Discovery and Characterization of a Small Molecule Inhibitor of the PDZ Domain of Dishevelled
2009; Elsevier BV; Volume: 284; Issue: 24 Linguagem: Inglês
10.1074/jbc.m109.009647
ISSN1083-351X
AutoresDavid K. Grandy, Jufang Shan, Xinxin Zhang, Sujata Rao, Shailaja Akunuru, Hongyan Li, Yanhui Zhang, Ivan Alpatov, Xin A. Zhang, Richard A. Lang, De-Li Shi, Jie Zheng,
Tópico(s)Cancer-related gene regulation
ResumoDishevelled (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 analysis, the receptor site was first defined as all of the residues in the PDZ domain within 7.0 Å of the Dpr peptide, and the core site (i.e. where the core fragment is to be placed during docking) was defined as all residues within 10.0 Å of the Thr (−2) in the Dpr peptide. In later stages of screening, the core site was redefined as a much smaller area made up of residues Ile-267, Ser-268, Ile-269, Leu-324, Arg-325, and Val-328, which make up the hydrophobic groove. This refined definition helped to prevent docked conformations that were likely to be incorrect. For example, when the larger core site was used, some docked compounds were almost entirely exposed to the solvent, although most of the molecules are hydrophobic. As many as 30 docked conformations were generated for each compound by FlexX. Docked conformations were scored by using the five consensus score functions included in the FlexX software. The Dpr peptide was used to calibrate docking parameters and to determine high (negative) values for each individual scoring function. The backbone root mean square deviation between the crystal structure conformation and the docked conformation of Dpr was 2.86 Å. Different forms of the Dvl PDZ domain protein were synthesized by using an Escherichia coli system as described (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, 15Shan J. Shi D.L. Wang J. Zheng J. Biochemistry. 2005; 44: 15495-15503Crossref PubMed Scopus (173) Google Scholar, 16London T.B. Lee H.J. Shao Y. Zheng J. Biochem. Biophys. Res. Commun. 2004; 322: 326-332Crossref PubMed Scopus (50) Google Scholar). To prepare TMR-labeled PDZ domain, a cysteine located outside of the binding site was mutated to alanine to increase solubility of the PDZ domain. A construct without a T328C mutation was made for use in fluorescence spectroscopy experiments. The fluorescent label 2-((5(6)-tetramethylrhodamine)carboxyamino)ethyl methanethiosulfonate (TMR) was covalently bound to this Cys residue, which is the only Cys in this PDZ domain. The PDZ solution was dialyzed overnight at 4 °C against 100 mm potassium phosphate buffer at pH 7.5 to remove dithiothreitol, which had been added to prevent disulfide bond formation. A 10-fold excess of TMR dissolved in DMSO was added dropwise to the PDZ solution while stirring. After 2 h at room temperature, the unbound TMR was removed from the solution by dialysis against 100 mm potassium phosphate buffer, pH 7.5, at 4 °C. All NMR studies used either a 600-MHz Varian INOVA spectrometer or a 600-MHz Bruker Avance spectrometer. 15N-labeled PDZ samples were prepared at a concentration of 0.3 mm in 100 mm KHPO4, 0.5 mm EDTA, and 10% D2O at pH 7.5. The compounds were dissolved either in the same buffer as the PDZ domain or in DMSO, depending on the aqueous solubility of the individual compound. Titration was carried out by adding small amounts of the compound to the PDZ domain and taking 15N-HSQC spectra of the mixture. Compound concentrations varied from 0.3 to 6.0 mm during the course of these titrations. All NMR spectra were processed with NMRPipe (17Delaglio F. Grzesiek S. Vuister G.W. Zhu G. Pfeifer J. Bax A. J. Biomol. NMR. 1995; 6: 277-293Crossref PubMed Scopus (11536) Google Scholar) software and analyzed with the Sparky program(18Goddard T.D. Kneller D.G. SPARKY 3.0. University of California, San Francisco, CA1998Google Scholar). All fluorescence measurements were obtained by using a Jobin-Yvon Fluorolog-3 spectrofluorometer (HORIBA Jobin-Yvon Inc., Edison, NJ) (15Shan J. Shi D.L. Wang J. Zheng J. Biochemistry. 2005; 44: 15495-15503Crossref PubMed Scopus (173) Google Scholar). Fluorescence anisotropy measurements of TMR-labeled PDZ domain were obtained for binding affinity calculations. Titrations of compounds to the solution of TMR-labeled PDZ domain were performed at 25 °C in 100 mm KHPO4, 0.5 mm EDTA buffer, at pH 7.5. The excitation wavelength for TMR-labeled PDZ domain was 551 nm with an entrance slit width of 5 nm. The maximum fluorescence emission wavelength was 578 nm with an exit slit width of 5 nm. The compounds were prepared to a concentration of ∼1.0–20.0 mm in the same buffer as that used for the protein. During titration, the range of concentrations of the compound was ∼100 nm–1.0 mm, depending on binding affinity for the particular compound. The anisotropy data were analyzed by fitting the data to the standard ligand binding curve in the program Prism (GraphPad Software Inc., San Diego, CA). Best-fit curves were obtained by using a global, non-linear regression model that assumed that the law of mass action was followed. Although changes of fluorescence anisotropy due to the compounds binding were small, we were able to obtain binding affinity values for the small molecule compounds. Under the same conditions, fluorescence polarization measurements of the ROX-labeled Dpr peptide (ROX-N-butyric-SGSLKMTTV-COOH) were also performed. The excitation wavelength for the ROX-labeled Dpr peptide was 578 nm with an entrance slit width of 5 nm. The maximum fluorescence emission wavelength was 605 nm with an exit slit width of 5 nm. By titrating the PDZ domain into 50 nm ROX-Dpr peptide solution in the absence and presence of 6 μm compound 3289-8625, respectively, we obtained the binding affinity (Kd) of Dpr and the apparent binding affinity (Kdapp) of the peptide in the presence of 3289-8625. The competition binding constant (Ki) between PDZ and 3289-8625 was calculated by the equation Kdapp = Kd (1 + [I]/Ki). Xenopus eggs were obtained from females that had been injected with 500 IU of human chorionic gonadotropin (Sigma-Aldrich) and had been artificially fertilized. Synthesis and microinjection of mRNAs were carried out as described previously (15Shan J. Shi D.L. Wang J. Zheng J. Biochemistry. 2005; 44: 15495-15503Crossref PubMed Scopus (173) Google Scholar, 19Umbhauer M. Djiane A. Goisset C. Penzo-Méndez A. Riou J.F. Boucaut J.C. Shi D.L. EMBO J. 2000; 19: 4944-4954Crossref PubMed Google Scholar). Briefly, for the luciferase assay, the siamois promoter-driven reporter DNA construct (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, 15Shan J. Shi D.L. Wang J. Zheng J. Biochemistry. 2005; 44: 15495-15503Crossref PubMed Scopus (173) Google Scholar) (400 pg) was injected alone or co-injected with Wnt3A mRNA (1 pg) into the animal pole region at the two-cell stage. Injected embryos were cultured in the absence or presence of the compound 3289-8625 at different concentrations, and animal cap explants were dissected at late blastula stage. For secondary axis assay, Wnt3A mRNA (1 pg) was injected in the ventro-vegetal region at the four-cell stage, and injected embryos were cultured in the absence or presence of the compound 3289-8625 until larval stage. The effect of the compound 3289-8625 on the PC-3 cells was examined by using the MTT cell proliferation assay (Promega, Madison, WI). In the experiments, PC-3 cells were plated in 24-well culture plates at a density of 1 × 104 cells/well and cultured overnight in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum plus penicillin and streptomycin at 37 °C. On the second day, compound 3289-8625 was added to the cells at different concentrations. Before being added to the cells, the compound was dissolved in DMSO at 200 mm. While adding the compound to the cells, the same amounts of DMSO were added to the control cells. After 68–69 h of treatment, 10% volume of MTT stock solution (5 mg/ml) was added to the cell culture. The cells were incubated at 37 °C until the total treatment time reached 72 h. The converted dye was then solubilized, and the absorbance was measured at 570 nm. Three sets of independent experiments were performed, and each data point was normalized against the control cells. PC-3 cells were seeded in 100-mm tissue culture dish, cultured in completed Dulbecco's modified Eagle's medium overnight, and then treated with 3289-8625 compound (final concentration 80 μm) or DMSO vehicle for 72 h. For the extraction of membrane and cytosolic proteins (20Zi X. Guo Y. Simoneau A.R. Hope C. Xie J. Holcombe R.F. Hoang B.H. Cancer Res. 2005; 65: 9762-9770Crossref PubMed Scopus (145) Google Scholar, 21Shimizu H. Julius M.A. Giarré M. Zheng Z. Brown A.M. Kitajewski J. Cell Growth Differ. 1997; 8: 1349-1358PubMed Google Scholar), cells were collected in TES suspension buffer and homogenized on ice. The lysate was centrifuged for 10 min at 500 × g. The crude supernatant was then fractionated at 100,000 × g for 90 min at 4 °C to generate a supernatant or cytosolic fraction and a membrane-rich pellet fraction. The membrane-enriched pellet was dissolved in phosphate-buffered saline buffer containing 1% Triton X-100 and 1% Nonidet P-40. Equal volumes of Laemmli buffer were added to protein solutions, and the samples were boiled for 5 min. The proteins were then separated by 10% SDS-PAGE electrophoresis under reducing condition, transferred to nitrocellulose membranes, blocked with 5% nonfat dry milk in phosphate-buffered saline with Tween, probed with antibody against β-catenin (Santa Cruz Biotechnology Inc., Santa Cruz, CA) and horseradish peroxidase-conjugated anti-rabbit IgG secondary antibody (Sigma-Aldrich), and visualized by a chemiluminescence detection system (PerkinElmer Life Sciences). Membranes were then stripped and reprobed with antibody against integrin α3 protein (22Zhang X.A. Bontrager A.L. Stipp C.S. Kraeft S.K. Bazzoni G. Chen L.B. Hemler M.E. Mol. Biol. Cell. 2001; 12: 351-365Crossref PubMed Scopus (53) Google Scholar) as equal loading control. To identify additional scaffolds of Dvl PDZ domain inhibitors, we carried out several rounds of new computational screening on the basis of our earlier studies (15Shan J. Shi D.L. Wang J. Zheng J. Biochemistry. 2005; 44: 15495-15503Crossref PubMed Scopus (173) Google Scholar, 23Shan J. Zheng J.J. J. Comput. Aided Mol. Des. 2009; 23: 37-47Crossref PubMed Scopus (29) Google Scholar, 24Lee H.J. Wang N.X. Shao Y. Zheng J.J. Bioorg. Med. Chem. 2009; 17: 1701-1708Crossref PubMed Scopus (24) Google Scholar). To identify possible PDZ binding compounds, we first used the UNITY module in the Sybyl software (Tripos Inc.) to screen data bases of drug-like compounds from the NCI (National Institutes of Health), ChemDiv, and Sigma-Aldrich. Hits returned from these searches were docked to the protein receptor site, and the conformations of the complexes were scored by using the FlexX module in Sybyl. The overall procedure of the computational screening was similar to that used in our earlier work (15Shan J. Shi D.L. Wang J. Zheng J. Biochemistry. 2005; 44: 15495-15503Crossref PubMed Scopus (173) Google Scholar), but many details were further refined in this study. 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 Scholar); we chose the ligand-based query approach, which was not 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. Distance and angle constraints between those atoms were used to run a three-dimensional flexible screen of the NCI (National Institutes of Health) and ChemDiv data bases; in addition, the Sigma-Aldrich data bases were searched by using the Sigma-Aldrich online two-dimensional search utility. The initial Unity query generated a list of several thousand compounds as potential hits. The program FlexX (25Kramer B. Rarey M. Lengauer T. Proteins. 1999; 37: 228-241Crossref PubMed Scopus (818) Google Scholar) was then used in the docking studies. This program uses five scoring functions to evaluate docking results: the standard FlexX scoring function F_score (26Böhm H.J. J Comput. Aided Mol. Des. 1992; 6: 61-78Crossref PubMed Scopus (778) Google Scholar, 27Böhm H.J. J Comput. Aided Mol. Des. 1994; 8: 243-256Crossref PubMed Scopus (985) Google Scholar, 28Klebe G. Mietzner T. J Comput. Aided Mol. Des. 1994; 8: 583-606Crossref PubMed Scopus (158) Google Scholar), the Chemscore function (29Eldridge M.D. Murray C.W. Auton T.R. Paolini G.V. Mee R.P. J Comput. Aided Mol. Des. 1997; 11: 425-445Crossref PubMed Scopus (1495) Google Scholar), the knowledge-based proton motive force (PMF) score (based entirely on protein-ligand atom pairs and their distances (30Muegge I. Martin Y.C. J Med. Chem. 1999; 42: 791-804Crossref PubMed Scopus (972) Google Scholar)), the G_score function (calculated from ligand-protein atom pair interactions based on values from the Tripos force field (31Jones G. Willett P. Glen R.C. Leach A.R. Taylor R. J. Mol. Biol. 1997; 267: 727-748Crossref PubMed Scopus (5365) Google Scholar)), and the D_score (based on the atom charges and the Van der Waals interactions between the ligand and protein (32Kuntz I.D. Blaney J.M. Oatley S.J. Langridge R. Ferrin T.E. J. Mol. Biol. 1982; 161: 269-288Crossref PubMed Scopus (1873) Google Scholar)). Data from our earlier studies (15Shan J. Shi D.L. Wang J. Zheng J. Biochemistry. 2005; 44: 15495-15503Crossref PubMed Scopus (173) Google Scholar) and from the additional experiments in this study revealed that the F_score and Chemscore functions were the most reliable in predicting binding to the PDZ domain. Therefore, in this study, we used only the F_score and Chemscore rather than the consensus score of all five scoring functions. The G_score function was used as an additional reference because in our experience, G_score can help to identify internal steric hindrance in a docked conformation. Approximately 50 high scoring compounds were then obtained from the NCI (National Institutes of Health) and from Sigma-Aldrich and further screened by using NMR spectroscopy. Before the NMR studies, the FlexX docked complexes of all selected compounds were visually inspected to confirm that the ligands were in the peptide binding groove of the PDZ domain and that there was no internal steric hindrance. To validate docking results, we performed 1H-15N correlated NMR spectroscopy. We obtained the 15N-HSQC spectra (15Shan J. Shi D.L. Wang J. Zheng J. Biochemistry. 2005; 44: 15495-15503Crossref PubMed Scopus (173) Google Scholar) by titrating various concentrations of the small molecules into samples of 15N-labeled mDvl1 PDZ domain. Examination of the spectra for chemical shift perturbations (33Wüthrich K. Nat. Struct. Biol. 2000; 7: 188-189Crossref PubMed Scopus (35) Google Scholar) revealed several small molecules that bound to the conventional C-terminal peptide binding groove of the PDZ domain; many of those NMR-confirmed hits showed a butyric acid substructure at one end. On the basis of this butyric acid group, we used the program Unity to perform a substructure search of the NCI (National Institutes of Health), Sigma-Aldrich, and ChemDiv compound data bases. The program FlexX was then used to dock and score the hits returned from the Unity screen. Several of the highest scoring compounds in the FlexX docking had a similar core structure that was predicted by FlexX to bind in the traditional binding groove of the PDZ domain. Those compounds were obtained from ChemDiv, Inc. and were tested by NMR spectroscopy. In the NMR experiments, compound 3289-5066 and compound 3289-8625 (Fig. 1) displayed the most significant chemical shift perturbations when titrated into the solution of 15N-labled Dvl PDZ domain. Their chemical shift patterns were generally similar and closely resembled that seen 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). Compound 3289-8625 bound more strongly to the Dvl PDZ domain, as judged by the chemical shift perturbations due to ligand binding (Fig. 2). When the weighted sums of the chemical shift data were plotted onto a tube representation of the mDvl1 backbone, compound 3289-8625 was seen to bind in the groove between the βB sheet and the αB helix (Fig. 2).FIGURE 2Interaction between the Dvl PDZ domain and compound 3289-8625. Shown are 15N-HSQC spectra of free PDZ domain (red) and PDZ domain with increasing concentrations of compound 3289-8625 (orange, green, cyan, purple, and blue, ligand:protein ratios of 1, 3, 5, 7, and 15). Upper inset, tube diagram of the PDZ domain with the weighted chemical shift intensities from the overlaid NMR spectra shown as regions of differing width and color. The widest (and red) regions contain the residues showing the greatest chemical shift. Lower inset, detail view of several peaks showing large chemical shift.View Large Image Figure ViewerDownload Hi-res image Download (PPT) To further assess the binding characteristics of the molecules identified in the virtual screen, we used fluorescence anisotropy to measure the binding affinities of the identified inhibitors to the Dvl PDZ domain. In these experiments, each small molecule inhibitor was titrated into a solution of TMR-labeled PDZ domain, and the anisotropy change due to ligand binding was used to determine the binding affinity of the inhibitor to the Dvl PDZ domain. To verify the accuracy of this method, we first determined the binding affinity between the PDZ domain and the Dpr peptide; this value (11.0 ± 1.4 μm) was consistent with the value obtained by other methods (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, 15Shan J. Shi D.L. Wang J. Zheng J. Biochemistry. 2005; 44: 15495-15503Crossref PubMed Scopus (173) Google Scholar). The fluorescence method yielded a measured binding affinity (Kd) value of 10.6 ± 1.7 μm between the PDZ domain and compound 3289-8625; the fluorescence data are plotted in Fig. 3. We also measured the binding of other, similar compounds identified in this study to the Dvl PDZ domain. As suggested by the NMR studies, all of these compounds had weaker binding affinities than did compound 3289-8625; for example, measured by the fluorescence anisotropy method, the binding affinity between compound 2372-2393 and the Dvl PDZ domain was 18.9 ± 2.1 μm. The binding affinity between compound 3289-8625 and the Dvl PDZ domain is comparable with the binding affinity of the Dpr peptide to the Dvl PDZ domain. To further demonstrate that compound 3289-8625 can compete with the Dpr peptide, we also measured the binding affinity between the Dpr peptide and the PDZ domain by monitoring the change of fluorescence polarization during titration of unlabeled Dvl PDZ into a solution of the fluorescent ROX-labeled Dpr peptide. This method yielded a measured binding affinity of 6.1 ± 0.4 μm., which is in good agreement with the value obtained by using TMR-labeled PDZ domain as described above (the slight difference may due to the effects of fluorescence tag labeling or experimental errors). By using the same assay, we showed that compound 3289-8625 inhibited the interaction between the Dpr peptide and the PDZ domain in the manner of classical competitive inhibition (KI values, 4.9 ± 1.7 μm, Fig. 3B). The data clearly show that compound 3289-8625 and the Dpr peptide competed for the same site on the surface of Dvl PDZ domain. Indeed, the conformation of PDZ-bound compound 3289-8625 as calculated by FlexX lies in this same region and closely resembles the crystal structure conformation of the MTTV motif of the Dpr peptide (10Cheyette B.N. Waxman
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