Novel broad-spectrum inhibitors of bacterial methionine aminopeptidase

Article abstract of DOI:10.1016/j.bmcl.2015.05.061

With increasing emergence of multi-drug resistant infections, there is a dire need for new classes of compounds that act through unique mechanisms. In this work, we describe the discovery and optimization of a novel series of inhibitors of bacterial methionine aminopeptidase (MAP). Through a high-throughput screening campaign, one azepinone amide hit was found that resembled the native peptide substrate and possessed moderate biochemical potency against three bacterial isozymes. X-ray crystallography was used in combination with substrate-based design to direct the rational optimization of analogs with sub-micromolar potency. The novel compounds presented here represent potent broad-spectrum biochemical inhibitors of bacterial MAP and have the potential to lead to the development of new medicines to combat serious multi-drug resistant infections.

Full text of DOI:10.1016/j.bmcl.2015.05.061

Bioorganic & Medicinal Chemistry Letters 25 (2015) 3301–3306
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Novel broad-spectrum inhibitors of bacterial methionine
aminopeptidase
Jonathan A. Rose a, Sushmita D. Lahiri b, David C. McKinney b, Rob Albert b, Marshall L. Morningstar b,
Adam B. Shapiro b, Stewart L. Fisher b, Paul R. Fleming b,
⇑
a Cleveland Clinic Lerner College of Medicine of Case Western Reserve University, 9500 Euclid Ave., Cleveland, OH 44195, USA
b Infection Innovative Medicines Unit, AstraZeneca R&D Boston, 35 Gatehouse Drive, Waltham, MA 02451, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
With increasing emergence of multi-drug resistant infections, there is a dire need for new classes of com-
pounds that act through unique mechanisms. In this work, we describe the discovery and optimization of
a novel series of inhibitors of bacterial methionine aminopeptidase (MAP). Through a high-throughput
screening campaign, one azepinone amide hit was found that resembled the native peptide substrate
and possessed moderate biochemical potency against three bacterial isozymes. X-ray crystallography
was used in combination with substrate-based design to direct the rational optimization of analogs with
sub-micromolar potency. The novel compounds presented here represent potent broad-spectrum bio-
chemical inhibitors of bacterial MAP and have the potential to lead to the development of new medicines
to combat serious multi-drug resistant infections.
Received 13 April 2015
Revised 20 May 2015
Accepted 22 May 2015
Available online 30 May 2015
Keywords:
Antibacterial
Methionine aminopeptidase
Azepinone
Structure-based design
Drug discovery
Ó 2015 Elsevier Ltd. All rights reserved.
Bacterial protein translation has been an abundant source of
antibacterial targets including many different classes of drugs that
treat serious clinical infections. With resistance to currently pre-
scribed antibiotics increasing, however, there is a need for new
classes of compounds that inhibit novel biochemical targets. In
prokaryotic organisms, protein translation is initiated with a
formylmethionine residue, which is converted to methionine dur-
ing protein elongation by the metalloenzyme, peptide deformylase
(PDF).1 Inhibitors of PDF containing metal chelating moieties have
shown in vivo efficacy, however concerns of toxicity remain.2 An
important step in the co- or posttranslational protein maturation
is hydrolysis of the N-terminal methionine by the metalloenzyme,
methionine aminopeptidase (MAP).3,4 MAP is a ubiquitous enzyme
found in all prokaryotes and gene deletion studies have shown it to
be essential for Escherichia coli5 and Salmonella typhimurium.6 Our
own unpublished gene deletion studies have confirmed this essen-
tiality in Streptococcus pneumoniae and Haemophilus influenza (data
not shown). Therefore, MAP is a validated target for the develop-
ment of broad-spectrum antibacterial drugs with a novel mecha-
nism of action that could provide therapeutic options to combat
multidrug-resistant bacterial pathogens.7
eukaryotes.8 In humans, type I and, in particular, type II MAP have
been considered as oncology targets, and type II MAP is known to
be inactivated by the anti-angiogenesis compound fumagillin.9
This compound and its subsequent analogs have been found to
inhibit both endothelial cell growth as well as tumor growth
in vivo.10 Similarity of human type I MAP to bacterial MAP is mod-
erate (30–40%), with even lower similarity observed for the type II
MAP (20%). Engineering selectivity for bacterial MAP should be
achievable given this sequence divergence and the fact that crystal
structures of both human11 and bacterial12 MAP enzymes have
been published.
MAP’s enzymatic hydrolysis of native proteins requires the
assistance of divalent metal ions that serve as a cofactor for the cat-
alytic reaction. MAP activation has been accomplished with Co(II),
Mn(II), Ni(II), and Fe(II).13 Although several MAPs were initially
Azepinone Amide
Eco 30 3 µM
Hin 60 9 µM
Spn 46 5 µM
Sau >200 µM
There are two major types of MAP enzymes. Type I enzymes are
found in eubacteria, while both type I and II enzymes are found in
Figure 1. High throughput screen azepinone amide hit (1). Resynthesis of the HTS
hit confirmed moderate biochemical activity against three MAP isozymes.
Eco = E. coli. Hin = H. influenzae. Spn = S. pneumoniae. Sau = S. aureus.
⇑
Corresponding author. Tel.: +1 617 248 5291; fax: +1 617 502 5291.
E-mail address: paulrobertfleming@gmail.com (P.R. Fleming).
http://dx.doi.org/10.1016/j.bmcl.2015.05.061
0960-894X/Ó 2015 Elsevier Ltd. All rights reserved.

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J. A. Rose et al. / Bioorg. Med. Chem. Lett. 25 (2015) 3301–3306
HO
Boc
N
H
MgCl
RCM
O
EDC, DCM, RT
Toluene, 80 °C
N
NH
N
THF, 0°C
67%
NH
5
6
51% of , 40% of
Boc
42%
O
O
4
3
2
O
H
N
+
HO
Boc
NH
Boc
NH
N
N
NH2
Boc
N
TFA, DCM, RT
85%
EDC, DCM, RT
O
O
92%
F
6
5
7
O
O
H
N
O
HO
F
NH2
N
H
Boc
N
N
H
N
TFA, DCM, RT
EDC, DCM, RT
41%, two steps
O
O
8
9
O
H
N
F
N
N
H
O
O
1
F
Scheme 1. Synthesis of azepinone amide hit (1).
Table 1
Structure–activity relationship of azepinone analogs
Compound number
Structure
Eco IC50
(lM
SE)
Hin IC50
(lM
SE)
Spn IC50
(l
M
SE)
O
O
H
N
O
10
8.1
1
11.3
>200
>200
1
7.4
1
N
H
N
N
O
O
O
H
N
11
12
>200
>200
>200
>200
N
H
O
O
H
N
O
H2N
O
N
H
O
O
H
N
O
13
14
15
16
17
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
N
N
H
O
O
O
O
O
H
N
O
N
H
N
O
O
O
O
N
O
N
H
N
N
N
O
O
O
H
N
O
N
H
O
O
H
N
O
102 18
132 27
71.4 13
N
H
HO
IC50 values are best-fit values standard error (SE).

J. A. Rose et al. / Bioorg. Med. Chem. Lett. 25 (2015) 3301–3306
3303
identified as Co(II) enzymes, later work has lead to the discovery of
inhibitors that can target the Co(II)-, Mn(II)-, and Fe(II)-forms of
E. coli MAP with high potency and selectivity.14,15 The inhibitors
of Fe(II)-MAP have been shown to be the only ones capable of bac-
terial growth inhibition against E. coli and Bacillus strains, suggest-
ing that Fe(II) may be the physiologically relevant metal in MAP.16
However, because the physiological metal cofactor has not been
determined conclusively there is a risk that potent inhibitors of
one metalloform may not inhibit other metalloforms and/or
translate to in vivo efficacy. One potential strategy for avoiding this
difficulty would be to find inhibitors that do not interact directly
with the divalent metal but interact solely with the protein amino
acid residues. In addition, by forgoing metal chelation as a strategy
for inhibition of MAP, concerns about selectivity versus other
important human metalloenzymes such as matrix metallopro-
teinases could potentially be minimized.
In this work, we describe an early drug discovery campaign that
led to the identification of a novel chemical series that inhibits
multiple bacterial MAP isozymes. We have obtained an X-ray
crystal structure of one of these inhibitors bound to the MAP active
site, which shows close interactions with the protein without
interacting directly with the divalent cation cofactor.
The project began with a high throughput screen (HTS) of over
1.2 million compounds from AstraZeneca’s corporate compound
collection using a novel high-throughput absorbance-based assay
for methionine published previously,17 in which Mn(II) was used
as the metal cofactor. A triage cascade was utilized that was based
on progressively more stringent criteria for hits. Over 1000 com-
pounds were tested for purity analysis and inhibition of enzyme
activity against E. coli MAP. From this set of actives, over 400 com-
pounds were tested for potency against MAP isozymes from H.
influenzae, S. pneumoniae, and Staphylococcus aureus. Of these only
one chemical series emerged with moderate potency against three
of the isozymes (Fig. 1). This hit contained an azepinone amide
backbone and resynthesis of the compound confirmed inhibition
of enzyme activity. Monocyclic and bicyclic azepinones have been
incorporated as dipeptide mimics in mercaptoacyldipeptides inhi-
bitors of angiotensin converting enzyme (ACE)18–20 including the
marketed ACE inhibitor benazepril.21,22 Medicinal chemistry effort
was subsequently deployed to achieve potency and physical prop-
erty optimization.
The synthesis of compound 1 was achieved in 6 steps from 1
commercially available methyl imine starting material (2) accord-
ing to Scheme 1. Grignard reaction with commercially available
vinyl magnesium chloride gave the allyl amine (3). Amide coupling
with EDC and commercially available carboxylic acid was used to
form the Boc-protected amide (4). Ring-closing metathesis (RCM)
was performed in toluene at 80 °C to afford a mixture of isomers
that were purified by column chromatography to give a decent
yield of the desired azepinone (5). Removal of the Boc-protecting
group with TFA in dichloromethane at room temperature was then
followed by amide coupling using EDC and commercially available
Boc-protected-(S)-alanine to generate the ala-Boc azepinone inter-
mediate (8). Removal of the Boc-protecting group with TFA in
dichloromethane at room temperature and amide coupling with
EDC and difluorobenzyl carboxylic acid gave the final compound
(1) in good yield.
Various azepinone analogs were synthesized in order to explore
the series’ structure–activity relationship (SAR). Table
1
summarizes the results of efforts to optimize enzyme inhibition
and physical properties of the hit compound. Testing of ala-Cbz
intermediate (10) resulted in a slight improvement in potency as
compared to the original hit, which showed that the RHS could
be varied to other lipophilic moieties. This also allowed compara-
tive Cbz analogs to be synthesized in fewer steps, enabling rapid
SAR generation. Synthesis of left-hand side (LHS) and right-hand
Figure 2. X-ray crystal structure of compound 1 in E. coli MAP. (a) There are several
strong polar interactions including hydrogen bonds with His79, Cys169, His171 and
His178 and indirect interaction with active site residues Asp97, Asp108, Glu235,
and Glu204 through a water bridge (W1), which is more clearly shown in (b). The
compound does not displace the primary metal, but instead interacts indirectly
through W1. (c) The electrostatic surface of the binding pocket shows the
positioning of the difluorobenzyl group deep within the hydrophobic pocket and
the bent conformation placing the two aromatic rings in close proximity.

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J. A. Rose et al. / Bioorg. Med. Chem. Lett. 25 (2015) 3301–3306
side (RHS) fragments (compound 11 and 12, respectively) showed
that both sides of the original backbone are required for enzyme
inhibition. LHS modification to compound 13 showed that the R-
stereochemistry of the phenyl substituent was required for
potency. Modifications of the amide linker also resulted in loss of
inhibition, including changing to the gem-dimethylamino acid 14,
removal of the amide hydrogen to give tertiary amide 15, and
change to include a bulky side chain like valine 16. Interestingly,
an intermediate side chain such as in serine analog 17 allowed
for retention of modest potency, likely reflecting a steric constraint
in that region of the binding pocket. All analogs demonstrated
limited specific interactions to the LHS group other than to the
backbone peptide. However, the outer lining of this pocket is
formed of aromatic groups such as Tyr168, Trp221, and Tyr62,
which provides favorable interactions to the phenyl substituent
of the LHS. Additionally, the structure clearly shows that the inhi-
bitor binds to the pocket in a bent conformation such that the phe-
nyl group of LHS is brought in close proximity to the difluorobenzyl
ring of RHS. An intramolecular hydrophobic contact provides some
favorable interactions to the otherwise exposed phenyl group of
the RHS.
As shown in the crystal structure, the nitrogen of the benzyl
amide is shown to be interacting with active site residues Asp97,
Asp108, and Glu204 through a water bridge W1 (Figs. 2a and 3a).
These residues are theorized to interact with the nitrogen of
methionine at the N-terminus of the natural substrate, allowing
proper stabilization for peptide hydrolysis.13,23 Therefore it was
hypothesized that the inhibitor resembles a substrate analog and
that addition of a benzyl amino group to the right-hand side would
mimic the methionine nitrogen. It was hypothesized that this
would result in improved potency through displacement of the
water molecule and direct hydrogen-bonding with these three
active site residues (Fig. 3b). Consistent with this hypothesis, syn-
thesis of compound 18 showed improved biochemical potency in
all three isozymes with sub-micromolar potency against S. pneu-
moniae (Table 2). A small library of benzyl amine compounds
showed consistent improvement in potency as compared to the
methylene analogs, and delivered the most active compound for
this series (22) with sub-micromolar inhibition against E. coli and
S. pneumoniae (Table 2). Unfortunately even with this improved
potency, compounds 10, 18, 21, and 22 failed to show antibacterial
aqueous solubility >200 lM with other physical properties of cer-
tain analogs similar to 1 (see Supplementary material).
An X-ray crystal structure of 1 in complex with E. coli MAP was
obtained at 1.8 angstrom resolution (PDB ID 4Z7M). The structure
showed one metal bound to the active site and a clear density for
compound 1 was in the vicinity (Fig. 2a). Several strong interac-
tions were observed between 1 and the peptide backbone, with
no direct interaction with the divalent metal cofactor. However,
the compound does not displace the primary metal, but instead
interacts with the water molecule that is bound to the metal
(Fig. 2b). There are multiple polar interactions observed in the
structure. The alanine carboxy oxygen was found to form a hydro-
gen bond with His79. The amino group of the azepinone ring made
a hydrogen bond with the carboxy oxygen of Cys169 and the car-
boxy oxygen of the azepinone ring made a hydrogen bond with
the backbone amide of Cys169. The carboxy oxygen of the benzyl
amide formed hydrogen bonds with the imidazole nitrogens of
His171 and His178, while the nitrogen of the benzyl amide formed
an indirect interaction with active site residues Asp97, Asp108,
Glu235 and Glu204 through a water bridge (W1). These interac-
tions are summarized in Figure 2a.
activity at concentrations up to 64 lg/ml against any of the diverse
collection of Gram-negative and Gram-positive pathogens in our
In addition to the polar interactions, there are also hydrophobic
residues that contribute to the binding mode. Most notable is the
benzyl ring interactions that are positioned in a pocket formed
by aromatic residues Phe177, Trp221, Tyr65, and Tyr62. This
pocket positions the N-terminal methionine that allows recogni-
tion and cleavage of this amino acid, and thus is hydrophobic
and enclosed (Fig. 2c). In contrast, the LHS azepinone ring is more
solvent exposed, as this part of the pocket binds to the rest of the
peptide chain with very little specificity for the amino acid
sequence other than the backbone peptide. As a result, there are
typical screening panel.
The structural modifications were able to improve biochemical
potency against E. coli and S. pneumoniae MAP, thus allowing the
design of a broad spectrum compound. However, the compounds
always lacked measurable S. aureus potency. Crystal structures of
S. aureus MAP are available in the public domain and provide a
basis for rationalizing this difference. When the E. coli structure
bound to compound 1 was overlaid on a previously-published S.
aureus MAP structure (1QXZ, Oefner et al),24 it was clear that all
key polar interactions and the residues surrounding the
Figure 3. Structure/substrate-based design. (a) X-ray crystal structure of compound 1 in E. coli MAP showing interaction of the benzyl amide nitrogen with Asp97, Asp108,
Glu235 through a water bridge. (b) Substrate-based design of lead compound hypothesizing that addition of a benzyl nitrogen would mimic the methionine nitrogen of the
natural substrate and interact with directly with active site residues.

J. A. Rose et al. / Bioorg. Med. Chem. Lett. 25 (2015) 3301–3306
3305
Table 2
Structure–activity relationship of benzyl amine analogs
Compound number
Structure
Eco IC50
1.4 0.3
(
lM
SE)
Hin IC50
6.7 0.7
(l
M
SE)
Spn IC50
(
l
M
SE)
O
NH2
NH2
H
N
18
0.12 0.01
4.0 0.4
>200
N
H
N
N
N
O
O
O
O
O
O
O
H
N
19
20
21
22
8.0 0.9
>200
15.6 1.0
>200
N
H
O
O
O
O
NH2
NH2
NH2
H
N
N
H
O
H
N
2.2 0.4
0.8 0.1
4.7 0.5
1.1 0.1
9.7
1
N
H
N
N
O
H
N
Cl
0.3 0.03
N
H
O
IC50 values listed as mean concentration standard error (SE).
We have described the discovery of a novel series of MAP inhibi-
tors with broad-spectrum biochemical potency against E. coli, S.
pneumoniae, and H. influenzae. High-throughput screening was used
to identify potential candidates from a large compound collection.
From this screening campaign, one azepinone amide hit was found
that resembled the native peptide substrate and possessed moder-
ate biochemical potency against three bacterial isozymes. An X-ray
crystal structure was obtained of this compound bound to E. coli
MAP, which showed several strong polar interactions between the
enzyme and compound backbone in addition to many non-specific
hydrophobic interactions. Most notably this structure showed no
direct interaction between the compound and divalent metal cofac-
tor. Rational medicinal chemistry and substrate/structure-based
design using this crystal structure along with an understanding of
MAP’s mechanism allowed for the synthesis of compound analogs
with sub-micromolar potency. Although S. aureus inhibition was
never achieved with this series, structural analysis revealed an
explanation for this difference in binding affinity and could direct
future programs to achieve potency against S. aureus. The com-
pounds presented here represent novel MAP inhibitors with the
potential to lead to the development of new medicines to treat seri-
ous multi-drug resistant infections.
Acknowledgments
Figure 4. X-ray crystal structure of compound 1 in E. coli MAP (green) overlaid with
S. aureus MAP (pink). The larger helix in the S. aureus structure creates a smaller
binding pocket for the RHS and may explain the lack of comparable binding
potency.
Kanayo Azogu, Bridget Reaney, Nancy DeGrace, Ying Liu, Ziling
Lu, Camil Joubran and Mark Zambrowski are thanked for analytical
support. Frank Kilty is thanked for bioinformatics support.
Supplementary data
compounds were conserved (Fig. 4). While the amino acids
interacting with the LHS moiety were not exactly same, the
nature of the substitution, such as Tyr168 replaced with Leu, or
Val223 replaced with Phe, remained unchanged and thus would
be expected to have minimal impact on binding potency.
Interestingly however, the benzyl pocket of the RHS was slightly
different in S. aureus compared to that of E. coli. The residues
Cys59, Tyr62, and Tyr65 are contributed by a small helix in E. coli
(H3 helix). This helix is larger in the S. aureus structure, and results
in a smaller pocket. We propose that the larger helix is suboptimal
for the binding of this scaffold to S. aureus, as the RHS binding
plane would influence the hydrogen bonding with the backbone
residues of the molecule.
Supplementary data (experimental details and spectroscopic
characterization of compounds involved in the synthesis, including
ADME characterization of select compounds) associated with this
article can be found, in the online version, at http://dx.doi.org/10.
1016/j.bmcl.2015.05.061.
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