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A key challenge in treating Mabs-PD is the lack of bactericidal antibiotics effective at therapeutically relevant concentrations, underscoring an urgent need for drug discovery. Targeting cell-wall synthesis is a promising approach, as evidenced by the success of broad-spectrum \u03b2-lactam antibiotics and the frontline antituberculosis drug isoniazid. However, these agents exhibit limited efficacy against Mabs, often requiring concentrations unachievable in lung tissues. Here, we used a bioluminescence-based whole-cell assay optimized to identify drugs targeting both cell-wall synthesis and the oxidative phosphorylation pathway. Screening a small drug library against Mabs revealed multiple hits, including \u03b2-lactam antibiotics, validating the effectiveness of this approach to identify cell wall-targeting agents. Among these, we identified a chemically tractable naphthalene scaffold with potent bactericidal activity. The optimized derivative GM47-1 targets MmpL3, disrupting cell wall integrity, inducing ATP leakage into the extracellular milieu, and uncoupling respiration, predominantly through the cytochrome\n                  <jats:italic>\n                    bcc:aa\n                    <jats:sub>3<\/jats:sub>\n                  <\/jats:italic>\n                  branch. Further chemical optimization resulted in a new derivative exhibiting a nanomolar minimum inhibitory concentration, with potent activity against intracellular Mabs and in a zebrafish model of infection. This study offers a promising scaffold for future therapeutic development and highlights the utility of this approach as a rapid assay platform for identifying bactericidal compounds against Mabs.\n                <\/jats:p>","DOI":"10.1101\/2025.10.26.684512","type":"posted-content","created":{"date-parts":[[2025,10,27]],"date-time":"2025-10-27T03:40:12Z","timestamp":1761536412000},"source":"Crossref","is-referenced-by-count":0,"title":["A bioluminescence-based chemical screen identifies a bactericidal naphthalene scaffold targeting MmpL3 in\n                  <i>Mycobacterium abscessus<\/i>"],"prefix":"10.64898","author":[{"ORCID":"https:\/\/orcid.org\/0000-0001-7075-931X","authenticated-orcid":false,"given":"Samsher","family":"Singh","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ria","family":"Sorayah","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yushu","family":"Chen","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Claire V.","family":"Mulholland","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Wassim","family":"Daher","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Carmen J. 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