Synthesis and biological evaluation of zinc chelating compounds as metallo-β-lactamase inhibitors

Article abstract of DOI:10.1039/c8md00578h

The syntheses of metallo-β-lactamase inhibitors comprising chelating moieties, with varying zinc affinities, and peptides partly inspired from bacterial peptide sequences, have been undertaken. The zinc chelator strength was varied using the following chelators, arranged in order of ascending binding affinity: dipicolylamine (DPA, tridentate), dipicolyl-1,2,3-triazolylmethylamine (DPTA, tetradentate) dipicolyl ethylenediamine (DPED, tetradentate) and trispicolyl ethylenediamine (TPED, pentadentate). The chosen peptides were mainly based on the known sequence of the C-terminus of the bacterial peptidoglycan precursors. Biological evaluation on clinical bacterial isolates, harbouring either the NDM-1 or VIM-2 metallo-β-lactamase, showed a clear relationship between the zinc chelator strength and restoration of meropenem activity. However, evaluation of toxicity on different cancer cell lines demonstrated a similar trend, and thus inclusion of the bacterial peptides did possess rather high toxicity towards eukaryotic cells.

Full text of DOI:10.1039/c8md00578h

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Andersen, C. Schnaars, A. Prandina, S. Radix, M. Le Borgne, L. P. Jordheim, T. Gjøen, A. Andresen, S.
Lauksund, C. Fröhlich, Ø. Samuelsen, P. Rongved and O. A. H. Åstrand, Med. Chem. Commun., 2019,
DOI: 10.1039/C8MD00578H.
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DOI: 10.1039/C8MD00578H
Journal Name
ARTICLE
Synthesis and biological evaluation of zinc chelating compounds
as metallo-β-lactamase inhibitors
a
a
a
b
b
Geir Kildahl-Andersen , Christian Schnaars , Anthony Prandina , Sylvie Radix , Marc Le Borgne , Lars
Received 00th January 20xx,
Accepted 00th January 20xx
c
d
d
e,f
Petter Jordheim , Tor Gjøen , Adriana Magalhães Santos Andresen , Silje Lauksund , Christopher
Fröhlich , Ørjan Samuelsen , Pål Rongved and Ove Alexander Høgmoen Åstrand
e,g
e,f
a
a*
DOI: 10.1039/x0xx00000x
www.rsc.org/
The syntheses of metallo--lactamase inhibitors comprising chelating moieties, with varying zinc affinities, and peptides
partly inspired from bacterial peptide sequences, have been undertaken. The zinc chelator strength was varied using the
following chelators, arranged in order of ascending binding affinity: dipicolylamine (DPA, tridentate), dipicolyl-1,2,3-
triazolylmethylamine (DPTA, tetradentate) dipicolyl ethylenediamine (DPED, tetradentate) and trispicolyl ethylenediamine
(
TPED, pentadentate). The chosen peptides were mainly based on the known sequence of the C-terminus of the bacterial
peptidoglycan precursors. Biological evaluation on clinical bacterial isolates, harbouring either the NDM-1 or VIM-2 metallo-
-lactamase, showed a clear relationship between the zinc chelator strength and restoration of meropenem activity.

However, evaluation of toxicity on different cancer cell lines demonstrated a similar trend, and thus inclusion of the bacterial
peptides did possess rather high toxicity towards eukaryotic cells.
Metallo-β-lactamases (MBLs) are a group of hydrolytic enzymes that
break down antibiotics of the -lactam class, including penicillins,
Introduction
Infectious diseases are a leading cause of death worldwide, including
cephalosporins and carbapenems. Carbapenems is a class of last-
resort antibiotics, and resistance towards these drugs are
threatening our abilitiy to treat serious infections. Worryingly, the
MBLs are already widespread, and their occurrence is increasing.6
There are currently no MBL inhibitors on the market, and a successful
inhibitor could extend the therapeutic lifetime of carbapenems. Even
newly approved β-lactamase inhibitors, such as avibactam and
vaborbactam, do not target MBLs.
1
more than half of all child mortality under the age of five. Currently,
there is a serious concern regarding new and re-emerging infectious
diseases, for which effective therapies are lacking.2-4 The emergence
of multidrug-resistant (MDR) strains of common Gram-negative
pathogens, such as Escherichia coli, are of increased concern.
Extensively drug-resistant or pandrug-resistant are now commonly
used terms to describe clinically important isolates of Pseudomonas
aeruginosa and Enterobacterales that are resistant to virtually all
clinically available antibiotic drugs.5
MBLs are zinc dependent enzymes, carrying one or two zinc atoms
within their active site. Several zinc-chelating compounds have
inhibitory activity towards MBLs and some of these inhibitors have
even reached clinical trials.7-11 By using chelators with moderately
soft donor atoms, nitrogen in our case, selectivity towards rather soft
a
Department of Pharmaceutical Chemistry, School of Pharmacy, University of Oslo,
PO Box 1068 Blindern, 0316 Oslo, Norway.
b
EA 4446 Bioactive Molecules and Medicinal Chemistry, Faculté de Pharmacie –
ISPB, Université de Lyon, Université Lyon 1, SFR Santé Lyon-Est CNRS UMS3453 -
INSERM US7, F-69373, Lyon cedex 08, France
2+
metal ions, such as Zn , can be achieved. Harder metal ions of
c
Université de Lyon, Université Claude Bernard Lyon 1, INSERM 1052, CNRS
UMR5286, Centre Léon Bérard, Centre de Recherche en Cancérologie de Lyon, 69008
Lyon, France.
Centre for Integrative Microbial Evolution (CIME), Faculty of Mathematics and
biological relevance, such as Ca2+ and Mg2+, are better chelated by
oxygen donor atoms.12 However, many zinc-chelating compounds
d
Natural Sciences, University of Oslo, Blindern, Oslo, Norway.
e
Norwegian National Advisory Unit on Detection of Antimicrobial Resistance,
suffer from low selectivity, low potency and/or high eukaryotic
toxicity. We have earlier studied the effect of tris-picolylamine (TPA)
derivatives as MBL inhibitors.13 In this work, we chose to vary the zinc
Department of Microbiology and Infection Control, University Hospital of North
Norway, 9038 Tromsø, Norway.
f
Department of Pharmacy, UiT – The Arctic University of Norway, 9037 Tromsø,
Norway.
g
The Norwegian Structural Biology Centre (NorStruct), Department of Chemistry,
1
4
UiT The Arctic University of Norway, 9037 Tromsø, Norway
binding strength by using N,N-dipicolylamine (DPA) , N,N-dipicolyl-
N-triazolylmethylamine (DPTA), N,N-dipicolyl ethylenediamine
*
Corresponding author: Tel: +47 22854478; e-mail: o.a.h.astrand@farmasi.uio.no †
Electronic Supplementary Information (ESI) available: Synthetic details, NMR
spectra and intrinsic bacterial activity of chelators. See DOI: 10.1039/x0xx00000x
This journal is © The Royal Society of Chemistry 20xx
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(
DPED) and trispicolyl ethylenediamine (TPED) as starting points. The
a)
O
O
O
View Article Online
H
DOI: 10.1039/C8MD00578H
N
chelators were attached to various peptides, using peptide coupling
reagents, to produce chelator-peptide constructs as putative MBL
inhibitors.
HO
N
H
OH
NH2
A
NH2
O
E
C
B
D
We hypothesized that chelators attached to the bacterial peptide
fragment D-Ala-D-Ala, could achieve affinity for bacterial penicillin-
binding proteins (PBP). In addition, we also explored several other
small peptides that could have an affinity to bacterial proteins such
as meso-diaminopimelic acid (m-DAP)-D-Ala-D-Ala, as well as
considering other attachment points (Fig 1a). For compounds of this
type, we chose to use L-Lys as a substitute for m-DAP, eliminating
one carboxylic acid in m-DAP for ease of synthesis, but preserving the
attachment points A and B (Fig. 1a). One can also imagine attaching
the chelator at point D (Fig. 1a), as evidence from the literature
suggests that compounds with larger substituents than methyl on
the ultimate D-amino acid are acceptable substrates for the
PBPs.15,16,17 We also created boronic acid-containing transition state
intermediates (BATSI), knowing that the last D-Ala unit is cleaved by
serine catalysis in the PBPs.18 For this reason, we prepared chelating
compounds with L-Lys-boroglycine as peptides. Compounds of this
type could potentially target either PBPs or even serine β-
lactamases, being aware that these proteins share an evolutionary
past.19 Also, peptidomimetic boron-containing compounds binding
to or inhibiting PBPs are well-known.20,21 As it would later turn out,
current evidence suggests this reasoning to be too simplistic and
such dual activity cannot be expected,22 but we have included such
b)
N
N
N
R
N
N
R
N
N
DPA
DPED
N
N
N
N
N
N
R
N
R
N
N
N
N
DPTA
TPED
Figure 1. a) Potential chelator attachment points A-E based on
tripeptide m-DAP-D-Ala-D-Ala. See main text for details. b) Chelators
included in this study: dipicolylamine (DPA), dipicolyl 1,2,3-
triazolylmethylamine (DPTA), dipicolyl ethylenediamine (DPED) and
trispicolyl ethylenediamine (TPED).
Results and discussion
Synthesis of compounds. The full details of the syntheses are
provided in the supplementary information. The final assembly of
the majority of the peptide-chelator compounds followed a common
strategy. The part containing the chelating group was prepared
either as a free acid (1-4), or as a free amine (5-7). The acids 1-4 were
attached, using standard peptide coupling methodology, to methyl
esters of dipeptides with free amine groups, available as either HCl
or TFA salts, whereas the amines 5-7 were coupled to N-Boc-
protected dipeptides containing a free acid. Then, coupling products
were deprotected in the final step, to yield the desired products 8-
compounds in this study for completeness. In a recent report, we
studied the linkers between the chelators and the peptides,23 but
found that the biological effect was mostly due to the strength of the
zinc chelator. We, therefore, have not performed a comprehensive
investigation of all possible chelator-linker-peptide combinations for
the chelators and peptides in the present work.
2
0 (Scheme 1). We included the previously reported compound 15
here for completeness.23 Note that methyl esters have been
preserved in 8-14, as the corresponding free acids have performed
poorly in biological assays (results not shown). All products 8-20
show an attachment of chelator moiety either at point C or point E
(Fig. 1a).
2
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Assembly of the chelating groups (1-7), from commercially available mesylate 26, followed by nucleophilic displacement witVhiewDPArAtictloe Ofonrlinme
DOI: 10.1039/C8MD00578H
precursors, was achieved as outlined in Scheme 2. The DPA amine 5 the ethyl ester 27. In situ hydrolysis of 27, using lithium hydroxide,
was prepared from N-Boc-protected 4-(2-aminoethyl)aniline as afforded 2, which was directly used in peptide coupling reactions.
previously described.23 Quantitative conversion to the acid 1 was The TPED chelators 3 and 7 were synthesized in two steps, from
achieved by reaction with succinic anhydride. For the dipicolyl 1,2,3- N,N,N'-tris-pyridin-2-ylmethylethane-1,2-diamine (28), available
triazolylmethylamine chelators (DPTA), the amine 6 was obtained by following literature procedures.27 First, the amine was alkylated with
copper(I)-mediated azide-alkyne cycloaddition (CuAAC) between either 29 or 31, in order to form the protected intermediates 30 and
tert-butyl 4-azidophenethylcarbamate (21),24 and N-propargyl-di(2- 32, which were subsequently deprotected to yield free acid (3) or
picolyl)amine (22),25 followed by deprotection with HCl. Preparation free amine (7). All chelators 1-3 and 5-7 contain a phenyl ring in the
of the acid 2 from ethyl (4-bromophenyl)acetate (23) required link to the peptide. For the DPED series, we decided to circumvent
conversion to the corresponding azide 24,26 and subsequent CuAAC the linker and directly couple the peptide to the acid 4, which was
reaction with propargyl alcohol to afford the 1,2,3-triazole synthesized with only minor deviations from previously published
containing alcohol 25. Assembly of the chelator was then methods.28
accomplished by converting the alcohol to the corresponding
O
O
O
H
OH
N
O
HN
HN
N
H
O
O
O
O
H
N
a
NH2
HN
NH3Cl
O
HN
HN
b
DPA
DPA
, 48%
O
O
8
1
HN
HN
DPA
DPA
O
O
O
OH
O
15, 64%
R
5
a
HN
NH
R
O
O
O
HN
DPAT
NH2
DPAT
O
NH
R
c
R
2
R = H: 9, 73%
R = CH3: 10, 50%
O
NH2
DPTA
R = H: 16, 37%
R = CH3: 17, 23%
DPTA
O
O
R
R'
O
6
H
N
OH
N
O
H
O
R
R'
a
O
R
NH2
R'
NH3Cl
H
N
N
H
O
NH
R
R'
O
O
NH
d
R = R' = H: 11, 42%
R = CH3, R' = H: 12, 40%
R = H, R' = CH3: 13, 20%
O
NH
TPED
TPED
3
O
NH
R= R' = H: 18, 35%
R = CH3, R' = H: 19, 28%
R = H, R' = CH3: 20, 14%
TPED
7
TPED
O
H
OH
a
N
O
DPED
DPED
N
H
O
O
O
14, 14%
4
Scheme 1. Preparation of products 8-20. Reagents: a. D-alanyl-D-alanine methyl ester hydrochloride (for 8, 10, 12, 14) or glycylglycine methyl
.
ester hydrochloride (for 9), HATU, N-methylmorpholine, DMF. Compound 11: glycylglycine methyl ester hydrochloride, EDC HCl, HOAt, N-
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methylmorpholine, ethanol/DMF; Compound 13:L-alanyl-L-alanine methyl ester hydrochloride, HATU, N-methylmorpholine,VDieMw AFr;ticble. O1n)liNne-
DOI: 10.1039/C8MD00578H
Boc-D-alanyl-D-alanoic acid, HATU, N-methylmorpholine, DCM, 2) TFA, DCM; c. 1) N-Boc-glycyl-glycine (for 16) or N-boc-D-alanyl-D-alanoic
acid (for 17), HATU, N-methylmorpholine, DMF, 2) TFA, DCM; d. 1) N-Boc-glycyl-glycine (for 18), N-Boc-D-alanyl-D-alanoic acid (for 19) or N-
Boc-alanyl-alanoic acid (for 20), HATU, N-methylmorpholine or DIPEA (20 only), DMF or ACN (18 only), 2) TFA, DCM.
O
OH
NH2
HN
NHBoc
H2N
O
O
O
1) CuSO4
O
N
N
Na-(+)-ascorbate
H2O : t-BuOH
N
Acetone
2) HCl, dioxane
0
>
°C - rt.
99%
65% over two steps
HN
HN
DPTA
N3
DPA
DPA
O
O
21
22
6
5
1
O
OEt
O
OR'
O
OEt
Propargyl alcohol
Cu(I)I
Di-(2-picolyl)amine
K2CO3
Acetonitrile
N
Acetonitrile
80%
96%
N
DPTA
N
X
2
7: R' = Et
R' = H
1) LiOH
H2O/THF quant.
2
3: X = Br
NaN3, Cu(I)I
Na-(+)-ascorbate
EtenEt, Et2O, H2O
RO
2
:
2) HCl
2
5: R = H
2
4: X = N3
8%
MsCl, Et N
DCM
3
6
2
6: R = Ms
64%
O
O
O
Br
N
H
1) LiOH, H2O/THF
OH
O
O
2) HCl
O
2
9
TPED
O
TPED
O
N
H
K2CO3, KI
ACN
quant.
N
H
76%
3
30
O
N
Br
NHBoc
N
N
1) TFA in DCM
) Basic work-up
NH2
H
O
O
NH
2
3
1
TPED
N
TPED
NHBoc
N
N
H
N
Et N, KI
3
62%
H
ACN
27%
28
32
7
Scheme 2. Synthesis of precursors 1-3, 6 and 7.
In addition to the syntheses outlined in Schemes 1 and 2, some chloride 34, prepared directly from the H-D-Ala-D-Ala-OMe
compounds were prepared using other routes. The DPA analogue 33, dipeptide (Scheme 3).
in which the linker was omitted, was obtained in two steps via the
4
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O
O
View Article Online
H
N
DOI: 10.1039/C
N
8MD00578H
Cl
N
N
ClH N
O
3
HO
RN3
CuSO4
Na-(+)-ascorbate
O
N
R
N
H O : t-BuOH
O
O
2
N
N
N
HATU, NMM
DMF
H
N
Cl
N
N
H
O
35-43
0°C - rt.
22
O
92%
3
4
O
O
O
O
O
O
H
H
N
N
N
O
O
O
N
O
O
O
H
H
DPA, DIPEA, KI(cat)
MeCN
H
N
O
O
DPA
N
H
O
3
5 (27%)
64%
O
3
3
O
H
N
39 (32%)
Scheme 3. Preparation of compound 33, omitting the linker.
N
H
O
O
R'
3
6 (38 %)
H
Finally, a majority of the DPTA compounds was prepared from azido
N
O
N
H
peptides, by the CuAAC reaction with N-propargyl-di(2-picolyl)amine
O
O
O
H
N
(
22) as either the final step in the case of 35-43 (38 excepted, Scheme
N
H
4
), or the penultimate step in the case of the potential BATSI
O
NH
R' = H: 40 (47%)
R' = CH : 41 (45%)
3
7 (62%)
compounds 44 and 45 (Scheme 5). In this manner, we were thus able
to provide compounds corresponding to attachment of chelator in
position A (40, 41, 45), position B (42-44) and position D (38-39), (cf.
Fig. 1a). The rather elaborate syntheses of the azides are described
in detail in the supporting information.
3
O
O
H
N
H N
O
2
O
R'
H
O
N
O
N
H
O
O
NH
Because the CuAAC reaction in these reactions created a powerful
chelator, we attempted different work-up schemes in order to
remove the copper from the reaction. No clear conclusion was
reached whether one work-flow performed better than the other
with regard to yield. Work-up with extraction from basic aqueous
O
NH
R' = H: 42 (41%)
R' = CH : 43 (44%)
38 (40%, including final
Fmoc deprotection of amine)
O
3
Scheme 4. Preparation of compounds 35-43 by way of CuAAC triazole
formation.
+
2+ 29
phases containing EDTA, in order to chelate Cu /Cu , gave, for
instance, 62% yield in the preparation of Fmoc-protected 38,
whereas only 27% yield of 48 was obtained. Solubility of the product
in the aqueous phase was detrimental, and the subsequent
purification on alumina column often yielded poorer separation than
desired. An alternative work-flow, in which Chelex 100 was used for
+
2+
complexing Cu /Cu , followed by purification of product on
reversed-phase SPE material, was employed, for instance, in the
synthesis of 37, yielded only 62% of the expected product. Both
methods were attempted for compounds 35 and 36, giving
comparative yields in both cases, thus it seems that the choice can
be a matter of convenience. More details can be found in the
experimental description.
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O
O
concentrations, interspersed with some of the less activVeiewcoAmrticpleoOunnlidnes
DOI: 10.1039/C8MD00578H
comprising the DPTA chelator (Table 2).
NH
O
NH
O
b
We have previously reported results on how variations in the linker
OH
B
H
N
5
3%
DPTA
O
between peptide fragment and zinc chelator influenced meropenem
MIC restoration and eukaryotic toxicity.13,23 In this present paper, we
N
N
OH
H
H
O
DPTA
O
4
8
4
4
have explored how variations in the peptide, the corresponding
attachment point, as well as differences in chelator strength,
influenced the effect on meropenem MIC and toxicity of these
compounds. As shown in Tables 1 and 2, data suggest a strong
dependence upon the strength of the chelator for reducing the MIC
of meropenem. It should also be noted that none of the reported
compounds displayed any antibiotic effect on their own (data shown
in supplementary information, Table S1). This includes the boronic
acid containing compounds 44 and 45, despite being modelled from
the bacterial peptide in the peptidoglycan cell wall. Overall,
compound activities seems to follow the chelating strength. For our
compounds, the order of chelating strength would be DPA < DPTA <
DPED ≈ TPED < TPEN. The DPA chelator performs weaker than those
containing DPTA, whereas compounds with DPED and TPED both
seem to perform equally well based on our data, and much better
than DPTA. This also clearly illustrates the rather low chelating
a
27%
NHR
O
R'HN
O
6 R = Ac, R' = -COCH N
4
4
2
3
7 R = -COCH N R' = Ac
2
3,
a
29%
DPTA
NH
O
DPTA
NH
O
O
b
O
OH
B
2
2%
H
N
O
N
N
H
OH
H
3
O
O
strength of N in 1H-1,2,3-triazoles, compared to the aliphatic amine
4
5
4
9
nitrogen present in DPED. The influence of the chelator strength was
tested at low inhibitor concentration (Table 2). For the DPTA
compounds, the inhibitory effect at 31 µM is almost completely
obliterated. Data also suggest no active role of the peptide for the
biological effect. Neither change of configuration from D-amino acids
to L-amino acids, change of attachment points nor addition of
boronic acids seem to be of consequence. It should also be noted
that testing of compound 44 and 45 against strain with reduced
Scheme 5. Preparation of potential BATSI compounds 44 and 45.
Reagents: a. Copper(II)sulfate, sodium-(+)-ascorbate, 22, water, tert-
butanol; b. 1) LiOH hydrate, water, THF, 2) neutralize with 1M HCl, 3)
bis-trimethylsilyl-aminoethaneboronate pinacol ester, HATU, N-
methylmorpholine, water, DMF.
MIC determination in combination with meropenem.
susceptibility to meropenem due to class
A and class D
All the prepared compounds, as well as some of the intermediates,
were tested in combination with the carbapenem meropenem
against two different clinical isolates of Pseudomonas aeruginosa
and Klebsiella pneumoniae expressing the VIM-2 (Verona Integron-
Mediated metallo-β-lactamase 2) and NDM-1 (New Delhi metallo-β-
lactamase 1) metallo-β-lactamases, respectively (Table 1). The
compounds were screened at a fixed concentration of either 50 or
carbapenemases (KPC-2 and OXA-48, respectively, data shown in
supplementary information, Table S2) revealed no potentiation of
meropenem in those assays, suggesting that they do not function as
transition-state inhibitors of those enzymes. This is perhaps
unsurprising when we keep in mind that the -lactamases need to be
inert against the endogenous peptides of the bacterium.
1
25 M, and MIC for meropenem was determined. In addition, a
Based on these data, a zinc-depletion mechanism seems to be the
most likely mode of action of our compounds. A similar mechanism
was concluded for the fungal natural product aspergillomarasmine
selection of the most active compounds was tested at reduced
6
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A, identified as a potent inhibitor of NDM-1 and other clinically a class of spiro-indoline-thiazole analogues working similarly.32
View Article Online
DOI: 10.1039/C8MD00578H
relevant metallo--lactamases.8, 30-31 There are also recent reports on
Table 1. MIC values of meropenem (MEM) in combination with the prepared chelators against P. aeruginosa and K. pneumoniae harbouring
VIM-2 and NDM-1, respectively. Assays were performed in duplicate, with at least three separate experiments. All prepared chelators were
tested at a fixed concentration of 125 µM, except for compounds marked with and asterisk (*), which were tested at 50 µM. The MIC values
of meropenem against both strains were determined to be 32-64 mg/L. TPEN = N,N,N’,N’-tetrapicolyl ethylenediamine.
Compound
MIC MEM (mg/L)
Compound
MIC MEM (mg/L)
P. aeruginosa
VIM-2
K. pneumoniae
P. aeruginosa
VIM-2
K. pneumoniae
NDM-1
NDM-1
DPA:
8
DPED:
14
1
*
0.125*
3
1
1
2
6
6
8-16
8
1
3
5
3
8-16
TPED:
11
DPTA:
9
1
0.125
0.125
12
1
8
8
8
4
*
*
*
*
4*
1
1
1
2
3
3
3
3
3
4
4
4
4
4
4
0
6
7
7
5
6
7
8
9
0
1
2
3
4
5
13
1
0.125
4*
18
precipitated
precipitated
0.125
4*
19
2
2
4*
20
0.125
1
6*
4*
28
0.5
<0.25
2
<0,5
8
2
4
2
TPEN
1
<0.5
precipitated
4
precipitated
1
16*
16*
32*
32*
2
4*
8*
8*
8*
0,5
0,5
2
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Table 2. MIC values of MEM (mg/L), in combination with lower concentrations of inhibitor (31 and 16 µM), against P. aeVrieuwgAinrtioclseaOnalninde
DOI: 10.1039/C8MD00578H
K. pneumoniae harbouring VIM-2 and NDM-1, respectively. The MIC values of meropenem against both strains were determined to be 32-
6
4 mg/L.
Compound
P. aeruginosa VIM-2
1 µM 16 µM
K. pneumoniae NDM-1
31 µM 16 µM
3
1
6
3
3
4
4
5
6
4
5
32
6
1
1
3
4
6
6
2
32
32
32
32
32
32
32
32
32
32
32
8
16
16
8
8
16
16
16
16
8
0
.125
2
1
1
4
1
1
-2
2
4
1
2
4
1
1
1
1
2
2
2
3
9
0
8
0.125
0.125
0.125
0.125
0.125
8
16
8
Toxicity in human cell lines. Toxicity of a selection of the compounds compared.25 The DPED chelator is given a value of 11.8, based on
was assessed in three different cancer cell lines, with IC50-values as structural similarities with the previously reported chelator DPESA.27
stated in Table 3. Also, included in the same table, are estimated Finally, TPED is estimated to 12.3, also based on structural similarity
values for the stability constants pK
D
of the corresponding zinc with the known chelator TPESA.27 We note that this value might be
complexes. The value for the DPA chelators are taken directly from underestimated, as another source put TPEN itself at 15.6, with a
the literature,33 whereas the DPTA chelators have been given a value corresponding statement that zinc is bound to TPEN in a
of 1.3 log units above DPA, based on data provided in a study where pentadentate manner, suggesting that the contribution of the fourth
coordinating and non-coordinating triazole moieties were pyridine ring in TPEN compared to TPED could be relatively minor.33
D
Table 3. Properties and intrinsic toxicity of selected zinc chelator constructs sorted by estimated pK .
Compound
cLogP
Peptide characteristics
Chelator characteristics
Intrinsic Toxicity (µM)‡
Attachment
MDA-MB
231
*
Sequence
Type
Est. pK
D
Mia-PaCa
Colo-357
point#
8
0.3
D-Ala-D-Ala
D-Ala-D-Ala
D-Ala-D-Ala
D-Ala-D-Ala
D-Ala-D-Ala
L-Ala-L-Ala
C
DPA
7.6
7.6
7.6
8.9
8.9
8.9
141  42
127  92
184  46
125  27
152  17
157  10
45  15
118  32
98  6
152  8
21  5
1
3
1
3
3
5
3
0
5
6
0.4
0.1
2.1
0.2
0.2
E
C
C
C
C
DPA
DPA
90  12
105  23
133  18
96  11
DPTA
DPTA
DPTA
119  21
116  9
105  16
8
| J. Name., 2012, 00, 1-3
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View Article Online
DOI: 10.1039/C8MD00578H
3
3
3
4
4
1
1
1
1
1
2
7
8
9
4
5
4
1
2
8
9
0
-0.8
-0.1
1.0
-3.5
-3.5
0.1
0.7
1.7
1.2
2.2
2.2
Gly-Gly
C
D
D
B
A
C
C
C
E
DPTA
DPTA
DPTA
DPTA
DPTA
DPED
TPED
TPED
TPED
TPED
TPED
8.9
168  77
127  55
131  15
101  21
128  44
176  11
137  78
D-Ala-D-Lys
D-Ala-D-Lys
L-Lys-boroGly
L-Lys-boroGly
D-Ala-D-Ala
Gly-Gly
8.9
106  10
128  8
66  13
119  3
114  3
101  15
112  33
82  6
8.9
8.9
8.9
11.8
12.3
12.3
12.3
12.3
12.3
4
7
4
1
4
5
.5  0.6
.8  0.4
.1  1.4
5  3
5.1  0.4
12  3
4.3  0.2
12  4
D-Ala-D-Ala
Gly-Gly
5.9  2.3
21  23
4.8  1.2
5.5  0.9
5.4  0.8
24  6
D-Ala-D-Ala
L-Ala-L-Ala
E
.9  0.7
.1  0.2
6.8  1.4
6.8  1.6
E
The IC50 values shown in Table 3, corresponding to toxicity in three What seems to have an effect is the changing the peptide from Ala-
different human cancer cell lines, separates clearly the compounds Ala, in any configuration, to Gly-Gly, as shown for 11, and even more
in two different groups, with the weaker chelators DPA and DPTA pronounced, for 18. The dominating effect of this switch to glycine
exhibiting lower toxicity than DPED and TPED. Thus, it would seem might be to make the compounds more hydrophilic, which in turn
that both ability to potentiate the activity of meropenem and points toward another strategy for tuning the toxicity of the
intrinsic toxicity correlate with each other through chelator strength. strongest chelators. We have in fact prepared DPED and TPED
Initially, we hoped for some visible detoxifying effect of the vector or compounds (50 and 51, Figure 2, syntheses given in supplementary
linker for the strongest chelator, but this seems not to be the case. information) taking this strategy to the extreme by the inclusion of
For instance, by comparison between 12, 19 and 20, we see that carbohydrates as “hydrophilic vectors”, and, compared their toxicity
attachment to C- or N-terminus of the peptide (attachment points E to their peptide counterparts 14 and 19 (Table 4). The human cells
and C, respectively, in Fig. 1a), or change from D-amino acids to L- are not the same as used in the first and large screen (Table 3), but
amino acids, have no effect on the toxicity with the TPED chelator. If allow comparing the interesting compounds with each other.
the obtained toxicity data are representative for normal human cells, However, no clear conclusion as to the hydrophilic vector could be
the MIC values of meropenem at a lower chelator concentration drawn, as compound 50 was 17-fold less toxic than 14, and
illustrates the dilemma of the strongest chelators as they have little compound 15 was 8-fold less toxic than 19. Further, in vitro and in
to no therapeutic window. Application of such compounds in the vivo testing of 50, and maybe 51 also, must be undertaken to fully
clinic may be viewed as balancing acts, wherein the chelator strength evaluate their clinical utility.
must be strong enough for significant potentiation, but not too
strong, in order to limit side effects. Consequently, we have in
parallel investigated the use of the TPA chelator in order to explore
this region of intermediate chelator strength between DPTA and
DPED.12
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DOI: 10.1039/C8MD00578H
Table 4. Direct comparison of MIC values of meropenem (MEM) and toxicity of two different chelators (DPED and TPED) with similar side
chains. The MIC values of meropenem against both strains were determined to be 32-64 mg/L.
Compound
cLogP
Chelator
MIC data towards MEM [mg/L] at 31 and 16 µM
Intrinsic toxicity
‡
IC50 (µM) HepG2
P. aeruginosa VIM-2
K. pneumoniae NDM-1
31 M 16 M
3
1 M
16 M
1
4
0
9
1
0.1
-1.7
2.2
0.7
DPED
DPED
TPED
TPED
6.83 ± 1.05
117.6 ± 1.11
13.56 ± 1.30
1.70 ± 6.17
1
-2
32
32
32
32
0.125
16
5
1
5
1
2
2
0.25
0.125
0.125
32
8
8
toxicity in human cells, did not hold up, as the corresponding
inhibitors containing L-Ala-L-Ala showed exactly the same results as
the inhibitor containing D-Ala-D-Ala. Instead, data for inhibitors
containing Gly-Gly point towards decreased lipophilicity as a path
towards lowering the toxicity for the strongest chelators. We
postulate that by choosing zinc chelators of intermediate strength,
toxicity might be limited, while preserving a therapeutic utility.
Compound 50, with its poly-alcohol side chain, looks promising and
it might seem these hydrophilic side chains have more potential than
peptides.
OH
OH
N
OH
N
O
N
N
OH
OH
N
N
N
N
N
N
N
N
N
O
HO
HO
OH
OH
51
5
0
Figure 2. Compounds containing strong chelators coupled with
carbohydrates.
Experimental
Chemical synthesis. The detailed experimental procedures for all
new compounds can be found in the supporting information, except
for commercially available TPEN and the previously reported
compound 15.23
Conclusion. In this work, we have prepared zinc chelators bound to
small peptide fragments, with theoretical dissociation constants for
bound zinc (K
d
) ranging from 10 to 10-14. In co-administration with
-8
meropenem towards meropenem-resistant bacterial strains
harbouring the MBLs VIM-2 or NDM-1, we observed potentiation of
meropenem for all chelator types. However, for the weakest chelator
variants, higher concentrations of chelator were needed to obtain
any effect, while stronger chelators were effective down to 31 M
concentration.
Meropenem MIC determination. The MICs of meropenem alone or
in combination with the compounds were determined using pre-
made broth microdilution plates, containing meropenem in 2-fold
dilution steps ranging from 0.03 to 64 mg/L (TREK Diagnostic
Systems/Thermo Fisher Scientific, East Grinstead, UK). The
compounds were initially screened at a final concentration of 125 or
5
0 µM. The MIC assays were performed using two MBL-producing
Toxicity of the new compounds was evaluated in vitro in three human
cancer cell lines. The data suggested a correlation between chelator
strength and increased toxicity. Our initial hypothesis that inclusion
of peptidde fragments of bacterial origin might lead to decreased
meropenem resistant strains, one NDM-1-producing K.
pneumoniae34 and one VIM-2-producing P. aeruginosa.35 In brief, the
compounds were diluted in MH-broth (Becton Dickinson, Franklin
Lakes, NJ USA) and 50 µL of the suspension was added to each well
1
0 | J. Name., 2012, 00, 1-3
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of the meropenem plate. Bacterial colonies were suspended in 0.9% X is log(concentration) analysis, and a four-paramVieewteArrticlleoOginsltiniec
DOI: 10.1039/C8MD00578H
saline buffer to 0.5 McFarland and further diluted 1:100 in MH broth regression from GraphPad Prism 6 (GraphPad Software Inc., USA).
(Thermo Fisher Scientific, East Grinstead, UK). 50 µL of the diluted cLogP calculations. The partition coefficient for all compounds were
bacterial suspension was added to the meropenem plates to a final calculated using PerkinElmer Informatics ChemDraw Professional
volume of 100 µL. The plates were incubated for 20 h at 37 °C. Each version 15.1.0.144.
MIC was determined in duplicate. To investigate any intrinsic Conflict of interest. OAHA, ØS, PR, GKA and CS have filed patents on
antibacterial activity of the compounds the MIC of each compound this discovery.
were determined in a 2-fold serial dilution series in MH broth (Becton
Dickinson, Franklin Lakes, NJ USA) ranging from 2-1000 µM. 50 µL of
Acknowledgements
O.A.H.Å is grateful for funding from The Research Council of
each dilution step was then mixed with 50 µL of a bacterial inoculum,
prepared as described above, in 96 well microtiter plates (Thermo
Fisher Scientific, Roskilde, Denmark). The plates were incubated, and
MIC determined as described above.
Norway (NFR) mobility grant (grant number: 240215). G.K-A and
C.S are grateful for a NFR Biotek2021 grant (244219) and a NFR
FORNY grant (273430). We also thank NOVO Pre Seed (NOVO
Nordisk Fonden ByGS/SSch) for a generous grant as well as
Cell viability assays. Human breast cancer cells (MDA-MB-231) and funding from the University of Oslo.
pancreatic cancer cells (MIA-PaCa2 and Colo-357), all obtained from
2
ATTC, were cultured at 37 °C with 5% CO in DMEM media,
Author Contributions
containing 10% fetal bovine serum and antibiotics. For cell survival
assays, cells were seeded in 96 well plates (3000 cells per well, 100
µL medium), adhered for 24 h and exposed to different
G.K-A. - Synthesis and characterization of compounds, project
supervision, manuscript composition and writing
C.S.
project supervision, manuscript writing
- Synthesis and characterization of compounds,
biological testing of compounds
- Synthesis and characterization of compounds,
concentrations (from 300 µM to 0.3 µM) of the Zn chelators A.P.
dissolved in DMSO) for an additional 72 h. Then, metabolically active
cells were quantified by the MTT assay by adding 100 ng MTT per
(
S.R., M.L.B.
L.P.J., C.F.
- Supervision of synthesis, manuscript writing
- Biological testing of compounds, manuscript
well, incubating cells for 2 h, replacing media with 100 µL writing
isopropanol/HCl/H20 (90/1/9, v/v/v) and determining optical density
T.G., A.M.S.A., S.L.
Ø.S. - Biological testing and supervision, data analysis,
manuscript writing
- Biological testing, data analysis
at 540 nm. IC50 values were determined using the CompuSyn
software. Results are expressed as mean values of IC50 ± SD (µM) of
three independent experiments performed in triplicate.
P.R. - Synthesis supervision, project conception, data analysis
and manuscript writing
O.A.H.Å. - Synthesis and characterization of compounds,
supervision of synthetic work, project conception, data analysis,
manuscript composition and writing
The zinc chelators were dissolved in DMSO at concentrations ranging
-3
-4
from 1 to 10 mM, for compounds 14 and 50, and from 0.1 to 10
mM, for compounds 19 and 51. They were added to white 96-well
plates (maximum DMSO concentration in wells lower than 1%)
containing 20.000 HepG2 cells/well, obtained from ATCC. Plates
Notes and references
#
‡
See figure 1a. * See main text for explanation. IC
5
0
values reported in µM with standard deviation.
2
were incubated for 24 h at 37 °C in a 5% CO atmosphere. After 24 h,
AlamarBlue cell viability reagent (Thermo Fisher, Carlsbad, CA, USA)
was added as a 10% solution, and plates were placed back in the
incubator for 4 h. AlamarBlue is a red-ox indicator yielding a
fluorescence signal proportional to the number of viable cells in each
well. The fluorescence signal was measured in a microplate reader
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Metallkomplexbildung
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