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Research Article. Received: September 2 2013; Accepted: October 30 2013; Published on line 1 November 2013.
ABSTRACT
Thymosin -1 belongs to the small molecule family of thymosines, which have been studied for a long time as
molecules with very high potential use as drugs. In particular Thymosin -1 is under intensive studies for its use
in different disorders and diseases such as respiratory distress syndrome, hepatitis C, hepatitis B, AIDS, cyto-
megallovirus infection etc. Thymosin -1 is implicated in the control of gene expression of MHC I, MHC II,
cytokines and other immune system regulators, but its molecular mechanism remains still unclear. On the other
hand several studies were conducted with the aim to identify the structure of the peptide. In fact it was found that
thymosin -1 presents an -helix conformation in fluorinated alcool/water mixtures but it is highly unstructured
in water solution. It is also presumed that very likely thymosin -1 presents an -helix conformation when bound
to the receptor. Here we present a MD simulation of a Poly Ethylen Oxide Diamine stapled thymosin -1 as a
new strategy for the stabilization of its -helix conformation in water solution. The PEO diamine stapled peptide
presented a good overall stability also at high temperatures offering new insights into the design of more potent
Thymosin -1 derivative molecules.
probably the first step toward the formation of a stable prepared according the protein preparation workflow
-helix conformation and may play an important role in Maestro and the Epik module [11].
in the mechanism of action [5,6]. Each peptide was then placed in a cubic cell, then
In general the -helix is a common element of water molecules and 0.150 M of NaCl was added
protein secondary structure often involved in using the System Builder module of the Desmond
regulatory protein-protein interaction [7]. It is then package [12,13]. The molecular dynamics simulations
straightforward the use of peptides for the were performed using the OPLS 2005 force field and
construction of stable stand-alone secondary the TIP3P model for water molecules [14]. The long-
structures such as -helix with possible uses as range electrostatic interactions were calculated with
agonists or antagonists bio-drugs presenting a better the particle-mesh Ewald method (PME) [15] with a
target recognition with respect to common small grid spacing of 0.8 Angstrom. On the other hand Van
molecule obtained from synthetic chemistry. der Waals and short range electrostatic interactions
Unfortunately the high conformational instability of were smoothly truncated at 9.0 Angstrom. The
peptides is a problem to overcome and often is the Desmond package offers a default protocol for system
major cause for target binding failure. Obviously, equilibration which was adopted in this work. Such
stabilizing the -helix conformation of Thymosin -1 protocol consists in a series of restrained
in water solution is very important in order to minimizations which are very important for system
understand the molecular mechanism of interaction minimization and which allow having an overall more
with its cellular target. Moreover as demonstrated by a realistic equilibrated system and a good starting point
huge research literature, secondary structure stabilized for the real and unrestrained molecular dynamics.
peptides present 5 to 5000 fold increase in terms of Briefly the procedure consist of two rounds of steepest
target affinity [8, 9] which will be of major interest in descent minimization and of four steps of molecular
terms of Thymosin -1 dosing. There have been dynamics simulations were the first three are run as
proposed several procedures for -helix stabilization restrained for system relaxation and the last one is the
which includes incorporation or modifications of unrestrained molecular dynamics for system
single aminoacids in order to introduce in the peptide equilibration. The first simulation consist of a 12 ps
the seeding of -helix nucleation. run at a temperature of 10 K maintaining constant the
Another very successful technique for -helix number of particles, volume, and temperature. The
stabilization is the all-hydrocarbon -helix staple. second run was a 12 ps simulation performed at 10K
Briefly in such technique an olefinic chain is maintaining constant the number of particles,
introduced between two modified aminoacids in the pressure, and temperature. The third run was a 24-ps
positions i, i+3; i, i+4 or i, i+7, which induce and simulation during this run the temperature was raised
stabilize the -helix conformation by macrocyclic to 300 K and the number of particles, pressure, and
bridge formation [10]. temperature was maintained constant. The last run
In this study we used Molecular Dynamics (MD) was a 24 ps simulation performed at 300 K
to access the stability of the stapled structures of maintaining constant the number of particles,
Thymosin -1. In contrast with the all-hydrocarbon - pressure, and temperature with all restraints removed.
helix staple, we propose the -helix stapling of After the system equilibration a long time molecular
thymosin -1 with Poly Ethylen Oxide (PEO) diamine dynamic run of 5 ns was performed for each peptide
for two important reasons: first PEOs high tissue (the Thymosin -1 and the stapled ones).
compatibility and second PEOs high water solubility
that may further limit Thymosin -1 in the REMD (Replica Exchange Molecular Dynamics).
extracellular compartment where this peptide is During the replica exchange molecular dynamics
presumed to interact with its molecular targets. (REMD) approach [16] a number of copies of the
Moreover here we describe a strategy for the chemical system in parallel evolves at different temperatures
synthesis of the PEO diamine stapled Thymosin -1. and periodically the configurations are exchanged
between trajectories i and j with the probability
MATERIAL AND METHODS
Pij = exp(-D) [Eq. 1]
Initial structures of Thymosin -1 were obtained from
the published structure at www.pdb.org code 2L9I. considering that D = (bi-bj) (Ej-Ei), where Ei is the
Thymosin -1 and stapled Thymosin analogs were potential energy at the temperature Ti and b=1 kBTi.
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The systems were prepared and relaxed as described Where fh represent the helix fraction and 1-fh
earlier. In each case, during the REMD experiment 9 represent the coil fraction calculated from each
replicas of the system were evolved in parallel for 15 REMD trajectory as previously described. The
ns at constant NVT, with temperatures varying evaluation of the stability of each peptide was
between 300 and 500 K. obtained from the helix temperature of melting (Tm)
calculated from:
Molecular Dynamics Analysis.
The produced trajectories were analyzed with the Tm = H/S [Eq. 6]
VMD software [17] and modules there implemented
able to read the Desmond output trajectories in order RESULTS
to evaluate alpha helix dihedral angles and hydrogen
bonds which were very important for the peptides The structures of the Thymosin -1 and its stapled
conformation in each temperature during for this analog peptides studied in this work are shown in the
study. The presence of the alpha helix conformation Fig.1. The PEO stapling was introduced strategically
during each temperature was evaluated both from i, on the peptide between residue Glu 18 and Glu 25 as
i+4 C=O....H bond within a distance of 3.8 Angstrom PEO diamines, HNCH2CH2OCH2CH2OCH2CH2NH
and a directional angle of 30 and by evaluating the or HNCH2CH2OCH2CH2NH guided from synthesis
Phi-Psi angles obtained during the REMD experiment. strategy needs and were indicated as, Thymosin-L (the
one presenting the long PEO diamine chain) and
Free Energy Landscape Thymosin-S (the one presenting the short PEO
The free energy landscape or Potential of Mean Force diamine chain).
[18] can be obtained by calculating the probability of
finding a structure in a determined state expressed as a
function of various reaction coordinates and where
n is any set of these reaction coordinates.
Psi (degree)
expected. In fact we observed again a helix to coil 0
100
second region (pink rectangle) lie in the range of Psi
50
angle between -30, 30 and Phi range angle between
Psi (degree)
-120, -150 , the third region (red rectangle) lie in the 0
range of Psi angle between 120, 170 and Phi range -50
200
A) 50
Psi (degree)
150
0
100
-50
50
Psi (degree)
-100
0
-150
-50 T=350 K
-200
-200 -150 -100 -50 0 50 100 150 200
-100
Phi (degree)
-150
T=300 K 200
-200
-200 -150 -100 -50 0 50 100 150 200
Phi (degree)
150 C)
100
200
50
Psi (degree)
150 B) 0
100
-50
50
Psi (degree)
-100
0
-150
-50
-200
T=400 K
-200 -150 -100 -50 0 50 100 150 200
-100
Phi (degree)
-150
T=350 K
-200
-200 -150 -100 -50 0 50 100 150 200
Fig. 3. Ramachandran plots for Thymosin-L at A) 300
Phi (degree) K; B) 350 K and C) 400 K. In green Region 1; in pink
Region 2; in red Region 3 and in purple Region 4.
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50 a) b) c)
Psi (degree)
-50
-100
3.0
2.8
A)
2.6
2.4
2.2
ln Keq
2.0
1.8
1.6
1.4
1.2
1.0
0.0020 0.0022 0.0024 0.0026 0.0028 0.0030
1/T (K)
1.8
1.6
B)
Fig. 5. Free Energy Landscape of Tymosin -1 at 300 K 1.4
1.2
and conformational states of the peptide.
1.0
ln Keq
0.8
Evaluation of the stability of the Thymosin -1 and 0.6
its stapled analogs from thermodynamic parameters. 0.4
In order to obtain the thermodynamic parameters and 0.2
to evaluate the stability of structures studied in this 0.0
work, the data were fitted to the Vant Hoff equation 0.0018 0.0020 0.0022 0.0024 0.0026 0.0028 0.0030
1/T (K)
[Eq.4] as described in Materials and methods. The
equilibrium evaluated was focused on the transition 1.6
from the folded state to the unfolded state. There was 1.4
entalpic one (numbers between parentheses in Tab. 1). Fig. 2 Vant Hoff plots for helix to coil transition of A)
Thymosin -1 (R2= 0.95) B) Thymosin-L (R2= 0.99) and C)
One may observe that the increased stability of the
Thymosin-S (R2= 0.98).
stapled peptides may derive from an enthropic term.
This result can be explained as due to the rigidity
Tab. 1
introduced by the stapling to the peptide which
Thermodynamic parameters for helix to coil
produces a more ordered structure, and in last
transition
analysis accounts for the increase of the peptide order.
Infact as we can observe in Tab.1 the entropic term is Molecule H (Kcal mol-1) S (Kcal K-1 mol-1) Tm
decreased of about 1.5 fold in the stapled peptides (K)
compared to Thymosin -1. Furthermore the Tm
calculated from Eq. 6 was 293 K , 338 K and 359 K Thymosin 4.11 0.014 293
respectively for Thymosin -1, Thymosin-L and Thymosin-L 3.18 (22.6%) 0.009 (35.7%) 338
Thymosin-S and the Tm between stapled and not
stapled forms of the Thymosine -1 was 45 K and 66 Thymosin-S 3.59 (12.9%) 0.010 (28.6%) 359
K, indicating a major stability of the -helix structure
for the stapled forms of the peptide compared with
Tab. 1 Thermodynamic parameters Calculated from the
Thymosin -1.
Vant Hoff plots for helix to coil transition.
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DISCUSSION CONCLUSIONS
Thymosin -1 as previously mentioned is under We are aware that further studies are needed to fully
different phases of clinical study in some countries explore the PEO diamine stapling as a stabilizing
and in use for some particular diseases in others [19]. methodology on small peptides in order to use them as
Giving the great interest for this molecule we here potential drugs. In fact it will be of great interest to
describe a possible method for its secondary structure explore the length of the stapling chain and its
stabilization in water solution which may be very implication in the stability of the secondary alpha
important in terms of drug dosage or drug stability. In helix structure on Thymosin -1 or in its biological
this work we demonstrated the stabilization of the effects. Moreover in vitro and cellular studies are
secondary structure of Thymosin -1 by PEO diamine needed to evaluate the biological effects of these new
stapling. The thermodynamic parameters, H and S, peptides. Anyway PEO diamine stapled peptide
calculated here have the correct sign and are overall presented a good overall stability also in high
consistent with an ideal helix to coil transition temperatures offering new insights into the design of
considering the simplified system. more potent Thymosin -1 derivative molecules. The
Infact this modification induced an increase of the of stapling strategy described here may find a broad use
Tm from 293 K (20 C) for Thymosin -1 to 338 K also on other peptides when needed to stabilize their
(65C) for Thymosin-L and 359 K (86 C) for secondary structure.
Thymosin-S. This characteristic makes them good
candidates for further evaluation in vitro or in cell ABBREVIATIONS
studies. Choosing of the PEO diamine stapling was DMPC, Dimyristoylphosphatidylcholine; DMPA,
derived from two principal needs, first the synthesis Dimyristoylphosphatidic acid; TFE Trifluoroethanol.
strategy and second the biological compatibility in MD, Molecular Dynamics. NVT, constant number of
terms of weak immunogenicity and biocompatibility. atoms N, Volume V and Temperature T.