Abstract

Indirubin is the active ingredient of Danggui Longhui Wan, a mixture of plants that is used in traditional Chinese medicine to treat chronic diseases. Here we identify indirubin and its analogues as potent inhibitors of cyclin-dependent kinases (CDKs). The crystal structure of CDK2 in complex with indirubin derivatives shows that indirubin interacts with the kinase's ATP-binding site through van der Waals interactions and three hydrogen bonds. Indirubin-3'-monoxime inhibits the proliferation of a large range of cells, mainly through arresting the cells in the G2/M phase of the cell cycle. These results have implications for therapeutic optimization of indigoids.

Introduction

Over four millennia, practitioners of traditional Chinese medicine have accumulated a considerable pharmacopoeia that is largely still used in modern China and in many other parts of the world. The last encyclopaedia of traditional Chinese medicine (1977) lists over 5,500 natural sources (82.8% of which are plants) that form the basis for the 100,000–500,000 prescriptions of traditional Chinese medicine ^{1, 2}. Of these, some are described as relieving 'cancer' (although this term may not always relate to the modern scientific definition of cancer). Several Chinese plants have provided compounds with significant antitumour activity; Camptotheca acuminata (camptothecin) and Cephalotaxus sp. (homoharringtonine/ harringtonine) are two famous examples ³.

Chronic myelocytic leukaemia (CML) has customarily been treated with the traditional Chinese recipe Danggui Longhui Wan, a mixture of 11 herbal medicines ⁴. In 1966, the Institute of Haematology of the Chinese Academy of Medical Sciences embarked on the identification of the active factor of this complex mixture ^{2, 3, 4, 5}. The activity was traced to one ingredient, Qing Dai (indigo naturalis), a dark blue powder prepared from the leaves of Baphicacanthus cusia (Acanthaceae), Polygonum tinctorium (Polygonaceae), Isatis indigotica (Brassicaceae), Indigofera suffrutticosa (Fabaceae) and Indigofera tinctoria (Fabaceae). This powder contained a high level of the blue dye indigo, but the antileukaemic activity was attributed to the red-coloured isomer of indigo, indirubin, a minor constituent of the mixture ^{6, 7, 8, 9, 10, 11}. Indigo (also known as indigotin) and indirubin (isoindigotin or indigo red) are both derived, by oxidation and dimerization, from indoxyl and isatin, which are themselves liberated from colourless precursor conjugates, indican (from Indigofera and Polygonum species amongst others) or isatan B (from Isatis tinctoria) (refs 12,13 and see Supplementary Information ).

Indirubin inhibits DNA synthesis in several cell lines ^{7, 14}, in a cell-free assay ¹⁵ and in vivo in rats with Walker-256 sarcoma ¹⁶. A weak binding of indirubin to DNA in vitro has been described ¹⁷. Alterations are seen in the surface morphology of white blood cells and in endoplasmic-reticulum, chromatin and nuclear-envelope structures of peripheral blood samples and bone-marrow leukocytes of patients treated with indirubin ¹⁸. Results of animal studies showed that indirubin exhibits limited toxicity; no effects of indirubin on bone marrow and the production of haematopoietic stem cells have been observed, even in long-term studies ^{19, 20, 21}. Indirubin shows poor solubility and absorption, so several analogues have been designed in an effort to improve these characteristics ²². Of these, 5-halogeno-indirubin ²³, N-ethyl-indirubin ²² and N-methylisoindigo ²⁴ exhibited a higher antitumour activity than did indirubin in animal models ²³. Indirubin-3'-monoxime was as active as indirubin in several tumour models ²⁵.

Synthetic indirubin ²⁶ exhibited good antitumour activity and only minor toxicity in animal models ¹⁹. In dogs given a dose of synthetic indirubin 25 times that used for human therapy (according to body weight) continuously for 6 months, diarrhoea and some damage to the liver were observed, with no change in haematopoiesis, electroencephalogram activity and renal functions ²⁷. In further animal studies, no suppression of haematopoietic stem-cell production by synthetic indirubin was observed ²⁸. Indirubin has been approved for clinical trials against chronic myelocytic and chronic granulocytic leukaemia ( refs 29–32; for reviews see ref. 3, 27, 33). In these studies, 26% of the 314 CML patients showed complete remission and 33% showed partial remission in response to indirubin treatment. Toxicity was low and side effects were limited to mild abdominal pain, diarrhoea, nausea and vomiting. There were three cases of reversible pulmonary arterial hypertension and cardiac insufficiency ³⁴. A comparative clinical study of CML patients showed that indirubin and busulfan exhibited similar efficiency, with a higher rate of complete remissions observed with busulfan ³². No crossresistance between the two drugs was observed.

CDKs are crucial in initiating and coordinating the phases of the cell-division cycle ³⁵. The search for chemical inhibitors of CDKs is mainly inspired by the prospect of identification of new antitumour agents, but such inhibitors may also have applications in other areas, such as psoriasis, cardiovascular diseases, glomerulonephritis, parasitism, viral infections and neurodegenerative disorders ³⁶.

Here, while searching for antineoplastic agents from plants used in traditional Chinese medicine ², we discovered that indirubin and its analogues selectively inhibit CDKs and block cell proliferation in the late-G1 and G2/M phases of the cell cycle. The crystal structures of CDK2 in complex with two indirubin derivatives reveal the atomic interactions of these inhibitors with the kinase's ATP-binding pocket. It remains to be determined how this molecular effect of indigoids contributes to their antileukaemic properties. Nevertheless, knowledge of this mechanism of action may allow optimization of indigoids as therapeutic antiproliferative agents.

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Results

Indirubin and analogues selectively inhibit CDKs. We purified the starfish CDK1–cyclin B complex by affinity chromatography on p9 ^CKShs1–Sepharose and assayed its kinase activity towards the substrate histone H1 in the presence of a final ATP concentration of 15 M, as described ³⁷. While screening a collection of natural products derived from traditional Chinese medicinal plants ², we noticed that indirubin exhibited some inhibitory activity towards CDK1–cyclin B (half-maximal inhibitory concentration (IC₅₀) 10 M) ( Fig. 1). In this assay, indigo and isatin were nearly inactive (IC₅₀ >1,000 M and 900 M, respectively) and isoindigo was only slightly active (IC₅₀ 80 M). Three previously described indirubin derivatives inhibited CDK1 efficiently, namely indirubin-3'-monoxime (IC₅₀ 0.18 M), 5-chloro-indirubin (IC₅₀ 0.4 M) and indirubin-5-sulphonic acid (IC₅₀ 0.055 M) ( Fig. 1). These IC₅₀ values are comparable to those of previously described CDK inhibitors³⁶, namely olomoucine (7 M), roscovitine (0.45 M), purvalanol (0.004 M), flavopiridol (0.400 M), staurosporine (0.006 M) and butyrolactone (0.600 M).

Figure 1: Inhibition of CDK1–cyclin B by various indigoids.

CDK1–cyclin-B kinase activity towards histone H1 was assayed in the presence of 15 M ATP and increasing concentrations of indigoids. Activity is presented as a percentage of maximal activity (in the absence of inhibitors).

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We next studied the kinase selectivity of these indigoids. We expressed and/or purified various kinases and assayed them with appropriate substrates as described ³⁷ in the presence of 15 M ATP and increasing concentrations of indigoids. Table 1 shows IC ₅₀ values. Most of the 25 kinases tested were not inhibited by indirubin, 5-chloro-indirubin and indirubin-5-sulphonic acid; the inhibitory activities of these indigoids were limited to CDKs. Indirubin, like flavopiridol, inhibited all CDKs equally, but other indigoids showed a preference for CDK1, CDK2 and CDK5 over CDK4. The 3'-monoxime substitution led to a slight reduction in selectivity.

Table 1: IC⁵⁰ values for kinase inhibition by indirubin and analogues.

Full table

Structures of complexes of CDK2 and indirubin derivatives. To understand the mechanism of action of indigoids better, we co-crystallized two of them with CDK2. The structures of the CDK–indigoid complexes were refined to yield the crystallographic statistics given in Table 2 . The structure of CDK2 represents the minimal catalytic kinase core, composed of two domains: a smaller, amino-terminal domain (of ~80 residues) formed mainly from -sheets, and a larger, predominantly -helical, carboxy-terminal domain (of ~210 residues). As described previously for both the apo-enzyme and other monomeric CDK2–inhibitor-complex structures ^{38, 39, 40, 41, 42, 43}, two regions of CDK2 are flexible in the CDK2–indirubin complex structures. The residues missing from the crystal structure (because they belong to these flexible regions) constitute the loop connecting strand 3 to helix C (residues 36–43) and the 'activation segment', which contains Thr 160, a residue that is phosphorylated in the fully active CDK2–cyclin-A complex ⁴⁴ ( Table 2). The electron density in the initial F_o-F _c maps showed that the indirubin derivatives bind CDK2 at the ATP-binding site. Fig. 2 shows the electron density for both indirubin derivatives in final 2F_o-F_c maps.

Figure 2: Binding of indirubin-3'-monoxime and indirubin-5-sulphonate to CDK2.

a, Binding of indirubin-3'-monoxime. b, Binding of indirubin-5-sulphonate. CDK2 residues are shown in green, and the indirubin derivatives are shown in yellow. CDK2 residues that contact the bound ligands, as calculated by the CCP4 program Contact ⁴⁸, are included. Clear electron density defined the residues that interact with the inhibitor molecule. Hydrogen bonds are drawn between the indirubin derivatives and the backbone oxygen of Glu 81 of CDK2, and between the indirubin derivatives and the backbone oxygen and nitrogen of Leu 83. The figure also includes (2 F_o-F_c)_calc electron density for indirubin-3'-monoxime and indirubin-5-sulphonate calculated at the end of refinement using map coefficient output from REFMAC, with resolution between 20 and 2.0 Å. The maps are contoured at a level of a, 0.19 e^- Å^-3 and b, 0.20 e^- Å^-3, corresponding in each case to 1.0 times the root-mean-square deviation of the maps from their mean values.

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Table 2: Statistics of the datasets used and of the refined structures.

Full table

Indirubin-3'-monoxime and indirubin-5-sulphonic acid share a similar binding mode: the lactam amide nitrogen of the inhibitor donates a hydrogen bond to the peptide oxygen of Glu 81 of CDK2; the NH group of Leu 83 of CDK2 donates a hydrogen bond to the lactam amide oxygen; and the cyclic nitrogen acts as a hydrogen-bond donor to the backbone oxygen of Leu 83 ( Fig. 2). In comparison with previously reported CDK2–inhibitor structures ^{38, 39, 40, 41, 42, 43}, the indirubin derivatives form an extra hydrogen bond with the CDK2 backbone. Like isopentenyladenine, staurosporine ( Fig. 3b) and ATP, the indirubin derivatives form hydrogen bonds with the backbone oxygen of Glu 81 and the NH group of Leu 83, but the indirubin derivatives also resemble olomoucine or roscovitine in that they also interact with the backbone oxygen of Leu 83 ( Fig. 3a). Although residues 80–84 of CDK2 constitute part of the hinge between the N- and C-terminal domains, the interactions with indirubin derivatives do not result in a significant change in relative domain orientation. The shape of the indirubin molecule complements the bent shape of the ATP-binding site in the hinge region. This shape complementarity is also observed in the CDK2–staurosporine and CDK2–roscovitine structures ( Fig. 3). Indirubin-3'-monoxime and indirubin-5-sulphonic acid make several apolar contacts with conserved residues that line CDK2's ATP-binding site ( Fig. 4). Both derivatives contact Phe 80 and fill the back of the cleft more completely than either ATP or staurosporine ( Fig. 3b). This extra interaction site is also exploited by roscovitine: the N9 isopropyl group packs against the sidechain of Phe 80 ( Fig. 3a) ⁴⁰.

Figure 3: Superposition of roscovitine and indirubin-5-sulphonate and staurosporine and indirubin-5-sulphonate bound to CDK2.

a, Superposition of roscovitine and indirubin-5-sulphonate bound to CDK2. Indirubin-5-sulphonate is shown in yellow, roscovitine in white and CDK2 in green. b, Superposition of staurosporine and indirubin-5-sulphonate bound to CDK2. Colouring as in a, with staurosporine in white. The CDK2 residues that contact indirubin-5-sulphonate, as calculated by the CCP4 program Contact, are included. Roscovitine ⁴⁰ and staurosporine ⁴² are shown as positioned after superimposing the respective protein structures on the CDK2–indirubin-5-sulphonate complex.

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Figure 4: The Connolly molecular surface of CDK2–indirubin-5-sulphonate, calculated with a probe radius of 1.5 Å.

The surface is coloured by electrostatic potential; blue represents a potential of +15 kT and red a potential of -15 kT. Superimposed on this colour scheme is colouring of hydrophobic potential; regions showing marked hydrophobicity are in yellow. a, View looking from the C-terminal domain of CDK2 onto the N-terminal domain. b, View from the N-terminal domain onto the C-terminal domain. c, Side view, with the CDK2 N-terminal domain above. See Supplementary Information . Ball-and-stick diagrams represent atoms of the CDK2 structure seen through the surface representation.

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The sulphonate group of indirubin-5-sulphonate occupies a site similar to that occupied by one of the rings of staurosporine's extended indole ring system, as seen in the structure of staurosporine bound to CDK2 ( ref. 42). The ring systems of these two bound inhibitors are not co-planar. Through its ability to make both charge and hydrogen-bond interactions, the sulphonate group is more complementary to the CDK2 structure in this region than is the staurosporine ring. A superposition of the structures of cyclic-AMP-dependent protein kinase (cAPK) ⁴⁵, the phosphorylated CDK2–cyclin-A complex ⁴⁴, the CDK2–ATP complex ⁴³ and the CDK2–indirubin-5-sulphonate complex shows that the sulphonate group does not precisely mimic one of the ATP phosphate groups, as occurs in the other CDK2 structures. However, the sulphonate group of indirubin-5-sulphonate and the -phosphate of ATP in the cAPK–Mg-ATP–PKI (where PKI is cAMP-dependent protein-kinase inhibitor) ternary complex ⁴⁵ interact with the sidechain amino group of Lys 33. In addition to this interaction, the sulphonate oxygens contact the backbone nitrogen of Asp 145 and the amide group of Asn 132. The Asp 145 side chain is displaced from its position in the CDK2–ATP structure and interacts with the amide group of Asn 132.

Binding of staurosporine to CDK2 causes changes to the main-chain conformation of residues Asp 145, Phe 146, Gly 147 and Leu 148 (Asp 145, Phe 146 and Gly 147 constitute the conserved 'DFG' motif found in most protein kinases). Movement of the DFG motif is required to avoid an unfavourable contact between Asp 145 and staurosporine. This change tends towards that seen in the active form of CDK2 bound to cyclin A ⁴⁴. Binding of indirubin-5-sulphonate to CDK2 causes a similar movement of the main chain at residues Asp 145 and Phe 146 as a result of unfavourable steric and charge effects between the sulphonate group and Asp 145 ( Fig. 5). Gly 147 can be located in the CDK2–indirubin-5-sulphonate complex electron-density maps, but the following residues that lead into the activation loop cannot be defined and the model resumes at Val 163. In contrast, the CDK2–indirubin-3'-monoxime structure is similar to the CDK2–ATP structure and shows a loop region that is ordered through to residue Ala 151 ( Fig. 5). The monoxime group of indirubin-3'-monoxime occupies the ATP-ribose-binding site and makes no direct interactions with CDK2.

Figure 5: Movement of the conserved DFG loop accompanies indirubin-5-sulphonate binding to CDK2.

The structures of CDK2–indirubin-3'-monoxime (green) and CDK2–indirubin-5-sulphonate (white) are superimposed; the ligands and residues Asp 145, Phe 146 and Gly 147 are shown here. The site is located within the overall CDK2 model, represented as a ribbon fold. As a result of steric and charge repulsion between the side chain of Asp 145 and the sulphonate group of indirubin-5-sulfonate, the DFG loop is rearranged into a conformation that differs from that observed in both the CDK2–indirubin-3'-monoxime and CDK2–ATP structures.

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Indirubin-3'-monoxime inhibits cell proliferation. We next investigated the effects of various indigoids on cell proliferation. The most potent indigoid, indirubin-5-sulphonic acid, as judged by our in vitro kinase assay, had limited effects on cell proliferation, probably because of its limited permeability. We therefore used indirubin-3'-monoxime to study the effects of indigoids on the T-lymphoblastic Jurkat cell line. Indirubin-3'-monoxime inhibited the proliferation of Jurkat cells in a concentration-dependent manner ( Fig. 6a). This arrest was associated with a modest increase in the apoptosis rate (see Methods; Fig. 6a). Analysis of the cell-cycle distribution showed a marked G1-phase arrest at low indirubin-3'-monoxime concentrations, whereas at higher concentrations cells accumulated in G2/M phase ( Fig. 6b). The G1 arrest was accompanied by a decrease in phosphorylation of the retinoblastoma protein ( Fig. 6c). We attribute this effect to inhibition of CDK2 rather than of CDK4–cyclin D (see Table 1), but it could also be an indirect effect (for example, induction of cellular CDK inhibitors such as those induced by DNA damage, or downregulation of cyclin D, whose induction probably involves several protein kinases).

Figure 6: Indirubin-3'-monoxime inhibits proliferation of Jurkat cells, accumulates cells in G1 and G2/M and reduces phosphorylation of the retinoblastoma protein.

a, Cells were treated for 30 h with various concentrations of indirubin-3'-monoxime and counted. Cell counts (from two independent experiments) are expressed as a percentage of the number of cells at time 0. Filled symbols, living cells; open symbols, apoptotic cells. b, Cell-cycle distribution of similarly treated cells, as a function of indirubin-3'-monoxime concentration. c, Western blot of total cell extracts with anti-phospho-retinoblastoma-protein antibodies following treatment with increasing concentrations of indirubin-3'-monoxime.

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To determine whether the mechanism of growth inhibition of the Jurkat cells was general or dependent on the cell phenotype, we analysed the cell-cycle distribution of various haematopoietic and non-haematopoietic cells following exposure to a range of indirubin-3'-monoxime concentrations. Proliferation of most cell lines was inhibited by indirubin-3'-monoxime in a concentration-dependent manner ( Fig. 7). Only the human adenocarcinoma cell line HT-29-18-C1 remained insensitive. Fluorescence-activated cell sorting (FACS) analysis of the cell-cycle distribution showed that indirubin-3'-monoxime, at concentrations over 10 M, induced arrest at G2/M phase in all the sensitive cell lines analysed. We observed, for the most sensitive cells, another cell-cycle block at lower indirubin-3'-monoxime concentrations (5–10 M); this block depended on the cell phenotype. For example, MCF-7 and Jurkat cells were arrested at G1 phase and CCL-39 cells were arrested at S phase. These in vitro studies confirm and extend our knowledge of the antiproliferative properties of indirubin-3'-monoxime.

Figure 7: Cell-cycle-phase distribution of various cell lines following exposure to indirubin-3'-monoxime.

Cells were treated for 30 h with various concentrations of indirubin-3'-monoxime and analysed by FACS (results shown are representative of three independent experiments for each cell line). Note the range in responses, from the most sensitive cell line, HBL-100, to the insensitive HT-29 line. Values on the y-axis show percentages of the total cell population.

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Acknowledgements

We thank N. Xiuren for his help with Chinese ideogram; the fishermen of the Station Biologique de Roscoff for collecting the starfish; D. Alessi, M. Cobb, W. Harper, F. Hofmann, J. Lahti, S. Lohmann, H. Mett, D. Morgan, L. Pinna and H. Y. L. Tung for providing reagents and purified enzymes; D. Louvard, M. Mareel, M. Crepin and J.P. Moulinoux for providing cell lines; the scientists at the X-ray diffraction beamline, Elettra, Trieste; W. Burmeister for assistance during data collection on ID14-3 at the ESRF; and S. Lee, R. Bryan, K. Measures and I. Taylor for assistance. This research was supported by grants from the Association pour la Recherche sur le Cancer and the Conseil Régional de Bretagne (to L.M.), from the BBSRC, MRC and the Royal Society (to J.A.E. and M.E.M.N.) and from the German Bundesministerium für Bildung, Wissenschaft, Forschung und Technologie (to G.E.).

Correspondence and requests for materials should be addressed to L.M. or G.E.

Supplementary Information is available on Nature Cell Biology's World-Wide Web site (http://cellbio.nature.com) or as paper copy from the London editorial office of Nature Cell Biology.

Received 8 February 1999; Accepted 29 March 1999.

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