Reactions of Arsenoplatin-1 with Protein Targets:
A Combined Experimental and Theoretical Study
Arsenoplatin-1 (AP-1) is a dual-action anticancer metallodrug with a promising pharmacological profile that features the simultaneous presence of a cisplatin-like center and an arsenite center. We investigated its interactions with proteins through a joint experimental and theoretical approach. The reactivity of AP-1 with a variety of proteins, including carbonic anhydrase (CA), superoxide dismutase (SOD), myoglobin (Mb), glyceraldehyde 3-phosphate dehydrogenase (GAPDH), and human serum albumin (HSA), was analyzed by means of electrospray ionization mass spectrometry (ESI MS) measurements. In accordance with previous observations, ESI MS experiments revealed that the obtained metallodrug–protein adducts originated from the binding of the [(AP-1)-Cl]+ fragment to accessible protein residues. Remarkably, in two cases, i.e., Mb and GAPDH, the formation of a bound metallic fragment that lacked the arsenic center was highlighted. The reactions of AP-1 with various nucleophiles side chains of neutral histidine, methionine, cysteine, and selenocysteine, in neutral form as well as cysteine and selenocysteine in anionic form, were subsequently analyzed through a computational approach. We found that the aquation of AP-1 is energetically disfavored, with a reaction free energy of +19.2 kcal/mol demonstrating that AP-1 presumably attacks its biological targets through the exchange of the chloride ligand. The theoretical analysis of thermodynamics and kinetics for the ligand-exchange processes of AP-1 with His, Met, Cys, Sec, Cys–, and Sec– side chain models unveils that only neutral histidine and deprotonated cysteine and selenocysteine are able to effectively replace the chloride ligand in AP-1.
Introduction<!><!>Introduction<!>Reactions
of AP-1 with a Few Representative
Proteins Analyzed by ESI MS<!><!>Reactions
of AP-1 with a Few Representative
Proteins Analyzed by ESI MS<!>Computational Studies<!><!>Computational Studies<!><!>Computational Studies<!><!>Computational Studies<!><!>Materials<!>Sample
Preparation<!>ESI MS Analysis: Final Dilutions<!>Instrumental
Parameters<!>Computational Methods<!>Conclusions<!><!>Author Contributions<!>
<p>Transition-metal
complexes are widely used in medicinal chemistry;1−4 the case of cisplatin as an anticancer
agent being the most representative.5 As
a matter of fact, there is a continuous interest
in the development of cisplatin derivatives with the objective of
ameliorating their antitumor potency while decreasing systemic toxicity.6−8 The toxicity of cisplatin originates from the relatively easy in vivo replacement of the chloride ligands by donor atoms
of endogenous targets; actually, the testing of a plethora of less
reactive Pt ligands in the place of chloride has permitted the production
of metallodrugs with lower systemic toxicity and a higher therapeutic
index.9−13 This proves that the design of new active Pt(II)-based compounds
should involve the structure-based control of the substitution reaction.14,15</p><p>Arsenoplatin-1 (AP-1) is a novel dual-action metallodrug characterized
by an antitumor effect based on the synergetic interplay of a square
planar Pt(II) center and the coordinated arsenic trioxide moiety16 (Figure 1), resulting in a superior antitumor activity in a majority
of cancer cell lines.16,17</p><!><p>Chemical structure of arsenoplatin-1.</p><!><p>The mechanism of action of AP-1 is not yet completely
comprehended
at the molecular level, although several experimental16−19 and computational20−22 investigations have been reported so far. AP-1 binding
to DNA was studied by inductively coupled plasma mass spectrometry
on AP-1-DNA adducts extracted from triple-negative breast MDA-MB-231
cancer cells, revealing that gradual and continuous release of the
As(OH)2 moiety inside the cell results in the augmented
toxicity of arsenoplatin-1 juxtaposed to cisplatin.16 Density-functional theory (DFT) calculations unveiled that
guanine is a more favored binding site than adenine for AP-1 as well
as for other platinum-containing compounds.20 It was also shown that the hydrolysis of AP-1 necessitates a higher
energy barrier than that for DNA platination, although the barrier
for aquation is lower than that of cisplatin because of the trans
effect of the arsenic moiety.20 A detailed
computational study of the metalation of the bovine pancreatic ribonuclease
(RNase A) by AP-1 revealed the binding of His to platinum(II), retaining
the Pt–As bond. The computations evidenced that the metalation
is more advantageous in water than in the protein milieu, consistent
with the character of the protein binding pocket residues.22</p><p>Interestingly, the replacement of chloride
in AP-1 with iodide
did not hamper its cytotoxicity, thus proving that the Pt–As
core is the "true" cytotoxic metal scaffold.23</p><p>Given the differences in the mechanism
of action of arsenoplatin
compared to cisplatin, it is plausible to assume that the interactions
with proteins may play a prominent part in the action mode of AP-1.
To the best of our knowledge, there are only two investigations of
the reactivity of AP-1 with proteins. The first study focuses on the
interactions of AP-1 with the small model proteins hen egg-white lysozyme
(HEWL) and bovine pancreatic ribonuclease (RNase A). The corresponding
crystal structures of AP-1-protein adducts revealed that the preferred
binding sites for AP-1 are the His side chains in both proteins.16 Unlike cisplatin and carboplatin, which target
the sulfurs of Met side chains of RNase A,24 AP-1 does not show any preference for Met side chains. Another evidence
of AP-1 targeting His residue was offered by a recent study, in which
AP-1 was placed into the apoferritin (AFt) nanocage; the resulting
X-ray structure revealed the coordination of the AP-1 fragment to
the side chain of a His residue.19</p><p>The present study has a twofold objective.</p><p>On one hand, we
aim to expand the knowledge of the reactions of
AP-1 with proteins by considering a larger and more representative
group of proteins including human carbonic anhydrase 1 (hCA1), bovine superoxide dismutase (SOD), horse heart myoglobin (Mb),
glyceraldehyde 3-phosphate dehydrogenase (GAPDH) from rabbit muscle,
and human serum albumin (HSA). These reactions and the associated
adduct formation were examined through the classical ESI MS strategy
developed in our laboratory.</p><p>On the other hand, the reactions
of the neutral AP-1 with water
and the histidine, methionine, cysteine, and selenocysteine side chains,
which are the main candidates for protein metalation by AP-1, are
investigated by means of DFT approaches to shed light on the observed
binding preferences of protein residues for AP-1. More specifically,
we have employed simple (short) models of these residues where each
side chain is modeled by the nucleophilic group, i.e., imidazole, CH3S–, HS–, and HSe– for His, Met, Cys, and Sec residues,
respectively, whereas the remainder of the chain is rendered by an
ethyl group (Scheme 1).</p><p>Moreover, we assume that the reaction of AP-1
with protein targets
occurs through the nucleophilic substitution on the Pt(II) center
of the labile chloride ligand by the entering X ligand via an associative
interchange mechanism as depicted in Scheme 2.</p><p>To gain a broader comprehension of the binding
mechanism of AP-1
with protein targets as well as of their binding preference, we have
evaluated both the thermodynamics and kinetics of the proposed mechanistic
hypotheses. Indeed, computational studies were frequently and auspiciously
employed for the characterization of the reactivity of metals and
metallodrugs with proteins.25−28 Understanding the binding preference of AP-1 would
be very beneficial to completely understand its mechanism of action in vivo and may be advantageous to design more efficacious
anticancer drugs.</p><!><p>The reactions of AP-1 with the
model proteins HEWL and RNase A were studied by ESI MS measurements
in our previous study.16 Those results
clearly showed that AP-1 binds both proteins through coordination
of an [(AP-1)-Cl]+ fragment after the release of the Cl– ligand. Complementary X-ray diffraction studies revealed
that this metallic fragment was coordinated at the level of His15
of HEWL and His105 and His119 residues in the case of RNase A.</p><p>Here, we have extended this type of approach to a larger number of
proteins some of them also being of a considerably greater size. Specifically,
the following proteins were employed for this new study: hCA1, SOD, Mb, GAPDH, and HSA.</p><p>The interactions of these proteins
with AP-1 were investigated
according to a standard experimental setup including preparation of
the protein solution in 2 × 10–3 M ammonium
acetate at pH 6.8; addition of a threefold excess of AP-1; incubation
delay; recording of the ESI MS spectra.</p><p>The resulting deconvoluted
ESI MS spectra are reported in Figure 2, with a direct comparison
of the proteins' spectra after and before the AP-1 addition.
Particularly, the metalation of all tested proteins already took place
after 3 h of AP-1 incubation.</p><!><p>(a) Deconvoluted mass spectrum of human carbonic
anhydrase 1 (hCA1)
overlapped to the deconvoluted mass spectrum of AP-1 incubated with hCA1, at 37 °C for 3 h in 1:3 protein-to-AP-1 ratio.
(b) Deconvoluted mass spectrum of superoxide dismutase 1 (SOD1) overlapped
with the deconvoluted mass spectrum of AP-1 incubated with SOD1, at
37 °C for 3 h in 1:3 protein-to-AP-1 ratio. (c) Deconvoluted
mass spectrum of glyceraldehyde 3-phosphate dehydrogenase (GAPDH)
overlapped to the deconvoluted mass spectrum of AP-1 incubated with
GAPDH, at 37 °C for 3 h in 1:3 protein-to-AP-1 ratio, with 0.1%
v/v of formic acid shortly prior to the infusion in the mass spectrometer.
(d) Deconvoluted mass spectrum of myoglobin (Mb) overlapped with the
deconvoluted mass spectrum of AP-1 incubated with Mb, at 37 °C
for 3 h in 1:3 protein-to-AP-1 ratio, with 0.1% v/v of formic acid
shortly prior to the infusion in the mass spectrometer.</p><!><p>The interpretation of these ESI MS spectra is quite straightforward.
In most cases, AP-1-protein adducts are formed as witnessed by the
appearance of new peaks of a greater mass; however, the amount of
the formed adducts is quite limited. In most cases, a peak characterized
by a mass shift of +419 is detected. This mass increase well matches
the mass of an [(AP-1)-Cl] fragment, or of its dimer (in the case
of hCA1), in line with previous observations.29</p><p>The spectra of the tested proteins, after
24 h incubation, typically
show a net decrease in the intensity of the adducts signal, suggesting
a progressive instability of the binding.</p><p>The spectrum of the
GAPDH protein is characterized by two major
signals; one, at 35 764 Da, assigned to the native protein,
and another, at 35 797 Da, probably due to the Cys150 sulfhydration;
this double signal is also detected in the spectrum of the AP-1 adducts.</p><p>Interestingly, in the case of both GAPDH and Mb, a new fragment
of a different mass, with a shift of 311 Da, is observed. Notably,
this mass shift well corresponds to the Pt(NHC(CH3)O)2 fragment. Observation of this fragment offers direct evidence
that arsenoplatin-1, upon interaction with certain proteins, may undergo
the breaking of the As–Pt bond and the detachment of the As(OH)2 group, an interesting feature that had not been observed
in the previous studies.</p><p>In addition, AP-1 has been tested with
HSA, the main plasma protein;
we found that AP-1 manifests the tendency to react again, forming
an adduct with the Pt(NHC(CH3)O)2 fragment.
In Figure S1, Supporting Information, the
mass spectra of HSA before and after the addition of AP-1 are presented.
The deconvoluted mass spectrum of metal-free HSA is marked by the
signals at 66 438 and 66 557 Da, corresponding to the
protein in its native and its cysteinylated forms, respectively, i.e., the protein with a Cys residue bound to the Cys34.
Interestingly, AP-1 upon reacting with HSA produces adducts with both
the native and the cysteinylated proteins, primarily with the native
protein as evidenced by the lower intensity of the native protein
signal with respect to the cysteinylated protein signal.</p><!><p>A preliminary
investigation on the AP-SCN and AP-1 complexes21 has shown that the range-corrected CAM-B3LYP density functional
with the LANL2DZ effective core potential and the 6-31+G* basis set
yields the minimized structures well matching the crystallographic
data17 with Pt–S and Pt–As
bond distances within 0.04 Å error. We extracted the AP-1-His
complex from the X-ray crystallographic data for the AP-1-HEWL adduct16 and made a comparison of geometrical parameters
obtained by experiment, optimization in the gas phase, and optimization
in water (Figure 3).
We can see that most of the calculated bond distances, including the
crucial metal center–ligand bond Pt–N, were estimated
within an error of 0.01 Å, whereas the calculated Pt–As
bond is within 0.1 and 0.08 Å for the optimization in the gas
phase and solvated phase, respectively.</p><!><p>Representation of the
experimentally characterized structure of
AP-1-His (4-His) complex.16 The reported
values of bond distances are experimentally measured (black), optimized
in the gas phase (red), and optimized in the solvated phase (blue).
All distances are in angstroms.</p><!><p>The thermodynamics and kinetics of the ligand substitution of chloride
by entering nucleophile molecules were analyzed via DFT computations.
The choice of modeling the investigated protein residues with the
simplified models shown in Scheme 1, instead of capped or free amino acids, requires some
further consideration.30 On one hand, the
capped forms of amino acids are linked to the nucleophilic groups
of side chains via hydrocarbon chains of different lengths, which
leads to considerable variation in the size of the ligand interacting
with metal complex and thus substantially affecting the computation
of solvation free energies. On the other hand, free amino acids contain
terminus carboxylic acid and amine groups in ionized zwitterionic
form, which do not exist in proteins. We assume all residues to exist
in their most stable protonation state at pH = 7.2: histidine and
methionine are neutral, whereas selenocysteine is anionic (however,
we included the neutral form of selenocysteine for completeness).
Both neutral and anionic forms of cysteine were considered since both
are present at neutral pH, although the anionic form can be found
only in low concentrations. Moreover, the anionic form of cysteine
might be stabilized in the vicinity of histidine and other basic residues.</p><p>We assume that the ligand-exchange reactions on AP-1 undergo an
associative interchange mechanism, with reactants and products forming
stable noncovalent adducts before and after the reaction. Thus, the
geometries of the reactants (R), reactant adducts (RA), transition
states (TS), product adducts (PA), and products (P) were calculated.
The activation enthalpies and free energies were calculated as the
difference between TS and the lowest between reactants and reactant
adducts, whereas the reaction enthalpies and free energies were calculated
as the difference between reagents and products infinitely apart.</p><p>Initially, we analyzed the hydrolysis reaction with the exchange
of chloride by a water molecule to test the stability of AP-1 in biological
fluids and consequently determine if the chlorido or the aquo form
of AP-1 might be the reactive species with protein targets. Indeed,
it is well known that cisplatin and other biologically active metal
complexes31−33 often go through hydrolytic activation, at which
the labile aquo ligand replaces at least one ligand of the metal center.
The calculated reaction free energy value for the aquation of AP-1
is 19.2 kcal/mol, a similar value to that calculated in ref (20). This value shows that
this reaction is thermodynamically unfavorable and is improbable to
take place at physiological temperature, indicating that AP-1 is expected
to attack the biomolecular targets with the metal center in its chlorido
form.</p><p>The reaction of AP-1 with models of the His, Met, Cys,
and Sec
side chains was consequently investigated assuming the associative
interchange mechanism in each case. The optimized geometries of transition
state structures allow us to obtain interesting insights into the
reactivity disclosed by the analyzed protein residues. For example,
the Pt–As bond in all calculated transition states is 2.28–2.30
Å, suggesting that the trans effect is not observed in the geometrical
structure with a trigonal bipyramidal or square pyramidal configuration.
The transition state geometries for the reaction of AP-1 with the
neutral side chain models are characterized by an approximately trigonal
bipyramidal coordination of the Pt center (Figure 4) with all entering ligand–Pt-leaving
Cl angles ranging from 81.5 to 85.6°. The Pt–N distances
of 2.52 and 2.55 Å are observed for 4-His and 5-His, respectively,
and the same Pt–Cl distance of 2.56 Å, which suggests
that both tautomers disclose almost the same reactivity. The Pt–S/Se
bond lengths of 2.67–2.72 Å correspond well with the 2.67–2.71
Å lengths of Pt–Cl bonds for the transition states of
neutral Cys, Sec, and Met, indicating that these transition states
are neither early nor late. Deprotonated cysteine and selenocysteine,
the only anionic nucleophiles in this study, form somewhat distorted
square pyramidal transition states with S–Pt–Cl and
Se–Pt–Cl angles of 90.1 and 92.7° for Cys– and Sec–, respectively. Pt–S/Se bonds are
2.99/3.04 Å, while the Pt–N distances are 2.50 Å,
this suggests very early transition states (Figure 4).</p><!><p>Transition states for the considered nucleophiles.
All distances
are in angstroms and all angles in degrees.</p><!><p>The computed values for the reaction enthalpy and free energy for
the ligand substitution reactions involving the exchange of chloride
with the investigated models (Table S1 and Figure 5) permit us to determine
the thermodynamic preferences for Pt(II) binding to the examined side
chains of protein residues. Calculations indicate that the reaction
of AP-1 with the neutral Cys, Sec, and Met is moderately endothermic
and endergonic, whereas the reaction with His and deprotonated Cys– and Sec– is exothermic and exergonic.
It is also worth noticing that 5-His and 4-His have very close reaction
and activation free energies. In accordance with the calculated thermodynamics,
we are able to finally establish the regiochemistry for the reaction
of AP-1 with the tested side chain models in the trend Cys– > Sec– > 5-His∼4-His > Met >
Sec > Cys,
hence noting the significance of the protonation state of the nucleophile.</p><!><p>Reaction
profiles for AP-1 reacting with the considered nucleophiles.
Values in kcal/mol are computed in solution at the CAM-B3LYP/LANL08(f)/6-311++G**//CAM-B3LYP/LANL2DZ/6-31+G*
level.</p><!><p>The activation free energies for
the reaction of AP-1 with 5-His
and 4-His are 19.5 and 18.9 kcal/mol, respectively, resulting in the
lowest barriers among the considered neutral residues. Moreover, the
reaction free energies of −0.7 and −0.8 kcal/mol for
5-His and 4-His, respectively, make them the only neutral protein
side chains targeted by AP in both exothermic and exergonic processes.
The reaction of AP-1 with either Met, Cys, or Sec model is affected
by a higher activation free energy, i.e., 20.6, 22.6, or 21.6 kcal/mol,
respectively, as well as higher reaction free energies of 8.6, 11.3,
and 10.0 kcal/mol in the same order, respectively. Our calculations
clearly indicate the chloride substitution by His results to be both
thermodynamically and kinetically most favorable compared to the other
neutral side chain models.</p><p>On the other hand, the reaction of
AP-1 with cysteine and selenocysteine
in their anionic forms results to be both thermodynamically and kinetically
the most favorable. Indeed, the computed activation free energy values
for the exchange of chloride ligand with Cys– and
Sec– are only 15.1 and 15.4 kcal/mol, respectively,
whereas the corresponding values for the reaction free energy are
−16.2 and −15.5 kcal/mol.</p><p>An overall insight into
both thermodynamics and kinetics of the
ligand substitution reactions investigated in the present study is
provided in Figure 5. The results suggest that AP is expected to preferentially bind
at His residues unless deprotonated cysteine or selenocysteine are
available.</p><p>The binding preference in the AP-1 protein targeting
obtained by
our computational models implicitly assumes the same steric accessibility
of these residues. However, protein side chains characterized by a
high solvent exposure are more reachable to the AP complex and thus
expected to be more reactive.</p><p>To better assess the targetability
of specific protein systems
by AP-1, the regioselectivity based on the nucleophilic substitution
must be paralleled by a study on the solvent exposure of the considered
protein residues. Hence, solvent-accessible surface (SAS) analyses
(Figure 6) were performed
on the X-ray structures of the hen egg-white lysozyme (HEWL) and the
bovine pancreatic ribonuclease (RNase A) proteins (pdb ids 5nj1 and 5nj7(16)), showing that the His residues that form adducts with
AP-1 are closer to the surface. Indeed, the residues His12 and His48
in the bovine pancreatic ribonuclease are not metallated and, after
a thorough investigation of the X-ray structure, we might conclude
that the only reason is solvent inaccessibility. Cysteines and methionines
are also situated in regions with less solvent accessibility, although
not very deep, and are also not metallated.</p><!><p>Solvent-accessible surface
(SAS) computed on the X-ray structures
of adducts of AP with two small model proteins, hen egg-white lysozyme
(HEWL), and bovine pancreatic ribonuclease (RNase A) (pdb entries 5nj1 and 5nj7). Circles, squares,
and diamonds indicate His, Met, and Cys protein side chains, respectively.
AP-1 bound amino acids are indicated with arrows.</p><!><p>Lyophilized human carbonic anhydrase (hCA
I), glyceraldehyde 3-phosphate dehydrogenase (GAPDH) from rabbit muscle,
bovine superoxide dismutase (SOD), myoglobin (Mb) from horse heart,
and human serum albumin (HSA) were acquired from Merck and utilized
without additional purification or manipulation. AP-1 was synthesized
in the MetMed laboratories at the Department of Chemistry, University
of Florence in accordance with already established procedures.13,14</p><p>Dimethyl sulfoxide (DMSO) was acquired from Fluka. Liquid
chromatography-mass spectrometry (LC-MS) materials (water and ammonium
acetate) were procured from Honeywell.</p><!><p>Stock solutions of hCA I 10–4 M, HSA 10–3 M, GAPDH 10–4 M,
SOD 5 × 10–3 M, and Mb were prepared by
dissolving the proteins and the peptide in H2O LC-MS grade.
Stock solutions 10–2 M of the AP-1 compound was
obtained dissolving the samples in DMSO.</p><p>For the experiments
with hCA I, solutions of the protein 10–5 M and
AP-1 at protein-to-metal ratio 1:3 were prepared and diluted with
ammonium acetate solution 2 × 10–3 M (pH 6.8).
The mixtures were then incubated at 37 °C up to 24 h.</p><p>For
the experiments with GADPH, aliquots of the stock solutions
were mixed with aliquots of AP-1 at protein-to-metal ratio 1:3 and
diluted with ammonium acetate solution 2 × 10–3 M (pH 6.8) to 10–5 M final protein concentration.
The mixtures were incubated at 37 °C up to 1 h.</p><p>For the
experiments with Mb and SOD, solutions of the protein 10–5 M and AP-1 at protein-to-metal ratio 1:3 were prepared
by diluting with ammonium acetate solution 2 × 10–3 M (pH 6.8). The mixtures were then incubated at 37 °C up to
24 h.</p><p>For the experiments with HSA, solution of the protein
10–4 M and AP-1 at the protein-to-metal ratio of
1:0.9 or 1:3 was prepared
and diluted with ammonium acetate solution 2 × 10–3 M (pH 6.8). The mixture was then incubated at 37 °C up to 24
h.</p><!><p>After the incubation
time, all solutions were sampled and diluted to a final protein concentration
of 5 × 10–7 M for hCA I, HSA, GAPDH, and 10–7 M for Mb and SOD using ammonium acetate solution
2 × 10–3 M (pH 6.8).</p><p>In the final Mb,
GAPDH and HSA solutions were also added with 0.1% v/v of formic acid
shortly prior to the infusion in the mass spectrometer.</p><!><p>The ESI mass study was carried
out utilizing a TripleTOF 5600+ high-resolution mass spectrometer
(Sciex, Framingham, MA) furnished with a DuoSpray interface operating
with an ESI probe. Respective ESI mass spectra were obtained via direct
infusion at a flow rate of 5 Î¼L/min.</p><p>The general ESI source
parameters optimized for each protein and peptide analysis were as
follows.</p><p>SOD parameters are positive polarity, ion spray voltage
floating
5500 V, temperature 0, ion source gas 1 (GS1) 25 L/min; ion source
gas 2 (GS2) 0, curtain gas (CUR) 15 L/min, collision energy (CE) 10
V, declustering potential (DP) 200 V, and range 1300–3400 m/z.</p><p>Mb parameters are positive polarity,
ion spray voltage floating
5500 V, temperature 0, ion source gas 1 (GS1) 40 L/min, ion source
gas 2 (GS2) 0, curtain gas (CUR) 15 L/min, collision energy (CE) 10
V, declustering potential (DP) 100 V, and range 700–2200 m/z.</p><p>GAPDH parameters are positive
polarity, ion spray voltage floating
5500 V, temperature 0, ion source gas 1 (GS1) 20 L/min, ion source
gas 2 (GS2) 0, curtain gas (CUR) 15 L/min, collision energy (CE) 10
V, declustering potential (DP) 100 V, and acquisition range 600–2000 m/z.</p><p>hCA I parameters are positive
polarity, ion spray voltage floating
5500 V, temperature 0, ion source gas 1 (GS1) 25 L/min, ion source
gas 2 (GS2) 0, curtain gas (CUR) 20 L/min, collision energy (CE) 10
V, declustering potential (DP) 300 V, and range 1500–3500 m/z.</p><p>HSA parameters are positive
polarity, ion spray voltage floating
5500 V, temperature 0, ion source gas 1 (GS1) 40 L/min, ion source
gas 2 (GS2) 0, curtain gas (CUR) 20 L/min, collision energy (CE) 10
V, declustering potential (DP) 200 V, and range 900–2600 m/z.</p><p>For acquisition, Analyst TF
software 1.7.1 (Sciex) was employed,
and deconvoluted spectra were attained by utilizing the Bio Tool Kit
microapplication v.2.2 embedded in PeakView software v.2.2 (Sciex).</p><!><p>All computations were carried
out with the Gaussian 09 A.0234 quantum
chemistry package. Optimizations and the determination of electronic
and solvation energies were performed in solvated phase (C-PCM)35,36 and by employing the density functionals as described below.</p><p>All geometrical optimizations were performed with the LANL2DZ effective
core potential for Pt atom37 and the 6-31+G*
basis set for other elements,38,39 while single-point
electronic and solvation energy computations were performed with the
LANL08(f) effective core potential for platinum37,40 and the 6-311++G** basis set for other elements.41−43 We used the
range-corrected DFT functional CAM-B3LYP44 for geometrical optimization and electronic and solvation energies
calculations. As we have shown elsewhere,21 the CAM-B3LYP/LANL08(f)/6-311++G**//CAM-B3LYP/LANL2DZ/6-31+G* functional-basis
set combination yields the best results for geometry and energy computations
for the aquation of arsenoplatin-1. DFT functionals are recognized
to produce adequate geometries and reaction profiles for transition-metal-containing
compounds45−48 including Pt-based anticancer compounds.49−51</p><p>Frequency
calculations were carried out to confirm the convergence
to the stationary points and to evaluate zero-point energy (ZPE) and
thermal corrections to thermodynamic properties. Intrinsic reaction
coordinate (IRC) computations were utilized to determine reactants
and products minima connected with the transition states for each
examined reaction step.</p><p>Single-point electronic energy computations
were performed on the
geometries optimized in the solution. The C-PCM continuum solvent
methodology was employed to account for solvation.35 It was demonstrated to yield significantly smaller discrepancies
than other continuum models for aqueous free energies of solvation
for cations, anions, and neutrals and to be especially efficacious
for the calculations of solution properties necessitating an enhanced
accuracy of solution free energies.52 Free
energies of solvation, considered as the difference between the solution
energies and the gas phase energies, were added to the gas phase enthalpies
and free energies values to have the corresponding values in the aqueous
solution.</p><p>The solvent-accessible surface (SAS) of each examined
amino acid
was calculated by employing the SAS option available in Gromacs software.53</p><!><p>This
study includes a combined experimental and theoretical investigation
of arsenoplatin-1 interactions with protein targets. The analysis
of the biomolecular interactions of AP-1 grounded on ESI MS measurements
was extended here to a larger number of proteins than in the past,
including carbonic anhydrase, superoxide dismutase, myoglobin, glyceraldehyde
3-phosphate dehydrogenase, and human serum albumin. The ESI MS results
reveal that AP-1 generates in most cases tight adducts with the studied
proteins containing the [(AP-1)-Cl]+ fragment, in nice
agreement with previous observations made on HEWL and RNase A, and
with the computational analysis carried out here. More in detail,
the computational studies have considered the reactions of AP-1 with
various nucleophiles, which mimic the side chains of neutral histidine,
methionine, cysteine, and selenocysteine in neutral form as well as
cysteine and selenocysteine in anionic form. The aquation of AP-1
is energetically disfavored with the reaction free energy of 19.2
kcal/mol, thus indicating that AP-1 presumably attacks its biomolecular
targets by the direct substitution of the chloride ligand. The theoretical
examination of thermodynamics and kinetics for the ligand substitution
processes of AP with His, Met, Cys, Sec, Cys–, and
Sec– side chain models revealed that only neutral
histidine and deprotonated cysteine and selenocysteine can effectively
replace the chloride ligand in AP-1.</p><p>Moreover, a different and
innovative result has been achieved here
through the ESI MS experiments when reacting AP-1 with GAPDH and Mb.
Indeed, in these latter cases, the adducts just contained a smaller
fragment where the [As(OH)2] moiety is lost. This result
is of particular interest as it provides direct evidence that arsenoplatin-1
may undergo degradation in the biological milieu, with the cleavage
of the As–Pt bond giving rise to a protein-bonded platinum-containing
fragment while releasing an arsenic-containing fragment.</p><p>Although
the mechanistic details of the [As(OH)2] detachment
from AP-1 were not expressly addressed in the present study, our calculations
showed that the Pt–As distance is not significantly affected
when replacing chloride by a nucleophilic protein ligand. This computational
outcome suggests that the [As(OH)2] release is probably
subsequent to protein metalation and may be kinetically influenced
by the protein environment surrounding the Pt(II) binding site.</p><!><p>S1: Deconvoluted
mass spectrum of human serum albumin
and incubated with AP-1. S2: Enthalpy and Gibbs free energy values
for the reaction of AP-1 with the investigated protein residue models
in solution (PDF)</p><p>ic1c03732_si_001.pdf</p><!><p>The manuscript
was written through contributions of all authors. All authors have
given approval to the final version of the manuscript.</p><!><p>L.M., L.Ma.,
and M.T. thank the Ente Cassa Risparmio Firenze (ECR) and AIRC for
funding the project "Advanced mass spectrometry tools for cancer
research: novel applications in proteomics, metabolomics, and nanomedicine"
(Multi-user Equipment Program 2016, ref code 19650). T.M. thanks Beneficentia
Stiftung, Vaduz (BEN2019/48) and the University of Pisa, "PRA
– Progetti di Ricerca di Ateneo" Institutional Research
Grants – Project no. PRA_2020_58 "Agenti innovative
e nanosistemi per target molecolari nell'ambito dell'oncologia
di precisione".</p><p>The authors declare no
competing financial interest.</p>