1. Inventi Rapid: Molecular Modeling Vol. 2014, Issue 4
[ISSN 2278-4071]
2014 pmm 14329, CCC: $10 © Inventi Journals (P) Ltd
Published on Web 04/10/2014, www.inventi.in
RESEARCH ARTICLE
INTRODUCTION
Cancer is uncontrolled growth of cells, which may cause by
physical, environmental metabolic, chemical and genetic
factors. WHO report 2007, says cancer cause human death
about 7.9 million per year in worldwide and it will increase
up to 12 million by 2030. TRAIL (Apo2L) is expressing on
various immune cells include natural killer (NK) cells, T
cells, natural killer T cells (NKT cells), dendritic cells and
macrophages. [1] TRAIL is very good anticancer candidate, it
induce apoptosis via binding with two receptors, namely
TRAIL-R1 and TRAIL2. [2,3] This binding attract FAAD
towards TRAIL-Rs, then FAAD recruits pro-caspase 8 and
form Death Induce Signaling complex (DISC), where pro-
caspase 8/10 become activate, caspase 8/10 activate the
effector caspase 3/7 and apoptosis is taking place, [4, 5] this
process know as extrinsic apoptosis. Some cases caspase
8/10 signal is not enough to active caspase 3/7, in that
condition caspase 8/10 is link with mitochondrial pathway
apoptosis by converting bid into tbid. [6, 7]
TRAIL selectively induces the apoptosis in cancer cells
and has no cytotoxicity towards the normal cells. Because
of its specificity TRAIL was expected to be potential anti
cancer drug. But, due to over expression of anti-apoptotic
proteints, all cancer cells became resistant to TRAIL, [8, 9]
hence it did give anticipated result in clinical trials. cFLIP is
one of the important anti-apoptotic protein which could
bind with FAAD and prevents pro-caspase 8 activation. [10,
11] So the current challenge is overcome the TRAIL
resistance by inhibiting the anti-apoptotic proteins like
cFLIP. The present investigation was aimed to Insilco
screen the fungal peptides for inhibiting cFLIP by using
Hex6.0 docking software.
MATERIALS AND METHODS
cFLIP PDB Protein Structure
1Centre of Advanced Study in Marine Biology, Annamalai University,
Parangipettai-608502, Tamilnadu, India.
E-mail: sathiishkumarbiotech@gmail.com
*Corresponding author
Crystal structure of cFLIP protein was retrieved from
Protein Data Bank (PDB) (http://www.rcsb.org/pdb) as a
pdb file (ID: 3H13).
Ligand Preparation
The fungal peptide structure was retrieved from Pubchem
database (http://www.ncbi.nlm.nih.gov/pccompound) as
sdf file (Table 1). This sdf file was converted into pdb
format by using Open Babel.
Hex 8.0.0 Docking Analysis
Docking studies were performed with help of Hex 8.0.0. [12]
Hex window was opened. Receptor and ligand molecules
were retrieved from the file menu, docking were activated
from option control and the following docking parameters
were used, correlation type – Shape only, Grid Dimension –
0.6, Receptor range – 180, Ligand Range – 180, Twist range
– 360, distance Range–40. Finally binding energy (ΔE) was
obtained and saved carefully. The docking complex was
saved separately in .pdb format for Ligplot analysis.
Ligplot Analysis
Ligplot was performed to find protein-ligand interface after
Hex 8.0.0 dock. [13] In Ligplot window, the receptor- protein
complex.pdb files was opened. Ligand was selected and
clicked the run button. Plot has been generated on screen
and binding interfaces were viewed in Pymol.
RESULTS
Hex Docking Confirmations of Fungal Peptides with
cFLIP
The Hex 8.0.0 analysis revealed that all four fungal peptides
showed very good binding affinity towards cFLIP.
Pneumocandin B0 showed highest negative value of
binding energies -455.18 among all other fungal peptides.
Aureobasidin G confirmed docking energy of -414.96,
whereas WF 11899A showed -377.18 energy value.
Echinocandin B expressed very low affinity -352.54 and -
342.59 respectively.
In-silco Screening of Fungal Peptides against Tumor Necrosis
Factor α (TNF-α) Related Apoptosis Inducing Ligand-Receptors
(TRAIL-Rs) Suppressing Protein CFLIP
Sathishkumar Arumugam1*, Mano Govindaraj1, Kalaiselvam Murugiyan1
Abstract: Cellular FLICE (FADD-like IL-1beta-converting enzyme)-inhibitory protein (c-FLIP) is major resistant and inhibitor
factor of TRAIL mediated apoptosis in cancer cells. Generally cFLIP is over expressed in all cancer cells and act as anti apoptotic
agent, hence it became one of the important targets for cancer therapy. Fungal peptides are having very good pharmaceutical
value and reported for various treatment. In present investigation, we described the binding affinity of fungal peptides with
cFLIP. Four fungal peptides Pneumocandin B0, Aureobasidin G, WF 11899A, Echinocandin B structures were retrieved pubchem
database and cFLIP protein structure was retrieved from Protein Data Bank. Docking studies were carryout by using Hex 8.0.0.
Their interactions and binding interface were identified by Ligplot 1.4.5. All four fungal peptides are having binding affinity with
cFLIP, among them Pneumocandin B0 showed highest binding energy value of -455.18, having two hydrogen and nine
hydrophobic bonds with cFLIP. Whereas, Aureobasidin G exposed energy value of -414.96, Ligpolt analysis showed three
hydrogen and seven hydrophobic bonds. WF 11899A binding energy was found to be -377.18, one hydrogen and eight
hydrophobic bonds were identified with cFLIP interaction. Echinocandin B was having least binding affinity -342.59 and having
only eight hydrophobic bonds. Further in-vitro and in-vivo study is needed to confirm their inhibitor effect on cFLIP.
1

2. Inventi Rapid: Molecular Modeling Vol. 2014, Issue 4
[ISSN 2278-4071]
2014 pmm 14329, CCC: $10 © Inventi Journals (P) Ltd
Published on Web 04/10/2014, www.inventi.in
RESEARCH ARTICLE
Fungal Peptides Interaction with cFLIP
Ligplot generated diagrams depicted interface of cFLIP
protein with fungal peptides (Table 2). Pneumocandin B0
showed two hydrogen bonds and nine hydrophobic bonds,
this is highest number of interactions with cFLIP, among
other tested proteins. Aureobasidin G had three hydrogen
bonds and seven hydrophobic bonds with receptor. One
hydrogen bond and eight hydrophobic bond were seen
between WF 11899A and cFLIP. Eight hydrophobic bonds
only observed between Echinocandin B and cFLIP (Figure 2).
DISCUSSION
Fungi are producing different kind of peptides including
Cyclic nonribosomal peptides (NRPs), lipopeptides, lectin
etc, they are pharmaceutically important for various
diseases. [14] Pneumocandin B0 was found to most potential
to bind with cFLIP, this belongs to lipopeptides family and
it’s reported for antifungal activity of drug resistant Candia
sp and Cryptococcus neoformans in-vitro study, also
reported in mouse model. [15, 16] Moreover Pneumocandin
B0 has antipneumocystis activity. [17]
Table 1: Details of Fungal Peptides from Pubchem Database
S. No.
Compound
Name
Fungal Source
Pubchem
ID
Molecular
Weight
Chemical Structure
1 Pneumocandin B0 Zalerion arboicola CID 462570 1108.2814
2 Aureobasidin G
Aureobasidium
pullulans
CID 3080245 1085.42008
3 WF 11899A
Coleophoma
empetri
CID 197367 1175.30278
4 Echinocandin B Aspergillus nidulans CID 171361 1064.2686
Table 2: cFLIP and Fungal Peptides Hex Docking Energy value and Binding Interface
S. No. Fungal Peptide Hex E-value
Ligplot Analysis of Binding Interface
No. of
Hydrogen
Bond
Residues in
Hydrogen
Bond
No. of
Hydrophobic
Bond
Residues in Hydrophobic Bond
1 Pneumocandin B0 -455.18 2
Gln291,
Arg338
9
His287, Ser290, Gly294, Cys298,
Leu331, His335, Met339, Ser344,
Tyr347.
2 Aureobasidin G -414.96 3
Asp269,
Val278,
Thr272.
7
Lys249, Pro250, Arg268, Ser273,
Gly275, TRY276, Glu277.
3 WF 11899A -377.18 1 Met246 8
Pro240, Glu242, Arg243, Tyr244,
Lys245, Lys247, Lys473, Tyr479.
4 Echinocandin B -342.59 - - 8
Pro240, Glu241, Glu242, Arg243,
Lys245, Lys247, Arg303.
2

3. Inventi Rapid: Molecular Modeling Vol. 2014, Issue 4
[ISSN 2278-4071]
2014 pmm 14329, CCC: $10 © Inventi Journals (P) Ltd
Published on Web 04/10/2014, www.inventi.in
RESEARCH ARTICLE
Figure 1: Hex docking confirmations between fungal peptides and cFLIP, (A) Pneumocandin B0, (B) Aureobasidin G, (C) WF 11899A,
(D) Echinocandin B
Figure 2: Ligplot illustration of cFLIP interactions with fungal peptides, (A) Pneumocandin B0, (B) Aureobasidin G, (C) WF 11899A,
(D) Echinocandin B
3

4. Inventi Rapid: Molecular Modeling Vol. 2014, Issue 4
[ISSN 2278-4071]
2014 pmm 14329, CCC: $10 © Inventi Journals (P) Ltd
Published on Web 04/10/2014, www.inventi.in
RESEARCH ARTICLE
Aureobasidin G is a cyclic peptides produce by
Aureobasidium pullulans showed second highest binding
affinity with cFLIP, Aureobasidin A is reported to act
against Toxoplasma gondii parasite associated with AIDS
patient. [18] WF 11899A is a water-soluble lipopeptides was
isolated from culture broth of Coleophoma empetri, it
related to echinocandins family. [19] It suppresses growth of
clinical isolated Candida albicans at 0.004 to 0.03 μg/ml,
also inhibit growth of Aspergillus fumigatus and A. niger.
[20] Echinocandin B was also reported to act against
Candida albicans and Cryptococcus neoformans, by both in-
vivo and in-vitro investigations. Significant reduction of
Candida CFU observed in kidneys at doses of 2.5 mg/kg.[21]
CONCLUSION
All four investigated fungal peptides are already reported
for very good anti fungal and anti parasite activity, but
potential of anti cancer property, especially to efficiency of
deciphering TRAIL-R is not studied so far. This present
Insilco investigations has identified their potential to be an
anti cancer molecule and deciphering TRAIL mediated
apoptosis. Even though, their exact biological activity to be
address through the in-vitro and in-vivo studies.
REFERENCES AND NOTES
1. Falschlehner C, Schaefer U, Walczak H. Following TRAIL’s path
in the immune system. Immunology, 127:145–154, 2009.
2. Pan G, O'Rourke K, Chinnaiyan AM, Gentz R, Ebner R, Ni J. The
receptor for the cytotoxic ligand TRAIL. Science,
276(5309):111-3, 1997.
3. Walczak H, Degli-Esposti M A, Johnson R S, Smolak P J, Waugh
J Y, Boiani N. TRAIL-R2: a novel apoptosis-mediating receptor
for TRAIL. EMBO Journal, 16(17):5386-97, 1997.
4. Kischkel F C, Lawrence D A, Chuntharapai A, Schow P, Kim K
J, Ashkenazi A. Apo2L/TRAIL-Dependent Recruitment of
Endogenous FADD and Caspase-8 to Death Receptors 4 and 5.
Immunity, 2(6):611-20, 2000.
5. Sprick M R, Weigand M A, Rieser E, Rauch C T, Juo P, Blenis J.
FADD/MORT1 and Caspase-8 Are Recruited to TRAIL
Receptors 1 and 2 and Are Essential for Apoptosis Mediated
by TRAIL Receptor 2. Immunity, 12(6):599- 09, 2000.
6. Ozoren N, Kim K, Burns T F, Dicker D T, Moscioni A D, El-Deiry
W S. The caspase 9 inhibitor Z-LEHD-FMK protects human
liver cells while permitting death of cancer cells exposed to
tumor necrosis factor-related apoptosis-inducing ligand.
Cancer Research, 60(22):6259-65, 2000.
7. 0zoren N and El-Deiry W S. Defining Characteristics of Types I
and II Apoptotic Cells in Response to TRAIL. Neoplasia,
4(6):551 – 57, 2002.
8. Ashkenazi A, Holland P, Eckhardt SG. Safety and antitumor
activity of recombinant soluble Apo2 ligand. Journal of Clinical
Investigation, 104(2):155-62, 1999.
9. Kelley S K, Harris L A, Xie D, Deforge L, Totpal K, Bussiere J.
Preclinical Studies to Predict the Disposition of Apo2L/Tumor
Necrosis Factor-Related Apoptosis-Inducing Ligand in
Humans: Characterization of in-vivo Efficacy,
Pharmacokinetics and Safety. Journal of Pharmacology and
Experimental Therapeutics, 299(2):31- 8, 2001.
10. Chan D W, Xing Z, Pan Y, Algeciras- Schimnich A, Branhart B C,
Yaish-ohad S, Peter ME, Yang X. CFLIP dual function regulator
for caspase 8 activation and CD95 mediated apoptosis. The
EMBO Journal, 21(4):3704-14, 2002.
11. Safa A R, Pollok K E. Targeting the Anti-Apoptotic Protein c-
FLIP for Cancer Therapy. Cancers, 3:1639- 71, 2011.
12. Dave Ritchie. Hex 6.12 User Manual Protein Docking Using
Spherical Polar Fourier Correlations: France: Vandoeuvre-les-
Nancy, 2012.
13. Laskowski R A, Swindells M B. LigPlot: multiple Ligand -
protein interaction diagrams for drug discovery. Journal of
Chemical Information and Modelling, 51:2778, 2011.
14. Gao X, Haynes S W, Ames B D, Wang P, Vien L P, Walsh C T,
Tang Y. Cyclization of Fungal Nonribosomal Peptides by a
Terminal Condensation-Like Domain. Nature Chemical
Biology, 8(10):823–30, 2012.
15. Bartizal K, Scott T, Abruzzo G K, Gill C J, Pacholok C, Lynch L,
Kropp H. In-vitro Evaluation of the Pneumocandin Antifungal
Agent L-733560, a New Water-Soluble Hybrid of L-705589
and L-731373. Antimicrobial Agents and Chemotherapy, 39
(5):1070–76, 1995.
16. Abruzzo G K, Flattery A M, Gill C J, Kong L, Smith J G, Krupa
D, Pikounis V B, Kropp H, Bartizal K. Evaluation of water-
soluble pneumocandin analogs L-733560, L-705589 and L-
731373 with mouse models of disseminated aspergillosis,
candidiasis and cryptococcosis. Antimicrobial Agents
Chemotherapy, 9(5):1077-81, 1995.
17. Schmatz D M, Powles M A, MCFadden D, Nollstadt K, Bouffard
FA, Dropinski J F, Liberator P, Andersen J. New semisynthetic
pneumocandins with improved efficacies Against Peumocystis
carinii in the rat. Antimicrobial Agents and Chemotherapy,
1320–23, 1995.
18. Sonda S, Sala G, Ghidoni R, Hemphill A. Inhibitory Effect of
Aureobasidin A on Toxoplasma gondii. Antimicrobial Agents
and Chemotherapy, 49(5)1794–1801, 2005.
19. Iwamoto T, Fujie A, Sakamoto K, Tsurumi Y, Shigematsu N,
Yamashita M, Hashimoto S, Okuhara M, Kohsaka M.
WF11899A, B and C, novel antifungal lipopeptides. I.
Taxonomy, fermentation, isolation and physico-chemical
properties. Journal of Antibiotics (Tokyo), 47(10):1084-91,
1994.
20. Iwamoto T, Fujie A, Nitta K, Hashimoto S, Okuhara M, Kohsaka
M. WF11899A, B and C, novel antifungal lipopeptides. II.
Biological properties. Journal of Antibiotics (Tokyo),
47(10):1092-7, 1994.
21. Bartizal K, Abruzzo G, Trainor C, Krupa D, Nollstadt K, Schmatz
D, Schwartz R, Hammond M, Balkovec J, Vanmiddlesworth F.
In vitro antifungal activities and in vivo efficacies of 1,3-beta-
D-glucan synthesis inhibitors L-671,329, L-646,991,
tetrahydroechinocandin B and L-687,781, a papulacandin.
Antimicrob Agents Chemother, 36(8):1648-57, 1992.
Cite this article as: Sathishkumar Arumugam, Mano
Govindaraj, Kalaiselvam Murugiyan. In-silco Screening of
Fungal Peptides against Tumor Necrosis Factor α (TNF-
α) Related Apoptosis Inducing Ligand-Receptors
(TRAIL-Rs) Suppressing Protein CFLIP. Inventi Rapid:
Molecular Modeling, 2014(4):1-4, 2014.
4