Download Table 4.3: Binding energy between KRAS protein and

An in vitro studies on the inhibition of lung cancer
proliferation using different natural antioxidants
By
1. Murtada M. Hussein (Lecturer of Biotechnology, Al-mustaqbal
University College, Babylon, Iraq, E-mail: [email protected])
2. Saif Qahtan Salman
(Lecturer of Biochemistry, College of
Biotechnology, Al-Qasim Green University, Babylon, Iraq, E-mail:
[email protected])
3. Ghufran A. Abdulraheem (Lecturer of
Zoology, College of
Biotechnology, Al-Qasim Green University, Babylon, Iraq, E-mail:
[email protected])
Al-Qasim Green University
Iraq, 2015
ABSTRACT
In this study, Kras protein sequence, stucture and functional analysis were performed.
The mutation at 12th position changing the existing amino acid to arginine has been
reported in lung cancer. The antioxidants selected for the study are Ascorbic acid,
3,7, dihydroxyflavone, isoflavone and quercetin. Antioxidants drug likeliness,
toxicity are studied. The chemical structures of those antioxidants were studied to be
used for binding to the mutational site of KRAS. Based on the binding efficiency the
best antioxidant which can be used in lung cancer treatment is determined.
Key words: Kras protein, antioxidants, ascorbic acid, 3,7, dihydroxyflavone,
isoflavone, quercetin.
INTRODUCTION
RAS Mutations in NSCLC:
The RAS gene family includes HRAS, KRAS and NRAS and encodes for membranebound 21-kd guanosine triphosphate (GTP) binding proteins regulating cell growth,
differentiation and apoptosis by interacting with multiple effectors including
mitogen-activated protein kinase (MAPK), phosphoinositide 3-kinase (PI3K) and
signal transducer and activator of transcription (STAT) cascades (Figure 1). RAS
proteins acquire transforming potential when a point mutation in the gene replaces an
amino acid at position 12, 13 or 61 (Bos., 1998). These mutations lead to forms of
RAS with impaired GTPase activity, causing a constitutive activation of RAS
signalling pathway. Mutations in K-RAS gene occur frequently in NSCLC
(Rodenhuis et al., 1987)., more frequently (20–30%) in adenocarcinoma and less
frequently (about 7%) in squamous-cell carcinoma (Jemal et al.,2008).
Figure 1: Ras activation/deactivation cycle by GEF (guanine exchange factors) and
GAP (GTPase activating proteins).
Types of antioxidants:
5.2.1 Ascorbic acid:
Ascorbic acid or "vitamin C" is a monosaccharide antioxidant found in both animals
and plants. As one of the enzymes needed to make ascorbic acid has been lost by
mutation during human evolution, it must be obtained from the diet and is a vitamin.
Most other animals are able to produce this compound in their bodies and do not
require it in their diets. In cells, it is maintained in its reduced form by reaction with
glutathione, which can be catalyzed by protein disulfide isomerase and glutaredoxins.
Ascorbic acid is a reducing agent and can reduce and thereby neutralize, reactive
oxygen species such as hydrogen peroxide.
Luteolin
Luteolin is a yellow crystalline compound. It is a flavonoid; to be specific, it is one of
the more common flavones. From preliminary research, it is thought to play a role in
the human body possibly as an antioxidant, a free radical scavenger, a promoter
of carbohydrate metabolism, or an immune system modulator . If applicable to the
human condition, these characteristics may inhibit cancer mechanisms. Basic
research results indicate luteolin as an anti-inflammatory agent. with other potential
effects on septic shock . It has been suggested for multiple sclerosis on the basis of in
vitro work.
THE CHEMISTRY OF ANTIOXIDANTS
It involves the mechanism of action of antioxidant. Two principle mechanisms of
action have been proposed for antioxidants. The first is a chain-breaking mechanism
by which the primary antioxidants donate electrons to the free radicals present in the
system, example lipid radicals. The second mechanism involves removal of ROS
(reactive oxygen species) and RNS (reactive nitrogen species) initiator by quenching
chain initiator catalyst.
REVIEW OF LITERATURE
EFFECTS OF ANTIOXIDANTS:
A healthy cell has a mortal enemy which is called a "free radical." Free radicals
constantly seek out healthy cells and attack their vulnerable outer membranes
eventually causing cellular degeneration and death. Free radicals scientists today,
carry out the actual destructive work in disease, in infection, in stress and in aging.
Additionally, free radicals can negatively affect athletic performance by slowing or
halting muscle growth and by lowering aerobic capacity. Further, free radicals are
known to cause defects in normal RNA as well as in life perpetuating DNA, the
genetic material of the cells (Warner et al., 2004).
Many nutraceutical and health food companies sell formulations of antioxidants as
dietary supplements and these are widely used in industrialized countries. These
supplements may include specific antioxidant chemicals, like resveratrol (from grape
seeds or knotweed roots), combinations of antioxidants, like the "ACES" products
that contain _-carotene (provitamin A), vitamin C, vitamin E and selenium, or herbs
that contain antioxidants - such as green tea and jiaogulan. Although some levels of
antioxidant vitamins and minerals in the diet are required for good health, there is
considerable doubt as to whether these antioxidant supplements are beneficial or
harmful (Warner et al., 2004).
Mutant KRAS inhibition by antioxidants The effects of the dietary phytochemicals
quercetin(Q), luteolin(L) and ursolic acid (UA) on cell proliferation and apoptosis in
two human CRC derived cell lines, HCT15 and CO115, harboring KRAS and BRAF
activating mutations, respectively. In KRAS mutated HCT15 cells, Q and L
significantly decreased ERK phosphorylation, whereas in BRAF mutated CO115
cells the three compounds decreased Akt phosphorylation but had no effect on
phospho-ERK. These natural compounds have ant proliferative and proapoptotic
effects and simultaneously seem to act on KRAS and PI3K but not on BRAF. These
results shed light on the molecular mechanisms of action of Q, L and UA and
emphasize the potential of dietary choices for the control of CRC progression
(Xavier et al., 2009). Therapeutic strategies that target tumors harboring these
mutations represent an unmet medical need. In this study, we investigated the
relationship between antifolate sensitivity and KRAS mutation/amplification status in
NSCLC. Human NSCLC cell lines (KRAS wild type, KRAS mutant non-amplified
and KRAS mutant amplified) were treated with Methotrexate (MTX) or Pemetrexed
(PEM) and assayed for proliferation. In these studies, KRAS wild type and KRAS
mutant amplified cells showed resistance to MTX treatment (IC50 >10μM). In
contrast, growth of all KRAS mutate non-amplified cell lines studied was inhibited
with MTX treatment (IC50 <100nM). Similar effects were observed for PEM in this
study (Moran et al., 2012).
MATERIALS AND METHODS
TOOLS AND DATABASES
PUBMED:
PubMed comprises more than 21 million citations for biomedical literature from
MEDLINE, life science journals, and online books covering portions of the life
sciences, behavioral sciences, chemical sciences, and bioengineering.It also provides
access to additional relevant web sites and links to the other NCBI molecular biology
resources.
PDB:
The Protein Data Bank (PDB) is a repository for the 3-D structural data of large
biological molecules, such as proteins and nucleic acids.It contains information about
experimentally-determined structures of proteins, nucleic acids, and complex
assemblies. RCSB PDB curates and annotates PDB data according to agreed upon
standards.
RCSB PDB is used to perform simple and advanced searches based on annotations
relating to sequence, structure and function, and to visualize, download, and analyze
molecules.
PubMed is a free resource that is developed and maintained by the National Center
for Biotechnology Information (NCBI), at the U.S. National Library of Medicine
(NLM), located at the National Institutes of Health (NIH).
STRUCTURAL ANALYSIS TOOLS
EXPASY
ExPASy is the SIB Bioinformatics Resource Portal which provides access to
scientific databases and software tools in different areas of life sciences including
proteomics, genomics, phylogeny, systems biology, population genetics,
transcriptomics.
1.PROTPARAM (Primary Structure Analysis)
ProtParam is a tool which allows the computation of various physical and chemical
parameters for a given protein stored in Swiss-Prot or TrEMBL or for a user entered
sequence. The computed parameters include the molecular weight, theoretical pI,
amino acid composition, atomic composition, extinction coefficient, estimated halflife, instability index, aliphatic index and grand average of hydropathicity (GRAVY).
2. SOPMA (Secondary structure analysis)
SOPMA (Self-Optimized Prediction Method with Alignment) is an improvement
of SOPM method. The improvent takes place in the fact that SOPMA takes into
account information from an alignment of sequences belonging to the same family. If
there are no homologous sequences the SOPMA prediction is the SOPM one.
Tertiary Structure Analysis
CPH Models :
CPHmodels 3.2 is a protein homology modeling server. The template recognition is
based on profile-profile alignment guided by secondary structure and exposure
predictions.
CPHmodels-3.0 is a web-server predicting protein 3D-structure by use of single
template homology modeling. The server employs a hybrid of the scoring functions
of CPHmodels-2.0 and a novel remote homology-modeling algorithm. A query
sequence is first attempted modeled using the fast CPHmodels-2.0 profile-profile
scoring function suitable for close homology modeling.
DRUG DESIGNING TOOLS
1. ARGUS LAB
ArgusLab is a free molecular modeling package that runs under windows, graphics. It
is a drug designing program with 3D builder, ab initio calculation modules, and
simple molecular mechanics. It also used for protein docking purposes.
2. PUBCHEM
PubChem provides information on the biological activities of small molecules.
PubChem includes substance information, compound structures, and BioActivity data
in three primary databases, Pcsubstance, Pccompound, andPCBioAssay, respectively.
It is maintained by NCBI. The compound structures and descriptive datasets can be
freely downloaded.
3. DUNDEE PRODRG SERVER
PRODRG takes a description of a small molecule (as PDB coordinates / MDL
Molfile / SYBYL Mol2 file / text drawing) and from it generates a variety of
topologies for use, as well as energy-minimized coordinates in a variety of formats.
4. ADME TOX
ADME an acronym in pharmacokinetics and pharmacology for absorption, distributio
n, metabolism,
and excretion,
and
describes
the
disposition
of
a pharmaceutical compound within an organism. The four criteria all influence the
drug levels and kinetics of drug exposure to the tissues and hence influence the
performance and pharmacological activity of the compound as a drug. Mobyle is a
system which provides an access to different software elements, in order to allow
users to perform bioinformatics analyses.
RESULTS AND DISCUSSIONS
Kras protein
MTEYKLVVVGAGGVGKSALTIQLIQNHFVDEYDPTIEDSYRKQVVIDGETCLLDILDTAG
QEEYSAMRDQYMRTGEGFLCVFAINNTKSFEDIHHYREQIKRVKDSEDVPMVLVGNKCDL
PSRTVDTKQAQDLARSYGIPFIETSAKTRQRVEDAFYTLVREIRQYRLKKISKEEKTPGC
VKIKKCIIM
Structure visualization using argus lab highlighting the mutational
site G12D
GEOMETRY
Optimization:
INFRENCE: using argus lab KRAS structure optimization was don. The energy of the
protein structure is 1151.78 kcal/mol.
BINDING RESULTS
3,7 Dihydroxyisoflavone:
Binding results showed the binding KRAS protein mutation with antioxidant (3, 7
dihydroxyisoflavone) closely through the use of simulation system Argus lab. The
high-energy showed the binding in this compound for two reasons, first, the high
receptor for KRAS protein mutation, site (12D), second The high susceptibility of the
3,7 dihydroxyisoflavone .
Figure 4.12: KRAS protein and 3,7dihydroxyisoflavone binding which are high
binding energy: -6.63067kcal/mol. This shape explain the binding between
3,7dihydroxyisoflavone (violet color) and KRAS protein in site 12D (yellow
color)
Isoflavone:
Isoflavone Binding results (Figure 4.14) showed the binding KRAS protein mutation
with antioxidant Flavones closely through the use of simulation system Argus lab.
The high-energy showed the binding in this compound for two reasons, first, the high
receptor for KRAS protein mutation, site (12D), second The high susceptibility of the
Flavones.
Figure4.14: KRAS protein and Isoflavone binding appear high energy binding: 6.23017kcal/mol. This shape explain the binding between flavones (green color)
and KRAS protein in site 12D (orange color)
Ascorbic acid
Ascorbic acid have high binding energy with KRAS protein in the activate 12D
which are responsible about KRAS mutation. This binding (figure 4.13) showed the
high ability for ascorbic acid to link with KRAS protein. Through used Agrus lab
showed clear binding occurring between the antioxidants compounds with KRAS
protein in the site 12D, This binding showed the antioxidants activity for inhibiting
the KRAS mutation.
Figure 4.13: KRAS protein and ascorbic acid binding appear high Binding
Energy: -5.78191kcal/mol. This shape explain the binding between ascorbic acid
(yellow color) and KRAS protein in site 12D (green color)
Quercitin
Quercitin have high binding energy with KRAS protein in the activate 12D which are
responsible about KRAS mutation. This binding (Figure 4.15) showed the high
ability for Quercitin to link with KRAS protein. Through used Agrus lab showed
clear binding occurring between the antioxidants compounds with KRAS protein in
the site 12D, this binding showed the antioxidants activity for inhibiting the KRAS
mutation.
Figure 4.15: KRAS and quercitin binding appear high
Energy: -5.89287kcal/mol. This shape explain the binding between quercitin
(violet color) and KRAS protein in site 12D (yellow color)
Table 4.3: Binding energy between KRAS protein and Antioxidants
compounds.
Antioxidant
3,7 dihydroxyisoflavone
Isoflavone
Quercetin
Ascorbic acid
Protein structure
Kras
Kras
Kras
Kras
Binding Energy
-6.63067 kcal/mol
-6.23017 kcal/mol
-5.89287 kcal/mol
-5.78191 kcal/mol
SUMMARY AND CONCLUSION
From the computational analysis, Kras protein was found to be hydrophilic,
Aliphatic, Stable, negatively charged having the PI 6.33 and molecular weight
21655.8 daltons. Antioxidants (Ascorbic acid, 3,7, dihydroxyflavone, Isoflavone and
Quercetin) were tested for drug likeliness and toxicity. From the binding energies
between the mutational site 12D and the various antioxidants, the antioxidant named
3,7, dihydroxyIsoflavone showed the efficient binding -6.63067 kcal/mol, when
compared with other antioxidants. Thus 3, 7 dihydroxyisoflavone can be taken as an
effective antioxidant in the inhibition of Lung cancer proliferation.
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