E. Petruzzella et al. / Inorganica Chimica Acta xxx (2016) xxx–xxx
5
The mixture was monitored until day 20, and no changes were
observed, thus the reaction was considered complete.
lower concentrations of reactants, that are closer to the intracellu-
lar concentrations of the Pt complexes [21,22].
At this point, an equivalent of GSH was added to the mixture
Fig. 6d). 11 days after the addition of GSH, a new peak appeared
at 11.3 min; this peak was not identified, while the kiteplatin-glu-
tathione 2:1 macrochelate adduct (B) and kiteplatin-glutathione
As a first step we investigated the stability of GSH and GMP, in
order to be able to recognize their degradation products. Then we
proceeded with the investigation of the reactions of kiteplatin with
the biomolecules (GSH and GMP).
(
2
:1 dimeric adduct (A) appeared at 13.0 and 13.5 min, respectively
The reaction between kiteplatin and GSH leads to the formation
of the kiteplatin-GSH adducts already reported in a previous work
[28]. The macrochelate adduct B is formed in greater amount using
buffered conditions than in unbuffered medium (in the latter case
the pH values decreased to ꢁ4 during the reaction) [28]. The higher
pH, 7.4, used in the present work could shift the equilibrium
toward the macrochelate B in which the deprotonated aminic
group of glutamate can coordinate to Pt.
(Fig. 6e). In the following days, peak D decreased in intensity, while
peak at 11.3 min increased (Fig. 6f). 28 days after the addition of
GSH, kiteplatin-GMP bis-adduct D was still present (Fig. 6g).
2
The above experiment was repeated under N atmosphere.
After the addition of GSH to the kiteplatin-GMP bis-adduct, the
intensity of the latter peak decreased slightly faster and it disap-
peared 23 days after the addition of GSH.
3.3. Reactivity of kiteplatin toward a mixture of GSH and GMP
4.1. Reactivity toward GMP of the interaction products between
kiteplatin and GSH
Finally, we investigated the competition between GMP
(
(
0.2 mM) and GSH (0.1 mM) for reaction with kiteplatin (0.1 mM)
Fig. 7). At time 0, the peaks belonging to GMP, GSH, kiteplatin
Addition of two equivalents of GMP to the previous solution
(kiteplatin and GSH) leads to formation of the kiteplatin-GMP
bis-adduct, confirming the results of a preliminary NMR experi-
ment carried out under different conditions (unbuffered medium
and 10-fold higher concentration of the reactants) [28]. The kite-
platin-GMP bis-adduct, formed slowly (over 10 days), strengthens
the hypothesis that kiteplatin-GSH adducts can act as drug reser-
voir [32,33], slowly releasing the Pt(cis-1,4-DACH) moiety, thus
making it available for interaction with DNA. Monitoring the reac-
tion for almost other 20 days, we observed a decrease in the con-
centration of the kiteplatin-GMP adduct D, which, however, was
present in the mixture for several days.
and some GSSG were detected (Fig. 7a). After 1 day, the GMP peak
decreased, the GSH and kiteplatin peaks also disappeared and a
new peak appeared at 9.6 min, which was assigned to guanosine
(
loss of the phosphoric group by GMP); the peak at 10.0 min was
assigned to the kiteplatin-GMP bis-adduct D. The peak at
1.5 min was not identified (Fig. 7b). The peak D increased in the
following days. This peak was prominent after 10 days at 37 °C
Fig. 7c). After 13 days, GMP had decreased considerably while
1
(
the peak of the guanosine at 9.6 min had increased (Fig. 7d), simi-
larly to what found in the stability study of GMP (Fig. S2). After
1
6 days, GMP was totally absent (Fig. 7e). The reaction was moni-
2
When the reaction was repeated under a N atmosphere, the
tored up to 30 days, and the most evident change was a slight
decrease of peak D (Fig. 7f).
formation of the kiteplatin-GMP bis-adduct was slower and its
intensity slightly increased in the first 15 days after the addition
of GMP, but it remained rather low. In the following 25 days, the
2+
concentration of bis-adduct [Pt(cis-1,4-DACH)(GMP)
until it almost disappeared. In a previous NMR experiment per-
formed under N atmosphere, the formation of the kiteplatin-
2
]
decreased
4
. Discussion
In the present work, HPLC was selected as analytical tool to ana-
2
GMP bis-adduct D [28] was not detected. This could be due to
two factors: (a) unbuffered medium and 10-fold higher concentra-
tion of reactants used in the previously reported NMR experiment;
lyze complex mixtures formed in the reaction between kiteplatin,
GSH and GMP. The formed products can be identified by comparing
their retention times with those of pure standards. Compared to
NMR spectroscopy used in the past for this type of study [12,29–
(
b) the greater sensitivity of HPLC as compared to NMR spec-
troscopy. Thus, with the concentration of [Pt(cis-1,4-DACH)
3
1], HPLC ensures higher sensitivity and allows one to work with
2
+
(
GMP)
atmosphere, it could not be detected by NMR spectroscopy.
The difference in terms of speed and yield of formation of [Pt
2
]
quite low when the reaction is carried out under N
2
f
2
+
(
cis-1,4-DACH)(GMP)
ditions, strengthens the hypothesis that the formation of GSSG,
that occurs in the presence of O , shifts the equilibrium from the
2
]
on passing from aerobic to anaerobic con-
e
guanosine
2
d
kiteplatin-GSH adducts to the kiteplatin-GMP adduct [28].
These experiments suggest that kiteplatin can platinate nuclear
DNA also after reacting with GSH present in the cytosol. This result
could be compared to the greater activity of kiteplatin in the cis-
platin-resistant A2780cisR cell line, whose resistance is connected
to elevated levels of glutathione [8]. In the case of cisplatin, it has
been proven that the interaction with cytosolic GSH leads to the
D
c
guanosine
D
B
formation of the [Pt(GS-S,N)] species, with the loss of both NH
and Cl ligands, that is a substrate for the efflux GS-X pump [21–
3]. In the case of kiteplatin, the dimer and/or the macrochelate
3
1
1.5
b
a
GMP
2
adducts, if they form inside the cell, could not be an ideal substrate
for the GS-X pump, due to their remarkably different structures
compared to [Pt(GS-S,N) ]. If so, they could act as a drug reservoir
2
permitting the platination of the nuclear DNA in a second instance.
After investigating the reactivity of GMP toward the interaction
products between kiteplatin and GSH, we proceeded with a sepa-
rate investigation of the reaction with GMP of the dimeric and
the macrochelate kiteplatin-GSH adducts. In this way we could,
GSH
GSSG
Kiteplatin
Fig. 7. HPLC chromatograms of the reaction between kiteplatin, GMP and GSH (a) at
time 0; (b) after 1 day; (c) 10 days; (d) 13 days; (e) 16 days; (f) 30 days.