Selective Targeting of the Zinc Finger Domain of HIV Nucleocapsid Protein NCp7 with Ruthenium Complexes

Article abstract of DOI:10.1002/chem.201803917

Nucleocapsid protein 7 (NCp7) is an attractive target for anti-HIV drug development. Here we found that ruthenium complexes are reactive to NCp7 and various Ru-agents exhibit significantly different reactivity. Interestingly, the zinc-finger domains of NCp7 also demonstrate different affinity to Ru-complexes; the C-terminal domain is much more reactive than the N-terminal domain. Each zinc-finger domain of NCp7 binds up to three Ru-motifs, and the ruthenium binding causes zinc-ejection from NCp7 and disrupts the protein folding. Therefore, ruthenium complexes interfere with the DNA binding of NCp7 and interrupt the protein function. The different reactivity of Ru-agents suggests a feasible strategy for improving the targeting of NCp7 by ligand design. This work provides an insight into the mechanism of ruthenium complex with NCp7, and suggests more potential application of ruthenium drugs.

Full text of DOI:10.1002/chem.201803917

A Journal of
Accepted Article
Title: Selective Targeting of the Zinc Finger Domain of HIV
Nucleocapsid Protein NCp7 with Ruthenium Complexes
Authors: Yaping Sheng, Kaiming Cao, Ji Li, Zhuanghao Hou, Siming
Yuan, Guangming Huang, Hongke Liu, and Yangzhong Liu
This manuscript has been accepted after peer review and appears as an
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of the final Version of Record (VoR). This work is currently citable by
using the Digital Object Identifier (DOI) given below. The VoR will be
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content of this Accepted Article.
To be cited as: Chem. Eur. J. 10.1002/chem.201803917
Link to VoR: http://dx.doi.org/10.1002/chem.201803917
Supported by

Chemistry - A European Journal
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COMMUNICATION
Selective Targeting of the Zinc Finger Domain of HIV
Nucleocapsid Protein NCp7 with Ruthenium Complexes
Yaping Sheng, Kaiming Cao, Ji Li, Zhuanghao Hou, Siming Yuan, Guangming Huang, Hongke Liu,*
Yangzhong Liu *
Abstract: Nucleocapsid protein 7 (NCp7) is an attractive target for
anti-HIV drug development. Here we found that ruthenium
complexes are reactive to NCp7 and various Ru-agents exhibit
significantly different reactivity. Interestingly, the zinc-finger domains
of NCp7 also demonstrate different affinity to Ru-complexes; the C-
terminal domain is much more reactive than the N-terminal domain.
Each zinc-finger domain of NCp7 binds upto three Ru-motifs, and
the ruthenium binding causes zinc-ejection from NCp7 and disrupts
the protein folding. Therefore, ruthenium complexes interfere with
the DNA binding of NCp7 and interrupt the protein function. The
different reactivity of Ru-agents suggests a feasible strategy for
improving the targeting of NCp7 by ligand design. This work
provides an insight into the mechanism of ruthenium complex with
NCp7, and suggests more potential application of ruthenium drugs.
nucleic acids, such as RNA SL2. In addition to the platinum and
gold agents, ruthenium complexes exhibit promising therapeutic
[
8]
activity in drug development, such as the anti-tumor potency.
Ru compounds have also shown high potency in anti-microbial
[
9]
and anti-HIV activities. Although Ru complexes are proposed
to bind to DNA in the action as antitumor agents, mechanism
study indicates that, in comparison to platinum complexes,
[
10]
ruthenium complexes more favor protein binding. Therefore, it
is interesting to explore whether Ru agents can react with NCp7,
and how ligands influence the reactivity of Ru compounds.
In this work, we investigated the reaction of NCp7 with
4
different Ru compounds. NAMI-A (ImH[trans-RuCl (DMSO)(Im)],
[
8c]
Im = imidazole), an anticancer drug in clinic trial, was chosen
in the reaction. Two arene Ru complexes (Ru-1 and Ru-2, see
Figure 1), which have shown high potency to tumor cells, were
[
8d]
Nucleocapsid protein 7 (NCp7) is a small zinc finger protein (55
also used. Two other di-nuclear Ru complexes (Ru-3 and Ru-
4) with the bridge of chloro or 1-(4-((1H-imidazol-1-
yl)methyl)benzyl)-1H-imidazole (p-bib) ligand were used for
[
1]
aa) that plays an important role in the HIV life cycle. NCp7
contains two highly conserved and mutationally intolerant CCHC
zinc finger structures, which is important for the protein function
[
8d, 9b, 9c, 11]
comparison.
The reaction of these ruthenium
[
1-2]
in the recognition of nucleic acid.
A number of studies have
compounds with NCp7 has been studied in detail, and the effect
of ligands on the reaction towards NCp7 has been discussed.
Furthermore, perturbation of the protein folding of NCp7 and its
recognition to nucleic acid have been investigated.
shown that the infectivity of HIV-1 is significantly suppressed
[
3]
upon the mutation of zinc binding residues of NCp7. The
inhibition of NCp7, typically through the zinc-ejection of zinc-
fingers, leads to the structure perturbation of NCp7 and inhibition
of infection of the virus. Therefore, NCp7 is a potential target for
[
3c, 4]
developing new antiviral agents.
Interference of the zinc
binding in NCp7 is a typical strategy for inhibitor design of this
small protein, for instance, by targeting cysteine residues at the
[
1, 3c, 4]
zinc binding site.
In this regard, metallodrugs could
demonstrate great advantages in drug design due to the
coordination properties.
A
[
5]
[5c, 6]
Several metal compounds, such as Pt(II), Au(III),
As(III)
[
7]
agents, have shown inhibition activity to NCp7. These metal
complexes bind to NCp7 through coordination substitution,
resulting in zinc-ejection from the protein by thiolate metalation.
Therefore, metallodrugs could disrupt the protein structure of
NCp7 and interfere with the recognition of NCp7 with target
B
C
[
a]
Y. Sheng, K. Cao, Z. Hou, S. Yuan, Prof. G. Huang, Prof. Y. Liu
CAS Key Laboratory of Soft Matter Chemistry, Department of
Chemistry, University of Science and Technology of China,
Hefei, Anhui 230026, China
E-mail: liuyz@ustc.edu.cn
[
b]
J. L, Prof. H Liu
Jiangsu Key Laboratory of Biofunctional Materials,
College of Chemistry and Materials Science, Nanjing Normal
University,
Nanjing, 210046, Jiang Su, P. R. China
E-mail: liuhongke@njnu.edu.cn
Figure 1. Scheme of ruthenium complexes and sequences of protein and
DNA. (A) Structure of five ruthenium complexes used in this work. (B)
Sequence and the two zinc-fingers of NCp7 (aa 12-55). The zinc binding
residues are highlighted in red. (C) The sequence of SL2 DNA that can be
recognized by NCp7.
Supporting information for this article is given via a link at the end of
the document.
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Chemistry - A European Journal
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COMMUNICATION
The reaction of five ruthenium complexes with NCp7 was
investigated by fluorescence spectroscopy. NCp7 contains a
tryptophan residue (Trp37); hence the protein exhibits intrinsic
order to verify this hypothesis, Ellman’s assay was conducted to
measure thiol content of NCp7 in the reaction with Ru
complexes. Ellman's reagent (5, 5'-dithiobis-(2-nitrobenzoic acid)
or DTNB) reacts with thiol groups to yield a yellow product TNB,
[
12]
fluorescence with excitation at 280 nm. The fluorescence of
NCp7 was quenched by the incubation with ruthenium
complexes, indicating their reaction with the protein (Figure 2A).
Notably, the five ruthenium complexes exhibited significantly
different efficiency in quenching the fluorescence of NCp7. While
Ru-3 completely quenched the fluorescence of NCp7, NAMI-A
reduced the fluorescence only to 42% (Figure 2A). The other
three complexes quenched the fluorescence of NCp7 to different
levels (Ru-1: 28%, Ru-2: 9%, and Ru-4: 60%). This result
indicates that the five ruthenium complexes possess different
reactivity towards NCp7, and Ru-3 exhibits the highest reactivity
among these compounds. To further confirm this conclusion, the
reaction was also conducted at different molar ratios of
[
14]
which has
Therefore, this absorption provides
a
characteristic UV absorption at 412 nm.
reliable method for
a
measuring free thiol of proteins. The reaction was performed on
NCp7 with different amount of Ru agents for 2 h. Then DTNB
was added and incubated for 10 min before UV measurements.
The result clearly showed that the absorbance decreased with
the increase of concentration of Ru-3, indicating the binding of
ruthenium to thiol of NCp7 (Figure 3A). In addition to the most
reactive complex Ru-3, the reactions of other compounds were
also analyzed (Figure 3B). With the same concentration, Ru-4 or
NAMI-A reduced the thiol content of NCp7 to a less degree than
Ru-3, indicating that Ru-4 and NAMI-A are less reactive to the
thiol groups of NCp7. No detectable reaction was observed on
the reaction of Ru-1 and Ru-2 under the same experimental
condition. The different reactivity of ruthenium compounds
measured by Ellman’s assay is consistent with the fluorescence
measurement.
[Ru]/[NCp7]. Data show that the fluorescence of NCp7 was
quenched by Ru-complexes in concentration-dependent
manner; nevertheless, the same order of the reactivity was
observed on these ruthenium complexes at all concentrations
a
(Figure 2B).
The reaction of NCp7 with ruthenium complexes was further
analyzed with fluorescence titration. NAMI-A, as the most
intensively investigated Ru agent, was firstly chosen for the
analysis. The titration of NAMI-A resulted in the gradual
fluorescence quench of NCp7; however, a large excessive
NAMI-A is needed to reach the complete reaction (Figure 2C).
In comparison, Ru-3 nearly completely quenched fluorescence
of NCp7 at the molar ratio of [Ru-3]/[NCp7] = 3 (Figure 2B).
These data confirmed that Ru-3 is highly reactive to NCp7,
whereas NAMI-A possesses only moderate reactivity to the
protein.
A 1.25
B
Ru-3
1
.2
Ru-1
Ru-2
NAMI-A
Ru-4
Ru-3
0
- 120 μM
1.00
0.75
0.50
0.25
0.00
1
.2
0.9
.6
0.9
0.6
0
0.3
0.0
0
.3
.0
0
2
4
6
8
10
Ru-3 / NCp7
0
3
75
450
525
600
0
3
6
9
Wavelength (nm)
Ru-agents / NCp7
Figure 3. Ellman’s assay of the thiol content of NCp7 in the reaction with
ruthenium complexes. (A) The reaction of NCp7 with Ru-3. NCp7 (15 µM) was
incubated with different concentration Ru-3 at 37°C for 2 h. UV spectra were
recorded after adding 150 M DTNB for 10 min. The inset shows the
absorption at 412 nm at various ratio of [Ru]:[NCp7]. (B) Reaction of NCp7
with different ruthenium complexes. The absorption at 412 nm measured at
different molar ratio of [Ru]:[NCp7].
It has been reported that the thiol group of cysteine residues,
including those at the zinc binding site, are the most reactive site
[
5, 7]
for metallodrugs, such as platinum and arsenic agents.
These thiol groups could also be the preferential binding site of
[
13]
ruthenium complexes based on the coordination chemistry. In
It has been reported that the binding of metallodrugs, such
A
6000
B
100
Control
Ru-2
Ru-1
NAMI-A
Ru-4
Ru-3
as Au(I) or Pt(II) agents, to the cysteine residues of zinc finger
5
4
3
2
1
000
000
000
000
000
0
8
6
0
0
[5c, 15]
protein can lead to zinc ejection from the protein.
To test
Ru-3
Ru-4
NAMI-A
Ru-1
Ru-2
whether the ruthenium binding can also disrupt the zinc binding
to NCp7, the zinc release assay was carried out using a Zn(II)
dye zincon (2-carboxy-2’-hydroxy-5’-sulfoformazylbenzene).
Zincon is a well-known colorimetric reagent for the detection of
zinc ions in aqueous solution. The formation of Zn-zincon
40
2
0
0
3
00
350
400
450
500
0
3
6
9
12 15 18
Wavelength (nm)
Ru-agents / NCp7
C
D 3750
[16]
3
3
2
1
750
000
250
500
complex exhibits a characteristic UV absorption at 620 nm.
0
0
NAMI-A
Ru-3
3
2
000
250
The assay was performed on NCp7 in the reaction with different
amount of Ru-3. After 2 h reaction, zincon was added and the
absorption was measured. The absorption at 620 nm clearly
showed the reaction of Ru-3 with NCp7 led to zinc release from
the protein in a concentration dependent manner (Figure 4A).
The control experiment indicates that zincon alone did not react
with NCp7, showing the strong binding of Zn(II) ions to the
protein. This result confirmed that the binding of Ru to the
cysteine residue of NCp7 results in zinc ejection from the protein.
Furthermore, the zinc release assay also showed that Ru-4 and
NAMI-A disrupted the zinc coordination to NCp7 less efficiently
than Ru-3, and nearly no reaction occurred on Ru-1 and Ru-2
9
0
1
200
1500
(μM)
(μM)
7
50
0
7
50
0
3
00
350
400
450
500
3
00
350
400
450
500
Wavelength (nm)
Wavelength (nm)
Figure 2. Fluorescence of NCp7 in the reaction with ruthenium complexes. (A)
The fluorescence spectra of NCp7 after the reaction with different ruthenium
complexes. Spectra were recorded on 50 µM NCp7 after the incubation with
ruthenium complexes (300 µM) at 37C for 2 h. (B) Fluorescence of NCp7
quenched by ruthenium complexes at different ratios of [Ru]:[NCp7].
Reactions were performed on 50 µM NCp7 for 2 h at 37C. F represents the
fluorescence intensity at 356 nm during the Ru titration, and F
fluorescence intensity. (C-D) Fluorescence titration of NAMI-A (C) and Ru-3
D). Spectra were recorded on 30 µM NCp7 after incubation with different
concentration of ruthenium complexes for 2 h at 37C.
0
is the initial
(
Figure 4B). Taken together, it can be concluded that the
reactivity of NCp7 with the five Ru-agents is in the order of Ru-3
> Ru-4 > NAMI-A > Ru-1 ~ Ru-2.
(
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Chemistry - A European Journal
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COMMUNICATION
This observation is consistent with recent report that two zinc-
fingers of NCp7 possess different dynamic properties; in which
the N-Zn knuckle is conformationally more stable, whereas the
coordination exchange of Cys36 makes the C-terminal zinc-
A 1.2
B
1.0
0.8
0.6
0.4
0.2
0.0
Ru-3
Ru-3
Ru-4
NAMI-A
Ru-1
Ru-2
0
.6
0.4
.2
0.0
0.8
0.4
0.0
[17-18]
0
2
4
6
8
finger more reactive.
Ru-3 / NCp7
0
The composition of ruthenium binding adducts of NCp7 was
characterized by electrospray ionization mass spectra (ESI-MS).
The reaction of Ru-3 showed that the bi-ruthenium binding
adduct was formed quickly during the reaction (3 min) (Figure 6).
The composition of this adduct, [NCp7 - 1Zn + 2(Ru-cym)]
600
700
800
900
0
1
2
3
4
Wavelength (nm)
Ru-agents / NCp-7
Figure 4. Zinc release assay on the reaction of NCp7 with ruthenium
complexes measured by UV spectroscopy. (A) Zinc release from NCp7 upon
the reaction of Ru-3. The inset shows the absorption at 620 nm at various ratio
of [Ru]:[NCp7]. (B) The comparison of zinc-release in the reaction of five Ru-
agents. The zinc release assay was measured on 21 μM NCp7 with the
reaction of ruthenium complexes at 37°C for 2 h. UV absorption at 620 nm
was measured in the presence of 400 μM Zincon.
6
(cym=η -p-cymene) indicates the binding of two Ru-cym to
NCp7. The presence of one zinc ion in the protein suggests that
a zinc-finger domain remains intact while the binding of two
ruthenium atoms. This observation is consistent with the NMR
result that the C-terminal zinc-finger domain is more reactive
than the N-terminal domain. After 30 min reaction, NCp7 nearly
completely reacted and the signal of bi-ruthenium adduct
became dominant in the spectra. In addition, a minor tri-Ru
adduct [NCp7 - 1Zn + 3(Ru-cym)] appeared on the spectra.
Another minor peak at m/z 1131.67 is corresponding to the
binding of one Ru-cym moiety to holo-NCp7, which could be
non-specific binding adduct, or the intermediate state of Ru
binding before zinc ejection.
Zinc coordination plays a crucial role in maintaining the
secondary structure of zinc-finger proteins; therefore, it is
expected that the zinc ejection induced by Ru reaction can
disrupt the structure of NCp7. The secondary structure of NCp7
was investigated by CD spectra (Figure 5A). In accordance with
the literature, the CD spectrum of NCp7 exhibits a positive
[
5a]
ellipticity at ~215 nm and a negative ellipticity at ~ 201 nm.
More Ru binding to the protein was observed after 12 h
reaction. Two major adducts, [NCp7 - 2Zn + 4(Ru-cym)] and
EDTA removed the Zn(II) from NCp7 and disrupted the protein
structure, switching the signal at 215 nm from positive to
[NCp7 - 2Zn + 5(Ru-cym)] indicated the binding of four or five
[
5a]
negative.
Incubation of NCp7 with Ru agents result in the
Ru-motifs to the protein. The composition of these adducts
indicate that the release of each zinc-finger tends to bind two or
three ruthenium motifs. A minor peak with 6-Ru binding was also
detected. Both zinc ions were released in these tetra- to hexa-
Ru binding adducts formation. These data indicate that each
zinc-finger domain of NCp7 protein can bind upto three
ruthenium atoms, and the cym ligand remains coordination with
Ru in all adducts. On the other hand, Ru-4 showed much less
reactive to NCp7; the signal of unreacted NCp7 is still very
intense after incubation 6-fold Ru-4 for 12 h, and only di-Ru
adduct [NCp7 - 1Zn + 2(Ru-cym)] was detected (Figure S3).
decrease of the positive peak at 215 nm and the negative peak
at 201 nm, indicating that Ru-agent can disrupt the protein
folding of NCp7. Consistent with the reactivity assays, Ru-3
induced more structure perturbation than NAMI-A.
1
15
2
D H- N HSQC NMR spectra of NCp7 were recorded to
further analyze the reaction of Ru-3 (Figure 5B). The well-
dispersed signal of NCp7 is consistent with literature report and
[
17]
indicates the well-folded protein. After incubation with Ru-3 for
h, a large number of signals disappeared and a series of
1
1
peaks appeared at 7.9~8.4 ppm on the H dimension, which is
[
7]
consistent with the protein unfolding. This result further
supports the conclusion that Ru binding disrupts the secondary
structure of NCp7. Interestingly, the signals of NCp7 that shifted
after Ru-3 reaction are almost all from C-terminal residues
-
2Zn + 4(Ru-cym) - 2Zn + 5(Ru-cym)
1246.11
1
1
1
00
1292.90
-
2Zn + 3(Ru-cym)
1199.50
75
50
25
0
12 h
- 2Zn + 6(Ru-cym)
(Arg29 and after), whereas those from N-terminal were very little
1339.30
perturbed (Figure S2). This result indicates that the C-terminal
zinc-finger domain is more reactive than the N-terminal domain.
00
-
Zn + (Ru-cym)
1118.88
75
50
25
0
-
Zn + 3(Ru-cym)
212.28
+
(Ru-cym)
1
30 min
1131.67
A
B
2
0
0
G35
H44
G40
Q45 C39
NCp7
1084.86
00
- Zn + 2(Ru-cym)
1165.48
K47
E42
T50
C36
7
5
-20
-40
-60
3
min
NCp7
K38
50
25
M46
E51
K33
+
+
+
NAMI-A
Ru-3
EDTA
R29
W37
0
1050
1100
1150
1200
1250
1300
1350
2
00
220
240
m/z
Wavelength (nm)
Figure 6. ESI-MS analysis of NCp7 in the reaction with Ru-3. The selected
region shows +5 charged signals of the spectra. The reaction was performed
on 10 μM NCp7 with 30 μM Ru-3 at 37°C in 100 mM ammonium acetate
buffer (pH 7.0). The spectra were recorded after incubation for 3 min, 30 min
or 12 h. The signals with Ru binding and zinc release are annotated in the
spectra. The peak labeled with NCp7 represents the two zinc ions bound
protein. Detailed peak assignments are listed in Table S1.
Figure 5. Characterization of protein folding of NCp7 upon Ru binding. (A) CD
spectra of NCp7 after incubation with ruthenium complexes in the molar ratio
of [Ru]:[NCp7] = 3:1. All reactions were performed on 50 µM NCp7 for 2 h at
1
15
37°C. (B) H, N-HSQC of NCp7 before (red) and after (green) Ru-3 reaction.
The reaction was conducted on 350 µM NCp7 with 700 µM Ru-3 at 25°C in 50
mM phosphate buffer (pH 7.0) for 1 h, the cross-peaks of the changed
residues are labeled in black.
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Chemistry - A European Journal
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COMMUNICATION
[
25]
NCp7 is a nucleoside chaperone that plays an important role
in the process of HIV viral replication by binding to RNA, which
cysteine residues and imidazole at histidine residues.
As to
this aspect, the zinc binding residues of zinc-finger proteins
could be potential binding target of Ru(II) complexes, while
cysteine demonstrates higher reactivity than histidine towards
In addition to the anti-cancer activity, Ru
compounds can inhibit HIV virus replication, and nucleic acids of
[
19]
through its highly conserved zinc-knuckle structure.
Given the
2+
fact that Ru-3 binding results in Zn ions ejection from NCp7
and disrupts the protein folding, it is interesting to know whether
Ru complex can interfere with the interaction of NCp7 with
nucleic acids. A 23-base single strand DNA (SL2, see Figure
[
23b]
Ru compounds.
[
9b]
the virus were also proposed to be drug target of Ru-agents.
1C), which is an analogue of RNA SL2 of the NCp7 binding
Since nucleocapsid proteins are important drug targets of HIV
sequence, was often used in the investigation of the interaction
virus, several metal complexes have been evaluated on the
[
5a, 19-20]
[5a, 9b]
of NCp7 protein.
Fluorescence polarization (FP) assay in
protein reaction and anti-virus activity.
Results in this work
this work confirmed the recognition of NCp7 to SL2 DNA (Figure
S4). Hence, SL2 sequence was employed for studying the
interaction with NCp7.
showed that ruthenium complexes exhibit significantly different
reactivity to the protein. Interestingly, the in vitro NMR titration
indicates that compound Ru-3 disrupts the folding of NCp7
much more effective than mercaptobenzamide-1,
a well-
The effect of ruthenium binding on the interaction of NCp7
with SL2 DNA was analyzed using EMSA. The result shows that
the presence of NCp7 resulted in a new band of DNA with lower
mobility, indicating the formation of the NCp7/SL2 complex
investigated zinc ejector with high activity of anti-HIV virus by
[15]
targeting NCp7.
The different reactivity indicates that ligands can largely
influence the reaction of Ru complexes. The two complexes, Ru-
1 and Ru-2 are nearly inert to NCp7, suggesting that the
chelation of ethylenediamine (en) significantly impedes the
binding of Ru complexes to the protein. It is well-known that
(Figure 7A). The reaction of NCp7 with Ru-3 clearly inhibited the
recognition of protein to SL2 DNA. The EMSA assay showed
that Ru-3 disrupted the formation of NCp7/SL2 complex in a
concentration dependent manner (Figure 7B). As Ru-DNA
adduct was detected in the ESI-MS measurement (Figure S5),
we further analyzed the effect of Ru-reactions of NCp7 and DNA
on the disruption of DNA/protein complex. EMSA results showed
that the Ru-binding to either NCp7 or DNA can interfere with the
formation of DNA/protein complex, while the NCp7 reaction
exhibited higher effect (Figure S6). The EMSA assay was also
performed using RNA; the result indicates that Ru-3 can also
disrupt the complexes of RNA/NCp7 (Figure S7). As RNA is
6
6
arene coordination (such as η -biphenyl and η -p-cymene in this
work) is very stable to Ru(II), and the chelation of
ethylenediamine cannot be substituted in the reaction of GSH or
[
26]
DNA. Therefore, both Ru-1 and Ru-2 act as monofunctional
Ru(II) complexes, which is probably the reason of low reactivity
to NCp7. By comparison, Ru-3 and Ru-4, which do not contain a
chelation ligand, are more reactive than Ru-1 and Ru-2. It can
be envisioned that the ligands of chloro and p-bib can be
substituted in the reaction of NCp7, so that Ru-3 and Ru-4 can
act as a bi- or tri-functional complexes. This assumption was
confirmed by ESI-MS analysis that only [Ru-cym] motif binds to
the NCp7 in the adducts (Figure S3). The higher reactivity of Ru-
3 than Ru-4 is probably because of different substitution rates of
two compounds. The p-bib ligand is certainly kinetically more
stable than chloro ligand, which results in the more reactive Ru-
3 than Ru-4. In addition, the hydrolysis rate of Ru-3 (t1/2 = 0.4 h)
is much higher than that of Ru-4 (t1/2 = 2.1 h) (Figure S9), which
[
21]
also a proposed target of metal complexes for anti-virus, the
reaction in this work suggest that the Ru compound could be a
dual-targeting agents. In addition, the effect of NAMI-A on the
formation of NCp7/SL2 was also measured. The result showed
that both Ru-3 and NAMI-A are able to disrupt the interaction of
NCp7 with SL2, while Ru-3 exhibited higher inhibition efficiency
since the band of NCp7/SL2 on the gel was reduced more by
Ru-3 than by NAMI-A at the same concentration (Figure S8).
[
22, 27]
could also contribute to their different reactivity.
Different
A
NCp7
B
Ru-3
from the other four Ru(II) compounds, NAMI-A is a Ru(III)
complex, so that it demonstrated moderate reactivity to NCp7
although it does not contain chelation ligand.
Complex
DNA
The different reactivity of Ru-agents to NCp7 suggests that
the more efficient inhibition could be achieved by modulation of
the ligand of Ru complexes. It has been reported that Ru
Lane
1
2
3
4
5
6
7
8
9
10
1
2
3
4
5
6
7
8 9
[8d]
compounds exhibited different reactivity to DNA or proteins;
particularly, the selectivity of Ru-agents varies dependent upon
Figure 7. Effect of Ru-3 on the interaction of NCp7 with DNA. (A) Interaction
of NCp7 with DNA. DNA was incubated with different concentrations of NCp7
for 30 min. Lane 1: DNA only; Lane 10, NCp7 only; Lane 2 - 9: DNA (5 μΜ) +
NCp7 (5, 10, 15, 20, 30, 40, 60 or 80 μΜ). (B) Incubation of the complex of
DNA (5 μΜ) and NCp7 (60 μΜ) with different amount of Ru-3. Lane 1: DNA
only; Lane 2 - 9: NCp7/DNA complex with Ru-3 (0, 0.72, 1.08, 1.44, 1.8, 2.16,
[
23b]
the target of biomolecules.
The different types of zinc finger
proteins, such as CCHH, CCHC, and CCCC domains, are
[7, 28]
known to have different reactivity to metallodrugs.
In addition,
the protein sequence and residues around zinc center also
influence the reactivity of the protein. For instance, the -
stacking between aromatic residues of proteins and ligand of
2
.52 or 2.88 mM).
[
5]
metal complexes can enhance their reactions. The reaction of
zinc-finger domains can further be modulated by residues
adjacent to the zinc binding site through H-bonding or charge
Ruthenium complexes have demonstrated promising
therapeutic activity in chemotherapy.
that, similar to platinum drugs, ruthenium agents induce cell
apoptosis also by targeting DNA.
of researches indicate that proteins could play more important
roles in the mechanism of Ru agents than that of Pt-drugs.
This hypothesis is reasonable according to the coordination
chemistry since Ru(II) highly favors the binding of thiol at
[
8d, 22]
It has been proposed
[
29]
transfer. These factors distinguish the reactivity of zinc-finger
proteins to various metallodrugs. Therefore, it is feasible to
develop more specific Ru inhibitors with high selectivity for NCp7,
particularly, by using ligands with incorporation of molecules that
[
8d, 22-23]
Nevertheless, a number
[
8d, 10a,
24]
[3c, 30]
can recognize NCp7 (such as SAMTs).
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Chemistry - A European Journal
10.1002/chem.201803917
COMMUNICATION
In summary, the reaction of NCp7 with ruthenium complexes
has been investigated in this work. The fluorescence
measurement indicates that ruthenium complexes exhibit
significantly different reactivity to the protein. Ruthenium
complexes are able to bind to the cysteine residue at zinc
coordination site and lead to the zinc-ejection from NCp7. All
these assays indicate that the reactivity of these Ru complexes
is in the order of Ru-3 > Ru-4 > NAMI-A > Ru-1 ~ Ru-2. ESI-MS
measurement on the reaction of Ru-3 showed that each zinc-
finger domain of NCp7 can bind upto three ruthenium motifs, in
which the arene ligand remaining coordination. Interestingly,
NMR measurements indicate that the C-terminal zinc-finger
domain of NCp7 is more reactive than the N-terminal domain.
Ru binding disrupts the protein folding and interferes with the
function of NCp7 in the DNA recognition. The different reactivity
of ruthenium complex suggests that targeting NCp7 could be
modulated by the modification of ligands in ruthenium
complexes. This work discovered a new type of inhibitors of
NCp7, and the results provide an insight into the reaction of
ruthenium complex with NCp7.
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This work was supported by the National Key R&D Program of
China (2017YFA0505400) and the National Science Foundation
of China (21573213, 21877103, 21420102002, 21771109), the
Major Program of Development Foundation of Hefei Center for
Physical Science and Technology (2018ZYFX004), and Suzhou
Science and Technology Projects (SYG201624), Jiangsu
Natural Science Foundation (BK20151238). A portion of this
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Collaborative Innovation Center of Suzhou Nano Science and
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This article is protected by copyright. All rights reserved.

Chemistry - A European Journal
10.1002/chem.201803917
COMMUNICATION
Entry for the Table of Contents
Layout 1:
COMMUNICATION
Ruthenium complexes are reactive to
NCp7 and exhibit significantly different
reactivity. Ruthenium binding results
in zinc-ejection from NCp7 and
disrupts recognition of the protein to
nucleic acids. In addition, the C-
terminal domain of NCp7 is much
more reactive than the N-terminal
domain to ruthenium complexes. This
work found a new type of inhibitors of
NCp7. The different reactivity of
ruthenium complex suggests that
targeting NCp7 could be modulated by
ligand modification.
Yaping Sheng, Kaiming Cao, Ji Li,
Zhuanghao Hou, Siming Yuan,
Guangming Huang, Hongke Liu,*
Yangzhong Liu *
Page No. – Page No.
Selective Targeting of the Zinc Finger
Domain of HIV Nucleocapsid Protein
NCp7 with Ruthenium Complexes
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