Atazanavir Resensitizes Candida auris to Azoles

Candida auris is an emerging multidrug-resistant fungus associated with high mortality rates worldwide (1). Azole antifungals have been the standard of care for the treatment of most fungal infections (2). However, the overuse of azoles has led to an upsurge of azole resistance in Candida species (3). Most C. auris strains have developed high-level azole resistance (4). Due to the increasing resistance and prevalence of infections, the CDC has classified drug-resistant C. auris as an urgent health threat which critically needs the development of new antifungals (5).

A promising strategy to thwart azole resistance and restore antimicrobial efficacy is using codrugs to potentiate the activity of azoles (3, 6–8). Repurposing FDA-approved drugs is a valuable approach for identifying new codrugs that reduces the cost and the time required for drug development (9–12). Utilizing both approaches, we identified the HIV protease inhibitor atazanavir as a codrug for azole antifungals against C. auris. The objective of this study was to assess the effects of atazanavir on the activity of azole antifungal drugs against C. auris in vitro and in vivo.

The activity of atazanavir in combination with fluconazole and itraconazole was evaluated against a panel of pathogenic C. auris strains. The atazanavir-itraconazole combination was also evaluated with a time-kill assay. Additionally, mechanistic studies were performed to determine the possible mechanism of action by which atazanavir restores the activity of azoles against C. auris. Finally, we evaluated the in vivo efficacy of the atazanavir-itraconazole combination, in the presence of ritonavir as a pharmacokinetic enhancer (13), in a murine model of disseminated C. auris infection.

Nineteen clinical isolates of C. auris (Table 1) were obtained from the Westerdijk Fungal Biodiversity Institute (Utrecht, the Netherlands), the CDC, and BEI Resources. Drugs used in the study were purchased commercially: itraconazole and ritonavir (TCI America), atazanavir (Ambeed), and fluconazole (Acros Organics). Media and reagents were obtained from commercial vendors.

The MICs of the drugs were identified following CLSI guidelines (14). We also evaluated the interactions between atazanavir and two azole drugs (fluconazole and itraconazole) against C. auris isolates using the checkerboard method, as described elsewhere (15–17). Atazanavir alone did not exhibit any antifungal activity (MICs, >128 μg/mL) against all tested C. auris isolates. When tested in combination with fluconazole, atazanavir interacted synergistically against 36.8% (7/19) of C. auris isolates, with a fractional inhibitory concentration index (ΣFICI) ranging from 0.19 to 0.38. Interestingly, atazanavir displayed the most potent synergistic interaction with itraconazole, with an effect against 100% (19/19) of C. auris isolates tested (ΣFICI, 0.09 to 0.38). These findings align with our earlier findings, where lopinavir displayed the most effective synergistic interaction with itraconazole (against 100% of C. auris isolates tested) (6). Because atazanavir interacted synergistically with itraconazole against all C. auris isolates tested, the focus for the rest of the work was to further investigate the atazanavir-itraconazole combination using several experiments, including the killing kinetics, a mechanistic study, and the in vivo efficacy against C. auris isolates.

A time-kill assay was used to investigate the killing kinetics of the atazanavir-itraconazole combination against C. auris AR0390, as described previously (3, 6, 18). As shown in Fig. 1A and B, atazanavir and itraconazole (16 and 0.5 μg/mL, respectively) exerted a fungistatic activity against the tested isolate, lowering the fungal burden by 4.5 and 4.9 log₁₀ after 24 and 48 h, respectively, compared to the negative control.

One of the key methods by which C. auris isolates withstand the activity of azole antifungals, resulting in treatment failure, is the overexpression of efflux pumps (3). Therefore, using an efflux pump inhibitor is a potential strategy for overcoming the microorganism’s resistance to antifungals. Encouraged by our previous work with lopinavir (6), we investigated the effect of atazanavir on the efflux activity, glucose utilization, and ATP synthesis of C. auris isolates. Glucose-induced Nile red efflux assays were performed using five isolates of C. auris, as reported previously (19–21). As shown in Fig. 2A, atazanavir (8 μg/mL) hindered the efflux of Nile red in all tested C. auris isolates, leading to an increase in Nile red fluorescence intensity by 40% to 50%.

A glucose-induced acidification assay was performed to assess the impact of atazanavir on glucose utilization (22). As expected, atazanavir (32 μg/mL) significantly obstructed the ability of C. auris cells to utilize glucose, and less reduction in the OD₅₉₀ values was detected (Fig. 2B).

Finally, the impact of atazanavir on the cellular ATP content of C. auris isolates was evaluated, as previously reported (23). Atazanavir (8 μg/mL) decreased the cellular ATP content in C. auris AR0390 by 20% (Fig. 2C). These results are consistent with our published data (6).

The efficacy of the itraconazole-atazanavir combination in the presence of the bioavailability enhancer ritonavir (13) was evaluated in an in vivo mouse model of disseminated candidiasis, as outlined in previous reports (24, 25). The animal experiment was conducted following the guidelines from the Virginia Tech Animal Care and Use Committee. Briefly, female immunocompromised CD-1 mice were infected with 3.33 × 10⁷ CFU/mouse C. auris AR0390 intraperitoneally. Treatments, which were administered orally 2 h after infection and continued for 48 h, included itraconazole (5 mg/kg), atazanavir plus ritonavir (90 and 30 mg/kg, respectively), and a combination of itraconazole with atazanavir-ritonavir. The mice were then euthanized, and the fungal counts in the kidneys were determined by plating onto yeast extract-peptone-dextrose (YPD) agar containing chloramphenicol (100 μg/mL). The itraconazole-atazanavir-ritonavir combination significantly decreased the fungal burden of C. auris in murine kidneys by 1.15 log₁₀ (93%) and 1.3 log₁₀ (95%) compared to the vehicle control and itraconazole alone, respectively (Fig. 3).

Our findings highlight that HIV protease inhibitors are a promising class of drugs that can be combined with azole antifungals to potentially treat fungal infections caused by challenging pathogens such as C. auris. It is noteworthy that patients with HIV are at higher risk for serious fungal infections, and protease inhibitors, such as atazanavir and ritonavir, are part of the routine therapy for them (26). Therefore, azole and HIV protease inhibitor combination therapy could be the best fit for such scenarios (27).

In conclusion, our results show that atazanavir is a promising drug that enhances the activity of azole antifungals against C. auris both in vitro and in vivo.

M.N.S. and E.A.S. are supported in part by the National Institutes of Health (R01AI141439). We declare no conflicts of interest.