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

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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.

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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.

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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

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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)

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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)

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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

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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.

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