AAC Accepts, published online ahead of print on 14 July...

Avibactam and class C β-lactamases: mechanism of inhibition, conservation of binding pocket 1

and implications for resistance. 2

3

S. D. Lahiri1δ, M. Johnstone1, P.L. Ross2, R. McLaughlin1, N. B. Olivier2 and R. A. Alm1 δ 4

5

1Infection Innovative Medicines, AstraZeneca R&D Boston, MA, USA 6

2Discovery Sciences Innovative Medicines, AstraZeneca R&D Boston, MA, USA 7

8

δ To whom correspondence should be addressed. E-mail: [email protected], [email protected]. 9

10

Abstract 11

Avibactam is a novel non-β-lactam β-lactamase inhibitor that inhibits a wide range of β-lactamases. These include 12

class A, class C and some class D enzymes, which erode the activity of β-lactam drugs in multi-drug-resistant 13

pathogens like Pseudomonas aeruginosa and Enterobacteriaceae. Avibactam is currently in clinical development in 14

combination with the β-lactam antibiotics ceftazidime, ceftaroline fosamil and aztreonam. Avibactam has the potential 15

to be the first β-lactamase inhibitor that could provide activity against class C-mediated resistance, which represents 16

a growing concern in both hospital- and community-acquired infections. Avibactam has an unusual mechanism of 17

action: it is a covalent inhibitor that acts via ring-opening but, in contrast to other currently used β-lactamase 18

inhibitors, this reaction is reversible. Here we present a high resolution structure of avibactam bound to a class C β-19

lactamase, AmpC, from P. aeruginosa that provided insight into the mechanism of both acylation and recyclization in 20

this enzyme class and highlighted the differences observed between class A and class C inhibition. Furthermore, 21

variants resistant to avibactam were isolated that identified the residues that are important for inhibition. Finally, the 22

structural information was used to predict effective inhibition by sequence analysis and functional studies of class C 23

β-lactamases from a large and diverse set of contemporary clinical isolates (P. aeruginosa and several 24

Enterobacteriaceae spp.) obtained from recent infections to understand any pre-existing variability in the binding 25

pocket that could affect inhibition by avibactam. 26

27

AAC Accepts, published online ahead of print on 14 July 2014Antimicrob. Agents Chemother. doi:10.1128/AAC.03057-14Copyright © 2014, American Society for Microbiology. All Rights Reserved.

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Introduction 28

The continual emergence of multi-drug resistance in Gram-negative bacteria has eliminated many former treatment 29

options. The β-lactam drug class, once the foundation of treatment regimens for many hospital- and community-30

acquired infections, is rapidly becoming obsolete due to the proliferation of β-lactamases (1-3). Even the 31

carbapenems, which for many years represented the last line of defense, are being eroded by the emerging 32

pandemic of carbapenemases such as metallo-β-lactamase containing pathogens (4, 5). The currently available β-33

lactamase inhibitors, such as clavulanic acid and sulbactam, are effective inhibitors of many of the class A β-34

lactamases, but are incapable of inhibiting any other classes, including class C (6). Chromosomally encoded class C 35

β-lactamases are found in many clinically important pathogens, such as P. aeruginosa and many Enterobacteriaceae 36

(7, 8). In many cases, the expression of this enzyme is inducible; however, they can get derepressed, and the 37

subsequent overexpression results in resistance to many β-lactam drugs. Furthermore, there is a myriad of class C 38

enzymes that are encoded on transferable plasmids that enable horizontal transfer of class C mediated β-lactam 39

resistance between bacterial species. 40

Avibactam (Fig 1A) is a novel non-β-lactam β-lactamase inhibitor that inhibits both class A and class C (and some 41

class D) enzymes, thus providing protection from a diverse range of β-lactamase-mediated resistance mechanism (9-42

11). It is currently in clinical development in combination with the cephalosporins ceftazidime and ceftaroline fosamil, 43

as well as with the monobactam aztreonam (Fig 1B), as alternative therapeutic options for the treatment of infections 44

caused by multi-drug resistant P. aeruginosa and Enterobacteriaceae (12-17). Avibactam is structurally distinct 45

from the clinically used β-lactamase inhibitors in that it does not contain a β-lactam core. In addition, it has a unusual 46

mechanism of inhibition. While the covalent inhibition proceeds in a similar fashion via the opening of the avibactam 47

ring, the reaction is reversible, whereby deacylation results in regeneration of the intact compound as opposed to 48

hydrolysis and turnover (9). This mechanistic difference from clinically used inhibitors contributes towards making 49

avibactam highly effective in providing protection to the β-lactam partner against hydrolysis by chromosomal and 50

plasmidic β-lactamases. 51

We have recently described the mechanism of covalent inhibition of class A enzymes by avibactam as well as a 52

medium resolution structural view of a class C co-complex to rationalize the broad spectrum activity (18). However, 53

the mechanism of inhibition of class C β-lactamases, which is a differentiating attribute of avibactam, could not be 54

confirmed. We now report high resolution P. aeruginosa AmpC structures in complex with avibactam in both the 55

ring-open and ring-closed form, as well as in complex with the monobactam β-lactam, aztreonam. The subsequent 56

analyses have enabled an understanding of the reversible deacylation in class C enzymes as well as a rationale for 57


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the more rapid recyclization observed with this class C enzyme in comparison to class A β-lactamases. In addition, 58

we also evaluated the conservation of the avibactam binding pocket to assess the risk of any pre-existing pool of 59

resistant class C enzymes by sequencing the chromosomal ampC gene from over 500 diverse clinical isolates. 60

61

Materials and Methods 62

Crystallization, Data Collection, Structure Determination, and Refinement. Crystals of AmpC were obtained as 63

described previously (18). Crystals were soaked with a final concentration of 10mM of avibactam or aztreonam for 15 64

minutes followed by flash freezing in liquid nitrogen. Data were collected at Advanced Photon Source in Chicago at 65

IMCA beamline BM16. Data processing and refinement statistics are provided in Supplementary Table S1. 66

Genome sequencing Total genomic DNA was prepared using the Genomic DNA Purification kit in the Maxwell 16 67

instrument (Promega, Madison, WI). DNA libraries were prepared using the Nextera library construction protocol 68

(Illumina, San Diego, CA) and sequenced on a MiSeq Sequencer (Illumina, San Diego, CA). 69

Data analysis Whole genome data were analyzed using the CLC Bio Workbench software v. 5.5.1 (CLC Bio, 70

Cambridge, MA). Multiple sequence alignments were generated using the Clustal X program. Multi-locus sequence 71

typing (MLST) was performed by comparing the assembled genome sequences of each strain to the database of 72

known P. aeruginosa MLST alleles available at www.pubmlst.org. ConSurf, a bioinformatics tool 73

(www.consurf.tau.ac.il), was used to map the multiple sequence alignment to the AmpC:avibactam structure to 74

visualize the 3-dimensional sequence diversity. Pymol (www.pymol.org) was used to display the diversity, and also to 75

model the residue substitutions identified in the resistant variants. 76

Resistance Selection and Antimicrobial Susceptibility Testing. Resistant variants were selected by plating a 77

bacterial suspension on agar plates containing two-fold increasing concentrations of aztreonam with avibactam held 78

constant at 4ug/mL. The minimum inhibitory concentration (MIC) values were determined by the broth microdilution 79

method following the guidelines established by the Clinical Laboratory Standards Institute. 80

Expression Analysis. RNA from mid-logarithmically grown strains was prepared using the Maxwell 16 LEV 81

simplyRNA purification kit from Promega (Fitchburg, WI). A total of 5 ng RNA was used in a RT-PCR assay using the 82

Qiagen Quantitect SYBR Green RT-PCR kit (Germantown, MD) using a BioRad CFX96™ instrument. The 83

oligonucleotides used to detect the expression of the chromosomal blaampC allele in C. freundii were 5’-84

CGAGGGGAAACCTTATTA-3’ and 5’- TGTATAGGTGGCTAAGTG-3’ and the control oligonucleotides to detect 85


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expression of the rpsL ribosomal gene were 5’- TAAAAAACCGAACTCCGCA-3’ and 5’- 86

GTCACTTCAAAACCGTTAG-3’. The level of rpsL expression was used for normalization. 87

88

Results 89

High resolution structure of avibactam bound to P. aeruginosa AmpC 90

Crystals of avibactam covalently bound to P. aeruginosa AmpC were obtained that diffracted to 1.1 Å. The higher 91

resolution provided improved understanding of avibactam binding, in particular the piperidine ring conformation, the 92

position of the carboxamide and sulfate groups, and most importantly, the accurate positions of the residues involved 93

in catalysis (Fig 2A-C). The high resolution allowed observation of an intramolecular connectivity between the amide 94

group and the N1 nitrogen of the piperidine ring (Fig 2A), suggestive of a short strong hydrogen bond (<2.5 Å) 95

between these two moieties. This interaction is novel to the class C binding mode of avibactam, as the bound 96

structure of a class A β-lactamase at a comparable resolution (18) showed an increased distance between these 97

atoms with no connectivity of electron density. Further, there was additional density observed between the cleaved 98

C7-N6 bonds of the pyrazolidine ring (Fig 2A). The most plausible explanation for this additional density is that a 99

small subpopulation of ring-closed or intermediate state of the inhibitor exists simultaneously in this position. 100

However, refinement in the presence of a closed form of avibactam could not convincingly distinguish the two forms 101

of avibactam and hence the model was refined with the open covalently linked inhibitor (Fig 2B). 102

The refined structure showed that eight residues provided three key contributions to the binding interactions with 103

avibactam (Fig 2C). The carboxamide group of avibactam interacted with the side chains of Asn152 and Gln120, the 104

sulfate moiety was positioned by Thr316, Lys315 and Asn346, whereas Tyr150 and Lys67 were positioned to 105

participate in catalytic roles to enable formation of the covalent bond with Ser64. Specifically, the Oη of Tyr150 was 106

equidistant, 3.3Å and 3.2Å respectively, from N6 of avibactam and Oγ of Ser64. The Nζ of Lys67 was 2.9Å from the 107

Oγ of Ser64 and 4.9 Å from N6 of avibactam. Comparison of the apo structure at a similar resolution showed a 108

minimal shift in the binding-pocket residues upon acylation with all residues superimposing exactly except Tyr150, 109

Lys67, and Asn152 (Fig 2D). In the avibactam bound form, the side chain of Tyr150 moves closer to Lys67 by 1.0 Å, 110

indicative of the formation of a new hydrogen bond upon acylation. In addition, a new hydrogen bond is also formed 111

between the carboxamide group of avibactam and Asn152 side chain. Asn152 maintains its hydrogen bond to Lys67 112

upon acylation, that results in a shift in the position of these residues. 113


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114

Comparison to a substrate binding mode 115

To understand the differences in interactions between a β-lactam drug and avibactam, the structure of aztreonam 116

bound to P. aeruginosa AmpC was solved to 1.3 Å (Fig 3A). Aztreonam contains a sulphonyl group in place of the 117

carboxylate group seen in other β-lactam antibiotics, which makes it structurally closer to avibactam than other β-118

lactams. In addition, whereas aztreonam is hydrolyzed by class C β-lactamases resulting in decreased susceptibility, 119

the rate of hydrolysis is slower than that observed for other β-lactams (6), making it feasible to capture the acyl-120

enzyme complex crystallographically. The structure showed that the position of the acylated aztreonam molecule in 121

P. aeruginosa AmpC was very similar to that previously observed in a Citrobacter freundii class C enzyme, with the 122

exception of the position of the oxyimino group (19). There was a significant similarity in the binding mode and 123

interacting residues between aztreonam and avibactam (Fig 3B). The sulfonyl group of aztreonam and the sulfate 124

group of avibactam were similarly located and interacted with Lys315, Thr316 and Asn346. Additionally, both groups 125

displace the deacylating water molecule (WAT) previously observed in crystal structures (PDB code 1IEL) (20). The 126

position of the N1 of aztreonam upon ring cleavage was in a similar location to N6 of avibactam, although the C3-C4 127

bond of the aztreonam β-lactam ring underwent a rotation of approximately 70⁰ upon ring opening, which displaced 128

the trajectory for recyclization. The side chains of Tyr150 and Lys67 were in identical positions in the two binding 129

modes, as were the interactions of Asn152 with both Lys67 and the ligands. However, in the case of aztreonam, 130

Asn152 formed a hydrogen bond with the carbonyl oxygen, which is opposite in polarity to the amide group of 131

avibactam. Overall, the hydrogen bonding patterns and the key interactions in the acylated forms were very similar 132

between the substrate (aztreonam) and the inhibitor (avibactam) despite the differences in the compound structures. 133

The major differences lay in the overall size and the limited rotational freedom of avibactam compared to aztreonam. 134

135

Conservation of Class C β-lactamase enzymes 136

Given the emerging resistance to β-lactams in P. aeruginosa and evidence that small sequence differences can affect 137

the substrate spectrum (7), there was a need to understand whether avibactam could be expected to inhibit all of the 138

AmpC enzyme variants in this species. There were 36 unique AmpC sequences from P. aeruginosa, representing 67 139

different isolates, available in the public domain (7, 21). An additional 464 isolates, obtained from 25 different 140

countries and isolated from multiple clinical indications from 2008 to 2012 were sequenced. Multi locus sequence 141

typing (MLST) analysis demonstrated a high level of diversity among the isolates. The 531 P. aeruginosa AmpC 142


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proteins were clustered into groups representing 72 unique sequences. The relative population within each cluster 143

varied, with the largest cluster representing 109 (20.5%) of the isolates examined, whereas 35 clusters were 144

represented by a single isolate (Supplementary section, Fig S1 and Table S2). Although amino acid variations were 145

observed in over 22% of the residues, the eight residues directly involved in avibactam binding were highly 146

conserved, with six of them being conserved in all proteins and the other two (Ser64 and Asn346) having one and 147

two variants, respectively (Table 1). Of these, the isolate containing a Ser64Leu substitution was expected to be non-148

functional because Ser64 is critical for covalent catalysis (22). The changes Asn346Thr and Asn346Ile were both to an 149

amino acids of a similar size, of which, the Asn346Ile substitution also changed the property of the substitution from a 150

polar to a hydrophobic group. 151

Given that an important attribute of avibactam is the inhibition of Class C β-lactamases, the conservation analyses 152

were extended to other Class C enzymes in clinically relevant bacterial pathogens, including the chromosomal AmpC 153

proteins of Escherichia coli, Enterobacter cloacae, Enterobacter aerogenes, and Citrobacter spp. and plasmidic 154

enzymes that can carried by multiple species (Table 1). In the case of E. cloacae, where derepession of the 155

chromosomal blaampC gene is a common resistance mechanism, 79 recent clinical isolates were sequenced, and their 156

chromosomal AmpC proteins, along with 19 sequences from the public domain, were clustered into 57 unique AmpC 157

sequences with almost 40% of the amino acid residues varying among these sequences (Supplementary section, Fig 158

S1 and Table S3). Similarly, unique chromosomal AmpC proteins from E. coli, E. aerogenes and Citrobacter spp. 159

along with 127 plasmidic class C β-lactamase enzymes from public and internal data were also included in the 160

conservation analysis. Despite the dramatic sequence variability observed between these class C enzymes (Fig 4A), 161

Lys67, Tyr150, Asn152 and Lys315 were completely conserved (Fig 4B). A small number of isolates contained 162

variations at Ser64 (n=1), Gln120 (n=2) and Thr316 (n=1). Asn346 was the most variable of the binding pocket 163

residues: three variants among the chromosomal enzymes and 20 plasmidic class C enzymes carried the Asn346Ile 164

substitution (Table 1). This modification had been previously attributed to an extended-spectrum cephalosporinase 165

activity (23, 24). 166

Microbiological susceptibility tests were performed in P. aeruginosa and E. cloacae isolates where the chromosomal 167

blaampC was derepressed and contained no other β-lactamases to enable clear interpretation of the ability of 168

avibactam to inhibit these chromosomal AmpC variants. The susceptibility data confirmed that the restoration of β-169

lactam activity by 4 μg/mL avibactam was not affected by variations in these enzymes and restored the ceftazidime 170

and aztreonam MIC values to susceptible range as defined by CLSI (representative MIC values shown in Table 2). 171

The only variations in the 8 key avibactam binding residues (Table 1) that could not be verified were the Gln120Lys, 172


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which was not available for testing and the chromosomal Thr316Met which was not derepressed in the isolate. Taken 173

together, these data suggest that there is not a significant pool of pre-existing class C-based resistance to avibactam 174

in clinical isolates of Gram-negative pathogens. 175

Resistance to avibactam in combination with a β-lactam 176

Given the conservation of the avibactam binding-pocket residues and the importance of these residues in β-lactam 177

recognition and catalysis (25-29), we investigated whether any variations in these could compromise the inhibition 178

potency of avibactam while still allowing hydrolysis of the β-lactam drug and thus result in resistance. Spontaneous 179

resistance frequency experiments were carried out in several isolates carrying class C β-lactamases, with initial focus 180

on Enterobacteriaceae spp. where aztreonam in combination with avibactam was more potent than in P. aeruginosa. 181

Resistant variants from a C. freundii and an E. coli isolate that carried multiple β-lactamase enzymes were obtained 182

at low frequencies of 6 x 10-10 and 6 x 10-9, respectively. These mutants had an 8- to 64-fold increase in the 183

aztreonam-avibactam MIC compared with the parental isolate (Table 3). Whole genome sequence analyses of the 184

parent and the daughter strains identified the mutations responsible for the loss of susceptibility. The variation in the 185

resistant C. freundii strains were either a Asn346Tyr or a Tyr150Ser change in the chromosomal AmpC protein, which 186

was confirmed by RT-PCR analysis to be stably derepressed in both the parent and daughter strains to equivalent 187

levels (Table 3 and supplementary Fig S2). The variation in the E. coli mutant was a Tyr150Cys substitution in the 188

plasmid-encoded class C enzyme (CMY-6). 189

Structural models of the AmpC mutations indicated that the Asn346Tyr substitution would result in a steric clash with 190

the sulfate group of avibactam, thus influencing the binding affinity of the inhibitor (Fig 5A). Whereas a similar impact 191

to the sulphonyl of aztreonam is expected, the slightly greater distance between this group and the side chain, as well 192

as the greater flexibility of aztreonam, could still accommodate binding and hydrolysis. While the Tyr150Ser 193

substitution maintains a similar polarity, the reduced size of the Ser residue increases the binding pocket volume 194

(Fig5B). The rigid binding mode of avibactam and the possible role of Tyr150 in the mechanism of inhibition 195

suggested that this change displaced a critical base from the vicinity of acylation. The hydrolysis of aztreonam by a 196

AmpCTyr150Ser variant is not unprecedented, as kinetic studies in laboratory generated mutants have shown that an 197

enzyme carrying this substitution can hydrolyze the substrate, albeit with reduced efficiency, likely by positioning a 198

water molecule in the mutant (30). To further explore this hypothesis, hydrolysis of aztreonam by the cell extract was 199

monitored using LC/MS in the presence and absence of avibactam. The rate of aztreonam hydrolysis was impaired in 200

the Tyr150Ser mutant compared to the parent. The addition of avibactam protected the hydrolysis of aztreonam by 36-201

fold in the parent strain; however, this protection was significantly reduced in the mutant strain, where only a 2-fold 202


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protection was obtained after 22 h incubation (supplementary Fig S3). A similar rationale is also applicable for the 203

Tyr150Cys E. coli resistant variant due to the similarity in size of the substitutions and their ability to position a water 204

molecule for hydrolysis. 205

206

Discussion 207

Inhibition of class C β-lactamases is one of the most significant and unique attributes of avibactam (31, 32). The 208

structures presented here provide a molecular rationale for the stability of avibactam from hydrolysis by class C 209

enzymes and a hypothesis of the mechanism of acylation and recyclization as well as for a faster rate of recyclization 210

(9, 33) in this class compared to class A enzymes. 211

The lack of hydrolysis can be explained by comparing the structure described here with the well defined mechanism 212

of decylation in class C enzymes (20, 34). In its covalent binding mode, the sulfate group of avibactam has 213

completely displaced the deacylating water molecule (PDB code 1IEL) and in its position is the N6 atom of the 214

scissile bond which remains in an optimal trajectory for the reverse covalent attack on the Ser64 carbamoyl linkage. 215

This, along with a stable carbamoyl covalent linkage of avibactam, explains why intramolecular recyclization of 216

avibactam is preferred over water-mediated hydrolysis. A similar hindrance is also provided by the sulfonate group of 217

aztreonam resulting in its weaker hydrolysis compared to other β-lactams, but the flexibility and shorter length of its 218

sulfonate arm prevents complete protection. In contrast, the limited rotational freedom and the additional length of 219

the sulfate group in avibactam effectively prevent hydrolysis (Fig 3B). This mechanism of protection from hydrolysis is 220

distinct from the class A β-lactamase mechanism, where the hydrolytic water, while still observed in the binding 221

mode, was rendered ineffective by the changed protonation state of the base Glu166 (18). 222

Based on the observed structure, a mechanism of reversible acylation and recyclization of avibactam could be 223

proposed for class C enzymes (Fig 6). The first step for acylation would require the deprotonation of Ser64 and a 224

subsequent protonation of the cleaved N6 nitrogen of avibactam. Both Tyr150 and Lys67 are within hydrogen-225

bonding distances to Ser64, suggesting that either or both of these residues could participate in proton abstraction. 226

However, there is only one residue, Tyr150, within proximity to N6 of avibactam to act as a general base for ring 227

opening. Similarly, in the reverse recyclization reaction, Tyr150 is the only general base in the vicinity that could 228

abstract a proton from N6, while OγSer64 could receive a proton from either Tyr150 or Lys67. Taken together, this 229

suggested that inhibition by avibactam could either proceed via a single base mechanism (Fig 6, I-III-IV-V), where 230

Tyr150 functions as the sole catalytic residue acting as both the general base and the general acid to shuttle the 231

proton from OγSer64 to N6 of avibactam. Alternatively, a conjugated acid-base mechanism can be envisioned, 232

where Lys67 functions as the general base and Tyr150 as the general acid during acylation, and vice versa during 233


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recyclization (Fig 6, II-III-IV-VI). Both mechanisms would result in a phenolated Tyr150 in the acylated form (Fig 6, 234

IV), which is supported by the structural data where the distance between Tyr150 and Lys67 changes from 3.8Å to 235

2.8Å when covalently linked to avibactam. While this structure cannot distinguish between the single base and the 236

conjugated acid-base mechanisms, insight to the preferred path of electron transfer can be obtained from the 237

resistant variants. The Tyr150Ser and Tyr150Cys substitutions, which increase the distance between the general base 238

and the OγSer64 (Fig 5B) cause weaker but not complete loss of inhibition by avibactam. This suggests that another 239

residue is involved in acylation, thus favoring the conjugated acid-base mechanism (Fig 6, II-III-IV-VI). Further 240

structural and kinetic experiments are needed to better understand the mechanistic complexities. 241

Lastly, the faster recyclization rate of avibactam in class C enzymes compared to A β-lactamases can be explained 242

by the difference in location of the catalytic residues involved in acylation and deacylation (Figure 7). In contrast to 243

class A enzymes (CTX-M-15) where Glu166 and Ser130, the general bases for acylation and recyclization, 244

respectively, were located on the opposite faces of the carbonyl plane (18), the equivalent residues in class C 245

enzymes are located on the same side. The opposite side in the class C binding pocket, equivalent to the Glu166-246

face in class A enzymes, is less polar and formed by the hydrophobic side chain of Tyr122. The lack of polarity and 247

hydrogen-bonding residues in this region likely facilitates the intramolecular hydrogen bond observed between the 248

avibactam carboxamide group and the N1 nitrogen of the piperidine ring (Fig 2A). This strong intramolecular 249

hydrogen bond helps to delocalize electrons from the carbonyl plane, thus making it more favorable for nucleophilic 250

attack by N6. The closed loop of hydrogen bonds between Tyr150-Lys67-Asn152-carboxamide NH2-piperidine N1 in 251

the AmpC binding mode links the two cleaved ends of the scissile bond, which is able to quickly responsive to 252

alterations in electronics between the ring-closed and ring-open forms. 253

Concluding remarks. Drug resistance can occur through subtle changes that maintain substrate recognition and 254

catalysis while no longer permitting optimal inhibitor functionality. Isolates that are resistant to class A β-lactamase 255

inhibitors in current clinical use have emerged that carry β-lactamases with minor modifications, often at positions 256

where the binding mode or the mechanism of the inhibitor differs from that of the substrate, defined as substrate 257

envelope theory (35). To date, the evolutionary pressure on class C β-lactamases has been to adapt to the 258

increasing size of later generations of β-lactam drugs (36). However, they have remained naïve to any selection 259

pressure that would require the enzyme to compromise catalysis in order to avoid inhibition. This is evident by the 260

extremely well conserved binding pocket near the catalytic core among a wide range of chromosomal and plasmidic 261

class C β-lactamases. Avibactam has preserved many of the key features of β-lactam recognition and acylation to 262

efficiently exploit the residues that are critical for β-lactam catalysis. The interactions of avibactam are limited to this 263


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conserved element, which suggests a very high probability of inhibiting all class C enzymes. In vitro selection 264

experiments hint that the lack of rotational freedom of avibactam could limit its capacity to inhibit certain variants, but 265

the ability of avibactam to mimic the key interaction of a β-lactam substrate combined with its tight binding is likely to 266

bestow a high “genetic barrier” to the development of resistance in the clinic. 267

Acknowledgments All authors are employees of AstraZeneca R&D Boston. The authors acknowledge Jim 268

Whiteaker, Kathy MacCormack and Veronica Kos from Infection, AstraZeneca R&D Boston, for their contributions in 269

whole genome sequencing. We acknowledge Joe Patel from Discovery Sciences, AstraZeneca R&D Boston, for his 270

help during crystallographic data collection. Lastly, we are thankful to Wright Nichols, Patricia Bradford, Dave 271

Ehmann and Thomas Durand-Reville from Infection, AstraZeneca R&D Boston, for careful reading and constructive 272

feedback during the preparation of this manuscript. 273

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102911. 364

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Table 1: Conservation analysis of key residues in chromosomal and plasmidic class C β-lactamase enzymes 366

Class C b-lactamase enzyme Number of

unique variants

Variants with conserved residues (changes observed)

Ser64 Lys67 Gln120 Tyr150 Asn152 Lys315 Thr316 Asn346

Chromosomal

P. 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) 57

E. 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 16

Citrobacter spp. AmpC 15 15 15 15 15 15 15 15 15

Plasmidic

CMY 84 84 84 83 (1-Lys) 84 84 84 84 79 (5-Ile)

DHA 6 6 6 6 6 6 6 6 6

FOX 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 6

ACT 13 13 13 13 13 13 13 13 13

CFE 1 1 1 1 1 1 1 1 1

LAT 1 1 1 1 1 1 1 1 1

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Table 2 Susceptibility analyses of Pseudomonas aeruginosa isolates carrying different AmpC variations 368

CAZ = ceftazidime; ATM = aztreonam; AVI = avibactam (constant concentration of 4 µg/mL) 369

a Denotes unique AmpC sequence, see supplementary data Figure S1 and Table S1 for details 370

b The AZPC-48 sequence carries the Asn346Ile substitution, in all other AZPC clusters listed all eight key residues that interact 371 with avibactam are fully conserved 372

373

Strain AmpC genotypea MIC (µg/mL)

CAZ CAZ-AVI ATM ATM-AVIATCC 27853 AZPC-5 2 1 4 4

PAO1 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-48b 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 1


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Table 3: Susceptibility and β-lactamase profiles of aztreonam- and aztreonam-avibactam-resistant 374

Enterobacteriaceae variants 375

Strain β-lactamase content MIC (µg/mL)

Chromosome Plasmid ATM ATM-AVI

C. freundii 3885 (parent)

AmpC TEM-1, SHV-5, CMY-2 512 1

C. freundii (variant 1)

AmpC[Asn346Tyr] TEM-1, SHV-5, CMY-2 512 8

C. freundii (variant 2)

AmpC[Tyr150Ser] TEM-1, SHV-5, CMY-2 >512 64

E. coli 3799 (parent)

AmpC TEM-1, CTX-M-15, OXA-2, NDM-3, CMY-6 >512 8

E. coli (variant 1)

AmpC TEM-1, CTX-M-15, OXA-2, NDM-3, CMY-6[Tyr150Cys] >512 128

ATM = aztreonam, ATM-AVI = aztreonam plus avibactam (at 4 μg/mL) 376

377

378 on June 26, 2018 by guesthttp://aac.asm

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379

380

381

Fig 1 Chemical structure of avibactam (A) and aztreonam (B) 382

383

384

385

386

387


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A 388

389

B 390


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C 391

D 392


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Fig 2: Avibactam binding properties in AmpC Avibactam binding pocket in AmpC. (A) The unbiased Fo-Fc 393

electron density (2.9-σ cutoff, green mesh) of avibactam (pink) prior to its addition into the refinement cycle. The 394

connectivity of electron density between carboxamide and pyperidine ring is highlighted in blue arrow while the 395

additional density between observed between the cleaved C7-N6 bonds of the pyrazolidine ring is shown in green 396

arrow (B) Avibactam (pink sticks) and the neighboring residues (gray stick) depicted along with the final refined 2Fo-397

Fc map (1.5-σ cutoff, blue mesh). (C) Distances of Tyr150 (green) and Lys67 (blue) from avibactam (pink) and Ser64 398

(grey). (D) Overlay of avibactam binding pocket (grey sticks) over apo pocket (yellow sticks). Only residues with 399

changed position in apo structure has been shown. Avibactam is depicted in pink sticks. Distances between residues 400

in avibactam structure are depicted in blue lines and labels, and those of apo in yellow. 401

402


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A 403

B 404

405


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Fig 3: AmpC crystal structure with aztreonam Aztreonam bound to AmpC (A) The unbiased Fo-Fc electron 406

density (2.9-σ cutoff, blue mesh) of aztreonam (dark green stick) covalently bound to AmpC active site Ser64 (light 407

green stick). (B) Overlay of aztreonam binding pocket (green) on avibactam binding pocket (pink). The ceftazidime 408

and deacylating water molecule (WAT) from the ceftazidime-AmpC crystal structure. 409

410


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411

412

A413

414

Fig 4: Conservation Conservation of avibactam binding pocket has been mapped on the AmpC crystal structure. (A) 415

Sequence conservation of Class C residues is depicted as a surface map where cyan represents variable regions 416

while purple represents conserved residues. Avibactam is depicted in white stick. (B) Residues in the binding pocket 417

of avibactam (white stick) interacting via their side chains are depicted in stick and color-coded based on 418

conservation. 419

420

421

422

423

424


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A 425

426

B 427

Fig 5: Resistant mutants. Structural model of resistant variants using crystal structures of avibactam (in pink stick) 428

and aztreonam (in green stick). (A) Asn346 in the avibactam crystal structure is depicted in light pink while modeled 429

Tyr346 change has been depicted in white stick. (B) Tyr150 in aztreonam-AmpC structure is depicted in light green, 430

and Tyr150 in avibactam-AmpC is shown in light pink. The modeled Ser150 change has been depicted in grey stick 431

and the resulting difference in pocket volume due to this change has been depicted in a surface view.. 432


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433

434


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Fig 6: Potential mechanism scheme Scheme of proposed mechanism: acylations can proceed via a single base 435

mechanism (I-III-IV-V) or a two base mechanism (II-III-IV-VI) where either Tyr150 alone or Tyr150 and Lys67 436

together, respectively, can participate as the general acids-general bases to acylate followed by deacylate via 437

recyclization. 438

439


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440

441

Fig 7: Class A vs. Class C Comparison of Class A and Class C binding pocket. Avibactam bound to AmpC (green) 442

has been overlaid on avibactam bound to CTX-M15 (pink) to show differences in residues interacting with the 443

inhibitor. The residues have been labeled in their respective colors. The water molecule observed at the deacylating 444

position in CTXM-15 is shown as a red sphere. 445

446


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AAC Accepts, published online ahead of print on 14 July...

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