The bis-indole indirubin is an active ingredient of Danggui Longhui Wan, a traditional Chinese medicine recipe used in the treatment of chronic diseases such as leukemias. The antitumoral properties of indirubin appear to correlate with their antimitotic effects. Indirubins were recently described as potent (IC50: 50–100 nm) inhibitors of cyclin-dependent kinases (CDKs). We report here that indirubins are also powerful inhibitors (IC50: 5–50 nm) of an evolutionarily related kinase, glycogen synthase kinase-3β (GSK-3β). Testing of a series of indoles and bis-indoles against GSK-3β, CDK1/cyclin B, and CDK5/p25 shows that only indirubins inhibit these kinases. The structure-activity relationship study also suggests that indirubins bind to GSK-3β's ATP binding pocket in a way similar to their binding to CDKs, the details of which were recently revealed by crystallographic analysis. GSK-3β, along with CDK5, is responsible for most of the abnormal hyperphosphorylation of the microtubule-binding protein tau observed in Alzheimer's disease. Indirubin-3′-monoxime inhibits tau phosphorylation in vitro and in vivo at Alzheimer's disease-specific sites. Indirubins may thus have important implications in the study and treatment of neurodegenerative disorders. Indirubin-3′-monoxime also inhibits the in vivophosphorylation of DARPP-32 by CDK5 on Thr-75, thereby mimicking one of the effects of dopamine in the striatum. Finally, we show that many, but not all, reported CDK inhibitors are powerful inhibitors of GSK-3β. To which extent these GSK-3β effects of CDK inhibitors actually contribute to their antimitotic and antitumoral properties remains to be determined. Indirubins constitute the first family of low nanomolar inhibitors of GSK-3β to be described. The bis-indole indirubin is an active ingredient of Danggui Longhui Wan, a traditional Chinese medicine recipe used in the treatment of chronic diseases such as leukemias. The antitumoral properties of indirubin appear to correlate with their antimitotic effects. Indirubins were recently described as potent (IC50: 50–100 nm) inhibitors of cyclin-dependent kinases (CDKs). We report here that indirubins are also powerful inhibitors (IC50: 5–50 nm) of an evolutionarily related kinase, glycogen synthase kinase-3β (GSK-3β). Testing of a series of indoles and bis-indoles against GSK-3β, CDK1/cyclin B, and CDK5/p25 shows that only indirubins inhibit these kinases. The structure-activity relationship study also suggests that indirubins bind to GSK-3β's ATP binding pocket in a way similar to their binding to CDKs, the details of which were recently revealed by crystallographic analysis. GSK-3β, along with CDK5, is responsible for most of the abnormal hyperphosphorylation of the microtubule-binding protein tau observed in Alzheimer's disease. Indirubin-3′-monoxime inhibits tau phosphorylation in vitro and in vivo at Alzheimer's disease-specific sites. Indirubins may thus have important implications in the study and treatment of neurodegenerative disorders. Indirubin-3′-monoxime also inhibits the in vivophosphorylation of DARPP-32 by CDK5 on Thr-75, thereby mimicking one of the effects of dopamine in the striatum. Finally, we show that many, but not all, reported CDK inhibitors are powerful inhibitors of GSK-3β. To which extent these GSK-3β effects of CDK inhibitors actually contribute to their antimitotic and antitumoral properties remains to be determined. Indirubins constitute the first family of low nanomolar inhibitors of GSK-3β to be described. cyclin-dependent kinase Alzheimer's disease hypotonic lysis buffer paired helical filaments protein kinase A (cAMP-dependent kinase) glycogen synthase kinase-3β electron impact 4-morpholinepropanesulfonic acid dithiothreitol bovine serum albumin polyacrylamide gel electrophoresis Indigoı̈ds are bis-indoles derived from various natural sources by fermentation, oxidation, and dimerization in the presence of light. The colorful indirubin (1) and indigo (5) originate from the dimerization of colorless precursors, indoxyl and isatin (4) (see Fig. 1). These indoles are released during the fermentation process from conjugates, the nature of which depends on the plant (indican, isatan B) or mollusc (indoxylsulfate) species from which the dyes are prepared (see Fig.1). The use of indigoı̈ds as textile dyes dates back to the Bronze age (−7000), but indigo (now synthetic) remains the most abundantly produced dye in the world (blue jeans, denims, etc.) (1Hurry J.B. The Woad Plant and Its Dye. Oxford University Press, Oxford, UK1930Google Scholar, 2Balfour-Paul J. Indigo. British Museum Press, London1998Google Scholar). Indigo-producing plants have also been used in traditional Chinese medicine (3Tang W. Eisenbrand G. Chinese Drugs of Plant Origin: Chemistry, Pharmacology, and Use in Traditional and Modern Medicine. Springer-Verlag, Heidelberg1992Crossref Google Scholar, 4Lee, H. (1993) in Human Medicinal Agents from Plants(Kinghorn, A. D., and Balandrin, M. F., eds) Chapter 12, pp. 170–190, ACS Symposium Series 534, American Chemical Society, Washington, DC.Google Scholar, 5Han R. J. Ethnopharmacol. 1998; 24: 1-17Crossref Scopus (56) Google Scholar). A well-studied example is Danggui Longhui Wan, a mixture of 11 herbal medicines traditionally utilized against certain types of leukemias. Only one of these ingredients, Qing Dai (Indigo naturalis), a dark blue powder originating from various indigo-producing plants, was found to carry the antileukemic activity (6Chen D.H. Xie J.X. Chinese Trad. Herbal Drugs. 1984; 15: 6-8Google Scholar). Although it is mostly constituted of indigo, a minor constituent, indirubin, was identified as the active component by the Chinese Academy of Medicine (7Institute of Haematology, Chinese Academy of Medical Sciences Chinese J. Intern. Med. 1979; 15: 86-88Google Scholar, 8Chen D.W. Li Y.F. Ye H.P. Comm. Chinese Herbal Med. 1979; 10: 7-9Google Scholar, 9Wu L.M. Yang Y.P. Zhu Z.H. Comm. Chinese Herbal Med. 1979; 9: 6-8Google Scholar). Preclinical studies performed with indirubin, and more soluble analogues, confirmed that these compounds exhibit good antitumor activity and only minor toxicity (10Ji X.J. Zhang F.R. Lei J.L. Xu Y.T. Acta Pharm. Sin. 1981; 16: 146-148Google Scholar, 11Sichuan Institute of Traditional Chinese Medicine Chinese Trad. Herb Drugs. 1981; 12: 27-29Google Scholar, 12Wang J.H. You Y.C. Mi J.X. Ying H.G. Acta Pharm. Sin. 1981; 2: 241-244Google Scholar, 13Ji X.J. Zhang F.R. Acta Pharm. Sin. 1985; 20: 137-139Google Scholar, 14Li C. Go Y. Mao Z. Koyano K. Kai Y. Kaneshisa N. Zhu Q. Zhou Z. Wu S. Bull. Chem. Soc. Jpn. 1996; 69: 1621-1627Crossref Scopus (27) Google Scholar). Clinical trials showed that indirubin has a definite efficiency against chronic myelocytic leukemia (5Han R. J. Ethnopharmacol. 1998; 24: 1-17Crossref Scopus (56) Google Scholar, 15Institute of Haematology, Chinese Academy of Medical Sciences Chinese J. Intern. Med. 1979; 18: 83-88PubMed Google Scholar, 16Cooperative Group of Clinical Therapy of Indirubin Chinese J. Intern. Med. 1980; 1: 132-135Google Scholar, 17Gan W.J. Yang T.Y. Wen S.D. Liu Y.Y. Tan Z. Deng C.A. Wu J.X. Liu M.P. Chinese J. Hematol. 1985; 6: 611-613Google Scholar, 18Zhang Z.N. Liu E.K. Zheng T.L. J. Trad. Chinese Med. 1985; 5: 246-248PubMed Google Scholar, 19Zhang Z.N. Liu E.K. Zheng T.L. Chinese J. Integrated Trad. Western Med. 1985; 5: 80-82Google Scholar, 20Ma M. Yao B. J. Tradit. Chinese Med. 1983; 3: 245-248PubMed Google Scholar, 21Chang C.N. Chang H.M. Yeung H.W. Tso W. Koo A. Advances in Chinese Medicinal Materials Research. World Scientific Publishing Company, Singapore1985: 369-376Google Scholar). Several mechanisms of action have been brought forward to explain the antimitotic and antitumoral properties of indirubins (22Wu G.Y. Fang F.D. Liu J.Z. Chang A. Ho Y.H. Chinese Med. J. 1980; 60: 451-454Google Scholar, 23Wu G.Y. Liu J.Z. Fang F.D. Zuo J. Sci. Sin. 1982; 25: 1071-1079Google Scholar, 24Zhang L. Wu G.Y. Qiu C.C. Acta Acad. Med. Sin. 1985; 7: 112-116Google Scholar). We recently reported that indirubins are potent inhibitors of cyclin-dependent kinases (CDKs)1(25Hoessel R. Leclerc S. Endicott J. Noble M. Lawrie A. Tunnah P. Leost M. Damiens E. Marie D. Marko D. Niederberger E. Tang W. Eisenbrand G. Meijer L. Nat. Cell Biol. 1999; 1: 60-67Crossref PubMed Scopus (751) Google Scholar), a family of key cell cycle regulators (26Dunphy, W. G. (ed) (1997) Methods Enzymol. 283, 1-67Google Scholar, 27Morgan D. Annu. Rev. Cell Dev. Biol. 1997; 13: 261-291Crossref PubMed Scopus (1834) Google Scholar, 28Vogt P.K. Reed S.I. Curr. Top. Microbiol. Immunol. 1998; 227: 1-169PubMed Google Scholar). Indirubins act by competing with ATP for binding to the catalytic site of the kinase. The kinase selectivity study showed that indirubins have a strong affinity for CDKs (IC50 values in the range of 50–100 nm) (25Hoessel R. Leclerc S. Endicott J. Noble M. Lawrie A. Tunnah P. Leost M. Damiens E. Marie D. Marko D. Niederberger E. Tang W. Eisenbrand G. Meijer L. Nat. Cell Biol. 1999; 1: 60-67Crossref PubMed Scopus (751) Google Scholar). Nevertheless, they are not totally devoid of activity toward a few kinases (IC50 values in the 1–10 μm range) (25Hoessel R. Leclerc S. Endicott J. Noble M. Lawrie A. Tunnah P. Leost M. Damiens E. Marie D. Marko D. Niederberger E. Tang W. Eisenbrand G. Meijer L. Nat. Cell Biol. 1999; 1: 60-67Crossref PubMed Scopus (751) Google Scholar). This rather loose selectivity, when compared with the high specificity of purine inhibitors of CDKs, led us to continue to investigate the selectivity of indirubins as kinase inhibitors. We report here that indirubins are very potent inhibitors (IC50 values in the 5–50 nm range) of glycogen synthase kinase-3β (GSK-3β). This kinase is an essential element of the WNT signaling pathway (29Willert K. Nusse R. Curr. Opin. Genet. Dev. 1998; 8: 95-102Crossref PubMed Scopus (672) Google Scholar). It is involved in multiple physiological processes, including cell cycle regulation by controlling the levels of cyclin D1 (30Diehl J.A. Cheng M. Roussel M.F. Sherr C.J. Genes Dev. 1998; 12: 3499-3511Crossref PubMed Scopus (1882) Google Scholar) and β-catenin (31Yost C. Torres M. Miller J.R. Huang E. Kimelman D. Moon R.T. Genes Dev. 1996; 10: 1443-1454Crossref PubMed Scopus (1034) Google Scholar), dorsal-ventral patterning during development (31Yost C. Torres M. Miller J.R. Huang E. Kimelman D. Moon R.T. Genes Dev. 1996; 10: 1443-1454Crossref PubMed Scopus (1034) Google Scholar, 32He X. Saint-Jeannet J.-P. Woodgett H.E. Varmus H.E. Dawid I.B. Nature. 1995; 374: 617-622Crossref PubMed Scopus (452) Google Scholar, 33Emily-Fenouil F. Ghiglione C. Lhomond G. Lepage T. Gache C. Development. 1998; 125: 2489-2498PubMed Google Scholar), insulin action on glycogen synthesis (34Cohen P. Philos. Trans. R. Soc. Lond-Biol. Sci. 1999; 354: 485Crossref PubMed Scopus (140) Google Scholar, 35Summers S.A. Kao A.W. Kohn A.D. Backus G.S. Roth R.A. Pessin J.E. Birnbaum M.J. J. Biol. Chem. 1999; 274: 17934-17940Abstract Full Text Full Text PDF PubMed Scopus (199) Google Scholar), axonal outgrowth (36Lucas F.R. Goold R.G. Gordon-Weeks P.R. Salinas P.C. J. Cell Sci. 1998; 111: 1351-1361Crossref PubMed Google Scholar), HIV-1 Tat-mediated neurotoxicity (37Maggirwar S.B. Tong N. Ramirez S. Gelbard H.A. Dewhurst S. J. Neurochem. 1999; 73: 578-586Crossref PubMed Scopus (151) Google Scholar), among others. Furthermore, GSK-3β and CDK5 are responsible for most of the abnormal hyperphosphorylation of the microtubule-binding protein tau observed in the paired helical filaments, which are diagnostic for Alzheimer's disease (AD) (38Imahori K. Uchida T. J. Biochem. 1997; 121: 179-188PubMed Google Scholar, 39Mandelkow E.-M. Mandelkow E. Trends Cell Biol. 1998; 8: 425-427Abstract Full Text Full Text PDF PubMed Scopus (445) Google Scholar). It was recently demonstrated that conversion of p35, the regulatory subunit of CDK5, to a truncated form, p25, deregulates CDK5 activity and promotes neurodegeneration (40Patrick G.N. Zukerberg L. Nikolic M. De la Monte S. Dikkes P. Tsai L.H. Nature. 1999; 402: 615-622Crossref PubMed Scopus (1343) Google Scholar). We here show that indirubins are very potent inhibitors of CDK5/p25. Furthermore, indirubin-3′-monoxime inhibits tau phosphorylation in vitro and in vivoat Alzheimer's disease-specific sites. Indirubins may thus constitute a lead compound in the study and treatment of neurodegenerative disorders involving abnormal phosphorylation of tau (“taupathies”). We here also show that indirubin-3′-monoxime inhibits phosphorylation of DARPP-32 by CDK5. DARPP-32 is a striatum protein acting downstream of dopamine action, which is either phosphorylated on Thr-34 by cAMP-dependent protein kinase (PKA) (it then acts as a phosphatase 1 inhibitor) or on Thr-75 by CDK5 (it then becomes a PKA inhibitor) (41Bibb J.A. Snyder G.L. Nishi A. Yan Z. Meijer L. Fienberg A.A. Tsai L.-H. Kwon Y.T. Girault J.-A. Czernik A.J. Huganir R.L. Hemmings Jr., H.C. Nairn A.C. Greengard P. Nature. 1999; 402: 669-671Crossref PubMed Scopus (498) Google Scholar). Finally, we also show that many, but not all, CDK inhibitors are potent GSK-3β inhibitors. Whether the antimitotic/antitumoral properties of indirubins (and other CDK inhibitors) derive from their dual inhibitory effects on GSK-3β and CDKs remains to be determined. Indigo (2) (Fluka), isatin (3) (Fluka), 5,5′,7,7′-indigotetrasulfonic acid potassium salt (25) (Fluka), 5,5′,7-indigotrisulfonic acid potassium salt (26) (Fluka), indigo carmine (27) (Fluka), 5-chloroisatin (30) (Lancaster), 5-fluoroisatin (29) (Aldrich), 5-bromoisatin (31) (Fluka), 5-methylisatin (32) (Sigma), isatin-5-sulfonic acid sodium salt dihydrate (33) (Fluka), 5-nitroisatin (34) (Acros), 1-methylisatin (35) (Acros), 1-phenylisatin (36) (Lancaster), indoxyl acetate (43) (Fluka), 5-bromoindoxyl acetate (44) (Fluka), and other solvents and reagents were obtained from commercial suppliers. They were at least of reagent grade and were used without further purification. Indirubin (1), 5-iodoindirubin (4), 5-bromoindirubin (5), 5-chloroindirubin (6), 5-fluoroindirubin (7), 5-methylindirubin (8), 5-nitroindirubin (9), indirubin-5-sulfonic acid (sodium salt) (10), 5′-bromoindirubin (11), 5,5′-dibromoindirubin (12), 5′-bromoindirubin-5-sulfonic acid (sodium salt) (13), indirubin-3′-monoxime (14), 5-iodoindirubin-3′-oxime (15), 6-iodoindirubin (16), 1-methylindirubin (17), 1-phenylindirubin (18), 3′-hydroxyiminoindirubin-5-sulfonic acid (sodium salt) (19), indirubin-5-sulfonamide (20), indirubin-5-sulfonic acid dimethylamide (21), indirubin-5-sulfonic acid (2-hydroxyethyl)amide (22), indirubin-5-sulfonic acid bis-(2-hydroxyethyl)amide (23), indirubin-5-sulfonic acid methylamide (24), 5-iodoisatin (28), isatin-5-sulfonic acid dimethylamide (37), isatin-5-sulfonic acid bis-(2-hydroxyethyl)amide (38), 6-iodoisatin (39), isoindigo (40), 2,2′-bi-indole (41), 3,3′-diphenyl-2,2′-bi-indole (42), isatin-5-sulfonamide (45), isatin-5-sulfonic acid (2-hydroxyethyl)amide (46), isatin-5-sulfonic acid methylamide (47), and 2-hydroxyimino-N-(3-iodophenyl)acetamide (48) were synthesized and purified as described in the Supplementary material section. Synthesis reactions involving oxygen or moisture-sensitive compounds were performed under a dry argon atmosphere. All reaction mixtures and column chromatographic fractions were analyzed by thin layer chromatography on plates (Alugram Sil G/UV254, purchased from Macherey & Nagel). Column chromatography was carried out using Silica Gel 60 (0.063–0.2 mm, Macherey & Nagel). Melting points of the non-indigoı̈d compounds were determined on a Büchi 510 melting point apparatus and were uncorrected. Melting points over 260 °C were determined on a Wagner and Munz Kupferblock. Elemental analyses were performed using a 2400 CHN elemental analyzer (PerkinElmer Life Sciences). Unless otherwise indicated, NMR spectra were recorded at room temperature.1H NMR spectra were recorded at 400 MHz, 13C NMR spectra at 100 MHz on a Bruker AMX 400, using tetramethylsilane, or Me2SO (δ = 39.4 ppm) as internal standard.J values are reported in hertz. Apparent multiplicities were designated as s, singlet; d, doublet; dd, double doublet; t, triplet; pt, pseudo-triplet; q, quartet; m, multiplet; b, broad. Mass spectra were taken in the positive ion mode under electron impact (EI 70 eV) using a Finnigan MAT 90 mass spectrometer. Gas chromatography/mass spectrometry was performed using a Hewlett-Packard, 5890 Series II gas chromatograph on a 25-m fused silica column (Hewlett-Packard HP-5, I.D. = 0.25 mm; 0.25 μm) and a Hewlett-Packard, HP 5971A mass-selective detector with the following temperature program: 80 °C (4 min), 25 °/min, 320 °C (16.4 min). All compounds were dissolved and stored as 10 mm stock solutions in Me2SO. They were diluted in aqueous buffers just prior use. Sodium orthovanadate, EGTA, EDTA, RNase A, Mops, β-glycerophosphate, phenylphosphate, sodium fluoride, glutathione-agarose, dithiothreitol (DTT), bovine serum albumin (BSA), nitrophenylphosphate, leupeptin, aprotinin, pepstatin, soybean trypsin inhibitor, benzamidine, and histone H1 (type III-S) were obtained from Sigma Chemical Co. [γ-32P]ATP (PB 168) was obtained from Amersham Pharmacia Biotech. The GS-1 peptide (YRRAAVPPSPSLSRHSSPHQSpEDEEE) was synthesized by the Peptide Synthesis Unit, Institute of Biomolecular Sciences, University of Southampton, UK. AT-8, AT-180, and AT-100 antibodies were obtained from Innogenetics, SA, Ghent, Belgium, PHF-1 was a gift from Dr. P. Davies (Bronx, NY), and K9JA was obtained from Dako (Hamburg, Germany). 60 mmβ-glycerophosphate, 15 mm p-nitrophenylphosphate, 25 mm Mops (pH 7.2), 15 mm EGTA, 15 mm MgCl2, 1 mm DTT, 1 mm sodium vanadate, 1 mmNaF, 1 mm phenylphosphate, 10 μg/ml leupeptin, 10 μg/ml aprotinin, 10 μg/ml soybean trypsin inhibitor, and 100 μm benzamidine. Buffer A consisted of 10 mmMgCl2, 1 mm EGTA, 1 mm DTT, 25 mm Tris-HCl, pH 7.5, 50 μg/ml heparin. Buffer C consisted of homogenization buffer but 5 mm EGTA, no NaF, and no protease inhibitors. This buffer consisted of 50 mm Tris, pH 7.4, 150 mm NaCl, and 0.1% Tween 20. HLB buffer consisted of 50 mm Tris-HCl, pH 7.4, 120 mm NaCl, 10% glycerol, 1% Nonidet-P40, 5 mm DTT, 1 mm EGTA, 20 mm NaF, 1 mm orthovanadate, 5 μm microcystin, and 100 μg/ml each of leupeptin, aprotinin, and pepstatin. Kinase activities were assayed in Buffer A or C (unless otherwise stated), at 30 °C, at a final ATP concentration of 15 μm. Blank values were subtracted, and activities were calculated as picomoles of phosphate incorporated for a 10-min incubation. The activities are usually expressed in percentage of the maximal activity, i.e. in the absence of inhibitors. Controls were performed with appropriate dilutions of dimethyl sulfoxide. In a few cases phosphorylation of the substrate was assessed by autoradiography after SDS-PAGE (see below). GSK-3β was expressed in and purified from insect Sf9 cells (42Hughes K. Pulverer B.J. Theocharous P. Woodgett J.R. Eur. J. Biochem. 1992; 203: 305-311Crossref PubMed Scopus (59) Google Scholar). It was assayed, following a 1/100 dilution in 1 mg/ml BSA, 10 mm DTT, with 5 μl of 40 μm GS-1 peptide as a substrate, in buffer A, in the presence of 15 μm[γ-32P]ATP (3000 Ci/mmol; 1 mCi/ml) in a final volume of 30 μl. After 30-min incubation at 30 °C, 25-μl aliquots of supernatant were spotted onto 2.5- × 3-cm pieces of Whatman P81 phosphocellulose paper, and, 20 s later, the filters were washed five times (for at least 5 min each time) in a solution of 10 ml of phosphoric acid/liter of water. The wet filters were counted in the presence of 1 ml of ACS (Amersham Pharmacia Biotech) scintillation fluid. CDK1/cyclin B was extracted in homogenization buffer from M phase starfish (Marthasterias glacialis) oocytes and purified by affinity chromatography on p9CKShs1-Sepharose beads, from which it was eluted by free p9CKShs1 as described previously (43Meijer L. Borgne A. Mulner O. Chong J.P.J. Blow J.J. Inagaki N. Inagaki M. Delcros J.G. Moulinoux J.P. Eur. J. Biochem. 1997; 243: 527-536Crossref PubMed Scopus (1222) Google Scholar, 44Borgne A. Ostvold A.C. Flament S. Meijer L. J. Biol. Chem. 1999; 274: 11977-11986Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar). The kinase activity was assayed in buffer C, with 1 mg/ml histone H1, in the presence of 15 μm [γ-32P]ATP (3000 Ci/mmol; 1 mCi/ml) in a final volume of 30 μl. After 10-min incubation at 30 °C, 25-μl aliquots of supernatant were spotted onto P81 phosphocellulose papers and treated as described above. CDK5/p25 was reconstituted by mixing equal amounts of recombinant mammalian CDK5 and p25 expressed in Escherichia coli as glutathione S-transferase fusion proteins and purified by affinity chromatography on glutathione-agarose (vectors kindly provided by Dr. J. H. Wang). (p25 is a truncated version of p35, the 35-kDa CDK5 activator.) Its activity was assayed in buffer C as described for CDK1/cyclin B. Sf9 cells (Invitrogen, San Diego, CA) were grown at 27 °C in monolayer culture Grace's medium (Life Technologies, Inc., Gaithersburg, MD) supplemented with 10% fetal bovine serum and 50 μg/ml gentamicin and 2.5 μg/ml amphotericin. BaculoGold was obtained from PharMingen (San Diego, CA), pVL1392 was obtained from Invitrogen. The gene for htau23, the shortest human tau isoform, was excised from the bacterial expression vector pNG2 (45Biernat J. Gustke N. Drewes G. Mandelkow E.-M. Mandelkow E. Neuron. 1993; 11: 153-163Abstract Full Text PDF PubMed Scopus (674) Google Scholar) with XbaI and BamHI and inserted into the baculovirus transfer vector pVL1392 cut with the same restriction endonucleases. The BaculoGold system was used to construct the tau baculovirus-containing vector. The BaculoGold DNA is a modified type of baculovirus containing a lethal deletion. Cotransfection of the BaculoGold DNA with a complementing baculovirus transfer vector rescued the lethal deletion of this virus DNA and reconstituted viable virus particles carrying the htau23 coding sequence. Plasmid DNA used for transfections was purified using Qiagen cartridges (Hilden, Germany). Sf9 cells grown in monolayers (2 × 106 cells in a 60-mm cell culture dish) were cotransfected with baculovirus DNA (0.5 μg of BaculoGold DNA) and with vector derivatives of pVL1392 (2 μg) using a calcium phosphate coprecipitation method. The presence of recombinant protein was examined in the infected cells 5 days post-infection by SDS-PAGE and Western blotting. To determine the effects of aminopurvalanol and indirubin-3′-monoxime on tau phosphorylation, Sf9 cells infected with baculovirus expressing htau23 protein were treated 36 h post-infection (when cells have already expressed levels of tau sufficient for the outgrowth of cell processes (46Biernat J. Mandelkow E.-M. Mol. Biol. Cell. 1999; 10: 727-740Crossref PubMed Scopus (115) Google Scholar)) with 20 μm inhibitors for 3 h before being harvested. Sf9 cells were infected with recombinant virus at a multiplicity of infection of 1–5. Cell lysates were prepared in hypotonic lysis buffer (HLB). After 15-min centrifugation at 16,000 × g, the supernatant was recovered and its NaCl concentration raised to 500 mm. It was then boiled for 10 min and recentrifuged at 16,000 ×g for 15 min. Proteins (3 μg) were resolved by SDS-PAGE, transferred to a polyvinylidene difluoride membrane, and Western-blotted with the following antibodies: AT-8 (1:2000), AT-180 (1:1000), AT-100 (1:1000), PHF-1 (1:600), and polyclonal anti-tau antibody K9JA. The immunostaining was visualized using the ECL chemiluminescence system (Amersham Pharmacia Biotech, Braunschweig, Germany). Tau phosphorylation in vitrowas performed using purified GSK-3β and recombinant human tau-32 (provided by Dr. M. Goedert) as a substrate. After 30-min incubation in the presence of various indirubin-3′-monoxime concentrations, under the GSK-3β assay conditions described above, the kinase reaction was stopped by addition of Laemmli sample buffer. Tau was resolved by 10% SDS-PAGE, and its phosphorylation level was visualized by autoradiography. Adult mouse brain striatal slices were prepared using standard methodology (47Nishi A. Snyder G.L. Greengard P. J. Neurosci. 1997; 17: 8147-8155Crossref PubMed Google Scholar). Following equilibration in Krebs' bicarbonate buffer oxygenated with continuous aeration (95% O2/5% CO2), slices were treated with various concentrations of indirubin-3′-monoxime or 10 μm roscovitine for 60 min or were left in Krebs' bicarbonate buffer for the same period of time. Slices were homogenized by sonication in boiling 1% SDS and 50 mm NaF. Protein concentrations were determined by the BCA method using a BSA standard curve. Equal amounts of protein (80 μg) were subjected to SDS-PAGE using a 15% acrylamide gel, electrophoretically transferred to nitrocellulose membrane, and immunoblotted with a phosphorylation state-specific antibody that selectively detects DARPP-32 phosphorylated at Thr-75 (41Bibb J.A. Snyder G.L. Nishi A. Yan Z. Meijer L. Fienberg A.A. Tsai L.-H. Kwon Y.T. Girault J.-A. Czernik A.J. Huganir R.L. Hemmings Jr., H.C. Nairn A.C. Greengard P. Nature. 1999; 402: 669-671Crossref PubMed Scopus (498) Google Scholar). In the course of studying the CDK inhibitory properties of indirubin, we synthesized a series of indole derivatives and dimers (TableI). While further investigating the kinase inhibition selectivity of indirubin-3′-monoxime, the indirubin used in our cellular studies, we noticed that this compound was a powerful inhibitor of GSK-3β (see below). Our collection of indoles/bis-indoles was further evaluated for inhibition against purified GSK-3β, CDK5/p25, and CDK1/cyclin B. Kinase activities were assayed with an appropriate substrate (GSK-3β, GS1 peptide; CDKs, histone H1) in the presence of 15 μm ATP and increasing concentrations of compounds. IC50 values were calculated from the dose-response curves and are presented in TableII. The GSK-3β and CDK inhibition activity was limited to the indirubins family. Neither indigo nor isatin, and their derivatives, displayed a significant effect on any of the three kinases. To compare the effects of active compounds on GSK-3β and CDKs, the IC50 values toward each enzyme were plotted against the IC50 values for the other two kinases (Fig. 2). This analysis shows that the efficacies of indirubins toward CDK1 and CDK5 are closely related, whereas the efficacies toward GSK-3β and CDKs are less so. This probably reflects the closer evolutionary proximity between CDK1 and CDK5 compared with that between GSK-3β and CDKs (48Hanks S.K. Hunter T. FASEB J. 1995; 9: 576-596Crossref PubMed Scopus (2322) Google Scholar). The dose-response curves for (a) the most active compound on the three kinases (5-iodo-indirubin-3′-monoxime), (b) the most GSK-3β-selective compound (5,5′-dibromoindirubin), (c) the most CDK-selective compound (5-sulfonic acid-indirubin-3′-monoxime), and (d) the most frequently used compound in the studies of indirubins' cellular effects (indirubin-3′-monoxime) are presented in Fig. 3.Table IStructure of the indoles and bis-indoles used in this study Open table in a new tab Table IIInhibition of GSK-3β, CDK1, and CDK5 by indoles and bis-indolesView Large Image Figure ViewerDownload Hi-res image Download (PPT)Numbers refer to structures shown in Table I. Enzyme activities were assayed as described under “Experimental Procedures,” in the presence of increasing concentrations of indole derivatives. IC50 values were calculated from the dose-response curves. ≤0.01 μm (solid black), 0.01–0.1 μm (dark gray), 0.1–1 μm(medium gray), 1–10 μm (light gray), >10 μm(white). Open table in a new tab Figure 2Comparisons of the inhibitory activity of indirubins on GSK-3β, CDK5/p25, and CDK1/cyclin B. GSK-3β and CDKs were assayed using the GS-1 peptide or histone H1 as substrates, respectively, with 15 μm ATP and in the presence of increasing concentrations of indirubins. IC50 values toward each enzyme, determined graphically, were plotted against the IC50 values for the other two kinases. The dose-response curves for compounds 12, 14, 15, and 19 are presented in Fig.3.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 3Inhibition of GSK-3β, CDK5/p25, and CDK1/cyclin B by indirubins. GSK-3β and CDKs were assayed using the GS-1 peptide or histone H1 as substrates, respectively, with 15 μm ATP and in the presence of increasing concentrations of indirubins. Activity is presented as the percentage of maximal activity (no inhibitors). Dose-response are shown curves for the most active inhibitor toward GSK-3β (5-iodo-indirubin-3′-monoxime) (15) (A), the most GSK-3β-selective compound (5,5′-dibromoindirubin) (12) (B), the most CDK-selective compound (5-sulfonic acid-indirubin-3′-monoxime) (19) (C), and the most frequently used compound in the studies of indirubins' cellular effects (indirubin-3′-monoxime) (14) (D).View Large Image Figure ViewerDownload Hi-res image Download (PPT) Numbers refer to structures shown in Table I. Enzyme activities were assayed as described under “Experimental Procedures,” in the presence of increasing concentrations of indole derivatives. IC5
