Kinetic and in silico analysis of the slow-binding inhibition of human poly(A)-specific ribonuclease (PARN) by novel nucleoside analogues

Article abstract of DOI:10.1016/j.biochi.2011.10.011

Poly(A)-specific ribonuclease (PARN) is a 3′-exoribonuclease that efficiently degrades poly(A) tails and regulates, in part, mRNA turnover rates. We have previously reported that adenosine- and cytosine-based glucopyranosyl nucleoside analogues with adequ

Full text of DOI:10.1016/j.biochi.2011.10.011

Biochimie 94 (2012) 214e221
Contents lists available at SciVerse ScienceDirect
Biochimie
journal homepage: www.elsevier.com/locate/biochi
Research paper
Kinetic and in silico analysis of the slow-binding inhibition of human
poly(A)-specific ribonuclease (PARN) by novel nucleoside analogues
,
b
a
a
a
Nikolaos Balatsos a , Dimitrios Vlachakis , Vassiliki Chatzigeorgiou , Stella Manta , Dimitri Komiotis ,
**
Metaxia Vlassi b, Constantinos Stathopoulos c
,
*
a Department of Biochemistry & Biotechnology, University of Thessaly, 26 Ploutonos st., 41 221 Larissa, Greece
b Institute of Biology, National Centre for Scientific Research “Demokritos”, 15 310 Aghia Paraskevi, Attikis, Greece
c Department of Biochemistry, School of Medicine, University of Patras, 1 Asklipiou st., 26 504 Rio-Patras, Greece
a r t i c l e i n f o
a b s t r a c t
Poly(A)-specific ribonuclease (PARN) is a 30-exoribonuclease that efficiently degrades poly(A) tails and
regulates, in part, mRNA turnover rates. We have previously reported that adenosine- and cytosine-based
glucopyranosyl nucleoside analogues with adequate tumour-inhibitory effect could effectively inhibit
PARN. In the present study we dissect the mechanism of a more drastic inhibition of PARN by novel
glucopyranosyl analogues bearing uracil, 5-fluorouracil or thymine as the base moiety. Kinetic analysis
Article history:
Received 23 June 2011
Accepted 17 October 2011
Available online 24 October 2011
Keywords:
PARN
showed that three of the compounds are competitive inhibitors of PARN with Ki values in the low mM
concentration and significantly lower (11- to 33-fold) compared to our previous studies. Detailed kinetic
analysis of the most effective inhibitor, the uracil-based nucleoside analogue (named U1), revealed slow-
binding behaviour. Subsequent molecular docking experiments showed that all the compounds which
inhibited PARN can efficiently bind into the active site of the enzyme through specific interactions. The
present study dissects the inhibitory mechanism of this novel uracil-based compound, which prolongs its
inhibitory effect through a slow-binding and slow-release mode at the active site of PARN, thus
contributing to a more efficient inhibition. Such analogues could be used as leading compounds for
further rationale design and synthesis of efficient and specific therapeutic agents. Moreover, our data
reinforce the notion that human PARN can be established as a novel molecular target of potential anti-
cancer agents through lowering mRNA turnover rates.
Deadenylation
Slow-binding inhibition
Nucleoside analogues
Ó 2011 Elsevier Masson SAS. All rights reserved.
1. Introduction
regulates PARN activity possibly by preventing the cap-binding of
the translation initiation factor eIF4E, and/or by competing with the
A critical step in mRNA degradation is the removal of the poly(A)
[1]. The process is catalyzed by deadenylases both in the nucleus
and in the cytoplasm [2,3]. Among those enzymes, poly(A)-specific
ribonuclease (PARN) holds a key role both in development [3] and
in mRNA 30-end processing upon DNA-damaging conditions [4].
PARN is a dimeric enzyme where each subunit is composed of three
characteristic structural motifs and belongs to the DEDD family of
nucleases. It is the only known deadenylase, so far to interact both
with the 50-cap structure and the poly(A) tail [5e7]. This interaction
nuclear cap-binding complex subunit CBP80 [8,9]. It is also known
that poly(A)-binding protein C (PABPC) inhibits PARN activity,
probably through binding and interfering with the poly(A)
substrate [10,11]. On the other hand, PARN-mediated poly(A)
degradation is promoted by AU-rich elements (AREs) and ARE-
associated proteins [6,12e14].
PARN can be modulated by small compounds, such as amino-
glycoside antibiotics [15], and natural purine nucleotides that
inhibit its degrading activity [16,17]. More recently, work from our
group has shown that PARN can be effectively inhibited by a novel
family of synthetic nucleoside analogues, which are based on glu-
copyranosyl rings as the sugar moiety [18]. These compounds have
been evaluated for their efficient anti-cancer and anti-viral potency
when tested in cell cultures. Such novel nucleoside analogues have
emerged as important therapeutic agents for the development of
anti-viral and antitumour drugs [19e21]. Various novel unsatu-
rated ketopyranonucleosides [20,22e26] have been developed and
Abbreviations: PARN, poly(A)-specific ribonuclease;
fluorouracil, respectively; U1, [1-(3΄-deoxy-3΄-fluoro-
FU1, [1-(3΄-deoxy-3΄-fluoro- -glucopyranosyl)] 5-fluorouracil.
U
or FU, uracil or 5-
b-D-glucopyranosyl)] uracil;
b-D
* Corresponding author. Tel.: þ30 2610 997932; fax: þ30 2610 969167.
** Corresponding author. Tel.: þ30 2410 565261; fax: þ30 2410 565290.
E-mail addresses: balatsos@bio.uth.gr (N. Balatsos), cstath@med.upatras.gr
(C. Stathopoulos).
0300-9084/$ e see front matter Ó 2011 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.biochi.2011.10.011

N. Balatsos et al. / Biochimie 94 (2012) 214e221
215
have been shown to effectively inhibit PARN, which represents
a promising molecular target [18]. Based on the properties of those
nucleosides analogues, and the significance of PARN for the regu-
lation of the mRNA lifecycle, we have been actively engaged in the
development and the study of various unsaturated ketopyranonu-
cleosides and their inhibitory effect on PARN activity.
formulae of the compounds used in the present study are shown
in Table 1.
2.3. Purification of recombinant PARN, activity assays and kinetic
analysis
In the present study, we investigated the inhibitory effect of
novel glycopyranosyl nucleosides on human PARN activity, through
detailed kinetic analysis and molecular docking. After testing several
compounds, we concluded that the most effective inhibitor which is
based on uracil (U1, Table 1) represents a less bulky, less conjugated
compound with much more flexible core and increased potency
when compared to the adenosine- and cytosine-based compounds
that we have tested and characterized so far [18]. These compounds
The plasmid encoding full-size 74 kDa human PARN (for
expression of N-terminal His6-tagged polypeptide) was trans-
formed into BL21(DE3) cells to express the recombinant protein as
described previously [27].
The enzymatic activity was determined by the methylene blue
assay as described before [28]. Methylene blue buffer was prepared
by dissolving 1.2 mg methylene blue into 100 ml Mops buffer (0.1 M
MOPS-KOH, pH 7.5, 2 mM EDTA). The standard reaction buffer
contained 50 mM KH2PO4/K2HPO4 (pH 7.0), 1.5 mM MgCl2, 100 mM
KCl, 0.2 mM EDTA, 0.25 mM DTT, 10% (v/v) glycerol and 0.1 U of
RNasin. All analogues were dissolved in reaction buffer prior to use.
represent drastic molecules at the mM range (11- to 33-fold more
effective when compared to our previous studies) that act through
a mechanism that allows slow-binding and slow-release on the
active site of the enzyme. Therefore they enhance significantly their
drastic effect since they prolong their inhibitory interactions with
the active site of the enzyme. Both our biochemical and in silico
analysis suggest that the effective inhibition by slow-binding
nucleoside analogues represents an adequate mechanism of PARN
inhibition at least in vitro. Since human PARN represents a suitable
and effective target for inactivation and for the development of anti-
cancer compounds, our results provide substantial evidence for
promising in vivo trials of such compounds that could effectively
inhibit PARN and thus, providing the means to lowering mRNA
turnover rates in de-regulated cell lines.
The reactions were performed using 10e15
mM of recombinant
PARN. To evaluate the effect of Mg(II) ions on PARN activity in the
presence of the analogues, deadenylation assays were performed as
described above. The concentration of Mg(II) varied from 0.15 mM
to 15 mM (10-fold below and above the standard assay concen-
tration). For kinetic analysis, the substrate concentration [poly(A)]
varied from 70 to 600
compounds tested. The final reaction volume was 100
reaction was performed at 30ꢀC for 10 min. The reaction was
terminated by mixing the reaction solution with 900 l methylene
mM [17], and from 10 to 500
mM for the
ml and the
m
blue buffer and the mixed solution was incubated at 30 ꢀC for
another 15 min in the dark in a water bath. The absorbance at
662 nm of 1 ml sample was measured on a Spectronic Genesys 20
spectrophotometer.
2. Materials and methods
2.1. Materials
2.4. Molecular docking
All chemicals including purine ribonucleotides and deoxy-
nucleotides, methylene blue, polyadenylic acid potassium salt
(average size 300 adenosines, A300) and polyuridine acid potassium
salt (average size 300 uracils, U300) were from Sigma-Aldrich.
The Schrödinger suite of programs and the program Glide1
(grid-based ligand docking with Energetics) was used for the
molecular docking experiments. The Maestro program2 was used
for molecular building and coordinate preparation prior to docking.
The coordinates of the crystal structure of PARN (Protein Data Bank
accession code 2A1R) were used as the receptor and the structure
was prepared using the Protein Preparation Wizard3 module of
Maestro. All crystallographic water molecules were removed from
the coordinate file prior to docking. Hydrogen atoms and partial
charges were added to the enzyme and the initial structure was
energy minimized using the OPLS-AA force field [29]. For docking,
the scoring grid file was generated through the Glide Receptor Grid
Preparation module, using the same active site of PARN as previ-
ously [18]. A docking box of 40 ꢁ 40 ꢁ 40 points with a van der
Waals scaling factor of 1.0, was placed around the active site of the
protein, for this purpose the compound optimization module of
Maestro, LigPre4 was used to prepare the ligand database. For each
compound, 32 stereoisomers were generated and their 3D-struc-
ture was minimized using the OPLS-AA force field. The best three
conformations of each compound were kept for docking. The Glide
XP (extra precision) method [30] was employed for all docking
calculations.The maximum number of poses was set to 1000 and
the number of runs was set to 1. The best-docked pose for each
2.2. Synthesis of nucleoside analogues
The desired fluoro-pyranosyl nucleosides were synthesized by
implementing the Vorbruggen glycosylation reaction, adapted
into a one-step 5 min/130 ꢀC microwave (MW)-assisted reaction,
followed by subsequent deprotection. As an extension of a meth-
odology recently developed by our group for the synthesis of
fluorinated nucleosides [20,24,25], we found that it can be more
easily prepared in an one-step reaction using elevated tempera-
tures generated by microwave heating and we observed that the
glycosylation reactions could be conducted satisfactorily in 5 min
at 130 ꢀC. Using this approach, reaction of 1,2,4,6-tetra-O-acetyl-
3-deoxy-3-fluoro-glucopyranose [20] with uracil, 5-fluorouracil,
thymine, N4-benzoyl cytosine, and N6-benzoyl adenine under MW
irradiation at 130 ꢀC for 5 min gave the protected 1-(2,4,6-tri-O-
acetyl-3-deoxy-3-fluoro-b-D-glucopyranosyl) nucleosides, in the
presence of trimethylsilyltrifluoromethane-sulfonate and tin
chloride, respectively. Deacetylation of uracil, 5-fluorouracil and
thymine derivatives with ammonia in methanol afforded nucle-
osides [1-(30-deoxy-30-fluoro-
[1-(30-deoxy-30-fluoro-
and [1-(30-deoxy-30-fluoro-
b-D
b
-
D
-glucopyranosyl)] uracil (U1),
-glucopyranosyl)] 5-fluorouracil (FU1)
-glucopyranosyl)] thymine (T1),
b-D
1
while deprotection of N4-benzoyl cytosine and N6-benzoyl
adenine derivatives with NaOHeethanolepyridine, yielded ben-
zoylated analogues A2 and A6. The chemical and physical prop-
erties of the fluoro-pyranosyl nucleosides were in agreement with
previous data [20,24,25]. The nomenclature and the chemical
Glide, version 5.5, Schrödinger, LLC, New York, NY, 2009.
Maestro, version 9.0, Schrödinger, LLC, New York, NY, 2009.
Schrödinger Suite 2009 Protein Preparation Wizard; Epik version 2.0, Schrö-
2
3
dinger, LLC, New York, NY, 2009; Impact version 5.5, Schrödinger, LLC, New York,
NY, 2009; Prime version.
4
LigPrep (LigPrep, version 2.3, Schrödinger, LLC, New York, NY, 2009).

216
N. Balatsos et al. / Biochimie 94 (2012) 214e221
Table 1
Synthetic nucleosides used in the present work and inhibition constants.
Nucleoside
Ki,
mM
Chemical formula
Nomenclature
Abbreviation
1-(30-deoxy-30-fluoro-
b
-D
-glucopyranosyl) uracil
U1
19 ꢂ 5
1-(30-deoxy-30-fluoro-
b
-D
-glucopyranosyl) 5-fluorouracil
FU1
98 ꢂ 12
1-(30-deoxy-30-fluoro-
b
-D
-glucopyranosyl) thymine
T1
135 ꢂ 18
9-(30,40, dideoxy-30-fluoro-
b-
D
-glucopyranosyl)-N6-benzoyl adenine
A2
510 ꢂ 52a
1-(30,40, dideoxy-30-fluoro-
b-
D
-glucopyranosyl)-N4-benzoyl adenine
A6
210 ꢂ 45a
3-deoxy-3-fluoro-glucopyranose
B6
NDa
a
Data from [18].

N. Balatsos et al. / Biochimie 94 (2012) 214e221
217
compound was selected as the one with the lowest Glide Score
[30]; the more negative the Glide Score the more favourable the
binding.
PARN activity and act as competitive inhibitors. In particular U1
inhibits PARN more efficiently than other analogues tested.
3.2. U1 is a slow-binding inhibitor of PARN
3. Results
The fact that the U1 is an efficient inhibitor of PARN and taking
into account that PARN degrades in vitro poly(U) in a much slower
rate than it degrades poly(A) [31], prompted us to examine the
inhibitory mechanism in more detail. When the initial rates of the
reactions in the presence of increasing amounts of U1 were plotted
near the origin, the slopes of the progress curves changed rapidly,
implying slow-binding inhibition [32]. The time-dependent inhi-
bition of U1 is illustrated in Fig. 2A, showing the first-order time
plots of poly(A) degradation in the presence or absence of the
compound. The control reaction (absence of inhibitor) is linear,
therefore the analysis of the inhibition is analysed by fitting prog-
ress curves to equation (1) (termed “burst equation”) [32e35]:
3.1. U1, FU1 and T1 are competitive inhibitors of PARN
Recently, we examined the effect of several fluoro-pyranosyl
nucleosides with established anti-cancer and anti-viral activity, as
potential inhibitors of human PARN [18]. The inhibition of PARN by
compounds bearing adenine has been already established from our
previous studies and therefore we extended our survey on novel
analogues bearing uracil and thymine as the base moiety in
a survey of improved inhibitors. It has been reported that although
PARN degrades preferably poly(A) tails in vivo, it can also recognise
uracil and can degrade poly(U), albeit approximately 10-fold less
effectively than poly(A) [31].The novel analogues bear modified
sugar moiety to fit into the active site, as we have previously pre-
dicted in silico [18]. After initial screening for PARN inhibition, we
performed detailed kinetic analysis which revealed that three
compounds U1, FU1 and T1 behave as more efficient competitive
inhibitors of PARN in vitro (Fig. 1) compared to the compounds A2
and A6 that exhibit the strongest inhibition (11- to 33-fold) among
the analogues bearing adenine [18]. The calculated Ki values were
ꢀ
ꢁ
vst þ ðv0 þ vsÞ 1 ꢄ eꢄk
t
obs
½Pꢃ ¼
(1)
kobs
where, [P] is the concentration of the product at time t, vo and vs are
the initial and steady-state rates, and kobs is the observed rate
constant, or the exponential decay constant (sꢄ1) for the approach
to the steady-state final rate [34]. For PARN activity assay the
calculations are based on the degradation rate of the substrate.
In order to further dissect the mechanism of inhibition and
determine whether U1 acts as rapidly or slowly reversible inhibitor,
we used a preincubation/dilution assay [36]. In brief, PARN was pre-
20 mM for U1, 98 mM for FU1 and 135 mM for T1. The compound B6
(3-deoxy-3-fluoro-glucopyranose), which contains only the same
sugar ring without the base moiety, showed no inhibition and in
the subsequent analysis was used as the negative control (Table 1).
Our data show that glucopyranosyl nucleoside analogues with
uracil or thymine as the base moiety are suitable suppressors of
incubated with U1 for 60 min at 10-fold its Ki value (200 mM) leading
to saturation of the active sites. The pre-incubated mixture was then
B
A
100
8
6
4
2
50
-0.02
100
0
0.02
[µg poly(A)/ml
0.06
0.10
0
50
-0.06
-50
100
-1
]
[U1], µM
C
D
200
100
50
200
[FU1], µM
300
-100
100
500
-250
0
250
0
[T1], µM
Fig. 1. Inhibition of PARN activity by novel nucleoside analogues. Double reciprocal plot 1/v versus 1/[substrate] for PARN activity in the presence of U1 (A). The U1 concentrations
were 0 mM (circle), 0.010 mM (square), 0.050 mM (rhombus) and 0.1 mM (inverted triangle). The representative slopes (KM/Vmax) of the double reciprocal plots versus the
nucleoside concentration for U1 (B), FU1 (C) and T1 (D) are shown.

218
N. Balatsos et al. / Biochimie 94 (2012) 214e221
in the presence of a rapidly reversible inhibitor is above 90% at the
1.50
1.20
0.90
0.60
0.30
A
same time period, a value that could not be observed for U1. Actually,
when we prolonged the assay time we could observe a very slow
restoration of PARN activity (up to 80%) after 180 min (3 h) (Fig. 2B).
These results strongly suggest that U1 is indeed a slowly reversible
inhibitor of PARN and the binding of U1 into the active site of PARN is
very stable.
Based on both the time-dependent inhibition of PARN by U1 and
the preincubation assay, a kinetic model that describes with accu-
racy the inhibition of PARN by U1 is proposed (Fig. 2C), together
with the respective kinetic data for each step. The non-covalent EI
complex (PARN-U1) is rapidly formed followed by a slower step,
where it is transformed to the more stable EI* complex. In this
process, Ki represents the equilibrium constant for the first step, Ki*
0
5
10
15
Time, min
100
the overall dissociation constant and k2, k the interconversion
ꢄ2
B
rate constants [32]. At each concentration of U1, the kobs values
were determined for the initial (k0obs) and close to the steady-state
(ksobs) according to equation 2:
80
60
40
20
100
100 ꢄ x
ln
¼ kobs$t
(2)
where, x is the percent of the poly(A) bound on PARN and t is the
reaction time (Equation. (1a)) [37]. The double reciprocal plots of
k0obs and ksobs versus several substrate concentrations are shown in
Fig. 3 (A and B respectively), and show that the type of inhibition is
competitive, as expected from the analysis using the initial rates
shown in Fig. 1A. In addition, the slopes of the lines were reploted
versus increasing U1 concentrations (Fig. 3C). Both replots are
linear and meet the vertical axis at the same point, which coincides
with the point of the corresponding control experiment
30
180
0
10
20
Time, min
k1
k-1
kcat
E + S
ES
E + P
C
([U1] ¼ 0
competitive mode of inhibition [40]. Both lines shown in Fig. 3C
when extrapolated meet the horizontal axis at 19.4 and 2.2 M,
representing Ki and the overall dissociation constant Ki*, respec-
tively. The isomerisation constant, k2/k can be determined
mM). The linearity of the replot also confirms the
+
I
m
Constant
Ki* (µM)
k2/k-2
k2, min-1
k-2, min-1
Ki
2.2 0.4
7.6 0.5
5.3 0.3
0.7 0.1
ꢄ2
according to equation (3) [32,37] and equals to 7.6. Finally, the
EI
apparent association rate constant (k2 þ kꢄ2)/Ki, of U1 binding, can
be calculated and equals to 5.2 ꢁ 103 Mꢄ1
s
ꢄ1. This value is much
k2 k-2
EI*
lower than 106 Mꢄ1 sꢄ1, which is considered the upper limit value
for the classification of a compound as slow-binding inhibitor
[33,37].
Fig. 2. A. Reaction progress curves in the presence of increasing concentrations of U1.
Poly(A) degradation was monitored in the presence of 0 mM (open circle), 0.01 mM
(inverted triangle), 0.025 mM (rhombus), 0.10 mM (square), 0.25 mM (triangle) and
Ki
Ki*
¼
(3)
k
k
þ2
ꢄ2
1
mM (filled circle) of U1. B. Reversibility data for U1. PARN (0.07
pre-incubated with 0.2 mM U1 for 60 min at 37 ꢀC. The mixture was then diluted
100-fold into deadenylation assay mixture containing 7 g poly(A)/ml. Reaction rates
mg/ml) was
1 þ
m
Thus, our detailed kinetic analysis, show that compound U1
behaves as a slow-binding inhibitor of PARN. The overall dissocia-
tion constant Ki* value is 10-fold lower than the Ki value (w2
versus 20 M) indicating that EI* complex is more stable than EI
were monitored for 30 min further and expressed as percentage of the rates of the
uninhibited (control) reaction. C. Proposed kinetic scheme for slow-binding inhibition
of PARN by U1 including the calculated equilibrium and kinetic constants of PARN
inhibition.
m
M
m
complex (Fig. 2C). None of the additional compounds tested in this
work (FU1, TI or B6) behaved as slow-binding inhibitor of PARN.
Although poly(U) is not the natural substrate of PARN in vivo, it
can be degraded by PARN in vitro [31]. Initial experiments were
performed also using poly(U) as substrate (data not shown). We
observed elevated KM value for poly(U) and slow reaction rate, in
agreement with current reports. In addition, the Vmax value was
unaffected. Based on the above, we concluded that using poly(A) as
substrate is more appropriate because it is the natural substrate of
PARN and it can be used in reduced amounts (comparing the KM
values). The use of either poly(A) or poly(U), does not significantly
alter the kinetic behaviour (and thus the kinetic data) of the
compounds tested in the present work (data not shown).
diluted 100-fold into the deadenylation assay mixture [containing
7 mg poly(A)/ml] and the activity rates were monitored for 30 min
further and compared to the rates of the control reaction. A rapidly
reversible inhibitor should dissociate from the enzyme to restore
more than approximately 90% of activity [36]. Fig. 2B summarizes
the results of the preincubation/dilution assay. The comparison of
the pre-incubated and uninhibited reactions revealed that upon
100-fold dilution of the pre-incubated mixture of PARN and U1 into
substrate-containing assay mixture, 7% of the enzymatic activity was
restored after 10 min and 20% of the enzymatic activity was restored
after 30 min. This observation shows that U1 is released from the
enzymeeinhibitor complex, albeit in a very slow rate. Moreover, as
mentioned above, the threshold for a recovered enzymatic activity
Biochemical studies have shown that Mg(II) ions are necessary
for PARN activity at least in vitro [11,38]. We have previously

N. Balatsos et al. / Biochimie 94 (2012) 214e221
219
3.3. Molecular docking of U1, T1 and FU1 in the active site of PARN
12
10
A
To shed light on the binding mode of the inhibitory compounds
that were tested, we performed in silico molecular docking exper-
iments using the information from known crystal structure of
a truncated form of PARN [44]. The best-docked conformation of
compounds U1, FU1 and T1, as indicated by the lowest Glide Score
for each compound, is shown in Fig. 4. The binding orientation of
U1 in the active site of PARN (Fig. 4A) is optimal; the establishment
of a hydrogen bond between the eOH group on the sugar C-20 and
Glu30 optimises the geometry of the other two eOH groups of the
sugar ring to donate electrons to two of the Asp residues of the
catalytic triad (Asp292 and Asp28). Moreover, one of the ]O atoms
on the phenyl ring interacts weakly with the backbone of Phe31,
further stabilising and defining the binding mode of U1 within the
active site of PARN, away from Phe115 (Fig. 4A). On the contrary, the
methyl group that has been added to the base moiety of the
compound T1 is attracted by a hydrophobic pocket formed by
residues Phe155, Leu116 and Ser112 (Fig. 4B). Only one of the three
eOH groups on the sugar ring of T1 interacts with Asp382 residue
of the catalytic triad suggesting that this compound may be a less
potent inhibitor of PARN compared to U1. This prediction is in
agreement with our kinetic data, which show a higher Ki value for
T1 compared to U1 (Table 1). On the other hand, the eOH group on
the C-20 atom of FU1 is again hydrogen bonded to Glu30 (Fig. 4C), as
in the case of the U1 compound. However, a new hydrogen bond
between one of the sugar’s eOH groups and Asn288, slightly tilts
the docking axis of FU1 offset. This orientation allows only one of
the three eOH groups of the sugar moiety to interact with only one
residue of the catalytic triad (Asp292). It is strongly suggested that
the hydrogen bonding to Asn288 and the slight tilt of the axis of this
compound are responsible for the lower inhibitory effect of FU1
compared to U1.
8
6
4
2
-0.02
0
0.02 0.04 0.06 0.08 0.1
1/[µg poly(A)/ml]
B
100
80
60
40
20
-0.02
0
0.02 0.04 0.06 0.08 0.1
1/[µg poly(A)/ml]
C
1000
800
600
400
200
4. Discussion
Few are known about the biological significance of PARN.
Recently, (and interestingly) PARN was reported to be involved in
the late cytoplasmic cell-cycle checkpoint control through a mech-
anism that regulates 30 end processing in response to DNA damage
[39]. It has been demonstrated that PARN interacts with CstF-50,
a factor involved in mRNA maturation and DNA repair, while the
tumour-suppressor BARD1 strongly activates deadenylation by
PARN in the presence of CstF-50. PARN along with the CstF/BARD1
participates in the regulation of endogenous transcripts under
DNA-damaging conditions. All those novel evidence pinpoint PARN
as a key player in a concerted and dynamic network between
factors involved in mRNA maturation, degradation and tumour-
suppression to prevent the expression of prematurely terminated
messages, thus contributing to control of gene expression [4]. It is
now evident that PARN is involved in cell-cycle checkpoint control.
Besides the two well-established protein kinase-signalling units
that modulate the key elements of checkpoint control [40],
a recently characterized kinase pathway, p38/MK2, elicits check-
point arrest [41e43].
Chemically stable enzyme inhibitors have received increasing
attention in the design of drugs due to their potential for potency and
selectivity. A slow rate of dissociation is important in drug design,
since it is expected to enhance the effectiveness of the inhibitor. This
quest for new compounds has led to the development of many
potent, reversible inhibitors. A key characteristic of slow-binding
inhibitors is that they do not rapidly establish equilibrium with
their target enzymes relative to enzymatic turnover of substrate and
exhibit a time-dependent inhibition following complex kinetics
[32e35]. Human PARN represents a potential target for drug
-20
0
20
40
60
80
100
[U1], µM
Fig. 3. Double reciprocal plots for PARN inhibition by U1. Panels A and B represent data
collected from the early (t < 30 s) and the late phases (t > 5 min) of progress curve
plots, respectively. U1 concentrations were 0 mM (open circle), 0.01 mM (triangle),
0.025 mM (square), 0.10 mM (rhombus), 0.25 mM (inverted triangle) and 1 mM (filled
circle) of U1. C. Slopes of the double reciprocal plots versus U1 concentration (data of
the double reciprocal plots were obtained from A and B). The replot represents the
slopes of the lines from the early phase (filled circle) and late phase (open circle) from
progress curves as shown in A and B, respectively. The linearity of the slope replots
confirms the competitive behavior of U1.
reported that increased Mg(II) concentrations could release the
inhibitory effect by natural di- and tri-phosphate nucleotides but
not the inhibition by mono-phosphate nucleotides [17]. In addition,
we have reported that inhibition of PARN by synthetic glucopyr-
anosyl nucleosides is independent of the local Mg(II) concentration
and the effect of these compounds can not be deprived by altering
Mg(II) levels [18]. To exclude the possibility that the observed PARN
inhibition could be rescued in the presence of Mg(II) excess, we
performed deadenylation assays in the presence of Mg(II)concen-
trations varying approximately 10-fold below and above the
concentration of the standard assay conditions, as described
previously [18].In the present study, we verified that inhibition of
PARN by either U1, FU1 or T1 was unaltered under all the Mg(II)
concentrations tested (data not shown), although Mg(II) ions are
important for enzymatic activity.

220
N. Balatsos et al. / Biochimie 94 (2012) 214e221
development that has not been extensively studied so far. We have
shown that fluoro-pyranosyl nucleosides with cytosine or modified
adenine as the base moiety behave as competitive inhibitors of PARN
and herein we report the mechanism of improved and drastic
inhibitors of the enzyme’s activity, based on both structural and
biochemical analyses. Preliminary deadenylation assays revealed
that the compounds U1, FU1 and T1 could efficiently inhibit PARN.
Kinetic analysis showed that these compounds behave as competi-
tive inhibitors, while the Ki values are significantly improved when
compared to our previous report (summarized in Table 1) [18]. The
preliminary analysis also showed that the inhibition of PARN by U1
was time-dependent. In-depth kinetic analysis revealed that U1
behaved as slow-binding competitive inhibitor. It should be noted
that a slow-binding inhibitor that conform to this mechanism (such
as U1) display a potential clinical advantage. In contrast to classic
inhibitors, its effectiveness is not abolished by increased substrate
concentration through feedback inhibition mechanism [33,34].
The higher potency of U1 can be also explained from the
molecular docking analysis, which outlines the accommodation of
U1 into the active site of PARN. It has been previously described that
Phe115 is a key residue in the 3D-structure of PARN [18,44]. The
side-chain of this residue may establish
p-stacking interactions
with any compound with conjugated enough base moieties, which
compete the hydrogen bonding interactions of the eOH groups on
the sugar moiety of the compounds with the Asp members of the
catalytic triad. Therefore it appears that the stronger the interaction
with Phe115, the weaker the interactions of the compound with the
catalytic triad. U1 forms hydrogen bonds with most of the Asp
residues of the active site than any other compound in Table 1,
while at the same time does not form and is not able to form any
stacking with Phe115 (Fig. 4).
p-
Taken together, our kinetic and molecular docking data suggest
that the uracil-based U1 compound is a novel efficient slow-
binding inhibitor of PARN. This feature of U1 allows a stable and
prolonged interaction with the active site of PARN [31,38]. Such
molecules could represent promising leading compounds for the
development of novel, effective and specific anti-cancer agents,
both in vitro and in vivo.
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