New Antibiotic Candidates against Helicobacter pylori

Article abstract of DOI:10.1021/jacs.5b06110

Helicobacter pylori is a Gram-negative bacterium that colonizes the gut of over 50% of the worlds population. It is responsible for most peptic ulcers and is an important risk factor for gastric cancer. Antibiotic treatment for H. pylori infections is challenging as drug resistance has developed to antibiotics with traditional mechanisms of action. H. pylori uses an unusual pathway for menaquinone biosynthesis with 5′-methylthioadenosine/S-adenosylhomocysteine nucleosidase (MTAN) catalyzing an essential step. We validated MTAN as a target with a transition-state analogue of the enzyme [Wang, S.; Haapalainen, A. M.; Yan, F.; et al. Biochemistry 2012, 51, 6892-6894]. MTAN inhibitors will only be useful drug candidates if they can both include tight binding to the MTAN target and have the ability to penetrate the complex cell membrane found in Gram-negative H. pylori. Here we explore structural scaffolds for MTAN inhibition and for growth inhibition of cultured H. pylori. Sixteen analogues reported here are transition-state analogues of H. pylori MTAN with dissociation constants of 50 pM or below. Ten of these prevent growth of the H. pylori with IC₉₀ values below 0.01 μg/mL. These remarkable compounds meet the criteria for potent inhibition and cell penetration. As a consequence, 10 new H. pylori antibiotic candidates are identified, all of which prevent H. pylori growth at concentrations 16-2000-fold lower than the five antibiotics, amoxicillin, metronidazole, levofloxacin, tetracyclin, and clarithromycin, commonly used to treat H. pylori infections. X-ray crystal structures of MTAN cocrystallized with several inhibitors show them to bind in the active site making interactions consistent with transition-state analogues.

Full text of DOI:10.1021/jacs.5b06110

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Article
New Antibiotic Candidates Against Helicobacter pylori
Shanzhi Wang, Scott A. Cameron, Keith Clinch, Gary B.
Evans, Zhimeng Wu, Vern L. Schramm, and Peter C. Tyler
J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.5b06110 • Publication Date (Web): 23 Oct 2015
Downloaded from http://pubs.acs.org on October 25, 2015
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New Antibiotic Candidates Against Helicobacter
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pylori
Shanzhi Wang2, Scott A. Cameron2, Keith Clinch1*, Gary B. Evans1, Zhimeng Wu1, Vern L.
Schramm2*, and Peter C. Tyler1*
1The Ferrier Research Institute, Victoria University of Wellington, New Zealand
2Department of Biochemistry, Albert Einstein College of Medicine, New York 10461, USA
KEYWORDS: enzyme transition state, H. pylori, MTAN, futalosine pathway, menaquinone.
ABSTRACT
Helicobacter pylori is a Gram-negative bacterium that colonizes the gut of over 50% of the
world’s population. It is responsible for most peptic ulcers and is an important risk factor for
gastric cancer. Antibiotic treatment for H. pylori infections is challenging as drug resistance has
developed to antibiotics with traditional mechanisms of action. H. pylori uses an unusual
pathway for menaquinone biosynthesis with 5ʹ-methylthioadenosine/S-adenosylhomocysteine
nucleosidase (MTAN) catalyzing an essential step. We validated MTAN as a target with a
transition-state analogue of the enzyme [Wang, S., Haapalainen, A. M., Yan, F., et al.
Biochemistry 2012, 51, 6892-6894]. MTAN inhibitors will only be useful drug candidates if they
can include both tight binding to the MTAN target and have the ability to penetrate the complex
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cell membrane complex found in Gram-negative H. pylori. Here we explore structural scaffolds
for MTAN inhibition and for growth inhibition of cultured H. pylori. Sixteen analogues reported
here are transition-state analogues of H. pylori MTAN with dissociation constants of 50 pM or
below. Ten of these prevent growth of the H. pylori with IC90 values below 0.01 µg/mL. These
remarkable compounds meet the criteria for potent inhibition and cell penetration. As a
consequence, ten new H. pylori antibiotic candidates are identified, all of which prevent H.
pylori growth at concentrations 16 to 2,000 fold lower than the five antibiotics, amoxicillin,
metronidazole, levofloxacin, tetracyclin, and clarithromycin, commonly used to treat H. pylori
infections. X-ray crystal structures of MTAN co-crystallized with several inhibitors show them to
bind in the active site making interactions consistent with transition-state analogues.
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INTRODUCTION
The bacterial 5ʹ-methylthioadenosine/S-adenosylhomocysteine nucleosidases (MTANs) are dual
substrate enzymes hydrolyzing 5ʹ-methylthioadenosine (MTA) and S-adenosylhomocysteine
(SAH) to produce 5-methylthioribose or S-ribosylhomocysteine, and adenine. In most bacteria
MTAN is involved in methionine recycling, polyamine synthesis and quorum sensing, but is not
essential for cell growth or survival.1-3 Recently Helicobacter pylori (Hp) and Campylobacter
jeujeuni (Cj) were reported to have an unusual biosynthetic pathway for menaquinone in which
the respective MTANs play essential roles.4,5 This pathway utilizes 6-amino-6-deoxyfutalosine
which is also hydrolyzed by the Hp and Cj MTANs (Figure 1). Menaquinone is essential for
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Figure 1. The role of MTAN in the biosynthesis of Menaquinone.
electron transport in most gram-positive and anaerobic gram-negative bacteria and a block
imposed on this pathway by inhibition of the MTANs would likely be lethal to the organisms.6-8
H. pylori is the most common worldwide bacterial infection. It is related to 85% of gastric and
95% of duodenal ulcers and is an important risk factor for gastric malignancies.9,10 Antibiotic
treatment for H. pylori infections is becoming more challenging, with even triple and quadruple
therapies being often unsuccessful as drug resistance has developed.11 In addition, the
combination of proton pump inhibitors and multiple antibiotics disrupt the normal gut
microbiome and this treatment is associated with the development of Clostridium difficile-
associated diarrhea, a disorder linked with 29,000 annual deaths in the US in 2011.12,13 New
antibiotics are needed for H. pylori infections with novel targets and mechanisms of action.
Transition-state-analogue inhibitors of the biosynthesis of menaquinone provide a unique
approach to the design of new antibiotics for H. pylori.
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Figure 2. The hydrolysis of MTA by bacterial MTANs is characterized by dissociative transition
states. Similar transition states will be in effect when S-adenosylhomocysteine and 6-amino-6-
deoxyfutalosine are substrates.
Transition-state analogues capturing important features of an enzyme transition state can
bind tightly with the potential for exceptionally potent inhibitors.14-16 Knowledge of the
transition-state structure provides a blueprint for inhibitor design. The transition states of several
bacterial MTANs have been analyzed and shown to have strong ribocationic character with little
involvement of the water nucleophile (Figure 2).17-20 A number of transition-state-analogue
inhibitors of these MTANs have been developed with many in the pM, and some in the fM
range.20-24 The HpMTAN has been compared to other bacterial MTANs and shown to be also
dissociative in character with, in this case, a relatively early dissociative transition state.20 The
inhibitors 1 and 2 were used in the comparison, with 1 better mimicking an early transition state
and 2 a late transition state. Subsequently 3 was shown to be a 36 pM inhibitor of the HpMTAN
and additionally to have an IC90 of <8 ng/mL for the growth of Helicobacter pylori without
arresting growth of other common bacterial species.25 This has demonstrated the potential utility
of HpMTAN transition-state-analogue inhibitors as new, and H. pylori-specific, antibiotics.
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Here we present the HpMTAN inhibition data and IC90 values for transition-
stateanalogues including novel and simplified structures. Sixteen analogues have dissociation
constants of 50 pM or below. Ten of these prevent growth of the H. pylori at concentrations 16 to
2,000 fold lower than the five antibiotics in current use to treat H. pylori infections. Additionally,
the x-ray crystal structures of HpMTAN containing bound inhibitors is presented showing that
all bind in the active site making interactions with the protein consistent with transition-state
analogues.
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RESULTS AND DISCUSSION
Synthesis of New Transition-State Analogues.
Detail of the synthesis of new H. pylori MTAN inhibitors is presented in the Schemes and
experimental procedures in the SI.
Biological Results.
Enzymes achieve the catalytic rate enhancement of reactions by lowering the activation energy
for the transition states.26 When the electronic and spatial characteristics of an enzyme transition
state are known they provide a template for the design of transition-state analogues which can be
powerful inhibitors if they incorporate important features of that transition state in a stable
molecule. Kinetic isotope effects have been used to determine enzyme transition states leading to
the design and synthesis of exceptionally potent inhibitors.14-32 The transition states of several
bacterial MTANs have all been shown to be dissociative in character, with the HpMTAN
displaying characteristics of an early dissociative transition state.17-20 Transition-state-analogue
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inhibitors such as 1 mimic an early transition state with a short distance between C-1’ and C-9
whereas inhibitors such as 2 resemble a late transition state with a much longer distance between
N-1’ and C-9. In keeping with this 1 is marginally a more potent inhibitor than 2 of the
HpMTAN. However, the hydroxypyrrolidine compounds (eg 2) in general have better overall
affinity for the MTANs regardless of the transition state. This may be due to a closer ion pair
formation with the water nucleophile, the higher pKa of the pyrrolidine and the geometric
freedom inherent in the methylene bridge between the leaving group and ribocation mimics.
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Enzyme Inhibition Results.
The HpMTAN shows broad substrate specificity, effecting the hydrolytic cleavage of the
adenine moiety from the substrates methylthioadenosine, S-adenosylhomocysteine and 6-amino-
6-deoxyfutalosine. These substrates differ only in their adenosyl 5’-substituent. When the
previously described MTAN inhibitors 1-2322,23,27-29 were assayed against HpMTAN, the results
(Table 1 and Supporting Information Table 1) confirmed that the enzyme accepts varied
substituents at the 5’-position and that the hydroxypyrrolidine structures (eg 3) provided a
number of powerful pM inhibitors. Some alkylthio- (3, 5, 11), cycloalkylthio- (12-15) and
desthio- (19) analogues showed good potency as enzyme inhibitors. Compounds 20-23 were
originally synthesized in response to the observation that polyethylene glycol was present in the
5’-alkylthio binding site of crystalline S. enterica MTAN.29 The PEG was from the
crystallization solvent. This suggested that the 5’-binding site might easily accommodate
ethylene glycol-type substituents, and 23 was indeed a 5 pM inhibitor of the SeMTAN. The H.
pylori and S. enterica MTANs are very similar at the active site. In both cases the active site
entrance (the 5'-binding site) are rather hydrophobic: Met11, Ile53, Leu105, Phe108, Pro116,
Phe154 and Phe209 in HpMTAN. In SeMTAN, all of these residues are the same except Leu105,
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which is a valine. Consistent with this rationale, 23 is a 15 pM inhibitor of the HpMTAN.
Results from the other 5’-thio analogues 24-27 and 32, 33 showed that it is possible to vary the
substituent at this position while retaining good inhibitor potency. Comparing compounds 21 and
35 demonstrates that the position of the sulfur is not important (and even can be removed as in
19) whereas replacing S with O (36 vs 38) demonstrated that hydrophobic groups (S and CH2)
are preferred over oxygen.
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Table 1. Data of selected compounds for the inhibition of H. pylori MTAN and IC90 values for
H. pylori growth on blood agar.
Compound H. pylori MTAN Inhibition
Inhibition
(nM)
of H.
pylori
growth
IC90
Ki
Ki*
(ng/mL)
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0.56
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0.43
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0.07
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0.04
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0.001
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0.005
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N/Ob
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0.030
0.003
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aThese errors were calculated based on the Km errors, to provide an upper limit to the errors to Ki based on the data
from all other inhibitor determination. bNo slow onset inhibition was observed.
Previous results with other N-ribosyltransferase enzymes have demonstrated that acyclic
aminoalcohol derivatives can also act as transition-state analogues by acting as mimics of the
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ribocation moiety at the transition state.21,31-33 However, acyclic inhibitors of bacterial MTANs
have been investigated without discovering potent inhibitors of interest.21 Here for the
HpMTAN, new substituted-thio analogues 40-44 all demonstrated improved potency over the
methylthio compound 39, in particular 40, 41 and 44. The results from a number of desthio
compounds 45-55 with hydrophobic side chains indicated that branched-chain compounds were
not well tolerated and the pentyl derivative 54 was exceptional amongst these compounds with
low pM potency.
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Exploring the geometry and hydrophobicity of the 5’-substituents in a family of potential
transition-state analogues for H. pylori MTAN has yielded sixteen analogues with slow-onset
inhibition properties and dissociation constants of 50 pM or below (Table 1). Included among
these powerful inhibitors are three analogues with dissociation constants below 10 pM. This
value is biologically significant, as it has been documented that transition-state analogues of 10
pM or below bind to their targets with inhibition times approaching or exceeding the lifetimes of
cells.34 This inhibitory potential is significant for H. pylori, a bacterial target of the stomach,
where exposure to oral drugs might be transitory. Given the inhibitory potential of these agents,
we explored the ability of them to penetrate cells, inhibit the target enzyme, and prevent growth
of cultured H. pylori.
Bacterial Growth Assays.
Candidate inhibitors were tested against H. pylori growth on 5% horse blood agar under
conditions described previously (Table 1 and Supporting Information Table 1).25 Poor inhibitors
had little or no effect on bacterial growth. Powerful inhibitors did not always show growth
inhibition at low concentrations, an indication of biological access to the cell interior. Among the
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potent MTAN inhibitors, ten gave H. pylori growth inhibition (IC90 values) ≤ 10 ng/mL (~20
nM) while others required much higher concentrations. H. pylori is a Gram-negative organism,
the group of bacteria known to be difficult to drug because of the complex membrane and cell
wall structure. In our study, we have evaluated both the action of inhibitor on the MTAN target
by analysis of kinetic constants, and biological access to the cell interior by measuring IC90
values (the inhibitor concentration reducing growth on blood agar plates by 90%). The
hydroxypyrrolidine series of compounds with butylthio- 3, propylthio- 11, hexylthio- 24,
hexynylthio- 25 and pyrazinylthio- 32 substituents demonstrated IC90 values ~10 ng/mL,
whereas in the acyclic aminoalcohol series those with pyrazinylthio- 44, and the desthio butyl-
53 and pentyl- 54 substituents showed similar potency. The pyrazinylthio substituent is common
to both, perhaps again reflecting improved access to the cell interior. It is instructive to compare
the IC90 values reported here with those of the current antibiotics used in multi-drug therapy for
H. pylori infections. Metronidazole (16,000 ng/mL), amoxicillin (125 ng/mL), clarithromycin
(8,000 ng/mL), levofloxacin (1,000 ng/mL), tetracycline (500 ng/mL) and the new agent,
moenomycin A (2,000 ng/mL) are weaker than the ten best MTAN inhibitors (~8 ng/mL) by 16
(amoxicillin) to 2,000-fold (metronidazole).35 We can conclude that the present compounds are
promising leads with potent antibacterial activity against H. pylori.
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Inhibitor-bound HpMTAN Crystal Structure Comparisons.
HpMTAN was co-crystallised with the hydroxypyrrolidine compounds 2, 22, 32, and 36 and the
acyclic aminoalcohol compound 54. All of the inhibitor-bound structures are homo-dimeric, with
the asymmetric units containing between one and four monomer units (eg Figure 3). This
oligomerisation state is consistent with all other MTAN structures in the PDB from 15 other
organisms. In PDB-reported MTAN structures, two of the active site residues strongly hydrogen
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bond to the adenine portion of the native substrates or inhibitors. The five inhibitors presented
herein have the same interactions (Figure 4). The carboxylate of Asp199 (conserved in all
deposited MTAN structures) hydrogen bonds to N-6 and N-7; and the backbone amide nitrogen
and oxygen atoms of Val155 hydrogen bond to N-1 and N-6, respectively. (See numbering in
Figure 3). The tertiary amine (N-1’) in the inhibitors is protonated at neutral pH and mimics the
ribocationic nature of the transition state. In the respective inhibitor-bound structures, the nearby
nucleophilic water molecule that would act to hydrolyze the native substrates is highly
coordinated, being hydrogen bonded to N-1’ of the inhibitor in addition to Arg195 and Glu13.
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With inhibitors 2, 22, 32 and 36 there is an additional hydrogen bond to the 3ʹ-hydroxy group,
which is in turn hydrogen bonded to Glu176. In contrast, the hydroxymethyl group in the acyclic
inhibitor 54 is hydrogen bonded only to Glu176. This acyclic inhibitor has greater
conformational freedom and has one fewer carbon between N-1ʹ and O-3ʹ compared to the other
inhibitors, and consequently is situated differently in the active site. This is exemplified by the
torsion angle C9–C10–N1’–C1’, which is -115° in 54 and -75 3° in inhibitors 2, 22, 32 and 36.
The active site entrance (also the 5’-binding site) is rather hydrophobic (Met11, Ile53, Leu105,
Phe108, Pro116, Phe154, Phe209) and spacious, and is able to accommodate a range of
substrates, including MTA, SAH and aminofutalosine. As the 5’-substituent of 2 is small, there is
extra room in the 5’-binding site for compounds with larger 5’-groups (Figure 3 and Figure 4).
Adenine-bound MTAN crystal structures sometimes have small molecules from crystallization
buffer or cryoprotectant present in this cavity. For
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Figure 3. HpMTAN with 2 bound. Left; Ribbon diagram showing homo-dimeric structure. The
expansion shows the binding pocket, with hydrogen bonding residues and “nucleophilic” water
shown. Right; Surface representation showing the active site entrance, colored based on B-
factors.
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HpMTAN with 2
HpMTAN with 22
HpMTAN with 32
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HpMTAN with 36
HpMTAN with 54
Figure 4. HpMTAN bound to various inhibitors. Left; electron densities greater than 1 RMSD
around the ligand, in maps calculated with 2 Fobs-Fcalc coefficients. Right; Surface representation
of protein residues at active site entrance (colored by B-factor), with respective inhibitors shown
as sticks.
instance, PEG chains, glycerol or ethylene glycol are observed near the ribose binding pocket or
active site entrance in the S. enterica and E. coli MTAN structures, PDB codes 4F1W and 1Z5P,
respectively. The synthesis of 36 was partly inspired by this observation. Given the largely
hydrophobic nature of the active site, no substantial interactions are made to the 5'-alkylthio
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substituents, until the exterior of the protein is reached. At the protein exterior the hydroxybutyl
and PEG fragment, in 32 and 36 respectively, are close enough to hydrogen bond to one of the
imidazole nitrogen atoms of His110, although this is only observed in the hydroxybutyl inhibitor
(N···O = 2.69 Å). Longer inhibitors could also interact with Glu13. The HpMTAN structure with
54 bound also features well-ordered fusion tags. Two of the histidine residues from the fusion tag
in one of the monomers, in addition to His42 from a second dimer and Glu211 from a third, form
a tetrahedral coordination complex with a zinc ion (see the SI Figure). The fusion tag from the
second monomer does not form such an interaction, but is also well ordered.
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CONCLUSIONS.
A new antibiotic approach to H. pylori infections utilizes transition-state-analogue inhibitors of
the H. pylori MTAN targeting the biosynthesis of menaquinone. The transition-state analogues
include sixteen with dissociation constants below 50 pM. Ten of these sixteen powerful
inhibitors also prevent growth of the H. pylori at ≤ 10 ng/mL - concentrations 16 to 2,000 fold
lower than the five antibiotics in current use to treat H. pylori infections.
SUPPORTING INFORMATION.
Full experimental details including compound syntheses, characterization and NMR spectra,
enzyme inhibition and cell culture assays, as well as protein crystallization details, CIF files and
the diffraction data statistics are provided.
AUTHOR INFORMATION.
Peter Tyler: Phone, +64 4 463 0064; email, peter.tyler@vuw.ac.nz; Address, Ferrier Research
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Institute, P O Box 33436, Petone, New Zealand.
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Keith Clinch: Phone, +64 4 463 0043; email, keith.clinch@vuw.ac.nz; Address, Ferrier
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Research Institute, P O Box 33436, Petone, New Zealand.
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Vern Schramm: Phone, +1 (718) 4302814; email, vern.schramm@einstein.yu.edu, Address,
Albert Einstein College of Medicine, 1300 Morris Park Ave, Bronx, New York, USA.
ACKNOWLEDGEMENTS.
This work was financially supported by the New Zealand Foundation for Research Science and
Technology Grant C08X0209 and by the National Institutes of Health Research Grant
GM041916. Crystallographic data for this study were measured at beamline X29A of the
National Synchrotron Light Source. Financial support comes principally from the Offices of
Biological and Environmental Research and of Basic Energy Sciences of the US Department of
Energy, and from the National Center for Research Resources (P41RR012408) and the National
Institute of General Medical Sciences (P41GM103473) of the National Institutes of Health.
Herbert Wong and Yinrong Lu are thanked for a quality NMR and Mass Spec service.
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TOC Graphic.
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