Synthesis, kinetics and inhibition of Escherichia coli Heptosyltransferase I by monosaccharide analogues of Lipid A

Article abstract of DOI:10.1016/j.bmcl.2018.01.040

Gram-negative bacteria comprise the majority of microbes that cause infections that are resistant to pre-existing antibiotics. The complex cell wall architecture contributes to their ability to form biofilms, which are often implicated in hospital-acquired infections. Biofilms promote antibiotic resistance by enabling the bacteria to survive hostile environments such as UV radiation, pH shifts, and antibiotics. The outer membrane of Gram-negative bacteria contains lipopolysaccharide (LPS), which plays a role in adhesion to surfaces and formation of biofilms. The main focus of this work was the synthesis of a library of glycolipids designed to be simplified analogues of the Lipid A, the membrane embedded portion component of LPS, to be tested as substrates or inhibitors of Heptosyltransferase I (HepI or WaaC, a glycosyltransferase enzyme involved in the biosynthesis of LPS). Fourteen analogues were synthesized successfully and characterized. While these compounds were designed to function as nucleophilic substrates of HepI, they all demonstrated mild inhibition of HepI. Kinetic characterization of inhibition mechanism identified that the compounds exhibited uncompetitive and mixed inhibition of HepI. Since both uncompetitive and mixed inhibition result in the formation of an Enzyme-Substrate-inhibitor complex, molecular docking studies (using AutoDock Vina) were performed, to identify potential allosteric binding site for these compounds. The inhibitors were shown to bind to a pocket formed after undergoing a conformational change from an open to a closed active site state. Inhibition of HepI via an allosteric site suggest that disruption of protein dynamics might be a viable mechanism for the inhibition of HepI and potentially other enzymes of the GT-B structural class.

Full text of DOI:10.1016/j.bmcl.2018.01.040

Accepted Manuscript
Synthesis, kinetics and inhibition ofEscherichia coliHeptosyltransferase I by
monosaccharide analogues of Lipid A
Noreen K. Nkosana, Daniel J. Czyzyk, Zarek S. Siegel, Joy M. Cote, Erika A.
Taylor
PII:
S0960-894X(18)30051-9
https://doi.org/10.1016/j.bmcl.2018.01.040
BMCL 25566
DOI:
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
24 October 2017
17 January 2018
21 January 2018
Please cite this article as: Nkosana, N.K., Czyzyk, D.J., Siegel, Z.S., Cote, J.M., Taylor, E.A., Synthesis, kinetics
and inhibition ofEscherichia coliHeptosyltransferase I by monosaccharide analogues of Lipid A, Bioorganic &
Medicinal Chemistry Letters (2018), doi: https://doi.org/10.1016/j.bmcl.2018.01.040
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Synthesis, kinetics and inhibition of Escherichia coli Heptosyltransferase I by
monosaccharide analogues of Lipid A†
Noreen K. Nkosana, Daniel J. Czyzyk, Zarek S. Siegel, Joy M. Cote and Erika A. Taylor*
Department of Chemistry, Wesleyan University, Middletown, CT, 06459
*Corresponding Author : Department of Chemistry, Wesleyan University, 52 Lawn Ave., Hall-
Atwater Laboratories, Room 142, Middletown, CT 06459, United States; Tel: 860-685-2739.
Fax: 860-685-2211; E-mail: eataylor@wesleyan.edu.
†This work was supported by NIH grants 1R15AI119907-01.

Highlights
• Alkylated monosaccharides inhibit Heptosyltransferase I
• Compounds are uncompetitive with sugar acceptor substrate
• Compounds have mixed inhibition with sugar donor substrate
• Docking revealed putative allosteric inhibition pocket
Keywords
LPS; Lipopolysaccharide; Heptosyltransferase; Glycosyltransferase; GT-B; Inhibitor Design;
AutoDock Vina; Inhibition Kinetics; Structural Rearrangement; Allosterism
Abbreviations
glycosyltransferase (GT), heptosyltransferase I (HepI), L-glycero-D-manno-heptose (Heptose),
ADP- L-glycero-D-manno-heptose (ADP-Heptose), lipopolysaccharide (LPS), O-deacylated E.
coli Kdo2-LipidA (ODLA), fully-deacylated E. coli Kdo2-LipidA (FDLA)
Graphical abstract
Allosteric
Inhibition
Site

Abstract
Gram-negative bacteria comprise the majority of microbes that cause infections that are resistant
to pre-existing antibiotics. The complex cell wall architecture contributes to their ability to form
biofilms, which are often implicated in hospital-acquired infections. Biofilms promote antibiotic
resistance by enabling the bacteria to survive hostile environments such as UV radiation, pH
shifts, and antibiotics. The outer membrane of Gram-negative bacteria contains
lipopolysaccharide (LPS), which plays a role in adhesion to surfaces and formation of biofilms.
The main focus of this work was the synthesis of a library of glycolipids designed to be
simplified analogues of the Lipid A, the membrane embedded portion component of LPS, to be
tested as substrates or inhibitors of Heptosyltransferase I (HepI or WaaC, a glycosyltransferase
enzyme involved in the biosynthesis of LPS). Fourteen analogues were synthesized successfully
and characterized. While these compounds were designed to function as nucleophilic substrates
of HepI, they all demonstrated mild inhibition of HepI. Kinetic characterization of inhibition
mechanism identified that the compounds exhibited uncompetitive and mixed inhibition of HepI.
Since both uncompetitive and mixed inhibition result in the formation of an Enzyme-Substrate-
inhibitor complex, molecular docking studies (using AutoDock Vina) were performed, to
identify potential allosteric binding site for these compounds. The inhibitors were shown to bind
to a pocket formed after undergoing a conformational change from an open to a closed active site
state. Inhibition of HepI via an allosteric site suggest that disruption of protein dynamics might
be a viable mechanism for the inhibition of HepI and potentially other enzymes of the GT-B
structural class.

As the number of drug resistant (and multiple drug resistant) bacteria continue to
increase, the need for novel drug targets becomes increasingly pressing.1 The outer membrane
(OM) of Gram-negative bacteria provides a crucial layer of protection against many antibiotics.2
Bacterial lipopolysaccharide (LPS), also known as endotoxin, is a major component of the OM.
LPS is important for interactions with other cells, as in its interaction with human
lipopolysaccharide binding protein to trigger a TLR-4 immune response,3-5 and it maintains
structurally similar architecture throughout Gram-negative bacteria (but with variation of the
specific sugars incorporated) regardless of the ecological niche or growth conditions of the
bacteria.6 LPS consists of three domains – a glycolipid known as Lipid A, which is covalently
linked to the core oligosaccharide, which is, in turn, linked to a polysaccharide known as the O-
antigen (Figure 1).7, 8 All three components of LPS are required for the virulence of Gram-
negative bacteria, but only Lipid A is required for survival of the bacteria. Studies have shown
that Lipid A is the minimal LPS structure required for growth of E. coli and other Gram-negative
bacteria.9, 10
Heptosyltransferase I (HepI, also known as WaaC) is a member of the GT-B structural
family and it is involved in the synthesis of the inner core of LPS.11 The enzyme is known to
catalyze the addition of the first L-glycero-D-manno-heptose (Hep) to the inner 3-deoxy-D-
manno-oct-2-ulosonic acid (Kdo) residue of the Kdo2-Lipid A molecule in LPS.9, 11-13 HepI is an
inverting glycosyltransferase, which utilizes a direct-displacement reaction mechanism.11, 14-16
HepI has been observed to undergo conformational changes associated with substrate binding
and like other GT-B enzymes it is predicted to undergo an open-to-closed conformational
transition before catalysis.11, 15-19

Gal
Gal
NGa
NGa
NGc
n = 4 - 40
Gal
Gal
O-antigen
NGa
NGc
NGc
NGa
Glc
Gal
Core oligosaccharide
Gal
Glc
Hep
Hep
Hep
O
P
O-
O
O
O
Cell exterior
P
+H3N
O
O
P
O
O-
KDO
KDO
O
KDO
+
NH3
O
O-
O
P
O
O
O
HO
HO
O
O
O
-O
O
O
NH
Cell interior
NH
O
O
O P
O
O
O
OH
O-
O
HO
HO
Lipid-A
Figure 1: Representative structure of LPS from E. coli. (Hep) L-glycero-D-manno-heptose;
(Gal) galactose; (Glc) glucose; (KDO) 3-deoxy-D-manno-oct-2-ulosonic acid; (NGa) N-acetyl-
galactosamine; (NGc) N-acetyl-glucosamine. Modified from Petsch and Anspach.20

Previous studies in E. coli, Campylobacter jejuni and other bacteria have shown that HepI
knockouts can produce viable mutant cell lines that synthesize a truncated LPS that lack Heptose
residues (and the subsequent sugars) and display a deep-rough phenotype.9, 11-13, 21 Increased
sensitivity to hydrophobic antibiotics, detergents and bile salts and decreased virulence for
pathogenic Gram-negative bacteria have been associated with this rough phenotype.9, 21, 22 Based
on these observations, HepI has been the target for drug design from multiple labs.23-26 No efforts
to date have identified potent inhibitors of HepI, leading our group to investigate the overall
chemical and dynamical mechanism.17, 27, 28
HepI has been shown to efficiently utilize a number of Kdo2-Lipid A analogue molecules
as acceptor substrates (nucleophiles), including a partially deacylated (ODLA) and a fully
deacylated (FDLA) Kdo2-Lipid A molecule.28 These findings raised questions on the diversity of
substrate nucleophiles that would be tolerated by HepI. In this work, a series of acylated
monosaccharide molecules were designed as Lipid A analogues (Figure 2) in the hope of
identifying a minimal substrate. The alkyl chains of the analogues were expected to mimic the
acyl chains of the Lipid-A molecule, while the sugar residues mimic the Kdo residues of Lipid
A. From a stereochemical perspective, galactose analogues were predicted to be the best
nucleophiles because they share the stereoconfiguration of Kdo at the C-4 position.

OH
HO
OH
B.
HO
OH
OH
O
O
HO
OH
HO
O
OH
O
O
O
O
R
HO
OH
O
O
A.
OH
O
O
HO
O
!
- glucose/galactose analogues
β-glucose/galactose
analogues
2-O
OPO3
O
NH
NH
O
2-
OPO3
O
O
O
O
HO
HO
O
C.
OH
O
HO
HO
OH
O
R
″
- glucose/galactose analogues
α-glucose/galactose
analogues
E. coli Kdo2-lipid A
Figure 2: Structural comparison of E. coli Kdo2-Lipid A (A) and Lipid A analogues (B and C).
The sugar residues of the analogues are labeled in red and they are expected to mimic the Kdo
residues of Kdo2-Lipid A, also labeled in red. The alkyl chains of the analogues are expected to
mimic one of the acyl chains of Kdo2-Lipid A. One of the acyl chains in A is also labeled in blue.
The synthesis of these compounds builds off of the literature on the synthesis of allyl glycosides,
with further functionalization of these compounds through metatheses of a series of 1-alkenes of
different length tails. Fourteen alkylated sugar compounds were successfully synthesized using
glucose and galactose as starting materials (Figure 3 and S1, S2). Each of the syntheses began
with per-acetylation of the sugar starting material in reactions of modest yield (47 % and 53 %
30
for Glucose pentaacetate (15) and galactose pentaacetate (16), respectively).29,
After
acetylation, nucleophilic bromide was used to displace acetate from the C-1 position using two

Figure 3: Complete list of simplified Lipid A analogues.
slightly different methods previously published by Rodebaugh and Fraser-Reid, and by Ella-
Menye, for the glucose (17) and galactose (18) forms, respectively.30, 31 Using the α-bromo sugar
substrates, two different sets of allylation conditions could be used to afford product with either
retention or inversion of configuration at the anomeric position. β-allyl glucose tetraacetate (19)
and β-allyl galactose tetraacetate (20) were synthesized according to a method by Talley et al.
after unsuccessful attempts using methods of Rodebaugh and Fraser-Reid and Yoon et al.31,32,33
The α-allyl glucose tetraacetate (21) and α-allyl galactose tetraacetate (22) were synthesized
following the protocol by Lemieux and Morgan,34 which used 17 or 18, 2,4,6-trimethylpyridine,

tetrabutylammonium bromide and ethanol to achieve displacement of the anomeric bromine by
the allylic alcohol nucleophile.
Various 1-alkenes (1-decene, 1-octene, 1-heptene and 1-hexene) were used for metathesis
reactions to elongate the allyl-containing alkyl chains in both glucose and galactose analogues.
The β-metathesis syntheses involved the reaction between compounds 19 - 22 and the
corresponding 1-alkene in the presence of Grubbs catalyst using the methods published by
Plettenburg et al.35 The 2nd generation Grubbs catalyst was used for reactions with 1-decene and
1-octene whereas the 1st generation Grubbs was used for all metatheses with 1-heptene and 1-
hexene. The α-metathesis reactions were performed using the same methods, however, the
reactions that were performed using the 2nd generation Grubbs catalyst produced very low yields.
Use of the 1st generation Grubbs catalyst significantly improved yields, perhaps due to less steric
crowding than with the 2nd generation Grubbs catalyst.
Deacetylation of the α- and β- metathesis products was performed successfully using a
3:3:1 solution of methanol, water, and triethylamine.35 The structures of all compounds were
13
confirmed by 1H, COSY, and C NMR. Additionally, compounds 1-12, 23-26 and 31- 34 were
1
13
also analyzed by electro-spray mass spectrometry (ES-MS). The H, COSY, and C NMR, as
well as the ES-MS spectra are shown in Supplemental Materials.
Once synthesized, all compound were first screened to determine if they could be used as
substrates by HepI. Having previously determined that HepI can utilize a tetrasaccharide as its
acceptor substrate (nucleophile), we wanted to ascertain if a monosaccharide could also be
utilized as a substrate.28 Prior to testing the alkylated monosaccharides, galactose and glucose as
well as α- and β-methyl glycosides were first used to see if the deacylated monosaccharides
could serve as a substrate. None of these commercial compounds promoted the HepI

glycosyltransferase reaction. The compound library, synthesized above, were then examined, and
there was also no detectable HepI activity for any of these compounds (data not shown).
Retrospectively, this is not surprising because multiple GTs, like many enzymes, had previously
16, 36
demonstrated high substrate utilization fidelity.15,
While glucose and galactose are
somewhat similar to KDO, these sugars lacks a carboxylic acid as well as C7 and C8 positions.
Additionally, glucose and galactose are both hydroxylated at the C2 position while KDO is
dehydroxylated at the corresponding ring carbon (the C2 position in glucose or galactose
corresponds to C3 in KDO).
The molecules were then screened to determine if they could inhibit HepI catalysis with
the substrates, ODLA (the partially deacylated Kdo2-Lipid A) and ADP-Heptose. Each molecule,
to some extent, showed inhibition of HepI activity (Table 1). Interestingly, side by side
comparison of each glucose compound with its galactose analogue revealed that in every case
except for the octene-β-glycoside, the glucose compounds demonstrated a higher level of
inhibition than the galactose compounds. Also of interest, the commercially available detergent
n-Octyl-β-D-glucopyranoside also showed a small degree of inhibition of HepI. Ki values were
determined for all compounds that exhibited inhibition ≥ 20% (Table 1). The smallest Ki values
were in the high micromolar range, with the best alkylated monosaccharide hexene-β-D-
glucopyranoside (7) giving rise to a Ki of 340 µM. D-glucose and methyl-α-D-glucopyranoside
demonstrated similar

Compound
% Inhibition
28.5
8.8
Ki (µM)
500 260
D-galactose
-
-
methyl-α-D-galactopyranoside
methyl-β-D-galactopyranoside
allyl-β-D-galactopyranoside
hexene-β-D-galactopyranoside
heptene-β-D-galactopyranoside
octene-α-D-galactopyranoside
octene-β-D-galactopyranoside
decene-α-D-galactopyranoside
s-β-D-galactopyranoside
7
-
10
14
8
22.6
7.8
570 240
6
8.8
12
4
6.3
NA
5
10
2
36
280 200
370 160
680 370
650 410
D-glucose
-
27.8
19.5
19.6
8.3
methyl-α-D-glucopyranoside
methyl-β-D-glucopyranoside
allyl-β-D-glucopyranoside
n-Octyl-β-D-glucopyranoside
hexene-β-D-glucopyranoside
heptene-β-D-glucopyranoside
octene-α-D-glucopyranoside
octene-β-D-glucopyranoside
decene-α-D-glucopyranoside
decene-β-D-glucopyranoside
-
-
13
-
29.7
11.9
16.7
4.2
340 150
7
5
11
3
NA
23.4
9
520 280
1
Table 1: Inhibition parameters for modified monosccharide compound library

levels of inhibition. All of these compounds bind with lower affinity than the substrates, which
have Km values between 3-10 µM.11, 27, 28 Since compound 7 was the acylated monosaccharide
with the lowest Ki, it was used to determine if these compounds are competitive, noncompetitive,
or uncompetitive inhibitors with either the donor (ADP-Heptose) or the acceptor (ODLA)
substrates of HepI. Inhibition by 7 displayed noncompetitive (mixed-competitive) inhibition
against ADP-Heptose as indicated by the decrease in Vmax as inhibitor concentration increases
and a characteristic double reciprocal plot (Figure 4A) where lines intersect at a point not on the
y-axis. A noncompetitive inhibitor by definition has binding affinity for both free enzyme and
enzyme substrate (ES) complex. The equation describing this inhibition, v =
vmax[S]ks(1+[I]ki)+[S](1+[I]αki), includes an additional term α used to distinguish if an
inhibitor has a preference for either free enzyme (α>1), ternary complex (α<1) or no preference
(α=1).37 As inhibitor concentration increases, the changes in Km (increase, decrease or
unaffected) can be used to distinguish if α is >1, <1, or =1, respectively. As concentration of 7
increase, Km decreases, thus α<1, suggesting that 7 prefers to bind to the ES complex (Table 2).
Figure 4: Lineweaver-Burk plots for compound 7 a) against ADP-Heptose and b) against
ODLA. Each point represents an average of a triplicate set of data.

ADP-Heptose
ODLA
[Inhibitor], µM
KM (µM)
kcat (s-1)
0.76 0.08
1.6 0.1
KM (µM)
kcat (s-1)
0.9 0.2
1000
100
10
38 10
135 19
277 40
65 27
108.3 0.6
114 4
1.3 0.004
1.4 0.03
2.8 0.3
Table 2: Kinetics parameters for compound 7.
Unlike ADP-Heptose, inhibition by 7 against ODLA resulted in a double reciprocal plot with
parallel lines diagnostic of uncompetitive inhibition; this is also evident by the decrease in both
Km and vmax but no change in vmax/Km (Figure 4B and Table 2). The uncompetitive inhibition
profile means that 7 binds exclusively to the ES complex when ODLA concentrations are varied.
Since inhibition experiments with both substrates suggest a preference for these compounds to
bind to the ES complex, this suggests that these compounds are binding to a putative allosteric
site in HepI.
To gain a better idea as to where the compounds bind on HepI, dockings of these alkylated
monosaccharides were conducted using AutoDock Vina v1.1.2. The library of compounds were
docked into (1) the “open” apo HepI structure (2GT1.pdb), (2) the “open” HepI•ADP-Heptose
complex structure (2H1H.pdb) and (3) a “closed” apo model, previously developed.1717 In
addition, four additional protein-ligand complexes were evaluated, where each contained one of
the substrate molecules (ADP-Heptose bound to the C-terminal domain or FDLA, the fully
deacylated Kdo2-Lipid A analogue which by virtue of the removal of the acyl chains is an easier
compound to use for docking, was docked into the N-terminal domain) complexed with the apo
form of 2H1H and the closed model of HepI. All dockings were performed within four grid

boxes: (a) containing the entire protein, (b) the part of the active site contacted by Lipid A (the
N-terminal domain), (c) the part contacted by ADP-Heptose, and (d) the entire active site (Table
S1). The active site boxes were designed as to include the entire ligand and any residues within
five Å of the ligand in question. Dockings to these structures were performed to assess the effect
of interactions with the endogenous substrates and observe any differences in binding that might
occur when the compounds are physically displaced from either binding site.
The average energies of the alkylated monosaccharides were generally in the range of −4.5
to −6 kcal/mol, with binding energy minimums only slightly lower (Tables S2 and S3). There
was a definite trend in energies that shorter-chained alkylated monosaccharides had somewhat
weaker binding affinities. It was also notable that the control dockings of substrates showed a
significantly higher affinity than any of the inhibitors; ADP-Heptose was the tightest binding
ligand (average energy: −8.0 kcal/mol; minimum energy: −8.9 kcal/mol), while FDLA also
showed lower affinity than the library compounds (average energy: −7.2 kcal/mol; minimum
energy: −8.4 kcal/mol). This observation is unsurprising as all compounds were already shown
experimentally to have lower affinity than the Lipid A analogues.
The binding energies and binding sites of the ligands bound are highly conserved
regardless of the structure to which they were docked. Figure S1 shows the general patterns for
docking to 2H1H. The majority of ligands, when docked, bound to the ADP-Heptose site.
Interestingly, this trend is unaffected by whether the starting protein PDB contained one of the
substrates. The results were substantially different when the protein used was the closed model
of HepI. Perhaps most significantly, a putative allosteric site was identified. This site, no
analogue of which exists in 2H1H or 2GT1, is a “tunnel” between the ADP-Heptose binding site
and the linker region between the N- and C-terminal domains of HepI, which forms during the

Figure 5: Putative Allosteric site in the closed model of HepI. When viewed from the side
with the “surface” view activated, the allosteric site appears as a tunnel between the linker
region (at the right side) and the spade between the two domains. No such gap exists in
the structures of 2H1H or 2GT1.
simulatated closure of HepI (where upon the two domains coming together to bring the
substrates proximal; Figure 5). The site was identified when super imposing images of dockings
to this closed structure showed significant clustering of docking poses in this site. It was
therefore included in the binding site analyses alongside the ADP-Heptose and FDLA sites.
Generally, longer-chain ligands preferentially bound in the allosteric site and shorter-chain
ligands bound more in the ADP-Heptose site, e.g. decene-α-D-glucopyranoside with 95 poses in
the ADP-Heptose site, 9 in FDLA site, and 340 in the allosteric site, versus D-galactose with 188
poses in the ADP-Heptose site, 1 in the FDLA site, and 81 in the allosteric site. The substrates,
understandably, defied these trends, with nearly 100% of FDLA poses binding in both the ADP-
Heptose and FDLA sites, and a negligible number in the allosteric site. ADP-Heptose bound
primarily (∼75%) in the ADP-Heptose site, with the remainder being split approximately half in
each the FDLA and allosteric sites.

Additionally, the putative allosteric site is also significant in understanding the role of
conformational change in the dynamics of HepI. Although there is no crystal structure of a
ternary HepI complex, the existence of such a conformational change is supported by analogous
changes in related GT-B enzymes.38, 39 Previous dynamic studies on HepI, have suggested that a
17, 27 28, 29
conformational change is induced by the binding of Lipid A.
Although the alkylated
monosaccharides were designed with the idea of creating a substrate or competitive inhibitor to
bind and displace Lipid A, the docking data suggest that they may primarily inhibiting the ES
complex in an uncompetitive (e.g. by binding to and somehow altering the closed conformation
such that catalysis in impeded) and/or mixed-competitive (both competitive and uncompetitive)
manner. These findings are consistent with the kinetics data, since both non-competitive and
uncompetitive inhibition was observed. Thus, the putative allosteric site represents a compelling
future direction in understanding the inhibition of HepI and other GT-B structural enzymes.
In conclusion, this work demonstrates through experimental and computational studies that
the library of compounds herein bind to HepI in a previously undescribed allosteric pocket. For
enzymes that undergo conformational changes during catalysis, this analysis highlights that the
disruption of protein dynamics can be an important mechanism for the design of inhibitors.
While these inhibitors were not particularly potent, it is understandable that inhibitors for this
allosteric site wouldn’t necessarily have the same structural features as the substrates. This result
is important for consideration when trying to inhibit HepI, GT-B structural enzymes, as well as
other enzymes that undergo conformational changed during chemistry.

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Allosteric
Binding Site
Graphical abstract

Highlights
• Alkylated monosaccharides inhibit Heptosyltransferase I
• Compounds are uncompetitive with sugar acceptor substrate
• Compounds have mixed inhibition with sugar donor substrate
• Docking revealed putative allosteric inhibition pocket