Published Ahead of Print 14 July 2014. 10.1128/AAC.03057-14.

2014, 58(10):5704. DOI:Antimicrob. Agents Chemother. N. B. Olivier and R. A. AlmS. D. Lahiri, M. R. Johnstone, P. L. Ross, R. E. McLaughlin, Resistancethe Binding Pocket, and Implications forMechanism of Inhibition, Conservation of

-Lactamases:βAvibactam and Class C

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Avibactam and Class C �-Lactamases: Mechanism of Inhibition,Conservation of the Binding Pocket, and Implications for Resistance

S. D. Lahiri,a M. R. Johnstone,a P. L. Ross,b R. E. McLaughlin,a N. B. Olivier,b R. A. Alma

Infection Innovative Medicines, AstraZeneca R&D Boston, Boston, Massachusetts, USAa; Discovery Sciences Innovative Medicines, AstraZeneca R&D Boston, Boston,Massachusetts, USAb

Avibactam is a novel non-�-lactam �-lactamase inhibitor that inhibits a wide range of �-lactamases. These include class A, classC, and some class D enzymes, which erode the activity of �-lactam drugs in multidrug-resistant pathogens like Pseudomonasaeruginosa and Enterobacteriaceae spp. Avibactam is currently in clinical development in combination with the �-lactam antibi-otics ceftazidime, ceftaroline fosamil, and aztreonam. Avibactam has the potential to be the first �-lactamase inhibitor thatmight provide activity against class C-mediated resistance, which represents a growing concern in both hospital- and commu-nity-acquired infections. Avibactam has an unusual mechanism of action: it is a covalent inhibitor that acts via ring opening, butin contrast to other currently used �-lactamase inhibitors, this reaction is reversible. Here, we present a high-resolution struc-ture of avibactam bound to a class C �-lactamase, AmpC, from P. aeruginosa that provided insight into the mechanism of bothacylation and recyclization in this enzyme class and highlighted the differences observed between class A and class C inhibition.Furthermore, variants resistant to avibactam that identified the residues important for inhibition were isolated. Finally, thestructural information was used to predict effective inhibition by sequence analysis and functional studies of class C �-lacta-mases from a large and diverse set of contemporary clinical isolates (P. aeruginosa and several Enterobacteriaceae spp.) obtainedfrom recent infections to understand any preexisting variability in the binding pocket that might affect inhibition by avibactam.

The continual emergence of multidrug resistance in Gram-neg-ative bacteria has eliminated many former treatment options.

The �-lactam drug class, once the foundation of treatment regi-mens for many hospital- and community-acquired infections, israpidly becoming obsolete due to the proliferation of �-lacta-mases (1–3). Even the effectiveness of carbapenems, which formany years represented the last line of defense, is being eroded bythe emerging pandemic of carbapenemases, such as metallo-�-lactamase-containing pathogens (4, 5). The currently available�-lactamase inhibitors, such as clavulanic acid and sulbactam, areeffective inhibitors of many of the class A �-lactamases but areincapable of inhibiting any other classes, including class C (6).Chromosomally encoded class C �-lactamases are found in manyclinically important pathogens, such as Pseudomonas aeruginosaand many Enterobacteriaceae spp. (7, 8). In many cases, the expres-sion of these enzymes is inducible; however, they can becomederepressed, and the subsequent overexpression results in resis-tance to many �-lactam drugs. Furthermore, there is a myriad ofclass C enzymes encoded on transferable plasmids that enablehorizontal transfer of class C-mediated �-lactam resistance be-tween bacterial species.

Avibactam (Fig. 1A) is a novel non-�-lactam �-lactamase in-hibitor that inhibits both class A and class C (and some class D)enzymes, thus providing protection from a diverse range of �-lac-tamase-mediated resistance mechanisms (9–11). It is currently inclinical development in combination with the cephalosporins cef-tazidime and ceftaroline fosamil and with the monobactamaztreonam (Fig. 1B) as alternative therapeutic options for thetreatment of infections caused by multidrug-resistant P. aerugi-nosa and Enterobacteriaceae spp. (12–17). Avibactam is structur-ally distinct from the clinically used �-lactamase inhibitors in thatit does not contain a �-lactam core. In addition, it has a unusualmechanism of inhibition. While the covalent inhibition proceedsin a similar fashion via the opening of the avibactam ring, the

reaction is reversible, whereby deacylation results in regenerationof the intact compound as opposed to hydrolysis and turnover (9).This mechanistic difference from the clinically used inhibitorscontributes to making avibactam highly effective in providingprotection to the �-lactam partner against hydrolysis by chromo-somal and plasmidic �-lactamases.

We have recently described the mechanism of covalent inhibi-tion of class A enzymes by avibactam as well as a medium-resolu-tion structural view of a class C cocomplex to rationalize thebroad-spectrum activity (18). However, the mechanism of inhibi-tion of class C �-lactamases, which is a differentiating attribute ofavibactam, was not confirmed. We now report high-resolution P.aeruginosa AmpC structures in complex with avibactam in boththe ring-open and ring-closed forms and in complex with themonobactam �-lactam, aztreonam. The subsequent analyses haveenabled an understanding of the reversible deacylation in class Cenzymes and a rationale for the more rapid recyclization observedwith this class C enzyme in comparison to class A �-lactamases. Inaddition, we also evaluated the conservation of the avibactambinding pocket to assess the risk of any preexisting pool of resis-tant class C enzymes by sequencing the chromosomal ampC genefrom �500 diverse clinical isolates.

Received 17 April 2014 Returned for modification 21 May 2014Accepted 7 July 2014

Published ahead of print 14 July 2014

Address correspondence to S. D. Lahiri, sushmita.lahiri@astrazeneca.com, or R. A.Alm, richard.alm@astrazeneca.com.

Supplemental material for this article may be found at http://dx.doi.org/10.1128/AAC.03057-14.

Copyright © 2014, American Society for Microbiology. All Rights Reserved.

doi:10.1128/AAC.03057-14

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MATERIALS AND METHODSCrystallization, data collection, structure determination, and refine-ment. Crystals of AmpC were obtained as described previously (18). Crys-tals were soaked with a final concentration of 10 mM avibactam or aztreo-nam for 15 min, followed by flash freezing in liquid nitrogen. Data werecollected at Advanced Photon Source in Chicago at Industrial Macromo-lecular Crystallography Association (IMCA) beamline BM16. Data pro-cessing and refinement statistics are provided in Table S1 in the supple-mental material.

Genome sequencing. Total genomic DNA was prepared using thegenomic DNA purification kit in the Maxwell 16 instrument (Promega,Madison, WI). DNA libraries were prepared using the Nextera libraryconstruction protocol (Illumina, San Diego, CA) and sequenced on aMiSeq sequencer (Illumina).

Data analysis. Whole-genome data were analyzed using Workbenchsoftware version 5.5.1 (CLC Bio, Cambridge, MA). Multiple sequencealignments were generated using the ClustalX program. Multilocus se-quence typing (MLST) was performed by comparing the assembled ge-nome sequences of each strain to the database of known P. aeruginosaMLST alleles available at www.pubmlst.org. ConSurf, a bioinformaticstool (http://consurf.tau.ac.il/), was used to map the multiple sequencealignment to the AmpC-avibactam structure to visualize the 3-dimen-sional sequence diversity. PyMOL (www.pymol.org) was used to displaythe diversity and also to model the residue substitutions identified in theresistant variants.

Resistance selection and antimicrobial susceptibility testing. Resis-tant variants were selected by plating a bacterial suspension on agar platescontaining 2-fold increasing concentrations of aztreonam with avibactamheld constant at 4 �g/ml. The MIC values were determined by the brothmicrodilution method following the guidelines established by the Clinicaland Laboratory Standards Institute (CLSI).

Expression analysis. RNA from strains grown to mid-log phase wasprepared using a Maxwell 16 LEV simplyRNA purification kit (Promega).A total of 5 ng RNA was used in a reverse transcription (RT)-PCR assayusing a Qiagen QuantiTect SYBR green RT-PCR kit (Germantown, MD)with a Bio-Rad CFX96 instrument. The oligonucleotides used to detectthe expression of the chromosomal blaampC allele in Citrobacter freundiiwere 5=-CGAGGGGAAACCTTATTA-3= and 5=-TGTATAGGTGGCTAAGTG-3= and the control oligonucleotides to detect expression of the rpsLribosomal gene were 5=-TAAAAAACCGAACTCCGCA-3= and 5=-GTCACTTCAAAACCGTTAG-3=. The level of rpsL expression was used for nor-malization.

RESULTSHigh-resolution structure of avibactam bound to P. aeruginosaAmpC. Crystals of avibactam covalently bound to P. aeruginosa

AmpC that diffracted to 1.1 Å were obtained. The higher resolu-tion provided improved understanding of avibactam binding, inparticular, the piperidine ring conformation, the positions of thecarboxamide and sulfate groups, and most importantly, the accu-rate positions of the residues involved in catalysis (Fig. 2A to C).The high resolution allowed observation of an intramolecularconnectivity between the amide group and the N1 nitrogen of thepiperidine ring (Fig. 2A), suggestive of a short strong hydrogenbond (�2.5 Å) between these two moieties. This interaction isnovel to the class C binding mode of avibactam, as the boundstructure of a class A �-lactamase at a comparable resolution (18)showed an increased distance between these atoms with no con-nectivity of electron density. Further, there was additional densityobserved between the cleaved C7-N6 bonds of the pyrazolidinering (Fig. 2A). The most plausible explanation for this additionaldensity is that a small subpopulation of ring-closed forms or anintermediate state of the inhibitor exists simultaneously in thisposition. However, refinement in the presence of a closed form ofavibactam did not convincingly distinguish the two forms ofavibactam, and hence, the model was refined with the open cova-lently linked inhibitor (Fig. 2B).

The refined structure showed that eight residues providedthree key contributions to the binding interactions with avibac-tam (Fig. 2C). The carboxamide group of avibactam interactedwith the side chains of Asn152 and Gln120, and the sulfate moietywas positioned by Thr316, Lys315, and Asn346, whereas Tyr150and Lys67 were positioned to participate in catalytic roles to en-able formation of the covalent bond with Ser64. Specifically, theO� of Tyr150 was equidistant, 3.3 Å and 3.2 Å, respectively, fromN6 of avibactam and O� of Ser64. The N� of Lys67 was 2.9 Å fromthe O� of Ser64 and 4.9 Å from the N6 of avibactam. Comparisonof the apo structure at a similar resolution showed a minimal shiftin the binding pocket residues upon acylation with all residuessuperimposing exactly except for Tyr150, Lys67, and Asn152 (Fig.2D). In the avibactam bound form, the side chain of Tyr150 movescloser to Lys67 by 1.0 Å, indicative of the formation of a newhydrogen bond upon acylation. In addition, a new hydrogen bondis also formed between the carboxamide group of avibactam andthe Asn152 side chain. Asn152 maintains its hydrogen bond toLys67 upon acylation, which results in a shift in the positions ofthese residues.

Comparison to a substrate binding mode. To understand the

FIG 1 Chemical structures of avibactam (A) and aztreonam (B).

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differences in interactions between a �-lactam drug and avibac-tam, the structure of aztreonam bound to P. aeruginosa AmpC wassolved to 1.3 Å (Fig. 3A). Aztreonam contains a sulfonyl group inplace of the carboxylate group seen in other �-lactam antibiotics,which makes it structurally closer to avibactam than other �-lac-tams. In addition, whereas aztreonam is hydrolyzed by class C�-lactamases, resulting in decreased susceptibility, the rate of hy-

drolysis is slower than that observed for other �-lactams (6), mak-ing it feasible to capture the acyl-enzyme complex crystallographi-cally. The structure showed that the position of the acylatedaztreonam molecule in P. aeruginosa AmpC was very similar tothat previously observed in a Citrobacter freundii class C enzyme,with the exception of the position of the oxyimino group (19).There was a significant similarity in the binding mode and inter-

FIG 2 Avibactam binding pocket in AmpC. (A) Unbiased Fo-Fc electron density (2.9- cutoff, green mesh) of avibactam (pink) prior to its addition into therefinement cycle. The connectivity of the electron density between the carboxamide and piperidine ring is indicated by the blue arrow while the additional densityobserved between the cleaved C7-N6 bonds of the pyrazolidine ring is shown by the green arrow. (B) Avibactam (pink sticks) and the neighboring residues (graysticks) depicted along with the final refined 2Fo-Fc map (1.5- cutoff, blue mesh). (C) Distances of Tyr150 (green) and Lys67 (blue) from avibactam (pink sticks)and Ser64 (gray sticks). (D) Overlay of the avibactam binding pocket (gray sticks) over the apo pocket (yellow sticks). Only residues with changed positions inthe apo structure are shown. Avibactam is depicted by pink sticks. Distances between residues in the avibactam structure are depicted by blue lines and labels, andthose of apo in yellow.

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acting residues between aztreonam and avibactam (Fig. 3B). Thesulfonyl group of aztreonam and the sulfate group of avibactamwere similarly located and interacted with Lys315, Thr316, andAsn346. Additionally, both groups displace the deacylating watermolecule (Wat) previously observed in crystal structures (ProteinData Bank [PDB] code 1IEL) (20). The position of the N1 ofaztreonam upon ring cleavage was in a location similar to that ofthe N6 of avibactam, although the C3-C4 bond of the aztreonam�-lactam ring underwent a rotation of approximately 70° uponring opening, which displaced the trajectory for recyclization. Theside chains of Tyr150 and Lys67 were in identical positions in thetwo binding modes, as were the interactions of Asn152 with bothLys67 and the ligands. However, in the case of aztreonam, Asn152formed a hydrogen bond with the carbonyl oxygen, which is op-posite in polarity to the amide group of avibactam. Overall, thehydrogen-bonding patterns and the key interactions in the acy-lated forms were very similar between the substrate (aztreonam)and the inhibitor (avibactam) despite the differences in the com-

pound structures. The major differences lay in the overall size andlimited rotational freedom of avibactam compared to those ofaztreonam.

Conservation of class C �-lactamase enzymes. Given theemerging resistance to �-lactams in P. aeruginosa and evidencethat small sequence differences can affect the substrate spectrum(7), there was a need to understand whether avibactam might beexpected to inhibit all of the AmpC enzyme variants in this species.There were 36 unique AmpC sequences from P. aeruginosa, rep-resenting 67 different isolates, available in the public domain (7,21). An additional 464 isolates, obtained from 25 different coun-tries and isolated from multiple clinical indications from 2008 to2012 were sequenced. Multilocus sequence typing (MLST) analy-sis demonstrated a high level of diversity among the isolates. The531 P. aeruginosa AmpC proteins were clustered into groups rep-resenting 72 unique sequences. The relative population withineach cluster varied, with the largest cluster representing 109(20.5%) of the isolates examined, whereas 35 clusters were repre-

FIG 3 AmpC crystal structure with aztreonam. (A) Unbiased Fo-Fc electron density (2.9- cutoff, blue mesh) of aztreonam (dark green sticks) covalently boundto the AmpC active site Ser64 (light green sticks). (B) Overlay of the aztreonam binding pocket (green sticks) on the avibactam binding pocket (pink sticks). Theceftazidime and deacylating water molecule (Wat) from the ceftazidime-AmpC crystal structure.

TABLE 1 Conservation analysis of key residues in chromosomal and plasmidic class C �-lactamase enzymes

Class C �-lactamase enzymeNo. of uniquevariants

No. of variants with conserved residues (changes observed)

Ser64 Lys67 Gln120 Tyr150 Asn152 Lys315 Thr316 Asn346

ChromosomalP. aeruginosa AmpC 72 71 (1 Leu) 72 72 72 72 72 72 70 (1 Thr; 1 Ile)E. cloacae AmpC 57 57 57 57 57 57 57 56 (1 Met) 57E. coli AmpC 102 102 102 102 102 102 102 102 101 (1 Ser)E. aerogenes AmpC 16 16 16 15 (1 Lys) 16 16 16 16 16Citrobacter species AmpC 15 15 15 15 15 15 15 15 15

PlasmidicCMY 84 84 84 83 (1 Lys) 84 84 84 84 79 (5 Ile)DHA 6 6 6 6 6 6 6 6 6FOX 9 9 9 9 9 9 9 9 0 (9 Ile)MOX 7 7 7 7 7 7 7 7 1 (6 Ile)MIR 6 6 6 6 6 6 6 6 6ACT 13 13 13 13 13 13 13 13 13CFE 1 1 1 1 1 1 1 1 1LAT 1 1 1 1 1 1 1 1 1

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sented by a single isolate (see Fig. S1 and Table S2 in the supple-mental material). Although amino acid variations were observedin �22% of the residues, the eight residues directly involved inavibactam binding were highly conserved, with six of them beingconserved in all proteins and the other two (Ser64 and Asn346)having one and two variants, respectively (Table 1). Of these, theisolate containing a Ser64Leu substitution was expected to be non-functional because Ser64 is critical for covalent catalysis (22). Thechanges of Asn346Thr and Asn346Ile were both to amino acids ofsimilar sizes, and the Asn346Ile substitution also changed the prop-erty of the substitution from a polar to a hydrophobic group.

Given that an important attribute of avibactam is the inhibi-tion of class C �-lactamases, the conservation analyses were ex-tended to other class C enzymes in clinically relevant bacterialpathogens, including the chromosomal AmpC proteins of Esche-richia coli, Enterobacter cloacae, Enterobacter aerogenes, and Citro-bacter spp. and plasmidic enzymes that can be carried by multiplespecies (Table 1). In the case of E. cloacae, where derepression ofthe chromosomal blaampC gene is a common resistance mecha-nism, 79 recent clinical isolates were sequenced, and their chro-mosomal AmpC proteins, along with 19 sequences from the pub-lic domain, were clustered into 57 unique AmpC sequences withalmost 40% of the amino acid residues varying among these se-quences (see Fig. S1 and Table S3 in the supplemental material).Similarly, unique chromosomal AmpC proteins from E. coli, E.aerogenes, and Citrobacter spp. along with 127 plasmidic class C�-lactamase enzymes from public and internal data were also in-cluded in the conservation analysis. Despite the dramatic se-quence variability observed between these class C enzymes (Fig.4A), Lys67, Tyr150, Asn152, and Lys315 were completely con-served (Fig. 4B). A small number of isolates contained variationsat Ser64 (n 1), Gln120 (n 2), and Thr316 (n 1). Asn346 wasthe most variable of the binding pocket residues: three variantsamong the chromosomal enzymes and 20 plasmidic class C en-zymes carried the Asn346Ile substitution (Table 1). This modifica-tion was previously attributed to an extended-spectrum cepha-losporinase activity (23, 24).

FIG 4 Conservation of the avibactam binding pocket mapped on the AmpC crystal structure. (A) Sequence conservation of class C residues is depicted as asurface map where cyan represents variable regions while purple represents conserved residues. Avibactam is depicted as cyan sticks. (B) Residues in the bindingpocket of avibactam (white sticks) interacting via their side chains are depicted as sticks and color coded based on conservation.

TABLE 2 Susceptibility analyses of Pseudomonas aeruginosa isolatescarrying different AmpC variations

StrainAmpCgenotypea

MIC (�g/ml) forb:

CAZ CAZ-AVI ATM ATM-AVI

ATCC 27853 AZPC-5 2 1 4 4PAO1 AZPC-1 2 2 4 4ARC4644 AZPC-1 32 8 32 32ARC4884 AZPC-3 64 4 32 8ARC3604 AZPC-5 128 8 256 64ARC2144 AZPC-6 16 1 32 2ARC3509 AZPC-8 128 8 128 16ARC4989 AZPC-11 64 16 64 32ARC2147 AZPC-15 64 4 32 8ARC4889 AZPC-16 32 4 16 8ARC4938 AZPC-19 32 4 16 16ARC4647 AZPC-24 64 4 16 8ARC4906 AZPC-30 32 8 8 4ARC4836 AZPC-31 64 16 64 32ARC5058 AZPC-35 128 4 128 16ARC3609 AZPC-36 32 4 16 8ARC3610 AZPC-37 128 8 128 16ARC3608 AZPC-39 128 8 128 8ARC3737 AZPC-40 32 2 8 0.5ARC3862 AZPC-41 64 2 64 4ARC4453 AZPC-44 64 4 16 16ARC4372 AZPC-46 128 16 64 32ARC2416 AZPC-48c 128 4 8 0.5ARC2415 AZPC-49 �256 8 64 8ARC2413 AZPC-50 �256 4 64 4ARC2151 AZPC-52 64 4 8 2ARC2156 AZPC-53 64 4 16 1ARC2142 AZPC-54 128 2 64 2ARC4847 AZPC-55 64 2 16 8ARC5064 AZPC-68 32 8 32 16ARC5366 AZPC-71 32 8 32 16ARC2154 AZPC-72 64 2 8 1a Unique AmpC sequences (see Fig. S1 and Table S1 in the supplemental material fordetails).b CAZ, ceftazidime; ATM, aztreonam; AVI, avibactam (constant concentration of 4 �g/ml).c The AZPC-48 sequence carries the Asn346Ile substitution; in all other AZPC clusterslisted, all eight key residues that interact with avibactam are fully conserved.

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Microbiological susceptibility tests were performed in P.aeruginosa and E. cloacae isolates where the chromosomal blaampC

was derepressed and contained no other �-lactamases to enableclear interpretation of the ability of avibactam to inhibit thesechromosomal AmpC variants. The susceptibility data confirmedthat the restoration of �-lactam activity by 4 �g/ml avibactam wasnot affected by variations in these enzymes and restored the cef-tazidime and aztreonam MIC values to susceptible ranges as de-fined by the CLSI (representative MIC values are shown in Table2). The only variations in the 8 key avibactam binding residues(Table 1) that were not verified were the Gln120Lys, which was notavailable for testing, and the chromosomal Thr316Met, which wasnot derepressed in the isolate. Taken together, these data suggestthat there is not a significant pool of preexisting class C-basedresistance to avibactam in clinical isolates of Gram-negativepathogens.

Resistance to avibactam in combination with a �-lactam.Given the conservation of the avibactam binding pocket residuesand the importance of these residues in �-lactam recognition andcatalysis (25–29), we investigated whether any variations in thesemight compromise the inhibition potency of avibactam while stillallowing hydrolysis of the �-lactam drug and thus result in resis-tance. Spontaneous resistance frequency experiments were car-ried out in several isolates carrying class C �-lactamases, with aninitial focus on Enterobacteriaceae spp. where aztreonam in com-

bination with avibactam was more potent than in P. aeruginosa.Resistant variants from a C. freundii isolate and an E. coli isolatethat carried multiple �-lactamase enzymes were obtained at lowfrequencies of 6 � 10�10 and 6 � 10�9, respectively. These mu-tants had 8- to 64-fold increases in aztreonam-avibactam MICscompared with that for the parental isolate (Table 3). Whole-genome sequence analyses of the parent and daughter strainsidentified the mutations responsible for the loss of susceptibility.The variation in the resistant C. freundii strains was either anAsn346Tyr or a Tyr150Ser change in the chromosomal AmpC pro-tein, which was confirmed by RT-PCR analysis to be stably dere-pressed in both the parent and daughter strains to equivalent lev-els (Table 3; see also Fig. S2 in the supplemental material). Thevariation in the E. coli mutant was a Tyr150Cys substitution in theplasmid-encoded class C enzyme (CMY-6).

Structural models of the AmpC mutations indicated that theAsn346Tyr substitution would result in a steric clash with the sul-fate group of avibactam, thus influencing the binding affinity ofthe inhibitor (Fig. 5A). Whereas a similar impact to the sulfonylgroup of aztreonam is expected, the slightly greater distance be-tween this group and the side chain, as well as the greater flexibilityof aztreonam, did still accommodate binding and hydrolysis.While the Tyr150Ser substitution maintains a similar polarity, thereduced size of the Ser residue increases the binding pocket vol-ume (Fig. 5B). The rigid binding mode of avibactam and the pos-

TABLE 3 Susceptibility and �-lactamase profiles of aztreonam- and aztreonam-avibactam-resistant Enterobacteriaceae variants

Strain

�-Lactamase content of: MIC (�g/ml) fora:

Chromosome Plasmid(s) ATM ATM-AVI

C. freundii 3885 (parent) AmpC TEM-1, SHV-5, CMY-2 512 1C. freundii (variant 1) AmpC[Asn346Tyr] TEM-1, SHV-5, CMY-2 512 8C. freundii (variant 2) AmpC[Tyr150Ser] TEM-1, SHV-5, CMY-2 �512 64E. coli 3799 (parent) AmpC TEM-1, CTX-M-15, OXA-2, NDM-3, CMY-6 �512 8E. coli (variant 1) AmpC TEM-1, CTX-M-15, OXA-2, NDM-3, CMY-6[Tyr150Cys] �512 128a ATM, aztreonam; ATM-AVI, aztreonam plus avibactam (at 4 �g/ml).

FIG 5 Resistant mutants. Structural models of resistant variants using the crystal structures of avibactam (pink sticks) and aztreonam (green sticks). (A) Asn346in the avibactam crystal structure is depicted in light pink, while modeled Tyr346 changes are depicted as white sticks. (B) Tyr150 in aztreonam-AmpC structureis depicted in light green, and Tyr150 in avibactam-AmpC is shown in light pink. The modeled Ser150 change is depicted as gray sticks, and the resultingdifference in pocket volume due to this change is depicted in a surface view.

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sible role of Tyr150 in the mechanism of inhibition suggested thatthis change displaced a critical base from the vicinity of acylation.The hydrolysis of aztreonam by a AmpCTyr150Ser variant is notunprecedented, as kinetic studies in laboratory-generated mu-tants have shown that an enzyme carrying this substitution canhydrolyze the substrate, albeit with reduced efficiency, likely bypositioning a water molecule in the mutant (30). To further ex-plore this hypothesis, hydrolysis of aztreonam by the cell extractwas monitored using liquid chromatography-mass spectrometry(LC-MS) in the presence and absence of avibactam. The rate ofaztreonam hydrolysis was impaired in the Tyr150Ser mutant com-pared to that in the parent. The addition of avibactam protectedthe hydrolysis of aztreonam by 36-fold in the parent strain; how-ever, this protection was significantly reduced in the mutantstrain, where only a 2-fold protection was obtained after 22 h ofincubation (see Fig. S3 in the supplemental material). A similarrationale is also applicable for the Tyr150Cys E. coli resistant vari-

ant due to the similarity in the sizes of the substitutions and theirability to position a water molecule for hydrolysis.

DISCUSSION

Inhibition of class C �-lactamases is one of the most significantand unique attributes of avibactam (31, 32). The structures pre-sented here provide a molecular rationale for the stability ofavibactam from hydrolysis by class C enzymes and a hypothesis ofthe mechanisms of acylation and recyclization and of a higher rateof recyclization (9, 33) in this class than those for class A enzymes.

The lack of hydrolysis can be explained by comparing thestructure described here with the well-defined mechanism ofdeacylation in class C enzymes (20, 34). In its covalent bindingmode, the sulfate group of avibactam has completely displaced thedeacylating water molecule (PDB code 1IEL), and in its position isthe N6 atom of the scissile bond, which remains in an optimaltrajectory for the reverse covalent attack on the Ser64 carbamoyl

FIG 6 Scheme of the proposed mechanism. Acylations can proceed via a single-base mechanism (I-III-IV-V) or a two-base mechanism (II-III-IV-VI) whereeither Tyr150 alone or Tyr150 and Lys67 together, respectively, can participate as the general acids-general bases to acylate followed by deacylation viarecyclization.

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linkage. This, along with a stable carbamoyl covalent linkage ofavibactam, explains why intramolecular recyclization of avibac-tam is preferred over water-mediated hydrolysis. A similar hin-drance is also provided by the sulfonate group of aztreonam, re-sulting in its weaker hydrolysis compared to that of other�-lactams, but the flexibility and shorter length of its sulfonatearm prevent complete protection. In contrast, the limited rota-tional freedom and additional length of the sulfate group inavibactam effectively prevent hydrolysis (Fig. 3B). This mecha-nism of protection from hydrolysis is distinct from the class A�-lactamase mechanism, where the hydrolytic water, while stillobserved in the binding mode, was rendered ineffective by thechanged protonation state of the base Glu166 (18).

Based on the observed structure, a mechanism of reversibleacylation and recyclization of avibactam is proposed for class Cenzymes (Fig. 6). The first step for acylation requires the deproto-nation of Ser64 and a subsequent protonation of the cleaved N6nitrogen of avibactam. Both Tyr150 and Lys67 are within hydro-gen-bonding distances to Ser64, suggesting that either or both ofthese residues might participate in proton abstraction. However,there is only one residue, Tyr150, within proximity to N6 ofavibactam to act as a general base for ring opening. Similarly, inthe reverse recyclization reaction, Tyr150 is the only general basein the vicinity that could abstract a proton from N6, whileO�Ser64 could receive a proton from either Tyr150 or Lys67.Taken together, these results suggested that inhibition by avibac-tam might proceed via a single base mechanism (Fig. 6, I-III-IV-V), where Tyr150 functions as the sole catalytic residue acting asboth the general base and the general acid to shuttle the protonfrom O�Ser64 to N6 of avibactam. Alternatively, a conjugatedacid-base mechanism might be envisioned, where Lys67 functionsas the general base and Tyr150 as the general acid during acylation

and vice versa during recyclization (Fig. 6, II-III-IV-VI). Bothmechanisms result in a phenolated Tyr150 in the acylated form(Fig. 6, IV), which is supported by the structural data where thedistance between Tyr150 and Lys67 changes from 3.8 Å to 2.8 Åwhen covalently linked to avibactam. While this structure cannotdistinguish between the single base and the conjugated acid-basemechanisms, insight into the preferred path of electron transfercan be obtained from the resistant variants. The Tyr150Ser andTyr150Cys substitutions, which increase the distance between thegeneral base and O�Ser64 (Fig. 5B), cause weaker but not com-plete loss of inhibition by avibactam. This suggests that anotherresidue is involved in acylation, thus favoring the conjugated acid-base mechanism (Fig. 6, II-III-IV-VI). Further structural and ki-netic experiments are needed to better understand the mechanis-tic complexities.

Lastly, the faster recyclization rate of avibactam in class C en-zymes than in class A �-lactamases can be explained by the differ-ence in location of the catalytic residues involved in acylation anddeacylation (Fig. 7). In contrast to class A enzymes (CTX-M-15)where Glu166 and Ser130, the general bases for acylation and re-cyclization, respectively, were located on the opposite faces of thecarbonyl plane (18), the equivalent residues in class C enzymes arelocated on the same side. The opposite side in the class C bindingpocket, equivalent to the Glu166 face in class A enzymes, is lesspolar and is formed by the hydrophobic side chain of Tyr122. Thelack of polarity and hydrogen-bonding residues in this regionlikely facilitates the intramolecular hydrogen bond observed be-tween the avibactam carboxamide group and the N1 nitrogen ofthe piperidine ring (Fig. 2A). This strong intramolecular hydrogenbond helps to delocalize electrons from the carbonyl plane, thusmaking it more favorable for nucleophilic attack by N6. Theclosed loop of hydrogen bonds between Tyr150-Lys67-Asn152-

FIG 7 Comparison of class A and class C binding pockets. Avibactam bound to AmpC (green sticks) is overlaid on avibactam bound to CTX-M-15 (pink sticks)to show differences in residues interacting with the inhibitor. The residues have been labeled in their respective colors. The water molecule observed at thedeacylating position in CTX-M-15 is shown as a red sphere.

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carboxamide NH2-piperidine N1 in the AmpC binding modelinks the two cleaved ends of the scissile bond, which is able toquickly respond to alterations in electronics between the ring-closed and ring-open forms.

In conclusion, drug resistance can occur through subtlechanges that maintain substrate recognition and catalysis while nolonger permitting optimal inhibitor functionality. Isolates resis-tant to class A �-lactamase inhibitors in current clinical use haveemerged and carry �-lactamases with minor modifications, oftenat positions where the binding mode or the mechanism of theinhibitor differs from that of the substrate (defined as the sub-strate envelope theory) (35). To date, the evolutionary pressure onclass C �-lactamases has been to adapt to the increasing size oflater generations of �-lactam drugs (36). However, they have re-mained naive to any selection pressure that would require theenzyme to compromise catalysis in order to avoid inhibition. Thisis evident by the extremely well-conserved binding pocket nearthe catalytic core among a wide range of chromosomal and plas-midic class C �-lactamases. Avibactam has preserved many of thekey features of �-lactam recognition and acylation to efficientlyexploit the residues that are critical for �-lactam catalysis. Theinteractions of avibactam are limited to this conserved element,which suggests a very high probability of inhibiting all class Cenzymes. In vitro selection experimental results hint that the lackof rotational freedom of avibactam could limit its capacity to in-hibit certain variants, but the ability of avibactam to mimic the keyinteraction of a �-lactam substrate combined with its tight bind-ing is likely to bestow a high “genetic barrier” on the developmentof resistance in the clinic.

ACKNOWLEDGMENTS

We acknowledge Jim Whiteaker, Kathy MacCormack, and Veronica Kosfrom Infection, AstraZeneca R&D Boston, for their contributions towhole-genome sequencing and Joe Patel from Discovery Sciences, Astra-Zeneca R&D Boston, for his help during crystallographic data collection.We thank Wright Nichols, Patricia Bradford, Dave Ehmann, and ThomasDurand-Reville from Infection, AstraZeneca R&D Boston, for carefulreading and constructive feedback during the preparation of the manu-script.

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