Structure-Based Design, Synthesis, and Biological Evaluation of Non-Acyl Sulfamate Inhibitors of the Adenylate-Forming Enzyme MenE

Article abstract of DOI:10.1021/acs.biochem.9b00003

N-Acyl sulfamoyladenosines (acyl-AMS) have been used extensively to inhibit adenylate-forming enzymes that are involved in a wide range of biological processes. These acyl-AMS inhibitors are nonhydrolyzable mimics of the cognate acyl adenylate intermediates that are bound tightly by adenylate-forming enzymes. However, the anionic acyl sulfamate moiety presents a pharmacological liability that may be detrimental to cell permeability and pharmacokinetic profiles. We have previously developed the acyl sulfamate OSB-AMS (1) as a potent inhibitor of the adenylate-forming enzyme MenE, an o-succinylbenzoate-CoA (OSB-CoA) synthetase that is required for bacterial menaquinone biosynthesis. Herein, we report the use of computational docking to develop novel, non-acyl sulfamate inhibitors of MenE. A m-phenyl ether-linked analogue (5) was found to be the most potent inhibitor (IC₅₀ = 8 μM; K_d = 244 nM), and its X-ray co-crystal structure was determined to characterize its binding mode in comparison to the computational prediction. This work provides a framework for the development of potent non-acyl sulfamate inhibitors of other adenylate-forming enzymes in the future.

Full text of DOI:10.1021/acs.biochem.9b00003

This is an open access article published under an ACS AuthorChoice License, which permits
copying and redistribution of the article or any adaptations for non-commercial purposes.
Article
pubs.acs.org/biochemistry
Cite This: Biochemistry XXXX, XXX, XXX−XXX
Structure-Based Design, Synthesis, and Biological Evaluation of
Non-Acyl Sulfamate Inhibitors of the Adenylate-Forming Enzyme
MenE
Christopher E. Evans,†,# Yuanyuan Si,§,∥,# Joe S. Matarlo,§,⊥ Yue Yin,§,∥ Jarrod B. French,§,∥,⊥
Peter J. Tonge,*,§,∥ and Derek S. Tan*,†,‡
‡
†Pharmacology Program, Weill Cornell Graduate School of Medical Sciences, and Chemical Biology Program, Sloan Kettering
Institute and Tri-Institutional Research Program, Memorial Sloan Kettering Cancer Center, New York, New York 10065, United
States
§Institute of Chemical Biology and Drug Discovery, ∥Department of Chemistry, and ⊥Department of Biochemistry and Cell Biology,
Stony Brook University, Stony Brook, New York 11794, United States
S
* Supporting Information
ABSTRACT: N-Acyl sulfamoyladenosines (acyl-AMS) have
been used extensively to inhibit adenylate-forming enzymes
that are involved in a wide range of biological processes.
These acyl-AMS inhibitors are nonhydrolyzable mimics of the
cognate acyl adenylate intermediates that are bound tightly by
adenylate-forming enzymes. However, the anionic acyl
sulfamate moiety presents a pharmacological liability that
may be detrimental to cell permeability and pharmacokinetic
profiles. We have previously developed the acyl sulfamate
OSB-AMS (1) as a potent inhibitor of the adenylate-forming
enzyme MenE, an o-succinylbenzoate-CoA (OSB-CoA)
synthetase that is required for bacterial menaquinone biosynthesis. Herein, we report the use of computational docking to
develop novel, non-acyl sulfamate inhibitors of MenE. A m-phenyl ether-linked analogue (5) was found to be the most potent
inhibitor of (IC50 = 8 μM; Kd = 244 nM), and its X-ray co-crystal structure was determined to characterize its binding mode in
comparison to the computational prediction. This work provides a framework for the development of potent non-acyl sulfamate
inhibitors of other adenylate-forming enzymes in the future.
genetic studies that have demonstrated the essentiality of men
genes in bacteria such as Bacillus subtilis and M. tuber-
culosis.25−28
he spread of drug-resistant pathogens such as multi-drug-
Tresistant and extensively drug-resistant Mycobacterium
tuberculosis and methicillin-resistant Staphylococcus aureus
(MRSA) is a major threat to human health and places a
significant burden on our healthcare system.1−5 Thus, the
development of novel antibiotics that circumvent existing
resistance mechanisms is urgently needed. The biological
redox cofactor menaquinone is the sole electron carrier in the
electron transport chain of Gram-positive bacteria, all bacteria
growing anaerobically, and mycobacteria.6,7 Although mam-
mals use menaquinone (vitamin K2) as a cofactor in many
enzymes such as γ-glutamyl carboxylase, this quinone is
acquired from the diet or from gut flora rather than through de
novo biosynthesis.8−11 Thus, inhibitors that target the bacterial
menaquinone biosynthesis pathway are a promising avenue for
future antibiotics to address drug-resistant pathogens.12
MenE, the acyl-CoA synthetase in the menaquinone
biosynthetic pathway, has been the primary focus of our
efforts in this field. MenE, a member of the ANL (acyl-CoA
synthetase, nonribosomal peptide synthetase adenylation
domain, luciferase) family,29 carries out a two-step reaction
involving the initial activation of o-succinylbenzoate (OSB) by
adenylation to form a tightly bound OSB-adenosine mono-
phosphate (AMP) intermediate, followed by a subsequent
thioesterification with CoA to form OSB-CoA (Figure 1).30,31
Our previous efforts to inhibit MenE have focused on the use
of acyl-AMS [5′-O-(N-acylsulfamoyl) adenosine] ana-
logues,32−35 inspired by natural products such as ascamy-
cin.36,37 Using this approach, we developed OSB-AMS (1), a
tight-binding, low-nanomolar inhibitor of the S. aureus, M.
tuberculosis, and Escherichia coli MenE enzymes.33 While
Menaquinone is derived from chorismate through a series of
at least nine distinct enzyme-catalyzed transformations (Figure
1).7,13,14 Inhibitors have been developed against most of
enzymes in the pathway, including MenD,15,16 MenC,17
MenE,18 MenB,19,20 and MenA.21−24 Many of these com-
pounds have significant antimicrobial activity, consistent with
Received: January 7, 2019
Revised: February 7, 2019
© XXXX American Chemical Society
A
DOI: 10.1021/acs.biochem.9b00003
Biochemistry XXXX, XXX, XXX−XXX

Biochemistry
Article
Figure 1. Classical menaquinone biosynthesis pathway and structure of MenE inhibitor OSB-AMS (1). At least nine enzymes catalyze the
formation of menaquinone from chorismate. The fifth enzyme, MenE, is an acyl-CoA synthetase that catalyzes the ATP-dependent ligation of CoA
to o-succinylbenzoate (OSB) via an OSB-AMP intermediate. OSB-AMS (1) is a stable analogue of OSB-AMP in which the acyl phosphate mixed
anhydride is replaced with an acyl sulfamate.
effective in biochemical assays, OSB-AMS has significant
pharmacological liabilities that limit its further preclinical
development. Both the carboxylate and the acyl sulfamate are
deprotonated at physiologic pH, and this is the likely source of
its poor cellular permeability.38,39 Negative charge is also
associated with high levels of plasma protein binding and rapid
renal clearance.40,41 Finally, some acyl sulfamates may be
metabolically unstable in vivo.42 The free carboxylate of OSB is
necessary for potent binding to MenE,34 but we have recently
been able to replace the OSB motif with a difluoroindanediol
that is neutral at physiologic pH.34,35 Thus, our current efforts
have focused on replacing the acyl sulfamate moiety to avoid
the pharmacological liabilities associated with this linker.
Previous attempts to identify alternative bioisosteres of the
native acyl phosphate motif have met with limited success, with
most substitutions resulting in significant loss of biochemical
activity against the target adenylate-forming enzyzme.43−50
Herein, we report the design of a virtual library of OSB-AMS
linker analogues and the use of computational docking to
prioritize compounds for synthesis. Several novel linker
analogues were discovered, the most promising of which was
further characterized through X-ray crystallography to
elucidate its mechanism of binding for comparison to the
computational prediction.
performed using OPLS_3 force field restrained minimization
to a heavy atom convergence of 0.3 Å.
Ligand Preparation. Ligand preparation was performed using
̈
LigPrep in the Schrodinger Suite (version 2017.2). Lowest-
energy conformers were obtained using OPLS_3 force field
optimization. Ionization and tautomeric states were generated
using Epik at pH 7.4.
̈
Grid Generation. Using the Schrodinger Suite (version
2017.2) receptor grid generator, the receptor-binding site
was defined as the area around the co-crystallized ligand with a
cube grid with a side length of 10 Å. Nonpolar parts of the
receptor were softened using van der Waals radius scaling
(factor of 1.0 with a partial cutoff of 0.25). No constraints were
defined, and rotations allowed for all hydroxyl groups in the
defined binding pocket.
̈
Docking Using Soft Receptor. Using Schrodinger Glide
(version 7.2), ligands were docked to MenE at Glide XP
docking precision. Flexible ligand sampling was used, and Epik
state penalties were applied to docking scores. Postdocking
minimization was performed for all poses.
Inhibitor Synthesis. See the Supporting Information for
complete experimental protocols and analytical data for all new
compounds.
Site-Directed Mutagenesis. The K437A mutation was
introduced using high-fidelity Phusion polymerase (New
England BioLabs) with the forward primer 5′-AACGGCGG-
TATTGCGATTTCACG-3′ and the T7 reverse primer.
Purification of Wild-Type and K437A E. coli MenE. E.
coli MenE (UniProtKB entry P37353) was purified as
described previously.33 Briefly, BL21(DE3) pLysS cells were
transformed with a pET15b plasmid containing E. coli MenE
with an N-terminal His6 tag, then the cells were grown
overnight in 10 mL of LB medium containing 200 μg/mL
ampicillin. Next, 1 L of LB media containing 200 μg/mL
ampicillin was inoculated with the 10 mL overnight culture and
the cells were grown until the OD600 reached 0.6. Protein
expression was induced by addition of 1 mM isopropyl β-D-1-
thiogalactopyranoside, and the culture was shaken overnight at
20 °C. Cells were harvested by centrifugation at 5000 rpm for
MATERIALS AND METHODS
■
Computational Docking: Protein Preparation. The
OSB-AMS (1)·MenE (R195K) co-crystal structure [Protein
Data Bank (PDB) entry 5C5H] was processed using the
̈
Protein Preparation Wizard in the Schrodinger Suite (version
2017.2). Bond orders were assigned, explicit hydrogens added,
and nonbridging waters >5 Å from the ligand deleted. The
protonation and tautomeric states of the protein−ligand
complex were generated using Epik at pH 7.4. Hydrogen
bond assignment and optimization were performed with
PROPKA to sample the hydrogen bonding and orientation
of water molecules. Nonbridging waters (fewer than two
hydrogen bonds) were removed. Geometric refinement was
B
DOI: 10.1021/acs.biochem.9b00003
Biochemistry XXXX, XXX, XXX−XXX

Biochemistry
Article
10 min at 4 °C. The cell pellet was resuspended in 30 mL of
His-binding buffer [20 mM Tris·HCl, 100 mM NaCl, and 5
mM imidazole (pH 8.0)], and the bacteria were disrupted by
sonication. Cell debris was removed by centrifugation at 40000
rpm for 60 min at 4 °C, and the clear supernatant was loaded
onto a His-bind column (1.5 cm × 15 cm) containing 4 mL of
His-bind resin (Novagen) that had been charged with 10 mL
of charge buffer (Ni2+). The column was washed with washing
buffer containing 60 mM imidazole, and the protein was eluted
from the column with elution buffer containing 500 mM
imidazole. Fractions containing protein were loaded onto a
size-exclusion column (Superdex 75, GE Healthcare) and
eluted using 20 mM Tris·HCl buffer (pH 8.0) containing 100
mM NaCl to remove imidazole. The purified protein was
>97% pure as determined by sodium dodecyl sulfate−
polyacrylamide gel electrophoresis and stored at −80 °C in
storage buffer consisting of 20 mM Tris·HCl (pH 8.0)
containing 100 mM NaCl.
Biochemical Assays. Enzymatic inhibition was assessed
using a MenE−MenB coupled assay in which the MenE-
catalyzed reaction is the rate-limiting step.32−35 The reaction
was initiated by addition of 50 nM wild-type E. coli MenE to a
100 μL reaction system containing 60 μM OSB, 240 μM ATP,
240 μM CoA, 2.5 μM M. tuberculosis MenB, and varying
concentrations of the inhibitor in a buffer consisting of 20 mM
NaHPO4 (pH 7.4), 150 mM NaCl, and 1 mM MgCl2. The
production of 1,4-dihydroxy-2-naphthoyl-CoA was monitored
at 392 nm (ε392 = 4000 M−1 cm−1).
Isothermal Titration Calorimetry (ITC). ITC was
performed using a VP-ITC instrument at 22 °C. Inhibitor (1
mM) and E. coli MenE(wt or K437A mutant) (30 μM) were
prepared in 20 mM NaHPO4 buffer (pH 7.4) containing 150
mM NaCl and 1 mM MgCl2. The inhibitor was titrated from
the injection syringe into the sample cell containing 1.8 mL of
a solution of MenE. The data were fit to a single-binding site
model with the Origin software package.
X-ray Crystallography. Wild-type E. coli MenE was
prepared as previously described.33 Co-crystallization of
analogue 5 with MenE was achieved using the hanging drop
diffusion technique, in which 2 μL of the complex solution [10
mg/mL E. coli MenE and 600 μM analogue 5 dissolved in 20
mM Tris·HCl buffer (pH 8.0) containing 100 mM NaCl] was
mixed with 2 μL of the reservoir solution [0.1 M Tris·HCl, 0.2
M MgCl2, and 20% PEG (pH 6.5)] and equilibrated against
300 μL of the reservoir solution. Crystals formed at 293.15 K
after 4−5 days. A single crystal was selected for diffraction by
flash-freezing directly from the crystallization solution.
Diffraction data were collected at 100 K using beamline 17-
ID-1 (AMX) at National Synchrotron Light Source II at
Brookhaven National Laboratory. Data were integrated using
XDS51 and scaled using Aimless.52 The structure was
determined by molecular replacement with MolRep,53 using
our previously determined E. coli MenE (R195K) structure
(PDB entry 5C5H) as a search model. The model was refined
through successive rounds of manual model building using
COOT54 and restrained refinement using REFMAC5.55
Electron density for analogue 5 bound in the active site was
clearly visible and was added directly to the difference Fourier
map after refinement converged. Ligand restraints were
generated using the PRODRG server.56 The data collection
and refinement statistics are listed in Table S2. Coordinates
have been deposited as PDB entry 6NJ0.
Figure 2. Structures of OSB-AMS (1) and linker analogues bound to
E. coli MenE (R195K). (a) X-ray co-crystal structure of OSB-AMS (1,
gray) bound in the active site (cyan) of MenE with key binding
interactions (green) (PDB entry 5C5H). Computationally docked
structures of linker analogues (purple) (b) acyl squaramide 3, (c)
alkyl sulfamide 4, (d) m-phenyl ether 5, and (e) α-hydroxytetrazole
(S)-7, overlaid with OSB-AMS (1, beige) from the co-crystal
structure, with predicted active site interactions (light blue).
To compare the binding poses of co-crystallized OSB-AMS
(1) (PDB entry 5C5H) and docked and co-crystallized m-
phenyl ether analogue 5, the N-terminal domains (residues 1−
351) of the structures were aligned using PyMOL (align
command, default parameters).57
Antibacterial Assays. Minimum inhibitory concentrations
(MICs) were determined using visual growth inspection of
cells grown in transparent 96-well plates. E. coli (MG1655),
MRSA (ATCC BAA-1762), and B. subtilis (ATCC 6057) were
grown to mid log phase (OD600 = 0.6−0.8) in cation-adjusted
Miller Hinton (CAMH) medium at 37 °C in an orbital shaker.
A final inoculum of 100 μL of 106 colony-forming units/mL of
cells was treated with the inhibitor with final concentrations
ranging from 0.2 to 100 μg/mL. The MIC was defined as the
minimum concentration at which a well showed no obvious
growth by visual inspection.
C
DOI: 10.1021/acs.biochem.9b00003
Biochemistry XXXX, XXX, XXX−XXX

Biochemistry
Article
RESULTS
Table 1. Computational Docking Scores for OSB-AMS (1),
Selected Virtual Library Members (2−6), and Additional
Analogues (7−9)
■
Design and Computational Docking of OSB-AMS
Linker Analogues. We began by designing a 78-member
virtual library of OSB-AMS analogues in which the acyl
sulfamate motif was replaced by a variety of other potential
linkers (Table S1).58 As this is the region of the molecule
directly relevant to MenE catalytic activity, there are many
crucial interactions between the inhibitor and the enzyme,
making substitution at this position difficult. Thus, the virtual
library design focused on decreasing polarity while attempting
to retain as many of these key interactions as possible as well as
the geometry and distance between OSB and adenosine
observed in our co-crystal structure of OSB-AMS with E. coli
MenE (R195K mutant)34 (Figure 2a).
The library members were docked with the E. coli MenE
̈
(R195K) crystal structure (Schrodinger Glide) (Table 1 and
Table S1).58 Briefly, the co-crystal structure (PDB entry
5C5H)34 was minimized (Protein Preparation Wizard), and
then OSB-AMS was removed. Analogues were energy
minimized (LigPrep), and probable tautomeric and proto-
nation states predicted (Epik) and then docked into the
binding pocket using a soft receptor model (Glide XP).
Analogues were ranked by docking score, and those with
scores above −10 kcal/mol removed from further consid-
eration. Synthetic targets 2−5 were then selected from the list
on the basis of combined considerations of docking score,
reasonable docking pose that retained key interactions in the
binding pocket (Figure 2b−d), likelihood of improving overall
physiochemical properties of the scaffold (e.g., elimination of
negative charge), synthetic accessibility, and ease of function-
alization for further optimization. Linkers that had previously
been reported to be ineffective against another ANL family
enzyme were also eliminated from consideration.44−46 p-
Phenyl ether analogue 6, which had a poor docking score, was
selected for synthesis as a negative control.
Synthesis of Selected OSB-AMS Linker Analogues.
Synthesis of acyl tetrazole analogue 2 began with lithiation of
aryl bromide 10, prepared as previously described,34 quenching
with dimethyl carbonate, and acid-catalyzed opening of the
methyl ketal to afford primary alcohol intermediate 11 (Figure
3). Dess−Martin oxidation and conversion of the resulting
aldehyde to the corresponding cyanohydrin 12 set the stage for
nitrile 1,3-dipolar cycloaddition to construct the tetrazole
motif. Attempted cycloadditions with azide at this stage led to
undesired side products stemming from spirocyclization of the
free hydroxyl onto the aryl ketone and lactonization of the
methyl ester. Accordingly, reprotection as the cyclic methyl
ketal 13 allowed efficient cycloaddition to afford the desired
tetrazole intermediate 14. Reopening of the cyclic ketal and
oxidation of the hydroxyl group afforded linear acyl tetrazole
intermediate 15. Coupling to the protected adenosine scaffold
16, prepared as previously described,32 was achieved by
Mitsunobu reaction. However, upon attempted deprotection of
the penultimate intermediate 17, the acyl tetrazole moiety
proved to be hydrolytically unstable in the presence of even
relatively mild basic and neutral conditions.
to the synthesis of these analogues. Cyclic ketal-protected
tetrazole 14 was coupled to protected adenosine 16 via
Mitsunobu reaction to afford disubstituted tetrazole 18. Global
deprotection under acidic conditions led to the formation of
spirolactone 19, but this was readily saponified to afford the
desired α-hydroxytetrazole 7 as a 1:1 mixture of diastereomers.
Next, synthesis of acyl squaramide analogue 3 was achieved
by initial acylation of protected 5′-aminodeoxyadenosine 20,
prepared as previously described,59 with dimethyl squarate
followed by quenching with ammonia to form squaramide 21
(Figure 5). N-Acylation with the aromatic benzyl ester of OSB
(22) followed by hydrogenolytic debenzylation and desilyla-
tion afforded squaramide analogue 3.
We envisioned that this hydrolytic instability could be
avoided using the corresponding α-hydroxytetrazole analogue
7 (Figure 4). Both diastereomers were retrospectively
evaluated in our docking model and had promising docking
scores [(S)-7, −13.36 kcal/mol; (R)-7, −12.79 kcal/mol
(Table 1)] and poses (Figure 2e). Thus, we diverted attention
D
DOI: 10.1021/acs.biochem.9b00003
Biochemistry XXXX, XXX, XXX−XXX

Biochemistry
Article
Figure 4. Synthesis of α-hydroxytetrazole analogue 7. Abbreviations:
Boc, tert-butoxycarbonyl; DIAD, Boc, tert-butyloxycarbonyl; Cbz,
carbobenzyloxy; diisopropyl azodicarboxylate; TFA, trifluoroacetic
acid; THF, tetrahydrofuran.
Figure 3. Synthetic approach to acyl tetrazole analogue 2.
Abbreviations: Boc, tert-butoxycarbonyl; DIAD, diisopropyl azodi-
carboxylate; THF, tetrahydrofuran; TMS, trimethylsilyl.
Alkyl sulfamide analogue 4 was synthesized via Mitsunobu
coupling of reduced OSB intermediate 23 and protected
AMSN scaffold 24, prepared as previously described,32
followed by two-step deprotection (Figure 6).
To access m-phenyl ether analogue 5, dimethyl phthalate
(26) was converted to vinyl ketone 28 in five highly scalable
steps (Figure 7). Heck coupling with aryl bromide 29 provided
phenol 30, which underwent Mitsunobu coupling to protected
adenosine scaffold 16 to afford phenyl ether 31. The side-chain
olefin was reduced using Stryker’s reagent, the TBS group
removed, and the primary alcohol oxidized to the correspond-
ing carboxylic acid with TPAP/NMO. Global deprotection
with TFA provided m-phenyl ether analogue 5.
Figure 5. Synthesis of acyl squaramide analogue 3. Abbreviations:
BnOSB (22), 4-{2-[(benzyloxy)carbonyl]phenyl}-4-oxobutanoic
acid; DMAP, 4-(dimethylamino)pyridine; DMF, dimethylformamide;
EDC, N-ethyl-N′-[3-(dimethylamino)propyl]carbodiimide hydro-
chloride; TASF, tris(dimethylamino)sulfur trimethylsilyl difluoride;
TBS, tert-butyldimethylsilyl..
−10 kcal/mol (Table 1)] from the corresponding 4-
bromophenol Heck partner.58
On the basis of our structural analysis of m-phenyl ether
analogue 5 (Figure 1d), we also designed a 3-trifluoromethyl-
substituted analogue 8 [docking score of −12.80 (Table 1)] to
probe the steric constraints of the linker region as well as the
electronics of a possible cation−π interaction between the aryl
ring and a conserved lysine K437. This analogue was accessed
via the analogous route from the corresponding aryl bromide
Heck coupling partner, 3-bromo-5-trifluoromethylphenol
(Figure 7).58 In addition, a p-phenyl ether analogue 6 was
synthesized as a negative control [docking score of greater than
We also designed a m-3-pyridyl ether analogue 9 [docking
score of −13.28 (Table 1)] to probe the electronics of the
possible cation−π interaction between the aryl ring and K437
without the potential steric clashes introduced by the
trifluoromethyl group of analogue 8. This required develop-
ment of an alternative synthetic route to avoid pyridine
oxidation during late-stage Ley oxidation, which also proved to
be more concise (Figure 8). Thus, initial Suzuki coupling of
commercially available aryl boronic acid 32 and vinyl bromide
33 afforded styrene 34. Ozonolysis and one-pot iodination−
E
DOI: 10.1021/acs.biochem.9b00003
Biochemistry XXXX, XXX, XXX−XXX

Biochemistry
Article
Figure 6. Synthesis of alkyl sulfamide analogue 4. Abbreviations: Boc,
tert-butoxycarbonyl; Cy = cyclohexyl; DIAD, diisopropyl azodicarbox-
ylate; TFA, trifluoroacetic acid.
elimination gave vinyl ketone 35. Heck coupling with pyridyl
bromide 36 provided phenol intermediate 37. Coupling to
protected adenosine 16 under Mitsunobu conditions gave
pyridyl ether 38. Finally, olefin reduction and two-step
deprotection provided m-3-pyridyl ether analogue 9.
Biochemical and Antibacterial Evaluation. With linker
analogues 3−9 in hand, we next evaluated their inhibition of E.
coli MenE using our previously reported MenE−MenB
coupled assay.32 Consistent with predictions from the docking
experiments, all but one of the analogues inhibited MenE,
albeit with modest IC50 values compared to that of OSB-AMS
(1) (Table 2). Only acyl squaramide analogue 3 was inactive,
contrary to prediction. The m-phenyl ether analogue 5 was the
most potent inhibitor, with an IC50 of 8.1 0.9 μM, while the
corresponding 3-trifluoromethyl-substituted analogue 8 and m-
3-pyridyl analogue 9 exhibited slightly weaker inhibition. In
contrast, the p-phenyl ether analogue 6 showed no inhibition
of MenE, consistent with its docking score of greater than −10
kcal/mol. Taken together, these results demonstrated that
computational docking could be used effectively to prioritize
OSB-AMS analogues for synthesis and biochemical evaluation
against MenE.
We next investigated the binding affinities of the two most
potent inhibitors 5 and 9 with E. coli MenE using ITC.58 The
measured Kd values of m-phenyl ether analogue 5 (244 11
nM) and m-3-pyridyl ether analogue 9 (505 14 nM) were
generally consistent with IC50 values determined in the
biochemical assay. In contrast, p-phenyl ether analogue 6,
which did not inhibit MenE, also showed no binding in the
ITC assay (Figure S1).
Figure 7. Synthesis of m-phenyl ether analogue 5. p-Phenyl ether
analogue 6 and trifluoromethyl-substituted analogue 8 were
synthesized by analogous routes from the corresponding Heck
coupling partners (cf., 29).58 Abbreviations: DIAD, diisopropyl
azodicarboxylate; DMA, dimethylacetamide; dtbpf, 1,1′-bis(di-tert-
butylphosphino)ferrocene; NMO, N-methylmorpholine N-oxide;
TBAF, tetrabutylammonium fluoride; TBS, tert-butyldimethylsilyl;
TFA, trifluoroacetic acid; THF, tetrahydrofuran; TPAP, tetrapropy-
lammonium perruthenate.
decreased biochemical potency of these linker analogues in
comparison to that of OSB-AMS (1), which may be improved
in the future through further optimization of these newly
discovered chemotypes.
X-ray Co-Crystal Structure of m-Phenyl Ether Ana-
logue 5 Bound to E. coli MenE. To understand the binding
of m-phenyl ether analogue 5 and to provide guidance for
further inhibitor design, the co-crystal structure of 5 bound to
wild-type E. coli MenE was determined at 1.8 Å resolution
(PDB entry 6NJ0).58 The structure was determined by
molecular replacement using our previously reported structure
of OSB-AMS (1)-bound E. coli MenE (R195K) (PDB entry
5C5H)34 as a search model. Data collection and refinement
statistics are listed in Table S2.
Next, all of the synthesized analogues were evaluated for
antibacterial activity against E. coli, MRSA, and B. subtilis.
Unfortunately, none of the compounds exhibited an MIC of
<100 μg/mL, compared to the MIC of 31.25 μg/mL observed
for OSB-AMS (1) against MRSA (ATCC BAA1762).34 We
posit that this lack of antimicrobial activity is due to the
Comparison of the structures of m-phenyl ether analogue 5
bound to wild-type MenE and of OSB-AMS (1) bound to
MenE (R195K) revealed differences in the orientation of the
small C-terminal domain relative to the large N-terminal
domain (Figure 9a). In the structure with m-phenyl ether
F
DOI: 10.1021/acs.biochem.9b00003
Biochemistry XXXX, XXX, XXX−XXX

Biochemistry
Article
Table 2. Enzyme Inhibition and Docking Scores of
Synthesized Compounds
Figure 8. Synthesis of m-3-pyridyl ether analogue 9. Abbreviations:
Boc, tert-butoxycarbonyl; Cy, cyclohexyl; DBU, 1,8-
diazabicyclo[5.4.0]undec-7-ene; DDQ, 2,3-dichloro-5,6-dicyano-1,4-
benzoquinone; DIAD, diisopropyl azodicarboxylate; DMA, dimethy-
lacetamide; dtbpf, 1,1′-bis(di-tert-butylphosphino)ferrocene; TFA,
trifluoroacetic acid; THF tetrahydrofuran.
analogue 5, the C-terminal domain is rotated by 22°
(determined using DynDom60) about a hinge residue D352,
away from the active site, making it “slightly open” relative to
the “closed” conformation seen in the structure with OSB-
AMS (1). Notably, apo structures of S. aureus MenE have been
reported previously, and the C-terminal domains are rotated
much more dramatically, by 144° and 151°, in a fully “open”
conformation (PDB entry 3IPL chains A and B).61 Both
“open” and “closed” states have been observed previously for
other members of the ANL family,29,62 with fully open states
typically observed only in unliganded structures.63−68 In the
closed conformations, a highly conserved lysine residue (K437
in E. coli MenE)69 serves as a linchpin that coordinates the acyl
adenylate intermediate via its acyl group carbonyl, phosphate
pro-S oxygen, ribose 4′-oxygen, and/or ribose 5′-oxy-
gen.34,64,65,70 In the OSB-AMS (1)·MenE (R195K) complex,
K437 is observed in the active site and binds OSB-AMS (1)
through these interactions. In contrast, in the complex of m-
phenyl ether analogue 5 bound to wild-type MenE, the loop
region from residue 434 to 438 that includes K437 is
disordered. We speculate that the loss of the K437−ligand
interaction may result in a decreased force holding the C-
terminal domain in the closed conformation, resulting in a
slight rotation of the C-terminal domain away from the active
site. In addition, without the interaction of K437 with the
ligand, the loop region becomes highly dynamic and
disordered.61
a
IC50 values were measured using a MenB−MenE coupled assay.32
b
All measurements were determined in triplicate. Kd = 244 11 nM
c
as determined by ITC. Kd = 505 14 nM as determined by ITC.
d
No binding detected by ITC.
Structures of B. subtilus MenE in apo form and in complex
with AMP, ATP, and OSB-AMP have also been reported
previously (PDB entries 5BUQ, 5BUS, 5BUR, and 5GTD,
respectively).71,72 In the liganded structures, the C-terminal
domain is also rotated 24° relative to that in the E. coli MenE
(R195K)·OSB-AMS (1) complex, but about a distinct axis of
rotation that allows the ligands to retain interactions with the
corresponding linchpin residue (K471 in B. subtilis MenE).
Notably, this region is again disordered in the corresponding
apo structure.
Active Site Interactions between m-Phenyl Ether
Analogue 5 and E. coli MenE. The difference in the relative
orientation of the C-terminal domain does not appear to
G
DOI: 10.1021/acs.biochem.9b00003
Biochemistry XXXX, XXX, XXX−XXX

Biochemistry
Article
Figure 9. continued
structure with analogue 5 (purple) compared to that with OSB-AMS
(1) (cyan), while larger 144° and 151° rotations of the C-terminal
domains are observed in the S. aureus apo structures (green and
yellow). The E. coli linchpin residue K437 (green) is observed in the
structure with OSB-AMS (1) but is disordered in the structure with
m-phenyl ether analogue 5. (b) Active site of wild-type MenE (cyan)
with m-phenyl ether analogue 5 (purple) bound, revealing binding
interactions (green) and a 1.5 Å shift of the ribose motif compared to
OSB-AMS (1) (gray). (c) Schematic of putative active site
interactions (green) of m-phenyl ether analogue 5 (purple) with
wild-type MenE (black).
impact the overall orientation of m-phenyl ether analogue 5 in
the MenE active site compared to that of OSB-AMS (1)
(Figure 9b). However, the geometric constraints of the phenyl
ether linker force the ribose moiety ≈1.5 Å deeper into the
adenine region of the binding pocket.
Enzyme−inhibitor interactions are highlighted in panels b
and c of Figure 9. Residues R195, S222, T277, G268
(carbonyl), D336, and R350 form hydrogen-bonding inter-
actions with m-phenyl ether analogue 5, and these residues are
conserved in E. coli, S. aureus, and M. tuberculosis MenE.34 In B.
subtilis MenE, R195 is replaced by K205, T277 by Q294, and
G268 by S285.71 E. coli residues R195, S222, and T277 are
clustered around the OSB aromatic carboxylate. Residues R195
and T277 interact directly with the carboxylate, and R195 also
engages in a second, water-mediated interaction via water-17,
which is coordinated by S222. We had previously proposed
that a direct interaction with R195 should be present on the
basis of our analysis of the OSB-AMS (1)·MenE (R195K)
crystal structure, in which the OSB carboxylate interacts with
K195 via two water-mediated interactions (Figure 2a). In
contrast, in the structure of wild-type MenE complexed with 5
reported here, the R195 guanidinium group engages the OSB
carboxylate via one direct interaction and one water-mediated
interaction. In the linker region, no direct interactions are
observed with the m-phenyl ether. Residue T272, which binds
to the pro-S oxygen of the sulfamate in OSB-AMS, remains
within 4 Å of the aromatic ring of m-phenyl ether analogue 5.
However, the linchpin K437, which interacts with multiple
atoms in the linker region of OSB-AMS, is disordered and not
observed in the structure of m-phenyl ether analogue 5. In the
ribose region, D336 interacts with the 2′- and 3′-hydroxyl
groups of m-phenyl ether analogue 5 in the same way as OSB-
AMS (1).
Binding of m-Phenyl Ether Analogue 5 to a MenE
(K437A) Linchpin Mutant. Crucially, we did not observe the
predicted interactions between K437 and the linker region of
m-phenyl ether analogue 5, because the loop of residues 434−
438 was disordered. While analogue 5 lacks the carbonyl and
phosphate pro-S oxygen of the cognate intermediate OSB-
AMP, we posited that K437 might still hydrogen bond with the
ribose 4′- and/or 5′-oxygens and could also engage in a
cation−π interaction with the phenyl ether ring, based on our
earlier docking predictions (Figure 2d).
To probe for these interactions experimentally, we compared
the binding affinity of m-phenyl ether analogue 5 for wild-type
E. coli MenE and the K437A mutant. ITC titration curves
(Figure S1) show very similar Kd values of 244 and 249 nM,
respectively, consistent with the lack of a productive binding
interaction of K437 with m-phenyl ether analogue 5.
Figure 9. X-ray co-crystal structure of m-phenyl ether analogue 5
bound to wild-type E. coli MenE. (a) Overlay of structures of E. coli
wild-type MenE with m-phenyl ether analogue 5 (gray and purple)
bound, E. coli MenE (R195K) with OSB-AMS (1) bound (gray and
cyan, PDB entry 5C5H), and S. aureus MenE apo structures (wheat
and green or yellow, PDB entry 3IPL chains A and B). Alignment of
the large N-terminal domains [E. coli residues 1−351, gray, root-
mean-square deviation (rmsd) of 0.49 Å; S. aureus residues 1−396,
wheat, rmsd of 1.64 Å relative to 5C5H] reveals a 22° rotation of the
small C-terminal domain about the E. coli hinge residue D352 in the
H
DOI: 10.1021/acs.biochem.9b00003
Biochemistry XXXX, XXX, XXX−XXX

Biochemistry
Article
Comparison of Docked and X-ray Co-Crystal Struc-
tures of m-Phenyl Ether Analogue 5 with MenE. To
evaluate the effectiveness of the docking prediction, we
compared the binding positions of m-phenyl ether analogue
5 when docked with MenE (R195K) or co-crystallized with
wild-type MenE, relative to that of OSB-AMS (1) from its co-
crystal structure with MenE (R195K)34 (Figure S2). Overall,
there was good overlap between the positions of the docked
and co-crystallized inhibitors (root-mean-square deviation of
1.26 Å), with the most significant differences in the aryl linker
(rmsd = 1.56 Å) and ribose (rmsd = 1.79 Å) regions. In the
docked structure of m-phenyl ether analogue 5, the adenosine
region of the inhibitor overlapped well with that of co-
crystallized OSB-AMS (1) (rmsd = 0.63 Å). However, the aryl
ether linker of analogue 5 protrudes farther from the
adenosine-binding pocket than the acyl sulfamate linker of
OSB-AMS (1). Nonetheless, conformational differences in the
succinyl chain allow the OSB aromatic rings to overlap well
(rmsd = 0.76 Å). In contrast, in the co-crystal structure of m-
phenyl ether analogue 5, both the OSB and linker regions
overlap well with co-crystallized OSB-AMS (1) (rmsd = 0.35 Å
for the OSB aromatic ring), resulting in the ribose motif being
pushed deeper into the adenosine-binding pocket (rmsd = 1.51
Å). Notably, this translation of the ribose ring deeper into the
adenosine-binding pocket moves the ribose 5′-oxygen by 2.0 Å
relative to OSB-AMS (1), out of position to hydrogen bond
productively with K437 as positioned in the co-crystal
structure with OSB-AMS (1). However, the linker aryl ring
of 5 appears to be positioned even more favorably for a
cation−π interaction with K437 than in the docked structure,
so it is still possible that this interaction may be captured in
future analogue designs.
chemotypes: acyl squaramide (3), alkyl sulfamide (4), phenyl
ether (5), and α-hydroxytetrazole (7). Three of four of these
analogues inhibited E. coli MenE with IC50 valuess in the low-
to mid-micromolar range, validating the overall effectiveness of
docking in prioritization of analogues. However, the docking
scores did not accurately predict the rank ordering of inhibitor
potency between chemotypes, and we posit that this is because
binding interactions with MenE are dominated by the OSB
and adenosine motifs, which were invariant across all virtual
library members. Thus, the docking simply served to predict
whether each linker provided suitable geometry for directing
these binding anchors to the appropriate pockets. Consistent
with this interpretation, p-phenyl ether analogue 6 was
predicted not to provide a suitable binding geometry, and
this was confirmed in biochemical evaluation of this analogue.
On the other hand, docking scores within the m-aryl ether
chemotype (5, 8, and 9) did correlate with the rank ordering of
experimentally determined IC50 values. Thus, it remains to be
seen if such more quantitative predictions can be made
robustly across a larger panel of analogues within a given linker
chemotype.
In the co-crystal structure of m-phenyl ether analogue 5 with
wild-type E. coli MenE, the C-terminal lobe adopts a “slightly
open” conformation relative to the large N-terminal domain.
This is in contrast to the “closed” conformation that we
observed previously in the co-crystal structure of OSB-AMS
(1) with E. coli MenE (R195K) and to the fully “open”
conformations observed in the apo structures of S. aureus
MenE.61 For OSB-AMS (1), the conserved linchpin residue
K437 binds the acyl sulfamate and may stabilize the closed
conformation. However, in the case of m-phenyl ether
analogue 5, both the co-crystal structure and the similar Kd
values observed for wild-type and K437A E. coli MenE suggest
that this analogue does not interact with K437, and this may
lead to the “slightly open” conformation and disorder of
residues 434−438 that comprise the loop that includes K437.
Comparison of the available MenE structures reveals how
the C- and N-terminal domains may move relative to each
other in the course of binding substrates or inhibitors. We
posit that the ligand binds to the protein in an open
conformation, and subsequently, the small C-terminal domain
rotates toward the active site of the large N-terminal domain,
leading to an interaction between the ligand and K437 that
stabilizes the closed state. Consistent with this interpretation,
within the ANL superfamily, fully “open” conformations have
also been reported previously for apo structures of luciferase63
and long chain fatty acyl-CoA synthetase,65 and in both cases,
co-crystallization with the cognate adenylate or a mimic
thereof yields the “closed” conformation.64,65 It must be noted,
however, that Gulick has reported a similar “slightly open”
conformation for 4-chlorobenzoate-CoA ligase in apo, 4-
chlorobenzoate substrate-bound, and adenylate intermediate-
bound forms, with the linchpin K492 side chain disordered in
all cases,83,84 and it is unclear why the fully closed
conformation was not observed in the adenylate-bound
structure. In addition, Guo has reported structures of B.
subtilis MenE in apo form and in complex with AMP, ATP, and
OSB-AMP, all adopting a similar “closed” conformation in
which the linchpin residue (K471 in B. subtilis MenE) interacts
with the ligand,71,72 highlighting the fact that not all apo
structures of ANL enzymes result in “open” conforma-
tions.85,86
DISCUSSION
■
Acyl-AMS intermediate analogues have been used widely to
inhibit ANL family members and other adenylate-forming
enzymes.73−81 Several of these compounds have advanced to in
vivo proof-of-concept studies in animal models.42,75,82 How-
ever, the acyl sulfamate moiety carries several pharmacological
liabilities, including a negative charge that can be associated
with decreased penetration of Gram-negative bacteria,38,39 high
plasma protein binding,41 rapid renal clearance,40 and potential
metabolic instability.42 Thus, replacement of the acyl sulfamate
linker with stable, neutral isosteres would represent an
important advance, providing a path toward pharmacologically
optimized inhibitors of the adenylate-forming enzyme.
We previously reported the synthesis and characterization of
OSB-AMS (1), an acyl adenylate analogue that inhibits E. coli
MenE with an IC50 of 24
3 nM.34 However, despite this
potent biochemical inhibition, OSB-AMS has a modest MIC of
31.25 μg/mL against MRSA. We speculated that the poor
antibacterial activity of OSB-AMS could result from the double
negative charge carried by this molecule at physiological pH.
Having recently discovered a difluoroindanediol analogue of
the OSB side chain that removes one of these negative
charges,35 we set out to develop a new generation of analogues
in which the acyl sulfamate linker was replaced with an
uncharged motif. As previous efforts to develop new acyl
phosphate bioisosteres have proven to be challenging,43−50 we
designed a virtual library of linker analogues and docked these
candidates to our previously reported structure of E. coli MenE
(R195K).34 We used the docking results to prioritize
compounds for synthesis, focusing on four main bioisostere
I
DOI: 10.1021/acs.biochem.9b00003
Biochemistry XXXX, XXX, XXX−XXX

Biochemistry
Article
Notably, Gulick has identified a distinct, ≈140° rotation
about the hinge residue that generates a second, distinct
“closed” conformation in co-crystal structures of acetyl-CoA
synthetase with the adenylate mimic propyl-AMP and the co-
substrate CoA, as well as of 4-chlorobenzoate-CoA synthetase
with a product analogue 4-chlorophenacyl-CoA.87,88 This
conformation has also been observed recently in the structure
of B. subtilis MenE with a stable OSB-CoA analogue.89 This
“domain alternation” introduces new residues into the active
site to catalyze thioesterification in the second half-reaction.
Thus, rotation of the C-terminal domain about the hinge
residue may be involved in both substrate binding and catalysis
of the first half-reaction as well as CoA binding and catalysis of
the second half-reaction.
Derek S. Tan: 0000-0002-7956-9659
Author Contributions
#C.E.E. and Y.S. contributed equally to this work.
Funding
This work was supported by the National Institutes of Health
(NIH) (R01 GM100477 to D.S.T., R01 GM102864 to P.J.T.,
R35 GM124898 to J.B.F., T32 GM073546-Gross to C.E.E.,
and Cancer Center Support Grant P30 CA008748 to C. B.
Thompson) and a W. Burghardt Turner Fellowship (to
J.S.M.). The National Synchrotron Light Source is supported
by National Institutes of Health Grant GM0080 (supplement
to a PSI program) and U.S. Department of Energy Contract
DE-AC02-98CH10886.
The structure reported in this work also reveals a direct
interaction between E. coli MenE R195 and the OSB
carboxylate group, as proposed previously.34 While the acyl
sulfamate moiety of OSB-AMS (1) is bound by the linchpin
residue K437 as well as T272, the phenyl ether linker of
analogue 5 does not interact with these residues. A cation−π
interaction with K437 appeared to be possible on the basis of
the docking model, but this was not observed in the co-crystal
structure or evident in ITC binding assays to a K437A mutant.
The loss of these two interactions may account for the ≈2 log
weaker IC50 value of m-phenyl ether analogue 5 compared to
that of OSB-AMS (1). Conversely, however, the m-phenyl
ether linker is uncharged, hydrophobic, and not susceptible to
hydrolysis, making it an attractive platform for further
optimization to develop more potent, non-acyl sulfamate
MenE inhibitors. Combination of these optimized linkers with
our difluoroindanediol replacement for the OSB side chain35
could then provide analogues in which both negative charges
have been removed, affording improved pharmacological
properties.
Notes
The authors declare the following competing financial
interest(s): D.S.T., P.J.T., C.E.E., and J.S.M. are co-inventors
on International Patent Application PCT/US2016/055136;
D.S.T., P.J.T., C.E.E., and Y.S. are co-inventors on U.S.
Provisional Patent Application 62/802,650.
ACKNOWLEDGMENTS
■
The authors thank Dr. Stephen Walker (Stony Brook
University) for assistance with BSL2 studies and Dr. George
Sukenick, Rong Wang, and Dr. Sylvi Rusli (Memorial Sloan
Kettering Cancer Center) for expert NMR and mass
̈
spectrometry support. Access to the Schrodinger Small-
Molecule Drug Discovery Suite was generously provided by
the Sanders Innovation & Education Initiative of the Tri-
Institutional Therapeutics Discovery Institute.
REFERENCES
■
(1) Brown, E. D., and Wright, G. D. (2016) Antibacterial drug
discovery in the resistance era. Nature (London, U. K.) 529, 336−343.
(2) Lewis, K. (2013) Platforms for antibiotic discovery. Nat. Rev.
Drug Discovery 12, 371−387.
(3) Centers for Disease Control and Prevention (2013) Antibiotic
Resistance Threats in the United States, 2013. http://www.cdc.gov/
drugresistance/threat-report-2013/.
In conclusion, our studies show that replacement of the acyl
sulfamate moiety found in most inhibitors of the adenylate-
forming enzyme is feasible and provide a path forward for the
development of new MenE inhibitors. This strategy of
analogue prioritization using computational docking may also
be applicable to other adenylate-forming enzymes that have
previously been targeted with acyl sulfamate-based inhibitors.
(4) World Health Organization (2014) Antimicrobial resistance:
Global report on surveillance 2014. http://www.who.int/
drugresistance/documents/surveillancereport/en/.
(5) The Pew Charitable Trusts (2016) A Scientific Roadmap for
Antibiotic Discovery. https://www.pewtrusts.org/en/research-and-
analysis/reports/2016/05/a-scientific-roadmap-for-antibiotic-
discovery.
(6) Meganathan, R., and Kwon, O. (2009) Biosynthesis of
menaquinone (vitamin K2) and ubiquinone (Coenzyme Q). EcoSal
Plus 3, 173−218.
(7) Meganathan, R. (2001) Biosynthesis of menaquinone (vitamin
K2) and ubiquinone (coenzyme Q): A perspective on enzymatic
mechanisms. Vitam. Horm. 61, 173−218.
(8) Walther, B., Karl, J. P., Booth, S. L., and Boyaval, P. (2013)
Menaquinones, bacteria, and the food supply: The relevance of dairy
and fermented food products to vitamin K requirements. Adv. Nutr. 4,
463−473.
(9) Dowd, P., Ham, S. W., Naganathan, S., and Hershline, R. (1995)
The mechanism of action of vitamin K. Annu. Rev. Nutr. 15, 419−440.
(10) Danziger, J. (2008) Vitamin K-dependent proteins, warfarin,
and vascular calcification. Clin. J. Am. Soc. Nephrol. 3, 1504−1510.
(11) Nakagawa, K., Hirota, Y., Sawada, N., Yuge, N., Watanabe, M.,
Uchino, Y., Okuda, N., Shimomura, Y., Suhara, Y., and Okano, T.
(2010) Identification of UBIAD1 as a novel human menaquinone-4
biosynthetic enzyme. Nature 468, 117−121.
ASSOCIATED CONTENT
* Supporting Information
■
S
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.bio-
chem.9b00003.
Complete experimental procedures and analytical data
for all new compounds (PDF)
Accession Codes
MenE, UniProtKB entry P37353.
AUTHOR INFORMATION
Corresponding Authors
*E-mail: peter.tonge@stonybrook.edu. Telephone: (631) 632-
7907.
■
*E-mail: tand@mskcc.org. Telephone: (646) 888-2234.
ORCID
Christopher E. Evans: 0000-0001-9777-4114
Jarrod B. French: 0000-0002-6762-1309
Peter J. Tonge: 0000-0003-1606-3471
J
DOI: 10.1021/acs.biochem.9b00003
Biochemistry XXXX, XXX, XXX−XXX

Biochemistry
Article
(12) Paudel, A., Hamamoto, H., Panthee, S., and Sekimizu, K.
(2016) Menaquinone as a potential target of antibacterial agents.
Drug Discoveries Ther. 10, 123−128.
P., Moir, A., Moriya, S., Nagakawa, E., Nanamiya, H., Nakai, S.,
Nygaard, P., Ogura, M., Ohanan, T., O’Reilly, M., O’Rourke, M.,
Pragai, Z., Pooley, H. M., Rapoport, G., Rawlins, J. P., Rivas, L. A.,
Rivolta, C., Sadaie, A., Sadaie, Y., Sarvas, M., Sato, T., Saxild, H. H.,
Scanlan, E., Schumann, W., Seegers, J. F., Sekiguchi, J., Sekowska, A.,
Seror, S. J., Simon, M., Stragier, P., Studer, R., Takamatsu, H., Tanaka,
T., Takeuchi, M., Thomaides, H. B., Vagner, V., van Dijl, J. M.,
Watabe, K., Wipat, A., Yamamoto, H., Yamamoto, M., Yamamoto, Y.,
Yamane, K., Yata, K., Yoshida, K., Yoshikawa, H., Zuber, U., and
Ogasawara, N. (2003) Essential Bacillus subtilis genes. Proc. Natl.
Acad. Sci. U. S. A. 100, 4678−4683.
(27) Akerley, B. J., Rubin, E. J., Novick, V. L., Amaya, K., Judson, N.,
and Mekalanos, J. J. (2002) A genome-scale analysis for identification
of genes required for growth or survival of Haemophilus influenzae.
Proc. Natl. Acad. Sci. U. S. A. 99, 966−971.
(28) Chen, W. H., Lu, G., Chen, X., Zhao, X. M., and Bork, P.
(2017) OGEE v2: An update of the online gene essentiality database
with special focus on differentially essential genes in human cancer
cell lines. Nucleic Acids Res. 45, D940−D944.
(29) Gulick, A. M. (2009) Conformational dynamics in the acyl-
CoA synthetases, adenylation domains of non-ribosomal peptide
synthetases, and firefly luciferase. ACS Chem. Biol. 4, 811−827.
(30) Meganathan, R., and Bentley, R. (1979) Menaquinone (vitamin
K2) biosynthesis: conversion of o-succinylbenzoic acid to 1,4-
dihydroxy-2-naphthoic acid by Mycobacterium phlei enzymes. J.
Bacteriol. 140, 92−98.
(31) Kwon, O., Bhattacharyya, D. K., and Meganathan, R. (1996)
Menaquinone (vitamin K2) biosynthesis: Overexpression, purification,
and properties of o-succinylbenzoyl-coenzyme A synthetase from
Escherichia coli. J. Bacteriol. 178, 6778−6781.
(32) Lu, X., Zhang, H., Tonge, P. J., and Tan, D. S. (2008)
Mechanism-based inhibitors of MenE, an acyl-CoA synthetase
involved in bacterial menaquinone biosynthesis. Bioorg. Med. Chem.
Lett. 18, 5963−5966.
(33) Lu, X., Zhou, R., Sharma, I., Li, X., Kumar, G., Swaminathan, S.,
Tonge, P. J., and Tan, D. S. (2012) Stable analogues of OSB-AMP:
Potent inhibitors of MenE, the o-succinylbenzoate-CoA synthetase
from bacterial menaquinone biosynthesis. ChemBioChem 13, 129−
136.
(13) Jiang, M., Chen, X., Guo, Z. F., Cao, Y., Chen, M., and Guo, Z.
(2008) Identification and characterization of (1R,6R)-2-succinyl-6-
hydroxy-2,4-cyclohexadiene-1-carboxylate synthase in the menaqui-
none biosynthesis of Escherichia coli. Biochemistry 47, 3426−3434.
(14) Begley, T. P., Kinsland, C., Taylor, S., Tandon, M., Nicewonger,
R., Wu, M., Chiu, H.-J., Kelleher, N., Campobasso, N., and Zhang, Y.
(1998) Cofactor biosynthesis: A mechanistic perspective. In Biosyn-
thesis, pp 93−142, Springer.
(15) Fang, M., Toogood, R. D., Macova, A., Ho, K., Franzblau, S. G.,
McNeil, M. R., Sanders, D. A., and Palmer, D. R. (2010)
Succinylphosphonate esters are competitive inhibitors of MenD that
show active-site discrimination between homologous α-ketoglutarate-
decarboxylating enzymes. Biochemistry 49, 2672−2679.
(16) Fang, M., Macova, A., Hanson, K. L., Kos, J., and Palmer, D. R.
(2011) Using substrate analogues to probe the kinetic mechanism and
active site of Escherichia coli MenD. Biochemistry 50, 8712−8721.
(17) Pulaganti, M., Banaganapalli, B., Mulakayala, C., Chitta, S. K.,
and Anuradha, C. M. (2014) Molecular modeling and docking studies
of o-succinylbenzoate synthase of M. tuberculosis − A potential target
for antituberculosis drug design. Appl. Biochem. Biotechnol. 172,
1407−1432.
(18) Tian, Y., Suk, D. H., Cai, F., Crich, D., and Mesecar, A. D.
(2008) Bacillus anthracis o-succinylbenzoyl-CoA synthetase: Reaction
kinetics and a novel inhibitor mimicking its reaction intermediate.
Biochemistry 47, 12434−12447.
(19) Li, X., Liu, N., Zhang, H., Knudson, S. E., Li, H. J., Lai, C. T.,
Simmerling, C., Slayden, R. A., and Tonge, P. J. (2011) CoA adducts
of 4-oxo-4-phenylbut-2-enoates: Inhibitors of MenB from the M.
tuberculosis menaquinone biosynthesis pathway. ACS Med. Chem. Lett.
2, 818−823.
(20) Li, X., Liu, N., Zhang, H., Knudson, S. E., Slayden, R. A., and
Tonge, P. J. (2010) Synthesis and SAR studies of 1,4-benzoxazine
MenB inhibitors: Novel antibacterial agents against Mycobacterium
tuberculosis. Bioorg. Med. Chem. Lett. 20, 6306−6309.
(21) Kurosu, M., and Crick, D. C. (2009) MenA is a promising drug
target for developing novel lead molecules to combat Mycobacterium
tuberculosis. Med. Chem. 5, 197−207.
(22) Dhiman, R. K., Mahapatra, S., Slayden, R. A., Boyne, M. E.,
Lenaerts, A., Hinshaw, J. C., Angala, S. K., Chatterjee, D., Biswas, K.,
Narayanasamy, P., Kurosu, M., and Crick, D. C. (2009) Menaquinone
synthesis is critical for maintaining mycobacterial viability during
exponential growth and recovery from non-replicating persistence.
Mol. Microbiol. 72, 85−97.
(34) Matarlo, J. S., Evans, C. E., Sharma, I., Lavaud, L. J., Ngo, S. C.,
Shek, R., Rajashankar, K. R., French, J. B., Tan, D. S., and Tonge, P. J.
(2015) Mechanism of MenE inhibition by acyl-adenylate analogues
and discovery of novel antibacterial agents. Biochemistry 54, 6514−
6524.
(35) Evans, C. E., Matarlo, J. S., Tonge, P. J., and Tan, D. S. (2016)
Stereoselective synthesis, docking, and biological evaluation of
difluoroindanediol-based MenE inhibitors as antibiotics. Org. Lett.
18, 6384−6387.
(36) Isono, K., Uramoto, M., Kusakabe, H., Miyata, N., Koyama, T.,
Ubukata, M., Sethi, S. K., and McCloskey, J. A. (1984) Ascamycin and
dealanylascamycin, nucleoside antibiotics from Streptomyces sp. J.
Antibiot. 37, 670−672.
(37) Ueda, H., Shoku, Y., Hayashi, N., Mitsunaga, J., In, Y., Doi, M.,
Inoue, M., and Ishida, T. (1991) X-ray crystallographic conforma-
tional study of 5′-O-[N-(L-alanyl)-sulfamoyl]adenosine, a substrate
analog for alanyl-tRNA synthetase. Biochim. Biophys. Acta, Protein
Struct. Mol. Enzymol. 1080, 126−134.
(38) Yoshimura, F., and Nikaido, H. (1985) Diffusion of β-lactam
antibiotics through the porin channels of Escherichia coli K-12.
Antimicrob. Agents Chemother. 27, 84−92.
(39) Richter, M. F., Drown, B. S., Riley, A. P., Garcia, A., Shirai, T.,
Svec, R. L., and Hergenrother, P. J. (2017) Predictive compound
accumulation rules yield a broad-spectrum antibiotic. Nature 545,
299−304.
(40) Varma, M. V. S., Feng, B., Obach, R. S., Troutman, M. D.,
Chupka, J., Miller, H. R., and El-Kattan, A. (2009) Physicochemical
determinants of human renal clearance. J. Med. Chem. 52, 4844−4852.
(23) Kurosu, M., and Begari, E. (2010) Vitamin K2 in electron
transport system: are enzymes involved in vitamin K2 biosynthesis
promising drug targets? Molecules 15, 1531−1553.
(24) Li, K., Schurig-Briccio, L. A., Feng, X., Upadhyay, A., Pujari, V.,
Lechartier, B., Fontes, F. L., Yang, H., Rao, G., Zhu, W., Gulati, A.,
No, J. H., Cintra, G., Bogue, S., Liu, Y. L., Molohon, K., Orlean, P.,
Mitchell, D. A., Freitas-Junior, L., Ren, F., Sun, H., Jiang, T., Li, Y.,
Guo, R. T., Cole, S. T., Gennis, R. B., Crick, D. C., and Oldfield, E.
(2014) Multitarget drug discovery for tuberculosis and other
infectious diseases. J. Med. Chem. 57, 3126−3139.
(25) Sassetti, C. M., Boyd, D. H., and Rubin, E. J. (2003) Genes
required for mycobacterial growth defined by high density muta-
genesis. Mol. Microbiol. 48, 77−84.
(26) Kobayashi, K., Ehrlich, S. D., Albertini, A., Amati, G., Andersen,
K. K., Arnaud, M., Asai, K., Ashikaga, S., Aymerich, S., Bessieres, P.,
Boland, F., Brignell, S. C., Bron, S., Bunai, K., Chapuis, J.,
Christiansen, L. C., Danchin, A., Debarbouille, M., Dervyn, E.,
Deuerling, E., Devine, K., Devine, S. K., Dreesen, O., Errington, J.,
Fillinger, S., Foster, S. J., Fujita, Y., Galizzi, A., Gardan, R., Eschevins,
C., Fukushima, T., Haga, K., Harwood, C. R., Hecker, M., Hosoya, D.,
Hullo, M. F., Kakeshita, H., Karamata, D., Kasahara, Y., Kawamura, F.,
Koga, K., Koski, P., Kuwana, R., Imamura, D., Ishimaru, M., Ishikawa,
S., Ishio, I., Le Coq, D., Masson, A., Mauel, C., Meima, R., Mellado, R.
K
DOI: 10.1021/acs.biochem.9b00003
Biochemistry XXXX, XXX, XXX−XXX

Biochemistry
Article
(41) Charifson, P. S., and Walters, W. P. (2014) Acidic and basic
drugs in medicinal chemistry: A perspective. J. Med. Chem. 57, 9701−
9717.
(42) Tiwari, D., Park, S. W., Essawy, M. M., Dawadi, S., Mason, A.,
Nandakumar, M., Zimmerman, M., Mina, M., Ho, H. P., Engelhart, C.
A., Ioerger, T., Sacchettini, J. C., Rhee, K., Ehrt, S., Aldrich, C. C.,
Dartois, V., and Schnappinger, D. (2018) Targeting protein
biotinylation enhances tuberculosis chemotherapy. Sci. Transl. Med.
10, eaal1803.
New results on citrate synthase and T4 lysozyme. Proteins: Struct.,
Funct., Genet. 30, 144−154.
(61) Patskovsky, Y., Toro, R., Dickey, M., Sauder, J. M., Chang, S.,
Burley, S. K., and Almo, S. C. (2009) Crystal structure of o-
succinylbenzoic acid-CoA ligase from Staphylococcus aureus subsp.
aureus Mu50. Protein Data Bank. DOI: 10.2210/pdb3IPL/pdb.
(62) Gulick, A. M. ANL Super Family Solved Structures. http://www.
acsu.buffalo.edu/~amgulick/RANLChart.html (Accessed on March 6,
2019).
(43) Soares da Costa, T. P., Tieu, W., Yap, M. Y., Pendini, N. R.,
Polyak, S. W., Sejer Pedersen, D., Morona, R., Turnidge, J. D.,
Wallace, J. C., Wilce, M. C. J., Booker, G. W., and Abell, A. D. (2012)
Selective inhibition of biotin protein ligase from Staphylococcus aureus.
J. Biol. Chem. 287, 17823−17832.
(44) Somu, R. V., Boshoff, H., Qiao, C. H., Bennett, E. M., Barry, C.
E., and Aldrich, C. C. (2006) Rationally designed nucleoside
antibiotics that inhibit siderophore biosynthesis of Mycobacterium
tuberculosis. J. Med. Chem. 49, 31−34.
(45) Vannada, J., Bennett, E. M., Wilson, D. J., Boshoff, H. I., Barry,
C. E., and Aldrich, C. C. (2006) Design, synthesis, and biological
evaluation of β-ketosulfonamide adenylation inhibitors as potential
antitubercular agents. Org. Lett. 8, 4707−4710.
(46) Qiao, C. H., Gupte, A., Boshoff, H. I., Wilson, D. J., Bennett, E.
M., Somu, R. V., Barry, C. E., and Aldrich, C. C. (2007) 5 ’-O-[(N-
Acyl)sulfamoyl]adenosines as antitubercular agents that inhibit MbtA:
An adenylation enzyme required for siderophore biosynthesis of the
mycobactins. J. Med. Chem. 50, 6080−6094.
(47) Engelhart, C. A., and Aldrich, C. C. (2013) Synthesis of
chromone, quinolone, and benzoxazinone sulfonamide nucleosides as
conformationally constrained inhibitors of adenylating enzymes
required for siderophore biosynthesis. J. Org. Chem. 78, 7470−7481.
(48) Dawadi, S., Boshoff, H. I. M., Park, S. W., Schnappinger, D.,
and Aldrich, C. C. (2018) Conformationally constrained cinnolinone
nucleoside analogues as siderophore biosynthesis inhibitors for
tuberculosis. ACS Med. Chem. Lett. 9, 386−391.
(49) Callahan, B. P., Lomino, J. V., and Wolfenden, R. (2006)
Nanomolar inhibition of the enterobactin biosynthesis enzyme, EntE:
Synthesis, substituent effects, and additivity. Bioorg. Med. Chem. Lett.
16, 3802−3805.
(63) Conti, E., Franks, N. P., and Brick, P. (1996) Crystal structure
of firefly luciferase throws light on a superfamily of adenylate-forming
enzymes. Structure 4, 287−298.
(64) Nakatsu, T., Ichiyama, S., Hiratake, J., Saldanha, A., Kobashi,
N., Sakata, K., and Kato, H. (2006) Structural basis for the spectral
difference in luciferase bioluminescence. Nature 440, 372−376.
(65) Hisanaga, Y., Ago, H., Nakagawa, N., Hamada, K., Ida, K.,
Yamamoto, M., Hori, T., Arii, Y., Sugahara, M., Kuramitsu, S.,
Yokoyama, S., and Miyano, M. (2004) Structural basis of the
substrate-specific two-step catalysis of long chain fatty acyl-CoA
synthetase dimer. J. Biol. Chem. 279, 31717−31726.
(66) Andersson, C. S., Lundgren, C. A. K., Magnusdottir, A., Ge, C.,
Wieslander, A., Molina, D. M., and Hoegbom, M. (2012) The
Mycobacterium tuberculosis very-long-chain fatty acyl-CoA synthetase:
Structural basis for housing lipid substrates longer than the enzyme.
Structure 20, 1062−1070.
(67) Vergnolle, O., Xu, H., Tufariello, J. M., Favrot, L., Malek, A. A.,
Jacobs, W. R., and Blanchard, J. S. (2016) Post-translational
acetylation of MbtA modulates mycobacterial siderophore biosyn-
thesis. J. Biol. Chem. 291, 22315−22326.
(68) Liu, Z., Ioerger, T. R., Wang, F., and Sacchettini, J. C. (2013)
Structures of Mycobacterium tuberculosis FadD10 protein reveal a new
type of adenylate-forming enzyme. J. Biol. Chem. 288, 18473−18483.
(69) Stachelhaus, T., Mootz, H. D., and Marahiel, M. A. (1999) The
specificity-conferring code of adenylation domains in nonribosomal
peptide synthetases. Chem. Biol. 6, 493−505.
(70) Conti, E., Stachelhaus, T., Marahiel, M. A., and Brick, P. (1997)
Structural basis for the activation of phenylalanine in the non-
ribosomal biosynthesis of gramicidin S. EMBO J. 16, 4174−4183.
(71) Chen, Y., Sun, Y., Song, H., and Guo, Z. (2015) Structural basis
for the ATP-dependent configuration of adenylation active site in
Bacillus subtilis o-succinylbenzoyl-CoA synthetase. J. Biol. Chem. 290,
23971−23983.
(72) Chen, Y., Jiang, Y., and Guo, Z. (2016) Mechanistic insights
from the crystal structure of Bacillus subtilis o-succinylbenzoyl-CoA
synthetase complexed with the adenylate intermediate. Biochemistry
55, 6685−6695.
(73) Finking, R., Neumueller, A., Solsbacher, J., Konz, D.,
Kretzschmar, G., Schweitzer, M., Krumm, T., and Marahiel, M. A.
(2003) Aminoacyl adenylate substrate analogues for the inhibition of
adenylation domains of nonribosomal peptide synthetases. Chem-
BioChem 4, 903−906.
(74) Branchini, B. R., Murtiashaw, M. H., Carmody, J. N., Mygatt, E.
E., and Southworth, T. L. (2005) Synthesis of an N-acyl sulfamate
analog of luciferyl-AMP: A stable and potent inhibitor of firefly
luciferase. Bioorg. Med. Chem. Lett. 15, 3860−3864.
(50) Bisseret, P., Thielges, S., Bourg, S., Miethke, M., Marahiel, M.
A., and Eustache, J. (2007) Synthesis of a 2-indolylphosphonamide
derivative with inhibitory activity against yersiniabactin biosynthesis.
Tetrahedron Lett. 48, 6080−6083.
(51) Kabsch, W. (2010) Xds. Acta Crystallogr., Sect. D: Biol.
Crystallogr. 66, 125−132.
(52) Evans, P. R., and Murshudov, G. N. (2013) How good are my
data and what is the resolution? Acta Crystallogr., Sect. D: Biol.
Crystallogr. 69, 1204−1214.
(53) Vagin, A., and Teplyakov, A. (2010) Molecular replacement
with MOLREP. Acta Crystallogr., Sect. D: Biol. Crystallogr. 66, 22−25.
(54) Emsley, P., and Cowtan, K. (2004) Coot: Model-building tools
for molecular graphics. Acta Crystallogr., Sect. D: Biol. Crystallogr. 60,
2126−2132.
(55) Murshudov, G. N., Vagin, A. A., and Dodson, E. J. (1997)
Refinement of macromolecular structures by the maximum-likelihood
method. Acta Crystallogr., Sect. D: Biol. Crystallogr. 53, 240−255.
(75) Kim, S., Lee, S. W., Choi, E. C., and Choi, S. Y. (2003)
Aminoacyl-tRNA synthetases and their inhibitors as a novel family of
antibiotics. Appl. Microbiol. Biotechnol. 61, 278−288.
(76) Koroniak, L., Ciustea, M., Gutierrez, J. A., and Richards, N. G.
(2003) Synthesis and characterization of an N-acylsulfonamide
inhibitor of human asparagine synthetase. Org. Lett. 5, 2033.
(77) Brown, P. H., Cronan, J. E., Grotli, M., and Beckett, D. (2004)
The biotin repressor: Modulation of allostery by corepressor analogs.
J. Mol. Biol. 337, 857−869.
(56) Schuttelkopf, A. W., and Van Aalten, D. M. (2004) PRODRG:
̈
A tool for high-throughput crystallography of protein−ligand
complexes. Acta Crystallogr., Sect. D: Biol. Crystallogr. 60, 1355−1363.
(57) PyMOL Molecular Graphics System, version 1.8 (2017)
̈
Schrodinger, LLC.
(58) See the Supporting Information for complete details.
(59) Ferreras, J. A., Ryu, J. S., Di Lello, F., Tan, D. S., and Quadri, L.
E. N. (2005) Small-molecule inhibition of siderophore biosynthesis in
Mycobacterium tuberculosis and Yersinia pestis. Nat. Chem. Biol. 1, 29−
32.
(78) Tuck, K. L., Saldanha, S. A., Birch, L. M., Smith, A. G., and
Abell, C. (2006) The design and synthesis of inhibitors of
pantothenate synthetase. Org. Biomol. Chem. 4, 3598−3610.
(60) Hayward, S., and Berendsen, H. J. C. (1998) Systematic
analysis of domain motions in proteins from conformational change:
L
DOI: 10.1021/acs.biochem.9b00003
Biochemistry XXXX, XXX, XXX−XXX

Biochemistry
Article
(79) Lu, X., Olsen, S. K., Capili, A. D., Cisar, J. S., Lima, C. D., and
Tan, D. S. (2010) Designed semisynthetic protein inhibitors of Ub/
Ubl E1 activating enzymes. J. Am. Chem. Soc. 132, 1748−1749.
(80) Olsen, S. K., Capili, A. D., Lu, X., Tan, D. S., and Lima, C. D.
(2010) Active site remodelling accompanies thioester bond formation
in the SUMO E1. Nature 463, 906−912.
(81) Brownell, J. E., Sintchak, M. D., Gavin, J. M., Liao, H., Bruzzese,
F. J., Bump, N. J., Soucy, T. A., Milhollen, M. A., Yang, X., Burkhardt,
A. L., Ma, J., Loke, H. K., Lingaraj, T., Wu, D., Hamman, K. B.,
Spelman, J. J., Cullis, C. A., Langston, S. P., Vyskocil, S., Sells, T. B.,
Mallender, W. D., Visiers, I., Li, P., Claiborne, C. F., Rolfe, M., Bolen,
J. B., and Dick, L. R. (2010) Substrate-assisted inhibition of ubiquitin-
like protein-activating enzymes: the NEDD8 E1 inhibitor MLN4924
forms a NEDD8-AMP mimetic in situ. Mol. Cell 37, 102−111.
(82) Lun, S., Guo, H., Adamson, J., Cisar, J. S., Davis, T. D.,
Chavadi, S. S., Warren, J. D., Quadri, L. E. N., Tan, D. S., and Bishai,
W. R. (2013) Pharmacokinetic and in vivo efficacy studies of the
mycobactin biosynthesis inhibitor salicyl-AMS in mice. Antimicrob.
Agents Chemother. 57, 5138−5140.
(83) Gulick, A. M., Lu, X., and Dunaway-Mariano, D. (2004) Crystal
structure of 4-chlorobenzoate:CoA ligase/synthetase in the un-
liganded and aryl substrate-bound states. Biochemistry 43, 8670−8679.
(84) Reger, A. S., Wu, R., Dunaway-Mariano, D., and Gulick, A. M.
(2008) Structural characterization of a 140° domain movement in the
two-step reaction catalyzed by 4-chlorobenzoate:CoA ligase. Bio-
chemistry 47, 8016−8025.
(85) May, J. J., Kessler, N., Marahiel, M. A., and Stubbs, M. T.
(2002) Crystal structure of DhbE, an archetype for aryl acid activating
domains of modular nonribosomal peptide synthetases. Proc. Natl.
Acad. Sci. U. S. A. 99, 12120−12125.
(86) Hu, Y., Gai, Y., Yin, L., Wang, X., Feng, C., Feng, L., Li, D.,
Jiang, X.-N., and Wang, D.-C. (2010) Crystal structures of a Populus
tomentosa 4-coumarate:CoA ligase shed light on its enzymatic
mechanisms. Plant Cell 22, 3093−3104.
(87) Gulick, A. M., Starai, V. J., Horswill, A. R., Homick, K. M., and
Escalante-Semerena, J. C. (2003) The 1.75 A crystal structure of
acetyl-CoA synthetase bound to adenosine-5’-propylphosphate and
coenzyme A. Biochemistry 42, 2866−2873.
(88) Reger, A. S., Carney, J. M., and Gulick, A. M. (2007)
Biochemical and crystallographic analysis of substrate binding and
conformational changes in acetyl-CoA synthetase. Biochemistry 46,
6536−6546.
(89) Chen, Y., Li, T. L., Lin, X., Li, X., Li, X. D., and Guo, Z. (2017)
Crystal structure of the thioesterification conformation of Bacillus
subtilis o-succinylbenzoyl-CoA synthetase reveals a distinct substrate-
binding mode. J. Biol. Chem. 292, 12296−12310.
M
DOI: 10.1021/acs.biochem.9b00003
Biochemistry XXXX, XXX, XXX−XXX