Polypyridine ligands as potential metallo-β-lactamase inhibitors

Article abstract of DOI:10.1016/j.jinorgbio.2020.111315

Bacteria have developed multiple resistance mechanisms against the most used antibiotics. In particular, zinc-dependent metallo-β-lactamase producing bacteria are a growing threat, and therapeutic options are limited. Zinc chelators have recently been investigated as metallo-β-lactamase inhibitors, as they are often able to restore carbapenem susceptibility. We synthesized polypyridyl ligands, N,N′-bis(2-pyridylmethyl)-ethylenediamine, N,N,N′-tris(2-pyridylmethyl)-ethylenediamine, N,N′-bis(2-pyridylmethyl)-ethylenediamine-N-acetic acid (N,N,N′-tris(2-pyridylmethyl)-ethylenediamine-N′-acetic acid, which can form zinc(II) complexes. We tested their ability to restore the antibiotic activity of meropenem against three clinical strains isolated from blood and metallo-β-lactamase producers (Klebsiella pneumoniae, Enterobacter cloacae, and Stenotrophomonas maltophilia). We functionalized N,N,N′-tris(2-pyridylmethyl)-ethylenediamine with D-alanyl-D-alanyl-D-alanine methyl ester with the aim to increase bacterial uptake. We observed synergistic activity of four polypyridyl ligands with meropenem against all tested isolates, while the combination N,N′-bis(2-pyridylmethyl)-ethylenediamine and meropenem was synergistic only against New Delhi and Verona integron-encoded metallo-β-lactamase-producing bacteria. All synergistic interactions restored the antimicrobial activity of meropenem, providing a significant decrease of minimal inhibitory concentration value (by 8- to 128-fold). We also studied toxicity of the ligands in two normal peripheral blood lymphocytes.

Full text of DOI:10.1016/j.jinorgbio.2020.111315

Journal of Inorganic Biochemistry 215 (2021) 111315
Contents lists available at ScienceDirect
Journal of Inorganic Biochemistry
journal homepage: www.elsevier.com/locate/jinorgbio
Polypyridine ligands as potential metallo-β-lactamase inhibitors
a
b
c
d
b
Luana La Piana , Valentina Viaggi , Luigi Principe , Stefano Di Bella , Francesco Luzzaro ,
e
e
a,f,*
Maurizio Viale , Nadia Bertola , Graziella Vecchio
a
Dipartimento di Scienze Chimiche, Universit a` degli Studi di Catania, V.le A. Doria 6, 95125 Catania, Italy
Clinical Microbiology and Virology Unit, A. Manzoni Hospital, Via dell’Eremo 9/11, 23900 Lecco, Italy
Clinical Pathology and Microbiology Unit, San Giovanni di Dio Hospital, Largo Bologna, 88900 Crotone, Italy
Clinical Department of Medical, Surgical and Health Sciences, Trieste University, strada di Fiume 447, 34149 Trieste, Italy
IRCCS Ospedale Policlinico San Martino, U.O. Bioterapie, L.go R. Benzi 10, 16132 Genova, Italy
b
c
d
e
f
Consorzio Interuniversitario di Ricerca in Chimica dei Metalli nei Sistemi Biologici (CIRCMSB), Piazza Umberto I 1, 70121 Bari, Italy
A R T I C L E I N F O
A B S T R A C T
Keywords:
Antibiotic
Bacteria
Meropenem
Pyridine
Synergy
Zinc
Bacteria have developed multiple resistance mechanisms against the most used antibiotics. In particular, zinc-
dependent metallo-β-lactamase producing bacteria are a growing threat, and therapeutic options are limited.
Zinc chelators have recently been investigated as metallo-β-lactamase inhibitors, as they are often able to restore
′
carbapenem susceptibility. We synthesized polypyridyl ligands, N,N -bis(2-pyridylmethyl)-ethylenediamine, N,
′
′
′
N,N -tris(2-pyridylmethyl)-ethylenediamine, N,N -bis(2-pyridylmethyl)-ethylenediamine-N-acetic acid (N,N,N -
′
tris(2-pyridylmethyl)-ethylenediamine-N -acetic acid, which can form zinc(II) complexes. We tested their ability
to restore the antibiotic activity of meropenem against three clinical strains isolated from blood and metallo-
β-lactamase producers (Klebsiella pneumoniae, Enterobacter cloacae, and Stenotrophomonas maltophilia). We func-
′
tionalized N,N,N -tris(2-pyridylmethyl)-ethylenediamine with D-alanyl-D-alanyl-D-alanine methyl ester with the
aim to increase bacterial uptake. We observed synergistic activity of four polypyridyl ligands with meropenem
′
against all tested isolates, while the combination N,N -bis(2-pyridylmethyl)-ethylenediamine and meropenem
was synergistic only against New Delhi and Verona integron-encoded metallo-β-lactamase-producing bacteria.
All synergistic interactions restored the antimicrobial activity of meropenem, providing a significant decrease of
minimal inhibitory concentration value (by 8- to 128-fold). We also studied toxicity of the ligands in two normal
peripheral blood lymphocytes.
1
. Introduction
Despite continuing progress in chemotherapeutics, antimicrobial
Bacteria have developed multiple resistance mechanisms against
β-lactams, mainly producing β-lactamase enzymes [3]. Metallo-β-lacta-
mases (MBLs)-producing Enterobacterales have increasingly been re-
ported worldwide with high mortality rates (> 30% for bacteremia) [4].
MBLs are zinc-dependent enzymes that catalyze the hydrolysis of almost
all β-lactams, including the carbapenems, which are commonly used as
the first choice treatment against multidrug-resistant (MDR) bacteria
resistance is an increasingly serious threat to public health. It has been
estimated that yearly deaths attributable to antimicrobial resistance will
rise from the current 700,000 to 10 million in 2050 [1]. The most used
antibiotics are β-lactams: penicillins, cephalosporins, carbapenems, and
monobactams [2].
[5]. The active-sites of MBLs contain one or two Zn2 ions. According to
+
′
′
Abbreviations: BLAST, Basic Local Alignment Search Tool; Bispicen, N,N -bis(2-pyridylmethyl)-ethylenediamine; BispicenA, N,N -bis(2-pyridylmethyl)-ethyl-
enediamine-N-acetic acid; CAMH, Cation-Adjusted Mueller-Hinton; CFU, Colony-Forming Unit; CLSI, Clinical and Laboratory Standards Institute; EDC, 1-ethyl-3 (3-
dimethylamino propyl) carbodiimide; EDTA, EthyleneDiamineTetraAcetic acid; EUCAST, European Committee on Antimicrobial Susceptibility Testing; FICI, Frac-
tional Inhibitory Concentration Index; HOBt, 1-Hydroxybenzotriazole; IC50, Concentrations inhibiting 50% proliferation; ICU, Intensive Care Unit; IMP, Imipene-
mase; IL2, Interleukin-2; MBL, Metallo-β-Lactamases; McF, McFarland; MEM, Meropenem; MHA, Mueller Hinton Agar; MIC, Minimum Inhibitory Concentration;
MTT, 3-(4 5-dimethylthiazol-2-yl)-2 5-diphenyltetrazolium bromide; NDM, New Delhi Metallo-β-lactamase; PBL, Peripheral Blood Lymphocytes; PHA, Phytohe-
′
′
′
′
moagglutinin; TPEN, N,N,N ,N -tetrakis (2- pyridymethyl)ethylenediamine; Trispicen, N,N,N -tris(2-pyridylmethyl)-ethylenediamine; TrispicenA, N,N,N -tris(2-pyr-
′
idylmethyl)-ethylenediamine-N -acetic acid; VIM, Verona integron-encoded metallo-β-lactamase.
*
Corresponding author at: Dipartimento di Scienze Chimiche, Universit a` degli Studi di Catania, V.le A. Doria 6, 95125 Catania, Italy.
E-mail address: gr.vecchio@unict.it (G. Vecchio).
https://doi.org/10.1016/j.jinorgbio.2020.111315
Received 17 June 2020; Received in revised form 16 November 2020; Accepted 16 November 2020
Available online 21 November 2020
0
162-0134/© 2020 Elsevier Inc. All rights reserved.

L. La Piana et al.
Journal of Inorganic Biochemistry 215 (2021) 111315
the primary structure, MBLs are classified into three subclasses (B1, B2,
and B3) [6] with different metal scaffolds [7,8]. The B1 subclass en-
zymes have emerged as the most clinically significant [9]. The main B1
MBLs are Verona integron-encoded metallo-β-lactamases (VIM), imipe-
nemase (IMP), and New Delhi MBL (NDM) types [5,10,11]. The Indian
subcontinent, the Balkan region, and the Middle East are the main
endemic areas for NDM enzymes [12]. However, this determinant is also
emerging in Africa, raising enormous concern due to considerable
migration flows [13]. Acquired MBLs are commonly encountered in
Enterobacteriaceae, Pseudomonas aeruginosa, and Acinetobacter baumannii
Based on the recent interest in zinc chelators, in this paper, we
synthesized polypyridyl multidentate ligands based on the ethylenedi-
′
amine backbone. Specifically, we synthesized N,N -bis(2-pyr-
′
idylmethyl)-ethylenediamine (Bispicen), N,N,N -tris(2-pyridylmethyl)-
′
ethylenediamine (Trispicen), N,N,N -tris(2-pyridylmethyl)-ethylenedi-
′
′
amine-N -acetic acid (TrispicenA), N,N -bis(2-Pyridylmethyl)-ethyl-
enediamine-N-acetic acid (BispicenA) to study their ability to restore
meropenem activity (Fig. 1) against three clinical MBL (VIM-1, NDM-1
and L1) -producing strains isolated from blood. They were compared
to TPEN. The ligands studied can form Zn complexes with the stability
constants ranging from logKZnL = 11.4 (L = Bispicen) to logKML = 15.6
(L = TPEN) [40–43]. The modulation of stability constants is important
for the selection of the ligand, as very strong ligands may indiscrimin-
ately bind to the metal of essential metalloproteins in the host cells [44].
Moreover, COOH groups could improve the interaction with the
active site of MBL, as found for other ligands containing carboxylate
groups [18,45]. TrispicenA was also functionalized with the D-Ala-D-
Ala-D-AlaOCH3 peptide with the aim to improve selectivity for bacteria.
In bacteria, D-amino acids are found within peptidoglycan. Recently, it
has been found that D-Ala derivatives can be incorporated into bacteria,
one reason D-amino acid-derivatives have been tested as a high-value
target for antibiotics [46,47].
[
14]. The mobile nature of acquired MBLs is of critical concern for public
health and the horizontal transfer of MBL genes among different bac-
terial species through mobile genetic elements (mostly transposons and
plasmids) promotes the global spread of these resistance determinants.
Notably, other bacterial species can intrinsically produce MBLs.
A strategy for combatting the increasing resistance mediated by
MBLs is the identification of MBL resistant β-lactams or MBL inhibitors,
thus protecting the β-lactam drugs from hydrolysis [3,15,16]. MBL in-
hibitors could act as an antibiotic adjuvant, restoring β-lactam antimi-
crobial activity.
The number of MBL inhibitors have hugely increased over the past
five years, with more than 900 inhibitors studied [17]. Recently an
interactive website on MBL inhibitors has also been developed with the
aim to facilitate the discovery of new, improved inhibitors [17]. The
search for MBL inhibitors is now crucial for public health [5,18], and no
MBL inhibitor has thus far been clinically approved.
2. Experimental section
2.1. Materials
Zinc chelators have been studied as one type of MBL inhibitor
[
3,14,15,18]. Although chelators have been used for diagnostic pur-
1-Hydroxybenzotriazole (HOBt), 1-ethyl-3 (3-dimethylamino pro-
pyl) carbodiimide (EDC), pyridine-2-carboxaldehyde and TPEN were
purchased from TCI (TOKYO CHEMICAL INDUSTRY CO., Tokyo), D-Ala-
D-Ala-D-Ala was purchased from Bachem (Tokyo, Japan) and modified
poses and chelation therapy [19–21], they have never been included in
any anti-MBL commercially available pharmacologic formulations.
Chelators have not been used thus far in the clinical setting for two
reasons: 1) infections due to MBL-producing bacteria are a relatively
recent clinical problem, and 2) the development of a single inhibitor to
neutralize MBLs has been deemed too technically challenging in part
due to the difficulty in overcoming in vivo toxicity associated with cross-
reactivity with human metalloenzymes [22]. Given this, provided their
activity, efficacy, and tolerability are confirmed in preclinical and
clinical studies, therapies including MBL inhibitors may become a
reasonable alternative to classical therapeutic approaches for infections
of MBL producers, especially when robust safety data become available.
Zn ligands can act as zinc “strippers” from MBL enzymes, or they can
form a ternary complex, usually displacing a hydroxide ion shared by
in methyl-ester (D-Ala-D-Ala-D-AlaOCH ). Bispicen, Trispicen, Bispi-
3
cenA, and TrispicenA have been synthesized as reported elsewhere
[48–52].
′
2.1.1. Synthesis of N,N -Bis(2-pyridylmethyl)-ethylenediamine (Bispicen)
Ethylenediamine (0.60 g, 10 mmol) was added to pyridine-2-
carboxaldehyde (2.14 g, 20 mmol) in 20 mL of methanol. The solution
was refluxed for 5 h, cooled at r.t. and NaBH (1.52 g, 40 mmol, 10 mL of
4
methanol) was added. The solution was refluxed overnight. The solvent
was evaporated, and the product was extracted from the residue with
CH Cl (150 mL × 3). The extract was dried over MgSO . After evapo-
2
2
4
2
+
two Zn ions at the MBL catalytic site [11,23–26]. Many zinc ligands
such as ethylenediaminetetraacetic acid (EDTA), 1,4,7-triazacyclono-
nane-1,4,7-triacetic acid, 1,4,7,10-tetraazacyclododecane-1,4,7,10-tet-
ration of the solvent, Bispicen (1.99 g) was obtained as an oil and was
freeze-dried. Yield 82%.
TLC: Rf = 0.72 (PrOH/AcOEt/H 0/NH 5:2:3:1).
2
3
′
′
1
raacetic acid, dithiocarbamates, N,N,N ,N -tetrakis (2-pyridymethyl)
ethylenediamine (TPEN), trispyridyl-amine, picolinic acid, dithiolo-
pyrrolones, peptide aspergillomarasmine A, disulfiram, hydroxyquino-
lines, and other ligands have been proven to inhibit MBLs [24–33]. More
often, the ligands have been studied on B1 MBLs.
H NMR (500 MHz, D O) δ: 8.19 (d, J = 4.6 Hz, 2H, H-6 Py), 7.58 (t,
2
J = 7.7 Hz, 2H, H-4 Py), 7.15 (d, J = 7.9 Hz, 2H, H-3 Py), 7.09 (dd, J =
5.3 Hz, J = 7.5 Hz 2H, H-5 Py), 3.57 (s, 4H, CH Py), 2.49 (s, 4H,
2
+
+
CH NH). ESI-MS: m/z = 243.1 [M + H] , 265.2 [M + Na] .
2
′
Recently some chelators have also been studied in vivo [34,35].
2.1.2. Synthesis of N,N,N -Tris(2-pyridylmethyl)-ethylenediamine
′
Zincophore ethylenediamine-N, N -disuccinic acid improved survival of
(Trispicen)
Klebsiella pneumoniae infected Galleria mellonella larvae when adminis-
trated in combination with imipenem, compared to imipenem mono-
therapy [36]. 1,10-phenanthroline and TPEN improved survival of
Aspergillus fumigatus infected mice alone and in combination with cas-
pofungin [34]. Recently, we tested TPEN and nitroxoline in vivo and
found that TPEN can restore meropenem activity in infected
G. mellonella larvae [28].
Pyridine-2-carboxaldehyde (0.66 g, 6.15 mmol) was added to Bis-
picen (1.49 g, 6.15 mmol) in diethyl ether (20 mL) and the solution was
stirred at r.t. overnight with CaCl2 protection. The white precipitate
formed was filtered and washed with diethyl ether. It was dissolved in
10 mL of CH OH and NaBH CN (0.096 g, 1.54 mmol) and 1 mL of
3
3
3
CF COOH were added. The solution was stirred at room temperature
with CaCl protection. After 8 h, NaOH (water solution 15%, 65 mL) was
2
Recently, even if the role of zinc complexation has not been high-
lighted, a combination of EDTA as a metal chelator and sulbactam (an
MBL inhibitor) has been approved as antibiotic adjuvants of β-lactam
ceftriaxone for the treatment of multiresistant septicemia [37,38].
Furthermore, some authors have suggested that endogenous zinc che-
lators in combination with conventional antibiotics could be used to
treat infections caused by MBL-expressing pathogens [39].
added and the solution stirred overnight. The solvent was evaporated
and the solid was extracted with CH Cl (150 mL × 3). Trispicen (0.44 g)
2
2
was obtained as a yellow oil and was freeze-dried. Yield 85%.
TLC: Rf = 0.63 (PrOH/AcOEt/H 0/NH 5:2:3:1).
2
3
1
H NMR (500 MHz, D O) δ: 8.19 (d, J = 5.0 Hz, 1H, H-6’ Py), 8.15 (d,
2
J = 5.0 Hz, 2H, H-6 Py), 7.58 (t, J = 7.7 Hz, 1H, H-4’ Py), 7.52 (t, J = 7.7
′
Hz, 2H, H-4 Py), 7.16 (d, J = 7.8 Hz, 2H, H-3 Py), 7.09 (m, 4H, H-3 , H-5
2

L. La Piana et al.
Journal of Inorganic Biochemistry 215 (2021) 111315
Fig. 1. Polypyridine ligands investigated.
′
and H-5 of Py), 3.44 (s, 6H, CH
2
Py), 2.44 (t, J = 6.1 Hz, 2H, CH
2
N), 2.33
8.0 Hz, 1H, H-4 Py B), 7.81 (d, J = 8.0 Hz, 1H, H-3 Py B), 7.72 (t, J = 8.0
Hz, 1H, H-5 of Py), 7.63 (dd, J = 15.0, 7.6 Hz, 2H, H-5 py and H-3 Py B),
4.65 (s, 2H, CH2Py), 4.23 (s, 2H, CH2Py), 3.53 (s, 2H, CH2CO), 3.43 (t,
+
(
t, J = 6.1 Hz, 2H, CH
2
N). ESI-MS: m/z = 334.2 [M + H] , 356.2 [M +
+
Na] .
J = 5.5 Hz, 2H, CH2N), 3.13 (t, J = 5.5 Hz, 2H, CH
2
N). ESI-MS: m/z =
′
+
+
2
.1.3. Synthesis of N,N -bis([2-Pyridylmethyl)-ethylenediamine-N-acetic
acid (BispicenA)
Tert-Butyl bromoacetate (0.42 g, 2.15 mmol) was added to Bispicen
0.52 g, 2.15 mmol) and K CO (0.50 mg, 3.6 mmol) in 5 mL of CH CN.
The solution was refluxed overnight under stirring. K CO was filtered
and washed with CH CN. The solvent was evaporated under vacuum
301.2 [M + H] , 323.2 [M + Na] .
′
′
2.1.4. Synthesis of N,N,N -tris([2-Pyridylmethyl)-ethylenediamine-N -
acetic acid (TrispicenA)
(
2
3
3
2
3
Trispicen (1.96 g, 5.88 mmol) was alkylated with tert-
3
2 3
Butylbromoacetate (1.15 g, 5.88 mmol) and K CO in 5 mL of acetoni-
and the residue obtained was purified by flash-chromatography (Silica
trile. The reaction mixture was purified with flash-chromatography
column); Ethyl acetate/methanol 0 → 60% was used as the eluent.
(Silica Column, eluent ethyl acetate/methanol 0 → 60%). The butyl
1
H NMR (500 MHz, D2O) δ 8.29 (d, J = 4.9 Hz, 1H, H-6 py), 8.20 (d,
3
ester was hydrolyzed with CF COOH (2 mL), under stirring. After 12 h,
J = 4.8 Hz, 1H, H-6 py B), 7.67 (t, J = 7.7 Hz, 1H, H-4), 7.62 (t, J = 7.7
Hz, 1H, H-4 py B), 7.27 (d, J = 7.8 Hz, 1H, H-3 Py B), 7.22 (d, J = 8.0 Hz,
the solvent was evaporated, and the product was purified by Sephadex
ꢀ
DEAE A-25 (HCO
3
form) column using water as the eluent. The final
1
H, H-3 Py), 7.19 (m, 1H, H-5), 7.15 (m, 1H, H-5 py B), 3.72 (s, 2H,
product was freeze-dried.
Yield: 0.450 g, 20%.
CH2Py), 3.63 (s, 2H, CH2Py B), 3.15 (s, 2H, CH2CO), 2.68 (t, J = 5.5 Hz,
H, CH2N), 2.55 (t, J = 5.5 Hz, 2H, CH NH), 1.23 (s, 9H, t-but). B ring is
pyridine bound to the alkylated amino group.
2
2
TLC: Rf = 0.62 (PrOH/AcOEt/H
2 3
0/NH 5:2:3:1).
1
H NMR (500 MHz, D
2
O) δ 8.10 (d, J = 5.0 Hz, 2H, H-6 of Py), 7.83
1
3
′
C NMR (125 MHz, D
56.0 (C-2 of Py), 146.5 (C-6 of Py), 146.1 (C-6 of Py B), δ 135.8 (C-4 of
Py), δ 135.7 (C-4 Py B), δ 121.3 (C-3 py B), 120.38 (C-3, C-5, C-5 B of Py),
1.3 (C(CH3)), 57.6 (CH Py), 54.2 (CH COOBut), 50.6 (CH N), 50.2
CH of Py), 42.5 (CH NH), 25.6 (CH ).
The butyl ester was hydrolyzed with CF
2
O) δ 172.8 (COObut); 157.4 (C-2 of Py B),
(d, J = 4.9 Hz, 1H, H-6 of Py), 7.50–7.40 (m, 3H, H-4 and H-4 Py), 7.15
′
1
(d, J = 7.9 Hz, 2H, H-3 of Py), 7.05 (d, J = 7.8 Hz, 1H, H-3 of Py), 7.02
′
(dd, J = 6.9, 5.6 Hz, 2H, H-5 of Py), 6.99 (dd, J = 7.5, 5.1 Hz, 1H, H-5 of
8
2
2
2
Py), 3.95 (s, 2H, CH
2
Py B), 3.24 (d, J = 11.1 Hz, 4H, CH
2
Py), 3.12 (s, 2H,
(
2
2
3
CH
2
COOH), 3.08 (t, J = 5.7 Hz, 2H, CH
N).
2
CH
2
N), 2.62 (t, J = 5.7 Hz, 2H,
3
COOH (2 mL), under stir-
NCH
2
3
1
ring. After 12 h, the solvent was evaporated, and the product was pu-
C NMR (125 MHz, D
2
O) δ 170.0 (COOH); 156.4 (C-2 of Py), δ 149.2
ꢀ
′
′
rified by Sephadex DEAE A-25 (HCO
3
form) column using water as the
(C-2 of Py), 148.6 (C-6 Py B), 147.9 (C-6 Py), 138.1 (C-4 of Py), 138.0
′
′
eluent. The final product was freeze-dried. Yield: 0.13 g, 20%.
(C-4 of Py), 124.2 (C-3 and C-5 of Py), δ 123.7 (C-3 of Py), 123.1 (C-5 of
TLC: Rf = 0.85 (PrOH/AcOEt/H20/NH3 5:2:3:1).
Py), δ 58.5 (CH
46.8 (CH
2
Py), 57.5 (CH
2
PyB), 55.0 (CH
2
COOH), 49.9 (NCH2CO),
1
+
+
H NMR (500 MHz, D2O) δ 8.48 (d, J = 5.7 Hz, 1H, H-6 Py), 8.36 (d,
2
N). ESI-MS: m/z = 392.2 [M + H] , 414.2 [M + Na] .
J = 5.9 Hz, 1H, H-6 Py B), 8.27 (t, J = 7.9 Hz, 1H, H-4 Py), 8.22 (t, J =
3

L. La Piana et al.
Journal of Inorganic Biochemistry 215 (2021) 111315
2
.1.5. Synthesis of D-Ala-D-Ala-D-AlaOCH
3
cultures. K. pneumoniae isolate (LC954/14) was isolated from blood in
2014 and has been previously described [13]. It expressed an acquired
NDM-1 determinant, belonged to ST11, and represented the first
described NDM-producing K. pneumoniae imported from Africa to Italy.
S. maltophilia (LC669/17) was isolated from blood in 2017 and
expressed the chromosomally encoded L1 carbapenemase. Both isolates
have previously been used to evaluate the in vitro activity of the chela-
tors, including TPEN in restoring carbapenem activity against MBL
producers [28]. E. cloacae (this work) was isolated from blood in 2019
and expressed an acquired VIM carbapenemase. VIM carbapenemase
was identified at group level (VIM-type), by the GeneXpert® System
using the Xpert Carba-R test (Cepheid,Sunnyvale, CA), and then detec-
ted as VIM-1 by conventional PCR and sequencing In brief, the blaVIM
gene was amplified by PCR using primers and conditions already
described [53]. Sequencing was performed using the Dye Terminator
DNA sequencing kit V1.1 (Applied BiosystemsTM) followed by purifi-
cation using the DyeEx 2.0 Spin Kit (Qiagen, Hilden, Germany). The
sequences obtained were corrected and analyzed using the 4Peaks
program and then exported in FASTA format. Sequence alignments were
performed by using BLAST (Basic Local Alignment Search Tool) and
UniProt programs. The bioinformatic program Swiss-Model ExPASy was
used to obtain the Protein Data Bank (PDB) format of VIM protein from
the FASTA format.
D-Ala-D-Ala-D-AlaOCH
3
was synthesized from tripeptide D-Ala-D-
◦
Ala-D-Ala (100 mg, 0.43 mmol) in 2 mL of CH
3
OH at 0 C. 1 mL of acetyl
chloride was added under stirring. After 4 h, the solvent was evaporated
and the product was dissolved in water and purified with chromatog-
raphy (DEAE Sephadex A-25). 1H NMR spectroscopy confirmed the
presence of the OCH
MHz, D
3
group on the newly-formed ester. 1H NMR (500
O) δ: 4.25 (q, J = 7.3 Hz, 1H, CH); 4.14 (q, J = 7.2 Hz, 1H, CH);
.59 (s, 3H, OCH
2
3
3
); 3.42 (q, J = 6.9 Hz, 1H, CH); 1.26 (d, J = 7.5 Hz, 3H,
CH
3
); 1.24 (d, J = 7.4 Hz, 3H, CH
3
); 1.14 (dd, J = 13.9, 6.7 Hz, 3H, CH
3
).
′
′
2
.1.6. Synthesis of N,N,N -tris([2-Pyridylmethyl)-ethylenediamine-N -
acetyl-D-alanyl-D-alanyl-D-alanine methyl ester (TrispicenDAla)
HOBt (93 mg, 6.2 mmol) and EDC (117 mg, 6.2 mmol) was added to
TrispicenA (159 mg, 4.1 mmol) in DMF under stirring. After 15 min, D-
Ala-D-Ala-D-AlaOCH
3
(100 mg, 4.1 mmol, in DMF) and Triethylamine
(
0,057 mL, 4.1 mmol) were added. The solution was stirred at r.t.. After
1
2 h, the reaction mixture was purified by flash-chromatography using
C18 column and a linear gradient of water/acetonitrile. The solid
an R
P
obtained was purified again in the same condition. The final product (20
mg) was freeze-dried.
TLC: Rf = 0.70 (PrOH/AcOEt/H
2 3
0/NH 5:2:3:1).
1
H NMR (500 MHz, D
2
O) δ 8.28 (d, J = 4.8 Hz, 2H, H-6 of Py), δ 7.66
′
(
q, J = 6.6 Hz, 3H, H-4 and H-6 of Py), δ 7.30 (d, J = 7.8 Hz, 2H, H-3 of
Bacterial identifications have been performed by MALDI-TOF (Vitek
MS, bioMerieux, Marcy l’Etoile, France), while antimicrobial suscepti-
bility tests have been carried out by broth microdilution method using a
dedicated TREK panel (DKMGN, Thermo Fisher Diagnostics, Milan,
Italy). Antibiotic susceptibility profiles were interpreted according to
the European Committee on Antimicrobial Susceptibility Testing
(EUCAST) v.7.1 clinical breakpoints. Minimum inhibitory concentration
(MIC) values of meropenem (MEM) were further confirmed by standard
broth microdilution (CLSI), using Cation-Adjusted Mueller-Hinton
(CAMH, Thermo Fisher Diagnostics) broth.
′
′
′
Py), δ 7.22 (m, 5H, H-4 , H-3 , H-5 and H-5 of Py), δ 4.24 (q, J = 7.2 Hz,
H, CH Ala), δ 4.13 (q, 7.1 Hz, 1H, CH Ala); 4.11 (q, J = 7.2 Hz, 1H, CH
Ala), δ 3.74 (s, 2H, CH CO), δ 3.66 (s, 4H, CH Py), δ 3.63 (s, 3H, OCH ),
δ 3.60 (s, 2H, CH Py), δ 2.55 (m, 2H, CH CH N), δ 2.52 (m, 2H,
CH CH
of D-Ala), δ 1.24 (d, J = 7.2
Hz, 3H, CH of D-Ala).
1
2
2
3
2
2
2
2
2
N), δ 1.28 (d, J = 7.3 Hz, 3H, CH
3
3
of D-Ala), δ 1.18 (d, J = 7.1 Hz, 3H, CH
3
1
3
C NMR (125 MHz, D
2
O) δ 174.4 (COOH), 157.0 (C-2 of Py), 148.2
′
′
(
C-2 of Py), 137.9 (C-4 and C-6 of py), δ 138.1 (C-6 of Py), δ 124.4 (C-3-
′
′
of Py), δ 124.1 (C-5 of Py), δ 123.1 (C-4’,C-3 , and C-5 ), δ 60.4 (CH
δ 59.9 (CH
2
CO),
N), δ
Ala), δ 16.3
2 3 2 2 2
Py), δ 52.8 (OCH ), δ 51.9 (CH CH N), δ 51.1(NCH
4
8.6 (CH Ala), δ 48.8 (CH Ala), δ 49.2 (CH Ala), δ 17.1 (CH
3
2.3.2. Antimicrobial activity related to zinc chelators
(
CH
3
Ala), δ 15.8 (CH
3
Ala).
Antimicrobial activity of six zinc chelators (TPEN, TrispicenDAla,
TrispicenA, BispicenA, Trispicen and Bispicen) against the bacterial
isolates have been investigated by standard broth microdilution (Clin-
ical and Laboratory Standards Institute, CLSI) using Cation-Adjusted
Mueller-Hinton (CAMH) broth to obtain MIC values.
+
+
ESI-MS: m/z = 619.4 [M + H] , 641.3 [M + Na] .
Elemental Analysis for C32
42 8 5
H N O : calc C, 62.12; H, 6.84; N, 18.11;
found C 61.92; H, 6.86; N, 18.05.
2
.2. Instrumentation
1H and 13C NMR spectra were recorded at 25 C with a Varian UNITY
2
.3.3. Synergy between meropenem and zinc ligands
◦
Interactions were determined by a preliminary qualitative double-
PLUS-500 spectrometer at 499.9 and 125 MHz, respectively. NMR
spectra were obtained by using standard pulse programs from the Varian
library. The 2D experiments (COSY, TOCSY, HSQCAD, HMBC, NOESY)
were acquired by using 1000 data points, 256 increments, and a relax-
ation delay of 1.2 s. The spectra were referred to the solvent signal.
ESI mass spectra were acquired with an API 2000– ABSciex
spectrometer.
disk diffusion test, and by subsequent quantitative checkerboard.
The double-disk diffusion method was performed with disks placed
at 15 and 20 mm center-to-center for each combination chelator-
meropenem, as previously described [28]. Standard 0.5 McF (McFar-
land) inoculums for each isolate were plated on Mueller Hinton agar
(MHA, bioMerieux). Ten l of a concentrated solution of each chelator
μ
and MEM (1024 mg/L) were placed on blank disks on MHA. Synergy
was defined as the presence of alterations in disk’s inhibition rings.
Positive antibiotic/chelator interactions in preliminary screening were
then investigated by the checkerboard assay, as previously described
[28]. The range of drug concentration used in the checkerboard analysis
was such that the dilution range encompassed the MIC for each drug
used in the analysis. Broth microdilution plates were inoculated with
Flash chromatography was carried out with a CombiFlash Auto-
mated Flash Chromatography System (Teledyne ISCO).
2
2
.3. In vitro microbiological experiments
.3.1. Bacterial isolates
6
We studied three MBL-producing clinical isolates (with acquired or
each bacterial isolate to yield ~10 CFU (Colony-Forming Unit)/mL in a
◦
chromosomally-encoded MBLs) responsible for invasive infections in
patients hospitalized at the “Alessandro Manzoni” Hospital (Lecco,
Italy). Bacterial isolates were selected on the basis of their origin
100-
μ
l final volume and incubated for 18 h at 37 C. Synergy has been
defined as requiring a fourfold reduction in the MIC of both antibiotics in
combination, compared with each used alone, measuring the fractional
inhibitory concentration index (FICI). The FICI was calculated for each
(
invasive, Intensive care unit (ICU) patients), resistance to carbapenems,
typology of carbapenemases (chromosomally encoded or acquired
MBLs), and availability of very few therapeutic alternatives. Bacterial
isolates were as follows NDM-producing Klebsiella pneumoniae (LC954/
combination using the following formula: FICI = FIC
FIC
A
+ FIC
B
, where
A
= MIC of drug A in combination/MIC of drug A alone, and FIC =
B
MIC of drug B in combination/MIC of drug B alone. The FICI was
interpreted as follows: synergy, FICI ≤0.5; indifference, 0.5 < FICI ≤4;
antagonism, FICI >4 [54]. All measurements were performed in
1
4), L1-producing Stenotrophomonas maltophilia (LC669/17) and VIM-
producing Enterobacter cloacae (LC1341/19) isolated from blood
4

L. La Piana et al.
Journal of Inorganic Biochemistry 215 (2021) 111315
triplicate assays for each method.
Trispicen, BispicenA and TrispicenA (Table 1) as reported elsewhere
[
57]. The calculation of logKML values was based on donor group
2
.3.4. Antiproliferative activity by 3-(4,5-dimethylthiazol-2-yl)-2,5-
additivity used successfully elsewhere [58]. In Table 1, we report the
calculated logKML for TPEN and Bispicen that are very similar to the
experimental values. The calculated constant of Trispicen is the same as
that found for the Trispicen methyl derivative (log KZnL = 13.3) [59].
We studied the complexation reaction of TrispicenA and Trispi-
diphenyltetrazolium bromide (MTT) assay
The antiproliferative effect of compounds TPEN Trispicen, Trispi-
cenA, Bispicen, BispicenA e TrispicenDala was examined on two normal
peripheral blood lymphocytes (PBL A and B) that were stimulated with
cenDAla with Zn2 by H NMR. Similar studies have already been re-
+
1
1
% phytohemoagglutinin (PHA) plus 100 U/mL Interleukin-2 (IL-2) IL-2
2
+
for a week. Stimulated PBLs were then exposed to compounds for 72 h.
Once plated in 96-well round-bottomed microtiter plates at 10000 cells/
well PBLs were centrifuged at 1100 rpm for 2 min. and compounds
added after 6–8 h. All compounds were administered in five concen-
trations (1:10 dilution ratio starting from 100 uM concentration). Each
ported for Bispicen [40], Trispicen [42], and TPEN [60]. Zn
coordination with pyridine and amino nitrogen atoms has been pro-
posed [61–63].
A solution of TrispicenA in D
2
O at pH = 7.4 was titrated with a ZnCl
2
solution (Fig. 2). When zinc was added, new signals representing a Zn
complex species appeared in the spectra together with the signals of the
free ligand (Fig. 2). In the aromatic region, new signals appeared at 8.74,
well finally contained 200
described elsewhere [55].
μ
L. The MTT test was performed after 72 h as
8
3
.08, 7.99, and 7.81 ppm. In the aliphatic region, new signals at 4.02,
.90, 1.10, and 1.02 ppm appeared. Spectra changed up to a Zn/L ratio
3
. Results and discussion
=
1. 2D experiments were conducted on the solution of [Zn
TrispicenA)] species (Fig. S14-S18) and the full H and 13C NMR
+
1
(
3
.1. Synthesis and characterization
spectra assignment was obtained (Table S1). The complexation reduced
conformational freedom of the ligand. In the spectrum, twelve signals
due to the protons of the pyridine rings appeared. The complexation
produced a shift of the signals, and all methylene protons appeared at
different chemical shifts because of the coordination of the metal.
Similarly, in the 13C NMR spectrum (Fig. S14), pyridine rings showed 15
signals, in addition to 6 signals due to methylene carbons and CO at 178
ppm.
Bispicen and Trispicen have been synthesized through a reductive
amination reaction, as reported elsewhere [48–50]. They were alkylated
using tert-butyl bromoacetate [48,51].
NMR spectra confirmed the identity of Bispicen, Trispicen, and their
1
alkylated derivatives (Fig. S1- S9). In the H NMR spectrum of Bispi-
cenA, the protons of the two pyridine rings show different chemical
shifts for the presence of the COOH group. The typical pattern of pyri-
dine derivatives can be seen for each pyridine. Furthermore, the protons
of the ethylene chain appeared as two triplets at 3.43 ppm and 3.13 ppm.
The three singlets at 3.53 ppm, 4.23 ppm, and 4.65 ppm are due to the
methylene protons.
+
NOESY spectra of [Zn(TrispicenA)] (Fig. S18) showed the correla-
tions among the protons of the different pyridine rings in the zinc
complex.
Spectra suggested the complexation of Zn2 with the pyridine and
+
ꢀ
2+
In the 1H NMR spectrum of TrispicenA, in addition to the pyridine
protons in the aromatic region, the characteristic signals of the ethylene
chain appear as two triplets. Three different singlets appeared due to
methylene protons linked to the pyridine rings. In the 1 C NMR spectrum
of TrispicenA at 170 ppm, the CO signal appeared (Fig. S8).
We functionalized TrispicenA with the tripeptide D-Ala-D-Ala-D-
ethylenediamine nitrogens and COO with Zn in an octahedral envi-
ronment, as found in the solid-state [63].
2 2
TripicenDAla was also titrated with ZnCl in D O. The shift of the
3
signals of the D-Ala moiety suggested the involvement of the chain in the
coordination of Zn2 . In this case, the spectra showed broad signals,
+
probably due to the presence of an equilibrium between complex species
(
Fig. S19). The spectra changed up to Zn/L = 1. In addition to the co-
3
AlaOCH , to improve its permeability in the bacterial cell. The func-
ordination of pyridine and amine groups, the coordination of amide
bonds may be suggested as found for zinc with peptides [33]. A stability
constant value of Zn2 complex can be estimated higher than Trispicen
tionalization of TrispicenA was achieved through a condensation reac-
tion in DMF using HOBt and EDC as condensing agents. The identity of
the product was confirmed by NMR spectra (Fig. S10-S13).
+
1
and similar to TrispicenA.
In the H NMR spectra (Fig. S10), the signals in the aromatic region
Competition experiments with TPEN were performed by 1H NMR
can be assigned to the pyridine protons. The CH protons of the three D-
2
+
spectroscopy (Fig. 3). TPEN is a commonly used Zn chelator in cells
Ala residues resonate at 4.24 ppm and 4.13 ppm while protons of CH
3
[
64]. Preliminarily, NMR spectra of [Zn(TPEN)]2 complexes were ac-
+
resonate at 1.28 ppm, 1.24 ppm, and 1.18 ppm as doublets. The protons
of the ethylene chain resonate as multiplets at 2.55 ppm and 2.52 ppm.
The signals of the methyl ester resonate at 3.63 ppm, while other
methylene protons resonate at 3.74 ppm, 3.66 ppm, and 3.60 ppm. The
quired at different Zn/TPEN molar ratios. When zinc was added, new
signals of the zinc complex appeared in the spectra. Four signals
appeared at 8.13, 7.87, 7.38 and 7.34 ppm, while the signals of the
ligand protons disappeared. The two doublets at 4.26 ppm and 4.01 ppm
due to the zinc complex protons also appeared (Fig. 3e).
1
3
C NMR spectrum of the TrispicenDAla was also assigned by HSQC and
HMBC spectra (Fig. S12, S13).
+
TPEN was added to the [Zn(TrispicenA)] solution. NMR signals due
+
+
to [Zn(TrispicenA)] did not change, suggesting that [Zn(TrispicenA)]
did not lose Zn2 when small amounts of TPEN were added. We found
+
3
.2. Zn complexes
The stability constants of the ligands with Zn2 have been reported
2
+
that NMR spectra showed the formation of the Zn(TPEN) complex
when the Zn/TPEN/TrispicenA ratio was 2/1/2 two hours after TPEN
+
+
for TPEN and Bispicen [41,56]. We calculated the stability constants of
addition. The signals of [Zn(TrispicenA)] decreased but did not
disappear in the spectra when Zn/TPEN/TrispicenA were 1/1/1 (Fig. 3).
NMR data indicated that TrispicenA bonded zinc ion with a stability
constant similar to that of TPEN. NMR data were used to compare the
stability of the two complexes.
Table 1
Stability constants (log KZnL) for complexes with TPEN, Bispicen, BispicenA,
Trispicen and TrispicenA.
TPEN
56]
Bispicen
[41]
BispicenA
Trispicen
TrispicenA
Considering the reaction
[
+
2+
[
Zn(TrispicenA) ] + TPEN = [Zn(TPEN) ] + TrispicenAꢀ
Log KML
exp
15.7
15.5
11.4
10.9
/
/
K = [ZnTPEN][TrispicenA] [ZnTrispicenA][TPEN] = KZnTPEN
K
ZnTrispicenA
Log KML
calc
13.3
13.3
15.6
5

L. La Piana et al.
Journal of Inorganic Biochemistry 215 (2021) 111315
1
2
Fig. 2. H NMR spectra in D O (pD 7.4): a) TrispicenA alone; b) TrispicenA/Zn 1:0.3; c) TrispicenA/Zn 1:0.5; d) TrispicenA/Zn 1:1.
1
+
+
+
+
Fig. 3. H NMR spectra in D
2
O (pD 7.4): a) [Zn(TrispicenA)] alone; b) [Zn(TrispicenA)] /TPEN 3:1; c) [Zn(TrispicenA)] /TPEN 2:1; d) [Zn(TrispicenA)] /TPEN
2
+
1
:1. e) [Zn(TPEN)]
.
[
ZnTPEN]/[TPEN] and [TrispicenA]/[ZnTrispicen] can be deter-
Table 2
mined from the integration of the signals in the NMR spectra. From the
spectra K ZnTPEN/K ZnTrispicenA ≈ 1.3. This data is consistent with the
calculated stability constant value for Zn-TrispicenA.
Concentrations ( M) of TPEN, Trispicen, TrispicenA, Bispicen, BispicenA, and
μ
TrispicenDala inhibiting 50% proliferation (IC50) of normal PBL stimulated with
PHA and IL-2.
Ligand
PBL A
PBL B
3
.3. Toxicity in human cells
TPEN
5.08 ± 0.23
>85
5.54 ± 0.41
>100
TrispicenDAla
Trispicen
TrispicenA
Bispicen
4.91 ± 0.12
5.17 ± 0.41
13.9 ± 1.7
>100
5.39 ± 0.21
5.39 ± 0.20
17.8 ± 4.1
>100
All the ligands were tested for their cytotoxicity in two normal pe-
ripheral blood lymphocytes. IC50 values are reported in Table 2. Four of
compounds showed a pharmacologically significant antiproliferative
activity with IC50 ranging from 4.91 ± 0.12 M to 17.8 ± 4.1 M. TPEN
toxicity values has been reported in cancer cells and the zinc chelation
6
BispicenA
μ
μ
The values express the mean ± SD of 4 data.
6

L. La Piana et al.
Journal of Inorganic Biochemistry 215 (2021) 111315
was proposed as the prevailing cytotoxicity mechanism [65–67]. Bis-
picen showed lower toxicity than Trispicen and TrispicenA.
BispicenA and TrispicenDala did not show cytotoxicity on stimulated
PBLs (the value chosen to arbitrarily define an efficient in vitro anti-
proliferative activity was 30
μ
M). Interestingly the functionalization
with the peptide D-Ala-D-Ala-D-AlaOCH
3
reduced the antiproliferative
activity of the ligand compared to TPEN or TrispicenA. The peptide
residue may modify the lipophilicity of the ligand and reduce its toxicity
as recently reported for similar derivatives [68].
3
3
.4. In vitro microbiological results
.4.1. Evaluation of antibiotic MIC values and antimicrobial activity of zinc
ligands
Bacterial isolates were resistant to several antibiotics, demonstrating
a multidrug-resistant profile. They showed high MIC values for the
majority of tested antimicrobials (Table S2). As expected, all isolates
were resistant to carbapenems (ertapenem, imipenem, and mer-
openem). Low MIC values were also observed for trimethoprim/sulfa-
methoxazole in S. maltophilia, for colistin in NDM-producing
K. pneumoniae, and amikacin in VIM-1 producing E. cloacae
(
Table S2). MIC values were high for MEM in NDM-producing
K. pneumoniae (128 mg/L) and S. maltophilia (256 mg/L), while in
VIM-1 producing E. cloacae the MIC value was 16 mg/L (Table 3).
Standard broth microdilution of polypyridine ligands showed high
MIC values of TPEN and TrispicenDAla (256 mg/L and > 256 mg/L,
respectively) for all strains studied, confirming the absence of antimi-
crobial activity against these strains. High MIC values were also
observed for TrispicenA (512 mg/L) and BispicenA (>512 mg/L) in all
strains, Trispicen (256 mg/L) and Bispicen (64 mg/L) in NDM-producing
K. pneumoniae, and VIM-producing E. cloacae. In S. maltophilia, Trispicen
and Bispicen MIC values were 64 mg/L and 32 mg/L, respectively
(
Table 3). Notably, these compounds showed higher antimicrobial ac-
tivity than MEM (256 mg/L) alone against S. maltophilia.
3
.4.2. Synergy tests
Double-disk diffusion tests showed alterations in disk inhibition rings
for combinations of TPEN-MEM and TrispicenA-MEM against all tested
isolates (Fig. 4). No alteration was observed for combinations of
BispicenA-MEM. For the combination of TrispicenDAla-MEM, synergy
was observed only in VIM-producing E. cloacae. Alterations in disk in-
hibition rings were observed for the combination of Trispicen-MEM and
Bispicen-MEM in NDM-producing K. pneumoniae and VIM-producing
E. cloacae. Synergistic activities between chelators and MEM against
VIM-producing E. cloacae are shown in Fig. 4.
Fig. 4. Synergistic activity evaluated by double-disk diffusion test between
MEM and A) TPEN, B) TrispicenDAla, C) TrispicenA, D) BispicenA, E) Trispicen,
F) Bispicen, against VIM-producing E. cloacae (15 and 20 mm center-center).
Arrows indicate alterations of the inhibition zone around the discs.
negative cell wall, enter the periplasm where the MBLs are located [23],
and inhibit MBL activity.
MIC values of MEM in the presence of the ligands (at approximately
Checkerboard analysis (Table 4) confirmed the presence of syner-
gistic interactions demonstrated by double-disk diffusion tests and
confirmed the absence of interaction for BispicenA-MEM in all strains
and Bispicen-MEM in S. maltophilia. Moreover, the analysis also showed
3
0 μM) decreased compared to MEM alone. In particular, several syn-
ergistic interactions restored the antimicrobial activity of MEM,
providing a decrease of MIC values 8- to 128-fold, meaningfully below
the susceptibility breakpoint (2 mg/L). Particularly, Trispicen and
TrispicenA were the most effective ligands, providing, at 16–32 mg/L, a
MIC value for MEM of 0.25 mg/L compared to 32 mg/L of MEM alone in
E. cloacae; Trispicen and TrispicenA also showed very low FICIs (<0.1).
Unlike some data reported elsewhere for other classes of ligands
the presence of synergistic interactions for
a combination of
TrispicenDAla-MEM in S. maltophilia and NDM-producing K. pneumoniae
and Trispicen-MEM in S. maltophilia.
Overall, we found a synergistic activity between MEM and Trispi-
cenDAla, TrispicenA, Trispicen, and TPEN against all tested isolates. In
contrast, the combination of Bispicen-MEM was synergistic only against
NDM-producing K. pneumoniae and VIM-producing E. cloacae.
The synergistic activity suggests that the ligands can cross the Gram-
[
45], we did not find any strong evidence of the advantage of the
carboxylate groups.
Bispicen did not show any synergistic activity in L1-producing
Table 3
Antimicrobial activity of Meropenem (MEM), TPEN, Bispicen, BispicenA, TrispicenDAla, Trispicen and TrispicenA.
Microorganisms
MIC values (mg/L)
MEM
TPEN
TrispicenDAla
TrispicenA
BispicenA
Trispicen
Bispicen
K. pneumoniae NDM-1
S. maltophilia L1
128
256
16
256
256
256
>256
>256
>256
512
512
512
>512
>512
>512
256
64
64
32
64
E. cloacae VIM-1
256
7

L. La Piana et al.
Journal of Inorganic Biochemistry 215 (2021) 111315
Table 4
Synergistic interaction for TPEN, Bispicen, BispicenA, Trispicen, TrispicenDAla and TrispicenA with Meropenem (MEM) determined by FICI versus NDM-1 producing
K. pneumoniae, L1-producing S. maltophilia and VIM-1 producing E. cloacae clinical strains.
Checkerboard microdilution assays and synergistic concentrations
Compound: TPEN
MEM in combination
Compound: TrispicenDAla
Microorganisms
Compound concentration
(mg/ L)
FICI
Microorganisms
MEM in combination
(mg/L)
Compound concentration
FICI
(
mg/L)
(mg/ L)
K. pneumoniae
32
16
16
16
32
32
32
32
16
32
32
32
32
32
16
16
0.31
0.18
0.25
0.18
0.15
0.14
0.31
0.25
0.18
0.15
0.14
0.13
0.31
0.18
K. pneumoniae
32
16
8
32
32
32
32
32
0.31
0.18
0.12
0.09
0.07
(
NDM-1)
(NDM-1)
1
6
8
4
4
2
2
S. maltophilia (L1)
E. cloacae (VIM-1)
64
S. maltophilia (L1)
64
32
64
0.37
0.37
3
1
2
6
128
8
4
2
4
2
E. cloacae (VIM-1)
4
2
32
32
0.31
0.18
Compound: TrispicenA
Compound: Trispicen
Microorganisms
MEM in combination
mg/L)
Compound concentration
FICI
Microorganisms
MEM in combination
(mg/L)
Compound concentration
(mg/ L)
FICI
(
(mg/ L)
K. pneumoniae
32
16
8
16
16
16
16
16
32
32
32
64
64
128
128
128
16
16
16
16
32
32
0.28
0.16
0.09
0.06
0.05
0.31
0.19
0.12
0.19
0.16
0.28
0.26
0.26
0.28
0.16
0.09
0.06
0.09
0.08
K. pneumoniae
32
16
8
16
16
16
16
16
16
16
0.31
0.19
0.12
0.09
0.07
0.50
0.37
(
NDM-1)
(NDM-1)
4
4
2
2
S. maltophilia (L1)
64
S. maltophilia (L1)
64
32
3
1
1
2
6
6
8
8
4
2
4
2
1
E. cloacae (VIM-1)
E. cloacae (VIM-1)
4
2
8
8
0.28
0.16
0.09
0.12
0.09
0.08
1
8
0
.5
.5
1
16
16
16
0
0.5
0.25
0
.25
Compound: Bispicen
MEM in combination
Compound: BispicenA
Microorganisms
Compound concentration
(mg/ L)
FICI
Microorganisms
MEM in combination
(mg/L)
Compound concentration
(mg/ L)
FICI
NI
(
mg/L)
K. pneumoniae
32
16
8
16
16
16
16
16
–
16
16
16
16
16
0.50
0.37
0.31
0.28
0.26
NI
K. pneumoniae
–
–
(
NDM-1)
(NDM-1)
4
2
S. maltophilia (L1)
E. cloacae (VIM-1)
–
4
S. maltophilia (L1)
E. cloacae (VIM-1)
–
–
–
–
NI
NI
0.5
2
0.37
0.31
0.28
0.26
1
0
.5
0
.25
NI = no synergistic interaction, FICI = FIC
A
+ FIC
B
, where FIC
A
= MIC of drug A in combination/MIC of drug A alone, and FIC = MIC of drug B in combination/MIC of
B
drug B alone.
S. maltophilia, which has a relatively impermeable cell membrane [69],
which potentially explains the lower effect of TrispicenA on
S. maltophilia and the lack of synergistic activity of Bispicen. Further-
more, efflux mechanisms may not be excluded. The lack of synergy can
also be related to the different MBL enzyme of S. maltophilia. Notably, L1
has a higher affinity for zinc of other MBLs, and it is also active as a Zn1
enzyme [8]. Indeed, Bispicen is the ligand with the lowest stability
constant among the tested ligands.
dependence of the synergistic effect of the ligand on the stability con-
stant of the zinc complex. Bispicen with logKZnL = 11.4 and Trispicen
with logKZnL = 13.3 are effective in restoring MEM activity in these
bacteria.
The moiety D-Ala-D-Ala-D-AlaOCH
improve the synergistic activity in the studied bacteria in comparison to
trispicenA. TrispicenDAla showed similar effect of TPEN in
3
in TrispicenDAla did not
a
K. pneumoniae and a slightly less effect in other clinical strains. Inter-
In VIM-1 and NDM-1 producing bacteria, we did not observe a strong
estingly, TrispicenDAla was not toxic in human cells and this data can be
8

L. La Piana et al.
Journal of Inorganic Biochemistry 215 (2021) 111315
important for the selection of the ligand to study in vivo experiments.
Microbiological data suggest that zinc polypyridine ligands are po-
tential candidates as adjuvants in antibiotic therapy with meropenem,
for infections caused by MBL producers. Nevertheless, cytotoxicity of the
compounds suggest that a further investigation is needed on these class
of ligands in vivo models for the selection of the best ligands. However,
the functionalization of the ligands is a promising strategy to modulate
the toxicity as reported for other ligands. Although the cytotoxic of
TPEN and other similar ligands is known, they have been used in vivo as
potential therapeutics. TPEN has been administrated as an antiasthma
agent and an antifungal agent in vivo [34,70] and to protect against
botulinum neurotoxin in mice [71]. Bispicen has also been evaluated in
vivo for its ability to form zinc complexes as a potential inhibitor of p53-
dependent apoptotic pathways [72]. Cytotoxicity of a drug may not
necessarily be a limitation to the clinical application. However, risk/
benefit should be carefully evaluated especially for life-threatening
disease and in vivo studied are important to highlight the potential of
polypyridyl ligands.
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