CoA adducts of 4-oxo-4-phenylbut-2-enoates: Inhibitors of MenB from the M. tuberculosis menaquinone biosynthesis pathway

Article abstract of DOI:10.1021/ml200141e

A high-throughput screen led to the discovery of 2-amino-4-oxo-4- phenylbutanoate inhibitors of the 1,4-dihydroxy-2-naphthoyl-CoA synthase (MenB) from the menaquinone biosynthesis pathway in Mycobacterium tuberculosis. However, these compounds are unstabl

Full text of DOI:10.1021/ml200141e

LETTER
pubs.acs.org/acsmedchemlett
CoA Adducts of 4-Oxo-4-phenylbut-2-enoates: Inhibitors of MenB
from the M. tuberculosis Menaquinone Biosynthesis Pathway
Xiaokai Li,† Nina Liu,† Huaning Zhang,† Susan E. Knudson,‡ Huei-Jiun Li,† Cheng-Tsung Lai,†
Carlos Simmerling,† Richard A. Slayden,*,‡ and Peter J. Tonge*,†
†Institute for Chemical Biology & Drug Discovery, Department of Chemistry, Stony Brook University, Stony Brook,
New York 11794-3400, United States
‡Department of Microbiology, Immunology and Pathology, Colorado State University, Fort Collins, Colorado 80523, United States
S Supporting Information
b
ABSTRACT:
A high-throughput screen led to the discovery of 2-amino-4-oxo-4-phenylbutanoate inhibitors of the 1,4-dihydroxy-2-naphthoyl-
CoA synthase (MenB) from the menaquinone biosynthesis pathway in Mycobacterium tuberculosis. However, these compounds are
unstable in solution and eliminate to form the corresponding 4-oxo-4-phenylbut-2-enoates that then react with CoA in situ to form
nanomolar inhibitors of MenB. The potency of these compounds results from interaction of the CoA adduct carboxylate with the
MenB oxyanion hole, a conserved structural motif in the crotonase superfamily. 4-Oxo-4-chlorophenylbutenoyl methyl ester has
minimum inhibitory concentrations of 0.6 and 1.5 μg/mL against replicating and nonreplicating M. tuberculosis, respectively, and it is
proposed that the methyl ester penetrates the cell where it is hydrolyzed and reacts with CoA to generate the active antibacterial. The
CoA adducts thus represent an important foundation for the development of novel MenB inhibitors and suggest a general approach
to the development of potent inhibitors of acyl-CoA binding enzymes.
KEYWORDS: Menaquinone, MenB, 1, 4-dihydroxy-2-naphthoyl-CoA synthase, CoA, HTS, o-succinylbenzoic acid
uberculosis is a global health threat, and efforts to combat
the spread of this disease are hindered by the emergence of
drug resistance, coupled with the ability of Mycobacterium
tuberculosis to survive in a latent, nonreplicating state for many
MenB, the 1,4-dihydroxy-2-naphthoyl-CoA (DHNA-CoA)
synthase that catalyzes an intramolecular Claisen condensation
(Dieckmann) reaction leading to the formation of DHNA-CoA
from o-succinylbenzoate (OSB) (Figure 1).8
T
years.1,2 Because latent mycobacteria are thought to consume
ATP to remain viable,3 compounds that interfere with bacterial
respiration hold the promise of targeting both replicating as well
as nonreplicating bacterial populations.4 M. tuberculosis utilizes
menaquinone (vitamin K2), a polyisoprenylated naphthoqui-
none, as the lipid-soluble redox cofactor in the electron
transport chain, and efforts are underway to validate enzymes in
the menaquinone biosynthesis pathway as targets for drug
discovery.5,6 The menaquinone biosynthesis pathway in M.
tuberculosis mirrors that found in Escherichia coli (Figure 1),7,8
and transposon site hybridization has identified menC, menD, and
menE as essential for growth of M. tuberculosis.9 Recently, Sassetti
and co-workers have shown that menB is also essential (Dr.
Sassetti, personal communication), while inhibitors of MenA are
active against both replicating and nonreplicating bacterial
populations.6 In the present study, we now report the discovery
of a series of 2-CoA-4-oxo-4-phenylbutanoic acids that target
The substrate for MenB is unstable, and consequently, we
used a coupled assay8 to screen 105 091 small molecules (see the
Supporting Information).10 Following the primary screen, in
which compounds were tested at a single concentration of 12.5
μg/mL, we obtained 455 hits that had at least 30% inhibition
relative to control. Within these hits, we identified seven
compounds (Figure 2) that possessed the backbone structure
of OSB, the substrate for MenE. We hypothesized that these
molecules could inhibit either MenE or MenB, and so, com-
pounds were first evaluated for their ability to inhibit MenE
directly.11 Using this assay, it was found that 1548L21 (Figure 2)
did not inhibit MenE (100 nM) up to a concentration of 300 μM
(data not shown).
Received: June 18, 2011
Accepted: August 26, 2011
Published: August 26, 2011
r
2011 American Chemical Society
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LETTER
Figure 1. Menaquinone biosynthesis pathway. This pathway converts chorismate to menaquinone in prokaryotes such as E. coli, S. aureus, B. subtilis,
and M. tuberculosis. Shown in detail are the reactions catalyzed by the OSB-CoA synthase MenE and the 1,4,-dihydroxy-2-naphthoyl-CoA
synthase MenB.
Figure 2. HTS hits containing the OSB core. These compounds were identified in the initial HTS screen conducted using the MenE/MenB
coupled assay.
Analogues of 1548L21 were synthesized (Scheme S1 in the
Supporting Information) and evaluated for their ability to inhibit
MenB as well as bacterial growth (Table 1 and the Supporting
Information). Although several compounds inhibited MenB with
IC50 values below 10 μM (4 and 5), there was generally a poor
correlation between enzyme inhibition and antibacterial activity.
For example, 1 is at least 10-fold more active in the antibacterial
assay than 4 [minimum inhibitory concentration (MIC) is 6.25
μg/mL as compared to 50 μg/mL] but is a significantly poorer
inhibitor of MenB (IC50 = 52 μM). Subsequent studies revealed
that the inhibitors undergo retro-Michael addition,12 leading to
the regeneration of the (E)-benzoylacrylic acid and the amine in
solution. The 2-aminobutanoates have half-lives of ∼10 min to
12 h at pH 7, which is significant since the MIC measurements
take at least 24ꢀ48 h to complete. In addition, the IC50 values
were determined using the same protocol as that developed for
the high-throughput screening (HTS) in which compounds were
preincubated in reaction buffer with all components except
MenE for 1 h prior to determining enzyme activity, suggesting
that compound stability was also a factor in the enzyme inhibition
studies. Indeed, these compounds were found not to inhibit
MenB if the reaction was initiated immediately after addition of
inhibitor (Table 1).
To strengthen the connection between compound stability
and enzyme inhibition, we synthesized a series of compounds
that were unable to undergo retro-Michael addition (Figure 1S in
the Supporting Information). This included analogues 1S and 2S
in which the α-protons were replaced with fluorines or methyl
groups, 3S in which the ketone was replaced with a hydroxyl
group, and 4S, 5S, and 6S in which the amine was replaced with
an amide, heterocycle, or sulfur, respectively. Compounds 1S to
6S were stable in solution and did not inhibit MenB up to a
concentration of 50 μM. In addition, we also synthesized the
monomethyl and deuterium analogues 7S and 8S, which were
still able to undergo elimination and, as a consequence, also
retained the ability to inhibit MenB. On the basis of these results,
we hypothesized that the inhibitory activity of the compounds
was related to their stability in solution.
Because the (E)-benzoylacrylic acid is a Michael acceptor, we
speculated that CoA in the reaction mixture might react with the
acid, and it was found that the IC50 value for inhibition of MenB
by compound 3a decreased as the concentration of CoA
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Table 1. Activity of the 2-Amino-4-oxo-4-phenylbutanoic Acids
a IC50 values for MenB inhibition were determined at a MenB concentration of 150 nM. Assays were initiated by the addition of MenE either
immediately after adding inhibitor to the assay (without incubation) or after a 1 h incubation (with incubation). b Antibacterial activity against
M. tuberculosis H37Rv. c Stability at pH 7.4 and 25 °C.
Table 2. Inhibition of MenB by the 2-CoA-4-oxo-4-phenylbutanoic Acids
increased (data not shown). This suggested that inhibition of
MenB might result from an adduct formed between the (E)-
benzoylacrylic acid and CoA. Subsequently, the (E)-benzoy-
lacrylic acid and CoA were incubated together at pH 7.0 for 2
h, and the major product was purified by high-performance liquid
chromatography. Analytical data confirmed that the CoA thiol
had added to the C2 carbon of the acid.
To generate structureꢀactivity relationship data for MenB
inhibition, a series of CoA adducts were synthesized and
evaluated (Table 2). In general, the addition of a bulky sub-
stituent at either the meta or the para position of the phenyl
group resulted in significant reduction in enzyme inhibition.
Additionally, incorporation of electron-donating substituents at
either the ortho or the para positions also decreased inhibitor
potency. In contrast, introduction of an electron-withdrawing
substituent into the aromatic ring resulted in an increase in
enzyme inhibition. Perusal of the data in Table 2 indicates that
the most potent compounds (10 and 16) have IC50 values of
∼100 nM, which is close to 1/2[Eo]. To gain further mechanistic
insight into the functioning of these compounds, we thus used
steady state kinetic methods to study the inhibition of MenB
by 7, 10 and 16. These compounds were found to be
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Figure 3. Proposed structure of a CoA adduct bound to MenB. (a) Proposed structure of the CoA adduct bound to MenB. The CoA adduct adopts a
structure that mimics the resonance-stabilized carbanion formed during the MenB-catalyzed reaction. (b) The 2,4-dichloro CoA adduct (16) modeled
into the active site of MenB. This model was built using the structure of acetoacetyl-CoA bound to MenB (1Q51.pdb)8 and shows the proposed
interaction between the adduct carboxylate and the MenB oxyanion hole (G105 and G161). Also shown are three conserved MenB residues (S190,
D192, and Y0287). The figure was made using pymol.23
noncompetitive (mixed) inhibitors of MenB. Consistent with
their respective IC50 values (106 and 470 nM, respectively), the
Ki and Ki0 values of the 2,4-diCl derivative 16 (49 and 290 nM,
respectively) were significantly lower than the corresponding
values for the 4-Cl analogue 7 (0.35 and 1.6 μM, respectively).
Clearly, there is a preference for an electron-withdrawing sub-
stituent ortho to the succinyl side chain, which of course is the
position normally occupied by the OSB carboxyl group. To
account for noncompetitive inhibition, we are currently explor-
ing the possibility that binding of inhibitor to one subunit in the
MenB homohexamer can modulate the activity of adjacent
subunits.
The affinity of 16 is noteworthy as, in our experience, substrate
or product analogues of CoA-dependent enzymes do not gen-
erally bind with high affinity to their respective enzymes. Indeed,
potent inhibitors of CoA-binding enzymes are largely unknown
except in a few specific cases such as 2-octynoyl-CoA, which is a
mechanism-based inhibitor of acyl-CoA dehydrogenase.13 How-
ever, analysis of the MenB reaction suggests an explanation for
the potent inhibition of MenB by the CoA adducts. A mechanism
for the MenB-catalyzed reaction has been proposed in which an
intramolecular proton abstraction by the OSB carboxylate leads
to the formation of a resonance stabilized carbanion.8 Model
building suggests that the CoA adducts adopt a bound structure
that resembles the intermediate required for α-proton abstrac-
tion (Figure 3), which may account for the high affinity of these
compounds for MenB. Of key importance is the location of the
free carboxylate of the adduct, which is bound in the oxyanion
hole formed by Gly-105 and Gly-161.8,14 The oxyanion hole is a
conserved structural feature within the crotonase superfamily
and plays a central role in catalysis by stabilizing the carbanion/
enolate formed during reactions catalyzed by this superfamily.15
Indeed, many other CoA binding enzymes outside the crotonase
superfamily also function to increase either thioester electro-
philicity or α-proton acidity through interactions with the acyl-
CoA thioester carbonyl, and thus, our data suggest a general
structural feature that may facilitate the development of inhibi-
tors of CoA-dependent drug targets.
To investigate the importance of the proposed interaction
between the oxyanion hole and the adduct carboxylate, we
synthesized two additional compounds 17 and 18 (Scheme S1
in the Supporting Information), which have similar structures to
the CoA adducts but lack a free carboxylate group. As compared
with the CoA adducts, the CoA thioester (17) has a significantly
lower affinity for MenB (Table 2) with an IC50 value of 2.2 μM as
compared to 100 nM for the corresponding adduct (16). In
addition, compound 18 displayed no significant inhibition up to a
concentration of 400 μM. These two compounds thus support
the importance of the free carboxylate for CoA adduct binding.
The CoA adducts were found to have limited antibacterial
activity (data not shown), presumably due to poor uptake by
the bacteria. We speculated that CoA addition could occur once
the benzoylacrylic acid had penetrated the cell, and while
the benzoylacrylic acid 3a had limited antibacterial activity,
the corresponding benzoylacrylic acid methyl ester 3b, as well
as the fluoro analogue 2b, displayed potent antibacterial activity
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Table 3. Antibacterial Activity of the 4-Oxo-4-phenylbut-2-
enoates against H37Rv
enzyme, presumably due to favorable interactions between the
enzyme and the halogens at the 2- and 4-positions. A current goal
of our research program is to develop butenoates with increased
affinity for the enzyme but reduced electrophilicity, thus improv-
ing the selectivity of MenB inhibition by increasing the likelihood
that adduct formation would occur on the enzyme. In this regard,
we note the recent discussions concerning the utility of covalent
enzyme inhibitors that contain appropriately tuned electrophilic
groups.21
Finally, in addition to providing a promising starting point for
the development of MenB inhibitors, the work described here
also serves to reinforce issues that can arise with leads identified
through HTS.22 Although the present leads are stable in DMSO,
their reactivity under aqueous conditions reinforces the necessity
to consider compound stability under the assay conditions
employed, which is of particular importance for measurements
that require prolonged incubation such as antibacterial assays. In
the present work, the use of a coupled assay that included free
CoA resulted in the discovery of CoA adducts that bind with high
affinity to MenB and that represent a promising foundation for
the development of novel antibacterial agents that target mena-
quinone biosynthesis in M. tuberculosis as well as other patho-
genic bacteria that have this pathway. Importantly, our data also
support the proposal that MenB may be an appropriate target in
nonreplicating populations of M. tuberculosis.
a MIC determined in the microplate dilution assay under aerobic
conditions. b MIC determined in the in low oxygen recovery assay
against NRP-MTB. ND, not determined. Cytoxicity assays indicated
c
that the selectivity index of 2b and 3b is greater than 20.
(Table 3, MIC = 0.64 μg/mL). Indeed, 3b also had potent
activity in a low oxygen recovery assay (LORA) against non-
replicating M. tuberculosis (NRP-MTB) (Table 3, MIC = 1.5 μg/
mL), which is promising given that apart from rifampicin (MIC =
0.4 μg/mL), many current drugs are inactive against NRP-
MTB.16 Interestingly, it has been reported that the menB gene
is upregulated when H37Rv is grown under oxygen-limiting
conditions,17 supporting the possibility that this enzyme is an
intracellular target for the CoA adducts. On the basis of these
results, we propose that protection of the acid aids the entry of
the compounds into the cell where they then undergo reaction-
(s), such as a Michael addition by CoA, which results in the
formation of the antibacterial species. Although we cannot rule
out the possibility that the compounds react with other nucleo-
philes in the cell, we observed that the addition of 1,4-dihydroxy-
2-naphthoic acid (DHNA; 100 μg/mL) to the media was able to
rescue the growth of M. tuberculosis H37Rv that had been treated
with 2b or 3b at 2ꢁ MIC. DHNA is a downstream product of the
MenB reaction that has previously been used in complementa-
tion experiments to elucidate the order and identity of enzymes
that comprise the menaquinone biosynthesis pathway.18 Thus,
the ability of DHNA to rescue growth supports our current
hypothesis that the Michael acceptors inhibit menaquinone
biosynthesis in M. tuberculosis.
’ ASSOCIATED CONTENT
S
Supporting Information. Detailed experimental proce-
b
dures for the synthesis of compounds and biological assays. This
material is available free of charge via the Internet at http://pubs.
acs.org.
’ AUTHOR INFORMATION
Corresponding Author
*Tel: 970-491-2902. E-mail: Richard.Slayden@colostate.edu
(R.A.S.). Tel: 631-632-7907. Fax: 631-632-7934. E-mail: peter.
tonge@sunysb.edu (P.J.T.).
Funding Sources
We thank NERCE/NSRB for assistance with compound screen-
ing (NIH Grant AI057159). This work was funded by the NIH
Grants AI044639, AI070383, and AI058785 to P.J.T.
Further studies are required to confirm that CoA addition and
MenB inhibition occur within the cell and to fully elucidate the
mechanism of action of these compounds. In addition, it will be
important to further improve the affinity of the adducts for MenB
and also to replace the CoA portion of the adducts by more
druglike groups, and in this regard, we note the studies by Pereira
et al. in which inhibitors of E. coli acetyltransferase GlmU were
identified that occupy the CoA binding site in this enzyme.19
Because the benzoylacrylic acid and CoA bind to adjacent sites
on MenB, we are also exploring the possibility that MenB
catalyzes adduct formation in an analogous fashion to that
observed, for example, in the assembly of inhibitors via in situ
click chemistry.20 It is interesting to note that the IC50 values for
enzyme inhibition by the parent 2-aminobutanoates (Table 1)
correlate with their stability and hence reactivity of the butenoate
that is formed. There is also a correlation between IC50 and Ki
values for inhibition of MenB by the resulting adducts, indicating
that the more electrophilic butenoates bind more tightly to the
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