Antibacterial Strategy against H. pylori: Inhibition of the Radical SAM Enzyme MqnE in Menaquinone Biosynthesis

Article abstract of DOI:10.1021/acsmedchemlett.8b00649

Aminofutalosine synthase (MqnE) catalyzes an important rearrangement reaction in menaquinone biosynthesis by the futalosine pathway. In this Letter, we report the identification of previously unreported inhibitors of MqnE using a mechanism-guided approach

Full text of DOI:10.1021/acsmedchemlett.8b00649

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Letter
An Antibacterial Strategy Against H. pylori: Inhibition of the
Radical SAM Enzyme - MqnE in Menaquinone Biosynthesis
Sumedh Joshi, Dmytro Fedoseyenko, Nilkamal Mahanta, Rodrigo
G. Ducati, Mu Feng, Vern L. Schramm, and Tadhg P. Begley
ACS Med. Chem. Lett., Just Accepted Manuscript • DOI: 10.1021/acsmedchemlett.8b00649 • Publication Date (Web): 15 Feb 2019
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An Antibacterial Strategy Against H. pylori: Inhibition of the Radical SAM
Enzyme – MqnE in Menaquinone Biosynthesis
Sumedh Joshi‡§, Dmytro Fedoseyenko‡§, Nilkamal Mahanta‡§, Rodrigo G. Ducati†, Mu Feng†, Vern L.
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Schramm†, Tadhg P. Begley‡*
‡Department of Chemistry, Texas A&M University, College Station, TX 77842
†Department of Biochemistry, Albert Einstein College of Medicine, Bronx, NY 10461
Supporting Information Placeholder
reported.15-19 The antibiotic potential of the other enzymes
on the futalosine pathway, including the two radical SAM
ABSTRACT: Aminofutalosine synthase (MqnE)
catalyzes an important rearrangement reaction in
enzymes – MqnE and MqnC – has not been explored. In this
menaquinone biosynthesis by the futalosine pathway. In
letter, we report the identification of a mechanism-based
this letter, we report the identification of previously
inhibitor of MqnE and demonstrate its antibacterial activity
unreported inhibitors of MqnE using a mechanism-
guided approach. The best inhibitor shows efficient
against H. pylori and C. jejuni.
inhibitory activity against H. pylori (IC50 = 1.8 ± 0.4 μM)
and identifies MqnE as a promising target for antibiotic
development.
KEYWORDS: Radical SAM enzyme, MqnE, bi-
substrate inhibitor, Helicobacter pylori, antibiotic
Menaquinone is a lipid-soluble, redox-active cofactor
involved in the transmembrane electron transport chain of
the majority of microbes.1 Humans use menaquinone
(Vitamin K) as an essential blood clotting vitamin,2-4 and
acquire it from dietary sources and from its biosynthesis in
the gut microbiome.5 Menaquinone biosynthesis is therefore
an attractive target for antibiotic development6 and inhibitors
against gram-positive organisms such as Mycobacterium
tuberculosis and Staphylococcus aureus have been
identified.7 The recent discovery of a new, futalosine-
dependent, menaquinone biosynthesis pathway has
presented new opportunities for antibacterial development8-
Figure 1: Mechanistic proposal for the MqnE-catalyzed
conversion of 1 to 8.
MqnE is a radical SAM enzyme20-21 in the futalosine-
dependent menaquinone biosynthesis pathway that catalyzes
a key C-C bond formation.22 We have previously reported
mechanistic studies on this enzyme with successful trapping
of the captodative radical 3 and the aryl radical anion 7
(Figure 1).23-24
9
because important human pathogens including
Helicobacter pylori (causes gastric ulcers and cancer),
Campylobacter jejuni (causes diarrhea), Chlamydia strains
(cause urethritis and respiratory tract infections),
Spirochetes (cause syphilis and Lyme disease) utilize this
pathway.10 The absence of this pathway in humans and in
most of the human gut bacteria potentially provides the
required selectivity for targeting this pathway without
affecting the commensal bacteria. Potent, transition-state
analog inhibitors against the 5′-methylthioadenosine
nucleosidase (MTAN) from H. pylori11-13 and C. jejuni14
have been developed and long chain fatty acids and
macrolides targeting the later steps of the pathway have been
Figure 2: Mechanistic proposal for the MqnE reaction with
9.
High throughput screening for inhibitors of radical SAM
enzymes is technically demanding because these enzymes
are extremely oxygen sensitive and have low turnover. We
therefore undertook a mechanism guided approach for the
development of an inhibitor of MqnE. The captodative
radical intermediate 3 is expected to be the most stable
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radical intermediate in the conversion of 1 to 8. We therefore
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was a weaker inhibitor of MqnE with an IC50 value of 839 ±
187 μM (Figure S13). This suggests that the enzyme
undergoes a major conformational change after the
formation of 10 resulting in reduced affinity of the enzyme
for 12 and avoiding product inhibition by 8.
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anticipated that a structural analog of this intermediate might
act as a substrate or transition state mimic and form bi-
substrate inhibitor of MqnE. A bi-substrate inhibitor is a
molecule that is chemically synthesized or enzymatically
generated by covalent linking of two substrates of a bi-
substrate enzyme reaction and mimics the ternary enzyme
substrate complex25. This inhibitor design strategy has been
demonstrated to be effective in achieving enhanced potency
and selectivity and has led to the development of FDA
approved therapeutics such as finasteride, mupirocin and
isoniazid25.
1.0
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We hypothesized that replacing the bridging oxygen of the
native substrate 1 with a methylene group (compound 9)
would block the conversion of 11 to 13/14 due to the
instability of a primary carbanion (or radical). This would
allow the accumulation of 10 which after hydrogen atom
abstraction would result in the formation of the shunt
product 12 – a potential bi-substrate inhibitor (Figure 2).
0
100
200
300
400
500
600
[methylene analog 9] in µM
Figure 3: Inhibition kinetics with the methylene analog 9
The methylene analog 9 was synthesized as shown in
26-27
Figure S1
and tested with the Thermus thermophilus
The human pathogens H. pylori and C. jejuni were
selected to test the antibiotic activity of the methylene analog
9 and the bi-substrate analog 12. The effect of these
inhibitors on C. jejuni and H. pylori growth was measured
using the 96-well plate liquid culture method.29-31 As shown
in Table 1, the IC50 for the methylene analog 9 and the bi-
substrate analog 12 on C. jejuni were 13.6 ± 1.5 µM and 83.3
± 3.4 µM, respectively. Gentamicin was used as a control
and had an IC50 value of 1.9 ± 0.2 µM (Table 1). The
measured IC50 values for methylene analog 9 and bi-
substrate analog 12 on H. pylori were 1.8 ± 0.4 μM and 16.1
± 3.9 μM, respectively. BTDIA, a transition state analog of
the H. pylori MTAN (Figure S14),12 was tested as a control
and displayed an IC50 of 0.012 ± 0.001 and 1.4 ± 0.3 M for
H. pylori and C. jejuni,14 respectively (Table 1).
ortholog of MqnE. HPLC analysis of the reaction mixture
indicated the formation of one major product that was absent
in the controls (Figure S2). This product had a molecular ion
m/z of 456 Da consistent with the mass of the shunt product
12 (Figure S3). This structure was confirmed using MS
fragmentation and NMR analysis (Figure S4-S9). On
running the reaction in 95% D2O buffer, this peak showed
one deuterium incorporation implying that the abstracted
proton in 12 originated from solvent or a solvent
exchangeable protein residue (Figure S3).
The T. thermophilus MqnE enzyme catalyzed >25
turnovers under our in vitro conditions with the native
substrate (Figure S10). The MqnE reaction was slow with
the methylene analog 9, providing a single turnover (Figure
S10). Encouraged by this result, we used competitive
inhibition experiments in which MqnE-[4Fe-4S]2+ was
preincubated with variable concentrations of the methylene
analog 9 in the presence of excess SAM and substrate 1.
Reactions were then initiated by reducing the enzyme with
Ti(III) citrate and the rate of aminofutalosine 8 formation
was followed by a discontinuous HPLC analysis. The
normalized relative initial reaction rates were plotted as a
function of inhibitor concentration to generate a dose-
response curve and an IC50 value of 38.7 ± 3.4 μM was
obtained. (Figure S11). Since this IC50 value was within 5-
fold of the enzyme concentration used, the dose-response
curve data was fitted to the Morrison equation for tight-
binding inhibition28 which gave an inhibition constant Ki of
3.1 ± 0.1 µM (Figure 3). Irreversible inhibition was
eliminated by demonstrating full restoration of enzyme
activity after the enzyme was preincubated with 9 for one
hour, followed by removal of the inhibitor by gel filtration
(Figure S12).
Table 1: IC50 values for the inhibitors tested against H.
pylori and C. jejuni
12
9
Gentamicin
BTDIA
IC50 (μM)
IC50 (μM)
IC50 (μM)
IC50 (μM)
C. jejuni 83.3  3.4 13.6  1.5 1.9  0.2
H. pylori 16.1  3.9 1.8  0.4
1.4  0.3
0.012  0.0001
0.26*
*Literature reported value32
Radical SAM enzymes are widespread in cofactor
biosynthesis pathways.21 While these enzymes are
reasonable targets for antibiotic development, technical
difficulties working with highly oxygen sensitive low
turnover enzymes has retarded the development of inhibitors
against this family of enzymes. The methylene analog 9 is a
potential lead compound as an antibiotic against H. pylori. It
has comparable antibacterial activity to amoxicillin and
clarithromycin, currently approved antibiotics in the
treatment of H. pylori infections.33 In addition, this
compound is resistant to acid hydrolysis making it a suitable
lead compound for the development of an orally available
antibiotic against an acidophile like H. pylori.
The bi-substrate analog 12 was enzymatically synthesized
and also tested as a competitive inhibitor. This compound
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In summary, we have identified methylene analog 9 as an
8.
Joshi, S.; Fedoseyenko, D.; Mahanta, N.; Manion,
1
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inhibitor of MqnE and have demonstrated its antibacterial
activity against H. pylori (IC50 = 1.8 ± 0.4 μM). These
studies set the stage for the future development of antibiotics
against H. pylori with MqnE as the target.
H.; Naseem, S.; Dairi, T.; Begley, T. P., Novel enzymology
in futalosine-dependent menaquinone biosynthesis. Curr.
Opin.Chem. Biol 2018, 47, 134-141.
9.
Hiratsuka, T.; Furihata, K.; Ishikawa, J.;
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Yamashita, H.; Itoh, N.; Seto, H.; Dairi, T., An alternative
menaquinone biosynthetic pathway operating in
microorganisms. Science 2008, 321 (5896), 1670-1673.
ASSOCIATED CONTENT
10.
Dairi, T., An alternative menaquinone biosynthetic
Supporting Information
pathway operating in microorganisms: an attractive target
for drug discovery to pathogenic Helicobacter and
Chlamydia strains. J. Antibiot. 2009, 62 (7), 347-352.
9
The procedures for the overexpression and purification of
MqnE, protocols for in vitro and in vivo inhibition studies,
synthetic procedures for compound 9, NMR and MS
characterization of compound 12 are available in the
supporting information.
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11.
Evans, G. B.; Tyler, P. C.; Schramm, V. L.,
Immucillins in Infectious Diseases. ACS Infect. Dis. 2018, 4
(2), 107-117.
The Supporting Information is available free of charge on
the ACS Publications website at DOI:
12.
Wang, S.; Haapalainen, A. M.; Yan, F.; Du, Q.;
Tyler, P. C.; Evans, G. B.; Rinaldo-Matthis, A.; Brown, R.
L.; Norris, G. E.; Almo, S. C.; Schramm, V. L., A Picomolar
Transition State Analogue Inhibitor of MTAN as a Specific
Antibiotic for Helicobacter pylori. Biochemistry 2012, 51
(35), 6892-6894.
AUTHOR INFORMATION
Corresponding Author
* begley@tamu.edu
13.
Wang, S.; Cameron, S. A.; Clinch, K.; Evans, G.
Author Contributions
B.; Wu, Z.; Schramm, V. L.; Tyler, P. C., New Antibiotic
Candidates against Helicobacter pylori. J. Am. Chem. Soc.
2015, 137 (45), 14275-14280.
§ These authors contributed equally
Notes
14.
Ducati, R. G.; Harijan, R. K.; Cameron, S. A.;
The authors declare no competing financial interests
Tyler, P. C.; Evans, G. B.; Schramm, V. L., Transition-State
Analogues of Campylobacter jejuni 5′-Methylthioadenosine
Nucleosidase. ACS Chem. Biol. 2018, 13 (11), 3173-3183.
ACKNOWLEDGEMENT
This research was supported by a grant from the National
Science Foundation (1507191) and by the Robert A. Welch
Foundation (A0034).
15.
Yamamoto, T.; Matsui, H.; Yamaji, K.; Takahashi,
T.; Øverby, A.; Nakamura, M.; Matsumoto, A.; Nonaka, K.;
Sunazuka, T.; Ōmura, S.; Nakano, H., Narrow-spectrum
inhibitors targeting an alternative menaquinone biosynthetic
pathway of Helicobacter pylori. J. Infect. Chemother. 2016,
22 (9), 587-592.
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