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Halogenated biscoumarins have been reported as versatile antimicrobial and antiviral agents. Here, we screened eleven halogenated biscoumarin derivatives against DENV and identified compounds 3 and 4\u2014bearing chlorine substitutions at the 3- or 4-position of the phenyl moiety\u2014as the most potent inhibitors of DENV2, with EC\n                    <jats:sub>50<\/jats:sub>\n                    values of 3.62\u20139.72 \u00b5M and 4.62\u201310.88 \u00b5M, respectively, and selectivity indices (SI) up to 20.97. Both compounds also inhibited all four DENV serotypes and Zika virus (ZIKV) with comparable efficacy. Mechanistic analyses demonstrated that compounds 3 and 4 significantly suppressed viral translation and RNA replication in infectious and replicon models. Long-term passaging generated mutations in NS4B, although these substitutions did not confer resistance, was involved in self-dimerization to curvature formation of ER-derived spherules, which may reflect adaptive changes linked to host interaction. In vitro enzymatic assays further indicated that the viral NS5 methyltransferase is a potential target, with IC\n                    <jats:sub>50<\/jats:sub>\n                    values of 4.60\u2009\u00b1\u20090.83 \u00b5M and 3.17\u2009\u00b1\u20090.25 \u00b5M for compounds 3 and 4, respectively, and both compounds inhibited NS3 protease activity by &gt;\u200989% at 50 \u00b5M. Collectively, these results identify chlorinated biscoumarins as promising antiviral scaffolds that impair flaviviral translation and replication, supporting their further optimization and in vivo evaluation.\n                  <\/jats:p>","DOI":"10.1038\/s41598-026-35654-8","type":"journal-article","created":{"date-parts":[[2026,1,16]],"date-time":"2026-01-16T11:25:25Z","timestamp":1768562725000},"update-policy":"https:\/\/doi.org\/10.1007\/springer_crossmark_policy","source":"Crossref","is-referenced-by-count":1,"title":["Chlorinated bis-4-hydroxycoumarins suppress flavivirus replication by inhibiting dengue virus type 2 translation and replication"],"prefix":"10.1038","volume":"16","author":[{"given":"Naphat","family":"Loeanurit","sequence":"first","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Thi-Hong-Truc","family":"Phan","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Kowit","family":"Hengphasatporn","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Thanchanok","family":"Chanachanvong","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Borwornlak","family":"Toopradab","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Phornphimon","family":"Maitarad","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Prangwalai","family":"Chanchaem","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Vorthon","family":"Sawaswong","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Sunchai","family":"Payungporn","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Ajirawadee","family":"Suwanchan","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Auwal Rabiu","family":"Auwal","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Kittikhun","family":"Wangkanont","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Thanyada","family":"Rungrotmongkol","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Yasuteru","family":"Shigeta","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Tanatorn","family":"Khotavivattana","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Warinthorn","family":"Chavasiri","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Siwaporn","family":"Boonyasuppayakorn","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"297","published-online":{"date-parts":[[2026,1,16]]},"reference":[{"key":"35654_CR1","unstructured":"Lindenbach, B., Murray, C., Thiel, H. & Rice, C. 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