Discovery and Characterization of a Small Molecule Inhibitor of the PDZ Domain of Dishevelled
Dishevelled (Dvl) is an essential protein in the Wnt signaling pathways; it uses its PDZ domain to transduce the Wnt signals from the membrane receptor Frizzled to downstream components. Here, we report identifying a drug-like small molecule compound through structure-based ligand screening and NMR spectroscopy and show the compound to interact at low micromolar affinity with the PDZ domain of Dvl. In a Xenopus testing system, the compound could permeate the cell membrane and block the Wnt signaling pathways. In addition, the compound inhibited Wnt signaling and reduced the levels of apoptosis in the hyaloid vessels of eye. Moreover, this compound also suppressed the growth of prostate cancer PC-3 cells. These biological effects suggest that by blocking the PDZ domain of Dvl, the compound identified in our studies effectively inhibits the Wnt signaling and thus provides a useful tool for studies dissecting the Wnt signaling pathways. Dishevelled (Dvl) is an essential protein in the Wnt signaling pathways; it uses its PDZ domain to transduce the Wnt signals from the membrane receptor Frizzled to downstream components. Here, we report identifying a drug-like small molecule compound through structure-based ligand screening and NMR spectroscopy and show the compound to interact at low micromolar affinity with the PDZ domain of Dvl. In a Xenopus testing system, the compound could permeate the cell membrane and block the Wnt signaling pathways. In addition, the compound inhibited Wnt signaling and reduced the levels of apoptosis in the hyaloid vessels of eye. Moreover, this compound also suppressed the growth of prostate cancer PC-3 cells. These biological effects suggest that by blocking the PDZ domain of Dvl, the compound identified in our studies effectively inhibits the Wnt signaling and thus provides a useful tool for studies dissecting the Wnt signaling pathways. The Wnt signaling pathways are regulated by a family of secreted Wnt glycoproteins. The canonical Wnt pathway, which is highly conserved, is best understood. In this pathway, Wnt molecules interact with the seven-transmembrane Frizzled (Fz) 2The abbreviations used are: FzFrizzledDvlDishevelledDprDapper (Dpr)TMR2-((5(6)-tetramethylrhodamine)carboxyamino)ethyl methanethiosulfonateDMSOdimethyl sulfoxideHSQCheteronuclear single quantum correlationMTT3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromideTES2-{[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]amino}ethanesulfonic acidTUNELterminal deoxynucleotidyltransferase-mediated dUTP nick end-labeling.2The abbreviations used are: FzFrizzledDvlDishevelledDprDapper (Dpr)TMR2-((5(6)-tetramethylrhodamine)carboxyamino)ethyl methanethiosulfonateDMSOdimethyl sulfoxideHSQCheteronuclear single quantum correlationMTT3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromideTES2-{[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]amino}ethanesulfonic acidTUNELterminal deoxynucleotidyltransferase-mediated dUTP nick end-labeling. proteins (1Bhanot P. Brink M. Samos C.H. Hsieh J.C. Wang Y. Macke J.P. Andrew D. Nathans J. Nusse R. Nature. 1996; 382: 225-230Crossref PubMed Scopus (1223) Google Scholar) by binding to an N-terminal cysteine-rich-domain (2Dann C.E. Hsieh J.C. Rattner A. Sharma D. Nathans J. Leahy D.J. Nature. 2001; 412: 86-90Crossref PubMed Scopus (372) Google Scholar). The signal is then transduced into the cell through an internal sequence of Fz, C-terminal to the seventh transmembrane domain, which binds directly to the PDZ (postsynaptic density-95/discs large/zonula occludens-1) domain of the cytoplasmic protein Dishevelled (Dvl) (3Wong H.C. Bourdelas A. Krauss A. Lee H.J. Shao Y. Wu D. Mlodzik M. Shi D.L. Zheng J. Mol. Cell. 2003; 12: 1251-1260Abstract Full Text Full Text PDF PubMed Scopus (392) Google Scholar). Dvl then transduces the Wnt signals to downstream components (4Wallingford J.B. Habas R. Development. 2005; 132: 4421-4436Crossref PubMed Scopus (374) Google Scholar). Three Dvl homologs (Dvl-1, -2, and -3) have been identified in humans; all are expressed in both embryonic and adult tissues, including brain, heart, lung, kidney, skeletal muscle, and others (4Wallingford J.B. Habas R. Development. 2005; 132: 4421-4436Crossref PubMed Scopus (374) Google Scholar). Up-regulation and overexpression of Dvl proteins have been reported in many cancers, including those of breast, colon, prostate, mesothelium, and lung (non-small cell) (5Uematsu K. He B. You L. Xu Z. McCormick F. Jablons D.M. Oncogene. 2003; 22: 7218-7221Crossref PubMed Scopus (285) Google Scholar, 6Uematsu K. Kanazawa S. You L. He B. Xu Z. Li K. Peterlin B.M. McCormick F. Jablons D.M. Cancer Res. 2003; 63: 4547-4551PubMed Google Scholar, 7Bui T.D. Beier D.R. Jonssen M. Smith K. Dorrington S.M. Kaklamanis L. Kearney L. Regan R. Sussman D.J. Harris A.L. Biochem. Biophys. Res. Commun. 1997; 239: 510-516Crossref PubMed Scopus (20) Google Scholar, 8Mizutani K. Miyamoto S. Nagahata T. Konishi N. Emi M. Onda M. Tumori. 2005; 91: 546-551Crossref PubMed Scopus (44) Google Scholar). Frizzled Dishevelled Dapper (Dpr) 2-((5(6)-tetramethylrhodamine)carboxyamino)ethyl methanethiosulfonate dimethyl sulfoxide heteronuclear single quantum correlation 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide 2-{[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]amino}ethanesulfonic acid terminal deoxynucleotidyltransferase-mediated dUTP nick end-labeling. Frizzled Dishevelled Dapper (Dpr) 2-((5(6)-tetramethylrhodamine)carboxyamino)ethyl methanethiosulfonate dimethyl sulfoxide heteronuclear single quantum correlation 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide 2-{[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]amino}ethanesulfonic acid terminal deoxynucleotidyltransferase-mediated dUTP nick end-labeling. The Dvl protein is made up of three conserved domains: an N-terminal DIX domain, a central PDZ domain, and a C-terminal DEP domain (9Wong H.C. Mao J. Nguyen J.T. Srinivas S. Zhang W. Liu B. Li L. Wu D. Zheng J. Nat. Struct. Biol. 2000; 7: 1178-1184Crossref PubMed Scopus (126) Google Scholar). The central PDZ domain is of particular interest because of its interaction with Fz and other Wnt pathway proteins (3Wong H.C. Bourdelas A. Krauss A. Lee H.J. Shao Y. Wu D. Mlodzik M. Shi D.L. Zheng J. Mol. Cell. 2003; 12: 1251-1260Abstract Full Text Full Text PDF PubMed Scopus (392) Google Scholar, 10Cheyette B.N. Waxman J.S. Miller J.R. Takemaru K. Sheldahl L.C. Khlebtsova N. Fox E.P. Earnest T. Moon R.T. Dev. Cell. 2002; 2: 449-461Abstract Full Text Full Text PDF PubMed Scopus (219) Google Scholar). The direct interaction between the PDZ domain and Fz peptides is relatively weak, and other factors may play a role to ensure the communication between the two molecules (3Wong H.C. Bourdelas A. Krauss A. Lee H.J. Shao Y. Wu D. Mlodzik M. Shi D.L. Zheng J. Mol. Cell. 2003; 12: 1251-1260Abstract Full Text Full Text PDF PubMed Scopus (392) Google Scholar). For example, several studies suggest that the DEP domain of Dvl has a membrane-targeting function that may facilitate PDZ-Fz interaction (11Axelrod J.D. Miller J.R. Shulman J.M. Moon R.T. Perrimon N. Genes Dev. 1998; 12: 2610-2622Crossref PubMed Scopus (540) Google Scholar, 12Axelrod J.D. Genes Dev. 2001; 15: 1182-1187PubMed Google Scholar, 13Boutros M. Paricio N. Strutt D.I. Mlodzik M. Cell. 1998; 94: 109-118Abstract Full Text Full Text PDF PubMed Scopus (662) Google Scholar, 14Rothbächer U. Laurent M.N. Deardorff M.A. Klein P.S. Cho K.W. Fraser S.E. EMBO J. 2000; 19: 1010-1022Crossref PubMed Scopus (241) Google Scholar). However, the weak PDZ-Fz interaction provides an opportunity to block Wnt signaling at the Dvl level by using a small molecule inhibitor. An earlier study in our laboratories used an NMR-assisted virtual ligand screening approach to identify a peptide mimic that can bind to the Dvl PDZ domain (15Shan J. Shi D.L. Wang J. Zheng J. Biochemistry. 2005; 44: 15495-15503Crossref PubMed Scopus (173) Google Scholar). We have now used an improved algorithm to conduct an additional structure-based virtual screen of the PDZ domain of Dvl and have discovered a group of drug-like compounds that bind to the PDZ domain with moderate to low micromolar affinity. One of these compounds effectively blocked Wnt signaling in vivo and reduced the growth rate of a prostate cancer cell line. All compound data bases were obtained from the NCI (National Institutes of Health), Chemical Diversity Inc. (ChemDiv, San Diego, CA), or Sigma-Aldrich. Three-dimensional coordinates for all compounds were generated by using the Optive Research Concord program (Tripos Inc., St. Louis, MO) and stored in the Sybyl mol2 format. The Unity module of the Sybyl software package (Tripos Inc.) was used to select the compounds in the three-dimensional small molecule data base that matched the known ligand of the Dvl PDZ domain. The first Unity query was based on three-dimensional distance constraints determined by analyzing the structure of the Dapper (Dpr) peptide-PDZ complex (10Cheyette B.N. Waxman J.S. Miller J.R. Takemaru K. Sheldahl L.C. Khlebtsova N. Fox E.P. Earnest T. Moon R.T. Dev. Cell. 2002; 2: 449-461Abstract Full Text Full Text PDF PubMed Scopus (219) Google Scholar), which was obtained from the Protein Data Bank (PDB code: 1L6O). The atoms of the bound Dpr peptide within hydrogen-bonding distance of suitable H-bond acceptors and donors on the backbone of the βB sheet of the PDZ domain were selected. The compounds selected from the Unity queries were docked to the Dpr peptide binding site of Dvl PDZ domain by using the FlexX module of the Sybyl software package. For the docking the receptor site was first all of the in the PDZ domain within of the Dpr and the site the is to was all within of the in the Dpr In of the site was a made up of and which up the to docked that were to For example, the site was docked compounds were to the of the molecules are many docked were generated for compound by were by using the in the FlexX The Dpr peptide was used to docking and to for The backbone between the structure and the docked of Dpr was of the Dvl PDZ domain protein were by using an (3Wong H.C. Bourdelas A. Krauss A. Lee H.J. Shao Y. Wu D. Mlodzik M. Shi D.L. Zheng J. Mol. Cell. 2003; 12: 1251-1260Abstract Full Text Full Text PDF PubMed Scopus (392) Google Scholar, J. Shi D.L. Wang J. Zheng J. Biochemistry. 2005; 44: 15495-15503Crossref PubMed Scopus (173) Google Scholar, Lee H.J. Shao Y. Zheng J. Biochem. Biophys. Res. Commun. PubMed Scopus Google Scholar). PDZ domain, a of the binding site was to to of the PDZ domain. a was made for in spectroscopy The 2-((5(6)-tetramethylrhodamine)carboxyamino)ethyl methanethiosulfonate was bound to this which is the in this PDZ domain. The PDZ was at at to which been to of in was to the PDZ at the was from the by at All NMR studies used a or a PDZ were at a of in and at The compounds were in the the PDZ domain or in on the of the was by small of the compound to the PDZ domain and of the from to the of these All NMR were with F. S. J. A. J. PubMed Scopus Google Scholar) software and with the T.D. of San Scholar). All were obtained by using a Inc., (15Shan J. Shi D.L. Wang J. Zheng J. Biochemistry. 2005; 44: 15495-15503Crossref PubMed Scopus (173) Google Scholar). of PDZ domain were obtained for binding affinity of compounds to the of PDZ domain were at in at The for PDZ domain was with an of The was with an of The compounds were to a of in the that used for the the of of the compound was on binding affinity for the particular The data were by the data to the ligand binding in the program Inc., San Diego, were obtained by using a that that the of was of to the compounds binding were we were to binding affinity for the small molecule the of the Dpr peptide were also The for the Dpr peptide was with an of The was with an of the PDZ domain into peptide in the and of compound we obtained the binding affinity of Dpr and the binding affinity of the peptide in the of The binding between PDZ and was by the Xenopus were obtained from that been with of and been and of were (15Shan J. Shi D.L. Wang J. Zheng J. Biochemistry. 2005; 44: 15495-15503Crossref PubMed Scopus (173) Google Scholar, M. A. A. J.C. Shi D.L. EMBO J. 2000; 19: PubMed Google Scholar). for the the (3Wong H.C. Bourdelas A. Krauss A. Lee H.J. Shao Y. Wu D. Mlodzik M. Shi D.L. Zheng J. Mol. Cell. 2003; 12: 1251-1260Abstract Full Text Full Text PDF PubMed Scopus (392) Google Scholar, J. Shi D.L. Wang J. Zheng J. Biochemistry. 2005; 44: 15495-15503Crossref PubMed Scopus (173) Google Scholar) was or with into the at the were in the or of the compound at and were at For was in the at the and were in the or of the compound The of the compound on the PC-3 was by using the cell In the PC-3 were in at a of and in and at the compound was to the at to the the compound was in at the compound to the the of were to the cells. of of was to the cell The were at the The was then and the was at Three of were and data was the cells. PC-3 were in in and then with compound or for For the of membrane and proteins Y. J. Cancer Res. 2005; PubMed Scopus Google Scholar, M.A. M. Zheng Z. J. 1997; Google Scholar), were in and on The was for at The was then at for at to a or and a The was in and of were to protein and the were for The proteins were then by to blocked with in with with Inc., and and by a were then and with protein A.L. Mol. Biol. Cell. 2001; 12: PubMed Scopus Google Scholar) identify additional of Dvl PDZ domain we several of screening on the of our earlier studies (15Shan J. Shi D.L. Wang J. Zheng J. Biochemistry. 2005; 44: 15495-15503Crossref PubMed Scopus (173) Google Scholar, J. Zheng J. Mol. PubMed Scopus Google Scholar, H.J. Wang Shao Y. Zheng PubMed Scopus Google Scholar). identify PDZ binding we first used the module in the Sybyl software (Tripos Inc.) to screen data bases of drug-like compounds from the NCI (National Institutes of Health), and Sigma-Aldrich. from these were docked to the protein receptor and the of the were by using the FlexX module in The of the screening was to that used in our earlier (15Shan J. Shi D.L. Wang J. Zheng J. Biochemistry. 2005; 44: 15495-15503Crossref PubMed Scopus (173) Google Scholar), many were in this The first Unity query was based on three-dimensional distance constraints determined by analyzing the structure of the Dpr peptide-PDZ complex (10Cheyette B.N. Waxman J.S. Miller J.R. Takemaru K. Sheldahl L.C. Khlebtsova N. Fox E.P. Earnest T. Moon R.T. Dev. Cell. 2002; 2: 449-461Abstract Full Text Full Text PDF PubMed Scopus (219) Google we the query which was used in our earlier study (15Shan J. Shi D.L. Wang J. Zheng J. Biochemistry. 2005; 44: 15495-15503Crossref PubMed Scopus (173) Google Scholar). The atoms of the bound Dpr peptide within hydrogen-bonding distance of suitable H-bond acceptors and donors on the backbone of the βB sheet of the PDZ domain were selected. and constraints between those atoms were used to a three-dimensional screen of the NCI (National Institutes of and data in addition, the data bases were by using the The Unity query generated a of several compounds The program FlexX B. M. T. PubMed Scopus Google Scholar) was then used in the docking program uses to docking the FlexX function H.J. Mol. PubMed Scopus Google Scholar, H.J. Mol. PubMed Scopus Google Scholar, T. Mol. PubMed Scopus Google Scholar), the function Mol. 1997; PubMed Scopus Google Scholar), the on and PubMed Scopus Google the function from based on from the P. R. J. Mol. Biol. 1997; PubMed Scopus Google and the on the and the between the ligand and protein J.M. R. J. Mol. Biol. 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Biol. 2000; 7: PubMed Scopus Google Scholar) several small molecules that bound to the C-terminal peptide binding of the PDZ many of those a acid at the of this acid we used the program Unity to a of the NCI (National Institutes of Health), and compound data The program FlexX was then used to and the from the Unity of the compounds in the FlexX docking a structure that was by FlexX to bind in the binding of the PDZ domain. compounds were obtained from Inc. and were by NMR In the NMR compound and compound the into the of Dvl PDZ domain. were and that with Dpr and Fz binding (3Wong H.C. Bourdelas A. Krauss A. Lee H.J. Shao Y. Wu D. Mlodzik M. Shi D.L. Zheng J. Mol. Cell. 2003; 12: 1251-1260Abstract Full Text Full Text PDF PubMed Scopus (392) Google Scholar). bound to the Dvl PDZ domain, by the to ligand binding the of the data were a of the compound was to bind in the between the βB sheet and the between the Dvl PDZ domain and compound are of PDZ domain and PDZ domain with of compound and of and of the PDZ domain with the from the NMR of and The the the of several the binding of the molecules identified in the virtual we used to the binding of the identified to the Dvl PDZ domain. In these small molecule was into a of PDZ domain, and the to ligand binding was used to the binding affinity of the to the Dvl PDZ domain. the of this we first determined the binding affinity between the PDZ domain and the Dpr this was with the obtained by other (3Wong H.C. Bourdelas A. Krauss A. Lee H.J. Shao Y. Wu D. Mlodzik M. Shi D.L. Zheng J. Mol. Cell. 2003; 12: 1251-1260Abstract Full Text Full Text PDF PubMed Scopus (392) Google Scholar, J. Shi D.L. Wang J. 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The a binding affinity of between the PDZ domain and compound the data are in We also the binding of compounds identified in this study to the Dvl PDZ domain. by the NMR all of these compounds binding compound for example, by the the binding affinity between compound and the Dvl PDZ domain was The binding affinity between compound and the Dvl PDZ domain is with the binding affinity of the Dpr peptide to the Dvl PDZ domain. that compound can with the Dpr we also the binding affinity between the Dpr peptide and the PDZ domain by the of of Dvl PDZ into a of the Dpr a binding affinity of which is in with the obtained by using PDZ domain may to the effects of or using the we that compound inhibited the interaction between the Dpr peptide and the PDZ domain in the of The data show that compound and the Dpr peptide for the site on the of Dvl PDZ domain. the of compound by FlexX in this and the structure of the of the Dpr peptide (10Cheyette B.N. Waxman J.S. Miller J.R. Takemaru K. Sheldahl L.C. Khlebtsova N. Fox E.P. Earnest T. Moon R.T. Dev. Cell. 2002; 2: 449-461Abstract Full Text Full Text PDF PubMed Scopus (219) Google Scholar). the of Dpr and this compound are to The on of the compound the of and the on the other of the compound in the the The C-terminal of Dpr and that of the compound in the can interact with the PDZ domain The PDZ domain of Dvl directly with the conserved sequence C-terminal to the seventh transmembrane of the Wnt receptor Fz (3Wong H.C. Bourdelas A. Krauss A. Lee H.J. Shao Y. Wu D. Mlodzik M. Shi D.L. Zheng J. Mol. Cell. 2003; 12: 1251-1260Abstract Full Text Full Text PDF PubMed Scopus (392) Google Scholar), and Wnt signaling can inhibited by blocking this interaction (3Wong H.C. Bourdelas A. Krauss A. Lee H.J. Shao Y. Wu D. Mlodzik M. Shi D.L. Zheng J. Mol. Cell. 2003; 12: 1251-1260Abstract Full Text Full Text PDF PubMed Scopus (392) Google Scholar, J. Shi D.L. Wang J. Zheng J. 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However, the is from a of the identified by in screening and additional are to these screening In our NMR spectroscopy was used to a to identify small molecules that approach has to in our The of the study was to identify and small molecule of the PDZ domain of Dvl, and using the approach of NMR-assisted virtual we identified several Wnt signaling an role in embryonic and the of cell and its has been in cancer and other S. M. S. W. Nature. 2005; PubMed Scopus Google Scholar, Biophys. 2003; PubMed Scopus Google Scholar, Nusse R. Dev. Biol. PubMed Scopus Google Scholar, R.T. A. Nat. PubMed Scopus Google Scholar). For this within the Wnt signaling pathways have useful for the of N. Nat. PubMed Scopus Google Scholar). The cytoplasmic protein Dvl the Wnt signal from the Wnt receptor to downstream through the interaction between its PDZ domain and Wnt receptor the ligand binding site on the of the PDZ domain provides a site for small molecule to block the Wnt signaling pathway, and small molecules may a tool to study Wnt In addition, it may also in to the of that can with Wnt signal that to The best Dvl PDZ domain identified in our compound has a binding affinity to Dvl, which is to that of Fz, a Dvl PDZ domain binding in the Wnt signaling pathway (3Wong H.C. Bourdelas A. Krauss A. Lee H.J. Shao Y. Wu D. Mlodzik M. Shi D.L. Zheng J. Mol. Cell. 2003; 12: 1251-1260Abstract Full Text Full Text PDF PubMed Scopus (392) Google Scholar). this compound may useful for blocking the Wnt signaling pathway at the interaction of in our studies that this compound have the of blocking Wnt signaling in For a small molecule that can used to an interaction the compound also has to to through the cell The of compound is In addition, the of the compound is and the of the molecules is All these that the compound is we have its membrane our studies with Xenopus and cell that the compound was to through the of Xenopus and PC-3 cells. in vivo studies also that the compound could the membrane of cells. In by screening data bases of small molecules that were we were to identify several PDZ binding compounds the for and of the ligand In addition, we also that of the best could effectively the Wnt signals in the identified small molecule can used for also a tool for of the Wnt signaling pathways. We for the and and the Protein at St. Research for and Zhang for with NMR for and for the