The development of selective inhibitors of NagZ: increased susceptibility of Gram-negative bacteria to β-lactams

Abstract

The increasing incidence of inducible chromosomal AmpC β-lactamases within the clinic is a growing concern because these enzymes deactivate a broad range of even the most recently developed β-lactam antibiotics. As a result, new strategies are needed to block the action of this antibiotic resistance enzyme. Presented here is a strategy to combat the action of inducible AmpC by inhibiting the β-glucosaminidase NagZ, which is an enzyme involved in regulating the induction of AmpC expression. A divergent route facilitating the rapid synthesis of a series of N-acyl analogues of 2-acetamido-2-deoxynojirimycin is reported here. Among these compounds are potent NagZ inhibitors that are selective against functionally related human enzymes. These compounds reduce minimum inhibitory concentration values for β-lactams against a clinically relevant Gram-negative bacterium bearing inducible chromosomal AmpC β-lactamase, Pseudomonas aeruginosa. The structure of a NagZ-inhibitor complex provides insight into the molecular basis for inhibition by these compounds.

Keywords: carbohydrates; enzymes; inhibitors; selectivity; synthesis.

Scheme 1

A) NagZ catalyzed hydrolysis of peptidoglycan cell-wall fragments releases the series of 1,6-anhydroMurNAc peptide inducer molecules that activate transcription of ampC expression (tripeptide shown). B) The putative transition state of the NagZ catalyzed hydrolysis of N-acetylglucosaminides (denoted by ≠). C) Structures of known inhibitors of NagZ enzymes.

Scheme 2

Reagents: a) ImSO₂N₃⋅HCl, K₂CO₃, CuSO₄, MeOH; b) i: TsCl, pyridine, CH₂Cl₂; ii: NaI, DMF; iii: Ac₂O, C₅H₅N; c) DBU, THF; d) i: PBu₃, THF, H₂O; ii: (RCO)₂O; e) i: 3-chloroperbenzoic acid, CH₂Cl₂, BnOH; ii: NaOMe, MeOH; f) NH₄OAc, Pd(OH)₂/C, H₂, MeOH/H₂O (15:1).

Figure 1

Crystal structure of StNagZ bound to 21. Active-site residues and 21 are drawn as sticks with oxygen and nitrogen atoms shown in red and blue, respectively. Carbon atoms of the enzyme are shown in grey, while the carbon atoms of the inhibitor are yellow. The hydrophobic surface formed by side chains of Ala129 and Ile130, against which the butyl chain sits, is shown in grey. The catalytic nucleophile, Asp248, is shown in stick format. Occupancy values for the dual conformations of Asp248 are 0.48/0.52, and 0.53/0.47 for Asp249 (flipped in or out towards the inhibitor, respectively). Hydrogen bonds are shown as yellow dashed lines. Electron density around 21 is a maximum-likelihood-weighted omit map (F_obs−F_calcd) contoured to 2.5σ.