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Binding of Cordycepin Monophosphate toAMP-Activated Protein Kinase and its Effecton AMP-Activated Protein Kinase Activation

Zhanli Wang1, Xing Wang2, Kai Qu2,

Ping Zhu2, Na Guo2, Ruiping Zhang2,

Zeper Abliz2, Hui Yu3 and Haibo Zhu2*

1College of Pharmaceutical Science, Zhejiang University ofTechnology, Hangzhou 310014, China2Key Laboratory of Bioactive Substances and Resources Utilization

of Chinese Herbal Medicine, Ministry of Education & Key

Laboratory of Natural Drugs Biosynthesis, Ministry of Health,

Institute of Materia Medica, Chinese Academy of Medical Sciences& Peking Union Medical College, Beijing 100050, China3Department of Laboratory Medicine, the Affiliated Tenth People's

Hospital, Tongji University, Shanghai 200072, China

*Corresponding author: Haibo Zhu, zhuhaibo@imm.ac.cnThese two authors contributed equally to this work.

It had been reported that cordycepin could acti-

vate AMP-activated protein kinase. One possible

mechanism is that cordycepin mediated AMP-acti-

vated protein kinase activation by conversion into

cordycepin monophosphate, which acts as an

AMP analog to activate AMP-activated protein

kinase. To confirm the aforementioned hypothesis,

we investigate the binding of cordycepin mono-phosphate to AMP-activated protein kinase using

molecular docking. The modeling results indicate

that cordycepin monophosphate binds to AMP-

activated protein kinase with high affinity. The

hydrogen bonds provide attractive forces between

molecules. Our results further identify the key res-

idues contributing to the interaction. Also, the

modeling results predict that cordycepin mono-

phosphate and AMP would have similar binding

modes with AMP-activated protein kinase. Further

investigation of AMP-activated protein kinase

activation in vitro provides the evidence that cord-

ycepin monophosphate functioned as an AMP

mimic to activate AMP-activated protein kinase.

Key words: adenosine monophosphate (AMP), AMP-activated pro-tein kinase, cordycepin, cordycepin monophosphate, molecular docking

Received 24 November 2009, revised 25 June 2010 and accepted forpublication 3 July 2010

The AMP-activated protein kinase (AMPK) has been proposed toact as a sensor of cellular energy status capable of regulating

vital metabolic pathways in the cell (1). Changes in AMPK activ-ity have been shown to regulate energy consuming biosyntheticpathways, such as fatty acid and sterol synthesis, and adenosinetriphosphate (ATP)-producing catabolic pathways, such as fattyacid oxidation. AMPK has therefore been proposed as a majortherapeutic target for obesity and obesity-linked metabolic disor-ders such as hyperlipidemia. AMPK is a heterotrimeric complexconsisting of a catalytic subunit (a) and two regulatory subunits(b and c). The c subunits of AMPK have been shown to contain

AMP-binding sites (2). Regulation of AMPK activity is typicallyaffected through the binding of AMP to allosteric sites. AsAMPK represent potential drug targets for metabolic diseases,efforts were initiated to discover AMP mimics that bind to AMP-binding sites with high affinity and high enzyme specificity (3).Numerous investigators have focused on the nucleoside analogsthat generate nucleoside monophosphates (NMPs) inside cells(4,5). For example, 5-aminoimidazole-4- carboxamide 1-bD-ribofur-anoside (AICAR), a nucleoside analog discovered in the late1980s, was one of the AMPK stimulators (6). AICAR mediatedAMPK activation by conversion into ZMP (AICAR monophosphate),which acts as an AMP analog to activate AMPK without affect-ing the cellular AMP:ATP ratio.

Cordycepin, a nucleoside analog 3-deoxyadenosine, is a bioactivecompound present in traditional Chinese medicine Cordyceps. Inrecent years, many pharmacological properties of cordycepin havebeen reported, including antiviral (7), antifungal (8) and antitumoractivity (9). Recently, cordycepin was shown to function as an acti-vator of the AMPK pathway (10). One possible mechanism is thatcordycepin mediated AMPK activation by conversion into cordycepinmonophosphate, which acts as an AMP analog to activate AMPK.Moreover, a structural similarity has been noted among AMP, ZMPand cordycepin monophosphate (Chart 1). Therefore, we hypothesizethat the aforementioned compounds and AMPK share common bind-ing strategy and cordycepin monophosphate may interact withAMPK.

According to our best knowledge, there are no studies evaluatingthe interaction between cordycepin monophosphate and AMPK. Inthis investigation, a docking simulation of the AMPK complexedwith cordycepin monophosphate was performed. We examinedwhether cordycepin monophosphate and AMP have similar bindingmodes with AMPK. We also determined the AMPK activation bycordycepin monophosphate. Our results suggest that AMPKemerged as a possible target of cordycepin monophosphate.

340

Chem Biol Drug Des 2010; 76: 340344

Research Article

2010 John Wiley & Sons A/S

doi: 10.1111/j.1747-0285.2010.01019.x

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Materials and Methods

Agents

Cordycepin used in this study was kindly provided by Professor PingZhu (Chinese Academy of Medical Sciences, Beijing, China). SAMSpeptide was purchased from Millipore. ATP and AMP were fromSigma. Whatman p81 filter paper was obtained from Yingjun Tech-nology (Beijing, China).

Docking studies

The binding conformations of cordycepin monophosphate bound toAMPK were modeled using AutoDock 3.05 (11) based on the pub-lished crystal structure of AMPK (PDB code: 2OOX) (12). For ligand,GasteigerMarsili partial charges were assigned, as implemented inAutoDockTools. We retained the 240th, 307th and 364th water mol-ecules at the receptor-binding site for docking studies, as it wasfound to stabilize the interaction of the ligand molecule. Fifty inde-pendent docking runs were performed in ligand, and the resultingconformations clustered using a root mean-squared deviation crite-rion of 0.5 in x, y, z positional coordinates. The compound cord-ycepin was also docked to the AMPK active site using dockingprotocol as described earlier. The docked molecules were furtherscored with eHiTS_Score program, which contained the statisticallyderived empirical scoring function to capture the interactions

between receptors and ligands (13).

Activation of AMPK protein

Analyses of the activities of AMPK were carried out in typical assayconditions of a 30-ll reaction mixture containing 1 mM dithiothreitol(DTT), 50 mM MgCl2, 0.4 mM ATP (0.4 lCi of [c

32P] ATP per reaction)and 90 lM SAMS peptide. The reaction was initiated by the additionof AMPK proteins treated with cordycepin or cordycepin monophos-phate, incubated at 30 C for 20 min and terminated by the additionof 50 lL of 1% H3PO4. Particulate matter was then transferred to p81filter paper and washed three times with 0.05% H3PO4. Radioactivitythat had been incorporated in the SAMS peptide was determined by

liquid scintillation counting in a Wallac Microbeta plate counter.

Results

Assessment of the Autodock ability to properly

dock AMP into AMPK

To validate the docking method, we first applied our technique tothe rebuilding of the crystallographic AMPAMPK complex by dock-ing the ligand extracted from the structures back into the binding

site. The docking result demonstrated that the predicted bindingmode was in excellent agreement with the experimentally observedstructure, with small Root-mean-square Deviation (RMSD) valuebetween the heavy atoms (0.41 ) (data not shown).

Interactions between AMPK and cordycepin

monophosphate

To determine the interactions between AMPK and cordycepin mono-phosphate, a possible docking model of AMPK and cordycepin mono-phosphate is generated by the AutoDock 3.05 (Figure 1). In this model,we can see that cordycepin monophosphate located in the center ofthe binding pocket. We observed that hydrophobic interaction andhydrogen bond were the stabilizing force in this proteinligand inter-action. The theoretical calculation indicated that compound formedhydrogen bonds with Arg139, Arg141, Ala196, Ser217, Ala218,Arg290, Asp308, the 240th water molecule and the 307th water mole-cule, respectively (Figure 2). Moreover, the binding mode of cordycepinmonophosphateAMPK is consistent with that found in the crystalstructure of AMPK complexed with AMP (Figure 3). A comparison andsuperposition of the docked structure of cordycepin with the crystalstructure of AMP revealed a RMSD value of 0.99 (Figure 4).

A B C

Chart 1: The structures of(A) AMP, (B) ZMP and (C)Cordycepin monophosphate.

Figure 1: The predicted three-dimensional structure of theAMPK complexed with cordycepin monophosphate. Different partsof AMPK are presented in different colors: the a subunit in blue,the b subunit in green and the c subunits in red. Compound cord-ycepin monophosphate is depicted in CPK representation.

Binding and Activating of Cordycepin Monophosphate to AMPK

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Cordycepin was also docked into the binding pockets of AMPK (Fig-ure S1). Furthermore, the eHiTS_Score program was used to evalu-ate relative affinities between AMPK and ligands. Lower dockingscore value indicates a higher binding affinity of a ligand. The dock-

ing score values obtained for AMP, cordycepin monophosphate andcordycepin were )2.153, )1.822 and )1.046, respectively, indicat-ing that cordycepin monophosphate has relative higher bindingaffinity compared with cordycepin.

The effects of cordycepin monophosphate on

AMPK activation

To determine whether cordycepin monophosphate stimulates AMPK,we investigated the effects of variety concentrations of cordycepin

monophosphate on AMPK. We found that cordycepin monophos-phate can directly activate AMPK in vitro, and cordycepin mono-phosphate stimulates AMPK in a dose-dependent manner (Figure 5).Moreover, results indicated that cordycepin had no effects on AMPK

activation (data not shown).

Discussion

AMPK is a direct target of AMP and has emerged as a potentiallykey signaling intermediary in the regulation of changes in glucoseand lipid metabolism (1416). ZMP acts as an AMP analog to acti-vate AMPK. Cordycepin monophosphate also showed structural sim-ilarity with AMP. Therefore, we hypothesize that cordycepin

Figure 2: Key binding inter-actions of cordycepin monophos-phate and AMPK. Hydrogen bondsare presented as dotted lines.

Figure 3: Key binding interac-tions of AMP and AMPK (PDBcode: 2OOX). Hydrogen bonds arepresented as dotted lines.

Wang et al.

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monophosphate may interact with AMPK. To test this assumption,we simulated the 3D structure of AMPK complexed with cordycepinmonophosphate by molecular docking strategy.

We obtained docking model of the complex between AMPK andcordycepin monophosphate. Docking result means that hydrogenbonds play an important role in the interactions. This binding modeis agreement with that of AMP, because local environment withinAMPK-binding site is highly dependent on hydrogen bond interac-tions (17). Compared with AMP, the purine base moiety retains thethree hydrogen bonds formed between the 6NH2 and 1N groups ofcordycepin monophosphate and residues Ala196 and Ala218. More-

over, the phosphonic acid group retains the seven hydrogen bondsformed between the cordycepin monophosphate and residues in thepositively charged phosphate-binding site. Ribose moiety retains theone hydrogen bond formed between the 2 hydroxyl group of cord-ycepin monophosphate and the 307th water molecule. Unlike the 3 hydroxyl group of AMP, ribose moiety of cordycepin monophosphatedid not form hydrogen bond with the 364th water molecule. Previ-ous studies showed that the hydrogen bonds between the 3 hydro-xyl of AMP and residues contributed little to the overall bindingaffinity (18,19). The results suggested that the cordycepin mono-

phosphate readily binds to AMPK with binding affinity very similarto that found with AMP. Moreover, the docking score valuesobtained for AMP and cordycepin monophosphate were )2.153 and)1.822, respectively, confirming that AMP and cordycepin mono-phosphate have similarly high binding affinity. In contrast, the valueobtained for cordycepin was )1.046, indicating a decreased bindingaffinity compared with AMP and cordycepin monophosphate. It waswell known that the loss of phosphate group might result in weaker

binding energy that contributes to decreased affinity of ligand toAMPK (17). Compared with cordycepin monophosphate, cordycepincould not form hydrogen bonds with the positively charged phos-phate-binding site of AMPK, and the interactions of cordycepin withAMPK were apparently weaker than that observed with cordycepinmonophosphate. Therefore, we hypothesize that the loss of sevenhydrogen bonds has significant effects on the binding potency ofcordycepin.

For the purpose of obtaining additional evidences that AMPK isactivated by cordycepin monophosphate, the phosphorylation ofSAMS peptide was used as indicator of AMPK activation. Analysesof the activities of AMPK provided the first experimental data sup-porting the concept and the preference for cordycepin monophos-phate. Consistent with the molecular docking data, the resultshowed that cordycepin monophosphate was a potential activatorof AMPK. The data support the concept that cordycepin mediatedAMPK activation by conversion into cordycepin monophosphate.

Conclusions

In summary, evidence that cordycepin monophosphate not onlybound to AMPK but also functioned as an AMP mimic was appar-ent from both docking results as well as analyses of the AMPKactivation. The results reported give support to the idea that cord-ycepin mediated AMPK activation by conversion into cordycepin

monophosphate.

Acknowledgments

This study was supported by grants from National Natural SciencesFoundation of China (NSFC), Grant Number (30873063; 30873455;30973527), the National 973 Fundamental Project of China, GrantNumber (2009CB523004), Shanghai's Health Bureau Science Foun-dation for Youths, Grant Number (2008Y020), Natural SciencesFoundation of Beijing, Grant Number (7092068) and the Key Projectof Youth Foundation of Institute of Materia Medica, Chinese Acad-emy of Medical Sciences and Peking Union Medical College, Grant

Number (2006QZH01). We are indebted to the grant from NationalS&T Major Project (2009ZX09303-003; 2009ZX09103-034).

References

1. Kahn B.B., Alquier T., Carling D., Hardie D.G. (2005) AMP-acti-vated protein kinase: ancient energy gauge provides clues tomodern understanding of metabolism. Cell Metab;1:1525.

Figure 5: Activation of AMPK by cordycepin monophosphate.

Figure 4: Comparison of docked structures of cordycepin mono-phosphate (green) and AMP (yellow).

Binding and Activating of Cordycepin Monophosphate to AMPK

Chem Biol Drug Des 2010; 76: 340344 343

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2. Xiao B., Heath R., Saiu P., Leiper F.C., Leone P., Jing C., WalkerP.A., Haire L., Eccleston J.F., Davis C.T. (2007) Structural basisfor AMP binding to mammalian AMP-activated protein kinase.Nature;449:496500.

3. Hardie D.G., Hawley S.A. (2001) AMP-activated protein kinase:the energy charge hypothesis revisited. Bioessays;23:11121119.

4. Cool B., Zinker B., Chiou W., Kifle L., Cao N., Perham M., Dickin-son R. et al. (2006) Identification and characterization of a small

molecule AMPK activator that treats key components of type 2diabetes and the metabolic syndrome. Cell Metab;3:403416.5. Yamanaka G., Wilson T., Innaimo S., Bisacchi G.S., Egli P., Rine-

hart J.K., Zahler R., Colonno R.J. (1999) Metabolic studies onBMS-200475, a new antiviral compound active against hepatitisB virus. Antimicrob Agents Chemother;43:190193.

6. Gruber H.E. US Patent 5,658,889, August 19, 1997.7. Mueller W.E.G., Weiler B.E., Charubala R., Pfleiderer W., Leserman

L., Sobol R.W., Suhadolnik R.J., Schroeder H.C. (1991) Cordycepinanalogues of oligoadenylate inhibit human immunodeficiency virusinfection via inhibition of reverse transcriptase. BiochemWash;30:20272033.

8. Sugar A.M., McCaffrey R.P. (1998) Antifungal activity of 3 -de-oxyadenosine (cordycepin). Antimicrob Agents Chemother;42:14241427.

9. Okoshi R., Ozaki T., Yamamoto H., Ando K., Koida N., Ono S.,Koda T., Kamijo T., Nakagawara A., Kizaki H. (2008) Activationof AMP-activated protein kinase induces p53-dependent apopto-tic cell death in response to energetic stress. J BiolChem;283:39793987.

10. Wong Y.Y., Moon A., Duffin R., Barthet-Barateig A., Meijer H.A.,Clemens M.J., de Moor C.H. (2010) Cordycepin inhibits proteinsynthesis and cell adhesion through effects on signal transduc-tion. J Biol Chem;285:26102621.

11. Morris G.M., Goodshell D.S., Halliday R.S., Huey R., Hart W.E.,Belew R.K., Olson A.J. (1998) Automated docking using aLamarckian genetic algorithm and empirical binding free energy

function. J Comput Chem;19:16391662.12. Townley R., Shapiro L. (2007) Crystal structures of the adenylate

sensor from fission yeast AMP-activated protein kinase. Sci-ence;315:17261729.

13. Zsoldos Z., Reid D., Simon A., Sadjad B.S., Johnson A.P. (2006)eHiTS: an innovative approach to the docking and scoring func-tion problems. Curr Protein Pept Sci;7:421435.

14. Ko H.J., Zhang Z., Jung D.Y., Jun J.Y., Ma Z., Jones K.E., ChanS.Y., Kim J.K. (2009) Nutrient stress activates inflammation andreduces glucose metabolism by suppressing AMP-activated pro-tein kinase in the heart. Diabetes;58:25362546.

15. Gruzman A., Babai G., Sasson S. (2009) Adenosine monophos-phate-activated protein kinase (AMPK) as a new target for an-tidiabetic drugs: a review on metabolic, pharmacological andchemical considerations. Rev Diabet Stud;6:1336.

16. Guo D., Hildebrandt I.J., Prins R.M., Soto H., Mazzotta M.M.,Dang J., Czernin J., Shyy J.Y., Watson A.D., Phelps M., RaduC.G., Cloughesy T.F., Mischel P.S. (2009) The AMPK agonistAICAR inhibits the growth of EGFRvIII-expressing glioblastomasby inhibiting lipogenesis. Proc Natl Acad Sci U S A;106:1293212937.

17. Erion M.D., Dang Q., Reddy M.R., Kasibhatla S.R., Huang J.,Lipscomb W.N., van Poelje P.D. (2007) Structure-guideddesign of AMP mimics that inhibit fructose-1, 6- bisphosphatasewith high affinity and specificity. J Am Chem Soc;129:1548015490.

18. Reddy M.R., Erion M.D. (2001) Calculation of relative bindingfree energy differences for fructose 1, 6-bisphosphatase inhibi-tors using the thermodynamic cycle perturbation approach. J AmChem Soc;123:62466252.

19. Reddy M.R., Erion M.D. (2007) Relative binding affinities of fruc-tose-1,6- bisphosphatase inhibitors calculated using a quantummechanics-based free energy perturbation method. J Am ChemSoc;129:92969297.

Supporting Information

Additional Supporting Information may be found in the online ver-sion of this article:

Figure S1. Key binding interactions of cordycepin and AMPK.Hydrogen bonds are presented as dotted lines.

Please note: Wiley-Blackwell is not responsible for the content orfunctionality of any supporting materials supplied by the authors.Any queries (other than missing material) should be directed to thecorresponding author for the article.

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