Human FAM111A inhibits vaccinia virus replication by degrading viral protein I3 and is antagonized by poxvirus host range factor SPI-1.

Zoonotic poxviruses such as mpox virus (MPXV) continue to threaten public health safety since the eradication of smallpox. Vaccinia virus (VACV), the prototypic poxvirus used as the vaccine strain for smallpox eradication, is the best-characterized member of the poxvirus family. VACV encodes a serine protease inhibitor 1 (SPI-1) conserved in all orthopoxviruses, which has been recognized as a host range factor for modified VACV Ankara (MVA), an approved smallpox vaccine and a promising vaccine vector. FAM111A (family with sequence similarity 111 member A), a nuclear protein that regulates host DNA replication, was shown to restrict the replication of a VACV SPI-1 deletion mutant (VACV-ΔSPI-1) in human cells. Nevertheless, the detailed antiviral mechanisms of FAM111A were unresolved. Here, we show that FAM111A is a potent restriction factor for VACV-ΔSPI-1 and MVA. Deletion of FAM111A rescued the replication of MVA and VACV-ΔSPI-1 and overexpression of FAM111A significantly reduced viral DNA replication and virus titers but did not affect viral early gene expression. The antiviral effect of FAM111A necessitated its trypsin-like protease domain and DNA-binding domain but not the PCNA-interacting motif. We further identified that FAM111A translocated into the cytoplasm upon VACV infection by degrading the nuclear pore complex via its protease activity, interacted with VACV DNA-binding protein I3, and promoted I3 degradation through autophagy. Moreover, SPI-1 from VACV, MPXV, or lumpy skin disease virus was able to antagonize FAM111A by prohibiting its nuclear export. Our findings reveal the detailed mechanism by which FAM111A inhibits VACV and provide explanations for the immune evasive function of VACV SPI-1.

Anticancer Activity and Molecular Mechanisms of an Ursodeoxycholic Acid Methyl Ester-Dihydroartemisinin Hybrid via a Triazole Linkage in Hepatocellular Carcinoma Cells.

Hepatocellular carcinoma is the third most common cause of cancer-related death according to the International Agency for Research on Cancer. Dihydroartemisinin (DHA), an antimalarial drug, has been reported to exhibit anticancer activity but with a short half-life. We synthesized a series of bile acid-dihydroartemisinin hybrids to improve its stability and anticancer activity and demonstrated that an ursodeoxycholic-DHA (UDC-DHA) hybrid was 10-fold more potent than DHA against HepG2 hepatocellular carcinoma cells. The objectives of this study were to evaluate the anticancer activity and investigate the molecular mechanisms of UDCMe-Z-DHA, a hybrid of ursodeoxycholic acid methyl ester and DHA via a triazole linkage. We found that UDCMe-Z-DHA was even more potent than UDC-DHA in HepG2 cells with IC50 of 1 µM. Time course experiments and stability in medium determined by cell viability assay as well as HPLC-MS/MS analysis revealed that UDCMe-Z-DHA was more stable than DHA, which in part accounted for the increased anticancer activity. Mechanistic studies revealed that UDCMe-Z-DHA caused G0/G1 arrest and induced reactive oxygen species (ROS), mitochondrial membrane potential loss and autophagy, which may in turn lead to apoptosis. Compared to DHA, UDCMe-Z-DHA displayed much lower cytotoxicity toward normal cells. Thus, UDCMe-Z-DHA may be a potential drug candidate for hepatocellular carcinoma.

Evaluation of the Anticancer Activity of a Bile Acid-Dihydroartemisinin Hybrid Ursodeoxycholic-Dihydroartemisinin in Hepatocellular Carcinoma Cells.

Hepatocellular carcinoma (HCC) is the most common primary liver malignancy in adults and accounts for 85-90% of all primary liver cancer. Based on the estimation by the International Agency for Research on Cancer in 2018, liver cancer is the fourth leading cause of cancer death globally. Dihydroartemisinin (DHA), the main active metabolite of artemisinin derivatives, is a well-known drug for the treatment of malaria. Previous studies have demonstrated that DHA exhibits antitumor effects toward a variety of human cancers and has a potential for repurposing as an anticancer drug. However, its short half-life is a concern and may limit the application in cancer therapy. We have reported that UDC-DHA, a hybrid of bile acid ursodeoxycholic acid (UDCA) and DHA, is ∼12 times more potent than DHA against a HCC cell line HepG2. In this study, we found that UDC-DHA was also effective against another HCC cell line Huh-7 with an IC50 of 2.16 µM, which was 18.5-fold better than DHA with an IC50 of 39.96 µM. UDC-DHA was much more potent than the combination of DHA and UDCA at 1:1 molar ratio, suggesting that the covalent linkage rather than a synergism between UDCA and DHA is critical for enhancing DHA potency in HepG2 cells. Importantly, UDC-DHA was much less toxic to normal cells than DHA. UDC-DHA induced G0/G1 arrest and apoptosis. Both DHA and UDC-DHA significantly elevated cellular reactive oxygen species generation but with different magnitude and timing in HepG2 cells; whereas only DHA but not UDC-DHA induced reactive oxygen species in Huh-7 cells. Depolarization of mitochondrial membrane potential was detected in both HepG2 and Huh-7 cells and may contribute to the anticancer effect of DHA and UDC-DHA. Furthermore, UDC-DHA was much more stable than DHA based on activity assays and high performance liquid chromatography-MS/MS analysis. In conclusion, UDC-DHA and DHA may exert anticancer actions via similar mechanisms but a much lower concentration of UDC-DHA was required, which could be attributed to a better stability of UDC-DHA. Thus, UDC-DHA could be a better drug candidate than DHA against HCC and further investigation is warranted.