Molecular Basis of Selective Inhibition and Slow Reversibility of

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Molecular basis of selective inhibition and slow reversibility of avibactam against Class D carbapenemases: a structure-guided study of OXA-24 and OXA-48 Sushmita D Lahiri, Stefano Mangani, Haris Jahic, Manuela Benvenuti, Thomas F Durand-Reville, Filomena De Luca, David E Ehmann, Gian Maria Rossolini, Richard A Alm, and Jean-Denis Docquier ACS Chem. Biol., Just Accepted Manuscript • DOI: 10.1021/cb500703p • Publication Date (Web): 19 Nov 2014 Downloaded from http://pubs.acs.org on November 29, 2014

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ACS Chemical Biology

Molecular basis of selective inhibition and slow reversibility of avibactam against Class D carbapenemases: a structure-guided study of OXA-24 and OXA-48

Sushmita D. Lahiri,1# Stefano Mangani,2#

Haris Jahić,1 Manuela Benvenuti,2 Thomas F.

Durand-Reville,3 Filomena De Luca,4 David E. Ehmann, 1 Gian Maria Rossolini, 1

A Alm and Jean-Denis Docquier.

4,5,6

Richard

4

1

Infection Biosciences, AstraZeneca R&D Boston, Waltham, MA 02451, USA

2

Department of Biotechnology, Chemistry and Pharmacy, University of Siena, Siena, I-53100, Italy

3

Infection Chemistry, AstraZeneca R&D Boston, Waltham, MA 02451, USA

4

Department of Medical Biotechnologies, University of Siena, Siena, I-53100, Italy

5

6

Department of Experimental and Clinical Medicine, University of Florence, and Clinical Microbiology

and Virology Unit, Florence Careggi University Hospital, Florence I-50134, Italy

#

corresponding authors: Sushmita D. Lahiri, E-mail: [email protected]; Stefano

Mangani E-mail: [email protected]

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Abstract The Class D (or OXA-type) β-lactamases have expanded to be the most diverse group of serine β-lactamases with a highly heterogeneous β-lactam hydrolysis profile and are typically resistant to marketed β-lactamase inhibitors. Class D enzymes are increasingly found in multi-drug resistant (MDR) Acinetobacter baumannii, Pseudomonas aeruginosa, and various species of the Enterobacteriaceae and are posing a serious threat to the clinical utility of β-lactams including the carbapenems, which are typically reserved as the drugs of last resort. Avibactam, a novel non-β-lactam β-lactamase inhibitor, not only inhibits all class A and class C β-lactamases, but also has the promise of inhibition of certain OXA enzymes, thus extending the antibacterial activity of the β-lactam used in combination to the organisms that produce these enzymes. X-ray structures of OXA-24 and OXA-48 in complex with avibactam revealed the binding mode of this inhibitor in this diverse class of enzymes and provide a rationale for selective inhibition of OXA-48 members. Additionally, various subunits of the OXA-48 structure in the asymmetric unit provide snapshots of different states of the inhibited enzyme. Overall, these data provide the first structural evidence of the exceptionally slow reversibility observed with avibactam in class D β-lactamases. Mechanisms for acylation and deacylation of avibactam by class D enzymes are proposed, and the likely extent of inhibition of class D β-lactamases by avibactam is discussed.


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Introduction The β-lactams are the most widely used antibiotics in clinical practice, but they are succumbing to emerging resistance, and thus are losing their effectiveness in the treatment of infections caused by increasingly prevalent multi-drug resistant (MDR) or extensively drug-resistant (XDR) pathogens1,

2, 3, 4

.

A major cause of resistance in Gram-negative

bacteria is the expression of β-lactamases. These β-lactamases include the serine hydrolase enzymes of molecular classes A, C and D, as well as the metallo β-lactamases of Class B5,

6, 7

. Among the serine β-lactamases, class D (or OXA-type) enzymes are of

particular concern, as some members in this class can hydrolyze carbapenems, which are often reserved as the last resort in the treatment of MDR pathogens, which can thus evolve toward worrying XDR, colistin-only susceptible (COS) or even pan-drug resistant (PDR) phenotypes

8, 9

Class D enzymes have the most amino acid diversity amongst the four

classes, with less than 20% sequence identity among the large numbers of enzymes identified to date. Furthermore, they are the most functionally diverse class of β-lactamases, containing enzymes that can selectively hydrolyze penicillins, extended spectrum cephalosporins, or carbapenems. Of these, the carbapenem-hydrolyzing class D βlactamases (CHDLs) are of particular concern because their ability to hydrolyze carbapenems, such as imipenem and meropenem, is making the clinical management of infections caused by pathogens producing these enzymes extremely difficult. The class D enzymes are structurally diverse. Their active site is unique among serine-βlactamases, where an N-carboxylated lysine plays a critical role in the β-lactam hydrolysis mechanism10. Among the clinically relevant CHDLs, OXA-24 and OXA-48 have been the paradigms of different mechanistic models for carbapenem hydrolysis. Studying OXA-24 and OXA-48 has revealed that specific functional attributes in this class of enzymes have evolved through distinct structural and mechanistic changes. In the case of OXA-24, carbapenem hydrolysis is enabled by improved substrate affinity mediated by a hydrophobic bridge that spans the active site pocket that allows the drug to remain in the productive conformation for a longer time11. In contrast, this bridge is absent in OXA-48, which employs an alternative strategy of positioning a large hydrophobic residue near the active site, which as a consequence prevents the carbapenem molecules from adopting an unproductive conformation that resists hydrolysis in non-carbapenemase class D enzymes12.Nearly 400 class D enzyme variants have been reported to date, observed in an array of major Gramnegative pathogens including Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacteriaceae8.This diversity is particularly concerning, as the majority of class D β3 ACS Paragon Plus Environment


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lactamases remain recalcitrant to inhibition by the β-lactamase inhibitors currently in clinical use. Avibactam is a non-β-lactam β-lactamase inhibitor of serine-β-lactamases that is currently in clinical development in combination with ceftazidime, ceftaroline fosamil,

13,

14

and

aztreonam. Unlike other clinically used β-lactamase inhibitors, avibactam demonstrates reversible deacylation via recyclization

15

. It possesses excellent inhibition properties against

many clinically relevant enzymes, including class A extended-spectrum β-lactamases (ESBL) and carbapenemases (KPC-type), as well as both chromosomally- and plasmidencoded class C β-lactamases16, 17. Avibactam also inhibits some class D enzymes and can mitigate β-lactam resistance in Enterobacteriaceae strains such as OXA-48-producing Escherichia coli and Klebsiella pneumoniae

18, 19

. In line with the substrate diversity, the

inhibition profile by avibactam varies amongst members of this class, particularly with regards to its acylation rate constants. For example, the acylation rate of OXA-48 was comparable to that of the class C enzymes whereas that of OXA-10 was considerably slower16. Interestingly, the deacylation rate constants are significantly slower against all class D enzymes studied thus far, with their half life values ranging in days as compared to minutes for classes A and C. This observation indicates that the deacylation mechanism via recyclization of avibactam varies across classes and that once covalently bound, avibactam is an essentially irreversible inhibitor of class D β-lactamases. To further build our understanding, here we report inhibition kinetics of avibactam against two additional CHDLs, OXA-23 and OXA-24, both of which are increasingly implicated for carbapenemase resistance. Structural studies with avibactam thus far have elucidated the binding mode of avibactam to both class A and class C β-lactamases using CTX-M-15 and Pseudomonas aeruginosa AmpC as representative enzymes, respectively

20, 21, 22

. These structures have

provided clear insights into the unique reversibility observed by this covalent inhibitor

20

and

a rationale for its broad spectrum inhibitory properties. Here we describe the crystal structures of avibactam bound to OXA-24 and OXA-48 β-lactamases. Whereas both enzymes possess a similar substrate profile, in that they are both carbapenemases, they differ in both their avibactam binding affinity and acylation rates

16

. The structural analyses

presented here provide a rationale for the differential inhibition properties of avibactam between OXA-24 and OXA-48 as well as a hypothesis for the slow deacylation rate observed in class D enzymes. In addition, sequence comparison of all class D OXA βlactamases enables a better understanding and prediction of the spectrum of avibactam inhibition in this diverse class of β-lactamases.


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Methods Purification and crystallization of OXA-48.

OXA-48 was produced using a T7-based

E. coli expression system and purified to homogeneity using ion-exchange and sizeexclusion chromatography 12. In all cases, the concentration of the protein was measured by Bio-Rad’s protein assay, and the purity of the enzyme was verified to be >95% as assessed by SDS-PAGE gels. Purified protein was aliquoted and stored at -80⁰C for a single use OXA48:avibactam crystals were obtained by co-crystallization in a sitting drop setup using 0.1 M HEPES (pH 7.5), 10% PEG8000, between 5% to 8% 1-butanol, 10 mg/mL OXA-48, and 3 mg/mL avibactam. Diffraction data were collected at the ID23-1 beamline at the ESRF (Grenoble, France).

An initial model was obtained by molecular replacement using the

native OXA-48 structure (PDB: 3HBR)12 as the search model. The final model was obtained after cycles of manual building and structure refinement using refmac and coot, as previously described 23, 24, 25. Purification and crystallization of OXA-24. The OXA-24 β-lactamase was produced in E. coli and purified by chromatography as previously described

26

and aliquots were stored

at -80⁰C for a single use. Crystals of OXA-24 in complex with avibactam were obtained using the vapor diffusion technique in a sitting drop setup. The OXA-24-avibactam bound crystals were obtained by co-crystallizing the enzyme (6 mg/mL) with avibactam (3 mg/mL) in 0.1 M MES buffer (pH 6.0) containing 2.4 M (NH4)2SO4 as the precipitant solution. Diffraction data were collected at the ID-29 beamline at the ESRF (Grenoble, France). Data were processed and scaled using the iMOSFLM and SCALA programs. An initial model of the OXA-24:avibactam complex was obtained by molecular replacement using the program MOLREP in the CCP4 suite24 and using the native OXA-24 structure (PDB: 2JC7) as the search model. The final model was obtained by iterative refinement processes using refmac and coot 23, 24, 25.

Purification of OXA-23. The OXA-23 expression plasmid pJT787 was constructed by replacing the native signal sequence for OXA-23 with the signal sequence for PelB during PCR. Protein was expressed in E. coli BL21(DE3) cells transformed with pJT787. Cell extracts were resuspended in 20 mM MES (pH 5.5). The crude extract was further purified using SP-Sepharose HP column followed by HiLoad 26/60 Superdex 200 column (GE Healthcare Life Sciences). The purified protein was finally eluted into 50mM HEPES (pH 7.5), 150 mM NaCl and aliquots were stored at -80⁰C.



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Biochemical studies Enzyme assays with OXA-23 and OXA-24 were performed at 37 °C. One liter of the assay buffer contained 50 millimoles of NaH2PO4, 50 millimoles of Na2HPO4, 100 mg of bovine serum albumin, and 50 millimoles of NaHCO3. Determination of kinetic parameters of avibactam inhibition was described previously

16

. For on-rate kinetics, onset of inhibition

progress curves were acquired using a Cary Bio UV-visual spectrophotometer (Varian, inc) outfitted with a temperature controller. Reactions were initiated in stirred 1 cm quartz cuvettes by adding 20 µL of 50x enzyme to 980 µL of 204 µM nitrocefin solution in the presence or absence of avibactam. The final enzyme concentrations were 0.025 nM OXA23, and 0.025 nM OXA-24. For off-rate kinetics, data acquisitions for off-rate experiments were performed using a jump-dilution technique. OXA-23 and OXA-24 inactivation was performed using 1 µM enzyme with 100 µM avibactam. Inactivation mixtures were diluted 40,000-fold. The recovery of enzyme activity was assessed after combining 20 µL of diluted inactivation mixture with 180 µL of 200 µM nitrocefin in the presence or absence of avibactam. Discontinuous sampling was applied due to exceptionally slow off-rates. Conservation Analysis The

OXA

β-lactamase

protein

sequences

as

defined

by

the

Lahey

site

(www.lahey.org/Studies) were extracted from GenBank and aligned using the Clustal X software. The diverse sequences were clustered into families and realigned separately to assess conservation. The overall conservation of the class D enzymes was assessed using the consurf algorithm (www.consurf.tau.ac.il/)27 and the multiple sequence alignment was mapped

onto the

OXA-24

structure,

and

visualized

using

28

www.pymol.org) .


Pymol (Schrodinger,

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Results and Discussion Avibactam OXA-48 co-crystal structure The structure of OXA-48 bound to avibactam was determined to 2.3Å resolution (statistics provided in Table 1). There are eight OXA-48 molecules in each asymmetric unit comprised of four physiological homodimers

12

. A clear

electron density of avibactam covalent acyl intermediate attached to the catalytic Ser70 residue is observed in all eight subunits. Avibactam is linked via a carbamoyl linkage as previously observed in class A and class C structures

20, 21, 22

. A strong additional electron

density beyond the Lys73 ɛ-amino nitrogen was observed in two of these eight subunits (Figure 1A and 1B), suggesting that these binding pockets contained the modified carboxylated form of Lys73. The crystallization buffer did not contain any bicarbonate ion, suggesting that either atmospheric CO2 is adequate for saturating microdrops, or this modification is maintained during protein purification. In another three subunits, an additional density in the vicinity of Lys73 was observed which can be attributed to CO2. The remaining three subunits did not contain any additional density, which suggests that neither a modified N-carboxy lysine nor a CO2 molecule exists in these binding sites (Figure 1A and 1B). Importantly, a water molecule (w1) is observed in the position of the deacylating water for βlactam hydrolysis in all subunits (Figure 1A i-iv).

The key polar interactions observed with avibactam in the OXA-48 binding pocket are the hydrogen bonds with the active site Ser118 (of the S-x-V motif), Thr213, Arg250 as well as with the conserved Lys208, Thr209, and Gly210 of the K-T-G motif (Figure 1C).

The

carbamate carbonyl of avibactam is positioned in the oxyanion hole formed by the backbone nitrogens of Tyr211 and Ser70. In addition, the carboxylate group of carboxy Lys73 or the ɛamino nitrogen of unmodified Lys73 is close to the carboxamide group of avibactam. In fact, the carboxylate moiety of carboxy Lys73 curves away from the catalytic serine and makes an indirect hydrogen bond with the carboxamide group of avibactam via the water molecule w1 (see Figure 1A, iii). The remainder of the binding pocket in the active site is mostly defined by hydrophobic residues characteristic of class D enzymes. Avibactam makes van der Waals interactions with the hydrophobic surface formed by Val120, Trp105, and Ile102 on one side, and Leu247, Tyr211, and Leu158 on the opposite side (Figure 1D). Leastsquare superposition of all subunits shows that they all adopt a similar conformation, with all of the sidechains that interact with avibactam oriented in a similar fashion with the exception of Leu158, and the unmodified Lys73 (Figure 1B). Comparison with the native OXA-48 structure (PDB:3HBR)12, where a carboxy Lys73 was observed, indicates that the binding of acylated avibactam does not induce major 7 ACS Paragon Plus Environment


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rearrangements of the active site nor of any loops that are adjacent to the pocket (Figure 1C). The most prominent movements observed are the shifts of several side-chains surrounding the pocket.

Of interest is the position of Ser118, which in the uninhibited

structure points towards Lys208, whereas in the two subunits of the OXA-48:avibactam structure with clear CO2 density (Figure 1A-ii) it may form a hydrogen bond with decarboxylated Lys73. The avibactam binding also changes the position of the Leu158 and Arg214 side-chains, both of which have been implicated in the carbapenemase activity of OXA-4829. Lastly, the side-chains of the α3-α4 loop residues, which contain the hydrophobic amino acids Val120, Trp105, and Ile102, shift by approximately 2.0Å upon inhibitor binding. Avibactam OXA-24 co-crystal structure. The structure of the avibactam-bound OXA-24 carbapenemase was determined to 2.4Å resolution (statistics in Table 1). Unlike OXA-48, OXA-24 is a monomer in solution 26. There is only one subunit in the asymmetric unit, which expectedly shows the inhibitor covalently bound via carbamoyl linkage to Ser81. The Fourier difference map from the refinement suggested that the Lys84 was in a decarboxylated form with a CO2 molecule observed in the pocket and a density for the deacylating water molecule implicated for β-lactam hydrolysis is also observed, albeit at a much weaker intensity (w1 in Figure 2A). Avibactam interacts with the key conserved residues, including the S-x-V (Ser128-Val130) and K-T-G motifs (Lys218-Gly220). The typical class D KTG triad is replaced by K-S-G in OXA-24, but the Ser219 in OXA-24 makes the same interaction with the oxygen atom of the sulfamate group of avibactam (Figure 2B) as the Thr209 makes in OXA-48 (Figure 1C). The bridge formed by the Tyr112 and Met223 residues of the Y-G-N motif interacts closely with avibactam and maintains the tunnel-like entrance of the OXA-24 active site. This bridge conformation formed by Tyr112 and Met223 was also observed in other OXA-24 structures including one bound to doripenem (PDB 3PAE)11.

This

“hydrophobic bridge” that is responsible for holding the substrate by van der Waals interactions remains intact upon avibactam binding. In the crystal structure, the ε-amino group of Lys84 interacts with the CO2 oxygen, which in turn also interacts with the deacylating water molecule in the pocket. The Ser128 in this case does not form a hydrogen bond with Lys84 as observed in some OXA-48 subunits. The water molecule is positioned by the indole nitrogen of Trp167 and is within hydrogen bonding distance of the carboxamide group of avibactam. There are very few additional polar interactions observed with the carboxamide group, which is surrounded primarily by the hydrophobic residues Leu168, Trp167, Val130, Trp115, Tyr112, and Met223 that form the hydrophobic bridge (Figure 2B and 2C). Of these, Met223 is less than 4Å removed from the carboxamide nitrogen of avibactam, suggesting this proximity may be energetically 8 ACS Paragon Plus Environment

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unfavorable. In contrast to OXA-48, the binding of avibactam to OXA-24 only slightly affects the orientation of the side chains of nearby residues and does not induce any major displacement of the protein backbone (Figure 2D). Rationale for the differences in inhibitory activity of avibactam for OXA-24 and OXA48. Acylation rates for avibactam have been previously reported for a variety of class A, C, and D β-lactamases16. Among the examined class D enzymes, OXA-48 was inhibited more efficiently than OXA-10. To expand the understanding of the ability of avibactam to inhibit class D carbapenemases, acylation and deacylation rates were measured for OXA-24 and OXA-23 using the same protocol (Table 2). Compared to OXA-48, the acylation rates for OXA-24 and OXA-23 were reduced by 28-fold and 5-fold, respectively. The off-rates for non-hydrolytic reversible deacylation were nearly equivalent for OXA-24, OXA-23, and OXA48, and therefore the improvement in affinity as calculated from the on and off rates of avibactam inhibition against OXA-48 appears to be solely due to the increased acylation rate. There was no hydrolysis of avibactam observed with any of these enzymes.

The comparison of the avibactam-bound active sites of OXA-24 and OXA-48 is depicted in Figure 2D. The overall binding conformation of avibactam is comparable between OXA-24 and OXA-48, and is similar to that observed in class A and class C β-lactamases

20

. Thus,

the differences inhibition between class A/C and class D could be attributed to the properties of the binding pocket. The residues lining the pocket are more hydrophobic in the class D βlactamases than that observed in class A and C enzymes. Whereas the polar interactions are fewer, avibactam is stabilized by a similar set of polar interactions in both OXA-24 and OXA-48. The sulfamate moiety interacts with the side chains of the conserved Arg261/250 (numbering used here and subsequently will be OXA-24/OXA-48 respectively) and the KT/S-G and S-x-V motifs. Similarly, the carbonyl oxygen occupies the oxyanion hole formed by the backbone atoms of the catalytic serine and Tyr221/211.

Apolar contacts in the

binding pocket are observed between the piperidine ring of avibactam and the side chain of a hydrophobic Leu168/158 that is reorientated upon binding. In addition, the hydrophobic bridge in OXA-24 makes close contact with the piperidine ring. Overall, the key interactions with avibactam are similar between the two class D structures and the differences between the unliganded and covalently-bound forms of each enzyme are minimal. However, the presence of the hydrophobic bridge in OXA-24 significantly restricts the accessibility of avibactam to this pocket when compared to the more open pocket configuration seen in OXA-48. In the case of OXA-48, the residue at this position is Thr213, which is not only smaller in size but also provides an additional hydrogen bond to the 9 ACS Paragon Plus Environment


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carboxamide group. Additionally, OXA-48 has more polar residues, such as Arg214 and Asp101 in the vicinity of the binding pocket when compared to OXA-24, and although they do not make direct contact with avibactam, they could facilitate the formation of the Michaelis complex and optimally orient avibactam to enable a faster reaction with the catalytic Ser (Figure 1D and 2C). In summary, the more hydrophobic nature of the OXA-24 binding pocket results in fewer polar interactions with avibactam when compared to OXA-48. This, along with an obstructed access to the binding site due to the hydrophobic bridge, results in reduced acylation rates in OXA-24 compared to OXA-48. An overlay of these structures with OXA-23 (PDB: 4JF6 and 4K0X) suggests that a similar rationale also applies for the weaker OXA-23 activity as it also contains the hydrophobic bridge like OXA-2430,

31

. However, in

OXA-23 the Tyr112 in OXA-24 is replaced by a Phe, which make the contact distance with the facing Met223 slightly “looser” compared to OXA-24, possibly explaining its intermediate acylation rate when compared to OXA-48 and OXA-24.

Rationale for lack of hydrolysis and slow reversibility. Although the acylation rates between OXA-24 and OXA-48 are different, it is noteworthy that the rates of deacylation are similar for these two enzymes and significantly slower when compared to those for class A and class C β-lactamases (Table 2). However, similar to the class A and C β-lactamases, no water-mediated hydrolysis of the avibactam acyl enzyme complex is observed for class D enzymes. The only pathway by which deacylation occurs is through recyclization that results in regeneration of an intact avibactam molecule. In the class C structure, the deacylating water molecule was displaced by the sulfamate group of avibactam

21

, whereas in the class

A structure this water was rendered ineffective by the protonated carboxylate of Glu166 20. In the case of class D enzymes described here, the deacylating water molecule is only observed in the proper position for attack when Lys is decarboxylated. We hypothesize that in the class D enzymes, the change in charge distribution in the vicinity of carboxy-Lys84/73 upon avibactam binding, results in a preferential decarboxylation of Lys84/70, which then removed the general base needed for deacylation by hydrolytic water. Given the lack of hydrolysis, the slow deacylation rate of class D enzymes can be attributed to the slow recyclization rate of avibactam. The catalytic mechanism in this class of enzymes involves the N-carboxylated lysine, which is formed and stabilized by the highly hydrophobic active-site environment. The structures of OXA-48 and OXA-24 described here suggest that, as with acylation by β-lactams, the acylation by avibactam is most likely facilitated by the carboxylate group of carboxy Lys acting as the general base to abstract a proton from the catalytic Ser. The acylation also requires a general acid to donate a proton to the aminosulfate nitrogen. The binding modes in the two enzymes suggest that the residue 10 ACS Paragon Plus Environment

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poised to play this role is Ser128/Ser118 assisted by Lys 218/208 as depicted in Figure 3A. This Ser128/118 is analogous to the Ser130 and Tyr150 proposed to act as general acid for avibactam acylation in class A and C enzymes, respectively 20, 21. The slow recyclization is likely indicative of a slow step of either protonation of catalytic Ser70 and/or deprotonation of the aminosulfate nitrogen. Any role of conformational change of avibactam upon acylation can be rules out, as the ring-open conformation of avibactam is very similar to that observed in class A and C β-lactamases. Of the two residues in our proposed

mechanism

of

recyclization

involving

the

concomitant

contribution

of

Ser128/Ser118 and carboxy Lys84/73 (protonated or water-mediated) as the general base and general acid respectively (Figure 3B), the presence of a carboxylated Lys was not always observed in the structures, suggesting that absence of a general acid is likely the reason for the slow recyclization. In the case of class A and class C enzymes, the residues participating as proton acceptor and donor, Ser130 and Glu166 respectively for class A and Tyr150 and Lys73 respectively for class C, are not modified during the reaction cycle upon avibactam binding, which might explain the faster recyclization rate. Studies with penam sulfone and penem inhibitors of OXA enzymes have also hypothesized that the carboxylation state of Lys73 and its rate of reloading with CO2 are determinants of hydrolytic deacylation rates towards inhibitors32, 33. The structural data here suggest that the decarboxylation of Lys84/73 can readily happen with avibactam covalently bound to the enzyme. This decarboxylations could be due to the greater access of a water molecule resulting from the movement of Leu168/158 upon avibactam binding

32

.

A second

contributor may be the slow recarboxylation of Lys73 following spontaneous decarboxylation due to the presence of a polar carboxamide residue close to the Lys amine, which could result in an unfavorable increase in the pKa of lysine that would prevent efficient recarboxylation. Conservation of binding pocket among class D carbapenemases. To understand the variation in the avibactam binding pocket and potentially predict its inhibitory activity, all known class D sequences were aligned and grouped into phylogenic clusters, similar to that described previously 7, 8, 9. A total of 286 OXA proteins were clustered into 16 distinct clusters (Table 3), leaving 24 singletons. The alignment of a single representative from each of the 16 clusters (Figure 4A) showed that all residues interacting with avibactam in OXA-24 and OXA-48 crystal structures were highly conserved, with the exception of the residues that form the hydrophobic bridge. Any deviation of members from within a cluster to its representative sequence is noted in Table 3. There were 24 singletons which, despite their 11 ACS Paragon Plus Environment


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large sequence diversity, still maintained a significant degree of conservation in the key avibactam binding residues. The alignment of all 310 unique OXA sequences was mapped onto the OXA-24 structure to visualize the conservation of the protein surface (Figure 4B). Although there are large differences in the outer parts of the pocket that could impact the observed functional differences in substrate selectivity,34, 35 most of the residues interacting with avibactam were conserved. These included the residues around the sulfamate binding region of avibactam which corresponded to the conserved K-x-G and S-x-V motifs as well as the catalytic residues from the S-x-x-K motif. When visualized in the surface map (Figure 4B), these changes, although typically to residues of similar hydrophobicity, are located in the carboxamide binding region or form the hydrophobic tunnel27. The conservative hydrophobic changes are likely to have minimal effect on the binding potency of avibactam. However, the presence or absence, and the composition of the residues forming the hydrophobic bridge would impact the accessibility of the inhibitor to the active site and might represent a thermodynamically unfavourable factor that could potentially impact the acylation rates of avibactam in these enzymes. These differences would not be predicted to impact the very slow recyclization, thus promoting “almost irreversible” inhibition. Based on the sequence conservation analysis, OXA-subgroups such as OXA-23, OXA-51, OXA-58, OXA213, might all have an OXA-24 like hydrophobic bridge that could hinder access to avibactam and diminish its inhibitory activity. Conclusions As carbapenems are becoming an increasingly preferred choice for the treatment of infections caused by multidrug-resistant nosocomial pathogens, resistance to carbapenems has been steadily increasing. The mechanisms of resistance include the emergence of various carbapenem-hydrolysing β-lactamases belonging to classes A, B and D. Avibactam is a promising new β-lactamase inhibitor as it possesses a broad spectrum of inhibition against these enzymes. It is currently in advanced stages of clinical development in combination with β-lactam partners with the potential of being able to treat infection caused by Gram negative pathogens producing class A and class C enzymes17. Although it does not inhibit the class B metallo β-lactamases, it does have the potential to inhibit a selection of class D enzymes. The structural data of avibactam bound to two phylogenetically and structurally distinct class D carbapenamases presented here gives insight into the molecular mechanism of avibactam inhibition in this significant class of β-lactamases. Our overall understanding suggests that whereas the inhibition properties of avibactam may be weaker towards certain enzymes of this class, this is primarily due to poorer access to the recognition elements of the binding pocket as opposed to the any variability in the residues that form the binding pocket. Additionally, conservation analysis across the reported class D 12 ACS Paragon Plus Environment

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enzymes indicates that key residues within the avibactam binding site are well conserved and that it may be possible to design an improved inhibitor using this scaffold that will effectively inhibit the majority of class D β-lactamases. Lastly, ceftazidime-avibactam, which is the combination of avibactam plus an antibiotic furthest along in clinical development, is impervious to most CHDLs, as these enzymes do not hydrolyze ceftazidime18, 36.

Acknowledgments. We would like to thank C. Pozzi for her help with data collection and refinement and acknowledge the European Synchrotron Radiation Facility (ESRF, Grenoble, France) and their staff for provision of synchrotron radiation facilities and for assistance with beamline usage. We acknowledge the technical contribution of J.M. Bruneau (formerly with Novexel S.A., Romainville, France). We also thank C. Miossec and M.T. Black (formerly with Novexel S.A., Romainville, France), who initially commissioned part of the structural study, for their constant interest in this work and for helpful discussion of the data. We also acknowledge the CIRMMP consortium (Consorzio Interuniversitario Risonanze Magnetiche di. Metalloproteine Paramagnetiche) for their financial support. Accession Numbers The coordinates and structure factors deposited into the Protein Data Bank are under the following codes: OXA24-Avibactam 4WM9; OXA48-Avibactam: 4WMC. Disclosures Authors S. D. L., H.J., T.F.D, D.E.E. and R. A. A. are employees of AstraZeneca.



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Table 1. Data collection and refinement statistics for OXA-48 and OXA-24 avibactam complexes. Data in parentheses refer to the high resolution shell. OXA-48-AVI

OXA-24-AVI

4WMC

4WM9

X-ray source

ESRF ID23-1

ESRF ID-29

Space group

P21

P41212

Cell dimensions (Å)

a=63.89,

PDB codes Data Collection

b=165.47, a=b=102.18,

c=87.92,

c=108.52, β=90.39

α=β=γ=90.00

Subunits/asu

8

1

Resolution (Å)

65.80-2.30 (2.42-2.30)

55.82-2.40 (2.53-2.40)

Reflections measured

309213 (38125)

64246 (9460)

Unique reflections

99733 (12691)

17188 (2517)

Completeness (%)

93.4 (88.0)

92.5 (94.4)

Rmerge (%)

6.8 (25.0)

11.1 (37.9)

Multiplicity

3.1 (3.0)

3.7 (3.8)

I/σ(I)

10.6 (5.9)

8.7 (4.0)

Resolution range (Å)

63.88-2.30 (2.36-2.30)

31.76 -2.40 (2.46-2.40)

Number of reflections

85927 (5821)

16268 (1190)

Rcryst (%)

21.05 (23.5)

17.88 (25.9)

Rfree (%)

27.25 (29.6)

22.93 (31.0)

Protein atoms

15288

1921

Ligand atoms

136 AVI, 9 CO2

17 AVI, 3 CO2 , 5 SO4, 20

Refinement

EDO Water molecules

256

81

Average B factors (Å )

28.71

28.73

r.m.s.d. bond lengths (Å)

0.014

0.019

r.m.s.d. bond angles (°)

1.695

2.167

r.m.s.d. planes (Å)

0.007

0.009

r.m.s.d. chiral centers (Å3)

0.127

0.154

r.m.s.d. atomic positions (Å)

0.201

0.136

2

*data in parentheses refers to the highest resolution shell


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Table 2 Inhibition kinetics of avibactam against class A, C and D β-lactamases Class

β-lactamase

Acylation kinact/Ki -1 -1

-

Deacylation koff (s )

5

A

CTXM-15*

1.3 ± 0.1 x 10

C

AmpC*

2.9 ± 0.1 x 10

D

OXA-48*

1.4 ± 0.1 x 10

D

OXA-24

5.2 ± 1.2 x 10

D

OXA-23

3.0 ± 0.2 x 10

3

3

1

2

Kd (µM)

t1/2 (min)

1

(M s )

Deacylation

-4

40 ± 10

0.002

-3

6±2

0.66

-5

1000 ± 300

0.009

-6

1823 ± 111

0.12

1436 ± 76

0.027

3.0 ± 1 x 10

1.9 ± 0.6 x 10

1.2 ± 0.4 x 10

6.3 ± 0.4 x 10

-6

8.0 ± 0.4 x 10

* data from references 15 for comparison.



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Table 3: Conservation of Avibactam binding residues among OXAs


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Cluster

N

OXA Members

Conservation

of

AVI

residues within clustera

Name OXA-1

8

1, 4, 30b, 31, 33, 47, 224, 320

OXA-2

17

2, 3, 15, 20, 21, 32, 34, 36, 37, 46, 53, 118, All conserved

All conserved

119, 141, 161, 205, 210 OXA-10

22

5, 7, 10, 11, 13, 14, 16, 17, 19, 28, 35, 56, OXA-145

–

Leu155

74, 101, 129, 142, 145, 147, 183, 240, 251, missing; 256 OXA-23

18

OXA5/129 – Phe208Tyr

23, 27, 49, 73, 102, 103, 105, 133, 146, 165, All conserved 166, 167, 168, 169, 170, 171, 225, 239

OXA-24

13

24, 25, 26, 40c, 72, 139, 143, 160, 182, 207, All conserved 231, 253, 255

OXA-42

4

42, 43, 57, 59

OXA-43 – Ser104Pro

OXA-48

12

48, 54, 162, 163, 181, 199, 204, 232, 244, OXA-247 245, 247, 370

OXA-51

95

–

Tyr211Ser;

OXA-162 – Thr213Ala

51, 64, 65, 66, 67, 68, 69, 70, 71, 75, 76, 77, OXA-202/248 – Ile129Met; 78, 79, 80, 82, 83, 84, 86, 87, 88, 89, 90, 91, OXA-83,

110,

312

–

92, 93, 94, 95, 98, 99, 100, 104, 106, 107, Ile129Leu; 108, 109, 110, 111, 112, 113, 115, 116, 117, OXA131/172/173 120, 128, 130, 131, 132, 138, 144, 148, 149, Ile129Val;

16

–

members

150, 172, 173, 174, 175, 176, 177, 178, 179, have Leu167Val; OXA-79 – 180, 194, 195, 196, 197, 200, 201, 202, 203, Trp222Gly;

OXA-

206, 208, 216, 217, 219, 223, 241, 242, 248, 172/173/200/250 249, 250, 254, 259, 260, 261, 262, 263, 312, Trp222Leu 313, 314, 315, 316, 317, 365, 371 OXA-58

4

58, 96, 97, 164

OXA-164 – Phe114Leu

OXA-63

4

63, 136, 137, 192

All conserved

OXA-134

22

134, 186, 187, 188, 189, 190, 191, 235, 236, All conserved 237, 276, 277, 278, 282, 283, 284, 285, 335, 360, 361, 362, 363

OXA-211

7

211, 212, 280, 281, 309, 333, 334

All conserved

OXA-213

31

213, 267, 268, 269, 270, 271, 272, 273, 304, OXA-269 – Trp221Leu 305, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 348, 349, 350, 351, 352, 353, 354, 357, 358, 359

OXA-214

4

214, 215, 264, 265

All conserved


–


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OXA-228

10

228, 229, 230, 257, 274, 275, 300, 301, 355, All conserved 356

OXA-286

15

266, 286, 287, 288, 291, 292, 293, 294, 295, All conserved 297, 298, 302, 303, 306, 307

a

Conservation of residues that are within hydrogen bonding distance or form hydrophobic contact with

avibactam. The residue alignment among clusters is depicted in Figure 4A. The changes here represent deviation from the parent sequence of the cluster. b

OXA-1 and OXA-30 have the same sequence.

c

OXA-24 and OXA-40 have the same sequence.


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Figure Legends: Figure 1: OXA-48 avibactam crystal structure. A: Electron density of the avibactambound OXA-48 active site depicting various configurations (i-iv). (i) Lys73 is unmodified. Electron density for a CO2 molecule, the deacylating water (W1) as well as a water close to the sulphate group (w3) is observed. The Lys73 is hydrogen-bonded to CO2 here. (ii) Lys73 is decarboxylated, with both CO2 as well as the deacylating water (w1) observed in the pocket. In this form, Lys73 is hydrogen-bonded to Ser118. (iii) Lys73 is carboxylated, with the deacylating water molecule (w1) density observed along with two additional water molecules, one interacting with carboxylysine (w2) and other close to Trp157 (w4). (iv) Lys73 is unmodified, but only the deacylating water (w1) is observed. Lys73 does not appear to be hydrogen-bonded to any residue in the vicinity and no CO2 molecule is observed. B. Superimposition of the binding pocket of the eight subunits of OXA-48 crystal structure. Each subunit is depicted in a distinct color, which are described in the table. The residues in the binding pocket as well as avibactam are depicted in stick model whereas the water molecules are depicted in spheres of the respective colors. C. Comparison of avibactambound OXA-48 with the native structure. Residues in the avibactam cocrystal structure are drawn in blue sticks, including avibactam (AVI), and the residues in the native form are depicted in white. Water molecules are drawn as spheres of the respective color. D. Polarity of the OXA-48 binding pocket. The surface map has been colored based on the residue type. The positively charged residues are colored blue, the negatively charged residues are colored red, the non-charged polar residues are colored light blue, the aromatic residues are colored pink and the apolar hydrophobic residues are colored grey. Avibactam is depicted in yellow stick model, and CO2 is depicted in red stick.. Figure 2: OXA-24 avibactam crystal structure. A. Electron density 2fofc map of avibactam bound to the binding pocket is depicted in grey mesh at 1.0σ. The residues in the pocket including avibactam are depicted in light brown sticks whereas the deacylating water molecule (w1) is shown as red sphere. B. Comparison of avibactam-bound OXA-24 bound to avibactam with the native structure. Residues in the avibactam bound structure are depicted in light brown sticks and that in the native structure are depicted in white sticks. The water molecules in the avibactam-bound structure are shown as red spheres and that in the native form are shown as white spheres. C. Polarity of the binding pocket. The surface map has been colored based on the residue type. The positively charged residues are colored blue, the negatively charged residues are colored red, the non-charged polar residues are colored light blue, and the aromatic residues are colored pink while the apolar hydrophobic residues are colored grey. Avibactam is depicted in yellow stick model, and CO2 is depicted 19 ACS Paragon Plus Environment


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in red stick. D. Comparison of the avibactam binding pocket in OXA-24 and OXA-48. The residues lining the pocket including avibactam are depicted in light brown stick for OXA-24 and blue stick for OXA-48. The deacylating water (w1) for each structure is shown in respective color.

Figure 3: A proposed mechanism of avibactam inhibition in class D enzymes during acylation (A) and deacylation (B). Residues participating in OXA-48 are labeled in blue and those in OXA-24 are labeled in orange. Figure 4: Conservation of avibactam binding pocket in class D enzymes. All class D enzymes available to date, 286 across 16 clusters plus 24 singletons were aligned. A. Alignment of a representative sequence from each of the 16 clusters is depicted, where the avibactam binding-pocket residues are highlighted in grey. The residues that interact with avibactam in the OXA-24 and OXA-48 complexed structures are in white text with black background. The two residues forming the hydrophobic bridge in OXA-24 are marked (ϕ). B. The sequence alignment of all 310 unique OXA enzymes was mapped onto the OXA-24 structure. The residues that are >90% conserved are depicted in purple, the residues that have

Molecular Basis of Selective Inhibition and Slow Reversibility of

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