Identification of non-substrate-like glycosyltransferase inhibitors from library screening: Pitfalls & hits

Article abstract of DOI:10.1039/c7md00550d

Bacterial glycosyltransferases are potential targets for the development of novel antibiotics and anti-virulence agents. Most existing glycosyltransferase inhibitors are substrate analogues with limited potential for drug development. The identification of alternative inhibitor chemotypes is therefore of great interest for medicinal chemistry, drug discovery and chemical glycobiology. We describe the application of a biochemical glycosyltransferase assay to screen a small compound library containing three distinct chemical scaffolds (nucleosides, steroids and 5-methyl pyrazol-3-ones) against the retaining α-1,4-galactosyltransferase LgtC from Neisseria meningitidis. While no genuine LgtC inhibitory activity was observed in the nucleoside and steroid series, the best hit compounds in the 5-methyl pyrazol-3-one series showed low micromolar activity. We adapted our assay protocol to develop initial structure-activity relationships in this series, and to establish the target selectivity of the most potent inhibitor over two other glycosyltransferases. Our results provide insights into the activity of this class of non-substrate-like glycosyltransferase inhibitors, and highlight important general pitfalls for inhibitor screening against this enzyme family. Key elements of our experimental design, including a validated single-concentration protocol for inhibitor screening, and our process for elimination of false positives, are, in principle, directly transferable to many other sugar-nucleotide-dependent glycosyltransferases.

Full text of DOI:10.1039/c7md00550d

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Identification of non-substrate-like glycosyltransferase inhibitors from library
screening: pitfalls & hits
Masaki Ema [1], Yong Xu [1], Sebastian Gehrke [2] & Gerd K. Wagner [1]*
[1]
King’s College London, Department of Chemistry, Faculty of Natural &
Mathematical Sciences, Britannia House, 7 Trinity Street, London, SE1 1DB, UK.
Phone: +44 (0)20 7848 1926; eꢀmail: gerd.wagner@kcl.ac.uk
[2]
King’s College London, Institute of Pharmaceutical Science, Faculty of Life
Sciences & Medicine
*Corresponding author
20 November 2017
Keywords: glycosyltransferase, inhibitor, screening, bacteria
1

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Abstract
Bacterial glycosyltransferases are potential targets for the development of novel
antibiotics and antiꢀvirulence agents. Most existing glycosyltransferase inhibitors are
substrate analogues with limited potential for drug development. The identification of
alternative inhibitor chemotypes is therefore of great interest for medicinal chemistry,
drug discovery and chemical glycobiology. We describe the application of a
biochemical glycosyltransferase assay to screen a small compound library
containing three distinct chemical scaffolds (nucleosides, steroids and 5ꢀmethyl
pyrazolꢀ3ꢀones) against the retaining αꢀ1,4ꢀgalactosyltransferase LgtC from
Neisseria meningitidis. While no genuine LgtC inhibitory activity was observed in the
nucleoside and steroid series, the best hit compounds in the 5ꢀmethyl pyrazolꢀ3ꢀone
series showed low micromolar activity. We adapted our assay protocol to develop
initial structureꢀactivity relationships in this series, and to establish the target
selectivity of the most potent inhibitor over two other glycosyltransferases. Our
results provide insights into the activity of this class of nonꢀsubstrateꢀlike
glycosyltransferase inhibitors, and highlight important general pitfalls for inhibitor
screening against this enzyme family. Key elements of our experimental design,
including a validated singleꢀconcentration protocol for inhibitor screening, and our
process for elimination of false positives, are, in principle, directly transferable to
many other sugarꢀnucleotideꢀdependent glycosyltransferases.
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Introduction
Gramꢀnegative bacterial pathogens such as Haemophilus and Neisseria use certain
glycan structures in their cell envelope as virulence factors, e.g. to protect against
the immune response of the human host. The biosynthetic machinery required for
the biosynthesis of these glycoconjugates therefore offers a rich source of targets for
the development of novel antibiotics and antiꢀvirulence agents. An important class of
enzymes that fall into this category are glycosyltransferases (GTs), enzymes that
catalyse the transfer of a monoꢀ or oligosaccharide from a glycosyl donor to a
specific acceptor [1]. Most existing inhibitors of bacterial GTs are substrate
analogues with limited potential for drug development [2]. An important strategy for
the identification of alternative GT inhibitor chemotypes is the screening of
compound libraries. Despite successful previous examples [3], the screening for GT
inhibitors is not trivial, and the identification both of suitable assay conditions, and of
suitable screening libraries can be challenging. This is particularly true if a
biochemical, rather than a binding, assay is used, due to the complex multiꢀsubstrate
mechanism of these enzymes.
A considerable number of biochemical GT assays have been developed,
including radiochemical, immunological, chromatographic, fluorimetric and
spectrophotometric formats [4,5]. However, many of these assay formats cannot
easily be adapted for inhibitor screening because of the requirement for expensive
specialized equipment, difficulty to access labelled donor or acceptor, or a general
unsuitability of the assay format for highꢀthroughput conditions [4]. We have recently
described an assay protocol for the identification of nonꢀsubstrateꢀlike inhibitors of
the retaining αꢀ1,4ꢀgalactosyltransferase LgtC from Neisseria meningitidis [6]. LgtC
catalyses the transfer of αꢀ
Dꢀgalactose from uridine diphosphate αꢀDꢀgalactose
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(UDPꢀGal) to terminal lactose residues on lipooligosaccharide (LOS) structures of
the outer core [7], resulting in increased serum resistance [8]. Inhibitors of LgtC may
therefore be useful as antiꢀvirulence agents and tool compounds for antiꢀvirulence
drug discovery. Our LgtC assay uses a phosphatase to cleave both phosphates off
the secondary reaction product UDP, followed by colorimetric detection of the
inorganic phosphate with Malachite Green (Fig. 1). The assay is inexpensive and
operationally simple, and can be carried out in a microplate format.
Fig. 1 The LgtC reaction, and the principle of the coupled biochemical LgtC assay
In this study, we have adapted this assay format to screen a small compound library
against LgtC, in order to identify new inhibitor chemotypes. Most existing LgtC
inhibitors are structurally derived from the donor substrate UDPꢀGal [7,9]. Our library
contained three distinct nonꢀsubstrateꢀlike chemical scaffolds: nucleosides, steroids
and pyrazolꢀ3ꢀones. Nucleosides were selected to investigate if they may be able to
act as uncharged mimics of the UDPꢀGal donor. Steroids and pyrazolꢀ3ꢀones were
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included because of previously reported activities of these structural classes against
other GTs. Thus, inhibitory activity against different mammalian and bacterial GTs
has been reported for several steroid derivatives, such as finasteride against human
UDPꢀglucuronosyltransferase UGT [10], murgocil against the peptidoglycan GT
MurG [11], and lithocholic acid derivatives such as AL10 against αꢀ2,3ꢀ
sialyltransferase [12,13]. Pyrazolꢀ3ꢀones have been reported as inhibitors of different
bacterial GTs, including MurG [3] and WaaC [14].
We have recently identified a single 5ꢀmethyl pyrazolꢀ3ꢀone as an inhibitor of
LgtC [15]. Herein, we report initial structureꢀactivity relationships (SAR) in this
inhibitor series. For these experiments, we have established a singleꢀconcentration
screening protocol for our assay, and validated it with full IC50 measurements. With
this approach, we identified several 5ꢀmethyl pyrazolꢀ3ꢀones with low micromolar
activity against LgtC. Following further adaptation of our assay protocol, we also
investigated the LgtC target selectivity of the most potent inhibitor over two other
glycosyltransferases. No genuine inhibitory activity against LgtC was observed, on
the other hand, in either the nucleoside or the steroid series. In the steroid series, we
did observe false positives, which prompted us to carefully investigate critical
parameters of our screening assay protocol. The results from this study therefore
provide not only initial SAR trends in a new class of nonꢀsubstrateꢀlike GT inhibitors,
but also highlight important general pitfalls for inhibitor screening against this
enzyme family.
Results
Assay adaptation. For the initial screening experiment, we selected 130 compounds
in total, including 20 nucleosides and 90 steroids from a commercial library of known
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drugs and bioactives, and 20 5ꢀmethyl pyrazolꢀ3ꢀones that had been synthesised in
our own laboratory (Table S1, ESI). Inhibitor candidates were initially tested at a
single concentration (50 ꢁM) against LgtC. For this experiment, we adapted our
recently reported colorimetric assay protocol [6]. LgtC, βꢀlactose acceptor, inhibitor
(in DMSO), calfꢀintestinal phosphatase (CIP), MnCl2, chicken eggꢀwhite lysozyme
(CEL) and Triton Xꢀ100 were premixed, and the reaction was started by addition of
UDPꢀGal donor. After incubation for 20 mins, the Malachite Green reagents were
added to stop the reaction, and absorbance was measured at 620 nm. With this setꢀ
up, 10 inhibitors could be examined, in duplicate, on a single 96ꢀwell microplate. To
accelerate screening and save on materials, we also attempted to miniaturise the
assay, using 384ꢀwell microplates. This would have allowed us to reduce the total
amount of reagent in each well from 210 ꢁM to 70 ꢁM. Unfortunately, this setꢀup
resulted in poor reproducibility, especially at higher concentrations of inhibitor (Fig.
S1, ESI). We therefore used the standard 96ꢀwell plate setꢀup for all screening
experiments.
Library screening. With suitable assay conditions in hand for screening, we tested
the three selected compound collections. First, 90 steroid derivatives were selected
from a commercial library of known drugs and bioactives. In the singleꢀconcentration
screen of these 90 steroid derivatives, two derivatives showed significantly higher
inhibitory activity than the remaining steroids (79: 38% @ 50 ꢁM; 90: 34% @ 50 ꢁM).
Both compounds belong to the class of aminosteroid muscle relaxants (Fig. S2, ESI).
To confirm their inhibitory activity, we determined their full IC50 curves. Encouragingly,
in these experiments both compounds showed concentrationꢀdependent activity at
concentrations above 100 ꢁM (Fig. S2, ESI).
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To investigate the possibility of false positives, e.g. from inhibition of the
phosphatase used in the assay and/or interference of the inhibitors with the
colorimetric assay readꢀout, we next carried out control experiments with 79 and 90.
First, the assay was repeated with only the phosphatase present, but no LgtC, and
the phosphatase substrate UDP instead of UDPꢀGal. Disappointingly, in this control
experiment both 79 and 90 gave a similar doseꢀresponse curve as in the IC50
experiments with LgtC, which indicated that the activity observed in the coupled
assay was not due to inhibition of LgtC (Fig. S2, ESI). To understand if these
compounds inhibited the phosphatase or interfered with the Malachite Green
detection reagent, we carried out a second control experiment. Inorganic phosphate
(Pi) at different concentrations was reacted with Malachite Green in the presence or
absence of the screening hits. Both 79 and 90 significantly reduced the absorbance
under these conditions (Fig. S2, ESI). Taken together, these results showed that the
activity observed for both compounds in the coupled LgtC assay was an assay
artefact resulting from interference of 79 and 90 with the colorimetric readꢀout of our
assay. These results highlight not only the need for stringent control experiments
with this type of coupled assay, but also the capacity of charged aminosteroids such
as 79 and 90 for nonꢀspecific aggregation with assay components.
Next, we repeated the screening experiment against LgtC with a collection of
20 nucleoside derivatives (Table S1, ESI), but did not observe any inhibitory activity.
Finally, we repeated the screening experiment against LgtC with 20 5ꢀmethyl
pyrazolꢀ3ꢀone derivatives (Table S1, ESI). In this series, diverse levels of activities
were observed, ranging from compounds that were practically inactive, such as 127,
to compounds such as 113, which were at least equipotent to our prototype inhibitor
CSG164 (Fig. 2A). For compounds that showed inhibition greater than 10% in the
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singleꢀconcentration screen, we determined full IC50 values. Pleasingly, we found a
good correlation between inhibitory activity in the singleꢀconcentration screen and in
full IC50 experiments (Fig. 2B). This result demonstrates that the singleꢀconcentration
assay format is suitable for discrimination between active and inactive molecules.
Importantly, and in contrast to results with aminosteroids 79 and 90, control
experiments against phosphatase, with inhibitor at 50 ꢁM, confirmed that for all
pyrazolꢀ3ꢀones 111ꢀ130, the activity observed in the coupled assay was due to
genuine inhibition of LgtC.
Fig. 2 A: Activity of 5ꢀmethyl pyrazolꢀ3ꢀones 111ꢀ130 against LgtC in the singleꢀ
concentration screening assay and in full IC50 experiments. Prototype inhibitor
CSG164 is included for direct comparison. B: Correlation of results from both assays.
C: Full IC50 curves for inhibitors 112 and 113.a
A
B
112
113
C
120
100
80
60
40
20
0
120
100
80
60
40
20
0
IC50 = 33 ± 6 µM
IC50 = 30 ± 1 µM
ꢀ8
ꢀ7
ꢀ6
ꢀ5
ꢀ4
ꢀ3
ꢀ8
ꢀ7
ꢀ6
ꢀ5
ꢀ4
ꢀ3
log c Inhibitor (M)
log c Inhibitor (M)
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aConditions for IC50 experiments: LgtC was incubated with 112 or 113 (0.1ꢀ100 µM),
UDPꢀGal (28 µM), MnCl2 (5 mM), CIP (10 U/mL), CEL (1 mg/mL), Triton (0.01%) and
lactose acceptor (2 mM) for 20 min at 30 oC in 13 mM HEPES buffer (pH 7.0). Donor
turnover (%) was 20% (112) and 29% (113), respectively. Data were analysed with
GraphPad Prism. Each experiment was carried out in triplicate.
Profiling of hit compound 113. Next, we further investigated the activity profile of the
most potent inhibitor identified from the screen, 5ꢀmethyl pyrazolꢀ3ꢀone 113. At
concentrations up to 100 ꢁM, 113 showed no activity in control experiments against
phosphatase, and did not affect absorbance readings (Fig. S3, ESI). This provided
further confirmation that 113 is a genuine LgtC inhibitor. To assess the target profile
of 113 for LgtC, we also tested this inhibitor against two other GTs, the αꢀ(2,3)ꢀ
sialyltransferase from Pasteurella multocida (SiaT) and the human
fucosyltransferase 2 (FUT2). Although both GTs differ from LgtC in their donor
substrate specificity (Table 1), our assay can, in principle, also be used for these
GTs, as it is based on the generic detection of inorganic phosphate (Pi), irrespective
of the nature of the sugarꢀnucleotide donor from which it is released [6]. The
application of the assay with SiaT and FUT2 is, however, complicated by the
significantly higher Km value of these two enzymes for their respective donor (Table
1). In our standard protocol, LgtC inhibition experiments are carried out with UDPꢀ
Gal concentrations slightly above the donor Km. To carry out the inhibition assay with
SiaT and FUT2 under comparable conditions, significantly higher donor
concentrations were therefore required. This complicated the application of the
assay, as its linear range for the correlation between absorbance and analyte
concentration is limited to Pi concentrations below 25 ꢁM [6].
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Table 1
Comparison of LgtC, Pasteurella multocida SiaT, and human FUT2
LgtC
m donor (ꢁM) 16 [6]
SiaT
FUT2
K
430 [16]
440 [17]
CMPꢀNeu5Ac
βꢀlactose
7.5ꢀ9.0
197 [18]
108 [19]
GDPꢀFuc
αꢀlactose
7.5
18 [7]
Donor
Acceptor
Optimal pH
UDPꢀGal
βꢀlactose
7.0
To keep the final Pi concentrations within the linear range of the colorimetric
detection step, we therefore adapted our assay protocol. For GTs with a higher
donor Km, we introduced a dilution step after the enzymatic reaction, in order to bring
the final Pi concentration within the linear range of the assay. To assess if the
introduction of such a dilution step might affect inhibition assay results, we first
carried out a validation experiment with LgtC and the positive control CSG164. In
two separate sets of reaction mixtures, inhibitor, LgtC and βꢀlactose acceptor were
preꢀmixed with MnCl2, CEL, CIP and Triton Xꢀ100, and the reactions were started by
addition of UDPꢀGal donor. After 20 minutes incubation, one set of reaction mixtures
was analysed as is, while the other was diluted 5ꢀfold with buffer before detection.
Upon addition of Malachite Green reagents and recording of the absorbance at 620
nm, no significant difference in the percentage inhibition of LgtC was observed in the
diluted sample, compared to the standard protocol (Fig. S4, ESI). At 100 ꢁM,
CSG164 showed 60% inhibition of LgtC both in the standard protocol and upon 5ꢀ
fold dilution of the assay solution prior to addition of the Malachite Green reagents.
With a reliable dilution protocol in hand, 113 was tested against SiaT and
FUT2. 113, SiaT or FUT2, acceptor (βꢀlactose for SiaT, αꢀlactose for FUT2), metal
chloride (MgCl2 for SiaT, MnCl2 for FUT2), CEL, CIP and Triton Xꢀ100 were preꢀ
mixed, and the reaction was started by addition of the respective donor. The reaction
mixture was incubated for 20 mins and diluted as required. Finally, Malachite Green
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was added and the absorbance was recorded at 620 nm. Under these conditions,
113 did not reduce the enzymatic activity of SiaT, and had only a modest effect, at
the highest concentration, against human FUT2 (Fig. 3A & B). Thus, 113 displays a
concentrationꢀdependent inhibitory effect only against LgtC, as the direct comparison
of results against all three GTs shows (Fig. 3C).
Fig. 3 A: Enzyme activity of Pasteurella sialyltransferase SiaT in the presence of
pyrazolꢀ3ꢀone 113. B: Enzyme activity of human fucosyltransferase FUT2 in the
presence of 113. C: Direct comparison of inhibitory activities of 113 against LgtC,
SiaT and FUT2. Percentage inhibition values at 10 ꢁM, 25 ꢁM and 100 ꢁM of 113
are based on residual enzyme activity in the presence of inhibitor, and are extracted
from figures 2C (for LgtC), 3A (for SiaT) and 3B (for FUT2), respectively.
A
B
C
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Conclusion
In this study, we have screened a small collection of nonꢀsubstrateꢀlike inhibitor
candidates against the bacterial glycosyltransferase LgtC, using a biochemical
coupled assay. Our results demonstrate that this assay format can be adapted for
screening, and highlight several important features for such an application: we show
that despite the addition of surfactant to the assay mixture, false positives can arise
from interference of inhibitor candidates with the colorimetric Malachite Green
reagent, but also that these assay artefacts can be readily identified through
appropriate control experiments. Results in the pyrazolꢀ3ꢀone series demonstrate
that a singleꢀconcentration setꢀup is suitable for the identification of potent GT
inhibitors, which will facilitate screening of larger libraries.
The vast majority of GTs releases a nucleoside diphosphate (NDP) as their
secondary reaction product. Our assay is based on the detection of this generic
reaction product. Key elements of our experimental design, including the singleꢀ
concentration screening protocol we have established and validated, and our
process for elimination of false positives, are therefore directly transferable to many
other GTs, beyond the enzymes in this study. It is useful, in this context, that our
assay protocol can be readily adapted for GTs with a significantly higher Km (donor)
than LgtC, as is common in this enzyme family. We demonstrate that the limited
linear range of the colorimetric detection step can be overcome by introducing a
dilution step after the enzymatic reaction. This modification is important because it
allows the profiling of inhibitor candidates not only with regard to their potency, but
also their GT target selectivity.
5ꢀMethyl pyrazolꢀ3ꢀone 113, which was identified from this screen, is one of
the most potent nonꢀsubstrateꢀlike inhibitors against LgtC reported to date. SAR
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trends in this series suggest that the ortho position of the benzylidene substituent
offers interesting opportunities to modulate inhibitory activity. Nonꢀpolar substituents
in this position such as 2ꢀCH3 (113) and 2ꢀchloro (112) are advantageous, while a
polar group is not (128, 10% inhibition at 50 uM). We have recently shown that the
prototype inhibitor CSG164 reacts covalently, through its Michael acceptor system,
with a nonꢀcatalytic cysteine residue in the LgtC active site [15]. Small, nonꢀpolar
substituents at the ortho position of the benzylidene substituent may contribute to
orienting the Michael acceptor system correctly in the LgtC active site, for example
by acting as a steric block that restricts rotation about the benzylidene single bond.
These results therefore provide an exciting starting point for further optimisation of
this new class of nonꢀsubstrateꢀlike GT inhibitors.
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Acknowledgement
We thank King’s College for a King’s China Award (to Y.X.), the EPSRC National
Mass Spectrometry Facility, Swansea, for the recording of mass spectra, and Mr
Hugo Leung and Dr Jiangqian Jiang for assistance with the synthetic chemistry. The
LgtC plasmid was a gift by Dr Warren Wakarchuk (Toronto). M.E. was supported by
a visiting fellowship from the Japanese Patent Office.
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Experimental section
General. Steroids 1ꢀ90 and nucleosides 91ꢀ108 were selected from the Prestwick
Chemical Library (www.prestwickchemical.com). Nucleosides 109ꢀ110 were
synthesised as previously reported [20]. 5ꢀMethyl pyrazolꢀ3ꢀones 111ꢀ130 were
synthesized by Knoevenagel condensation of a given 5ꢀmethyl pyrazolꢀ3ꢀone
derivative and the requisite aldhehyde according to our recently reported protocol [15]
1
and fully characterised by H NMR, 13C NMR and mass spectrometry (ESI). LgtC
was expressed and purified as previously reported [9]. Recombinant αꢀ(2,3)ꢀ
sialyltransferase from Pasteurella multocida was purchased from Sigma.
Recombinant human fucosyltransferase 2 (FUT2) was purchased from R&D
Systems. Assays were carried out on 96ꢀwell plates, unless otherwise stated, and
analysed at 620 nm on a Polarstar Optima plate reader (BMG Labtech).
Inhibition experiments with LgtC.
Singleꢀconcentration screening assay. All concentrations are final concentrations.
LgtC was activated with 5 mM dithiothreitol (DTT) for 30 minutes at 30 °C. Aliquots
(15 ꢁL) of LgtC, MnCl2 (5 mM), chicken eggꢀwhite lysozyme (CEL, 1 mg/mL), calfꢀ
intestinal phosphatase (CIP, 10 U/mL), Triton Xꢀ100 (0.01%), and HEPES buffer (13
mM, pH 7.0) were combined with βꢀlactose acceptor (30 ꢁL, 2 mM) or buffer (30 ꢁL,
control). Inhibitor (50 ꢁM) in DMSO (15 ꢁL, 10% final DMSO concentration) or
DMSO only (15 ꢁL, control) was added. The reaction was started by addition of
UDPꢀGal donor (15 ꢁL, 28 ꢁM) and incubated for 20 mins at 30°C. Malachite Green
reagent A (30 ꢁL) was added, and the microplate was shaken carefully. Malachite
Green reagent B (30 ꢁL) was added, and the absorbance was recorded at 620 nm.
Each experiment was carried out at least in duplicate.
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Determination of IC50 values. The same protocol as for the screening assay was
used, but with each individual inhibitor at a range of concentrations from 0ꢀ100 ꢁM
(in DMSO, 10% final DMSO concentration).
Validation of the dilution step. All concentrations are final concentrations. LgtC was
activated with 5 mM dithiothreitol (DTT) for 30 minutes at 30 °C. Aliquots (15 ꢁL) of
activated LgtC, MnCl2 (5 mM), chicken eggꢀwhite lysozyme (CEL, 1 mg/mL), calfꢀ
intestinal phosphatase (CIP, 10 U/mL), Triton Xꢀ100 (0.01%), and HEPES buffer (13
mM, pH 7.0) were combined with βꢀlactose acceptor (30 ꢁL, 2 mM) or buffer (30 ꢁL,
control). CSG164 (100 ꢁM) in DMSO (15 ꢁL, 10% final DMSO concentration) or
DMSO only (15 ꢁL, control) was added. The reaction was started by addition of
UDPꢀGal donor (15 ꢁL, 28 ꢁM) and incubated for 20 mins at 30°C. Samples from
requisite wells were diluted 5ꢀfold with HEPES buffer (13 mM, pH 7.0) or used
without dilution (control). Malachite Green reagent A (30 ꢁL) was added to each well,
and the microplate was shaken carefully. Malachite Green reagent B (30 ꢁL) was
added, and the absorbance was recorded at 620 nm. The experiment was carried
out in duplicate.
Inhibition experiments with other GTs.
Sialyltransferase assay. All concentrations are final concentrations. Aliquots (15 ꢁL)
of the sialyltransferase, MgCl2 (5 mM), chicken eggꢀwhite lysozyme (CEL, 1 mg/mL),
calfꢀintestinal phosphatase (CIP, 15 U/mL), Triton Xꢀ100 (0.01%), and HEPES buffer
(13 mM, pH 8.0) were combined with βꢀlactose acceptor (30 ꢁL, 2 mM) or buffer (30
ꢁL, control). 113 (0ꢀ100 ꢁM) in DMSO (15 ꢁL, 10% final DMSO concentration), or
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DMSO only (15 ꢁL, control) was added. The reaction was started by addition of
CMPꢀNeu5Ac (15 ꢁL, 448 ꢁM), and incubated for 20 mins at 30°C. Reactions in
each well were diluted 10ꢀfold with the reaction mixture (5 mM MgCl2, 1 mg/mL CEL,
0.01% Triton Xꢀ100, 10% DMSO, 13 mM HEPES pH 8.0). Malachite Green reagent
A (30 ꢁL) was added, and the microplate was shaken carefully. Malachite Green
reagent B (30 ꢁL) was added, and the absorbance was recorded at 620 nm. The
experiment was carried out in duplicate.
Fucosyltransferase assay. All concentrations are final concentrations. Aliquots (15
ꢁL) of FUT2, MnCl2 (5 mM), chicken eggꢀwhite lysozyme (CEL, 1 mg/mL), calfꢀ
intestinal phosphatase (CIP, 10 U/mL), Triton Xꢀ100 (0.01%), and HEPES buffer (13
mM, pH 7.0) were combined with αꢀlactose acceptor (30 ꢁL, 2 mM) or buffer (30 ꢁL,
control). 113 (0ꢀ100 ꢁM) in DMSO (15 ꢁL, 10% final DMSO concentration), or DMSO
only (15 ꢁL, control) was added. The reaction was started by addition of GDPꢀFuc
(15 ꢁL, 200 ꢁM), and incubated for 20 mins at 30°C. Reactions in each well were
diluted 5ꢀfold with the reaction mixture (5 mM MnCl2, 1 mg/mL CEL, 0.01% Triton Xꢀ
100, 10% DMSO, 13 mM HEPES pH 7.0). Malachite Green reagent A (30 ꢁL) was
added, and the microplate was shaken carefully. Malachite Green reagent B (30 ꢁL)
was added, and the absorbance was recorded at 620. The experiment was carried
out in duplicate.
Control experiments.
Inhibition of phosphatase. All concentrations are final concentrations. Aliquots (15 ꢁL)
of MnCl2 (5 mM), chicken eggꢀwhite lysozyme (CEL, 1 mg/mL), Triton Xꢀ100 (0.01%),
and 60 ꢁL of HEPES buffer (13 mM, pH 7.0) were combined with calfꢀintestinal
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phosphatase (CIP, 15 ꢁL, 10 U/mL) or buffer (15 ꢁL, control). Inhibitor (50 ꢁM for
singleꢀconcentration experiments, 0ꢀ100 ꢁM for IC50 experiments) in DMSO (15 ꢁL,
10% final DMSO concentration) was added. The reaction was started by addition of
UDP (15 ꢁL, 6.25 ꢁM), and incubated for 20 minutes at 30°C. Malachite Green
reagent A (30 ꢁL) was added, and the microplate was shaken carefully. Malachite
Green reagent B (30 ꢁL) was added, and the absorbance was recorded at 620 nm.
Each experiment was carried out in duplicate.
Interference with colorimetric readꢀout. All concentrations are final concentrations.
Aliquots (15 ꢁL) of MnCl2 (5 mM), chicken eggꢀwhite lysozyme (CEL, 1 mg/mL), and
Triton Xꢀ100 (0.01%) were combined with HEPES buffer (75 ꢁL, 13 mM, pH 7.0).
Inhibitor (79 and 90: 1 mM; 113: 50 ꢁM) in DMSO (15 ꢁL, 10% final DMSO
concentration) or DMSO only (15 ꢁL, control) was added. The reaction was started
by addition of KH2PO4 (0ꢀ20 ꢁM, 15 ꢁL), and incubated for 20 minutes at 30°C.
Malachite Green reagent A (30 ꢁL) was added, and the microplate was shaken
carefully. Malachite Green reagent B (30 ꢁL) was added, and the absorbance was
recorded at 620 nm. The experiment was carried out in duplicate.
Synthesis and characterization of compound 113: (Z)ꢀ5ꢀmethylꢀ4ꢀ(2ꢀ
methylbenzylidene)ꢀ2ꢀphenylꢀ2,4ꢀdihydroꢀ3Hꢀpyrazolꢀ3ꢀone.
5ꢀMethylꢀ2ꢀphenylꢀ2,4ꢀdihydroꢀ3Hꢀpyrazolꢀ3ꢀone (174 mg, 1.0 mmol) and 2ꢀ
methylbenzaldehyde (240 mg, 2.0 mmol) were dissolved in glacial acetic acid. The
reaction mixture was stirred at 110ºC until TLC showed complete consumption of the
starting material. The target compound was obtained as an orange solid in 45% yield
(120 mg) after column chromatography. 1HꢀNMR (400 MHz, CDCl3, ppm) δ: 2.39 (s,
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3H), 2.51 (s, 3H), 7.20 (t, J = 7.4 Hz, 1H), 7.32 (m, 1H), 7.35 (d, J = 7.4 Hz, 1H),
7.38ꢀ7.47 (m, 3H), 7.74 (s, 1H), 7.96 (d, J = 7.7 Hz, 2H), 8.65 (d, J = 7.6 Hz, 1H).
13CꢀNMR (100 MHz, CDCl3, ppm) δ: 13.3, 20.2, 118.9, 124.8, 126.0, 127.7, 128.8,
130.5, 130.9, 131.9, 132.6, 138.3, 139.4, 144.3, 150.7, 161.7. ESIꢀMS: m/z 277.1
(100%) [M+H]+; HRꢀMS: m/z 277.1339 [M+H]+, 277.1335 calculated for [C18H17N2O]+.
Conflict of interest
The authors declare no competing interests.
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ToC
5-Methyl pyrazol-3-ones are genuine, non-substrate-like inhibitors of the
bacterial glycosyltransferase LgtC, but aminosteroids are not