4-amino-1,2,4-triazole-3-thione as a promising scaffold for the inhibition of serine and metallo-β-lactamases

Article abstract of DOI:10.3390/ph13030052

The emergence of bacteria that co-express serine-and metallo-carbapenemases is a threat to the efficacy of the available β-lactam antibiotic armamentarium. The 4-amino-1,2,4-triazole-3-thione scaffold has been selected as the starting chemical moiety in the design of a small library of β-Lactamase inhibitors (BLIs) with extended activity profiles. The synthesised compounds have been validated in vitro against class A serine β?Lactamase (SBLs) KPC-2 and class B1 metallo β?Lactamases (MBLs) VIM-1 and IMP-1. Of the synthesised derivatives, four compounds showed cross-class micromolar inhibition potency and therefore underwent in silico analyses to elucidate their binding mode within the catalytic pockets of serine-and metallo-BLs. Moreover, several members of the synthesised library have been evaluated, in combination with meropenem (MEM), against clinical strains that overexpress BLs for their ability to synergise carbapenems.

Full text of DOI:10.3390/ph13030052

pharmaceuticals
Article
4-Amino-1,2,4-triazole-3-thione as a Promising
Scaffold for the Inhibition of Serine and
Metallo-β-Lactamases
Pasquale Linciano 1, Eleonora Gianquinto 2, Martina Montanari 1, Lorenzo Maso 3
,
Pierangelo Bellio 4 , Esmeralda Cebrián-Sastre 5, Giuseppe Celenza 4, Jesús Blázquez 5,
Laura Cendron 3, Francesca Spyrakis 2,
*
and Donatella Tondi 1,
*
1
Department of Life Sciences, University of Modena and Reggio Emilia, Via G. Campi 103, 41125 Modena,
Italy; p.linciano@unimore.it (P.L.); 186334@studenti.unimore.it (M.M.)
2
3
4
5
Department of Drug Science and Technology, University of Turin, Via P. Giuria 9, 10125 Turin, Italy;
eleonora.gianquinto@unito.it
Department of Biology, University of Padua, Viale G. Colombo 3, 35121 Padua, Italy;
lorenzo.maso@unipd.it (L.M.); laura.cendron@unipd.it (L.C.)
Department of Biotechnological and Applied Clinical Sciences, University of L’Aquila, via Vetoio 1,
67100 L’Aquila, Italy; pierangelo.bellio@univaq.it (P.B.); giuseppe.celenza@univaq.it (G.C.)
National Center of Biotechnology-CSIC, Calle Darwin 3, 28049 Madrid, Spain;
esmeralda.cebrian@cnb.csic.es (E.C.-S.); blazquez@cnb.csic.es (J.B.)
*
Correspondence: francesca.spyrakis@unito.it (F.S.); donatella.tondi@unimore.it (D.T.)
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
Received: 11 February 2020; Accepted: 21 March 2020; Published: 24 March 2020
Abstract: The emergence of bacteria that co-express serine- and metallo- carbapenemases is a threat to
the efficacy of the available
scaffold has been selected as the starting chemical moiety in the design of a small library of
β
-lactam antibiotic armamentarium. The 4-amino-1,2,4-triazole-3-thione
-Lactamase
β
inhibitors (BLIs) with extended activity profiles. The synthesised compounds have been validated
in vitro against class A serine β−Lactamase (SBLs) KPC-2 and class B1 metallo β−Lactamases (MBLs)
VIM-1 and IMP-1. Of the synthesised derivatives, four compounds showed cross-class micromolar
inhibition potency and therefore underwent in silico analyses to elucidate their binding mode within
the catalytic pockets of serine- and metallo-BLs. Moreover, several members of the synthesised
library have been evaluated, in combination with meropenem (MEM), against clinical strains that
overexpress BLs for their ability to synergise carbapenems.
Keywords: 4-amino-1,2,4-triazole-3-thione; bacterial resistance; structure-based drug design;
non-covalent inhibition; thione/thiol tautomerism; broad-spectrum activity
1. Introduction
The spread of multidrug-resistant (MDR) Gram-negative bacteria is a recognised public-health
issue, compromising the efficacious treatment of bacterial infections, and becoming a leading cause of
death worldwide [
new targets, mechanisms and molecular entities have been addressed [
investment in antibiotic-drug development is necessary. At present,
commonly used antibiotics in bacterial infections worldwide. However, their misuse in humans and
animals exposes bacteria to a massive pressure, leading to the selection of MDR microorganisms [ ].
Among the several mechanisms that bacteria adopt to inactivate -lactam antibiotics, the over-expression
of the hydrolytic enzymes β-lactamases (BLs) remains the most relevant [6,7].
1
]. Although there has recently been a resurgence in antibiotic drug discovery and
], the need for continuous
-lactams remain the most
2–4
β
5
β
Pharmaceuticals 2020, 13, 52; doi:10.3390/ph13030052
www.mdpi.com/journal/pharmaceuticals

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According to Ambler’s classification,
β
-lactamases are divided into four classes, A, B, C and D [
8].
Classes A, C and D are serine-based enzymes (SBLs), whereas class B includes metallo-
(MBLs) [
around the world since its discovery, only a few years ago, becoming endemic in some countries [
β
-lactamases
6
]. KPC-2 (Klebsiella pneumoniae carbapenemase-2) is the most prevalent SBL and has spread
9
].
The clinical relevance of KPC-2 and its several variants relies on its capability to hydrolyse a broad
variety of β-lactams, including last-resort carbapenems [10].
Subclass B1 is the most clinically relevant of the MBLs subclasses, and includes the IMP-
(imipenemase) and VIM-types (Verona integrin encoded MBL), which are targets in our study, as
well as the NDM-type (New Delhi MBL) [11,12]. The MBL IMP variants have been reported with
increasing frequency since their first detection in a Pseudomonas aeruginosa isolate in Japan in 1990 [13].
VIM-1 was first identified in Italy a few years later, in 1997, in a P. aeruginosa strain. This was followed,
soon after, by the isolation of an allelic variant (VIM-2) in France. VIM-1 possesses the broadest range
of substrate hydrolysis and can degrade nearly all
and carbapenems [14], and thus represents the first cause for the failure of carbapenem treatment
in bacterial infections [15 16]. MBLs can hydrolyse all the classes of -lactams with the exception of
β-lactams, including cephamycins, oxacephamycins,
,
β
monobactams. In addition, the genes that encode MBLs are carried on plasmids, thus allowing them to
spread to other pathogens through horizontal gene transfer [17].
While several inhibitors for SBLs are currently available in therapy, no inhibitors for MBLs have
been approved so far, and only a few molecules are now in clinical trials [18
emphasize that, in clinical strains, BLs of several classes, SBLs as well as MBLs, are often coproduced,
thus expanding bacterial resistance to all available -lactams and leaving few therapeutic options
available [21]. Cross-class BL inhibitors (BLIs) that can synergise -lactams against several BLs would
–20]. It is important to
β
β
undoubtedly be beneficial in bacterial resistant infections and would help the rehabilitation of many
antibiotics that are now ineffective. However, despite its attractiveness, the design of cross-classes
BLIs is complicated by the structural and mechanistic differences that characterize SBLs and MBLs.
Their binding site architectures are different, challenging the design of broad-spectrum inhibitors.
In MBLs’ binding site two zinc ions, Zn1 and Zn2, are generally present and maintained in place
by an extensive coordination network. In the apo form of both VIM-1 and IMP-1, a catalytic water
(wat1) bridges Zn1 to Zn2, while another conserved water molecule (wat2) coordinates the Zn2 atom
(Figures S1 and S2). Conversely, in KPC-2, like in all SBLs, the process involves the catalytic Ser70,
which performs a nucleophilic attack on the
in the active site by an extensive network of interactions [22
β
-lactam core. The β−lactam substrate is further stabilized
–
24]. The first inhibitor active against both
SBLs and MBLs classes has, only recently, reached clinical trials [19,20].
Among metallo-BLIs (MBLIs) so far designed, bisthiazolidines, thiols, tetrazoles, sulphonamides,
succinic acids, hydroxamates, and boronic acids are recurrent moieties [11,25–29]. In particular,
sulfur-containing molecules that are able to interfere, in the binding site, with the hydrolytic water and
the Zn-ion network of interactions represent chemical entities that have been widely characterized as
MBLIs. Moreover, triazole-thio based compounds have frequently been identified in virtual screening
campaigns as micromolar inhibitors of NDM-1 [30,31], IMP-1 [32] and VIM-2 [33].
Interestingly, despite the effectiveness of this scaffold and the pressing need to strike SBLs and
MBLs simultaneously, there has been no concerted effort to extend the inhibitory profile of triazole-thiol
derivatives against other BL classes, i.e., class A carbapenemases, thus far.
With this in mind, we have selected the 4-amino-1,2,4-triazole-3-thione as the starting scaffold for
the design and synthesis of a small library of new possible MBLIs [30–35]. The 14 compounds in the
library were designed to extend their activity towards class A SBL KPC-2, while maintaining their
affinity vs MBLs.
The broad-spectrum profile of the obtained derivatives was validated in vitro against one SBL
representative, class A carbapenemases KPC-2, and against two representatives of the MBLs subclass
B1, VIM-1 and IMP-1. Moreover, several compounds in the synthesized library have been evaluated for
their ability to synergize β-lactams in biological tests against clinical strains overexpressing targeted BLs.

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2. Results and Discussion
2.1. Compounds Design and Synthesis
A library of 14 new compounds that share the 4-amino-2,4-dihydro-3H-1,2,4-triazole-3-thione
scaffold has been designed and synthesized. Our approach recognizes that, while the thione is
fundamental for binding to MBLs through the coordination of the Zn ions (the triazole-thio moiety is
common in several MBLs inhibitors [30–33]), the triazole itself may provide the KPC-2 binding site
with the necessary electron-donor requirements for its interactions with key residues such as Ser70,
Lys73, Ser130 and Asn132. Moreover, triazole is a bioisostere of the carboxylic group and is, thus,
able to make favourable interactions in the deepest part of the class A BL binding site [36–39].
The small series of amines (1a–g) and the corresponding imine (2a–g) derivatives, variously
decorated to reach additional active-site residues in SBLs and MBLs, were obtained according to
Scheme 1.
HN N
S
a
b
c
H2N NH2
H2N
NH2
S
N
N
N
H
H
NH2
4
3
a.
R = Ph-
HN N
HN N
d
b. R = 4-Br-Ph-
c.
d.
S
S
N
N
N
R = 2-NO2-Ph-
R = 4-(N,N-CH2CH3)Ph-
R
R
HN
e. R = 4-OH-3-CH3O-Ph-
f.
R = Benzodioxo-5-yl
g. R = Indol-3-yl
2a-g
1a-g
Scheme 1. Reagents and conditions: (a) CS2, H2O, refl., 3h, 50% yield; (b) formic acid (1 mL per gram
of ), refl., 45 min, 87% yield; (c) aryl carboxaldehyde (1.1 eq.), 35% HCl (cat.), EtOH, refl., 4–12h, yield:
4
76% (for 2a), 85% (for 2b), 67% (for 2c), 62% (for 2d), 87% (for 2e), 79% (for 2f), 70% (for 2g); (d) NaBH4
(8 eq.), 80% EtOH (v/v), 0 ◦C to r.t. 6–24h, yield: 90% (for 1a), 87% (for 1b), 75% (for 1c), 83% (for 1d),
75% (for 1e), 87% (for 1f), 81% (for 1g).
The 4-amino-2,4-dihydro-3H-1,2,4-triazole-3-thione (
reaction of aqueous hydroxylamine with carbon disulfide in refluxing water for 3 h to give
hydrazinecarbothiohydrazide ( ) first. This was followed by cyclization with formic acid under
reflux for 45 min. The condensation of with the appropriate aryl carboxaldehyde in refluxing ethanol
3) moiety was synthesized via the
4
3
in presence of hydrochloric acid, used as catalyst, gave the corresponding imines 2a–g in good yields.
Bidimensional NMR analysis (NOESY) confirmed an E-configuration at the imine double bond (see
Supporting Information). Finally, the reduction of the imine 2a–g with NaBH4 in 80% aqueous ethanol
for 6–24 h, led to the respective final amines 1a–g in good yields, and avoided the oxidation of thiol to
disulphide, as previously reported (Scheme 1) [40]. Both imines 2a–g and amines 1a–g were obtained
and isolated in the 1,2,4-triazole-3-thione tautomeric form as confirmed by 1H NMR spectroscopy.
Our compounds showed the characteristic proton signals for NH groups in the 13–14 ppm range, in
accordance with the spectroscopic thiol-thione tautomeric studies reported in the literature [41–44].
2.2. Compounds In-Vitro Validation vs. SBLs and MBLs
The compounds were screened in vitro for their inhibitory activity against carbapenemase MBLs
VIM-1 and IMP-1, and SBL KPC-2, using a spectrophotometric assay (Table S1). Compounds 1d, 1f, 2b
and 2g showed a weak, but promising broad-spectrum inhibitory activity against the three targeted
BLs (Table 1). The detected inhibitory activities against MBLs and SBLs define the compounds in the

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library as micromolar but, also, as able to target cross-class BLs, that are notably mechanistically and
structurally different. The obtained results clearly indicate that the candidates follow the intended
direction of designing broad-spectrum inhibitors.
Table 1. Binding affinity of the most promising broad-spectrum inhibitors against VIM-1, IMP-1
and KPC-2.
VIM-1
IMP-1
KPC-2
Code
Structure
Ki (µM) a
Ki (µM) a
Ki (µM) a
1d
224 ± 11
120 ± 4
173 ± 6
125 ± 5
263 ± 13
138 ± 3
1f
2b
2g
58 ± 2
95 ± 3
285 ± 13
104 ± 4
158 ± 7
139 ± 4
a
Estimated Ki as per competitive inhibitor [45]. All the experiments were performed in triplicate.
2.3. Molecular Docking and Molecular Dynamics
The four compounds were docked in the protein active site, providing valuable indications on
the orientation of thio-triazole candidates in carbapenemases (Figure 1). Even if the compounds were
isolated as thiones, we assumed, as detailed hereafter, that the presence of the zinc ions in MBLs binding
site favours the shifting of the tautomeric equilibrium towards the thiol form and the consequent
deprotonation, with the generation of a thiolate able to properly coordinate the Zn ions. Differently,
in SBLs the thione form was accounted as the most favourite.
2.3.1. MBLs
The binding pose of these scaffolds in MBL binding site is conserved. The zinc-chelating power
derives from their anionic nature: the delocalization of the negative charge between the 2-nitrogen and
the non-ring sulfur may indicate the prevalence of a thiolate, rather than a thione, as supported by
crystallographic evidence [40,41] and by recent works [34,46]. These observations led us to simulate the
compounds as thiolates when docked in the MBLs. All four compounds present a common orientation
in MBLs, with the triazole nitrogen in position 2 and the thiolate occupying the position of wat1 and
wat2, thus coordinating Zn1 and Zn2, respectively (Figure 1a–h). As mentioned previously, this motif
had already been observed in the crystallographic structures of L1 di-zinc MBL, in complex with
1,2,4-triazol-3-thiol (PDB code 2hb9 and 5dpx) [31,47]. Indeed, Zn1 is coordinated by one nitrogen,
while Zn2 is coordinated by the deprotonated sulfur atom in both structures (Figure S3). Other than
the coordination of Zn1 and Zn2, the interactions with the pocket are mainly hydrophobic and involve
residues located in the L3 loop or flanking the binding site, namely Phe62, Tyr67, Trp87 and His240 in
VIM-1, and Val25, Trp28, Phe51 and His197 in IMP-1. In particular, in VIM-1, compound 1d establishes
π-π interactions with Tyr67 and His240 through the aromatic substituent (Figure 1a). Compound 1f
maintains the same orientation (Figure 1b), as do 2b and 2g, which, however, orient the aromatic
substituents more towards Tyr67 than His240 (Figure 1c,d, respectively). In this pose, a substantial

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portion of the ligand points towards the solvent-exposed side of the hinge, allowing quite bulky
substituents to be accommodated. Interestingly, the most active compounds 2b and 2g both present an
imine double bond in the E configuration, which possibly provides the aromatic group with the proper
orientation to form closer π-π interactions with Tyr67.
In order to better investigate the binding pose and the binding path of the most promising
candidate, 2b, in VIM-1, we performed 15 Molecular Dynamics (MD) docking replicas (20 ns each).
Even though the MD docking shed light on the dynamic path the ligand experiences before binding
VIM-1, no relevant information was added to the rigid docking results: the width/openness of the
binding cavity offered straightforward access to 2b, and hydrophobic interactions with Tyr67 and
His240 were principally responsible for stabilising the ligand in the binding site. The MD docking in
VIM-1 therefore almost identically reflected the interactions and observations that had already been
reported in rigid docking studies, and hence corroborate the reliability of the static analyses in MBLs.
The most probable pose assumed by the ligands in IMP-1 binding site largely resembled the one
in VIM-1. Again, the zinc ions are coordinated by the thiolate and by the triazole nitrogen, and the rest
of the ligand forms
π-π interactions with Trp28, which replaces Tyr67 in VIM-1 (Figure 1e–h). The best
inhibition was obtained for compound 2g, which forms an almost perfect contact with Trp28.
Figure 1. Docking pose of the compounds in VIM-1, IMP-1 and KPC-2. (
and 2g in VIM-1 binding site, respectively. ( ) Compounds 1d 1f 2b and 2g in IMP-1 binding site,
respectively. ( ) Compounds 1d 1f 2b and 2g in KPC-2 binding site, respectively. The position
a–d) Compounds 1d, 1f, 2b
e
–h
,
,
i
–
l
,
,
originally occupied by the catalytic water wat1 in KPC-2 has been indicated. Even when the water
was not retained in the docking simulations, none of the compounds occupied its position, nor was it
able to generate valuable contact with it. Proteins are displayed as cartoon (VIM-1, magenta; IMP-1,
light blue; KPC-2 dark yellow), zinc atoms are solid grey spheres and their coordination bonds are
represented as grey dashed lines. The side chains of the relevant residues are shown as sticks and are
labelled accordingly; ligands are depicted in thick, bright-coloured sticks. Hydrogen bonds formed by
the ligand with the residues lining the pocket are shown as yellow dashed lines. The catalytic water in
KPC-2 is labelled and shown as a red sphere. For clarity, residues have been numbered according to
the PDB.

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2.3.2. KPC-2
Given the absence of the zinc ions, the compounds were modelled in the thione form when
docked in the KPC-2 binding site. In accordance with the architecture that this binding cavity presents,
the docking returned a ligand orientation in which the triazole moiety always sinks into the active site,
while the substituents introduced at position 4 points towards the opening of the binding site, which is
delimited by Trp105 (Figure 1i–l). Compound 1d, the least active, only forms a π-π interaction with
Trp105. On the other hand, compound 1f loses this contact with Trp105, but H-bonds to Asn132 through
the triazole nitrogens at positions 1 and 2, and to Thr235 and Thr237 by means of a benzodioxole oxygen.
Compounds 2b and 2g also show similar inhibition activity and a similar binding mode. They both
form good π-π interactions with Thr235; with that of 2g, possibly being stronger because of the larger
aromatic system. Compound 2b also H-bonds Asn132, similarly to 1f. In general, the similarities of the
poses satisfactorily explain the comparable inhibition activity of the four compounds. The H-bonds
formed by some of them with the residues lining the pocket open the way for the optimization of these
derivatives to provide them with substituents able to interact more extensively within KPC-2 active
site. Indeed, while the hydrophobic requirements of the binding site are well met by the compound
aromatic regions, the number of polar interactions should be increased to further improve the binding
affinity. In particular, compounds could be functionalized to contact Arg220 via a stronger electrostatic
interaction. Furthermore, polar substituents could be attached to the aromatic portion to better reach
the residues lining the oxyanion hole, that is Thr235 and Thr237.
2.4. Determination of Minimum Inhibitory Concentration (MIC) against Clinical Strains
To investigate the ability of the compounds to reach the periplasmic space, where BLs are secreted
and concentrated in Gram-negative bacteria, and to synergically protect β-lactam antibiotics from
BLs hydrolysis, the minimum inhibitory concentration (MIC) values were determined against clinical
strains overexpressing BLs targets of our studies (Table S2). Unfortunately, the obtained MIC showed
no synergistic effect for none of the tested compounds, except for 1a, 1c, 2c and 2g which very slightly
potentiated meropenem (MEM) activity.
It is important to consider that, in clinical strains, multiple mechanisms of resistance are co-present
and employed by bacteria, thus complicating the interpretation of the obtained results. At the same
time the necessity to validate designed compounds against their clinical bacterial targets is of critical
importance for the further effective selection of candidates for hit-to-lead optimization.
3. Conclusions
A library of 14 derivatives has been designed, synthesized and tested, both, in vitro and in
biological tests against MBLs and SBLs.
The most active compounds showed weak, but cross-class, inhibitory activity against all targeted
BLs, confirming the possibility to introduce broad-spectrum activity on recurrent moieties targeting
only one class. Starting from a scaffold that is widely recognised to bind to the MBLs active site,
extended affinity against SBLs was achieved, although only at micromolar potency. Molecular modeling
and dynamics experiments have been performed to analyse the binding orientations responsible for
the inhibitory activity of the most active compounds. The obtained results may serve as a guide in the
necessary hit-to-lead optimization of these derivatives, as the most opportune substitutions that can
lead to more extensive interactions within BLs active sites will be sought out.
So far, the broad-spectrum inhibitors that have been designed to be active against SBLs and MBLs
have mimicked the tetrahedral intermediates that are common to all BL classes [18–20].
This often implies that a covalent mechanism of inhibition is involved, at least for SBLs [26,48].
In our case, the developed molecules possess a micromolar affinity towards SBLs and MBLs and were
non-covalent inhibitors of SBLs. This was possible thanks to detailed 3D structural information, known
mechanisms of action and computational methodologies.

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The best molecules will now undergo X-ray crystallographic studies to support further hit-to-lead
optimisation processes.
4. Materials and Methods
4.1. Chemistry
All commercially available chemicals and solvents were reagent grade and were used without
further purification unless otherwise specified. Reactions were monitored by thin-layer chromatography
on silica gel plates (60F-254, E. Merck) and visualized with UV light, cerium ammonium sulphate or
alkaline KMnO4 aqueous solution. The following solvents and reagents have been abbreviated: ethyl
ether (Et2O), dimethyl sulfoxide (DMSO), ethyl acetate (EtOAc), dichloromethane (DCM), dimethyl
formamide (DMF), methanol (MeOH), and ethanol (EtOH). NMR spectra were recorded on a Bruker
1
400 spectrometer with H at 400.134 MHz and 13C at 100.62 MHz. Proton chemical shifts were
referenced to the solvent residue peak. Chemical shifts are reported in parts per million (ppm,
δ units).
Coupling constants are reported in Hertz (Hz). Splitting patterns are designed as s, singlet; d, doublet;
t, triplet; q quartet; dd, double doublet; m, multiplet; b, broad. Mass analysis was performed on
an Agilent 1200 series LC system coupled to an Agilent 6310A Ion Trap mass spectrometer (LC/MS)
(Agilent Technologies, Milan, Italy). Melting points were recorded on a Stuart, SMP3 (Barloworld
Scientific Limited Stone, Staffordshire, UK) and are uncorrected. Elemental analysis was performed on
a C,H,N,S CE Instruments EA 1110. All the compounds showed a level of purity above 95% by NMR,
1
ESI-MS analysis, melting point and elemental analysis. H and 13C NMR Spectra of all compounds are
reported in Supporting Information.
4.1.1. General Procedure for the Synthesis of Amines 1a–g
To a stirred solution of imine 2a–g (1 eq.) in 80% aqueous ethanol, NaBH4 (8 eq.) was added at
0 ◦C. The mixture was stirred at room temperature, in a close vessel, until disappearance of the starting
materials as stated by TLC (12–72 h). The solvent was removed under reduced pressure and the crude
triturated in acetate buffer (pH 5). The solid obtained was collected by filtration, dried and purified by
crystallization or column chromatography.
4.1.2. General Procedure for the Synthesis of Imines 2a–g
To a stirred suspension of 3 (1 eq.) in EtOH, the appropriate aryl carboxaldehyde (1.1 eq.) and a
drop of 37% aqueous HCl as catalyst were added. The suspension was refluxed until disappearance of
the starting materials as stated by TLC (4–12 h). The solution was cooled at room temperature and the
solvent partially concentrated to 1–2 mL. By standing overnight at 0 ◦C, a solid precipitate from the
mother solution. The solid was collected by filtration, rinsed with cold EtOH and dried to give the
pure desired product.
4.1.3. Spectroscopic Data
4-(benzylamino)-2,4-dihydro-3H-1,2,4-triazole-3-thione (1a)
Crystalized from Et2O. Pale yellow solid (90% yield). M.p. [138.8–140.2 ◦C]. 1H NMR (DMSO-d6)
δ
4.26 (d, J = 4.3 Hz, 2H), 6.00 (t, J = 4.5 Hz, 1H), 7.23–7.33 (m, 5H), 7.80 (s, 1H), 12.53 (bs, 1H). 13
C
NMR (DMSO-d6)
δ
55.70, 127.64, 128.03 (
×
2), 129.00 (
2), 136.76, 142.49, 160.25. ESI-MS m/z [M + H]+
×
Calcd. for C9H11N4S+: 207.0. Found: 207.1.
4-((4-bromobenzyl)amino)-2,4-dihydro-3H-1,2,4-triazole-3-thione (1b) ◦
1
Crystalized from Et2O. White solid (87% yield). M.p. [131.3–132.7 C]. H NMR (400 MHz,
DMSO-d6)
(s 1H). 13C NMR (DMSO-d6)
[M + H]+ Calcd. for C9H10BrN4S+: 285.0. Found: 285.1.
4-((2-nitrobenzyl)amino)-2,4-dihydro-3H-1,2,4-triazole-3-thione (1c)
δ
4.28 (s, 2H), 6.53 (bs, 1H), 7.29 (d, 2H, J = 8.0 Hz), 7.53 (d, 2H, J = 8.0 Hz), 8.17 (s, 1H), 13.70
δ
52.34, 121.21, 131.70 ( 2), 131.79 ( 2), 136.54, 142.92, 165.63. ESI-MS m/z
×
×

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Purified by column chromatography using DCM:Et2O 8:2 as mobile phase. Yellow solid (75%
yield). M.p. [140.1–141.2 ◦C]. 1H NMR (DMSO-d6)
4.62 (d, 2H, J = 4.57 Hz), 6.95 (t, 1H, J = 4.46 Hz),
7.58 (t, 1H, J = 8.0 Hz), 7.62 (d, 1H, J = 8.0 Hz), 7.69 (dt, 1H, J = 7.72, 1.0 Hz), 8.01 (dd, 1H, J = 8.0 Hz,
δ
1.0 Hz), 8.18 (s, 1H), 13.70 (bs, 1H). 13C NMR (DMSO-d6)
δ 50.23, 125.17, 129.64, 131.90, 132.04, 133.95,
142.43, 149.47, 165.66. ESI-MS m/z [M + H]+ Calcd. for C9H11N5O2S+: 252.0. Found: 252.1.
4-((4-(diethylamino)benzyl)amino)-2,4-dihydro-3H-1,2,4-triazole-3-thione (1d)
Crystalized from Et2O. Yellow solid (83% yield). M.p. [140 ◦C with dec.]. H NMR (DMSO-d6)
δ
1
1.14 (t, 6H, J = 8.0 Hz), 3.36–3.39 (m, 4H), 4.62 (d, 2H), 6.67 (d, 2H, J = 8.0 Hz), 6.94–6.97 (m, 3H), 7.18 (s,
1H), 13.69 (bs, 1H). 13C NMR (DMSO-d6)
δ
12.29 (
×
2), 45.30 (
×
2), 55.81, 115.65 (
×
2), 121.86 ( 2), 138.13,
×
142.47, 148.34, 160.22. ESI-MS m/z [M + H]+ Calcd. for C13H20N5S+: 278.1. Found: 278.2. Anal. Calcd
for C13H19N5S: C, 56.29; H, 6.90; N, 25.25; S, 11.56. Found: C, 56.32; H, 6.87; N, 25,20; S, 11.70.
4-((4-hydroxy-3-methoxybenzyl)amino)-2,4-dihydro-3H-1,2,4-triazole-3-thione (1e)
Purified by column chromatography using DCM:Et2O 8:2 as mobile phase. White solid (75%
yield). M.p. [131.1–132.3 ◦C]. 1H NMR (DMSO-d6)
δ
3.75 (s, 3H), 4.16 (s, 2H), 6.55 (bs, 1H), 6.63 (dd,
1H, J = 1.9, 8.0 Hz), 6.69 (d, 1H, J = 8.0 Hz), 6.88 (d, 1H, J = 1.9 Hz), 8.08 (d, 1H, J = 1.9 Hz), 8.94 (s, 1H),
13.67 (s, 1H). 13C NMR (DMSO-d6)
δ 53.07, 55.94, 113.33, 115.64, 122.00, 127.54, 142.80, 146.53, 147.98,
165.44. ESI-MS m/z [M + H]+ Calcd. for C10H13N5O2S+: 253.1. Found: 253.2.
4-((benzo[d][1,3]dioxol-5-ylmethyl)amino)-2,4-dihydro-3H-1,2,4-triazole-3-thione (1f)
Crystalized from Et2O. White solid (87% yield). M.p. [160.2–161.7 ◦C]. 1H NMR (DMSO-d6)
δ
4.18 (d, 2H, J = 4.2 Hz), 6.00 (s, 1H), 6.63 (t, 1H, J = 4.2 Hz), 6.70 (d, 1H, J = 7.8 Hz), 6.84 (d, 1H, J = 7.9
Hz), 6.92 (s, 1H), 8.14 (s, 1H), 13.67 (bs, 1H). 13C NMR (DMSO-d6)
δ 52.91, 101.40, 108.50, 109.67, 122.83,
130.71, 142.85, 147.17, 147.79, 165.50. ESI-MS m/z [M + H]+ Calcd. for C10H11N4O2S+: 251.0. Found:
251.1. Anal. Calcd for C10H10N4O2S: C, 47.99; H, 4.03; N, 22.39; S, 12.81. Found: C, 47.85; H, 4.10; N,
22.45; S, 12.77.
4-(((1H-indol-3-yl)methyl)amino)-2,4-dihydro-3H-1,2,4-triazole-3-thione (1g)
Crystalized from MeOH:Et2O. Brown solid (81% yield). M.p. [171.5–173.2 ◦C]. 1H NMR (DMSO-d6)
δ
4.53 (d, 2H, J = 4.2 Hz), 6.62 (t, 1H, J = 4.2 Hz), 7.01–7.05 (m, 3H), 7.40 (d, 1H, J = 8.0 Hz), 7.54 (d, 1H,
J = 8.0 Hz), 8.14 (s, 1H), 8.25 (s, 1H), 13.60 (bs, 1H). 13C NMR (DMSO-d6)
δ 49.79, 110.86, 112.91, 119.5,
119.80, 121.86, 123.59, 127.33, 136.35, 139.37, 167.21. ESI-MS m/z [M + H]+ Calcd. for C11H12N5S+:
246.1. Found: 246.2.
4-(benzylideneamino)-2,4-dihydro-3H-1,2,4-triazole-3-thione (2a)
White solid (76% yield). M.p. [175.3–176.7 ◦C], lit. [175–176 ◦C] [49]. 1H NMR (DMSO-d6)
δ:
7.56–7.63 (m, 3H), 7.86–7.90 (m, 2H), 8.95 (s, 1H), 9.47 (s, 1H), 13.95 (bs, 1H). 13C NMR (DMSO-d6)
δ
: 129.02 (
×
2), 129.65 (
2), 132.98, 136.62, 138.65, 156.84, 161.54. ESI-MS m/z [M + H]+ Calcd. for
×
C9H9N4S+: 205.1. Found: 205.2.
4-((4-bromobenzylidene)amino)-2,4-dihydro-3H-1,2,4-triazole-3-thione (2b)
1
Pale yellow solid (85% yield). H NMR (CDCl3)
δ
: 7.78–7.83 (m, 4H), 8.95 (s, 1H), 9.46 (s, 1H),
13.96 (s,1H). 13C NMR (CDCl3)
δ
: 128.78, 130.77 (
×
2), 132.30, 132.78 ( 2), 138.43, 159.97, 163.55. ESI-MS
×
m/z [M + H]+ Calcd. for C9H8BrN4S+: 283.0. Found: 283.1. Anal. Calcd for C9H7BrN4S: C, 38.18; H,
2.49; N, 19.79; S, 11.32. Found: C, 38.12; H, 2.55; N, 19.80; S, 11.30.
4-((2-nitrobenzylidene)amino)-2,4-dihydro-3H-1,2,4-triazole-3-thione (2c)
1
White solid (67% yield). M.p. [172.6–174.1 ◦C], lit. [175 ◦C] [50]. H NMR (DMSO-d6)
δ: 7.85 (t,
1H, J = 8.0 Hz), 7.94 (t, 1H, J = 8.0 Hz), 8.12 (d, 1H, J = 8.0 Hz), 8.21 (d, 1H, J = 8.0 Hz), 8.94 (s, 1H),
10.19 (s, 1H), 14.05 (bs, 1H). 13C NMR (DMSO-d6)
δ 125.46, 127.42, 129.64, 133.31, 134.75, 139.22, 149.15,
155.78, 163.44. ESI-MS m/z [M + H]+ Calcd. for C9H9N5O2S+: 250.0. Found: 250.1.
4-((4-(diethylamino)benzylidene)amino)-2,4-dihydro-3H-1,2,4-triazole-3-thione (2d)
Bright yellow solid (62% yield). M.p. [151.7–153.1 ◦C]. 1H NMR (DMSO-d6)
δ
1.14 (t, J = 8.0 Hz,
6H), 3.43 (q, J = 8.0 Hz, 4H), 6.78 (d, 2H, J = 8.0 Hz), 7.66 (d, 2H, J = 8.0 Hz), 8.80 (s, 1H), 9.05 (s, 1H),
13.80 (bs, 1H). 13C NMR (DMSO-d6)
12.85 ( 2), 44.34 ( 2), 111.51 ( 2), 118.39, 131.19, 139.01 ( 2),
151.09, 162.78, 163.06. ESI-MS m/z [M + H]+ Calcd. for C13H18N5S+: 276.1. Found: 276.2.
δ
×
×
×
×

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4-((4-hydroxy-3-methoxybenzylidene)amino)-2,4-dihydro-3H-1,2,4-triazole-3-thione (2e)
White solid (87% yield). H NMR (DMSO-d6)
δ 3.84 (s, 3H), 6.93 (d, J = 7.5 Hz, 1H), 7.30 (dd, 1H, J
= 8.3, 2.0 Hz), 7.44 (s, 1H), 8.84 (s, 1H), 9.26 (s, 1H), 10.02 (bs, 1H), 13.87 (bs, 1H). 13C NMR (DMSO-d6)
δ
56.09, 110.62, 116.12, 123.74, 124.99, 139.13, 148.61, 151.78, 162.84, 162.95. ESI-MS m/z [M + H]+ Calcd.
for C10H11N5O2S+: 251.1. Found: 251.2.
4-((benzo[d][1,3]dioxol-5-ylmethylene)amino)-2,4-dihydro-3H-1,2,4-triazole-3-thione (2f)
1
White solid (79% yield). H NMR (DMSO-d6)
δ
6.11 (s, 2H), 7.01 (d, J = 7.9 Hz, 1H), 7.20 (dd, 1H, J
= 8.12, 1.54 Hz), 7.34 (d, 1H, J = 1.54 Hz), 8.21 (s, 1H), 10.41 (s, 1H). 13C NMR (DMSO-d6)
δ 102.09,
105.57, 108.96, 124.77, 128.93, 140.26, 148.50, 150.11, 154.50, 161.55. ESI-MS m/z [M + H]+ Calcd. for
C10H9N4O2S+: 249.0. Found: 249.1.
4-(((1H-indol-3-yl)methylene)amino)-2,4-dihydro-3H-1,2,4-triazole-3-thione (2g)
1
White solid (70% yield). M.p. [251.3–252.8 ◦C], lit [250–253 ◦C] [51]. H NMR (DMSO-d6)
δ: 7.23
(t, 1H, J = 6.65 Hz), 7.28 (t, 1H, J = 7.39 Hz), 7.52 (d, 1H, J = 8.0 Hz), 8.09 (s, 1H), 8.25 (d, 1H, J =
8.0 Hz), 8.86 (s, 1H), 9.38 (s, 1H), 12.01 (bs, 1H) 13.80 (bs, 1H); 13C NMR (DMSO-d6)
δ 110.46, 112.78,
121.90, 122.59, 123.75, 124.64, 135.32, 137.76, 139.06, 159.23, 162.71. ESI-MS m/z [M + H]+ Calcd. for
C11H10N5S+: 244.1. Found: 244.2. Anal. Calcd for C11H9N5S: C, 54.31; H, 3.73; N, 28.79; S, 13.18.
Found: C, 54.25; H, 3.70; N, 28.83; S, 13.25.
4-amino-2,4-dihydro-3H-1,2,4-triazole-3-thione (3)
A stirred suspension of
4 (1 g, 9.4 mmol) in formic acid (1 mL per gram of 4) is refluxed for 45 min.
Thereafter, the suspension is filtered still hot and the filtrate is slowly cooled at room temperature
overnight. A grey solid separate from the solution. The solid is collected by filtration and re-crystalized
from ethanol to give 926 mg of pink crystals (85% yield). M.p. [165.2–166.7 ◦C], lit. [167–168 ◦C] [52].
1H NMR (400 MHz, DMSO-d6)
δ 5.70 (s, 2H), 8.45 (s, 1H), 13.65 (s, 1H). ESI-MS m/z [M + H]+ Calcd.
for C2H5N4S+: 117.0. Found: 117.1.
Hydrazinecarbothiohydrazide (4)
To a stirred solution of hydrazine hydrate (20 mL) in water (60 mL), carbon disulphide (6 mL) was
added dropwise. The mixture was refluxed for 3 h and then slowly chilled down at room temperature
overnight. A yellow solid separate from the solution which was collected by filtration and re-crystalized
from water to give 2.5 g of yellow-greenish needle crystals. The physical aspect and the melting point
of the crystal is in accordance with the literature. [53] ESI-MS m/z [M + H]+ calcd. for C7H7N4S+:
107.2. Found: 107.3.
4.2. Static Docking
Rigid docking studies were carried out with GOLD software version 5.5 (www.ccdc.cam.ac.uk),
using default parameters for the genetic algorithm. The CHEMPLP scoring function was then selected
to rank the best conformations of ligands in BLs. For each compound, a maximum of 30 poses was
generated in VIM-1, IMP-1 and KPC-2 binding sites, respectively. Protomers and tautomers were
calculated with MoKa [54] and chosen according to their relative abundance at pH 7.4: in MBLs,
the most abundant tautomeric form was the deprotonated, thiolate form, in agreement with reported
evidences [31,46,47,55–58]; in KPC-2 binding site, 1,2,4-triazole-3-thione was the preferred tautomeric
state. The binding site in KPC was centred on the hydroxyl atom of residue S70, with a radius of 7 Å.
For MBLs, the docking site comprised all residues distant no more than 12 Å from the Zn2 atom.
VIM-1. The available structures of VIM-1 co-crystallized with hydrolyzed meropenem was
selected as the target structure (PDB code 5n5i) [59]. The docking simulations were guided by the
recently solved crystallographic structure of one thio-triazole derivative complexed with VIM-2 (PDB
code 6tgi). The similarity of the protein binding site justifies the approach (Figure S1).
Accordingly, the coordination length was set in a 1.5 Å–1.9 Å range for triazole nitrogen and Zn1
and in a 1.5 Å–2.2 Å range for sulfur and Zn2. Solvent and hydrolyzed substrate were omitted.

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IMP-1. X-ray resolved structure of IMP-1 bound to an inhibitor was retrieved from the Protein
Data Bank (PDB code 1jjt) [60]. Waters, ligand and maximum separation settings were treated as
reported before for VIM-1.
KPC-2. X-ray resolved structure of apo-KPC-2 was retrieved from the Protein Data Bank (PDB
code 5ul8), all waters were omitted in docking calculations [23].
4.3. MD Docking
The most promising candidate 2b was submitted to a MD docking in VIM-1 (PDB code 5n5i),
to investigate which is the mechanism of binding and which are the residues involved in it. MD
docking was set up and performed following the original protocol developed by BiKi Technologies [61]:
two subsets (A and B) were defined, with A being the ligand and B the zinc atoms. 15 replicas of 20
ns each were set up, and for each run velocities were reassigned according to a Maxwell-Boltzmann
distribution at 300 K. An electrostatic bias was applied until subset A reached a distance of 4 Å from
subset B, then the bias was switched-off and the production was carried out as a plain MD until the
end of the simulation time.
4.4. Proteins Production and Purification
4.4.1. KPC-2
Expression and purification of recombinant KPC-2 have been performed as already reported [62].
4.4.2. VIM-1
The enzyme was purified from E. coli BL21 (DE3) transformed with the overexpre◦ssion vector
pet24a(+) carrying blaVIM-1 gene (pBPAQND2009). The bacteria were grown at 37 C in 1 L of
Terrific Broth (Biolife) supplemented with 50 µg/mL of kanamycin (Sigma-Aldrich) until the O.D.600
reached 0.6. The cells were further grown at 27 ◦C overnight. Afterwards the bacterial suspension
was harvested by centrifugation and washed 3 times with 30 mM sodium cacodylate buffer ◦(pH 6.3)
◦
(Sigma Aldrich). The dried pellet was subjected to 6 cycles of freeze (
−
80 C) / thaw (37 C) and
then resuspended in 115 mL (7 mL/g of cell pellet) of the same buffer supplemented with 100
ZnCI2. The cells were disrupted by sonication 10 times for 60 s at 80% power and 50% cycle (UP50H
sonicator) and centrifuged 105,000
g for 60 min at 4 ◦C. The clarified supernatant was loaded on a
High Q column (2.5 20 cm; GE Healthcare) equilibrated in 30 mM sodium cacodylate buffer (pH
6.3) supplemented with 100 M ZnCI2 (CBZ). The bound protein was eluted using a linear NaCI
gradient, (0 to 1 M) in CBZ at a flow rate of 3 mL/min. The fractions containing -lactamase activity
were dialyzed against 30 mM HEPES (pH 7.5) supplemented with 100 M ZnCI2 and concentrated
15-fold by Millipore Amicon 10000 kD centrifuge tubes. The concentrated fractions were loaded on a
Superdex 75 column (16 100 cm; GE Healthcare, Chicago, IL, USA) previously equilibrated with 30
mM HEPES (pH 7.5) (Sigma-Aldrich, St. Louis, MO, USA) supplemented with 100 M ZnCI2. The
eluted fractions containing VIM-1 -lactamase was concentrated to 9 mg/mL by centrifugal filtration
and stored at
80 ◦C. The protein concentration was determined by Bradford assay and purity by
µM
×
×
µ
β
µ
×
µ
β
−
sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).
4.4.3. IMP-1
The codon-optimized wild type blaIMP-1 gene of Pseudomonas aeruginosa was purchased by
Eurofins Genomics, and directly cloned into pETite N-His SUMO Kan Vector. Gene design and cloning
was carried out according to the kit protocol of Expresso® T7 SUMO Cloning and Expression System
(Lucigen, Middleton, WI, USA). The correct sequence of the resulting recombinant construct was
confirmed by Sanger DNA sequencing (Eurofins Genomics, Ebersberg Germany). Overexpression of
the N-His SUMO IMP-1 protein was performed transforming E. coli Lemo21(DE3) cells (NEB). 20 mL
of LB medium (25 mg/L kanamycin, 17 mg/L chloramphenicol) were inoculated with fresh colonies

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and grown at 37 ◦C overnight. The overnight culture was used to inoculate 1 L of LB medium (25 mg/L
kanamycin, 17 mg/L chloramphenicol) grown at 37 ◦C with shaking. The cells were grown till 0.5
O.D. was reached, induced with 0.2 mM Isopropyl-β-D-1thiogalactopyranoside (IPTG) and incubated
overnight at 25 ◦C. Cell pellet was resuspended in lysis buffer (20 mM Tris-HCl pH 7.5, 150 mM NaCl,
1 mM ZnCl2, 10% glycerol and 20 mM imidazole) supplemented with protease inhibitors cocktail
(Roche, Basel, Switzerland), and lysed by French press. The supernatant fractions were isolated from
cell debris by centrifugation and N-His SUMO IMP-1 was purified through IMAC (HisTrap HP 1
mL column, GE Healthcare). After an extensive washing with buffer A (20 mM Tris-HCl pH 7.5, 150
mM NaCl, 10% glycerol and 20 mM imidazole), the His-tagged protein was eluted with an increasing
gradient of buffer B (20 mM Tris-HCl pH 7.5, 150 mM NaCl, 10% glycerol and 0.5 M imidazole).
Fractions containing N-His SUMO IMP-1 were evaluated by SDS-PAGE, and buffer exchanged with
PD10 desalting columns (GE Healthcare) in buffer C (20 mM Tris-HCl pH 7.5, 150 mM NaCl and 10%
glycerol), to remove imidazole before digestion with SUMO protease. N-His SUMO removal was
performed through cleavage of the purified protein, added with 2 mM DTT, with SUMO express
Protease (Lucigen), for 2 h at 30 ◦C. Cleaved IMP-1 was isolated from N-His SUMO, SUMO express
Protease (His-tagged) and uncleaved N-His SUMO IMP-1 by IMAC. Most pure fractions of IMP-1
recovered from the flow through, as revealed by SDS-PAGE, were pooled together and concentrated by
centrifugal filters (Vivaspin® Turbo 4 10000 MWCO, Sartorius, Göttingen, Germany). A final step of
purification was performed by size exclusion chromatography (Superose12 10/300 GL, GE Healthcare)
in buffer 30 mM Hepes, 150 mM NaCl pH 7.1.
4.5. In Vitro Enzyme Inhibition Assays Against KPC-2, VIM-1 and IMP-1
The half-maximal inhibitory concentration (IC50) of synthesized derivatives was determined as
follows. Reactions were monitored using a Jasco V-730 spectrophotometer at 485 nm wavelength.
Compounds were dissolved in dimethyl sulfoxide (DMSO) to a concentration of 20 mM and stored at
−
20 ◦C. Each compound was tested at 5 different concentrations for inhibitory activity vs full-length
KPC-2 enzyme, VIM-1 and IMP-1. For class A carbapenemase KPC-2, tests were conducted in 50 mM
of PB + 50 mM KCl at pH 7.4 at 25 ◦C with 0.01% v/v Triton X-100, to avoid compound aggregation and
promiscuous inhibition [62
+ 100 mM NaCl and ZnSO4 10
proteins, reported substrate nitrocefin was used at 50
for VIM-1 and at 25 (Km 25 M) for IMP-1. All the experiments were performed in triplicate. The
,
63]. For MBLs VIM-1 and IMP-1 tests were conducted in 20mM of HEPES
M at pH 7.4 at 25 ◦C with 0.01% v/v Triton X-100. For all targeted
M, (Km 36 M) for KPC-2, at 25 (Km 22 µM)
µ
µ
µ
µM
µ
reaction was typically initiated by adding the enzyme to the reaction buffer last. The IC50 values were
determined by measuring the rate of hydrolysis of a reporter substrate in the presence of five different
inhibitor concentrations at
λ 482 nm. The binding affinity Ki was estimated from the determined IC50
by Cheng–Prusoff equation as per competitive inhibition (Table 1 and Table S1) [45]. For targeted
enzyme a known, in house broad spectrum inhibitor, was used as control [26,28].
4.6. MICs Assays
Susceptibility testing was performed in Mueller Hinton broth and interpreted following the
guidelines of the National Committee for Clinical Laboratory Standards [64]. To test the inhibitory
activity, the compounds were dissolved in DMSO and further dilutions were done using growth
medium. In all cases tests were performed at a final DMSO concentration below 5%, avoiding unwanted
DMSO inhibition effect on strains growth (blank test control with DMSO were performed). The MICs of
MEM alone and in association were determined against several relevant Gram-negative clinical isolates,
selected from the pathogen collection of the Spanish Network for Research in Infectious Pathology
(REIPI; Sevilla, Spain). The selection was based on strains capability of producing
interest for the present study. The MEM: compounds ratio was 1:1 (molar).
β-Lactamases of

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Supplementary Materials: The following are available online at http://www.mdpi.com/1424-8247/13/3/52/s1,
Figure S1: Catalytic site of VIM-1; Figure S2: Comparison of VIM-1 and IMP-1 binding site; Figure S3:
Crystallographic orientation of a 1,2,4-triazole-3-thiol compound in L1 MBL; Table S1: Enzymatic inhibitor activity
of compounds 1a-g and 2a-g against VIM-1, IMP-1 and KPC-2; Table S2: MICs of meropenem in combination with
compounds 1a-g and 2a-g; 1H and 13C NMR spectra of compounds 1a-g and 2a-g. (PDF).
Author Contributions: P.L. synthesized and characterized the compounds. M.M. validated the compounds
in vitro. E.G. and F.S. performed the in-silico analyses. P.B., G.C., L.M. and L.C. produced VIM-1 and IMP-1;
J.B. and E.C.-S. performed biological studies. D.T., P.L. and F.S. wrote the manuscript. D.T. and F.S. were responsible
for data curation. D.T. conceived and supervised the project. All authors contributed to the preparation and
editing of the manuscript. All authors have read and agreed to the published version of the manuscript.
Funding: This research was funded by Grant FAR2014 and FAR2017 (Finanziamento di Ateneo per la Ricerca)
from the University of Modena and Reggio Emilia to D.T. supporting independent research on carbapenemases
and bacterial resistance.
Acknowledgments: We kindly acknowledge the Centro di Competenza sul Calcolo Scientifico (C3S) at the
University of Turin (c3s.unito.it) for providing the computational time and resources, and BiKi Technologies
for providing the BiKi LiFe Sciences suite.We thank the CIGS (Centro Interdipartimentale Grandi Strumenti,
University of Modena and Reggio Emilia, UNIMORE).
Conflicts of Interest: The authors declare no conflict of interest.
Abbreviations
BLs:
β
-lactamases; BLI:
β
-lactamase inhibitor; EtOH: ethanol; IMP: imipenemase; KPC-2: Klebsiella pneumoniae
carbapenemase 2; MBL: metallo-
β
-lactamases, MBLI: metallo- -lactamase inhibitor; MD: molecular dynamic,
β
MDR: Multi drug resistant, MIC: minimum inhibitory concentration, MEM: meropenem, NOESY: Nuclear
Overhauser Effect Spectroscopy, SBLs: serine
Integrin-encoded MBL.
β-lactamases, TLC: thin layer chromatography, VIM: Verona
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