Small Molecule Inhibitors of a Glycoside Hydrolase Attenuate Inducible AmpC-mediated β-Lactam Resistance

Abstract

The increasing spread of plasmid-borne ampC-ampR operons is of considerable medical importance, since the AmpC ␤-lactamases they encode confer high level resistance to many third generation cephalosporins. Induction of AmpC ␤-lactamase from endogenous or plasmid-borne ampC-ampR operons is mediated by a catabolic inducer molecule, 1,6-anhydro-Nacetylmuramic acid (MurNAc) tripeptide, an intermediate of the cell wall recycling pathway derived from the peptidoglycan. Here we describe a strategy for attenuating the antibiotic resistance associated with the ampC-ampR operon by blocking the formation of the inducer molecule using small molecule inhibitors of NagZ, the glycoside hydrolase catalyzing the formation of this inducer molecule. The structure of the NagZ-inhibitor complex provides insight into the molecular basis for inhibition and enables the development of inhibitors with 100-fold selectivity for NagZ over functionally related human enzymes. These PUGNAc-derived inhibitors reduce the minimal inhibitory concentration (MIC) values for several clinically relevant cephalosporins in both wild-type and AmpC-hyperproducing strains lacking functional AmpD.The expression of inducible chromosomal AmpC ␤-lactamases (1, 2) is one increasingly problematic resistance mechanism seen in many Gram-negative bacteria, including clinically opportunistic pathogens, such as Pseudomonas aeruginosa and Citrobacter freundii. These AmpC ␤-lactamases inactivate a broad range of ␤-lactam antibiotics, thereby conferring resistance to clinically important cephamycins, cephalosporins, and even monobactams, next generation ␤-lactams designed to be stable against ␤-lactamases (3). One alarming development resulting from continued ␤-lactam use is the spread of genetically diverse, plasmid-borne ampC, including those that are under inducible control. Indeed, the movement of ampC genes onto plasmids (4 -8) as well as other transposable elements has greatly increased the prevalence of this type of resistance mechanism in Gram-negative organisms (9). The regulation of ampC gene expression can vary between genera of Gram-negative bacteria. In some, chromosomal ampC is expressed constitutively, although high level expression has been suggested to decrease bacterial fitness and virulence, at least in Salmonella enterica (10). Often, however, chromosomal ampC ␤-lactamase is inducible as found in various clinically relevant opportunistic pathogens, such as Enterobacter spp., and P. aeruginosa (11,12).A major achievement in the field of antibiotic research has been the discovery of ␤-lactamase inhibitors. These inhibitors have been shown, in some cases, to reverse antibiotic resistance mediated by certain classes of ␤-lactamase. There are currently three main ␤-lactamase inhibitors available on the market, each having the same ␤-lactam core found in ␤-lactam antibiotics. One of these inhibitors, clavulanic acid, however has been found to be of limited use against AmpC (13, 14). The other two, sulbactam and tazobactam, demonstrate effectiveness against...

Insight into a strategy for attenuating AmpC‐mediated β‐lactam resistance: Structural basis for selective inhibition of the glycoside hydrolase NagZ

“…16,28 Moreover, we have demonstrated that AmpC mediated b-lactam resistance can be attenuated by inhibiting NagZ with these selective inhibitors. Attenuation has been achieved in a model system of E. coli harboring a plasmid encoding an ampC-ampR operon, 16 and more recently in the opportunistic pathogen P. aeruginosa. 29 The most selective inhibitor found to date is $100-fold selective for NagZ over the human b-hexosaminidase isoenzymes and O-GlcNAcase.…”

“…Although GH3 enzymes, including NagZ, are functionally related to the GH20 human b-hexosaminidase isoenzymes and GH84 O-GlcNAcase, recent kinetic [17][18][19] and structural studies 16,[20][21][22] have revealed that GH3 enzymes use a catalytic mechanism that differs from that used by GH20 and GH84 enzymes. This distinction should therefore offer a tractable route to generating selective inhibitors of NagZ, and clear comparisons of these enzymes at a structural level would greatly accelerate these efforts.…”

“…One approach, which we have taken, is to generate inhibitors of NagZ that act to hinder the production of AmpC and therefore render bacteria that harbour AmpC more sensitive to b-lactams. 16 One challenge facing this approach however is that inhibitors used for this purpose need to be selective for NagZ and not potently inhibit the functionally related human enzymes.…”

“…1(C)] has been shown to be a non-selective yet potent inhibitor of NagZ, human b-hexosaminidases, and O-GlcNAcase (Table I). 16,18,28 To generate inhibitors specific for NagZ, we exploited the structural differences between NagZ, GH20 human b-hexosaminidases, and GH84 OGlcNAcase and generated 2-N-acyl derivatives of PUGNAc. 16,28 Moreover, we have demonstrated that AmpC mediated b-lactam resistance can be attenuated by inhibiting NagZ with these selective inhibitors.…”

“…16,18,28 To generate inhibitors specific for NagZ, we exploited the structural differences between NagZ, GH20 human b-hexosaminidases, and GH84 OGlcNAcase and generated 2-N-acyl derivatives of PUGNAc. 16,28 Moreover, we have demonstrated that AmpC mediated b-lactam resistance can be attenuated by inhibiting NagZ with these selective inhibitors. Attenuation has been achieved in a model system of E. coli harboring a plasmid encoding an ampC-ampR operon, 16 and more recently in the opportunistic pathogen P. aeruginosa.…”