Repositioning Lopinavir, an HIV Protease Inhibitor, as a Promising Antifungal Drug: Lessons Learned from Candida albicans—In Silico, In Vitro and In Vivo Approaches

Abstract

The repurposing strategy was applied herein to evaluate the effects of lopinavir, an aspartic protease inhibitor currently used in the treatment of HIV-infected individuals, on the globally widespread opportunistic human fungal pathogen Candida albicans by using in silico, in vitro and in vivo approaches in order to decipher its targets on fungal cells and its antifungal mechanisms of action. Secreted aspartic proteases (Saps) are the obviously main target of lopinavir. To confirm this hypothesis, molecular docking assays revealed that lopinavir bound to the Sap2 catalytic site of C. albicans as well as inhibited the Sap hydrolytic activity in a typically dose-dependent manner. The inhibition of Saps culminated in the inability of C. albicans yeasts to assimilate the unique nitrogen source (albumin) available in the culture medium, culminating with fungal growth inhibition (IC50 = 39.8 µM). The antifungal action of lopinavir was corroborated by distinct microscopy analyses, which evidenced drastic and irreversible changes in the morphology that justified the fungal death. Furthermore, our results revealed that lopinavir was able to (i) arrest the yeasts-into-hyphae transformation, (ii) disturb the synthesis of neutral lipids, including ergosterol, (iii) modulate the surface-located molecules, such as Saps and mannose-, sialic acid- and N-acetylglucosamine-containing glycoconjugates, (iv) diminish the secretion of hydrolytic enzymes, such as Saps and esterase, (v) negatively influence the biofilm formation on polystyrene surface, (vi) block the in vitro adhesion to epithelial cells, (vii) contain the in vivo infection in both immunocompetent and immunosuppressed mice and (viii) reduce the Sap production by yeasts recovered from kidneys of infected animals. Conclusively, the exposed results highlight that lopinavir may be used as a promising repurposing drug against C. albicans infection as well as may be used as a lead compound for the development of novel antifungal drugs.

From Naturally-Sourced Protease Inhibitors to New Treatments for Fungal Infections

“…For instance, secreted aspartic proteases (SAPs), are involved in several virulence processes, including tissue invasion, growth, and immune system evasion among the important human fungal pathogens, Candida albicans and C. neoformans [34,35]. Additionally, SAPs have been assessed as antifungal targets using protease inhibitors with promising results for further exploration [36][37][38]. Other important anti-virulence mechanisms include cell wall disruption or membrane pore formation initiated by protease inhibitors to deregulate ion flow and/or membrane disruption to cause leakage of internal cellular components, affecting cell viability [30,31].…”

“…ATBI binds within the active site of the HIV-1 protease (competitive inhibition), leading to inactivation of the enzyme, and thereby suggesting pharmaceutical potential as a drug for the treatment of AIDS. As described above, compounds such as HIV-1-protease inhibitors (e.g., indinavir or ritonavir) possess antifungal properties [36][37][38]99], highlighting the need for more research using ATBI against human fungal pathogens, such as Candida spp. and C. neoformans.…”

“…As identified here, important areas for further exploration include the search for natural compounds that mimic current synthetic compounds with anti-HIV activity. For example, the beneficial effects of anti-HIV protease inhibitors on the incidence of disease and the subsequent outcome of opportunistic fungal infections, such as candidiasis [36,37,98] and cryptococcosis [38,99,100,107,108]. This includes the off-target effects of anti-HIV aspartic protease inhibitors (e.g., saquinavir, indinavir and ritonavir) against hydrolytic enzymes (e.g., SAPs in C. albicans), which correspond with reduced fungal infections in HIVinfected patients [36,37,98,109].…”

“…For example, the beneficial effects of anti-HIV protease inhibitors on the incidence of disease and the subsequent outcome of opportunistic fungal infections, such as candidiasis [36,37,98] and cryptococcosis [38,99,100,107,108]. This includes the off-target effects of anti-HIV aspartic protease inhibitors (e.g., saquinavir, indinavir and ritonavir) against hydrolytic enzymes (e.g., SAPs in C. albicans), which correspond with reduced fungal infections in HIVinfected patients [36,37,98,109]. Additionally, the HIV aspartic protease inhibitor, indinavir, selectively inhibits the production of proteases and urease by C. neoformans, interfering with capsule formation and resulting in heightened susceptibility of fungal cells to intracellular killing by natural effector cells [99].…”

Special Issue: Alternative Therapeutic Approaches of Candida Infections

“…Drug repurposing may be a potential alternative therapeutic approach in certain types of fungal infections [13]. Santos et al (2021) examined the effects of lopinavir, an aspartic protease inhibitor in HIV therapy, on C. albicans by using in silico, in vitro, and in vivo approaches [14]. Based on their findings, lopinavir inhibits the yeast-to-hyphae transition, disturbs the ergosterol synthesis, blocks the adhesion to epithelial cells, and reduces the secreted aspartyl proteinase production by Candida cells [14].…”