In vitro combination effects and mechanisms of Revaprazan with Triazole antifungal drugs on Aspergillus

Potassium-competitive acid blockers (P-CABs), such as revaprazan (REV), act by competitively inhibiting potassium (K⁺) binding on the proton pump (H⁺/K⁺-ATPase) and are reversible K⁺ antagonists. This study evaluated REV combined with triazoles (itraconazole, ITR; voriconazole, VOR; posaconazole, POS) against 30 clinical Aspergillus isolates (14 A. fumigatus, 12 A. flavus, 4 A. terreus) using MIC and FICI assays. REV demonstrated dose-dependent synergy (FICI ≤ 0.5) with POS in 90% of isolates (27/30), reducing MIC values by 4-8fold, while synergy with ITR occurred in 20% (6/30). Species-specific analysis revealed highest efficacy against A. flavus (91.7%, 11/12) and A. terreus (100%, 4/4). No synergy was observed with VOR. Furthermore, the mechanisms underlying the synergistic effects of REV with triazoles were investigated using an A. fumigatus major facilitator superfamily (MFS) transporter gene-deficient strains. Knockout strains ΔAF-MFS32 and ΔAF-MFS35, selected from our MFS transporter gene-deficient strains, showed a abolishment of synergistic interaction when combined with REV and the triazoles ITR and POS. Suggesting a functional link between these transporters and the drug interaction. Disk diffusion and efflux pump assays corroborated these findings (P < 0.05). These in vitro results identify REV + POS as a promising combination against Aspergillus, though in vivo studies and mechanistic validation of transporter inhibition are warranted.Overall, this study shows that the potassium-competitive acid pump antagonist REV exhibits synergistic antifungal activity with triazoles in treating Aspergillus infections. The MFS transporter AF-MFS32 and AF-MFS35 are implicated as possible targets mediating the synergistic interaction between REV and these antifungal drugs.