Reversibility of Covalent, Broad-Spectrum Serine Î²-Lactamase

Article Cite This: ACS Infect. Dis. 2017, 3, 833-844

pubs.acs.org/journal/aidcbc

Reversibility of Covalent, Broad-Spectrum Serine β‑Lactamase Inhibition by the Diazabicyclooctenone ETX2514 Adam B. Shapiro,*,† Ning Gao,‡ Haris Jahić,§ Nicole M. Carter,† April Chen,† and Alita A. Miller† †

Entasis Therapeutics, 35 Gatehouse Drive, Waltham, Massachusetts 02451, United States Discovery Sciences, AstraZeneca R&D Boston, 35 Gatehouse Drive, Waltham, Massachusetts 02451, United States § Infection Innovative Medicines, AstraZeneca R&D Boston, 35 Gatehouse Drive, Waltham, Massachusetts 02451, United States ‡

S Supporting Information *

ABSTRACT: ETX2514 is a non-β-lactam serine β-lactamase inhibitor in clinical development that has greater potency and broader spectrum of βlactamase inhibition than the related diazabicyclooctanone avibactam. Despite opening of its cyclic urea ring upon acylation, avibactam can recyclize and dissociate intact from certain β-lactamases. We investigated reversibility of ETX2514 acylation of 10 serine β-lactamases representing Ambler classes A, C, and D. Dissociation rate constants varied widely between enzymes and were lowest for class D. For most enzymes, the covalent adduct mass was that of ETX2514 (277 Da). OXA-10 was acylated with 277 and 197 Da adducts, consistent with loss of the sulfate moiety. KPC-2 showed only the 197 Da adduct. ETX2514 recyclized and dissociated intact from AmpC, CTX-M-15, P99, SHV-5 and TEM-1 but not from KPC-2, OXA-10, OXA-23, OXA-24, or OXA-48. Inactivation partition ratios were 1 for all enzymes except KPC-2, for which it increased to 3.0 after 2 h. This result and mass spectrometry showed that KPC-2 very slowly degraded ETX2514. Nevertheless, ETX2514 restored β-lactam activity to equal potency against isogenic Pseudomonas aeruginosa strains each overexpressing one of the 10 β-lactamases. KEYWORDS: β-lactamase, β-lactamase inhibitor, antibacterial, antibiotic, Acinetobacter baumannii, avibactam ETX2514 is a diazabicyclooctenone β-lactamase inhibitor in clinical development, related to avibactam but containing an endocyclic double bond and a methyl substituent (Figure 1).

M

uch attention has been paid in recent years to the need to develop new antibacterial drugs to combat drugresistant infections.1−4 One important cause of drug resistance in bacterial pathogens is the spread of the genes for numerous β-lactamase enzymes.5−8 These enzymes degrade β-lactam antibacterial drugs, a class that includes penicillins, cephalosporins, carbapenems, and monobactams.9 One key strategy to counter this mechanism of resistance is to combine a βlactam antibacterial drug with a β-lactamase inhibitor.9−12 Several β-lactam/β-lactamase inhibitor combinations are in current clinical use, including amoxicillin/clavunic acid, ampicillin/sulbactam, piperacillin/tazobactam, ceftolozane/tazobactam, and ceftazidime/avibactam.9 β-Lactamases have been grouped into classes A, B, C, and D based on their amino acid sequence relationships.13,14 Classes A, C, and D are referred to as serine β-lactamases because of the involvement of a nucleophilic serine residue in the catalytic active site. Class B enzymes are zinc-dependent metallo-βlactamases. Clavulanic acid, sulbactam, and tazobactam, which are themselves β-lactams, are inhibitors of class A β-lactamases, predominantly. The recently introduced diazabicyclooctanone (DBO) derivative avibactam is the first approved non-β-lactam β-lactamase inhibitor. Avibactam inhibits class A and C βlactamases and the class D OXA-48 enzyme,15 a significant cause of carbapenem resistance in Enterobacteriaceae pathogens.16 © 2017 American Chemical Society

Figure 1. Chemical structure of avibactam (left) and ETX2514 (right).

These differences enhance its potency as a β-lactamase inhibitor compared with avibactam and extend its utility to inhibition of a broad range of class D β-lactamases17 that contribute to βlactam resistance, especially in Acinetobacter baumannii.8 Avibactam was previously shown to have a reversible covalent mechanism of inhibition with certain β-lactamases, in which the carbamoylated enzyme is able to recyclize to the cyclic urea of avibactam, allowing the intact compound to dissociate from the enzyme.18 With some β-lactamases, the avibactam adduct loses an 80 Da moiety, presumably caused by loss of SO3.15 In this Received: July 25, 2017 Published: August 23, 2017 833

DOI: 10.1021/acsinfecdis.7b00113 ACS Infect. Dis. 2017, 3, 833−844

ACS Infectious Diseases

Article

Figure 2. Recovery at 22 °C of β-lactamase activity after jump dilution of ETX2514-enzyme complexes. Solid lines, enzyme without ETX2514. Dashed lines, ETX2514-enzyme complexes.

classes A, C, and D. Partial loss of uninhibited enzyme activity was observed in each case as nonlinearity of the reaction progress curve. Possible reasons for this include inhibition of the enzymes by the product of the reaction with nitrocefin, loss of the enzyme due to adsorption to the assay plate surface, and thermal instability of the enzyme. When the effect was also seen with the ETX2514-inhibited enzyme, the off-rate constant was calculated based on less than the full 2-h period. A wide range of off-rate constants was observed (Table 1). The most rapid

paper, we investigate the reversibility of ETX2514 acylation of a set of β-lactamases representing classes A, C, and D.

■

RESULTS AND DISCUSSION β-Lactamase off-Rate Constants. Recovery during a 2-h continuous measurement at ambient temperature of βlactamase activity after jump dilution of ETX2514-enzyme complexes is shown in Figure 2. Off-rate constants (koff) were measured, when possible, for 10 β-lactamases representing 834



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Table 1. ETX2514 koff Measurements for β-Lactamases from Jump Dilution Experimentsa class A A A A C C D D D D

enzyme CTX-M-15 KPC-2 SHV-5 TEM-1 P. aeruginosa AmpC E. cloacae P99 OXA-10 OXA-23 OXA-24 OXA-48

μM enzyme in preincubation for Figure 2 expt 5 27.5 15 2 7.5 4 10 5 5 5

koff (s−1) (±SD) 2.2 1.0 5.5 1.4 4 3.4 3.4 1.1 1.7 2.5

−4

(±0.5) × 10 (±0.1) × 10−3 (±0.3) × 10−4 (±0.2) × 10−3 (±1) × 10−3 (±0.5) × 10−4 (±0.1) × 10−6 (±0.04) × 10−5 (±0.1) × 10−5 (±0.3) × 10−5

kinact/Ki (M‑1s‑1)17 7 9.3 6.4 1.4 9 2.3 9 5.1 9 8

(±2) × 106 (±0.6) × 105 (±0.5) × 106 (±0.6) × 107 (±5) × 105 (±0.4) × 106 (±2) × 103 (±0.2) × 103 (±2) × 103 (±2) × 105

a

Averages and standard deviations (in parentheses) are for 3-4 separate experiments for class A and C enzymes and for triplicates within a single experiment for class D enzymes.

Figure 3. Recovery at 37 °C of class D β-lactamase activity after jump dilution of ETX2514-enzyme complexes. Replicates are shown in black, red, and blue.

recovery of activity was seen with TEM-1 and P. aeruginosa AmpC. In Figure 2, it appears that recovery of the activity of some of the β-lactamases (CTX-M-15, KPC-2, SHV-5, P99) was incomplete, despite sufficient time elapsing. This was due to the fact that the dilution of the enzyme-ETX2514 mixtures was insufficient to ensure that no significant inhibition of the enzyme would occur at the final inhibitor and enzyme concentrations, as observed in the raw data used to calculate kinact/Ki in ref 17 (data not shown). Greater dilution was impractical, since that would result in such low enzyme concentrations that their activity could not be measured. For CTX-M-15, KPC-2, SHV-5, and P99, partial inhibition persisted even 24 h after 250,000-fold dilution (data not shown).

There was little or no recovery of activity of the 4 class D enzymes OXA-10, OXA-23, OXA-24, and OXA-48 during the 2-h period at 22 °C. Only slight recovery was observed after 24 h (data not shown). In order to measure off-rate constants for these enzymes, initial rates were sampled at various times over a more prolonged period of up to 70 h at 37 °C (Figure 3). The higher temperature was likely responsible for the somewhat faster recovery of activity than would have been expected based on the results shown in Figure 2. Values of koff for ETX2514 with the class D enzymes measured by this technique were nevertheless approximately 2 orders of magnitude lower than for class A and C enzymes. Lahiri et al.28 explained that slow recyclization of avibactam in class D enzymes, combined with exclusion of hydrolytic water from the active site, is responsible for the low koff of 835



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Figure 4. Acylation exchange experiment between ETX2514-TEM-1 complex and CTX-M-15 after 1 h.

Figure 5. Acylation exchange experiment between ETX2514-KPC-2 complex and CTX-M-15. (A) Samples after 19 h of incubation. (B) % of KPC-2 acylated as a function of incubation time in the absence of acceptor enzyme. Markers and error bars represent the means and standard deviations, respectively, of measurements made on triplicate samples. % acylation was calculated based on peak areas of the two KPC-2 peaks.

The values of koff obtained here for ETX2514 are within 10fold, in each case, of the koff values measured for avibactam with the same β-lactamases reported by Ehmann et al.15 Acylation Exchange. Kinetic measurements do not distinguish between dissociation of the intact inhibitor, as was

avibactam from these enzymes. The authors suggested several possible enzyme structure−function correlates that could contribute to the slow rate of recyclization. The exploration of these correlates to the low koff of ETX2514 is a topic for future research. 836



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Figure 6. Acylation exchange experiment between ETX2514-P99 complex and CTX-M-15. (A) Samples after 19 h of incubation. (B) Timedependent changes in the sample with CTX-M-15 added to the ETX2514-P99 complex.

observed with avibactam with some β-lactamases,15,18 or dissociation of a modified product. To further investigate the disposition of the enzyme-bound inhibitor, we performed acylation exchange experiments similar to those reported for avibactam.15,18 In this experiment, the donor enzyme was acylated with ETX2514, the free ETX2514 was removed, and an acyl acceptor enzyme was added. If ETX2514 dissociated intact, it could be transferred to the acceptor enzyme, which was detected by protein mass spectrometry. The ETX2514acylated enzymes tested were CTX-M-15, SHV-1, TEM-1, KPC-2, P. aeruginosa AmpC, Enterobacter cloacae P99, OXA-10, OXA-23, OXA-24, and OXA-48. The acceptor enzyme in most cases was CTX-M-15, which was chosen because of its high acylation rate and low koff (Table 1). When CTX-M-15 was the donor, OXA-48 was used as the acceptor. TEM-1, a class A β-lactamase, treated with ETX2514 had the expected +277 Da adduct mass for the covalent addition of the compound to the enzyme (Figure 4). When CTX-M-15 was added to the ETX2514-TEM-1 covalent complex from which unincorporated ETX2514 had been removed, the inhibitor was exchanged between the enzymes, resulting in CTX-M-15 with the +277 Da adduct. This result is consistent with the ability of ETX2514 to dissociate intact from TEM-1, with reclosing of the urea ring that opens when the compound acylates the enzyme. The phenomenon was also observed with avibactam.18 The exchange of ETX2514 from TEM-1 to CTX-M-15 occurred within 1 h, consistent with the relatively high koff of

the ETX2514-TEM-1 complex (Table 1). There was no evidence for loss of mass from ETX2514 when in complex with TEM-1 or CTX-M-15. Thus, koff for the ETX2514-TEM-1 complex represents, at least in part, dissociation of intact ETX2514. (Dissociation of modified ETX2514 would not be detected by this method.) A similar result was obtained with another class A β-lactamase, SHV-5, although it required about 2 h for the exchange to reach equilibrium, consistent with the slower koff of SHV-5 compared with TEM-1 (Supplemental Figure S1). Because of the low koff for dissociation of ETX2514 from CTX-M-15 (Table 1), when CTX-M-15 was the donor enzyme, substantial exchange of ETX2514 between CTX-M-15 and the acceptor enzyme OXA-48 required 18 h (Supplemental Figure S2). Note that time courses for acylation exchange are not simply related to koff. Acylation exchange rates are also affected by rebinding to the donor, the relative rate constants for acylation of donor and acceptor, and the relative concentrations of donor and acceptor. When the class A β-lactamase KPC-2 was used as the donor in the acylation exchange experiment, the result was quite different from that seen with TEM-1 and SHV-5 (Figure 5A). The adduct mass with KPC-2 was +197 Da instead of +277 Da. The difference of 80 Da suggests a loss of SO3. This was also observed with avibactam acylation of KPC-2.15 The proportion of the peaks for the +197 Da KPC-2 adduct to unacylated KPC2 decreased with time (Figure 5B), indicating gradual deacylation of the enzyme consistent with the koff measurement 837



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Figure 7. Acylation exchange experiment between ETX2514-OXA-10 complex and CTX-M-15. (A) Time-dependent changes in the ETX2514OXA-10 complex. (B) Samples after 17 h of incubation.

Figure 8. Acylation exchange experiment between ETX2514-OXA-24 complex and CTX-M-15 after 17 h of incubation.

which reached a final rate within 2 h that was lower than the rate of the uninhibited enzyme. When 20 μM ETX2514 was incubated for 2 h at 37 °C with 6 μM KPC-2, there was no remaining free ETX2514, showing that KPC-2 degrades ETX2514. No 197 Da product (loss of SO3) or 181 Da product (loss of SO4) was observed. The product(s) of KPC-2-catalyzed degradation of ETX2514 has (have) not yet been identified.

(Table 1). No transfer of intact ETX2514, or any derivative of ETX2514, to CTX-M-15 was seen. Thus, koff for ETX2514KPC-2 represents loss from the enzyme of one or more products derived from ETX2514. The observation that a substantial portion of the KPC-2 retained acylation with a +197 Da adduct even after 19 h (Figure 5) is consistent with the kinetics of recovery of catalytic activity by KPC-2 (Figure 2), 838



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Acylation of E. cloacae P99, a class C β-lactamase, with ETX2514 resulted in a major peak with an adduct mass of +277 Da and a minor peak with an adduct mass of +197 Da (Figure 6A). Both products were stable over time in the absence of an acceptor enzyme, and the relative amount of the two products was also stable with time. When CTX-M-15 was added as an acceptor, however, ETX2514 was transferred intact from P99 to CTX-M-15, as shown by the accumulation over time of apo-P99 and CTX-M-15 with a +277 Da adduct (Figure 6B). No +197 Da adduct of CTX-M-15 was seen, and the +197 Da adduct of P99 remained throughout the full incubation time. Thus, ETX2514 can slowly dissociate intact from the ETX2514-P99 complex, consistent with the measured koff. The minor +197 Da adduct seems not to be dissociable. A very similar result was obtained with P. aeruginosa AmpC (Figure S3), including the appearance of a minor +197 component. In the case of AmpC, however, exchange was complete within 1 h, consistent with the higher koff compared to P99 (Table 1). It may thus be concluded that koff for these class C enzymes represents, at least in part, dissociation of intact ETX2514. Acylation of OXA-10 with ETX2514 resulted in +277 Da and +197 Da adducts with similar abundance and some unacylated enzyme 1 h after removal of excess ETX2514 (Figure 7A). Over time, the proportion of the +277 Da adduct decreased while the proportion of the unacylated enzyme increased, whereas the abundance of the +197 Da adduct remained essentially unchanged. By 17 h, essentially no +277 adduct remained. No acylation exchange to CTX-M-15 was observed at any time (Figure 7B). Thus, deacylation of OXA-10 results in the release of modified ETX2514 that is unreactive with CTXM-15. The experiment does not reveal the mass of the released ETX2514 product. The observation of partial recovery of unacylated enzyme after 17 h in Figure 7 is consistent with the observation in Figure 3 that recovery of OXA-10 activity was biphasic, with about a third of the activity recovered in the first 18 h. OXA-10 bearing the +197 adduct may be an end point from which deacylation does not occur. The acylation exchange experiment with OXA-24 showed a +277 Da adduct and a very minor amount of a +197 Da adduct (Figure 8). A small amount of deacylation was observed in the absence of acceptor enzyme after 17 h. In the presence of CTXM-15 acceptor, exchange of the +277 Da adduct was detectable to a very slight extent after a 17 h incubation, although this is not visible in Figure 8. The stability of the ETX2514-OXA-24 complex and low level of exchange are consistent with the very low koff (Table 1 and Figure 2). With OXA-23, only the +277 Da adduct was seen, with no detectable deacylation or exchange within 18 h (Figure S4). With OXA-48, the +277 adduct was observed. There was no +197 adduct, no deacylation, and no exchange observed within 18 h (Figure S5). Effect of OXA-10 Concentration on Rate of Activity Recovery. The lack of recovery of OXA-10 catalytic activity 2 h after jump dilution (Figure 2) appears inconsistent with the regeneration of unacylated enzyme observed between 1 and 2 h in the acylation exchange experiment (Figure 7A) and the partial recovery of activity in the discontinuous jump dilution experiment (Figure 3). In the latter case, the higher temperature of the discontinuous versus continuous jump dilution experiment (37 °C versus 22 °C, respectively) is probably responsible for the greater recovery of activity seen in the discontinuous experiment. In the acylation exchange experiment, on the other hand, the micromolar OXA-10 concentration, versus the picomolar concentration in the

continuous jump dilution experiment, probably plays a role in the different degree of deacylation. OXA-10 is known to be in equilibrium between dimer and monomer.19,20 Danel et al.19 showed that OXA-10 is 50% dimeric at about 0.1 μM enzyme. In the absence of added divalent transition metal cations, the enzyme should be mostly dimeric under the conditions of the acylation exchange experiment (approximately 4 μM) but monomeric under the conditions of the continuous jump dilution experiment (20 pM). The dimer has been shown to be more active than the monomer, and it is possible that the dimer also has a higher rate of ETX2514 deacylation than the monomer. This hypothesis was supported by the observation that the rate of recovery of activity of ETX2514-acylated OXA10 increased with increasing OXA-10 concentration (Figure S6). Partition Ratio Measurements. The partition ratio, also referred to as the turnover number, is defined as kcat/kinact, where kcat is the rate constant for the catalytic hydrolysis of the inhibitor and kinact is the rate constant for the formation of the enzyme−inhibitor complex. For a stable, irreversible inhibitor, the partition ratio is 1, such that each reaction of the enzyme with the inhibitor results in inactivation of the enzyme. Since some β-lactamase inhibitors are themselves β-lactams, they may be hydrolyzed by the enzymes to some extent, resulting in a partition ratio greater than 1.33 For example Papp-Wallace et al.21 reported partition ratios for inhibition of KPC-2 by clavulanic acid, sulbactam, and tazobactam of 2500, 1,000 and 500, respectively. In contrast, partition ratios of 1 were reported for avibactam with KPC-2,22 CMY-2,23 and SHV-1.24 We measured the partition ratios for ETX2514 with several βlactamases. Values of approximately 1 were obtained with CTXM-15, OXA-10, OXA-23, OXA-24, OXA-48, E. cloacae P99, and SHV-5 (Figure S7). Small deviations from precisely 1 likely resulted from error in the measurement of protein concentrations due to comparison with bovine serum albumin as the standard. For KPC-2, the partition ratio increased with increasing time of incubation of the enzyme with ETX2514 prior to dilution into the nitrocefin assay (Figure 9). This demonstrates that KPC-2 slowly catalyzed ETX2514 hydrolysis, consistent with the acylation exchange experiment. For OXA-10, which also showed evidence of ETX2514 hydrolysis during prolonged incubation in the acylation exchange experiment (Figure 7), the partition ratio increased from 0.91 ± 0.02 after a 1-h incubation to 0.96 ± 0.02 after 4 h and 1.01 ± 0.02 after 19 h. This slight increase in partition ratio after 19 h with OXA-10 shows that spontaneous (i.e., nonenzymatic) degradation of ETX2514 cannot be responsible for the larger time-dependent increase of the partition ratio for KPC-2. Restoration of β-Lactam Activity in Bacteria. In order to understand the physiological relevance of the biochemical characteristics of ETX2514 as described above, we tested its ability to restore the activity of piperacillin, aztreonam, ceftazidime, or imipenem against an isogenic panel of P. aeruginosa strains overexpressing the 10 serine β-lactamases characterized above, as compared to avibactam. Each βlactamase was expressed from a plasmid in a strain of P. aeruginosa PAO1 where the chromosomal ampC and poxB βlactamases were disrupted. Therefore, the only β-lactamase activity in each strain was derived from the gene encoded on the plasmid. The susceptibility of each strain to piperacillin or imipenem alone or in the presence of either ETX2514 or 839



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piperacillin activity to all strains expressing class A or C βlactamases, and OXA-48 of the class D enzymes, as previously described15 (Table 2). The greater effectiveness of ETX2514 toward class D enzymes, as compared to avibactam, was also observed when the two β-lactamase inhibitors were paired with imipenem. Avibactam and ETX2514 were both effective in restoring piperacillin and imipenem activity against P. aeruginosa strains expressing KPC-2. The very low but detectable rate of degradation of ETX2514 by KPC-2 could not overcome inhibition of the enzyme by ETX2514. The fate of ETX2514 covalently bound to the 10 serine βlactamases examined in this study was quite varied. Rapid release with minimal modification of the compound was seen with the class C enzyme P. aeruginosa AmpC, such that the behavior of the compound was barely distinguishable from equilibrium binding (Figure 2, Figure S3). A similar result was seen with the class A enzyme TEM-1, although the koff was somewhat slower (Figure 2 and Figure 4). Another class C enzyme, E. cloacae P99 had a substantially lower koff than AmpC (Figure 2). Two other class A enzymes, SHV-5 and CTX-M-15, had lower koff than TEM-1 (Figure 2). In none of these other examples was release of ETX2514 from an enzyme necessarily concomitant with its degradation, as demonstrated by the transfer of acylation between enzymes (Figure 6, Figure S1, Figure S2, Figure S5). In contrast to the above, the class A enzyme KPC-2 released a chemically modified ETX2514 that was not able to acylate an acceptor enzyme. This enzyme was also observed to be acylated by an adduct with mass 80 Da lower than that of ETX2514 (Figure 5). It cannot be ascertained from these experiments whether or not it was the +197 Da adduct that was released. A similar result was seen with the class D OXA-10 (Figure 7), although the rate of recovery of OXA-10 activity was much lower than that of KPC-2 (Figure 2). The other class D enzymes tested, OXA-23, OXA-24, and OXA-48, all had very low values of koff like OXA-10 (Figure 2), did not undergo substantial loss of 80 Da from the adduct, and showed no acylation exchange (Figure 8, Figure S4, Figure S5). Recovery of enzyme activity in the jump dilution experiments was only observed at elevated temperature after prolonged incubation. Thus, inhibition of these enzymes by ETX2514 is essentially irreversible. It is interesting to note the differences and similarities between ETX2514 and the closely related compound avibactam (Figure 1) in these experiments. Ehmann et al.18 observed acylation exchange of avibactam to TEM-1 from CTX-M-15, AmpC, and P99, consistent with our ETX2514 observations. In contrast to our results, however, Ehmann et al.18 saw exchange of avibactam from KPC-2 to TEM-1 and loss of 80 Da from the

Figure 9. Partition ratio for ETX2514 with KPC-2 as a function of incubation time. (A) % of control versus [ETX2514]/[KPC-2] plots at various incubation times. The incubation times were 15 (black), 30 (red), 60 (green), and 120 (red) minutes. The best-fit values and standard deviations of the partition ratios are 1.47 ± 0.05 at 15 min, 1.80 ± 0.05 at 30 min, 2.14 ± 0.05 at 60 min, and 2.98 ± 0.06 at 120 min. (B) Partition ratio versus incubation time.

avibactam (fixed concentration of 4 μg/mL) was measured (Table 2). The MIC values of piperacillin and piperacillin plus ETX2514 versus the parental strain (i.e., in the absence of any β-lactamase activity) were both 2 μg/mL. The MIC of piperacillin alone increased by ≥32-fold for each β-lactamaseexpressing strain relative to the parental strain, indicating that the presence of each plasmid resulted in overexpression of the β-lactamase gene that it encoded. Addition of 4 μg/mL ETX2514 to piperacillin decreased the MIC by >8-fold relative to the MIC of piperacillin alone against every strain, including those expressing class D enzymes. Avibactam restored

Table 2. MIC Values (mg/L) of β-Lactams Alone or in the Presence of 4 μg/mL β-Lactamase Inhibitors ETX2514 or Avibactam against an Isogenic Panel of β-Lactamases Expressed in P. aeruginosaa parent

class C

class D

β-lactam

inhibitor

PAO1 ampC-poxB-

CTX-M-15

KPC-2

SHV-2a

TEM-1

AmpC

P99

OXA-10

OXA-23

OXA-24

OXA-48

piperacillin

none ETX2514 avibactam none ETX2514 avibactam

2 2 4 0.25 0.13 0.25

>64 4 4 0.25 0.25 0.25

>64 4 8 >64 0.25 0.5

>64 4 8 0.25 0.25 0.25

>64 4 8 0.25 0.25 0.25

>64 4 8 0.5 0.25 0.25

>64 4 4 0.5 0.25 0.25

>64 8 >64 0.5 0.25 0.5

>64 8 64 16 2 8

>64 8 >64 64 2 64

>64 4 8 64 0.25 1

imipenem

a

class A

The MICs of ETX2514 and avibactam alone were 32 and >64 μg/mL, respectively. 840



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dilutions. The 250,000-fold dilutions (25 μL) were then mixed in quadruplicate with 25 μL of 200 μM nitrocefin in the same buffer in a clear 384-well polystyrene assay plate. The absorbance at 490 nm (A490) was monitored at 30-s intervals for 1−2 h at 22 °C with a Spectramax 384 Plus plate reader (Molecular Devices, Sunnyvale, CA). The average baseline for the buffer sample was subtracted from each of the other progress curves. For class A and C β-lactamases, the average of 4 replicate baseline-subtracted progress curves for the ETX2514-treated enzymes was fit to the equation

avibactam adduct of TEM-1. Loss of 80 Da from a small portion of the avibactam adduct with TEM-1 and P99 was observed by Stachyra et al.25 The koff measurements for avibactam reported by Ehmann et al.15 were similar to our measurements for ETX2514 with TEM-1, CTX-M-15, P. aeruginosa AmpC, and OXA-48. Additionally, the koff value of 1.5 × 10−4 s−1 for avibactam-CTX-M-15 as reported by King et al.26 was quite similar to the value we obtained with ETX2514 (Table 1). For KPC-2 and P99, however, the ETX2514 koff was 7- and 9-fold higher, respectively, than the avibactam koff. The OXA-10 koff was given as

Reversibility of Covalent, Broad-Spectrum Serine Î²-Lactamase

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