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Therefore, agents active against both replicating and non-replicating mycobacteria remain important to explore. Here, we investigated secondary metabolites from the Indonesian marine-derived fungus Aspergillus ostianus for activity against Mycobacterium smegmatis, a BSL-1 mycobacterial model, under aerobic and hypoxia-induced non-replicating conditions, and examined the underlying mechanism. Bioassay-guided fractionation and spectroscopic analysis identified three hydroxamate pyrazinones: neohydroxyaspergillic acid (NHAA), hydroxyaspergillic acid (HAA), and neoaspergillic acid (NAA). The MIC values were 1.56 \u00b5g\/mL for NHAA and 3.13 \u00b5g\/mL for HAA and NAA under both aerobic and hypoxic atmospheres. Time-kill kinetics showed \u22653-log10 CFU reductions within 24\u201372 h at 4\u20138\u00d7 MIC under aerobic conditions and within 48\u201396 h at 4\u20138\u00d7 MIC under hypoxia, with no regrowth at the final sampling point. Scanning electron microscopy and release of UV-absorbing intracellular material at OD260\/OD280 were consistent with envelope disruption in both atmospheres. Antimycobacterial activity was attenuated in a concentration-dependent manner by exogenous Fe3+ and was reversed at 100 \u00b5M FeCl3, whereas isoniazid activity was unaffected, supporting iron-reversible and pyrazinone-specific killing. Together with the established Fe3+-binding hydroxamate pharmacophore shared by this compound class, these findings support iron sequestration as a plausible mechanism and identify fungal hydroxamate pyrazinones as scaffolds that retain bactericidal activity against hypoxia-adapted non-replicating mycobacteria, warranting further evaluation in M. tuberculosis models.<\/jats:p>","DOI":"10.3390\/md24070236","type":"journal-article","created":{"date-parts":[[2026,7,3]],"date-time":"2026-07-03T16:15:53Z","timestamp":1783095353000},"page":"236","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Iron-Reversible Bactericidal Activity of Marine-Derived Aspergillus ostianus Hydroxamate Pyrazinones Against Replicating and Hypoxia-Induced Non-Replicating Mycobacterium smegmatis"],"prefix":"10.3390","volume":"24","author":[{"ORCID":"https:\/\/orcid.org\/0000-0003-3966-582X","authenticated-orcid":false,"given":"Muhammad","family":"Azhari","sequence":"first","affiliation":[{"name":"Department of Pharmacochemistry, School of Pharmacy, Institut Teknologi Bandung, Bandung 40132, Indonesia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Shinnosuke","family":"Isshiki","sequence":"additional","affiliation":[{"name":"Laboratory of Natural Products for Drug Discovery, Graduate School of Pharmaceutical Sciences, The University of Osaka, Suita 565-0871, Japan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Riku","family":"Horinouchi","sequence":"additional","affiliation":[{"name":"Laboratory of Natural Products for Drug Discovery, Graduate School of Pharmaceutical Sciences, The University of Osaka, Suita 565-0871, Japan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Marlia","family":"Singgih","sequence":"additional","affiliation":[{"name":"Department of Pharmacochemistry, School of Pharmacy, Institut Teknologi Bandung, Bandung 40132, Indonesia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-2771-1948","authenticated-orcid":false,"given":"Masayoshi","family":"Arai","sequence":"additional","affiliation":[{"name":"Laboratory of Natural Products for Drug Discovery, Graduate School of Pharmaceutical Sciences, The University of Osaka, Suita 565-0871, Japan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Afrillia Nuryanti","family":"Garmana","sequence":"additional","affiliation":[{"name":"Department of Pharmacology and Clinical Pharmacy, School of Pharmacy, Institut Teknologi Bandung, Bandung 40132, Indonesia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Rika","family":"Hartati","sequence":"additional","affiliation":[{"name":"Department of Pharmaceutical Biology, School of Pharmacy, Institut Teknologi Bandung, Bandung 40132, Indonesia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-8087-9152","authenticated-orcid":false,"given":"Yuni Elsa","family":"Hadisaputri","sequence":"additional","affiliation":[{"name":"Department of Biological Pharmacy, Faculty of Pharmacy, Universitas Padjadjaran, Sumedang 45363, Indonesia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Nunung","family":"Yuniarti","sequence":"additional","affiliation":[{"name":"Department of Pharmacology and Clinical Pharmacy, Faculty of Pharmacy, Universitas Gadjah Mada, Yogyakarta 55281, Indonesia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-5504-6140","authenticated-orcid":false,"given":"Elin","family":"Julianti","sequence":"additional","affiliation":[{"name":"Department of Pharmacochemistry, School of Pharmacy, Institut Teknologi Bandung, Bandung 40132, Indonesia"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2026,7,3]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","unstructured":"Salina, E.G., and Makarov, V. 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