Antiviral activity of shikonin ester derivative PMM-034 against enterovirus 71 in vitro

Abstract Human enterovirus 71 (EV71) is the major causative agent of hand, foot, and mouth disease (HFMD), particularly in infants andchildren below 4 years of age. Shikonin is a bioactive compound with anti-inflammatory, antiviral, and antibacterial activitiesderived from the roots of the Chinese medicinal herb Lithospermum erythrorhizon. This study aimed to examine the antiviralactivity of PMM-034, a shikonin ester derivative, against EV71 in rhabdomyosarcoma (RD) cells. Cytotoxicity of PMM-034 onRD cells was determined using WST-1 assay. Dose- and time-dependent effects of PMM-034 on EV71 replication in RD cellswere determined using plaque reduction assay. mRNA expression levels of EV71/VP1 and pro-inflammatory cytokines (IL-1b,IL-6, IL-8, and TNF-a) were determined by real-time RT-PCR, and EV71/VP1 and phospho-p65 protein expressions weredetermined by western blot analysis. PMM-034 exhibited only weak cytotoxicity against RD cells. However, PMM-034 exhibitedsignificant antiviral activity against EV71 in RD cells with 50% inhibitory concentration of 2.31 mg/mL. The VP1 mRNA andprotein levels were significantly reduced in cells treated with PMM-034. Furthermore, relative mRNA expression levels of IL-1b,IL-6, IL-8, and TNF-a significantly decreased in the cells treated with PMM-034, while the phospho-p65 protein expression wasalso significantly lower in the treated cells. These results indicated that PMM-034 suppressed the expressions of pro-inflammatory cytokines in RD cells, exhibiting antiviral activity against EV71, as evidenced by the reduced VP1 mRNA andprotein levels in PMM-034-treated cells. Thus, PMM-034 is a promising candidate for further development as an EV71 inhibitor.Key words: EV71; VP1; Shikonin ester derivatives PMM-034; Rhabdomyosarcoma cells; NF-kB