Repurposing approach identifies pitavastatin as a potent azole chemosensitizing agent effective against azole-resistant Candida species

Abstract

the limited number of antifungals and the rising frequency of azole-resistant Candida species are growing challenges to human medicine. Drug repurposing signifies an appealing approach to enhance the activity of current antifungal drugs. Here, we evaluated the ability of Pharmakon 1600 drug library to sensitize an azole-resistant Candida albicans to the effect of fluconazole. The primary screen revealed 44 non-antifungal hits were able to act synergistically with fluconazole against the test strain. Of note, 21 compounds, showed aptness for systemic administration and limited toxic effects, were considered as potential fluconazole adjuvants and thus were termed as "repositionable hits". A follow-up analysis revealed pitavastatin displaying the most potent fluconazole chemosensitizing activity against the test strain (ΣFICI 0.05) and thus was further evaluated against 18 isolates of C. albicans (n = 9), C. glabrata (n = 4), and C. auris (n = 5). Pitavastatin displayed broad-spectrum synergistic interactions with both fluconazole and voriconazole against ~89% of the tested strains (ΣFICI 0.05-0.5). Additionally, the pitavastatin-fluconazole combination significantly reduced the biofilm-forming abilities of the tested Candida species by up to 73%, and successfully reduced the fungal burdens in a Caenorhabditis elegans infection model by up to 96%. This study presents pitavastatin as a potent azole chemosensitizing agent that warrant further investigation. Candida species are the most common nosocomial fungal pathogens and are a major cause of healthcare-associated bloodstream infections 1-3. In the USA, Candida species are the fourth-leading cause of bloodstream infections 4,5. Diseases caused by Candida species can range from self-limited uncomplicated superficial lesions to a deadly form of disseminated invasive infection that is often associated with a high mortality rate (42-65%) 6. Available epidemiological data derived from several independent surveillance studies portray C. albicans and C. glabrata as the two major causes of Candida-related infections in North America and Europe 7-9. However, the recent emergence of C. auris has become a global health concern, considering its unique multidrug resistance nature, the efficient ability to colonize human tissues and to provoke several global outbreaks 10,11. Thus, C. auris was recently categorized by the US Centers for Disease Control and Prevention (CDC) as an urgent health threat 12. Treatment of systemic Candida infections is currently limited to only three major drug classes; azoles, polyenes, and echinocandins 13,14. The limited toxicity, oral bioavailability, and broad-spectrum of antifungal activities made azoles the most commonly prescribed drugs for treating and controlling Candida infections 14,15. Azoles

Antifungal Drug Repurposing

“…Related antifungal “chemosensitization” has been developed recently as a new intervention strategy, where co-application of a repurposed compound (chemosensitizer), such as food additives, with conventional drugs enhanced the antifungal efficacy of the co-applied drugs [ 77 , 233 ]. A chemosensitizer causes the target pathogen to be more susceptible to the co-applied conventional drug via the modulation of the pathogen’s defense system, such as the oxidative stress signaling system or cell-wall integrity pathway.…”

Combination Therapy to Treat Fungal Biofilm-Based Infections

“…In C. albicans, expression of CDR1 , CDR2 and MDR1 , FLU1 is often determined as these genes encode efflux pumps of the ABC superfamilies and MFS pumps, respectively, which are the main azole efflux pumps of this yeast [ 137 , 145 , 147 , 222 ]. Additionally, the red fluorescent dye Rhodamine 6G [ 223 ] is frequently used to measure yeast efflux pump activity as this dye uses the same transporters as azoles in yeast [ 137 , 144 , 145 , 147 ]. In this assay, cells are incubated in the presence of rhodamine 6G, which is taken up by the cells in starvation conditions, and the tested compounds.…”

“…In this assay, cells are incubated in the presence of rhodamine 6G, which is taken up by the cells in starvation conditions, and the tested compounds. After a period of efflux, the remaining fluorescence of intracellular rhodamine 6G as compared to the culture before treatment can be measured by flow cytometry or rhodamine 6G fluorescence in the supernatants can be determined using a microplate reader or a microscope [ 137 , 144 , 145 , 147 ]. Alternatively, similar assays using other dyes, like Rh123 or Nile Red efflux assays, can be performed [ 224 , 225 ].…”

“…A biofilm-active combination can affect both virulence factors and tolerance mechanisms simultaneously. Through screening of a drug repositioning library, Eldesouky and colleagues identified antihyperlipidemic statin drugs as novel fluconazole potentiators against azole-resistant C. albicans and pitavastatin was the most promising hit [ 144 ]. Although discovered in a planktonic setup, the pitavastatin-fluconazole combination appeared to reduce biofilm formation of C. albicans isolates, C. glabrata isolates and isolates of emerging pathogen C. auris.…”

“…However, this combination was not active against mature C. albicans biofilms. Apparently, pitavastatin interfered with ABC transporter efflux pumps [ 144 ]. Furthermore, a synergistic effect of dexamethasone and fluconazole against fluconazole-resistant C. albicans planktonic cultures and biofilms in early state of biofilm development has been observed [ 145 ].…”