Amoxicillin Inactivation by Thiol-Catalyzed Cyclization Reduces Protein Haptenation and Antibacterial Potency

Article abstract of DOI:10.3389/fphar.2020.00189

Serum and cellular proteins are targets for the formation of adducts with the β-lactam antibiotic amoxicillin. This process could be important for the development of adverse, and in particular, allergic reactions to this antibiotic. In studies exploring protein haptenation by amoxicillin, we observed that reducing agents influenced the extent of amoxicillin-protein adducts formation. Consequently, we show that several thiol-containing compounds, including dithiothreitol, N-acetyl-L-cysteine, and glutathione, perform a nucleophilic attack on the amoxicillin molecule that is followed by an internal rearrangement leading to amoxicillin diketopiperazine, a known amoxicillin metabolite with residual activity. Increased diketopiperazine conversion is also observed with human serum albumin but not with L-cysteine, which mainly forms the amoxicilloyl amide. The effect of thiols is catalytic and can render complete amoxicillin conversion. Interestingly, this process is dependent on the presence of an amino group in the antibiotic lateral chain, as in amoxicillin and ampicillin. Furthermore, it does not occur for other β-lactam antibiotics, including cefaclor or benzylpenicillin. Biological consequences of thiol-mediated amoxicillin transformation are exemplified by a reduced bacteriostatic action and a lower capacity of thiol-treated amoxicillin to form protein adducts. Finally, modulation of the intracellular redox status through inhibition of glutathione synthesis influenced the extent of amoxicillin adduct formation with cellular proteins. These results open novel perspectives for the understanding of amoxicillin metabolism and actions, including the formation of adducts involved in allergic reactions.

Full text of DOI:10.3389/fphar.2020.00189

ORIGINAL RESEARCH
published: 04 March 2020
doi: 10.3389/fphar.2020.00189
Amoxicillin Inactivation by Thiol-
Catalyzed Cyclization Reduces
Protein Haptenation and Antibacterial
Potency
María A. Pajares1, Tahl Zimmerman1†, Francisco J. Sánchez-Gómez1, Adriana Ariza2,3
,
Edited by:
Sabina Passamonti,
María J. Torres2,3,4, Miguel Blanca5, F. Javier Cañada1, María I. Montañez2,3
*
and Dolores Pérez-Sala1
*
University of Trieste, Italy
Reviewed by:
Xiaoli Meng,
1 Department of Structural and Chemical Biology, Centro de Investigaciones Biológicas (CSIC), Madrid, Spain, 2 Allergy
Research Group, Instituto de Investigación Biomédica de Málaga-IBIMA, Hospital Civil, Málaga, Spain, 3 Nanostructures for
University of Liverpool,
United Kingdom
Diagnosing and Treatment of Allergic Diseases Laboratory, Andalusian Center for Nanomedicine and Biotechnology-
BIONAND, Malaga, Spain, 4 Allergy Unit, Hospital Regional Universitario de Málaga, Hospital Civil, Málaga, Spain, 5 Servicio
́
Giancarlo Aldini,
de Alergología, Hospital Infanta Leonor, Madrid, Spain
University of Milan, Italy
*Correspondence:
María I. Montañez
Serum and cellular proteins are targets for the formation of adducts with the b-lactam
antibiotic amoxicillin. This process could be important for the development of adverse, and
in particular, allergic reactions to this antibiotic. In studies exploring protein haptenation by
amoxicillin, we observed that reducing agents influenced the extent of amoxicillin-protein
adducts formation. Consequently, we show that several thiol-containing compounds,
including dithiothreitol, N-acetyl-L-cysteine, and glutathione, perform a nucleophilic attack
on the amoxicillin molecule that is followed by an internal rearrangement leading to
amoxicillin diketopiperazine, a known amoxicillin metabolite with residual activity.
Increased diketopiperazine conversion is also observed with human serum albumin but
not with L-cysteine, which mainly forms the amoxicilloyl amide. The effect of thiols is
catalytic and can render complete amoxicillin conversion. Interestingly, this process is
dependent on the presence of an amino group in the antibiotic lateral chain, as in
amoxicillin and ampicillin. Furthermore, it does not occur for other b-lactam antibiotics,
including cefaclor or benzylpenicillin. Biological consequences of thiol-mediated
amoxicillin transformation are exemplified by a reduced bacteriostatic action and a
lower capacity of thiol-treated amoxicillin to form protein adducts. Finally, modulation of
the intracellular redox status through inhibition of glutathione synthesis influenced the
extent of amoxicillin adduct formation with cellular proteins. These results open novel
perspectives for the understanding of amoxicillin metabolism and actions, including the
formation of adducts involved in allergic reactions.
maribel.montanez@ibima.eu
Dolores Pérez-Sala
dperezsala@cib.csic.es
†Present address:
Tahl Zimmerman,
North Carolina Agricultural and
Technical State University,
Greensboro, NC, United States
Specialty section:
This article was submitted to Drug
Metabolism and Transport,
a section of the journal
Frontiers in Pharmacology
Received: 23 September 2019
Accepted: 10 February 2020
Published: 04 March 2020
Citation:
Pajares MA, Zimmerman T,
Sánchez-Gómez FJ, Ariza A,
Torres MJ, Blanca M, Cañada FJ,
Montañez MI and Pérez-Sala D (2020)
Amoxicillin Inactivation by
Thiol-Catalyzed Cyclization
Reduces Protein Haptenation
and Antibacterial Potency.
Front. Pharmacol. 11:189.
Keywords: amoxicillin, b-lactam antibiotics, inactivation mechanism, redox regulation, protein adducts, thiol
doi: 10.3389/fphar.2020.00189
groups, thiol-containing molecules, bacterial growth
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Mechanism of Amoxicillin Inactivation by Thiols
GRAPHICAL ABSTRACT
recognized by the immune system and induce an allergic
response (Ariza et al., 2015) (Figure 1).
INTRODUCTION
Allergic reactions to drugs and to antibiotics in particular,
constitute important medical problems (Blanca et al., 2003;
Torres et al., 2014). These reactions can be life-threatening and
limit the therapeutic tools against infections. Binding of b-lactam
antibiotics to endogenous protein carriers is considered an
important step in the development of allergic reactions
(Faulkner et al., 2014). Nevertheless, our knowledge of this
process is still limited. It is generally accepted that the low
molecular weight of antibiotics and other drugs prevents them
from being recognized by the immune system on their own, for
which they require association with larger structures
(Landsteiner and Jacobs, 1935). Importantly, numerous drugs
or their metabolites are able to bind covalently to proteins, a
process known as haptenation (Ariza et al., 2011; Tailor et al.,
2016), which renders structures with the potential of being
Recent work of our group identified several serum proteins as
potential targets for haptenation by amoxicillin (AX) (Ariza
et al., 2012). In addition, intracellular proteins are also
susceptible of covalent modification by this antibiotic (Ariza
et al., 2014; Sánchez-Gómez et al., 2017). Nucleophilicity and a
favorable environment appear to be important for the selective
modification of proteins on certain residues. In vitro, incubation
of human serum albumin (HSA) with AX, results in the
modification of several lysine residues of which, Lys190 and
Lys195 appear to be the most reactive (Ariza et al., 2012).
Interestingly, our own studies have found only Lys190 as the
modified residue after oral administration of AX during 24 h
(Garzon et al., 2014). Adduction of Lys190, together with
modification of additional residues, has been reported in later
studies, after administration of AX in combination with
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FIGURE 1 | Effect of various compounds on amoxicillin-protein adduct formation. (A) Scheme of the formation of amoxicillin-protein adducts. Purified human serum
albumin (HSA) (B) at 10 mg/ml in phosphate buffer saline (PBS) or total human serum (C) was preincubated with vehicle, 1 mM DTT, 2.5 mM GSH or GSSG for 6 h
at 37°C, after which, amoxicillin (AX) from a stock solution in bicarbonate buffer was added at a final concentration of 0.5 mg/ml, as indicated, and incubation was
continued overnight at 37°C. Aliquots from the incubation mixtures containing 2 µg of protein were separated by SDS-PAGE. The formation of AX-HSA adducts was
monitored by immunoblot with monoclonal anti-amoxicillin AO3.2 antibody. Band intensity was estimated by image scanning and average results SEM from three
independent assays are shown in the corresponding graphs (*p ≤ 0.05).
clavulanic acid during more prolonged regimens, in higher doses
or after intravenous administration (Garzon et al., 2014; Meng
et al., 2016). Apart from the chemical reactivity of the antibiotic
and the protein residues, little is known about other factors
influencing protein haptenation by AX in biological settings.
Previous studies with the antibiotic sulfamethoxazole have
shown that oxidative stress can potentiate the formation of
protein adducts in cells (Lavergne et al., 2009). This alteration
of the redox status concurs with many diseases and is involved in
their pathogenesis or perpetuation. Therefore, oxidative stress is
a concomitant factor in numerous pathological situations
requiring antibiotic administration. Oxidative stress can
influence drug protein-adduct formation by multiple
mechanisms, including induction of conformational alterations
or oxidative modifications that can either increase or blunt the
reactivity of the modified residues towards the drug, as we have
recently reviewed (González-Morena et al., 2016). While
exploring the impact of oxidants or reducing agents on protein
modification by AX, we observed that the presence of exogenous
thiol-containing molecules influenced the extent of AX-protein
adduct formation. Investigation into the basis of this effect has
unveiled the capability of thiol-containing compounds to
catalytically transform AX. These results shed new light into
the factors determining AX bioavailability and, therefore, its
antimicrobial potency and adduct formation capacity, with
potential implications for the understanding of drug allergy.
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(DKP) as well as oxidized and reduced forms of DTT. The
resulting crude was analyzed by 1H-NMR, attenuated total
reflection–Fourier transform infrared (ATR-FTIR)
spectroscopy and electrospray ionization-mass spectrometry
(ESI-MS). The conversion of AX to DKP was quantitative.
1H-NMR signals corresponding to DKP were the following:
1H-NMR (400 MHz, D2O) d ppm: 7.18 (d, 2H, J = 8.1Hz), 6.83
(d, 2H, J = 8.1Hz), 5.19 (s, 1H, CHPh), 5.16 (d, 1H, J = 2.6Hz, H-
6), 4.02 (d, 1H, J = 2.6 Hz, H-5), 3.37 (s, 1H, H-3), 1.52 (s, 3H,
CH3), 1.15(s, 3H, CH3).
Mass spectra were acquired as described above. Solid samples
were dissolved in MilliQ water for the analysis of AX and in a
MilliQ water-methanol mixture for the analysis of the resulting
product (DKP) (Ariza et al., 2016). Detection was carried out
with positive polarity, in selected ion monitoring (SIM) mode,
selecting the 366 ion and MS/MS mode for confirmation of
analytes. ESI-MS m/z (%) were as follows: 366 (M+, 20), 207 (16),
160 (100).
MATERIALS AND METHODS
Reagents
Dithiothreitol (DTT), glutathione (GSH), 2-mercaptoethanol, b-
NADPH, 5,5-dithio-bis-(2-nitrobenzoic acid), ethanothiol,
triethanolamine, 2-vinylpyridine, sulfosalicylic acid, bovine
serum albumin (BSA), buthionine sulfoximine (BSO), tris(2-
carboxyethyl)phosphine (TCEP), N-acetyl-L-cysteine, L-
cysteine, cefaclor, and cephadroxil were products of Sigma.
HSA, was from Sigma or purified from serum as described
below. Glutathione reductase (GSR) and Triton X-100 were
purchased from Roche and Calbiochem, respectively. Microcon
cells and Immobilon-P Transfer Membrane were obtained from
Amicon and Millipore, respectively. The bicinchoninic acid
(BCA) kit for protein determination was from Pierce.
Amoxicillin (Clamoxil) was from Beecham. Ampicillin (AMP),
benzylpenicillin (BP), and cefuroxime were from Laboratorios
Normon (Madrid, Spain). Deuterium oxide (D2O) for proton-
nuclear magnetic resonance (1H-NMR) studies was from VWR.
ECL was purchased from GE Healthcare.
In Vitro Incubations of b-Lactam Antibiotics
for 1H-NMR and LC-MS/MS Studies
Instrumental Analysis
Incubations of AX with DTT, other thiol-containing compounds
or antioxidants were carried out at 37°C and physiological pH in
phosphate-buffered saline solution (PBS) using different ratios of
reactants and times. The resulting species were compared with a
reference control solution containing only AX kept under the
same conditions, following two methodologies: (i) after
incubation in PBS in inert atmosphere, mixtures were
lyophilized and the resulting solids were dissolved in D2O and
analyzed by 1H-NMR; and (ii) incubations were monitored
1H-NMR spectra were recorded at 400 MHz using a NMR
Bruker Ascend™ 400 MHz for 1H, and D2O or deuterated-
phosphate buffer saline (PBS-d) as solvents. ATR-FTIR spectra
were recorded with a FTIR spectrometer (VERTEX 70) equipped
with a Golden Gate Single Reflection Diamond ATR System
from Specac. Mass spectra were acquired on a High Resolution
Mass Spectrometer Orbitrap, Q-Exactive (Thermo Fisher
Scientific) equipped with electrospray ionization (H-ESI-II),
coupled to a Liquid Chromatographer Dionex Ultimate 3000
HPLC system (Thermo Fisher Scientific) equipped with a
Accucore RP-MS column (50 × 2.1 mm ID, 2.6 mm) (Thermo
Scientific). The chromatographic separation was performed in
the following conditions: Column temperature: 20°C;
autosampler temperature: 10°C; injection volume: 10 ml; flow:
0.2 ml/min. The column was equilibrated with acetonitrile/
ammonium formate, pH 2.5 (2/98). Samples were eluted with
isocratic flow 2/98 (acetonitrile/ammonium formate, pH 2.5) for
2 min, followed by gradient from 2/98 to 70/30 in 1 min, then
isocratic mobile phase 70/30 (acetonitrile/ammonium formate,
pH 2.5) for 2 min, and a final gradient from 70/30 to 2/98
(acetonitrile/ammonium formate, pH 2.5) for 3 min. Detection
was carried out with positive polarity, in selected reaction
monitoring (SRM) mode, selecting the following ions: AX
366.4, 114.0 (22eV), 349.0 (6eV); Amoxicilloic Acid 384.4,
189.0 (19eV), 323.1 (13eV); and DKP 366.4, 114.1 (31eV),
160.1 (14eV); and MS/MS mode for confirmation of analytes.
1
directly in PBS-d by H-NMR at different times. DKP NMR
signals in PBS (400 MHz, PBS-d) d ppm: 7.27 (d, 2H, J = 8.6Hz),
6.93 (d, 2H, J = 8.6Hz), 5.28 (s, 1H, CHPh), 5.26 (d, 1H, J =
2.6Hz, H-6), 4.12 (d, 1H, J = 2.6 Hz, H-5), 3.47 (s, 1H, H-3), 1.62
(s, 3H, CH3), 1.26(s, 3H, CH3).
Freshly prepared AX in PBS was incubated in the absence or
presence of purified HSA purified from serum for 16 h at 37°C
(final concentrations of HSA and AX in incubation mixture were 3
mg/ml and 2.5 mg/ml, respectively). Then, samples were filtered
through Amicon filter devices, 3 kDa MWCO (Millipore), and
filters were washed three times with H2O. Filtrates (<3 kDa
fractions) were recovered for analysis by LC-MS/MS.
To assess the effect of N-acetyl-L-cysteine in serum, freshly
prepared AX in PBS was incubated with human serum at a final
concentration of 25 µg/ml, in the absence or presence of 100 µM
N-acetyl-L-cysteine for 16 h at 37°C. Then, samples were filtered
through Amicon filter devices, as above and filtrates (<3 kDa
fractions) were used for LC-MS/MS analysis.
Incubations of other b-lactams (BP, AMP, cefaclor,
cephadroxil or cefuroxime; 0.05 mmol) with DTT (0.1 mmol)
at 37°C were carried out either at physiological (PBS-d, 0.6 ml) or
at basic pH (Na2CO3 in D2O, pD = 11, 0.6 ml), and compared
with a reference control solution containing the b-lactam under
the same conditions of pH and temperature. Solutions were
Synthesis and Characterization of
Diketopiperazine Derived From AX
Sodium AX (19 mg, 0.05 mmol) and DTT (15 mg, 0.1 mmol)
were dissolved in 1.5 ml of degassed H2O under N2 atmosphere
and stirred overnight at room temperature. The mixture was
lyophilized to yield a white solid containing diketopiperazine
1
monitored directly by H-NMR at different times.
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Mechanism of Amoxicillin Inactivation by Thiols
Formation of AX-HSA Adducts
Electrophoresis and Western blot
Mixtures of AX (5 mg/ml) with vehicle or antioxidants (1 mM
TCEP, 1 mM Trolox, or 100 mM DTT in PBS, pH 7.4) were
preincubated for 24 h at 37°C or prepared fresh. Both types of
mixtures were diluted 1:1 (v/v) with HSA (Sigma) at 0.6 mg/ml
in PBS and further incubated for 24 h more at 37°C, after which,
aliquots containing 4 µg of protein were loaded on 12% SDS-
PAGE gels and analyzed by western blot.
HSA (Sigma), serum or cellular extracts modified with AX or
AX-treated with reducing or oxidizing agents and the
corresponding unmodified controls were incubated in Laemmli
buffer for 5 min at 95°C. Protein samples (1–15 µg) were loaded
on SDS-PAGE gels and electrotransferred to Immobilon-P
Transfer membranes for immunoblotting. Membranes were
stained with Ponceau S (Sigma) as a control of protein loading
and transfer. After destaining, membranes were incubated with
1:200 (v/v) mouse monoclonal anti-AX AO3.2 (Mayorga et al.,
1995) and 1:2,000 (v/v) anti-mouse IgG-HRP (Dako
Cytomation) diluted in 1% (w/v) BSA in TTBS. Detection was
carried out by chemiluminescence using the ECL system and the
signal quantified utilizing image scanning and Scion
Image software.
Colony Formation
Mixtures of AX preincubated with vehicle or the reducing agents
as above, were diluted with LB agar to prepare plates with a
theoretical antibiotic concentration of 100 µg/ml. Equivalent
volumes of PBS were added to control plates. E. coli DH5a
were seeded on the plates with the resulting mixtures and colony
formation was assessed by visual inspection and counting after
overnight incubation at 37°C.
Determination of Glutathione
Concentrations
RPMI 8866 cells (106) were treated as above. Cells were harvested
by centrifugation and washed three times with PBS. The pellets
were kept frozen at −80°C until use. The levels of glutathione
reduced (GSH) and oxidized (GSSG) forms were measured using
the enzymatic method of Tietze as described by (Rahman et al.,
2006). The extracts (250 µl) were diluted 1:10 (v/v) or 1:2 (v/v)
for detection of GSH or GSSG, respectively.
Bacterial Growth Curves
A stock solution of the antibiotic (137 mM) was incubated with
the selected thiol reagents (DTT, GSH, 2-mercaptoethanol) at
several molar ratios (1:0 up to 1:20) for 24 hours at 37°C in a final
volume of 43 µl in PBS (AX inactivation reactions), whereas in
control reactions AX was substituted by PBS. In parallel, E. coli
DH5a were grown in LB medium (10 ml) overnight at 37°C and
the A620 of the culture measured before the experiment. Putative
AX inactivation was then evaluated following E. coli growth in
the presence of the treated antibiotic using multi-well plates. For
this purpose, appropriate volumes of the AX inactivation or
control reactions were added to each well to obtain a theoretical
antibiotic concentration of 75 µg/ml in a final volume of 50 µl.
The growth curve (150 µl per well) started by addition of 100 µl
of the diluted bacterial culture (A620 ∼0.1) and was followed by
Isolation of HSA From Serum
The Andalusian Public Health System Biobank has coordinated
the collection, processing, management, and assignment of the
biological samples used in this study, according to the standard
procedures established for this purpose. Venous blood (9 ml) was
collected from healthy volunteers using Vacuette Greiner Bio-
One tubes (Kremsmnster, Austria), and serum was obtained by
̈
centrifugation at 4,000 rpm for 5 min. The study was conducted
according to the Declaration of Helsinki principles and was
approved by the local Ethics Committee (Comité de Ética para
la Investigación Provincial de Málaga, Spain). All subjects were
informed and signed the corresponding informed consent form.
HSA was isolated from the serum by affinity chromatography on
Affi-Gel Blue Gel (Bio-Rad), as previously described (Garzon
et al., 2014). Briefly, an aliquot of serum (50 ml) was diluted with
150 ml of sodium phosphate dibasic 20 mM, pH 7.1 (buffer A),
and kept at room temperature for 15 min. Meanwhile, a Sigma
Prep Spin column was filled with 500 ml of Affi-Gel Blue Gel, and
the gel was activated as indicated by the manufacturer. Aliquots
of diluted serum (200 ml) were loaded onto the column and
washed three times with buffer A. Albumin was eluted by adding
two aliquots of 250 ml of NaCl 1.4 M (buffer B) to the column.
HSA concentration was determined spectrophotometrically at
280 nm as in (Garzon et al., 2014).
A620 at 20 min intervals for 350 min. Measurements were carried
out in triplicate for each condition and bacterial growth analyzed
using GraphPad Prism software.
Cell Culture
Human lymphocyte RPMI 8866 cells were grown in DMEM
supplemented with 10% (v/v) fetal bovine serum (FBS, PAA
Laboratories). Cells (106) were treated with vehicle or BSO (50 or
100 µM) for 24 h in complete medium without antibiotics, after
which, cells were washed with serum free medium and incubated in
this medium in the absence or presence of BSO or AX (2.5 mg/ml)
for 16 h. Cells were harvested by centrifugation and washed with
PBS before lysis using 10 mM Tris/HCl pH 7.5 containing 0.1 mM
EDTA, 0.1 mM EGTA, 0.1 mM 2-mercaptoethanol, 320 µg/ml
Pefablock, 2 µg/ml trypsin inhibitor, 2 µg/ml aprotinin, 2 µg/ml
leupeptin, 0.1 mM sodium orthovanadate, 50 mM sodium fluoride,
and 0.5% (w/v) SDS and several passages through 26G 1/2 needles.
Homogenates were then centrifuged 5 min at 13,000 × g and 4°C to
obtain the supernatants. Protein concentration was measured with
the BCA kit.
Statistical Analysis
GraphPad Prism v. 5.0 (GraphPad Software) was used for
statistical analysis of the data. Data are shown as average
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Mechanism of Amoxicillin Inactivation by Thiols
values from at least three experiments
SEM. Experiments
when AX was incubated under the same conditions without DTT.
This observation, together with the fact that 1 equivalent of DTT is
not consumed in the reaction, indicated that intramolecular amide
formation by nucleophilic attack of the AX side chain amino
group to its electrophilic b-lactam carbonyl, under these
conditions, occurs only in the presence of DTT. Moreover, the
reducing agent acts in a catalytic manner through the formation of
an intermediate thioester, as depicted in the mechanism
schematized in Figure 3A (see below). Nevertheless, DKP is a
known metabolite of AX that can arise as well by thiol-
independent mechanisms, including the acidic degradation of
AX (Nagele and Moritz, 2005).
containing two groups of data were analyzed using Student's t-
test for independent samples; differences were considered
significant when p < 0.05.
RESULTS
Effect of Oxidants or Reducing Agents on
AX-Protein Adduct Formation
Oxidative stress can act as a concomitant factor or as a mediator
of drug adverse effects and influence drug-protein adduct
formation (Ariza et al., 2012; González-Morena et al., 2016).
We have previously shown that AX forms adducts with HSA and
other serum proteins (Ariza et al., 2012). Therefore, we were
interested in the effect of oxidants and reducing agents on
protein adduct formation by AX. Preincubation of purified
HSA with DTT prior to AX addition led to a reduction in the
levels of AX-HSA adducts, as detected by immunoblot with the
anti-AX antibody (Figure 1B). In contrast, preincubation with
GSH did not significantly affect adduct formation, whereas
incubation with GSSG increased adduct levels. When the assay
was carried out with total serum, DTT tended to reduce, whereas
GSH and GSSG tended to increase, AX-HSA adduct formation,
although these effects were not statistically significant
(Figure 1C). These observations suggested that formation of
AX-protein adducts in vitro can be modulated by several redox-
active agents. Therefore, we set out to study these effects in
more detail.
Next, the incubation experiments were carried out in PBS
buffer at 37°C using different AX : DTT ratios (Table 1), and the
1
progress of the reaction was monitored by H-NMR using the
chemical shifts for AX b-lactam ring protons (H5 and H6). In all
cases, we observed a time-dependent reduction in AX signals
together with the appearance of signals consistent with the
formation of the DKP cyclic product (H5 and H6, at 4.12 and
5.26 ppm, respectively) (Figure S1). The amoxicilloyl thioesther
intermediate might undergo an intramolecular aminolysis
favored by the formation of the six-membered heterocycle as
soon as it is formed. Moreover, DKP formation was shown to be
time- and DTT concentration-dependent. We also considered
the possibility that the reaction, instead of being catalyzed by the
amoxicilloyl thioesther formation, could be favored by the redox
properties of DTT. With the aim of investigating this potential
mechanism, the fate of AX incubated in the presence of
ethanothiol, as the nucleophile, was studied. This treatment
resulted in virtually complete transformation of AX into DKP
(Figure S2), which confirmed the thiol-catalyzed intramolecular
aminolysis of the AX b-lactam. Of note, as the DKP produced
possesses the same mass than AX, the cyclization of the antibiotic
in the presence of thiol-containing compounds would remain
undetected if monitored only by MS (Reyns et al., 2008).
Based on these data, we propose the cyclization mechanism
represented in Figure 3A, in which the formation of DKP
would be dependent on the presence of the amino group in the
lateral chain of AX. To confirm this, the reactivity of other
penicillins (with identical nuclear bicycle structure, and
similar or different side chains) and cephalosporins (with
different nuclear bicycle structure and identical, similar or
different side chains) was studied in the presence of DTT (2
equiv) at neutral or basic pH. Among aminopenicillins, AMP
was chosen since it shares the nuclear bicycle structure of AX
and the presence of amino functionality in its side chain. AMP
incubation with DTT at neutral pH for 24 h resulted in the
formation of its DKP-AMP, showing the same reactivity than
AX through intramolecular cyclization (Figure 3A and
Figure S3). Next, BP was used as a model penicillin with the
same nuclear bicycle structure, but without amino side chain
functionality. In contrast to AX and AMP, the spectrum of BP
suffered no modification after incubation with DTT at neutral
pH (Figure S4). However, addition of DTT at basic pH clearly
elicited BP hydrolysis to the benzylpenicilloic acid and the
appearance of small amounts of the thioesther intermediate.
Thiol-Containing Compounds Induce a
Cyclization of AX With the Formation of
AX-DKP
Although some of the effects observed in Figure 1 could be due to
the influence of redox agents on the structure or conformation of
the proteins themselves (see Discussion), we also considered the
possibility that these compounds might have an effect on the AX
molecule. Therefore, the integrity of AX (1 equivalent) in the
presence of DTT (2 equivalents) was investigated after overnight
incubation in aqueous media using several characterization
techniques (Figure 2). The ATR-FTIR spectra of the mixture
lacked the typical high infrared frequency b-lactam carbonyl band
(C=O stretching >1780 cm−1) (Figure 2A), which indicated
cleavage of the b-lactam ring. ESI-MS analysis of the resulting
mixture showed the same molecular ion as AX, but different
fragmentations, providing evidence of an intramolecular
1
rearrangement (Figure 2B). The H-NMR spectra showed the
disappearance of the doublet signals corresponding to the b-
lactam ring protons (H5 and H6) of AX at 5.39 and 5.35 ppm
(J5,6 = 3.9 Hz), and their appearance at 4.02 and 5.17 ppm (J5,6
=
2.6 Hz) (Figure 2C). The shift of two proton bands is in agreement
with the formation of the DKP cyclic product in quantitative
yields. In addition, the presence of oxidized and reduced DTT was
detected. Moreover, the singlets corresponding to methyl groups
of the C2 thiazolidine shifted from 1.41 and 1.35 ppm in AX to
1.52 and 1.16 ppm in DKP. This rearrangement was not observed
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FIGURE 2 | Monitoring amoxicillin reactivity in the presence of Dithiothreitol (DTT) in aqueous solution. (A) Attenuated total reflection-Fourier transform infrared (ATR-
FTIR) spectra of the starting b-lactam, amoxicillin (AX, blue) and the resulting diketopiperazine (DKP, red) after DTT incubation. The band corresponding to C=O
stretching of b-lactam (>1780 cm−1) in the AX molecule disappeared after reaction with DTT, which indicated its cleavage, in agreement with the formation of DKP.
(B) Electrospray ionization-mass spectrometry (ESI-MS) of the starting b-lactam AX m/z (%): 366 (M+, 5), 349 (100), 208 (66), upper panel, and of the resulting DKP
m/z (%): 366 (M+, 22), 207 (16), 160 (100), showing the same molecular ion peak than AX, but different fragmentation pattern and base peak, lower panel. (C) 1H-
NMR spectrum of the starting b-lactam AX (in D2O), upper panel, and of the resulting DKP obtained after incubation of DTT (2 equivalents) with AX (1 equivalent) in
H2O overnight at room temperature, lower panel. The resonances of b-lactam protons (H5 and H6) were used to monitor the reaction, shifting from 5.39 and 5.35
ppm to 4.02 and 5.17 ppm. The DKP spectrum also shows peaks corresponding to reduced and oxidized forms of DTT in the 2.5 to 3.7 ppm range.
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Mechanism of Amoxicillin Inactivation by Thiols
FIGURE 3 | Reactivity of different penicillins in the presence of thiols. (A) Nucleophilic ring opening of aminopenicillins—amoxicillin (AX) and ampicillin (AMP)—by
thiols and subsequent intramolecular aminolysis resulting in their corresponding diketopiperazine (DKP). (B) Hydrolysis of benzylpenicillin (BP) catalyzed by thiols, with
formation of the benzylpenicilloate thioester intermediate (BP-SR), yielding the benzylpenicilloate (BPO).
TABLE 1 | Reactivity of penicillins in presence of Dithiothreitol (DTT) in different
conditions.
Interaction of Biologically or
Pharmacologically Relevant Thiol-
Penicillin pH Penicillin : DTT Incubation Cyclization product (DKP)
Containing Molecules With AX
ratio
time
% of total
In view of the data obtained with DTT, we explored whether
other relevant thiol-containing compounds could induce AX
transformation (Table 2). The antioxidant N-acetyl-L-cysteine,
which is the active principle of widely used mucolytic
pharmacological preparations, also promoted DKP formation
when incubated in PBS. Incubation of AX in the presence of HSA
purified from serum resulted in increased DKP formation (from
5.4% to 36.6% DKP formation in the absence and presence of
HSA, respectively). Nevertheless, incubation of AX with total
serum or with serum spiked with N-acetyl-L-cysteine at
concentrations in the range of those occurring after
intravenous administration did not result in consistent
detection of DKP formation. Notably, incubation of AX with
L-cysteine (with the nonprotected-amino group) did not afford
DKP, but mainly, the product corresponding to the aminolysis of
the b-lactam ring, which remained conjugated to the amino
group of cysteine through an amide bond (Figure S8).
Importantly, GSH, the main low molecular weight intracellular
thiol antioxidant, effectively induced cyclization of AX. In
contrast, nitro-benzyl-glutathione, a GSH analog in which the
thiol group is protected, and GSSG, did not elicit this effect.
AXa
AXb
5.8
7.4
1:2
1:1.13
14 h
6 h
30 h
4 h
7 h
24 h
4 h
100
62
93
43
60
99
28
34
99
85
60
0
AXb
7.4
7.4
1.32:1
3.46:1
AXb
7 h
24 h
14 h
14 h
24 h
24 h
24 h
24 h
AXa
7.4
7.4
7.4
11
7.4
11
1.32:1
3.46:1
1:1
1:1
1:1
AXa
BPb
BPb
0
95
95
AMPb
AMPb
1:1
Incubations were carried out at 37°C for the time stated in each case in (a) distilled water or
phosphate buffer saline (PBS) and then lyophilized; or (b) deuterated solvent (D2O, PBS or
carbonate). In both cases, 1H-NMR was employed for identifying and quantifying the
resulting cyclization product.
Thus, the mechanism of the reaction in this case would
correspond to that depicted in Figure 3B. In contrast, no
reactivity was observed upon addition of DTT to
cephalosporins bearing identical side chains than AX
(cephadroxil) or AMP (cefaclor) at neutral or basic pH
(Figure 4 and Figures S5 and S6). Additionally, incubation
of cefuroxime, a cephalosporin without free amino
functionality in the side chain, in the presence of DTT at
physiological pH resulted in only a small increase of the rate of
hydrolysis compared to the incubation without the thiol
compound. Moreover, the extent of the hydrolysis did
increase at basic pH when using DTT (Figure 4 and
Figure S7).
Effect of Nonthiol-Based Antioxidants on
AX Availability
In view of the above results it was of interest to explore whether
nonthiol-based antioxidants commonly used in protein
biochemistry and cell biology could interact with AX. This
knowledge will be necessary for any subsequent studies
involving the interaction of AX with proteins, requiring the
presence of an antioxidant. The antioxidant Trolox did not
alter AX (Table 2). In contrast, the widely used antioxidant
TCEP, induced decomposition of AX into several products,
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Mechanism of Amoxicillin Inactivation by Thiols
FIGURE 4 | Structure and reactivity of cephalosporins in the presence of thiols. (A) Structure of different cephalosporins included in the study with highlighted R1
and R2 substituents. (B) Scheme of proposed reactivity of cephalosporins (CP) with a good R2 leaving group in presence of thiols, resulting in the thioester
intermediate (CP-SR3) and the subsequent cephalosporoate (CPO), whose expulsion of the leaving group at C3' affords the exo-methylene compound.
including DKP, amoxicilloate, and other nonidentified
substances, which accounted for up to 80% of the resulting
mixture. In addition, it should be taken into account that TCEP
itself can suffer some decomposition in phosphate buffers at
neutral pH (Wingfield, 2001). Therefore, Trolox appears as a
suitable nonthiol antioxidant for AX-protein studies.
that had been preincubated for 24 h at 37°C (24 h) were
combined with a solution of HSA and the formation of AX-
HSA adducts was monitored 24 h later. As observed in Figure
5A, AX preincubation with PBS or Trolox did not significantly
reduce AX-HSA adduct formation with respect to the adducts
formed by the freshly prepared mixtures, whereas preincubation
with TCEP showed a tendency towards decreasing adduct levels.
This behavior is consistent with the lack of interaction of Trolox
with AX and the complex interaction exhibited by TCEP with the
AX Transformation Into DKP Implies a Loss
of Reactivity and Antibacterial Potency
1
antibiotic as revealed by H-NMR (Table 2). Remarkably, the
DKP is a known metabolite of AX, considered to have lower or
residual reactivity and bacteriostatic activity (Reyns et al., 2008),
which has been detected in humans, in experimental animals and
in the environment (Connor et al., 1994; Reyns et al., 2008; Gozlan
et al., 2013). To confirm the reduced DKP reactivity its interaction
with butylamine, as a model of a nucleophilic compound
formation of adducts by the freshly prepared DTT-AX mixture
was clearly lower than adducts formed by AX in PBS (control).
Moreover, preincubation of AX with DTT further diminished
the capacity of AX to form adducts with HSA (Figure 5A).
Interaction of AX with thiols also reduced its antibacterial
potency, as evidenced using two different approaches, namely,
evaluation of colony formation and of the growth of suspension
cultures of AX-sensitive E. coli. Preincubation of AX with PBS or
the nonthiol antioxidants TCEP and Trolox did not impair its
antibacterial effect since strong inhibition of colony formation
was observed (Figure 5B). In contrast, incubation with DTT
relieved this inhibition, thus indicating its interference with the
bacteriostatic action of AX (Figure 5B). Additionally, assessment
of the effect of thiol-containing compounds on the antibacterial
action of AX by following E. coli growth in turbidity assays
showed that all the thiol-containing compounds, namely, DTT,
GSH, and 2-mercaptoethanol, attenuated the bacteriostatic effect
of AX in a concentration-dependent manner (Figure 6). DTT
was the most potent, blocking AX effect at equimolar ratios
(Figure 6A), as expected from its higher reduction potential and
the presence of two thiols in the molecule. On the other hand,
GSH and 2-mercaptoethanol displayed similar potency, needing
1
susceptible to adduction by AX, was explored. H-NMR analysis
of incubation mixtures containing equimolar amounts of DKP
and butylamine revealed quantitative preservation of DKP (95% of
the starting amount), thus confirming its lack of reactivity.
Next, the effect of preincubating AX with DTT or nonthiol
antioxidants on its capacity to covalently modify HSA was
monitored. For this purpose, antioxidant concentrations within
the range used in cellular or biochemical applications were
included in the assays. In particular, low micromolar to
millimolar concentrations of Trolox are used in cell culture
and in vivo applications (Messier et al., 2013), whereas low
millimolar TCEP concentrations are mostly utilized in protein
preparations (Getz et al., 1999). DTT is generally included at low
millimolar levels in most cell lysis and protein extraction
protocols and at high millimolar concentrations in protein
reduction methods. Therefore, freshly prepared mixtures of AX
with vehicle (PBS) or the indicated antioxidants (0 h) or mixtures
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Mechanism of Amoxicillin Inactivation by Thiols
TABLE 2 | Reactivity of amoxicillin (AX) with thiol (protected, reduced or oxidized forms) and nonthiol antioxidants.
Compound
Formed products
DKP (%) AXO (%)
Remaining
AX (%)
Other products (%)
None
DTT
9
9
82
41
50
27
9
8
–
–
N-acetyl-cysteine
L-cysteine
65
0
2
12
86
(Amoxicilloyl-Cys conjugate)
L-Glutathione reduced (GSH)
L-Glutathione oxidized (GSSG)
29
9
8
9
63
82
–
–
S-para-nitro-benzyl glutathione
9
9
82
–
Trolox
TCEP
9
9
82
20
–
20
10
50 (related nonidentified amoxicilloyl products)
Incubations were carried out using equimolar amounts of AX and the thiol functional-reagent or nonthiol antioxidant, in phosphate buffer saline (PBS) at 37°C for 24 h and lyophilized.
Integration of 1H-NMR signals was employed for calculating the quantity of obtained products.
thiol:AX ratios above 4:1 to obtain a complete abolition of
antibiotic effects (Figures 6B, C).
pretreated with BSO before AX addition, a drastic decrease in
both GSH and the GSH/GSSG ratio was observed compared to
both control and AX-treated cells (Figure 7A). As mentioned
before, AX can form adducts with cellular proteins that can be
detected by immunological methods (Ariza et al., 2014; Sánchez-
Gómez et al., 2017). Interestingly, a decrease of GSH levels and the
GSH/GSSG ratio in RPMI 8866 cells by pretreatment with BSO
correlated with a significant increase in the formation of AX-
protein adducts (Figure 7B). Nevertheless, whether this effect is
related to an improved availability of AX due to decreased GSH
levels requires further study.
Importance of AX-Thiol Interaction in
Biological Settings
The interactions between AX and thiols could be highly relevant
for the action of this antibiotic in biological environments, in
which levels of thiol-containing compounds have a very important
role in the control of the redox status. Among them, GSH reaches
millimolar concentrations in cells. GSH synthesis can be blocked
by BSO, an inhibitor of g-glutamylcysteine synthetase (Griffith,
1999), the rate limiting enzyme of this pathway, thus reducing
cellular GSH levels and inducing oxidative stress. In order to
explore the interplay between redox status and AX action, we
treated RPMI 8866 cells with AX alone or in combination with
BSO and the effect on GSH concentrations was measured. AX
alone did not alter cellular glutathione levels, neither the GSH/
GSSG ratios (Figure 7A). In contrast, when cells had been
DISCUSSION
AX is a widely used antibiotic and one of the most frequently
involved in allergic reactions. Therefore, AX reactivity and
conjugation to carrier molecules have been the subject of
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Mechanism of Amoxicillin Inactivation by Thiols
FIGURE 5 | Effect of various antioxidants on the capacity of amoxicillin to form protein adducts and inhibit bacterial colony formation. (A) Mixtures of amoxicillin (AX)
at 5 mg/ml (14 mM) and 100 mM DTT, 1 mM TCEP, or 1 mM Trolox in phosphate buffer saline (PBS) were either prepared fresh or preincubated for 24 h at 37°C.
All mixtures were combined with an equal volume of HSA at 0.6 mg/ml in PBS and subsequently incubated for 24 h at 37°C. Finally, aliquots containing 4 µg of HSA
were separated by SDS-PAGE and the formation of AX-HSA adducts was detected by western blot using anti-amoxicillin AO3.2. The blot shown corresponds to a
representative experiment out of three with similar results. The histogram depicts the mean SEM of the intensity of the AX-HSA adduct signal obtained with every
preincubated mixture with respect to that obtained with the freshly prepared mixture (100%) for each of the agents tested.(*p ≤ 0.05). (B) AX was incubated in the
presence of vehicle or reducing agents as above, and incubation mixtures were used to prepare LB-agar plates so that the theoretical final concentration of AX
would be 100 µg/ml. E. coli DH5a were plated and allowed to grow for 24 h at 37°C, after which, colonies were imaged on a transilluminator and counted. Results
show the number of colonies observed as percentage of those grown in the absence of AX, and are from a typical experiment out of three carried out.
thorough investigation (Torres et al., 2010; Ariza et al., 2016).
The redox status is a major determinant of drug action and
oxidative stress is a well-recognized coadjuvant factor in drug
adverse reactions (González-Morena et al., 2016). Moreover, the
redox status and oxidative modifications can influence drug
binding to carrier proteins. Control of redox status is brought
about by a complex interplay of enzymatic mechanisms and
small molecule antioxidants. The tripeptide GSH, which bears a
strong nucleophilic thiol group, plays a key role in this regulation
in cells, and it also participates in drug detoxification (Sánchez-
Gómez et al., 2016). Besides GSH, drugs can encounter a variety
of thiol-containing molecules along their administration or
distribution route in the body, including peptides, proteins or
other drugs, like mucolytic agents containing N-acetyl-L-
cysteine. Here we report the interaction of thiol-containing
molecules with AX. Our results constitute the first evidence for
the catalytic inactivation of this antibiotic, and potentially other
b-lactams of similar characteristics, by thiols. This finding could
have both pharmacological and biotechnological applications.
Initial assays preincubating proteins with reducing agents
before treatment with AX showed a clear decrease in AX-protein
adduct formation in the presence of DTT, whereas the effect of
GSH was less consistent (Figure 1). This could be potentially due
to instability of GSH under these conditions. Subsequent assays
preincubating AX in the presence of the thiol-containing
compounds (Figures 5 and 6) clearly showed their ability to
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Mechanism of Amoxicillin Inactivation by Thiols
decrease AX availability or potency. Nevertheless, it should be
taken into account that several factors can influence the
formation of adducts in the presence of these compounds. The
possibility of protein conformational alterations induced by the
reducing agents, resulting in decreased adduct formation, cannot
be excluded. Nevertheless, our results show that, for the same
concentration of reducing agent, the extent of the decrease in
adduct formation is dependent on the time that it is preincubated
with AX, thus supporting the importance of the inactivation
of AX.
From the chemical point of view, our data show that, in the
presence of thiols, AX is transformed into its DKP. This indicates
an intramolecular amidation subsequent to thiolysis
(Figure 3A). On the one hand, the chemical reactivity of
penicillins is related to the electrophilic properties of the b-
lactam carbonyl, which is associated with the high tension within
the b-lactam ring fused to the thiazolidine ring (Perez-Inestrosa
et al., 2005). Additionally, the protonated form of the thiol group
is not particularly reactive, whereas the thiolate anion is
nucleophilic and therefore can participate in reactions with
electrophilic reactive species (Wall et al., 2012). In this context,
interaction between AX and thiol molecules may stablish an
acid-base equilibrium, which generates catalytic amounts of
thiolate anion. This initiates the reaction with the b-lactam
ring towards the formation of an intermediate thioester, the
geometry of which should favor further intramolecular
amidation/cyclization (by the side chain amino group)
(Montañez et al., 2011). To the best of our knowledge, the
thiol catalysis for intramolecular reactivity forming cyclizated
structures characterized here has not been previously reported
for b-lactam antibiotics.
The existence of interactions between thiols and b-lactam
antibiotics, both in vitro and in vivo, has long been known.
Early studies detected a loss of the antibacterial activity of
penicillin induced by cysteine, which was attributed to
chemical reactions involving the thiol and/or amino groups of
the amino acid (Chow and McKee, 1945; Nakken et al., 1960;
Bakalov and Lilov, 1964). In agreement with this, our results have
showed a complete inactivation of AX by L-cysteine, but this
involves a chemical reaction with amino groups rather than with
the thiol groups of cysteine. More recent studies have described
in detail the thiol-catalyzed hydrolysis of BP (Llinas et al., 2000;
Garcías et al., 2005), in which the catalytically reactive form of
the thiol is the thiolate anion (Llinas et al., 2000). This result is in
agreement with our observations, which indicate a higher
reactivity between BP and the thiol at basic pH. The penicillin
thioester, previously identified by 1H-NMR, is the first detectable
product in the reaction, which subsequently undergoes
hydrolysis to the benzylpenicilloic acid (Figure 3B)
(Llinas et al., 2000). Therefore, the opening of the strained
four-membered ring is faster through thiolysis than hydrolysis,
due to the higher nucleophilicity of the thiolate anion in
comparison with the oxygen anion. Moreover, the minute
amount of the thioester intermediate detected supports its low
stability and high reactivity towards hydroxyl anions. (Llinas
et al., 2000).
FIGURE 6 | Effects of amoxicillin inactivation by reducing agents on bacterial
growth. The ability of glutathione (A), 2-mercaptoethanol (B) and DTT (C) to
inactivate amoxicillin (AX) was determined after incubation for 24 hours at 37°C
with the reducing agent vs. AX molar ratios indicated in the figure. The turbidity
assay was carried out on a multiwell plate using E. coli DH5a and a final
theoretical AX concentration of 25 µg/ml per well. Controls of bacterial growth
without the antibiotic, with 25 µg/ml AX or appropriate concentrations of each
reducing agent were also included; only controls with the highest concentrations
of reducing agents are shown for clarity. The figure depicts results of a typical
experiment (n = 3) carried out in triplicate (mean SEM).
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Mechanism of Amoxicillin Inactivation by Thiols
FIGURE 7 | Effects of amoxicillin and glutathione availability in a cellular context. (A) RPMI 8866 cells (106) were pretreated with phosphate buffer saline (PBS)
(control), or buthionine sulfoximine (BSO 50 or 100 µM) for 24 h, after which, either PBS or amoxicillin (AX), as indicated, were added for 16 h, and the intracellular
concentrations of reduced (GSH) and oxidized glutathione (GSSG) at the end of the incubation measured. The figure shows results (mean SEM) of five
independent experiments that were measured in triplicate. The resulting GSH/GSSG ratio is also depicted. (B) Impact of inhibiting GSH synthesis in RPMI 8866 cells
with 50 µM BSO on the formation of AX-protein adducts. Cells were treated with or without BSO and AX as above, harvested and the incorporation of AX into
proteins analyzed by western blot using anti-amoxicillin AO3.2. The AX signal of each lane was quantified by densitometric scanning and corrected using actin levels
as loading control. The graph shows the mean SEM of seven independent experiments (*p < 0.05 vs control AX).
The six-membered ring to which the b-lactam is fused in
cephalosporins, results in lower tension within the b-lactam ring
compared to those of penicillins, and therefore less reactivity
against thiolate anions (Perez-Inestrosa et al., 2005). Thiol-
catalyzed hydrolysis of cephalosporins has been described for a
series of these antibiotics with kinetics that depend on the
basicity of the thiol (Llinas et al., 2004). Our observations
indicate that the chosen aminocephalosporins, cephadroxil and
cefaclor, are not reactive towards thiols at either neutral or basic
pH. Results obtained with cephadroxil corroborate a previous
publication stating that the nature of the C3 substituent
influences thiolysis. The R2 chemical structure can modulate
this reactivity depending on its ability to polarize the electronic
binding (Perez-Inestrosa et al., 2005). In cephadroxil, position 3
is occupied by a methyl group, whose nonpolar nature can
explain the lack of effect we observe on the kinetic hydrolysis
of the b-lactam ring (Figure 4 and Figure S5). Cefaclor has a
chlorine atom at position 3, whose high electronegativity should
facilitate the opening of the b-lactam ring by electronic
induction. Although in basic media we observe a low degree of
hydrolysis of cefaclor leading to unidentified degradation
products, none of them can be attributed to the thiol reactivity
(Figure 4 and Figure S6). Cefuroxime shows reactivity of the b-
lactam group in the presence of thiols, resulting in its eventual
hydrolysis and further degradation (Figure 4 and Figure S7).
This increased b-lactam reactivity is explained by the R2
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Mechanism of Amoxicillin Inactivation by Thiols
substituent that may act as a leaving group (Perez-Inestrosa et al.,
2005), which is in agreement with previous observations in
cephalosporins with a good leaving group at this position
(Llinas et al., 2004).
adducts, as those detected in vitro and in the serum of patients
(Jenkins et al., 2009; Ariza et al., 2012), and facilitate the
development of allergic reactions.
In contrast, plasmatic GSH concentrations are low (<4 µM)
(Michelet et al., 1995), whereas intracellular concentrations reach
1–5 mM in most cell types and 5–10 mM in hepatocytes (Griffith,
1999; Lu, 2000; Wu et al., 2004). Hence, a major role of GSH on
AX availability could be envisioned in intracellular
environments, where additional antioxidant systems could
contribute to DKP production. In fact, the normal GSH/GSSG
ratios of 100:1 will favor the reductive and conjugating activities
of glutathione, whereas decreases in this ratio due to oxidative
stress will impair these activities potentially enhancing the
availability of drugs and AX (Martinez-Chantar and Pajares,
1996; Couto et al., 2016). Additionally, it could be hypothesized
that cellular proteins with thiol-disulfide active sites, such as
GSR, could contribute not only to reduce GSSG levels and to the
normalization of the GSH/GSSG ratio, but also to decrease AX
availability by a thiol-mediated mechanism. Importantly, the
distribution of GSH and of protein antioxidant systems is not
uniform inside cells. For instance, in HeLa cells, glutathione
redox potentials are more reducing in the mitochondrial matrix
than in the cytoplasm (Couto et al., 2016), whereas the activity of
glutathione transferases can differ between cellular
compartments, including cytosol, mitochondria, and nucleus
(Raza, 2011). Therefore, some of these protein antioxidant
systems, together will glutathione, may contribute thiols to
reduce the effective concentration of AX within the cell and
both, the system involved and the ratio of conversion may
depend on the subcellular compartment where the reaction
occurs. Indeed, we have observed that inhibiting cellular GSH
synthesis with BSO produces a strong depletion of GSH and a
marked decrease in the GSH/GSSG ratio, indicative of a drastic
decrease in the availability of free thiols in cells. These effects
associate with an increase in the formation of AX-protein
adducts and could be related to a lower extent of thiol-induced
transformation of AX into DKP under these conditions. Notably,
free radicals have also been reported to destroy b-lactam
antibiotics in aqueous solution (Song et al., 2008).
Nevertheless, whether this can occur under pathophysiological
conditions needs to be ascertained.
In biological settings and biochemical research, low molecular
weight thiols are important antioxidants, which act through a
variety of mechanisms. Among them, their role as components
of the general thiol/disulfide redox buffer, metal chelators, radical
quenchers, substrates for specific redox reactions (GSH), and
specific reductants of protein disulfide bonds (thioredoxin) can
be highlighted (Deneke, 2000). Thiol groups need to be
deprotonated to form charged thiolates with higher reactivity
compared to thiols. Thus, the inherent reactivity of low
molecular weight thiol compounds depends on the acid
dissociation constant (pKa) of their thiol groups, i.e., the lower
the pKa the higher the reactivity of the thiol (Wall et al., 2012).
Besides DTT, we have studied the ability of several biochemically
or pharmacologically relevant antioxidants to catalyze AX
inactivation. Both GSH and N-acetyl-L-cysteine bear thiol
groups with similar pKa values (in the range of 9.2–9.6).
Moreover, these pKa values are close to those of thiol groups
in DTT (9.2 and 10.1). Hence, differences observed in vitro for
AX inactivation with thiol-containing antioxidants can be
ascribed to the different number of thiol groups in the
molecules used, one in GSH and N-acetyl-L-cysteine and two
in DTT. Consistent with these facts, our assays demonstrate a
more efficient inactivation of AX by DTT than with either GSH
or N-acetyl-cysteine (Table 2).
In physiological settings, the nature and abundance of the
thiol-containing molecules that AX will encounter depends on
the environment (e.g. plasma or tissues), the cell type and even
the nutritional status (e.g. cysteine content after a protein-rich
ingestion). Among them, HSA is one of the main antioxidants in
plasma, where its concentration reaches 4.6 g/dl (Levitt and
Levitt, 2016). HSA represents more than 50% of the antioxidant
capacity of this fluid in normal subjects (Taverna et al., 2013). In
addition to its role as scavenger of reactive species (Aldini et al.,
2015), the state of Cys34 of HSA is considered the major
determinant of plasma redox status and represents about 80%
of the free thiol groups in blood (Regazzoni et al., 2013).
Importantly, the redox state of HSA is altered under various
pathophysiological conditions, including diabetes, renal disease,
and during exercise, in which the proportion of reduced HSA
(mercaptalbumin, thiol group of Cys34 in its reduced form)
versus oxidized HSA (cysteinylated), is decreased. Therefore, it
could be speculated that the state of oxidation of HSA could
influence AX availability. Nevertheless, the interactions between
AX and HSA are far more complex, since HSA is a major target
for haptenation by AX (Ariza et al., 2012). Thus, reactive lysine
residues in HSA could also have an impact on AX availability by
covalently biding the antibiotic. HSA is synthesized in the liver,
where its production attains 10.5 g/day, and has a turnover time
of ∼25 days (Levitt and Levitt, 2016). According to these data,
only 8.5% of HSA is renewed daily, thus allowing a long exposure
time to drugs, which can favor the formation of drug-HSA
The interaction of thiol-containing drugs and b-lactam
antibiotics has been also studied in vivo. Available evidence is
conflicting and both potentiating and interfering effects have
been described. Cysteine-containing compounds have been
reported to reduce the potency of several antibiotics, including
AX (Kikuchi et al., 1980; Alfredsson et al., 1987). In clinical
settings, the presence of thiol-containing compounds such as N-
acetyl-L-cysteine could interfere with the antibiotic itself
diminishing its efficacy. Nevertheless, N-acetyl-L-cysteine could
also potentiate antibiotics' effects by favoring their access to the
sites of infection due to its mucolytic action or its ability to
interfere with biofilms (Zhao and Liu, 2010; Blasi et al., 2016).
Therefore, the clinical benefit or interference of thiol compounds
with the bacteriostatic effect of AX needs to be evaluated in every
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Mechanism of Amoxicillin Inactivation by Thiols
specific setting. Administration through different routes or in an
asynchronous way, as recommended in practice, appears to be an
advisable strategy.
AUTHOR CONTRIBUTIONS
MP, MM, and DP-S designed the study, performed experiments,
analyzed data, prepared figures, and wrote the manuscript. AA,
FS-G, and TZ performed experiments, analyzed data, and
prepared figures. MT, FC, and MB provided feedback,
interpreted data, and discussed the manuscript.
In summary, our results indicate that thiols may alter AX
availability, both in vitro and in cellular environments, thus
constituting a major determinant for AX action under
pathophysiological conditions. Additionally, the oxidative
stress concurrent with many pathological situations could be
an important contributor to the formation of the larger amounts
of AX-protein adducts observed under this type of stress and that
seem involved in AX adverse reactions. These finding can be
relevant for understanding the mechanisms involved in
hypersensitivity reactions to b-lactams.
FUNDING
This work was supported by grant SAF2015-68590-R from
MINECO/FEDER, RTI2018-097624-B-I00 and RETIC Aradyal
from ISCIII/FEDER RD16/0006/0021 to DP-S; RD16/0006/0001
to MT, RD16/0006/0024 to MB; grants CP15/00103 and PI17/
01237 from ISCIII/ERDF and PI-0179-2014 from Andalusian
Regional Ministry Health to MM. AA holds a “Sara Borrell”
research contract (CD17/0146) supported by ISCIII from
MINECO [confounded by the European Social Fund (ESF)].
We acknowledge support of the publication fee by the CSIC
Open Access Publication Support Initiative through its Unit of
Information Resources for Research (URICI).
DATA AVAILABILITY STATEMENT
The datasets generated for this study are available on request to
the corresponding authors.
ETHICS STATEMENT
SUPPLEMENTARY MATERIAL
The studies involving human participants were reviewed and
approved by Comité de Ética para la Investigación Provincial de
Málaga, Spain. The patients/participants provided their written
informed consent to participate in this study.
The Supplementary Material for this article can be found online
at: https://www.frontiersin.org/articles/10.3389/fphar.2020.
00189/full#supplementary-material
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(2016). The effect of N-acetylcysteine on biofilms: implications for the
treatment of respiratory tract infections. Respir. Med. 117, 190–197. doi:
10.1016/j.rmed.2016.06.015
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Copyright © 2020 Pajares, Zimmerman, Sánchez-Gómez, Ariza, Torres, Blanca,
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