Discovery and Structural Optimization of Spirodioxynaphthalene-Based Mdr1 Efflux Pump Inhibitor to Combat Azole Resistance in <i>Candida albicans</i>

Overexpression of efflux pump genes drives azole resistance in <i>Candida albicans</i>, highlighting the need for efflux pump inhibitors. We identified pleopcin A (<b>PA</b>), a novel spirodioxynaphthalene from <i>Pleopunctum brunnescens</i>, which reverses Mdr1-mediated azole resistance. To enhance potency, we achieved its first total synthesis and designed 82 derivatives via target-ligand analysis. Among them, <b>PA36-2</b> emerged as the most potent lead compound, showing 64-fold greater activity than <b>PA</b> in combination with fluconazole against <i>MDR1</i>-overexpressing <i>C. albicans</i> strains. Mechanistic studies confirmed <b>PA36-2</b> as a specific Mdr1 inhibitor. In addition, <b>PA36-2</b> combined with fluconazole exhibited potent in vivo activity against <i>Galleria mellonella</i> and mice infected with <i>MDR1</i>-overexpressing <i>C. albicans</i> strain. These findings support further pharmaceutical investigation of the potential value of <b>PA36-2</b> as an Mdr1 inhibitor for overcoming azole resistance in <i>C. albicans</i> infections.