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Repurposing approach identifies phenylpentanol derivatives as potent azole chemosensitizing agents effective against azole-resistant Candida species

The limited number of systemic antifungals and the emergence of azole-resistant Candida species constitute a growing challenge to human medicine. Combinatorial drug therapy represents an appealing approach to enhance the activity of, or restore the susceptibility to current antifungals. Here, we evaluated the fluconazole chemosensitization activity of the Pharmakon 1600 drug library against azole-resistant Candida albicans. We identified 33 non-antifungal drugs that were able to restore susceptibility to fluconazole in an azole-resistant C. albicans. Structural investigation of identified hits revealed phenylpentanol scaffold as a valuable pharmacophore for re-sensitizing azole-resistant Candida species to the effect of current azole antifungal drugs. All phenylpentanol derivatives displayed potent fluconazole chemosensitizing activities (ΣFICI 0.13-0.28) and were able to reduce fluconazole’s MIC by 15-31 fold against the tested strain. Particularly pitavastatin displayed the most potent fluconazole chemosensitizing activity (ΣFICI 0.06-0.50). The pitavastatin-fluconazole combination displayed a broad-spectrum synergistic relationship against 90% of the tested strains, including strains of C. albicans, C. glabrata, and C. auris. Moreover, pitavastatin restored the susceptibility of the multidrug-resistant C. auris to the antifungal activities of itraconazole and voriconazole. Additionally, the pitavastatin-fluconazole combination significantly reduced the biofilm-forming abilities of the tested Candida species and successfully reduced the fungal burdens in a Caenorhabditis elegans infection model. Both pitavastatin and the plain phenylpentanol scaffold were able to interfere significantly with Candida’s efflux activities as demonstrated by Nile Red efflux assays and flow cytometry. This study presents phenylpentanol derivatives as potent azole chemosensitizers that warrant further investigation.


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Candidiasis is a leading cause of healthcare-associated bloodstream infections and is 50 associated with high morbidities and mortalities. Infections caused by Candida species can range 51 from uncomplicated self-limited superficial infections of mucosal membranes to deadly 52 disseminated bloodstream and deep-seated tissue infections often associated with a mortality rate 53 of 42 -65% [1]. Current epidemiological data portrays C. albicans and C. glabrata as the major 54 causes of Candida-related infections [2][3][4][5]. However, the recent emergence of new Candida 55 species, such as multidrug-resistant C. auris is expected to impact the current epidemiological 56 trends [6][7][8]. 57 Additionally, Candida species are known for their remarkable capabilities of forming 58 robust adherent structures (i.e., biofilms) on surfaces of different abiotic surfaces, such as 59 catheters, and medical implants [9][10][11]. Biofilms limit the penetration of antifungal drugs and can 60 contribute to treatment failure and chronic infections [12]. Fungal cells residing in biofilms have 61 been reported to have increased expression of efflux genes [13,14]. Biofilms were also reported 62 to trigger the formation of Candida persisters, which can tolerate very high doses of antifungal 63 agents [15]. Collectively, these factors contribute significantly to the remarkable ability of 64 Candida's biofilms to resist the effect of antifungal drugs, especially azoles [16,17]. 65 Unfortunately, treatment of systemic Candida infections is currently limited to only three 66 major drug classes; azoles, polyenes, and echinocandins [18,19]. Azoles act as inhibitors to the 67 14α-demethylase enzyme, Erg11, which is vital for ergosterol biosynthesis. Interference with the 68 ergosterol biosynthesis pathway significantly compromises the functions of fungal cell 69 membranes. The limited toxicity, oral bioavailability, and broad-spectrum of antifungal activities 70 made azoles the most commonly prescribed drugs for controlling and treating Candida infections 71 [19,20]. The high dependence of clinicians on azole antifungal agents has been associated with 72 the emergence of azole-resistant Candida strains [21,22]. 73 Given the clinical importance of azole antifungals, there is a pressing need for potent co-74 drugs that would augment the antifungal effect of azole drugs, particularly against Candida 75 biofilms and azole-resistant strains. In this study, we explored the azole chemosensitizing activity 76 of ~1600 approved drugs and clinical molecules from the Pharmakon drug library. We identified 77 33 non-antifungal drugs that were able to restore susceptibility to fluconazole in an azole-resistant 78 C. albicans strain. A more in-depth structural investigation of identified hits revealed the 79 phenylpentanol scaffold as a valuable pharmacophore for re-sensitizing azole-resistant Candida 80 species to the effect of current azole antifungal drugs. The most potent phenylpentanol derivative 81 (pitavastatin) was further investigated in combination with azole drugs against strains of C. 82 albicans, C. glabrata, and the multidrug-resistant C. auris was evaluated for the ability to inhibit 83 Candida biofilm formation and was assessed for the ability to reduce Candida burdens in infected 84 nematodes. Furthermore, the effect of pitavastatin and the plain phenylpentanol scaffold on the 85 efflux activities of Candida strains with known efflux mechanisms was evaluated.

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Identification of drugs with fluconazole chemosensitizing activity. The Pharmakon 1600 drug 88 library was initially screened at 16 µM concentration against C. albicans NR-29448, a strain that 89 displayed high-level resistance to several azole antifungal drugs including fluconazole, 90 itraconazole, and voriconazole. In order to explore the azole chemosensitizing activities of the 91 Pharmakon 1600 drug library, two rounds of screening were performed either in the presence or 92 absence of a sub-inhibitory concentration of fluconazole (32 µg/ml). This high concentration of 93 fluconazole was selected to maximize the initial pool of active hits. Drugs that inhibited the growth 94 of C. albicans, only in the presence of fluconazole, were identified as "positive hits.". Our initial 95 screening identified 33 drugs that exhibited synergistic interactions with fluconazole against the 96 azole-resistant strain C. albicans NR-29448 (Table-1). The initial screening identified novel 97 fluconazole chemosensitizers that have never been reported before, such as bufexamac, 98 apomorphine, and sulfaquinoxaline. As expected, several drugs with reported fluconazole 99 chemosensitization activity were identified, such as the calcineurin inhibitors cyclosporin, 100 tacrolimus, sirolimus, nisoldipine, benazepril, and estrogen receptor modulators [23][24][25]. Also, 101 several antibacterial agents with reported fluconazole chemosensitization activities were 102 identified, such as sulfa drugs, doxycycline, and clofazimine [25][26][27]. However, structural analysis 103 of the identified hits revealed a common phenylpentanol core present in many of these structurally 104 diverse hit compounds. Phenylpentanol, or its saturated cyclohexylpentanol, was observed in nine 105 drugs, namely; apomorphine, atorvastatin, lovastatin, simvastatin, cholecalciferol, doxycycline, 106 quinestrol, testosterone, and norgestimate (Figure-1).

Synergistic interactions between fluconazole and phenylpentanol derivatives against C.
108 albicans NR-29448. Microdilution checkerboard assays were used to assess the synergistic 109 interactions between phenylpentanol derivatives and fluconazole against the azole-resistant C. 110 albicans NR29448 strain. Expectedly, as shown in Table- Table-3).

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Effect of the pitavastatin-fluconazole combination on the growth kinetics of Candida species. 140 The checkerboard microdilution assays revealed a significant synergistic relationship between 141 pitavastatin and fluconazole that was effective against C. albicans, C. glabrata, and C. auris. To    The azole chemosensitization activity displayed by pitavastatin is attributed, at least in part, to its 250 ability to interfere with Candida's efflux machinery. Furthermore, the efflux inhibitory activity

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The Pharmakon 1600 drug library was screened against the azole-resistant C. albicans NR-29448. 276 An overnight culture of C. albicans NR-29448 was diluted to approximately 0.5 -2.5 × 10 3 cells/ml  Biofilm inhibition assay. Three Candida species, C. albicans NR-29448, C. glabrata HM-1123, 298 and C. auris 385 demonstrated prominent ability to form robust adherent biofilms. As such, these 299 strains were used to study the antibiofilm activity of the pitavastatin-fluconazole combination. The 300 microtiter biofilm formation assay using crystal violet was used, as previously described [26].