Insilico Evaluation of Inhibitory Profiles of Phytochemicals
for ACC Domains
A. Rajasekaran*, S. Santhosh, K.S.G. Arulkumaran and M.
Periyasamy
KMCH College of Pharmacy, Coimbatore, Tamilnadu.
*Corresponding Author E-mail: rsekaran2001in@yahoo.co.in
ABSTRACT:
Evaluating acetyl-CoA
carboxylase (ACC) as the target enzyme for the treatment of
hyperlipidemia can lead to the detection of new inhibitors that can potentially
be optimized as lipid lowering agents and could also form a novel method of
screening a large number of plant constituents. Inhibition
of ACC2 may prevent lipid-induced insulin resistance and type 2 diabetes,
making the enzyme an attractive pharmaceutical target. The crystal structures
of the biotin carboxylase (BC) domain of human ACC2 (PDB ID: 3GLK) and carboxyl transferase (CT) domain of human ACC2 (PDB
ID: 3FF6) have been selected for molecular
targets for Insilico studies of some potent phytochemicals like hydroxycitric
acid, terrestrosin, forskolin and EGC. The interactions between the compounds
and crystal structures of the enzymes have been
performed using Accelrys Discovery Studio 2.1 Ligandfit protocol. Findings from
the docking procedure indicate different binding modes of the compounds ,
further more the studies reveal that EGC was found to be a ideal inhibitor for
both ACC2 Domains, thus can be suggested as a better inhibitor for lipid
lowering .
KEYWORDS: Phytochemicals; Inhibitory profiles; ACC domains
INTRODUCTION:
Adjunctive therapy in dyslipidemia can serve as a means
to achieve a more comprehensive control of elevated lipid levels. Plant based
drugs are being explored to decrease the plasma cholesterol and
triglycerides in hypercholesterolemia patients by increasing the
expression of the hepatic low density lipoprotein receptor. The
immediate precursor for fatty acid synthesis is the acetyl co A, the major
building block for long chain fatty acid synthesis. Inhibition of Acetyl-CoA
carboxylase (ACC), results in inhibition of fatty acid synthesis and
stimulation of fatty acid oxidation, thereby beneficially affecting the
metabolic syndrome. The malonyl-CoA product of acetyl-CoA carboxylase is used
as a building block to extend the chain length of fatty acids in two carbon
increments, a process catalyzed by fatty acids synthase1. Herbs
have been widely used as medicines to treat hypercholesterolemia and few plants
with diterpenes alkaloids, hydroxycitric acid and sterones are found to possess
cholesterol-lowering properties.
In human, ACC1 and ACC2 have
central roles in fatty acid biosynthesis and fatty acid oxidation, making these
enzymes potent targets for the development of mono or adjunct therapy against
obesity and other manifestations of the metabolic syndrome2-4
observed a correlation between the reduced levels of malonyl-CoA and ACC
activity. Very few potent, small molecules that are inhibitors of mammalian
ACCs are currently known. Compound CP-640186 has IC50 values of
about 50mM against ACC1 and ACC2 that was reported to5
reduce tissue malonyl-CoA levels, inhibit fatty acid biosynthesis, stimulate
fatty acid oxidation and reduced body fat mass and body weight3.
Nanomolar inhibitors against the plant ACCs have been developed using the
haloxyfop binding site, and nanomolar inhibitors against the mammalian ACCs
have been developed using the CP-640186 binding site5. It may also
be possible that amino acid difference between ACC1 and ACC2 outside the active
site region can indirectly affect the conformation of the active site, which
could help enhance the selectivity of inhibitors.
In the present study, attempts were made to screen
plant compounds with computational tools to evaluate and identify the potent
compounds. The binding energies of the active constituents Hydroxycitric acid,
Terrestrosin, forskolin and EGC are compared to select the potent compounds for
pharmacological studies. The protein inhibitory potential of the compounds
compared with standard compounds using the Accelrys Software that could provide
inputs for development of plant based drugs as structure-based lead
optimization approaches are increasingly playing a role in the drug-discovery
process. Specific conformational changes are essential for the function of the
protein and the binding of a protein and ligand is often coupled to a conformational
change in the protein that makes the binding site more complementary to the
ligand, usually through specific interactions with one or more additional
ligands.
MATERIALS AND METHODS:
Protein Preparation:
Molecular models were based on
crystallographic PDB entries of Human ACC2 of BC and CT Domains (PDB
ID: and 3FF6) with released coordinates and those with lowest resolution,
sequence identity was selected for this study .The ligands and crystallographic
water molecules were removed from the protein and the chemistry of the protein
was corrected for missing hydrogen. Crystallographic disorders and unfilled
valence atoms were corrected using alternate conformations and valence monitor
options.
Ligand Preparation:
The three dimensional structures of phytochemical
compounds were downloaded in .sdf format from Pubchem database. Hydrogen Bonds
were added and the energy was minimized using CHARMm force field. Molecular
weight, log P and number of Hydrogen-bond donors and acceptors for the
active principles were noted. The Structures are shown in Figure 1 (A-EGC,
B- Terrestrosin, C- Forskolin, D-Hydroxycitric acid)
A-EGC
B-Terrestrosin
C-Forskolin
D-Hydroxy citric acid
Fig. 1. Three
dimensional structures of EGC, Terrestrosin, Forskolin and Hydroxy citric acid
Docking Study:
Docking study of the above compounds on the biotin carboxylase (BC) domain of human ACC2 was
carried out on the Ligandfit protocol of Accelrys Discovery Studio 2.1 and
compounds dock score were obtained .These compounds were also docked towards
carboxyl transferase (CT) domain of human ACC2. The determination of
the Ligand binding affinity was calculated using Dock Score.
RESULTS AND DISCUSSION:
Molecular Docking continues to holds great
promise in the field of Computer based drug design which screens small
molecules by orienting and scoring them in the binding site of a protein. As a
result novel ligands for receptors of known structure were designed and their
interaction energies were calculated using the scoring functions6.
There exists a need to identify natural compounds that activates or inactivates
the vital steps of important metabolic pathways. In the present study, a
protocol is described that incorporates Accelrys Discovery Studio Ligand fit
tool as a mode to identify the effective compounds. The evaluation of plant
based ACC inhibitors were investigated with the help of computational tools and
compared with the results of two domains of ACC2 which enable rapid
screening of a wide range of plant based compounds for their hypolipidemic
properties. Protein ligand binding has immense importance in the biological
systems since all organisms have a mechanism of interaction with its
environment. A ligand binds at a site on the protein which is complementary to
the ligand in size, shape, charge, hydrophobic and hydrophilic character.
Furthermore, the interaction is specific as the protein can discriminate among
the thousands of different molecules in its environment and selectively bind
only one or a few and has separate binding sites for several different ligands.
Structure-based screening involved docking the selected active constituents
onto a target binding site, identify the molecules that have a complementary
fit onto the target binding site that is scored with binding energy, reflecting
the inhibitory property of the compound. The screening of the active
constituents of the plants such as Hydroxycitric acid, Terrestrosin Forskolin
and EGC for assessing its inhibitory potential on a key enzyme of fatty acid
synthesis, was carried out as it is involved in a multitude of risk factors
associated with obesity and insulin resistance syndrome a clinical disorder
that is defined as the presence of increased insulin concentration in
association with visceral obesity, dyslipidemia and hyperglycemia.
Docking score:
As a result of docking there were 10
different conformations were generated for Hydroxycitric acid, Terrestrosin,
Forskolin and EGC. But only for top ranked docked complex the scores were
copied from the table browser view of Discovery studio for binding affinity
analysis. The 3D structures of protein-ligand complexes, scores are calculated
by summing pairwise interaction terms over all interatomic pairs of the
receptor-ligand complex, A higher score indicates a stronger receptor-ligand
binding affinity)7-8 and dockscore (Candidate ligand poses are
evaluated and prioritized according to the DockScore function) . It is
estimated that docking programs currently dock 70 – 80% of ligands correctly9.
The binding modes of the compounds have shown in the Figure 2 and 3. The Figure
2 shows the binding affinities of the Compounds A-EGC, B-Terrestrosin,
C-Forskolin and D-Hydroxy citric acid towards the biotin
carboxylase (BC) domain of human ACC2) and Figure 3 shows the binding
affinities of the Compounds A-EGC, B-Terrestrosin, C-Forskolin and D-Hydroxy
citric acid towards the carboxyl transferase (CT) domain
of human ACC2.
A-EGC
B-Terrestrosin
C-Forskolin
D-Hydroxy citric acid
Fig.2: Docking models of EGC, Terrestrosin,
Forskolin and Hydroxy citric acid with biotin
carboxylase (BC) domain of human ACC2. The green dot lines
denoted the hydrogen bonds. Amino acid residues involved in molecular
interactions with Ligands were shown in green color.
Hydrogen Bonds Information:
A close view of the bonding interactions of
ACC, BC and CT domains with the selected compounds Hydroxycitric acid,
Terrestrosin, Forskolin and EGC are shown in Table 1 and 2.
Lipinkis rule:
While focusing on the drug likeness and
dockscore, the compound EGC had the best dockscore (79.071 towards CT domain
and75.955 towards BC domain) among all the compounds and shown positive values
in Lipinkis rule. After docking of the Hydroxycitricacid, Forskolin and EGC,
Terrestrosin has also shown dual inhibition effect towards both the domains but
Terrestrosin render unsatisfactory result on Lipinkis parameter evaluation.
The four ligands that targeted the ACC2 BC and CT domains were
screened and ranked individually based on their dockscore. The Lipinkis rule of
five helped in the identifying of more suitable inhibitor are shown in Table 3.
CONCLUSION:
This analysis involves a comparative
investigation of binding affinities of the selected 4 compounds towards the BC
and CT domains of human ACC2. It is clear that the EGC (Had Highest
dockscore on both domains and more number of H-bonds interaction) satisfied
almost all properties like highest dockscore, more number of H-bonds
interaction and lower Log P values. Thus EGC can be treated as a potential
inhibitor for both BC and CT domains of human ACC2, thus can be considered as a
good for lipid lowering and suggested for further clinical testing.
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