Discovery of novel bacterial elongation condensing enzyme inhibitors by virtual screening

Article abstract of DOI:10.1016/j.bmcl.2014.03.033

The elongation condensing enzymes in the bacterial fatty acid biosynthesis pathway represent desirable targets for the design of novel, broad-spectrum antimicrobial agents. A series of substituted benzoxazolinones was identified in this study as a novel class of elongation condensing enzyme (FabB and FabF) inhibitors using a two-step virtual screening approach. Structure activity relationships were developed around the benzoxazolinone scaffold showing that N-substituted benzoxazolinones were most active. The benzoxazolinone scaffold has high chemical tractability making this chemotype suitable for further development of bacterial fatty acid synthesis inhibitors.

Full text of DOI:10.1016/j.bmcl.2014.03.033

Bioorganic & Medicinal Chemistry Letters xxx (2014) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Discovery of novel bacterial elongation condensing enzyme
inhibitors by virtual screening
Zhong Zheng a, Joshua B. Parsons b, Rajendra Tangallapally a, Weixing Zhang c, Charles O. Rock b,
Richard E. Lee a,
⇑
a Department of Chemical Biology and Therapeutics, St Jude Children’s Research Hospital, Memphis, TN 38105, USA
b Department of Infectious Diseases, St Jude Children’s Research Hospital, Memphis, TN 38105, USA
c Department of Structural Biology, St Jude Children’s Research Hospital, Memphis, TN 38105, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
The elongation condensing enzymes in the bacterial fatty acid biosynthesis pathway represent desirable
targets for the design of novel, broad-spectrum antimicrobial agents. A series of substituted benzoxazoli-
nones was identified in this study as a novel class of elongation condensing enzyme (FabB and FabF)
inhibitors using a two-step virtual screening approach. Structure activity relationships were developed
around the benzoxazolinone scaffold showing that N-substituted benzoxazolinones were most active.
The benzoxazolinone scaffold has high chemical tractability making this chemotype suitable for further
development of bacterial fatty acid synthesis inhibitors.
Received 2 January 2014
Revised 7 March 2014
Accepted 12 March 2014
Available online xxxx
Keywords:
Virtual screening
Fatty acid synthesis
Condensing enzymes
Antibiotics
Ó 2014 Elsevier Ltd. All rights reserved.
Bacterial fatty acid biosynthesis has emerged as an appealing
target for the development of novel antibacterial chemotherapeu-
tics.1 Mammals utilize a single multifunctional enzyme complex
that is structurally distinct from the dissociated bacterial system
(FASII).2 The catalytic steps are the same in both systems, but sig-
nificant structural differences between the mammalian and bacte-
rial system can be exploited to design specific inhibitors.1,2
Multiple inhibitors of the enoyl–acyl carrier protein (ACP) reduc-
tase step (FabI) have been described.3 For example, AFN-1252 is
a nanomolar FabI inhibitor that eradicates Staphylococcus aureus
infections.4–6 However, FabI inhibitors are not broad spectrum
agents because many important pathogens express structurally
distinct enoyl–ACP reductases (FabK, FabL or FabV) that are refrac-
tory to FabI inhibitors.3 Our work is focused on the elongation con-
densing enzymes (3-ketoacyl-ACP synthase) because they are
ubiquitously expressed in bacteria and the two subgroups (FabB
and FabF) have superimposable active sites.7 These targets are
essential in Gram-negative bacteria. Although this group of
bacteria can incorporate extracellular fatty acids into phospholipid,
FASII is required to produce the acyl chains in the lipopolysaccha-
ride of the outer membrane.1 Some Gram-positive bacteria
(Streptococci) can circumvent FASII inhibitors by incorporating
extracellular fatty acids, but others (Staphylococci) require FASII
even when environmental fatty acids are present8 The natural
products cerulenin, thiolactomycin (TLM) and platensimycin target
both FabB and FabF.9,8 These inhibitors are broad-spectrum agents
with in vivo efficacy against Gram-positive and Gram-negative
bacteria. However, they have severe limitations, including substan-
dard pharmacokinetic properties and limited synthetic access.10
New chemical scaffolds are clearly needed, and this paper de-
scribes a virtual screening approach to discover a novel class of
elongation condensing enzyme inhibitors using Escherichia coli
FabB as the model.
The high resolution crystal structure of the FabB–TLM binary
complex11 was used as the template to identify the key pharmaco-
phore features to be incorporated into the design of new condens-
ing enzyme inhibitors. TLM binds non-covalently adjacent to the
active site residue Cys163 (Fig. 1).11 The carbonyl group forms
hydrogen bonds with both His298 and His333 in the active site,
and the isoprenoid moiety slides into a tight hydrophobic pocket
sandwiched between Gly391/Phe392 and Ala271/Pro272. A stan-
dard molecular dynamics simulation using AMBER12 with a pro-
duction run of 5 ns was carried out to gain a dynamic picture of
TLM binding as well to optimize hydrogen positions. The chain A
in the co-crystal structure of TLM bound to E. coli FabB (PDB:
2VB8)13 was used as the starting conformation. The complex sys-
tem was solvated in explicit water molecules with counter ions
to neutralize the system. Energy minimization was performed first
with solute constrained then released. The system temperature
⇑
Corresponding author. Tel.: +1 9015956617; fax: +1 9015955715.
E-mail address: richard.lee@stjude.org (R.E. Lee).
http://dx.doi.org/10.1016/j.bmcl.2014.03.033
0960-894X/Ó 2014 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Zheng, Z.; et al. Bioorg. Med. Chem. Lett. (2014), http://dx.doi.org/10.1016/j.bmcl.2014.03.033

2
Z. Zheng et al. / Bioorg. Med. Chem. Lett. xxx (2014) xxx–xxx
Figure 1. Binding modes of TLM and compound 14 to FabB. (A) Cocrystal structure of TLM in complex with FabB from E. coli (PDB: 2vb8). TLM is shown in spheres. (B) A close-
up view of the interactions between TLM and the binding site. (C) Compound 14 (green) docked into the binding site superimposed with TLM (purple).
was slowly heated from 0 to 300 K followed by equilibration and
production simulation. The observed binding mode of TLM in the
crystal structure was highly stable and all key interactions were
maintained through simulation. A free energy analysis was exe-
cuted to provide residue-based energy contribution to the TLM
binding (Fig. 2A).14,15 This analysis showed that His298, Phe392,
Thr302, Phe390, Val270, Pro272, Thr300, Gly391 and His333 con-
tribute to TLM binding.
HTVS followed by Glide SP.20 Preference was given in docking to
compounds that could form the desired hydrogen bonds with
His298 and His333. The top ranking 500 compounds were pre-
served for further examination. After consideration of binding pose
by eye, chemical diversity and tractability, a total of 31 compounds
were prioritized and acquired for in vitro testing (see Supplemen-
tary Fig. 1). TLM was incorporated in the virtual screening library
from the beginning as a positive control. This compound passed
the pharmacophore search filter and was ranked very high in the
subsequent docking study.
The purchased compounds were all analyzed by LCMS to con-
firm purity and identity. All 31 compounds passed this step and
were then screened using NMR wLOGSY assay21 to evaluate FabB
binding (see Supplementary Fig. 1). The eight compounds that
exhibited binding in the NMR wLOGSY assay were further tested
as FabB inhibitors using the condensing enzyme radiochemical as-
say previously described11 except myristoyl-ACP, which was
substituted by lauryl-ACP synthesized using the Vibrio harveyii
acyl-ACP synthetase enzyme.22 Initial analysis of inhibition was
A two-step virtual screen was performed against E.coli FabB
using a total of ꢀ1.1 million compounds from the Enamine (Ad-
vanced Collection) and Chembridge (EXPRESS-Pick Collection Stock
and CORE Library Stock) libraries. A single-conformation UNITY16
database was created and 3D conformations were generated for
each compound by Concord. Compound sets were filtered for a
molecular weight cut off of 350 to search for lead-like inhibitors17
that allows for facile further modification. Using the key binding
elements identified from MD simulation, a UNITY pharmacophore
query was established including a hydrogen bond acceptor atom
connected to a five-member ring that could form a bidentate inter-
action with His298 and His333 (Fig. 2B).18 Spatial constrains were
applied to constrain the three-dimensional conformation of phar-
macophore features. This pharmacophore query search was ap-
plied to remove inactives and to simplify the subsequent
computationally expensive docking step. Compounds that success-
fully passed the filter (ꢀ250, 000 compounds) were advanced into
docking experiments using the Virtual Screening Workflow from
Schrödinger.19 Compounds were filtered to remove those with
reactive functional groups. Compounds were docked with Glide
conducted at a single final inhibitor concentration of 200
screens revealed that compound 1 exhibited binding in NMR and
showed 24% inhibition at 200 M in the enzymatic assay. Dose re-
sponse experiments established an IC50 of 800 M (Table 1). The
lM. Both
l
l
IC50 for compound 1 was confirmed by resynthesis and retesting
in the enzyme assay.
A similarity search performed in SciFinder uncovered 11 ana-
logues of compound 1 that were ordered and tested. The scifinder
analysis was complemented by a medicinal chemistry effort to fill
Figure 2. The TLM pharmacophore model. (A) Decomposed free energy contribution per residue to TLM binding calculated from MD simulation. (B) Pharmacophore model
developed in UNITY.
Please cite this article in press as: Zheng, Z.; et al. Bioorg. Med. Chem. Lett. (2014), http://dx.doi.org/10.1016/j.bmcl.2014.03.033

Z. Zheng et al. / Bioorg. Med. Chem. Lett. xxx (2014) xxx–xxx
3
Table 1
The structures and measured FabB inhibition of compounds from procurement and chemical synthesis arranged for SAR purposes
#
1
X
O
R1
H
R2 R3
% Inhibition IC50
#
X
O
R1 R2 R3
% Inhibition IC50
#
X
O
R1 R2 R3
% Inhibition IC50
@ 200
lM
(l
M)
@ 200
l
M
(lM)
@ 200
lM
(lM)
Cl
24.1
800
ND
ND
11
Cl
H
H
H
37.7
335
ND
ND
21
H
H
H
H
H
Cl
30
ND
ND
ND
2
3
O
O
H
H
H
24
12
13
O
O
Cl
Cl
26.4
23.8
22
23
O
O
37.8
38
Cl
17.7
4
5
6
O
O
O
H
Br
H
17.7
40.1
36.7
ND
ND
350
14
15
16
O
O
O
H
H
H
H
Cl
H
33.5
32.1
5
235
ND
ND
24
25
26
O
O
O
Cl
H
H
Cl
H
30.4
36.4
37.1
415
ND
CH3
Cl
H
Cl
305
7
O
H
Cl
31.7
ND
17
O
H
Cl
28.3
ND
27
O
Cl
H
55.4
260
8
O
O
O
H
H
H
Cl
Br
Cl
24.3
32.7
38.8
ND
600
ND
18
19
20
O
O
O
Cl
H
H
H
41.4
23.6
30
ND
ND
ND
28
29
30
O
O
S
Cl
H
H
H
31.9
42.1
36.1
ND
9
Cl
Cl
CN
Cl
253
303
10
All compounds with stereochemistry were tested as racemates.
in the SAR through synthesis of 18 additional compounds. Com-
pounds were synthesized in one step from appropriately substi-
tuted benzoxazolinones and structurally unique secondary
amines using Mannich chemistry (Supplementary Scheme 1).23
The inhibitory activities of compounds from both sets are listed
and grouped by structure in Table 1.
spiro-tetrahydropyran moieties (10 and 11) which maintained
inhibitory activity. Compound 11 had an IC50 of 335 M, on par
l
with the best tetrahydrofuran analogue (6). The pyrrolidine ring
in 13 was well tolerated in comparison with tetrahydrofuran com-
pound 1. In the second sub set (14–22), an aryl group was intro-
duced into the side chain in place of the tetrahydrofuran group
found in 1. The most potent analog of this set was the simple phe-
nyl compounds 14 and 18 with a hydroxyl group on the meta-po-
sition. Replacing phenyl group with 2-pyridyl (20) and 3-pyridyl
(19) groups did not improve potency. However, bioisosteric phenyl
replacement of 14 with a thiophene ring (21 and 22) was well tol-
erated. The third subseries was designed to conformationally re-
strict the ter-amine side chain (set 23–30) in which the benzyl
group of compound 14 was attached by a 4- to 6-membered ring.
Compounds 27 and 29 both with a 5-membered pyrrolidine ring
and a bromo substitution on meta-position of the phenyl ring
Compared with the initial hit (1), many compounds in Table 1
showed improved enzymatic inhibition at a single inhibitor con-
centration of 200
response experiments producing IC50 values ranging from
235–625 M (Table 1). Various benzoxazolinone substitutions
lM. Selected compounds were measured in dose
l
were tested with R1 and R2 (5 and 6 position) halogen substitution
producing the most potent analogs. It is notable that moving the
chlorine atom from the 6 position in 1 to the 5 position in 6 sub-
stantially increased inhibitory activity. Most structural divergence
in this series was introduced via R3 N-substitution to the benzox-
azolinone core, and divergent secondary and cyclic amines were
used to probe for improved sidechain moieties in three subseries.
First, a series of similar aliphatic amine side chains (2–13) were
examined. Substituting tetrahydrofuran with a cyclopentyl ring
in compounds 3 and 4 resulted in loss of activity. Tetrahydropyran
system (7 and 8) was well tolerated as well as corresponding
showed the best activity with IC50’s of 260 and 253 lM, respec-
tively. The cyclopropane group (22) was also a better substitutent
than methyl (21). Substituting 5-chloro of 27 with 5-cyano in 29,
or changing benzoxazole with benzothiozole 30 yielded compara-
ble IC50 values. The expectation that the benzoxazolinones would
also be FabF inhibitors was tested by examining the IC50 of
Please cite this article in press as: Zheng, Z.; et al. Bioorg. Med. Chem. Lett. (2014), http://dx.doi.org/10.1016/j.bmcl.2014.03.033

4
Z. Zheng et al. / Bioorg. Med. Chem. Lett. xxx (2014) xxx–xxx
Acknowledgments
We would like to thank Laura Wells and Protein Production
Shared Resource for their expert technical assistance. This study
was supported by the National Institutes of Health Grant
5R01GM034496, Cancer Center Support Grant CA21765 and the
American Lebanese Syrian Associated Charities (ALSAC).
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2014.
03.033.
References and notes
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Figure 3. Compound 14 inhibition of E. coli FabB and S. aureus FabF in vitro.
Maximum activities were: FabB, 4.7 nmoles/min/mg; FabF, 4.0 nmoles/min/mg.
compound 14 against FabF from S. aureus. The IC50 of compound 14
6. Yao, J.; Maxwell, J. B.; Rock, C. O. J. Biol. Chem. 2013.
against E. coli FabB was 235
against S. aureus FabF, indicatingthe potential for broad-spectrum
activity of this scaffold. (Fig. 3)
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The docking of compound 14 into the TLM binding site (Fig. 1C)
provides a rationale for understanding the structural basis of benz-
oxazolinones binding. The superimposition of compound 14 with
TLM bound to the E. coli FabB enzyme showed that the ketone
group on the oxazole ring mimics the bidentate binding mode with
the catalytic His298 and His333, and the methylaniline group
occupied the same binding pocket as the isoprene side chain of
TLM forming favorable van der Waals interactions. Other active
compounds were also modeled to study their potential binding
conformations. Compound 6 and 27 adopted very similar poses
to each other and 14 (Supplementary Fig. 2A and B). They both
formed two hydrogen bonds with His298 and His333. The respec-
tive cyclopropane (6) and pyrrolidine (27) side chains occupy the
same cavity above H298 that was originally partially filled by the
chiral 5-methyl group on thiophene ring in TLM, while the tetrahy-
drofuran (6) and bromobenzene (27) rings are located in the same
binding pocket as the methylaniline group in 14. Although an ac-
tive inhibitor, compound 11 showed less favorable docking, as
binding requires reorientation of the TLM binding site to fit the big-
ger azaspiro side chain, losing one hydrogen bond with His298
(Supplementary Fig. 2C).
18. Martin, Y. C. J. Med. Chem. 1992, 35, 2145.
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20. Repasky, M. P.; Shelley, M.; Friesner, R. A. Curr. Protoc. Bioinf. 2007. Chapter 8,
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Compared to the FabB natural product inhibitors, the benzoxaz-
olinones show selective inhibition of the condensing enzymes
along with improved physicochemical properties and easy
synthetic access. This new inhibitor class is being further explored
to increase target affinity.
Please cite this article in press as: Zheng, Z.; et al. Bioorg. Med. Chem. Lett. (2014), http://dx.doi.org/10.1016/j.bmcl.2014.03.033