Inhibition of the class C beta-lactamase from Acinetobacter spp.: insights into effective inhibitor design

Abstract

The need to develop beta-lactamase inhibitors against class C cephalosporinases of Gram-negative pathogens represents an urgent clinical priority. To respond to this challenge, five boronic acid derivatives, including a new cefoperazone analogue, were synthesized and tested against the class C cephalosporinase of Acinetobacter baumannii [Acinetobacter-derived cephalosporinase (ADC)]. The commercially available carbapenem antibiotics were also assayed. In the boronic acid series, a chiral cephalothin analogue with a meta-carboxyphenyl moiety corresponding to the C(3)/C(4) carboxylate of beta-lactams showed the lowest K(i) (11 +/- 1 nM). In antimicrobial susceptibility tests, this cephalothin analogue lowered the ceftazidime and cefotaxime minimum inhibitory concentrations (MICs) of Escherichia coli DH10B cells carrying bla(ADC) from 16 to 4 microg/mL and from 8 to 1 microg/mL, respectively. On the other hand, each carbapenem exhibited a K(i) of <20 microM, and timed electrospray ionization mass spectrometry (ESI-MS) demonstrated the formation of adducts corresponding to acyl-enzyme intermediates with both intact carbapenem and carbapenem lacking the C(6) hydroxyethyl group. To improve our understanding of the interactions between the beta-lactamase and the inhibitors, we constructed models of ADC as an acyl-enzyme intermediate with (i) the meta-carboxyphenyl cephalothin analogue and (ii) the carbapenems, imipenem and meropenem. Our first model suggests that this chiral cephalothin analogue adopts a novel conformation in the beta-lactamase active site. Further, the addition of the substituent mimicking the cephalosporin dihydrothiazine ring may significantly improve affinity for the ADC beta-lactamase. In contrast, the ADC-carbapenem models offer a novel role for the R(2) side group and also suggest that elimination of the C(6) hydroxyethyl group by retroaldolic reaction leads to a significant conformational change in the acyl-enzyme intermediate. Lessons from the diverse mechanisms and structures of the boronic acid derivatives and carbapenems provide insights for the development of new beta-lactamase inhibitors against these critical drug resistance targets.

Figure 1

Schemes illustrating the interactions of a serine β-lactamase with: (A) the β-lactam cephalosporin ceftazidime; (B) the boronic acid ceftazidime analog, compound 2; and (C) the carbapenem imipenem.

Figure 2

Chemical structures of: (A) commercially available inhibitors and cephalosporin substrate cephalothin; (B) boronic acid derivatives; and (C) carbapenems used in this study. Cephalothin structure is labeled with accepted ring numbering system. The C₆ hydroxyethyl group of imipenem, which may be eliminated after formation of the acyl-enzyme, is circled in dashed red lines.

Figure 3

Overlay of the molecular coordinates for the E. coli AmpC covalently bound to cephalothin substrate (colored by atom, PDB entry 1KVM) and boronic acid chiral cephalothin analog, compound 5 (colored green, PDB entry 1MXO). The position of cephalothin’s dihydrothiazine ring and C₄ carboxylate is shown relative to the meta-carboxyphenyl group of compound 5, which is designed to mimic in stereochemistry and geometry the conserved β-lactam carboxylate.

Figure 4

Deconvoluted mass spectra of: (A) ADC β–lactamase alone; (B) ADC after 15 min incubation with compounds 2 and 5; and (C) ADC β-lactamase after 15 min incubation with imipenem, ertapenem, doripenem, and meropenem. The peak in each ADC: boronate spectrum corresponds to the unmodified ADC enzyme. The major peak in each of the ADC: carbapenem spectrum indicates covalent attachment of the β-lactam with a minor additional peak corresponding to the acyl-enzyme without the carbapenem’s C₆ hydroxyethyl substituent. All measurements have an error of ± 3 atomic mass units (amu).

Figure 4

Deconvoluted mass spectra of: (A) ADC β–lactamase alone; (B) ADC after 15 min incubation with compounds 2 and 5; and (C) ADC β-lactamase after 15 min incubation with imipenem, ertapenem, doripenem, and meropenem. The peak in each ADC: boronate spectrum corresponds to the unmodified ADC enzyme. The major peak in each of the ADC: carbapenem spectrum indicates covalent attachment of the β-lactam with a minor additional peak corresponding to the acyl-enzyme without the carbapenem’s C₆ hydroxyethyl substituent. All measurements have an error of ± 3 atomic mass units (amu).

Figure 5

Proposed mechanism of the retroaldolic reaction leading to elimination of C₆ hydroxethyl substituent from the β-lactamase: carbapenem acyl-enzyme. Glu272, supported by Lys315, may serve as the base to deprotonate the alcoholic function β–hydroxy carbonyl moiety of the C₆ substituent. Alternatively, Glu272 may abstract a proton from Lys315 which subsequently deprotonates the C₆ group (mechanism shown in red). The negative charge on the β-lactam carbonyl could be supported by Tyr150 and a Lys67.

Figure 6

Overlay of molecular coordinates for the E. coli AmpC: 5 complex in yellow (PDB entry 1MXO) and generated ADC: 5 model colored by atom. The position of α–helices and β–sheets is generally preserved between the two proteins, but deviations are observed in the strand turns between these secondary structures. Active site differences are illustrated by the altered conformation of 5 (colored green) in ADC as compared to 5 (colored yellow) bound to the E. coli AmpC.

Figure 7

Comparison of the binding site interactions between E. coli AmpC: 5 (left panel) and ADC: 5 (right panel). Figures have a perspective view to show positions of the residues in relation to the inhibitor. The boronic acid derivative is bound to Ser64 in both structures, but the relative rotation of the inhibitor in ADC changes the relationships with other active site residues. Specifically, the boronic acid oxygens interact with Ala318 and Tyr150 in E. coli AmpC, but the hydrogen bond with Ser318 is lost in ADC. Also in ADC, the meta-carboxylate of the dihydrothaizine ring analog has no clear interaction with previously identified carboxylate binding residues (e.g, Asn346, Arg349, or like Asn289 in E. coli AmpC). Instead, the group may form a long hydrogen bond with Asn287. The carbonyl oxygen of the R₁ cephalothin analog interacts with Asn152 in E. coli AmpC, but is reoriented towards Lys67 in ADC. Lastly, the R₁ thiophene ring sulfur in ADC: 5 is moved towards Tyr150 as compared to the E. coli AmpC: 5 structure. Overall, these significant active site differences suggest that while ADC may possess novel architecture, the ability to recognize inhibitors and substrates is preserved because of the versatile functions of the binding site residues.

Figure 8

Molecular representation of: (A) ADC: imipenem acyl-enzyme model; and (B) ADC: meropenem acyl-enzyme model. The intact carbapenem is shown in green and the carbapenem without the C₆ hydroxyethyl group in orange. Hydrogens are not shown except on the carbapenem C₆ hydroxyethyl which is likely deprotonated by Glu272, leading to elimination of the group. Removal of this C₆ substituent precipitates significant reorientation of the compound in the active site. Specifically, the β-lactam carbonyl moves back towards the oxyanion hole formed by the backbone nitrogens of Ser 64 and Ser318, approximately 90° rotation for imipenem and entirely into the hole for meropenem. Also after C₆ elimination, the R₂ group of imipenem is repositioned from outside of the binding pocket into a network of interactions with Tyr150, Asn152, Lys67 and Gln120. Less change is observed for the R₂ group of meropenem, which may have implications for the differing K_is of these carbapenems.

Figure 8

Molecular representation of: (A) ADC: imipenem acyl-enzyme model; and (B) ADC: meropenem acyl-enzyme model. The intact carbapenem is shown in green and the carbapenem without the C₆ hydroxyethyl group in orange. Hydrogens are not shown except on the carbapenem C₆ hydroxyethyl which is likely deprotonated by Glu272, leading to elimination of the group. Removal of this C₆ substituent precipitates significant reorientation of the compound in the active site. Specifically, the β-lactam carbonyl moves back towards the oxyanion hole formed by the backbone nitrogens of Ser 64 and Ser318, approximately 90° rotation for imipenem and entirely into the hole for meropenem. Also after C₆ elimination, the R₂ group of imipenem is repositioned from outside of the binding pocket into a network of interactions with Tyr150, Asn152, Lys67 and Gln120. Less change is observed for the R₂ group of meropenem, which may have implications for the differing K_is of these carbapenems.