Resistance gene-guided genome mining identifies HB-35018, a novel spiro-cis-decalin tetramic acid from Aspergillus terreus that functions as a potent, broad-spectrum antifungal through covalent inhibition of acetolactate synthase (ALS) via an unprecedented lysine-targeting mechanism involving strain-relieving bond fragmentation, demonstrating superior efficacy compared to known ALS inhibitors against pathogenic fungi including azole-resistant isolates and validating ALS as essential for Aspergillus fumigatus virulence in invasive aspergillosis.

Invasive fungal infections claim over two million lives annually, a problem exacerbated by rising resistance to current antifungal treatments and an increasing population of immunocompromised individuals. Despite this, antifungal drug development has stagnated, with few novel agents and fewer novel targets explored in recent decades. Here, we validate acetolactate synthase (ALS), an enzyme critical for branched-chain amino acid biosynthesis and absent in humans, as a promising target for new therapeutics. Using resistance gene-guided genome mining, we discovered a biosynthetic gene cluster in Aspergillus terreus encoding HB-35018 (1), a novel spiro-cis-decalin tetramic acid that potently inhibits ALS. Biochemical and antifungal assays demonstrate that 1 surpasses existing ALS inhibitors in efficacy against Aspergillus fumigatus and other pathogenic fungi. Structural studies via cryo-electron microscopy reveal a unique covalent binding interaction between compound 1 and ALS, distinct from known inhibitors and finally, we demonstrate that ALS is essential for the virulence in a mouse model of invasive aspergillosis. These findings position ALS as a promising target for antifungal development and demonstrate the potential of resistance gene-guided genome mining for antifungal discovery.