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  • Growth inhibition assays are the most commonly used to

    2023-01-31

    Growth-inhibition assays are the most commonly used to identify antifungal small molecules. However, they have several facts that limit their use since many pathogenic fungi grow as filaments making difficult a correlation between growth and OD (optical density). They are not useful for identifying molecules active against fungal biofilms and they cannot differentiate molecules that inhibit growth and those that kill the microorganism [8]. Recently, to address such limitations, new high throughput screening assays have been developed. In many of them, the approach has been to asses loss of cellular integrity using as markers of cell lysis the cytoplasmic enzyme adenylate kinase [99], or dyes such as AlamarBlue®, XTT and resazurine that are converted to fluorescent molecules when they are metabolized by viable Kaempferol-3-rutinoside organisms [100], [101].
    Mechanisms of antifungal resistance The mechanisms of antifungal resistance have been reported at the molecular level for most antifungal agents and fungal pathogens [102], [103], [104]. To counteract the fungicidal or fungistatic effects of all antifungal classes, microorganisms develop three major mechanisms of resistance [7], [104]. The first is based on decreasing the effective drug concentration, the second is drug target alterations and the third is due to modifications of metabolism to divert the toxic effects exerted by some antifungal agents. CDR1 and CDR2 are the major transporters involved in azole resistance in C. albicans. Upregulation enhances drug efflux and reduces their accumulation in the cells [105], [106]. Many others ABC transporters involved in azole resistance have been described in C. glabrata (CgCDR1, CgCDR2, CgSNQ2) and AFR1 in C. neoformans. In A. fumigatus and A. nidulans ABC transporters have been identified as a resistance mechanism to azole. MDR1 is the MFS involved in the development of azole resistance in clinical isolates form C. albicans and C. dubliniensis and its upregulation results in increased azole efflux [107]. FLU1 from C. albicans is another MFS involved in fluconazole resistance [108]. Upregulation of ABC and MFS transporters is mediated by specific regulations in resistant fungal pathogens. Point mutations defined as gain-of-function mutations (GOF) in these regulators confer an inherent high expression level of the transporters in the drug-resistance strains [7]. GOF mutations in the transcription factor Upc2p [109] caused increased fluconazole resistance in C. albicans. In the same way, upregulation of Cyp51A in A. fumigatus lead to azole resistance mediated by duplication of 34 and 42bp in the Cyp51A promoter [110]. Another way to decrease the effective drug concentration is overexpression of the ERG11 (gene encoding 14-α sterol demethylase) and/or other genes of sterol biosynthesis, such as ERG1, ERG3, ERG7, ERG9 or ERG25 which encode enzymes downstream C14α-demethylase such upregulation leads to azole resistance [[99], [111], [112], [113]]. Another mechanism to reduce the effective drug concentration in some medically relevant fungi including Candida, Aspergillus, Cryptococcus, Trichosporon, Coccidioides and Pneumocystis[114], [115] is the ability to form biofilms increasing their resistance to several drugs including azoles, pyrimidine analogs and polyenes [116]. Biofilms are multicellular structures in which cells form a dense network that is covered by a matrix of polysaccharides, carbohydrates, proteins and signaling molecules that restricts the penetration of drugs by formation of a diffusion barrier sequestering the antifungal agents [117], [118], [119], [120]. Only amphotericin B and the echinocandins (e.g. caspofungin and micafungin) have demonstrated consistent in vitro activity against C. albicans biofilms [121]. However, even with these two agents, Candida biofilm-related infections are extremely difficult, if not impossible, to eradicate [122]. Target drug alteration is another mechanism by which fungal pathogens are able to develop resistance. This mechanism has been reported for azoles and echinocandins. Mutations in ERG11 in C. albicans decrease affinity for azoles leading to resistance. Many of these point mutations decreased affinity to fluconazole and have a moderate effect on posaconazole [123]. In A. fumigatus single mutations in Cyp51A are the most frequent mechanism to confer high level resistance to azoles in clinical isolates of this species [124].