PO.ET06.05 · 实验与分子治疗

通过虚拟筛选鉴定阻断肿瘤特异性调幅射频电磁场治疗作用的FDA批准药物

Virtual screening identification of FDA approved drugs blocking the therapeutic action of tumor-specific amplitude-modulated radiofrequency electromagnetic fields

编号 5712 展板 1 时间 4/21 02:00–05:00 区域 Section 13 主讲 Hugo Jimenez, PhD

分会场 Molecular Targets 2

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作者与单位 Authors & Affiliations

Hugo Jimenez¹, Ping Dou¹, Aaban A. Azmi¹, Elyas Khan¹, Alhussein Elshebiny¹, Yusuf Fateh¹, Nathan Elias¹, Cameron Osenkowski¹, Liyue Zhang¹, Grayson Barker¹, Allan M. Johansen¹, Ravi Paluri², Janaka S. S. Liyanage¹, Mohammed N. Al Hallak¹, Anthony F. Shields¹, Muhammad W. Saif¹, Husain Y. Khan¹, Amro Aboukameel¹, Md Hafiz Uddin¹, Sahar F. Bannoura¹, Ramzi M. Mohammad¹, Alexandre Barbault³, Boris C. Pasche¹

¹Oncology, Wayne State University School of Medicine, Barbara Ann Karmanos Cancer Institute, Detroit, MI,²Medicine, Atrium Health Wake Forest Baptist Hospital, Winston-Salem, NC,³TheraBionic GmbH, Ettlingen, Germany

摘要 Abstract

中文摘要

背景：经由置于患者舌上的勺形天线递送的调幅27.12 MHz射频电磁场（AM RF EMF）可使晚期肝细胞癌（HCC）患者的原发和转移肿瘤缩小。该治疗已获FDA批准，用于一线和二线治疗失败的晚期HCC患者（H220001）。AM RF EMF发挥直接抗增殖作用的机制依赖于细胞外Ca2+经CaV3.2（CACNA1H）T型电压门控钙通道（VGCC）流入癌细胞。在此我们表明，HCC特异性AM RF EMF（HCCMF）的治疗作用受到与ethosuximide（乙琥胺）结合谱相同的药物的抑制。方法：使用AlphaFold蛋白质结构数据库（AFDB登录号：AF-O95180-F1-v4）进行的计算对接研究显示，ethosuximide结合于由CACNA1H结构域1中游离N端片段的氨基酸（AAs）Val79和Phe80以及S2-S3连接区的Leu160和Phe161所形成的一个胞内疏水口袋。为确认ethosuximide结合口袋（EBP）的氨基酸[Val79、Phe80、Leu160和Phe161]对于抑制HCCMF的重要性，我们对FDA药物库进行虚拟筛选（VS），寻找与ethosuximide结合方式相似的药物。随后在HCC细胞系中对经VS预测/鉴定的药物进行检测（集落形成、qPCR和Fluo-4 Ca2+染料），评估其对HCCMF介导的细胞增殖的影响。结果：Tadalafil（他达拉非）和abiraterone acetate（醋酸阿比特龙）是通过VS发现并检测的、能抑制HCCMF的前两种药物。在Huh7细胞系中，就集落形成和Ki-67/Cyclin D1表达而言，tadalafil阻断了HCCMF介导的细胞增殖。Tadalafil还在Huh7和HepG2细胞系中阻断了以Fluo-4评估的Ca2+流入。有趣的是，我们发现sildenafil（西地那非）虽与tadalafil同属磷酸二酯酶抑制剂类，却未阻断HCCMF介导的Ca2+流入。这一点通过在Huh7和HepG2中的Fluo-4 Ca2+染色得以显示。具体而言，即使存在sildenafil，HCCMF处理组的平均荧光强度（MFI）仍更高。与SHAM（无处理对照）相比，HCCMF-MFI在Huh7中增加了33.07%（p值：0.0162，n=5），在HepG2中增加了31.86%（p值：0.0012，n=5）。结论：EBP氨基酸的可及性对于HCCMF介导的HCC细胞增殖抑制是必需的。经实验室实验证实的理论建模表明，药物与该口袋的结合会干扰Ca2+流入并阻断HCCMF的抗癌效应。这些结果支持使用计算建模来预测和鉴定所有可能在癌症治疗期间使用、因而应在接受HCCMF治疗的患者中避免使用的FDA药物。

查看英文原文 English abstract

Background: Amplitude-modulated 27.12 MHz radiofrequency electromagnetic fields (AM RF EMF) delivered via a spoon-shaped antenna placed on the patient's tongue result in shrinkage of the primary and metastatic tumors in patients with advanced hepatocellular carcinoma (HCC). Treatment is FDA-approved for patients with advanced HCC who fail 1 st line and 2 nd line therapy (H220001). The mechanism by which AM RF EMF has a direct antiproliferative effect depends on the influx of extracellular Ca 2+ into the cancer cell via Ca V 3.2 ( CACNA1H ) T-type voltage-gated calcium channel (VGCC). Here we show that HCC-specific AM RF EMF's (HCCMF) therapeutic action is inhibited by drugs with the same binding profile as ethosuximide. Methods: Computational docking study, using AlphaFold Protein Structure Database (AFDB accession: AF-O95180-F1-v4) showed that ethosuximide binds to an intracellular, hydrophobic pocket formed by amino acids (AAs) Val79 and Phe80 of the free N-terminal segment and Leu160 and Phe161 of S2-S3 linker of CACNA1H Domain 1. To confirm the ethosuximide binding-pocket (EBP) AAs [Val79, Phe80, Leu160, and Phe161] are important for the inhibition of HCCMF, we virtually screened (VS) the FDA library for drugs that would bind similarly to ethosuximide. Drugs predicted / identified via VS where then tested (colony formation, qPCR, and Fluo-4 Ca 2+ dye) HCCMF-mediated cell proliferation in HCC cell lines. Results: Tadalafil and abiraterone acetate are the first two drugs found, via VS and tested, to inhibit HCCMF. In the Huh7 cell line, tadalafil blocked HCCMF-mediated cell proliferation with respect to colony formation and Ki-67/Cyclin D1 expression. Tadalafil also blocked Ca 2+ influx assessed by Fluo-4 in Huh7 and HepG2 cell lines. Interestingly, we found that sildenafil, while belonging to the same class of phosphodiesterase inhibitors as tadalafil, did not block HCCMF-mediated Ca 2+ influx. This was shown via Fluo-4 Ca 2+ staining in both Huh7 and HepG2. Specifically, mean fluorescent intensity (MFI) was higher in the HCCMF treated group even when sildenafil was present. HCCMF-MFI increased by 33.07% ( p -value: 0.0162, n=5) in Huh7 and by 31.86% ( p -value: 0.0012, n=5) in HepG2 compared to SHAM (no treatment control). Conclusions: The availability of the EBP AAs is necessary for HCCMF-mediated inhibition of HCC cell proliferation. Theoretical modelling confirmed by laboratory experiments demonstrate that binding of drugs to this pocket interferes with Ca 2+ influx and block the anti-cancer effects of HCCMF. These results support the use of computational modeling to predict and identify all FDA drugs which could be used during cancer therapy and should thereby be avoided in patients receiving HCCMF treatment.

利益披露 Disclosure

H. Jimenez, None.. P. Dou, None.. A. A. Azmi, None.. E. Khan, None.. A. Elshebiny, None.. Y. Fateh, None.. N. Elias, None.. C. Osenkowski, None.. L. Zhang, None.. G. Barker, None.. A. M. Johansen, None.. R. Paluri, None.. J. S. S. Liyanage, None.. M. N. Al Hallak, None.. A. F. Shields, None.. M. W. Saif, None.. H. Y. Khan, None.. A. Aboukameel, None.. M. H. Uddin, None.. S. F. Bannoura, None.. R. M. Mohammad, None. A. Barbault, TheraBionic GmbH Employment, Stock, Patent, Other Intellectual Property. TheraBionic Employment, Stock, Patent, Other Intellectual Property. B. C. Pasche, TheraBionic GmbH Employment, g., Board of Directors, non-salaried role), Stock, Patent, Other Intellectual Property. TheraBionic Employment, g., Board of Directors, non-salaried role), Stock, Patent, Other Intellectual Property.

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