Mechanism-based inhibitors of MenE, an acyl-CoA synthetase involved in bacterial menaquinone biosynthesis

Article abstract of DOI:10.1016/j.bmcl.2008.07.130

Menaquinone (vitamin K₂) is an essential component of the electron transfer chain in many pathogens, including Mycobacterium tuberculosis and Staphylococcus aureus, and menaquinone biosynthesis is a potential target for antibiotic drug discovery. We report herein a series of mechanism-based inhibitors of MenE, an acyl-CoA synthetase that catalyzes adenylation and thioesterification of o-succinylbenzoic acid (OSB) during menaquinone biosynthesis. The most potent compound inhibits MenE with an IC₅₀ value of 5.7 μM.

Full text of DOI:10.1016/j.bmcl.2008.07.130

Bioorganic & Medicinal Chemistry Letters 18 (2008) 5963–5966
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Mechanism-based inhibitors of MenE, an acyl-CoA synthetase involved
in bacterial menaquinone biosynthesis q
a,
Xuequan Lu a, , Huaning Zhang b, , Peter J. Tonge b, , Derek S. Tan
*
*
a Molecular Pharmacology & Chemistry Program and Tri-Institutional Research Program, Memorial Sloan–Kettering Cancer Center,
1275 York Avenue, Box 422, New York, NY 10065, USA
b Institute of Chemical Biology & Drug Discovery, Department of Chemistry, Stony Brook University, Stony Brook, NY 11794, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Menaquinone (vitamin K2) is an essential component of the electron transfer chain in many pathogens,
including Mycobacterium tuberculosis and Staphylococcus aureus, and menaquinone biosynthesis is a
potential target for antibiotic drug discovery. We report herein a series of mechanism-based inhibitors
of MenE, an acyl-CoA synthetase that catalyzes adenylation and thioesterification of o-succinylbenzoic
acid (OSB) during menaquinone biosynthesis. The most potent compound inhibits MenE with an IC50
Received 26 June 2008
Revised 28 July 2008
Accepted 29 July 2008
Available online 12 August 2008
value of 5.7 lM.
Keywords:
Ó 2008 Elsevier Ltd. All rights reserved.
Acyl-CoA synthetase
Mycobacterium tuberculosis
Staphylococcus aureus
Mechanism-based inhibitor
Antibiotic
The growing incidence of drug-resistant strains of pathogens
such as Mycobacterium tuberculosis and Staphylococcus aureus
poses a serious threat to human health and necessitates the devel-
opment of novel antibiotics.1 While humans and some bacteria use
ubiquinone as the lipid-soluble electron carrier in the electron
transport chain, this function is fulfilled solely by menaquinone
(vitamin K2) in M. tuberculosis, most Gram positive bacteria,
including S. aureus, and some Gram negative organisms.2 Although
menaquinone plays an important role in the mammalian blood
clotting cascade,3 humans lack the biosynthetic pathway for gen-
erating this compound and instead obtain it from the diet or intes-
tinal bacteria. Thus, bacterial menaquinone biosynthesis is an
attractive target for drug discovery.4 Toward this end, we report
herein a series of mechanism-based inhibitors of MenE, an acyl-
CoA synthetase used in menaquinone biosynthesis.
and Bacillus subtilis, and the pathway is best understood in E. coli,
where the first six enzymes are present in an operon. These and
other genetic experiments delineated many of the components of
the pathway and also demonstrated the essential role menaquin-
one plays in bacterial viability.5b,6
Our initial efforts to target this pathway have focused on MenE,7
an acyl-CoA synthetase (ligase) that is essential in M. tuberculosis.6b
MenE converts o-succinyl-1-benzoate (OSB) to OSB-CoA via a two-
step process involving initial ATP-dependent adenylation of OSB to
form a reactive OSB-AMP intermediate, followed by thioesterifica-
tion with CoA to form OSB-CoA.
Acyl-CoA synthetases8 belong to a superfamily of structurally
and mechanistically related adenylate-forming enzymes that also
includes non-ribosomal peptide synthetase (NRPS) adenylation
domains9 and firefly luciferase.10 Analogous adenylation reactions
are also catalyzed by structurally unrelated aminoacyl-tRNA syn-
thetases.11 We and others have used 50-O-(N-acylsulfamoyl)adeno-
sines (acyl-AMS) and related compounds to inhibit such
adenylate-forming enzymes by mimicking the cognate, tightly
bound acyl-AMP intermediates.10,12–14 These molecules were in-
spired by a class of sulfamoyladenosine natural products that in-
cludes nucleocidin and ascamycin.15 To avoid potential liabilities
of the aromatic carboxylate moiety with respect to cell permeabil-
ity or chemical instability via spirodilactone formation (observed
for OSB-CoA), we posited that it might be replaced with a neutral
methyl ester, since this carboxylate is not directly involved in the
Menaquinone is biosynthesized from chorismate by the action
of at least eight enzymes (Fig. 1).5 The first studies on menaquin-
one biosynthesis focused on Escherichia coli, Mycobacterium phlei,
q
Dedicated to Professor Benjamin F. Cravatt, in honor of his outstanding
contributions to chemical biology and his receipt of the 2008 Tetrahedron Young
Investigator Award.
* Corresponding authors. Tel.: +1 631 632 7907; fax: +1 631 632 7960 (P.J.T.); tel.:
+1 646 888 2234; fax: +1 646 422 0416 (D.S.T.).
E-mail addresses: peter.tonge@sunysb.edu (P.J. Tonge), tand@mskcc.org (D.S.
Tan).
These authors contributed equally to this work.
0960-894X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.07.130

5964
X. Lu et al. / Bioorg. Med. Chem. Lett. 18 (2008) 5963–5966
MenF
MenD
NH2
N
NH2
N
O
O
HS–CoA
HS–CoA
–O2C
O
N
N
N
N
O–
O–
O
O O–
P
O O
S
O
YfbB
MenC
O
O
HO
O–
N
N
R
O
O
R
N
H
H+
O
chorismate
OSB (o-succinyl-1-benzoate)
HO
OH
HO
OH
NH2
Figure 3. Mechanism of covalent inhibition. Left: The CoA thiol nucleophile attacks
the carbonyl group in the acyl-AMP intermediate during the second half-reaction
catalyzed by acyl-CoA synthetases. Right: A vinyl sulfonamide Michael acceptor is
appropriately positioned to trap the incoming nucleophile and form a covalent
adduct.
O
O
N
N
O–
O
O O–
P
MenE
ATP PPi
MenE
N
N
O
O
O
CoA AMP
HO
OH
OH
OSB-AMP
O
O
OMe
O
O
8
O–
O
B(OH)2
O
MenB
H2O
S
CoA
OMe O
Pd(PPh3)4, K3PO4
Br
O
S
CoA
O
O
dioxane, 85 °C, 24 h
CH2
O
OH
DHNA-CoA
(1,4-dihydroxy-
2-napthoyl-CoA)
85%
CH2
OSB-CoA
7
9
1:2 TFA/CH2Cl2
rt, 2 h, 86%
MenA
UbiE
H
8
O
O
O
1:2 TFA/
CH2Cl2
O3, PPh3
Me
OMe O
OMe O
O
CH2Cl2
–78 °C, 2 h
81%
rt, 2 h
88%
O
OH
menaquinone (vitamin K2)
X
10
11: X = O
12: X = CH2
Figure 1. Bacterial biosynthesis of menaquinone from chorismate. The acyl-CoA
synthetase MenE catalyzes initial adenylation of OSB (o-succinyl-1-benzoate) to
form an OSB-AMP intermediate, followed by transthioesterification with CoA to
form an OSB-CoA thioester adduct. MenB then catalyzes Dieckmann condensation
to form DHNA-CoA, which is ultimately converted to menaquinone.
Figure 4. Synthesis of MeOSB (11) and the corresponding exo-methylene analog 12.
bis(acylimidazole), followed by methanolysis.16 To provide more
efficient and flexible access to OSB and analogs thereof, we devel-
oped a new synthesis from the known vinyl bromide 7, prepared
by alkylation of tert-butyl acetate with 2,3-dibromopropene
(Fig. 4).21 Suzuki cross-coupling with aryl boronate 8 provided sty-
rene 9. Ozonolysis of the vinyl group afforded the orthogonally pro-
tected OSB diester 10. Acid deprotection of the tert-butyl ester then
yielded the desired aromatic monoester MeOSB (11). This modular
approach should provide access to a wide range of OSB analogs. In-
deed, the exo-methylene intermediate 9 provided immediate access
to the corresponding OSB analog 12, which we envisioned would al-
low us to remove the potentially enolizable ketone functionality in
OSB-AMPanalogs4–6 (Fig. 2)andtoassessitsimportanceinbinding.
The corresponding vinyl sulfonyl chlorides 20 and 21 were also
prepared by a similar route (Fig. 5), featuring selective Horner–
Wadsworth–Emmons coupling of ketoaldehyde 15 with sulfonyl
phosphonate 1722 to afford the vinyl sulfonate 18. The exo-methy-
lene analog 19 was similarly prepared from 16. The esters were
purified and converted to vinyl sulfonyl chlorides 20 and 21, which
were used without further purification.
reaction mechanism.16 Thus, we envisioned that MeOSB-AMS (1)
or its sulfamide analog MeOSB-AMSN (2) might be effective inhib-
itors of MenE and menaquinone biosynthesis (Fig. 2).
We also considered that the corresponding vinyl sulfonamide
MeOSB-AVSN (3) might inhibit MenE through covalent binding to
the incoming CoA thiol nucleophile during the second half-reaction
(Fig. 3), forming a mimic of the tetrahedral intermediate. Michael
acceptors have been used extensively to inhibit cysteine prote-
ases,17 and also to target protein thiol nucleophiles in polyketide
and non-ribosomal peptide synthetases.18 Based on studies of
Roush and coworkers on the inherent reactivities of various sulfo-
nyl-based Michael acceptors,19 we selected the vinyl sulfonamide
moiety to provide the requisite balance of reactivity and selectivity
to bind CoA in the MenE active site without reacting promiscu-
ously with other nucleophiles.
Synthesis of these inhibitors began with the preparation of
MeOSB (11, Fig. 4). OSB was first synthesized by Roser in 1884 from
phthalic anhydride and succinic acid.20 MeOSBhas also been synthe-
sized by selective monohydrolysis of the corresponding CDI-derived
NH2
NH2
N
NH2
N
O
X
O
X
O
X
N
N
N
N
N
N
N
OMe O
OMe O
OMe
O O
S
O O
S
O O
S
O
O
O
N
N
N
N
O
N
N
N
H
H
HO
OH
HO
OH
HO
OH
1, MeOSB-AMS (X = O)
4 (X = CH2)
2, MeOSB-AMSN (X=O)
5 (X = CH2)
3, MeOSB-AVSN (X = O)
6 (X = CH2)
Figure 2. Structures of designed inhibitors of MenE. The sulfamate (1, 4) and sulfamide (2, 5) functionalities (red) are designed to mimic the phosphate group in the cognate
OSB-AMP reaction intermediate. The vinyl sulfonamide moiety (3, 6) is designed to trap the incoming CoA nucleophile covalently. The corresponding exo-methylene analogs
(4–6) are designed to probe the importance of the aromatic ketone functionality (green) for binding.

X. Lu et al. / Bioorg. Med. Chem. Lett. 18 (2008) 5963–5966
5965
It is interesting to note that the vinyl sulfonamide analog
MeOSB-AVSN (3) is the most potent inhibitor of MenE. In contrast
to the sulfamate and sulfamide analogs 1 and 2, this compound
lacks the carbonyl and adjacent heteroatom of the acyl phosphate
group in OSB-AMP, which may be involved in hydrogen bonding
interactions, based on the cocrystal structure of a related fatty
acyl-CoA synthetase with myristoyl-AMP.8d These results also con-
trast with the relative potencies of related inhibitors of the NRPS
salicylate adenylation enzyme MbtA.18b This may be due to a vari-
ety of factors, including possible structural differences between
these enzymes,24 different binding requirements for the inhibitors
or resulting covalent adducts, and/or the different thiol nucleo-
philes involved: CoA in the case of MenE and a protein (MbtB)
phosphopantetheine group in the case of MbtA. Our results also
suggest that the OSB ketone group is required for inhibition, as
shown by the complete lack of activity in exo-methylene analogs
4–6.
In conclusion, we have designed, synthesized, and evaluated a
series of mechanism-based inhibitors of the OSB-CoA synthetase
MenE, which is used in bacterial menaquinone biosynthesis. This
work expands the scope of sulfonyladenosine-based inhibitors to
the acyl-CoA synthetase class of the adenylate-forming enzyme
superfamily and sets the stage for future assessment of these
inhibitors and additional analogs in cellular and animal models
of infection to evaluate the potential of targeting MenE in antibac-
terial drug discovery.
O
8, Pd(PPh3)4
K3PO4
DIBAL
Br
OMe
7
OH
dioxane
85 °C, 24 h
39%
THF
–78 0 °C
4h, 92%
OH
CH2
13
14
O
P
O
O
17
EtO
S
O
O
X
OEt
Dess–
Martin
EtO
n-BuLi
OMe H
OMe
O O
S
CH2Cl2
rt, 2 h
95%
THF
O
OEt
–78 °C, 2.5 h
47% (18)
72% (19)
X
O3, PPh3
15: X = O
18: X = O
19: X = CH2
CH2Cl2, –78 °C 16: X = CH2
2 h, 76%
O
1) Bu4NI, acetone
56 ˚C, 24 h
OMe
O O
S
20: X = O
21: X = CH2
2) SOCl2, PPh3
CH2Cl2, rt, 5 h
Cl
X
Figure 5. Synthesis of vinyl sulfonyl chloride reagents 20 and 21.
With these OSB analogs in hand, MeOSB-AMS (1) and its exo-
methylene analog 4 were synthesized by analogy to our estab-
lished procedures,14h via N-acylation of a protected 50-O-sulfa-
moyladenosine derivative with 11 and 12, respectively, followed
by deprotection.23 Sulfamide analogs 2 and 5 were synthesized
similarly from a protected 50-N-sulfamoylaminodeoxyadenosine.23
The vinyl sulfonamide analogs 3 and 6 were prepared by acylation
Acknowledgments
We thank Dr. George Sukenick, Hui Liu, Hui Fang, and Sylvi Rusli
(MSKCC Analytical Core Facility) for expert mass spectral analyses.
D.S.T. is an Alfred P. Sloan Research Fellow. Financial support from
the NIH (R01 AI068038 to D.S.T.; R01 AI044639 and R21 AI058785
to P.J.T.), NYSTAR Watson Investigator Program (D.S.T.), William H.
Goodwin and Alice Goodwin and the Commonwealth Foundation
for Cancer Research, and MSKCC Experimental Therapeutics Center
is gratefully acknowledged.
of
a
protected 50-aminodeoxyadenosine with 20 and 21,
respectively.23
To test these compounds for inhibition of MenE, we used a cou-
pled assay with MenE and MenB, the DHNA-CoA synthetase that
follows MenE in the biosynthetic pathway.4,23 E. coli MenE and
M. tuberculosis MenB were separately cloned and expressed with
N-terminal His6-tags in E. coli (BL21) cells, then purified to homo-
geneity using affinity chromatography. Reactions were initiated by
adding MenE (final concentration 20 nM) to a solution containing
Supplementary data
MenB (7.2
inhibitor (0–200
392 nm, and IC50 values were determined.
We were gratified to find that both the sulfamate MeOSB-AMS
(1) and sulfamide MeOSB-AMSN (2) were effective inhibitors of
MenE (Table 1). Moreover, the vinyl sulfonamide analog MeOSB-
AVSN (3) proved to be the most potent inhibitor, with an IC50 of
5.7 0.7 lM; kinetic analysis indicated that this compound is a
slow-binding inhibitor, suggesting a conformational change during
l
M), ATP (240
lM), CoA (240 lM), OSB (240 lM) and
Experimental procedures and analytical data for all new com-
pounds are provided. Supplementary data associated with this arti-
cle can be found, in the online version, at doi:10.1016/
j.bmcl.2008.07.130.
lM). Formation of DHNA-CoA was monitored at
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Inhibition of MenE by designed inhibitors 1–6
Compound
IC50
(
l
M)a
Compound
IC50 (lM)
1
2
3
38.0 3.0
34.1 2.8
5.7 0.7
4
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a
Values are means of three experiments with standard deviation indicated.

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