Structure-based development of (1-(3′-Mercaptopropanamido)methyl)boronic Acid Derived Broad-Spectrum, Dual-Action Inhibitors of Metallo- And Serine-β-lactamases

Article abstract of DOI:10.1021/acs.jmedchem.9b00735

The emergence and spread of bacterial pathogens acquired metallo-β-lactamase (MBL) and serine-β-lactamase (SBL) medicated β-lactam resistance gives rise to an urgent need for the development of new dual-action MBL/SBL inhibitors. Application of a pharmacophore fusion strategy led to the identification of (2′S)-(1-(3′-mercapto-2′-methylpropanamido)methyl)boronic acid (MS01) as a new dual-action inhibitor, which manifests broad-spectrum inhibition to representative MBL/SBL enzymes, including the widespread VIM-2 and KPC-2. Guided by the VIM-2:MS01 and KPC-2:MS01 complex structures, further structural optimization yielded new, more potent dual-action inhibitors. Selectivity studies indicated that the inhibitors had no apparent inhibition to human angiotensin-converting enzyme-2 and showed selectivity across serine hydrolyases in E. coli and human HEK293T cells labeled by the activity-based probe TAMRA-FP. Moreover, the inhibitors displayed potentiation of meropenem efficacy against MBL- or SBL-positive clinical isolates without apparent cytotoxicity. This work will aid efforts to develop new types of clinically useful dual-action inhibitors targeting MBL/SBL enzymes.

Full text of DOI:10.1021/acs.jmedchem.9b00735

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Article
Structure-Based Development of (1-(3'-Mercaptopropanamido)methyl)boronic
Acid Derived Broad-Spectrum, Dual-Action
Inhibitors of Metallo- and Serine-#-Lactamases
Yao-Ling Wang, Sha Liu, Zhu-Jun Yu, Yuan Lei, Meng-Yi Huang, Yu-Hang Yan, Qiang Ma, Yang Zheng,
Hui Deng, Ying Sun, Chengyong Wu, Yamei Yu, Qiang Chen, Zhenling Wang, Yong Wu, and Guo-Bo Li
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.9b00735 • Publication Date (Web): 03 Jul 2019
Downloaded from http://pubs.acs.org on July 3, 2019
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Structure-Based Development of (1-(3'-Mercaptopropanamido)methyl)boronic
Acid Derived Broad-Spectrum, Dual-Action Inhibitors of Metallo- and Serine-β-
Lactamases
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†
,§
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Yao-Ling Wang, Sha Liu, Zhu-Jun Yu, Yuan Lei, Meng-Yi Huang, Yu-Hang
†
†
†
‡
‡
‡
‡
Yan, Qiang Ma, Yang Zheng, Hui Deng, Ying Sun, Chengyong Wu, Yamei Yu,
‡
‡
,†
,†
Qiang Chen, Zhenling Wang, Yong Wu,* Guo-Bo Li*
†
Key Laboratory of Drug-Targeting and Drug Delivery System of the Education
Ministry, Sichuan Engineering Laboratory for Plant-Sourced Drug and Sichuan
Research Center for Drug Precision Industrial Technology, West China School of
Pharmacy, Sichuan University, Sichuan 610041, China.
‡
State Key Laboratory of Biotherapy and Cancer Center, West China Hospital, West
China Medical School, Sichuan University, Chengdu, 610041, China
§
Y.-L. Wang and S. Liu are co-first authors.
*
Correspondence: liguobo@scu.edu.cn (G.-B. Li); wyong@scu.edu.cn (Y. Wu)
ABSTRACT
The emergence and spread of bacterial pathogens acquired both metallo-β-lactamase
MBL) and serine-β-lactamase (SBL) medicated β-lactam resistance gives rise to an
(
urgent need for the development of new dual-action MBL/SBL inhibitors. Application
of a pharmacophore fusion strategy led to the identification of (2'S)-(1-(3'-mercapto-2'-
methylpropanamido)methyl)boronic acid (MS01) as a new dual-action inhibitor, which
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manifests broad-spectrum inhibition to representative MBL/SBL enzymes, including
the widespread VIM-2 and KPC-2. Guided by the VIM-2:MS01 and KPC-2:MS01
complex structures, further structural optimization yielded new more potent dual-action
inhibitors. Selectivity studies indicated that the inhibitors had no apparent inhibition to
human angiotensin-converting enzyme-2, and showed selectivity across serine
hydrolyases in E. coli and human HEK293T cells labeled by the activity-based probe
TAMRA-FP. Moreover, the inhibitors displayed potentiation of meropenem efficacy
against MBL- or SBL-positive clinical isolates without apparent cytotoxicity. This
work will aid efforts to develop new types of clinically useful dual-action inhibitors
targeting MBL/SBL enzymes.
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INTRODUCTION
The β-lactam antibiotics are the most widely used class of broad-spectrum antibacterial
agents, however, which have been plagued by the ongoing problems of resistance.1-5
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Resistance to β-lactam antibiotics is mainly due to the production of β-lactamases,
which can be classified into two categories according to their hydrolytic mechanisms.6-8
The first are serine-β-lactamases (SBLs; ambler classes A, C, and D), which employ an
active site nucleophilic serine to destroy the β-lactam ring (Figure 1A); clinically
important SBL families include KPC, TEM, SHV, CTX-M, GES, SME, AmpC, OXA,
and others.4,9-13 The second are metallo-β-lactamases (MBLs; ambler class B), which
use one or two active site zinc ions to activate a nucleophilic water molecule and
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thereby cleave the β-lactam ring (Figure 1A). MBLs are further divided into subclass
B1, B2, B3, and somewhere B4, primarily by difference in active site zinc coordination
features.15,16 The B1 MBLs (e.g. NDM, VIM, and IMP) and B3 MBLs (e.g. GOB, AIM,
SMB, and L1) are catalyticlly active with two zinc ions and show a broad substrate
profile, while the mono-zinc B2 MBLs, such as CphA and Sfh-I, have a narrow
specificity profile, solely hydrolysing carbapenems. Among them, the di-zinc B1 MBLs,
such as NDM-1, are of widespread clinical significance.14,17-20
Several SBL inhibitors have been used in combination with a β-lactam partner to
overcome resistance, as, for example, clavulanate/amoxicillin, tazobactam/piperacillin,
sulbactam/ampicillin, and avibactam/ceftazidime.2,4 Recently vaborbactam, a boron-
heterocyclic SBL inhibitor, was approved for use with meropenem for treating
complicated urinary tract infections and pyelonephritis.21,22 By contrast, there are no
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equivalent inhibitors for MBLs, which can hydrolyze almost all β-lactam antibiotics
including carbapenems (i.e., “last resort” antibiotics) as well as the β-lactam SBL
inhibitors (e.g., clavulanate, tazobactam, and sulbactam).18,19,23 More concerning is the
continuous emergence and spread of multidrug-resistant pathogens acquired both MBL
and SBL genes,24,25 leading to an urgent need for the development of broad-spectrum,
dual-action MBL/SBL inhibitors to overcome MBL or MBL/SBL-mediated resistance.
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(A)
R2
R2
R1
R2
R1
R1
R3
+
SBL
R3
R3
N
N
OH
O
N
H
O
O
O
OH
OH
O
O
..
SBL-Ser-OH
O
SBL-Ser
H O
Carbapenems
+
MBL
2
Zn(II) , n=1,2
SBL
n
R2
R2
N
R1
O
R1
R3
R2
R1
R3
R3
N
O
OH
OH
OH
HO
N
OH
O
O
MBL
H
H
O
O
OH
O
H
Zn2
Zn2
Zn1
β
-Amino acid
Inactive form'
Zn1
H O
2
'
R2
(B)
R
O
R1
Cl
HN
Cl
S
N
O
HO
HO
B
S
B
P OH
O
O
OH
P OH
H
OH
O
OH
HO
OH
O
O
OH
Cyclic boronates
Noncyclic boronic acids
Cyclobutanones
Bisphosphonates
O
R2
O
3
'
3
'
(
S)
OH
OH
1'
N
1
B
HS
2'
R1
1' N
H
1
B
HS
2
'
H
OH
OH
MS01
Thiol-containing acyclic boronates
Figure 1. (A) The mechanisms for metallo- (MBL) and serine-β-lactamase (SBL)
catalysed hydrolysis of carbapenems. (B) The dual-action MBL/SBL inhibitors.
Until recently, there have been a few reports of dual-action MBL/SBL inhibitors
(Figure 1B). Cyclic boronates are the most desirable dual-action inhibitors, which can
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potently inhibit all classes of MBL and SBL enzymes via a mechanism involving
mimicking of their common natural tetrahedral intermediates as revealed by
crystallographic analyses.26,27 The benzo[b]thiophen-2-ylboronic acids28,29 and
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cyclobutanones (Figure 1B) inhibit multiple MBL and SBL enzymes by an analogous
manner to cyclic boronates (i.e. mimicking the tetrahedral intermediates). Using
combined computational and experimental assays, we recently identified aromatic
bisphosphonates and rosmarinic acid as dual-action MBL/SBL inhibitors,31,32 but which
are only active against some types of MBL and SBL enzymes. Here, we report
structure-based development of (1-(3'-mercaptopropanamido)methyl)boronic acid
derived new broad-spectrum, dual-action MBL/SBL inhibitors.
Considering the structural and mechanistic difference between MBL and SBL
enzymes, we proposed a pharmacophore fusion strategy to obtain dual-action
MBL/SBL inhibitors. Application of this strategy led to the identification of the
compound (2'S)-(1-(3'-mercapto-2'-methylpropanamido)methyl)boronic acid (MS01,
Figure 1B), which manifested broad-spectrum inhibition to all classes of representative
MBL and SBL enzymes, including subclass B1 VIM-2, subclass B1 NDM-1, subclass
B2 Sfh-I, subclass B3 GOB-18, class A KPC-2, class A TEM-1, class C AmpC, and
class D OXA-48 enzymes; with the exception of TEM-1 and AmpC, these enzymes are
carbapenemases that are able to inactivate carbapenems and other β-lactams.11,33
Crystallographic analyses reveal insights into the dual-action inhibition modes of MS01
with VIM-2 and KPC-2, providing important basis for structural optimization. The
subsequent multi-target structure-activity relationship (SAR) studies identified new (1-
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(3'-mercaptopropanamido)methyl)boronic acid derivatives as broad-spectrum, dual-
action inhibitors. Some of them were observed by crystallographic analyses to bind
with VIM-2 and KPC-2 via unexpected binding modes. The inhibitors were further
evaluated for their selectivity to human angiotensin-converting enzyme (ACE)-2 and
to the serine hydrolyases in E. coli and human HEK293T cells by activity-based protein
profiling using TAMRA-FP, as well as their efficacy to potentiate meropenem activity
against MBL- or SBL-positive clinical isolates.
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RESULTS AND DISCUSSIONS
Design of (2'S)-(1-(3'-mercapto-2'-methylpropanamido)methyl)boronic Acid
(MS01) as a Dual-Action MBL/SBL Inhibitor. By analyses of crystal structures of
MBL enzymes in complex with small-molecule inhibitors,21,34 we observed that most
of MBL inhibitors involve direct active site metal chelation via metal-binding
pharmacophores (MBPs),35 such as (S)-3-mercapto-2-methylpropanamido,36-38 5-
aminothiazole-4-carboxylic acid,39 (Z)-2-mercapto-3-phenylacrylic acid,40 2-
(
mercaptomethyl)benzoic acid,41 and phthalic acid.42 Comparatively, most of SBL
inhibitors are positioned to covalently bind with the active site nucleophilic serine, e.g.,
via the boronic acid group.29,43-47 With the aim to obtain dual-action inhibitors targeting
MBL and SBL enzymes, we came up with a strategy of integrating the key
pharmacophores of MBL and SBL inhibitors. As an initial attempt, we designed and
synthesized
the
compound
(2'S)-(1-(3'-mercapto-2'-
methylpropanamido)methyl)boronic acid (MS01, Figure 1B and Scheme 1). The (2'S)-
3'-mercapto-2'-methylpropanamido moeity of MS01, as a key motif of captopril (an old
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drug used in clinical for more than 30 years), may have potential biocompatibility and
achieve broad-spectrum inhibition to multiple MBL enzymes, particularly for B1 MBL
enzymes (e.g., NDM-1 and VIM-2), whereas the α-amino boronic acid moiety of MS01
is expected to target the catalytic serine residue of SBL enzymes. Meanwhile, we
synthesized the non-thiol analogues MS02 and MS03 (Table 1 and Scheme 1) to test
whether thiol is essential for MBL inhibition, and the non-boronic acid compound
MS04 (Table 1 and Scheme 1), bearing a carboxylic acid group instead of boronic acid,
to examine the necessity of the boronic acid group for SBL inhibition.
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To investigate the MBL/SBL inhibitory activity of MS01-MS04, we selected a
representative panel of clinically relevant MBLs, including VIM-2 (subclass B1),
NDM-1 (subclass B1), Sfh-I (subclass B2), and GOB-18 (subclass B3), and SBLs,
including KPC-2 (class A), TEM-1 (class A), AmpC (class C), and OXA-48 (class D).
All of these recombinant enzymes were expressed and purified from E. coli DH5α and
BL21 cultures; the details are described in the Experimental section. Since FC5 has
been demonstrated as a robust fluorescent substrate for the di-zinc B1 MBLs (e.g.,
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VIM-2 and NDM-1), we here examined whether FC5 could be used as a substrate for
activity test for other classes of β-lactamases. The enzyme kinetic analyses revealed
that all of the tested β-lactamases exhibited substantial hydrolytic activity towards FC5
resulting in measurable fluorescent signals, albeit with apparent difference in their
kinetic parameters (Figure S1 and Table S1), indicating that FC5 was a suitable
substrate for these enzymes. Consequently, we determined IC values of MS01-MS04
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the enzyme kinetics, we calculated the inhibition constant (K ) values from the
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corresponding IC valuesusing the Cheng-Prusoff equation to compare the inhibitory
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potency for the compounds (for details see the Experimental Section).
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As anticipated, MS01 displayed inhibitory activity against all of the tested MBL
and SBL enzymes (Table 1 and Figure S2). For the subclass B1 MBLs, MS01 showed
K values of 0.76 and 67.13 μM to the widespread VIM-2 and NDM-1, respectively,
i
comparable to L-captopril, a well-known MBL inhibitor (Table 1), while the non-thiol
compounds MS02 and MS03, bearing a boronic acid group, had low inhibition to VIM-
2 and NDM-1 (Table 1), suggesting the importance of the thiol group (rather than
boronic acid) for B1 MBL inhibition. Another compound MS04, which contains a thiol
group but no boronic acid, displayed potent inhibition to these two B1 MBLs as MS01
(Table 1), probably owing to the contributions of both the thiol and carboxylate group,
similar as previously reported inhibitors.36-38 Notably, MS01 can also inhibit subclass
B2 Sfh-I and B3 GOB-18 with K values of 7.34 and 2.23 μM, respectively (Table 1).
i
The non-thiol compound MS02 exhibited comparable inhibition as MS01 to Sfh-I and
GOB-18; conversely, the thiol compounds MS04 and L-captopril did not show marked
inhibition against Sfh-I and GOB-18 (Table 1), indicating that the thiol group is likely
not essential for inhibiting these B2/B3 enzymes.
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3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Table 1. The inhibitory potency (K , μM) of MS01-MS04 to the representative MBL
and SBL enzymes.a
i
MBLs
Subclass Subclass
SBLs
Class C Class D
Subclass B1
Class A
ID
Structure
B2
B3
GOB-18 KPC-2 TEM-1 AmpC
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
VIM-2 NDM-1
Sfh-I
OXA-48
O
3'
(S)
OH
MS01
MS02
MS03
MS04
L-cap
0.76
>300
>300
1.68
0.36
67.13
>300
>300
22.50
5.45
7.34
5.78
2.23
12.14
>300
>300
>300
2.70
40.97
6.46
58.45
291.10
>300
70.19
101.61
>300
HS
1' N
H
1
B
2
'
OH
O
OH
38.15 100.28
N
B
H
OH
O
OH
>300
>300
>300
86.07
140.03
>300
36.26
>300
>300
N
B
H
OH
O
(
S)
OH
>300
HS
N
H
O
O
OH
O
(S)
(S)
>300
>300
HS
N
O
H
N
O
S
N
N
Tazo
N
128.24
>300
62.23
56.59
0.20
0.19
18.75
15.74
O
OH
O
a
The method for measuring IC /K values is described in Experimental Section, and
5
0
i
the dose–response curves of compounds inhibiting the selected enzymes are given in
Supporting Information Figure S2.
For the tested SBLs, MS01 manifested K values of 2.70, 40.97, 6.46, and 70.19
i
μM, respectively, to class A SBL KPC-2 and TEM-1, class C AmpC, and class D OXA-
4
8 (Table 1 and Figure S2). Similarly, the boronic acid compounds MS02 and MS03
displayed inhibitory activity against these SBL enzymes, whereas structurally similar
non-boronic acid compound MS04 had no or weak SBL inhibition, suggesting that the
boronic acid group is likely important for SBL inhibition. These SAR results
demonstrated feasibility of integrating the key pharmacophores of MBL and SBL
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inhibitors to develop broad-spectrum, dual-action inhibitors.
X-ray Structure Determination of the VIM-2:MS01 and KPC-2:MS01 Complexes.
We then conducted crystallographic analyses for the VIM-2:MS01 and KPC-2:MS01
complexes to investigate the dual inhibition modes of MS01. Details of crystallization
and structure determinations are described in Experimental section and Table S2. Co-
crystallization experiments yielded high-resolution crystal structures of the VIM-
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
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3
3
3
3
3
4
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4
4
4
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5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
2
:MS01 (PDB ID 6J8R) and KPC-2:MS01 (PDB ID 6J8Q) complexes. VIM-2:MS01
5
0
crystallized in the previously described space group P2 22 , with one molecule in the
1
1
asymmetric unit (ASU) (Table S3). In VIM-2:MS01 structure, the observed clear Fo–
Fc electron density adjacent to the two zinc ions allow unambiguous modelling of
MS01 (Figure 2A and S3A). KPC-2:MS01 crystallized in space group P321 with four
molecules in the ASU, different from those previously reported45,51,52 (Table S4); MS01
could also be well-modelled into clearly defined electron density in the active site of
KPC-2 (Figure 2B and S3B).
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2
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5
5
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5
5
6
Figure 2. Crystallographic analyses reveals compound MS01 binding modes to VIM-
2 MBL and KPC-2 SBL. (A) The mode of MS01 binding as defined by the mF -DF
o
c
electron density maps observed in the VIM-2:MS01 structure (PDB ID 6J8R),
indicating that MS01 fits well with the electron density, and chelates to and bridges
with the two active site zinc ions. (B) The mode of MS01 binding as defined by the
mF -DF electron density maps observed in the KPC-2:MS01 structure (PDB ID 6J8Q),
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
o
c
indicating that MS01 fits well with the electron density, and forms a covalent bond with
the residue Ser . (C) View from the VIM-2:MS01 structure (PDB ID 6J8R) reveals
7
0
that the inhibitor binds to form hydrophobic and electrostatic interactions with residues
on the L3 and L10 loops (e.g., hydrogen-bonding interactions with Asn233 and Arg228,
and hydrophobic interactions with Phe ). (D) View from the KPC-2:MS01 structure
6
1
(PDB ID 6J8Q) reveals that the inhibitor binds to make hydrogen-bonding interactions
with the residues Asn , Glu , Asn , and Thr in the active site.
1
32
166
170
237
The VIM-2:MS01 structure reveals that MS01 works via a thiol-participating
metal chelating mode of inhibition, similar to that observed for previously reported
thiol-based MBL inhibitors.36-38,53 In VIM-2:MS01, the thiol binds to displace the zinc-
bridging nucleophilic hydroxide that is observed in the native VIM-2 active site (PDB
36
ID: 4BZ3) , and chelate with the two active site zinc ions (2.2~2.3 Å) (Figure 2C),
demonstrating the importance of the thiol for VIM-2 inhibition, consistent with the SAR
data shown in Table 1. The amido-carbonyl group of MS01 is positioned to make
hydrogen-bonding interactions with the residue Asn233 (3.0 Å) on the L10 loop of VIM-
2
(Figure 2C), a conserved residue observed in active site of subclass B1 MBLs (e.g.
36
NDM-1 Asn , IMP-1 Asn , and BcII Asn ) , and its boronic acid form hydrogen
2
33
233
233
bonds with Arg228 (3.5 Å) (Figure 2C), which is structurally equivalent to the catalytic
important positively charged residue on L10 loop of B1 MBLs, for example, NDM-1
3
6
Lys , IMP-1 Lys , and BcII Lys . This may indicate that MS01 has a similar
2
11
224
224
inhibition mode to subclass B1 MBLs, including NDM-1. By contrast, for B2 MBL
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Shf-I and B3 MBL GOB-18, MS01 appears to bind via boronic acid (rather than thiol)-
involving zinc ion contacts as predicted by molecular docking analyses (Figure S4),
similar as that observed in crystal structures of B2 Sfh-I:L-CS319 (a thiol containing
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
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3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
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8
9
0
1
2
3
4
5
6
7
8
9
0
5
3
54
compound) (PDB ID: 5EW0) and B2 CphA:D-Captopril (PDB ID: 2QDS) ; this
distinct inhibition mode of MS01 to B2/B3 MBLs may explain the differences in the
SAR trends (e.g., MS01 vs MS02 and MS01 vs MS04; see Table 1).
The KPC-2:MS01 structure reveals continuous electron density connecting the
boron atom of MS01 with the terminal hydroxyl group of the catalytic Ser (Figure 2B
7
0
and S3B), demonstrating that MS01 binds with KPC-2 via forming a covalent adduct
between its boronic acid and Ser (Figure 2D), consistent with the SAR results in Table
7
0
1; this inhibition mode is similar as that of previously reported boronic acid-based SBL
inhibitors.22,46,47,55,56 The KPC-2:MS01 structure represents a covalent inhibition mode
(i.e. the formed tetrahedral boron atom) that mimics the tetrahedral transition state of
4
3
substrate binding, which is transiently present on the hydrolytic pathway. Moreover,
MS01 binding is further stabilized by multiple hydrogen-bonding interactions with the
catalytically important residues Ser (2.8 Å), Thr (2.8 Å), Asn170 (2.7 Å) Glu166 (2.7
7
0
237
Å), and Asn132 (2.9 Å) (Figure 2D). Superimposing structures of KPC-2 with TEM-1
57
58
59
(
PDB ID 1M40), AmpC (PDB ID 1FSY), and OXA-48 (PDB ID 5FAQ) revealed
that despite differences in their active sites, MS01 may have a similar binding mode
with these SBL enzymes (Figure S5).
The VIM-2:MS01 and KPC-2:MS01 structures not only reveal the substrate-
competitive, dual-action MBL/SBL inhibition modes but also provide important basis
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for further structural optimization. On superimposing VIM-2:MS01 with other crystal
structures of VIM-2 complexed with 3-mercaptopropanamido-containing inhibitors36-
38,
hydrophobic interactions with Phe and Tyr on the flexible L3 loop that is important
we observed that comparing to other inhibitors, MS01 only makes weak
0
1
2
3
4
5
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9
0
1
2
3
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9
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9
0
1
2
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9
0
6
1
67
for inhibitor capture as previously described36-38,50 (Figure S6). Further comparison of
KPC-2:MS01 and reported KPC-2 structures45,56,60 revealed that MS01 did not occupy
the subpocket around the residues Trp , Ser , and Thr (Figure S7). These results
1
05
130
235
suggested that introduction of a substituent at the 1-position of MS01 might be able to
improve the potency to VIM-2 and KPC-2 as well as other MBL/SBL enzymes.
Structure-Based Optimization of (1-(3'-Mercaptopropanamido)methyl)boronic
Acid Derivatives. Based on the VIM-2:MS01 and KPC-2:MS01 structures, we
performed fragment-growing analyses on the 1-position using the LEADOPT program,
61
a tool for structure-based lead optimization. The results suggested that the introduced
fragments in (R)-configuration might be better for binding with VIM-2 and KPC-2.
According to the synthetic accessibility, we synthesized a compound MS05, bearing a
benzyl group at 1-position in (R)-enantiomer (Table 2), via the route shown in Scheme
2
. MS05 displayed potent inhibition to VIM-2 (K = 0.36 μM), comparable to MS01
i
(
K = 0.76 μM), while it showed slightly less potent inhibition to KPC-2 (K = 6.85 μM)
i
i
as compared with MS01 (K = 2.70 μM) (Table 2 and Figure S8). MS05 also manifested
i
inhibition to subclass B1 MBL NDM-1, B2 Sfh-I, B3 GOB-18, and class A SBL TEM-1,
class C AmpC, and class D OXA-48, with K values of 3.88, 3.48, 9.03, 15.41, 7.87,
i
and 25.83 μM, respectively; by comparison with MS01, MS05 had slightly improved
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potency to NDM-1, TEM-1, and OXA-48 (Table 2 and Figure S8).
Table 2. The inhibitory potency (K , μM) of MS05-MS12, with modifications of the
i
(
S)-3'-mercapto-2'-methylpropanamido moiety, to the MBL and SBL enzymes.a
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
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0
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2
3
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0
MBLs
Subclass Subclass
SBLs
Subclass B1
Class A
Class C Class D
ID
Structure
B2
B3
GOB-18 KPC-2 TEM-1 AmpC
VIM-2 NDM-1
Sfh-I
OXA-48
25.83
O
MS05
3'
(R)
0.36
3.00
3.88
6.87
3.48
5.79
9.03
6.85
15.41
7.87
(
S)
OH
HS
HS
1'
N
H
1
B
2'
OH
O
(R)
OH
MS06
MS07
14.26
13.70 114.07
11.56
>300
>300
N
H
B
OH
O
(R)
(S)
OH
HS
O
N
H
O
B
2.41
5.42
57.71
14.81
7.76
4.17
32.15
11.55
8.65
3.86
32.82
28.02
27.39
11.49
N
OH
O
(
R)
(
S)
OH
HS
N
H
B
MS08
50.22
NH
OH
O
MS09
MS10
MS11
(R)
4.04
3.34
>300
190.53
165.00
>300
0.89
0.48
9.21
1.64
1.69
19.38
12.41
15.84
18.73
>300
1.14
1.56
246.79
50.51
>300
OH
HS
N
H
B
OH
O
(R)
HS
OH
N
B
H
OH
O
(R)
23.54
106.79
33.41
OH
N
H
B
OH
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O
MS12
>300
>300
152.3
~252
>300
>300
158.49
>300
(
R)
OH
N
B
H
OH
a
The dose–response curves of compounds inhibiting the selected enzymes are given in
Supporting Information Figure S8.
0
1
2
3
4
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7
8
9
0
1
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9
0
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9
0
We obtained a crystal structure of KPC-2:MS05 (PDB ID 6JN3) to 2.2 Å resolution
(
Table S2 and S4), in which MS05 could be confidently fitted with the electron density
maps in the KPC-2 active site (Figure S9). Similar to MS01, MS05 was observed to
form a covalent bond with the catalytic residue Ser and make multiple hydrogen bonds
70
with the residues Ser (3.0 Å), Thr (2.7 Å), Asn170 (2.6 Å), Glu166 (2.7 Å), and Asn132
7
0
237
(3.1 Å) (Figure 2B and 3A). The introduced benzyl group of MS05 bound to the
subpocket located between the L3 and L5 loops, and made edge-to-face aromatic
interactions with Trp105 (Figure 3A). By superimposing the structures of KPC-2:MS05
and KPC-2:MS01, we observed that the binding of MS05 resulted in substantial
rotation of the side chain of Trp105 to accommodate the introduced benzyl group,
providing evidence for flexibility in the conformation of Trp105 and suggesting its
important role in substrate/inhibitor capture (Figure 3B and Figure S7).45,56,60
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0
Figure 3. Crystallographic analyses reveals MS05 binding mode to KPC-2. (A) View
from the KPC-2:MS05 structure (PDB ID 6JN3) reveals that the inhibitor binds to form
a covalent bond with the catalytic residue Ser , make hydrogen-bonding interactions
7
0
with the residues Ser , Thr , Asn , Glu , and Asn , and makes edge-to-face
7
0
237
170
166
132
aromatic interactions with Trp . (B) Comparison of KPC-2:MS05 and KPC-2:MS01
1
05
(PDB ID 6J8Q) reveals evidence for the important role of Trp105 in substrate/inhibitor
capture.45,56,60
Exploring the Modifications of (2'S)-3'-mercapto-2'-methylpropanamide Motif. We
then explored modifications of the (2'S)-3'-mercapto-2'-methylpropanamido moiety,
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1
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2
2
2
2
2
2
2
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with fixing the benzyl group at 1-position (Table 2), to examine whether it is possible
to improve the inhibitory activity. The replacement of the 2'-methyl group of (2'S)-3'-
mercapto-2'-methylpropanamido with relatively larger chemical groups, including the
benzyl (MS06), 1,3-dioxoisoindolin-2-yl (MS07), and benzylamino (MS08) group
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
(
Scheme 3), had no improvement in the inhibitory potency to the tested MBL and SBL
enzymes as compared with MS05, and even led to a decrease in ligand efficiency (Table
and Figure S8). In contrast, the removal of the 2'-methyl group (MS09) appeared to
improve the inhibitory activity for suclass B2 Sfh-I (K = 0.89 μM), B3 GOB-18 (K =
2
i
i
1.64 μM), and class C AmpC (K = 1.14 μM), but reduce the potency to suclass B1
i
VIM-2 (K = 4.04 μM) and NDM-1 (K = 190.53 μM) (Table 2 and Figure S8),
i
i
compared with MS05. Similarly, MS10, with 2'-mercaptoacetamide instead of (2'S)-3'-
mercapto-2'-methylpropanamide, exhibited decreased activity to VIM-2 (K = 3.34 μM)
i
and NDM-1 (K = 165 μM), and comparable activity to other tested MBL and SBL
i
enzymes as MS05 and MS09 (Table 2 and Figure S8). The non-thiol analogues MS11
and MS12 displayed low inhibitory activity to VIM-2 and NDM-1, and also had
decreased inhibitory activity to the tested SBL enzymes (Table 2), similar as that
observed for MS02 and MS03 (Table 1). These results indicated that the (2'S)-3'-
mercapto-2'-methylpropanamide moiety was a relatively optimal moiety to achieve
broad-spectrum inhibition to the representative MBL and SBL enzymes.
Substitutions at 1-Position Improve the Dual-Action MBL/SBL Inhibitory Activity.
With fixing the optimal (2'S)-3'-mercapto-2'-methylpropanamide group, we further
explored the influences of substitutions and enantiomers at 1-position to the dual-action
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MBL/SBL inhibition. Compounds MS13-MS31 were synthesized via the routes shown
in Scheme 2, and their SARs with the representative MBLs and SBLs are given in Table
3
. Compared with MS05, the (1S)-enantiomer MS13, with a benzyl group at 1-position,
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
showed comparable inhibition to subclass B1 VIM-2 (K = 1.04 μM) and NDM-1 (K =
i
i
1
6.86 μM), B2 Sfh-I (K = 1.00 μM), and B3 GOB-18 (K = 4.38 μM) (Table 3). In
i i
contrast, for the tested SBLs, MS13 showed decreased potency to class A KPC-2/TEM-
and class D OXA-48 (Table 3), probably due to the benzyl group in (1S)-configuration
1
making certain influences for the formation of a covalent bond between the boronic
acid and active site catalytic Ser . The enantiomer pairs MS14 vs MS15, MS16 vs
7
0
MS17, and MS18 vs MS19 were observed to have similar inhibition profiles to the
tested SBL enzymes as MS05 and MS13, e.g., the (1R)-enantiomers MS14, MS16, and
MS18 showing comparable or slightly better (< 5-fold) potency to KPC-2, TEM-1, and
AmpC than the corresponding (1S)-enantiomers MS15, MS17, and MS19 (Table 3 and
Figure S10). Notably, the (1R)-enantiomer MS18, bearing a (benzofuran-3-yl)methyl
substituent at 1-position, displayed broad-spectrum inhibition to all of the tested
MBL/SBL enzymes with the exception of class D OXA-48; in particular, it manifested
nanomolar potent inhibition to subclass B1 VIM-2 (K = 0.44 μM), B2 Sfh-I (K = 0.26
i
i
μM), B3 GOB-18 (K = 0.13 μM), class A KPC-2 (K = 0.61 μM), and class C AmpC
i
i
(K = 0.11 μM) (Table 3 and Figure S10). Compared with KPC-2, TEM-1, and AmpC,
i
OXA-48 bears a distinct active site,57-59 e.g., with several hydrophic residues (Val ,
1
20
Leu , Ile , and Trp ) and the lack of a structurally equivalent residue to KPC-
1
58
102
105
2
/TEM-1 Asn132 and AmpC Asn152 (Figure S5) to form hydrogen bonds with the amido-
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carbonyl of MS18 (see subsequent crystallographic analyses for MS22 and MS23),
partly explaining why MS18 does not potently inhibit OXA-48. More interestingly, the
(
1S)-enantiomer MS19 inhibited the subclass B1 MBLs VIM-2 (K = 0.07 μM) and
i
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
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9
0
NDM-1 (K = 6.06 μM) more potently (> 6-fold) than the (1R)-enantiomer MS18 (with
i
K values of 0.44 μM and 37.95 μM to VIM-2 and NDM-1, respectively) (Table 3 and
i
Figure S10). To investigate the binding mode of MS19 with VIM-2, a high-quality
crystal structure of the VIM-2:MS19 complex to 1.60 Å resolution was obtained (Table
S2 and S3). MS19 was well-fitted with the electron density maps found in the VIM-2
active site (Figure S11).
Table 3. The inhibitory potency (Ki, μM) of 1-substituted (1-(3'-
mercaptopropanamido)-methyl)boronic acid derivatives MS13-MS31 and MS32-
MS33 against the selected MBL and SBL enzymes.a
MBLs
Subclass Subclass
SBLs
Subclass B1
Class A
Class C Class D
ID
Structure
B2
B3
GOB-18 KPC-2 TEM-1 AmpC
VIM-2 NDM-1
Sfh-I
OXA-48
O
MS05
MS13
MS14
MS15
3'
(R)
0.36
1.04
0.26
0.48
3.88
16.86
22.81
17.42
3.48
1.00
0.19
0.77
9.03
4.38
1.01
6.33
6.85
40.61
4.82
15.41
>300
4.16
7.87
2.47
0.77
1.98
25.83
>300
85.75
>300
(
S)
OH
HS
1'
N
H
1
B
2'
OH
O
(S)
(S)
OH
HS
N
H
B
OH
F
O
O
(R)
(S)
OH
HS
N
B
H
OH
F
14.64 178.98
(
S)
(S)
OH
HS
N
H
B
OH
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OMe
O
MS16
0.18
0.56
27.22
7.22
0.51
0.74
1.72
2.50
1.96
3.36
1.23
1.18
171.50
>300
(
R)
(
S)
OH
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
HS
HS
N
H
B
OH
OMe
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
O
MS17
MS18
11.12
80.72
(S)
(S)
OH
N
H
B
OH
O
OH
0.44
0.07
37.95
6.06
0.26
0.72
0.13
0.61
1.22
10.77
32.70
0.11
3.96
>300
O
(
R)
(
S)
HS
N
H
B
OH
O
OH
MS19
10.98
200.83
O
(
S)
(S)
HS
N
H
B
OH
O
MS20
MS21
(R)
0.73
2.69
26.43
46.42
0.08
2.45
1.11
5.45
1.67
7.76
6.37
0.43
7.03
>300
(S)
OH
HS
N
H
B
OH
O
(S)
68.59
257.25
(S)
OH
HS
N
H
B
OH
F
MS22
MS23
MS24
0.41
0.65
3.28
14.73
21.12
16.70
0.33
0.28
0.51
0.33
1.02
2.78
2.02
7.08
4.35
14.17
67.94
2.41
0.64
1.57
1.99
>300
>300
>300
O
(R)
(S)
OH
HS
N
H
B
OH
F
O
(S)
(S)
OH
HS
N
H
B
OH
O
(R)
(S)
OH
HS
N
H
B
OH
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1
1
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1
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1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
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3
3
3
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5
5
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MS25
MS26
MS27
O
1.28
0.72
7.54
15.92
13.07
0.35
11.07
9.75
2.75
17.91
23.46
11.50
0.78
13.50
2.03
2.03
20.16
25.29
>300
(
S)
(
S)
OH
HS
N
H
B
OH
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
>300
>300
O
(
R)
(S)
OH
HS
N
H
B
OH
100.54
11.95 215.72
O
O
O
(S)
(S)
(S)
OH
HS
HS
HS
N
H
B
OH
(
R)
OH
MS28
MS29
0.51
0.70
36.80
25.58
9.14
11.50
57.97
5.35
165.78
>300
11.51
>300
>300
>300
N
H
B
OH
(
S)
(S)
OH
109.54
158.26
N
H
B
OH
O
O
MS30
MS31
(S)
(R)
0.85
1.13
22.81
37.59
6.11
1.91
11.10
4.25
36.12 170.70
17.73
3.07
>300
>300
OH
HS
HS
N
H
B
OH
(S)
18.37
5.56
70.55
35.74
(S)
OH
N
H
B
OH
O
MS32
MS33
0.33
16.49
10.20
0.63
0.12
2.50
1.67
4.47
0.97
>300
O
(S)
OH
HS
N
H
B
OH
O
42.75
0.50
6.84
217.29
O
(S)
HS
OH
N
B
H
OH
a
The dose–response curves of compounds inhibiting the selected enzymes are given in
Supporting Information Figure S10.
The VIM-2:MS19 structure revealed that similar to MS01, the (2'S)-3'-mercapto-
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2'-methylpropanamido moiety of MS19 was positioned to chelate with the two zinc
ions and form hydrogen-bonding interactions with Asn233 (3.0 Å) that is a conserved
residue on the L10 loop in subclass B1 MBLs;36,50 the boronic acid made hydrogen
bonds with the residue Arg228 (2.8 Å) (Figure 4A) that is structurally equivalent to B1
MBLs, e.g. NDM-1 Lys , IMP-1 Lys , and BcII Lys , which are important to
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
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3
3
3
3
3
3
3
4
4
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4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
2
11
224
224
recognize β-lactam 2-C carboxylic acid.36,50 Unexpectedly, the structure revealed an
unusual binding mode of the benzofuran moiety of MS19, i.e., which was positioned to
make orthophoric face-to-face π-π stacking interactions with residue Phe on the VIM-
6
1
2 L3 loop (Figure 4A), partly explaining why MS19 had very potent inhibition to VIM-
(K = 0.07 μM). Notably, superimposition of the VIM-2:MS19 and VIM-2:MS01
2
i
structures revealed that the L3 loop of VIM-2 was positioned away from the active zinc
ions (2.8 Å and 0.8 Å movements of the C-α atoms of Phe and Tyr respectively,
6
1
67
Figure 4B) when MS19 binding to VIM-2, providing further evidence for the proposal
that the L3 loop contributes to the highly efficient nature of MBL catalysis by capturing
the potential substrates and delivering them to the zinc ions for hydrolysis.50
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9
1
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1
1
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2
2
2
2
2
2
3
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3
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0
1
2
3
4
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9
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1
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9
0
Figure 4. Crystallographic analyses reveals an unexpected mode of MS19 binding to
VIM-2. (A) Close-up view from the VIM-2:MS19 structure (PDB ID 6JN6) reveals
that the inhibitor binds to form hydrophobic and electrostatic interactions with residues
on the L3 and L10 loops (e.g., hydrogen-bonding interactions with Asn233 and Arg228,
and, unexpectedly, π-π stacking interactions with Phe ). (B) Superimposition of VIM-
6
1
2:MS19 and VIM-2:MS01 (PDB ID 6J8R) reveals evidence for flexibility in the
conformation of Phe and Tyr on the L3 loop, suggesting they may play important
61
67
roles in capturing substrates and delivering them to the zinc ions for hydrolysis.50
Further comparison of the enantiomeric pairs, with the phenyl (MS20 vs MS21),
para-fluorophenyl (MS22 vs MS23), para-methylphenyl (MS24 vs MS25), and
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naphthyl (MS26 vs MS27) moiety at 1-position (Table 3), revealed that the (1R)-
enantiomeric compounds MS20, MS22, and MS24 had comparable inhibition as the
corresponding (1S)-enantiomeric compounds MS21, MS23, and MS25 to the tested
B1/B2/B3 MBL enzymes; (1R)-enantiomer MS26, with a rigid substitent of naphthyl,
inhibited subclass B1 VIM-2 and NDM-1 more potently than (1S)-enantiomer MS27,
while MS26 had comparable potency to B2 Sfh-I and B3 GOB-18 as MS27 (Table 3
and Figure S10). By comparison, for the SBL enzymes KPC-2, TEM-1, and AmpC,
these (1R)-enantiomeric compounds generally displayed more potent inhibitory
activities compared with their corresponding (1S)-enantiomeric compounds, similar as
that observed for other enantiomeric pairs in Table 3. Consequently, we conducted
crystallographic analyses for KPC-2 in complex with the (1R)-enantiomer MS22 and
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
(1S)-enantiomer MS23, with the aim to investigate the difference in their binding
modes. The crystal structures of KPC-2:MS22 (PDB ID 6JN4) and KPC-2:MS23 (PDB
ID 6JN5) were obtained and solved to 1.90 Å and 1.97 Å resolution, respectively (Table
S4); both compounds were well-modelled into the observed electron density maps in
the active site (Figure S12 and S13).
The structures revealed that both MS22 and MS23 bound to form a covalent bond
with the catalytic residue Ser and make multiple hydrogen bonds with the active site
7
0
residues Ser , Thr , Asn , Glu , and Asn (Figure 5A-C), similar as that observed
7
0
237
170
166
132
for MS01 and MS05 (Figure 2D and 3A). Similar to MS05, MS22 and MS23 were
positioned to occupy the subpocket between the L3 and L5 loops, and made
hydrophobic interactions with the flexible residue Trp105 (Figure 5A-B). Notably, in
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1
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2
2
2
2
2
2
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KPC-2:MS22 structure, the amido group adopts a trans-conformation and makes
hydrogen-bonding interactions with Thr237 (2.9 Å) and Asn132 (3.0 Å) (Figure 5A),
same as that observed in the structure of (1R)-enantiomer MS05 complexed with KPC-
2 (Figure 3A and S14). By contrast, the amido group of the (1S)-enantiomer MS23 was
unexpectedly observed to bind in a cis-conformation and only form hydrogen bonds
with Asn132 (2.9 Å and 3.5 Å) (Figure 5B). These differences may, in part, explain why
0
1
2
3
4
5
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7
8
9
0
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0
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4
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8
9
0
(1R)-enantiomers inhibit the SBL enzymes more potently than (1S)-enantiomers.
Additionally, these structures provided important structural basis for developing chiral
α-amino boronic acid-based SBL inhibitors.
Figure 5. Crystallographic analyses reveals similar binding modes of (1R)-enantiomer
MS22 and (1S)-enantiomer MS23 with KPC-2. (A) Close-up view from a structure of
MS22 complexed with KPC-2 (PDB ID 6JN4). (B) Close-up view from a structure of
MS23 complexed with KPC-2 (PDB ID 6JN5). (C) Superimposition of KPC-2:MS22
and KPC-2:MS23 reveals MS22 and MS23 bind in similar modes involving covalent
bonding and hydrogen-bonding interactions. Note that unlike MS22, MS23 is bound
with cis-amide conformation.
We then compared the MBL/SBL inhibitory activity for compounds MS28-MS31,
which bear small moieties at 1-position (Table 3). We observed that for the tested MBL
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enzymes, the (1R)-enantiomeric compounds MS28 and MS30 have comparable activity
as the corresponding (1S)-enantiomeric compounds MS29 and MS31, with the
exception of MS29; MS29 have relatively weak inhibition (5-fold lower) to subclass
B2 Shf-I and B3 GOB-18. By comparison with MS18, compounds MS28-MS31
mostly had less potent inhibition to the SBL enzymes. Interestingly, to class A KPC-
2/TEM-1 and class C AmpC, the (1R)-enantiomer MS28 was more potent than (1S)-
enantiomer MS29, whereas (1R)-enantiomer MS30 had less inhibitory activities than
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
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5
6
7
8
9
0
1
2
3
4
5
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9
0
(1S)-enantiomer MS31 (Table 3 and Figure S10). Comparison of the dual-action
inhibition potencies of MS32/MS33 with MS19 further demonstrated the importance
of the (2'S)-3'-mercapto-2'-methylpropanamido group for achieving broad-spectrum
MBL inhibition, particularly for the subclass B1 VIM-2 and NDM-1.
The overall SAR studies of various thiol- and/or boronic acid-containing
compounds with the representative MBL/SBL enzymes (Table 1-3 and Figure S15)
revealed the substantial influences of the thiol moiety and substitutions/enantiomers at
1
-position on the dual-action MBL/SBL inhibition. To subclass B1 MBLs, the (2'S)-3'-
mercapto-2'-methylpropanamide moiety was demonstrated to be an optimal MBP for
targeting VIM-2 and NDM-1, similar as previously reported,36-38 while the inhibitory
activities were synthetically affected by the 1-position substituents and enantiomeric
conformations (Table 3). By contrast, for subclass B2 Shf-I and B3 GOB-18, no
significant difference were observed between the tested enantiomer pairs (Table 3),
implying that the inhibitors may have different inhibition modes to subclass B1 and
B2/B3 MBLs. To class A KPC-2/TEM-1 and class C AmpC, the (1R)-enantiomeric
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compounds, in most cases, displayed better activities than the corresponding (1S)-
enantiomeric compounds (Table 3 and Figure S15), possibly owing to the difference in
their amino binding conformations as observed by crystallographic analyses (Figure 5).
Yet, almost all of the tested compounds had no or weak inhibition to the structurally
different class D OXA-48 (Figure S5). Notably, the crystallographic analyses revealed
that the flexible loops in the active sites of MBL and SBL enzymes played important
roles in capturing various inhibitors, which may be one of critical aspects to understand
the relatively complicated multi-target SAR data. More importantly, the SAR studies
identified new more potent dual-action inhibitors compared with the starting compound
MS01. For example, MS18 manifested potent nanmolar inhibition to several tested
MBL and SBL enzymes, including the clinically widespread carbapenemases subclass
B1 VIM-2 (K = 0.44 μM) and class A KPC-2 (K = 0.61 μM); MS18 was observed to
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be a reversible inhibitor to both VIM-2 and KPC-2, particularly which had a relatively
slow dissociation rate for KPC-2 compared with other tested inhibitors, including
Tazobactam (Figure S16).
Selectivity to Metalloenzyme ACE-2 and Serine Hydrolyases. Since these dual-
action inhibitors have the (2'S)-3'-mercapto-2'-methylpropanamido moiety, which is a
key scaffold of captopril, an angiotensin-converting enzyme (ACE) inhibitor, we tested
the dual-action inhibitors MS01, MS05, MS06, and MS13-MS33, for examining their
selectivity to the commercially available human ACE-2 (hACE-2) enzyme. With the
exception of MS06, all these compounds have no apparent inhibitory activity to hACE-
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Journal of Medicinal Chemistry
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of 63.2% and 44.6% to hACE-2 at 100 μM and 50 μM, respectively (Table S7 and
Figure S17).
We also examined the selectivity of the representative inhibitors MS01, MS05,
MS06, MS13, MS18, MS19, MS20, and MS21 across serine hydrolases in E. coli cells
and human HEK293T cells by competitive activity-based protein profiling using
TAMRA-FP, since they bear an α-amino boronic acid moiety. TAMRA-FP is a well-
characterized broad-spectrum serine hydrolase activity-based probe, which provides
selectivity profile assays across a broad array of serine hydrolases. As shown in Figure
6, the tested inhibitors have weak inhibitory activity against most of the labeled serine
hydrolases in E. coli and HEK293T cells. Particularly, MS01 showed high selectivity
with no significant off-targets (enzyme inhibition are all below 50%, Table S8)
identified in both E.coli and HEK293T cell lysates (Figure 6 and Table S8). Of note,
the control compound AN2728, a FDA-approved boronate-containing drug, exhibited
less selectivity compared with most of the tested inhibitors with several off-targets
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(Figure 6 and Table S8). These results indicate that the dual-action inhibitors are
selective among serine hydrolases in E. coli and HEK293T cells labeled by TAMRA-
FP.
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