Iminodiacetic Acid as a Novel Metal-Binding Pharmacophore for New Delhi Metallo-β-lactamase Inhibitor Development

Article abstract of DOI:10.1002/cmdc.202000123

The fungal natural product aspergillomarasmine A (AMA) has been identified as a noncompetitive inhibitor of New Delhi metallo-β-lactamase-1 (NDM-1) that inhibits by removing Zn^II from the active-site. The nonselective metal-chelating properties and difficult synthesis and derivatization of AMA have hindered the development of this scaffold into a potent and selective inhibitor of NDM-1. Iminodiacetic acid (IDA) has been identified as the metal-binding pharmacophore (MBP) core of AMA that can be leveraged for inhibitor development. Herein, we report the use of IDA for fragment-based drug discovery (FBDD) of NDM-1 inhibitors. IDA (IC₅₀=120 μM) was developed into inhibitor 23 f (IC₅₀=8.6 μM, K_i=2.6 μM), which formed a ternary complex with NDM-1, as evidenced by protein thermal-shift and native-state electrospray ionization mass spectrometry (ESI-MS) experiments. Combining mechanistic analysis with inhibitor derivatization, the use of IDA as an alternative AMA scaffold for NDM-1 inhibitor development is detailed.

Full text of DOI:10.1002/cmdc.202000123

Accepted Article
Title: <p>Iminodiacetic Acid as a Novel Metal-binding Pharmacophore
for New Delhi Metallo-&amp;beta;-lactamase Inhibitor
Development</p>
Authors: Allie Y Chen, Caityn Thomas, Pei W Thomas, Kundi Yang,
Zishuo Cheng, Walter Fast, Michael W Crowder, and Seth
Mason Cohen
This manuscript has been accepted after peer review and appears as an
Accepted Article online prior to editing, proofing, and formal publication
of the final Version of Record (VoR). This work is currently citable by
using the Digital Object Identifier (DOI) given below. The VoR will be
published online in Early View as soon as possible and may be different
to this Accepted Article as a result of editing. Readers should obtain
the VoR from the journal website shown below when it is published
to ensure accuracy of information. The authors are responsible for the
content of this Accepted Article.
To be cited as: ChemMedChem 10.1002/cmdc.202000123
Link to VoR: https://doi.org/10.1002/cmdc.202000123
01/2020

10.1002/cmdc.202000123
ChemMedChem
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Iminodiacetic Acid as a Novel Metal-binding Pharmacophore for
New Delhi Metallo-β-lactamase Inhibitor Development
Allie Y. Chen,[a] Caitlyn Thomas,[b] Pei W. Thomas,[c] Kundi Yang,[b] Zishuo Cheng,[b] Walter Fast,*[c]
Michael W. Crowder,*[b] Seth M. Cohen*[a]
[a]
Allie Y. Chen, Prof. Seth M. Cohen
Department of Chemistry and Biochemistry
University of California, San Diego
La Jolla, CA 92093, United States
[b]
Caitlyn Thomas, Kundi Yang, Prof. Michael W. Crowder
Department of Chemistry and Biochemistry
Miami University
Oxford, OH 45056, United States
[c]
Dr. Pei W. Thomas, Prof. Walter Fast
Division of Chemical Biology & Medicinal Chemistry, College of Pharmacy
University of Texas, Austin
Austin, TX 78712, United States
Abstract: The fungal natural product Aspergillomarasmine A (AMA)
has been identified as a non-competitive inhibitor of New Delhi
Metallo-β-lactamase-1 (NDM-1) that inhibits via active site Zn(II)
removal. The non-selective metal-chelating properties and the
difficult synthesis and derivatization of AMA have hindered the
development of this scaffold into a potent and selective inhibitor of
NDM-1. Iminodiacetic acid (IDA) has been identified as the metal-
binding pharmacophore (MBP) core of AMA that can be leveraged for
inhibitor development. Herein, we report the utilization of IDA for the
fragment-based drug discovery (FBDD) of NDM-1 inhibitors. IDA
(IC50 = 122 μM) was developed into inhibitor 23f (IC50 = 8.6 μM, Ki =
2.6 μM) and displayed the formation of a ternary complex with NDM-
1, as evidenced by protein thermal shift and native state electrospray
ionization mass spectrometry (ESI-MS) experiments. Combining
mechanistic analysis in tandem with inhibitor derivatization, the
utilization of IDA as an alternative AMA scaffold for NDM-1 inhibitor
development is detailed.
after the introduction of penicillin in the 1940s, the first observed
MBL was reported.[5] Currently, there are >80 unique MBL
families.[3] With their wide substrate profile (able to hydrolyze
virtually all clinically used bicyclic β-lactam antibiotics), MBLs
have risen to become one of the most problematic resistance
mechanisms.[6]
elsewhere.[7]
Detailed reviews on MBLs can be found
Depending on protein sequence homology and number of
Zn(II) ions in the catalytic site, MBLs are divided into three
subclasses (B1, B2, and B3). The most prevalent members
belong to subclass B1 and include IMiPenemase (IMP), Verona
Integron-encoded Metallo-β-lactamase (VIM), and New Delhi
Metallo-β-lactamase (NDM).[6, 8] NDM is the most recent member
of the trio, with its genetic and biochemical characterization first
reported in 2009 upon isolation from carbapenem-resistant
Klebsiella pneumoniae.[9] The rapid spread of NDM is attributed
to many factors, including the ability for blaNDM-gene bearing
plasmids to undergo horizontal gene transfer between different
species of microorganisms and to co-harbor genes that encode
for other resistance factors.[9-10] In contrast to other MBLs (which
are soluble periplasmic proteins), NDM is a lipoprotein that
anchors to bacterial outer membrane and displays increased
protein stability and secretion.[11] Additionally, NDM variants (>24
reported to date) have evolved to overcome metal scarcity and
increased thermal stability.[11c, 12] The NDM active site bears two
Zn(II) ions, with Zn1 ligated by H116, H118, H196, and a bridging
hydroxide in a tetrahedral coordination geometry, and Zn2 ligated
by D120, C221, H263, the bridging hydroxide, and an apical H2O
in a trigonal bipyramidal coordination geometry (standard BBL
numbering, Figure 1).[13] The binding pocket of NDM has a
volume of 591 Å3, which is nearly 2-fold larger in comparison to
that of IMP (303 Å3) and almost 4-fold larger compared to that of
Introduction
A recent report published by the Centers for Disease Control
and Prevention (CDC) estimated that antibiotic-resistant bacteria
and fungi cause >2.8 million cases of infections in the United
States each year, with >35,000 of those cases resulting in
death.[1] Resistance mechanisms (including mutation of penicillin-
binding proteins, production of efflux pumps, and expression of β-
lactamases) evolved and employed by pathogens are a prime
example of bacterial adaptability and pose an urgent threat to the
public health.[2] The most valuable class of drugs for combating
bacterial infections include β-lactam antibiotics. This class of
antibiotics acts as a substrate analogue to obstruct peptidoglycan
chain cross-linking in bacterial cell wall biosynthesis and accounts
for ~65% of all injectable antibiotics prescribed in the United
States.[3] However, the over-use of β-lactams has led to the
evolution of β-lactamases, enzymes that hydrolyze the β-lactam
ring to render the drug ineffective. Three classes of β-lactamases,
Ambler class A, C, and (serine-β-lactamases, SBLs) utilize an
active site serine residue for hydrolysis, while one class of β-
lactamase, Ambler class B (metallo-β-lactamase, MBL) utilizes a
metal center to initiate ring cleavage.[2a, 4] Merely two decades
VIM (140 Å3).[14]
This highly plastic and large cavity
accommodates a wide range of antibiotic substrates and allows
for the efficient hydrolysis of nearly all β-lactam antibiotics.[15]
1
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10.1002/cmdc.202000123
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FULL PAPER
development for the sequestration of Zn(II) in IDA-modified
human lysozyme (IDA-hLys) against Zn(II)-mediated Aβ-
aggregation for the treatment of Alzheimer’s disease.[24]
Utilization of IDA for inhibitor development allows for greater
synthetic accessibility of derivatives to probe the NDM-1 active
site and to develop inhibitors that form stable ternary complexes.
In addition, IDA bears structural resemblance to the hydrolyzed
antibiotic β-lactam ring, suggesting the potential for the
development of transition-state analogue inhibitors. Notably, IDA
is an aliphatic derivative of the previously investigated dipicolinic
acid inhibitor,[19d] further justifying its use as a scaffold for novel
NDM-1 inhibitor development (Figure 2, highlighted in bold).
Herein, we report the utilization of IDA as a novel metal-
binding pharmacophore (MBP) for NDM-1 inhibitor development.
Through fragment-based drug discovery (FBDD), the IDA MBP
Figure 1. Scheme of the NDM active site and
mechanism of the β-lactam antibiotic penicillin.
a proposed hydrolysis
There are currently >500 distinct NDM inhibitors reported in
literature (representative structures show in Figure 2).[16] An
important class of compounds bear a sulfhydryl-motif (including
D/L-captopril and bisthiazolidines) that act via a competitive
inhibition mechanism by displacing the bridging hydroxide ion to
form a μ-bridging species between the Zn(II) ions.[17] Another
important class of inhibitors includes the cyclic boronates, which
have been shown to successfully pan-inhibit SBLs and MBLs via
(IC50 = 122 μM) was developed into the lead inhibitor 23f (IC50
=
8.6 μM) against NDM-1. Protein thermal shift and native state
electrospray ionization mass spectrometry (ESI-MS) experiments
revealed 23f, and related derivatives, inhibits NDM-1 via the
formation of a stable NDM-1:Zn(II):23f ternary complex. This
work demonstrates the potential in the IDA scaffold for NDM-1
inhibitor development and provides a roadmap for future IDA
derived inhibitors.
a tetrahedral anionic transition state mimetic.[18]
taniborbactam (VNRX-5133) is the only candidate to have
advanced to the clinic (currently in phase III clinical trials).[18c]
Notably,
Results and Discussion
IDA MBP Design and Synthesis. To verify IDA as a potential
MBP lead for inhibitor design, a small library of MBP compounds
bearing structural similarity to IDA was assembled and their
inhibitory activity against NDM-1 was assessed (Table 1). This
library included compounds that have at least one nitrogen atom
and one carboxylate functional group for bidentate metal-binding
(N,O-donor atoms). The N-donor atom in the MBPs were either
a tertiary amine (1), secondary amine (IDA, 2 – 4), or aromatic
amine (5 – 10). These MBPs were screened at a single
concentration of 200 μM via an enzymatic assay which monitored
the NDM-1 mediated hydrolysis of the substrate meropenem.[25]
Evaluation of this library revealed IDA to be the only MBP with a
secondary amine N-donor atom to yield significant inhibition
activity (48%). MBPs 2 – 4, where the secondary amine is a part
of the saturated ring, did not display any appreciable inhibition
(≤8%). A methylated IDA derivative (1), was the most potent of
this library, reaching 80% inhibition. Some MBPs bearing the
aromatic amine motif showed some inhibitory activity, with 8
displaying the second highest inhibition value (57%). Data from
this small set of MBPs suggest a preference for a tertiary or
aromatic heterocycle N-donor atom. These findings verified the
IDA MBP as a viable scaffold for NDM-1 inhibitor development.
Based on these findings, MBP 1 was chosen for further
investigation.
Figure 2. Representative inhibitors of NDM-1.
The last class includes compounds that bear metal-
chelating motifs.[19]
Of these, the fungal natural product
Aspergillomarasmine A (AMA, IC50 = 4 – 7 μM, Figure 2), an
aminopolycarboxylic acid, has gained attention due to its ability to
restore meropenem activity in a mouse infected with NDM-1
expressing Klebsiella pneumoniae.[19e,
20]
AMA inhibitor
development has focused on modification of the carboxylic acid
functional groups through removal or conversion to an ester motif,
or exploration of related aminocarboxylic acid analogues.[20b, 21]
AMA-1 and AMA-2 (Figure 2), where one of the carboxylate
groups is replaced with a methyl substituent yielded weaker
inhibition (IC50 = 22 and 94 μM, respectively) compared to that of
AMA, validating the requirement of free carboxylic acids for
enzyme inactivation and supporting the mechanism of action of
AMA is via non-selective Zn(II) sequestration (similar to that of
EDTA). The metal-chelating properties of AMA along with difficult
synthesis and derivatization routes has hindered the development
of this motif into NDM-1 inhibitors.[21a, 22] Structural comparison of
AMA and EDTA reveals iminodiacetic acid (IDA) as a privileged
scaffold that could be leveraged for NDM-1 inhibitor development
(Figure 2, highlighted in bold). IDA is a strong tridentate metal
chelator (via its O,N,O-donor atoms), as evidenced by its role in
immobilized metal affinity chromatography (IMAC)[23] and its
Table 1. Percent Inhibition of IDA derived MBPs (at 200 μM) against NDM-1.[a]
Compound
% Inhibition
48±2
Compound
% Inhibition
3±10
O
O
O
O
H
N
N
H
HO
O
IDA
6
HO
OH
O
O
CH3
N
O
80±4
7
0
1
N
H
HO
OH
OH
2
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10.1002/cmdc.202000123
ChemMedChem
FULL PAPER
O
N
IDA Derivative Synthesis and Inhibitory Activity. Compounds
1 and 11 were selected as scaffolds for inhibitor development, and
additional IDA derivatives with various substituents were
HO
OH
8±7
0
8
57±4
2
N
H
N
H
OH
O
O
O
OH
OH
OH
N
prepared.
This sublibrary was prepared using a double
9
28±1
33±5
N
H
3
N
H
O
substitution reaction of various primary amines with t-butyl 2-
bromoacetate to yield compounds 21a – 21m (Scheme 4, Table
O
O
O
OH
0
10
3).
The sublibrary was screened at a single inhibitor
N
H
4
5
HO
OH
O
N
NH
concentration of 250 μM. The majority of the compounds in this
sublibrary inhibited NDM-1 at an appreciable level (~60%);
however, no clear SAR could be elucidated. Compared to the
percent inhibition of 11 (65%, Table 2), modification via an ethyl-
linker (21a) or a bi-phenyl substituent (21b and 21c) did not result
in substantial inhibition improvements (56 – 76%). Notably,
compounds bearing a phenyl- or benzyl-sulfonamide motif (21j
and 21k) displayed a complete loss of activity (most likely due to
the reduced basicity of the central nitrogen); however, substitution
with a thiophene substituent (21l and 21m) restored activity by
~20%. In addition, 21i stood out as the most potent inhibitor of
this sublibrary with almost complete inhibition against NDM-1
(~99%). When the heterocyclic oxygen is swapped out for a sulfur
(21h), the inhibition activity is reduced to 64%, showing a
preference for the furan substituent.
O
O
4±4
N
H
HO
OH
[a] Values are the average of triplicates experiments with standard deviations
shown.
A second-generation of IDA derivatives (Table 2) were
synthesized according to Scheme 1 – Scheme 3. Compounds in
this sublibrary incorporated a benzyl substituent, as aromatic
rings have previously been shown to form favorable hydrophobic
interactions with the NDM β-hairpin loop.[16] Utilizing the concept
of bioisosteric replacement, IDA derivatives where one carboxylic
acid was modified (13a, 13b, 15, 17) were prepared to determine
if both carboxylic acids were necessary for inhibition and if
alternative MBPs could achieve increase potency. Compounds
where the methyl- or benzyl-substituent (19a – 19d) was placed
at the α-carbon were explored as well. The compounds were
screened against at a single concentration of 250 μM against
NDM-1. The methylated-IDA (1) remained the most potent of the
series, exhibiting 90% inhibition at 250 μM. The benzylated-IDA
R
R
R
O
O
O
a
b
t-BuO
N
HO
N
HN
Ot-Bu
OH
Ot-Bu
O
O
13a, R = H,
13b, R = Ph
12a, R = H
12b, R = Ph
R = H or Ph
(11) was the second most potent (65%).
Interestingly,
Scheme 1. Synthesis of 13a and 13b. Reagents and conditions: (a) t-butyl
bioisosteres with a propionic acid motif (13a and 13b, which are
most structurally similar to AMA) displayed a complete loss of
activity. While the phosphate isostere (15) showed no inhibition
against NDM-1, the less acidic tetrazole isostere (17) showed
inhibition that was comparable to that of 11. Notably, there was
no preference for R- or S- stereoisomers at the α-carbon position
of IDA, as evident by similar inhibitory values (51 – 65%)
displayed by derivatives 19a – 19d.
acrylate, TEA, EtOH, 65 °C, 16 h, 43 – 90%; (b) TFA:CH2Cl2, 25 °C, 16 h, ~99%.
O
O
a
b
O
O
N
N
P
O
P
OH
O
HO
O
OH
14
15
O
HN
O
Ethyl benzylglycinate
c
d
N
N
O
O
N
N
N
N
N
H
OH
O
16
17
Table 2. Percent Inhibition of IDA derivatives (at 250 μM) against NDM-1.[a]
Scheme 2. Synthesis of IDA inhibitors 15 and 17. Reagents and conditions:
(a) diethyl(2-bromoethyl)phosphonate, K2CO3, KI, ACN, 82 °C, 16 h, 40%; (b)
HCl, 100 °C, 16 h, ~99%; (c) 2-bromoacetonitrile, K2CO3, KI, CH3CN, 25 °C, 16
h, 74%; (d) NaN3, NH4Cl, DMF, 110 °C, 16 h; then 1:1:1 MeOH:THF:1 M NaOH,
60 °C, 16 h; two steps 19%.
Compound
% Inhibition
90±1
Compound
% Inhibition
O
CH3
N
O
N
N
N
O
43±1
1
17
HO
OH
N
N
H
OH
R
R
CH3
R
b
a
O
O
HO
OH
O
O
O
N
H
N
H
H2
N
65±1
0
19a
19b
53±2
51±1
N
11
O
O
O
O
O
O
H
O
OH
OH
N
HO
OH
18a, R = R -CH3
18b, R = S -CH3
18c, R = R - Bz
18d, R = S - Bz
19a, R = R -CH3
19b, R = S -CH3
19c, R = R - Bz
19d, R = S - Bz
R = R -CH3
= S -CH3
= R - Bz
CH3
N
O
CH3
O
O
O
= S - Bz
N
H
OH
13a
OH
OH
OH
Scheme 3. Synthesis of 19a – 19d. Reagents and conditions: (a) t-butyl 2-
bromoacetate, TEA, DMF, 0 – 25 °C, 16 h, 40 – 54%; (b) TFA, CH2Cl2, 25 °C,
16 h, ~99%.
O
0
0
19c
19d
54±1
65±1
13b
15
O
O
HO
N
N
OH
H
OH
OH
O
a
b or c
O
R
N
O
O
R
N
O
R
NH2
HO
OH
O
O
O
O
20a - 20m
22a - 22h
21a - 21m
23a - 23h
O
O
N
N
H
P
OH
HO
OH
OH
OH
[a]
Values are the average of triplicates experiments with standard deviations
shown.
Scheme 4. Synthesis of IDA inhibitors 21a – 21m and 23a – 23h. Reagents
and conditions: (a) t-butyl 2-bromoacetate, KHCO3, THF, 25 °C, 16 h, 25 –
98%; (b) TFA, CH2Cl2, 25 °C, 16 h or (c) MeOH:THF:1M NaOH, 100 °C, 16 h,
29 – 99%.
3
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10.1002/cmdc.202000123
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action.
The Cheng–Prusoff relationship[27] for competitive
Table 3. Inhibitory activity of 21a – 21m (at 250 μM) against NDM-1.[a]
inhibitors enables calculation of Ki of 2.6±0.3 μM for 23f. Two
methods, thermal shift and native state ESI-MS, were utilized to
interrogated the mode of inhibition of selected inhibitors.
Compared to alternative methods (such as NMR, crystallography,
equilibrium dialysis, and others), thermal shift assay and native
state ESI-MS utilize relatively lower protein and inhibitor
concentrations, and are more amenable to high-throughput
analysis, making them a suitable approach for initial mechanistic
studies.
O
R
N
O
HO
OH
Compound
% Inhibition
64±2
Compound
% Inhibition
70±1
S
21a
21h
O
56±2
21i
99±7
Table 4. Inhibitory activity of IDA derivatives 23a – 23h against NDM-1.[a]
21b
O
R
N
O
HO
OH
O
S
O
76±6
66±2
36±2
21j
21k
21l
0
0
21c
21d
21e
Compound
IC50 (μM)
25±2
Compound
IC50 (μM)
120±10
OH
O
O
H
CH3
S
IDA
1
S
O
Cl
S
O
O
172±8
66±3
21i
32±3
22±1
21h
23a
20±5
S
O
S
CN
S
23b
O
74±2
21m
21±8
O
21f
S
O
S
N
N
N
H
N
91±8
51±2
23d
47±4
23c
67±2
21g
S
O
23f
8.6±0.2
[a]
23e
23g
Values are the average of triplicates experiments with standard deviations
shown.
Br
Cl
To further develop inhibitors against NDM-1 and investigate
the difference in inhibitory activity between the furan and
thiophene substituents, a second library bearing analogues of
21h and 21i were synthesized and evaluated (23a – 23h, Table
4). In general, all derivatives bearing a furan motif exhibited a
lower IC50 value compared to that of the corresponding thiophene
derivative. These results validate a preference for an oxygen
heteroatom. While the 1,2-furan (23b, IC50 = 22 μM) displayed a
S
S
23h
32±2
46±2
[a]
Values are the average of triplicate experiments with fitting errors shown.
Protein Thermal Shift Assay. Protein thermal shift assay
detects ligand-induced protein stabilization, and has emerged as
a valuable tool for hit-identification and validation methodology in
[28]
lowered IC50 value compared to that of the 1,3-furan (21i, IC50
=
drug discovery.
Herein, we utilize this general method to
32 μM), the introduction of a methyl substituent at the 5-positon
(23d, IC50 = 47 μM) resulted in poorer activity. Extension from a
methyl-linker (23b) to an ethyl-linker (23f) resulted in a 2.5-fold
fold improvement in inhibitory activity and resulted in the most
potent inhibitor of this sublibrary (IC50 = 8.6 μM). It is predicted
that the ethyl linker allows for the furan substituent to make more
favorable interactions with the base of the L3 β-hairpin loop of
NDM-1, as observed in the crystal structure of hydrolyzed
antibiotic cefuroxime complexed with NDM-1 (PDB 5O2E);[24]
however, further experiments are required to confirm the specific
validate IDA derivatives as inhibitors of NDM-1 and evaluate their
propensity to remove Zn(II) from the active site of NDM-1. In this
assay, a fluorescent dye is utilized to monitor the difference in the
unfolding temperature of the native protein versus the inhibitor-
bound protein. The inhibitor-bound protein generally has greater
protein stability and increases the melting temperature (positive
[29]
∆Tm, as observed with L-captopril, Table 5).
In contrast, the
removal of Zn(II) has been observed to destabilize the protein and
[12b]
results in a negative ΔTm (as seen with DPA).
It is important
to note that while reported thermal shift data have revealed a good
[30]
correlation between the observed IC50 value and ∆Tm,
this
ligand-protein interactions.
The corresponding thiophene
[29, 31]
derivatives displayed the same trends, albeit with poorer inhibition
values. Due to the strong affinity IDA has for Zn(II) ions (Kd =
3.2×10-5 M)[26] and the analysis of inhibition mechanism (vide
infra), we propose that 23f binds via coordination to the Zn(II) ions
at the NDM-1 protein active site via a competitive mechanism of
correlation has not been observed for inhibitors of NDM-1.
In the case of the compounds tested here, ∆Tm did not correlate
with IC50 values. All tested compounds displayed a range of
positive ∆Tm values, with IDA, 21h, 23c, and 23h yielding ∆Tm on
par with, or better than, that of L-captopril. Although no correlation
4
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was observed, the small, positive ∆Tm shifts exhibited by all
derivatives represents the absence of Zn(II)-chelation and is
suggestive evidence for the formation of ternary complexes.
2,848 m/z and a more intense +8 peak at 3,202 m/z, both of which
correspond to the mass of di-Zn(II) NDM-1:23f (25,612 Da, Figure 3).
Table 5. Protein thermal shift of selected compounds against NDM-1.[a]
Table 6. Summary of the native ESI-MS experimental results for NDM-1.[a]
Compound
L-Captopril
IDA
∆Tm (°C)
4.61±0.07
4.39±0.04
4.43±0.07
3.1±0.1
Compound
DPA
1
∆Tm (°C)
-14.5±0.2
1.9±0.2
3.4±0.3
1.6±0.4
3.4±0.2
1.5±0.1
4.2±0.2
NDM-1
+
Inhibitor
Complex
Mass
Peak
Charge
(+)
Predicted
Peak
(m/z)
Observed
Peak
(m/z)
Sample
NDM-1
Complex
(Da)
21h
21i
2Zn:
NDM-1
23a
23b
25,385
25,385
25,255
25,320
25,385
25,532
9
10
9
2,821
2,539
2,807
2,814
2,821
2,838
2,822
2,540
2,807
2,814
2,821
2,837
23c
5.2±0.1
23d
2Zn:
NDM-1
23e
1.7±0.2
23f
NDM-1
+ DPA
0Zn:
NDM-1
23g
3.0±0.3
23h
[a]
Values are the average of eight replicates with standard deviations shown.
1Zn:
NDM-1
Native NDM-1 was observed to melt at TM = 55.95±0.06 °C.
9
Native State Electrospray Ionization Mass Spectrometry.
Native state ESI-MS was used to further investigate the formation
of an NDM-1:Zn(II):inhibitor ternary complex of derivatives 1 and
23c – 23f against NDM-1 and VIM-2. This method allows for the
rapid determination of the changes in NDM-1:Zn(II):inhibitor
stoichiometry.[32]. The major advantage of ESI-MS is in its ability
to analyze for the formation of a ternary complex using near
physiological concentrations of enzyme and inhibitor. Briefly,
NDM-1 and VIM-2 (at 10 μM) were incubated for approximately 5
min with each inhibitor (50 μM) prior to analysis. The control
spectra of native NDM-1 revealed a dominant +9 peak at 2,822
m/z, corresponding to the mass of di-Zn(II) NDM-1 (25,385 Da,
Table 6, Figure S1). In contrast, incubation of NDM-1 with DPA,
a metal chelator, resulted in +9 peaks at 2,807 m/z, 2,814 m/z,
and 2,822 m/z, corresponding to the presence of apo-, mono-
Zn(II) and di-Zn(II) NDM-1, respectively. These results were
similar to the observed spectra of NDM-1 with EDTA, which
showed a dominant peak corresponding to metal free NDM-1
(unpublished data). It is important to note that the current native
ESI-MS experiment procedures do not generate quantitative
results. Higher relative peak intensities (dominant peaks) do not
indicate higher concentrations of the solution species, but rather
the species that is best ionized by the mass analyzer.[33]
The spectra of NDM-1 incubated with inhibitors 1 and 23c – 23f
all showed the presence of ternary complexes, with the predicted and
observed peaks summarized in Table 6 (Figure S1). In all of these
experiments, in addition to the dominant di-Zn(II) NDM-1 peak, an
additional peak corresponding to the mass of di-Zn(II) NDM-1 plus
inhibitor was observed. Protein incubation with derivative 1 revealed
a less dominant di-Zn(II) NDM-1:1 +9 peak at 2,837 m/z (25,532 Da).
Incubation of protein with 23c yielded +9 and +8 peaks at 2,850 m/z
and 3,210 m/z, respectively, corresponding to the di-Zn(II) NDM-1:23c
complex (25,628 Da). Similarly, incubation of protein with 23d yielded
+9 and +8 peaks at 2,846 m/z and 3,202 m/z, corresponding to the di-
Zn(II) NDM-1:23d complex. Inhibitor 23e produced a significantly less
intense +9 and +8 peaks at 2,847 m/z and 3,205 m/z, corresponding
to the di-Zn(II) NDM-1:23e complex (25,628 Da). Lastly, incubation
of NDM-1 with lead inhibitor 23f revealed a less intense +9 peak at
2Zn:
NDM-1
9
NDM-1
+ 1
2Zn:
NDM-1:1
9
2Zn:
NDM-
1:23c
NDM-1
+ 23c
25,628
25,612
25,628
25,612
9
8
9
8
9
8
9
8
2,848
3,204
2,847
3,202
2,848
3,204
2,847
3,202
2,850
3,210
2,846
3,202
2,847
3,205
2,848
3,202
2Zn:
NDM-
1:23c
2Zn:
NDM-
1:23d
NDM-1
+ 23d
2Zn:
NDM-
1:23d
2Zn:
NDM-
1:23e
NDM-1
+ 23e
2Zn:
NDM-
1:23e
2Zn:
NDM-
1:23f
NDM-1
+ 23f
2Zn:
NDM-
1:23f
[a] Percent error was calculated by subtracting the expected peak value from the
actual peak value, dividing by the expected peak value and multiplying by 100
and all observed to be <0.2%.
5
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10.1002/cmdc.202000123
ChemMedChem
FULL PAPER
ternary complex, respectively. This data is evident that the
representative IDA inhibitors form ternary complexes with VIM-2,
and represents a promising scaffold for future development
against other MBLs.
Table 7. Summary of the native ESI-MS experimental results for VIM-2.[a]
NDM-1
+
Inhibitor
Complex
Mass
Peak
Charge
(+)
Predicted
Peak
(m/z)
Observe
d Peak
(m/z)
Sample
VIM-2
Complex
(Da)
2Zn:
VIM-2
25,972
25,972
26,010
26,010
26,119
26,215
26,199
26,215
26,199
9
8
9
8
9
8
8
8
8
2,887
3,247
2,891
3,252
2,903
3,278
3,276
3,278
3,276
2,886
3,247
2,891
3,253
2,902
3,277
3,276
3,277
3,278
2Zn:
VIM-2
VIM-2
+ DPA
0Zn:
VIM-2:DPA
0Zn:
VIM-2:DPA
VIM-2
+ 1
2Zn:
VIM-2:1
VIM-2
+ 23c
2Zn:
VIM-2:23c
VIM-2
+ 23d
2Zn:
VIM-2:23d
VIM-2
+ 23e
2Zn:
VIM-2:23e
VIM-2
+ 23f
2Zn:
VIM-2:23f
Figure 3. Native state ESI-MS of lead inhibitor 23f with: NDM-1 (top) and VIM-
2 (bottom).
[a] Percent error was calculated by subtracting the expected peak value from the
actual peak value, dividing by the expected peak value and multiplying by 100
and all observed to be <0.07%.
Previous work has shown that different mechanisms of
inhibition can be observed for the same inhibitors against varying
MBLs (unpublished data). To verify that the IDA inhibitors are
able to form ternary complexes with other MBLs and have the
potential to be developed into pan-MBL inhibitors, additional ESI-
MS experiments were performed for inhibitors 1 and 23c – 23f
with VIM-2. Control spectra of VIM-2 revealed dominant +9 and
+8 peaks at 2,886 m/z and 3,247 m/z, respectively, corresponding
to the mass of di-Zn(II) VIM-2 (25,972 Da, Table 7, Figure S2).
VIM-2 incubated with DPA revealed dominant +9 and +8 peaks at
2,891 m/z and 3,253 m/z, respectively, corresponding most
closely with the mass of apo-VIM-2 with 1 equivalent of DPA
bound (26,010 Da).
Conclusion
Since the discovery of AMA as an effective inhibitor against
NDM-1 capable of restoring the efficacy of antibiotic meropenem
in mouse models, the synthesis and development of this
compound into a more potent and selective inhibitor have been of
interest.[19e, 22a, 22b] AMA, similar to EDTA, is a non-competitive
inhibitor that deactivates NDM-1 via active site Zn(II) metal
sequestration.[20a]
Inhibitor development of AMA through
Similar to the previously reported NDM-1:inhibitor
complexes, spectra of VIM-2 incubated with inhibitors 1 and 23c
– 23f all revealed the presence of ternary complexes. In addition
to the dominant di-Zn(II) VIM-2 peak, additional di-Zn(II) VIM-
2:inhibitor peaks were observed. The predicted and observed
peaks are summarized in Table 7 (Figure S2). Incubation of VIM-
2 with 1 revealed a less dominant +9 peak at 2,902 m/z,
corresponding to di-Zn(II) VIM-2:1 complex (26,119 Da). The
native MS of VIM-2 incubated with inhibitors 23c – 23f displayed
similar secondary peaks at 3,277 m/z (26,215 Da), 3,276 m/z
(26,199 Da), 3,277 m/z (26,215 Da), and 3,278 m/z (26,199 Da)
representing the formation of the di-Zn(II) VIM-2:23c, di-Zn(II)
VIM-2:23d, di-Zn(II) VIM-2:23e, and di-Zn(II) VIM-2:23f (Figure 3)
modification of the carboxylic acid functional groups has been
unsuccessful, as that motif is necessary for metal-chelation.[21a]
Herein, we report the FBDD of IDA as a novel MBP for NDM-1
inhibitor development. IDA is a simplified analogue of AMA and
EDTA, allowing for more facile inhibitor derivatization to probe the
NDM-1 active site pocket. Reducing the number of carboxylates
should also reduce the affinity of these compounds for free Zn(II)
ions, thereby reducing their metal-stripping propensity. From a
preliminary screen of a small library of MBPs, IDA and 1 were
verified as novel hits for inhibitor development. Upon rounds of
library design, synthesis, and mechanistic analysis IDA (IC50
=
122 μM) was developed into inhibitor 23f (IC50 = 8.6 μM). To study
the mode of inhibition, protein thermal shift and native state ESI-
6
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10.1002/cmdc.202000123
ChemMedChem
FULL PAPER
3-((Carboxymethyl)(methyl)amino)propanoic acid (13a). White solid,
MS were utilized. Both experiments revealed 23f and related
quantitative yield (102 mg, 0.40 mmol).
derivatives inhibited NDM-1 via the formation of stable ternary
complexes.
Additional studies are currently underway to
tert-Butyl
3-(benzyl(2-(tert-butoxy)-2-oxoethyl)amino)propanoate
elucidate the precise protein-inhibitor binding interactions;
however, similarities are observed with optimized dipicolinic acid
derivatives.[19d] Each scaffold is optimized through the addition of
a central hydrophobic substituent that includes a hydrogen-bond
partner that appears to require precise positioning, presumably
reflecting the binding interactions made with the beta-hairpin loop
neighboring the di-nuclear Zn(II) ion site of NDM-1. While lead
compound 23f displayed an inhibitory value similar to that of AMA
(IC50 = 4 – 9 μM), rational inhibitor design integrated with detailed
mechanistic studies has allowed for the development of an AMA-
inspired alternative that displays the formation of a NDM-
1:Zn(II):inhibitor ternary complex with a Ki of 2.6 μM. This work
represents the benefit of performing mechanistic analysis hand-
in-hand with inhibitor derivation for the development of inhibitors
with a mode of inhibition more suitable for drug development. By
utilizing a novel IDA MBP scaffold, traditional metal chelators
(such as AMA and EDTA) not viewed as candidates for inhibitor
development can be elaborated into potent inhibitors that form
favorable ternary complexes. Our findings provide a path for the
development of IDA-based inhibitors against NDM-1 and other
clinically relevant MBLs. Upon the development of advance
inhibitors with greater potency and selectivity, detailed
spectroscopy and microbiology studies can be performed to
further validate the mechanism of action.
(12b). Clear colorless oil, yield: 43% (239 mg, 0.67 mmol).
3-(Benzyl(carboxymethyl)amino)propanoic acid (13b). White solid,
quantitative yield (168 mg, 0.47 mmol).
Ethyl N-benzyl-N-(2-(diethoxyphosphoryl)ethyl)glycinate (14). Clear
colorless oil, yield: 40% (150 mg, 0.42 mmol).
N-Benzyl-N-(2-phosphonoethyl)glycine (15). White solid, yield: 99%
(114 mg, 0.42 mmol).
Ethyl N-benzyl-N-(cyanomethyl)glycinate (16). Clear colorless oil,
yield: 74% (447 mg, 1.92 mmol).
N-((1H-tetrazol-5-yl)methyl)-N-benzylglycine (17). Yellow solid, yield:
18% (40.0 mg, 0.16 mmol).
General procedures for the synthesis of 19a – 19d
To a solution of the corresponding amine (1.1 equivalents) and TEA (2
equivalents) in DMF (10 mL) was added t-butyl 2-bromoacetate (1
equivalent) dropwise at 0°C. The reaction was allowed to warm to 25 °C
and stirred for additional 16 h. After completion of the reaction, the salts
were removed via vacuum filtration.
concentrated in vacuo and the residue was purified via flash column
chromatograph to afford the desired intermediates 18a 18d.
The collected filtrate was
–
Intermediates were dissolved in TFA:CH2Cl2 (4:1 mL) and the reaction
mixture was stirred at 25 °C for 16 h. The excess TFA removed via co-
evaporation with copious amounts of MeOH under reduced pressure. The
product was purified via reverse phase column chromatography using
MeOH in H2O (w/ 0.1% formic acid) as eluent to afford the title compounds
19a – 19d.
Experimental Section
Inhibitors 1 – 11, IDA, reagents, and solvents were obtained from
commercial sources and used without further purification. All reactions,
unless otherwise stated, were performed at room temperature under a
nitrogen atmosphere. Flash column chromatography was performed using
a Teledyne ISCO CombiFlash Rf system using hexanes, ethyl acetate,
dichloromethane, and methanol as eluents with prepacked silica cartridges.
Reverse phase column chromatography was performed on the same
instrument using methanol and water (w/ 0.1% formic acid) as eluents with
high-performance Gold C18 columns. Column separation was monitored
via Teledyne ISCO RF+ PurIon ESI-MS. 1H and 13C NMR spectra were
recorded at ambient temperature on a 400 Varian Mercury Plus or 500
Varian VX NMR instrument located in the Department of Chemistry and
Biochemistry at the University of California, San Diego. Mass spectra were
obtained at the Molecular Mass Spectrometry Facility (MMSF) in the
Department of Chemistry and Biochemistry at the University of California,
San Diego. The purity of all compounds used in assays was determined
to be ≥95% by high-performance liquid chromatography. Enzymatic
assays were performed via monitoring the hydrolysis of substrate
meropenem on Synergy H4 Hybrid Microplate Reader using 96-well UV-
transparent microplates #3635 (Corning) according to previously
published procedures.[25] Thermal shift assays were performed on
QuantStudio 3 real-time PCR machines (Applied Biosystems) using 96-
well 0.2 mL optical MicroAmp thermocycler plates and SYPRO orange
Thermal Shift dye (ThermoFisher). Native state ESI-MS experiments were
tert-Butyl (2-(tert-butoxy)-2-oxoethyl)-D-alaninate (18a). Clear oil,
yield: 40% (175 mg, 0.68 mmol).
(Carboxymethyl)-D-alanine (19a). Clear oil, yield: 99% (93 mg, 0.63
mmol).
tert-Butyl (2-(tert-butoxy)-2-oxoethyl)-L-alaninate (18b). Clear oil,
yield: 40% (176 mg, 0.68 mmol).
(Carboxymethyl)-L-alanine (19b). White solid, yield: 99% (72 mg, 0.49
mmol).
tert-Butyl (2-(tert-butoxy)-2-oxoethyl)-D-phenylalaninate (18c). Clear
oil, yield: 52% (296 mg, 0.88 mmol).
(Carboxymethyl)-D-phenylalanine (19c). White solid, yield: 98% (116
mg, 0.52 mmol).
tert-Butyl (2-(tert-butoxy)-2-oxoethyl)-L-phenylalaninate (18d). Clear
oil, yield: 54% (308 mg, 0.92 mmol).
performed on
a LTQ Orbitrap XL hybrid ion trap-orbitrap mass
spectrometer (ThermoScientific).
(Carboxymethyl)-L-phenylalanine (19d). White solid, yield: 98% (110
mg, 0.49 mmol).
Synthesis
General procedures for the synthesis of 21a – 21m and 23a – 23h
tert-Butyl 3-((2-(tert-butoxy)-2-oxoethyl)(methyl)amino)propanoate
(12a). Clear colorless oil, yield: 90% (676 mg, 2.47 mmol).
The synthesis of compounds 21a – 21m and 23a – 23h were adapted from
literature reported procedures.[34] To a solution of the corresponding
7
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10.1002/cmdc.202000123
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FULL PAPER
amine (1 equivalent) and KHCO3 (4 equivalents) in THF or DMF (10 mL)
was added t-butyl 2-bromoacetate (2.25 equivalents), and the reaction
was stirred at 25 °C for 16 h. After completion of the reaction, as indicated
by TLC, the salts were removed via vacuum filtration. The collected filtrate
was concentrated in vacuo and the residue was purified via flash column
chromatograph using hexane/ethyl acetate as eluent to afford the desired
intermediates 20a – 20m and 22a – 22h. Compounds 21a – 21m and 23a
– 23h were obtained through the following deprotection procedures: A)
Intermediate was dissolved in a mixture of TFA:CH2Cl3 (4:1 mL) and the
reaction was stirred at 25 °C for 16 h. The excess TFA was removed under
reduced pressure and co-evaporated with copious amounts of MeOH. The
acid product was purified via reverse phase column chromatography with
MeOH and H2O (w/ 0.1% formic acid) as eluent to afford the desired
products; or B) Intermediate was dissolved in 1M NaOH:THF:MeOH (3:1:1
mL) and the reaction mixture was stirred at 65 °C for 16 h. THF and MeOH
was removed under reduced pressure and the aqueous solution was
acidified to pH 5 with 4M HCl. The precipitate was collected via vacuum
filtration.
Di-tert-butyl 2,2'-((furan-3-ylmethyl)azanediyl)diacetate (20i). Clear
colorless oil, yield: 29% (230 mg, 0.71 mmol).
2,2'-((Furan-3-ylmethyl)azanediyl)diacetic acid (21i). Deprotection
procedure A. White solid, yield: 99% (179 mg, 0.84 mmol).
Di-tert-butyl 2,2'-((phenylsulfonyl)azanediyl)diacetate (20j). White
crystalline solid, yield: 70% (273 mg, 0.71 mmol).
2,2'-((Phenylsulfonyl)azanediyl)diacetic acid (21j).
Deprotection
Procedure A. White solid, yield: 100% (115 mg, 0.42 mmol).
Di-tert-butyl 2,2'-((benzylsulfonyl)azanediyl)diacetate (20k). White
solid, yield: 76% (310 mg, 0.78 mmol).
2,2'-((Benzylsulfonyl)azanediyl)diacetic acid (21k).
Deprotection
Procedure A. White solid, yield: 99% (90 mg, 0.31 mmol).
Di-tert-butyl 2,2'-(phenethylazanediyl)diacetate (20a). Viscous clear
Di-tert-butyl 2,2'-((thiophen-2-ylsulfonyl)azanediyl)diacetate (20l).
colorless oil, yield: 84% (708 mg, 2.03 mmol).
White crystalline solid, yield: 77% (308 mg, 0.79 mmol).
2,2'-(Phenethylazanediyl)diacetic acid (21a). Deprotection procedure A.
2,2'-((Thiophen-2-ylsulfonyl)azanediyl)diacetic
acid
(21l).
Deprotection Procedure A. Off-white solid, yield: 90% (105 mg, 0.38
mmol).
White solid, yield: 99% (104 mg, 0.44 mmol).
Di-tert-butyl
2,2'-(([1,1'-biphenyl]-4-ylmethyl)azanediyl)diacetate
(20b). White fluffy solid, yield: 69% (680 mg, 1.65 mmol).
Di-tert-butyl 2,2'-((benzo[b]thiophen-2-ylsulfonyl)azanediyl)diacetate
(20m). Off-white crystalline solid, yield: 70% (312 mg, 0.71 mmol).
2,2'-(([1,1'-Biphenyl]-4-ylmethyl)azanediyl)diacetic
acid
(21b).
Deprotection procedure A. White solid, yield: 99% (72 mg, 0.24 mmol).
2,2'-((Benzo[b]thiophen-2-ylsulfonyl)azanediyl)diacetic acid (21m).
Deprotection Procedure A. Yellow solid, yield: 99% (101 mg, 0.31 mmol).
Di-tert-butyl
2,2'-(([1,1'-biphenyl]-3-ylmethyl)azanediyl)diacetate
Di-tert-butyl 2,2'-((thiophen-2-ylmethyl)azanediyl)diacetate (22a).
(20c). Clear colorless oil, yield: 98% (967 mg, 2.35 mmol).
Clear colorless oil, yield: 87% (717 mg, 2.10 mmol).
2,2'-(([1,1'-Biphenyl]-3-ylmethyl)azanediyl)diacetic
acid
(21c).
Deprotection procedure A. White solid, yield: 98% (141 mg, 0.47 mmol).
2,2'-((Thiophen-2-ylmethyl)azanediyl)diacetic acid (23a). Deprotection
Procedure A. White solid, yield: 70% (130 mg, 0.57 mmol).
Di-tert-butyl 2,2'-((4-hydroxybenzyl)azanediyl)diacetate (20d). White
solid, yield: 63% (530 mg, 1.51 mmol).
Di-tert-butyl 2,2'-((furan-2-ylmethyl)azanediyl)diacetate (22b). Yellow
oil, yield: 82% (646 mg, 1.99 mmol).
2,2'-((4-Hydroxybenzyl)azanediyl)diacetic acid (21d). Deprotection
2,2'-((Furan-2-ylmethyl)azanediyl)diacetic acid (23b). Deprotection
procedure A. White solid, yield: 99% (105 mg, 0.44 mmol).
Procedure A. Yellow oil, yield: 99% (160 mg, 0.75 mmol).
Di-tert-butyl 2,2'-((4-chlorobenzyl)azanediyl)diacetate (20e). White
crystalline solid, yield: 82% (727 mg, 1.97 mmol).
Di-tert-butyl 2,2'-(((5-methylthiophen-2-yl)methyl)azanediyl)diacetate
(22c). Clear colorless oil, yield: 82% (700 mg, 1.97 mmol).
2,2'-((4-Chlorobenzyl)azanediyl)diacetic acid (21e).
Deprotection
procedure A. White solid, yield: 98% (120 mg, 0.48 mmol).
2,2'-(((5-Methylthiophen-2-yl)methyl)azanediyl)diacetic acid (23c).
Deprotection Procedure A. Yellow solid, yield: 97% (113 mg, 0.46 mmol).
Di-tert-butyl 2,2'-((4-cyanobenzyl)azanediyl)diacetate (20f). White
Di-tert-butyl
2,2'-(((5-methylfuran-2-yl)methyl)azanediyl)diacetate
crystalline solid, yield: 75% (1.30 g, 3.60 mmol).
(22d). Clear colorless oil, yield: 64% (522 mg, 1.54 mmol).
2,2'-((4-Cyanobenzyl)azanediyl)diacetic acid (21f). White solid, yield:
94% (67 mg, 0.27 mmol).
2,2'-(((5-Methylfuran-2-yl)methyl)azanediyl)diacetic
acid
(23d).
Deprotection Procedure B. Yellow solid, yield: 53% (70 mg, 0.31 mmol).
Di-tert-butyl 2,2'-((4-(1H-tetrazol-5-yl)benzyl)azanediyl)diacetate (20g).
Pale yellow solid, yield: 46% (130 mg, 0.32 mmol).
Di-tert-butyl 2,2'-((2-(thiophen-2-yl)ethyl)azanediyl)diacetate (22e).
Light yellow oil, yield: 85% (726 mg, 2.04 mmol).
2,2'-((4-(1H-Tetrazol-5-yl)benzyl)azanediyl)diacetic
acid
(21g).
2,2'-((2-(Thiophen-2-yl)ethyl)azanediyl)diacetic
acid
(23e).
Deprotection procedure B. Beige solid, yield: 54% (50 mg, 0.17 mmol).
Deprotection Procedure A. White solid, yield: 75% (109 mg, 0.45 mmol).
Di-tert-butyl 2,2'-((thiophen-3-ylmethyl)azanediyl)diacetate (20h).
Yellow oil, yield: 62% (506 mg, 1.48 mmol).
Di-tert-butyl 2,2'-((2-(furan-2-yl)ethyl)azanediyl)diacetate (22f). Yellow
oil, yield: 84% (683 mg, 2.01 mmol).
2,2'-((Thiophen-3-ylmethyl)azanediyl)diacetic acid (21h). Deprotection
procedure A. White solid, yield: 80% (118 mg, 0.52 mmol).
8
This article is protected by copyright. All rights reserved.

10.1002/cmdc.202000123
ChemMedChem
FULL PAPER
2,2'-((2-(Furan-2-yl)ethyl)azanediyl)diacetic acid (23f). Deprotection
Procedure A. Yellow solid, yield: 33% (24 mg, 0.11 mmol).
XL™ hybrid ion trap-orbitrap mass spectrometer. The major parameters
were set as follows: capillary temperature, 180 °C; sheath gas, 0; auxiliary
gas, 0; sweep gas, 0; spray voltage, 1.1-1.9kV; tube-lens, 150 V; capillary
voltage, 35 V; full scan ranging 1000-4000 (m/z); and resolution set to
30,000. The automated gain control was set as follows: full scan, 3x104;
Di-tert-butyl 2,2'-(((5-bromothiophen-2-yl)methyl)azanediyl)diacetate
(22g). Yellow crystalline solid, yield: 90% (908 mg, 2.16 mmol).
SIM, 1x104; and MSn 1x105 for Fourier-transform.
Making slight
modifications, the nESI source was equipped with an offline unite (Catalog
number ES260) which was constructed based on previously published
material.[37] To construct the source, a platinum white (0.25 mm diameter)
was inserted into the center of the offline unit. The glass capillaries (inner
tip diameter 0.8 mm, outer tip diameter 1.5 mm) were produced in-house
using a micropipette puller (model P-87 Flaming/Brown Micropipette Puller,
Sutter Instrument Inc., USA), 5 μL of sample was loaded into the pulled
glass capillary. The platinum wire was inserted into the capillary and the
capillary position was adjusted approximately 3 mm from the MS inlet.
2,2'-(((5-Bromothiophen-2-yl)methyl)azanediyl)diacetic acid (23g).
Deprotection Procedure A. White crystalline solid, yield: 98% (108 mg,
350 mmol).
Di-tert-butyl 2,2'-(((5-chlorothiophen-2-yl)methyl)azanediyl)diacetate
(22h). Yellow solid, yield: 80% (510 mg, 1.36 mmol).
2,2'-(((5-Chlorothiophen-2-yl)methyl)azanediyl)diacetic acid (23h).
Deprotection Procedure A. Dark yellow solid, yield: 98% (107 mg, 0.41
mmol).
Acknowledgements
Inhibition screening and IC50 determination
We thank Dr. Yongxuan Su for mass spectrometry sample
analysis at The Molecular Mass Spectrometry Facility (MMSF) at
UC San Diego. This work was supported by the National
Institutes of Health (Grant GM111926, GM098435, and
GM134454) and by the Robert A. Welch Foundation (Grant F-
1572 to W.F.). S.M.C. is a cofounder of and has an equity interest
in Cleave Therapeutics and Forge Therapeutics, companies that
may potentially benefit from the research results. S.M.C. also
serves on the Scientific Advisory Board for these companies. The
terms of this arrangement have been reviewed and approved by
the University of California, San Diego in accordance with its
conflict of interest policies.
A soluble truncation of NDM-1 was over-expressed and purified as
described previously.[35]
IC50 values were determined using 11
concentrations of compound that span the IC50 value, and were assayed
using meropenem as described previously[25] except that total assay
volumes were increased to 200 μL. Final DMSO concentrations (derived
from compound stock solutions) were 1% (v/v). For initial screening of
compounds, the percent inhibition at 200 μM and 250 μM for each
compound was determined using a similar procedure. The NDM-1
catalyzed hydrolysis rate in the absence of added inhibitor (adjusted for
constant DMSO concentration) was set at 100% activity (0% inhibition),
and the relative rates determined in the presence of inhibitors were used
to calculate percent inhibition with respect to that control (e.g. 90% activity
is reported as 10% inhibition). Briefly, each compound (357 μM) was
incubated with NDM-1 (3.6 nM) for 20 min at 25 °C and diluted upon
addition of the meropenem substrate to initiate the reaction. Final
concentrations: NDM-1 (2.5 nM), compound (200 μM and 250 μM),
meropenem (180 μM), CHAPS (2 mM), HEPES (50 mM), DMSO (0.5 %)
at pH 7. Assays were completed as described for the IC50 determinations
above.
Keywords:
Metal-binding
Pharmacophore
(MBP)
•
Aspergillomarasmine A (AMA) • Iminodiacetic Acid (IDA) • Metal
Chelator • New Delhi Metallo-β-lactamase (NDM) • antibiotic
resistance
Thermal Shift Assay
To each well of a 96-well 0.2 mL optical MicroAmp (ThermoFisher)
thermocycler plate was added 9.5 μL of buffer (50 mM HEPES pH 7.5), 4
μL of NDM-1 in buffer (25 μM), 4 μL of inhibitor in buffer (1 mM), and 2.5
μL of SYPRO orange Thermal Shift dye (ThermoFisher) in buffer. Each
well contained a final concentration of 5 μM NDM-1 and 200 μM inhibitor.
Thermocycler plate wells were sealed prior to analysis, and the plate was
then heated in a thermocycler from 25 to 99 °C at a ramp rate of
0.05 °C/sec. Each thermal shift measurement was taken in eight replicates.
References
[1]
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Entry for the Table of Contents
Iminodiacetic acid (IDA) was identified as a novel lead fragment for New Delhi Metallo-β-lactamase (NDM) inhibitor development.
Through a series of fragment-based drug design, synthesis, and mechanistic analysis, 23f was identified as a potent inhibitor. This
inhibitor represents the potential to convert traditional metal chelators to one that displays the formation of a ternary complex. The
IDA fragment and inhibitors reported provide a roadmap for future metallo-β-lactamase inhibitor development.
11
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