A mechanism-based GlcNAc-inspired cyclophellitol inactivator of the peptidoglycan recycling enzyme NagZ reverses resistance to β-lactams in Pseudomonas aeruginosa

Article abstract of DOI:10.1039/c8cc05281f

The development of a potent mechanism-based inactivator of NagZ, an enzyme critical to the production of inducible AmpC β-lactamase in Gram-negative bacteria, is presented. This inactivator significantly reduces MIC values for important β-lactams against

Full text of DOI:10.1039/c8cc05281f

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Winogrodzki, A. Debowski, Z. Madden, D. Vocadlo, B. L. Mark and K. Stubbs, Chem. Commun., 2018, DOI:
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A mechanism‐based GlcNAc‐inspired cyclophellitol inactivator of
the peptidoglycan recycling enzyme NagZ reverses resistance to ‐
lactams in Pseudomonas aeruginosa
Received 00th January 20xx,
Accepted 00th January 20xx
Louisa A. Ho,a Judith L. Winogrodzki,b Aleksandra W. Debowski,a,c Zarina Madden,d David J.
DOI: 10.1039/x0xx00000x
Vocadlo,d Brian L. Mark*b and Keith A. Stubbs*a
www.rsc.org/
The development of a potent mechanism‐based inactivator of
NagZ, an enzyme critical to the production of inducible AmpC ‐
lactamase in Gram‐negative bacteria, is presented. This inactivator
significantly reduces MIC values for important ‐lactams against a
clinically relevant strain of Pseudomonas aeruginosa.
Medical organisations have highlighted the urgent need for
new antibiotic strategies and have proposed a list of priority
antimicrobial resistant pathogens, including several Gram‐
negative bacteria.1,2 One particularly worrisome area is the
increasing prevalence of resistance to
‐lactam antibiotics,
which are heavily relied upon to treat Gram‐negative bacterial
infections.3 β‐Lactamases, which are enzymes that deactivate
β‐lactams by cleaving the cyclic amide moiety, are the most
problematic.4 A class of these enzymes that pose a significant
clinical problem are the inducible chromosomal AmpC β‐
lactamases,5 which have a broad spectrum activity against β‐
lactams.6 AmpC enzymes are generally resistant to classical β‐
lactam‐based β‐lactamase inhibitors such as clavulanic acid;5
however, non‐β‐lactam based inhibitors effective against
AmpC are now reaching the market.7
In contrast to the classical approach of directly blocking ‐
lactamase activity, one can also aim to block ampC gene
expression. Expression of inducible AmpC depends on
peptidoglycan (PG) metabolism and recycling.8 During normal
growth a large amount of PG is degraded and recycled
(Scheme 1A).8,9 PG degradation fragments, GlcNAc‐1,6‐
anhydroMurNAc‐peptides, are transported into the cytoplasm
by AmpG, and further degraded to produce a series of 1,6‐
anhydroMurNAc‐tri‐, tetra‐ and pentapeptides (Scheme 1A).10
One or more of these PG metabolites induces ampC by
Scheme 1. (A) The peptidoglycan recycling pathway shows the important
biological activity of NagZ in the production of 1,6‐anhydroMurNAc peptides
(pentapeptide shown) to induce AmpC expression. Inset: The catalytic
mechanism of NagZ (B) The inhibition by Cyclophellitol of retaining
‐
glucosidases that use two catalytic carboxylic acid residues. A GlcNAc‐based
cyclophellitol would allow for the formation of a covalent adduct with the
enzymic nucleophile of NagZ, leading to its irreversible inhibition. Structures of
the known epoxide‐based inactivators Cyclophellitol and Conduritol B epoxide
and the compounds of interest in this study.
binding to its transcriptional regulator, AmpR.11,12 Another
a. School of Molecular Sciences, University of Western Australia, Crawley, WA 6009, molecule, that acts as a repressor of AmpR is UDP‐MurNAc‐
Australia. E‐mail: keith.stubbs@uwa.edu.au
pentapeptide; a PG building block that is formed by further
metabolism of the 1,6‐anhydroMurNAc peptides.11,13,14
Overall, the regulation of AmpC expression, and the ability for
bacteria to sense β‐lactams is controlled by the relative
See concentrations of these PG ligands that modulate AmpR
activity.15,16
b.Department of Microbiology, University of Manitoba, Winnipeg, Manitoba,
R3T2N2, Canada. Email: Brian.mark@umanitoba.ca
c.School of Biomedical Sciences, University of Western Australia, Crawley, WA 6009,
Australia.
d.Department of Chemistry, Simon Fraser University, Burnaby, BC V5A1S6, Canada
Electronic
Supplementary
Information
(ESI)
available:
DOI: 10.1039/x0xx00000x
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key enzyme in PG recycling is the
Journal Name
A
‐N‐
View Article Online
DOI: 10.1039/C8CC05281F
acetylglucosaminidase, NagZ, which cleaves the non‐reducing
GlcNAc residue from GlcNAc‐1,6‐anhydroMurNAc‐peptides to
produce the 1,6‐anhydroMurNAc‐peptides that activate AmpR
(Scheme 1A).17,18 Genetic studies have demonstrated that this
enzyme is important for induction of AmpC β‐lactamase.19‐21
Strategies to inhibit this enzyme using small molecules are
therefore of interest since they could block formation of the
AmpR inducer molecules. This approach to potentiate co‐
administered
‐lactams has received some in vitro
validation,19,22‐26 and structural insight into inhibition of NagZ
has enabled the design of improved NagZ inhibitors.27,28 Yet,
none of the inhibitors have been able to recapitulate the
effects seen when deleting the NagZ gene.19‐21
NagZ is a member of glycoside hydrolase family 3 (GH3) Scheme 2. a) MCPBA, CH2Cl2; b) PPh3, DPPA, DEAD, toluene; c) i. PMe3, THF:H2O
(20:1); ii. (RC(O))2O, C5H5N, CH2Cl2; d) MeOH; e) Pd(OH)2/C, H2, MeOH.
and uses a two‐step, double‐displacement mechanism. Two
residues play key catalytic roles. A histidine residue acts as a
strategy of Shing39 allowed for the hydroxy‐directed MCPBA
epoxidation of which gave us exclusively the epoxide in
good yield. This material was then exposed to Mitsunobu
conditions which afforded us the azide . We then treated the
azide to Staudinger conditions and trapped the intermediate
amine with either acetic anhydride to give the acetamide or
trifluoroacetic anhydride to give . With these compounds in
hand all that was required was removal of the protecting
general catalytic acid/base29,30 and an aspartate acts as a
catalytic nucleophile to enable the formation and breakdown
of a covalent glycosyl‐enzyme intermediate (Scheme 1A).10,30‐
33† Generation of NagZ inhibitors has centred on mimicking
the oxocarbenium ion‐like transition states that are thought to
flank the covalent glycosyl‐enzyme intermediate to yield
competitive inhibitors of the enzyme (ESI, Fig. S1).
3
4
5
5
6
7
One approach to NagZ inhibitors that has not been
pursued, is to exploit the nucleophilicity of the catalytic
nucleophile that acts to form the glycosyl‐enzyme
intermediate. Such mechanism‐based inhibitors form covalent
adducts with the catalytic nucleophile of the enzyme. One
notable set of these mechanism‐based inhibitors are the
groups to give the desired epoxides
Unfortunately, dissolution of the acetamide
1
and
2
6
respectively.
in methanol
resulted in its spontaneous conversion to the oxazoline
8 in
almost quantitative yield. We found this also occurred in other
protic solvents (ethanol, isopropanol) as well as aprotic
solvents including tetrahydrofuran and acetonitrile. These
results were disappointing, but we thought that the
epoxide‐containing cyclitols, of which Cyclophellitol34,
a
natural product, and Conduritol B epoxide35 are the best
known (Scheme 1B).36,37 These compounds and their
derivatives have received attention for their ability to
inactivate a wide range of glycoside hydrolases and have
demonstrated utility.38 We accordingly felt that the GlcNAc‐
trifluoroacetamide
conversion due to the reduced nucleophilicity of the carbonyl
group of the amide. Gratifyingly, the trifluoroacetamide was
stable in methanol and thus, after palladium‐catalysed
7 would be far more resistant to this
7
hydrogenolysis, we obtained the desired polyol
We then tested the ability of compound
2.
2
based cyclophellitols
1 and 2, would be excellent candidates
to inactivate
for mechanism‐based inactivation of NagZ. We noted studies
of competitive NagZ inhibitors have shown that replacement
of the acetamido group with a trifluoroacetamido moiety
increases potency of inhibitors toward NagZ.26
Here, we present a significantly improved strategy for
inhibiting NagZ‐type enzymes as compared to previous
reversible NagZ inhibitors that have had only limited success at
reversing antibiotic resistance mediated by inducible AmpC.
NagZ enzymes using BcNagZ, a model NagZ from Burkholderia
cenocepacia that has been used in other NagZ inhibitor
studies.26,28 Incubation of BcNagZ with
2 results in rapid time‐
dependent inactivation of the enzyme (Fig. 1A). We
determined the kinetic parameters governing inactivation (ki
and Ki) by plotting kobs versus [I] (Fig. 1B). In all cases the
inactivation data fitted well to a single exponential decay
equation. Compound
ki value of 0.17 ± 0.01 min‐1. We also confirmed that the
inactivation of BcNagZ by was irreversible (ESI, Fig S2).
2 shows a Ki value of 8.0 ± 1.0 M and a
These epoxides in combination with
susceptibility of a clinically relevant Gram‐negative bacterium
possessing inducible AmpC to ‐ lactams, far beyond the
‐lactams, increases the
2

One important caveat in the development of NagZ
inhibitors for eventual clinical use is the potential concomitant
inhibition of functionally related human enzymes. These
effects seen previously with competitive inhibitors and
provides the first clear validation that NagZ can be targeted
with small molecules to yield effects matching those seen
when deleting the NagZ gene.
enzymes include the GH84


‐glucosaminidase human O‐
GlcNAcase,40 the GH20 human
‐hexosaminidases41 and GH89
Starting from
D
‐(‐)‐quinic acid using established literature
in good overall

‐N‐acetylglucosaminidase NAGLU.42 Early competitive
procedures39 we obtained the allylic alcohol
3
inhibitors suffered from a lack of selectivity and thus
derivatives were required to obtain selectivity for NagZ (ESI,
Fig. S1). An important feature of these enzymes is that they
yield (Scheme 2). At this point, we decided to first install the
epoxide and later perform the required epimerization to give
the correct stereochemistry at C2. Using the epoxidation
2 | J. Name., 2012, 00, 1‐3
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use different catalytic mechanisms as compared to NagZ‐like
enzymes. The GH2032 and GH8443 enzymes use substrate‐
assisted catalysis involving the 2‐acetamido group as a
View Article Online
DOI: 10.1039/C8CC05281F
nucleophile and the GH89 enzymes act on
‐GlcNAc linked
moieties44. None of these enzymes form a NagZ‐like covalent
glycosyl‐enzyme intermediate and therefore they should not
be inactivated using the approach described here. Moreover,
inspection of the active sites of these enzymes suggest that
they are less likely to accommodate the N‐trifluoroacetamido
Figure 1. Time‐dependent inactivation of BcNagZ by
represent BcNagZ activity of control samples (without
BcNagZ: 0 ( ), 1 ( ), 5 ( ), 10 (◊), 25 ( ), 40 ( M. B) Curves are the nonlinear
fits of data to a single exponential decay equation; B) Plot of the inactivation rate
constants (kobs) as a function of the concentration of for BcNagZ.
2
: A) Experimental data
group of compound 2 27
. To confirm that epoxide 2 did not act
2
) over time. [ ] used with
2
●
□
■
▲
♦) 
as a time‐dependent inhibitor of these enzymes we incubated
them with a large inhibitor concentration for extensive time
periods. No significant time dependent inactivation was
observed for all enzymes tested (ESI, Fig. S3) suggesting that
the compound likely acts as a poor reversible inhibitor of these
2
as high as 1 mM. Notably, MICs obtained in the presence of
are similar to MICs obtained for the nagZ knockout of this
strain,20,46 which is a marked improvement over competitive
NagZ inhibitors that have been evaluated as ‐lactam
potentiators. We speculate that the improved activity of
epoxide represents stems from it being an irreversible
2

enzymes. Moreover, analysis of the data shows that epoxide
2

inactivates NagZ on a useful timescale Given the high
conservation of GH3 NagZ among Gram‐negative bacteria,28
2
this approach could potentiate
‐lactams against numerous
inactivator of NagZ, which makes it reasonable that the effects
seen here reflect genetic inactivation of NagZ. Moreover, it
pathogens carrying an NagZ‐mediated inducible ampC gene.
To gain insight into the molecular basis for inhibition of
may also be that the structural similarity of
can be scavenged by many bacteria, may enable its active
transport into the bacterial cytosol. Accordingly, we believe
2 to GlcNAc, which
NagZ by
complex with
(ESI, Table S1, PDB 6DTE). Electron density for
within the enzyme active site, where a covalent bond between
C1 of and the enzymic nucleophile of the BcNagZ (Asp253) is
clearly observed, confirming the basis for the mechanism‐
based inactivation of NagZ enzymes by (Fig. 2A). The adduct
causes the hexose ring of to sit lower in the active site, closer
to Asp 253 as compared to the product of the normal reaction,
GlcNAc (PDB 4GNV, Fig. 2B). Aside from this difference, and
2
, we determined the X‐ray structure of BcNagZ in
by molecular replacement to 1.9 Å resolution
was observed
2
2
that
2 and potential analogues, or other covalent inhibitors,
represent an exciting new strategy to advance this adjunct
antimicrobial approach.
2
2
2
2
GlcNAc bind similarly. Further, the catalytic loop containing the
general acid/base His183, which is typically disordered in the
absence of substrate or product28,30 adopts a catalytically
competent conformation in the presence of
NagZ binds very similarly, in overall geometry, to GlcNAc.
We next set out to test if epoxide could increase the
susceptibility of bacteria harbouring inducible AmpC
lactamase to ‐lactams. Pseudomonas aeruginosa is
nosocomial Gram‐negative pathogen that harbours an
inducible ampC.45 Previous work has shown that genetic
inactivation of nagZ in clinically problematic AmpC‐
derepressed mutants of P. aeruginosa markedly increases
2. In summary,
2
2
Figure 2. Crystal structure of BcNagZ bound to
weighted difference map (mFo‐DFc) contoured at 4
covalently bound to Asp253 in the BcNagZ active site. B) BcNagZ active site
bound to (green carbon atoms) with the BcNagZ product complex PDB 4GNV
2
. A) Electron density (sigma‐A

‐

) for (green carbon atoms)
2

a
2
superposed. The GlcNAc product is shown with yellow carbon atoms.
In summary, there is a pressing need for new strategies to
overcome inducible chromosomal AmpC β‐lactamase
susceptibility to
‐lactams supporting the validity of this
resistance to

‐lactams. Here we describe a new class of NagZ
‐lactams
target.19,46
inhibitor that provides chemical potentiation of

To determine if
important ‐lactams, ceftazidime, aztreonam and imipenem,
were assayed against the most clinically relevant AmpC
derepressed mutant of P. aeruginosa (
dacB)46 (Table 1).
Minimal inhibitory concentration (MIC) assays demonstrated
that treating cultures with (100 M) restored the efficacy of
2 potentiates ‐lactams, clinically
against clinically problematic P. aeruginosa that is equivalent
to genetic inactivation of the enzyme. This strategy accordingly

opens a new chemical approach to suppressing
resistance in clinical pathogens that carry NagZ‐mediated
inducible AmpC ‐lactamase.
‐lactam


2

The authors wish to thank the CMCA at The University of
Western Australia, which is supported by University, State and
Federal Government funding. KAS thanks the Australian
Research Council for funding. DJV holds a Tier I Canada
Research Chair in Chemical Glycobiology. BLM and DJV thank
Cystic Fibrosis Canada and the Canadian Institutes of Health
Research for financial support (PJT‐148496).
ceftazidime and aztreonam, reducing MICs to below their
resistance breakpoints.47 Susceptibility to imipenem also
increased slightly; however, imipenem is already effective
against this strain since it is a poor AmpC substrate. As
expected,
2 does not exhibit any inherent antibacterial or
bacterostatic properties on its own, even at concentrations
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There are no conflicts to declare.
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