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5-Fluorouracil Mediated Anti-Cancer Activity in Colon Cancer Cells is through the
Induction of Adenomatous Polyposis Coli: Implication of the Long-Patch Base Excision
Repair Pathway
Dipon Das1, Ranjan Preet1, Purusottam Mohapatra1, Shakti Ranjan Satapathy1, Sumit
Siddharth1, Tigist Tamir2, VaibhavJain3, Prasad V. Bharatam3, Michael D. Wyatt2, Chanakya
Nath Kundu1*
1KIIT School of Biotechnology, KIIT University, Campus-11, Patia, Bhubaneswar, Orissa,
751024, India.
2Department of Drug Discovery and Biomedical Sciences, South Carolina College of
Pharmacy, University of South Carolina, Columbia, SC, USA
3Department of Pharmacoinformatics, National Institute of Pharmaceutical Education and
Research (NIPER), Sector 67, S.A.S. Nagar (Mohali), Punjab-160062, India
To whom correspondence should be addressed: *Chanakya Nath Kundu, KIIT School of
Biotechnology, KIIT University, Campus-11, Patia, Bhubaneswar, Orissa, 751024, India.
Tel : +91-674-272-5466 ; Fax: +91-674-272-5732 ; E-mail: cnkundu@gmail.com
1
Supplementary Information
Molecular modelling and docking studies: Methodology
The three dimensional (3D) structure of a few sub domains of APC are available in Protein
Data Bank (PDB), but no 3D structure of the DRI domain from X-ray crystallography, NMR,
or homology modelling has yet been reported. Thus, in order to create a reliable structure of
the DRI domain, an extended chain of amino acids 1141-1330 of APC was considered and a
homology model was constructed. The specific APC protein sequence (1141-1330) was
retrieved from the UniProt database (Uniprot ID: P25054) (Magrane, 2011). The query
sequence was subjected to the BLASTp program (Altschul et al, 1990) using BLOSUM62
matrix against Protein Data Bank (PDB) (keeping Expect value cut off of 100) in order to
search suitable templates for homology modelling. This resulted in various sequences
showing significant alignment with parts of the query sequence. Three protein crystal
structures; PDB ID: 3DY5 (Gilbert et al, 2008) (chain A, identity-34% and similarity-53%),
PDB ID: 2BNH (Kobe and Deisenhofer, 1996) (chain A, identity-26% and similarity-40%)
and PDB ID: 3A2S (Tanabe et al, 2010) (chain X, identity-34% and similarity-48%) were
chosen as templates based on their identity, similarity, and the region aligned with the query
sequence (N-terminal, central, and C-terminal regions respectively). Subsequently, a
homology model of the APC protein (1141-1330) was constructed using the EasyModeller
2.0 programme (Kuntal et al, 2010), which is a graphical user interface of the MODELLER
software (Marti-Renom et al, 2000). This homology model built from 3 different templates
was crude and possesses poor sterochemical parameters as assessed by PROCHECK statistics
(Laskowski et al, 1993) and Errat plots (Colovos and Yeates, 1993). The constructed model
is mostly composed of loops, which are highly flexible in nature. The secondary structure 2
prediction of the query protein APC (1141-1330) using PSIPRED server
(http://bioinf.cs.ucl.ac.uk/psipred/) also showed the maximum occupancy of loops in the
structure.
In order to obtain a refined and more realistic structure of the APC protein (1441-1330), full
atomic molecular dynamics (MD) simulations were performed in TIP3P with water as the
explicit solvent using AMBER11 program package (Case et al, 2010), implementing
AMBERff99SB force field. After long run MD simulations (2 ns equilibration and 15 ns
production), 10 random snapshots representing the minimum energy of the system were
extracted from the equilibrated trajectories (last 5 ns). The best refined model of APC protein
(1441-1330) was selected on the basis of PROCHECK statistics (Ramachandran plot) and
ERRAT plot. The MD refined homology model of APC was prepared for the docking study
using the Protein Preparation Wizard tool implemented in Maestro version 9.2 (Schrödinger
Inc. 2011). The 3D structure of the ligand 5-FU was built using the Maestro interface of the
Schrödinger molecular modelling suit. OPLS-2005 charges were assigned to the atoms of the
ligand and energy minimization was carried out using the Ligprep module of Maestro
interface by implementing OPLS-2005 force field. An interaction grid was generated for the
MD refined, modelled structure of APC by using the receptor grid generation wizard of
GLIDE 5.5 (Grid Based Ligand docking with Energetics). The grid box was defined by
selecting the centroid of the residues involved in DRI domain (1245-1273) and further
extended to 20 Å in all the directions. All docking calculations were performed at the
Standard Precision (SP) mode and Glide scoring function (Friesner et al, 2004) was used to
assess the binding affinity of a ligand with the protein. OPLS-2005 force field was
implemented for docking simulations, while keeping all other parameters as default. 10 poses
per ligand were generated and all of them were analysed for their Glide Score, E-model score
and intermolecular interactions with the residues of the protein.
3
Homology model refinement using molecular dynamics simulations: Methodology
Molecular dynamics (MD) simulations are well established methodology for the refinement
and assessing the stability of a protein during dynamic conditions (Karplus and McCammon,
2002). They can also be used to obtain the realistic structure of a protein (if its 3D structure is
not known) by allowing the time evolution of a system and conformational changes that
occur during the course of simulation under mimicked physiological environment (Fan and
Mark, 2004).
In the present study MD simulations were performed on a crude homology modeled structure
of the APC protein (amino acids 1141-1330) using AMBER11 programme package (Case
and Darden, 2010). Structure of the protein was solvated with TIP3P water model (Jorgensen,
1983) with solvent buffer being extended to 10 Å in each direction of the solute forming a
cube. In addition, some of the water molecules were replaced by Na+ counterions to
neutralize the negative charge build on the protein. To calculate the non-bonded interactions,
the cut-off distance was kept at 10 Å and long-range electrostatic interactions were treated by
the Particle-Mesh Ewald (PME) method (Darden et al, 1993). The minimization of the
system was carried out in 3 consecutive steps. In the first step, the protein was restrained with
a force constant of 3 kcal/mol/Å2 and only solvent phase was minimized with 500 cycles of
each steepest descent and conjugate gradient methods. This allowed the TIP3P water
molecules to reorganize themselves to eliminate any bad contacts with the protein. In the
second step, the restraint weight was reduced to 1 kcal/mol/Å2 to maintain gradual decrease
and 250 cycles of minimization was carried with both methods. In the third step, the whole
system was minimized for 1000 cycles of steepest descent and 24000 cycles of conjugate
gradient without any restraints in order to relax the system.. The minimized system was 4
subjected to heating under NVT ensemble for 50 ps where the system was gradually heated
from 0 to 300 K by putting restraint on the protein with a force constant of 2 kcal/mol/Å2.
Subsequently, 50 ps of density equilibration was carried out under NPT ensemble with weak
restraints of 2 kcal/mol/Å2, followed by 100 ps of the same with restraints of 1 kcal/mol/Å2 on
the protein and then 50 ps of density equilibration without any restraints. Further, constant
pressure equilibration (NPT) was performed for 2 ns at 300 K and 1 atm pressure with
pressure relaxation time of 2.0 ps, followed by final production run for about 15 ns by using
the same simulation parameters used in equilibration run. During the whole simulation run,
periodic boundary condition was enabled and all the covalent bonds containing hydrogen
atoms were constrained using SHAKE algorithm (Ryckaert et al, 1977). Equations of motion
were solved using the Verlet leapfrog algorithm (Verlet, 1967). The integration time step was
set to 2 fs and the trajectory was recorded in every 2 ps. A Langevin thermostat and barostat
was used for temperature and pressure coupling with a collision frequency of 2.0.
5
Figure. S1 Fluctuations in the Cα backbone RMSD of the homology model of APC protein
(amino acids 1141-1330) during the whole simulation run (17 ns)
Figure. S2 Secondary structure of the final MD refined homology model of APC protein
(1141-1330). The DRI domain (1250-1269) of APC is characterized by short stretch of alpha
helix (1258-1266).
Ramachandran and Errat plots of the APC homology model
The Ramachandran plot of the MD refined final homology model of APC showed 79.4% of
the residues in the allowed region, 18.3% in the additionally allowed region, 1.7% in the
generously allowed region, while only 0.6% i.e., one residue, Ser1223 (away from the region
of our interest) was found in the disallowed region (Fig. S3). These statistics indicate that the
6
backbone dihedral angles (phi and psi) distribution in the amino acids of the APC homology
model is reasonably accurate. In addition, the model also showed acceptable ERRAT plot
with the overall quality factor of 90.0% (Fig. S4). The validation statistics thus confirm that
the model developed has an acceptable structure and can be used for molecular docking
studies.
Figure. S3 Ramachandran plot statistics of the final MD refined homology model of APC
protein (amino acids 1141-1330)
7
Figure. S4 Errat plot of the final MD refined homology model of APC (amino acids 1141-
1330). The DRI domain (1250-1269) is particularly well modelled as indicated by the Errat
plot (no steric clashes in the side chain and low error value)
8
Figure. S5 2D interaction profile of 5-FU with the final MD refined homology model of APC
protein (1141-1330). This figure was generated by the Maestro interface of Schrödinger
molecular modeling suit
9
Figure. S6 Western blot of whole cell extracts of HCT-116 cells expressing GFP (WT
control) or expressing siRNA against POLβ (KD). The cells were a kind gift from R. Sobol,
University of Pittsburgh.
Figure. S7 Viability of HCT-116 cells expressing GFP (WT control) or expressing siRNA
against POLβ (KD) and treated with 5-FU + LV. Viabilty was performed by MTT as
described in the Materials and Methods. Data is the mean ± SD of three different experiments
10
% V
iabi
lity
Pol β
β-actin
HCT-116KD WT
Figure. S8 A schematic diagram showing the action of 5-FU and APC-mediated inhibition of
LP-BER. Steps 1-7 show the normal LP-BER process and steps 8-11 show 5-FU mediated
LP-BER inhibition and cell death in CRC. 5-FU induces APC protein levels. When 5-FU
binds to APC, it displaces FEN1 from the DNA repair complex, which then promotes
proteasome-mediated degradation of FEN1. Lack of FEN1 activity impairs flap removal,
which prevents completion of repair and ultimately leads to apoptosis.
References
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