Three decades of beta-lactamase inhibitors

Abstract

Since the introduction of penicillin, beta-lactam antibiotics have been the antimicrobial agents of choice. Unfortunately, the efficacy of these life-saving antibiotics is significantly threatened by bacterial beta-lactamases. beta-Lactamases are now responsible for resistance to penicillins, extended-spectrum cephalosporins, monobactams, and carbapenems. In order to overcome beta-lactamase-mediated resistance, beta-lactamase inhibitors (clavulanate, sulbactam, and tazobactam) were introduced into clinical practice. These inhibitors greatly enhance the efficacy of their partner beta-lactams (amoxicillin, ampicillin, piperacillin, and ticarcillin) in the treatment of serious Enterobacteriaceae and penicillin-resistant staphylococcal infections. However, selective pressure from excess antibiotic use accelerated the emergence of resistance to beta-lactam-beta-lactamase inhibitor combinations. Furthermore, the prevalence of clinically relevant beta-lactamases from other classes that are resistant to inhibition is rapidly increasing. There is an urgent need for effective inhibitors that can restore the activity of beta-lactams. Here, we review the catalytic mechanisms of each beta-lactamase class. We then discuss approaches for circumventing beta-lactamase-mediated resistance, including properties and characteristics of mechanism-based inactivators. We next highlight the mechanisms of action and salient clinical and microbiological features of beta-lactamase inhibitors. We also emphasize their therapeutic applications. We close by focusing on novel compounds and the chemical features of these agents that may contribute to a "second generation" of inhibitors. The goal for the next 3 decades will be to design inhibitors that will be effective for more than a single class of beta-lactamases.

FIG. 1.

Chemical structures of compounds discussed in the text. Compounds 1 to 7, a representative penicillin (compound 1), an extended-spectrum cephalosporin (compound 2), a monobactam (compound 3), and carbapenems (compounds 4 to 7). The numbering scheme for penicillins, cephalosporins, and monobactams is shown. Compounds 8 to 10, β-lactamase inhibitors in clinical practice. Compounds 11 to 38, investigational β-lactamase inhibitors: monobactam derivatives (compounds 11 to 14), a penicillin derivative (compound 15), penems (compounds 16 to 20), penam sulfones (compounds 21 to 24), a boronic acid transition state analog (compound 25), non-β-lactams (compounds 26 to 28), and metallo-β-lactamase inhibitors (compounds 29 to 38).

FIG. 1.

Chemical structures of compounds discussed in the text. Compounds 1 to 7, a representative penicillin (compound 1), an extended-spectrum cephalosporin (compound 2), a monobactam (compound 3), and carbapenems (compounds 4 to 7). The numbering scheme for penicillins, cephalosporins, and monobactams is shown. Compounds 8 to 10, β-lactamase inhibitors in clinical practice. Compounds 11 to 38, investigational β-lactamase inhibitors: monobactam derivatives (compounds 11 to 14), a penicillin derivative (compound 15), penems (compounds 16 to 20), penam sulfones (compounds 21 to 24), a boronic acid transition state analog (compound 25), non-β-lactams (compounds 26 to 28), and metallo-β-lactamase inhibitors (compounds 29 to 38).

FIG. 2.

“Family portrait” of β-lactamase enzymes. (A) Class A SHV-1; (B) class B IMP-1; (C) class C E. coli AmpC; (D) class D OXA-1. For classes A, C, and D, the active-site serine is shown in yellow; for class B, the two Zn²⁺ ions are shown. (Based on PDB entries 1SHV, 1DDK, 2BLS, and 1M6K, respectively.)

FIG. 3.

Proposed reaction mechanism for a penicillin β-lactam substrate and a class A serine β-lactamase enzyme in which Glu166 participates in activating a water molecule for both acylation and deacylation (additional evidence exists to suggest that this acylation scheme may exist in competition with a mechanism where Lys73 acts as the general base to activate Ser70) (261). Dashed lines represent hydrogen bonds, and the number labels are included to help the reader appreciate the general order of events, not as absolute and discrete steps. Following activation of the hydroxyl group, Ser70 performs a nucleophilic attack on the carbonyl group of the β-lactam antibiotic, resulting in a high-energy acylation intermediate. Protonation of the β-lactam nitrogen leads to cleavage of the C-N bond and formation of the covalent acyl-enzyme, which adopts a lower energy state. Attack by a catalytic water leads to a high-energy deacylation intermediate, with subsequent hydrolysis of the bond between the β-lactam carbonyl and the oxygen of Ser70. Deacylation regenerates the active enzyme and releases the inactive β-lactam.

FIG. 4.

Schematic representation of the Zn²⁺-binding site of dinuclear subclass B1 metallo-β-lactamases such as B. cereus BcII.

FIG. 5.

Proposed mechanism of inhibition of class A β-lactamases by clavulanate, showing the different acyl-enzyme fragmentation products (expressed in daltons), that have been experimentally observed. (Based on data from references , , , , and .)

FIG. 6.

Imipenem tautomers hypothesized to form after acylation of carbapenems by serine β-lactamases. The deacylation rate of the Δ¹-pyrroline is significantly lower than that of the Δ²-pyrroline, which is thought to play a large role in the inhibitory activity of these compounds (407, 458).

FIG. 7.

Molecular representation of SHV-1/meropenem acyl-enzyme, based on PDB coordinates 2ZD8. The conformation with the carbonyl of the acyl-serine bond in the oxyanion hole (formed by NH amides of Ser70 and Ala237) is colored teal, while the conformation with the carbonyl flipped out is colored purple. Residues 244, 130, and 105, which demonstrate significant orientation changes compared to the apo SHV-1 β-lactamase, are labeled. Glu166 and Asn132, important for making hydrogen bonds with the deacylation water (the approximate location is represented as a red sphere) and C-6 hydroxyethyl substituent, respectively, are shown. The large R′ carbamoyl-pyrrolidinyl group of meropenem was disordered and thus is only partially illustrated.

FIG. 8.

Molecular representation of TEM-1 active site, showing residues that are most frequently implicated in the development of inhibitor-resistant TEM enzymes. Based on PDB coordinates 1TEM.

FIG. 9.

Schematic representation of the proposed Michaelis preacylation complex of TEM-1 and clavulanate.

FIG. 10.

Proposed reaction mechanisms for the inactivation of a serine β-lactamase by BRL 42715, showing formation of the seven-membered thiazepine ring (A), and by LN-1-255, showing intermolecular capture by the pyridyl nitrogen leading to a bicyclic aromatic intermediate (B). (Based on data from references , , , and .)

FIG. 11.

Scheme illustrating the interactions of a serine β-lactamase with a cefotaxime substrate (A) compared to a cefotaxiime-like boronic acid TSA (B). Notice that the reaction with the inhibitor is reversible.

FIG. 12.

Molecular representation of cross-linked active-site residues Ser64 and Lys315 (shown in yellow) formed after aminolysis of an O-aryloxycarbonyl hydroxamate inhibitor in E. cloacae P99 β-lactamase. (Based on PDB 2P9V and data from references and .)