The potential role of anibamine, a natural product CCR5 antagonist,

The potential role of anibamine,a natural product CCR5antagonist,

and its analogues as leads toward development of anti-ovarian cancer agents

Yan Zhang a ,b ,?,Christopher K.Arnatt a ,Feng Zhang a ,Jiannan Wang a ,Kendra M.Haney a ,Xianjun Fang b ,c

a

Department of Medicinal Chemistry,Virginia Commonwealth University,Richmond,VA 23298,USA b

Massey Cancer Center,Virginia Commonwealth University,Richmond,VA 23298,USA c

Department of Biochemistry,Virginia Commonwealth University,Richmond,VA 23298,USA

a r t i c l e i n f o Article history:

Received 12April 2012Revised 22May 2012Accepted 29May 2012

Available online 16June 2012Keywords:

Ovarian cancer Chemokines CCR5

Anibamine

a b s t r a c t

Chemokines and their receptors play important roles in the development of primary tumors and their metastases.Particularly CC chemokine receptor 5(CCR5)and its ligand CC chemokine ligand 5(CCL5/RANTES)seem to be critical in proliferation and invasion of ovarian cancer,the leading cause of death from gynecological malignancies in the United States.Anibamine,the ?rst natural product CCR5antagonist,and its analogues were examined for their effects on proliferation of the OVCAR-3ovarian cancer cells in order to validate their candidacy as leads to develop novel anti-ovarian cancer agents.Act-ing as CCR5antagonists,anibamine and its analogues signi?cantly suppressed CCL5-induced intracellular Ca 2+?ux.The compounds also inhibited the proliferation of OVCAR-3at micromolar to submicromolar range.Moreover,anibamine and several analogues did not show signi?cant cytotoxicity in NIH 3T3cells at concentrations up to 20l M.Based on these results,anibamine and one of its synthetic analogues were de?ned as potential leads to develop novel agents against ovarian cancer.

Published by Elsevier Ltd.

Ovarian cancer is the second most common (affecting about every one out of seventy women)and the deadliest (1%of all women die of it)gynecological cancer in the United States,and it is also the ?fth leading cause of cancer related deaths in women.1,2Ovarian cancer is called a ‘silent killer’because symptoms normally won’t be evident until the disease has advanced and the chance of cure or remission becomes very slim.3Therefore,identi?cation of factors and pathways responsible for the ovarian cancer development and progression is of critical importance and such effort may lead to development of novel therapeutic agents.Tumor cell growth can be directly regulated,among others,by chemokines,a family of small proteins inducing directed cell migration (chemotaxis),via speci?c G-protein coupled recep-tors.4–10Initially,chemokines are considered to be pro-in?amma-tory which can be induced during an immune response as regulating leukocyte recruitment at sites of in?ammation.During the last decade,it has become increasingly clear that chemokines also have the capacity to mediate several other functions and therefore are more than simple traf?cking controllers.Recently,a number of reports demonstrated that a complex network of che-mokines and their receptors in?uence the development of primary tumors and their metastasis.11–15For example,the expression of chemokines was detected in several types of human and murine tumors.16–22In some cases,chemokines were found to be autocrine factors produced by tumor cells that are essential to tumor cell proliferation or survival.23–26In view of their chemotactic proper-ties,it has been suggested that chemokines may mediate the recruitment of tumor-associated leukocytes to tumor sites,27–34a process postulated to accelerate the progression of several malig-nant diseases.

The C–C chemokine receptor CCR5is a G-protein coupled recep-tor.The ligands of this receptor include monocyte chemoattractant protein 2(MCP-2),macrophage in?ammatory protein 1alpha (MIP-1alpha),macrophage in?ammatory protein 1beta (MIP-1beta),and RANTES (Regulated on activation normal T expressed and secreted protein),which is also known as CCL5.Along with other chemokine receptors,CCR5has been characterized for its po-tential role in cancer development,35particularly,in ovarian onco-genesis.For instance,analysis of the levels of the chemokine in plasma of patients at different stages of the disease revealed an association between CCL5and ovarian carcinoma progression while it appeared that CCL5protein levels were higher in ovarian cancer patients than in patients diagnosed with benign ovarian cysts,and elevated in stages III–IV of ovarian cancer compared to stages I–II.36,37This suggested that intrinsic CCL5levels could be useful for detection of malignant ovarian tumors.Another study showed that interleukin-12(IL-12)induced the regression of xeno-grafts of the OV-HM ovarian carcinoma cells,which was mediated by the chemokine receptor CCR5,while the CCR5antagonist TAK-779in tumor-bearing mice prevented IL-12induced T-cell migration.38This certainly suggested a therapeutic opportunity of

0960-894X/$-see front matter Published by Elsevier Ltd.https://www.sodocs.net/doc/ad8651589.html,/10.1016/j.bmcl.2012.05.127

Corresponding author.

E-mail address:yzhang2@https://www.sodocs.net/doc/ad8651589.html, (Y.Zhang).

using CCR5antagonist in ovarian cancer.Meanwhile ovarian tu-mors have been observed to over-express CCR5and inhibition of CCL11(one of the CCR5endogenous agonists)signaling by the combination of neutralizing antibodies against CCL11and its receptors signi?cantly increased the ovarian carcinoma cell sensi-tivity to cisplatin.39Overall,these?ndings suggested that develop-ment of appropriate chemokine receptor CCR5antagonists could provide a novel strategy to treat ovarian cancer.

Anibamine,a unique pyridine quaternary alkaloid recently isolated from Aniba panurensis,was found to bind to CCR5with an IC50of1l M in competition with125I-gp120,an HIV viral envel-op protein.40As the?rst natural product CCR5antagonist,anib-amine provides a structural skeleton that is remarkably different from all previously identi?ed lead CCR5antagonists(Fig.1)derived from extensive high throughput screening studies.The chemokine receptor CCR5has mainly been employed in AIDS therapies since it was?rst cloned more than a decade ago.41–44To date only one drug,maraviroc,has been approved by the FDA in2007while con-cerns remained that maraviroc could be associated with increased risks of liver damage,lymphoma,infections and heart attack.45 Apparently,there is an urgent need to explore new chemical struc-tures and templates with a wider range of structural features to de-velop novel https://www.sodocs.net/doc/ad8651589.html,pared to those high-throughput screening hits,lead compounds derived from natural products,of-ten contain more diverse skeletons with wider ranges of shape, chemical features,and speci?c biological activities.46,47Thus,natu-ral products are desirable and useful resources for drug discovery and development.

Recent studies showed that anibamine inhibited proliferation of prostate cancer cell lines at micromolar to submicromolar concen-trations and prevented adhesion and invasion of the highly meta-static M12prostate cancer cell line.48Meanwhile the total synthesis of anibamine has been reported a couple of years ago49 while an updated synthetic route was made available very re-cently.50A homology modeling based docking study of anibamine and other known CCR5antagonists further veri?ed the possible binding mode of anibamine in the CCR5.51The development of these synthetic pathways and modeling studies provided the opportunity to prepare diversi?ed anibamine derivatives in order to investigate the structure–activity relationship of anibamine as a CCR5antagonist and potential anti-cancer agent.

Characterization of anibamine as the lead for anti-ovarian cancer agent.To validate anibamine’s function as a CCR5antagonist,its ef-fect on the intracellular calcium mobilization stimulated by CCL5 (RANTES)in MOLT4/CCR5cells was?rst examined by following the reported procedure50since the inhibitory effects of CCR5 antagonists in chemokine-induced calcium ion mobilization have been demonstrated to correlate well with their af?nity in radioli-gand competition binding assays.52,53Anibamine showed moder-ate inhibition of calcium?ux at an IC50of5.4l M,which was in line with its reported binding af?nity to the CCR5receptor.40 Anibamine was then evaluated for its ability to inhibit growth of the OVCAR-3ovarian cancer cells through a cell proliferation as-say.In the initial experiments,CCL5was not added in the protocol in the consideration of avoiding possible non-speci?c stimulatory effect of CCL5on other chemokine receptors.It turned out that anibamine effectively inhibited the proliferation of OVCAR3cells at an IC50around1l M,which again was in line with its binding af?nity to the CCR5receptor and the inhibitory effect on the CCL5stimulated calcium?ux(Table1).

Since it has been reported that CCR5and CCL5were expressed on OVCAR3cells,39it would be interesting to see if CCL5could act as a cancer cell growth stimulator.Therefore,different concentra-tions of CCL5were applied to the OVCAR3cells and its effect on cell proliferation was determined as illustrated in Figure3.Interest-ingly,at the lower concentrations(0.3nM–3nM),CCL5signi?-cantly promoted the growth of OVCAR3cells by at least50% (P<0.01)while higher concentrations of CCL5tested(10nM and 30nM)showed some but not very signi?cant effect(Fig.2).Such observation was in coincidence with the reported stimulatory pat-tern of CCL5on several prostate cancer cells.22

To examine whether the inhibitory effect of anibamine on the OVCAR3cells observed previously was in correlation to its antago-nist activity on the CCL5/CCR5system,its inhibitory effect on the proliferation of OVCAR-3cells was further tested in the presence of CCL5(1nM).Under such condition,anibamine showed rela-tively stronger inhibitory effect on the OVCAR3cell proliferation (IC50at0.9l M)than the condition without CCL5stimulation, which implied that the inhibitory effect of anibamine on ovarian cancer cell growth may be mediated through its antagonist activity on the CCR5receptor(Table1).

Afterwards,the basal cytotoxicity assay was performed using the NIH3T3cells under similar proliferation conditions(WST-1 as the proliferation agent)to determine whether the inhibitory effect of anibamine on cancer cells was due to its basal cytotoxic-ity.It turned out that much higher concentration of anibamine (IC50=23.5l M)was required to inhibit the growth of3T3cells, suggesting a very promising selectivity of anibamine in inhibiting the growth of cancer cells.

Application of‘deconstruction-reconstruction-elaboration’method to study the structure–activity relationship of anibamine as anti-ovar-ian cancer lead compound.The initial structural modi?cation of anibamine was conducted by following the‘deconstruction-recon-struction-elaboration’method.This method has been successfully

5094Y.Zhang et al./Bioorg.Med.Chem.Lett.22(2012)5093–5097

applied to improve the pharmacological activities of both synthetic agents and natural products.54–57In our case(Fig.3),each struc-tural component of the lead compound,anibamine,was‘removed’one at a time in order to test the in?uence of that component on its CCR5antagonism and anti-ovarian cancer activity.Once the essen-tial structural components were de?ned,they were retained in the core structure.Further modi?cation was then done on the core structure to improve its activities.

Total fourteen analogues of anibamine were prepared following the synthetic route of anibamine with appropriate modi?cations (See the Supplementary data for details).49,50Similar to the charac-terization of anibamine,all of its analogues were?rst evaluated in a CCL5-induced Ca2+mobilization experiment to see if they act as antagonists to the CCR5receptor and inhibit the calcium ion?ux induced by CCL5.These analogues were then tested in the cell pro-liferation assay against OVCAR3cells in the absence or presence of CCL5.Similarly,3T3cells were adopted to characterize the basal cytotoxicity of these analogues.

Anibamine deconstruction analogues and their activity studies. Two side chain deconstruction analogues of anibamine,compound 2and3,were prepared to study the necessity of the side chains for the binding to the CCR5receptor and potential anti-ovarian cancer activity(Table1).The fact that both of them showed signi?cantly decreased inhibition to the receptor function of calcium?ux com-pared to the parent anibamine indicated that both side chains should be retained to facilitate the binding to the receptor complementarily.

On the other hand,compound2and3showed similar or some-what higher activity than the parent natural product in the anti proliferation assay in OVCAR3cells.Considering their relatively lower af?nity to the receptor CCR5as indicated by the calcium mobilization inhibition activity,such improved anti-proliferative effect on OVCAR-3cells may be conferred in part by interaction with other off-target proteins.This was further supported by their signi?cantly higher cytotoxicity in the3T3cells compared with the parent lead compound.

To test if the core ring system would be essential,compound4 and5were prepared(Fig.3).In compound4,a methyl group was retained at position2to minimize the steric hindrance alteration due to the removal of the fused ring part.The second molecule, compound5,was designed to retain the same number of the car-bon atoms from the aliphatic core ring system in order to minimize the change of hydrophobicity.To facilitate the synthesis,a methox-yl group was applied to cap the end of the new side chain.The fact that these two core ring deconstruction analogues carried no sig-ni?cant antagonism to the receptor CCR5(calcium?ux inhibition IC50higher than100l M,Table1)demonstrated that the positively charged nitrogenous center was critical for binding to the receptor, which was consistent with the observation from the molecular modeling studies reported previously.48Therefore this ring system needs to be retained for the future operation.

Anibamine elaboration analogues activity study.Four analogues of anibamine were prepared to evaluate the possible in?uence of double bond con?gurations to the recognition of the receptor and potential anti cancer activity(Fig.3).Among these four com-pounds,6through9,it seemed that in general,the double bond con?gurations(either trans,cis,or saturated)on the two side chains did not in?uence the functional inhibition to the receptor signi?cantly based on the calcium mobilization assay results.They also showed either similar or slightly higher anti-proliferation activity as indicated by their IC50values compared with the parent anibamine.In the presence of CCL5stimulation,all of them showed signi?cantly higher anti-proliferation activity against the OVCAR3 cells,indicating that they inhibited OVCAR3cells through targeting the CCR5receptor.On the other hand,their higher basal cytotoxic-ity compared with the parent natural product was somehow dis-couraging(Table1).

To further evaluate the core ring size in?uence on the receptor CCR5af?nity as indicated by their functional inhibition to calcium ?ux as well as the potential anti-ovarian cancer activity,six core ring size modi?ed analogues were prepared and studied(Fig.3,

Table1

Biological characterization of anibamine and its analogs

Compound CCR5antagonism MOLT-4/CCR5IC50(l M)Anti-proliferation OVCAR3IC50

(l M)

Basal cytotoxicity NIH3T3TC50(l M)

w/o CCL5w/CCL5

1,Aanibamine 5.4±0.9 1.1±0.30.86±0.123.5±2.4

29.2±0.4 1.3±0.20.9±0.1 6.9±1.6

316.3±4.20.4±0.1 1.0±0.17.8±1.5

4>100ND a ND ND

5>100ND ND ND

6 6.5±1.80.8±0.30.7±0.2 4.4±0.6

77.8±1.40.6±0.10.3±0.1 5.1±2.4

810.1±3.90.9±0.10.5±0.1 3.3±0.3

99.2±0.60.9±0.10.6±0.1 4.0±0.3

1010.0±0.4 1.7±0.40.3±0.129.1±0.6

118.4±0.9 1.2±0.30.7±0.110.1±1.1

12 4.6±1.6 2.4±1.2 3.4±2.017.5±4.6

1348.1±21.7 3.6±0.90.4±0.2 4.8±0.6

1415.2±7.9 1.7±0.60.4±0.1 3.8±0.5

1537.6±5.4 2.1±0.40.8±0.111.7±1.5

a ND,not determined.

Y.Zhang et al./Bioorg.Med.Chem.Lett.22(2012)5093–50975095

and Table1).In general,it seemed that six-member ring analogues of anibamine(compound10,12and14)showed relatively higher receptor af?nity than the seven-member ring analogues(com-pound11,13,and15).By comparing their anti-proliferative activ-ity against OVCAR3cells,it seemed that they all showed very similar pro?le.Given the fact that the relatively lower receptor af?nity and higher basal cytotoxicity pattern,the seven-member ring analogues probably interacted with some off-target protein(s).

A closer look at compound10and12,both of which showed rel-atively lower basal cytotoxicity pro?le with reasonable anti-prolif-erative activity against OVCAR3cells,provided some insight into the potential role of the ring size combined with the side chain con?https://www.sodocs.net/doc/ad8651589.html,pound10,which showed the lowest basal cytotoxicity among all the analogues,also carried a similar anti-proliferative effect compared with the natural product lead, anibamine.More importantly,the presence of CCL5dramatically increased its anti-proliferative activity,indicating its speci?c antagonistic interaction with the CCR5.For compound12,no sig-ni?cant difference was observed in its anti-proliferative activity with or without the stimulation of CCL5.Therefore compound10 was considered our next generation lead compound based on its biological activity pro?le.

In summary,the natural product CCR5antagonist,anibamine, was characterized as a lead to develop novel anti-ovarian cancer

5096Y.Zhang et al./Bioorg.Med.Chem.Lett.22(2012)5093–5097

agent.Its biological pro?le seemed to be encouraging.It exhibited signi?cant anti-proliferative activity against the OVCAR3cells with much lower basal cytotoxic effect in the nontransformed NIH3T3 cells.The application of‘deconstruction-reconstruction-elabora-tion’concept on anibamine structure–activity relationship charac-terization revealed that the core ring system was critical for the binding af?nity to the receptor CCR5while both side chains seemed to be important for the binding af?nity to the receptor and selective inhibition of the chemokine CCL5correlated cancer cell proliferation.The ring size modi?cation of the core ring system provided us a new lead,compound10,with reasonable receptor binding af?nity and promising anti-ovarian cancer proliferative activity while no signi?cant basal cytotoxicity was observed at a concentration up to29l M.The identi?cation of anibamine together with compound10showed promise in development of novel chemokine receptor CCR5antagonist for therapeutic inter-vention of ovarian cancer.

Acknowledgments

We are grateful to the funding support from US Army Prostate Cancer Research Program PC073739.The content is solely the responsibility of the authors and does not necessarily represent the of?cial views US Army Prostate Cancer Research Program. We thank NIH AIDS Research and Reference Reagent Program for providing the MOLT-4/CCR5cell line.

Supplementary data

Supplementary data associated with this article can be found,in the online version,at https://www.sodocs.net/doc/ad8651589.html,/10.1016/j.bmcl.2012.05. 127.

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