{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"institution":[{"name":"bioRxiv"}],"indexed":{"date-parts":[[2026,7,30]],"date-time":"2026-07-30T13:18:28Z","timestamp":1785417508325,"version":"3.56.0"},"posted":{"date-parts":[[2026,7,10]]},"group-title":"Biophysics","reference-count":64,"publisher":"openRxiv","license":[{"start":{"date-parts":[[2026,7,10]],"date-time":"2026-07-10T00:00:00Z","timestamp":1783641600000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"award":["U19AI171110"],"award-info":[{"award-number":["U19AI171110"]}],"id":[{"id":"https:\/\/ror.org\/01cwqze88","id-type":"ROR","asserted-by":"publisher"}]},{"name":"Alberta Ministry of Technology and Innovation","award":["Striving for Pandemic Preparedness\u2014The Alberta Research Consortium"],"award-info":[{"award-number":["Striving for Pandemic Preparedness\u2014The Alberta Research Consortium"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"accepted":{"date-parts":[[2026,7,10]]},"abstract":"<jats:title>ABSTRACT<\/jats:title>\n                <jats:p>\n                  Enterovirus D68 (EV-D68) is a non-polio picornavirus that has caused increasing rates of severe respiratory illness and acute flaccid myelitis in children worldwide this century. There are no approved vaccines or antivirals for EV-D68. Thus, we conducted a crystallographic fragment screening (CFS) and a high-throughput screening (HTS) biochemical assay against the EV-D68 RNA-dependent RNA polymerase 3D (3D\n                  <jats:sup>pol<\/jats:sup>\n                  ) to identify ligandable sites and non-nucleoside compounds that can spearhead anti-enteroviral drug discovery. The CFS, involving 650 fragments, identified 68 hit compounds (~10% hit rate) distributed across 3D\n                  <jats:sup>pol<\/jats:sup>\n                  , including the functionally relevant sites RNA template channel, Active site, and RNA primer channel, and the previously unknown \u201cThumb site II\u201d and \u201cIndex-middle finger pocket\u201d. Inhibition assays confirmed that compounds binding to each site can inhibit EV-D68 3D\n                  <jats:sup>pol<\/jats:sup>\n                  activity. The HTS, a fluorescence-based PicoGreen biochemical assay, permitted screening 50,000 compounds of the ChemBridge Premium Library (0.77% hit rate). After a second-round dose-response screening, we identified 5-aminoindazole as a promising scaffold that inhibits EV-D68 3D\n                  <jats:sup>pol<\/jats:sup>\n                  , including hit-to-lead compound 727590, which displayed an IC\n                  <jats:sub>50<\/jats:sub>\n                  value of 25 \u03bcM and preliminary structure-activity relationships. These hits offer amenable starting points for discovery and development of non-nucleoside inhibitors and provide opportunities for structure-based drug design against enteroviruses.\n                <\/jats:p>\n                <jats:sec>\n                  <jats:title>GRAPHICAL ABSTRACT<\/jats:title>\n                  <jats:fig id=\"ufig1\" position=\"float\" orientation=\"portrait\" fig-type=\"figure\">\n                    <jats:caption>\n                      <jats:p>\n                        Created with\n                        <jats:ext-link xmlns:xlink=\"http:\/\/www.w3.org\/1999\/xlink\" ext-link-type=\"uri\" xlink:href=\"http:\/\/biorender.com\">biorender.com<\/jats:ext-link>\n                        and PyMOL Molecular Graphics System, version 2.5.0. Schr\u00f6dinger, LLC.\n                      <\/jats:p>\n                    <\/jats:caption>\n                    <jats:graphic xmlns:xlink=\"http:\/\/www.w3.org\/1999\/xlink\" xlink:href=\"737532v1_ufig1\" position=\"float\" orientation=\"portrait\"\/>\n                  <\/jats:fig>\n                <\/jats:sec>","DOI":"10.64898\/2026.07.09.737532","type":"posted-content","created":{"date-parts":[[2026,7,11]],"date-time":"2026-07-11T11:20:37Z","timestamp":1783768837000},"source":"Crossref","is-referenced-by-count":0,"title":["Discovery of non-nucleoside inhibitors of the enterovirus D68 3D polymerase through crystallographic fragment and high-throughput biochemical screening"],"prefix":"10.64898","author":[{"ORCID":"https:\/\/orcid.org\/0000-0001-8686-9687","authenticated-orcid":false,"given":"Iman","family":"Biswas","sequence":"first","affiliation":[{"name":"Center for Advanced Biotechnology and Medicine, Rutgers, the State University of New Jersey, Piscataway, NJ 08854, USA"},{"name":"Department of Chemistry and Chemical Biology, Rutgers, the State University of New Jersey, Piscataway, NJ 08854, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-1223-8193","authenticated-orcid":false,"given":"Qinghui","family":"Wang","sequence":"additional","affiliation":[{"name":"Department of Pharmaceutical Chemistry, UCSF, CA 94158, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-8812-829X","authenticated-orcid":false,"given":"Jack T.","family":"McCann","sequence":"additional","affiliation":[{"name":"Department of Pharmaceutical Chemistry, UCSF, CA 94158, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-1698-2961","authenticated-orcid":false,"given":"Egor P.","family":"Tchesnokov","sequence":"additional","affiliation":[{"name":"Department of Medical Microbiology and Immunology, University of Alberta, Edmonton, Alberta T6G 2E1, Canada"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Linh","family":"Nguyen","sequence":"additional","affiliation":[{"name":"Department of Pharmaceutical Chemistry, UCSF, CA 94158, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-2242-3218","authenticated-orcid":false,"given":"Manisha","family":"Saini","sequence":"additional","affiliation":[{"name":"Center for Advanced Biotechnology and Medicine, Rutgers, the State University of New Jersey, Piscataway, NJ 08854, USA"},{"name":"Department of Chemistry and Chemical Biology, Rutgers, the State University of New Jersey, Piscataway, NJ 08854, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jocelyn","family":"Cantero","sequence":"additional","affiliation":[{"name":"Department of Pharmaceutical Chemistry, UCSF, CA 94158, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-8794-6784","authenticated-orcid":false,"given":"Jezrael Lafuente","family":"Revalde","sequence":"additional","affiliation":[{"name":"Department of Pharmaceutical Chemistry, UCSF, CA 94158, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-6213-5806","authenticated-orcid":false,"given":"Matthias","family":"G\u00f6tte","sequence":"additional","affiliation":[{"name":"Department of Medical Microbiology and Immunology, University of Alberta, Edmonton, Alberta T6G 2E1, Canada"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Adam R.","family":"Renslo","sequence":"additional","affiliation":[{"name":"Department of Pharmaceutical Chemistry, UCSF, CA 94158, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-2247-9345","authenticated-orcid":false,"given":"R. 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