Optimization of amino acid thioesters as inhibitors of metallo-β-lactamase L1

Article abstract of DOI:10.1016/j.bmcl.2016.08.048

The emergence of antibiotic resistance caused by metallo-β-lactamases (MβLs) is a global public health problem. Recently, we found amino acid thioesters to be a highly promising scaffold for inhibitors of the MβL L1. In order to optimize this series of inhibitors, nine new amino acid thioesters were developed by modifying the substituents on the N-terminus of the thioesters and the groups representing the amino acid side chain. Biological activity assays indicate that all of them are very potent inhibitors of L1 with an IC₅₀value range of 20–600?nM, lower than those of most of the previously reported inhibitors of this scaffold. Analysis of structure–activity relationship reveals that big hydrophobic substituents on the N-terminus and a methionine amino acid side chain improves inhibitory activity of the thioesters. All these inhibitors are able to restore antibacterial activity of a β-lactam antibiotic against Escherichia coli BL21(DE3) cells producing L1 to that against E. coli cells lacking a β-lactamase. Docking studies reveal that a large N-terminal hydrophobic group results in a slightly different binding mode than smaller hydrophobic groups at the same position.

Full text of DOI:10.1016/j.bmcl.2016.08.048

Bioorganic & Medicinal Chemistry Letters 26 (2016) 4698–4701
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Optimization of amino acid thioesters as inhibitors of metallo-b-
lactamase L1
a
a
a
a
Xiao-Long Liu a, Ke-Wu Yang a, , Yue-Juan Zhang , Ying Ge , Yang Xiang , Ya-Nan Chang ,
⇑
Peter Oelschlaeger b
a Key Laboratory of Synthetic and Natural Functional Molecule Chemistry of Ministry of Education, Chemical Biology Innovation Lab, College of Chemistry and Materials
Science, Northwest University, Xi’an 710127, PR China
b Department of Pharmaceutical Sciences, College of Pharmacy, Western University of Health Sciences, 309 East Second Street, Pomona, CA 91766, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
The emergence of antibiotic resistance caused by metallo-b-lactamases (MbLs) is a global public health
problem. Recently, we found amino acid thioesters to be a highly promising scaffold for inhibitors of
the MbL L1. In order to optimize this series of inhibitors, nine new amino acid thioesters were developed
by modifying the substituents on the N-terminus of the thioesters and the groups representing the amino
acid side chain. Biological activity assays indicate that all of them are very potent inhibitors of L1 with an
IC50 value range of 20–600 nM, lower than those of most of the previously reported inhibitors of this
scaffold. Analysis of structure–activity relationship reveals that big hydrophobic substituents on the
N-terminus and a methionine amino acid side chain improves inhibitory activity of the thioesters. All
these inhibitors are able to restore antibacterial activity of a b-lactam antibiotic against Escherichia coli
BL21(DE3) cells producing L1 to that against E. coli cells lacking a b-lactamase. Docking studies reveal that
a large N-terminal hydrophobic group results in a slightly different binding mode than smaller hydropho-
bic groups at the same position.
Received 22 April 2016
Revised 11 August 2016
Accepted 18 August 2016
Available online 22 August 2016
Keywords:
Antibiotic resistance
Metallo-b-lactamase
L1
Inhibitor
Mercaptoacetic acid thioester
Ó 2016 Elsevier Ltd. All rights reserved.
b-Lactam-containing drugs, such as, penicillins, cephalosporins
and carbapenems are among the most important and frequently
used antimicrobial agents, which prevent transpeptidase-mediated
cross-linking of adjacent peptidoglycan strands to inhibit cell wall
biosynthesis.1,2 Over the past 70 years, the overuse of b-lactam-
containing antibiotics has resulted in a large number of bacteria
that are resistant to almost all antibiotics. Most commonly, bacte-
ria become resistant to b-lactam antibiotics by producing b-lacta-
mases, which hydrolyze the C–N bond in the four-membered
ring of the b-lactam antibiotics.3 b-Lactamases consist of four
classes of enzymes (A–D) based upon DNA sequence similarity.
The groups A, C and D are called serine b-lactamases (SbLs), which
exhibit biological activity by utilizing an active-site serine as a
nucleophile to attack the b-lactam carbonyl.4 The group B enzymes
require 1–2 Zn(II) ions to hydrolyze b-lactams and thus are called
metallo-b-lactamases (MbLs). Now MbLs are of increasing clinical
concern, because they hydrolyze almost all b-lactam antibiotics.5
However, to date there are no inhibitors of the MbLs available in
the clinic.6,7 MbLs have been further subdivided into three sub-
classes, B1, B2, and B3. The MbL L1, a representative of B3 subclass
enzymes, requires two metal ions in the active site for full activity:
Zn1 has His116, His118, His196, and a bridging hydroxide as
ligands; Zn2 has Asp120, His121, His263, the bridging hydroxide,
and a terminally bound H2O as ligands.8
Facing the increasing challenge of antibiotic resistance, a num-
ber of MbL inhibitors have been reported, including b-lactam
derivatives,9–12 thiol carboxylates,13–15 and sulfonamides.16 Also,
aspergillomarasmine A (AMA)5 and ebselen17 as inhibitors of
New Delhi metallo-b-lactmase-1 (NDM-1) have been described.
Recently, we found that mercaptoacetic acid thioester, found in
compounds A, B, and C (Fig. 1), is a highly promising scaffold for
the development of specific L1 inhibitors.18 In order to further
increase the inhibitory activity of these compounds against L1,
our idea was to change the substituent R1 into a non-polar group
as shown in Figure 1. In addition, the structure–activity relation-
ship gained in our recent work implies that methionine and tryp-
tophan side chains as R2 groups are favorable for inhibitory
activity,18 suggesting to retain these amino acid side chains. Based
on these ideas, we designed and synthesized compounds 1–3 and
7–9. The series 4–6 with R1 groups corresponding to those in
1–3, but with a phenyl group derived from phenylalanine as the
R2 group was hypothesized to exhibit less inhibition. Thus, these
compounds were synthesized and tested for comparison. Thioester
⇑
Corresponding author.
http://dx.doi.org/10.1016/j.bmcl.2016.08.048
0960-894X/Ó 2016 Elsevier Ltd. All rights reserved.

X.-L. Liu et al. / Bioorg. Med. Chem. Lett. 26 (2016) 4698–4701
4699
S
O
R2
NH
O
O
O
S
CO2H
S
CO2H
S
CO2H
N
H
S
CO2H
S
R1
N
H
N
H
S
N
H
S
O
O
O
O
A
B
C
Amino acid thioester scaffold
IC50=0.018uM, Ki=0.11uM
IC50=2.9uM, Ki=0.95uM
IC50=0.29uM, Ki=0.17uM
Figure 1. Amino acid thioester scaffold and structures of specific compounds A–C reported previously.18
10 lacking an amino acid side chain was also included as a
reference.
nyl methyl is larger than the benzyl, and the benzyl is larger than
the pentyl group. Within the series with a constant 4-diphenyl
methyl R1 group, 1 and 7 are more potent than 4, while in the ser-
ies with a constant benzyl R1 group, 2 and 8 are more potent than
5, implying that the tryptophan and methionine amino acid side
chains (R2) are more favorable than the phenylalanine side chain,
which is in agreement with previous results.18 With the aliphatic
pentyl R1 group, the tryptophan side chain as R2 in 3 did not result
in higher potency than the phenylalanine side chain in 6. 9 had
almost identical potency as 8, indicating that the phenyl R1 group
is nearly equivalent to the benzyl R1 group. Furthermore, com-
pared with the previously reported compounds B and C,18 the inhi-
bitory activities of 1 and 4 are increased, indicating that the 4-
diphenyl methyl R1 group is superior to the 2-thiophenyl methyl
group.
The synthetic pathway of the ten thioesters is shown in
Scheme 1. Firstly, hexanoic acid, phenylacetic acid, 4-bipheny-
lacetic acid, and benzoic acid, respectively, were refluxed with sul-
fur dichloride to get the corresponding acyl chlorides.19,20
Secondly, racemic mixtures of amino acids reacted respectively
with the four kinds of acyl chloride to give amides 1b–9b. Finally,
the amides reacted with mercaptoacetic acid to give the target
products mercaptoacetic acid thioesters 1–9. The synthetic process
of 10b and 10 is similar to that of 1b–9b and 1–9. The structures of
the synthesized mercaptoacetic acid thioesters are shown in Fig-
ure 2. These synthesized thioesters were all characterized by 1H
and 13C NMR and confirmed by MS (see Supporting information).
Due to racemic mixtures of amino acids being used, the products
are not expected to be chiral.
To test whether the inhibitory activities of these mercaptoacetic
acid thioesters against L1 have improved, L1 was over-expressed
and purified as previously described.21 In vitro, the inhibitory
activities of all compounds prepared were tested against L1 on
an Agilent UV8453 spectrometer as described by Bush et al. using
cefazolin as the substrate.22 The substrate concentration was
The capacity of the mercaptoacetic acid thioesters to restore the
antibacterial activity of cefazolin against Escherichia coli cells
expressing L1 was investigated by determining the minimum inhi-
bitory concentrations (MICs) of cefazolin in the absence and pres-
ence of 16 lg/mL 1–10. MIC values were determined by using the
Clinical and Laboratory Standards Institute (CLSI) macrodilution
(tube) broth method.23 The bacterial strain of E. coli BL21(DE3)
containing plasmids pET26b-L1, as well as an E. coli BL21(DE3)
control without plasmid, were used to assess these inhibitors.
60
lM, and inhibitor concentrations were varied between 0.01
and 1
l
M. Enzyme and inhibitor were pre-incubated for 30 min
before starting the kinetic assays. The inhibitor concentrations
causing 50% decrease of enzyme activity (IC50) were calculated
based on the kinetic data. The IC50 values of the ten compounds
against L1 with cefazolin as substrate are listed in Table 1. It can
be observed that the thioesters 1–9 exhibited strong inhibition of
The final concentration of inhibitor was 16 lg/mL. The data listed
in Table 2 shows that inhibitors 1–9 resulted in at least 4-fold
reduction of MIC for E. coli BL21(DE3) expressing L1, effectively
restoring the MIC observed with E. coli not expressing L1, indicat-
ing successful inhibition of L1 in vivo (inside the bacterial cells).
Inhibitor 10 did not decrease the MIC relative to the blank control,
indicating no inhibition of L1, which is consistent with the absence
of any inhibition in vitro.
L1 with an IC50 value range of 0.02–0.6 lM, while compound 10
had no inhibitory activity. A possible reason is that the amino acid
in its structure was replaced by 4-aminobenzoic acid. Clearly, the
IC50 values of 1, 4 and 7 are smaller than those of 2, 5 and 8, respec-
tively, revealing that the 4-diphenyl methyl as R1 results in stron-
ger inhibitory activity against L1 than the benzyl. IC50 values of 2
and 5 are lower than the corresponding values of 3 and 6, respec-
tively, indicating that the aromatic R1 improves inhibitory activity
compared to the aliphatic pentyl group. This activity relationship is
related to the space sizes of these substitutes, that is, the 4-diphe-
In order to clarify why the introduction of 4-biphenyl methyl as
R1 can enhance inhibitory activity, 4, 5 and 6 were docked into the
active site of the L1 crystal structure (PDB code 2AIO)24 using the
same procedure as reported previously.25 We chose these com-
pounds because of a significant improvement of their inhibitory
effect compared to inhibitor C (IC50 = 2.9
formations (the top ranked conformations) of 4, 5, and 6 docked
l
M).18 Low-energy con-
R2
O
R2
O
R2
H2N
CO2H
c
b
S
CO2H
CO2H
R1
N
H
CO2H
R1
N
H
O
O
a
O
R1 Cl
1-9
R1 OH
1b-9b
O
d
e
Cbz
HN
S
H2N
CO2H
CO2H
Cbz
,
N
H
10b
10
R1=
NH
R2
(CH2)4
,
,
,
S
,
Scheme 1. Synthetic route of mercaptoacetic acid thioesters. Reagents and conditions: (a) SOCl2, DMF, 80 °C, 3 h; (b) NaOH, diethyl ether, HCl; (c) mercaptoacetic acid, ethyl
chloroformate, Et3N, AcOEt/DMF; (d) CbzCl, NaOH, diethyl ether, HCl; (e) mercaptoacetic acid, ethyl chloroformate, Et3N, DMF.

4700
X.-L. Liu et al. / Bioorg. Med. Chem. Lett. 26 (2016) 4698–4701
NH
NH
NH
H
H
N
N
H
N
O
O
O
O
S
CO2H
O
S
CO2H
CO2H
O
S
CO2H
3
2
1
H
N
H
H
N
N
O
O
S
CO2H
O
O
O
S
CO2H
O
S
4
6
5
S
S
S
H
N
H
H
N
N
O
O
O
S
CO2H
O
S
CO2H
CO2H
O
O
S
CO2H
7
8
9
O
O
S
O
N
H
10
Figure 2. Structures of the synthesized mercaptoacetic acid thioesters.
Table 1
amide nitrogen and the thioester oxygen interacts with Zn1
(Fig. 3B). The R1 group occupies the same position as R1 in com-
pounds 5 and 6; however, the central portion of the molecule
seems to be less extended and rather folded, probably due to the
larger R1 group. As a result, the orientation of the amide resembles
more that of the amide in hydrolyzed moxalactam, including the
hydrogen bond with Tyr32 via the amide oxygen rather than the
nitrogen (Fig. 3A and B).
In conclusion, in further optimizing the inhibitor scaffold
against L1, ten new mercaptoacetic acid thioesters were designed
and synthesized. Biological activity assays indicate that nine thioe-
sters containing amino acids exhibited IC50 values ranging from
Inhibitory activities of mercaptoacetic acid thioesters against metallo-b-lactamase L1
Inhibitor
IC50
(
lM)
Inhibitor
IC50 (lM)
1
2
3
4
5
0.08
0.21
0.60
0.14
0.25
6
7
8
9
0.33
0.02
0.09
0.08
—
10
Cefazolin was used as substrate; inhibitor concentrations were varied between 0.01
and 1 M.
l
—No inhibition observed at the concentrations tested.
0.02 to 0.6 lM, which are lower than those of the inhibitors
into the L1 active site with binding energies of À12.9, À12.3 and
À11.5 kcal molÀ1, are shown in Figure 3, panels (B), (C), and (D),
respectively. These binding energies showed a trend corresponding
reported previously.18 All these inhibitors were able to decrease
MIC values of cefazolin using E. coli BL21(DE3) cells producing L1
to levels when using the same cells not expressing any b-lacta-
mase, thus effectively restoring the antimicrobial activity of the
antibiotic by inhibiting L1. Analysis of structure–activity relation-
ship reveals that replacement of 2-thiophenyl methyl18 or benzyl
R1 groups by 4-diphenyl methyl improves the inhibitory activity
of the thioesters. Docking studies revealed that the introduction
of 4-diphenyl methyl as R1 altered the orientation of the inhibitor
slightly, resulting in a binding fashion that is more similar to that
of a moxalactam hydrolysis product co-crystallized with L1.24 As
previously observed,18 using methionine in the amino acid thioe-
sters yielded the highest L1 binding affinity.
to the experimental IC50 values (0.14, 0.25, and 0.33 lM, respec-
tively). The binding mode of 5 and 6 is the same as that of the pre-
viously docked compounds A and B, which is that one of the
carboxylate oxygens bridges the two Zn(II) ions, while the other
oxygen coordinates Zn2 and hydrogen bonds with Ser221, while
the amide nitrogens form hydrogen bonds with the Tyr32 side
chain (Fig. 3C and D).18 The phenylalanine side chain (R2) is located
in a position corresponding to that of the methionine and trypto-
phan side chains of compounds A and B18 and the 4-hydrox-
yphenyl ring of a co-crystalized moxalactam hydrolysis product
(Fig. 3A).24 The R1 groups of 5 and 6 as well as A and B18 are located
in a space unoccupied by hydrolyzed moxalactam next to Tyr32.
For inhibitor 4, the big 4-biphenyl group changes the binding mode
slightly. The position of the carboxylate is similar to that of 5 and 6,
but the amide oxygen hydrogen bonds with Tyr32 rather than the
Acknowledgment
This work was supported by grants 81361138018, 21272186
and 21572179 (to K.W.Y.) from the National Natural Science Foun-
dation of China.
Table 2
MICs of cefazolin (lg/mL) in the presence and absence of mercaptoacetic acid
thioester inhibitors at a concentration of 16 lg/mL observed in E. coli cells expressing
L1 or no b-lactamase
Supplementary data
Inhibitor
E. coli
E. coli-L1
Inhibitor
E. coli
E. coli-L1
Supplementary data (detailed synthesis procedure, NMR and
ESI mass data for target compounds; methods for enzyme overex-
pression and purification; inhibition assay; determination of MIC
and IC50; and docking study) associated with this article can be
found, in the online version, at http://dx.doi.org/10.1016/j.bmcl.
2016.08.048.
Blank
4
4
4
4
4
4
16
4
4
4
2
6
7
8
9
4
4
4
4
4
2
4
4
4
1
2
3
4
5
10
16
2

X.-L. Liu et al. / Bioorg. Med. Chem. Lett. 26 (2016) 4698–4701
4701
A
Mox-L1
B
Compound 4
C
D
Compound 6
Compound 5
Figure 3. Hydrolyzed moxalactam co-crystallized with L1 (A).24 Low energy conformations of 4 (B), 5 (C) and 6 (D) docked into the active site of L1. The enzyme backbone is
shown as a cartoon in green and selected residues are shown as sticks colored by atom (C, magenta; N, blue; O, red). The Zn(II) ions are shown as blue spheres. Hydrolyzed
moxalactam and three inhibitors are shown as sticks colored by atom (C, yellow; N, blue; O, red; S, pale yellow). Key interactions between the inhibitors and protein residues
are indicated by dashed lines. The upper one is Zn1 and the lower one is Zn2. These figures were generated with PyMOL.
14. Concha, N. O.; Janson, C. A.; Rowling, P.; Pearson, S.; Cheever, C. A.; Clarke, B. P.;
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