Design, synthesis, and evaluation of peptide-imidazo[1,2-a]pyrazine bioconjugates as potential bivalent inhibitors of the VirB11 ATPase HP0525

Article abstract of DOI:10.1002/psc.3353

Helicobacter pylori (H. pylori) infections have been implicated in the development of gastric ulcers and various cancers: however, the success of current therapies is compromised by rising antibiotic resistance. The virulence and pathogenicity of H. pylori is mediated by the type IV secretion system (T4SS), a multiprotein macromolecular nanomachine that transfers toxic bacterial factors and plasmid DNA between bacterial cells, thus contributing to the spread of antibiotic resistance. A key component of the T4SS is the VirB11 ATPase HP0525, which is a hexameric protein assembly. We have previously reported the design and synthesis of a series of novel 8-amino imidazo[1,2-a]pyrazine derivatives as inhibitors of HP0525. In order to improve their selectivity, and potentially develop these compounds as tools for probing the assembly of the HP0525 hexamer, we have explored the design and synthesis of potential bivalent inhibitors. We used the structural details of the subunit–subunit interactions within the HP0525 hexamer to design peptide recognition moieties of the subunit interface. Different methods (cross metathesis, click chemistry, and cysteine-malemide) for bioconjugation to selected 8-amino imidazo[1,2-a]pyrazines were explored, as well as peptides spanning larger or smaller regions of the interface. The IC₅₀ values of the resulting linker-8-amino imidazo[1,2-a]pyrazine derivatives, and the bivalent inhibitors, were related to docking studies with the HP0525 crystal structure and to molecular dynamics simulations of the peptide recognition moieties.

Full text of DOI:10.1002/psc.3353

Received: 8 January 2021
DOI: 10.1002/psc.3353
Revised: 11 May 2021
Accepted: 13 May 2021
R E S E A R C H A R T I C L E
Design, synthesis, and evaluation of peptide-imidazo[1,2-a]
pyrazine bioconjugates as potential bivalent inhibitors of the
VirB11 ATPase HP0525
James R. Sayer1,2 | Karin Walldén3,4 | Hans Koss1,5
| Helen Allan1
|
Tina Daviter6,7 | Paul J. Gane8,9
| Gabriel Waksman3 | Alethea B. Tabor1
1Department of Chemistry, UCL, London, UK
2MedPharm Ltd, Guildford, UK
Helicobacter pylori (H. pylori) infections have been implicated in the development of
gastric ulcers and various cancers: however, the success of current therapies is com-
promised by rising antibiotic resistance. The virulence and pathogenicity of H. pylori
is mediated by the type IV secretion system (T4SS), a multiprotein macromolecular
nanomachine that transfers toxic bacterial factors and plasmid DNA between bacte-
rial cells, thus contributing to the spread of antibiotic resistance. A key component of
the T4SS is the VirB11 ATPase HP0525, which is a hexameric protein assembly. We
have previously reported the design and synthesis of a series of novel 8-amino
imidazo[1,2-a]pyrazine derivatives as inhibitors of HP0525. In order to improve their
selectivity, and potentially develop these compounds as tools for probing the assem-
bly of the HP0525 hexamer, we have explored the design and synthesis of potential
bivalent inhibitors. We used the structural details of the subunit–subunit interactions
within the HP0525 hexamer to design peptide recognition moieties of the subunit
interface. Different methods (cross metathesis, click chemistry, and cysteine-
malemide) for bioconjugation to selected 8-amino imidazo[1,2-a]pyrazines were
explored, as well as peptides spanning larger or smaller regions of the interface. The
IC50 values of the resulting linker-8-amino imidazo[1,2-a]pyrazine derivatives, and
the bivalent inhibitors, were related to docking studies with the HP0525 crystal
structure and to molecular dynamics simulations of the peptide recognition moieties.
3Institute of Structural and Molecular Biology,
UCL, London, UK
4Department Biochemistry and Biophysics,
Stockholm University, Solna, Sweden
5Department of Biochemistry and Molecular
Biophysics, Columbia University, New York,
New York, USA
6Department of Biological Sciences, ISMB
Biophysics Centre, London, UK
7The Institute of Cancer Research, London, UK
8Wolfson Institute for Biomedical Research,
UCL, London, UK
9Abcam, Cambridge, UK
Correspondence
Alethea B. Tabor, Department of Chemistry,
UCL, 20, Gordon Street, London WC1H 0AJ,
UK.
Email: a.b.tabor@ucl.ac.uk
Present address
James R. Sayer, MedPharm Ltd, R&D Centre,
Unit 3/Chancellor Court, 50 Occam Road,
Surrey Research Park, Guildford, GU2 7AB, UK
Karin Walldén, Department Biochemistry and
Biophysics, Stockholm University,
Tomtebodavägen 23A, Solna, SE-171 65,
Sweden
K E Y W O R D S
antimicrobial, ATPase inhibitor, bivalent inhibitor, docking, protein–protein interaction (PPI)
Hans Koss, Department of Biochemistry and
Molecular Biophysics, Columbia University,
701 West 168th Street, New York, NY, 10032,
US
Tina Daviter, The Institute of Cancer Research,
237 Fulham Road, London, SW3 6JB, UK
Paul J. Gane, Abcam, Discovery Drive
Biomedical Campus, Cambridge, CB2 0AX, UK
This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium,
provided the original work is properly cited.
© 2021 The Authors. Journal of Peptide Science published by European Peptide Society and John Wiley & Sons Ltd.
J Pep Sci. 2021;e3353.
wileyonlinelibrary.com/journal/psc
1 of 13
https://doi.org/10.1002/psc.3353

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SAYER ET AL.
Funding information
Wellcome Trust, Grant/Award Numbers:
082227, 096617/Z/11/Z; Biotechnology and
Biological Sciences Research Council, Grant/
Award Number: BBS/SE/2006/1326/9
1
|
INTRODUCTION
to act as a molecular switch that controls the export of DNA and the
assembly of the T4SS pilus. Hence, there has been much interest in
the structure and function of this protein. There have been extensive
studies into the crystal structures of the H. pylori VirB11 homolog
HP0525 bound to both ADP23 and the non-hydrolysable ATPγS as
well as the nucleotide-free, apo-form.24 HP0525 forms double
hexameric ring structures where each subunit monomer consists of
328 amino acid residues comprising the N-terminal domain (NTD) and
C-terminal domain (CTD). Each of the NTDs and CTDs form two sepa-
rate ring structures (Figure 1a) with the CTD ring forming a six-clawed
grapple mounted on the hexameric ring formed by the NTD creating a
cylindrical chamber. The structures of ADP- and ATPγS-HP0525 are
virtually identical; however, apo-HP0525 exists as an asymmetric
hexamer, very different to that of the nucleotide bound forms. Exami-
nation of the crystal structures of HP0525 showed that nucleotide
binding and not hydrolysis is responsible for ATP-induced conforma-
tional changes23,24 and allowed a mechanism for the mode of action
of the ATPase to be proposed (Figure 1b).
Since its isolation in 1983,1 Helicobacter pylori (H. pylori) has been
identified as the most common human bacterial infection, present in
approximately half of the world's population.2 This type of Gram-
negative bacteria is found in the human stomach and causes illnesses
such as gastric ulcers, gastritis, and various cancers including mucosa-
associated lymphoid tissue (MALT)-lymphoma and gastric adenocarci-
noma.3,4 Although the majority of those infected are asymptomatic,
H. pylori-positive patients have a 10%–20% lifetime risk of developing
ulcer disease and a 1%–2% risk of developing gastric cancer,5 and as
such, the bacteria have been classified as a category 1 carcinogen.6 It
has been estimated that between 2008 and 2015, the proportion of
noncardiac gastric cancer attributable to H. pylori increased from
74.7% to 89.0%.7 The current standard treatment of H. pylori infec-
tions is based upon triple therapy,8,9 consisting of a proton pump
inhibitor and a choice of two antibiotics or quadruple therapy8–10 in
which bismuth compounds are also used. The success of these thera-
pies is unfortunately under pressure due to rising antibiotic resistance
and off-target effects caused by prolonged antibiotic treatment.11
Multi-drug resistant H. pylori (resistance to ≥3 antibiotics of different
classes) ranges from ≤10% in Europe to >20% in India and >40% in
Peru.12 So far, no eradication therapy can provide high eradication
rates (>90%), and a variety of approaches to targeting H. pylori includ-
ing antivirulence therapeutics, mucolytic agents, and antibacterial
agents are currently being investigated.13
Selective inhibitors of VirB11 have the potential to combat the
proteins and pathways that lead to pathogenic, symptomatic coloniza-
tion of the stomach, and may also be useful chemical biology tools to
elucidate the pathways of assembly of the T4SS macromolecular com-
plex. However, only a small number of small molecule inhibitors of
H. pylori HP0525 have so far been reported.15 Thiadiazolidine-3,-
5-diones26 and the non-competitive inhibitor 4-(5-methylpyridin-2-yl)
oxybenzoic acid27 have been shown to inhibit VirB11 ATPase,
whereas heterocyclic 2-pyridone inhibitors28 have been shown to
disrupt T4SS apparatus biogenesis by attenuating the delivery of
peptidoglycan and CagA to host cells. Unsaturated 2-alkynoic fatty
acids have also been shown to inhibit conjugation in TrwD, a VirB11
homolog.29
Gram-negative bacteria have evolved a range of secretion sys-
tems to transport substrates across their cell membranes. They can
release small molecules, proteins, and DNA into extracellular space or
can inject these substrates into a target cell.14 Seven classes of double
membrane-spanning secretion systems, Type I–Type VII, have so far
been identified. The Type IV secretion system (T4SS) is of particular
interest, as it mediates the transfer of plasmid DNA between bacterial
cells, thus contributing to the spread of antibiotic resistance genes.
Inhibitors of the T4SS are therefore of interest as antimicrobial agents
with the potential to slow the development of antimicrobial
resistance.15
H. pylori can be grouped into two classes,16,17 and the more viru-
lent type I strains contain the cytotoxin-associated genes pathogenic-
ity island (cagPAI)18 and are referred to as CagA+ strains. The cagPAI
consists of 31 genes, the majority of which code for T4SS,5,16 which
in H. pylori is responsible for penetrating the gastric epithelial cells and
facilitating the translocation of toxic bacterial factors into host
cells.19–21 T4SS are multifunctional macromolecular nanomachines,
incorporating 12 different types of protein subunit with specific roles
in the complex.22 The VirB11 ATPase HP0525 is a key component of
this complex, which provides energy to power the system and is
required to drive CagA secretion and delivery. It is also hypothesized
We have previously reported the synthesis and high throughput
screen of a series of novel 8-amino imidazo[1,2-a]pyrazine derivatives
against VirB11 ATPase HP0525.30 Biochemical evaluation showed
moderate to good potency, highlighting these compounds as competi-
tive inhibitors of HP0525 and potential antibacterial agents. However,
as these molecules probably bind at the ATPase active site, to avoid
inhibiting other ATPases in mammalian cells, we wished to try to
improve their selectivity for VirB11 ATPase. We were also motivated
to exploit the potential of these compounds as chemical biology tools
for probing the assembly of the HP0525 hexamer. In order to achieve
both of these aims, in this paper, we have explored the design and
synthesis of potential bivalent inhibitors of the VirB11 ATPase
HP0525.
Affinity and specificity of an inhibitor can be greatly enhanced by
taking advantage of hydrophobic/hydrophilic features near the
enzyme active site. By linking an active site binding compound to a
moiety that interacts with these features on the enzyme surface, it is

SAYER ET AL.
3 of 13
FIGURE 1 (A) Illustration of the three major contacts in the VirB11 ATPase HP0525 subunit–subunit interface. Blue: Subunit A (dark
blue = NTD, light blue = CTD), green: Subunit B (dark green = NTD, light green = CTD); 1: NTD-NTD interactions, 2: NTD-CTD interactions, 3:
CTD-CTD interactions. Image generated using chimera.25 (B)4hematic representation for the mode of action of VirB11 ATPases. NTD (light pink),
CTD (light blue). ATP binding (blue) and ADP (yellow). In 1 (nucleotide-free form), the CTD maintains the pseudo-scaffold and the NTD is mobile.
Binding of three ATP molecules then locks three of the subunits into a rigid conformation (state 2). Hydrolysis of the first three ATPs to ADP
together with binding of a further three ATP molecules to the remaining subunits leads to state 3. Hydrolysis of the final ATP molecules leads to a
rigid hexamer (state 4). Finally, release of the six ADPs allows the hexamer to revert to its nucleotide-free asymmetric form (state 1). Reproduced
with permission (The EMBO Journal) from Savvides et al.24
possible to differentiate among enzymes. The resulting compounds
are known as bivalent inhibitors. Bivalent molecules comprising a
small molecule and a peptide have been frequently studied, as this
approach has been particularly valuable in designing selective kinase
inhibitors,31 in which an ATP-binding site directed small molecule is
tethered to a protein substrate site of the protein kinase. Examples of
potent and selective bivalent inhibitors include ATPγS-IRS727 peptide
bioconjugates as insulin receptor protein tyrosine kinase (IRK) inhibi-
tors32 and high-affinity nonselective kinase inhibitor K252a- protein
kinase inhibitor protein (PKI) bioconjugates as inhibitors of protein
kinase A (PKA).33 Bivalent inhibitors can also be designed to selec-
tively disrupt protein–protein interactions (PPIs).34
characterizations of all other compounds are reported in the
supporting information.38
2.2
|
Solid phase peptide synthesis
Amino acids and resins for peptide synthesis were purchased from
Novobiochem, UK. HPLC grade solvents for peptide synthesis and
HPLC purification were purchased from Sigma, UK and VWR. All
peptides were synthesized on a MultiSynTech Syro I automated
system. Either pre-loaded Fmoc-Asp (OtBu)-NovaSyn® TGT resin
(0.200 mmol/g, 100 mg, 20.0 μmol) or pre-loaded Fmoc-Leu-NovaSyn®
TGT resin (0.210 mmol/g, 100 mg, 21.0 μmol) were used in all cases.
All resins were pre-swelled in DMF for at least 30 min prior to
synthesis start. Standard Fmoc solid phase peptide synthesis (SPPS)
was employed. The total volume of all reagents in each step was 1.5 ml.
All reagents were dissolved in HPLC grade DMF.
In this work, we have used the imidazo[1,2-a]pyrazine com-
pounds previously synthesized to target the ATP binding site of
HP0525, whereas a peptide sequence has been rationally designed to
disrupt the peptide–peptide interactions of the subunit–subunit inter-
face and, in doing so, disrupting hexamer formation.
2
|
MATERIALS AND METHODS
2.2.1
|
Fmoc removal
2.1
synthesis
|
General methods, reagents, and chemical
The reaction syringe containing N-terminal Fmoc-protected peptide
was added piperidine in DMF (40% v/v, 1.5 ml). The mixture was
agitated for 20 s every min for a total of 3 min. The reagents were
removed by filtration under vacuum, and the resin washed with
DMF (4 Â 1.5 ml). Piperidine in DMF solution (40% v/v, 0.75 ml)
was added to the reaction syringe followed by DMF (0.75 ml) to
make an overall 20% v/v solution of piperidine in DMF. This
mixture was agitated for 20 s every min for a total of 10 min. The
General methods for chemical synthesis are included in the
supporting information. All chemicals were of commercial quality
and have been used without additional purification; 19,35 20,36 21,37
and 2530 were synthesized according to literature procedures.
The synthetic procedures, purification methods, and compound

4 of 13
SAYER ET AL.
reagents were removed by filtration under vacuum, and the resin
using Star Chromatography Workstation software Version 1.9.3.2.
ES-MS analysis was performed on a Waters Acquity Ultra Perfor-
mance LC/MS machine. MALDI analysis was carried out using a
Waters MALDI MICRO MX, Micromass Technologies.
washed with DMF (6 Â 1.5 ml).
2.2.2
|
Amino acid coupling
Analytical HPLC traces, ES+, and MALDI spectra of peptides 1–9
can be found in the supporting information.
The reaction syringe was added Fmoc-protected amino acid
(0.600 ml, 0.140 M in DMF, 4 eq.), HBTU (0.600 ml, 0.140 M in DMF,
4 eq.), and DIPEA (0.300 ml, 0.560 M in DMF, 8 eq.). The mixture was
agitated for 20 s every 3 min for a total of 40 min. The reagents were
removed by filtration under vacuum, and the resin washed with DMF
(4 Â 1.5 ml).
2.3
|
Conjugation
2.3.1
|
Cysteine-maleimide conjugation (solution)
The purified peptide was taken up in DMF (approximately 2 mg/ml).
An aliquot of a stock solution of 17 in DMF (2.87 mM) was added
such that the volume added corresponded to 3 eq, and the reaction
mixture stirred at RT for 3 h. The solvent was removed in vacuo and
the crude material purified via reverse phase preparative HPLC.
2.2.3
|
Peptide cleavage and side chain
deprotection
The resin was washed with CH2Cl2 (3 Â 3 ml), MeOH (3 Â 3 ml) and
Et2O (3 Â 3 ml) and dried (desiccator) followed by adding TFA/EDT/
H2O/TIPS (88:5:4:2; 3 ml) to the reaction syringe. The syringe was
then agitated for 3 h at RT. The cleavage cocktail was drained from
the vessel under vacuum and Et2O (ꢀ10–15 ml) added to the filtrate.
The resultant precipitate in solution was stored at À20ꢁC for 30 min
prior to being spun at 4000 rpm for 10 min at 4ꢁC to produce a crude
peptide pellet. The supernatant Et2O was decanted off, and the
peptide washed a further three times with Et2O. The crude peptide
pellet was then re-dissolved in minimum water and freeze-dried for
storage prior to purification.
2.3.2
|
“Click” chemistry (on resin)
The peptides were synthesized according to standard SPPS, with all
side chain and the terminal Fmoc protecting groups left on. The
peptide was also left bound to the resin, and all subsequent reactions
were carried out in the syringe; 164 (43.3 mg, 0.080 mmol, 4 eq) in
DMF (2 ml) was added to the reaction syringe followed by the addi-
tion of a suspension of CuI (76.0 mg, 20 eq) and sodium ascorbate
(158 mg, 40 eq) in H2O/tBuOH (2:1; 750 μl) to give a total solvent
composition of DMF/H2O/tBuOH (8:2:1). The mixture was agitated
at RT for 16 h, followed by evacuating the solvent and washing with
DMF (3 Â 3 ml), MeOH (2 Â 3 ml), CH2Cl2 (3 ml), and DMF
(2 Â 3 ml). The terminal Fmoc was removed, followed by cleavage of
the peptide from the resin and deprotection of the side-chain
protecting groups using conditions stated above.
2.2.4
|
General peptide purification
The peptides were analyzed and purified via reverse phase HPLC
using a Varian ProStar system with a Model 210 solvent delivery
module and a Model 320 UV detector. The preparative purification
was performed using either a Discovery®BIO Wide Pore C18 (Varian;
100 Â 21.2 mm, 5 μm beads, flow rate of 10 ml/min), Discovery®BIO
Wide Pore C18 (Varian; 25 cm  21.2 mm, 10 μm beads, flow rate of
8 ml/min) or an Onyx Monolithic Semi-Prep C18 (Phenomenex®;
100 Â 10 mm, 2 μm macropore size, 13 nm mesopore size, flow rate
10 ml/min), loaded with 200–400 μl aliquots of a 10–20 mg/ml solu-
tion of peptide dissolved in 0.1% TFA containing H2O. The mobile
phase was a decreasing gradient of H2O/0.1% TFA (A) in MeCN/0.1%
TFA (B). Precise gradients are reported for each peptide. The fractions
containing the correct peak were pooled, and the solvent was
removed under reduced pressure to approximately 2 ml, and the
solution freeze-dried.
2.3.3
|
Cysteine-maleimide conjugation (on resin)
The peptides were synthesized according to standard SPPS, with all
side chain and the terminal Fmoc protecting groups left on. The
peptide was also left bound to the resin, and all subsequent
reactions were carried out in the syringe. The StBu protecting group
on R240C was selectively deprotected by: washing the resin-bound
peptide with CH2Cl2 (3 Â 3 ml); soaking in EtOH/CH2Cl2/H2O (4:6:1;
3 ml); purging with Ar; adding nBu3P (50.0 μl, 0.200 mmol, 10 eq); and
agitating for 3 h at RT. The syringe was evacuated and washed with
CH2Cl2 (2 Â 3 ml), MeOH (2 Â 3 ml), CH2Cl2 (2 Â 3 ml), and DMF
(2 Â 3 ml). Maleimide conjugation to the free thiol in R240C was
carried out by adding 5 (42.0 mg, 0.060 mmol, 3 eq) in DMF (3 ml)
and agitating at RT for 16 h. The syringe was evacuated and washed
with DMF (3 Â 3 ml). The terminal Fmoc was removed, followed by
cleavage of the peptide from the resin and deprotection of the
side-chain protecting groups using conditions stated above.
The purified peptide was analyzed by analytical HPLC using an
Discovery®BIO Wide Pore C18 (Varian; 25 cm  4.6 mm, 10 μm
beads, flow rate of 1 ml/min) or Onyx monolithic C18 column
(Phenomenex®; 100 Â 3.0 mm,
2 μm macropore size, 13 nm
mesopore size, flow rate 1.0 ml/min). Precise gradients reported for
each peptide. The analysis of the chromatograms was conducted

SAYER ET AL.
5 of 13
Analytical HPLC traces, ES+, and MALDI spectra of conjugated
peptides 28, 29, and 30 can be found in the supporting information.
2.5
|
Molecular modelling
AutoDock 4.040 and Vina41 were used for the in silico docking of the
compounds into .pdb file: 1G6O (ADP-HP0525). All heteroatoms
(H2O, PEG, and ADP molecules) were removed from the .pdb file,
which was converted into .pdbqt (includes partial charges and
autodock atom types) format using AutoDock Tools (ADT), with the
addition of polar hydrogens and Kollman charges. AutoDock 4.0
requires the following files: enzyme.pdbqt, ligand.pdbqt, GridFile.gpf
(grid parameter file), and DockingFile.dpf (docking parameter file).
AutoDock Vina requires the enzyme.pdbqt, ligand.pdbqt, and configu-
ration.txt files. ADT was used to generate the ligand.pdbqt file from a .
pdb or .mol2 input, which was subjected to no initial energy minimiza-
tions. When using PyRx (Open Source software with intuitive user
interface for docking), the program automatically converts .pdb or .
mol2 files to .pdbqt files. The size of the grid box used is as follows:
x (14 Å), y (16 Å), and z (24 Å). Coordinates of the grid box used for
the docking into sites A (À40.36, 53.652, and 22.3) and B (À12.034,
24.627, and 22.363) of 1G6O. In the case of AutoDock 4.0, the grid
file and docking file were generated using ADT. In the case of
AutoDock Vina, the coordinates were used in the configuration text
file, and a value of 8 was used for the exhaustiveness. Linux was used
to run the AutoDock 4.0 program, and either cmd or PyRx was
used to run AutoDock Vina. The output file from 4.0 was in .dlg for-
mat (docked log files), which can be converted to .pdbqt using ADT
and viewed in PyMOL. In order to visualize the docking file using
chimera, the file needs to be converted further to the .pdb, using
PyMOL. Vina exports the docking files as .pdbqt.
2.4
|
Biological assays
The HP0525 protein was produced in the E. coli strain BL21 Star
(DE3) (Invitrogen) as described previously.39 The protein concentra-
tion was estimated spectroscopically using a NanoDrop (Thermo
Scientific) and a calculated extinction coefficient at 280 nm, based on
the amino acid composition. The ATPase activity of HP0525 was
measured, with and without a specific amount of compound present,
using an in vitro ATPase colorimetric assay kit (Innova Biosciences).
The assay was performed in 96-well ELISA microplates (Greiner
Bio-One), using a multipipette/robot.
A volume of 49 μl of a substrate/buffer solution (200 mM
tris(hydroxymethyl)aminomethane [TRIS], pH 7.5; 5 mM MgCl2;
250 μM ATP; and 10% DMSO) was added to each assigned well,
followed by the addition of 1 μl of compound (at 0.5; 5 or 50 mM to
achieve the final concentrations of 5; 50 and 500 μM in the reaction,
respectively) in DMSO (or 1 μl DMSO to controls). The solutions were
mixed carefully by pipetting. The reaction was started by the addition
of 50 μl of 0.106 μM HP0525 to each well (except the negative con-
trol, see text below), and the reaction plate was directly transferred to
37ꢁC for 30 min of incubation. The reaction was stopped by the addi-
tion of the gold mix according to the standard protocol of the kit. The
absorbance at 620 nm was measured after 30 min at RT. For each
compound, the percentage of absorbance relative to non-inhibited
HP0525 was calculated, after subtracting the absorbance value of the
negative control. In the negative control, the protein was added after
the gold mix, as described in the standard protocol of the kit, which
when used as a blank corrects for all free Pi not produced by the
enzyme during the 30 min incubation at 37ꢁC. A known inhibitor of
HP0525 (CHIR02)26 was used as a control inhibitor. All measurements
were made in duplicate.
MOE 2009 was used for evaluation of the bifunctional
reagents.42 The R240 residue was mutated to the relevant residue,
and the molecule was built using the “Build Molecule” function. For
example, select residue, add H, convert to C, add H, convert to C,
add H, and so forth.
The software package GROMACS v. 4.5.4 was used for
molecular dynamics simulations.43 Three peptide fragments from
the HP0525 crystal structure24 1NLZ were extracted as .pdb
structure files:
A selection of the compounds was assayed as above at additional
concentrations ranging between 1 and 50 μM (the measuring points
were optimized to cover the range to fit a sigmoidal dose response
curve, and at the same time ensure compounds were not precipitat-
ing) from which IC50 values were calculated (Figure S1). Each
compound was screened on three separate plates, and each plate was
read twice, and a mean was calculated for each concentration. The
data were normalized by subtracting the negative control (0% active)
and relating it to the positive control (100% active). The software
GraphPad Prism 5 was used to generate two dose–response curves
(Log [inhibitor] vs. normalized response [standard and variable slope]),
and the IC50 values were calculated from each.
1. αF: subunit F - residues 229–242
2. αF-β10 subunit F - residues 229–246
3. β9-αF-β10 subunit F - residues 218–246
The dynamics of these peptides were simulated in the following way:
the peptide structures were converted into
a model with an
AMBER99SB-ILDN43 forcefield. The peptide was then placed in a
40 Å Â 40 Å Â 40 Å box. After energy minimization (steepest
descent, dt = 0.002 ps, 20,000 steps), the system was filled with SPC-
water. Water molecules were recursively substituted by NaCl until c
(NaCl) = 0.15 M. The system was minimized again using the same
conditions as mentioned above. The actual atomistic molecular
dynamics simulation of this system was performed on a desktop
computer with two cores using the following conditions:
The same assay format was used for the enzyme kinetics
experiments, where the activity of HP0525 was tested at various
concentrations of ATP (0–500 μM). The data were plotted and fitted
to the Michaelis–Menten equation to obtain Vmax, from which Km can
be calculated.

6 of 13
SAYER ET AL.
duration = 10 ns; dt = 0.002 ps; temperature coupling: Berendsen
thermostat, T = 300 K, τT = 0.1 ps; pressure coupling: isotropic
Parinello–Rahman, p = 1.0 bar, τT = 1.0 ps, compressibility = 4.6Á10–
5 bar-1. The system had 6494/6492/6463 elements in αF/αFβ10/
β9-αF-β10. The resulting trajectory was progressively fit to the first
(centered) frame. VMD44 was used for visualization and for calculat-
ing Ramachandran plots.
3
|
RESULTS AND DISCUSSION
| Bivalent inhibitor design
3.1
To design bivalent inhibitors that would be selective for the
VirB11 ATPase, we sought to combine the structural insights gained
from the PPIs between the subunits of the hexameric HP0525
complex24 with the active site-directed small molecule inhibitors
previously reported.
2.6
|
Circular dichroism spectroscopy
The HP0525 monomer consists of two domains: the NTD from
residues 6 to 136, and the CTD from residues 137 to 328, linked
via a short loop between residues 134 and 141 (Figure 2a). The
nucleotide binding site is located between the NTD and CTD, with
residues from both domains contributing to the binding site. In the
hexameric structure of HP0525, each subunit interacts with two
flanking subunits, and NTD-CTD interactions make up the majority
of subunit–subunit interfaces. Most of the solvent accessible
surfaces of the β-sheet of the NTD make contact with residues of
the β9 sheet, αF helix, αF-β10 loop, and αE-β8 loop of the CTD
(Figure 2a). There is also a cluster of acidic and basic residues within
the NTD-CTD interface that plays a pivotal role in maintaining the
hexameric structure of HP0525. Glu47 from the NTD makes ionic
interactions with Arg238 from the adjacent αF-β10 loop in the CTD.
However, Arg113 and Arg133 from the NTD and Arg240 from the
αF-β10 loop in the CTD would clash electrostatically if it were not
for the presence of a nucleotide neutralizing these like-charges. In
the absence of nucleotide, these clashes would have a destabilizing
effect on the interface, causing the NTD to be released and swivel
into an open conformation.
The experiments were performed at the ISMB Biophysics Centre.
Circular dichroism spectra were measured with Jasco J-720
a
spectropolarimeter. Three different peptides were weighted first:
αF-loop 1 0.5 0.1 mg 14 residues, MW/residue = 113.9 g/mol;
αF-β10 2 0.4 0.1 mg 18 residues, MW/residue = 116.1 g/mol;
β9-αF-β10 3 1.2 0.1 mg 29 residues, MW/residue = 114.9 g/mol.
The peptides were dissolved in water; their CD spectra were
recorded thrice at 1 mg/ml (β9-αF-β10 3 at 0.5 mg/ml) in a Quartz
Suprasil cell (pathlength: 0.1 mm). Data were processed with
CDTool45 and Dichroweb46. Outliers were excluded, spectra
zeroed, averaged, and converted from millidegree units into Δε
units, which is a molar unit; the weighed amount of the sample is
important for this conversion. Given the weighting errors stated
above, data processing was performed for the corresponding range
of different sample concentrations. Different algorithms for analysis
of the results were tested in Dichroweb.44 CONTIN was chosen as
the data analysis algorithm. SP175_S3 was chosen as a reference
set (others were tested before making this choice). Given α-helical
content was calculated for different protein concentrations, the
results are provided as a range.
In order to design a mimic of the PPIs at the subunit interface, we
aimed to rationally design a peptide that resembles the αF helix and
FIGURE 2 (A) Crystal structure of ATPγS bound to HP0525 (1NLY), highlighting the targeted peptide region at the subunit–subunit interface.
ATPγS is in yellow.24 (B) Structure of the β9-αF-β10 peptide region showing the different secondary structures and the potential first point of
attachment for conjugation. Image generated using PyMOL45

SAYER ET AL.
7 of 13
neighbouring residues (Figure 2a). We envisaged that by anchoring
the small molecule in the nucleotide binding site, the peptide part of
the chimera would interact with the subunit–subunit interface, possi-
bly displacing the native segment and in the process disrupting
hexamer formation. The peptide sequence highlighted consists of the
β9 sheet followed by a loop into the αF helix followed by a further
loop into the β10 sheet (Figure 2b). It was unclear what length of pep-
tide would be required to interact with the native peptide region, and
therefore, three peptides with varying lengths were chosen for inves-
tigation: αF-loop 1, αF-β10 2, and β9-αF-β10 3 (Table 1). Furthermore,
as Arg240 is in close proximity to the nucleotide binding site, this
seemed to be the ideal site for conjugation to the imidazo[1,2-a]
pyrazine partner. By substituting this arginine for other amino acids
capable of conjugating to other moieties, a range of bivalent inhibitors
could be synthesized. We therefore prepared variants of these three
peptides with Arg240 substituted by Cys (4, 5, 6), S-allyl cysteine
(SAC) (7, 8, 9), or azidolysine (AzLys) (10, 11, 12).
region. A sulfonamide group is also a common feature of this series of
inhibitors, and in 13, docking indicated that this sulfonamide moiety
might occupy the phosphate binding region, pointing towards the
adjacent subunit and the center of the hexameric chamber.30 This
suggested that this region of these inhibitors might be an appropriate
point for conjugation to the peptide recognition partner. Furthermore,
modifications to this region of the inhibitors by attachment of a linker
presented an opportunity to further explore the structure–activity
relationships. We elected to modify the toluoyl moiety by adding a
carbamate group and then linking the inhibitor to the peptide via an
intermediate PEG chain. We reasoned that this would be more stable
than an ester in vivo and would provide more hydrogen bonding
possibilities in the phosphate binding/subunit–subunit interface
region than an amide linkage. This in turn might potentially increase
the potency and selectivity of this series of inhibitors. Our previous
docking studies of another inhibitor in this series, 14, had suggested
that in this case, the sulfonamide points away from the phosphate
region and out into the extra pocket (Figure S2). This leads to a differ-
ence in binding affinity and accounts for the drop in potency com-
pared with 13. However, the synthetic route to 14 was more direct
and higher yielding than the route to 13 and easier to adapt for the
preparation of carbamate-linked bioconjugates. Moreover, the
addition of the carbamate-PEG linker was expected to afford
bioconjugates of a similar length and spacing. We therefore elected to
use 14 as the base imidazo[1,2-a]pyrazine unit for initial investigation
into bivalent HP0525 inhibitors.
We have previously reported a number of small molecule inhibi-
tors of HP0525, based on the imidazo[1,2-a]pyrazine scaffold
(Figure 3), and carried out molecular docking studies on selected
structures to elucidate their interactions with the active site.30
Docking studies of one of the best inhibitors, 13, suggested that this
series of inhibitors are deeply buried within the enzyme active site.
Within the ATP-binding site, the naphthalene ring occupies the purine
binding region towards the solvent side (outside) of the enzyme, with
the core imidazo[1,2-a]pyrazine moiety occupying the ribose binding
TABLE 1 Table indicating the amino
acid sequence for each of the target
peptides
Peptide
αF-loop
αF-β10
Sequence
WT
1
R240C
R240SAC
R240AzLys
SADCLKSCLRMRPD
4
5
6
7
8
9
10
11
12
SADCLKSCLRMRPDRIIL
YTQLFFGGNITSADCLKSCLRMRPDRIIL
2
β9-αF-β10
3
Note: Letters in red indicate an α-helix; blue indicates a β-sheet; black indicates turns and loops; R240 is
highlighted in yellow.
Abbreviations: AzLys, azidolysine; SAC, S-allyl cysteine.
FIGURE 3 Structure of previously developed inhibitors
and carbamate PEG linker design

8 of 13
SAYER ET AL.
Three of the most common approaches for peptide conjugation
3.2.1
|
Small molecule linker
to the PEG chain were considered. Cross metathesis46 with the
R240SAC peptides 7, 8, 9 incorporating S-allyl cysteine could be
carried out with 15. Click chemistry47 requires azido lysine-
substituted peptidesR240AzLys 10, 11, 12 and could be achieved
using alkyne 16. Finally, maleimide coupling48 could be carried out
between the cysteine-substituted peptides 4, 5, 6 (R240C) and the
maleimide 17. The length of the PEG chain is also an important
factor with the design of the bivalent inhibitor reagents.
Preliminary docking studies suggested that a single PEG unit should
be of sufficient length to extend from the carbamate linked to the
toluoyl moiety, to the peptide sequence that will make interactions
with the adjacent subunit.
The synthesis of the allyl and alkyne linked PEGylated imidazo[1,2-a]
pyrazines (15, 16) is shown in Scheme 1. The PEG-linker was attached
using a Curtius rearrangement of 18 (synthesized from the commer-
cially available acid precursor)49 with PEG-linkers 19, 20, 21 to give
22, 23, or 24, respectively. The imidazo[1,2-a]pyrazine fragment
(25)30 was then incorporated using nucleophilic aromatic substitution.
The Buchwald–Hartwig coupling used previously in the synthesis of
14, to couple the sulfonamide portion to the chloro-imidazo[1,2-a]
pyrazine fragment,30 was not suitable due to the presence of the
alkene and alkyne.
PEGylated-maleimide 17 was synthesized in an analogous manner
to the alkyne and alkene linkers. Direct attachment of the maleimide
to the PEG chain with an ethyl linker was unsuccessful, so an elon-
gated carbamate structure was synthesized. The Buchwald–Hartwig
coupling was low yielding due to the poor solubility of 24 (7% yield).
Again, switching to nucleophilic displacement conditions (NaH and
DMF) gave the desired product (26) in 57% yield. Removal of the Boc
group afforded the free amine 27, which was isolated as the TFA salt
as decomposition was observed when the salt was basified. Finally,
coupling of 27 to N-maleoyl-β-alanine gave target maleimide 17.
Thus, our bivalent inhibitor reagents were designed to consist of
a small molecule inhibitor, based on imidazo[1,2-a]pyrazine, that will
bind to the nucleotide binding site, linked (via a PEG chain) to a
peptide, based on the αF helix of HP0525, that could then substitute
the native αF helix and disrupt the opening and closing mechanism of
the hexameric portal (Figure 1b).
3.2
|
Synthesis
There were a number of possible approaches to the synthesis of these
bivalent inhibitors. We adopted the strategy of first synthesizing the
imidazo[1,2-a]pyrazine inhibitor, attached via a carbamate linker to a
PEG chain primed with the conjugation moiety. These PEGylated-
imidazo[1,2-a]pyrazines would then be regioselectively attached to a
specific residue on the target peptides.
3.2.2
|
Peptide synthesis
Initially, it was envisaged that chemoselective bioconjugation of the allyl
(15) and maleimide (17) linked PEGylated imidazo[1,2-a]pyrazines to
appropriately substituted peptides would be carried out in solution.
SCHEME 1 Synthetic route to PEGylated-imidazo[1,2-a]
pyrazines 15, 16, and 17

SAYER ET AL.
9 of 13
Peptides were therefore synthesized (Table 1) using Fmoc SPPS
(Fmoc-Asp (OtBu)-NovaSyn®TGT and Fmoc-Leu-NovaSyn®TGT
resins), incorporating either cysteine (4, 5, 6) or S-allyl cysteine
(SAC) (7, 8, 9) in the R240 position. Peptides 1, 2, and 3,
corresponding to the native sequence, were also synthesized in order
to establish whether they adopted the predicted secondary structures.
In the case of R240C peptides a non-acid labile protecting group
(acetamidomethyl - Acm)50,51 was used on the other cysteine residues
so that the peptide produced has only one thiol free for maleimide
conjugation.
followed by standard Fmoc removal and deprotection/resin
cleavage. This procedure was successful for the (αF-loop peptide,
giving 28 (Figure 4), but unfortunately no product was isolated
when the longer peptides (αF-β10 and β9-αF-β10) were used.
Despite trialling
a
range of conditions, the on-resin cross
metathesis of R240SAC peptides and 15 was unsuccessful.
Greater success was seen with the alkyne tethered imidazo[1.2-α]
pyrazine 16. The three desired peptides (αF-loop 10, αF-β10 11, and
β9-αF-β10 12) were synthesized on resin with azido lysine at the
R240 position. The click reaction between R240AzLys peptides and
16 was carried out using excess CuI (20 eq) and sodium ascorbate
(40 eq) in DMF/H2O/tBuOH (8:2:1) at room temperature for 16 h.
After Fmoc removal and resin cleavage, both the conjugated αF-loop
(29) and αF-β10 (30) peptides were isolated (Figure 4). However,
no product could be isolated from the click reaction between
resin-bound β9-αF-β10 12 and 16.
3.2.3
|
Bivalent inhibitor synthesis
Before attempting to conjugate malemide 17 to the target
peptides, reaction conditions were optimized using a model system.
Reaction of Fmoc-Cys-OH with hexaethyleneglycol malemide in
DMF gave the desired conjugated product in 81% yield. When
these conditions were applied to the conjugation of 17 with
peptides 4, 5, and 6, MALDI analysis indicated that the desired
conjugates had formed (see supporting information). However, not
enough material was isolated after purification to continue with
3.3
|
Biological results
Initially, the activity of the PEGylated-imidazo[1,2-a]pyrazines were
tested for inhibition of HP0525 using an in vitro ATPase colorimet-
ric assay.30 The IC50 data are shown in Table 2. PEGylated-imidazo
the Acm protecting group removal, and
a large amount of
unreacted 17 was recovered. Cross metathesis conditions were
developed using S-allyl cysteine and 2-(allyloxy)ethanol (Grubbs' II
t
catalyst52 [6 mol%] in BuOH/H2O at 32ꢁC for 3 h, 100% conver-
TABLE 2 IC50 data for the PEGylated imidazo[1,2-a]pyrazines
synthesized
sion). However, when these conditions were applied to 15 and
peptides 7 and 8, no desired product (or starting peptides) was
isolated. In order to overcome these difficulties, it was decided to
conjugate the PEGylated imidazo[1.2-a]pyrazines to the peptides
on resin. The full length peptides were synthesized as before, but
without the removal of the final Fmoc protecting group or global
TFA deprotection/resin cleavage. In contrast to the solution
phase synthesis, a trityl protecting group was used on the other
cysteine residues, in place of the Acm group so that global
deprotection would occur in the resin cleavage step. For the
malemide coupling, the tBu protecting group on R240C was
selectively removed using PBu3 in EtOH/CH2Cl2/H2O (4:6:1) for
3 h at RT.53 Conjugation of the free cysteine to 17 was then
achieved by agitating in DMF at room temperature for 16 h,
Compound
PEG chain (X)
IC50 (μM)
14
15
Carbamate replaced by CH3
88
46
16
27
17
61
137
18
FIGURE 4 Structure of peptide conjugates synthesized

10 of 13
SAYER ET AL.
[1,2-a]pyrazines 15, 16, and 17 show improved inhibition of
HP0525 compared with the parent compound 14. In order to
establish whether the extra H-bonding possibilities provided by the
carbamate moiety (and in the case of 17, the amide and maleimide
groups) had led to greater interactions with the active site, we car-
ried out further docking studies (Figure 5). Indeed, when studying
the in silico lowest energy binding modes within the nucleotide
binding site, they appear to adopt a conformation similar to that of
13 rather than 14. When looking at 17 (Figure 5A), the molecule
shifts so that the carbamate mimics the same sulfonamide interac-
tions as in 13; the sulfonamide is now in the ribose region, and
the imidazo[1,2-a]pyrazine is located in the purine region. This
means that the naphthalene moiety extends further into the
enzyme active site, possibly picking up the further π-stacking
interactions; 15 and 16 have similar IC50 values, and this can
be attributed to similar conformations observed within the
nucleotide binding site (Figure 5B,C). It is interesting that 27 shows
poor inhibition because in silico studies indicate similar
conformations as 15 and 16 (Figure 5D), and therefore, a similar
activity might have been expected.
3.3.1
|
Bivalent inhibitor reagents
The three peptide-imidazo[1,2-a]pyrazine conjugates (28, 29, 30)
were tested at concentrations of 5, 50, and 500 μM, and the % absor-
bance at 620 nm analyzed to give an indication of their inhibitory
effect (Table 3). The results clearly indicate that the peptide
conjugates only inhibit very weakly at a high concentration.
3.3.2
|
Wild-type peptides
The stability of the peptide fold might be important for both activity
and reactivity. In silico stability testing on the three WT peptides using
molecular dynamics showed only that the β9-αF-β10 peptide 3 was
FIGURE 5 In silico images of 13 (red) and ATPγS (yellow) in ATPγS-HP0525 with (A) 17, (B) 15, (C) 16, (D) 27. PDB: 1NLY. Image generated
using PyMOL45
TABLE 3 Percentage absorbance values for each of the peptide conjugates at 500, 50, and 5 μM
% absorbance (620 nm)
Entry
Peptide conjugate
500 μM
93
50 μM
100
5 μM
100
100
100
1
2
3
28
29
30
91
100
90
100

SAYER ET AL.
11 of 13
FIGURE 6 Smoothed CD spectra for the
three different peptides, indicating the level of
α-helicity: αF-loop 1: 6%–7%, αF-β10 2: 9%–10%,
β9-αF-β10 3: 8%–9%. Spectra were processed
with Dichroweb
stable to the simulations (Figure S3); the αF-loop peptide 1 unfolded
immediately, and the αF-β10 peptide 2 unfolded, but at a slower rate.
Circular dichroism (CD) spectroscopy, however, indicated that none of
the WT peptides synthesized form α-helices (Figure 6).
inhibitors. This was particularly disappointing, as functionalization of
the 8-amino imidazo[1,2-a]pyrazine fragment with the PEG-
carbamate linkers actually improved their inhibition relative to the
parent compound 14. One possible explanation for this is that the
linker between the 8-amino imidazo[1,2-a]pyrazine and the peptide
fragment has the wrong length and/or structure to correctly orient
the peptide fragment for binding to the next subunit. Whereas further
detailed docking studies might help to identify improved linkers, a
more powerful approach would undoubtably be to use a peptide
aptamer “Trojan Horse”57 or an enzyme-templated fragment elabora-
tion strategy,58,59 in which the enzyme itself selects the correct com-
bination of linker and peptide fragment for effective bivalent binding
from a small library. A second explanation is that the β9-αF-β10 seg-
ment of HP0525, and smaller fragments, do not fold in such a way as
to form effective mimics of the HP0525 subunit interface, or that this
segment cannot compete with the complete protein during assembly
of the hexamer. This is partly supported by our CD and MD studies.
Approaches such as helix stabilization by stapling60 might improve the
binding, although introduction of a constraint to preorganize peptides
to a particular secondary structure does not always increase the
binding potency to the target.61 Finally, peptide sequences with
N-terminal capping and C-terminal amide modifications should be
synthesized, in order to avoid introducing additional charges to the
sequence. All of these additional peptide modifications would then
need to be tested separately for their ability to fold in solution, access
the subunit–subunit interface, and to inhibit hexamer assembly. How-
ever, the initial studies presented in this paper will enable both an
enzyme-templated fragment elaboration strategy, and the pathway of
the HP0525 hexameric complex assembly, to be further studied.
4
|
CONCLUSION
Improving the selectivity profiles of ATPase or kinase inhibitors that
bind to the ATP binding site is crucial to avoiding toxicity due to
off-target effects in vivo. A bivalent inhibitor approach, whereby the
tight binding of ATP mimics is combined with targeting elements that
bind outside the ATP binding site, has considerable potential.
Designing a peptide or a small molecule that can mimic or disrupt PPIs
is challenging, and the parameters that will lead to a successful
inhibitor are not well-understood.54
In this work, we have succeeded in improving the activity of a
lead 8-amino imidazo[1,2-a]pyrazine inhibitor of the VirB11 ATPase
HP0525 by attachment of a PEG moiety, primed for bioconjugation.
We have established effective methodology for the bioconjugation of
the 8-amino imidazo[1,2-a]pyrazine-PEG to short peptides, via
either cysteine-maleimide reaction or alkyne/azide click chemistry. A
possible reason for the conjugation failing for the longer length
peptides could be the accessibility of the relevant amino acid. Where
the conjugation was carried out on the solid phase, the R240 amino
acid is close to the C-terminus and therefore close to the resin. If the
longer length peptides are extensively folded on-resin, the thiol, allyl,
or azide moieties might be considerably buried from their conjugation
partner. The use of a lower loaded resin,55 or of protecting groups
such as Hmb or pseudoproline moieties designed to disrupt folding on
resin of “difficult sequences,56 could be beneficial, as could using
alkyne/azide click chemistry for bioconjugation in solution.
ACKNOWLEDGEMENTS
The authors would like to thank the Biotechnology and
However, conjugation to peptide fragments designed to mimic or
disrupt the interactions between the subunits in the VirB11 ATPase
HP0525 hexameric complex did not result in effective enzyme
Biological Sciences Research Council for a PhD studentship
(BBS/SE/2006/1326/9) to J. S. We also thank the Wellcome Trust for
a research grant (082227) to K. W. and for a PhD studentship

12 of 13
SAYER ET AL.
16. Selbach M, Moese S, Backert S, Jungblut PR, Meyer T. The
Helicobacter pylori CagA protein induces tyrosine dephosphorylation
of ezrin. Proteomics. 2004;4(10):2961-2968. https://doi.org/10.
1002/pmic.200400915
(096617/Z/11/Z) to H. K. We also wish to thank Dr Frederick
Campbell for the synthesis of azido lysine.
ORCID
17. Blaser MJ, Berg DE. Helicobacter pylori genetic diversity and risk of
human disease. J Clin Invest. 2001;107(7):767-773. https://doi.org/
10.1172/JCI12672
Hans Koss
Helen Allan
Paul J. Gane
https://orcid.org/0000-0003-0493-169X
https://orcid.org/0000-0002-7186-4370
https://orcid.org/0000-0002-9153-5240
18. Tegtmeyer N, Wessler S, Backert S. Role of the cag-pathogenicity
island encoded type IV secretion system in Helicobacter pylori patho-
genesis. FEBS j. 2011;278(8):1190-1202. https://doi.org/10.1111/j.
1742-4658.2011.08035.x
Gabriel Waksman
Alethea B. Tabor
https://orcid.org/0000-0003-0708-2726
https://orcid.org/0000-0001-8216-0347
19. Bourzac KM, Guillemin K. Helicobacter pylori-host cell interactions
mediated by type IV secretion. Cell Microbiol. 2005;7(7):911-919.
https://doi.org/10.1111/j.1462-5822.2005.00541.x
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