Synthesis and evaluation of inhibitors of E. coli PgaB, a polysaccharide de-N-acetylase involved in biofilm formation

Article abstract of DOI:10.1039/c2ob26105g

Many medically important biofilm forming bacteria produce similar polysaccharide intercellular adhesins (PIA) consisting of partially de-N-acetylated β-(1 → 6)-N-acetylglucosamine polymers (dPNAG). In Escherichia coli, de-N-acetylation of the β-(1 → 6)-N-

Full text of DOI:10.1039/c2ob26105g

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PAPER
Synthesis and evaluation of inhibitors of E. coli PgaB, a polysaccharide
de-N-acetylase involved in biofilm formation†
Anthony Chibba,a Joanna Poloczek,a Dustin J. Little,b P. Lynne Howellb and Mark Nitz*a
Received 7th June 2012, Accepted 23rd July 2012
DOI: 10.1039/c2ob26105g
Many medically important biofilm forming bacteria produce similar polysaccharide intercellular adhesins
(PIA) consisting of partially de-N-acetylated β-(1 → 6)-N-acetylglucosamine polymers (dPNAG). In
Escherichia coli, de-N-acetylation of the β-(1 → 6)-N-acetylglucosamine polymer (PNAG) is catalysed by
the carbohydrate esterase family 4 deacetylase PgaB. The de-N-acetylation of PNAG is essential for
productive PNAG-dependent biofilm formation. Here, we describe the development of a fluorogenic
assay to monitor PgaB activity in vitro and the synthesis of a series of PgaB inhibitors. The synthesized
inhibitors consist of a metal chelating functional group on a glucosamine scaffold to target the active site
metal ion of PgaB. Optimal inhibition was observed with N-thioglycolyl amide (Ki = 480 μM) and
N-methyl-N-glycolyl amide (Ki = 320 μM) glucosamine derivatives. A chemoenzymatic synthesis of an
N-thioglycolyl amide PNAG pentasaccharide led to an inhibitor with an improved Ki of 280 μM.
Introduction
Surface associated, matrix embedded colonies of bacteria,
known as biofilms, are responsible for approximately 65% of all
chronic human bacterial infections.1 Bacteria residing in biofilms
have a high tolerance to antibiotics and are sheltered from the
host’s immune system making biofilm associated infections chal-
lenging to eradicate.2,3 During biofilm formation bacteria excrete
an extracellular matrix to facilitate the adherence between bac-
teria and the colonized surface. Exopolysaccharides form an
essential component of these biofilm matrices.4,5 Many medi-
Fig. 1 PgaB catalyzed de-N-acetylation. Polymeric PNAG is tens to
cally important biofilm forming bacterial strains, including
Staphylococcus epidermidis6 and aureus,7 Escherichia coli,8
Acinetobacter baumannii,9 Bordetella species,10,11 Actinobacillus
pleuropneumoniae,12 Burkoholderia species13 and Yersinia pestis,14
generate similar partially de-N-acetylated β-(1 → 6)-D-N-acetyl-
glucosamine homopolymers (dPNAG), also known as the poly-
saccharide intercellular adhesin (PIA), as a key biofilm matrix
exopolysaccharide.
In gram negative and gram positive bacteria, partial de-N-
acetylation of the β-(1 → 6)-D-N-acetylglucosamine homopoly-
mer (PNAG) is essential for the function of the polysaccharide
in biofilm formation (Fig. 1). Deletion of the PNAG de-N-
hundreds of monosaccharides in length and the degree of de-N-acetyl-
ation varies between bacterial species (10–40%).8
acetylase, IcaB, in S. epidermidis results in secretion of the
PNAG exopolysaccharide into the growth media rather than
localization of the exopolysaccharide at the bacterial surface.15
The ΔicaB S. epidermidis strain was less virulent and displayed
an attenuated biofilm formation phenotype.16 In E. coli disrup-
tion of the PNAG de-N-acetylase, PgaB, results in a PNAG
polysaccharide that fails to be exported from the periplasm and
abolished bacterial biofilm formation.8,17 These studies suggest
that PNAG de-N-acetylases are good targets for therapeutic inter-
vention in biofilm related infections.
From sequence homology PNAG de-N-acetylases have been
classified as members of the family 4 carbohydrate esterases
(CE4) in the CAZY database18 (http://www.cazy.org). CE4
family enzymes are metal dependent hydrolases having a His-
His-Asp metal coordinating triad as well as conserved catalytic
residues. Recently, we have solved the first structure of a PNAG
aDepartment of Chemistry, University of Toronto, Toronto, Ontario M5S
3H6, Canada. E-mail: mnitz@chem.utoronto.ca
bProgram in Molecular Structure and Function, The Hospital of Sick
Children and Department of Biochemistry, University of Toronto,
Ontario M5G 1X8, Canada
†Electronic supplementary information (ESI) available: Full experimen-
tal details, NMR spectra and Lineweaver–Burk plots. See DOI:
10.1039/c2ob26105g
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de-N-acetylase, PgaB from E. coli.19,20 The N-terminal de-N-
acetylase domain of PgaB is structurally homologous to other
well characterized CE4 family members such as the Colletotri-
chum lindemuthianum chitin de-N-acetylase, ClCDA, and the
Streptococcal peptidoglycan de-N-acetylase, SpPgdA.21,22
Despite the structural similarities, PgaB is specific for PNAG,
and shows no activity on chitin (β-(1 → 4)-N-acetylglucosamine)
substrates. PgaB also has unique metal dependence, showing
optimal activity with Fe2+, Ni2+, and Co2+ in contrast to the Zn2+
dependency observed for other de-N-acetylases in the CE4 family.
To probe the active site of PgaB and to develop an inhibitor of
PNAG de-N-acetylation, we have developed a chromogenic
assay for PgaB activity, synthesized a series of potential inhibi-
tors and assayed these against PgaB. The inhibitors are based on
Inhibitor design
Many inhibitors have been developed for metal dependent
N-acetylamidases, such as the histone de-N-acetylases, and other
carbohydrate processing de-N-acetylases, such as lipopolysac-
charide de-N-acetylase LpxC.25,26 A number of potential inhibi-
tors (IC50 = 100–500 μM) have also been identified for the CE4
Streptococcal peptidoglycan de-N-acetylase through a large
in silico library screen.27 Recently, Urbaniak et al. have explored
the inhibition of N-acetylglucosamine-phosphatidylinositol
de-N-acetylase from Trypanosoma brucei using a series of
GlcNAc derivatives.28 The common feature of the N-acetyl-
amidase inhibitors identified is a metal coordinating functional
group, which ligates the de-N-acetylase active site metal ion.
Based on the success of the glucose based hydroxamic acid
inhibitor (TU-514) against lipopolysaccharide de-N-acetylases,
we installed a range of metal coordinating groups on a glucos-
amine scaffold with the aim of identifying a PgaB inhibitor
(Fig. 3).29 In addition, methylation of the amide nitrogen was
evaluated, as similar modifications of metal chelating groups led
to higher affinity inhibitors for Zn metalloproteases, which are
proposed to act by a similar mechanism to PgaB.30 Although
analysis has shown that monosaccharides are not substrates of
PgaB,19 it was envisaged that once an optimal metal chelating
functional group was identified it could be installed on a larger
PNAG oligosaccharide via a chemoenzymatic synthesis to yield
an improved inhibitor.
a
methyl 2-deoxy-2-amino-β-D-glucopyranoside scaffold in
which the amino group displays a range of potential metal che-
lating functional groups. One of the optimal metal chelating
functional groups, a thioglycolyl amide, was chemoenzymati-
cally installed on a PNAG pentasaccharide scaffold yielding the
highest affinity inhibitor (Ki = 280 μM).
Results and discussion
Assay development
The catalytic efficiency for de-N-acetylation by PgaB on syn-
thetic PNAG oligomers is low (kcat/Km for pentasaccharide =
0.25 M−1 s−1), and saturation of enzymatic activity cannot be
achieved due to the limited solubility of the substrate.19 Further-
more, the previous assay used to monitor the de-N-acetylation
reaction of the natural substrate required a cumbersome discon-
tinuous fluorescamine assay. A simpler continuous assay was
developed to allow for more rapid analysis of PgaB inhibition and
to allow full kinetic characterization of the enzymatic activity.
A series of chromogenic acetyl esters were evaluated as PgaB
substrates (Fig. 2). The compounds were dissolved in DMSO
and added (0.10–10 mM) to PgaB (10 μM) in HEPES buffer
(100 mM, pH 7.5). Acetate hydrolysis was monitored by absor-
bance or fluorescence spectroscopy. p-Nitrophenyl acetate
(pNPA), 5-acetoxymethylquinolinium (5-AMQ)23 and 7-acetoxy-
methylquinolinium (7-AMQ)23 were substrates for PgaB but
these substrates suffered from high rates of background hydro-
lysis. 4-Methylumbelliferyl acetate (AMC) and 3-carboxyumbel-
liferyl acetate (ACC)24 showed good stability to hydrolysis and
also served as substrates for PgaB. The limiting solubility of
AMC (∼1 mM) prevented saturation of the enzymatic activity;
however, ACC was soluble up to 10 mM concentrations allowing
Inhibitor synthesis
Methyl β-D-2-deoxy-2-aminoglucopyranoside (2) served as the
starting scaffold for synthesis of the monosaccharide based
inhibitors. Using the protecting-group-free chemistry based on
glycosyl tosylhydrazides, the methyl glycoside 1 could be syn-
thesized in two steps from N-acetylglucosamine. De-N-acetyl-
ation of 1 with hydrazine hydrate gave 2 in excellent yield.31
Alternatively, via direct methanolysis of 3,4,6-triacetyl-2-deoxy-
2-aminoglucosyl bromide, the methyl glycoside 2 could be gen-
erated and subsequently deacetylated.32 The free amine of 2 was
then condensed with benzyloxyacetic acid and Cbz-Gly-OH
using HBTU, or via direct acylation with pentafluorophenyl
S-acetylthioglycolate (SAMA-OPFP) to provide compounds 4, 6
and 8 after deprotection. The hydroxylurea 9 could be accessed
by acylation of 2 with 1-(4-nitrophenol)-N-hydroxycarbamate.33
Compound 2 was also sulfonylated with mesylchloride to
provide compound 10 (Scheme 1).
Benzyl protected methyl 2-aminomethyl-2-deoxy-β-D-gluco-
side (14) was synthesized in four steps from 1. Initial
for the determination of the kinetic parameters of PgaB (kcat
=
0.013 0.002 s−1, Km = 1.2 0.2 mM).
Fig. 3 Structure of TU-514 a potent lipopolysaccharide de-N-acetylase
(LpxC) inhibitor (left) and the proposed inhibitor structures (right)
where X, Y, Z represent atoms forming a bidentate metal chelate.
Fig. 2 Acetyl esters evaluated as substrates for PgaB.
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per-O-benzylation followed by N-carbamylation with Boc2O and
catalytic DMAP gave the protected mixed imide 12 as pre-
viously reported.34 De-N-acylation of 12 with catalytic NaOMe
followed by reduction with LiAlH4 gave the protected methyl-
amine 14 in good yield. The secondary amine was then functio-
nalized as has been described above (Scheme 1). After final
deprotection potential inhibitors 20–24 were isolated in good
yield (Scheme 2). Rotamers were observed in the cases of the
per-O-benzylated species in both 13C NMR and 1H NMR
spectra at room temperature, as expected by literature pre-
cedent.35 In most cases only a single rotamer was observed for
the final N-Me derivatives by 1H NMR in D2O at room tempera-
ture; however, some signal broadening was observed, and dis-
tinct rotamers could be seen with compound 21.
The synthesis of the sulfamate derivative 27 and the N-
methylthioglycolyl amide derivative 30 required slightly
modified schemes. Due to the reactivity of the sulfamylating
agent (benzyl N-(chlorosulfonyl)carbamate) (BNCC)36 hydroxyl
protecting groups were required. Thus, 14 was treated with TFA
to liberate the amine which reacted smoothly to form the Cbz-
protected sulfamate 26. Global deprotection of this compound
by hydrogenation yielded 27. The incompatibility of the thiogly-
colyl amide with hydrogenation conditions required deprotection
of 14 to give 28 prior to acylation. After final acetolysis com-
pound 30 was isolated as the symmetrical disulfide (Scheme 3).
Inhibitor evaluation
The potential de-N-acetylase inhibitors (4, 6, 8, 9, 10, 20–24, 27,
30) were tested for their inhibitory potency against Ni2+ loaded
PgaB in a competitive assay with the coumarin substrate ACC.
We have previously shown PgaB to also be active in its Fe2+
form but this enzyme is unstable under ambient conditions and
was not evaluated.19 None of the inhibitors were substrates of
1
Scheme 1 Synthesis of inhibitors 4, 6, 8–10. (a) Tosyl hydrazide
(1.1 equiv.), DMF : H2O : AcOH (5 : 1 : 0.1), rt, 24 h, 92%. (b) NBS
(2.4 equiv.), MeOH (20 equiv.), DMF, rt, 1 h, 78%. (c) NH2NH2·H2O,
110 °C, 24 h, 96%. (d) HBTU (1 equiv.), benzyloxyacetic acid
(1 equiv.), TEA (1.2 equiv.), DMF, rt, 12 h, leading to 3, 84%. (e)
HBTU (1 equiv.), Cbz-Gly-OH (1 equiv.), TEA (1.2 equiv.), DMF, rt,
12 h, leading to 5, 76%. (f) SAMA-OPFP (1 equiv.), TEA (1.2 equiv.),
DMF, rt, 12 h, leading to 7, 78%. (g) 1-(4-Nitrophenol)-N-hydroxycar-
bamate (1 equiv.), TEA (1.2 equiv.), DMF, rt, 12 h, leading to 9, 54%.
(h) MsCl (1 equiv.), TEA (2 equiv.), DMF, rt, 12 h, leading to 10, 58%.
(i) 10% Pd/C, 1 atm H2, MeOH, rt, 12 h, 81–89%. ( j) NaOMe
(0.1 equiv.), MeOH, rt, 10 min, 92%.
PgaB as determined by H NMR after overnight incubation of
the compounds at 10 mM under the assay conditions. Inhibitors
containing a disulfide (8, 30) were preincubated with DTT prior
to assay onset to reduce the disulfide. Compounds showing
greater than 10% inhibition of coumarin deacetylation in the
presence of 10 mM inhibitor were subjected to full kinetic analy-
sis. In all fully characterized inhibitors, competitive inhibition
was observed with the coumarin substrate. Methyl 2-deoxy-2-
amino-glucopyranoside (2) showed no inhibition of PgaB at
1 mM, whereas at higher concentrations the free amine caused a
significant increase in background signal during the assay. The
results of the inhibitor assay are summarized in Table 1.
Of the primary amide derivatives (4, 6, 8, 9, 10, and 27) only
the reduced thioglycolyl amide, 8, showed significant inhibitory
activity against PgaB (Ki = 480 μM). Thioglycolyl amides are
known to be good inhibitors for many metalloamidases.37 Sur-
prisingly compounds 9 and 27 showed little activity, as inhibitors
Scheme 2 Synthesis of inhibitors 20–24. (a) BnBr (3.5 equiv.), NaH
(3.5 equiv.), DMF, 0 °C, 16 h, 76%. (b) Boc2O (2 equiv.), DMAP
(0.25 equiv.), THF, 60 °C, 12 h. (c) NaOMe (0.2 equiv.), MeOH, rt, 1 h,
72% over 2 steps. (d) LiAlH4 (3 equiv.), THF, 60 °C, 6 h, 68%. (e) DCC
(1 equiv.), benzyloxyacetic acid (1 equiv.), TEA (0.2 equiv.), EtOAc :
DCM (1 : 1), rt, 12 h, leading to 15, 89%. (f) DCC (1 equiv.), Cbz-Gly-
OH (1 equiv.), TEA (0.2 equiv.), EtOAc : DCM (1 : 1), rt, 12 h, leading to
16, 92%. (g) 1-(4-Nitrophenol)-N-hydroxycarbamate (1 equiv.), TEA
(0.2 equiv.), DMF, rt, 12 h, leading to 17, 72%. (h) MsCl (1 equiv.), TEA
(1.2 equiv.), EtOAc, rt, 12 h, leading to 18, 88%. (i) TEA (1.2 equiv.),
DCM, rt, 3 h, 88%. (j) 10% Pd/C, 1 atm H2, MeOH, 12 h, 75–82%.
Scheme 3 Synthesis of inhibitors 27 and 30. (a) 1 : 1 TFA : DCM, rt,
30 min, 81%. (b) BNCC (1.5 equiv.), TEA (1.2 equiv.), DCM, rt, 3 h,
88%. (c) 10% Pd/C, 1 atm H2, MeOH, 12 h, 75%. (d) 10% Pd/C, 1 atm
H2, MeOH, 12 h, 78%. (e) SAMA-OPFP (1 equiv.), TEA (1.2 equiv.),
DMF, rt, 12 h, 69%. (f) NaOMe (0.1 equiv.), MeOH, rt, 10 min, 64%.
This journal is © The Royal Society of Chemistry 2012
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Table 1 Inhibition of PgaB
Residual activitya (%)
Inhibitor
Ki (μM)
Inhibitor
Residual activitya (%)
17
Ki (μM)
92
3
—
2
320 10
98 5c
—
96 4c
—
24
2
4
480 10
52
3
3
920 20
106
—
103
—
93
91
3
2
—
—
82
33
3
4
5800 100
680 20
a Remaining PgaB deacetylase activity with 10 mM inhibitor. b Inhibitor incubated with DTT (1 equiv.) prior to assay. c Remaining PgaB deacetylase
activity with 1 mM inhibitor.
Fig. 4 Preferred conformers of N-acylglucosamine amides.
containing these metal chelating groups have shown promising
activity with the histone de-N-acetylases.37,38
The N-methyl derivatives displayed dramatically improved
inhibitory activity over the N–H derivatives. Conformational
analysis of methyl 2-N-methyl-N-acetamido-2-deoxy-β-D-gluco-
pyranoside has shown a significant population of the acetyl
group in the (E)-anti conformation in comparison with the
primary amide of methyl 2-acetamido-2-deoxy-β-D-glucopyrano-
side which adopts primarily a (Z)-anti conformation (Fig. 4).35,39
The improved affinity observed with the N-methyl derivatives
suggests that the preferred bound conformation of the acyl metal
chelates may be (E)-anti. Interestingly the best inhibitor of the
N-Me derivatives, 20, is a metal chelating group that has not
Scheme 4 Synthesis of thioglycolyl amide pentasaccharide 32.
been previously reported as an inhibitor of amidases.
productive metal chelating groups identified above into a larger
oligosaccharide would yield a higher affinity inhibitor.
Chemoenzymatic synthesis
PgaB shows no measurable de-N-acetylation activity towards
methyl β-D-2-acetamido-2-deoxyglucopyranoside.19 However,
PgaB shows increased catalytic activity against larger PNAG
substrates with measurable de-N-acetylation of trisaccharide and
increasing activity on oligomers up to pentasaccharide in length.
Thus, it was hypothesized that introduction of one of the
Based on the thioglycolyl amide inhibitor 8 an improved
inhibitor was synthesized using a chemoenzymatic approach
(Scheme 4). PgaB was used to de-N-acetylate a PNAG pentasac-
charide.40 Previous analysis of the de-N-acetylation products
produced by PgaB showed that the predominant product of a
pentasaccharide substrate was mono-de-N-acetylation at the
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central glucosamine residue.19 Due to the low catalytic activity
of the enzyme complete de-N-acetylation was not achieved and a
mixture of the starting material and the mono-de-N-acetylated
pentasaccharide was carried forward to the acylation step. Acyla-
tion with S-octanoylthioglycolate N-hydroxysuccinamide ester
provided a product which could be readily separated from the
fully acetylated pentasaccharide by RP-HPLC. The octanoyl
thioester could be cleaved under basic conditions and the free
octanoic acid removed by acidic extraction (Scheme 4).
Analysis of the inhibition of compound 32, after reduction of
the disulfide bond, against PgaB was determined using the same
protocol as described for the monosaccharide inhibitors. The
pentasaccharide 32 showed a two-fold increase in inhibitory
(Ki = 280 μM) activity over compound 8. A greater gain in
affinity was expected given the observed improvement in sub-
strate turnover by PgaB on larger PNAG oligomers. Further
work is required to discern the binding mode of compounds 8
and 32 to the enzyme to explain this result with confidence.
Likely the optimal binding orientation of the thioglycolyl amide
8 is different from the orientation of a monosaccharide within a
larger oligomer.
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Conclusion
In conclusion, we have synthesized a series of glucosamine
derivatives bearing potential metal chelating groups as inhibitors
against PgaB. N-Methylated chelators showed consistently stron-
ger inhibition than their primary amide counterparts, yielding
high micromolar inhibitors. Using a chemoenzymatic synthesis it
was possible to generate a thioglycolyl amide functionalized
pentasaccharide inhibitor with a Ki value of 280 μM against
PgaB. Not surprisingly preliminary biofilm inhibition assays
with S. epidermidis 1457 using compounds 8, 20, 24, at a con-
centration of 1 mM, showed no inhibition likely due to the weak
activity of these compounds.
Acknowledgements
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This work is supported by research grants from the Canadian
Institutes of Health Research (CIHR) (#43998 and #89708 to
P.L.H. and M.N., respectively). A.C., J.P. and D.J.L. have been
supported by graduate scholarships from the University of
Toronto. P.L.H. is the recipient of
a Canada Research
Chair. M. Otto and colleagues are acknowledged for their help in
preliminary biofilm assays.
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