{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"institution":[{"name":"bioRxiv"}],"indexed":{"date-parts":[[2026,6,29]],"date-time":"2026-06-29T13:45:17Z","timestamp":1782740717294,"version":"3.54.5"},"posted":{"date-parts":[[2026,6,26]]},"group-title":"Biochemistry","reference-count":79,"publisher":"openRxiv","license":[{"start":{"date-parts":[[2026,6,26]],"date-time":"2026-06-26T00:00:00Z","timestamp":1782432000000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/www.biorxiv.org\/about\/FAQ#license"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"accepted":{"date-parts":[[2026,6,26]]},"abstract":"<jats:title>Abstract<\/jats:title>\n                <jats:p>\n                  Scrub typhus is a potentially fatal infectious disease caused by the obligate intracellular bacterium\n                  <jats:italic>Orientia tsutsugamushi<\/jats:italic>\n                  . While antibiotic treatment is generally effective, it requires extended treatment, and drug resistance and treatment failures have emerged.\n                  <jats:italic>O. tsutsugamushi<\/jats:italic>\n                  encodes a deubiquitylating enzyme, OtDUB, which interferes with host ubiquitin-dependent pathways. OtDUB cleaves ubiquitin from various substrates, but whether this activity can be selectively targeted by small molecules is unknown. Here we have screened a chemically diverse small-molecule library using a fluorescence-based deubiquitylation assay to identify potential inhibitors of OtDUB. Two compounds, gentisic acid and amiloride hydrochloride, inhibited OtDUB activity at low dosage, with little effect on the related\n                  <jats:italic>Wolbachia<\/jats:italic>\n                  CidB or yeast Ulp1 enzymes. Computational docking predicted the compounds engage regions near the OtDUB catalytic pocket, suggesting a competitive mode of inhibition; this was supported by enzyme kinetic analyses. Neither compound caused detectable cytotoxicity in mammalian cells. Amiloride hydrochloride treatment reduced both total cellular deubiquitylating activity and the\n                  <jats:italic>O. tsutsugamushi<\/jats:italic>\n                  bacterial load in infected cells. While the identified compounds are not optimized inhibitors, they establish that bacterial pathogen-encoded deubiquitylating enzymes can be targeted by small molecules. Overall, our results provide a framework for using selective inhibitors as tools to study DUB function in genetically intractable intracellular bacteria and as potential treatments for scrub typhus.\n                <\/jats:p>","DOI":"10.64898\/2026.06.25.734012","type":"posted-content","created":{"date-parts":[[2026,6,26]],"date-time":"2026-06-26T13:45:20Z","timestamp":1782481520000},"source":"Crossref","is-referenced-by-count":0,"title":["Small-molecule inhibition of the\n                  <i>Orientia tsutsugamushi<\/i>\n                  deubiquitylating enzyme OtDUB impairs bacterial reproduction"],"prefix":"10.64898","author":[{"ORCID":"https:\/\/orcid.org\/0009-0009-4119-4191","authenticated-orcid":false,"given":"Min Jae","family":"Lee","sequence":"first","affiliation":[{"name":"Department of Molecular Biophysics & Biochemistry Yale University, New Haven, CT 06520"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jason R.","family":"Hunt","sequence":"additional","affiliation":[{"name":"Department of Microbiology and Immunology Virginia Commonwealth University Medical Center, School of Medicine, Richmond, VA 23298"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Soyoung","family":"Cho","sequence":"additional","affiliation":[{"name":"Department of Molecular Biophysics & Biochemistry Yale University, New Haven, CT 06520"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-1378-9162","authenticated-orcid":false,"given":"Travis J.","family":"Chiarelli","sequence":"additional","affiliation":[{"name":"Department of Microbiology and Immunology Virginia Commonwealth University Medical Center, School of Medicine, Richmond, VA 23298"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Christofer N.","family":"Perry","sequence":"additional","affiliation":[{"name":"Department of Microbiology and Immunology Virginia Commonwealth University Medical Center, School of Medicine, Richmond, VA 23298"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-6166-501X","authenticated-orcid":false,"given":"Jason A.","family":"Carlyon","sequence":"additional","affiliation":[{"name":"Department of Microbiology and Immunology Virginia Commonwealth University Medical Center, School of Medicine, Richmond, VA 23298"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-1131-5484","authenticated-orcid":false,"given":"Mark","family":"Hochstrasser","sequence":"additional","affiliation":[{"name":"Department of Molecular Biophysics & Biochemistry Yale University, New Haven, CT 06520"},{"name":"Department of Molecular, Cellular and Developmental Biology Yale University, New Haven, CT 06520"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"54368","reference":[{"key":"2026062905550442000_2026.06.25.734012v1.1","doi-asserted-by":"publisher","DOI":"10.3201\/eid2609.200918"},{"key":"2026062905550442000_2026.06.25.734012v1.2","doi-asserted-by":"publisher","DOI":"10.1002\/ptr.6573"},{"key":"2026062905550442000_2026.06.25.734012v1.3","doi-asserted-by":"publisher","DOI":"10.3201\/eid3111.250763"},{"key":"2026062905550442000_2026.06.25.734012v1.4","doi-asserted-by":"publisher","DOI":"10.1128\/iai.00469-22"},{"key":"2026062905550442000_2026.06.25.734012v1.5","doi-asserted-by":"publisher","DOI":"10.1038\/s41467-024-52119-6"},{"key":"2026062905550442000_2026.06.25.734012v1.6","doi-asserted-by":"publisher","DOI":"10.1128\/iai.00559-24"},{"key":"2026062905550442000_2026.06.25.734012v1.7","unstructured":"Allen, P. 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