Photon Factory Activity Report 2014 #32 (2015) B

AR-NE3A /2014G563

Structural and computational study on inhibitory compounds for endonucleaseactivity of influenza virus polymerase
Tyuji Hoshino1,* Satoshi Fudo 1 and Michiyoshi Nukaga 2
1
Graduate School of Pharmaceutical Sciences, Chiba University,
1-8-1 Inohana, Chuo-ku, Chiba 260-8675, Japan
2
Faculty of Pharmaceutical Sciences, Josai International University,
Gumyo 1, Togane-shi Chiba 283-8555, Japan
1 Introduction
Seasonal epidemics and occasional pandemics caused
by influenza viruses are global threats to humans. Since
the efficacy of currently approved drugs is limited by theemerging resistance of the viruses, the development of
new antiviral drugs is still demanded. Endonucleaseactivity, which lies in the influenza polymerase acidic
protein N-terminal domain (PAN), is a potent target for
novel antiviral agents. Here, we report the identificationof some novel inhibitors for PAN endonuclease activity.
The binding mode of one of the inhibitory compounds to
PAN was investigated in detail by means of X-ray crystal
structure analysis and molecular dynamics (MD)simulation [1].
2 Experiment
Chemical screening for PAN endonuclease inhibitors
was conducted with compound stock in our research team.
Single-stranded DNA plasmid M13mp18 (5 ng/µl) wasincubated in the reaction buffer (20 mM Tris-HCl pH 7.5,
100 mM NaCl, 10 mM β-mercaptoethanol, and 1.0 mM
MgCl2) in the presence of 1.0 mg/mL PAN and 100 μM ofeach compound from our library for 60 min at 37 °C. The
reaction was stopped by adding 20 mM EDTA. The
reaction products were loaded on a 0.8% agarose gel forelectrophoresis and stained with ethidium bromide.
Crystals of PANΔloop were grown by vapor diffusion in
hanging drops containing 1.0 µL of 9.9 mg/mL protein
solution including 4.0 mM MnCl2 and 1.0 µL of wellsolution (100 mM MES [pH 5.8], 1.1 M ammonium
sulfate, 0.1 M potassium chloride and 9% (v/v) trehalose)
at 18°C. Crystals were soaked with well solution
containing 10.0 mM MnCl2 and 4.0 mM compound 1 for
2.0 hours. The crystals were then cryo-protected by brief
immersion in well solution containing 22.5% (v/v)
glycerol followed by flash-freezing in liquid nitrogen.
X-ray diffraction data were collected at 100 K with awavelength of 1.0 Å on the AR-NE3A beamline ofphotonfactory (PF, Tsukuba, Japan). The diffraction data
were indexed, scaled and merged with HKL2000.
Intensities were converted into structure factors and 5%of the reflections were flagged for Rfree calculations. The
PANΔloop structure was determined by molecularreplacement using the program MOLREP with the crystal
structure 4M5Q (PDB code) as a search model. Structurerefinement and model building were carried out using
PHENIX and COOT.

3 Results and Discussion
We first carried out an in vitro chemical screening forfinding endonuclease inhibitors with approximately 410
compounds. The screening resulted in the identificationof several PAN endonuclease inhibitors including
compounds 1, 2, and 3 (Fig 1).

Figure 1: Structures of compounds identified as inhibitors
of PAN endonuclease activity.
Compounds bearing a trihydroxyphenyl group havealready been suggested to show inhibitory activity against
PAN. Compounds 1 and 3 contain a trihydroxyphenyl
moiety, but they have not been reported as inhibitors forPAN. Compound 2 has a different scaffold. We carriedout a FRET-based endonuclease assay for measurementof the IC50 values of these compounds (Fig. 2).

Figure 2: Measurement of IC50 values of compounds 1-3
using a FRET-based assay. Vm was measured in triplicate
at each concentration for the respective compounds.
In protein crystallization, we used truncated PAN(PANΔloop) and obtained the co-crystal structure with one

Photon Factory Activity Report 2014 #32 (2015) Bof the inhibitors, compound 1, in the P41212 space groupwith one protein molecule in an asymmetric unit. It was
shown in the crystal structure that three molecules of theinhibitor were bound to different positions in the active
site (Figs. 3A, 3B).

Figure 3: Crystal structure of PANΔloop in complex withcompound 1. (A) Overall structure of the complex.
Protein backbone is depicted in cartoon (green), twomanganese ions are in spheres (magenta), and threemolecules of compound 1 are in the ball-and-stick modelcolored yellow (carbon), red (oxygen), and palecyan(fluorine). Three inhibitory molecules are labeled 1A, 1B,and 1C. (B) Surface representation of the protein coloredgray (carbon), red (oxygen), and blue (nitrogen). (C-E)
Magnified images of the complex, in which theinteractions of the protein with 1A (C), 1B (D), and 1C(E) are depicted. The residues of the protein near theinhibitory molecules are represented in the ball-and-stickmodel colored cyan (carbon), red (oxygen), and blue(nitrogen) with the labels of their residue names, whileother residues are shown in cartoon (gray). Interactions
among the protein, metal ions, and compound 1 aredepicted by black dotted lines. (F) Conformationalchange of R124 induced by the binding of compound 1.
The complex structure in this work (palegreen) is
superimposed on chain A of 4AWG (PDB code,lightpink) for comparison. Only R124 is represented in
the ball-and-stick model for 4AWG. It should be notedthat the side chain of R124 is oriented in almost theopposite direction.
Molecule 1A was coordinated to two manganese ions in
the active center by its trihydroxyphenyl group, and pfluorophenyl group of 1A was extended toward thepocket made by Arg84, Trp88, Phe105, and Leu106 (Fig.
3C). The trihydroxyphenyl group of molecule 1B made aπ-π interaction with His41 and formed several hydrogenbonds with the target protein. The p-fluorophenyl groupof 1B was bound to the pocket formed by Thr20, Tyr24,Glu26, Lys34, and Ile38 (Fig. 3D). Compared with these
two molecules, molecule 1C had a unique binding mode,in which its p-fluorophenyl group was deeply inserted
into the highly hydrophobic pocket (Fig. 3E). This pocket
was comprised of Val122, Arg124, Phe150, Trp188,Phe191, Arg192, and Glu195 and was formed mainly due
to the drastic conformational change of Arg124 asdepicted in Fig. 4F. Originally, this arginine residue
buried the pocket, making the protein surface positively
charged (A representative structure is shown in lightpink
in Fig. 3F.), and was sometimes utilized for polarinteraction with some inhibitory compounds. However, inthe case of molecule 1C, the side chain of the arginine
was oriented in almost the opposite direction andinteracted with a sulfate ion (structure shown in palegreen
in Fig. 3F), which allowed the compound to be fit for thehydrophobic pocket. This conformational change ofArg124 has not been observed in any previously reported
crystal structures of PAN. Some of those crystal structures
were obtained under the crystallization conditions that
include ammonium sulfate. Hence, the presence ofammonium sulfate had little influence on theconformational change of R124, which suggests that notsulfate ion but inhibitor molecule is an essential factor
induce the conformational change. The three hydroxygroups and a carbonyl group at the opposite side of 1C
seem not to strongly interact with the residues of PAN,
while one hydrogen bond is observed between the 3hydroxy group of 1C and the side chain of E31 at adistance of 3.2 Å. Possibly, these polar moieties
contribute to the binding affinity by covering thehydrophobic part of the compound and the protein,
thereby increasing the polar surface area of the complex.
The binding site for molecule 1C was not observed in
the previous crystallographic studies. Hence, the stabilityof inhibitor binding was examined by performing 100-ns
MD simulation. During the MD simulation, the three
inhibitor molecules fluctuated at the respective bindingsites at different amplitudes, while all of the molecules
maintained interactions with the protein. Molecularmechanics/generalized Born surface area (MM/GBSA)
analysis suggested that the molecule 1C has a higher
affinity than the others. Structural information obtained inthis study will provide a hint for designing anddeveloping novel potent agents against influenza viruses.
References[1] S. Fudo, N. Yamamoto, M. Nukaga, T. Odagiri, M.
Tashiro, S. Neya, T. Hoshino : Structural andcomputational study on inhibitory compounds forendonuclease activity of influenza virus polymerase,
Bioorganic & Medicinal Chemistry, in press (2015).
[2] PDB code : 4YYL, 4ZHZ, 4ZI0, 4ZQQ
* hoshino@chiba-u.jp