N	B-chemical
-	I-chemical
acylhydrazone	I-chemical
inhibitors	O
of	O
influenza	B-taxonomy_domain
virus	B-taxonomy_domain
PA	B-protein
endonuclease	B-protein_type
with	O
versatile	O
metal	O
binding	O
modes	O

Influenza	B-taxonomy_domain
virus	B-taxonomy_domain
PA	B-protein
endonuclease	B-protein_type
has	O
recently	O
emerged	O
as	O
an	O
attractive	O
target	O
for	O
the	O
development	O
of	O
novel	O
antiviral	O
therapeutics	O
.	O

This	O
is	O
an	O
enzyme	O
with	O
divalent	O
metal	O
ion	O
(	O
s	O
)	O
(	O
Mg2	B-chemical
+	I-chemical
or	O
Mn2	B-chemical
+)	I-chemical
in	O
its	O
catalytic	B-site
site	I-site
:	O
chelation	B-bond_interaction
of	O
these	O
metal	O
cofactors	O
is	O
an	O
attractive	O
strategy	O
to	O
inhibit	O
enzymatic	O
activity	O
.	O

Here	O
we	O
report	O
the	O
activity	O
of	O
a	O
series	O
of	O
N	B-chemical
-	I-chemical
acylhydrazones	I-chemical
in	O
an	O
enzymatic	B-experimental_method
assay	I-experimental_method
with	O
PA	B-protein
-	O
Nter	B-structure_element
endonuclease	B-protein_type
,	O
as	O
well	O
as	O
in	O
cell	B-experimental_method
-	I-experimental_method
based	I-experimental_method
influenza	I-experimental_method
vRNP	I-experimental_method
reconstitution	I-experimental_method
and	O
virus	B-experimental_method
yield	I-experimental_method
assays	I-experimental_method
.	O

Several	O
N	B-chemical
-	I-chemical
acylhydrazones	I-chemical
were	O
found	O
to	O
have	O
promising	O
anti	O
-	O
influenza	B-taxonomy_domain
activity	O
in	O
the	O
low	O
micromolar	O
concentration	O
range	O
and	O
good	O
selectivity	O
.	O

Computational	B-experimental_method
docking	I-experimental_method
studies	I-experimental_method
are	O
carried	O
on	O
to	O
investigate	O
the	O
key	O
features	O
that	O
determine	O
inhibition	O
of	O
the	O
endonuclease	B-protein_type
enzyme	O
by	O
N	B-chemical
-	I-chemical
acylhydrazones	I-chemical
.	O

Moreover	O
,	O
we	O
here	O
describe	O
the	O
crystal	B-evidence
structure	I-evidence
of	O
PA	B-protein
-	O
Nter	B-structure_element
in	B-protein_state
complex	I-protein_state
with	I-protein_state
one	O
of	O
the	O
most	O
active	O
inhibitors	O
,	O
revealing	O
its	O
interactions	O
within	O
the	O
protein	O
’	O
s	O
active	B-site
site	I-site
.	O

Influenza	B-taxonomy_domain
virus	B-taxonomy_domain
is	O
an	O
enveloped	B-taxonomy_domain
virus	I-taxonomy_domain
with	O
a	O
segmented	O
negative	B-chemical
-	I-chemical
oriented	I-chemical
single	I-chemical
-	I-chemical
stranded	I-chemical
RNA	I-chemical
genome	O
,	O
belonging	O
to	O
the	O
Orthomyxoviridae	B-taxonomy_domain
.	O

Seasonal	O
influenza	B-taxonomy_domain
A	I-taxonomy_domain
and	O
B	B-taxonomy_domain
viruses	B-taxonomy_domain
affect	O
each	O
year	O
approximately	O
5	O
–	O
10	O
%	O
of	O
the	O
adult	O
and	O
20	O
–	O
30	O
%	O
of	O
the	O
paediatric	O
population	O
,	O
and	O
there	O
is	O
a	O
permanent	O
risk	O
of	O
sudden	O
influenza	B-taxonomy_domain
pandemics	O
,	O
such	O
as	O
the	O
notorious	O
‘	O
Spanish	O
flu	O
’	O
in	O
1918	O
and	O
the	O
swine	O
-	O
origin	O
H1N1	B-species
pandemic	O
in	O
2009	O
.	O

Two	O
classes	O
of	O
anti	O
-	O
influenza	B-taxonomy_domain
virus	B-taxonomy_domain
drugs	O
are	O
available	O
,	O
acting	O
on	O
the	O
viral	B-taxonomy_domain
M2	B-protein_type
ion	I-protein_type
-	I-protein_type
channel	I-protein_type
(	O
amantadine	B-chemical
and	O
rimantadine	B-chemical
)	O
or	O
on	O
the	O
viral	B-taxonomy_domain
neuraminidase	B-protein_type
(	O
zanamivir	B-chemical
and	O
oseltamivir	B-chemical
).	O

The	O
M2	B-protein_type
inhibitors	O
have	O
limited	O
clinical	O
utility	O
due	O
to	O
their	O
central	O
nervous	O
system	O
side	O
effects	O
and	O
widespread	O
resistance	O
,	O
as	O
in	O
the	O
case	O
of	O
the	O
2009	O
pandemic	O
H1N1	B-species
virus	B-taxonomy_domain
;	O
resistance	O
is	O
also	O
a	O
growing	O
concern	O
for	O
oseltamivir	B-chemical
.	O

The	O
influenza	B-taxonomy_domain
virus	B-taxonomy_domain
polymerase	B-protein_type
complex	O
is	O
composed	O
of	O
three	O
subunits	O
:	O
PB1	B-protein
,	O
PB2	B-protein
and	O
PA	B-protein
.	O

The	O
PA	B-protein
subunit	B-structure_element
performs	O
the	O
‘	O
cap	O
-	O
snatching	O
’	O
endonuclease	B-protein_type
reaction	O
,	O
the	O
PB2	B-protein
subunit	B-structure_element
is	O
responsible	O
for	O
initial	O
binding	O
of	O
the	O
capped	B-chemical
RNAs	I-chemical
,	O
while	O
the	O
actual	O
RNA	B-chemical
synthesis	O
is	O
performed	O
by	O
the	O
PB1	B-protein
protein	O
.	O

Given	O
its	O
crucial	O
role	O
in	O
the	O
viral	B-taxonomy_domain
life	O
cycle	O
,	O
the	O
influenza	B-taxonomy_domain
virus	B-taxonomy_domain
polymerase	B-protein_type
is	O
widely	O
recognized	O
as	O
a	O
superior	O
target	O
for	O
antiviral	O
drug	O
development	O
and	O
,	O
in	O
particular	O
,	O
inhibition	O
of	O
the	O
PA	B-protein
endonuclease	B-protein_type
has	O
deserved	O
much	O
attention	O
in	O
recent	O
years	O
.	O

The	O
endonuclease	B-protein_type
catalytic	B-site
site	I-site
resides	O
in	O
the	O
N	B-structure_element
-	I-structure_element
terminal	I-structure_element
domain	I-structure_element
of	O
PA	B-protein
(	O
PA	B-protein
-	O
Nter	B-structure_element
;	O
residues	O
1	B-residue_range
~	I-residue_range
195	I-residue_range
).	O

It	O
comprises	O
a	O
histidine	B-residue_name
(	O
His41	B-residue_name_number
)	O
and	O
a	O
cluster	O
of	O
three	O
strictly	B-protein_state
conserved	I-protein_state
acidic	B-protein_state
residues	O
(	O
Glu80	B-residue_name_number
,	O
Asp108	B-residue_name_number
,	O
Glu119	B-residue_name_number
),	O
which	O
coordinate	B-bond_interaction
(	O
together	O
with	O
Ile120	B-residue_name_number
)	O
one	O
,	O
two	O
,	O
or	O
three	O
manganese	B-chemical
or	O
magnesium	B-chemical
ions	O
.	O

Since	O
the	O
intracellular	O
concentration	O
of	O
Mg2	B-chemical
+	I-chemical
is	O
at	O
least	O
1000	O
-	O
fold	O
higher	O
than	O
that	O
of	O
Mn2	B-chemical
+,	I-chemical
magnesium	B-chemical
may	O
be	O
more	O
biologically	O
relevant	O
.	O

A	O
controversy	O
about	O
number	O
and	O
type	O
of	O
metal	O
ions	O
exists	O
also	O
for	O
the	O
active	B-site
site	I-site
of	O
HIV	B-species
-	I-species
1	I-species
integrase	B-protein_type
.	O

HIV	B-species
-	I-species
1	I-species
integrase	B-protein_type
inhibitors	O
are	O
a	O
paradigm	O
for	O
the	O
innovative	O
drug	O
concept	O
that	O
is	O
based	O
on	O
coordination	O
with	O
the	O
metal	B-chemical
cofactor	O
(	O
s	O
)	O
of	O
viral	B-taxonomy_domain
enzymes	O
:	O
similarly	O
,	O
several	O
PA	B-protein
-	O
binding	O
agents	O
with	O
metal	O
-	O
chelating	O
properties	O
have	O
been	O
identified	O
as	O
influenza	B-taxonomy_domain
endonuclease	B-protein_type
inhibitors	O
(	O
Fig	O
.	O
1	O
),	O
including	O
2	B-chemical
,	I-chemical
4	I-chemical
-	I-chemical
dioxobutanoic	I-chemical
acid	I-chemical
derivatives	O
,	O
flutimide	B-chemical
and	O
its	O
derivatives	O
,	O
2	B-chemical
-	I-chemical
hydroxyphenyl	I-chemical
amide	I-chemical
derivatives	O
,	O
as	O
well	O
as	O
tetramic	B-chemical
acids	I-chemical
,	O
5	B-chemical
-	I-chemical
hydroxypyrimidin	I-chemical
-	I-chemical
4	I-chemical
-	I-chemical
one	I-chemical
derivatives	O
,	O
marchantins	B-chemical
and	O
green	B-taxonomy_domain
tea	I-taxonomy_domain
catechins	B-chemical
,	O
like	O
epigallocatechin	B-chemical
-	I-chemical
3	I-chemical
-	I-chemical
gallate	I-chemical
(	O
EGCG	B-chemical
,	O
Fig	O
.	O
1	O
).	O

In	O
recent	O
years	O
,	O
we	O
focused	O
our	O
research	O
on	O
chemical	O
scaffolds	O
that	O
are	O
able	O
to	O
chelate	O
metal	O
ions	O
of	O
PA	B-protein
-	O
Nter	B-structure_element
,	O
resulting	O
in	O
inhibition	O
of	O
influenza	B-taxonomy_domain
virus	B-taxonomy_domain
replication	O
.	O

N	B-chemical
-	I-chemical
acylhydrazones	I-chemical
represent	O
an	O
appealing	O
class	O
of	O
chelating	O
ligands	O
with	O
a	O
broad	O
spectrum	B-evidence
of	O
biological	O
activities	O
,	O
such	O
as	O
activity	O
against	O
HIV	B-taxonomy_domain
,	O
hepatitis	B-taxonomy_domain
A	I-taxonomy_domain
,	O
vaccinia	B-taxonomy_domain
and	O
influenza	B-taxonomy_domain
virus	B-taxonomy_domain
.	O

In	O
the	O
present	O
work	O
,	O
we	O
report	O
the	O
biological	O
activity	O
of	O
a	O
series	O
of	O
N	B-chemical
-	I-chemical
acylhydrazones	I-chemical
(	O
Fig	O
.	O
2	O
),	O
as	O
determined	O
in	O
an	O
enzymatic	B-experimental_method
assay	I-experimental_method
with	O
PA	B-protein
-	O
Nter	B-structure_element
endonuclease	B-protein_type
as	O
well	O
as	O
in	O
cell	B-experimental_method
-	I-experimental_method
based	I-experimental_method
influenza	I-experimental_method
viral	I-experimental_method
ribonucleoprotein	I-experimental_method
(	I-experimental_method
vRNP	I-experimental_method
)	I-experimental_method
reconstitution	I-experimental_method
and	O
virus	B-experimental_method
yield	I-experimental_method
assays	I-experimental_method
.	O

Several	O
N	B-chemical
-	I-chemical
acylhydrazones	I-chemical
were	O
found	O
to	O
have	O
promising	O
anti	O
-	O
influenza	B-taxonomy_domain
activity	O
with	O
50	B-evidence
%	I-evidence
effective	I-evidence
concentration	I-evidence
values	O
(	O
EC50	B-evidence
)	O
in	O
the	O
range	O
of	O
3	O
–	O
20	O
μM	O
and	O
good	O
selectivity	O
(	O
Table	O
1	O
and	O
Fig	O
.	O
3	O
).	O

Computational	B-experimental_method
docking	I-experimental_method
studies	I-experimental_method
of	O
two	O
candidate	O
ligands	O
in	O
the	O
PA	B-protein
-	O
Nter	B-structure_element
active	B-site
site	I-site
gave	O
information	O
about	O
the	O
features	O
that	O
could	O
determine	O
inhibition	O
of	O
endonuclease	B-protein_type
activity	O
.	O

Moreover	O
,	O
we	O
describe	O
the	O
X	B-evidence
-	I-evidence
ray	I-evidence
crystal	I-evidence
structure	I-evidence
of	O
PA	B-protein
-	O
Nter	B-structure_element
in	B-protein_state
complex	I-protein_state
with	I-protein_state
one	O
of	O
the	O
most	O
active	O
inhibitors	O
.	O

N	B-chemical
-	I-chemical
acylhydrazones	I-chemical
1	B-chemical
–	I-chemical
27	I-chemical
(	O
Fig	O
.	O
2	O
)	O
were	O
prepared	O
in	O
high	O
yields	O
by	O
following	O
literature	O
methods	O
(	O
Fig	O
.	O
2A	O
);	O
they	O
were	O
characterized	O
by	O
spectroscopic	O
tools	O
,	O
mass	B-experimental_method
spectrometry	I-experimental_method
and	O
elemental	B-experimental_method
analysis	I-experimental_method
.	O

Even	O
if	O
isomerism	O
around	O
the	O
C	O
=	O
N	O
bond	O
is	O
possible	O
,	O
1	B-chemical
–	I-chemical
27	I-chemical
are	O
present	O
in	O
the	O
E	O
form	O
in	O
solution	O
,	O
as	O
evidenced	O
by	O
the	O
chemical	O
shift	O
values	O
of	O
the	O
HC	O
=	O
N	O
and	O
NH	O
protons	O
in	O
the	O
1H	B-experimental_method
-	I-experimental_method
NMR	I-experimental_method
spectrum	B-evidence
.	O

Exceptions	O
are	O
represented	O
by	O
the	O
alkyl	O
-	O
derivatives	O
3	B-chemical
and	O
4	B-chemical
(	O
2	O
:	O
1	O
and	O
5	O
:	O
3	O
E	O
:	O
Z	O
ratio	O
,	O
respectively	O
).	O

If	O
R	O
’	O
(	O
Fig	O
.	O
2A	O
)	O
is	O
a	O
2	O
-	O
hydroxy	O
substituted	O
phenyl	O
ring	O
,	O
the	O
corresponding	O
acylhydrazones	B-chemical
can	O
coordinate	B-bond_interaction
one	O
or	O
,	O
depending	O
on	O
denticity	O
,	O
two	O
metal	O
centers	O
(	O
modes	O
A	O
and	O
B	O
in	O
Fig	O
.	O
4	O
).	O

Starting	O
from	O
N	B-chemical
’-(	I-chemical
2	I-chemical
,	I-chemical
3	I-chemical
-	I-chemical
dihydroxybenzylidene	I-chemical
)-	I-chemical
semicarbazide	I-chemical
(	O
1	B-chemical
)	O
and	O
its	O
methoxy	O
-	O
analogue	O
(	O
2	B-chemical
),	O
we	O
modified	O
the	O
acylhydrazonic	O
substituent	O
R	O
”	O
(	O
3	B-chemical
–	I-chemical
8	I-chemical
,	O
18	B-chemical
,	O
19	B-chemical
,	O
Fig	O
.	O
2A	O
).	O

In	O
18	B-chemical
and	O
19	B-chemical
,	O
also	O
the	O
gallic	B-chemical
moiety	O
can	O
be	O
involved	O
in	O
the	O
chelation	B-bond_interaction
of	O
the	O
metal	O
cofactors	O
(	O
mode	O
C	O
,	O
Fig	O
.	O
4	O
).	O

In	O
order	O
to	O
investigate	O
the	O
role	O
of	O
hydroxyl	O
substituents	O
9	B-chemical
–	I-chemical
11	I-chemical
,	O
13	B-chemical
–	I-chemical
17	I-chemical
,	O
20	B-chemical
–	I-chemical
23	I-chemical
and	O
27	B-chemical
were	O
also	O
synthesized	O
.	O

Compound	O
12	B-chemical
was	O
synthesized	O
in	O
order	O
to	O
confirm	O
the	O
crucial	O
influence	O
of	O
the	O
gallic	B-chemical
moiety	O
.	O

Finally	O
,	O
26	B-chemical
was	O
here	O
considered	O
,	O
because	O
it	O
is	O
an	O
inhibitor	O
of	O
HIV	B-taxonomy_domain
RNase	B-protein
H	I-protein
,	O
another	O
enzyme	O
with	O
two	O
magnesium	B-chemical
ions	O
in	O
its	O
active	B-site
site	I-site
.	O

Since	O
the	O
inhibitory	O
activity	O
of	O
the	O
N	B-chemical
-	I-chemical
acylhydrazones	I-chemical
could	O
be	O
related	O
to	O
chelation	B-bond_interaction
of	O
the	O
divalent	O
metal	B-chemical
cofactor	O
(	O
s	O
)	O
in	O
the	O
influenza	B-taxonomy_domain
PA	B-protein
-	O
Nter	B-structure_element
active	B-site
site	I-site
,	O
we	O
investigated	O
the	O
coordination	O
properties	O
of	O
one	O
model	O
ligand	O
(	O
i	O
.	O
e	O
.	O
19	B-chemical
,	O
H2L	B-chemical
)	O
towards	O
Mg2	B-chemical
+.	I-chemical

Different	O
reaction	O
conditions	O
were	O
used	O
(	O
1	O
:	O
1	O
and	O
1	O
:	O
2	O
metal	O
to	O
ligand	O
ratio	O
,	O
up	O
to	O
4	O
equivalents	O
of	O
triethylamine	B-chemical
),	O
but	O
in	O
any	O
case	O
the	O
same	O
chemical	O
species	O
Mg	B-chemical
(	I-chemical
HL	I-chemical
)	I-chemical
2	I-chemical
∙	I-chemical
4H2O	I-chemical
was	O
recovered	O
and	O
conveniently	O
characterized	O
.	O

The	O
use	O
of	O
a	O
coordinating	O
solvent	O
as	O
d6	B-chemical
-	I-chemical
DMSO	I-chemical
causes	O
partial	O
decoordination	O
of	O
the	O
ligand	O
,	O
but	O
the	O
1H	B-experimental_method
-	I-experimental_method
NMR	I-experimental_method
spectrum	B-evidence
in	O
MeOD	O
,	O
instead	O
,	O
shows	O
only	O
the	O
signals	O
attributable	O
to	O
the	O
complex	O
.	O

In	O
the	O
13C	B-experimental_method
-	I-experimental_method
NMR	I-experimental_method
spectrum	B-evidence
,	O
the	O
signal	O
of	O
the	O
C	O
=	O
O	O
quaternary	O
carbon	O
is	O
practically	O
unaffected	O
by	O
complexation	O
,	O
suggesting	O
that	O
the	O
C	O
=	O
O	O
group	O
is	O
weakly	O
involved	O
in	O
the	O
coordination	O
to	O
the	O
metal	O
ion	O
.	O

This	O
is	O
confirmed	O
,	O
in	O
the	O
IR	B-experimental_method
spectrum	B-evidence
,	O
by	O
the	O
shift	O
of	O
about	O
20	O
cm	O
−	O
1	O
of	O
the	O
C	O
=	O
O	O
absorption	O
,	O
while	O
a	O
shift	O
of	O
30	O
–	O
50	O
cm	O
−	O
1	O
is	O
expected	O
when	O
the	O
carbonylic	O
oxygen	O
is	O
tightly	O
bound	O
to	O
the	O
metal	O
ion	O
.	O

ESI	B-experimental_method
-	I-experimental_method
mass	I-experimental_method
spectra	B-evidence
and	O
elemental	B-experimental_method
analysis	I-experimental_method
confirmed	O
the	O
formula	O
Mg	B-chemical
(	I-chemical
HL	I-chemical
)	I-chemical
2	I-chemical
∙	I-chemical
4H2O	I-chemical
.	O

The	O
interaction	O
between	O
the	O
N	B-chemical
-	I-chemical
acylhydrazone	I-chemical
ligands	O
and	O
the	O
magnesium	B-chemical
cation	O
was	O
investigated	O
also	O
by	O
means	O
of	O
UV	B-experimental_method
-	I-experimental_method
visible	I-experimental_method
spectroscopy	I-experimental_method
(	O
UV	B-experimental_method
-	I-experimental_method
visible	I-experimental_method
titrations	I-experimental_method
of	O
23	B-chemical
and	O
19	B-chemical
with	O
increasing	B-experimental_method
amount	I-experimental_method
of	O
Mg	B-chemical
(	I-chemical
CH3COO	I-chemical
)	I-chemical
2	I-chemical
are	O
shown	O
in	O
Figure	O
S1	O
).	O

The	O
spectrum	B-evidence
of	O
19	B-chemical
includes	O
a	O
band	O
at	O
313	O
nm	O
assignable	O
to	O
n	O
-	O
π	O
*	O
transitions	O
of	O
the	O
C	O
=	O
N	O
and	O
C	O
=	O
O	O
groups	O
.	O

By	O
adding	O
increasing	O
equivalents	O
of	O
Mg	B-chemical
(	I-chemical
CH3COO	I-chemical
)	I-chemical
2	I-chemical
,	O
the	O
absorption	O
around	O
400	O
nm	O
increases	O
,	O
and	O
a	O
new	O
band	O
appears	O
with	O
a	O
maximum	O
at	O
397	O
nm	O
.	O

When	O
the	O
same	O
experiment	O
was	O
performed	O
with	O
23	B-chemical
,	O
a	O
different	O
behavior	O
was	O
observed	O
.	O

Increasing	O
concentration	O
of	O
Mg2	B-chemical
+,	I-chemical
in	O
fact	O
,	O
caused	O
a	O
diminution	O
in	O
the	O
maximum	O
absorption	O
,	O
an	O
isosbestic	O
point	O
is	O
visible	O
at	O
about	O
345	O
nm	O
,	O
but	O
a	O
new	O
band	O
at	O
400	O
nm	O
does	O
not	O
appear	O
.	O

Ligands	O
19	B-chemical
and	O
23	B-chemical
coordinate	B-bond_interaction
the	O
Mg2	B-chemical
+	I-chemical
ions	O
in	O
different	O
ways	O
:	O
19	B-chemical
chelates	O
the	O
metal	O
ion	O
by	O
using	O
the	O
deprotonated	O
salicyl	O
oxygen	O
and	O
the	O
iminic	O
nitrogen	O
,	O
while	O
for	O
23	B-chemical
,	O
the	O
gallic	O
moiety	O
is	O
supposed	O
to	O
be	O
involved	O
(	O
Fig	O
.	O
4A	O
,	O
B	O
versus	O
C	O
),	O
leading	O
to	O
different	O
,	O
less	O
extensive	O
,	O
modifications	O
of	O
the	O
UV	B-experimental_method
spectrum	B-evidence
.	O

Inhibition	O
of	O
the	O
PA	B-protein
-	O
Nter	B-structure_element
enzyme	O

All	O
the	O
compounds	O
were	O
tested	O
for	O
their	O
ability	O
to	O
inhibit	O
the	O
influenza	B-taxonomy_domain
endonuclease	B-protein_type
in	O
an	O
enzymatic	B-experimental_method
plasmid	I-experimental_method
-	I-experimental_method
based	I-experimental_method
assay	I-experimental_method
with	O
recombinant	O
PA	B-protein
-	O
Nter	B-structure_element
,	O
as	O
well	O
as	O
in	O
cell	B-experimental_method
-	I-experimental_method
based	I-experimental_method
influenza	I-experimental_method
methods	I-experimental_method
(	O
i	O
.	O
e	O
.	O
virus	B-experimental_method
yield	I-experimental_method
and	I-experimental_method
vRNP	I-experimental_method
reconstitution	I-experimental_method
assays	I-experimental_method
).	O

The	O
results	O
are	O
shown	O
in	O
Table	O
1	O
and	O
summarized	O
in	O
Fig	O
.	O
3	O
to	O
visualize	O
the	O
structure	O
-	O
activity	O
relationships	O
;	O
Figure	O
S2	O
shows	O
the	O
dose	B-evidence
-	I-evidence
response	I-evidence
curves	I-evidence
for	O
three	O
representative	O
compounds	O
(	O
i	O
.	O
e	O
.	O
10	B-chemical
,	O
13	B-chemical
and	O
23	B-chemical
)	O
in	O
either	O
the	O
PA	B-experimental_method
-	I-experimental_method
enzyme	I-experimental_method
or	I-experimental_method
vRNP	I-experimental_method
reconstitution	I-experimental_method
assay	I-experimental_method
.	O

The	O
moderate	O
activity	O
(	O
IC50	B-evidence
=	O
24	O
μM	O
)	O
of	O
N	B-chemical
’-	I-chemical
2	I-chemical
,	I-chemical
3	I-chemical
-	I-chemical
dihydroxybenzylidene	I-chemical
semicarbazide	I-chemical
(	O
1	B-chemical
)	O
was	O
completely	O
lost	O
when	O
the	O
NH2	O
moiety	O
was	O
replaced	O
by	O
a	O
hydrophobic	O
heptyl	O
chain	O
(	O
3	B-chemical
),	O
but	O
it	O
is	O
less	O
affected	O
when	O
a	O
phenyl	O
or	O
a	O
2	O
-	O
hydroxyphenyl	O
is	O
present	O
(	O
5	B-chemical
and	O
7	B-chemical
,	O
IC50	B-evidence
=	O
84	O
and	O
54	O
μM	O
,	O
respectively	O
).	O

When	O
the	O
hydroxyl	O
in	O
position	O
3	O
on	O
R1	O
(	O
2	B-chemical
,	I-chemical
3	I-chemical
-	I-chemical
dihydroxybenzylidene	I-chemical
)	O
was	O
replaced	O
by	O
a	O
methoxy	O
group	O
(	O
2	B-chemical
-	I-chemical
hydroxy	I-chemical
-	I-chemical
3	I-chemical
-	I-chemical
methoxybenzylidene	I-chemical
),	O
the	O
activity	O
disappeared	O
(	O
compounds	O
2	B-chemical
,	O
4	B-chemical
,	O
6	B-chemical
and	O
8	B-chemical
).	O

The	O
activity	O
is	O
unaffected	O
(	O
IC50	B-evidence
values	O
ranging	O
from	O
45	O
to	O
75	O
μM	O
)	O
when	O
going	O
from	O
two	O
hydroxyls	O
in	O
R1	O
(	O
7	B-chemical
)	O
to	O
compounds	O
with	O
three	O
hydroxyls	O
(	O
i	O
.	O
e	O
.	O
9	B-chemical
,	O
10	B-chemical
and	O
11	B-chemical
).	O

Similarly	O
,	O
11	B-chemical
(	O
R1	O
=	O
3	O
,	O
4	O
,	O
5	O
-	O
trihydroxyphenyl	O
,	O
R2	O
=	O
2	O
-	O
hydroxyphenyl	O
)	O
had	O
comparable	O
activity	O
as	O
27	B-chemical
(	O
R1	O
=	O
3	O
,	O
4	O
,	O
5	O
-	O
trihydroxyphenyl	O
,	O
R2	O
=	O
NH2	O
).	O

Within	O
the	O
series	O
carrying	O
a	O
2	O
-	O
hydroxyphenyl	O
R2	O
group	O
,	O
the	O
activity	O
of	O
11	B-chemical
is	O
particularly	O
intriguing	O
.	O

11	B-chemical
does	O
not	O
have	O
the	O
possibility	O
to	O
chelate	O
in	O
a	O
tridentate	O
ONO	O
fashion	O
(	O
mode	O
A	O
in	O
Fig	O
.	O
4	O
),	O
but	O
it	O
can	O
coordinate	B-bond_interaction
two	O
cations	O
by	O
means	O
of	O
its	O
three	O
OH	O
groups	O
in	O
R1	O
(	O
mode	O
C	O
,	O
Fig	O
.	O
4	O
).	O

Note	O
that	O
a	O
similar	O
chelating	O
mode	O
was	O
observed	O
in	O
a	O
crystal	B-evidence
structure	I-evidence
,	O
solved	O
by	O
Cusack	O
and	O
coworkers	O
,	O
of	O
PA	B-protein
-	O
Nter	B-structure_element
endonuclease	B-protein_type
in	B-protein_state
complex	I-protein_state
with	I-protein_state
the	O
inhibitor	O
EGCG	B-chemical
.	O

The	O
PA	B-protein
-	O
Nter	B-structure_element
inhibitory	O
activity	O
strongly	O
depends	O
on	O
the	O
number	O
and	O
position	O
of	O
hydroxyl	O
substituents	O
in	O
R1	O
and	O
R2	O
:	O
this	O
is	O
clearly	O
highlighted	O
by	O
the	O
data	O
obtained	O
with	O
compounds	O
13	B-chemical
–	I-chemical
23	I-chemical
,	O
in	O
which	O
R2	O
is	O
a	O
3	O
,	O
4	O
,	O
5	O
-	O
trihydroxyphenyl	O
(	O
gallic	O
)	O
group	O
,	O
the	O
most	O
active	O
scaffold	O
in	O
our	O
series	O
.	O

The	O
analogue	O
carrying	O
an	O
unsubstituted	O
aromatic	O
ring	O
as	O
R1	O
(	O
compound	O
13	B-chemical
)	O
had	O
moderate	O
activity	O
(	O
IC50	B-evidence
=	O
69	O
μM	O
).	O

When	O
one	O
OH	O
was	O
added	O
at	O
position	O
2	O
of	O
the	O
R1	O
ring	O
(	O
14	B-chemical
),	O
the	O
activity	O
was	O
lost	O
.	O

Adding	O
a	O
second	O
OH	O
substituent	O
at	O
position	O
5	O
resulted	O
in	O
strong	O
activity	O
(	O
compound	O
15	B-chemical
,	O
IC50	B-evidence
=	O
9	O
μM	O
);	O
medium	O
activity	O
for	O
a	O
3	O
-	O
OH	O
(	O
18	B-chemical
;	O
IC50	B-evidence
=	O
83	O
μM	O
),	O
and	O
marginal	O
activity	O
when	O
the	O
second	O
OH	O
is	O
at	O
position	O
4	O
(	O
17	B-chemical
,	O
IC50	B-evidence
≥	O
370	O
μM	O
).	O

The	O
addition	O
of	O
a	O
3	O
-	O
methoxy	O
group	O
(	O
19	B-chemical
)	O
abolished	O
all	O
inhibitory	O
activity	O
.	O

This	O
cannot	O
be	O
related	O
to	O
variations	O
in	O
the	O
chelating	O
features	O
displayed	O
by	O
the	O
R1	O
moiety	O
,	O
since	O
compounds	O
14	B-chemical
–	I-chemical
19	I-chemical
all	O
have	O
,	O
in	O
theory	O
,	O
the	O
capacity	O
to	O
chelate	O
one	O
metal	O
ion	O
through	O
the	O
ortho	O
-	O
OH	O
and	O
iminic	O
nitrogen	O
(	O
mode	O
A	O
in	O
Fig	O
.	O
4	O
).	O

Moreover	O
,	O
compound	O
18	B-chemical
can	O
,	O
in	O
principle	O
,	O
chelate	O
the	O
two	O
M2	B-chemical
+	I-chemical
ions	O
in	O
the	O
active	B-site
site	I-site
according	O
to	O
mode	O
B	O
(	O
Fig	O
.	O
4	O
),	O
yet	O
it	O
(	O
IC50	B-evidence
=	O
83	O
μM	O
)	O
has	O
nine	O
-	O
fold	O
lower	O
activity	O
than	O
15	B-chemical
,	O
that	O
does	O
not	O
possess	O
this	O
two	O
-	O
metal	O
chelating	O
feature	O
.	O

Therefore	O
,	O
we	O
hypothesized	O
that	O
the	O
inhibitory	O
activity	O
of	O
the	O
series	O
containing	O
the	O
gallic	O
moiety	O
is	O
determined	O
by	O
:	O
(	O
i	O
)	O
the	O
capacity	O
of	O
the	O
moiety	O
R2	O
to	O
chelate	O
two	O
metal	O
ions	O
in	O
the	O
active	B-site
site	I-site
of	O
the	O
enzyme	O
,	O
according	O
to	O
mode	O
C	O
(	O
Fig	O
.	O
4	O
);	O
and	O
(	O
ii	O
)	O
the	O
presence	O
and	O
position	O
of	O
one	O
or	O
more	O
hydroxyl	O
substituents	O
in	O
R1	O
,	O
which	O
may	O
possibly	O
result	O
in	O
ligand	O
-	O
protein	O
interactions	O
(	O
e	O
.	O
g	O
.	O
through	O
hydrogen	B-bond_interaction
bonds	I-bond_interaction
).	O

This	O
assumption	O
was	O
supported	O
by	O
molecular	B-experimental_method
docking	I-experimental_method
calculations	I-experimental_method
and	O
X	B-experimental_method
-	I-experimental_method
ray	I-experimental_method
analysis	I-experimental_method
of	O
inhibitor	O
23	B-chemical
in	B-protein_state
complex	I-protein_state
with	I-protein_state
PA	B-protein
-	O
Nter	B-structure_element
(	O
vide	O
infra	O
).	O

Substitution	O
of	O
the	O
5	O
-	O
hydroxyl	O
in	O
15	B-chemical
by	O
a	O
methoxy	O
group	O
(	O
16	B-chemical
)	O
causes	O
a	O
dramatic	O
drop	O
in	O
activity	O
(	O
IC50	B-evidence
=	O
9	O
and	O
454	O
μM	O
for	O
15	B-chemical
and	O
16	B-chemical
,	O
respectively	O
).	O

In	O
particular	O
,	O
all	O
the	O
compounds	O
with	O
a	O
trihydroxylated	O
phenyl	O
group	O
as	O
R1	O
(	O
i	O
.	O
e	O
.	O
20	B-chemical
,	O
21	B-chemical
,	O
22	B-chemical
and	O
23	B-chemical
)	O
were	O
able	O
to	O
inhibit	O
PA	B-protein
-	O
Nter	B-structure_element
quite	O
potently	O
.	O

The	O
lowest	O
IC50	B-evidence
values	O
were	O
obtained	O
for	O
21	B-chemical
and	O
23	B-chemical
(	O
IC50	B-evidence
=	O
13	O
and	O
7	O
μM	O
,	O
respectively	O
),	O
which	O
both	O
have	O
one	O
of	O
their	O
three	O
hydroxyl	O
groups	O
at	O
position	O
5	O
.	O

The	O
most	O
active	O
compound	O
in	O
this	O
series	O
was	O
23	B-chemical
,	O
which	O
lacks	O
the	O
hydroxyl	O
group	O
at	O
position	O
2	O
of	O
R1	O
,	O
further	O
confirming	O
that	O
this	O
function	O
is	O
undesirable	O
or	O
even	O
detrimental	O
for	O
inhibitory	O
activity	O
against	O
PA	B-protein
-	O
Nter	B-structure_element
,	O
as	O
already	O
noticed	O
above	O
for	O
14	B-chemical
.	O

Consistent	O
with	O
a	O
crucial	O
role	O
of	O
the	O
R2	O
gallic	O
moiety	O
in	O
metal	O
chelation	B-bond_interaction
,	O
the	O
strong	O
activity	O
of	O
15	B-chemical
was	O
completely	O
lost	O
in	O
its	O
3	O
,	O
4	O
,	O
5	O
-	O
trimethoxy	O
analogue	O
12	B-chemical
.	O

On	O
the	O
other	O
hand	O
,	O
the	O
R2	O
gallic	O
containing	O
compounds	O
displayed	O
moderate	O
activity	O
(	O
IC50	B-evidence
values	O
around	O
40	O
μM	O
)	O
when	O
R1	O
was	O
absent	O
(	O
i	O
.	O
e	O
.	O
the	O
3	B-chemical
,	I-chemical
4	I-chemical
,	I-chemical
5	I-chemical
-	I-chemical
trihydroxybenzohydrazide	I-chemical
28	B-chemical
,	O
Fig	O
.	O
2	O
),	O
or	O
composed	O
of	O
an	O
extended	O
ring	O
system	O
(	O
26	B-chemical
)	O
or	O
a	O
pyrrole	O
ring	O
(	O
25	B-chemical
).	O

Still	O
lower	O
activity	O
was	O
seen	O
with	O
the	O
pyridine	O
analogue	O
24	B-chemical
.	O

Evidently	O
,	O
the	O
3	O
,	O
4	O
,	O
5	O
-	O
trihydroxybenzyl	O
moiety	O
at	O
R2	O
is	O
fundamental	O
but	O
not	O
sufficient	O
to	O
ensure	O
potent	O
PA	B-protein
-	O
Nter	B-structure_element
endonuclease	B-protein_type
inhibition	O
,	O
since	O
the	O
interactions	O
of	O
R1	O
with	O
the	O
amino	O
acid	O
side	O
chains	O
of	O
the	O
protein	O
appear	O
crucial	O
in	O
modulating	O
activity	O
.	O

Inhibition	O
of	O
vRNP	B-complex_assembly
activity	O
or	O
virus	B-taxonomy_domain
replication	O
in	O
cells	O

To	O
determine	O
the	O
anti	O
-	O
influenza	B-taxonomy_domain
virus	B-taxonomy_domain
activity	O
of	O
compounds	O
1	B-chemical
–	I-chemical
28	I-chemical
in	O
cell	O
culture	O
,	O
we	O
performed	O
an	O
influenza	B-experimental_method
vRNP	I-experimental_method
reconstitution	I-experimental_method
assay	I-experimental_method
in	O
human	B-species
embryonic	O
kidney	O
293	O
T	O
(	O
HEK293T	O
)	O
cells	O
,	O
then	O
subjected	O
the	O
active	O
compounds	O
(	O
i	O
.	O
e	O
.	O
EC50	B-evidence
<	O
100	O
μM	O
)	O
to	O
a	O
virus	B-experimental_method
yield	I-experimental_method
assay	I-experimental_method
in	O
influenza	B-taxonomy_domain
virus	B-taxonomy_domain
-	O
infected	O
Madin	O
-	O
Darby	O
canine	O
kidney	O
(	O
MDCK	O
)	O
cells	O
(	O
Table	O
1	O
and	O
Fig	O
.	O
3	O
).	O

For	O
some	O
N	B-chemical
-	I-chemical
acylhydrazone	I-chemical
compounds	O
,	O
we	O
observed	O
quite	O
potent	O
and	O
selective	O
activity	O
in	O
the	O
vRNP	B-experimental_method
reconstitution	I-experimental_method
assay	I-experimental_method
.	O

This	O
indicates	O
that	O
they	O
are	O
able	O
to	O
inhibit	O
viral	B-taxonomy_domain
RNA	B-chemical
synthesis	O
and	O
suggests	O
that	O
they	O
could	O
be	O
classified	O
as	O
original	O
PA	B-protein
inhibitors	O
.	O

Values	O
for	O
EC50	B-evidence
(	O
vRNP	B-complex_assembly
)	O
or	O
EC90	B-evidence
(	O
virus	B-taxonomy_domain
yield	O
)	O
in	O
the	O
range	O
of	O
0	O
.	O
4	O
–	O
18	O
μM	O
were	O
obtained	O
for	O
compounds	O
15	B-chemical
and	O
20	B-chemical
–	I-chemical
23	I-chemical
,	O
which	O
all	O
carry	O
a	O
3	O
,	O
4	O
,	O
5	O
-	O
trihydroxyphenyl	O
as	O
R2	O
,	O
and	O
possess	O
either	O
two	O
(	O
15	B-chemical
)	O
or	O
three	O
(	O
20	B-chemical
–	O
23	B-chemical
)	O
hydroxyl	O
substituents	O
in	O
the	O
R1	O
moiety	O
.	O

As	O
in	O
the	O
enzymatic	B-experimental_method
PA	I-experimental_method
-	I-experimental_method
Nter	I-experimental_method
assays	I-experimental_method
,	O
the	O
compounds	O
having	O
R2	O
as	O
a	O
gallic	O
moiety	O
(	O
Fig	O
.	O
3	O
:	O
21	B-chemical
,	O
22	B-chemical
and	O
23	B-chemical
)	O
showed	O
slightly	O
higher	O
activity	O
than	O
the	O
compounds	O
carrying	O
a	O
2	O
-	O
hydroxyl	O
R2	O
group	O
(	O
9	B-chemical
,	O
10	B-chemical
and	O
11	B-chemical
);	O
10	B-chemical
and	O
22	B-chemical
have	O
substantially	O
the	O
same	O
EC50	B-evidence
in	O
the	O
vRNP	B-experimental_method
reconstitution	I-experimental_method
assay	I-experimental_method
in	O
HEK293T	O
cells	O
.	O

The	O
hydrazide	B-chemical
28	B-chemical
displayed	O
weak	O
(	O
virus	B-taxonomy_domain
yield	O
)	O
to	O
moderate	O
(	O
vRNP	B-experimental_method
reconstitution	I-experimental_method
)	O
activity	O
,	O
albeit	O
less	O
than	O
the	O
most	O
active	O
molecules	O
in	O
the	O
3	O
,	O
4	O
,	O
5	O
-	O
trihydroxyphenyl	O
series	O
(	O
i	O
.	O
e	O
.	O
18	B-chemical
and	O
21	B-chemical
–	I-chemical
23	I-chemical
).	O

Even	O
if	O
there	O
are	O
no	O
data	O
indicating	O
that	O
the	O
compounds	O
reported	O
in	O
the	O
paper	O
are	O
subject	O
to	O
hydrolysis	O
,	O
the	O
activity	O
of	O
28	B-chemical
could	O
raise	O
the	O
concern	O
that	O
for	O
some	O
N	B-chemical
-	I-chemical
acylhydrazones	I-chemical
the	O
antiviral	O
activity	O
in	O
cell	O
culture	O
may	O
be	O
related	O
to	O
their	O
intracellular	O
hydrolysis	O
.	O

However	O
,	O
this	O
is	O
unlikely	O
,	O
since	O
the	O
antiviral	O
potency	O
showed	O
large	O
differences	O
(	O
i	O
.	O
e	O
.	O
EC50	B-evidence
values	O
between	O
0	O
.	O
42	O
and	O
29	O
μM	O
)	O
for	O
compounds	O
with	O
the	O
same	O
R2	O
but	O
different	O
R1	O
groups	O
,	O
meaning	O
that	O
R1	O
does	O
play	O
a	O
role	O
in	O
modulating	O
the	O
antiviral	O
effect	O
.	O

Most	O
compounds	O
carrying	O
as	O
R1	O
a	O
2	B-chemical
,	I-chemical
3	I-chemical
-	I-chemical
dihydroxybenzylidene	I-chemical
(	O
i	O
.	O
e	O
.	O
3	B-chemical
,	O
5	B-chemical
and	O
7	B-chemical
)	O
or	O
2	B-chemical
-	I-chemical
hydroxy	I-chemical
-	I-chemical
3	I-chemical
-	I-chemical
methoxybenzylidene	I-chemical
moiety	O
(	O
i	O
.	O
e	O
.	O
4	B-chemical
,	O
6	B-chemical
and	O
8	B-chemical
)	O
showed	O
relatively	O
high	O
cytotoxicity	O
in	O
the	O
vRNP	B-experimental_method
assay	I-experimental_method
,	O
with	O
CC50	B-evidence
values	O
below	O
50	O
μM	O
and	O
a	O
selectivity	B-evidence
index	I-evidence
(	O
ratio	O
of	O
CC50	B-evidence
to	O
EC50	B-evidence
)	O
below	O
8	O
.	O

Two	O
notable	O
exceptions	O
are	O
18	B-chemical
and	O
19	B-chemical
(	O
containing	O
a	O
2	B-chemical
,	I-chemical
3	I-chemical
-	I-chemical
dihydroxybenzylidene	I-chemical
or	O
2	B-chemical
-	I-chemical
hydroxy	I-chemical
-	I-chemical
3	I-chemical
-	I-chemical
methoxybenzylidene	I-chemical
R1	O
,	O
respectively	O
)	O
which	O
were	O
not	O
cytotoxic	O
at	O
200	O
μM	O
and	O
displayed	O
favorable	O
antiviral	O
selectivity	O
.	O

Some	O
N	B-chemical
-	I-chemical
acylhydrazone	I-chemical
compounds	O
were	O
devoid	O
of	O
activity	O
in	O
the	O
enzymatic	B-experimental_method
assay	I-experimental_method
,	O
yet	O
showed	O
good	O
to	O
moderate	O
efficacy	O
in	O
cell	O
culture	O
(	O
e	O
.	O
g	O
.	O
14	B-chemical
and	O
19	B-chemical
,	O
having	O
EC50	B-evidence
values	O
of	O
2	O
.	O
2	O
and	O
7	O
.	O
1	O
μM	O
,	O
respectively	O
).	O

For	O
most	O
of	O
the	O
active	O
compounds	O
(	O
i	O
.	O
e	O
.	O
9	B-chemical
,	O
11	B-chemical
,	O
13	B-chemical
,	O
15	B-chemical
–	I-chemical
21	I-chemical
,	O
23	B-chemical
,	O
24	B-chemical
and	O
26	B-chemical
)	O
a	O
fair	O
correlation	O
was	O
seen	O
for	O
the	O
two	O
cell	B-experimental_method
-	I-experimental_method
based	I-experimental_method
assays	I-experimental_method
,	O
since	O
the	O
EC50	B-evidence
values	O
obtained	O
in	O
the	O
vRNP	B-experimental_method
assay	I-experimental_method
were	O
maximum	O
5	O
-	O
fold	O
different	O
from	O
the	O
EC90	B-evidence
values	O
in	O
the	O
virus	B-experimental_method
yield	I-experimental_method
assay	I-experimental_method
.	O

On	O
the	O
other	O
hand	O
,	O
this	O
difference	O
was	O
8	O
-	O
fold	O
or	O
more	O
for	O
7	B-chemical
,	O
10	B-chemical
,	O
14	B-chemical
,	O
22	B-chemical
,	O
25	B-chemical
and	O
28	B-chemical
.	O

Some	O
N	B-chemical
-	I-chemical
acylhydrazone	I-chemical
compounds	O
showed	O
good	O
to	O
moderate	O
efficacy	O
in	O
the	O
vRNP	B-experimental_method
assay	I-experimental_method
(	O
e	O
.	O
g	O
.	O
14	B-chemical
and	O
19	B-chemical
,	O
having	O
EC50	B-evidence
values	O
of	O
2	O
.	O
3	O
and	O
5	O
.	O
7	O
μM	O
,	O
respectively	O
),	O
yet	O
were	O
devoid	O
of	O
activity	O
in	O
the	O
enzymatic	B-experimental_method
assay	I-experimental_method
.	O

This	O
observation	O
suggests	O
that	O
they	O
may	O
inhibit	O
the	O
viral	B-taxonomy_domain
polymerase	B-protein_type
in	O
an	O
endonuclease	B-protein_type
-	O
independent	O
manner	O
.	O

To	O
achieve	O
a	O
clear	O
insight	O
into	O
the	O
antiviral	O
profile	O
of	O
the	O
N	B-chemical
-	I-chemical
acylhydrazones	I-chemical
,	O
specific	O
mechanistic	O
experiments	O
are	O
currently	O
ongoing	O
in	O
our	O
laboratory	O
,	O
in	O
which	O
we	O
are	O
analyzing	O
in	O
full	O
depth	O
their	O
effects	O
on	O
virus	B-taxonomy_domain
entry	O
,	O
polymerase	B-protein_type
-	O
dependent	O
RNA	B-chemical
synthesis	O
or	O
the	O
late	O
stage	O
(	O
maturation	O
and	O
release	O
)	O
of	O
the	O
virus	B-taxonomy_domain
replication	O
cycle	O
.	O

Docking	B-experimental_method
studies	I-experimental_method

In	O
order	O
to	O
explore	O
the	O
possible	O
binding	O
mode	O
of	O
the	O
synthesized	O
compounds	O
,	O
docking	B-experimental_method
simulations	I-experimental_method
by	O
GOLD	B-experimental_method
program	I-experimental_method
were	O
performed	O
by	O
using	O
the	O
structural	O
coordinates	O
(	O
PDB	O
code	O
4AWM	O
)	O
for	O
the	O
PA	B-protein
-	O
Nter	B-structure_element
endonuclease	B-protein_type
in	B-protein_state
complex	I-protein_state
with	I-protein_state
EGCG	B-chemical
.	O

Considering	O
that	O
the	O
position	O
of	O
the	O
side	O
-	O
chains	O
of	O
some	O
residues	O
changes	O
depending	O
on	O
which	O
pocket	O
the	O
ligand	O
is	O
occupying	O
,	O
we	O
superimposed	B-experimental_method
some	O
X	B-evidence
-	I-evidence
ray	I-evidence
structures	I-evidence
of	O
complexes	O
between	O
PA	B-protein
-	O
Nter	B-structure_element
endonuclease	B-protein_type
and	O
known	O
active	O
ligands	O
.	O

It	O
was	O
observed	O
that	O
the	O
side	O
-	O
chain	O
of	O
amino	O
acid	O
Tyr24	B-residue_name_number
shows	O
greater	O
movement	O
than	O
the	O
other	O
residues	O
and	O
for	O
this	O
reason	O
we	O
considered	O
it	O
as	O
a	O
flexible	B-protein_state
residue	O
during	O
the	O
docking	B-experimental_method
procedure	I-experimental_method
.	O

First	O
,	O
test	B-experimental_method
docking	I-experimental_method
calculations	I-experimental_method
,	O
using	O
EGCG	B-chemical
,	O
L	B-chemical
-	I-chemical
742	I-chemical
,	I-chemical
001	I-chemical
and	O
2	B-chemical
-(	I-chemical
4	I-chemical
-(	I-chemical
1H	I-chemical
-	I-chemical
tetrazol	I-chemical
-	I-chemical
5	I-chemical
-	I-chemical
yl	I-chemical
)	I-chemical
phenyl	I-chemical
)-	I-chemical
5	I-chemical
-	I-chemical
hydroxypyrimidin	I-chemical
-	I-chemical
4	I-chemical
(	I-chemical
3H	I-chemical
)-	I-chemical
one	I-chemical
(	O
Fig	O
.	O
1	O
),	O
were	O
carried	O
out	O
to	O
compare	O
experimental	O
and	O
predicted	O
binding	O
modes	O
and	O
validate	O
docking	B-experimental_method
procedure	I-experimental_method
.	O

Their	O
best	O
docking	O
poses	O
agreed	O
well	O
with	O
the	O
experimental	O
binding	O
modes	O
(	O
rmsd	B-evidence
values	O
of	O
0	O
.	O
8	O
,	O
1	O
.	O
2	O
and	O
0	O
.	O
7	O
,	O
respectively	O
).	O

Next	O
,	O
docking	B-experimental_method
of	O
several	O
N	B-chemical
-	I-chemical
acylhydrazones	I-chemical
was	O
performed	O
and	O
this	O
generated	O
a	O
number	O
of	O
possible	O
binding	O
conformations	O
,	O
highlighting	O
that	O
the	O
active	B-site
site	I-site
cavity	I-site
of	O
the	O
PA	B-protein
endonuclease	B-protein_type
is	O
quite	O
spacious	O
,	O
as	O
already	O
demonstrated	O
by	O
crystallographic	B-experimental_method
studies	I-experimental_method
,	O
and	O
confirming	O
the	O
ability	O
of	O
this	O
scaffold	O
to	O
chelate	O
the	O
two	O
M2	B-chemical
+	I-chemical
ions	O
in	O
different	O
ways	O
(	O
Mode	O
A	O
-	O
C	O
in	O
Fig	O
.	O
4	O
).	O

Figure	O
5	O
displays	O
the	O
first	O
(	O
panel	O
A	O
)	O
and	O
second	O
(	O
panel	O
B	O
)	O
GOLD	B-experimental_method
cluster	I-experimental_method
docked	I-experimental_method
solutions	O
for	O
compound	O
23	B-chemical
.	O

These	O
two	O
complex	O
structures	B-evidence
represent	O
the	O
largest	O
clusters	O
with	O
similar	O
fitness	O
values	O
(	O
59	O
.	O
20	O
and	O
58	O
.	O
65	O
,	O
respectively	O
).	O

In	O
both	O
cases	O
,	O
23	B-chemical
appears	O
able	O
to	O
coordinate	B-bond_interaction
the	O
two	O
M2	B-chemical
+	I-chemical
ions	O
in	O
the	O
active	B-site
site	I-site
through	O
the	O
three	O
contiguous	O
OH	O
groups	O
(	O
Fig	O
.	O
5	O
).	O

In	O
addition	O
,	O
23	B-chemical
was	O
predicted	O
to	O
form	O
two	O
hydrogen	B-bond_interaction
bonding	I-bond_interaction
interactions	I-bond_interaction
,	O
i	O
.	O
e	O
.	O
with	O
the	O
catalytic	B-protein_state
Lys134	B-residue_name_number
on	O
the	O
one	O
side	O
and	O
Glu26	B-residue_name_number
on	O
the	O
other	O
side	O
.	O

Furthermore	O
,	O
in	O
these	O
two	O
different	O
binding	O
modes	O
,	O
23	B-chemical
forms	O
π	B-bond_interaction
–	I-bond_interaction
π	I-bond_interaction
interactions	I-bond_interaction
with	O
the	O
aromatic	O
ring	O
of	O
Tyr24	B-residue_name_number
,	O
in	O
a	O
fashion	O
similar	O
to	O
that	O
described	O
for	O
other	O
endonuclease	B-protein_type
inhibitors	O
,	O
i	O
.	O
e	O
.	O
EGCG	B-chemical
and	O
L	B-chemical
-	I-chemical
742	I-chemical
,	I-chemical
001	I-chemical
.	O

The	O
best	O
docked	O
conformation	O
for	O
compound	O
15	B-chemical
(	O
Fig	O
.	O
6	O
,	O
fitness	B-evidence
value	I-evidence
68	O
.	O
56	O
),	O
which	O
has	O
an	O
activity	O
slightly	O
lower	O
than	O
23	O
,	O
reveals	O
a	O
different	O
role	O
for	O
the	O
gallic	O
moiety	O
.	O

The	O
ligand	O
seems	O
to	O
form	O
two	O
hydrogen	B-bond_interaction
bonding	I-bond_interaction
interactions	I-bond_interaction
with	O
Tyr130	B-residue_name_number
as	O
well	O
as	O
a	O
cation	B-bond_interaction
–	I-bond_interaction
π	I-bond_interaction
interaction	I-bond_interaction
with	O
Lys134	B-residue_name_number
.	O

Tyr130	B-residue_name_number
lies	O
in	O
a	O
pocket	B-site
that	O
also	O
contains	O
Arg124	B-residue_name_number
,	O
a	O
residue	O
that	O
was	O
proposed	O
to	O
have	O
a	O
crucial	O
role	O
in	O
binding	O
of	O
the	O
RNA	B-chemical
substrate	O
.	O

Compound	O
15	B-chemical
appears	O
further	O
stabilized	O
by	O
hydrogen	B-bond_interaction
bonding	I-bond_interaction
interactions	I-bond_interaction
between	O
two	O
hydroxyl	O
groups	O
and	O
Arg82	B-residue_name_number
and	O
Asp108	B-residue_name_number
.	O

In	O
this	O
case	O
,	O
chelation	B-bond_interaction
of	O
the	O
two	O
M2	B-chemical
+	I-chemical
ions	O
is	O
carried	O
out	O
by	O
involving	O
the	O
imine	O
group	O
(	O
mode	O
A	O
in	O
Fig	O
.	O
4	O
).	O

It	O
is	O
important	O
to	O
highlight	O
that	O
compounds	O
23	B-chemical
and	O
15	B-chemical
,	O
although	O
in	O
different	O
ways	O
,	O
both	O
are	O
able	O
to	O
chelate	O
the	O
metal	O
cofactors	O
and	O
to	O
establish	O
interactions	O
with	O
highly	B-protein_state
conserved	I-protein_state
aminoacids	O
(	O
Tyr24	B-residue_name_number
,	O
Glu26	B-residue_name_number
,	O
Arg124	B-residue_name_number
,	O
Tyr130	B-residue_name_number
and	O
Lys134	B-residue_name_number
)	O
that	O
are	O
very	O
important	O
for	O
both	O
endonuclease	B-protein_type
activity	O
and	O
transcription	O
in	O
vitro	O
.	O

The	O
crucial	O
role	O
of	O
such	O
interactions	O
is	O
underlined	O
by	O
the	O
differences	O
in	O
activity	O
between	O
15	B-chemical
(	O
IC50	B-evidence
=	O
9	O
.	O
0	O
μM	O
)	O
and	O
19	B-chemical
(>	O
500	O
μM	O
):	O
their	O
coordinating	O
features	O
are	O
similar	O
,	O
since	O
both	O
coordinate	B-bond_interaction
to	O
the	O
divalent	O
metal	O
ion	O
through	O
the	O
phenolic	O
oxygen	O
,	O
the	O
iminic	O
nitrogen	O
and	O
the	O
carbonylic	O
oxygen	O
(	O
mode	O
A	O
in	O
Fig	O
.	O
4	O
),	O
but	O
the	O
biological	O
activity	O
could	O
be	O
related	O
to	O
their	O
different	O
ability	O
to	O
engage	O
interactions	O
with	O
the	O
protein	O
environment	O
.	O

Crystallographic	B-experimental_method
Studies	I-experimental_method

Attempts	O
were	O
made	O
to	O
co	B-experimental_method
-	I-experimental_method
crystallize	I-experimental_method
PA	B-protein
-	O
Nter	B-structure_element
with	O
15	B-chemical
,	O
20	B-chemical
,	O
21	B-chemical
and	O
23	B-chemical
in	O
one	O
to	O
four	O
molar	O
excess	O
.	O

While	O
crystals	B-evidence
appeared	O
and	O
diffracted	O
well	O
,	O
upon	O
data	O
processing	O
,	O
no	O
or	O
very	O
little	O
electron	B-evidence
density	I-evidence
for	O
the	O
inhibitors	O
was	O
observed	O
.	O

Attempts	O
to	O
soak	O
apo	B-protein_state
crystals	B-evidence
in	O
crystallization	O
solution	O
containing	O
5	O
mM	O
inhibitor	O
overnight	O
also	O
did	O
not	O
result	O
in	O
substantial	O
electron	B-evidence
density	I-evidence
for	O
the	O
inhibitor	O
.	O

As	O
a	O
last	O
resort	O
,	O
dry	O
powder	O
of	O
the	O
inhibitor	O
was	O
sprinkled	O
over	O
the	O
crystallization	O
drop	O
containing	O
apo	B-protein_state
crystals	B-evidence
and	O
left	O
over	O
night	O
.	O

This	O
experiment	O
was	O
successful	O
for	O
compound	O
23	B-chemical
,	O
the	O
crystals	B-evidence
diffracted	O
to	O
2	O
.	O
15	O
Å	O
and	O
diffraction	O
data	O
were	O
collected	O
(	O
PDB	O
ID	O
5EGA	O
).	O

The	O
refined	O
structure	B-evidence
shows	O
unambiguous	O
electron	B-evidence
density	I-evidence
for	O
the	O
inhibitor	O
(	O
Table	O
S1	O
and	O
Fig	O
.	O
7	O
).	O

The	O
complex	B-evidence
structure	I-evidence
confirms	O
one	O
of	O
the	O
two	O
binding	O
modes	O
predicted	O
by	O
the	O
docking	B-experimental_method
simulations	I-experimental_method
(	O
Fig	O
.	O
5	O
,	O
panel	O
B	O
).	O

The	O
galloyl	O
moiety	O
chelates	O
the	O
manganese	B-chemical
ions	O
,	O
while	O
the	O
trihydroxyphenyl	O
group	O
stacks	O
against	O
the	O
Tyr24	B-residue_name_number
side	O
chain	O
.	O

It	O
is	O
interesting	O
to	O
note	O
that	O
two	O
of	O
these	O
hydroxyl	O
groups	O
are	O
in	O
position	O
to	O
form	O
hydrogen	B-bond_interaction
bonds	I-bond_interaction
with	O
the	O
side	O
chain	O
of	O
Glu26	B-residue_name_number
and	O
Lys34	B-residue_name_number
(	O
Fig	O
.	O
7	O
).	O

These	O
interactions	O
suggest	O
that	O
other	O
functional	O
groups	O
,	O
e	O
.	O
g	O
.	O
halogens	O
,	O
could	O
be	O
used	O
in	O
place	O
of	O
the	O
hydroxyl	O
groups	O
for	O
better	O
interactions	O
with	O
Glu26	B-residue_name_number
and	O
Lys34	B-residue_name_number
side	O
chains	O
,	O
and	O
the	O
inhibitory	O
potency	O
of	O
these	O
compounds	O
could	O
be	O
further	O
improved	O
.	O

Chemical	O
structures	O
of	O
some	O
prototype	O
inhibitors	O
of	O
influenza	B-taxonomy_domain
virus	B-taxonomy_domain
endonuclease	B-protein_type
.	O

Inhibitor	O
activity	O
in	O
enzymatic	B-experimental_method
assays	I-experimental_method
(	O
IC50	B-evidence
,	O
μM	O
)	O
as	O
reported	O
in	O
:	O
aref	O
.,	O
bref	O
.,	O
cref	O
.,	O
dref	O
..	O

General	O
synthesis	O
for	O
N	B-chemical
-	I-chemical
acylhydrazones	I-chemical
1	B-chemical
–	I-chemical
27	I-chemical
and	O
hydrazides	B-chemical
28	B-chemical
and	O
29	B-chemical
(	O
A	O
).	O

Chemical	O
structures	O
of	O
compounds	O
1	B-chemical
–	I-chemical
27	I-chemical
(	O
B	O
).	O

Overview	O
of	O
the	O
structure	O
-	O
activity	O
relationship	O
for	O
compounds	O
1	B-chemical
–	I-chemical
27	I-chemical
.	O

Scheme	O
of	O
possible	O
binding	O
modes	O
of	O
the	O
studied	O
N	B-chemical
-	I-chemical
acylhydrazones	I-chemical
.	O

First	O
(	O
A	O
)	O
and	O
second	O
(	O
B	O
)	O
GOLD	B-experimental_method
cluster	I-experimental_method
docked	I-experimental_method
solutions	O
of	O
compound	O
23	B-chemical
(	O
orange	O
and	O
cyan	O
,	O
respectively	O
)	O
in	B-protein_state
complex	I-protein_state
with	I-protein_state
PA	B-protein
endonuclease	B-protein_type
.	O

Key	O
residues	O
of	O
the	O
pocket	B-site
are	O
presented	O
using	O
PyMOL	O
[	O
http	O
://	O
www	O
.	O
pymol	O
.	O
org	O
]	O
and	O
LIGPLUS	B-experimental_method
[	O
Laskowski	O
,	O
R	O
.	O
A	O
.;	O
Swindells	O
,	O
M	O
.	O
B	O
.	O
Journal	O
of	O
chemical	O
information	O
and	O
modeling	O
2011	O
,	O
51	O
,	O
2778	O
].	O

Hydrogen	B-bond_interaction
bonds	I-bond_interaction
are	O
illustrated	O
by	O
dotted	O
lines	O
,	O
while	O
the	O
divalent	O
metal	O
ions	O
are	O
shown	O
as	O
purple	O
spheres	O
.	O

Schematic	O
drawings	O
of	O
the	O
interactions	O
of	O
the	O
first	O
(	O
C	O
)	O
and	O
second	O
(	O
D	O
)	O
GOLD	B-experimental_method
cluster	I-experimental_method
docked	I-experimental_method
solutions	O
generated	O
using	O
LIGPLUS	B-experimental_method
.	O

Dashed	O
lines	O
are	O
hydrogen	B-bond_interaction
bonds	I-bond_interaction
and	O
‘	O
eyelashes	O
’	O
show	O
residues	O
involved	O
in	O
hydrophobic	B-bond_interaction
interactions	I-bond_interaction
.	O

(	O
A	O
)	O
Binding	O
mode	O
of	O
compound	O
15	B-chemical
(	O
orange	O
)	O
in	B-protein_state
complex	I-protein_state
with	I-protein_state
PA	B-protein
endonuclease	B-protein_type
.	O

Hydrogen	B-bond_interaction
bonds	I-bond_interaction
are	O
illustrated	O
by	O
dotted	O
lines	O
while	O
the	O
divalent	O
metal	O
ions	O
are	O
shown	O
as	O
purple	O
spheres	O
.	O

(	O
B	O
)	O
Schematic	O
drawing	O
of	O
the	O
interactions	O
of	O
compound	O
15	B-chemical
generated	O
using	O
LIGPLUS	B-experimental_method
.	O

Crystal	B-evidence
structure	I-evidence
of	O
PANΔLoop	B-mutant
in	B-protein_state
complex	I-protein_state
with	I-protein_state
compound	O
23	B-chemical
.	O

Active	B-site
site	I-site
residues	O
are	O
shown	O
in	O
sticks	O
with	O
green	O
carbons	O
,	O
manganese	B-chemical
atoms	O
are	O
shown	O
as	O
purple	O
spheres	O
and	O
water	B-chemical
molecules	O
as	O
red	O
spheres	O
.	O

Compound	O
23	B-chemical
is	O
shown	O
in	O
sticks	O
with	O
yellow	O
carbons	O
.	O

2Fo	B-evidence
-	I-evidence
Fc	I-evidence
electron	I-evidence
density	I-evidence
map	I-evidence
contoured	O
at	O
1σ	O
is	O
shown	O
as	O
blue	O
mesh	O
.	O

Hydrogen	B-bond_interaction
bonds	I-bond_interaction
and	O
metal	B-bond_interaction
coordination	I-bond_interaction
are	O
shown	O
with	O
dotted	O
lines	O
.	O

The	O
H	B-bond_interaction
-	I-bond_interaction
bond	I-bond_interaction
distances	O
from	O
the	O
side	O
chain	O
carboxyl	O
group	O
of	O
Glu26	B-residue_name_number
to	O
p	O
-	O
OH	O
and	O
m	O
-	O
OH	O
of	O
the	O
trihydroxyphenyl	O
group	O
of	O
the	O
inhibitor	O
are	O
2	O
.	O
7	O
Å	O
and	O
3	O
.	O
0	O
Å	O
,	O
respectively	O
.	O

The	O
H	B-bond_interaction
-	I-bond_interaction
bond	I-bond_interaction
distance	O
from	O
the	O
side	O
chain	O
of	O
Lys34	B-residue_name_number
to	O
p	O
-	O
OH	O
of	O
the	O
trihydroxyphenyl	O
group	O
is	O
3	O
.	O
6	O
Å	O
.	O
The	O
H	B-bond_interaction
-	I-bond_interaction
bond	I-bond_interaction
distance	O
to	O
the	O
water	B-chemical
molecule	O
from	O
m	O
-	O
OH	O
of	O
the	O
galloyl	O
moiety	O
is	O
3	O
.	O
0	O
Å	O
,	O
which	O
in	O
turn	O
is	O
H	B-bond_interaction
-	I-bond_interaction
bonded	I-bond_interaction
to	O
the	O
side	O
chain	O
of	O
Tyr130	B-residue_name_number
with	O
a	O
distance	O
of	O
2	O
.	O
7	O
Å	O
.	O
Crystal	B-evidence
structure	I-evidence
has	O
been	O
deposited	O
in	O
the	O
RCSB	O
Protein	O
Data	O
Bank	O
with	O
PDB	O
ID	O
:	O
5EGA	O
.	O

Inhibitory	O
activity	O
of	O
the	O
N	B-chemical
-	I-chemical
acylhydrazones	I-chemical
1	B-chemical
–	I-chemical
27	I-chemical
and	O
hydrazide	B-chemical
28	B-chemical
in	O
the	O
enzymatic	B-experimental_method
assay	I-experimental_method
with	O
influenza	B-taxonomy_domain
virus	B-taxonomy_domain
PA	B-protein
-	O
Nter	B-structure_element
endonuclease	B-protein_type
,	O
or	O
in	O
cellular	B-experimental_method
influenza	I-experimental_method
virus	I-experimental_method
assays	I-experimental_method
.	O

Compound	B-experimental_method
Enzyme	I-experimental_method
assay	I-experimental_method
with	O
PA	B-protein
-	O
Ntera	O
Virus	B-experimental_method
yield	I-experimental_method
assay	I-experimental_method
in	O
influenza	B-taxonomy_domain
virus	B-taxonomy_domain
-	O
infected	O
MDCK	O
cellsb	O
vRNP	B-experimental_method
reconstitution	I-experimental_method
assay	I-experimental_method
in	O
HEK293T	O
cellsc	O
Antiviral	O
activity	O
Cytotoxicity	O
SId	O
Activity	O
Cytotoxicity	O
IC50	B-evidence
EC99	B-evidence
EC90	B-evidence
CC50	B-evidence
EC50	B-evidence
CC50	B-evidence
(	O
1	O
)	O
24	O
NDf	O
ND	O
ND	O
107	O
>	O
200	O
(	O
2	O
)	O
>	O
500	O
ND	O
ND	O
ND	O
>	O
100	O
>	O
200	O
(	O
3	O
)	O
>	O
500	O
ND	O
ND	O
>	O
200	O
5	O
.	O
9	O
48	O
(	O
4	O
)	O
>	O
500	O
ND	O
ND	O
>	O
200	O
6	O
.	O
3	O
33	O
(	O
5	O
)	O
67	O
>	O
25	O
>	O
25	O
≥	O
146	O
2	O
.	O
6	O
10	O
(	O
6	O
)	O
>	O
500	O
>	O
50	O
>	O
50	O
>	O
200	O
15	O
14	O
(	O
7	O
)	O
54	O
172	O
100	O
>	O
200	O
>	O
2	O
.	O
0	O
3	O
.	O
2	O
8	O
.	O
9	O
(	O
8	O
)	O
>	O
500	O
>	O
12	O
.	O
5	O
>	O
12	O
.	O
5	O
>	O
200	O
1	O
.	O
9	O
15	O
(	O
9	O
)	O
34	O
16	O
5	O
.	O
3	O
>	O
200	O
>	O
38	O
5	O
.	O
5	O
>	O
200	O
(	O
10	O
)	O
68	O
14	O
8	O
.	O
5	O
111	O
>	O
13	O
0	O
.	O
40	O
132	O
(	O
11	O
)	O
45	O
30	O
12	O
>	O
200	O
>	O
17	O
5	O
.	O
6	O
>	O
200	O
(	O
12	O
)	O
>	O
500	O
>	O
12	O
.	O
5	O
>	O
12	O
.	O
5	O
>	O
200	O
20	O
39	O
(	O
13	O
)	O
69	O
71	O
34	O
>	O
200	O
>	O
5	O
.	O
9	O
6	O
.	O
3	O
>	O
200	O
(	O
14	O
)	O
>	O
500	O
63	O
37	O
>	O
200	O
>	O
5	O
.	O
4	O
2	O
.	O
3	O
>	O
200	O
(	O
15	O
)	O
8	O
.	O
9	O
18	O
7	O
.	O
5	O
≥	O
172	O
≥	O
23	O
14	O
>	O
200	O
(	O
16	O
)	O
454	O
67	O
28	O
>	O
200	O
>	O
7	O
.	O
1	O
5	O
.	O
2	O
>	O
200	O
(	O
17	O
)	O
482	O
21	O
8	O
.	O
1	O
>	O
200	O
>	O
25	O
7	O
.	O
1	O
>	O
200	O
(	O
18	O
)	O
83	O
6	O
.	O
2	O
2	O
.	O
2	O
>	O
200	O
>	O
91	O
3	O
.	O
3	O
>	O
200	O
(	O
19	O
)	O
>	O
500	O
53	O
26	O
>	O
200	O
>	O
7	O
.	O
7	O
5	O
.	O
7	O
>	O
200	O
(	O
20	O
)	O
18	O
35	O
11	O
>	O
200	O
>	O
18	O
2	O
.	O
2	O
>	O
200	O
(	O
21	O
)	O
13	O
8	O
.	O
3	O
3	O
.	O
6	O
>	O
200	O
>	O
56	O
2	O
.	O
5	O
>	O
200	O
(	O
22	O
)	O
75	O
7	O
.	O
4	O
3	O
.	O
4	O
>	O
200	O
>	O
59	O
0	O
.	O
42	O
>	O
200	O
(	O
23	O
)	O
8	O
.	O
7	O
11	O
3	O
.	O
5	O
>	O
200	O
>	O
57	O
3	O
.	O
1	O
>	O
200	O
(	O
24	O
)	O
131	O
58	O
26	O
>	O
200	O
>	O
7	O
.	O
7	O
25	O
>	O
200	O
(	O
25	O
)	O
40	O
132	O
70	O
>	O
200	O
>	O
2	O
.	O
9	O
4	O
.	O
1	O
>	O
200	O
(	O
26	O
)	O
30	O
36	O
13	O
>	O
200	O
>	O
15	O
5	O
.	O
5	O
>	O
200	O
(	O
27	O
)	O
36	O
ND	O
ND	O
ND	O
21	O
>	O
200	O
(	O
28	O
)	O
40	O
158	O
85	O
>	O
200	O
>	O
2	O
.	O
4	O
7	O
.	O
2	O
>	O
200	O
DPBAe	O
5	O
.	O
3	O
ND	O
ND	O
ND	O
ND	O
ND	O
Ribavirin	O
ND	O
13	O
8	O
.	O
5	O
>	O
200	O
>	O
24	O
9	O
.	O
4	O
>	O
200	O

aRecombinant	O
PA	B-protein
-	O
Nter	B-structure_element
was	O
incubated	B-experimental_method
with	O
the	O
ssDNA	B-chemical
plasmid	O
substrate	O
,	O
a	O
Mn2	B-chemical
+-	I-chemical
containing	O
buffer	O
and	O
test	O
compounds	O
.	O

The	O
IC50	B-evidence
represents	O
the	O
compound	O
concentration	O
(	O
in	O
μM	O
)	O
required	O
to	O
obtain	O
50	O
%	O
inhibition	O
of	O
cleavage	O
,	O
calculated	O
by	O
nonlinear	B-experimental_method
least	I-experimental_method
-	I-experimental_method
squares	I-experimental_method
regression	I-experimental_method
analysis	I-experimental_method
(	O
using	O
GraphPad	O
Prism	O
software	O
)	O
of	O
the	O
results	O
from	O
2	O
–	O
4	O
independent	O
experiments	O
.	O

bMDCK	O
cells	O
were	O
infected	O
with	O
influenza	B-taxonomy_domain
A	I-taxonomy_domain
virus	B-taxonomy_domain
(	O
strain	O
A	O
/	O
PR	O
/	O
8	O
/	O
34	O
)	O
and	O
incubated	O
with	O
the	O
compounds	O
during	O
24	O
h	O
.	O
The	O
virus	B-taxonomy_domain
yield	O
in	O
the	O
supernatant	O
was	O
assessed	O
by	O
real	B-experimental_method
-	I-experimental_method
time	I-experimental_method
qPCR	I-experimental_method
.	O

The	O
EC99	B-evidence
and	O
EC90	B-evidence
values	O
represent	O
the	O
compound	O
concentrations	O
(	O
in	O
μM	O
)	O
producing	O
a	O
2	O
-	O
log10	O
or	O
1	O
-	O
log10	O
reduction	O
in	O
virus	B-taxonomy_domain
titer	O
,	O
respectively	O
,	O
determined	O
in	O
2	O
–	O
3	O
independent	O
experiments	O
.	O

The	O
cytotoxicity	O
,	O
assessed	O
in	O
uninfected	O
MDCK	O
cells	O
,	O
was	O
expressed	O
as	O
the	O
CC50	B-evidence
value	O
(	O
50	O
%	O
cytotoxic	O
concentration	O
,	O
determined	O
with	O
the	O
MTS	B-experimental_method
cell	I-experimental_method
viability	I-experimental_method
assay	I-experimental_method
,	O
in	O
μM	O
).	O

cHEK293T	O
cells	O
were	O
co	B-experimental_method
-	I-experimental_method
transfected	I-experimental_method
with	O
the	O
four	O
vRNP	B-complex_assembly
-	O
reconstituting	O
plasmids	O
and	O
the	O
luciferase	O
reporter	O
plasmid	O
in	O
the	O
presence	B-protein_state
of	I-protein_state
the	O
test	O
compounds	O
.	O

The	O
EC50	B-evidence
represents	O
the	O
compound	O
concentration	O
(	O
in	O
μM	O
)	O
producing	O
50	O
%	O
reduction	O
in	O
vRNP	B-complex_assembly
-	O
driven	O
firefly	O
reporter	O
signal	O
,	O
estimated	O
at	O
24	O
h	O
after	O
transfection	O
.	O

The	O
EC50	B-evidence
value	O
was	O
derived	O
from	O
data	O
from	O
2	O
–	O
4	O
independent	O
experiments	O
,	O
by	O
nonlinear	B-experimental_method
least	I-experimental_method
-	I-experimental_method
squares	I-experimental_method
regression	I-experimental_method
analysis	I-experimental_method
(	O
using	O
GraphPad	O
Prism	O
software	O
).	O

The	O
CC50	B-evidence
(	O
in	O
μM	O
),	O
i	O
.	O
e	O
.	O
the	O
50	O
%	O
cytotoxic	O
concentration	O
,	O
was	O
determined	O
in	O
untransfected	O
HEK293T	O
cells	O
by	O
MTS	B-experimental_method
cell	I-experimental_method
viability	I-experimental_method
assay	I-experimental_method
.	O

dSI	B-evidence
,	O
selectivity	B-evidence
index	I-evidence
,	O
defined	O
as	O
the	O
ratio	O
between	O
the	O
CC50	B-evidence
and	O
EC90	B-evidence
.	O

eDPBA	B-chemical
,	O
2	B-chemical
,	I-chemical
4	I-chemical
-	I-chemical
dioxo	I-chemical
-	I-chemical
4	I-chemical
-	I-chemical
phenylbutanoic	I-chemical
acid	I-chemical
.	O