Design, synthesis and biological evaluation of indole derivatives as Vif inhibitors

Article abstract of DOI:10.1016/j.bmcl.2017.07.026

The crystal structure of viral infectivity factor (Vif) was reported recently, which makes it possible to design new inhibitors against Vif by structure-based drug design. Through analysis of the protein surface of Vif, the C2 pocket located in the N-terminal was found, which is suit for developing small molecular inhibitors. Then, in our article, fragment-based virtual screening (FBVS) was conducted and a series of fragments was obtained, among which, Zif-1 bearing indole scaffold and pyridine ring can form H-bonds with Tyr148 and Ile155. Subsequently, 19 derivatives of Zif-1 were synthesized. Through the immune-fluorescence staining and Western blot assays, Zif-15 shows potent activity in inhibiting Vif-mediated A3G degradation. Further docking experiment shows that Zif-15 form H-bond interactions with residues His139, Tyr148 and Ile155. Therefore, Zif-15 is a promising lead compound against Vif that can be used to treat AIDS.

Full text of DOI:10.1016/j.bmcl.2017.07.026

Accepted Manuscript
Design, Synthesis and biological evaluation of indole derivatives as Vif inhib-
itors
Chunlan Pu, Rong-Hua Luo, Mengqi Zhang, Xueyan Hou, Guoyi Yan, Jiang
Luo, Yong-Tang Zheng, Rui Li
PII:
S0960-894X(17)30723-0
DOI:
http://dx.doi.org/10.1016/j.bmcl.2017.07.026
BMCL 25139
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
21 April 2017
12 June 2017
8 July 2017
Please cite this article as: Pu, C., Luo, R-H., Zhang, M., Hou, X., Yan, G., Luo, J., Zheng, Y-T., Li, R., Design,
Synthesis and biological evaluation of indole derivatives as Vif inhibitors, Bioorganic & Medicinal Chemistry
Letters (2017), doi: http://dx.doi.org/10.1016/j.bmcl.2017.07.026
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Design, Synthesis and biological evaluation of indole derivatives
as Vif inhibitors
Chunlan Pu a, 1, Rong-Hua Luob, 1, Mengqi Zhanga, 1, Xueyan Hou a, Guoyi Yan a, Jiang Luoa, Yong-Tang Zheng
b,*, Rui Li a,*
a
Cancer Center, West China Hospital, Sichuan University, and Collaborative Innovation Center for Biotherapy,
Chengdu, 610041 Sichuan, P. R. China. E. mail: lirui@scu.edu.cn
b Key Laboratory of Bioactive Peptides of Yunnan Province/Key Laboratory of Animal Models and Human Disease
Mechanisms of the Chinese Academy of Sciences, Kunming Institute of Zoology, Chinese Academy of Sciences,
Kunming, Yunnan 650223, China. E. mail: zhengyt@mail.kiz.ac.cn
1 These authors contributed equally to this work.
* Corresponding author.
Abstract: The crystal structure of viral infectivity factor (Vif) was reported recently, which makes
it possible to design new inhibitors against Vif by structure-based drug design. Through analysis
of the protein surface of Vif, the C2 pocket located in the N-terminal was found, which is suit for
developing small molecular inhibitors. Then, in our article, fragment-based virtual screening
(FBVS) was conducted and a series of fragments was obtained, among which, Zif-1 bearing
indole scaffold and pyridine ring can form H-bonds with Tyr148 and Ile155. Subsequently, 19
derivatives of Zif-1 were synthesized. Through the immune-fluorescence staining and western blot
assays, Zif-15 shows potent activity in inhibiting Vif-mediated A3G degradation. Further docking
experiment shows that Zif-15 form H-bond interactions with residues His139, Tyr148 and Ile155.
Therefore, Zif-15 is a promising lead compound against Vif that can be used to treat AIDS.
Keywords: APOBEC3G degradation; Vif inhibitor; FBVS; indole derivatives
Today, AIDS is one of the biggest public health problems in the world. In clinical, the
existing 28 anti-human immunodeficiency virus (HIV) drugs can effectively delay AIDS
progression, but cannot cure this disease and need lifelong medication.1,2 Scientists are attempting
to find a safe and effective HIV-1 vaccine that can prevent HIV-1 infection, but the result is
disappointing.3,4 Nowadays, considerable progress has been made in treating HIV-infected patients
by using highly active antiretroviral therapy (HAART).5 However, with the increasing incidence
of drug resistant viruses and drug toxicity, it is urgent to find more anti-HIV drugs with new
.

mechanisms.
Apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like 3G (APOBEC3G, A3G)
is a cytidine deaminase.6 As one of the A3G family members, A3G is a major cellular host defense
factor. It was believed that A3G is able to protect the host cell from retroviruses and severely
weaken the infectivity of HIV-1.7-9 The antiviral effect of A3G is not only through restricting DNA
replication, but also via the deaminase-independent mechanisms.10 However, A3G can be
degradated by Vif through ubiquitin-mediated pathway.11-15 Therefore, inhibition of Vif is an
appealing way to treat HIV-1.16,17 Several small molecule inhibitors of Vif inhibitor (RN-18,
MM-1, IMB-26 and benzimidazoles) were reported (Fig. 1).18-22 In this study, we designed and
synthesized a series of indole derivatives and found Zif-15 as an effective Vif inhibitors, which
may targeting the Vif–ElonginC interaction (Fig. 2).
Fig. 1. Structures of HIV-1 Vif inhibitors.
Fig. 2. Schematic illustration of the mechanism of Vif inhibitor Zif-15. Zif-15 binding to the C2
pocket of Vif, which might block the interaction of Vif with ElonginC (EloC), thereby inhibiting the
formation of the E3 complex and thus impeding the degradation of A3G.
23
The crystal structure of viral infectivity factor (Vif) was reported recently , which makes it
.

possible to design new inhibitors against Vif by structure-based drug design. Firstly, to find the
appropriate binding pocket, the crystal structure of Vif complex (PDB ID: 4N9F) was obtained
from the Protein Data Bank (Fig. 3A). By using Discovery Studio 3.1 packages, five possible
binding sites (C1~C5) were identified. As can be seen in Fig. 3B, C3~C5 are smaller in space that
cannot accommodate small molecules (Table 1). Although the size of C1 was appropriate, ligands
binding to this site may not be helpful in impeding the formation of E3 complex. C2 pocket,
located in the N-terminal of Vif, is in vicinity of the ElonginC, hence, ligands binding to this
pocket may inhibit the interaction of Vif with ElonginC, thus leading to the dissociation of E3
complex and prevention of A3G from degradation. Therefore, C2 pocket was used for the further
FBVS campaign.
Fig. 3. A. Crystal structure of the Vif-CBFbeta-Cullin5-EloB-EloC pentameric complex (the
yellow transparent sphere represents the location of C2 pocket of Vif). B. Possible binding sites of
Vif protein C1~C5.
Table 1. The location, size and function of the five binding sites.
binding site
C1
X
Y
Z
volume
57.50
43.25
27.13
14.50
13.75
color
red
function
70.989
72.881
67.09
-87.494
-69.58
-180.097
-181.951
˗
C2
C3
C4
C5
blue
interact with ElonginC
-100.832 -181.877
yellow
green
gray
˗
55.443
72.837
-97.504
-100.88
-187.186
-188.206
˗
interact with CBF-β
Based on the virtual screening of the ZINC fragment-based database, a series of fragments
that can bind to the C2 pocket of Vif were discovered (Fig. 4A), and the structures of these
fragments were provided in supporting information. Most of the fragments show attractive binding
modes, among which Zif-1 bearing the indole and pyridine ring can form hydrogen bonds with
Tyr148 and Ile155 (Fig. 4B). Therefore, 19 novel derivatives of Zif-1 were designed and
.

synthesized.
Fig. 4. A. Overlapping image of the molecules obtained from the fragment-based virtual screening
against Vif C2 pocket. B. Binding mode of Zif-1 in C2 pocket of Vif.
The general preparation method of 5-substituted indole derivatives was outlined in Scheme 1.
Briefly, 5-bromoindole was reacted with bis(pinacolato)diboron at 100 °C to prepare compound a.
Compound
b
was obtained through the acylation of 6-bromonicotinic acid with
1,1'-carbonyldiimidazole (CDI) in THF.24 The target compounds were generated by Suzuki
cross-coupling reactions25-27 of compound a with bromine substituted aromatic compounds. The
structures of the all synthesized compounds were summarized in Table 2. The detailed reaction
conditions, processing procedures, MS, NMR and yield data are provided in supporting
information.
Scheme 1: Reagents and conditions: (i) PdCl2(dppf)CH2Cl2, K2CO3, 1,4-dioxane, ~60 °C, 4~5 h,
50~70%. (ii) CDI, THF, NH4OH, rt, > 85%. (A represents phenyl or pyridyl moiety)
Table 2. The structures, biological activities and yields of the target compounds 1~20.
.

Compound
A#
R
H
MFI
˗
A3G restoring level
lowest
medium
low
Yields (%)
63
pyridin-2-yl
pyridin-2-yl
pyridin-2-yl
pyridin-3-yl
pyridin-3-yl
pyridin-2-yl
pyridin-2-yl
C6H5
1
3-NH2
++
+
64
2
2-NH2
52
3
2-NH2
+
low
54
4
6-NH2
++
+
medium
low
60
5
2-CHO
3-CHO
4-CHO
2-CHO
3-CHO
2-CN
63
6
˗
lowest
lowest
low
59
7
˗
57
8
C6H5
+
76
9
C6H5
+
low
56
10
11
12
13
14
15
16
17
18
19
20
C6H5
˗
lowest
medium
low
55
C6H5
4-SO2CH3
6-OCH3
3-CONH2
3-COOH
4-COOH
4-CH2COOH
4-COOH
3-CONH2
4-CONH2
++
+
61
pyridin-3-yl
pyridin-2-yl
pyridin-2-yl
C6H5
72
++
+++
++
˗
medium
high
24
42
medium
lowest
medium
low
32
C6H5
59
pyridin-2-yl
pyridin-2-yl
pyridin-3-yl
++
+
46
35
++
medium
42
# A represents benzene ring or pyridine ring.
To determinate their biological activities, the Tet-On system is used in regulating the
expression of Vif in TREX-hvif-15 cell line.28 The intensity of fluorescence was shown in Fig. 5A.
Comparing with A3G+, Vif－ group, the fluorescence of A3G+, Vif+ was dramatically decreased
(Fig. 5B). Next, the 20 compounds were added to the A3G+, Vif+ system and the results show that
the fluorescence intensity of Zif-15 group is the strongest, similar to that of A3G+, Vif－ group.
Compounds Zif-2, 5, 12, 14, 16, 18 and 20 have the moderate fluorescence, while compounds
Zif-3, 4, 6, 9, 10, 13 and 19 had relatively weak fluorescence. Besides, there is almost no
fluorescence observed in compounds Zif-1, 7, 8, 11 and 17.
.

Fig. 5. The mean fluorescent intensity (MFI) analysis of the effect of compound 1~20 on the
.

intensity of A3G. # The fold change of A3G MFI. The intensity was described as following: ˗, no or
*
less than 1 fold change; +, 1~2 fold change; ++, 2~3 fold change; +++, >3 fold change. The
concentration of compounds was 50 µM. A3G+: TREX-hvif-15 cells were transfected with
EYFP-N1-hA3G. Vif +: the protein of Vif was induced by 1 μg/mL Dox.
Next, to detect the variation of the amount of the Vif and A3G protein when treated by the 20
compounds, western blot experiments were done. The results are illustrated in Fig. 6. Zif-15
restored the high level of expression of A3G. Compounds Zif-2, 5, 12, 14, 16 and 18 showed the
medium restoration level of expression of A3G, while other compounds Zif-3, 4, 6, 9, 10, 13 and
19 had a low A3G levels. Zif-1, 7, 8, 11 and 17 show no activity in restoring the A3G levels. The
fluorescence intensity and the restoring level of A3G are shown in Table 2.
Fig. 6. Effects of Compounds 1~20 on the expression of A3G and Vif. TREX-hvif-15 cells were
transfected with EYFP-N1-hA3G, and Vif was induced by 0.1 μg/mL Dox. The cells were treated
with or without compounds 1~20 in concentration of 20 μM, respectively.
According to the structures and biological activities of compounds 1~20 (Table 2), it can be
found that when the aromatic rings "A" is the pyridine-2-yl ring without any substituent group,
Zif-1 shows no activity. According to the interaction mode of Zif-1 with C2 pocket of Vif (Fig.
4B), the substituent group with hydrogen donor might form the H-bond interaction with residue
His139, thus increase the binding affinity. Hence, an amino group was introduced to C-3′ position
of the pyridin-2-yl (Zif-2) and it increased the activity. When the amino group was introduced to
the C-2′ position, the activity was dropped, which can be ascribed to the increased distance
between amino group and His139. Next, changing the position of pyridine N atom to pyridin-3-yl
(Zif-4) leads to the reduced activity, while further changing the amino position to para-position of
the indole ring (Zif-5) leads to the partial restoration of the activity, which can be attributed to
formation of H-bond with His139. According to the Zif-1 binding mode, introduction of
substituent group with hydrogen acceptor would not be helpful in binding. To validate our
.

assumption, compounds substituted with aldehyde group (Zif-6~Zif-10), cyano group (Zif-11) and
methoxy group (Zif-13) were synthesized, and they all show low to no activity, independent of the
location of pyridine N atom and the substituent group. It should be noted that Zif-12, with
methylsulfonyl substituted at the C-4′ position still retain some activity. Finally, compounds
substituted by the groups with both hydrogen donor and acceptor, like acylamino group and
carboxyl group, were synthesized (Zif-14~16, Zif-18, Zif-20) and their activity are all good,
especially Zif-15 shows the best activity. The binding mode of Zif-15 with C2 pocket of Vif was
provided in Fig.7. Specifically, the N atom of the pyridine and the H atom of -NH- on the indole
ring of Zif-15 form H-bonds with Tyr148 and Ile155, respectively. Apart from that, the H atom of
the carboxyl group forms H-bond with His139. Besides, the indole core forms the hydrophobic
interaction with Pro157. Due to loss of the H-bond of pyridine with Tyr148, the activity of Zif-16
was decreased. When a methylene group was inserted to the carboxyl group, Zif-17 shows almost
no activity, which may be ascribed to the steric hindrance. As for the acylamino group,
pyridine-2-yl (Zif-14, 20) is superior to pyridine-3-yl (Zif-19) in activity, which is similar to the
abovementioned Zif-4.
Fig. 7. The binding mode of the ligand Zif-15 in Vif C2 pocket.
In summary, through analysis of the protein surface of Vif, the C2 pocket which is suit for
developing small molecular inhibitors was found. Then FBVS was conducted to find a series of
promising fragments. According to the binding mode of Zif-1 with C2 pocket of Vif, 19 indole
derivatives was designed and synthesized. Quantitative structure-activity relationship (QSAR)
shows that the pyridine-2-yl and hydrogen donor group at the para-position is favorable, and
indole core is indispensable for the potency. Among them, through fluorescence-based primary
screening and western blot, Zif-15 shows the best activity, and is deserved to be further optimized.
Acknowledgment
The Vif stably expressed cell line TREX-hvif-15 and APOBEC3G expressed plasmid
.

EYFP-N1-hA3G were kindly donated by Prof. Guangxia Gao (Institute of Biophysics, Chinese
Academy of Sciences). We also thank the support of the National Natural Science Foundation of
China (81472780, 81102483) and the National Key Program of China during the 12th Five-Year
Plan Period (Grant 2012ZX09103101-022)
Supporting Information
Experimental procedures and characterization data of compounds.
Conflicts of Interest
The authors declare no conflict of interest.
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Graphical abstract:
FBVS was conducted against the C2 pocket of Vif, and Zif-1 was selected for further
optimization. Through the immune-fluorescence staining and western blot assays,
Zif-15 shows potent activity in inhibiting Vif-mediated A3G degradation.
.

Highlights
1. C2 pocket of Vif was found which is suit for developing small molecular inhibitors.
2. FBVS was conducted against C2 pocket, and a series of fragments were obtained.
Zif-15 shows potent activity in inhibiting Vif-mediated A3G degradation.
.