Novel DNA Aptamer for CYP24A1 Inhibition with Enhanced Antiproliferative Activity in Cancer Cells
Novel DNA Aptamer for CYP24A1 Inhibition with Enhanced Antiproliferative Activity in Cancer Cells
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DOI：
10.1021/acsami.1c22965
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发表时间：
2022-04-27
影响因子：
9.5
通讯作者：
Biyani, Manish
中科院分区：
文献类型：
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作者：
Biyani, Madhu;Yasuda, Kaori;Biyani, Manish
维生素D3失活酶CYP 24 A1（细胞色素P450家族24亚家族，下文称为CYP 24）的过度表达可引起慢性肾病、骨质疏松症和几种类型的癌症。因此，CYP 24抑制被认为是一种潜在的治疗方法。维生素D3模拟物和小分子抑制剂已被证明是有效的，但非特异性结合，耐药性和潜在的毒性限制了它们的有效性。我们利用基于竞争的适体选择策略，以CYP 24为阳性靶蛋白，以CYP 27 B1（催化活性维生素D3产生的酶）为反靶蛋白，筛选出一种新的基于70-nt DNA适体的CYP 24抑制剂。其中一种已鉴定的适体Apt-7与CYP 24的结合亲和力是类似竞争对手CYP 27 B1的5.8倍。有趣的是，Apt-7选择性抑制CYP 24（相对CYP 24活性降低39.1 +/- 3%，几乎不抑制CYP 27 B1）。此外，Apt-7通过内吞作用在CYP 24过表达的A549肺腺癌细胞中显示细胞内化，并在癌细胞中诱导内源性CYP 24抑制的抗增殖活性。我们还采用了高速原子力显微镜实验和分子对接模拟，以提供一个基于适配体的CYP 24抑制机制的单分子解释。在这项研究中发现的新型适体为生物医学研究提供了一个产生识别和抑制CYP 24的新探针的机会，并可能有助于癌症的诊断和治疗。
Overexpression of the vitamin D3-inactivating enzyme CYP24A1 (cytochrome P450 family 24 subfamily and hereafter referred to as CYP24) can cause chronic kidney diseases, osteoporosis, and several types of cancers. Therefore, CYP24 inhibition has been considered a potential therapeutic approach. Vitamin D3 mimetics and small molecule inhibitors have been shown to be effective, but nonspecific binding, drug resistance, and potential toxicity limit their effectiveness. We have identified a novel 70-nt DNA aptamer-based inhibitor of CYP24 by utilizing the competition-based aptamer selection strategy, taking CYP24 as the positive target protein and CYP27B1 (the enzyme catalyzing active vitamin D3 production) as the countertarget protein. One of the identified aptamers, Apt-7, showed a 5.8-fold higher binding affinity with CYP24 than the similar competitor CYP27B1. Interestingly, Apt-7 selectively inhibited CYP24 (the relative CYP24 activity decreased by 39.1 +/- 3% and showed almost no inhibition of CYP27B1). Furthermore, Apt-7 showed cellular internalization in CYP24-overexpressing A549 lung adenocarcinoma cells via endocytosis and induced endogenous CYP24 inhibition-based antiproliferative activity in cancer cells. We also employed high-speed atomic force microscopy experiments and molecular docking simulations to provide a single-molecule explanation of the aptamer-based CYP24 inhibition mechanism. The novel aptamer identified in this study presents an opportunity to generate a new probe for the recognition and inhibition of CYP24 for biomedical research and could assist in the diagnosis and treatment of cancer.