| Summary: | 碩士 === 國立陽明大學 === 醫學生物技術暨檢驗學系 === 101 === Enterovirus 71 (EV71), a member of the genus Enterovirus of the family Picornaviridae, is a common pathogen often associated with mild foot-and-mouth disease and occasionally severe neurological manifestations in young children. However, there have been no anti-EV71 drugs available for clinical use. Several strategies for anti-EV71 drug development have been employed, ranging from viral target-based design, compound screening, and compound series generated from antiviral developments against related poliovirus and human rhinoviruses. All these efforts exclusively focused on viral targets, which likely develop drug resistance. Viral infection can induce host genes and cellular signaling pathways that are essential for virus propagation. Therefore, we proceeded with a screen from the Library of Pharmacologically Active Compound (LOPAC; Sigma–Aldrich) that consist of 1280 small molecules with well-defined bioactivities against diverse cell functions for hits that could intervene the activated, virus-supporting cellular genes/pathways. Using fluorescence resonance energy transfer (FRET), our laboratory previously developed a genetically engineered, FRET-based biosensor achieved by stable expression of a fusion substrate construct composed of the green fluorescent protein 2 and red fluorescent protein 2, with a cleavage motif of the EV71 2A protease connected in between. The HeLa cell based-FRET biosensor showed a real-time and quantifiable impairment of FRET proportional to EV71 replication. We first assessed the adaptability of the FRET-biosensor as a high-throughput drug screen platform, and showed its rapidity and robustness with a Z’ value at 0.53. Among fifteen compounds that significantly (>50%) elevated the FRET ratio, E compound, a clinical drug for treating amebiasis, was chosen for further study. The 50% effective concentration (EC50) and 50% cytotoxicity concentration (CC50) are 1.19 μM and >4096 μM, respectively, making the selectivity index >3439.1. E compound significantly protected the infected cells from the cytopathic effect and cell death caused by EV71 in a dose-dependent manner. From the time-of-addition experiment, we identified the E compound action time at about 0~5 h pi. We further showed that the compound effectively intervened the virus lifecycle at the stages of the viral protein synthesis, genome replication but not the viral translation initiation and protease activity. This new use of E compound may provide new perspectives for therapeutic strategies for controlling EV71 infections and their consequences.
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