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The limited antifungal arsenal and emerging multidrug-resistant species necessitate the development of new therapies. One strategy for combating drug-resistant pathogens is the administration of molecules that restore fungal susceptibility to approved drugs. Accordingly, we carried out a screen to identify small molecules that could restore the susceptibility of pathogenic\n                    <jats:italic>Candida<\/jats:italic>\n                    species to azole antifungals. This screening effort led to the discovery of novel 1,4-benzodiazepines that restore fluconazole susceptibility in resistant isolates of\n                    <jats:italic>Candida albicans<\/jats:italic>\n                    , as evidenced by 100\u20131,000-fold potentiation of fluconazole activity. This potentiation effect was also observed in azole-tolerant strains of\n                    <jats:italic>C. albicans<\/jats:italic>\n                    and in other pathogenic\n                    <jats:italic>Candida<\/jats:italic>\n                    species. The 1,4-benzodiazepines selectively potentiated different azoles, but not other approved antifungals. A remarkable feature of the potentiation was that the combination of the compounds with fluconazole was fungicidal, whereas fluconazole alone is fungistatic. Interestingly, the potentiators were not toxic to\n                    <jats:italic>C. albicans<\/jats:italic>\n                    in the absence of fluconazole, but inhibited virulence-associated filamentation of the fungus. We found that the combination of the potentiators and fluconazole significantly enhanced host survival in a\n                    <jats:italic>Galleria mellonella<\/jats:italic>\n                    model of systemic fungal infection. Taken together, these observations validate a strategy wherein small molecules can restore the activity of highly used anti-infectives that have lost potency.\n                  <\/jats:p>\n                  <jats:sec>\n                    <jats:title>IMPORTANCE<\/jats:title>\n                    <jats:p>\n                      In the last decade, we have been witnessing a higher incidence of fungal infections, due to an expansion of the fungal species capable of causing disease (e.g.,\n                      <jats:italic>Candida auris<\/jats:italic>\n                      ), as well as increased antifungal drug resistance. Among human fungal pathogens,\n                      <jats:italic>Candida<\/jats:italic>\n                      species are a leading cause of invasive infections and are associated with high mortality rates. Infections by these pathogens are commonly treated with azole antifungals, yet the expansion of drug-resistant isolates has reduced their clinical utility. In this work, we describe the discovery and characterization of small molecules that potentiate fluconazole and restore the susceptibility of azole-resistant and azole-tolerant\n                      <jats:italic>Candida<\/jats:italic>\n                      isolates. Interestingly, the potentiating 1,4-benzodiazepines were not toxic to fungal cells but inhibited their virulence-associated filamentous growth. Furthermore, combinations of the potentiators and fluconazole decreased fungal burdens and enhanced host survival in a\n                      <jats:italic>Galleria mellonella<\/jats:italic>\n                      model of systemic fungal infections. Accordingly, we propose the use of novel antifungal potentiators as a powerful strategy for addressing the growing resistance of fungi to clinically approved drugs.\n                    <\/jats:p>\n                  <\/jats:sec>","DOI":"10.1128\/mbio.00479-23","type":"journal-article","created":{"date-parts":[[2023,6,16]],"date-time":"2023-06-16T10:09:07Z","timestamp":1686910147000},"update-policy":"https:\/\/doi.org\/10.1128\/asmj-crossmark-policy-page","source":"Crossref","is-referenced-by-count":20,"title":["Small molecules restore azole activity against drug-tolerant and drug-resistant\n                    <i>Candida<\/i>\n                    isolates"],"prefix":"10.1128","author":[{"given":"Philip E.","family":"Alabi","sequence":"first","affiliation":[{"name":"Department of Chemistry, Brown University , Providence, Rhode Island, USA"},{"name":"Department of Pharmaceutical Chemistry, University of California San Francisco , San Francisco, California, 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