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

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.