| 研究課題/領域番号 |
17K15017
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| 研究機関 | 筑波大学 |
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研究代表者 |
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| 研究期間 (年度) |
2017-04-01 – 2019-03-31
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| キーワード | in vitro selection / macrocyclic peptides / ALK1 receptor / tumor angiogenesis / cancer signaling |
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| 研究実績の概要 |
ALK1 is thought to promote tumor angiogenesis, and direct regulation of aberrant ALK1 activity is hypothesized to inhibit tumor progression. We had previously isolated a promising macrocyclic peptide using in vitro selection and proceeded with its characterization. We first examined at its strength of binding to ALK1 in vitro. Although the selection assay suggested that there was interaction between ALK1-3R6-5 and ALK1, our physical measurement of the interaction did not indicate strong binding. To assist with the characterization, we produced chemically modified variants of ALKL1-3R6-5. We synthesized a biotinylated ALK1-3R6-5 for measurement of the binding strength and a fluorescein-labelled ALK-3R6-5 for cell staining. Testing of these derivatives is underway.
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| 現在までの達成度 (区分) |
現在までの達成度 (区分)
3: やや遅れている
理由
The characterization of the macrocyclic peptide was delayed due to the establishment of methods for detecting binding for detecting bioactivity. We discovered that the method that we used for the detection of binding strength is not sensitive enough for the detection of peptide binding to surface-bound protein. It is, however, capable of detecting protein binding to surface-bound peptide. While not difficult, this forced us to resynthesize and purify our peptide(s) with a biotin tag to facilitate binding to the biosensor surface.
For the detection of bioactivity, we need a reporter system to observe the inhibition of ALK1 signaling from whole cells. We are currently detecting what could be false positives and the failure of the fluorescein-labelled macrocyclic peptides supports this.
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| 今後の研究の推進方策 |
As we were only able to isolate a single clone from the initial selection and strong binding has yet to be observed, we plan to reselect using a new macrocyclic peptide library. In addition, since the extracellular domain of ALK1 that is used in the selection is very small (14 kDa) by our selection standards, we will compensate by using randomized libraries of larger biomolecules. Specifically, we are developing libraries based on triple helix bundles, known as affibodies. We surmise that the increased ligand surface area will promote stronger binding to ALK1. The established assay methods will be applied to the next generation of isolated ligands and their characterizations will be expedited as we are now more experienced with these methods.
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