| 研究課題/領域番号 |
21K15239
|
|---|
| 研究機関 | 金沢大学 |
|---|
研究代表者 |
Biyani Madhu 金沢大学, ナノ生命科学研究所, 特任助教 (30882245)
|
|---|
| 研究期間 (年度) |
2021-04-01 – 2023-03-31
|
| キーワード | CYP24 / Vitamin D3 / Aptamer / HS-AFM / Molecular docking |
|---|
| 研究実績の概要 |
In the first year, to predict and investigate the possible binding sites and interaction mechanism between Aptamer and CYP24, we analyzed the binding sites of Aptamer in CYP24 by molecular docking. The top-ranking docking models that can inhibit the enzyme activity were selected and analyzed. The docking results revealed that the two short arms of Y-shaped Apt-7 embedded in the binding sites, and sterically covers the substrate binding site of CYP24 and adrenodoxin (ADX) binding site.
Parallelly, we observed the real-times dynamics of Aptamer and CYP24 binding to Aptamer by HS-AFM and the spatio-temporal analysis of captured images enabled us to characterize the binding interaction of Aptamer and CYP24A1.
To test the prediction accuracy and reliability of molecular docking results by experiments, CYP24-dependent activity was evaluated in the presence or absence of Aptamer, and inhibition was calculated using Lineweaver-Burk plots. Our obtained results clearly shown that the maximum velocity (Vmax) of the enzyme is unchanged, and this is seen in the Lineweaver-Burk plot as changing the 1/S intercept but not affecting the 1/v intercept. So, this result strongly suggested that Apt-7 depict a competitive inhibition of CYP24 activity.
|
| 現在までの達成度 (区分) |
現在までの達成度 (区分)
1: 当初の計画以上に進展している
理由
In the first year, we set the goal to utilize HS-AFM platform that has been developed by our collaborator in Kanazawa University and visualize the real time images of Aptamer with and without CYP24. In addition, parallel study by molecular docking was planned to obtain the atomic information essential for binding event.
I am very thankful to the cooperation of HS-AFM and molecular docking collaborators for providing their continuous guidance and support to this research work and I could be able to make a smooth progress toward our preset goals in the first year. Now we can expect that the information obtained from HS-AFM and molecular docking analysis will be useful to optimize aptamer molecules and use them in cellulo and vivo studies.
|
| 今後の研究の推進方策 |
Based on the information obtained from HS-AFM and molecular docking, the optimized length and sequence of Aptamer will be evaluated for improving the inhibition capability and specificity of the CYP24 by in vitro enzymatic and cell-based assays. After successful optimization of aptamer, we will engage to the in vivo experiment, which is evaluating the plasma concentration of 1,25-D3 in mice administrated 1,25-D3. Then, we will investigate the inhibitory effects of aptamer using tumor-bearing mice.
|
| 次年度使用額が生じた理由 |
In this year, I plan to have the business trip to the the Toyama Prefectural University for discussion about the characterization of aptamers in cell-based assays. I also plan to attend the domestic conferences. However due to spread of Corona infection, the most of conferences and meetings cancelled and some held online. So, I did not have a business trip. Thus, the plan is changed, there is incurring amount to be used next fiscal year.
Usage Plan: I plan to have business trip to Toyama Prefectural University for discussion about the characterization and how to deliver aptamers for in vivo applications. I also plan to attend the domestic and international conferences. I intend the incurring amount to be used next fiscal year.
|
