews et al. that Arg244 played an importantrole in the recognition of the conserved substrate C-3/C-4 carbox-ylate (179, 267). Further studies by Mobashery’s laboratory alsorevealed that Arg244Ser was more resistant to inactivation byclavulanate (179). Interestingly, the first IR class A �-lactamasewas isolated from a clinical isolate of E. coli in the same year. Theenzyme was determined to be related to TEM-1 or TEM-2 andhence was designated inhibitor-resistant TEM (IRT-1) (29, 427).Sequencing information revealed an Arg-to-Cys or -Ser substitu-tion at residue 244 (23, 427).

Since that time in the early 1990s, the number of clinicallyidentified IRTs has expanded to include more than 35 uniqueenzymes (Table 4) (www.lahey.org/studies/webt.asp). In addi-tion to amoxicillin-clavulanate-resistant E. coli, IRTs havebeen identified in Klebsiella, Proteus, Shigella, and Citrobacterspp. (385). IR SHVs have also been isolated from K. pneu-moniae, with a total of five IR SHVs presently described (Table5) (109). Operationally, inhibitor resistance refers to resistanceto amoxicillin-clavulanate and may, but does not necessarily,include resistance to sulbactam and tazobactam (410). More-over, we stress that this is a “relative resistance”; provided thatsufficient clavulanate concentrations are used, these IR en-zymes can be inactivated.

Currently, reports describing non-TEM, non-SHV family IRenzymes are becoming more common. The CTX-M ESBLs,which typically hydrolyze penicillins and extended-spectrumcephalosporins (preferentially cefotaxime), have not yet shown

significant resistance to the �-lactamase inhibitors (32, 68).Present studies indicate that the KPC-2 �-lactamase is notinactivated by clavulanate, sulbactam, and tazobactam, as in-dicated by high MICs and turnover numbers of 2,500, 1,000,and 500, respectively (319a). The ability of KPC-2 to avoidinactivation by the currently available inhibitors may representa novel class of highly IR class A enzymes. The rapid emer-gence of Enterobacteriaceae harboring these carbapenemases isa significant public health concern (290).

Epidemiology and Detection of �-LactamaseInhibitor Resistance

Resistance to �-lactam–�-lactamase inhibitor combinationschallenges the ability to successfully treat serious urinary tract,respiratory tract, and bloodstream infections (154, 219, 376).The prevalence of organisms expressing inhibitor-resistant en-zymes varies throughout the world. In 1993, a French study of2,972 E. coli isolates from urinary tract infections (UTIs) foundthat 25% and 10% of hospital and community isolates, respec-tively, showed amoxicillin-clavulanate MICs of �16/2 �g/ml(160). Characterization of these isolates, including MIC pro-files (amoxicillin-clavulanate MIC90 of �1,042 �g/ml andcephalosporin MIC of �32 �g/ml), isoelectric focusing, andDNA-DNA hybridization, suggested that 27.5% and 45% ofhospital and community isolates, respectively, were TEM-1derived and had substitutions at previously described amino acidpositions that confer the IRT phenotype. In this survey, 4.9% ofE. coli isolates from UTIs were IRT producing. In 1998, a geri-atric department of a French hospital reported an outbreak ofamoxicillin-clavulanate-resistant isolates which all produced thesame IR TEM �-lactamase, TEM-30 (Arg244Ser) (141). In astudy published in 2000, Leflon-Guibout et al. determined themolecular mechanism of amoxicillin-clavulanate resistance (de-fined by MICs of �16/2 �g/ml) in E. coli isolates from threeFrench hospitals from 1996 to 1998 (218). The overall resistancerate was 5%, and the majority of these resistant organisms werefrom patients with respiratory tract infections. Production of IRTswas implicated in resistance in 30 to 41% of the isolates over thetime period, which was only slightly less frequent than the hyper-production of chromosomal AmpC enzymes. Two independentSpanish studies reported production of IRTs in 5.4% and 9.5% ofamoxicillin-clavulanate-resistant E. coli isolates (266, 331).

The first IRT identified in the United States was not reporteduntil 2004 in a 14-month survey of E. coli isolates from a north-eastern tertiary care clinical microbiology laboratory (198). Kayeet al. found that 24% of the 283 isolates classified as ampicillin-sulbactam resistant by disk diffusion and MIC testing were alsoresistant to amoxicillin-clavulanate as determined by disk diffu-sion zone diameters of �13 mm. Of the amoxicillin-clavulanate-resistant isolates, 83% were from community-acquired infections.Many of these 69 isolates were recovered from UTIs of outpa-tients, and two expressed the IR TEM-34 (Met 69Val) and onethe TEM-122 (Arg275Gln) enzyme. Incidentally, also reported in2004 was a KPC-2-producing K. pneumoniae isolate from NewYork City that contained IRT-2 (41).

Isolates producing IRTs are more frequently reported inEurope than in the United States. The explanation for thisdiscrepancy is not clear and probably reflects a combinationof environmental, methodological, and clinical practice fac-



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tors. The phenomenon does not appear to be related tolarge differences in the use of �-lactam–�-lactamase inhib-itor combinations, as these agents are widely used in bothEurope and the United States and likely produce similarselective pressures on bacteria (68, 159).

The detection of IRTs presents a significant number of op-erational challenges. Disagreements between the results ofMIC testing and disk diffusion assays are often seen, especiallyamong isolates with intermediate resistance phenotypes (68,

198, 301). Currently, an international standard for the amountof �-lactamase inhibitor that should be combined with thepartner �-lactam for detection of IR enzymes is not in use, anddifferent combinations can significantly alter the assigned results(301, 409). For example, the use of the 2:1 ratio for amoxicillin-clavulanate appears to underestimate the presence of IR �-lac-tamases compared to employing the fixed concentration of 2�g/ml clavulanate (409). Further, organisms producing low levelsof IR enzymes may be undetectable at the 2:1 ratio (302). As a

TABLE 4. Clinically identified inhibitor-resistant TEM (IRT) �-lactamasesa

�-LactamaseAmino acid at positionb:

21 39 69 104 127 130 165 182 221 238 244 261 265 275 276

TEM-1 Leu Gln Met Glu Ile Ser Trp Met Leu Gly Arg Val Thr Arg AsnTEM-30 (IRT-2) SerTEM-31 (IRT-1) CysTEM-32 (IRT-3) Ile ThrTEM-33 (IRT-5) LeuTEM-34 (IRT-6) ValTEM-35 (IRT-4) Leu AspTEM-36 (IRT-7) Val AspTEM-37 (IRT-8) Ile AspTEM-38 (IRT-9) Val LeuTEM-39 (IRT-10) Leu Arg AspTEM-40 (IRT-11) IleTEM-44 (IRT-13) Lys SerTEM-45 (IRT-14) Leu GlnTEM-51 (IRT-15) HisTEM-54 LeuTEM-58 Ser IleTEM-59 (IRT-17) Lys GlyTEM-65 (IRT-16) Lys CysTEM-67 Ile Lys CysTEM-73 (IRT-18) Phe Cys MetTEM-74 (IRT-19) Phe Ser MetTEM-76 (IRT-20) GlyTEM-77 (IRT-21) Leu SerTEM-78 (IRT-22) Val Arg AspTEM-79 GlyTEM-80 (IRT-24) Leu Val AspTEM-81 Leu ValTEM-82 Val GlnTEM-83 Leu Cys GlnTEM-84 AspTEM-103 (IRT-28) LeuTEM-122 GlnTEM-145 Met HisTEM-159 Phe IleTEM-160 Lys Val Thr

a Based on data from www.lahey.org/studies/webt.asp.b Unless otherwise indicated, the amino acid is the same as in the non-IR TEM-1.

TABLE 5. Clinically identified inhibitor-resistant SHV �-lactamasesa


35 69 130 140 146 187 192 193 234 238 240

SHV-1 Leu Met Ser Ala Ala Ala Lys Leu Lys Gly GluSHV-10 Gly Arg Asn Val Ser LysSHV-26 ThrSHV-49 IleSHV-56 Gln ArgSHV-72 Val Arg

a Based on data from www.lahey.org/studies/webt.asp.b Unless otherwise indicated, the amino acid is the same as in the non-IR SHV-1.



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result of the methodological complications of identifying IR�-lactamase producers, the actual IRT prevalence may be under-estimated (68). Definitive identification requires specific enzymekinetic testing, determination of isoelectric points, and molecularcharacterization (60, 72, 77). Routine susceptibility tests may notaccurately detect strains expressing IRTs. A direct correlationbetween the level of resistance (MIC) and the predicted clinicaloutcome is not yet known.

Substitutions in Class A EnzymesConferring Inhibitor Resistance

The amino acid substitutions associated with clavulanateresistance are different from those that produce the ESBLphenotype, and thus IRTs have unique hydrolytic profiles com-pared to ESBLs. For example, the E. coli isolates that containIRTs may be more susceptible to cephalosporins, carbapen-ems, and monobactams (35). However, among TEM and SHV�-lactamases, substitutions that confer an IR phenotype aresometimes found in pairs or are combined with amino acidchanges that also increase the ability of the enzyme to hydro-lyze oxyimino-cephalosporins. These variants are designatedcomplex mutants of TEM (CMTs) and may foreshadow theemergence of a new class of versatile �-lactamases with evenbroader hydrolytic and resistance profiles (Table 6) (369).

Table 7 summarizes kinetic properties of representativeclass A IR enzymes that have been identified clinically or

generated in the laboratory. IRTs, when derived from mutatedwild-type bla genes, typically display a reduction in �-lactamcatalytic efficiency (kcat/Km), mediated by lower kcat and in-creased Km values for penicillins compared to the wild-typeenzymes (35). IR �-lactamases have increased KIs and IC50sfor clavulanate and sulbactam; smaller increases are observedfor tazobactam. In general, the MICs for organisms expressingIR enzymes are lowered for penicillins and increased for cla-vulanate and sulbactam. MICs may remain in the susceptiblerange for tazobactam when obtained with the combination ofpiperacillin-tazobactam. This susceptibility is likely due to pip-eracillin’s greater potency and rapid cell entry (see above) aswell as the relative preservation of tazobactam’s KI (36). Ofsignificant interest is the recent description of the kinetic prop-erties of SHV-72 (Ile8Phe, Ala146Val, Lys234Arg) (258). Incontrast to most other IR enzymes, this unique IR SHV dem-onstrates increased catalytic efficiency for penicillins.

Amino acid substitutions in TEM and SHV result in resis-tance to clavulanate by two primary mechanisms: (i) alter-ations in the geometry of shape of the oxyanion hole and (ii)changes in the position of Ser130 or Arg244 (33, 73, 101, 120,408, 411, 416). The individual residues that are most commonlysubstituted in IR TEM enzymes are Arg244, the nearbyAsn276 and Arg275, Met69, and the active-site Ser130 (www.lahey.org/studies/webt.asp) (Fig. 8).

TABLE 6. Clinically identified complex mutants of TEM (CMT) �-lactamasesa


39 69 104 130 164 237 238 240 244 265 275 276 284

TEM-1 Gln Met Glu Ser Arg Ala Gly Glu Arg Thr Arg Asn AlaTEM-50 (CMT-1) Leu Lys Ser AspTEM-68 Ser Lys Met LeuTEM-89 (CMT-3) Lys Lys Gly SerTEM-109 (CMT-5) Leu Lys His CysTEM-121 (CMT-4) Lys Lys Ser Thr Lys SerTEM-151 Val His Asp GlyTEM-152 Val His Lys AspTEM-154 Leu SerTEM-158 (CMT-9) Leu Ser Asp

a Based on data from www.lahey.org/studies/webt.asp.b Unless otherwise indicated, the amino acid is the same as in the non-IR TEM-1.

TABLE 7. Kinetic properties of representative class A inhibitor-resistant and wild-type enzymesa

�-LactamaseClavulanate Sulbactam Tazobactam

Reference(s)KI (�M) kinact (s�1) tn KI (�M) kinact (s�1) tn KI (�M) kinact (s�1) tn

TEM-1 0.1 0.02 160 1.6 0.002 10,000 0.01 �0.02 140 73, 180TEM Met69Leu 1.65 0.002 2,800 22 0.0009 7,000 0.20 0.004 2,900 73TEM Ser130Gly 105 0.003 1,600 220 0.03 1,900 95 0.04 630 408TEM Arg244Ser 33 44 0.00001 7,800 179, 180TEM Asn276Asp 15.6 0.01 250 378TEM Met69Leu, Asn276Asp 27 49 0.6 386SHV-1 1 0.04 60 8.6 0.056 13,000 0.07 0.1 5 153, 410, 411SHV Arg244Ser 63 0.09 50 240 0.13 100 410, 411SHV Ser130Gly 47 0.03 40 4.2 0.06 5 153OHIO-1 0.4 0.01 80 17 0.001 40,000 0.30 0.01 400 229OHIO Met69Ile 10 0.002 2,000 68 0.0004 200,000 18 0.0016 2,000 229




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Arginine 244. To date, 12 IRT variants with substitutions atresidue 244 have been reported. The guanidinium group(CH5N3

�) of Arg244 contributes to substrate affinity throughhydrogen bonding to the conserved �-lactam carboxylate(436). Structural and computational studies of the mechanismof clavulanate resistance in TEM-1 reveal that a hydrogen onthe N�2 atom of the guanidinium group of Arg244 and thecarbonyl oxygen atom of the backbone of Val216 coordinate awater molecule (Fig. 9) (179, 411, 436, 457). When clavulanateis bound, this water molecule is the likely donor of a protonthat saturates the double bond at the C-2 position, facilitatingthe opening of the five-membered ring, formation of an inter-mediate, and eventual �-lactamase inactivation (179). Loss ofthe guanidinium group of Arg244 displaces this water moleculeand may explain the inhibitor resistance and catalytic impair-ment that is observed in the TEM Arg244 variants. Withoutprotonation of the double bond, elimination of oxygen at C-1is thermodynamically unfavored, which may lead to increasedinhibitor turnover (179).

Interestingly, we have not found a clinical report of an SHVenzyme that has the 244 substitution. However, mutagenesis ofSHV at Arg244 revealed that the 244Leu and -Ser substitu-tions at this residue also confer resistance to amoxicillin-clav-ulanate (138, 411). The biochemical correlates of these clav-ulanate-resistant phenotypes are different for SHV and TEM

and are related to slight differences in the inactivation mech-anisms for these enzymes (179, 410, 411, 436, 457).

Methionine 69. Met69 is the most commonly substituted res-idue in inhibitor-resistant enzyme isolates, appearing either as asingle substitution or in combination with additional amino acidreplacements in 15 of the 36 presently defined IRTs. Despite theproximity to enzyme nucleophilic Ser70, the Met69 side chain isoriented away from the catalytic serine and not part of the sub-strate binding site (191). Residue 69 forms the back wall of theoxyanion hole or “electrophilic center” that polarizes the �-lac-tam for nucleophilic attack by Ser70 and, in the case of inhibitors,possibly Ser130 as well (206). The residue is not highly conservedin class A �-lactamases, and TEM can tolerate many substitutionsat the site and still retain activity against most substrates (10, 101).The SHV Met69Phe, -Lys, and -Tyr variants have increased MICsand hydrolytic activity for the extended-spectrum ceftazidime(155). While the residue displays some flexibility in its role insubstrate binding, certain substitutions in TEM and SHV conferresistance to the inhibitors.

The TEM variants, Met69Ile, -Val, and -Leu, each show anapproximately 10-fold increase in KI and 10-fold decrease inkinact, leading to a 100-fold decrease in inhibitory efficiency(kinact/KI) (73). Evidence from X-ray crystallography and mo-lecular dynamic studies has revealed that each substitutionintroduces subtle active-site changes that perturb inhibitor rec-ognition and enzyme catalysis (262, 438). Similarly, SHVMet69Ile, -Val, and -Leu show increased clavulanate MICsand IC50s (155). Reduced inhibitor acylation efficiency in SHVwas elucidated by observed changes in the oxyanion hole in thecrystal structures of SHV Met69Val/Glu166Ala in complexwith clavulanate, sulbactam, and tazobactam (PDB 2H0T,2H0Y, and 2H10, respectively) (416).

Asparagine 276. Fourteen TEM variants with substitutionsat Asn276 have been discovered; seven demonstrate an IRphenotype. Ambler position 276 remains unchanged in cur-rently identified SHV variants. Despite its relative distance (8to 10 Å) from the enzyme active site, the crystallographicstructure of TEM Asn276Asp (PDB 1CK3) reported bySwaren et al. confirmed the importance of the electrostaticenvironment surrounding residue 276 (400). In TEM, aminoacid 276 is located on the enzyme’s C-terminal �-helix H11,which lies behind the �-sheet including Arg244. TheAsn276Asp substitution creates new interactions with Arg244and significant displacement of �-helices H1, H10, and H11.The Asp at residue 276 is displaced only 0.75 Å compared tothe wild-type Asn, but this movement results in a tighter at-traction between Arg244 and 276Asp.

This enhanced interaction has two major effects (378, 400).First, there is an effective “neutralization” of the Arg244 con-tribution. This decreased positive charge may lower the localaffinity for the C-3 carboxylate of clavulanate and penicillins(400). Charge changes in the active site may have a moreprofound consequence for the inhibitors, which are positionedas small penicillin substrates and rely heavily on the interac-tions with Arg244 for binding affinity (179, 378, 457). Second,the TEM Asn276Asp variant’s increased clavulanate kcat val-ues may be explained by the displacement of the water mole-cule necessary for secondary ring opening of clavulanate, asdiscussed above for TEM Arg244 variants (378, 400).

Similar to the case for Arg244, IR SHV 276 variants have

FIG. 8. Molecular representation of TEM-1 active site, showingresidues that are most frequently implicated in the development ofinhibitor-resistant TEM enzymes. Based on PDB coordinates 1TEM.

FIG. 9. Schematic representation of the proposed Michaelispreacylation complex of TEM-1 and clavulanate.



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not yet reached clinical attention (411). Molecular modeling ofSHV Asn276Asp showed that novel electrostatic interactionsare formed between Arg244N�2 and both 276AspO�1 andO�2, reminiscent of the TEM Asn276Asp crystal structure(108). Additional data from both kinetic studies and timedelectrospray ionization mass spectrometry (ESI-MS) of clavu-lanate-inhibited SHV-1 and SHV Asn276Asp suggest that theAsn276Asp phenotype is produced by decreased affinity forthe C-3 carboxylate of clavulanate and not by a change in thereaction mechanism. These results are consistent with the no-tion advanced by Thomson et al., suggesting that clavulanateinactivation in SHV may not rely on a water molecule coordi-nated by Arg244 and Val216, as in TEM enzymes (191, 210,411). These important differences in TEM and SHV mecha-nisms support a divergent evolution of these enzymes vis-a-vissubstrate and inhibitor profiles.

Serine 130. Ser130 is a conserved amino acid among all classA �-lactamases but is replaced by a Gly residue in two IRTs(TEM-59 and -76) and one IR SHV enzyme (SHV-10). Themultiple roles of this residue include anchoring �-lactams toand stabilizing the active site through hydrogen bonding withthe C-3/C-4 carboxylate of the inhibitors and substrates andfacilitating proton transfer to the �-lactam nitrogen duringacylation, leading to �-lactam ring opening (179, 180, 214,426). Ser130 also serves as a second nucleophile which attacksthe imine intermediate and becomes covalently cross-linked toSer70 in the terminal inactivation of the mechanism-based inhib-itors (209). Removing the cross-linking residue seems a clear wayto overcome inactivation, but inhibitors, and particularly tazobac-tam, retain efficacy against the Ser130Gly enzyme. Based on thesemechanistic roles, how does the Ser130Gly variant maintain hy-drolytic activity and why does it confer resistance to inhibitors(408)? In response to the first part of the question, evidence fromX-ray crystallography of SHV and TEM Ser130Gly (PDB 1TDLand 1YT4, respectively) confirmed that a relocated water mole-cule, initially predicted by Helfand et al., compensates for the lossof the Ser130 hydroxyl group by donating a proton to the �-lac-tam nitrogen (153, 157, 180, 408, 438). The decreased catalyticefficiencies seen in the TEM and SHV variants are likely a resultof perturbations caused by the repositioned water molecule.These perturbations vary somewhat between SHV and TEMSer130Gly (e.g., reorientation of Ser70 and Lys73 in SHV andTEM Ser130Gly, respectively), but both involve changes in hy-drogen bonding networks in the active site, which are modified toaccommodate the new water molecule (397, 408).

In regard to the second part of the question concerninginhibitor resistance, the data on Ser130Gly variants argue thatcross-linking is not essential for enzyme inactivation. Despiteincreased MICs for the inhibitors, the turnover numbers forclavulanate and tazobactam by SHV Ser130Gly are equivalentto those by the wild-type enzyme (153). This “paradox of in-hibitor resistance” illustrates that despite replacement of a keycatalytic residue, SHV Ser130Gly is still capable of inactivationby the mechanism-based inhibitors (153, 157, 311, 397). Theresistant phenotype presents primarily in whole-cell assays,where bacteria are exposed to a limited inhibitor concentra-tion. Structural perturbations created by the Ser130Gly substi-tution discourage formation of the preacylation complex (in-creased inhibitor KIs), which ultimately leads to fewerinactivation events (157). Thus, while the enzymatic machinery

for inhibition is intact, the decreased accumulation of acyl-enzyme intermediates and inactivation products can lead toinhibitor resistance in vivo.

Arginine 275. Similar to observations regarding Arg244 andAsn276, the Arg275 variant has been identified only in TEM.Before description of the IR variants TEM-103 and TEM-122,which possess only the Arg275Leu and -Gln changes, respec-tively, substitution at 275 was seen only in combination withthe Met69Val and -Leu substitutions (9, 198). Hence, the con-tribution of Arg275 to the IR phenotype was not appreciated,and rigorous kinetic and structural studies of this variant havenot yet been completed. However, from crystal structures ofthe wild-type enzymes and a better understanding of propertiesat nearby Arg244 and Asn276, Chaibi et al. postulated thatsubstitutions at Arg275 (like those at Asn276) disrupt localelectrostatic interactions and displace the water molecule thatis key to inactivation by clavulanate (74).

Lysine 234. The recent definition of two clinical IR SHV�-lactamases with Lys234Arg substitutions draws attention tothis important active-site residue (110, 258). Previously, TEMor SHV variants obtained from the clinic had not been iden-tified as having changes at this highly conserved class A resi-due. Site-directed mutagenesis of Lys234 to Arg in TEM-1leads to a 10-fold-decreased affinity for penicillins, presumablybecause of the hydrogen-bonding role of Lys234 to the C-3carboxylate (220) (Fig. 3). However, for the clinically isolatedSHV-72 enzyme (Ile8Phe, Ala146Val, Lys234Arg), Km valuesfor penicillins were not significantly changed and IC50s forclavulanate increased 10-fold compared to those of SHV-1(258). Interestingly, the kcat values for the penicillins wereincreased, up to 5.8-fold for ticarcillin. Molecular dynamicssimulations show that Lys234Arg induces movement of theSer130 oxygen away from Ser70. Mendonca et al. proposedthat the mechanism of IR in SHV-72 may be due to movementof Ser130 based on its role in both hydrolysis and terminalinactivation by cross-linking with Ser70 (258).

THE PROMISE OF NOVEL �-LACTAMASE INHIBITORS

This section reviews compounds that have demonstratedfavorable inactivation properties against �-lactamases and in-troduces newer compounds showing promise as “second-gen-eration” inhibitors. Table 8 provides Ki (or KI) values and IC50sfor representative inhibitors against the different �-lactamaseclasses to illustrate the challenge of achieving broad-spectruminhibition.

Monobactam Derivatives

Monocyclic, N-sulfonated �-lactams are a family of antibi-otic compounds produced by bacteria (178, 404, 405). Struc-ture-activity studies of these monobactams led to the develop-ment of the synthetic compound aztreonam, which interactedwith the PBPs of a wide range of aerobic Gram-negative bac-teria, including P. aeruginosa, and was introduced into clinicalpractice in 1984 (151, 168, 403). In addition, aztreonam (Fig. 1,compound 3) resists hydrolysis by many plasmid-mediated�-lactamases such as TEM-1 and -2, OXA-2, and SHV-1, be-having as a very poor substrate with low affinity (e.g., Km of 2.9



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mM for TEM-2) (403). With class C enzymes, such as E.cloacae P99, aztreonam forms a stable acyl-enzyme that is veryslowly hydrolyzed (low kcat) (128, 403). Aztreonam also exhib-its very low Ki values for chromosomally encoded cephalospo-rinases (e.g., 1.9 nM for P99) and serves as a transient inacti-vator (56, 128). In vivo, where periplasmic �-lactamaseconcentrations can be in the low mM range, clinically signifi-cant catalytic efficiencies may be obtainable (128).

Further modification of the monobactam nucleus was em-barked upon to capitalize on the inhibitory activity of aztreo-nam. The 1,5-dihydroxy-4-pyridone monobactam Syn 2190(Fig. 1, compound 11) possesses a novel C-3 side chain thatmay utilize the iron uptake pathway to enter Gram-negativebacteria. Syn 2190 displayed IC50s in the nM range for cepha-losporinases (e.g., 6 nM for E. cloacae P99) and synergy withceftazidime and cefpirome in MIC testing of clinical strains,including AmpC-derepressed variants of P. aeruginosa (15,289). In addition, Syn 2190 in combination with ceftazidime orcefpirome (at a 1:1 ratio) showed improved efficacy comparedto ceftazidime-tazobactam in murine models of systemic infec-tions with cephalosporin-resistant P. aeruginosa. Syn 2190 hasalso shown utility for the challenging clinical task of identifyingproduction of plasmid-mediated AmpC �-lactamase in Kleb-siella spp. In combination with cefotetan, Syn 2190 achieved100% specificity and 91% sensitivity for AmpC producers (28).However, Syn 2190 has lower affinity than tazobactam for classA enzymes and could not restore susceptibility to cephalospo-rins in bacteria expressing these enzymes. This limitationagainst class A enzymes may partly explain the absence offurther development of this compound.

The design of bridged monobactam derivatives was guidedby the crystal structure of the monobactam aztreonam in com-plex with the class C �-lactamase from Citrobacter freundii(PDB 1FR1) (152, 299). Studies of the structure demonstratedthat before deacylation, aztreonam had to rotate around theC-3–C-4 bond to allow a hydrolytic water access to the esterbond. This rearrangement requires breaking and reforming ofthe active-site hydrogen bond network and leads to rate-limit-ing deacylation (205). The introduction of a bridge between

C-3 and C-4 limits this rotation and stabilizes the acyl-enzymeagainst hydrolysis by AmpC enzymes (99, 171).

Heinze-Krauss et al. synthesized and evaluated of a panel ofbridged monobactams that exhibited very favorable inhibitionof the class C C. freundii and P. aeruginosa �-lactamases (IC50sof as low as 10 nM) but lower affinities for the class A TEM-3(IC50s of �100 �M) (152). At sufficiently high concentrations,class A �-lactamases were acylated rapidly, but the rate ofdeacylation was higher, leading to high hydrolysis rates and lowactive-site occupancy. Comparison of the crystal structures ofclass A TEM-1, PC1, the �-lactamase from Bacillus lichenifor-mis 749/C, and the class C �-lactamase from C. freundii re-vealed that in class A enzymes, hydrolysis occurs on the oppo-site side of the bridged monobactam ester by a water moleculeactivated by hydrogen bond networks not present in class Cenzymes (152, 166, 191, 267, 299, 392). Rotation about theC-3–C-4 bond is less critical to the inhibition mechanism inclass A enzymes, and restricting this rotation does less to sta-bilize the acyl-enzyme. For example, the bridged monobactamRo 48-1256 (Fig. 1, compound 12) (at 4 and 8 �g/ml) effec-tively potentiated the activity of imipenem against both induc-ible and derepressed class C-expressing P. aeruginosa isolates(238). The activities of piperacillin and ceftazidime were alsopotentiated by the combination with Ro 48-1256 against thestrains expressing elevated quantities of AmpC enzyme. How-ever, Ro 48-1256 did not improve the activity of piperacillin,ceftazidime, or carbapenems against organisms expressingclass A, B, or D �-lactamases.

Modification of the monobactam structure at the �-lactamnitrogen has had some success against class A �-lactamases(50, 171). Analysis of a series of N-sulfonyloxy-�-lactammonobactam derivatives demonstrated rapid acylation of theTEM-1 active site and resistance to deacylation, as evidentfrom turnover numbers ranging from 2 to 8 (50). Additionalanalysis of one of these derivatives (Fig. 1, compound 13)revealed that following enzyme acylation in the class A car-bapenemase NMC-A, the tosylate group is eliminated andgives rise to two acyl-enzyme species (273). These inhibited en-zyme species are trapped in local energy minima which yield low

TABLE 8. Kinetic properties of selected inhibitors against different �-lactamase Ambler classesa

Inhibitor (compound no. in Fig. 1) Kineticparameter

Value (�M) for Ambler class:Reference(s)

A B C D

Aztreonam (3) Km or Ki 200 �1,000 0.0019 56, 217, 337BAL30072 siderophore monobactam (14) IC50 1.7 �100 0.09 134ATMO-penicillin (15) IC50 0.90 418BRL 42715 methylidene penem (16) IC50 0.008 0.008 0.004 87BLI-489 methylidene penem IC50 0.009 0.062 294Novel tri- and bicyclic methylidene

penems (including 17 and 18)IC50 0.0004–0.0031 0.014–0.260 0.0015–0.0048 0.012 25, 441

LK-157 tricyclic carbapenems (20) IC50 0.055 0.062 344Ro 48-1220 penam sulfone (21) IC50 0.05–1.07 24–200 0.21–3.4 0.10–0.43 367, 422LN-1-255 penam sulfone (23) KI or IC50 0.100 0.026 0.70 63, 322; Drawz et al.,

unpublished dataBoronic acid meta-carboxyphenyl

cephalosporin analog (25)Ki 3.9 0.001 272, 411

NXL104 (26) IC50 0.008 0.080 31J-111,225 1-�-methylcarbapenem (33) Ki 11.1 0.18 0.93 281Phosphonates Ki 0.08–76 0.016–12.1 0.094 4, 244C-6-mercaptomethyl penicillnate (38) IC50 6.8 0.10 10.5 61




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deacylation rates. Based on molecular simulations, Mourey et al.postulated that the acyl-enzyme alternates between two differentconformations that move in and out of the oxyanion hole (273).This motion requires breaking and reforming of hydrogen bondsbetween the acyl-enzyme ester and carbonyl oxygen atoms andthe oxyanion hole backbone nitrogens and Ser130 oxygen atom.In both conformations, the water implicated in the deacylationreaction is positioned poorly for hydrolysis, offering a structuralexplanation for the irreversible inhibition.

A series of monobactam derivatives was recently developedto target pathogens harboring multiple �-lactamases. Some ofthese monobactam analogues act as very effective bactericidalagents for difficult-to-treat Gram-negative pathogens, whileothers are bridged monobactams, such as BAL29880, that areinhibitors of AmpC enzymes (99, 102, 116a, 317). Additionalderivatives include monobactams bearing a siderophore sidechain which can enhance cell entry through bacterial iron up-take systems (49). Of this siderophore type, BAL19764 andBAL30072 (Fig. 1, compound 14) have potent antibacterialactivity against carbapenem-resistant P. aeruginosa, Acineto-bacter spp., S. maltophilia, and S. marcescens as well as againstGram-negative bacilli expressing VIM and IMP �-lactamases(37, 102). Additionally, BAL19764 is stable to hydrolysis byMBLs (314). Further, these siderophore compounds are po-tent enzyme inhibitors; BAL30072 has low �M IC50s forTEM-3 (1.7 �M), and the AmpCs from C. freundii and P.aeruginosa (0.09 and 15 �M, respectively) (134). By combiningBAL19764 or BAL30072 with clavulanate and a bridgedmonobactam (all at a fixed concentration of 4 �g/ml), theMICs of the siderophore compounds were improved for arange of Gram-negative bacteria (including P. aeruginosa) pro-ducing AmpCs as well as carbapenemases or overexpression ofefflux systems (315, 316). However, the activity of carbapenemsis still superior to that of these new monobactams for manyESBL-producing isolates (e.g., K. pneumoniae CTX-M andSHV-5, E. coli, and E. cloacae) (38, 102, 173). The dual anti-bacterial/inhibitory action of these monobactam derivativesmakes them important leads, and the further development ofthe siderophore compound BAL30072 seems likely.

ATMO Derivatives

Certain cephems adopt catalytically incompetent conforma-tions in the active site, effectively “trapping” the enzyme.Evidence from the crystal structure of an AmpC enzyme com-plexed with ceftazidime (PDB 1IEL) suggested that the pres-ence of the 2-amino-4-thiazolyl methoxyimino (ATMO) R1

side group, which is common among extended-spectrum ceph-alosporins (e.g., cefotaxime and ceftazidime), destabilized theformation of the deacylation transition state (350). Synthesis ofinhibitors containing this ATMO side chain on a penicillin(Fig. 1, compound 15) and carbacephem backbone led toAmpC IC50s of 900 nM and 80 nM, respectively (418). Subse-quent examination of the crystal structure of the ATMO-pen-icillin in complex with the AmpC (PDB 1LLB) showed that itresembled the acyl-enzyme formed by ceftazidime. TheATMO groups were located in very similar positions, in bothcases leading to steric hindrance between the deacylating waterand the �-lactam ring nitrogen, accounting for inhibition. Thisdesign highlights the potential of incorporating destabilizing

groups onto conserved substrate skeletons, which will facilitaterecognition by the enzyme (418).

Penems

BRL 42715 and Syn 1012. Methylidene penems are potentinhibitors in which the inhibitory properties of the penemstructure are enhanced by introduction of a double bond at C-6and a sulfide at penem position 1 (260). These compoundsoffer advantages over the traditional �-lactamase inhibitors,including (i) a large R1 side chain that may aid in cell perme-ability and contribute to active-site affinity and (ii) a novelinactivation mechanism. The parent compound from thisgroup, BRL 42715 C-6-(N1-methyl-1,2,3-triazolylmethyl-ene)penem (Fig. 1, compound 16), is an effective inhibitor ofclass A, C, and D �-lactamases (119, 250). IC50s of BRL 42715were 10- to 100-fold lower than those of clavulanate, tazobac-tam, and sulbactam for class A TEM-1 and SHV-1, class C P99,class D OXA-1, and the S. aureus �-lactamase NCTC 11561(87). However, BRL 42715 was a substrate for class B metallo-�-lactamases from Aeromonas hydrophila and S. maltophiliaand for BcII (250). In susceptibility testing, BRL 42715 con-centrations of 0.25 �g/ml or lower were able to restore amoxi-cillin MICs to �16 �g/ml for TEM-1- and OXA-1-producingorganisms (87). Further, amoxicillin-BRL 42715 combinationswere more effective than amoxicillin-clavulanate for E. coli, K.pneumoniae, H. influenzae, S. aureus, and N. gonorrhoeaestrains producing plasmid-mediated �-lactamases (mostlyTEM and SHV expressors) and for K. oxytoca, Proteus vulgaris,and P. mirabilis strains producing chromosomal �-lactamases.Kinetic studies of BRL 42715 and NCTC 11561, TEM-1, andP99 showed rapid, irreversible inactivation with an inhibitionstoichiometry of 1:1, or turnover numbers of 0 (119).

Initial studies to elucidate the BRL 42715 inactivation productusing UV difference spectroscopy for both enzyme- and base-catalyzed hydrolysis of the methylidene penem suggested thepresence of a dihydrothiazepine rearrangement product (46, 250).Accordingly, mass spectrometry studies demonstrated that themethylidene penem inhibition pathway does not involve fragmen-tation in the active site (119, 250, 406). When TEM-1, P99, andthe class A �-lactamase from B. cereus I were inactivated by BRL42715, spectra showed a mass increases equivalent to the molec-ular masses of the inhibitor plus the enzyme (119). The crystalstructure of the class C �-lactamase E. cloacae 908R in complexwith BRL 42715 (PDB 1Y54) confirmed the formation of a stableseven-membered thiazepine ring covalently attached to Ser64(263). Studies of both BRL 42715 and related penems support themechanism involving formation of an imine intermediate fromthe acyl-enzyme, which then undergoes rearrangement, and even-tually endo-trig cyclization to form the seven-membered thiaz-epine ring end product of inactivation for class A, C, and Denzymes (Fig. 10) (250, 263, 294, 428).

Crystallization of BLI-489, a related heterocyclic O C-6-substi-tuted penem with IC50s of 9.0 and 6.2 nM for SHV-1 and class CGC1, respectively (see below), revealed the common seven-mem-bered thiazepine ring when complexed with the SHV-1 and GC1enzymes (PDB 1ONG and 1ONH, respectively) (294). Interest-ingly, the orientation of the ring in SHV-1 and GC1 differed by180° about the bond to the acylated serine. This rotameric pref-erence may be due to the relative “openness” of the class C



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binding site at the bottom of the b3 �-sheet. Crystallization of atricyclic 6-methylidene derivative in complex with SHV-1 andGC1 (PDB 1Q2P and 1Q2Q, respectively) also showed the com-mon seven-membered thiazepine ring covalently attached toSer70 and Ser64, respectively (429). The SHV-1/penem complexwas similar to what was observed with BLI-489, where the ringhad R stereochemistry at the new C-7 moiety (294). However, inthe GC1/penem complex, both the R and S chiral forms of theintermediate were found.

Taken together, these findings suggest that the 6-methylidenepenems’ acyl-enzyme stability is likely due to the low occupancyor disorder of the hydrolytic water molecule and, in part, to theconjugation of the ester with the ring system. The nucleophilicattack of the double bond at C-6 appears to be important to theinhibition mechanism of the methylidene penems, and thesenovel inhibitors seem to share a similar inactivation mechanismfor different �-lactamase classes (429).

Most �-lactamases appear to follow this linear pathway toinactivation by the penem compounds, i.e., formation of anacyl-enzyme and rearrangement to the stable dihydrothia-zepine ring adduct (119, 250). However, some class A en-zymes, such as the �-lactamases from Staphylococcus albus(Staphylococcus epidermidis) and K. pneumoniae K1, exhibitmore complex kinetics (46, 250). A branched pathway leadsto an acyl-enzyme hydrolysis product, which also rapidlyrearranges to the dihydrothiazepine species.

While BRL 42715 and the structurally related investiga-tional penem Syn 1012 showed promising in vitro kineticsagainst a large spectrum of Gram-positive and Gram-negativeisolates, they were relatively unstable in human and mouseplasma (342). Further, serum levels of BRL 42715 and Syn1012 were undetectable 1 h after intravenous administration ina rabbit model. Thus, the successful elements of BRL 42715have been applied to additional drug development efforts di-rected at improving in vivo stability (see below).

BRL 42715 derivatives. Derivatives of BRL 42715, includingtricyclic and bicyclic heterocyclic methylidene penems, have beenthe focus of a large body of recent structure-activity studies andcontinue to show promising inhibition properties in investiga-tional studies. The bicyclic heterocycles are synthesized as6-methylidene penems attached to thiophene, imidazole, and pyr-azole rings (428). Extension of the second heterocyclic ring yieldsthe tricyclic derivatives, which show improved stability in solutionand increased lipophilicity in in vivo studies (429). As a group,these compounds have proven to be potent inhibitors of class A,C, and D �-lactamases (25, 156, 335, 428, 441).

A panel of one tricyclic and six bicyclic penem compoundsdisplayed IC50s of 0.4 to 3.1 nM for TEM-1 (class A), 7.8 to 72nM for IMI-1 (class A), 1.5 to 4.8 nM for AmpC (class C), and14 to 260 nM for a CcrA (class B) (441). Compared to tazobac-tam IC50s, these penems were 100- to 56,000-fold more activeagainst TEM-1 and the AmpC enzymes. Each of the sevenpenems, at a constant concentration of 4 �g/ml, was combinedwith piperacillin, which significantly lowered MICs in ESBLproducers and restored susceptibility in piperacillin-resistantEnterobacter spp., Citrobacter spp., and Serratia spp. In a mousemodel of infection caused by E. coli and Enterobacter aerogenesclass A (including ESBL) and C �-lactamase-producing organ-isms, piperacillin-penem combinations decreased the overall50% effective dose (ED50) of piperacillin from 1.6- to 8-fold(441). However, class B metallo-�-lactamase producers werenot reported for susceptibility tests or the murine model, andthey warrant further attention for clinical translation.

Of the panel of tricyclic and bicyclic penem compoundstested, the bicyclic BLI-489 was chosen as a lead compound tobe combined with piperacillin (335). With 4 �g/ml of BLI-489,92% of non-piperacillin-susceptible clinical strains (including ex-pressors of representative �-lactamases from all four classes) hadpiperacillin–BLI-489 MICs of �16 �g/ml, in contrast to 66% forpiperacillin-tazobactam. Furthermore, the BLI-489 combination

FIG. 10. Proposed reaction mechanisms for the inactivation of a serine �-lactamase by BRL 42715, showing formation of the seven-memberedthiazepine ring (A), and by LN-1-255, showing intermolecular capture by the pyridyl nitrogen leading to a bicyclic aromatic intermediate (B).(Based on data from references 263, 294, 322, and 428.)



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demonstrated improved activity over piperacillin-tazobactamagainst ESBL- and AmpC-expressing strains, as well as against apanel of staphylococcal and streptococcal isolates. Notably, BLI-489 did not improve the activity of piperacillin against the entero-cocci and penicillin-intermediate and -resistant strains of S. pneu-moniae (which may reflect that the piperacillin resistancemechanism is not �-lactamase mediated) (335). While these prac-tical studies help to bring closer the possible clinical introductionof these novel inhibitors, only a small number of the isolates wereKPC or IRT producers. These KPC- and IRT-producing strainsalso expressed up to three other �-lactamases, which complicatesthe interpretation of activity against these enzymes. Thus, onecannot yet endorse the efficacy of this inhibitor combination forthese relevant resistance threats.

Two of these bicyclic investigational penems (Fig. 1, com-pounds 17 and 18), containing either a dihydropyrazolo[1,5-c][1,3]thiazole or a dihydropyrazolo[5,1-c][1,4]thiazine moiety,are extremely effective inactivators of the class D OXA-1 �-lac-tamase, with nM affinity and low turnover values compared totazobactam (tn 0 and 350, respectively) (25). Additionally,these penems demonstrate efficacy with IR class A �-lactama-ses. Against the SHV Arg244Ser variant, the penem inhibitorsshowed significantly lower KI values than clavulanate and sul-bactam (penems 1 and 2, 1.0 to 4.4 �M; clavulanate and sul-bactam, 63 and 240 �M, respectively) and extremely low turn-over numbers (tn 0, 50, and 100 for the penems, clavulanate,and sulbactam, respectively) (410).

Oxapenems. Oxapenems are derivatives of clavulanic acid,possessing an oxygen atom at penem position 1. Their synthesiswas first described in the 1970s, and Cherry et al. introduced anoxapenem that had improved in vitro activity over clavulanateagainst the E. cloacae AmpC and staphylococcal �-lactamases(86). However, the compound was unstable and did not haveactivity in whole-cell assays. In 1993, Pfaendler et al. improvedthe stability of the compounds by adding bulky substituents atthe C-2 position (339). The lead compound from this panel ofoxapenems, the zwitterionic AM-112 (Fig. 1, compound 19),has shown potent �-lactamase-inhibitory properties, particu-larly for class C and D enzymes (189, 190). Against class Aenzymes, IC50s were 10- to 20-fold higher for AM-112 com-pared to clavulanate (190). However, the IC50s of AM-112 forthe class C enzymes from E. cloacae, S. marcescens, and P.aeruginosa, as well as OXA-1 and OXA-5 from E. coli, were1,000- to 100,000-fold lower than those of clavulanate. AM-112in combination with ceftazidime at 1:1 or 1:2 reduced ceftazi-dime MICs against class A ESBL producers and the class Cenzyme hyperproducers. For the class D enzyme producersand P. aeruginosa strains tested, AM-112 did not enhance theactivity of ceftazidime. The susceptibility results were similarfor AM-112’s protection of cefepime, ceftriaxone, and ce-foperazone (189). In a mouse sepsis model, ceftazidime pro-tection was maintained against E. cloacae P99 and K. pneu-moniae SHV-5 (190). The preclinical development of AM-112has been led by Amura Ltd., and it warrants further examina-tion as a potential multi-class �-lactamase inhibitor.

Tricyclic carbapenems (trinems). Rational structure-baseddesign informed the synthesis of novel tricyclic carbapenemsbuilt by the fusion of a cyclohexane ring onto a carbapenemscaffold (90, 91). LK-157 (Fig. 1, compound 20) was derived byintroduction of a methoxy substituent at position C-4 and

shows nM affinity for both the class A TEM-1 and class C E.cloacae 908R �-lactamases (431). The IC50s of clavulanate andLK-157 for TEM-1 were similar (0.030 �M and 0.055 �M,respectively), but that of LK-157 was over 2000-fold better forE. cloacae AmpC (136.2 �M and 0.062 �M, respectively)(344). In combination with ampicillin, LK-157 (at 30 �g/ml)restored susceptibility for the AmpC-overexpressing E. cloacaeP99 strain in MIC testing (431). Against a large panel ofbacteria producing class A ESBLs (excepting KPC andCTX-M) and class C �-lactamases, LK-157 in combinationwith cephalosporins showed improved potency compared toclavulanate, tazobactam, and sulbactam (324). The lowestMICs (ranging from �0. 025 to 1.6 �g/ml) were achieved forcefepime or cefpirome in combination with LK-157 at 4 �g/ml.Despite these promising data with class A and C �-lactamase-producing Gram-negative bacteria, LK-157 behaves as a sub-strate with the carbapenem-hydrolyzing enzymes BcII andNMC-A (431). Further, while the inhibitor displays nM affinityfor and rapidly inactivates the class D OXA-10 �-lactamase,the acyl-enzyme is unstable and rapidly hydrolyzed (431).

The crystal structure of LK-157 in complex with the E. clo-acae P99 (PDB 2Q9N), together with spectroscopic data forreaction intermediates, suggests that after deacylation at theactive-site Ser64, the C-4 methoxy group is eliminated (344,431). Further, the catalytic water molecule presumed to beresponsible for deacylation in class C enzymes is not observedin the AmpC/LK-157 crystal structure. Rotation about theopened �-lactam ring after acylation likely leads to displace-ment of this water molecule and subsequent enzyme inhibition(344). The C-10 ethyldiene group is probably not bulky enoughto induce the conformational change seen with inhibition ofclass A enzymes by carbapenems, but resonance stabilizationof the acyl-enzyme through the acyl carbonyl and ethyldienegroup may contribute to stability of the intermediate (255,344). Trinems are worthy of attention for the potential to serveas leads for additional structure-based inhibitor design, andLek Pharmaceuticals continues to investigate LK-157. Devel-oped for parental use, the half-life (t1/2) of LK-157 in rats issimilar to those of other �-lactams after intravenous adminis-tration (354). Additional studies have focused on developingan oral agent with improved permeability over LK-157, andwhile prodrug esters have good solubility, the intestinal per-meability remains low.

1-�-Methylcarbapenems. A panel of carbapenem deriva-tives bearing a methyl group at C-1 and various substituents atC-2 were screened for inhibitory activity against the MBLIMP-1 (282). The study revealed a compound, J-110,441, thathad potent inhibitory activity against several MBLs but alsolow Ki values for class A TEM and class C E. cloacae enzymes(2.54 �M and 0.037 �M, respectively). Susceptibility testing ofthe AmpC-expressing E. cloacae demonstrated an MIC de-crease from 64 to 4 �g/ml with imipenem and J-110,411 at 4�g/ml. Further work with these carbapenem derivatives hasfocused on their potential as MBL inhibitors and will be dis-cussed below (see Inhibition of Metallo-�-Lactamases).

Penicillin and Cephalosporin Sulfone Derivatives

Following the success of sulbactam and tazobactam, medic-inal chemists have focused much effort on the development of



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penicillin and cephalosporin sulfones with functionalities thatmay improve inhibitor efficiency. One of the first series ofinvestigational sulfones, the C-7 alkylidene cephems, had leadcompounds that were potent inhibitors of class C �-lactamases(65). Subsequent reports have explored the roles of C-2 andC-6 penam and C-3 cephem substituents. The findings for eachwill be discussed in the context of the derivative group.

C-2/C-3-substituted penicillin and cephalosporin sulfones.�-Lactamase inhibitors with C-2 substitutions have shown ef-ficacy against TEM- and SHV-type enzymes, including ESBLs(367, 422). The acrylonitrile derivative Ro 48-1220 (Fig. 1,compound 21) achieves an IC50 for TEM-1 (0.08 �M) that iscomparable to that of clavulanate (0.10 �M), but the IC50 forSHV-1 (0.27 �M) is slightly higher than that of clavulanate(0.05 �M) (422). Inhibition with Ro 48-1220 was as effective astazobactam for TEM-1 and SHV-1, with IC50s within 0.03 �Mfor both enzymes. Most class C enzymes tested were inhibitedat lower concentrations of Ro 48-1220 than of tazobactam(IC50 range approximately 2- to 30-fold lower) (367, 422). TheIC50s for X. maltophilia and Bacteroides fragilis class B enzymeswere 24 and 200 �M, in comparison to 4 and �10 mM fortazobactam (367). Ro 48-1220 (at 4 �g/ml) performed well insusceptibility testing, protecting the activity of ceftriaxone andceftazidime against class C- and ESBL-producing organisms(422). Central to the potency of this sulfone inhibitor was thelow enzyme recovery rate compared to tazobactam, especiallyfor class C �-lactamases from C. freundii and E. coli (367). UVabsorption spectroscopy and X-ray crystallography revealedthe identity of a linear enamine formed by elimination of theSO2 group, ring opening, and rearrangement into a linearconjugated system (367). This particularly stable intermediatepresumably accounted for enhanced inhibitor potency.

The design of the C-2-substituted penicillin sulfone SA2-13(Fig. 1, compound 22) drew on the insight that the tazobactamimine is the common intermediate, or “gatekeeper,” in bothpermanent interaction and enzyme regeneration (58, 309, 453).If the more stable enamine, the trans-enamine, can be favored,then the reaction will be “trapped” in transient inhibition. Infact, IR enzymes do exhibit decreased trans-enamine forma-tion, as in the case of the SHV Met69Val/Glu166Ala variantreacted with tazobactam and clavulanate (416). SA2-13 in-cludes a negatively charged carboxyl group attached to a linkerwhich is designed to stabilize the trans-enamine conformationin close proximity to conserved active-site residues. Interest-ingly, the crystal structure of SA2-13 in complex with SHV-1(PDB 2H5S) showed an additional salt bridge with Lys234 andhydrogen bonds with Ser130 and Thr235. Compared to SHV-1inhibited by tazobactam, SA2-13 had a 10-fold-lower deacyla-tion rate, presumably mediated by the stabilization of the trans-enamine species (309). However, the dissociation constant forSA2-13 was 17-fold lower than that for tazobactam (0.1 versus1.7 �M, respectively), and there was 47% 24-hour recovery ofactivity of the SA2-13/SHV-1 mixture versus complete inacti-vation with tazobactam.

The 6-alkylidene-2�-substituted penicillin sulfone LN-1-255(Fig. 1, compound 23), attains nM KIs for the class A SHV-1 andESBL SHV-2 and class D oxacillinase-, ESBL-, and even carbap-enemase-type OXA enzymes (63, 322) (S. M. Drawz et al., un-published data). Moreover, LN-1-255 restores the activity of pip-eracillin against E. coli IR SHV enzyme producers and of

ceftazidime and cefpirome against E. coli and K. pneumoniaeexpressing CTX-M, OXA, and CMY �-lactamases (63, 322).Meropenem MICs were lowered from 32 to 1 �g/ml with 4 �g/mlof LN-1-255 against S. marcescens possessing SME-1. The C-3hydrophobic catechol moiety of this investigational compoundappears to improve both its entry into cells via siderophore ironchannels and its affinity for �-lactamase active sites (75).

X-ray crystallography of LN-1-255 in complex with SHV-1(PDB 3D4F) revealed that the inhibitor’s C-6 (heteroaryl)alkylidene group plays an important role in the formation of aplanar bicyclic aromatic intermediate (322). The pyridyl nitro-gen of the C-6 substituent acts as a base and promotes intramo-lecular capture, leading to the formation of a bicyclic species(Fig. 10B). The acyl-enzyme ester carbonyl of the intermediateis resonance stabilized by the conjugated � system. Addition-ally, the carbonyl group of the intermediate is positioned out-side the oxyanion hole. The decreased deacylation rates of thisspecies are likely due to resonance stabilization and the loca-tion of the carbonyl, at an increased distance from the hydro-lytic water and improperly positioned for nucleophilic attack(322).

By expanding the five-membered penicillin sulfone design toa six-membered cephalosporin sulfone, Buynak et al. devel-oped synthetic methodologies allowing modification of C-3substituents (62, 64). The different functional groups intro-duced at C-3 had significant impact on whether the inhibitorwas selective for either class A or class C �-lactamases, withsome features, such as electronegativity, increasing inhibitionof both classes. DVR-II-41S (Fig. 1, compound 24) has boththe C-7 (heteroaryl)alkylidene group of LN-1-255 and a C-3vinyl substituent, similar to nitrocefin, an excellent substratefor nearly all classes of �-lactamases (62, 64). This cephalo-sporin sulfone demonstrated IC50s of 90 nM for class A TEM-1and 10 nM for class C GC1. The crystal structure of GC1/DVR-II-41S (PDB 1GA0) revealed a bicyclic aromatic systemreminiscent of the LN-1-255 intermediate (94). Additional sta-bility results from the placement of the inhibitor’s anionicsulfinate group between Tyr105 and the acyl-enzyme bond,likely blocking the approach of the deacylating water molecule.

C-6-substituted penicillin sulfones. C-6-substituted penicil-linates are nM to low �M inhibitors of serine �-lactamases,with the sulfone oxidation state at the penam thiazolidinesulfur showing improved affinities over the sulfide state (27, 61,374, 375). The stereochemistry and specific functionality of theC-6 substituents can affect the specificity of inhibition. Forexample, � isomer alkenyl derivatives, as opposed to � isomerderivatives, show improved IC50s for class B �-lactamases, andderivatives with thiazole rings, particularly with fluorine,amino, or hydroxyl groups, show potency for class A �-lacta-mases (374). Further, compounds with a mercaptomethylgroup at C-6, as opposed to hydroxymethyl, are better inhibi-tors of class B enzymes, while C-6-mercapto-penicillinates arerelatively less active inhibitors of classes A, B, and C (61).

The C-6-hydroxyakylpenicillinates have been employed astools for studying the mechanism of �-lactamase hydrolysis andinhibition (143). The crystal structure of 6-�-hydroxymeth-ylpenicillanate complexed to TEM-1 (PDB 1TEM) suggeststhat while C-6-substituted penicillin sulfones are acylated inthe same way as �-lactam substrates, the hydroxymethyl groupat C-6 impedes the approach of the hydrolytic water and pro-



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longs the lifetime of the acyl-enzyme species, effectively inhib-iting the enzyme (254). However, the same compound acted asa substrate for the class A NMC-A. Crystal structure determi-nation of NMC-A complexed with 6-�-[(1-hydroxy-1-methyl)ethyl]penicillanate (PDB 1BUL) showed that the repositioningof residue Asn132 in NMC-A, compared to TEM-1, enlargedthe substrate binding cavity and allowed Asn132 to make newhydrogen bonds with the C-6 substituent (274). These interac-tions permit approach by the hydrolytic water and facilitatedeacylation of the acyl-enzyme intermediate for the 6-�-hy-droxymethylpenicillanate and C-6-substituted carbapenemssuch as imipenem in NMC-A. Upon introduction of a bulkierC-6 hydroxypropyl group, deacylation was impeded, in a mech-anism very similar to that for the inhibition of TEM-1 byimipenem (407).

Generalization of these and other findings leads to the con-clusion that when the hydrolytic water molecule approachesthe acyl-enzyme from the “� direction,” as with class A en-zymes, �-hydroxyalkylpenicillinates are effective inhibitors.Conversely, �-hydroxyalkylpenicillinates inhibit the “� ap-proach” in class C enzymes (143). A group at Wyeth Researchfound that a 6-�-hydroxymethyl penicillin sulfone has the bestactivity against both class A and C enzymes, with IC50s of 8 nMfor TEM-1 and 1.2 �M for AmpC (26). Presumably, the in-hibitor worked like tazobactam or sulbactam in class A en-zymes, but the specific crowding of the �-face of the esterprevents approach of the hydrolytic water in class C �-lacta-mases (51). The class D OXA-10 enzyme showed inhibition byboth C-6 �- and �-hydroxyisopropylpenicillinates (252). Whileonly the �-isomer could be crystallized with OXA-10 (PDB1K54), this structure and computational models of the �-iso-mer suggest related, but different, mechanisms of inactivationbased on the C-6 stereochemistry of the inhibitor. However,both mechanisms involved displacing the hydrolytic water nec-essary for deacylation.

Non-�-Lactam Inhibitors

Boronic acid transition state analogs (TSAs). In the 1970s,boronic acid compounds were described as forming reversible,dative covalent bonds with serine proteases and inhibitingthese enzymes by assuming tetrahedral reaction intermediates(30, 230). Kiener and Waley then applied the chemistry to theclass A �-lactamase from B. cereus and showed that boronicacid derivatives, as well as the phenyl and m-aminophenylderivatives, were mM inhibitors of the enzyme (201). Furtherstudy by other groups demonstrated that these compounds,which lack the �-lactam motif, form adducts that resemble thegeometry of the tetrahedral transition state of the �-lactamasehydrolytic reaction (22, 82, 95) (Fig. 11). By modifying thesetransition state analogs to contain the R1 side chains of naturalsubstrates, affinities in the nM range for both class A TEM-1and ESBL-type SHV and class C enzymes have been deter-mined (70, 392, 394, 412). Weston et al. used the crystallo-graphic structure of E. coli AmpC �-lactamase in complex withm-aminophenylboronic acid (PDB 3BLS) as a model for de-signing boronic acid-based inhibitors specifically for the class Cactive site (425, 443). The compound showing the greatestaffinity was benzo[b]thiophene-2-boronic acid, which had a Ki

for the E. coli AmpC of 27 nM.In addition to their demonstrated potential as effective in-

hibitors, these compounds have been used to probe the impor-tant interactions between the inhibitor and the enzyme activesite, thereby informing the design of future TSAs. Using theindividual crystal structures for nine TSAs and four �-lactamscomplexed with the E. coli AmpC �-lactamase, computationalanalyses have generated consensus binding pockets in theAmpC active site (352). The functionalities recognized by cer-tain residues led to the development of new glycylboronic acidTSAs, including a compound containing the R1 side chain ofcephalothin. One of these cephalothin analogs achieved a Ki of1 nM for the E. coli AmpC (Fig. 1, compound 25), a 300-foldimprovement over the parent compound, which lacked themeta-carboxylphenyl substituent (272). The substituent was de-signed to resemble both the dihydrothiazine ring and the C-4carboxylate of the cephalosporin nucleus. In the crystal struc-ture of this cephalothin meta-carboxyphenyl analog complexedwith an AmpC (PDB 1MY8), the inhibitor carboxylate inter-acted with Asn289, a residue not previously implicated in struc-tural or modeling studies as interacting with the cephalosporin’sC-4 carboxylate. Asn289 is not a highly conserved residueamong class C �-lactamases, but the low Ki is maintained whendifferent AmpCs with other residues at position 289 are tested(e.g., Kis of 29 nM for E. cloacae P99 and 11 nM for A.baumannii (107a, 241, 371). The versatility of this inhibitor mayreflect an important flexibility in AmpC active sites that couldaffect the recognition of both substrates and inhibitors.

High-affinity inhibitors will be clinically useful only if theycan effectively permeate the bacterial cell membrane and re-store susceptibility to partner �-lactams. The boronic acid de-rivatives enter both S. aureus producing class A �-lactamasesand Enterobacteriaceae such as E. coli, C. freundii, E. cloacae,and P. aeruginosa expressing class C �-lactamases, and theyeffectively protect ampicillin and ceftazidime (70, 272, 349,430, 436). Boronates have not yet been developed for clinicaluse in combination with a �-lactam, in part because of con-cerns about the toxicity of boron. The safety of boron-contain-ing compounds is currently being addressed by clinical studiessponsored by Anacor Pharmaceuticals. Anacor develops boroncompounds for medical uses and has current trials for boron-

FIG. 11. Scheme illustrating the interactions of a serine �-lacta-mase with a cefotaxime substrate (A) compared to a cefotaxiime-likeboronic acid TSA (B). Notice that the reaction with the inhibitor isreversible.



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based topical treatments of fungal, bacterial, and anti-inflam-matory diseases, as well as research stage data on a systemicantibacterial agent.

Phosphonates. Phosphonate monoester derivatives canacylate the active-site serines of class A, C, and D �-lactama-ses, leading to effective inhibition (4, 223, 243, 244, 353). Thesecompounds exhibit branched kinetic pathways, in some casesreflecting inhibition by the products that are formed. Acylphosphonates inhibit class C enzymes, while cyclic acyl phos-phonates inhibit both class A and C �-lactamases. For diacylphosphonates, hydrophobic substituents decrease the inhibi-tor’s Ki value, and the acylation rates of these compoundsfollow the order class D � class C � class A (243, 244).Majumdar et al. posit that this relative inhibitor efficiencyreflects the general hydrophobicity of the enzyme active sitesand that rational design of diacyl phosphate side chains couldlead to nM inhibition of all serine �-lactamase classes.

Crystal structures with these phosphonic acids have also offeredimportant insight into the reaction mechanisms of class C en-zymes (297). The ability of the E. cloacae GC1 �-lactamase tohydrolyze extended-spectrum cephalosporins was elucidated bytrapping of the enzyme in complex with a phosphonate cefo-taxime transition state analog (297). A three-residue insertioninto the �-loop of the enzyme, compared to the C. freundii �-lac-tamase/transition state analog complex, allowed for a better fit ofcefotaxime’s side chain in the enzyme active site and facilitatedthe activation of the hydrolytic water molecule. The clinical po-tential of phosphonates has been limited by their poor stability inaqueous solution and susceptibility to phosphodiesterases.

NXL104. NXL104 (also known as AVE1330A) (Fig. 1,compound 26) is a bridged diazabicyclo[3.2.1]octanone non-�-lactam inhibitor with very promising activity against classA, C, and D �-lactamases (31, 116, 240, 390). The majorityof the published work on NXL104 describes MIC testing,although IC50s of 8, 38, and 80 nM have been reported forclass A TEM-1 and KPC-2 and class C P99 enzymes, respec-tively (31, 390). A remarkable property of NXL104 is theprolonged deacylation rate. The 50% enzyme recovery timepoint was 7 days for both TEM-1 and P99 (compared to 7min for TEM-1 and clavulanate and 290 min for P99 andtazobactam), suggesting the presence of a very stable andlong-lived intermediate species (31). The formation of acovalent complex has been supported by an ESI-MS study ofTEM-1 and NXL104 (391). Currently, the only reportedcrystal structure of the inhibitor is in complex with CTX-M-15, and it shows opening of the NXL104 ring upon acylation,interaction with several conserved active-site residues, anddisplacement of the putative deacylation water by approxi-mately 1 Å (104).

With ceftazidime as a partner �-lactam in a 4:1 ratio(ceftazidime at 4 �g/ml and NXL104 at 1 �g/ml), NXL104lowered the MICs for a series of Enterobacteriaceae strainsat least 8-fold compared to those of ceftazidime alone, to �4�g/ml for E. coli, K. pneumoniae, Citrobacter diversus, and P.mirabilis strains and to 2 �g/ml for E. cloacae and Serratiasp. strains (31). Against class A �-lactamase producers (pri-marily TEM and SHV types), the ceftazidime-NXL104 com-bination was comparable to ceftazidime-clavulanate butsuperior to amoxicillin-clavulanate and piperacillin-tazobac-tam. Ceftazidime-NXL104 was more potent than piperacil-

lin-tazobactam for class C enzyme-producing Enterobacteri-aceae, including both ceftazidime-resistant and -susceptiblestrains of C. freundii and E. cloacae. The NXL104 concen-tration of 4 �g/ml restored MICs into the susceptible rangefor a collection of clinical P. aeruginosa isolates (21% non-susceptible to ceftazidime versus 6% nonsusceptible toceftazidime-NXL104) (380). In in vivo murine studies, in-cluding models of sepsis with CTX-M Enterobacteriaceaeproducers, pneumonia with AmpC and SHV-11 K. pneu-moniae producers, and thigh infection and sepsis withKPC-2, TEM-1, and SHV-11 K. pneumoniae producers, theceftazidime-NXL104 combination has demonstrated prom-ising therapeutic efficacy (221, 259, 442).

NXL104 also enhanced the activity of cefotaxime in a studyof Enterobacteriaceae expressing CTX-M, KPC, or OXA-48,where 4 �g/ml of the inhibitor lowered MICs to �1 �g/ml. Forisolates with ertapenem resistance due to combinations of�-lactamases (ESBLs or AmpCs) and impermeability, theMICs were still �2 �g/ml. While cefotaxime-NXL104 loweredthe MIC of one S. marcescens isolate expressing SME-1 from0.5 to 0.12 �g/ml, MICs for K. pneumoniae and E. coli express-ing class B IMP and VIM metallo-�-lactamases were not low-ered by either cephalosporin with NXL104.

Two recent studies examined more closely the ability ofNXL104 to restore susceptibility to �-lactams for KPC en-zyme-producing clinical isolates (116, 390). Stachyra et al.demonstrated that NXL104 at 4 �g/ml restored susceptibilityof six KPC-2 or -3 producers to piperacillin, ceftazidime, ceftri-axone, and imipenem (390). Endimiani et al. tested NXL104 atvarious concentrations (1, 2, and 4 �g/ml) against a panel of 42K. pneumoniae isolates with �3 bla genes each and MIC90s of�128 �g/ml for all �-lactams tested (116). All of these K.pneumoniae strains were susceptible to NXL104 at 2 �g/mlwith cefotaxime, ceftazidime, cefepime, and aztreonam and at4 �g/ml with piperacillin. Collectively, these data suggest thatNXL104, in combination with several cephalosporins, aztreo-nam, and even imipenem, may be an excellent candidate forrestoring susceptibility to difficult-to-treat emerging carbapen-emase- and ESBL-producing organisms.

NXL104, manufactured by Novexel Inc., is currently inphase II clinical trials in combination with ceftazidime fortreatment of complicated urinary tract and intra-abdominalinfections (www.clinicaltrials.gov). Additionally, Forest Labo-ratories Inc. intends to begin phase I clinical trials withNXL104 in combination with the novel broad-spectrum ceph-alosporin ceftaroline (which is currently in phase III trials asmonotherapy for complicated skin infections and community-acquired pneumonia). The results of clinical trials are awaited.

Hydroxamates. O-Aryloxycarbonyl hydroxamate is an irre-versible inhibitor of the class C E. cloacae P99 �-lactamase thatutilizes a mechanism of action which differs from those of thecurrently available inhibitors (Fig. 1, compound 27) (330, 450).The proposed inhibition mechanism involves rate-limitingacylation of the hydroxamate. However, the inhibitor is stabi-lized not by the traditional oxyanion hole comprised of thebackbone nitrogens of Ser64 and Ser318 but rather by the sidechains of Tyr150 and Lys315 (330). As with clavulanate andtazobactam, the acyl-enzyme proceeds to either hydrolysis orinactivation. The inactivation mechanism leads to aminolysis ofLys315 and a covalent cross-linking of Ser64 and Lys315, as



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seen in the crystal structure of the inhibited P99 (Fig. 12) (PDB2P9V) (450). TEM-2 and OXA-1 �-lactamases were also in-hibited by O-aryloxycarbonyl hydroxamates, and the MBLGIM-1 was inhibited by a “reverse” hydroxamate (hydroxy-lamino replacing hydroxylamine) conjugated to a cephalospo-rin nucleus (131, 330). Additional mechanistic studies of hy-droxamates and their derivatives, as well as in vitro activitydata, will be of much interest.

Other non-�-lactam inhibitors. Work by Conrath et al. ex-plored the �-lactamase-inhibitory potential of soluble frag-ments derived from the variable region of heavy-chain drom-edary antibodies (89). IC50s of 35 �M and 300 �M wereobtained for TEM-1 and the B. cereus MBL BcII, but thedevelopment of this strategy would require innovative tech-niques such as increasing the permeativity of these 15-kDapolypeptides to outer membranes of Gram negative �-lacta-mase producers.

Inhibitors with a sulfonamide core and varied substituentswere developed by structure-based optimization (351, 413).This non-�-lactam design approach is intended to avoid bothhydrolysis by �-lactamases and the upregulation of �-lactama-ses induced by some �-lactam-based inhibitors. Starting from amap of binding “hot spots” developed from crystal structuresof 13 different ligands in complex with the E. coli AmpC �-lac-tamase, 200,000 commercially available small molecules weredocked into the active site of the enzyme by computer simu-lation (351, 352). Based on these results, lead compounds werescreened in vitro, and the molecule with the highest affinity (26�M) was crystallized in complex with the AmpC (PDB 1L2S)(Fig. 1, compound 28). The structure closely resembled theprediction of the docking simulation, revealing the plasticity ofthe enzyme active site to recognize functional groups differentfrom �-lactams. This catalyzed the optimization of a novelinhibitor, and replacement of the chloride substituent with a

carboxylate yielded a compound with a 26-fold-improved Ki (1�M) (413). In MIC testing, the inhibitor restored the suscep-tibility to ceftazidime (at a 1:1 ratio) in the AmpC producers E.cloacae and C. freundii (but not P. aeruginosa) and, in contrastto clavulanate, did not upregulate �-lactamase expression in aninducible strain of E. cloacae.

�-Sultams are the sulfonyl analogs of �-lactams, and N-acyl�-sultams (Fig. 1, compound 29) inactivate the P99 enzyme (318,419). ESI-MS studies suggest that the �-sultams sulfonylate theserine residue to form a sulfonate ester. Elimination of the sul-fonate anion leads to C-O bond fission and formation of a dehy-droalanine at the previous Ser64 residue (419). These compoundsdo not show inhibition of class B MBLs, and there are currentlyno studies examining their activities in class A or D enzymes.

BLIP is a 165-amino-acid protein isolated from the sameorganism that produces clavulanic acid, Streptomyces clavulig-erus (105). Studies of BLIP’s inhibitory activity have revealedaffinities for class A enzymes that vary from pM to �M (theenzymes with the highest affinities for BLIP are KPC-2 and theP. vulgaris �-lactamases) (147, 393). Many of the individualclass A residues which contribute to BLIP binding have beenidentified through crystallography and mutagenesis (363, 456,460). While the clinical viability of a peptide inhibitor based onthis protein is limited because of protein degradation in vivo,the high-affinity interactions offer direction for the design ofnovel �-lactamase inhibitors.

The mixture of vanadate and catechol compounds in aque-ous solution produces vanadate-catechol complexes which caninhibit class A TEM-2, class C P99, and class D OXA-1 �-lac-tamases with apparent Kis of 62, 0.58, and 1.5 �M, respectively(5). A crystal structure with the complex has not been attained,but molecular modeling suggests that the active-site serine isbound to a penta- or hexa-coordinated vanadium. Vandate-catechols represent another group of compounds that are use-ful as tools for novel inhibitory active-site interactions, but lackclear clinical potential.

INHIBITION OF METALLO-�-LACTAMASES

We note that designing and/or discovering inhibitors toMBLs presents special challenges. The diversity of class Benzyme subclasses (i.e., B1, B2, and B3) and the mechanisticcomplexities regarding the role of one or two Zn2� ions haveimpeded effective inhibitor design. In addition, indiscrimi-nately inhibiting or chelating metal ions can have untowardeffects on natural or endogenous metalloenzymes. For exam-ple, similarities between the active-site architectures of MBLsand essential mammalian enzymes (e.g., human glyoxalase II)complicate the design of an inhibitor that would be safe forhuman use (97). As a class, the MBLs are resistant to all themechanism-based inhibitors of the serine enzymes (the inhib-itory activity of the methylidene penems against class B �-lac-tamases may be an important exception), and few investiga-tional inhibitors demonstrate sub-�M efficacy (335, 441). Here,we provide a brief survey of the more promising agents indevelopment.

FIG. 12. Molecular representation of cross-linked active-site resi-dues Ser64 and Lys315 (shown in yellow) formed after aminolysis of anO-aryloxycarbonyl hydroxamate inhibitor in E. cloacae P99 �-lacta-mase. (Based on PDB 2P9V and data from references 330 and 450.)



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Thiol Derivatives

Due to catalytic dependence on Zn2� ions, the metalloen-zymes are inhibited by thiols or chelating agents that have anaffinity for the hydrophobic pocket of the enzyme active siteand bind to, or interfere with, the bonding network betweenthe hydrolytic water and the Zn2� ions (170, 227, 312). Thiolderivatives, such as thiomandelic acids, typically show low-�MKi inhibition of B1 (e.g., BcII, IMP-1, and VIM-2) and B3 (e.g.,L1) subclasses but over 100-fold-higher Ki values for the sub-class B2 CphA MBL (268). Structural analysis of thiol-basedinhibitors complexed with the three MBL subclasses suggeststhat the thiol group chelates the Zn2� ions for subclasses B1and B3 (227). For example, the crystal structure of IMP-1 fromP. aeruginosa bound to a thiol inhibitor (Fig. 1, compound 30)(PDB 1DD6) showed interactions with active-site residues,allowing the thiol of the inhibitor to bridge the two catalyticZn2� molecules and displace the bridging water important forhydrolysis (88). A similar inhibition mechanism was resolvedfrom the crystal structure of the B1 VIM-2 complexed to amercaptocarboxylate compound (452). However, this bindingmode was not observed in the crystal structure of the thiolateD-captopril bound to the subclass B2 CphA from A. hydrophila(227). Instead, the inhibitor’s main interactions with the en-zyme were via the thiolate carboxylate group (227).

Mass spectrometric data suggest that mercaptoacetic estersmay act as mechanism-based inhibitors for BcII by generatingmercaptoacetic acid in situ and forming a disulfide linkage witha cysteine residue in the enzyme active site (327). However, aswith other thiols, the IC50s of these mercaptoacetic estersvaried from low �M to mM across MBL subclasses.

Pyridine Dicarboxylates

Recent work demonstrates that pyridine carboxylates, inparticular 2-picolinic and pyridine-2,4-dicarboxylic acids (Fig.1, compound 31), are competitive inhibitors of CphA (a sub-class B2 MBL), with Ki values of 5.7 and 4.5 �M, respectively(170). The pyridine carboxylate derivatives showed little inhib-itory activity for B1 and B3 MBLs. In the crystal structure ofCphA/pyridine-2,4-dicarboxylic acid (PDB 2GKL), the cata-lytic Zn2� ion is coordinated by the nitrogen and one of thecarboxylate oxygens of the inhibitor (170). The Zn2� ion is alsoengaged in electrostatic interactions with residues Asp120,Cys221, and His263. Hydrophobic interactions with Asn233,Trp87, and Val67 help to further stabilize the inhibitor in theactive site. The larger and more flexible active sites of subclassB1 and B3 MBLs may not permit comparable stabilizing con-tacts with the pyridine carboxylates, offering an explanation forthe B2 specificity.

Trifluoromethyl Ketones and Alcohols

Reactive trifluoromethyl ketones and alcohols, conju-gated to a penicillin-like phenyl side chain, effectively inhibitMBLs from B. cereus, P. aeruginosa, S. maltophilia, and A.hydrophila in vitro, presumably by binding the active-siteZn2� ion (433, 434). The crystal structure of a biphenyltetrazole inhibitor, L-159,061 (Fig. 1, compound 32), incomplex with the B. fragilis enzyme (PDB 1A8T) indicates

that the tetrazole portion of the molecule interacts directlywith the active-site Zn2� ions (414). IC50 and Ki values inthe low �M range were observed, and the incorporation ofsubstituents on the phenyl groups and their interactions withspecific residues were essential to the strong binding of thebiphenyl tetrazoles (414). These inhibitors do appear toenter cells, as the addition of imipenem or penicillin G tobiphenyl tetrazole compounds increased inhibition zones foran imipenem-resistant B. fragilis isolate (414).

Carbapenem Analogs

1-�-Methylcarbapenems with cyclic C-2 substituents, such asJ-110,441, achieve sub-�M Kis for the IMP-1, CcrA, LI, andBcII MBLs as well as representative class A and C enzymes(282). For several IMP-1-producing S. marcescens and P.aeruginosa isolates, imipenem MICs were lowered to 4 �g/mlby the addition of J-110,441. The further development ofJ-111,225 included a trans-3,5-disubstituted pyrrolidinylthiosubstituent at the C-2 position (Fig. 1, compound 33) (281).J-111,225 maintained inhibitory activity against IMP-1 (Ki of0.18 �M) and synergy with imipenem but had moderatelyimproved MICs as a single agent against IMP-1-expressing S.marcescens and P. aeruginosa (MIC range of 4 to 32 �g/ml).However, J-111,225 acted as a substrate for other chromo-somally encoded class B MBLs, including CcrA, L1, and BcII.

Tricyclic Natural Products

Natural product screening for inhibitors to BcII led to thediscovery of extracts from Chaetomium funicola which demon-strated IC50s ranging from 389 to 0.3 �M for BcII, P. aerugi-nosa IMP-1, and B. fragilis CfiA MBLs (329). The “lead com-pound” of the three tricyclic compounds, SB238569 (Fig. 1,compound 34), also showed low Ki values for the same en-zymes (79, 17, and 3.4 �M, respectively). In combination withmeropenem, 8 �g/ml of SB238569 restored susceptibility to B.fragilis CfiA. The crystal structure of CfiA and SB236050 (PDB1KR3) identified important polar (Lys184, Asn194, andHis162) and ring-stacking (Trp49) interactions which likelycontribute to the inhibitory activity. Comparison of this struc-ture with that of IMP-1/mercaptocarboxylate (PDB 1DD6)suggests that the binding site created by the loop precedingLys184 is shorter in IMP-1 and may leave a larger portion ofthe compound exposed to solvent (88). The decreased inhibi-tory activity of the tricyclic inhibitor for S. maltophilia L1 mayalso be explained by active-site variations, including differencesin the positions of the Lys184 and Asn194 equivalents.

Succinate Derivatives

The IMP-1 enzyme is encoded by a transferable blaIMP-1

gene and represents an increasing resistance threat, and thusthe development of inhibitors for this target is a priority. Suc-cinic acid compounds have been screened as IMP-1 inhibitors,and several derivatives have restored meropenem susceptibilityof IMP-1-expressing E. coli (269, 415). Compound 35 (Fig. 1)belongs to a series of 2,3-(S,S)-disubstituted succinic acidswhich inhibit IMP-1 with nM IC50s (415). Crystal structure



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analysis revealed that the inhibitors’ carboxylate groups inter-act with both Zn2� ions and active-site residues while displac-ing the bridging water (PDB 1JJE and 1JJT) (415). Disubsti-tuted succinic acids have also been described as inhibitors ofthe L1 MBL and display binding modes similar to those inIMP-1 (284, 303).

IMP-1 is also inhibited by N-arylsulfonyl hydrazone com-pounds, with IC50s as low as 1.6 �M (Fig. 1, compound 36)(384). Structure-activity relationship studies revealed thatbulky aromatic substituents on both sides of the sulfonyl hy-drazone backbone improved the activity of these inhibitors.

Lienard et al. demonstrated the ability of dynamic combina-torial mass spectrometry (DCMS) to identify potent BcII in-hibitor leads (228). A meso-2,3-dimer-captosuccinic acid wasbound to the MBL active site via one thiol group, while thesecond thiol group was linked in a disulfide bond to a thiol-containing compound from a screening library. ESI-MS re-vealed which compounds bound the enzyme, and subsequentstructure optimization led to the development of a mercapto-carboxylate that inhibits BcII with a Ki of 740 nM (Fig. 1,compound 37).

C-6-Mercaptomethyl Penicillinates

Penicillin derivatives with C-6-mercaptomethyl substituents(Fig. 1, compound 38) show promising kinetics against class A,B, and C �-lactamases (IC50s of 6.8, 0.10, and 10.5 �M, re-spectively, against representative enzymes) (61). These peni-cillinates, in combination with piperacillin, lowered MICsagainst MBL-producing strains of P. aeruginosa. Building in-hibitors on the backbone of the natural substrate for all �-lac-tamases, the penicillin nucleus, may help bridge the gap be-tween these diverse enzyme classes.

LESSONS LEARNED

We dedicate the penultimate section of this review to sum-marizing the “lessons learned” from our consideration of the�-lactamase inhibitors. From the perspective of clinicians, wepropose that several important features must be regarded inorder to optimize the development of �-lactamase inactivators.

(i) High affinity for the active site of the target �-lactamase.It is axiomatic that in order for an inhibitor to work efficiently,it must exhibit high affinity for its target. This principle isemphasized by the development of clavulanate resistance inTEM-1 and SHV-1 �-lactamases mediated by substitution ofamino acid residues 69, 130, 244, and 276, leading to increasedinhibitor KIs and/or IC50s (73, 153, 378, 411). Particularlypromising are those inhibitors that maintain high affinitiesacross different groups of �-lactamases, such as the C-2-sub-stituted sulfone LN-1-255, the methylidene penems, and phos-phonates which achieve nM inhibition of class A, C, and D (4,25, 63, 245, 322, 441) (Drawz et al., unpublished data).

(ii) Mimicking the “natural substrate.” The available inhib-itors are effective primarily against class A serine �-lactamases,and the structures of class A �-lactamase inhibitors are similarto those of penicillin, bearing a fused four- or five-memberedring system. Similarly, data on transition state analogs suggestthat class C �-lactamases have high affinity for inhibitors that

resemble the natural cephalosporin substrates (272). A guide-line for inhibitor design may be “penicillin analogs for penicil-linases, and cephalosporin analogs for cephalosporinases.”This principle also ensures that the inhibition mechanism fol-lows the substrate mechanism—save for hydrolysis. While�-lactamases continue to develop resistance to all developed�-lactams, designing inhibitors that follow the hydrolytic mech-anism may help to thwart the emergence of resistant isolates,as the enzyme must find novel ways to preserve function butevade inhibition. We hasten to add that this approach may notbe practical in all cases.

(iii) Stabilizing interactions in the active site. Stabilizing orprolonging the acyl-enzyme intermediate is central to success-ful inactivation by the currently available class A inhibitors (81,396). Promising novel inhibitors, such as the methylidene pen-ems (e.g., BRL 42715) and LN-1-255 are designed to capitalizeon and enhance this stabilization. One approach is to prolongthe acylation step (k2). Alternatively, acyl-enzyme intermedi-ates can be stabilized by multiple interactions in the active site,as observed for SHV-1 with tazobactam and SA2-13 (308, 309).The inhibition of NMC-A by the monobactam derivative 13(Fig. 1) is enhanced by the incorporation of chemical compo-nents that confer structural flexibility to the inhibitor, allowingthe enzyme-substrate species to adopt multiple conformationsand trap the enzyme in energy minima unfavorable for hydro-lysis (273, 308, 445). Another viable approach is to designinhibitors that induce conformational changes in the enzymeor displace key water molecules important for hydrolysis, suchas seen with the large seven-membered intermediate formedfrom the methylidene penems (60, 294, 429).

(iv) Reaction chemistry that slows deacylation and favorsinactivation over inhibitor hydrolysis. Features of an inhibitorwhich slow deacylation and drive toward terminal inactivation,such as acyl-enzyme stabilization (see above) or the presenceof a good leaving group at C-1, contribute to effective inhibi-tion. Inhibitors that can delay deacylation for longer than 15 to20 min (koff rate of �0.001 s�1) may outlast the bacterialgeneration time, allowing the partner �-lactam time to disruptthe cell wall synthesis necessary for cell division (396, 410). Forexample, the bridged monobactams delay deacylation by lim-iting the C-3–C-4 rotation necessary for approach of the hy-drolytic water in class C enzymes (152, 238). Additionally,relatively minor changes introduced by the acylation of mero-penem by SHV-1, including minor displacements to active-siteresidues (overall root mean square deviation [RMSD] of 0.29Å) and a restructured hydrogen bonding network, lead to per-sistent complex formation (295).

(v) Rapid cell penetration. �-Lactamases are typically foundin the periplasmic space of Gram-negative bacteria, and therapidity with which inhibitors can access their target is a pre-requisite for successful inhibition. Certain �-lactams are zwit-terionic and rapidly penetrate the bacterial outer membrane(e.g., cefepime). The same principle extends to the inhibitors;e.g., the enhanced cell entry of LN-1-255 and the novelmonobactams BAL19764 and BAL30072 is likely due to up-take through siderophore channels (75, 316). Attention mustalso be paid to designing “permeable drugs” that are not hin-dered as they pass through porin channels or quickly returnedinto the medium by multidrug efflux pumps, an important



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resistance determinant independent of �-lactamases (seeabove) (287).

(vi) Low propensity to induce �-lactamase production. Suc-cessful inhibitors need to avoid inducing expression of theenzymes that they are designed to inhibit, particularly amongAmpC �-lactamases. Examples of this approach include thenon-�-lactam inhibitors with sulfonamide cores designed toevade hydrolysis as well as to avoid induction of AmpC enzymeexpression (413).

(vii) Identification of measureable biological correlates of�-lactamase inhibition in the cell. Regardless of how attractivean inhibitor’s laboratory characteristics may be (e.g., nM Ki

values, low IC50s, high kinacts, or low MICs), a successful in-hibitor must prove its worth in the clinical setting. In vivoefficacy requires an integration of many complex features, in-cluding those listed above, partnership with an appropriate�-lactam, sufficient serum and periplasm concentrations, andfactors less well understood (e.g., serum protein binding andindividual clearance rates). Many microbiological properties(time kill, mutant prevention concentration, and synergy) areimportant parameters that need to be considered. There is nocertain consensus on how best to identify and measure theseattributes in the early research on candidate inhibitors. Previ-ous studies with �-lactam antibiotics have demonstrated thatMICs may be poorly correlated with the sensitivity of PBPs forinactivation (213). Finding reliable biological correlates of in-hibition is a substantial and relevant challenge, and our currentmethodological outcome measurements may benefit from re-examination in this vein.

(viii) In the meantime, use what we have. While scientistsand physicians continue struggling to stay ahead, or at leastabreast, of the abilities of �-lactamases, we must utilize intel-ligently and productively the agents that are currently avail-able. For example, we can commit to applying the research thatdemonstrates the potential of novel combinations of �-lactamsand �-lactamase inhibitors, such as clavulanate with cefepime,cefpirome, or meropenem, to expand and enhance inhibitors’abilities to protect partner �-lactams (172, 239). Several sig-nificant hurdles stand in the way of these novel combinations,such as corporate relationships affecting the funding of thetrials necessary to demonstrate clinical efficacy, as well as op-timization of the pharmacokinetics, pharmacodynamics, andsafety of the components. The �-lactamase-inhibitory activityof carbapenems should not be overlooked in the face of in-creasing �-lactam resistance. Carbapenems, for the time being,remain effective against many difficult-to-treat infections andteach us that the distinction between an inhibitor and a slowsubstrate may be blurred.

A PERSPECTIVE

From our vantage point, the main challenge in �-lactamaseinhibitor development is discovering novel inhibitors with ac-tivity against a broad spectrum of inhibitor-susceptible and-resistant enzymes from multiple classes (17, 410). New inhib-itors must also target carbapenemases (serine and metallo-), ascarbapenems are still the most potent �-lactams available forclinical use. Future advances in inhibitor design against versa-tile �-lactamases must incorporate strategies that address each

of the key structural features of these diverse proteins, keepingin mind that mechanism-based inhibitors may engage inunique reaction chemistry with �-lactamases. Compounds withthe chemical features of the “ideal inhibitor,” such as en-hanced permeability through the cell membrane, affinity forthe active site of the �-lactamase, formation of intermediatesthat “trap” the enzyme, displacement of critical water mole-cules, and prolonged time to enzyme recovery, many of whichare illustrated by the investigational complexes highlightedherein, may have a significant impact on the development of“second-generation” inhibitors that target resistant �-lactamases.

We also acknowledge the magnitude of this challenge. In 30years of targeted research on �-lactamase inactivation, new in-hibitors have been only slowly introduced into clinical trials.There have been many starts, but few agents cross the finish line.This is a testament to the versatility and complexity of �-lactama-ses and the incredible evolutionary ability of the organisms har-boring them. Is the perfect �-lactamase inhibitor an unattainablegoal? Perhaps. Yet, we anticipate with excitement the achieve-ments in the next 3 decades and suspect that better chemistriesand combinations will ultimately be found.

ACKNOWLEDGMENTS

This work was supported in part by the Department of VeteransAffairs Merit Review Program and National Institutes of Health grant1RO1 A1063517-01. R.A.B. is also supported by the Veterans Inte-grated Service Network (VISN) 10 Geriatric Research, Education, andClinical Center (GRECC). S.M.D. was supported in part by NIH grantT32 GM07250 and the Case Medical Scientist Training Program.

We thank the four anonymous reviewers and A. Endimiani andM. G. P. Page for careful reading and suggestions regarding thisreview.

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Sarah M. Drawz received her bachelor’s de-gree from Amherst College magna cumlaude. She is currently an M.D./Ph.D. stu-dent at Case Western Reserve UniversitySchool of Medicine in the Department ofPathology. Her Ph.D. dissertation work fo-cuses on the inhibition of �-lactamase en-zymes. She plans to return to her medicalstudies in the coming year.

Robert A. Bonomo received his M.D. fromthe Case Western Reserve UniversitySchool of Medicine. He trained in Inter-nal Medicine and Infectious Diseases atUniversity Hospitals of Cleveland and re-ceived a Certificate of Added Qualifica-tion in Geriatrics. Dr. Bonomo served assection chief of the infectious disease di-vision at the Cleveland Veterans AffairsMedical Center before becoming Directorof the Veterans Integrated Service Net-work 10 Geriatric Research, Education, and Clinical Center(GRECC). He is a Professor of Medicine at Case Western ReserveUniversity School of Medicine and also holds appointments in theDepartments of Pharmacology and Molecular Biology and Micro-biology. Dr. Bonomo’s research focuses on microbial resistance toantibiotics, especially �-lactams.



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