In vitro validation of acetyltransferase activity of GlmU as an antibacterial target in Haemophilus influenzae

Article abstract of DOI:10.1074/jbc.M111.274068

GlmU is a bifunctional enzyme that is essential for bacterial growth, converting D-glucosamine 1-phosphate into UDP-GlcNAc via acetylation and subsequent uridyl transfer. A biochemical screen of AstraZeneca's compound library using GlmU of Escherichia coli identified novel sulfonamide inhibitors of the acetyltransferase reaction. Steady-state kinetics, ligand-observe NMR, isothermal titration calorimetry, and x-ray crystallography showed that the inhibitors were competitive with acetyl-CoA substrate. Iterative chemistry efforts improved biochemical potency against Gram-negative isozymes 300-fold and afforded antimicrobial activity against a strain of Haemophilus influenzae lacking its major efflux pump. Inhibition of precursor incorporation into bacterial macromolecules was consistent with the antimicrobial activity being caused by disruption of peptidoglycan and fatty acid biosyntheses. Isolation and characterization of two different resistant mutant strains identified the GlmU acetyltransferase domain as the molecular target. These data, along with x-ray co-crystal structures, confirmed the binding mode of the inhibitors and explained their relative lack of potency against Gram-positive GlmU isozymes. This is the first example of antimicrobial compounds mediating their growth inhibitory effects specifically via GlmU.

Full text of DOI:10.1074/jbc.M111.274068

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 286, NO. 47, pp. 40734–40742, November 25, 2011
© 2011 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.
In Vitro Validation of Acetyltransferase Activity of GlmU as an
Antibacterial Target in Haemophilus influenzae*
Received for publication, June 20, 2011, and in revised form, September 22, 2011 Published, JBC Papers in Press, October 7, 2011, DOI 10.1074/jbc.M111.274068
Ed T. Buurman‡1, Beth Andrews‡, Ning Gao‡, Jun Hu‡, Thomas A. Keating‡, Sushmita Lahiri‡, Ludovic R. Otterbein§2,
Arthur D. Patten‡, Suzanne S. Stokes‡, and Adam B. Shapiro‡
From ‡AstraZeneca R&D Boston, Infection Innovative Medicines Unit, Waltham, Massachusetts 02451 and §Discovery Enabling
Capabilities and Sciences, AstraZeneca, Alderley Park, Macclesfield, Cheshire SK10 4TG, United Kingdom
Background: A search was initiated to identify inhibitors of the acetyltransferase domain of GlmU that could be exploited
as starting points for new antimicrobials.
Results: Sulfonamide inhibitors were identified that upon chemical modification displayed antimicrobial activity mediated via
GlmU.
Conclusion: Enzymatic inhibition of GlmU can lead to antimicrobial activity.
Significance: For the first time, GlmU was validated as an antimicrobial target in vitro.
GlmU is a bifunctional enzyme that is essential for bacterial
growth, converting D-glucosamine 1-phosphate into UDP-
GlcNAc via acetylation and subsequent uridyl transfer. A bio-
chemical screen of AstraZeneca’s compound library using
GlmU of Escherichia coli identified novel sulfonamide inhibi-
tors of the acetyltransferase reaction. Steady-state kinetics,
ligand-observe NMR, isothermal titration calorimetry, and
x-ray crystallography showed that the inhibitors were competi-
tive with acetyl-CoA substrate. Iterative chemistry efforts
improved biochemical potency against Gram-negative isozymes
300-fold and afforded antimicrobial activity against a strain of
Haemophilus influenzae lacking its major efflux pump. Inhibi-
tion of precursor incorporation into bacterial macromolecules
was consistent with the antimicrobial activity being caused by
disruption of peptidoglycan and fatty acid biosyntheses. Isola-
tion and characterization of two different resistant mutant
strains identified the GlmU acetyltransferase domain as the
molecular target. These data, along with x-ray co-crystal struc-
tures, confirmed the binding mode of the inhibitors and
explained their relative lack of potency against Gram-positive
GlmU isozymes. This is the first example of antimicrobial com-
pounds mediating their growth inhibitory effects specifically via
GlmU.
gets have been optimized further to circumvent resistance
mechanisms, e.g. ceftaroline (2). Alternatively, novel targets
have been explored because presumably their antibacterial
effectiveness has not been eroded by accumulation of mecha-
nism-based resistance mutations in clinical strains. Inhibitors
of new antibacterial targets have started to enter the initial
phases of clinical testing (3).
GlmU is a bifunctional enzyme involved in the synthesis of
UDP-N-acetylglucosamine (UDP-GlcNAc). UDP-GlcNAc is a
key intermediate in the synthesis of peptidoglycan, which is
essential for growth for nearly all eubacteria. GlmU contains
two separate catalytic sites, an acetyltransferase and a uridyl-
transferase site, responsible for yielding N-acetylglucosamine
1-phosphate and UDP-GlcNAc in consecutive steps (4). As syn-
thesis of UDP-GlcNAc is essential for peptidoglycan formation
and, in Gram-negative species, synthesis of lipopolysaccha-
rides, inhibition of GlmU leads to a loss of cell wall integrity,
resulting in cell death (5, 6). Quinazolines and aminopiperi-
dines are the first reported inhibitors of the uridyl transferase
domain of Haemophilus influenzae GlmU (Protein Data Bank
codes 2WOV and 2WOW) (7). Inhibitors of the acetyltrans-
ferase of Escherichia coli GlmU have also been identified (8).
This report describes the identification of novel sulfonamide
inhibitors of the acetyltransferase of E. coli GlmU that are com-
petitive with acetyl-CoA. A subsequent iterative chemistry
effort improved the biochemical potency of these inhibitors
and afforded compounds with antimicrobial activity against a
strain of H. influenzae that lacks efflux via the AcrB-TolC efflux
pump (9). Mode-of-action studies showed that the compound
acts via GlmU, thus providing for the first time in vitro valida-
tion of the target, i.e. showing that chemical inhibition of GlmU
results in inhibition of bacterial growth.
The increasing medical need for new antibacterial agents
resulting from primary resistance of emerging pathogens and
acquired resistance among established pathogens has been well
documented (1). To address this need, a two-pronged approach
has emerged. Existing scaffolds inhibiting well established tar-
* All authors are, or were, employees of AstraZeneca.
The atomic coordinates and structure factors (code 3TWD) have been deposited
in the Protein Data Bank, Research Collaboratory for Structural Bioinformat-
ics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).
1 To whom correspondence should be addressed: AstraZeneca R&D Bos-
ton, Infection Innovative Medicines Unit, 35 Gatehouse Dr., Waltham,
MA 02451. Tel.: 781-839-4592; Fax: 781-839-4600; E-mail: Ed.Buurman@
astrazeneca.com.
EXPERIMENTAL PROCEDURES
Strains
Bacterial strains used in this study for both susceptibility
testing and as a source of genomic DNA for cloning work were
Streptococcus pneumoniae NCTC7466, Staphylococcus aureus
RN4220 (10), E. coli ATCC 27325, and H. influenzae
2 Present address: Valeocon Management Consulting, 11 William Mews, Lon-
don SW1X 9HF, United Kingdom.
40734 JOURNAL OF BIOLOGICAL CHEMISTRY
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Antimicrobial Inhibitors of GlmU Acetyltransferase
ATCC51907. The latter two were parental strains of E. coli
⌬tolC::Tn10 and H. influenzae acrB::cat, respectively.
H. influenzae GlmU—pBA750 was transformed into E. coli
BL21(DE3) (Novagen) and plated on LB agar containing 25
␮g/ml kanamycin (Acros Organics). After overnight growth at
37 °C, several transformants were inoculated into 3 liters of LB
broth containing 25 ␮g/ml kanamycin and grown at 37 °C with
aeration to mid-logarithmic phase (A600 ϭ 0.5), and then iso-
propyl ␤-D-thiogalactopyranoside was added to a final concen-
tration of 1 mM. After 3 h at 37 °C, the cells were harvested by
centrifugation at 5000 ϫ g for 15 min at 25 °C. Cell paste was
stored at Ϫ20 °C, and protein expression and solubility were
checked by SDS-PAGE.
Construction of Expression Vectors
All glmU genes were amplified by PCR using genomic DNA
isolated from the respective pathogens (Wizard Genomic Prep,
Promega, Madison WI) as templates and the following primer
pairs: E. coli, 5Ј-ACGTCATATGTTGAATAATGCTATG
AGC-3Ј and 5Ј-ACGTGAATTCTCACTTTTTCTTTACC
GGACG-3Ј; H. influenzae, 5Ј-AACTACATATGACAAAAA-
AAGCATTAAGTGCGG-3Ј and 5Ј-GTCTTGCTGCCTTTT-
GTTCG-3Ј; S. aureus, 5Ј-ACTACATATGCGAAGACCACG-
CGATAATTTTGGC-3Ј and 5Ј-ACTAAGTCGACGGATTA-
TCCTTTTATCCTAGCC-3Ј; and S. pneumoniae, 5Ј-ACTAA-
CATATGTCAAATTTTGCCATTATTTTAGCAGCG-3Ј and
5Ј-ACTAAGTCGACTCATCACTGGTTCTTAGGATGAT-
GAGG-3Ј. Amplifications were performed using High Fidelity
PCR Master (Roche Applied Science, Indianapolis, IN), and
PCR products were purified using a QuickStep 2 PCR purifica-
tion kit (EDGE Biosystems, Gaithersburg, MD). The 1.4-kb
E. coli glmU PCR product was digested with NdeI/EcoRI and
cloned into similarly digested pMAL-p2x vector (New England
Biolabs) to generate pLH734. E. coli glmU was subcloned from
pLH734 as an NdeI/SalI fragment into NdeI/XhoI-digested
pZT-73.3 (11) to generate pBA738. The 1.7-kb H. influenzae
glmU PCR product was cloned into pCR4-TOPO (Invitrogen)
to generate pBA742 and then subcloned as an NdeI/EcoRI frag-
ment from pBA742 into similarly digested pET30a (Novagen,
Madison, WI) to generate pBA750. The 1.4-kb S. aureus glmU
PCR product was cloned into pCR4-TOPO to generate
pBA986. The glmU gene was isolated from pBA986 by SalI
digest followed by partial NdeI digest, and the fragment was
cloned into NdeI/XhoI-digested pZT7–3.3 to generate
pBA987. The 1.4-kb S. pneumoniae glmU PCR product was
digested with NdeI/SalI and cloned into NdeI/XhoI-digested
pZT7–3.3 to generate pBA989. DNA sequences of the cloned
glmU genes were confirmed by sequencing on an ABI PRISM
3100 DNA sequencer (Applied Biosystems) using Big Dye Ter-
minator cycle sequencing kit (Applied Biosystems). Computer
analyses of DNA sequences were performed with Sequencher
(Gene Codes Corp., Ann Arbor, MI).
S. aureus GlmU—pBA987 was transformed into E. coli
BL21(DE3) (Novagen) and plated on LB agar containing 10
␮g/ml tetracycline. After overnight growth at 37 °C, several
transformants were inoculated into 3 liters of LB broth contain-
ing 10 ␮g/ml tetracycline and grown at 30 °C with aeration to
mid-logarithmic phase (A600 ϭ 0.5), and then isopropyl ␤-D-
thiogalactopyranoside was added to a final concentration of 1
mM. After 3 h at 30 °C, the cells were harvested by centrifuga-
tion at 5000 ϫ g for 15 min at 25 °C. Cell paste was stored at
Ϫ20 °C, and protein expression and solubility were checked by
SDS-PAGE.
S. pneumoniae GlmU—pBA989 was transformed into E. coli
HMS174(DE3) (Novagen) and plated on LB agar containing 10
␮g/ml tetracycline. After overnight growth at 37 °C, several
transformants were inoculated into 3 liters of LB broth contain-
ing 10 ␮g/ml tetracycline and grown at ambient temperature
with aeration to mid-logarithmic phase (A600 ϭ 0.5), and then
isopropyl ␤-D-thiogalactopyranoside was added to a final con-
centration of 1 mM. After 18 h of induction at ambient temper-
ature, the cells were harvested by centrifugation at 5000 ϫ g for
15 min at 25 °C. Cell paste was stored at Ϫ20 °C, and protein
expression and solubility were checked by SDS-PAGE.
Purification of GlmU Isozymes
The frozen cell pastes from 3 liters of cell culture were sus-
pended in 50 ml of lysis buffer consisting of 50 mM Tris-HCl
(pH 8.0), 2 mM EDTA, 2 mM DTT, 10% (v/v) glycerol, 50 mM
NaCl, and 1 protease inhibitor mixture tablet (Roche Diagnos-
tics). Cells were disrupted by French press at 18,000 psi twice at
4 °C, and the crude extract was centrifuged at 25,000 rpm (45Ti
rotor, Beckman-Coulter, Brea, CA) for 30 min at 4 °C. The super-
natantwasappliedataflowrateof2.0ml/minontoa20-mlQ-Sep-
harose HP (HR16/10) column (GE Healthcare) pre-equilibrated
withBufferA, consistingof50mM Tris-HCl(pH8.0), 2mM EDTA,
2 mM DTT, 50 mM NaCl, and 10% (v/v) glycerol. The column was
then washed with Buffer A, and the protein was eluted by a linear
gradient from 0 to 1 M NaCl in Buffer A. Fractions containing
GlmU were pooled, and solid (NH4)2SO4 was added to a final con-
centration of 1 M. The sample was applied at a flow rate of 2.0
ml/min onto a 20-ml Phenyl Sepharose HP (HR16/10) column
pre-equilibrated with Buffer B consisting of 50 mM Tris-HCl (pH
8.0), 2 mM EDTA, 2 mM DTT, 10% (v/v) glycerol, and 1 M
(NH4)2SO4. The column was washed with Buffer B, and the pro-
tein was eluted by a linear gradient from 1 to 0 M (NH4)2SO4 in
Buffer B. Fractions containing GlmU were pooled, and solid
(NH4)2SO4 (0.4 g/ml) was added to precipitate all the proteins,
mixed, and kept on ice for 1 h. The sample was centrifuged at
GlmU Overexpression
E. coli GlmU—pBA738 was transformed into E. coli
HMS174(DE3) (Novagen, Madison, WI) and plated on Luria-
Bertani (LB)3 agar containing 10 ␮g/ml tetracycline (Fisher Sci-
entific). After overnight growth at 37 °C, several transformants
were inoculated into 3 liters of LB broth containing 10 ␮g/ml
tetracycline and grown at 37 °C with aeration to mid-logarith-
mic phase (A600 ϭ 0.5) after which isopropyl ␤-D-thiogalactopy-
ranoside (Acros Organics, Geel, Belgium) was added to a final con-
centration of 1 mM. After 2 h at 37 °C, the cells were harvested by
centrifugationat5000ϫgfor15minat25 °C. Cellpastewasstored
at Ϫ20 °C, and protein expression and solubility were checked by
SDS-PAGE.
3 The abbreviations used are: LB, Luria-Bertani; DMSO, dimethyl sulfoxide;
MIC, minimum inhibitory concentration(s).
NOVEMBER 25, 2011•VOLUME 286•NUMBER 47
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Antimicrobial Inhibitors of GlmU Acetyltransferase
TABLE 1
20,000 rpm for 30 min at 4 °C, and the pellet was then dissolved in
5 ml of Buffer A. The 5-ml sample was applied at a flow rate of 1.5
ml/min onto a 320-ml Sephacryl S-200 (HR 26/60) column (GE
Healthcare Life Sciences) pre-equilibrated with Buffer C consist-
ingof50mM Tris-HCl(pH8.0), 2mM EDTA, 2mM DTT, 10%(v/v)
glycerol, and 150 mM NaCl. The fractions containing GlmU were
pooled and dialyzedagainst1 literofstorage buffer consisting of50
mM Tris-HCl (pH 8.0), 2 mM EDTA, 2 mM DTT, 150 mM NaCl,
and 20% (v/v) glycerol. The protein was characterized by SDS-
PAGE analysis and analytical LC-MS. The protein was stored at
Ϫ80 °C. The same procedure was used to purify H. influenzae
GlmU, S. aureus GlmU, and S. pneumoniae GlmU.
Biochemical and antimicrobial properties of sulfonamide inhibitors of
GlmU acetyl transferase
No antimicrobial activity, i.e. MIC Ͼ 64 ␮g/ml, was observed with wild type strains
of S. aureus (Sau), S. pneumoniae (Spn), H. influenzae (Hin), and E. coli (Eco) and E.
coli tolC::Tn10.
IC50 Measurements
Compounds were dissolved in dimethyl sulfoxide (DMSO)
and 2-fold serial dilutions were prepared with DMSO, with a
top concentration of 10 mM. Two ␮l of compound dilutions
were added to clear polystyrene 96-well plates (Corning, Corn-
ing, NY). Enzyme reactions were performed in a 100-␮l volume
containing 225 ␮M acetyl-CoA (Sigma), 225 ␮M D-glucosamine
1-phosphate (Sigma), and 110 pM E. coli GlmU, 2.7 nM H. influ-
enzae GlmU, 260 pM S. pneumoniae GlmU, or 6 nM S. aureus
GlmU in assay buffer consisting of 50 mM MOPS-NaOH (pH
7.35), 75 mM potassium acetate, 10 mM MgCl2, and 0.005%
Tween 20. Reactions were quenched after 30 min with 50 ␮l of
1.5 mM Ellman’s reagent (Sigma) in 0.1 M sodium phosphate
(pH 7.2). The absorbance at 412 nm was measured after 5 min
with a Spectramax plate reader (Molecular Devices, Sunnyville,
CA). The baseline absorbance for 100% inhibition was mea-
sured for reactions inhibited by pretreatment of the enzyme for
30 min with 200 ␮M ␥-maleimidobutyric acid (Sigma). Because
the background absorbance, which is due to the presence of
CoA thiol as a contaminant of the acetyl-CoA, was suppressed
by reaction with ␥-maleimidobutyric acid, the absorbance was
corrected by supplementation with 12 ␮M 4-nitrophenol
(Sigma). Percent inhibition was calculated using the equation:
% inhibition ϭ 100(1 Ϫ (A412 Ϫ Min)/(Max Ϫ Min)), where
TABLE 2
Results of best-fit nonlinear least squares regression of the kinetic
mode of inhibition data for the inhibitor to the steady-state ordered
rate equation including substrate inhibition by both substrates and
competition by compound 1 with both substrates
The data were fit to models in which acetyl-CoA or D-glucosamine 1-phosphate is
substrate A, i.e. the first substrate to bind. The difference in ␹2 values for the two
regressions did not reach statistical significance (p ϭ 0.22).
Substrate A
D-Glucosamine
Kinetic constant
max (⌬A412
Acetyl-CoA
1-phosphate
V
K
K
K
K
K
K
K
)
1.49 Ϯ 0.03
1.48 Ϯ 0.03
ia (␮M)
24 Ϯ 2
25 Ϯ 3
(␮M)
398 Ϯ 15
311 Ϯ 11
2050 Ϯ 190
881 Ϯ 36
0.79 Ϯ 0.02
none
290 Ϯ 2
mA
mB (␮M)
423 Ϯ 16
965 Ϯ 40
2400 Ϯ 280
7.1 Ϯ 0.5
0.86 Ϯ 0.02
0.44
siA (␮M)
siB (␮M)
I1 (␮M) (substrate A competitive)
I2 (␮M) (substrate B competitive)
2
␹
0.48
A412 is the absorbance in the test well, Min is the absorbance of
globally by nonlinear least squares regression to kinetic rate equa-
tions using the program Grafit (Erithacus Software).
the fully inhibited reaction, and Max is the absorbance of the
uninhibited reaction. The compound concentration resulting
in 50% inhibition (IC50) was calculated by nonlinear least-
squares regression to the equation: % inhibition ϭ 100[I]b/
(IC50 ϩ [I]b), where [I] is the inhibitor concentration, and b is
the Hill slope (typically 1.0).
The kinetic mechanism of the acetyltransferase activity of
GlmU has not been elucidated. Therefore, the data from the above
experiment were fit to rate equations for a set of plausible mecha-
nisms, taking into account the observation of substrate inhibition
by both acetyl-CoA and D-glucosamine 1-phosphate. The purpose
of the fitting was to determine whether the inhibitor was likely to
be competitive with acetyl-CoA or D-glucosamine 1-phosphate or
both, rather than to support a particular kinetic mechanism for the
enzyme. The rate equations allowed for competitive inhibition by
the inhibitor with both of the substrates with different potencies
and exclusive of simultaneous binding with the substrate inhibitor
occupying the same substrate binding site. The steady-state
ordered bisubstrate rate equation is as follows (12).
Kinetic Mode of Inhibition
Enzyme activity measurements for analysis of the kinetic
mode of inhibition were performed in assay buffer in 384-well
clear polystyrene assay plates (Corning). The reaction volume
of 50 ␮l contained 2% (v/v) DMSO from the inhibitor stock
solution. Reactions contained 0–4 ␮M inhibitor, 0–1400 ␮M
D-glucosamine 1-phosphate, 60–1000 ␮M acetyl-CoA, and 100
pM E. coli GlmU. After 30 min, the reactions were quenched with
25 ␮l of 3 mM Ellman’s reagent in 100 mM sodium phosphate (pH
7.2). The absorbance at 412 nm was measured after 8 min with a
Spectramax plate reader. The averages and standard deviations
V ϭ Vmax[A][B]/{͑KiaKmB ϩ KmA[B]͒͑1 ϩ [B]/KsiB ϩ [I]/KI1͒
ϩ KmB[A]͑1 ϩ [A]/KsiA ϩ [I]/KI2͒ ϩ [A][B]} (Eq. 1)
were calculated for 4 replicate measurements. The data were fit The Ping Pong bisubstrate rate equation is as follows.
40736 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 286•NUMBER 47•NOVEMBER 25, 2011

Antimicrobial Inhibitors of GlmU Acetyltransferase
FIGURE 1. A, the steady-state ordered bisubstrate kinetic mechanism in which acetyl-CoA is the first substrate, both substrates cause substrate inhibition, and
the inhibitor competes with acetyl-CoA is one plausible kinetic mechanism for the E. coli GlmU acetyltransferase activity based on the data herein. B, compar-
ison of the experimental data to the best-fit non-linear least squares regression for the mechanism in A.
binding to the enzyme after its reaction with substrate A and
V ϭ Vmax[A][B]/{͑KmA[B]͑1 ϩ [B]/KsiB ϩ [I]/KI1͒ ϩ KmB[A]͑1
dissociation of the first product.
ϩ [A]/KsiA ϩ [I]/KI2͒ ϩ [A][B]} (Eq. 2)
Susceptibility Testing—Minimum inhibitory concentrations
(MIC) were determined using broth microdilution according to
the guidelines of the Clinical and Laboratory Standards Institute
(13). In the case of the GlmU overexpression strains and com-
pound-resistant strains, the differences in MIC were too small to
be measured reliably with 2-fold serially diluted compounds.
Instead, arithmetic dilutions were used either starting at a maxi-
mum concentration of 100 ␮g/ml in steps of 10 ␮g/ml or 200
␮g/ml in steps of 20 ␮g/ml maintaining a final DMSO concentra-
tion of 2% (v/v).
Vis the reaction rate. Vmax is the theoretical maximal reaction rate
when the enzyme is saturated with substrates. [A] and [B] are the
concentrations of substrates A and B, respectively. KmA and KmB
aretheMichaelisconstantsforsubstratesAandB, respectively. Kia
is the dissociation constant of substrate A. KsiA and KsiB are the
substrate inhibition constants for substrates A and B, respec-
tively. KI1 and KI2 are the inhibition constants for the inhib-
itor when binding to the enzyme without bound substrate
and the enzyme with substrate A bound, respectively, in the
steady-state ordered kinetic mechanism. In the ping-pong
H. influenzae GlmU Overexpression Strains—glmU was
mechanism, KI2 is the inhibition constant for the inhibitor amplified from H. influenzae acrB::cat genomic DNA using
NOVEMBER 25, 2011•VOLUME 286•NUMBER 47
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Antimicrobial Inhibitors of GlmU Acetyltransferase
5Ј-AACTACATATGACAAAAAAAGCATTAAGTGCGG-3Ј
and 5Ј-ACTAAGTCGACTTATTTTTTCTTTATTGGTCG-
TTGCC-3Ј. The 1.7-kb fragment was digested with NdeI and
SalI and cloned in similarly digested pASK5 (14), yielding
pBA756. A TetAR cassette was generated by amplification of
pASK6 (14) using 5Ј-TGCTGCAGTTAAGACCCACTTTCA-
CATTTAAGTTGTTTTTCTAATCCGCAAATGATCAAT-
TCAAGGCC-3Ј and 5Ј-TTGCCCGGGAAGCTTGCATGCT-
GCAGGCTATTTACCGCGGCTTTTTATTGAGC-3Ј, and
the resulting 2.1-kb product was digested with PstI and cloned
into similarly digested pBA756, which replaces the chloram-
phenicol resistance marker with a tetracycline resistance
marker. Using 5Ј-CGTACGGACGTCAAGCGTATTCAGC-
AAGCG-3Ј and 5Ј-CTCGCAAAGCTTGCCAAATGGCTTG-
TTGAAATGCG-3Ј, the Omp-GlmU-TetAR was amplified
and transformed into H. influenzae acrB::cat. Similarly, the
control cassette was amplified from pASK6 and transformed
into H. influenzae acrB::cat. This yielded strains H. influenzae
ARC2347 and ARC2348, respectively.
NMR—All NMR spectra were acquired at 298 K with a 600
MHz NMR instrument (Bruker, Billerica, MA) with an
AVANCE III console and a triple-resonance cryogenic probe.
In the WaterLOGSY experiment (15), the first water-selective
180° Sinc pulse was 6 ms long, and a weak rectangular pulse field
gradient was applied during the mixing time (1.8 s). A gradient
recovery time of 2 ms was introduced after the mixing time.
Water suppression was achieved by the excitation sculpting
scheme (16), and the water-selective 180° Sinc shape pulse was
3 ms long. The data were collected with a sweep width of 9157
Hz, 0.45 s of aquisition time, and 1.8 s for the relaxation delay.
64 scans were recorded for each experiment, requiring 5 min
per spectrum. The data were zero-filled to 32,768 complex
points and multiplied by an exponential function (line broad-
ening 3 Hz) prior to Fourier transformation.
FIGURE 2. Isothermal titration calorimetry studies of compound 2 bind-
ing to E. coli GlmU (Kd ؍ 1.9 ␮M). Observed heats evolved over time are
shown in the top, and the corresponding fitted one-site binding curve is
shown at the bottom.
Binding Studies—Binding of substrates and an inhibitor to
GlmU was investigated with isothermal titration calorimetry
(MicroCal VP-ITC, GE Healthcare). E. coli GlmU was dialyzed
Isolation of Resistant Mutants—Mutants of H. influenzae
into buffer consisting of 50 mM MOPS-NaOH (pH 7.35), 75 mM acrB::cat were isolated as described previously (17). In short,
potassium acetate, 10 mM magnesium chloride, and 2% DMSO. 100 ␮l of freshly prepared Haemophilus test medium (13)
Substrates acetyl-CoA and D-glucosamine 1-phosphate, and containing 104–108 cfu from an overnight culture were
compound 2 were dissolved in identical buffer. For titrations, plated on Haemophilus test medium agar plates containing
500 ␮M acetyl-CoA was injected into 15 ␮M GlmU, 200 ␮M filter-sterilized hematin (Sigma), filter-sterilized nicotina-
compound 2 was injected into 18.8 ␮M GlmU, and 500 ␮M mide adenine dinucleotide (Sigma), and various 2-fold serial
D-glucosamine 1-phosphate was injected into 25 ␮M GlmU. dilutions of compound 5, ranging from 16–128 ␮g/ml, were
Collected data were normalized and fit to a one-site binding all added immediately prior to pouring the plates. Resistant
model using Origin software (version 7).
colonies appeared after incubation for 72 h at 37 °C with 5%
Crystallization and Structure Solution—Crystals were (v/v) CO2 at the plate MIC compound concentration of 32
grown at ambient temperature by the hanging drop method. ␮g/ml at a rate of 10Ϫ6–10Ϫ7. None were found at higher
The protein in 50 mM Tris-HCl, pH8.0, 150 mM NaCl, 2 mM compound concentrations, suggesting a resistance rate of
EDTA, 2 mM Tris(2-carboxylethyl)phosphine was equili- Ͻ10Ϫ8. Resistant colonies were purified on plates identical
brated against a reservoir solution containing 19–22% (w/v) to those from which they were isolated. About half of the
PEG3350, 100 mM propionic acid/cacodylate/Bis-trispro- colonies grew up again and were subsequently stored in Hae-
pane system (pH 5.5), and 400 mM ammonium sulfate. Com- mophilus test medium containing 50% (v/v) glycerol at
plexes were obtained by co-crystallization of the protein Ϫ80 °C prior to further analysis.
with compound 3 to a final concentration of 4 mM. Crystals
Inhibition of Macromolecule Biosynthesis—Measurements of
were flash frozen, and diffraction data were collected to 1.9 Å inhibition of the incorporation into growing cells of various
in a home source. Refinement was performed using refmac in radiolabeled metabolic precursors was performed according to
CCP4 and Coot.
Hilliard et al. (18), with changes as described below. Specific
40738 JOURNAL OF BIOLOGICAL CHEMISTRY
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Antimicrobial Inhibitors of GlmU Acetyltransferase
FIGURE 3. One-dimensional WaterLOGSY spectral comparison of 200 ␮M acetyl-CoA binding to 10 ␮M H. influenzae GlmU with (bottom) and without
(top) the addition of 200 ␮M compound 4 (left) or 5 (right). Signals of proton a, b, and c from acetyl-CoA are labeled and compared in the WaterLOGSY
spectra. The signals in the dashed boxes are from the compounds. The NMR samples were prepared in 50 mM HEPES (pH 7.5), 5 mM MgCl2, 5 mM DTT, 0.1 mM
EDTA, and 5% D2O. The spectra were acquired at 298 K.
radioactivities and final concentrations were as follows: 315 gressed linearly. Plates were stored at 4 °C overnight after
Ci/mol L-[U-14C]leucine, final concentration of 5 ␮Ci/ml; 28 which well contents were transferred using a Filtermate har-
Ci/mmol L-[3,4(n)-3H]valine, final concentration of 5 ␮Ci/ml; vester (Packard, Downers Grove, IL) onto Packard GF/B
50 Ci/mol N-acetyl-D-[1-14C]glucosamine, final concentration 96-well Unifilters. Wells and filters were washed 10 times
of 1 ␮Ci/ml; 58 Ci/mol [2-14C]acetic acid, final concentration of with 200 ␮l of water to remove unincorporated precursors.
5 uCi/ml plus 50 mg/liter unlabeled sodium acetate; 25 These filters were dried, and 40 ␮l of scintillation fluid
Ci/mmol [3H]uridine, final concentration of 5 ␮Ci/ml plus 120 (Microscint 20, PerkinElmer Life Sciences, Waltham, MA)
mg/liter unlabeled uridine (Sigma); and 25 Ci/mmol [methyl- was added to each well, and radioactivity was measured with
3H]thymidine, final concentration of 5 ␮Ci/ml plus 5 mg/liter a TopCount scintillation counter (Packard).
unlabeled thymidine (Alfa Aesar, Ward Hill, MA). Radiolabeled
Compounds—The chemical synthesis of compounds 1-5 was
precursors were from GE Healthcare. H. influenzae was har- performed in-house and will be presented elsewhere. Com-
vested when in exponential phase (A600 ϭ 0.4, 250 ml of culture pound purity was determined by LC/MS shortly before biolog-
in a 1-liter flask, shaking at 100 rpm, 30 °C), centrifuged at ical testing and was Ͼ90%.
ambient temperature for 10 min at 3500 ϫ g, and resuspended
RESULTS
at the same cell density in fresh, prewarmed Haemophilus test
medium. A volume of 40 ␮l was added to wells of a pre-
A high throughput screen of the AstraZeneca compound col-
warmed 96-well plate containing 20 ␮l of 2-fold serially lection was performed using E. coli GlmU by detecting inhibi-
diluted compound in Mueller Hinton broth (12) (plus tion of its acetyltransferase activity, resulting in the identifica-
DMSO (2% v/v final) plus 40 ␮l of Mueller Hinton broth tion of compound 1 (Table 1). Its inhibitory activity was
containing the radiolabeled precursor. The incorporation retained upon resynthesis of the compound. The compound
was stopped with one volume of ice-cold stopping reagent was similarly potent against H. influenzae GlmU but did not
(20% (w/v) trichloroacetic acid with BactoTM casamino acids inhibit GlmU of Gram-positive pathogens S. pneumoniae and
(Bacto, Liverpool, UK), uridine, thymidine, N-acetylgluco- S. aureus at concentrations Ͻ200 ␮M.
samine, and sodium acetate, each at 1% (w/v)) after 0, 30, and
Using E. coli GlmU, the mode of inhibition of compound 1
60 min of incubation, during which incorporation pro- was investigated with steady-state kinetics. Kinetic constants
NOVEMBER 25, 2011•VOLUME 286•NUMBER 47
JOURNAL OF BIOLOGICAL CHEMISTRY 40739

Antimicrobial Inhibitors of GlmU Acetyltransferase
were determined by non-linear least-squares global fitting of
the data from the mode of inhibition experiment to steady-state
ordered bisubstrate rate equations in which either acetyl-CoA
or D-glucosamine 1-phosphate is the first substrate to bind
(Table 2). Both substrates caused substrate inhibition. The
equations allowed for competition by compound 1 with both
substrates with different KI values. The results for the Ping
Pong bisubstrate model fitting are not shown because the fit-
ting to the ordered models was significantly better (p Ͻ 0.01).
The difference in ␹2 values between the fits to the two ordered
models is not statistically significant (p ϭ 0.22). The results
show that compound 1 is predominantly competitive with
acetyl-CoA. The fit of the data to the ordered bisubstrate rate
equation with acetyl-CoA as the first substrate is shown in Fig.
1. The Michaelis constants for acetyl-CoA and D-glucosamine
1-phosphate are similar to apparent Km values reported in the
literature for E. coli GlmU acetyltransferase. Mengin-Lecreulx
and van Heijenoort (4) reported apparent Km values of 600 and
150 ␮M for acetyl-CoA and D-glucosamine 1-phosphate,
respectively. Gehring et al. (19) reported Km values of 320 and
250 ␮M for acetyl-CoA and D-glucosamine 1-phosphate,
respectively.
Isothermal titration calorimetry was carried out to assess the
binding of substrates acetyl-CoA, glucosamine 1-phosphate,
and a representative inhibitor to the free form of the enzyme.
Because the solubility of compound 1 was insufficient, a more
soluble but equipotent analog, compound 2, was used (Table 1).
Acetyl-CoA bound to GlmU with Kd ϭ 15 ␮M and a stoichiom-
etry of 0.6, whereas no evidence of binding of D-glucosamine
1-phosphate was observed (upper limit of detection ϳ 30 ␮M)
(data not shown). Compound 2 bound to GlmU with Kd ϭ 1.9
␮M and a stoichiometry of 0.6, very similar to that of acetyl-CoA
(Fig. 2). The Kd of acetyl-CoA agrees well with the Kia from
steady-state kinetics with acetyl-CoA as the first substrate to
bind (Table 2). Likewise, the binding of compound 2 to free
GlmU supports the kinetic conclusion of inhibitor competition
with acetyl-CoA. This result was confirmed by WaterLOGSY
NMR using H. influenzae GlmU with more potent compounds
4 and 5 (Fig. 3). Reduction of acetyl-CoA signals in the presence
of compound 4 or 5 clearly demonstrates that both compounds
compete with acetyl-CoA binding (Fig. 3). In addition, greater
acetyl-CoA signal reduction in the presence of compound 5 by
comparison with compound 4 indicates compound 5 is a stron-
ger acetyl-CoA competitor than compound 4, which is consis-
tent with the IC50 data of these compounds against H. influen-
zae GlmU (Table 1).
FIGURE 4. A, a view of the inhibitor binding pocket in the E. coli GlmU trimer. B,
electron density(2Fo Ϫ Fc map at 1.2␴)of compound 3 incomplex withGlmU.
C, binding interactions of compound 3 and acetyl-CoA. Compound 3 has
been depicted in thick yellow sticks, and acetyl-CoA is shown in thin gray lines.
The three monomers are colored pink, gray, and cyan. The residues mutated
intheresistantstrainsarehighlightedindarkershadesoftherespectivemon-
omer color. Hydrogen bonds are depicted as dotted lines.
the pocket, whereas the sulfonamide side chain faces the
bulk solvent.
An iterative chemistry effort led to compound 5 that dis-
played a 300-fold lower IC50 against the GlmU isozymes from
E. coli and H. influenzae (Table 1). Measurable inhibition could
also be detected against the Gram-positive GlmU isozyme from
S. pneumoniae but not S. aureus. Routine testing of antimicro-
bial activity showed MIC Ͼ 64 ␮g/ml against any wild type
strains and an E. coli strain lacking the TolC subunit of the
major efflux transporter AcrABTolC. However, growth of an
H. influenzae strain missing AcrB was sensitive and showed an
MIC of 32 ␮g/ml (Table 1).
An x-ray crystallographic system was developed with E. coli
GlmU that allowed inhibitors to be co-crystallized with the
protein. The overall structure of the protein chain was iden-
tical between the acetyl-CoA-bound and inhibitor-bound
forms. Clear electron density could be observed for bound
compound 3, allowing all atoms to be assigned (Fig. 4B). The
inhibitor is bound in each of the three acetyl-CoA binding
sites formed by the interfaces of two of the three chains of the
trimer and the carboxyl-terminal loop of the remaining
monomer (Fig. 4A) (20). This is in agreement with the NMR
To determine the mode of action against H. influenzae
acrB::cap, inhibition of incorporation of radiolabeled pre-
cursors into cellular macromolecules was measured at vari-
ous concentrations of compound 5. Incorporation of
N-acetylglucosamine into peptidoglycan and acetate into
lipids was inhibited by 50% at 16–32 ␮g/ml compound 5,
which is similar to the MIC value (Table 1). Inhibition of
incorporation of thymidine into DNA, uridine into RNA,
and valine and leucine into protein, required at least 64
studies. The aliphatic carboxylate group is buried deeper in ␮g/ml of compound 5 (Fig. 5).
40740 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 286•NUMBER 47•NOVEMBER 25, 2011

Antimicrobial Inhibitors of GlmU Acetyltransferase
A GlmU overexpression strain of H. influenzae acrB, which removed a stabilizing ␲-stacking interaction between
ARC2347, was constructed that was isogenic to its parent, the tryptophan side chain and the aromatic sulfonamide side
ARC2348, but contained a copy of glmU under control of the
ompP1 promotor. A comparison of glmU and omp1 transcript
levels in H. influenzae acrB::cat suggested that expression of
glmU under control of ompP1 would result in a relatively small,
8-fold, increase in GlmU (data not shown). As a result, the MIC
value against ARC2347 for compounds 4 and 5 increased by
50% relative to the parental strain (133 Ϯ 27 ␮g/ml versus 80 Ϯ
18 ␮g/ml (n ϭ 6, p ϭ 0.003) and 57 Ϯ 8 ␮g/ml versus 38 Ϯ 4
␮g/ml (n ϭ 7, p ϭ 0.0001), respectively).
chain. The other mutation, T420I, sterically alters the orienta-
tion of Trp-449 and thus indirectly alters the same ␲-stacking
interaction. In Gram-positive GlmU isozymes, leucine is pres-
ent in this position (Fig. 6), which likely prevents the ␲-stacking
interaction, and thus efficient binding of the inhibitors, in sim-
ilar fashion.
DISCUSSION
In a search for chemical starting points to exploit GlmU
acetyltransferase for the development of new antibacterials,
a high-throughput screen of AstraZeneca’s library was per-
formed. To maximize success both quality and diversity of
the screened compound set and sensitivity of the assay are
important. Because the aim was to identify inhibitors to
either the acetyl CoA or the D-glucosamine 1-phosphate
binding site, both the kinetic mechanism and the Km values
for each substrate were determined. Following the method of
Cheng and Prusoff (22), two equations were derived to cal-
culate the IC50/Ki ratio for the GlmU acetyltransferase assay
for dead-end inhibitors competitive either with acetyl-CoA
or glucosamine 1-phosphate. The basis for the derivations
was the steady-state ordered bisubstrate rate equation given
above, with acetyl-CoA as the first substrate to bind and
substrate inhibition by both substrates. The kinetic con-
stants from Table 2 and concentration of 225 ␮M for both
substrates were used for the calculations. The IC50/Ki ratio
for the high-throughput screen and IC50 assay were 2.6 for
acetyl-CoA competitive inhibitors and 3.6 for glucosamine
1-phosphate competitive inhibitors. Thus, the high-
throughput screen was only slightly biased toward acetyl-
CoA competitive inhibitors at the expense of glucosamine
1-phosphate competitive inhibitors. The 1.9 ␮M Kd of com-
pound 2 measured by isothermal titration calorimetry and
corresponding IC50 of 3.3 ␮M are consistent, within experi-
mental error, with the IC50/Ki ratio for an acetyl-CoA-com-
petitive inhibitor.
Three strains of H. influenzae acrB::cat resistant to com-
pound 5 were isolated with a frequency of 10Ϫ6–10Ϫ7 at a com-
pound concentration that equaled the MIC value 32 ␮g/ml.
None were found at higher compound concentrations suggest-
ing a relatively small increase in MIC. Arithmetic dilution of
compound in broth confirmed an increase in MIC from 30
␮g/ml to 50 (1 strain) or 60 ␮g/ml (2 strains). The glmU gene
and its promoter of all three strains were sequenced and
revealed a single mutation in the glmU genes in the two more
resistant strains. Both mutations were located in the region
encoding the acetyltransferase domain (Fig. 5). One mutation
resulted in a W449G substitution (E. coli numbering (21)),
X-ray crystallography confirmed binding of compound 3 in
the acetyl-CoA binding site. The carboxamide hydrogen bonds
with the peptide backbone of Tyr-387 and Ser-405 and the sul-
fonyl oxygens hydrogen bond with the Ala-423 backbone and
the guanidine side chain of Arg-440. This binding mode is sim-
ilar to that of the pantothenate moiety of CoA (23). The itera-
tive chemistry effort led to 300-fold improvement in IC50
against the Gram-negative isozymes and introduced activity
against the isozyme of S. pneumoniae but not S. aureus. This is
mainly due to the extension of the amide substituent with an
aliphatic carboxylate, which mimics the ␤-mercaptoethyl-
FIGURE 5. Compound 5 preferentially inhibits N-acetylglucosamine and
acetic acid incorporation into macromolecules of H. influenzae acrB::cat.
Inhibition of synthesis of protein (leucine and valine incorporation; not
shown), RNA (uridine incorporation; circles), fatty acids (acetate incorpora-
tion; triangles), peptidoglycan (N-acetylglucosamine incorporation; squares),
and DNA (thymidine incorporation; not shown) was measured as described
under “Experimental Procedures.” The incorporation rates of added precur-
sors into uninhibited cells were 21, 65, 1100, 7600, 3.1, and 35 ␮mol/h/A600
,
respectively. Inhibition of leucine, valine, thymidine, and uridine all showed
similar modest inhibition and stayed below 50% even at the highest com-
pound concentration tested. For clarity, only uridine is shown as a represent-
ativeofthesefour. Dataareaveragesoftwoindependentmeasurements;S.D.
are ϳ10%.
FIGURE 6. Amino acid sequences of the carboxyl-terminal end of four GlmU isozymes, numbered according to the sequence of the E. coli
isozyme. Thr-420 and Trp-449, which when mutated to Ile and Gly respectively led to resistance of H. influenzae acrB::cat against compound 5, are
highlighted.
NOVEMBER 25, 2011•VOLUME 286•NUMBER 47
JOURNAL OF BIOLOGICAL CHEMISTRY 40741

Antimicrobial Inhibitors of GlmU Acetyltransferase
amine extension of the pantothenate moiety of CoA and activity of these inhibitors was shown to be caused by inhi-
reaches to Ala-380 and Asn-377. The aromatic side chain on bition of GlmU acetyltransferase transferase. Although clin-
the sulfonamide is stabilized by ␲-stacking with Trp-449. The ical drug candidates acting via GlmU are still a long time
importance of this interaction is illustrated by the W449G away, this work for the first time provides in vitro validation
mutant that confers resistance to compound 5 and by weaker of this enzyme activity as an antibacterial target.
activity against S. pneumoniae GlmU. S. pneumoniae GlmU
Acknowledgments—We thank the AstraZeneca R&D Boston clinical
activity could be measured only after the initial hit was
susceptibility group for MIC determinations and thank Rob Albert,
extended with larger groups in this region, thus providing addi-
Jason Breed, Amy Kutschke, Valerie Laganas, Stephania Livchak,
tional interactions which compensated for the absence of ␲-␲
Kathy MacCormack, and Wei Yang for technical expertise with var-
interactions. Sequence analysis also suggested that S. aureus
ious parts of this study.
GlmU contains a shorter carboxyl-terminal end, lacking resi-
dues forming this part of the pocket (Fig. 6). This may explain
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VOLUME 286•NUMBER 47•NOVEMBER 25, 2011