22nd International Symposium on Plasma Chemistry
July 5-10, 2015; Antwerp, Belgium
Mutation of P-glycoprotein by plasma-generated OH radicals
N. Khosravian, E.C. Neyts, B. Kamaraj and A. Bogaerts
Research Group PLASMANT, Department of Chemistry, University of Antwerp, 2610 Antwerpen-Wilrijk, Belgium
Abstract: Multidrug resistance (MDR) in the treatment of a wide number of cancers is
attributed to the function of P-glycoprotein. In this work, the effect of OH radicals – as
important plasma species on the transmembrane (TM) segment 6 of human P-glycoprotein
is studied using the density functional tight binding approach (DFTB). Furthermore,
non-reactive molecular dynamic simulations are employed to investigate the structural
behaviour of TM6 after reaction with OH radicals.
Keywords: plasma medicine, OH radicals, Cancer, DFTB, molecular dynamics
1. Introduction
The application of non-thermal atmospheric pressure
plasma (NTAPP) is emerging as an efficient technique in
medicine. It includes sterilization [1], treatment of skin
diseases [2], and cancer treatment [3-7], so-called
“plasma oncology”. An overview of many studies in
plasma oncology was published in a recent review paper
[3]. Moreover, a recent study reveals that pre-treatment
of glioblastoma (GBM), which is the most common and
aggressive brain tumor in adults, by plasma will reduce
the resistance of this tumor cell against chemotherapy [8].
The anti-cancer treatment by NTAPP is mainly due to
the unique properties of plasma-generated reactive species
such as neutral species (molecules, atoms, radicals and
excited species), free electrons and (positive and negative)
ions, as well as photons. The advantage of plasmas is
their operation at body temperature and atmospheric
pressure, as well as their ability to attack cancer cells,
without damaging healthy cells.
In general, it is accepted that reactive oxygen and
nitrogen species (ROS/RNS/RONS) formed in the plasma
(e.g., OH, HO 2 , H 2 O 2 , O 3 , NO, NO 2 , ONOO-) play a
crucial role in plasma oncology. They are able to react
with bio-molecules (i.e., DNA, proteins, and lipids), and
thereby oxidize proteins, lipids and DNA, which may
eventually lead to apoptosis.
Although many studies show promising results in the
application of plasma in cancer treatment, the underlying
mechanisms and their biological effects still remain
unclear.
In the context of plasma-mediated cancer treatment, it is
very important to also elucidate the molecular basis of the
plasma effects. In this regard, the interaction of plasma
agents (ROS/RONS/RNS) with biomolecules must be
investigated.
Among the proteins, which are targeted in cancer
treatment, P-glycoprotein, as one of the membrane
proteins, attracted most attention. This protein is highly
expressed in tumor cells and causes reduction in access to
cytotoxic drugs. Therefore, tumours resist to various
anticancer drugs.
It is believed that the treatment failure in over 90% of
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patients with metastatic cancer is mainly due to a failure
in response to chemotherapy by acquiring multidrug
resistance [9] that is related to the function of
P-glycoprotein.
P-glycoprotein is composed of 1280 amino acids and is
expressed as a 170 - 180 kDa plasma membrane. This
protein has two symmetrical nucleotide binding domains
(NBDs) and transmembrane domains (TMs) inclusive of
six 𝛼-helical membrane-spanning domains. The active
pore, through which drugs are exported, has been formed
by twelve TM segments and NBDs oriented in the
cytoplasm. TM 1, 5, 6, 11, and 12 have critical roles in
the binding of substrates to P-glycoprotein.
The drug binding pocket of p-glycoprotein is made of
hydrophobic and aromatic residues, such as phenylalanine
(Phe) and Tyrosine (Tyr). It is known that these residues
act as binding sites of P-glycoprotein, which strongly
interact with ligand molecules [10].
Blocking P-glycoprotein in cancer cells and increasing
its susceptibility to drug has become a major challenge in
cancer treatment. In this regard, deactivating the active
domain of P-glycoprotein by introducing the antibodies
that can bind to the active site of P-glycoprotein and block
the motions needed for pumping was studied [11].
Also, it has been reported that substitution of
phenylalanine with alanine at position 335 in TM6 can
reduce the affinity of this protein to vinblastine and
actinomycin D in comparison with that of the wild-type
P-glycoprotein [12]. In short, vinblastine is an antimicrotubule drug used to treat certain kinds of cancer,
including Hodgkin's lymphoma, non-small cell lung
cancer, breast cancer, head and neck cancer, and testicular
cancer.
With respect to the role of TM6 in drug binding, the
interaction of OH radicals with active amino acids of
TM6 (i.e., Phe 332 and Phe 339) will be studied. For this
purpose, atomistic simulations are employed to gain
insight into the fundamental aspects of reaction
mechanisms of OH radicals with this active site of
P-glycoprotein.
1
2. Methodology
An approximative quantum-mechanical simulation
method, named the self-consistent charge density
functional tight binding (SCC-DFTB) method, was used
to investigate the reaction mechanisms of the impact of
OH radicals on TM6 in gas phase. SCC-DFTB is based
on a second-order expansion of the Kohn-Sham total
energy with respect to charge density variations δρ,
relative to a chosen ρ 0 reference density, in the LCAO
(tight-binding) framework [13]. This method has been
used for some small biological systems, such as peptides
[14, 15]. Also, DFTB has shown good description for
DNA base geometry [16]. Moreover, it has been tested in
the QM part of the QM/MM approach for peptides and
proteins in MD simulations [17-20].
In our simulation, TM6 is first minimized and then
equilibrated at 300 K. After thermalization, OH radicals
are inserted in the simulation box. The structure of TM6
is shown in Fig. 1.
by PME (Particle Mesh Ewald) method. At the end, we
performed simulations for 100 ns. We then computed the
comparative root-mean-square deviation (RMSD)
(RMSD) and root-mean-square fluctuation (RMSF)
analysis of the structural significance in native and mutant
TM6 of P-glycoprotein.
3. Result and discussion
The results obtained by DFTB indicate that the OH
radicals can react with the active sites of phenylalanine
that are represented in Fig. 2.
Fig. 2. Structure of phenylalanine.
Fig. 1. Structure of TM6 represented in ball and stick
form. The blue, red, grey and small white balls indicate
nitrogen, oxygen, carbon and hydrogen atoms,
respectively.
In order to investigate the flexibility of TM6 after
reaction with OH radicals, long time MD simulations are
required. Since MD simulations in the nanosecond time
scale using DFTB are computationally demanding, nonreactive MD simulations using GROMACS 4.6.1 [21]
with the OPLS all-atom force field [22] are performed to
compare the flexibility of native and mutant TM6 (i.e., the
reaction product of the interaction of OH radicals with
TM6).
Wild type and mutant TM6 were used as initial
configurations for the MD simulations. The simulation
setup adopted in this work is as follows.
Molecules are located at the center of the simulation
box with dimensions of 55.32×55.32×55.32 Å3. The
simulation box was filled with TIP3P water molecules.
Afterwards, steepest descent was employed to minimize
the system for 5000 iterations. After minimization, the
system is equilibrated in NVT at 300 K using
the Berendsen thermostat, and subsequently in NPT at 1
atm with an allowed compressibility range of 4.5 x 105
atm, by employing the Berendsen barostat for 1000 ps.
The time step is chosen as 2 fs. During the equilibration,
position restrain is applied on the solute molecule (TM6),
therefore the solvent can equilibrate around the protein
without the added variable of structural changes in the
protein. Long range electrostatic interactions are treated
2
The reaction path consists of H-abstraction from the
alpha and beta site and OH addition to the i, o, m and p
sites of phenylalanine. The letters i, o, m and p stands for
ipso, ortho, meta and para sites, respectively.
We performed RMSD and RMSF analysis for both the
native and mutant TM6 of p-glycoprotein. In the RMSD
plot (Fig. 3), from the start to 22 ns, the native and mutant
structures show a similar deviation. After that, the mutant
structure shows fewer deviations than the native structure
until the end of the simulation. Form the RMSD plot, we
observe that, due to the mutations, TM6 of P-glycoprotein
loses its stability and alters its overall structural
conformation.
Fig. 3. RMSD result of native and mutant TM6.
The conformational changes of the TM6 of
P-glycoprotein upon mutation were further supported by
RMSF analysis. The RMSF values of native and mutant
structures are shown in Fig. 4. In the RMSF plot, a higher
degree of flexibility is observed in the native structure
than in the mutant structure. It confirms that due to the
mutation, the structure loses its flexibility and becomes
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more rigid. These structural changes may affect the
function of TM6 of P-glycoprotein.
Fig. 4. RMSF result of native and mutant TM6.
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