Journal of Enzyme Inhibition and Medicinal Chemistry

ISSN: 1475-6366 (Print) 1475-6374 (Online) Journal homepage: https://www.tandfonline.com/loi/ienz20

Molecular dynamic simulations and structure-

based pharmacophore development for
farnesyltransferase inhibitors discovery

N. S. Hari Narayana Moorthy, Sergio F. Sousa, Maria J. Ramos & Pedro A.

Fernandes

To cite this article: N. S. Hari Narayana Moorthy, Sergio F. Sousa, Maria J. Ramos & Pedro
A. Fernandes (2016) Molecular dynamic simulations and structure-based pharmacophore
development for farnesyltransferase inhibitors discovery, Journal of Enzyme Inhibition and
Medicinal Chemistry, 31:6, 1428-1442, DOI: 10.3109/14756366.2016.1144593

To link to this article: https://doi.org/10.3109/14756366.2016.1144593

Published online: 18 Feb 2016.

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ISSN: 1475-6366 (print), 1475-6374 (electronic)

J Enzyme Inhib Med Chem, 2016; 31(6): 1428–1442

! 2016 Informa UK Limited, trading as Taylor & Francis Group. DOI: 10.3109/14756366.2016.1144593

RESEARCH ARTICLE

Molecular dynamic simulations and structure-based pharmacophore

development for farnesyltransferase inhibitors discovery
N. S. Hari Narayana Moorthy, Sergio F. Sousa, Maria J. Ramos, and Pedro A. Fernandes

UCIBIO, REQUIMTE, Departamento de Quı́mica e Bioquı́mica, Universidade do Porto, 687, Rua do Campo Alegre, Porto, Portugal

Abstract Keywords
Farnesyltransferase is one of the enzyme targets for the development of drugs for diseases, Binding free energy, cancer, farnesyltransfer-
including cancer, malaria, progeria, etc. In the present study, the structure-based pharmaco- ase, molecular dynamic simulations, PLIF,
phore models have been developed from five complex structures (1LD7, 1NI1, 2IEJ, 2ZIR and structure-based pharmacophore
2ZIS) obtained from the protein data bank. Initially, molecular dynamic (MD) simulations were
performed for the complexes for 10 ns using AMBER 12 software. The conformers of the History
complexes (75) generated from the equilibrated protein were undergone protein–ligand
interaction fingerprint (PLIF) analysis. The results showed that some important residues, such as Received 9 July 2015
LeuB96, TrpB102, TrpB106, ArgB202, TyrB300, AspB359 and TyrB361, are predominantly present Revised 18 January 2016
in most of the complexes for interactions. These residues form side chain acceptor and surface Accepted 18 January 2016
(hydrophobic or –) kind of interactions with the ligands present in the complexes. The Published online 16 February 2016
structure-based pharmacophore models were generated from the fingerprint bits obtained
from PLIF analysis. The pharmacophore models have 3–4 pharmacophore contours consist of
acceptor and metal ligation (Acc & ML), hydrophobic (HydA) and extended acceptor (Acc2)
features with the radius ranging between 1–3 Å for Acc & ML and 1–2 Å for HydA. The excluded
volumes of the pharmacophore contours radius are between 1–2 Å. Further, the distance
between the interacting groups, root mean square deviation (RMSD), root mean square
fluctuation (RMSF) and radial distribution function (RDF) analysis were performed for the MD-
simulated proteins using PTRAJ module. The generated pharmacophore models were used to
screen a set of natural compounds and database compounds to select significant HITs. We
conclude that the developed pharmacophore model can be a significant model for the
identification of HITs as FTase inhibitors.

Introduction such as P. falciparum-resistant malaria, trypanosomatid infections

(African sleeping sickness), Chagas disease, Toxoplasmosis and
Cancer is still one of the life-threatening diseases and the second
Leishmaniasis and as antiviral agents7–14.
leading cause of death in the world. Cancer accounted for 8.2
FTase modifies the oncogene Ras through the farnesyl
million deaths in 2012 and is expected that the annual cancer cases
pyrophosphate (FPP) intermediate by the farnesylation of a
will rise from 14 million in 2012 to 2022 within the next two
carboxyl terminus protein in the CAAX (tetrapeptide motif) (‘‘C’’
decades1,2. The development of anticancer agents for the success-
refers to the cysteine, ‘‘A’’ to any aliphatic amino acid often
ful treatment of cancer remains a challenging goal, because of its
methionine, and ‘‘X’’ to any amino acid, glutamine or serine in
nonselectivity, toxicity and poor pharmacokinetic profiles. Hence,
FTase and leucine or phenylalanine in GGTase-1). The G-protein,
medicinal chemists are working on different biological targets to
Ras plays an important role in mediating cellular responses to
develop novel molecules as anticancer agents3.
growth signals and the appearance of its oncogenic mutants is
Farnesyltransferase (FTase) is one of three prenyltransferase
associated to a high percentage of human cancers6,15. It could be
enzymes (farnesyltransferase (FTase), geranylgeranyltransferase-I
related to the development of cancer by mutations in these
(GGTase-I) and geranylgeranyltransferase-II (GGTase-II)) used
proteins. The inhibition of FTase enzyme activity can be a better
by normal and malignant cells to catalyze covalent attachment of
target for anticancer drug development and some FTase inhibitors,
prenyl groups to 4300 polypeptides in the human proteome4–6.
such as tipifarnib, lonafarnib, BMS214 662, L7, 78123 and SCH4,
FTase has become a major target in the development of potential
4342 are currently being assessed in clinical trials for the
anticancer drugs and has also been used as an effective target for
treatment of human cancers6,16,17. Scientists have been working
the development of drugs against Progeria and parasites diseases,
on the drug discovery field, using different approaches for drug
discovery. Computational methods are one of the prominent
methods used nowadays to reduce the cost and time in the drug
Address for correspondence: N. S. Hari Narayana Moorthy, Department
of Chemistry and Biochemistry, University of Porto, 687, Rua de Campo development program. Our research group has been working on
Alegre, Porto, Porto, 4169-007 Portugal. Tel: +351-220 402 506. E-mail: this target for the last 10 years for the development of FTase
hari.moorthy@fc.up.pt or hari.nmoorthy@gmail.com inhibitors using computational-based approaches3,6,18.
DOI: 10.3109/14756366.2016.1144593 MD simulations and structure-based pharmacophore development 1429

Generally, computer-aided drug design techniques, such as the from human FTase and 1NI1 (resolution 2.30 Å), 2ZIR (resolution
structure-based (docking) and ligand-based (pharmacophore, 2.40 Å) and 2ZIS (resolution 2.60 Å) were derived from rat
QSAR, etc.) approaches are used to screen a large set of FTase29 (Supplementary Table S1). These complexes were used
compounds (database libraries) to identify significant HIT for MD simulations, Gibbs binding free energy calculation,
compounds for a particular target. Structure-based drug design protein–ligand interactions fingerprint (PLIF) analysis, structure-
method does require a three-dimensional (3D) structure of the based pharmacophore development and screening of compounds.
protein, acquired by means of several techniques, including
electron microscopy, atomic force microscopy, X-ray crystallog- MD simulations
raphy and NMR spectroscopy, or by computational methods, for
The AMBER 12 molecular dynamics package30 was used in all
example, homology modeling. The molecular docking is one of
the MD simulations performed. Conventional protonation states
the well-known methods used in structure-based drug design
for all amino acids at pH 7 were considered. All the hydrogen
approaches. The ligand-based drug design approaches are used
atoms were added and counter-ions (Na+) were employed
when 3D structure of the protein is not available; however, it
to neutralize the highly negative charges of the systems.
considers compounds (especially active compounds) those have
The Leap program was used for this purpose. Each of these
activity on the particular target. The QSAR, pharmacophore
systems was then placed in rectangular boxes containing a
analyses are best-suited techniques applied for ligand-based
minimum distance of 12 Å of TIP3P water molecules between the
analysis19–26. In the present investigation, we have applied both
enzyme and the box side. The size of these five systems was of ca
structure- and ligand-based drug design approaches to generate
100 000 atoms.
structure-based pharmacophore models to identify the FTase
A set of parameters specifically designed to allow a reliable
inhibitors from data set of natural compounds.
treatment of the Zn2+ coordination sphere formed during catalysis
As per IUPAC, pharmacophores are defined as ‘‘an ensemble
and based on DFT (B3LYP) and molecular mechanical calcula-
of steric and electronic features, that is, necessary to ensure the
tions31, crystallographic data32–34, extended X-ray absorption fine
optimal supramolecular interactions with a specific biological
structure (EXAFS) results35 and on several other more recent
target and to trigger (or block) its biological response’’. Basically,
mechanistic studies36,37 were applied to the systems in study.
this pharmacophore approach adheres to the premise that related
These parameters are described in detail elsewhere38 and have
chemical groups (i.e., hydrogen bond donors/acceptors, aromatic
been already used with success in the study of FTase38–40. The
rings, and so forth) for a set of known active molecules that are
AMBER ff12SB parameters were employed to describe the
analyzed in 3D space which are responsible for specific drug–
remaining of the proteins, while the ANTECHAMBER module of
receptor interactions27,28. The drawback of the ligand-based
AMBER and the General AMBER Force Field (GAFF) were used
method is that they do not provide detailed structural information
to parameterize the inhibitors, and the charges derived with RESP
(receptor binding) to help medicinal chemists to design new
at the HF/6–31G(d) level of theory30,41.
molecules.
All systems were subjected to a four stages refinement
The structure or receptor-based pharmacophore models pro-
protocol using the SANDER module of AMBER 12, in which
vide an efficient alternative to docking-based virtual screening of
the constraints on the enzyme were gradually removed. In the first
small molecules, still representing specific ligand–protein inter-
stage (2000 steps) 50 kcal/mol/Å2 harmonic forces were used to
actions19–26. The generation of pharmacophore models directly
restrain the positions of all atoms in the systems except those from
from the complex crystal structures is more reliable because it
the water molecules. In the second stage (4000 steps), these
imposes the necessary constraints required for interaction and
constraints were applied only to the heavy atoms and in the third
selectivity20. The protein–ligand-derived pharmacophores was
stage (8000 steps) were limited to the Ca and N-type atoms
applied by Langer et al. in order to identify potential targets of
(backbone a-carbon and nitrogen atoms). This process ended in a
bioactive ligands in a series of retrospective screening experi-
full-energy minimization (4th stage, maximum 50 000 steps) until
ments focusing on small protein–ligand matrices21,22. The
the rms gradient was smaller than 0.0002 kcal/mol.
advanced experimental techniques, such as X-ray crystallography
MD simulations were carried out using the PMEMD module
and NMR, are available for the determination of a protein
of AMBER 12 and considering periodic boundary conditions to
structure, and hence, the reliable and robust techniques to
simulate a continuous system. The SHAKE algorithm was applied
construct pharmacophores from a receptor structure is import-
to fix all bond lengths involving a hydrogen atom, permitting a 2-
ant23,24. The addition of exclusion volume spheres to the
fs time step. The Particle-Mesh Ewald (PME) method was used to
structure-based pharmacophore models onto the coordinates
include the long-range interactions, and a nonbond interaction
defined by protein side chain atoms to characterize inaccessible
cutoff radius of 10 Å was considered. Following a 500 ps
areas for any potential ligand26. With the consideration of this
equilibration procedure, 10 ns MD simulations were carried out
concept, in the present investigation, we have developed robust
at 310 K using the Langevin temperature coupling scheme at
structure-based pharmacophore models from five X-ray crystal-
constant pressure (1 atm) with isotropic molecule-based scaling,
lographic complexes obtained from protein data bank. The
resulting in a total simulation time of 50 ns. The MD trajectory
conformers of the complexes were generated from the equilibrated
was sampled every 2.0 ps. All of the MD simulations were
state of the complexes obtained through MD simulations. These
analyzed with the PTRAJ module of AMBER 12, with values
conformers were considered for the structure-based pharmaco-
retrieved from the last 8 ns of the simulation.
phore model generation and those pharmacophore models were
used to virtual screen a data set of compounds. This work has
PLIF analysis
been performed as per the flow chart provided in Figure 1.
The MD simulations of the proteins were performed till the
Experimental backbone a-carbons (Ca) were stable (10 ns MD simulations).
The conformers (75–80 conformers for each complex) created
Protein preparation
from the MD simulated proteins at the stable time (equilibration)
The X-ray crystallographic structure of protein–ligand complexes were collected using VMD software42 and those conformers were
of FTases were obtained from protein data bank. The complexes used for the PLIF analysis. Other than the conformers of the
1LD7 (resolution 2 Å) and 2IEJ (resolution 1.8 Å) were obtained complexes, the PLIF was analyzed on the raw X-ray
1430 N. S. H. N. Moorthy et al. J Enzyme Inhib Med Chem, 2016; 31(6): 1428–1442

Protein-Ligand Complexes (PDB)

(1LD7, 1NI1, 2IEJ, 2ZIR and 2ZIS)

Refining the Complex

Molecular Dynamic Simulations

Statistical analysis of the MD simulated

Generation of Conformer of the trajectories
Binding Free Energy Complexes
Calculation
(MMGBSA )
• RMSD analysis.
• Distance analysis between important
Protein Ligand Interaction functional groups/atoms (based on
Fingerprint (PLIF) Analysis PLIF results).
• RMSF analysis on important
residues have interaction with
ligands.
• RDF of functional groups/atoms in
Structure Based Pharmacophore residues and ligands
Query Generation Based on
PLIF results

Virtual screening of database

(NPs)

Figure 1. Flow chart of the work.

crystallographic structure (protein–ligand complexes) obtained
from protein data bank. The PLIF module in MOE software43 was
used for the analysis. It provides important protein–ligand
interaction residues commonly present in the superimposed/
overlaid complexes/conformers obtained from the complex struc-
tures. PLIF possesses a composition of seven visible finger-
print bits (side-chain hydrogen bond donor (D), side-chain
hydrogen bond acceptor (A), backbone hydrogen bond donor
(d), backbone hydrogen bond acceptor (a), solvent hydrogen
bond (O), ionic attraction (I) and surface contact (C)). The
hydrogen bond fingerprints are calculated using a method
based on protein contact statistics, whereby a pair of atoms is
scored by distance and orientation. Ionic interactions are
scored by calculating the inverse square of the distance between
atoms with opposite formal charge (e.g. a carboxylate oxygen
atom and a protonated amine). Surface contact interactions are
determined by calculating the solvent-exposed surface area of the
residue, first in the absence of the ligand, then in presence of the
ligand44,45.
Structure-based pharmacophore generation
Structure-based pharmacophore queries were developed for the
complexes (all five complexes) and species (rat and human)
individually from the PLIF results. It considers actual structural
information (fingerprint bits) obtained directly from the protein–
ligand interactions in 3D space. The pharmacophore query
generator tool in the module operates on the principle that a
modest selection of poses with a homogeneous set of interaction
fingerprints, most likely possess a homogeneous set of pharma-
cophore feature points. The pharmacophore feature points can be
groups and filtered according to which they interact with
and those with a sufficiently tight grouping can be con-
verted into a pharmacophore query feature. The excluded
volume of the pharmacophore query points was also included in
the structure-based virtual screening of the molecules. The details
of the query points generated and used in the screening are
provided in Table 1. The following pharmacophore query struc-
tures were developed; the conformers of individual complexes (Ph-
1LD7, Ph-1NI1, Ph-2IEJ, Ph-2ZIR and Ph-2ZIS), proteins from rat
FTase (Ph-rat), human FTase (Ph-human) and both rat and human
FTase complexes (Ph-all). The developed pharmacophore query
models were validated using FTase inhibitors in clinical use
(tipifarnib, lonafarnib and L7,78123) and some known natural
product based FTase inhibitors. Further, the validated pharmaco-
phore models were used to screen a data set comprised of 100
natural products compounds. Additionally 1000 synthetic FTase
inhibitors from literatures and a data set compounds from binding
database (http://www.bindingdb.org/bind/index.jsp) were screened
on the developed pharmacophore models.
FTase-binding assay
In a 96-well black plate, 10 mL of stock solution of FPP,
recombinant FTase (100 nM) and compound (1–50 mM) separ-
ately. Adding 150 mL of Tris buffer (pH ¼ 7) to the well and
mixed well to initiate the FTase reaction. The time course
fluorescence change at 520 nm (ex: 340 nm) was monitored at
30  C using a microplate reader.
Results and discussion
In the present study, MD simulations and structure-based
pharmacophore analysis were performed on five crystallographic
structures of human (1LD7 and 2IEJ) and rat (1NI1, 2ZIR and
2ZIS) FTase enzymes. The MD simulations were performed using
AMBER 12 software for 10 ns for each complex. The simulated
systems (equilibrated) were used for conformer generation, PLIF
analysis and structure-based pharmacophore generation. These
DOI: 10.3109/14756366.2016.1144593 MD simulations and structure-based pharmacophore development 1431
Table 1. Details of the pharmacophore query points in the structure-based pharmacophore models.

Ph-1LD7 Ph-1NI1 Ph-2IEJ Ph-2ZIR Ph-2ZIS Ph-Rat Ph-Human Ph-All

Code PhC R PhC R PhC R PhC R PhC R PhC R PhC R PhC R
F1 A 1.5 B 3 A 2 A 1 A 2 B 2 B 2 B 2
F2 B/A 2 A 2.5 B 1.5 B 1.5 B 2 B 1.5 B 1.5 B 1.5
F3 A 1.5 A 2.5 B 1.5 B 1.5 A 2 A 2 A 1.5 A 1.5
F4 – – A 3 A 2 – – C 2 A 1.5 A 1.5 A 2
+V1 Excl V 1.8 V 1 V 1 V 1 V 1.7 V 1 V 1 V 1

A: Acc&ML, B: HydA, C: Acc2 and V: Excluded volume, PhC: Pharmacophore contour, R: radius in Å.

α of Protein
RMSD of Cα RMSD of ligands
2.5 2.5

2 2
RMSD (Å)

RMSD (Å)
1.5 1.5

1 1

0.5 0.5

0 0
0 2000 4000 6000 8000 10000 0 2000 4000 6000 8000 10000
Time (ps) Time (ps)
1LD7 1NI1 2IEJ 2ZIR 2ZIS 1LD7 1NI1 2IEJ 2ZIR 2ZIS

RMSD of Zn
2

1.5
RMSD (Å)

0.5

0
0 2000 4000 6000 8000 10000
Time (ps)
1LD7 1NI1 2IEJ 2ZIR 2ZIS

2+
Figure 2. RMSD of protein, ligand and Zn .

pharmacophore models were used for virtual screening of a data
set made of natural compounds.
The X-ray crystallographic complexes obtained from the
protein data bank composed of the protein, substrate and the
inhibitors. The complexes, such as 1LD7, 2ZIR and 2ZIS, are
having FPP as substrate and other complexes, such as 1NI1 and
2IEJ, have a-hydroxy farnesylphosphonic acid and [(3,7,11-
trimethyl-dodeca-2,6,10-trienyloxycarbamoyl)-methyl]-phos-
phonic acid, respectively, as substrates. The MD-simulated
complexes were statistically analyzed with root mean square
deviation (RMSD), root mean square fluctuation (RMSF),
distances and radial distribution function (RDF).
RMSD analysis performed for Ca, substrates, Zn2+ and
inhibitors illustrate that the Ca in proteins and the ligand
molecules (inhibitors) showed variation in the RMSD values
with time in all the MD simulations performed. The results
established that the studied systems are well equilibrated after
the initial 2 ns MD simulations (Figure 2). In agreement with
this observation, all simulations were taken into consideration
for the calculation of the statistical mechanical values of the
systems. The last 8 ns which are equilibrated after the MD
simulations were used to derive different conformer of the
complexes for the PLIF analysis. In order to investigate the
binding interactions of the best conformers of the inhibitors, the
average protein structure was calculated from the created MD
simulations. The conformers that showed lowest RMSD values
were used to analyze the binding interactions of the molecules.
The best conformers of the ligands in the complexes exhibited
the RMSD values of 0.64, 0.80, 0.60, 0.63 and 0.62 Å for 1LD7,
1NI1, 2IEJ, 2ZIR and 2ZIS respectively (Supplementary Figure
S1). These best conformers showed that the TyrB361 have
hydrogen-bonding interaction with the nitrile (CN) group and
have surface type interaction with the imidazole or phenyl rings
present in the inhibitors. The nitrogen atoms in the imidazole or
methyl imidazole ring form acceptor interaction with the
Zn2+ present in the protein. Further, the binding mode of the
ligands was analyzed detail by generating conformers of the
ligands at different snaps of the MD simulated proteins. These
developed conformers were used for PLIF analysis and to
generate structure (receptor) based pharmacophore generation.
1432 N. S. H. N. Moorthy et al. J Enzyme Inhib Med Chem, 2016; 31(6): 1428–1442

PLIF analysis

AspB359, TyrB361
LeuB96, TrpB102,

Tyr166, ArgB202,
The PLIF analysis is a method to study the interaction between

AspB359
the residues in the protein and the ligands on a superimposed

TyrB361
All
structure of the complexes. The derived fingerprint bits are used
to investigate the common residues that interact with the ligand
and those were used for the development of structure based
pharmacophore queries (models). In this study, we were used five
FTase–ligand complexes obtained from protein data bank (as
mentioned earlier), for MD simulations. Among these complexes,

TrpB102,

ArgB202
TyrB361
LeuB96,

Tyr166,
Human
three complexes are belonging to rat FTase and two complexes
belong to human FTase enzymes. The PLIF analysis was
performed on the five complexes together (the raw complex
structures from data bank) and individually the complexes of the

AspB359, TyrB361
LeuB96, TrpB106,

Tyr166, ArgB202,
Tyr166, TyrB93,
rat and the human FTases. The conformers of the ligands were
derived from the MD simulated complex from the last 8 ns

AspB359
TyrB361
simulation time on the equilibrated state. The PLIF analysis

Rat
performed on the 75 conformers for each complex derived from
MD simulations (Table 2).
The PLIF analysis performed on the rat protein and human
protein complexes (only the coordinate conformers from X-ray
crystallographic structures) showed that the ligand in both kind of

ArgB202, TyrB300,
TrpB102, TrpB106,

His201 ArgB202,
complexes (rat and human) have interaction commonly with

His201, LeuB96,
Tyr166, ArgB202 and TyrB361 through surface and side-chain

TyrB361

HisB362
2ZIS
acceptor interactions. The rat FTase additionally showed specific
interactions with TyrB93, LeuB96, TrpB106 and AspB359 by
surface interactions and the later one (AspB359) provided side-
chain acceptor interaction. The human FTase showed in addition
to the common residues provided earlier, LeuB99 and TrpB102
have surface interactions (with phenyl group or aliphatic chain)

TrpB106, TyrB300,

ArgB202 HisB362
with the ligand molecules. The PLIF analysis performed with the

LeuB96, TrpB102
His201, TyrB93,
superimposed complexes of both enzymes (rat and human)

TyrB361
His201,
provided the same type of interaction fingerprints those were

2ZIR
obtained from the individual type of proteins (rat and human).
The original residual number and its corresponding common
number are provided in Table 3.
In order to analyze further, 75 conformers created from the
MD-simulated complexes of each protein were used to analyze
Table 2. important residues and its type of interactions obtained from the PLIF analysis.

TrpB106, TyrB300,
specific type of interactions for individual compounds. The AspB359, TyrB361
LeuB96, TrpB102,
Lys164, Tyr166,

conformers were overlaid separately for the interaction analysis TyrB361

and the results are provided in Table 2 and graphically represented
2IEJ

in Figure 3. The results showed that some important residues,

such as LeuB96, TrpB102, TrpB106, ArgB202, TyrB300,
AspB359 and TyrB361, are predominantly present in most of
the complexes for interactions. The LeuB96 makes surface
interaction (solvent exposed surface) type of interaction with the
ligand in all the complexes. The residue TrpB106 also provided
AspB359, TyrB361

PheB360, TyrB361
LeuB96, TyrB300,
Lys164, TyrB93

interaction fingerprints with all the ligands, which form side-chain

ArgB202

acceptor (the OH groups of this residue) and surface type of

1NI1

interaction with the ligands (with aromatic ring). Another

tryptophan residue, TrpB102 has interacted with the ligands in
2ZIR, 2IEJ and 2ZIS through surface interaction on the aromatic
rings or aliphatic chains present in the ligands and the residues.
TyrB300 and TyrB361 are forming surface and side-chain
acceptor interactions with the ligands in all the complexes
TyrB258, TyrB361
LeuB96, TrpB106,

SerB99, ArgB202
Ala129, CysB95,

(except 2IEJ for TyrB300). The later residue (TyrB361) also

TyrB258

provided an interaction fingerprints on the backbone acceptor

1LD7

interactions.
ArgB202 has interacted with all the ligands in the complexes
except 2IEJ and the nitrogen atoms (guanidinium) in the residue
make side-chain acceptor interaction with the ligands and also
observed a surface interaction on this functional group. AspB359
formed surface interaction with the ligand and this interaction is
interaction

interaction

absent in 1LD7. The interaction residues revealed that the

Type of

Surface

ChAcc

BkAcc

complexes, such as 1NI1, 2ZIR and 2ZIS, are belonging to the

FTase enzyme of rat and which showed the same type of
DOI: 10.3109/14756366.2016.1144593 MD simulations and structure-based pharmacophore development 1433
Table 3. Summary of the common residue names and its type of interactions.

Residue number
Type of interaction Residue No. common 1NI1 2ZIR 1LD7 2IEJ 2ZIS
Surface Ala129 – – 75 – 75
Surface Lys164 110 – – 110 110
Surface, ChAcc Tyr166 112 – – 112 –
Surface, ChAcc His201 – 147 – – 147
Surface, ChAcc TyrB93 383 387 – – –
Surface LeuB96 386 390 393 397 389
BkDon, Surface CysB95 – 389 392 – 388
Surface TrpB102 – 396 – 403 395
Surface, ChAcc TrpB106 396 400 399 407 399
ChAcc, Surface SerB99 – – 396 – 392
ChAcc, Surface ArgB202 492 496 499 – 495
ChDon, BkAcc, ChAcc, Surface TyrB258 – – 548 – 544
ChDon, Ionic AspB297 – – – – 590
Surface, ChAcc TyrB300 590 594 – 601 593
Surface AspB352 642 – – – –
Surface AspB359 649 653 – 660 652
BkAcc PheB360 650 – – – –
BkAcc, ChAcc, Surface TyrB361 651 655 658 662 654
Surface, ChAcc HisB362 652 656 – – 655

ChDon: Side-chain donor, ChAcc: Side-chain acceptor, BkAcc: Backbone acceptor.

Figure 3. PLIF results: Graphical representation of protein-ligand interactions.

interaction with 90% of the residues. HisB362 is a residue has residues (LeuB96, TrpB106 and TyrB361) for the interaction as
coordination bonding with the Zn2+ and has interacted with the the rat enzyme. Additionally, the 2IEJ complex interacted with
ligands present in rat FTase. The ligands from the complexes of Lys164 and Tyr166 residues by surface and side-chain acceptor
human enzyme (1LD7 and 2IEJ) possessed limited common interactions. The conformers obtained from 1LD7 provided
1434 N. S. H. N. Moorthy et al. J Enzyme Inhib Med Chem, 2016; 31(6): 1428–1442

interaction specifically with Ala129 through surface interaction. Distance analysis

Furthermore, these conformers have interacted with CysB95,
In order to understand the interactions between the specific
SerB99 and TyrB258 through side-chain acceptor and surface
groups/atoms in the ligands, those have shown protein–ligand
interactions, which are also observed on the ligand in 2ZIS. The
interaction fingerprint bits with the residues, the distance between
PLIF analysis performed on the conformers derived from the MD
them were analyzed on the MD simulated proteins using PTRAJ
simulated complex showed that some common residual inter-
module of AMBER 12 (Figure 4)30,46. The distance between the
actions observed on both rat and human FTase enzymes.
Zn2+ and the nitrogen atom present in the imidazole or methyl
The interaction points of the residues in the ligands are
imidazole rings were analyzed which showed that the distance
graphically represented in Table 2. The Tyr166, ArgB202 and
between these atoms are almost stable below the distance of 3 Å
TyrB361 residues form hydrogen-bonding interaction with the
throughout the MD-simulated time for 1NI1, 2ZIR and 2ZIS
nitrogen atom of the nitrile groups in the molecules. The
complexes. The distance between these atoms are varying lot for
imidazole or methyl imidazole rings form – interaction (surface
those complexes from human enzyme (1LD7 and 2IEJ), while it is
or hydrophobic interactions) with the aromatic rings in the
stable for the rat FTase complex. It is showing that the ligands in
aromatic amino acids (especially, Tyr166 and TyrB361). The
the complexes (1NI1, 2ZIR and 2ZIS) have a stable interaction
phenyl rings present in the ligands form – interaction with the
with the Zn2+ ion than the human FTase. The distance between the
hydrophobic or aromatic rings in the amino acids (leucine and
hydrogen atom in guanidinium group in the ArgB202 with the
tyrosine). Ketone groups and other polar groups (SO2, NO, OH,
nitrogen atom in nitrile groups in the ligands such as 1NI1
etc.) present in the molecules have some hydrogen-bonding
(interact with both hydrogen atoms in NH of the guanidinium
interactions with polar residues (oxygen and nitrogen atoms)
group) because it also has surface type interactions and 2ZIS
present in the proteins.
(the ligand have two nitrile groups). The figure showed that the

Distance between ligand and Zn Distance between ligand and ArgB202

11 11

9 9
Distance (Å)
Distance (Å)

7 7

5 5

3 3

1 1
0 2000 4000 6000 8000 10000 0 2000 4000 6000 8000 10000
Time (ps) Time (ps)
1NI1-N1 & N2 1NI1-N1 1NI1-N2 2ZIS-N1 2ZIS-N2
1LD7 1NI1 2IEJ 2ZIR 2ZIS

Distance between ligand and TyrB361 Distance between ligand and TyrB300
8
11
7
6 9
Distance (Å)

Distance (Å)

5
7
4
5
3
2 3

1 1
0 2000 4000 6000 8000 10000 0 2000 4000 6000 8000 10000
Time (ps) Time (ps)
1LD7-Surf 1NI1-Surf 1NI1-Acc 2IEJ-Surf
2ZIR-Surf 2ZIS-Surf1 2ZIS-Surf2 1NI1-Surf1 1NI1-Surf2 2IEJ-Surf 2IEJ-Acc 2ZIR-Surf

Distance between ligand and LeuB96 Distance between ligand and some residues
8 12

10
7
Distance (Å)

8
Distance (Å)

6 6

5 4

2
4
0
3 0 2000 4000 6000 8000 10000
0 2000 4000 6000 8000 10000 Time (ps)
Time (ps) 1LD7-SerB99-Acc 1LD7-TyrB258-Surf 1NI1-PheB360-Acc
2ZIR-TrpB106-Surf 2ZIS-TrpB106-Surf 2ZIR-HisB362-Acc
1NI1 2IEJ 2ZIR 2ZIS 2ZIS-HisB362-Acc 2IEJ-Lys164-Surf 2IEJ-Tyr166-Surf

Figure 4. Distance between protein and ligands from MD simulations.

DOI: 10.3109/14756366.2016.1144593 MD simulations and structure-based pharmacophore development 1435

nitrile group 1 has better interaction with the ArgB202 and the
nitrile group 2 has little interaction with the hydrogen atom of
the guanidinium group. The nitrile group in the 1NI1 complex has
interacted with the hydrogen atoms (in NH) of the guanidinium
group with equal distance. The figure reveals that the distance
between the atoms have been increased after 7 ns from the
distance of 2 Å. This result reveals that when a ligand has two
nitrile groups one nitrile group interacts with the ArgB202 and the
other do not (other nitrile group interact with OH group present in
TyrB300 or TyrB361).
The distance between the phenyl ring in the TyrB361 with
the aromatic ring in the ligand was calculated, because these
compounds showed surface interaction in the PLIF analysis.
The average distance between the atoms (carbon) in phenyl ring
of the TyrB361 and the atoms in the aromatic rings in the
ligands are ranging between 4 and 7 Å. Additionally, the
distance between the nitrogen atom in the nitrile group of
the ligand (in 1NI1) with the hydrogen atom in hydroxyl group
of the tyrosine moiety (TyrB361). The acceptor type of
interaction observed between these groups is stable at the
distance below 3 Å. The distance calculated for the complexes
except 1LD7 with the aliphatic chain of LeuB96 and the atoms
in aromatic rings or the hydrophobic atoms are between 4–6 Å.
There is an acceptor kind of interaction observed between the
hydrogen atom in the hydroxyl group of TyrB300 with the
nitrogen atom in the imidazole ring of the ligand in complex
2IEJ, which has showed the distance below 3 Å after 6 ns
simulation. However, the same residue has also show surface
interaction between the atoms in phenyl ring and the atoms in
ArgB202, TyrB300, AspB359, TyrB361 and HisB362, have
significant interaction with the ligands in all the complexes. The
RMSF values of these residues for all the complexes except 1NI1
is below 2 Å. The complex 1NI1 showed large variation of
flexibility especially in the TyrB93, LeuB96 and CysB95 residues
than other residues (Figure 5). The PLIF results for these residues
showed that the interaction with CysB95 is absent and LeuB96 is
significant. Also, the RMSF values for the atoms in the phenyl
nitrile groups of the ligands are analyzed. The results provided in
figure showed that the ligands in complexes, such as 1LD7, 1NI1
and 2ZIS (2 phenyl nitrile groups), have the RMSF values less
than 2 Å. The second phenyl nitrile group in the ligand in the 2ZIS
has larger RMSF value than other ligands. The nitrogen atom in
the nitrile group has changed the flexibility than the phenyl ring.
In the same way, the RMSF values of the atoms in the imidazole/
methyl imidazole rings were also calculated. The methyl imid-
azole rings in the 1NI1 is having the RMSF value between 2 and
4 Å. The nitrogen and the carbon atoms in the imidazole ring have
low RMSF values than the hydrogen atoms in the ring. The
methyl group (C4) connected with the imidazole ring have larger
RMSF value than the carbon atoms of the imidazole ring in the
ligands.
α of protein
RMSF of Cα
6
5
RMSF (Å)

the aromatic ring of the ligands in 1NI1, 2IEJ and 2ZIS 4

complexes. The results showed that the ligand in 1NI1 has 3
larger distance than other complexes (5–9 Å). 2
The oxygen atoms in some functional groups such as C¼O, 1
SO2, NO, OH and other polar groups in the ligands have shown
0
acceptor type interactions with the polar groups (OH, NH2, –NH,
S, etc.) in some residues. In the complex 1LD7, the distance
between the oxygen atom in keto group of the ligand with the Residues
hydrogen atom in hydroxyl group of the SerB99 showed 53 Å 1LD7 1NI1 2IEJ 2ZIR 2ZIS

and is 52 Å after 8 ns. A surface interacting group such as atoms RMSF of phenyl nitrile in ligands
in the pyrrolidine ring with the atoms in the aromatic ring of the 4
TyrB258 showed the distance 45 Å. In 1NI1, the distance 3.5
between the nitrogen atom in nitrile group in the ligand with the 3
hydrogen atom in amino group in the PheB360 is 53 Å. In case
RMSF (Å)

2.5
of 2IEJ, the distance between the aliphatic group in the Leu164 2
with the atoms in the phenyl ring of the ligand and the atoms in 1.5
phenyl ring in the Tyr166 with morpholine ring in the ligand are 1
between 5–8 Å. The distance between the hydrogen atom in the 0.5
NH group (in ring) in HisB362 of the complexes such as 2ZIR
0
and 2ZIS were calculated with the nitrogen atom in the C1 C2 C3 C4 C5 C6 H1 H2 H3 H4 C7 N1
imidazole ring, which is 54 Å. Further, the atoms in the Atoms in Ligands
1LD7 INI1 2ZIS-1 2ZIS-2
phenyl ring in the TrpB106 of 2ZIR and 2ZIS were analyzed
with the atoms in the phenyl ring in the ligands. The distance RMSF of methyl imidazole in ligands
4.5
analysis results reveal that the average distance on the surface
4
interactions (aromatic rings) are between 4 and 10 Å and the 3.5
acceptor groups provided the distances below 3.5 Å throughout 3
RMSF (Å)

the MD-simulated time. 2.5

2
1.5
RMSF analysis
1
RMSF analysis allows an identification of the more flexible 0.5
regions and also a comparison of the relative flexibility of 0
different regions of a system46,47. In this analysis, we have N1 C1 N2 C2 C3 C4 H1 H2 H3 H4 H5
Atoms in Ligands
analyzed some important residues, which have interaction with
1LD7 1NI1 2IEJ-1 2IEJ-2 2ZIR 2ZIS
the ligand molecules on different complexes (analyzed through
PLIF studies). The important residues, such as Lys164, Tyr166, Figure 5. RMSF of important residues in protein and interaction
His201, TyrB93, LeuB96, CysB95, TrpB106, TrpB102, SerB99, functional groups in ligands.
1436 N. S. H. N. Moorthy et al. J Enzyme Inhib Med Chem, 2016; 31(6): 1428–1442

RDF analysis 2ZIS and N+ in 1LD7. It is interesting that all the functional
groups have interactions with the hydrogen or oxygen atom in the
The RDF (or pair correlation function) g(r) in a system of
water molecules surrounded them. These groups show peak
particles (atoms, molecules, colloids and so forth), describes how
between 2.5 and 2.8 Å with varying RDF values. The NO group
density varies as a function of distance from a reference
and the OH groups in the molecules have large RDF value than
particle46–48. In this analysis, the RDF of water around some
the SO2 group. The keto groups also showed significant RDF
important functional groups in the ligands and the residues has
values with sharp peek at the distance specified earlier. These
shown interactions as studied through PLIF (Figure 6). A more
functional groups such as nitro (NO) interact with the Asp590,
comprehensive analysis of figure and all the structural elements
ketone groups interact with tyrosine or serine or leucine residues
considered at this level present a possible relation with the RDF
and the oxygen atoms in the OH group interact with the tyrosine
analysis of water around some polar functional groups (CN, SO2,
residues. The RDF analysis showed that these functional groups
C ¼ O, NO, N+) and OH groups in TyrB361 and NH in
also have significant interaction with the water molecules, which
guanidinium group in ArgB202. Generally, these functional
are surrounded in the active position.
groups possessed acceptor type of interaction.
The residues, such as ArgB202 and TyrB361, are exhibited
The density of water around the nitrile groups in the ligand and
polar interaction with the complimentary functional group or
weather they interact with the water molecules were analyzed
atoms (polar groups) in the ligand. Hence, the RDF for the OH
through RDF calculation. The figure developed for the RDF of
group in TyrB361 and NH group of guinidinium in the ArgB202
nitrile groups in the molecules showed that the ligands in 1LD7,
were analyzed and the results are presented in Figure 5. The OH
1NI1 and 2ZIS are having nitrile group in their structures and
group in TyrB361 in 1LD7 has provided significant RDF value
showed a sharp peek at 2.8 Å for the nitrile groups in the ligands
(0.9) at 2.8 Å. Other complexes do not provide significant RDF
in 1LD7 and 1NI1. The second nitrile group in the ligand in 1NI1
values reveal that this moiety present in 1LD7 has interaction with
has shown no interaction with the water molecule. In the same
the water molecules surrounded on the residues. The two
way, the second nitrile group in the ligand present in 2ZIS
guanidinium nitrogen atoms present in the ArgB202 showed
complex also do not have any interaction with the water
prominent peeks at 2.8 Å distance. It reveals that these nitrogen
molecules. These results revealed that there is a specific
atoms also have interactions with the water molecules surrounded
interaction exists between the hydrogen atoms of the water
on the residues. These water molecules may cause bridge
molecules and the nitrile groups of the ligand. This reveals that
formation between the nitrogen atoms with the functional
the nitrile groups in the molecules projected into the water
groups in the ligands.
accessible area for polar interactions.
These nitrile groups also interact with the hydrogen atom
Structure-based pharmacophore generation
present in the OH groups of the tyrosine residues as revealed by
the PLIF analysis. It reveals that the water molecules present in The structure-based pharmacophore analysis is one of the
the space between the hydroxyl group of the tyrosine and the important techniques used nowadays to screen large data set to
nitrile group on the ligands. Further, the ligands have some other identify HIT molecules. Generally, these pharmacophore points
polar functional groups in their structures, such as SO2 in 2IEJ, are generated directly from the protein–ligand complex, consider-
keto group in 1LD7, 2IEJ and 2ZIR, OH in 2ZIS and 2ZIR, NO in ing both ligand and the protein structures19–26. In this analysis,

1 10 2 10
Nitrile groups NH of ArgB202
0.8 8 8
1.5

No. of water molecules

No of water molcules
RDF g(r)

0.6 6
6
RDF g(r)

1
0.4 4
4

0.2 2 0.5
2

0 0
0 2 4 6 8 10 0 0
Distance (Å)
0 2 4 6 8 10
1LD7-CN 1LD7-CN 1NI1-CN-1 1NI1-CN-1 1NI1-CN-2 Distance (Å)
1NI1-CN-2 2ZIS-CN-1 2ZIS-CN-1 2ZIS-CN-2 2ZIS-CN-2 1LD7-NH1-Arg 1LD7-NH1-Arg 1LD7-NH2-Arg 1LD7-NH2-Arg 1NI1-NH1-Arg
1NI1-NH1-Arg 1NI1-NH2-Arg 1NI1-NH2-Arg 2IEJ-NH1-Arg 2IEJ-NH1-Arg
2IEJ-NH2-Arg 2IEJ-NH2-Arg 2ZIR-NH1-Arg 2ZIR-NH1-Arg 2ZIR-NH2-Arg
2ZIR-NH2-Arg 2ZIS-NH1-Arg 2ZIS-NH1-Arg 2ZIS-NH2-Arg 2ZIS-NH2-Arg
1 10
OH of TyrB361 2 10
Functional groups
0.8 8 in ligands 8
1.5
No. of water molecules

No. of water molecules

RDF g(r)

0.6 6
RDF g(r)

6
1
0.4 4 4

0.5
0.2 2 2

0 0
0 0 0 2 4 6 8 10
0 2 4 6 8 10 Distance (Å)
Distance (Å)
2IEJ-SO2-1 2IEJ-SO2-1 2IEJ-SO2-2 2IEJ-SO2-2 1LD7-Keto
1LD7-OH-Tyr 1LD7-OH-Tyr 1NI1-OH-Tyr 1NI1-OH-Tyr 1LD7-Keto 2IEJ-Keto 2IEJ-Keto 2ZIR-Keto 2ZIR-Keto
2IEJ-OH-Tyr 2ZIR-OH-Tyr 2ZIR-OH-Tyr 2ZIS-OH-Tyr 1LD7-N+ 1LD7-N+ 2ZIS-NO 2ZIS-NO 2ZIR-OH
2ZIS-OH-Tyr 2IEJ-OH-Tyr 2ZIR-OH 2ZIS-OH 2ZIS-OH

Figure 6. RDF analysis of functional groups in residues and ligands.

DOI: 10.3109/14756366.2016.1144593 MD simulations and structure-based pharmacophore development
protein–ligand interaction fingerprint bits created through PLIF
analysis was used to generate the pharmacophore queries. The
details of the fingerprint bits created in the PLIF analysis are
provided in Table 2 and are discussed in earlier section. In order
to perform pharmacophore analysis, eight pharmacophore queries
were generated from the PLIF created from the analysis. These
queries made of the conformers of the individual complexes (Ph-
1LD7, Ph-1NI1, Ph-2IEJ, Ph-2ZIR and Ph-2ZIS), only complexes
of rat (Ph-rat) and human (Ph-human) and all the complexes (Ph-
all) (Figure 7 and Table 1).
The important interaction fingerprint bits (425% abundance)
obtained from the PLIF analysis were used for the pharmacophore
query development. The pharmacophore contours generated from
the analysis are provided in Table 1. The excluded volumes of the
pharmacophore contours are included in the pharmacophore
models, those are used to screen the data set. Each pharmacophore
Figure 7. Graphical representation of the
structure based pharmacophore models.
1437
models have 3–4 pharmacophore contours consist of acceptor &
metal ligand (Acc&ML), hydrophobic (HydA) and extended
acceptor (Acc2) features with the radius ranging between 1 and
3 Å for Acc&ML and 1–2 Å for HydA. The excluded volume of
the pharmacophore contours radius is between 1 and 2 Å. The
developed pharmacophore models with the interacting residues
are graphically represented in Figure 6. In these models, the
pharmacophore contours are generated specifically through the
interacting residues. The functional groups/atoms in ligands and
the residues causing the type of the interactions are discussed
earlier. Eventhough, it confirms that the aromatic residues, such
as tyrosine and tryptophan and the aliphatic residue leucine, are
majorly caused for the hydrophobic interactions. The hydroxyl
group in the tyrosine, the nitrogen atoms in the arginine and side
chains polar groups in other important residues (cysteine, serine
and histidine) causes the Acc&ML pharmacophore contour
1438 N. S. H. N. Moorthy et al. J Enzyme Inhib Med Chem, 2016; 31(6): 1428–1442

Table 4. Virtual screening results of the identified HITs.

Pharmacophore models and RMSD values (Å)

Comp Code Ph-1LD7 Ph-1NI1 Ph-2IEJ Ph-2ZIR Ph-2ZIS Ph-Rat Ph-Human Ph-All IC50 nM
Tipifarnib 1.3532 1.2619 1.3452 1.2331 1.8493 1.0384 1.2672 1.3732 0.86
Lonafarnib 1.2034 1.6791 1.5321 1.2355 1.3547 1.4508 1.3009 1.3291 1.90
L-778123 – 1.8912 1.3212 1.4661 1.5531 1.3683 – 1.2345 2.00
Nat1 1.3302 1.7070 1.4757 1.1816 1.8286 1.3408 1.2691 1.2873 230
Nat2 1.3334 1.3757 1.3135 1.0710 1.7353 1.3624 – 1.2324 50
Nat3 1.3014 – 1.5053 – 1.7480 1.4074 – – 50
Nat4 1.5113 – – 1.1707 – 1.5530 – 1.5040 6000
Nat5 – – 1.4053 – – 1.4789 1.3066 1.5223 16800
Nat6 1.3177 – – 1.1240 – 1.6346 – 1.4706 NA
Nat7 1.3135 – 1.0715 – 1.3624 – 1.3629 1.2972 NA
Comp-1 1.8765 – 1.9450 – – – – 1.9233 2540
Comp-2 1.3982 1.3032 – 1.3029 – – 1.4652 1.2354 1300
Comp-3 1.6783 – – – 1.5674 – – – 410000
Comp-4 1.3221 – – 1.3457 – – – 1.4560 1320

NA is not active.

Table 5. Structure of the inhibitors used for the validation of the pharmacophore models and the identified HITs.

Comp Code Compound name Structures

Ref-1 Tipifarnib
Cl
N

NH2

Cl

Ref-2 Lonafarnib
Br

Cl
N

Br

O
N

N
O NH2

Ref-3 L778123
N
O HN

N N

Cl N

(continued )
DOI: 10.3109/14756366.2016.1144593 MD simulations and structure-based pharmacophore development 1439

Comp Code Compound name Structures

Nat1 Actinoplanic acid A
O
C

O COOH

O O

O COOH OH

COOH

OH O

Nat2 Actinoplanic acid B

O
COOH

O COOH

O O

O COOH OH

COOH

OH O

Nat3 Zaragonic acid

O
HO
O

O O
COOH
O
O
OH

COOH
COOH

Ph

Nat4 CP-225917
O

O O

O OH
HOOC H O

HO H

H
O

Nat5 Citreohybridones A CH3

O
CH3
O

CH3

O O
OAc
O OCH3
O
H3C H3C CH3

(continued )
1440 N. S. H. N. Moorthy et al. J Enzyme Inhib Med Chem, 2016; 31(6): 1428–1442

Table 5. Continued

Comp Code Compound name Structures

Nat6 Pentapeptide
O NH

O O O
H H H
O N N N
N N NH2
H H
O O O CH2
OBzl
NH
N

Nat7 2-Desacetyl-8-epixanthumanol-4-O-b-Dgalactopyranoside
OH
HO OH

HO
O

AcO
O

Comp-1
O
OH
O

O
H O

Comp-2
Br

N O
N

S N
N

Comp-3 BDBM50128063 CH2

O N
S

N CH3

Br

(continued )
DOI: 10.3109/14756366.2016.1144593 MD simulations and structure-based pharmacophore development
Comp Code Compound name
Comp-4 BDBM50226160
generation. Hence, these interacting groups generated the hydro-
phobic type of pharmacophore contours in the studied complexes
or the conformers.
The validation of the developed pharmacophore is important
before going to virtual screen large data set. In order to perform
validation, a set of reference compounds (tipifarnib, lonafarnib
and L7,78,123 and natural compounds exhibited FTase inhibitory
activities were screened. The results obtained from the validation
analysis are provided in Table 4. It shows that the reference
compounds tipifarnib and lonafarnib have been identified as
significant HIT through all the pharmacophore models. The
compound L7,78,123 has not identified byPh-1LD7 and human
pharmacophore models. It is considered that the RMSD value for
the compounds identified through these models are 52 Å.
Additionally, the developed structure-based pharmacophore
models were validated by screening a data set of natural
compounds exhibiting FTase inhibitory activities and some
compounds do not have FTase inhibitory activities. The important
compounds identified through virtual screening are provided in
Table 4. The results showed that the natural compounds Nat1 and
Nat2 have been identified as significant compounds through all
the pharmacophore models. These compounds have reported
FTase inhibitory activity (IC50) of 230 and 50 nM, respectively.
The compound Nat3 has been identified by four models, which
have the experimental FTase inhibitory activity of 50 nM.
The RMSD values of these molecules also 52 Å. These results
confirm that the developed structure-based pharmacophore
models are significant for virtual screening of new molecules.
Further, the virtual screening of 100 natural compounds, 1000
synthetic compounds from literatures and a data set from data
base (binding database). Those molecules identified by more than
three models and its RMSD values 52 Å was selected as
significant hits and the novel hits identified through the
structure-based pharmacophore analysis are also provided in
Table 4. There are two molecules identified from four pharma-
cophore models with the RMSD values 52 Å. The structures of
the molecules selected from the analysis are provided in Table 5.
From the literature data set and the binding database, four
compounds were identified through 2–3 models. Among the six
compounds selected from all the data set three compounds
(compounds 1, 2 and 4) have significant activities such as 2540,
1441
Structures
NH2
H
N
N
O
O
NH O
HN
NH O
O
HO
OH
O
1300 and 1320 nM, respectively. These compounds can be used as
lead molecules for the development of novel FTase inhibitors.
Conclusion
In this analysis, the structure-based pharmacophore models were
generated for some complexes from protein data bank. In contrast
to the existing methods, in this study, we have used the
conformers of the ligands, generated through MD simulations
for the pharmacophore query generation through PLIF analysis.
This method provides a significant pharmacophore model with
major interaction fingerprints of the conformers than a single
conformer used in normal method. The validated pharmacophores
models identify the HITs effectively. The statistical mechanics of
the MD simulations results provided significant output in terms of
RMSD, RMSF, RDF and distance analysis. This reveals that the
developed structure based pharmacophore model is significant,
because generally, the structure based pharmacophore models
generated on the ligand coordination in the X-ray complex. But in
this analysis, along with the coordinated ligand, different confor-
mers from the same ligands were utilized. We conclude that the
developed pharmacophore model can be a significant model for
the identification of HITs as FTase inhibitors. Hopefully, this
protocol can be an alternative for some structure-based virtual
screening of data sets.
Declaration of interest
The authors report no conflicts of interest. The authors alone are
responsible for the content and writing of this article.
One of the Authors (N.S.H.N. Moorthy) gratefully acknowledges
the Fundaçao para a Ciencia e Technologia (FCT), Portugal, for a
Postdoctoral Grant (SFRH/BPD/44,,469/2008). N.S.H.N. Moorthy
is also thankful to RGPV, Bhopal, India, for providing support in
this work.
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