Midkine inhibitors: application of a simple assay procedure to screening of inhibitory compounds
© Matsui et al. 2010
Received: 20 April 2010
Accepted: 21 June 2010
Published: 21 June 2010
Midkine is a heparin-binding cytokine and is involved in etiology of various diseases. Thus, midkine inhibitors are expected to be helpful in treatment of many diseases.
We developed a simple assay for midkine activity based on midkine-dependent migration of osteblastic cells. Midkine inhibitors were searched as materials that inhibit this midkine activity. To develop peptides that inhibit midkine activity, we constructed models in which C-terminal half of midkine interacted with α4β1-integrin. Low molecular weight compounds which are expected to bind to midkine with high affinity were searched by in silico screening with the aid of Presto-X2 program.
Among peptides in putative binding sites of midkine and the integrin, a peptide derived from β1-integrin and that derived from the first β sheet of the C-terminal half of midkine significantly inhibited midkine activity. Two low molecular weight compounds found by in silico screening exhibited no toxicity to target cells, but inhibited midkine activity. They are trifluoro compounds: one (PubChem 4603792) is 2-(2,6-dimethylpiperidin-1-yl)-4-thiophen-2-yl-6-(trifluoromethy)pyrimidine, and the other has a related structure.
The assay procedure is helpful in screening midkine inhibitors. All reagents described here might become mother material to develop clinically effective midkine inhibitors.
Midkine is a heparin-binding cytokine of molecular weight 13 kDa [1–3]. It enhances growth, survival and migration of various target cells. Midkine has around 50% sequence identity with pleiotrophin, and the two factors exhibit overlapping roles in many cases [1, 4]. Midkine is also involved in initiation or progression of many pathological status, such as tumor invasion  rheumatoid arthritis , experimental autoimmune encephalitis , adhesion after surgery , neointima formation of the blood vessel , hypertension , and renal injury after ischemia , exposure to chemotherapeutic reagent  and diabetes . Antisense oligonucleotides or siRNAs to midkine exhibit therapeutic effects in animal experiments concerning tumor growth [5, 14–16], ischemic renal failure , neointima formation [18, 19], adhesion after surgery , and antibody-induced arthritis  Polyclonal antibodies to midkine inhibit growth of Wilms' tumor cells in vitro . Peptides or low molecular weight compounds are also expected to inhibit midkine activities, and in many cases these inhibitors might be superior to antisense oligoDNA or siRNA because of the ease in the administration methods.
In the present investigation, at first we developed a convenient method to screen midkine inhibitors. Then, applying the assay method, we tried to develop midkine inhibitors, namely peptides and other low molecular weight compounds.
Midkine is composed of two domains held by disulfide bridges. The C-terminal half domain is principally responsible for midkine activity and its heparin binding capability . Two heparin binding sites are present in the C-terminal half . Especially, Arg 81 has been identified to be important in heparin binding and midkine-induced neurite outgrowth and migration of neurons [24, 25]. Thus, we searched for peptides and low molecular weight compounds, which bind to the C-terminal half.
A rationale to obtain midkine inhibitory peptide should be based on midkine-receptor interaction. Midkine receptor is a molecular complex containing proteoglycans such as receptor-like protein tyrosine phosphatase ζ, low density lipoprotein receptor-related protein  and integrin α4β1 or α6β1 . A peptide sequence derived from low density lipoprotein receptor-related protein has already been utilized to develop an inhibitor of midkine . In this study we were interested in interaction of midkine with α4β1 integrin.
Human midkine was produced in yeast as described before . Candidate inhibitory peptides were synthesized by Peptide Institute, Osaka, Japan, as trifluoroacetate salts. Low molecular weight compounds for screening of midkine inhibitors were purchased from ChemDiv (San Diego, CA).
Assay of midkine activity
Midkine activity was determined by promotion of migration of UMR106 (ATTC No. 1661) cells . Chemotaxicell (pore size 8 micron, Kurabo Industries Ltd., Japan) was coated with 20 μg/ml of midkine at the lower surface of the filter for 1 h, and washed with Dulbecco's phosphate-buffered saline (PBS) twice. UMR106 cells (2 × 105 cells in 0.2 ml of Dulbecco's modified Eagle's medium with 0.3% bovine serum albumin) were added to the upper chamber. The lower chamber contained 0.5 ml of the same medium. Cells were cultured for 4 h. The inner Chemotaxicell was cleaned with PBS, and cells were fixed with 100% methanol at room temperature for 20 min. The inner cell was cleaned with cotton bar, and the migrated cells were stained with 1% crystal violet at room temperature for 30 min., and were washed with H2O. The cells were extracted with 0.2 ml of 1% SDS with 1% Triton X-100 for 1 h upon agitation. Then extract of 0.15 ml was transferred to a 96 well plate and OD 590 nm was determined. Upon inhibition assay using low molecular weight compounds, they were dissolved in ethanol and were added to the lower chamber so that the final ethanol concentration was 2%. The control contained also 2% ethanol in this occasion. Inhibition assay using peptides was performed by adding them dissolved in H2O. Assay was done in duplicate.
Binding activity to midkine was evaluated in the BIACORE 3000 System (GE Healthcare, UK Ltd). A CM5 sensor chip coated with carboxymethyldextran was activated by injecting a solution (1:1) of 200 mM N-ethyl-N'-(3-dimethylaminopropyl) carbodiimide hydrochloride and 100 mM N-hydroxysuccinimide at a flow rate of 10 μl/min. A 70 μl aliquot of midkine solution (50 μg/ml in 10 mM acetate buffer, pH 5.0) was injected for immobilization. The remaining activated N-hydroxysuccinimide ester groups were blocked by injecting 1 M ethanolamine hydrochloride/NaOH, pH 8.5, and washed with 10 μl of 1 M NaCl. Peptides (5 μg/ml) in the running buffer (10 mM HEPES-NaOH, pH 7.4, containing 0.15 M NaCl and 0.0005% Tween 20) were injected into the flow cell, and the change in resonance units was recorded. Binding assays were performed at 25°C with a constant flow rate of 10 μl/ml in both association and dissociation phases.
Docking of α4β1-integrin with midkine was performed by the Greenpepper program (Chuden CTI) and docking of low molecular weight compounds in ZINC library http://zinc.docking.org/ with midkine was performed by the Presto-X2 program (National Institute of Advanced Industrial Science and Technology, Tsukuba, Japan).
Design of midkine inhibitory peptides based on interaction of midkine with integrin
Binding of integrin-derived peptides to midkine
Binding constant to midkine (μM)
The expected binding sites in the midkine C-terminal half to α4 β1 integrin were mostly in the three anti-pararel β-sheets . Thus the following peptides in the β-sheets were selected as candidate peptide inhibitors derived from midkine: YKFENWGACDGG (YK12), GTGTKVRQGTL (GT11), and QETIRVTKPC (QE10).
By surface plasmon assay, all of the integrin-derived peptides were found to have significant binding activity to midkine (Table 1). Then, we tested whether these peptides inhibited midkine activity.
Inhibitory activities of synthetic peptides and low molecular weight compounds to midkine-dependent migration.
Migration activity (%)
44.0 ± 10.5
156 ± 6
99.5 ± 10.6
107 ± 15
99.5 ± 6.9
107 ± 2
105 ± 24
43.7 ± 12.8
113 ± 10.1
69.4 ± 46.1
Low molecular weight compounds
43.8 ± 10.2
53.6 ± 4.1
Search of low molecular weight compounds with inhibitory activity
A convenient method has been developed to survey materials that inhibit midkine activity. This method has been effective to screen peptides and low molecular weight compounds with the inhibitory activity. An aptamer, which has midkine inhibitory activity and inhibits development of experimental autoimmune encephalitis, was also found using the method .
Concerning peptides and low molecular weight compounds with midkine binding and midkine inhibitory activity, these are expected to bind mainly to the first β-sheet of the C-terminal half domain. Probably, this strand is exposed and is easier to be bound by the inhibitors, and through this binding, R81, which is a key amino acid in midkine activity and comprises a heparin binding site [23, 24], is expected to be covered by the inhibitor. In addition, a possibility is not excluded that the first β-sheet itself plays a key role in midkine activity, such as in biding to integrins. Indeed a peptide derived from the first β-sheet was found to inhibit midkine activity. It is noted that the first β-sheet has Trp 69, which forms βC1 bulge with Arg81, the key residue in heparin-binding, which is in the centrally located β-sheet .
The inhibitory activity of peptides and low molecular weight compounds described here is not potent, and further elaboration is required to obtain materials of potential clinical utility. However, we think that the present material might even become seed materials. As an example, we noted that a methyl group in Compound I, a midkine inhibitory material, is expected to be located near to Arg 81 (marked with an arrow as 1 in Fig. 2), a key amino acid in midkine activity. Thus, conversion of the methyl group to carboxyl group might enhance the affinity of the compound to midkine, and increases the inhibitory activity.
A simple assay procedure was developed for screening of midkine inhibitors. Inhibitory materials, especially low molecular weight compounds found by the present study are expected to become mother compounds to develop clinically applicable midkine inhibitors.
Dulbecco's phosphate-buffered saline
The Presto-X2 program was kindly made available by Dr. K. Fukunishi and Prof. H. Nakamura, National Institute of Advanced Industrial Science and Technology. This work was supported in parts by grants from the Ministry of Education, Culture, Sports, Science and technology of Japan.
- Muramatsu T: Midkine and pleiotrophin: two related proteins involved in development, survival, inflammation and tumorigenesis. J Biochem 2002, 132:359–371.PubMedGoogle Scholar
- Kadomatsu K, Muramatsu T: Midkine and pleiotrophin in neural development and cancer. Cancer Lett 2004, 204:127–143.View ArticlePubMedGoogle Scholar
- Muramatsu T: Midkine, a heparin-binding cytokine with multiple roles in development, repair and diseases. Proc Japan Acad Ser B 2010, 86:410–425.View ArticleGoogle Scholar
- Muramatsu H, Zou P, Kurosawa N, Ichihara-Tanaka K, Maruyama K, Inoh K, Sakai T, Chen L, Sato M, Muramatsu T: Female infertility in mice deficient in midkine and pleiotrophin, which form a distinct family of growth factors. Genes Cells 2006, 11:1405–1417.View ArticlePubMedGoogle Scholar
- Takei Y, Kadomatsu K, Matsuo S, Itoh H, Nakazawa K, Kubota S, Muramatsu T: Antisense oligodeoxynucleotide targeted to Midkine, a heparin-binding growth factor, suppresses tumorigenicity of mouse rectal carcinoma cells. Cancer Res 2001, 61:8486–8491.PubMedGoogle Scholar
- Maruyama K, Muramatsu H, Ishiguro N, Muramatsu T: Midkine, a heparin-binding growth factor, is fundamentally involved in the pathogenesis of rheumatoid arthritis. Arthritis Rheum 2004, 50:1420–1429.View ArticlePubMedGoogle Scholar
- Wang J, Takeuchi H, Sonobe Y, Jin S, Mizuno T, Miyakawa S, Fujiwara M, Nakamura Y, Kato T, Muramatsu H, Muramatsu T, Suzumura A: Inhibition of midkine alleviates experimental autoimmune encephalomyelitis through the expansion of regulatory T cell population. Proc Natl Acad Sci USA 2008, 105:3915–3920.View ArticlePubMedGoogle Scholar
- Inoh K, Muramatsu H, Ochiai K, Torii S, Muramatsu T: Midkine, a heparin-binding cytokine, plays key roles in intraperitoneal adhesions. Biochem Biophys Res Commun 2004, 317:108–113.View ArticlePubMedGoogle Scholar
- Horiba M, Kadomatsu K, Nakamura E, Muramatsu H, Ikematsu S, Sakuma S, Hayashi K, Yuzawa Y, Matsuo S, Kuzuya M, Kaname T, Hirai M, Saito H: Neointima formation in a restenosis model is suppressed in midkine-deficient mice. J Clin Invest 2000, 105:489–495.View ArticlePubMedGoogle Scholar
- Hobo A, Yuzawa Y, Kosugi T, Kato N, Sato W, Maruyama S, Ito Y, Kobori H, Ikematsu S, Nishiyama A, Matsuo S, Kadomatsu K: The growth factor midkine regulates the rennin-angiotensin system in mice. J Clin Invest 2009, 119:1616–1625.View ArticlePubMedGoogle Scholar
- Sato W, Kadomatsu K, Yuzawa Y, Hotta N, Matsuo S, Muramatsu T: Midkine is involved in neutrophil infiltration into the tubulointerstitium in ischemic renal injury. J Immunol 2001, 167:3463–3469.PubMedGoogle Scholar
- Kawai H, Sato W, Yuzawa Y, Kosugi T, Matsuo S, Takei Y, Kadomatsu K, Muramatsu T: Lack of the growth factor midkine enhances survival against cisplatin-induced renal damage. Am J Pathol 2004, 165:1603–1612.View ArticlePubMedGoogle Scholar
- Kosugi T, Yuzawa Y, Sato W, Kawai H, Matsuo S, Takei Y, Muramatsu T, Kadomatsu K: Growth factor midkine is involved in the pathogenesis of diabetic nephropathy. Am J Pathol 2006, 168:9–19.View ArticlePubMedGoogle Scholar
- Takei Y, Kadomatsu K, Goto T, Muramatsu T: Combinational antitumor effect of siRNA against midkine and paclitaxel on growth of human prostate cancer xenografts. Cancer 2006, 107:864–873.View ArticlePubMedGoogle Scholar
- Dai LC, Wang X, Yao X, Lu YL, Ping JL, He JF: Enhanced therapeutic effects of combined chemotherapeutic drugs and midkine antisense oligonucleotides for hepatocellular carcinoma. World J Gastroenterol 2007, 13:1989–1994.PubMedGoogle Scholar
- Dai LC, Wang X, Yao X, Min LS, Ping JL, He JF: Antisense oligonucleotides targeting midkine inhibit tumor growth in an in situ human hepatocellular carcinoma model. Acta Pharmacol Sin 2007, 28:453–458.View ArticlePubMedGoogle Scholar
- Sato W, Takei Y, Yuzawa Y, Matsuo S, Kadomatsu K, Muramatsu T: Midkine antisense oligodeoxyribonucleotide inhibits renal damage induced by ischemic reperfusion. Kidney Int 2005, 67:1330–1339.View ArticlePubMedGoogle Scholar
- Hayashi K, Banno H, Kadomatsu K, Takei Y, Komori K, Muramatsu T: Antisense oligodeoxyribonucleotide as to the growth factor midkine suppresses neointima formation induced by balloon injury. Am J Physiol Heart Circ Physiol 2005, 288:H2203–2209.View ArticlePubMedGoogle Scholar
- Banno H, Takei Y, Muramatsu T, Komori K, Kadomatsu K: Controlled release of small interfering RNA targeting midkine attenuates intimal hyperplasia in vein grafts. J Vasc Surg 2006, 44:633–641.View ArticlePubMedGoogle Scholar
- Yamamoto H, Muramatsu H, Nakanishi T, Natori Y, Sakuma S, Ishiguro N, Muramatsu T: Midkine as a molecular target: comparison of effects of chondroitin sulfate E and siRNA. Biochem Biophys Res Commun 2006, 351:915–919.View ArticlePubMedGoogle Scholar
- Muramatsu H, Shirahama H, Yonezawa S, Maruta H, Muramatsu T: Midkine, a retinoic acid-inducible growth/differentiation factor: immunochemical evidence for the function and distribution. Dev Biol 1993, 159:392–402.View ArticlePubMedGoogle Scholar
- Muramatsu H, Inui T, Kimura T, Sakakibara S, Song X, Murata H, Muramatsu T: Localization of heparin-binding, neurite outgrowth and antigenic regions in midkine molecule. Biochem Biophys Res Commun 1994, 203:1131–1139.View ArticlePubMedGoogle Scholar
- Iwasaki W, Nagata K, Hatanaka H, Inui T, Kimura T, Muramatsu T, Tasumi M, Inagaki F: Solution structure of midkine, a new heparin-binding growth factor. EMBO J 1997, 16:6936–6946.View ArticlePubMedGoogle Scholar
- Asai T, Watanabe K, Ichihara-Tanaka K, Kaneda N, Kojima S, Iguchi A, Inagaki F, Muramatsu T: Identification of heparin-binding sites in midkine and their role in neurite-promotion. Biochem Biophys Res Commun 1 997, 236:66–70.View ArticleGoogle Scholar
- Maeda N, Ichihara-Tanaka K, Kimura T, Kadomatsu K, Muramatsu T, Noda M: A receptor-like protein-tyrosine phosphatase PTPζ/RPTPβ binds a heparin-binding growth factor midkine. Involvement of arginine 78 of midkine in the high affinity binding to PTPζ. J Biol Chem 1999, 274:12474–12479.View ArticlePubMedGoogle Scholar
- Muramatsu H, Zou K, Sakaguchi N, Ikematsu S, Sakuma S, Muramatsu T: LDL receptor-related protein as a component of the midkine receptor. Biochem Biophys Res Commun 2000, 270:936–941.View ArticlePubMedGoogle Scholar
- Muramatsu H, Zou P, Suzuki H, Oda Y, Chen GY, Sakaguchi N, Sakuma S, Maeda N, Noda M, Takada Y, Muramatsu T: α-4β1 and α-6β1 integrins are functional receptors for midkine, a heparin-binding growth factor. J Cell Sci 2004, 117:5405–5415.View ArticlePubMedGoogle Scholar
- Chen S, Bu G, Takei Y, Sakamoto K, Ikematsu S, Muramatsu T: Midkine and LDL-receptor-related protein 1 contribute to the anchorage-independent cell growth of cancer cells. J Cell Sci 2007, 120:4009–4015.View ArticlePubMedGoogle Scholar
- Ikematsu S, Yano A, Aridome K, Kikuchi M, Kumai H, Nagano H, Okamoto K, Oda M, Sakuma S, Aikou T, Muramatsu H, Kadomatsu K, Muramatsu T: Serum midkine levels are increased in patients with various types of carcinomas. Br J Cancer 2000, 83:701–706.View ArticlePubMedGoogle Scholar
- Qi M, Ikematsu S, Maeda N, Ichihara-Tanaka K, Sakuma S, Noda M, Muramatsu T, Kadomatsu K: Haptotactic migration induced by midkine. Involvement of protein-tyrosine phosphatase ζ, mitogen-activated protein kinase, and phosphatidylinositol 3-kinase. J Biol Chem 2001, 276:15868–15875.PubMedGoogle Scholar
- Xiong JP, Stehle T, Diefenbach B, Zhang R, Dunker R, Scott DL, Joachimiak A, Goodman SL, Arnaout MA: Crystal structure of the extracellular segment of integrin αVβ3. Science 2001, 294:339–345.View ArticlePubMedGoogle Scholar
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