Screening using a Bank of Molecules of the Amazon Region

against functional NS3-4A protease-helicase enzyme of

Hepatitis C virus
Alan Sena Pinheiroa, Jaqueline Bianca Carvalho Duarte a, Cludio Nahum Alvesb,
Fbio Alberto de Molfettaa*
1

Laboratrio de Modelagem Molecular,Instituto de Cincias Exatas e Naturais, Universidade Federal do Par,

CP 11101, 60075-110, Belm, PA, Brazil
2
Planejamento e Desenvolvimento de Frmacos, Instituto de Cincias Exatas e Naturais, Universidade Federal do
Par, CP 11101, 60075-110, Belm, PA, Brazil
*
Corresponding author: Fbio Alberto de Molfetta, E-mail address: fabioam@ufpa.br

Abstract
Hepatitis C virosis (HCV) is a disease that reaches approximately 3% of the world population and requires new
therapeutic agents without the inconveniences the current anti-HCV ones involve for its treatment. This paper reports
on a study of a virtual screening and a molecular dynamics simulation of compounds of natural products from the
Amazon region potentially effective against the NS3-4A enzyme of HCV, which plays an important role in the
replication process of the Hepatitis C virus. According to the results of the molecular docking calculations and
consecutive consensual analysis, the best scored compounds showed interactions between hydrogen and residues of
the catalytic triad as well as interactions with residues that guide the ligands regarding the active site of the enzyme.
They also showed stability in the molecular dynamics simulation, as the structures preserved important interactions in
the active site of the enzyme and the RMSD values were stabilized at the end of the simulation time. Such
compounds are considered promising for the therapy against HCV.
Key-words: Bank of Molecules of the Amazon (BMA), Hepatitis C virus, NS3-4A protease-helicase, Molecular
docking, consensual analysis, Molecular dynamics

1. Introduction
HCV is a human pathogen that has infected approximately 170 million individuals on the planet, of
whom over 350 thousand die every year from diseases related to such a virus [1,2]. The translation
process of the RNA of HCV codifies a poliprotein of more than 3000 residues of aminoacids, which is
further cleaved by viral and cellular proteases, originating ten mature viral proteins: structural proteins
(E1, E2 and Core), p7 and non-structural proteins (NS2, NS3, NS4A, NS4B, NS5A and NS5B) [2,3].
The infections by HCV cause acute symptomatic diseases in the liver, which can advance to more
severe diseases, such as hepatic cirrhosis and liver cancer [1,4]. The current standard therapy based on the
combination of interferon- (IFN- ) and ribavirine causes several side effects and the response to the
treatment has shown unsatisfactory to most patients [1,3]. Therefore, more effective and less harmful
therapies to the patients are necessary [5].
On of the most promising and most studied targets in the development of drugs against HCV is protein
NS3, which contains a domain with serine-protease activity and another with helicase activity. Both
activities are modeled by the presence of NS4A cofactor, which acts as an activating cofactor. Component
NS3-NS4A is a multifactor enzymatic complex whose catalytic triad is constituted by residues of
aminoacids His57, Asp81 and Sere139. It also exhibits an oxianionic cavity common in members of the
serine-protease family. Such a complex is essential for the replication process of the virus [2,4,6,7].
Several studies that employ molecular docking with NS3-4A target have been conducted [4,6,8-10] and
most of them involve peptide-like compounds. However, such compounds exhibit poor pharmacokinetic
properties and low bioavailability [8,9], which shows the importance of investigating the potentiality of
compounds from natural products as a source of new anti-HCV agents [8]. Molecules from natural
products are usually recognized by their higher susceptibility to being absorbed by the human organism in
comparison to synthetic substances [11].
The Molecular Dynamics technique has extensively contributed to various stages of the biological
process in the drug design, once in chemical reactions of biological interest the introduction of the effect
of the environment is important be taken into account in computational calculations. Moreover, the use of
Molecular Dynamics by the hybrid QM/MM method enables the prediction of the breaking and formation
of chemical bonds of interest within the biological system [12,13,14].
This study comprises a virtual screening that employed a database of compounds with 664 molecular
structures from natural products extracted from vegetal species of the Amazon region, an area of richest

biodiversity in the world and an important source of therapeutically effective compounds [11]. A
consensual analysis from different docking programs was further applied to the result of the virtual
screening. Furthermore, a combined quantum mechanics/molecular mechanics (QM/MM) approach was
employed for a detailed study of the best candidates from the natural products selected in the analysis.
The results can be used for the selection of more promising structures that can inhibit bifunctional
enzyme NS3-NS4A of HCV.
2. Methods
2.1- Molecular Docking
The techniques of molecular docking aim at geometrically and energetically predicting the
conformation and orientation of the ligand with the binding site of the target. The search starts by the
application of docking algorithms, which allocate the molecules in the site of the receptor. Scoring
functions are then implemented so that all the components that contribute to the free energy value of the
ligand-receptor complex formed [15] can be counted.
All the molecular structures used in this study were obtained from theses, dissertations and scientific
papers on natural products extracted from vegetal species of the Amazon region. Such a set of compounds
has been called Bank of Molecules of the Amazon (BMA). The structures were designed in bidimensional
representations on MarvinSketch 5.9.3 software [16] and then converted into tridimensional
representations by OMEGA 2.2 program [17], which employs DREIDING field force [18]. Subsequently
a virtual screening with BMA was conducted on Molegro Virtual Docker 5.0.0 [19] and DOCK 6.3 [20]
programs.
Molegro Virtual Docker (MVD) contains a hybrid heuristic search algorithm, called guided differential
evolution algorithm, which combines a differential evolution optimization technique [21] with a cavityprediction algorithm. MVD employs MolDock scoring function derived from Piecewise Linear Potential
(PLP) [22] and extended on GEMDOCK [23] and has included a term regarding the directionality of the
hydrogen bond [19].
On the other hand, DOCK 6.3 program is characterized by the use of an incremental construction
algorithm and the scoring functions that guide the ligands to the target are based on a grid of potential
energy, where the van der Waals interactions are assessed by the Lennard-Jones potentials and the
electrostatic interactions are evaluated by means of time-dependent dielectric functions [20,24].
The docking calculations were performed with the crystallographic coordinates of the NS3-NS4A
protease-helicase bifunctional enzyme of hepatitis C (PDB code: 1CU1) [25] recovered from Protein Data
Bank (PDB). As the structure utilized is homodimeric, only one enzyme chain was chosen for the
calculations. The zinc and phosphate ions as well as all water molecules of the chain chosen were
removed by Chimera software [26].
The hydrogens were added to the ligands and the loads were calculated by AM1-BCC method [27] for
the calculations on DOCK 6.3. The hydrogens were removed from the crystallographic model and a box
of 10 size was generated and calculated by dms, SPHGEN, grid and SHOWBOX [28-30] programs. An
extra margin of 6 and 0,3 resolution was used. Its center was placed on the ligand, which was
positioned in the center of the active site described in the literature (a region of 9 between residues
Ala621 and Thr631, which corresponds to the C-terminal cleavage product). Such residues were deleted
for the docking of the ligands [4,31]. After the structures of the receptor and the ligands had been
prepared, the docking and subsequent minimization of the ligands were started on the binding site of the
target [20,32].
In MVD, the hydrogens were added to the ligands and the structures of the receptor and the atomic
charges were assigned by the standard automated preparation module of the program. The binding cavity
was then computed in a 10 space from coordinates x=34.65, y=69.63 and z=114.72 with a 0,3 grid
resolution, which provided total flexibility to the residues of His57, Asp81, Lys136, Gly137, Ser139,
Arg155 and Ala157 amino acids (critical residues for the interaction of natural substrate with NS3-4A
enzyme of HCV). The standard values of the other parameters of the program were kept during the
process [19,33].
The fact that each scoring function implemented in the docking programs provides different results in
the scoring of a same molecule configures a problem faced when only one docking program is used in
virtual screening. To improve the results of a virtual screening, a strategy that employs several programs
with distinct scoring functions is generally applied. Such an approach, called consensual analysis,
improves the accuracy of the results and compensates the deficiencies found in each scoring function. The
consensual analysis consists in combining the results of each different scoring and classifying them again

according to such a combination, which provides a more accurate estimate and represents an important
tool for studies of protein-ligand intermolecular interactions [34,35].
The consensual analysis can be applied by three distinct methods: scaled-rank-by-number, rank-byrank and rank-by-vote [36,37]. In our study we chose scaled-rank-by-number, as it has provided the best
success rates in the prediction of active molecules in virtual screening with collections of compounds
[37].
The scaled-rank-by-number is employed by scoring the energy values predicted for all compounds in
the molecular docking with the different programs, according to the following equation
Xranked= (X - Xmin)/(Xmax Xmin)
(1)
where Xranked is the scored value obtained and Xmax and Xmin are, respectively, the maximum and minimum
values of the set utilized. Xmax corresponds to the most favorable affinity energy (lowest energy value), i.e.
Xranked equals 1, and the least favorable affinity energy value (highest energy value) is assigned 0, i.e. Xranked
= 0. The respective values scored for the compounds are then summed in each program and the final rank
of the compounds best scored by different scoring functions is obtained [36].
2.2- Molecular Dynamics
The molecular dynamics simulations combined with the hybrid QM/MM method have been widely used
in studies of inhibitors/protein interactions and enzymatic catalytic mechanism [38]. In this study, the
initial coordinates used for the calculations of the QM/MM molecular dynamics were obtained from
enzyme NS3-4A of HCV retrieved from PDB with code 1CU1.
Firstly, the protonation states of amino acid residues at pH = 7 were accurately assigned by recalculating
the standard pKa values by the propKa 3.1 program [39], because the standard values of pKa of ionizable
groups can be displaced by the local enzyme environment. Hydrogen atoms were added to the structure
and a series of optimization algorithms (gradient in conjunction, steepest descent and L-BFGS-B) [40]
was applied. The optimized protein was then placed in a cubic box of pre-equilibrated waters (80 side),
using the center of mass of the inhibitor as the geometrical center. To avoid the denaturation of the protein
structure, all heavy atoms of both protein and inhibitor were restricted by means of a cartesian harmonic
umbrella with a force constant of 1000 kJ.mol-1.-2. The system was totally relaxed, however the
peptide backbone was restricted with constant force of 100 kJ.mol-1.-2. Afterwards, 100 ps of hybrid
QM/MM Langevin-Verlet molecular dynamics at a temperature of 300 K and in a canonical
thermodynamic ensemble (NVT) were used to equilibrate the model [41,42].
For the calculations of the molecular dynamics using the QM/MM method, the atoms of the ligand and
amino acid residues His57 and Ser139 were selected to be treated by QM using a semi-empirical AM1
method [43]. The rest of the system (enzyme plus water molecules) was described by OPLS-AA [44]
force fields and TIP3P [45], respectively, as implemented in the DYNAMO library [46]. Due to the
large amount of degrees of freedom, any residue 20 apart from any of the atoms of the initial inhibitor
was selected to be kept frozen in the remaining calculations so that the model could be computationally
feasible. Cutoffs for the non-bonding interactions were applied by a switching scheme, within a 14 to 16
radius range. As the system was pre-balanced, 2000 ps of QM/MM molecular dynamics were
performed for each of the two systems, with steps of 0,001 ps [38,47,48].
3. Results and discussion
3.1- Molecular Docking
The consensual analysis of the calculations of the virtual screening with BMA resulted in the selection of
five compounds (263, 394, 393, 250, 396) with their respective chemical structures and autoscaled values
(Table 1). Table A.1 of Appendix A provides the energy values obtained by docking, the scored values
obtained by Eq. (1) and the final rank obtained by the scaled-rank-by-number method for all compounds
on both programs.
In a next step, a visual inspection for the elimination of the compounds that might display structural
characteristics similar to peptides revealed the molecular mass of compounds 393 and 394 was very high
and similar to that of peptides, which would cause bioavailability problems [9]. Although the molecular
mass of compound 396 is not high, it shows no hydrogen interactions with the residues of the site (Fig.
A.1 of Appendix A), which hampers the molecular recognition process and destabilizes the complex
formed with the enzyme [49]. Therefore, those three compounds were eliminated by the above-mentioned
evaluation criteria.

The conformation obtained by the molecular docking for compound 250 showed hydrogen bonds with
the residues of amino acids His57 and Ser139 (Fig. 1 (a)), which are part of the catalytic triad of enzyme
NS3/4A of HCV. Therefore, such bonds play a fundamental role in the enzyme activity and must be
strictly related to the inhibition process of the biological target [50].
The conformation predicted for compound 263 (Fig. 1 (b)) also shows hydrogen interactions with the
catalytic residues of His57 and Ser139, besides hydrogen interactions with residues of Arg155 and
Gln526. The inhibitors hydrogen interactions with the residue of Arg155 have been commonly reported in
studies of molecular docking of 1CU1 structure [4,51-53]. Such an interaction is highly important in the
orientation of the ligand regarding the proximity to the oxianionic hole (formed by residues of Gly137
and Ser139), a region where there occurs a cleavage of the natural substrate of serine-proteases. Moreover
the absence of conditions favorable for the hydrogen interactions with the residue of Arg155 can
influence the loss of the compounds inhibition potency, resulting in a weak interaction with the
oxianionic cavity [51]. The hydrogen bonds with the residue of Gln526 are considerably significant in the
inhibition process of the target as they integrate one of the potential sites of molecular recognition of the
enzyme [52].
3.2- Molecular dynamics
The simulations of molecular dynamics enabled the analysis of the behavior of the two models obtained
through molecular docking calculations in the enzyme-ligand complex in the biological environment.
They generated graphs of RMSD (Root Mean Square Desviation) [54], shown in Figures 2 and 3, through
which a comparison between the structure of the initial receptor-ligand complex (before simulation) and
the final structure of the receptor-ligand complex (after the simulation) can be performed.
The graph of RMSD of carbon- atoms of the system showed that compound 250 ((-)-6-trans-methoxykhellactona) (Figure 2) remained stable at a simulation time of 1000 ps. This compound suffered only
mild structural fluctuations and remained below 0.7 . Compound 263 (surinamensin) (Figure 3) showed
structurally stable after 1000 ps of simulation and kept an RMSD value of approximately 0.45 during a
large part of the simulated time, with small deviations, which suggests the stabilization of both molecules
in the active site of the complex.
In this study, we used the QMMM hybrid and MD simulations to verify the contribution of each residue
to the total interaction between the selected ligands and the enzyme structure. Calculations of interaction
energy per residue were performed during the last 500 ps of simulation of each system by the AM1/MM
method [40]. The graphics of interaction energy per residue are shown in Figures 4 and 5, where the
positive values correspond to interactions of repulsive forces and negative values correspond to
interactions that contribute to the stabilization of the ligand in the complex [41,42].
The results of the calculations of interaction energy per residue for compound 250 (Figure 4) showed
more significant contributions of residues Gln41, His57, Ser139, Asp168 and Asp527. Interactions of
hydrogen with catalytic residues His57 and Ser139 were observed in our molecular docking results. The
contributions of residues Asp168 (belonging to the domain of protease enzyme) and Asp527 (the helicase
domain of the enzyme) are considered important for the molecular recognition process, collaborating for
the binding affinity of known inhibitors of NS3-4A of HCV [55]. The contribution of repulsive residue
Asp81 was observed in QM/MM calculations performed by Rodriguez and coworkers [3], who used the
three main natural substrates NS5A/ 5B, NS4B/5A and NS4A/ 4B in the active site of protease NS3-4A of
HCV to clarify details on the reaction mechanism of this enzyme.
The results of energies for the attractive interactions of residue Gln41 and repulsive interactions (Figure
4) revealed interactions of molecular dynamics simulations and calculations of interaction energy per
residue with enzyme NS3-4A.Therefore, these results can be exploited for the design of new compounds
and the understanding of the iterative process, contributing to the achievement of relevant structural
information on the catalytic mechanism of enzyme NS3-4A.
In Figure 5, the graph of interaction energy per residue of compound 263 shows effective contributions
of residues His57, Asp79, Ala156, Ser139, Asp487, Cys525 and Gln526 to the interaction energy of the
molecule, highlighting the contributions of catalytic residues His57 and Ser139, also found for compound
250, besides residue Gln526, both observed in our molecular docking results. Other residues, such as
Asp79 and Ala156 enable interactions for the stabilization of both oxianionic holes, because they are
important for the stabilization of the substrate during its process of cleavage [3, 55]. The significant
attractive contributions of interaction energy between ligand and protein identified for the residues of
Asp487 and Cys52 (belonging to the helicase domain) are consistent with the proposal that inhibitors of
this enzyme should interact with residues of protease and helicase domains to prevent the activity of the
enzyme [55,56]. On the other hand, the highlighted interactions of repulsive forces of residues Arg155,
Arg481 and Phe486 also shown in Figure 5, but not mentioned in the works related to the contributions of

interaction in the enzyme, can serve as a basic element for the performance of such residues in the
extensive and detailed process of inhibition of the enzyme.
Therefore, as the simulations of QM/MM molecular dynamics are a more robust method than the
molecular docking, they showed support for a more detailed analysis of the behavior of ligands in the
NS3-4A enzyme connection. They have also corroborated the literature data and are in accordance with
our molecular docking results obtained with different programs.
4- Conclusions
This paper has reported on the identification of promising compounds that act against enzyme NS3-4A
of HCV by the calculations of molecular docking and consecutive consensual analysis. The two
compounds selected ((-)-6-transmetoxi-khellactona and surinamensin) showed interactions with key
residues for the processes of molecular recognition and inhibition of the target. Simulations of molecular
dynamics coupled to the hybrid QM/MM method were performed and showed good stability of the
compounds selected in the active site of the enzyme. The results of the individual contribution of the
residues for the interaction energy showed a well detailed network of interactions that encompasses
important residues, as His57, Ala156, Ser139, Asp168, Gln526 and Asp527, which play a substantial role
in studies of inhibitors of the target.
The results of this study are extremely useful for the planning and development of new bioactive
compounds against the virus of Hepatitis C. They show the potential of small compounds from natural
products for the inhibition of NS3-4A, without the inconveniences of the pharmacokinetic properties and
bioavailability of the conventional peptides and peptidomimetics inhibitors.
Acknowledgements
The authors are indebted to CNPq for the financial support provided to this research.
Appendix A: Supplementary data
Supplementary data associated with this article can be found in the online version, at
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