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ATM (ataxia telangiectasia mutated) ºÐ¼® ¼­ºñ½º

°³¹ßÇϽŠlead compounds¿¡ ´ëÇÑ ATM (ataxia telangiectasia mutated) activity¸¦ ELISA ¹æ¹ýÀ¸·Î ÃøÁ¤ÇØ µå¸³´Ï´Ù.

Eurofins Discovery
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    • ATM (ataxia telangiectasia mutated) Àº phosphatidylinositol 3-kinase related kinase (PIKK) family¿¡ ¼ÓÇÏ´Â serine/threonine kinase ÀÔ´Ï´Ù. ÀÌ kinases ±×·ìÀº mTOR, ATR, DNA-PK¸¦ Æ÷ÇÔÇÕ´Ï´Ù.
    • Cell cycle arrestÀÇ °á°ú·Î½á, DNA ¼Õ»ó°ú °ü·ÃµÈ ATM ´Ü¹éÁúÀÇ È°¼ºÀ» ¾ïÁ¦ÇÏ´Â inhibitor¸¦ ºÐ¼®ÇÕ´Ï´Ù.
    • ¼¼Æ÷ ³»¿¡¼­ ATM¿¡ ´ëÇÑ Å¸°Ù substrate Áß ÇϳªÀÎ p53ÀÇ ÀλêÈ­¸¦ ¸ð´ÏÅ͸µÇÔÀ¸·Î½á, ATM activity¸¦ ÃøÁ¤ÇÕ´Ï´Ù.
    • ºÐ¼® ¼­ºñ½º´Â ÇÑ´Þ¿¡ Çѹø ÁøÇàµÇ¸ç, °á°ú ¹ß¼Û±îÁö 5-6ÁÖ ¼Ò¿äµË´Ï´Ù.
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ATM activity can be detected by ELISA. Following the incubation with c-Myc-tagged p53 and ATP, the products are captured onto a microplate coated with anti-c-Myc antibody. The reaction components are washed away leaving just the p53 on the plate. Detection of phosphorylated p53 is achieved using a phospho-specific antibody to Ser-15 of p53.
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Figure 1. ELISA signal is specific to purified ATM. ATM was titrated to demonstrate p53 phosphorylation activity. Negative controls were derived from cells transfected with empty vector, or with a kinase which does not phosphorylate Ser-15 of p53.
Figure 2. Western blot also demonstrates p53 phosphorylation at Ser-15. Left hand panel is the ponceau stain of the nitrocellulose membrane before blotting. Right hand panel is the western blot. Lanes (L-R) are: markers; ATM + p53 (no ATP); ATM + ATP (no p53); ATM + ATP + p53; p53 only.
Figure 3. The ATM assay can be used to screen inhibitors. ATM was assayed by ELISA in the presence of selected small molecule inhibitors at 1 and 10 μM.
Figure 4. Dose responses for selected inhibitors. EC50s are: KU-55933, 7 nM; PI-103, 450 nM, PIK-90, 500 nM.
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