S3.4a The repurposing approach identifies pitavastatin (calcium) making fluconazole fungicidal by inhibiting ergosterol synthesis

Abstract

 S3.4 Free oral paper session, September 21, 2022, 4:45 PM - 6:15 PMObjectivesMaking fluconazole (FLC) fungicidal in combination with adjuvants is a promising strategy to avoid the emergence of FLC resistance and eliminate the persistence and recurrence of fungal infections. To address this question, we combined in vitro screening of a library of FDA-approved drugs to identify compounds for making FLC fungicidal.MethodsWe performed a high-throughput screen of an FDA-approved compound library (HY-L022, MCE®), which contains 2372 drugs, to identify potentially novel FLC synergistic lethal adjuvants using broth microdilution and dose-matrix titration assays. The abilities of candidate drugs to turn FLC from fungistatic to fungicidal were further investigated by FLC disk diffusion assays carried out by four tested strains with different FLC tolerance levels (SC5314, SN152, cmp1-aid∆/cmp1-aid∆, and ADH1p-UPC2 strains). We determined the median lethal dose (LD50) of Candidate compounds by the Up-and-Down procedure (UDP) (OECD 425, 2008) via the intraperitoneal route in adult mice and used cyclosporine A and geldanamycin as control drugs to screen FLC synergistic lethal adjuvants with lower toxicity. Finally, we constructed heterozygous deletion mutants for ergosterol synthesis-related genes to identify the mechanism of action of the synergistic lethality of pitavastatin (calcium) (PIT) and FLC (Fig. 1a).ResultsWe found that 200 compounds (≤100 μm) could make FLC (4 μg/ml) fungicidal and further confirmed that 30 compounds turned FLC (4 μg/ml) from fungistatic to fungicidal at a concentration lower than 12.5 μm by broth microdilution assays (Fig. 1b). We further identified that 12/30 compounds (≤3.125 μm) can make FLC fungicidal (≤4 μg/ml) using dose-matrix titration assays. Among these compounds, PIT can make FLC fungicidal at as low as 0.78 μm (Fig. 1c). In the FLC disk diffusion assay, we identified 8 compounds (5 μm) that were superior to or equivalent to the abilities of the control drugs to eliminate the FLC tolerance of four tested strains. It was worth noting that PIT could make FLC fungicidal against all four tested strains (Fig. 1d). The LD50 value of PIT is 103.6 mg/kg and the highest of the tested compounds. Spot assay results showed feeding exogenous 100 μm ergosterol counteracted the antifungal activity of PIT (16 μm) (Fig. 2a), but did not restore the growth defect of Tet-HMG1/hmg1∆ mutant, in which the HMG1 gene expression would be inhibited by tetracycline (Fig. 2b). This paradoxical phenotype suggests that Hmg1 is unlikely to be a target of PIT. We found four heterozygous deletion mutants increased susceptibility to PIT (Fig. 2c) and the growth defect of ERG8/erg8∆ and IDI1/idi1∆ mutants exposed to PIT can be restored by ergosterol (Fig. 2d), suggesting that Erg8 and Idi1 are potential targets of PIT.ConclusionsOur study demonstrates that PIT has a better synergistic lethal effect with FLC by inhibiting ergosterol synthesis and higher safety and suggests that PIT may be repurposed as a promising adjuvant to make FLC fungicidal and enhance the efficacy of FLC in treating invasive fungal infections caused by pathogenic fungi with high FLC tolerance.