Structural insights into the activity of carbapenemases: understanding the mechanism of action of current inhibitors and informing the design of new carbapenem adjuvants

Abstract

Antimicrobial resistance challenges the effectiveness of carbapenem antibiotics as last-line therapy, due to the production of both serine and metallo-β-lactamase enzymes. β-Lactamase inhibitors currently available on the market include clavulanic acid, sulbactam, tazobactam, avibactam, relebactam and vaborbactam but, while they are active against serine β-lactamases, they are inactive against the zinc-containing metallo-β-lactamases. This review aims to discuss the distinctive structural qualities of β-lactamase enzymes and to summarise the efficacy of clinically approved and emerging β-lactamase inhibitors against clinically significant carbapenemases.

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Introduction

Since their discovery in the early 20th century, β-lactam antibiotics have revolutionised treatment outcomes for bacterial infections; they include penicillins, cephalosporins, carbapenems and monobactams, which are structurally related by a central β-lactam ring (Fig. 1).^1–3 This central pharmacophore is necessary for the irreversible acylation of penicillin binding proteins (PBPs), which catalyse peptidoglycan formation during the dynamic assembly and disassembly process involved in cell wall maintenance.² PBP inhibition prevents peptidoglycan formation, causing a net movement of water into the bacterial cell via osmosis, and eventually a bactericidal effect.³

Fig. 1 Core pharmacophore of β-lactam antibiotics including carbapenems, penicillins, cephalosporins and the monobactam, aztreonam. The conserved β-lactam ring is essential for PBP inhibition.

The overuse and misuse of antibiotics has imposed a significant selection pressure on bacterial pathogens and has given rise to antimicrobial resistance (AMR)⁴ and the proliferation of resistant strains has limited the effectiveness of currently available antibiotics. One prominent resistance mechanism that contributes to the loss of β-lactam efficacy involves antibiotic hydrolysing enzymes known as β-lactamases (BLs).^4,5 When the carbapenem antibiotics were first developed they were reserved as last-line agents for highly resistant bacterial infections due to their potential to resist hydrolysis by many BLs.⁶ Carbapenemases are a specific broad class of BLs, which are able to hydrolyse and inactivate carbapenems, penicillins and cephalosporins via either a divalent cation [metallo-β-lactamases (MBLs)] or serine residue [serine β-lactamases (SBLs)] within their active site.

The global increase in carbapenemase dissemination is challenging the use of these last-line antibiotics;⁷ in 2024, the World Health Organization (WHO) released an updated list of priority pathogens to guide antibacterial research and development, and four carbapenem-resistant Gram negative bacteria feature among the top ten (Table 1).⁸ The WHO uses a number of criteria to stratify priority pathogens, including the transmissibility of the pathogenic resistance mechanisms, the associated disease burden and severity, and the availability of drugs to which these pathogens are susceptible.⁸ Since the introduction of the fluoroquinolones, no new antibacterials have entered clinical practice which are effective against these pathogens,⁹ confirming the inadequacy of the current pipeline of agents for the treatment of infections caused by carbapenem-resistant bacteria. The discovery of drug-inactivating enzyme inhibitors has become a priority as an alternative method of targeting carbapenemase-producers¹⁰ however, despite strong activity against SBLs, the structural heterogenicity, particularly of MBLs, makes the development of broad-spectrum carbapenemase inhibitors challenging. The only currently available treatment options for MBL-producers, such as those expressing New Delhi metallo-β-lactamase (NDM) are colistin, tigecycline and fosfomycin.¹¹ These agents too are limited in terms of their clinical availability, recommended indications and tendency to produce adverse effects.¹¹ This review aims to highlight the distinctive structural qualities of carbapenemase enzymes and to summarise their effects on the currently available serine BL inhibitors and MBL inhibitors in development. An increased understanding of the structural determinants of carbapenem resistance may highlight new strategies for overcoming this resistance in the priority pathogens listed in Table 1.

Table 1 Carbapenem-resistant pathogens in the WHO Bacterial Priority Pathogens List, 2024 (ref. 8)

Prioritization	Carbapenem-resistant pathogen
1st	Klebsiella pneumoniae
3rd	Acinetobacter baumannii
5th	Escherichia coli
10th	Pseudomonas aeruginosa
12th	Enterobacter species

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Serine β-lactamases (ambler β-lactamase classes A, C and D)

The hydrolytic activity of the serine β-lactamases (SBLs) is dependent upon a serine residue in the active site and the SBLs include enzymes from ambler classes A, C and D. The conserved serine residue in the active site acts as a nucleophile, attacking the carbapenem amide carbonyl and forming an unstable tetrahedral intermediate, which collapses to form an acyl-enzyme intermediate.³ An activated water molecule then attacks the carbonyl group of the acyl-enzyme adduct, producing a second unstable tetrahedral intermediate, before deacylation releases the inactive hydrolysed carbapenem from the active site (Scheme 1).³ Ampicillin β-lactamases (AmpC), which belong to ambler class C, are not typically considered carbapenemases but contribute to carbapenem resistance when co-occurring with reduced outer membrane porin function and efflux pump over-expression;^1,12 this review will limit discussion to SBLs with documented carbapenemase activity, such as Klebsiella pneumoniae carbapenemases (KPC) and oxacillinases (OXA).

Scheme 1 SBLs attack the amide bond of the carbapenem ring via the hydroxyl group of the conserved serine residue in the active site, forming a tetrahedral intermediate 1. This intermediate 1 collapses to form an acyl-enzyme adduct 2. Attack by an activated water molecule then forms a second tetrahedral intermediate 3, before deacylation releases the inactive carbapenem hydrolysis product 4 from the active site (KPC [ambler class A] active site residue identities shown; serine 70, lysine 73, and glutamic acid 166).

Klebsiella pneumoniae carbapenemases (KPCs)

KPCs are class A carbapenemases which, as the name suggests, were initially isolated from K. pneumoniae and have since been identified in other Klebsiella species, as well as in Escherichia coli, Proteus mirabilis, Enterobacter cloacae, Acinetobacter baumannii, Pseudomonas aeruginosa and Citrobacter freundii.^13–15 The secondary structure of KPC consists of an α-helical domain and a five-stranded β-sheet flanked by α-helices. The active site is surrounded by the Ω-loop, the α3–α4 loop and the hinge region, all containing residues necessary for optimal binding interactions with carbapenem substrates and active site conformational stability (Fig. 2).¹⁶ The key KPC catalytic residue, Ser70, is located adjacent to the amide bond of the carbapenem substrate so is optimally positioned to facilitate hydrolysis. Deprotonation of a water molecule is enabled by Glu166 and is necessary for the deacylation step (Scheme 1),^17,18 which is stabilised by hydrogen bonds with Ser70 and Thr237.¹⁸

Fig. 2 (a) Secondary structure of KPC-2 (PDB: 7TI2).²³ The α-subdomain is formed by several α-helices. The β-subdomain is formed by the β-sheet and various α-helices. Three key loops surround the KPC-2 active site: the Ω-loop, the α3-α4 loop and the hinge region; (b) the key KPC catalytic residues, Ser70 and Glu166.

These amino acid residues may suggest potential structural targets for new inhibitor development by impairing their role in catalysis and thus helping to overcome carbapenem hydrolysis (Table 2).^19,20

Table 2 Examples of natural KPC variants and their effect on their capacity to hydrolyse carbapenems^19,26

KPC variant	Mutation	Resulting effect on carbapenemase activity
KPC-2	Wild type (N/A)	Normal carbapenemase activity
KPC-3	His272Tyr	Unchanged carbapenemase activity
KPC-5	Pro104Arg	Reduced carbapenemase activity
KPC-6	Val240Gly	Unchanged activity against meropenem and ertapenem
		Reduced activity against imipenem
KPC-11	Pro104Leu	Reduced carbapenemase activity
KPC-12	Leu168Met	Reduced carbapenemase activity
KPC-29	ins269: Lys–Asp–Asp	Reduced activity against meropenem and ertapenem
		Unchanged activity against imipenem
KPC-31	His272Tyr, Asp179Tyr	Reduced carbapenemase activity
KPC-33	Asp179Tyr	Reduced carbapenemase activity
KPC-41	ins267: Pro–Asn–Lys	Reduced activity against meropenem and ertapenem
		Unchanged activity against imipenem
KPC-44	ins261: Ala–Val–Tyr–Thr–Arg–Ala–Pro–Asn–Lys–Asp–Asp–Lyz–His–Ser–Glu	Reduced activity against meropenem and ertapenem
		Unchanged activity against imipenem
KPC-45	Thr93Lys	Increased activity against imipenem
		Reduced activity against meropenem
		Unchanged activity against ertapenem
KPC-50	ins275: Glu–Ala–Val	Reduced activity against meropenem and ertapenem
		Unchanged activity against imipenem
KPC-54	Ala62Ser	Increased activity against imipenem
		Unchanged activity against ertapenem and meropenem
KPC-59	Gly89Asp	Increased activity against imipenem
		Reduced activity against meropenem
		Unchanged activity against ertapenem
KPC-77	Arg164Pro	Reduced carbapenemase activity
KPC-81	del1731l	Reduced carbapenemase activity

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Single amino acid mutations to these catalytic residues and structural loops significantly impact the ability of KPC to hydrolyse carbapenems. For example, a Glu166Ala substitution completely eradicates the activity of KPC towards all β-lactam substrates by impairing the essential activating role of Glu166. Mutations in the Ω-loop, particularly at key hydrogen bonding sites Arg164 and Asp179, are known to eliminate the required interactions between the KPC and β-lactamase inhibitors (Table 2).²¹ Mutations to Cys238 and Cys68, which are adjacent to the key Ser70, disrupt their role in forming a disulphide bridge, which is essential for the folding of KPC, thus leading to a decrease in carbapenemase activity.¹⁹

Conserved hydrophobic networks among class A β-lactamases exist between the α- and β-subdomains¹⁶ and have been demonstrated to play an essential role in KPC-induced carbapenem hydrolysis, through a comparison of the activity of BLs containing these hydrophobic networks to variants containing hydrophilic residues, using a combination of computational methods and site-directed mutagenesis (Tables 2 and 3).¹⁶ As these hydrophobic networks are essential to the KPC-2 active site structure, their disruption leads to a breakdown in the structural integrity of the active site, the loss of the KPC-2 mediated resistance phenotype, and the restoration of carbapenem susceptibility.¹⁶ Residues which are distant from the active site may also impact activity; for example, the Gly147Lys mutation has been shown to impact the shape of the α6 helix and, therefore, the conformation of the active site.¹⁹ All current BL inhibitors exert their activity by binding to the active site so there is potential for the development of allosteric modulators which bind within the α-network and disrupt it, thus indirectly impairing carbapenem hydrolysis. Investigations of the role of key residues within the α-network have shown that the orientation of hydrophobic residues is also essential for enzymatic activity. In particular, the indole ring of Trp105 forms π–π stacking and van der Waals interactions within the active site and these are known to enhance enzyme stability and catalytic activity (Fig. 3).²² Trp105 predominantly occupies a ‘flipped-out’ orientation in the resting KPC-2 enzyme, in which the indole ring is not permanently engaged in hydrophobic interactions. A disruption in the α-network causes the ‘flipped in’ conformation of the indole ring to be adopted only when required in order to compensate for a loss of structural integrity in the active site which can occur during substrate catalysis.¹⁶ If this hydrophobic residue can be targeted, there may be potential for a novel inhibitor to destabilise the active site and thus contribute to a loss of catalytic activity, by permanently preventing this dynamic ‘flipping in’ of tryptophan. This hypothesis is supported by an in vitro study which showed a decrease in the minimum inhibitory concentration (MIC) of meropenem, imipenem and ertapenem of at least 8-fold in E. coli TOP10 expressing just a Trp105Gly mutation in KPC-2.¹⁹

Table 3 Examples of possible amino acid mutations in KPC-2, their location within the enzyme structure and the proposed effect on carbapenemase activity and β-lactam/β-lactamase inhibitor (BL/BLI) combination susceptibility

Variants/amino acid mutations	Location within KPC structure	Proposed effect on carbapenemase activity or BL/BLI susceptibility	Ref.
Wild type	N/A	Normal carbapenemase activity and normal carbapenem susceptibility	16, 18
Leu102Thr	α3 helix	Increased carbapenem susceptibility and hydrolytic activity	16
Ile108Asn	α4 helix
Leu138Asn	α7 helix	Enzyme misfolding, resulting in no enzyme expression therefore no carbapenemase activity	16
Leu199Arg	α9–α10 loop
Phe72Tyr	Active site pocket	New hydrogen bond formed between hydroxyl group on tyrosine and Glu166, which lowers basicity and impairs catalytic water molecule activation	18
		Dramatically reduced carbapenemase activity
Thr237Ala	β-3 sheet	Decreased acylation rate and improved carbapenem binding	17, 18
		Impaired carbapenemase activity
Thr215Pro	Glu214–Arg220 loop	Dramatically reduced carbapenemase activity	18
Glu128His	Interacts with the Glu214–Arg220 loop	Impaired carbapenemase activity	18
Arg220His	Glu214–Arg220 loop	Decreased acylation rate and improved carbapenem binding	17, 18
		Impaired carbapenemase activity
Thr216Pro	Glu214–Arg220 loop	Impaired carbapenemase activity	18
Ser106Pro	103–106 loop	Impaired carbapenemase activity	18
Ala126Thr	α-Helix near Ser130	Impaired carbapenemase activity	18
Asn170Ala	Ω-Loop	Decreased deacetylation rate and significantly decreased carbapenemase activity	17
Asp179Asn	Ω-Loop	Increased carbapenemase activity	25, 27

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Fig. 3 (a) Trp105 occupies a flipped-out position in the KPC-2 enzyme, with minimal hydrophobic interactions with the substrate and surrounding active site residues (PDB: 3RXX).²⁴ (b) Trp105 occupies a flipped-in conformation in the KPC-2 enzyme in which hydrophobic π–π stacking interactions and hydrophobic bonds are formed between the indole ring and the active site substrate, as well as hydrogen bonds with surrounding active site residues (PDB: 7TC1).²⁵

Oxacillinases (OXAs)

OXAs are class D BLs and ten sub-classes with carbapenemase activity have been defined (Table 4), most of which are produced by A. baumannii isolates.²⁸ The secondary structure of OXA includes an α-subdomain of seven α-helices, and a β-domain of a six-stranded antiparallel β-sheet flanked by two α-helices, with the active site located between these domains (Fig. 4). All OXAs are unique in their spontaneous and reversible formation of a carbamylated lysine residue which, like Glu166 in KPC, acts as a general base to deprotonate a water molecule and facilitate a nucleophilic attack on the acyl-enzyme intermediate produced during acylation.^29–31 The structural diversity (Table 5) among OXAs enables these enzymes to display unique carbapenem affinity and hydrolytic activity.

Table 4 OXA enzymes with carbapenemase activity³²

OXA family	Bacterial species isolated from	Sequence divergence within family
OXA-23, OXA-27, OXA-49	A. baumannii	2–5 amino acids
OXA-24, OXA-25, OXA-26, OXA-40, OXA-72	A. baumannii	1–5 amino acids
OXA-51, OXA-64 to -66, OXA-68 to -71, OXA-75 to -78	A. baumannii	1–15 amino acids
OXA-58	A. baumannii	N/A
OXA-55, OXA-SHE	Shewanella algae	1–5 amino acids
OXA-48	K. pneumoniae	20 amino acids
OXA-54	Shewanella oneidensis
OXA-50 cluster	P. aeruginosa	1–5 amino acids
OXA-60 cluster	Ralstonia pickettii	1–21 amino acids

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Fig. 4 (a) Secondary protein structure of OXA-23 with α-helices and a β-sheet (PDB: 4JF6);⁴⁰ (b) the key catalytic residue, Ser79, and the carbamylated lysine reside, Lys82, in the OXA-23.

Table 5 Summary of the secondary structures of OXA-23, OXA-24/40, OXA-51, OXA-58, and OXA-143, their catalytic serine and carbamylated lysine residues, and residues involved in hydrophobic bridge formation

OXA-variant	Catalytic serine residue	Carbamylated lysine residue	Residues involved in hydrophobic bridge formation	Ref.
OXA-23	Ser79	Lys82	Hydrophobic bridge formed by Phe110 and Met221	40
OXA-24/40	Ser81	Lys84	Hydrophobic bridge formed by Tyr112 and Met223	34
OXA-51	Ser80	Lys83	Despite the presence of the aromatic residues Phe111 and Trp222, the hydrophobic bridge is weakly formed/not present	37
OXA-58	Ser83	Lys86	Despite the presence of the aromatic residues Phe114 and Met225, the hydrophobic bridge is weakly formed/not present	35
OXA-143	Ser81	Lys84	Despite the presence of the aromatic residues Tyr112 and Met223, the hydrophobic bridge is weakly formed/not present	41

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OXA-23 and OXA-24/40 contain a hydrophobic bridge over their active site cleft, formed by hydrophobic residues facing towards the active site (Fig. 5).^28,31,33 Various hydrophobic residues (such as Thr111, Trp115, and Trp221 in OXA-24) contribute to the stable, non-polar properties of the surface of this tunnel. The tunnel-like architecture of carbapenemase OXAs has been suggested to be a potential reason for their specificity for carbapenems over oxacillin, in comparison to other OXA enzymes which have high oxacillin-hydrolysing capabilities.³⁴ In comparison to the large, sterically hindered methylphenylisoxazolyl group present in oxacillin, the smaller side chains of carbapenems, such as the hydroxyethyl side chains in meropenem and imipenem (Fig. 6), may result in a reduction in the restriction encountered by these substrates upon entry into the OXA-carbapenemase active site and thus account for their rapid hydrolysis. OXA-48, OXA-51 and OXA-58 have similar hydrophobic residues at the entry to the active site, but have a weak hydrophobic bridge structure (Fig. 5);³⁵ although the hydrophobic bridge is not essential in OXA enzymes, it has a clear role in enhancing carbapenem hydrolysis efficiency.³⁶

Fig. 5 (a) Gaussian surface representation of the hydrophobic properties of OXA-23 (hydrophobic: green, charged/polar: red). The hydrophobic residues, Phe110 and Met221 which form the hydrophobic bridge are labelled (PDB: 4JF4 (https://www.rcsb.org/3d-view/4JF4/1)).⁴² Meropenem is depicted within the active site tunnel; (b) Gaussian surface representation of the hydrophobic properties of OXA-58 (hydrophobic: green, charged/polar: red). The hydrophobic residues, Phe114, Phe113 and Met225 are labelled (PDB: 7VX3 (https://www.rcsb.org/3d-view/7VX3/1)).⁴³ Hydrolysed meropenem is bound within the active site.

Fig. 6 Structures of oxacillin, imipenem and meropenem.

In addition to some large structural differences, OXA carbapenemases may differ by single amino acid mutations; OXA-51 is unique in the presence of a tryptophan residue, Trp222, which takes the place of methionine in other OXA carbapenemases (Table 5).³⁷ In contrast to these methionine-containing variants, an overlapping interaction between the indole ring of tryptophan and the pyrroline of an incoming carbapenem substrate results in significant steric hindrance. This is further complicated by a network of hydrophobic interactions created by the β4–β5 loop, which increase the energy barrier for the binding of the incoming substrate.³⁷ The presence of tryptophan at this position possibly accounts for the relatively low carbapenemase activity of OXA-51 in comparison to OXA-24/40, OXA-23, and OXA-58.³⁷ In addition, single amino acid mutations to Trp222 have been identified in clinically isolated strains of OXA-51 which have enhanced carbapenem affinity.³⁸ For example, the Michaelis constant (K_m) and dissociation constant of the enzyme–substrate complex (K_s) for OXA-234, a Trp222Leu mutant of OXA-51,³⁹ are both significantly decreased in comparison to OXA-51, indicating the greater substrate affinity of this mutant. This enhancement in activity can largely be explained by the presence of a smaller leucine side chain, which eliminates the steric clash at the entrance to the active site.³⁹ Other amino acid mutations in clinically isolated OXA-51 variants commonly occur at Leu167, Ile129, and Pro130,³⁸ with variable effects upon carbapenemase activity (Table 6). A similar effect is observed with an Ile129Val substitution, where the steric hindrance between the side chain of isoleucine and the hydroxyethyl group of a carbapenem substrate is eliminated by replacement with valine.³⁹ The unique properties of OXA-51 may have resulted in its decreased propensity to cause carbapenem resistance, but the evolution and dissemination of OXA-51 mutants, such as those harbouring a Trp222 substitution, holds the potential to transform these relatively weak hydrolytic enzymes into clinically important carbapenemases.

Table 6 Examples of amino acid mutations which enhance carbapenem affinity and increase carbapenemase activity of OXA enzymes

OXA variant	Amino acid mutation	OXA mutant	Ref.
OXA-51	Trp222Leu	OXA-200	29, 30
OXA-51	Trp222Gly	OXA-79	39
OXA-51	Trp222Met	OXA-51 M	37
OXA-51	Leu167Val	OXA-219	38, 44
OXA-66	Leu167Val	OXA-82	38, 44
OXA-69	Leu167Val	OXA-107	38, 44
OXA-71	Leu167Val	OXA-113	38, 44
OXA-66	Pro130Gln	OXA-109	39
	Gain of activity specific for carbapenem substrates
OXA-66	Trp222Leu	OXA234	39
	Gain of activity is not as limited to carbapenem substrates like the Pro130Gln substitution
OXA-66	Ile129Val/Trp222Leu	OXA-172	39
	Gain of activity specific for carbapenem substrates
OXA-66	Ile129Val/Trp222Leu/Pro226Leu	OXA-173	39
	Unusually broad activity against penicillins, cephalosporins, carbapenems and aztreonam
OXA-66	Pro130Gln/Trp222Leu	Unnumbered variant	39
	Greater gain of activity against carbapenem substrates compared with either substitution alone

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Serine β-lactamase inhibitors

Clavulanic acid (Fig. 7) was the first clinically used BL inhibitor, and has activity against some class A BLs, but not against KPCs,⁴⁵ class D OXAs or class B MBLs. The semi-synthetic BL inhibitors sulbactam⁴⁶ and tazobactam⁴⁷ (Fig. 7) are active against a similar spectrum of penicillinases but a broader range of cephalosporinases; KPC and OXA carbapenemases are, however, resistant to inhibition as they are able to hydrolyse these inhibitors.^45,48

Fig. 7 Structure of clavulanic acid, the sulphone-BL inhibitors, and the non-β-lactam-BL inhibitors.

Avibactam

Mechanism of action

Avibactam (Fig. 7) was the first non-β-lactam BL inhibitor to complete clinical trials and, upon binding to a serine BL enzyme, it undergoes acylation to form a unique carbamoyl–enzyme complex.⁴⁹ Uniquely, deacylation then regenerates the unhydrolysed form of avibactam due to recyclisation being favoured over hydrolysis of the avibactam-enzyme complex (Scheme 2).⁴⁹ Thus, avibactam remains capable of binding to new enzymatic targets as opposed to being hydrolysed and deactivated.^1,50,51 An exception to this unique recyclisation is observed when avibactam acts on the KPC-2 enzyme, as this normal, slow recyclisation competes with a process which hydrolyses and fragments avibactam. If avibactam becomes fragmented upon release from KPC-2, it is permanently inactivated.^50,52 The hydrolysis of avibactam has a minimal effect upon its inhibitory activity, as only a small proportion is fragmented but the evolution of KPC-2 mutants which rapidly fragment and inactivate this inhibitor, without allowing the ring closure mechanism to occur, may challenge its future clinical usefulness.⁵²

Scheme 2 Recyclisation and hydrolysis pathways for avibactam.

Spectrum of activity and clinical use

Avibactam has therapeutic advantages over sulfone BL inhibitors and is a potent inhibitor of class A, C, and some class D carbapenemases, including KPC-2 and OXA-48.^50,51,53 Avibactam-resistant KPC-producers are reported to harbour unique mutations to the Ω-loop (Table 2),^21,25,54 which is usually responsible for substrate stabilization and orientation within the active site. Interestingly, avibactam/aztreonam is unique in its activity against SBL and MBL-producing bacteria, despite the lack of inhibitory activity of avibactam alone on either of these enzyme classes (Table 7).⁵⁵ Aztreonam is resistant to hydrolysis by MBLs, but is susceptible to hydrolysis by SBLs.⁵⁶ When combined, avibactam inhibits SBLs, protecting aztreonam's antibacterial activity and allowing the combination to exhibit a full range of activity against SBL and MBL-producing Enterobacterales.⁵⁷P. aeruginosa displays resistance to avibactam/aztreonam, which could be due to co-existing resistance mechanisms, such as extended-spectrum BLs (which are poorly inhibited by avibactam and able to hydrolyse aztreonam), and efflux pump overexpression.⁵⁸ Another combination, ceftazidime/avibactam, is active against SBL carbapenemase-producers, provided these bacteria do not co-express MBLs (Table 7).⁵⁵

Table 7 BL/BLI combinations which are currently approved by the Food and Drug Administration (FDA) and/or European Medicines Agency (EMA) and/or Therapeutic Goods Administration (TGA)

Combination	Main SBL carbapenemase targets			Approving authorities	Current approved indications
	KPC	AmpC	OXA
Avibactam
Aztreonam	✓	✓	✓	FDA, EMA, TGA	Complicated intra-abdominal infection with metronidazole in adults with limited to no alternative options against susceptible Gram negatives
					Hospital-acquired bacterial pneumonia
					Ventilator-associated bacterial pneumonia
Ceftazidime	✓	✓	✓	FDA, EMA, TGA	Complicated urinary tract infection including pyelonephritis and complicated intra-abdominal infection with metronidazole in adults with limited to no alternative options against susceptible Gram negatives
					Hospital-acquired bacterial pneumonia
					Ventilator-associated bacterial pneumonia
Vaborbactam
Meropenem	✓	✓	✗	FDA, EMA	Complicated urinary tract infections including pyelonephritis
					Hospital-acquired bacterial pneumonia
					Ventilator-associated bacterial pneumonia
Relebactam
Imipenem and cilastin	✓	✓	✗	FDA, EMA	Complicated urinary tract infection
					Complicated intra-abdominal infection
					Hospital-acquired bacterial pneumonia
					Ventilator-associated bacterial pneumonia and associated bacteriaemia

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Vaborbactam

Mechanism of action

Vaborbactam (Fig. 7) is a cyclic boronate SBL inhibitor; the central boron moiety forms a covalent bond to the serine hydroxyl group in the enzyme's active site, producing a structure which mimics the high energy, tetrahedral transition state analogue formed during the normal catalytic hydrolysis process (Scheme 1). This covalent bond prevents the breakdown of the tetrahedral intermediate and thus traps the enzyme and prevents further substrate hydrolysis.

Spectrum of activity and clinical use

Vaborbactam is a broad spectrum SBL inhibitor, with potent activity against class A (including KPC) and C enzymes,⁵¹ but clinically insignificant inhibitory activity against class D carbapenemases;^59,60 it is inactive against MBLs.⁵⁹ Structural studies have revealed that even, in the presence of KPC Ω-loop mutations, strong interactions exist between vaborbactam and KPC, with minimal disturbance to the Glu166 or Asn170 residues which are essential for catalysis.²⁵ This allows vaborbactam to maintain susceptibility against KPC mutants which are resistant to ceftazidime/avibactam combinations (Table 7). Some resistance has been reported to the vaborbactam/meropenem combination due to low expression of outer membrane porins, particularly OmpK35 and OmpK36, as ample expression of these porins is necessary to facilitate vaborbactam's entry into bacterial cells.^61,62 Although vaborbactam is able to overcome aspects of ceftazidime/avibactam resistance caused by Ω-loop mutations, cross-resistance to meropenem/vaborbactam has been noted in KPC producing K. pneumoniae strains, which have the potential to significantly challenge the effective treatment outcomes of BL/BLI combination use against some carbapenemase producing bacteria.

Relebactam

Mechanism of action

Relebactam (Fig. 7) is a diazabicyclooctane BL inhibitor, which displays binding that similar to that of avibactam.⁵¹ The carbonyl group of the diazabicyclooctane core is susceptible to nucleophilic attack by the catalytic serine residue within the active site of SBLs. A reversible covalent bond is formed within the active site, resulting in enzymatic acylation. Relebactam displays a long residence time within the active site, during which time it effectively inhibits β-lactam antibiotic substrate binding and inactivation, and this is followed by a slow-off deacylation process.⁶³

Spectrum of activity and clinical use

Relebactam is a potent inhibitor of class A (including KPCs)⁶⁴ and class C AmpC, but has variable activity against class D BLs,⁶⁵ with OXA-48 carbapenemase-producing bacteria, such as A. baumannii showing inconsistent susceptibility to relebactam.⁶⁶ Relebactam is completely inactive against MBLs.^51,65 Relebactam can successfully restore imipenem susceptibility against KPC-producers, but not against OXA-48 producers (Table 7).⁶⁶ Additionally, relebactam remains active in the presence of porin mutations and efflux pump overexpression, offering a therapeutic advantage over vaborbactam's activity provided the bacteria do not produce class D carbapenemases or MBLs.^51,67

Ambler class B metallo-β-lactamases

MBLs are zinc-dependant, ambler class B enzymes with either one (B2) or two (B1 and B3) divalent zinc ions in their active site to facilitate hydrolysis via zinc chelation.² MBLs display hydrolytic activity towards all carbapenems.⁶⁸ The monocyclic structure of monobactams was previously believed to be non-conducive to MBL binding, resulting in a decrease in their potential to be hydrolysed by these enzymes.⁶⁹ However, recent studies have illustrated the potential for NDM-1 to hydrolyse aztreonam,⁷⁰ thus removing the advantage which this monobactam previously held regarding its activity against the MBL classes.

In B1 MBLs, the Zn1 site is coordinated to three histidine residues, His120, His122, and His189 (NDM-1 numbering), whereas the Zn2 site is coordinated to Asp124, Cys208 and His250 (NDM-1 numbering).^68,71 MBLs containing two zinc ions interact with an incoming β-lactam substrate via the β-lactam ring carbonyl group at the Zn1 site, and the amide nitrogen with the Zn2 site. These interactions optimally orientate the β-lactam carbonyl group for attack by the zinc-coordinated hydroxide ion within the active site, forming a tetrahedral intermediate. This unstable intermediate undergoes hydrolysis at the carbonyl-carbon producing an anionic nitrogen which coordinates to Zn2,⁷² but which is quickly protonated and released from the active site having rendered the β-lactam substrate inactive (Scheme 3).⁷³ B1 MBLs display the broadest spectrum of activity towards penicillins, cephalosporins and carbapenems⁶⁸ with the most clinically relevant B1 MBLs being the imipenemases (IMP), NDM and Verona integron (VIM) classes.

Scheme 3 Mechanism of hydrolysis by zinc dependent, MBL enzymes on carbapenems.

New Delhi metallo-β-lactamase

NDM-1 was first isolated from K. pneumoniae and variants then emerged rapidly and have been disseminated globally.⁷⁴ MBL enzymes are composed of a conserved αβ/βα fold, and two adjacent β-sheets forming a ‘β-sandwich’, surrounded by five α-helices (Fig. 8). Contained between the ‘β-sandwich’, the NDM-1 active site is shallow and wide, surrounded by the L3 and L10 loops.⁷¹ The L3 loop is composed of hydrophobic amino acids Leu65, Met67, Phe70, and Val73, which aid in favourable carbapenem–enzyme interactions and exhibit a high level of flexibility to accommodate rigid carbapenem substrates.⁷¹ The L10 loop, composed of Cys280, Lys211 and Asn220, accommodates Zn²⁺ at the Zn2 site, and facilitates active site substrate binding.⁷¹

Fig. 8 (a) Secondary protein structure of NDM-1 with α-helices and a β-sandwich in complex with a hydrolysed imipenem (PDB: 5YPL (https://www.rcsb.org/3d-view/5YPL/1));⁸¹ (b) within the active site, NDM-1 contains two catalytic Zn²⁺ ions, located in the Zn1 and Zn2 sites.

Among NDM mutants, the Met154Leu substitution is the most common and is associated with the greatest carbapenem resistance.⁷⁵ Interestingly, this substitution does not directly interact with Zn²⁺ or the β-lactam substrate,⁷⁶ but with the leucine residue which stabilizes the Zn2 site and usually gives up its Zn²⁺ ion in zinc-deprived conditions.^72,76,77 It is not surprising that the ability for NDM to retain its di-zinc core is catalytically beneficial and resistance-promoting, as the clinically relevant B1 MBLs are most active and therefore inactivate carbapenems readily, in their di-zinc forms.⁷⁷ The decrease in catalytic activity observed when the NDM active site is stripped of zinc, in zinc-deprived conditions, provides a rationale for the interest in MBL inhibitors which act as zinc-chelators, such as ethylenediaminetetraacetic acid (EDTA).^11,78 Translating the usefulness of the metal-chelating mechanism of action into clinically useful inhibitors remains challenging, however, as over one third of human proteins, e.g. the angiotensin-converting enzyme, are metalloproteins⁷⁹ and the potential for off-target effects due to interactions with other human metalloenzymes is high.⁷⁹ Efforts to develop selective metalloenzyme inhibitors still presents a promising future opportunity in advancing the armoury against MBL-producers.

Amino acid mutations in L3 and L10 loops, zinc coordination sites or distal solvent-exposed regions of NDM-1 also distinctly impact catalytic activity. In zinc coordination sites, His122Gln and Asp124Ala substitutions have been shown to reduce the binding contacts between imipenem and the NDM-1 active site.⁸⁰ The same is true for the His122Arg mutant, with meropenem as the ligand.⁸⁰ Interestingly, both mutants display significantly decreased affinity for their respective carbapenem substrates due to the formation of fewer contacts with active site residues, despite conserved interactions with Zn coordination sites.⁸⁰ While it was concluded that these mutations do affect carbapenem binding ability, however, further studies were needed to conclude the effects on binding free energy.⁸⁰ Solvent exposed residues of NDM-1, such as Arg270, also have a clear role in carbapenemase activity, albeit indirectly. The carbapenemase activity of NDM-20 has been shown to be lower than NDM-5, despite these isoforms differing by only an Arg270His substitution. It is probable that the Arg270His mutation has an impact on protein folding and structural conformation, rather than on the dynamics of the catalytic active site. Similar enzyme-misfolding effects which impair carbapenemase activity have been observed in KPC-2 enzymes, due to Leu138Asn and Leu199Arg substitutions (Table 3). However, unlike the detrimental effects of the mentioned KPC-2 mutations, NDM-20 displays stronger catalytic activity than the wild-type NDM-1 enzyme, probably due to co-occurring gain-of-function mutations in this variant (Table 8). Clearly, modifications within and distal to the active site have the potential to influence catalysis. Establishing the surrounding structural residues with the greatest susceptibility to change active site dynamics or overall protein structure may guide the targeted development of MBL inhibitors which bypass the selectivity issues and potential pharmacologically adverse effects of metal-binding inhibitors.

Table 8 Amino acid chain substitutions of various natural NDM variants, compared with the NDM-1, wild-type enzyme

NDM variants	Substitution mutation harboured by variant compared with NDM-1	Effect of substitution on activity	Ref.
NDM-1	N/A	Normal carbapenemase activity	82
NDM-2	Pro28Ala	Similar activity to NDM-1	83
NDM-4	Met154Leu	Increased carbapenemase activity relative to NDM-1	84
NDM-5	Val88Leu	Increased carbapenemase activity relative to NDM-1	76, 82
	Met154Leu
NDM-7	Asp130Asn	Increased carbapenemase activity relative to NDM-1	85
	Met154Lys
NDM-8	Asp130Gly	Similar activity to NDM-1	86
	Met154Lys
NDM-9	Glu149Lys	Similar carbapenemase activity to NDM-1. Resistant to taniborbactam inhibition	87
NDM-14	Asp130Gly	Increased carbapenemase activity relative to NDM-1	85
NDM-20	Val88Leu	Increased carbapenemase activity relative to NDM-1, decreased relative to NDM-5	88
	Met154Leu
	Arg270His
NDM-60	Val88Leu	Increased carbapenemase activity relative to NDM-5	82
	Met154Leu
	Asp202Asn
NDM-1 mutant	Lys125Ala	Decreased carbapenemase activity	80
NDM-1 mutant	Asp192Ala	Decreased carbapenemase activity	80
NDM-1 mutant	Asp212Ala	Decreased carbapenemase activity	80

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Metallo-β-lactamase inhibitors

Taniborbactam (VNRX-5133)

Mechanism of action. Taniborbactam (Fig. 7) is a 3rd-generation BL inhibitor which displays pan-inhibition of MBLs and SBLs. The zinc-bound hydroxide within the active site acts as a nucleophile, attacking taniborbactam's boron atom and forming a boron-oxygen covalent bond (Scheme 4). This process is aided by the aromatic carboxylic acid group, which optimally positions the boronate ring towards the nucleophilic moiety, mimicking the carboxylate group of β-lactam antibacterial substrates. For SBL inhibition, a reversible covalent bond is formed between the boron atom and the catalytic serine in the active site. Both MBL and SBL inhibition proceed via the formation of a tetrahedral conformation about the boron atom, mimicking the tetrahedral transition state formed otherwise by the enzyme and an antibiotic substrate (Scheme 4).⁸⁹

Scheme 4 Mechanism of SBL inhibition by taniborbactam, producing a boronic transition state analogue.

Spectrum of activity and clinical use. Taniborbactam inhibits enzymes from all Ambler classes including KPCs, OXAs (except OXA-48), AmpC, and B1 class MBLs VIM-1, VIM-2, and NDM-1.^9,90–92 MBL inhibition relies on taniborbactam forming hydrogen bonds with conserved active site residues. Substitution mutations to these residues, such as Glu149Lys in NDM-9 (ref. 87) and an aspartic acid at this position in IMP-1, are suggested to account for the lack of inhibition of these MBL enzymes by taniborbactam,^9,87,89,93 due to the side-chains of these residues being less conducive to hydrogen-bond formation.⁸⁹ Ionic interactions are also essential to taniborbactam's activity, between anionic residues in B1 MBL enzymes, and the conjugate acid of the primary amine of taniborbactam's (2-aminoethyl)cyclohexylamine tail.⁹⁴ Conserved anionic residues in B1 MBLs are necessary for the interaction with this chain, which enhances membrane permeability and correctly positions the boron atom distant from this tail, towards the nucleophile in the MBL active site. The substitution of conserved anionic residues with cationic or uncharged residues, such as Glu149Lys in NDM-9, Asp236Tyr in NDM-30, and Glu149Lys in VIM-83, abolishes ionic interactions between these enzymes and taniborbactam, allowing them to evade inhibition.⁹⁴ Despite the rarity of these mutations, they present a potentially detrimental challenge to the successful use of cyclic boronate BL inhibitors against MBL producers. This is particularly concerning when considering the scarcity of inhibitors with activity against these enzymes.

With regards to the SBLs, the combination of cefepime/taniborbactam retains activity against KPC-3 enzymes, which display resistance to ceftazidime/avibactam due to Ω-loop mutations.^9,90 Most notably, taniborbactam can restore the susceptibility of cefepime against Enterobacterales and P. aeruginosa, which produce NDM-1 and VIM-2 respectively, a significant therapeutic advantage in comparison to the previous BL/BLI combinations, which completely lack MBL inhibitory coverage.⁹ Although only cefepime has been investigated in clinical trials with taniborbactam, experimental studies have provided evidence that it can also restore meropenem's activity in carbapenem-resistant strains.^95,96 The cefepime/taniborbactam combination is yet to receive new drug approval by the FDA, and is currently halted in Phase III clinical trials whilst awaiting additional data from the manufacturing company (Table 9).⁹⁷

Table 9 Clinical trials involving the cefepime/taniborbactam or ceftibuten/xeruborbactam combinations

Investigational combination	ClinicalTrials.gov ID	Trial objectives	Trial phase and status
Cefepime/taniborbactam	NCT04951505 (ref. 98)	Assess the intrapulmonary pharmacokinetics of cefepime/taniborbactam in healthy adults	Phase 1: completed (September 2021)
	NCT03840148 (ref. 99)	Assess the safety, efficacy, and tolerability of cefepime/taniborbactam versus meropenem in adult patients with complicated urinary tract infection	Phase 3: completed (December 2021)
	NCT06168734 (ref. 100)	Assess the safety and efficacy of cefepime/taniborbactam versus meropenem in adult patients with ventilated hospital-acquired bacterial pneumonia or ventilator-associated bacterial pneumonia	Phase 3: withdrawn (estimated completion in December 2027)
Ceftibuten/xeruborbactam oral prodrug (QPX7831)	NCT06079775 (ref. 101)	Assess safety, tolerability, and pharmacokinetics of both agents alone and in combination, in healthy adults	Phase 1: in progress
Ceftibuten/xeruborbactam oral prodrug (QPX7831)	NCT06157242 (ref. 102)	Assess safety, tolerability, and pharmacokinetics of the combination in renal impairment	Phase 1: in progress
Cefiderocol/xeruborbactam	NCT06547554 (ref. 103)	Assess safety and pharmacokinetics of the combination in healthy adults	Phase 1: in progress

---

Xeruborbactam (QPX7728)

Mechanism of action. Xeruborbactam (Fig. 7) is a competitive, broad-spectrum cyclic boronate inhibitor which, like taniborbactam, also displays pan-inhibitory activity against MBLs and SBLs. MBL inhibition proceeds by a catalytic water molecule within the active site covalently binding to the boron atom of xeruborbactam, forming a Michaelis–Menten enzyme–inhibitor complex, followed by the rapid dissociation of the substrate from the enzyme active site.¹⁰⁴ In contrast, xeruborbactam inhibits SBLs by binding to the catalytic serine residue within the active site. A common feature of both MBL and SBL inhibition is the formation of a tetrahedral boron configuration.¹⁰⁴ The rigidity of xeruborbactam, due to its cyclic structure and compactness, which allow a good fit within the BL active site, may account for its strong inhibitory activity.¹⁰⁴

Spectrum of activity and clinical use. Xeruborbactam is a potent inhibitor of B1 MBLs, including NDM-1, IMP, and VIM-1,¹⁰⁵ with superior activity against IMP and NDM-9, against which taniborbactam is inactive.¹⁰⁵ Although little to no inhibition is displayed against class B2 and B3 MBLs,^104,106 these classes are significantly less widespread and therefore, less associated with resistance to last-line antibiotics in comparison to B1 MBLs. Xeruborbactam also inhibits class A KPCs, with greater inhibitory activity than vaborbactam, relebactam and avibactam, class D OXAs, and AmpC. Xeruborbactam increases the susceptibility of MBL and SBL-producing carbapenem resistant Enterobacterales (and other bacteria, including P. aeruginosa) to meropenem with in vitro evidence suggesting this combination displays greater potency against these Gram negative bacteria than ceftazidime/avibactam, meropenem/vaborbactam and imipenem/relebactam.¹⁰⁶ However, xeruborbactam is not yet clinically approved for use, either alone or in combination, although three Phase 1 clinical trials are currently underway (Table 9).

Conclusions

Carbapenemase production by Gram negative bacteria is a prelude to the possibility of a post-antibiotic era, as the susceptibility to last line carbapenem antibiotics continues to decline. This review highlights the unique structural differences among prominent BL enzymes which confer resistance to carbapenems, and where they have been defined in current literature, their effects on enzymatic efficiency and susceptibility to inhibition by available BL inhibitors. The recent development of serine BL inhibitors, such as those of the diazabicyclooctane class, has improved the clinical efficacy of β-lactam antibiotics and elucidated the potential to overcome treatment resistance caused by class A, C and D BLs, including those with a carbapenem-hydrolyzing phenotype. Only pan-inhibitors, taniborbactam and xeruborbactam, have the potential to overcome resistance posed by both MBLs and SBLs but they are yet to obtain regulatory approval. A continued structural and mechanistic understanding of the NDM active site is necessary to guide the development of inhibitors that are both target-specific, and sufficiently stable to overcome the strong catalytic tendency of the MBL class of enzymes. Such efforts would help to ensure an effective clinical resource for the treatment of MBL-producing infections.

Conflicts of interest

There is no conflict of interest to declare.

Data availability

No primary research results, software or code have been included and no new data were generated or analysed as part of this review.

Acknowledgements

We would like to acknowledge the custodianship of the Gadigal and Dharug people of the Eora Nation as the traditional owners of the lands where the work on this literature review was conducted. We acknowledge support from the Sydney Anti-Bacterial Accelerator (SABA), co-funded by Sydney Infectious Diseases Institute and The University of Sydney Centre for Drug Discovery Innovation.

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