Design, synthesis, and characterization of rhein analogs as novel inhibitors of scavenger receptor A

Article abstract of DOI:10.1016/j.bmcl.2016.11.029

Scavenger receptor A (SRA) has been known as an immunosuppressive factor and therefore therapeutic inhibition of SRA may be potentially exploited for cancer immunotherapy. Our previously work suggested that rhein may act as an inhibitor of SRA in reversin

Full text of DOI:10.1016/j.bmcl.2016.11.029

Accepted Manuscript
Design, Synthesis, and Characterization of Rhein Analogs as Novel Inhibitors
of Scavenger Receptor A
Yi Zheng, Xia Li, Piyusha P. Pagare, Yunyun Yuan, Xiang-Yang Wang, Yan
Zhang
PII:
S0960-894X(16)31172-6
DOI:
http://dx.doi.org/10.1016/j.bmcl.2016.11.029
BMCL 24424
Reference:
Bioorganic & Medicinal Chemistry Letters
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Revised Date:
Accepted Date:
27 October 2016
8 November 2016
11 November 2016
Please cite this article as: Zheng, Y., Li, X., Pagare, P.P., Yuan, Y., Wang, X-Y., Zhang, Y., Design, Synthesis, and
Characterization of Rhein Analogs as Novel Inhibitors of Scavenger Receptor A, Bioorganic & Medicinal Chemistry
Letters (2016), doi: http://dx.doi.org/10.1016/j.bmcl.2016.11.029
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Design, Synthesis, and Characterization of Rhein Analogs as Novel
Inhibitors of Scavenger Receptor A
Yi Zhenga,#, Xia Lib,#, Piyusha P. Pagarea, Yunyun Yuana, Xiang-Yang Wangb,c,d, *, Yan
Zhanga,d,*
a Department of Medicinal Chemistry, Virginia Commonwealth University, 800 E.
Leigh Street, Richmond, Virginia 23298, United States
b Department of Human and Molecular Genetics, Virginia Commonwealth University,
Richmond, Virginia 23298, United States
c Institute of Molecular Medicine, Virginia Commonwealth University, Richmond, VA
23298, United States
d Massey Cancer Center, Virginia Commonwealth University, Richmond, VA, 23298,
United States
# Equal contributors
* Corresponding authors:
Yang Zhang, Department of Medicinal Chemistry, Virginia Commonwealth
University. Email address: yzhang2@vcu.edu
Xiang-Yang Wang, Department of Human and Molecular Genetics, Virginia
Commonwealth University. Email address: xywang@vcu.edu

Abstract
Scavenger receptor A (SRA) has been known as an immunosuppressive factor and
therefore therapeutic inhibition of SRA may be potentially exploited for cancer
immunotherapy. Our previously work suggested that rhein may act as an inhibitor of
SRA in reversing immunosuppression of SRA during T cells activation. Herein, three
deconstruction analogs of rhein, compound 1, 2, and 3, were further studied as
inhibitors of SRA. These three compounds, particularly compound 1, also known as a
natural product danthron, enhanced T cells activation, indicated by increased
transcriptional activation of interleukin 2 (Il2) gene, production of IL-2 protein, and
proliferation of T cells. Additionally, the interaction between these compounds and
SRA was studied by molecular modeling. Compound 1 showed a favorable binding
mode with the cysteine rich domain of SRA protein compared to compound 2 and 3.
Collectively, those results would provide insight for future design and development of
next generation rhein derivatives as SRA inhibitors.
Keywords: scavenger receptor A, rhein, danthron, cancer immunotherapy

Scavenger receptor A (SRA), also namely CD204 or macrophage scavenger
receptor, is mainly expressed in myeloid cells (e.g., dendritic cells, macrophages), and
has multiplex structures [1]. The activity of SRA in lipid metabolism was firstly
discovered by Goldstein et al [2], and SRA was also the first member of scavenger
receptor family that was cloned [3, 4]. Due to its ligand-binding feature, the activity
of SRA in lipid metabolism, host defense [5, 6], atherosclerosis [7], and pathogen
recognition [8, 9] have been extensively studied. We have demonstrated that SRA
plays a role as an immunosuppressive regulator in restricting dendritic cell-induced
immune responses during vaccination against tumor [10-15], suggesting that
development of small molecule inhibitors for SRA may provide therapeutic potential
in immunotherapy of cancer.
Two natural products, namely sennoside B (Fig. 1) and tannic acid, were previously
identified as SRA inhibitors by Mamula et al [16]. Particularly, sennoside B, a
macromolecular natural product, showed dose dependent binding to the SRA [16]. We
subsequently used the ‘deconstruction-reconstuction-elaboration’ approach to identify
critical molecular component for its biological activity [17, 18]. Through this
deconstruction strategy, a new lead compound, namely rhein or cassic acid (Fig. 1),
was identified [19]. Rhein is a known natural product isolated from a traditional
Chinese medicinal plant rhubarb [20]. Compared with sennoside B, rhein showed a
higher inhibition activity of SRA in rescuing T cell activation.
Figure 1. The design of three rhein analogs.
Herein, rhein was further deconstructed in order to define the necessary functional
groups in this molecule. In this process, each of three functional groups on the
anthraquinone skeleton was removed one by one and three rhein analogs were
obtained. One of them (compound 1, Fig. 1) is commercially available. Compound 1
was known as a natural product, namely danthron. Danthron was also isolated from
the traditional Chinese medicinal plant, rhubarb [21]. The other two compounds
(compound 2 and 3, Fig. 1) were synthesized through multi-step chemical synthesis

(compound 2, Scheme 1; compound 3, Scheme 2). Then, the biological activity of
these three analogs were tested and related docking study based on SRA cysteine rich
domain was conducted.
Scheme 1. Synthesis of compound 2.20 Regents and conditions: (a) TFA,
o
Hexamethylenetetramine, reflux, 90-95 C; (b) Diethyl succinate, NaH, Toluene,
o
EtOH, N2, 55 C; (c) Ac2O, NaOAc, N2, reflux; (d) Ammonium cerium(IV) nitrate,
acetonitrile, rt; (e) Buta-1,3-dien-1-yl-acetate, EtOH, reflux; (f) 10% NaOH.
Scheme 2. Synthesis of compound 3.21, 22 Regents and conditions: (a) TMSCl, TEA,
ZnCl2, Hydroquinone, benzene, 4 h, 70 oC; (b) HOAc, CrO3, Benzene, reflux 40 h; (c)
11, THF, rt, 40 h; (d) i. Ac2O, Pyridine; ii. CrO3, HOAC, Ac2O.
First, to test the activity of these three analogs in rescuing T cell activation,
β-Galactosidase (β-Gal) assays was used with B3Z T cells. As we reported previously
[19], β-Gal was encoded by the structure of lacZ and lacZ came from IL2 gene [22].
Once B3Z cells were activated, IL2 gene in cells would be triggered and produce lacZ
and β-Gal. In this experiment, anti-CD3/CD28 antibodies were used to induce the
activation of B3Z T cells and as a mark of β-Gal increase. Besides, SRA protein was
used as an immunosuppressive inhibitor of T cells. As our forward report [23],

anti-CD3/CD28 antibodies could efficiently activate B3Z T cells, induce thee increase
of β-Gal, and the activatio of T cells induced by anti-CD3/CD28 antib dies was
n
oo
distinctly reduced by the presence of SRA protein (Fig. 2). When B3Z T ccells were
treated with rhein analogs, the suppressive effect of SRA protein was evidently
reversed (Fig. 2). Compared to the control, compound 1, 2, and 3 displayedd a distinct
activity of activating T cellls during reversion of the suppression of SRAA protein.
Compound 1 showed the
potency compared with that
on these, compound 1 and 3 wwere chosen to carry on for further studies.
mmost significant activity. Compound 3 showed similar
o
o
f anti-CD3/CD28 antibodies in activating T ceells. Based
Figure 2. β-Gal assay of rhhein derivatives in T cell activation. B3Z T ccells were
seeded 2 x 106/well in 12-well plate in the presence of anti-CD3/CD28 sttimulation
combined with SRA protein and compound 1, 2, and 3 (50 µM), respectivelly, for 5 h.
Cells without any treatment served as control. β-Gal assay was performed to detect
the activation of Il2 gene promoter after incubation at 37 °C for 1 h. The exxperiments
were repeated at least three times with similar results. *, p < 0.05; **, p < 0.001.
Enzyme linked immuno
two compounds in antagonizing the immunosuppressive actiivity of
CD11b+Ly6ChighLy6G- myel
id-derived suppressive cells (MDSCs) that express SRA
ssorbent assay (ELISA) was then used to further test these
oo
[24], by assaying the llevel of interleukin (IL-2) secreted by T cells.
CD11b+Ly6ChighLy6G- cells were considered to be one kind of immunosuppressive
cells derived from myeloi
cytokines product by macrophages to adjust the responses of immun
promote the growth and spread of tumor [24, 25]. Here, MDSCs were treateed with or
without various compounds prior to co-culture with splenocytes in the p esence of
d
d
. It was reported that MDSCs through regullating the
e
system,
r

anti-CD3/CD28 antibodies. As expected, MDSCs strongly suppressed T cell
activation, indicated by IL-2 production. Compared to the control, both compound 1
and rhein significantly enhanced T cells activation at indicated doses (Fig. 3). It is
evident that compound
1
exhibited its activity in a dose-dependent fashion. At a
was even more potent than the parent natural
appeared to be modest compared to others.
and , especially compound , is able to
higher dose (20 µM), compound
1
product rhein. The effect of compound
These results indicated that compound
3
1
3
1
reverse the immunosuppressive activity of CD11b+Ly6ChighLy6G- MDSCs and
promote T cells activation.
Figure 3. Rhein and its derivatives reversed immunosuppressive activity of
CD11b+Ly6ChighLy6G- MDSCs. Mice were subcutaneously injected 2 x 105
RM1-OVA tumor cells. Ten days later, spleen-derived CD11b+Ly6ChighLy6G- cells
were sorted using a Flow cytometer. MDSCs were pretreated with rhein, compound 1
or compound
co-cultured with purified MDSCs at 1:1 ratio in the presence of 1
anti-CD3 mAb, 0.5 g/mL soluble anti-CD28 mAb and rhein or the other two
3
at indicated doses for 2 h. Splenocytes from naïve mice were
µg/mL plate-bound
µ
derivatives with indicated doses in a 96-well flat-bottom plate. Supernatant was
collected 56 h after culture, and levels of cytokines IL-2 was determined by ELISA.
The experiments were repeated at least three times with similar results. *,
< 0.01.
p < 0.05; **,
p
To confirm the ability of the two compounds to block the immunosuppressive
activity of CD11b+Ly6ChighLy6G- MDSCs, T cells proliferation was assessed using
[3H] thymidine ([3H]TdR, 0.5 µ Ci/well) incorporation test [12]. As shown in Fig. 4,
rhein, compound
1, and 3 enhanced T cell proliferation in the presence of

immunosuppressive MDSCs. Consistent with IL-2 production in Fig. 3, T cells,
co-cultured with compound 1-treated MDSCs, showed higher [3H]TdR uptake than
the other two at both doses.
Figure 4. [3H]thymidine incorporation assays for the proliferation of T cells. MDSCs
sorted from tumor-bearing mice were pretreated with rhein or its derivatives
compound 1 or 3 at indicated doses for 2 h. Splenocytes from naïve mice were
co-cultured with purified MDSCs at 1:1 ratio in the presence of 1 µg/mL plate-bound
anti-CD3 mAb, 0.5 µg/mL soluble anti-CD28 mAb and rhein or the other two
derivatives with indicated doses in a 96-well flat-bottom plate for 72 h. Cells were
pulsed with 0.5 µ Ci/well [3H]thymidine for the last 16 h of incubation. Proliferation
was measured based on [3H]thymidine incorporation in triplicate wells. The
experiments were repeated at least three times with similar results. *, p < 0.05; **, p <
0.01.
Finally, we used the β-Gal assay to assess the effect of compound 1 and 3 on
promoter activity of Il2 gene in B3Z T cells. As expected, anti-CD3/CD28 antibodies
effectively induced activation of Il2 gene promoter, indicated by increased level of
β-Gal (Fig. 5). Treatment of cells with SRA protein markedly decreased β-Gal level
upon stimulation with anti-CD3/CD28 antibodies. When compound 1 or 3 was added,
the promoter activity of Il2 gene impaired by SRA was clearly restored during T cell
stimulation.

Figure 5. β-Gal assay of compound 1 and compound 3 in restoring SRA-impaired
transcription of Il2 gene during T cell stimulation. B3Z T cells were seeded 2 x
106/well in 12-well plate and stimulated with anti-CD3/CD28 antibodies in the
presence of SRA protein (10 µg/mL) combined with compound 1, or compound 3 (50
µM each), respectively, for 5 h. β-Gal assay was performed to detect the activation of
Il2 gene promoter. The experiments were repeated at least three times with similar
results. *, p < 0.05; **, p < 0.01.
The results above demonstrated that these rhein analogs, especially compound 1,
could act as inhibitors of SRA in rescuing T cells activation. As mentioned above,
compound 1 was known as a natural product, danthron. The documented biological
activities of danthron included genotoxicity [26], DNA damage and anti-cancer [27],
and apoptosis [28]. It was also reported that danthron could promote the cytotoxic
activity of immune cells in leukemic mouse model [29]. In the current study, we
revealed that enhanced activity of immune cells following danthron treatment may be
attributed to its inhibitory activity toward SRA.
In addition to biological assays, molecular modeling was applied to gain insight on
the interaction between these analogs and the SRA protein in order to understand why
these structurally similar compounds had different biological activities in reversing
immunosuppression during T cells activation.
All three molecules were docked into the SRA model following similar procedure
previously [19]. Results from clustering of the docking poses of rhein analogs showed
very similar binding modes for compound 2 and 3 (Fig. 6b and 6c). The carboxylic
acid group in these ligands appeared to be involved in electrostatic interactions with
ARG2 of chain A. One of the carbonyl group (the one on the opposite side of
hydroxyl group) showed hydrogen bonding with the backbone nitrogen of VAL1 of
chain A. Both molecules also presented hydrophobic interactions with ILE16 and
LEU17 of chain B.

However, compound 1 showed a different binding mode as compared to the other
two (Fig. 6a). Hydroxyl groups in compound 1 displayed strong hydrogen bonding
interaction networks with SER76 and SER41 of chain B. The carbonyl group on the
opposite side of the ring appeared to be involved in hydrogen bonding interactions
with the backbone nitrogen of VAL1 of chain A. VAL1, ILE16, and LEU17 form a
hydrophobic cavity that interacted with the anthraquinone backbone of compound 1.
Thus, the residues in the binding pocket provided potential hydrogen bonding
interactions and hydrophobic force to stabilize the ligand in the binding pocket. On
the other hand, the COOH group and the carbonyl group next to it in compound 2 and
3 seemed to fix the ligands in a predominate pose in the protein binding site
irrespective of other functional groups on the molecules. Due to this, Compound 2
and 3 bound to the protein in a way that the hydroxyl groups in both ligands were
placed away from the serine residues and thus couldn’t participate in forming
hydrogen bonding networks as seen in binding interactions with Compound 1. Such
result from the docking study showed that compound 1 had a beneficial binding pose
with the cysteine rich domain in SRA protein compared with compound 2 and 3.
Furthermore, the molecular interaction between three rhein analogs and SRA
protein were evaluated by applying different scoring systems. Among them, GOLD
and ChemPLP scoring systems have been generally used for characterizing the
binding poses of ligand in the active pocket of protein based on factors such as
hydrogen bonding energy, van der Waals energy, binding angle and distance [30]
while HINT scoring system has been applied to understand the interaction and
binding affinity between ligands and their active pocket of protein mainly based on
free energy and non-covalent interactions in biological surroundings [31-33].
Interestingly, no remarkable difference was observed between the GOLD and
ChemPLP scores for all three compounds (Table 1). One possible explanation for such
a similarity would be that all three compounds were deconstruction products from
rhein, which shared the same rigid structural skeleton, and such a structural feature
mainly decided the binding poses for all three compounds as GOLD and ChemPLP
Scores indicated even in somehow different binding environments. On the other hand,
the HINT scores of three compounds were clearly different with compound 1 the
highest, compound 3 the second, and compound 2 the lowest. Such a result was in a
good agreement with the sequence of those compounds’ biological activity observed.
Comparing compound 2 and 3 (both in a similar binding pocket), while the
carboxylate moiety served as an anchor to recognize the basic side chain in ARG2 of
the protein, the hydroxyl group on the same aromatic ring in compound 2 seemed not
in favorable interaction with LEU17 which lead to reduction of its HINT score.
Meanwhile compound 1 seemed to be able to settle in a nearby binding locus through
a much more favorable interaction between its aromatic skeleton and a hydrophobic
pocket formed mainly by VAL1, ILE16, and LEU17, which apparently contributed to
its higher HINT score. Such a different binding mode may provide explanations for
their biological activity as inhibitors of scavenger receptor A.

Figure 6. Docking study of rhein analogs in cysteine rich domain of SRA protein.
Most preferred docking mode of compound 1 (yellow), compound 2 (pink), and
compound 3 (green) in the cysteine rich domain of SRA protein. Amino acid residues

involved in putative interactions are shown in grey sticks representation.
Table 1. GOLD, ChemPLP and HINT Scores for the interaction between rhein
analogs and the SRA protein cysteine rich domain.
Ligands
GOLD
Score
35.30
37.38
38.45
ChemPLP Score HINT Score
Compound 1
Compound 2
Compound 3
35.13
37.26
38.48
195.3719
62.3885
163.1161
In conclusion, three rhein analogs, compound 1, 2, and 3, were designed and
obtained by applying deconstruction strategy and tested as inhibitors of SRA in
rescuing T cells activation. The biological studies showed these analogs, especially
compound 1, were able to enhance T cell activation by inhibiting immune suppression
of SRA protein, evidenced by increased production of IL-2, transcriptional activation
of Il2 gene and the proliferation of T cells. Docking study suggested that compound 1
displayed a different binding mode in the cysteine rich domain compared to
compound 2 and 3. The relative position of two hydroxyl groups and carbonyl groups
in compound 1 made it in an advantageous position to bind in a hydrophobic pocket
in the protein through a hydrogen bonding network. These results would guide future
molecular design of rhein analogs as SRA inhibitors.
Acknowledgements
The research was supported by Virginia Commonwealth University Massey Cancer
Center Pilot Project Grant and by National Institutes of Health (NIH) Grant
CA175033.
Supporting information
Brief statement in format listing the contents of the materials supplied as
Supporting Information. Results of analytic parameters, experimental materials and
methods for rhein analogs were available.
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Graphic Abstract
OH
O
OH
O
O
OH
HO
OH
OH
O
O
OH
COOH
rhein (cassic acid)
COOH
COOH
H
H
OH
O
OH
OH
HO
O
O
O
OH
OH
O
OH
Compound 1 (danthron)
Sennoside B